Enginnering cardiac tissue using human induced pluripotent stem cell derivatives: Proteomic characterization of co-cultures of cardiomyocytes and endothelial cells

Prediction of cardiac toxicity effect is extremely relevant in the development of new drugs for different medical applications. In this way, it is important to develop more predictable human cell-based models which physiologically better mimic the human heart and allow the prediction of this toxic effect as well as establish the tools that enable the characterization of these complex cell models. To recreate engineered cardiac tissue, it is essential to reproduce the complexity of the heart by resorting to different cell types. Cardiomyocytes (CMs) are functional contractile units of the heart, and it is known that their communication with endothelial cells (ECs) is crucial for cardiac homeostasis. The aim of this study is to recreate a human pluripotent stem cells (hiPSC)-based cardiac tissue model and evaluate the impact of communication between both cell types on the phenotype of CMs. Co-cultures of hiPSC-CM and hiPSC-EC were established and maintained for 12 days as confirmed by immunofluorescence microscopy. Quantitative whole-proteome analysis was performed using SWATH Mass Spectrometry tools to compare the conditions of hiPSC-CM mono-culture and the co-culture of hiPSC-CM and hiPSC-EC. Our data showed relative increase of expression ratios of morphological maturation-related cardiac proteins in hiPSC-CM co-cultures. In particular, the expression ratios of MYH7/MYH6, MYL2/MYL7, TNNI3/TNNI1 increased 2.4-, 5.1-, and 5-fold, respectively, when compared to the mono-culture condition, indicating that in the presence of hiPSC-EC, hPSC-CM display a more adult- and ventricular- like phenotype. Changes in the extracellular matrix composition were also observed, especially related with the increased expression of ECM proteins in co-culture condition namely, collagens I and III (8.6-fold and 6-fold, respectively), fibronectin (3.5-fold) and thrombospondin-4 (2.5-fold). Other growth factors attributed to the extracellular space (e.g. CTGF, PAI1, CRTAP, IGFBP7, and NPPB) that may be responsible for the communication between both cell types have also shown to be up-regulated in the co-culture condition. The presence of a SMA+ (myofibroblast-like) population in the co-culture condition was observed by immunofluorescence microscopy images, which is in agreement with the more complex and fibrotic extracellular matrix found by whole proteome analysis. Ultrastructure characterization of CMs was carried out by transmission electron microscopy. In both conditions, hiPSC-CM displayed aligned myofibrils composed by sarcomeres with organized Z-disks, A- and I-bands, intercalated discs between adjacent cells as well as abundant mitochondria. Noteworthy, sarcomere length was higher in hiPSC-CM cultured with hiPSC-EC, suggesting structural changes associated with cardiomyocyte maturation. Calcium imaging is being performed to evaluate calcium handling of hiPSC-CMs and their response to drugs. All together our data revealed that promoting the communication of hiPSC-CM and hiPSC-EC induced structural changes in hiPSC-CM associated with maturation. This study provides important insights towards the development of more complex cardiac tissues and establishes potent analytical tools for the characterization of these models. This work was supported by Fundação para a Ciência e Tecnologia (FCT)-funded project CARDIOSTEM (MITP-TB/ECE/0013/2013); and iNOVA4Health UID/Multi/04462/2013, a program supported by FCT/Ministério da Educação e Ciência, through national funds and cofounded by FEDER under the PT2020 Partnership Agreement. BA. was supported by FCT Grant SFRH/BD/52475/201

Production of high-quality SARS-CoV-2 antigens for vaccine development and serological assays implementation

The development of novel and/or improved vaccines as well as the establishment of tools to monitor vaccine responses are two key factors to control COVID-19 pandemic, often requiring the manufacturing of significant amounts of high-quality SARS-CoV-2 antigens. In this work, we produced SARS-CoV-2 Spike (S) and the receptor binding domain (RBD) proteins in human or insect cell lines to be further used in (i) the implementation of serological assays for detection of antibodies against SARS-CoV-2 virus in the Portuguese population, and (ii) the development of a virosome-based COVID-19 vaccine candidate. Please click Download on the upper right corner to see the full abstract

