Efficiently generate functional hepatic cells from human pluripotent stem cells by complete small-molecule strategy.

In vitro differentiation of human pluripotent stem cells into hepatic cells and in vivo transplantation into mice

“We demonstrated that small-molecule cocktail CIP (including CHIR99021, IDE1, and PD0332991) efficiently induced definitive endoderm (DE) formation via increased endogenous TGF-β/Nodal signaling.”

Human pluripotent stem cells (iPSC line UC15 and ESC line H1) and immune-deficient NOD-SCID-IL2RG−/− mice

“Human PSC lines, including iPSC line (UC15) and ESC line (H1), were cultured in mTeSR1 medium (Stem Cell Technologies) on 100-fold-diluted Matrigel matrix (Growth Factor Reduced, BD Bioscience).”

n = 3 for in vitro experiments; 9 mice per group for in vivo survival analysis

“Data are presented as mean ± SEM, n = 3.”

In vitro differentiation and expansion over multiple days and passages; in vivo observation for 4 weeks

“Four weeks post-transplantation, Di1 and hALB co-positive cells were observed more widespread in HBs transplanted mice liver (Fig. 7G).”

The small-molecule cocktail CIP efficiently induced definitive endoderm formation by increasing endogenous TGF-β/Nodal signaling.

“We demonstrated that small-molecule cocktail CIP (including CHIR99021, IDE1, and PD0332991) efficiently induced definitive endoderm (DE) formation via increased endogenous TGF-β/Nodal signaling.”

The combination of Vitamin C, Dihexa, and Forskolin (VDF) substituted growth factors to induce hepatic specification with efficiency comparable to growth factor controls.

“Furthermore, we identified that combining Vitamin C, Dihexa, and Forskolin (VDF) could substitute growth factors to induce hepatic specification.”

Small-molecule-derived hepatoblasts expanded and matured into functional hepatocyte-like cells that repopulated injured liver in vivo.

“The small-molecule-derived HLCs displayed typical functional characteristics as mature hepatocytes in vitro and repopulating injured liver in vivo.”

Previous small-molecule protocols generally lacked hepatocyte-specific functional analyses in vitro and in vivo, and had lower differentiation efficiency than growth-factor-based approaches.

“So far, complete small-molecule-derived HLCs protocols have been reported in several works of the literature, with a lower hepatic differentiated efficiency than growth-factor-based approaches [6, 14–16].”