Enriched Population of PNS Neurons Derived from Human Embryonic Stem Cells as a Platform for Studying Peripheral Neuropathies

BACKGROUND: The absence of a suitable cellular model is a major obstacle for the study of peripheral neuropathies. Human embryonic stem cells hold the potential to be differentiated into peripheral neurons which makes them a suitable candidate for this purpose. However, so far the potential of hESC to differentiate into derivatives of the peripheral nervous system (PNS) was not investigated enough and in particular, the few trials conducted resulted in low yields of PNS neurons. Here we describe a novel hESC differentiation method to produce enriched populations of PNS mature neurons. By plating 8 weeks hESC derived neural progenitors (hESC-NPs) on laminin for two weeks in a defined medium, we demonstrate that over 70% of the resulting neurons express PNS markers and 30% of these cells are sensory neurons. METHODS/FINDINGS: Our method shows that the hNPs express neuronal crest lineage markers in a temporal manner, and by plating 8 weeks hESC-NPs into laminin coated dishes these hNPs were promoted to differentiate and give rise to homogeneous PNS neuronal populations, expressing several PNS lineage-specific markers. Importantly, these cultures produced functional neurons with electrophysiological activities typical of mature neurons. Moreover, supporting this physiological capacity implantation of 8 weeks old hESC-NPs into the neural tube of chick embryos also produced human neurons expressing specific PNS markers in vivo in just a few days. Having the enriched PNS differentiation system in hand, we show for the first time in human PNS neurons the expression of IKAP/hELP1 protein, where a splicing mutation on the gene encoding this protein causes the peripheral neuropathy Familial Dysautonomia. CONCLUSIONS/SIGNIFICANCE: We conclude that this differentiation system to produce high numbers of human PNS neurons will be useful for studying PNS related neuropathies and for developing future drug screening applications for these diseases

Hereditary sensory and autonomic neuropathies: types II, III, and IV

The hereditary sensory and autonomic neuropathies (HSAN) encompass a number of inherited disorders that are associated with sensory dysfunction (depressed reflexes, altered pain and temperature perception) and varying degrees of autonomic dysfunction (gastroesophageal reflux, postural hypotention, excessive sweating). Subsequent to the numerical classification of four distinct forms of HSAN that was proposed by Dyck and Ohta, additional entities continue to be described, so that identification and classification are ongoing. As a group, the HSAN are rare diseases that affect both sexes. HSAN III is almost exclusive to individuals of Eastern European Jewish extraction, with incidence of 1 per 3600 live births. Several hundred cases with HSAN IV have been reported. The worldwide prevalence of HSAN type II is very low. This review focuses on the description of three of the disorders, HSAN II through IV, that are characterized by autosomal recessive inheritance and onset at birth. These three forms of HSAN have been the most intensively studied, especially familial dysautonomia (Riley-Day syndrome or HSAN III), which is often used as a prototype for comparison to the other HSAN. Each HSAN disorder is likely caused by different genetic errors that affect specific aspects of small fiber neurodevelopment, which result in variable phenotypic expression. As genetic tests are routinely used for diagnostic confirmation of HSAN III only, other means of differentiating between the disorders is necessary. Diagnosis is based on the clinical features, the degree of both sensory and autonomic dysfunction, and biochemical evaluations, with pathologic examinations serving to further confirm differences. Treatments for all these disorders are supportive

Mutations in DCHS1 Cause Mitral Valve Prolapse

SUMMARY Mitral valve prolapse (MVP) is a common cardiac valve disease that affects nearly 1 in 40 individuals1–3. It can manifest as mitral regurgitation and is the leading indication for mitral valve surgery4,5. Despite a clear heritable component, the genetic etiology leading to non-syndromic MVP has remained elusive. Four affected individuals from a large multigenerational family segregating non-syndromic MVP underwent capture sequencing of the linked interval on chromosome 11. We report a missense mutation in the DCHS1 gene, the human homologue of the Drosophila cell polarity gene dachsous (ds) that segregates with MVP in the family. Morpholino knockdown of the zebrafish homolog dachsous1b resulted in a cardiac atrioventricular canal defect that could be rescued by wild-type human DCHS1, but not by DCHS1 mRNA with the familial mutation. Further genetic studies identified two additional families in which a second deleterious DCHS1 mutation segregates with MVP. Both DCHS1 mutations reduce protein stability as demonstrated in zebrafish, cultured cells, and, notably, in mitral valve interstitial cells (MVICs) obtained during mitral valve repair surgery of a proband. Dchs1+/− mice had prolapse of thickened mitral leaflets, which could be traced back to developmental errors in valve morphogenesis. DCHS1 deficiency in MVP patient MVICs as well as in Dchs1+/− mouse MVICs result in altered migration and cellular patterning, supporting these processes as etiological underpinnings for the disease. Understanding the role of DCHS1 in mitral valve development and MVP pathogenesis holds potential for therapeutic insights for this very common disease

An Empirical Comparison of EM Initialization Methods and Model Choice Criteria for Mixtures of Skew-Normal Distributions Una comparación empírica de algunos métodos de inicialización EM y criterios de selección de modelos para mezclas de distribuciones normales asimetricas

