DIFFERENTIATION AND DERIVATION OF LINEAGECOMMITTED CHONDROPROGENITORS AND CHONDROGENIC CELLS FROM HUMAN EMBRYONIC STEM CELLS FOR CARTILAGE TISSUE ENGINEERING AND REGENERATION TOH WEI SEONG (M Sc National University of Singapore, Singapore) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF ORAL AND MAXILLOFACIAL SURGERY NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENTS I am most grateful to my supervisors: Associate Professor Cao Tong, Vice-Dean (Research), Department of Oral & Maxillofacial Surgery, Faculty of Dentistry, National University of Singapore, and Professor Lee Eng Hin, Director of Graduate Medical Studies, Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore for their countless encouragement, guidance, assistance and patience during my PhD program I would also like to express my heartfelt gratitude to Dr Andre Choo, Senior Research Scientist, Stem Cell Group, Bioprocessing Technology Institute, A*STAR, for his guidance and critical discussion of my work, as well as Dr Guo Xi-min, Research Scientist, Department of Tissue Engineering & Regenerative Medicine, Beijing Institute of Basic Medical Sciences for his help in animal transplantation studies Last but not least, I would also like to express my sincere thanks to Assistant Professor Jerry Chan, Experimental Fetal Medicine Group, Yong Loo Lin School of Medicine, National University of Singapore, for his advice on human chimerism studies and assistance in manuscript preparation Thanks to all my trusted colleagues in Stem Cell Lab, Department of Oral & Maxillofacial Surgery- Liu Hua, Fu Xin, Lu Kai, Li Mingming and Vinoth Kumar S/O Jayaseelan for their support and endless concern throughout the course of my work Not forgetting to mention my colleagues and friends in NUS Tissue Engineering Program- Hossein Nejadnik, Yeow Chen Hua, See Kwee Hua, Angela Tan Hwee San, i Afizah Hassan and Julee Chan Without them, my research would not have been so enjoyable Last but not least, I am grateful to my families, my parents and parent-in-laws, my wife Saw Tzuen Yih for their understanding, patience and great support during the years of my PhD pursuit ii TABLE OF CONTENTS ACKNOWLEDGEMENTS……………………………………………………… i TABLE OF CONTENTS………………………………………………… iii SUMMARY……………………………………………………………………… xi LIST OF TABLES…………………………………………………………………xiv LIST OF FIGURES……………………………………………………………… xv LIST OF ABBREVIATIONS…………………………………………………… xvii CHAPTER 1 INTRODUCTION…………………………………………………………1 1.1 Objectives………………………………………………………………… CHAPTER 2 LITERATURE REVIEW…………………………………………………4 2.1 Articular cartilage and its associated clinical problems…………………… 2.2 Human Embryonic Stem Cells (hESCs)…………………………………… 2.2.1 2.3 Expansion of hESCs……………………………………………… Differentiation of hESCs into chondrogenic lineage……………………… 10 2.3.1 Direct chondrogenic differentiation with EB formation…………… 11 2.3.1.1 Growth factor induction…………………………………………… 12 2.3.1.2 Co-culture………………………………………………………… 13 2.3.1.3 Challenges in the EB differentiation system……………………… 14 iii 2.3.2 Direct chondrogenic differentiation without EB formation……… 15 2.3.2.1 Growth factor induction…………………………………………… 15 2.3.2.2 Genetic manipulation……………………………………………… 15 2.3.2.3 Co-culture and conditioned medium……………………………… 16 2.3.3 Indirect differentiation………………………………………….