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

Abstract

Over the last several years, a method has emerged that endows adult hepatocytes with in vitro proliferative capacity, producing chemically induced liver progenitors (CLiPs). However, there is a growing controversy regarding the origin of these cells. Here, we provide lineage tracing-based evidence that adult hepatocytes acquire proliferative capacity in vitro using rat and mouse models. 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 epithelial cell-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 injured mouse livers, reinforcing our earlier argument that CLiPs can be a cell source for liver regenerative medicine. This study advances our understanding of the origin of CLiPs and motivates the application of this technique in liver regenerative medicine.

Keywords: biliary epithelial cell; cell of origin; cell transplantation therapy; cellular plasticity; chemically induced liver progenitors; hepatocyte; repopulation; reprogramming.

Graphical abstract

Figure 1

In vitro lineage tracing of rat hepatocytes (A) Schematic representation of in vitro lineage tracing of rat hepatocytes. (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. Scale bars: 100 μm.

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. Scale bars: 100 μm. (C) Existence of the contaminated BECs becomes evident around 5 days after plating. Scale bars: 100 μm. (D) YFP− epithelial cells express both HNF1B and EPCAM, while YFP+ cells express only HNF1B. Scale bars: 100 μm. (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 hepatic induction of hepCLiPs and bilCLiPs. Images obtained for one of the hepCLiP clones are shown as a representative example. Scale bars: 100 μm. (D) Gene set enrichment analysis (GSEA) comparing hepCLiPs cultured under 2D and 3D conditions using sets of hepatocyte-enriched genes (n = 3,432) compared with BECs and BEC-enriched genes (n = 2,048) compared with hepatocytes (Merrell et al., 2021) (n = 3 hepCLiP clones established from two donors; n = 3 bilCLiP clones established from one donor). (E) PCA mapping of hepCLiPs and bilCLiPs cultured in 2D and 3D (n = 3) 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 (n = 3,432) and BEC (n = 2,048) marker genes as assessed by RNA-seq with clonal hepCLiPs (n = 3 clones from two donors) and bilCLiPs (n = 3 clones from one donor) along with DDC-induced in vivo reprogrammed cells (n = 3).

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. Global p value among the three groups was calculated by the Kruskal-Wallis test. (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. Global p value among the three groups was calculated by the Kruskal-Wallis test. (E) Stitched image of an H&E-stained hepCLiP-repopulated FRG mouse liver tissue (left), and the CFP IF and DAPI counterstained images of the corresponding region are shown (right). A TrypLE-dissociated hepCLiP_3D-transplanted sample is shown as a representative image. Similar staining patterns are observed for the other two groups as shown in Figure S5. Scale bars: 1 mm. (F) A higher magnification image of the region indicated by the rectangle in the H&E staining of Figure 4E. Scale bar: 50 μm.

Figure 5

Histological characterization of hepCLiP-repopulated FRG mouse livers IF images of (A) CFP, FAH, and KRT19, where arrows indicate atypically expanding ductal cells (known as ductular reaction); (B) CFP, GLUL, and ABCB11 in a pericentral region; (C) CFP, GLUL, and ABCB11 in a periportal region; (D) CFP and CYP2E1; (E) CFP and SOX9, where arrows indicate hepatocytes weakly expressing SOX9. The nuclei of all the IF samples are counterstained with DAPI. All the images shown are taken from the hepCLiP_3D_TrypLE samples (Figure 5A), but we confirmed similar staining patterns for both hepCLiP_2D_TrypLE and hepCLiP_3D_Accutase samples (data not shown). CV, central vein; PV, portal vein. Scale bars: 50 μm.