A brief very-low oxygen tension regimen is sufficient for the early chondrogenic commitment of human adipose-derived mesenchymal stem cells.

PURPOSE: The aim of this study was to evaluate the effects exerted over chondrogenic commitment of human adipose-derived mesenchymal stem cells (ADSCs) by a very low oxygen tension (<1% pO2). MATERIALS/METHODS: Cell morphology, mRNA levels of chondrocyte-specific marker genes and the involvement of p38 MAPK signalling were monitored in human ADSCs under a very low oxygen tension. RESULTS: Cell morphology was significantly changed after two days of hypoxic preconditioning when they featured as elongated spindle-shaped cells. SRY-box containing gene 9, aggrecan and collagen type II mRNA levels were enhanced under severe hypoxic culture conditions. Moreover, the inhibition of p38 MAPK resulted in a substantial reduction in transcription of the above-mentioned specific genes, proving the pivotal role of this pathway in the transcriptional regulation of chondrogenesis. CONCLUSIONS: Here, we propose a protocol showing the early commitment of stem cells towards the chondrogenic phenotype in only 2 days of culture via a very low hypoxic environment, in the absence of growth factors added in the culture medium.

Doxorubicin and α-Mangostin oppositely affect luminal breast cancer cell stemness evaluated by a new retinaldehyde-dependent ALDH assay in MCF-7 tumor spheroids.

According to cancer stem cell theory, only a limited number of self-renewing and cloning cells are responsible for tumor relapse after a period of remittance. The aim of the present study was to investigate the effects of Doxorubicin and α-Mangostin, two antiproliferative drugs, on both tumor bulk and stem cells in multicellular tumor spheroids originated from the luminal MCF-7 breast cancer cell line. A new and original fluorimetric assay was used to selectively measure the activity of the retinaldehyde-dependent isoenzymes of aldehyde dehydrogenase (RALDH), which are markers of a subpopulation of breast cancer stem cells. The administration of 5 µg/ml (12.2 µM) α-Mangostin for 48 h provoked: i) a marked disaggregation of the spheroids, leading to a doubling of their volume (p < 0.01), ii) a 40 % decrease in cell viability (p < 0.01), evaluated by the acid phosphatase assay, and iii) a reduction by more than 90 % of RALDH activity. By contrast, Doxorubicin given for 48 h in the range of 0.1-40 µM did not significantly reduce cell viability and caused only a modest modification of the spheroid morphology. Moreover, 40 µM Doxorubicin increased RALDH activity 2.5-fold compared to the untreated sample. When the two drugs were administered together using 5 µg/ml α-Mangostin, the IC50 of Doxorubicin referred to cell viability decreased six-fold and the RALDH activity was further reduced. In conclusion, the combined administration of Doxorubicin and α-Mangostin provoked a significant cytotoxicity and a remarkable inhibition of RALDH activity in MCF-7 tumor spheroids, suggesting that these drugs could be effective in reducing cell stemness in luminal breast cancer.

Deferoxamine Protects Stromal/Stem Cells of "Lull pgm System"-Processed Lipoaspirates Against Damages Induced by Mitochondrial Respiration Inhibition.

BACKGROUND: The ischemic environment of the receiving area compromises the outcome of autologous fat grafts. The aim of this study was to isolate and expand the stromal vascular fraction from patient lipoaspirates and investigate the gain in cell viability exerted by some protective agents against the blockage of mitochondrial respiration. METHODS: The aspirates were (1) washed, using the "Lull pgm system," (2) centrifuged and (3) decanted. The corresponding stromal vascular fractions were isolated, and after cell adherence selection, the stromal/stem cell subpopulations were exposed to Antimycin A for 1 h. Then, the protection induced by cell pretreatment with deferoxamine, diazoxide and IGF-1 was evaluated. RESULTS: The residual cell viability of the "Lull pgm system"-washed samples was greater than that of the centrifuged samples (p < 0.05), and this advantage was maintained during the following 12 days of culture. The administration of 400 µM deferoxamine before Antimycin A treatment increased the number of viable cells from 56.5 to 80.8% (p < 0.05). On the contrary, the pretreatment with 250 µM diazoxide or 0.1 µg/ml IGF-1 did not exert any significant pro-survival action. Echinomycin abolished the positive effect of deferoxamine, suggesting that its protection involved HIF-1α. CONCLUSIONS: Adipose-derived stromal-stem cells survive the inhibition of mitochondrial respiration better if the lipoaspirate is washed using the "Lull pgm system" rather than centrifuged. Moreover, a significant contribution to cell survival can be obtained by preconditioning stromal-stem cells with deferoxamine. In a clinical perspective, this drug could be safely administered before surgery to patients undergoing autologous fat transfer. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Complete Disaggregation of MCF-7-derived Breast Tumour Spheroids with Very Low Concentrations of α-Mangostin Loaded in CD44 Thioaptamer-tagged Nanoparticles.

