Background/Purpose: Mesenchymal stromal cells (MSCs) are non-hematopoietic multipotent cells with immunomodulatory, proangiogenic, and antifibrotic properties. MSC functions are mediated by paracrine soluble factors and small vesicles present in the MSC conditioned medium (CM). The three pathogenic axes in systemic sclerosis (SSc) are autoimmunity, vasculopathy, and fibrosis. We hypothesized that functional abnormalities of MSCs contributed to SSc pathogenesis.We aim to characterize the phenotype, transcriptomic signatures, and key functional properties of adipose-derived MSCs from SSc patients (SSc MSC).

Methods: MSCs were isolated from the adipose tissue associated with two 4 mm punch biopsies from the forearm of 15 SSc and 15 age- and sex-matched healthy controls (Ctrl). MSCs were characterized according to the International Society for Cell and Gene Therapy (ISCT) minimal criteria. RNA sequencing was performed on 8 SSc and 8 Ctrl MSCs. Proliferation (doubling time), stemness (fibroblast colony forming units: CFU-F), and immunopotency (flow cytometry) were assessed in vitro. MSC-CM cytokine profiling was done with the Luminex xMAP® platform.

Results: SSc and Ctrl MSC met ISCT criteria (i.e., adhered to plastic, differentiated into three lineages, and exhibited specific surface markers). Compared to Ctrl, SSc MSCs had 151 upregulated and 16 downregulated genes. Gene Set Enrichment Analysis (GSEA) documented the enrichment of proinflammatory and profibrotic pathways in MSCs recovered from SSc patients. However, SSc MSCs retain the capacity to inhibit the proliferation of activated T cells in vitro. SSc MSC depicted a myofibroblast-like phenotype, characterized by increased α–SMA gene and protein expression, and elevated levels of the profibrotic cytokine CCL2 in MSC-CM. SSc MSC had reduced proliferation and clonogenicity, and were enriched in senescence-associated gene sets.

Conclusion: SSc MSCs differ from Ctrl MSCs in their transcriptomic and functional properties. SSc MSCs have profibrotic and senescent phenotypes, which could contribute to amplify or perpetuate SSc pathogenic mechanisms. Future studies will evaluate the effect of MSC modulation on SSc severity.

Figure 1. SSc MSCs display a myofibroblast-like phenotype A. Gene Set Enrichment Analysis (gene list: Zhu et al., 2024) B. Quantification of alpha-SMA gene expression (n=14, *p < 0.05). C. Western blot representative images and quantification of alpha-SMA and procollagen protein levels(n=8, *p < 0.05; ns: non-significant).

Figure 2. SSc MSCs have a senescent transcriptomic imprint underlying their reduced proliferation and clonogenic capacity A. Gene Set Enrichment Analysis plot for two gene lists (Gao et al., 2023, Psaroudis et al., 2022). B. MSCs population doubling (n=15) and C. Clonogenicity (n=15). *p < 0.05 **p < 0.01.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/mesenchymal-stromal-cells-in-systemic-sclerosis-are-dysfunctional-and-have-a-profibrotic-and-senescent-phenotype/