Factors Influencing Propagation Of Cultured Cord Blood-Derived Mesenchymal Stem Cells

Transcription

1 Factors Influencing Propagation Of Cultured Cord Blood-Derived Mesenchymal Stem Cells Thesis Submitted for Fulfillment of the Master Degree in Clinical & Chemical Pathology By Dina Samir Abd El-Hady M.B.B.CH Supervisors Prof. Dr. Taghreed Mohamed Gaafar Professor of Clinical &Chemical Pathology, Faculty of Medicine,Cairo University Prof. Dr. Zeinab Abou Bakr Demerdash Professor of Immunology, Theodor Bilharz Research Institute Faculty of Medicine Cairo University 2012

2 ACKNOWLEDGEMENT First of all, Thanks to GOD who helped me to achieve and complete this work. I would like to express my deepest appreciation and gratitude to Prof. Dr. Taghreed Mohamed Gafar, Professor of Clinical Pathology, Cairo University, for her great efforts, guidance and support throughout the work. I am greatly honored to express my sincere gratitude and faithful appreciation to Prof. Dr. Zeinab Abou Bakr Demerdash Professor of Immunology, Head of immunology lab, Theodor Bilharz Research Institute who kindly directed me, pushed me forward and encouraged me to do the best, I would like to thank her for her great guidance, supervision and generous help. I would like to extend my thanks and gratefulness to Prof. Dr. Zeinab Shaker, previous head of Immunology department, Theodor Bilharz Research Institute for her great advice and perfect guidance. I would like to thank Prof. Dr. Hanan Gamal El-Baz, Professor of Immunology, Head of immunology and Therapeutic Evaluation Division, Theodor Bilharz Research Institute, for her kind care and continuous encouragement I am sincerely grateful to Prof. Dr. Salwa Hassan & Prof. Dr. Faten Salah Professors of Immunology, Theodor Bilharz Research Institute who kindly helped me to fulfill this work, I would like to thank them for their great efforts, perfect guidance and faithful advice. I would like to thank Dr. Tamer Fouad Taha, Assistant Professor of reproductive biology department in National Research Center for his great efforts, cooperation, and for the facilities he offered for initiation of this work. I would like to thank Dr. Hoda Abou Taleb, Lecturer of Environmental Science, Environmental Research Department, Theodor Bilharz Research Institute, for her generous help, continuous support and perfect assistance.

3 A lot of Thanks to the staff of immunology department and to my dear colleagues especially Dr. Ali Bayioumi, Dr. Shimaa Atteya, and Dr. Marwa Hassan in Theodor Bilharz Research Institute for their great support, cooperation, and generous help. I would like to dedicate this work to the soul of Prof. Dr. Azza El Bassiouny, Professor of Immunology, Immunology Department, Theodor Bilharz Research Institute. A very special dedication to my dear parents, beloved husband and lovely daughter who helped me and allowed me to complete this work. Dina Samir Abd El Hady 2012

4 ABSTRACT Background Mesenchymal stem cells (MSCs) are considered to be a promising source of stem cells in the field of regenerative therapy. They are multipotent population of cells capable of differentiating into multiple cell types including adipocytes, chondrocytes, and osteocytes. They have the ability to adhere to plastic surface displaying spindle shaped morphology. They exhibit positive expression of CD73, CD90 and CD105 with a concomitant absence of CD34, CD45 and HLA-DR expression. Bone marrow (BM) is the most frequent source of MSCs however BM aspiration is an invasive painful procedure. Umbilical cord (UC) is a readily accessible, more consistent and richer source of MSC than BM. For optimization of in vitro expansion of MSCs for cell therapy approaches, several parameters were tried including culture media, growth factors and plating density. Culture media of MSCs are traditionally supplemented with fetal bovine serum (FBS) however there is a growing interest to avoid the use of FBS due to potential xenogeneic immune reactions and a high lot-to-lot variability. Serum free media provides robust expansion of cells especially when supplemented with growth factors that act synergistically. Low plating density results in higher yield and faster expansion of cells furthermore low seeding density preserves the proliferative capacity and the stemness of MSCs. Aim of the work Determination of the factors driving CB MSCs population to expand sufficiently to generate cell number suitable for clinical application. Materials and Methods MSCs were generated from 2 cord blood samples, the mononuclear cells (MNC) were isolated by Ficoll according to density gradient. MSCs were cultured in Dulbecco's modified Eagle's medium (DMEM) and allowed to expand. Immunophenotyping of MSCs was done by flowcytometry. Differentiation potential of MSCs was estimated by osteogenic differentiation. MSCs at P3 were retrieved from the liquid nitrogen, allowed to reach confluency then they were harvested and cultured in 3 different media (DMEM/ 30%FBS, DMEM/ 2%FBS, EGM-2 and Mesencult) with 3 different seeding densities (5, 50, 500 cells/ cm 2 ), Population doubling (PD) was calculated, statistical analysis was performed comparing between the 3 different media and the 3 different seeding densities.

