神经退行性疾病干细胞移植治疗目前研究现状与未来展望.pdf

目前研究现状与未来展望神经退行性疾病干细胞移植治疗胎儿神经干细胞治疗帕金森氏病临床研究发展历程Timeline of landmarks in neural transplantation in PD19801990200020101998 Generation of hESC-derrved cd I lines20062007iPSC methods first descnbed.2)12Akst TRANSEURO transplants scheduled1988First f/M grafts prfomed1992-1999Positn/G results from several openlabel trials2001,2003 Double-blind fVM graft Inals negatrve first reports GIDS2010 TRANSEURO beginsEvans JR,Mason SL,Barker RA.Prog Brain Res、2012;200:169-98Transplanted fetal mesencephalic dopaminergic neurons(11-16 years)developed alpha-synuclein-positive Lewy bodies in grafted neuronsLindvall 0,et a/,Nat Med、2008 May;14(5):501-3AGrafted nigral neurons were found to have Lewy body-like inclusions14 years after transplantation into the striatum of an individual with PDSynuclfein-Hostein-Grafted NeuronsOlanow CW、et al Nat MedsUbiquintinnGrafted Newons:2008May;14(5):504-6sTransplanted dopamine neurons in people with PD do not contain Lewy bodies p e-q n sauq一 35DuwnbsnUbiquitin-synuclein OverlayTHSubject 5Subjeci 6b idSubject 4GraftSNGraftSN GraltMendez,Isacson et al,，NATURE MEDICINE VOLUME 14(5):507-509,2008Freed CR,J Nucl Med.2010 Jan；51(1)：7-15 Long term Study 33 of the original trial participants who were followed for 2 years after transplantation and 15 of these subjects who were followed for 2 additional years.These results suggest that clinical benefit and graft viability are sustained up to 4 y after transplantationFreed CR,Neurotherapeutics(2011)8:549-561人体胚胎干细胞分化的多巴胺神经元移植改善小鼠,大鼠和猴子帕金森氏病的运动障碍abTH NCAM TH FOXA2NCAM/TH/FOXA2G D L 2诉*TH/NA/DAPI TH/NCAKVDAPI22/29 DECEMBER 2011|VOL 480|NATURE|547,Lorenz Studer,et al Memorial Sloan-Kettering Cancer CenterImproved Cell Therapy Protocol for Parkinsons Disease Based on Differentiation Efficiency and Safety of hESC-,Hipsc and Non-Human Primate iPSC-Derived DA Neuronsc181614Q1064 7O-24Isacson et al,5 Stem Cells、2013;31(8):1548-62Dopamine release from transplanted neural stem cells in Parkinsonian rat striatum in vivoElution Time(min)Zhou z5 et al,Proc Natl Acad Sci USA、2014 Nov 4;111(44):15804-9iPSC-Derived Dopamine Neurons function after Transplantation in a Non-Human Primate Model of Parkinsons DiseasePre-MPTP Post-MPTP Post-transplantationTyrosine hydroxylaseDopamine transporterNON-TRANSPLANT SIDE TRANSPLANT SIDEcc CaulakiLVIcSepUmPuanen 学/LGPg.RCell Stem Cell、2015 Mar 5;16(3):26974、Ole Isacson et al,Harvard Stem Cell InstituteStem cell-based Clinical Trials for(ALS)一 Nuralstem,In c v the first Phase I clinical trial for a stem cell-based treatment of ALS.-Initiated in 2010 and pleted in 2013,involved the transplan-tation of human spinal cord-derived NSCs into the spinal cord of 15 late to mid-stage ALS patientsNeuralstem clinical trial patient summary.Pluse IEach injection administeied to patients contained approximately 100,000 cells in an 85-10 pL volume.GroupSpinal cord region Disease condition Total#of injectionsAl A2 B C DE,Unilateral./luinbar(L2-L4)Noiumbulatoiy 5Bilateral/lumbar(L2-L4)Noiumbulatory 10Unilateral.lumbar(L2-L4)Ambulatory 5Bilateral/lumbar(L2-L4)Ambulatory 10UnilateralXeivical(C3-C5)Ambulatory 5Unilateraljtervical(C3-5)Ambulatory 5,Patients in Group E were the same patients as in Group C.receiving additional injections at a later time point,for a total#of 15 injections.Phase IIAll patients in Pluse H a re ambulatory,early-stage with arm weakness,but!K)t paralysis,receiving bilateral injections of varying cell doses suspended in an 85-10 pL volume-GroupSpinX cord region Total#of cells Total#of injectionsA B C D ECeivic C3-C4 2.000,000 10(200,000cell$/injection)Ceivic C3-C5 4.00QOOO 20(200,000 cell$/injection)Ceivic C3-C5 6.000.000 20(300,000 cell$/injection)Ceivic C3-C5 S.OOGOOO 20(400.000 cell$/injection)Lumbar L2-L5,followed 4-12 weeks later with cervical C3-C5 160,000,000 