br Results To investigate the
Results To investigate the dynamics of hCNS-SCns engraftment, we quantified SC121, a human cytoplasmic marker, in conjunction with bromodeoxyuridine (BrdU) or KI67, markers for mitotically active cells, or cleaved caspase 3 (CC3), a marker for cellular apoptosis. Lineage-specific differentiation of hCNS-SCns was determined by double-labeling immunohistochemistry for SC121 in combination with OLIG2 (oligodendrocytic) or DCX (neuronal), or single labeling for SC123 (human astrocytic). Representative images of coronal sections for human special info with proliferating or apoptotic cells are shown in Figures 2 and S1 (available online). Stereological quantification for these markers was performed on all animals.
Discussion In the SCI field, subacute NSC transplantation time points (7–10 dpi) have been postulated to increase survival by avoiding acute inflammation (Okano et al., 2003) and have persisted as a paradigm for the majority of preclinical studies (Tetzlaff et al., 2011). The data presented here demonstrate that although the injured microenvironment at subacute time points may play a role in survival, the majority of subacutely transplanted cells did not survive at 1 dpt, even in an uninjured spinal cord. Recent studies have identified a prominent role for biomechanical disruption during the injection process in determining early survival of injected cells (Aguado et al., 2012). Taken together, these data suggest that early survival of transplanted NSC populations may be more dependent on transplantation parameters than on postinjury transplantation timing. However, differences in the injured microenvironment at different postinjury time points may still regulate the engraftment dynamics. Between 1 dpt and 98 dpt, the total number of hCNS-SCns increased dramatically in both the injured and uninjured spinal cords (15-fold and 20-fold, respectively) when calculated relative to the number of surviving cells and not transplantation dose. Despite this observation, hCNS-SCns proliferation in the injured spinal cord was delayed for the first 28 dpt, an effect that may be influenced by multiple factors. For example, inflammation, including toll-like receptor (TLR) activation, can inhibit NSC proliferation (Das and Basu, 2008; Ekdahl et al., 2009; Okun et al., 2010; Taylor et al., 2010) and is altered in a spatiotemporal manner after SCI (Anderson, 2002; Popovich and Jones, 2003; Anderson et al., 2004; Fleming et al., 2006; Kigerl et al., 2007; Kigerl and Popovich, 2009; Beck et al., 2010). Similarly, chondroitin sulfate proteoglycans (CSPGs) in the injured spinal cord have been shown to diminish the proliferation of both transplanted (Karimi-Abdolrezaee et al., 2010) and endogenous (Karimi-Abdolrezaee et al., 2012; Karus et al., 2012) NSCs. Between 28 dpt and 98 dpt, hCNS-SCns number increased 8-fold in the injured spinal cord, compared with 2-fold in the uninjured spinal cord. Again, these data could be the result of multiple nonmutually exclusive mechanisms. First, although hCNS-SCns proliferation was equivalent at both 28 dpt and 98 dpt, proliferation may have been differentially regulated in the interim period, which would not have been detected in the cumulative BrdU incorporation paradigm employed in the 98 dpt cohort. Alternatively, there could have been decreased long-term survival of hCNS-SCns in the uninjured spinal cord during this period. Many newborn cells in the dentate gyrus and subventricular zone die within 4 weeks after birth, presumably as a result of failing to functionally integrate into circuitry (Zhao et al., 2008). Thus, it is possible that hCNS-SCns in the uninjured spinal cord, which in contrast to the injured spinal cord may lack appropriate sites for integration, were pruned in the period between 28 dpt and 98 dpt. Future studies utilizing intermediate time points or advances in noninvasive cell-tracking methods (Li et al., 2010) could elucidate these mechanisms. Despite robust proliferation, there was no evidence of tumor formation in any animal that received hCNS-SCns. At 98 dpt, we identified that ∼10% of hCNS-SCns expressed KI67, which is slightly higher than values reported in previous studies using similar transplantation paradigms (1%–5%) (Yan et al., 2007; Ogawa et al., 2009; Nori et al., 2011). However, those studies did not utilize stereological sampling methods, which preclude direct comparisons with the work presented here. Furthermore, the majority of transplanted cells in those studies exhibited neuronal lineage differentiation. In contrast, we found that the majority of hCNS-SCns differentiated along the oligodendroglial lineage, and that human cells expressing KI67+ also exhibited labeling for OLIG2, supporting the lineage commitment of this cell population by 98 dpt. Oligodendrocyte precursor cells proliferate extensively in both intact and injured spinal cords (Barnabé-Heider et al., 2010; Payne et al., 2013), which may suggest that transplanted hCNS-SCns respond to similar cues.