Specific Lysis for Large-Volume, High-Purity Endothelial Progenitor Cell Isolates

This work develops Antigen-Specific Lysis (ASL) for the large scale isolation of progenitor cells for impact in progenitor cell clinical research and the widespread treatment of acute myocardial infarctions and other ischemic conditions. Despite the growing evidence supporting the use of a purified progenitor population in revascularization of ischemic and damaged tissues, there is no scalable technical approach for high throughput, high purity isolation of these endothelial progenitor cells (EPCs). The execution of the proposed research will i) develop a novel mode of cell isolation which circumvents fundamental throughput and purity limitations of conventional sorting methods, ii) demonstrate the isolation of >99% pure EPCs from whole blood, iii) demonstrate a single pass isolation from >1010 white cells in 99%) and simplicity (easily achieved in basic lab settings) to be unrivaled by fluorescent or magnetic sorting of CD133+ and CD34+ cells. We have previously demonstrated the formation of polymer coatings on cells in a wide variety of polymerization chambers. Based on this experience, we expect the throughput of ASL to scale with the irradiation area, enabling the lab-scale isolation of batches in excess of 200 mL and commercial isolation at even greater batch sizes. We will support this hypothesis by sorting >1010 white cells for CD133+, and into culture in under an hour. The lab-scale prototype system is based on inexpensive light sources and the cells will be processed exclusively in standard polystyrene labware, eliminating time consuming sterilization procedures. This approach also allows the simultaneous processing of independent batches in separate dishes, permitting the parallel isolation of samples in the same system without contamination. We expect batch size to have minimal impact on the previously demonstrated high-purity and viability of the isolate, and to enable individual PIs to isolate EPCs in-lab on a scale consistent with therapeutic doses for vasculogenesis. Aim 2. Isolation and characterization of pure and viable endothelial progenitor cells. Previous research from the Balasubramaniam lab has shown that there are quantifiable numbers of endothelial progenitor cells in the cord blood of newborn infants. These cells can be quantified through flow cytometric isolation, magnetic bead sorting and through direct culture of cord blood mononuclear cells. Cord blood derived EPCs consist of two distinct types that share some endothelial characteristics, but differ in their angiogenic function. Early outgrowth EPCs are the more common type of cell and represent approximately 3% of the cord blood mononuclear population. These cells express both angiogenic and hematopoietic markers, have a very low proliferative rate and cannot form blood vessels de novo. Despite this, they have been found to be pro-angiogenic in their function. These cells seem to require the much rarer population of cells, the late outgrowth EPC, in order to form blood vessels de novo. The late outgrowth EPC compromises only 0.01% of the cord blood mononuclear cell fraction, which translates roughly into one to two cells per ml of cord blood. Although rare, late outgrowth EPCs are highly proliferative and exhibit the clonal characteristics of a progenitor cell. The challenge with conventional isolation techniques for this rare cell population is that purity is hard to achieve with magnetic bead sorting, and that these cells are exquisitely sensitive to stress induced by flow cytometry that affects their viability. This aim will compare both the in vitro viability and function of isolated late outgrowth EPCs from the cord blood between ASL versus magnetic bead isolation versus flow cytometric isolation. We will examine the viability, growth, tube formation and migration of isolated cells in vitro. We will also examine the efficiency of these cells to form vessels in vivo in a subcutaneous matrigel plug system in an immunotolerant mouse. We expect ASL to provide exceptional viability, functionality, and purity when contrasted with magnetic or fluorescent sorting.