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Engineering Ex Vivo–Expanded Marrow Stromal Cells to Secrete Calcitonin Gene–Related Peptide Using Adenoviral Vector
Calcitonin gene–related peptide (CGRP) is a target for cardiovascular gene therapy. Marrow stromal cells (MSCs) hold promise for use in adult stem cell–based cell and gene therapy. To determine the feasibility of adenoviral‐mediated CGRP gene transfer into ex vivo–expanded MSCs, rat MSCs were isolat...
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Published in: | Stem cells (Dayton, Ohio) Ohio), 2004-01, Vol.22 (7), p.1279-1291 |
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description | Calcitonin gene–related peptide (CGRP) is a target for cardiovascular gene therapy. Marrow stromal cells (MSCs) hold promise for use in adult stem cell–based cell and gene therapy. To determine the feasibility of adenoviral‐mediated CGRP gene transfer into ex vivo–expanded MSCs, rat MSCs were isolated, ex vivo expanded, and transduced with adenoviruses. Adprepro‐CGRP and AdntlacZ, adenoviral vectors containing prepro‐CGRP or nuclear‐targeted β‐galactosidase reporter gene ntlacZ under the control of Rous sarcoma virus promoter, were used. In this study, it can be shown that transduction efficiency of adenoviral‐mediated gene transfer into ex vivo–expanded MSCs is dose dependent, transgene expression persists for more than 21 days in culture, and adenoviral transduction does not alter the proliferation or viability of MSCs. Transduced MSCs retain multipotentiality and transgene expression after cell differentiation. The expression and secretion of CGRP by Adprepro‐ CGRP–transduced MSCs was confirmed by Western blot analysis and enzyme immunoassay. The secretion of CGRP by Adprepro‐CGRP–transduced MSCs is dose dependent, and the transduced cells release as much as 9.5 ± 0.4 pmol CGRP/1 × 106 cells/48 hours (mean ± standard error of mean, n = 3) into culture medium at a multiplicity of infection of 300. Furthermore, culture supernatant from Adprepro‐CGRP–transduced MSCs increases intracellular cyclic AMP levels in pulmonary artery smooth muscle cells in culture. These findings suggest that replication‐deficient recombinant adenovirus can be used to gene engineer ex vivo–expanded MSCs and that high‐level secretion of biologically active CGRP can be achieved, underscoring the clinical potential of using this novel adult stem cell–based cell and gene therapy strategy for the treatment of cardiovascular diseases. |
doi_str_mv | 10.1634/stemcells.2004-0032 |
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Marrow stromal cells (MSCs) hold promise for use in adult stem cell–based cell and gene therapy. To determine the feasibility of adenoviral‐mediated CGRP gene transfer into ex vivo–expanded MSCs, rat MSCs were isolated, ex vivo expanded, and transduced with adenoviruses. Adprepro‐CGRP and AdntlacZ, adenoviral vectors containing prepro‐CGRP or nuclear‐targeted β‐galactosidase reporter gene ntlacZ under the control of Rous sarcoma virus promoter, were used. In this study, it can be shown that transduction efficiency of adenoviral‐mediated gene transfer into ex vivo–expanded MSCs is dose dependent, transgene expression persists for more than 21 days in culture, and adenoviral transduction does not alter the proliferation or viability of MSCs. Transduced MSCs retain multipotentiality and transgene expression after cell differentiation. The expression and secretion of CGRP by Adprepro‐ CGRP–transduced MSCs was confirmed by Western blot analysis and enzyme immunoassay. The secretion of CGRP by Adprepro‐CGRP–transduced MSCs is dose dependent, and the transduced cells release as much as 9.5 ± 0.4 pmol CGRP/1 × 106 cells/48 hours (mean ± standard error of mean, n = 3) into culture medium at a multiplicity of infection of 300. Furthermore, culture supernatant from Adprepro‐CGRP–transduced MSCs increases intracellular cyclic AMP levels in pulmonary artery smooth muscle cells in culture. These findings suggest that replication‐deficient recombinant adenovirus can be used to gene engineer ex vivo–expanded MSCs and that high‐level secretion of biologically active CGRP can be achieved, underscoring the clinical potential of using this novel adult stem cell–based cell and gene therapy strategy for the treatment of cardiovascular diseases.