Background: α-Mangostin (αMG) is a natural substance that exerts a wide range of antitumor effects. Recently, we described that free αMG was able to dissociate multicellular tumour spheroids (MCTSs) generated from breast carcinoma cells and to reduce their cellular viability and motility. Here, αMG was encapsulated into lipidic nanoparticles (NPs), conjugated or not to a CD44 thioaptamer, and the anticancer action evaluated against MCF-7 breast MCTSs. Methods: NPs containing αMG were formulated with a core of polylactic-co-glycolyc acid. Some of them were decorated with a CD44 thioaptamer using as catalysts 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide. Both size and density of MCF-7-derived MCTSs were monitored during 72 h of treatment with NPs carrying 0.1, 0.5 and 1.0 µg/ml final concentrations of αMG. MCTSs were cultured on Matrigel or gelatine to better simulate the extracellular environment. Results: The NPs without thioaptamer and conveying 0.1 µg/ml αMG caused a significant dissociation of the MCTSs grown in gelatine after 24 h of treatment (p < 0.01). The most significant disaggregation of MCTSs was obtained using NPs carrying 0.5 µg/ml αMG (p < 0.01). A similar dissociating effect was observed when MCTSs were cultured in Matrigel under the same conditions for 48 - 72 h. By contrast, only concentrations over 1.0 µg/ml of free αMG were able to provoke a damage to MCTSs, consisting in a substantial reduction in their size (p < 0.05). Since the MCTS dissociation induced by αMG-loaded NPs occurred only in the presence of Matrigel or gelatine, an impairment of cell contacts to collagen fibres was likely responsible of this effect. Finally, the treatment of MCTSs with αMG-loaded NPs that were conjugated to the CD44 thioaptamer caused a similar decrease in density but a lower expansion of the spheroid, suggesting that a significant number of cells were died or arrested in cycle. Conclusion: Very low concentrations of αMG delivered by lipidic NPs are sufficient to provoke a substantial disaggregation of MCF-7 MCTSs that involves cell-to-collagen contacts. Similarly, the treatment of MCTSs with NPs conjugated to a CD44 thioaptamer leads to MCTS dissociation but through a more damaging action that causes also a reduction in cell number.

Effects of α-Mangostin on Viability, Growth and Cohesion of Multicellular Spheroids Derived from Human Breast Cancer Cell Lines.

Background: α-Mangostin (αMG) is extracted from Garcinia mangostana Linn and exerts antiproliferative activities. Although several researches on αMG were performed using cell monolayers, the in vitro pharmacological effects on 3D cancer models have never been investigated. Aim of the present study was to find new anticancer properties of αMG by evaluating the changes that this compound provokes in multicellular tumour spheroids (MCTSs). Methods: MCTSs were generated from MDA-MB-231 and MCF-7 breast tumour cell lines and then treated with 0.1÷30 µg/ml αMG for 24 and 48 h. MCTS size, density, and cell migration were determined by software elaboration of phase contrast images captured by a digital camera. Cell viability was evaluated by resazurin and acid phosphatase assays, while cell apoptosis was assessed by a fluorescent assay of caspase activity. The distribution of living cells inside MCTSs was shown by live/dead fluorescence staining. Results: A dose-dependent decrease in cell viability was obtained by treating MDA-MB-231 spheroids with αMG for 48 h (IC50 = 0.70-1.25 µg/ml). A significant reduction in spheroid volume, paralleled by its increased compactness, was observed only at concentration of 30 µg/ml, but not with lower doses of αMG. By contrast, αMG in the range of 5-15 µg/ml increased the size of MCTSs due to a parallel reduction in cell aggregation. The same window of concentrations was also able to stimulate cell apoptosis in a dose-dependent manner. Bimodal volumetric effects were also obtained by treating the spheroids generated from the MCF-7 cells with 0.1÷30 µg/ml αMG for 48 h. Finally, doses higher than 5 µg/ml caused a progressive impairment in cell migration from the edge of MDA-MB-231 MCTSs. Conclusion: After exposure at doses of αMG just above IC50, MDA-MB-231 spheroids showed a significant reduction in cell adhesion that did not stimulate cell migration but, on the contrary, blunted cell motility. These findings suggest a novel anticancer feature of αMG that could be taken into consideration to improve conventional drug penetration into the tumour bulk.

