Autologous Transplantation Of Ex Vivo Expanded Bone Marrow Stromal Cells And Endothelial Progenitor Cells For Treatment Of Focal Cerebral Ischemia

Stroke is the third leading cause of death,ranking behind diseases of the heart and all forms of cancer.At present,thrombolytic therapy inducing recanalization of the occluded vessels in the cerebral infarcted area is a commonly used therapeutic strategy.However,only a minority of patients have timely access to this kind of therapy.Thus,most of stroke causes a disturbance of neuronal circuitry and disruption of the blood-brain-barrier that can lead to functional disabilities.However,no treatment currently exists to restore lost neurological function after stroke.Remodeling the brain,like remodeling a house,may require new plumbing, such as new brain cells;new blood vessels;new electrical connections or new synapses in order to regain function.A growing number of studies highlight the potential of stem cell transplantation as a novel therapeutic approach for stroke.Bone marrow stromal cells(BMSCs),obtained from postnatal bone marrow, contain progenitors able to differentiate into several mesenchymal lineages,and has been successful transdifferentiate into neual cells by combination of growth factors and substrates.Series of reports that BMSCs can ameliorate recovery after focal cerebral ischemia in rodents have been published,which suggests that BMSCs are one of the most promising cells resource for repairing of stroke.However,because of their low frequency in vivo,to obtain a sufficient number of cells for cell therapy, a step of extensive in vitro expansion is required,which could significantly reduce the cells ability of pluripotent differentiation,proliferation and homing.BMSCs exhibit senescence-associated growth arrest during long-term in vitro culture.Endothelial progenitor cells(EPCs),another stem cells originating from bone marrow,play an important role in therapeutic neovascularization.The low levels of EPCs,which correlated with the Framingham coronary risk score,play a role in the pathophysiology of cerebrovascular disease.EPCs can be recruited and contribute to neovascularization after stroke.Thus,infusion of ex vivo expanded EPCs may provide a novel therapeutic option for cerebral ischemia.However,a critical limitation for the therapeutic application of postnatal EPCs is their low number in the circulation.This low cell number is further reduced in patients with cardiovascular risk factors.Therefore,the main theme of the research is to explore strategies for enrichment and expansion of BMSCs and EPCs,and to investigate the effect and potential mechanism of autologous transplantation of BMSCs and EPCs for treatment of focal cerebral ischemia.The research may have favorable prospects of clinical application, since both the two cells can be expanded from patients themselves without serious problems of ethic or immunological rejection.The literature consists of three chapters: Chapterâ… .Expansion and differentiation of bone marrow stromal cells in vitroSection 1.Lack of Telomerase activity in rabbit bone marrow stromal cells during differentiation along neural pathwayObjective:To investigate telomerase activity in rabbit bone marrow stromal cells (BMSCs)during their committed differentiation in vitro along neural pathway;and to investigate the effect of glial cell line-derived neurotrophic factor(GDNF)on the expression of telomerase.Methods:BMSCs were acquired from rabbit marrow and divided into "Control" group,"glial cell line-derived neurotrophic factor(GDNF, 10ng/ml)" group."Cytiokine·NSCs medium"(prepared by our lab,Patent No.ZL02134314.4)supplemented with 10%fetal bovine serum(FBS)were used to induce BMSCs differentiate along neural pathway.Fluorescent immunocytochemistry was employed to identify the expression of Nestin,NSE,and GFAP.The growth curves of the cells and the status of cell circle were analyzed, respectively.During the differentiation,telomerase activitys were detected using the Telomeric Repeat Amplification Protocol(TRAP)-ELISA assay.Result: BMSCs were successfully induced to differentiate along neural pathway;and expressed specific markers of fetal neural epithelium and mature neuron and glial cells.Telomerase activities were not detectable in BMSCs,or in them thereafter during differentiation along neural pathway.Similar evolvement of cell growth curves,cell circle status and telomerase expression were obtained in the two groups. Conclusion:Rabbit BMSCs do not display telomerase activity during differentiation along neural pathway.GDNF show little impact on proliferation and telomerase activity of BMSCs.Section 2.Effect of EGFP nucleofection on differentiation of rabbit primary bone marrow stromal cells along neuron pathwayObjective To approach the effect of transfecting the plasmids encoding enhanced green fluorescent protein(EGFP)with recently developed nucleofection technique on the differentiation of