Fig. 1: Human iPS-MSPCs undergo continuous maturation towards reacquisition of immune modulatory potential.

a Experimental setup: I. MSPCs derived from bone marrow (BM) and umbilical cord blood (UCB) were reprogrammed into iPSCs before mesoderm induction and maturation into mesodermal iPS-MSPCs to enable sequential gene expression and functional profiling. II. DOCK-2 mutated patient fibroblast (FB) lines were compared to healthy controls for phenotype and immunomodulation. III. Healthy iPSCs were used for CRISPR/Cas9-mediated bi-allelic DOCK-2 knockout, subsequent DOCK-2^-/- iPS-MSPC propagation and function comparison. IV. Healthy control FB and iPS-MSPCs were subjected to si-RNA-mediated DOCK2 knockdown to determine the immediate effect of reduced DOCK2 GEF presence in stromal cells during immunomodulation. b Cluster analysis of single cell-based flow cytometry marker profiling during differentiation of MSPC-derived iPSCs back into iPS-MSPCs. Mean ΔMFI values (specific antibody staining minus isotype control) from passage 0 (p0) to p8, compared to parental MSPCs. c Immunomodulatory potential of iPS-MSPCs inhibiting T-cell mitogenesis at 1:3 ratio iPSCs or MSPCs:immune cells increased with differentiation from early to late passages compared to parental primary MSPCs and iPSCs. (BM: iPSC n = 10, iPS-MSPC p1 – 2 n = 6, iPS-MSPC p4 – 6 n = 8, iPS-MSPC p ≥ 8 = 7, MSPC n = 9; UCB: iPSC n = 7, iPS-MSPC p1 – 2 n = 5, iPS-MSPC p4 – 6 n = 8, iPS-MSPC p ≥ 8 n = 7, MSPC n = 7; PBMC:MSPC ratio 1:3; one-way ANOVA including Tukey´s multiple comparison test: ****p < 0.0001 for BM and UCB, BM: df = 70, F = 24.16; UCB: df = 58, F = 25.48). Percent inhibition after normalizing mean T cell proliferation values of individual assays shown. Symbol and color-code as indicated. Error bars represent standard deviation.