Efficient Differentiation Of Motor Neurons From Human Induced Pluripotent Stem Cells In Vivo

Amyotrophic lateral sclerosis(ALS)is a devastating motor neuron disease with no effective treatment at present,which is characterized by extensive motor neuron death.human induced Pluripotent Stem Cells(hi PSCs)can be reprogrammed from somatic cells,greatly expanded in culture and differentiated into specific subtype of neurons,creating new hope for the treatment of ALS.However,certain key challenges of stem cell therapy still remain,including insufficient sources,uncontrollable differentiation,and poor survival of transplanted cells in vivo.To address these problems,we made multi-gene modification on hi PSCs:(1)Use the Tet-On system to conditionally express Ngn2,Isl1 and Lhx3 gene with or without tetracycline treatment,which not only retains the high proliferation rate of hi PSCs,but also achieve the availability of induced motor neuron differentiation.(2)transfer anti-apoptosis gene Bcl-x L into hi PSCs,to guarantee long-term survival of transplanted cells.After selection with blasticidin,proliferative,inducible and anti-apoptosis hi PSCs lines(hi PSCs-NIL-BSD+Bcl-LG)were established.These genetically modified hi PSCs were GFP-positive with high luciferase activity.Following tetracycline induction,hi PSCs-NIL-BSD+Bcl-LG expressed transgenic protein Isl1/2.After identification of these cell colonies,tetracycline was added to the culture medium to activate the transgenes and initiate the motor neuron differentiation process.As expected,hi PSCs-NIL-BSD+Bcl-LG were effectively and rapidly converted to neuron-like cells with condensed nuclei and long axons.To characterize the properties of hi PSCs-NIL-BSD+Bcl-LG induced motor neurons,we performed immunofluorescence and functional analysis.We found that induced motor neurons expressed markers for motor neurons,including MAP2,HB9 and Ch AT,and exhibited ion currents,indicating that hi PSCs-NIL-BSD+Bcl-LG are differentiated to electrophysiologically mature motor neurons after tetracycline treatment.Furthermore,the survival rate of hi PSCs-NIL-BSD+Bcl-LG induced motor neurons was statistically higher than that of hi PSCs-NIL-BSD both in replating-induced stressmodel and glutamate toxicity assay,suggesting an anti-apoptosis function of Bcl-x L.Subsequently,hi PSCs-NIL-BSD+Bcl-LG were subcutaneously injected into immunodeficient mice directly.After induction with tetracycline,the transplanted cells were differentiated into neuron-like cells,as indicated by staining with ?-tubulin,and no tumor formation was noted,compared to PBS control group.After ensuring the safety of transplantation of these modified hi PSCs,they then were injected into the cerebral lateral ventricles and spinal cords of immunodeficient mice.The transplanted cells survived for more than 4-week,and directionally differentiated into motor neurons in vivo after tetracycline treatment,which were consistent with the in vitro results.For the first time,we established the hi PSCs-NIL-BSD+Bcl-LG line by multi-gene modification,which not only retains the high proliferation rate of hi PSCs,providing sufficient sources for stem cell transplantation.But also,it ensured the controllability of differentiation direction and the survival rate of stem cells after transplantion.Moreover,these genetically modified hi PSCs were injected into immunodeficient mice directly,which can be directionally differentiated into motor neurons by a drug-inducible system,provide a novel therapeutic strategy for neurodegenerative diseases such as ALS.