The Experimental Study Of Trunk Neural Crest Stem Cell Isolated, Purified And Induced To Differentiating Into Schwann Cells

Peripheral nerve injuries are common and results in incomplete or no functional recovery,particularly after a complete transaction.The regeneration in the peripheral nervous system is incomplete and the treatment of severe lesions with nerve tissue loss is primarily aimed at recreating nerve continuity.The development of tissue engineering has provided a new strategy for the reparation of damaged nerves,the scaffold and seed cells are critical for the construction of tissue engineering nerve.Schwann cell which derided from neural crest of early embryo is the mainly glia cell in the peripheral neural system and promotes nerve regeneration and recovery of function.It is seen as the candidate cell in the cell transplantation.While it is difficult to preserve the autoallergic Scs proliferate massively and steady characters,moreover it needs two times surgery;The allograft may cause the immunological rejection in the host.So it is necessity to looking for other cells more fit the transplantation continuously.The neural crest cell is located in the neuroectoderm of early embryo and migrates from the neural tube to its destinations.It is regarded as the cell with the most differentiation potential for differentiating into neuron,glial and diverse noneural cells.The trunk neural crest stem cell can differentiate into neuron and glia which contribute to PNS.So it is considered as the primordium of PNS.Therefore,it is necessary to study the neural crest stem cell's biological character for understanding the formation of PNS.Furthermore,it is thought as the candidate of tissue engineering for its pluripotency.Although we have known that neural crest stem cell can be induced to differentiate into neuron,glia and osteoblast in vivo,the same results can not be easily gained in vitro.There are some difficulties about the isolation and culture of the cell.And it differentiates into its derivations easily in ordinary medium in vitro. There are some reports about the survival,differentiation and effects about the NCC. But the mechanism and the inducing factors of the neural crest stem cells and the effects between the Sos and neural crest stem cells are known little.The search for how to realize a advantageous environment for the regeneration of damaged peripheral nerves,how to gain sufficient Sos for the transplantation,how promote the survival of the NSC in transplantation areas and the neurons amount differentiated from the NCSC,would give more efficient strategies for treating the diseases in the PNS.All above this,we design this experiment:We isolate and culture the trunk neural crest stem cell using tissue culture of neural tube in the E10.5 Wistar rat embryo in defined medium,then observing its' biological properties,such as the survive,proliferation,pluripotent ability and directed differentiation of the neural crest stem cells;We cultured the NCSC into SCs conditioned medium;Using MTT,cell count and ICC to investigating the optimism conditions of gaining sufficient Schwann cells to provide theoretic and experimental evidence for the treating the PNS diseases through cell transplantation.Result:We can observe that cells migrated from the edge of the neural after 24h.The cells array around the neural tube with fusiform shaped.The immuocytochemistry results show that the cell are nestin positive and low affinity neural growth factor receptor(LNGFR) p75 positive which are NCSC characteristic antibodies.After serial culture,the cell show clonal growth.We gain the TNCSC doubling time-34.2h through growth curve.We have studies the differentiation of TNCSC using FCS.The result shows that after 7 days culture in 10%FCS medium,the cells can differentiate into neuron,glial cell and smooth music-like cell.These cells respectively appear to be MAP2,GFAP,α-SMA positive through immuocytoehemistry.In the culture system including TNCSC and SC conditioned medium,the neural crest stem cell were attached quicker than the control group.After 36 hours the emigrating cells were round and full and two to three branches were observed.These cells were like the mature neurons morphologically.The Schwann cells were not marked changes early. After persistent culture,they were arranged sarciniform and the double-track formation were observed in the bright field of the microscopic.In the control groups the cells grow badly and few attached the wall or attached unstable and no living cells were observed a week later.The ratio of the MAP-2 to GFAP positive cells in the experimental groups was 0.75:1.There were no positive cells in the control groups. MTT shows that the experimental group promote the survive and proliferation of the Schwann cells(P＜0.01).Parts of the neurites were MBP positive along the neurites by immunocytochemistry.This demonstrates that the neural glia derived from TNCSC could be induced to differentiate towards the myelined Schwann cells by the neurons derived from the neural crest stem cells.MBP was detected in the culturing system and the Schwann cells but the content was less than the nerve tissue.Conclusion:We have successfully isolated and cultured the trunk neural crest stem cell and observed its properties as stem cell.Schwann cells conditioned medium in which the neural crest stem cells cultured is fit for the neural crest stem cells surviving and differentiating into neurons and the neural glial,the neurons are matured enough to induce the TNCSC derived Schwann cells to differentiating to myelinating Schwann cells and expressing MBP.