Human Hepatic Progenitor Cells

ERα and ERβ Promote Hepatic Growth and Differentiation through Interaction with Proteins and Regulation of Downstream Target Genes

Improving liver regeneration (LR) capacity and thereby liver function reserve is a critical bridging strategy for managing liver failure patients. The correlation between an increase of nuclear estrogen receptor (ER) with the onset of DNA synthesis in LR has been reported for decades. This paper demonstrated that estrogen signals orchestrate hepatic repopulation and differentiation viadistinct transcriptome patterns governed by Erα or ERβ.

To examine the role of ERβ in hepatic differentiation, human hepatic progenitor cells (HepRG) were treated with E2, PPT, or the ERβ-specific ligand DPN for 2 and 4 days. Hepatocyte (Alb, AFP, G6PD, GST) and cholangiocyte (KRT19, CECAM1, TMSb4x) markers, along with the liver progenitor marker HNF4α, were monitored. After 2 days, DPN significantly upregulated these markers, unlike E2 and PPT (Fig. 1c). After 4 days, only DPN showed a marked increase in expression (Fig. 1d). Moreover, knocking down Ube3a (one unique ERβ-interacting protein) reduced Ifna5 and albumin expression, while DPN treatment dramatically upregulated Ifna5 (Fig. 1e, lane 1 vs. lane 2). Ube3a knockdown also nullified DPN-induced gene expression (Fig. 1e, lane 3 vs. lane 4), indicating Ube3a's role in ERβ-specific gene expression and hepatocyte differentiation.

Overall, estrogenic signals either promote cell growth via the ERα-Chd1 axis or facilitate hepatic differentiation via the ERβ-Ube3a axis (Fig. 1). Thus, ERα and ERβ are crucial for ensuring tissue quality during liver regeneration.

Fig. 1. The ERα→Chd1 axis for cell proliferation and the ERβ→Ube3a axis for liver function/differentiation in vitro. (Kao TL., Kuan YP., et al., 2018)

Isolation and Characterization of Extracellular Vesicles Derived from Human Hepatic Progenitor Cells

Frostbite is a cold-induced tissue injury that occurs when the temperature falls below the freezing point of tissue, and severe cases can result in amputation. Promoting deep frostbite wound healing can reduce the risk of amputation. Currently, there have been reports suggesting that stem cell-derived extracellular vesicles might aid in wound healing, but related research is limited. Zhang's team aims to investigate the potential effect of human hepatic progenitor cell-derived extracellular vesicles (EVs) on the healing of deep frostbite wounds.

Human hepatic progenitor cells (hHPCs) at passage 5 exhibited a fascicular and whorled shape (Fig. 2A). To obtain the target extracellular vesicles, cultured hHPCs were subjected to gradient centrifugation to sequentially remove cells, cell debris, and contaminating proteins. The pelleted EVs were then washed with PBS and sterilized by filtration. Then, the hHPC-derived EVs were examined by nanoparticle tracking analysis (NTA), transmission electron microscope (TEM) and Western blotting for biomarkers. As demonstrated by NTA results, the diameter of EVs ranged from 30 to 150 nm (Fig. 2B). Typical cyathiform structure was observed under the TEM scope (Fig. 2C). In addition, EVs markers, including CD9 and Alix, were also detected by Western blotting (Fig. 2D). The above results proved the successful isolation of EVs from hHPCs. Finally, the internalization ability of EVs by HSFs was examined by fluorescent dying. To this end, EVs were labeled with PKH67 (green) and co-cultured with HSFs for 4 h. Under the fluorescence microscope, green fluorescence points were detected among HSFs (DAPI, blue), which indicated that the EVs were absorbed by HSFs (Fig. 2E).

Fig. 2. Characteristics of EVs derived from hHPCs (Zhang, N., Yu, X., et al., 2022).