Characterization of Monocyte Subpopulations (CD14⁺CD16⁻ and CD14⁺⁺CD16⁺) generated from Cord Blood Haematopoietic Progenitor CD34⁺

Introduction. In the past decade significant advances in ex vivo expansion of haematopoietic stem cells were made. Protocols of differentiating CD34⁺ cells into cells from hematopoietic linages, especially dendritic cells, were described. However, little is known about in vitro differentiation of CD34⁺ cells to monocytes. In peripheral blood there are two main populations of monocytes CD14⁺⁺CD16⁻ and CD14⁺CD16⁺. They represent about 10 percent of total blood monocytes. Recently we have showed that expansion and differentiation of cord blood CD34⁺ cells to monocytes/macrophages leads to generation of CD14⁺CD16⁻ and CD14⁺⁺CD16⁺ monocyte subpopulations (Stec et al, J Leukoc Biol. 2007).

Aim of study. To compare two monocyte subpopulation: CD14⁺CD16⁻ and CD14⁺⁺CD16⁺ obtained from our expansion and differentiation protocol of CD34⁺ cord blood cells.

Materials and methods. After 3–10 days expansion in X-VIVO10 medium with FBS, SCF, IL-3, FLT-3L, TPO and then 7–10 days differentiation in IMDM medium with FBS, M-CSF, FLT-3L, IL-3, SCF, CD14⁺CD16⁻ and CD14⁺⁺CD16⁺ subpopulations were isolated by FACS sorting (FACSVantage). Release of IL-6, IL8, IL-12, IP-10, MIP1 α, MIP-1 β, RANTES, VEGF and FGF after LPS/INFγ or cancer cells stimulation were analysed using CBA (Cytometric Bead Array) method. Cytotoxic activity against cancer cells were assessed by MTT test and migration capacity was assesed with uncoated porous filters (8 μm) in a 24-well Boyden chamber. The adhesion capacity of subpopulations on human umbilical vein endothelial cells (HUVEC) were assessed with 5(6)-CFDA, SE (Molecular Probes) application and fluorescent microscope. NOD-SCID mice were used to evaluate the influence of subpopulations on angiogenesis and tumour growth.

Results. CD14⁺⁺CD16⁺ monocytes released more IL-8, IL-12, IP-10, MIP1-α, MIP1-β, and RANTES than CD14⁺CD16⁻ monocytes after LPS/INFγ or cancer cells stimulation. After 24 hours culture in medium without cytokines and than stimulation with LPS/INFγ or cancer cells, CD14⁺⁺CD16⁺ subpopulation released more RANTES, TNF, MIP1-α, IL10 than CD14⁺CD16⁻cells. CD14⁺CD16⁻ subpopulation exhibited greater chemotaxis to SDF-1 and MIP1-α than CD14⁺⁺CD16⁺ (12,2%±5,5% versus 2,2%±1%). At the same time CD14⁺⁺CD16⁺ monocytes showed significantly higher cytotoxic activity against tumour cells in vitro than CD14⁺CD16⁻ (34,04±17,45% versus 15,56±13,66%). CD14⁺⁺16⁺ population showed increased adhesion to HUVEC than CD14⁺16⁻ population (79±20 cells versus 30±13 cells). In in vivo tests in NODSCID mice both subpopulations together inhibited angiogenesis, but CD14⁺CD16⁻ subpopulation inhibited stronger tumour growth than CD14⁺⁺CD16⁺ subpopulation.

Conclusions. Using our expansion protocol it was possible to obtain two subpopulations of monocytes: CD14⁺CD16⁻ and CD14⁺⁺CD16⁺ which showed the differences in the release of cytokines/chemokines following stimulation with LPS/INFγ or tumour cells, chemotactic and antiangiogenic activity and cytotoxicity and are clearly distinct from known main subpopulations of blood monocytes CD14⁺CD16⁺ and CD14⁺⁺. Additional tests, which are still in progress in our lab will provide evidence if both populations are functional monocytes.