ИСТИНА

Question Delivery of cells for therapeutic application is a well-established approach to trear limb ischemia and a range spectrum of other disorders. Still efficacy of cell therapy is hindered in a number of trials due to a number of factprs including low survival of transplanted cells. This obstacle can be circumvented by application of tissue-engieered constructs, e.g. cell sheets (CS), which comprise of cellular elements along with their extracellular matrix. Methods We established and effective and rapid protocol for generation of CS from adipose-derived stromal cells (ADSC) of human and animal origin without appliaction of thermoresponsive dishes. Detached CS were transplanted to mice with induced limb ischemia (n=8-10 per group) by subcutaneous attachment after placement of exposed skeletal muscle. In some animals CS were transduced using a hybrid baculoviral system to express VEGF165, which enahnces ADSC therapeutic potential. Suspended ADSC were also deliverd via injections to serve as reference method. Limb perfusion was assessed via laser Doppler and histology inclided routine stains and analysis vascular density (stains for CD3 and a-SMA), proliefration (Ki-67) and apoptosis (caspase-3) in muscle and CS. Results In all specimen analzyed after animal euthanasia we found CS to be engrafted, vascularized and infiltarted by CD68+ monocytes at 7 and 14 days post delivery. Vacularization of CS comprised of CD31+ capillaries and a-SMA+ arterioles indicating graft-host interactions. Approximately 10% of cells within CS were caspase-3 positive indicating apopotosis and a number of Ki-67+ nuclei was found reflecting proflierating cells. These changes were accompanied by significant improvement of perfusion, vascular density and necrosis reduction indicating ischemia relief comapred to untreated control. In our study CS delivery was superior to injection of suspended ADSC in equivalent amount. In these animals we found significant engraftment of ADSC, which can be described as intramuscular infiltrates yet perfusion was lower, than in CS group. Still attempting to further enhance method’s efficacy we expressed VEGF165 is CS prior to delivery and found that VEGF165-expressing CS had even stronger impact of limb perfusion reaching up to 65% per cent vs. 50-55% in unmodified CS and approximately 40% in susp. Moreover we found VEGF165-expressing CS to induce angiogenesis in a more efficient way and further stimulate capillarogenesis. Conclusions Overall, our data indicates that delivery of CS from ADSC is an effective way for tissue protection and stimulation of angiogenesi in ischemic limb. Delivery of CS was superior to suspension in terms of limb perfusion and angiogenesis stimulation. Still viral expression of VEGF165 has enhanced therapeitic potential of CS and allowed to robustly restore limb perfusion and reduce necrotic change to minimal extent.