Lee-Lee Ong

Bcl‐2 Engineered MSCs Inhibited Apoptosis and Improved Heart Function

Engraftment of mesenchymal stem cells (MSCs) derived from adult bone marrow has been proposed as a potential therapeutic approach for postinfarction left ventricular dysfunction. However, limited cell viability after transplantation into the myocardium has restricted its regenerative capacity. In this study, we genetically modified MSCs with an antiapoptotic Bcl‐2 gene and evaluated cell survival, engraftment, revascularization, and functional improvement in a rat left anterior descending ligation model via intracardiac injection. Rat MSCs were manipulated to overexpress the Bcl‐2 gene. In vitro, the antiapoptotic and paracrine effects were assessed under hypoxic conditions. In vivo, the Bcl‐2 gene‐modified MSCs (Bcl‐2‐MSCs) were injected after myocardial infarction. The surviving cells were tracked after transplantation. Capillary density was quantified after 3 weeks. The left ventricular function was evaluated by pressure‐volume loops. The Bcl‐2 gene protected MSCs against apoptosis. In vitro, Bcl‐2 overexpression reduced MSC apoptosis by 32% and enhanced vascular endothelial growth factor secretion by more than 60% under hypoxic conditions. Transplantation with Bcl‐2‐MSCs increased 2.2‐fold, 1.9‐fold, and 1.2‐fold of the cellular survival at 4 days, 3 weeks, and 6 weeks, respectively, compared with the vector‐MSC group. Capillary density in the infarct border zone was 15% higher in Bcl‐2‐MSC transplanted animals than in vector‐MSC treated animals. Furthermore, Bcl‐2‐MSC transplanted animals had 17% smaller infarct size than vector‐MSC treated animals and exhibited functional recovery remarkably. Our current findings support the premise that transplantation of antiapoptotic gene‐modified MSCs may have values for mediating substantial functional recovery after acute myocardial infarction.

Is the intravascular administration of mesenchymal stem cells safe?: Mesenchymal stem cells and intravital microscopy

We investigated the kinetics of human mesenchymal stem cells (MSCs) after intravascular administration into SCID mouse cremaster vasculature by intravital microscopy. MSCs were injected into abdominal aorta through left femoral artery at two different concentrations (1 x 10(6) or 0.2 x 10(6) cell). Arterial blood velocity decrease by 60 and 18% 1 min after high/low dose MSCs injection respectively. The blood microcirculation was interrupted after 174+/-71 and 485+/-81 s. Intravital microscopy observation and histopathologic analysis of cremaster muscles indicated MSCs were entrapped in capillaries in both groups. 40 and 25% animals died of pulmonary embolism respectively in both high and low MSCs dose groups, which was detected by histopathologic analysis of the lungs. Intraarterial MSCs administration may lead to occlusion in the distal vasculature due to their relatively large cell size. Pulmonary sequestration may cause death in small laboratory animals. MSCs should be used cautiously for intravascular transplantation.

Cell Origin of Human Mesenchymal Stem Cells Determines a Different Healing Performance in Cardiac Regeneration

The possible different therapeutic efficacy of human mesenchymal stem cells (hMSC) derived from umbilical cord blood (CB), adipose tissue (AT) or bone marrow (BM) for the treatment of myocardial infarction (MI) remains unexplored. This study was to assess the regenerative potential of hMSC from different origins and to evaluate the role of CD105 in cardiac regeneration. Male SCID mice underwent LAD-ligation and received the respective cell type (400.000/per animal) intramyocardially. Six weeks post infarction, cardiac catheterization showed significant preservation of left ventricular functions in BM and CD105+-CB treated groups compared to CB and nontreated MI group (MI-C). Cell survival analyzed by quantitative real time PCR for human GAPDH and capillary density measured by immunostaining showed consistent results. Furthermore, cardiac remodeling can be significantly attenuated by BM-hMSC compared to MI-C. Under hypoxic conditions in vitro, remarkably increased extracellular acidification and apoptosis has been detected from CB-hMSC compared to BM and CD105 purified CB-derived hMSC. Our findings suggests that hMSC originating from different sources showed a different healing performance in cardiac regeneration and CD105+ hMSC exhibited a favorable survival pattern in infarcted hearts, which translates into a more robust preservation of cardiac function.

