In Vivo Differentiation Of Oligodendrocyte Progenitor Cells From Human Induced Pluripotent Stem Cells And Its Neural Protective Effects

With the growth of population and social development,more and more countries are facing the aging problem,which is expected to occur in most regions and countries in 2030.Neurodegenerative diseases are the second most common geriatric diseases after stroke(about 27.4%),and place a considerable burdenon society and families.One of the most striking hallmarks of neurodegenerativediseases is extensive neuronal death.Due to the irreproducibility of neurons,thereare no effective strategies to reverse neural death in clinic so far.Stem cells can be differentiated into specificsubtype of neurons,creating new hope for the treatment of neurodegenerativediseases.However,certainkey challenges of stem cell therapy still remain,including insufficient sources,uncontrollable differentiation,and poor survival of transplanted cellsin vivo.To address these problems,we have established a proliferative,anti-apoptotic and directionally inducible genetically engineered hi PSCs line(NILB-hi PSCs),which conditionally express motor neuron specific transcription factors(Ngn2,Isl1 and Lhx3),and constitutively express anti-apoptosis gene Bcl-x L.These cells showed normal proliferative capacity,resistance toapoptosis inducerand directional differentiation into motor neurons both in vitro and in vivo after Doxycycline(Dox)induction.Previousstudies have demonstrated that it will benecessary to ensure that the transplanted cells survive to differentiate into enough functional neurons to formnew neural circuits.Oligodendrocytes are an attractive population for neurodegenerative diseases because they can produce sphingomyelin to form myelin sheath,and then protect nerve fibers.Therefore,myelin sheath plays an important role in the structure and function of axons.Olig2 and Sox10 arekey transcription factor in the differentiation and maturation of oligodendrocytes.Based on our previous experience,we made multi-gene modification on hi PSCs: use the Tet-On system to conditionally express Olig2 and Sox10 gene,and transfer Bcl-x L into hi PSCs,to establish Olig2-SOX10-engineered,oligodendrocyte-producing and anti-apoptotic hi PSCs lines,and further investigatedtheir protective effects on neurons.Two Piggy Bac transposon vectorswere constructed.The first vector,named as PBOS,contains Dox-inducible polycistronic genes Olig2 and Sox10.The other vector,named as PB-BLG,contains an anti-apoptotic gene Bcl-x L,and two trackergenes,firefly luciferase and Enhanced Green Fluorescent Protein(EGFP).hi PSCsweretransfected with both PB-OS and PB-BLG,and selected by blasticidin(BSD),resulting in a stable hi PSCsline,named as OSB-hi PSCs.After treated with Dox,glialike cells withlong spindlewere formed.These induced cellsstained positive for PDGFR-? and O4,suggesting that Olig2 and Sox10 can program hi PSCs into oligodendrocyte progenitorsin vitro.To initially evaluate the differentiation direction of these engineeredhi PSCs in vivo,OSB-hi PSCs were subcutaneously injected into immunodeficient mice.In control group(without treatment of Dox),the luciferase activity and size of the gtafts increased rapidly with time,and formed teratoma-like structures.While in Dox treated group,size of the gtafts was much smaller than that of control group.Furthermore,these transplanted cells also expressed PDGFR-? and O4.After ensuring thesafety of OSBhi PSCs,they were mixed with NILB-hi PSCs and then subcutaneously injectedinto the mice.Both in vivo imaging and immunofluorescence staining showed that the luciferase activity and surviving neuron number in mixed group were higher than that of NILB-hi PSCs group.Thus,a mixture of NILB-hi PSCs and OSB-hi PSCs was injected into the ventricles of mice.Similar to subcutaneously injection,the luciferase activity in mixed group was higher than that of NILB-hi PSCs group.In addition,these mixed transplanted cells both were positive for ?-tubulin and BMP,indicating that oligodendrocyte progenitors derived from OSB-hi PSCs improved the survival of NILB-hi PSCs induced neurons.In summary,we describe a highly effective method that generates an enrichedpopulation of oligodendrocytesvia Dox induction in vivo.These resulting oligodendrocytes produced BMP and improved the survival of neurons.Our method is expected to solve the poor survival and function of transplanted stem cells,which will provide a novel therapeutic strategy for neurodegenerative diseases.