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Ashwini Ketkar-Atre,1 Tom Struys,1,2 Stefaan J Soenen,3 Ivo Lambrichts,2 Catherine M Verfaillie,4 Marcel De Cuyper,5 Uwe Himmelreich1 1Biomedical MRI/MoSAIC, Department of Imaging and Pathology, Biomedical Sciences Group, Katholieke Universiteit Leuven, Leuven, Belgium; 2Lab of Histology, Biomedical Research Institute, Hasselt University, Campus Diepenbeek, Agoralaan, Diepenbeek, Belgium; 3Lab for General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Ghent, Belgium; 4Interdepartmental Stem Cell Institute, O&N IV, Katholieke Universiteit Leuven, Leuven, Belgium; 5Laboratory of BioNanoColloids, Interdisciplinary Research Centre, Katholieke Universiteit Leuven, Kortrijk, Belgium Abstract: The need to track and evaluate the fate of transplanted cells is an important issue in regenerative medicine. In order to accomplish this, pre-labelling cells with magnetic resonance imaging (MRI) contrast agents is a well-established method. Uptake of MRI contrast agents by non-phagocytic stem cells, and factors such as cell homeostasis or the adverse effects of contrast agents on cell biology have been extensively studied, but in the context of nanoparticle (NP)-specific parameters. Here, we have studied three different types of NPs (Endorem®, magnetoliposomes [MLs], and citrate coated C-200) to label relatively larger, mesenchymal stem cells (MSCs) and, much smaller yet faster proliferating, multipotent adult progenitor cells (MAPCs). Both cell types are similar, as they are isolated from bone marrow and have substantial regenerative potential, which make them interesting candidates for comparative experiments. Using NPs with different surface coatings and sizes, we found that differences in the proliferative and morphological characteristics of the cells used in the study are mainly responsible for the fate of endocytosed iron, intracellular iron concentration, and cytotoxic responses. The quantitative analysis, using high-resolution electron microscopy images, demonstrated a strong relationship between cell volume/surface, uptake, and cytotoxicity. Interestingly, uptake and toxicity trends are reversed if intracellular concentrations, and not amounts, are considered. This indicates that more attention should be paid to cellular parameters such as cell size and proliferation rate in comparative cell-labeling studies. Keywords: cell labeling, MR contrast agents, transmission electron microscopy, mesenchymal stem cells, multipotent adult progenitor cells, magnetic resonance imaging, nanoparticles, iron oxide

Increased dosage of methyl-CpG-binding protein-2 (MeCP2) results in a dramatic neurodevelopmental phenotype with onset at birth. We generated induced pluripotent stem cells (iPSCs) from patients with the MECP2 duplication syndrome (MECP2dup), carrying different duplication sizes, to study the impact of increased MeCP2 dosage in human neurons. We show that cortical neurons derived from these different MECP2dup iPSC lines have increased synaptogenesis and dendritic complexity. In addition, using multi-electrodes arrays, we show that neuronal network synchronization was altered in MECP2dup-derived neurons. Given MeCP2 functions at the epigenetic level, we tested whether these alterations were reversible using a library of compounds with defined activity on epigenetic pathways. One histone deacetylase inhibitor, NCH-51, was validated as a potential clinical candidate. Interestingly, this compound has never been considered before as a therapeutic alternative for neurological disorders. Our model recapitulates early stages of the human MECP2 duplication syndrome and represents a promising cellular tool to facilitate therapeutic drug screening for severe neurodevelopmental disorders.

Objective: It is believed that an organism remains normoglycemic despite an increase in the beta-cell mass because of decreased insulin production by beta-cells on a per-cell basis. However, some transgenic mouse models with beta-cell hyperplasia suggest that insulin production remains excessive and that normoglycemia is maintained by insulin resistance.Methods: Here, we investigated the effect of an increased beta-cell mass on glycemia and insulin resistance by grafting excess normal islets in normoglycemic mice, as well as using targeted PLAG1 expression in beta-cells, which leads to beta-cell expansion.Results: In both models, fasting plasma insulin levels were increased, even though animals were normoglycemic. After an intraperitoneal glucose tolerance test, plasma insulin levels increased, which was associated with improved glucose clearing. Under these conditions, normoglycemia is maintained by hepatic insulin resistance as demonstrated by hyperinsulinemic euglycemic clamp experiments.Conclusions: In conclusion, we demonstrate that when excess beta-cells are grafted, insulin production on a per beta-cell basis is not sufficiently decreased, leading to hyperinsulinemia and hepatic insulin resistance. This observation might be important for the design of stem cell-based islet replacement therapies. [ABSTRACT FROM AUTHOR]

Provides information on a way to determine hematopoietic stem cell content in blood or marrow grafts. Method used to assess progenitors and hematopoietic stem cells; Overview of phenotypic characterization of primitive cells; Assessment of the absolute frequency of primitive progenitors in a given cell population.

