Head-to-head comparison of relevant cell sources of small extracellular vesicles for cardiac repair: Superiority of embryonic stem cells.

Small extracellular vesicles (sEV) derived from various cell sources have been demonstrated to enhance cardiac function in preclinical models of myocardial infarction (MI). The aim of this study was to compare different sources of sEV for cardiac repair and determine the most effective one, which nowadays remains limited. We comprehensively assessed the efficacy of sEV obtained from human primary bone marrow mesenchymal stromal cells (BM-MSC), human immortalized MSC (hTERT-MSC), human embryonic stem cells (ESC), ESC-derived cardiac progenitor cells (CPC), human ESC-derived cardiomyocytes (CM), and human primary ventricular cardiac fibroblasts (VCF), in in vitro models of cardiac repair. ESC-derived sEV (ESC-sEV) exhibited the best pro-angiogenic and anti-fibrotic effects in vitro. Then, we evaluated the functionality of the sEV with the most promising performances in vitro, in a murine model of MI-reperfusion injury (IRI) and analysed their RNA and protein compositions. In vivo, ESC-sEV provided the most favourable outcome after MI by reducing adverse cardiac remodelling through down-regulating fibrosis and increasing angiogenesis. Furthermore, transcriptomic, and proteomic characterizations of sEV derived from hTERT-MSC, ESC, and CPC revealed factors in ESC-sEV that potentially drove the observed functions. In conclusion, ESC-sEV holds great promise as a cell-free treatment for promoting cardiac repair following MI.

Selective induction of human renal interstitial progenitor-like cell lineages from iPSCs reveals development of mesangial and EPO-producing cells.

Recent regenerative studies using human pluripotent stem cells (hPSCs) have developed multiple kidney-lineage cells and organoids. However, to further form functional segments of the kidney, interactions of epithelial and interstitial cells are required. Here we describe a selective differentiation of renal interstitial progenitor-like cells (IPLCs) from human induced pluripotent stem cells (hiPSCs) by modifying our previous induction method for nephron progenitor cells (NPCs) and analyzing mouse embryonic interstitial progenitor cell (IPC) development. Our IPLCs combined with hiPSC-derived NPCs and nephric duct cells form nephrogenic niche- and mesangium-like structures in vitro. Furthermore, we successfully induce hiPSC-derived IPLCs to differentiate into mesangial and erythropoietin-producing cell lineages in vitro by screening differentiation-inducing factors and confirm that p38 MAPK, hypoxia, and VEGF signaling pathways are involved in the differentiation of mesangial-lineage cells. These findings indicate that our IPC-lineage induction method contributes to kidney regeneration and developmental research.

A simeprevir-inducible molecular switch for the control of cell and gene therapies.

Chemical inducer of dimerization (CID) modules can be used effectively as molecular switches to control biological processes, and thus there is significant interest within the synthetic biology community in identifying novel CID systems. To date, CID modules have been used primarily in engineering cells for in vitro applications. To broaden their utility to the clinical setting, including the potential to control cell and gene therapies, the identification of novel CID modules should consider factors such as the safety and pharmacokinetic profile of the small molecule inducer, and the orthogonality and immunogenicity of the protein components. Here we describe a CID module based on the orally available, approved, small molecule simeprevir and its target, the NS3/4A protease from hepatitis C virus. We demonstrate the utility of this CID module as a molecular switch to control biological processes such as gene expression and apoptosis in vitro, and show that the CID system can be used to rapidly induce apoptosis in tumor cells in a xenograft mouse model, leading to complete tumor regression.

The critical role of apoptosis in mesenchymal stromal cell therapeutics and implications in homeostasis and normal tissue repair.

Mesenchymal stromal cells (MSCs) have been extensively tested for the treatment of numerous clinical conditions and have demonstrated good safety but mixed efficacy. Although this outcome can be attributed in part to the heterogeneity of cell preparations, the lack of mechanistic understanding and tools to establish cell pharmacokinetics and pharmacodynamics, as well as the poorly defined criteria for patient stratification, have hampered the design of informative clinical trials. We and others have demonstrated that MSCs can rapidly undergo apoptosis after their infusion. Apoptotic MSCs are phagocytosed by monocytes/macrophages that are then reprogrammed to become anti-inflammatory cells. MSC apoptosis occurs when the cells are injected into patients who harbor activated cytotoxic T or NK cells. Therefore, the activation state of cytotoxic T or NK cells can be used as a biomarker to predict clinical responses to MSC treatment. Building on a large body of preexisting data, an alternative view on the mechanism of MSCs is that an inflammation-dependent MSC secretome is largely responsible for their immunomodulatory activity. We will discuss how these different mechanisms can coexist and are instructed by two different types of MSC "licensing": one that is cell-contact dependent and the second that is mediated by inflammatory cytokines. The varied and complex mechanisms by which MSCs can orchestrate inflammatory responses and how this function is specifically driven by inflammation support a physiological role for tissue stroma in tissue homeostasis, and it acts as a sensor of damage and initiator of tissue repair by reprogramming the inflammatory environment.

