Abstract
BackgroundPluripotent stem cells (PSCs) such as embryonic stem cells and induced pluripotent stem cells are promising target cells for cell regenerative medicine together with recently advanced technology of in-vitro differentiation. However, residual undifferentiated stem cells (USCs) during in-vitro differentiation are considered a potential risk for development of cancer cells and nonspecific lineage cell types. In this study we observed that USCs still exist during hepatic differentiation, consequently resulting in poor quality of the hepatic population and forming teratoma in vivo. Therefore, we hypothesized that effectively removing USCs from in-vitro differentiation could improve the quality of the hepatic population and guarantee safety from risk of teratoma formation.MethodsHuman PSCs were differentiated to hepatocytes via four steps. YM155, a known BIRC5 inhibitor, was applied for removing the residual USCs on the hepatic differentiation. After YM155 treatment, hepatocyte development was evaluated by measuring gene expression, immunostaining and hepatic functions at each stage of differentiation, and forming teratomas were confirmed by cell transplantation with or without YM155.ResultsThe selected concentrations of YM155 removed USCs (NANOG+ and OCT4+) in a dose-dependent manner. As a result, expression of endodermal markers (SOX17, FOXA2 and CXCR4) at stage II of differentiation and hepatic markers (ALB, AFP and HNF4A) at stage III was up-regulated by YM155 treatment as well as the hepatic population (ALB+), and functions (ALB/urea secretion and CYP450 enzyme activity) were enhanced at the final stage of differentiation (stage IV). Furthermore, we demonstrated that NANOG and OCT4 expression remaining until stage III (day 15 of differentiation) completely disappeared when treated with YM155 and teratoma formation was effectively prevented by YM155 pretreatment in the in-vitro study.ConclusionsWe suggest that the removal of USCs using YM155 could improve the quantity and quality of induced hepatocytes and eliminate the potential risk of teratoma formation.
Highlights
Pluripotent stem cells (PSCs) such as embryonic stem cells and induced pluripotent stem cells are promising target cells for cell regenerative medicine together with recently advanced technology of in-vitro differentiation
Hepatic differentiation of human PSCs with the modified protocol To achieve hepatic differentiation, human PSCs were differentiated to hepatocytes for 20 days via four sequential stages; definitive endoderm (5 days), hepatic endoderm (5 days), hepatic specification (5 days) and hepatic maturation (5 days) (Fig. 1a)
Transcription factor GATA-4 (GATA4), Sex determining region Y-Box 17 (SOX17), Forkhead box protein area 2 (A2) (FOXA2) and C-X-C chemokine receptor type 4 (CXCR4) as definitive endodermal markers were highly expressed at stage I, and expression diminished thereafter
Summary
Pluripotent stem cells (PSCs) such as embryonic stem cells and induced pluripotent stem cells are promising target cells for cell regenerative medicine together with recently advanced technology of in-vitro differentiation. The residual USCs exist long term in both in-vitro and in-vivo differentiation [10] This is a considerably critical concern in cell replacement therapy because the residual stem cells have inherent problems (i.e., teratoma formation) [11,12,13]. Small molecules have been suggested to effectively eliminate the risk of teratomas [17, 18] Of these molecules, YM155 is one of the inhibitors targeting the BIRC5 (Survivin) gene, an anti-apoptotic factor, which is highly overexpressed in ESCs/iPSCs and cancer stem cells. YM155 is one of the inhibitors targeting the BIRC5 (Survivin) gene, an anti-apoptotic factor, which is highly overexpressed in ESCs/iPSCs and cancer stem cells This molecule can induce selective apoptotic cell death of USCs [18]. The compound has been introduced in clinical applications for cancer therapy and interruption of teratoma formation after stem cell therapy [19]
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