Most studies on stem cell transplantation therapy on myocardially infarcted animal models and phase-I human clinical trials have focused on the use of undifferentiated stem cells. There is a strong ...possibility that some degree of cardiomyogenic differentiation of stem cells in vitro prior to transplantation would result in higher engraftment efficiency, as well as enhanced myocardial regeneration and recovery of heart function. Additionally, this may also alleviate the probability of spontaneous differentiation of stem cells into undesired lineages and reduces the risk of teratoma formation, in the case of embryonic stem cells. The development of efficient protocols for directing the cardiomyogenic differentiation of stem cells in vitro will also provide a useful model for molecular studies and genetic manipulation. This review therefore critically examines the various techniques that could possibly be used to direct and control the cardiomyogenic differentiation of stem cells in vitro.
This study aimed to test the hypothesis that branchial osmoregulatory acclimation involved increased apoptosis and replacement of mitochdonrion-rich cells (MRCs) in the climbing perch, Anabas ...testudineus, during a progressive acclimation from freshwater to seawater. A significant increase in branchial caspase-3/-7 activity was observed on day 4 (salinity 20), and an extensive TUNEL-positive apoptosis was detected on day 5 (salinity 25), indicating salinity-induced apoptosis had occurred. This was further supported by an up-regulation of branchial mRNA expression of p53, a key regulator of cell cycle arrest and apoptosis, between day 2 (salinity 10) and day 6 (seawater), and an increase in branchial p53 protein abundance on day 6. Seawater acclimation apparently activated both the extrinsic and intrinsic pathways, as reflected by significant increases in branchial caspase-8 and caspase-9 activities. The involvement of the intrinsic pathway was confirmed by the significant increase in branchial mRNA expression of bax between day 4 (salinity 20) and day 6 (seawater). Western blotting results revealed the presence of a freshwater Na(+)/K(+)-ATPase (Nka) α-isoform, Nka α1a, and a seawater isoform, Nka α1b, the protein abundance of which decreased and increased, respectively, during seawater acclimation. Immunofluorescence microscopy revealed the presence of two types of MRCs distinctly different in sizes, and confirmed that the reduction in Nka α1a expression, and the prominent increases in expression of Nka α1b, Na(+):K(+):2Cl(-) cotransporter 1, and cystic fibrosis transmembrane conductance regulator Cl(-) channel coincided with the salinity-induced apoptotic event. Since modulation of existing MRCs alone could not have led to extensive salinity-induced apoptosis, it is probable that some, if not all, freshwater-type MRCs could have been removed through increased apoptosis and subsequently replaced by seawater-type MRCs in the gills of A. testudineus during seawater acclimation.
Myoblast-based cardiac repair: Xenomyoblast versus allomyoblast transplantation Guo, Changfa, MD; Haider, Husnain Kh., PhD; Shim, Winston S.N., PhD ...
Journal of thoracic and cardiovascular surgery/The Journal of thoracic and cardiovascular surgery/The journal of thoracic and cardiovascular surgery,
11/2007, Letnik:
134, Številka:
5
Journal Article
Recenzirano
Odprti dostop
Objective We sought to investigate immune cell kinetics in relation to skeletal myoblast survival and heart function improvement after nonautologous skeletal myoblast transplantation in a rat model ...of myocardial infarction. Methods One week after myocardial infarction, 208 Wistar rats were grouped into group 1 (n = 24, receiving 150 μL of medium only), group 2 (n = 24, receiving 150 μL of medium and cyclosporine INN: ciclosporin), group 3 (n = 40, human skeletal myoblast transplantation), group 4 (n = 40, human skeletal myoblast transplantation with cyclosporine treatment), group 5 (n = 40, rat skeletal myoblast transplantation), and group 6 (n = 40, rat skeletal myoblast transplantation with cyclosporine treatment). The hearts were harvested at 10 minutes and 1, 4, 7, and 28 days after cell transplantation. Skeletal myoblast survival was confirmed by means of immunohistochemical studies and quantified by using real-time polymerase chain reaction. Host immune responses were assessed by immunostaining for macrophages and CD4+ and CD8+ lymphocytes. Heart function was evaluated by means of echocardiographic analysis. Results The majority of macrophages and lymphocytes infiltrated in the acute phase (from day 1 to day 7) and then subsided by day 28. The donor skeletal myoblasts survived and differentiated well in all skeletal myoblast transplantation groups. Allogeneic skeletal myoblasts showed a superior survival rate than xenogeneic skeletal myoblasts ( P < .01). Cyclosporine inhibited the infiltration of the immunocytes, enhanced skeletal myoblast survival, and improved heart performance compared with that seen in the groups not receiving cyclosporine treatment ( P < .05). Conclusions Allomyoblasts survive better than do xenomyoblasts after transplantation into infarcted myocardium. After inhibition of immunocyte infiltration by means of immunosuppressive treatment, skeletal myoblast survival is enhanced, with improved heart performance. These findings suggest the feasibility of nonautologous myoblast transplantation with immunosuppressive treatment.
