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Reparative response by bone marrow (BM)-derived progenitor cells (PCs) to ischemia is a multistep process that comprises the detachment from the BM endosteal niche through activation of osteoclasts and proteolytic enzymes (such as matrix metalloproteinases (MMPs)), mobilization to the circulation, and homing to the injured tissue. We previously showed that intramyocardial nerve growth factor gene transfer (NGF-GT) promotes cardiac repair following myocardial infarction (MI) in mice. Here, we investigate the impact of cardiac NGF-GT on postinfarction BM-derived PCs mobilization and homing at different time points after adenovirus-mediated NGF-GT in mice. Immunohistochemistry and flow cytometry newly illustrate the temporal profile of osteoclast and activation of MMP9, PCs expansion in the BM, and liberation/homing to the injured myocardium. NGF-GT amplified these responses and increased the BM levels of active osteoclasts and MMP9, which were not observed in MMP9-deficient mice. Taken together, our results suggest a novel role for NGF in BM-derived PCs mobilization/homing following MI.

Rationale: Optimization of cell therapy for cardiac repair may require the association of different cell populations with complementary activities., Objective: Compare the reparative potential of saphenous vein-derived pericytes (SVPs) with that of cardiac stem cells (CSCs) in a model of myocardial infarction, and investigate whether combined cell transplantation provides further improvements., Methods and Results: SVPs and CSCs were isolated from vein leftovers of coronary artery bypass graft surgery and discarded atrial specimens of transplanted hearts, respectively. Single or dual cell therapy (300 000 cells of each type per heart) was tested in infarcted SCID (severe combined immunodeficiency)-Beige mice. SVPs and CSCs alone improved cardiac contractility as assessed by echocardiography at 14 days post myocardial infarction. The effect was maintained, although attenuated at 42 days. At histological level, SVPs and CSCs similarly inhibited infarct size and interstitial fibrosis, SVPs were superior in inducing angiogenesis and CSCs in promoting cardiomyocyte proliferation and recruitment of endogenous stem cells. The combination of cells additively reduced the infarct size and promoted vascular proliferation and arteriogenesis, but did not surpass single therapies with regard to contractility indexes. SVPs and CSCs secrete similar amounts of hepatocyte growth factor, vascular endothelial growth factor, fibroblast growth factor, stem cell factor, and stromal cell-derived factor-1, whereas SVPs release higher quantities of angiopoietins and microRNA-132. Coculture of the 2 cell populations results in competitive as well as enhancing paracrine activities. In particular, the release of stromal cell-derived factor-1 was synergistically augmented along with downregulation of stromal cell-derived factor-1-degrading enzyme dipeptidyl peptidase 4., Conclusions: Combinatory therapy with SVPs and CSCs may complementarily help the repair of infarcted hearts., (© 2015 American Heart Association, Inc.)

We have developed a quantitative approach to eventually enable precise and multiplexing protein analysis of very small systems, down to a single or a few cells. Through DNA-directed immobilization of DNA-protein conjugates we immobilized antibodies specific for a certain protein of interest, on a complementary DNA nanoarray fabricated by means of nanografting, a nanolithography technique based on atomic force microscopy (AFM). The proof of concept was realized for glial fibrillary acidic protein (GFAP), a biomarker crucial in cell's differentiation of astrocytes, and functional to grade classification of gliomas, the most common of primary malignant brain tumors. The efficiency of the nano-immuno sensing was tested by obtaining the immobilization of purified recombinant GFAP protein at different concentration in a standard solution then in a cellular lysate. A comparison of sensitivity between our technique and conventional ELISA assays is provided at the end of the paper., From the Clinical Editor: This team developed a quantitative approach to enable precise and multiplexing protein analysis of very small systems, down to a single or a few cells, demonstrating the utility of this DNA-based nano-immunoassay in the detection of GFAP., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Introduction: Autologous fat grafting is commonly used to correct soft-tissue contour deformities. However, results are impaired by a variable and unpredictable resorption rate. Autologous adipose-derived stromal cells in combination with lipoinjection (cell-assisted lipotransfer) seem to favor a long-term persistence of fat grafts, thus fostering the development of devices to be used in the operating room at the point of care, to isolate the stromal vascular fraction (SVF) and produce SVF-enhanced fat grafts with safe and standardized protocols. Focusing on patients undergoing breast reconstruction by lipostructure, we analyzed a standard technique, a modification of the Coleman's procedure, and three different commercially available devices (Lipokit, Cytori, Fastem), in terms of 1) ability to enrich fat grafts in stem cells and 2) clinical outcome at 6 and 12 months., Methods: To evaluate the ability to enrich stem cells, we compared, for each patient (n=20), the standard lipoaspirate with the respective stem cell-enriched one, analyzing yield, immunophenotype and colony-forming capacity of the SVF cells as well as immunophenotype, clonogenicity and multipotency of the obtained adipose stem cells (ASCs). Regarding the clinical outcome, we compared, by ultrasonography imaging, changes at 6 and 12 months in the subcutaneous thickness of patients treated with stem-cell enriched (n=14) and standard lipoaspirates (n=16)., Results: Both methods relying on the enzymatic isolation of primitive cells led to significant increase in the frequency, in the fat grafts, of SVF cells as well as of clonogenic and multipotent ASCs, while the enrichment was less prominent for the device based on the mechanical isolation of the SVF. From a clinical point of view, patients treated with SVF-enhanced fat grafts demonstrated, at six months, a significant superior gain of thickness of both the central and superior-medial quadrants with respect to patients treated with standard lipotransfer. In the median-median quadrant the effect was still persistent at 12 months, confirming an advantage of lipotransfer technique in enriching improving long-term fat grafts., Conclusions: This comparative study, based on reproducible biological and clinical parameters and endpoints, showed an advantage of lipotransfer technique in enriching fat grafts in stem cells and in favoring, clinically, long-term fat grafts.

Active cell migration and invasion is a peculiar feature of glioma that makes this tumor able to rapidly infiltrate into the surrounding brain tissue. In our recent work, we identified a novel class of glioma-associated-stem cells (defined as GASC for high-grade glioma--HG--and Gasc for low-grade glioma--LG) that, although not tumorigenic, act supporting the biological aggressiveness of glioma-initiating stem cells (defined as GSC for HG and Gsc for LG) favoring also their motility. Migrating cancer cells undergo considerable molecular and cellular changes by remodeling their cytoskeleton and cell interactions with surrounding environment. To get a better understanding about the role of the glioma-associated-stem cells in tumor progression, cell deformability and interactions between glioma-initiating stem cells and glioma-associated-stem cells were investigated. Adhesion of HG/LG-cancer cells on HG/LG-glioma-associated stem cells was studied by time-lapse microscopy, while cell deformability and cell-cell adhesion strengths were quantified by indentation measurements by atomic force microscopy and single cell force spectroscopy. Our results demonstrate that for both HG and LG glioma, cancer-initiating-stem cells are softer than glioma-associated-stem cells, in agreement with their neoplastic features. The adhesion strength of GSC on GASC appears to be significantly lower than that observed for Gsc on Gasc. Whereas, GSC spread and firmly adhere on Gasc with an adhesion strength increased as compared to that obtained on GASC. These findings highlight that the grade of glioma-associated-stem cells plays an important role in modulating cancer cell adhesion, which could affect glioma cell migration, invasion and thus cancer aggressiveness. Moreover this work provides evidence about the importance of investigating cell adhesion and elasticity for new developments in disease diagnostics and therapeutics.

