Dihydrotestosterone Augments the Angiogenic and Migratory Potential of Human Endothelial Progenitor Cells by an Androgen Receptor-Dependent Mechanism.

Endothelial progenitor cells (EPCs) play a critical role in cardiovascular regeneration. Enhancement of their native properties would be highly beneficial to ensuring the proper functioning of the cardiovascular system. As androgens have a positive effect on the cardiovascular system, we hypothesized that dihydrotestosterone (DHT) could also influence EPC-mediated repair processes. To evaluate this hypothesis, we investigated the effects of DHT on cultured human EPCs' proliferation, viability, morphology, migration, angiogenesis, gene and protein expression, and ability to integrate into cardiac tissue. The results showed that DHT at different concentrations had no cytotoxic effect on EPCs, significantly enhanced the cell proliferation and viability and induces fast, androgen-receptor-dependent formation of capillary-like structures. DHT treatment of EPCs regulated gene expression of androgen receptors and the genes and proteins involved in cell migration and angiogenesis. Importantly, DHT stimulation promoted EPC migration and the cells' ability to adhere and integrate into murine cardiac slices, suggesting it has a role in promoting tissue regeneration. Mass spectrometry analysis further highlighted the impact of DHT on EPCs' functioning. In conclusion, DHT increases the proliferation, migration, and androgen-receptor-dependent angiogenesis of EPCs; enhances the cells' secretion of key factors involved in angiogenesis; and significantly potentiates cellular integration into heart tissue. The data offer support for potential therapeutic applications of DHT in cardiovascular regeneration and repair processes.

Biotechnological advances and applications of human pluripotent stem cell-derived heart models.

In recent years, significant biotechnological advancements have been made in engineering human cardiac tissues and organ-like models. This field of research is crucial for both basic and translational research due to cardiovascular disease being the leading cause of death in the developed world. Additionally, drug-associated cardiotoxicity poses a major challenge for drug development in the pharmaceutical and biotechnological industries. Progress in three-dimensional cell culture and microfluidic devices has enabled the generation of human cardiac models that faithfully recapitulate key aspects of human physiology. In this review, we will discuss 3D pluripotent stem cell (PSC)-models of the human heart, such as engineered heart tissues and organoids, and their applications in disease modeling and drug screening.

Inflammatory status of the pancreas in NOD mice that do not develop overt diabetes.

Type 1 diabetes (T1D) is an autoimmune disease in which immune cells target the pancreatic islets and destroy the ß-cells, resulting in hyperglycemia and decreased plasmatic insulin levels. The non-obese diabetic (NOD) mouse is the most used animal model for studying diabetes because it spontaneously develops T1D and shares similarities with the human disease. A hallmark feature of this model is the appearance of insulitis, defined as an inflammatory cell infiltration of the pancreatic islets. However, a small percentage of NOD mice do not develop overt diabetes even after 28-35 weeks of age. Thus, we questioned the status of the pancreatic islets in these non-diabetic NOD mice, with particular focus on islet inflammation and plasmatic insulin levels, in comparison to pre-diabetic (11 weeks old) and new-onset diabetic mice. Diabetes progression was evaluated by assessing blood glucose and pancreas histology. The inflammatory score was determined on Hematoxylin-Eosin (HE)-stained sections of pancreas. Plasma insulin was detected by enzyme-linked immunosorbent assay (ELISA). The results showed that inflammation increased in an age-dependent manner in all mice, irrespective of their diabetic status. Mostly affected within the analyzed groups were the 28 weeks old non-diabetic NOD mice, in which insulin production was reduced and inversely correlated with the inflammatory status. We conclude that in NOD mice, pancreatic inflammation progresses independently of diabetes onset and clinical signs of disease. Most likely, the NOD females that do not develop overt diabetes preserve a small mass of functional ß-cells, which is able to provide the physiological insulin levels and avoid diabetes onset.

Proteomic Analysis of Estrogen-Mediated Enhancement of Mesenchymal Stem Cell-Induced Angiogenesis In Vivo.