Low-level transgene efficiency is one of the main obstacles in ex vivo nonviral vector-mediated gene transfer into primary human skeletal myoblasts (hSkMs). We optimized the cholesterol:N-1-(2, ...3-dioleoyloxy)propyl-N, N, N-trimethylammonium methylsulfate liposome (CD liposome) and 22-kDa polyethylenimine (PEI22)- and 25-kDa polyethylenimine (PEI25)-mediated transfection of primary hSkMs for angiogenic gene delivery. We found that transfection efficiency and cell viability of three nonviral vectors were cell passage dependent: early cell passages of hSkMs had higher transfection efficiencies with poor cell viabilities, whereas later cell passages of hSkMs had lower transfection efficiencies with better cell viabilities. Trypsinization improved the transfection efficiency by 20% to 60% compared with adherent hSkMs. Optimum gene transfection efficiency was found with passage 6 trypsinized hSkMs: transfection efficiency with CD lipoplexes was 6.99 +/- 0.13%, PEI22 polyplexes was 18.58 +/- 1.57%, and PEI25 polyplexes was 13.32 +/- 0.88%. When pEGFP (a plasmid encoding the enhanced green fluorescent protein) was replaced with a vector containing human vascular endothelial growth factor 165 (phVEGF(165)), the optimized gene transfection conditions resulted in hVEGF(165) expression up to Day 18 with a peak level at Day 2 after transfection. This study demonstrated that therapeutic angiogenic gene transfer through CD or PEI is feasible and safe after optimization. It could be a potential strategy for treatment of ischemic disease for angiomyogenesis.
We sought to compare host immune cell kinetics, survival profile of donor skeletal myoblasts, and skeletal myoblast graft efficacy after autologous and allogeneic skeletal myoblast transplantation ...into a rat model of myocardial infarction.
One week after myocardial infarction, 128 animals were divided into four groups: group 1 (n = 24, receiving medium only), group 2 (n = 24, receiving medium and cyclosporine), group 3 (n = 40, autologous skeletal myoblast transplantation), and group 4 (n = 40, allogeneic skeletal myoblast transplantation with cyclosporine treatment). Rats were euthanized 10 minutes, 1 day, and 4, 7, and 28 days later. Host immune cell kinetics were assessed by immunohistochemical studies for macrophages, and CD4+ and CD8+ lymphocytes. Donor skeletal myoblast survival was confirmed by tracking prelabeled signals, and quantified by beta-gal assay. Heart function was evaluated by echocardiography.
A transient immune cell infiltration was demonstrated in group 3, with macrophage infiltration on day 1 and day 4, CD8+ cell infiltration on day 4 and day 7, and CD4+ cell infiltration on day 4. In group 4, immunocyte infiltration was slightly more severe than that in group 3. Automyoblasts and allomyoblasts showed no significant difference of survival from day 1 to day 7 (p > 0.10); however, on day 28, automyoblasts showed better survival than allomyoblasts (p < 0.05). Transplantation of allomyoblasts increased systolic heart function and limited heart dilation after myocardial injury to a similar degree as automyoblasts (p > 0.10).
The use of allomyoblasts is feasible and effective for cardiac repair with immunosuppressive treatment as compared with automyoblasts.
The study aims to use cholesterol (Chol) + DOTAP liposome (CD liposome) based human vascular endothelial growth factor‐165 (VEGF165) gene transfer into skeletal myoblasts (SkMs) for treatment of ...acute hind limb ischaemia in a rabbit model. The feasibility and efficacy of CD liposome mediated gene transfer with rabbit SkMs were characterized using plasmid carrying enhanced green fluorescent protein (pEGFP) and assessed by flow cytometry. After optimization, SkMs were transfected with CD lipoplexes carrying plasmid‐VEGF165 (CD‐pVEGF165) and transplanted into rabbit ischaemic limb. Animals were randomized to receive intramuscular injection of Medium199 (M199; group 1), non‐transfected SkM (group 2) or CD‐pVEGF165 transfected SkM (group 3). Flow cytometry revealed that up to 16% rabbit SkMs were successfully transfected with pEGFP. Based on the optimized transfection condition, transfected rabbit SkM expressed VEGF165 up to day 18 with peak at day 2. SkMs were observed in all cell‐transplanted groups, as visualized with 6‐diamidino‐2‐phenylindole and bromodeoxyuridine. Angiographic blood vessel score revealed increased collateral vessel development in group 3 (39.7 ± 2.0) compared with group 2 (21.6 ± 1.1%, P < 0.001) and group 1 (16.9 ± 1.1%, P < 0.001). Immunostaining for CD31 showed significantly increased capillary density in group 3 (14.88 ± 0.9) compared with group 2 (8.5 ± 0.49, P < 0.001) and group 1 (5.69 ± 0.3, P < 0.001). Improved blood flow (ml/min./g) was achieved in animal group 3 (0.173 ± 0.04) as compared with animal group 2 (0.122 ± 0.016; P= 0.047) and group 1 (0.062 ± 0.012; P < 0.001). In conclusion, CD liposome mediated VEGF165 gene transfer with SkMs effectively induced neovascularization in the ischaemic hind limb and may serve as a safe and new therapeutic modality for the repair of acute ischaemic limb disease.
