Human pancreatic islets of Langerhans contain five distinct endocrine cell types, each producing a characteristic hormone. The dysfunction or loss of the insulin-producing β cells causes diabetes ...mellitus, a disease that harms millions. Until now, β cells were generally regarded as a single, homogenous cell population. Here we identify four antigenically distinct subtypes of human β cells, which we refer to as β1-4, and which are distinguished by differential expression of ST8SIA1 and CD9. These subpopulations are always present in normal adult islets and have diverse gene expression profiles and distinct basal and glucose-stimulated insulin secretion. Importantly, the β cell subtype distribution is profoundly altered in type 2 diabetes. These data suggest that this antigenically defined β cell heterogeneity is functionally and likely medically relevant.
The erythropoietin receptor (EpoR) is widely expressed but its renoprotective action is unexplored. To examine the role of EpoR in vivo in the kidney, we induced acute kidney injury (AKI) by ...ischemia-reperfusion in mice with different EpoR bioactivities in the kidney. EpoR bioactivity was reduced by knockin of wild-type human EpoR, which is hypofunctional relative to murine EpoR, and a renal tubule-specific EpoR knockout. These mice had lower EPO/EpoR activity and lower autophagy flux in renal tubules. Upon AKI induction, they exhibited worse renal function and structural damage, more apoptosis at the acute stage (<7 days), and slower recovery with more tubulointerstitial fibrosis at the subacute stage (14 days). In contrast, mice with hyperactive EpoR signaling from knockin of a constitutively active human EpoR had higher autophagic flux, milder kidney damage, and better renal function at the acute stage but, surprisingly, worse tubulointerstitial fibrosis and renal function at the subacute stage. Either excess or deficient EpoR activity in the kidney was associated with abnormal peritubular capillaries and tubular hypoxia, creating a "U-shaped" relationship. The direct effects of EpoR on tubular cells were confirmed in vitro by a hydrogen peroxide model using primary cultured proximal tubule cells with different EpoR activities. In summary, normal erythropoietin (EPO)/EpoR signaling in renal tubules provides defense against renal tubular injury maintains the autophagy-apoptosis balance and peritubular capillary integrity. High and low EPO/EpoR bioactivities both lead to vascular defect, and high EpoR activity overides the tubular protective effects in AKI recovery.
Novel cell surface-reactive monoclonal antibodies generated against extrahepatic biliary cells were developed for the isolation and characterization of different cell subsets from normal adult human ...gallbladder. Eleven antigenically distinct gallbladder subpopulations were isolated by fluorescence-activated cell sorting. They were classified into epithelial, mesenchymal, and pancreatobiliary (PDX1+SOX9+) subsets based on gene expression profiling. These antigenically distinct human gallbladder cell subsets could potentially also reflect different functional properties in regards to bile physiology, cell renewal and plasticity. Three of the novel monoclonal antibodies differentially labeled archival sections of primary carcinoma of human gallbladder relative to normal tissue. The novel monoclonal antibodies described herein enable the identification and characterization of antigenically diverse cell subsets within adult human gallbladder and are putative tumor biomarkers.
•Novel surface-reactive monoclonal antibodies (mAbs) were made from adult human gallbladder.•Antigenically distinct gallbladder subpopulations were isolated using these mAbs by FACS.•Three mAbs may be used to identify biomarkers for gallbladder carcinoma.
► Novel monoclonal antibodies marking murine pancreatic cell types are described. ► Live cell isolation of alpha, beta and exocrine cells by FACS is demonstrated. ► DGKB, GPM6A, TTR, DPPIV show alpha ...or beta cell selective expression. ► Labeling changes from E14.5−E18.5 show early or late developmental marking.
Tools permitting the isolation of live pancreatic cell subsets for culture and/or molecular analysis are limited. To address this, we developed a collection of monoclonal antibodies with selective surface labeling of endocrine and exocrine pancreatic cell types. Cell type labeling specificity and cell surface reactivity were validated on mouse pancreatic sections and by gene expression analysis of cells isolated using FACS. Five antibodies which marked populations of particular interest were used to isolate and study viable populations of purified pancreatic ducts, acinar cells, and subsets of acinar cells from whole pancreatic tissue or of alpha or beta cells from isolated mouse islets. Gene expression analysis showed the presence of known endocrine markers in alpha and beta cell populations and revealed that TTR and DPPIV are primarily expressed in alpha cells whereas DGKB and GPM6A have a beta cell specific expression profile.
