CHARACTERIZATION OF HUMAN ADIPOSE DERIVED ADULT MULTIPOTENT PRECURSOR CELLS LEONG TAI WEI DAVID NATIONAL UNIVERSITY OF SINGAPORE 2006 i CHARACTERIZATION OF HUMAN ADIPOSE DERIVED ADULT MULTIPOTENT PRECURSOR CELLS LEONG TAI WEI DAVID (Bachelor of Chemical Engineering, Hons) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2006 ii PREFACE This thesis is submitted for the degree of Doctorate of Philosophy in the Department of Biological Sciences at the National University of Singapore No part of this thesis has been submitted for any other degree or equivalent to another university or institution All the work in this thesis is original unless references are made to other works Parts of this thesis had been published or presented in the following: International Refereed Journal Publications Cover pages of some of the following papers are found in the appendix Leong TW, Chew FT, Hutmacher DW Isolating bone marrow stem cells using sieve technology Stem Cells 2004; 22:1123-1125 *Ng KW, *Leong DT, Hutmacher DW The challenge to measure cell proliferation in three dimensions Tissue Engineering 2005; 11:182-191 Leong DT, Hutmacher DW, Chew FT, Lim TC Viability and adipogenic potential of human adipose tissue processed cell population obtained from pump-assisted and syringe-assisted liposuction Journal of Dermatological Science J Dermatol Sci 2005 37:169-176 Leong DT, Khor WM, Chew FT, Lim TC, Hutmacher DW Characterization of osteogenically induced adipose tissue derived precursor cells in 2-dimensional and 3-Dimensional environments Cells Tissues Organs 2006; 182(1):1-11 Leong DT, Abraham MC, Rath SN, Lim T-C, Chew FT and Hutmacher DW Investigating the effects of preinduction on human adipose derived precursor cells in an athymic rat model Differentiation In press iii Leong DT et al Absolute quantification of gene expression with real time PCR Biomaterials 2007; 28(2):203-210 *Gupta A, *Leong DT, Bai HF, SB Singh, Chew FT, Hutmacher DW The combined action of 1α, 25–dihydroxyvitamin D3, β–glycerophosphate and ascorbic acid is essential for the osteo – maturation of adipose derived stem cells Manuscript under preparation Leong DT, Hutmacher DW, Chew FT Genome wide gene expression revealed activating transcription factor (ATF5) to be responsible for maintaining stemness in adipose derived stem cells Manuscript under prep * contributed equally Book Chapters Hutmacher DW, Leong DT, Chen Fulin Polysaccharides in Tissue Engineering Applications, Handbook of Carbohydrate Engineering In press Intellectual Competition Finalist in Tan Kah Kee Young Inventors Awards 04 Making bone from fat International and Local Conferences Presentations and Awards Poster Presentation Preliminary studies on human adipose derived stem cells obtained from pump- assisted liposuction 4th Sino-Singapore Conference on Biotechnology, National University of Singapore, Singapore, November 11-13th, 2003 Leong TW, Chew FT, Hutmacher DW, Lim TC Oral Presentation Preliminary studies on human adipose derived stem cells obtained from pump- assisted liposuction 2nd prize for 8th Biological Sciences Graduate Congress, National University of Singapore, iv Singapore, December 3-5th, 2003 Leong TW, Chew FT, Hutmacher DW, Lim TC Oral Presentation Osteogenesis of human adipose derived stem cells The Asian Surgical Association - 14th Biennial Congress & Scientific Meeting, Kota Kinabalu, Malaysia December 4-6 2003 Leong TW, Chew FT, Hutmacher DW, Lim TC Poster Presentation: Viability of human adipose tissue stem cells from pump- vs syringe-assisted liposuction The Asian Surgical Association 14th Biennial Congress & Scientific Meeting, Kota Kinabalu, Malaysia December 4-6 2003 Leong TW, Chew FT, Hutmacher DW, Lim TC Poster Presentation The behavior of human adipose tissue derived precursor scaffolds cells Tissue seeded on Engineering polycaprolactone-tricalcium Society International, phosphate th Annual International Conference and Exposition, Orlando Florida, United States of America December 10-13, 2003 Leong TW, Khor WM, Chew FT, Hutmacher DW Poster Presentation Osteoprogenitor Cells induced from Adipose derived Mesenchymal Cells 8th National University of Singapore-National