Therapeutic angiogenesis by transplantation of human embryonic stem cell-derived CD133+ endothelial progenitor cells for cardiac repair.

OBJECTIVE: This study aim to enhance endothelial differentiation of human embryonic stem cells (hESCs) by transduction of an adenovirus (Ad) vector expressing hVEGF(165) gene (Ad-hVEGF(165) ). Purified hESC-derived CD133(+) endothelial progenitors were transplanted into a rat myocardial infarct model to assess their ability to contribute to heart regeneration. METHODS: Optimal transduction efficiency with high cell viability was achieved by exposing differentiating hESCs to viral particles at a ratio of 1:500 for 4 h on three consecutive days. RESULTS: Reverse transcription-PCR analysis showed positive upregulation of VEGF, Ang-1, Flt-1, Tie-2, CD34, CD31, CD133 and Flk-1 gene expression in Ad-hVEGF(165) -transduced cells. Additionally, flow cytometric analysis of CD133, a cell surface marker, revealed an approximately fivefold increase of CD133 marker expression in Ad-hVEGF(165)-transduced cells compared with the nontransduced control. Within a rat myocardial infarct model, transplanted CD133(+) endothelial progenitor cells survived and participated, both actively and passively, in the regeneration of the infarcted myocardium, as seen by an approximately threefold increase in mature blood vessel density (13.62 +/- 1.56 vs 5.11 +/- 1.23; p < 0.01), as well as significantly reduced infarct size (28% +/- 8.2% vs 76% +/- 5.6%; p < 0.01) in the transplanted group compared with the culture medium-injected control. There was significant improvement in heart function 6 weeks post-transplantation, as confirmed by regional blood-flow analysis (1.72 +/- 0.612 ml/min/g vs 0.8 +/- 0.256 ml/min/g; p < 0.05), as well as echocardiography assessment of left ventricular ejection fraction (60.855% +/- 7.7% vs 38.22 +/- 8.6%; p < 0.05) and fractional shortening (38.63% +/- 9.3% vs 25.2% +/- 7.11%; p < 0.05). CONCLUSION: hESC-derived CD133(+) endothelial progenitor cells can be utilized to regenerate the infarcted heart.

Stable therapeutic effects of mesenchymal stem cell-based multiple gene delivery for cardiac repair.

AIMS: We have previously shown that transplantation of mesenchymal stem cells (MSCs) co-overexpressing angiopoietin-1 (Ang-1) and Akt prevented cell apoptosis, enhanced angiogenesis, and improved left ventricular heart function. The present study was designed to determine the persistence of therapeutic benefits on longer term basis. METHODS AND RESULTS: Acute myocardial infarction model was developed in 30 young female Fischer-344 rats by permanent ligation of the left anterior descending coronary artery. The animals were grouped (n = 10) to receive 70 microL Dulbecco's modified Eagle's medium (DMEM) without cells (DMEM group 1) or containing 3 x 10(6) non-transduced male MSCs (MSC group 2) or transduced MSCs co-overexpressing Ang-1 and Akt (MAA group 3). The injections were carried out intramyocardially in the free wall of left ventricle at multiple sites. Three months after cell transplantation, real-time polymerase chain reaction for the rat sry gene, confocal imaging, and immunohistochemical studies revealed the extensive survival and myogenic differentiation of the PKH67-labelled cell graft. Blood vessel density was significantly higher in the MAA group (P < 0.05) at 3 months compared with the other groups. Blood vessel maturation index as determined by double-fluorescent immunostaining for vWFactor VIII and smooth muscle actin showed that most of the newly formed vessels matured to develop a smooth muscle covering in MAA group. Sonographic assessment of heart function showed that heart function deteriorated in the DMEM group, whereas the functional benefits were stable over a period of 3 months following transplantation of transfected cells. CONCLUSION: Engraftment of genetically modified MSCs co-overexpressing Ang-1 and Akt produced long-term histological and functional benefits in an infarcted heart.

Directing endothelial differentiation of human embryonic stem cells via transduction with an adenoviral vector expressing the VEGF(165) gene.

