Dielectric spectroscopy monitoring of a bioreactor process for hiPSC expansion and differentiation

Bioprocessing strategies using 3D cell culturing approaches, such as cell aggregates, are promising solutions to achieve efficient and scalable bioprocesses for stem cell expansion and differentiation. However, tracking viable and total cell numbers in such culture systems is not straightforward. It requires cell detachment, disaggregation or disruption, which results in measurements that are laborious, biased and with high variability. In this work, we used a commercially available capacitance probe to explore the applicability of dielectric spectroscopy for in situ monitoring of a multistep process for expansion and differentiation of human induced pluripotent stem cells (hiPSC) cultivated as cell aggregates. After 5 days of cell expansion in a bioreactor, the hepatic differentiation step was integrated by addition of different levels of specific soluble factors at various stages of the process to promote growth and generate populations successively enriched for definitive endoderm, hepatoblasts, hepatocyte progenitors and mature hepatocytes. While this differentiation procedure has been previously validated for monolayer cultures, this was the first time it was carried out in a stirred tank bioreactor operated in perfusion mode. Phenotype analysis confirmed a marked increase in key hepatic differentiation markers culminating at day 21 of differentiation. Our data shows a good correlation between total volume of the cell aggregates and permittivity measured by the probe (R2 = 0.84). However, there was a delay between changes in cell concentration and the permittivity signal. This suggests that cell expansion requires a few days to result in increased volume of the cell aggregates and that each aggregate behaves as one overall inducible dipole. The β-dispersion curve shape also appears to change over culture time and could eventually be used as an indicator for differentiation progression. Dielectric spectroscopy has been used successfully to monitor viable cell concentration in different single-cell suspension cultures, but there are few published applications to 3D cultures. Our results demonstrate the potential of dielectric spectroscopy to monitor complex bioprocesses for human stem cell aggregates in stirred cultures. Acknowledgements: Funding provided by ERA-NET/E-Rare3 programme through research project ERAdicatPH (E-Rare3/0002/2015). The authors acknowledge Dr Juan Rodriguez-Madoz (University of Navarra, Spain) and Dr Anders Aspegren (Takara Bio Europe – Cellartis AB, Sweden) for helpful discussions on hepatic differentiation of hiPSC.

An Intelligent Safety System for Human-Centered Semi-Autonomous Vehicles

Nowadays, automobile manufacturers make efforts to develop ways to make cars fully safe. Monitoring driver's actions by computer vision techniques to detect driving mistakes in real-time and then planning for autonomous driving to avoid vehicle collisions is one of the most important issues that has been investigated in the machine vision and Intelligent Transportation Systems (ITS). The main goal of this study is to prevent accidents caused by fatigue, drowsiness, and driver distraction. To avoid these incidents, this paper proposes an integrated safety system that continuously monitors the driver's attention and vehicle surroundings, and finally decides whether the actual steering control status is safe or not. For this purpose, we equipped an ordinary car called FARAZ with a vision system consisting of four mounted cameras along with a universal car tool for communicating with surrounding factory-installed sensors and other car systems, and sending commands to actuators. The proposed system leverages a scene understanding pipeline using deep convolutional encoder-decoder networks and a driver state detection pipeline. We have been identifying and assessing domestic capabilities for the development of technologies specifically of the ordinary vehicles in order to manufacture smart cars and eke providing an intelligent system to increase safety and to assist the driver in various conditions/situations.Comment: 15 pages and 5 figures, Submitted to the international conference on Contemporary issues in Data Science (CiDaS 2019), Learn more about this project at https://iasbs.ac.ir/~ansari/fara

Increasing Therapy Related Myeloid Neoplasms in Multiple Myeloma

© 2018 Stichting European Society for Clinical Investigation Journal Foundation. This document is made available under the CC-BY-NC 4.0 license http://creativecommons.org/licenses/by-nc /4.0/ This document is the submitted version of a published work that appeared in final form in European Journal of Clinical Investigation.Background: Despite the longer survival achieved in multiple myeloma (MM) patients due to new therapy strategies, a concern is emerging regarding an increased risk of secondary primary malignancies (SPMs) and how to characterize those patients at risk. We performed a retrospective study covering a 28‐year follow‐ up period (1991‐2018) in a tertiary single institution. Material and Methods: Data of 403 MM patients were recorded and compared with the epidemiologic register of the population area covered by our centre, calculating the standardize incidence ratio (SIR) for the different types of SPMs diagnosed in the MM cohort. Fine and Gray regression models were used to identify risk factors for SPMs. Results: Out of the 403 MM patients, 23 (5.7%) developed SPMs: 13 therapyrelated myeloid (TRM) malignancies (10 of them (77%) myelodysplastic syndrome (MDS), 1 acute lymphoid leukaemia and 9 solid neoplasms. In the MM cohort, the relative risk of MDS was significantly higher than in the general population. Survival of patients with TRM malignancies was poor with a median of 4 months from the diagnosis, and most of them showed complex karyotype. Within the MM subset, multivariable analysis showed a higher risk of TRM malignancies in patients that previously received prolonged treatment with lenalidomide (>18 months). Conclusions: Though the improvement in MM outcome during the last decades is an unprecedented achievement, it has been accompanied by the rise in TRM malignancies with complex cytogenetic profile and poor prognosis that are in the need of an improved biologic and therapeutic approach

Un caso de seudología.