……18 2.3.3.1 Chondrogenic differentiation of hESC-derived MSCs…………… 18 2.3.3.2 Chondrogenic differentiation of hESC-derived mesenchymal cells 19 2.3.4 Biomaterial-assisted chondrogenic differentiation………………… 21 2.3.4.1 Cartilage tissue engineering using hydrogels……………………… 21 2.3.4.2 Cartilage tissue engineering using polymeric scaffolds…………… 23 2.4 Cartilage formation and regeneration using ESCs………………………… 24 2.4.1 2.4.2 Delivery strategy and biomaterial choice………………………… 26 2.4.3 2.5 Homogeneity and differentiation of ESCs………………………… 25 Site of transplantation and host cell interference………………… 28 Animal models………………………………………………………………31 CHAPTER 3 MATERIALS AND METHODS………………………………………… 33 3.1 Reagents, chemicals, culture media and labware consumables…………… 33 3.2 Experimental design……………………………………………………… 33 3.3 Cell differentiation………………………………………………………… 34 3.3.1 Culture of hESCs………………………………………………… 34 3.3.2 Chondrogenic differentiation via embryoid body outgrowth culture.35 iv 3.3.3 Chondrogenic differentiation via high-density micromass culture… 35 3.3.4 Isolation and expansion of hESC-derived chondrogenic cells…… 37 3.3.4.1 Culture of hESC-derived chondrogenic cells on various ECM substratum 38 3.3.5 3.3.6 3.4 In vitro cartilage-like tissue formation …………………………… 39 Multi-lineage differentiation analysis……………………………….40 Cellular assays and cytogenetics…………………………………………… 41 3.4.1 3.4.2 Cell cycle analysis………………………………………………… 41 3.4.3 Surface marker analysis…………………………………………… 42 3.4.4 3.5 Growth kinetics…………………………………………………… 41 Multi-color fluorescence in situ hybridization (mFISH)………… 43 Molecular biology assays………………………………………………… 43 3.5.1 3.5.2 3.6 Total RNA extraction and cDNA synthesis……………………… 43 RT-PCR and real-time PCR quantitative analysis………………… 44 Biochemical assays………………………………………………………… 46 3.6.1 Sulfated glycosaminoglycan quantification……………………… 46 3.6.2 Collagen quantification…………………………………………… 48 3.6.3 Collagen II quantification………………………………………… 48 3.6.3.1 Sample preparation………………………………………………… 49 3.6.3.2 Collagen II ELISA………………………………………………… 49 3.6.4 3.6.5 3.7 DNA quantification………………………………………………… 50 Alkaline phosphatase (ALP) activity assay……………………… 50 Histochemical and fluorescence staining techniques……………………… 51 v 3.7.1 Staining of cell cultures…………………………………………… 51 3.7.1.1 Alcian blue staining……………………………………………… 51 3.7.1.2 Alkaline phosphatase (ALP) staining……………………………… 51 3.7.1.3 Alizarin red S staining…………………………………………… 52 3.7.1.4 Oil red-O staining………………………………………………… 52 3.7.1.5 Immunofluorescence (IF) staining………………………………… 53 3.7.2 Staining of tissue specimens……………………………………… 54 3.7.2.1 Processing of tissue specimens…………………………………… 54 3.7.2.2 Haematoxylin and eosin staining………………………………… 55 3.7.2.3 Alcian blue staining……………………………………………… 56 3.7.2.4 Safranin-O staining………………………………………………… 56 3.7.2.5 Masson’s trichrome staining……………………………………… 57 3.7.2.6 Immunohistochemical staining…………………………………… 57 3.8 Animal studies…………………………………………………………… 59 3.8.1 3.8.2 Osteochondral defect model……………………………………… 60 3.8.3 3.8.4 Post-operative procedures………………………………………… 61 A Micro-computational tomography (micro-CT)…………………… 62 3.8.5 3.9 In vivo implantation assay………………………………………… 59 Human cell chimerism…………………………………………… 62 Statistical analysis………………………………………………………… 63 CHAPTER 4 RESULTS………………………………………………………………… 65 vi 4.1 PHASE I: MODEL SYSTEM…………………………………………… 65 4.1.1 Pluripotency of human embryonic stem cells……………………… 65 4.1.2 Effects of culture conditions in modulation of chondrogenesis…….66 4.1.3 Effects