Pediatric oral ranula : clinical follow-up study of 57 cases

Objective: To present 57 cases of oral ranula in children, analyzing the clinical characteristics, treatment and outcome of these lesions. Methods: The clinical histories of patients diagnosed with oral ranula, seen between 1998 and 2008 at the Oral and Maxillofacial Surgery Unit of a reference Children?s Hospital (0-14 years) were reviewed. All patients with clinical diagnosis of oral ranula were included. Results: Fifty-seven patients, 21 boys and 36 girls, with a mean age of 5.1 years were included in the study. Thirtytwo cases were located on the left side of the floor of the mouth. The lesion diameter varied between 1 and 3 cm in 27 cases, 22 were less than 1 cm, and 8 were larger than 3 cm. Fifty-four cases were asymptomatic and 3 ranulas had pain on swallowing. Twenty-two cases were resolved by opening with a tract dilator and 35 by marsupialization. Seven cases recurred at a mean of 12 months after treatment, three of these from the marsupialization group. Conclusion: The majority of the oral ranulas occurred in females, asymptomatic, on the left side of the floor of the mouth, with a mean size of 1 to 3 cm; all lesions were treated by surgery, of which 7 recurred

Spanish Cell Therapy Network (TerCel) : 15 years of successful collaborative translational research

In the current article we summarize the 15-year experience of the Spanish Cell Therapy Network (TerCel), a successful collaborative public initiative funded by the Spanish government for the support of nationwide translational research in this important area. Thirty-two research groups organized in three programs devoted to cardiovascular, neurodegenerative and immune-inflammatory diseases, respectively, currently form the network. Each program has three working packages focused on basic science, pre-clinical studies and clinical application. TerCel has contributed during this period to boost the translational research in cell therapy in Spain, setting up a network of Good Manufacturing Practice-certified cell manufacturing facilities- and increasing the number of translational research projects, publications, patents and clinical trials of the participating groups, especially those in collaboration. TerCel pays particular attention to the public-private collaboration, which, for instance, has led to the development of the first allogeneic cell therapy product approved by the European Medicines Agency, Darvadstrocel. The current collaborative work is focused on the development of multicenter phase 2 and 3 trials that could translate these therapies to clinical practice for the benefit of patients

One-step in vitro generation of ETV2-null pig embryos

Each year, tens of thousands of people worldwide die of end-stage organ failure due to the limited availability of organs for use in transplantation. To meet this clinical demand, one of the last frontiers of regenerative medicine is the generation of humanized organs in pigs from pluripotent stem cells (PSCs) via blastocyst complementation. For this, organ-disabled pig models are needed. As endothelial cells (ECs) play a critical role in xenotransplantation rejection in every organ, we aimed to produce hematoendothelial-disabled pig embryos targeting the master transcription factor ETV2 via CRISPR-Cas9-mediated genome modification. In this study, we designed five different guide RNAs (gRNAs) against the DNA-binding domain of the porcine ETV2 gene, which were tested on porcine fibroblasts in vitro. Four out of five guides showed cleavage capacity and, subsequently, these four guides were microinjected individually as ribonucleoprotein complexes (RNPs) into one-cell-stage porcine embryos. Next, we combined the two gRNAs that showed the highest targeting efficiency and microinjected them at higher concentrations. Under these conditions, we significantly improved the rate of biallelic mutation. Hence, here, we describe an efficient one-step method for the generation of hematoendothelial-disabled pig embryos via CRISPR-Cas9 microinjection in zygotes. This model could be used in experimentation related to the in vivo generation of humanized organs

Adipose-derived mesenchymal stromal cells for the treatment of patients with severe SARS-CoV-2 pneumonia requiring mechanical ventilation. A proof of concept study

