Abstract 17229: Quantity and Quality Culture Restores Diabetic Endothelial Progenitor Cell Dysfunction for Wound Healing
Background: The quality and quantity of endothelial progenitor cells (EPC), i.e., CD34+ cells is known to be impaired in diabetic patients, thereby raising declined tissue repair in autologous EPC therapy. Recently, we have disclosed the newly developed quantity and quality control culture (QQc) system to potentiate the vasculogenic property of EPCs for tissue repair. Herein, we investigate the therapeutic application of QQc on clinical diabetic patient’s CD34+ cells for wound healing.
Methods CD34 + cells were isolated from 50 ml of peripheral blood in diabetic patients by auto MACS system, then underwent QQc for 7 days using STEMSPAN serum free medium with VEGF, Flt-3 ligand, TPO, IL-6 and SCF. The vascular regeneration capability of CD34 + cells pre- or post QQc was evaluated with EPC colony forming assay (EPC-CFA), FACS, or qRT-PCR. Then, 2x104/wound pre- or post- QQc CD34 + cells were transplanted (Tx) into the bed of the stented 6-mm wounds in Balb/c nude mice. Morphometric analysis of % wound closure, granulation area or angio-vasculogenesis by co-staining of the antiboides for CD31 and/or human specific mitochondria antigen (HMA), was assessed for wound healing.
Results: EPC-CFA disclosed the predominant generation of functional definitive EPC-CFU in post QQc CD34+ cells vs pre QQc CD34+ cells (dEPC-CFU No./1000 cells: 31±10 vs 1.2 ±0.2, P < 0.05, n=4) with higher RNA transcripts of VEGF, Ang1, and Ang2. Tx of post QQc CD34+ cells consecutively unveiled the greater closure compared with that of pre QQc CD34+ cell Tx (% wound closure post- vs pre- QQc Tx at day 21: 94.64±2.2 vs 61.45±5.2, p<0.05, n=4). Further, post- QQc CD34+ cells promoted wound granulation and maturation as well as vascularity compared to pre- QQc CD34+ cells at day 21 (post- vs pre- QQc Tx for wound granulation pixels/ hpf: 386837±84945 vs 224295±17688; for CD31+ cells/ hpf: 34.64±4.2 vs.43±2.6; p < 0.05, n=4). Co-staining for HMA and CD31 revealed vasculogenesis of Tx cells.
Conclusion: QQc ystem of autologous CD34+ cell provides the methodological clue to overcome the insufficient efficacy of naïve CD34+ cell therapy for wound healing in diabetic patients.
- © 2012 by American Heart Association, Inc.