It has been reported that LPC
It has been reported that LPC-like A-443654 Supplier were established from hESCs/hiPSCs (Takayama et al., 2013; Yanagida et al., 2013; Zhao et al., 2009), and these cells were shown to proliferate and differentiate into hepatocyte-like cells or cholangiocyte-like cells. These LPCs were either isolated by cell sorting using a combination of specific cell surface markers or generated by adenovirus-mediated gene transfer to promote hepatic lineage differentiation. To develop an efficient culture system for large-scale production of mature functional hepatocytes, our aim was to identify a specific cell surface marker for isolating hiPSC-derived LPCs. In this study, we identified carboxypeptidase M (CPM) as a cell surface marker for hepatoblasts. CPM was also upregulated in hiPSC-derived cells during hepatic differentiation, and the sorted CPM+ cells exhibited features typical of hepatoblasts. Moreover, we developed a highly efficient and reliable culture system for hiPSC-derived LPCs capable of proliferating and differentiating into both hepatocytes and cholangiocytes in vitro.
Discussion Generation of mature hepatocytes and cholangiocytes from iPS/ES cells requires time-consuming multiple processes with expensive cytokines. Therefore, it would be beneficial to derive expandable precursor cells to simplify the procedure. Because LPCs are able to proliferate extensively in vitro and differentiate to both hepatocytes and cholangiocytes (Suzuki et al., 2000; Tanimizu et al., 2003, 2007), they are ideal cells to use in developing an efficient protocol for large-scale production of mature liver cells. In order to isolate LPCs, we first tested the expression of DLK1 and CXCR4, which are well known to be expressed in hepatoblasts. However, neither of these markers was appropriate for enriching LPCs from hiPSC-derived cells, as described above. We therefore searched for another marker and showed that CPM is a cell surface antigen expressed on hepatoblasts in mouse fetal liver between E11.5 and E17.5, although its expression is dramatically downregulated in mature hepatocytes and cholangiocytes. CPM is a member of the carboxypeptidase family, expressed on the cell surface to catalyze the release of C-terminal arginine or lysine residues of peptides and proteins (Skidgel et al., 1989). While its role in hepatoblasts is currently unknown, we found that the expression of CPM was gradually upregulated along with hepatic differentiation from hiPSC-like liver development. The simple method of single step cell sorting based on CPM expression made it possible to enrich the LPC fraction after induction of hiPSCs to the immature hepatocyte stage. These CPM+ cells exhibited a high proliferative potential and the expanded cells could be cryopreserved. Moreover, they expressed various liver progenitor markers (Figure 2H). While most CPM+ cells expressed HNF4α (Figure 2E), expression of midgut/hindgut markers such as CDX2 and PDX1 was also detected by RT-PCR (data not shown), suggesting that CPM+ cells may contain non-liver progenitors. However, after induction of hepatic differentiation, almost all cells became hepatocytes as shown by their morphology and ALB expression (Figures 3B, 3C, and S3A). If such non-hepatic progenitors were present in the CPM+ cells, they did not affect hepatocyte differentiation. CPM+ LPCs were able to differentiate in a single step culture to either hepatocyte-like cells or cholangiocyte-like cells depending on the culture condition. Furthermore, hepatocytes derived from CPM+ cells exhibited a significantly higher level of metabolic activity compared to the hiPSC-derived hepatocytes using a conventional differentiation protocol. Importantly, these hepatocyte-like cells remain phenotypically stable for more than 2 weeks (Figure S3B). Thus, CPM+ LPCs derived from hiPSCs will be useful for developing a reliable long-term hepatocyte culture system, and this simplified purification method will contribute to basic and clinical research related to liver diseases. Although CPM+ hepatocytes highly expressed mature hepatic genes involved in glucogenesis (G6PC, PCK1) and the urea cycling (CPS1), they exhibited variable levels of CYP expression compared with cultured primary human hepatocytes (Figure S3C). It is well known that the capacity to metabolize drugs is variable due to genetic polymorphisms in CYPs (Ingelman-Sundberg et al., 2007). Hepatocyte-like cells differentiated from iPSCs are highly variable due to retention of donor-specific metabolic capacity (Takayama et al., 2014), suggesting that the expression of CYPs in CPM+ Hepatocytes may be affected by a donor’s genetic background.