Neural Cell 3D Microtissue Formation Is Marked by Cytokines' Up-Regulation

Cells cultured in three dimensional (3D) scaffolds as opposed to traditional two-dimensional (2D) substrates have been considered more physiologically relevant based on their superior ability to emulate the in vivo environment. Combined with stem cell technology, 3D cell cultures can provide a promising alternative for use in cell-based assays or biosensors in non-clinical drug discovery studies. To advance 3D culture technology, a case has been made for identifying and validating three-dimensionality biomarkers. With this goal in mind, we conducted a transcriptomic expression comparison among neural progenitor cells cultured on 2D substrates, 3D porous polystyrene scaffolds, and as 3D neurospheres (in vivo surrogate). Up-regulation of cytokines as a group in 3D and neurospheres was observed. A group of 13 cytokines were commonly up-regulated in cells cultured in polystyrene scaffolds and neurospheres, suggesting potential for any or a combination from this list to serve as three-dimensionality biomarkers. These results are supportive of further cytokine identification and validation studies with cells from non-neural tissue

Extracellular Vesicles, Apoptotic Bodies and Mitochondria: Stem Cell Bioproducts for Organ Regeneration

Purpose of Review In the current work, we will present the characterization of the main different stem cell-derived vesicular bio-products with potential application in organ regeneration. Recent Findings The therapeutic effects of stem cell therapy in organ repair, specifically those utilizing mesenchymal stromal cells, are largely dependent on the cells' release of different bio-products. Among these bio-products, extracellular vesicles (EVs) appear to play a major role due to their ability to carry and deliver bioactive material for modulation of cellular pathways in recipient cells. Concurrently, mitochondria transfer emerged as a new mechanism of cell communication, in which the bioenergetics of a damaged cell are restored. Finally, apoptotic bodies released by dying apoptotic stem cells contribute to stimulation of the tissue's stem cells and modulation of the immune response. Summary Exploitation of isolated extracellular vesicles, mitochondria and apoptotic bodies in preclinical models of organ damage shows promising results. Here, we describe the results of the pre-clinical applications of stem cell vesicular products, as well as the first clinical trials approaching artificial administration of extracellular vesicles and mitochondria in human subjects and their possible benefits and limitations. This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Actions, grant agreement No 813839, Innovative Training Network RenalToolBox and 765274, Innovative Training Network iPlacenta

Predicting proximal tubule failed repair drivers through regularized regression analysis of single cell multiomic sequencing

Renal proximal tubule epithelial cells have considerable intrinsic repair capacity following injury. However, a fraction of injured proximal tubule cells fails to undergo normal repair and assumes a proinflammatory and profibrotic phenotype that may promote fibrosis and chronic kidney disease. The healthy to failed repair change is marked by cell state-specific transcriptomic and epigenomic changes. Single nucleus joint RNA- and ATAC-seq sequencing offers an opportunity to study the gene regulatory networks underpinning these changes in order to identify key regulatory drivers. We develop a regularized regression approach to construct genome-wide parametric gene regulatory networks using multiomic datasets. We generate a single nucleus multiomic dataset from seven adult human kidney samples and apply our method to study drivers of a failed injury response associated with kidney disease. We demonstrate that our approach is a highly effective tool for predicting key cis- and trans-regulatory elements underpinning the healthy to failed repair transition and use it to identify NFAT5 as a driver of the maladaptive proximal tubule state

Extracellular matrix-based hydrogels obtained from human tissues: a work still in progress

Purpose of reviewThe current review summarizes contemporary decellularization and hydrogel manufacturing strategies in the field of tissue engineering and regenerative medicine.Recent findingsDecellularized extracellular matrix (ECM) bioscaffolds are a valuable biomaterial that can be purposed into various forms of synthetic tissues such as hydrogels. ECM-based hydrogels can be of animal or human origin. The use of human tissues as a source for ECM hydrogels in the clinical setting is still in its infancy and current literature is scant and anecdotal, resulting in inconclusive results.SummaryThus far the methods used to obtain hydrogels from human tissues remains a work in progress. Gelation, the most complex technique in obtaining hydrogels, is challenging due to remarkable heterogeneity of the tissues secondary to interindividual variability. Age, sex, ethnicity, and preexisting conditions are factors that dramatically undermine the technical feasibility of the gelation process. This is contrasted with animals whose well defined anatomical and histological characteristics have been selectively bred for the goal of manufacturing hydrogels

Comprehensive characterization of the human pancreatic proteome for bioengineering applications

Interactions between the pancreatic extracellular matrix (ECM) and islet cells are known to regulate multiple aspects of islet physiology, including survival, proliferation, and glucose-stimulated insulin secretion. Recognizing the essential role of ECM in islet survival and function, various engineering approaches have been developed that aim to utilize ECM-based materials to recreate a native-like microenvironment. However, a major impediment to the success of these approaches has been the lack of a robust and comprehensive characterization of the human pancreatic proteome. Herein, by combining mass spectrometry (MS) and multiplex ELISA, we have provided an improved workflow for the in-depth profiling of the proteome, including minor constituents that are generally underrepresented. Moreover, we have further validated the effectiveness of our detergent-free decellularization protocol in the removal of cellular proteins and retention of the matrisome. It has also been established that the decellularized ECM and its derivatives can provide more tissue-specific cues than traditionally used biological scaffolds and are therefore more physiologically relevant for the development of hydrogels, bioinks and medium additives, in order to create a pancreatic niche. The data generated in this study would contribute significantly to the efforts of comprehensively defining the ECM atlas and also serve as a standard for the human pancreatic proteome to provide further guidance for design and engineering strategies for improved tissue engineering scaffolds. [Display omitted

