An Experimental Study Of Skin-derived Precursors (SKPs)Differentiation In Vitro And Delaying Denervated Muscle Atrophy

Part1Adult rat skin-derived precursors (SKPs) differentiated into neuronal-like phenotype and express a variety neurotrophins in vitroObjective:The purpose of this study is to (1) isolate, expand, and characterize of skin-derived precursor from skin of adult rat,(2) differentiate SKPs into neuronal-like phenotype cells in vitro,(3) compare the expression of the neurotrophins in vitro in SKPs and neuronal-like phenotype cells derived from SKPs.Methods:The SKPs isolated from back skin of the adult rat and grew in suspension as spheres in the presence of the mitogens fibroblast growth factor2and epidermal growth factor. After three passages, the SKPs were transferred to neuronal induction media including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3(NT-3) for2-4weeks. Reverse transcription-polymerase chain reaction (RT-PCR) and immunostain were used to determine the expression of BDNF, NGF, GDNF (glial-derived neurotrophic fator) and CNTF (ciliary neurotrophic factor) in SKPs and neuronal-like phenotype cells derived from SKPs.Results:SKPs can be isolated by a technique devised to generate neutral stem cells form the bain. The adult spheres were immunostained nestin-a marker of neural stem cell positive. SKPs expressed neuronal marker nestin and β-tubulinⅢ. SKPs also produced fibronectin, a protein that produced by bone marrow mesenchymal stem cells. They did not express neuronal crest stem cell marker p75neurotrophin receptor (p75NTR) and polysialic acid NCAM (PSA-NCAM). SKPs co-express nestin and fibronection. After three passages, more than60%of cells produce nestin. After cultured adult epidermis, dermis and sciatic nerve, only dermis generated spheres of proliferating cells, and also nestin and fibronectin-prositive cells are present within adult dermis before culture. SKPs did not express vimentin, cytokeratin, they are distinct from mesenchymal stem cells. Rat SKPs could differentiate into cells with the phenotype peripheral neurons, a cell type never seen in skin. The SKPs and neuronal-like phenotype cells derived from SKPs both expressed neurotrophins (BDNF, NGF, GDNF, CNTF) in vitro.Conclusions:SKPs are an endogenous precursor cells, nestin-positive, fibronectin-positive stem cells (SKPs) can be generated from adult skin, that these precursors derive from the dermis, and that they are distinct from mesenchymal stem cells. Under proper conditions, SKPs can be trans-differentiated into neurons. The SKPs and neuronal-like phenotype derived from SKPs have an expression profile of neurotrophins. SKPs and represent a novel adult stem cell that easily generates neurons.Part2Study of survival of skin-derived precursors and neuronal-like phenotype cells derived from SKPs in peripheral nerveObjective:To explore (1) the survival rate of SKPs after transplanting in different micro circumstance in different time point;(2) the survival of SKPs from GFP transgenic rats and neuronal-like phenotype derived from SKPs after transplanting in injured peripheral nerve; and (3) the expression of the neurotrophins in vivo in SKPs transplanted group and neuronal-like phenotype cells derived from SKPs transplanted group.Methods:Dived by the micro environment cells were transplanted, the Lewis rats were distribute to tibial nerve uninjured group and injured group at random. We established the animal model of Lewis rat with transversely section of the tibial nerve in lcm above gastrocnemius muscle, and0.5cm section was removed before capped two stumps by rubber cups.In tibial nerve injured group, there were4sub-groups (Group1:SKPs inject immediately after nerve section; Group2:median inject1week after nerve section; Group3:SKPs inject1week after nerve section; Group4: neuronal-like phenotype cells derived from SKPs inject1week after nerve section). At4,8and12weeks after cell transplantation, the distal stump of the tibial nerves were harvest. Then immunostain was used to determine the expression of BDNF, NGF, GDNF and CNTF in SKPs and neuronal-like phenotype cells derived from SKPs in vivo.Results:Survival of SKPs was particularly poor in cells transplanted into intact nerves versus transected nerves. To acute nerve injury, the chronically denervated nerve appeared to be poorly supportive for SKP survival. After GFP-SKPs and neuronal-like phenotype cells derived from SKPs were transplanted in tibial nerve distal stump, two kinds of cells both survived. While survival of neuronal-like phenotype cells derived from SKPs was greater than that of SKPs. At each time point after surgery, the percent survival differed from each other. The survival rate decrease from4weeks to12weeks after cell transplantation. At the twelfth week, the SKPs and neuronal-like phenotype cells derived from SKPs still survive and both expressed neurotrophins (BDNF, NGF, GDNF, CNTF) in vivo.Conclusions:SKPs demonstrated variable survival within peripheral nerve, depending on the nerve injury environment. SKPs and neuronal-like phenotype cells derived from SKPs can survival for at least12weeks after transplantation. The neuronal-like phenotype cells pre-differentiated of SKPs improve their survival in nerve lesions site. SKPs and neuronal-like phenotype cells derived from SKPs can express neurotrophins in vivo. The result maybe valuable for further research about SKPs and neuronal-like phenotype cells derived from SKPs therapeutic purpose in improving muscle reinnervation or delaying denervated muscle atrophy.Part3Study of skin-derived precursors and neuronal-like phenotype cells derived from SKPs in delaying muscular atrophyObjective:To evaluate the effect of reversing post-denervation muscle atrophy after SKPs and neuronal-like phenotype derived from SKPs transplanting in injured peripheral nerve.Methods:Each Lewis rats was denervated by cutting the tibial nerve lcm before the nerve join the muscle, then0.5cm section was removed before capped two stumps by rubber cups. After1week of muscle denervation, animals were divided into three groups at random (media injection, SKPs injection, neuronal-like phenotype derived from SKPs injection). The animals were euthanized at4、8and12weeks after cell transplantation the walking analysis was performed, after that nerve tissue and gastrocnemius muscle sample were harvested. The muscle strength, gastrocnemius muscle wet weight, Mallory staining, muscle fiber area, axon number in nerve fiber, percent of neural tissue, G ratio were measured.Results:SKPs and neuronal-like phenotype cells both could improve the function of denervated gastronemius muscles when cells transplanted to the distal stump of the tibial nerve. SKPs and the neuronal-like phenotype cells pre-differentiated treatment delayed denervated muscular atrophy when compared to media control. Neuronal-like cells pre-differentiated treated nerves demonstrated a histological profile better than that had been transplanted by naive SKPs. SKPs and neuronal-like phenotype cells improved muscle reinnervation compared to media control.Conclusions:Skin derived precursors and neuronal-like phenotype cells could delay denervated muscular atrophy in a rat model. Although the detailed mechanism by which SKPs and neuronal-like phenotype cells delaying denervated muscular atrophy is being investigated in our lab, our results suggest that skin derived precursors differentiated into neuronal-like cells in vitro before transplantation represent powerful therapeutic potential in delaying denervated muscular atrophy.