Optimization of culture conditions to enhance transfection of human CD34+ cells by electroporation
The ability to culture CD34+ stem cells, while maintaining their pluripotency, is essential for manipulations such as gene transfection for therapeutic trials. Human peripheral blood (PB) CD34+ cells (> or = 90% purity) were cultured for up to 4 days in serum-free culture medium supplemented with...
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| Veröffentlicht in: | Bone marrow transplantation (Basingstoke) 2001-06, Vol.27 (11), p.1201-1209 |
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| creator | WU, M. H SMITH, S. L DANET, G. H LIN, A. M WILLIAMS, S. F LIEBOWITZ, D. N DOLAN, M. E |
| description | The ability to culture CD34+ stem cells, while maintaining their pluripotency, is essential for manipulations such as gene transfection for therapeutic trials. Human peripheral blood (PB) CD34+ cells (> or = 90% purity) were cultured for up to 4 days in serum-free culture medium supplemented with thrombopoietin (TPO), stem cell factor (SCF), Flt-3 ligand (Flt-3L), with or without PIXY321 (IL-3/GM-CSF fusion protein) and human serum. The CD34 mean fluorescence intensity (MFI) and cell cycle status were evaluated daily using flow cytometry and hypotonic propidium iodide. Prior to culture (day 0), 97.0 +/- 0.9%, 1.9 +/- 0.3% and 1.0 +/- 0.6% of the selected CD34+ cells were in G0-G1, S-phase, or G2-M, respectively. After 2-4 days in culture with TPO/SCF/Flt-3L, there was an increase in the percent of cells in S-phase to 26.4 +/- 0.1% without significant loss of CD34 MFI. The addition of PIXY321 increased.the percentage of CD34+ cells in S-phase to 36.3 +/- 4.0%, but the CD34 MFI and numbers of CFU (colony-forming units) were significantly decreased at day 3 when cultured with PIXY321 or various recombinant cytokine combinations that included IL-3 and IL-6. There is an increase from day 0 to day 4 in the percentages of CD34+ with CD38-, HLA-DR-, and c-kit(low), but not Thy-1+ cells. Electroporation with EGFP reporter gene showed that 1-2 days of pre-stimulation in X-VIVO 10 supplemented with TPO/SCF/Flt-3L was necessary and sufficient for efficient transfection. Flow cytometry analysis demonstrated that 22% of the viable cells are CD34+/EGFP+ 48 h post electroporation. The introduced reporter gene appears to be stable as determined by EGFP+/LTC-IC (long-term colony-initiating cells), at 30-40 positive colonies (16 +/- 7%) per 1 x 10(5) electroporated CD34+ cells. |
| doi_str_mv | 10.1038/sj.bmt.1703054 |
| format | Article |
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H ; SMITH, S. L ; DANET, G. H ; LIN, A. M ; WILLIAMS, S. F ; LIEBOWITZ, D. N ; DOLAN, M. E</creator><creatorcontrib>WU, M. H ; SMITH, S. L ; DANET, G. H ; LIN, A. M ; WILLIAMS, S. F ; LIEBOWITZ, D. N ; DOLAN, M. E</creatorcontrib><description>The ability to culture CD34+ stem cells, while maintaining their pluripotency, is essential for manipulations such as gene transfection for therapeutic trials. Human peripheral blood (PB) CD34+ cells (> or = 90% purity) were cultured for up to 4 days in serum-free culture medium supplemented with thrombopoietin (TPO), stem cell factor (SCF), Flt-3 ligand (Flt-3L), with or without PIXY321 (IL-3/GM-CSF fusion protein) and human serum. The CD34 mean fluorescence intensity (MFI) and cell cycle status were evaluated daily using flow cytometry and hypotonic propidium iodide. Prior to culture (day 0), 97.0 +/- 0.9%, 1.9 +/- 0.3% and 1.0 +/- 0.6% of the selected CD34+ cells were in G0-G1, S-phase, or G2-M, respectively. After 2-4 days in culture with TPO/SCF/Flt-3L, there was an increase in the percent of cells in S-phase to 26.4 +/- 0.1% without significant loss of CD34 MFI. The addition of PIXY321 increased.the percentage of CD34+ cells in S-phase to 36.3 +/- 4.0%, but the CD34 MFI and numbers of CFU (colony-forming units) were significantly decreased