40(400,000celk/injection)-Glass,Feldman,E.L、5 2012、Lumbar intraspinal injection of neural stem cells in patients with amyotrophic lateral sclerosis:results of a phase I trial in 12 patients、Stem Cells 30(6),1144-1151、-Riley,J、5 Feldman,E、L、5 2014、“Intraspinal stem cell transplantation in ALS:a phase I trial,cervical microinjection and final surgical safety outes5 Neurosurgery 74(1),77-87Intraspinal stem cell transplantation in ALS:a phase I trial,2014 RESULTS:Unilateral cervical(group D,n=3)and cervical plus thoracolumbar(group E,n=3)microinjections to the ventral horn have been pleted in ambulatory patientss One patient developed a postoperative kyphotic deformity prompting pletion of a laminoplasty in subsequent patientss Another required reoperation for wound dehiscence and infection.The solitary patient with bulbar amyotrophic lateral sclerosis required perioperative reintubations CONCLUSION:Delivery of a cellular payload to the cervical or thoracolumbar spinal cord was well tolerated by the spinal cord in this vulnerable population.This encouraging finding supports consideration of this delivery approach for neurodegenerative,oncologic,and traumatic spinal cord afflictionssI P S c e=s W e r e a n dBNCF，1NCF.2NCF6PDLPD，2G e n e r a t e d f r o m PD p a e n.s N o r m a.C O M r o-s5B+sodium ButyratehES medium+sodEm ButyrateTransduction 一5i Removal of W retroviruses 5；I32 Splitingthe 3 fibroblasts on MEF feeder8iPS cell Colonyo pickingE n d o,o a 4T 9-S O X 2T g，9mycT9KK4GAPDHRat PD Model6-OHDA-inducedHuman IPS cells Integrated to the Host Brain of 6-OHDA-induced Rat PD ModelTmJ-1(S)OEZJ。suo-EnQGFARBCHan F,Wang W5 Chen C5 Duan J,et al Cytotherapy 2015分化的胎脑神经干细胞移植治疗PD一一区3 Tuj10 GFAP 日 MAP2 ED TH建立大鼠SCI损伤模型A.暴露和部分横切脊髓外科手术。B、T7横断损伤产生后 肢瘫痪。C、无脊髓损伤的正常大鼠。RT-PCR to Detect the MicroRNA Expression in Rat SCI Model/Real-Time RT-PCR to Detect the MicroRNA Expression in SCI干细胞移植修复脊髓神经损伤移植神经干细胞分化的神经轴索与宿主脊髓神经细胞及其树突形成突触连接GFP/GFAP NeuN.Electrophysiology Stim Site of RecordRe-TransectionC7 T3-Graft T6Lu P et al,Cell.2012 September 14;150(6):1264-1273Bone Marrow Stromal Cell Intraspinal Transplants Fail to Improve Motor Outes in a Severe Model of SCIJournal of Neurotrauma 2015,Tuszynski MH To determine whether local mechanisms mediate BMSC neuroprotective actions grafted allogeneic BMSCs to sites of severe,pressive spinal cord injury(SCI)in Sprague Dawley rats、Cells were administered 48 hours after the original injury Additional animals received allogeneic MSCs that were genetically modified to secrete BDNF,to further determine whether a locally administered neurotrophic factor provides or extends neuroprotection.two months post-injury in a clinically relevant model of severe SCI,BMSC grafts with or without BDNF secretion failed to improve motor outes、Thus,allogeneic grafts of BMSCs do not appear to act through local mechanisms,and future clinical trials that acutely deliver BMSCs to actual sites of injury within days are unlikely to be beneficialIntraspinal Stem Cell Transplantation in Amyotrophic Lateral Sclerosis:A Phase I Safety Trial,Technical Note,and Lumbar Safety Outes NEUROSURGERY VOLUME 71|NUMBER 2|AUGUST 2012Department of Neurosurgery,Emory University 3 Atlanta 3 Georgia;Department of Neurology,Emory University,Atlanta,Georgia;Department of Neurology,University of Michigan,Ann Arbor,Michigan神经干细胞移植方法-Each microinjection series prised 5 injections(10mL/injection)separated by 4 mm.-Each injection:100 000 neural stem cells derived from a fetal spinal cord.