Plasticité post-lésionnelle des réseaux neurovégétatifs centraux : cas du réseau respiratoire. Récupération fonctionnelle après lésion médullaire et transplantation de cellules gliales gainantes olfactives

Ce travail doctoral a étudié, après hémisection cervicale chez le rat adulte, l'incidence de la stratégie de transplantation post-traumatique de cellules gliales olfactives sur la plasticité post-lésionnelle d'un réseau neurovégétatif central (réseau respiratoire) et sur la récupération fonctionnelle qui en résulte. Trois à 6 mois post-lésionnels, une récupération fonctionnelle conséquente des activités phrénique et diaphragmatique ipsilatérales à la lésion (80% et 73% de l'activité controlatérale ; Animaux Témoins : 29% et 10%) est obtenue chez les animaux Transplantés. 57% de l'activité phrénique relevée chez ces animaux persiste après élimination de toute incidence d'origine controlatérale. Aucune activité ipsilatérale semblable n'a pu être mise en évidence chez les Témoins. L'obtention de réponses phréniques post-synaptiques ipsilatérales à des stimulations sus-lésionnelle, chez les transplantés uniquement, révèle la présence d'une réinnervation phrénique imputable à la stratégie.AIX-MARSEILLE3-BU Sc.St Jérô (130552102) / SudocSudocFranceF

A new model of upper cervical spinal contusion inducing a persistent unilateral diaphragmatic deficit in the adult rat

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Optogenetically controlled human functional motor endplate for testing botulinum neurotoxins

International audienceAbstract Background The lack of physiologically relevant and predictive cell-based assays is one of the major obstacles for testing and developing botulinum neurotoxins (BoNTs) therapeutics. Human-induced pluripotent stem cells (hiPSCs)-derivatives now offer the opportunity to improve the relevance of cellular models and thus the translational value of preclinical data. Methods We investigated the potential of hiPSC-derived motor neurons (hMNs) optical stimulation combined with calcium imaging in cocultured muscle cells activity to investigate BoNT-sensitivity of an in vitro model of human muscle-nerve system. Results Functional muscle-nerve coculture system was developed using hMNs and human immortalized skeletal muscle cells. Our results demonstrated that hMNs can innervate myotubes and induce contractions and calcium transient in muscle cells, generating an in vitro human motor endplate showing dose-dependent sensitivity to BoNTs intoxication. The implementation of optogenetics combined with live calcium imaging allows to monitor the impact of BoNTs intoxication on synaptic transmission in human motor endplate model. Conclusions Altogether, our findings demonstrate the promise of optogenetically hiPSC-derived controlled muscle-nerve system for pharmaceutical BoNTs testing and development

Recurrent genomic instability of chromosome 1q in neural derivatives of human embryonic stem cells

Human pluripotent stem cells offer a limitless source of cells for regenerative medicine. Neural derivatives of human embryonic stem cells (hESCs) are currently being used for cell therapy in 3 clinical trials. However, hESCs are prone to genomic instability, which could limit their clinical utility. Here, we report that neural differentiation of hESCs systematically produced a neural stem cell population that could be propagated for more than 50 passages without entering senescence; this was true for all 6 hESC lines tested. The apparent spontaneous loss of evolution toward normal senescence of somatic cells was associated with a jumping translocation of chromosome 1q. This chromosomal defect has previously been associated with hematologic malignancies and pediatric brain tumors with poor clinical outcome. Neural stem cells carrying the 1q defect implanted into the brains of rats failed to integrate and expand, whereas normal cells engrafted. Our results call for additional quality controls to be implemented to ensure genomic integrity not only of undifferentiated pluripotent stem cells, but also of hESC derivatives that form cell therapy end products, particularly neural lines

Human iPSC-derived neurons reveal early developmental alteration of neurite outgrowth in the late-occurring neurodegenerative Wolfram syndrome

International audienceRecent studies indicate that neurodegenerative processes that appear during childhood and adolescence in individuals with Wolfram syndrome (WS) occur in addition to early brain development alteration, which is clinically silent. Underlying pathological mechanisms are still unknown. We have used induced pluripotent stem cell-derived neural cells from individuals affected by WS in order to reveal their phenotypic and molecular correlates. We have observed that a subpopulation of Wolfram neurons displayed aberrant neurite outgrowth associated with altered expression of axon guidance genes. Selective inhibition of the ATF6α arm of the unfolded protein response prevented the altered phenotype, although acute endoplasmic reticulum stress response—which is activated in late Wolfram degenerative processes—was not detected. Among the drugs currently tried in individuals with WS, valproic acid was the one that prevented the pathological phenotypes. These results suggest that early defects in axon guidance may contribute to the loss of neurons in individuals with WS