at day 3 when cultured with PIXY321 or various recombinant cytokine combinations that included IL-3 and IL-6. There is an increase from day 0 to day 4 in the percentages of CD34+ with CD38-, HLA-DR-, and c-kit(low), but not Thy-1+ cells. Electroporation with EGFP reporter gene showed that 1-2 days of pre-stimulation in X-VIVO 10 supplemented with TPO/SCF/Flt-3L was necessary and sufficient for efficient transfection. Flow cytometry analysis demonstrated that 22% of the viable cells are CD34+/EGFP+ 48 h post electroporation. The introduced reporter gene appears to be stable as determined by EGFP+/LTC-IC (long-term colony-initiating cells), at 30-40 positive colonies (16 +/- 7%) per 1 x 10(5) electroporated CD34+ cells.</description><identifier>ISSN: 0268-3369</identifier><identifier>EISSN: 1476-5365</identifier><identifier>DOI: 10.1038/sj.bmt.1703054</identifier><identifier>PMID: 11551032</identifier><identifier>CODEN: BMTRE9</identifier><language>eng</language><publisher>Basingstoke: Nature Publishing Group</publisher><subject>Antigens, CD34 - blood ; Biological and medical sciences ; Biotechnology ; Bone marrow ; Bone marrow transplantation ; CD34 antigen ; CD38 antigen ; Cell culture ; Cell Culture Techniques - methods ; Cell cycle ; Cell Cycle - drug effects ; Colonies ; Culture ; Cytokines ; Electroporation ; Electroporation - methods ; Flow Cytometry ; Fluorescence ; Fundamental and applied biological sciences. Psychology ; Fusion protein ; Genetic engineering ; Genetic technics ; Granulocyte-macrophage colony-stimulating factor ; Granulocyte-Macrophage Colony-Stimulating Factor - pharmacology ; Hematopoietic Stem Cells - drug effects ; Hematopoietic Stem Cells - immunology ; Hematopoietic Stem Cells - metabolism ; Histocompatibility antigen HLA ; Humans ; Immunophenotyping ; Interleukin 3 ; Interleukin 6 ; Interleukin-3 - pharmacology ; Iodides ; Membrane Proteins - pharmacology ; Methods. Procedures. Technologies ; Optimization ; Peripheral blood ; Pluripotency ; Propidium iodide ; Recombinant Fusion Proteins - pharmacology ; Reporter gene ; Stem cell factor ; Stem Cell Factor - pharmacology ; Stem cell transplantation ; Stem cells ; Thrombopoietin ; Thrombopoietin - pharmacology ; Transfection ; Transfection - methods ; Transplantation ; Vectors (cloning, transfer, expression). Insertion sequences and transposons</subject><ispartof>Bone marrow transplantation (Basingstoke), 2001-06, Vol.27 (11), p.1201-1209</ispartof><rights>2001 INIST-CNRS</rights><rights>Copyright Nature Publishing Group Jun 2001</rights><rights>Macmillan Publishers Limited 2001.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c415t-d08766e22c6c555ae6a2964a0141e359d3f38a954e1aa2927e89f39b5fac20bb3</citedby><cites>FETCH-LOGICAL-c415t-d08766e22c6c555ae6a2964a0141e359d3f38a954e1aa2927e89f39b5fac20bb3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27923,27924</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=1103099$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/11551032$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>WU, M. H</creatorcontrib><creatorcontrib>SMITH, S. L</creatorcontrib><creatorcontrib>DANET, G. H</creatorcontrib><creatorcontrib>LIN, A. M</creatorcontrib><creatorcontrib>WILLIAMS, S. F</creatorcontrib><creatorcontrib>LIEBOWITZ, D. N</creatorcontrib><creatorcontrib>DOLAN, M. E</creatorcontrib><title>Optimization of culture conditions to enhance transfection of human CD34+ cells by electroporation</title><title>Bone marrow transplantation (Basingstoke)</title><addtitle>Bone Marrow Transplant</addtitle><description>The ability to culture CD34+ stem cells, while maintaining their pluripotency, is essential for manipulations such as gene transfection for therapeutic trials. Human peripheral blood (PB) CD34+ cells (> or = 90% purity) were cultured for up to 4 days in serum-free culture medium supplemented with thrombopoietin (TPO), stem cell factor (SCF), Flt-3 ligand (Flt-3L), with or without PIXY321 (IL-3/GM-CSF fusion protein) and human serum. The CD34 mean fluorescence intensity (MFI) and cell cycle status were evaluated daily using flow cytometry and hypotonic propidium iodide. Prior to culture (day 0), 97.0 +/- 0.9%, 1.9 +/- 0.3% and 1.0 +/- 0.6% of the selected CD34+ cells were in G0-G1, S-phase, or G2-M, respectively. After 2-4 days in culture with TPO/SCF/Flt-3L, there was an increase in the percent of cells in S-phase to 26.4 +/- 0.1% without significant loss of CD34 MFI. The addition of PIXY321 increased.the percentage of CD34+ cells in S-phase to 36.3 +/- 4.0%, but the CD34 MFI and numbers of CFU (colony-forming units) were significantly decreased at day 3 when cultured with PIXY321 or various recombinant cytokine combinations that included IL-3 and IL-6. There is an increase from day 0 to day 4 in the percentages of CD34+ with CD38-, HLA-DR-, and c-kit(low), but not Thy-1+ cells. Electroporation with EGFP reporter gene showed that 1-2 days of pre-stimulation in X-VIVO 10 supplemented with TPO/SCF/Flt-3L was necessary and sufficient for efficient transfection. Flow cytometry analysis demonstrated that 22% of the viable cells are CD34+/EGFP+ 48 h post electroporation. The introduced reporter gene appears to be stable as determined by EGFP+/LTC-IC (long-term colony-initiating cells), at 30-40 positive colonies (16 +/- 7%) per 1 x 10(5) electroporated CD34+ cells.</description><subject>Antigens, CD34 - blood</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>Bone marrow</subject><subject>Bone marrow transplantation</subject><subject>CD34 antigen</subject><subject>CD38 antigen</subject><subject>Cell culture</subject><subject>Cell Culture Techniques - methods</subject><subject>Cell cycle</subject><subject>Cell Cycle - drug effects</subject><subject>Colonies</subject><subject>Culture</subject><subject>Cytokines</subject><subject>Electroporation</subject><subject>Electroporation - methods</subject><subject>Flow Cytometry</subject><subject>Fluorescence</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Fusion protein</subject><subject>Genetic engineering</subject><subject>Genetic technics</subject><subject>Granulocyte-macrophage colony-stimulating factor</subject><subject>Granulocyte-Macrophage Colony-Stimulating Factor - pharmacology</subject><subject>Hematopoietic Stem Cells - drug effects</subject><subject>Hematopoietic Stem Cells - immunology</subject><subject>Hematopoietic Stem Cells - metabolism</subject><subject>Histocompatibility antigen HLA</subject><subject>Humans</subject><subject>Immunophenotyping</subject><subject>Interleukin 3</subject><subject>Interleukin 6</subject><subject>Interleukin-3 - pharmacology</subject><subject>Iodides</subject><subject>Membrane Proteins - pharmacology</subject><subject>Methods. Procedures. Technologies</subject><subject>Optimization</subject><subject>Peripheral blood</subject><subject>Pluripotency</subject><subject>Propidium iodide</subject><subject>Recombinant Fusion Proteins - pharmacology</subject><subject>Reporter gene</subject><subject>Stem cell factor</subject><subject>Stem Cell Factor - pharmacology</subject><subject>Stem cell transplantation</subject><subject>Stem cells</subject><subject>Thrombopoietin</subject><subject>Thrombopoietin - pharmacology</subject><subject>Transfection</subject><subject>Transfection - methods</subject><subject>Transplantation</subject><subject>Vectors (cloning, transfer, expression). Insertion sequences and transposons</subject><issn>0268-3369</issn><issn>1476-5365</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNp10c1LwzAYBvAgis7p1aMEFS_Sme82R5mfMNhFzyFNU9bSNjNpD_OvN3MVRfAUSH55eJMHgDOMZhjR7DbUs7ztZzhFFHG2ByaYpSLhVPB9MEFEZAmlQh6B4xBqhDBjiB-CI4w5j9fJBOTLdV-11YfuK9dBV0IzNP3gLTSuK6rtZoC9g7Zb6c5Y2HvdhdKab70aWt3B-T1lN9DYpgkw30DbRODd2vmv1BNwUOom2NNxnYK3x4fX-XOyWD69zO8WiWGY90mBslQIS4gRhnOurdBECqbj0NhSLgta0kxLzizW8YSkNpMllTkvtSEoz-kUXO9y1969Dzb0qq3CdijdWTcElcZXZ1yyCC__wNoNvouzKSIYIQhzLKO6-FdhISii6RbNdsh4F4K3pVr7qtV-ozBS24JUqFUsSI0FxQvnY-qQt7b44WMjEVyNQAejmzJ-uKnCLxdzpKSf2ZWYSw</recordid><startdate>20010601</startdate><enddate>20010601</enddate><creator>WU, M. 