MIF Regulates The Differentiation Of Human Embryonic Stem Cells Into Dopaminergic Neural Progenitor Cells In The Ventral Midbrain

Objective: On the basis of the “dual SMAD channel inhibitor” differentiation scheme,the effects of different concentrations of MIF and its inhibitor ISO-1 on the differentiation of human embryonic stem cells into mesencephalic dopaminergic progenitor cells were observed by adding different concentrations of MIF and its inhibitor ISO-1 in the medium,so as to find a new way to solve the heterogeneity of the process of human embryonic stem cell differentiation into mesencephalic dopaminergic.Methods:1.Intervention of human embryonic stem cell differentiation by adding MIF and ISO-1.The entire study was divided into non-intervention group,MIF group and ISO-1group.In this study,based on the differentiation scheme of “double SMAD channel inhibitor”,3ng/m L,10ng/m L,30ng/m L MIF,0.2 ?M,0.6 ?M and 1.8 ?M ISO-1 were added to differentiate human embryonic stem cells into DA-ergic neural progenitor cells.The differentiation cycle was 16 days,starting from induction,using N2 basal medium.During the first eight days of differentiation,0.8 ?M CHIR99021,10 ?M SB431542,10 ?M Y-27632,100 ng/m L Noggin and 400 ng/m L SHH-C24 II were added to synergistically promote the differentiation of human embryonic stem cells into ventral and caudal hemispheres.At the same time,MIF and ISO-1 were added to intervene.On day 9 of differentiation,100 ng / m L FGF8 b was added.From the 11 th day of differentiation,B27 basal medium was added with 10 ?M Y-27632,100 ng/m L FGF8 b,0.2 m M L-ascorbic acid and 20 ng/m L BDNF to co-promote the differentiation of human embryonic stem cells towards the regionalization and stereotyping of dopaminergic progenitor cells in the substantia nigra of the midbrain.2.Detection of cell samples.1)Cell immunofluorescence was used to detect the expression of MIF-related factors,including MIF,CD44,CD74 and CXCR7 in human embryonic stem cells.2)The effects of MIF and ISO-1 on the differentiation of human embryonic stem cells into mesencephalic DA neurons were analyzed by optical microscope.3)Real-time fluorescent quantitative polymerase chain reaction was used to detect LIM homeobox transcription factor 1A(LMX1A),homeobox gene EN1(EN1),and forkhead box transcription factor A2(FOXA2).Orthodenticle homologous frame 2(OTX2),a common marker of midbrain and forebrain;Forkhead box transcription factor G1(FOXG1);Heterotypic frame gene A2(HOXA2)of posterior brain marker;Paired box 6(PAX6)of supracortical markers;STN neuronal marker BARH homologous box 1(BARHL1).The effects of MIF and ISO-1 on the differentiation of human embryonic stem cells into DA-ergic neural progenitor cells in the midbrain were analyzed at the gene expression level.The analysis was done by statistical software SPSS.Significant differences were found if P<0.05.4)Cell immunofluorescence and Western blotting were used to detect the related factors,such as LMXIA,EN1,FOXA2;the common marker of mesencephalon and forebrain,OTX2;the marker of forebrain,FOXG1;the marker of HOXA2;the marker of PAX6;STN neuron,the marker of neuron,BARHL1 and so on.The effects of MIF and ISO-1 on the differentiation of human embryonic stem cells into mesencephalic DA neural progenitor cells were analyzed from the level of protein expression.Through the statistical software SPSS to do the analysis.If P < 0.05,there is significant difference.Results:1)Immunofluorescence results showed that a large number of MIF+/ CD44+/CXCR7+ human embryonic stem cells,nearly 100%,but human embryonic stem cells did not express CD74.2)In terms of morphology,human embryonic stem cells gradually changed from typical stem cell morphology to neural progenitor cell morphology : the appearance of human embryonic stem cells is paving stone,high nuclear-to-plasm ratio,large nucleus,less cytoplasm,cell processes and shorter,the cell size is more average,its aggregation growth,and fusion into pieces.After nerve induction,the cell morphology gradually changed into spindle shape,the nuclear-to-mass ratio gradually changed,the cytoplasm content increased,the long protrusions extended,the cell shape became smaller and rounded,and the intercellular space became increasingly close.The growth rate and morphological changes of cells in MIF and ISO-1intervention group were similar to those in normal differentiation group.There was no significant morphological difference between the intervention group regulated by MIF and ISO-1.3)Cellular immunofluorescence results showed that at day 16 of differentiation,we could see a large number of LMX1+/FOXA2+/EN1+/OTX2+ ventral midbrain dopaminergic neural progenitor cells,with a conversion rate of approximately 43% in the non-intervention group to the ventral as well as effective regions of the midbrain,compared to 1.8 times the conversion rate in the MIF group and 1.6 times the conversion rate in the ISO-1 group times.4)Western Blot results showed that compared with the undifferentiated group,the expression of LMX1 A / FOXA2 / EN1 was high in the MIF 30 ng group,while the expression of BARHL1 / HOXA2 / FOXG1 was low(P< 0.05).The expression of FOXA2 / SOX6 was higher in ISO-1 0.2 ?M group,while the expression of BARHL1 / PAX6 / FOXG1 was lower(P< 0.05).5).Real-time fluorescence quantitative PCR(q PCR)results showed high expression of LMX1A/FOXA2 and low expression of FOXG1/PAX6 in the MIF 30 ng group compared to the undifferentiated group(P<0.05).High expression of FOXA2 / SOX6 and low expression of PAX6 / LMXIA / FOXA2 were observed in ISO-1 0.2 ?M group(P< 0.05).Conclusions:1.Human embryonic stem cells secrete MIF,which is secreted into the culture medium in an autocrine form and participates in the differentiation of stem cells.2.The "dual SMAD channel inhibitor" differentiation protocol was used to induce neurally human embryonic stem cells.After MIF intervention,it has no effect on the morphological changes,proliferation and survival of the cells in the process of neural induction.It can successfully induce dopaminergic neural progenitor cells and dopaminergic neurons located in the origin of the midbrain floor plate,and differentiate within a certain concentration range.Progenitor cells are more concentrated in the midbrain region,and inhibit the expression of miscellaneous cells such as forebrain and hindbrain,without concentration dependence.3.MIF plays an important role in the dopaminergic differentiation potential of h ESCs and the fate determination of the ventral midbrain.The intervention of MIF further optimizes the localization of neural induction and accelerates the transformation of this program into clinical practice.However,the mechanism of high expression of SOX6 and inhibition of EN1 after ISO-1 intervention remains to be studied.