CRISPR gene editing in pluripotent stem cells reveals the function of MBNL proteins during human in vitro myogenesis

International audienceAlternative splicing has emerged as a fundamental mechanism for the spatiotemporal control of development. A better understanding of how this mechanism is regulated has the potential not only to elucidate fundamental biological principles, but also to decipher pathological mechanisms implicated in diseases where normal splicing networks are misregulated. Here, we took advantage of human pluripotent stem cells to decipher during human myogenesis the role of muscleblind-like (MBNL) proteins, a family of tissue-specific splicing regulators whose loss of function is associated with myotonic dystrophy type 1 (DM1), an inherited neuromuscular disease. Thanks to the CRISPR/Cas9 technology, we generated human-induced pluripotent stem cells (hiPSCs) depleted in MBNL proteins and evaluated the consequences of their losses on the generation of skeletal muscle cells. Our results suggested that MBNL proteins are required for the late myogenic maturation. In addition, loss of MBNL1 and MBNL2 recapitulated the main features of DM1 observed in hiPSC-derived skeletal muscle cells. Comparative transcriptomic analyses also revealed the muscle-related processes regulated by these proteins that are commonly misregulated in DM1. Together, our study reveals the temporal requirement of MBNL proteins in human myogenesis and should facilitate the identification of new therapeutic strategies capable to cope with the loss of function of these MBNL proteins

CRISPR gene editing in pluripotent stem cells reveals the function of MBNL proteins during human in vitro myogenesis

International audienceAlternative splicing has emerged as a fundamental mechanism for the spatiotemporal control of development. A better understanding of how this mechanism is regulated has the potential not only to elucidate fundamental biological principles, but also to decipher pathological mechanisms implicated in diseases where normal splicing networks are misregulated. Here, we took advantage of human pluripotent stem cells to decipher during human myogenesis the role of muscleblind-like (MBNL) proteins, a family of tissue-specific splicing regulators whose loss of function is associated with myotonic dystrophy type 1 (DM1), an inherited neuromuscular disease. Thanks to the CRISPR/Cas9 technology, we generated human-induced pluripotent stem cells (hiPSCs) depleted in MBNL proteins and evaluated the consequences of their losses on the generation of skeletal muscle cells. Our results suggested that MBNL proteins are required for the late myogenic maturation. In addition, loss of MBNL1 and MBNL2 recapitulated the main features of DM1 observed in hiPSC-derived skeletal muscle cells. Comparative transcriptomic analyses also revealed the muscle-related processes regulated by these proteins that are commonly misregulated in DM1. Together, our study reveals the temporal requirement of MBNL proteins in human myogenesis and should facilitate the identification of new therapeutic strategies capable to cope with the loss of function of these MBNL proteins

