Induced pluripotent stem cell modeling of Gaucher's disease: what have we learned?

Gaucher's disease (GD) is the most frequently inherited lysosomal storage disease, presenting both visceral and neurologic symptoms. Mutations in acid beta-glucocerebrosidase disrupt the sphingolipid catabolic pathway promoting glucosylceramide (GlcCer) accumulation in lysosomes. Current treatment options are enzyme replacement therapy (ERT) and substrate reduction therapy (SRT). However, neither of these approaches is effective in treating the neurological aspect of the disease. The use of small pharmacological compounds that act as molecular chaperones is a promising approach that is still experimental. In recent years, an association between GD and Parkinson like synucleinopathies has been discovered. Since 1992, a number of mouse models of GD have been the developed and partially reproduce phenotype of the disease. More recently, the discovery of direct reprograming has allowed the derivation of induced pluripotent stem cells (iPSc) from fibroblasts obtained from GD patients. iPSc can be expanded indefinitely in vitro and differentiated to macrophages and neurons, the main relevant cell types involved in GD. In this work, we review iPSc models of GD and summarize what we have learned from this system.Program for Regenerative Medicine PhD Fellowship; Genzyme Corporationinfo:eu-repo/semantics/publishedVersio

Matrisomal components involved in regenerative wound healing in axolotl and acomys: implications for biomaterial development

Achieving regeneration in humans has been a long-standing goal of many researchers. Whereas amphibians like the axolotl (Ambystoma mexicanum) are capable of regenerating whole organs and even limbs, most mammals heal their wounds via fibrotic scarring. Recently, the African spiny mouse (Acomys sp.) has been shown to be injury resistant and capable of regenerating several tissue types. A major focal point of research with Acomys has been the identification of drivers of regeneration. In this search, the matrisome components related to the extracellular matrix (ECM) are often overlooked. In this review, we compare Acomys and axolotl skin wound healing and blastema-mediated regeneration by examining their wound healing responses and comparing the expression pattern of matrisome genes, including glycosaminoglycan (GAG) related genes. The goal of this review is to identify matrisome genes that are upregulated during regeneration and could be potential candidates for inclusion in pro-regenerative biomaterials. Research papers describing transcriptomic or proteomic coverage of either skin regeneration or blastema formation in Acomys and axolotl were selected. Matrisome and GAG related genes were extracted from each dataset and the resulting lists of genes were compared. In our analysis, we found several genes that were consistently upregulated, suggesting possible involvement in regenerative processes. Most of the components have been implicated in regulation of cell behavior, extracellular matrix remodeling and wound healing. Incorporation of such pro-regenerative factors into biomaterials may help to shift pro-fibrotic processes to regenerative responses in treated wounds.European Union’s Horizon 2020 ;Grant Agreement No. 955722info:eu-repo/semantics/publishedVersio

Trophoblast attachment to the endometrial epithelium elicits compartment-specific transcriptional waves in an in-vitro model

Implantation is a major bottleneck in human reproduction (Polanski et al., 2014). The average implantation rate for an embryo ranges from 30% to 40% (Coughlan et al., 2014). Recurrent implantation failure (RIF) is estimated to occur in approximately 4% of IVF cycles (Koot et al., 2012), although estimates vary because there are several somewhat different definitions of RIF in the literature. Implantation of the blastocyst in the receptive endometrium is a sequential process involving apposition, attachment and invasion that precedes the establishment of pregnancy (Wang and Dey, 2006). Successful implantation requires embryo competence and endometrial receptivity, both of which are dynamic and highly regulated states (Wang and Dey, 2006). In addition to genetic disorders (which are a major cause of implantation failure and miscarriage), embryo competence, quality and ultimately developmental potential depend on the embryo achieving the correct regulatory, signalling and metabolic states (Fu et al., 2009; Hourvitz et al., 2006; Lundin et al., 2001; Simon and Laufer, 2012; Sjoblom et al., 2006). A key determinant of these embryonic states is their underlying transcriptional dynamics; for instance, waves of embryonic transcriptional activation direct early development and the symmetry breaking needed for cell fate specification (Shi et al., 2015; Vassena et al., 2011).info:eu-repo/semantics/publishedVersio

