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

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It is clear from animal studies that transplantation of bone marrow-derived stromal cells (MSC) promotes functional recovery after SCI^2-5.  However, there is also evidence to suggest that SCI in humans may influence the activity of other bone marrow cells.  SCI is known to depress natural and adaptive immunity and in vitro studies have demonstrated not only decreased lymphocyte function^{13, 14} but also reduced long-term colony formation of haemopoietic stem cells¹³.  A clear question, therefore, is whether these cells can be isolated in humans with SCI and how they behave in culture.  There is little published data characterising MSC in humans with SCI, although one recent report stated that bone marrow harvests from SCI donors gave rise to ‘fibroblast-like mesenchymal cells’ in just 75% of cases¹⁵.

Using standard protocols for MSC isolation, we found no significant differences in the expansion of stromal cells from the bone marrow of SCI or non-SCI donors, successfully growing cells with MSC characteristics through to passage II and beyond in all cases.  Furthermore, no differences were seen in the proliferative rate of the MSC generated from either group, based on their culture DT after first passage.  MSC population doublings in non-SCI humans have previously been found to relate to age, where cultures established from pediatric donors proliferated to generate nearly twice as many cells as those derived from young adults within the same time span¹⁶.  In addition, differences in the number of mononuclear cells present in bone marrow biopsies taken from SCI donors with cervical versus thoracic injuries have been reported, where donors with cervical lesions had larger cell counts/ ml of marrow¹⁵.  We found that the number of MSC generated at passage I as a proportion of mononucleated cells originally isolated (i.e., the MSC Index) was inversely related to age in SCI donors, but not non-SCI donors.  We observed no differences in the MSC Index or culture DT of SCI donors in comparison with non-SCI donors. In contrast, MSC from individuals with cervical SCI lesions proliferated more slowly than those with thoracic lesions.  Given the small sample sizes involved, and that fact that 2/4 of the cervical samples were female whereas no females were included in the thoracic group, this difference may relate in some way to sex.  However, no significant differences were seen in either the MSC Index or MSC culture DT in female versus male donors when the SCI and non-SCI groups were pooled. These findings may indicate differences in the behaviour of MSC from SCI donors, as the MSC Index is a combined indication of cell isolation, cell adhesion and cell proliferation.  However, larger donor sample groups (of increased donor age range) and further study are required to ascertain (i) whether the influence of age on MSC proliferation is more pronounced in individuals with SCI compared with non-SCI donors, or (ii) why MSC from SCI donors with cervical lesions appear to proliferate more slowly than those from donors with thoracic lesions.  One possible explanation for the differences in proliferation rate is that although more mononucleated cells are present in the bone marrow of SCI donors with cervical lesions¹⁵, these may contain proportionally fewer MSC.  If this were the case, then fewer MSC will have been initially seeded into the culture flasks and these will have undergone increased rounds of proliferation to reach passage I, resulting in a reduced proliferation rate thereafter compared to cells undergoing fewer rounds of division prior to first passage. We report no significant relationships in the MSC index or culture DT and the period between injury onset (5 to 272 months) and when the bone marrow sample was collected in SCI donors. We realise that this donor population excludes patients in the acute phase of SCI which may well influence the behaviour of MSC in a manner that differs to the chronic setting. However, following local research ethical committee guidelines, bone marrow aspirates could only be harvested from individuals with a complete, long-term Frankel grade A SCI, 3+ months post injury.

The number of cells available for transplantation may well be limiting to the success of cell transplantation therapies for SCI, especially when scaling up in lesion size from animal models to humans.  Using an in vitro model, and supporting this assumption, we have recently reported that cell number relates directly to the capacity of MSC to diminish the nerve-inhibitory effects of molecules found in the glial scar after SCI⁸.  Here, we show that seeding MSC at lower densities from passages I-III significantly reduced the normal culture DT and greatly increased overall MSC yield.  In addition, we found that this proliferative response did not lead to increased cell senescence, as delineated by SA-b gal activity, which is generally deleterious to cell function.  It is unclear how MSC seeding density relates to proliferative rate.  Cell contact inhibition induces growth arrest in C3H10T1/2 cells at confluence, which is a murine fibroblast cell line with similar phenotypical characterisitics to MSC¹⁷; this growth arrest of C3H10T1/2 is reversed by re-seeding the cells at lower density¹⁸.  Hence, establishing MSC cultures at lower cell seeding densities may similarly reduce the effects of contact inhibition, maintaining a greater proportion of cells in cycle.  If there is a window of opportunity in the acute phase of SCI when MSC grafting is therapeutic, then generating sufficient MSC from autologous bone marrow over relatively short time periods is potentially of great importance.

In summary, we have shown that MSC can be successfully isolated from bone marrow biopsies of donors with SCI and subsequently expanded in culture.  In parallel studies, we have also demonstrated that MSC from SCI donors promote nerve growth⁸.  Together, these findings support and validate previous animal studies suggesting that MSC transplantation may provide therapeutic benefits to humans with SCI.