Derivation Of Neuroepithelial Progenitors And Dopaminergic Neurons From Human Embryonic Stem Cells

Human embryonic stem cells (hESCs), isolated from the inner cell mass (ICM) of preimplanted blastocyst, possessing the ability of pluripotency and unlimited self-renewal, were considered as potential seed cell sources for cell replacement therapy, as well as novel materials for researches on human embryo developmental mechanism, gene functional assays and pharmacological screening. Neurodegenerative diseases were caused by lack or death of neural cells which could not be effectively regenerated by human body to replace the original ones, such as Parkinson Disease, Alzheimer Disease and Huntington Disease. These diseases, with a high rate of incidence and long period of process, lacked effective clinical treatments; hESCs derived neural cells-based replacement therapy might provide new prospects for these refractory diseases.In order to make hESCs function as seed cells, we should first establish methods to direct them to differentiate into committed specific cells, and to induce highly purified specific neurons in vitro with proper biological functions. Present methods for induction of hESCs into neural epithelium and neurons had their individual merits and drawbacks, but systematic comparisons between them had not been carried out yet. Our purpose of the study, using several self-established hESC lines, was to perform neural induction in vitro and establish a simple, efficient, serum free and animal cell free culture system for neuroepithelial progenitors (NEP) and dopaminergic neurons (DAN), and to provide technical platforms and research materials for early neural development researches as well as for cell replacement therapy for Parkinson Disease.The first chapter introduced the establishment of simple and efficient inductive system of hESCs to NEP. Comparison between human embryonic fibroblasts (HEF) and mouse embryonic fibroblasts (MEF) found that both of them could induce hESCs into NEP with typical rosette structures and expression of Musashi, Nestin and Pax6 under serum free conditions, and inductive efficiency was even higher on HEF. Compared with the floating culture strategy, the direct adherent strategy proved to be more efficient. With or without addition of exogenetic fibroblast growth factor 2 (FGF2) in the culture medium, the inductive efficiency of NEP were not affected. Using feeder free (FF) culture system could directly induce rosette structures expressing Musashi, Nestin and Pax6, along with 34% SSEA4 positive cells. The assay of bone morphogenetic proteins (BMP) signalling pathway related gene expression in HEF and FF inductive systems found that FF system had higher expression of BMP4, while HEF system almost no expression, indicating lower BMP signalling activities in HEF system was more propitious for NEP induction. The second chapter continued to establish systems for HEF and FF system derived NEP to proliferate and expand and differentiate into DAN. FGF2 was found to promote the proliferation and expansion of neurospheres and replated adherent NEP, the function of which had a dose-dependent relationship with FGF2. NEP could be culture for at least 6 passages maintaining the NEP related marks and potentials for neuronal and glial differentiation. With the neural pattern specification factors fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH), NEP could be induced into Otx2 and En1 positive midbrain dopaminergic neuronal precursors, and further into tyrosine hydroxylase (TH) positive DAN. The proportion of Tuj1 and TH positive cells in HEF or FF induced terminally differentiated cells had no difference, and no SSEA4 positive cells remained, which indicated that efficiency of DAN production was similar in both systems. As FF system was independent on feeder cells, it could be more useful in the researches on nervous system development and industrialization of future neural cell differentiation system.The third chapter focused on the efficiency and safety of parthenogenetic human embryonic stem cells (phESCs) inducted into NEP and DAN. PhESCs, as well as normal human embryonic stem cells (nhESCs), could produce En1, TH and Tuj1 positive cells, which indicated the similar ability of phESCs to differentiate into DAN. Comparative genome hybridization assay found no change of genome chromosome copies. Expression of imprinted genes, such as H19, IGF2 and Snrpn indicated that NEP had basically maintained the expression pattern of imprinted genes. Thus, phESCs with the ability to differentiate into NEP and DAN as well as to maintain the stability of chromosomes and expression of imprinted genes, could be served as sources for cell replacement therapy.With those results we concluded that HEF system was a highly efficient NEP inductive system for hESCs; and that FGF and BMP signalling pathways were essential for NEP induction; and that FF system derived NEP could produce DAN; and that phESCs possessed normal differentiating potentials for neural cells. These NEP and DAN inductive systems we established could provide basement for hESCs clinical and basic researches, as well as possibility for future neural cell replacement therapy.