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Exp Hematol. 2008 Sep;36(9):1167-75. doi: 10.1016/j.exphem.2008.04.009. Epub 2008 Jun 11.

Homogeneous monocytes and macrophages from human embryonic stem cells following coculture-free differentiation in M-CSF and IL-3.

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Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, UK.



To develop a simple and efficient method for producing homogeneous populations of monocytes and macrophages from human embryonic stem cells (hES).


Human embryonic stem cell lines KCL001, KCL002, and HUES-2 were differentiated into monocytes by coculture-free differentiation with two growth factors using a three-step method. The method involved embryoid body (EB) formation in hES media, directed differentiation with macrophage colony-stimulating factor and interleukin (IL)-3, and harvest of nonadherent monocytes from the culture supernatants. hES monocytes (esMCs) were analyzed by microscopy, flow cytometry, transcriptome analysis, and tested for the ability to differentiate into macrophages. hES monocyte-derived macrophages (esMDM) were analyzed for phagocytosis and endocytosis by microscopy and flow cytometry, cytokine secretion by multiplex cytokine assay, and for interferon (IFN)-gamma and IL-4 activation by flow cytometry.


Homogeneous esMCs (>90% CD14-positive) that did not require any additional purification steps were produced after 18.7 +/- 7.7 days (mean +/- SD, n = 19). Production continued for several months when growth factors were replaced, with a total yield of 3.4 x 10(5) +/- 2.0 esMCs (mean +/- SD, n = 9) per EB. Transcriptome analysis of the esMC and the esMDM revealed a distinct myeloid signature that correlated with primary adult blood-derived monocytes and spleen tissue samples but not with other tissue samples tested. We found that esMCs and esMDMs expressed well-defined markers of the mononuclear phagocyte system including PU-1, C/EBPalpha, EMR1, and EMR2, MPEG1, CD1c, CD4, CD18, CD32, CD33, CD68, cathepsins and serine carboxypeptidase. Finally, esMCs differentiated into functional macrophages that could endocytose acetylated low-density lipoprotein, phagocytose opsonized yeast particles, secrete specific cytokines in response to lipopolysaccharide, and be activated differentially with IFN-gamma and IL-4.


We have developed a simple and efficient method for producing homogeneous populations of monocytes and macrophages from hES cells. esMCs have a myeloid signature and can differentiate into functional macrophages. The method should prove useful in answering experimental questions regarding monocyte and macrophage development and biology.

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