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H ; SMITH, S. L ; DANET, G. H ; LIN, A. M ; WILLIAMS, S. F ; LIEBOWITZ, D. N ; DOLAN, M. E</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c415t-d08766e22c6c555ae6a2964a0141e359d3f38a954e1aa2927e89f39b5fac20bb3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2001</creationdate><topic>Antigens, CD34 - blood</topic><topic>Biological and medical sciences</topic><topic>Biotechnology</topic><topic>Bone marrow</topic><topic>Bone marrow transplantation</topic><topic>CD34 antigen</topic><topic>CD38 antigen</topic><topic>Cell culture</topic><topic>Cell Culture Techniques - methods</topic><topic>Cell cycle</topic><topic>Cell Cycle - drug effects</topic><topic>Colonies</topic><topic>Culture</topic><topic>Cytokines</topic><topic>Electroporation</topic><topic>Electroporation - methods</topic><topic>Flow Cytometry</topic><topic>Fluorescence</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Fusion protein</topic><topic>Genetic engineering</topic><topic>Genetic technics</topic><topic>Granulocyte-macrophage colony-stimulating factor</topic><topic>Granulocyte-Macrophage Colony-Stimulating Factor - pharmacology</topic><topic>Hematopoietic Stem Cells - drug effects</topic><topic>Hematopoietic Stem Cells - immunology</topic><topic>Hematopoietic Stem Cells - metabolism</topic><topic>Histocompatibility antigen HLA</topic><topic>Humans</topic><topic>Immunophenotyping</topic><topic>Interleukin 3</topic><topic>Interleukin 6</topic><topic>Interleukin-3 - pharmacology</topic><topic>Iodides</topic><topic>Membrane Proteins - pharmacology</topic><topic>Methods. Procedures. Technologies</topic><topic>Optimization</topic><topic>Peripheral blood</topic><topic>Pluripotency</topic><topic>Propidium iodide</topic><topic>Recombinant Fusion Proteins - pharmacology</topic><topic>Reporter gene</topic><topic>Stem cell factor</topic><topic>Stem Cell Factor - pharmacology</topic><topic>Stem cell transplantation</topic><topic>Stem cells</topic><topic>Thrombopoietin</topic><topic>Thrombopoietin - pharmacology</topic><topic>Transfection</topic><topic>Transfection - methods</topic><topic>Transplantation</topic><topic>Vectors (cloning, transfer, expression). Insertion sequences and transposons</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>WU, M. H</creatorcontrib><creatorcontrib>SMITH, S. L</creatorcontrib><creatorcontrib>DANET, G. H</creatorcontrib><creatorcontrib>LIN, A. M</creatorcontrib><creatorcontrib>WILLIAMS, S. F</creatorcontrib><creatorcontrib>LIEBOWITZ, D. N</creatorcontrib><creatorcontrib>DOLAN, M. 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H</au><au>SMITH, S. L</au><au>DANET, G. H</au><au>LIN, A. M</au><au>WILLIAMS, S. F</au><au>LIEBOWITZ, D. N</au><au>DOLAN, M. E</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Optimization of culture conditions to enhance transfection of human CD34+ cells by electroporation</atitle><jtitle>Bone marrow transplantation (Basingstoke)</jtitle><addtitle>Bone Marrow Transplant</addtitle><date>2001-06-01</date><risdate>2001</risdate><volume>27</volume><issue>11</issue><spage>1201</spage><epage>1209</epage><pages>1201-1209</pages><issn>0268-3369</issn><eissn>1476-5365</eissn><coden>BMTRE9</coden><abstract>The ability to culture CD34+ stem cells, while maintaining their pluripotency, is essential for manipulations such as gene transfection for therapeutic trials. Human peripheral blood (PB) CD34+ cells (> or = 90% purity) were cultured for up to 4 days in serum-free culture medium supplemented with thrombopoietin (TPO), stem cell factor (SCF), Flt-3 ligand (Flt-3L), with or without PIXY321 (IL-3/GM-CSF fusion protein) and human serum. The