title: Both interleukin-3 and interleukin-6 are necessary for better ex vivo expansion of CD133+ cells from umbilical cord blood.
abstract: Umbilical cord blood (UCB), an ideal source for transplantable hematopoietic stem cells (HSC), is readily available and is rich in progenitor cells. Identification of conditions favoring UCB-HSC ex vivo expansion and of repopulating potential remains a major challenge in hematology. CD133+ cells constitute an earlier, less-differentiated HSC group with a potentially higher engraftment capacity. The presence of SCF, Flt3-L, and TPO are essential for CD133+ and/or CD34+ cells ex vivo expansion; however, IL-3 and IL-6 influence has not yet been clearly established. We investigated this influence on CD133+ cells from UCB ex vivo expansion and the effect of these cytokines upon cell phenotype. Immediately after isolation an 85% of CD133+ cell purity was obtained, diminishing after 4 and 8 days of ex vivo expansion. CD133+ fold-increase was higher using IMDM with SCF, Flt3-L, and TPO (BM)+IL-3 or BM+IL-3+IL-6 on day 8 (13.83- and 17.47-fold increase, respectively). BM+IL-6 presented no significant difference from BM alone. We demonstrated that 5.1% of the CD133+ cells expressed IL-6 receptor (IL-6R) after isolation. After 4 and 8 days in culture, the percentage of CD133+ cells that expressed IL-6R was as follows: BM alone (9.8% and 22.02%, respectively); BM+IL-3 (8.33% and 16.74%); BM+IL-6 (9.2% and 17.67%); and BM+IL-3+IL-6 (12.5% and 61.20%). Cell cycle analysis revealed quiescent cells after isolation, 95.5% CD133+ cells in the G0/G1 phase. Regardless of culture period or cytokine incubation, CD133+ cell cycle altered to 70% of CD133+ in the G0/G1 phase. Colony-forming unit (CFU) doubled in BM+IL-3+IL-6 after 8 days of incubation compared with BM group. SOX-2 and NANOG-relative gene expression was detected on day 0 after isolation. BM+IL-6 prevented the decrease in NANOG and SOX-2 gene expression level compared to BM+IL-3 or BM+IL-3+IL-6 incubated cells. Our results indicated that UCB-isolated CD133+ cells were better ex vivo expanded in the presence of SCF, Flt3-L, TPO, IL-3+IL-6. IL-3 probably promotes higher CD133+ cell expansion and IL-6 maintains immature phenotype.
authors: Center of Haematology and Hemotherapy, University of Campinas, Campinas, São Paulo Brazil.
authors: Pedro
authors: P
authors: Bordeaux-Rego
authors: Angela
authors: A
authors: Luzo
authors: Fernando Ferreira
authors: FF
authors: Costa
authors: Sara Teresinha
authors: ST
authors: Olalla Saad
authors: Daniella Pereira
authors: DP
authors: Crosara-Alberto
mesh_terms: D000071916:AC133 Antigen; D015703:Antigens, CD / Q000097:blood; D018952:Antigens, CD34 / Q000097:blood; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects*; D002469:Cell Separation; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D004357:Drug Synergism; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects; D006023:Glycoproteins / Q000097:blood; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D007231:Infant, Newborn; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D007377:Interleukin-3 / Q000494:pharmacology*; D015850:Interleukin-6 / Q000494:pharmacology*; D000071317:Nanog Homeobox Protein; D010455:Peptides / Q000097:blood; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors / Q000235:genetics; D013997:Time Factors
chemical_list: D000071916:AC133 Antigen; D015703:Antigens, CD; D018952:Antigens, CD34; D006023:Glycoproteins; D018398:Homeodomain Proteins; D036341:Intercellular Signaling Peptides and Proteins; D007377:Interleukin-3; D015850:Interleukin-6; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; C000606023:PROM1 protein, human; D010455:Peptides; D055748:SOXB1 Transcription Factors