Abstract
Hepatic Stem/progenitor cells (HSPCs) have gained a large amount of interest for treating acute liver disease. However, the isolation and identification of HSPCs are unclear due to the lack of cell-specific surface markers. To isolate adult HSPCs, we used cell surface-marking antibodies, including CD49f and Sca-1. Two subsets of putative HSPCs, Lin−CD45−Sca-1−CD49f+ (CD49f+) and Lin−CD45−Sca-1+CD49f− (Sca-1+) cells, were isolated from adult mice liver by flow cytometry. Robust proliferative activity and clonogenic activity were found in both CD49f+ and Sca-1+ cells through colony-forming tests and cell cycle analyses. Immunofluorescence staining revealed that CD49f+ cells expressed ALB and CK-19 while Sca-1+ cells expressed only ALB, indicating that CD49f+ cells were bipotential and capable of differentiating into hepatocyte and cholangiocyte. Consequently, PAS stain showed that differentiated CD49f+ and Sca-1+ cells synthesised glycogen, indicating they could differentiate into functional hepatocytes. mRNA expression profile indicated that both CD49f+ and Sca-1+ cells showed differential expression of genes that are associated with liver progenitor function such as Sox9 and EpCam. Moreover, two subsets of putative HSPCs were activated by DDC and we found that their abundance and proliferation increased with age. In summary, we hypothesized that CD49f+ cells were a type of potential HSPCs and may be utilised for clinical stem cell therapy.
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Funding
This study was supported by Grants from National Natural Science Foundation of China (81972700, 61827819, 32160159), National Science Foundation of Guangxi (2018GXNSFBA281146, 2018GXNSFBA281115, 2018GXNSFAA138004) and Middle-aged and Young Teachers’ Basic Ability Promotion Project of Guangxi (2019KY0052). We thank International Science Editing for editing this manuscript.
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Supplementary file1 Supplemental Figure 1. Identification and characterization of HSPCs in CD49f+ population. (a) Isolation of CD133- and CD133+ cells from CD49f+HSPCs from the liver tissue of normal mice. (b) Morphological features of CD133- and CD133+ cells by phase-contrast microscopy over time. (c) Expression of ALB and CK-19 in differentiated freshly-isolated HSPCs after 14 days of culture in DMEM-F medium by immunofluorescent staining. Scale=100μm. (TIF 5035 kb)
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Supplementary file2 Supplemental Figure 2. The gene expression of ALB, CK-19, CK-18, AFP, and GADPH was measured by q-PCR. HSPCs were cultured in DMEM-F medium for 14 days. *P<0.05, ** P<0.01. (JPG 41 kb)
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Supplementary file3 Supplemental Figure 3. Localization of HSPCs in mice liver. (a) Immunofluorescent staining of CD49f+HSPCs in livers of normal and DDC mice: Green=CD49f+, Blue=DAPI. (b) HE staining for liver tissue of normal and DDC mice. Scale=100μm. (TIF 11048 kb)
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Guo, Z., Pu, S., Li, Y. et al. Functional characterization of CD49f+ hepatic stem/progenitor cells in adult mice liver. J Mol Histol 53, 239–256 (2022). https://doi.org/10.1007/s10735-022-10063-z
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DOI: https://doi.org/10.1007/s10735-022-10063-z