The Reliability Of Differentiation From Bone Marrow Stromal Cell Into Schwann Cells In Vitro

1.Aim and MeaningSpinal cord injury(SCI) is a severe trauma of the nervous system.With the progress of society,mainly from traffic accidents and fall injury there is an upward incidence of SCI year after year.To the family and society SCI are a heavy burden.It is firmly held that the injured mammalian brain and spinal cord could not regeneration after spinal cord injury by the traditional concept.However,in recent years the experimental study confirmed that the central nervous system have the ability of regeneration after injury,and then people come to realize that the limited capacity of the central nervous system's regeneration is because of unsuitable local micro-environment.The main repair problems after SCI are:(1) glial scar constitute an mechanical barriers to the growth of axons;(2) primary and secondary neuronal apoptosis(3) the lack of neurotrophic factors;(4) local harmful factors that inhibit axon regeneration such as Nogo,myelin-associated glycoprotein and so on.In recent years,cell transplantation in the treatment of experimental SCI has showed good results for the treatment of SCI.By changing the pathological process after SCI,cell transplantation can promote the functional restoration of spinal cord,including:(1) graft site can be filled with bridge-forming cells,consequently,it is possible to provide a mechanical or biological channel for the regeneration of axons;(2) the secretion growth factors can for provide necessary conditions for the regeneration of axons or neurons;(3) these transplanted cells can replace or provide new neurons to form new neural loop.Because of the advantages all above provided by cell transplanted,the method has been used in many SCI experimental studies.At present, several kinds of cell are used in cell transplantation for the treatment for SCI.Such as Schwann cells(SCs),olfactory ensheathing cells,neural stem cells,bone marrow stromal stem cells and umbilical cord blood stem cells.Autologous cell transplantation doesn't include negative rejection,therefore SCs has broad application prospects in the spinal cord injury repair.As the most important glial cells in the peripheral nervous system which SCs originated in the neural crest,and proliferated and migrated simultaneously along with the growth of axons,thereby supporting and providing nutrition for nerve fibers. One SCs envelops one axon in myelinated nerve fibers;While in non-myelinated nerve fibers several axons can be enveloped by a SCs.SCs shows homogeneity,that is,different parts of the cytological characteristics of SCs is basically the same.When axons are damaged,the bodies produce Waller's degeneration,in the distal end of the injury,SCs perform great proliferation and envelope the damaged axons in order to repair the injured neurons.SCs,as the most important one of seed cells for tissue engineering,would play an important role in in repair of peripheral nerve and in central nervous system.Even though Schwann cells are the major peripheral nerve structural and functional cells,and play an important role in nerve regeneration and functional recovery,however,Schwann cells belong to terminal cells,it is difficult to carry out mitosis,and to amplify Schwann cells largely.Also because of its antigenicity,SCs' applications in peripheral nerve and central nervous system repair is greatly limited. In 2001 Dezawa induced bone marrow stromal cells(BMSCs) in vitro to differentiate into,and Schwann-like cells express SCs' specific proteins such as p75,S100 and GFAP.However,this obtained cell induced from bone marrow stromal cells (dBMSCs)is not is not exactly the same in morphology as SCs from peripheral nerve. There has not been report whether dBMSCs can secrete neurotrophic factors or not, and whether dBMSCs could maintain their Stability when cell passage have been performed is not reported in the literature.In this study,SCs is used as a positive control to appraise the method in the whole process.And the stability of related properties of dBMSCs has been also assessed.2.MethodsThere are two parts of this expreiment2.1.1 Culture of BMSCsIsolation of adult SD rat MSCs was performed according to the method described by Mari Dezawa et al.(2001).In brief,rats were killed with an overdose of pentobarbital,and tibias and femurs were dissected out.The marrow was then extruded with 10 mL of alpha-MEM and cultured in alpha-MEM supplemented with 15%fetal bovine serum(FBS),2mM L-glutamine and 100mg/mL kanamycin, incubated at 37℃,humidity 95%and CO₂ 5%.After 48 h,the nonadherent cells were removed by replacing the medium.MSCs were subcultured four times,and finally brought to the following experiments.2.1.2 Purification of SCsIsolation of neonatal rat SCs was performed according to the method described by He Jing et al.