rabbit primary bone marrow stromal cells(BMSCs) along neuron pathway.Methods Rabbit bone marrow stromal cells were harvested by means of density gradient centrifugation following a thighbone puncture.The primary BMSCs were cultured and either transfected with pEGFP-C₂ by nucleofector technology(as EGFP group)or left uninfected(as control)in vitro. "Cytiokine·NSCs medium"(prepared by our lab,Patent No.ZL02134314.4) supplemented with 10%fetal bovine serum(FBS)were used to induce BMSCs differentiated along neuron pathway.FACS was employed to assess the The transfection efficient.Compared with the Control,the cellular growth curves and expression of Nestin,NSE,and GFAP of the labeled cells were analyzed, respectively.Result:EGFP were successfully expressed at 24h after nucleofection.The efficient was assessed by FACS 48h after transfection,with about 38.4%of EGFP-positive cells in the total BMSCs.Similar morphology evolvement, growth curves were obtained in the two groups.There is no significant difference in the proportion of NSE or GFAP positive cells between the treated group and the control group.Conclusion:Nucleofection is an efficient nonviral gene transfer method for the introduction of genes into primary rabbit BMSCs.EGFP played an important role in stable gene marking of rabbit BMSCs.Neuclofection of pEGFP-C₂ shows no significant effect on the proliferation and differentiation along neuron pathway of rabbit BMSCs.Section 3.In vitro expansion of human marrow stromal cells for application in nervous system diseases Objective:To explore expansion strategies of human marrow stromal cells for application in nervous system diseases.Methods:BMSCs were acquired from healthy donors and divided into 4 groups:FBS(L)(culture medium supplemented with 2%low concentration FBS),FBS(H)(10%hige concentrationFBS),bFGF+FBS(L)(10ng/ml bFGF+2%FBS),and bFGF+FBS(H) (10ng/ml bFGF+10%FBS).The proliferative activity of each groups were assessed by analysis of the growth curves,status of cell circle,and telomerase activitys.Cells' differentiation potentials to neural lineage of each groups were also evaluated.The mineralized matrix was visualized by von Kossa staining,and adipocytes were dected using the oil red O staining.Results:Human BMSCs contained two morphologically distinct cell types:spindle-shaped rapidly self-renewing cells and large fiat cells.The former possess greater potential for proliferation and multipotential differentiation.Both FBS and bFGF supported growth of human BMSCs.However,High concentration of FBS showed to promote cells differentiating into mesenchymal cells,including osteoblasts and adipocytes,and to inhibit nestin expression,whereas bFGF was favorable to maintain the cells' differentiation potential to neural lineage.Conclusion:We established a strategy for expansion of human marrow stromal cells based on bFGF and low serum for application in nervous system diseases.Chapterâ…¡.Expansion and differentiation of human endothelial progenitor cells in vitroSection 1.Expansion of endothelial-like cells derived from bone marrow in vitro Objective:To investigate the feasibility of enrichment and expansion of endothelial progenitor cells from unselected attached bone marrow mononuclear cells,and to establish strategy for expansion of human endothelial-like cells derived from bone marrow.Methods:Human bone marrow mononuclear cells(MNCs) were incubated in fibronectin-coated plastic dish.Dulbecco's modified Eagle's Medium-Low Glucose(DMEM-LG)[containing 3.97mM GLUTAMAX^TM(the dipeptide L-Alanyl-Glutamine)and 110g/L sodium pyruvate]+ 1×Insulin-Transferrin-Selenium-X(ITS)+10^-8M dexamethasone+2%Fetal bovine serum+ antibiotic" were use as "isolation medium",and the isolation medium supplemented with "20ng/ml Vascular endothelial growth factor(VEGF)+5ng/ml Basic fibroblast growth factor(bFGF)+ 5ng/ml Insulin like growth factor(IGF)" were used as "differentiation medium".Changes of cells morphology and surface antigen were observed.Hoechst 33342/PI double staining was carried out to identify apoptotic cells.Results:The adherent cells cultured in isolation medium were present as marrow stromal cells.However,when the MNCs were seeded in differentiation medium,some non-adherent cells differentiated into adherent cells until day 7 in culture.Cord-like structures,Blood island-like cells clusters and endothelial cobblestone monolayer were observed.The cells gradually matured to express endothelial linage markers,such as KDR,vWF and UEA-1,and could be expanded in vitro for over 20 passages.Less than 1%of the total cells were undergoing apoptosis.Conclusion:We developed established a simple and reliable strategy to enrich and expand endothelial cells precursors derived from attached bone marrow MNCs for potential clinical application.Section 2.Function analysis of endothelial-like cells derived from bone marrowObjective:To study the function of endothelial-like cells derived from bone marrow mononuclear