Human Embryonic and Rat Adult Stem Cells with Primitive Endoderm-Like Phenotype Can Be Fated to Definitive Endoderm, and Finally Hepatocyte-Like Cells

Abstract

Stem cell-derived hepatocytes may be an alternative cell source to treat liver diseases or to be used for pharmacological purposes. We developed a protocol that mimics mammalian liver development, to differentiate cells with pluripotent characteristics to hepatocyte-like cells. The protocol supports the stepwise differentiation of human embryonic stem cells (ESC) to cells with characteristics of primitive streak (PS)/mesendoderm (ME)/definitive endoderm (DE), hepatoblasts, and finally cells with phenotypic and functional characteristics of hepatocytes. Remarkably, the same protocol can also differentiate rat multipotent adult progenitor cells (rMAPCs) to hepatocyte-like cells, even though rMAPC are isolated clonally from cultured rat bone marrow (BM) and have characteristics of primitive endoderm cells. A fraction of rMAPCs can be fated to cells expressing genes consistent with a PS/ME/DE phenotype, preceding the acquisition of phenotypic and functional characteristics of hepatocytes. Although the hepatocyte-like progeny derived from both cell types is mixed, between 10–20% of cells are developmentally consistent with late fetal hepatocytes that have attained synthetic, storage and detoxifying functions near those of adult hepatocytes. This differentiation protocol will be useful for generating hepatocyte-like cells from rodent and human stem cells, and to gain insight into the early stages of liver development.

NKX2-1 Activation by SMAD2 Signaling After Definitive Endoderm Differentiation in Human Embryonic Stem Cell

“…In vivo this occurs by signaling via Nodal. As has been demonstrated by others and us [23,24], we here demonstrate that transcripts for the mesendoderm (ME)/DE marker genes, including BRACHYURY, GSC, EOMES, and SOX17 were expressed maximally on d4 of differentiation in the presence of Activin-A (n = 3) (Supplementary Fig. 2B).…”

“…2B). In vivo, gastrulation and induction of foregut endoderm is also supported by canonical Wnt signaling [24,25]. Although addition of Wnt3a from d0 to d2 or d2 to d4, improved ME/DE marker gene expression, the differences with Activin-A alone were non-significant (n = 3) ( Fig.…”

Antagonism of Nodal signaling by BMP/Smad5 prevents ectopic primitive streak formation in the mouse amnion

“…H9 cells were induced to undergo initial steps of mesendoderm/liver differentiation as described previously (Roelandt et al, 2010). At day 4 of differentiation, cells were stimulated with 50 ng/ml activin (R&D) or 50 ng/ml activin and 50 ng/ml rhBMP4 for 90 minutes.…”

“…To obtain H9 cells that were fully BMP4 and activin responsive, cells were differentiated towards mesendoderm using a liver differentiation protocol (Roelandt et al, 2010). On day 4 of the differentiation, cells were co-stimulated with activin and BMP4, or with activin alone for 90 minutes.…”

Stem Cell Therapy for Liver Diseases

“…Adult stem cells are more capable of transdifferentiation than other cells. Liver regeneration is mainly an endogenous process, driven by hepatocytes and resident hepatic stem/progenitor cells [84]. It has been observed that certain populations of extra hepatic Adult Stem Cells can migrate into the liver and contribute to its repopulation and turnover [85].…”