Comparison Of Mesenchymal Stem Cells From Human Bone Marrow And Adipose Tissue For The Treatment Of Spinal Cord Injury

Spinal cord injury (SCI) is serious trauma of the central nervous system(CNS) primarily affects young adults and consequent disabilities usually persist throughout the rest of their lives and SCI has been described as the major cause of morbidity and mortality in human. Acute SCI affects140,000individuals annually in the world. In bejing, China, SCI occurs with a incidence of60cases per million people in2002. The patients survived from the injury present with a range of functional impairmentS,their quality of life seriously deteriorated. These brought heavy economic burden to the society and families. It is still a severe situation for the emergency treatment of SCI in our country because the change of industrial and agricultural production style and the relative lagging of medical rescue network development. SCI seriously influence people’s life and health with high disability and cause huge economic loss for individual, family and society, developing new treatment strategy for the SCI has become a research hotspot.The pathophysiology of SCI is extremely complicated. The spinal cord is subject to mechanical compression, tissue tears, and distortions by the vertebrae displaced after various traumatic events. These primary insults are accompanied by a disruption of local circulation, and ensuing ischemia, edema, and petechial hemorrhage contribute to further damage to neural cells in the spinal cord. Impaired ionic homeostasis in the injured spinal cord tissue, especially an increase in intra cellularcalcium concentration, results in abnormalities in neural transmission through spinal axons. Degeneration of axons in the white matter also occurs primarily due to changes in concentration of calcium and sodium ions. Another class of growth inhibitory elements is associated with glial scars. Reactive astrocytes at the injury sites produce one of the extracellular matrix proteins, chondroitin sulfate proteon such glycans (CSPGs), which also contribute to a nonpermissive environment for axonal regeneration. Furthermore, neutrophil and macrophage in the the peripheral circulatory system leaks into the CNS immediately after blood-brain barrier disruption or vascular damage, accompany with resident microglial further contribute to inhibit the axonal regeneration. In order to treat SCI, some therapeutic strategies have been developed, including drug administration, neurothrophic factors, gene therapy and cell transplantation, etc.Stem cell transplantation is proved to be a promising strategy to treat SCI as research continues. Several kinds of stem cells have been grafted into different injury animal model and even been used in phase Ⅰ and Ⅱ clinical trial. Theoretically, embryonic stem cells are presumably the best candidate for treating SCI because their pluripotency, although embryonic stem cells can proliferate and differentiate into virtually any cell types, danger of teratoma formation and ethical concerns limit their use; Neural stem cells derived from CNS decrease in neurogenesis and undergo replicative senescence over time and also be limited by ethical issues; induced pluripotent stem cells(iPS) are a promising cell source for SCI treatment, but the endeavor to obtain highly purified and large amount of iPS is limited by current technology; Schwann cells and Olfactory ensheathing cells are associated with well therapeutic effects, but it is very difficult to obtain sufficient cells. Adult mesenchymal stem cells(MSCs) can be detected in various adult tissues and may contribute to tissue repair and regeneration. Compared witt stem cells from the embryo or fetus, adult MSCs ale easy to obtain and handle, and can be used as the carrier of genes. Moreover, MSCs are suggested to be immunoprivileged, such that allogeneic MSCs can be transplanted when necessary. Therefore, MSCs may become the most appropriate cells source for transplantation.MSCs has been assessed in experimental models of CNS injury and disease for two deeates. Although some encouraging results have been reported, it is difficult to get further functional recvovery by using current approaches. Why? Except the extremely complicated pathophysiology of SCI, single therapy is difficult to affect simultaneously multiple injury mechanisms. Basing on previous studies, we throught the following key pionts hindered the cell transplantation to