Evidence for in vitro extensive proliferation of adult hepatocytes and biliary epithelial cells

Abstract

Over the last several years, a method has emerged which endows adult hepatocytes with in vitro proliferative capacity, producing chemically-induced liver progenitors (CLiPs). However, a recent study questioned the origin of these cells, suggesting that resident liver progenitor cells, but not hepatocytes, proliferate. Here, we provide lineage tracing-based evidence that adult hepatocytes acquire proliferative capacity in vitro . Unexpectedly, we also found that the CLiP method allows biliary epithelial cells to acquire extensive proliferative capacity. Interestingly, after long-term culture, hepatocyte-derived cells (hepCLiPs) and biliary-derived cells (bilCLiPs) become similar in their gene expression patterns, and they both exhibit differentiation capacity to form hepatocyte-like cells. Finally, we provide evidence that hepCLiPs can repopulate chronically injured mouse livers, reinforcing our earlier argument that CLiPs can be a cell source for liver regenerative medicine. Moreover, this study offers bilCLiPs as a potential cell source for liver regenerative medicine.

Figure 1.. In vitro lineage tracing of rat MHs.

(A) Schematic representation of in vitro lineage tracing of rat MHs. (B) Phase contrast and the corresponding tdTomato fluorescent images of rat hepatocytes cultured with or without YAC at the designated time points. Arrows indicate the single tdTomato-labeled hepatocyte identified at Day 1 in this microscopic field.

Figure 2.. Mouse hepatocytes proliferate and undergo partial biliary reprogramming, while contaminated BECs gradually dominate the population.

(A) Schematic representation of in vitro lineage tracing of mouse hepatocytes. (B) Confirmation of biphenotypic marker expression of YAC-treated proliferative hepatocytes. (C) Existence of the contaminated BECs becomes evident around 5 days after plating. (D) YFP− epithelial cells express both Hnf1b and Epcam, while YFP+ cells express only Hnf1b. (E) Quantification of YFP+ hepatocyte-derived population by flow cytometry throughout the continued passages. Date represent mean ± SEM (n = 4 donors). (F) Quantification of BEC marker expression in hepatocyte-derived YFP+ cells and that in contaminated YFP− BECs by flow cytometry. Fresh BECs are defined as cells positive for Epcam or Cd24, and the positive values are set to 100%. Date represent mean ± SEM (n = 4 donors).

Figure 3.. Both hepCLiPs and bilCLiPs exhibit BEC-like phenotypes under 2D culture, while they become more hepatocyte-like under the 3D culture.

(A) Schematic of strategy to establish hepCLiPs and bilCLiPs. (B) Heatmap of hepatic and BEC markers as assessed by qRT-PCR with clonal hepCLiPs (n = 23), bilCLiPs (n = 11), fresh hepatocytes (n = 6), and fresh BECs (n = 5). (C) Schematic of 3D culture-based hepatocyte induction of hepCLiPs and bilCLiPs. Images obtained for one of the hepCLiP clones are shown as a representative example. (D) Gene set enrichment analysis (GSEA) comparing hepCLiPs cultured under 2D and 3D conditions using sets of hepatocyte-enriched genes compared with BECs and BEC-enriched genes compared with hepatocytes (n = 2 independent donor-derived hepCLiPs). (E) PCA mapping of hepCLiPs cultured in 2D and 3D (n = 2) along with in vivo reprogrammed cells (n = 3). In vivo samples were harvested from normal mouse livers and those under hepatobiliary reprogramming induced by challenging the mice with 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). For in vivo reprogramming experiments, the cells were harvested by FACS. (F) Heatmap of hepatic and BEC markers as assessed by qRT-PCR with clonal hepCLiPs (n = 6 clones from 2 donors) and bilCLiPs (n = 6 clones from 1 donor).

Figure 4.. Mouse hepCLiPs repopulate chronically injured mouse livers.

(A) Schematic of experimental design for the establishment of CFP-labeled clonal hepCLiPs and the repopulation assay for one of these CFP+ hepCLiP clones using the FRG mouse system. (B) Macroscopic brightfield and fluorescent images of the FRG mouse livers. Livers were harvested from the host mice which were treated with the nitisinone cycle for 3.8 months. (C) Estimation of repopulation efficiency based on the gross fluorescent images described in (B). The horizontal bars indicate the mean values. (D) Number of nodules visible on the liver surface were counted on each image shown in (B) and represented as “per transplanted 5 × 10⁵ cells” (note that 5 × 10⁵ cells/mouse were transplanted for 2D_TrypLE and 3D_TrypLE groups, while 2.5 × 10⁵ cells/mouse were transplanted for 3D_Accutase. See Experimental Procedures for details). The horizontal bars indicate the mean values.