Small molecule-mediated reprogramming of human hepatocytes into bipotent progenitor cells.

Currently, much effort is directed towards the development of new cell sources for clinical therapy using cell fate conversion by small molecules. Direct lineage reprogramming to a progenitor state has been reported in terminally differentiated rodent hepatocytes, yet remains a challenge in human hepatocytes. Human hepatocytes were isolated from healthy and diseased donor livers and reprogrammed into progenitor cells by 2 small molecules, A83-01 and CHIR99021 (AC), in the presence of EGF and HGF. The stemness properties of human chemically derived hepatic progenitors (hCdHs) were tested by standard in vitro and in vivo assays and transcriptome profiling. We developed a robust culture system for generating hCdHs with therapeutic potential. The use of HGF proved to be an essential determinant of the fate conversion process. Based on functional evidence, activation of the HGF/MET signal transduction system collaborated with A83-01 and CHIR99021 to allow a rapid expansion of progenitor cells through the activation of the ERK pathway. hCdHs expressed hepatic progenitor markers and could self-renew for at least 10 passages while retaining a normal karyotype and potential to differentiate into functional hepatocytes and biliary epithelial cells in vitro. Gene expression profiling using RNAseq confirmed the transcriptional reprogramming of hCdHs towards a progenitor state and the suppression of mature hepatocyte transcripts. Upon intrasplenic transplantation in several models of therapeutic liver repopulation, hCdHs effectively repopulated the damaged parenchyma. Our study is the first report of successful reprogramming of human hepatocytes to a population of proliferating bipotent cells with regenerative potential. hCdHs may provide a novel tool that permits expansion and genetic manipulation of patient-specific progenitors to study regeneration and the repair of diseased livers. Human primary hepatocytes were reprogrammed towards hepatic progenitor cells by a combined treatment with 2 small molecules, A83-01 and CHIR99021, and HGF. Chemically derived hepatic progenitors exhibited a high proliferation potential and the ability to differentiate into hepatocytes and biliary epithelial cells both in vitro and in vivo. This approach enables the generation of patient-specific hepatic progenitors and provides a platform for personal and stem cell-based regenerative medicine.

Kim Y ，Kang K ，Lee SB ，Seo D ，Yoon S ，Kim SJ ，Jang K ，Jung YK ，Lee KG ，Factor VM ，Jeong J ，Choi D ... -  《-》

Conversion of Terminally Committed Hepatocytes to Culturable Bipotent Progenitor Cells with Regenerative Capacity.

A challenge for advancing approaches to liver regeneration is loss of functional differentiation capacity when hepatocyte progenitors are maintained in culture. Recent lineage-tracing studies have shown that mature hepatocytes (MHs) convert to an immature state during chronic liver injury, and we investigated whether this conversion could be recapitulated in vitro and whether such converted cells could represent a source of expandable hepatocytes. We report that a cocktail of small molecules, Y-27632, A-83-01, and CHIR99021, can convert rat and mouse MHs in vitro into proliferative bipotent cells, which we term chemically induced liver progenitors (CLiPs). CLiPs can differentiate into both MHs and biliary epithelial cells that can form functional ductal structures. CLiPs in long-term culture did not lose their proliferative capacity or their hepatic differentiation ability, and rat CLiPs were shown to extensively repopulate chronically injured liver tissue. Thus, our study advances the goals of liver regenerative medicine.

Katsuda T ，Kawamata M ，Hagiwara K ，Takahashi RU ，Yamamoto Y ，Camargo FD ，Ochiya T ... -  《-》

Human chemically-derived hepatic progenitors (hCdHs) as a source of liver organoid generation: Application in regenerative medicine, disease modeling, and toxicology testing.