MicroRNAs in embryonic stem cell function and fate

Since their discovery in the early 1990s, microRNAs (miRs) have gone from initially being considered an oddity to being recognized as a level of gene expression regulation that is integral to the normal function of cells and organisms. They are implicated in many if not all biological processes in animals, from apoptosis and cell signaling to organogenesis and development. Our understanding of cell regulatory states, as determined primarily by transcription factor (TF) profiles, is incomplete without consideration of the corresponding miR profile. The miR complement of a cell provides robust and redundant control over the output of hundreds of possible targets for each miR. miRs are common components of regulatory pathways, and in some cases can constitute on–off switches that regulate crucial fate decisions. In this review, we summarize our current knowledge about the biogenesis and regulation of miRs and describe their involvement in the pathways that regulate cell division, pluripotency, and reprogramming to the pluripotent state

CRE recombinase-inducible RNA interference mediated by lentiviral vectors

Recently, several systems designed to trigger RNA interference by using small hairpin RNA driven by polymerase III promoters have been described. Here, we report a lentiviral-mediated small interfering RNA delivery system that can be induced by CRE recombinase. The system consists of a lentiviral vector carrying a mouse U6 promoter that is separated from a small hairpin RNA by a random DNA stuffer sequence flanked by modified loxP sites. The silencing cassette is not expressed until activated by addition of CRE recombinase delivered by a lentiviral vector. We have used this system to show specific down-regulation of GFP and two endogenous genes (the tumor suppressor p53 and the NF-κB transcription factor subunit p65) in vitro. Furthermore, down-regulation of both p53 and p65 resulted in the expected effect on downstream genes and cellular phenotype. We foresee multiple applications of this system both in vitro and in vivo to down-regulate specific targets in a tissue-specific and localized manner

Pancreatic polypeptide: a review of its involvement in neuro-endocrine reflexes, islet-acinar interactions and ethanol-evoked physiopatologic pancreatic gland changes

l péptido regulador PP (polipéptido pancreático) es secre - tado por células insulares PP, principalmente en islotes del "processus uncinatus" ("gancho") del páncreas (esbozo ven - tral de la glándula) y, también, por células "dispersas" en el epitelio glandular. En su mecanismo secretorio se hallan involucrados dos reflejos neuroendocrinos (RNE): uno "cor - to" (RNE-C) y otro "largo" (RNE-L). El primero es des - encadenado desde el duodeno por un componente, aún no precisado, de la secreción bilio-pancreática. El del segundo, se centra en un arco reflejo vago-vagal que da lugar a una depresión del tono colinérgico intrapancreático. Éste, por vía humoral, frena la descarga de impulsos parasimpáticos por parte del "complejo nuclear dorsal del vago". Una con - secuencia bien establecida de lo precedente es la caída del rol permisivo ejercido por el tono parasimpático sobre los efectos secretorios en los "pancreones" por parte, sobre todo, de la hormona secretina. El PP, probablemente en conjunción con otros agentes, ejerce un rol de "freno" que trata de prevenir en los pancreocitos acinares el desencadenamiento de un pro - ceso de "estimulación ecbólica supramáxima". Su rol modu - lador, lo demuestra, a través del incremento de su nivel en plasma en la primera semana de un episodio de pancreatitis aguda. En esta última entidad, subsecuente a una "comi - da copiosa", el sobrepaso de la capacidad de "freno" de la glándula tendría una influencia fisiopatogénica clave. En el alcoholismo crónico, tanto experimental como clínico, una neuropatía vagal que compromete a las "fibras inhibitorias" de Pavlov es el fundamento que explica, teniendo por pivote una depresión de la secreción del PP, la reactividad exacer - bada de los reflejos duodeno-pancreáticos. Lo precedente con - duce a un "hipertono colinérgico intrapancreático" y a una disfunción asociada de la papila de Vater. Estos cambios, acoplados a una respuesta incrementada de los pancreocitos a la hormona CCK, constituyen la médula del proceso que puede encuadrarse dentro del término que hemos concebido como de "estimulación supramáxima" del "pancreón". Éste suele conducir a la desorganización organelar y consecuente atrofia o muerte de la célula acinar. El hipertono colinérgico intrapancreático, la incrementada reactividad a la CCK de los pancreocitos y el aumento de la resistencia al flujo secreto - rio por parte de la región esfinteriana del Oddi, explican la influencia agravante del alcoholismo crónico en un episodio de pancreatitis aguda biliar. Se concluye que la secreción del péptido regulador PP, esencialmente desencadenado por la secretina, CCK, alimentos y la hipoglucemia insulínica, en la condición antes esbozada se halla deprimida; ello, para - dójicamente, en presencia de un aumento en el número de su célula endocrina. Esto se constata en pacientes alcohólicos crónicos, en aquellos con fibrosis quística y también en pe - rros con ligadura ductal. Se ha inferido que en todas estas circunstancias aparte de un compromiso de las fibras inhibi - torias vagales de Pavlov ("freno pancreonal") se acopla, sea próximo y/o en la propia célula efectora, un defecto en la vía final común de los estímulos antes descriptos. Una conca - tenación de modificaciones neuroendocrinas muy próximas a las precedentes es la que se comprueba en pacientes con insuficiencia renal crónica. Éstas logran ser mimetizadas experimentalmente en la rata efectuando la exéresis de un 85% de la masa renal. Un hallazgo a enfatizar es el que se constata en diabéticos insulino-dependientes. En esta enti - dad, como en el caso del alcoholismo crónico, cabe considerar una perturbada transmisión de impulsos nerviosos siguiendo el trayecto de las fibras inhibitorias vagales de Pavlov