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix (ECM) Production

Abstract Type 2 diabetic cardiomyopathy features Ca 2+ signaling abnormalities, notably an altered mitochondrial Ca 2+ handling. We here aimed to study if it might be due to a dysregulation of either the whole Ca 2+ homeostasis, the reticulum–mitochondrial Ca 2+ coupling, and/or the mitochondrial Ca 2+ entry through the uniporter. Following a 16-week high-fat high-sucrose diet (HFHSD), mice developed cardiac insulin resistance, fibrosis, hypertrophy, lipid accumulation, and diastolic dysfunction when compared to standard diet. Ultrastructural and proteomic analyses of cardiac reticulum–mitochondria interface revealed tighter interactions not compatible with Ca 2+ transport in HFHSD cardiomyocytes. Intramyocardial adenoviral injections of Ca 2+ sensors were performed to measure Ca 2+ fluxes in freshly isolated adult cardiomyocytes and to analyze the direct effects of in vivo type 2 diabetes on cardiomyocyte function. HFHSD resulted in a decreased IP3R–VDAC interaction and a reduced IP3-stimulated Ca 2+ transfer to mitochondria, with no changes in reticular Ca 2+ level, cytosolic Ca 2+ transients, and mitochondrial Ca 2+ uniporter function. Disruption of organelle Ca 2+ exchange was associated with decreased mitochondrial bioenergetics and reduced cell contraction, which was rescued by an adenovirus-mediated expression of a reticulum-mitochondria linker. An 8-week diet reversal was able to restore cardiac insulin signaling, Ca 2+ transfer, and cardiac function in HFHSD mice. Therefore, our study demonstrates that the reticulum–mitochondria Ca 2+ miscoupling may play an early and reversible role in the development of diabetic cardiomyopathy by disrupting primarily the mitochondrial bioenergetics. A diet reversal, by counteracting the MAM-induced mitochondrial Ca 2+ dysfunction, might contribute to restore normal cardiac function and prevent the exacerbation of diabetic cardiomyopathy

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix (ECM) Production

Abstract Type 2 diabetic cardiomyopathy features Ca 2+ signaling abnormalities, notably an altered mitochondrial Ca 2+ handling. We here aimed to study if it might be due to a dysregulation of either the whole Ca 2+ homeostasis, the reticulum–mitochondrial Ca 2+ coupling, and/or the mitochondrial Ca 2+ entry through the uniporter. Following a 16-week high-fat high-sucrose diet (HFHSD), mice developed cardiac insulin resistance, fibrosis, hypertrophy, lipid accumulation, and diastolic dysfunction when compared to standard diet. Ultrastructural and proteomic analyses of cardiac reticulum–mitochondria interface revealed tighter interactions not compatible with Ca 2+ transport in HFHSD cardiomyocytes. Intramyocardial adenoviral injections of Ca 2+ sensors were performed to measure Ca 2+ fluxes in freshly isolated adult cardiomyocytes and to analyze the direct effects of in vivo type 2 diabetes on cardiomyocyte function. HFHSD resulted in a decreased IP3R–VDAC interaction and a reduced IP3-stimulated Ca 2+ transfer to mitochondria, with no changes in reticular Ca 2+ level, cytosolic Ca 2+ transients, and mitochondrial Ca 2+ uniporter function. Disruption of organelle Ca 2+ exchange was associated with decreased mitochondrial bioenergetics and reduced cell contraction, which was rescued by an adenovirus-mediated expression of a reticulum-mitochondria linker. An 8-week diet reversal was able to restore cardiac insulin signaling, Ca 2+ transfer, and cardiac function in HFHSD mice. Therefore, our study demonstrates that the reticulum–mitochondria Ca 2+ miscoupling may play an early and reversible role in the development of diabetic cardiomyopathy by disrupting primarily the mitochondrial bioenergetics. A diet reversal, by counteracting the MAM-induced mitochondrial Ca 2+ dysfunction, might contribute to restore normal cardiac function and prevent the exacerbation of diabetic cardiomyopathy

Bioengineering of the digestive tract: approaching the clinic

The field of regenerative medicine is developing technologies that, in the near future, will offer alternative approaches to either cure diseases affecting the gastrointestinal tract or slow their progression by leveraging the intrinsic ability of our tissues and organs to repair after damage. This article will succinctly illustrate the three technologies that are closer to clinical translation?namely, human intestinal organoids, sphincter bioengineering and decellularization, whereby the cellular compartment of a given segment of the digestive tract is removed to obtain a scaffold consisting of the extracellular matrix. The latter will be used as a template for the regeneration of a functional organ, whereby the newly generated cellular compartment will be obtained from the patient?s own cells. Although clinical application of this technology is approaching, product development challenges are being tackled to warrant safety and efficacy. ? 2021 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved