Instructive roles of DRG neurons on the fate choice of bone marrow derived glial cells

Abstract

Transplantation of autologous Schwann cells into sites of lesion in patients with demyelinating diseases or nerve injury promises improvements in axonal regrowth and remyelination as well as functional recovery. The bone marrow stroma (BM) can be tapped as an autologous source of neural progenitor cells from which Schwann cell-like cells (SCLCs) can be derived. Commitment to the Schwann cell fate was however not achieved until the BM-derived SCLCs acquired contact-mediated signals from embryonic DRG neurons as in coculture. In the search for membrane-associated cues, purified DRG neurons from embryonic rats (E14.5) were found immunopositive for the Notch ligands, DLL1 and Jagged1. Coincidentally, SCLCs were immunopositive for the cell-surface receptor, Notch-1. Coculture of BM-derived SCLCs with the purified DRG neurons effected Notch ligand-receptor interactions resulting in endoproteolytic release and nuclear translocation of the Notch intracellular domain in SCLCs; the effect was no longer observable when Notch signaling was blocked with the use of the γ-secretase inhibitor, DAPT. Treatment of co-cultures with DAPT also resulted in significant decrease in ErbB2 as revealed by immunocytochemistry and Western blot analysis, and consequently, decrease in fate-committed Schwann cells. Transient over-expression of NICD in SCLCs was sufficient to bring about up-regulation of ErbB2. These suggest γ-secretase activity on membrane-bound Notch-1 as a key event that accomplished ligand triggered Notch signaling in SCLCs. We then checked for up-regulated expression of ErbB2/3 as possible effectors of Notch signaling. Both immunocytochemistry and RT-PCR indicated increased expression in synchrony with the progression of SCLCs to the Schwann cell fate in the coculture with DRG neurons. Reinforced by the finding of neuregulin 1-type III (Nrg1-III) on DRG neurons, we suggest that DRG neurons present membrane-bound ligands that instruct Notch and ErbB2/3 signalling and that Nrg1-III-ErbB2/3 interaction is a consequence of Notch signaling in the progression of SCLCs to the Schwann cell fate.

As expected, the SCLCs that failed to contact DRG neurons in the co-culture failed to achieve the Schwann cell fate. The soluble factors in the co-culture environment nevertheless were sufficient to direct differentiation of these cells into oligodendrocyte precursor cells. In our protocol for in vitro derivation of glia from BM stromal cells, contact with DRG neurons is therefore important in both providing positive cues that direct differentiation of BM-derived neurosphere cells along the Schwann cell lineage and interfering with differentiation along the oligodendroglial lineage.