Research Proposal Enhancement of Angiogenesis through Genetically Engineered Human Stem Cells.

Supervised By Dr. Muhammad Ayaz Khan

By Shams-uz-Zaman

GOMAL CENTRE OF BIOCHEMISTRY AND BIOTECHNOLOGY GOMAL UNIVERSITY D.I.KHAN

TABLE OF CONTENTS S.No 1 2 3 4 5 6 Title Introduction Review Of Literature Objectives Benefits Methodology Reference Page No. 1 2 2 2 3 4

Enhancement of Angiogenesis through Genetically Engineered Human Stem Cells Research proposal on Enhancement of Angiogenesis through Genetically Engineered Human Stem Cells

Introduction Controlled angiogenesis is an important component of successful tissue regeneration (1) as well as the treatment of ischemic diseases. Differentiated cells such as hematopoietic cells (2) and myoblasts (3) have been shown to induce vessel formation in limb or myocardial ischemic model by expressing angiogenic factors. However, the clinical application of differentiated cells is hindered by the difficulty in obtaining a large cell number, their lack of ability to expand in vitro and poor engraftment efficiency to target tissue sites. Stem cells are promising therapeutics for revascularization because of their capability of self-renewal, relative ease of isolation, and ability to migrate toward the ischemic tissues (4). Stem cells can contribute to angiogenesis directly, by participating in new vessel formation (5, 6), or indirectly by secreting a broad spectrum of angiogenic and antiapoptotic factors (7, 8). Furthermore, stem cells possess a homing capacity that allows them to migrate toward and engraft into the sites of ischemia or injury. Several factors such as stromal-derived factor 1 (SDF-1) and CXCR4 play a key role during the stem cell homing process, and overexpression of these chemokines contributes to enhanced homing to the target tissues (9). Genetic modification of stem cells to express angiogenic factors is a promising approach to further enhance the efficacy of stem cells for therapeutic angiogenesis. Virally modified, VEGF-overexpressing mesenchymal stem cells (MSCs) were reported to enhance angiogenesis (1) in vivo and improve myocardial function (10). Genetic modification of MSCs with Akt or Bcl-2 gene also improved the therapeutic efficacy of cell transplantation in treating myocardium (11, 12). However, previous studies have largely relied on viral vectors to deliver these therapeutic genes to stem cells, which are associated with safety concerns. Nonviral delivery systems, such as polyethylenimine and Lipofectamine, offer an alternative (3, 13) but are often associated with toxicity and typically provide significantly lower transfection efficiency than a viral-based approach. Here, we will attempt to develop a biodegradable polymerDNA nanoparticles to engineer stem cells to efficiently express angiogenic factors for the purpose of promoting angiogenesis in vivo. Compared with the methods of using nonviral gene therapy alone, this combined polymerstem cells approach takes advantage of the stem cells ability to target to the ischemic sites. Bone marrow-derived human mesenchymal stem cells (hMSCs) and human embryonic stem cell-derived cells (hESdCs) will be modified with angiogenic factor (VEGF) DNA by using poly(- amino esters), a family of hydrolytically biodegradable polymers that can condense DNA to form nanoparticles (14). This study is a description of a VEGF high-expressing stem cell therapy for angiogenesis using biodegradable polymer-DNA nanoparticles, Liposomal and Biolistic Method. The technology described herein may have utility as a tool for promoting therapeutic angiogenesis and treating ischemic disease.