Several types of human stem cells from embryonic (ESCs) and induced pluripotent (iPSCs) to adult tissue-specific stem cells are commonly used to generate 3D liver organoids for modeling tissue physiology and disease. We have recently established a protocol for direct conversion of primary human hepatocytes (hPHs) from healthy donor livers into bipotent progenitor cells (hCdHs). Here we extended this culture system to generate hCdH-derived liver organoids for diverse biomedical applications. To obtain hCdHs, hPHs were cultured in reprogramming medium containing A83-01 and CHIR99021 for 7 days. Liver organoids were established from hCdHs (hCdHOs) and human liver cells (hLOs) using the same donor livers for direct comparison, as well as from hiPSCs. Organoid properties were analyzed by standard in vitro assays. Molecular changes were determined by RT-qPCR and RNA-seq. Clinical relevance was evaluated by transplantation into FRG mice, modeling of alcohol-related liver disease (ARLD), and in vitro drug-toxicity tests. hCdHs were clonally expanded as organoid cultures with low variability between starting hCdH lines. Similar to the hLOs, hCdHOs stably maintained stem cell phenotype based on accepted criteria. However, hCdHOs had an advantage over hLOs in terms of EpCAM expression, efficiency of organoid generation and capacity for directed hepatic differentiation as judged by molecular profiling, albumin secretion, glycogen accumulation, and CYP450 activities. Accordingly, FRG mice transplanted with hCdHOs survived longer than mice injected with hLOs. When exposed to ethanol, hCdHOs developed stronger ARLD phenotype than hLOs as evidenced by transcriptional profiling, lipid accumulation and mitochondrial dysfunction. In drug-induced injury assays in vitro, hCdHOs showed a similar or higher sensitivity response than hPHs. hCdHOs provide a novel patient-specific stem cell-based platform for regenerative medicine, toxicology testing and modeling liver diseases.

Salas-Silva S ，Kim Y ，Kim TH ，Kim M ，Seo D ，Choi J ，Factor VM ，Seo HR ，Song Y ，Choi GS ，Jung YK ，Kim K ，Lee KG ，Jeong J ，Shin JH ，Choi D ... -  《-》

Hepatic patch by stacking patient-specific liver progenitor cell sheets formed on multiscale electrospun fibers promotes regenerative therapy for liver injury.

Recently, use of cell sheets with bio-applicable fabrication materials for transplantation has been an attractive approach for the treatment of patients with liver failure. However, renewable and scalable cell sources for engineered tissue patches remain limited. We previously reported a new type of proliferating bipotent human chemically derived hepatic progenitor cells (hCdHs) developed by small molecule-mediated reprogramming. Here, we developed a patient-specific hepatic cell sheet constructed from liver biopsy-derived hCdHs on a multiscale fibrous scaffold by combining electrospinning and three-dimensional printing. Analysis of biomaterial composition revealed that the high-density electrospun sheet was superior in increasing the functional properties of hCdHs. Furthermore, the hepatic patch assembled by multilayer stacking with alternate cell sheets of hCdHs and human umbilical vein endothelial cells (HUVECs) recapitulated a liver tissue-like structure, with histological and morphological shape and size similar to those of primary human hepatocytes, and exhibited a significant increase in hepatic functions such as albumin secretion and activity of cytochrome P450 during in vitro hepatic differentiation compared with that in hCdH cells cultured in a two-dimensional monolayer. Interestingly, in the hepatic patch, the induction of functional hepatocytes was associated with both the electrospun fibrous-facilitated oncostatin M signaling and selective activation of AKT signaling by HUVECs. Notably, upon transplantation into a mouse model of therapeutic liver repopulation, the hepatic patch effectively repopulated the damaged parenchyma and induced the restoration of liver function with healthy morphology in the lobe and an improved survival rate (>70%) in mice. Overall, these results suggested that liver biopsy-derived hCdHs can be an efficient alternative source for developing hepatic cell sheets and patches with potential clinical applications in tissue engineering to advance liver regeneration.

Kim Y ，Kim YW ，Lee SB ，Kang K ，Yoon S ，Choi D ，Park SH ，Jeong J ... -  《-》

Generation of Hepatic Progenitor Cells from the Primary Hepatocytes of Nonhuman Primates Using Small Molecules.

Hee Hong D ，Lee C ，Kim Y ，Lee SB ，Han SC ，Kim SJ ，Yang HM ，Choi D ，Jeong J ，Ryu K ... -  《-》