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Germinal matrix hemorrhages (GMH) and the consequent posthemorrhagic hydrocephalus (PHH) are among the most common and severe neurological complications of preterm birth that require lifelong complex neurosurgical care. GMH and PHH provoke disruption of neuroepithelium/ependyma development, a key structure implicated in brain development and homeostasis. Neuroepithelial/ependymal damage causes lifelong cognitive and motor deficits; however, no therapy is directed to recover the damaged ependyma. This study is aimed to test the possibilities of ependymal repair in GMH/PHH using neural stem cells (NSCs) or ependymal progenitors (EpPs). Thus, it sets the basis for a therapeutic approach to treating ependymal damage and preventing brain developmental deficits. GMH/PHH was induced in 4-day-old mice using different experimental procedures involving collagenase, blood, or blood serum injections. PHH severity was characterized using magnetic resonance, immunofluorescence, and protein expression quantification with mass spectrometry. Additionally, a newexvivoapproach using ventricular walls from mice developing moderate and severe GMH/PHH was generated to study ependymal restoration and wall regeneration after stem cell treatments. NSCs or EpPs obtained from newborn mice were transplanted in the explants, and pretreatment with mesenchymal stem cells (MSCs) was tested. Ependymal differentiation and the effect of MSC-conditioned microenvironment were investigated in both explants and primary cultures. In the animals, PHH severity was correlated with the extension of GMH, ependymal disruption, astroglial/microglial reactions, and ventriculomegaly. In the explants, the severity and extension of GMH hindered the survival rates of the transplanted NSCs/EpPs. In the explants affected with GMH, new multiciliated ependymal cells could be generated from transplanted NSCs and, more efficiently, from EpPs. Blood and TNFα negatively affected ciliogenesis in cells expressing Foxj1. Pretreatment with mesenchymal stem cells (MSC) improved the survival rates of EpPs and ependymal differentiation while reducing the edematous and inflammatory conditions in the explants. In conclusion, in GMH/PHH, the ependyma can be restored from either NSC or EpP transplantation, being EpPs in an MSC-conditioned microenvironment more efficient for this purpose. Modifying the neuroinflammatory microenvironment by MSC pretreatment positively influenced the success of the ependymal restoration.

Aquaporin-4 (AQP4) plays a crucial role in brain water circulation and is considered a therapeutic target in hydrocephalus. Congenital hydrocephalus is associated with a reaction of astrocytes in the periventricular white matter both in experimental models and human cases. A previous report showed that bone marrow-derived mesenchymal stem cells (BM-MSCs) transplanted into the lateral ventricles of hyh mice exhibiting severe congenital hydrocephalus are attracted by the periventricular astrocyte reaction, and the cerebral tissue displays recovery. The present investigation aimed to test the effect of BM-MSC treatment on astrocyte reaction formation. BM-MSCs were injected into the lateral ventricles of four-day-old hyh mice, and the periventricular reaction was detected two weeks later. A protein expression analysis of the cerebral tissue differentiated the BM-MSC-treated mice from the controls and revealed effects on neural development. In in vivo and in vitro experiments, BM-MSCs stimulated the generation of periventricular reactive astrocytes overexpressing AQP4 and its regulatory protein kinase D-interacting substrate of 220 kDa (Kidins220). In the cerebral tissue, mRNA overexpression of nerve growth factor (NGF), vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1 (HIF1α), and transforming growth factor beta 1 (TGFβ1) could be related to the regulation of the astrocyte reaction and AQP4 expression. In conclusion, BM-MSC treatment in hydrocephalus can stimulate a key developmental process such as the periventricular astrocyte reaction, where AQP4 overexpression could be implicated in tissue recovery.