Angiogenesis and myogenesis using human skeletal myoblasts for cardiac repair 2

Chapter Results 104 3.1 In vitro characterization of skeletal myoblast Fresh culture of human skeletal myoblasts was obtained from Cell Transplants Singapore Pte. Ltd. Singapore, for every transplantation experiment. The cells were grown and cultured in the laboratory using Super-medium to achieve the required number. 3.1.1 Cell culture characteristics of skeletal myoblast Skeletal myoblasts obtained from human male donor had a typical morphological appearance. Freshly cultured human skeletal myoblasts were small spheres of about 12 µm in diameter (Figure 1a). They transformed into elongated, spindle-shaped cells and started to grow as an adherent culture after 12 hours on collagen coated tissue culture flasks (Figure 1b). They reached to 50% of confluence at 48 hours (Figure 1c) and > 90% of confluence at 120 hours (Figure 1d) after isolation. Their doubling time cultured in Supermedium supplemented with 10% FBS, incubated at 37oC in 5% CO2 incubator was less than 18 hours. Skeletal myoblasts were regularly passaged every 48-72 hours to prevent their pre-mature fusion and differentiation in vitro. 3.1.2 Purity of skeletal myoblast culture Skeletal myoblast culture was more than 98 % of purity as shown by desmin or CD56 expression (Figure 2a & b). Dual fluorescent immunostaining demonstrated that >95% of skeletal myoblasts simultaneously expressed desmin and CD56 (Figure 2c) using human fibroblasts as a negative control (Figure 2d). Cytofluorimetric study further confirmed high purity of skeletal myoblast culture. The result showed that 96% of skeletal myoblasts were positive for desmin expression when 1% of autofluorescence was gated by non-stained skeletal myoblasts (Figure 3a & b), while 92% of skeletal myoblasts were positive for CD56 expression (Figure 3d & e) and 91% of skeletal myoblasts co-expressed desmin and CD56 (Figure 3g & h) using human flbroblast as negative control (Figure 3c, f & i). 105 a b c d Figure 1a-d: Phase contrast photomicrographs of human skeletal myoblasts showing in vitro growth profile. Skeletal myoblast culture at (a) 0, (b) 12, (c) 48 and (d) 120 hours after isolation. (Magnification: a= 200x; b-d = 100x) 106 a b c d Figure 2a-d: Dual fluorescent immunostaining of human skeletal myoblasts for desmin and CD56 expression. (a) Desmin expression (green fluorescence = FITC) (b) CD56 expression (red fluorescence = PE). (c) Superimposition of pictures (a) and (b) to show coexpression of desmin and CD56. (d) Human fibroblasts were used as a negative control. The cells were counterstained with DAPI (blue fluorescence) to show the nuclei (Magnification = 200x). 107 a c b 0.97% d 97.79% f e 1.06% g 93.95% 3.36% i h 0.8% 0.32% 91.9% 2.6% Figure 3a-i: Cytofluorimetry of human skeletal myoblasts for desmin and CD56 expression. Non-stained human skeletal myoblasts (missing primary antibody) for (a) desmin (d) CD56 and (g) co-expression of desmin and CD56 were used as a baseline for autofluorescence. The results showed that (b) 96% of skeletal myoblasts expressed desmin whereas (e) 92% of skeletal myoblast expressed CD56, while (h) 91% of skeletal myoblasts co-expressed desmin and CD56. Human fibroblasts were used as negative controls (c) for desmin (f) for CD56 and (i) for co-expression of desmin and CD56. 108 3.1.3 The effect of multiple transduction on skeletal myoblast Fluorescent immunostaining and cytofluorimetric analysis for desmin and CD56 expression demonstrated that viral vector transduction with skeletal myoblasts did not change the phenotype of skeletal myoblasts. Dual fluorescent immunostaining showed that >95% of skeletal myoblasts expressed desmin or CD56, or co-expressed desmin and CD56 (Figure 4a-c). Cytofluorimetric analysis further confirmed these results. It was observed that 83% of skeletal myoblasts expressed desmin (Figure 5b), while 95% of them expressed CD56 (Figure 5d) when 1% of autofluorescence was gated by non-stained skeletal myoblasts (Figure 5a & c). Co-expression of desmin and CD56 was observed to be 82% (Figure 5f). 