Development of human iPSC-derived quiescent hepatic stellate cell-like cells for drug discovery and in vitro disease modeling.

Hepatic stellate cells (HSCs) play a central role in the progression of liver fibrosis by producing extracellular matrices. The development of drugs to suppress liver fibrosis has been hampered by the lack of human quiescent HSCs (qHSCs) and an appropriate in vitro model that faithfully recapitulates HSC activation. In the present study, we developed a culture system to generate qHSC-like cells from human-induced pluripotent stem cells (hiPSCs) that can be converted into activated HSCs in culture. To monitor the activation process, a red fluorescent protein (RFP) gene was inserted in hiPSCs downstream of the activation marker gene actin alpha 2 smooth muscle (ACTA2). Using qHSC-like cells derived from RFP reporter iPSCs, we screened a repurposing chemical library and identified therapeutic candidates that prevent liver fibrosis. Hence, hiPSC-derived qHSC-like cells will be a useful tool to study the mechanism of HSC activation and to identify therapeutic agents.

Impaired differentiation of fetal hepatocytes in homozygous jumonji mice.

Homozygous jumonji (jmj(-)/jmj(-)) mice were previously shown to exhibit hepatic hypoplasia and defective hematopoiesis in the liver and die at around embryonic day 15.5 (E15.5), suggesting that jmj is essential for liver development. In order to gain insight into the mechanism of liver development, we analyzed the expression and function of jmj in fetal hepatocytes. The number of hepatocytes in jmj(-)/jmj(-) mice was markedly reduced in comparison with control mice and the expression of jmj in hepatocytes increased along with development. As jmj(-)/jmj(-) embryos die by E15.5, we employed an in vitro culture system in which fetal hepatocytes differentiate in response to oncostatin M. The proliferation potential of jmj(-)/jmj(-) hepatocytes was comparable to that of wild type cells in vitro, however maturation of hepatocytes as evidenced by the expression of liver enzymes such as tyrosine amino transferase was severely impaired by the jmj gene inactivation. These results suggested that jmj plays a pivotal role in the development of mid-fetal hepatocytes to the neonatal stage.

Monolayer culture conditions suitable for primitive erythroid cell differentiation from embryonic stem cells in vitro.

Embryonic stem (ES) cells effectively differentiated into primitive erythroid/mesodermal cells when grown in the absence of both a feeder layer and leukemia inhibitory factor (LIF). The formation of a three-dimensional structure, exogenous mesoderm induction factors and exogenous hematopoietic growth factors were not essential for their differentiation. Primitive erythroid cells were first detected on day 5 in the differentiation-permissive cultures. Differentiation into other mesodermal cells was always preceded by that into primitive erythroid cells. Precursor cells of erythroid cells but of other hematoid cells were also detected in this system. This model system is useful for studying the early steps of mesoderm formation in mouse embryogenesis.