Endothelial progenitors derived from human embryonic stem cells (hESCs) hold much promise in clinical therapy. Conventionally, lineage-specific differentiation of hESCs is achieved through supplementation of various cytokines and chemical factors within the culture milieu. Nevertheless, this is a highly inefficient approach that is often limited by poor replicability. An alternative is through genetic modulation with recombinant DNA. Hence, this study investigated whether transduction of hESCs with an adenoviral vector expressing the human VEGF(165) gene (Ad-hVEGF(165)) can enhance endothelial-lineage differentiation. The hESCs were induced to form embryoid bodies (EBs) by culturing them within low-attachment plates for 7 days, and were subsequently trypsinized into single cells, prior to transduction with Ad-hVEGF(165). Optimal transduction efficiency with high cell viability was achieved by 4-h exposure of the differentiating hESCs to viral particles at a ratio of 1 : 500 for three consecutive days. ELISA results showed that Ad-hVEGF(165)-transduced cells secreted human vascular endothelial growth factor (hVEGF) for more than 30 days post-transduction, peaking on day 8, and the conditioned medium from the transduced cells stimulated extensive proliferation of HUVEC. Real-time PCR analysis showed positive upregulation of VEGF, Ang-1, Flt-1, Tie-2, CD34, CD31, CD133 and Flk-1 gene expression in Ad-hVEGF(165)-transduced cells. Additionally, flow cytometric analysis of CD133 cell surface marker revealed an approximately 5-fold increase in CD133 marker expression in Ad-hVEGF(165)-transduced cells compared to the non-transduced control. Hence, this study demonstrated that transduction of differentiating hESCs with Ad-hVEGF(165) facilitated expression of the VEGF transgene, which in turn significantly enhanced endothelial differentiation of hESCs.

Possible advantages of stem cell transfusion into the peripheral circulation, as opposed to localized transplantation in situ.

Studies to date have overlooked the possible limitations of transplanting stem cells locally (in situ), directly at the site of tissue or organ damage. This approach is contrary to the intrinsic physiological process of tissue and organ regeneration in vivo, which is thought to involve the activation of stem cells resident within the transplanted tissues or their mobilization from ectopic sites, in particular the bone marrow. Signaling pathways and other molecular processes within stem cells transplanted in situ may not be primed to achieve optimal tissue regeneration and may even be confused by the sudden rapid transition in the cellular microenvironment encountered during transplantation. Moreover, there is a risk of the transplanted cells giving rise to undesired lineages at the transplantation site. A novel alternative would be to transfuse stem cells into the peripheral blood circulation, followed by induced homing to the site of tissue damage. This could better replicate the natural physiological process of tissue repair in vivo. Transfusion into the peripheral blood circulation could be a strategy to augment the inadequate mobilization of endogenous adult stem cells from ectopic sites for tissue repair, which may be the case for older patients. The transfused stem cells can then be induced to home in on a damaged tissue or organ, via the controlled release of specific cytokines or chemokines (i.e., stromal cell derived factor-1) emanating from that particular tissue or organ. The gradient of released cytokines/ chemokines within the peripheral blood circulation would then direct the chemotactic migration and homing of the transfused stem cells to the target tissue or organ.

Combining transfusion of stem/progenitor cells into the peripheral circulation with localized transplantation in situ at the site of tissue/organ damage: a possible strategy to optimize the efficacy of stem cell transplantation therapy.

Several studies have demonstrated the efficacy of localized in situ transplantation of stem/progenitor cells for tissue/organ regeneration. However, the possible limitations of such an approach have largely been overlooked. This is contrary to the intrinsic physiological process of tissue/organ regeneration in vivo, which is thought to involve the mobilization of stem/progenitor cells resident within the tissue/organ itself, as well as from ectopic sites, in particular the bone marrow. Signaling pathways and other molecular processes within stem/progenitor cells transplanted in situ may not be primed to achieve optimal tissue/organ regeneration, and may even be confused by the sudden rapid transition in the cellular microenvironment encountered during transplantation. To overcome these putative limitations, a possible strategy may be to combine transfusion of stem/progenitor cells into the peripheral circulation with localized transplantation in situ at the site of tissue/organ damage. This could better replicate the natural physiological process of tissue/organ repair in vivo. Possible synergistic interactions between the transplanted stem/progenitor cells in situ with migratory transfused cells from the peripheral circulation may further enhance tissue/organ regeneration. The transfused stem/progenitor cells may be induced to home in on a damaged tissue/organ, via the controlled release of specific cytokines or chemokines (i.e., SDF-1) emanating from that particular tissue/organ. There are a number of possible ways to achieve this. For example, the transplanted cells may be delivered on tissue-engineered scaffolds that are designed for the controlled release of specific homing factors such as SDF-1. Another alternative may be to stimulate or genetically modulate the transplanted cells to copiously secrete homing factors such as SDF-1, to encourage the migration and homing of transfused cells within the peripheral circulation. At the same time, it may also be advantageous to pre-stimulate the transfused cells to strongly express surface receptors specific to homing factors such as SDF-1, in particular CXCR-4. More rigorous investigations should be carried out on the possible strategy of combining in situ transplantation of stem/progenitor cells with transfusion into the peripheral circulation, together with induced homing of the transfused cells to the site of organ/tissue damage. This may possibly result in better efficacy for some, but not all models of tissue/organ regeneration.

