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Cell therapy has developed as a complementary treatment for myocardial regeneration. While both autologous and allogeneic uses have been advocated, the ideal candidate has not been identified yet. Amniotic fluid-derived stem (AFS) cells are potentially a promising resource for cell therapy and tissue engineering of myocardial injuries. However, no information is available regarding their use in an allogeneic context. c-kit-sorted, GFP-positive rat AFS (GFP-rAFS) cells and neonatal rat cardiomyocytes (rCMs) were characterized by cytocentrifugation and flow cytometry for the expression of mesenchymal, embryonic and cell lineage-specific antigens. The activation of the myocardial gene program in GFP-rAFS cells was induced by co-culture with rCMs. The stem cell differentiation was evaluated using immunofluorescence, RT-PCR and single cell electrophysiology. The in vivo potential of Endorem-labeled GFP-rAFS cells for myocardial repair was studied by transplantation in the heart of animals with ischemia/reperfusion injury (I/R), monitored by magnetic resonance imaging (MRI). Three weeks after injection a small number of GFP-rAFS cells acquired an endothelial or smooth muscle phenotype and to a lesser extent CMs. Despite the low GFP-rAFS cells count in the heart, there was still an improvement of ejection fraction as measured by MRI. rAFS cells have the in vitro propensity to acquire a cardiomyogenic phenotype and to preserve cardiac function, even if their potential may be limited by poor survival in an allogeneic setting.

In the last 10 years an increasing interest has been devoted to the study of endothelial progenitor cells (EPCs), a subtype of immature cells involved in endothelial repair and neoangiogenesis. EPCs have been discovered as a novel integrated part of the cardiovascular system, which plays a comprehensive role in tissue homeostasis. Consistently, alterations and/or reduction of the circulating EPC pool have been associated with different manifestations of cardiovascular disorders and atherosclerosis. This is why, the extent of the EPC pool is now considered a mirror of vascular health, while EPC reduction has become a surrogate biomarker of cardiovascular risk and of the ongoing vascular damage. Unfortunately, the methods used to study EPCs still lack standardization, and this is significantly decelerating progress in the field. In this review, we focus on some aspects related to the two methods used to assess circulating EPCs: flow cytometry and cell culture. We uncover the many traps hidden in the choice of the right protocol, and suggest the best solutions on the basis of evidence and background theories.

Background: The enhancement of circulating endothelial progenitor cells (EPCs) obtained by exercise training can be beneficial to patients with cardiac disease. Changes in the levels and differentiation of CD34(pos)/KDR(pos) EPCs, as well as the plasma concentration of vascular endothelial growth factor (VEGF) and stromal cell-derived factor (SDF)-1 EPC-mobilizing cytokines, were evaluated in patients with chronic heart failure after 8 weeks of supervised aerobic training (SAT) and 8 weeks of subsequent discontinued SAT (DSAT)., Methods and Results: The levels of circulating EPC and EPC differentiation potential of 22 patients who underwent SAT were studied by fluorescence-activated cell sorter analysis and colony forming-unit assay, respectively. The plasma levels of VEGF and SDF-1 were measured by enzyme-linked immunosorbent assay. In response to SAT, the levels of both EPC and VEGF/SDF-1 markedly increased (P < .001 vs baseline) but returned to the baseline levels after DSAT. A similar change was observed with the EPC clonogenic potential, but on DSAT the baseline level was incompletely attained., Conclusions: In response to SAT, patients with chronic heart failure show enhanced EPC levels and clonogenic potential that is mirrored by increased plasma VEGF and SDF-1 levels. DSAT can interfere with the maintenance of training-acquired VEGF/SDF-1-related EPC levels and clonogenic potential.

Atherosclerotic diseases are responsible for a significant part of morbidity and mortality in western countries. According to the classical views, atherosclerotic lesions develop as the result of an inflammatory process initiated by endothelial damage. The discovery that bone marrow-derived cells participate in endothelial repair and new vessel growth has changed the pathogenetic models of cardiovascular disease. These cells, termed endothelial progenitor cells (EPCs), represent the endogenous endothelial regenerative capacity and the ability to form new collateral vessels. In this review we describe how quantitative and qualitative alterations of EPCs have a significant role in virtually all stages of the atherosclerotic process and in the clinical manifestations of the diseases: starting from the impact of risk factors on EPCs, through the mechanisms that link EPC reduction/dysfunction to plaque formation, and finally to the clinical syndromes. An attempt to diverge our attention from the vessel wall to the bloodstream reveals a central role of EPCs in atherogenesis.

