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.
Highlights
Many groups are investigating alternative sources of functional hepatocyte-like cells to alleviate the shortage of human hepatocytes needed for cell replacement therapies and for pharmaceutical applications [1,2,3,4,5]
We evaluated if this protocol would support hepatic differentiation of adult cell types with higher differentiation potential, such as rat multipotent adult progenitor cells. rMAPC are isolated clonally from cultured rat bone marrow (BM). rMAPC are characterized by the expression of genes associated with pluripotency such as Oct4, Rex-1 and Sall4, genes used to generate iPSC such as Klf2/4, n/c-Myc and Lin28, and genes typical for primitive endoderm (PrE), such as Foxa2, Sox7, Sox17, Gata4, Gata6 [11,12,13]
Some studies have concluded that differentiation of hESC leads to a more homogeneous population of albumin protein expressing hepatocyte-like cells than we demonstrate here, the functional properties we demonstrate for non-purified hESC progeny, including albumin secretion [31,32,33,34,35], urea secretion [34,35,36] and cytochrome P450 activity [33,34,36,37] are in line, or even more robust than observed in reportedly more homogeneous progeny
Summary
Many groups are investigating alternative sources of functional hepatocyte-like cells to alleviate the shortage of human hepatocytes needed for cell replacement therapies and for pharmaceutical applications [1,2,3,4,5]. Few studies have addressed if differentiation to hepatocyte-like cells in vitro occurs via similar developmental steps as liver development in vivo, nor have they proven to be applicable to pluripotent cells from different species, or for adult stem cell differentiation. Embryogenesis requires that the many differentiated cells generated from totipotent stem cells are correctly assembled in the different embryonic as well as extra-embryonic tissues. This occurs in a well orchestrated stepwise process, whereby totipotent stem cells are first fated to trophectoderm that differentiate solely to the extra-embryonic trophoblast, or to pluripotent cells in the inner cell mass (ICM)[9]. For a cell to become a terminally differentiated cell type, it must undergo all consecutive steps of development to be able to respond to the surrounding differentiation cues
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