Caudalization And Ventralization Differentiation Patterns Induce Human Embryonic Stem Cells Into Mesencephalic Dopaminergic Neurons

Objective:Differentiation of human embryonic stem cells(h ESCs)into A9 dopaminergic neurons was induced using a differentiation protocol of " Caudalization " and "Ventralization " modes.We observed the feasibility and effectiveness of the protocol in order to establish a protocol that specifically differentiates human embryonic stem cells into dopaminergic neurons in the substantia nigra compacta.This program has high efficiency and relatively low cultivation cost,and is suitable for mass production,which can meet the needs of clinical transplantation,drug screening,and in vitro model research Methods:1.Differentiation of Human Embryonic Stem Cells Induced by Differentiation Scheme of "ventralization" and "caudalization " Models1)The first stage is the differentiation of h ESCs into mesencephalic dopaminergic progenitor cells.Human embryonic stem cells(H9 and HN4)were cultured on the culture plate coated with matrigel and human recombinant laminin-111 for 16 days.The basic nerve medium was N2 and B27.The addition factors were 10 ?M Y-27632,10?M SB431542 and 100 ng/m L Noggin.In order to promote its "ventralization" and " caudalization ",300 ng/m L Shh-C24 II and 0.7 ?M CHIR99021 were added to the culture medium.On the 9th day of differentiation,100ng/m L FGF8 b was added.On the 11 th day of differentiation,20 ng/m L BDNF,0.2 m M AA,100 ng/m L FGF8 b were added to promote the stereotyping and regionalization of midbrain dopaminergic progenitor cells.2)The second stage is the differentiation and maturation of midbrain dopaminergic progenitor cells.On the 16 th day of differentiation,20 ng/m L BDNF,0.2 m M AA,10ng/m L GDNF,500?M c AMP and 1 ?M DAPT were added,and then cultured for 42 days to ensure their differentiation into mature dopaminergic neurons.2.Detection of Cell Samples at Different Culture Stages1)Cell morphological changes were detected by inverted phase contrast microscopy.2)Cell immunofluorescence assay was used to detect the expression of TH,MAP2,FOXA2,LMX1,OTX2,GIRK2 and Calbindin1,which are related markers of midbrain dopaminergic neurons.3)q PCR was used to detect the expression of TH,FOXA2,LMX1,A9(substantia nigra compacta)dopaminergic neuron markers GIRK2,A10(ventral tegmental area)dopaminergic neuron markers Calbindin1,BARHL1 and other related genes.4)The expression of TH,FOXA2,LMX1,GIRK2 and other related proteins were detected by Western blotting.Results:1.Under the same directional induction conditions,the number,survival and cell yield of adherent cells on the matrigel-coated culture plate were more than those on human recombinant laminin-111.2.The whole process of this experiment was carried out on the matrigel-coated culture plate.Cell morphology gradually differentiated from typical stem cell morphologypaving stone-like to typical cluster dopaminergic neuron morphology-spindle or polygon.3.The results of cellular immunofluorescence showed that on the 16 th day of differentiation,a large number of ventral mesencephalic progenitor cells of FOXA2+/LMX1+/OTX2+ were observed,accounting for about 35% of the total cells.On the 42 nd day of differentiation,a large number of GIRK2+/TH+,FOXA2+/LMX1+,TH+/MAP2+,GIRK2+/LMX1+ cells were observed.4.Compared with h ESCs,the expression of FOXA2,LMX1,BARHL1 and TH in the STN neuron markers were higher on the 16 th day of differentiation(P < 0.05),while the expression of GIRK2 and Calbindin1 was slightly higher(P < 0.05).Compared with the 16 th day,FOXA2,LMX1 and BARHL1 expression decreased on the 42 nd day of differentiation(P < 0.05),but GIRK2,Calbindin1 and TH increased significantly(P< 0.05).5.The results of Western blotting showed that on the 42 nd day of differentiation,TH,FOXA2 and LMX1 were all expressed in midbrain dopaminergic neurons,and GIRK2 expression was significantly increased.6.Regulating the concentration of CHIR99021 in a certain range showed that with the increasing concentration of CHIR99021,the number of FOXA2 positive cells increased continuously,but had little effect on the number of LMX1+/OTX2+ cells.The number of FOXA2 positive cells was the highest when the concentration of CHIR99021 was 0.8 ?M.Conclusions:1.Matrigel as coating agent can be used to induce directional differentiation of h ESCs.Its effect of promoting cell adhesion and cell survival is better than that of recombinant human laminin-111,and it has certain economic value.2.Human embryonic stem cells can be differentiated into a large number of A9 dopaminergic neurons by "ventralization" and " caudalization " differentiation schemes,and the characteristics of brain dopaminergic neurons can be confirmed by morphological changes,protein level and gene level,which proves that the scheme is effective and feasible.3.The concentration of " caudalization " inducible factor CHIR99021 was closely related to the positive rate of ventral mesencephalic progenitor cells,and increased with the increase of CHIR99021 concentration in a certain range.4.A9 dopaminergic neurons are the main neurons obtained in this study,but a small number of subthalamic nucleus neurons cells and A10 dopaminergic neurons cells are still included,which indicates that this scheme is still not perfect and has the possibility of further optimization.