Human amniotic fluid-derived stem (AFS) cells, similarly to embryonic stem cells, could possess privileged immunological characteristics suitable for a successful transplantation even in a discordant xenograft system. We investigated whether AFS cells could be fruitfully used in a rat model of myocardial infarction. c-kit immunomagnetic-sorted AFS cells were characterized by flow cytometric analysis and cytospins as well as reverse-transcription polymerase chain reaction, Western blotting and immunocytochemistry for cardiovascular differentiation markers. In vitro, AFS cell phenotypic conversion was assayed by cardiovascular-specific induction media or co-cultured with rat neonatal cardiomyocytes. AFS cells showed mRNAs and/or protein for endothelial (angiopoietin, CD146) and smooth muscle (smoothelin) cells, and cardiomyocyte (Nkx2.5, MLC-2v, GATA-4, beta-MyHC) markers. Acquisition of a cardiomyocyte-like phenotype in rare AFS cells could be seen only in co-cultures with rat neonatal cells. In vivo, AFS cells xenotransplantated in a rat model of myocardial infarction, with or without cyclosporine treatment, or in intact heart from immuno-competent or immuno-deficient animals were acutely rejected due to the different recruitment of recipient CD4(+), CD8(+) T and B lymphocytes, NK cells and macrophages. This reaction is most likely to be linked to expression of B7 co-stimulatory molecules CD80 and CD86 as well as macrophage marker CD68 on AFS cells. Xenotransplanted AFS cells gave also rise in some animals to cell masses in the subendocardium and myocardium suggestive of a process of chondro-osteogenic differentiation. Despite AFS cells in vitro can differentiate to some extent to cells of cardiovascular lineages, their in vivo use in xenotransplantation for cell therapy of myocardial infarction is hampered by their peculiar immunogenic properties and phenotypic instability.

Objective: Peripheral arterial disease (PAD) is a threatening complication of diabetes. As endothelial progenitor cells (EPCs) are involved in neovasculogenesis and maintenance of vascular homeostasis, their impairment may have a role in the pathogenesis of diabetic vasculopathy. This study aimed to establish whether number and function of EPCs correlate with PAD severity in type 2 diabetic patients., Methods and Results: EPCs were defined by the expression of CD34, CD133 and KDR, and quantified by flow cytometry in 127 diabetic patients with and without PAD. PAD severity has been assessed as carotid atherosclerosis and clinical stage of leg atherosclerosis obliterans. Diabetic patients with PAD displayed a significant 53% reduction in circulating EPCs versus non-PAD patients, and EPC levels were negatively correlated with the degree of carotid stenosis and the stage of leg claudication. Moreover, the clonogenic and adhesion capacity of cultured EPCs were significantly lower in diabetic patients with PAD versus patients without., Conclusions: This study demonstrates that EPC decrease is related to PAD severity and that EPC function is altered in diabetic subjects with PAD, strengthening the pathogenetic role of EPC dysregulation in diabetic vasculopathy. EPC count may be considered a novel biological marker of peripheral atherosclerosis in diabetes.

Purpose: Wound healing of the cryo-injured bladder can bring about organ remodeling because of incomplete reconstitution of depleted smooth muscle cells. Stem cell transplantation could be beneficial to improve smooth muscle cell regeneration and/or modulate the remodeling process. The repair of bladder injury using adult-type stem cells would be useful for adult urological patients but unsuited for neonatal patients, in whom major benefits are likely to derive from fetal-type stem cells. Materials and Methods: The smooth muscle cell differentiation potential of fetal-type vs adult-type stem cells was evaluated by injecting green fluorescent protein labeled mesenchymal stem cells from rat amniotic fluid or bone marrow, respectively, in cryo-injured rat bladder walls. Results: At 30 days after transplantation only a few fetal-type or adult-type mesenchymal stem cells gave rise to enteric or vascular smooth muscle cells, whereas most mesenchymal stem cells appeared incapable of specific differentiation. In vitro co-culture experiments of smooth muscle cells with fetal-type or adult-type mesenchymal stem cells selectively labeled with distinct fluorochromes showed the presence of hybrid cells, suggesting that some mesenchymal stem cells can undergo cell fusion. Surprisingly the major effect of rat bone marrow or amniotic fluid mesenchymal stem cell transplantation seemed to be preventing cryo-injury induced hypertrophy of surviving smooth muscle cells. Conclusions: In this model stem cell transplantation has a limited effect on smooth muscle cell regeneration. Instead it can regulate post-injury bladder remodeling, possibly via a paracrine mechanism. [Copyright &y& Elsevier]