Both interleukin-3 and interleukin-6 are necessary for better ex vivo expansion of CD133+ cells from umbilical cord blood.
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.
Bordeaux-Rego
Pedro
P
Center of Haematology and Hemotherapy, University of Campinas, Campinas, São Paulo Brazil.
Luzo
Angela
A
Costa
Fernando Ferreira
FF
Olalla Saad
Sara Teresinha
ST
Crosara-Alberto
Daniella Pereira
DP
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
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