Single nuclei transcriptomics of the in situ human limbal stem cell niche.

The corneal epithelium acts as a barrier to pathogens entering the eye; corneal epithelial cells are continuously renewed by uni-potent, quiescent limbal stem cells (LSCs) located at the limbus, where the cornea transitions to conjunctiva. There has yet to be a consensus on LSC markers and their transcriptome profile is not fully understood, which may be due to using cadaveric tissue without an intact stem cell niche for transcriptomics. In this study, we addressed this problem by using single nuclei RNA sequencing (snRNAseq) on healthy human limbal tissue that was immediately snap-frozen after excision from patients undergoing cataract surgery. We identified the quiescent LSCs as a sub-population of corneal epithelial cells with a low level of total transcript counts. Moreover, TP63, KRT15, CXCL14, and ITGß4 were found to be highly expressed in LSCs and transiently amplifying cells (TACs), which constitute the corneal epithelial progenitor populations at the limbus. The surface markers SLC6A6 and ITGß4 could be used to enrich human corneal epithelial cell progenitors, which were also found to specifically express the putative limbal progenitor cell markers MMP10 and AC093496.1.

Time-resolved single-cell RNAseq profiling identifies a novel Fabp5+ subpopulation of inflammatory myeloid cells with delayed cytotoxic profile in chronic spinal cord injury.

Traumatic spinal cord injuries (SCI) are a group of highly debilitating pathologies affecting thousands annually, and adversely affecting quality of life. Currently, no fully restorative therapies exist, and SCI still results in significant personal, societal and financial burdens. Inflammation plays a major role in the evolution of SCI, with myeloid cells, including bone marrow derived macrophages (BMDMs) and microglia (MG) being primary drivers of both early secondary pathogenesis and delayed wound healing events. The precise role of myeloid cell subsets is unclear as upon crossing the blood-spinal cord barrier, infiltrating bone marrow derived macrophages (BMDMs) may take on the morphology of resident microglia, and upregulate canonical microglia markers, thus making the two populations difficult to distinguish. Here, we used time-resolved scRNAseq and transgenic fate-mapping to chart the transcriptional profiles of tissue-resident and -infiltrating myeloid cells in a mouse model of thoracic contusion SCI. Our work identifies a novel subpopulation of foam cell-like inflammatory myeloid cells with increased expression of Fatty Acid Binding Protein 5 (Fabp5) and comprise both tissue-resident and -infiltrating cells. Fabp5+ inflammatory myeloid cells display a delayed cytotoxic profile that is predominant at the lesion epicentre and extends into the chronic phase of SCI.

Bioinformatics analysis identified apolipoprotein E as a hub gene regulating neuroinflammation in macrophages and microglia following spinal cord injury.

Macrophages and microglia play important roles in chronic neuroinflammation following spinal cord injury (SCI). Although macrophages and microglia have similar functions, their phagocytic and homeostatic abilities differ. It is difficult to distinguish between these two populations in vivo, but single-cell analysis can improve our understanding of their identity and heterogeneity. We conducted bioinformatics analysis of the single-cell RNA sequencing dataset GSE159638, identifying apolipoprotein E (APOE) as a hub gene in both macrophages and microglia in the subacute and chronic phases of SCI. We then validated these transcriptomic changes in a mouse model of cervical spinal cord hemi-contusion and observed myelin uptake, lipid droplets, and lysosome accumulation in macrophages and microglia following SCI. Finally, we observed that knocking out APOE aggravated neurological dysfunction, increased neuroinflammation, and exacerbated the loss of white matter. Targeting APOE and the related cholesterol efflux represents a promising strategy for reducing neuroinflammation and promoting recovery following SCI.

Single cell biology-a Keystone Symposia report.

Single cell biology has the potential to elucidate many critical biological processes and diseases, from development and regeneration to cancer. Single cell analyses are uncovering the molecular diversity of cells, revealing a clearer picture of the variation among and between different cell types. New techniques are beginning to unravel how differences in cell state-transcriptional, epigenetic, and other characteristics-can lead to different cell fates among genetically identical cells, which underlies complex processes such as embryonic development, drug resistance, response to injury, and cellular reprogramming. Single cell technologies also pose significant challenges relating to processing and analyzing vast amounts of data collected. To realize the potential of single cell technologies, new computational approaches are needed. On March 17-19, 2021, experts in single cell biology met virtually for the Keystone eSymposium "Single Cell Biology" to discuss advances both in single cell applications and technologies.

Metabolic Control of Smoldering Neuroinflammation.

Compelling evidence exists that patients with chronic neurological conditions, which includes progressive multiple sclerosis, display pathological changes in neural metabolism and mitochondrial function. However, it is unknown if a similar degree of metabolic dysfunction occurs also in non-neural cells in the central nervous system. Specifically, it remains to be clarified (i) the full extent of metabolic changes in tissue-resident microglia and infiltrating macrophages after prolonged neuroinflammation (e.g., at the level of chronic active lesions), and (ii) whether these alterations underlie a unique pathogenic phenotype that is amenable for therapeutic targeting. Herein, we discuss how cell metabolism and mitochondrial function govern the function of chronic active microglia and macrophages brain infiltrates and identify new metabolic targets for therapeutic approaches aimed at reducing smoldering neuroinflammation.

