High-efficient generation of VCAM-1+ mesenchymal stem cells with multidimensional superiorities in signatures and efficacy on aplastic anaemia mice

Abstract

Objective: Longitudinal studies have indicated VCAM-1⁺ mesenchymal stem/stromal cells (MSCs) as promising resources in regenerative medicine, yet the abundance in gene expression is far from adequate in the advantaged and "discarded" hUC-MSCs. Thus, high-efficient preparation and systematic dissection of the signatures and biofunctions of the subpopulation is the prerequisite for large-scale clinical applications.

Materials and methods: We primarily took advantage of a cytokine-based programming strategy for large-scale VCAM-1⁺ hUC-MSC generation (III-MSCs). Thereafter, we conducted multifaceted analyses including cytomorphology, immunophenotype, cell vitality, multilineage differentiation, whole-genome analysis, tube formation and Matrigel plug assay, lymphocyte activation and differentiation, and systemic transplantation for aplastic anaemia (AA) treatment.

Results: III-MSCs with high-proportioned VCAM-1 expression were obtained by combining IL-1β, IL-4 with IFN-γ, which exhibited comparable immunophenotype with untreated hUC-MSCs (NT-MSCs) but revealed multidimensional superiorities both at the cellular and molecular levels. Simultaneously, systemic infusion of III-MSCs could significantly ameliorate clinicopathological features and finally help facilitate haematopoietic reconstruction and immunoregulation in AA mice.

Conclusions: We have established a high-efficient procedure for large-scale generation of III-MSCs with preferable signatures and efficacy upon aplastic anaemia in mice. Our findings suggested that III-MSCs were advantageous sources with multifaceted characteristics for regenerative medicine.

Keywords: Aplastic anaemia; VCAM-1; genetic alteration; hUC-MSCs; immunoregulation; proangiogenesis.

FIGURE 1

High‐efficient generation of III‐mesenchymal stem/stromal cells (MSCs) with high expression level of VCAM‐1. A and B, FCM analysis of VCAM‐1 expression in hUC‐MSCs with single (A) or combined (B) cytokine treatment. C and D, Immunofluorescent images (C) and Mean fluorescence intensity (MFI) (D) of VCAM‐1 in NT‐ and III‐MSCs (scale bar = 40 μm). E, Immunofluorescent images of Phalloidin in NT‐ and III‐MSCs. F and G, FCM diagram (F) and statistical analysis (G) of surface marker expression in NT‐ and III‐MSCs. H, Karyotype analysis of NT‐ and III‐MSCs. Data were shown as mean ± SEM (n = 3). *P < .05; **P < .01; ***P < .001; ****P < .0001; NS, not significant

FIGURE 2

The distribution of differentially expressed genes and mutation spectrum in NT‐mesenchymal stem/stromal cells (MSCs) and III‐MSCs. A, Gene expression distribution of total genes in the genome of NT‐ and III‐MSCs. B, Volcano plot analysis of total genes. C and D, GO analysis of upregulated (C) and downregulated (D) genes (P < .05, log₂ FC > 1). E, GSEA shows enrichment plot of the indicated subsets in NT‐ and III‐MSCs. F and G, Distribution of SNPs (F) and fusion genes (G) in the genome of NT‐ and III‐MSCs

FIGURE 3

III‐mesenchymal stem/stromal cells (MSCs) with comparable cell vitality but enhanced CFU‐F formation potential. A, Heatmap analysis of gene sets in NT‐ and III‐MSCs. B, Pd assay of NT‐ and III‐MSCs for 17 passages. C, Proliferation assay with CCK‐8 kit. D and E, Distributions of apoptotic population in NT‐ and III‐MSCs as shown by FCM diagram (D) and statistical analysis (E). F and G, The proportion of apoptotic population as shown by FCM diagram (F) and statistical analysis (G). H and I, Images of total CFU‐Fs (H) and representative colonies (I) (scale bar = 500 μm). J, Statistical analysis of CFU‐F numbers. Data were shown as mean ± SEM (n = 3). *P < .05, **P < .01; NS, not significant

FIGURE 4

III‐mesenchymal stem/stromal cells (MSCs) exhibited superior characteristics in migration and vasculo‐angiogenic capacity in vitro and in vivo. A GSEA showed enrichment plot of ECM‐receptor interaction and crosslinking of collagen fibrils. B and C, Representative images (B) and statistical analysis (C) of cell migration in NT‐ and III‐MSCs. Scale bar = 200 μm. D and E, Representative images (D) and statistical analysis (E) of in vitro capillary tube‐like structures. Scale bar = 200 μm. F, Matrigel plug assay showed the in vivo vasculo‐angiogenic ability of NT‐ and III‐MSCs. Scale bar = 1 μm or 2 μm. G, Statistical analysis of vascular density of Matrigel plugs with H&E staining. Data were shown as mean ± SEM (n = 3). *P < .05, **P < .01

FIGURE 5

III‐mesenchymal stem/stromal cells (MSCs) displayed preferable immunoregulatory ability over NT‐MSCs in vitro. A and B, qRT‐PCR analysis of multiple immunoregulation‐associated cytokines (A) and IDO‐1 (B) in NT‐MSCs and III‐MSCs. C, ELISA analysis of secreted cytokines in the supernatant. D‐K, FCM and statistical analyses of the differentiated Th1 (D and E), Th2 (F and G), Th17 (H and I) and Treg (J and K) cells by coculturing CD4⁺ T cells with NT‐ or III‐MSCs. Data were shown as mean ± SEM (n = 3). *P < .05, **P < .01, ***P < .001, ****P < .0001; NS, not significant

FIGURE 6

The haemogram and clinicopathologic features of AA mice were alleviated by III‐mesenchymal stem/stromal cell (MSC) transplantation. A, Schematic of the AA mice model. B, Body weight loss of mice (%) in the indicated groups. C, Statistical analysis of nucleated cells and splenocytes in bone marrow (BM) and spleen (SP) of mice. D and E, Statistical analysis of subpopulations in the blood of recipient mice including RBC, WBC (D) and reticulocyte (RET) (E). F, Pathological sections of sterna with H&E staining in the indicated groups. Scale bar = 1 μm. Data were shown as mean ± SEM (n = 5). *P < .05, **P < .01, ***P < .001

FIGURE 7

The immunodysfunction of lymphocytes in bone marrow of AA mice was ameliorated by systemic infusion of III‐mesenchymal stem/stromal cells (MSCs). A and B, The distributions of the CD4⁺ and CD8⁺ subpopulations in BM of the indicated mice as shown by the FCM diagram (A) and statistical analysis (B). C and D, Statistical analyses of the CD4⁺ IFN‐γ⁺ Th1 cells (C), CD8⁺ IFN‐γ⁺ Tc1 cells (D), CD4⁺ CD25⁺ FoxP3⁺ Treg cells (E) in the BM and SP of mice, respectively. Data were shown as mean ± SEM (n = 3). *P < .05, **P < .01, ***P < .001, ****P < .0001