Metabolism not only produces energy necessary for the cell but is also a key regulator of several cellular functions, including pluripotency and self-renewal. Nucleotide sugars (NSs) are activated... Show moreMetabolism not only produces energy necessary for the cell but is also a key regulator of several cellular functions, including pluripotency and self-renewal. Nucleotide sugars (NSs) are activated sugars that link glucose metabolism with cellular functions via protein N-glycosylation and O-GlcNAcylation. Thus, understanding how different metabolic pathways converge in the synthesis of NSs is critical to explore new opportunities for metabolic interference and modulation of stem cell functions. Tracer-based metabolomics is suited for this challenge, however chemically-defined, customizable media for stem cell culture in which nutrients can be replaced with isotopically labeled analogs are scarcely available. Here, we established a customizable flux-conditioned E8 (FC-E8) medium that enables stem cell culture with stable isotopes for metabolic tracing, and a dedicated liquid chromatography mass-spectrometry (LC-MS/MS) method targeting metabolic pathways converging in NS biosynthesis. By C-13(6)-glucose feeding, we successfully traced the time-course of carbon incorporation into NSs directly via glucose, and indirectly via other pathways, such as glycolysis and pentose phosphate pathways, in induced pluripotent stem cells (hiPSCs) and embryonic stem cells. Then, we applied these tools to investigate the NS biosynthesis in hiPSC lines from a patient affected by deficiency of phosphoglucomutase 1 (PGM1), an enzyme regulating the synthesis of the two most abundant NSs, UDP-glucose and UDP-galactose. Show less
The intrinsic capacity of human hematopoietic stem cells (hHSCs) to reconstitute myeloid and lymphoid lineages combined with their self-renewal capacity hold enormous promises for gene therapy as a... Show moreThe intrinsic capacity of human hematopoietic stem cells (hHSCs) to reconstitute myeloid and lymphoid lineages combined with their self-renewal capacity hold enormous promises for gene therapy as a viable treatment option for a number of immune-mediated diseases, most prominently for inborn errors of immunity (IEI). The current development of such therapies relies on disease models, both in vitro and in vivo, which allow the study of human pathophysiology in great detail. Here, we discuss the current challenges with regards to developmental origin, heterogeneity and the subsequent implications for disease modeling. We review models based on induced pluripotent stem cell technology and those relaying on use of adult hHSCs. We critically review the advantages and limitations of current models for IEI both in vitro and in vivo. We conclude that existing and future stem cell-based models are necessary tools for developing next generation therapies for IEI. Show less
Pulmonary arterial hypertension (PAH) patients eventually die of right heart failure (RHF). Currently, there is no suitable pre-clinical model to study PAH. Therefore, we aim to develop a right... Show morePulmonary arterial hypertension (PAH) patients eventually die of right heart failure (RHF). Currently, there is no suitable pre-clinical model to study PAH. Therefore, we aim to develop a right heart dysfunction (RHD) model using the 3-dimensional engineered heart tissue (EHT) approach and cardiomyocytes derived from patient-induced pluripotent stem cells (iPSCs) to unravel the mechanisms that determine the fate of a pressure-overloaded right ventricle. iPSCs from PAH and healthy control subjects were differentiated into cardiomyocytes (iPSC-CMs), incorporated into the EHT, and maintained for 28 days. In comparison with control iPSC-CMs, PAH-derived iPSC-CMs exhibited decreased beating frequency and increased contraction and relaxation times. iPSC-CM alignment within the EHT was observed. PAH-derived EHTs exhibited higher force, and contraction and relaxation times compared with control EHTs. Increased afterload was induced using 2x stiffer posts from day 0. Due to high variability, there were no functional differences between normal and stiffer EHTs, and no differences in the hypertrophic gene expression. In conclusion, under baseline spontaneous conditions, PAH-derived iPSC-CMs and EHTs show prolonged contraction compared with controls, as observed clinically in PAH patients. Further optimization of the hypertrophic model and profound characterization may provide a platform for disease modelling and drug screening. Show less