Mesenchymal stem/stromal cells (MSCs) are the precursors of various cell types that compose both normal and cancer tissue microenvironments. In order to support the widely diversified parenchymal cells and tissue organization, MSCs are characterized by a large degree of heterogeneity, although available analyses of molecular and transcriptional data do not provide clear evidence. We have isolated MSCs from high-grade serous ovarian cancers (HG-SOCs) and various normal tissues (N-MSCs), demonstrated their normal genotype and analyzed their transcriptional activity with respect to the large comprehensive FANTOM5 sample dataset. Our integrative analysis conducted against the extensive panel of primary cells and tissues of the FANTOM5 project allowed us to mark the HG-SOC-MSCs CAGE-seq transcriptional heterogeneity and to identify a cell-type-specific transcriptional activity showing a significant relationship with primary mesothelial cells. Our analysis shows that MSCs isolated from different tissues are highly heterogeneous. The mesothelial-related gene signature identified in this study supports the hypothesis that HG-SOC-MSCs are bona fide representatives of the ovarian district. This finding indicates that HG-SOC-MSCs could actually derive from the coelomic mesothelium, suggesting that they might be linked to the epithelial tumor through common embryological precursors., (© 2014 AlphaMed Press.)

Cardiac stem cells (CSC) from explanted decompensated hearts (E-CSC) are, with respect to those obtained from healthy donors (D-CSC), senescent and functionally impaired. We aimed to identify alterations in signaling pathways that are associated with CSC senescence. Additionally, we investigated if pharmacological modulation of altered pathways can reduce CSC senescence in vitro and enhance their reparative ability in vivo. Measurement of secreted factors showed that E-CSC release larger amounts of proinflammatory cytokine IL1β compared with D-CSC. Using blocking antibodies, we verified that IL1β hampers the paracrine protective action of E-CSC on cardiomyocyte viability. IL1β acts intracranially inducing IKKβ signaling, a mechanism that via nuclear factor-κB upregulates the expression of IL1β itself. Moreover, E-CSC show reduced levels of AMP protein kinase (AMPK) activating phosphorylation. This latter event, together with enhanced IKKβ signaling, increases TORC1 activity, thereby impairing the autophagic flux and inhibiting the phosphorylation of Akt and cAMP response element-binding protein. The combined use of rapamycin and resveratrol enhanced AMPK, thereby restoring downstream signaling and reducing IL1β secretion. These molecular corrections reduced E-CSC senescence, re-establishing their protective activity on cardiomyocytes. Moreover ex vivo treatment with rapamycin and resveratrol improved E-CSC capacity to induce cardiac repair upon injection in the mouse infarcted heart, leading to reduced cardiomyocyte senescence and apoptosis and increased abundance of endogenous c-Kit(+) CSC in the peri-infarct area. Molecular rejuvenation of patient-derived CSC by short pharmacologic conditioning boosts their in vivo reparative abilities. This approach might prove useful for refinement of CSC-based therapies., (© 2014 AlphaMed Press.)

Aim: Circulating mesenchymal cells increase in heart failure (HF) patients and could be used therapeutically. Our aim was to investigate whether HF affects adipose tissue derived mesenchymal cell (adMSC) isolation, functional characteristics and Notch pathway., Methods and Results: We compared 25 patients with different degrees of HF (11 NYHA classes I and II and 14 NYHA III and IV) with 10 age and gender matched controls. 100% adMSC cultures were obtained from controls, while only 72.7% and 35.7% from patients with mild or severe HF (p<0.0001). adMSC from HF patients showed higher markers of senescence (p16 positive cells: 14±2.3% in controls and 35.6±5.6% (p<0.05) and 69±14.7% (p<0.01) in mild or severe HF; γ-H2AX positive cells: 3.7±1.2%, 19.4±4.1% (p<0.05) and 23.7±3.4% (p<0.05) respectively), lower proliferation index (Ki67 positive cells: 21.5±4.9%, 13.2±2.8% and 13.7±3.2%, respectively), reduced pluripotency-associated genes (Oct4 positive cells: 86.7±4.9%, 55±12% (p<0.05) and 43.3±8.7% (p<0.05), respectively; NANOG positive cells: 89.8±3.7%, 39.6±14.4% (p<0.01) and 47±8.1%, respectively), and decreased differentiation markers (α-sarcomeric actin positive cells: 79.8±4.6%, 49±18.1% and 47±12.1% (p<0.05) and CD31-positive endothelial cells: 24.5±2.9%, 0.5±0.5% (p<0.001) and 2.3±2.3% (p<0.001), respectively). AdMSC from HF patients also showed reduced Notch transcriptional activity (lowered expression of Hey 1 and Hey 2 mRNAs). Stimulation with TNF-α of adMSC isolated from controls affected the transcription of several components of the Notch pathway (reduction of Notch 4 and Hes 1 mRNAs and increase of Notch 2 and Hey 1 mRNAs)., Conclusions: In HF yield and functionality of adMSC are impaired and their Notch signaling is downregulated., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Unlabelled: Low-to-moderate levels of reactive oxygen species (ROS) govern different steps of neurogenesis via molecular pathways that have been decrypted only partially. Although it has been postulated that redox-sensitive molecules are involved in neuronal differentiation, the molecular bases for this process have not been elucidated yet. The aim of this work was therefore to study the role played by the redox-sensitive, multifunctional protein APE1/Ref-1 (APE1) in the differentiation process of human adipose tissue-derived multipotent adult stem cells (hAT-MASC) and embryonic carcinoma stem cells (EC) towards a neuronal phenotype., Methods and Results: Applying a definite protocol, hAT-MASC can adopt a neural fate. During this maturation process, differentiating cells significantly increase their intracellular Reactive Oxygen Species (ROS) levels and increase the APE1 nuclear fraction bound to chromatin. This latter event is paralleled by the increase of nuclear NF-κB, a transcription factor regulated by APE1 in a redox-dependent fashion. Importantly, the addition of the antioxidant N-acetyl cysteine (NAC) to the differentiation medium partially prevents the nuclear accumulation of APE1, increasing the neuronal differentiation of hAT-MASC. To investigate the involvement of APE1 in the differentiation process, we employed E3330, a specific inhibitor of the APE1 redox function. The addition of E3330, either to the neurogenic embryonic carcinoma cell line NT2-D1or to hAT-MASC, increases the differentiation of stem cells towards a neural phenotype, biasing the differentiation towards specific subtypes, such as dopaminergic cells. In conclusion, during the differentiation process of stem cells towards a neuroectodermic phenotype, APE1 is recruited, in a ROS-dependent manner, to the chromatin. This event is associated with an inhibitory effect of APE1 on neurogenesis that may be reversed by E3330. Therefore, E3330 may be employed both to boost neural differentiation and to bias the differentiation potential of stem cells towards specific neuronal subtypes. These findings provide a molecular basis for the redox-mediated hypothesis of neuronal differentiation program.