Background: Identification of effective treatments in severe cases of COVID-19 requiring mechanical ventilation represents an unmet medical need. Our aim was to determine whether the administration of adipose-tissue derived mesenchymal stromal cells (AT-MSC) is safe and potentially useful in these patients. Methods: Thirteen COVID-19 adult patients under invasive mechanical ventilation who had received previous antiviral and/or anti-inflammatory treatments (including steroids, lopinavir/ritonavir, hydroxychloroquine and/or tocilizumab, among others) were treated with allogeneic AT-MSC. Ten patients received two doses, with the second dose administered a median of 3 days (interquartile range-IQR- 1 day) after the first one. Two patients received a single dose and another patient received 3 doses. Median number of cells per dose was 0.98 × 106 (IQR 0.50 × 106) AT-MSC/kg of recipient's body weight. Potential adverse effects related to cell infusion and clinical outcome were assessed. Additional parameters analyzed included changes in imaging, analytical and inflammatory parameters. Findings: First dose of AT-MSC was administered at a median of 7 days (IQR 12 days) after mechanical ventilation. No adverse events were related to cell therapy. With a median follow-up of 16 days (IQR 9 days) after the first dose, clinical improvement was observed in nine patients (70%). Seven patients were extubated and discharged from ICU while four patients remained intubated (two with an improvement in their ventilatory and radiological parameters and two in stable condition). Two patients died (one due to massive gastrointestinal bleeding unrelated to MSC therapy). Treatment with AT-MSC was followed by a decrease in inflammatory parameters (reduction in C-reactive protein, IL-6, ferritin, LDH and d-dimer) as well as an increase in lymphocytes, particularly in those patients with clinical improvement. Interpretation: Treatment with intravenous administration of AT-MSC in 13 severe COVID-19 pneumonia under mechanical ventilation in a small case series did not induce significant adverse events and was followed by clinical and biological improvement in most subjects. Funding: None.We would like to acknowledge the Instituto de Salud Carlos III (ISCIII) through the project “RD16/0011: Red de Terapia Celular”, from the sub-program RETICS, integrated in the “Plan Estatal de I+D+I 2013-2016” and co-financed by the European Regional Development Fund “A way to make Europe”, groups RD16/0011/0001, -/0002, -/005, -/0013, -/0015, -/0029), the Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, Spain and AvanCell-CM (Red de Investigación de Terapia Celular de la Comunidad de Madrid, Spain), for supporting some personnel and networking activities

Fabrication of human myocardium using multidimensional modelling of engineered tissues

Biofabrication of human tissues has seen a meteoric growth triggered by recent technical advancements such as human induced pluripotent stem cells (hiPSCs) and additive manufacturing. However, generation of cardiac tissue is still hampered by lack of adequate mechanical properties and crucially by the often unpredictable post-fabrication evolution of biological components. In this study we employ melt electrowriting (MEW) and hiPSC-derived cardiac cells to generate fibre-reinforced human cardiac minitissues. These are thoroughly characterized in order to build computational models and simulations able to predict their post-fabrication evolution. Our results show that MEW-based human minitissues display advanced maturation 28 post-generation, with a significant increase in the expression of cardiac genes such as MYL2, GJA5, SCN5A and the MYH7/MYH6 and MYL2/MYL7 ratios. Human iPSC-cardiomyocytes are significantly more aligned within the MEW-based 3D tissues, as compared to conventional 2D controls, and also display greater expression of C ×43. These are also correlated with a more mature functionality in the form of faster conduction velocity. We used these data to develop simulations capable of accurately reproducing the experimental performance. In-depth gauging of the structural disposition (cellular alignment) and intercellular connectivity (C ×43) allowed us to develop an improved computational model able to predict the relationship between cardiac cell alignment and functional performance. This study lays down the path for advancing in the development of in silico tools to predict cardiac biofabricated tissue evolution after generation, and maps the route towards more accurate and biomimetic tissue manufacture

In vivo screening characterizes chromatin factor functions during normal and malignant hematopoiesis

Bulk ex vivo and single-cell in vivo CRISPR knockout screens are used to characterize 680 chromatin factors during mouse hematopoiesis, highlighting lineage-specific and normal and leukemia-specific functions. Cellular differentiation requires extensive alterations in chromatin structure and function, which is elicited by the coordinated action of chromatin and transcription factors. By contrast with transcription factors, the roles of chromatin factors in differentiation have not been systematically characterized. Here, we combine bulk ex vivo and single-cell in vivo CRISPR screens to characterize the role of chromatin factor families in hematopoiesis. We uncover marked lineage specificities for 142 chromatin factors, revealing functional diversity among related chromatin factors (i.e. barrier-to-autointegration factor subcomplexes) as well as shared roles for unrelated repressive complexes that restrain excessive myeloid differentiation. Using epigenetic profiling, we identify functional interactions between lineage-determining transcription factors and several chromatin factors that explain their lineage dependencies. Studying chromatin factor functions in leukemia, we show that leukemia cells engage homeostatic chromatin factor functions to block differentiation, generating specific chromatin factor-transcription factor interactions that might be therapeutically targeted. Together, our work elucidates the lineage-determining properties of chromatin factors across normal and malignant hematopoiesis

