Human UCB CD133⁺ Cell Infusion In NOD-SCID Mice with Acute Hind Limb Ischemia Improves Reperfusion of An Ischemic Bed

Abstract 4697

Clinical trials have shown intracoronary or intramuscular injections of hematopoietic stem cells (HSC) ameliorate vascular ischemia. HSCs are isolated from bone marrow (BM), umbilical cord blood (UCB), and adult peripheral blood (AB). Critical limb ischemia patients, injected with autologous HSC into ischemic tissue had improved perfusion and reduced amputations. However, autologous HSC effectiveness declines with age. We hypothesized that UCB CD133⁺ HSCs’ are more efficacious than autologous HSC at revascularization for an ischemic bed.

Immune reactive surface antigens on UCB CD133⁺ cells were determined using multi-parameter flow cytometry. UCB CD133⁺ allogeneic immune responses from 72 h co-cultures were analyzed using a mixed lymphocyte culture (MLR) proliferation assays, and induced cytokine production found using TH1(inflammatory)/TH2(anti-inflammatory) cytometric bead assays (CBA) (BD Biosciences (San Jose, CA). In vivo studies were done using NOD/SCID mice treated with low-dose irradiation (200cGy) then femoral ligation. Study mice were divided into 3 treatment groups and underwent intracoronary infusions of; 1×10⁶ UCB CD133⁺, 0.5×10⁶ UCB mononuclear cells (MNCs), or EGM media alone. Day 3 post-ligation and cell infusion, quadriceps from 3 mice in each group were harvested, total RNA isolated using Trizol™ and RNA for each treatment group pooled. Pooled RNA was converted to cDNA then assayed using mouse ABI Affymetrix™ Gene chip. Computational pathway analyzes were run for significant transcriptome changes. Gene array data was confirmed on select genes using quantitative reverse transcriptase real time PCR (qPCR). Treated mice were followed for ischemic bed reperfusion by Laser Doppler imaging for 28 days post-ligation. UCB CD133⁺ cells express MHC class I and II, but lack CD80 and CD86. Allogeneic MLR assays using UCB and AB MNCs showed UCB CD133⁺ cells were stimulatory to MNC proliferation. The MNCs alloresponse to UCB CD133⁺ cells produced high concentration of interleukin 4 (IL-4) and IL-10 indicative of a TH2 response. These results show allogeneic UCB CD133⁺ cells induce a TH2 immune response due to an absence of co-stimulation of immune cells. To verify whether the results are recapitulated in vivo, we treated acute limb ischemia in NOD/SCID mice with human UCB CD133⁺ cells and monitored revascularization by blood flow and Affymetrix mouse gene array. Computational analysis of Affymetrix mouse gene arrays from comparing all 3 treatment protocols indicated that ischemic quadriceps treated with UCB CD133⁺ cells exhibited a significant enrichment of anti-angiogeneic, chemotaxic, cytokine, and immune response genes. UCB CD133⁺ treated mice had increase expression of anti-inflammatory, and anti-angiogeneic genes versus controls. Conversely media and UCB MNC treated mice had equal to or higher pro-inflammatory gene expression. The anti-inflammatory gene response was confirmed for 2 of the 3 genes, arginase 1, CXCL7, and tumor growth factor beta binding protein 2 (TGFβBP2) comparing CD133⁺ treated mice to UCB MNC treated mice. TGFβBP2 was not observed to change in vivo. UCB CD133⁺ cells treated mice also had improved blood perfusion of the ischemic bed at 28 days post-ligation versus control protocols. The in vivo mouse model supports the in vitro observation, but demonstrates improved reperfusion over controls.

Currently there are 2 non-exclusive hypotheses for the function of CD133⁺ cells in revascularization. First the CD133⁺ cells integrate and differentiate into new vascular endothelial cells and the second is that the CD133⁺ cells augment revascularization via a paracrine effect which does not require integration into the host tissue. In vitro studies suggest that UCB CD133⁺ cells promote an allogeneic TH2 type response because of a lack of co-stimulatory immune surface antigens, resulting in high concentrations of IL-4 and IL-10. This skewed TH2 response inhibits inflammation, the subsequent tissue damage, and may augment revascularization. Although these studies cannot exclude a role for UCB CD133⁺ cellular integration, results support the paracrine hypothesis as infusion of selected CD133⁺ cells appears to promote an anti-inflammatory, anti-angiogenic profile at day 3 post-ligation and at 1 month improved reperfusion compared with non-selected MNC.