Purpose: Adipose-derived stem cells (ADSC) are multipotent, safe, non-immunogenic and can differentiate into functional keratocytes in situ. The topical use of ADSC derived from human processed lipoaspirate was investigated for treating injured rat cornea., Methods: A total of 19 rats were used. Six animals initially underwent corneal lesion experiments with 0.5 N NaOH (right eye) and 0.2 N (left). The 0.2 NaOH protocol was then used in 13 rats. All 26 eyes of 13 rats eyes received topical azythromycin bid for 3 d and divided into five treatment groups (n = 5 eyes/group), which included: control, stem cells, serum, stem + serum and adipose (raw human lipoaspirate). The four treatment groups received topical treatment three times daily for 3 d. Stem cells were isolated and harvested from human lipoaspirate. Topical eye drops were prepared daily with 1 × 10(5) cells/treatment. Fluorescein positive defect area and light microscope assessment was performed at 20, 28, 45, 50 and 74 h. Animals were sacrificed at 74 h for histological evaluation. Data were statistically analyzed for differences amongst groups., Results: The stem cell-treated eyes had significantly smaller epithelial defects at each time point compared to control- and adipose-treated eyes (p < 0.05). This group showed slightly better epithelium healing than the serum and combined group, yet not significantly different. Histology showed that stem cell-treated corneas had complete re-epithelization, with less inflammatory cells and limited fibroblast activation structure compared with the control eyes., Conclusions: Our preliminary results show that topical treatment with ADSC seems to improve corneal wound healing.

Rationale: The impact of diabetes mellitus on bone marrow (BM) structure is incompletely understood., Objective: Investigate the effect of type-2 diabetes mellitus (T2DM) on BM microvascular and hematopoietic cell composition in patients without vascular complications., Methods and Results: Bone samples were obtained from T2DM patients and nondiabetic controls (C) during hip replacement surgery and from T2DM patients undergoing amputation for critical limb ischemia. BM composition was assessed by histomorphometry, immunostaining, and flow cytometry. Expressional studies were performed on CD34(pos) immunosorted BM progenitor cells (PCs). Diabetes mellitus causes a reduction of hematopoietic tissue, fat deposition, and microvascular rarefaction, especially when associated with critical limb ischemia. Immunohistochemistry documented increased apoptosis and reduced abundance of CD34(pos)-PCs in diabetic groups. Likewise, flow cytometry showed scarcity of BM PCs in T2DM and T2DM+critical limb ischemia compared with C, but similar levels of mature hematopoietic cells. Activation of apoptosis in CD34(pos)-PCs was associated with upregulation and nuclear localization of the proapoptotic factor FOXO3a and induction of FOXO3a targets, p21 and p27(kip1). Moreover, microRNA-155, which regulates cell survival through inhibition of FOXO3a, was downregulated in diabetic CD34(pos)-PCs and inversely correlated with FOXO3a levels. The effect of diabetes mellitus on anatomic and molecular end points was confirmed when considering background covariates. Furthermore, exposure of healthy CD34(pos)-PCs to high glucose reproduced the transcriptional changes induced by diabetes mellitus, with this effect being reversed by forced expression of microRNA-155., Conclusions: We provide new anatomic and molecular evidence for the damaging effect of diabetes mellitus on human BM, comprising microvascular rarefaction and shortage of PCs attributable to activation of proapoptotic pathway.

Aims: Diabetes impinges upon mechanisms of cardiovascular repair. However, the biochemical adaptation of cardiac stem cells to sustained hyperglycaemia remains largely unknown. Here, we investigate the molecular targets of high glucose-induced damage in cardiac progenitor cells (CPCs) from murine and human hearts and attempt safeguarding CPC viability and function through reactivation of the pentose phosphate pathway., Methods and Results: Type-1 diabetes was induced by streptozotocin. CPC abundance was determined by flow cytometry. Proliferating CPCs were identified in situ by immunostaining for the proliferation marker Ki67. Diabetic hearts showed marked reduction in CPC abundance and proliferation when compared with controls. Moreover, Sca-1(pos) CPCs isolated from hearts of diabetic mice displayed reduced activity of key enzymes of the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD), and transketolase, increased levels of superoxide and advanced glucose end-products (AGE), and inhibition of the Akt/Pim-1/Bcl-2 signalling pathway. Similarly, culture of murine CPCs or human CD105(pos) progenitor cells in high glucose inhibits the pentose phosphate and pro-survival signalling pathways, leading to the activation of apoptosis. In vivo and in vitro supplementation with benfotiamine reactivates the pentose phosphate pathway and rescues CPC availability and function. This benefit is abrogated by either G6PD silencing by small interfering RNA (siRNA) or Akt inhibition by dominant-negative Akt., Conclusion: We provide new evidence of the negative impact of diabetes and high glucose on mechanisms controlling CPC redox state and survival. Boosting the pentose phosphate pathway might represent a novel mechanistic target for protection of CPC integrity.

Cellular senescence processes affecting tissue resident stem cells are considered, at present, an hallmark of both aging and age-related pathologies. Therefore it is mandatory to address this problem with adequate techniques that could highlight the molecular alterations associated with this complex cellular response to stressors. Here we describe methods to characterize cardiac stem cell (CSC) senescence from a molecular and functional standpoint.

Stem cells are characterized by the ability to renew themselves and to differentiate into specialized cell types, while stem cell therapy is believed to treat a number of different human diseases through either cell regeneration or paracrine effects. Herein, an in vivo and ex vivo near infrared time domain (NIR TD) optical imaging study was undertaken to evaluate the migratory ability of murine adipose tissue-derived multipotent adult stem cells [mAT-MASC] after intramuscular injection in mice. In vivo NIR TD optical imaging data analysis showed a migration of DiD-labelled mAT-MASC in the leg opposite the injection site, which was confirmed by a fibered confocal microendoscopy system. Ex vivo NIR TD optical imaging results showed a systemic distribution of labelled cells. Considering a potential microenvironmental contamination, a cross-validation study by multimodality approaches was followed: mAT-MASC were isolated from male mice expressing constitutively eGFP, which was detectable using techniques of immunofluorescence and qPCR. Y-chromosome positive cells, injected into wild-type female recipients, were detected by FISH. Cross-validation confirmed the data obtained by in vivo/ex vivo TD optical imaging analysis. In summary, our data demonstrates the usefulness of NIR TD optical imaging in tracking delivered cells, giving insights into the migratory properties of the injected cells.