Human pancreatic islets of Langerhans contain five distinct endocrine cell types, each producing a characteristic hormone. The dysfunction or loss of the insulin-producing β cells causes diabetes ...mellitus, a disease that harms millions. Until now, β cells were generally regarded as a single, homogenous cell population. Here we identify four antigenically distinct subtypes of human β cells, which we refer to as β1-4, and which are distinguished by differential expression of ST8SIA1 and CD9. These subpopulations are always present in normal adult islets and have diverse gene expression profiles and distinct basal and glucose-stimulated insulin secretion. Importantly, the β cell subtype distribution is profoundly altered in type 2 diabetes. These data suggest that this antigenically defined β cell heterogeneity is functionally and likely medically relevant.
The human endocrine pancreas consists of multiple cell types and plays a critical role in glucose homeostasis. Here, we apply mass cytometry technology to measure all major islet hormones, ...proliferative markers, and readouts of signaling pathways involved in proliferation at single-cell resolution. Using this innovative technology, we simultaneously examined baseline proliferation levels of all endocrine cell types from birth through adulthood, as well as in response to the mitogen harmine. High-dimensional analysis of our marker protein expression revealed three major clusters of beta cells within individuals. Proliferating beta cells are confined to two of the clusters.
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•Mass cytometry promotes high-throughput phenotyping of islets at a single-cell level•Alpha cells maintain higher basal proliferation and are more responsive to mitogens•Beta cells exist in distinct states
Wang et al. use innovative mass cytometry to examine proliferation and markers of signaling pathways at single-cell resolution in human islets. The basal proliferation rate of endocrine cells declines with age, with alpha cells maintaining higher replication potential. Beta cells cluster into three distinct groups, two of which include proliferating beta cells.
In multiple sulfatase deficiency (MSD), a human inherited disorder, the activities of all sulfatases are impaired due to a defect in posttranslational modification. Here we report the identification, ...by functional complementation using microcell-mediated chromosome transfer, of a gene that is mutated in MSD and is able to rescue the enzymatic deficiency in patients' cell lines. Functional conservation of this gene was observed among distantly related species, suggesting a critical biological role. Coexpression of
SUMF1 with sulfatases results in a strikingly synergistic increase of enzymatic activity, indicating that
SUMF1 is both an essential and a limiting factor for sulfatases. These data have profound implications on the feasibility of enzyme replacement therapy for eight distinct inborn errors of metabolism.
Metachromatic leukodystrophy (MLD) is a demyelinating lysosomal storage disorder for which new treatments are urgently needed. We previously showed that transplantation of gene-corrected ...hematopoietic stem progenitor cells (HSPCs) in presymptomatic myeloablated MLD mice prevented disease manifestations. Here we show that HSC gene therapy can reverse neurological deficits and neuropathological damage in affected mice, thus correcting an overt neurological disease. The efficacy of gene therapy was dependent on and proportional to arylsulfatase A (ARSA) overexpression in the microglia progeny of transplanted HSPCs. We demonstrate a widespread enzyme distribution from these cells through the CNS and a robust cross-correction of neurons and glia in vivo. Conversely, a peripheral source of enzyme, established by transplanting ARSA-overexpressing hepatocytes from transgenic donors, failed to effectively deliver the enzyme to the CNS. These results indicate that the recruitment of gene-modified, enzyme-overexpressing microglia makes the enzyme bioavailable to the brain and makes therapeutic efficacy and disease correction attainable. Overall, our data provide a strong rationale for implementing HSPC gene therapy in MLD patients.
Fanconi anemia (FA), a genetic disorder affecting DNA repair, is characterized by bone marrow failure and cancer susceptibility. In FA mouse models the biguanide metabolic agent metformin improves ...blood counts and delays tumor development. We conducted a single institution pilot study of metformin in non-diabetic patients with FA to assess feasibility and tolerability of metformin treatment and to determine whether metformin could improve blood counts. Fourteen of 15 patients with at least 1 cytopenia (hemoglobin <10g/dL, platelet count <100K cells/µL, or an absolute neutrophil count <1K cells/µL) were eligible to receive metformin for 6 months. Median patient age was 9.4 years (range 6.0-26.5), and 8/14 were male (57%). Thirteen of 14 subjects (93%) tolerated maximal dosing for age; one subject had dose reduction for grade 2 gastrointestinal symptoms. No subjects developed hypoglycemia or metabolic acidosis. No subjects had dose interruptions due to toxicity, and no grade 3 or higher adverse events attributed to metformin were observed. Hematologic response (HR) based on modified MDS IWG criteria was observed in 4 of 13 evaluable patients (30.8%, 90% CI:11.3 to 57.3). Median time to response was 84.5 days (range 71-128). Responses were noted in neutrophils (n=3), platelets (n=1), and red blood cells (n=1). No subjects met criteria for disease progression or relapse during treatment. Correlative studies explored potential mechanisms of metformin activity in FA. Plasma proteomics showed reduction in inflammatory pathways with metformin. We conclude that metformin is safe and tolerable in non-diabetic patients with FA and may provide therapeutic benefit. This trial is registered at www.clinicaltrials.gov as NCT03398824.
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