The Induction Of Rat Hair Follicle Neural Crest Stem Cells Into Motor Neuron

Purpose：Motor neuron disease is a set of common clinical nervous system disease, which iscaused by chronic progressive degeneration of motor neuron. There are no effectiveclinical treatments for the higher morbidity of motor neuron disease, such as AmyotrophicLateral Sclerosis (ALS), progressive Spinal Muscular atrophy (pSMA), Progressive bulbarpalsy (PBP) and Primary Lateral Sclerosis (PLS), etc. And the stem cell transplantationtherapy is a hot spot in the present study.There are many problems are not well resvoled in the stem cell transplantationaltherapy of motor neuron disease, such as the source of stem cells, the methods of stem cellsdifferentiation into motor neuron and stem cell-derived neurons in the body function andstability, etc. Therefore, it will have important theoretical value and practical significanceto these problems in-depth study and evaluate the feasibility.Hair follicles neural crest stem cells (hfNCSCs) widely exist in hair follicle jugadepartment, which belongs to the neural crest stem cells, and they can differented intoneural cells under the natural ondition. It has been successful induced into neuron-like cellsin our laboratory preliminary work, and the hfNCSCs-derived neuron-like cells can betransplanted to repair different distance of peripheral nerve defects. For the hfNCSCs cancome from self, easily to obtain, good active, and the low chance in forming tumors, so itis worthy to study and investigate as a stem cell source.It has been widely studied in the induction and differentiation of motor neuron in therecent studies. The inductive substance and methods involved in these studies such asRetinoic Acid (RA), Sonic hedgehog (Shh), Trichostatin A (TSA),8-Br-cAMP, Rolipram(Rp) and RG108, etc. Each have advantages and disadvantages, and there is no an idealdifferentiation scheme of motor neuron.To solve the problem of the source of motor neurons, this study intends to use ratshfNCSCs as seed cells, application of combined the TSA, RA, Shh,8-Br-cAMP, Rp andRG108induce hfNCSCs into motor neuron. Then research the cell morphology, theexpression of motor neuron specific genes and proteins, and the stability in the body andwhether they can carry on the function of motor neurons, and discuss the feasibility of thestem cell transplantation in the treatment of motor neuron disease. Methods:⑴The isolation, culture, amplification and identification of rat hfNCSCs: usingpiece-sticking method to isolate, culture, and amplify rat hfNCSCs, and test the expressionof Nestin (a neural stem cell surface marker), Sox10(a neural crest cell marker).⑵The differentiation of rat hfNCSCs into motor neurons: Using RA, Shh as well assome small molecule compounds, such as8-Br-cAMP, Rp, TSA, and RG108, to induce rathfNCSCs after amplification, and test the expression of motor neuron markers such asβ-Tubulin Ⅲ, MAP2, ChAT, HB9, Olig2and Islet-1,etc.⑶The function and stability tests of the rat hfNCSCs source-derived motor neuronlike cells in vivo:rat sciatic nerve injury models are constructed and grouped, respectively:rat hfNCSCs source-derived motor neuron like cells group (induced cell group, injects thehfNCSCs-derived motor neurons), hfNCSCs group (injects hfNCSCs) and PBS group(injects PBS). According to the grouping processing, test and record respectively in2weeks and4weeks,8and12weeks after operation, and analyzed.Results:⑴We can get plenty of good active, fast multiplication, morphology consistency andhigh fine purified rat hfNCSCs after the isolate, culture, and amplification usingpiece-sticking method. The positive expression of Nestin is97.78%, while the positiveexpression of Sox10is92.67%.⑵After the combinational differentiation of RA, Shh, and8-Br-cAMP, Rp, TSA,RG108, rat hfNCSCs show a polysynaptic motor neuron shape, and the results ofimmunofluorescence show the cells express some motor neuron specific makers, such asHB9、ChAT、Islet-1、Olig2,etc. And it can be verified by the results of Real-Time PCR,and Western blot at the same time. And there are about50%of rat hfNCSCs are inducedinto motor neuron-like cell.⑶The rat hfNCSCs source-derived motor neuron like cells can effectively repair ratsciatic nerve injury, and they can proliferation, survival, and maintain the function andcharacteristics of motor neurons in sciatic nerve.Conclusion:⑴The hfNCSCs can be used as a source of the stem cell-derived motor neuron forclinical transplantation. ⑵The combined application of RA,Shh as well as the four small molecularcompounds,8-Br-cAMP, Rp, TSA, and RG108can effectively induce rat hfNCSCs intomotor neuron.⑶The rat hfNCSCs source-derived motor neuron like cells can proliferation,survival, and maintain the function and characteristics of motor neurons in peripheralnerve.