Issue 3

THE STEM CELL Issue 3 Dec 2012

F Co ree py

ΜϚϋϓ Όϋϒϒ Ηϋϝϙ ΏϘϕϓ ΎύϟϖϚ

News From Japan Changes the World!

The Era of iPs cells

New Pluripotent Cell Shinya Yamanaka , MD , Ph.D

Director , center for iPS cell research center and application (CiRA) Professor, institute for frontier Medical sciences , Kyoto University

Nobel Prize Winner 2012

N

O

B

E

L

P

R

I

Z

E

Shinya Yamanaka The nobel prize winner in Science 2012 due to his discoveries in the field of stem cells , the induced pluripotent stem cell (IPs) Yamanka in the USA news papers after the prize

UCSF Researcher Wins Nobel Award SF Chronicle, October 8, 2012 Nobel Prize in Medicine Awarded to Sir John Gurdon, Shinya Yamanaka CNN, October 8, 2012

Good Eggs The Economist, October 8, 2012 Nobel Winners Unlocked Cells' Unlimited Potential NPR, October 8, 2012

Scientists Win Nobel Prize for Stem-Cell Work Wall Street Journal, October 8, 2012 Cloning and Stem Cell Discoveries Earn Nobel in Medicine New York Times, October 8, 2012

Stem-Cell Pioneers Gurdon, Yamanaka Win Nobel Prize Bloomberg Businessweek, Nobel Prize for Medicine Awarded toOctober 8, 2012 Gurdon, Yamanaka for Stem Cell Discoveries October 8, 2012

The Career Shinya Yamanaka, MD, PhD, a senior investigator at the Gladstone Institutes — which is affiliated with UCSF — has won the 2012 Nobel Prize in Physiology or Medicine for his discovery of how to transform ordinary adult skin cells into cells that, like embryonic stem cells, are capable of developing into any cell in the human body.

Yamanaka, who works in both San Francisco and Kyoto, is also the director of the Center for iPS Cell Research and Application (CiRA) and a principal investigator at the Institute for Integrated Cell-Material Sciences (iCeMS), both at Kyoto University. The former orthopedic surgeon trained in biomedical research at Gladstone in the 1990s, before returning to San Francisco in 2007 as a Gladstone senior investigator and a UCSF anatomy professor. Dr. Yamanaka’s story is a thrilling tale of creative genius, focused dedication and successful cross-disciplinary science,” said R. Sanders Williams, MD, president of Gladstone, a leading and independent biomedical-research The Stem Cells

“When

I saw the embryo, I suddenly realized there was such a small difference between it and my daughters. I thought, we can't keep destroying embryos for our research. There must be another way.” organization. “These traits, nurtured during Dr. Yamanaka’s postdoctoral training at Gladstone, have led to a breakthrough that has helped propel the San Francisco Bay Area to the forefront of stem cell research.

Dozens of labs — often supported by organizations such as the California Institute for Regenerative Medicine (CIRM) and the

Roddenberry Foundation – have adopted his technology. Altogether, hundreds of scientists around the world are employing the ‘Yamanaka factors’ and related techniques to search for solutions to a host of relentless illnesses — including those on which Gladstone focuses: diseases of the heart, diseases of the brain and diseases caused by deadly viruses.”

Six years ago, Yamanaka discovered that by adding just four genes into adult skin cells in mice, he could induce the cells to become like embryonic stem cells. He called them induced pluripotent stem cells, or iPS cells. In 2007, he announced that he had done the same with human adult skin cells

issue 3 December 2012

S

H

I

N

Y

A

Y

A

M

A

N

A

K

A

Shinya Yamanaka “I like the freedom of research. Plus, if I fail in science, I know I can always survive because I have an M.D. This has been my insurance policy. There is no way now to get around some use of embryos. But my goal is to avoid using them. “ Read more at Embryonic stem cells — which are “pluripotent” because they can develop into any type of cell — hold tremendous promise for regenerative medicine, in which damaged organs and tissues can be replaced or repaired. Many in the science community consider the use of stem cells to be key to the future treatment and eradication of a number of diseases, such as diabetes, blindness and Parkinson's disease.

But the use of embryonic stem cells has long been controversial — which is one reason why Yamanaka's discovery of an alternate way to obtain human stem cells, without the use of embryos, is so important.

