Vsel literature oristem june2013 v 1 0 by Bruce Cameron

LITERATURE REVIEW Very Small Embryonic-Like (VSEL) Cells

Highlights include:

A study from 2013 demonstrating that collagen sponges seeded with VSEL cells have the capacity to facilitate new bone formation following implant into cranial defects (skull bone).

Research from 2012 which demonstrated that, following acute limb ischaemia (oxygen deprivation) VSEL cells are mobilised into the blood stream, potentially playing a role in encouraging repair and regeneration in tissues deprived of oxygen.

Another study looking at heart function after coronary occlusion concluded that VSEL cells expanded in culture retain the ability to alleviate left ventricular dysfunction, substantially improved heart function. The results support the potential therapeutic utility of VSEL cells for cardiac repair.

In a study from 2011, Americian scientists we able to demonstrate pluripotency of VSEL cells which were induced down a hematopoietic lineage. Purified from bone marrow, VSEL cells were co-cultured with OP9 stroma cell line and tested for their hematopoietic potential. The authors found that VSEL cells were able to differentiate along the hematopoietic lineage in a similar way as embryonic stem cells or inducible pluripotent stem cells.

Clinical evidence that VSEL cells are mobilized into peripheral blood in patients after stroke was provided in 2009. The authors demonstrated that following a stroke, there is a significant increase in the number of circulating VSEL cells which had been mobilised from the bone marrow. They concluded that stroke triggers the mobilisation VSEL cells potentially as part of the repair and regeneration process.

CONTENTS: Very small embryonic-like cells: Biology and function of these potential endogenous pluripotent stem cells in adult tissues ----------------------------------------------------------------------------------------------------------------------------------------------- 7 Human very small embryonic-like cells generate skeletal structures, in vivo -------------------------------------------------------------- 7 Very small embryonic-like stem cells: Implications in reproductive biology --------------------------------------------------------------- 8 Murine and human very small embryonic-like cells: a perspective --------------------------------------------------------------------------- 8 In vivo ischemic limb injury activates proliferation and mobilization of pluripotent bone marrow-derived very small embryonic-like stem cells ------------------------------------------------------------------------------------------------------------------ 9 Prolonged strenuous exercise expands the population of developmentally early stem cells in bone marrow and mobilizes them into peripheral blood-novel evidence that strongly supports a positive effect of physical activity on extension of life span at the level of stem cells -------------------------------------------------------------------------------------- 9 Novel evidence that a quiescent murine population of bone marrow (BM)-residing, developmentally early, very small SCA-1+LIN-CD45-cells is highly responsive to prolonged bleeding by in vivo proliferation and differentiation into CD45+ hematopoietic stem/progenitor cells (HSPCS) ---------------------------------------------------------------- 10 A novel view of bone marrow as a â&#x20AC;&#x153;stem cell sensorâ&#x20AC;? of tissue/organ damage-evidence that in vivo exposure to the neurotoxin kainic acid (KA) induces proliferation and neural specification of developmentally early stem cells directly in bone marrow before they are mobilized into peripheral blood ------------------------------------------------- 12 Development of a highly efficient method for isolating very small embryonic-like stem cells identified in adult mouse bone and their stem cell characteristics ------------------------------------------------------------------------------------------ 13 Peripheral blood stem cells: phenotypic diversity and potential clinical applications ------------------------------------------------- 14 Global gene expression analysis of very small embryonic-like stem cells reveals that the Ezh2-dependent bivalent domain mechanism contributes to their pluripotent state ------------------------------------------------------------------------ 14 Human umbilical cord is a unique and safe source of various types of stem cells suitable for treatment of hematological diseases and for regenerative medicine ------------------------------------------------------------------------------------ 15 Mobilization of very small embryonic-like stem cells by G-CSF in MACO mice ---------------------------------------------------------- 15 Mobilization of OCT-4+ very small embryonic like stem cells, hematopoietic stem cells and endothelial progenitor cells in children with congenital heart disease undergoing cardiac surgery ----------------------------------------------- 16 Mobilization of various types of pluripotent progenitor and stem cells is surrogate prognostic marker of mucosal healing in early clinical remission in Crohn's disease ------------------------------------------------------------------------------- 16 Response of healthy volunteers to granulocyte-colony stimulating factor --------------------------------------------------------------- 17

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Single cell level genome-wide gene expression analysis of bone marrow-derived oct-4 + very small embryonic-like stem cells (VSELs) revealed that a polycomb group protein Ezh2 regulates VSELs pluripotency by maintaining bivalent domains at promoters of important homeodomain-containing developmental transcription factors ---------------------------------------------------------------------------------------------------------------- 18 Identification of SSEA-4+ very small embryonic-like stem cells in human myocardium ----------------------------------------------- 19 Aldehyde dehydrogenase (ALDH) - a promising new candidate for use in preclinical and clinical selection of pluripotent very small embryonic-like stem cells (VSEL SCs) of high long-term repopulating hematopoietic potential----------------------------------------------------------------------------------------------------------------------------------------------------- 20 Transplantation of expanded bone marrow-derived very small embryonic-like stem cells (VSEL-SCs) improves left ventricular function and remodelling after myocardial infarction -------------------------------------------------------------------------- 20 Clinical evidence for mobilization of very small embryonic/epiblast like stem cells (VSEL-SCS) in patients with inflammatory bowel disease --------------------------------------------------------------------------------------------------------------------------- 21 Circulating stem cells in patients with gastric malignancies ---------------------------------------------------------------------------------- 22 The mobilization of very small embryonic-like stem cells (VSELs) and multipotent mesenchymal stem cells (MSCs) into peripheral blood in pancreatic cancer patients correlates with tumor stage -------------------------------------------- 23 Identification and isolation from either adult human bone marrow or G-CSF-mobilized peripheral blood of CD34(+)/CD133(+)/CXCR4(+)/ Lin(-)CD45(-) cells, featuring morphological, molecular, and phenotypic characteristics of very small embryonic-like (VSEL) stem cells ------------------------------------------------------------------------------- 23 Effect of granulocyte-colony stimulating factor on mobilization and recruitment of very small embryonic-like stem cells --------------------------------------------------------------------------------------------------------------------------------------------------- 24 The role of pluripotent embryonic-like stem cells residing in adult tissues in regeneration and longevity ---------------------- 25 Optimal recovery of SSEA-4+/OCT-4+/CDI33+/CXCR4+/LIN-/ CD45- very small embryonic-like (VSEL) stem cells from umbilical cord blood (CB) using plasma depletion/reduction (PDR) compared to red cell reduction -------------- 25 Adult murine bone marrow-derived very small embryonic-like stem cells differentiate into the hematopoietic lineage after coculture over OP9 stromal cells --------------------------------------------------------------------------------------------------- 26 Stem cells for neural regeneration--a potential application of very small embryonic-like stem cells ----------------------------- 27 A novel paradigm in stem cell trafficking: The ratio of peripheral blood sphingosine-1 phosphate (S1P) to bone marrow ceramide-1 phosphate (C1P) regulates mobilization and homing of hematopoietic stem cells ----------------- 27 Intermittent hypoxia mobilizes bone marrow-derived very small embryonic-like stem cells and activates developmental transcriptional programs in mice ------------------------------------------------------------------------------------------------ 28 Characterisation of circulating stem and progenitor cells in type 2 diabetic patients with foot ulceration --------------------- 29 Molecular characterization of isolated from murine adult tissues very small embryonic/epiblast like stem cells (VSELs) ----------------------------------------------------------------------------------------------------------------------------------------- 30

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Treatment viability of stem cells in ophthalmology --------------------------------------------------------------------------------------------- 30 Cells expressing stem cells (sc) and early gastrointestinal markers are mobilized into peripheral blood in patients with inflammatory bowel disease -prognostic and therapeutic implications --------------------------------------------- 31 Bone marrow transplantation temporarily improves pancreatic function in streptozotocin-induced diabetes: potential involvement of very small embryonic-like cells ------------------------------------------------------------------------------------- 32 Resident bone marrow stem cells are recruited to peripheral circulation in children with OSA: Relevance to endothelial function ---------------------------------------------------------------------------------------------------------------------------------- 33 In vitro and in vivo evidence that umbilical cord blood (UCB)-derived CD45-/SSEA-4+/OCT-4+/CD133+/ CXCR4+/lin - very small embryonic/epiblast like stem cells (VSELs) do not contain clonogenic hematopoietic progenitors but are highly enriched in more primitive stem cells - Novel view on hierarchy of UCB stem cell compartment ---------------------------------------------------------------------------------------------------------------------------------------------- 33 Novel evidence that very small embryonic like stem cells (vsels) are mobilized into peripheral blood in patients with inflammatory bowel diseases - Correlation with young age and severity of disease -------------------------------- 34 CD45-/Lin-/CD133+/ALDH-low VSEL stem cells isolated from cord blood-as potential long term repopulating hematopoietic stem cells (LT-HSC) ------------------------------------------------------------------------------------------------------------------- 35 An evidence that CD45-lin-sca-1 + oct-4 + VSEL stem cells are embryonic remnants and are present in embryonic tissues during development ------------------------------------------------------------------------------------------------------------ 36 Novel epigenetic mechanisms that control pluripotency and quiescence of adult bone marrow-derived Oct4(+) very small embryonic-like stem cells ----------------------------------------------------------------------------------------------------- 37 Stem cell-based therapy in central nervous system diseases --------------------------------------------------------------------------------- 37 Potential application of adult stem cells in retinal repair--challenge for regenerative medicine ----------------------------------- 38 Circulating Oct-4+SSEA-4+ very small embryonic-like cells and improvement of LVEF in patients with acute myocardial infarction ---------------------------------------------------------------------------------------------------------------------------- 38 Identification of small Sca-1(+), Lin(-), CD45(-) multipotential cells in the neonatal murine retina -------------------------------- 39 Society for Cardiovascular Angiography and Interventions' 32nd Annual Scientific Sessions --------------------------------------- 39 Evidence of mobilization of pluripotent and very small embryonic-like (VSEL) stem cells in patients with myocardial ischemia: A potential therapeutic target ------------------------------------------------------------------------------------------- 40 Characterization of endogenous stem cells from the mouse penis that express an embryonic stem cell gene and undergo differentiation into several cell lineages ----------------------------------------------------------------------------------------- 40 A multi-instrumental approach to identify and purify very small embryonic like stem cells (VSELs) from adult tissues -------------------------------------------------------------------------------------------------------------------------------------------------------- 41 Clinical evidence that very small embryonic-like stem cells are mobilized into peripheral blood in patients after stroke -------------------------------------------------------------------------------------------------------------------------------------------------------- 41

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Mobilization of bone marrow-derived Oct-4+ SSEA-4+ very small embryonic-like stem cells in patients with acute myocardial infarction ---------------------------------------------------------------------------------------------------------------------------- 42

Mobilization of Bone Marrow-Derived Oct-4 + SSEA-4 + Very Small Embryonic-Like Stem Cells in Patients with Acute Myocardial Infarction --------------------------------------------------------------------------------------------------------------------------- 43 Fetal Liver Very Small EmbryonicEpiblast Like Stem Cells Follow Developmental Migratory Pathway of Hematopoietic Stem Cells ------------------------------------------------------------------------------------------------------------------------------ 44 A population of serum deprivation-induced bone marrow stem cells (SD-BMSC) expresses marker typical for embryonic and neural stem cells ---------------------------------------------------------------------------------------------------------------- 44 Mobilization of stem/progenitor cells from bone marrow into peripheral blood during stress related to administration of sodium fluoride ------------------------------------------------------------------------------------------------------------------- 45 Circulating very small embryonic-like stem cells and the recovery of the LVEF in patients with acute myocardial infarction ---------------------------------------------------------------------------------------------------------------------------------------------------- 45 Stem cells in adult retina--current state of research, future therapeutic prospects --------------------------------------------------- 46 Novel epigenetic mechanisms that control pluripotency and quiescence of adult bone marrow-derived Oct4 + very small embryonic-like stem cells -------------------------------------------------------------------------------------------------------------- 46 Very small embryonic-like stem cells in adult tissues-potential implications for aging ------------------------------------------------ 47 Gene networks and biological pathways in bone marrow-derived Very Small Embryonic Stem Cells (VSEL) from mice following Intermittent Hypoxia (IH) --------------------------------------------------------------------------------------------------- 47 Very small embryonic like (VSEL) stem cells: Characterization, biological significance, and potential applications ----------- 48 Very small embryonic-like (VSEL) stem cells in adult organs and their potential role in rejuvenation of tissues and longevity ----------------------------------------------------------------------------------------------------------------------------------------------- 48 Evidence that very small embryonic-like stem cells are mobilized into peripheral blood -------------------------------------------- 49 Phenotypic and functional characterization of hematopoietic stem cells ---------------------------------------------------------------- 49 Very small embryonic-like (VSEL) stem cells: purification from adult organs, characterization, and biological significance ------------------------------------------------------------------------------------------------------------------------------------------------- 50 Transplantation of bone marrow-derived very small embryonic-like stem cells attenuates left ventricular dysfunction and remodeling after myocardial infarction -------------------------------------------------------------------------------------- 50 Bone marrow - Home of versatile stem cells ------------------------------------------------------------------------------------------------------ 51 Hunt for pluripotent stem cell - Regenerative medicine search for almighty cell ------------------------------------------------------- 51 Identification of very small embryonic like (VSEL) stem cells in bone marrow ---------------------------------------------------------- 52 An efficient two-step method to purify very small embryonic-like (VSEL) stem cells from umbilical cord blood (UCB). ------------------------------------------------------------------------------------------------------------------------------------------------ 52

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Morphological characterization of very small embryonic-like stem cells (VSELs) by ImageStream system analysis ---------- 53 Bone marrow-derived very small embryonic-like stem cells: Their developmental origin and biological significance ------- 53 Adult marrow-derived very small embryonic-like stem cells and tissue engineering -------------------------------------------------- 54 Morphological and molecular characterization of novel population of CXCR4+ SSEA-4+ Oct-4+ very small embryonic-like cells purified from human cord blood: preliminary report --------------------------------------------------------------- 54 Physiological and pathological consequences of identification of very small embryonic like (VSEL) stem cells in adult bone marrow ----------------------------------------------------------------------------------------------------------------------------------- 55 The pleiotropic effects of the SDF-1-CXCR4 axis in organogenesis, regeneration and tumorigenesis ----------------------------- 55 A population of very small embryonic-like (VSEL) CXCR4 + SSEA-1 + Oct-4 + stem cells identified in adult bone marrow ---------------------------------------------------------------------------------------------------------------------------------------------- 56 The developmental deposition of epiblast/germ cell-line derived cells in various organs as a hypothetical explanation of stem cell plasticity? ------------------------------------------------------------------------------------------------------------------ 56

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Very small embryonic-like cells: Biology and function of these potential endogenous pluripotent stem cells in adult tissues Author: Kassmer, Susannah H; Krause, Diane S Publication info: Molecular reproduction and development (Feb 25, 2013). Abstract: Very small embryonic-like cells (VSELs), found in murine bone marrow and other adult tissues, are small, non-hematopoietic cells expressing markers of pluripotent embryonic and primordial germ cells. A similar cell type in humans has begun to be characterized, though with a slightly different phenotype and surface markers. Consistent with expression of pluripotency genes, murine VSELs differentiate into cell types from three germ layer lineages in vitro, though pluripotency has yet to be shown at the single cell level or in vivo. VSELs appear to be quiescent under steady state conditions, apparently due to partially erased imprinting and overexpression of cell cycle inhibitory genes. In vivo, VSELs can enter the cell cycle under stress conditions, but which factors regulate quiescence versus proliferation and self-renewal versus differentiation are as yet unknown, and in vitro conditions that induce proliferation and self renewal have yet to be defined. Future experiments are needed to address whether a VSEL niche actively regulates quiescence in vivo or quiescence is cell autonomous under steady state conditions. Insights into these mechanisms may help to address whether or not VSELs could play a role in regenerative medicine in the future. Mol. Reprod. Dev. ÂŠ 2013 Wiley Periodicals, Inc. _______________________________________________________________

Human very small embryonic-like cells generate skeletal structures, in vivo Author: Havens, Aaron M.; 1; Shiozawa, Yusuke; 2; Jung, Younghun; 2; Sun, Hongli; 3; Wang, Jincheng; 2; McGee, Samantha; 2; Mishra, Anjali; 4; Taichman, L. Susan; 2; Danciu, Theodora; 2; Jiang, Yajuan; 5; Yavanian, Gregory; 5; Leary, Elizabeth; 5; Krebsbach, Paul H.; 3; Rodgerson, Denis; 5; Taichman, Russell S.; 2; 1Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, 1011 North University Avenue, Ann Arbor, MI 48109-1078, United States, Department of Orthodontics; and Pediatric Dentistry, University of Michigan School of Dentistry, United States; 2Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, 1011 North University Avenue, Ann Arbor, MI 48109-1078, United States rtaich@umich.edu; 3Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, MI, United States; 4Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, 1011 North University Avenue, Ann Arbor, MI 48109-1078, United States, Department of Biologic and; Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, MI, United States; 5NeoStem, Inc., New York, NY, United States Publication info: Stem Cells and Development 22. 4 (Feb 15, 2013): 622-630. Abstract: Human very small embryonic-like (hVSEL) cells are a resident population of multipotent stem cells in the bone marrow involved in the turnover and regeneration of tissues. The levels of VSEL cells in blood are greatly increased in response to injury, and they have been shown to repair injured tissues. Adult hVSEL cells, SSEA-4+/CD133+/CXCR4 +/Lin-/CD45-, express the pluripotency markers (Oct-4 and Nanog) and may be able to differentiate into cells from all 3 germ lineages. hVSEL cells isolated from blood by apheresis following granulocyte-colony-stimulating factor mobilization were fractionated and enriched by elutriation and fluorescence activated cell sorting. Collagen sponge scaffolds containing 2,000-30,000 hVSEL cells were implanted into cranial defects generated in SCID mice. Analysis by microcomputed tomography showed that a Very Small Embryonic Like Cell References

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cell population containing VSEL cells produced mineralized tissue within the cranial defects compared with controls at 3 months. Histologic studies showed significant bone formation and cellular organization within the defects compared with cellular or scaffold controls alone. Antibodies to human leukocyte antigens demonstrated that the newly generated tissues were of human origin. Moreover, human osteocalcin was identified circulating in the peripheral blood. There was evidence that some level of hVSEL cells migrated away from the defect site, using quantitative real-time polymerase chain reaction to detect for human-specific Alu sequences. This study demonstrates that hVSEL cells are able to generate human bone tissue in a mouse model of skeletal repair. These studies lay the foundation for future cell-based regenerative therapies for osseous and connective tissue disorders, including trauma and degenerative conditions, such as osteoporosis, fracture repair, and neoplastic repair. ÂŠ Copyright 2013, Mary Ann Liebert, Inc. 2013. _______________________________________________________________

Very small embryonic-like stem cells: Implications in reproductive biology Author: Bhartiya, Deepa; 1; Unni, Sreepoorna; 1; Parte, Seema; 1; Anand, Sandhya; 1; 1Stem Cell Biology Department, National Institute for Research in Reproductive Health (ICMR), Mumbai, Maharashtra 400 012, India deepa.bhartiya@yahoo.in; poornaunni@gmail.com; seema.parte@gmail.com; sandhya0810@gmail.com Publication info: BioMed Research International 2013 (2013). Abstract: The most primitive germ cells in adult mammalian testis are the spermatogonial stem cells (SSCs) whereas primordial follicles (PFs) are considered the fundamental functional unit in ovary. However, this central dogma has recently been modified with the identification of a novel population of very small embryonic-like stem cells (VSELs) in the adult mammalian gonads. These stem cells are more primitive to SSCs and are also implicated during postnatal ovarian neo-oogenesis and primordial follicle assembly. VSELs are pluripotent in nature and characterized by nuclear Oct-4A, cell surface SSEA-4, and other pluripotent markers like Nanog, Sox2, and TERT. VSELs are considered to be the descendants of epiblast stem cells and possibly the primordial germ cells that persist into adulthood and undergo asymmetric cell division to replenish the gonadal germ cells throughout life. Elucidation of their role during infertility, endometrial repair, superovulation, and pathogenesis of various reproductive diseases like PCOS, endometriosis, cancer, and so on needs to be addressed. Hence, a detailed review of current understanding of VSEL biology is pertinent, which will hopefully open up new avenues for research to better understand various reproductive processes and cancers. It will also be relevant for future regenerative medicine, translational research, and clinical applications in human reproduction. ÂŠ 2013 Deepa Bhartiya et al. _______________________________________________________________

Murine and human very small embryonic-like cells: a perspective Author: Heider, Andreas; Danova-Alt, Ralitza; Egger, Dietmar; Cross, Michael; Alt, RĂźdiger Publication info: Cytometry. Part A : the journal of the International Society for Analytical Cytology 83. 1 (Jan 2013): 72-5. Abstract: In 2006, very small embryonic-like (VSEL) stem cells were described as a pluripotent population of prospectively isolated stem cells in adult murine bone marrow (mBM) and human umbilical cord blood (hUCB). While rigorous proof of pluripotency is still lacking, murine VSEL cells have been shown to overlap with an independently identified population of neural crest derived mesenchymal stem cells (MSC). The presence of primitive mesenchymal precursors within the VSEL cell population may partially explain the findings that have Very Small Embryonic Like Cell References

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led to the concept of an "embryonic-like" stem cell in mBM. However, our own studies on human VSEL cells revealed very little similarity between murine VSEL cells and their reportedly equivalent population in hUCB. On the contrary, our data strongly suggest that human VSEL cells are an aberrant and inactive population that cannot expand in vitro and has neither embryonic nor adult stem cell like properties. Here we critically re-examine the data supporting stemness and pluripotency of murine and human VSEL cells, respectively. _______________________________________________________________

In vivo ischemic limb injury activates proliferation and mobilization of pluripotent bone marrow-derived very small embryonic-like stem cells Author: Labedz-Maslowska, Anna; 1; Kamycka, Elzbieta; 1; Ratajczak, Mariusz Z.; 2; Madeja, Zbigniew; 1; Zuba-Surma, Ewa K.; 1; 1Dept of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian Univ, Poland; 2Stem Cell Biology Institute, Univ of Louisville, United States Publication info: Circulation, suppl. SUPPL. 1 126. 21 (Nov 20, 2012). Abstract: Very small embryonic-like stem cells (VSELs) were identified as rare quiescent, nonhematopoietic (CD45-/Lineage-) and pluripotent Oct-4+ stem cells in adult murine and human tissues including bone marrow (BM). Murine BM- derived VSELs were shown to differentiate into cells from all three germ layers and to participate in heart repair after injection into infarcted myocardium by enhancing perfusion and angiogenesis. However, their activation during physiological or pathological events in vivo have not been reported. In this study, we examined if acute ischemic injury may stimulate both proliferation of quiescent VSELs in BM and their mobilization into peripheral blood (PB). Wild-type C57BL/6 mice (9-week old) underwent hind limb ischemia by permanent proximal femoral artery occlusion. Mice were injected with bromodeoxyuridine (BrdU, 1mg/mouse/i.p.)every two days and were scarified at 2, 7, 14 and 28 days following ischemia (N=5/group). PB and BM from non- and ischemic limbs were collected from individual animals. Healthy non-ischemic mice were used as control groups in each time point. The presence of proliferating (BrdU+) VSELs (CD45-/Lineage-/Sca-1+), endothelial progenitor cells (EPCs; CD45-dim/Lin-/Sca-1+/Flk-1+) and hematopoietic stem cells (HSCs; CD45+/Lineage-/Sca-1+) in PB and BM was evaluated by multiparameter flow cytometry and ImageStream system. The expression of genes related to the VSEL and EPC presence was examined by real time RT-PCR. We established that the content of VSELs was significantly increased in BM of ischemic mice after 7 days post injury. We observed elevated number of BrdU+ VSELs in BM and circulating in PB of the injured animals indicating vast impact of acute ischemia on activation of these cells. Similar results were obtained for BM- derived and circulating EPCs. We conclude that acute tissue injury such as limb ischemia may provide stimulatory agents to activate proliferation of quiescent VSELs in BM and their mobilization into PB. Thus, the ischemic injury may recruit the normally quiescent pluripotent stem cell pools to enhance endogenous mechanisms of tissue repair including stem/ progenitor cell- dependent angiogenesis which we currently investigate. _______________________________________________________________

