Analysis of local extracellular matrix identifies different aetiologies behind bicuspid and tricuspid aortic valve degeneration and suggests therapies.

Aortic valve degeneration (AVD) is a life-threatening condition that has no medical treatment and lacks individual therapies. Although extensively studied with standard approaches, aetiologies behind AVD are unclear. We compared abundances of extracellular matrix (ECM) proteins from excised valve tissues of 88 patients with isolated AVD of normal tricuspid (TAV) and congenital bicuspid aortic valves (BAV), quantified more than 1400 proteins per ECM sample by mass spectrometry, and demonstrated that local ECM preserves molecular cues of the pathophysiological processes. The BAV ECM showed enrichment with fibrosis markers, namely Tenascin C, Osteoprotegerin, and Thrombospondin-2. The abnormal physical stress on BAV may cause a mechanical injury leading to a continuous Tenascin C-driven presence of myofibroblasts and persistent fibrosis. The TAV ECM exhibited enrichment with Annexin A3 (p = 1.1 × 10-16 and the fold change 6.5) and a significant deficit in proteins involved in high-density lipid metabolism. These results were validated by orthogonal methods. The difference in the ECM landscape suggests distinct aetiologies between AVD of BAV and TAV; warrants different treatments of the patients with BAV and TAV; elucidates the molecular basis of AVD; and implies possible new therapeutic approaches. Our publicly available database (human_avd_ecm.surgsci.uu.se) is a rich source for medical doctors and researchers who are interested in AVD or heart ECM in general. Systematic proteomic analysis of local ECM using the methods described here may facilitate future studies of various tissues and organs in development and disease.

Spatiotemporal extracellular matrix modeling for in situ cell niche studies.

Extracellular matrix (ECM) components govern a range of cell functions, such as migration, proliferation, maintenance of stemness, and differentiation. Cell niches that harbor stem-/progenitor cells, with matching ECM, have been shown in a range of organs, although their presence in the heart is still under debate. Determining niches depends on a range of in vitro and in vivo models and techniques, where animal models are powerful tools for studying cell-ECM dynamics; however, they are costly and time-consuming to use. In vitro models based on recombinant ECM proteins lack the complexity of the in vivo ECM. To address these issues, we present the spatiotemporal extracellular matrix model for studies of cell-ECM dynamics, such as cell niches. This model combines gentle decellularization and sectioning of cardiac tissue, allowing retention of a complex ECM, with recellularization and subsequent image processing using image stitching, segmentation, automatic binning, and generation of cluster maps. We have thereby developed an in situ representation of the cardiac ECM that is useful for assessment of repopulation dynamics and to study the effect of local ECM composition on phenotype preservation of reseeded mesenchymal progenitor cells. This model provides a platform for studies of organ-specific cell-ECM dynamics and identification of potential cell niches.

Diversity of respiratory parameters and metabolic adaptation to low oxygen tension in mesenchymal stromal cells.

Objective: Cell metabolism has been shown to play an active role in regulation of stemness and fate decision. In order to identify favorable culture conditions for mesenchymal stromal cells (MSCs) prior to transplantation, this study aimed to characterize the metabolic function of MSCs from different developmental stages in response to different oxygen tension during expansion. Materials and methods: We cultured human fetal cardiac MSCs and human adult bone-marrow MSCs for a week under hypoxia (3% O2) and normoxia (20% O2). We performed mitochondrial characterization and assessed oxygen consumption- and extracellular acidification-rates (OCR and ECAR) in addition to oxygen-sensitive respiration and mitochondrial complex activities, using both the Seahorse and Oroboros systems. Results: Adult and fetal MSCs displayed similar basal respiration and mitochondrial amount, however fetal MSCs had lower spare respiratory capacity and apparent coupling efficiency. Fetal MSCs expanded in either hypoxia or normoxia demonstrated similar acidification rates, while adult MSCs downregulated their aerobic glycolysis in normoxia. Acute decrease in oxygen tension caused a higher respiratory inhibition in adult compared to fetal MSCs. In both sources of MSCs, minor changes in complex activities in normoxic and hypoxic cultures were found. Conclusions: In contrast to adult MSCs, fetal MSCs displayed similar respiration and aerobic glycolysis at different O2 culture concentrations during expansion. Adult MSCs adjusted their respiration to glycolytic activities, depending on the culture conditions thus displaying a more mature metabolic function. These findings are relevant for establishing optimal in vitro culturing conditions, with the aim to maximize engraftment and therapeutic outcome.

