Int J Stem Cells.  2021 Aug;14(3):262-274. 10.15283/ijsc20002.

Umbilical Cord Mesenchymal Stem Cells for Inhibiting the Fibrosis and Autoimmune Development in HOCl-Induced Systemic Scleroderma Mouse Model

• Jin X
• Hou J
• Zheng K
• Wei D
• Zhang A
• Wang S
• Mei H
• Li C
• Cheng L
• Sun X

• KMID: 2519369
• DOI: http://doi.org/10.15283/ijsc20002

Abstract

Background and Objectives
Systemic scleroderma (SSc) is a rare and serious connective tissue disease, an autoimmune disease, and a rare refractory disease. In this study, preventive effect of single systemic human umbilical cord mesenchymal stem cells (UC-MSCs) transfusion on SSc was preliminarily explored.
Methods and Results
SSc mouse model was established by daily intradermal injection of Hypochlorite (HOCl). SSc mice were treated by single transfusion of UC-MSCs at 0.625×10 ⁵ , 2.5×10⁵ and 1×10⁶ respectively. At the 42nd day of intradermal injection of HOCl, the symptoms showed up by skin and alveolar wall thickening, lymphocytic infiltration, increased collagen in skin/lung, and the increased proportion of CD3 ^＋ CD4^＋ CD25^＋ FoxP3^＋ cells (a Treg subset) in spleen. After UC-MSCs transfusion, the degree of skin thickening, alveolar wall thickening and lymphocyte infiltration were decreased, the collagen sedimentation in skin/lung was decreased, and the proportion of CD3^＋ CD4^＋ CD25^＋ FoxP3^＋ cells was decreased.
Conclusions
UC-MSC can achieve a preventive effect in SSc mice by fibrosis attenuation and immunoregulation.

Keyword

Mesenchymal stem cells; Immunoregulation; Regulatory T cell; Systemic scleroderma

Figure

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  Fig. 1 Protocol of animal experiment. In the induced SSc mouse model, HOCl (400 μl/d) was injected intradermally for 42 days in 8-weeks-old female BALB/c mice. In control group, PBS was injected intradermally at equivalent dose. In UC-MSCs treatment groups, at Day 0 of modeling, 100 μl UC-MSCs were infused once in tail vein of mice at the doses of 0.625×105 cells (low dose group), 2.5×105 cells (medium dose group) and 1×106 cells (high dose group). The skin thickness of mice was measured weekly in each group. At Day 42 (i.e. Week 6), the mice were sacrificed, and specimens were collected for histopathological examination and analysis of lymphocyte subsets in splenocytes.

• 

  Fig. 2 UC-MSCs regulated the proportion of lymphocyte subsets. (A) UC-MSCs inhibited the proliferation of T cells (PBMCs/MSCs ratio was 1,000：1, 100：1 and 10：1 respectively). (B) The proportion of CD3＋ T cells in total lymphocytes. (C) The proportion of CD3＋CD4＋ helper T cells in CD3＋ T cells. (D) The proportion of CD3＋CD8＋ cytotoxic T cells in CD3＋ T cells. (E) The proportion of CD3＋CD4＋CD25＋CD127− Treg cells in CD3＋ T cells. (F) The proportion of Treg cells in CD3＋CD4＋ T cells. (G) The count of Treg cells. The concentration of cell factors in the supernatant of culture medium: (H) IFN-γ, (I) TNF-α, (J) IL-2, (K) IL-17, (L) IL-10. *p<0.05, **p<0.01 and ***p<0.001.

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  Fig. 3 The representative FACS cytograms of vitro experiment. The effect of (A) different ratios of UC-MSCs (PBMCs/MSCs ratio was 1,000 and 1 and 10：1 respectively) on T cell proliferation, and the effect of UC-MSCs on the proportion of (B) CD3＋ T cells (C) CD3＋CD4＋ helper T cells, (D) CD3＋CD8＋ cytotoxic T cells, (E) CD3＋CD4＋CD25＋ Treg cells, (F) CD3＋CD4＋CD25＋CD127− Treg cells with or without PHA stimulation.

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  Fig. 4 Therapeutic effect of UC-MSCs in SSc mouse model. (A) The skin thickness of mice (n=15). (B) Compared to the skin thickness of mice before modeling, the weekly increase percentage of skin thickness (n=15). (C) The increase percentage of skin thickness at Day 42 (n=15). (D) The proportion of mice with an increase, no change or decrease of skin thickness at Day 42 (n=15). (E) Dermis thickness of skin sections; HE staining on the representative sections of (F) skin and (G) lung obtained at Day 42 (amplification ratio 200×). *p<0.05, **p<0.01 and ***p<0.001.

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  Fig. 5 Staining of collagen in the sections of skin and lung tissues. Representative sections of (A) skin and (B) lung obtained at Day 42 (amplification ratio 200×), sections were stained with Masson Trichrome staining. Analysis of Masson Trich-rome staining on (C) skin and (D) lung sections by Image J software and calculation of collagen volume fraction (n=15). *p<0.05, **p<0.01 and ***p<0.001.

• 

  Fig. 6 The immunoregulatory effect of UC-MSCs in vivo. (A) Immunofluorescence staining on representative sections of skin obtained at Day 42 (TGF-β1＋: green, α-SMA＋: red, F4/80＋: green, DAPI: blue), amplification ratio 100× for left diagram, amplification ratio 400× for right diagram of each group; Analysis of immunofluorescence staining on skin sections by Image J software (n=6) and calculation of fluorescent area percentage of (B) TGF-β1＋, (C) α-SMA＋ and (D) F4/80＋ in the skin. The proportion of (E) CD3＋ T cells and (F) CD19＋ B cells in the splenocytes; the proportion of (G) CD3＋CD4＋ helper T cells and (H) CD3＋CD8＋ killer T cells in CD3＋ T cells; the proportion of (I) CD3＋CD4＋CD25＋FoxP3＋ Treg cells in CD3＋CD4＋ T cells; and the count of (J) Treg cells. *p<0.05, **p<0.01 and ***p<0.001.

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  Fig. 7 The representative FACS cytograms of vivo experiment. The proportion of (A) CD3＋ T cells, (B) CD19＋ B cells, (C) CD3＋CD4＋ helper T cells and CD3＋CD8＋ killing T cells, (D) CD3＋CD4＋CD25＋FoxP3＋ Treg cells in splenocytes of SSc mice treated with different doses of UC-MSCs.

Reference

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