University Hospital Annual Meeting, October 7-8, 2004 Lim TC, Leong TW, Chew FT, Hutmacher DW Poster Presentation In vivo characterization of adipose derived osteoprogenitors within polycaprolactone-tricalcium phosphate scaffolds Regenerate 2005, June 1-3 2005, Atlanta Georgia Leong TW, Lim TC, Chew FT, Hutmacher DW v Acknowledgements “Therefore, since we are surrounded by such a great cloud of witnesses, let us throw off everything that hinders …, and let us run with perseverance the race marked out for us.” Hebrews Chapter 12, verse 1, The Holy Bible This thesis and its corresponding research work was a race, a race that would have been impossible for me to finish if not for the people that God had placed in my life I would like to thank them with my deepest gratitude Prof Chew Fook Tim, I will always remember how you believed in a stranger to the field of biology I hope that the many life lessons you have passed down to me had been and would continue to be useful to me Thank you for teaching me some skills that would ensure that I survived in this competitive arena Thank you for these many years of patience and huge funding for my experiments I hope that I would be as good as you, or even better, in research work, as you have once so expressed Thank you for making this experience fondly memorable Prof Dietmar Hutmacher, you are a well-spring of innovative ideas I hope that some of your creativity had rubbed off on me Thank you for looking out for me and nurturing me Thank you for your encouraging support to listen to my crazy ideas and actually funding some of them You have taught me so much over these four years I know that I will be able to give to my students in the future just as you have given to me Thank you Prof Lim Thiam Chye, many thanks for providing us with the adipose tissues Without which, it would be impossible for me to work Many thanks for giving your clinical inputs in my project To my good friends in TE laboratory, Kee Woei, Mohan, Suman and Amy Thanks for the many hours of tea sessions discussing our ideas and aspirations I will always treasure those times we had Kee Woei thank you for teaching me at the beginning of my thesis work It would not be as smooth if not for your help To my colleagues in TE laboratory, many thanks for your help over these four years Last but certainly not least, to the dearest people in my life, my wife, Shirley Ting – you are my light at the end of the long tunnel Thank you for your long-suffering patience I would never have made it without your constant encouragement Thank you love and my mom, Teo Siok Lew who never failed to support me in what I love to Thank you for teaching me perseverance in my growing up years Thank you mom Both of you share the degree with me Thank you for your support, sacrifice, patience and love during this period I love you This thesis is dedicated to the memory of my late father, Leong Fook Wing Above all, to God be the glory David Leong July 31, 2006 vi Table of Contents Page Preface iii Acknowledgements vi Table of Contents vii Summary x List of Tables xii List of Figures xiii Chapter – Introduction 1.1 Background 1.1.1 Clinical need requiring bone healing or regenerative intervention 1.1.2 The use of stem cells in regeneration of bone 1-1 1-1 1-2 Chapter – Literature Review 2.1 Description of the adipose tissue 2.2 Adipose tissue – a possible alternative to bone marrow as a source of adult mesenchymal stem cells 2.3 Adult mesenchymal stem cells or precursor cell populations 2-1 2-3 2-4 2.3.1 ADSC characteristics 2-5 2.3.2 Cell Surface Characterization 2-6 2.3.3 General information on ADSC 2-7 2.3.4 Specific differentiation capacity of ADSC 2-8 2.4 Scaffolds based bone engineering 2-12 2.5 Other potential applications in regenerative medicine 2-16 2.6 Gene delivery vehicles 2-17 vii Chapter – Research Program 3.1 Overview 3-1 3.2 Four study phases 3-2 3.2.1 Phase I: Procurement of adipose tissue and their viability (Chapter 5) 3.2.2 Phase II: Characterization of ADSC in culture plastic and polycaprolactone environments (Chapter and Chapter 7) 3.2.3 Phase III: Characterization of ADSC in in vivo environments (Chapter 8) 3.2.4 Phase IV: