The stem cell therapy for treating ischemic diseases is promising; however, the limited availability and compromised quality of progenitor cells in aged and diseased patients limit its therapeutic use. Here we report a nanofiber-based ex vivo stem cell expansion technology and proangiogenic growth factors overexpression of human umbilical cord blood (UCB)-derived progenitor cells to enhance angiogenic potential of therapeutic stem cells. The progenitor cells were expanded ~225-fold on nanofiber-based serum-free ex vivo expansion culture technique without inducing differentiation. The expanded cells express high levels of stem cell homing receptor, CXCR4, and adhesion molecule, LFA-1. The nanofiber-expanded stem cells uptake AcLDL effectively, and migrate efficiently in an in vitro transmigration assay. These expanded cells can also differentiate into endothelial and smooth muscle cells in vitro. In a NOD/SCID mouse hind limb vascular injury model, nanofiber-expanded cells were more effective in blood flow restoration and this effect was further augmented by VEGF164 and PDGF-BB, growth factor overexpression. The data indicate that nanofiber-based ex vivo expansion technology can provide an essential number of therapeutic stem cells. Additionally, proangiogenic growth factors overexpression in progenitor cells can potentially improve autologous or allogeneic stem cell therapy for ischemic diseases.

BackgroundTherapeutic potential was evaluated in a rat model of myocardial infarction using nanofiber-expanded human cord blood derived hematopoietic stem cells (CD133+/CD34+) genetically modified with VEGF plus PDGF genes (VIP).Methods and FindingsMyocardial function was monitored every two weeks up to six weeks after therapy. Echocardiography revealed time dependent improvement of left ventricular function evaluated by M-mode, fractional shortening, anterior wall tissue velocity, wall motion score index, strain and strain rate in animals treated with VEGF plus PDGF overexpressed stem cells (VIP) compared to nanofiber expanded cells (Exp), freshly isolated cells (FCB) or media control (Media). Improvement observed was as follows: VIP>Exp> FCB>media. Similar trend was noticed in the exercise capacity of rats on a treadmill. These findings correlated with significantly increased neovascularization in ischemic tissue and markedly reduced infarct area in animals in the VIP group. Stem cells in addition to their usual homing sites such as lung, spleen, bone marrow and liver, also migrated to sites of myocardial ischemia. The improvement of cardiac function correlated with expression of heart tissue connexin 43, a gap junctional protein, and heart tissue angiogenesis related protein molecules like VEGF, pNOS3, NOS2 and GSK3. There was no evidence of upregulation in the molecules of oncogenic potential in genetically modified or other stem cell therapy groups.ConclusionRegenerative therapy using nanofiber-expanded hematopoietic stem cells with overexpression of VEGF and PDGF has a favorable impact on the improvement of rat myocardial function accompanied by upregulation of tissue connexin 43 and pro-angiogenic molecules after infarction.

Background Clinical trials using intracoronary (IC) delivery of cells have addressed efficacy but the optimal delivery technique is unknown. Our study aimed to determine whether transvenous intramyocardial (TVIM) approach was advantageous for cellular retention in AMI. Methods Domestic pigs (n=4) underwent catheterization with coronary angiography and ventriculography prior to infarction and pre- and post-cells. Pigs underwent 90 minute balloon occlusion of the left anterior descending artery (LAD). After one week they were prepared for IC (n=2) or TVIM (n=2) delivery of bone marrow mononuclear cells (MNC) labeled with GFP. IC infusion used an over-the-wire catheter to engage the LAD and balloon inflation to prevent retrograde flow. Venography via the coronary sinus was used for TVIM delivery. The anterior interventricular vein was engaged with a guide wire allowing use of the TransAccess™ catheter, which is outfitted with an ultrasound tip for visualization. Animals were sacrificed one hour after delivery and tissue was analyzed. Results Procedures were performed without complication and monitoring was uneventful. 1 × 108 MNC’s were isolated from each BM preparation and 1 × 107 MNC delivered. Ventriculography at one week revealed wall motion abnormalities consistent with an anterior AMI. TVIM and IC delivery revealed mean 452 cells/section and 235 cells/section on average, respectively in the infarct zone (p = 0.01). Conclusion We have demonstrated that TVIM approach for cell delivery is feasible and safe. Moreover, this approach may provide an advantage over IC infusion in retention of the cellular product, however, larger studies will be necessary.

Background: Umbilical cord blood (UCB) and marrow-derived CD133+ cells have been shown to mediate encouraging effects on therapeutic angiogenesis in both animal models and early clinical trials. Low numbers of CD133+ cells derived from a single donor have been a limitation of use of these cells in cardiovascular therapy. We hypothesized that an ex vivo aminated nanofiber system combined with cytokine supplementation would provide optimized topographical and biochemical signals to allow the expansion and potential functional augmentation of CD133+ cells without promoting terminal differentiation. Methods and Results: Human UCB derived CD133+ progenitor cells were isolated by MACS sorting and ex vivo expanded on aminated nanofiber plates with cytokine rich media. Cells harvested 10 days after expansion demonstrated a 225X increase in total number. Flow cytometric analysis demonstrated CD133–24%, CD34–93%, CXCR4–97%, LFA-97% surface expression. The expanded cells can uptake AcLDL efficiently and demonstrate a 2.3X increase in transwell migration to SDF-1 as compared to fresh UCB CD133+ cells. In vitro analysis revealed that expanded cells have potential to differentiate into endothelial or smooth muscle phenotype as demonstrated with CD31, vWF, VCAM-1 and F-pholloidin, α-actin, and SM myosin heavy chain immunocytochemistry when re-cultured for 14d in EGM2 or SMBM respectively. RT-QPCR analysis of 1% O 2 exposed (hypoxic) cells demonstrated a 2X increase in VEGF and 3X increase in IL-8 gene expression compared to normoxic control. In vivo functionality in a NOD/SCID mouse hind limb ischemic model demonstrated that mice treated with 5 x 10 6 expanded cells (n=7) augmented blood flow ratio (ischemic/control limb) as compared to mice treated with CD133+ cells (n=7) and control (n=7) at 28d. (control 0.32±.02 vs. UCB133+ 0.37±.02 vs. expanded cells 0.50±.04 p<0.01) Capillary density in ischemic hind-limb was increased at 28d (control 62.5±5.4 vs. expanded cell 97.6±2.5 p< 0.01) Conclusions: These studies demonstrate successful high level expansion of UCB derived CD133+ cells into functionally potent stem cells which have the capacity to differentiate into vascular cells and promote in vivo neovascularization.