GLA-modified RNA treatment lowers GB3 levels in iPSC-derived cardiomyocytes from Fabry-affected individuals

Recent studies in non-human model systems have shown therapeutic potential of nucleoside-modified messenger RNA (modRNA) treatments for lysosomal storage diseases. Here, we assessed the efficacy of a modRNA treatment to restore the expression of the galactosidase alpha (GLA), which codes for α-Galactosidase A (α-GAL) enzyme, in a human cardiac model generated from induced pluripotent stem cells (iPSCs) derived from two individuals with Fabry disease. Consistent with the clinical phenotype, cardiomyocytes from iPSCs derived from Fabry-affected individuals showed accumulation of the glycosphingolipid Globotriaosylceramide (GB3), which is an α-galactosidase substrate. Furthermore, the Fabry cardiomyocytes displayed significant upregulation of lysosomal-associated proteins. Upon GLA modRNA treatment, a subset of lysosomal proteins were partially restored to wild-type levels, implying the rescue of the molecular phenotype associated with the Fabry genotype. Importantly, a significant reduction of GB3 levels was observed in GLA modRNA-treated cardiomyocytes, demonstrating that α-GAL enzymatic activity was restored. Together, our results validate the utility of iPSC-derived cardiomyocytes from affected individuals as a model to study disease processes in Fabry disease and the therapeutic potential of GLA modRNA treatment to reduce GB3 accumulation in the heart.</p

Sex-specific control of human heart maturation by the progesterone receptor

Background: Despite in-depth knowledge of the molecular mechanisms controlling embryonic heart development, little is known about the signals governing postnatal maturation of the human heart. Methods: Single nucleus RNA-sequencing (snRNA-seq) of 54,140 nuclei from 9 human donors was used to profile transcriptional changes in diverse cardiac cell types during maturation from fetal stages to adulthood. Bulk RNA-sequencing and the assay for transposase-accessible chromatin using sequencing (ATAC-seq) were used to further validate transcriptional changes and to profile alterations in the chromatin accessibility landscape in purified cardiomyocyte nuclei from 21 human donors. Functional validation studies of sex steroids implicated in cardiac maturation were performed in human pluripotent stem cell-derived cardiac organoids and mice. Results: Our data identify the progesterone receptor as a key mediator of sex-dependent transcriptional programs during cardiomyocyte maturation. Functional validation studies in human cardiac organoids and mice demonstrate the progesterone receptor drives sex-specific metabolic programs and maturation of cardiac contractile properties. Conclusions: These data provide a blueprint for understanding human heart maturation in both sexes and reveal an important role for the progesterone receptor in human heart development.Choon Boon Sim, Belinda Phipson, Mark Ziemann, Haloom Rafehi, Richard J. Mills, Kevin I. Watt ... et al

A reference human induced pluripotent stem cell line for large-scale collaborative studies

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field

Stem Cells, Self-Renewal, and Lineage Commitment in the Endocrine System

The endocrine system coordinates a wide array of body functions mainly through secretion of hormones and their actions on target tissues. Over the last decades, a collective effort between developmental biologists, geneticists, and stem cell biologists has generated a wealth of knowledge related to the contribution of stem/progenitor cells to both organogenesis and self-renewal of endocrine organs. This review provides an up-to-date and comprehensive overview of the role of tissue stem cells in the development and self-renewal of endocrine organs. Pathways governing crucial steps in both development and stemness maintenance, and that are known to be frequently altered in a wide array of endocrine disorders, including cancer, are also described. Crucially, this plethora of information is being channeled into the development of potential new cell-based treatment modalities for endocrine-related illnesses, some of which have made it through clinical trials

Alpha-protein kinase 3 (ALPK3)-truncating variants are a cause of autosomal dominant hypertrophic cardiomyopathy.

AIMS: The aim of this study was to determine the frequency of heterozygous truncating ALPK3 variants (ALPK3tv) in patients with hypertrophic cardiomyopathy (HCM) and confirm their pathogenicity using burden testing in independent cohorts and family co-segregation studies. METHODS AND RESULTS : In a discovery cohort of 770 index patients with HCM, 12 (1.56%) were heterozygous for ALPK3tv [odds ratio(OR) 16.01, 95% confidence interval (CI) 7.89-29.74, P < 8.36e-11] compared to the Genome Aggregation Database (gnomAD) population. In a validation cohort of 2047 HCM probands, 32 (1.56%) carried heterozygous ALPK3tv (OR 16.17, 95% CI 10.31-24.87, P < 2.2e-16, compared to gnomAD). Combined logarithm of odds score in seven families with ALPK3tv was 2.99. In comparison with a cohort of genotyped patients with HCM (n = 1679) with and without pathogenic sarcomere gene variants (SP+ and SP-), ALPK3tv carriers had a higher prevalence of apical/concentric patterns of hypertrophy (60%, P < 0.001) and of a short PR interval (10%, P = 0.009). Age at diagnosis and maximum left ventricular wall thickness were similar to SP- and left ventricular systolic impairment (6%) and non-sustained ventricular tachycardia (31%) at baseline similar to SP+. After 5.3 ± 5.7 years, 4 (9%) patients with ALPK3tv died of heart failure or had cardiac transplantation (log-rank P = 0.012 vs. SP- and P = 0.425 vs. SP+). Imaging and histopathology showed extensive myocardial fibrosis and myocyte vacuolation. CONCLUSIONS : Heterozygous ALPK3tv are pathogenic and segregate with a characteristic HCM phenotype

