The liver is a crucial human organ with a complex architecture. Although the liver has great regeneration potential, deadly liver diseases are associated with irreversible hepatocytes damage. Currently, a liver transplant is the only treatment for liver failure. A shortage of donors forced extensive research for alternative treatments. The most promising hepatocyte source could be obtained from the differentiation of induced pluripotent stem cells (iPSCs). This technology can give us great amounts of pluripotent cells without ethical restrictions, which could be available in a variety of haplotypes to minimize the possibility of rejection. There are many reprogramming protocols available. However, there is still no standardised method to obtain clinical grade iPSCs. From those stem cells, it is possible to obtain hepatic-like cells (HLCs) by direct differentiation in vitro. HLCs express multiple hepatocyte-specific features, but their names signal that they still show fetal liver identity. A variety of hepatic differentiation protocols were described, although the process of hepatic differentiation must be improved in order to be translated into the clinic. Along with genes, microRNA (miRNA) is the well-known controller of cell fate.
MiRNA is a type of non-coding RNA (ncRNA) which can influence gene transcription by inhibiting gene expression. In contrast to genes, many of the miRNA can affect up to thousands of genes simultaneously. Another group of ncRNA, which is a subject of potential differences are small nucleolar RNA (snoRNA). SnoRNA are involved in RNA chemical modifications by acting as a guide, mostly for ribosomal RNA (rRNA), but some of them have additional functions. In this study, a new iPSCs line was generated from skin fibroblasts using lipotransfection of episomal vectors. This method is free from exogene integration and shows low cytotoxicity. A pluripotency of generated cells was confirmed by morphological assessment, immunocytochemical staining, and spontaneous differentiation assay. To be sure that the genome of the cells was not changed, karyotype analysis was performed. Next, HLCs were derived from those iPSCs using a four-stage hepatic differentiation protocol. The obtained HLCs were characterised using, among others, a hepatic gene expression analysis. Cells after differentiation express mature and fetal hepatic markers, which is consistent with previous results. The attempt to improve differentiation using transient overexpression of master hepatic transcription factor – HNF4α, was not sufficient, as shown by gene expression analysis and whole slide scanning. Previous studies failed to point out the genetic inhibitors of hepatic maturation and non-coding RNA (ncRNA) profiles of iPSCs – derived HLCs were not investigated. In this study, the sequencing of ncRNA was performed in order to compare the expression profiles of HLCs on two stages of differentiation (Day 20 and 24) with mature hepatocytes. The obtained results indicate that HLCs express miRNA, which control hepatic differentiation and maintain their fetal liver character. In comparison to mature hepatocytes, differentially expressed miRNAs in HLCs control the pathways of fatty acid metabolism and synthesis, proteoglycan in cancer, the Hippo signaling pathway, ECM-receptor interaction and adherens junction. Some of those highly expressed miRNAs can potentially block maturation by inhibiting epithelial-mesenchymal transition (EMT) which has an impact that is essential during hepatic differentiation. However, this should be resolved in future research. In this work, differentially expressed snoRNA were also identified. A total of 68% of differentially expressed snoRNAs was C/D box class. This is interesting because this snoRNa class was previously indicated as capable to be processed by an miRNA processing pathway. Many of the differentially expressed snoRNAs belong to the imprinted loci,
