Browsing by Subject "Liver cells--Laboratory manuals."

Now showing 1 - 1 of 1

Unknown
Differentiation of hepatocyte like cells from immortalized mouse embryonic fibroblasts harboring large T antigen
(2015) Keleş, Umur
Genetic and acquired liver diseases are generally progressive and life threatening with limited curative therapy options. Although organ transplantation is the most potent treatment, number of patients waiting for organ transplant far outnumbers the potential suitable donors. Recently, new alternative methods have been developed to generate functional hepatocytes which can directly be administered to patients. Generating hepatocytes from di erent cells derived from patient has been one of the most promising alternative. Direct conversion of terminally di erentiated cells into hepatocyte like cells has been reported previously. However, hepatocyte di erentiation from SV40 Large-T antigen expressing immortalized Mouse Embryonic Fibroblasts has not been reported. To this end, rst we have evaluated the e ects of individual and combined retroviral expression of liver lineage determining transcription factors: Hnf4 , Foxa2 and Foxa3. Single factor transduced immortal MEFs gave little or no signi cant epithelial marker expression. These conditions were also insu cient to induce liver speci c phenotype. However, combined expression of either Hnf4 +Foxa2 or Hnf4 +Foxa3 have resulted in an increased epithelial and liver speci c characteristics such as albumin expression and glycogen storage. To elucidate epigenetic background of this process we genotyped transgenic mouse strains with conditional knockout alleles of histone variants. Histone variant H3.3A conditional knockout immortal MEFs were also infected with Cre expressing retroviral vectors. Our studies indicated that, Large-T antigen immortalized MEFs can be transdifferentiated by using the protocol designated for primary MEFs. Additionally, by isolating and immortalizing genetically determined MEFs, we have established cell lines ready for understanding the roles of histone variants on trans differentiation. That will be the foundation of subsequent studies delineating e effects of histone variants on hepatocyte differentiation from MEFs.