Abstract
The field of vascular regenerative medicine is rapidly growing and the demand for cell-based therapy is high. In our studies, human embryonic stem cells (hESCs) were differentiated via coculture with M2-10B4 mouse bone marrow derived stromal cells for 13–15 days. At this time, CD34+ were isolated using an immunomagnetic separation technique and further phenotyped. As shown by flow cytometric analysis, the population co-expressed typical endothelial cell surface antigens such as CD31 and Flk. Upon culture of these CD34+ cells in endothelial culture medium containing VEGF, bFGF, IGF-1, EGF, and heparin, the cells assumed a endothelial cell morphology, formed vascular like networks when placed in Matrigel, and expressed CD31, Flk1, CD146, Tie2, eNOS, vWF, and VE-cadherin (each confirmed by quantitative real time PCR, immunohistochemistry, and flow cytometry). Transmission electron micrograph images of these cells, termed hESC-ECs, showed a defined cortical filamentous rim as seen in other endothelial cells and a significant number of micro-particles being released from the cell surface. Additionally, permeability studies revealed these cells exhibit trans-electrical resistance of 1200Ω, consistent with basal barrier properties exhibited by conduit endothelial cells. These hESC-ECs also proved capable of further differentiation into smooth muscle cells, hESCSMCs. When culture conditions were changed to support SMC growth (DMEM + PDGFBB and TGF-β1), cells assumed SMC morphology including intracellular fibrils, down regulated endothelial gene transcript and protein expression, and began to express α-SMC actin, calponin, SM22, smoothelin, myocardin. Also, concomitant increases in expression of APEG-1 and CRP2/SmLIM, expressed preferentially by arterial SMCs, was found. In contrast, HUVECs placed under these SMC conditions did not display SMC characteristics. Additional studies evaluated intracellular calcium release in hESC-ECs and hESC-SMCs subjected to various pharmacological agonists. The hESC-SMC population preferentially responded to bradykinin, oxytocin, endothelin-1, histamine, and ATP, while hESC-ECs responsed to endothelin-1, histamine, bradykinin, and carbachol. Functional studies were initially done by in vitro culture of these cell populations in Matrigel. hESC-SMCs placed in Matrigel alone did not form a vascular like network. However, an improved vascular structure was seen when hESC-ECs were placed in Matrigel along with hESC-SMCs. Together, these cells formed a dense, more robust vascular network composed of thicker tube structures, indicating a more physiologically relevant model of vasculogenesis. Next in vivo studies have been initiated utilizing a mouse myocardial infarct model. NOD/SCID mice were anesthetized and subjected to ligation of the left anterior descending artery. By assessing cardiac function 3 weeks post infarction, we found that mice receiving an hESC-EC injection (1×106 cells directly into infarction sight) showed greater vascular repair and increased ejection fraction when compared to mice that did not receive an hESCEC injection [untreated control ejection fraction= 14.3% vs hESC-EC treated= 21.3%]. Currently, studies are underway evaluating combined use of hESC-ECs and hESC-SMCs in this infarct model, as we hypothesize that combined use of these cells will be more beneficial for vascular development and repair than either one population alone. Together, the phenotypic and functional studies of these hESC-derived CD34+ cells suggest these cells can act as pericytes with dual endothelial cell and SMC developmental potential and these hESC-derived pericytes can provide an important resource for developing novel cellular therapies for vascular repair.
173 EFFECT OF CULTURE SYSTEM FOR IVM-IVF PIG EMBRYOS ON THE ICMS ABILITY TO PRODUCE OUTGROWTHS FOR EMBRYONIC STEM CELL DERIVATION