5 Abstract Results MSCs generated from 2 cord blood samples displayed spindle shaped morphology forming a homogenous monolayer of cells after 2 weeks. They were expanded up to 10 and 12 passages respectively with gradual decrease of proliferative potential at late passages. MSCs from P3 and P8 displayed similar immunophenotypic markers, they displayed positive expression of CD90, CD105, CD73 and negative expression of CD45, CD34 and HLA-DR. Osteogenic differentiation at P3 was detected by Alizarin red stain showing red area of bone mineralization. MSCs from P3 were harvested after reaching confluency then they were seeded in 3 different culture media in 3 different seeding density. MSCs cultured in DMEM/ 2% FBS supplemented with EGF and FGF-4 didn't achieve confluency at any of the 3 different concentrations, MSCs cultured in DMEM/ 30% FBS and in EGM-2 acquired higher PD at concentration 5 than concentration 50 and 500 (p< 0.05 & p< 0.01 respectively) while MSCs cultured in Mesencult acquired the highest PD at concentration 50. DMEM grew with higher PD than EGM-2 and Mesencult when plated at the 3 different concentrations (5, 50, 500 cells/ cm 2 ). There was no change in cell morphology among MSCs cultured in the 3 different media. Conclusion From this study we concluded that MSCs lose their proliferative potential gradually with late passages. Immunophenotyping of MSCs showed no marked differences of markers expression between early and late passages. For optimal culture conditions, the conventional serum complete medium (DMEM/ 30%FBS) is the media of choice regarding proliferation potential and growth kinetics when compared with EGM-2 (2%FBS) and Mesencult (serum-free, xeno-free medium). Concentration 5 cells/ cm 2 is the optimal seeding density that allows for in vitro expansion of MSCs with more folds increase than concentration 50 & 500 cells/ cm 2 to be used in therapeutic approaches. Key words Mesenchymal stem cells, Bone marrow, Umbilical cord, Fetal bovine serum, Serum complete media, Population doubling.

6 TABLE OF CONTENTs Page INTRODUCTION AND AIM OF THE WORK. 1 REVIEW OF LITERATURE.. 3 AN OVERVIEW OF STEM CELLS.. 3 Definition 3 Properties of Stem Cells 3 Classification of Stem Cells.. 4 A- Classification according to stem cell potency. 4 1-Toitipotent stem cells Pluripotent stem cells Multipotent stem cells. 5 4-Oligopotent stem cells Unipotent stem cells. 5 B- Classification according to source of stem cells. 7 1-Embryonic stem cells 7 2-Fetal and amniotic stem cells 8 3-Umbilical cord stem cells. 8 4-Adult stem cells. 9 C- Classification according to the type of stem cells Hematopoietic stem cells Mesenchymal stem cells Endothelial progenitor cells Multipotent Adult Progenitor Cells Unrestricted somatic stem cells 14 Relation between USSCs and MSCs 14 6-Induced pluripotent stem cells.. 15 Somatic cell nuclear transfer, cellular fusion, and exposure to cellular extract 16 Stem Cell Plasticity. 16 Stem Cell Lines. 19 Therapeutic Applications of Stem Cells. 20 MESENCHYMAL STEM CELLS.. 23 Definition 23 Sources of MSCs General Characteristics of MSCs 24 Heterogeneous Population within MSCs Preparations 25 Umbilical Cord Derived Mesenchymal Stem Cells Versus Bone Marrow Mesenchymal Stem Cells The Human Umbilical Cord 26 Circulation and Niches of MSCs.. 27 Characterization of MSCs Phenotypic Heterogeneity of MSCs 29 Homing and Engraftment of Transplanted MSCs 30 Immune Regulation by MSCs. 30 Immunomodulatory Properties of MSCs 31 Immunomodulatory effect of mesenchymal stem

