Immunosuppressive therapy based on stem cells from fat

Nephrology and Transplantation Laboratory Department of Internal Medicine Erasmus Medical Centre Rotterdam Pascal Gunsch & Willem-Jan de Voogd Erasmus MC Junior Med School 4 August 2014 – 29 August 2014

Supervisors Franka Luk, Tanja Strini, Samantha de Witte, Sander Korevaar, Dr Carla Baan & Dr Martin Hoogduijn

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Immunosuppressive therapy based on stem cells from fat

Pascal Gunsch pascal.gunsch@gmail.com PENTA college CSG Jacob van Liesveldt (Hellevoetsluis) Supervisor: I.M. van Benschop & P.P. Dijkwel Willem-Jan de Voogd wj_voogd@hotmail.com Calvijn college (Goes) Supervisor: B.P. Pors Number of words: 13305 Number of pages: 57 © 2014. All rights reserved.

· Abstract ·

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ABSTRACT Each year, hundreds of lives are saved by organ transplantations. But despite decades of transplantation medicine, immune suppressive therapy still causes severe side effects and has a major impact on the lives of patients. Promising experimental and clinical studies demonstrate the immunomodulatory properties of mesenchymal stem cells (MSC). Further development of an immune suppressive therapy based on MSC needs more evidence about functional properties and other characteristics of different cell-lines in order to select the best cells. We investigated MSC isolated from perirenal fat and subcutaneous fat in some of their properties.

MSC were isolated from perirenal and subcutaneous fat, cultured and induced by specific medium to differentiate into osteoblasts, chondrocytes and adipocytes. The effect of interferon-(IFN)γ on expression of markers was tested using flow cytometry and immunosuppression was determined by measuring proliferation of mismatched lymphocytes cultured with MSC. MSC isolated from perirenal fat differentiated better than MSCs from subcutaneous fat. We also found that upon stimulation with IFNγ, MSC began to express elevated levels of markers associated with immune suppression and higher concentration of L-kynurenine in the medium.

In addition, the results showed that MSC suppressed proliferation of mismatched lymphocytes in a dosedependent way.

Our results suggest differences between MSC from perirenal fat and subcutaneous fat. Preliminary studies to functional properties support our findings. Collectively, these data provide support for the development of a better immunosuppressive therapy and suggests differences in properties between cells with various fat origins.

· Abstract ·

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SAMENVATTING Transplantaties redden elk jaar de levens van honderden mensen, toch zijn er nog altijd grote problemen met medicijnen die afstoting moeten voorkomen. Deze medicijnen hebben veel bijwerkingen met een grote impact op het leven van de patiënten. Veelbelovende experimentele en klinische onderzoeken tonen de immuunregulatoire eigenschap van mesenchymale stamcellen (MSC). Voor het ontwikkelen van een immuun onderdrukkende therapie gebaseerd op MSC is het belangrijk dat de beste cellijn geselecteerd wordt. Wij onderzochten de verschillen in functionele eigenschappen tussen MSC geïsoleerd van perirenaal en subcutaan vet.

MSC werden geïsoleerd van perirenaal en subcutaan vet, gekweekt en geïnduceerd tot naar vetcellen, botcellen en kraakbeencellen. Het effect van interferon-(IFN)γ op marker expressie werd getest met behulp van flowcytometrie en immuunsuppressie werd bepaald door proliferatie te meten van gemismatchte lymfocyten gekweekt met MSC. De MSC geïsoleerd uit perirenaal vet differentieerden beter dan MSC van subcutaan vet. Tevens zagen we dat stimulatie met IFNγ zorgde verhoogde expressie van markers geassocieerd met immuunsuppressie en een verhoogde concentratie van L-kynurenine in het medium.

Daarbij laten onze resultaten zien dat MSC de proliferatie van lymfocyten dosisafhankelijk onderdrukken.

Onze resultaten suggereren dat er verschillen zijn tussen MSC van perirenaal vet en MSC van subcutaan vet.

Voormalige studies naar de eigenschappen van MSC ondersteunen onze resultaten. Samenvattend, onze resultaten bieden ondersteuning voor het ontwikkelen van een betere immunosuppressieve therapie en suggereren verschil in eigenschappen tussen cellen met een verschillende vetorigine.

· Samenvatting ·

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TABLE OF CONTENTS ABSTRACT ............................................................................... 3 SAMENVATTING ..................................................................... 4 TABLE OF CONTENTS ............................................................. 5 PREFACE ................................................................................. 6 INTRODUCTION ....................................................................... 7 Kidneys and kidney failure · The immune system · Transplant rejection · Mesenchymal stem cells · Immunosuppressive capacity of MSC METHODS .............................................................................. 20 Source of MSC · Isolation of MSC from adipose tissue · MSC culture · Differentiation assay · Immunosuppressive capacity of MSC · Flow cytometry · IDO-assay · Mixed-lymphocyte reactions · Isolation of PBMC from spleen tissue · Statistical analysis RESULTS ................................................................................ 24 MSC isolation and culture · Differentiation assay · Flow cytometry · IDO-assay · Mixed-lymphocyte reactions DISCUSSION .......................................................................... 32 Conclusions REFERENCES ........................................................................ 38 PROTOCOLS .......................................................................... 43 APPENDICES ......................................................................... 52

· Table of Contents ·

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PREFACE In addition to the regular vwo (high school) programme, we got the chance to follow the so-called Junior Med School, a two-year course at the Erasmus MC in Rotterdam, aimed at students interested in biology, the human body and biomedical research. The programme consisted of a two-week summer school and ten occasional meetings, covering most traditional fields of medicine, including cardiology, oncology, genetics, neurology, endocrinology, pharmacology, radiology and transplantation medicine.

We want to thank our respected professors for the great colleges and experiments we did, and the coordinators, prof.dr.ir. A.P.N. Themmen and prof.dr. M.A. Frens, for providing us with the opportunity to follow this challenging and prestigious programme.

The second part of the course was a four-week research internship at one of the departments. The research had to be presented in a report and orally, on a presentation day.

This is our research report. We did our research internship at the Nephrology and Transplantation Laboratory.

One of the main things they look at is immunosuppression during transplant rejection. The research in this area focusses on the immunosuppressive characteristics of a special type of stem cell: the mesenchymal stem cell. It is relatively easy to isolate these cells from fat, which makes them a promising research target.

We want to thank our supervisors, Samantha de Witte, Tanja Strini, Franka Luk and Dr Martin Hoogduijn for their indispensable coaching during the four weeks of academic internship. But they were not the only ones, and we want to thank everyone else who supported us during the time of research and college days.

Pascal Gunsch & Willem-Jan de Voogd October 2014

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INTRODUCTION Perhaps the most innovative and promising advancement in modern medicine is transplantation medicine. Equally, probably no other field of current medical practice asks for such an interdisciplinary approach in order to solve the many complex and challenging problems physicians face.

The idea of transplantation first appeared during the Middle Ages. The first successful transplantation, a kidney transplantation between two identical twins, was in 1954.1 Still, it was only during the second half of the twentieth century that people began to understand that certain factors on the transplant cells caused the immune system to reject the organ (except for identical twins). This is because the factors were recognised as intruders. These factors are now called human leukocyte antigens (HLA).

The growing interest and research in this field led to the identification of the three main pillars of transplantation: HLA-classification, immunosuppressive medication and organ preservation. New research with focus on these principals paved the way for successful transplantations, and as a result of these improvements, in the Netherlands alone, there are approximately 1200 lifesaving organ transplantations a year.

The vast majority of transplantations, approximately 80%, are kidney transplantations.2 In 2013, 954 patients were transplanted, of which 520 received a kidney from a living donor (table 1). The organs from living donors perform better than those from deceased donors: five years after transplantation, 83% of the organs from deceased donors still function, while by that time, 92% of the organs from living donors still function.

kidney donors living deceased

2009 814 417 397

2010 867 472 395

2011 860 440 420

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2012 961 485 476

2013 954 520 434

 table 1 – The amount of living

and deceased kidney donors over the last five years.2

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Kidneys and kidney failure The kidneys are two bean-shaped organs located at the back of the abdominal cavity (figure 1), just under the diaphragm. There is one kidney on each side of the vertebral column, of which the left kidney lies a bit higher than the right kidney. The kidneys and the adrenal glands, resting on top of each kidney, are protected by the lower ribs, some muscular tissues and a layer of fat called the perirenal fat.

Their main function is to filter waste products from the blood and to excrete them via urine. They play a vital role in homeostasis, as they reabsorb essential nutrients like water and glucose, regulate electrolyte concentrations and maintain acid-base balance and control blood pressure.

 figure 1 –

Schematic representation of the location and external and internal morphology of the kidneys.

When kidney failure occurs, waste products accumulate in the body. Other consequences are electrolyte imbalance, hypertension and bone disease.

The two types of kidney failure are acute kidney failure and chronic kidney failure. Acute kidney failure is usually caused by trauma, interrupted blood supply or high doses of toxins. It can be reversible, but if not treated properly, it will result in chronic kidney failure.