Submitted By: Shams-uz-Zaman

Enhancement of Angiogenesis through Genetically Engineered Human Stem Cells Literature Review Asahara et.al (15) reported that, Putative endothelial cell (EC) progenitors or angioblasts were isolated from human peripheral blood by magnetic bead selection on the basis of cell surface antigen expression. they suggest that EC progenitors may be useful for augmenting collateral vessel growth to ischemic tissues (therapeutic angiogenesis) and for delivering anti- or pro-angiogenic agents, respectively, to sites of pathologic or utilitarian angiogenesis Stamm et.al (16) studied that, Implantation of bone-marrow stem cells in the heart might be a new method to restore tissue viability after myocardial infarction. they believe that implantation of AC133+ stem cells to the heart is safe and might induce angiogenesis, thus improving perfusion of the infarcted myocardium. Certain applications of angiogenesis research are now in clinical trial. These fall into three areas: diagnostic applications, acceleration of angiogenesis in wound healing, and inhibition of angiogenesis in neoplasia. Quantitation of angiogenesis in biopsy specimens of breast cancer provides an independent marker of future metastatic risk (17). Topical application of bFGF in chronic wounds accelerates angiogenesis and wound healing (18). Life-threatening hemangiomas can be successfully treated by utilizing the antiangiogenic property of a-interferon (19). Other applications are being prepared for clinical trial. The quantitation of elevated angiogenic factors in the blood and urine of cancer patients is being evaluated to determine prognosis and to guide therapy. Orally delivered bFGF which accelerates angiogenesis and healing in duodenal ulcers (20) is being studied for future clinical trial in patients with refractory gastrointestinal ulceration. A potent fungal-derived angiogenesis inhibitor is under study for eventual use as an anti-cancer agent (21). Continued advances in understanding the mechanism of the angiogenic process at the biochemical and molecular levels may provide further diagnostic and therapeutic benefits in a variety of diseases.

Benefits The role of angiogenesis in atherosclerosis and other cardiovascular diseases has emerged as a major unresolved issue. Angiogenesis has attracted interest from opposite perspectives. Angiogenic cytokine therapy has been widely regarded as an attractive approach both for treating ischemic heart disease and for enhancing arterioprotective functions of the endothelium; conversely, a variety of studies suggest that neovascularization contributes to the growth of atherosclerotic lesions and is a key factor in plaque destabilization leading to rupture.

Objective of this Study Genetic Engineering of Stem cell by inserting and expression of VEGF plasmid for Angiogenesis. Transplantation of Stem cell in Mouse for in vivo expression of VEGF Optimizing Condition for Expression of VEGF in vivo and In Vitro Submitted By: Shams-uz-Zaman 2

Enhancement of Angiogenesis through Genetically Engineered Human Stem Cells

Materials and Methods A. Synthesis of Biodegradable Polymeric Nanoparticles. B. Poly(-Amino Ester) synthesize as describe by Zugates et al(22).

Human Stem Cell Culture Bone marroe derived hMSCs in Growth Medium Consisting of DMEM supplemented with 10% FBS, 100mM Sodium Pyruvate. Human embryonic Stem cell-derived cells (hESdCs) will be grown in inactiviated mouse embryonic fibroblast (MEFs) in hESC growth medium consists of 80% Knockout DMEM supplemented with 20% knockout serum replacement,4ng/mL basic fibroblast factor (bFGF).

C.

Transfection Through Biodegradable Polymeric Nanoparticles Cell(hMSCs & hESdCs) transfection withVEGF plasmid and EGFP as reporter gene in growth medium containing optimized Poly(-Amino ester) Transfection condition.

Through Liposomal Method Through Biolistic Method ELISA for the detection of VEGF Production from Human stem cells in medium supernatant. Reverse Transcrition Polymerase Chain Reaction(RT-PCR). To test the presence of human stem cells in mouse tissues. PCR analysis for human chromosome 17 satellite region. Quantitative Real-Time PCR. To Examine VEGF mRNA expression in Human Stem cell after Transfection. Fabrication of Cylindrical PLGA/PLLA Scaffolds (10mmdiameter and Height 2mm) by using solvent casting and Particulate leaching method. Cell of each type would be seeded into scaffold before 24hours of implantation, and then implanted into stem cell space in the dorsal region of Mice. Hind Limb Ischemia will be induced as describe by Cho et.al(23). After 4 weeks after treatment mice will be killed and, and limb of the ischemic sides will be analyzed. To asses the hind limb muscle viability and Morphology.

D.

Subcutaneous Implantation of Stem Cell-seeded Scaffolds.

E.

Transplantation of stem cell into a Mouse Ischemic Hindlimb Model.

F.

Triphenyltetrazolium Chloride (TTC) Staining.

G.

Estimation of Micro vessels Density

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Enhancement of Angiogenesis through Genetically Engineered Human Stem Cells Using immunohistochemistry staining Techniques.

