Effects of various stimulating factors on proliferation of cord blood hematopoietic stem/progenitor cellsin vitro

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Abstract

BACKGROUND:

Cord blood in vitro amplification supported by cytokines may be more safe and easier to be used in clinic.

OBJECTIVE:

To compare the effects of leukaemia inhibitory factor, interleukin-6 and lipopolysaccharide on in vitro proliferation of cord blood hematopoietic stem/progenitor cells.

DESING, TIME AND SETTING:

The cytological in vitro controlled study was performed at the Central Laboratory, Hospital Affiliated to Guangdong Medical College from August 2003 to May 2004.

MATERIALS:

Ten normal cord blood samples were obtained from the Department of Gynaecology and Obstetrics, Hospital Affiliated to Guangdong Medical College, and Department of Gynaecology and Obstetrics, Zhanjiang Health Center for Women and Children. Leukaemia inhibitor factor (Dake, China), interleukin-6 (Jingmei, China), and lipopolysaccharide (made by Professor Li Yan-ping) were used for this study.

METHODS:

Cord blood mononuclear cells were in vitro isolated by the density gradient method. Cord blood CD34+ cells were extracted by the magnetic sorting machine and monoclonal antibody immunomagnetic beads (positive) sorting. Liquid culture system was created, which was RPMI 1640 medium containing 40% fetal bovine serum, 20 g/L bovine serum albumin, 20 μg/L granulocyte-macrophage colony-stimulating factor, 20 μg/L stem cell factor, and 20 μg/L interleukin-3. Purified CD34+ cells (2×108/L) were incubated in 50 μL above-mentioned liquid medium. There were 8 groups. Cells in the group 1 were treated with RPMI 1640 medium as controls. Cells in the groups 2-4 were respectively treated with lipopolysaccharide, leukaemia inhibitory factor, and interleukin-6. Cells in the groups 5-7 were subjected to pairwise mixed liquor of lipopolysaccharide, leukaemia inhibitory factor, and interleukin-6. Cells in the group 8 were given the mixed liquor of lipopolysaccharide, leukaemia inhibitory factor and interleukin-6. The final concentrations of lipopolysaccharide, leukaemia inhibitory factor and interleukin-6 were respectively 10 U/mL, 10 μg/L and 10 mg/L.

MAIN OUTCOME MEASURES:

MTT assay was used to detect effects of various stimulating factors on cord blood CD34+ cell proliferation in liquid medium.

RESULTS:

Compared with controls, at 3 days leukaemia inhibitory factor alone could promote proliferation of cord blood CD34+ cells (F=3.33, P < 0.01), but the effects of lipopolysaccharide or interleukin-6 alone were not significant (F=2.14, 1.83, P > 0.05). Pairwise combination of stimulating factors and combination of three stimulating factors could accelerate proliferation of cord blood CD34+ cells (F=3.54-4.06, P < 0.01). The effects of combination of three stimulating factors were the most significant.

CONCLUSION:

Under a special liquid medium, leukaemia inhibitory factor alone significantly enhances proliferation of cord blood hematopoietic stem/progenitor cells in vitro. Under combination conditions, interleukin-6, lipopolysaccharide and leukaemia inhibitory factor have synergistic effects on promoting proliferation.

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