Prolonged strenuous exercise expands the population of developmentally early stem cells in bone marrow (BM) and mobilizes them into peripheral blood-novel evidence that strongly supports a positive effect of physical activity on extension of life span at the level of stem cells Author: Tarnowski, Maciej; 1; Piotrowska, Katarzyna; 1; Grymula, Katarzyna; 1; Suszynska, Malwina; 1; Poniewierska, Agata; 1; Ratajczak, Mariusz Z.; 2; 1Physiology, Pomeranian Medical University, Poland; 2 Medicine, Stem Cell Institute at James Graham Brown Cancer Center, University of Louisville, United States Publication info: Blood 120. 21 (Nov 16, 2012). Very Small Embryonic Like Cell References

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Abstract: Background.: Bone marrow (BM) contains a variety of stem cells, including hematopoietic stem/progenitor cells (HSPCs), endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs), and the developmentally most primitive very small embryonic-like stem cells (VSELs). Evidence has accumulated that HSPCs (Circ J. 2008;72:897) and EPCs (J Am Coll Cardiol. 2004;43:2314) are mobilized into PB during strenuous exercise. Therefore, we became interested in whether a pool of bone marrow (BM)-residing developmentally more primitive, highly quiescent, early stem cells (VSELs) would respond to stimuli related to prolonged, strenuous exercise. As we have demonstrated previously, these cells play an important role in tissue/organ rejuvenation and their number positively correlates with life span in experimental animals (Age 2012 in press doi:10.1007/s11357-011-9364-8). Hypothesis.: We hypothesized that the positive effect of physical activity may be explained by the expansion of a developmentally early pool of highly quiescent stem cells that play a role in tissue/organ rejuvenation. Experimental strategies.: To test the effect of exercise on the BM pool of stem cells, we employed C57Bl6 mice that were exposed to short (1 day), mid-term (14 days), and long- term (6 months) strenuous exercise in rotating wheels. These mice were subsequently sacrificed 1h after removal from the rotating wheels, and we i) measured changes in peripheral blood cell counts, ii) enumerated the number of VSELs and HSPCs both in BM and PB of sacrificed animals by FACS analysis, iii) performed clonogeneic in vitro methylocellulose assays to enumerate the number of CFU-GM and BFU-E mobilized into PB and the number of these cells residing in the BM microenvironment, iv) employed the in vivo bromodeoxyuridine (BrdU) incorporation assay to evaluate the number of VSELs and HSPCs in BM undergoing cell division, and finally v) performed a molecular analysis of the expansion and mobilization of VSELS by measuring the expression of genes regulating stem cell pluripotency at the mRNA (RQ-PCR) and protein levels (immunofluorescence of cells fixed on cytospin slides). Results.: Our data confirmed that strenuous exercise mobilizes Sca-1+Lin-CD45+ HSPCs into PB, and, for the first time, we show that prolonged, enforced exercise of mice in rotating wheels is associated with expansion in BM and mobilization into PB of the most primitive population of stem cells, Sca-1+Lin-CD45- VSELs. The expansion of VSELs in BM and their mobilization into PB was confirmed by FACS analysis, immunohistochemical staining, and RQ-PCR analysis for expression of genes that regulate pluripotency (e.g., Oct-4 and Nanog) in small Sca-1+Lin-CD45-cells sorted from BM and PB. The proliferation of VSELs, which are a quiescent population of BM-residing stem cells, has been confirmed by BrdU incorporation. In contrast to VSEL expansion, we did not observe significant changes in the number of BM-residing HSPCs. As expected, mice exposed to prolonged exercise exhibited a significant increase in skeletal muscle and a decrease in abdominal fat. Conclusions.: BM-residing stem cells respond to prolonged strenuous exercise by expansion and subsequent mobilization into PB of BM-residing VSELs, which, as proposed by us and others, may play an important role in tissue/organ rejuvenation and thus positively affect life span. Therefore, our data for the first time demonstrates a positive correlation between exercise and the expansion of a most primitive pool of stem cells in BM. _______________________________________________________________

Novel evidence that a quiescent murine population of bone marrow (BM)-residing, developmentally early, very small SCA-1+LIN-CD45-cells is highly responsive to prolonged bleeding by in vivo proliferation and differentiation into CD45+ hematopoietic stem/progenitor cells (HSPCS) Author: Ratajczak, Mariusz Z.; 1; Mierzejewska, Kasia; 1; Ratajczak, Janina; 1; Kucia, Magdalena; 1; 1Medicine, Stem Cell Institute, University of Louisville, United States Publication info: Blood 120. 21 (Nov 16, 2012).

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Abstract: Background.: Several phenotypes have been proposed for long-term repopulating hematopoietic stem cells (LT-HSCs) in murine bone marrow (BM). However, evidence from our and other laboratories has accumulated that adult murine tissues contain a population of developmentally early, so-called very small embryonic-like stem cells (VSELs), which we have proposed as playing an important role as precursors of LT-HSCs (Exp. Hematology 2011;39:225, Leukemia 2012;25,1278). As we reported, these cells are kept quiescent in the BM microenvironment by erasure of the somatic imprint in differentially methylated regions (DMRs) of some developmentally crucial, paternally imprinted genes (Igf2-H19, RasGRF1, and p57Kip2), which proper expression is required for proliferation and expansion of pluripotent stem cells (e.g., embryonic stem cells) (Leukemia 2009;23:2042). However, we also demonstrated that these cells may be specified into the hematopoietic lineage in vitro in co-cultures over OP9 stromal cells. Hypothesis.: We hypothesized that these very small cells, which can be specified into the hematopoietic lineage ex vivo in an “artificial” OP9 microenvironment, should also be able to become specified into HSPCs in vivo in the normal BM microenvironment in situations of hematopoietic stress that promote the formation of new HSPCs. Experimental strategies.: Normal C57Bl6 mice were bled (twice a week, 200 μl/bleeding) for 4 weeks, and by the end of each week were injected with bromodeoxyuridine (BrdU) to label cells that are in the cell cycle. These mice were subsequently sacrificed and BM cells, flushed from BM cavities as well as from crushed/collagenase-treated bones to recover cells associated with endosteal niches, were obtained from both control and bled mice. In these cell suspensions, we measured i) the total number of Sca-1+Lin-CD45+ HSPCs and small Sca-1+Lin-CD45- VSELs by FACS and ii) the number of cycling BrdU+ HSPCs and VSELs. Moreover, by employing RQ-PCR, we measured the expression of genes regulating the early stages of hematopoiesis and imprinted genes that keep VSELs quiescent in the cell cycle. We also tested the ability of VSELs from control and bled mice to differentiate into CD45+ HSPCs in OP9 co-cultures and their ability to reconstitute hematopoiesis in lethally irradiated mice. Salient results.: We observed that the number of cycling BrdU+ VSELs increased from ∼1 ± 0.03% (control) to ∼26 ± 4% and ∼32 ± 6% among BM cells derived from flushed and crushed bones, respectively. Furthermore, in comparison with control animals, BM VSELs isolated from mice after chronic bleeding expressed lower levels of pluripotency markers such as Oct-4 and Nanog, upregulated expression of pro-proliferative mRNA whose expression is regulated by paternal imprinting (Igf2, IGF-1R, and RasGRF1), and downregulated expression of mRNA for paternally imprinted, proliferation-inhibiting H19 and p57Kip2genes. At the same time, the number of BM HSPCs increased from ∼17 ± 3% to 35 ± 7% and 1 ± 0.02% to 40 ± 5% in flushed and crushed bone-derived cells, respectively. Most importantly, we observed that VSELs isolated from bled mice highly upregulated the expression of genes involved in early stages of hematopoiesis, including Ikaros, Lmo2, GATA-2, HoxB4, PU.1, Scl and c-myb, and this correlated with their accelerated ability to become specified into CD45+ HSPCs in co-cultures over OP9 stroma. Finally, VSEL-derived CD45+ HSPCs, when isolated from OP9 cultures, grew methylocelulose colonies from all major hematopoietic lineages and were able to reconstitute hematopoiesis in lethally irradiated recipients. Conclusions.: Our data, obtained in an in vivo murine model of hematopoietic stress from chronic bleeding, strongly support the notion that developmentally early murine Sca-1+Lin-CD45- VSELs represent a population of quiescent stem cells in BM that become specified into the hematopoietic lineage in vivo. We propose that, in order to establish the relationship of these cells to other LT-HSC phenotypes described in BM as well as to construct a complete developmental hierarchy, their hematopoietic potential should be compared side-by-side with other BM-derived stem cells isolated using different phenotypic criteria. _______________________________________________________________

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A novel view of bone marrow as a “stem cell sensor” of tissue/organ damage-evidence that in vivo exposure to the neurotoxin kainic acid (KA) induces proliferation and neural specification of developmentally early stem cells directly in bone marrow before they are mobilized into peripheral blood Author: Grymula, Katarzyna; 1; Tarnowski, Maciej; 1; Suszynska, Malwina; 1; Piotrowska, Katarzyna; 1; Borkowska, Sylwia; 1; Mierzejewska, Kasia; 2; Kucia, Magdalena; 2; Ratajczak, Mariusz Z.; 2; 1Physiology, Pomeranian Medical University, Poland; 2Medicine, Stem Cell Institute, University of Louisville, United States Publication info: Blood 120. 21 (Nov 16, 2012). Abstract: Background.: It is well known that various stem cells become mobilized into peripheral blood (PB) in response to tissue/organ injuries (e.g., heart infarct, stroke, or bleeding); however, the data on the immediate response of stem cells in BM during organ injuries are somewhat limited. We and others have demonstrated the presence of developmentally early stem cells in BM that we have named very small embryonic-like stem cells (VSELs). These Oct-4+SSEA-1+Sca-1+Lin-CD45- cells are kept quiescent in BM in the G0 phase of the cell cycle by erasure of the somatic imprint in the differentially methylated regions (DMRs) of some crucial paternally imprinted genes, (Igf2-H19, RasGRF1, and p57Kip2) that regulate proliferation of embryonic stem cells (Leukemia 2009;23:2042). These cells are mobilized into peripheral blood, for example, during heart infarct (J Am Coll Cardiol 2009;6:1-9.), stroke (Stroke 2009;40:1237-44.), or skin burns (Stem Cell Rev. 2012;8:184-94.). Hypothesis.: We hypothesized that this population of BM-residing, small, quiescent, pluripotent cells should be able to respond to organ injury induced by a known neurotoxin, kainic acid (KA), in a brain damage model. We hypothesized that these quiescent cells would began to proliferate, expand, and become specified into the neural lineage. Experimental strategies.: C57Bl6 mice were injected with increasing doses of KA and at various time intervals mice were sacrificed to harvest BM, PB samples, and brains for analysis. Brain damage was confirmed by histological analysis. The number of Sca-1+Lin-CD45- VSELs and Sca-1+Lin-CD45+ HSPCs was evaluated in BM and PB by FACS. The cell cycle status of VSELs and HSPCs was evaluated by FACS in cells isolated from mice that received bromodeoxyuridine (BrdU) after KA injection. By employing RQ-PCR, we also measured the expression of genes that regulate stem cell pluripotency (Oct-4, Nanog, Sox2, and Rex1) and regulate neuronal development (Nestin, βIII-tubulin, Olig1, Olig2, and GFAP). The expression of these genes was subsequently confirmed in sorted cells by immunohistochemical staining. The numbers of clonogenic CFU-GM and BFU-E progenitors residing in BM and circulating in PB were tested in methylcellulose cultures. Results.: We found that 12 hrs after administration of KA (25 mg/kg bw) quiescent VSELs residing in BM enter the cell cycle: ∼2 ± 1% for control vs. 37 ± 6% for KA-treated cells. Interestingly, at the same time we did not observe significant changes in the proliferation rate of HSPCs (15±5% for control vs. 17±4% for KA-treated cells). The elevated number of VSELs in the cell cycle remained detectable for a few days and returned to control values (∼2%) after 1 week after KA administration. Furthermore, an increase in the number of cycling VSELs correlated with an increase in expression of pluripotent markers, according to RQ-PCR analysis. In parallel, 48 hrs after KA administration we observed the release from BM into PB of Sca-1+Lin-CD45-VSELs highly enriched for mRNAs characteristic of neural differentiation. Interestingly, while we observed a significant increase in VSEL number in BM and PB after KA-induced brain damage, no significant changes were observed for both BM-residing and circulating HSPCs. Conclusions.: For the first time, we provide evidence that the compartment of developmentally early stem cells residing in BM responds robustly to brain damage induced by a neurotoxin. This effect seems to be specific for VSELs, as no significant changes were observed for HSPCs. The kinetics of changes in BM revealed that BM VSELs enter the cell cycle and, after they become specified into the neural lineage, egress from BM and enter the PB. Thus, our data provide novel Very Small Embryonic Like Cell References

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evidence that developmentally early stem cells in BM “sense” the damage to brain tissue and respond to this type of organ injury. In parallel, we are studying the specificity of the response of BM-residing VSELs and HSPCs to other types of organ damage, such as heart infarct and acute limb ischemia. _______________________________________________________________

Development of a highly efficient method for isolating very small embryonic-like stem cells identified in adult mouse bone and their stem cell characteristics Author: Iwaki, Ryuji; 1; Nakatsuka, Ryusuke; 2; Matsuoka, Yoshikazu; 2; Takahashi, Masaya; 2; Fujioka, Tatsuya; 2 ; Sasaki, Yutaka; 2; Asano, Hiroaki; 3; Uemura, Yasushi; 4; Kwon, A.-Hon; 1; Sonoda, Yoshiaki; 2; 1Department of Surgery, Kansai Medical University, Japan; 2Department of Stem Cell Biology and Regenerative Medicine, Kansai Medical University, Japan; 3School of Nursing, Kyoto Prefectural University of Medicine, Japan; 4Division of Immunology, Aichi Cancer Center Research Institute, Japan Publication info: Blood 120. 21 (Nov 16, 2012). Abstract: Background: Ratajczak and his colleagues identified a unique population of very small embryonic-like (VSEL) stem cells in adult mouse bone marrow (BM) (Leukemia 2006:20;857). These VSELs are; 1) very small (∼4 μm); 2) express pluripotent stem cell markers, such as Oct4, Nanog, SSEA-1, and Rex-1; 3); responsive to a SDF-1 gradient; 4) possess large nuclei that contain euchromatin. It is very interesting to note that VSELs possess the potential to differentiate into 3 germ layers in vitro and in vivo, thereby contributing to tissue/organ regeneration. These VSELs were isolated as lineage-negative (Lin-), Sca-1-positive (Sca-1+), CD45-negative (CD45-) cells by FACS. However, the incidence of VSELs in BM-derived mononuclear cells is ∼0.01%. Therefore, it is difficult to isolate VSELs very effectively. This study describes our recently developed highly efficient method for isolating VSELs using enzymatic treatment of murine bone. Materials and Methods: Murine BM nucleated cells (BMNC) were isolated from BM flushed from the pairs of femurs and tibiae of 8 week-old C57BL/6 mice. Erythrocytes were removed using a hypotonic solution. Then the remaining bone tissues were thoroughly washed using PBS- with 2% FCS. These bone tissue specimens were crushed in a mortar and then incubated in cell dissociation buffer containing a-medium with 5% FCS supplemented with 1.5 mg/ml type I collagenase and 2 mg/ml dispase at 37°C for 1 hour. Next, the BMNCs and bone-derived nucleated cells (BDNCs) were stained with various monoclonal antibodies, including anti-lineages, anti-CD45, anti-Sca-1, anti-CXCR4, anti-CD133, and anti-PDGFRα, and then were used for subsequent FACS analyses. Results: The R1 gate was set on the FSC channel using 4 and 10 μm synthetic beads, based on the predicted very small size of VSELs. The VSELs were isolated from BMNCs and BDNCs by multicolor FACS, as a population of Lin-Sca-1+CD45- cells (Fig. 1A). The incidences of VSELs in the BMNCs and BDNCs were 0.001% and 0.1%, respectively. Therefore, the enzymatic treatment of bone tissues yielded about 100 times the efficiency for the isolation of VSELs (Fig. 1B). The bone-derived (BD) VSELs were small (< 5 μm) and possessed a relatively large nucleus surrounded by a narrow rim of cytoplasm. They expressed CD133, but not PDGFRα. However, they weakly expressed CXCR4. The gene expression profiles were analyzed using real time quantitative PCR (RQ-PCR) to evaluate the expression of ES cell markers (Oct4, Nanog, Rex1, Dppa3), HSC (KSL) markers (c-kit, Tal1, GATA2), and MSC markers (Nestin, Ang1, CXCL12, VE-Cadherin). Unexpectedly, BD VSELs expressed high levels of Nestin and Cadherin. However, they expressed weak levels of Oct4 and Nanog. The gene expression profile of the BD VSELs was clearly distinct from the well-defined populations of ES cells, KSL cells, and MSCs. Interestingly, the number of these BD VSELs significantly increased after the induction of liver injury by carbon tetrachloride administration. They were then most likely mobilized into the peripheral blood (PB). G-CSF did mobilize KSL cells into PB, as previously reported. However, G-CSF did not mobilize the BD VSELs. The effects Very Small Embryonic Like Cell References

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of sRANKL on the mobilization of BD VSELs were examined in vivo. Interestingly, the number of BD VSELs significantly increased 2-3 days after the administration of sRANKL. However, the number of VSELs in PB did not increase. These results suggest that BD VSELs actively proliferated after liver injury and bone resorption. Conclusion: The present data suggest that the majority of the Lin-Sca-1+CD45- cells reside in the bone tissue. BD VSELs resemble BM-derived VSELs. However, a RQ-PCR analysis revealed that the gene expression profile of BD VSELs was different from those of the previously reported BM-derived VSELs. Further studies will therefore be required to elucidate their stem cell characteristics and the potential relationship between BD VSELs and BM-derived VSELs. (Figure presented). _______________________________________________________________

Peripheral blood stem cells: phenotypic diversity and potential clinical applications Author: Zhang, Yichi; Huang, Bing Publication info: Stem cell reviews 8. 3 (Sep 2012): 917-25. Abstract: A small proportion of cells in peripheral blood are actually pluripotent stem cells. These peripheral blood stem cells (PBSCs) are thought to be heterogeneous and could be exploited for a variety of clinical applications. The exact number of distinct populations is unknown. It is likely that individual PBSC populations detected by different experimental strategies are similar or overlapping but have been assigned different names. In this mini review, we divide PBSCs into seven groups: hematopoietic stem cells (HSCs), CD34- stem cells, CD14+ stem cells, mesenchymal stem cells (MSCs), very small embryonic-like (VSEL) stem cells, endothelial progenitor cells (EPCs), and other pluripotent stem cells. We review the major characteristics of these stem/progenitor cell populations and their potential applications in ophthalmology. _______________________________________________________________

Global gene expression analysis of very small embryonic-like stem cells reveals that the Ezh2-dependent bivalent domain mechanism contributes to their pluripotent state Author: Shin, Dong-Myung; 1; Liu, Rui; 2; Wu, Wan; 2; Waigel, Sabine J.; 3; Zacharias, Wolfgang; 4; Ratajczak, Mariusz Z.; 5; Kucia, Magda; 5; 1Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, 500 South Floyd Street, Louisville, KY 40202, United States, Department of Medicine, Graduate School,; University of Ulsan, Pungnap-2 dong, Songpa-gu Seoul 138-736, South Korea d0shin03@amc.seoul.kr; 2 Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, 500 South Floyd Street, Louisville, KY 40202, United States; 3Microarray Facility, University of Louisville, Louisville, KY, United States; 4 Department of Medicine, University of Louisville, Louisville, KY, United States, Department of Pharmacology and Toxicology, University of Louisville, Louisville, KY, United States; 5Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, 500 South Floyd Street, Louisville, KY 40202, United States, Department of Physiology, Pomeranian Medical; University, Szczecin, Poland mzrata01@louisville.edu Publication info: Stem Cells and Development 21. 10 (Jul 1, 2012): 1639-1652. Abstract: Recently, we identified a population of Oct4+Sca-1 +Lin-CD45- very small embryonic-like stem cells (VSELs) in murine and human adult tissues. VSELs can differentiate in vitro into cells from all 3 germ layers and in vivo tissue-committed stem cells. Open chromatin structure of core pluripotency transcription factors (TFs) supports the pluripotent state of VSELs. However, it has been difficult to determine how primitive VSELs maintain pluripotency, owing to their limited number in adult tissues. Here, we demonstrate by genome-wide gene-expression analysis with a small number of highly purified murine bone marrow-derived VSELs that Oct4 Very Small Embryonic Like Cell References

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VSELs (i) express a similar, yet nonidentical, transcriptome as embryonic stem cells (ESCs), (ii) highly express cell cycle checkpoint genes, (iii) express at a low level genes involved in protein turnover and mitogenic pathways, and (iv) highly express enhancer of zeste drosophila homolog 2 (Ezh2), a polycomb group protein. Furthermore, as a result of high expression of Ezh2, VSELs, like ESCs, exhibit bivalently modified nucleosomes (trimethylated H3K27 and H3K4) at promoters of important homeodomain-containing developmental TFs, thus preventing premature activation of the lineage-committing factors. Notably, spontaneous or RNA interference-enforced downregulation of Ezh2 during VSEL differentiation removes the bivalent domain (BD) structure, which leads to de-repression of several BD-regulated genes. Therefore, we suggest that Oct4+ VSELs, like other pluripotent stem cells, maintain their pluripotent state through an Ezh2-dependent BD-mediated epigenetic mechanism. Furthermore, our global survey of VSEL gene expression signature would not only advance our understanding of biological process for their pluripotency, differentiation, and quiescence but should also help to develop better protocols for ex vivo expansion of VSELs. ÂŠ 2012 Mary Ann Liebert, Inc. _______________________________________________________________

Human umbilical cord is a unique and safe source of various types of stem cells suitable for treatment of hematological diseases and for regenerative medicine Author: Pelosi, Elvira; Castelli, Germana; Testa, Ugo Publication info: Blood cells, molecules & diseases 49. 1 (Jun 15, 2012): 20-8. Abstract: Cord blood (CB) is a rich source of hematopoietic stem cells (HSCs) and for this reason CB transplantation has been used successfully for the treatment of some malignant and nonmalignant diseases. However, this technique is limited by the relatively low number of HSCs present in each CB unit and by the delayed engraftment of platelets and neutrophils. To bypass these obstacles efforts have been made to develop strategies to expand CB HSCs in vitro for transplantation. CB is also an important source of other stem cells, including endothelial progenitors, mesenchymal stem cells (MSCs), very small embryonic/epiblast-like (VSEL) stem cells, and unrestricted somatic stem cells (USSC), potentially suitable for use in regenerative medicine. For some of these stem cell populations, such as MSCs, clinical studies have been started and for other stem cell populations potential clinical applications have been identified and clinical studies will follow. In addition to CB, other parts of umbilical cord, such as the Wharton's jelly, or tissues strictly linked such as the placenta are also rich sources of stem cells. _______________________________________________________________ Mobilization of very small embryonic-like stem cells by G-CSF in MACO mice Author: Zheng, Min; 1; Wang, Jian; 2; 1Department of Histology and Embryology, Lab of Stem Cell, Chongqing Medical University, China; 2Second Affiliated Hospital, Chongqing Medical University, China Publication info: FASEB Journal, suppl. Meeting Abstracts 26 (Apr 2012). Abstract: Objective: To investigate the effect of G-CSF on neural restoration and on mobilization of very small embryonic-like stem cells(VSEL-SC) in bone marrow of focal cerebral ischemia mouse. Methods: MCAO model was produced by filament occlusion method. 50|Ă&#x152;g/(kg.d) G-CSF were given by subcutaneous injection for 5 days. Neurological scale was evaluated. The number of VSEL-SC (CXCR4+Lin-CD45-) in peripheral blood was checked by fluorescence-activated cell sorting analysis (FACS). Level of stromal derived factor-1(SDF-1) in plasma and infarct cerebral tissue were checked by ELISA. Expression of SDF-1 in ischemia area was checked by immunohistochemical staining. Results: G-CSF treatment significantly decreased the neurological scale of Very Small Embryonic Like Cell References

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MCAO model mice compared with controls. G-CSF could mobilize VSEL-SC in the bone marrow of focal cerebral ischemia mice into peripheral blood. The concentration of SDF-1 in plasma and in cerebral tissue significantly increased in G-CSF treated group. Expression of SDF-1 in infarct region significantly increased in G-CSF treated group. Conclusion: The beneficial effect of G-CSF may be related to the mobilization of VSEL-SC and the promotion of SDF-1 expression in the infarct tissue of MACO mice. _______________________________________________________________