Bioprocess intensification for the continuous expansion of 3D human induced pluripotent stem cell aggregates in bioreactors

Human induced pluripotent stem cells (hiPSC) are attractive tools for drug screening and disease modeling and promising candidates for cell therapy applications. However, to achieve the high numbers of cells required for these purposes, scalable and clinical-grade technologies must be established. In this study, we use environmentally controlled stirred-tank bioreactors operating in perfusion as a powerful tool for bioprocess intensification of hiPSC production. Firstly, we demonstrate the importance of controlling the dissolved oxygen concentration at low levels (4% oxygen) and perfusion at 1.3 day-1 dilution rate to improve hiPSC growth as 3D aggregates in xeno-free medium (Cellartis® DEF-CS™ 500 Xeno-Free Culture Medium). This strategy allowed for increased cell specific growth rate, maximum volumetric cell concentrations (4.7x106 cell/mL) and expansion factors (approximately 19), resulting in an overall improvement of 2.6-fold in cell yields. Extensive cell characterization, including whole proteomic analysis was performed to confirm that the pluripotent phenotype was maintained during culture. Secondly, we have tested different chemical and mechanical strategies for hiPSC aggregate dissociation, revealing similar viable cell recovery yields (approximately 50%). However, only the mechanical dissociation strategies enabled the re-aggregation of hiPSC in stirred conditions, with the mechanical dissociation using a 70 μm pore size nylon mesh allowing a higher expansion factor after dissociation. Finally, a scalable protocol for continuous expansion of hiPSC aggregates in bioreactors was implemented using the mechanical dissociation for aggregate disruption/passaging. A total expansion factor of 1100 in viable cells was obtained in 11 days of culture after 3 sequential passages in bioreactors, while cells maintained their proliferation capacity, pluripotent phenotype and potential as well as genomic stability. To our knowledge, this is the highest expansion factor reported for hiPSC for such a short culture time frame. The strategy described herein for continuous expansion of hiPSC provides important insights towards up-scaling the production of hiPSC. Integrative biomanufacturing processes using this continuous strategy are now being pursued for hiPSC expansion and differentiation towards cardiac lineages in order to recreate cardiac models for drug discovery, toxicity testing and disease modeling. Acknowledgments: This work was supported by Fundação para a Ciência e Tecnologia (FCT)-funded projects CARDIOSTEM (MITP-TB/ECE/0013/2013) and CardioRegen (HMSP-ICT/0039/2013); and iNOVA4Health UID/Multi/04462/2013, a program supported by FCT/Ministério da Educação e Ciência, through national funds and cofounded by FEDER under the PT2020 Partnership Agreement. BA. was supported by FCT Grant SFRH/BD/52475/2013. The work was also funded by a Vinnova Grant, registration number 2014-00310 (Cell therapies via large scale expansion of pluripotent stem cells)

Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: Bioprocess intensification and scaling-up approaches

Human induced pluripotent stem cells (hiPSC) are attractive tools for drug screening and disease modeling and promising candidates for cell therapy applications. However, to achieve the high numbers of cells required for these purposes, scalable and clinical-grade technologies must be established. In this study, we use environmentally controlled stirred-tank bioreactors operating in perfusion as a powerful tool for bioprocess intensification of hiPSC production. We demonstrate the importance of controlling the dissolved oxygen concentration at low levels (4% oxygen) and perfusion at 1.3 day-1 dilution rate to improve hiPSC growth as 3D aggregates in xeno-free medium (Cellartis® DEF-CS™ 500 Xeno-Free Culture Medium). This strategy allowed for increased cell specific growth rate, maximum volumetric cell concentrations (4.7x106 cell/mL) and expansion factors (approximately 19), resulting in an overall improvement of 2.6-fold in cell yields. Extensive cell characterization, including whole proteomic analysis was performed to confirm that the pluripotent phenotype was maintained during culture. Furthermore, a scalable protocol for continuous expansion of hiPSC aggregates in bioreactors was implemented using mechanical dissociation protocols for aggregate disruption and cell passaging. A total expansion factor of 1100 in viable cells was obtained in 11 days of culture (Figure 1), while cells maintained their proliferation capacity, pluripotent phenotype and potential as well as genomic stability after 3 sequential passages in bioreactors. To our knowledge, this is the highest expansion factor reported for hiPSC for such a short culture time frame. The strategy described herein for continuous expansion of hiPSC provides important insights towards up-scale production of hiPSC. This will strengthen the utility of hiPSC in cell therapy, drug discovery, toxicity testing and disease modeling. Please click Additional Files below to see the full abstract