“This is a wonderful day for Dr. Yamanaka, UCSF, the Gladstone The Stem Cells

Institutes, Kyoto University and the world,” said UCSF Chancellor Susan Desmond-Hellmann, MD, MPH. “Dr. Yamanaka’s work exemplifies the potential of basic research to transform our understanding of human cell and molecular biology, and to use this knowledge to work toward the development of treatments for currently intractable diseases. He has opened up a whole new field of discovery, and our scientists are working hard to advance the research.”

flies or mice for disease research, iPS technology allows human stem cells to be created from patients with a specific disease. As a result, the cells contain a complete set of the genes that resulted in that disease — representing the potential of a farsuperior human model for studying disease and testing new drugs and treatments. In the future, iPS cells could be used in a Petri dish to test both drug safety and efficacy for an individual patient

In addition to avoiding the controversial use of embryonic stem cells, iPS cell technology also represents an entirely new platform for fundamental studies of human disease — and the development of therapies to overcome them. Rather than using models made in yeast, issue 3 December 2012

S

H

iPS Cells Gain Momentum Initially, the simplicity of Yamanaka’s technology was met with skepticism. But he made his data and the DNA of his work publicly available to enable any scientist to work with these new cells. Within months of the 2006 breakthrough, scientists around the world had reproduced and adopted this new approach to generating and studying stem cells.

I

N

Y

A

Y

A

M

A

N

A

Yadong Huang, MD, PhD, announced the use of a single genetic factor to transform skin cells into cells that develop on their own into an interconnected, functional network of brain cells. Both announcements offer new hope in the fight against neurodegenerative conditions such as Alzheimer's disease and Huntington’s disease.

In July, Yamanaka’s own lab at Gladstone discovered that environmental factors critically “The impact of Dr. Yamanaka’s discovery is immense,” said Deepak influence the growth of iPS cells — Srivastava, MD, who leads stem cell offering newfound understanding of how these cells form. These results and cardiovascular research at Gladstone. “It suggested that human are crucial for future studies of how iPS cells grow and mature — while adult cells retain a greater ability to be modified than previously thought all of these research results point to the significance of Yamanaka’s — and could potentially be altered discovery for the future creation of into whatever cell type might be treatments for some of the world’s desired.” most debilitating diseases.

The impact can be seen at Gladstone “The best part about this prize is that where, for example, it will bring attention to — and will the Roddenberry Center for Stem likely spur — the important stem cell Cell Biology and Medicine — work that scientists around the launched at Gladstone last October world are conducting,” said with a generous gift from the Yamanaka, who is also the L.K. Roddenberry Foundation — Whittier Foundation Investigator in collaborates with Kyoto’s CIRA on Stem Cell Biologyat Gladstone. “This the use of iPS technology in patient iPS technology is for patients — and therapeutics to improve human the more scientists who build on it, health. the faster we can help those who live Scientific results that Gladstone has with chronic or life-threatening announced over the past six months diseases.” underscore the value of that collaboration. In April, for example, Leading up to Monday’s Nobel prize announcement, Yamanaka has Srivastava announced that his lab had reprogramed cardiac fibroblasts received a host of other honors recognizing the importance of his — the heart’s connective tissue — directly into beating cardiac-muscle iPS discovery, including the Albert Lasker Basic Medical Research cells in animals.

Award, the Wolf Prize in Medicine, In June, scientists in the lab of the Shaw Prize and the Kyoto Prize Gladstone Investigator Steve for Advanced Technology. In 2011, Finkbeiner, MD, PhD, announced the Yamanaka was elected to the U.S. creation of a human model of National Academy of Sciences, Huntington's disease from the skin garnering one of the highest honors cells of patients with the disease. available for U.S. scientists and Earlier that same month, scientists in engineers. In June, Yamanaka won the lab of Gladstone Investigator the Millennium Technology Award The Stem Cells

K

A

Grand Prize — the world's largest and most prominent technology award — along with Linus Torvalds, the creator of Linux software.

Yamanaka will be a speaker at the ISSCR-Roddenberry International Symposium on Cellular Reprogramming being held at Gladstone later this month, on October 24 and 25.

Gladstone is an independent and nonprofit biomedical-research organization dedicated to accelerating the pace of scientific discovery and innovation to prevent, treat and cure cardiovascular, viral and neurological diseases.

Dr. Yamanaka’s work exemplifies the potential of basic research to transform our understanding of human cell and molecular biology, and to use this knowledge to work toward the development of treatments for currently intractable diseases. He has opened up a whole new field of discovery, and our scientists are working hard to advance the research.”

UCSF Chancellor Susan Desmond-Hellmann

issue 3 December 2012

Jan, 2012

IPS BY CELL SAFE

ISSCR Yokohama 2012 Cell Safe bank in collaboration with NSA and MSA university presented a poster in the annual meeting with a title of IPS derived stem cells from adult testis differentiated into mesoderm ,endoderm and ectoderm Registration n= T3023

IPS from germ cell and re-differentiation to mesoderm , endoderm and ectoderm

Sherif N Amin , Hisham Issa , Nora Khan, Rima Akid, Omar Akram , Suzan Aly, Mahmoud Rozaykat , Andrew Mikhael. ( NSA, Cell Safe Bank and MSA)

One of the hottest topics in stem cell research today is the study of induced pluripotent stem cells (iPS). These are adult cells that are engineered or reprogrammed to become pluripotent. Behave like an embryonic stem cell. While these iPS cells share many of the same characteristics of embryonic stem cells including the ability to give rise to all the cell types in the body, it is important to understand that they are not identical. The original iPS cells were produced by using viruses to insert extra copies of three to four genes known to be important in embryonic stem cells into the specialized cell. It is not yet completely understood how those three to four reprogramming genes are able to

1

induce pluripotency this question is the focus of ongoing research.

