PDGFRα and c-Kit blockade prevents tPA-induced PαS-MSC expansion in EC and PαS-MSCs cocultures. (A) Experimental setting: mouse PαS-MSCs and ECs (mouse CD45⁻CD31⁺ or human HUVECs) were cocultured with or without recombinant tPA for 3 days and separated from each other using a transwell chamber system. (B-C) The absolute numbers of PαS-MSCs per well after the addition of recombinant tPA or vehicle in cocultures with (B) HUVECs or (C) mouse ECs as determined by hemocytometer counting (n = 3/group). (D) HUVECs or (E) mouse CD45⁻CD31⁺ ECs were cocultured with or without recombinant tPA: relative mRNA expression of (D) human and (E) mouse FGF2 and PDGF-BB (n = 3/group). Data are normalized to GAPDH. (F) Relative mRNA expression of mouse KitL and mouse PDGFRα derived from mouse PαS-MSCs in MSC-HUVEC cocultures (n = 3/group). Data are normalized to GAPDH. (G) Original gel and (H) densitometric quantification of active mouse MMP-2 and MMP-9 after gelatin zymography in PαS-MSCs from MSC-HUVEC cocultures (n = 3). B6 mouse plasma was used as positive and MMP-9 KO plasma as negative control. (I) Absolute numbers of PαS-MSCs per well in single cultures treated with vehicle, tPA, ACK2, PDGFRα inhibitor, or Gleevec as determined by hemocytometer counting (n = 3/group). (J) The absolute numbers of MSCs per well in MSC-HUVEC coculture with vehicle (white) or tPA (black) with or without ACK2, PDGFRα inhibitor, or Gleevec as determined by hemocytometer counting (n = 4/group). Mean ± SEM. *P < .05; **P < .01; ***P < .001 by Student t test or ANOVA with Tukey HSD tests for multigroup analysis. Data are representative of 3 independent experiments.