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Abstract
The objective of this study was to develop angiopoietin-1 (Ang1)-expressing genetically modified human adipose tissue derived stem cells (hASCs) for myocardial therapy. For this, an efficient gene delivery system using recombinant baculovirus complexed with cell penetrating transactivating transcriptional activator TAT peptide/deoxyribonucleic acid nanoparticles (Bac-NP), through ionic interactions, was used. It was hypothesized that the hybrid Bac- NPAng1 system can efficiently transduce hASCs and induces favorable therapeutic effects when transplanted in vivo. To evaluate this hypothesis, a rat model with acute myocardial infarction and intramyocardially transplanted Ang1-expressing hASCs (hASC-Ang1), genetically modified by Bac-NPAng1, was used. Ang1 is a crucial pro-angiogenic factor for vascular maturation and neovasculogenesis. The released hAng1 from hASC-Ang1 demonstrated profound mitotic and anti-apoptotic activities on endothelial cells and cardiomyocytes. The transplanted hASC-Ang1 group showed higher cell retention compared to hASC and control groups. A significant increase in capillary density and reduction in infarct sizes were noted in the infarcted hearts with hASC-Ang1 treatment compared to infarcted hearts treated with hASC or the untreated group. Furthermore, the hASC-Ang1 group showed significantly higher cardiac performance in echocardiography (ejection fraction 46.28% ± 6.3%, P < 0.001 versus control, n = 8) than the hASC group (36.35% ± 5.7%, P < 0.01, n = 8), 28 days post-infarction. The study identified Bac-NP complex as an advanced gene delivery vehicle for stem cells and demonstrated its potential to treat ischemic heart disease with high therapeutic index for combined stem cell-gene therapy strategy.
Acknowledgments
The authors gratefully acknowledge the assistance received from the Canadian Institutes of Health Research (MOP #64308) to S Prakash, and the Natural Sciences and Engineering Research Council of Canada to S Prakash and D Shum-Tim. A Paul acknowledges the Alexander Graham Bell Post Graduate Scholarship and Michael Smith Foreign Study Award – Doctoral from the Natural Sciences and Engineering Research Council of Canada.
Disclosure
The authors report no conflicts of interest in this work.
Supplementary information
Characterization of baculovirus (Bac)-nanoparticle (NP) hybrid complex
The particle size and zeta potential of the NP, Bac, and Bac-NP hybrid particles were measured by the technique of electrophoretic laser Doppler anemometry using a Zeta Potential Analyzer (ZetaPlus; Brookhaven Instruments Corporation, Holtsville, NY). ZetaPlus Particle Sizer Software (v 4.11; Brookhaven) was used to determine the size distribution of the particles and Zeta Potential Analyzer software (v 3.57; Brookhaven) was used for zeta potential analysis. Both particle size and zeta potential were measured for three independent preparations and each measurement was obtained after taking the average of the three runs. Transmission electron microscopy (TEM) was used to obtain the size characterization. The NPs were suspended in 1× phosphate buffered saline and analyzed on CM200 FEG-TEM (Royal Philips Electronics, Markham, ON, Canada). Results are presented in Figure S1.
The scanning electron microscope (SEM; S4700 FEG-SEM Hitachi, Oakville, ON, Canada) and atomic force microscope (AFM; Digital Instruments, Palo Alto, CA) photomicrographs in confirm the formation of NP by transactivating transcriptional activator (TAT) and DNA complexation. The formed NPs were further studied by TEM and zeta potential analyzer, as demonstrated in Figure S1C–F. The Bac-NP complex was first characterized by measuring the zeta potential of the nanocomplex with laser Doppler electrophoresis (Figure S1C). At its natural pH 6.8, Bac is negatively charged with zeta potential of −6.5 ± 1.4 mV. At physiological pH 7.4, Bac had a zeta potential of −12.8 ± 3.1 mV. On the other hand, prepared TAT/DNA NPs showed high positive charge of 26.5 ± 3.2 mV at N/P ratio of 3. The positively charged TAT/DNA NPs, upon conjugation with the negatively charged Bac, formed positively charged (5.1 ± 2.7 mV) Bac-NP hybrid nanocomplexes.
