Screen of nuclear receptors for the enhanced and alternative generation of induced pluripotent stem cells

SCREEN OF NUCLEAR RECEPTORS FOR THE ENHANCED AND ALTERNATIVE GENERATION OF INDUCED PLURIPOTENT STEM CELLS DOMINIC HENG JIAN CHIEN NATIONAL UNIVERSITY OF SINGAPORE 2012 SCREEN OF NUCLEAR RECEPTORS FOR THE ENHANCED AND ALTERNATIVE GENERATION OF INDUCED PLURIPOTENT STEM CELLS DOMINIC HENG JIAN CHIEN (B.Sc (Hons), NTU) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2012 Acknowledgements First and foremost, I would like to thank my supervisor, Dr Ng Huck Hui for allowing me to work in his laboratory back in 2008 I am also grateful for his invaluable guidance, insights and direction throughout my PhD stint Graduating from his laboratory has not only made me a more critical, analytical, resourceful, intelligent and independent researcher but also a more, determined and team-playing individual I would also like to thank Professor Davor Solter and Dr Thomas Lufkin, wonderful members of my thesis advisory committee They have been incredible with their advice, support and time I thank Jiang Jianming for help with the ChIP, and ChIP-sequencing experiments, Feng Bo for her help with the imaging of the teratoma tissue, Yuriy Orlov for ChIPseq analysis, Petra Kraus and Han Jianyong for relevant mouse work, Lim Seong Soo for karyotyping, and Ng Jia Hui for help with the microarray analysis I also thank Kyle M Loh for his wonderful intelligent discourse over scientific papers and concepts as well as his unfailing help to vet through all my manuscripts and papers A very big thank you goes out to Felicia Hong for her faithful love, unwavering support and countless encouragement that she has showered upon me throughout all these years She will always be in my heart forever Last but not least, I thank God for his bountiful grace for granting me the strength and wisdom to undertake this challenging PhD stint i Table of Contents Acknowledgements……………………………………………………………………i Table of Contents…………………………………………………………………….ii Summary……………………………………………………………… ………….viii List of Tables………………………………………………………………………….x List of Figures………………………………………………………… …………xi List of Symbols…………………………………………………………….……… xv Introduction……………………………………………………………………… 1.1 A plethora of cell types governed by transcriptional and epigenetic regulation… 1.2 Embryonic stem cell, the common precursor cell……………………… ……….3 1.3 Important transcription factors governing the ESC fate: Oct4, Sox2 and Nanog 1.4 The first derivations of mouse and human ESCs………………………… …… 1.5 The issues that plague the utilisation of human ESCs for regenerative medicine 1.6 Differentiation was previously conceived as an irreversible process……… …….8 1.7 Methodologies to reprogram somatic cells to a state of pluripotency……… … 10 1.7.1 Cell fusion………………………………………………………… .10 1.7.2 Somatic cell nuclear transfer………………………………………… 11 1.7.3 Cell explantation…………………………………………….………….12 ii 1.7.4 Reprogramming with transcription factors…………………… ………13 1.8 The canonical reprogramming factors………………………………….……… 14 1.8.1 Oct4 in reprogramming………………………………….…………… 14 1.8.2 Sox2 in reprogramming………………………………………… …….14 1.8.3 Klf4 in reprogramming………………………………… ……….…….15 1.8.4 c-Myc in reprogramming……………………………………………….16 1.9 Advantages of iPSCs………………………………………………… …………17 1.10 The biology of reprogramming to iPSCs………………………………….……18 1.11 The rapid development of the field of iPSC………………………… ……… 21 1.12 Various cell types from various species reprogrammed……………………….23 1.13 Various techniques to generate iPSCs………………………………………….24 1.14 Screen for reprogramming factors……………………………………… ……26 1.14.1 Initial screen for factors that could reprogram………………….…….26 1.14.2 Screen for factors that could reprogram human fibroblasts………… 29 1.14.3 Screen for factors that could enhance the generation of human iPSCs………………………………………………………………… …….31 1.14.4 Screen for factors that could replace exogenous Klf4 in reprogramming…………………………………………………… ……… 33 1.14.5 Screen of human transcription factor library………………… …….35 iii 1.14.6 Other players in reprogramming: non-coding RNAs…………………36 1.14.6.1 MicroRNAs in reprogramming…………………………… 36 1.14.6.2 LincRNAs in reprogramming…………………….