| Regenerative medicine is a field of research involving the repair, replacement orregeneration of damaged or diseased cells, tissues and organs by transplanting into thepatient with stem cells from a compatible donor or the patient’s own stem cells. However,the generation of induced pluripotent stem cells (iPS cells) from the somatic cells hasbrought another potential source of stem cells for cellular therapy, studying diseasepathogenesis and testing new compounds, and even they can circumvent the ethical issues.Attention has recently focused on exploring the field of embryonic stem cells (ES cells)and induced pluripotent stem cells, regarding their enormous role in studying andtherpying human diseases. More and more studies have shown that the regulation ofepigenetic modification plays a crucial role in maintaining the “stemness” state of the EScells, controlling the lineage-specific decisions and reprogramming somatic cells. Thehistone modifications may serve as "switches" to activate or repress gene expression,wherein the acetylation is one of the most important ways. Steady-state levels ofacetylation of the core histones results from the balance between the opposing activities ofhistone acetyltransferases (HATs) and histone deacetylases (HDACs).HDACs play an important role in mediating a wide range of biological functionsincluding growth, death, differentiation, development and immune reaction. MammalianHDACs are classified into four groups. In particular, HDAC5, a member of class IIaHDACs, acting as a transcriptional repressor, has been implicated in smooth and skeletalmuscle differentiation, cardiac hypertrophy, angiogenesis and immune responses.Objective:Since HDAC5plays such an important role in cell differentiation and developmentprocess, then what role does it play in the early development of ES cells, regarding themaintenance of self-renewal and pluripotency? Exploring this issue will help us to betterunderstand some molecular regulation procedures involved in early embryonicdevelopment, and lay the foundation for application of stem cells.In addition, iPS cells are expected to be used as a substitute for ES cells, regarding theirbiological similarity. However, eliminating inherent epigenetic markers of somatic cellsand creating new ones for pluripotency in the process of reprogramming is needed, so wetry to investigate whether HDAC5participates in the regulation of somatic cellreprogramming? Exploring this issue will help us to better understand epigeneticmechanisms in reprogramming procedures, and put forward some methods to current problems.Therefore, this study is divided into two parts, exploring the role of HDAC5inmaintaining pluripotency, lineage commitment of ES cells and somatic reprogramming.Methods:1. Observe the influence of HDAC5on the self-renewal of mouse ES cells.In this study, mouse ES-E14TG2a cell line was used as a research model. First, weestablished lentivirus-mediated stably HDAC5knockdown or overexpression cell lines.Then we detected some of the indicators in evaluation of self-renewal capacity of ES cells,including the positive results of AP staining, expression of self-renewal genes andmaintaining normal clonogenic capacity.2. Observe the influence of HDAC5on the lineage commitment of mouse ES cells.To assess the developmental potential, HDAC5knockdown or overexpression cell lineswere used to detect the ability of vitro and vivo differentiation. In vitro differentiation,these cells were induced to form embryoid bodies (EBs), and then were assayed for theexpression of cell-type specific markers; As a more stringent pluripotency test, a teratomamodel was used to evaluate the in vivo differentiation capacity of these cells.3. Evaluation the role of HDAC5in somatic reprogramming.We employed the classic Yamanaka factors with the HDAC5shRNA to inducereprogramming and compared the induction efficiency with control group.4. Observe the influence of HDAC5on the MET in the process of reprogramming.To evaluate the role of HDAC5in reprogramming, we compared the expression ofmarker genes related to MET in control and experimental groups.Results:1. The influence of HDAC5on the self-renewal of mouse ES cells.Experiments revealed that HDAC5knockdown or overexpression did not affect theexpression of the known self-renewal genes, such as Oct4and Sox2. These cells were alsoable to form alkaline phosphatase positive colonies as efficiently as control ES cells andtheir ability of clonogenic capacity remained largely unaltered.2. The influence of HDAC5on the lineage commitment of mouse ES cells.As expected, the expression of lineage-specific markers was generally upregulated incontrol group of vitro or vivo experiments. In HDAC5knockdown group, the expressionof ectoderm markers was largely abolished, while mesoderm markers were induced andexpressed at a higher level; When HDAC5is overexpressed, we can observe the opposite phenomenon.Meanwhile endoderm markers were not influenced in either HDAC5knockdown or overexpress groups.3. The role of HDAC5in somatic reprogramming.Knocking down HDAC5using shRNA vectors largely reduced the reprogrammingefficiency with Yamanaka factors. Therefore, endogenous HDAC5are required forreprogramming.4. The influence of HDAC5on the MET in the process of reprogramming.Detecting MET marker genes, it shows that knocking down HDAC5will inhibit theexpression of epithelial genes and maintain the abnormal activation status of mesenchymalgenes in early reprogramming.Conclusion:1. HDAC5is not required for ES cells self-renewal.2. HDAC5is a regulator of lineage commitment.3. HDAC5is necessary for somatic reprogramming.4. HDAC5regulates somatic reprogramming through the MET. |
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