一 Twelve patients were treated with either unilateral or bilateral injections一 Patients are followed clinically and radiologically to assess potential toxicity of the procedure.Lumbar LaminectomyMicroinjection platform applicationPostoperative imaging progressionFIGURE 6.Po stuptrative imaging pro prstio tL Sagittal T2-wcinctlMRI scans centered at th e bruel o f T11/12 arc dano fistratrtL Th b patient received a subfascial trpulural drain.A.prto perative baseline inulling.B.inuiging taken at 1 mo nth po sto pcrittively.Siiificant epiduralpseuilu，nenM即rlr abutting th e lumbar enlitrgnwnt is ubsentd.as a a maidartifaa 而 a muh tl/rvni micruinjeaio n ntrdlrpldccmcnt.C,inutging utkm in 16 nwntbspo stvpcratiiTly.Tbo racuhhh ir ca，uilsttfio sis Ims impro vedto war reso luutm o f epidural pseudsnrni八gac山.Nfetal artifaa dh niniJyed but rrmah ts present.RHeyy Js,Feldman Es Ls 2014s Intraspinalstem cell transplantation in ALS:a phase/trial,cervical microinjection and final surgical safety outes、Neurosurgery 74(1)77-87FIGURE 6.Vo uLir fingerprint,grdji uitntifieatio n.Pnclinieal and ci nival experienee a 趣的 Mriab/ttiuuLir pattern obred o n th e do rsalsurface o f 而 spinal card.W2 h am th is vaidar pa urn m pratide land-nutrks dttring graft$”e t/ienttfiaiMn.A.th is intrdo peratit inuigt.takefi with 血 o fratio e miefo uo pe,ih o ws a h igh ly ipecifii vaxutar pawm and/所 straps eo rdfnetratim uK由 th e fbatlngiannula,m lefio f etnur in th e bkak insa.B.th e 娟咏 h i艰)specifu:iwhL”fh ittem is ubxrtwd in th e kime end ifctnun after renewal at at”呼”.Th is o!1m$reo rienidtim s(h e exaa$ig o f co rd penaraucn and graft delh ry.Clinical Trials using ESCs and iPSCsTable 1.ESC TrialsTrial Sponsor(Location)Disease TargetCell TherapyNo.PatientsPhaseChabiotech Co.Ltd.(S.Korea)macular degenerationhuman-ESC-derived RPE12phase l/llOcata Therapeutics(MA,USA)Stargardts macular dystrophyhuman-ESC-derived RPE16phase l/llmacular degenerationhuman-ESC-derived RPE16phase l/llmyopic macular degenerationhuman-ESC-derived RPEunknownphase l/llPfizer(UNmacular degenerationhuman-ESC-derived RPE10phase ICell Cure Neurosciences Ltd.(Israel)macular degenerationhuman-ESC-derived RPE15phase l/llViaCyte(CA,USA)type I diabetes mellitushuman-ESC-derived pancreatic endoderm cell40phase l/llAssistance Publique-Hopitaux de Paris(France)heart failurehuman-ESC-derived CD15+lsl-1+progenitors6phase IInternational Stem Cell Corp.(Australia)Parkinsons diseasehuman parthenogenetic-derived neural stem cellsunknownphase l/llAsterias Biotherapeutics(CAt USA)spinal cord injuryhuman-ESC-derived oligodendrocyte precursor cells13phase l/ll-There is also a report of one Japanese patient who received a transplant of a-sheet of iPSC-derived RPESummary on Molecular Mechanism of Stem Cell Transplantation for Neurological Diseases Transplanted cells survive,differentiate to neurons,astrocytes?oligodendrocyte precursors(hESC9 hiPSC,NSC)and release neurological transmittors such as dopamineAch Release of neurotrophic factors(GDNF,GDNEJGF0 to increase the functions of the endogenous neural stem cells Release of immuno-regulatory factors such as IL-2,6,8,10 to play immuno-modulation and attenuation of the inflammatory process,such as MSC.The transplanted cells formed synapse with host cells.Others such as delaying the onset and prolonging survival of SOD1 rats Increasing host neurosenesis今后干细胞治疗神经退行性疾病的临床研究需要考虑的问题1、Cell Sources:Neural projenitors,MSC,hES cells,iPS cells2、SC grafting should be conducted to ensure 100,000 dopaminergic neurons(PD)survive per transplantation site、3、SC grafts should exhibit regulated release of dopamine in line with that of endogenous dopaminergic neurons s4、By reestablishing the striatal dopaminergic system,grafts should show the capacity to restore functional connectivity within the basal ganglia and at extra-striatal locis5、Long-lasting and significant symptom-relief must be achieved(over 2-3 years)s6、Evaluation System(Symptoms,Dopamine release,Levodopa-response),Unified Parkinsons Disea se Ra ting Seal e(UPDRS),Biomarkers7、Adverse effects must be minimals This include s the absence of tumor formation and GIDs(graf t-induced dyskinesia)throughout long-term follow-up periods s-感谢您的聆听!