exibit their therapeutic effects. First, the exact mechanisms underlying MSCs repair SCI is far from clear. Cell replacement? Neurothrophic factors? Anti-apoptosis? or anti-inflammation. Sencond, the issues of cell source. MSCs were originally isolated from human bone marrow(bone marrow derived messenchymal stem cells, BMSC), and most of studies associated with MSCs transplantation for CNS injury treatment were conducted with BMSC. However, preparing a sufficient number of BMSC is relatively difficult in clinical settings. It make the patients suffered from dangerous, such as infection, pain. Furthermore, the cells show senescence after passaged7-8times, the onset of senescence was associated with a significant reduction in ability of secretion. However, it is unclear, at present, whether MSCs isolated from different tissue sources have similar therapeutic potential and which source or isolation protocol is optimal for therapeutic purposes. Third, the transdifferentiation and corelated mechanisms of MSCs are still unclear now. Although MSCs are derived from mesoderm, it has been reported that MSCs can not only differentiate into cells in mesoderm, but also can differentiate into cells express neural and glial markers. It is still controversial whether it is necessary to induce the MSCs before transplantation.Although various studies have been published demonstrating the thrapeutic effects of BMSC in SCI repair, the harvest of bone marrow is a highly invasive procedure and the number, differentiation potential, and maximal life span of MSCs from bone marrow decline with increasing age. Therefore, alternative sources from which to isolate MSCs are subjected to intensive investigation. In the last decade, MSCs with characteristics similar to BMSC have been obtained from adipose tissue, umbilical cord blood, placenta and dental pulp. Among these MSCs, adipose tissue-derived mesenchymal stem cells (ADSC) in particular are considered to be an attractive alternative to MSCs from bone marrow because of their abundant availability and excellent expansion and proliferation capacities. ADSC have now been sufficiently characterized, and their differentiation potential in vitro and in vivo has been described extensively, but there is still a lack of comprehensive data comparing the regenerative potential of ADSC with that of BMSC in an SCI animal model. Therefore, the aim of this study was to compare the effects of ADSC and BMSC in the treatment of SCI in rats.Two studies from different countries have been performed to compare the different therapeutic effect of ADSC and BMSC in the infarcted heart model and ischemic stroke model, all of these studies were conducted with MSCs derived from murine. However, the activity between human MSCs and rat MSCs may be quite different, translation of the results across these different types of cells is not possible. To our best knowledge, there is no report of comparing the regenerative potential of human ADSC (hADSC) with those of human BMSC (hBMSC) in an SCI animal model. Recently, more and more studies focus on transplantation of human derived MSCs into animal models of SCI. Such as human adult dental pulp stem cells, unrestricted somatic stem cells, human umbilical mesenchymal stem cells, human-induced pluripotent stem-cell, human amniotic epithelial cell even human neural stem cell,etc. To facilitate comparing our data with these papers, we chose human BMSC and human ADSC. Furthermore, based on clinic application in the future, human MSCs is superior to rat MSCs.Part Ⅰ:Isolatiton and culture of human bone marrow mesenchymal stem cells and human adipose mesenchymal stem cells and comparisons of the biological characteristics of these two types of MSCs.Obiectives:Isolating the human bone marrow mesenchymal stem cells(hBMSC) and human adipose mesenchymal stem cells(hADSC), comparing the characteristics of the two types of cells.Methods:This study was approved by the institutional review board of Zhujiang hospital (GuangZhou,China). Five patients undergoing total hip arthroplasty in our department (aged45-60years) were enrolled in the study. The human tissues that were used for isolation of MSCs were the leftover materials from the surgical protocols. Bone marrow and subcutaneous adipose tissue samples were obtained from the same donor after informed consent was obtained. To minimize the potential variation resulting from experimental artifacts, such as serum concentration and cell passaging