Optimisation of transfection conditions of CD34+ hematopoietic cells derived from human umbilical cord blood
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Human umbilical cord blood is frequently used as a source of transplantable hematopoietic cells and more recently as a target of gene therapy - a new approach for treatment of various disorders. The aim of our study was optimisation of the transfection conditions of cord blood-derived CD34+ hematopoietic cells. Mononuclear cells fraction was isolated from cord blood samples by density gradient centrifugation. Subsequently, CD34+ hematopoietic cells were separated on immunomagnetic MiniMACS columns. Pure population of CD34+ cells was incubated in a serum free medium supplemented with thrombopoietin, stem cell factor and Flt-3 ligand for 48 h and then transfected with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene. We studied the influence of various pulse settings and DNA concentrations on the transfection efficiency, measured by flow cytometry as the fluorescence of target cells due to the expression of EGFP. The optimal settings were as follows: 4 mm cuvette, 1600 μF, 550 V/cm, and 10 μg of DNA per 500 μl. With these settings we obtained a high transfection frequency (41.2%) without a marked decrease of cell viability. An increase of the pulse capacitance and/or of DNA concentration resulted in a greater electroporation efficiency, but also in a decrease of cell viability. In conclusion, the results described here allow one to recommend electroporation as an efficient method of gene delivery into CD34+ hematopoietic cells derived from human umbilical cord blood.
- Baum C, Eckert HG, Stockschlader M, Just U, Hegewisch-Becker S, Hildinger M, Uhde A, John J, Ostertag W. (1996) Improved retroviral vectors for hematopoietic stem cell protection and in vivo selection. J Hematother.; 5: 323-9.
- Bierhuizen MFA, Westerman Y, Visser TP, Dimjati W, Wognum AW, Wagemaker G. (1997) Enhanced green fluorescent protein as selectable marker of retroviral-mediated gene transfer in immature hematopoietic bone marrow cells. Blood.; 90: 3304-15.
- Brenner MK. (1997) Emerging application of gene transfer in the hematopoietic cancers. J Pediatr Hematol Oncol.; 19: 1-6.
- Capel B, Hawley RG, Mintz B. (1990) Long- and short-lived murine haematopoietic stem cell clones individually identified with retroviral integration markers. Blood.; 75: 2267-82.
- Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. (1994) Green fluorescent protein as a marker of gene expression. Science.; 263: 802-5.
- Corrias MV, Scuderi F, Pasino M, Biglino P, Bocca P, Marotta F, Figini E, Pistoia V, Mori PG. (1998) Growth factors increase retroviral transduction but decrease clonogenic potential of umbilical cord blood CD34+ cells. Haematologica.; 83: 580-6.
- Frey BM, Hackett NR, Bergelson JM, Finberg R, Crystal RG, Moore MA, Rafii S. (1998) High-efficiency gene transfer into ex vivo expanded human hematopoietic progenitor and precursor cells by adenovirus vectors. Blood.; 91: 2781-92.
- Gluckman E. (2000) Current status of umbilical cord blood hematopoietic stem cell transplantation. Exp Hematol.; 28: 1197-205.
- Gluckman E, Broxmeyer HE, Auerbach AD, Friedman HS, Douglas GW, Devergie A, Esperou H, Thierry D, Socie G, Lehn P, Cooper S, English D, Kurtzberg J, Bard J, Boyse EA. (1989) Hematopoietic reconstitution in a patient with Fanconi's anemia by means of umbilical cord blood from an HLA-identical sibling. N Engl J Med.; 321: 1174-82.
- Hows JM, Bradley BA, Marsh JC, Luft T, Coutinho L, Testa NG, Dexter TM. (1992) Growth of human umbilical cord blood in long-term haematopoietic cultures. Lancet.; 340: 73-6.
- von Kalle C, Kiem HP, Goehle S, Darovsky B, Heimfeld S, Torok-Storb B, Storb R, Schuening FG. (1994) Increased gene transfer into human hematopoietic progenitor cells by extended in vitro exposure to a pseudotyped retroviral vector. Blood.; 84: 2890-7.
- Li LH, McCarthy P, Hui SW. (2001) High-efficiency electrotransfection of human primary hematopoietic stem cells. FASEB J.; 15: 586-8.
- Limon A, Briones J, Puig T, Carmona M, Fornas O, Cancelas JA, Nadal M, Garcia J, Rueda F, Barquinero J. (1997) High-titer retroviral vectors containing enhanced green fluorescent protein (EGFP) for efficient expression in hematopoietic cells. Blood.; 90: 3316-21.
- Mayani H, Lansdorp PM. (1998) Biology of human umbilical cord blood-derived hematopoietic stem/progenitor cells. Stem Cells.; 16: 153-65.
- Mayani H, Gutierrez-Rodriguez M, Espinoza L, Lopez-Chalini E, Huerta-Zepeda A, Flores E, Sanchez-Valle E, Luna-Bautista F, Valencia I, Ramirez OT. (1998) Kinetics of hematopoiesis in Dexter-type long-term cultures established from human umbilical cord blood cells. Stem Cells.; 16: 127-35.
- Persons DA, Allay JA, Allay ER, Smeyne RJ, Ashmun RA, Sorrentino BP, Nienhuis AW. (1997) Retroviral-mediated transfer of the green fluorescent protein gene into murine hematopoietic cells facilitates scoring and selection of transducted progenitors in vitro and identification of genetically modified cells in vivo. Blood.; 90: 1777-86.
- Pettengell R, Luft T, Henschler R, Hows JM, Dexter TM, Ryder D, Testa NG. (1994) Direct comparison by limiting dilutions analysis of long-term culture-initiating cells in human bone marrow, umbilical cord blood and blood stem cells. Blood.; 84: 3653-9.
- Toneguzzo F, Keating A. (1986) Stable expression of selectable genes introduced into human hematopoietic stem cells by electric field-mediated DNA transfer. Proc Natl Acad Sci U S A.; 83: 3496-9.
- Traycoff CM, Abboud MR, Laver J, Clapp DW, Srour EF. (1994) Rapid exit from G0/G1 phases of cell cycle in response to stem cell factor confers on umbilical cord blood CD34+ cells an enhanced ex vivo expansion potential. Exp Hematol.; 22: 1264-72.
- Van Tendeloo VF, Willems R, Ponsaerts P, Lenjou M, Nijs G, Vanhove M, Muylaert P, Van Cauwalaert P, Van Broeckhoven C, Van Bockstaele DR, Berneman ZN. (2000) High-level transgene expression on primary human T lymphocytes and adult bone marrow CD34+ cells via electroporation-mediated gene delivery. Gene Ther.; 7: 1431-7.
- Wang JCY, Doedens M, Dick JE. (1997) Primitive human hematopoietic cells are enriched in cord blood compared with adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell assay. Blood.; 89: 3919-24.
- Wu MH, Liebowitz DN, Smith SL, Williams SF, Dolan ME. (2001a) Efficient expression of foreign genes in human CD34+ hematopoietic precursor cells using electroporation. Gene Ther.; 8: 384-90.
- Wu MH, Smith SL, Danet GH, Lin AM, Williams SF, Liebowitz DN, Dolan ME. (2001b): Optimization of culture conditions to enhance transfection of human CD34+ cells by electroporation. Bone Marrow Transplant.; 27: 1201-9.
- Wu MH, Smith SL, Dolan ME. (2001c): High efficiency electroporation of human umbilical cord blood CD34+ hematopoietic precursor cells. Stem Cells.; 19: 492-9.
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