The cell culture expansion of bone marrow stromal cells from humans with spinal cord injury: implications for future cell transplantation therapy

Abstract

Study design: Previous studies have shown that transplantation of bone marrow stromal cells (MSCs) in animal models of spinal cord injury (SCI) encourages functional recovery. Here, we have examined the growth in cell culture of MSCs isolated from individuals with SCI, compared with non-SCI donors. Setting: Centre for Spinal Studies, Midland Centre for Spinal Injuries, RJAH Orthopaedic Hospital, Oswestry, UK. Methods: Bone marrow was harvested from the iliac crest of donors with long-term SCI (43 months, n ¼ 9) or from non-SCI donors (n ¼ 7). Mononuclear cells were plated out into tissue culture flasks and the adherent MSC population subsequently expanded in monolayer culture. MSC were passaged by trypsinization at 70% confluence and routinely seeded into new flasks at a density of 5 Â 10 3 cells per cm 2 . Expanded cell cultures were phenotypically characterized by CD-immunoprofiling and by their differentiation potential along chondrocyte, osteoblast and adipocyte lineages. The influence of cellseeding density on the rate of cell culture expansion and degree of cell senescence was examined in separate experiments. Results: In SCI, but not in non-SCI donors the number of adherent cells harvested at passage I was age-related. The proliferation rate (culture doubling times) between passages I and II was significantly greater in cultures from SCI donors with cervical lesions than in those with thoracic lesions. There was no significant difference, however, in either the overall cell harvests at passages I or II or in the culture doubling times between SCI and non-SCI donors. At passage II, more than 95% of cells were CD34Àve, CD45Àve and CD105 þ ve, which is characteristic of human MSC cultures. Furthermore, passage II cells differentiated along all three mesenchymal lineages tested. Seeding passage I-III cells at cell densities lower than 5 Â 10 3 cells per cm 2 significantly reduced culture doubling times and significantly increased overall cell harvests while having no effect on cell senescence. Conclusion: MSCs from individuals with SCI can be successfully isolated and expanded in culture; this is encouraging for the future development of MSC transplantation therapies to treat SCI. Age, level of spinal injury and cell-seeding density were all found to relate to the growth kinetics of MSC cultures in vitro, albeit in a small sample group. Therefore, these factors should be considered if either the overall number or the timing of MSC transplantations post-injury is found to relate to functional recovery.

Bridging defects in chronic spinal cord injury using peripheral nerve grafts combined with a chitosan-laminin scaffold and enhancing regeneration through them by co-transplantation with bone-marrow-derived mesenchymal stem cells: Case series of 14 patients

“…93 A fourth possible explanation is that mesenchymal stem cells obtained from spinal cord injured patients not from healthy volunteers might be associated with inferior results. 58 A fifth possible explanation is spontaneous or treatment-induced anatomical neural plasticity, the adaptive reorganization of the neural pathways occurring after injury, and acting to restore some of the lost function. 94 Complications in this study include haematoma and seroma formation.…”

“…A fourth unresolved issue is whether to obtain stem cells from patients with spinal cord injury or from healthy volunteers. 58 Our choice for using autologous mesenchymal stem cells obtained from the iliac crest has been based on the overwhelming experimental evidence [59][60][61][62] in support of the efficacy of mesenchymal stem cells. They are more easily obtained from various tissues such as the bone marrow and adipose tissue 63 ; they have been reported to stimulate neurite outgrowth over neural proteoglycans, myelin-associated glycoprotein, and Nogo-A 64 ; they can differentiate both in vitro and in vivo into cells expressing neuronal markers.…”

Neural stem cells for spinal cord repair

“…MSC can be easily isolated and expanded in vitro and used as autologous cell grafts (Keating 2006;Wright et al 2008). Initial reports indicated that MSC can differentiate into neural cells (Sanchez-Ramos et al 2000;Woodbury et al 2000Woodbury et al , 2002Tondreau et al 2004).…”

Artificial collagen-filament scaffold promotes axon regeneration and long tract reconstruction in a rat model of spinal cord transection

“…In most cases of human SCI, there is significant loss of spinal cord tissue and hence the resulting cavity formation is an important obstacle for axonal regeneration [1]. Several experimental strategies have recently demonstrated the capacity for neurologic recovery from functional deficit in SCI [2][3][4][5][6][7][8][9]. Various tissues or cells were transplanted into damaged spinal cord tissue to provide a permissive environment for the regeneration of CNS axons [10].…”