Generation Of Human Down Syndrome-induced Pluripotent Stem Cells And Their Potential For Neural Differentiation

BACKGROUND:21Trisomy（T21）syndrome or Down’s syndrome（DS）,which is the leading genetic causeof intellectual disabilities and related diseases.In China, about1/600to1/800of live births carry atrisomy21, which causes Down’s syndrome.The genes on chromosome21are most likely to beassociated with Down’s syndrome, but the detailed mechanism are difficult due to high geneticcomplexity and phenotypic variability of Down’s syndrome. Despite progress in mouse models forDown’s syndrome, there remains a need for better ways to understand the underlying cell anddevelopmental pathology of human Down’s syndrome.Induced pluripotent stem cells or iPSCstechnology by using specific transcription factors could reprogram the differentiated cell intopluripotent stem cell.The disease-specific iPSCs maintaining their cell populations by self-renewdivision and carrying genetic disorder genes during the whole proliferation.They could help us toexplore the pathogenesis of many diseases.The iPSCs have the potential of differentiating intodifferent types of cells,which would be a good cellular model for drug screening and genetherapeutic evaluation.OBJECTIVE:Generation of DS-specific induced pluripotent stem cells from human amniotic fluid-derivedcells and dermal fibroblasts.Comparison of the efficiency of iPSCs from different types ofcells.Differentiate T21-iPSCs into neurons,astrocytes and oligodendrocytes.Using inducedpluripotent stem cells technology for modeling Down syndrome in vitro will hold great promisefor advancements in developmental biology, and study pathogenesis of Down’s syndome disease.METHODS:1.we have generated DS-specific induced pluripotent stem cells from human amnioticfluid-derived cells and dermal fibroblast cells through lentiviral delivery of four humantranscription factors(Oct4/Sox2/Klf4/c-Myc) and identified their pluripotency by using APstaining,immunofluorescent staining,qPCR experiment.2.we used floating cultivation to form embryoid bodies(EBs) and detected the expression ofspecific genes by semiquantitatice PCR to evaluate the vitro differentiation ability.To confirm thepluripotency of iPSCs with DS in vivo,we injected the T21-iPSCs into SCID mice for teratomaformation,6months later,teratoma were harvested and processed with hematoxylin and eosinstaining.3.Karyotype analysis was used to detect karyotype of each cell line after10passages.4.Compare the time of T21-iPSCs cloning formation and the positive efficiency after beingAP staining at the first passage. 5.We have differentiated T21-iPSCs into neural stem cells and three kinds of neural cells andcomparison of the efficency of differentiation by immunofluorescent staining.RESULTS:1.we have successfully generated DS-specific induced pluripotent stem cells(iPSCs) fromhuman amniotic fluid-derived cells and dermal fibroblast cells through lentiviral delivery of fourhuman transcription factors(Oct4/Sox2/Klf4/c-Myc).The T21-iPSCs showed characteristicssimilar to those of human embryonic stem cells,particularly the morphology,pluripotent-specifictranscription-factor (Oct4,Nanog)expression.The pluripotency of T21-iPSCs was also tested invitro and in vivo.Embryoid bodies and teratomas were formed and showed the expression ofdifferentiated markers for three germ layers.The T21-iPSCs showed a good self-renewal conditionand maintain the karyotypes after long-term culturing in vitro.2.After reprogramming, the amniotic fluid cells emerge ES-like clones cost nearly7days andthe AP staining positive efficiency was0.285%,the dermal fibroblast cells cost nearly15days toemerge ES-like clones, AP staining positive efficiency was0.027%,The data was analyzed bychi-square test, the efficiency had statistical difference between amniotic fluid cells and dermalfibroblast cells.3.Differentiated neural stem cells from T21-iPSCs and control cells which could expressmarker Nestin, the difference between three groups was not significant. Differentiated the neuralstem cells can further form into neurons, astrocytes and oligodendrocytes. They express neuralmarkers Tuj1,GFAP and O4.The efficiency between three groups in differentiation was notsignificant.CONCLUSION：1.We have successfully derivated induced pluripotent cells(iPSCs)of Down syndrome, TheDS-iPSCs showed a good self-renewal condition and maintain the karyotypes and pluripotent afterlong-term culturing in vitro.2.It was found that generation of iPSCs from human amniotic fluid cells were more rapid andefficient than dermal fibroblast cells.Amniotic fluid cells may be a preferred tissue for generatingT21-iPSCs.3.We have generated T21-iPSCs neural differentiated system preliminarily and show thatT21-iPSCs can differentiate into many type of cells.The differentiation system of neural stem cellof T21-iPSCs was feasible and effective.