Lull pgm system: A suitable technique to improve the regenerative potential of autologous fat grafting.

Autologous fat grafting and methods of purification of harvested tissue have become one of the most current themes in regenerative medicine. The aim of this study was to evaluate the in vitro regenerative potential of abdomen lipoaspirates subjected to a combined washing-decantation purifying procedure, the Lull pgm System (Lull). Blood cells and stromal-vascular fraction (SVF) cells contained in the aspirates were investigated and compared with those obtained through more conventional fat-processing methods, that is, the decantation and Coleman's centrifugation techniques. The lowest number of erythrocytes, which are proinflammatory cells, was observed in the Lull samples, corresponding to about 50% of those isolated by decantation and centrifugation. The highest amount of SVF cells were isolated from the Lull samples whose number of colony forming units, representative of the amount of adipose-derived stem cells (ADSCs), was about fourfold and sixfold higher than in the decantation and centrifugation samples, respectively. Adipocyte and osteoblast commitment of SVF cells obtained from all the three procedures also confirmed that the subpopulation of ADSCs was actively represented in the processed aspirates. Moreover, the growth rate of the SVF cells was more accentuated in the samples obtained from decantation and Lull than centrifugation. In conclusion, Lull seems to be the best processing technique for adipose tissue graft with respect to decantation and centrifugation, because it clears more efficiently the fat from proinflammatory blood cells and provides the greatest number of proliferating SFV cells and ADSCs.

* An Engineered Multiphase Three-Dimensional Microenvironment to Ensure the Controlled Delivery of Cyclic Strain and Human Growth Differentiation Factor 5 for the Tenogenic Commitment of Human Bone Marrow Mesenchymal Stem Cells.

At present, injuries or rupture of tendons are treated by surgical repair or conservative approaches with unpredictable clinical outcome. Alternative strategies to repair tendon defects without the undesirable side effects associated with the current options are needed. With this in mind, a tissue engineering approach has gained considerable attention as a promising strategy. Here we investigated a synthetic three-dimensional (3D) microenvironment able to interact with stem cells and inducing, via coupled biochemical and physical signals, their early commitment toward the tenogenic lineage. This multiphase 3D construct consisted of a braided hyaluronate elastic band merged with human bone marrow mesenchymal stem cells (hBMSCs) and poly-lactic-co-glycolic acid microcarriers loaded with human growth differentiation factor 5 (hGDF-5) by means of fibrin hydrogel. The multiphase structure allowed hBMSC culture under cyclic strain within a microenvironment where a controlled amount of hGDF-5 was regularly delivered. The cooperative biochemical and physical stimuli induced significantly increased expression of tenogenic markers, such as collagen type I and III, decorin, scleraxis, and tenascin-C, within only 3 days of dynamic hBMSC culture. This approach opens exciting perspectives for future development of engineered tendon tissue substitutes.

Mechanical Actuation Systems for the Phenotype Commitment of Stem Cell-Based Tendon and Ligament Tissue Substitutes.