In addition recent studies have focused on alternative ways of reprogramming cells using methods that are safer for in clinical setting. One o f the major advantages of iPS cells and one of the reasons that researchers are very interested in studying them is that they are a very good way to produce pluripotent stem cell lines that are specific to disease or even to an individual patient. Disease specific stem cells are powerful tools for studying the cause of a particular disease and then for testing drugs or discovering other approaches to treat or cure that disease. The development of patient specific stem cells is also very attractive for cell therapy as

these cell lines are from the patient themselves and may minimize some of the serious complications of rejection and immunosuppression that can occur following transplants.

Pluripotency induction A number of techniques have been developed over the years to induce pluripotency in somatic cells These include somatic cell transfer, cell fusion, reprogramming through cell extracts and direct reprogramming.

The direct reprogramming is currently being widely investigated as it is possible to reprogram a mature nucleus by introducing a known set of genes

IPS by cell safe jan 2012

Pluripotency induction

cultured germ cell before pluripotency induction

Initially retroviruses were developed to introduce these genes into the nucleus. However, more recent attention has shifted to other similar techniques to perform direct reprogramming.

These techniques include the use of lentivirus, adenovirus, plasmid transfection and the piggyback transposition system

by cell safe

Disadvantage of those techniques

Somatic Transfer : This technique has not been demonstrated in humans as it's a technical challenge to transfer this procedure from a mouse to human model.

Cell fusion: no technique has been devised to remove the embryonic stem cell chromosomal components which limit real time application of this technique.

Cell Extracts: perfect in-vitro but failed Cells were injected intra-testicular via in-vivo its done through cell culture. image guiding Direct reprogramming : It has been Unfortunately we had a partial germ shown that Oct4, Sox2 and Klf4 work cell growth followed by a seminoma together in combination to control a set of gene expression and repression programs to maintain a pluripotent cell. Protocol Dedifferentiation of Germ But this technique either led to oncogenisis , or transfection. cells into Embryonic-Like

stem cells (iPScs Germ Cells As a source of stem cells

One of the adult stem cells sources with pluripotent like character In a different research cell safe induced germ cell deficiency by intraabdominal chemotherapy injection.

After proving stem cell deficiency , MSC were extracted from Wharton’s jelly

Mouse testis dissected, and germ cells were extracted by trypsinization and a single cell suspension is obtained , and was processed within 24 hours.

Germ cells were Cultured in culture flasks containing 10 ml DMEM high glucose medium containing 15% knockout serum re-placement for ES cells, 2 ml 1-glutamine, 0.5 ml β-

mercaptoethanol, 2ml nonessential amino acids, 2ml antibiotic (penicillinstreptomycin), and 10 ng/mL bFGF.

Preparation Embryonic-Like stem cells (iPScs

Samples were staged to run for multiparameter flow cytometric analysis of cells. Prior to flow cytometry. Half of the media within the culture flask were poured out and addition of 1ml collagenase to each culture flask followed by light tabbing to allow the cells to detach from the walls of the culture flask.

objective

IPS c after one month of culture

2

IPS FORMATION

TERATOMA TESTING

Germ cell extraction from mice testis followed by culture expansion then transforming to embryonic stem cells using knock out serum and IP growth factors TRI-LINEAGE DIFFERENTIATION After confirmation of pluripotency by flowcytometry , cells were redifferentiated into mesoderm , endoderm and ectoderm

IPS were tested for teratoma transformation and were confirmed negative

IPS by cell safe jan 2012

“Lorem Ipsum Dolor Set Ahmet In Condinmentum. Nullam Wisi Acru Suscpit Consectetuer viviamus Lorem Ipsum Dolor Set Ahmet. Lorem Ipsum Dolor Set Ahmet In Wisi Acru Suscpit Consectetuer viviamus.�

Leo Praesen

IPS re-differentiation Into pancreatic cells

By Cell Safe , Rima Akid , Gihan Hammad

10 mice were induced to become diabetic through alloxan injection in the tail vein After one month of non reversed diabetes status mice were divided into two groups One treated with IPS differentiated into Pancreatic stem cells IPS were differentiated to pancreatic cells using bFGF & fibronectin Confirmation of Pancreatic differentiation was done by IHC Islet cell Ab Cells were injected in the tail vein Blood sugar monitored through a one month span showing the following : A decline in blood glucose level in a period of Five weeks Figure 2.1 A, B, C, D: display a camera image of several 2.1 A, B, C, a camera image fields Figure of the initial form of D: the display Mesenchymal stem cellsofupon several fields of of the the media initial form of the Mesenchymal stem the addition for differentiation, under 40X cells upon the addition of the media for differentiation, magnification. under 40X magnification.

