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. Author manuscript; available in PMC: 2018 Jan 1.
Published in final edited form as: Kidney Int. 2016 Sep 29;91(1):129–143. doi: 10.1016/j.kint.2016.07.037

Figure 3.

Figure 3

Characterization of renal artery-derived progenitor cell culture (A)[i] Isolation of RAPC by single-cell sorting. Representative fluorescent intensity (FI) histogram due to binding of non-specific IgG2a-κ isotype control control antibody (0.0%), anti-human CD184 monoclonal antibody labelled with APC (94.7%) and anti-human CD309 antibody labelled with APC (85.3%) on sorted cells derived from non-confluent monolayer prior to VEGF exposure (dashed line linked to CD184 and CD309 bar graph), cell count=10,000 per condition, [ii] RAPC cultured for seven days in basal media compared to RAPC treated with VEGF (n=22), CD184 pre- vs post-VEGF (91.3±4.3%, vs 37.0±5.2%), CD309 pre- vs post-VEGF (85.2±4.3% vs 34.6±4.2%), CD14 pre- vs post-VEGF (48.0±5.1% vs 27.6±4.58%), CD202 pre- vs post-VEGF (42.9±4.0% vs 38.4±4.0%), CD73 pre- vs post-VEGF (2.9±1.2% vs91.5%±3.8%), CD105 pre- vs post-VEGF (1.5±1.3 vs 88.9±4.5%), CD90 pre- vs post-VEGF (0.8±0.7% vs 2.6±1.9%), CD144 pre- vs post-VEGF (1.6±1.3% vs 27.7±3.9), CD31 (1.4±1.8% vs 29.2±3.2%), CD133 pre- vs post-VEGF (0.5±0.6% vs 0.96±1.1), CD45 (0.9±0.8% vs 0.6±0.9%) CD163 (0.8% ± 0.9%), respectively; (*, p<0.001). (B) Relative CD105, CD73, CD202, and CD31 mRNA expression by quantitative PCR in RAPC+VEGF (n=10 separate patients), RAPC (n=10 separate patients), human EC (n=10) and human VSMC (n=10) (4 groups compared pairwise). CD105, RAPC+VEGF vs RAPC (1.00±0.00 vs 0.01±0.01)(*p<0.001, CI=0.89–1.10); CD105, RAPC+VEGF vs VSMC (1.00±0.00 vs 0.00±0.00)(**, p<0.001, CI=0.89–1.10); CD73, RAPC+VEGF vs RAPC (1.00±0.00 vs 0.01±0.01)(*p<0.001, CI=0.89–1.10); CD73, RAPC+VEGF vs VSMC (1.00±0.00 vs 0.00±0.00)(**, p<0.001, CI=0.89–1.10); CD202, RAPC+VEGF vs RAPC (1.00±0.00 vs 1.11±0.11)(*p=0.002, CI=−0.19–0.03); CD202, RAPC+VEGF vs EC (1.00±0.00 vs 0.18±0.06)(**, p<0.001, CI=0.88–1.04); CD202, RAPC+VEGF vs VSMC (1.00±0.00 vs 0.04±0.04)(***, p<0.001, CI=0.89–1.10); CD31, RAPC+VEGF vs RAPC (1.00±0.00 vs 0.02±0.01)(*p=0.001, CI=−0.91–1.04); CD31, RAPC+VEGF vs VSMC (1.00±0.00 vs 0.02±0.01)(**, p<0.001, CI=0.91–1.05); (C) Fluorescent intensity (arbitrary units, AU) of trazolofluorescein was measured to assess for NOS activity, before (RAPC) and after (RAPC+VEGF) VEGF exposure (1.16±0.35 [AU][n=10] vs 8.05±2.03 [n=10]; *, p<0.001). NOS was also measured in human vascular smooth muscle cells (scale bar = 200 micrometers). (D) Representative immunoblots of eNOS (1.16 ± 0.02 AU [n=7] vs 1.48 ± 0.07 [n=7]; p=0.01) and vWF protein (1.10± 0.04 [n=8] vs 17.70 ± 3.71 [n=8]; p<0.001) protein expression in RAPC cultured without VEGF supplementation and 7 days after VEGF supplementation, respectively.