7 cells in innate immunity 32 Immunomodulatory effect of mesenchymal stem cells in adaptive immunity Growth and Expansion of MSCs.. 34 Differentiation Potential of MSCs 34 In vitro Senescence of MSCs.. 37 Therapeutic Potential and Clinical Applications of MSCs 37 ENHANCING MSC PROLIFERATION 42 Introduction.. 42 Factors Influencing Proliferation of MSCs Influence of Cell Seeding Density on MSC Proliferation 44 Influence of Culture Media & Growth Factors on MSC Proliferation 47 MATERIALS AND METHODS.. 54 RESULTS. 64 DISCUSSION 99 SUMMARY AND CONCLUSIONS 111 RECOMMENDATION REFERENCES Arabic Summary

8 List of Figures Figure No: page Figure (1): Potency of stem cells. 6 Figure (2): Scheme of the hierarchical origin of MSCs Figure (3): Hematopoietic and stromal stem cell differentiation Figure (4): Stem cell division and differentiation. 19 Figure (5): Potential uses of stem cells. 22 Figure (6): Cross section of an umbilical cord. 28 Figure (7): MSCs mesenchymal differentiation in vitro. Figure (8): Morphological difference between low density and high density cultures of MSCs Figure (9): CB-MSC after 10 days of culture. 65 Figure (10): CB-MSC after 14 days of culture. 66 Figure (11): MSCs of the 12th passage showing senescence. Figure (12): Flow cytometry analysis of MSCs expression markers at (P3). Figure (13): Flow cytometry analysis of MSCs expression markers at (P8). Figure (14): Control culture of MSCs before Alizarin red staining. Figure (15): Osteogenic differentiation of MSCs before Alizarin red staining

9 Figure (16): Control culture of MSCs stain negative for Alizarin red stain Figure (17): Osteogenic differentiation of MSCs after Alizarin red staining Figure (18): Spindle shape of MSCs. 82 Figure (19): Flat monolayer of MSCs. 83 Figure (20): MSCs at 500 cells/ cm 2 in 2% FBS/ DMEM supplemented with FGF-4 & EGF. Figure (21): PD of MSCs cultured at 5 cells density/ cm 2 in different culture media Figure (22): PD of MSCs cultured at 50 cells density/ cm 2 in different culture media Figure (23): PD of MSCs cultured at 500 cells density/ cm 2 in different culture media

10 List of Tables Table NO Table (1): The difference between MSCs cultured at low density level and those cultured at high density. Table (2): Expansion profile of USSCs isolated from CB sample 1. Table (3): Expansion profile of MSCs isolated from CB sample 2. Table (4): Percentage of immunophenotype markers expression of CB MSCs. Page Table (5): PD and culture days of MSCs cultured in 30% FBS/ DMEM at 5, 50, and 500 cells/ cm 2. Table (6): PD and culture days of MSCs cultured in EGM at 5, 50, and 500 cells/ cm 2. Table (7): PD and culture days of MSCs cultured in Mesencult at 5, 50, and 500 cells/ cm 2. Table (8): MSCs cultured at 5 cells/ cm2 in different culture media. Table (9): MSCs cultured at 50 cells /cm2 in different culture media. Table (10): MSCs cultured at 500 cells /cm2 in different culture media. Table (11): Hypothetical expansion potential of 10 6 MSCs. cultured in 30% FBS/ DMEM at a density of 5 cells/ cm 2, 50 cells/ cm 2, and 500 cells/ cm 2 using T25 cm 2 flasks