3

IPS by cell safe jan 2012

Diabetes subculture results Glucose monitor curve

800"

700"

600"

1st"mouse"

500"

2nd"mouse" 400" 3rd"mouse" 300"

4"th"mouse"

200"

100"

0" 5+Jun"

7+Jun"

9+Jun"

6+Nov"

13+06"

15+06"

Figure 2.12: Illustrative graph demonstrating the monitoring period for the Mice, each line represents the depreciation of glucose levels throughout a 10-day period; the control mice are demonstrated in purple. The injected mice are in Red and Green. The male injected mouse is in green and the female injected mouse is in Red. IHC Anti b langerhan cell Anti bodies

Figure 2.2 A, B, C, D display a camera image of several fields of 2 days after the addition of the media to the MSCs for differentiation, showing cellular growth, under 40X magnification

Figure 2.3 A, B, C, D display a camera image of several fields of 4 days after the addition of the media to the MSCs for differentiation, showing cellular growth, under 40X magnification

FASHIONMONTHLY March 3, 2013

IPS into corneal endothelium injected in rabbit cornea By cell safe

Destruction of corneal layer A thin insulin syringe was used for the destruction of the lining of the cornea; the result of the scratching is shown in figure 3.1.1and 3.1.2

Figure 3.1.1 – Rabbit’s cornea: The destruction of the corneal lining, it was similar in the control and the rabbit being treated.

Follow up after 1 day

Figure 3.2.1: Monitored rabbit eye…edema and opaqueness in the cornea Figure 3.2.2: Injected rabbit eye…edema and opaqueness was also persistent

A mixture of IPS with EGF for 21 days Cells were tested using IHC and pathological assessment

FASHIONMONTHLY March 3, 2013

A week later The final week of monitoring the progression with both rabbits, was monitored, and showed the following results in both, persistent corneal opaqueness, demonstrates no regeneration of damaged cornea. cell safe property

A full research with the following contributors NSA , Cell Safe Bank , MSA , and Cairo university

By Prof Sherif N Amin ,A Prof Hisham Issa ,Nora Khan , Dr Abeer Shaalan ,Prof Sahar Daoud , Prof Ahmed Tawfik Urology Prof Rania Sobhy Optha ,Prof Sherif Gamal opth ,Prof Amr Karamany Vet , Suzan Saad , Rima Akid, Mahmoud rozaykat,

omar Akram, Andro mikhail and Tamer Reda

December 24, 2012

G ERM C EL L S P s

l

u t

r

e

i

p

m I

o c

P

’

t e

e l

n l

t s

s

Cellular Origin of Testis-Derived Pluripotent Stem Cells: A Case for Very Small Embryonic-Like Stem Cells Deepa Bhartiya, Sandhya Kasiviswananthan, and Ambreen Shaikh It has been suggested that testicular germ stem cells represent the only adult body stem cells that dedifferentiate and reprogram into a Toma Genetic lab pluripotent state without any genetic modification. Emerging debate The Best genetic lab in about the authenticity of embryonic stem cell (ES)-like cells derived from adult testicular tissue has prompted us europe now in Egypt.

The lab is highly to put forth this letter. We wish to reinforce specialized in genetic our findings that pluripotent very small medicine with full scope all embryonic-like stem cells (VSELs) exist as advanced of generic a small population in adult mammalian testing. testis and may result in ES-like colonies.

Toma Lab has collaborated with NSA laboratories and Because of their small size, it is felt that Cell safe to offer advanced VSELs could be contaminating the initial cells used for seeding, although efforts were genetic testing in the made to place a single germ cell per well in middle east.

For more info call NSA a 96-well plate for clonal expansion, or laboratories magnetic activated cell sorting (MACS)tel : 02-33456251 sorted a6 integrin positive cells were used. On a similar note, it is felt that the presence of VSELs in various tissues along with mesenchymal stem cells (MSCs) may provide an alternative explanation The Stem Cells

• Issue 3 • www.cellsafebank.com • call1138

December 24, 2012

G ERM C EL L S cells reported by Conrad et al. [6] on the basis that the microarray results extracted by them from Conrad’s data and western blot results of Conrad’s group do not match.