Periosteum contains mesenchymal stem cells (Pe-MSCs) that contribute to normal bone growth, healing, and turnover; understanding Pe-MSC capabilities may shed light over the treatment of bone defects using tissue engineering. Bone tissue regeneration needs in vitro bone precursors or stem cell coculture onto specific scaffolds but, despite extensive research in the field, very little is known about the matrix structure of the tissue-engineered tissues and the scaffold's effects on cell differentiation. To this purpose we have selected a clonal population (murine Pe-MSCs) that was seeded and differentiated onto an acellular bone scaffold. Cell differentiation was assessed after 3 months and 1 year by molecular, histological, biochemical, and biophysical analyses and results were compared with the same osteoinduced clonal cells cultured as cellular aggregates. Our data show that Pe-MSCs cultured onto acellular bone scaffold develop a complex three-dimensional matrix and an osteoblastic phenotype but do not produce hydroxyapatite (HA); moreover, they seem able to reabsorb the colonized bone scaffold. On the contrary, cells cultured as three-dimensional aggregates differentiate and produce osteoblastic markers and HA nanocrystals.

The term "cellular senescence" denotes a cellular response to several stressors that results in irreversible growth arrest, alterations of the gene expression profile, epigenetic modifications, and an altered secretome, all of which eventually impair the reparative properties of primitive cells, adding a layer of complexity to the field of regenerative medicine. The purpose of this review is to illustrate how cellular senescence could affect tissue repair and to propose interventions that aim at interfering with it.

Although the role played by the core transcription factor network, which includes c-Myc, Klf4, Nanog, and Oct4, in the maintenance of embryonic stem cell (ES) pluripotency and in the reprogramming of adult cells is well established, its persistence and function in adult stem cells are still debated. To verify its persistence and clarify the role played by these molecules in adult stem cell function, we investigated the expression pattern of embryonic and adult stem cell markers in undifferentiated and fully differentiated dental pulp stem cells (DPSC). A particular attention was devoted to the expression pattern and intracellular localization of the stemness-associated isoform A of Oct4 (Oct4A). Our data demonstrate that: Oct4, Nanog, Klf4 and c-Myc are expressed in adult stem cells and, with the exception of c-Myc, they are significantly down-regulated following differentiation. Cell differentiation was also associated with a significant reduction in the fraction of DPSC expressing the stem cell markers CD10, CD29 and CD117. Moreover, a nuclear to cytoplasm shuttling of Oct4A was identified in differentiated cells, which was associated with Oct4A phosphorylation. The present study would highlight the importance of the post-translational modifications in DPSC stemness maintenance, by which stem cells balance self-renewal versus differentiation. Understanding and controlling these mechanisms may be of great importance for stemness maintenance and stem cells clinical use, as well as for cancer research.

Adult stem cells have been proposed as an alternative to embryonic stem cells to study multilineage differentiation in vitro and to use in therapy. Current culture media for isolation and expansion of adult stem cells require the use of large amounts of animal sera, but animal-derived culture reagents give rise to some questions due to the real possibility of infections and severe immune reactions. For these reasons a clinical grade substitute to animal sera is needed. We tested the isolation, proliferation, morphology, stemness related marker expression, and osteoblastic differentiation potential of Dental Pulp Stem Cells (DPSC) in a chemically defined medium containing a low percentage of human serum, 1.25%, in comparison to a medium containing 10% Fetal Bovine Serum (FBS). DPSCs cultured in presence of our isolation/proliferation medium added with low HS percentage were obtained without immune-selection methods and showed high uniformity in the expression of stem cell markers, proliferated at higher rate, and demonstrated comparable osteoblastic potential with respect to DPSCs cultured in 10% FBS. In this study we demonstrated that a chemically defined medium added with low HS percentage, derived from autologous and heterologous sources, could be a valid substitute to FBS-containing media and should be helpful for adult stem cells clinical application.

Rationale: Pericytes are key regulators of vascular maturation, but their value for cardiac repair remains unknown., Objective: We investigated the therapeutic activity and mechanistic targets of saphenous vein-derived pericyte progenitor cells (SVPs) in a mouse myocardial infarction (MI) model., Methods and Results: SVPs have a low immunogenic profile and are resistant to hypoxia/starvation (H/S). Transplantation of SVPs into the peri-infarct zone of immunodeficient CD1/Foxn-1(nu/nu) or immunocompetent CD1 mice attenuated left ventricular dilatation and improved ejection fraction compared to vehicle. Moreover, SVPs reduced myocardial scar, cardiomyocyte apoptosis and interstitial fibrosis, improved myocardial blood flow and neovascularization, and attenuated vascular permeability. SVPs secrete vascular endothelial growth factor A, angiopoietin-1, and chemokines and induce an endogenous angiocrine response by the host, through recruitment of vascular endothelial growth factor B expressing monocytes. The association of donor- and recipient-derived stimuli activates the proangiogenic and prosurvival Akt/eNOS/Bcl-2 signaling pathway. Moreover, microRNA-132 (miR-132) was constitutively expressed and secreted by SVPs and remarkably upregulated, together with its transcriptional activator cyclic AMP response element-binding protein, on stimulation by H/S or vascular endothelial growth factor B. We next investigated if SVP-secreted miR-132 acts as a paracrine activator of cardiac healing. In vitro studies showed that SVP conditioned medium stimulates endothelial tube formation and reduces myofibroblast differentiation, through inhibition of Ras-GTPase activating protein and methyl-CpG-binding protein 2, which are validated miR-132 targets. Furthermore, miR-132 inhibition by antimiR-132 decreased SVP capacity to improve contractility, reparative angiogenesis, and interstitial fibrosis in infarcted hearts., Conclusion: SVP transplantation produces long-term improvement of cardiac function through a novel paracrine mechanism involving the secretion of miR-132 and inhibition of its target genes.

