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Changes in gut microbiota composition and a diverse role of B cells have recently been implicated in multiple sclerosis (MS), a central nervous system (CNS) autoimmune disease. Immunoglobulin A (IgA) is a key regulator at the mucosal interface. However, whether gut microbiota shape IgA responses and what role IgA+ cells have in neuroinflammation are unknown. Here, we identify IgA-bound taxa in MS and show that IgA-producing cells specific for MS-associated taxa traffic to the inflamed CNS, resulting in a strong, compartmentalized IgA enrichment in active MS and other neuroinflammatory diseases. Unlike previously characterized polyreactive anti-commensal IgA responses, CNS IgA cross-reacts with surface structures on specific bacterial strains but not with brain tissue. These findings establish gut microbiota-specific IgA+ cells as a systemic mediator in MS and suggest a critical role of mucosal B cells during active neuroinflammation with broad implications for IgA as an informative biomarker and IgA-producing cells as an immune subset to harness for therapeutic interventions.

Multiple sclerosis (MS) is a neuroinflammatory disease with a relapsing-remitting disease course at early stages, distinct lesion characteristics in cortical grey versus subcortical white matter and neurodegeneration at chronic stages. Here we used single-nucleus RNA sequencing to assess changes in expression in multiple cell lineages in MS lesions and validated the results using multiplex in situ hybridization. We found selective vulnerability and loss of excitatory CUX2-expressing projection neurons in upper-cortical layers underlying meningeal inflammation; such MS neuron populations exhibited upregulation of stress pathway genes and long non-coding RNAs. Signatures of stressed oligodendrocytes, reactive astrocytes and activated microglia mapped most strongly to the rim of MS plaques. Notably, single-nucleus RNA sequencing identified phagocytosing microglia and/or macrophages by their ingestion and perinuclear import of myelin transcripts, confirmed by functional mouse and human culture assays. Our findings indicate lineage- and region-specific transcriptomic changes associated with selective cortical neuron damage and glial activation contributing to progression of MS lesions.

BACKGROUND: The authors have developed a novel protocol for isolating adipose-derived stem cells from human lipoaspirate. In this study, they compare their new method to a previously published standard protocol. METHODS: Human adipose-derived stem cell isolation was performed using two methods to compare cell yield, cell viability, cell proliferation, and regenerative potential. The new and conventional isolation methods differ in two key areas: die collagenase digestion buffer constituents and the use of an orbital shaker. The osteogenic and adipogenic potential of adipose-derived stem cells isolated using both protocols was assessed in vitro, and gene expression analysis was performed. To assess the ability of the isolated cells to generate bone in vivo, die authors created critical-size calvarial defects in mice, which were treated with adipose-derived stem cells loaded onto hydroxyapatite-coated poly(lactic-co-glycolic acid) scaffolds. To test die ability of the isolated cells to enhance adipogenesis, die cells were added to lipoaspirate and placed beneath the scalp of immunocompromised mice. Fat graft volume retention was subsequently assessed by serial computed tomographic volumetric scanning. RESULTS: The new method resulted in a 10-fold increased yield of adipose-derived stem cells compared with die conventional method. Cells harvested using die new method demonstrated significantly increased cell viability and proliferation in vitro (p < 0.05). New method cells also demonstrated significantly enhanced osteogenic and adipogenic differentiation capacity in vitro (p < 0.05) in comparison with die conventional method cells. Both cell groups demonstrated equivalent osteogenic and adipogenic regenerative potential in mice. CONCLUSIONS: The authors have developed a protocol that maximizes the yield of adipose-derived stem cells derived from lipoaspirate. The new method cells have increased osteogenic and adipogenic potential in vitro and are not inferior to conventional method cells in terms of their ability to generate bone and fat in vivo. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Significant advances in our understanding of human obesity, endocrinology, and metabolism have been made possible by murine comparative models, in which anatomically analogous fat depots are utilized; however, current research has questioned how truly analogous these depots are. In this study, we assess the validity of the analogy from the perspective of cellular architecture. Whole tissue mounting, confocal microscopy, and image reconstruction software were used to characterize the three-dimensional structure of the inguinal fat pad in mice, gluteofemoral fat in humans, and subcutaneous adipose tissue of the human abdominal wall. Abdominal and gluteofemoral adipose tissue specimens from 12 human patients and bilateral inguinal fat pads from 12 mice were stained for adipocytes, blood vessels, and a putative marker for adipose-derived multipotent progenitor cells, cluster of differentiation 34 (CD34). Samples were whole-mounted and imaged with laser scanning confocal microscopy. Expectedly, human adipocytes were larger and demonstrated greater size heterogeneity. Mouse fat displayed significantly higher vascular density compared with human fat when normalized to adipocyte count. There was no significant difference in the concentration of CD34-positive (CD34(+)) stromal cells from either species. However, the mean distance between CD34(+) stromal cells and blood vessels was significantly greater in human fat. Finally, mouse inguinal fat contained larger numbers of brown adipocytes than did human gluteofemoral or human abdominal fat. Overall, the basic architecture of human adipose tissue differs significantly from that of mice. Insofar as human gluteofemoral fat differs from human abdominal adipose tissue, it was closer to mouse inguinal fat, being its comparative developmental analog. These differences likely confer variance in functional properties between the two sources and thus must be considered when designing murine models of human disease.

BACKGROUND Cell-assisted lipotransfer has shown much promise as a technique for improving fat graft take. However, the concentration of stromal vascular fraction cells required to optimally enhance fat graft retention remains unknown. METHODS Human lipoaspirate was processed for both fat transfer and harvest of stromal vascular fraction cells. Cells were then mixed back with fat at varying concentrations ranging from 10,000 to 10 million cells per 200 μl of fat. Fat graft volume retention was assessed by means of computed tomographic scanning over 8 weeks, and then fat grafts were explanted and compared histologically for overall architecture and vascularity. RESULTS Maximum fat graft retention was seen at a concentration of 10,000 cells per 200 μl of fat. The addition of higher number of cells negatively impacted fat graft retention, with supplementation of 10 million cells producing the lowest final volumes, lower than fat alone. Interestingly, fat grafts supplemented with 10,000 cells showed significantly increased vascularity and decreased inflammation, whereas fat grafts supplemented with 10 million cells showed significant lipodegeneration compared with fat alone CONCLUSIONS : The authors' study demonstrates dose dependence in the number of stromal vascular fraction cells that can be added to a fat graft to enhance retention. Although cell-assisted lipotransfer may help promote graft survival, this effect may need to be balanced with the increased metabolic load of added cells that may compete with adipocytes for nutrients during the postgraft period.