An investigation into the characteristics and potential therapeutic application of human bone marrow-derived mesenchymal stromal cells in experimental spinal cord injury

Abstract

Transplanted mesenchymal stromal cells (MSC) have been reported to improve functional recovery after spinal cord injury (SCI). The mechanism(s) responsible for this effect are, however, largely unknown. The first studies about MSC transplantation into spinal cord lesions reported differentiation of the donor cells to a neural lineage. This so-called transdifferentiation capacity has aroused great excitement as well as great scepticism. Other studies suggested that the beneficial effects were due to a paracrine mechanism. This thesis was designed to provide insights into the properties of MSC as well as into their fate and function after transplantation into spinal cord lesions. Human MSC (hMSC) were found to constitutively express several neural-related markers, a property which may have led other groups to conclude that hMSC were capable of transdifferentitation to a neural lineage. Interestingly, these markers demonstrated a donor-dependent variability which may have influenced the widely differing opinions about hMSC transdifferentiation. Clinical application of hMSC requires the expansion of isolated cells to relevant numbers without supplementation of animal- or donor-derived serum. Conventional expansion, however, includes the employment of 10% fetal bovine serum (FBS) which might transfer infectous agents or might lead to the immune rejection of transplanted cells. The isolation and expansion of hMSC in the absence of serum supplementation was attempted in the present thesis but could not be accomplished. However, significantly better proliferation could be achieved with supplementation of only 2% FBS and certain growth factors compared to the conventional medium. The multipotent capacity of hMSC was found to be unaffected by the employment of this serum-reduced medium. Since the beneficial effects of hMSC transplantation into SCI were thought to be due to paracrine mechanisms, the expression patterns for several growth factors and cytokines were investigated. A donor-dependent variability of expression could be demonstrated in untreated hMSC as well as in hMSC that had been exposed to lipopolysaccharide (LPS). To investigate the possibility that hMSC could change their specific expression pattern when implanted into debris-laden spinal cord lesions, hMSC were co-cultivated with tissue homogenates from normal and injured rat spinal cords. To determine if this possibility might be tissue specific, homogenates from normal and infarcted rat heart were also applied. None of the above homogenates were found to change the individual donor-dependent patterns of growth factor- or cytokine expression suggesting that indivual hMSC expression patterns remained unaltered following implantation into the lesioned spinal cord. However, such donor-dependent variability might have a profound influence on the degree of subsequent functional recovery and it has to be considered that some donor samples might not have the appropriate growth factor and cytokine expression profiles for optimal tissue repair. A major aspect of the present thesis was the evaluation of an hMSC-based tissue engineering strategy to promote orientated axonal re-growth and functional tissue repair in an experimental animal model of acute SCI. Therefore, cooperation partners transduced hMSC to express green fluorescent protein (GFP) to enable identification of transplanted cells. These cells were seeded into oriented 3D collagen scaffolds and implanted into thoracic spinal cord hemisections. Already 1 week after implantation, animals which received hMSC-seeded scaffolds demonstrated better functional improvement than the control animals, which was even significant after four weeks. This difference, however, was no longer significant at 8 weeks. Immunohistochemistry revealed that only small numbers of transplanted hMSC had survived up to the termination date of 8 weeks after implantation. Nonetheless, animals receiving hMSC-seeded scaffolds demonstrated increased numbers of regenerating axons as well as reduced astrocytic and inflammatory responses. Interestingly, donor hMSC were found to express chondroitin sulphate proteoglycans (CSPG) and although few viable cells could be detected, CSPG deposits could be observed throughout the scaffold. In conclusion, it is likely that hMSC exert their regenerative properties by the expression of growth factors, cytokines and extracellular matrix molecules which are capable of influencing resident and invading cells