cells.Methods:Endothelial-like cells were acquired according to the methods established in the former section.Histamine-mediated release of vWF,acLDL uptake,binding of UEA-1,in vitro capillary formation,were analyzed,respectively.Ultrastructures of the cells were detected by transmission electron microscopy(TEM).Finally,rabbit models of hindlimb ischemia were used to determine whether autologous transplantation of culture-expanded endothelial-like cells contribute to angiogenesis in vivo.Results:These cells expressed various endothelial cells functions in vitro,such as release of vWF mediated by histamine, acLDL uptake,binding of UEA-1 and in vitro capillary formation.TEM showed that the cells acquired important ultrastructural and physiological properties of endothelial cells in functional vessels as they contained Weibel-Palade bodies, Plasmalemmal vesicle,Cytoplasmic processes,dilated rough endoplasmic reticulum, enlarged Golgi complex and mitochondria with homogeneous mitochondrial matrix as well as regular arrangement of cytoskeletal filaments.In animal models of ischemia,autologous enthelial-like cells incorporated into sites of active angiogenesis.Conclusion:Functional endothelial cells can be expanded from unselected bone marrow mononuclear cells.Chapterâ…¢.Autologous transplantation of bone marrow stromal cells and endothelial progenitor cells for treatment of middle cerebral artery occlusion models in rabbitsSection 1.Establishment of a permanent middle cerebral artery occlusion model in rabbit Objective:To establish a permanent middle cerebral artery occlusion model in rabbit,for provide a foundation for further cell transplantation study.Methods: MCA occlusion was induced by a modified Transorbital approach.The ischemic areas were visualized by triphenyltetrazolium chloride(TTC)staining. Hematoxylin-eosin staining,Nissl staining and electron microscopy were employed for analysis of ischemic cell damage.DNA fragmentation at 2h,48h and 2 weeks after MCAO was measured with the use of a Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling(TUNEL)and Hoechst 33342 staining.Results: Occlusion of MCA resulted in a reliable and well-delineated lesion in the cerebral cortex of the MCA territory.Accompanying neuronal necrosis,apoptosis were found at each of the three time points,at which,the number of apoptotic cells at 48h after MCAO showed the largest.Conclusion:The permanent MCAO model in rabbit was successfully established;Presence of apoptotic cells in the described model of cerebral ischemia persists at least for 2 weeks.Section 2.Autologous transplantation of bone marrow stromai cells and endothelial progenitor cells for treatment of cerebral ischemia in rabbitsObjective:To investigate the effect and potential mechanism of autologous transplantation of bone marrow stromal cells and endothelial progenitor cells for treatment of focal cerebral ischemia in rabbits.Methods:18 rabbits were randomly divided into 3 groups:Vehicle,BMSCs,EPCs groups(n=6,respectively), and BMSCs(BMSCs group)and EPCs(EPCs group)were expanded respectively. 30 days later,animals were subjected to middle cerebral artery occlusion.Models were treated with intravenous injection of 3×10⁷ autologous BMSCs or EPCs or PBS (Vehicle group)1 day after ischemia.Neurological scores,cerebral infarct volume, number of apoptotic cells and microvessels density of the three groups were compared 14 days after transplantation.Immunohistochemistry was used to detect the differentiation of DiO labeled BMSCs and DiI labeled EPCs in another 4 models. Another animal was subjected to intracerebroventricular injection of mixed BMSCs and EPCs,and then the cells distribution was observed.Results:Compared with the Vehicle group,BMSCs group exhibited statistically significant improvement on the neurological severity scores,significantly fewer apoptotic cells and higher microvessels density in the ischemic boundary area(all P＜0.05).And there were significant improvement in the functional outcome,less infarct volume,fewer apoptotic cells and higher microvessels density in EPCs group as compared to Vehicle rabbits(all P＜0.05).BMSCs and EPCs localized to the ipsilateral ischemic hemisphere.A few BMSCs(＜1%)expressed protein marker phenotypic neural cells, while most of EPCs(90%)bound to UEA-1,some of which had incorporated into capillaries.The BMSCs and EPCs,injected into the contralateral ventricle, preferentially migrated into the injured hemisphere,and could incorporate with ependyma,and migrated toward infarct core.Conclusion:Intravenous administration of autologous BMSCs fosters functional improvement,reduces apoptosis and promotes neovascularization after cerebral ischemia;Intravenous transplantation of autologous EPCs improves functional outcome,reduces infarct volume and apoptosis,and promotes neovascularization after cerebral ischemia; BMSCs and EPCs injected intracerebroventricularly preferentially migrate toward infarct core.