Generation of Self-Renewing Hepatoblasts From Human Embryonic Stem Cells by Chemical Approaches

Abstract

Somatic stem cells play crucial roles in organogenesis and tissue homeostasis and regeneration and may ultimately prove useful for cell therapy for a variety of degenerative diseases and injuries; however, isolation and expansion of most types of somatic stem cells from tissues are technically challenging. Human pluripotent stem cells are a renewable source for any adult cell types, including somatic stem cells. Generation of somatic stem cells from human pluripotent stem cells is a promising strategy to get these therapeutically valuable cells. Previously, we developed a chemically defined condition for mouse hepatoblast self-renewal through a reiterative screening strategy. In the present study, we efficiently generated hepatoblasts from human embryonic stem cells by a stepwise induction strategy. Importantly, these human embryonic stem cell-derived hepatoblasts can be captured and stably maintained using conditions previously established for mouse hepatoblast self-renewal, which includes basal media supplemented with insulin, transferrin, sodium selenite, epidermal growth factor, glycogen synthase kinase 3 inhibitor, transforming growth factor b receptor inhibitor, lysophosphatidic acid, and sphingosine 1-phosphate. The cells can stably retain hepatoblast phenotypes during prolonged culture and can differentiate into mature hepatocytes through in vitro provision of hepatocyte lineage developmental cues. After being embedded into three-dimensional Matrigel, these cells efficiently formed bile duct-like structures resembling native bile duct tissues. These human embryonic stem cell-derived hepatoblasts would be useful as a renewable source for cell therapy of liver diseases. STEM CELLS TRANSLATIONAL MEDICINE 2015;4:1275-1282 SIGNIFICANCE Somatic stem cells have been proposed as promising candidates for cell-based therapy; however, isolation of somatic stem cells from adult tissues is usually invasive and technically challenging. In the present study, hepatoblasts from human embryonic stem cells were efficiently generated. These human hepatoblasts were then stably captured and maintained by a growth factor and small molecule cocktail, which included epidermal growth factor, glycogen synthase kinase 3 inhibitor, transforming growth factor b receptor inhibitor, lysophosphatidic acid, and sphingosine 1-phosphate. These human embryonic stem cell-derived hepatoblasts would be useful as a renewable source for cell therapy of liver diseases.

Synergistic modulation of signaling pathways to expand and maintain the bipotency of human hepatoblasts

“…In this study, we demonstrated that CHIR could improve the HB growth significantly, which was in line with recent reports [14, 25, 27]. However, CHIR treatment led to loss of HB identity and reversing the gene expression pattern from hepatic to an intestinal endoderm pattern.…”

“…In order to establish an optimized condition that promotes the expansion of HBs without diminishing their hepatic characteristics, as observed in our results and reported elsewhere [14, 17, 20], we screened for synergistically regulated signaling transductions (Fig. 3a).…”

“…An ideal strategy would be to enable directed differentiation of human iPSCs to HBs with simultaneous cell expansion, which would enable up-scaling hepatic cell production. The expansion of HBs derived from hPSCs has been previously reported [14–18]. However, these studies relied mainly only on biomarker analyses to evaluate the bipotency and lacked many of the functional analyses and confirmation through animal transplantation was limited.…”

“…Recent studies identified small molecules which are used in the expansion of human HBs in defined chemical conditions [17, 20, 21]. However, these proliferative HBs were refractory to mature hepatocyte induction, which results in low ALB-positive cells and poor metabolic activity [14]. One possible explanation for the inability to derive functional hepatocytes is unstable maintenance of HB phenotype during expansion, which may be due to an ambiguous mechanism.…”

Stem Cells Applications in Regenerative Medicine and Disease Therapeutics

“…These hepatocytes are excellent source for the endpoint screening of drugs for accurate prediction of clinical outcomes [ 20 ]. Generation of hepatic cells from ESCs can be achieved in multiple ways, as serum-free differentiation [ 21 ], chemical approaches [ 20 , 22 ], and genetic transformation [ 23 , 24 ]. These ESCs-derived hepatocytes are long lasting source for treatment of liver injuries and high throughput screening of drugs [ 20 , 23 , 24 ].…”