Genome-wide transcriptome analysis of ADSC under induction gave insights into differentiation behavior of ADSC (Chapter 9) 3-2 3-3 3-3 3-5 Chapter – General Materials and Methods Chapter – Viability and adipogenic potential of human adipose tissue derived stem cells isolated from tissue aspirated with a vacuum pump or syringe 5.1 Chapter Abstract 5-1 5.2 Introduction 5-2 5.3 Specific Materials And Methods 5-4 5.4 Results 5-6 5.5 Discussion 5-12 5.6 Conclusions 5-15 Chapter – The combined action of 1,25–dihydroxyvitamin D3, β– glycerophosphate and ascorbic acid was essential for the osteogenic induction of ADSC 6.1 Chapter Abstract 6-1 6.2 Introduction 6-3 6.3 Specific Materials And Methods 6-6 6.4 Results 6-7 6.5 Discussion 6-16 6.6 Conclusions 6-18 viii Chapter – Characterization of osteogenically induced adipose tissue derived precursor cells in 2D and 3D environments 7.1 Chapter Abstract 7-1 7.2 Introduction 7-2 7.3 Specific Materials And Methods 7-4 7.4 Results 7-8 7.5 Discussion 7-17 7.6 Conclusion 7-23 Chapter – Investigating the effects of preinduction on human adipose derived precursor cells in an athymic rat model 8.1 Chapter Abstract 8-1 8.2 Introduction 8-2 8.3 Specific Materials And Methods 8-5 8.4 Results 8-9 8.5 Discussion 8-21 8.6 Conclusion 8-24 Chapter – Genome wide analysis revealed ATF5 as potential transcription factor for downstream expression of a stemness gene in adipose derived stem cells 9.1 Chapter Abstract 9-1 9.2 Introduction 9-2 9.3 Specific Materials And Methods 9-7 9.4 Results And Discussion 9-13 9.5 Conclusion 9-29 Chapter 10 – Conclusions and Future Work 10.1 Summary Of Work 10-1 10.2 Further Work 10-5 Chapter 11 – References Chapter 12 – Appendix ix Summary of Thesis This thesis was motivated by the hypothesis that there is a subpopulation of cells in human adipose tissue which possesses multipotential differentiation properties for applications in regenerative medicine applications A sub-population of cells termed by Zuk et al (2002) as “adipose derived stem cells” (ADSC) adhered to tissue culture plastics and was able to proliferate well in standard tissue culture conditions Further characterizations were carried out on several fronts, from their responses to osteogenic induction on tissue culture conditions to 3-D environments in an athymic mouse model The characterization of ADSC for this thesis ended with the eludication of a potential stemness marker based on robust global gene expression array analysis from an in-house depository of donors’ adipose derived stem cells In this thesis, it was shown that ADSC isolated from adipose tissue aspirated with the vacuum pump were viable for subsequent cellular studies The osteogenic differentiation was chosen as a proof of principle of the multipotential properties of ADSC It was shown that 1,25–dihydroxyvitamin D3 worked synergistically with β-glycerophosphate and ascorbic acid to induce ADSC towards the osteogenic lineage ADSC were able to express bone matrix proteins and mineralization of its matrix under such conditions Progressing from culture plastics systems to 3dimensional studies using polycaprolactone-tricalcium phosphate scaffolds, ADSC were still able to express bone characteristics when induced Similar characteristics of ADSC were confirmed in an in vivo model x Chapter 12 References Hicok KC, Du Laney TV, Zhou YS, Halvorsen YD, Hitt DC, Cooper LF, Gimble JM Human adipose-derived adult stem cells produce osteoid in vivo Tissue Eng 2004; 10(3-4):371-380 Hong JH, Hwang 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71(1):18-27 12 – 22 .. .CHARACTERIZATION OF HUMAN ADIPOSE DERIVED ADULT MULTIPOTENT PRECURSOR CELLS LEONG TAI WEI DAVID (Bachelor of Chemical Engineering, Hons) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY... of stem cells in regeneration of bone 1-1 1-1 1-2 Chapter – Literature Review 2.1 Description of the adipose tissue 2.2 Adipose tissue – a possible alternative to bone marrow as a source of adult. .. Reliable procurement of autologous ADSC • Characterization of cells in in vitro culture plastic environment • Characterization of cells in in vitro 3-D environment • Characterization of cells in in vivo