Identification of storage conditions stabilizing extracellular vesicles preparations.

Extracellular vesicles (EVs) play a key role in many physiological and pathophysiological processes and hold great potential for therapeutic and diagnostic use. Despite significant advances within the last decade, the key issue of EV storage stability remains unresolved and under investigated. Here, we aimed to identify storage conditions stabilizing EVs and comprehensively compared the impact of various storage buffer formulations at different temperatures on EVs derived from different cellular sources for up to 2 years. EV features including concentration, diameter, surface protein profile and nucleic acid contents were assessed by complementary methods, and engineered EVs containing fluorophores or functionalized surface proteins were utilized to compare cellular uptake and ligand binding. We show that storing EVs in PBS over time leads to drastically reduced recovery particularly for pure EV samples at all temperatures tested, starting already within days. We further report that using PBS as diluent was found to result in severely reduced EV recovery rates already within minutes. Several of the tested new buffer conditions largely prevented the observed effects, the lead candidate being PBS supplemented with human albumin and trehalose (PBS-HAT). We report that PBS-HAT buffer facilitates clearly improved short-term and long-term EV preservation for samples stored at -80°C, stability throughout several freeze-thaw cycles, and drastically improved EV recovery when using a diluent for EV samples for downstream applications.

Lentiviral-mediated HoxB4 expression in human embryonic stem cells initiates early hematopoiesis in a dose-dependent manner but does not promote myeloid differentiation.

The variation of HoxB4 expression levels might be a key regulatory mechanism in the differentiation of human embryonic stem cell (hESC)-derived hematopoietic stem cells (HSCs). In this study, hESCs ectopically expressing high and low levels of HoxB4 were obtained using lentiviral gene transfer. Quantification throughout differentiation revealed a steady increase in transcription levels from our constructs. The effects of the two expression levels of HoxB4 were compared regarding the differentiation potential into HSCs. High levels of HoxB4 expression correlated to an improved yield of cells expressing CD34, CD38, the stem cell leukemia gene, and vascular epithelium-cadherin. However, no improvement in myeloid cell maturation was observed, as determined by colony formation assays. In contrast, hESCs with low HoxB4 levels did not show any elevated hematopoietic development. In addition, we found that the total population of HoxB4-expressing cells, on both levels, decreased in developing embryoid bodies. Notably, a high HoxB4 expression in hESCs also seemed to interfere with the formation of germ layers after xenografting into immunodeficient mice. These data suggest that HoxB4-induced effects on hESC-derived HSCs are concentration-dependent during in vitro development and reduce proliferation of other cell types in vitro and in vivo. The application of the transcription factor HoxB4 during early hematopoiesis from hESCs might provide new means for regenerative medicine, allowing efficient differentiation and engraftment of genetically modified hESC clones. Our study highlights the importance of HoxB4 dosage and points to the need for experimental systems allowing controlled gene expression. Disclosure of potential conflicts of interest is found at the end of this article.

Human Fetal Cardiac Mesenchymal Stromal Cells Differentiate In Vivo into Endothelial Cells and Contribute to Vasculogenesis in Immunocompetent Mice.

Mesenchymal stromal cells (MSCs) have shown great potential as a treatment for systemic inflammatory diseases, but their local regenerative properties are highly tissue- and site specific. Previous studies have demonstrated that adult human MSCs respond to inflammatory cytokines through the release of paracrine factors that stimulate angiogenesis, but they do not themselves differentiate into vascular structures in vivo. In this study, we used human fetal cardiac MSCs (hfcMSCs) harvested during the first trimester of heart development and injected them into the subcutaneous tissue of normal immunocompetent mice treated with short-term costimulation blockade for tolerance induction. When hfcMSCs were transplanted subcutaneously together with Matrigel matrix, they contributed to vasculogenesis through differentiation into endothelial cells and generation of the basal membrane protein Laminin α4. These characteristics of hfcMSCs are similar to the mesodermal progenitors giving rise to the developing heart and they may be useful for treatment of ischemic injuries.

