9 research outputs found
Differentiation of hepatocyte like cells from immortalized mouse embryonic fibroblasts harboring large T antigen
Ankara : The Department of Molecular Biology and Genetics and the Graduate School of Engineering and Science of Bilkent University, 2015.Thesis (Master's) -- Bilkent University, 2015.Includes bibliographical references leaves 53-63.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.Keleş, UmurM.S