Human neural progenitor cells (hNPC) and human mesenchymal stromal cells (hMSC) have both been reported to be able to promote improved functional recovery after grafting into experimental spinal cord injuries. Transplantation of pre-differentiated neural progenitor cells has been shown to be an effective strategy to support repair of the spinal cord. In particular, the implantation of astrocyte restricted precursors may represent a means of promoting spinal cord recovery through the maintenance and support of axonal regeneration. Although it has been demonstrated by our group that foetal hNPC, pre-differentiated to type I astrocyte restricted precursors (hNP-AC), promote more extensive axon regeneration by adult rat dorsal root ganglia (DRG) neurons in vitro than non-differentiated hNPC or hMSC, the mechanisms of action to support neuritic outgrowth remained unclear. Therefore, hNPC were characterised before and after in vitro differentiation to hNP-AC to provide a clearer definition of the changes that the cells had undergone. Furthermore, possible contributions of released trophic factors (i.e. present in conditioned culture medium) or substrate-mediated mechanisms via the extracellular matrix (ECM) of hMSC and hNP-AC to support neuritic outgrowth were investigated. In the first part of this study, the in vitro immunocytochemical- and mRNA expression profile of selected marker gens in hNPC and in the differentiated hNP-AC was quantified. Furthermore, proliferative activity was estimated with a metabolic and a morphological assay, whereby the latter, by measuring the increase in volume over time, provided an indirect indicator of proliferation within intact neurospheres. The hNPC grew as neurospheres, which were immunoreactive for the stem/progenitor cell related markers nestin (91.0 3.4%), SOX2 (89.2 2.7%) as well as musashi (91.8 2.0%) and expressed CD133- and nestin mRNA. They showed spontaneous differentiation capacity into MAP2ab+ and TuJ1+ neuronal phenotypes as well as S100b+ (17.6 i 2.1%), GFAP+ (23.7 2.3%) and vimentin+ (96.8 1.1%) astroglial precursors. Whereas the differentiated hNP-AC demonstrated a similar immunocytochemical expression profile for the stem/ progenitor cell related markers nestin (89.2 3.8%), SOX2 (88.0 3.6%), musashi (89.2 3.8%) and expressed CD133- and nestin mRNA in similar proportions to the hNPC, the expression of astroglial markers increased. A significantly higher proportion of hNP-AC were immunoreactive for S100b (79.4 1.7%), GFAP (92.5 3.4%) and CD44. Likewise, hNP-AC demonstrated significantly increased CD44- and GFAP mRNA expression than the non-differentiated hNPC. Estimation of proliferative activity of hNPC and hNP-AC showed a reduced doubling time (DT) in hNP-AC with a value of 5.1 days (morphological assay) and 3.2 days (metabolic assay), contrasting to hNPC prior to in vitro differentiation with a DT of 3.46 days (morphological assay) and 1.68 days (metabolic assay). In the second part of this study, the contributions of trophic factors present in cell-conditioned medium (produced by either hNP-AC or hMSC) and their ECM in supporting DRG axon regeneration were investigated in vitro. Conditioned media from both cell types strongly supported neurite outgrowth over a defined PLL/laminin substrate. The ECM produced by the two cell types, however, demonstrated contrasting effects: hNP-AC ECM promoted substantial axon regeneration, but hMSC ECM supported relatively little axon growth. The combined application of hNP-AC ECM and hMSC conditioned media induced the greatest degree of axon outgrowth. The combination of hMSC ECM and hNP-AC conditioned medium, however, did not result in any enhanced axon growth over the values determined using hMSC ECM and control growth medium. Thus, although hNP-AC conditioned medium demonstrated potent trophic support for DRG axon growth, its effect was not supported by signals derived from hMSC ECM. This raises interesting questions for future experiments regarding the cross-talk between the intracellular signalling cascades activated by diffusible trophic factors and those activated by ECM-related molecules.weiterlesen