method, all MSCs populations were cultured under identical conditions in vitro. The mononuclear cells from the bone marrow were separated by centrifugation in a Ficoll-Hypaque gradient (density=1.077g/cm3; Sigma), suspended in DMEM containing10%fetal bovine serum and seeded at a concentration of1×106cells/cm2. To isolate hADSCs, adipose tissues were washed extensively with PBS. On removal of debris, tissues were then enzymatically dissociated with0.1%collagenase type I under gentle agitation for60min at37℃. The digested tissues were centrifuged at1500×g for10min to separate the floating adipocytes from the stromal vascular fraction. The pellet of stromal cells was resuspended in DMEM with10%fetal bovine serum. Cells were maintained at37℃in5%CO2. Cell between passages3and5were used in this study. These MSCs were characterized using flow cytometry, cholecystokinin (CCK)-8reagent, immunocytochemistry, real-time polymerase chain reaction and enzyme-linked immunosorbent assay. All data in this study are presented as means±standard deviation (SD)(x±SD). Data from more than two groups were analyzed using one-way analysis of variance, followed by Bonferroni post-hoc testing. The differences between two groups were analyzed using Student’s t-tests. Differences were deemed statistically significant at P<0.05.Results:hADSCs and hBMSCs were successfully obtained. The adherent cultured hADSCs and hBMSCs exhibited a fibroblast-like, spindle-shaped morphology when observed under a light microscope. No morphological differences were observed between the two cell types. Flow cytometric analysis revealed that both populations were positive for mesenchymal markers and were negative for endothelial and hematopoietic lineage markers. Immunocytochemistry analyses confirmed that the majority of hADSCs and hBMSCs expressed vimentin, laminin and fibronectin, known markers for MSCs, and markers for stem cells nestin; The percentages of hADSCs and hBMSCs expressing Ki67were57.22%±5.76%and46.02%±3.18%, respectively(P<0.05). hBMSCs appear to senesce by passage7-8, much earlier than hADSCs (by passage30). CCK-8test showed that, at days1and2, the results of the optical density analysis indicated that there were no significant differences between the two types of MSCs. From day3, however, the hADSCs showed a significantly higher optical density than that of hBMSCs. At the end of the6-day growth study, the optical density of hADSCs was approximately2-fold higher than that of hBMSCs. RT-PCR results showed that, compared with hBMSCs, hADSCs expressed significantly higher mRNA levels of HGF, VEGF and BDNF(p<0.05). We performed ELISA to detect the protein expression of HGF, VEGF and BDNF in hADSCs and hBMSCs. The concentration of BDNF and VEGF was significantly higher in hADSCs than in hBMSCs(p<0.05).Conclusions:These results demonstrate the limited passage numbers and low secretes ability of cultured hBMSCs available for use in research and suggest that adipose tissue may be preferable for tissue banking purposes.Part Ⅱ:Functional recovery in acute traumatic spinal cord injury after transplantation of human bone marrow mesenchymal stem cells and human adipose mesenchymal stem cellsObjective:The aim of the present study was to investigated whether human adipose tissue-derived mesenchymal stem cells (hADSCs) transplanted into a rat model of SCI would lead to similar or improved neurologic effects compared with human bone marrow-derived mesenchyrnal stem cells (hBMSCs).Methods:After anesthesia by intraperitoneal administration of3.6%chloral hydrate, a total of108female Sprague-Dawley rats underwent a bilateral dorsal laminectomy at the ninth thoracic vertebral level (T9), the dura was opened and the bilateral dorsal corticospinal tract (CST), rubrospinal tract and dorsal columns were cut with a pair of micro scissors to the depth of the central canal, were averagely divided into three experimental conditions:(i) SCI followed by injection of PBS without donor cells (control group),(ii) SCI followed by transplant of hBMSCs (hBMSC group) and (iii) SCI followed by transplant of hADSCs (hADSC group). After injury, a total of2×105cells, divided into two dosages, were transplanted into the injured spinal cord as previously described. Each injection was made at2mm rostral and2mm caudal from the lesion epicenter, respectively, at a depth of1.2mm. At each site,2mL of a cell suspension containing105cells or vehicle was injected. All animals received cyclosporine