CD34 mean fluorescence intensity (MFI) and cell cycle status were evaluated daily using flow cytometry and hypotonic propidium iodide. Prior to culture (day 0), 97.0 +/- 0.9%, 1.9 +/- 0.3% and 1.0 +/- 0.6% of the selected CD34+ cells were in G0-G1, S-phase, or G2-M, respectively. After 2-4 days in culture with TPO/SCF/Flt-3L, there was an increase in the percent of cells in S-phase to 26.4 +/- 0.1% without significant loss of CD34 MFI. The addition of PIXY321 increased.the percentage of CD34+ cells in S-phase to 36.3 +/- 4.0%, but the CD34 MFI and numbers of CFU (colony-forming units) were significantly decreased at day 3 when cultured with PIXY321 or various recombinant cytokine combinations that included IL-3 and IL-6. There is an increase from day 0 to day 4 in the percentages of CD34+ with CD38-, HLA-DR-, and c-kit(low), but not Thy-1+ cells. Electroporation with EGFP reporter gene showed that 1-2 days of pre-stimulation in X-VIVO 10 supplemented with TPO/SCF/Flt-3L was necessary and sufficient for efficient transfection. Flow cytometry analysis demonstrated that 22% of the viable cells are CD34+/EGFP+ 48 h post electroporation. The introduced reporter gene appears to be stable as determined by EGFP+/LTC-IC (long-term colony-initiating cells), at 30-40 positive colonies (16 +/- 7%) per 1 x 10(5) electroporated CD34+ cells.</abstract><cop>Basingstoke</cop><pub>Nature Publishing Group</pub><pmid>11551032</pmid><doi>10.1038/sj.bmt.1703054</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0268-3369 |
| ispartof | Bone marrow transplantation (Basingstoke), 2001-06, Vol.27 (11), p.1201-1209 |
| issn | 0268-3369 1476-5365 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_71158594 |
| source | MEDLINE; Nature Journals Online; EZB-FREE-00999 freely available EZB journals; SpringerLink Journals - AutoHoldings |
| subjects | Antigens, CD34 - blood Biological and medical sciences Biotechnology Bone marrow Bone marrow transplantation CD34 antigen CD38 antigen Cell culture Cell Culture Techniques - methods Cell cycle Cell Cycle - drug effects Colonies Culture Cytokines Electroporation Electroporation - methods Flow Cytometry Fluorescence Fundamental and applied biological sciences. Psychology Fusion protein Genetic engineering Genetic technics Granulocyte-macrophage colony-stimulating factor Granulocyte-Macrophage Colony-Stimulating Factor - pharmacology Hematopoietic Stem Cells - drug effects Hematopoietic Stem Cells - immunology Hematopoietic Stem Cells - metabolism Histocompatibility antigen HLA Humans Immunophenotyping Interleukin 3 Interleukin 6 Interleukin-3 - pharmacology Iodides Membrane Proteins - pharmacology Methods. Procedures. Technologies Optimization Peripheral blood Pluripotency Propidium iodide Recombinant Fusion Proteins - pharmacology Reporter gene Stem cell factor Stem Cell Factor - pharmacology Stem cell transplantation Stem cells Thrombopoietin Thrombopoietin - pharmacology Transfection Transfection - methods Transplantation Vectors (cloning, transfer, expression). Insertion sequences and transposons |
| title | Optimization of culture conditions to enhance transfection of human CD34+ cells by electroporation |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-08T07%3A15%3A45IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_cross&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Optimization%20of%20culture%20conditions%20to%20enhance%20transfection%20of%20human%20CD34+%20cells%20by%20electroporation&rft.jtitle=Bone%20marrow%20transplantation%20(Basingstoke)&rft.au=WU,%20M.%20H&rft.date=2001-06-01&rft.volume=27&rft.issue=11&rft.spage=1201&rft.epage=1209&rft.pages=1201-1209&rft.issn=0268-3369&rft.eissn=1476-5365&rft.coden=BMTRE9&rft_id=info:doi/10.1038/sj.bmt.1703054&rft_dat=%3Cproquest_cross%3E998061111%3C/proquest_cross%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=216630379&rft_id=info:pmid/11551032&rfr_iscdi=true |