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.. .GENERATION OF PORCINE INDUCED PLURIPOTENT STEM CELLS AND THEIR DIFFERENTIATION INTO CARDIAC LINEAGES TAN WAN CHIU GRACE (B.Eng.(Hons.), NTU) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF. .. generate porcine induced pluripotent stem cells (IPSCs) from porcine fibroblast cells and differentiate such cells into cardiac lineages to provide a model to study the development of cardiac cells. .. Pluripotent Stem Cells …………………………………………………………………… 45 4.9 Culture of cells transitioning from fibroblasts to Induced Pluripotent Stem Cells 45 iv 4.10 Culture of Porcine Induced Pluripotent Stem
GENERATION OF PORCINE INDUCED PLURIPOTENT STEM CELLS AND THEIR DIFFERENTIATION INTO CARDIAC LINEAGES TAN WAN CHIU GRACE (B.Eng.(Hons.), NTU) NATIONAL UNIVERSITY OF SINGAPORE 2014 GENERATION OF PORCINE INDUCED PLURIPOTENT STEM CELLS AND THEIR DIFFERENTIATION INTO CARDIAC LINEAGES TAN WAN CHIU GRACE (B.Eng.(Hons.), NTU) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING DEPARTMENT OF BIOENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2014 Declaration I hereby declare that this thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously Tan Wan Chiu Grace July 2014 i ACKNOWLEDGEMENTS I would like to thank the following people who have helped me complete this master’s thesis Firstly, I would like to thank A/ Prof Tong Yen Wah for his support in the entire Master’s programme I would like to thank him for his patience throughout the course I would also like to thank Dr Winston Shim and Dr Wei Heming for their guidance throughout my time at National Heart Centre They have provided great help in the design of the experiments and the technical aspects of the project I would also like to thank my colleagues at National Heart Centre who have helped me in one way or another ii Table of Contents Introduction 13 Aims and objectives 14 Literature Review 15 3.1 Human Embryonic Stem Cells (hESCs) 15 3.2 Porcine Embryonic Stem Cells 16 3.3 Generation of Induced Pluripotent Stem Cells 17 3.3.1 Retroviral/ Lentiviral Reprogramming 18 3.3.2 Episomal Reprogramming 20 3.3.3 mRNA reprogramming 21 3.3.4 Chemical reprogramming 21 3.3.5 Genes that regulate pluripotency in Human Embryonic Stem Cells………………… 22 3.3.6 OCT4………………………………………………………………23 3.3.7 SOX2………… 24 3.3.8 NANOG 24 3.3.9 KLF4…………… 25 3.3.10 c-myc……… 26 3.3.11 Lin28………… 26 3.4 Cardiac differentiation of Human IPS cell lines 27 3.5 Generation of Porcine Induced Pluripotent Stem Cells 29 3.5.1 Factors used for reprogramming of porcine cells 29 3.5.2 Reprogramming techniques of porcine cells 29 3.5.3 Retroviral and Lentiviral reprogramming 30 3.5.4 Episomal reprogramming of Porcine IPS cells 30 3.5.5 Reprogramming with decreased number of transcription factors………… 31 iii 3.6 Characterization and Definition of Porcine IPS cell lines 32 3.7 Difference in culture methods of Porcine IPSCs 34 3.7.1 Culture media 34 3.7.2 Culture on Matrigel 34 3.8 Differentiation protocol that has been published for pigs 35 3.8.1 Neural………… 35 3.8.2 Liver………… 36 3.8.3 Cardiac differentiation protocol 36 Materials and Methods 37 4.1 Culture of Mouse Embryonic Fibroblast (MEF) cells as feeder cells for hESCs 37 4.2 Culture of Human Embryonic Stem Cells (hESCs) 38 4.2.1 Culture of Human Embryonic Stem Cells on MEF feeders 38 4.2.2 Feeder- free Culture of Human Embryonic Stem Cells 39 4.2.3 Coating of Matrigel ™ 40 4.2.4 Enzymatic passaging of Human IPSCs 40 4.3 Differentiation of Human IPS cells into cardiomyocytes 40 4.4 Patch clamp recordings of human IPS derived cardiomyocytes 42 4.5 Culture of Porcine Fibroblast Cells 42 4.6 Preparation of Plasmids for transfection 42 4.7 Packaging of Lentiviruses for the induction of Pluripotent Stem Cells………………… 44 4.8 Infection of fibroblast cells to form Induced Pluripotent Stem Cells …………………………………………………………………… 45 4.9 Culture of cells transitioning from fibroblasts to Induced Pluripotent Stem Cells 45 iv 4.10 Culture of Porcine Induced Pluripotent Stem Cells 45 4.10 Characterization of Porcine Induced Pluripotent Stem Cells 46 4.10.1 Immunocytochemical staining for Porcine IPSCs 47 4.10.2 Polymerase Chain Reaction for Porcine IPSCs 49 4.10.3 Teratoma formation 50 4.10.4 Karyotyping 50 4.10.5 Alkaline Phosphatase Staining 50 4.11 Differentiation 51 4.11.1 In vitro teratoma 51 4.11.2 Differentiation into fat and cardiomyocytes 51 4.11.3 Staining of fat globules 52 Results……… 53 5.1 Morphology of hESC 53 5.2 Morphology of iPSCs 54 5.3 Differentiation of hESCs / hIPSCs into cardiomyocytes 55 5.4 Action potential recording of IPS derived cardiomyocytes 56 5.5 Culture of porcine fibroblast cells 57 5.6 Reprogramming process of porcine fibroblast cells 58 5.6 Formation of Porcine IPS colonies and the continuous culture of the colonies 60 5.7 Characterization of Porcine IPS cells 65 5.7.1 Immunocytochemical Staining of Porcine IPS colonies 65 5.7.2 PCR for exogenous genes in Porcine IPS 72 5.7.3 PCR for endogenous genes in porcine IPS 75 5.7.4 Teratoma formation 76 5.7.5 Karyotyping of IPS clones 77 v 5.7.6 Alkaline Phosphatase Staining of Porcine IPS colonies 78 5.8 Withdrawal of bFGF from the culture media 79 5.9 In vitro teratoma formation 82 5.10 Differentiation of porcine IPS cells into fat and cardiomyocytes 84 Discussion 89 6.1 Morphology of porcine IPS cells 89 6.2 Expression of pluripotent genes in porcine IPS cells 89 6.3 Function of LIF in the culture of porcine IPS cells 90 6.4 Importance of doxycycline in the culture media 91 6.5 Differentiation of porcine IPS cells into cardiomyocytes 92 6.6 Use of porcine cells for other downstream work 92 Future work 94 Conclusion 95 References ……………………………………………………………96 vi SUMMARY Heart disease is a major