Fear-of-intimacy-mediated zinc transport controls fat body cell dissociation through modulating Mmp activity in Drosophila

Matrix metalloproteinases (Mmps) are pivotal extracellular proteinases that have been implicated in tumour invasion and metastasis. Drosophila fat body is important for energy storage and utilization, as well as biosynthetic and metabolic activities. The fat body undergoes remodelling during metamorphosis which is characterized by the dissociation of the fat body into individual cells. Mmps play important roles in the regulation of fat body cell dissociation. Here we show that a zinc transporter fear-of-intimacy (foi) is necessary for the cell dissociation of fat body in Drosophila. The progression of fat body cell dissociation was delayed by fat body-specific foi knockdown while it was accelerated by foi overexpression (OE). In essence, these phenotypes are closely associated with intracellular zinc homeostasis, which can be modulated by dietary zinc intervention or genetic modulation of other zinc transporters. Further study indicated that Mmp1 and Mmp2 levels could be transcriptionally regulated by zinc in vivo. Consistently, the retarded fat body cell dissociation caused by Mmp1 or Mmp2 RNAi could be regulated by modulating the expression of foi. Further, by using Drosophila models of malignant tumour RafGOFscrib−/− and RasV12lgl−/−, we showed that the tumour growth, invasion and migration could be markedly inhibited by foi knockdown. These findings demonstrate a close connection between zinc levels and cell dissociation in vivo, and also suggest that manipulation of zinc levels may provide a novel therapeutic strategy for cancer.

Human Thymus single cell dissociation protocol - Teichmann Lab v1

This Protocol is intended for human Thymus single cell dissociation. It includes tissue preservation and handling, Enzymatic dissociation, FACS an MACS enrichments. Developed In the Teichmann lab at the Sanger institute, Wellcome Gemone Campus, UK VK, CS and JP contributed equally

Current Methodological Challenges of Single-Cell and Single-Nucleus RNA-Sequencing in Glomerular Diseases

Single-cell RNA-sequencing (scRNA-seq) and single-nucleus RNA-sequencing (snRNA-seq) allow transcriptomic profiling of thousands of cells from a renal biopsy at single-cell resolution. Both methods are promising tools to unravel the underlying pathophysiology of glomerular diseases. This review provides an overview of the technical challenges that should be addressed when designing single-cell transcriptomics experiments that focus on glomerulopathies. The isolation of glomerular cells from core needle biopsies for single-cell transcriptomics remains difficult and depends upon five major factors. First, core needle biopsies generate little tissue material and several samples are required to identify glomerular cells. Second, both fresh and frozen tissue samples may yield glomerular cells, although every experimental pipeline has different (dis)advantages. Third, enrichment for glomerular cells in human tissue prior to single-cell analysis is challenging as no effective standardized pipelines are available. Fourth, the current warm cell dissociation protocols may damage glomerular cells and induce transcriptional artefacts, which can be minimized by using cold dissociation techniques at a cost of less efficient cell dissociation. Finally, snRNA-seq methods may be superior to scRNA-seq in isolating glomerular cells, although its efficacy on core needle biopsies remains to be proven. The field of single-cell omics is rapidly evolving and the integration of these techniques in multi-omics assays will undoubtedly create new insights in the complex pathophysiology of glomerular diseases.

Paediatric gastric organoids as a tool for disease modelling and clinical translation

Purpose Knowledge of gastric epithelial homeostasis remains incomplete, lacking human-specific models for study. This study establishes a protocol for deriving gastric epithelial organoids from paediatric gastric biopsies, providing a platform for modelling disease and developing translational therapies. Methods Full-thickness surgical samples and endoscopic mucosal biopsies were obtained from six patients. Gastric glands were isolated by a chemical chelation protocol and then plated in 3D culture in Matrigel® droplets in chemically defined medium. After formation, organoids were passaged by single cell dissociation or manual disaggregation. Cell composition and epithelial polarity of organoids were assessed by bright field microscopy and immunofluorescence analysis, comparing them to native paediatric gastric tissue. Results Gastric glands were successfully isolated from all six patients who were aged 4 months to 16 years. Gastric glands from all patients sealed to form spherical gastric organoids. These organoids could be passaged by manual disaggregation or single cell dissociation, remaining proliferative up to 1 year in culture. Organoids retained normal epithelial cell polarity, with the apical surface orientated towards the central lumen. Organoids expressed markers of mature gastric epithelial cell types, except for parietal cells. Conclusion Gastric organoids can be reliably generated from paediatric biopsies and are a representative in vitro model for studying gastric epithelium.

Alternative method for trypsin-based cell dissociation using poly (amino ester) coating and pH 6.0 PBS

To maintain the cellular functions of a stem cells for therapeutic tissue engineering, an advanced cell culture method for safe cell dissociation is necessary. We developed a novel cell dissociation method by applying pH-responsive bioreducible polymer on the surface of tissue culture plates (TCPs). We applied acid-responsive bioreducible poly (amino ester) (PAE) as a new candidate for surface coating method to develop alternative cell dissociation method against conventional enzyme (trypsin-ethylene diamine tetra acetic acid (EDTA)) treatment. Human adipose derived stem cells (hADSCs) were cultured on and dissociated from PAE-coated TCPs to compare cell adhesion, cell proliferation, cell viability, and functionality to those of the cells cultured on and dissociated with trypsin-EDTA from normal TCPs without PAE coating. To confirm the in vivo therapeutic efficacy of the hADSCs retrieved from PAE-coated TCPs compared to that of the cells retrieved from normal TCPs with trypsin-EDTA, we induced skin defects at the dorsal area of mice and injected the cells collected from both conditions. With the PAE coating method, cell adhesion, cell proliferation, cell viability, and functionality, especially the angiogenic efficacy, were well preserved when compared to those of the cells treated with trypsin-EDTA. In addition to in vitro results, injecting hADSCs retrieved from PAE-coated TCPs showed similar in vivo angiogenesis and wound closing efficiency compared to those of injecting hADSCs retrieved from normal TCPs with trypsin-EDTA treatment at 2 weeks after the transplantation into mouse skin wound models. We proposed the alternative method for the cell dissociation with pH-responsive bioreducible polymer, PAE. This PAE coating method may lead to the development of alternative cell dissociation method without using enzyme for future regenerative medicine and stem cell therapy.