Systematic Methodological Evaluation of a Multiplex Bead-Based Flow Cytometry Assay for Detection of Extracellular Vesicle Surface Signatures.

Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids. EVs can be conceptually classified based on their size and biogenesis as exosomes and microvesicles. Nowadays, it is however commonly accepted in the field that there is a much higher degree of heterogeneity within these two subgroups than previously thought. For instance, the surface marker profile of EVs is likely dependent on the cell source, the cell's activation status, and multiple other parameters. Within recent years, several new methods and assays to study EV heterogeneity in terms of surface markers have been described; most of them are being based on flow cytometry. Unfortunately, such methods generally require dedicated instrumentation, are time-consuming and demand extensive operator expertise for sample preparation, acquisition, and data analysis. In this study, we have systematically evaluated and explored the use of a multiplex bead-based flow cytometric assay which is compatible with most standard flow cytometers and facilitates a robust semi-quantitative detection of 37 different potential EV surface markers in one sample simultaneously. First, assay variability, sample stability over time, and dynamic range were assessed together with the limitations of this assay in terms of EV input quantity required for detection of differently abundant surface markers. Next, the potential effects of EV origin, sample preparation, and quality of the EV sample on the assay were evaluated. The findings indicate that this multiplex bead-based assay is generally suitable to detect, quantify, and compare EV surface signatures in various sample types, including unprocessed cell culture supernatants, cell culture-derived EVs isolated by different methods, and biological fluids. Furthermore, the use and limitations of this assay to assess heterogeneities in EV surface signatures was explored by combining different sets of detection antibodies in EV samples derived from different cell lines and subsets of rare cells. Taken together, this validated multiplex bead-based flow cytometric assay allows robust, sensitive, and reproducible detection of EV surface marker expression in various sample types in a semi-quantitative way and will be highly valuable for many researchers in the EV field in different experimental contexts.

Spatial detection of fetal marker genes expressed at low level in adult human heart tissue.

Heart failure is a major health problem linked to poor quality of life and high mortality rates. Hence, novel biomarkers, such as fetal marker genes with low expression levels, could potentially differentiate disease states in order to improve therapy. In many studies on heart failure, cardiac biopsies have been analyzed as uniform pieces of tissue with bulk techniques, but this homogenization approach can mask medically relevant phenotypes occurring only in isolated parts of the tissue. This study examines such spatial variations within and between regions of cardiac biopsies. In contrast to standard RNA sequencing, this approach provides a spatially resolved transcriptome- and tissue-wide perspective of the adult human heart, and enables detection of fetal marker genes expressed by minor subpopulations of cells within the tissue. Analysis of patients with heart failure, with preserved ejection fraction, demonstrated spatially divergent expression of fetal genes in cardiac biopsies.

Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells.

After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts.

Endothelin-1 supports clonal derivation and expansion of cardiovascular progenitors derived from human embryonic stem cells.

Coronary arteriogenesis is a central step in cardiogenesis, requiring coordinated generation and integration of endothelial cell and vascular smooth muscle cells. At present, it is unclear whether the cell fate programme of cardiac progenitors to generate complex muscular or vascular structures is entirely cell autonomous. Here we demonstrate the intrinsic ability of vascular progenitors to develop and self-organize into cardiac tissues by clonally isolating and expanding second heart field cardiovascular progenitors using WNT3A and endothelin-1 (EDN1) human recombinant proteins. Progenitor clones undergo long-term expansion and differentiate primarily into endothelial and smooth muscle cell lineages in vitro, and contribute extensively to coronary-like vessels in vivo, forming a functional human-mouse chimeric circulatory system. Our study identifies EDN1 as a key factor towards the generation and clonal derivation of ISL1(+) vascular intermediates, and demonstrates the intrinsic cell-autonomous nature of these progenitors to differentiate and self-organize into functional vasculatures in vivo.

