We report here the remarkable neuroregenerative effect of rBMSCs cultured in simulated microgravity on functional recovery after SCI; to our knowledge, this is the first study in which rBMSCs cultured in simulated microgravity were transplanted into an SCI model.
Previously, BMSCs have been reported to exhibit distinct effects on the recovery of injured spinal cords and to improve locomotion in SCI rats; moreover, cavity formation in the spinal cord was reported to be reduced when BMSCs were infused into the cerebrospinal fluid with acute SCI [24, 25]. To examine possible future clinical applications of cells grown in simulated microgravity, we used a spinal-contusion model involving a weight-dropping method. In this study, grafted rBMSCs promoted functional improvement, which was accompanied by the suppression of cavity formation 3 weeks after SCI. As previously reported , functional recovery of the spinal-contusion models by using a weight-dropping method was less prominent and reached a plateau during the third week after SCI. In this study, we transplanted cells immediately after a surgical procedure to examine the therapeutic benefits of the transplanted cells concerning the multiple pathogenic signals that function synergistically during the early phase after SCI. The present study confirmed possibilities that rBMSCs cultured under simulated microgravity have the pluripotency and the advantage as grafting cells for the early phase of SCI repair.
Previous studies have shown that microgravity suppresses the differentiation of human osteoblast cells , human hematopoietic progenitor cells , and rat myoblasts . Simulated microgravity also allows novel culture methods for mouse ES cells that do not require leukemia inhibitory factor or animal-derived supplements . The 3D-clinostat is a device that allows generation of a multidirectional g force, resulting in an environment with an average of 10-3
g[14, 16]. Our previous study showed that cells cultured in a 3D-clinostat exhibit suppressed cell differentiation [16, 17, 26] and induction of growth inhibition through reduction of mitochondrial activity ; moreover, simulated microgravity enhanced the chemosensitivity of malignant glioma cells .
In the present study, simulated microgravity was considered to induce growth inhibition and to maintain the undifferentiated state of rBMSCs, because the rBMSCs cultured under simulated microgravity were smaller and had a dome-like shape, and the expression of Oct-4 mRNA was observed only in group CL cells.
A recent study showed that when undifferentiated ES cells are transplanted, they act in a neuroprotective manner and exert antinociceptive and therapeutic effects after excitotoxic SCI . Another study showed that transplantation of murine ES cells that had been undifferentiated into GABAergic neurons significantly induced recovery of sensorimotor function after traumatic brain injury, whereas animals transplanted with astrocytes did not show any recovery . Several studies have shown the superiority of using undifferentiated stem cells for transplantation after SCI for facilitating functional recovery [29–31]; the results of the present study were consistent with the findings of other transplantation studies, because simulated microgravity induced an undifferentiated state in the grafted rBMSCs.
By using the specific SDF-1 receptor (CXCR4), we found significant expression of the chemokine stromal-cell-derived factor-1 (SDF-1) in rBMSCs cultured under simulated microgravity. The interaction of SDF-1 with CXCR4 mediates the homing of hematopoietic stem cells to the bone marrow ; SDF-1 is also known to induce migration of neural cells [30, 33]. In this study, the number of transplanted rBMSCs that had migrated into the SCI lesion was markedly greater in group CL and the cavity formation after SCI was markedly reduced in group CL compared with group 1G. Our results suggest that simulated microgravity mediated the extended expression of CXCR4 in the rBMSCs, and that the interaction of SDF-1 with CXCR4 then facilitated the trafficking of rBMSCs to the SCI site.
Several studies have shown that BMSCs differentiate into neural cells, including astrocytes and neurons [6, 34, 35]; conversely, other studies have indicated that transplanted BMSCs do not differentiate into neural cells in the spinal cord [12, 20]. Our previous study, by using a mouse model of cerebral contusion, showed that the efficacy of grafting mouse BMSCs was attributed not only to the cells differentiating into neuronal cells but also to the factors released by grafted cells that suppressed the formation of a glial scar and that enhanced the elongation of axons . It has been suggested that transplanted BMSCs may not survive long enough to be integrated into the spinal cord tissue [12, 36] and that BMSCs, despite their limited survival time, enhance tissue matrix formation and axonal outgrowth in SCI by releasing diffusible neuroprotective factors [6, 25, 30, 34, 37]. This probably contributed to the distinct improvement of locomotor behavior as well as the reduction of the cavity formation noted in our study. Although, in our study, the transplanted rBMSCs expressed the differentiation marker GFAP, it is unlikely that the efficacy of rBMSCs for improving the injured spinal cord relates to these cells forming spinal cord tissue, because of the small number of rBMSCs used for transplantation.
It has been shown that stem cell transplantation can improve neurologic function by several mechanisms, some of which are neuroprotective effects on host neurons from trophic factors secreted by transplanted cells, and the reestablishment of functional neural networks through the integration of transplanted cells [38, 39]. All things considered, the beneficial effect may rather relate to the release of neuroprotective factors by the BMSCs [40–42].
Neurotrophins are a family of proteins that are best characterized by their modulation of survival, differentiation, and apoptosis of cells in the nervous system, and exogenously applied trophic factors, including BDNF, IGF-1, NT-3, GDNF, and HGF, have been shown to be effective for SCI. Although the trophic factors derived from BMSCs promote neurite extension and survival in vitro, their roles in the functional recovery of SCI are still largely unknown. In this study, we investigated the expression of NGF and BDNF mRNAs in graft cells, but found no difference between groups 1G and CL with regard to the expression of these mRNAs. The mRNA expressions of NGF and BDNF are only by a semiquantitative method. Therefore, to investigate the trophic effect of the grafted rBMSCs, we analyzed apoptosis markers in the SCI lesions, because SCI induces a series of endogenous biochemical changes that lead to secondary degeneration, including apoptosis. Mitochondrial apoptosis mediated by p53 is likely to be an important mechanism of cell death in SCI. Expression of p53 was observed in neurons, oligodendrocytes, and astrocytes after SCI, and upregulation of phospho-p53 and Bax, and downregulation of Bcl2, were detected after SCI . In this study, the fact that the expression of the apoptosis-promoting factor Bax significantly decreased and that of the apoptosis-inhibitory factor survivin significantly increased in group 1G and CL rats compared with group Control, demonstrated the trophic, antiapoptotic effect of the grafted rBMSCs. Apoptosis and free-radical damage are the prominent processes involved in secondary degeneration after SCI [43, 44]. Our results suggest that the grafted BMSCs immediately after injury prevented the secondary degeneration and enhanced the proliferation of the axons to a greater extent in group CL rather than in group 1G.