Abstract
ABSTRACTDifferentiation of stem cells to hepatocytes provides an unlimited supply of human hepatocytes and therefore has been vigorously studied. However, to date, the stem cell-derived hepatocytes were suggested to be of immature features. To obtain matured hepatocytes from stem cells, we tested the effect of culturing human-induced pluripotent stem (hiPS) cell-derived endoderm cells on collagen vitrigel membrane and compared with our previous reported nanofiber matrix. We cultured hiPS cell-derived endoderm cells on a collagen vitrigel membrane and examined the expression profiles, and tested the activity of metabolic enzymes. Gene expression profile analysis of hepatocytic differentiation markers revealed that upon culture on collagen vitrigel membrane, immature markers of AFP decreased, with a concomitant increase in the expression of mature hepatocyte transcription factors and mature hepatocyte markers such as ALB, ASGR1. Mature markers involved in liver functions, such as transporters, cytochrome P450 enzymes and phase II metabolic enzymes were also upregulated. We observed the upregulation of the liver markers for at least 2 weeks. Gene array profiling analysis revealed that hiPS cell-derived hepatocyte-like cells (hiPS-hep) resemble those of the primary hepatocytes. Functions of the CYP enzyme activities were tested in multi-institution and all revealed high CYP1A, CYP2C19, CYP2D6, CYP3A activity, which could be maintained for at least 2 weeks in culture. Taken together, the present approach identified that collagen vitrigel membrane provides a suitable environment for the generation of hepatocytes from hiPS cells that resemble many characteristics of primary human hepatocytes.
Highlights
The human liver is an important organ that performs many complex functions in metabolism and biosynthesis, the storage of essential nutrients, and biotransformation, including detoxification and bioactivation of drugs
Functional analysis of the metabolic enzyme activities of the human-induced pluripotent stem (hiPS)-derived hepatocytes Since CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 are the main cytochrome P450 enzymes that are involved in drug metabolism in the liver, we examined their enzymatic activities in the hiPS-hep
The hiPS-endo cultured on the collagen vitrigel (CV) membrane gradually downregulated the expression of immature markers of AFP or APOA4, upregulated mature hepatocyte transcription factors, such as Prospero homeobox 1 (PROX1), Hematopoietically expressed homeobox (HHEX), and nuclear receptors such as nuclear receptor subfamily 1H4 (NR1H4), and nuclear receptor 1I3 (NR1I3)
Summary
The human liver is an important organ that performs many complex functions in metabolism and biosynthesis, the storage of essential nutrients, and biotransformation, including detoxification and bioactivation of drugs. The hiPS cell-derived hepatocytes grown on the nanofiber matrix expressed higher levels of differentiation markers.
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