Mesenchymal stem cells alleviate renal injury of lupus nephritis mice by reconstructing the immune microenvironment of renal tissue

Figure 1 Umbilical cord mesenchymal stem cells morphology and identification. A: Umbilical cord mesenchymal stem cells (UCMSCs) morphology is displayed at 50 × magnification; B: UCMSCs morphology is displayed at 200 × magnification; C and D: The effective differentiation of UCMSCs into osteoblasts was confirmed by alizarin red staining. At a magnification of 100 × representative pictures of the control group (C) and induction group (D) were captured; E and F: Oil Red O staining was used to successfully differentiate UCMSCs into adipocytes. At a magnification of 100 ×, representative pictures of the control group (E) and induction group (F) were captured; G and H: Alcian blue staining was used to identify the chondrocytes that were successfully differentiated from UCMSCs. At 200 × magnification, representative pictures of the control group (G) and induction group (H) were captured; I-L: Using flow cytometry, the surface markers of UCMSCs, such as CD90, CD105, CD73, CD45, CD34, CD11b, CD19, and HLA-DR, were examined. Of them, more than 99% of the cells were positive for CD90, CD105, and CD73. The other genes’ expression was nearly negligible.

Figure 2 Comparing the results of biochemical measurements changes to renal function, serum dsDNA and proteinuria levels. A: Schematic illustration of umbilical cord mesenchymal stem cells treatment in MRL/Lpr mice; B: Serum dsDNA level (U/mL); C: Urinary albumin/urinary creatinine ratio level (mg/mmoL); D: Blood urea nitrogen level (mmol/L); E: Serum creatinine level (μmol/L). All data was expressed as mean ± SEM, ^aP < 0.05 vs lupus nephritis group, ^bP < 0.01 vs lupus nephritis group, ^cP < 0.001 vs lupus nephritis group. LN: Lupus nephritis; UCMSCs: Umbilical cord mesenchymal stem cells; uALB: Urinary albumin; uCr: Urinary creatinine.

Figure 3 Results of renal tissue pathology and electron microscopy. A: Pathological and electron microscopy results of renal tissue. Hematoxylin and eosin staining (1^st line, 400 ×), periodic acid-Schiff staining (2^nd line, 400 ×), Masson’s trichrome staining (3^rd line, 400 ×) and electron microscopy (4^th line, 12000 ×). Normal group (1^st column), lupus nephritis group (2^nd column), and umbilical cord mesenchymal stem cells group (3^rd column) were used; B: Immunohistochemical staining and statistical results of CD3, CD68 and B220 in mice kidney tissue. All data was expressed as mean ± SEM, ^bP < 0.01 vs lupus nephritis group, ^cP < 0.001 vs lupus nephritis group. LN: Lupus nephritis; UCMSCs: Umbilical cord mesenchymal stem cells; HE: Hematoxylin and eosin; PAS: Periodic acid-Schiff.

Figure 4 Changes in immune cells in the spleens and kidney tissues of MRL/Ipr mice. A-D: Multiple immunofluorescence and quantitative analysis of regulatory T (Treg) (CD4+CD25+Foxp3+) (A and C); multiple immunofluorescence and quantitative analysis of T helper 17 (IL-17A+CD4+) (B and D); E: Flow cytometric analysis of the percentages of Treg (CD4+CD25+Foxp3+) in the spleen; F: Quantitative analysis of the percentages of Treg in the spleen. All data were expressed as mean ± SEM, ^aP < 0.05 vs lupus nephritis group, ^bP < 0.01 vs lupus nephritis group. LN: Lupus nephritis; UCMSCs: Umbilical cord mesenchymal stem cells; IL: Interleukin.

Figure 5 Macrophage infiltration in mice kidney tissue. A: Multiplex immunofluorescence and quantitative analysis of M1 macrophages (F4/80+ iNOS+); B: Multiplex immunofluorescence and quantitative analysis of M2 macrophages (F4/80+ CD206+). LN: Lupus nephritis; UCMSCs: Umbilical cord mesenchymal stem cells; iNOS: Inducible nitric oxide synthase.

Figure 6 Changes in the percentages of immune cells after coculture of umbilical cord mesenchymal stem cells and peripheral blood mononuclear cells. A: Flow cytometry was used to analyse the proliferation of Carboxy Fluorescein Succinimidyl Ester-labelled lymphocytes and changes in the percentages of T helper 1 (TH1), TH17, and regulatory T cells after coculture of umbilical cord mesenchymal stem cells and peripheral blood mononuclear cell; B: Quantitative analysis of the percentages of TH1 cells, TH17 cells and regulatory T after co-culture of umbilical cord mesenchymal stem cells and peripheral blood mononuclear cell. All data was expressed as mean ± SEM, ^aP < 0.05 vs lupus nephritis group, ^cP < 0.001 vs lupus nephritis group. TH1: T helper 1; Treg: Regulatory T; IFN: Interferon; IL: Interleukin; CFSE: Carboxy Fluorescein Succinimidyl Ester; UCMSCs: Umbilical cord mesenchymal stem cells.