cause of death worldwide As a result of myocardial infarction, functional cardiomyocytes are lost and this results in a heart that does not contract properly The pig model is a good model to study cardiac functions in humans, as the size of the heart in the pig is similar to that of the human In this project, we aim to convert porcine fibroblast cells into porcine cardiomyocytes, in the hope of creating new functional cardiomyocytes to replace the ones that are no longer functional after myocardial infarction As no porcine embryonic stem cells have been isolated, induced pluripotent stem cells for the pig must be used as a source of pluripotent cells Hence, fibroblasts from the pig is taken, and induced into pluripotent cells These cells are in turn differentiated into cardiomyocytes, where more downstream work can be done to characterize them and also to use these cells as form of therapy for the failing heart In this project, we isolated fibroblasts from the pig’s thigh region, and used lentiviruses to induce the cells to pluripotent cells These pluripotent cells are studied and characterized The pluripotent stem cells exhibited morphology and genomic markers of pluripotency These cells were also finally differentiated into cardiomyocytes Even though the differentiated cells did not beat, there were cardiac genes found to be expressed in the form of mRNA This shows that the differentiation protocol is successful to a certain extent More work can be done to streamline to protocol to achieve greater efficiency in the production of porcine cardiomyocytes vii List of Tables Table 3.1 The applications, reprogramming methods and the advantages of using each method for somatic cell reprogramming (Gonzalez, Boue, & Izpisua Belmonte, 2011) 18 Table 3.2 Various pluripotent characteristics which defined porcine IPS cells 33 Table 4.1 Materials required for complete media 37 Table 4.2 Materials required for hESC media 39 Table 4.3 Materials required for CARM media 41 Table 4.4 Materials required for Mouse ESC media 46 Table 4.5 Primary Antibodies used for immunocytochemical staining 47 Table 4.6 Secondary Antibodies used for Immunocytochemical Staining 48 Table 4.7 Protocol for Polymerase Chain Reaction 49 Table 5.1 PCR primers for exogenous transcription factors 73 Table 5.2 PCR primers for endogenous transcription factors 75 Table 5.3 Primers used for determination of the presence of germ layers 83 Table 5.4 PCR primers to determine the presence of cardiomyocytes 87 viii of pluripotent cells The activation of endogenous genes were also observed through the use of RT-PCR Although the continuous expression of exogenous genes was necessary for the pluripotency of the porcine IPS cells, there was also the activation of endogenous genes However, we postulate that the expression of the selected endogenous genes may not be sufficient to sustain the cells for prolonged culture This may be due to the lack of other critical growth factors in the culture media Further work may be necessary to define the culture conditions best suited to porcine IPS cells 6.5 Differentiation of porcine IPS cells into cardiomyocytes Thus far, we were not able to differentiate the porcine IPSC to beating cardiomyocytes in our culture However, we were able to detect cardiac markers that were present in the culture The expression of these cardiac markers could come from precursors of cardiomyocytes and hence are cells that may not beat This may explain why no beating cells were observed Also, there may be the possibility of the presence of beating cardiomyocytes, but the efficiency of the production of cardiomyocytes was extremely low, and the beating cells were too few to be observed visually Though their observed differentiation towards fat cells suggested that mesodermal differentiation was initiated, but directing the lineage towards cardio-mesoderm and subsequent cardiomyocytes remains to be elucidated 6.6 Use of porcine cells for other downstream work Although the porcine IPS cells were not able to differentiate into cardiomyocytes, it is possible to consider the use of the porcine IPS cells for other downstream experiments, such as the differentiation of the cells into mesenchymal stem cells Studies 92 have shown that through the differentiation of IPS cells, MSCs can also be obtained alongside the IPS cells (Wei et al., 2012) Differentiated cells which not exhibit cardiomyocte behavior can be harvested and characterized to determine the nature of such cells 93 Future work The understanding of the developmental biology of porcine embryonic stem cells will enable us to understand the signaling pathway of such cells and the culture conditions to maintain the pluripotency of porcine pluripotent stem cells There have been several groups that each have their own definition of porcine pluripotent cells, and it is important to standardize such standards to ensure that the porcine IPS cell lines generated are of good quality and of good differentiation potential The differentiation of porcine IPS cells into cardiomyocytes has only been shown by one group The results have not been reproduced by other laboratories and we similarly failed to differentiate the porcine IPSC following that published protocol Further investigations will need to be done to improve the cardiac differentiation protocol The potential divergent of signaling pathways for cardiomyocytes differentiation in pig and human should be investigated in detail as cardiac differentiation protocols that worked in human IPSC failed to elicit similar outcome from the porcine IPSC 94 Conclusion We have successfully generated multiple lines of porcine IPS cells with pluripotent 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