To reconfirm the successful formation of the Bac-NP complexes, particle sizes of each complex were measured. Free Bac had an average size of 238 ± 10 nm, whereas free TAT/DNA NP showed an average size of around 72 ± 4.6 nm. The Bac-NP hybrid complexes had an average size of 480 ± 18.2 nm. This significant increase in size of Bac-NP particles, compared to that of free Bac and NPs, indicates the efficient production of the nanobiohybrid complexes, generated by strong electrostatic interactions of Bac with the NPs.
In order to look for the morphological evidence for successful conjugation of the budded Bac particles with the NPs, TEM was used. Electron micrographs showed the well-dispersed NPs (Figure S1D). On coming in contact with free Bac (Figure S1E; the rod-shaped particles with a length of 200–250 nm), there was an instant virus-NP complex formation by the negatively charged Bac with the positively charged NP, as indicated in Figure S1F to form the hybridized Bac-NP complex. The images of Bac-NP complexes also confirm the proper retention of the typical rod-shaped morphological appearance and envelope structure of Bac, suggesting that Bac were able to sustain their morphological integrity even after hybridization with NPs.
Optimization of viral dose for Bac-NP mediated human adipose tissue-derived stem cell (hASC) transduction
In order to achieve maximum transduction, the effect of multiplicity of infection (defined as plaque forming units per cell) and their combinatorial effects on the hybrid Bac-NP system were optimized. For this, multiplicity of infection ranging from 100 to 400 and N/P ratio of 3 were used. Initially, hASCs were seeded in six-well plates at 0.5 × 106 cells/well and incubated overnight at 5% carbon dioxide and 37°C. Following this, an appropriate volume of transduction solutions from different experimental groups (LacZ-carrying Bac, LacZ-carrying NP, Bacnull-LacZ-carrying NP, LacZ-carrying Bac, LacZ-carrying NP, LacZ-carrying Bac-NPnull), suspended in phosphate buffered saline, was added to each well according to varied multiplicity of infection, and incubated for 4 hours at 25°C. Bacnull and NPnull represent delivery systems carrying DNA with no gene of interest. The wells were replenished with fresh media and grown 37°C in carbon dioxide incubator. After 24 hours, the cells were stained with X-gal to detect the transduced LacZ-expressing cells. Results are presented in Figure S2.
Bac-NP has no toxic effects on hASCs: cytotoxicity assay
For the cytotoxicity assay, 2 × 104 hASCs/well were seeded in triplicate for each sample in 96-well plates. After culturing overnight, the cells were washed twice with phosphate buffered saline. NP, Bac, and Bac-NP particles suspended in culture media (200 μL) were added to the corresponding set of wells. Nontransduced cells were used as control. After 24 hours, absorbance of each well were measured at 490 nm using CellTiter® 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega, Fitchburg, WI) in a plate reader. The percentage of viable cells in different experimental groups was quantified. All the experiments were performed in triplicate. Results are presented in Figure S3.
Bac-NP transduced hASCs retain their differentiation potential
Bac-NP-transduced hASCs were then seeded in a 24-well plate at a high confluency of 6 × 104 cells/well. Nontransduced cells were used as control. After 24 hours, the medium was replaced with either adipogenic or osteogenic differentiation medium (Invitrogen Life Technologies, Carlsbad, CA). Osteogenic differentiation was assessed using Alizarin Red S staining (Invitrogen) after a 21-day period of induction towards this lineage. After a 15-day period, adipogenic differentiation was evaluated by LipidTOX™ red neutral lipid staining (Invitrogen). Results are presented in Figure S4. The presence of calcium deposits following osteogenic induction, as well as lipid vacuoles following adipogenic induction, is indicative of the transduced hASCs’ ability for multilineage differentiation.
