Abstract
SummaryStem cell-derived somatic cells represent an unlimited resource for basic and translational science. Although promising, there are significant hurdles that must be overcome. Our focus is on the generation of the major cell type of the human liver, the hepatocyte. Current protocols produce variable populations of hepatocytes that are the product of using undefined components in the differentiation process. This serves as a significant barrier to scale-up and application. To tackle this issue, we designed a defined differentiation process using recombinant laminin substrates to provide instruction. We demonstrate efficient hepatocyte specification, cell organization, and significant improvements in cell function and phenotype. This is driven in part by the suppression of unfavorable gene regulatory networks that control cell proliferation and migration, pluripotent stem cell self-renewal, and fibroblast and colon specification. We believe that this represents a significant advance, moving stem cell-based hepatocytes closer toward biomedical application.
Highlights
Significant advances in cell-based therapies, in the liver, represent promising alternatives to whole-organ transplantation
Differentiation procedures have advanced over the last decade, and efficient protocols to generate stem cell-derived hepatocyte-like cells (HLCs) from either human embryonic stem cells or induced pluripotent stem cells exist (Lavon et al, 2004; Hay et al, 2007, 2008, 2011; Cai et al, 2007; Duan et al, 2007; Basma et al, 2009; Sullivan et al, 2010; Touboul et al, 2010; Si-Tayeb et al, 2010; Rashid et al, 2010; Payne et al, 2011; Zhou et al, 2012, 2014; Medine et al, 2013; Takayama et al, 2013a; Szkolnicka et al, 2014a)
Differentiation of Pluripotent Stem Cells Toward Hepatocytes human embryonic stem cells (hESCs) were replated onto Matrigel and wells coated with pure laminin 521 and the laminin 521:111 mix (1:3 ratio, hereafter referred to as laminin 111 mix [L111]). hESCs were differentiated using our standard protocol, outlined in Figure 1, and analyzed at key time points. hESCs on all three matrices adhered, proliferated, and differentiated into HLCs
Summary
Significant advances in cell-based therapies, in the liver, represent promising alternatives to whole-organ transplantation. Differentiation procedures have advanced over the last decade, and efficient protocols to generate stem cell-derived hepatocyte-like cells (HLCs) from either human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) exist (Lavon et al, 2004; Hay et al, 2007, 2008, 2011; Cai et al, 2007; Duan et al, 2007; Basma et al, 2009; Sullivan et al, 2010; Touboul et al, 2010; Si-Tayeb et al, 2010; Rashid et al, 2010; Payne et al, 2011; Zhou et al, 2012, 2014; Medine et al, 2013; Takayama et al, 2013a; Szkolnicka et al, 2014a). These prototype systems have provided confidence in pluripotent stem cell technologies, their amenability to defined scale-up with clinical-grade hESC lines has not been achieved
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