Design of a Stem Cell-Based Therapy for Ependymal Repair in Hydrocephalus Associated With Germinal Matrix Hemorrhages.

Abstract

In preterm birth germinal matrix hemorrhages (GMHs) and the consequent posthemorrhagic hydrocephalus (PHH), the neuroepithelium/ependyma development is disrupted. This work is aimed to explore the possibilities of ependymal repair in GMH/PHH using a combination of neural stem cells, ependymal progenitors (EpPs), and mesenchymal stem cells. GMH/PHH was induced in 4-day-old mice using collagenase, blood, or blood serum injections. PHH severity was characterized 2 weeks later using magnetic resonance, immunofluorescence, and protein expression quantification with mass spectrometry. Ependymal restoration and wall regeneration after stem cell treatments were tested in vivo and in an ex vivo experimental approach using ventricular walls from mice developing moderate and severe GMH/PHH. The effect of the GMH environment on EpP differentiation was tested in vitro. Two-tailed Student t or Wilcoxon-Mann-Whitney U test was used to find differences between the treated and nontreated groups. ANOVA and Kruskal-Wallis tests were used to compare >2 groups with post hoc Tukey and Dunn multiple comparison tests, respectively. PHH severity was correlated with the extension of GMH and ependymal disruption (means, 88.22% severe versus 19.4% moderate). GMH/PHH hindered the survival rates of the transplanted neural stem cells/EpPs. New multiciliated ependymal cells could be generated from transplanted neural stem cells and more efficiently from EpPs (15% mean increase). Blood and TNFα (tumor necrosis factor alpha) negatively affected ciliogenesis in cells committed to ependyma differentiation (expressing Foxj1 [forkhead box J1] transcription factor). Pretreatment with mesenchymal stem cells improved the survival rates of EpPs and ependymal differentiation while reducing the edematous (means, 18% to 0.5% decrease in severe edema) and inflammatory conditions in the explants. The effectiveness of this therapeutical strategy was corroborated in vivo (means, 29% to 0% in severe edema). In GMH/PHH, the ependyma can be restored and edema decreased from either neural stem cell or EpP transplantation in vitro and in vivo. Mesenchymal stem cell pretreatment improved the success of the ependymal restoration.