Chronic kidney failure develops slowly, starting mildly and ending in end-stage renal disease (ESRD). More common causes of ESRD are diabetes mellitus and long-term hypertension. Overuse of common drugs, such as aspirin, paracetamol and ibuprofen, may also cause ESRD. When ESRD is very severe, renal replacement therapy is the only possible treatment option.

The two types of renal replacement therapy are dialysis and kidney transplantation. Dialysis is the artificial replacement of the kidney function. The blood is either filtered through a machine (haemodialysis), or waste products are filtered from the body using a glucose solution in the abdominal cavity (peritoneal dialysis).

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Kidney transplantation Dialysis is very heavy and demanding and can only take over 10% of the kidney function. Half of the dialysis patients live no longer than six years.

Kidney transplantation is the only fully effective treatment of ESRD (figure 2).

One kidney is enough for patients to be independent of dialysis.

But unfortunately, donor kidneys are rare.

Most of the time, patients have to wait four years to receive a transplantation.

Dialysis is then used as an ‘on hold’, until the transplantation takes place.

Patient survival rates living donor

deceased donor

100% 100 80% 80 60% 60 40% 40 20% 20 0% 0 0 0

5 10 5 initiating treatment 10 years after

Therefore, it is necessary to make the transplantation as successful as possible. Outside the body, this is done by preserving and cooling the organ and reducing the time the organ is outside the body. To reduce the immune system’s response and ensure transplant survival inside the body, it is essential that there is a match for blood type, human leukocyte antigen (HLA), and cross reactivity.

 figure 2 –

15 15

Patient survival rates of dialysis and kidney transplantation with a living or a deceased donor.

The blood types are A, B, AB and 0. Donor and recipient are compatible when the blood type is the same, the donor has blood type 0 or the recipient has blood type AB. HLA is the human version of the major histocompatibility complex (MHC). It is popularly referred to as ‘tissue type’, because of its analogy to blood type. Each person inherits six genes, which have a lot of different variations. Class I (HLA-I) is made up of HLA-A, -B and -C, and class II (HLA-II) of HLA-DR, -DQ and -DP. Especially HLA-A, -B and -DR have a lot of variation and are important for transplantations.

Before transplantation, the recipient is examined for the presence of pre-existing antibodies against the donor HLA in a cross-match test. If the recipient has antibodies against the donor HLA, they are mismatched. These antibodies were created after earlier exposure to the specific HLA, for example during blood transfusion, and the immune system will react immediately to them. When the test is negative, the transplantation can be started.

· Introduction ·

dialysis

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The immune system Normally, the immune system efficiently defends the body from harmful organisms (pathogens), using a principle called allorecognition. This means that the body is able to ‘see’ the difference between its own or self antigens, which could be any molecule present on a body cell, and non-self antigens, which could be anything that is not self.

This enables immune cells to recognise and eliminate viruses and bacteria, but also to recognise their own cells and leave them. However, since the donor kidney cells are non-self, the immune system will start eliminating them.

The immune system can be divided in two ‘departments’.

The innate immune system is non-specific and responds to all pathogens (and non-self cells). The adaptive immune system comprises cells and substances that only react to a specific antigen. Though it requires some time to activate, the adaptive immune system will ‘remember’ the antigens and be able to recognise them in the future.

The innate immune system The innate immune system is the body’s first line of defence. It includes the physical and chemical barriers present in the skin and mucus. If pathogens still manage to get inside the body, the immune system responds by producing cytokines. Cytokines are produced by immune cells and some other cells (such as endothelial cells), and have different functions. They can cause inflammation, induce proliferation and differentiation of specific immune cells, attract immune cells to sites of infection, change the expression of cell membrane molecules, increase antibody production and regulate the production of other cytokines.

Important proinflammatory cytokines are histamine, interferon gamma (IFNγ), interleukin 1 and 8 (IL-1 and IL8) and tumour necrosis factor alpha (TNFα).

There are various cell types associated with the innate immune system. Neutrophils, for instance, generally arrive first at sites of infection and either release granules containing cytotoxic factors or phagocytise (engulf and ingest) pathogens. Natural killer (NK) cells constantly survey cell membranes for abnormal proteins and kill cells with abnormal proteins by releasing granules with toxins.

· Introduction ·

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Antigen presentation Some phagocytising cells, such as dendritic cells (DC) and macrophages, do not only ingest the pathogen, but also display small parts of the pathogen as antigens on their cell surface using the HLA-II complex. This is why they are also called antigen presenting cells (APC). All other cells (except red blood cells) present small peptide chains of proteins they produce themselves using HLA-I.3 When a naive T-cell recognises the antigens on HLA-II by binding to it with CD4 and their T-cell receptor (TCR), a chain of reactions involving receptors from both the APC and T-cell is started (figure 3).4 This leads to the activation of the T-cell by changing it into a T-helper cell (Th-cell).

Unlike the cells from the innate immune system, T-cells are specific: they can only recognise one antigen, and if that antigen is not presented, there will be no reaction.

 figure 3 –

T-cell activation is a complex reaction involving many different stimulatory and costimulatory molecules. Note that signal 1 is the presentation of the antigen on HLA-II. Adaptive immunity Different types of Th-cells will then initiate both the cellular and humoral response of the adaptive immune system by activating other immune cells. The different Th-cells are Th1-cells, Th2-cells and regulatory T-cells (Treg-cells).5 The Th1-cells start activating other immune cells. One of these cells is the cytotoxic T-cell (Tc-cell). Tc-cells are CD8+, which means they can only interact with HLA-I. They are activated when they recognise their specific antigen on HLA-I and they are stimulated by Th1-cells by the cytokines IL-2 and IFNγ. Since virus infected cells and tumour cells produce abnormal proteins and display them on their HLA-I, the Tc-cells will recognise them as non-self and kill the cells, just like the NK-cells did.6

· Introduction ·

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B-cells are special APC that have their cell surface covered with a specific antigen. When the antigen attaches to the antibody, it may be recognised by the specific Th2-cell (figure 4). The Th2-cell will activate the B-cell by producing cytokines such as IL-4 and IL-5. Upon activation, it will differentiate into the antibody producing plasma cells. The antibodies, also known as immunoglobulins, enter the bloodstream and attach to antigens to inactivate them and facilitate phagocytosis by phagocytes.7,8

 figure 4 – General

overview of the immune system. Note that MHC is HLA. Ag = antigen; Th0 = Th-cell; CTL (cytotoxic T lymphocyte) = Tc-cell.

Treg-cells are essential in the immune response since they regulate and suppress other immune cells. They inhibit Tc-cell activity, Th-cell proliferation and DC maturation and activation. This way, they prevent autoimmune diseases and stop inflammation after the pathogen was disabled.

Some Tc-cells, Th-cells and B-cells form memory cells instead of effector cells. These memory cells will remain in the body, even after the infection is over. This way, when a new infection with the same pathogen occurs, the memory cells will be able to recognise it and respond immediately by killing the cells and producing antibodies.

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Transplant rejection After transplantation, the immune system of the recipient, also known as the host, will recognise the cells from the transplant as non-self and start to eliminate these cells by killing them and producing antibodies against them. There are two main ways the cells from the transplant can be recognised by the host’s immune system (figure 5).9 Just like all other cells, the donor cells present amino acid chains that were part of the proteins they produce on their HLA-I molecules. Also, there will be some APC from the donor in the transplant that do the same. However, due to the large variation in HLA, the donor HLA will be recognised as nonself by the host’s T-cells. This process is called direct presentation. This happens especially during the first weeks after the transplantation, when it leads to acute rejection.10,11 When cells from the transplant are destroyed or die, their HLA will be presented as antigens on the HLA-II of the host’s APC. These antigens are recognised by the T-cells, which will active Tc-cells and B-cells and cause further rejection. This is also known as indirect presentation.

This form of antigen presentation happens some weeks after the transplantation. This process will continue and will lead to chronic rejection.

 figure 5 –

Direct antigen presentation is facilitated by the donor cells, while indirect presentation happens when the host’s APC presents a donorderived peptide.

It is also possible that exactly the opposite reaction occurs.

This happens, for example, in bone marrow transplants.

The white blood cells from the transplant recognise the host’s HLA as non-self and start rejecting the host’s body.

The skin, intestines and lungs are usually the most affected by this condition, which is also known as Graftversus-Host-Disease (GvHD).12,13 Despite careful matching between donor and recipient, transplantation almost always causes the host’s immune system to react to and reject the transplant. To prevent this, patients receive immunosuppressive medication.

· Introduction ·

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Immunosuppressive therapy There are many different types of immunosuppressive medication. From these, corticosteroids are the most commonly prescribed medicine.14,15 These suppress the immune system by inhibiting certain activation pathways and help to slow down transplant rejection.

However, these medications have numerous side effects.

Because it weakens the immune system, the medication can cause infections in various organs or the development of tumours. Changes in appearance, such as weight gain and development of facial hair or acne, can occur. Certain medicines may cause cataracts, diabetes, hypertension, anaemia, bone disease and coronary artery disease.