…………38 1.15 Nuclear receptors……………………………………………………………… 39 Materials and Methods………………………………………………… ………42 2.1 Cell Culture…………………………………………………………… ……… 42 2.1 Transfection………………………………………………………………………42 2.3 Mouse genetics………………………………………………… ………………42 2.4 Packing of retroviruses and infection…………………………………………….43 2.5 PCR genotyping of retroviral integration into the genome………… ………… 48 2.6 Bisulphite genomic DNA sequencing…………………………………….….… 48 2.7 Embryoid body formation and in vitro differentiation……………… ………….49 2.8 Teratoma formation assay………………………………………………….…….49 2.9 TUNEL apoptosis assay………………………………………………………….50 2.10 Western blot analysis…………………………………………………… …….50 2.11 Southern blot analysis……………………………………………………… …51 2.12 Immunofluorescence and alkaline phosphatase staining……………………… 51 2.13 RNA extraction, reverse transcription and quantitative real-time PCR… ……52 2.14 G-banded karyotyping……………………………………………… …………55 iv 2.15 Microarray experiment and analysis……………………………………………55 2.16 ChIP assay………………………………………………………… ………….56 2.17 ChIP sequencing and analysis………………………………………………… 56 2.18 Gene targeting of POU5F1 locus by homologous recombination………… …57 Results ……………………………………………………………… …….…… 59 3.1 Setting up of the reprogramming assay………………………………….……… 59 3.2 Screen of nuclear receptors in reprogramming reveals Nr5a2 and Nr1i2 as enhancers…………………………………………………………………………… 59 3.3 Nr5a2 enhances both the efficiency and kinetics of reprogramming 63 3.4 Nr5a2 can replace exogenous Oct4 in reprogramming………………………… 66 3.5 Nr5a2-reprogrammed cells fulfil pluripotent assays……………………… ……71 3.6 Epigenetic and transcriptional profiles of Nr5a2-reprogrammed cells are akin to ESCs………………………………………………………………………………….74 3.7 The other nuclear receptors are unable to replace exogenous Oct4 in reprogramming………………………………………………………… ………… 78 3.8 Nr5a1, the close family member of Nr5a2 possess similar reprogramming capabilities as its counterpart…………………………………………….………… 78 3.9 Other transcription factors that bind to Oct4 regulatory region are unable to replace exogenous Oct4 in reprogramming………………………………………… 82 3.10 Nr5a2 sumoylation mutants can further enhance the efficiency of reprogramming…………………………………………………………….…………83 v 3.11 Genome wide binding analysis of Nr5a2 in mouse ESCs reveals that it shares many common target genes as its reprogramming counterparts, Sox2 and Klf4….…86 3.12 Nr5a2 works in part through Nanog in reprogramming…………………… …88 3.13 Nr5a2 works in a p53-pathway inhibition-independent fashion…………….….91 3.14 Nr5a2 does not enhance or replace exogenous Oct4 in human reprogramming…………………………………………………………………… 93 Discussion……………………………………………………………… ……… 98 4.1 The identification of more nuclear receptor factors associated with reprogramming…………………………………………………………….…………98 4.2 Nr5a2, the first factor reported that can replace exogenous Oct4 in reprogramming…………………………………………………………………….…99 4.3 The parallel importance of Nr5a2 in mouse ESCs, early embryogenesis and reprogramming……………………………………………………….…………… 101 4.4 Nr5a2 works in part through Nanog to mediate reprogramming……… …… 102 4.5 Nr5a2 additionally reprograms mouse EpiSCs besides mouse somatic cells…………………………………………………………………………….……104 4.6 Species-specific actions of Nr5a2 in reprogramming……………….………….105 4.7 Mouse ESC-like human ESCs with Nr5a2…………………… ……………….106 4.8 Possible role of Nr5a2 in transdifferentiation………………………………… 108 vi Conclusion…………………………………………………………….…………109 References……………………………………………………………………….110 vii Summary Differentiated cells are typified by their lineage restriction Nevertheless, a pluripotent state of unrestricted multilineage differentiation potential may be experimentally endowed upon differentiated cells via the process of pluripotential reprogramming in which the lineage restriction of differentiated cells is undone The resultant cells, known colloquially as induced pluripotent stem cells (iPSCs), become akin to pluripotent embryonic stem cells (ESCs) and pluripotent cells of the early embryo, thus gaining their characteristic developmental potential and other characteristics diagnostic of pluripotent cells Conferral of pluripotency upon differentiated cells is achieved by overexpressing pluripotency-associated transcription factors in these cells, such as Oct4, Sox2, Klf4, and c-Myc Here, I have investigated whether a heretofore underappreciated class of transcription factors, known as nuclear receptors, can function similarly to conventional pluripotency transcription factors to reprogram mouse