The first definitive hematopoietic progenitors emerge through the process of endothelial-to-hematopoietic transition in vertebrate embryos. With molecular regulators for this process worked out, the role of metabolic pathways used remains unclear. Here, we performed nano-LC-MS/MS-based proteomic analysis and predicted a metabolic switch from a glycolytic to oxidative state upon hematopoietic transition. Mitochondrial activity, glucose uptake, and glycolytic flux analysis supported this hypothesis. Systemic inhibition of lactate dehydrogenase A (LDHA) increased oxygen consumption rate in the hemato-endothelial system and inhibited the emergence of intra-aortic hematopoietic clusters. These findings were corroborated using Tie2-Cre -mediated deletion of Ldha that showed similar effects on hematopoietic emergence. Conversely, stabilization of HIF-1α via inhibition of oxygen-sensing pathway led to decreased oxidative flux and promoted hematopoietic emergence in mid-gestation embryos. Thus, cell-intrinsic regulation of metabolic state overrides oxygenated microenvironment in the aorta to promote a glycolytic metabolic state that is crucial for hematopoietic emergence in mammalian embryos.

Terminally differentiated cells are commonly regarded as the most stable cell state in adult organisms, characterized by growth arrest while fulfilling their specialized functions. A better understanding of the mechanisms involved in promoting cell cycle exit will improve the ability to differentiate pluripotent cells into mature tissues for both pharmacological and therapeutic use. Here, it demonstrates that a hyperosmolar environment enforces a protective p53-independent quiescent state in immature hepatoma cells and in pluripotent stem cell-derived models of human hepatocytes and endothelial cells. Prolonged culture in hyperosmolar conditions stimulates changes in gene expression promoting functional cell maturation. Interestingly, hyperosmolar conditions do not only trigger growth arrest and cellular maturation but are also necessary to maintain this maturated state, as switching back to plasma osmolarity reverses the changes in expression of maturation and proliferative markers. Transcriptome analysis revealed sequential stages of osmolarity-regulated growth arrest followed by cell maturation, mediated by activation of NF-κВ, and repression of WNT signaling, respectively. This study reveals that a modulated increase in osmolarity serves as a biochemical signal to promote long-term growth arrest and cellular maturation into different lineages, providing a practical method to generate differentiated hiPSCs that resemble their mature counterpart more closely., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Pathological abnormalities in the tau protein give rise to a variety of neurodegenerative diseases, conjointly termed tauopathies. Several tau mutations have been identified in the tau-encoding gene MAPT , affecting either the physical properties of tau or resulting in altered tau splicing. At early disease stages, mitochondrial dysfunction was highlighted with mutant tau compromising almost every aspect of mitochondrial function. Additionally, mitochondria have emerged as fundamental regulators of stem cell function. Here, we show that compared to the isogenic wild-type triple MAPT -mutant human-induced pluripotent stem cells, bearing the pathogenic N279K, P301L, and E10+16 mutations, exhibit deficits in mitochondrial bioenergetics and present altered parameters linked to the metabolic regulation of mitochondria. Moreover, we demonstrate that the triple tau mutations disturb the cellular redox homeostasis and modify the mitochondrial network morphology and distribution. This study provides the first characterization of disease-associated tau-mediated mitochondrial impairments in an advanced human cellular tau pathology model at early disease stages, ranging from mitochondrial bioenergetics to dynamics. Consequently, comprehending better the influence of dysfunctional mitochondria on the development and differentiation of stem cells and their contribution to disease progression may thus assist in the potential prevention and treatment of tau-related neurodegeneration.

