Establishment Of Urine-derived Cell Lines From SMA Patients And Exploration Of IPS Reprogramming And Direct Conversion

Background and ObjectiveSpinal muscular atrophy(SMA)is a group of common autosomal recessive inherited diseases amoung infancy and children,pathologically characterized by selective degeneration and loss of motor neurons in spinal cord,giving rise to progressive and symmetrical muscle weakness and atrophy.No effective treatment is available now,and SMA is one of the most common lethal genetic diseases among infancy.The scarcity of patient-derived and SMA-related motor neurons in vitro has largely hampered the pathogenesis research and drug screening for SMA.Induced pluripotent stem cells(i PS),a kind of special stem cell reprogrammed from adult body cells,possess the ability to differentiate to different tissues from three germ layers.Besides,the adult body cells could also be directly converted to another type of body cells by overexpression of some special lineage transduction factors.The development of i PS cells and direct conversion technology offer a new avenue for the obtaining of patient-derived motor neurons.In the present study,we aim to establish the urine-derived cell lines from SMA patients,and then reprogram those urine cells to i PS cells.Besides,we also attempt to convert the urine cells to neurons directly.Finally,the treatment of histone deacetylase inhibitors is performed on SMA urine cells and induced neurons in vitro.Methods1.Fresh urine was collected from SMA patients and control individuals,centrifuged,and then cultured the urine sediment in vitro.Reverse transcription-PCR,Western Blot,immunofluorescence staining and restricted fragment length polymorphisms(RFLP)were employed to identify the component of urine cells,and detect SMN gene mutation,as well as the amount and localization of SMN protein in urine cells.2.Four lenti-plasmids carrying OCT4,SOX2,c-MYC and KLF4 were constructed.Urine cells were infected by lentivirus with different MOI.Urine-derived i PS cells were identified by morphological characteristics,alkaline phosphatase(AP)staining,molecular imprinting detection,immunofluorescence staining,and teratoma formation.3.Eight lenti-plasmids carrying ASCL1,BRN2,MYT1 L,NEUROD1,ISL1,HB9,LHX3 and NGN2 were constructed.Urine cells and fibroblasts were infected by lentivirus.Induced neurons and motor neurons were identified by morphological characteristics,immunofluorescence staining,and cell patch clamp recording.The difference between SMA fibroblast-derived neurons and control ones were observed as well.4.Urine cells and induced neurons were further treated by histone deacetylase inhibitors in a dose and time dependent manner.The SMN protein level and growth condition of induced neurons were observed before and after drug intervention.Results1.We have established about 50 urine cell lines from different neurological genetic disease patients and healthy controls,including 13 SMA patients with different clinical phenotype.Most of the urine cell lines were composed of fusiform-and oval-shaped cells,which were confirmed to be podocyte,urinary epithelium and urine-derived mesenchymal stem cells.The urine cells growed robustly in vitro and could stably subculture about 6 to 10 passages.Besides,the urine cell lines carried SMN gene defect natively and SMN protein could be detected both in nucleus and cytoplasm.2.Urine cells were successfully reprogrammed into i PS cells by OCT4,SOX2,c-MYC and KLF4.The urine-derived i PS cells expressed a high AP activity and the typical markers of embryonic stem cells(ES),including NANOG,SSEA3,SSEA4,Tra-1-60 and Tra-1-81.Besides,the i PS cells possessed the ability to differentiate into different tissues of three germ layers in vivo.3.The urine cells and fibroblasts could be convered into neurons and motor neurons by the different combination of 8 transduction factors.The conversion procedure could be divided into 3 stages;risk stage(day 1-6),neuron induction stage(day 7-15)and neuron formation and maturation stage(day 16~).The induced neurons showed typical neuron-like morphologies and expressed the hallmark of Tuj1,CHAT and HB9;some induced neurons were electro-physiologically functional.Besides,the SMA-induced neurons exhibited significant reduced axonal outgrowth rate and survival in culture compared with non-SMA control one.4.Histone deacetylase inhibitors(HDACi)could up-regulate the amount of SMN protein in different urine cell lines from SMA patients and controls.Suberoylanilide hydroxamic acid(SAHA),a second generation of HDACi could significantly increase the number of Gems loci in nucleus,and seemed to sustain the growth of dendrites and delay the apotosis of induced neurons in vitro.Conclusion1.As a simple,non-invasive and highly acceptable procedure,urine cell culture technology is an effective avenue for obtaining cell samples from SMA patients.The urine cell lines were composed of different types of cells,mainly including podocyte,urinary epithelium and urine-derived mesenchymal stem cells.2.By overexpression of typical Yamanaka factors,the urine cells could be effectively reprogrammed to i PS cells,which were pluripotent in vitro and in vivo.3.The urine cells and fibroblasts could also be directly convered to neurons and motor neurons by expression of ASCL1,BRN2,MYT1 L,NEUROD1,ISL1,HB9,LHX3 and NGN2.4.Histone deacetylase inhibitors(HDACi)could significantly up-regulate the amount of SMN protein in different urine cell lines,especially SAHA,and seemed to sustain the growth of induced neurons in vitro.