Ironically enough, some immunosuppressants are toxic and damage the transplanted organs.16 Furthermore, not all patients react the same to the different types of medications, some even don’t react at all. Therefore, doctors constantly look for a balance, by giving enough medication to prevent severe transplant reaction, but keeping the dose as low as possible to lower the risk of developing side effects.

Because of the afore mentioned complications, scientists have long been looking for alternative ways to control transplant rejection. The last years, the main focus has been growing new organs from stem cells instead of using donor organs for transplantations.

Regenerative medicine Stem cells are the precursors of all cells. They have the unique ability to on the one hand self-renew and form identical daughter cells, and on the other hand, to create daughter cells that differentiate into various cell types.17 Embryonic stem cells (ESC), which are found in embryos, can differentiate into all the cell types. This ability might make it possible that are induced to form new organs.

However, there are various ethical issues concerning ESC, since are only present in embryos and though there are many scientists working on producing ESC from already differentiated cells, major results in growing new organs from ESC have yet to be achieved.

· Introduction ·

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In adults, there still are stem cells,18 which constantly form new cells to replace damaged or old ones. These stem cells, however, can only differentiate into a selective range of cells types. For instance, hematopoietic stem cells (HSC) in the bone marrow give rise to the different lineages of blood cells. Another type of stem cells that can be found in the bone marrow, but can be identified from all tissues,19 but mainly in connective tissue, like fat and muscle, are multipotent mesenchymal stromal cells, also known as mesenchymal stem cells (MSC).20 Mesenchymal stem cells In the embryo, the mesenchyme is another name for the part of the mesoderm that will develop into the connective tissue. Hence, MSC can become cells of the different tissues that developed from the mesenchyme: bone (osteoblasts), cartilage (chondrocytes), fat (adipocytes), tendon/ligament, stromal cells and muscle cells.18,21 The specific characteristics of MSCs, as determined by The International Society for Cellular Therapy (ISCT),22 are (1) adherence to plastic flasks and proliferation under standard culture conditions, (2) the expression of certain markers and (3) the potency to differentiate in vitro to adipocytes, osteoblasts and chondrocytes.

Unfortunately, MSC don’t have specific markers (also known as epitopes, which are the parts of antigens that are recognised by antibodies and the immune system) to define them, nor do they all express the same markers.23 Their immunophenotype (the markers they express) is generally positive for CD105, CD73 and CD90, and negative for CD45, CD31, CD34, CD14 or CD11b, CD79α or CD19 and HLA class II.22,24–26 However, it is important to know that cultured MSC and non-cultured MSC differ in immunophenotype.27 One of these differences is the loss of expression of CD34, and the upregulation of CD105, CD146 and CD271, which might give them their adhesive properties.28 Another difference is that the MSCs change from small, round shaped cells to relatively large spindle shaped cells, thus with a fibroblast-like appearance.26,27

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Because of their ability to differentiate into different types of connective tissue (figure 6), MSC could be a possible therapy for many diseases of the connective tissue by using cell replacement therapy.17,21 This entails the use of healthy MSC with unaffected genes to migrate to the affected areas, to replace the affected cells and take over their function. Using this technique, MSC could cure various diseases, such as fracture non-union, osteogenesis imperfecta, a genetic disease that results in fragile bones, and hypophosphatasia, a rare heritable condition that causes defective bone mineralization.

 figure 6 –

MSC give rise to different lineages of connective tissue cells.

Immunosuppressive capacity of MSC A fourth characteristic of MSC is their immunosuppressive capacity. It has been shown that they produce cytokines and the express associated receptors,25,29,30 which allow a direct interaction between MSC and white blood cells,23 enabling them to migrate to higher gradients of cytokines and go to sites of injury, to assist in tissue repair.20,31 Furthermore, MSC have been demonstrated to influence and suppress cell types from the innate and the adaptive immune system32–34 by the use of both soluble factor mechanisms and cell-to-cell interactions.35

· Introduction ·

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Many substances secreted by MSC, including transforming growth factor beta (TGFβ), hepatocyte growth factor (HGF), IL-10, prostaglandin E2 (PGE2) and HLA-G have been proposed as mediators that inhibit T-cell proliferation.36 In addition to these, indoleamine-2,3-dioxygenase (IDO) is often proposed as an important inhibitor of T-cells.

IDO is an enzyme that converts tryptophan to kynurenine, and both depletion of tryptophan and high concentrations of kynurenine, are responsible for T-cell inhibition.37,38 It has also been shown that MSC inhibit DC activation and maturation with IL-6 and PGE2, which on their turn inhibited the T-cell activation. Furthermore, it has been demonstrated that Treg-cell proliferation increased as a result of IL-10 and TGFβ production.24,33,34,37 In addition to soluble factor mechanisms, MSC also interact with the immune system by cell-to-cell contact.

MSC can, for instance, bind to the PD1 receptor on T-cells with PD-L1, which is expressed on their cell surface, and thereby inhibit T-cell proliferation.26,37 MSC react to inflammatory conditions by expressing very high levels of immunosuppressive factors.24 This makes them more immunosuppressive by changing both their immunophenotype and the soluble factors they produce.

This reaction can be evoked using proinflammatory cytokines, such as IFNγ and TNFα.26,39–41

 figure 7 –

An overview of the known mechanism by which MSC suppress the immune system. Arrows stand for production or stimulation, dashed arrows for inhibition.

· Introduction ·

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Immunosuppression in vitro and in vivo Many of the mechanisms by which MSCs suppress the immune system are still poorly understood. Most of the effects have only been shown in vitro, but that does not necessarily imply that the workings in vivo are alike.

In vitro and in vivo studies show that MSC migrate to inflammatory zones in response to cytokine signals.2 However, it has also been demonstrated that conditions of culturing have influence on the migration of MSC in vivo.42 Studies show that after infusion, the bulk of cultured MSC are trapped in the pulmonary capillaries, which are approximately four times smaller than MSC,19,27 and lose their efficacy in 48 hours.43–45 The homing of cultured MSC can be improved by insertion close to the injured or inflamed tissue.46 In general, MSC are not recognised by the immune system and therefore considered as immunoprivileged.

This is probably because their immunophenotype is specified as lacking full expression of HLA-I and HLA-II and lacking expression of costimulatory molecules.23,47 Their immunosuppressive ability may also contribute to the impaired immune recognition.48 On the other hand, in vivo studies show that MSC are able to create a T-cells response, which indicates that MSC are not totally immunoprivileged.49 Nonetheless, MSC are a promising target of regenerative and immunosuppressive medicine. As many as 421 clinical trials involving MSC have been registered,50 which are exploring the immunosuppressive characteristics of MSC as a potential clinical use in Crohn’s disease, graftversus-host disease, bone marrow transplants and solid organ transplantations.51–56

· Introduction ·

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To develop a successful immunosuppressive therapy based on MSC, it is important to know whether there are differences between the stem cell subpopulations. The markers that are used to distinguish between the MSC subpopulations may contribute to the immunosuppressive capacity of MSC, but might as well make MSC easier to recognise for the immune system.

Multiple studies have reported differences in potential between MSC from different tissue sources. We want to know if MSC also exhibit tissue specific characteristics, since these could possibly be important for defining the immunosuppressive ability of MSC. We investigated MSC isolated from perirenal fat and subcutaneous fat.

Furthermore, pre-activation of MSC by stimulation with IFNγ gives MSC different characteristics. We investigated if these differences make MSC more immunosuppressive and if the difference did not interfere with other properties.

Therefore aim of this study is to determine which type of adipose derived MSCs is the most suitable for developing an immunosuppressive therapy. To assess this, we looked at their differentiation capacity, reaction to inflammation and ability to suppress the immune system.

· Introduction ·

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METHODS Source of MSC MSC were isolated from surgically removed perirenal (PV) and subcutaneous fat (SV) from two healthy donors for kidney donation (table 2). These donors gave their written informed consent. The samples were named and either directly cultured, or frozen for later use.

Donor A Donor B

gender male female

age 49 49

sample names frozen SV1, PV1 yes SV2, PV2 no

Isolation of MSC from adipose tissue Adipose tissue was mechanically disrupted and, after they were washed twice with phosphate-buffered saline (PBS), enzymatically digested with sterile 0.5 mg/mL collagenase type IV in Roswell Park Memorial Institute 1640 medium (RPMI-1640) for 30 minutes at 37 °C. MSC medium (alpha minimum essential medium (MEM-α) with 2 mM L-glutamine (L-glut), 100 U/mL penicillin and 100 μg/mL streptomycin (1% p/s) and 15% fetal bovine serum (FBS)) was added to stop the reaction and after centrifuging, the pellet was incubated for 10 minutes at room temperature in red blood cell lysis buffer to lyse the red blood cells.

Then, MSC medium was added to stop the lysis and the cells were centrifuged again. The cells were then resuspended in MSC medium and filtered through a 70 μm cell strainer and transferred to culture flasks.

