The Use Of Human Embryonic Stem Cells Hescs To -Books Pdf

The use of Human embryonic stem cells hESCs to
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Dedication, To my family Mum Dad Daniel and Chad you are my world. All my love Trish,STATEMENT OF AUTHENTICATION, This is a signed statement to the effect that the work has not been submitted for a. higher degree at any other institution and an undertaking that the work is original and. as a result of the candidates own research endeavour. Patricia A Murphy,ACKNOWLEDGMENTS, First and foremost my thanks go to my supervisor Dr Michael O Connor for his. expertise tireless guidance and lengthy discussions on this project to make my PhD. experience productive and stimulating even during the tough times I am grateful to. him for his supervision and mentorship I believe he made an immense impact on my. personal and academic evolution and for that I offer my thanks. To everyone who has helped me in some way throughout my PhD candidature I will. always be grateful I appreciate the initial lab training I received from Dr Elisabeth. Wederell and Dr Christine Chiu To Prof Jens Coorssen and Dr Joanne Lind for. both generously providing access to their laboratories and facilities To Dr Michael. O Connor Prof John Morley and Dr Victoria Gauci who took the time out of their. busy schedule to read some or all of my thesis and for their helpful comments To Dr. Joanne Lind and Dr David Mahns for your guidance and wisdom I gratefully. acknowledge the funding sources that made my PhD work possible I was awarded. the Australian Postgraduate Award and UWS Higher Degree Research Candidate. My time at UWS was made enjoyable in large part due to the many friends that. became a part of my life To the members of the Regenerative Research Laboratory. Seaky and Mel I will miss getting to spend hours in the lab laughing together thank. you for keeping me balanced To Victoria and Daunia your love and support have not. gone unnoticed, Thanks to Mum and Dad for supporting me through all these years of study and. encouraging me to be passionate and driven Thanks Dan for telling me straight and. giving me a hug when I needed it Last but not least to Chad thank you for all your. love encouragement and patience,CONFERENCE ABSTRACTS AWARDS AND PATENT.
RESULTING FROM THIS THESIS, 1 Optimising the differentiation of human pluripotent stem cells to lens epithelial. cells PA Murphy MD O Connor 4th annual ASSCR meeting Leura October. 2 Development of an Excel based tool and associated pipeline facilitates. identification of novel molecular biology within the lens and gut MD. O Connor1 2 PA Murphy1 Presented at the Sino Australian Cell Therapy. Summit July 2013 Gansu province China, 3 Development of an Excel based tool and associated pipeline facilitates the. identification of novel molecular biology within the lens and gut PA Murphy. MD O Connor 6th annual ASSCR meeting Brisbane October 2013. 1 University of Western Sydney Forum Best in Session July 2012. 2 UWS 3 minute thesis finalist August 2012, 3 The National Stem Cell Foundation of Australia NSCFA travel grant The. Australasian Society for Stem Cell Research ASSCR 6th meeting Brisbane. Provisional patent, A Method for the Purification of Eye Lens Cells Australian Provisional Patent. Application Number 2014900269, Applicant University of Western Sydney Filing Date 29 January 2014.
Inventors PA Murphy and MD O Connor,PCT submission occurred 29 1 15 PCT AU2015 000046. Abstracts and Papers from PhD not published within thesis. An additional experimental chapter was obtained during this thesis although using. similar methods it was centered on the gut For continuity it has been left out. 1 Discovery of candidate novel molecular regulators of interstitial cells of Cajal. using a simple Excel based macro and associated bioinformatics pipeline P. Murphy MD O Connor V Ho Presented at the Australian Gastrointestinal. week meeting October 2013 Melbourne Australia, 2 PA Murphy and MD O Connor Excel based analysis of gastrointestinal data. identifies novel candidate hypothesis for the molecular control of gut motility. In preparation for submission to The Journal of Gastroenterology. TABLE OF CONTENTS,THESIS ASTRACT 1, CHAPTER ONE Introduction Stem cells and the ocular lens Implications for. cataract research and therapy 3,1 1 ABSTRACT 4,1 2 INTRODUCTION 5. 1 2 1 Cataract and its global impact 7,1 2 2 Causes of age related cataracts 9.