Further, since Oct-4, Nanog, and Sox2 expression in the pluripotent stem cells cultured by Conrad’s group were 100-, 10-, and 1,000-fold interesting discussion has less, respectively, compared with ES cells; a need was felt to critically reevaluate claims made by Conrad’s emerged in the literature as a result group. In reply to their comments, of the recent publications in Stem Conrad’s group [10] argued that the Cells [1,2] and in Stem Cell Review culture procedures were different and Reports [3] focused on the and the pluripotent stem cells derivation of pluripotent stem cells reported by them were closer ES from adult testicular tissue. Authors cells than are testicular fibroblasts, claim that the testicular germ stem and that microarray data across cells (GSCs) rep- resent the only studies cannot be compared. adult body stem cell that can spontaneously reprogram (ie, However, the discrepancy between dedifferentiate) into pluripotent state the microarray and western results without any genetic modification. could have occurred because Oct-4 The interest is obvious since the exists as two transcripts, Oct-4A and human pluripotent embryonic stem Oct-4B, and the polyclonal ancells derived from the inner cell tibody used did not differentiate mass of spare human embryos, between the 2. Oct-4 has indeed although they possess maximum confused stem cell biologists, as regenerative potential, have previously suggested [11,12]. It is associated ethical issues and two crucial to differentiate between main limitations, namely, risk of Oct-4A and Oct- 4B, since only immune rejection and teratoma Oct-4A is expressed in pluripotent formation. On the other hand, the cells and Oct-4B, though more pluripotent stem cells isolated from abundant, has no role in maintaining adult testicular tissue represent an pluripotent state of a cell. But what autologous source with no we found intriguing was the fact that associated ethical is- sues. Several various groups working on mouse groups have attempted to grow these [3–6] and human [7–9] testicular testicular cultures using adult mice tissue speculated that testicular [3–6] and human [7–9] testicular spermatogonial stem cells (SSCs) tissues; however, the process of dedifferentiate and acquire derivation of ES-like cultures from pluripotent characteristics. adult testicular tissue remains highly inefficient.

Isolation and culture of pluripotent stem cells from adult mice testicular Ko et al. [10] and Tapia et al. [1] stem cells was carried out by clonal recently questioned the pluripotency derivation of GSCs [6,13]. of human testis–derived ES-like Pluripotency of the stem cells grown

to the transdifferentiation potential of MSCs. We conclude that like Oct-4 biology, presence of VSELs in adult body tissues has somewhat surprised stem cell biologists.

An

The Stem Cells

in culture was confirmed by studying their differentiation potential into the three germ layers, germ cell contribution and chimera formation, teratoma development, etc. They used testes of Oct-4 green fluorescent protein (GFP) transgenic mice with GFP expression under the control of Oct-4 promoter for the derivation. Briefly, they cultured the testicular GSCs for 7 days in Stempro medium with varying growth factor combinations that resulted in grape-like colonies that were maintained for up to 37 passages. Seven days of culturing resulted in 15 Oct-4-GFP positive GSC colonies from 192 wells plated with single GSCs. These colonies were c-kit negative, indicating their primitive nature, and were further plated on mouse embryonic feeder (MEF) for expansion of pluripotent stem cells. They proved the unipotency and functionality of the cultured GSCs by transplanting them into the seminiferous tubules of germ cell-depleted w/w mutant mice, which resulted in restoration of spermatogenesis without any teratoma formation. One thousand GSCs were plated per well in a 24well dish on mouse embryonic feeder layer in Dulbecco’s modified Eagle medium with 15% fetal bovine serum. Interestingly, after 3– 4 weeks, a few cells appeared, grew rapidly, and formed round ES-like colonies. Based on these results, the authors concluded that GSCs converted to pluripotent stem cells (PSCs) and deny the presence of a pluripotent subpopulation which could have given rise to ES-like colonies based on the following two arguments: (1) not even a single ESlike colony appeared when GSCs were cultured, and (2) if a

• Issue 3 • www.cellsafebank.com • call1138

December 24, 2012

G ERM C EL L S subpopulation of PSCs exists, colonies should form in 2–3 days rather than after 3–4 weeks. Recently the group succeeded to achieve similar success using testicular biopsy rather than whole testis [3], thus highlighting the usefulness of the technology for regenerative medicine in humans.

But the conclusion that there was no subpopulation of pluripotent stem cells in the initial seeding of GSCs could have an alternative explanation. We have recently reported the presence of a distinct subpopulation of pluripotent stem cells [14] and stage-specific localization of c-kit shows in adult human testis, with Adark SSCs being negative for c-kit [15]. Presence of pluripotency network in adult testis was confirmed by using Oct-4 antibodies from 3 different sources, exon-specific primer sets for studying Oct-4A, and by in situ hybridization results. As expected, Oct-4A transcripts were detected in low abundance by quantitative polymerase chain reaction (Q-PCR) analysis. Oct-4A was immunolocalized in the nuclei of VSELs, whereas Oct-4B was in abundance and immunolocalized in the cytoplasm of slightly bigger Adark spermatogonial stem cells with dark stained nuclei, which underwent rapid clonal expansion and also exhibited the presence of cytoplasmic bridges. Based on the results we have proposed a revised model for premeiotic expansion of stem cells during spermatogenesis in humans. We propose that adult human testis has two distinct populations of stem cells: (1) quiescent and pluripotent VSELs with nuclear Oct-4A and (2) rapidly dividing progenitor stem cells with cytoplasmic Oct-4 (ie, Adark SSCs which undergo further differentiation and meiosis to differentiate into haploid sperm). We further discussed that the dark stained nuclei of Adark SSCs may reflect a simple stem cell phenomenon in vivo, wherein the open euchromatin of pluripotent VSELs gets compacted (hence appears dark), undergoes remodeling, gets heavily methylated, and is reprogrammed for differentiation into a particular lineage.