HLA type HLA-A2;68 B5;51 DR13 HLA-A3;68 B44;55 DR4;7  table 2 – Information about the donors used for this study.

MSC culture The cells were cultured at 37 °C, 5% CO2 and 95% humidity. After 3 days, non-adherent cells were removed and new MSC medium was added. The cultures were refreshed with new MSC medium twice a week. At 90% confluence, the adherent cells were removed by incubation in 0.05% trypsin-ethylenediamine tetraacetic acid (EDTA) at 37 °C. Then the MSC were counted using a counting chamber, and either reseeded in new culture flasks, or used for further experiments.

· Methods ·

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Differentiation assay The isolated cells from each sample were seeded and cultured for a day in MSC medium. For adipogenic and osteogenic differentiation, half of them were refreshed with differentiation medium and cultured for 21 days, refreshing medium twice a week.

Adipogenic medium consisted of MEM-α with 2 mM L-glut, 1% p/s and 15% heat inactivated FBS (FBS-HI), supplemented with 50 μg/mL L-ascorbic acid phosphate, 500 μM 3-isobutyl-1-methylxanthine, 60 μM indomethacin and 10 nM dexamethasone. After 21 days of culture, lipid vesicles were indicated with Oil Red O staining. Cells were washed with PBS, fixed in 60% isopropanol for 1 minute, and incubated with Oil Red O for 30 minutes. After three more washes with PBS, the cells were photographed.

Osteogenic medium consisted of MEM-α with 2 mM L-glut, 1% p/s and 15% FBS-HI, supplemented with 50 μg/mL L-ascorbic acid phosphate, 5 mM β-glycerophosphate and 10 nM dexamethasone. After 21 days of culture, calcium deposits were indicated with Alizarin Red staining. Cells were washed twice with PBS, fixed for 60 minutes in cold 70% ethanol at 4 °C. Then, the cells were incubated with Alizarin Red solution for 10 minutes, washed thrice with distilled water, and photographed.

For chondrogenic differentiation, the cells were seeded in 15 mL polypropylene tubes and centrifuged to pellets. The pellet was cultured in chondrogenic medium, consisting of Dulbecco’s Modified Eagle Medium High Glucose (DMEM HG) with 1% p/s, 2 mM L-glut, 1 mM sodium pyruvate, 40 μg/mL L-proline and ITS, supplemented with 10 ng/mL TGFβ1, 10 nM dexamethasone and 10 μM L-ascorbic acid phosphate. The cells were cultured for 21 days and the pellets were tapped loose before refreshing the medium.

After 21 days of culture, the cartilage matrix was indicated with Thionine staining. The pellets were fixed with paraffin, cut in small sections. These were deparaffinised and incubated with Thionine for 5 minutes, then differentiated by incubating for 30 seconds with 70% ethanol and rinsed once with ethanol, twice with xylene, and mounted and dried for an hour, and then the cells were photographed.

· Methods ·

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Immunosuppressive capacity of MSC To look at their reaction to inflammatory conditions, MSC were cultured in MSC medium with 50 ng/mL interferon gamma (IFNγ). After 7 days, cells were used for flow cytometry, mixed-lymphocyte reactions or restimulated with 50 ng/mL IFNγ for 5 days to perform an IDO-assay.

Flow cytometry The effect of IFNγ on the expression of markers on cell membranes was analysed using flow cytometry. MSC were trypsinised, counted and diluted, washed with FACSFlow and incubated with monoclonal antibodies with attached fluorochromes (CD31-Pacific Blue, CD45-APC-Cy7, CD105FITC, CD73-PE, CD90-APC, CD271-PE-Cy7, HLA-I-Pacific Blue, HLA-II-PerCP, ORB-APC, PD-L1-PE and CD13-PE-Cy7) for 30 minutes in the dark at 4 °C. The cells were washed twice with FACSFlow and analysed with the flow cytometer.

IDO-assay The medium was removed from the MSC after 12 days of culture. The medium was then incubated with 30% trichloroacetic acid (TCAA) for 30 minutes at 50 °C. Excess proteins were removed by centrifugation. Ehrlich reagent was added and absorbance was measured at 490 nm.

Mixed-lymphocyte reactions In order to look at the effects of MSC on peripheral blood mononuclear cell (PBMC) proliferation, mixed-lymphocyte reactions (MLR) was used. MSC were trypsinised, counted, and seeded in a 96-wells plate in different concentrations (20000, 10000 and 5000 cells). The next day, responder PBMC from Buffy A were labelled with CFSE and 50000 cells added to each well. 50000 gamma-irradiated (40 Gy) PBMC from Buffy B were also added. The PBMC were mismatched (Buffy A: HLA-A2 B16(38);16(39) DR13(6);8, Buffy B: HLA-A3 B60(40);47 DR4;7) and cultured 7 days in MEM-α with 2 mM L-glut, 1% p/s and 10% human serum (HuS). PBMC proliferation was measured using flow cytometry. PBMC were incubated with monoclonal antibodies with attached fluorochromes (CD3-PerCP, CD4Pacific Blue, CD8-APC and CD19-PE-Cy7) for 30 minutes in the dark at 4 °C. The cells were washed twice with FACSFlow and analysed with the flow cytometer.

· Methods ·

· 22 ·

Isolation of PBMC from spleen tissue Spleen tissue was obtained from a deceased heart donor.

The spleen was cut in smaller pieces, mashed and sifted twice through a sterile sieve and collected in DNase medium (RPMI-1640 with 1% p/s and 1% DNase solution), to reduce the clumping of cells. Then the cell suspension was filtered through a 70 μm cell strainer. The PBMC were isolated from the cell suspension by density gradient centrifugation using Ficoll Isopaque (δ = 1.077). The cells were collected in DNase medium with 10% FBS-HI, counted and diluted, and frozen at −150 °C until use.

Statistical analysis All data is represented as mean, unless otherwise stated.

Statistical analysis of the results was carried out with different tests. Differences were considered statistically significant with p-values of p < 0.05 For analysis of L-kynurenine concentration, the means of each group were compared after analysing the variances.

Data of the MLR were analysed with linear regression to look at dose dependence. CD4 and CD8 quantities were compared and further analysed using the sign-test and Student’s t-distribution. The control group was examined with the F-test for variances and Student’s t-test for comparison of means.

· Methods ·

· 23 ·

RESULTS MSC isolation and culture MSC were isolated from both perirenal and subcutaneous adipose tissue from two different donors and successfully expanded in culture. Cells derived from Donor B showed more proliferation than those from Donor A.

A

B

C

D

E

F

During culture, a clear morphological change occurred.

The freshly isolated MSC were small and round shaped.

After some days of culture, the cells had adhered to the flask and were more spindle shaped (figure 8A). They grew and stretched over the bottom of the flask and proliferated until full confluence was reached.

There also was a difference between MSC isolated from subcutaneous fat and perirenal fat. The first looked like thin spindles, and expanded organised in a nice linear pattern, which was less evident for latter, which looked more robust and disordered (figure 8C,F).

· Results ·

 figure 8 –

Culture of MSC derived from subcutaneous fat after (A) 4 days, (B) 12 days and (C) 19 days, and from perirenal fat after (D) 4 days, (E) 12 days and (F) 19 days. Representive experiments are shown (n = 2).

Microscope (50×).

· 24 ·

Differentiation assay MSC were cultured for 21 days in differentiation medium to induce adipogenic, osteogenic and chondrogenic differentiation. Differentiation cultures did not reach full confluence but changed in morphology. After staining the samples, differentiation was confirmed.

Adipogenic Adipogenic differentiation was successfully induced, as on day 8, lipid vesicles were visible. After 21 days, formation of lipid vesicles was indicated with Oil Red O staining (figure 9). MSC isolated from perirenal fat showed more differentiation than those isolated from subcutaneous fat.

 figure 9 –

The culture of MSC after (A) 8 days, (B) 15 days and (C) 21 days (Oil Red O) versus the culture of adipogenic MSC derived from perirenal fat after (D) 8 days, (E) 15 days and (F) 21 days (Oil Red O) and from subcutaneous fat after (G) 8 days, (H) 15 days and (I) 21 days (Oil Red O). Representive experiments are shown (n = 2).

Microscope (50×).

A

B

C

D

E

F

G

H

I

· Results ·

· 25 ·

Osteogenic Osteogenic differentiation was successfully induced by culturing MSC in osteogenic medium. After 21 days of culture, calcium deposits in the matrix were indicated with Alizarin Red staining (figure 10).

A

B

On day 11, calcification and matrix forming could be seen in the osteogenic culture. There was more calcium deposit formation in cell cultures with cells from Donor B and more for cultures with cells derived from perirenal fat.

C

 figure 10 –

Osteogenic MSC after (A) 11 days, (B) 21 days (Alizarin Red) (Representive image is shown (n = 4). Microscope (50×)) and (C) an overview of cell cultures after 21 days (Alizarin Red), upper row: osteogenic differentiation, lower row: negative controls. From left to right: PV2, SV2, SV1, PV1.