in which a different expression in a human were analysed before. In obteined dataset, copies of SNORD115 were upregulated in a liver, but not in HLCs, which is consistent with an earlier comparison of a liver and other endoderm organs. Additionally, an analysis of obtained sequencing data allowed for a discovery of 19 novel snoRNA genes. In summary, this work shows a new approach to the reprogramming of a fibroblast and investigates the involvement of miRNAs and snoRNAs in the dynamics of hepatic differentiation. This study has shed a light on the molecular and regulatory mechanisms that underlie the complex process of liver differentiation and will hopefully allow existing problems with the use of in vitro derived hepatocytes to be overcome. A dataset generated here can be the foundation for a hepatic-specialised rybosomes theory and enabled to discover novel snoRNA genes.:1. INTRODUCTION 11
1.1. PLURIPOTENT STEM CELLS 11
1.1.1. Pluripotency 11
1.1.2. IPSCs 13
1.1.3. Reprogramming methods 14
1.1.4. IPSCs as an alternative cell source for disease modelling and regenerative medicine 16
1.2. LIVER 18
1.2.1. Liver anatomy and function 18
1.2.2. Liver embryonal development 20
1.3. HEPATIC DIFFERENTIATION OF IPSCS IN VITRO 22
1.3.1. HLCs 22
1.3.2. Differentiation protocols into hepatocytes 24
1.4. NCRNA 25
1.4.1. MiRNA 26
1.4.2. SnoRNA 28
2. AIMS 31
3. MATERIALS 32
3.1. EQUIPMENT 32
3.2. SOFTWARE 32
3.3. ENZYMES, KITS AND TRANSFECTION REAGENTS 33
3.4. SOLUTIONS AND REAGENTS 33
3.5. CELL LINES 34
3.6. CELL CULTURE MEDIA AND CYTOKINES 34
3.7. PLASMIDS 35
3.8. PCR REAGENTS AND PRIMERS 35
3.8.1. PCR reagents 35
3.8.2. PCR primers 35
3.9. ANTIBODIES 36
4. METHODS 37
4.1. CELL BIOLOGY 37
4.1.1. Derivation and culture of primary human foreskin fibroblasts 37
4.1.2. Counting cells 37
4.1.3. Cryo-preservation of cells 37
4.1.4. Thawing of cryo-preserved cells 38
4.1.5. Cell reprogramming 38
4.1.6. Cultivation and expansion of iPSCs 39
4.2. IMMUNOCYTOCHEMISTRY 39
4.3. IN VITRO SPONTANEOUS DIFFERENTIATION 39
4.4. KARYOTYPE ANALYSIS 40
4.5. RNA ISOLATION 40
4.6. QUANTITATIVE PCR 40
4.7. PERIODIC ACID-SCHIFF (PAS) STAINING 41
4.8. INDOCYANINE GREEN UPTAKE AND RELEASE 41
4.9. PLASMID TRANSFECTION 42
4.10. HEPATIC DIFFERENTIATION 42
4.11. WHEAT GERM AGGLUTININ STAINING 42
4.12. VALIDATION OF HEPATIC DIFFERENTIATION EFFICIENCY 43
4.13. RNA ISOLATION AND SEQUENCING 43
4.14. BIOINFORMATIC ANALYSIS 44
4.14.1. Sequencing quality and mapping 44
4.14.2. Analysis of differential expressed ncRNAs 44
4.14.3. Target pathways prediction of differentially expressed miRNAs 44
4.14.4. Identification of novel ncRNAs candidates 45
5. RESULTS 46
5.1. GENERATION OF IPSCS USING EPISOMAL VECTORS 46
5.1.1. Cell transfection 46
5.1.2. Establishment of iPSCs line 48
5.2. PLURIPOTENCY CHARACTERISATION OF THE IPSCS 49
5.2.1. Pluripotency markers 49
5.2.2. Spontaneous differentiation assay 50
5.2.3. Karyotype 52
5.3. HEPATIC DIFFERENTIATION OF IPSCS AND HLCS CHARACTERISATION 53
5.3.1. iPSCs hepatic differentiation 53
5.3.2. Expression of hepatic markers 54
5.3.3. Hepatic gene expression in HLCs 56
5.3.4. Hepatic functions in HLCs 58
5.4. HNF4A OVEREXPRESSION DURING DIFFERENTIATION 59
5.4.1. Cell transfection during differentiation 59
5.4.2. Comparison of hepatic differentiation efficiency 60
5.4.3. Whole slide scanning 62
5.5. NON-CODING RNA ANALYSIS 64
5.5.1. Non-coding RNA sequencing quality 64
5.5.2. MicroRNA analysis 68
5.5.3. SnoRNA analysis 79
5.5.4. Short reads snoRNA analysis 84
5.5.5. New gene candidates 85
6. DISCUSSION 88
6.1. METHODICAL STRATEGY 88
6.2. CHARACTERISATION OF GENERATED IPSCS 89
6.3. HEPATIC DIFFERENTIATION OF IPSCS 89
6.3.1. Characterisation of HLCs 89
6.3.2. Protocol with HNF4a overexpression 90
6.3.3. Differentially expressed miRNA 90
6.3.4. Differentially expressed snoRNA 93
6.4. NOVEL SNORNA GENES 95
7. SUMMARY 96
8. REFERENCES 99
9. APPENDIX 118
ERKLÄRUNG ÜBER DIE EIGENSTÄNDIGE ABFASSUNG DER ARBEIT 122.
ACKNOWLEDGEMENTS 123
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:37503 |
| Date | 15 January 2020 |
| Creators | Skrzypczyk, Aniela |
| Contributors | Universität Leipzig |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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