Heterogeneous fate choice of genetically modulated adult neural stem cells in gray and white matter of the central nervous system

Abstract

Apart from dedicated oligodendroglial progenitor cells, adult neural stem cells (aNSCs) can also give rise to new oligodendrocytes in the adult central nervous system (CNS). This process mainly confers myelinating glial cell replacement in pathological situations and can hence contribute to glial heterogeneity. Our previous studies demonstrated that the p57kip2 gene encodes an intrinsic regulator of glial fate acquisition and we here investigated to what degree its modulation can affect stem cell‐dependent oligodendrogenesis in different CNS environments. We therefore transplanted p57kip2 knockdown aNSCs into white and gray matter (WM and GM) regions of the mouse brain, into uninjured spinal cords as well as in the vicinity of spinal cord injuries and evaluated integration and differentiation in vivo. Our experiments revealed that under healthy conditions intrinsic suppression of p57kip2 as well as WM localization promote differentiation toward myelinating oligodendrocytes at the expense of astrocyte generation. Moreover, p57kip2 knockdown conferred a strong benefit on cell survival augmenting net oligodendrocyte generation. In the vicinity of hemisectioned spinal cords, the gene knockdown led to a similar induction of oligodendroglial features; however, newly generated oligodendrocytes appeared to suffer more from the hostile environment. This study contributes to our understanding of mechanisms of adult oligodendrogenesis and glial heterogeneity and further reveals critical factors when considering aNSC mediated cell replacement in injury and disease.

Secretome Analysis of Mesenchymal Stem Cell Factors Fostering Oligodendroglial Differentiation of Neural Stem Cells In Vivo

“…In the brain, grey and white matter can differently affect OPC differentiation [55], and we previously showed that white matter localization promotes differentiation towards myelinating oligodendrocytes at the expense of astrocyte generation when transplanted aNSC were depleted from the intrinsic oligodendrogenesis inhibitor p57kip2 [26]. However, in both studies, regional differences of the brain showed no impact on cell survival, which was confirmed by the here-described brain transplantation experiments.…”

“…Importantly, such myelinated segments and nodal structures were only observed at MSC-CM pre-treated NSCs, thus providing strong evidence that mesenchymal factors contribute to the generation of myelinated segments in vivo. Moreover, ultrastructural analysis of transplanted MSC-CM pre-treated cells by means of immunoelectron microscopy (as established in [26]; transplanted cells were identified by chromogenic anti-GFP immunohistochemistry resulting in dark precipitates), demonstrated that these cells established myelin sheaths around axons ( Figure 5C) and also revealed GFP-immunoreactive signals in paranodal loops ( Figure 5C'). In order to analyze the functionality of MBP-expressing cells and to identify myelinated segments, we performed co-staining for neurofilament and contactin-associated protein (Caspr), a marker for paranodal axonal regions of nodes of Ranvier, after 14 days of cell transplantation into rat spinal cords as previously established for other stem cell transplantation approaches [27,28].…”

“…To investigate to what degree MSC-CM-stimulated aNSCs differentiate and integrate within different brain environments, we performed transplantations into either grey (GM) or white matter (WM) regions of young adult mouse brains as previously described [26]. Moreover, we tested different MSC-CM stimulation periods prior to transplantation.…”

“…Immunohistochemical staining was performed as previously described [26]. Briefly, defrosted brain and spinal cord sections were air-dried for at least 15 min at room temperature (RT), and were afterwards rehydrated for 5 min in distilled water, transferred to −20 • C acetone (5 min), and washed in 1x tris-buffered saline (TBS) (pH 7.6) and 1x TBS-T (TBS containing 0.02% Triton) for 5 min each.…”

“…Immunoelectron microscopy was performed as previously described [26], with slight modifications. Briefly, adult rats were perfused with 4% PFA in 1 M cacodylate buffer (Carl Roth GmbH + Co. KG, Karlsruhe, Germany), and spinal cords were harvested and post-fixed overnight in 4% PFA in 1M cacodylate buffer.…”