1 2 3 Limitations of current cataract treatments 10. 1 2 4 Mammalian embryonic lens development 12,1 2 5 Embryonic and postnatal lens growth 13. 1 2 6 Lens cell characteristics determine lens function 15. 1 2 7 Lens stem cells and lens regeneration 16,1 2 7 1 Evidence for lens stem cells 16. 1 2 8 PCO and lens stem cells 18, 1 2 9 In vitro cataract models from lens stem cells 19. 1 2 10 Human pluripotent stem cells for lens research 20. 1 2 11 Lens differentiation methods for human pluripotent cells 21. 1 2 12 Human ES cell derived lens cells for identification of developmental. mechanisms 23, 1 2 12 1 Human ES cell derived lens cells to identify anti cataract drugs 23. 1 2 13 Summary 25, CHAPTER TWO Growth factor optimisation for production of lens epithelial cells.
from human embryonic stem cells 26,2 1 ABSTRACT 27. 2 2 INTRODUCTION 29,2 3 METHODS 33,2 3 1 Cells 33,2 3 2 Reagents and consumables 33. 2 3 3 General cell culture 33, 2 3 4 Preparing human ES cells to be plated as single cells 34. 2 3 5 Stage 1 Differentiation towards neuroectoderm Day 0 to day 5 34. 2 3 6 Stage 2 Generation of lens placode cells Day 5 to Day 18 35. 2 3 7 Stage 3 Generation of LEC and fibre cell types Day 18 to Day 35 35. 2 3 8 Lentoid counts 35,2 3 9 RNA collection 37, 2 3 10 cDNA synthesis via reverse transcription 37. 2 3 11 Primer design 38,2 3 12 PCR amplification of mRNA transcripts 38.
2 3 13 Real time PCR 40,2 3 14 Analysis of real time PCR data 41. 2 4 RESULTS 42, 2 4 1 Stage 1 modification optimisation of neuroectoderm production 43. 2 4 2 Stage 2 modification optimising lens placode cell production 43. 2 4 3 Stage 3 modification optimisation of LEC maintenance 52. 2 5 DISCUSSION 58, CHAPTER THREE Identification of ELK1 as a novel lens transcription factor 65. 3 1 ABSTRACT 66,3 2 INTRODUCTION 68,3 3METHODS 71,3 3 1 Cells and general cell culture 71. 3 3 2 Development of an Excel based macro 71,3 3 3 GO analysis 72.
3 3 4 PASTAA analysis and GenePaint 72,3 3 5 RNA collection and cDNA synthesis 73. 3 3 6 Primer design and PCR amplification of mRNA transcripts 74. 3 3 7 Protein collection 75,3 3 8 Protein concentration an quantification 75. 3 3 9 Protein electrophoresis and staining 76,3 3 10 Western blotting 77. 3 3 11 Statistical analysis 77,3 4 RESULTS 79, 3 4 1 Comparative analyses of gene expression profiles 79. 3 4 2 PASTAA analysis to identify transcriptional regulators 79. 3 4 3 Assessment of FHL124 lens cell line for expression of ELK1 82. 3 4 4 GO analysis of TF targets 85,3 5 DISCUSSION 92.
CHAPTER FOUR Establishment of a novel LEC purification method via a new. bioinformatic tool 99,4 1 ABSTRACT 100,4 2 INTRODUCTION 102. 4 3 METHODS 104,4 3 1 Cells 104,4 3 2 Reagents and consumables 104. 4 3 3General FHL124 cell culture 104, 4 3 4 Human ES cell maintenance cell harvest and differentiation 105. 4 3 5 RNA collection and cDNA synthesis via reverse transcription 106. 4 3 6 Primer design 108,4 3 7 PCR amplification of mRNA transcripts 110. 4 3 8 Development of an Excel based macro 110,4 3 9 GO analysis 111.
