Rhepo

Neurospheres in the range of 100–200 μm were seeded on coverslips coated with as before to evaluate the influence of coating concentration on expansion of the neurosphere as a measurement of migration. Alternatively, fixed concentrations of 50 μg/ml of either PDL or PornT was used to evaluate the response of the spheres to proteins known to influence the migration and differentiation of NPCs depending on the coating of choice. In the latter case neurospheres were stimulated with 500 ng/μl of stromal-cell derived factor-1alpha (SDF-1a, R&D systems, Germany) or 50 IU/ml of rhEpo (NeoRecormon, Roche, Switzerland). Expansion was tracked for 8 h on a Zeiss inverted microscope equipped with a cell culture chamber. Images were taken every 10 min with a 10x phase contrast objective. Expansion of the spheres was analyzed using custom designed macros on ImageJ. The increase in area occupied by the neurosphere was calculated as $\mathit{r}\mathit{A}\mathit{=}\frac{\mathit{f}\mathit{A}}{\mathit{i}\mathit{A}}$ , where, rA is the ratio of the sphere area, fA is the area at the end of the recording, and iA is the area at the beginning of the recording. Data is represented as ratio of the respective control (Figures 7 and 8) or as rA (Supplementary Figure S1). At least 10 neurospheres were tracked in a total of 3–4 experiments per evaluated condition.

Single cells were sorted and cultured in 96-well plates in StemSpan SFEM medium (STEMCELL Technologies, Cat. 09600) supplemented with 20 ng/ml rmSCF (Peprotech, 250-03), 20 ng/ml rmTPO (eBioscience, 34-8686-63), 20 ng/ml rmIl3 (Peprotech, 213-13), and 5 U/ml rhEpo (Roche) and cultivated for 12 days at 37 °C with 5% CO₂.

Animals showing injury on initial MRI were selected and randomized to NaCl group, injected intraperitoneally with NaCl 0.9% or to EPO group, injected intraperitoneally with recombinant human EPO (rhEPO, Roche, Basel, Switzerland) 10 U/g body weight/day during the first week after HI (P3 to P10) and then 5 U/g body weight 3×/week until P25. A Control group, which was not subjected to surgery and injection, was also studied.