CPM Is a Useful Cell Surface Marker to Isolate Expandable Bi-Potential Liver Progenitor Cells Derived from Human iPS Cells

To develop a culture system for large-scale production of mature hepatocytes, liver progenitor cells (LPCs) with a high proliferation potential would be advantageous. We have found that carboxypeptidase M (CPM) is highly expressed in embryonic LPCs, hepatoblasts, while its expression is decreased along with hepatic maturation. Consistently, CPM expression was transiently induced during hepatic specification from human-induced pluripotent stem cells (hiPSCs). CPM+ cells isolated from differentiated hiPSCs at the immature hepatocyte stage proliferated extensively in vitro and expressed a set of genes that were typical of hepatoblasts. Moreover, the CPM+ cells exhibited a mature hepatocyte phenotype after induction of hepatic maturation and also underwent cholangiocytic differentiation in a three-dimensional culture system. These results indicated that hiPSC-derived CPM+ cells share the characteristics of LPCs, with the potential to proliferate and differentiate bi-directionally. Thus, CPM is a useful marker for isolating hiPSC-derived LPCs, which allows development of a large-scale culture system for producing hepatocytes and cholangiocytes

Investigation of the hepatic development in the coculture of hiPSCs-derived LSECs and HLCs in a fluidic microenvironment

Interactions between the different liver cell types are critical to the maintenance or induction of their function in vitro. In this work, human-induced Pluripotent Stem Cells (hiPSCs)-derived Liver Sinusoidal Endothelial Cells (LSECs) and Hepatocytes-Like Cells (HLCs) were cultured and matured in a microfluidic environment. Both cell populations were differentiated in Petri dishes, detached, and inoculated in microfluidic biochips. In cocultures of both cell types, the tissue has exhibited a higher production of albumin (3.19 vs 5.31 μg/mL/106 cells in monocultures and cocultures) as well as a higher inducibility CYP450 over monocultures of HLCs. Tubular-like structures composed of LSECs and positive for the endothelial marker PECAM1, as well as a tissue more largely expressing Stabilin-2 were detected in cocultures only. In contrast, monocultures exhibited no network and less specific endothelial markers. The transcriptomic analysis did not reveal a marked difference between the profiles of both culture conditions. Nevertheless, the analysis allowed us to highlight different upstream regulators in cocultures (SP1, EBF1, and GATA3) and monocultures (PML, MECP2, and NRF1). In cocultures, the multi-omics dataset after 14 days of maturation in biochips has shown the activation of signaling related to hepatic maturation, angiogenesis, and tissue repair. In this condition, inflammatory signaling was also found to be reduced when compared to monocultures as illustrated by the activation of NFKB and by the detection of several cytokines involved in tissue injury in the latter. Finally, the extracted biological processes were discussed regarding the future development of a new generation of human in vitro hepatic models

Investigation of the hepatic development in the coculture of hiPSCs-derived LSECs and HLCs in a fluidic microenvironment

International audienceInteractions between the different liver cell types are critical to the maintenance or induction of their function in vitro. In this work, human-induced Pluripotent Stem Cells (hiPSCs)-derived Liver Sinusoidal Endothelial Cells (LSECs) and Hepatocytes-Like Cells (HLCs) were cultured and matured in a microfluidic environment. Both cell populations were differentiated in Petri dishes, detached, and inoculated in microfluidic biochips. In cocultures of both cell types, the tissue has exhibited a higher production of albumin (3.19 vs 5.31 μg/mL/106 cells in monocultures and cocultures) as well as a higher inducibility CYP450 over monocultures of HLCs. Tubular-like structures composed of LSECs and positive for the endothelial marker PECAM1, as well as a tissue more largely expressing Stabilin-2 were detected in cocultures only. In contrast, monocultures exhibited no network and less specific endothelial markers. The transcriptomic analysis did not reveal a marked difference between the profiles of both culture conditions. Nevertheless, the analysis allowed us to highlight different upstream regulators in cocultures (SP1, EBF1, and GATA3) and monocultures (PML, MECP2, and NRF1). In cocultures, the multi-omics dataset after 14 days of maturation in biochips has shown the activation of signaling related to hepatic maturation, angiogenesis, and tissue repair. In this condition, inflammatory signaling was also found to be reduced when compared to monocultures as illustrated by the activation of NFKB and by the detection of several cytokines involved in tissue injury in the latter. Finally, the extracted biological processes were discussed regarding the future development of a new generation of human in vitro hepatic models