The Experimental Study On Adipose-derived Stem Cells For Transplantation In Rat Stroke Model

Background The discovery of neural stem cell have changed the traditionary viewpoints that neurons can't regenerate whenever it is injured,which lead a brand new cognition to the structure and function of nervous system.Cultured neural stem cells represent not only a useful model for development studies,but also a potential cell source for transplantation to cure nervous system diseases in clinic,but it clinic utility was greatly limited because of limitation in number and ethical problems.Adipose-derived stem cells(ADSCs)have characteristics of rich in number,easily obtain,quickly proliferation in vitro and ability of accesss to autologous cell transplantation,which suggested the more advantages of this kind of stem cells in clinic application.It was reported that ADSCs could be induced into neural cell-like cells,and may be able to contribut to neural repair through directly replacing lost neurons or other mechanisms,including modification of the environment surrounding a lesion by production of trophic factors,anti-oxidants,or matrix metalloproteases. Objective To study if special neural stem cell culture medium could be used to induce ADSCs differentiate alone the neural pathway,and express specific markers of neuraI precursor cells such as Nestin and specific markers of neuron such as neurone specific enolase(NSE),Investigate the feasibility that ADSCs could be labeled with Feridex-poly-1-lysine(FE-PLL)complex and tracked by MRI after transplantation into brain;Explore the mechanism of ADSCs transpalntation to improve neurological functional recovery after stroke in rats.Methods Inguinal adipose tissues were harvested after sacrifice of the adult rats.ADSCs were isolated after the adipose tissue was dissociated mechanically,digested using collagenase typeâ… . The third generation ADSCs were cultured in DMEM/F12(1:1) supplemented with 2%B27,20ng/mlbFGF and 20ng/ml EGF.DMEM/F12(1:1)only supplemented with 10%fetal bovine serum was used to induce ADSCs differentiate alone the neural pathway.Immunocytochemistry was employed to identify the expressions of Nestin,neurone specific enolase(NSE).The third generation ADSCs was also labeled with Feridex-poly-1-lysine(PLL) complex,Prussian blue staining and typan-blue exclusion test were used to evaluate the labeling efficiency of FE-PLL complex labeled ADSCs and cell viability,respectively.Meanwhile,MR imaging was performed for cell suspensions and in vivo tracing labeled ADSCs after transplantation into rat brain.All animals were randomly divided into ischemia control group,PBS group,ADSCs group.The neurological severity score(NSS)was used to evaluate neurological function recovery of rats at definite time following the transplantation.The biodistribution of grafted ADSCs was monitored noninvasively by magnetic resonance (MR)imaging.The transplanted ADSCs were analyzed in recipient rat brains by immunohistochemical method.Results The primary ADSCs proliferated rapidly.Typical neurosphere formation were observed 3 days after induction.After separating the cellular spheres into single cells and then plating them,we found the islets-shaped cellular spheres appeared again.Most of neurospheres expressed specific markers of neuraI precursor cells such as Nestin.ADSCs were successfully induced to diferentiate along neural pathway.Following differentiation of the NSCs spheres,some of them formed small buds,which then developed further into long projectsconnecting each other.Most of the cells with long projects showed positive NSE.Numerous intracytoplasticiron particles were stained with prussian blue.Typan-blue exclusion test showed that the viability of the labeled cells after labeling had no significantly difference with that of nonlabeled cells at 24h,1 week,2 week and 3 week(P＞0.05). In vitro MRI showed the suspension of labeled ADSCs by FE-PLL complex made T₂ signal intensity decrease significantly.The implanted labeled ADSCs were both visible on T₂WI and T₂^*WI as a hypointense area at the injection site,and T₂^*WI is the most sensitive sequence to detect the labeled cells.Significant improvement in NSS was observed in rats receiving ADSCs transplantation compared with control groups and PBS group after 3 weeks(P＜0.05)The transplanted ADSCs preferentially engrafted migrated toward the injured brain and expressed the neuronal marker NSE and vascular endothelial cell marker CD31. MR imagining showed remarkable hypointense signal in the region of transplanted ADSCs labeled with PLL-FE complex especially in T₂ GRE sequence and FE-labeled ADSCs migrated toward the edge of ischemic brain through corpus callosum.Conclusions It is possible that ADSCs could be induced into NSCs under certain experimental conditions.And also it implied the feasibility and validity that ADSCs might be used as the seed-cells of the neural stem cells.Magnetic labeling of rat ADSCs with FE-PLL complex is safe and efficient;Neurological and functional improvement was observed in rats with ADSCs transplantation following ischemia brain injury.The new generated neurons and neovascularity may promote the functional recovery following ischemia brain injuries after ADSCs transplantation.In vivo MRI tracking of SPIO-PPL complex labeled ADSCs could evaluate the therapeutic effect following transplantation.