Mobilization of OCT-4+ very small embryonic like stem cells, hematopoietic stem cells and endothelial progenitor cells in children with congenital heart disease undergoing cardiac surgery Author: Wojakowski, Wojciech; 1; Jacek, Kolcz; 1; Ewa, Zuba-Surma; 1; Justyna, Drukala; 1; Ratajczak, Mariusz; 1; Tendera, Michal; 1; Skalski, Janusz; 1; 1Medical University of Silesia, Poland, Jagiellonian University, Poland Publication info: Journal of the American College of Cardiology, suppl. SUPPL. 1 59. 13 (Mar 27, 2012): E968. Abstract: Background: Very small embryonic-like cells (VSEL) is rare population of cells expressing pluripotent markers (Oct-4,Nanog, SSEA-4). VSEL in adult humans are functionally and phenotypically different from hematopoietic stem cells (HSC) and endothelial progenitor cells (EPC). Aim was to assess the mobilization of VSEL, HSC and EPC in children undergoing cardiac surgery. Methods: 24 children (age 8 days to 10 y) with congenital heart disease were enrolled. Blood samples were drawn before surgery (1), during induction of hypothermia after initiation of cardiopulmonary bypass (2), in hypothermia (3), during restoration of normal temperature (4) and after surgery and weaning from the bypass (5). Number of VSEL (lin-CD133+CD45-), HSC (Lin-CD133+ CD45+) and two populations of EPC (Lin-CD133+ CD45-KDR+ and Lin-CD133- CD45-KDR+) was measured by FACS. Results: (Figure 1) The number of VSEL, HSC and EPC I-II was highest before initiation of surgical procedure and were subsequently reduced throughout the procedure reaching niveau during restoration of normothermia. After weaning from the cardiopulmonary bypass the number of cells rapidly returned to the preoperative levels. Same pattern of mobilization was observed for HSC and EPC. Conclusions: Cardiac surgery with cardiopulmonary bypass and hypothermia leads to transient suppression of stem cell mobilization. The cell release is markedly different than observed in adults with acute myocardial injury and might reflect homing of cells to the myocardium. (Figure presented). _______________________________________________________________

Mobilization of various types of pluripotent progenitor and stem cells is surrogate prognostic marker of mucosal healing in early clinical remission in Crohn's disease Author: Marlicz, W.; 1; Zuba-Surma, E.; 2; Blogowski, W.; 3; Kucia, M.; 4; Starzynska, T.; 1; Ratajczak, M.; 4; 1 Pomeranian Medical University, Department of Gastroenterology, Poland; 2Jagiellonian University, Department of Biochemistry, Poland; 3Pomeranian Medical University, Department of Laboratory Diagnostics and Molecular Medicine, Poland; 4University of Louisville, Stem Cell Graham Brown Cancer Center, United States Publication info: Journal of Crohn's and Colitis, suppl. SUPPL. 1 6 (Feb 2012): S120. Abstract: Background: Our group has reported that the number of very small embryonic/epiblast like stem cells (VSEL-SCs) mobilized into peripheral blood (PB) increases in active inflammatory bowel disease (IBD) (JCC 2010;4.1/Mo1815, DDW 2011). Currently we investigated the correlation of various types of stem/progeniotor cells mobilization phenomenon in Crohn's disease (CD) with clinical and endoscopic scores (ES). The activation of the complement cascade (CC) and factors involved in stem cell trafficking and their association with SCs Very Small Embryonic Like Cell References

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mobilization into PB in IBD were studied. Methods: 35 active/inactive CD and 30 healthy individuals were enrolled into the study. CD patients were classified according to clinical (Crohns Deasease Activity Index CDAI) and endoscopic (Simple Endoscopic Score for Crohn's Disease-SES-CD) criteria. CDAI/SES-CD were assessed at week 0 and 10 of treatment with 5-ASA/AZA and/or moanti- TNF-alfa. PB samples were collected, and by employing fluorescence-activated cell sorting (FACS) the numbers of (i) VSELs-SCs (CD45-CXCR4+/CD133+/Lin-), (ii) Mesenchymal SCs (MSC) (CD45-/STRO 1+/CD105+/CD29+), (iii) Endothelial Progenitor Cells (EPC) (CD45-/CD31+/CD34+/KDR+) and iv) Hematopoietic SCs (HSC) (Lin-/CD45+/CD133+) were counted and/or isolated. Immunofluorescence staining of sorted cells and RT-qPCR analysis of expression of mRNA for embryonic SCs markers (Oct-4, SSEA-4, Nanog), and markers of intestinal differentiation: Lgr-5, ASCL2, MSI1, DCLK1, BMI1 was evaluated in sorted VSEL-SCs. Plasma levels of C3a, C5a, membrane attack complex (C5b-9/MAC), VEGF, HGF and SDF-1 by ELISA were measured. Results: In IBD patients we found an increase in the number of circulating MSC, EPC and small primitive cells expressing the VSEL phenotype. The number of circulating progenitor/stem cells correlated with SES-CD (p <0.05) but not CDAI. SESCD also better correlated with plasma VEGF concentration (R=0.49; p <0.03) and HGF (R = 0.51; p <0.02). Active disease was associated with strong mRNA up-regulation of intestinal early differentiation markers (lgr-5 and ASCL-2) in circulating PB mononuclear cells. Higher plasma C5a levels in IBD in comparison to controls were noted (61.7±19.7 vs 45.8±11.2; p = 0.024 respectively). No correlation between SDF-1 and the number of VSEL-SC were found. Conclusions: Enumeration of circulating stem/progenitor cells could serve as surrogate parameter in accessing treatment efficacy in IBD. _______________________________________________________________

Response of healthy volunteers to granulocyte-colony stimulating factor Author: Clough, Lee F.; 1; Prentice, Bryan; 1; Rodgerson, Denis O.; 1; 1NeoStem, Inc, United States Publication info: Journal of Clinical Apheresis 27. 1 (2012): 14. Abstract: Background: Information on the mobilization of stem cells by granulocyte-colony stimulating factor (G-CSF) in healthy individuals is limited and the establishment of an optimum or minimum effective quantity for celldose of stem cells has not been determined. The increasing use of cells more diverse and defined than those for which CD34 positivity serves as a surrogate requires more detailed knowledge of the mobilization of these cells. Prior research has noted that very small embryonic-like (VELs) stem cells are contained within the bone marrow and can be mobilized to the peripheral blood, thus allowing collection of VSELs by apheresis. VSELs are different than hematopoietic stem cells and have shown to have several characteristics that are found in embryonic stem cells. VSELs have been proven to be pluripotent-they have the potential to develop into liver, pancreas, neuronal, skeletal muscle, and heart tissues cells. With the discovery of VSELS and their pluripotency, the future holds many options for treatments utilizing autologous VSELS and the potential to develop organs. In preparation for these studies, it is important to know how the apheresis collection and selection of VSELS is impacted by the sex and age of the donor. Study Design: Both male and female healthy donors with varying age groups were recruited. Donors underwent mobilization with GCSF either 480 mcg/day × 2 days or GSCF 10 mcg/kg/day × × days. Mobilization change was due to an attempt to increase the amount of MNCs for collection and therefore an increase in VSELs via selection. The apheresis process was established by collecting at varying collect line Hct until the best selection of VSEL was determined. Once the stem cells were collected, the lab staff then selected the cells using Elutra to select out the VSELs. Results: Twelve males and = females, 21-30 years old were treated with 10 lg/kg of G-CSF on three consecutive days. Total nucleated cells (TNC) were increased by a mean of 586% in the males and 555% in the females. CD34+ cells were Very Small Embryonic Like Cell References

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increased 1,390% in the males and 1,137% in the females. VSEL which are essentially nondetectable in premobilized peripheral blood were increased to a mean of 62/μL for males (range 20-146 VSEL/μL) and 46/ll for females (range 4.7-109 VSEL/μL). Sixteen males and 8 females, 21-30 years old were treated with 480 lg of G-CSF on two consecutive days. TNC were increased by a mean of 455% in the males and 198% in the females. CD34+ cells were increased 529% in the males and 839% in the females. VSEL were increased to a mean of 34/μL for males (range 0.94-140 VSEL/μL) and 24/ lL for females (range 1.7-67 VSEL/μL). Eight males and 7 females, 40-50 years old were treated with 480 lg of G-CSF on two consecutive days. TNC were increased by a mean of 470% in the males and 547% in the females. CD34+ cells were increased 564% in the males and 338% in the females. VSEL were increased to a mean of 17/μL for males (range 5.7- 47.4 VSEL /μL) and 9.7/μL for females (range 2.4-21 VSEL/μL. Conclusion: These data would suggest that significant mobilization of CD34+ cells and VSEL can be achieved in healthy individuals with lower doses than previously reported. However there appears to be very considerable person to person variation that does not appear to be related to age (between 21 and 50 years) or gender. The use of three days mobilization using 10 μg/kg G-CSF seems to offer advantages for both CD34+ cells and VSEL. However, the extreme inconsistency of the results indicates the need for more extensive studies including other mobilization agents. _______________________________________________________________

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BDs is characteristic for pluripotent stem cells (e.g., ESCs) and as result of Ezh2 overexpression, VSELs, like ESCs, exhibit BDs - bivalently modified nucleosomes (trimethylated H3K27 and H3K4) at promoters of important homeodomain-containing developmental transcription factors (Sox21 Nkx2.2 Dlx1 Zfpm2 Irx2 Lbx1h Hlxb9 Pax5 HoxA3). Of note, spontaneous (as seen during differentiation) or RNA interference-enforced down-regulation of Ezh2 removes BDs what, results in lose of their plurioptentiality and de-repression of several BD-regulated genes that control their tissue commitment. In conclusion, Our results show for first time that in addition to the expression of pluripotency core transcription factor Oct-4, VSELs, like other pluripotent stem-cells, maintain their pluripotent state through an Ezh2-dependent BD-mediated epigenetic mechanism. Based on this our genome-wide gene expression study not only advances our understanding of biological processes that govern VSELs pluripotency, differentiation, and quiescence but will also help to develop better protocols for ex vivo expansion of these promising cells for potential application in regenerative medicine. _______________________________________________________________

Identification of SSEA-4+ very small embryonic-like stem cells in human myocardium Author: Wojakowski, W.; 1; Zuba-Surma, E.K.; 2; Syzdol, M.; 1; Kowalowka, A.; 1; Drukala, J.; 3; Bachowski, R.; 1; Wo, S.; 1; Dulak, J.; 2; Ratajczak, M.; 4; Tendera, M.; 1; 1Slaski Uniwersytet Medyczny w Katowicach, Poland; 2 Jagiellonian University, Department of Medical Biotechnology, Poland; 3Jagiellonian University, Poland; 4Stem Cell Institute, United States Publication info: European Heart Journal, suppl. SUPPL. 1 32 (Aug 2011): 958. Abstract: Very small embryonic-like cells (VSELs) is rare population of cells expressing pluripotent stem markers (Oct-4,Nanog, SSEA-4). The adult bone marrowderived VSELs may be expanded and differentiatiated into all three germ layers. VSELs were identified in murine bone marrow and solid organs, umbilical cord blood and peripheral blood in humans. Aim: To assess the endogenous presence of VSELs in human hearts and their mobilization induced by cardiac surgery. Methods: Patients with CAD undergoing CABG were enrolled. Fragments of right atrial appendage (RAA), internal mammary artery and BM samples were harvested. Blood samples (VSELs, EPCs, SDF-1, HGF, SCF, VEGF) were taken before, after weaning from cardiopulmonary bypass, after 24 hrs and 5-7 days. Fragments of RAAwere cut (0.5-1.0 cm3), and enzymatically digested in collagenase I (2 mg/ml) for 30-45min in 37oC. Cell suspension was stained with antibodies against stem cell markers, CD34 (FITC), CD133 (APC), SSEA-4 (PE),and CD45 (APC-Cy7) for 30min. Cell were washed, fixed in 2% paraformaldehyde and resuspended in PBS. Nuclei were stained with 10mM of Hoechst 33342 10min before the analysis. Analyses were carried out on ImageStream X cytometer which is a platform combining classical flow cytometer and fluorescence microscope and collects real images of identified cells. Results: Cardiac tissue contains very small (4.8Âą0.6 Îźm) roundish (aspectratio 1:1) nucleated VSEL cells expressing embryonic antigens. ImageStreamsystem showed the presence of early developmental marker SSEA-4 on the surfaceof these cells. The cells express CD34 and CD133 stem cellmarkers and lack CD45 indicating their non-hematopoietic origin. Conclusion: We demonstrated for the first time the presence of VSEL cellsexpressing embryonic marker SSEA-4 in adult human myocardium. _______________________________________________________________

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Aldehyde dehydrogenase (ALDH) - a promising new candidate for use in preclinical and clinical selection of pluripotent very small embryonic-like stem cells (VSEL SCs) of high long-term repopulating hematopoietic potential Author: Paczkowska, Edyta; Kawa, Milosz; Klos, Patrycja; Staniszewska, Marzena; Sienko, Jerzy; Dabkowska, Elzbieta Publication info: Annals of transplantation : quarterly of the Polish Transplantation Society 16. 3 (Jul 2011 Sep 2011): 59-71. Abstract: BACKGROUND In the field of stem cells (SCs) biology there is great need for a universal marker that can effectively identify the rare populations of SCs in order to characterize and isolate them for research and therapeutic purposes. In line with this idea, the measurement of aldehyde dehydrogenase (ALDH) activity shows promising potential as a common marker for the identification of SCs. Recently discovered very small embryonic-like stem cells (VSEL SCs) have a pluripotent nature and high long-term repopulating hematopoietic potential. The aim of the present study was to determine whether VSEL SCs isolated from umbilical cord blood (CB) and different murine organs express high ALDH activity. MATERIAL/METHODS To address this issue, 12 CB units were analyzed by employing flow cytometry to detect the following populations: (i) CXCR4(+)/Lin(-)/CD45(-)/ALDH(high), (ii) CD34(+)/Lin(-)/CD45(-)/ALDH(high) and (iii) CD133(+)/Lin(-)/CD45(-)/ALDH(high). Additionally, selected organs from 16 5-week-old female inbred C57BL/6J mice and 16 7-month-old female C57BL/6J mice were analyzed for detection of the following populations: (i) Sca-1(+)/Lin(-)/CD45(-) and (ii) Sca-1(+)/Lin(-)/CD45(+). All these populations were assessed for ALDH activity. RESULTS We found that CB contains VSEL SCs with high ALDH activity. We also observed that ALDH(high) cells constitute a modest percentage of VSEL SCs present in selected murine organs. CONCLUSIONS We demonstrated that CB and adult murine organs possess a subpopulation of ALDH(high) VSEL SCs. Above all, the observed high level of ALDH activity can be considered a functional marker of organ-derived pluripotent SCs and allows for simple, efficient isolation of cells with primitive features for their utility in targeted cell therapies. _______________________________________________________________

Transplantation of expanded bone marrow-derived very small embryonic-like stem cells (VSEL-SCs) improves left ventricular function and remodelling after myocardial infarction Author: Zuba-Surma, Ewa K.; 1; Guo, Yiru; 2; Taher, Hisham; 2; Sanganalmath, Santosh K.; 3; Hunt, Greg; 2; Vincent, Robert J.; 3; Kucia, Magda; 4; Abdel-Latif, Ahmed; 2; Tang, Xian-Liang; 2; Ratajczak, Mariusz Z.; 4; Dawn, Buddhadeb; 3; Bolli, Roberto; 2; 1Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Crakow, Poland; 2Institute of Molecular Cardiology, University of Louisville, Louisville, KY, United States; 3Division of Cardiovascular Diseases and Cardiovascular Research Institute, University of Kansas Medical Center and the University of Kansas Hospital, Kansas City, KS, United States; bdawn@kumc.edu; 4Stem Cell Institute, University of Louisville, Louisville, KY, United States Publication info: Journal of Cellular and Molecular Medicine 15. 6 (Jun 2011): 1319-1328. Abstract: Adult bone marrow-derived very small embryonic-like stem cells (VSEL-SCs) exhibit a Sca-1+/Lin-/CD45- phenotype and can differentiate into various cell types, including cardiomyocytes and endothelial cells. We have previously reported that transplantation of a small number (1 Ă&#x2014; 10 6) of freshly Very Small Embryonic Like Cell References

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isolated, non-expanded VSEL-SCs into infarcted mouse hearts resulted in improved left ventricular (LV) function and anatomy. Clinical translation, however, will require large numbers of cells. Because the frequency of VSEL-SCs in the marrow is very low, we examined whether VSEL-SCs can be expanded in culture without loss of therapeutic efficacy. Mice underwent a 30 min. coronary occlusion followed by reperfusion and, 48 hrs later, received an intramyocardial injection of vehicle (group I, n= 11), 1 × 105 enhanced green fluorescent protein (EGFP)-labelled expanded untreated VSEL-SCs (group II, n= 7), or 1 × 105 EGFP-labelled expanded VSEL-SCs pre-incubated in a cardiogenic medium (group III, n= 8). At 35 days after myocardial infarction (MI), mice treated with pre-incubated VSEL-SCs exhibited better global and regional LV systolic function and less LV hypertrophy compared with vehicle-treated controls. In contrast, transplantation of expanded but untreated VSEL-SCs did not produce appreciable reparative benefits. Scattered EGFP+ cells expressing α-sarcomeric actin, platelet endothelial cell adhesion molecule (PECAM)-1, or von Willebrand factor were present in VSEL-SC-treated mice, but their numbers were very small. No tumour formation was observed. We conclude that VSEL-SCs expanded in culture retain the ability to alleviate LV dysfunction and remodelling after a reperfused MI provided that they are exposed to a combination of cardiomyogenic growth factors and cytokines prior to transplantation. Counter intuitively, the mechanism whereby such pre-incubation confers therapeutic efficacy does not involve differentiation into new cardiac cells. These results support the potential therapeutic utility of VSEL-SCs for cardiac repair. © 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd. _______________________________________________________________

Clinical evidence for mobilization of very small embryonic/epiblast like stem cells (VSEL-SCS) in patients with inflammatory bowel disease Author: Marlicz, Wojciech M.; Blogowski, Wojciech; Paczkowska, Edyta; Kucia, Magda; Lawniczak, Malgorzata; Dolegowska, Barbara; Starzynska, Teresa; Ratajczak, Mariusz Z. Publication info: Gastroenterology, suppl. SUPPL. 1 140. 5 (May 2011): S656. Abstract: INTRODUCTION: The evidence is emerging that stem cells (SCs) play an important role in the pathogenesis of inflammatory bowel diseases (IBD). Our team has recently identified in murine and human bone marrow (BM) population of very small embryonic/epiblast like stem cells (VSEL-SCs), capable of differentiating into cells from all three germ layers [Leukemia 2006;20(11)]. We hypothesize that under steady state conditions circulating VSEL-SCs play a significant role in turnover of tissue specific/committed SCs in various organs. We previously demonstrated that these cells are mobilized into peripheral blood (PB) in patients with myocardial infarction [JACC 2009;53(1)]and stroke [Stroke 2009;40(4)]. Currently we focused on identification and detailed characterization of similar population of SCs in PB of IBD patients. MATERIAL AND METHODS: There were enrolled 50 IDB and 30 healthy individuals. The severity of IBD was classified according to clinical/endoscopic indexes. PB samples were collected on admission and using fluorescence-activated cell sorting (FACS) VSELs-SCs (CD45-/CXCR4+/CD133+/Lin-) were counted/isolated and phenotyped for expression of TLR2 and TLR4 receptors. PB SCs were evaluated by employing direct immunofluorescence staining and RT-qPCR analysis for expression of embryonic SCs and intestinal differentation markers. Plasma levels of potential SCs chemoattractants (VEGF,HGF,SDF- 1,S1P) were also measured. RESULTS: In IBD patients we observed a significant increase in the number of circulating small primitive cells expressing VSEL phenotype (CXCR4+,CD133+,lin-,CD45-) in comparison to healthy individuals (p<0.001). The number of VSEL-SCs correlated with the serum levels of HGF and VEGF but not with SDF-1. We also found that IBD PBMNC are strongly enriched for mRNA of GI lineage markers (lgr- 5,ASCL-2,BMI-1,DSI-1,DCLK-1)and pluripotent stem cell markers (Oct-4, SSEA-4, Nanog). Of interest by employing FACS we were able to detect higher expression of Very Small Embryonic Like Cell References

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TLR receptors on mobilized and circulating VSEL-SCs in patients with active IDB as compared to those in remission (p<0.05). Their levels correlated with several plasma inflammatory molecules. CONCLUSION: Our multiparameter FACS analysis and molecular studies revealed that VSEL-SCs are mobilized and circulate in PB of IBD patients. The number of circulating VSEL-SCs is associated with activity of selected protective/inflammatory molecules and with clinical intensity of IBD. This strongly supports the hypothesis that circulating pluripotent VSEL-SCs might be involved in the pathogenesis of IBD. The biological and clinical consequences of this phenomenon require further investigations. _______________________________________________________________

Circulating stem cells in patients with gastric malignancies Author: Starzynska, Teresa; Blogowski, Wojciech; Zuba-Surma, Ewa; Marlicz, Wojciech M.; Nowak, Witold; Lawniczak, Malgorzata; Madej-Michniewicz, Anna; Deskur, Anna; Ratajczak, Mariusz Z. Publication info: Gastroenterology, suppl. SUPPL. 1 140. 5 (May 2011): S831. Abstract: Introduction: There is accumulating evidence to suggest that malignancy is a stem cell disorder. It was recently proven in an animal model of Helicobacter induced gastric cancer that bone-marrow derived cells may be mobilized into peripheral blood (PB), migrate to and repopulate the stomach tissue gradually transforming into cancer cells. Our team has recently identified a population of very small embryonic-like stem cells (VSELs) that as we hypothesize may give rise to certain types of tumors as well as are involved in cancer progression by providing vessels and stroma support for growing cancer (Leukemia 2006;20,857-69). AIMS &METHODS: In the current study we examined whether VSELs, Mesenchymal Stem Cells (MSCs), and Hematopoietic Stem Cells (HSCs) are mobilized into PB in patients with gastric malignancy. Using fluorescence-activated cell sorter (FACS), VSELs (Lin-/CD45-/CD133+/CD184+/CXCR4+), MSCs (CD45-/STRO-1+/CD105+/CD90+/ CD29+), ESC (CD45-/CD31+/CD34+/CD133+/KDR+) and HSCs(Lin/CD45+/CD34+/ CD133+) were enumerated and sorted from PB samples derived from 32 patients with newly diagnosed gastric malignancy (cancer n=20, GISTs n=9, NET n=2, lymphoma n=1) and 17 controls. By employing RT-qPCR we evaluated expression of embryonic (Oct4, Nanog) and mesenchymal (Brachyury, CD105) SCs' markers. The morphology of sorted cells was evaluated by ImageStream analysis, and expression of VSEL and MSC specific genes was performed by RQ-PCR. In addition, we measured plasma levels of i) SDF-1 (pro-metastatic chemokine), ii)VEGF (angiopoietic factor) and iii) C-reactive protein, complement C3 and C4 (markers of inflammation). Results: Significantly higher numbers of circulating VSELs and MSCs were detected in patients with gastric malignancy than in controls (P<0.05 and P<0.0001 respectively). The mobilization was irrespective of tumor histology. Significantly lower numbers of circulating HSCs were observed, as well as lower plasma SDF-1 levels were detected in the study group (P<0.05 for both). Conclusions: Our study shows for a first time that VSELs and MSCs are mobilized into PB in patients with gastric malignancy. Interestingly, however both VSELs and MSCs express CXCR4 receptor the serum level of SDF-1 does not seem to correlate with increased mobilization of these cells. Thus, further studies are required to identify factors (e.g., blood plasma bioactive lipids) that are responsible for SCs mobilization, and the role of mobilized SCs in development and progression of gastric malignancy. _______________________________________________________________

