In the liver, unlike mature hepatocytes (MHs), it is known that a subpopulation of hepatocytes, called small hepatocytes (SHs), can proliferate in vitro1. SHs are a candidate cell source in transplantation therapy, but their proliferative capacity is still limited.
Our group has succeeded in culturing stem/progenitor cells, including rat embryonic stem (ES) cells2, rat p63+/Ck14+ mammary gland progenitor cells, and multipotent mammary tumor cells3, with the use of four small molecules, Y-27632 (ROCK inhibitor), PD0325901 (MAPK inhibitor), A-83-01 (TGF-beta receptor inhibitor), and CHIR99021 (GSK3 inhibitor). This experience led us to investigate whether any combinations of these small molecules would allow stable culture of SHs, a putative hepatic progenitor cells.
We investigated whether rat SHs, as well as MHs as negative control, would increase proliferative capacity in the presence of all the possible combinations of the four small molecules. To our surprise, in the presence of several combinations of the small molecules allowed proliferation not only in SHs but also in MHs. Of these combinations, Y-27632, A-83-01 and CHIR99021 (termed YAC) endowed MHs with the highest proliferative capacity, and thus we focused on the MH-derived proliferative cells induced by YAC.
YAC-induced cells resembled fetal liver progenitor cells (LPCs) named hepatoblasts in morphology. Indeed, YAC-induced cells exhibited a bidirectional differentiation into both hepatocytes and biliary epithelial cells in response to differentiation stimuli, which recapitulated the characteristics of LPCs. In addition, YAC-induced cells could be stably cultured for a long term through serial passages. Thus, we named these cells as chemically induced liver progenitors (CLiPs). We also confirmed that mouse MHs could be converted to CLiPs with YAC.
To evaluate their regenerative capacity, we transplanted rat CLiPs to immunodeficient mice with chronic liver injury. Eight weeks after transplantation, we confirmed that 75-90% of the mouse livers was replaced with rat hepatocytes without any tumorigenic symptoms. Moreover, we confirmed that CLiP-derived rat hepatocytes showed gene expression profiles indistinguishable from those of primary rat hepatocytes.
Finally, we determined the origin of rat CLiPs. We sorted rat MHs into 3 subpopulations based on their ploidy status, namely, 2c, 4c and 8c, and investigated their proliferative capacity. Whereas 8c MHs did not form colonies, 2c and 4c clearly showed colony forming capacity. However, the extensive proliferative capacity was observed only in 2c MHs, demonstrating that rat CLiPs were derived from 2c MHs.
The present study will contribute not only to progress in regenerative medicine, but also to basic liver biology. Between 2013 and 2014, there was a sensational finding in liver biology4–7. In liver with chronic injury, proliferating LPCs are often observed both in humans and rodents. Given that MHs decrease their proliferative capacity during continuous injury, it was believed for several decades that chronic injury activates the proliferation of a small population of LPCs which are kept dormant in normal livers. Contrary to this hypothesis, recent studies have demonstrated that such LPCs are derived from MHs which are reprogrammed during regeneration under chronic liver injury4–7. Our in vitro study has provided direct evidence for this observation. In addition, considering that in vitro experimental setting makes it easier to manipulate gene expression (e.g. knockdown / overexpression) or intracellular signaling activity (e.g. pathway inhibition), our study may help mechanistic understanding of this reprogramming phenomenon.
Written by: Takeshi Katsuda and Takahiro Ochiya
Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
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