Functional characterisation of hepatocyte-like cells generated from human amniotic epithelial cells

2019-08-22T00:44:23Z (GMT) by Vaghjiani, Vijesh Gopal
Chronic liver diseases and hepatocellular carcinoma are major global health burdens and liver transplantation is the only curative option currently available when these conditions become end stage liver diseases. Due to the severe shortage of suitable donor organs, hepatocyte transplantation is being trialled for patients with end stage disease as well as acute liver failure. However, obtaining sufficient numbers of human hepatocytes from liver re-sections, cadavers and aborted fetuses for transplantation is the principal limiting factor. Alternate sources of cells are being sought for transplant purposes, drug and toxicity screening, and seeding of bio-artificial liver devices. Human amniotic epithelial cells (hAEC) from term placenta are a good alternative source of stem cells to human hepatocytes. In my studies, I show that differentiation of hAEC into hepatocyte like cells (HLC) results in expression of key hepatic transcription factors. Differentiated HLCs performed the hepatocytes functions including urea synthesis, drug metabolising cytochrome P450 (CYP)3A4 activity, indocyanine green(ICG) uptake, low-density lipoprotein (LDL) uptake and glutathione anti-oxidant capacity. A number of hepatocyte genes involved in fat, cholesterol, bile acid and xenobiotic metabolism were also expressed in the HLCs. When HLCs were encapsulated, they remained viable for 7 days in-vitro and continued to express genes involved in fat, cholesterol, bile acid and xenobiotic metabolism. Furthermore, encapsulated HLCs showed increased glutathione anti-oxidative capacity, CYP3A4 activity and urea synthesis MtDNA copy did not increase with HLC differentiation, which, in turn, is marked by increased levels of DNA methylation at exon 2 of POLGA. Treatment with 5-azacytidine increased mtDNA copy number and reduced DNA methylation at exon 2 of POLGA compared to untreated controls. MtDNA depleted hAEC did not reconstitute their mtDNA copy number when they were allowed to recover for 7 and 14 days. The data were correlated with DNA methylation of POLGA at exon 2. Gene expression patterns indicated expression of some hepatocyte specific markers as well as pluripotency markers. I performed RNAseq to evaluate the transcriptome profile of hAEC, HLC and encapsulated HLC. When I compared hAEC to HLC, I found 1722 differentially regulated genes, of which 695 genes were upregulated and 1027 genes down regulated. I performed network analysis of the differentially regulated genes and found that the top networks to be affected were cell cycle control and increased interferon signalling. When I compared hAEC to encapsulated HLC, I found 1325 differentially regulated genes with 705 genes upregulated and 620 genes down regulated. Analysis of HLC and encapsulated HLC indicated 286 genes were differentially regulated of which 210 genes were upregulated and 76 genes were down regulated. Analysis of canonical pathway showed up regulation of FXR/RXR activation and down regulation of pattern recognition and interferon signalling. Network analysis exhibited increased cellular development, cellular movement and lipid metabolism. Overall, hAEC can be differentiated into functional HLC. However, hAEC are unable to synchronously modulate their mtDNA copy number during differentiation and become immunogenic. Encapsulation of HLC increases their functional capacity and reduces their inflammatory response, thus improved differentiation is achieved when HLC are encapsulated.