Induce IPSCs To Differentiate Into Motor Neurons In Different Systems

Background and purposeHuman induced pluripotent stem cells(iPSCs),which have the ability of unlimited proliferation,self-renewal,multipotent differentiation and maintaining their undifferentiated state,are currently widely used in medical research.Various somatic cells derived from iPSCs can be used to establish in vitro disease models.In the study of motor neuron diseases,the batch culture of iPSCs and the induction of their differentiation into motors neurons for clinical research and treatment are necessary.The culture system can be divided into feeder layer and feeder-free system based on the presence or absence of feeder layer.The feeder layer culture system is the traditional stem cell culture system,and the most current research on neural differentiation programs are based on this system,but there are the risks of heterologous contamination in this system.Feeder-free culture systems have gradually emerged as a culture system for the application of stem cells in clinics in recent years,which can avoid the contamination of allogeneic cells,but the characteristics of iPSCs after long-term culture under this system need to be further explored.In this experiment,iPSCs were cultured in these two systems.Refer to previous motor neuron induction programs in feeder systems,iPSCs were induced to differentiate into motor neurons in feeder-free culture system and differentiation efficiency are compared,which provides the basis for later optimization of differentiation.Materials and Methods1.Experimental groups: there are two different groups:(1)Feeder layer culture system: mouse embryonic fibroblast(MEF)serves as feeder layer cells to support the growth of iPSCs;(2)Feeder free culture system : matrigel serves as the medium to promote adherence of iPSCs and support cell growth,and mTeSR is culture medium..2.Culture of iPSCs: iPSCs were cultured on MEF or Matrigel.After continuous culture for 5 generations,the morphology of the cells was observed under the light microscope.Immunofluorescence technique was used to detect the expression of pluripotent markers SOX2 and OCT4.3.Differentiation of iPSCs to motor neurons: iPSCs were induced by different combinations of small molecule compounds for 28 days to obtain mature motor neurons(MN).Cellular immunofluorescence was used to identify cells in different differentiation stages: neuroepithelial progenitors(NEP),motor neuron precursor cells(MNP),MN,then NEP and MNP amount was counted;the transcription levels of NEP and MNP related genes were detected and compared by qRT-PCR.Result1.In two culture systems,the iPSCs showed different cell clone morphology,and they maintained their typical cell morphology through subculture,and highly expressed the pluripotency markers SOX2,OCT4.2.Under the two culture systems,NEPs,MNPs,and MNs differentiated by iPSCs expressed their specific markers.3 The expression of gene SOX1,HOXA3 of NEP and of gene OLIG2,OCT4 of MNP cells in feeder layer system were significantly higher than that of feeder layer(P<0.01),and the expression of PAX6 and SOX2 showed no statistical difference(P>0.05).Conclusion:1.Under these two culture systems,iPSCs can proliferate for a long time and maintain their pluripotency.2.iPSCs cultured in the feeder-free culture system can effectively differentiate into motor neurons,but their differentiation efficiency is lower than that of the feeder system.3.Feeder cells may secrete some factors lacking in the feeder-free system to promote neural differentiation.Further study of these factors will further optimize the feeder-free culture system.