Fig 2: CD49a-high HSCs can be isolated from the db/db mouse liver and the human HCC tissue. (A) Application of the cold isolation protocol for obtaining HSCs from db/db mice (n = 3). The average body weight (BW) of mice was 51.8 g (range, 48?57 g). The sorted CD49a-high and CD49a-low cells among CD31-/CD45- cells were subjected to qPCR analysis for Lrat expression. Data were obtained in triplicate using the samples of 3 different mice and are presented as fold-change difference in comparison with pre-sorted HSCs. Statistical significance was determined using Bonferroni’s multiple comparisons test (see the raw data table by the bar graph). (B) The histology of post-sustained virologic responce (hepatitis C virus) human HCC tissue (hematoxylin and eosin staining, scale bar, 200 µm) and the patient’s information. (C) Method for isolating HSCs from T-hHSCs. (D) Flow cytometry analysis and qPCR analysis of T-hHSCs. The rectangle shows the double-positive fraction of CD49a expression and retinoid content. T-hHSCs, LI90 (human HSC line), or HepG2 (human HCC cell line) cells were subjected to qPCR analysis for evaluating ACTA2 expression. Data were obtained in triplicate using these samples. Statistical significance was determined using Bonferroni’s multiple comparisons test (see the raw data table by the bar graph).

Fig 3: CD49a can be a marker for HSC isolation. (A) Unsupervised t-SNE clustering of the sorted CD31-/CD45.2- cells revealed the expression of a cholangiocyte marker (Krt19) and hepatocyte marker (Hnf4a). (B) The gene expression in the HSC clusters (ND cluster 0 and 6; NT cluster 1; T cluster 2) was compared and analyzed using log2-Fold change (logFC). The top 11 genes encoding cell surface markers are illustrated in an ascending order of fluctuation. (C) Flow cytometry plots of HSCs from ND and HFD mice. The average body weight of mice: ND, 32.6 g (range, 31?34 g; n = 3); HFD, 58.5 g (range, 55?61 g; n = 3).