Application Of Human Pluripotent Stem Cells In Mechanism Research And Prevention Of NTDs

IntroductionNeural tube defects (neural tube defects, NTDs) are the most common and severe birthdefects. It is due to the abnormal in neural tube closure at blastocyst stage. The incidenceof NTDs is second only to congenital heart disease worldwide. Moreover, the rate ofChina is the highest in the world. Except for fatal NTDs, closed NTDs will not only causelifelong congenital disabilities and severe mental defects of infected children, but also puta heavy financial burden on their families and society.With the folic acid (FA) supplements during pregnancy, the prevalence of NTDs isremarkably decreased. However, large-scale studies showed that there is still30-50%ofNTDs which can not be prevented by FA supplements. Excessive complement FA alsomay cause a series of non-treatment effects including neonatal insulin resistance orobesity. These initiated an extensive controversy in the field including the role of the FAsupplement in NTDs prevention, whether it should become mandatory food additives andwhat dose is appropriate in pregnancy.In addition to folic acid deficiency, the application of the first-line antiepileptic drugValproic acid (VPA) is one of other participating factors. A mouse model study showedthat the VPA might lead to NTDs through the FA pathway and FA supplement may playa protective role in NTDs prevention caused by VPA. Scholars against VPA emphasizeits teratogenic effects on fetus, but there is no other better choice besides VPA for somecommon type of epilepsy. And this makes it difficult to change VPA to others duringpre-pregnancy or pregnancy.Neural tube closure occurs from the20th day after fertilization, and is a consistentlydeveloping process. This makes the previous studies encounter a lot of difficulties inobservation. As a special study object, especially considering the ethical issues of humanresearch, direct experiments on human embryos in vivo is impossible. In addition, due tothe irreversibility of development, we can not gain the information from the real processof NTDs in human. The only evidence can be obtained is through retrospective analysisindirectly. This makes the information form animal models hard to be verified in human.Therefore, building a human NTDs vitro model is very important for future study. Our research explores the use of embryonic stem cells (ES) and induced pluripotent stemcells (iPS) from somatic cells. Combined with in vitro induced differentiation of neurallinage, we setup human pluripotent stem cell-derived neural tube models. To investigatethe role of the FA and VPA during neural tube formation and nervous systemdevelopment, we applied different dug groups in our neural tube model developmentsystem. Meanwhile based on these studies, we want to initiate some application researchon drug screening, also to validate theory from animal models systems in human.Section one: Human pluripotent stem cell-derived in vitro neural tube modelMethods: Human pluripotent stem cell lines, H1, H9and iPS4were cultured andpassaged. Induced neural linage differentiation: Removed bFGF in suspension cultureand induced EB formation; EB were adherently cultured in NSC medium to induceRosette like structure (RS) formation; Immunofluorescence staining analysis of NSCmarkers; Further directly induced the NSCs ifferentiate into different cell types: defaultdifferentiation to large pyramidal cells of the cerebral cortex; ventral and caudalizeddifferentiation to motor neuron of the spinal cord; induced differentiation to glial cells;induced differentiation to neural crest cells; Immunofluorescence staining analysis of celltype-specific markers.Results: Human pluripotent stem cell lines cultured and passaged in vitro still expressedOct4, and Nanog; they are able to form EB; all three Lines can form the RS structureunder inducing conditions. And the cells in RS were Pax6, Nestin positive; Furtherinduced differentiation: large pyramidal cells of the cerebral cortex is Pax6positive;γ-aminobutyric acid neurons is GABA positive; Dopaminergic neurons is TH positive;Motor neurons of the spinal cord is ChAT positive; Astrocytes is GFAP and S100positive; Neural crest cells is P75, Sox10and SMA positive.Conclusion: Through induced differentiation of three human pluripotent stem cell lines,we first proposed the establishment of a human stem cell-derived neural tube-likestructure model in vitro. This model simulates the development of the neural tube in vivo,in the aspects of developmental time, cellularity and differentiation potential. It providesa novel potential in vitro system for drug screening and mechanism research of NTDs.Section two: the influence of Folate deficiency and supplement on neural tube formation and neurodevelopmentMethods: Based on the human pluripotent stem cell-derived RS model in section one, weselect iPS4as the object of study. Using the PRIM1640/woFA medium as a