High tensile forces transmitted by tendons and ligaments make them susceptible to tearing or complete rupture. The present standard reparative technique is the surgical implantation of auto- or allografts, which often undergo failure.Currently, different cell types and biomaterials are used to design tissue engineered substitutes. Mechanical stimulation driven by dedicated devices can precondition these constructs to a remarkable degree, mimicking the local in vivo environment. A large number of dynamic culture instruments have been developed and many appealing results collected. Of the cells that have been used, tendon stem cells are the most promising for a reliable stretch-induced tenogenesis, but their reduced availability represents a serious limitation to upscaled production. Biomaterials used for scaffold fabrication include both biological molecules and synthetic polymers, the latter being improved by nanotechnologies which reproduce the architecture of native tendons. In addition to cell type and scaffold material, other variables which must be defined in mechanostimulation protocols are the amplitude, frequency, duration and direction of the applied strain. The ideal conditions seem to be those producing intermittent tension rather than continuous loading. In any case, all physical parameters must be adapted to the specific response of the cells used and the tensile properties of the scaffold. Tendon/ligament grafts in animals usually have the advantage of mechanical preconditioning, especially when uniaxial cyclic forces are applied to cells engineered into natural or decellularized scaffolds. However, due to the scarcity of in vivo research, standard protocols still need to be defined for clinical applications.

Enhanced engraftment and repairing ability of human adipose-derived stem cells, conveyed by pharmacologically active microcarriers continuously releasing HGF and IGF-1, in healing myocardial infarction in rats.

One of the main cause of ineffective cell therapy in repairing the damaged heart is the poor yield of grafted cells. To overcome this drawback, rats with 4-week-old myocardial infarction (MI) were injected in the border zone with human adipose-derived stem cells (ADSCs) conveyed by poly(lactic-co-glycolic acid) microcarriers (PAMs) releasing hepatocyte growth factor (HGF) and insulin-like growth factor-1 (IGF-1) (GFsPAMs). According to treatments, animals were subdivided into different groups: MI_ADSC, MI_ADSC/PAM, MI_GFsPAM, MI_ADSC/GFsPAM, and untreated MI_V. Two weeks after injection, a 31% increase in ADSC engraftment was observed in MI_ADSC/PAM compared with MI_ADSC (p < 0.05). A further ADSC retention was obtained in MI_ADSC/GFsPAM with respect to MI_ADSC (106%, p < 0.05) and MI_ADSC/PAM (57%, p < 0.05). A 130% higher density of blood vessels of medium size was present in MI_ADSC/GFsPAM compared with MI_ADSC (p < 0.01). MI_ADSC/GFsPAM also improved, albeit slightly, left ventricular remodeling and hemodynamics with respect to the other groups. Notably, ADSCs and/or PAMs, with or without HGF/IGF-1, trended to induce arrhythmias in electrically driven, Langendorff-perfused, hearts of all groups. Thus, PAMs releasing HGF/IGF-1 markedly increase ADSC engraftment 2 weeks after injection and stimulate healing in chronically infarcted myocardium, but attention should be paid to potentially negative electrophysiological consequences.

Cancer stem cells and mesenchymal stem cells in the hypoxic tumor niche: two different targets for one only drug.

Putative cancer stem cells (CSCs) reside in a hypoxic microenvironment where mesenchymal stem cells (MSCs) are also present. In this niche MSCs seem to promote the generation of CSCs and sustain tumor progression. Therefore, it may assume clinical relevance to produce a drug which kills not only CSCs but also MSCs. We hypothesized that bifunctional nanoparticles, loaded with a HIF-1α inhibitor and conjugated with an aptamer targeting a common receptor of CSCs and MSCs, may fulfill this strategy. The nanoparticle should ensure that: (1) the conveyed drug is less susceptible to degradation, (2) the common receptor of CSCs and MSCs is recognized by a superselective aptamer, and (3) receptor-mediated internalization is the main process to enter target cells. Small RNA or DNA aptamers represent an advantage over antibodies because do not cause immune reactions, are better internalized into the target cell, are more resistant to degradation, their cost of production are lower, and the purity of the oligonucleotide ligand is extremely elevated. Concerning the drugs to be delivered, we suggest to employ those exerting an anti-HIF-1α activity because they should be harmful for hypoxic CSCs and MCSs in their tumor niche but provide very limited toxicity, or even none, to well-oxygenated normal cells. Corresponding experimental approaches to perform pre-clinical studies and verify this hypothesis are also addressed.

Adipose-derived stem cell adhesion on laminin-coated microcarriers improves commitment toward the cardiomyogenic lineage.

