Cardiac myocytes undergo apoptosis under condition of ischemia. Little is known, however, about the molecular pathways that mediate this response. We show that serum deprivation and hypoxia, components of ischemia in vivo, resulted in apoptosis of rat ventricular myoblast cells H9c2. Hypoxia alone did not induce signi¢cant apoptosis for at least 48 h, but largely increased the proapoptotic action of serum deprivation. H9c2 cells apoptosis is evidenced by an increase in terminal (TdT)-mediated dUTP nick end-labeling-positive nuclei and by activation of caspases 3, 6, 7 and 9, and loss of mitochondrial functions. In this model of simulated ischemia, represented by serum deprivation plus hypoxia, cardiomyoblasts apoptosis was associated with a p53-independent Bax accumulation and with a down-regulation of Bcl-xL, whereas the levels of cIAP-1, cIAP-2 and X-IAP proteins did not change. Phorbol-12-myristate-13-acetate signi¢cantly reduced the induction of apoptosis, inhibiting caspase 3 cleavage, Bax accumulation, Bcl-xL downregulation as well as restoring cell viability. ß

SummaryThe fabrication of biodegradable 3-D scaffolds enriched with multipotent stem cells seems to be a promising strategy for the repair of irreversibly injured tissues. The fine mechanisms of the interaction of rat mesenchymal stem cells (rMSCs) with a hyaluronan-based scaffold, i.e. HYAFF®11, were investigated to evaluate the potential clinical application of this kind of engineered construct. rMSCs were seeded (2 × 10 6 cells cm -2 ) on the scaffold, cultured up to 21 days and analysed using appropriate techniques. Light (LM), scanning (SEM) and transmission (TEM) electron microscopy of untreated scaffold samples showed that scaffolds have a highly porous structure and are composed of 15-μ m-thick microfibres having a rough surface. As detected by trypan blue stain, cell adhesion was high at day 1. rMSCs were viable up to 14 days as shown by CFDA assay and proliferated steadily on the scaffold as revealed by MTT assay. LM showed rMSCs in the innermost portions of the scaffold at day 3. SEM revealed a subconfluent cell monolayer covering 40 ± 10% of the scaffold surface at day 21. TEM of early culture showed rMSCs wrapping individual fibres with regularly spaced focal contacts, whereas confocal microscopy showed polarized expression of CD44 hyaluronan receptor; TEM of 14-day cultures evidenced fibronexus formation. Immunohistochemistry of 21-day cultures showed that fibronectin was the main matrix protein secreted in the extracellular space; decorin and versican were seen in the cell cytoplasm only and type IV collagen was minimally expressed. The expression of CD90, a marker of mesenchymal stemness, was found unaffected at the end of cell culture. Our results show that HYAFF®11 scaffolds support the adhesion, migration and proliferation of rMSCs, as well as the synthesis and delivery of extracellular matrix components under static culture conditions without any chemical induction. The high retention rate and viability of the seeded cells as well as their fine modality of interaction with the substrate suggest that such scaffolds could be potentially useful when wide tissue defects are to be repaired as in the case of cartilage repair, wound healing and large vessel replacement.

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.

In order to assess, in a controlled in vitro model, the differentiation potential of adult bone marrow derived stem cells we have developed a coculture procedure using adult rat cardiomyocytes and mesenchymal stem cells (MSCs) from transgenic GFP positive rats. We investigated in the cocultured MSCs the time course of cellular processes that are difficult to monitor in in vivo experiments. Adult rat cardiomyocytes and adult rat MSCs were cocultured for up to 7 days and analyzed by confocal microscopy. Several markers were studied by immunofluorescence technique. The fluorescent ST-BODIPY-Dihydropyridine was used to label calcium channels in living cells. Intracellular calcium was monitored with the fluorescent probe X-Rhod-1. Immunofluorescence experiments showed the presence of connexin-43 between cardiomyocytes and MSCs and between MSCs, while no sarcomeric structures were observed at any time of the coculture. We looked at the expression of calcium channels and development of voltage-dependent calcium signaling in cocultured MSCs. MSCs showed a time-dependent increase of labeling of ST-BODIPY-Dihydropyridine, reaching a relatively strong level after 72 h of coculture. The treatment with a non-fluorescent DHP, Nifedipine, completely abolished ST-BODIPY labeling. We investigated whether depolarization could modulate intracellular calcium. Depolarization-induced calcium transients increased in MSCs in relation to the coculture time. We conclude that MSCs cocultured with adult cardiomyocytes present preliminary evidence of voltage-dependent calcium modulation uncoupled with the development of nascent or adult myofibrils, thus showing a limited lineage specification and a low plasticity to differentiate in a full cardiomyocyte-like phenotype.

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-014-0100-4) contains supplementary material, which is available to authorized users.

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.

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.

The aim of this study was to compare different cell sources and culture conditions to obtain endothelial progenitor cells (EPCs) with predictable antigen pattern, proliferation potential and in vitro vasculogenesis. Pig mononuclear cells were isolated from blood (PBMCs) and bone marrow (BMMCs). Mesenchymal stem cells (MSCs) were also derived from pig bone marrow. Cells were cultured on fibronectin in the presence of a high concentration of VEGF and low IGF-1 and FGF-2 levels, or on gelatin with a lower amount of VEGF and higher IGF-1 and FGF-2 concentrations. Endothelial commitment was relieved in almost all PBMCs and BMMCs irrespective of the protocol used, whilst MSCs did not express a reliable pattern of EPC markers under these conditions. BMMCs were more prone to expand on gelatin and showed a better viability than PBMCs. Moreover, about 90% of the BMMCs pre-cultured on gelatin could adhere to a hyaluronan-based scaffold and proliferate on it up to 3 days. Pre-treatment of BMMCs on fibronectin generated well-shaped tubular structures on Matrigel, whilst BMMCs exposed to the gelatin culture condition were less prone to form vessel-like structures. MSCs formed rough tubule-like structures, irrespective of the differentiating condition used. In a relative short time, pig BMMCs could be expanded on gelatin better than PBMCs, in the presence of a low amount of VEGF. BMMCs could better specialize for capillary formation in the presence of fibronectin and an elevated concentration of VEGF, whilst pig MSCs anyway showed a limited capability to differentiate into the endothelial cell lineage.