Diabetic Cardiomyopathy Modelling Using Induced Pluripotent Stem Cell Derived Cardiomyocytes: Recent Advances and Emerging Models.

The global burden of diabetes has drastically increased over the past decades and in 2017 approximately 4 million deaths were caused by diabetes and cardiovascular complications. Diabetic cardiomyopathy is a common complication of diabetes with early manifestations of diastolic dysfunction and left ventricular hypertrophy with subsequent progression to systolic dysfunction and ultimately heart failure. An in vitro model accurately recapitulating key processes of diabetic cardiomyopathy would provide a useful tool for investigations of underlying disease mechanisms to further our understanding of the disease and thereby potentially advance treatment strategies for patients. With their proliferative capacity and differentiation potential, human induced pluripotent stem cells (iPSCs) represent an appealing cell source for such a model system and cardiomyocytes derived from induced pluripotent stem cells have been used to establish other cardiovascular related disease models. Here we review recently made advances and discuss challenges still to be overcome with regard to diabetic cardiomyopathy models, with a special focus on iPSC-based systems. Recent publications as well as preliminary data presented here demonstrate the feasibility of generating cardiomyocytes with a diabetic phenotype, displaying insulin resistance, impaired calcium handling and hypertrophy. However, capturing the full metabolic- and functional phenotype of the diabetic cardiomyocyte remains to be accomplished.

Humanizing Miniature Hearts through 4-Flow Cannulation Perfusion Decellularization and Recellularization.

Despite improvements in pre-clinical drug testing models, predictability of clinical outcomes continues to be inadequate and costly due to poor evidence of drug metabolism. Humanized miniature organs integrating decellularized rodent organs with tissue specific cells are translational models that can provide further physiological understanding and evidence. Here, we evaluated 4-Flow cannulated rat hearts as the fundamental humanized organ model for cardiovascular drug validation. Results show clearance of cellular components in all chambers in 4-Flow hearts with efficient perfusion into both coronary arteries and cardiac veins. Furthermore, material characterization depicts preserved organization and content of important matrix proteins such as collagens, laminin, and elastin. With access to the complete vascular network, different human cell types were delivered to show spatial distribution and integration into the matrix under perfusion for up to three weeks. The feature of 4-Flow cannulation is the preservation of whole heart conformity enabling ventricular pacing via the pulmonary vein as demonstrated by noninvasive monitoring with fluid pressure and ultrasound imaging. Consequently, 4-Flow hearts surmounting organ mimicry challenges with intact complexity in vasculature and mechanical compliance of the whole organ providing an ideal platform for improving pre-clinical drug validation in addition to understanding cardiovascular diseases.

The effects of PPAR-γ inhibition on gene expression and the progression of induced osteogenic differentiation of human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) can differentiate into several cell types, such as osteoblasts and adipocytes, both in vitro and in vivo. Although these two differentiation pathways are distinct from each other, cross-communication between cells of the two lineages exists both systemically and peripherally in the tissue. The transcription factor PPAR-γ, the main switch in adipogenic differentiation of MSCs, has previously been described to have a negative effect on osteogenic differentiation. The aim of this study was to investigate the effect of PPAR-γ inhibition on osteogenic differentiation of human MSCs, in vitro. Extracellular matrix analysis and quantification of osteogenic markers, revealed how these cells respond when the adipogenic differentiation pathway is blocked during induction of osteogenic differentiation. The inhibition leads to a significant increase in mineralization of the extracellular matrix, as well as an increased activity or up-regulated gene expression of alkaline phosphatase, the key enzyme involved in matrix mineralization. Furthermore, it was also demonstrated by microarray analysis, that PPAR-γ inhibition during osteogenic induction leads to a significant up-regulation of a number of genes related to both osteogenesis and adipogenesis such as c10orf10, leptin, GDF5 and KLF15. In conclusion, inhibition of PPAR-γ during induction of osteogenesis leads to increased osteogenic differentiation of human MSCs.

Novel markers of osteogenic and adipogenic differentiation of human bone marrow stromal cells identified using a quantitative proteomics approach.

Today, the tool that is most commonly used to evaluate the osteogenic differentiation of bone marrow stromal cells (BMSCs) in vitro is the demonstration of the expression of multiple relevant markers, such as ALP, RUNX2 and OCN. However, as yet, there is no single surface marker or panel of markers which clearly defines human BMSCs (hBMSCs) differentiating towards the osteogenic lineage. The aim of this study was therefore to examine this issue. Stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics was utilized to investigate differently expressed surface markers in osteogenically differentiated and undifferentiated hBMSCs. Labeled membrane proteins were analyzed by mass spectrometry (MS) and 52 proteins with an expression ratio above 2, between osteogenically differentiated and undifferentiated cells, were identified. Subsequent validation, by flow cytometry and ELISA, of the SILAC expression ratios for a number of these proteins and investigations of the lineage specificity of three candidate markers were performed. The surface markers, CD10 and CD92, demonstrated significantly increased expression in hBMSCs differentiated towards the osteogenic and adipogenic lineages. In addition, there was a slight increase in CD10 expression during chondrogenic differentiation. Furthermore, the expression of the intracellular protein, crystalline-αB (CRYaB), was only significantly increased in osteogenically differentiated hBMSCs and not affected during differentiation towards the chondrogenic or adipogenic lineages. It has been concluded from the present results that CD10 and CD92 are potential markers of osteogenic and adipogenic differentiation and that CRYaB is a potential novel osteogenic marker specifically expressed during the osteogenic differentiation of hBMSCs in vitro.

