Resource:A Cellular Developmental Taxonomy of the Bone Marrow Mesenchymal Stem Cell Population in Mice

Mesenchymal stem cells (MSCs) play pivotal roles in tissue (re)generation. In the murine bone marrow, they are thought to reside within the Sca-1(+) CD51(+) bone marrow stromal cell population. Here, using scRNAseq, we aimed to delineate the cellularheterogeneity of this MSC-enriched population throughout development. At the fetal stage, the MSC population is relatively homogeneous with subsets predicted to contain stem/progenitor cells, based on transcriptional modeling and marker expression. These subsets decline in relative size throughout life, with postnatal emergence of specialized clusters, including hematopoietic stem/progenitor cell (HSPC) niches. In fetal development, these stromal HSPC niches are lacking, but subsets of endothelial cells express HSPC factors, suggesting that they may provide initial niches for emerging hematopoiesis. This cellular taxonomy of the MSC population upon development is anticipated to provide a resource aiding the prospective identification of cellular subsets and molecular mechanisms driving bone marrow (re)generation

Functional tests to guide management in an adult with loss of function of type-1 angiotensin II receptor

BACKGROUND: Genetic loss of function of AGT (angiotensinogen), REN (renin), ACE (angiotensin-converting enzyme), or AGTR1 (type-1 angiotensin II receptor) leads to renal tubular dysgenesis (RTD). This syndrome is almost invariably lethal. Most surviving patients reach stage 5 chronic kidney disease at a young age. METHODS: Here, we report a 28-year-old male with a homozygous truncating mutation in AGTR1 (p.Arg216*), who survived the perinatal period with a mildly impaired kidney function. In contrast to classic RTD, kidney biopsy showed proximal tubules that were mostly normal. During the subsequent three decades, we observed evidence of both tubular dysfunction (hyperkalemia, metabolic acidosis, salt-wasting and a urinary concentrating defect) and glomerular dysfunction (reduced glomerular filtration rate, currently ~30 mL/min/1.73 m(2), accompanied by proteinuria). To investigate the recurrent and severe hyperkalemia, we performed a patient-tailored functional test and showed that high doses of fludrocortisone induced renal potassium excretion by 155%. Furthermore, fludrocortisone lowered renal sodium excretion by 39%, which would have a mitigating effect on salt-wasting. In addition, urinary pH decreased in response to fludrocortisone. Opposite effects on urinary potassium and pH occurred with administration of amiloride, further supporting the notion that a collecting duct is present and able to react to fludrocortisone. CONCLUSIONS: This report provides living proof that even truncating loss-of-function mutations in AGTR1 are compatible with life and relatively good GFR and provides evidence for the prescription of fludrocortisone to treat hyperkalemia and salt-wasting in such patients. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00467-021-05018-7

Recurrent FXYD2 p.Gly41Arg mutation in patients with isolated dominant hypomagnesaemia

Background Magnesium (Mg2+) is an essential ion for cell growth, neuroplasticity and muscle contraction. Blood Mg2+ levels <0.7 mmol/L may cause a heterogeneous clinical phenotype, including muscle cramps and epilepsy and disturbances in K+ and Ca2+ homeostasis. Over the last decade, the genetic origin of several familial forms of hypomagnesaemia has been found. In 2000, mutations in FXYD2, encoding the γ-subunit of the Na+-K+-ATPase, were identified to cause isolated dominant hypomagnesaemia (IDH) in a large Dutch family suffering from hypomagnesaemia, hypocalciuria and chondrocalcinosis. However, no additional patients have been identified since then. Methods Here, two families with hypomagnesaemia and hypocalciuria were screened for mutations in the FXYD2 gene. Moreover, the patients were clinically and genetically characterized. Results We report a p.Gly41Arg FXYD2 mutation in two families with hypomagnesaemia and hypocalciuria. Interestingly, this is the same mutation as was described in the original study. As in the initial family, several patients suffered from muscle cramps, chondrocalcinosis and epilepsy. Haplotype analysis revealed an overlapping haplotype in all families, suggesting a founder effect. Conclusions The recurrent p.Gly41Arg FXYD2 mutation in two new families with IDH confirms that FXYD2 mutation causes hypomagnesaemia. Until now, no other FXYD2 mutations have been reported which could indicate that other FXYD2 mutations will not cause hypomagnesaemia or are embryonically letha

Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric neuromuscular disorders, highlighting the importance to understand the molecular mechanisms underlying GI development and dysfunction. In this study, we present a method for gut isolation from zebrafish larvae at 5 days post fertilization to obtain live, viable cells which can be used for single-cell RNA sequencing (scRNA-seq) analysis. This protocol is based on the manual dissection of the zebrafish intestine, followed by enzymatic dissociation with papain. Subsequently, cells are submitted to fluorescence-activated cell sorting, and viable cells are collected for scRNA-seq. With this method, we were able to successfully identify different intestinal cell types, including epithelial, stromal, blood, muscle, and immune cells, as well as enteric neurons and glia. Therefore, we consider it to be a valuable resource for studying the composition of the GI tract in health and disease, using the zebrafish

Efficient generation of ETX embryoids that recapitulate the entire window of murine egg cylinder development