Se estudia un caso de seudología fantástica, trastorno de la personalidad incluido en los trastornos no especificados (DSM-III-R) y que podría ser definido, siguiendo a CASTILLA DEL PINO, como una caracterosis impositiva del self intelectual. El síntoma fundamental, en el caso que comentamos, está constituido por la personalidad del paciente, que se cimenta en un un falso self -expresado éste en los numerosos engaños y mentiras que giran, principalmente, alrededor de su identidad intelectual-. Como consecuencia de estas conductas mendaces, el paciente manifiesta, además, conductas desadaptivas (alcoholismo, endeudamientos en cadena, etc). A diferencia del fantasioso, el seudólogo opta por la acción: su intención es imponer el self fantaseado a los demás, por lo que no le basta la simple fantasía si ésta no es llevada a la práctica. Asímismo, es preciso diferenciar el cuadro seudólogo de las neurosis histéricas, estados hipomaníacos y psicosis paranoides crónicas de exaltación intelectual. El abordaje psicoterapéutico suele verse dificultado por la actitud del paciente

Un caso de seudología.

Se estudia un caso de seudología fantástica, trastorno de la personalidad incluido en los trastornos no especificados (DSM-III-R) y que podría ser definido, siguiendo a CASTILLA DEL PINO, como una caracterosis impositiva del self intelectual. El síntoma fundamental, en el caso que comentamos, está constituido por la personalidad del paciente, que se cimenta en un un falso self -expresado éste en los numerosos engaños y mentiras que giran, principalmente, alrededor de su identidad intelectual-. Como consecuencia de estas conductas mendaces, el paciente manifiesta, además, conductas desadaptivas (alcoholismo, endeudamientos en cadena, etc). A diferencia del fantasioso, el seudólogo opta por la acción: su intención es imponer el self fantaseado a los demás, por lo que no le basta la simple fantasía si ésta no es llevada a la práctica. Asímismo, es preciso diferenciar el cuadro seudólogo de las neurosis histéricas, estados hipomaníacos y psicosis paranoides crónicas de exaltación intelectual. El abordaje psicoterapéutico suele verse dificultado por la actitud del paciente

Low frequency of CD4(+) CD25(+) Treg in SLE patients: a heritable trait associated with CTLA4 and TGF gene variants

Background: CD4(+)CD25(+) regulatory T cells play an essential role in maintaining immune homeostasis and preventing autoimmunity. Therefore, defects in Treg development, maintenance or function have been associated with several human autoimmune diseases including Systemic Lupus Erythematosus (SLE), a systemic autoimmune disease characterized by loss of tolerance to nuclear components and significantly more frequent in females

Intensifying the manufacture of hiPSC therapy products through metabolic and process understanding

In vitro differentiation of human induced pluripotent stem cells into specific lineages such as cardiomyocytes (hPSC-CM) and hepatocytes (hPCS-Hep) is a crucial process to enable their application in cell therapy and drug discovery. Nevertheless, despite the remarkable efforts over the last decade towards the implementation of protocols for hPSC expansion and differentiation, there are some technological challenges remaining include the low scalability and differentiation yields. Additionally, generated cells are still immature, closely reminiscent of fetal/embryonic cells in what regards phenotype and function. In this study, we aim to overcome this hurdle by devising bioinspired and integrated strategies to improve the generation and functionality of these hiPSC-derivatives. We also applied robust multi-parametric techniques including proteomics, transcriptomics, metabolomics and fluxomics as complementary analytical tools to support bioprocess optimization and product characterization. We cultured hiPSC as 3D aggregates in stirred-tank bioreactors (STB) operated in perfusion and used a capacitance probe for in situ monitoring of cell growth/differentiation. After cell expansion, the hepatic differentiation step was integrated by addition of key soluble factors and controlling the dissolved oxygen concentration at various stages of the process to generate populations enriched for definitive endoderm, hepatocyte progenitors and mature hepatocytes. The analyses of hepatic markers expression throughout the stages of the differentiation confirmed that hepatocyte differentiation was improved in 3D spheroids when compared to 2D culture. Noteworthy, these hiPSC-HLC exhibited functional characteristics typical of hepatocytes (albumin production, glycogen storage and CYP450 activity). We also demonstrate the potential of dielectric spectroscopy to monitor cell expansion and hepatic differentiation in STB. For CM differentiation, we relied on the aggregation of hPSC-derived cardiac progenitors to establish a scalable differentiation protocol capable of generating highly pure CM aggregate cultures. We assessed if alteration of culture medium composition to mimic in vivo substrate usage during cardiac development improved further hPSC-CM maturation in vitro. Our results showed that shifting hPSC-CMs from glucose-containing to galactose- and fatty acid-containing medium promotes their fast maturation into adult-like CMs with higher oxidative metabolism, transcriptional signatures closer to those of adult ventricular tissue, higher myofibril density and alignment, improved calcium handling, enhanced contractility, and more physiological action potential kinetics. “-Omics” analyses showed that addition of galactose to culture medium and culturing the cells under perfusion improves total oxidative capacity of the cells and ameliorates fatty acid oxidation. This study demonstrated that metabolic shifts during differentiation/maturation of hPSC-CM are a cause, rather than a consequence, of the phenotypic and functional alterations observed. The metabolic-based strategy established herein holds technical and economic advantages over the existing protocols due to its scalability, simplicity and ease of application. Funding: This work was supported by FCT-funded projects NETDIAMOND (SAICTPAC/0047/2015), MetaCardio (Ref.032566) and FCT/ERA-Net (ERAdicatPH; Ref. E-Rare3/0002/2015). iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344) is also acknowledged