Therapeutic use of mesenchymal stem cells (MSCs) for tissue repair has great potential. MSCs from multiple sources, including those derived from human umbilical matrix, namely Wharton's jelly, may serve as a resource for obtaining MSCs. However, low in vivo engraftment efficacy of MSCs remains a challenging limitation. To improve clinical outcomes using MSCs, an in-depth understanding of the mechanisms and factors involved in successful engraftment is required. We recently demonstrated that 17ß-estradiol (E2) improves MSCs in vitro proliferation, directed migration and engraftment in murine heart slices. Here, using a proteomics approach, we investigated the angiogenic potential of MSCs in vivo and the modulatory actions of E2 on mechanisms involved in tissue repair. Specifically, using a Matrigel® plug assay, we evaluated the effects of E2 on MSCs-induced angiogenesis in ovariectomized (OVX) mice. Moreover, using proteomics we investigated the potential pro-repair processes, pathways, and co-mechanisms possibly modified by the treatment of MSCs with E2. Using RT-qPCR, we evaluated mRNA expression of pro-angiogenic molecules, including endoglin, Tie-2, ANG, and VEGF. Hemoglobin levels, a marker for blood vessel formation, were increased in plugs treated with E2 + MSCs, suggesting increased capillary formation. This conclusion was confirmed by the histological analysis of capillary numbers in the Matrigel® plugs treated with E2 + MSC. The LC-MS screening of proteins obtained from the excised Matrigel® plugs revealed 71 proteins that were significantly altered following E2 exposure, 57 up-regulated proteins and 14 down-regulated proteins. A major result was the association of over 100 microRNA molecules (miRNAs) involved in cellular communication, vesicle transport, and metabolic and energy processes, and the high percentage of approximately 25% of genes involved in unknown biological processes. Together, these data provide evidence for increased angiogenesis by MSCs treated with the sex hormone E2. In conclusion, E2 treatment may increase the engraftment and repair potential of MSCs into tissue, and may promote MSC-induced angiogenesis after tissue injury.

Mechanism of 17ß-estradiol stimulated integration of human mesenchymal stem cells in heart tissue.

Scarcity of gender specific donor hearts highlights the importance of mesenchymal stem cells (MSCs) as a therapeutic tool for heart repair. However, inefficient incorporation, retention, and activity of MSCs in cardiac tissue remain an obstacle. Since surges in follicular estradiol (E2; µmolar-range) trigger tissue remodeling (e.g. ovulation) and E2 exerts beneficial actions on the cardiovascular system, we hypothesized that E2 may promote/improve MSC-mediated cardiac repair processes. Using Wharton's jelly (WJ)-derived MSCs we assessed the effects of E2 on MSC proliferation, directed migration, and engraftment in murine heart slices (using xCELLigence real-time cell-impedance system, DNA quantification, and microscopy) and on MSC-induced angiogenesis in vivo (matrigel plug assay). Protein expression was assessed by Western blotting, ELISA/Luminex, and proteomic analysis; whereas mRNA expression was assessed by qRT-PCR. MSCs expressed estrogen receptors (ERs) -alpha and -beta. E2 promoted MSC proliferation and up-regulated mRNA and protein expression of ER-alpha, ER-beta, extracellular matrix metalloproteinase inducer (EMMPRIN), and matrix metalloproteinase (MMP) -9, yet down-regulated MMP-2 expression. Moreover, E2 up-regulated expression of vascular endothelial growth factor (VEGF)-A, VEGFR-2, vascular cell adhesion protein-1 (VCAM-1), and angiogenin (ANG) and stimulated nitric oxide (NO) production via ER. Proteomic analysis of MSCs showed that E2 up-regulated 47 proteins, down-regulated 7 proteins, and increased the expression of key biochemical components/pathways involved in tissue repair. In MSCs co-cultured with murine heart-slices, E2 significantly induced MSC migration in an ER-alpha-dependent fashion and significantly increased the secretion of MMP-2, MMP-9, ANG, and VEGF. In an in vivo matrigel assay, E2-treated MSCs increased angiogenesis and hemoglobin content. In conclusion, E2-treatment increases the incorporation of MSCs in heart slices and promotes MSC-induced angiogenesis. These beneficial effects are mediated via increases in molecules/pathways involved in tissue remodeling and angiogenesis. We speculate that E2 may enhance MSC ability to repair/regenerate cardiac tissue.

Dihydrotestosterone induces pro-angiogenic factors and assists homing of MSC into the cardiac tissue.