4 3 10 Magnetic activated cell sorting 111,4 3 11 Flow cytometry 112. 4 3 12 Antibody staining with ROR1 and CRYAB 113, 4 3 13 Flow cytometry data acquisition and data analysis 114. 4 3 14 Culture of MACS purified cells 115, 4 3 15 Toxicology assays and immunocytochemistry 115. 4 3 16 Statistical analysis 115,4 4 RESULTS 117,4 4 1 Defining receptors in the lens 117. 4 4 2 Assessment of receptor locations 117,4 4 3 Assessment of expression in culture 118.
4 4 4 Cell purification 118,4 4 5 Cell culture post MACS 122. 4 4 6 Toxicology anti cataract screening using ROR1 LECs 125. 4 5 DISCUSSION 129,CHAPTER FIVE General Discussion 135. 5 1 Limitations of current lens cell differentiation strategies 136. 5 2 Advances in LEC production and biological insight from this thesis 136. 5 3 Future technical developments 137, 5 4 Future applications for purified human LECs to understand lens and cataract. development 138,5 6 Concluding remarks 140,REFERENCES 142. LIST OF FIGURES, Figure 1 Schematic diagram of the lens within the eye.
Figure 2 1 Stage 1 modification increased LEC production. Figure 2 2 Stage 2 modifications with Activin A did not increase LEC production. Figure 2 3 Stage 2 modifications with SFRP1 did not increase LEC production. Figure 2 4 Stage 2 modifications with SFRP2 did not increase LEC production. Figure 2 5 Stage 2 modifications with SFRP3 did not increase LEC production. Figure 2 6 Stage 2 modifications with SFRP4 did not increase LEC production. Figure 2 7 Stage 2 modifications with DKK1 did not increase LEC production. Figure 2 8 Stage 2 modifications with DKK3 did not increase LEC production. Figure 2 9 Stage 2 modifications with DKK4 did not increase LEC production. Figure 2 10 Stage 3 modification of low FGF2 increased LEC production. Figure 2 11 Mixed morphologies found in human ES cell lens differentiation. Figure 3 1 Excel equation used to determine duplicate genes between 3P lens lists. Figure 3 2 Macro facilitates the assessment of publically available gene datasets by. bioinformatic tools, Figure 3 3 Promoter analysis of 3P lens gene lists via PASTAA reveals known. transcriptional regulators of the lens, Figure 3 4 Promoter analysis of 3P lens transcripts reveals members of the PAX. Figure 3 5 Promoter analysis of 3P lens gene lists via PASTAA predicted. transcription factor ELK1 in all proximal analysis. Figure 3 6 Biological validation of ELK1 predictions in human foetal lens cell line. Figure 4 1 Macro facilitates the identification of LEC specific surface proteins. Figure 4 2 ROR1 and GPR161 are expressed in lens culture conditions. Figure 4 3 ROR1 enabled purification of human ES cell derived LEC cells. Figure 4 4 Seeding densities of ROR1 cells, Figure 4 4 Proof of principal the culture of MACS purified LECs enables lens. drug and toxicology screening,LIST OF TABLES,Table 2 1 Culture and experimental media. Table 2 2 List of primers used to assess lens differentiation strategies. Table 3 1 Gene ontology analysis of BACH2 targets,Table 3 2 Gene ontology analysis of ELK1 targets.
Table 4 1 Stage 1 2 of the Published 3 stage differentiation medium and their. components, Table 4 2 List of primers used to assess lens differentiation strategies. SUPPLEMENTARY INFORMATION ON CD,Supplementary File 3 1 Macro code. Supplementary File 3 2 Macro sorted lists and PASTAA analysis. Supplementary File 3 3 GO analysis of ELK1 and BACH2 Targets. Supplementary File 4 1 Surface proteins within the lens.


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