Stem Cell Therapies for Progressive Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system characterized by demyelination and axonal degeneration. MS patients typically present with a relapsing-remitting (RR) disease course, manifesting as sporadic attacks of neurological symptoms including ataxia, fatigue, and sensory impairment. While there are several effective disease-modifying therapies able to address the inflammatory relapses associated with RRMS, most patients will inevitably advance to a progressive disease course marked by a gradual and irreversible accrual of disabilities. Therapeutic intervention in progressive MS (PMS) suffers from a lack of well-characterized biological targets and, hence, a dearth of successful drugs. The few medications approved for the treatment of PMS are typically limited in their efficacy to active forms of the disease, have little impact on slowing degeneration, and fail to promote repair. In looking to address these unmet needs, the multifactorial therapeutic benefits of stem cell therapies are particularly compelling. Ostensibly providing neurotrophic support, immunomodulation and cell replacement, stem cell transplantation holds substantial promise in combatting the complex pathology of chronic neuroinflammation. Herein, we explore the current state of preclinical and clinical evidence supporting the use of stem cells in treating PMS and we discuss prospective hurdles impeding their translation into revolutionary regenerative medicines.

Harnessing the Neural Stem Cell Secretome for Regenerative Neuroimmunology.

Increasing evidence foresees the secretome of neural stem cells (NSCs) to confer superimposable beneficial properties as exogenous NSC transplants in experimental treatments of traumas and diseases of the central nervous system (CNS). Naturally produced secretome biologics include membrane-free signaling molecules and extracellular membrane vesicles (EVs) capable of regulating broad functional responses. The development of high-throughput screening pipelines for the identification and validation of NSC secretome targets is still in early development. Encouraging results from pre-clinical animal models of disease have highlighted secretome-based (acellular) therapeutics as providing significant improvements in biochemical and behavioral measurements. Most of these responses are being hypothesized to be the result of modulating and promoting the restoration of key inflammatory and regenerative programs in the CNS. Here, we will review the most recent findings regarding the identification of NSC-secreted factors capable of modulating the immune response to promote the regeneration of the CNS in animal models of CNS trauma and inflammatory disease and discuss the increased interest to refine the pro-regenerative features of the NSC secretome into a clinically available therapy in the emerging field of Regenerative Neuroimmunology.

Combination of In Situ Lcn2 pRNA-RNAi Nanotherapeutics and iNSC Transplantation Ameliorates Experimental SCI in Mice.

Spinal cord injury (SCI) is a debilitating neurological condition characterized by different cellular and molecular mechanisms that interplay in exacerbating the progression of the pathology. No fully restorative therapies are yet available, and it is thus becoming recognized that combinatorial approaches aimed at addressing different aspects of SCI will likely results in greater functional outcomes. Here we employed packaging RNA-mediated RNA interference (pRNA-RNAi) nanotherapeutics to downregulate in situ the expression of lipocalin 2 (Lcn2), a known mediator of neuroinflammation and autocrine mediator of reactive astrogliosis, and to create a more amenable niche for the subsequent transplantation of induced neural stem cells (iNSCs). To our knowledge, this is the first approach that takes advantage of the modular and multifunctional pRNA three-way junction platform in the SCI niche, while also exploiting the therapeutic potential of immune-compatible and feasible iNSC transplants. We show the combination of such treatments in a mouse model of contusion thoracic SCI leads to significant improvement of locomotor function, albeit not better than single pRNA-RNAi treatment, and results in synergistic histopathological effects, such as reduction of glial scar volume, diminished pro-inflammatory response, and promotion of neuronal survival. Our results provide evidence for a novel combinatorial approach for treating SCI.

Characterization of Human iPSC-derived Spinal Motor Neurons by Single-cell RNA Sequencing.

Human induced pluripotent stem cells (iPSCs) offer the opportunity to generate specific cell types from healthy and diseased individuals, allowing the study of mechanisms of early human development, modelling a variety of human diseases, and facilitating the development of new therapeutics. Human iPSC-based applications are often limited by the variability among iPSC lines originating from a single donor, as well as the heterogeneity among specific cell types that can be derived from iPSCs. The ability to deeply phenotype different iPSC-derived cell types is therefore of primary importance to the successful and informative application of this technology. Here we describe a combination of motor neuron (MN) derivation and single-cell RNA sequencing approaches to generate and characterize specific MN subtypes obtained from human iPSCs. Our studies provide evidence for rapid and robust generation of MN progenitor cells that can give rise to a heterogenous population of MNs. Approximately 58% of human iPSC-derived MNs display molecular characteristics of lateral motor column MNs, with a number of molecularly distinct subpopulations present within this MN group. Roughly 19% of induced MNs resemble hypaxial motor column MNs, while ∼6% of induced MNs have features of median motor column MNs. The present study has the potential to improve our understanding of iPSC-derived MN subtype function and dysfunction, possibly leading to improved iPSC-based applications for the study of human MN biology and disease.