Intracardiac injection of erythropoietin induces stem cell recruitment and improves cardiac functions in a rat myocardial infarction model

Erythropoietin (EPO) protects the myocardium from ischaemic injury and promotes beneficial remodelling. We assessed the therapeutic efficacy of intracardiac EPO injection and EPO‐mediated stem cell homing in a rat myocardial infarction (MI) model. Following MI, EPO (3000 U/kg) or saline was delivered by intracardiac injection. Compared to myocardial infarction control group (MIC), EPO significantly improved left ventricular function (n= 11–14, P< 0.05) and decreased right ventricular wall stress (n= 8, P< 0.05) assessed by pressure‐volume loops after 6 weeks. MI‐EPO hearts exhibited smaller infarction size (20.1 ± 1.1%versus 27.8 ± 1.2%; n= 6–8, P< 0.001) and greater capillary density (338.5 ± 14.7 versus 259.8 ± 9.2 vessels per mm; n= 6–8, P< 0.001) than MIC hearts. Direct EPO injection reduced post‐MI myocardial apoptosis by approximately 41% (0.27 ± 0.03%versus 0.42 ± 0.03%; n= 6, P= 0.005). The chemoattractant SDF‐1 was up‐regulated significantly assessed by quantitative realtime PCR and immunohistology. c‐Kit+ and CD34+ stem cells were significantly more numerous in MI‐EPO than in MIC at 24 hrs in peripheral blood (n= 7, P< 0.05) and 48 hrs in the infarcted hearts (n= 6, P< 0.001). Further, the mRNAs of Akt, eNOS and EPO receptor were significantly enhanced in MI‐EPO hearts (n= 7, P< 0.05). Intracardiac EPO injection restores myocardial functions following MI, which may attribute to the improved early recruitment of c‐Kit+ and CD34+ stem cells via the enhanced expression of chemoattractant SDF‐1.

Enhanced thoracic gene delivery by magnetic nanobead-mediated vector

Systemic gene delivery is limited by the adverse hydrodynamic conditions on the collection of gene carrier particles to the specific area. In the present study, a magnetic field was employed to guide magnetic nanobead (MNB)/polymer/DNA complexes after systemic administration to the left side of the mouse thorax in order to induce localized gene expression.

Endothelial NOS is required for SDF-1α/CXCR4-mediated peripheral endothelial adhesion of c-kit+ bone marrow stem cells

In the era of intravascular approaches for regenerative cell therapy, the underlying mechanisms of stem cell migration to non-marrow tissue have not been clarified. We hypothesized that next to a local inflammatory response implying adhesion molecule expression, endothelial nitric oxide synthase (eNOS)-dependent signaling is required for stromal- cell-derived factor-1 alpha (SDF-1α)-induced adhesion of c-kit+ cells to the vascular endothelium. SDF-1α/tumor necrosis factor-alpha (TNF-α)-induced c-kit+-cell shape change and migration capacity was studied in vitro using immunohistochemistry and Boyden chamber assays. In vivo interaction of c-kit+ cells from bone marrow with the endothelium in response to SDF-1α/TNF-α stimulation was visualized in the cremaster muscle microcirculation of wild-type (WT) and eNOS (−/−) mice using intravital fluorescence microscopy. In addition, NOS activity was inhibited with N-nitro-L-arginine-methylester-hydrochloride in WT mice. To reveal c-kit+-specific adhesion behavior, endogenous leukocytes (EL) and c-kit+ cells from peripheral blood served as control. Moreover, intercellular adhesion molecule-1 (ICAM-1) and CXCR4 were blocked systemically to determine their role in inflammation-related c-kit+-cell adhesion. In vitro, SDF-1α enhanced c-kit+-cell migration. In vivo, SDF-1α alone triggered endothelial rolling—not firm adherence—of c-kit+ cells in WT mice. While TNF-α alone had little effect on adhesion of c-kit+ cells, it induced maximum endothelial EL adherence. However, after combined treatment with SDF-1α+TNF-α, endothelial adhesion of c-kit+ cells increased independent of their origin, while EL adhesion was not further incremented. Systemic treatment with anti-ICAM-1 and anti-CXCR4-monoclonal antibody completely abolished endothelial c-kit+-cell adhesion. In N-nitro-L-arginine-methylester-hydrochloride-treated WT mice as well as in eNOS (−/−) mice, firm endothelial adhesion of c-kit+ cells was entirely abrogated, while EL adhesion was significantly increased. The chemokine SDF-1α mediates firm adhesion c-kit+ cells only in the presence of TNF-α stimulation via an ICAM-1- and CXCR4-dependent mechanism. The presence of eNOS appears to be a crucial and specific factor for firm c-kit+-cell adhesion to the vascular endothelium.

