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Myotonic dystrophy type 1 (DM1) is an RNA-mediated disorder caused by a non-coding CTG repeat expansion that, in particular, provokes functional alteration of CUG-binding proteins. As a consequence, several genes with misregulated alternative splicing have been linked to clinical symptoms. In our search for additional molecular mechanisms that would trigger functional defects in DM1, we took advantage of mutant gene-carrying human embryonic stem cell lines to identify differentially expressed genes. Among the different genes found to be misregulated by DM1 mutation, one strongly downregulated gene encodes a transcription factor, ZNF37A. In this paper, we show that this defect in expression, which derives from a loss of RNA stability, is controlled by the RNA-binding protein, CUGBP1, and is associated with impaired myogenesis-a functional defect reminiscent of that observed in DM1. Loss of the ZNF37A protein results in changes in the expression of the subunit α1 of the receptor for the interleukin 13. This suggests that the pathological molecular mechanisms linking ZNF37A and myogenesis may involve the signaling pathway that is known to promote myoblast recruitment during development and regeneration.
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Empalme Alternativo/genética , Factores de Transcripción de Tipo Kruppel/genética , Desarrollo de Músculos/genética , Distrofia Miotónica/genética , Expansión de Repetición de Trinucleótido/genética , Línea Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Madre Embrionarias , Humanos , Subunidad alfa1 del Receptor de Interleucina-13/genética , Subunidad alfa1 del Receptor de Interleucina-13/metabolismo , Mutación , Distrofia Miotónica/fisiopatología , Transducción de Señal/genéticaRESUMEN
BACKGROUND: Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine. However, the safety of cell therapy using differentiated hPSC derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. To date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods. RESULTS: We used lentivectors encoding green fluorescent protein (GFP) driven by the liver-specific apoliprotein A-II (APOA-II) promoter to purify human hepatic progenitors. We evaluated both integrating and integration-defective lentivectors in combination with an HIV integrase inhibitor. A human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. Subsequently, cells were transduced and sorted at day 16 of differentiation to obtain a cell population enriched in hepatic progenitor cells. After sorting, more than 99% of these APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin 19. When further cultured for 16 days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. Moreover, they were devoid of vector DNA integration. CONCLUSIONS: We have developed an effective strategy to purify human hepatic cells from cultures of differentiating hPSCs, producing a novel tool that could be used not only for cell therapy but also for in vitro applications such as drug screening. The present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells.
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Diferenciación Celular , Separación Celular/métodos , Células Madre Embrionarias/citología , Vectores Genéticos/genética , Hepatocitos/citología , Lentivirus/genética , Integración Viral/fisiología , Apolipoproteína A-II/genética , Biomarcadores/metabolismo , Línea Celular , Citocromo P-450 CYP3A/metabolismo , ADN Viral/metabolismo , Citometría de Flujo , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Hepatocitos/metabolismo , Humanos , Hígado/citología , Especificidad de Órganos , Regiones Promotoras Genéticas/genética , Transducción GenéticaRESUMEN
Identifying and validating a biomarker with high specificity in early-stage dementia with Lewy bodies (DLB) using a feasible method is crucial to enhance the current suboptimal diagnostic procedure. Previous research revealed abnormalities, including hypoperfusion in the right anterior insular cortex at group level, in prodromal DLB. Exploring hypoperfusion of the right anterior insula, at an individual-level and assessing its relevance as a potential imaging biomarker in early DLB, has, to our knowledge, not been investigated. Our preliminary study aims to assess the feasibility of the technique and to provide a methodological framework for further investigation. We assessed the feasibility and accuracy of the hypoperfusion of the right anterior insula per arterial spin labelling magnetic resonance imaging (ASL-MRI) as a diagnostic biomarker in early DLB and provided rough estimates of its sensitivity and specificity. Defining the region of interest based on previous research, we established the biomarker as the hypoperfusion of the right anterior insula. Discriminative and analytical performances were assessed in comparison to a control group of treatment-resistant depression patients. Bayesian diagnostic reasoning was employed to assess the biomarker diagnostic usability in early DLB in two scenarios: healthy elderly controls and mild cognitive impairment. Additionally, we updated probabilities by integrating data from the Mayo-clinic cognitive fluctuations scale and real-time quaking-induced conversion (RT-QuIC) α-synuclein data. Lastly, a whole-brain perfusion analysis of DLB patients was conducted to identify further brain regions with discriminative abilities. We successfully replicated the right anterior insular hypoperfusion (RAI-Hypo) in all DLB patients at the individual level. The overall sensitivity of the biomarker was 96%, and the specificity was 92%. Bayesian testing revealed the biomarker's highest performance in early-stage DLB with cognitive fluctuations, showcasing a diagnostic potential associated with a high precision and moderate accuracy. In a cognitively non-impaired population, the RAI-Hypo showed a limited usability and lacked in selectivity to qualify as a screening tool. The exploratory whole-brain analysis revealed perfect discriminative capacities in the bilateral anterior insulae and the left inferior parietal lobule. Further studies are needed to confirm our preliminary results. If performance is maintained in subsequent studies and is compared to a more suitable control population, the proposed biomarker may be eventually sufficient to discriminate early-stage DLB from non-DLB.