We have investigated the blood levels of sub-classes of stem cells (SCs) [mesenchymal stem cells (MSCs), haematopoietic stem cells (HSCs), endothelial progenitor cells/circulating endothelial cells (EPCs/CECs) and tissue-committed stem cells (TCSCs)] in heart failure (HF) patients at different stage of pathology and correlated it with plasmatic levels of proangiogenic cytokines. Peripheral blood level of SCs were analysed in 97 HF patients (24 in NYHA class I, 41 in class II, 17 in class III and 15 in class IV) and in 23 healthy controls. Plasmatic levels of PDGF-BB, bFGF, HGF, vascular endothelial growth factor (VEGF), SDF-1α, TNF-α and NTproBNP were also measured. Compared with healthy individuals, MSC, and in particular the sub-classes CD45(-) CD34(-) CD90(+) , CD45(-) CD34(-) CD105(+) and CD45(-) CD34(-) CXCR4(+) were significantly enhanced in NYHA class IV patients (16.8-, 6.4- and 2.7-fold, respectively). Level of CD45(-) CD34(-) CD90(+) CXCR4(+) cells progressively increased from class II to class IV (fold increases compared with controls: 8.5, 12 and 21.5, respectively). A significant involvement of CXCR4(+) subpopulation of HSC (CD45(+) CD34(+) CD90(+) CXCR4(+) , 1.4 versus 13.3 cells/μl in controls and NYHA class III patients, respectively) and TCSC (CD45(-) CD34(+) CXCR4(+) , 1.5 cells/ μl in controls versus 12.4 and 28.6 cells/μl in NYHA classes II and IV, respectively) were also observed. All tested cytokines were enhanced in HF patients. In particular, for PDGF-BB and SDF-1α we studied specific ligand/receptors pairs. Interestingly, the first one positively correlated with TCSCs expressing PDGFR (r = 0.52, P = 0.001), whereas the second one correlated with TCSCs (r = 0.34, P = 0.005) and with MSCs CD90(+) expressing CXCR4 (r = 0.39, P = 0.001). HF is characterized by the increase in the circulating levels of different MSC, HSC, EPC and TCSC subsets. Both the entity and kinetic of this process varied in distinct cell subsets. Specifically, differently from HSCs and EPCs/CECs, MSCs and TCSCs significantly increased with the progression of the disease, suggesting a possible distinct role of these cells in the pathophysiology of HF., (© 2011 The Authors Journal compilation © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Currently, it is unknown whether defects in stem cell growth and differentiation contribute to myocardial aging and chronic heart failure (CHF), and whether a compartment of functional human cardiac stem cells (hCSCs) persists in the decompensated heart. To determine whether aging and CHF are critical determinants of the loss in growth reserve of the heart, the properties of hCSCs were evaluated in 18 control and 23 explanted hearts. Age and CHF showed a progressive decrease in functionally competent hCSCs. Chronological age was a major predictor of five biomarkers of hCSC senescence: telomeric shortening, attenuated telomerase activity, telomere dysfunction-induced foci, and p21(Cip1) and p16(INK4a) expression. CHF had similar consequences for hCSCs, suggesting that defects in the balance between cardiomyocyte mass and the pool of nonsenescent hCSCs may condition the evolution of the decompensated myopathy. A correlation was found previously between telomere length in circulating bone marrow cells and cardiovascular diseases, but that analysis was restricted to average telomere length in a cell population, neglecting the fact that telomere attrition does not occur uniformly in all cells. The present study provides the first demonstration that dysfunctional telomeres in hCSCs are biomarkers of aging and heart failure. The biomarkers of cellular senescence identified here can be used to define the birth date of hCSCs and to sort young cells with potential therapeutic efficacy., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Rationale: Studies in transgenic mice showed the key role of (Pim-1) (proviral integration site for Moloney murine leukemia virus-1) in the control of cardiomyocyte function and viability., Objective: We investigated whether Pim-1 represents a novel mechanistic target for the cure of diabetic cardiomyopathy, a steadily increasing cause of nonischemic heart failure., Methods and Results: In streptozotocin-induced type 1 diabetic mice, Pim-1 protein levels declined during progression of cardiomyopathy, along with upregulation of Pim-1 inhibitors, protein phosphatase 2A, and microRNA-1. Moreover, diabetic hearts showed low levels of antiapoptotic B-cell lymphoma-2 (Bcl-2) protein and increased proapoptotic caspase-3 activity. Studies on adult rat cardiomyocytes and murine cardiac progenitor cells challenged with high glucose confirmed the in vivo expressional changes. In rescue studies, anti-microRNA-1 boosted Pim-1 and Bcl-2 expression and promoted cardiomyocyte and cardiac progenitor cell survival under high glucose conditions. Similarly, transfection with Pim-1 plasmid prevented high glucose-induced cardiomyocyte and cardiac progenitor cell apoptosis. Finally, a single intravenous injection of human PIM-1 via cardiotropic serotype-9 adeno-associated virus (1 × 10(10) or 5 × 10(10) plaque-forming units per animal) at 4 weeks after diabetes induction led to sustained cardiac overexpression of Pim-1 and improved diastolic function and prevented left ventricular dilation and failure. Histological examination showed reduced cardiomyocyte apoptosis and fibrosis in association with increased c-kit(+) cells and cardiomyocyte proliferation, whereas molecular analysis confirmed activation of the prosurvival pathway and conservation of sarcoendoplasmic reticulum Ca(2+)-ATPase and α-myosin heavy chain in Pim-1-treated hearts., Conclusions: Pim-1 downregulation contributes in the pathogenesis of diabetic cardiomyopathy. Systemic delivery of human PIM-1 via cardiotropic adeno-associated virus serotype-9 represents a novel and effective approach to treat diabetic cardiomyopathy.

Tooth morphogenesis requires sequential and reciprocal interactions between the cranial neural crest-derived mesenchymal cells and the stomadial epithelium, which regulate tooth morphogenesis and differentiation. We show how mesenchyme-derived single stem cell populations can be induced to transdifferentiate in vitro in a structure similar to a dental bud. The presence of stem cells in the adipose tissue has been previously reported. We incubated primary cultures of human adipose tissue-derived stem cells in a dental-inducing medium and cultured the aggregates in three-dimensional conditions. Four weeks later, cells formed a three-dimensional organized structure similar to a dental bud. Expression of dental tissue-related markers was tested assaying lineage-specific mRNA and proteins by RT-PCR, immunoblot, IHC, and physical-chemical analysis. In the induction medium, cells were positive for ameloblastic and odontoblastic markers as both mRNAs and proteins. Also, cells expressed epithelial, mesenchymal, and basement membrane markers with a positional relationship similar to the physiologic dental morphogenesis. Physical-chemical analysis revealed 200-nm and 50-nm oriented hydroxyapatite crystals as displayed in vivo by enamel and dentin, respectively. In conclusion, we show that adipose tissue-derived stem cells in vitro can transdifferentiate to produce a specific three-dimensional organization and phenotype resembling a dental bud even in the absence of structural matrix or scaffold to guide the developmental process., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Tumor associated fibroblasts (TAFs) are considered a microenvironmental element critical for tumor growth and progression. Experimental studies suggest that their origin could be from mesenchymal stem cells (MSCs) derived from the bone marrow. However, the role played by TAFs in cirrhosis, hepatocellular carcinoma development, and progression is largely unknown, and in vitro human models are missing. This paper for the first time demonstrates that (1) human neoplastic livers possess a population of multipotent adult stem cells (MASCs) with properties of TAFs; (2) a population of MASC-derived TAFs is already present in cirrhotic, not yet neoplastic, livers; (3) MASCs isolated from nonneoplastic and noncirrhotic liver scan acquire a TAF phenotype when grown in a medium conditioned by tumor cell lines, supporting the notion that TAF could originate from resident primitive cells (MASCs), possibly through a paracrine mechanism.