</description><identifier>ISSN: 1066-5099</identifier><identifier>EISSN: 1549-4918</identifier><identifier>DOI: 10.1634/stemcells.2004-0032</identifier><identifier>PMID: 15579646</identifier><language>eng</language><publisher>Bristol: John Wiley & Sons, Ltd</publisher><subject>Adenoviridae - genetics ; Adenovirus ; Adipocytes - cytology ; Adipocytes - metabolism ; Animals ; Avian Sarcoma Viruses - genetics ; beta-Galactosidase - metabolism ; Blotting, Western ; Bone Marrow Cells - cytology ; Bone Marrow Cells - metabolism ; Calcitonin Gene-Related Peptide - genetics ; Calcitonin Gene-Related Peptide - secretion ; Calcitonin gene–related peptide ; Cell Differentiation ; Cell Proliferation ; Cell Survival ; Cyclic AMP - metabolism ; Differentiation ; Dose-Response Relationship, Drug ; Gene expression ; Gene therapy ; Gene Transfer Techniques ; Genes, Reporter ; Genetic Vectors ; Humans ; Immunoenzyme Techniques ; Male ; Marrow stromal cells ; Osteoblasts - cytology ; Osteoblasts - metabolism ; Rats ; Rous sarcoma virus ; Stromal Cells - cytology ; Stromal Cells - metabolism ; Time Factors ; Transgenes</subject><ispartof>Stem cells (Dayton, Ohio), 2004-01, Vol.22 (7), p.1279-1291</ispartof><rights>Copyright © 2004 AlphaMed Press</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c3869-4b86a276954d49358682cf7f09fbdf53e048760f1ec4d0d5f3ba3c58f4fc7dec3</citedby><cites>FETCH-LOGICAL-c3869-4b86a276954d49358682cf7f09fbdf53e048760f1ec4d0d5f3ba3c58f4fc7dec3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15579646$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Deng, Weiwen</creatorcontrib><creatorcontrib>Bivalacqua, Trinity J.</creatorcontrib><creatorcontrib>Chattergoon, Natasha N.</creatorcontrib><creatorcontrib>Jeter, James R.</creatorcontrib><creatorcontrib>Kadowitz, Philip J.</creatorcontrib><title>Engineering Ex Vivo–Expanded Marrow Stromal Cells to Secrete Calcitonin Gene–Related Peptide Using Adenoviral Vector</title><title>Stem cells (Dayton, Ohio)</title><addtitle>Stem Cells</addtitle><description>Calcitonin gene–related peptide (CGRP) is a target for cardiovascular gene therapy. Marrow stromal cells (MSCs) hold promise for use in adult stem cell–based cell and gene therapy. To determine the feasibility of adenoviral‐mediated CGRP gene transfer into ex vivo–expanded MSCs, rat MSCs were isolated, ex vivo expanded, and transduced with adenoviruses. Adprepro‐CGRP and AdntlacZ, adenoviral vectors containing prepro‐CGRP or nuclear‐targeted β‐galactosidase reporter gene ntlacZ under the control of Rous sarcoma virus promoter, were used. In this study, it can be shown that transduction efficiency of adenoviral‐mediated gene transfer into ex vivo–expanded MSCs is dose dependent, transgene expression persists for more than 21 days in culture, and adenoviral transduction does not alter the proliferation or viability of MSCs. Transduced MSCs retain multipotentiality and transgene expression after cell differentiation. The expression and secretion of CGRP by Adprepro‐ CGRP–transduced MSCs was confirmed by Western blot analysis and enzyme immunoassay. The secretion of CGRP by Adprepro‐CGRP–transduced MSCs is dose dependent, and the transduced cells release as much as 9.5 ± 0.4 pmol CGRP/1 × 106 cells/48 hours (mean ± standard error of mean, n = 3) into culture medium at a multiplicity of infection of 300. Furthermore, culture supernatant from Adprepro‐CGRP–transduced MSCs increases intracellular cyclic AMP levels in pulmonary artery smooth muscle cells in culture. These findings suggest that replication‐deficient recombinant adenovirus can be used to gene engineer ex vivo–expanded MSCs and that high‐level secretion of biologically active CGRP can be achieved, underscoring the clinical potential of using this novel adult stem cell–based cell and gene therapy strategy for the treatment of cardiovascular diseases.