intraperitoneally at a dosage of10mg/kg daily beginning1day before transplant until the animals were killed. Locomotor function, cell survival and differentiation, spinal cord tissue morphology and brain-derived neurotrophic factor (BDNF) expression were compared between groups. Statistical analysis was performed using SPSS13.0(Chicago, IL, USA) for Windows. All data in this study are presented as means±standard deviation (SD). Data from more than two groups were analyzed using one-way analysis of variance, followed by Bonferroni post-hoc testing or two-way ANOVA. The differences between two groups were analyzed using Student’s t-tests. Differences were deemed statistically significant at P<0.05.Results:(1) hBMSCs and hADSCs survive and engraft within the injured spinal cord. Grafted human stem cells could be clearly detected by immunohistochemical staining using a specific anti-human nuclei antibody (hNU, MAB1281). We found that1week post-transplant, there was extensive human cell survival (hBMSCs,33.87%±4.45%; hADSCs,35.59%±5.38%; P>0.05; N=3). The majority of surviving cells were observed at the lesion epicenter, which shows very little immunoreactivity for the pan-axonal marker neurofilament. This suggests that the grafted cells migrated from the injection site toward the injury sites. Fewer cells were found to be scattered in distant areas rostral and caudal to the lesion. The distribution and migration patterns of the two types of MSCs did not show apparent differences. The percentage of surviving cells versus total transplanted cells28days post-injection was0.87%±0.25%and0.95%±0.33%for the hBMSC and hADSC groups, respectively (P>0.05, n=3for each group).(2) Functional recovery. After SCI, animals displayed complete hind limb paralysis. The BBB score gradually increased to scores between4and5at1week post-injury in all groups. hADSC transplant immediately after SCI led to significant recovery of locomotor functions, which began2weeks after transplant and was measurable up to the end of the follow-up, compared with PBS-treated controls. Four weeks after injury, the mean score of rats with hADSC grafts was significantly higher than that for the other groups. Four weeks post-transplant, the BBB scores were12.5±0.51,11.2±0.6, and9.2±0.3for the hADSCs, hBMSCs and control groups, respectively.(3) To examine whether transplanted hBMSC and hADSC affect the axonal growth, we performed immunohistochemical analyses with anti-NF200antibodies4weeks after SCI. Compared with the PBS control group, the hBMSC group exhibited a significant increase in the NF200+fiber area at the lesion epicenter, and the hADSC group showed an even greater enhancement(P<0.05). The same results were also obtained in5-HT+fibers. In all groups, BDA-labeled fibers were detected in the rostral stump of the spinal cord, but there were no BDA-labeled fibers in the caudal stump in any rats.(4) BDNF expression. By3days post-injury, Western blot analysis revealed that the levels of BDNF were significantly increased in MSCs-treated groups compared with the level in the PBS control group. Notably, the hADSC group showed a higher BDNF concentration than the hBMSC group, we further investigated the time course of the expression of BDNF in the injured spinal cord by using quantitative ELISA on tissue extracts. Indeed, the concentration of BDNF in the hADSC group was strikingly increased at day3following SCI compared with the control and hBMSC groups, and a significant increase was sustained until day7post-SCI. There was no significant difference of the concentration of BDNF among three groups at28days post-transplant(5) Inflammation and apoptosis. Initially, we observed intergroup differences among the three treatment groups in the inflammatory/apoptotic cell counts including ED-1 and caspase-3(P<0.05, ANOVA-Bonferroni), and further analyzed the values by intergroup comparison. Quantification of ED1-immunoreactive area fractions showed that the areas of ED1-positive cells were significantly reduced by treatment with hBMSCs and hADSCs1week post-transplant (60.88%±6.25%,63.45%±5.33%and85.65%±9.35%for the hADSC, hBMSC and control groups). An analysis of active caspase-3+apoptotic cell numbers showed that hADSC group and hBMSC group had lower counts than the control group (all P<0.05), and the hADSC group had lowest counts than hBMSC group (P<0.05).Conclusions:The present results suggest that hADSC would be more appropriate for transplant to treat SCI than hBMSC.