Cellular senescence is a specialized form of growth arrest, confined to mitotic cells, induced by various stressful stimuli and characterized by a permanent growth arrest, resistance to apoptosis, an altered pattern of gene expression and the expression of some markers that are characteristic, although not exclusive, to the senescent state. Senescent cells profoundly modify neighboring and remote cells through the production of an altered secretome, eventually leading to inflammation, fibrosis and possibly growth of neoplastic cells. Mammalian aging has been defined as a reduction in the capacity to adequately maintain tissue homeostasis or to repair tissues after injury. Tissue homeostasis and regenerative capacity are nowadays considered to be related to the stem cell pool present in every tissue. For this reason, pathological and patho-physiological conditions characterized by altered tissue homeostasis and impaired regenerative capacity can be viewed as a consequence of the reduction in stem cell number and/or function. Last, cellular senescence is a double-edged sword, since it may inhibit the growth of transformed cells, preventing the occurrence of cancer, while it may facilitate growth of preneoplastic lesions in a paracrine fashion; therefore, interventions targeting this cell response to stress may have a profound impact on many age-related pathologies, ranging from cardiovascular disease to oncology. Aim of this review is to discuss both molecular mechanisms associated with stem cell senescence and interventions that may attenuate or reverse this process., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Grafts of tissue-engineered bone represent a promising alternative in the treatment of large and small bone defects. Current approaches are often badly tolerated by patients because of invasiveness, ethical problems, culture, and possibility of infection. Autologous grafts have been indicated as a solution to such problems. Because of tissue availability, many have proposed the use of cultured cells derived from bone marrow expanded in culture and induced to differentiate in bone tissue. Data reported in the literature show that it is possible to produce tissue substitutes in vitro indeed, but results are not always concordant regarding the in vitro produced bone quality. In the present work, we investigated bone formation in aggregates of human bone marrow-derived mesenchymal stem cells induced to differentiate in bone. After osteoinduction we characterized the mineral matrix produced using Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, and X-ray powder diffraction. Cells were obtained from bone marrow, subjected to immunodepletion for CD3, CD11b, CD14, CD16, CD19, CD56, CD66b, and glycophorin A using RosetteSep and cultured in a new formulation of medium for four passages and then were allowed to form spontaneous aggregates. At the end of proliferation before aggregation, cells were analyzed by fluorescent activated cell sorting (FACS) for markers routinely used to characterize expanded mesenchymal stem cells and were found to be remarkably homogeneous for CD29 (99% ± 1%), CD73 (99% ± 1%), CD90 (95% ± 4%), CD105 (96% ± 4%), and CD133 (0% ± 1%) expression. Our results show that not only aggregated cells express the major markers of osteogenic differentiation, such as osteocalcin, osteonectin, osteopontin, and bone sialoprotein, but also the inorganic matrix is made of an apatite structurally and morphologically similar to native bone even without a scaffold.

Rationale: The turnover of cardiomyocytes in the aging female and male heart is currently unknown, emphasizing the need to define human myocardial biology., Objective: The effects of age and gender on the magnitude of myocyte regeneration and the origin of newly formed cardiomyocytes were determined., Methods and Results: The interaction of myocyte replacement, cellular senescence, growth inhibition, and apoptosis was measured in normal female (n=32) and male (n=42) human hearts collected from patients 19 to 104 years of age who died from causes other than cardiovascular diseases. A progressive loss of telomeric DNA in human cardiac stem cells (hCSCs) occurs with aging and the newly formed cardiomyocytes inherit short telomeres and rapidly reach the senescent phenotype. Our data provide novel information on the superior ability of the female heart to sustain the multiple variables associated with the development of the senescent myopathy. At all ages, the female heart is equipped with a larger pool of functionally competent hCSCs and younger myocytes than the male myocardium. The replicative potential is higher and telomeres are longer in female hCSCs than in male hCSCs. In the female heart, myocyte turnover occurs at a rate of 10%, 14%, and 40% per year at 20, 60, and 100 years of age, respectively. Corresponding values in the male heart are 7%, 12%, and 32% per year, documenting that cardiomyogenesis involves a large and progressively increasing number of parenchymal cells with aging. From 20 to 100 years of age, the myocyte compartment is replaced 15 times in women and 11 times in men., Conclusions: The human heart is a highly dynamic organ regulated by a pool of resident hCSCs that modulate cardiac homeostasis and condition organ aging.

Background: This study was designed to evaluate the biological significance of simultaneous changes in the circulating levels of osteoprotegerin (OPG) and TNF-related apoptosis inducing ligand (TRAIL) in patients with coronary artery disease (CAD), and, in particular, with acute myocardial infarction (AMI)., Methods: Total levels of OPG and TRAIL were measured by ELISA in patients with AMI (n=113), unstable angina (UA, n=21) and healthy controls (n=120)., Results: Since OPG was elevated during the acute phase (first 12-24-48h) after AMI and in patients with UA with respect to healthy controls, while TRAIL was decreased in acute AMI patients, CAD patients were characterized by an increased OPG/TRAIL ratio. Moreover, the OPG/TRAIL ratio was significantly (p<0.05) higher in the acute AMI patients who developed heart failure (HF) than in those who did not develop HF in the follow-up., Conclusions: An impaired OPG/TRAIL ratio after AMI is related to a higher risk of HF., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

Background: Clinical trials in ischemic patients showed the safety and benefit of autologous bone marrow progenitor cell transplantation. Non-bone marrow progenitor cells with proangiogenic capacities have been described, yet they remain clinically unexploited owing to their scarcity, difficulty of access, and low ex vivo expansibility. We investigated the presence, antigenic profile, expansion capacity, and proangiogenic potential of progenitor cells from the saphenous vein of patients undergoing coronary artery bypass surgery., Methods and Results: CD34-positive cells, negative for the endothelial marker von Willebrand factor, were localized around adventitial vasa vasorum. After dissection of the vein from surrounding tissues and enzymatic digestion, CD34-positive/CD31-negative cells were isolated by selective culture, immunomagnetic beads, or fluorescence-assisted cell sorting. In the presence of serum, CD34-positive/CD31-negative cells gave rise to a highly proliferative population that expressed pericyte/mesenchymal antigens together with the stem cell marker Sox2 and showed clonogenic and multilineage differentiation capacities. We called this population "saphenous vein-derived progenitor cells" (SVPs). In culture, SVPs integrated into networks formed by endothelial cells and supported angiogenesis through paracrine mechanisms. Reciprocally, endothelial cell-released factors facilitated SVP migration. These interactive responses were inhibited by Tie-2 or platelet-derived growth factor-BB blockade. Intramuscular injection of SVPs in ischemic limbs of immunodeficient mice improved neovascularization and blood flow recovery. At 14 days after transplantation, proliferating SVPs were still detectable in the recipient muscles, where they established N-cadherin-mediated physical contact with the capillary endothelium., Conclusions: SVPs generated from human vein CD34-positive/CD31-negative progenitor cells might represent a new therapeutic tool for angiogenic therapy in ischemic patients.