For tissue-engineering studies of the infarcted heart it is essential to identify a source of cells that may provide cardiomyocyte progenitors, which is easy to amplify, accessible in adults, and allowing autologous grafts. Preclinical studies have shown that human adipose-derived stem cells (ADSCs) can differentiate into cardiomyocyte-like cells and improve heart function in myocardial infarction. We have developed pharmacologically active microcarriers (PAMs) which are biodegradable and biocompatible polymeric microspheres conveying cells on their biomimetic surface, therefore providing an adequate three-dimensional (3D) microenvironment. Moreover, they can release a growth factor in a prolonged manner. In order to implement ADSCs and PAMs for cardiac tissue engineering we first defined the biomimetic surface by studying the influence of matrix molecules laminin (LM) and fibronectin (FN), in combination with growth factors present in the cardiogenic niche, to further enhance the in vitro cardiac differentiation of ADSCs. We demonstrated that LM increased the expression of cardiac markers (Nkx2.5, GATA4, MEF2C) by ADSCs after 2 weeks in vitro. Interestingly, our results suggest that the 3D support provided by PAMs with a LM biomimetic surface (LM-PAMs) further enhanced the expression of cardiac markers and induced the expression of a more mature contractile protein, cardiac troponin I, compared with the 2D differentiating conditions after only 1 week in culture. The enrichment of the growth-factor cocktail with TGF-ß1 potentiated the cardiomyogenic differentiation. These results suggest that PAMs offering a LM biomimetic surface may be efficiently used for applications combining adult stem cells in tissue-engineering strategies of the ischemic heart.

Nitric oxide regulates multiple functions and fate of adult progenitor and stem cells.

Nitric oxide is an endogenous gas which exerts autocrine/paracrine actions by different signaling pathways and/or direct interactions with intracellular compounds and structures. Several processes are regulated by nitric oxide in stem cells including self-renewal, viability, migration, proliferation, and differentiation. The modulation of cell functions depends on its concentrations because opposite effects can be observed when low and high levels of nitric oxide are compared. Here, the responses to nitric oxide of adult stem/progenitor cells which are often used in regenerative medicine, including mesenchymal stem cells, hematopoietic stem cells, neural stem cells, endothelial progenitor cells, satellite cells, and fibro-adipogenic precursor cells, are reviewed. Therapeutic strategies which employ drugs releasing nitric oxide or modulating nitric oxide intracellular pathways are suggested to perform new ex vivo preconditioning or in vivo treatments suitable for stem/progenitor cell therapy and tissue engineering applications.

Hyaluronan and cardiac regeneration.

Hyaluronan (HA) is abundantly expressed in several human tissues and a variety of roles for HA has been highlighted. Particularly relevant for tissue repair, HA is actively produced during tissue injury, as widely evidenced in wound healing investigations. In the heart HA is involved in physiological functions, such as cardiac development during embryogenesis, and in pathological conditions including atherosclerosis and myocardial infarction. Moreover, owing to its relevant biological properties, HA has been widely used as a biomaterial for heart regeneration after a myocardial infarction. Indeed, HA and its derivatives are biodegradable and biocompatible, promote faster healing of injured tissues, and support cells in relevant processes including survival, proliferation, and differentiation. Injectable HA-based therapies for cardiovascular disease are gaining growing attention because of the benefits obtained in preclinical models of myocardial infarction. HA-based hydrogels, especially as a vehicle for stem cells, have been demonstrated to improve the process of cardiac repair by stimulating angiogenesis, reducing inflammation, and supporting local and grafted cells in their reparative functions. Solid-state HA-based scaffolds have been also investigated to produce constructs hosting mesenchymal stem cells or endothelial progenitor cells to be transplanted onto the infarcted surface of the heart. Finally, applying an ex-vivo mechanical stretching, stem cells grown in HA-based 3D scaffolds can further increase extracellular matrix production and proneness to differentiate into muscle phenotypes, thus suggesting a potential strategy to create a suitable engineered myocardial tissue for cardiac regeneration.

Pharmacologically active microcarriers associated with thermosensitive hydrogel as a growth factor releasing biomimetic 3D scaffold for cardiac tissue-engineering.