11 LIST OF ABBREVIATIONS AFSC AKI Ang APCs BDGF b-fgf BM BMMSCs BMPs CB CCR CD CFU-F CLI CTL DC DKK DLK-1/ PREF DMEM DMSO D.W. ECM EF EGF EGM EPCs ES FBS FGF FITC Frzb-1 GA-1000 GMP GVHD HA HAS HBsAg HCVAb HGF HIVAb HLA hmscs HPL HSCs Amniotic fluid stem cell Acute kidney injury Angiomotin Antigen presenting cells Brain-derived growth factor Basic fibroblast growth factors Bone marrow Bone marrow Mesenchymal stem cells Bone morphogenetic proteins Cord blood Chemokine receptor Cluster of differentiation Colony forming unit fibroblast Critical limb ischemia Cytotoxic T lymphocyte Dendritic cell Dickkopf-related protein Delta-like preadipocyte factor Dulbecco's modified Eagle's medium Dimethyl sulfoxide Distilled water Extracellular matrix Ejection fraction Epidermal growth factor Endothelial growth medium Endothelial progenitor cells Embryonic stem cell Fetal bovine serum Fibroblast growth factor Fluorescein isothiocyanate Frizzled gene Gentamicin and amphotericin-b Good manufacturing practice Graft versus host disease Hyaluronic acid Human autologous serum Hepatitis B surface antigen Hepatitis C virus antibodies Hepatocyte growth factor Human immunodeficiency virus antibodies Human leukocyte antigen Human mesenchymal stem cells Human platelet lysate Hematopoietic stem cells

12 huc-mscs Human umbilical cord mesenchymal stem cells hucbs Human umbilical cord blood serum IBMX Isobutyl methyl xanthine ICAM Intercellular adhesion molecule IDO Indoleamine 2,3- dioxygenase IFN Interferon IGF Insulin-like growth factor IL Interleukin ips Induced pluripotent stem cell ISCT International society for cellular therapy KLF Kruppel-like factor LIF Leukemia inhibitory factor MACS Magnetic activated cell sorting MAPC Multipotent adult progenitor cell MCP Monocyte chemoattractant protein MET Mesenchymal-to-epithelial transition MHC Major histocompatibility complex MLR Mixed lymphocyte reaction MNCs Mononuclear cells MPB Mobilized peripheral blood MSCs Mesenchymal stem cells NK Natural killer cells NKT Natural killer T cells NO Nitric oxide OCT 3/4 Octamer binding transcription factor 3/4 P Passage PAD Peripheral arterial disease PD Population doubling PD-1 Programmed cell death protein-1 PDEGF Platelet derived epidermal growth factor PDGF Platelet-derived growth factor PDT Population doubling time PE Phycoerthrin PGE-2 Prostaglandin E2 PTHrP Parathyroid hormone related peptide ROS Reactive oxygen species RS-cell Rapidly self-renewing cell SCF Stem cell factor SCs Stem cells SFM Serum free media SFRP1 Secreted frizzled-related protein 1 shla-g Soluble human leukocyte antigen G SLE Systemic lupus erythematosis SOX SRY related high-mobility group box protein SSEA Stage specific embryonic antigen TGF-β Transforming growth factor-beta TNF- Tumor necrosis factor alfa Treg Regulatory T cell

13 TSG-6 UC UCB UC-MSCs USSCs VCAM VEGF WJ XF Tumor necrosis factor-inducible gene 6 protein Umbilical cord Umbilical cord blood Umbilical cord Mesenchymal stem cells Unrestricted somatic stem cells Vascular cell adhesion molecule Vascular endothelial growth factor Wharton jelly Xeno-free