The number of circulating mesenchymal stem cells (MSC), analyzed after acute myocardial infarction (AMI), was lower in AMI patients who developed heart failure (HF) in the follow-up. Conversely, the circulating levels of tumor necrosis factor (TNF)-alpha, and osteoprotegerin (OPG) were higher in AMI patients who developed HF with respect to the patients who did not develop HF. In vitro exposure to TNF-alpha enhanced the migration of MSC in response to TNF-related apoptosis-inducing ligand (TRAIL) and significantly increased the release of OPG by endothelial cells. On the contrary, OPG dose-dependently neutralized the in vitro pro-migratory activity of TRAIL. Thus, TNF-alpha exhibits opposite effects on MSC migration driven by TRAIL: it is capable of potentiating MSC migration as well as of inhibiting MSC migration as an indirect consequence of OPG induction, which might result in a suboptimal recruitment of circulating MSC after AMI in those patients who develop HF in the follow-up.

In the last few years, pluripotent stem cells have been the objective of intense investigation efforts. These cells are of paramount therapeutic interest, since they could be utilized: as in vitro models of disease, for pharmaceutical screening purposes, and for the regeneration of damaged organs. Over the years, pluripotent cells have been cultured from teratomas, the inner cell mass, and primordial germ cells. Accumulating informations have partially decrypted the molecular machinery responsible for the maintenance of a very primitive state, permitting the reprogramming of differentiated cells. Although the debate is still open, an extreme excitement is arising from two strictly related possibilities: pluripotent cells could be obtained from adult tissues with minimal manipulations or very rare pluripotent cells could be identified in adult tissues. This intriguing option will trigger new researches aimed both at identifying the possible biological role of pluripotent adult stem cells and at exploiting their potential clinical use. The present review article will summarize current knowledge of the molecular cues for pluripotency but also discusses whether pluripotent stem cells could be obtained from adult tissues.

To determine whether the peripheral blood in humans contains a population of multipotent progenitor cells (MPCs), products of leukapheresis were obtained from healthy donor volunteers following the administration of granulocyte colony-stimulating factor. Small clusters of adherent proliferating cells were collected, and these cells continued to divide up to 40 population doublings without reaching replicative senescence and growth arrest. MPCs were positive for the transcription factors Nanog, Oct3/4, Sox2, c-Myc, and Klf4 and expressed several antigens characteristic of mesenchymal stem cells. However, they were negative for markers of hematopoietic stem/progenitor cells and bone marrow cell lineages. MPCs had a cloning efficiency of approximately 3%, and following their expansion, retained a highly immature phenotype. Under permissive culture conditions, MPCs differentiated into neurons, glial cells, hepatocytes, cardiomyocytes, endothelial cells, and osteoblasts. Moreover, the gene expression profile of MPCs partially overlapped with that of neural and embryonic stem cells, further demonstrating their primitive, uncommitted phenotype. Following subcutaneous transplantation in nonimmunosuppressed mice, MPCs migrated to distant organs and integrated structurally and functionally within the new tissue, acquiring the identity of resident parenchymal cells. In conclusion, undifferentiated cells with properties of embryonic stem cells can be isolated and expanded from human peripheral blood after granulocyte colony-stimulating factor administration. This cell pool may constitute a unique source of autologous cells with critical clinical import.

A significant fraction of the interneurons added in adulthood to the glomerular layer (GL) of the olfactory bulb (OB) are dopaminergic (DA). In the OB, DA neurons are restricted to the GL, but using transgenic mice expressing eGFP under the tyrosine hydroxylase (TH) promoter, we also detected the presence of TH-GFP+ cells in the mitral and external plexiform layers. We hypothesized that these could be adult-generated neurons committed to become DA but not yet entirely differentiated. Accordingly, TH-GFP+ cells outside the GL exhibit functional properties (appearance of pacemaker currents, synaptic connection with the olfactory nerve, intracellular chloride concentration, and other) marking a gradient of maturity toward the dopaminergic phenotype along the mitral-glomerular axis. Finally, we propose that the establishment of a synaptic contact with the olfactory nerve is the key event allowing these cells to complete their differentiation toward the DA phenotype and to reach their final destination.

The best therapy for hepatocellular carcinoma (HCC) is still debated. Hepatic resection (HR) is the treatment of choice for single HCC in Child A patients, whereas liver transplantation (LT) is usually reserved for Child B and C patients with single or multiple nodules. The aim of this study was to compare HR and LT for HCC within the Milan criteria on an intention-to-treat basis. Forty-eight patients were treated by LT and 38 by HR. The median time on the waiting list for transplantation was 118 days. The estimated overall survival was significantly higher (P = 0.005) in the LT group than in the HR one. The estimated freedom from recurrence was also significantly higher (P < 0.0001) for LT patients than for HR ones. Indeed, the probability of HCC recurrence after resection was higher than after transplantation achieving 31% and 76% for HR and 2% and 2% for LT at 3 and 5 years after surgery. Multivariate analysis confirmed that transplantation was superior to resection in terms of patient's survival and risk of HCC recurrence. We conclude that LT is superior to HR for small HCC in cirrhotic patients assuming that LT should be performed within 6-10 months after listing to reduce the dropouts for reasons of tumor progression.

The aims of our study were to verify whether it was possible to generate in vitro, from different adult human tissues, a population of cells that behaved, in culture, as multipotent stem cells and if these latter shared common properties. To this purpose, we grew and cloned finite cell lines obtained from adult human liver, heart, and bone marrow and named them human multipotent adult stem cells (hMASCs). Cloned hMASCs, obtained from the 3 different tissues, expressed the pluripotent state-specific transcription factors Oct-4, NANOG, and REX1, displayed telomerase activity, and exhibited a wide range of differentiation potential, as shown both at a morphologic and functional level. hMASCs maintained a human diploid DNA content, and shared a common gene expression signature, compared with several somatic cell lines and irrespectively of the tissue of isolation. In particular, the pathways regulating stem cell self-renewal/maintenance, such as Wnt, Hedgehog, and Notch, were transcriptionally active. Our findings demonstrate that we have optimized an in vitro protocol to generate and expand cells from multiple organs that could be induced to acquire morphologic and functional features of mature cells even embryologically not related to the tissue of origin.

The identification of cardiac progenitor cells in mammals raises the possibility that the human heart contains a population of stem cells capable of generating cardiomyocytes and coronary vessels. The characterization of human cardiac stem cells (hCSCs) would have important clinical implications for the management of the failing heart. We have established the conditions for the isolation and expansion of c-kit-positive hCSCs from small samples of myocardium. Additionally, we have tested whether these cells have the ability to form functionally competent human myocardium after infarction in immunocompromised animals. Here, we report the identification in vitro of a class of human c-kit-positive cardiac cells that possess the fundamental properties of stem cells: they are self-renewing, clonogenic, and multipotent. hCSCs differentiate predominantly into cardiomyocytes and, to a lesser extent, into smooth muscle cells and endothelial cells. When locally injected in the infarcted myocardium of immunodeficient mice and immunosuppressed rats, hCSCs generate a chimeric heart, which contains human myocardium composed of myocytes, coronary resistance arterioles, and capillaries. The human myocardium is structurally and functionally integrated with the rodent myocardium and contributes to the performance of the infarcted heart. Differentiated human cardiac cells possess only one set of human sex chromosomes excluding cell fusion. The lack of cell fusion was confirmed by the Cre-lox strategy. Thus, hCSCs can be isolated and expanded in vitro for subsequent autologous regeneration of dead myocardium in patients affected by heart failure of ischemic and nonischemic origin.