fibroblasts into iPSCs I have identified two nuclear receptors, Nrli2 and Nr5a2, that can enhance the efficiency of iPSC generation by about 3- to 4-fold, respectively Saliently, Nr5a2 can fully replace the need for exogenous Oct4 to generate mouse iPSCs, making it the first known factor capable of “replacing” Oct4 in iPSC reprogramming Its close family member Nr5a1 functions similarly in substituting for Oct4 Piqued by how Nr5a2 can replace the singularly important Oct4 in iPSC generation, I have furthermore found that Nr5a2 is endogenously required for iPSC generation—bereft of it, few iPSCs form Moreover, in reprogramming fibroblasts, Nr5a2 directly binds the Nanog enhancer and upregulates expression of the dominant pluripotency factor Nanog In brief, my study illuminates an unexpected role for nuclear receptors in iPSC generation, identifies Nr5a2 as the first factor that viii overexpression of an ESC-important factor within EpiSCs, which totally lacks that factor to begin with187, could account for such a reprogramming feat Nonetheless, it is not surprising that the Nr5a factors are able to reprogram EpiSCs which are derived from the post-implantation stage embryo and hence their expected greater malleability to be converted (as compared to MEFs, which are typically derived from E13.5 mouse embryos, and can also be reprogrammed by Nr5a2) to iPSCs Regardless, this discovery ultimately corroborates the reprogramming capabilities of the Nr5a factors in the reprogramming of mouse cells as shown by the study herein, albeit of different developmental time point 4.6 Species-specific actions of Nr5a2 in reprogramming As the canonical reprogramming code of Oct4, Sox2, Klf4 and c-Myc can be employed to generate both mouse and human iPSCs48,49, it is of course significant to investigate if Nr5a2 also exhibits reprogramming capabilities in human cells However, what was demonstrated in murine reprogramming for Nr5a2 could not be recapitulated in human reprogramming (Figure 41) Not only was there no enhancement in the reprogramming of human fibroblasts, exogenous NR5A2 was also unable to replace exogenous OCT4 in the generation of human iPSCs (Figure 41) These results show that there is in fact some species-specific reprogramming factors, unlike what was previously thought as evidenced by the canonical reprogramming quartet However, this result is not surprising as unlike mouse ESCs, human ESCs express very minimal levels of NR5A2189 and hence introducing NR5A2, a nonhighly expressed human ESC transcription factor may not necessarily promote the 105 reversion of human somatic cells to a human ESC state Notably, human ESCs, similar to mouse EpiSCs, are also regarded to be in a primed state of pluripotency as they share many defining traits including their flattened morphology as well as their growth factor requirements in culture Incidentally, as mentioned above, EpiSCs, which are murine equivalents to human ESCs also not express Nr5a2 187 and these Nr5a2-less EpiSCs must require the exogenous introduction of this transcription factor to create a mouse ESC-like state within themselves during reprogramming187 Interestingly, in an expression profiling study of nuclear receptors performed in both mouse and human ESCs, the authors claimed species-specific functions of Nr5a2, Esrrb and Dax1, nuclear receptors that were implicated in mouse ESC biology189 These nuclear receptors that are highly expressed in mouse ESCs have different expression patterns in human ESCs Moreover, the authors also show that while Nr5a2 tends to be reduced upon differentiation in mouse ESCs, the opposite is true in human ESCs, in which its expression tends to increase upon differentiation 189 Nonetheless, it is interesting to speculate that human ESCs might contain other Nr5a2-like factors (not necessarily Nr5a2) that orchestrate cognate mechanisms as shown in mouse ESCs Taken together, it is interesting to report herein a nuclear receptor that might have a species-specific function, in which it enhances and replaces Oct4 in murine reprogramming but not exhibit the same characteristics in the context of human reprogramming 4.7 Mouse ESC-like human ESCs with Nr5a2 106 On this note, it is tempting to speculate if the overexpression of Nr5a2 might induce a mouse ESC-like state in human ESCs since this nuclear receptor can reprogram