Chondrogenic Differentiation of MSC in to chondrocytes was successfully induced. On day 11, the pellets of chondrogenic cultures (figure 11A) were visibly larger than the pellets of control cultures. After 21 days, formation of cartilage matrix was indicated with Thionine staining (figure 11B,C). Cartilage formed from subcutaneous fat derived MSC looked more structured than perirenal fat derived MSC.

A

· Results ·

 figure 11 –

Chondrogenic (A) pellets (arrows) after 11 days (PV1 and PV2), and after 21 days (Thionine), overview of (B) pellet and (C) cells (Microscope (50×)). Representive images are shown (n = 4).

B

C

· 26 ·

Flow cytometry MSC were analysed using flow cytometry in two stain panels and one unstained, as a negative control. For both panels, the CD45− subpopulation (figure 12) was gated from the cells, and used for further analysis.

Immunophenotype MSC were positive for CD13, CD73, CD90, CD105 and CD271, and negative for CD31, CD45, ORB, PD-L1, HLA-I and HLA-II. MSC derived from perirenal fat expressed more CD105 (figure 13E), while those from subcutaneous fat expressed higher levels of CD90 (figure 13D). Donor A was more positive for CD73, CD105 (figure 13C,E) and CD13 than Donor B, while Donor B had higher expression of CD271.

 figure 12 –

From the viable cells, the CD45− subpopulation was gated to be used for further analyses.

Flow cytometry markers unstained

SV1

PV1

SV2

PV2

A CD31

B CD45

C CD73

D CD90

E CD105

F CD271

 figure 13 – Flow cytometric characteristics of MSC.

· Results ·

· 27 ·

Reaction to inflammatory conditions MSC stimulated with IFNγ not only changed the expression of most markers, but also began to express new markers.

They were more positive for most markers. This was especially the case for CD73 (figure 14A). However, the cells were less positive for CD90 and for CD13, for which MSC were negative after IFNγ stimulation.

After stimulation with IFNγ, MSC began to express ORB, HLA-I, HLA-II and PD-L1 (figure 14C,D,E,F).

The MSC derived from the perirenal fat of Donor A (PV1) seemed particularly stimulated, as the level of expression of CD73, CD105, ORB and HLA-II was approximately double that of the other samples.

Mean fluorescence intensity unstained

unstimulated MSC

IFNγ stimulated MSC

30000

12000

1200

25000

10000

1000

20000

8000

800

15000

6000

600

10000

4000

400

5000

2000

200

0

0 SV1 SV2 PV1 PV2

0 SV1 SV2 PV1 PV2

SV1 SV2 PV1 PV2

A CD73

B CD105

C ORB

500

12000

1200

400

10000

1000

8000

800

6000

600

4000

400

2000

200

300 200 100 0

0 SV1 SV2 PV1 PV2 D HLA-I

0 SV1 SV2 PV1 PV2 E HLA-II

 figure 14 – The mean fluorescence intensity (MFI) of

stimulated, especially for ORB, HLA-II and PD-L1.

· Results ·

SV1 SV2 PV1 PV2 F PD-L1

various epitopes. Expression of all epitopes was

· 28 ·

IDO-assay The medium of IFNγ stimulated cells contained four to five times more L-kynurenine than the medium of unstimulated cells (figure 15). Medium of the IFNγ stimulated cells of Donor B contained significantly more L-kynurenine than medium of the IFNγ stimulated cells of Donor A (p < 0,05).

L-kynurenine

unstimulated MSC

concentration IFNγ stimulated MSC

60

concentration (µM)

50 40 30 20 10

 figure 15 – Concentration of L-

0 SV1

SV2

PV1

PV2

kynurenine (in µM) in the medium after culture.

Mixed-lymphocyte reactions To measure the immunosuppressive effect of MSC on the proliferation of PBMC, mixed-lymphocyte reactions were used. PBMC were cultured for seven days with MSC in a concentration of 1:2.5, 1:5 or 1:10. Using flow cytometry, the proliferation of B-cells (CD19+), Th-cells (CD4+) and Tc-cells (CD8+) was measured. These subpopulations (figure 16) were gated from the proliferated cell (CFSE−) subpopulation, which was gated from the viable cells.

These subpopulations were used for further analyses.

PBMC of Buffy A alone did not proliferate (figure 17A: A).

Proliferation of Buffy A was induced when exposed to the irradiated PBMC of Buffy B (figure 17A: AxB~). When the PBMC were cultured with MSC, PBMC proliferation was suppressed. This was the case for SV1, SV2 and PV2.

PBMC cultured with PV1, however, did not suppress, but rather stimulated proliferation. The suppression of PBMC was higher for MSC isolated from Donor B than for MSC isolated from Donor A.

· Results ·

· 29 ·

When PBMC were cultured with a higher concentration of MSC, the proliferation was lower. The immunosuppression followed a dose-response relationship. This was the case for SV1, SV2 and PV2. However, for the IFNγ stimulated MSC from SV2, the dose-response relationship was inversed (figure 17B: SV2). Except for this, there was no difference in proliferation between unstimulated cells and IFNγ stimulated cells.

 figure 16 –From

the viable cells, proliferated cells were obtained by gating for CFSE− cells. B-cells (CD19+), Th-cells (CD4+) and Tc-cells (CD8+) were gated from the CFSE− subpopulation.

For each well PBMC of Buffy A were added. After 7 days of culturing we found that only a small amount of B-cells (CD19+) had proliferated, compared to large amounts of T-cells (CD3+). Th-cells proliferated significantly more than Tc-cells (p < 0.001). We also saw that samples seeded with MSC from Donor A contained more T-cells than samples seeded with MSC from Donor B. NEXT PAGE figure 17 –

The immunosuppressive regulation of MSC as demonstrated by mixed-lymphocyte reactions for both unstimulated and IFNγ stimulated MSC.

· Results ·

· 30 ·

Mixed-lymphocyte reactions control

MSC 1:2.5

MSC 1:5

MSC 1:10

100 90 80

proliferation (%)

70 60 50 40 30 20 10 0 A

AxB~ AxA~

SV1

SV2

PV1

PV2

PV1

PV2

A unstimulated MSC

100 90 80

proliferation (%)

70 60 50 40 30 20 10 0 A

AxB~ AxA~

SV1

SV2

B IFNγ stimulated MSC

· Results ·

· 31 ·

DISCUSSION Mesenchymal stem cells are present in several tissue types, including fat, and accumulating data shows the ability of these cells to suppress the immune system via both soluble factors and cell-to-cell contact. Furthermore, it is relatively easy to isolate them from fat and culture them. These characteristics suggest the potential to form an alternative immunosuppressive therapy. Therefore studies to the various groups of MSC are necessary to produce a specific treatment for transplant rejection.

In this study we investigated differences between different groups of MSC in the capacity to differentiate into various cell types in a differentiation assay, the reaction to inflammatory conditions by performing flow cytometry and an IDO-assay on IFNγ stimulated cells and the ability to suppress the immune system as evaluated in MLR. We looked at which group of MSC, those isolated from perirenal fat or subcutaneous fat, those that were frozen for later use or directly cultured and those that were unstimulated or IFNγ stimulated, was the most suitable for the development of an immunosuppressive therapy.

In brief, our findings show that MSC from perirenal fat differentiate better than MSC from subcutaneous fat. The data also show that IFNγ stimulated cells express more immunosuppressive markers and exhibit more IDO activity than unstimulated cells. Furthermore, we found that the MSC suppressed the proliferation of immune cells in a dose-dependent way. MSC derived from perirenal fat was better at suppressing the immune cell proliferation than MSC derived from subcutaneous fat.

The adherence to plastic flasks and expansion in normal culture conditions, the expression of specific markers and the ability of the cells to differentiate in adipocytes, osteoblasts and chondrocytes, were consistent with the ISCT criteria for defining MSC.22

· Discussion ·

· 32 ·

There was a difference in the amount of viable cells of Donor A and Donor B. Donor A had less cells, which is probably due to the freezing to preserve the cells. Cells from Donor B were freshly isolated and directly cultured.

However, these observations are based on the confluence reached and cell counts prior to trypsinisation. These methods are not very reliable, since the differences could also have other causes. The cells could have had more time to grow, or were seeded in a lower density. To confirm this hypothesis, a colony forming unit assay should have been used with a larger amount of samples.

Our results of the differentiation assay show that MSC derived from perirenal fat exhibit more adipogenic and osteogenic differentiation capacity than MSC derived from subcutaneous fat. Subcutaneous fat, on the other hand, seemed to favour chondrogenic differentiation more than MSC from perirenal fat. The latter, though, was less evident due to the formation of only small amounts of chondrocytes and the loss of the control samples and two chondrocyte pellets when refreshing the medium. Due to limitations in time, these experiments could not be redone to obtain more accurate results. These results are based on analysis of microscopy images, whereas true methods of quantification would be more accurate to base these results on. Furthermore, since we analysed one sample per group, this study could be made more reliable by increasing the number of samples.