The challenge of tissue engineering of the infarcted heart is how to improve stem cell engraftment, survival, homing, and differentiation for myocardial repair. We here propose to integrate human adipose-derived stem cells (ADSCs) and pharmacologically active microcarriers (PAMs), a three-dimensional (3D) carrier of cells and growth factors, into an injectable hydrogel (HG), to obtain a system that stimulates the survival and/or differentiation of the grafted cells toward a cardiac phenotype. PAMs are biodegradable and non-cytotoxic poly(lactic-co-glycolic acid) (PLGA) microspheres conveying cells on their 3D surface that deliver continuously and in a controlled manner a growth factor (GF) acting on the transported cells and on the microenvironment to improve engraftment. The choice of the appropriate GF and its protection during the formulation process and delivery are essential. In this study two GFs, hepatocyte growth factor (HGF) and insulin-like growth factor (IGF-1), have been encapsulated under a solid state in order to limit their interaction with the polymer and conserve their integrity. GF precipitation conditions and release profile from PAMs have been first investigated before combining them to ADSCs. The released IGF-1 and HGF induced the protein synthesis of cardiac differentiation markers GATA4, Nkx2.5, cTnI and CX43 after 1week in vitro. Moreover, the GFs accelerated cell cycle progression, as suggested by the increased expression of Cyclin D1 mRNA and the widespread distribution of Ki67 protein. Integrating PAMs within the thermosensitive P407 hydrogel increased their elastic properties but decreased the transcription of most cardiac markers. In contrast, CX43 expression increased in ADSC-PAM-GF complexes embedded within the hydrogel compared to the ADSCs cultured alone in the absence of P407. These results suggest that particulate scaffolds releasing HGF and IGF-1 may be beneficial for applications in tissue-engineering strategies for myocardial repair and the association with a P407 hydrogel can increase substrate elasticity and junction connections in ADSCs.

Strategies affording prevascularized cell-based constructs for myocardial tissue engineering.

The production of a functional cardiac tissue to be transplanted in the injured area of the infarcted myocardium represents a challenge for regenerative medicine. Most cell-based grafts are unviable because of inadequate perfusion; therefore, prevascularization might be a suitable approach for myocardial tissue engineering. To this aim, cells with a differentiation potential towards vascular and cardiac muscle phenotypes have been cocultured in 2D or 3D appropriate scaffolds. In addition to these basic approaches, more sophisticated strategies have been followed employing mixed-cell sheets, microvascular modules, and inosculation from vascular explants. Technologies exerting spatial control of vascular cells, such as topographical surface roughening and ordered patterning, represent other ways to drive scaffold vascularization. Finally, microfluidic devices and bioreactors exerting mechanical stress have also been employed for high-throughput scaling-up production in order to accelerate muscle differentiation and speeding the endothelialization process. Future research should address issues such as how to optimize cells, biomaterials, and biochemical components to improve the vascular integration of the construct within the cardiac wall, satisfying the metabolic and functional needs of the myocardial tissue.

An innovative stand-alone bioreactor for the highly reproducible transfer of cyclic mechanical stretch to stem cells cultured in a 3D scaffold.

Much evidence in the literature demonstrates the effect of cyclic mechanical stretch in maintaining, or addressing, a muscle phenotype. Such results were obtained using several technical approaches, useful for the experimental collection of proofs of principle but probably unsuitable for application in clinical regenerative medicine. Here we aimed to design a reliable innovative bioreactor, acting as a stand-alone cell culture incubator, easy to operate and effective in addressing mesenchymal stem cells (MSCs) seeded onto a 3D bioreabsorbable scaffold, towards a muscle phenotype via the transfer of a controlled and highly-reproducible cyclic deformation. Electron microscopy, immunohistochemistry and biochemical analysis of the obtained pseudotissue constructs showed that cells 'trained' over 1 week: (a) displayed multilayer organization and invaded the 3D mesh of the scaffold; and (b) expressed typical markers of muscle cells. This effect was due only to physical stimulation of the cells, without the need of any other chemical or genetic manipulation. This device is thus proposed as a prototypal instrument to obtain pseudotissue constructs to test in cardiovascular regenerative medicine, using good manufacturing procedures.

Molecular mechanisms of ischemic preconditioning and postconditioning as putative therapeutic targets to reduce tumor survival and malignancy.