Reference 1. Jabbarzadeh E, et al. (2008) Induction of angiogenesis in tissue-engineered scaffolds designed for bone repair: A combined gene therapy-cell transplantation approach.Proc Natl Acad Sci USA 105:1109911104. Iba O, et al. (2002) Angiogenesis by implantation of peripheral blood mononuclear cells and platelets into ischemic limbs. Circulation 106:20192025. Ye L, et al. (2007) Transplantation of nanoparticle transfected skeletal myoblasts overexpressing vascular endothelial growth factor-165 for cardiac repair. Circulation116:I113I120. Franz WM, Zaruba M, Theiss H, David R (2003) Stem-cell homing and tissue regeneration in ischaemic cardiomyopathy. Lancet 362:675676. Kocher AA, et al. (2001) Neovascularization of ischemic myocardium by human bonemarrow derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 7:430436. Cho SW, et al. (2007) Improvement of postnatal neovascularization by human embryonic stem cell derived endothelial-like cell transplantation in a mouse model of hindlimb ischemia. Circulation 116:24092419. Kinnaird T, et al. (2004) Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ Res 94:678685. Crisostomo PR, et al. (2008) Embryonic stem cells attenuate myocardial dysfunction and inflammation after surgical global ischemia via paracrine actions. Am J Physiol 295:H1726H1735. Chavakis E, Urbich C, Dimmeler S (2008) Homing and engraftment of progenitor cells: A prerequisite for cell therapy. J Mol Cell Cardiol 45:514522. Matsumoto R, et al. (2005) Vascular endothelial growth factor-expressing mesenchymal stem cell transplantation for the treatment of acute myocardial infarction. Arterioscler Thromb Vasc Biol 25:11681173. Gnecchi M, et al. (2005) Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells. Nat Med 11:367368. Li W, et al. (2007) Bcl-2 engineered MSCs inhibited apoptosis and improved heart function. Stem Cells 25:21182127. Elmadbouh I, et al. (2007) Ex vivo delivered stromal cell-derived factor-1alpha promotes stem cell homing and induces angiomyogenesis in the infarcted myocardium. JMol Cell Cardiol 42:792803. Anderson DG, Akinc A, Hossain N, Langer R (2005) Structure/property studies of polymeric gene delivery using a library of poly(beta-amino esters). Mol Ther 11:426434.

2. 3.

4. 5.

6.

7.

8.

9. 10.

11. 12. 13.

14.

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Enhancement of Angiogenesis through Genetically Engineered Human Stem Cells 15. Asahara, T., T. Murohara, A. Sullivan, M. Silver, R. Zee, T. Bernhard Witzenbichler, G. Schatteman, J. Isner (1997). Isolation of Putative Progenitor Endothelial Cells for Angiogenesis. Science: 275( 5302) pp. 964-966. Stamm, C., B. Westphal, H. Kleine, M. Petzsch, C. Kittner, H. Klinge, C. Schmichen, C. Nienaber, M. Freund, G. Steinhoff (2003). Autologous bonemarrow stem-cell transplantation for myocardial regeneration. Lancet 2003; 361: 4546. Weidner, N., Semple, J. P., Welch, W. R., and Folkman, J. (1991) N. Engl.J. Med. 324, l-8. Robson, M. C., Phillips, L. G., Lawrence, W. T., Bishop, J. B., Youngerman,J. S., Hayward, P. G., Broemeling, L. D., and Heggers, J. P.(1992). Ann. Surg., in press. Ezekowitz, R. A,, Mulliken, J. B., and Folkman, J. (1992) N. Engl. J. Med. Folkman; J.; Szabo, S:, StovroffP'M., 'McNeil, Pl, Li, W., and Shing, Y.(1991) Ann. Surg. 214,414-427. Ingber, D., Fujita, T., Kishimoto, S., Sudo, K., Kanamaru, T., Brem, H. and Folkman, J. (1990) Nature 348,555-557. Zugates GT, et al. (2007) Rapid optimization of gene delivery by parallel endmodification of poly (beta-amino ester)s. Mol Ther 15:13061312. Cho SW, et al. (2007) Improvement of postnatal neovascularization by human embryonic stem cell derived endothelial-like cell transplantation in a mouse model of hindlimb ischemia. Circulation 116:24092419.

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17. 18.

19. 20. 21. 22. 23.

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