The Direct Differentiation Of Mouse Parthenogenetic Stem Cells Into Neural Cells Without Embryoid Body Culture In Vitro

Embryonic stem cells are pluripotent cell lines derived from the inner cell mass of the mammalian blastocyst.They have abilities to self-renew indefinitely in culture and to develop into any cell types in adult body. Therefore,these cells have important value in basic research and in regenerative medicine. Neuro degenerative disease such as injury Alzheimer's, Parkinson's disease and neural damage following stroke are frequently encountered disease. Scientists found neural stem cells could be transplanted into central nervous system and replace dead or degenerative neurons.Significant symptomatic relief can be obtained after cell replacement. This finding is a milestone to the treatment of neuro degenerative disease and neural damage. Therefore, neural diferentiation of ES cells has been a popular investigation in science field. However,there are many obstacles that hinder the development of these cell-based transplantation therapies.One such obstacle is immune rejection. Embryonic stem cells can be derived from four kinds of embryos: One is normally fertilized eggs; Second is derived from genital ridge of early embryos.Third is blastocyst derived from nuclear transfer. Fourth is parthenogenetically activated embryos .The embryonic stem cells form the fourth approach is called parthenogenetic stem cells. Parthenogenetic stem cells have unique value in basic research and application realm. Normal embryonic stem cells derived from fertilized eggs may cause immunological rejection because of notable difference of MHC genes between individuals.A transplant can be much less immunogenic when its two MHC haplotypes are identical, resulting in a much higher probability of tissue matching.And some literature reported that mammal parthenogenetic stem cells have the ability to differentiate into all three embryonic germ layers.So the parthenogenetic stem cells may become another source of cell-based therapies.Objective: To investigate the feasibility of directly differentiation of mouse parthenogenetic stem cells into neural cells without embryoid body culture in vitro.Methods : These mouse pathenogenetic stem cells which were established in our lab is homozygous in genotype.These stem cells are positive for telomerase activity and are immunoreactive for alkaline phosphatase,octamer-binding transcriptionfactor4(Oct-4) ,stage-specific embryonic antigen-4(SSEA-4) and stage-specific embryonic antigen-1 are negtive.They have a normal chromosome karyotype and when these stem cell were injected into severe combined immunodeficient mice,teratomas with derivatives from three embryonic germ layers were obtained. The method of phase induction was used to induce direct differentiation from parthenogenetic stem cells to neural precursors which were identified by nestin immunofluorescence staining. Sequential culture was applied for the further differentiation into neural cells with the removal of mitogen and addition of 5% fetal bovine serum.The characteristics of the further induced cells were identified by immunofluorescence staining.Results: Large amounts of parthenogenetic stem cells were differentiated into neural precursors in the selected medium on the 3th day which were nestin-positive.The appearance of these cells are small round,most of them have two cyto-process which have the typical morphologic characteristics of neural precurosor. With the removal of mitogen and addition of 5% fetal bovine serum for further culture.Abour 10 days later, neural epithelium -like cells were observable. The characteristics of the further induced cells were identified by immunocytochemically staining and these cells areβ-Ⅲ-tubline-positive cells.Conclusion: (1)The mouse pathenogenetic stem cells (mPGES) which were established in our lab can be successful induced into early neuron in vitro.(2)When we induced mouse parthenogenetic stem cells into neurons, the mathod of direct differentaition which was without embryoid body culture in vitro can be uesd .It is more easier than EB culture method.(3) The parthenogenetic stem cells may become another source of cell-based therapies for neuro degenerative disease.