Tackling Ischemic Reperfusion Injury With the Aid of Stem Cells and Tissue Engineering

Ischemia is a severe condition in which blood supply, including oxygen (O), to organs and tissues is interrupted and reduced. This is usually due to a clog or blockage in the arteries that feed the affected organ. Reinstatement of blood flow is essential to salvage ischemic tissues, restoring O, and nutrient supply. However, reperfusion itself may lead to major adverse consequences. Ischemia-reperfusion injury is often prompted by the local and systemic inflammatory reaction, as well as oxidative stress, and contributes to organ and tissue damage. In addition, the duration and consecutive ischemia-reperfusion cycles are related to the severity of the damage and could lead to chronic wounds. Clinical pathophysiological conditions associated with reperfusion events, including stroke, myocardial infarction, wounds, lung, renal, liver, and intestinal damage or failure, are concomitant in due process with a disability, morbidity, and mortality. Consequently, preventive or palliative therapies for this injury are in demand. Tissue engineering offers a promising toolset to tackle ischemia-reperfusion injuries. It devises tissue-mimetics by using the following: (1) the unique therapeutic features of stem cells, i.e., self-renewal, differentiability, anti-inflammatory, and immunosuppressants effects; (2) growth factors to drive cell growth, and development; (3) functional biomaterials, to provide defined microarchitecture for cell-cell interactions; (4) bioprocess design tools to emulate the macroscopic environment that interacts with tissues. This strategy allows the production of cell therapeutics capable of addressing ischemia-reperfusion injury (IRI). In addition, it allows the development of physiological-tissue-mimetics to study this condition or to assess the effect of drugs. Thus, it provides a sound platform for a better understanding of the reperfusion condition. This review article presents a synopsis and discusses tissue engineering applications available to treat various types of ischemia-reperfusions, ultimately aiming to highlight possible therapies and to bring closer the gap between preclinical and clinical settings

Costs and benefits of automation for astronomical facilities

The Observatorio Astrof\'isico de Javalambre (OAJ{\dag}1) in Spain is a young astronomical facility, conceived and developed from the beginning as a fully automated observatory with the main goal of optimizing the processes in the scientific and general operation of the Observatory. The OAJ has been particularly conceived for carrying out large sky surveys with two unprecedented telescopes of unusually large fields of view (FoV): the JST/T250, a 2.55m telescope of 3deg field of view, and the JAST/T80, an 83cm telescope of 2deg field of view. The most immediate objective of the two telescopes for the next years is carrying out two unique photometric surveys of several thousands square degrees, J-PAS{\dag}2 and J-PLUS{\dag}3, each of them with a wide range of scientific applications, like e.g. large structure cosmology and Dark Energy, galaxy evolution, supernovae, Milky Way structure, exoplanets, among many others. To do that, JST and JAST are equipped with panoramic cameras under development within the J-PAS collaboration, JPCam and T80Cam respectively, which make use of large format (~ 10k x 10k) CCDs covering the entire focal plane. This paper describes in detail, from operations point of view, a comparison between the detailed cost of the global automation of the Observatory and the standard automation cost for astronomical facilities, in reference to the total investment and highlighting all benefits obtained from this approach and difficulties encountered. The paper also describes the engineering development of the overall facilities and infrastructures for the fully automated observatory and a global overview of current status, pinpointing lessons learned in order to boost observatory operations performance, achieving scientific targets, maintaining quality requirements, but also minimizing operation cost and human resources.Comment: Global Observatory Control System GOC

Improving Embryonic Stem Cell Expansion through the Combination of Perfusion and Bioprocess Model Design

