Glial cells expressing neuron‐glial antigen 2 (NG2), also known as oligodendrocyte progenitor cells (OPCs), play a critical role in maintaining brain health. However, their ability to differentiate after ischemic injury is poorly understood. The aim of this study was to investigate the properties and functions of NG2 glia in the ischemic brain. Using transgenic mice, we selectively labeled NG2‐expressing cells and their progeny in both healthy brain and after focal cerebral ischemia (FCI). Using single‐cell RNA sequencing, we classified the labeled glial cells into five distinct subpopulations based on their gene expression patterns. Additionally, we examined the membrane properties of these cells using the patch‐clamp technique. Of the identified subpopulations, three were identified as OPCs, whereas the fourth subpopulation had characteristics indicative of cells likely to develop into oligodendrocytes. The fifth subpopulation of NG2 glia showed astrocytic markers and had similarities to neural progenitor cells. Interestingly, this subpopulation was present in both healthy and post‐ischemic tissue; however, its gene expression profile changed after ischemia, with increased numbers of genes related to neurogenesis. Immunohistochemical analysis confirmed the temporal expression of neurogenic genes and showed an increased presence of NG2 cells positive for Purkinje cell protein‐4 at the periphery of the ischemic lesion 12 days after FCI, as well as NeuN‐positive NG2 cells 28 and 60 days after injury. These results suggest the potential development of neuron‐like cells arising from NG2 glia in the ischemic tissue. Our study provides insights into the plasticity of NG2 glia and their capacity for neurogenesis after stroke.

Wnt signaling plays an important role in the self-renewal, fate-commitment and survival of the neural stem/progenitor cells (NS/PCs) of the adult central nervous system (CNS). Ischemic stroke impairs the proper functioning of the CNS and, therefore, active Wnt signaling may prevent, ameliorate, or even reverse the negative effects of ischemic brain injury. In this review, we provide the current knowledge of Wnt signaling in the adult CNS, its status in diverse cell types, and the Wnt pathway’s impact on the properties of NS/PCs and glial cells in the context of ischemic injury. Finally, we summarize promising strategies that might be considered for stroke therapy, and we outline possible future directions of the field.

Ischemic brain injury and Alzheimer’s disease (AD) both lead to cell death in the central nervous system (CNS) and thus negatively affect particularly the elderly population. Due to the lack of a definitive cure for brain ischemia and AD, it is advisable to carefully study, compare, and contrast the mechanisms that trigger, and are involved in, both neuropathologies. A deeper understanding of these mechanisms may help ameliorate, or even prevent, the destructive effects of neurodegenerative disorders. In this review, we deal with ischemic damage and AD, with the main emphasis on the common properties of these CNS disorders. Importantly, we discuss the Wnt signaling pathway as a significant factor in the cell fate determination and cell survival in the diseased adult CNS. Finally, we summarize the interesting findings that may improve or complement the current sparse and insufficient treatments for brain ischemia and AD, and we delineate prospective directions in regenerative medicine.

NG2 glia, also known as oligodendrocyte precursor cells (OPCs), exhibit unexpected plasticity in the adult brain after injury, yet the molecular cues guiding their fate remain poorly defined. Wnt signaling contributes to tissue responses following ischemic stroke, but its precise role in post-injury glial remodeling is not fully understood. Here, we define how Wnt/β-catenin signaling shapes NG2 glial behavior after focal cerebral ischemia. Using genetic mouse models enabling cell-specific Wnt pathway activation or inhibition, combined with single-cell RNA sequencing, immunohistochemistry, and electrophysiological recording, we disclosed a central role for Wnt signaling in post-injury fate specification. We identified 12 transcriptionally distinct subpopulations within the oligodendroglial lineage, including a subset with an astrocyte-like transcriptional profile. Wnt signaling strongly influenced the balance between OPC proliferation and differentiation: pathway hyperactivation impaired oligodendrocyte maturation and expanded astrocyte-like NG2-derived cells, likely through concomitant activation of the Notch pathway. Remarkably, Wnt hyperactivation also induced the appearance of cholinergic neuron-like cells derived from NG2-expressing cells exclusively in the somatosensory cortex; these cells generated action potentials and exhibited sodium conductance characteristic of functional neurons. Together, these findings demonstrate that NG2 glia undergo distinct fate transitions after stroke and that their lineage plasticity is highly sensitive to Wnt pathway dynamics. Targeted fine-tuning of Wnt/β-catenin signaling may enable directed redirection of NG2 glia toward specific reparative outcomes, including neuronal reprogramming, in the injured adult brain.

Single-cell transcriptomics has revealed the central role of microglia in brain development, homeostasis, and disease, particularly in the context of neuroinflammation. While single-cell RNA-sequencing enables targeted microglial analysis from fresh tissue, studying these cells in cryopreserved or archival samples remains challenging due to the lack of a protocol for their specific enrichment and RNA-sequencing. In this study, we developed a method for profiling microglial nuclei from fresh-frozen tissue using Smart-seq3xpress. This approach relies on PU.1-based enrichment, made possible by a brief formaldehyde fixation step to preserve the antigen. To ensure compatibility with Smart-seq3xpress, we utilized a Thermolabile Proteinase K treatment to reverse cross-links, thereby maintaining high transcriptomic sensitivity. We benchmarked the method in a mouse model of ischemic stroke, evaluating both technical performance and its ability to capture biologically meaningful microglial states. Compared to standard single-nucleus protocols, our approach yielded higher gene and UMI counts and a greater proportion of coding reads. Transcriptomic profiles closely matched those from whole-cell RNA-sequencing, including the detection of activation markers and diverse microglial subpopulations. This approach enables high-resolution transcriptomic analysis of microglia from fresh-frozen or archival brain samples, overcoming major limitations of single-nucleus RNA sequencing. It broadens access to cellular insights from biobanked material, supporting both basic research and translational studies of neuroinflammatory disease. Graphical Abstract A modified Smart-seq3xpress protocol was developed for microglial profiling by combining formaldehyde fixation and PU.1-based nuclei enrichment from fresh-frozen brain tissue. The method yields high gene and UMI counts alongside strong coding coverage, enabling high-resolution characterization of diverse microglial activation states following stroke.

Single-cell RNA sequencing has advanced our understanding of glial diversity, yet tissue dissociation remains an important technical variable. We revealed that a widely used commercial dissociation protocol can underrepresent reactive astrocytes in an acute ischemic stroke model, with preliminary observations indicating a comparable trend in a chronic tauopathy model. Benchmarking against an optimized, low-shear workflow demonstrates that different tissue dissociation workflows can substantially alter the recovery and apparent transcriptomic profile of disease-associated astrocytes, selectively losing signatures of morphological remodeling. Our findings indicate that workflow-dependent recovery biases affecting reactive astrocytes may be more prevalent than recognized, underscoring the need for pathology-tailored, gentle isolation workflows to avoid distortions in downstream neuropathological interpretations and the prioritization of therapeutic targets.