14 INTRODUCTION AND AIM OF THE WORK Mesenchymal stem cells (MSCs) are defined by the International Society for Cellular Therapy (ISCT) as multipotent stromal cells, also referred to as connective tissue progenitor cells (Dominici et al. 2006). Mesenchymal stem cells (MSCs) were first reported by Fridenstein et al. (1976). The interest in MSCs rapidly grew with expanding knowledge about their exceptional characteristics and usefulness in the clinic (Reiser et al. 2005; Kallis et al. 2007; Le Blanc et al. 2008; Siniscalco et al. 2008). MSCs are multipotent stem cells that are able to differentiate into different lineages including mesodermal, ectodermal, and endodermal type cells (Kopen et al. 1999; Pittenger et al. 1999; Sato et al. 2005; Caplan 2009). Phenotypically, MSCs exhibit the expression of CD90, CD105, STRO-1, CD73 and CD44, with a concomitant absence of CD14, CD19, CD34, CD45 and HLA-DR expression (Dominici et al. 2006). MSCs can be easily isolated by their ability to adhere to plastic generating single cell-derived colonies (Castro-Malaspina et al. 1980; Colter et al. 2001) that can be expanded to obtain high numbers of cells for clinical use in cell and gene therapy for a number of human diseases (Caplan 2009). The main source of MSCs is bone marrow. They constitute, however, only a small percentage of the total number of bone marrow cells. Although bone marrow is considered the gold standard for MSCs isolation, it is a painful invasive procedure. Therefore the use of umbilical cord for MSCs isolation is encouraged since it is a readily accessible source of MSCs; moreover the human UC-MSCs have higher number and stronger proliferation ability, which make it possible to obtain substantial number of cells required for clinical transplantation in a short time (Petsa et al. 2009). Identification of optimal culture conditions of in vitro cell culture expansion is highly needed. Low seeding density results in higher yields and a faster expansion of MSCs (Colter et al. 2001; Prockop et al. 2001; Sekiya et al. 2001; Sotiropoulou et al. 2006a) without losing differentiation potential. Page 1

15 Introduction and Aim of the work Furthermore low density culture system results in more purified MSCs due to the colonogenic property of MSCs (Eslaminejad and Nadri 2009). The use of serum complete media (DMEM) allows the expansion and propagation of MSCs since fetal bovine serum (FBS) and fetal calf serum (FCS) contain a lot of factors that stimulate cell growth (nutrients, growth factors, etc.) (Osipova et al. 2011), however, the use of FBS is controversial for a number of reasons: serum is subjected to batch-to-batch variations, the use of animal serum carries the risk of transmission of infectious agents as well as the possibility of immune rejection of the transplanted cells in response to bovine serum (van der Valk et al. 2004; Schiff 2005; Muller et al. 2006). Therefore there is a strong motivation to use serum-free media supplemented with growth factors e.g. endothelial growth media (EGM-2) and Mesencult (Tamama et al. 2010). The addition of growth factors e.g. FGF and EGF enhance the proliferation of MSCs and maintain their multipotentiality (Hutley et al. 2004). Their combination with each other provides a robust synergistic effect on MSCs expansion without losing differentiation potential (Ng et al. 2008). Aim of Work Determination of the factors driving CB MSCs population to expand sufficiently to generate cell number suitable for clinical application. Page 2

16 REVIEW OF LITERATURE AN OVERVIEW OF STEM CELLS Definition Stem cells are biological cells found in all multicellular organisms, that can divide (through mitosis) and differentiate into diverse specialized cell types (Harting 2011). Stem cells are defined as being self-renewing, pluri- or multi-potent, and clonogenic. Clonogenic cells are single stem cells that are able to generate a line of genetically identical cells thereby maintaining their self-renewal and differentiation potential (Allen et al. 2005; Roobrouck et al. 2008). Progenitor cells are cells that are direct descendants of stem cells, are less potent than stem cells, and have diminished capacity for self-renewal relative to stem cells, but retain the ability to become at least one, if not multiple, cell types (Harting 2011). Properties of Stem Cells Stem cells, regardless of their source, have three general properties; they are capable all of dividing and renewing themselves for long periods, they are unspecialized, and they can give rise to specialized cell types (Friedenstein et al. 1974a; 1976). Self-renewal is the ability to go through numerous cycles of cell division while maintaining the undifferentiated state. Stem cells are capable of dividing and renewing themselves for long periods. Unlike muscle cells, blood cells, or nerve cells, which do not normally replicate themselves, stem cells may replicate many times. When cells replicate themselves many times over it is called proliferation (Kumar and Bakrudeen 2009). A starting population of stem cells that proliferates for many months in the laboratory can yield millions of cells. If the resulting cells continue to be Page 3

