| Background: The efficient acqtion of primary neurons from different neurodegeneration is a major challenge in neurobiology and neurological disorder.Human pluripotent stem cells(h PSCs)include human embryonic stem cells(h ESC)and human induced pluripotent stem cells(hi PSCs).h PSCs can differentiate into neural progenitor cells(NPCs)and can further differentiate into neurons and glial cells,neurodegeneration plays an important role in the study of pathogenesis,screening of disease markers,drug screening and cell transplantation,it provides an excellent source of cells for the study of neural development,cell therapy,disease modeling and drug screening.Therefore,it is very important to establish a robust and efficient method of neural differentiation.At present,many researchers have established methods of neural differentiation,but many methods have low differentiation efficiency.Therefore,we improved a dual suppression strategy of SMAD pathway,and compared it with the method of RA induced NPC,i PSCs were induced into long-term cryopreserved NPC,NPC was further induced to differentiate into primary neurons.Methods:(1)To get NPC,i PSC were treated for 7 days with RA/MGCD or dual SMAD inhibitor,and the morphology of i PSC was compared during induction,and the shape and size of NSP were generated.(2)NPC induced by these two methods were detected by Q-PCR and immunofluorescence staining,such as PAX6,NESTIN,SOX2.(3)NPC was induced by the combination of different transcription factors in vitro.The main neurons were stained by immunostaining for the expression of specific markers,such as CHAT,DARPP32,etc..(4)The right striatum of SCID mice was modeled by stereotaxic injection of quinolinic acid(QA)to show HD-like symptoms.(5)NPCs induced by transcription factors for 3 days were implanted into the right striatum of the SCID,and the mice were taken 2-3 months after transplantation to detect whether NPCs were successfully differentiated into MSN in vivo by immunofluorescence staining,to make synaptic connections to the mice peripheral neurons.(6)To determine whether HD-like symptoms of transplanted NPC mice can be ameliorated by means of behavioral examination(once a month after NPC injection)of the above-mentioned SCID mice and NPC mice.Results: In this study,We completed the rapid differentiation strategy of h PSCs into NPCs within 18 days by combining cell-adherent culture with NSP suspension technique,and by introducing a specific combination of transcription factors at about 21 days,specific master neurons(> 90%)were efficiently generated from NPCs.We compared retinoic acid(RA)-induced and dual SMAD pathway inhibition to induce neural differentiation.Both methods can quickly and efficiently generate cryopreserved NPCs,but the NPCs induced by the two methods have different regional identities.NPC induced by these two methods can differentiate into most excitatory and inhibitory neurons at the same time,and the purity is not high,and there is almost no MSN,MN and other specific main neurons,therefore,we further differentiate NPC into specific master neurons by over-expression of specific transcription factor sets in NPC and by addition of small molecular inhibitors.Conclusions:(1)We used two groof inducing factors,RA(trans retinoic acid)and dual SMAD inhibitor differentiation,both of which produced high-purity NPC,and RA/MGCD induced NPC with more ventral phenotypes.(2)NPC can be induced to differentiate into different types of neurons and astrocytes.(3)Proneural factor ASCL1 and NGN2 can induce neurons rapidly and efficiently,while mi R-9/124 and sg PTBP1 have lower efficiency.(4)Different combination of transcription factors can induce NPC to generate different subtypes of primary neurons.In particular,we verify that the induction efficiency of MN,MSN can reach more than 95%.9 figures,16 tables,and 52 references... |
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