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The mobilization of very small embryonic-like stem cells (VSELs) and multipotent mesenchymal stem cells (MSCs) into peripheral blood in pancreatic cancer patients correlates with tumor stage Author: Starzynska, Teresa; Blogowski, Wojciech; Zuba-Surma, Ewa; Marlicz, Wojciech M.; Nowak, Witold; Binkowska-Borgosz, Iza; Ratajczak, Mariusz Z. Publication info: Gastroenterology, suppl. SUPPL. 1 140. 5 (May 2011): S831. Abstract: INTRODUCTION: Mounting evidence accumulates that malignancy arises in stem cell compartment and that cancer stem cells are responsible for tumor growth, its re-growth after unsuccessful treatment and metastases. Our team has recently identified a population of very small embryonic-like stem cells (VSELs) that as we hypothesize may give rise to certain types of tumors, as well as, are involved in cancer progression (Leukemia 2006;20,857-69). AIMS &METHODS: In the current study we examined whether VSELs, Mesenchymal Stem Cells (MSCs), and Hematopoietic Stem Cells (HSCs) are mobilized into peripheral blood (PB) in pancreatic cancer patients. Furthermore the correlation between the number of mobilized stem cells and tumor stage was assessed. Using fluorescence-activated cell sorter (FACS),VSELs(Lin-/CD45-/CD133+/CXCR4+),MSCs(CD45-/STRO1+/CD105+/CD90+/ CD29+and HSCs (Lin-/CD45+/CD133+) were enumerated and sorted from PB samples derived from 17 newly diagnosed pancreatic cancer patients and 17 controls. By employing RT-qPCR we evaluated expression of embryonic (Oct4, Nanog) and mesenchymal (Brachyury, CD105) SCs' markers. The morphology of sorted cells was evaluated by ImageStream analysis, and expression of VSEL and MSC specific genes was performed by RQ-PCR. In addition, plasma levels of i) SDF-1 (pro-metastatic chemokine), ii) VEGF (angiopoietic factor) and iii) C-reactive proteins (marker of inflammation) were measured. RESULTS: We observed significantly higher mean number of circulating VSELs and MSCs in cancer patients, comparing to controls (P<0.05 and P<0.0001, respectively). Moreover, number of circulating VSELs and MSCs positively correlated with presence of distal metastases (R=0.77; P=0.00001) and plasma level of C-reactive protein (R=0.43; P=0.03). To our surprise, significantly lower number of circulating HSCs, and lower plasma SDF-1 levels were found in cancer patients (P<0.05 for both). CONCLUSIONS: Our study shows for a first time that VSELs and MSCs are mobilized into PB in patients with pancreatic cancer, as well as, their number correlates with metastatic spread. Interestingly, however both VSELs and MSCs express CXCR4 receptor, the serum level of SDF-1 does not seem to correlate with increased mobilization of these cells. Thus, further studies are required to identify factors (e.g., blood plasma bioactive lipids) that are responsible for SCs mobilization, and the role of mobilized SCs in pancreatic cancer development and progression. _______________________________________________________________

Identification and isolation from either adult human bone marrow or G-CSF-mobilized peripheral blood of CD34(+)/CD133(+)/CXCR4(+)/ Lin(-)CD45(-) cells, featuring morphological, molecular, and phenotypic characteristics of very small embryonic-like (VSEL) stem cells Author: Sovalat, Hanna; Scrofani, Maurice; Eidenschenk, Antoinette; Pasquet, Stéphanie; Rimelen, Valérie; Hénon, Philippe Publication info: Experimental hematology 39. 4 (Apr 2011): 495-505. Abstract: OBJECTIVE - Recently, we demonstrated that normal human bone marrow (hBM)-derived CD34(+) cells, released into the peripheral blood after granulocyte colony-stimulating factor mobilization, contain cell Very Small Embryonic Like Cell References

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subpopulations committed along endothelial and cardiac differentiation pathways. These subpopulations could play a key role in the regeneration of post-ischemic myocardial lesion after their direct intracardiac delivery. We hypothesized that these relevant cells might be issued from very small embryonic-like stem cells deposited in the BM during ontogenesis and reside lifelong in the adult BM, and that they could be mobilized into peripheral blood by granulocyte colony-stimulating factor. MATERIALS AND METHODS - Samples of normal hBM and leukapheresis products harvested from cancer patients after granulocyte colony-stimulating factor mobilization were analyzed and sorted by multiparameter flow cytometry strategy. Immunofluorescence and reverse transcription quantitative polymerase chain reaction assays were performed to analyze the expression of typical pluripotent stem cells markers. RESULTS - A population of CD34(+)/CD133(+)/CXCR4(+)/Lin(-) CD45(-) immature cells was first isolated from the hBM or from leukapheresis products. Among this population, very small (2-5 Îźm) cells expressing Oct-4, Nanog, and stage-specific embryonic antigen-4 at protein and messenger RNA levels were identified. CONCLUSIONS - Our study supports the hypothesis that very small embryonic-like stem cells constitute a "mobile" pool of primitive/pluripotent stem cells that could be released from the BM into the peripheral blood under the influence of various physiological or pathological stimuli. In order to fully support that hBM- and leukapheresis product-derived very small embryonic-like stem cells are actually pluripotent, we are currently testing their ability to differentiate in vitro into cells from all three germ layers. _______________________________________________________________

Effect of granulocyte-colony stimulating factor on mobilization and recruitment of very small embryonic-like stem cells Author: Guo, Hui-Juan; 1; Wang, Jian; 1; Zhang, Xing-Xiu; 1; Zheng, Min; 2; 1Department of Neurology, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China guohuijia88@163.com; 2 Department of Basic Medical Sciences, Chongqing Medical University, Chongqing 400016, China zmdwj625@gmail.com Publication info: Journal of Clinical Rehabilitative Tissue Engineering Research 15. 14 (Apr 2011): 2530-2534. Abstract: BACKGROUND: A population of very small embryonic-like stem cells (VSEL-SCs) is a kind of non-hemopoietic stem cells, which has similar biological characteristics to embryonic stem cells. Currently, most studies concerning VSEL-SCs after myocardial infarction, but mobilization of VSEL-SCs and its effect on repair damaged tissues after acute cerebral infarction remain poorly understood. OBJECTIVE: To investigate the effects of granulocyte-colony stimulating factor (G-CSF) on mobilization of bone marrow-derived VSEL-SCs, recruitment and mechanism after mouse acute cerebral infarction. METHODS: Mouse middle cerebral artery occlusion (MACO) model was induced by using the filament occlusion method. G-CSF and normal saline were separately injected to MACO model. Neurological scales were evaluated. The number of VSEL-SCs mobilized into peripheral blood was checked by fluorescence-activated cell sorting analysis. The level of stromal cell derived factor-1 (SDF-1) in plasma and cerebral tissue were determined by ELISA assay. Positive expression of SDF-1 in ischemic regions was detected by immunohistochemistry. RESULTS AND CONCLUSION: Compared with the normal saline group, the neurological behavioral scale of G-CSF treated group was significantly lower at 108 hours after operation (P <0.05). G-CSF groups mobilized more VSEL-SCs into peripheral blood compared with the operated control group at 72 and 108 hours (P <0.05). The SDF-1 density significantly increased in the plasma and brain tissues of the G-CSF group than that of the normal saline group (P <0.05), and positive correlation can be seen between the number of VSEL-SCs mobilized into peripheral blood and the SDF-1 Very Small Embryonic Like Cell References

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plasma concentration. The positive expression of SDF-1 in the G-CSF group was greater than that of the normal saline group. The results indicated that G-CSF mobilized VSEL-SCs in adult MACO mouse bone marrow into peripheral blood. The mechanism of beneficial effects of G-CSF possibly related to increase of SDF-1 expression in the infracted tissues and recruitment of more VSEL-SCs through CXCR4/SDF-1 axis. _______________________________________________________________

The role of pluripotent embryonic-like stem cells residing in adult tissues in regeneration and longevity Author: Ratajczak, Mariusz Z.; 1; Liu, Rui; 1; Ratajczak, Janina; 2; Kucia, Magda; 2; Shin, Dong-Myung; 1; 1Stem Cell Institute at James Graham Brown Cancer Center, University of Louisville, Louisville, KY 40202, United States mzrata01@louisville.edu; 2Stem Cell Institute at James Graham Brown Cancer Center, University of Louisville, Louisville, KY 40202, United States, Department of Physiology, Pomeranian Medical University, Szczecin,; Poland Publication info: Differentiation 81. 3 (Mar 2011): 153-161. Abstract: From the point of view of regenerative potential, the most important cells are pluripotent stem cells (PSCs). Such cells must fulfill certain in vitro as well as in vivo criteria that have been established by work with PSCs isolated from embryos, which are known as embryonic stem cells (ESCs). According to these criteria, pluripotent stem cells should: (i) give rise to cells from all three germ layers, (ii) complete blastocyst development, and (iii) form teratomas after inoculation into experimental animals. Unfortunately, in contrast to immortalized embryonic ESC lines or induced PSCs (iPSCs), these last two criteria have thus far not been obtained in a reproducible manner for any potential PSC candidates isolated from adult tissues. There are two possible explanations for this failure. The first is that PSCs isolated from adult tissues are not fully pluripotent; the second is that there are some physiological mechanisms involved in keeping these cells quiescent in adult tissues that preclude their "unleashed proliferation", thereby avoiding the risk of teratoma formation. In this review we present an evidence that adult tissues contain remnants from development; a population of PSCs that is deposited in various organs as a backup for primitive stem cells, plays a role in rejuvenation of the pool of more differentiated tissue-committed stem cells (TCSCs), and is involved in organ regeneration. These cells share several markers with epiblast/germ line cells and have been named very small embryonic-like stem cells (VSELs). We suggest that, on one hand, VSELs maintain mammalian life span but, on the other hand, they may give rise to several malignancies if they mutate. We provide an evidence that the quiescent state of these cells in adult tissues, which prevents teratoma formation, is the result of epigenetic changes in some of the imprinted genes. ÂŠ 2011 International Society of Differentiation. _______________________________________________________________

Optimal recovery of SSEA-4+/OCT-4+/CDI33+/CXCR4+/LIN-/ CD45- very small embryonic-like (VSEL) stem cells from umbilical cord blood (CB) using plasma depletion/reduction (PDR) compared to red cell reduction (RCR) Author: Chow, R.; 1; Tonai, R.; 1; Klich, I.; 2; Wang, B.C.; 1; Chow, M.; 1; Ratajczak, J.; 2; Petz, L.D.; 1; Ratajczak, M.Z.; 2; 1StemCyte International Cord Blood Center, United States; 2University of Louisville, United States Publication info: Biology of Blood and Marrow Transplantation, suppl. SUPPL. 1 17. 2 (Feb 2011): S219. Abstract: We previously reported the presence of VSEL stem cells (SC) in CB which are capable of tri-lineage differentiation, and the techniques to identify and isolate these cells (Leukemia 2007:21:297-303). Because of Very Small Embryonic Like Cell References

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their small size (< 6 mm) and different cell density, 42.5± 12.6% of these VSEL SC in CB are lost upon red cell reduction (RCR) processing with AXPTM AutoXpress technology, and significant percentages of VSEL SC are also lost with Ficoll-Paque and red cell lysis techniques (Europ. J. Hematology 2009:84:3446). Moreover, after thawing, 82.7±17.3% of the VSELs cyopreserved after RCR are recovered. Since these CB VSELs may be important for regenerative medicine applications and contribute to CB hematopoiesis and possess potential of long term repopulating hematopoietic stem cells (LT-HSCs), we sought to optimize recovery of these cells with different CB volume reduction techniques. Previously, it was reported that plasma depletion/reduction (PDR) of CB loses less than 0.01% of nucleated cells in the discarded plasma portion (Biol. Blood Marrow Transplant 2007;13:1346-57), so we performed 3 parallel comparisons using CB products that were divided into two equal portions processed using either hetastarch (HES) RCR or PDR. As can be seen in the table below, there is essentially no loss of VSEL SCs that we can detect after PDR processing, compared to 44.7±3.9% VSEL SC loss with RCR. Post-thaw, the two types of products appear to yield similar recoveries of VSELs compared to post-processing and pre-freeze samples (85.2% PDR versus 86.8% RCR). We conclude that (1) we confirmed our previous observations of significant CB VSEL SC loss with standard RCR techniques, this time using a manual HES RCR method, (2) PDR offers essentially 100% recovery of VSEL SCs in CB, and (3) even after thawing, PDR retains 85.2% of the collected VSELs compared to 50.9% for HES RCR. Whether these observations extend to the outstanding transplant-related mortality (TRM), overall (OS) and disease free survival (DFS) seen with PDR CB transplants, and whether VSELs are responsible for the effect seen with post-thaw wash versus direct infusion on chronic GvHD, TRM, OS and DFS are the subjects of current investigations. (Table Presented). _______________________________________________________________

Adult murine bone marrow-derived very small embryonic-like stem cells differentiate into the hematopoietic lineage after coculture over OP9 stromal cells Author: Ratajczak, Janina; 1; Wysoczynski, Marcin; 2; Zuba-Surma, Ewa; 3; Wan, Wu; 2; Kucia, Magda; 2; Yoder, Mervin C.; 4; Ratajczak, Mariusz Z.; 2; 1Stem Cell Institute at James Graham Brown Cancer Center, University of Louisville, Louisville, KY, United States, Department of Physiology, Pomeranian Medical University, Szczecin, Poland; j0rata01@louisville.edu; 2Stem Cell Institute at James Graham Brown Cancer Center, University of Louisville, Louisville, KY, United States; 3Stem Cell Institute at James Graham Brown Cancer Center, University of Louisville, Louisville, KY, United States, Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics; and Biotechnology, Jagiellonian University, Krakow, Poland; 4Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN, United States Publication info: Experimental Hematology 39. 2 (Feb 2011): 225-237. Abstract: Objective: We recently identified a population of small Sca-1+/Lin-/CD45- cells in adult murine bone marrow that express several epiblast/germ line and pluripotent stem cell markers (e.g., Oct-4 and SSEA-4) that we named "very small embryonic-like stem cells" (VSELs). In this report, we test the hypothesis that VSELs can differentiate along the hemato/lymphopoietic lineage. Materials and Methods: Purified from bone marrow, VSELs were primed/cocultured over OP9 stroma cell line and subsequently tested in vitro and in vivo assays for their hematopoietic potential. In parallel, cells derived from VSELs were evaluated for expression of hematopoietic genes and surface markers. Results: Although we observed that freshly isolated VSELs do not exhibit in vitro and in vivo hematopoietic potential, they may, after coculture over OP9 stromal cells, differentiate along the hematopoietic lineage in a similar way as embryonic stem cells or inducible pluripotent stem cells. "OP9-primed," VSEL-derived cells acquired expression of several hemato/lymphopoiesis-specific Very Small Embryonic Like Cell References

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genes and markers, gave rise to hematopoietic colonies in vitro, and protected lethally irradiated mice in both primary and secondary transplant models on transplantation. We also observed that, compared to hematopoietic stem/progenitor cells, VSELs are highly resistant to total body irradiation. Conclusions: Based on these observations, we postulate that VSELs are the most primitive murine bone marrow-residing population of stem cells that have the potential to become specified into the hematopoietic lineage and may share some of the characteristics of long-term repopulating HSCs. ÂŠ 2011 ISEH - Society for Hematology and Stem Cells. _______________________________________________________________

Stem cells for neural regeneration--a potential application of very small embryonic-like stem cells Author: Ratajczak, J; Zuba-Surma, E; Paczkowska, E; Kucia, M; Nowacki, P; Ratajczak, M Z Publication info: Journal of physiology and pharmacology : an official journal of the Polish Physiological Society 62. 1 (Feb 2011): 3-12. Abstract: The goal of regenerative medicine is to ameliorate irreversible destruction of brain tissue by harnessing the power of stem cells in the process of neurogenesis. Several types of stem cells, including mesenchymal stem cells, hematopoietic stem cells, as well as neural cells differentiated from embryonic stem cell lines, have been proposed as potential therapeutic vehicles. In this review paper we will discuss a perspective of stem cell therapies for neurological disorders with special emphasis on potential application of cells isolated from adult tissues. In support of this our group found that murine bone marrow contains a mobile population of Oct-4+CXCR4+SSEA-1+Sca-1+linâ&#x2C6;&#x2019;CD45â&#x2C6;&#x2019; very small embryonic-like stem cells (VSELs) that are mobilized into peripheral blood in a murine stroke model. The number of these cells in circulation increases also after pharmacological mobilization by administration of granulocyte colony stimulating factor (G-CSF). Recently we found that VSELs are present in various non-hematopoietic adult organs and, interestingly, our data indicate that the brain contains a high number of cells that display the VSEL phenotype. Based on our published data both in human and mice we postulate that VSELs are a mobile population of epiblast/germ line-derived stem cells and play an important role as an organ-residing reserve population of pluripotent stem cells that give rise to stem cells committed to particular organs and tissues--including neural tissue. In conclusion human VSELs could be potentially harnessed in regenerative medicine as a source of stem cells for neurogenesis. _______________________________________________________________

A novel paradigm in stem cell trafficking: The ratio of peripheral blood sphingosine-1 phosphate (S1P) to bone marrow ceramide-1 phosphate (C1P) regulates mobilization and homing of hematopoietic stem cells Author: Kim, Chihwa; 1; Wan, Wu; 1; Liu, Rui; 2; Kucia, Magdalena; 2; Laughlin, Mary J.; 3; Ratajczak, Janina; 1; Ratajczak, Mariusz Z.; 1; 1Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, United States; 2Stem Cell Biology, University of Louisville, United States; 3Hematopoietic Stem Cell Program, University of Virginia, United States Publication info: Blood 116. 21 (Nov 19, 2010). Abstract: The stromal derived factor-1 (SDF-1)-CXCR4 axis plays an unquestioned role in developmental migration of hematopoietic stem cells (HSPCs) and their retention in the bone marrow (BM). However, changes in the SDF-1 gradient between BM and peripheral blood (PB) do not always support its having a Very Small Embryonic Like Cell References

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crucial role as chemoattractant for mobilization or homing of HSPCs. As demonstrated by others (e.g., Bone Marrow Transplantation 2003; 31:651-654, and Transfus Apher Sci 2009;40:159) and us (Leukemia 2010;24:976-985) the plasma SDF-1 level does not correlate with mobilization of HSPCs. On the other hand, there is increasing doubt about an exclusive role for SDF-1 in homing of HSPCs in BM. This is based on evidence that i) CXCR4-/- fetal liver HSPCs may home to BM in an SDF-1-independent manner (Immunity 1999;10:463-471), ii) homing of murine HSPCs made refractory to SDF-1 by incubation and co-injection with a CXCR4 receptor antagonist is normal or only mildly reduced (Science 2004;305:1000), and finally iii) HSPCs in which CXCR4 has been knocked down by means of an SDF-1 intrakine strategy also engraft in lethally irradiated recipients (Blood 2000;96:2074-,2080). All this strongly suggests the existence of other factors involved in the mobilization and homing of HSPCs. Moreover, while SDF-1 is a potent chemoattractant for HSPCs when employed at supraphysiological concentrations in vitro, as a peptide it is highly susceptible to degradation by proteases that are elevated, for example, in PB during stem cell mobilization or in the BM microenvironment after myeloablative conditioning for transplantation. Employing ELISA for detection in the present study, we observed insignificant changes in SDF-1 level both in PB during mobilization and in BM after myeloablative conditioning. We also found that mobilized PB (mPB) plasma as well as conditioned media (CM) from lethally irradiated mice chemoattract HSPCs in an SDF-1-independent manner as demonstrated by i) normal chemotaxis of AMD3100 pre-treated cells and ii) preservation of chemotactic activity of plasma and BM-derived CM following heat inactivation. However, the chemotactic activity of mPB plasma and BM CM was inhibited after stripping by activated charcoal. This suggested the involvement of small molecule bioactive lipids. It is known that sphingolipids, which are important components of cell membranes, give rise to two bioactive derivatives, sphingosine-1 phosphate (S1P) and ceramide-1 phosphate (C1P), with S1P already identified as a chemoattractant for HSPCs (Ann N Y Acad Sci. 200;1044:84-89). To our surprise, we found that C1P is also a strong chemoattractant for human and murine HSPCs. In addition, we observed that at physiological concentrations both these bioactive lipids i) activate phosphorylation of MAPKp42/44 and AKT in HSPCs, ii) induce expression of matrix metalloproteinases (MMPs), and iii) modulate adhesion to stroma and endothelium. Interestingly, by employing ELISA and/or mass spectophotometry we found that, while the S1P level increases in PB during mobilization, the C1P level increases in BM after myeloablative conditioning for transplantation. Based on these findings, we propose a new paradigm in which the S1P:C1P ratio plays a role in mobilization and homing of HSPCs. While S1P is a major chemoattractant that directs egress of HSPCs from BM into PB, C1P released from damaged cells in BM after myeloablative conditioning creates a homing gradient for circulating HSPCs. We also postulate that the S1P:C1P ratio plays a more universal role and is involved in regulating migration of other types of stem cells, such as circulating mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic-like (VSEL) stem cells. Accordingly, while S1P plays a role in egress of stem cells into PB, C1P released from damaged cells (e.g., in infarcted myocardium or brain tissue after stroke) chemoattracts circulating stem cells for potential repair. _______________________________________________________________

Intermittent hypoxia mobilizes bone marrow-derived very small embryonic-like stem cells and activates developmental transcriptional programs in mice Author: Gharib, Sina A; Dayyat, Ehab A; Khalyfa, Abdelnaby; Kim, Jinkwan; Clair, Heather B; Kucia, Magdalena; Gozal, David Publication info: Sleep 33. 11 (Nov 2010): 1439-46.