of culture conditions on hypertrophic development……… 71 4.1.4 Modulation of chondrogenesis in different EB seeding densities… 74 4.1.5 Effects of culture conditions in lineage selection during chondrogenesis…………………………………………………… 75 4.2 PHASE II: GROWTH FACTOR MODULATION……………………… 76 4.2.1 4.2.2 Growth factor modulation of matrix synthesis…………………… 78 4.2.3 Growth factor modulation of chondrogenic commitment………… 81 4.2.4 4.3 Growth factor modulation of chondrogenesis………………………76 TGFβ1 induction of chondrogenic cells…………………………… 83 PHASE III: ISOLATION OF CHONDROGENIC CELLS……………… 86 4.3.1 4.3.2 Expansion of hESC-derived chondrogenic cells……………………88 4.3.3 Differentiation capability of hESC-derived chondrogenic cells…… 88 4.3.4 Characterization of hESC-derived chondrogenic cell line (TC1)… 90 4.3.5 4.4 Derivation of hESC-derived chondrogenic cells………………… 86 ECM modulation of hESC-derived chondrogenic cells…………….96 PHASE IV: FUNCTIONALITY………………………………………… 98 4.4.1 Cartilage tissue engineering using hESC-derived chondrogenic cells………………………………………………………………… 98 4.4.1.1 Optimal growth factor induction for cartilage tissue engineering… 98 vii 4.4.1.2 Effects of 3D HA hydrogel encapsulation in cartilaginous tissue development……………………………………………………… 98 4.4.1.3 Human ESC-derived chondrogenic cell-engineered cartilage (HCCEC)………………………………………………………… 100 4.4.2 Cartilage regeneration in osteochondral defect…………………… 103 4.4.2.1 Comparison of hESC-derived chondrogenic cells and HCCEC in cartilage repair…………………………………………………… 103 4.4.2.2 HCCEC in cartilage regeneration………………………………… 106 4.4.2.3 Human cell chimerism…………………………………………… 116 4.4.3 Phenotypic stability of hESC-derived chondrogenic cells………….119 CHAPTER 5 DISCUSSION…………………………………………………………… 121 5.1 PHASE I: MODEL SYSTEM…………………………………………… 121 5.1.1 Human ESCs as a model system to study chondrogenesis………… 121 5.1.1.1 Chondrogenic differentiation in EB outgrowth…………………… 121 5.1.1.2 Effects of high-density microenvironment on chondrogenic differentiation……………………………………………………… 123 5.1.1.3 Effects of high-density microenvironment on hypertrophic maturation………………………………………………………… 125 5.1.1.4 Effects of higher EB seeding numbers on chondrogenic differentiation……………………………………………………… 126 viii 5.1.1.5 Effects of high-density microenvironment on other lineage differentiation………………………………………………… 127 5.2 PHASE II: GROWTH FACTOR MODULATION……………………… 129 5.2.1 5.2.2 Effects of TGFβ1 on other lineage differentiation………………… 131 5.2.3 5.3 Growth factor modulation of chondrogenesis………………………129 Pluripotency vs chondrogenesis - Role of TGFβ1………………… 131 PHASE III: ISOLATION OF CHONDROGENIC CELLS……………… 133 5.3.1 Human ESC-derived chondrogenic cells………………………… 133 5.3.1.1 Effects of growth factors and ECM on hESC-derived chondrogenic cells………………………………………………………………… 133 5.4 PHASE IV: FUNCTIONALITY 136 5.4.1 Human ESC-derived chondrogenic cells in cartilage tissue engineering………………………………………………………… 136 5.4.1.1 Human ESC-derived chondrogenic cell-engineered cartilage 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Joint Surg Am 80(12): 1745-1757 Zhang ZY, Teoh SH, Chong MS, Lee ES, Tan LG, Mattar CN, Fisk NM, Choolani M, Chan J (2010) Neo-vascularization and bone formation mediated by fetal mesenchymal stem cell tissueengineered bone grafts in critical-size femoral defects Biomaterials 31(4):608-620 zur Nieden NI, Kempka G, Rancourt DE, Ahr HJ (2005) Induction of