Generation of kisspeptin-responsive GnRH neurons from human pluripotent stem cells

© 2017 Elsevier B.V. GnRH neurons are fundamental for reproduction in all vertebrates, integrating all reproductive inputs. The inaccessibility of human GnRH-neurons has been a major impediment to studying the central control of reproduction and its disorders. Here, we report the efficient generation of kisspeptin responsive GnRH-secreting neurons by directed differentiation of human Embryonic Stem Cells and induced-Pluripotent Stem Cells derived from a Kallman Syndrome patient and a healthy family member. The protocol involves the generation of intermediate Neural Progenitor Cells (NPCs) through long-term Bone morphogenetic protein 4 inhibition, followed by terminal specification of these NPCs in media containing Fibroblast Growth Factor 8 and a NOTCH inhibitor. The resulting GnRH-expressing and -secreting neurons display a neuroendocrine gene expression pattern and present spontaneous calcium transients that can be stimulated by kisspeptin. These in vitro generated GnRH expressing cells provide a new resource for studying the molecular mechanisms underlying the development and function of GnRH neurons

Expression of progenitor markers is associated with the functionality of a bioartificial adrenal cortex

This work was in part supported by the International Fund Congenital Adrenal Hyperplasia (IFCAH) (to SRB), by the Deutsche Forschungsgemeinschaft (CRC-TRR 127 (to SRB and BL), CRC-TRR 205 (to SRB, BL and CS), IRTG 2251 (to SRB, BL and CS), and CRU 252 (to SRB and BL)), the German Academic Exchange Service (DAAD) (to GRB) and The TransCampus initiative between TU Dresden and King’s College London (to SRB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Modeling Congenital Adrenal Hyperplasia and Testing Interventions for Adrenal Insufficiency Using Donor-Specific Reprogrammed Cells.

Adrenal insufficiency is managed by hormone replacement therapy, which is far from optimal; the ability to generate functional steroidogenic cells would offer a unique opportunity for a curative approach to restoring the complex feedback regulation of the hypothalamic-pituitary-adrenal axis. Here, we generated human induced steroidogenic cells (hiSCs) from fibroblasts, blood-, and urine-derived cells through forced expression of steroidogenic factor-1 and activation of the PKA and LHRH pathways. hiSCs had ultrastructural features resembling steroid-secreting cells, expressed steroidogenic enzymes, and secreted steroid hormones in response to stimuli. hiSCs were viable when transplanted into the mouse kidney capsule and intra-adrenal. Importantly, the hypocortisolism of hiSCs derived from patients with adrenal insufficiency due to congenital adrenal hyperplasia was rescued by expressing the wild-type version of the defective disease-causing enzymes. Our study provides an effective tool with many potential applications for studying adrenal pathobiology in a personalized manner and opens venues for the development of precision therapies.Biotechnology and Biological Sciences Research Counci

Predicted Benign and Synonymous Variants in CYP11A1 Cause Primary Adrenal Insufficiency Through Missplicing.

Primary adrenal insufficiency (PAI) is a potentially life-threatening condition that can present with nonspecific features and can be difficult to diagnose. We undertook next generation sequencing in a cohort of children and young adults with PAI of unknown etiology from around the world and identified a heterozygous missense variant (rs6161, c.940G>A, p.Glu314Lys) in CYP11A1 in 19 individuals from 13 different families (allele frequency within undiagnosed PAI in our cohort, 0.102 vs 0.0026 in the Genome Aggregation Database; P A, Thr330 = ; c.1173C>T, Ser391 =). Although p.Glu314Lys is predicted to be benign and showed no loss-of-function in an Escherichia coli assay system, in silico and in vitro studies revealed that the rs6161/c.940G>A variant, plus the c.990G>A and c.1173C>T changes, affected splicing and that p.Glu314Lys produces a nonfunctional protein in mammalian cells. Taken together, these findings show that compound heterozygosity involving a relatively common and predicted "benign" variant in CYP11A1 is a major contributor to PAI of unknown etiology, especially in European populations. These observations have implications for personalized management and demonstrate how variants that might be overlooked in standard analyses can be pathogenic when combined with other very rare disruptive changes.Medical Research Council UK Project (grant MR/K020455/1 to L.A.M.).J.C.A. is a Wellcome Trust Senior Research Fellow in Clinical Science (grants 098513/Z/12/Z and 209328/Z/17/Z) with research support from Great Ormond Street Hospital Children’s Charity (grant V2518) and the National Institute for Health Research, Great Ormond Street Hospital Biomedical Research Centre (grant IS-BRC-1215-20012).Funding also included support from The Mater Medical Research Institute (to M.H.) and National Institutes of Health (grant R01GM086596 to R.J.A.)