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Over two decades, most cancer vaccines failed clinical development. Key factors may be the lack of efficient priming with tumor-specific antigens and strong immunostimulatory signals. MVX-ONCO-1, a personalized cell-based cancer immunotherapy, addresses these critical steps utilizing clinical-grade material to replicate a successful combination seen in experimental models: inactivated patient's own tumor cells, providing the widest cancer-specific antigen repertoire and a standardized, sustained, local delivery over days of a potent adjuvant achieved by encapsulated cell technology. We conducted an open-label, single-arm, first-in-human phase I study with MVX-ONCO-1 in patients with advanced refractory solid cancer. MVX-ONCO-1 comprises irradiated autologous tumor cells coimplanted with two macrocapsules containing genetically engineered cells producing granulocyte-macrophage colony-stimulating factor. Patients received six immunizations over 9 weeks without maintenance therapy. Primary objectives were safety, tolerability, and feasibility, whereas secondary objectives focused on efficacy and immune monitoring. Data from 34 patients demonstrated safety and feasibility with minor issues. Adverse events included one serious adverse event possibly related to investigational medicinal product and two moderate-related adverse events. More than 50% of the patients with advanced and mainly nonimmunogenic tumors showed clinical benefits, including partial responses, stable diseases, and prolonged survival. In recurrent/metastatic head and neck squamous cell carcinoma, one patient achieved a partial response, whereas another survived for more than 7 years without anticancer therapy for over 5 years. MVX-ONCO-1 is safe, well tolerated, and beneficial across several tumor types. Ongoing phase IIa trials target patients with advanced recurrent/metastatic head and neck squamous cell carcinoma after initial systemic therapy. SIGNIFICANCE: This first-in-human phase I study introduces a groundbreaking approach to personalized cancer immunotherapy, addressing limitations of traditional strategies. By combining autologous irradiated tumor cells as a source of patient-specific antigens and utilizing encapsulated cell technology for localized, sustained delivery of granulocyte-macrophage colony-stimulating factor as an adjuvant, the study shows a very good safety and feasibility profile. This innovative approach holds the promise of addressing tumor heterogeneity by taking advantage of each patient's antigenic repertoire.
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Vacunas contra el Cáncer , Inmunoterapia Activa , Neoplasias , Medicina de Precisión , Humanos , Persona de Mediana Edad , Masculino , Femenino , Neoplasias/terapia , Neoplasias/inmunología , Vacunas contra el Cáncer/uso terapéutico , Vacunas contra el Cáncer/administración & dosificación , Anciano , Medicina de Precisión/métodos , Inmunoterapia Activa/métodos , Adulto , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéuticoRESUMEN
Encapsulated cell therapy holds a great potential to deliver sustained levels of highly potent therapeutic proteins to patients and improve chronic disease management. A versatile encapsulation device that is biocompatible, scalable, and easy to administer, retrieve, or replace has yet to be validated for clinical applications. Here, we report on a cargo-agnostic, macroencapsulation device with optimized features for protein delivery. It is compatible with adherent and suspension cells, and can be administered and retrieved without burdensome surgical procedures. We characterized its biocompatibility and showed that different cell lines producing different therapeutic proteins can be combined in the device. We demonstrated the ability of cytokine-secreting cells encapsulated in our device and implanted in human skin to mobilize and activate antigen-presenting cells, which could potentially serve as an effective adjuvant strategy in cancer immunization therapies. We believe that our device may contribute to cell therapies for cancer, metabolic disorders, and protein-deficient diseases.
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[This corrects the article DOI: 10.1016/j.omtm.2022.07.017.].
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Despite many promising results obtained in previous preclinical studies, the clinical development of encapsulated cell technology (ECT) for the delivery of therapeutic proteins from macrocapsules is still limited, mainly due to the lack of an allogeneic cell line compatible with therapeutic application in humans. In our work, we generated an immortalized human myoblast cell line specifically tailored for macroencapsulation. In the present report, we characterized the immortalized myoblasts and described the engineering process required for the delivery of functional therapeutic proteins including a cytokine, monoclonal antibodies and a viral antigen. We observed that, when encapsulated, the novel myoblast cell line can be efficiently frozen, stored, and thawed, which limits the challenge imposed by the manufacture and supply of encapsulated cell-based therapeutic products. Our results suggest that this versatile allogeneic cell line represents the next step toward a broader development and therapeutic use of ECT.
