Ra-treated Sequential Method Induced Embryonic Stem Cells Differentiation Into Neural Cells And The Expression Change Of Protein 4.1 After The Induction

Embryonic stem cells(ESCs) which derived from the inner cell mass(ICM) of blastocyst-stage embryos have the ability to proliferate indefinitely in vitro and the pluripotency to differentiate into any cell type derived from ectoderm,mesoderm or endoderm.Lots of reports indicated that ESCs could be directionally differentiated into neural cells in specific induced system which provided practical evidences for the transplantation of ESCs to treat center nervous system diseases.So far,the methods used to directionally induce ESCs differentiation into neural cells include 4-/4+ assay, five-step method,SDIA(a stromal cell-derived inducing activity),adherent monoculture,gene methods,and so on.4-/4+ assay is the most classical method. However,ESCs must form embryonic bodies(EB) before inducted by RA which makes it difficult to control and analyze the process of induction.Moreover,this method requires long culture time,complicated manipulation and strict culture condition including cell density,nutrition and equipments.Searching for a new induction method to shorten the induction period and simplify manipulation will improve the study on ESCs culture and differentiation in vitro.It is uncertain about the mechanism of ESCs differentiation into neural cells presently.Many researchers found that the expression of some protein increased(i.e.PKC_δand PKC_ε) or decreased(i.e.notchl) during neural cells differentiation.Study about rat PC12 cell differentiation and mouse cerebellum neuron development revealed that the expression amount,alternative splicing and location of protein 4.1 changed along with the differentiation of neural cells.But it's unclear about the expression change of 4.1 protein in the process of differentiation ESCs into neural cells.Objective1.To develop a new method to induce ESCs differentiate into neural cells in vitro, thereby shorten differentiated time and simplify manipulation.2.To study the expression of protein 4.1N and 4.1B during differentiation of ESCs into neural cells in vitro.MethodsESCs were offered by Zhengzhou University stem cells laboratory.After anabiosis,ESCs were seeded on a mitonycin C-arrested MEF monolayer and cultured with the MEF conditioned medium containing LIF.As ESCs growth stably,replant them on culture plastic dishes at a density (10⁵/ml).Three groups were set as follows:(1) Control group:ESCs were seeded in EB culture medium and cultured without an additional revulsant.(2) 4-/4+ induced group:ESCs were suspended in EB culture medium for 4 d to form EB,during which shook every 2-3 h to avoid EB adhesion.On 5^th d the cells were collected and seeded on glutin-coated culture plastic dishes in.induction medium(containing 10^-6 mol/L RA).Exchange with EB culture medium after 4 d.(3) RA-treated sequential induced group:ESCs were seeded in induction medium(containing 10^-6 mol/L RA) directly. After 48 h,every dish was added 3 ml neural stem cells culture medium(containing 20μg/L bFGF,2%B27,1%N2,DMEM/F12 without FBS).After 2 d,every dish was added 1 ml neural cell culture medium(containing 2%B27,15%FBS,DMEM/F12) every day.On 7^th d the medium was exchange with neural cell culture medium.Inverted microscope was used to observe the shape of ESCs during differentiation.Immunocytochemical staining was applied to calculate the positive percen,tage of NSE and GFAP positive cells respectively.Western-blot was used to study the 4.1N and 4.1B expression amount.ResultsUnder inverted microscope,MEF was fusiform and adherent.The undifferentiated ESCs grew in colonies with high refraction.After differentiation for 10 d,the cells in control group were almost epithelial-like cells,surrounded by a few fusiform cells.In 4-/4+ induced group,after 12 h adhesion on 5^th d,some fusiform cells migrated from undifferentiated ESCs colonies;on 10^th d,neural-like cells were observed with slender processes to form network.In RA-treated sequential induced group,neural-like cells with slender processes were observed on 5^th d.Subsequently, the number of neural-like cells increased obviously.On 7^th d the cells displayed thin long bipolar or multipolar processes to form network.After differentiation for 10 d,NSE positive cell rates of control group,4-/4+ induced group and RA-treated sequential induced group were 11.8%±1.8%,50.6%±2.7%and 57.6%±1.4%.GFAP positive cell rates were 19.0%±2.9%,36.0%±2.5%and 33.0%±2.0%,separately.Statistics analysis showed that there are prominent difference between control and either induction group(P＜0.01) while no prominent difference between the two induction groups(P＞0.05).Western-blot showed that 4.1N and 4.1B were expressed in undifferentiated ESCs and both them increased after induction.Conclusions1.RA-treated sequential induced method can get neuron-like cells on 7^th d,3 d before 4-/4+ assay.NSE and GFAP positive cell percentages have no difference between the two groups.So RA-treated sequential induced method can be used to replace classical 4-/4+assay.2.The level of 4.1N and 4.1B in ESCs increases during differentiation into.neural cells.