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Abstract: BACKGROUND - obstructive sleep apnea is a prevalent disorder associated with cognitive dysfunction and cardiovascular and metabolic morbidity and is characterized by recurrent episodes of hypoxia during sleep. Bone marrow-derived very small embryonic-like (VSEL) pluripotent stem cells represent a recruitable pool that may play an important role in organ repair after injury. We hypothesized that exposure to intermittent hypoxia (IH) can mobilize VSELs from the bone marrow (BM) to peripheral blood (PB) in mice and can activate distinct transcriptional programs. METHODS - adult mice were exposed to IH or normoxia for 48 hours. VSELs were sorted from BM and PB using flow cytometry. Plasma levels of stem cell chemokines, stromal cell derived factor-1 (SDF-1), hepatocyte growth factor (HGF), and leukemia inhibitory factor (LIF) were measured. Transcriptional profiling of VSELs was performed, and differentially expressed genes were mapped to enriched functional categories and genetic networks. RESULTS - exposure to IH elicited migration of VSELs from BM to PB and elevations in plasma levels of chemokines. More than 1100 unique genes were differentially expressed in VSELs in response to IH. Gene Ontology and network analysis revealed the activation of organ-specific developmental programs among these genes. CONCLUSIONS - exposure to IH mobilizes VSELs from the BM to PB and activates distinct transcriptional programs in VSELs that are enriched in developmental pathways, including central nervous system development and angiogenesis. Thus, VSELs may serve as a reserve mobile pool of pluripotent stem cells that can be recruited into PB and may play an important role in promoting end-organ repair during IH. _______________________________________________________________

Characterisation of circulating stem and progenitor cells in type 2 diabetic patients with foot ulceration Author: Koblik, T.; 1; Zuba-Surma, E.K.; 2; Witek, P.; 1; Nowak, W.; 2; Jozkowicz, A.; 2; Malecki, M.T.; 1; Dulak, J.; 2 1 ; Department of Metabolic Diseases, Jagellonian University of Krakow, Poland; 2Department of Medical Biotechnology, Jagellonian University of Krakow, Poland Publication info: Diabetologia, suppl. SUPPL. 1 53 (Sep 2010): S460. Abstract: Background and aims: Type 2 diabetes (T2DM) is a major health problem in Europe due to severe complications, for example the diabetic foot syndrome (DFS). Ulcerations that are often difficult to heal. Their pathogenesis may be associated not only with metabolic abnormalities but also with alterations in stem and progenitor cell mobilization. It has been shown that both the functionality and the number of endothelial progenitor cells (EPC) circulating in peripheral blood (PB) are altered in diabetic patients. However, other stem cell (SC) populations potentially involved in wound healing and regeneration such as i) mesenchymal SC (MSC) and ii) adult pluripotent SC (PSC), including very small embryonic-like (VSEL) SC, have never been examined in T2DM. Aim of the study was to examine the phenotype and level of SC circulating in PB in T2DM subjects with and without DFS as compared to healthy controls. Materials and methods: Three groups of subjects were included: a) T2DM patients without DFS (n=9); b) T2DM with DFS with ulceration(s) (n=3), c) controls without diabetes (n=4). We employed flow cytometry to evaluate the presence of the following SC populations based on the surface markers: i) EPC (CD31+CD133+CD45-, CD31+KDR+CD34+CD45-); ii) MSC (STRO1+CD105+CD45-, CD90+CD29+CD45-) and PSC (Lin-CD45-CD133+, Lin-CD45-Tra1.81+). Results: We found that when compared to healthy subjects, the number of EPC was decreased in both T2DM groups (w/o and with Very Small Embryonic Like Cell References

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DFS) (9.6±2.1, 5.3±0.7∗ and 3.4±2.3 of CD31+CD133+CD45- cells/1ml PB, respectively). Similarly, the number of MSC were smaller in both T2DM groups (6.4±1.1, 3.9±0.5∗ and 2.2±0.4 of STRO-1+CD105+CD45- cells/ 1ml PB, respectively). Interestingly, the numbers of PSC was slightly decreased in healthy controls as compared to T2DM patients (0,27±0.7, 0.30±0.08 and 0.38±0.07of Lin- CD45-CD133+ cells/ 1ml PB, respectively). ∗ P<0.05 vs. control. Conclusion: For the first time we characterized several subsets of SC circulating in the blood of T2DM patients. Our initial results indicate that as compared to healthy controls, their mobilization may be different, particularly in the subgroup with DFS and ulceration; a potential pathogenic role of this phenomenon should be considered. _______________________________________________________________

Molecular characterization of isolated from murine adult tissues very small embryonic/epiblast like stem cells (VSELs) Author: Shin, Dong-Myung; 1; Liu, Rui; 1; Klich, Izabela; 1; Ratajczak, Janina; 2; Kucia, Magda; 2; Ratajczak, Mariusz Z.; 2; 1Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, Louisville, United States; 2Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, Louisville, United States, Department of Physiology, Pomeranian Medcial University, Szczecin, Poland; mzrata01@louisville.edu Publication info: Molecules and Cells 29. 6 (Jun 2010): 533-538. Abstract: Pluripotent very small embryonic/epiblast derived stem cells (VSELs) as we hypothesize are deposited at begin of gastrulation in developing tissues and play an important role as backup population of pluripotent stem cells (PSCs) for tissue committed stem cells (TCSCs). We envision that during steady state conditions these cells may be involved in tissue rejuvenation and in processes of regeneration/repair after organ injuries. Molecular analysis of adult bone marrow (BM)-derived purified VSELs revealed that they i) express pluripotent stem cells markers e.g., Oct4, Nanog, Klf-4, SSEA-1 ii) share several markers characteristic for epiblast as well as migratory primordial germ cells (PGCs), and iii) possess a unique pattern of genomic imprinting (e.g., erasure of differently methylated regions at Igf2-H19 and Rasgrf1 loci and hypermethylation at KCNQ1 and Igf2R loci). This supports that VSELs are related to epiblast-derived migrating PGC-like cells and, despite their pluripotent stem cell character, changes in the epigenetic signature of imprinted genes keep these cells quiescent in adult tissues and prevent them from teratoma formation. In contrast epigenetic changes/mutations that lead to activation of imprinted genes could potentially lead to tumor formation by these cells. Mounting evidence accumulates that perturbation of expression of imprinted genes is a common phenomenon observed in developing tumors. © 2010 The Korean Society for Molecular and Cellular Biology and Springer Netherlands. _______________________________________________________________

Treatment viability of stem cells in ophthalmology Author: Jeganathan, V. Swetha E.; 1; Palanisamy, Muthusamy; 2; 1Tun Hussein Onn National Eye Hospital, Lorong Utara B, 46200 Petaling Jaya, Selangor Darul Ehsan, Malaysia, Centre for Eye Research Australia, University of Melbourne, VIC, Australia; vswetha@ausdoctors.net; 2Department of Ophthalmology, University Sabah Malaysia, Kota Kinabalu, Malaysia Publication info: Current Opinion in Ophthalmology 21. 3 (May 2010): 213-217. Abstract: Purpose of Review: Adult ocular stem cells have the potential to restore vision in patients previously Very Small Embryonic Like Cell References

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deemed incurable. This review summarizes strides in stem cell research and stumbling blocks that must be overcome to enable treatment viability in ophthalmology. Recent Findings: Stem/progenitor cells located in different regions of the eye are capable of differentiating enabling cell repopulation and tissue regeneration. At present, limbal epithelial stem transplantation is the sole ocular cell-based therapy being implemented into clinical practice. Research performed in animal models gives hope for using similar strategies to treat a wide range of ocular diseases in humans. The essential step toward successful therapeutic exploitation is to unravel regulators that control their cell proliferation and renewal pathways. The recently identified very small embryonic-like stem cells (VSEL-SCs) present in the bone marrow could potentially be harvested for regeneration from cord blood via ex-vivo expansion and differentiation protocols Summary: Although numerous impediments remain, the use of bioengineered stem cells is promising and may epitomize the future for replacement and regeneration of ocular tissues in various previously incurable ocular disorders. ÂŠ 2010 Wolters Kluwer Health | Lippincott Williams &Wilkins. _______________________________________________________________

Cells expressing stem cells (sc) and early gastrointestinal markers are mobilized into peripheral blood in patients with inflammatory bowel disease -prognostic and therapeutic implications Author: Marlicz, W.; 1; Blogowski, W.; 1; Paczkowska, E.; 2; Machalinski, B.; 2; Kucia, M.; 3; Starzynska, T.; 1; Ratajczak, M.; 4; 1Department of Gastroenterology, Pomeranian Medical University, Poland; 2Department of Physiopathology, Pomeranian Medical University, Poland; 3Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, United States; 4Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, United States, Department of Physiology, Pomeranian Medical University, Poland Publication info: Journal of Crohn's and Colitis Supplements 4. 1 (Apr 2010): 23-24. Abstract: Introduction: We hypothesized that circulating pluripotent stem cells (PSC) could play an important role in the pathogenesis of inflammatory bowel diseases (IBD), being able to ameliorate damage of intestinal epithelium. Recently, our group has identified in murine and human BM as well as other organs a population of very small embryonic like stem cells (VSELs) (Leukemia 2006, 20, 857-69). These cells are deposited in BM during development and differentiate into cells from all three germ layers. We hypothesize that under steady state conditions VSELs play an important role in turnover of tissue-specific/committed stem cells in various organs. We have also reported that the number of VSELs circulating in peripheral blood (PB) increases during stress and tissue/organ injury e.g., in patients after stroke (Stroke 2009, 40, 1237-1244) and myocardial infarction (JACC 2009, 53, 1-9). In this study we become interested if similar population of stem cells could be identified in peripheral blood of patients with IBD. Aims and Methods: We evaluated the mobilization of VSELs into PB in patients with various forms of IBD. There were enrolled thirty treated and untreated patients (n = 30) with mild to moderate ulcerative colitis (truelove and Witt's criteria) and Crohn's disease (Crohns Disease Activity Index) as well as age-matched healthy subjects (n = 20). Blood was sampled on admission, erythrocytes were lysed and CXCR4+ CD133+ CD34+ lin- CD45- VSELs were evaluated in PB by fluorescence-activated cell sorting analysis (FACS), direct immunofluorescence staining and real-time quantitative polymerase chain reaction to detect expression of developmentally early genes. Results: In IBD patients, we found an increase in the number of circulating cells expressing stem cells-associated antigens such as CD133, CD34, and CXCR4. More important, we found an increase in the number of circulating small primitive cells expressing the VSEL phenotype (CXCR4+ CD133+ lin- CD45- cells) median 3.7 [range 0.9 to 8.9] cells/ml; p <0.001) in comparison to healthy individuals (median 1.33 [range 0.2-1.45] cells/ml; p <0.001 ([Mann-Whitney test Friedman's AN0VAfollowed by Wilcoxon signed-rank test]). The mobilization of VSELs Very Small Embryonic Like Cell References

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was higher in patients with active Crohn's disease. 0f note, their mobilization was more effective in patients with de novo diagnosed untreated disease. We found that in IBD patients circulating cells are significantly enriched for mRNA for gastrointestinal lineage (lgr-5 and ASCL-2) and pluripotent stem cell markers (0ct-4, SSEA-4, Nanog) as well as CXCR4 receptor. The number of mobilized/circulating VSELs correlated in IBD patients with elevated serum levels of stromal derived factor-1 (SDF-1) as well as hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF). Conclusion: 0ur multiparameter FACS analysis and molecular studies reveal that VSELs are mobilized and circulate in PB of patients with IBD. This strongly supports the hypothesis that circulating pluripotent stem cells might be involved in the regeneration process of damaged intestinal epithelium. However, the biological and clinical significance as well as true regenerative potential of these cells in regeneration of injured gut tissue needs further investigations. _______________________________________________________________

Bone marrow transplantation temporarily improves pancreatic function in streptozotocin-induced diabetes: potential involvement of very small embryonic-like cells Author: Huang, Yiming; Kucia, Magda; Hussain, Lala-Rukh; Wen, Yujie; Xu, Hong; Yan, Jun; Ratajczak, Mariusz Z; Ildstad, Suzanne T Publication info: Transplantation 89. 6 (Mar 27, 2010): 677-85. Abstract: BACKGROUND - The role of bone marrow (BM)-derived cells in pancreatic beta-cell regeneration remains unresolved. We examined whether BM-derived cells are recruited to the site of moderate pancreatic injury and contribute to beta-cell regeneration. METHODS - Low-dose streptozotocin (STZ) treatment was used to induce moderate pancreatic damage and hyperglycemia. Enhanced green fluorescent protein-positive (EGFP) BM chimeras were evaluated for beta-cell regeneration after STZ treatment. RESULTS - To test the hypothesis that pancreatic tissue injury induces a stromal cell-derived factor (SDF)-1 gradient to chemoattract the stem cells, we evaluated the expression of mRNA for SDF-1 in damaged pancreatic tissue. SDF-1 was significantly increased in the pancreas after damage, peaking at day 10. The majority of BM cells expressing mRNA for pancreatic development markers were detected in the subpopulation of CD45/Sca-1/Lin very small embryonic-like (VSEL) cells. VSEL cells mobilized from BM to peripheral blood in response to pancreatic damage, peaking in peripheral blood at day 5, and were enriched in the pancreas 10 to 15 days after STZ treatment. To confirm a role for BM-derived cells in pancreatic beta-cell regeneration, we prepared EGFP-->B6 chimeras. In the EGFP chimeras, EGFP cells were detected around duct and islets and were positive for insulin after STZ treatment. However, STZ-induced hyperglycemia was reduced only transiently (49-77 days) after pancreatic injury. CONCLUSIONS - These data suggest that VSEL cells are mobilized into injured pancreatic tissue and contribute to beta-cell regeneration. Transplantation of BM-derived cells improves the function of injured pancreas, although the response is not sufficient to restore sustained normoglycemia. _______________________________________________________________

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Resident bone marrow stem cells are recruited to peripheral circulation in children with OSA: Relevance to endothelial function Author: Gozal, L.K.; 1; Bhattacharjee, R.; 2; Kim, J.; 1; Clair, H.; 2; Gozal, D.; 1; 1Pediatrics, University of Chicago, United States; 2Pediatrics, University of Louisville, United States Publication info: Sleep, suppl. SUPPL. 1 33 (2010): A313. Abstract: Introduction: Endothelial dysfunction is a potential complication of obstructive sleep apnea syndrome (OSAS) in children, and has been ascribed to systemic inflammatory changes. However, not all children with OSAS will manifest endothelial dysfunction. We hypothesized that the variability in endothelial function in OSAS may be related to the ability to recruit repair mechanisms such as bone marrow derived stem cells (BMSC). Methods: Pre-pubertal non-hypertensive children with or without polysomonographically-confirmed OSAS were recruited. Endothelial function was assessed in a morning fasted state, using a modified hyperemic test involving cuff-induced occlusion of the radial and ulnar arteries. Blood was drawn and 3 types of BMSC were assessed by flow cytometry, namely endothelial progenitor cells (EPC), hematopoietic stem cells (HSC), and very small embryonic-like stem cells (VSEL). Results: 25 children with OSAS (mean age 7.6 Âą 1.5 years, mean BMI z-score: 1.23 Âą 0.6) and 10 age-, gender-, ethnicity-, and BMI-matched controls (CO) were studied. Compared to CO, significant delays to peak capillary reperfusion after occlusion release (Tmax) occurred in the OSAS children as a group, but substantial individual variability was present. Similarly, EPC, HSC, and VSEL counts were significantly higher in OSAS children compared to CO (P <0.01). However, Tmax was significantly and inversely correlated with EPC (P <0.01), but not with HSC or VSEL. Conclusion: OSAS in children is associated with increases in BMSC in peripheral blood. Endothelial dysfunction is a frequent, yet not universal consequence of OSAS in children. The variance in endothelial functional phenotype in pediatric OSAS appears to be associated with the ability to recruit BMSC, and more specifically EPC. _______________________________________________________________

In vitro and in vivo evidence that umbilical cord blood (UCB)-derived CD45-/SSEA-4+/OCT-4+/CD133+/CXCR4+/lin - very small embryonic/epiblast like stem cells (VSELs) do not contain clonogenic hematopoietic progenitors but are highly enriched in more primitive stem cells - Novel view on hierarchy of UCB stem cell compartment Author: Zuba-Surma, Ewa K.; 1; Klich, Izabela; 1; Wysoczynski, Marcin; 1; Greco, Nicholas J.; 2; Laughlin, Mary J.; 3 ; Ratajczak, Mariusz Z.; 1; Ratajczak, Janina; 1; 1James Graham Brown Cancer Center, Stem Cell Institute, University of Louisville, United States; 2Case Medical Center, DBA Cleveland Cord Blood Center, Abraham J amp; Phyllis Katz Cord Blood Foundation, United States; 3University Hospitals of Cleveland, Case Medical Center, United States Publication info: Blood 114. 22 (Nov 20, 2009). Abstract: Recently, we identified in umbilical cord blood (UCB) a population of very small embryonic/epiblast-like (VSEL) stem cells (Leukemia 2007;21:297-303) that are i) smaller than erythrocytes, ii) SSEA-4+/Oct-4+/CD133+/CXCR4+/Lin-/CD45-, iii) respond to SDF-1 gradient and iv) possess large nuclei containing primitive euchromatin. We have demonstrated in vitro that UCB-derived VSELs did not reveal hematopoietic activity freshly after isolation, but grow hematopoietic colonies following co-culture/activation over OP-9 cells. To investigate the hierarchy of UCB-derived, CD45 negative VSELs, we employed staining with Aldefluor - detecting aldehyde dehydrogenase (ALDH), the enzyme expressed in primitive hematopoietic cells. Very Small Embryonic Like Cell References

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Subsequently, we sorted CD45-/CD133+/ALDHhigh and CD45-/CD133+/ALDHlow sub-fractions of VSELs from UCB samples and established that freshly sorted from UCB VSELs in contrast to sorted CD45+/ CD133+/ALDHhigh and CD45+/CD133+/ALDHlow hematopoietic stem cells (HSC) did not grow colonies in vitro. However, when CD45VSELs were activated/expanded over OP-9 stroma cells, they exhibit hematopoietic potential and grew in routine methylcellulose cultures hematopoietic colonies composed of CD45+ cells. Interestingly, while CD45-/CD133+/ALDHhigh VSELs gave raise to hematopoietic colonies after the first replating, the formation of colonies by CD45-/CD133+//ALDHlow VSELs was somehow delayed, what suggest that they needed more time to acquire hematopoietic commitment. Thus our in vitro data indicate that both populations of CD45 -cells may acquire hematopoietic potential; however hematopoietic specification is delayed for CD45-/CD133+/ALDHlow cells, suggesting their more primitive nature. In parallel, real time PCR analysis confirmed that while freshly isolated CD45-/CD133+/ALDHhigh VSELs express more hematopoietic transcripts (e.g., c-myb, 80.2±27.4 fold difference), CD45-/CD133+/ALDHlow exhibit higher levels of pluripotent stem cell markers (e.g., Oct-4, 119.5±15.5 fold difference as compared to total UCB mononuclear cells) (Figure 1 panel A). Next hematopoietic potential of UCB-derived VSELs was tested in vivo after transplantation into NOD/SCID mice (Figure 1 panel B and C). We noticed that both CD45-/CD133+ and CD45-/CD133+/ALDHhigh VSELs, give rise to human lympho-hematopoietic chimerism in lethally irradiated NOD/SCID mice as assayed 4-6 weeks after transplantation. The level of human hematopoietic CD45+ cells in murine peripheral blood (PB), bone marrow (BM) and spleen (SP) were comparable for both transplanted UCB-VSELs fractions - 7.1±2.9% (PB), 23.2±0.2% (SP) and 25.2±1.0% (BM). In conclusion, our data suggest that freshly isolated very small CD45 negative UCB-VSELs are depleted from clonogeneic progenitors, however they are highly enriched for primitive HSC. Based on our in vitro and in vivo data we postulate following hierarchy of hematopoietic stem cells in UCB (from most primitive to more differentiated) i) CD45-/CD133+/ALDHlow, ii) CD45-/CD133+/ALDHhigh , iii) CD45+/CD133+/ALDHlow and iv) CD45-//CD133+//ALDHhigh. We also postulate that as we have already shown for murine BM-derived VSELs, human UCB-derived CD45 negative VSELs correspond to a population of most primitive long term repopulating HSC (LT-HSC). Of note, we also found that currently employed, routine UCB processing strategies may lead up to ∼50% unwanted loss of these small cells that are endowed with such remarkable hematopoietic activity. _______________________________________________________________

Novel evidence that very small embryonic like stem cells (vsels) are mobilized into peripheral blood in patients with inflammatory bowel diseases - Correlation with young age and severity of disease Author: Marlicz, Wojciech; 1; Paczkowska, Edyta; 2; Halasa, Maciek; 2; Machaliñski, Boguslaw; 3; Starzynska, Teresa; 1; Ratajczak, Mariusz Z.; 4; 1Gastroenterology, Pomeranian Medical University, Poland; 2Department of Physiopathology, Pomeranian Medical University, Poland; 3Department of Physiopathology, Pomeranian Medical University, Poland, Poland; 4James Graham Brown Cancer Ctr., Stem Cell Institute, University of Louisville, United States Publication info: Blood 114. 22 (Nov 20, 2009). Abstract: Introduction: We have hypothesized that circulating pluripotent stem cells could play a protective role in the pathogenesis of inflammatory bowel diseases (IBD) and participate in regeneration of damaged intestinal eipthelium. Recently, our group has identified in murine and human BM as well as other organs a population of very small embryonic like stem cells (VSELs) (Leukemia 2006 20, 857-69). These are cells deposited in BM during development, differentiate into cells from all three germ layers and are capable of long term repopulation of hematopoiesis. They also play an important role in turnover of Very Small Embryonic Like Cell References

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tissue-specific/committed stem cells in various organs. We have also shown that the number of VSELs circulating in peripheral blood (PB) increases during stress and tissue/organ injury e.g., in patients after stroke (Stroke 2009, 40, 1237-1244) and myocardial infarction (JACC 2009, 53, 1-9). It is currently unknown whether VSELs are mobilized into PB in patients with IBD. Materials and Methods: We evaluated the mobilization of VSELs into PB in patients with active IBD. There were twenty patients (n=20) enrolled with de novo diagnosed or untreated flare-ups with mild to moderate ulcerative colitis (Truelove and Witt's criteria) and Crohn's disease (Crohns Disease Activity Index) as well as age-matched healthy subjects (n=10). Blood was sampled on admission, erythrocytes were lysed and CXCR4+ CD133+ lin- CD45- VSELs were evaluated in PB by fluorescence-activated cell sorting analysis (FACS), direct immunofluorescence staining and real-time quantitative polymerase chain reaction to detect expression of developmentally early genes. Results: In IBD patients, we found an increase in the number of circulating cells expressing stem cellsassociated antigens such as CD133, CD34, and CXCR4. More important, we found an increase in the number of circulating primitive cells expressing the VSEL phenotype (CXCR4+ CD133+ in- CD45- cells - smaller.than erythrocytes) (median 3.7 [range 0.9 to 8.9] cells/ml; p <0.001) in comparison to healthy individuals (median 1.33 [range 0.2 - 1.45] cells/ml; p <0.001 [Mann-Whitney test Friedman's ANOVA followed by Wilcoxon signed-rank test]). The mobilization of VSELs was higher in patients with active Crohn's disease and younger patients. Of note antimicrobial treatment also influenced the mobilization process. Circulating VSELs express pluripotent stem cell markers (Oct-4, SSEA-4, Nanog), CXCR4 receptor and respond robust to stromal derived factor-1 (SDF-1) gradient. We also noticed that the number of mobilized/circulating VSELs correlated in IBD patients with elevated serum level of CXCR4 receptor ligand - stromal derived factor -1 (SDF-1). Conclusions: Using multiparameter analysis, we have demonstrated for the first time that VSELs could be detected in circulating PB of patients with active IBD. Thenumber of this cell positively correlated with intensity of disease and young age. This strongly supports a potential involvement of circulating pluripotent stem cells in regeneration of damaged intestinal epithelium. However, the biological significance as well as potential application of these cells in regeneration of damaged gut tissue needs further investigations and is currently tested in animal models in our laboratories. _______________________________________________________________

CD45-/Lin-/CD133+/ALDH-low VSEL stem cells isolated from cord blood-as potential long term repopulating hematopoietic stem cells (LT-HSC) Author: Zuba-Surma, Ewa; 1; Klich, Izabela; 2; Greco, Nicholas; 3; Paul, Philip; 3; Laughlin, Mary; 3; Ratajczak, Janina; 2; Ratajczak, Mariusz; 2; 1Department of Medical Biotechnology, Jagiellonian University, Poland ewa.zuba-surma@uj.edu.pl; 2Stem Cell Biology Program, University of Louisville, United States; 3Case Western University, Cleveland Cord Blood Center, United States Publication info: Human Gene Therapy 20. 11 (Nov 1, 2009): 1469-1470. Abstract: Recently, we identified in human cord blood (CB) a population of very small embryonic-like (VSEL) stem cells that are i) smaller than erythrocytes, ii) SSEA-4 + Oct-4 + CD133 + CXCR4 + Lin-CD45-, iii) express embryonic markers. Similar population from murine bone marrow (BM) reveals hematopoietic activity after co-culture over OP-9 cells and possess potential of long term repopulating hematopoietic stem cells (LT-HSC). To investigate hematopoietic capacity of CB- derived VSEL, we employed staining with Aldefluor detecting aldehyde dehydrogenase (ALDH)- the enzyme of primitive hematopoietic cells and we sorted CD133 + CD45-ALDHhigh (VSEL/ALDHhigh) and CD133 + CD45-ALDHlow (VSEL/ALDHlow) subfractions of VSEL from CB samples. Both freshly sorted VSEL populations did not grow hematopoietic colonies in vitro. However, when Very Small Embryonic Like Cell References

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expanded over OP-9 cells, they exhibit hematopoietic potential and initiate colonies composed of CD45 + cells when replated into methylcellulose cultures. Furthermore, VSEL/ALDHhigh gave raise to such colonies faster than VSEL-ALDHlow. Real time RT-PCR revealed that freshly isolated VSEL/ALDHhigh express more hematopoietic transcripts (c-myb, 80.2 ±27.4 fold difference), while VSEL-ALDHlow more pluripotent markers (Oct-4,119.5 ± 15.5 fold difference) as compared to total CB cells. Moreover, both VSEL/ALDHhigh and VSEL/ALDHlow populations - freshly isolated or expanded on OP-9 cells - reconstituted hematopoiesis in lethally irradiated SCID mice after 1 month post trans-plantation. In conclusion, our data suggest that CBderived VSEL that are CD133 + CD45-ALDHlow are enriched for most primitive population of LT-HSC. These cells may be responsible for long term CB engraftment and should be expanded into HSCs for potential clinical applications. _______________________________________________________________