blank controlgroup, we grouped the experiment according to the FA concentration:0.02uM,0.2uM,2uM,20uM,80uM,160uM. iPS4cells were differentiated in PRIM1640/woFA mediumadding corresponding concentration of FA. Compare the following aspects among groups:EB formation; the mRNA and protein expression levels of pluripotent markers as Oct4and Nanog; RS formation; the mRNA and protein expression levels of NSC markers inRS as Pax6and Nestin; The differentiation rate in neurons, glial cells and neural crestcells.Results: Lack of FA groups (0-2μM): EB formation rate is lower (P <0.05), and theexpression levels of pluripotent factors decreased; RS formation was significantly lessthan high dosage groups (P <0.05); The mRNA and protein expression levels of Pax6,and Nestin were down regulated; Differentiation rates of neuron and neural crest reducedin a dose-dependent way. FA supplemented groups (20-160uM): EB formation ratesincreased, pluripotent factors expression levels were similar with iPS cells; RS formationincreased (P <0.05), the expression of markers of NSCs unregulated and thedifferentiation rates of neuron and neural crest increased. There is no statisticalsignificance among FA supplemented groups. The glial cell differentiation had nosignificant difference among all groups.Conclusion: During human embryonic development, the absence or lack of FA willaffect early neurodevelopment. FA deficiency inhibited the NSC differentiation. Theresulting RS formation inhibition will affect further differentiation of neuron and neuralcrest lineages. This then leads to a series of birth defects and syndrome. For thepreconceptional women with normal FA metabolism, keeping the FA concentrationaround normal or slightly higher levels (20uM-80uM in serum, by taking FA rich diet or400ug/day supplement dose) will be conducive to neuronal differentiation and theformation of the neural tube. There is no need to have an even higher dose of FA (160uMor600ug/day) which will bring unnecessary non-therapeutic side effects.Section three: Influence of VPA and folic acid on the development of the nervoussystem and their interactionsMethods: Select iPS4as the object of study in Section three, using DMEM/F12+N2 medium as a blank control group. And the experimental groups were divided into: VPAtreatment group (1mM of VPA) and VPA+FA group (1uM VPA+160mM FA).Neuronal induced differentiation was preformed in different groups. Compare the resultsin: RS formation; the mRNA and protein expression levels of NSC markers; Analysis ofNSC proliferation and apoptosis; Cell cycle Analysis of NSCs; The mRNA and proteinexpression levels of the FA receptor Forl expression; Long-term neuronal differentiationrate.Results: VPA group: RS formation reduced significantly (P <0.01); Pax6and Nestinexpression lowered (P<0.001) in mRNA and protein; Proliferation of NSCs deseased(41.01±3.53%, P <0.01); Apoptosis increased significantly (21.18%vs.3.57%, P <0.01);The proportion of cells in G2-S phase declined (35.69%VS.63.37%, P <0.05) whileG0-G1phase increased (64.31%vs.36.63%, P <0.05); Forl expression lowered;Long-term neuron differentiation rate reduced. VPA+FA group: Compared with VPAgroup, RS formation increased; Pax6and Nestin expression are significantly higher (P<0.01); NSC proliferation and cells in G2-S (43.69%VS.35.69%) increased(58.0±4.67%, P <0.05); Compared with the control group, cell death (15.71%vs.3.57%, P<0.01) increased; percentage of G0-G1phase is still high (56.31%vs36.63%, P <0.05);Forl expression lowered; Long-term neuron differentiation rate still reduced.Conclusion: The results confirm that therapeutic dose of VPA will inhibit in vitro neuraldifferentiation of human pluripotent stem cells. It works through the inhibition of cellproliferation; activation of apoptosis; interference of FA metabolism and destruction ofnormal neural tube-like structure formation. In the presence of other teratogens, FAsupplementation plays some role in protecting early neural embryo development, incompensation for cell proliferation, inhibition of apoptosis. But there is still a certainabnormality compared with the control. This provides a new research platform to solvethe clinical contradiction and could be useful in drug screening.Summary and innovationFor the first time, our subject applied in vitro human pluripotent stem cells derived neuraltube model in research of human birth defects as NTDs. Based on the establishment ofthe neural tube model in vitro of the human source of stem cells, we preliminary appliedthe FA and VPA treatment screening. We firstly verified the theories from animal modelsin human differentiation and development system. This also laid the foundation for generation of patients’ individualized differentiation of neural tube model, study of NTDspathogenesis, screening of virulence gene and for optimizing prevention recipe. And ithas taken a new step on the road to improve the population quality, and to prevent NTDsultimately.