Adult Human Blood Leukocytes as a Source of Tissue-Committed Progenitors for Cellular Therapy of Cardiovascular Disease.

Significant progress has been made in recent years in developing therapeutic strategies for the treatment of a variety of cardiovascular disorders, mainly using bone marrow-derived progenitor cells. We hypothesized that blood leukocytes can also serve as a source for a wide range of clinical protocols. We report here the generation in vitro of both angiogenic cell precursors (ACP) and cardiomyocyte (CMC) progenitors from a newly discovered blood-derived multipotent cell population, termed synergetic cell population (SCP), and their function in vitro and in vivo.

Progenitor cells were purified from healthy donor blood samples using density-based gradients. SCP-derived ACPs grown in the presence of autologous serum and VEGF exhibited an elongated, spindle-shaped morphology and expressed the stem cell markers CD34 (an average of 23.1% of cells), CD133 (10.2%), and CD117 (10.8%), and the endothelial markers KDR (8.9%), Tie-2 (24.8%), CD144 (41.2%), and CD31 (83.1%). Up to 30% of the cells exhibited Dil-Ac-LDL uptake, typical of endothelial cells. In vitro, ACPs showed organization into capillary tube structures when plated on extracellular matrix gels. An average of 50x10⁶ ACPs were generated from 450 ml blood. CMC progenitors, which resulted from culturing SCP cells in medium containing autologous serum and bFGF followed by activation in a medium containing 5-azacytidine, appeared elongated with dark cytoplasm and expressed the cardiomyocyte markers desmin and troponin (on 19.7% and 52.3% of cells, respectively). The therapeutic potential of blood derived ACPs is currently being evaluated in patients with severe angina pectoris. Seventeen patients on maximal drug therapy have so far been prospectively enrolled, based upon identifying ischemic but viable myocardium in distribution of the coronary arteries that were totally occluded. ACPs (25x10⁶, SE=4.9) were injected via a catheter into the coronary artery. Preliminary results demonstrate safety and improved clinical symptoms at 3 months vs. baseline. Mean Canadian Cardiovascular Scale for angina severity decreased from 1.8±0.8 to 1.06±0.3 (P=0.062) and exercise capacity measured by metabolic equivalents increased from 6.3±2.3 to 7.4 ±2.8 (P= 0.0083). One patient died two weeks after the treatment due to acute myocardial infarction. However, coronary angiography demonstrated acute occlusion of an artery not treated with cells. These results suggest the treatment is safe with preliminary short term beneficial effect. Continued follow-up is currently being conducted to determine the long-term effects of this therapy in a larger number of patients.

In order to examine the functional mechanisms underlying the therapeutic effects of ACPs and CMC progenitors, an in-vivo experiment is also being carried out in a nude rat acute myocardial infarction model. We demonstrate here that a newly-discovered multipotent cell population which we term SCP can be isolated from peripheral blood and differentiated into therapeutically effective tissue-committed progenitor cells. The SCP contains hematopoietic stem cells and supportive cells enabling differentiation into various lineages, such as ACPs, cardiomyocyte and neural progenitors (the latter reported in a separate abstract by our group) which have thus been generated.