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Current classification systems use the terms "catatonia" and "psychomotor phenomena" as mere a-theoretical descriptors, forgetting about their theoretical embedment. This was the source of misunderstandings among clinicians and researchers of the European collaboration on movement and sensorimotor/psychomotor functioning in schizophrenia and other psychoses or ECSP. Here, we review the different perspectives, their historical roots and highlight discrepancies. In 1844, Wilhelm Griesinger coined the term "psychic-motor" to name the physiological process accounting for volition. While deriving from this idea, the term "psychomotor" actually refers to systems that receive miscellaneous intrapsychic inputs, convert them into coherent behavioral outputs send to the motor systems. More recently, the sensorimotor approach has drawn on neuroscience to redefine the motor signs and symptoms observed in psychoses. In 1874, Karl Kahlbaum conceived catatonia as a brain disease emphasizing its somatic - particularly motor - features. In conceptualizing dementia praecox Emil Kraepelin rephrased catatonic phenomena in purely mental terms, putting aside motor signs which could not be explained in this way. Conversely, the Wernicke-Kleist-Leonhard school pursued Kahlbaum's neuropsychiatric approach and described many new psychomotor signs, e.g. parakinesias, Gegenhalten. They distinguished 8 psychomotor phenotypes of which only 7 are catatonias. These barely overlap with consensus classifications, raising the risk of misunderstanding. Although coming from different traditions, the authors agreed that their differences could be a source of mutual enrichment, but that an important effort of conceptual clarification remained to be made. This narrative review is a first step in this direction.
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Catatonia , Neurociencias , Trastornos Psicóticos , Catatonia/diagnóstico , Catatonia/terapia , Consenso , Humanos , Desempeño Psicomotor , Trastornos Psicóticos/diagnósticoRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide pandemic with at least 3.8 million deaths to date. For that reason, finding an efficient vaccine for this virus quickly became a global priority. The majority of vaccines now marketed are based on the SARS-CoV-2 spike protein that has been described as the keystone for optimal immunization. In order to monitor SARS-CoV-2 spike-specific humoral responses generated by immunization or infection, we have developed a robust and reproducible enzyme-linked immunosorbent assay (ELISA) protocol. This protocol describes a method for quantitative detection of IgG antibodies against the SARS-CoV-2 spike protein using antigen-coated microtiter plates. Results showed that antibodies could be quantified between the range of 1.953 ng/mL to 500 ng/mL with limited inter- and intra-assay variability.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide pandemic with recurrences. Therefore, finding a vaccine for this virus became a priority for the scientific community. The SARS-CoV-2 spike protein has been described as the keystone for viral entry into cells and effective immune protection against SARS-CoV-2 is elicited by this protein. Consequently, many commercialized vaccines focus on the spike protein and require the use of an optimal adjuvant during vaccination. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has demonstrated a powerful enhancement of acquired immunity against many pathogens when delivered in a sustained and local manner. In this context, we developed an encapsulated cell-based technology consisting of a biocompatible, semipermeable capsule for secretion of GM-CSF. In this study, we investigated whether murine GM-CSF (muGM-CSF) represents a suitable adjuvant for SARS-CoV-2 immunization, and which delivery strategy for muGM-CSF could be most beneficial. To test this, different groups of mice were immunized with intra-dermal (i.d.) electroporated spike DNA in the absence or presence of recombinant or secreted muGM-CSF. Results demonstrated that adjuvanting a spike DNA vaccine with secreted muGM-CSF resulted in enhancement of specific cellular and humoral immune responses against SARS-CoV-2. Our data also highlighted the importance of delivery strategies to the induction of cellular and humoral-mediated responses.
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Chordoma is a rare tumor of notochordal origin, currently principally treated by surgery and/or irradiation. Here, we describe the clinical outcome of 3 consecutive patients with metastatic and locally advanced chordoma, treated with different immunotherapeutic approaches. All patients presented fast growing tumors and failure of standard therapies. One was treated with a tumor-based vaccine, the 2 others with anti-PD1 antibodies, all with impressive clinical and radiological responses. We therefore propose that chordoma is an immunogenic tumor and thus that translational and clinical research is necessary to develop rationally designed immunotherapy approaches.
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In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength. Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.
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Células Madre Embrionarias/citología , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Nanopartículas/química , Regeneración/fisiología , Comunicación Celular , Diferenciación Celular/fisiología , Humanos , Imagenología Tridimensional/métodos , Miocitos Cardíacos/citología , Niobio/química , Óxidos/química , Potasio/química , Medicina RegenerativaRESUMEN
Myotonic dystrophy type 1 (DM1) is a multisystem disorder affecting a variety of organs, including the central nervous system. By using neuronal progeny derived from human embryonic stem cells carrying the causal DM1 mutation, we have identified an early developmental defect in genes involved in neurite formation and the establishment of neuromuscular connections. Differential gene expression profiling and quantitative RT-PCR revealed decreased expression of two members of the SLITRK family in DM1 neural cells and in DM1 brain biopsies. In addition, DM1 motoneuron/muscle cell cocultures showed alterations that are consistent with the known role of SLITRK genes in neurite outgrowth, neuritogenesis, and synaptogenesis. Rescue and knockdown experiments suggested that the functional defects can be directly attributed to SLITRK misexpression. These neuropathological mechanisms may be clinically significant for the functional changes in neuromuscular connections associated with DM1.