An evidence that CD45-lin-sca-1 + oct-4 + VSEL stem cells are embryonic remnants and are present in embryonic tissues during development Author: Zuba-Surma, Ewa; 1; Yoshimoto, Momoko; 2; Kucia, Magdalena; 3; Ratajczak, Janina; 3; Yoder, Mervin; 2 ; Ratajczak, Mariusz; 3; 1Department of Medical Biotechnology, Jagiellonian University, Poland ewa.zuba-surma@uj.edu.pl; 2Herman B Wells Center for Pediatric Research, Indiana University, School of Med., United States; 3Stem Cell Biology Program, University of Louisville, United States Publication info: Human Gene Therapy 20. 11 (Nov 1, 2009): 1493. Abstract: Recently, we identified in murine adult bone marrow (BM) a population of very small embryonic-like (VSEL) stem cells that are i) smaller than erythrocytes, ii) SSEA-1 + Oct-4 + Sca-1 + CXCR4 + Lin-CD45-, iii) express pluripotent markers. We also found VSEL in other adult organs and in fetal liver. Due to the expression of epiblast antigens and unique methylation status of genes, we hypothesized their embryonic origin. Therefore, we examined if VSEL are present in embryonic tissues and if they develop in embryo or migrate from extra embryonic tissue. We used flow cytometric and Image Stream analysis of VSEL presence in embryo in stages: 8.5 dpc (3-7sp), 9.5 dpc (19-24sp), 10dpc, 11dpc, 12-13dpc and 15-16dpc. We established that very small (4.13 ± 0.32μm) Oct-4 + Sca-1 + Lin-CD45- VSEL are present in embryo in all stages of development including 8.5dpc (before cellular migration from yolk sac). Interestingly, absolute number of VSEL in stages 8.5dpc and 9.5dpc was very low (6.1 ± 1.8 and 1.6±0.1K cells, respectively) and then increased peaking at 11dpc (312.5 ± 15.9 K). The total number of VSELs per embryo was established on the level observed in adult body at 15-16dpc (163.2 ±5.9 K). Real-time RT PCR showed high expression of pluripotent genes (Oct-4, Nanog, Fgf5, Lit1 and Eras), confirming primitive nature of these cells. In conclusion, our data suggest that Oct-4 + Sca-1 + Lin-CD45- VSEL detected in adult tissues are originated from embryonic tissues. The number of VSEL increases during development due to proliferation or developmental migration. They may play a potential role in formation of the embryonic body and to establish the future pools of VSEL deposited in different organs which survive until adulthood as embryonic remnants-adult pluripotent stem cells. _______________________________________________________________

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Novel epigenetic mechanisms that control pluripotency and quiescence of adult bone marrow-derived Oct4(+) very small embryonic-like stem cells Author: Shin, D M; Zuba-Surma, E K; Wu, W; Ratajczak, J; Wysoczynski, M; Ratajczak, M Z; Kucia, M Publication info: Leukemia 23. 11 (Nov 2009): 2042-51. Abstract: Recently, we identified in adult tissues a population of Oct4(+)SSEA-1(+)Sca-1(+)lin(-)CD45(-) very small embryonic-like stem cells (VSELs). First, to address recent controversies on Oct4 expression in cells isolated from adult organs, we show here evidence that Oct4 promoter in bone marrow (BM)-derived VSELs has an open chromatin structure and is actively transcribed. Next, to explain VSELs quiescence and lack of teratoma formation, we demonstrate a unique DNA methylation pattern at some developmentally crucial imprinted genes, showing hypomethylation/erasure of imprints in paternally methylated and hypermethylation of imprints in maternally methylated ones. These epigenetic characteristics leading to upregulation in VSELs of H19 and p57 (KIP2) (also known as Cdkn1c) and repression of Igf2 and Rasgrf1 explain VSEL's quiescent status. Interestingly, this unique pattern in imprinted gene methylation is reverted in cocultures with a C2C12 supportive cell-line when VSELs are induced to form VSEL-derived spheres (VSEL-DSs) enriched for stem cells able to differentiate into all three germ layers. Therefore, we suggest that the proliferative/developmental potential of Oct4(+) VSELs is epigenetically regulated by expression of Oct4 and some imprinted genes, and postulate that restoring the proper methylation pattern of imprinted genes will be a crucial step for using these cells in regenerative medicine. _______________________________________________________________

Stem cell-based therapy in central nervous system diseases Author: Paczkowska, Edyta; Dabkowska, Elzbieta; Nowacki, Przemysław; Machaliński, Bogusław Publication info: Neurologia i neurochirurgia polska 43. 6 (Nov 2009 - Dec 2009): 550-8. Abstract: Much of the current research into stem cell biology is focused on its potential for regeneration of various tissues and organs. Stem cell-based therapy with autologous bone marrow stem cells could provide an attractive alternative to the classical therapeutic approach in the foreseeable future. The possibility of nervous tissue regeneration in neurodegenerative disorders of the central nervous system generates a special challenge for researchers and clinicians involved in that field of medicine. Very small embryonic-like stem cells (VSEL SCs), recently discovered in murine bone marrow and human umbilical cord blood, arouse great hope. VSEL SCs display several features typical for embryonic stem cells, such as a large nucleus surrounded by a narrow rim of cytoplasm, euchromatin, and expression of pluripotent markers (Oct-4, Nanog, SSEA-4). Application of these cells in regenerative medicine could have considerable advantages over strategies using embryonic stem cells, since ethical concerns might be naturally solved. Thus, these cells can become a recommended source of stem cells for cell therapy as compared to those isolated from developing embryos. _______________________________________________________________

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Potential application of adult stem cells in retinal repair--challenge for regenerative medicine Author: Machalińska, Anna; Baumert, Bartłomiej; Kuprjanowicz, Leszek; Wiszniewska, Barbara; Karczewicz, Danuta; Machaliński, Bogusław Publication info: Current eye research 34. 9 (Sep 2009): 748-60. Abstract: Stem cells (SCs) maintain the balance among somatic cell populations in various tissues and are responsible for organ regeneration. The remarkable progress of regenerative medicine in the last few years indicates promise for the use of SCs in ophthalmic disorder treatment. This review describes the current view on hierarchy in the SC compartment and presents the latest attempts to use adult SCs in the regeneration of the retina. Research performed primarily in animal models gives hope for using similar strategies in humans. However, the search for the optimal source of SCs for cell therapy continues. We briefly discuss various potential sources of adult SCs that could be employed in regenerative medicine, particularly focusing on recently identified, very small embryonic-like SCs (VSEL-SCs). These cells are even present in the bone marrow and adult tissues of older patients and could be harvested from cord blood. We believe that VSEL-SCs, after the establishment of ex vivo expansion and differentiation protocols, could be harnessed for retina regeneration. _______________________________________________________________

Circulating Oct-4+SSEA-4+ very small embryonic-like cells and improvement of LVEF in patients with acute myocardial infarction Author: Wojakowski, W.; 1; Ciosek, J.; 1; Kucia, M.; 2; Paczkowska, E.; 3; Kazmierski, M.; 1; Buszman, P.; 1; Ochala, A.; 1; Machalinski, B.; 3; Ratajczak, M.Z.; 2; Tendera, M.; 1; 1Slaski Uniwersytet Medyczny w Katowicach, Poland; 2Stem Cell Institute, United States; 3Pomeranian Medical University, Poland Publication info: European Heart Journal, suppl. SUPPL. 1 30 (Sep 2009): 497. Abstract: Acute myocardial infarction (MI) is associated with rapid mobilization of bone marrow stem cells including rare population of very small embryonic-like stem cells (VSEL), expressing markers of embryonic pluripotent stem cells (PSC), early cardiac markers and displaying following imunophenotype: lin-CD133+CD34+CD45- CXCR4+. We recently showed that mobilization of VSELs is compromised in patients with acute MI and reduced LVEF. AIM of the study was to evaluate the association between mobilization of VSEL and improvement of LVEF in patients with acute MI in 6 months follow-up Methods: 30pts with acute MI and 30 healthy subjects (CTRL) were enrolled. Blood (20 mL) was sampled 24 hours after primary PCI and after 6 months. Isolation of VSELs: erythrocytes were lysed and CD34+CXCR4+lin-CD133+CD45- cells were isolated using live cell sorting system (FACSAria). LVEF was measured using echocardiography. Results: In healthy subjects number of circulating VSEL is very low (1,1±0,2 cells/μL). In acute MI the number of VSELs increased significantly (4,95±1,1; p<0,001). Circulating VSELs were enriched in mRNA of PSC markers (Oct-4; Nanog) and cardiac lineage (GATA-4, Nkx2.5/Csx, MEF2C) markers. Number of circulating VSELS in acute MI was significantly correlated with absolute increase of LVEF in 6 month follow-up (r=0.53, p<0.01). Conclusion: Mobilization of very small embryonic-like stem cells expressing pluripotent markers is significantly correlated with improvement of LVEF in 6 months follow-up. _______________________________________________________________

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Identification of small Sca-1(+), Lin(-), CD45(-) multipotential cells in the neonatal murine retina Author: Liu, Yongqing; Gao, Ling; Zuba-Surma, Ewa K; Peng, Xiaoyan; Kucia, Magdalena; Ratajczak, Mariusz Z; Wang, Wei; Enzmann, Volker; Enzman, Volker; Kaplan, Henry J; Dean, Douglas C Publication info: Experimental hematology 37. 9 (Sep 2009): 1096-107, 1107.e1. Abstract: OBJECTIVE: Bone marrow contains a subset of stem cells that give rise to nonhematopoietic lineages. These nonhematopoietic stem cells appear heterogeneous and contain cells committed to mesenchymal and endothelial lineages, as well as more primitive multipotential cells resembling progenitors of germ cells and very small embryonic/epiblast-like stem cells (VSELs). Nonhematopoietic stem cells can be mobilized from the bone marrow in response to tissue injury, and cells with similar properties have been found in cord blood and normal adult organs. However, the relationship between bone marrow cells and these adult organ stem cells is still unclear. The differentiation potential of some adult stem cells is organ-restricted, but other populations appear to retain multipotential capacity. MATERIALS AND METHODS: A population of small Sca-1(+), lineage-negative (Lin(-)), CD45(-) cells resembling VSELs were isolated from neonatal mouse retina by cell sorting. Differentiation of the cells in culture was achieved by exposure to embryonic stem cell differentiation protocols. RESULTS: VSEL-like cells comprise 1.5% of the neonatal mouse retina. They remain quiescent during retinal differentiation, and thus they do not contribute to normal retinal development. However, they display eye cell differentiation potential in culture and they are also multipotential and can give rise to cells representative of all three embryonic layers. CONCLUSIONS: The neonatal retina is an abundant postnatal source of multipotential VSEL-like cells that can differentiate in culture into a variety of lineages. _______________________________________________________________

Society for Cardiovascular Angiography and Interventions' 32nd Annual Scientific Sessions Publication info: Catheterization and Cardiovascular Interventions, suppl. SUPPL. 1 73 (Jun 1, 2009). Abstract: The proceedings contain 230 papers. The topics discussed include: arstasis approach for arterial access and sealing: first-in-man clinical experience; when does door-to-balloon time matter? analysis from the HORIZONS-AMI and CADILLAC trials; visualization of the left atrial appendage to plan percutaneous appendage closure; safety, feasibility and cost-effectiveness of bivalirudin bolus without infusion during percutaneous coronary intervention; evidence of mobilization of pluripotent and very small embryonic-like (VSEL) stem cells in patients with myocardial ischemia: a potential therapeutic target; six-year outcomes after sirolimus-eluting stent implantation (SIRIUS study); a novel and versatile percutaneous life support system with LVAD and ECMO capability in high-risk PCI and refractory shock; and left main coronary artery stenosis: a meta analysis of stents versus coronary artery bypass grafting. _______________________________________________________________

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Evidence of mobilization of pluripotent and very small embryonic-like (VSEL) stem cells in patients with myocardial ischemia: A potential therapeutic target Author: Abdel-Latif, Ahmed; 1; Hamdalla, Hussam; 1; Kucia, Magdalena; 2; Mukherjee, Debabrata; 1; Ratajczak, Mariusz; 2; Smyth, Susan; 1; Syed, Mushaber; 1; Ziada, Khaled; 1; Zuba-Surma, Ewa; 2; 1University of Kentucky, United States; 2University of Louisville, United States Publication info: Catheterization and Cardiovascular Interventions, suppl. SUPPL. 1 73 (Jun 1, 2009): S4-S5. Abstract: Background: Studies have shown that predestined bone marrowderived stem cells are mobilized in acute myocardial infarction (MI) and different scenarios of ischemia. In contrast, the mobilization of pluripotent stem cells (PSCs), including Very Small Embryonic- Like stem cells (VSELs) and their hematopoetic counterparts (HSCs), in MI and other ischemic conditions, has not been demonstrated. Methods: In this study, we sought to investigate whether PSCs such as VSELs and HSCs are mobilized into peripheral blood (PB) in various clinical conditions of myocardial ischemia using a multidimensional approach employing flow cytometric, ImageStream system (ISS), and RQ-PCR analyses. PB was collected from patients with: chronic ischemic heart disease (IHD), non ST-segment elevation MI (NSTEMI), ST-segment elevation MI (STEMI), and controls. Results: By flow cytometry, PSCs, hematopoetic stem cells, and VSELs' content was lowest in controls and highest in patients with STEMI, particularly at the time of presentation (BSL) (Fig.1A). A similar pattern of expression of the pluripotency marker Oct-4 was observed by ISS (Fig. 1A and 1B) and, by RQ-PCR analyses, the expression of pluripotency markers (Oct-4 and Nanog), cardiac markers (Nkx-2.5 and GATA-4), endothelial markers (vWF and VECadherin), and CXCR-4 was highest at BSL in STEMI patients (Fig. 1C). Mobilization of PSCs and HSCs correlated positively with the extent of myocardial injury as estimated by cardiac enzymes and negatively with age. Conclusions: This is the first comprehensive and systematic report that demonstrates that PSCs and HSCs are mobilized in patients with ischemic heart disease. Mobilization of PSCs with cardiogenic potential in myocardial ischemia suggests that these cells may play a role in repair of infarcted myocardium.(Table presented) (Figure presented). _______________________________________________________________

Characterization of endogenous stem cells from the mouse penis that express an embryonic stem cell gene and undergo differentiation into several cell lineages Author: Vernet, Dolores; 1; Heydarkhan, Sanaz; 1; Kovanecz, Istvan; 1; Lue, Yan-H.; 1; Rajfer, Jacob; 1; Gonzalez-Cadavid, Nestor F.; 1; 1, United States Publication info: Journal of Urology, suppl. SUPPL. 1 181. 4 (Apr 2009): 43. Abstract: INTRODUCTION AND OBJECTIVE: Very small embryonic-like (VSEL) stem cells expressing embryonic markers, specifically Oct 4, have recently been found in many adult organs. We have previously isolated endogenous multipotent cells from the human penile tunica albuginea, and detected cells in the rat tunica albuginea and corpora cavernosa that express stem cell markers. We and others have demonstrated that stem cells from other organs implanted in the rat corpora cavernosa regenerate smooth muscle cells (SMC) and neural cells and correct erectile dysfunction. The current work determined whether VSEL are present in penile tissues, and by using a transgenic mouse that expresses gfp under the control of the Oct 4 promoter (Oct 4 Pr-gfp mouse) we isolated and tested these cells for multipotency both in vitro and in vivo. METHODS: Shaft penile tissue sections from the rat and wild type (WT) mouse were immunostained for Oct 4, and fresh tissues were subjected to RT/PCR and western blot for Oct4 and other embryonic stem cell markers. Penile shaft and crura frozen sections from the Oct 4 Pr-gfp mouse were examined for cellular green fluorescence, and fresh Very Small Embryonic Like Cell References

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tissues were subjected to a modified stem cell isolation procedure. pP1 to pP6 and related cultures from the “pre-plating” procedure were assayed in several media for multiple cell differentiation by avidin-Texas red immunofluorescence for α-smooth muscle actin, calponin, vimentin, troponin T, myosin heavy chain-II, and other markers. DAPI-labeled cultures were injected into the corpora cavernosa (0.3×105) of WT mice and penile tissues were excised at 7 and 14 days to check for cell differentiation. RESULTS: Oct 4 + cells and Oct 4 mRNA and protein were detected in the rat and WT mouse tunical and corporal tissues. Very small, easily detachable green fluorescent cells with a large nuclei, that eventually formed embryonic-like spheroids were shown to differentiate into SMC, myofibroblasts, and cardiomyocytes. Differentiation slowly turned off Oct 4 expression, but it remained active in many cells concurrently with the differentiated marker. Implanted cells also differentiated in vivo in the WT mouse corpora cavernosa. CONCLUSIONS: This is the first report on the isolation and characterization of embryonic-like endogenous stem cells from penile tissues. Dormant endogenous stem cells are potential targets for pharmacological activation aimed to non-invasive repair of penile tissue to treat erectile dysfunction and Peyronie's disease. _______________________________________________________________

A multi-instrumental approach to identify and purify very small embryonic like stem cells (VSELs) from adult tissues Author: Ratajczak, Mariusz Z.; 1; Kucia, Magda; 1; Ratajczak, Janina; 1; Zuba-Surma, Ewa K.; 1; 1Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, 500 South Floyd Street, Louisville, KY 40202, United States, Department of Physiopathology, Pomeranian; Medical University, 70-111 Szczecin, Poland mzrata01@louisville.edu; ekzuba01@louisville.edu Publication info: Micron 40. 3 (Apr 2009): 386-393. Abstract: We employed several complementary cell image analytical methods including ImageStream system (ISS analysis) and molecular approaches to identify and purify from adult murine organs a population of very small embryonic like stem cells (VSELs). These cells are (i) small in size, (ii) possess high cytoplasmic/nuclear ratio, (iii) contain primitive unorganized euchromatin, (iv) in mice are found among Sca-1+ Lin- CD45- cells and in humans among CD133+ CXCR4+ CD34+ Lin- CD45- cells and (v) express embryonic markers such as Oct-4 protein in nuclei and SSEA antigens on the surface. In mice the highest number of these cells resides in brain, kidney, pancreas and bone marrow. Data from our laboratory indicate that VSELs are most likely a population of germ line/epiblast-derived pluripotent stem cells, that is deposited during organogenesis in developing tissues as a source of tissue committed stem cells and that the number of these cells decreases with the age. We believe that VSELs could be harnessed as a source of pluripotent stem cells for regenerative medicine. © 2008 Elsevier Ltd. All rights reserved. _______________________________________________________________

Clinical evidence that very small embryonic-like stem cells are mobilized into peripheral blood in patients after stroke Author: Paczkowska, Edyta; 1; Kucia, Magda; 2; Koziarska, Dorota; 3; Halasa, Maciej; 1; Safranow, Krzysztof; 4; Masiuk, Marek; 5; Karbicka, Anna; 3; Nowik, Marta; 3; Nowacki, Przemyslaw; 3; Ratajczak, Mariusz Z.; 6; Machalinski, Boguslaw; 7; 1Department of Physiopathology, Pomeranian Medical University, Szczecin, Poland; 2 Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, Louisville, KY; 3Clinic of Neurology, Department of Biochemistry, Pomeranian Medical University, Szczecin, Poland; 4Medical Chemistry, Pomeranian Medical University, Szczecin, Poland; 5Department of Pathology, Pomeranian Medical University, Szczecin, Poland; 6Department of Physiopathology, Pomeranian Medical University, Szczecin, Very Small Embryonic Like Cell References

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Poland, Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, 580 S Preston St, Baxter II,; Louisville, KY 40202 mzrata01@louisville.edu; 7Department of Physiopathology, Pomeranian Medical University, Szczecin, Poland, Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, Louisville, KY, Department; of Physiopathology, Pomeranian Medical University, 72 Powstancow Wlkp Str, 70-111 Szczecin, Poland machalin@sci.pam.szczecin.pl Publication info: Stroke 40. 4 (Apr 1, 2009): 1237-1244. Abstract: Background and Purpose - In a murine model of stroke, we identified a population of very small embryonic-like (VSEL) stem cells (SCs) in adult murine bone marrow that could be mobilized into peripheral blood (PB). This raised the question of whether a similar population of cells is mobilized in human stroke patients. Methods - We evaluated a number of cells that corresponded to VSEL SCs in the PB of 44 stroke patients and 22 age-matched controls. After each patient's stroke, PB samples were harvested during the first 24 hours, on day +3, and on day +7 and then compared with normal controls. The circulating human cells with the phenotype of VSEL SCs were evaluated in PB by real-time quantitative polymerase chain reaction, fluorescence-activated cell sorting analysis, and direct immunofluorescence staining. In parallel, we also measured the serum concentration of stromal derived factor-1 by ELISA. Results - In stroke patients, we found an increase in the number of circulating cells expressing SC-associated antigens, such as CD133, CD34, and CXCR4. More important, we found an increase in the number of circulating primitive cells expressing the VSEL phenotype (CXCR4+lin-CD45- small cells), mRNA for Octamer-4 and Nanog, and Octamer-4 protein. All changes were accompanied by an increased serum concentration of stromal derived factor-1. Additionally, we found a positive correlation between stroke extensiveness, stromal derived factor-1 concentration in serum, and the number of CXCR4+ VSEL SCs circulating in the PB. Conclusions - We conclude that stroke triggers the mobilization of CXCR4+ VSEL SCs that have potential prognostic value in stroke patients. However, the potential role of these mobilized cells in brain regeneration requires further study. © 2009 American Heart Association, Inc. _______________________________________________________________

Mobilization of bone marrow-derived Oct-4+ SSEA-4+ very small embryonic-like stem cells in patients with acute myocardial infarction Author: Wojakowski, Wojciech; Tendera, Michał; Kucia, Magda; Zuba-Surma, Ewa; Paczkowska, Edyta; Ciosek, Joanna; Hałasa, Maciej; Król, Marek; Kazmierski, Maciej; Buszman, Paweł; Ochała, Andrzej; Ratajczak, Janina; Machaliński, Bogusław; Ratajczak, Mariusz Z Publication info: Journal of the American College of Cardiology 53. 1 (Jan 6, 2009): 1-9. Abstract: OBJECTIVES This study sought to assess of the mobilization of nonhematopoietic very small embryonic-like stem cells (VSELs) in acute myocardial infarction (MI). BACKGROUND Acute MI induces mobilization of bone marrow stem cells. Recently, a rare population of VSELs, expressing markers of embryonic pluripotent stem cells (PSCs), was identified in adult murine bone marrow and human umbilical cord blood. METHODS Thirty-one patients with acute MI and 30 healthy subjects were enrolled. Blood was sampled on admission, after 24 h, and 5 days later. Erythrocytes were lysed and lin(-)CD45(-) VSELs were isolated using a live cell sorting system (FACSAria, Beckton Dickinson, San Jose, California). Very Small Embryonic Like Cell References

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RESULTS In healthy subjects the median number of circulating VSELs was very low (median 0.8 [range 0 to 1.3]) cells/microl. In acute MI, VSELs were mobilized early (median 2.7 [range 0.2 to 3.9] cells/microl; p <0.001) and remained elevated after 24 h and 5 days (median 4.7 [range 0.2 to 6.4] cells/microl; p <0.003, and median 2.6 [range 0.3 to 3.6] cells/microl; p <0.03, respectively). The mobilization of VSEL was significantly reduced in patients older than 50 years and with diabetes in comparison with younger and nondiabetic patients. Circulating VSELs were small (7 to 8 microm) and enriched in the messenger ribonucleic acid of PSC markers (Oct-4, Nanog), cardiac lineage (GATA-4, Nkx2.5/Csx, MEF2C), and endothelial (VE-cadherin) markers. The presence of PSC markers (Oct-4, SSEA-4) and the chemokine receptor CXCR4 in circulating VSELs was confirmed at the protein level by immunofluorescent staining and ImageStream system (Amnis Corporation, Seattle, Washington) analysis. CONCLUSIONS Acute MI induced mobilization of VSELs expressing pluripotent markers, early cardiac and endothelial markers, and chemokine receptor CXCR4. _______________________________________________________________