The use of mesenchymal stem cells (MSC) as a therapeutic tool in cardiovascular diseases is promising. Since androgens exert some beneficial actions on the cardiovascular system, we tested our hypothesis that this hormone could promote MSC-mediated repair processes, also. Cultured MSCs isolated from Wharton's jelly were exposed to 30 nM dihydrotestosterone (DHT) for 1 or 4 days and the effects of the hormone on their growth/migration/adhesion and the underlying mechanisms were assessed. Results were obtained by real-time cell impedance measurements, and DNA quantification showed that DHT increased MSC proliferation by ~30%. As determined by xCELLigence system, DHT augmented (~2 folds) the migration of MSC toward cardiac tissue slices (at 12 h), and this effect was blocked by flutamide, an androgen receptor (AR) antagonist. Exposure of cells to DHT, upregulated the gene and protein expression of AR, EMMPRIN and MMP-9 and downregulated the expression of MMP-2 DHT significantly induced the release of nitric oxide by MSC (≥2-fold) and flutamide blocked this effect. When MSCs were co-cultured with cardiac slices, immunohistochemical analysis and qRT-PCR showed that the integration of DHT-stimulated MSC was significantly higher than that of in controls. In conclusion, our findings provide the first evidence that DHT promotes MSC growth, migration and integration into the cardiac slices. The modulating effects of DHT were associated with upregulation of ARs and of key molecules known to promote tissue remodeling and angiogenesis. Our findings suggest that priming of MSC with DHT may potentially increase their capability to regenerate cardiac tissue; in vivo studies are needed to confirm our in vitro findings.

Testosterone stimulates proliferation and preserves stemness of human adult mesenchymal stem cells and endothelial progenitor cells.

Human adult stem and progenitor cells are promising cell types widely studied for their clinical benefits. A reduced number of stem cells present in the human body are associated with numerous dysfunctions. Since androgens have a profound effect on different cell types, we questioned whether testosterone (T), one of the main androgens, influence and are involved in the proliferation of stem cells and÷or affect their stemness potential. Isolated mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) were cultured and then stimulated with different concentration of testosterone (10-100 nM). The cellular proliferation rate, adhesion, and viability were measured in real-time using xCELLigence system and DNA-cell proliferation assay. The immunophenotype of the stimulated cells versus non-stimulated cells was determined by flow cytometry. The maximal effect on MSCs and EPCs proliferation was obtained at 40 nM testosterone; this concentration was used in further experiments. The cellular index measured in real-time by impedance-based dynamic measurements revealed that 40 nM testosterone had a proliferative effect on both MSCs and EPCs, having a proliferative index of ˜50% above the control (non-stimulated) cells. Furthermore, flow cytometry assay indicated that testosterone stimulation did not alter the phenotype of MSCs and EPCs, both cell types preserving the expression of the characteristic surface markers. Testosterone stimulation increases the proliferation and preserves stemness of MSCs and EPCs suggesting that, besides other factors, the hormone may engineer these cells and increase their therapeutic potential.

Isolation method and xeno-free culture conditions influence multipotent differentiation capacity of human Wharton's jelly-derived mesenchymal stem cells.

INTRODUCTION: Human Wharton's jelly (WJ) has become a preferred source of mesenchymal stem cells (MSCs) whose clinical applications are limited by the use of adequate xeno-free (XF), in vitro manipulation conditions. Therefore, the objective of our study was to characterize WJ-derived MSCs (WJ-MSCs), isolated by different methods and cultured in a commercially available, MSC XF medium, not least of all by investigating their endothelial differentiation capacity. METHODS: WJ explants and enzymatically dissociated WJ cells were cultured in a defined, XF medium for MSCs. Adherent cells at passages 2 and 5 were characterized as MSCs by flow cytometry, MTT, real-time quantitative reverse transcription PCR, and functional multipotent differentiation assays. The endothelial differentiation capacity of MSCs isolated and expanded until passage 2 in the MSC XF medium, and then subcultured for five passages in a commercially available endothelial growth medium (group A), was assessed over serial passages, as compared to adherent WJ-derived cells isolated and expanded for five consecutive passages in the endothelial medium (group B). RESULTS: The MSC phenotype of WJ explant- and pellet-derived cells, isolated and expanded in the MSC XF medium, was proven based on the expression of CD44/CD73/CD90/CD105 surface markers and osteo-/adipo-/chondrogenic multipotent differentiation potential, which differed according to the isolation method and/or passage number. Upon exposure to endothelial differentiation cues, cells belonging to group A did not exhibit endothelial cell characteristics over serial passages; by contrast, WJ pellet-derived cells belonging to group B expressed endothelial characteristics at gene, protein and functional levels, potentially due to culture conditions favoring the isolation of other stem/progenitor cell types than MSCs, able to give rise to an endothelial progeny. CONCLUSIONS: The use of defined, MSC XF media for isolation and expansion of human WJ-MSCs is a prerequisite for the establishment of their real endothelial differentiation capacity, as candidates for clinical therapy applications. Thus, the standardization of WJ-MSCs isolation and culture expansion techniques in defined, MSC XF media, for their accurate characterization, would be a priority in the stem cell research field.