Monocyte exosomes stimulate the osteogenic gene expression of mesenchymal stem cells.

Inflammation and regeneration at the implant-bone interface are intimately coupled via cell-cell communication. In contrast to the prevailing view that monocytes/macrophages orchestrate mesenchymal stem cells (MSCs) and progenitor cells via the secretion of soluble factors, we examined whether communication between these different cell types also occurs via exosomes. LPS-stimulated human monocytes released exosomes, positive for CD9, CD63, CD81, Tsg101 and Hsp70, as determined by flow cytometry and Western blot. These exosomes also contained wide size distribution of RNA, including RNA in the size of microRNAs. The exosomes were shown to interact with human mesenchymal stem cells. After 24 h of culture, a considerable portion of the MSCs had internalised PKH67-labelled exosomes. Furthermore, after 72 h, the gene expression of the osteogenic markers runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein-2 (BMP-2) had increased in comparison with control medium, whereas no significant difference in osteocalcin (OC) expression was demonstrated. The present results show that, under given experimental conditions, monocytes communicate with MSCs via exosomes, resulting in the uptake of exosomes in MSCs and the stimulation of osteogenic differentiation. The present observations suggest that exosomes constitute an additional mode of cell-cell signalling with an effect on MSC differentiation during the transition from injury and inflammation to bone regeneration.

Virtual ligand-based screening reveals purmorphamine analogs with the capacity to induce the osteogenic differentiation of human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) have extensive proliferative capacity, are able to self-renew and have the potential to differentiate into cells of the connective tissue lineages. These properties make them a putative cell type for tissue engineering applications, as well as a possible in vivo target for the pharmaceutical modulation of the differentiation processes. The aim of this study was to find one or more small-molecule substances that would enhance the osteogenic differentiation of hMSCs in vitro. The strategy used here was ligand-based virtual screening for substances similar to the previously suggested osteoinductive purmorphamine followed by an in vitro screening of the selected analogs in hMSCs isolated from bone marrow. We investigated the osteoinductive capacity of several purmorphamine analogs by determining the protein and gene expression of markers for osteogenic differentiation as well as the extracellular matrix (ECM) mineralization of these cells. Treatment with two candidate substances or purmorphamine resulted in increased levels of alkaline phosphatase (ALP) activity compared to the control. Other purmorphamine analogs demonstrated higher calcium deposition in the ECM after 5 weeks of osteogenic differentiation, compared to both purmorphamine and the control condition. The resulting substances, which had positive effects on the osteogenic differentiation, are promising as possible modes of treatment for bone-related diseases or defects that target and enhance the osteogenic differentiation of MSCs, in vitro or in vivo. Furthermore, the concept of combining the virtual ligand-based screening method with in vitro screening, using human adult stem cells as a possible strategy for drug discovery, is demonstrated.

The stimulation of an osteogenic response by classical monocyte activation.

The monocyte/macrophage system plays a central role in host defense, wound healing and immune regulation at biomaterial surfaces. Monocytes can be classically and alternatively activated, and can be stimulated differently in response to variations in biomaterial surface properties. In this study, human monocytes, cultured on polystyrene surfaces (Ps), were activated either classically, by lipopolysaccharide (LPS), or alternatively, by interleukin-4 (IL-4). Monocytes were also cultured on anodically oxidized (Ox) and machined (Ma) titanium surfaces, with and without LPS stimulation. Cells were cultured for 1 and 3 days and their conditioned media (CM) were collected. The osteogenic response of hMSCs to the monocyte CM was determined by analyzing the gene expression of key osteogenic markers. The CM from classically activated monocytes increased the hMSCs expression of runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP). Furthermore, CM from monocytes cultured on Ox surface resulted in a modest increase of the expression of bone morphogenetic protein-2 (BMP-2). LPS stimulation of the surface-seeded monocytes overwhelmed the effect of the surface properties and resulted in significant upregulation of BMP-2 and Runx2 for all samples. The results show that human monocytes, cultured on different surfaces and/or under different activation pathways, communicate pro-osteogenic signals to hMSCs. The signals involve regulation of autologous BMP-2 in the hMSCs. The classical activation results in profound and prolonged osteogenic effect compared to the effect of the investigated surface properties.