primed EpiSCs to an earlier pluripotent derivative187 Already, several groups have demonstrated the generation of mouse ESC-like human ESCs, which can be passaged as single cells, display shiny dome-shaped morphologies very akin to mouse ESCs that self-renew in culture conditions permissive for mouse ESCs190-192 These groups introduce various canonical reprogramming factors and/or a cocktail of chemical inhibitors in the presence of LIF to achieve such a reprogrammed state in human ESCs However, it should be noted that Nr5a2 appears to display species-specific roles and what is observed in the murine system may not necessarily be similarly translated for the human system In addition, Nr5a2 is also highly expressed in endodermal-derived organs and an elevation of Nr5a2 in human ESCs may induce them towards differentiation Interestingly, a group showed that NR5A2 in combination with another nuclear receptor (RARγ), in addition with OSKM, are able to reprogram human somatic cells to iPSCs that resemble mouse ESCs with respect to their morphologies, gene expression as well as culture condition requirements178 These 6-factor-reprogrammed human cells were able to be grown in LIF in the presence of 2i Notably, when the authors removed LIF and supplemented the culture media with FGF, the iPSCs assumed a hESC-like morphology instead All in all, this study demonstrates that Nr5a2 is not only able to contribute to the reprogramming of mouse somatic cells and EpiSCs to a mouse ESC-like state but also human somatic cells as well, hence these reflect the reproducibility of Nr5a2 in naïve state reprogramming 107 4.8 Possible role of Nr5a2 in transdifferentiation The potency of Nr5a2 in mediating universal reprogramming of both mouse and human cells to a mouse ESC-like state is indeed remarkable86,178,187 Incidentally, Nr5a2 is not only expressed in mouse ESCs and the early developing embryo but also in endodermal-derived adult tissues such as the liver, pancreas and intestines193-195 Besides endodermal cells, Nr5a2 is also expressed in steroidogenic tissue such as the testes and ovaries196 Hence, Nr5a2 plays a dual role in establishing pluripotency as well as inducing myriad avenues of differentiation In this respect, it is tempting to speculate the possibility of Nr5a2 in converting somatic cells such as fibroblasts into an endodermal or steroidogenic cell type It should be noted that even if it were able to cells into an endodermal derivative, there are many endodermal cell types in which Nr5a2 is expressed, and a heterogenous mixture of cells may eventually be derived Nonetheless, specialised cell culture media with relevant supplements to solely derive one endodermal cell type can be utilised Even if that is not possible, a multipotent endodermal precursor cell which Nr5a2 can create from its overexpression in fibroblast would be somewhat remarkable Fortuitously, transdifferentiation of cells to endodermal or steroidogenic tissues have yet to be demonstrated and it would therefore be interesting to investigate Nr5a2’s capabilities in orchestrating directed conversion 108 Conclusions Altogether, a screen of nuclear receptors in their potential ability to augment the generation of mouse iPSCs reveals nuclear receptors Nr1i2 and Nr5a2 as enhancers of reprogramming Besides being able to enhance the number of iPSC colonies generated, Nr5a2 is also able to enhance the kinetics of reprogramming Strikingly, Nr5a2 can replace exogenous Oct4 in the reprogramming of MEFs This finding is novel as no transcription factor, let alone factor have been previously shown to be able to substitute for exogenous Oct4 in the reprogramming process Interestingly, besides commonly known to work through Oct4, Nr5a2 is herein shown to also work in part through Nanog to mediate the successful reprogramming of murine somatic cells 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THESIS SUBMITTED FOR THE DEGREE OF DOCTOR... akin to pluripotent embryonic stem cells (ESCs) and pluripotent cells of the early embryo, thus gaining their characteristic developmental potential and other characteristics diagnostic of pluripotent. .. cells via the process of pluripotential reprogramming in which the lineage restriction of differentiated cells is undone The resultant cells, known colloquially as induced pluripotent stem cells