FIG. 1. Immunolocalization and relative expression of Oct-4 and Oct-4A transcripts in adult mice testis. Images from confocal microscopy on testicular smears show (A) nuclear Oct-4 positive very small embryonic-like stem cells and (B) cytoplasmic Oct- 4 positive, bigger progenitor stem cells with propidium iodide as a counterstain. Magnification, 63 ·5 · zoom. (C) Immunohistochemical localization of nuclear Oct-4 positive VSELs is next to basement membrane of seminiferous tubules (arrow), whereas adjacent larger germ cells exhibit cytoplasmic Oct-4. Magnification, 40 · . (D) Graph shows relative expression levels of Oct-4 and Oct-4A in adult mice testis.

Please note that Oct-4A transcript responsible for pluripotency is much less compared with total Oct-4.

Presence of similar pluripotency network–comprising VSELs with nuclear Oct-4 and slightly bigger progenitor stem cells with cytoplasmic Oct-4B has been recently re- ported by our group in adult rabbit, sheep, monkey, and human perimenopausal ovary [16] and also in cord blood, bone marrow, and umbilical cord tissue [17] (Table 1). We have observed similar biology (ie, two distinct populations of stem cells in adult mice testicular tissue) by immuno-localization studies and Q-PCR (Fig. 1). Very small cells have nuclear Oct-4 and slightly larger cells have cytoplasmic Oct-4, and as expected, Q-PCR data shows abundance of Oct-4B.

The Stem Cells

• Issue 3 • www.cellsafebank.com • call1138

December 24, 2012

G ERM C EL L S The presence of two distinct stem cell populations in various tissues is in agreement with the results of Li and Clevers [18], who have also reported presence of a relatively quiescent and actively dividing stem cell populations in hair follicle, gut epithelium, and in bone marrow.

Rataczak and his group were the first to report and publish extensively on VSELs [19,20]. VSELs exist in various body tissues of both mice and humans, including bone marrow and cord blood [21]; can be isolated by flow cytometry; and are pluripotent in nature, possibly arising very early during development at epiblast stage [19].

The epiblast-derived primordial germ cells (PGCs) during migration through the embryo proper on their way to the genital ridges are also understood to migrate and settle in various other body organs [22].

They have suggested that the VSELs could be the normal stem cells that get transformed into cancer stem cells resulting in cancer [23].

Various groups have failed to detect the presence of VSELs in normal testicular tissue because mostly germ cell markers have been used in various studies, whereas Oct-4 is a pluripotent stem cell marker, and because of shortcomings in immunolocalization protocols, including choice of fixative, antigen retrieval, et cetera.[14]. Similarly, Oosterhuis et al. [24] have reported that although Oct-4 is an extremely sensitive and specific marker for human malignant germ cell tumors, it may be false negative in biopsies fixed in Bouin’s or Stieve’s fixative.

We propose that the VSELs present in adult testicular tissue of both mice

The Stem Cells

and men may be responsible for ESlike colonies rather than dedifferentiation of GSCs. The VSELs, under yet not wellunderstood conditions, get transformed and multiply rapidly to give rise to germ cell tumors, as suggested earlier [23]. Similarly, emerging literature on the involvement of Oct-4–positive stem cells in ovarian cancers has been recently reviewed by Virant-Klun et al. [25].

Our reservations with the study reported by Conrad and group: As pointed out by Ko et al. [10] many of the discrepancies in the results are because Conrad’s group did not use specific primer sets to amplify Oct-4A responsible for pluripotency and rather amplified both Oct-4A and Oct-4B [7]. Alpha6 integrin–sorted cells were used to initiate the cultures. The pluripotent stem cell profile reported in supplementary Fig 3 has several problems. If the cells are truly pluripotent they are not expected to exhibit DAZL and VASA (germ cell markers). Also, SSEA-4 in the figures is cytoplasmic and cells have fibroblast like morphology, whereas the pluripotent stem cells are expected to be spherical in shape with a high nucleo-cytoplasmic ratio and with a ring of SSEA-4 on the surface. In contrast, the nuclei are elliptical in some figures and round in some, and most importantly, the cells have abundant cytoplasm— which should actually be minimal in pluripotent stem cells. Thus, the colonies reported by Con- rad’s group are a heterogeneous mix of cells and not a pure population of true pluripotent stem cells. No explanation is offered by the group

that no Oct-4 transcripts are detected in normal testicular tissue but isolated SSCs are Oct-4 positive by reverse transcriptase-polymerase chain reaction (RT-PCR). Getting both germ cell and PSC markers by RT-PCR suggests that the sample is actually a mix of PSCs and germ cells. How- ever, presence of CD133 in all 3 samples is a very interesting result shown in their Fig 3a. It is a marker for VSELs raising a possibility that they had contaminating VSELs in the testicular biopsies which remained unnoticed because of their size.

