XENO-Free production and recovery of human pluripotent stem cells using synthetic dissolvable microcarriers

The implementation of scalable culture platforms for the large-scale production of human pluripotent stem cells (hPSC) and their derivatives is mandatory to fulfill the requirement of obtaining large numbers of these cells for cell therapies and other in vitro biomedical applications, such as drug screening, toxicology assays and disease modeling. Recent progress includes the development of chemically-defined culture conditions for manufacturing of hPSC and their derivatives, namely the development of xeno-free microcarrier platforms to meet good manufacturing practice (GMP) quality requirements [1]. One challenge that remains to be addressed is the establishment of a robust, scalable, and cost-effective downstream processing for cell recovery and removal of the microcarriers. Since hPSC have the tendency to create multilayers of cells on the microcarriers, often forming very large cell-microcarrier aggregates, the process of cell recovery can be technically challenging and time consuming. In this work, we developed a robust and efficient platform for large-scale production of hPSC using synthetic dissolvable microcarriers, which can be quickly dissolved by a non-proteolytic enzyme. This allows an easy cell recovery without the need of the microcarrier separation step, facilitating the downstream processing. Moreover, these synthetic microcarriers are sterile and ready-to-use, and are functionalized with the Synthemax® surface, based on a peptide-acrylate matrix designed for long-term support of hESC self-renewal [2]. hPSC were able to attach and grow on the dissolvable microcarriers and the expansion process was evaluated in a scalable stirred culture system. The cells growth performance on these microcarriers was comparable with the ones obtained when culturing hPSCs in non-dissolvable microcarriers (backbone of polystyrene coated with different ECM molecules), being possible to obtain 1.3x106 cells/mL during 5 days. Importantly, hPSCs cultured on these novel microcarriers were efficiently recovered without the need of the filtration step to separate the microcarriers from the cells and maintained their typical colony morphology and pluripotency-associated marker-expression after re-plating on tissue culture plates. Moreover, their potential for spontaneous differentiation into cells of the three embryonic germ layers was demonstrated through formation of embryoid bodies containing cells expressing typical markers of endoderm, ectoderm and mesoderm. These novel synthetic dissolvable microcarriers allow an easy and efficient downstream processing for hPSCs recovery after expansion/differentiation, without compromising the quality of the cells (viability, potency and functionality), which are a major process breakthrough for stem cell manufacturing. [1] Badenes SM, et al., “Defined Essential 8™ Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems”, PlosOne (2016), 11(3):e0151264. [2] Melkoumian Z, et al, “Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells”, Nat Biotechnol (2010), 28(6): 606-10. Acknowledgements: We acknowledge CORNING Incorporated for supplying the dissolvable microcarriers

Regulation of embryonic neurogenesis by germinal zone vasculature

In the adult rodent brain, new neurons are born in two germinal regions that are associated with blood vessels, and blood vessels and vessel-derived factors are thought to regulate the activity of adult neural stem cells. Recently, it has been proposed that a vascular niche also regulates prenatal neurogenesis. Here we identify the mouse embryo hindbrain as a powerful model to study embryonic neurogenesis and define the relationship between neural progenitor cell (NPC) behavior and vessel growth. Using this model, we show that a subventricular vascular plexus (SVP) extends through a hindbrain germinal zone populated by NPCs whose peak mitotic activity follows a surge in SVP growth. Hindbrains genetically defective in SVP formation owing to constitutive NRP1 loss showed a premature decline in both NPC activity and hindbrain growth downstream of precocious cell cycle exit, premature neuronal differentiation, and abnormal mitosis patterns. Defective regulation of NPC activity was not observed in mice lacking NRP1 expression by NPCs, but instead in mice lacking NRP1 selectively in endothelial cells, yet was independent of vascular roles in hindbrain oxygenation. Therefore, germinal zone vascularization sustains NPC proliferation in the prenatal brain