Vaginal microbiota profile at the time of embryo transfer does not affect live birth rate in IVF cycles with donated oocytes

Research question: What is the relationship between the vaginal microbiota profile at the time of embryo transfer and live birth rates in women undergoing IVF/intracytoplasmic sperm injection (ICSI) with donated oocytes? Design: One hundred and fifty Caucasian women receiving donated oocytes were prospectively included in the study from March 2017 to January 2018. Samples of vaginal fluid were taken immediately before transfer of a fresh single blastocyst and genomic DNA (gDNA) was extracted. Bacterial load as well as the presence of four lactobacilli (L. crispatus, L. gasseri, L. jensenii and L. iners) and species associated with bacterial vaginosis (Gardnerella vaginalis, Atopobium vaginae, Mycoplasma hominis and Prevotella spp. - here collectively termed BVB) were determined by quantitative polymerase chain reaction. Vaginal microbiota profiles for each patient were characterized and correlated with reproductive results. Results: Although bacterial load was variable, a majority of samples were dominated by a single species (80.7%, 121/150). Most samples (76.7%, 115/150) were dominated by Lactobacillus spp., while 23.3% (35/150) were dominated by bacteria associated with bacterial vaginosis. The distribution of microbiota profiles among women who achieved a live birth and women who did not was similar (P = 0.43). Interestingly, we found a significantly higher proportion of samples dominated by L. crispatus- in women achieving live birth compared with those who did not (P = 0.021)this correlation was also statistically significant for biochemical pregnancy (P = 0.039) and clinical pregnancy (P = 0.015). Conclusions: Our data suggest that bacterial vaginosis-like vaginal microbiota at the time of embryo transfer does not directly affect the live birth rate.intramural funding of Clinica EUGINMinistry of Economy and Knowledge of the Government of Catalonia [GENCAT 2015 DI 048]info:eu-repo/semantics/publishedVersio

Human oocyte meiotic maturation is associated with a specific profile of alternatively spliced transcript isoforms

The transition from a transcriptionally active state (GV) to a transcriptionally inactive state (mature MII oocytes) is required for the acquisition of oocyte developmental competence. We hypothesize that the expression of specific genes at the in vivo matured (MII) stage could be modulated by posttranscriptional mechanisms, particularly regulation of alternative splicing (AS). In this study, we examined the transcriptional activity of GV oocytes after ovarian stimulation followed by oocyte pick-up and the landscape of alternatively spliced isoforms in human MII oocytes. Individual oocytes were processed and analyzed for transcriptional activity (GV), gene expression (GV and MII), and AS signatures (GV and MII) on HTA 2.0 microarrays. Samples were grouped according to maturation stage, and then subgrouped according to women's age and antral follicular count (AFC); array results were validated by quantitative polymerase chain reaction. Differentially expressed genes between GV and MII oocytes clustered mainly in biological processes related to mitochondrial metabolism. Interestingly, 16 genes that were related to the regulation of transcription and mitochondrial translation showed differences in alternatively spliced isoform profiles despite not being differentially expressed between groups. Altogether, our results contribute to our understanding of the role of AS in oocyte developmental competence acquisition.Secretary for Universities and Research of the Ministry of Economy and Knowledge of the Government of Catalonia GENCAT 2015 DI 048.info:eu-repo/semantics/publishedVersio