Mechanisms by which K(ATP) channel openers produce acute and delayed cardioprotection.

Mitochondria are being increasingly studied for their critical role in cell survival. Multiple diverse signaling pathways have been shown to converge on the K+-sensitive ATP channels as the effectors of cytoprotection against necrosis and apoptosis. The role of potassium channel openers in regulation and transformation of cell membrane excitability, action potential and electrolyte transfer has been extensively studied. Cardiac mitoK(ATP) channels are the key effectors in cardioprotection during ischemic preconditioning, as yet with an undefined mechanism. They have been hypothesized to couple myocardial metabolism with membrane electrical activity and provide an excellent target for drug therapy. A number of K(ATP) channel openers have been characterized for their beneficial effects on the myocardium against ischemic injury. This review updates recent progress in understanding the physiological role of K(ATP) channels in cardiac protection induced by preconditioning and highlights relevant questions and controversies in the light of published data.

Reprogramming autologous skeletal myoblasts to express cardiomyogenic function. Challenges and possible approaches.

Cell transplantation therapy is emerging as a promising mode of treatment following myocardial infarction. Of the various cell types that can potentially be used for transplantation, autologous skeletal myoblasts appear particularly attractive, because this would avoid issues of immunogenicity, tumorigenesis, ethics and donor availability. Additionally, skeletal myoblasts display much higher levels of ischemic tolerance and graft survival compared to other cell types. There is some evidence for improvement in heart function with skeletal myoblast transplantation. However, histological analysis revealed that transplanted myoblasts do not transdifferentiate into functional cardiomyocytes in situ. This is evident by the lack of expression of cardiac-specific antigens, and the absence of intercalated disc formation. Instead, there is differentiation into myotubes that are not electromechanically coupled to neighboring cardiomyocytes. This could in turn limit the clinical efficacy of treatment. This review would therefore examine the various challenges faced in attempting to reprogram autologous skeletal myoblast to express cardiomyogenic function, together with the various possible strategies that could be employed to achieve this objective.

Comments about possible use of human embryonic stem cell-derived cardiomyocytes to direct autologous adult stem cells into the cardiomyogenic lineage.

Several studies have shown that cell-transplantation therapy following myocardial infarction has some efficacy in aiding myocardial repair and subsequent recovery of heart function. Large-scale production of human embryonic stem cell-derived cardiomyocytes can potentially provide an abundant supply of donor cells for myocardial transplantation. There are, however, immunological barriers to their use in human clinical therapy.A novel approach would be to look at utilizing human embryonic stem cell-derived cardiomyocytes to reprogram autologous adult stem cells to express cardiomyogenic function, instead of using these directly for transplantation. This could be achieved through a number of novel techniques. Enucleated cytoplasts generated from human embryonic stem cell-derived cardiomyocytes could be fused with autologous adult stem cells to generate cytoplasmic hybrids or cybrids. Adult stem cells could also be temporarily permeabilized and exposed to cytoplasmic extracts derived from these cardiomyocytes. Alternatively, intact cells or enucleated cytoplasts from human embryonic stem cell-derived cardiomyocytes could be co-cultured with adult stem cells in vitro, to provide the cellular contacts and electrical coupling that might enable some degree of trans-differentiation to take place. This review would therefore examine the potential advantages and disadvantages of such a novel approach, in comparison to other more conventional techniques such as the use of exogenous cytokines/growth factors or the use of genetic modulation.

An overview and synopsis of techniques for directing stem cell differentiation in vitro.

The majority of studies on stem cell differentiation have so far been based in vivo, on live animal models. The usefulness of such models is limited, since it is much more technically challenging to conduct molecular studies and genetic manipulation on live animal models compared to in vitro cell culture. Hence, it is imperative that efficient protocols for directing stem cell differentiation into well-defined lineages in vitro are developed. The development of such protocols would also be useful for clinical therapy, since it is likely that the transplantation of differentiated stem cells would result in higher engraftment efficiency and enhanced clinical efficacy, compared to the transplantation of undifferentiated stem cells. The in vitro differentiation of stem cells, prior to transplantation in vivo, would also avoid spontaneous differentiation into undesired lineages at the transplantation site, as well as reduce the risk of teratoma formation, in the case of embryonic stem cells. Hence, this review critically examines the various strategies that could be employed to direct and control stem cell differentiation in vitro.