In Vitro Pluripotent Stem Cell Differentiation to Hepatocyte Ceases Further Maturation at an Equivalent Stage of E15 in Mouse Embryonic Liver Development

Abstract

Hepatocyte-like cells (HLCs) can be derived from pluripotent stem cells (PSCs) by sequential treatment of chemical cues to mimic the microenvironment of embryonic liver development. However, these HLCs do not reach the full maturity level of primary hepatocytes. In this study, we carried out a meta-analysis of cross-species transcriptome data of in vitro differentiation of human PSCs to HLCs and in vivo mouse embryonic liver development to identify the developmental stage at which HLC maturation was blocked at. Systematic variations were found associated with the data source and removed by batch correction. Using principal component analysis, HLCs from different stages of differentiation were aligned with mouse embryonic liver development chronologically. A "unified developmental time" (DT) scale was developed after aligning in vitro HLC differentiation and in vivo embryonic liver development. HLCs were found to cease further maturation at an equivalent stage of mouse embryonic day (E)13-15. Genes with discordant time dynamics were identified by aligning in vivo and in vitro data set onto a common DT scale. These genes may be targets of genetic intervention for enhancing the maturity of PSC-derived HLCs.

FOXA1/2 depletion drives global reprogramming of differentiation state and metabolism in a human liver cell line and inhibits differentiation of human stem cell-derived hepatic progenitor cells

“…Interestingly, these data are consistent with a meta-analysis of hPSC-liver differentiation studies in terms of factors that are aberrantly upregulated during liver differentiation (Raju, Chau et al 2018). Among the top 25 downregulated genes include those involved in energy metabolism (PPARGC1A (PPARG coactivator 1alpha)), liver development and liver function (albumin), and drug and steroid metabolism (AKRC12 (Aldo-Keto Reductase Family 1 Member C2)).…”

“…Further, we also observed evidence of changes in genes associated with metabolism (i.e. PPARGC in Table 1) which were also identified in the metanalysis (Raju, Chau et al 2018). A final connection between our study and the metanalysis is the fact that we observed increased Cdx2 expression in the shFoxa1/2 (-/-) liver cells (Fig.…”

“…2D). Thus, engineering and analyzing shFoxa1/2 (-/-) cells, we are recapitulating several aspects of a phenotype seen in all major seminal studies of human stem cell differentiation (Raju, Chau et al 2018). One way of interpreting this surprising connection is that the data suggests that Foxa1/2 mis-regulation is tied to problems in hPSC-liver differentiation by activation of inappropriate developmental factors in adult cells, loss of metabolic functions, and increased intestinal gene expression.…”

“…Overall, this suggests that the downregulation of shFoxa1/2 resulted in a downregulation of liver GRN, and resulted in a global block liver differentiation, while greatly altering differentiation state. The alteration of differentiation was characterized by clear activation of genes for alternate cell fates, as observed in a recent metanalysis of human stem-cell derived hepatic cells (Raju, Chau et al 2018). We wanted to know if any key downregulated genes were controlled by the hepatic nuclear factor network.…”

“…Another key finding of the Foxa1/2 -/-phenotype in our study is that it provides some relevance for our study for human stem cell research, which was also unexpected. Although there have been a series of key studies in the field that have advanced our knowledge of stem cell differentiation, a meta-analysis of 8 seminal studies hPSC-liver differentiation lists several major unsolved problems (Raju, Chau et al 2018): 1) the resulting hPSC-HEPS are fetal-like, 2) they express a set of undesired genes, including cardiac mesoderm and neural/eye development 3) they are not fully functional metabolically, 4) they are missing signals from liver development, and 5) they express increased amount of intestinal master factors. Interestingly, the shFoxa1/2 (-/-) phenotype in stable liver cells also demonstrates upregulation of genes cardiac and neural/eye morphogenesis (Fig.…”