Prior studies report differences in differentiation capacity between MSC from different tissue sources.57,58 The differences in differentiation capacity can be used for distinguishing cell-lines in order to develop a specific immune suppressive therapy. Another reason for the importance of defining the differentiation capacity is that the different properties may have influence on the function of cells. We found significant differences between MSC derived from subcutaneous fat and perirenal fat.

Because MSC are present in many different tissues, it is reasonable to assume that they have a different function.

Therefore, the tissue the MSC were isolated from could have an impact on their function. Further investigation is needed to study the capacities of tissue-specific MSC.

路 Discussion 路

路 33 路

In this study, we also evaluated immunophenotypic differences using flow cytometry. We found that MSC were positive for CD13, CD73, CD90, CD105 and CD271, and negative for CD31, CD45, ORB, PD-L1, HLA-I and HLA-II, which was also found in other studies.22,24,25 We also saw a difference in the expression of the markers CD73, CD90, CD105, CD13 and CD271, although for CD13 and CD271 mean fluorescence intensity was low and therefore the differences less evident.

Donor A was more positive for CD73, CD105 and CD13, and Donor B had higher expression of CD271. It was also observed that MSC derived from perirenal fat were more positive for CD105, while those derived from subcutaneous fat were more positive for CD90.

Multiple studies show variation in properties of MSCs from different donors.59 The latest studies suggest that differentiation capacity is phenotype related, while differences in phenotype are affected by gender and age.60 In our study we used MSC from two donors with the same age, but of opposite gender. However, the capacity to differentiate was the same, which suggests that any differences, related to gender or preservation, had no influence on differentiation capacity. These conclusions are taken from a small population and to confirm this, larger populations should be used.

The expression of specific markers is an important characteristic of MSC and lies at the basis of selecting a subpopulation. The function of most markers, however, is often only partially known, which makes it difficult to establish a relation between a marker and its properties.

Nonetheless, we looked for a possible correlation between expression of specific markers and properties in order to interpret the results.

CD73, otherwise known as ecto-5’-nucleotidase, catalyses the reaction that changes adenosine monophosphate into adenosine. High levels of adenosine are shown to inhibit T-cell responses.61 This function may contribute to the immunosuppressive characteristics of MSC.

· Discussion ·

· 34 ·

The function of CD90 or Thy-1 is not totally revealed, but the marker is considered as useful for MSC selecting and to play a role as differentiation marker.62 CD105 or endoglin is part of the TGFβ complex and has influence on several properties. TGFβ also plays a vital role in the function of regulatory T-cells.63 These cells might play a role in the immunosuppressive effect of MSC, although our data showed no differences in suppression between perirenal and subcutaneous fat.

We saw that during culturing perirenal fat derived MSC varied in morphology from subcutaneous derived MSC. Since upregulation of CD105 and CD271 are related to improved motility and adherence to plastic,28 dissimilarity might also explain differences in morphology. But since our results are limited to small sample sizes, there is a lack of underlining for this idea. Therefore, more flow cytometry tests during culturing and mRNA expression tests are needed to confirm this.

After IFNγ stimulation, expression of all markers changed, which is consistent with earlier literature.39–41 The MSC also started to express high levels of specific markers related to immunosuppression. These markers were ORB, HLA-I, HLA-II and PD-L1.

In earlier studies, it was demonstrated that ORB, also known as syndecan-2, inhibits T-cell activation.64,65 PD-L1 serves as an inhibitory co-stimulatory molecule.66 The increased expression of these markers therefore contributes to the immunosuppressive characteristics of MSC. Another molecule that plays an important role in the immunomodulatory function of MSC is IDO.37 This enzyme converts tryptophan into kynurenine, and these soluble factors suppresses the T-cell proliferation. In the current study, kynurenine was found in high doses in the medium of IFNγ stimulated cells. This is consistent with earlier findings.37,38 After IFNγ stimulation, it was present in higher doses for Donor B. To understand why Donor B reacted better to IFNγ, other markers should be analysed.

· Discussion ·

· 35 ·

The effect size of IFNγ stimulation was large compared to unstimulated cells, but we could not find a statistical difference of kynurenine concentration in medium of perirenal and subcutaneous fat. However, due to the small sample size, retrospective power analysis has be used to confirm this is the case, but we could not employ these methods because programs for this analysis were not available. Future studies should include larger sample size to make statistical analyses more representative.

We also investigated the immunosuppressive capacity of the different groups of MSC. Our results show that MSC suppress the immune system in a dose-dependent way, which is in agreement with prior studies.32,67 We also saw that there is no difference between IFNγ stimulated and unstimulated MSC, which is contrary to other studies.39,40 The cause of this difference is unclear, since the further characteristics the MSC display are close to identical.

We found a difference in suppression between cells of Donor A and Donor B. Donor B showed more suppression.

The only difference Donor B showed in the markers we assessed was increased CD271 expression. CD271, or p75NTR, is associated with apoptosis,68 There is, however, no known function in immunosuppression. The result might therefore be caused by other factors we did not focus on, or be a coincidence. Including more markers and more experiments may help to answer these questions.

 figure 18 –

Dose-dependence of stimulated SV2 was exactly opposite of unstimulated SV2 as demonstrated by linear regression.

· Discussion ·

Dose-dependence unstimulated SV2 liv ing dnor

IFNγ stimulated SV2

70 60 Proliferation (%)

There were two remarkable results.

Firstly, IFNγ stimulated MSC from SV2 induced PBMC proliferation in a reversed dose-dependent manner. This relationship was exactly the opposite of the relation found for unstimulated SV2 (figure 18). We attribute this to a technical error. To confirm this, the experiment has to be repeated with more wells of this sample.

50 40 30 20 10 0 0

1:2.5

1:5

1:7.5

· 36 ·

1:10

Secondly, MSC from PV1 stimulated proliferation rather than inducing it, because proliferation was approximately 10% higher than for the control samples. Flow cytometry has indicated that PV1 has elevated levels of expression of CD73, CD105, ORB and HLA-II. Except for HLA-II, these markers are all known as immunosuppressive factors.28 Furthermore, the HLA of Donor A is more similar to that of Buffy A than Donor B, which means that for Donor A, the reaction should be less than for Donor B. However, other studies indicated that the reaction of PBMC to MSC is independent of HLA-type.69 None of the examined markers can account for why PV1 stimulated PBMC proliferation.

It might be caused by uninvestigated MSC-derived factors.

Further investigation involving more markers and including larger sample sizes is necessary to be able to explain the increased PBMC proliferation.

The proliferation of different PBMC was measured. We found that Th-cell proliferation was more than Tc-cell proliferation, both in control samples and samples cultured with MSC. Ratio of Tc-cells to Th-cell remained the same, suggesting that proliferation of both types of PBMC are consistently inhibited by MSC, which is consistent with other studies.24,32 Conclusions In clinical practice, MSC can be used for both regenerative and immunomodulatory purposes.24,25 We compared these various characteristics of different groups of MSC. In conclusion, our findings show that MSC from perirenal fat differentiate better than MSC from subcutaneous fat.

IFNγ stimulated cells express more immunosuppressive markers and exhibit more IDO activity than unstimulated cells. Furthermore, we found that the MSC suppressed the proliferation of immune cells in a dose dependent way. For all experiments, MSC from Donor B performed better than those from Donor A. This is why we would say that the perirenal fat of Donor B, that is, PV2, is the best MSC group to develop an immunosuppressive therapy.

· Discussion ·

· 37 ·

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48. Nauta AJ, Westerhuis G, Kruisselbrink AB, Lurvink EG, Willemze R, Fibbe WE. Donorderived mesenchymal stem cells are immunogenic in an allogeneic host and stimulate donor graft rejection in a nonmyeloablative setting. Blood 2006;108:2114-20.

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52. Reinders ME, de Fijter JW, Roelofs H, Bajema IM, de Vries DK, Shaapherder AF, et al. Autologous Bone Marrow-Derived Mesenchymal Stromal Cells for the Treatment of Allograft Rejection After Renal Transplantation: Results of a Phase I Study. Stem Cells Transl Med 2013;2:107-11.

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58. Phinney DG, Kopen G, Righter W, Webster S, Tremain N, Prockop DJ. Donor variation in the growth properties and osteogenic potential of human marrow stromal cells. J Cell Biochem 1999;75:424-36.

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59. Siegel G, Kluba T, Hermanutz-Klein U, Bieback K, Northoff H, Schäfer R. Phenotype, donor age and gender affect function of human bone marrow-derived mesenchymal stromal cells. BMC Med 2013;11:146.

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61. Chen XD, Qian HY, Neff L, Satomura K, Horowitz MC. Thy-1 Antigen Expression by Cells in the Osteoblast Lineage. J Bone Miner Res 1999;14:362-75.

62. Belghith M, Bluestone JA, Barriot S, Mégret J, Bach JF, Chatenoud L. TGF-βdependent mechanisms mediate restoration of self-tolerance induced by antibodies to CD3 in overt autoimmune diabetes. Nat Med 2003;9:1202-8.

