Figure 2: Effect of prospective hASC selection and co-morbidities on ASC subpopulation dynamics to inform cell source decisions.

(a,b) Enrichment for the transcriptionally identified hASC subpopulation enhances cell survival following exposure to an in vitro apoptotic stimulus (Fas ligand; measuring caspase activation (red)), (c,d) increases cell proliferation and clonogenecity and (e) prolongs stemness marker (CD34) expression. (f,g) The transcriptionally identified ASC subpopulation is significantly depleted and possesses deregulation of critical signalling pathways visible on single-cell analysis in the setting of both diabetes and aging. Gene expression presented as fold change from median (yellow—high expression, 32-fold above median to blue—low expression, 32-fold below median; grey—no expression). (h) Principal component projections of individual cells (left) and genes (right) demonstrating considerable segregation among phenotypes, driven largely by vascular/tissue remodelling genes. (i) Single-cell transcriptional analysis of healthy, aged and diabetic mASCs reveals that the depletion/dysfunction of cluster 1 cells in these states is not a the result of cell SM loss and redistribution to other clusters (expression profiles of subpopulation-defining SMs and tissue remodelling genes highlighted). (j) Flow cytometric analysis demonstrating dynamic DPP4/CD55 subpopulation increases in wild-type wounds, supporting their role in the wound healing process. The DPP4/CD55 subpopulation was also elevated in diabetic and aged wounds as compared with uninjured skin, with a trend toward compensatory overrecruitment consistent with an impaired cellular functionality. *indicates P≤0.05 via one-way ANOVA or Student’s t-test (healthy versus aged or diabetic in f; day 7 versus respective controls in j). indicates P≤0.05 for positive versus negative selection via Student’s t-test. Error bars represent s.e.m. Scale bar, 50 μm.