Bone Marrow Stem Cell Transplantation Combined With Erythropoietin Treatment Of Ischemic Cardiovascular Disease,

Section oneCulture in vitro and primary identification of rat marrow mesenchymalstem cellsObjective To explore the feasibility of the clutivation of rat marrowmesenchymal stem cells (MSC) in vitro and analyse their phenotypicalproperties, to investigate the possibility of a new cell source for ischemiccardiovascular diseases trasnsplantation. Methods The differential adherenceto plastic was employed to separate MSC. MTT method was employed tocharacterize the potent of proloferation and self-renewal of MSC. Preliminarilyindentification of mesenchymal stem cells was used byflowcytometer. Adipogenic medium was used as induction for thedifferentiation of passage 4 MSC into adipocytes. The differentiated cells wereidentified by oil red O immunohistochemistry stain. Results The purified MSCshowed the morphology of firoblasts increased in the medium. The MSC coulddirectionally into adipocytes induced by adipogenic medium. Flowcytometryshowed that it is positive CD44, CD29, CD13 and negative CD34, CD45,CD33. Conclusion The purified MSC can be harvested by method ofdifferential adherence to plastic. These MSC have the potent of proliferationand self-renewal. The method is easy to operate and simple. Section twoEffect of erythropoietin on in-vitro proliferation and cell circle stage of ratbone marrow mesenchymal stem cellsObjective To determine the effect of erythropoietin (EPO) on in-vitroproliferation of mesenchymal stem cells(MSC). Methods Under serum freeculture media, MSC incubated with different doses of EPO for 24h, and thenproliferation of MSC were measured by MTT assay. Cell circle stage wasmeasured by flow cytometry assays after incubating MSC incubated with10U/ml EPO for 72h.Results MTT assay showed that EPO could enhance MSC proliferation in dosedepend manner. There was significant difference among EPO treatment groupsand control group, except that 0.25U/ml group. The effect on proliferation ofMSC was highest in 10U/ml group among all groups. Furthermore, flowcytometry assays showed that EPO could decrease G0/G1 ratio, increase S stageand G2/M stage ratio. There was significant difference between EPO group andcontrol group. Conclusions EPO could enhance MSC proliferation in dosedepend manner.Section ThreeStudy of erythropoietin inhibitting apoptosis and enhancing paracrineaction of rat bone marrow mesenchymal stem cells Objective To determine the effect of erythropoietin (EPO) on in-vitroapoptosis and paracrining vascular endothelial growth factor (VEGF) ofmesenchymal stem cells(MSC). Methods MSC apoptosis was detected by flowcytometry following culture in serum-free medium for 24h with or without EPO.Release of vascular endothelial growth factor (VEGF) by MSC incubated withdifferent doses of EPO was assayed using ELISA. Results EPO inhibited MSCapoptosis induced by serum-free medium and increased vascular endothelialgrowth factor released by MSC.Conclusions EPO could inhibited MSCapoptosis and enhanced MSC paracrining action.Section FourErythropoietin enhances the therapeutic potency of bone marrowmesenchymal stem cells for myocardial infarction in ratsObjective Erythropoietin (EPO) induces angiogenesis and inhibits cell apoptosisthrough the phosphatidylinositol 3-kinase/Akt pathway. Transplantation ofmesenchymal stem cells (MSC) has been shown to improve heart perfusion andfunctions after myocardial infarction. We investigated whether EPO enhancesthe therapeutic potency of MSC transplantation. Methods 32 rats were dividedinto four groups: acute myocardial infarction group (MI), EPO group, MSCgroup and MSC-EPO group. Myocardial infarction model was made by ligatingthe left anterior descending coronary artery. MSC was injected into the infacted area after ligation both in group MSC and group MSC-EPO. EPO wasadministered i. p. both in group EPO and group MSC-EPO at the dose of 3000U/kg body weight three days around the ligation and three days after fourteendays. Cardiac function was measured by 2D-Echocardiography 2 days and 21days after surgery respectively. Western blot and immunohistologicalassessments were performed to examine the effects of these treatments. ResultsThere was no difference in LVEF among all groups 2 days after MI. After 21days a decrease LVEF was observed in control group while the LVEF markedlyimproved both in EPO group and in MSC group. MSC-EPO group demonstrateda further improvement in LVEF.Cardiac infarct size was significantly smallerboth in MSC and EPO groups than that in control group. A combination of EPOinfusion and MSC transplantation demonstrated a further decrease in infarct size.EPO and MSC significantly increased capillary density in ischemic myocardium,suggesting the angiogenic potency of EPO and MSC. EPO infusion plus MSCtransplantation demonstrated a further increase in capillary density comparedwith EPO or MSC alone. EPO and MSC improved cell survival parameters asshown by increased the ratio of p-AKt/AKt and the level of Bcl-2 and reducedthe level of Bax at 21 days after MI. EPO infusion plus MSC transplantationdemonstrated a further improvement. MSC could transdifferentiate intoendothelial cells, smooth muscle cells and cardiac cells. Conelusions Our resultsdemonstrated that MSC transplantation and EPO infusion could improve heartfunction. EPO enhanced the angiogenic potency of MSC transplantation and improved cardiac function in rats with myocardial infarction. This beneficialeffect may be mediated partly by the angiogenic property of EPO itself and byits antiapoptotic effect both on MSC and cardiomyocytes.Section FiveErythropoietin enhances the angiogenic potency of bone marrow stromalcells in a mouse hindlimb ischemia modelObjectives: Previous studies have shown that erythropoietin inhibits vascularendothelial cell apoptosis and induces angiogenesis. We investigated whethererythropoietin enhances bone marrow stem cells induced angiogenesis. MethodsImmediately after hindlimb ischemia was created, mice were randomized toreceive erythropoietin(EPO) infusion plus bone marrow stem cells(MSC)transplantation (EPO-MSC group), EPO infusion alone (EPO group), MSCtransplantation alone (MSC group), or vehicle infusion (control group). ResultsThe levels of VEGF protein in the ischemic hindlimb were significantly higher7 days after combination treatment with MSC and EPO than with MSC orEPO or vihichle treatment alone .The blood flow rate in the ischemic hindlimbwas significantly higher 3 wk later in the EPO and MSC groups than in thecontrol group, which suggests the angiogenic potency of EPO and MSC.Importantly, improvement in blood perfusion was marked in the EPO-MSC group. Capillary density was highest in the EPO-MSC group, followed by theEPO and MSC groups. In vitro, EPO inhibited MSC apoptosis. In vivo, EPOadministration enhanced the differentiation of MSC into endothelialcells. Conclusions A combination of EPO infusion and MSC transplantationcaused significantly greater improvement in hindlimb ischemia than MSCtransplantation alone. This effect may be mediated in part by the angiogenicpotency of EPO itself and the beneficial effects of EPO on the survival anddifferentiation of transplanted MSC.