Cell Therapy of Ischemia
Development of the Technology for Cell Therapy of Ischemia with Adipose Tissue Stem Cells and Recombinant Human Alpha-Fetoprotein
Tech Area / Field
- MED-DRG/Drug Discovery/Medicine
8 Project completed
Senior Project Manager
Melnikov V G
Institute of Immunological Engineering, Russia, Moscow reg., Lyubuchany
- Moscow State University / Faculty of Basic Medicine, Russia, Moscow
- IDI Farmaceutici S.p.A., Italy, Rome\nUniversity of Turku / Finnish-Russian Joint Biotechnology Laboratory, Finland, Turku
Project summaryFast developing medical biotechnology basing on the principles of stem cell therapy pays a great attention to searching for the protein and peptide factors increasing viability of tissue and cellular materials in the all steps of preparation for transplantation including in vitro cell culture and cell survival in vivo.
The solution of this problem is of particular importance for the cell therapy of cardiovascular diseases, which are the general cause of morbidity and mortality in the industrial countries. Cell therapy offers a modern solution of the problem of curing the cardiovascular pathology and enhancement of the efficacy of medication of impaired myocardium via stimulation of cell regeneration or vascular reconstruction due to stem cells transplantation. The most available and potent source of autologous stem cells is adipose tissue, from which the cells analogous to bone marrow mesenchymal stem cells can be isolated. Recent experimental results have shown that adipose tissue stem cells (ATSCs), when cultured in vitro, were secreting growth factors promoting endothelial cell survival and stimulating vascular growth. ATSCs can be successfully used for restoration of rat heart function following myocardial infarction and for correction of blood flow disorder in ischemic mice limb. To date, the experimental study and clinical trials are performing for the ATSC usage for treatment of the human cardiovascular pathology. At the same time, to use ATSCs for cell therapy, it is necessary to search for the new pharmacological agents promoting the increase of the yield of progenitor cells in the in vitro culture as well as to promote cell viability during their transplantation into the ischemic area.
Present project is designed to develop the new technology for adipose tissue-derived stem cell propagation with the usage of recombinant and peptide analogues of embryonic growth stimulator, immunoregulator and differentiating factor - human alpha-fetoprotein (rhAFP), which is isolated from the culture liquid of recombinant yeast producer strain Sacharomyces cerevisiae.
AFP is a major mammalian embryonic serum protein belonging to the albumin family of proteins. This protein is a natural embryonic stem cell growth and differentiation factor having the ability to stimulate growth of immature cells of bone marrow, immune system, nervous tissue, liver and a number of other cells in developing embryo. AFP operates as a molecular carrier for biologically active hydrophobic ligands including polyunsaturated fatty acids, fat-soluble vitamins, steroids or metals, to deliver them inside developing cells. Embryonic stem cells and immature cells of immune system express specific membrane receptors for this protein, which supply directivity of AFP action. It has been shown recently that AFP is able to regulate the programmed cell death (apoptosis) in developing cells of immature phenotype including embryonic and cancerous ones. The active site of the AFP molecule, which is responsible for regulation of apoptosis, was identified, and it was shown that synthetic peptides modeling the AFP pro-apoptotic site are able to abolish spontaneous death of developing immature cells in vitro. The project is focused on studying of the effects of rhAFP and its synthetic peptide derivatives on the apoptotic processes arising during the ATSC culturing, as well as on their differentiation, migration and proliferation. It is also planned to study effects of rhAFP and its peptides on the process of apoptosis during the in vivo cell implantation, to increase implanted cell viability and therapeutic efficacy of the ischemia treatment. A possibility for such an application is based on the ability of AFP to affect the inflammatory process (via suppression of inflammatory mediator production by macrophages) and angiogenesis (via potentiation of endothelial cell activity). The complex of biological properties of the AFP, availability of its gene-engineering analogue as well as the advantages of the usage of adult adipose tissue as a source of stem cells are the decisive arguments for developing the efficient technology for its potential application for the treatment of cardiovascular pathology.