</description><subject>Adenoviridae - genetics</subject><subject>Adenovirus</subject><subject>Adipocytes - cytology</subject><subject>Adipocytes - metabolism</subject><subject>Animals</subject><subject>Avian Sarcoma Viruses - genetics</subject><subject>beta-Galactosidase - metabolism</subject><subject>Blotting, Western</subject><subject>Bone Marrow Cells - cytology</subject><subject>Bone Marrow Cells - metabolism</subject><subject>Calcitonin Gene-Related Peptide - genetics</subject><subject>Calcitonin Gene-Related Peptide - secretion</subject><subject>Calcitonin gene–related peptide</subject><subject>Cell Differentiation</subject><subject>Cell Proliferation</subject><subject>Cell Survival</subject><subject>Cyclic AMP - metabolism</subject><subject>Differentiation</subject><subject>Dose-Response Relationship, Drug</subject><subject>Gene expression</subject><subject>Gene therapy</subject><subject>Gene Transfer Techniques</subject><subject>Genes, Reporter</subject><subject>Genetic Vectors</subject><subject>Humans</subject><subject>Immunoenzyme Techniques</subject><subject>Male</subject><subject>Marrow stromal cells</subject><subject>Osteoblasts - cytology</subject><subject>Osteoblasts - metabolism</subject><subject>Rats</subject><subject>Rous sarcoma virus</subject><subject>Stromal Cells - cytology</subject><subject>Stromal Cells - metabolism</subject><subject>Time Factors</subject><subject>Transgenes</subject><issn>1066-5099</issn><issn>1549-4918</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2004</creationdate><recordtype>article</recordtype><recordid>eNqNkc1u1DAURiMEoqXlCZCQV-xS7PgvFqtqFNpKrUCdtlvLY19XRkk82J52uuMd-oY8SRPNCJaw8l1837nyPVX1geATIij7nAsMFvo-nzQYsxpj2ryqDglnqmaKtK-nGQtRc6zUQfUu5x8YE8bb9m11QDiXSjBxWG278T6MACmM96jborvwEH__eu62azM6cOjKpBQf0bKkOJgeLeZ9qES0BJugAFqY3oYSxzCiMxhhql5Db8rU_A7rEhyg2zyjTx2M8SGkiXEHtsR0XL3xps_wfv8eVbdfu5vFeX357exicXpZW9qK6SerVphGCsWZY4ryVrSN9dJj5VfOcwqYtVJgT8Ayhx33dGWo5a1n3koHlh5Vn3bcdYo_N5CLHkKez2ZGiJushSRUKSX_GSRSCjnddQrSXdCmmHMCr9cpDCY9aYL1bEb_MaNnM3o2M7U-7vGb1QDub2evYgp82QUeQw9P_8PUy5vuqmlIIxV9Adgxor0</recordid><startdate>20040101</startdate><enddate>20040101</enddate><creator>Deng, Weiwen</creator><creator>Bivalacqua, Trinity J.</creator><creator>Chattergoon, Natasha N.</creator><creator>Jeter, James R.</creator><creator>Kadowitz, Philip J.</creator><general>John Wiley & Sons, Ltd</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QO</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>20040101</creationdate><title>Engineering Ex Vivo–Expanded Marrow Stromal Cells to Secrete Calcitonin Gene–Related Peptide Using Adenoviral Vector</title><author>Deng, Weiwen ; Bivalacqua, Trinity J. ; Chattergoon, Natasha N. ; Jeter, James R. ; Kadowitz, Philip J.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c3869-4b86a276954d49358682cf7f09fbdf53e048760f1ec4d0d5f3ba3c58f4fc7dec3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2004</creationdate><topic>Adenoviridae - genetics</topic><topic>Adenovirus</topic><topic>Adipocytes - cytology</topic><topic>Adipocytes - metabolism</topic><topic>Animals</topic><topic>Avian Sarcoma Viruses - genetics</topic><topic>beta-Galactosidase - metabolism</topic><topic>Blotting, Western</topic><topic>Bone Marrow Cells - cytology</topic><topic>Bone Marrow Cells - metabolism</topic><topic>Calcitonin Gene-Related Peptide - genetics</topic><topic>Calcitonin Gene-Related Peptide - secretion</topic><topic>Calcitonin gene–related peptide</topic><topic>Cell Differentiation</topic><topic>Cell Proliferation</topic><topic>Cell Survival</topic><topic>Cyclic AMP - metabolism</topic><topic>Differentiation</topic><topic>Dose-Response Relationship, Drug</topic><topic>Gene expression</topic><topic>Gene therapy</topic><topic>Gene Transfer Techniques</topic><topic>Genes, Reporter</topic><topic>Genetic Vectors</topic><topic>Humans</topic><topic>Immunoenzyme Techniques</topic><topic>Male</topic><topic>Marrow stromal cells</topic><topic>Osteoblasts - cytology</topic><topic>Osteoblasts - metabolism</topic><topic>Rats</topic><topic>Rous sarcoma virus</topic><topic>Stromal Cells - cytology</topic><topic>Stromal Cells - metabolism</topic><topic>Time Factors</topic><topic>Transgenes</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Deng, Weiwen</creatorcontrib><creatorcontrib>Bivalacqua, Trinity J.