Liver transplantation is currently the only curative therapy for patients with acute or chronic liver failure. However, a dramatic gap between the number of available liver grafts and the number of patients on the transplantation waiting list emphasizes the need for valid liver substitutes. Whole-organ engineering is an emerging field of tissue engineering and regenerative medicine. It aims to generate transplantable and functional organs to support patients on transplantation waiting lists until a graft becomes available. It comprises two base technologies developed in the last decade; (1) organ decellularization to generate a three-dimensional (3D) extracellular matrix scaffold of an organ, and (2) scaffold recellularization to repopulate both the parenchymal and vascular compartments of a decellularized organ. In this review article, recent advancements in both technologies, in relation to liver whole-organ engineering, are presented. We address the potential sources of hepatocytes and non-parenchymal liver cells for repopulation studies, and the role of stem-cell-derived liver progeny is discussed. In addition, different cell seeding strategies, possible graft modifications, and methods used to evaluate the functionality of recellularized liver grafts are outlined. Based on the knowledge gathered from recent transplantation studies, future directions are summarized.

Interaction with microenvironmental factors is crucial for the regulation of hematopoietic stem cell (HSC) function. Stroma derived factor (SDF)-1α supports HSCs in the quiescent state and is central to the homing of transplanted HSCs. Here, we show that integrin signaling regulates Sdf-1α expression transcriptionally. Systemic deletion of Periostin, an Integrin-αv ligand, showed increased expression of Sdf-1α in bone marrow (BM) niche. Pharmacological inhibition or CRISPR-Cas9-mediated deletion of SRC, resulted in a similar increase in the chemokine expression in vitro . Importantly, systemic SRC-inhibition led to increase in SDF-1α levels in BM plasma. This resulted in a robust increase (14.05 ± 1.22% to 29.11 ± 0.69%) in the homing efficiency of transplanted HSCs. In addition, we observed enhancement in the recovery of blood cell counts following radiation injury, indicating an enhanced hematopoietic function. These results establish a role of SRC-mediated integrin signaling in the transcriptional regulation of Sdf-1α . This mechanism could be harnessed further to improve the hematopoietic function., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

Duchenne muscular dystrophy (DMD) is a progressive muscle disorder caused by mutations in the Dystrophin gene. Cardiomyopathy is a major cause of early death. We used DMD-patient-specific human induced pluripotent stem cells (hiPSCs) to model cardiomyopathic features and unravel novel pathologic insights. Cardiomyocytes (CMs) differentiated from DMD hiPSCs showed enhanced premature cell death due to significantly elevated intracellular reactive oxygen species (ROS) resulting from depolarized mitochondria and increased NADPH oxidase 4 (NOX4). CRISPR-Cas9 correction of Dystrophin restored normal ROS levels. ROS reduction by N-acetyl-L-cysteine (NAC), ataluren (PTC124), and idebenone improved hiPSC-CM survival. We show that oxidative stress in DMD hiPSC-CMs was counteracted by stimulating adenosine triphosphate (ATP) production. ATP can bind to NOX4 and partially inhibit the ROS production. Considering the complexity and the early cellular stress responses in DMD cardiomyopathy, we propose targeting ROS production and preventing detrimental effects of NOX4 on DMD CMs as promising therapeutic strategy., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The pharmaceutical industry is in high need of efficient and relevant in vitro liver models, which can be incorporated in their drug discovery pipelines to identify potential drugs and their toxicity profiles. Current liver models often rely on cancer cell lines or primary cells, which both have major limitations. However, the development of human induced pluripotent stem cells (hiPSCs) has created a new opportunity for liver disease modeling, drug discovery and liver toxicity research. hiPSCs can be differentiated to any cell of interest, which makes them good candidates for disease modeling and drug discovery. Moreover, hiPSCs, unlike primary cells, can be easily genome-edited, allowing the creation of reporter lines or isogenic controls for patient-derived hiPSCs. Unfortunately, even though liver progeny from hiPSCs has characteristics similar to their in vivo counterparts, the differentiation of iPSCs to fully mature progeny remains highly challenging and is a major obstacle for the full exploitation of these models by pharmaceutical industries. In this review, we discuss current liver-cell differentiation protocols and in vitro iPSC-based liver models that could be used for disease modeling and drug discovery. Furthermore, we will discuss the challenges that still need to be overcome to allow for the successful implementation of these models into pharmaceutical drug discovery platforms.