To reach the Project objective, a collaborative group of high professional scientists is created integrating the personnel of the Faculty of Basic Medicine, Moscow Lomonosov’s State University, having the great experience in the field of fundamental and applied study of cardiovascular pathology, and personnel of the Institute of Immunological Engineering having more than 10-year experience of the study in the field of AFP properties and molecular mechanisms of apoptosis. The implementators of the Project are the authors of a number of inventions in the field of modern biotechnology and have a successful experience of collaboration with ISTC (Projects ##1878, 2915, 401).
A Goal of the Project: Elaboration of the clinically applicable technique of the in vitro adipose stem cell culturing in the presence of rhAFP and/or AFP-peptides aimed to an increase of stem cell viability, proliferation activity, and differentiation potential. As well as a development of experimental protocols for the combined administration of stem cells with rhAFP and AFP- peptides into the ischemic myocardium and skeletal muscles in order to improve cell survival and functional efficacy of transplanted cells is also planned.
Main Tasks of the Project: 1. Production of the experimental lots of the yeast recombinant AFP and synthetic AFP- derived peptides for project execution and functional studies. 2. Study of regulatory effects of rhAFP and synthetic peptide AFP-derivatives on the differentiation potential of adipose tissue stem cells (ATSC). 3. Study of the effects of rhAFP and AFP-derived synthetic peptides on the apoptosis of the ATSC in vitro. 4. Development of the new technology of the in vitro adipose stem cell preparation aimed to increase its proliferation, invasive and migratory potential. 5. Study of the effects of rhAFP and AFP-derived synthetic peptides on the viability of ATSC and vascular cells in vitro in conditions of a hypoxia and an inflammation. 6. Study of the effects of rhAFP and AFP-derived synthetic peptides on the therapeutic potential of ATSC in experimental models of ischemia in vivo.
Anticipated Results: In the scope of the project, the results of fundamental significance will be obtained: the experimental data characterizing the effects of rhAFP and AFP-derived peptides on the in vitro stem cell proliferation, migration and differentiation; the experimental data showing molecular mechanisms underlying the activities related to regulation of stem cell growth and differentiation; the active sites of the AFP molecule, which are responsible for regulation of stem cell growth and differentiation will be identified.
The results of applied and commercial significance are the following: the protocols of using of rhAFP and AFP-derived peptides in vitro for significant improving of the yield of ATSCs; high technology commercial products on the base of rhAFP and AFP-derived peptides for medical purposes; protocols for in vivo implantation of adipose-derived stem cells in combination with rhAFP and/or AFP-derived peptides to increase the tissue-protecting effects of ATSCs; experimental evidence for application of rhAFP and AFP-derived peptides to improve the blood supply in ischemic tissue and to restore the function of damaged tissue.
Technical Approach and Methodology: Upon cultivation of the recombinant yeast strains, the fermentation apparatus with the automatic monitoring of the temperature and pH regimens will be utilized. Recombinant AFP will be isolated from the culture medium of the yeast strain Saccharomyces cerevisiae transfected with the gene of human AFP by liquid gel-chromatography, ion exchange chromatography and affinity chromatography. The peptides will be synthesized by solid phase peptide synthesis. Characterization of rhAFP and AFP-peptides will be performed by HPLC chromatography, PAAG-electrophoresis, and immune blot analysis. Adipose tissue stem cells will be isolated from the adult human liposuction products by treatment by the mixture of collagenase type I and dispase. To analyse the biological activity of rhAFP and its peptides, the methods of the in vitro cell culture as well as molecular biology methods and experimental models in vivo will be used. To study cell differentiation, survival and apoptosis, methods of light microscopy, phase contrast microscopy and fluorescent microscopy will be applied. Analysis of the molecular mechanisms of apoptosis will be done by using specific substrates for active caspases in the cytosolic lysates and expression of pro- and antiapoptotic markers in culturing cells in vitro. Signal transduction mechanisms will be studied by methods of ligand blotting, inhibitory analysis, Western immunoblotting, ELISA, PCR and immune precipitation. To evaluate therapeutic potential of progenitor cells in vivo, the models of mice hind limb ischemia, vascular growth in 3D Matrigel and artificial myocardial infarction will be used. Evaluation of angiogenesis will be performed by counting of the number of vessels stained by specific antibodies to endothelial and smooth muscle cell antigens.