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Titolo:
Optimization of culture conditions to enhance transfection of human CD34(+) cells by electroporation
Autore:
Wu, MH; Smith, SL; Danet, GH; Lin, AM; Williams, SF; Liebowitz, DN; Dolan, ME;
Indirizzi:
Univ Chicago, Dept Med, Hematol Oncol Sect, Chicago, IL 60637 USA Univ Chicago Chicago IL USA 60637 matol Oncol Sect, Chicago, IL 60637 USA Univ Chicago, Ctr Canc Res, Chicago, IL 60637 USA Univ Chicago Chicago ILUSA 60637 go, Ctr Canc Res, Chicago, IL 60637 USA Univ Penn, Howard Hughes Med Inst, Philadelphia, PA 19104 USA Univ Penn Philadelphia PA USA 19104 Med Inst, Philadelphia, PA 19104 USA Univ Penn, Abramson Family Canc Res Inst, Philadelphia, PA 19104 USA Univ Penn Philadelphia PA USA 19104 Res Inst, Philadelphia, PA 19104 USA
Titolo Testata:
BONE MARROW TRANSPLANTATION
fascicolo: 11, volume: 27, anno: 2001,
pagine: 1201 - 1209
SICI:
0268-3369(200106)27:11<1201:OOCCTE>2.0.ZU;2-N
Fonte:
ISI
Lingua:
ENG
Soggetto:
EX-VIVO EXPANSION; HEMATOPOIETIC PROGENITOR CELLS; EFFICIENCY GENE-TRANSFER; EARLY-ACTING CYTOKINES; MEDIATED DNA TRANSFER; BLOOD STEM-CELLS; FLT3 LIGAND; SELF-RENEWAL; CORD-BLOOD; IN-VITRO;
Keywords:
CD34 cells; TPO; SCF; Flt-3L; cell cycle; EGFP;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Clinical Medicine
Life Sciences
Citazioni:
58
Recensione:
Indirizzi per estratti:
Indirizzo: Dolan, ME Univ Chicago, Dept Med, Hematol Oncol Sect, 5841 S Maryland Ave,Box MC2115, Chicago, IL 60637 USA Univ Chicago 5841 S Maryland Ave,Box MC2115 Chicago IL USA 60637
Citazione:
M.H. Wu et al., "Optimization of culture conditions to enhance transfection of human CD34(+) cells by electroporation", BONE MAR TR, 27(11), 2001, pp. 1201-1209

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 (greater than or equal to 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 statuswere 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 GO-GI, 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 inS-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 PIXY321or 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(-) withCD38(-), 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.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 08/04/20 alle ore 12:10:42