Non-Asthmatic Patients Show Increased Exhaled Nitric Oxide Concentrations

OBJECTIVE: Evaluate whether exhaled nitric oxide may serve as a marker of intraoperative bronchospasm. INTRODUCTION: Intraoperative bronchospasm remains a challenging event during anesthesia. Previous studies in asthmatic patients suggest that exhaled nitric oxide may represent a noninvasive measure of airway inflammation. METHODS: A total of 146,358 anesthesia information forms, which were received during the period from 1999 to 2004, were reviewed. Bronchospasm was registered on 863 forms. From those, three groups were identified: 9 non-asthmatic patients (Bronchospasm group), 12 asthmatics (Asthma group) and 10 subjects with no previous airway disease or symptoms (Control group). All subjects were submitted to exhaled nitric oxide measurements (parts/billion), spirometry and the induced sputum test. The data was compared by ANOVA followed by the Tukey test and Kruskal-Wallis followed by Dunn's test. RESULTS: The normal lung function test results for the Bronchospasm group were different from those of the asthma group (p <0.05). The median percentage of eosinophils in induced sputum was higher for the Asthma [2.46 (0.45-6.83)] compared with either the Bronchospasm [0.55 (0-1.26)] or the Control group [0.0 (0)] (p <0.05); exhaled nitric oxide followed a similar pattern for the Asthma [81.55 (57.6-86.85)], Bronchospasm [46.2 (42.0 -62.6] and Control group [18.7 (16.0-24.7)] (p< 0.05). CONCLUSIONS: Non-asthmatic patients with intraoperative bronchospasm detected during anesthesia and endotracheal intubation showed increased expired nitric oxide

Eficácia do herbicida glyphosate no controle de Brachiaria decumbens na cultura da guariroba (Syagrus oleracea)

 This study was carried out to analyze the use, in total area, of different doses of glyphosate in guariroba crop to control Brachiaria decumbens in post-emergence. The commercial product Roundup Transorb (648 g of glyphosate 1L) was applied at the doses of: 972,1215,1458,1701 and 1944 g/ha. The experimental design was arandom blocks, with four repetitions. The volume of solution applied was 2001 L/ha, at a temperature of26°C and relative humidity of 84%, by the use of a manual sprayer, pressured by C02 at 32 lb/in', with six flat nozzles TI 110.02. Phytotoxicity and the control of Brachiaria decumbens were analyzed 6, 12,21 and 31 days after chemical application (DAA).1t was conclued that the species Syagrus oleracea was highly tolerant to the herbicide glyphosate, even at the highest studied dose and the herbicide glyphosate independent1y of the studied dose, was very efficient in the control of young Brachiaria decumbens plants, exerting 100% control.Este trabalho foi realizado com objetivo de avaliar o efeito, em área total, de diferentes doses de glyphosate sobre a cultura da guarirobae no controle de Brachiaria decumbens em pós-emergência. Foram utilizadas as doses de 972, 1215, 1458, 1701 e 1944 g/ha. O delineamento experimental foi o de blocos casualizados, com quatro repetições. O volume de calda aplicado foi de 200 L/ha, sob temperatura de 26°C e umidade relativa de 84%, utilizando-se pulverizador manual, pressurizado por CO2 a 2,2 kgf/cm/, com seis bicos tipo leque de jato plano TI 110.02. Avaliou-se a fitotoxicidade e o controle de B. decumbens aos 6, 12, 21 e 31 dias após a aplicação (DAA). Concluiu-se que a espécie Syagrus oleracea é altamente tolerante ao herbicida glyphosate, mesmo na dose de 1944 g/ha. Independentemente da dose estudada, o glyphosate foi muito eficaz no controle de plantas jovens de Brachiaria decumbens, uma vez que exerceu um controle de 100%.

Loss of Prox1

Correct regulation of troponin and myosin contractile protein gene isoforms is a critical determinant of cardiac and skeletal striated muscle development and function, with misexpression frequently associated with impaired contractility or disease. Here we reveal a novel requirement for Prospero-related homeobox factor 1 (Prox1) during mouse heart development in the direct transcriptional repression of the fast-twitch skeletal muscle genes troponin T3, troponin I2, and myosin light chain 1. A proportion of cardiac-specific Prox1 knockout mice survive beyond birth with hearts characterized by marked overexpression of fast-twitch genes and postnatal development of a fatal dilated cardiomyopathy. Through conditional knockout of Prox1 from skeletal muscle, we demonstrate a conserved requirement for Prox1 in the repression of troponin T3, troponin I2, and myosin light chain 1 between cardiac and slow-twitch skeletal muscle and establish Prox1 ablation as sufficient to cause a switch from a slow- to fast-twitch muscle phenotype. Our study identifies conserved roles for Prox1 between cardiac and skeletal muscle, specifically implicated in slow-twitch fiber-type specification, function, and cardiomyopathic disease