Intracardiac Erythropoietin Injection Reveals Antiinflammatory Potential and Improved Cardiac Functions Detected by Forced Swim Test

Systemic administration of erythropoietin (Epo) protects the myocardium from an ischemic insult and promotes beneficial remodeling. We hypothesized that intracardiac injection of Epo may exhibit cardioprotective potential with reduced systemic toxicity. Following myocardial infarction (MI), Epo was injected directly into the border of the infarction. Six weeks after an MI, we evaluated infarction size, angiogenesis, and pathologic effects of the treatment. Myocardial performance was assessed with a Forced Swim Test adapted to the study. Anti-inflammatory and cellular proliferative effects of Epo were analyzed by measuring expression of integrin-beta and CdK4 by reverse transcriptase-polymerase chain reaction (RT-PCR). The findings indicated improved cardiac status with direct Epo administration. Exercise capacity detected by the Forced Swim Test was significantly increased. There was radical reduction of absolute infarction size, ventricular dilatation, and hypertrophy in the Epo group. Integrin-beta was down-regulated and CdK4 expression was increased significantly with Epo. In conclusion, the study demonstrated that intramyocardial Epo injection, following MI, reduced inflammation, enhanced angiogenesis and proliferation, improved myocardial functions, and did not lead to intramural thrombus formation.

Localized SDF-1alpha gene release mediated by collagen substrate induces CD117 stem cells homing.

Stromal cell‐derived factor‐1α (SDF‐1α) mediated mobilization and homing of stem cells showed promising potential in stem cell based tissue engineering and regenerative medicine. However local and sustained release of SDF‐1α is indispensable for stem cell mediated regenerative process due to its short half‐life under inflammatory conditions. In this study, a gene activated collagen substrate (GAC) was formed via assembly of plasmid encoding SDF‐1α into a collagen substrate to create a microenvironment favoring stem cell homing. Local release of SDF‐1α from the transfected cells on GAC and its effect on CD117+ stem cell homing were investigated. Non‐viral poly‐ethyleneimine (25kDa PEI)/DNA complexes were mixed with rat tail collagen solution to form the GAC. Optimization of GAC was carried out based on collagen effects on the PEI/DNA complexes, viability and luciferase expression of COS7 cells on GAC. CD117+ stem cells homing in response to SDF‐1α local expression from transfected cells on GAC were investigated in a flow chamber in vitro and in a mouse hind limb model in vivo. The gene expression, migration of CD117+ stem cells and the induced inflammation were investigated with immunostaining, reverse transcription polymerase chain reaction (RT‐PCR) and H&E staining. The optimized parameters for GAC were DNA dosage 10 μg/cm2, molar ratio of PEI nitrogen in primary amine to DNA phosphate (N/P ratio) 4 and mass ratio of collagen to DNA (C/D ratio) 1.0. It kept cell viability above 75% and transfection efficiency around 5.8 × 105 RLU/mg protein. GAC allowed the sustained gene release up to 60 days. GAC mediated SDF‐1α gene release induced migration and homing of CD117+ stem cells in vitro and in vivo significantly, and the inflammation of GAC reduced significantly two weeks after transplantation. GAC is a promising stem cell based therapeutic strategy for regenerative medicine.

Retraction: Mesenchymal Stem Cells: Are They Suitable for Systemic Injection? An In Vitro Study

This article is being retracted because another article was published using the same data, but under a different title, in Microvascular Research.