63. Teixé T, Nieto-Blanco P, Vilella R, Engel P, Reina M, Espel E. Syndecan-2 and -4 expressed on activated primary human CD4+ lymphocytes can regulate T cell activation. Mol Immunol 2008;45:2905-19.

64. Rovira-Clavé X, Angulo-Ibáñez M, Noguer O, Espel E, Reina M. Syndecan-2 can promote clearance of T-cell receptor/CD3 from the cell surface. Immunology 2012;137:214-25.

65. Roemeling-van Rhijn M, Mensah FK, Korevaar SS, Leijs MJ, van Osch GJ, Ijzermans JN, et al. Effects of Hypoxia on the Immunomodulatory Properties of Adipose TissueDerived Mesenchymal Stem cells. Front Immunol 2013;4:203.

66. Le Blanc K. Immunomodulatory effects of fetal and adult mesenchymal stem cells.

Cytotherapy 2003;5(6):485-489 67. Wong ST, Henley JR, Kanning KC, Huang KH, Bothwell M, Poo MM. A p75(NTR) and Nogo receptor complex mediates repulsive signaling by myelin-associated glycoprotein. Nat Neurosci 2002;5:1302-8.

68. Le Blanc K, Tammik L, Sundberg B, Haynesworth SE, Ringdén O. Mesenchymal Stem Cells Inhibit and Stimulate Mixed Lymphocyte Cultures and Mitogenic Responses Independently of the Major Histocompatibility Complex. Scand J Immunol 2003;57:11-20. figure 1 figure 2 figure 3 figure 4 figure 5 figure 6 figure 7

http://www.webmd.com/urinary-incontinence-oab/picture-of-the-kidneys http://www.transplantatiestichting.nl/cijfers/organen-jaarcijfers2 Halloran PF. Immunosuppressive Drugs for Kidney Transplantation. N Engl J Med 2004;351:2715-29.4 http://www.intechopen.com/books/immunodeficiency/-wrapped-up-vaccinesin-the-context-of-hiv-1-immunotherapy http://www.sciamsurgery.com/sciamsurgery/institutional/figTabPopup. action?bookId=ACS&linkId=part10_ch12_fig2&type=fig http://www.discoverymedicine.com/Tracey-L-Bonfield/files/2010/04/ bonfield_caplan_no47_figure_1.jpg Abdi R, Fiorina P, Adra CN, Atkinson M, Sayegh MH. Immunomodulation by Mesenchymal Stem Cells: A Potential Therapeutic Strategy for Type 1 Diabetes. Diabetes 2008;57:1759-67.37

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PROTOCOLS Used mediums  MSC medium MEM-α with 2 mM L-glut, 1% p/s 15% FBS-HI  Freeze medium MEM-α with 10% dimethyl sulfoxide (DMSO) 10% FBS-HI  DNAse medium RPMI-1640 with 1% p/s 1% DNAse stock  Routine serum (for washing)  Human culture medium (HCM) MEM-α with 2 mM L-glutamine, 1% p/s 10% human serum (HuS))  Adipogenic medium MEM-α with 2 mM L-glut, 1% p/s 15% FBS-HI 50 μg/mL L-ascorbic acid phosphate 500 μM 3-isobutyl-1-methylxanthine 60 μM indomethacin 10 nM dexamethasone  Osteogenic medium MEM-α with 2 mM L-glut, 1% p/s 15% FBS-HI 50 μg/mL L-ascorbic acid phosphate 5 mM β-glycerophosphate 10 nM dexamethasone  Chondrogenic medium, DMEM HG with 2 mM L-glut, 1% p/s 1 mM sodium pyruvate 40 μg/mL L-proline ITS 10 ng/mL TGFβ1 10 nM dexamethasone 10 μM L-ascorbic acid phosphate

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Protocol 1: Isolation of MSC from adipose tissue  Mechanically disrupt the adipose tissue with scalpel knifes on a petridish  Transfer the adipose tissue to a 50 mL tube (using a sterile pincet, then flush petridish with 40 mL PBS)  Centrifuge for 5 minutes at 2000 rpm  Remove and pellet by sticking a 10 mL pipet through adipose layer  Add 40 mL PBS, centrifuge for 5 minutes at 2000 rpm  Remove PBS, add 10 mL sterile filtered 0.5 mg/mL collagenase type IV (weigh 5 mg collagenase in the tube with orange cap, add 10 mL sterile RPMI-1640, put through 20 µm sieve) to tissue for 30 minutes at 37 °C at 200 rpm.  Add 8-10 mL of MSC medium to stop the reaction.  Centrifuge for 10 minutes at 1700 rpm  Flush the tube with MEM-α, use a new 50 mL tube every step to remove liquid fat  Remove medium and floating adipose tissue, leave the pellet in situ  Use 10 mL pipet to resuspend pellet in 5 ml red blood cell lysis buffer and incubate 10 min at room temperature to lyse the red blood cells  Add 5 mL MSC medium to stop reaction  Centrifuge for 10 minutes at 1700 rpm  Resuspend the pellet in 4 mL MSC medium.  Filter through a 70 µm cell strainer (to remove klontjes)  Transfer to cell culture flask in 20 mL MSC medium

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Protocol 2: Culture of MSC  Culture cells at 37 °C, 5% CO2 and 95% humidity.

Refresh medium every 3 days.  When more than 90% confluence is reached, cells have to be trypsinised, counted, and frozen or used for further experiments, and, when necessary, stimulated with IFNγ, Trypsinisation  Remove the old medium from the culture flasks  Wash the cells twice with 10 mL PBS  Add 2.5 mL of 0.05% trypsin-EDTA solution  Incubate for 5 minutes at 37 °C  Tap the flask and check under the microscope if the cells are loose  Add 6 mL of MSC medium and rinse some times Counting  Add 20 µL of tryptan blue dye to 20 µL cell suspension  Fill a Thiefe-Bürker counting chamber with the solution  Count the cells in 16 squares (n) and use the formula: concentration (cells/mL) = n/16 × 25 × 2 × 104 Freezing  Trypsinise the cells  Centrifuge for 5 minutes at 2000 rpm  Add 1 mL of MSC medium to dissolve the pellet  Count the cells and dilute to 200 000 cells/mL  Fill a freeze vial with 1 mL cell suspension and 1 mL freeze medium IFNγ stimulation  Add 50 ng/mL of IFNγ to the cells

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Protocol 3: MSC Differentiation assay  Seed 4 wells with 500000 cells/well in 2 mL of MSC medium in a 6-wells plate.  Resuspend cells to a concentration of 200000 cells/0,5 ml in DMEM. Do the same for chondrogenic medium.  Put 0,5 ml of suspension per 15 mL conical tube  Centrifuge for 8 minutes at 1000 rpm, carefully place in incubator for 24 hours Day 2  For each sample, keep 2 wells as a control and of the other two, add adipogenic medium to one, and osteogenic medium to the other (then do this every 3 days)  Gently loosen cell pellet by gently tapping after 24 hours, and refresh medium (then do this every 3 days) Day 22  For Oil Red O staining of adipogenic cultures: – Wash cells 1x with PBS – Fix in 60% isopropanol for 1 minute – Incubate with Oil Red O working solution for 30 minutes at room temperature – Wash 3x in PBS – Photograph  For Alizarin Red staining of osteogenic cultures: – Wash cells 2x with PBS – Fix for 60 minutes in cold 70% ethanol at 4 °C – Incubated with Alizarin Red solution for 10 minutes – Wash 3x with distilled water – Photograph  For Thionine staining of chondrogenic cultures: – Fix pellets with paraffin – Cut in small slides – Deparaffinise the slides – Incubate with Thionine for 5 minutes – Differentiated by incubating for 30 seconds with 70% ethanol – Wash with ethanol, then 2x with xylene – Mount with Entallan while slides are wet from xylene – Let slides dry for 60 minutes – Photograph

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Protocol 4: Flow cytometry  Trypsinise the cells  Add 6 mL of MSC medium to the culture flask, transfer the cell suspension to a 15 mL tube and rinse the flask  Centrifuge for 5 minutes at 2000 rpm  Count and dilute to 100000 cells per FACS tube  Add 2 mL of FACSFlow  Centrifuge for 5 minutes at 2000 rpm  Prepare antibody mix: – To make mix 1 (for 8 tubes), add: CD31-Pacific Blue 80 μL CD45-APC-H7 8 μL CD105-FITC 80 μL CD73-PE 40 μL CD90-APC 8 μL CD271-PE-Cy7 40 μL FACSFlow 544 μL – To make mix 2 (for 8 tubes), add: HLA-I-Pacific Blue 40 μL HLA-II-PerCP 80 μL ORB-APC 16 μL PD-L1-PE 24 μL CD13-PE-Cy7 8 μL CD45-APC-H7 8 μL FACSFlow 624 μL  Pour off supernatant and add 100 μL of either mix 1, mix 2 or FACSFlow (for the negative controls) to the designated tube  Incubate for 30 minutes in the dark at 4 °C  Add 2 mL of FACSFlow and centrifuge for 5 minutes at 2000 rpm  Pour off supernatant and add 100 μL of FACSFlow  Analyse using the flow cytometer