Mn-based methacrylated gellan gum hydrogels for MRI-guided cell delivery and imaging

This work aims to engineer a new stable injectable Mn-based methacrylated gellan gum (Mn/GG-MA) hydrogel for real-time monitored cell delivery into the central nervous system. To enable the hydrogel visualization under Magnetic Resonance Imaging (MRI), GG-MA solutions were supplemented with paramagnetic Mn2+ ions before its ionic crosslink with artificial cerebrospinal fluid (aCSF). The resulting formulations were stable, detectable by T1-weighted MRI scans and also injectable. Cell-laden hydrogels were prepared using the Mn/GG-MA formulations, extruded into aCSF for crosslink, and after 7 days of culture, the encapsulated human adipose-derived stem cells remained viable, as assessed by Live/Dead assay. In vivo tests, using double mutant MBPshi/shi/rag2 immunocompromised mice, showed that the injection of Mn/GG-MA solutions resulted in a continuous and traceable hydrogel, visible on MRI scans. Summing up, the developed formulations are suitable for both non-invasive cell delivery techniques and image-guided neurointerventions, paving the way for new therapeutic procedures.Sílvia Vieira acknowledges the FCT Ph.D. scholarship (SFRH/BD/102710/2014). J. Miguel Oliveira and J. Silva-Correia acknowledge the FCT grants under the Investigator FCT program (IF/01285/2015 and IF/00115/2015, respectively). The authors also acknowledge the funds provided under the project NanoTech4ALS, funded under the EU FP7 M-ERA.NET program, and ESF (POWR.03.02.00-00-I028/17-00)

Hierarchical HRP-crosslinked silk fibroin/ZnSr-doped TCP nancocomposites towards osteochondral tissue regeneration: Biomechanical performance and in vivo assessment

[Excerpt] Introduction. Recent investigations highlight promising regenerative strategies for osteochondral (OC) tissue treatment, such as hierarchical nanocomposite scaffolds containing ionic dopants.1,2 They allow cell infiltration and ECM formation throughout the engineered cartilage and subchondral tissues. The biomechanical behavior, antibacterial properties, and in vivo performance of hierarchical nanostructures combining enzymatically crosslinked silk fibroin (SF) and ZnSr-doped β-tricalcium phosphate (ZnSrTCP) for OC tissue regeneration is herein assessed. [...]Thanks to the Portuguese Foundation for Science and Technology for M-era-Net/0001/2014 project, and for the distinctions (IF/01285/2015) and (CEECIND/03673/2017)

Magnetic behaviour of perovskite compositions derived from BiFeO3

The phase content and sequence, the crystal structure, and the magnetic properties of perovskite solid solutions of the (1−y)BiFeO3–yBiZn0.5Ti0.5O3 series (0.05 ≤ y ≤ 0.90) synthesized under high pressure have been studied. Two perovskite phases, namely the rhombohedral R3c and the tetragonal P4mm, which correspond to the structural types of the end members, BiFeO3 and BiZn0.5Ti0.5O3, respectively, were revealed in the as-synthesized samples. The rhombohedral and the tetragonal phases were found to coexist in the compositional range of 0.30 ≤ y ≤ 0.90. Magnetic properties of the BiFe1−y [Zn0.5Ti0.5]yO3 ceramics with y < 0.30 were measured as a function of temperature. The obtained compositional variations of the normalized unit-cell volume and the Néel temperature of the BiFe1−y [Zn0.5Ti0.5]yO3 perovskites in the range of their rhombohedral phase were compared with the respective dependences for the BiFe1−yB 3+yO3 perovskites (where B 3+ = Ga, Co, Mn, Cr, and Sc). The role of the high-pressure synthesis in the formation of the antiferromagnetic states different from the modulated cycloidal one characteristic of the parent BiFeO3 is discussed.publishe

