Action of human interleukin‐4 and stem cell factor on erythroid and mixed colony formation by peripheral blood‐derived CD34⁺c‐kit^high or CD34⁺c‐kit^low cells

Abstract

We studied the interaction of interleukin (IL)‐4 and other burst‐promoting activity (BPA) factors, such as IL‐3, granulocyte/macrophage colony‐stimulating factor (GM‐CSF), IL‐9 and stem cell factor (SCF), on erythroid burst‐forming unit (BFU‐E) and erythrocyte‐containing mixed (CFU‐Mix) colony formation in serum‐free culture. IL‐4 alone did not support mixed colony formation in the presence of erythropoietin (Epo). However, IL‐4 showed weak but significant BPA when peripheral blood (PB)‐derived CD34+c‐kitlow cells were used as the target population. The BPA of IL‐4 was much weaker than that of IL‐3, which exerted the most potent activity, as previously reported. When CD34+c‐kithigh cells were used as the target, four factors known to have BPA, IL‐3, GM‐CSF, IL‐9 and SCF, could express BPA. In contrast, IL‐4 alone failed to support erythroid burst formation. Interestingly, IL‐4 showed a remarkable enhancing effect with SCF in promoting the development of erythroid burst and erythrocyte‐containing mixed colonies from CD34+c‐kitlow and CD34+c‐kithigh cells. Delayed addition of SCF + Epo or IL‐4+Epo to the cultures initiated with either IL‐4 or SCF alone clearly demonstrated that SCF was a survival factor for both BFU‐E and CFU‐Mix progenitors. In contrast, the survival effect of IL‐4 was much weaker than that of SCF, and appeared to be more important for progenitors derived from CD34+c‐kitlow cells than for those derived from CD34+c‐kithigh cells. It was recently reported that CD34+c‐kitlow cells represent a more primitive population than CD34+c‐kithigh cells. Taken together, these results suggest that IL‐4 helps to recruit primitive progenitor cells in the presence of SCF.

Interleukin 6 receptor expression by human cord blood‐ or peripheral blood‐derived primitive haematopoietic progenitors implies acquisition of different functional properties

“…These results were highly consistent with those of our previous studies using two‐colour flow cytometric analysis and cell sorting ( Sonoda et al , 1994 , 1997; Kimura et al , 1997 ; Sakabe et al , 1997 ) and again suggested that IL‐6R expression by primitive progenitors combined with negativity for the c‐ kit receptor promoted myeloid differentiation. Conversely, a high level of c‐ kit receptor expression and the loss of IL‐6R expression induced erythroid differentiation.…”

“…Limiting dilution analysis of long‐term culture‐initiating cells (LTC‐ICs) Co‐cultures were established by incubating sorted CD34 + IL‐6R + or IL‐6R − cells in 96‐well plates precoated with a murine stromal cell line, MS‐5 (kindly provided by Dr Kazuhiro J. Mori, Niigata University, Niigata, Japan) ( Itoh et al , 1989 ), instead of allogeneic BM stromal cells, as reported elsewhere ( Sakabe et al , 1997 , 1998). In these limiting dilution experiments, each CD34 + subfraction was seeded by an Automated Cell Deposition Unit (Clone‐Cyt), as reported previously ( Kimura et al , 1997 ; Sonoda et al , 1997 ; Tanimukai et al , 1997 ; Sakabe et al , 1998 ). Seeding was carried out at four different initial cell concentrations (from 2 to 100 cells/well), using a mean of 87 ± 37 (CB) to 108 ± 51 (PB) replicate wells for 2–10 cells and 30 ± 1 (CB) to 36 ± 0 (PB) wells for 20–100 cells.…”

Haematopoietic action of flt3 ligand on cord blood‐derived CD34‐positive cells expressing different levels of flt3 or c‐kit tyrosine kinase receptor: comparison with stem cell factor

“…Dishes were incubated at 37°C in a fully-humidified atmosphere flushed with a combination of 5% CO,, 5% O,, and 90% N,. On d 12-14 of culture, all colonies were identified under an inverted microscope based on their typical morphological features, as reported previously (16,19,(22)(23)(24). The colony types identified included granulocyte (CFU -G ) , macrophage (CFU -M), granulocyte/ macrophage (CFU-GM), erythroid burst (BFU-E), eosinophil (CFU-Eo) and erythrocyte-containing mixed (CFU-mix) colonies.…”

“…Immediately after staining, cells were separated using a FACStar Plus (BD) equipped with an argon laser tuned at 488 nm, as reported elsewhere (16,19,22,23). Briefly, sorting gates were established for both forward-scatter and side-scatter.…”

“…Serum-free cultures were carried out in 35-mm Lux suspension culture dishes (No. 171099, Nunc Inc., Naperville, IL, USA), as reported elsewhere (16,(22)(23)(24). One millilitre of culture medium contained 150-350 sorted cells, 1.2% of 1500 centipoise meth-ylcellulose (MTC, Shinetsu Chemical, Tokyo, Japan), 1 YO globulin-free bovine serum albumin (BSA, Sigma Chemical Co., St Louis, MO, USA) that had been crystallized and deionized, ASF 104 medium (Ajinomoto Co., Ltd, Tokyo, Japan) (25), 5~1 0 -~ mol/l2-mercaptoethanol(2-ME, Sigma), 300 pg/ml fully iron-satulated human transferrin (Sigma), 10 pg/ml lecithin (Sigma), 6 pg/ml cholesterol (Sigma) and various combinations of CSFs.…”