Combining Hypoxia and Bioreactor Hydrodynamics Boosts Induced Pluripotent Stem Cell Differentiation Towards Cardiomyocytes

Cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) hold great promise for patient-specific disease modeling, drug screening and cell therapy. However, existing protocols for CM differentiation of iPSCs besides being highly dependent on the application of expensive growth factors show low reproducibility and scalability. The aim of this work was to develop a robust and scalable strategy for mass production of iPSC-derived CMs by designing a bioreactor protocol that ensures a hypoxic and mechanical environment. Murine iPSCs were cultivated as aggregates in either stirred tank or WAVE bioreactors. The effect of dissolved oxygen and mechanical forces, promoted by different hydrodynamic environments, on CM differentiation was evaluated. Combining a hypoxia culture (4 % O(2) tension) with an intermittent agitation profile in stirred tank bioreactors resulted in an improvement of about 1000-fold in CM yields when compared to normoxic (20 % O(2) tension) and continuously agitated cultures. Additionally, we showed for the first time that wave-induced agitation enables the differentiation of iPSCs towards CMs at faster kinetics and with higher yields (60 CMs/input iPSC). In an 11-day differentiation protocol, clinically relevant numbers of CMs (2.3 × 10(9) CMs/1 L) were produced, and CMs exhibited typical cardiac sarcomeric structures, calcium transients, electrophysiological profiles and drug responsiveness. This work describes significant advances towards scalable cardiomyocyte differentiation of murine iPSC, paving the way for the implementation of this strategy for mass production of their human counterparts and their use for cardiac repair and cardiovascular research. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12015-014-9533-0) contains supplementary material, which is available to authorized users

26Al kinematics: superbubbles following the spiral arms? : Constraints from the statistics of star clusters and HI supershells

High energy resolution spectroscopy of the 1.8 MeV radioactive decay line of 26Al with the SPI instrument on board the INTEGRAL satellite has recently revealed that diffuse 26Al has large velocities in comparison to other components of the interstellar medium in the Milky Way. 26Al shows Galactic rotation in the same sense as the stars and other gas tracers, but reaches excess velocities up to 300 km s−1Peer reviewe

Soul's Tools

This paper explores the various ways Aristotle refers to and employs “heat and cold” in his embryology. In my view, scholars are too quick to assume that references to heat and cold are references to matter or an animal’s material nature. More commonly, I argue, Aristotle refers to heat and cold as the “tools” of soul. As I understand it, Aristotle is thinking of heat and cold in many contexts as auxiliary causes by which soul activities (primarily “concoction”) are carried out. This, as I argue, is what it means to call them “tools” of soul. An upshot of this investigation is the fuller picture of Aristotle’s conception of efficient causation it provides in general, and the better understanding of the efficient causal operation of an organism’s nature or soul it provides in particular

From Early Embryonic to Adult Stage: Comparative Study of Action Potentials of Native and Pluripotent Stem Cell-Derived Cardiomyocytes

Cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPS-CMs) are promising candidates for cell therapy, drug screening, and developmental studies. It is known that iPS-CMs possess immature electrophysiological properties, but an exact characterization of their developmental stage and subtype differentiation is hampered by a lack of knowledge of electrophysiological properties of native CMs from different developmental stages and origins within the heart. Thus, we sought to systematically investigate action potential (AP) properties of native murine CMs and to establish a database that allows classification of stem cell-derived CMs. Hearts from 129S2PasCrl mice were harvested at days 9-10, 12-14, and 16-18 postcoitum, as well as 1 day, 3-4 days, 1-2 weeks, 3-4 weeks, and 6 weeks postpartum. AP recordings in left and right atria and at apical, medial, and basal left and right ventricles were performed with sharp glass microelectrodes. Measurements revealed significant changes in AP morphology during pre-and postnatal murine development and significant differences between atria and ventricles, enabling a classification of developmental stage and subtype differentiation of stem cell-derived CMs based on their AP properties. For iPS-CMs derived from cell line TiB7.4, a typical ventricular phenotype was demonstrated at later developmental stages, while there were electrophysiological differences from atrial as well as ventricular native CMs at earlier stages. This finding supports that iPS-CMs can develop AP properties similar to native CMs, but points to differences in the maturation process between iPS-CMs and native CMs, which may be explained by dissimilar conditions during in vitro differentiation and in vivo development

Murine transgenic iPS cell line for monitoring and selection of cardiomyocytes

We report here a transgenic murine induced pluripotent stem cell (iPSC) line expressing puromycin N-acetyl-transferase (PAC) and enhanced green fluorescent protein (EGFP) under the control of a-myosin heavy chain promoter. This transgenic cell line reproducibly differentiates into EGFP-expressing cardiomyocytes (CMs) which can be generated at high purity with puromycin treatment and exhibit molecular and functional properties of immature heart muscle cells. This genetically modified iPSC line can be used for assessment of the utility of CMs for myocardial repair, pharmacological and toxicological applications and development of improved cardiac differentiation protocols. (C) 2016 The Authors. Published by Elsevier B.V

Salicylic diamines selectively eliminate residual undifferentiated cells from pluripotent stem cell-derived cardiomyocyte preparations

Clinical translation of pluripotent stem cell (PSC) derivatives is hindered by the tumorigenic risk from residual undifferentiated cells. Here, we identified salicylic diamines as potent agents exhibiting toxicity to murine and human PSCs but not to cardiomyocytes (CMs) derived from them. Half maximal inhibitory concentrations (IC50) of small molecules SM2 and SM6 were, respectively, 9- and 18-fold higher for human than murine PSCs, while the IC50 of SM8 was comparable for both PSC groups. Treatment of murine embryoid bodies in suspension differentiation cultures with the most effective small molecule SM6 significantly reduced PSC and non-PSC contamination and enriched CM populations that would otherwise be eliminated in genetic selection approaches. All tested salicylic diamines exerted their toxicity by inhibiting the oxygen consumption rate (OCR) in PSCs. No or only minimal and reversible effects on OCR, sarcomeric integrity, DNA stability, apoptosis rate, ROS levels or beating frequency were observed in PSC-CMs, although effects on human PSC-CMs seemed to be more deleterious at higher SM-concentrations. Teratoma formation from SM6-treated murine PSC-CMs was abolished or delayed compared to untreated cells. We conclude that salicylic diamines represent promising compounds for PSC removal and enrichment of CMs without the need for other selection strategies

Aristotle on the Powers of Thermic Equilibrium

Popa focuses on the role of the two basic active dunameis, the hot and the cold, in Aristotle, Meteorology IV. There, the dispositional properties of the homoeomers are often defined and explained in virtue of bodies’ reactions to heat and cold: some homogeneous bodies are solidifiable by heat, others by cold. Popa argues that Meteor. IV aims to account for the coming about and persistence of uniform stuffs by appealing to what he calls “thermic equilibrium” (summetria, logos). The main purpose of this chapter is to clarify the ways in which Aristotle puts this notion to work in his study of homogeneous materials (especially in Meteor. IV) and, through this clarification, to contribute to a better understanding of the role played by proper or internal natural heat in his scientific works