Biocomposite macrospheres based on strontium-bioactive glass for application as bone fillers

Traditional bioactive glass powders are typically composed of irregular particles that can be packed into dense configurations presenting low interconnectivity, which can limit bone ingrowth. The use of novel biocomposite sphere formulations comprising bioactive factors as bone fillers are most advantageous, as it simultaneously allows for packing the particles in a 3-dimensional manner to achieve an adequate interconnected porosity, enhanced biological performance, and ultimately a superior new bone formation. In this work, we develop and characterize novel biocomposite macrospheres of Sr-bioactive glass using sodium alginate, polylactic acid (PLA), and chitosan (CH) as encapsulating materials for finding applications as bone fillers. The biocomposite macrospheres that were obtained using PLA have a larger size distribution and higher porosity and an interconnectivity of 99.7%. Loose apatite particles were observed on the surface of macrospheres prepared with alginate and CH by means of soaking into a simulated body fluid (SBF) for 7 days. A dense apatite layer was formed on the biocomposite macrospheresâ surface produced with PLA, which served to protect PLA from degradation. In vitro investigations demonstrated that biocomposite macrospheres had minimal cytotoxic effects on a human osteosarcoma cell line (SaOS-2 cells). However, the accelerated degradation of PLA due to the degradation of bioactive glass may account for the observed decrease in SaOS-2 cells viability. Among the biocomposite macrospheres, those composed of PLA exhibited the most promising characteristics for their potential use as fillers in bone tissue repair applications.This work was funded by grant #2019/15960-6, São Paulo Research Foundation in Brazil (FAPESP) and the Portuguese Foundation for Science and Technology (FCT), Reference UID/CTM/50025/2019 and FCT/Minister of Science, Technology and Higher Education in Portugal (MCTES) and by European Regional Development Fund (FEDER) funds through the COMPETE 2020 Program in the framework of ORAiDEA project (ref n° 39985 - AAC 31/SI/2017). The authors would also like to acknowledge Materials Research Center (CENIMAT) of the Associated Laboratory Institute of Nanostructures, Nanomodeling and Nanofabrication (i3N), NOVA University of LisbonCENIMAT|i3N and National Council for Scientific and Technological Development in Brazil CNPq (303149/2018-3). Ibrahim Fatih Cengiz acknowledges the FCT distinction attributed to him under the “Estímulo ao Emprego Científico” program (2021.01969.CEECIND). The authors thank the financial support provided under the projects: “HEALTH-UNORTE: Setting-up biobanks and regenerative medicine strategies to boost research in cardiovascular, musculoskeletal, neurological, oncological, immunological, and infectious diseases”, reference NORTE-01-0145-FEDER-000039, funded by the Norte Portugal Regional Coordination and Development Commission (CCDR-N), under the NORTE2020 Program; Projects LA/P/0037/2020, UIDP/50025/2020, and UIDB/50025/ 2020 of the Associate Laboratory i3N financed by national funds from FCT

Apoio institucional a projetos de inovação: criando cenários de aproximação ao ‘Scholarship of Teaching and Learning’

O Programa de Apoio a Projetos de Inovação e Desenvolvimento do Ensino e da Aprendizagem dinamizado pelo Centro IDEA-UMinho procura aproximar a inovação ao ‘Scholarship of Teaching and Learning’ (SoTL), o que pressupõe que os docentes desenvolvam estratégias de intervenção e analisem as suas práticas, construam conhecimento útil à profissão e o disseminem junto dos pares. O estudo aqui apresentado, desenvolvido pelos autores enquanto membros do Centro, focou-se na análise qualitativa das 10 candidaturas selecionadas na 1ª edição do Programa, lançada em finais de 2018. Foram consideradas na análise três dimensões da inovação relacionadas com o SoTL: Problematização, Indagação e Transferibilidade. Os resultados indicam que o Programa favorece o SoTL, observando-se algumas limitações na explicitação dos processos investigativos e apontandose linhas de ação futura. O estudo contribui para uma reflexão sobre o papel das estruturas de apoio ao ensino na promoção da inovação nas instituições de ensino superior em Portugal