3.1.4 Skeletal myoblast viability Cell viability at the time of cell transplantation was more than 99% as determined by Trypan blue dye exclusion test. 3.2 Ad-vector titer The adenoviral vector titer determined by end-point assay revealed that the viral titer was ~1x1010 PFU/ ml for Ad-null, ~8x109 PFU/ ml for Ad-VEGF165, ~7x109 PFU/ ml for Ad-Ang-1 and ~5x108 PFU/ ml for Ad-Bic. All of the viral vectors were found replication deficient when tested for replication competence. 3.3 Skeletal myoblast transduction with Ad-VEGF165 After optimizing the transduction procedure, qualitative and quantitative assessments were carried out to characterize Ad-VEGF165 transduced myoblasts for hVEGF165 expression in vitro. 109 a b c d Figure 4a-d: Dual fluorescent immunostaining of human skeletal myoblasts for desmin and CD56 expression after multiple viral vector transduction. (a) Desmin expression (green fluorescence = FITC), (b) CD56 expression (red fluorescence = PE). (c) Superimposition of pictures (a) and (b) to show co-expression of desmin and CD56. (d) Human fibroblasts were used as a negative control. The cells were counterstained with DAPI (blue fluorescence) to show the nuclei (Magnification = 200x). 110 a b 1.06% 84.5% d c 96.09% 1.02% e f 0.0% 82.4% Figure a-f: Cytofluorimetry of human skeletal myoblasts for desmin and CD56 expression. Non-stained human skeletal myoblasts (missing primary antibody) for (a) desmin, (c) CD56 and (e) co-expression of desmin and CD56 were used as a baseline for autofluorescence. The results showed that (b) 83% of skeletal myoblasts expressed desmin and (d) 95% of skeletal myoblast expressed CD56, while (f) 82% of skeletal myoblasts coexpressed desmin and CD56. 111 3.3.1 Optimization of transduction condition A dose dependent relation between the Ad-VEGF165 vector titer and skeletal myoblast number was revealed by human VEGF ELISA. With the increase in the number of viral particles, transduction efficiency also increased (Figure 6a). The highest transduction efficiency was, however, achieved at 1000PFU/ myoblast. The exposure time of skeletal myoblast to Ad-VEGF165 vector showed a causal relation with transduction efficiency (Figure 6b). Repeated transduction (in triplicate) of skeletal myoblasts improved transduction efficiency (Figure 6b). At higher viral titer (>1000 PFU) or a longer time (>8 hours) exposure, however, the ill effects of adenoviral vector towards skeletal myoblasts were observed. Thus, the optimum transduction conditions were achieved at a ratio of 1000PFU/ myoblast when transduction was carried out for hours in triplicate with 24 hours interval. 3.3.2 Qualitative assessment of hVEGF165 expression from hVEGF165-myoblasts Immunohistochemical staining of hVEGF165-myoblasts revealed high transduction efficiency. Both the DAB substrate (Figure 7a & c) and FITC fluorescent (Figure 7d) immunostaining showed > 95% of Ad-VEGF165 transduced myoblasts expressing hVEGF165, using Ad-null transduced myoblasts as a negative control (Figure 7b & e). The strong signal demonstrated that hVEGF165 was actively expressed by Ad-VEGF165 transduced myoblasts. RT-PCR for hVEGF165 expression revealed that Ad-VEGF165 transduced myoblasts expressed hVEGF165 for up to 30 days of observation in vitro (Figure 8). The presence of mRNA encoding for hVEGF165 as detected by hVEGF165 specific primers demonstrated that Ad-VEGF165 transduced myoblasts had the highest level of hVEGF165 gene expression compared with non-transduced and Ad-null transduced myoblasts. The peak expression level was achieved at day after multiple transductions. 112 C on cen tration s of h V E GF 165 (p g/m l) 2000 1500 1000 500 0 500 a 1000 1500 2000 2500 3000 3500 4000 C on cen tration s of h V E GF 165 (n g/m l ) Ad-VEGF165 vector to myoblast (x :1) b 30 20 10 PBS Nontransduced myoblast Ad-null myoblast 2h 4h 8h 24 h Transduction efficiency based on transduction time (hours) Figure 6a & b: Optimization of skeletal myoblast transduction conditions using AdVEGF165. (a) Efficiency of skeletal myoblast transduction as a function of Ad-VEGF165 titer. Optimum transduction efficiency was achieved at a ratio of 1000 Ad-vector : one myoblast. (b) Expression of hVEGF165 from hVEGF165-myoblast as a function of transduction time. Transduction was repeated three times. 