Traditional toxicity risk assessment approaches have until recently focussed mainly on histochemical readouts for cell death. Modern toxicology methods attempt to deduce a mechanistic understanding of pathways involved in the development of toxicity, by using transcriptomics and other big data-driven methods such as high-content screening. Here, we used a recently described optimised method to differentiate human induced pluripotent stem cells (hiPSCs) to hepatocyte-like cells (HLCs), to assess their potential to classify hepatotoxic and non-hepatotoxic chemicals and their use in mechanistic toxicity studies. The iPSC-HLCs could accurately classify chemicals causing acute hepatocellular injury, and the transcriptomics data on treated HLCs obtained by TempO-Seq technology linked the cytotoxicity to cellular stress pathways, including oxidative stress and unfolded protein response (UPR). Induction of these stress pathways in response to amiodarone, diclofenac, and ibuprofen, was demonstrated to be concentration and time dependent. The transcriptomics data on diclofenac-treated HLCs were found to be more sensitive in detecting differentially expressed genes in response to treatment, as compared to existing datasets of other diclofenac-treated in vitro hepatocyte models. Hence iPSC-HLCs generated by transcription factor overexpression and in metabolically optimised medium appear suitable for chemical toxicity detection as well as mechanistic toxicity studies.

Chronic liver injury, as observed in non-alcoholic steatohepatitis (NASH), progressive fibrosis, and cirrhosis, remains poorly treatable. Steatohepatitis causes hepatocyte loss in part by a direct lipotoxic insult, which is amplified by derangements in the non-parenchymal cellular (NPC) interactive network wherein hepatocytes reside, including, hepatic stellate cells, liver sinusoidal endothelial cells and liver macrophages. To create an in vitro culture model encompassing all these cells, that allows studying liver steatosis, inflammation and fibrosis caused by NASH, we here developed a fully defined hydrogel microenvironment, termed hepatocyte maturation (HepMat) gel, that supports maturation and maintenance of pluripotent stem cell (PSC) derived hepatocyte- and NPC-like cells for at least one month. The HepMat-based co-culture system modeled key molecular and functional features of TGFβ-induced liver fibrosis and fatty-acid induced inflammation and fibrosis better than monocultures of its constituent cell populations. The novel co-culture system should open new avenues for studying mechanisms underlying liver steatosis, inflammation and fibrosis as well as for assessing drugs counteracting these effects., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Hematopoietic stem and progenitor cell (HSPC) engraftment after transplantation during anticancer treatment depends on support from the recipient bone marrow (BM) microenvironment. Here, by studying physiological homing of fetal HSPCs, we show the critical requirement of balanced local crosstalk within the skeletal niche for successful HSPC settlement in BM. Transgene-induced overproduction of vascular endothelial growth factor (VEGF) by osteoprogenitor cells elicits stromal and endothelial hyperactivation, profoundly impacting the stromal-vessel interface and vascular architecture. Concomitantly, HSPC homing and survival are drastically impaired. Transcriptome profiling, flow cytometry, and high-resolution imaging indicate alterations in perivascular and endothelial cell characteristics, vascular function and cellular metabolism, associated with increased oxidative stress within the VEGF-enriched BM environment. Thus, developmental HSPC homing to bone is controlled by local stromal-vascular integrity and the oxidative-metabolic status of the recipient milieu. Interestingly, irradiation of adult mice also induces stromal VEGF expression and similar osteo-angiogenic niche changes, underscoring that our findings may contribute targets for improving stem cell therapies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Frontotemporal dementia (FTD) is a neurodegenerative disease, leading to behavioral changes and language difficulties. Heterozygous loss-of-function mutations in progranulin ( GRN ) induce haploinsufficiency of the protein and are associated with up to one-third of all genetic FTD cases worldwide. While the loss of GRN is primarily associated with neurodegeneration, the biological functions of the secreted growth factor-like protein are more diverse, ranging from wound healing, inflammation, vasculogenesis, and metabolic regulation to tumor cell growth and metastasis. To date, no disease-modifying treatments exist for FTD, but different therapeutic approaches to boost GRN levels in the central nervous system are currently being developed (including AAV-mediated GRN gene delivery as well as anti-SORT1 antibody therapy). In this review, we provide an overview of the multifaceted regulation of GRN levels and the corresponding therapeutic avenues. We discuss the opportunities, advantages, and potential drawbacks of the diverse approaches. Additionally, we highlight the therapeutic potential of elevating GRN levels beyond patients with loss-of-function mutations in GRN ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Terryn, Verfaillie and Van Damme.)

