Establishment Of Amyotrophic Lateral Sclerosis Induced Pluripotent Stem Cell Lines And Neuronal Differentiation

Objective: Amyotrophic lateral sclerosis(ALS)is a motor neuron disease that seriously threatens the health of patients.It is caused by complex environmental and genetic factors,and the mechanism of action is unclear.There is no effective treatment.At present,most studies have applied animal or single gene intervention cell lines as ALS disease models,but they cannot accurately reflect the pathological characteristics of ALS.IPSC(induced pluripotent stem cells)is a technology that uses patient's somatic cells to obtain embryonic like stem cells through reprogramming induced by transcription factors,which can best mimic lesions and is used to reveal the mechanism of ALS and develop therapeutic drugs.In the process of i PSC reprogramming,the selection of somatic cells and the optimization of conditions are critical to the progress of i PSC culture.The most widely used source of somatic cells is skin fibroblasts.However,the traditional method of isolation and culture of skin fibroblasts requires long time,low tissue utilization and high cost.Therefore,in this study,skin tissue was used to optimize fibroblast culture methods by integrating a variety of culture conditions,in order to establish a somatic cell culture and expansion process for the group,and provide seed cells for subsequent rapid and efficient programming.Subsequently,the skin fibroblasts of healthy people and patients with amyotrophic lateral sclerosis obtained by the optimized method were reprogrammed to induce pluripotent stem cells,and the neural stem cells were further differentiated.The difference of differentiation rate between ALS patient and healthy subject neural stem cells was studied preliminarily,which provided technical basis and experimental evidence for the further study of disease mechanism.Methods:1.The sterile skin tissue were divide into four parts and treated with four methods.(1)Directly plant the suspension after digesting minced skin tissue for 1h with 0.25% trypsin(try1h group);(2)directly plant tissue on plate(cut group);(3)digest skin tissue for 1h with 0.25% trypsin(try1h-c group);(4)digest skin tissue for 2h with 0.25% trypsin(try2h-c group);the latter two groups' tissues were adherence after digestion.2.Use microscope to record the daily changes of tissues and cells,and compare the appearance time of cells,cell morphology,and number of cells in each group.3.Immunofluorescence,RT-PCR,Western-Blot,and cell counting were used to compare the differences in protein expression and proliferative capacity of the four groups of cells;Wound Healing was used to compare the migration and healing capabilities of the four groups of cells.4.The reprogramming kit was used to transfer four transcription factors(h-Myc,Se V,KOS,Klf4)into fibroblasts,and the fibroblasts obtained from a healthy person and a patient with ALS were reprogrammed into induced pluripotent stem cells(iPSCs).5.RT-PCR,immunofluorescence and cell flow cytometry were used to characterized the ability of proliferation and differentiation.6.Differentiate the induced pluripotent stem cells into neural stem cells and perform cell immunofluorescence staining to compare the differentiation ratio between the two groups.Results:1.Microscopic observation results showed that cells appeared relatively early and had the shortest cell culture period in the adherent tissues after 2 hour of enzymatic digestion group.2.Immunofluorescence,Western Blot,and Wound healing showed that fibroblasts isolated by the optimized method had no differences in vimentin expression,proliferation and migration ability with cells isolated by the traditional method.3.After reprogramming,the cells showed typical human embryonic stem cell-like morphology.After manual picked,the stem cells have the ability to proliferate.4.Immunofluorescent staining and flow cytometry analysis showed the cell lines express the pluripotency markers with a purity of over 90%.Immunofluorescence results showed that induced pluripotent stem cells can differentiate into three germ layer cells,suggesting the multiple differentiation potential of induced pluripotent stem cells.5.After differentiation,the cells showed the typical morphology of neural stem cells,and immunofluorescence showed that the specific protein of neural stem cells was strongly positive.6.By calculating the positive protein expression rate of NSC,there was no statistical difference in the ratio of i PSC differentiation to NSC in healthy and ALS patients.Conclusions:1.By comparison,the treatment method of 0.25% trypsin digestion for 2h(try2h-c group)can successfully isolate a sufficient number of fibroblasts from a very small amount of skin tissue in the shortest time,which is the most cost-effective cultivation method with high tissue utilization and success rate.2.Fibroblasts isolated and cultured by optimized methods can be reprogrammed into non-toxic induced pluripotent stem cells of the same origin as fibroblasts,with proliferative capacity and multiple differentiation potentials,providing a basis for further differentiation of neural stem cells.3.After the cultivation of small molecule inducers,i PSC can successfully differentiate into NSC,and there is no significant statistical difference in the differentiation ratio of diseased and healthy cells.