Asiakastyytyväisyys ja kehittämiskohteet : case Matkailukeskus Rauhalahti

Tämän opinnäytetyön tarkoituksena on selvittää Matkailukeskus Rauhalahden kesän 2015 asiakastyytyväisyyskyselyiden pohjalta Matkailukeskus Rauhalahden kehitystarpeet ja asiakasprofiili. Lisäksi työssäni vertaan tekemääni asiakasprofiilia vuoden 2012 opinnäytetyönä tehtyyn Matkailukeskus Rauhalahden asiakasprofiilitutkimukseen. Asiakaskyselylomakkeita jaettiin asiakkaille kesän aikana noin 5000 kpl. Palautuneista lomakkeista 800 oli suomenkielisiä. Suomenkielisistä lomakkeista jouduttiin valitettavasti hylkäämään 329 kappaletta, joten jäljelle jäi 471 lomaketta tutkittavaksi. Asiakastyytyväisyyslomakkeen lopussa asiakas pystyi täyttämään yhteystietonsa ja osallistumaan huvilalahjakortin arvontaan. Tarkoituksena on kartoittaa Matkailukeskus Rauhalahden kehittämiskohteet asiakkaiden näkökulmasta ja esittää niihin mahdollisia ratkaisuja. Lisäksi tavoitteena on verrata tutkimuksen pohjalta tehtyä asiakasprofiilia vuoden 2012 asiakasprofiiliin ja huomioida mahdolliset muutokset. Lisäksi analysoin asiakastyytyväisyyskyselyn tuloksia, jotta voidaan nähdä, mikä Matkailukeskus Rauhalahdessa on asiakkaiden mielestä hyvää ja mikä huonompaa. Opinnäytetyöni teoriaosuus koostuu asiakasprofiiliin ja asiakastyytyväisyyteen liittyvistä asioista, kuten esimerkiksi asiakaskokemuksesta. Työni kertoo, kuinka tärkeää esimerkiksi asiakkaiden segmentointi on yritykselle ja miten yritys voi hyötyä asiakaskokemuksen parantamisesta. Kerron myös erilaisista asiakastyytyväisyystutkimuksista ja niiden hyödyistä yrityksille. Tutkimus on kvantitatiivinen eli määrällinen. Se on tehty asiakastyytyväisyyslomakkeilla ja tutkimuksessa on huomioitu vain suomenkieliset lomakkeet. Tutkimus suoritettiin Matkailukeskus Rauhalahden kesäkauden aikana, eli toukokuun 2015 lopusta elokuun 2015 loppuun. Kyselyssä kysyttiin asiakkaiden taustatietoja, kuten ikää, sukupuolta ja yhteiskunnallista asemaa. Lisäksi kysyttiin, mistä asiakas on saanut tietonsa Matkailukeskus Rauhalahdesta ja mikä vaikutti asiakkaan päätökseen majoittua Matkailukeskus Rauhalahteen. Myös asiakkaiden majoitusmuotoa ja majoitusöiden lukumäärää tiedusteltiin lomakkeessa. Opinnäytetyöni tutkimusosio on tehty kehittämisnäkökulmasta.The purpose of this thesis was to find the needs of development and the customer profile for Matkailukeskus Rauhalahti. The research was made from customer satisfaction questionnaire that was made during summer 2015. In my thesis I compare my customer profile to the one made in 2012 by Sarianne Suominen. There were about 5000 questionnaires given to the customers in summer 2015 and there were 800 Finnish questionnaires that came back. I had to disqualify 329 of them because of incomplete questionnaires, so I had 471 questionnaires to make my research with. Matkailukeskus Rauhalahti tempted customers to fill the forms in with a lottery, where customers could win a gift card to stay in a holiday villa. The purpose was to present the developing needs from the customers’ aspect and provide solutions to them. I also compare the customer profile I made to the one made in 2012 and pay attenton to possible changes. I also analyze the results of the questionnaire, so we can see what customers think is good and what is bad in Matkailukeskus Rauhalahti. The theory part in this thesis is made of things that relate to customer profiles and customer satisfaction, such as for example customer experience. My thesis tells how important customer segmentation is for the companies and how a company can profit from making their customer experience better. I also tell about different customer satisfaction researches and how they can profit a company. The research in this thesis is quantitative. It is made with customer satisfactory questionnaires in Finnish. Research was made from May 2015 to August 2015. There were questions about customers’ age, sex and their social status. There were also questions about how the customer got his/hers information about Matkailukeskus Rauhalahti and which factors affected their decision to stay in Matkailukeskus Rauhalahti. The questionnaire also asked about how many nights the customer stayed and in what kind of accommodation. The research was made from the customers’ point of view

Additional file 7: Figure S3. of Dp412e: a novel human embryonic dystrophin isoform induced by BMP4 in early differentiated cells

The new DMD exon 1 belongs to a retrovirus-like sequence. (a) Alignment of the new exon 1 region (highlighted in light blue) among 100 vertebrate species with the conserved upstream/downstream sequence marked by red arrows ( https://genome.ucsc.edu ). (b) Schematic representation of the approximately 8 kb region in the DMD gene that is conserved among a sub-group of anthropoids. It is composed of simple repeats, Alu sequences and the whole human endogenous retrovirus-like sequence HuERS-P1 ( http://www.dfam.org/entry/DF0000214 ) flanked by two LTR8 elements

Additional file 1: Table S1. of Dp412e: a novel human embryonic dystrophin isoform induced by BMP4 in early differentiated cells

Human embryonic and induced pluripotent stem cell line list. Details concerning the stem cell lines used in the present study