Development of Functional Thyroid C Cell-like Cells from Human Pluripotent Cells in 2D and in 3D Scaffolds

Medullary thyroid carcinoma contributes to about 3–4% of thyroid cancers and affects C cells rather than follicular cells. Thyroid C cell differentiation from human pluripotent stem cells has not been reported. We report the stepwise differentiation of human embryonic stem cells into thyroid C cell-like cells through definitive endoderm and anterior foregut endoderm and ultimobranchial body-like intermediates in monolayer and 3D Matrigel culture conditions. The protocol involved sequential treatment with interferon/transferrin/selenium/pyruvate, foetal bovine serum, and activin A, then IGF-1 (Insulin-like growth factor 1), on the basis of embryonic thyroid developmental sequence. As well as expressing C cell lineage relative to follicular-lineage markers by qPCR (quantitative polymerase chain reaction) and immunolabelling, these cells by ELISA (enzyme-linked immunoassay) exhibited functional properties in vitro of calcitonin storage and release of calcitonin on calcium challenge. This method will contribute to developmental studies of the human thyroid gland and facilitate in vitro modelling of medullary thyroid carcinoma and provide a valuable platform for drug screening

Multiple endocrine neoplasia type 2B: modelling the disease in human cells and avian embryos

© 2017 Dr Kwaku Dad Abu-BonsrahPaediatric cancer initiation is difficult to study because the early stages are often prenatal. Patient cells at the time of detection already have complex genetic (including epigenetic) changes, and the cancer cell population is heterogeneous. One way to model cancer initiation at the organism level is to engineer the specific initiating mutation into the appropriate cell lineage of an experimental animal embryo. For the human cell context, an ideal cell model would start with the normal human cell of origin and create candidate initiating mutations. Multiple Endocrine Neoplasia type 2B (MEN2B) is an autosomal dominant complex oncologic disease of the neural crest (NC) cell lineage, a so-called neurocristopathy. It presents with i) multiple mucosal ganglioneuromas including hyperplasia of the enteric (gut) nervous system leading to gastrointestinal disorders, ii) pheochromocytoma, with sympathoadrenal (SA) hyperplasia and catecholamine disturbance, and iii) medullary thyroid carcinoma with C-cell hyperplasia, elevated calcitonin and calcium metabolism disturbance. In addition, patients have marfanoid facial features. MEN2B results from de novo germline gain-of-function mutations in the gene RET, most often M918T. RET codes for the signalling receptor for the growth factor ligand GDNF, hence in MEN2B cells, RET signalling is divorced from GDNF availability. MEN2B is rare but it is often misdiagnosed especially early in life. This is due to the nature and diversity of the lineages affected; SA and enteric NC-lineage cells and thyroid C-cells, the latter being of foregut endodermal entero-endocrine lineage. We were the first to successfully use CRISPR/Cas9 to mutate the developing chicken embryo in vivo, showing phenotypic abnormality. This included creating a single point mutation by homology directed repair in vivo in NC cells at the avian MEN2B homologous site (M910T). For the human cell context, we combined the CRISPR/Cas9 technology and knowledge of embryo development and cell differentiation to create MEN2B M918T cells using the human embryonic stem cell (hESC) lines H9, HES3 and MEL2. We modified a hESC differentiation protocol to produce enteric NC-like cells, showing in vitro that these cells upregulated key NC and enteric genes. Functionally we also showed higher proliferation and greater axon production in the MEN2B mutant cells: this is consistent with the ganglioneuroma phenotype. In addition, we developed a new differentiation protocol to produce human SA progenitors and medullary chromaffin-like cells, as marked by expression of key catecholamine genes TH and PNMT, and expression of adrenaline and noradrenaline by HPLC. These cells are affected in MEN2B patient pheochromocytoma. We then developed a novel differentiation protocol for thyroid C-cell-like cells from hESCs via Definitive Endodermal Cells. These cells produce Calcitonin and we validated their functionality by ELISA assay and compared the MEN2B clones with the control hESCs. These cells are affected in MEN2B patients resulting in medullary thyroid carcinoma