RNA Profiling of Mouse Ependymal Cells after Spinal Cord Injury Identifies the Oncostatin Pathway as a Potential Key Regulator of Spinal Cord Stem Cell Fate

Abstract

Ependymal cells reside in the adult spinal cord and display stem cell properties in vitro. They proliferate after spinal cord injury and produce neurons in lower vertebrates but predominantly astrocytes in mammals. The mechanisms underlying this glial-biased differentiation remain ill-defined. We addressed this issue by generating a molecular resource through RNA profiling of ependymal cells before and after injury. We found that these cells activate STAT3 and ERK/MAPK signaling post injury and downregulate cilia-associated genes and FOXJ1, a central transcription factor in ciliogenesis. Conversely, they upregulate 510 genes, seven of them more than 20-fold, namely Crym, Ecm1, Ifi202b, Nupr1, Rbp1, Thbs2 and Osmr—the receptor for oncostatin, a microglia-specific cytokine which too is strongly upregulated after injury. We studied the regulation and role of Osmr using neurospheres derived from the adult spinal cord. We found that oncostatin induced strong Osmr and p-STAT3 expression in these cells which is associated with reduction of proliferation and promotion of astrocytic versus oligodendrocytic differentiation. Microglial cells are apposed to ependymal cells in vivo and co-culture experiments showed that these cells upregulate Osmr in neurosphere cultures. Collectively, these results support the notion that microglial cells and Osmr/Oncostatin pathway may regulate the astrocytic fate of ependymal cells in spinal cord injury.

Regulating Endogenous Neural Stem Cell Activation to Promote Spinal Cord Injury Repair

“…Similar to NSCs found in the brain, the majority of NSCs in the spinal cord are definitive NSCs [ 57 ], while a rare population of primitive NSCs have also been identified [ 48 ]. Most recently, single-cell sequencing (scRNA-seq) and proteomics, have facilitated a comprehensive profile of PVZ heterogeneity and identified a novel, MSX1+ population of NSCs [ 58 , 59 ]. Finally, recent work has demonstrated that NSC properties are elicited from a population of spinal cord niche cells known as cerebrospinal fluid contacting neurons (CSFcNs) in vitro.…”

“…Increased resolution pertaining to the identification and potential of distinct NSC populations is fundamental to exploiting these populations for neural repair in mammals [ 59 ]. Here, we provide a current and more detailed description of the spinal cord NSC pools ( Figure 1 ).…”

“…A conditional MSX1-CreER T2 mouse crossed to a tdTomato reporter (MSX1-Cre:tdTom) was injected with tamoxifen on E11.5 and tdTomato+ cells were identified in vivo when tissue was examined at E13. MSX1+ NSCs are restricted to the dorsal region of the PVZ and share several molecular markers with dNSCs including Nestin, GFAP, Sox2 and FoxJ1 [ 58 , 59 ]. Further, MSX1+ cells express cRET (GDNF and NTN growth factor receptors) which are found on hematopoetic stem cell populations and Id4 which is highly enriched in quiescent NSCs from the forebrain [ 80 ].…”

“…As such, it is important to look beyond marker expression and examine cell behavior when identifying stem cell populations, including the fundamental properties of self-renewal and multipotency. Important recent work has begun to unravel the distinct characteristics in the mammalian (including human) spinal cord niche [ 58 , 59 ].…”

“…Ciliated ependymal cells lining the central canal, identified by their expression of CD133 (Prominin1), FoxJ1 and Sox2 [ 109 , 110 , 111 ], play an important role in the circulation of cerebrospinal fluid [ 112 ]. Ciliated gene expression is affected by SCI (including expression of FOXJ1) and culture conditions, including the neurosphere assay [ 59 ]. Interestingly, ependymal cells have been ascribed as a source of multipotent NSCs that are activated following injury or following in vitro isolation [ 29 , 30 , 113 ].…”