To date, there is no representative in vitro model for liver sinusoidal endothelial cells (LSECs), as primary LSECs dedifferentiate very fast in culture and no combination of cytokines or growth factors can induce an LSEC fate in (pluripotent stem cell (PSC)-derived) endothelial cells (ECs). Furthermore, the transcriptional programmes driving an LSEC fate have not yet been described. Here, we first present a computational workflow (CenTFinder) that can identify transcription factors (TFs) that are crucial for modulating pathways involved in cell lineage specification. Using CenTFinder, we identified several novel LSEC-specific protein markers, such as FCN2 and FCN3, which were validated by analysis of previously published single-cell RNAseq data. We also identified PU.1 (encoded by the SPI1 gene) as a major regulator of LSEC-specific immune functions. We show that SPI1 overexpression (combined with the general EC TF ETV2) in human PSCs induces ECs with an LSEC-like phenotype. The ETV2-SPI1-ECs display increased expression of LSEC markers, such as CD32B and MRC1, as well as several of the proposed novel markers. More importantly, ETV2-SPI1-ECs acquire LSEC functions, including uptake of FSA-FITC, as well as labelled IgG. In conclusion, we present the CenTFinder computational tool to identify key regulatory TFs within specific pathways, in this work pathways of lineage specification, and we demonstrate its use by the identification and validation of PU.1 as a master regulator for LSEC fating.

The fact that monogenic diseases are related to mutations in one specific gene, make gene correction one of the promising strategies in the future to treat genetic diseases or alleviate their symptoms. From this perspective, and along with recent advances in technology, genome editing tools have gained momentum and developed fast. In fact, clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs) are regarded as novel technologies which are able to correct a number of genetic aberrations in vitro and in vivo. The number of ongoing clinical trials employing these tools has been increased showing the encouraging outcomes of these tools. However, there are still some major challenges with respect to the safety profile and directed delivery of them. In this paper, we provided updated information regarding the history, nature, methods of delivery, and application of the above-mentioned gene editing tools along with the meganucleases (an older similar tool) based on published in vitro and in vivo studies and introduced clinical trials which employed these technologies., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

We earlier showed that outside-in integrin signaling through POSTN-ITGAV interaction plays an important role in regulating adult hematopoietic stem cell (HSC) quiescence. Here, we show that Itgav deletion results in increased frequency of phenotypic HSCs in fetal liver (FL) due to faster proliferation. Systemic deletion of Postn led to increased proliferation of FL HSCs, albeit without any loss of stemness, unlike Vav-Itgav -/- HSCs. Based on RNA sequencing analysis of FL and bone marrow HSCs, we predicted the involvement of DNA damage response pathways in this dichotomy. Indeed, proliferative HSCs from Postn-deficient FL tissues showed increased levels of DNA repair, resulting in lesser double-strand breaks. Thus POSTN, with its expression majorly localized in the vascular endothelium of FL tissue, acts as a regulator of stem cell pool size during development. Overall, we demonstrate that the duality of response to proliferation in HSCs is developmental stage dependent and can be correlated with DNA damage responses., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Predicting drug-induced liver injury in a preclinical setting remains challenging, as cultured primary human hepatocytes (PHHs), pluripotent stem cell-derived hepatocyte-like cells (HLCs), and hepatoma cells exhibit poor drug biotransformation capacity. We here demonstrate that hepatic functionality depends more on cellular metabolism and extracellular nutrients than on developmental regulators. Specifically, we demonstrate that increasing extracellular amino acids beyond the nutritional need of HLCs and HepG2 cells induces glucose independence, mitochondrial function, and the acquisition of a transcriptional profile that is closer to PHHs. Moreover, we show that these high levels of amino acids are sufficient to drive HLC and HepG2 drug biotransformation and liver-toxin sensitivity to levels similar to those in PHHs. In conclusion, we provide data indicating that extracellular nutrient levels represent a major determinant of cellular maturity and can be utilized to guide stem cell differentiation to the hepatic lineage.