The murine embryonic–trophoblast–extra-embryonic endoderm (ETX) model is an integrated stem cell–based model to study early postimplantation development. It is based on the self-assembly potential of embryonic, trophoblast, and hypoblast/primitive/visceral endoderm-type stem cell lines (ESC, TSC, and XEN, respectively) to arrange into postimplantation egg cylinder–like embryoids. Here, we provide an optimized method for reliable and efficient generation of ETX embryoids that develop into late gastrulation in static culture conditions. It is based on transgenic Gata6-overproducing ESCs and modified assembly and culture conditions. Using this method, up to 43% of assembled ETX embryoids exhibited a correct spatial distribution of the three stem cell derivatives at day 4 of culture. Of those, 40% progressed into ETX embryoids that both transcriptionally and morphologically faithfully mimicked in vivo postimplantation mouse development between E5.5 and E7.5. The ETX model system offers the opportunity to study the murine postimplantation egg cylinder stages and could serve as a source of various cell lineage precursors

Unbiased characterization of the larval zebrafish enteric nervous system at a single cell transcriptomic level

Summary: The enteric nervous system (ENS) regulates many gastrointestinal functions including peristalsis, immune regulation and uptake of nutrients. Defects in the ENS can lead to severe enteric neuropathies such as Hirschsprung disease (HSCR). Zebrafish have proven to be fruitful in the identification of genes involved in ENS development and HSCR pathogenesis. However, composition and specification of enteric neurons and glial subtypes at larval stages, remains mainly unexplored. Here, we performed single cell RNA sequencing of zebrafish ENS at 5 days post-fertilization. We identified vagal neural crest progenitors, Schwann cell precursors, and four clusters of differentiated neurons. In addition, a previously unrecognized elavl3+/phox2bb-population of neurons and cx43+/phox2bb-enteric glia was found. Pseudotime analysis supported binary neurogenic branching of ENS differentiation, driven by a notch-responsive state. Taken together, we provide new insights on ENS development and specification, proving that the zebrafish is a valuable model for the study of congenital enteric neuropathies

Deficiency of the ribosome biogenesis gene Sbds in hematopoietic stem and progenitor cells causes neutropenia in mice by attenuating lineage progression in myelocytes

Shwachman-Diamond syndrome is a congenital bone marrow failure disorder characterized by debilitating neutropenia. The disease is associated with loss-of-function mutations in the SBDS gene, implicated in ribosome biogenesis, but the cellular and molecular events driving cell specific phenotypes in ribosomopathies remain poorly defined. Here, we established what is to our knowledge the first mammalian model of neutropenia in Shwachman-Diamond syndrome through targeted downregulation of Sbds in hematopoietic stem and progenitor cells expressing the myeloid transcription factor CCAAT/enhancer binding protein a (Cebpa). Sbds deficiency in the myeloid lineage specifically affected myelocytes and their downstream progeny while, unexpectedly, it was well tolerated by rapidly cycling hematopoietic progenitor cells. Molecular insights provided by massive parallel sequencing supported cellular observations of impaired cell cycle exit and formation of secondary granules associated with the defect of myeloid lineage progression in myelocytes. Mechanistically, Sbds deficiency activated the p53 tumor suppressor pathway and induced apoptosis in these cells. Collectively, the data reveal a previously unanticipated, selective dependency of myelocytes and downstream progeny, but not rapidly cycling progenitors, on this ubiquitous ribosome biogenesis protein, thus providing a cellular basis for the understanding of myeloid lineage biased defects in Shwachman-Diamond syndrome

Comparing approaches to normalize, quantify, and characterize urinary extracellular vesicles

Background Urinary extracellular vesicles (uEVs) are a promising source for biomarker discovery, but optimal approaches for normalization, quantification, and characterization in spot urines are unclear.  Methods Urine samples were analyzed in a water-loading study, from healthy subjects and patients with kidney disease. Urine particles were quantified in whole urine using nanoparticle tracking analysis (NTA), time-resolved fluorescence immunoassay (TR-FIA), and EVQuant, a novel method quantifying particles via gel immobilization.  Results Urine particle and creatinine concentrations were highly correlated in the water-loading study (R2 0.96) and in random spot urines from healthy subjects (R2 0.47-0.95) and patients (R2 0.41-0.81). Water loading reduced aquaporin-2 but increased Tamm-Horsfall protein (THP) and particle detection by NTA. This finding was attributed to hypotonicity increasing uEV size (more EVs reach the NTA size detection limit) and reducing THP polymerization. Adding THP to urine also significantly increased particle count by NTA. In both fluorescence NTA and EVQuant, adding 0.01% SDS maintained uEV integrity and increased aquaporin-2 detection. Comparison of intracellular- and extracellular-epitope antibodies suggested the presence of reverse topology uEVs. The exosome markers CD9 and CD63 colocalized and immunoprecipitated selectively with distal nephron markers.  Conclusions uEV concentration is highly correlated with urine creatinine, potentially replacing the need for uEV quantification to normalize spot urines. Additional findings relevant for future uEV studies in whole urine include the interference of THP with NTA, excretion of larger uEVs in dilute urine, the ability to use detergent to increase intracellular-epitope recognition in uEVs, and CD9 or CD63 capture of nephron segment-specific EVs