Primary Study On Neuroepithelial Cells And Dopamine Neurons In Vitro From The Human Embryonic Stem Cells

Part 1 Comparison of the Two Systems of Inducing Human Embryonic Stem Cells into Neuroectoderm CellsObjective: 1.We aimed to compare the difference between the efficiency of two systems for inducing hESCs into neuroectoderm cells in order to establish a highly effective inducing system. 2. We tried to reveal if the differentiating procedure under both inductive systems accorded to the rules of early neural development in vivo.Methods: Human embryonic stem cells were cultured on human fibroblast feeder cells. Cells in group A were directly induced on human feeder cells while cells in group B underwent an embryoid-like floating stage. 16 days later, RT-PCR was used for testing the expression of pluripotent genes, early neural development-related genes, mesoderm and endoderm genes, and regional genes on day 0, day 6, day 10 and day 16, and immunofluorescence used for testing the expression of the hESCS pluripotent markers and neuroectoderm-related markers.Results: 1.Under both inductive systems, we obtained high production of neuroectoderm cells expressing Nestin and Musashi; 2. Counting of Pax6 and Sox1 positive cells indicated that group A had a higher inductive efficiency than group B; 3. Cells of both groups were Pax6+Soxl- rosette structures on day 10, and on day 16 were Pax6+Soxl+ neural tube-like structures; 4. Cells in both groups had similar gene expression profiles, pluripotent genes down-regulated and neural-related genes up-regulated; 5. Cells in group A had a higher expression of forebrain markers, while cells in group B had a higher expression of midbrain and hindbrain markers.Part 2 Induction of Neuroectoderm Cells into Dopaminergic NeuronsObjective: 1.We aimed to establish an induction system for inducing neuroectoderm cells into dopaminergic neurons and provide cell sources for Parkinson Disease; 2.We compared the difference of inducing neuroectoderm cells from different differentiating stages into dopaminergic neurons.Methods: Neuroectoderm cells obtained at day 10 and day 16 were used as materials for early-treated group and late-treated group respectively, and were cultured until day 22 in induction medium containing 100ng/ml FGF8 and 200ng/ml SHH. The control group used SHH and FGF8 free medium. RT-PCR was used for testing the expression of midbrain dopaminergic neuron-related genes En1 and Nkx6.1, and immunofluorescence for testing midbrain dopaminergic neuron specific marker TH. Results: 1. Cells in early-treated group had a higher expression of En1 and Nkx6.1 than late-treated and control group on day 22; 2.There were large number of TH positive clones in early-treated group, while only few of them were in late-treated group.Part 3 Establishment of Parkinson Disease Rat ModelObjective: We aimed to establish a stable and reliable rat model of Parkinson Disease, which could be the appropriate animal models for cell transplantation therapy.Methods: 2ul of 4ug/ul 6-Hydroxydopamine were stereotaxically injected into the right medial forebrain bundle and substantia nigra pars compacta of adult Sprague-Dawley rats. Apomorphine was used to induce rotations, and we evaluated the behavior of rats. The successful rat models were perfused and sectioned for immunochemistry of tyrosine hydroxylase.Results: 1. The Parkinson Disease model rats showed typical rotations, 5 among 10 rats with a rotation speed exceeding 210 rounds in 30 minutes which persisted for the following continuous 4 weeks. 2. Immunochemistry of tyrosine hydroxylase indicated significant loss of tyrosine hydroxylase-positive cells in the injected part of substantia nigra pars compacta. Conclusions: 1.We established a humanized culture system for inducing hESCs into neuroectoderm cells, and found that direct induction was more efficient than EB stage undergoing method; 2. The neural inductive process in vitro accorded to the early neural development in vivo; 3. The cell fate of neuroectoderm on day 10 of induction was not decided, which provided itself a starting time-point for further induction; 4. FGF8 and SHH could induce the day 10 neuroectoderm into midbrain dopaminergic neurons; 5. Using the method of unilateral double-sited injection of 6-OHDA could successfully make stable and reliable unilateral Parkinson Disease rat models.