New Horizons: Novel Adrenal Regenerative Therapies

Adrenal insufficiency requires lifelong corticoid replacement therapies. However, current therapies are not able to replace the physiological circadian pattern of the adrenal cortex and are associated with many metabolic, vascular, neuroendocrine and mental perturbations. Therefore, regenerative and more curative strategies would be desirable. In the current perspective, we describe emerging new regenerative therapies for the treatment of adrenal insufficiency. Especially, we discuss gene therapy and cell replacement strategies. Furthermore, we discuss how adrenal cells might be used as a source for regenerative therapies of non-adrenal neurodegenerative diseases such as Parkinson's disease

Novel methods in adrenal research: A metabolomics approach.

Metabolic alterations have implications in a spectrum of tissue functions and disease. Aided by novel molecular biological and computational tools, our understanding of physiological and pathological processes underpinning endocrine and endocrine-related disease has significantly expanded over the last decade. Herein, we focus on novel metabolomics-related methodologies in adrenal research: in situ metabolomics by mass spectrometry imaging, steroid metabolomics by gas and liquid chromatography-mass spectrometry, energy pathway metabologenomics by liquid chromatography-mass spectrometry-based metabolomics of Krebs cycle intermediates, and cellular reprogramming to generate functional steroidogenic cells and hence to modulate the steroid metabolome. All four techniques to assess and/or modulate the metabolome in biological systems provide tremendous opportunities to manage neoplastic and non-neoplastic disease of the adrenal glands in the era of precision medicine. In this context, we discuss emerging clinical applications and/or promising metabolic-driven research towards diagnostic, prognostic, predictive and therapeutic biomarkers in tumours arising from the adrenal gland and extra-adrenal paraganglia as well as modern approaches to delineate and reprogram adrenal metabolism

Role of IGSF10 mutations in self-limited delayed puberty

Background Abnormal timing of puberty affects over 4% of adolescents and is associated with adverse health and psychosocial outcomes. Previous studies estimate that 60–80% of variation in the timing of pubertal onset is genetically determined. However, little is known about the genetic control of human puberty. Self-limited delayed puberty segregates in an autosomal dominant pattern; our study aimed to identify novel genetic regulators of disease in these patients. Methods We performed whole-exome sequencing in 18 families with self-limited delayed puberty from our cohort, followed by candidate gene sequencing in a further 42 families. The functional consequences of the identified mutations in one candidate gene were interrogated via expression of wild type and mutant proteins in mammalian cells. For this gene we defined tissue expression in human and mouse embryos. The effects of gene knockdown were assessed via in-vitro neuronal migration assays, and in vivo with a transgenic zebrafish model. Findings In ten unrelated families, we identified four rare mutations in IGSF10 in individuals with self-limited delayed puberty (adjusted p value after rare variant burden testing=3·4 × 10–2). The identified mutations were in evolutionarily conserved positions, and two mutations resulted in intracellular retention with failure in secretion of the N-terminal fragment of the protein. IGSF10 mRNA was strongly expressed in the nasal mesenchyme in mouse and human embryos during migration of gonadotropin-releasing hormone (GnRH) neurons from their nasal origin towards the hypothalamus. IGSF10 knockdown caused reduced migration of immature GnRH neurons in the in-vitro analysis, and perturbed migration and extension of GnRH neurons in the zebrafish model. Interpretation Our findings strongly support the contention that mutations in IGSF10 cause delayed puberty in human beings, through misregulation of GnRH neuronal migration during embryonic development

Whole exome sequencing identifies deleterious rare variants in CCDC141 in familial self-limited delayed puberty

Developmental abnormalities of the gonadotropin-releasing hormone (GnRH) neuronal network result in a range of conditions from idiopathic hypogonadotropic hypogonadism to self-limited delayed puberty. We aimed to discover important underlying regulators of self-limited delayed puberty through interrogation of GnRH pathways. Whole exome sequencing (WES) data consisting of 193 individuals, from 100 families with self-limited delayed puberty, was analysed using a virtual panel of genes related to GnRH development and function (n = 12). Five rare predicted deleterious variants in Coiled-Coil Domain Containing 141 (CCDC141) were identified in 21 individuals from 6 families (6% of the tested cohort). Homology modeling predicted all five variants to be deleterious. CCDC141 mutant proteins showed atypical subcellular localization associated with abnormal distribution of acetylated tubulin, and expression of mutants resulted in a significantly delayed cell migration, demonstrated in transfected HEK293 cells. These data identify mutations in CCDC141 as a frequent finding in patients with self-limited delayed puberty. The mis-localization of acetylated tubulin and reduced cell migration seen with mutant CCDC141 suggests a role of the CCDC141-microtubule axis in GnRH neuronal migration, with heterozygous defects potentially impacting the timing of puberty

Stress-inducible-stem cells: a new view on endocrine, metabolic and mental disease?

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