113 Smooth muscle actin vWF-VIII a merged b c e f h i Normal pig d DMEM group g Ad-null myoblast group j k l Non-transduced myoblast group (Continued on next page) 172 m n o q r t u hVEGF165-myoblast group p Ang-1 myoblast group s Bic-myoblast group Figure 54a-u: Visualization of blood vessel in pig heart (low power magnification 100x) at 12 weeks after cell transplantation. Dual fluorescent immunostaining for vWF-VIII (red fluorescence=TRITC) and SMA (green fluorescence= FITC) was carried out to visualize blood vessels. 173 Blood vessel density (number of blood vessels per microscopic field) based on vWF-VIII expression at low power (100x) was 16.18± 0.91 at weeks for DMEM group which decreased to 13.44± 0.90 at 12 weeks (Figure 55). For Ad-null myoblast and nontransduced myoblast groups, blood vessel density was 26.57± 2.09 and 25.2 ± 0.94 at weeks respectively, as compared with 26.86± 2.15 and 24.1± 1.69 at 12 weeks. The highest blood vessel density was achieved by hVEGF165-myoblast transplantation at weeks (57.13±4.19). However, it significantly declined to 32.1± 1.74 (p=0.001) at 12 weeks. The progressive increase in blood vessel density was only seen in the Ang-1 myoblast and Bicmyoblast groups where it continuously increased from 39.9± 3.09 and 45.2± 5.87 at weeks to 45.14± 1.75 (p=0.365) and 53.5± 5.87 (p=0.234) at 12 weeks respectively. For inter-group comparison, vWF-VIII immunostaining showed that the blood vessel density of hVEGF165-myoblast, Ang-1 myoblast and Bic-myoblast groups were significantly higher than any other group at weeks (p[...]... (Figure 23 c) A scar was formed in the infarct region in the control animals treated with basal DMEM without skeletal myoblast therapy as visualized by Masson Trichrome staining (Figure 23 d) 3.7.3 Survival of skeletal myoblasts in rat heart X-gal staining for nLac-z expression showed survival of human skeletal myoblasts in rat heart at 2 days, 2 and 6 weeks after myoblast transplantation (Figure 24 a-c)... oil immersion) 124 1 day 1 hVEGF165 Human GAPDH 2 3 4 5 6 8 days 18 days 7 8 9 10 30 days 11 12 13 Ang-1 Figure 17 RT-PCR of Ad-Bic transduced myoblasts for co-expression of hVEGF165 and Ang-1 in vitro Lane 1 = DNA ladder Lane 2 & 3 = Non-transduced myoblasts Lane 4 & 5 = Ad-null transduced myoblasts Lane 6 ~13 = Ad-Bic transduced myoblasts at 1, 8, 18 and 30 days after transduction 125 Western blot... expression (c) co-expression of hVEGF165 and Ang-1 127 Concentration of hVEGF165 (ng/ml) Nontransduced myoblast Ad-null myoblast 40 35 30 25 20 Bic-myoblast 15 10 5 0 0 1 2 4 6 8 10 12 14 16 18 22 26 30 Time (days) after Ad-Bic transduction Figure 19: Expression of hVEGF165 from Ad-Bic transduced myoblasts as a function of time 128 4 HUVEC cell number (10 ) 6 Day-0 * Day-4 4 # 2 0 B C D a F G H 3 Scintillation... Histochemistry for nLac-z expression in (a) FLY-A4 packaging cells and (b) human skeletal myoblasts The figures show nuclear localized green color signal after X-gal staining (c) Immunostaining of human skeletal myoblasts for BrdU localization The antigen-antibody reaction was visualized by alkaline phosphatase using nitroblue tetrazolium substrate (magnification: a & b= 100x, c= 20 0x) 1 32 3.7 In vivo... counts/ 10 minutes for HUVEC cultured with supernatant from hVEGF165 -myoblasts was highest ( 422 3.6 ± 301.4 counts, p . non-stained skeletal myoblasts (Figure 3a & b), while 92% of skeletal myoblasts were positive for CD56 expression (Figure 3d & e) and 91% of skeletal myoblasts co-expressed desmin and CD56. 20 0x). 110 a b 1.06% 84.5% d c 96.09% 1. 02% f e 0.0% 82. 4% Figure 5 a-f: Cytofluorimetry of human skeletal myoblasts for desmin and CD56 expression. Non-stained human. and lysate from Ad-VEGF 165 transduced myoblasts for hVEGF 165 expression in vitro. 0 5 10 15 20 25 30 35 40 45 0 1 2 4 6 8 10 12 14 16 18 22 26 30 Time (days) after Ad-VEGF 165 transduction