Controle de hiperglicemia intra-hospitalar em pacientes críticos e não-críticos

Hiperglicemia é uma condição frequente encontrada no ambiente intra-hospitalar. Quando presente em pacientes críticos internados pode levar a maior morbi-mortalidade, comprometendo o tratamento da doença de base. Essa revisão tem por objetivo padronizar a conduta terapêutica na hiper e hipoglicemias nos pacientes internados em ambiente hospitalar, de forma a reduzir tempo de internação e mortalidadeHyperglycemia is a common condition found in-hospital environment. When present in critically ill patients admitted can lead to increased morbidity and mortality, affecting the treatment of the underlying disease. This protocol aims to standardize the therapeutic approach in hyper-and hypoglycemia in patients admited to hospital in order to reduce length of stay and mortality.

Iliac artery false aneurysm twelve years after allograft nephrectomy

We report a case of a non-infected right external iliac artery false aneurysm. The patient was a 44 year-old woman on chronic peritoneal dialysis had had an allograft nephrectomy 12 years before and who presented with acute abdominal pain. Ultrasound and CT-scan showed a saccular aneurysm arising from the right external iliac artery. A large false aneurysm was identified during surgery, from donor arterial vessel remaining in situ after graft nephrectomy. Resection of the false aneurysm, with ligation of the right external iliac artery and interposition of a femorofemoral graft was successfully performed, with an uneventful post-operative recovery. False aneurysms after renal allograft nephrectomy are very rare. To our knowledge, this is the longest reported period of time between na nephrectomy and clinical evidence of a false aneurysm.info:eu-repo/semantics/publishedVersio

Mesoscale and Nutrient Conditions Associated with the Massive 2008 Cochlodinium polykrikoides Bloom in the Sea of Oman/Arabian Gulf

Cochlodinium polykrikoides formed large blooms in the coastal waters of Oman from October 2008 through mid-January 2009, and satellite images from Aqua-MODIS and region-wide reports suggest that this bloom was found throughout the Arabian Gulf and Sea of Oman for more than 10 months. The unusual occurrence of this species appears to have supplanted the more regularly occurring bloom species, Noctiluca scintillans, in 2008–2009. For the first 2 weeks of the coastal Omani bloom, C. polykrikoides abundance was near monospecific proportions, with cell densities ranging from 4.6 × 103 to 9 × 106 cells L−1 and very high levels of chlorophyll a (78.0 μg L−1) were also recorded. The regional progression of the bloom likely began with stronger than normal upwelling along the Iranian and northern Omani coasts during the southwest monsoon in late summer, followed by discharge of unusually warm coastal plume water along the coast of Oman with the reversal of monsoonal winds in late October. The occurrence and persistence of high densities of C. polykrikoides in Oman coastal water were also significantly influenced by an elevated nutrient load and warmer than normal temperatures. Concentrations of nutrients, especially NH4 +, urea, PO4 3−, and organic nitrogen and phosphorus, were manyfold higher than observed in the year prior or since. These findings suggest that mesoscale features were important in bloom dynamics more regionally, but locally the bloom was sustained by nutrient enrichment supplemented by its mixotrophic capabilities