Factors from Human Embryonic Stem Cell-derived Fibroblast-like Cells Promote Topology-dependent Hepatic Differentiation in Primate Embryonic and Induced Pluripotent Stem Cells*

Abstract

The future clinical use of embryonic stem cell (ESC)-based hepatocyte replacement therapy depends on the development of an efficient procedure for differentiation of hepatocytes from ESCs. Here we report that a high density of human ESC-derived fibroblast-like cells (hESdFs) supported the efficient generation of hepatocyte-like cells with functional and mature hepatic phenotypes from primate ESCs and human induced pluripotent stem cells. Molecular and immunocytochemistry analyses revealed that hESdFs caused a rapid loss of pluripotency and induced a sequential endoderm-to-hepatocyte differentiation in the central area of ESC colonies. Knockdown experiments demonstrated that pluripotent stem cells were directed toward endodermal and hepatic lineages by FGF2 and activin A secreted from hESdFs. Furthermore, we found that the central region of ESC colonies was essential for the hepatic endoderm-specific differentiation, because its removal caused a complete disruption of endodermal differentiation. In conclusion, we describe a novel in vitro differentiation model and show that hESdF-secreted factors act in concert with regional features of ESC colonies to induce robust hepatic endoderm differentiation in primate pluripotent stem cells.

Characteristic Expression of Major Histocompatibility Complex and Immune Privilege Genes in Human Pluripotent Stem Cells and Their Derivatives

“…4 and Table 3). The stages of hepatocyte-like cell differentiation from stem cells were based on the previous report (24). The percentages of cells expressing HLA-II, HLA-G, and HLA-E remained low even after differentiation for 30 days in these cell lines (Tables 3-5).…”

“…The medium was changed every 3 days, and the cells were collected at predetermined time points. The details about stem cell-derived hepatocytes have recently been published by us (24), in which the progressive differentiation of stem cell-derived hepatocytes and the expression of hepatocytespecific markers in comparison to normal hepatocytes were included. The stem cells were also treated with interferon-g (IFN-g; 25 ng/ml for 48 h; Sigma) before or after differentiation in some experiments.…”

“…However, reports on hiPSCs and the comparison between multiple stem cell lines are scarce. This study therefore aimed to examine the immunogenicity of several hESC and hiPSC lines and their derivatives, including pluripotent stem cell-derived hepatocyte-like cells (24). The data from this report suggest that derivatives of hESCs or hiPSCs likely will induce an immune response, although characteristics of immune privilege could occur through lower expression levels of MHC, antigen-presenting cells, and the modulation of immune privilege genes.…”

“…We examined the MHC expression of two hESCs (H9 and NTU1 cells) and five hiPSC lines, respectively, from foreskin fibroblasts (iCFB46 and iCFB50) (24), granulosa cells (iGra1 and iGra2), and dermal papilla cells (iDPC3) (24), by microarray analysis (Fig. 1).…”

Mesenchymal Stem Cells: Possibilities of New Treatment Options

“…Moreover, cell fate specifi cation and maturation can be selectively altered via manipulation of endogenous developmental signaling pathways (Rathjen and Rathjen 2003 ;Meyer et al 2009 ) . The ability to differentiate iPS cells in vitro to specifi c lineages effi ciently and reproducibly has been achieved using those described protocols with certain modifi cations (Dimos et al 2008 ;Narazaki et al 2008 ;Tateishi et al 2008 ;Wernig et al 2008 ;Ebert et al 2009 ;Hu and Zhang 2009 ;Jin et al 2009 ;Karumbayaram et al 2009 ;Pfannkuche et al 2009 ;Senju et al 2009 ;Tanaka et al 2009 ;Taura et al 2009b ;Zhang et al 2009b ;Carvajal-Vergara et al 2010 ;Comyn et al 2010 ;Dick et al 2010 ;Gamm and Meyer 2010 ;Huang et al 2010 ;Kaichi et al 2010 ;Lamba et al 2010 ;Lee et al 2010 ;Martinez-Fernandez et al 2010 ;Parameswaran et al 2010 ;Swistowski et al 2010 ;Teramura et al 2010 ;Zhou et al 2010 ) .…”