We compare the effectiveness of direct adenoviral angiopoietin-1 (Ad-Ang-1) injection with transplantation of skeletal myoblasts (SkMs) over-expressing angiopoietin-1 (Ang-1) for angiogenic response ...and improvement of heart function in an experimental porcine model of myocardial infarction (MI).
Methods
Ad-Ang-1 was used for intramyocardial injection or transduction of SkMs. Three weeks after coronary artery ligation in 32 female pigs, animals were grouped to receive multiple intramyocardial injections of DMEM without cells (group-1; n=7), or containing 3∑108
Lac-z labelled SkMs transduced with Ad-Null vector carrying no gene (group-2; n=7), or 1∑1010 PFU Ad-Ang-1 (group-3; n=9), or 3∑108
Lac-z labelled SkMs transduced with Ad-Ang-1 (group-4; n=9). The animals were immunosuppressed for 6-weeks. After euthanasia, their heart tissue was processed for histological studies.
Results
Extensive survival of Lac-z positive SkMs was observed in and around the infarct 6 and 12-weeks after transplantation. Fluorescent immunostaining for vWF-VIII at 6-weeks revealed increased blood vessel density (∑100) in group-4 (p<0.05) as compared with other groups. Regional blood flow (ml/g/min) in the peri-infarct area was improved in group-4 (2.7; p<0.05) as compared with group-1 (1.2±0.1), group-2 (1.1±0.4) and group-3 (1.7±0.1) at 6-weeks. Similarly, ejection fraction was significantly higher in group-4 (49.2±5.9%, p=0.03) as compared with group-1 (36.8±3%) at 6 weeks.
Conclusion
SkMs mediated Ang-1 delivery is associated with improved angiogenic response, regional myocardial perfusion and heart function as compared with direct Ad-Ang-1 administration.
Objective
To achieve angiogenic interaction between VEGF165 and angiopoietin-1 (Ang-1) using a novel adenoviral bicistronic vector (Ad-Bic) encoding the two factors and delivered ex vivo using ...sex-mismatched human skeletal myoblasts.
Methods and results:
A myocardial infarction model was developed in 29 female pigs; randomised into four groups: DMEM (group-1, n=6); Adenovirus null (Ad-null) vector-myoblast (group-2, n=5); Ad-Ang-1 myoblast (group 3, n=7) and Ad-Bic-myoblast (group-4, n=11). Three weeks later, 5 ml DMEM without myoblasts or containing 3 × 108 myoblasts carrying lac-z gene and transduced with Ad-null, Ad-Ang-1 or Ad-Bic were injected intra-myocardially in and around the infarct. 2D-echocardiography and fluorescent microsphere studies 6- and 12-weeks post-treatment revealed significantly improved cardiac performance and regional blood flow in groups 3 and 4. Histological studies and Y-chromosome analysis revealed extensive survival of lac-z positive myoblasts staining positive for human proteins in the pig heart. ELISA, immunostaining and RT-PCR revealed that Ad-Bic transduced myoblasts concomitantly but transiently expressed hVEGF165 and Ang-1 both in vitro and in vivo. Double fluorescent immunostaining of the tissue sections for vWFactor-III and smooth muscle actin showed significantly higher vascular density of mature blood vessels per low power microscopic field in groups 3 and 4 at 6- and 12-weeks.
Conclusion:
Our combined approach led to enhanced angiogenesis with a greater percentage of functionally mature blood vessels in a porcine heart.
The aim of this study was to compare the body temperature measurements at tympanic, forehead and temporal sites using infrared thermometers. A total of 1576 consecutive visitors to Singapore General ...Hospital at two entry locations were included in this study. Pearson correlation and Bland–Altman mean difference between sites (95% confidence interval for limits of agreement) were calculated for the relationship between the three different sites of temperatures recorded (i.e. temporal, forehead and tympanic). Of all the visitors, 27 (1.7%) had fever. Moderate positive correlation was found between temporal and forehead temperature readings (r=0.602, mean difference (temporal – forehead), (95% limits of agreement) = 0.1 (−0.8, 0.7)), and there was very weak positive correlation between tympanic and temporal temperature readings (r=0.177, mean difference (temporal – tympanic), (95% limits of agreement) = −0.3 (−1.7, 1.1)). Sensitivity for temporal temperature readings (⩾37.5°C) to detect febrile visitors was 3.7%, specificity was 99.6%, positive predictive value was 14.3% and negative predictive value was 98.3%. Our results demonstrate that tympanic temperature readings should be used for fever screening instead of temporal or forehead readings.
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