Comparison Of Adult Neural Stem Cells From Different Origin For Treatment Of Rat Spinal Cord Injury

BackgroundsSpinal cord injury(SCI) belongs to one of the serious diseases of central nervous system(CNS).The limited regenerative ability of spinal cord results in significant neurological dysfunction and disability,including muscle atrophy,faster contractile properties,and increased fatigability.The traditional therapeutic interventions could only relieve the trauma partially.Actually,there are still no effective measures for repairing the damaged spinal cord in clinic.Recently,it may become possible to repair the injured spinal cord with the discoveries of neural plasticity and the ability of adult CNS regeneration.Nowadays, neural stem cells(NSCs) have been widely considered as one of the most promising tools for repairing SCI.The therapeutic effects of NSCs on the injury CNS are involved in replacing the damaged neurons and glia,excreting neural trophic factors (NTFs);protecting the damaged nerve cells and forming the cellular bridge to guide the nerve cells regeneration,etc.Although NSCs have been widely used in the repairment of CNS injury,the clinic application of NSCs remains to be limited because of its resources and cell number scale.Now,we could isolated NSCs from adult tissues including neural and non-neural ones,which are named as adult stem cells.The neural tissue-derived adult stem cells can be gotten from subventricular zone,the dentate gyrus of hippocampus and spinal cord,and so on.The non-neural tissue-derived adult stem cells,however, can be harvested from bone marrow(BM),adipose tissue(AD) and skin,etc.The adult NSCs from different sources of tissues present the similar morphologies and cellular phenotypes,and differentiate into neurons and glia in vitro and in vivo.NSCs from adult tissue can also secrete many kinds of cytokines and growth factors which can promote axonal regeneration and neural survival.Partially functional recovery has been reported following different types of NSCs transplantation into the injured spinal cord in established animal models.However,do the adult NSCs from different sources of tissues possess the same therapeutic potentials?In this study,we intended to isolated NSCs from subventricular zone (SVZ-NSCs,as the positive control),bone marrow(BM-NSCs) and adipose tissue (AD-NSCs) from the same rat and investgated their neural differentiation ability and therapeutic efficacies in vitro and in vivo.Neural differentiation ability was detected by immunocytochemical and flow cytometric analysis.The ability of secreting nerve growth factor(NGF) and brain-derived neurotrophic factor(BDNF) was examined by ELISA.Spinal cord injury(SCI) animal models were established to investigate the therapeutic effect of SVZ-NSCs,BM-NSCs and AD-NSCs on reparing them further. The full text consists of the two sections as following.Part One:Isolation and comparison the biological characteristics of the neural stem cells from different origins in vitroPurposes:To isolate and compare the NSCs from rat suventricular zone (SVZ-NSCs),adipose tissue(AD-NSCs) and bone marrow(BM-NSCs) in the same SD rat in terms of their morphologies,phenotypes,and the potential of neural differentiation and the ability to secrete neurotrophins(NTs).Methods:SVZ-NSCs,AD-NSCs and BM-NSCs were harvested from suventricular zone,adipose tissue and bone marrow respectively,from the same SD rat.The abilities of proliferation and differentiation in the three groups were compared.Immunocytochemistry and Western blot were performed to detect the expression of surface markers of neurons and glia,including Nestin,β-tubulinⅢ, galactocerebroside C(GalC) and glial fibrillary acidic protein(GFAP).NTFs was detected by ELISA.Results:There was no obvious difference in morphology between SVZ-NSCs and BM-NSCs or AD-NSCs.In the stage of neurospheres,at the 15 day after inducement,the proliferation ability of SVZ-NSCs(61.00±6.24) showed that stronger than that of BM-NSCs(22.60±7.19) or AN-NSCs(38.00±6.16).The percentage of Nestin-positive cells in the SVZ-NSCs(84.41±6.18) was significantly higher than that in the BM-NSCs(69.18±7.33) or AD-NSCs(74.24±5.86),which in the AD-NSCs was significantly higher than the one in the BM-NSCs.After neurospheres differentiation, there was no obvious difference in expression ofβ-tubulinⅢ,GFAP,and GalC