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. 2020 Sep 25;10:15808. doi: 10.1038/s41598-020-72875-x

Figure 3.

Figure 3

Adipose-derived, lineage-marked Myh11+ mural cells give rise to mesenchymal stem cells (MSCs) during adaptation and growth in vitro. (A) Immunostained epididymal adipose tissue from Myh11-eYFP mice indicated eYFP+ (green) lineage marker is expressed in microvascular smooth muscle cells (vSMCs) (arrowhead) and microvascular pericytes (PCs) (asterisk) along lectin + blood vessels. Scale bar, 50 µm. (B) Immunostained adipose tissue revealed vSMC “tire tread” pattern on larger arterioles. Scale bar, 25 µm. (C) PCs are wrapped around adipose capillary microvasculature. Scale bar, 10 µm. (D) Flow cytometry analysis showed adipose Myh11+ mural cells collected from the SVF have relatively low endogenous expression of CD73, CD90, CD105, and CD146, however, after isolation via fluorescence activated cell-sorting (FACS), cultured, passage 3–5 Myh11+ mural cells significantly increased expression of CD73, CD90, CD105, and CD146 in vitro (three independent flow analyses per panel). (E) Graphical representation of flow cytometry analysis demonstrated significant increase of MSC surface antigens in Myh11+ mural cells after isolation from the SVF and cultured in vitro. (F) Flow cytometry analysis also revealed FAC-sorted and cultured passage 3–5 Myh11+ mural cells lacked expression for hematopoetic, endothelial, and macrophage markers CD11b, CD19, CD34, CD31, and CD45 (three independent flow analyses per panel). (G,H) Protein and genetic analysis of passage 2 Myh11+ mural cells when cultured in adipogeneic, chondrogenic, or osteogenic media for 14 days. (G) Increase in FABP4, Collagen II, and Osteopontin was observed by immunohistochemistry in Myh11+ mural cells undergoing tri-differentiation. Scale bar, 50 µm. (H) qPCR showed mRNA expression of protein markers and transcription factors involved in adipogenesis, chondrogenesis, and osteogenesis were significantly upregulated in Myh11+ mural cells following tri-differentiation (n = 3 biological replicates). Relative expression is normalized to GAPDH expression in each sample. Results are represented as mean ± standard error of mean (SEM). Data were analyzed using multiple unpaired t tests followed by the Holm–Sidak post-hoc comparisons to correct for multiple comparisons (E), or a ratio paired t-test (H). *p < 0.05, **p < 0.01, ***p<0.001. Immunohistochemistry images were captured through randomly sampling of microvasculature tissue and culture wells. Tissue and cultured cells were isolated from Myh11-eYFP mice 10–12 weeks of age.