Search

Efficient isolation of neurons and glia from the human enteric nervous system (ENS) is challenging because of their rare and fragile nature. Here, we describe a staining panel to enrich ENS cells from the human intestine by fluorescence-activated cell sorting (FACS). We find that CD56/CD90/CD24 co-expression labels ENS cells with higher specificity and resolution than previous methods. Surprisingly, neuronal (CD24, TUBB3) and glial (SOX10) selective markers appear co-expressed by all ENS cells. We demonstrate that this contradictory staining pattern is mainly driven by neuronal fragments, either free or attached to glial cells, which are the most abundant cell types. Live neurons can be enriched by the highest CD24 and CD90 levels. By applying our protocol to isolate ENS cells for single-cell RNA sequencing, we show that these cells can be obtained with high quality, enabling interrogation of the human ENS transcriptome. Taken together, we present a selective FACS protocol that allows enrichment and discrimination of human ENS cells, opening up new avenues to study this complex system in health and disease.

Pompe disease is an inherited metabolic myopathy caused by deficiency of acid alpha-glucosidase (GAA), resulting in lysosomal glycogen accumulation. Residual GAA enzyme activity affects disease onset and severity, although other factors, including dysregulation of cytoplasmic glycogen metabolism, are suspected to modulate the disease course. In this study, performed in mice and patient biopsies, we found elevated protein levels of enzymes involved in glucose uptake and cytoplasmic glycogen synthesis in skeletal muscle from mice with Pompe disease, including glycogenin (GYG1), glycogen synthase (GYS1), glucose transporter 4 (GLUT4), glycogen branching enzyme 1 (GBE1), and UDP-glucose pyrophosphorylase (UGP2). Expression levels were elevated before the loss of muscle mass and function. For first time, quantitative mass spectrometry in skeletal muscle biopsies from five adult patients with Pompe disease showed increased expression of GBE1 protein relative to healthy controls at the group level. Paired analysis of individual patients who responded well to treatment with enzyme replacement therapy (ERT) showed reduction of GYS1, GYG1, and GBE1 in all patients after start of ERT compared to baseline. These results indicate that metabolic changes precede muscle wasting in Pompe disease, and imply a positive feedforward loop in Pompe disease, in which lysosomal glycogen accumulation promotes cytoplasmic glycogen synthesis and glucose uptake, resulting in aggravation of the disease phenotype.

Bone marrow-derived mesenchymal stem/stromal cells (BMSCs) are fundamental to bone regenerative therapies, tissue engineering, and postmenopausal osteoporosis. Donor variation among patients, cell heterogeneity, and unpredictable capacity for differentiation reduce effectiveness of BMSCs for regenerative cell therapies. The cell surface glycoprotein CD24 exhibits the most prominent differential expression during osteogenic versus adipogenic differentiation of human BMSCs. Therefore, CD24 may represent a selective biomarker for subpopulations of BMSCs with increased osteoblastic potential. In undifferentiated human BMSCs, CD24 cell surface expression is variable among donors (range: 2%-10%) and increased by two to fourfold upon osteogenic differentiation. Strikingly, FACS sorted CD24 pos cells exhibit delayed mineralization and reduced capacity for adipocyte differentiation. RNAseq analysis of CD24 pos and CD24 neg BMSCs identified a limited number of genes with increased expression in CD24 pos cells that are associated with cell adhesion, motility, and extracellular matrix. Downregulated genes are associated with cell cycle regulation, and biological assays revealed that CD24 pos cells have reduced proliferation. Hence, expression of the cell surface glycoprotein CD24 identifies a subpopulation of human BMSCs with reduced capacity for proliferation and extracellular matrix mineralization. Functional specialization among BMSCs populations may support their regenerative potential and therapeutic success by accommodating cell activities that promote skeletal tissue formation, homeostasis, and repair.