chondro-, osteo- and adipogenesis in embryonic stem cells by bone morphogenetic protein-2: effect of cofactors on differentiating lineages BMC Dev Biol 5: 1-15 167 PUBLICATIONS Some data and protocols of this thesis have been published in original research articles, book chapters, reviews and conferences International Referred Publications Toh WS, Lee EH, Guo XM, Chan JKY, Yeow CH, Choo AB, Cao T (2010) Cartilage repair using hyaluronan hydrogel-encapsulated human embryonic stem cell-derived chondrogenic cells Biomaterials (Accepted in press) Yang Z, Sui L, Toh WS, Lee EH, Cao T (2009) Stage-Dependent Effect of TGFβ1 on Chondrogenic Differentiation of Human Embryonic Stem Cells Stem Cells & Development 18(6) 929-940 Toh WS, Guo XM, Choo AB, Lu K, Lee EH, Cao T (2009) Differentiation and Enrichment of expandable chondrogenic cells from Human Embryonic Stem Cells in Vitro Journal of Cellular and Molecular Medicine 13(9B): 3570-3590 Toh WS, Yang Z, Liu H, Heng BC, Lee EH, Cao T (2007) Effects of BMP2 and culture conditions on the extent of chondrogenesis from human embryonic stem cells Stem Cells 25(4): 950-960 Book Chapters Toh WS, Lee EH, Richards M, Cao T (2010) ‘In Vitro Derivation of Chondrogenic cells from Human Embryonic Stem Cells’ Methods in Molecular Biology, vol 584: Human Embryonic Stem Cell Protocols 2nd Edition pp 317-331 Humana Press Toh WS, Yang Z, Heng BC, Cao T (2007) ‘Differentiation of Human Embryonic Stem Cells towards the Chondrogenic Lineage’ Methods in Molecular Biology, vol 407: Stem Cell Assays pp 333-349 Humana Press Reviews Toh WS, Yang Z, BC Heng, T Cao (2006) New perspectives in chondrogenic differentiation of stem cells for cartilage repair ScientificWorldJournal 6: 361-364 (Invited review) 168 Presentations and Conferences Toh WS, Guo XM, Choo AB, Lu K, Liu H, Lee EH, Cao T Human embryonic stem cell-derived chondrogenic cells: potential for regenerative dentistry 2nd Meeting of IADR Pan Asian Pacific Federation (PAPF) and the 1st Meeting of IADR Asia/Pacific Region (APR) 2009, Wuhan, China Toh WS, Guo XM, Choo AB, Lu K, Liu H, Lee EH, Cao T Efficient generation of expandable chondrogenic cells from human embryonic stem cells for cartilage tissue engineering 7th Annual Meeting of the International Society for Stem cell Research (2009), Barcelona, Spain Toh WS, Lee EH, Choo AB, Cao T Differentiation and derivation of chondrogenic cells from human embryonic stem cells Workshop on Engineering Surfaces for Regulating Cell Behaviour (28th Feb 2008) Discovery Theatrette, Biopolis, Singapore Yang Z, Toh WS, Heng BC, Liu H, Lee EH, Cao T Different culture conditions affect the extent of human embryonic stem cell chondrogenesis 4th Annual Meeting of the International Society for Stem cell Research (2006), Toronto, Ontario, Canada 169 ... plating of hESC-derived chondrogenic cells Fig 17 Chondrogenic differentiation capability of hESC-derived chondrogenic cells Fig 18 Analysis of pluripotency and lineage- restriction of hESC-derived chondrogenic. .. explored for their potential as viable cell sources for cartilage tissue engineering (Chung et al., 2008) Human embryonic stem cells (hESCs) are stem cell lines of embryonic origin, isolated from. .. biomaterial and biophysical stimulations have been employed to harness the chondrogenic potential of hESCs for cartilage regeneration and tissue engineering 2.2.1 Expansion of hESCs Human embryonic stem