Experimental And Clinical Study On Transplantation Of Purified CD34~+Cells From Peripheral Blood In The Treatment Of Critical Limb Ischemia

Part ⅠThe culture, identification and biological characteristics of human primary CD34+cells from human peripheral bloodObjective:To evaluate the culture, amplification, identification, cryopreservation, resuscitation and biological characteristics of Primary CD34+cells in vitro, isolated from human peripheral blood by density gradient centrifugation and immunomagnetic beads separation technique, and to prepare another transplantation for the same patient.Methods:CD34+cells were extracted from peripheral blood by Density gradient centrifugation and immunomagnetic beads separation technique. And then CD34+cells were cultured and amplified in high glucose DMEM medium and X-VIVO medium containing growth factors. Morphology and growth characteristics were observed by light microscope, division and migration were recorded by automatic living cell observation and analysis system (cell IQ), the surface markers CD34and CD133were identified by immunofluorescence technique, cell activity was calculated after cryopreservation for1year and resuscitation.Results:The primary CD34+cells in X-VIVO medium were grown in suspension, colonies were formatted in3days, a small number of adherent cells grew in5days, spindle-shaped cells increased in7days, cell debris appeared in9days, and then suspension cells and adherent cells were cultured separately with X-VIVO and high glucose DMEM, most suspension cells were death and adherent cell colonies were formatted in14days with X-VIVO medium. The average time of cells division was90minutes and almost all suspended cells moved to the Central by Cell-IQ dynamic observation. The surface marker CD34and CD133were both positive by immunofluorescence. Cell viability were (89.0±2.58)%in Cryostor and (84.9±3.28)%in DMSO (p=0.006) after storing at-80℃for12months and resuscitation. Conclusion:Purified CD34+cells culture and amplification in vitro are feasible. The impact on the cells vitality is little after cryopreservation and resuscitation. It may provide sufficient stem cells for patients’ transplantation again. Part IIThe experiments of CD34+cell transplantation in the treatment of the nude lower extremity ischemia disordersObjective:To explore the effect of human peripheral blood CD34+cell transplantation in the treatment of the nude lower extremity arterial ischemia and its possible mechanism.Methods:36nude mice were randomly divided into CD34group, X-VIVO control group and sham operation control (n=12), CD34group and X-VIVO control group mice were stripped left whole femoral artery and observed the state of limb ischemia, sham group does not peel off blood vessels. CD34group were injected0.1ml X-VIVO medium containing CD34+cells (1×105cells) to left limb ischemic muscle tissue, X-VIVO groups were injected only0.1ml X-VIVO medium, and sham group were injected only saline0.1ml in the7th day after operation. Nude mice adductor and semimembranosus specimens were acquired after two weeks of injection. Anti-CD31and anti-CD34factor immunohistochemical staining showed vascular endothelial cells and vascular density; Real time PCR detected mRNA of VEGF and bFGF expression;Results:Immunohistochemical staining showed that vascular density in CD34group was significantly increased than X-VIVO group and sham group, RT-PCR examination showed mRNA expression of VEGF and bFGF were strongest in CD34group, followed by X-VIVO group (P<0.05), sham group was the lowest (P<0.05).Conclusion:CD34+cell transplantation can significantly promote angiogenesis, the formation of collateral vessels and increased lower limb ischemic tissue perfusion. Part Ⅲ Transplantation of Purified CD34+Cells in Treatment of No-option Critical Limb IschemiaObjective: To investigate the feasibility, safety and efficacy of intramuscular injection of CD34+cells isolated from peripheral blood mononuclear cells (PB-MNC) mobilized by granulocyte colony stimulating factor (G-CSF) for management of no-option critical limb ischemia (CLI).Methods:From May2009to July2011,25patients were enrolled with25lower and3upper extremities treated, mean age44±12years. After subcutaneous administration of G-CSF for5days with the dose of5-10μg/kg, apheresis and immunomagnetic separation were performed to acquire the isolated CD34+cells which were then intramuscularly injected into the ischemic sites. All cases were divided into3groups:low dose,105/kg; mid dose,(5×105)/kg; and high dose,106/kg. Both the overall outcomes among all cases and comparison between3groups were evaluated.Results:During the follow-up from6to33months, the overall outcomes showed: Wong-Baker FACES pain rating scale score decreased from7±2to3±3(p=0.0000) and1±2(p=0.0000) at1and2months, respectively; at respective3and6months, peak pain-free walking time increased from4±3min to13±7min (p=0.0005) and18±6min (p=0.0000), ankle-brachial index from0.46±0.21to0.60±0.17(p=0.003) and0.67±0.15(p=0.001), and transcutaneous partial oxygen pressure from27±10mmHg to41±11mmHg (p=0.0001) and55±12mmHg (p=0.0000); ulcer was healed in10out of the14cases;4cases had above-or below-knee amputation within3months; the Kaplan Meier estimate of the rate of freedom from major amputation at6months was84%(95%confidence interval,0.63to0.94). The comparison among3different groups (low dose,5cases; mid,11; high,9) revealed no significant difference, except that WFPRSC improvement at1month from baseline in high-dose group (6.3±1.7) was significantly superior to that in either the low-dose (3.2±3.3, p=0.0487) or the mid-dose group (3.7±2.8,p=0.0352).Conclusions:Transplantation of CD34+cells isolated from G-CSF-mobilized PB-MNC appears to be feasible, safe and effective in treatment of no-option CLI.