<div><p>Background</p><p>High proliferative and differentiation capacity renders embryonic stem cells (ESCs) a promising cell source for tissue engineering and cell-based therapies. Harnessing their potential, however, requires well-designed, efficient and reproducible expansion and differentiation protocols as well as avoiding hazardous by-products, such as teratoma formation. Traditional, standard culture methodologies are fragmented and limited in their fed-batch feeding strategies that afford a sub-optimal environment for cellular metabolism. Herein, we investigate the impact of metabolic stress as a result of inefficient feeding utilizing a novel perfusion bioreactor and a mathematical model to achieve bioprocess improvement.</p><p>Methodology/Principal Findings</p><p>To characterize nutritional requirements, the expansion of undifferentiated murine ESCs (mESCs) encapsulated in hydrogels was performed in batch and perfusion cultures using bioreactors. Despite sufficient nutrient and growth factor provision, the accumulation of inhibitory metabolites resulted in the unscheduled differentiation of mESCs and a decline in their cell numbers in the batch cultures. In contrast, perfusion cultures maintained metabolite concentration below toxic levels, resulting in the robust expansion (>16-fold) of high quality ‘naïve’ mESCs within 4 days. A multi-scale mathematical model describing population segregated growth kinetics, metabolism and the expression of selected pluripotency (‘stemness’) genes was implemented to maximize information from available experimental data. A global sensitivity analysis (GSA) was employed that identified significant (6/29) model parameters and enabled model validation. Predicting the preferential propagation of undifferentiated ESCs in perfusion culture conditions demonstrates synchrony between theory and experiment.</p><p>Conclusions/Significance</p><p>The limitations of batch culture highlight the importance of cellular metabolism in maintaining pluripotency, which necessitates the design of suitable ESC bioprocesses. We propose a novel investigational framework that integrates a novel perfusion culture platform (controlled metabolic conditions) with mathematical modeling (information maximization) to enhance ESC bioprocess productivity and facilitate bioprocess optimization.</p></div

Gene expression differences between batch and perfusion cultures.

<p>The accumulation of metabolic stress within batch cultures (a) leads to the down-regulation of the expression levels of <i>Rex1</i> and <i>Dppa3</i> accompanied by the up-regulation of the <i>Fgf5</i>. Perfusion feeding (b) removes the metabolic stress resulting in the up-regulation of <i>Rex1</i> and <i>Dppa3</i> and the down-regulation of the differentiation marker <i>Fgf5</i>. Results were normalised with fresh (day 0) mESCs.</p

Batch culture growth kinetics, viability and metabolism.

<p>a) Total number of viable ESCs; experimental (◊) and model predictions (–); b) Simulation results of the different ‘naïve’ (<i>X_U</i>) and ‘primed’ (<i>X_D</i>) mESC sub-populations; c) Micrographs of the mESCs encapsulated in the alginate hydrogels at day 9 (i, ii); Live (green)/dead (red) fluorescence micrographs showing live cells (iii) forming colonies of <200 µm in diameter as well as a high proportion of dead cells (iv) at day 9; d) experimental (symbols) and simulation (lines) glucose and lactate concentration profiles; e) experimental (symbols) and simulation (lines) glutamine and ammonia concentration profiles. Experimental values represent mean±SD, N = 3.</p

Pluripotency-related gene expression in batch cultures.

<p>Gene expression levels of a) <i>Rex1</i>, b) <i>Oct3/4</i>, c) <i>Fgf5</i>, d) <i>Sox2</i>, e) <i>Dppa3</i> and f) <i>Nanog</i>. Model simulation results are predicted for <i>Rex1</i>, <i>Fgf5</i> and <i>Dppa3</i> (line). *<i>P</i><0.05, one-way ANOVA. Experimental values represent mean±SD, N = 3.</p

Schematic of the experimental design.

<p>mESCs were encapsulated in alginate hydrogels, as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081728#pone.0081728-Hwang1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081728#pone.0081728-Randle1" target="_blank">[22]</a>, and cultured within a batch operated HARV bioreactor and a custom-built perfusion bioreactor.</p

Subset of significant parameter (identified by GSA) values re-estimated from perfusion culture.

<p>Subset of significant parameter (identified by GSA) values re-estimated from perfusion culture.</p

Pluripotency-related gene expression in perfusion cultures.

<p>Gene expression levels of a) <i>Rex1</i>, b) <i>Oct3/4</i>, c) <i>Fgf5</i>, d) <i>Sox2</i>, e) <i>Dppa3</i> and f) <i>Nanog</i>. Model simulation results are predicted for <i>Rex1</i>, <i>Fgf5</i> and <i>Dppa3</i> (line). *<i>P</i><0.05, one-way ANOVA. Experimental values represent mean±SD, N = 3.</p

LIF concentration and associated gene expression in batch cultures.

<p>a) LIF growth concentration levels over the 6 day culture period remain significantly higher than the half maximal and differentiation threshold levels. b) Gene expression levels of <i>LIF-Stat3</i> signalling (<i>Socs3</i>, <i>Stat3</i>, <i>Klf4</i>) and <i>BMP-ID</i> signalling (<i>Sox1</i>, <i>Id1</i> and <i>Id3</i>) for control (day 0) and day 6 batch culture values. *<i>P</i><0.05, Student t-test. Experimental values represent mean±SD, N = 3.</p