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. 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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Free PMC article

Homogeneous Monocytes and Macrophages From Human Embryonic Stem Cells Following Coculture-Free Differentiation in M-CSF and IL-3

Karl R Karlsson et al. Exp Hematol. .
Free PMC article

Abstract

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

Materials and methods: 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.

Results: 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.

Conclusions: 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.

Figures

Figure 1
Figure 1
A three-step method for the differentiation of human embryonic stem (hES) cells into monocytes. (A) Human hES cells were removed from mouse embryonic fibroblasts either by microdissecting colonies or treating with trypsin. Patches were cultured with hES medium in suspension for spontaneous embryoid body (EB) formation for 3 days (Step 1). Adherent EBs were next differentiated in macrophage colony-stimulating factor (M-CSF) and interleukin (IL)-3 (Step 2). hES monocytes (esMCs) were produced and harvested from the supernatant of cultures around day 18, and growth factors replaced for continuous esMC production (Step 3). Arrow head in Step 3 show part of an EB. Images are from hES cell line KLC002 and representative of KCL001 and HUES-2. (B) Eosin-Methylene blue staining of harvested esMCs from cytospins. (C) Morphology of esMCs by transmission electron microscopy. (D) Forward-side scatter plot of esMCs from hES cell line HUES-2. (E) CD14 surface expression on esMCs from hES cell line HUES-2. Histograms show antibody staining (in black) relative to isotype-matched control (in gray). (F) Accumulated esMCs yield and production kinetics from separate experiments from hES cell line HUES-2. Mean number of EBs used per experiment was 30 ± 13 EBs (range, 19–55, n = 9). (G) Phase contrast image of hES monocyte-derived macrophages (esMDMs) differentiated in M-CSF for 7 days and the esMDM expression of CD68 and CD163. Histograms show antibody staining (in black) relative to isotype-matched control (in gray). All scale bar units are in micrometers.
Figure 2
Figure 2
Transcriptome analysis of human embryonic stem (hES) monocytes (esMCs) and hES monocyte–derived macrophages (esMDM) reveal a distinct myeloid gene signature. Transcriptome analysis of esMCs using the GeneChip Human Gene 1.0 ST Array (Affymetrix) containing a total of ∼29,000 transcripts. (A) Pearson correlation analysis of RNA expression levels corresponding to esMCs, hES monocyte-derived macrophages (esMDMs), blood–derived monocytes (bMCs), and 11 other major tissues samples. Pearson correlation levels >0.9 are visualized in red, and <0.9 in blue. (B) Hierarchical clustering of 42 selected genes expressed in the esMC, esMDM, bMC, and spleen tissue samples. The 42 selected genes were obtained by performing a Euclidian distance complete hierarchical clustering with an adjusted Bonferroni corrected p value <0.05. (C) Confirmation of expression data of six selected proteins by flow cytometry.
Figure 3
Figure 3
Human embryonic stem (hES) monocytes (esMCs) differentiate into functional macrophages. (A) hES monocyte–derived macrophages (esMDM) endocytose acetylated low-density lipoprotein (LDL). esMDMs and blood monocyte–derived macrophages (bMDM) were pretreated in medium alone, with the scavenger receptor inhibitor poly inosinic acid, or control poly cytidylic acid and cells incubated with acetylated LDL. Images show immunofluorescence with the corresponding phase-contrast image inserted. Inserted values show ratio of endocytosis uptake calculated by dividing the mean fluorescence intensity (MFI) with the background signal from cells cultured without acetylated low-density lipoprotein (acLDL). (B) esMDMs phagocytose opsonized yeast particles. esMDMs and bMDMs were incubated with zymosan particles opsonized with human serum at a 1:50 cell-to-particle ratio. Frequency distribution histogram show number of particles per TEM section comparing esMDMs (in black) with bMDMs (in gray). Inserted images show binding and internalization in esMDMs by SEM (top) and TEM (bottom). Scale bar units are in μm. Results are representative of two separate experiments. (C) esMDMs upregulate major histocompatibility complex (MHC) class II and mannose receptors after alternative activation with interleukin (IL)-4. esMDMs and bMDMs were cultured in media alone or activated with interferon (IFN)-γ (20 U/mL) or IL-4 (20 ng/mL) for 72 hours and assessed for MHC class II and mannose receptor surface expression by flow cytometry. Histograms show antibody staining (in black) relative to isotype-matched control (in gray). Inserted values show the ratio of surface receptor expression relative to isotype-matched control. (D) esMDMs produce chemokines and cytokines in response to lipopolysaccharide (LPS). esMDMs were cultured with LPS (1 μg/mL) and supernatants harvested after 2, 8, and 10 hours of stimulation and assessed for cytokine and chemokine production. Culture media contained less than the lower detection limit for all proteins tested. Results are mean values from experimental triplicates.

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