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Protocol 5: IDO-assay  Label tubes and prepare plate  Prepare 2 mL 30% trichloroacetic acid (TCA)  Prepare Ehrlich reagent: add 100 mg DMBA to 5 mL acetic acid  Prepare L-kynurenine standards: – Dilute L-kynurenine stock solution [0.24 M] from freezer to 200 μM: add 4.2 μL stock [0.24 M] to 4.996 mL MSC medium – Make standards by : Medium L-kynurenine St0 0 μM 200 μL 200 μL remove St2.5 2.5 μM 200 μL of St5 200 μL St5 5 μM 200 μL 200 μL of St10 St10 10 μM 200 μL 200 μL of St20 St20 20 μM 200 μL 200 μL of St40 St40 40 μM 200 μL 200 μL of St80 St80 80 μM 200 μL 200 μL of St160 800 μL stock remove St160 160 μM 200 μL 200 μM 600 μL Add 200 μL of sample to each tube Add 100 μL 30% TCA to each sample/standard Vortex Incubate for 30 minutes in 50 °C water bath Centrifuge tubes for 5 minutes at 12000 rpm Add 150 μL of sample in the first well of the duplo, resuspend (2x), and aliquoat 75 μL to the second well  Add 75 μL Ehrlich reagent with the multichannel to each well  Measure absorbance at 490 nm      

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Protocol 6: Mixed-lymphocyte reactions  Add 20000 (1:2.5), 10000 (1:5) or 5000 (1:10) MSC in MSC medium to a 96-wells plate (see plate layout) Day 2  Add 5 mL DNAse medium and 1 mL routine serum in a 15 mL tube  Get the vials with PBMCs from the -190 °C freezer and put them in dry ice  Melt the ampuls in a water bath (37 °C) until there is only a little ice left  Add the cell suspension from the vial to the 15 mL tube (with 5 mL DNAse medium + 1 mL routine serum).

Wash the vial with 1.5 mL DNAse medium.  Centrifuge for 5 minutes at 2000 rpm  Pour off supernatant and resuspend the pellet in 5 mL DNase medium + 1 mL routine serum.  Centrifuge for 5 minutes at 2000 rpm  Pour off supernatant and resuspend the pellet in 5 mL human culture medium.  Count the cells and dilute to 1 × 106 cells/mL  Irradiate cells: – Add 1 × 106 cells of Buffy A in 2 mL of HCM in a tube and add 6 × 106 cells of Buffy B in 12 mL of HCM in a tube – Put the tubes in a plastic bag and put a 25 Gy control sticker on the bag – Irradiate the cells at 40 Gy with the gamma-irradiator  Colour 9 × 106 cells of Buffy A with CFSE: – Dilute CSFE in PBS: 20 µL per 1 mL PBS, until 10 million cells: 1 mL CFSE in PBS – Add CFSE to the cells and resuspend, colour for 7 minutes in the dark – Add medium until the solution is 10 mL – Centrifuge for 5 minutes at 2000 rpm – Count the cells and put the cells in MEM 10% HuS solution to a concentration of 1 × 106 cells / mL  Make 2 µg/ml PHA (in well diluted to 1:1, 100 µL cell suspension + 100 µL PHA 2 µg/mL)  Centrifuge the irradiated cells for 5 minutes at 2000 rpm, take them in a solution of 12 mL HCM (Buffy A) or 2 mL HCM (Buffy B)  Add to 96-wells plate (see plate layout)

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MLR plate MLR 1 2

3

4

5

6 7

8

9 10 11 12

A

Buffy A

A x A irradiated

B

A x B irradiated

A uncoloured

C

A x B~ + PHA

D

SV1 1:2.5

E

SV1 + IFNγ 1:2.5 SV1 + IFNγ 1:5 SV1 + IFNγ 1:10

F

PV1 1:2.5

G

PV1 + IFNγ 1:2.5 PV1 + IFNγ 1:5 PV1 + IFNγ 1:10

SV1 1:5

SV1 1:10

PV1 1:5

PV1 1:10

H MLR 1

2

3

4

5

6 7

8

9 10 11 12

A

Buffy A

A x A irradiated

B

A x B irradiated

A uncoloured

C

A x B~ + PHA

D

SV2 1:2.5

E

SV2 + IFNγ 1:2.5 SV2 + IFNγ 1:5 SV2 + IFNγ 1:10

F

PV2 1:2.5

G

PV2 + IFNγ 1:2.5 PV2 + IFNγ 1:5 PV2 + IFNγ 1:10

SV2 1:5 PV2 1:5

SV2 1:10 PV2 1:10

H Day 8  Add all wells from one group to a tube  Add 2 mL of FACSFlow  Centrifuge for 5 minutes at 2000 rpm  Prepare antibody mix: – To make mix (for 32 tubes), add: CD3-PerCP 160 μL CD4-Pacific Blue 32 μL CD8-APC 3,2 μL CD19-PE-Cy7 8 μL FACSFlow 2996,8 μL  Pour off supernatant and add 100 μL of the mix  Incubate for 30 minutes in the dark at 4 °C  Add 2 mL of FACSFlow and centrifuge for 5 minutes at 2000 rpm  Pour off supernatant and add 100 μL of FACSFlow  Analyse using the flow cytometer

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Protocol 7: Isolation of PBMC from spleen tissue  Get the spleen from the refrigerator on 11Z OK thorax  Pour some hot DNase to a petri-dish, lay here the part of the spleen  In case of a large piece of the spleen, rip the capsule with two tweezers  Pipet some DNase medium into one of the spleen pots and put a strainer on top  Pound small pieces of the spleen through the strainer  Wash the strainer occasionally with DNase medium  Put a strainer on the second spleen pot  Pipet the mixture of spleenpot 1 through the strainer of spleenpot 2  Flush the remains, except the capsules. The volume in the spleenpot must be less than a half.  Put the strainers and tweezers in soap. Flush them later with water, put them after drying in autoclave packets for sterilisation  Pipet the supernatant from the cell suspension through the 70 µm Falcon strainer in a 50 mL tube. After that, the cell suspension must be pipetted on the strainer.  Put the Ficoll above the cell suspension. (15 mL Ficoll, 25-30 mL cell suspension)  Centrifuge for 20 minutes at 2000 rpm brake 0  Remove the Ficoll till ± 10 mL of the interphase  Pipet the interphase with the ± 10 ml in another 50 mL tube (1 tube per gradient)  Supplement the tube with DNase medium and centrifuge for 7 minutes at 2200 rpm brake 9  Wash for another 2 times with DNase medium and centrifuge for 5 minutes at 2000 rpm brake 9  Put the pellet in a 10 mL medium solution (DNase medium + 10% FBS-HI) and count the cells with CASY  Cells must be in a concentration of 20 × 106 cells per mL medium (DNase + 10% FBS HI)  Dilute the cell suspension with freeze medium and fill the whole concentration in freeze ampoules

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APPENDICES Appendix 1 – Flow cytometry markers The following table contains an overview of the markers we looked at during flow cytometry of the MSC. name aminopeptidase N

cell types MSC MSC + IFNγ CD13 mono, DC, endo, + + epi, fibro CD31 platelet endothelial cell mono, T, B, NK, − − adhesion molecule (PECAM-1) plat, endo CD45 leukocyte common antigen (LCA) leuko − − CD73 ecto-5’-nucleotidase DC, T, B, endo, + + epi, neuro, MSC CD90 Thy-1 HSC, fibro, + + neuro, MSC CD105 endoglin mono, erythro, + + endo, MSC CD271 p75NTR B, neuro, + + melano PD-L1 Programmed death-ligand 1 DC, T, B, NK, epi − + (CD274) ORB syndecan-2 (CD362) mono, endo, epi − + HLA-I Human Leukocyte Antigen ABC nucleated cells − + HLA-II Human Leukocyte Antigen DR APC − +  table 3 – The presence of the markers we analysed with flow cytometry on different cell types. mono = monocyte, DC = dendritic cell, T = T-cell, B = B-cell, NK = natural killer cell, plat = platelet, leuko = leukocyte, erythro = erythrocyte, endo = endothelial cell, epi = epithelial cell, neuro = neuronal cell, melano = melanocyte, APC = antigen presenting cell.

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Appendix 2 – Statistical analyses For analysis of L-kynurenine concentration in different MSC samples, variances and means were analysed with consecutive tests. Although variances in mathematics are considered to be the same for similar objects, we proved it statistically. By comparing samples of PV and SV, donor A and donor B a two-tailed student’s test for equal variances was used. Only for samples of PV174+ and SV117+ student’s test for unequal variances was used. S-pooled was used to calculate effect size (ES).

For these analyses, the database names of the samples were used. SV117 is referred to in the report as SV1, SV150 as SV2, PV174 as PV1 and PV206 as PV2. A ‘+’ after the name indicates IFNγ stimulation.

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