New strategies are urgently needed for the treatment of terminal heart-failure. Recently, new therapeutic strategies were developed to induce cardiac regeneration and thereby enhancing myocardial performance by using the patients own cell pool. In the quest for adequate donor cells skeletal myoblasts, bone marrow (BM) derived cells, endothelial progenitor cells (EPCs) and resident cardiac stem cells but also cytokine application were examined. This review critically discusses the recent findings in the field of endogenous adult progenitor cells and proposes new areas for future research.

In this study, we tested whether the human heart possesses a cardiac stem cell (CSC) pool that promotes regeneration after infarction. For this purpose, CSC growth and senescence were measured in 20 hearts with acute infarcts, 20 hearts with end-stage postinfarction cardiomyopathy, and 12 control hearts. CSC number increased markedly in acute and, to a lesser extent, in chronic infarcts. CSC growth correlated with the increase in telomerase-competent dividing CSCs from 1.5% in controls to 28% in acute infarcts and 14% in chronic infarcts. The CSC mitotic index increased 29-fold in acute and 14-fold in chronic infarcts. CSCs committed to the myocyte, smooth muscle, and endothelial cell lineages increased approximately 85-fold in acute infarcts and approximately 25-fold in chronic infarcts. However, p16(INK4a)-p53-positive senescent CSCs also increased and were 10%, 18%, and 40% in controls, acute infarcts, and chronic infarcts, respectively. Old CSCs had short telomeres and apoptosis involved 0.3%, 3.8%, and 9.6% of CSCs in controls, acute infarcts, and chronic infarcts, respectively. These variables reduced the number of functionally competent CSCs from approximately 26,000/cm3 of viable myocardium in acute to approximately 7,000/cm3 in chronic infarcts, respectively. In seven acute infarcts, foci of spontaneous myocardial regeneration that did not involve cell fusion were identified. In conclusion, the human heart possesses a CSC compartment, and CSC activation occurs in response to ischemic injury. The loss of functionally competent CSCs in chronic ischemic cardiomyopathy may underlie the progressive functional deterioration and the onset of terminal failure.

A concept of impossibility of appearance of novel cardiomyocytes in the heart of adult men in exchange for those lost due to cardiovascular diseases had dominated medicine and biology for many long decades. However ability of human myocardium to regenerate was demonstrated during recent years in multiple studies. This dictated necessity to reconsider previously generally accepted concept. At present researchers and practicing physicians actively discuss possibility of the use of transplantation of bone marrow stem cells, proper cardiac stem cells, skeletal muscle myoblasts or precursors of endothelial cells in patients with myocardial infarction and heart failure in order to restore normal cardiac structure and function. Another potential method of restoration of the myocardium in patients with cardiovascular diseases is the use of cytokines which stimulate migration of stem cells into myocardium and their differentiation into cardiomyocytes.

The notion of the adult heart as terminally differentiated organ without self-renewal potential has been undermined by the existence of a subpopulation of replicating myocytes in normal and pathological states. The origin and significance of these cells has remained obscure for lack of a proper biological context. We report the existence of Lin(-) c-kit(POS) cells with the properties of cardiac stem cells. They are self-renewing, clonogenic, and multipotent, giving rise to myocytes, smooth muscle, and endothelial cells. When injected into an ischemic heart, these cells or their clonal progeny reconstitute well-differentiated myocardium, formed by blood-carrying new vessels and myocytes with the characteristics of young cells, encompassing approximately 70% of the ventricle. Thus, the adult heart, like the brain, is mainly composed of terminally differentiated cells, but is not a terminally differentiated organ because it contains stem cells supporting its regeneration. The existence of these cells opens new opportunities for myocardial repair.

The effects of using different algorithms to estimate the time constant of changes in oxygen uptake at the onset of square-wave 120 W cycloergometric exercise were evaluated in seven subjects. The volume of oxygen taken up at the alveoli (VO(2Ai)) was determined breath-by-breath (BB) from the volume of O(2) transferred at the mouth (VO(2mi)) minus the corresponding volume changes in O(2) stores in the alveoli: VO(2Ai)= VO(2mi)-[V(Ai-1)(FO(2Ai)- FO(2Ai-1))+ FO(2Ai) x Delta V(Ai)], where V(Ai-1) is the alveolar volume at the end of the previous breath, FO(2Ai) and FO(2Ai-1) are estimated from the fractions of end-tidal O(2) in the current and previous breaths, respectively, and Delta V(Ai) is the change in volume during breath i. These quantities can be measured BB, with the exception of V(Ai-1) which must be assumed. The respiratory cycle has been defined as the time elapsing between identical fractions of expiratory gas in two successive breaths. Using this approach, since FO(2Ai)= FO(2Ai-1), any assumption regarding V(Ai-1) becomes unnecessary. In the present study, VO(2Ai) was calculated firstly, by using this approach, and secondly by setting different V(Ai-1) values (from 0 to FRC+0.5 l, where FRC is the functional residual capacity). Values for alveolar O(2) flow (VO(2Ai)), as calculated from the quotient of VO(2Ai) divided by breath duration, were then fitted bi-exponentially. The time constant of the phase II kinetics of VO(2Ai) (tau(2)) was linearly related to V(Ai-1), increasing from 36.6 s (V(Ai-1)=0) to 46.8 s (V(Ai-1)=FRC+0.5 l) while tau(2) estimated using the first approach amounted to 34.3 s. We concluded that, firstly, the first approach allowed us to calculate O(2A) during transitions in step exercise; and secondly, when using methods wherein V(Ai-1) must be assumed, tau(2) depended on V(Ai-1).

Cell death has been questioned as a mechanism of ventricular failure. In this report, we tested the hypothesis that apoptotic death of myocytes, endothelial cells, and fibroblasts is implicated in the development of the dilated myopathy induced by ventricular pacing. Accumulation of reactive oxygen products such as nitrotyrosine, potentiation of the oxidative stress response by p66(shc) expression, formation of p53 fragments, release of cytochrome c, and caspase activation were examined to establish whether these events were coupled with apoptotic cell death in the paced dog heart. Myocyte, endothelial cell, and fibroblast apoptosis was detected before indices of severe impairment of cardiac function became apparent. Cell death increased with the duration of pacing, and myocyte death exceeded endothelial cell and fibroblast death throughout. Nitrotyrosine formation and p66(shc) levels progressively increased with pacing and were associated with cell apoptosis. Similarly, p50 (DeltaN) fragments augmented paralleling the degree of cell death in the failing heart. Moreover, cytochrome c release and activation of caspase-9 and -3 increased from 1 to 4 weeks of pacing. In conclusion, cardiac cell death precedes ventricular decompensation and correlates with the time-dependent deterioration of function in this model. Oxidative stress may be critical for activation of apoptosis in the overloaded heart.