Figure S1 Characterization of the Bac-NP hybrid nanocomplex. (A) Scanning electron microscope photograph of transactivating transcriptional activator/deoxyribonucleic acid nanoparticles with subset showing a magnified image. (B) Atomic force microscope photograph of NPs demonstrating their surface topography. (C) Zeta potential of free Bac at pH 6.8 (pH of insect cells media), free Bac at pH 7.4 (pH of mammalian cell culture), free NP (phosphate buffered saline: pH 7.4), and hybrid Bac-NP nanocomplexes at pH 7.4, with N/P ratio of 3. The complexes were prepared from 1 μg deoxyribonucleic acid (complexed with transactivating transcriptional activator) per 109 plaque forming units Bac. Transmission electron microscope images of (D) NP with N/P ratio of 3, (E) Bac, and (F) Bac-NP suspended in phosphate buffered saline.
Notes: Arrows indicate NPs hybridized on Bac surface. Scale bar: 100 nm.
Abbreviations: Bac, baculovirus; Bac-NP, baculovirus-nanoparticle complex; NP, nanoparticle.
Figure S2 Optimization of transduction condition in human adipose tissue-derived stem cells with Bac-NPLacZ. In each well of 96-well plate, 2 × 104 cells were seeded and cultured overnight. The nanobiohybrid complexes were prepared from 1 μg deoxyribonucleic acid (complexed with transactivating transcriptional activator) per 109 plaque forming units baculovirus. Effect of baculovirus MOI on baculovirus-nanoparticle complex-mediated cell transduction. Cells were transduced with the prepared baculovirus-nanoparticle complex at constant N/P ratio of 3 with MOI of 100, 200, and 400 and stained with X-gal, 24 hours post-transduction, to determine the percentage cells transduced with LacZ expression. Three independent experiments were performed and data expressed as mean ± standard deviation. Bac-NPLacZ with MOI of 200 showed highest transduction as compared to other groups.
Abbreviations: BacLacZ, LacZ-carrying baculovirus; BacLacz-NPnull, LacZ-carrying baculovirus-nanoparticle (carrying no gene of interest) complex; Bac-NPLacZ, baculovirus-LacZ-carrying nanoparticle complex; Bacnull-NPLacZ, baculovirus (carrying no gene of interest)-LacZ-carrying nanoparticle complex; MOI, multiplicity of infection.
Figure S3 Cytotoxic effects of baculovirus-nanoparticle hybrid complexes on human adipose tissue-derived stem cells. In each well of 96-well plate, 2 × 104 cells were seeded and cultured overnight. The cells were incubated with nanoparticles (N/P ratio 3) only, baculovirus (multiplicity of infection 200) only, and baculovirus-nanoparticle complex (multiplicity of infection 200 and N/P ratio 3) for 12 hours followed by cell toxicity analysis.
Notes: Three independent experiments were performed and data expressed as mean ± standard deviation. There were no significant differences in percentage viability between the groups, confirming that the baculovirus-nanoparticle complex did not have any toxic effect on the human adipose tissue-derived stem cells.
Abbreviations: BacLacZ, LacZ-carrying baculovirus; BacLacZ-NPLacZ, LacZ-carrying baculovirus-LacZ-carrying nanoparticle complex; BacLacz-NPnull, LacZ-carrying baculovirus-nanoparticle (carrying no gene of interest) complex; Bacnull-NPLacZ, baculovirus (carrying no gene of interest)-LacZ-carrying nanoparticle complex; NPLacZ, LacZ-carrying nanoparticle.
Figure S4 Adipogenic and osteogenic differentiation of baculovirus-nanoparticle complex-transduced human adipose tissue-derived stem cells. Transduced adipose tissue-derived stem cells were cultured in either adipogenic or osteogenic differentiation media. Adipogenic differentiation was assessed via LipidTOX™ Red neutral lipid staining of lipid vacuoles and 4′,6-diamidino-2-phenylindole staining of nucleus: (i) transduced differentiated and (ii) nontransduced differentiated. Osteogenic differentiation was determined by Alizarin Red staining of calcium deposits: (i) transduced differentiated and (ii) nontransduced differentiated.
Notes: Arrows show the cell differentiated area. Results confirm that human adipose tissue-derived stem cells retain their multilineage differential potential even after baculovirus-nanoparticle complex transduction.