among the groups.Western blot was performed to demonstrate the results in the protein levels.By means of ELISA,BDNF and NGF in the SVZ-NSCs(103.85±8.28 and 117.64±14.85 ) were significantly higher than that in BM-NSCs(22.82±4.43 and 44.96±8.81 ) and AD-NSCs(45.78±8.82 and 62.38±13.99).Conclusions:SVZ-NSCs,AD-NSCs and BM-NSCs showed the similar morphological and phenotypic characteristics.However,SVZ-NSCs as the positive control,presented the more powerful in proliferation,differentiation and secretion of NTFs than AD-NSCs and BM-NSCs did.The results in this experiment indicated that SVZ-NSCs were with the much more of potentials repairing SCI than BM-NSCs and AD-NSCs,beside of other problems such as immuno-repulsion,ethic,etc.The AD-NSCs appeared to achieve better potentiality compared with the BM-NSCs.Part Two:Comparison the therapeutic effects of neural stem cells from different origins after being grafted into the injured rat spinal cordPurposes:To investigate the therapeutic effects of SVZ-NSCs,BM-NSCs or AD-NSCs in the contusive injured spinal cord of adult rats further,basing on the experiment in vivo.Methods:One hundred of adult SD rats which were invided 5 of groups including SVZ-NSCs,AD- NSCs,BM- NSCs,Saline control and Sham control,were used in this study.SCI animal models were established by dropping a 10g impact rod from a 25mm height onto the exposed dorsal surface of the spinal cord.One week following injury,6×10~5 NSCs isolated from subventricular zone,bone marrow and adipose tissue,were grafted into the injury sites and we labeled cells with BrdU before transplantation.Locomotor function of the hindlimbs was evaluated according to the Basso,Beattie,and Bresnahan(BBB) locomotor rating scale. Immunohistochemistry was used to examined the BrdU/β-tubulinⅢ+,BrdU/GFAP+ and BrdU/GalC+ to show the survival and differentiation of the grafted cells.Every eighth of sections from the spinal cords of rats killed at 8 weeks after transplantation were processed for HE staining.The area of cavitation of each section was traced using Image J software,and then calculated the percentage of cavitation in SCI tissue.Results:Acooding to the BBB scale,all of the treatment groups with three different origins of NSCs showed an early increment over Saline controls from the 2th to the 8th weeks,and became the apparent improvement at the 9th week after stem cells graft.SVZ-NSs-treated group appeared better hindlimb movement ability comparing with BM-NSs-treated group and AD-NSs-treated group,although there were no statistical differences among them.Most of the surviving cells were positively located around the lesion site.Double immunoreactivity for positive BrdU andβ-tubulin,GFAP or GalC showed that the grafted cells mainly differentiated into neurons and glials.At the 8th week after surgery,only few of BrdU/β-tubulin+ and BrdU/GFAP+ cells were examined.In general,there were no differences in the percentage of BrdU/β-tubulin+,BrdU/ GalC+ and BrdU/GFAP+ cells among the NSs-treated groups at end of the 8th week following cells graft.Conclusions:SVZ-NSCs,BM-NSCs or AD-NSCs could improve the locomotor functional recovery of SCI partially.However,the therapeutic effect of SVZ-NSCs was more significant than those of AD-NSCs and BM-NSCs on the injured spinal cord.AD-NSs-treated group appeared better hindlimb movement comparing with BM-NSs-treated group,although there was no statistical difference between them.It was concluded that SVZ-NSCs presented the better therapeutic effects in intreatment of SCI than AD-NSCs and BM-NSCs did.Cellular replacement might not be the main responsible for the functional recovery following rat SCI because the rat of cavitation in SVZ-NSCs showed lower than the one in both of AD-NSCs and BM-NSCs.Summary:In conclusion,SVZ-NSCs,AD-NSCs and BM-NSCs possessed the similar morphological and phenotypic characteristics.All of them could improve locomotor functional recovery partially during the treatment of SCI.SVZ-NSCs presented better therapeutic effects than AD-NSCs and BM-NSCs did.However,the application of brain-derived NSCs will not be so optimistic because of the limitation of ethics.Therefore,AD-NSCs and BM-NSCs offered a more clinically feasible source than brain-derived stem cells.Because adipose tissue are abundant, reproducible,and easily accessed,AD-NSCs may be considered as the better choice.