We investigate, via simulation study, the performance of the EM algorithm for maximum likelihood estimation in finite mixtures of skew-normal distributions with component specific parameters. The study takes into account the initialization method, the number of iterations needed to attain a fixed stopping rule and the ability of some classical model choice criteria to estimate the correct number of mixture components. The results show that the algorithm produces quite reasonable estimates when using the method of moments to obtain the starting points and that, combining them with the AIC, BIC, ICL or EDC criteria, represents a good alternative to estimate the number of components of the mixture. Exceptions occur in the estimation of the skewness parameters, notably when the sample size is relatively small, and in some classical problematic cases, as when the mixture components are poorly separated.El presente artículo muestra un estudio de simulación que evalúa el desempeño del algoritmo EM utilizado para determinar estimaciones por máxima verosimilitud de los parámetros de la mezcla finita de distribuciones normales asimétricas. Diferentes métodos de inicialización, así como el número de interacciones necesarias para establecer una regla de parada especificada y algunos criterios de selección del modelo para permitir estimar el número apropiado de componentes de la mezcla han sido considerados. Los resultados indican que el algoritmo genera estimaciones razonables cuando los valores iniciales son obtenidos mediante el método de momentos, que junto con los criterios AIC, BIC, ICL o EDC constituyen una eficaz alternativa en la estimación del número de componentes de la mezcla. Resultados insatisfactorios se verificaron al estimar los parámetros de simetría, principalmente seleccionando un tamaño pequeño para la muestra, y en los casos conocidamente problemáticos en los cuales los componentes de la mezcla están suficientemente separados

Una comparación empírica de algunos métodos de inicialización em y criterios de selección de modelos para mezclas de distribuciones normales asimetricas

El presente artículo muestra un estudio de simulación que evalúa el desempeño del algoritmo EM utilizado para determinar estimaciones por máxima verosimilitud de los parámetros de la mezcla finita de distribuciones normales asimétricas. Diferentes métodos de inicialización, así como el número de interacciones necesarias para establecer una regla de parada especificada y algunos criterios de selección del modelo para permitir estimar el número apropiado de componentes de la mezcla han sido considerados. Los resultados indican que el algoritmo genera estimaciones razonables cuando los valores iniciales son obtenidos mediante el método de momentos, que junto con los criterios AIC, BIC, ICL o EDC constituyen una eficaz alternativa en la estimación del número de componentes de la mezcla. Resultados insatisfactorios se verificaron al estimar los parámetros de simetría, principalmente seleccionando un tamaño pequeño para la muestra, y en los casos conocidamente problemáticos en los cuales los componentes de la mezcla están suficientemente separados.We investigate, via simulation study, the performance of the EM algorithm for maximum likelihood estimation in finite mixtures of skew-normal distributions with component specific parameters. The study takes into account the initialization method, the number of iterations needed to attain a fixed stopping rule and the ability of some classical model choice criteria to estimate the correct number of mixture components. The results show that the algorithm produces quite reasonable estimates when using the method of moments to obtain the starting points and that, combining them with the AIC, BIC, ICL or EDC criteria, represents a good alternative to estimate the number of components of the mixture. Exceptions occur in the estimation of the skewness parameters, notably when the sample size is relatively small, and in some classical problematic cases, as when the mixture components are poorly separated

Loss of Mouse Ikbkap, a Subunit of Elongator, Leads to Transcriptional Deficits and Embryonic Lethality That Can Be Rescued by Human IKBKAP▿ †

Familial dysautonomia (FD), a devastating hereditary sensory and autonomic neuropathy, results from an intronic mutation in the IKBKAP gene that disrupts normal mRNA splicing and leads to tissue-specific reduction of IKBKAP protein (IKAP) in the nervous system. To better understand the roles of IKAP in vivo, an Ikbkap knockout mouse model was created. Results from our study show that ablating Ikbkap leads to embryonic lethality, with no homozygous Ikbkap knockout (Ikbkap−/−) embryos surviving beyond 12.5 days postcoitum. Morphological analyses of the Ikbkap−/− conceptus at different stages revealed abnormalities in both the visceral yolk sac and the embryo, including stunted extraembryonic blood vessel formation, delayed entry into midgastrulation, disoriented dorsal primitive neural alignment, and failure to establish the embryonic vascular system. Further, we demonstrate downregulation of several genes that are important for neurulation and vascular development in the Ikbkap−/− embryos and show that this correlates with a defect in transcriptional elongation-coupled histone acetylation. Finally, we show that the embryonic lethality resulting from Ikbkap ablation can be rescued by a human IKBKAP transgene. For the first time, we demonstrate that IKAP is crucial for both vascular and neural development during embryogenesis and that protein function is conserved between mouse and human

Therapeutic potential and mechanism of kinetin as a treatment for the human splicing disease familial dysautonomia.

Mutations that affect the splicing of pre-mRNA are a major cause of human disease. Familial dysautonomia (FD) is a recessive neurodegenerative disease caused by a T to C transition at base pair 6 of IKBKAP intron 20. This mutation results in variable tissue-specific skipping of exon 20. Previously, we reported that the plant cytokinin kinetin dramatically increases exon 20 inclusion in RNA isolated from cultured FD cells. The goal of the current study was to investigate the nature of the FD splicing defect and the mechanism by which kinetin improves exon inclusion, as such knowledge will facilitate the development of future therapeutics aimed at regulating mRNA splicing. In this study, we demonstrate that treatment of FD lymphoblast cell lines with kinetin increases IKBKAP mRNA and IKAP protein to normal levels. Using a series of minigene constructs, we show that deletion of a region at the end of IKBKAP exon 20 disrupts the ability of kinetin to improve exon inclusion, pinpointing a kinetin responsive sequence element. We next performed a screen of endogenously expressed genes with multiple isoforms resulting from exon skipping events and show that kinetin's ability to improve exon inclusion is not limited to IKBKAP. Lastly, we highlight the potential of kinetin for the treatment of other human splicing disorders by showing correction of a splicing defect in neurofibromatosis