In tumors intermittent hypoxia has been reported to be more representative than normoxia or continuous exposure to low oxygen concentrations. Intermittent hypoxia is thought to increase tumor resistance against both anti-cancer therapy and the sustained ischemia that randomly occurs because of the dynamic nature of tumor vasculature. Here, we hypothesize that the molecular mechanisms underlying intermittent hypoxia in tumor cells share some triggers, modulators, and end-effectors of the intermittent episodes of ischemia and reperfusion that characterize ischemic preconditioning and postconditioning. These are among the most effective maneuvers protecting cells from ischemia-reperfusion injury. If this hypothesis were confirmed, several well-investigated molecular mediators of pre/post-conditioning could be explored as therapeutic targets against tumor malignancy. For examples, drugs that completely block the cardioprotection induced by ischemic preconditioning, such as mitochondrial potassium ATP channel inhibitors or mitochondrial permeability transition pore openers, could be extraordinarily efficient in counteracting the adaptations of tumor cells and cancer stem cells to intermittent hypoxia. As a consequence, this strategy should be effective in blunting tumor capacity to progress toward malignancy and survive in ischemic conditions.

Mechanostimulation protocols for cardiac tissue engineering.

Owing to the inability of self-replacement by a damaged myocardium, alternative strategies to heart transplantation have been explored within the last decades and cardiac tissue engineering/regenerative medicine is among the present challenges in biomedical research. Hopefully, several studies witness the constant extension of the toolbox available to engineer a fully functional, contractile, and robust cardiac tissue using different combinations of cells, template bioscaffolds, and biophysical stimuli obtained by the use of specific bioreactors. Mechanical forces influence the growth and shape of every tissue in our body generating changes in intracellular biochemistry and gene expression. That is why bioreactors play a central role in the task of regenerating a complex tissue such as the myocardium. In the last fifteen years a large number of dynamic culture devices have been developed and many results have been collected. The aim of this brief review is to resume in a single streamlined paper the state of the art in this field.

Priming adult stem cells by hypoxic pretreatments for applications in regenerative medicine.

The efficiency of regenerative medicine can be ameliorated by improving the biological performances of stem cells before their transplantation. Several ex-vivo protocols of non-damaging cell hypoxia have been demonstrated to significantly increase survival, proliferation and post-engraftment differentiation potential of stem cells. The best results for priming cultured stem cells against a following, otherwise lethal, ischemic stress have been obtained with brief intermittent episodes of hypoxia, or anoxia, and reoxygenation in accordance with the extraordinary protection afforded by the conventional maneuver of ischemic preconditioning in severely ischemic organs. These protocols of hypoxic preconditioning can be rather easily reproduced in a laboratory; however, more suitable pharmacological interventions inducing stem cell responses similar to those activated in hypoxia are considered among the most promising solutions for future applications in cell therapy. Here we want to offer an up-to-date review of the molecular mechanisms translating hypoxia into beneficial events for regenerative medicine. To this aim the involvement of epigenetic modifications, microRNAs, and oxidative stress, mainly activated by hypoxia inducible factors, will be discussed. Stem cell adaptation to their natural hypoxic microenvironments (niche) in healthy and neoplastic tissues will be also considered.

Epigenetic signature of early cardiac regulatory genes in native human adipose-derived stem cells.

Adipose-derived stem cells (ADSCs) are stromal mesenchymal stem cells isolated from lipoaspirates, and they display a broad potential to differentiate toward different lineages. The role of epigenetics in regulating the expression of their lineage-specific genes is under evaluation, however till date virtually nothing is known about the relative significance of cardiac-specific transcription factor genes in human ADSCs. The aim of this study was to investigate DNA promoter methylation and relevant histone modifications involving MEF-2C, GATA-4, and Nkx2.5 in native human ADSCs. CpG sites at the transcription start in their promoters were found unmethylated using methylation-specific PCR. Chromatin immunoprecipitation assay showed low levels of total acetylated H3 histone (acH3) and high levels of trimethylated lysine 27 in H3 histone (H3K27me3) which were associated with both GATA-4 and Nkx2.5 promoters, indicating their transcriptional repressive chromatin arrangement. On the other hand, the opposite was apparent for MEF-2C promoter. Accordingly, MEF-2C-but not GATA-4 and Nkx2.5-transcripts were evidenced in native human ADSCs. These results suggest that the chromatin arrangement of these early cardiac regulatory genes could be explored as a level of intervention to address the differentiation of human ADSCs toward the cardiac lineage.