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.

Complex interactions in EML cell stimulation by stem cell factor and IL-3

“…In particular, SCF promotes the expansion of early hematopoietic precursor cells, whereas IL-3 is a multilineage cytokine that stimulates growth of early, partly differentiated cells (7,22,23). The effects of these cytokines on EML cells is consistent with this difference, because the population of lineage-negative, CD34…”

Current Strategies to Improve Engraftment in Cord Blood Transplantation

“…In liquid culture, isolated CD34 + or CD133 + HSC are expanded in the presence of selected cytokines and growth factors, including stem cell factor (SCF), Thrombopoietin (TPO), Granulocyte Colony Stimulating Factor (GCSF), and/or fmslike tyrosine kinase 3 ligand (FLT-3-L) [88,89]. The optimal milieu of cytokines and growth factors remains uncertain but several groups have shown improved expansion by the addition if IL-3 and/or IL-6 [90]. Shpall et al [88] performed a feasibility study in which CD34 + cells were isolated from a fraction (40-60%) of the UCB unit and expanded in liquid culture with SCF, GCSF, TPO, and megakaryocyte growth and differentiation factor.…”

In-vitro Behavior of Human Umbilical Cord Blood Stem Cells Towards Serum Based Minimal Cytokine Growth Conditions

“…The high concentration of HSCs, i.e. CD34?/CD38-, with its rapid proliferation ability, makes the cord blood as more attractive for therapeutic application [5,6]. The clinical results after transplantation have confirmed that either engraftment or survival significantly correlate with cell dose infused.…”