Mobilization of Bone Marrow-Derived Oct-4 + SSEA-4 + Very Small Embryonic-Like Stem Cells in Patients with Acute Myocardial Infarction Author: Wojakowski, Wojciech; 1; Tendera, Michał; 1; Kucia, Magda; 2; Zuba-Surma, Ewa; 2; Paczkowska, Edyta; 3 ; Ciosek, Joanna; 1; Hałasa, Maciej; 3; Król, Marek; 4; Kazmierski, Maciej; 1; Buszman, Paweł; 5; Ochała, Andrzej; 1 ; Ratajczak, Janina; 2; Machaliński, Bogusław; 3; Ratajczak, Mariusz Z.; 6; 1Third Division of Cardiology, Medical University of Silesia, Katowice, Poland; 2Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, Louisville, KY, United States; 3Department of Pathology and Pathophysiology, Pomeranian Medical University, Szczecin, Poland; 4American Heart of Poland, Ustron, Poland; 5Third Division of Cardiology, Medical University of Silesia, Katowice, Poland, American Heart of Poland, Ustron, Poland; 6Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, Louisville, KY, United States, Department of Pathology and Pathophysiology, Pomeranian Medical University,; Szczecin, Poland mzrata01@louisville.edu Publication info: Journal of the American College of Cardiology 53. 1 (Jan 6, 2009): 1-9. Abstract: Objectives: This study sought to assess of the mobilization of nonhematopoietic very small embryonic-like stem cells (VSELs) in acute myocardial infarction (MI). Background: Acute MI induces mobilization of bone marrow stem cells. Recently, a rare population of VSELs, expressing markers of embryonic pluripotent stem cells (PSCs), was identified in adult murine bone marrow and human umbilical cord blood. Methods: Thirty-one patients with acute MI and 30 healthy subjects were enrolled. Blood was sampled on admission, after 24 h, and 5 days later. Erythrocytes were lysed and lin-CD45- VSELs were isolated using a live cell sorting system (FACSAria, Beckton Dickinson, San Jose, California). Results: In healthy subjects the median number of circulating VSELs was very low (median 0.8 [range 0 to 1.3]) cells/μl. In acute MI, VSELs were mobilized early (median 2.7 [range 0.2 to 3.9] cells/μl; p <0.001) and remained elevated after 24 h and 5 days (median 4.7 [range 0.2 to 6.4] cells/μl; p <0.003, and median 2.6 [range 0.3 to 3.6] cells/μl; p <0.03, respectively). The mobilization of VSEL was significantly reduced in patients older than 50 years and with diabetes in comparison with younger and nondiabetic patients. Circulating VSELs were small (7 to 8 μm) and enriched in the messenger ribonucleic acid of PSC markers (Oct-4, Nanog), cardiac lineage (GATA-4, Nkx2.5/Csx, MEF2C), and endothelial (VE-cadherin) markers. The presence of PSC markers (Oct-4, SSEA-4) and the chemokine receptor CXCR4 in circulating VSELs was confirmed at the protein level by immunofluorescent staining and ImageStream system (Amnis Corporation, Seattle, Washington) analysis. Conclusions: Acute MI induced mobilization of VSELs expressing pluripotent markers, early cardiac and endothelial markers, and Very Small Embryonic Like Cell References

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Fetal Liver Very Small EmbryonicEpiblast Like Stem Cells Follow Developmental Migratory Pathway of Hematopoietic Stem Cells Author: Zuba-Surma, Ewa K.; 1; Kucia, Magda; 1; Rui, Liu; 1; Shin, Dong-Myung; 1; Wojakowski, Wojtek; 1; Ratajczak, Janina; 1; Ratajczak, Mariusz Z.; 1; 1Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, 500 Floyd St., Louisville, KY 40202, United States mzrata01@louisville.edu Publication info: United States: Blackwell Publishing Inc, 2009. Abstract: Fetal liver (FL) has been described as a source of both hematopoietic and nonhematopoietic stem cells. Recently we have purified from murine adult bone marrow (BM) a population of CXCR4 +Oct-4+SSEA-1 + Sca-1+Lin-CD45- very small embryonicepiblast-like stem cells (VSELs). By employing several complementary imaging and molecular strategies, we report in this study that VSELs, like hematopoietic stem cells (HSCs), are highly enriched in murine FL during the second trimester of gestation. Subsequently, at the beginning of the third trimester of gestation their number decreases, which corresponds to the time when HSCs egress FL and follow the stromal derived factor-1 (SDF-1) gradient in order to colonize developing BM. Thus, our data support the hypothesis that VSELs are a mobile pool of primitive stem cells that respond to similar chemotactic gradients as HSCs and follow their developmental migratory route. © 2009 New York Academy of Sciences. _______________________________________________________________

A population of serum deprivation-induced bone marrow stem cells (SD-BMSC) expresses marker typical for embryonic and neural stem cells Author: Sauerzweig, Steven; 1; Munsch, Thomas; 2; Leßmann, Volkmar; 2; Reymann, Klaus G.; 1; Braun, Holger; 3 1 ; Leibniz Institute for Neurobiology (IfN), Projectgroup Neuropharmacology, Brenneckestr. 6, 39118 Magdeburg, Germany, Research Institute for Applied Neuroscience (FAN gGmbH), Leipziger Str.; 44, 39120 Magdeburg, Germany sauerzweig@zenit-magdeburg.de; 2Institute for Physiology, Medizinische Fakultät, Otto-von-Guericke-University, Leipziger Str. 44, 39120 Magdeburg, Germany; 3Leibniz Institute for Neurobiology (IfN), Projectgroup Neuropharmacology, Brenneckestr. 6, 39118 Magdeburg, Germany Publication info: Experimental Cell Research 315. 1 (Jan 1, 2009): 50-66. Abstract: The bone marrow represents an easy accessible source of adult stem cells suitable for various cell based therapies. Several studies in recent years suggested the existence of pluripotent stem cells within bone marrow stem cells (BMSC) expressing marker proteins of both embryonic and tissue committed stem cells. These subpopulations were referred to as MAPC, MIAMI and VSEL-cells. Here we describe SD-BMSC (serum deprivation-induced BMSC) which are induced as a distinct subpopulation after complete serum deprivation. SD-BMSC are generated from small-sized nestin-positive BMSC (S-BMSC) organized as round-shaped cells in the top layer of BMSC-cultures. The generation of SD-BMSC is caused by a selective proliferation of S-BMSC and accompanied by changes in both morphology and gene expression. SD-BMSC up-regulate not only markers typical for neural stem cells like nestin and GFAP, but also proteins characteristic for embryonic cells like Oct4 and SOX2. We hypothesize, that SD-BMSC like MAPC, MIAMI and VSEL-cells represent derivatives from a single pluripotent stem cell fraction within BMSC exhibiting characteristics of embryonic and tissue committed stem cells. The complete removal of serum might offer a simple way to specifically enrich this Very Small Embryonic Like Cell References

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Mobilization of stem/progenitor cells from bone marrow into peripheral blood during stress related to administration of sodium fluoride Author: Dziedziejko, Violetta; 1; Baśkiewicz-Masiuk, Magdalena; 2; Paczkowska, Edyta; 2; Stecewicz, Iwona; 2; Machalińska, Anna; 3; Walczak, Mieczysław; 2; Machaliński, Bogusław; 2; 1Department of Biochemistry and Chemistry, Pomeranian Medical University, Szczecin, Poland; 2Department of General Pathology, Pomeranian Medical University, Al. Powstancow Wlkp. 72, 70-111 Szczecin, Poland machalin@sci.pam.szczecin.pl; 3 Department of Histology and Embryology, Pomeranian Medical University, Szczecin, Poland Publication info: Fluoride 42. 1 (Jan 2009 - Mar 2009): 17-22. Abstract: Accumulating evidence demonstrates that stem/progenitor cells circulating in peripheral blood (PB) play a role in the regeneration of damaged organs. The potential toxic effects of fluoride (F) on various tissues have been widely studied. It was investigated here whether stress related to intravenous injection of NaF triggers the pathophysiological mobilization of different populations of stem/progenitor cells from bone marrow to peripheral blood, which could subsequently contribute to the regeneration of injured tissues. To address this issue, six-month-old female inbred BALB/C mice were injected via the orbital venous plexus with 3.33 mg NaF/kg bw). The peripheral blood samples were harvested from the mice 1, 3, and 7 days after the NaF injection. Cells of the mice were analyzed by employing flow cytometry (FACS Aria, BD) for the populations of: i) lin-Sca-1+CD45- (enriched in very small embryonic-like stem cells, VSEL), ii) lin-Sca-1 +CD45+ (enriched in hematopoietic stem cells, HSC), and iii) CD144+/VEGFR2+ cells (early endothelial cells). Additionally, at the same points in time, the concentration of stromal-derived factor 1 (SDF-1) was measured in PB. Samples of PB were also harvested from healthy untreated mice as well as mice injected with PBS instead of NaF and used as controls. It was found that stress related to NaF injection triggered the mobilization of all the analyzed cell populations from the bone marrow to the peripheral blood. The egress of stem/progenitor cells correlated with increased SDF-1 levels in the NaF-treated mice. These circulating stem/progenitor cells may contribute to the regeneration of damaged tissues. Nevertheless, this hypothesis requires further studies. Copyright © 2009 The International Society for Fluoride Research Inc. _______________________________________________________________

Circulating very small embryonic-like stem cells and the recovery of the LVEF in patients with acute myocardial infarction Author: Wojakowski, Wojciech; 1; Kucia, Magda; 1; Zuba-Surma, Ewa; 1; Kazmierski, Maciej; 1; Syzdol, Marcin; 1; Smolka, Grzegorz; 1; Cybulski, Wieslaw; 1; Paczkowska, Edyta; 1; Krol, Marek; 1; Ochala, Andrzej; 1; Machalinski, Boguslaw; 1; Ratajczak, Mariusz Z.; 1; Buszman, Pawel; 1; Tendera, Michal; 1; 1Medical University of Silesia, Poland, Stem Cell Institute, United States Publication info: Journal of the American College of Cardiology 53. 10 (2009): A315. Abstract: Background: In patients with acute myocardial infarction (MI) a significant mobilization of bone marrow-derived non-hematopoietic very small embryonic-like cells (VSELs) occurs. VSELs are small (7-8 um), negative for lineage and CD45 markers, enriched for markers of embryonic pluripotent stem cells (Oct4, Nanog) and express CD133 and CXCR4. VSEL mobilization is reduced in older, diabetic patients with significantly reduced left ventricular ejection fraction (LVEF). Aim was to assess the correlation between the Very Small Embryonic Like Cell References

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recovery of the (LVEF) and mobilization of VSELs in patients with acute myocardial infarction. Methods: 40 patients with anterior MI and 30 healthy controls (CTRL) were enrolled. Number of VSELs was measured 24 hours after primary PCI and after 1 year. After lysis of erythrocytes population of lin-CD45-CD133+CXCR4+ VSELs was isolated using a live cell sorting system (FACSAria). VSELS were characterized using immunofluorescence, FACS and ImageStream and RQ-PCR. MRI was used for measurement of LVEF and volumes. Results: In acute MI there was a significant mobilization of VSELs [4.9 (0.1-7.3); p <0.001] enriched in pluripotent (Oct-4, Nanog) and cardiac lineage markers (GATA-4, Nkx2.5, MEF2C). Number of VSELs after 1 year was comparable to CTRL [0.7 (0.1-3.3) vs 0.8 (0-1.3) cells/μL; p <0.53]. VSELs mobilization in MI was significantly lower in patients with reduced (< 40%) LVEF in the acute MI as well as in patient with persistently reduced LVEF <40% after 1 year of follow-up [3.2 (0.1-4.9) vs. 4.8 (0.5-6.4) cells/μL]. Mobilization of VSELs was significantly positively correlated with absolute increase of LVEF during 1-year follow-up (R = 0.51; p = 0.01). Patients with better mobilization of VSELs (> median) were more likely to have significant (> 5%) absolute increase of LVEF after 1-year [OR 0,2 (0,04-0,5), p = 0.006]. Conclusion: Mobilization of small non-hematopoietic embryonic-like stem cells is positively correlated with the recovery of LVEF in patients with acute MI treated with primary PCI. Conclusion: Acute MI induced mobilization of VSEL SCs expressing pluripotent markers, early cardiac and endothelial markers, and chemokine receptor CXCR4. _______________________________________________________________

Stem cells in adult retina--current state of research, future therapeutic prospects Author: Machalińska, Anna; 1; Zuba-Surma, Ewa K; 1; 1Z Katedry i Załladu Histologii i Embriologii Pomorskiej Akademii Medycznej w Szczecinie. Publication info: Klinika oczna 111. 7-9 (2009): 253-257. Abstract: The latest research reports revealed the presence of stem/progenitor cells located in different regions of matured eye. They are able to differentiate into retinal pigment epithelium cells as well as neural structure of retina. These cells were identified in neurosensory retina, pigment epithelium and within cilliary body and iris epithelium. Moreover, it has been proved that Muller glia possess the potential of differentiation into retinal cells. These findings indicate the presence of potential mechanisms enabling retinal cell repopulation and retinal tissue regeneration. In the present work, the recent reports documenting the presence of different stem cell populations in eye have been reviewed, particularly focusing on recently identified very small embryonic-like stem cells (VSEL-SCs). The potential clinical applications of the residing stem cells and limitations of such therapeutic strategies have been also discussed. _______________________________________________________________

Novel epigenetic mechanisms that control pluripotency and quiescence of adult bone marrow-derived Oct4 + very small embryonic-like stem cells Author: Shin, D.M.; 1; Zuba-Surma, E.K.; 1; Wu, W.; 1; Ratajczak, J.; 1; Wysoczynski, M.; 1; Ratajczak, M.Z.; 1; Kucia, M.; 1; 1Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, Louisville, KY, United States Publication info: Leukemia 23. 11 (2009): 2042-2051. Abstract: Recently, we identified in adult tissues a population of Oct4+ SSEA-1+Sca-1+lin-CD45- very small embryonic-like stem cells (VSELs). First, to address recent controversies on Oct4 expression in cells isolated from adult organs, we show here evidence that Oct4 promoter in bone marrow (BM)-derived VSELs has an Very Small Embryonic Like Cell References

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open chromatin structure and is actively transcribed. Next, to explain VSELs quiescence and lack of teratoma formation, we demonstrate a unique DNA methylation pattern at some developmentally crucial imprinted genes, showing hypomethylation/erasure of imprints in paternally methylated and hypermethylation of imprints in maternally methylated ones. These epigenetic characteristics leading to upregulation in VSELs of H19 and p57KIP2 (also known as Cdkn1c) and repression of Igf2 and Rasgrf1 explain VSEL's quiescent status. Interestingly, this unique pattern in imprinted gene methylation is reverted in cocultures with a C2C12 supportive cell-line when VSELs are induced to form VSEL-derived spheres (VSEL-DSs) enriched for stem cells able to differentiate into all three germ layers. Therefore, we suggest that the proliferative/developmental potential of Oct4+ VSELs is epigenetically regulated by expression of Oct4 and some imprinted genes, and postulate that restoring the proper methylation pattern of imprinted genes will be a crucial step for using these cells in regenerative medicine. _______________________________________________________________

Very small embryonic-like stem cells in adult tissues-potential implications for aging Author: Zuba-Surma, E K; Wu, W; Ratajczak, J; Kucia, M; Ratajczak, M Z Publication info: Mechanisms of ageing and development 130. 1-2 (Jan 2009 - Feb 2009): 58-66. Abstract: Recently our group identified in murine bone marrow (BM) and human cord blood (CB), a rare population of very small embryonic-like (VSEL) stem cells. We hypothesize that these cells are deposited during embryonic development in BM as a mobile pool of circulating pluripotent stem cells (PSC) that play a pivotal role in postnatal tissue turnover both of non-hematopoietic and hematopoietic tissues. During in vitro co-cultures with murine myoblastic C2C12 cells, VSELs form spheres that contain primitive stem cells. Cells isolated from these spheres may give rise to cells from all three germ layers when plated in tissue specific media. The number of murine VSELs and their ability to form spheres decreases with the age and is reduced in short-living murine strains. Thus, developmental deposition of VSELs in adult tissues may potentially play an underappreciated role in regulating the rejuvenation of senescent organs. We envision that the regenerative potential of these cells could be harnessed to decelerate aging processes. _______________________________________________________________

Gene networks and biological pathways in bone marrow-derived Very Small Embryonic Stem Cells (VSEL) from mice following Intermittent Hypoxia (IH) Author: Gozal, D.; 1; Gharib, S.; 2; Dayyat, E.; 1; Boazza, M.; 1; Clair, H.; 1; Kucia, M.; 1; Khalyfa, A.; 1; 1Pediatrics, University of Louisville, United States; 2Center for Lung Biology, University of Washington, United States Publication info: Sleep, suppl. Suppl. S 32 (2009): A94. Abstract: Introduction: A murine BM homogenous population of rare Sca-1+ lin- CD45- cells that express markers of pluripotent stem cells, and highly express Rif-1 telomerase protein was identified. These cell express neural and vascular lineage markers and form neurospheres and endothelium in vitro. VSEL are recruited from BM during IH and may play a role in repair mechanisms following IH-induced end-organ injury. Methods: Adult CB57BL mice (n=60) were exposed to either IH (cycling of 5.7% or 21% oxygen every 3 min) or to room air for 6 hours. VSELs were extracted and sorted from BM using flow cytometry, and total RNA was isolated from VSELs and hybridized onto mouse whole genome oligonucleotide-microarrays. After filtering and normalization of the microarray data, differentially expressed genes in VSEL during IH relative to RA were identified and mapped to enriched functional catego-ries based on Gene Ontology (GO) classification. Next, Very Small Embryonic Like Cell References

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we integrated our pathway-focused approach with genetic network analysis to explore the details of putative mechanisms activated by IH in VSELs. Results: IH induced significant changes in the expression of 637 unique genes (FDR <0.01) in VSELs. GO analysis revealed that the vast majority of these differentially expressed genes could be incorporated into a selected number of functionally relevant pathways underlying anatomical structure development and morphogenesis, regulation of growth, insulin-like growth factor binding, extracellular matrix, cell differentiation, response to axon injury, glycosaminoglycan and lipoprotein binding, angiogenesis, cell proliferation and nervous system development. Conclusion: Exposure to IH elicits not only end-organ injury, but can also induce repair mechanisms involving recruitment of pluripotent stem cells such as VSELs from the BM. Using novel computational methods, we show that IH activates critical biological pathways and their respective transcriptional networks in VSELs, supporting the concept that VSELs serve as a reserve mobile pool of stem cells that can be mobilized into peripheral blood, and play an important role in end-organ regeneration during IH. _______________________________________________________________

Very small embryonic like (VSEL) stem cells: Characterization, biological significance, and potential applications Author: Ratajczak, Mariusz; 1; 1Stem Cell Biology, University of Louisville, United States Publication info: The FASEB Journal 23. S1 (2009). Abstract: Bone marrow (BM) was for many years primarily envisioned as the “home organ” of hematopoietic stem cells (HSC). Augmenting evidence demonstrates, however that BM in addition to HSC also contains a heterogeneous population of non-hematopoietic stem cells (non-HSC). Recently our group identified in BM and other adult tissues including brain, kidney, pancreas, skeletal muscles, liver, spleen and lungs a population of very small embryonic like stem cells (VSELs) which express several markers characteristic for pluripotent stem cells (PSC) that are characteristic for epiblast/germ line- derived stem cells (Leukemia 2006;20:857-869 and Leukemia 2007;21:297-303). We hypothesize that VSELs are a population of epiblast-derived cells that are deposited during early gastrulation in developing tissues/organs and play an important role in turnover of tissue specific/committed stem cells. In this context for example VSELs deposited in BM may give rise to long term repopulating HSC. We noticed that VSELs could be also mobilized into peripheral blood (PB) and their number of these cells circulating in PB increases during stress and tissue/organ injuries (e.g., G-CSF administration, heart infarct and stroke). However, the number of these cells decreases with the age. Our recent data indicate that VSELs could be harnessed as a source of pluripotent stem cells for regenerative medicine. Finally, we envision that in pathological situations VSELs are involved in development of some malignancies (e.g., teratomas, germinal tumors, pediatric “small round blue cell” sarcomas). _______________________________________________________________

Very small embryonic-like (VSEL) stem cells in adult organs and their potential role in rejuvenation of tissues and longevity Author: Ratajczak, Mariusz Z.; 1; Zuba-Surma, Ewa K.; 1; Shin, Dong-Myung; 1; Ratajczak, Janina; 1; Kucia, Magda; 1; 1Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, 500 South Floyd Street, Louisville, KY 40202, United States, Department of Physiopathology, Pomeranian; Medical University, Szczecin, Poland mzrata01@louisville.edu Publication info: Experimental Gerontology 43. 11 (Nov 2008): 1009-1017.

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Abstract: Recently, we purified rare CXC chemokine receptor 4 expressing (CXCR4+) small stem cells (SCs) from the murine bone marrow (BM) that express markers characteristic for embryonic (E)SCs, epiblast (EP)SCs, and primordial germ cells (PGCs). We named these primitive cells very small embryonic-like (VSEL) SCs (VSELs). Our data indicate that VSELs are also present in many other organs in mice and that they may differentiate into cells from all three germ layers. Similar SCs were also isolated from human cord blood (CB) and mobilized peripheral blood (mPB). We hypothesize that VSELs are deposited during gastrulation and organogenesis in developing organs/tissues of mammals as a population of pluripotent stem cells (PSCs) that give rise to tissue committed monopotent SCs and that their number decreases with age. Therefore VSELs could play a pivotal role in normal rejuvenation of adult tissues as well as involvement in regeneration of damaged organs. Thus, these cells are potential SCs candidates for regenerative medicine and we envision that the regenerative potential of these cells could be harnessed to decelerate the aging processes. ÂŠ 2008 Elsevier Inc. All rights reserved. _______________________________________________________________

Evidence that very small embryonic-like stem cells are mobilized into peripheral blood Author: Kucia, Magda J; Wysoczynski, Marcin; Wu, Wan; Zuba-Surma, Ewa K; Ratajczak, Janina; Ratajczak, Mariusz Z Publication info: Stem cells (Dayton, Ohio) 26. 8 (Aug 2008): 2083-92. Abstract: Recently, we identified in murine adult tissues, including bone marrow, a population of very small embryonic-like (VSEL) stem cells. Here, we provide further evidence that under steady-state conditions these cells circulate at very low levels in peripheral blood (PB) ( approximately 100-200 cells/ml) and could be additionally mobilized during pharmacological granulocyte-colony-stimulating factor-induced or stress-related mobilization, as demonstrated in a model of toxic liver or skeletal muscle damage induced by injection of carbon tetrachloride or cardiotoxin, respectively. The number of circulating VSEL stem cells under steady-state conditions in PB of 2-month-old animals was five times higher than that in 1-year-old mice. In conclusion, this study supports a hypothesis that VSEL stem cells are a mobile pool of primitive stem cells that could be released from the stem cell niches into PB. Further studies are needed, however, to see whether the level of these cells circulating in PB could become a prognostic indicator to assess the regenerative potential of an adult organism and/or clinical outcome from an injury. Disclosure of potential conflicts of interest is found at the end of this article. _______________________________________________________________

Phenotypic and functional characterization of hematopoietic stem cells Author: Ratajczak, Mariusz Z.; 1; 1Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, Louisville, KY 40202, United States, Stem Cell Institute, James Graham Brown Cancer Center, University of; Louisville, 500 S. Floyd Street, Louisville, KY 40202, United States mzrata01@louisville.edu Publication info: Current Opinion in Hematology 15. 4 (Jul 2008): 293-300. Abstract: Purpose of Review: This article summarizes recent progress in understanding the developmental hierarchy of the stem cell compartment in hematopoietic organs and discusses assays and phenotypic markers that allow their identification. Recent Findings: In the past few years, we have witnessed significant progress in understanding the complexity of the embryonic origin of the hematopoietic system and the developmental migration of hematopoietic stem cells, which involves multiple anatomical sites in an embryo before adult Very Small Embryonic Like Cell References

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hematopoiesis is established in bone marrow. New data shed more light on the developmental hierarchy of the stem cell compartment in bone marrow and there is growing evidence that bone marrow may also contain a pluripotent stem cell that gives rise to the long-term repopulating of hematopoietic stem cells, mesenchymal stromal cells and endothelial progenitors. Furthermore, rare populations of primitive hematopoietic stem cells have been identified in bone marrow that engraft after intrabone injection only. New strategies to isolate/purify hematopoietic stem cells are developed based on employing surface markers and metabolic properties. Noteworthy, expression of some surface markers may change during their activation and isolation. Summary: Hematopoietic stem cells are heterogeneous and differ in the expression of both surface markers and the assays by which they can be detected and quantified. ÂŠ 2008 Wolters Kluwer Health | Lippincott Williams &Wilkins. _______________________________________________________________

Very small embryonic-like (VSEL) stem cells: purification from adult organs, characterization, and biological significance Author: Ratajczak, Mariusz Z; Zuba-Surma, Ewa K; Machalinski, Bogdan; Ratajczak, Janina; Kucia, Magda Publication info: Stem cell reviews 4. 2 (Jul 2008 - Sep 2008): 89-99. Abstract: In this review, we discuss current views of the bone marrow (BM) stem cell (SC) compartment and present data showing that BM contains heterogeneous populations of hematopoietic (H)SCs and non-HSCs. These cells are variously described in the literature as: endothelial progenitor cells (EPCs); mesenchymal (M)SCs; multipotent adult progenitor cells (MAPCs); marrow-isolated adult multilineage inducible (MIAMI) cells; and multipotent adult (MA)SCs. In some cases, it is likely that similar or overlapping populations of primitive SCs in the BM detected using various experimental strategies were assigned different names. Recently, we purified rare CXC chemokine receptor 4 expressing (CXCR4(+)) small SCs from the murine BM that express markers characteristic for embryonic (E)SCs, epiblast (EP)SCs, and primordial germ cells (PGCs). We named these primitive cells very small embryonic-like (VSEL) SCs. Our data indicate that VSELs may differentiate into cells from all three germ layers. _______________________________________________________________