Simple Summary:& nbsp;Despite improvements in the treatment of several cancer types, the extremely poor prognosis of pancreatic cancer patients has remained unchanged over the last decades.... Show moreSimple Summary:& nbsp;Despite improvements in the treatment of several cancer types, the extremely poor prognosis of pancreatic cancer patients has remained unchanged over the last decades. Therefore, new therapeutic regimens for pancreatic cancer are highly needed. In this review, we will discuss the potential of induced pluripotent stem cells (iPSCs) to generate representative pancreatic cancer models that can aid the development of novel diagnostics and therapeutic strategies. Furthermore, the potential of iPSCs as pancreatic cancer vaccines or as a basis for cellular therapies will be discussed. With promising preclinical results and ongoing clinical trials, the potential of iPSCs to further the treatment of pancreatic cancer is being explored and, in turn, will hopefully provide additional therapies to increase the poor survival rates of this patient population.Advances in the treatment of pancreatic ductal adenocarcinoma (PDAC) using neoadjuvant chemoradiotherapy, chemotherapy, and immunotherapy have had minimal impact on the overall survival of patients. A general lack of immunogenic features and a complex tumor microenvironment (TME) are likely culprits for therapy refractoriness in PDAC. Induced pluripotent stem cells (iPSCs) should be explored as a means to advance the treatment options for PDAC, by providing representative in vitro models of pancreatic cancer development. In addition, iPSCs could be used for tailor-made cellular immunotherapies or as a source of tumor-associated antigens in the context of vaccination. Show less
Pericytes (PCs) have been reported to contribute to the mechanoregulation of the capillary diameter and blood flow in health and disease. How this is realized remains poorly understood. We designed... Show morePericytes (PCs) have been reported to contribute to the mechanoregulation of the capillary diameter and blood flow in health and disease. How this is realized remains poorly understood. We designed several models representing basement membrane (BM) in between PCs and endothelial cells (ECs). These models captured a unique protein organization with micron-sized FN patches surrounded by laminin (LM) and allowed to obtain quantitative information on PC morphology and contractility. Using human induced pluripotent stem cell-derived PCs, we could address mechanical aspects of mid-capillary PC behavior in vitro. Our results showed that PCs strongly prefer FN patches over LM for adhesion formation, have an optimal stiffness for spreading in the range of EC rigidity, and react in a non-canonical way with increased traction forces and reduced spreading on other stiffness then the optimal. Our approach opens possibilities to further study PC force regulation under well-controlled conditions. Show less
Gene editing permits changing specific DNA sequences within the vast genomes of human cells. Stem cells are particularly attractive targets for gene editing interventions as their self-renewal and... Show moreGene editing permits changing specific DNA sequences within the vast genomes of human cells. Stem cells are particularly attractive targets for gene editing interventions as their self-renewal and differentiation capabilities consent studying cellular differentiation processes, screening small-molecule drugs, modeling human disorders, and testing regenerative medicines. To integrate gene editing and stem cell technologies, there is a critical need for achieving efficient delivery of the necessary molecular tools in the form of programmable DNA-targeting enzymes and/or exogenous nucleic acid templates. Moreover, the impact that the delivery agents themselves have on the performance and precision of gene editing procedures is yet another critical parameter to consider. Viral vectors consisting of recombinant replication-defective viruses are under intense investigation for bringing about efficient gene-editing tool delivery and precise gene-editing in human cells. In this review, we focus on the growing role that adenoviral vectors are playing in the targeted genetic manipulation of human stem cells, progenitor cells, and their differentiated progenies in the context of in vitro and ex vivo protocols. As preamble, we provide an overview on the main gene editing principles and adenoviral vector platforms and end by discussing the possibilities ahead resulting from leveraging adenoviral vector, gene editing, and stem cell technologies. Show less