(2006).In brief,rats were anaesthetized with ether,and under sterile conditions,bilateral sciatic nerves were dissected out,and placed into 35mm uncoated tissue culture dishes.Every week nerve segments were transferred to new 35mm dishes until,after 3 weeks in culture,the nerve segments(essentially depleted of fibroblasts) were enzymatically and mechanically dissociated before transferring to new dishes for SC expansion.After repeating for two to three times,the cells have been mainly SCs,and finally brought to the following experiments.2.1.3 Differentiation of MSCsSubconfluent cultures of MSCs were incubated for 24h with alpha-MEM containing 1mM beta-mercaptoethanol(BME),the media were removed,washed with phosphate-buffered saline(PBS)(pH7.4) and replaced with new media consisting of alpha-MEM,10%FBS and 35 ng/mL all-trans-retinoic acid(RA) for 3 days.Cells were washed with PBS and transferred to alpha-MEM containing 10% FBS,5mM forskolin(FSK),10ng/mL recombinant human basic-fibroblast growth factor(bFGF),5ng/mL recombinant human platelet derived growth factor-AA (PDGF) and 200ng/mL recombinant human heregulin-betal(HRG);they were then incubated for 7 days.2.2 induced secretion of cell surface markers and ability to detect2.2.1.Immunofluorescence StainingInduced BMSCs were washed in PBS and fixed with 4%formaldehyde in PBS at room temperature for 20 minutes.Immunocytochemistry was performed to discriminate the differentiation characteristics using the following primary antibodies against S100,p75,and GFAP.Primary antibodies were detected with rhodamine (TRITC)- conjugated,affinipure labelled,goat anti-rabbit IgG(H+L) antibody.A double-stranded DNA specific fluorescent dye,Hoechst33342,was used for cell nuclei counterstaining.2.2.2.Real-time PCRTo detect the expression of p75,CD104 and S100,all being known as markers of Schwann cells,Real-time PCR was performed.Crossing point values were measured with the Stratagene MxPro QPCR analysis software and percent changes were calculated relative to untreated lymphocytes as 2-△△CT.Three replicate reactions were performed for each individual per gene analyzed and values were normalized to the housekeeping gene GAPDH.2.2.3.PCRIn order to evaluate the ability of the induced cells to secrete neurotrophic factors,the PCR was performed to detect the expression of mRNA including Nerve Growth Factor,neurotrophin-3,Brain Derived Grouth Factor,glial cell line-derived neurotrophic factors.3,Results3.1,The dynamic change in morphology of BMSCs after induction After addition of BME,cytoplasm of the large,flat BMSCs began to retract and produced a halation,leaving a contracted cell body with a few of process-like extensions that were cross-linked to form networks.On the other hand,the processes of some other BMSCs were drawn back,causing the cells to float until death.At day 3 after addition of RA,further retraction of the cell body of BMSCs was observed. Most of BMSCs showed spindle-shape and a few of BMSCs retracted,giving rise to elongated processes,while some of BMSCs died.Seven d after administration of FSK,bFGF,PDGF,and HRG,the BMSCs became the spindle-shaped with stereovision and some of cells interconnected through their processes,displaying an ordered arrangement with cell endings adjacent to each other.3.2,The morphology of BMSCs,dBMSCs,passaged dBMSCs and SCs.Undifferentiated BMCs,under phase contrast microscopy,display a flattened fibroblast-like morphology;at day 7 post-differentiation,,show a bipolar, spindle-shaped morphology;after passage the dBMSCs return to BMSCs' morphology.(Figure 2).3.3,Flow cytometry and immunofluorescence stainingFlow cytometry and immunohistochemistry indicated that MSCs expressed CD44,CD29 and CD90,but not CD34 and CD45(Figure 3).Our results showed that dBMSCs were positive for GFAP and S100,SCs were positive for GFAP,S100 and p75(Figure 3).3.4,Real-time PCR detectionThe Real-time PCR results showed that after the end of the induced the expression of CD104 mRNA and S100 mRNA reached the level of the SCs,while, after the passage was performed,the expression of CD104 and S100 drop to the level of expression of undifferentiated BMCs;at the whole procedure of induction,the expression of p75 still have a wide gap with the expression of SCs(Figure 5).3.5,PCRThe results of PCR indicate both BMSCs and SCs can express the mRNA of NGF, BDNF,GDNF,NT-3.After induction,dBMSCs loss the capacity to secrete NT-3,the expression of NT-3 was negative(Figure 5). 4,Conclusion Through the method of repeated adherentce,bone marrow stromal cells can be purified.The use of pre-induce coupled with compound inducing factors make BMSCs morphologically similar with Schwann cells.Immunofiuorescence staining show that cells induced express the Schwann cells' surface protein S100,GFAP,but the Real-time PCR results show that the expression of p75 mRNA still have a wide gap with the expression of SCs.The results of PCR indicate dBMSCs loss the capacity to secrete NT-3.Cells induced will restore to bone marrow stromal cells' morphology.5,Statistical analysis:All data used SPSS 13.0 statistical software for statistical analysis,the conventional test for homogeneity of variance,test of normality.Measurement data to the experimental data mean±standard deviation(x±SD) said.Univariate data comparisons between the two groups using t tests,multiple comparisons between data using one-way ANOVA to P＜0.05 for difference has statistical significance.