Transplantation of bone marrow-derived very small embryonic-like stem cells attenuates left ventricular dysfunction and remodeling after myocardial infarction Author: Dawn, Buddhadeb; 1; Tiwari, Sumit; 2; Kucia, Magdalena J.; 3; Zuba-Surma, Ewa K.; 3; Guo, Yiru; 2; Sanganalmath, Santosh K.; 2; Abdel-Latif, Ahmed; 2; Hunt, Greg; 2; Vincent, Robert J.; 2; Taher, Hisham; 2; Reed, Nathan J.; 2; Ratajczak, Mariusz Z.; 3; Bolli, Roberto; 1; 1Institute of Molecular Cardiology, University of Louisville, Louisville, KY, United States, Institute of Molecular Cardiology, University of Louisville, Louisville, KY 40292, United States; buddha@louisville.edu; rbolli@louisville.edu; 2Institute of Molecular Cardiology, University of Louisville, Louisville, KY, United States; 3Stem Cell Biology Program, University of Louisville, Louisville, KY, United States Publication info: Stem Cells 26. 6 (Jun 2008): 1646-1655. Abstract: Adult bone marrow (BM) contains Sca-1+/Lin-/CD45-very small embryonic-like stem cells (VSELs) that express markers of several lineages, including cardiac markers, and differentiate into cardiomyocytes in vitro. We examined whether BM-derived VSELs promote myocardial repair after a reperfused myocardial infarction (MI). Mice underwent a 30-minute coronary occlusion followed by reperfusion and received Very Small Embryonic Like Cell References

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intramyocardial injection of vehicle (n = 11), 1 × 105 Sca-1+/Lin-/CD45+ enhanced green fluorescent protein (EGFP)-labeled hematopoietic stem cells (n = 13 [cell control group]), or 1 × 104 Sca-1+/Lin-/CD45EGFP-labeled cells (n = 14 [VSEL-treated group]) at 48 hours after MI. At 35 days after MI, VSEL-treated mice exhibited improved global and regional left ventricular (LV) systolic function (echocardiography) and attenuated myocyte hypertrophy in surviving tissue (histology and echocardiography) compared with vehicle-treated controls. In contrast, transplantation of Sca-1+/Lin-/CD45+ cells failed to confer any functional or structural benefits. Scattered EGFP+ myocytes and capillaries were present in the infarct region in VSEL-treated mice, but their numbers were very small. These results indicate that transplantation of a relatively small number of CD45+ VSELs is sufficient to improve LV function and alleviate myocyte hypertrophy after MI, supporting the potential therapeutic utility of these cells for cardiac repair. ©AlphaMed Press. _______________________________________________________________

Bone marrow - Home of versatile stem cells Author: Ratajczak, Mariusz Z.; 1; Zuba-Surma, Ewa K.; 2; Wojakowski, Wojtek; 3; Ratajczak, Janina; 2; Kucia, Magda; 2; 1Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, United States, Department of Pathophysiology, Medical University Szczecin, Poland, Department of Cancer; Biology, Stem Cell Institute, University of Louisville, 500 South Floyd Street, Louisville, KY 40202, United States mzrata01@louisville.edu; 2Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, United States; 3Department of Cardiology, Medical University of Silesia, Poland Publication info: Transfusion Medicine and Hemotherapy 35. 3 (Jun 2008): 248-259. Abstract: Bone marrow (BM) has been for many years primarily envisioned as the 'home organ' of hematopoietic stem cells (HSC). In this review we will discuss current views of the BM stem cell compartment and present data showing that BM in addition to HSC also contains a heterogeneous population of non-hematopoietic stem cells. These cells have been variously described in the literature as i) endothelial progenitor cells (EPC), ii) mesenchymal stem cells (MSC), iii) multipotent adult progenitor cells (MAPC), iv) marrow-isolated adult multilineage inducible (MIAMI) cells, v) multipotent adult stem cells (MACS) and vi) very small embryonic-like (VSEL) stem cells. It is likely that in many cases similar or overlapping populations of primitive stem cells in the BM were detected using different experimental strategies and hence were assigned different names. Copyright © 2008 S. Karger AG. _______________________________________________________________

Hunt for pluripotent stem cell - Regenerative medicine search for almighty cell Author: Ratajczak, Mariusz Z.; 1; Zuba-Surma, Ewa K.; 1; Wysoczynski, Marcin; 1; Wan, Wu; 1; Ratajczak, Janina; 1 ; Wojakowski, Wojciech; 2; Kucia, Magda; 1; 1Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, 500 South Floyd Street, Louisville, KY 40202, United States mzrata01@louisville.edu; 2 Department of Cardiology, Silesian Medical University, Katowice, Poland Publication info: Journal of Autoimmunity 30. 3 (May 2008): 151-162. Abstract: Regenerative medicine and tissue engineering are searching for a novel stem cell based therapeutic strategy that will allow for efficient treatment or even potential replacement of damaged organs. The pluripotent stem cell (PSC), which gives rise to cells from all three germ lineages, seems to be the most ideal candidate for such therapies. PSC could be extracted from developing embryos. However, since this source of stem cells for potential therapeutic purposes remains controversial, stem cell researchers look for PSC that Very Small Embryonic Like Cell References

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could be isolated from the adult tissues or generated from already differentiated cells. True PSC should possess both potential for multilineage differentiation in vitro and, more importantly, also be able to complement in vivo blastocyst development. This review will summarize current approaches and limitations to isolate PSC from adult tissues or, alternatively, to generate it by nuclear reprogramming from already differentiated somatic cells. ÂŠ 2007 Elsevier Ltd. All rights reserved. _______________________________________________________________

Identification of very small embryonic like (VSEL) stem cells in bone marrow Author: Kucia, M; Wysoczynski, M; Ratajczak, J; Ratajczak, M Z Publication info: Cell and tissue research 331. 1 (Jan 2008): 125-34. Abstract: Bone marrow (BM) develops in mammals by the end of the second/beginning of the third trimester of gestation and becomes a major hematopoietic organ in postnatal life. The alpha-chemokine stromal derived factor-1 (SDF-1) to CXCR4 (G ai-protein-coupled seven transmembrane-spanning chemokine receptor) axis plays a major role in BM colonization by stem cells. By the end of the second trimester of gestation, BM becomes colonized by hematopoietic stem cells (HSC), which are chemoattracted from the fetal liver in a CXCR4-SDF-1-dependent manner. Whereas CXCR4 is expressed on HSC, SDF-1 is secreted by BM stroma and osteoblasts that line BM cavities. Mounting evidence indicates that BM also contains rare CXCR4(+) pluripotent stem cells (PSC). Recently, our group has identified a population of CXCR4(+) very small embryonic like stem cells in murine BM and human cord blood. We hypothesize that these cells are deposited during development in BM as a mobile pool of circulating PSC that play a pivotal role in postnatal tissue turnover, both of non-hematopoietic and hematopoietic tissues. _______________________________________________________________

An efficient two-step method to purify very small embryonic-like (VSEL) stem cells from umbilical cord blood (UCB). Author: Halasa, Maciej; 1; Baskiewicz-Masiuk, Magdalena; 1; Dabkowska, Elzbieta; 1; Machalinski, BogusĹ&#x201A;aw; 1; 1 Department of General Pathology, Pomeranian Medical University, Szczecin, Poland. Publication info: Folia histochemica et cytobiologica / Polish Academy of Sciences, Polish Histochemical and Cytochemical Society 46. 2 (2008): 239-243. Abstract: The identification in murine bone marrow (BM) of very small embryonic-like (VSEL) stem cells, possessing several features of pluripotent stem cells, encouraged us to investigate if similar population of cells could be also isolated from the human umbilical cord blood (UCB). Here our approach to purify VSEL from human UCB is described by employing a two-step isolation strategy based on i) hypotonic lysis of erythrocytes followed ii) by multi-parameter FACS sorting. Accordingly, first, erythrocytes are removed from the UCB samples by hypotonic ammonium chloride solution and next, the UCB mononuclear cells (UCB MNC) are stained with monoclonal antibodies against all hematopoietic lineages including the common leukocyte antigen CD45. The cells carrying these markers (lin+CD45+) are eliminated from the sort by electronic gating. At the same time the antibodies against CXCR4, CD34 and CD133 are employed as positive markers to enrich the UCB MNC for VSEL. This combined two step approach enables to purify VSEL stem cells, which are small and express mRNA for pluripotent stem cells (PSC) (Oct-4 and Nanog) and tissue-committed stem cells (TCSC) (Nkx2.5/Csx, VE-cadherin and GFAP) similarly to those isolated from the adult BM (3-5 micron cells with large nuclei). Very Small Embryonic Like Cell References

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Morphological characterization of very small embryonic-like stem cells (VSELs) by ImageStream system analysis Author: Zuba-Surma, Ewa K.; 1; Kucia, Magdalena; 2; Abdel-Latif, Ahmed; 3; Dawn, Buddhadeb; 3; Hall, Brian; 4; Singh, Rajesh; 5; Lillard Jr., James W.; 5; Ratajczak, Mariusz Z.; 1; 1Stem Cell Biology Institute, University of Louisville, Louisville, KY, United States, Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, 500 Floyd St.,; Louisville, KY 40202, United States ekzuba01@louisville.edu; mzrata01@louisville.edu; 2Stem Cell Biology Institute, University of Louisville, Louisville, KY, United States; 3 Institute of Molecular Cardiology, University of Louisville, Louisville, KY, United States; 4Amnis Corporation, Seattle, WA 98121, United States; 5Brown Cancer Center, University of Louisville, Louisville, KY, United States Publication info: Journal of Cellular and Molecular Medicine 12. 1 (Jan 2008 - Feb 2008): 292-303. Abstract: Recently, our group purified a rare population of primitive Sca1 +/Lin-/CD45- cells from murine bone marrow by employing multiparameter cell sorting. Based on flow cytometric and gene expression analysis, these cells have been shown to express several markers of embryonic stem cells and were accordingly termed Very Small Embryonic-Like stem cells (VSELs). In order to better characterize VSELs, we focused on their morphological parameters (e.g. diameter, nuclear to cytoplasmic ratio, cytoplasmic area) as well as expression of Oct-4. To examine the morphological features of VSELs, we employed a multi-dimensional approach, including (i) traditional flow cytometry, (ii) a novel approach, which is ImageStream (IS) cytometry and (iii) confocal microscopy. We demonstrate by all of the sensitive and precise methods employed, that VSELs are a population of very small cells, which are significantly smaller than haematopoetic stem cells (HSC) (3.63 ± 0.09 versus 6.54 ±0.17 μm in diameter). They also exhibit higher nuclear to cytoplasmic ratio and lower cytoplasmic area as compared with HSCs and mature granulocytes. Besides confirming the size characteristics, confocal microscopic analysis also confirmed that VSELs express Oct-4, a marker of pluripotent embryonic stem cells. Morphological examination reveals that VSELs are unusually small eukaryotic cells that posses several characteristics of embryonic cells. Thus, FACS-based sorting strategies should consider that adult tissues harbour small primitive cells that are larger than platelets and smaller than erythrocytes. © 2008 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd. _______________________________________________________________

Bone marrow-derived very small embryonic-like stem cells: Their developmental origin and biological significance Author: Kucia, M.; 1; Wu, W.; 1; Ratajczak, Mariusz Z.; 2; 1Stem Cell Biology Program, James Graham Brown Cancer Center, University of Louisville, Louisville, KY, United States; 2Stem Cell Biology Program, James Graham Brown Cancer Center, University of Louisville, Louisville, KY, United States, Stem Cell Institute, James Graham Brown Cancer Center, University of; Louisville, 580 S. Preston Street, Baxter II, Louisville, KY 40202, United States mzrata01@louisville.edu Publication info: Developmental Dynamics 236. 12 (Dec 2007): 3309-3320. Abstract: Data from our and other laboratories provide evidence that bone marrow (BM) contains a population of stem cells that expresses early developmental markers such as (1) stage-specific embryonic antigen (SSEA) and (2) transcription factors Oct-4 and Nanog. These are the markers characteristic for embryonic stem cells, epiblast stem cells, and primordial germ cells (PGC). The presence of these stem cells in Very Small Embryonic Like Cell References

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adult BM supports the concept that this organ contains some population of pluripotent stem cells that is deposited in embryogenesis during early gastrulation. We hypothesize that these cells could be direct descendants of the germ lineage that, to pass genes on to the next generations, has to create soma and, thus, becomes a "mother lineage" for all somatic cell lineages present in the adult body. Germ potential is established after conception in totipotent zygotes and retained in blastomeres of morula, cells from the inner cell mass of blastocyst, epiblast, and population of PGC. We will present a concept that SSEA + Oct-4+ Nanog+ cells identified in BM could be descendants of epiblast cells as well as some rare migrating astray PGC. ÂŠ 2007 Wiley-Liss, Inc. _______________________________________________________________

Adult marrow-derived very small embryonic-like stem cells and tissue engineering Author: Kucia, Magda; 1; Zuba-Surma, Ewa K.; 1; Wysoczynski, Marcin; 1; Wu, Wan; 1; Ratajczak, Janina; 1; Machalinski, Boguslaw; 2; Ratajczak, Mariusz Z.; 1; 1University of Louisville, Stem Cell Institute, James Graham Brown Cancer Center, Louisville, KY 40202, United States, Department of Cancer Biology, Stem Cell Institute, University of; Louisville, Stem Cell Institute, James Graham Brown Cancer Center, 500 South Floyd Street, Louisville, KY 40202, United States mzrata01@louisville.edu; 2University of Louisville, Stem Cell Institute, James Graham Brown Cancer Center, Louisville, KY 40202, United States, Department of Cancer Biology, Stem Cell Institute, Pomeranian Medical; University, Department of Pathophysiology, Szczecin, Poland Publication info: Expert Opinion on Biological Therapy 7. 10 (Oct 2007): 1499-1514. Abstract: A population of CXCR4+ lin- CD45- cells that express SSEA, Oct-4 and Nanog has been identified in adult bone marrow. These cells are very small and display several features typical for primary embryonic stem cells such as: i) a large nuclei surrounded by a narrow rim of cytoplasm; ii) open-type chromatin (euchromatin); and iii) high telomerase activity. These cells were named very small embryonic-like stem cells (VSEL-SC). The authors hypothesized that they are direct descendants of the germ lineage. Germ lineage, in order to pass genes on to the next generation, has to create soma and thus becomes a 'mother lineage' for all somatic cell lineages present in the adult body. Germ potential is established after conception in a totipotent zygote and retained subsequently during development in blastomers of morula, cells form the inner cell mass of blastocyst, epiblast and population of primordial germ cells. The authors envision that VSEL-SC are epiblast-derived pluripotent stem cells and could potentially become a less-controversial source of stem cells for regeneration. _______________________________________________________________

Morphological and molecular characterization of novel population of CXCR4+ SSEA-4+ Oct-4+ very small embryonic-like cells purified from human cord blood: preliminary report Author: Kucia, M; Halasa, M; Wysoczynski, M; Baskiewicz-Masiuk, M; Moldenhawer, S; Zuba-Surma, E; Czajka, R; Wojakowski, W; Machalinski, B; Ratajczak, M Z Publication info: Leukemia 21. 2 (Feb 2007): 297-303. Abstract: Recently, we purified from adult murine bone marrow (BM) a population of CXCR4(+), Oct-4(+) SSEA-1(+), Sca-1(+) lin(-) CD45(-) very small embryonic-like (VSEL) stem cells and hypothesized that similar cells could be also present in human cord blood (CB). Here, we report that by employing a novel two-step isolation procedure -- removal of erythrocytes by hypotonic lysis combined with multiparameter sorting -- we could isolate from CB a population of human cells that are similar to murine BM-derived VSELs, described previously Very Small Embryonic Like Cell References

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by us. These CB-isolated VSELs (CB-VSEL) are very small (3-5 micro m) and highly enriched in a population of CXCR4(+)AC133(+)CD34(+)lin(-) CD45(-) CB mononuclear cells, possess large nuclei containing unorganized euchromatin and express nuclear embryonic transcription factors Oct-4 and Nanog and surface embryonic antigen SSEA-4. Further studies are needed to see if human CB-isolated VSELs similar to their murine BM-derived counterparts are endowed with pluripotent stem cell properties. _______________________________________________________________

Physiological and pathological consequences of identification of very small embryonic like (VSEL) stem cells in adult bone marrow Author: Kucia, M.; 1; Zuba-Surma, E.; 1; Wysoczynski, M.; 1; Dobrowolska, H.; 1; Reca, R.; 1; Ratajczak, J.; 1; Ratajczak, Mariusz Z.; 2; 1Stem Cell Biology Program, James Graham Brown Cancer Center, University of Louisville, Louisville, KY 40202, United States; 2Stem Cell Biology Program, James Graham Brown Cancer Center, University of Louisville, Louisville, KY 40202, United States, Department of Physiology, Pomeranian Medical University,; Szczecin, Poland, Stem Cell Biology Program, University of Louisville, 580 S. Preston St., Baxter II, Louisville, KY 40202, United States mzrata01@louisville.edu Publication info: Journal of Physiology and Pharmacology 57. SUPPL. 5 (Nov 2006): 5-18. Abstract: Bone marrow (BM) contains a population of self-renewing hematopoietic stem cells (HSC) that give rise to cells from all hemato-lymphopoietic lineages. The concept that HSC could also be plastic and be able to transdifferentiate into stem/progenitor cells for different non-hematopoietic tissues became one of the most controversial issues of modern stem cell biology. Accumulating experimental evidence suggests that contribution of BM-derived stem cells to organ/tissue regeneration could be explained not by plasticity (transdifferentiation) of HSC but rather by the presence of non-hematopoietic stem cells in BM. In this review new evidence will be presented, that adult BM contains a small population of pluripotent very small embryonic-like (VSEL) stem cells. These cells are deposited in BM early during ontogenesis and could be mobilized from BM and circulate in peripheral blood during tissue/organ injury in an attempt to regenerate damaged organs. However, if these cells are mobilized at the wrong time and migrate to the wrong place they may contribute to the development of several pathologies, including tumor formation. _______________________________________________________________

The pleiotropic effects of the SDF-1-CXCR4 axis in organogenesis, regeneration and tumorigenesis Author: Ratajczak, M.Z.; 1; Zuba-Surma, E.; 1; Kucia, M.; 1; Reca, R.; 1; Wojakowski, W.; 2; Ratajczak, J.; 1; 1Stem Cell Biology Program, James Graham Brown Cancer Center, University of Louisville, Louisville, KY, United States; 2Third Division of Cardiology, Silesian School of Medicine, Katowice, Poland Publication info: Leukemia 20. 11 (Nov 2006): 1915-1924. Abstract: Proper response of normal stem cells (NSC) to motomorphogens and chemoattractants plays a pivotal role in organ development and renewal/ regeneration of damaged tissues. Similar chemoattractants may also regulate metastasis of cancer stem cells (CSC). Growing experimental evidence indicates that both NSC and CSC express G-protein-coupled seven-transmembrane span receptor CXCR4 and respond to its specific ligand Îą-chemokine stromal derived factor-1 (SDF-1), which is expressed by stroma cells from different tissues. In addition, a population of very small embryonic-like (VSEL) stem cells that express CXCR4 and respond robustly to an SDF-1 gradient was recently identified in adult tissues. VSELs express several markers of embryonic and primordial germ cells. It is proposed that these cells are deposited early in the development as Very Small Embryonic Like Cell References

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a dormant pool of embryonic/pluripotent NSC. Expression of both CXCR4 and SDF-1 is upregulated in response to tissue hypoxia and damage signal attracting circulating NSC and CSC. Thus, pharmacological modulation of the SDF-1-CXCR4 axis may lead to the development of new therapeutic strategies to enhance mobilization of CXCR4+ NSC and their homing to damaged organs as well as inhibition of the metastasis of CXCR4+ cancer cells. _______________________________________________________________

A population of very small embryonic-like (VSEL) CXCR4 + SSEA-1 + Oct-4 + stem cells identified in adult bone marrow Author: Kucia, M.; 1; Reca, R.; 1; Campbell, F.R.; 1; Zuba-Surma, E.; 1; Majka, M.; 1; Ratajczak, J.; 1; Ratajczak, M.Z.; 1; 1Stem Cell Biology Program, James Graham Brown Cancer Center, University of Louisville, Louisville, KY 40202, United States mzrata01@louisville.edu Publication info: Leukemia 20. 5 (May 2006): 857-869. Abstract: By employing multiparameter sorting, we identified in murine bone marrow (BM) a homogenous population of rare (∼0.02% of BMMNC) Sca-1+lin-CD45- cells that express by RQ-PCR and immunohistochemistry markers of pluripotent stem cells (PSC) such as SSEA-1, Oct-4, Nanog and Rex-1. The direct electronmicroscopical analysis revealed that these cells are small (∼2-4 μm), posses large nuclei surrounded by a narrow rim of cytoplasm, and contain open-type chromatin (euchromatin) that is typical for embryonic stem cells. In vitro cultures these cells are able to differentiate into all three germ-layer lineages. The number of these cells is highest in BM from young (∼ 1-month-old) mice and decreases with age. It is also significantly diminished in short living DBA/2J mice as compared to long living B6 animals. These cells in vitro respond strongly to SDF-1, HGF/ SF and LIF and express CXCR4, c-met and LIF-R, respectively, and since they adhere to fibroblasts they may be coisolated with BM adherent cells. We hypothesize that this population of Sca-1+ lin-CD45- very small embryonic-like (VSEL) stem cells is deposited early during development in BM and could be a source of pluripotent stem cells for tissue/organ regeneration. © 2006 Nature Publishing Group. All rights reserved. _______________________________________________________________

The developmental deposition of epiblast/germ cell-line derived cells in various organs as a hypothetical explanation of stem cell plasticity? Author: Kucia, Magda; 1; Machalinski, Boguslaw; 2; Ratajczak, Mariusz Z.; 3; 1Stem Cell Biology Program, James Graham Brown Cancer Center, University of Louisville, Louisville, KY 40202, United States; 2Department of Physiology, Pomeranian Medical University, Szczecin, Poland; 3Stem Cell Biology Program, James Graham Brown Cancer Center, University of Louisville, Louisville, KY 40202, United States, Department of Physiology, Pomeranian Medical University,; Szczecin, Poland mzrata01@louisville.edu Publication info: Acta Neurobiologiae Experimentalis 66. 4 (2006): 331-341. Abstract: The embryo develops from germ cell line (fertilized oocyte) and precursors of primordial germ cells (PGC) are the first population of stem cells that are specified in mice at the beginning of gastrulation in proximal primitive ectoderm (epiblast) - region adjacent to the extraembryonic ectoderm. These founder cells subsequently move through the primitive streak and give rise to several extra-embryonic mesodermal lineages and to germ cells. By day 7.25 of embryonic development, a cluster of PGC is visible at the basis of allantois. Subsequently PGC migrate through the embryo proper and colonize genital ridges, where they finally differentiate into sperm and oocytes. We hypothesize that during early development epiblast/germ Very Small Embryonic Like Cell References

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line-derived cells including PGC become a founder populations of pluripotent stem cells (PSC). These cells are deposited during embryogenesis in various organs and may persist in these locations into adulthood - for example in bone marrow (BM). To support this, we recently identified in BM a population of veiy small embryonic-like (VSEL) stem cells that express epiblast/germ line-derived cells transcription factor Oct-4 and several other PGC markers. Similarly, cells expressing Oct-4 were also identified in several adult tissues by other investigators. Thus, pluripotent epiblast/PGC may persist beyond embryogenesis in neonatal and adult tissues. Their fate is defined by several mechanisms which regulate cell proliferation and affect status of somatic imprint on selected genes responsible for pluripotency. We hypothesize that these cells play an important role in tissue/organ regeneration and their presence in adult tissues may explain phenomenon of stem cell plasticity. In pathological situations, however they may undergo malignant transformation and give rise to tumors.

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