EPCs were obtained from mononuclear cells isolated from peripheral blood (100ml) by density gradient method (38 (link)). “Late” EPCs were obtained from cells collected 3 weeks after RAS induction, and subsequently cultured for 3 weeks, while “early” EPCs were obtained from cells collected and cultured 1 week before infusion (2 (link)).
EPCs were characterized by fluorescence activated cell sorting (FACS) analysis after immunostaining with monoclonal antibodies against the progenitor markers CD133 (R&D Systems, MN, Cat# AF3890, NS0-derived rpCD34 1:50) and kinase-insert domain receptor (KDR, Santa-Cruz, CA, Cat# sc-504, Clone: C-1158 1:50), as previously described (10 (link)).
MSCs were isolated from adipose tissue (5–10g) collected from pigs during RVH induction or sham. Tissue was processed for MSC isolation with standard protocol (32 (link)), and cultured with advanced minimum-essential-medium (Gibco/Invitrogen) supplemented with 5% platelet lysate (Mayo Clinic Transfusion Medicine) in 37°/5% CO2. FACS was used to determine cellular phenotype for the MSC markers CD44 (abcam Cat#: ab10558 1:100) and CD90 (BD Pharmigen Cat#:55593 1:100). Before delivery EPCs and MSCs were labeled with a fluorescent membrane dye (CM-DiI, CellTracker™, Catalog #: C7001, Life Technologies) and kept in cell recovery medium at −80°C for transplantation. Then, 6 weeks after RVH or sham induction, 10^6 cells/mL of EPC (an equal mix of early and late) or MSCs suspended in 10ml of PBS (Life Technologies, # 10010-023) were injected slowly through a balloon catheter (OPTA® Pro PTA Dilatation Catheter, Cordis, New Jersey) placed in the renal artery proximal to the stenosis.
Fluorescent-labeled cells were subsequently manually counted ex-vivo under fluorescence microscopy (ZEN® 2012 blue edition, Carl ZEISS SMT, Oberkochen, Germany) in 5µm LV cross-sections and 5µm renal tissue sections stained with cytokeratin (AbD Serotec, Cat# MCA1907), and their number per field averaged (7 (link),8 (link),34 (link)). Furthermore, EPC and MSC distribution was evaluated by immunofluorescence staining with the distal tubular marker peanut agglutinin (PA, Vector Lab, Cat# FL-1071, 1:500), the proximal tubular marker phaseolus vulgaris erythroagglutinin (PHA-E, Vector Lab, Cat# FL-1121, 1:500), the endothelial marker CD31 (AbD Serotec, Cat# MCA 1747, dilution 1:50), and the proliferating cell nuclear antigen (PCNA, Abcam, Cambridge, MA; Cat# ab29,1:100).
EPCs were characterized by fluorescence activated cell sorting (FACS) analysis after immunostaining with monoclonal antibodies against the progenitor markers CD133 (R&D Systems, MN, Cat# AF3890, NS0-derived rpCD34 1:50) and kinase-insert domain receptor (KDR, Santa-Cruz, CA, Cat# sc-504, Clone: C-1158 1:50), as previously described (10 (link)).
MSCs were isolated from adipose tissue (5–10g) collected from pigs during RVH induction or sham. Tissue was processed for MSC isolation with standard protocol (32 (link)), and cultured with advanced minimum-essential-medium (Gibco/Invitrogen) supplemented with 5% platelet lysate (Mayo Clinic Transfusion Medicine) in 37°/5% CO2. FACS was used to determine cellular phenotype for the MSC markers CD44 (abcam Cat#: ab10558 1:100) and CD90 (BD Pharmigen Cat#:55593 1:100). Before delivery EPCs and MSCs were labeled with a fluorescent membrane dye (CM-DiI, CellTracker™, Catalog #: C7001, Life Technologies) and kept in cell recovery medium at −80°C for transplantation. Then, 6 weeks after RVH or sham induction, 10^6 cells/mL of EPC (an equal mix of early and late) or MSCs suspended in 10ml of PBS (Life Technologies, # 10010-023) were injected slowly through a balloon catheter (OPTA® Pro PTA Dilatation Catheter, Cordis, New Jersey) placed in the renal artery proximal to the stenosis.
Fluorescent-labeled cells were subsequently manually counted ex-vivo under fluorescence microscopy (ZEN® 2012 blue edition, Carl ZEISS SMT, Oberkochen, Germany) in 5µm LV cross-sections and 5µm renal tissue sections stained with cytokeratin (AbD Serotec, Cat# MCA1907), and their number per field averaged (7 (link),8 (link),34 (link)). Furthermore, EPC and MSC distribution was evaluated by immunofluorescence staining with the distal tubular marker peanut agglutinin (PA, Vector Lab, Cat# FL-1071, 1:500), the proximal tubular marker phaseolus vulgaris erythroagglutinin (PHA-E, Vector Lab, Cat# FL-1121, 1:500), the endothelial marker CD31 (AbD Serotec, Cat# MCA 1747, dilution 1:50), and the proliferating cell nuclear antigen (PCNA, Abcam, Cambridge, MA; Cat# ab29,1:100).