AG5 is a potent non-steroidal anti-inflammatory and immune regulator that preserves innate immunity

12 pages, 5 figures.-- This is an open access article under the CC BY-NC-ND licenseAn archetypal anti-inflammatory compound against cytokine storm would inhibit it without suppressing the innate immune response. AG5, an anti-inflammatory compound, has been developed as synthetic derivative of andrographolide, which is highly absorbable and presents low toxicity. We found that the mechanism of action of AG5 is through the inhibition of caspase-1. Interestingly, we show with in vitro generated human monocyte derived dendritic cells that AG5 preserves innate immune response. AG5 minimizes inflammatory response in a mouse model of lipopolysaccharide (LPS)-induced lung injury and exhibits in vivo anti-inflammatory efficacy in the SARS-CoV-2-infected mouse model. AG5 opens up a new class of anti-inflammatories, since contrary to NSAIDs, AG5 is able to inhibit the cytokine storm, like dexamethasone, but, unlike corticosteroids, preserves adequately the innate immunity. This is critical at the early stages of any naïve infection, but particularly in SARS-CoV-2 infections. Furthermore, AG5 showed interesting antiviral activity against SARS-CoV-2 in humanized miceThis work has been supported by NextGenerationEU Recovery and Resilience Facility (RRF) through the PTI+ Global Health Platform of Spanish National Research Council, grants SGL2103023 (PBA), SGL2103053 (MMA) and SGL2103015 (MM); by Spanish National Research Council through the program “Ayudas extraodinarias a proyectos de investigacion en el marco de las medidas urgentes extraodinarias para hacer frente al impacto económico y social del COVID-19”, grants CSIC-COV19-093 (PBA) and CSIC-COV19-117 (MM); by Generalitat Valenciana through the program “Ayudas urgentes para proyectos de investigación, desarrollo tecnológico e innovación (I+D+i) por la COVID-19”, grant GVA-COVID19/2021/059 (PBA); by the Conference of Rectors of the Spanish Universities, Spanish National Research Council and Banco Santander through the FONDO SUPERA COVID-19, grant CAPriCORn (JSM, JMB); by Severo Ochoa center of excellence program (grant CEX2021-001230-S) (PBA)Peer reviewe

Isolation methods of large and small extracellular vesicles derived from cardiovascular progenitors: A comparative study.

Since the discovery of the beneficial therapeutical effects of extracellular vesicles (EVs), these agents have been attracting great interest as next-generation therapies. EVs are nanosized membrane bodies secreted by all types of cells that mediate cell-cell communication. Although the classification of different subpopulations of EVs can be complex, they are broadly divided into microvesicles and exosomes based on their biogenesis and in large and small EVs based on their size. As this is an emerging field, current investigations are focused on basic aspects such as the more convenient method for EV isolation. In the present paper, we used cardiac progenitor cells (CPCs) to study and compare different cell culture conditions for EV isolation as well as two of the most commonly employed purification methods: ultracentrifugation (UC) and size-exclusion chromatography (SEC). Large and small EVs were separately analysed. We found that serum starvation of cells during the EV collecting period led to a dramatic decrease in EV secretion and major cell death. Regarding the isolation method, our findings suggest that UC and SEC gave similar EV recovery rates. Separation of large and small EV-enriched subpopulations was efficiently achieved with both purification protocols although certain difference in sample heterogeneity was observed. Noteworthy, while calnexin was abundant in large EVs, ALIX and CD63 were mainly found in small EVs. Finally, when the functionality of EVs was assessed on primary culture of adult murine cardiac fibroblasts, we found that EVs were taken up by these cells, which resulted in a pronounced reduction in the proliferative and migratory capacity of the cells. Specifically, a tendency towards a larger effect of SEC-related EVs was observed. No differences could be found between large and small EVs. Altogether, these results contribute to establish the basis for the use of EVs as therapeutic platforms, in particular in regenerative fields