Wnt/ß-Catenin Stimulation and Laminins Support Cardiovascular Cell Progenitor Expansion from Human Fetal Cardiac Mesenchymal Stromal Cells.

The intrinsic regenerative capacity of human fetal cardiac mesenchymal stromal cells (MSCs) has not been fully characterized. Here we demonstrate that we can expand cells with characteristics of cardiovascular progenitor cells from the MSC population of human fetal hearts. Cells cultured on cardiac muscle laminin (LN)-based substrata in combination with stimulation of the canonical Wnt/ß-catenin pathway showed increased gene expression of ISL1, OCT4, KDR, and NKX2.5. The majority of cells stained positive for PDGFR-α, ISL1, and NKX2.5, and subpopulations also expressed the progenitor markers TBX18, KDR, c-KIT, and SSEA-1. Upon culture of the cardiac MSCs in differentiation media and on relevant LNs, portions of the cells differentiated into spontaneously beating cardiomyocytes, and endothelial and smooth muscle-like cells. Our protocol for large-scale culture of human fetal cardiac MSCs enables future exploration of the regenerative functions of these cells in the context of myocardial injury in vitro and in vivo.

Costimulation blockade induces foxp3(+) regulatory T cells to human embryonic stem cells.

Transplantation of human embryonic stem cells (hESCs), like other allogeneic cellular transplants, require immunomodulation or immunosuppression in order to be maintained in the recipient. Costimulation blockade applied at the time of transplantation inhibits costimulatory signals in the immunological synapse leading to a state of anergy in the donor reactive T-cell population and a state of immunological tolerance in the host. In models of solid organ transplantation, tolerance is maintained by the infiltration of Foxp3(+) regulatory T cells into the graft. In order to study if regulatory T cells could be generated to hESC transplants, costimulation blockade (CTLA4Ig, anti-CD40L, anti-LFA-1) was administered for the first week after transplantation of two different hESC lines implanted under the kidney capsule of wild-type mice. hESC transplants were maintained indefinitely, and when harvested at long-term follow-up, Foxp3(+) T-cells were found surrounding the graft, implying the maintenance of tolerance through the induction of regulatory T cells. These results imply that costimulation blockade could be a useful treatment strategy for the induction of tolerance to hESC transplants and may down-modulate immune responses locally around the graft.

TK.007: A novel, codon-optimized HSVtk(A168H) mutant for suicide gene therapy.

Conditional elimination of infused gene-modified alloreactive T cells, using suicide gene activation, has been shown to be an efficient strategy to abrogate severe graft-versus-host disease (GvHD) in the context of adoptive immunotherapy. To overcome shortcomings of the most widely used suicide gene, wild-type (splice-corrected) herpes simplex virus thymidine kinase (scHSVtk), we generated two new variants: the codon-optimized coHSVtk and, by introducing an additional mutation (A168H), the novel TK.007. We transduced human hematopoietic cell lines and primary T cells with retroviral "sort-suicide vectors" encoding combinations of selection markers (tCD34 and OuaSelect) with one of three HSVtk variants. In vitro we observed higher expression levels and sustained long-term expression of TK.007, indicating lower nonspecific toxicity. Also, we noted significantly improved kinetics of ganciclovir (GCV)-mediated killing for TK.007-transduced cells. In an experimental (murine) allogeneic transplantation model, TK.007-transduced T cells mediated severe GvHD, which was readily abrogated by application of GCV (10 mg/kg). Last, we established a modified allotransplantation model that allowed quantitative comparison of the in vivo activities of TK.007 versus scHSVtk. We found that TK.007 mediates both significantly faster and higher absolute killing at low GCV concentrations (10 and 25 mg/kg). In summary, we demonstrate that the novel TK.007 suicide gene combines better killing performance with reduced nonspecific toxicity (as compared with the frequently used splice-corrected wild-type scHSVtk gene), thus representing a promising alternative for suicide gene therapy.

Derivation of human skin fibroblast lines for feeder cells of human embryonic stem cells.

After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arouse. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures of research grade human skin fibroblast lines. We also describe how to make feeder layers for hESC using these fibroblasts.