Human Liver MSCs Retain Their Basic Cellular Properties in Chronically Inflamed Liver Tissue

Abstract

Every 25th death worldwide is associated with liver pathology. The development of novel approaches to liver diseases therapy and protocols for maintaining the vital functions of patients on the liver transplant waiting list are urgently needed. Resident mesenchymal stem cells (MSCs) play a significant role in supporting liver tissue integrity and improve the liver condition after infusion. However, it remains unclear whether MSCs isolated from chronically inflamed livers are similar in their basic cellular properties to MSCs obtained from healthy livers. We applied a large array of tests to compare resident MSCs isolated from apparently normal liver tissue and from chronically inflamed livers of patients with fibrosis, cirrhosis, and viral hepatitis. Chronic inflammatory environment did not alter the major cellular characteristics of MSCs, including the expression of MSC markers, stem cell markers, adhesion molecules, and the hallmarks of senescence, as well as cell proliferation, migration, and secretome. Only the expression of some immune checkpoints and toll-like receptors was different. Evidently, MSCs with unchanged cellular properties are present in human liver even at late stages of inflammatory diseases. These cells can be isolated and used as starting material in the development of cell therapies of liver diseases.

Keywords: cell phenotype; cell therapy; cellular senescence; cirrhosis; fibrosis; liver mesenchymal stem cell; migration; proliferation; secretome.

Figure 1

The rates of the expression of markers by NL-MSCs (green) and PL-MSCs (red) represented as the relative fluorescence intensities (RFI). (A) Mean (±SD) of RFI for markers without statistically significant differences between NL-MSCs and PL-MSCs groups. (B) Mean (±SD) of RFI for markers with statistically significant differences between NL-MSCs and PL-MSCs groups. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Figure 2

Flow cytometric analysis of mesenchymal cell markers expressed by NL-MSCs and PL-MSCs. Blue peak—autofluorescence of the unstained cells, orange peak—fluorescence of the antibody-stained cells. Representative data.

Figure 3

Flow cytometric analysis of the pluripotency markers expressed by NL-MSCs and PL-MSCs. Blue peak—autofluorescence of unstained cells, orange peak—fluorescence of the antibody-stained cells. Representative data.

Figure 4

Flow cytometric analysis of the adhesion-associated markers expressed by NL-MSCs and PL-MSCs. Blue peak—autofluorescence of unstained cells, orange peak—fluorescence of antibody-stained cells. Representative data.

Figure 5

Flow cytometric analysis of the complement-protection markers expressed by NL-MSCs and PL-MSCs. Blue peak—autofluorescence of unstained cells, orange peak—fluorescence of the antibody-stained cells. Representative data.

Figure 6

Flow cytometric analysis of immune markers expressed by NL-MSCs and PL-MSCs without different expression. Blue peak—autofluorescence of unstained cells, orange peak—fluorescence of the antibody-stained cells. Representative data.

Figure 7

Flow cytometric analysis of immune markers expressed by NL-MSCs and PL-MSCs with different expression. Blue peak—autofluorescence of unstained cells, orange peak—fluorescence of the antibody-stained cells. Representative data.

Figure 8

Proliferative activities of NL-MSCs (green, n = 3), PL-MSCs (red, n = 7), and human dermal fibroblasts (blue, n = 3). (A) Cell cultures growth curves. Different symbols represent data for the individual cell cultures (mean ± SD). (B) Time intervals necessary for reaching confluence by cell cultures (mean ± SD). There are no statistically significant differences between any groups.

Figure 9

Cell migration ability of MSCs from normal and pathologic liver tissue. (A) Wound confluence curves with mitomycin c treatment (NL-MSCs—green, n = 5; PL-MSC—red, n = 5; HDFs—blue, n = 3). (B) Wound confluence curves without mitomycin c treatment (NL-MSCs—green, n = 4; PL-MSC—red, n = 4; HDFs—blue, n = 3). Different symbols represent individual cell cultures. (C) Mean (±SD) time necessary for reaching confluence by cell cultures in the wound healing assay with and without mitomycin c treatment. There were no any statistically significant differences between NL-MSCs, PL-MSCs, and HDFs groups analyzed separately for mitomycin c treatment cells and without it. (D,E) Time-lapse brightfield images of wound healing with confluence masks for two NL-MSCs (Liver16 and Liver20) and two PL-MSCs (Liver18 and Liver25) cultures with mitomycin c treatment. The blue mask represents initial wound area, the orange mask represents cell confluence, and merging of both colors represents the initial wound area gradually covered by migrated cells during the healing process. Scale bar—100 µm.

Figure 10

Senescence markers of the MSCs isolated from liver. (A) Representative FACS-analysis histograms of senescence-associated beta-galactosidase (SA-β-gal) expression in NL-MSCs, PL-MSCs, and HDFs. (B) Mean (±SD) of RFI for SA-β-gal expression in NL-MSCs (n = 3), PL-MSCs (n = 4), and HDFs (n = 3). (C) SA-β-gal activity in NL-MSCs (n = 5), PL-MSCs (n = 3), two human dermal fibroblast cultures—HDF2 and HDF3, and MSCs from placenta (PL) and umbilical cord (UC). For facilitation of graph readability, statistical significance is shown only for the NL-MSCs and PL-MSCs groups. (D) Representative FACS-analysis histograms of H2A.X-histone expression in NL-MSCs, PL-MSCs, and HDFs. (E) Mean (±SD) of RFI for H2A.X-histone expression in NL-MSCs (n = 4), PL-MSCs (n = 4), and HDFs (n = 3). (F) Representative ImageStream distribution of nucleus sizes in NL-MSCs, PL-MSCs, and HDFs. (G) Mean values (± SD) of the nucleus area in NL-MSCs (n = 5), PL-MSCs (n = 3), and HDFs (n = 3). * p ≤ 0.05; *** p ≤ 0.001.

Figure 11

The heatmap representation of the multiplex evaluation of the cytokine (top heatmap) and chemokine (bottom heatmap) secretion by NL-MSCs and PL-MSCs. Different concentrations of molecules are represented by colors: low concentration is represented by blue gradient, high concentration is represented by yellow gradient, highest concentration is represented by red.