| Introduction:Stroke is seriously threatening human’s health worldwide with high morbidity,mortality, high recurrence and disability rate. Among it, intracerebral hemorrhage(ICH) is more serious and people survived usually have sustained neurologicaldysfunction. The current treatments to ICH, including medicine and surgery, aremostly supportive. In most severe cases, there is little therapeutic effect since lackingof specific method toward the pathophysiological injuries.During the injury of ICH, the ability of self-repair of nervous system is limitedbecause the endogenous neural stem cells (NSCs) are rare and neural cells can notself renew. Researchers are now paying more attention to stem cell transplantation inorder to recover or reestablish the damaged central nervous system.The newly emerging technology of induced pluripotent stem cells (iPSCs)provides novel hopes for ICH patients. The patient specific, personalized iPSCs andiPSCs-derived NSCs transplantation has more potential for the treatment of tractableICH without the ethic and immune rejection problems.The purposes of this study are firstly to reprogramme the skin fibroblasts of one severe ICH patient into iPSCs in vitro. Subsequently, NSCs would be induced fromthese skin fibroblasts-derived iPSCs. Then the iPSCs-derived NSCs are transplantedinto the rat ICH models to assess their treating effects and the possible mechanisms.These results would further facilitate the application of ICH patient-specific iPSCsand iPSC-derivevd NSCs in treating refractory hemorrhagic stroke.Part I The induction and identification of iPSCsObjective:To reprogramme the skin fibroblasts of one severe ICH patient into iPSCs invitro and identify the stem cell features of iPSCs.Methods:①After informed consent by patient and the family, skin tissue sample withfour square millimeter was obtained from one ICH patient by a medical skindrilling apparatus;②The skin fibroblasts were separated by enzyme digestion and cultured in themedium;③Then the fibroblasts were transfected by lentivirus carrying OCT4, SOX2,KLF4and c-Myc genes to reprogramme into iPSCs-like cell clones；④These cell clones were identified with AP staining, immunofluorescentstaining to detect the expressions of hESCs specific proteins of Oct4, SOX2,SSEA4, TRA-1-60and Nanog, their multilineage potential by EB formingexperiment in vitro and Teratoma formation in vivo.Results：①The skin fibroblasts of ICH patient was induced into ESCs-like cell clones;②These ESCs-like cell clones were AP positive, showed similar morphological features and sustained the expression of multi potentialmarkers such as Nanog, OCT4, SOX2, SSEA-4and TRA-1-60, which issimilar to that of hESCs；③ESCs-like cell clones were similar to hESCs with the ability of multipotentdifferentiation They formed EB in vitro and expressed the markers of AFP,a-SMA, Desmin, β-Ⅲ Tubulin and GFAP that represent the three germlayers. In SCID rat muscles, the transplanted ESCs-like cell clones formedTeratoma；④iPSCs have the ability of self renewal.Conclusion：The skin fibroblasts from one ICH patient were successfully reprogrammed intoiPSCs in vitro.Part II Induction of iPSCs into NSCs in vitroObjective:To induce reprogrammed iPSCs derived from an ICH patient skin fibroblastsinto NSCs in vitro.Methods:iPSCs were firstly cultured to form Embryoid bodys (EBs). Then the EBs wereinduced into neural tube-like rosettes in neural induction medium. NSCs mediumwere used for cell clumps floating cultivation to form neurospheres. Neurosphereswere trypsinized into single cells and differentiated into neurons and astrocytes indifferentiation medium. Nestin for neural stem cell-specific marker, β-III Tubulin forneuron marker and GFAP for astrocyte marker were detected byimmunocytochemistry. Results：iPSCs were cultured in EB medium for3days to form simple EBs. These EBsgradually formed the early neural rosette structures in the center of the attached EBs.Numerous neural tube-like structures containing mature neuroepithelial cells werefinally formed. Morphologically, neurospheres were translucent and cells inneurospheres expressed NSC-specific marker proteins (Nestin). These iPSC-derivedNES cells can be propagated many passages without losing their stem cellcharacteristics. NES cells differentiated into neurons and astrocytes indicated by β-IIITubulin and GFAP staining respectively.Conclusion：Reprogrammed iPSCs derived from an ICH patient skin fibroblasts wereinduced into NSCs in vitro.Part III Transplantation of iPSCs-derived NSCsimproves the neural function of ICH model ratsObjective:To investigate the therapeutic effects of grafted iPSCs derived NSCs on thetreatment of rat experimental ICH and its possible mechanisms.Methods：The iPSC-derived NSCs were incubated with20mmol/L BrdU for48h beforetransplantation. BrdU-conjugated NSCs with90–95%viability were used fortransplantation. At24h post-ICH, rats were localized in a stereotactic frame andinjected with BrdU-conjugated NSCs into the perihematoma. The neural function of each rat was evaluated using both the modified neurological severity score (mNSS)and the modified limp placing test (MLPT) by a blinded investigator at days1,3,7,14and28post-ICH. The tissues surrounding the hematoma were sectioned andprocessed for immunofluorescent staining with antibodies specific for BrdU, Nestin,β-III Tubulin, and GFAP to identify the transplanted cells, neural stem cells, neuronsandastrocytes respectively.Results：Periodic evaluation disclosed that the neurological function of rats treated withiPSC-derived NSCs was significantly improved compared with the PBS-treatedcontrols by day14(P <0.05) and persisted for up to28days (P <0.05).iPSC-derived NSCs differentiated into neural-like cells after transplantation. Byimmunostaining, BrdU-conjugated cells were found in the surrounding area ofhematoma. Double-fluorescence revealed that iPSC-derived NSCs were able todifferentiate into neuron-like (BrdU+/β-III Tubulin+) and glia-like cells(BrdU+/GFAP+) in vivo.Conclusion：Transplantation of iPSCs-derived NSCs improved the neural function of ICHrats and the possible mechanisms may involve the neural cell replacement. |
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