17 Chapter 1 An Overview of Stem Cells unspecialized, like the parent stem cells, the cells are said to be capable of longterm self-renewal (Kumar and Bakrudeen 2009). One of the fundamental properties of a stem cell is that they do not have any tissue-specific structures that allow them to perform specialized functions. However, unspecialized stem cells can give rise to specialized cells, including heart muscle cells, blood cells, or nerve cells. When unspecialized stem cells give rise to specialized cells, the process is called differentiation (Harting 2011). Scientists are just beginning to understand the signals inside and outside cells that trigger stem cell differentiation, signals are controlled by cell genes. The internal signals are interspersed across long strands of DNA, and carry coded instructions for all the structures and functions of a cell. The external signals for cell differentiation include chemicals secreted by other cells, physical contact with neighboring cells, and certain molecules in the microenvironment (Saxena et al. 2010). Classification of Stem Cells A- Classification according to stem cell potency: Potency specifies the differentiation potential (the potential to differentiate into different cell types) of the stem cell (Schöler 2007). Embryonic stem cells are the most potent since they give rise to every type of cell in the body. According to their potency, stem cells are classified into (Fig.1): 1-Totipotent stem cells: The ability to differentiate into all possible cell types. Such cells can construct a complete, viable organism (Schöler 2007). These cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent (Mitalipov and Wolf 2009). Examples are the zygote formed at egg fertilization and the first few cells that result from the division of the zygote. Page 4

18 Chapter 1 An Overview of Stem Cells 2-Pluripotent stem cells: They are the descendants of totipotent cells and can differentiate into nearly all cells i.e. cells derived from any of the three germ layers; mesoderm, endoderm, and ectoderm (Ulloa-Montoya et al. 2005; Schöler 2007). 3-Multipotent stem cells: Stem cells can differentiate into a number of cells, but only those of a closely related family of cells (Schöler 2007). Examples include hematopoietic (adult) stem cells that can become red and white blood cells or platelets. 4-Oligopotent stem cells: stem cells can differentiate into only a few cells, such as lymphoid or myeloid stem cells (Schöler 2007). 5-Unipotent stem cells : Cells can produce only one cell type, their own but have the property of self-renewal, which distinguishes them from non-stem cells (e.g., muscle stem cells) (Schöler 2007). Page 5

19 Chapter 1 An Overview of Stem Cells Figure (1): Potency of stem cells. Pluripotent, embryonic stem cells originate as inner mass cells within a blastocyst. Only the morula's cells are totipotent, able to become all tissues and a placenta ( Page 6

20 Chapter 1 An Overview of Stem Cells B-Classification according to source of stem cells: 1-Embryonic stem cells Embryonic stem cells (ES), as their name suggests, are derived from embryos that develop from in vitro fertilized eggs, in an in vitro fertilization clinic, when a male's sperm fertilizes a female's ovum (egg) to form a single cell called a zygote and then donated for research purposes with informed consent of the donors. They are not derived from eggs fertilized in a woman's body. The single zygote cell then begins a series of divisions, forming 2, 4, 8, 16 cells, etc. After four to six days before implantation in the uterus, this mass of cells is called a blastocyst. The blastocyst consists of an inner cell mass (embryoblast) and an outer cell mass (trophoblast). The outer cell mass becomes part of the placenta and the inner cell mass is the group of cells that will differentiate to become all the structures of an adult organism (Graves and Moreadith 1993). This latter mass is the source of embryonic stem cells; totipotent cells (cells with total potential to develop into any cell in the body) (Boheler et al. 2002). ES cells, being pluripotent cells, require specific signals for correct differentiation. If injected directly into another body, ES cells will differentiate into many different types of cells, causing a teratoma (Cibelli et al. 2002). A human embryonic stem cell is defined by the presence of several transcription factors and cell surface proteins required for self-renewal and pluripotency including the transcription factors: Octamer binding transcription factor 4 (Oct4), Nanog, and SRY related high-mobility group box protein (SOX)2. (Boyer et al. 2005; Saxena et al. 2010). The cell surface antigens most commonly used to identify human ES cells are the glycolipids stage specific embryonic antigen (SSEA3) and (SSEA4) and the keratan sulfate antigens Tra-1-60 and Tra-1-81(Adewumi et al. 2007). Page 7