Our reservations with the study reported by Ko and group: The unipotency of transplanted GSCs reported by Ko et al. [6] was expected, because in the testis, even though the VSELs may contaminate the GSCs at time of transplantation, it is not expected that being pluripotent they will differentiate into multiple lineages in the testicular microenvironment. They will most probably exist as a relatively quiescent res- ervoir of stem cells to maintain homeostasis and as a source to give rise to GSCs. Thus although they mention clonal expansion of cells to derive ES-like cultures, we feel that a high probability existed of VSELs contaminating their initial GSC cultures because of their very small size. Moreover, their reasoning that the pluripotent stem cells should have given rise to at least a few colonies in GS cell culture conditions may not be valid since it is possible that the culture conditions used by them may not be conducive for ES-like cultures.

• Issue 3 • www.cellsafebank.com • call1138

December 24, 2012

G ERM C EL L S It is only when the cells get plated on mouse feeders in medium for culturing embryonic stem cells that a few cells in a clump start to expand and form ES-like colonies. Their argument that it takes 3 weeks for the colonies to appear and if a subpopulation of pluripotent stem cells is present, they should appear within 2–3 days of culture under ESlike conditions could possibly be because the VSELs are relatively quiescent and are different from embryonic stem cells in their epigenetic status [26]. They do not divide easily like embryonic stem cells, and attempts in our lab over the last year have failed to expand them in culture. It is most likely that these cells take time (about 3 weeks in culture under ES- like culture conditions) to get reprogrammed and then start behaving like ES-like cells in culture. If dedifferentiation and reprogramming of GSCs occurs to give rise to pluripotent stem cells, one could counter argue by observing the inefficiency in the process, as only 1–2 cells in a group give rise to the ES-like colony—in spite of all cells being exposed to same environment.

material and gave rise to ES-like colonies in culture. The inefficiency of the process is just because the VSELs are present in very few numbers. We reported similar alkaline phosphatase positive colonies using adult ovarian tissue culture [16], but the process is highly inefficient and the colonies could not be expanded further on feeder support. Furthermore, use of Oct-4 GFP mice with GFP expression under the control of Oct-4 promoter fails to discriminate between cells with nuclear and cytoplasmic Oct- 4 (ie, the pluripotent and progenitor stem cells since both will express GFP and may result in more ambiguity).

VSELs, mesenchymal stem cells, and iPS cells: Our results suggest that the presence of a subpopulation of VSELs in various body tissues has somewhat surprised the stem cell biologists just like Oct-4 biology. Presence of VSELs with nuclear Oct-4 in umbilical cord tissue sections along with mesenchymal cells with cytoplasmic Oct-4 [17] can provide an explanation to a large body of existing ‘‘controversial’’ literature that MSCs are pluripotent and have VSELs may be giving rise to ES-like the ability to transdifferentiate. The colonies: MSCs are not pluripotent but The VSELs are very small in actually progenitor stem cells with size, are not easily detected, and cytoplasmic Oct-4. Transdiffermay ‘‘hide’’ or adhere with the entiation of MSCs (because of their bigger cells by a phenomenon pluripotent nature and give rise to all termed ‘‘emperipolesis,’’ as reported 3 germ layers) is controversial, and earlier [19,27]. Thus, although they may not necessarily Conrad’s group used a6 integrin differentiate, especially into positive MACS sorted cells to ectoderm and endoderm. However, initiate their cultures and Ko’s group MSCs are multipotent, committed cultured a single germ stem cell per to- wards mesodermal lineage, and well, it is very likely that VSELs may give rise to various mesodermal were contaminating their starting lineages like osteoblast, adipocytes, The Stem Cells

• Issue 3 •

and chon- drocytes. Interestingly, reports are available that MSCs in undifferentiated state also express neural and endodermal transcripts (we believe that this is because of contaminating VSELs). Osonoi et al.

[28] reported that human dermal fibroblasts are able to differentiate directly to all 3 germ layer derivatives [ie, neurons (ectodermal), skeletal myocytes (mesodermal) and insulin-producing cells (endodermal)]. They exhibit nestin, desmin, and insulin when exposed to specific cocktail of growth factors. Thus it is felt that achieving transdifferentiation on the basis of im- munolocalization or presence of transcripts may not suffice. Rather, functional maturation needs to be demonstrated.

