Laminin-511 and laminin-521-based matrices for efficient hepatic specification of human pluripotent stem cells

Abstract

Human pluripotent stem cells (hPSCs) have gained a solid foothold in basic research and drug industry as they can be used in vitro to study human development and have potential to offer limitless supply of various somatic cell types needed in drug development. Although the hepatic differentiation of hPSCs has been extensively studied, only a little attention has been paid to the role of the extracellular matrix. In this study we used laminin-511, laminin-521, and fibronectin, found in human liver progenitor cells, as culture matrices for hPSC-derived definitive endoderm cells. We observed that laminin-511 and laminin-521 either alone or in combination support the hepatic specification and that fibronectin is not a vital matrix protein for the hPSC-derived definitive endoderm cells. The expression of the laminin-511/521-specific integrins increased during the definitive endoderm induction and hepatic specification. The hepatic cells differentiated on laminin matrices showed the upregulation of liver-specific markers both at mRNA and protein levels, secreted human albumin, stored glycogen, and exhibited cytochrome P450 enzyme activity and inducibility. Altogether, we found that laminin-511 and laminin-521 can be used as stage-specific matrices to guide the hepatic specification of hPSC-derived definitive endoderm cells.

Differences in definitive endoderm induction approaches using growth factors and small molecules

“…We earlier used the two hPSC lines WA07 and iPS(IMR90)‐4 in hepatic differentiation studies by using M1 media for 6 days in generation of DE (Kanninen, Harjumaki, et al, ; Kanninen, Porola, et al, ). We had also tested M5, but due to massive cell death we could not obtain enough DE cells for hepatic specification.…”

“…All the used primers and TaqMan Gene Expression Assay mixes are listed in Table . All primers were designed by Primer Express v2.0 software (Applied Biosystems) (Kanninen, Harjumaki, et al, ), except the primers for OCT4 (Yu et al, ) and HNF3B (D'Amour et al, ), and they were synthesized by Oligomer Oy (Helsinki, Finland). The relative quantification of each target gene in comparison with the housekeeping gene was made by a standard curve method based on a published mathematical model (Pfaffl, ).…”

“…Differentiation experiments were performed three times and analyzed on differentiation days 0, 1, 4, and 6. (Kanninen, Harjumaki, et al, 2016), except the primers for OCT4 (Yu et al, 2007) and HNF3B (D'Amour et al, 2005), and they were synthesized by Oligomer Oy (Helsinki, Finland). The relative quantification of each target gene in comparison with the housekeeping gene was made by a standard curve method based on a published mathematical model (Pfaffl, 2001).…”

Differential response to hepatic differentiation stimuli of amniotic epithelial cells isolated from four regions of the amniotic membrane

“…In the current study, a previously published protocol for the differentiation of hAEC into hepatocyte‐like cells was adapted for the use with commercially available culture substrates. Laminin 521, which is a major component of basal membranes, has been shown to successfully maintain hepatic function in human hepatocytes cultures, and to promote differentiation and maintenance of iPS‐derived hepatocytes . Here, we showed that the hepatic differentiation of hAEC cultured on laminin 521 was enhanced when compared to laminin 411 or Geltrex.…”

“…More recently, recombinant laminins, major components of basal membranes, have been shown to successfully maintain hepatic function in human hepatocytes in vitro . Moreover, laminin 411 and 521 were used for the differentiation and maintenance of induced pluripotent stem cells (iPS)‐derived hepatocytes . We tested the efficacy of laminin 411, 521 and Geltrex as a culture substrate for the differentiation of hAEC into hepatocyte‐like cells.…”