Regulation of Maturation and Proliferation of Liver Progenitor Cells During Embryonic Development

Fetal hepatic progenitor cells, or hepatoblasts, demonstrate limited metabolic activities but support the hematopoietic cells (Kinoshita et al. 1999). Oncostatin M (OSM), an interleukin 6 family cytokine, can promote hepatic maturation, as determined by the induction of metabolic enzymes, accumulation of glycogen and lipids, and detoxification of ammonia. In particular, OSM was expressed in CD45+ hematopoietic cells in the mid-fetal livers, whereas the OSM receptor was mainly detected in hepatic cells (Kamiya et al. 1999). The interaction between extracellular matrix and integrin is important for terminal maturation of fetal hepatic cells (Kamiya et al.

2002). These results suggest that OSM produced from hematopoietic cells and extracellular matrices produced from non-parenchymal liver cells play pivotal roles in fetal liver development. However, these maturation factors, i.e., OSM and extracellular matrices could not induce high-level maturation of hepatic progenitor cells. Expression of several hepatic genes, in the progenitor-derived mature cells, stimulated by these factors was significantly lower than that in the adult hepatocytes (Kamiya et al. 1999; Si-Tayeb et al. 2010). It is thus suggested that unknown factors are required to induce high level of differentiation into mature hepatocytes.

Transcription factors are usually involved in maturation of stem/progenitor cells during embryonic development. Recently, we found that basic helix-loop-helix (bHLH) transcription factor Mist1 regulates the maturation of fetal hepatic progenitor cells in vitro (Chikada et al. 2015). Mist1 was identified as one of the bHLH transcription factors that binds to an E-box sequence and is known to play an important role in pancreatic acinar cell organization (Lemercier et al. 1997; Pin et al.

2001). At first, we analyzed expressional changes of Mist1 using our hepatic progenitor cell culture. Mist1 mRNA decreased in this culture without hepatic maturation factors, OSM and extracellular matrices. In contrast, this decrease was partially attenuated by the induction of hepatic maturation, suggesting that induction of Mist1 expression by OSM and extracellular matrices is involved in maturation of hepatic progenitor cells. Next, we analyzed the function of Mist1 for maturation of hepatic progenitor cells using the retrovirus-derived gene overexpression vector. Overexpression of Mist1 induced the expression of hepatic functional genes such as Cps1, Cytochrome P450 (Cyp) 3a11, Cyp2b9, and Cyp2b10. In contrast, the overexpression of Mist suppressed the expression of cholangiocytic markers such as Sox9, Sox17, cytokeratin (Ck) 19, and Grhl2. Next, we directly analyzed metabolism of a CYP3A-target substrate using mass spectrometry. The substrate of CYP3A, midazolam, was added into hepatic progenitor cell culture and the amounts of midazolam and the metabolite of midazolam, 1-hydroxymidazolam, were measured using liquid chromatography-tandem mass spectrometry. Overexpression of Mist1 in combination with OSM and extracellular matrices significantly increased the amount of the metabolite 1-hydroxymidazolam. These results suggested that hepatocyte-like cells with Mist1 overexpression have high expression and activity of drug metabolic enzymes in vitro.

During in vivo liver development, hepatic progenitor cells highly expand and differentiate into a large number of hepatocytes and cholangiocytes (Watanabe et al.

2002). These in vivo characteristics of hepatic progenitor cells have been elucidated by several transplantation experiments (Kakinuma et al. 2009; Oertel et al. 2008). However, high proliferative potential of hepatic progenitor cells is inhibited during in vitro culture. It is reported that genetic modification is required for long-term expansion of hepatic progenitor cells in the conventional culture system (Chiba et al. 2010). When purified hepatic progenitor cells are cultured at a low density, they can form several colonies on extracellular matrix-coated dishes. Fetal hepatic progenitor cells cultured on collagen-coated dishes mainly differentiated into CK19-positive cholangiocytic cells and the efficiency of colony formation is low. In contrast, when these cells were co-cultured with mesenchymal cells such as mouse embryonic fibroblasts (MEFs), most hepatic progenitor cells differentiated into albumin-positive hepatocyte-like cells and the efficiency of colony formation increased owing to the co-culturing with MEFs (Fig. 1.1). We also found that the addition of MEK inhibitor, PD0325901, can induce proliferation of hepatic progenitor cells in long-term culture. Primary hepatic progenitor cells derived from mid-fetal livers express several cell cycle related genes, such as p27cdkn1b, p57cd- kn1c, p18cdkn2c, and p19cdkn2d. Expression of p21cdkn1a and p16/19cdkn2a is barely detected in primary hepatoblasts. However, expression of these cdk inhibitors is highly induced during the expansion in vitro, indicating that the up-regulation of these inhibitors is involved in the low proliferative ability of hepatic progenitor cells in long-term in vitro culture. We found that the up-regulation of these inhibitors is induced by MEK-ERK signaling pathway. Thus, the addition of PD0325901 can induce long-term proliferative ability through the suppression of these cdk

Colony formation by hepatic progenitor cells co-cultured with mesenchymal cells

Fig. 1.1 Colony formation by hepatic progenitor cells co-cultured with mesenchymal cells. Expression of albumin (Alb, red) and CK19 (green) was detected. E13.5 CD45-Ter119-c-Kit- Dlk+CD133+ progenitor cells were cultured in the conventional H-CFU-C culture system (collagen coated) or MEF co-culture system (MEF co-cultured). After 6 days of culture, the cells were immunostained with anti-albumin and -CK19 antibodies. Nuclei were counterstained with DAPI (blue). (Reprinted with permission from ref. Kamiya et al. 2015 in STEM CELLS AND DEVELOPMENT, 2015, published by Mary Ann Liebert, Inc., New Rochelle, NY) inhibitors (Kamiya et al. 2015). Primary hepatic progenitor cells can expand and differentiate into functional hepatocytes by transplantation into pre-conditioned recipient mouse livers. However, the transplantation and recovery of the injured liver usually requires many donor cells because liver is a large organ. A few hepatic progenitor cells have been expanded, using our co-culture system with PD0325901, and transplanted into the injured livers. Progenitor cells with PD0325901 maintain their proliferative ability after in vitro expansion and can proliferate in the recipient livers. Using this culture system, 100 mouse hepatic progenitor cells can proliferate into almost 3 x 105 cells in the long-term culture. It is thus suggested that this technique allows a small number of hepatic progenitor cells to be used for cell transplantation for future regenerative therapies in severe liver diseases. MEK-ERK pathway is known to be involved in the progression of G0-G1 cell cycle progression (Coutant et al. 2002; Fremin et al. 2012). In contrast, it is concluded that high activation of MEK-ERK pathway in fetal hepatic progenitor cells induced cell cycle arrest through the accumulation of p16/19cdkn2a and its downstream target p21cdkn1a.

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