LETTER TO THE EDITOR

Table 1. Stem Cells and Progenitors Identified on the Basis of Oct-4 Immunolocalization Studies Tissue Testis Ovary Bone marrow Cord blood Umbilical cord tissue

Stem cells with Progenitors with nuclear Oct-4 cytoplasmic Oct-4 References VSELs VSELs VSELs VSELs VSELs

Adark SSCs OGSCs HSCs HSCs MSCs

[14] [16] [14] [14] [14]

HSCs, hematopoietic stem cells; MSCs, mesenchymal stem cells; OGSCs, ovarian germ stem cells; VSELs, very small embryonic-like stem cells.

quiescent and actively dividing stem cell populations in hair follicle, gut epithelium, and in bone marrow. Rataczak and his group were the first to report and publish extensively on VSELs [19,20]. VSELs exist in various body tissues of both mice and humans, including bone marrow and cord blood [21]; can be isolated by flow cytometry; and are pluripotent in nature, possibly arising very early during development at epiblast stage [19]. The epiblast-derived primordial germ cells (PGCs) during migration through the embryo proper on their way to the genital ridges are also understood to migrate and settle in various other body organs [22]. They have suggested that the VSELs could be the normal stem cells that get transformed into cancer stem cells resulting in cancer [23].

Various groups have failed to detect the presence of VSELs in normal testicular tissue because mostly germ cell markers have been used in various studies, whereas Oct-4 is a pluripotent stem cell marker, and because of shortcomings in immunolocalization protocols, including choice of fixative, antigen retrieval, et cetera.[14]. Similarly, Oosterhuis et al. [24] have reported that although Oct-4 is an extremely sensitive and specific marker for human malignant germ cell tumors, it may be false negative in biopsies fixed in Bouin’s or Stieve’s fixative. We propose that the VSELs present in adult testicular tissue of both mice and men may be responsible for ES-like colonies rather than dedifferentiation of GSCs. The VSELs, under yet not well-understood conditions, get transformed and multiply rapidly to give rise to germ cell tumors, as suggested earlier [23]. Similarly, emerging literature on the involvement of Oct-4–positive stem cells in ovarian cancers has been recently reviewed by Virant-Klun et al. [25].

Our reservations with the study reported by Conrad and group: As pointed out by Ko et al. [10] many of the discrepancies in the results are because Conrad’s group did not use specific primer sets to amplify Oct-4A responsible for pluripotency and both Oct-4A www.cellsafebank . crather o mamplified • ca ll 1 1 3and 8 Oct-4B [7]. Alpha6 integrin–sorted cells were used to initiate the cultures. The pluripotent stem cell profile reported in supplementary Fig 3 has several problems. If the cells are truly pluripotent they are not expected to exhibit DAZL and VASA (germ cell

pluripo rad’s g popula offered normal by reve Getting that the ever, p result s possibil biopsies Our unipote was ex may co not exp into m They w ervoir o to give expans high pr GSC cu their re given r ditions conditi culture in med in a clu argume if a sub should conditi tively q in their embryo year ha that the like cu behavin reprogr stem ce ciency the ESenviron VSE very sm adhere ‘‘emper Conrad to initi germ s contam

December 24, 2012

G ERM C EL L S

Our basic understanding of the presence of a pluripotent stem cell network in various adult body tissues forces us to conclude that the need to reprogram somatic cells to plu- ripotent state by induced pluripotent stem cells (iPS) technology requires further justification.

The derivation process remains highly inefficient and there was a debate when this technology was first reported and still continues on whether it is really reprogramming of somatic cells or there exists a subpopulation of pluripotent stem/ progenitor cells that grow in culture [29]. The somatic cells used to derive iPS cells may not be a good starting material since they accumulate mutations over time and have short telomeres. In contrast, VSELs are relatively healthy, pluripotent stem cells (which exist in adult body tissues) with intact genome and long telomeres (because of their quiescent nature).

The 3 recent publications in Nature [30–32] and commentaries on them [33–35] do not surprise us at all.

Further justification is required to

reprogram somatic cells by iPS technology.

possible to derive and expand autologous pluripotent stem cells from testicular biopsies and that Indeed, VSELs may be the almighty transplantation of cultured GSCs stem cells for regenerative (which we believe are contaminated medicine, as suggested earlier [36], with VSELs) do not form teratoma. for the several reasons. Like This holds lot of promise as an embryonic stem cells, they are autologous source of pluripotent pluripotent with maximum stem cells for regenerative medicine regenerative potential; however, in future. they do not form teratoma, can be isolated from an autologous source, For References Contact Cell Safe and have no associated ethical bank/ CME department issues. They are naturally occurring pluripotent stem cells in adult body tissues with healthy, intact genome, have long telomeres, get mobilized under disease conditions, and should exhibit maximum regenerative potential.

To conclude, whether the pluripotent stem cells during culture of mammalian testicular arise from unipotent GSCs or from a subpopulation of VSELs will get resolved in due time, but more important are the results showing that it is

!

Member and partner with

!

!

!

The Stem Cells

• Issue 3 • www.cellsafebank.com • call1138