</creatorcontrib><creatorcontrib>Chattergoon, Natasha N.</creatorcontrib><creatorcontrib>Jeter, James R.</creatorcontrib><creatorcontrib>Kadowitz, Philip J.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Biotechnology Research Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Stem cells (Dayton, Ohio)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Deng, Weiwen</au><au>Bivalacqua, Trinity J.</au><au>Chattergoon, Natasha N.</au><au>Jeter, James R.</au><au>Kadowitz, Philip J.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Engineering Ex Vivo–Expanded Marrow Stromal Cells to Secrete Calcitonin Gene–Related Peptide Using Adenoviral Vector</atitle><jtitle>Stem cells (Dayton, Ohio)</jtitle><addtitle>Stem Cells</addtitle><date>2004-01-01</date><risdate>2004</risdate><volume>22</volume><issue>7</issue><spage>1279</spage><epage>1291</epage><pages>1279-1291</pages><issn>1066-5099</issn><eissn>1549-4918</eissn><abstract>Calcitonin gene–related peptide (CGRP) is a target for cardiovascular gene therapy. Marrow stromal cells (MSCs) hold promise for use in adult stem cell–based cell and gene therapy. To determine the feasibility of adenoviral‐mediated CGRP gene transfer into ex vivo–expanded MSCs, rat MSCs were isolated, ex vivo expanded, and transduced with adenoviruses. Adprepro‐CGRP and AdntlacZ, adenoviral vectors containing prepro‐CGRP or nuclear‐targeted β‐galactosidase reporter gene ntlacZ under the control of Rous sarcoma virus promoter, were used. In this study, it can be shown that transduction efficiency of adenoviral‐mediated gene transfer into ex vivo–expanded MSCs is dose dependent, transgene expression persists for more than 21 days in culture, and adenoviral transduction does not alter the proliferation or viability of MSCs. Transduced MSCs retain multipotentiality and transgene expression after cell differentiation. The expression and secretion of CGRP by Adprepro‐ CGRP–transduced MSCs was confirmed by Western blot analysis and enzyme immunoassay. The secretion of CGRP by Adprepro‐CGRP–transduced MSCs is dose dependent, and the transduced cells release as much as 9.5 ± 0.4 pmol CGRP/1 × 106 cells/48 hours (mean ± standard error of mean, n = 3) into culture medium at a multiplicity of infection of 300. Furthermore, culture supernatant from Adprepro‐CGRP–transduced MSCs increases intracellular cyclic AMP levels in pulmonary artery smooth muscle cells in culture. These findings suggest that replication‐deficient recombinant adenovirus can be used to gene engineer ex vivo–expanded MSCs and that high‐level secretion of biologically active CGRP can be achieved, underscoring the clinical potential of using this novel adult stem cell–based cell and gene therapy strategy for the treatment of cardiovascular diseases.</abstract><cop>Bristol</cop><pub>John Wiley & Sons, Ltd</pub><pmid>15579646</pmid><doi>10.1634/stemcells.2004-0032</doi><tpages>13</tpages></addata></record> |
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subjects | Adenoviridae - genetics Adenovirus Adipocytes - cytology Adipocytes - metabolism Animals Avian Sarcoma Viruses - genetics beta-Galactosidase - metabolism Blotting, Western Bone Marrow Cells - cytology Bone Marrow Cells - metabolism Calcitonin Gene-Related Peptide - genetics Calcitonin Gene-Related Peptide - secretion Calcitonin gene–related peptide Cell Differentiation Cell Proliferation Cell Survival Cyclic AMP - metabolism Differentiation Dose-Response Relationship, Drug Gene expression Gene therapy Gene Transfer Techniques Genes, Reporter Genetic Vectors Humans Immunoenzyme Techniques Male Marrow stromal cells Osteoblasts - cytology Osteoblasts - metabolism Rats Rous sarcoma virus Stromal Cells - cytology Stromal Cells - metabolism Time Factors Transgenes |
title | Engineering Ex Vivo–Expanded Marrow Stromal Cells to Secrete Calcitonin Gene–Related Peptide Using Adenoviral Vector |
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