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

Abstract

ObjectiveTo develop a simple and efficient method for producing homogeneous populations of monocytes and macrophages from human embryonic stem cells (hES).Materials and MethodsHuman 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)-γ and IL-4 activation by flow cytometry.ResultsHomogeneous 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 × 105 ± 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/EBPα, 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-γ and IL-4.ConclusionsWe 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.

Generation of mature human myelomonocytic cells through expansion and differentiation of pluripotent stem cell–derived lin–CD34+CD43+CD45+ progenitors

“…Recently, methods for obtaining neutrophils, macrophages, and DCs from hESCs have been described as well (31)(32)(33)(34)(35). However, the progenitors giving rise to cells of myeloid lineage are often obscure, especially in differentiation systems employing ES cells.…”

Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells

“…The hPSC H1 cell line was used to generate the PB-CRISPR/Cas hPSCs by piggyBac transposon-mediated gene engineering and subsequently differentiated into monocyte/macrophages through spin-EB generation using a feeder-free protocol, which was modified from previous publications 27,28 . Cells form spin EBs on 96-well low attachment plates supplemented with cytokines (SCF, VEGF and BMP4) and were grown for 4 days.…”

“…4d). These CRISPR/Cas9-hPSCs can be differentiated into monocytes/macrophages using a feeder-free protocol 27,28 ( Fig. 4e).…”

Controlled embryoid body formation via surface modification and avidin–biotin cross-linking

“…Consequently, ES cells provide a renewable source of cellular material for tissue engineering and regenerative medicine (Ungrin et al 2008). However, generation of specified homogeneous cultures for clinical application remains problematic Karlsson et al 2008). …”