CoChat
Sign Up
Cited by 360Pluripotent Stem Cells ResearchMesenchymal stem cell researchHematopoietic Stem Cell Transplantation

The DOI matches its Crossref record; 44 of the 44 references it cites were found in a registry or an index.

Efficient, Long Term Production of Monocyte-Derived Macrophages from Human Pluripotent Stem Cells under Partly-Defined and Fully-Defined Conditions

Open access
Wilgenburg, Bonnie van, Browne, Cathy, Vowles, Jane, Cowley, Sally A.·2013·PLoS ONE
DOI 10.1371/journal.pone.0071098PMC PMC3741356
Open this paper in CoChat — free accountOpen PDF

Abstract

Human macrophages are specialised hosts for HIV-1, dengue virus, Leishmania and Mycobacterium tuberculosis. Yet macrophage research is hampered by lack of appropriate cell models for modelling infection by these human pathogens, because available myeloid cell lines are, by definition, not terminally differentiated like tissue macrophages. We describe here a method for deriving monocytes and macrophages from human Pluripotent Stem Cells which improves on previously published protocols in that it uses entirely defined, feeder- and serum-free culture conditions and produces very consistent, pure, high yields across both human Embryonic Stem Cell (hESC) and multiple human induced Pluripotent Stem Cell (hiPSC) lines over time periods of up to one year. Cumulatively, up to ∼3×10(7) monocytes can be harvested per 6-well plate. The monocytes produced are most closely similar to the major blood monocyte (CD14(+), CD16(low), CD163(+)). Differentiation with M-CSF produces macrophages that are highly phagocytic, HIV-1-infectable, and upon activation produce a pro-inflammatory cytokine profile similar to blood monocyte-derived macrophages. Macrophages are notoriously hard to genetically manipulate, as they recognise foreign nucleic acids; the lentivector system described here overcomes this, as pluripotent stem cells can be relatively simply genetically manipulated for efficient transgene expression in the differentiated cells, surmounting issues of transgene silencing. Overall, the method we describe here is an efficient, effective, scalable system for the reproducible production and genetic modification of human macrophages, facilitating the interrogation of human macrophage biology.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1371/journal.pone.0071098

Cites

44 of 44 references verified0 citation errors · 0 not checkable

Statistics

Figures

Figure 1 Protocols for the long-term production of PSC-MDM which are genetically modifiable. A) Diagram outlining the defined and quick protocols for the differentiation of MDM from PSC. Using the defined protocol, PSC colonies are cultured on feeder-free Synthemax™ tissue culture plates and uniform EBs are generated using centrifugation of PSC in a 96-well plate (to isolate single EBs) or in a AggreWell™ plate. The defined EBs are formed in mTeSR™-1 medium and require the addition of three growth factors. Using the quick protocol, PSC colonies are cultured on inactivated feeder cells and EBs are generated using mechanical dissociation and culture in PSC media in ultra-low adherence plates. After 4 days of EB culture, EBs are transferred to regular tissue culture plates and differentiated in X-VIVO™ 15 media, supplemented with M-CSF and IL-3. Non-adherent PSC-MC can be harvested from the supernatant of cultures and plated in X-VIVO™ 15 media+M-CSF without serum, or in RPMI media +10% serum+M-CSF, to generate PSC-MDM. A pure population of genetically modified PSC-MDM can be obtained by transducing PSC with a self-inactivating lentiviral construct (in this case expressing RFP-IRES-PuromycinR under control of the EF1alpha promoter). Scale bar, 200 µM. Detailed protocol described in materials and methods. B) Characteristics of the defined and quick protocol. C) Histogram represents RFP expression by PSC-MDM differentiated from PSC transduced with EF1a-RFP-IRES-PuromycinR (black line) or control PSC-MDM (shaded gray).
Figure 1 Protocols for the long-term production of PSC-MDM which are genetically modifiable. A) Diagram outlining the defined and quick protocols for the differentiation of MDM from PSC. Using the defined protocol, PSC colonies are cultured on feeder-free Synthemax™ tissue culture plates and uniform EBs are generated using centrifugation of PSC in a 96-well plate (to isolate single EBs) or in a AggreWell™ plate. The defined EBs are formed in mTeSR™-1 medium and require the addition of three growth factors. Using the quick protocol, PSC colonies are cultured on inactivated feeder cells and EBs are generated using mechanical dissociation and culture in PSC media in ultra-low adherence plates. After 4 days of EB culture, EBs are transferred to regular tissue culture plates and differentiated in X-VIVO™ 15 media, supplemented with M-CSF and IL-3. Non-adherent PSC-MC can be harvested from the supernatant of cultures and plated in X-VIVO™ 15 media+M-CSF without serum, or in RPMI media +10% serum+M-CSF, to generate PSC-MDM. A pure population of genetically modified PSC-MDM can be obtained by transducing PSC with a self-inactivating lentiviral construct (in this case expressing RFP-IRES-PuromycinR under control of the EF1alpha promoter). Scale bar, 200 µM. Detailed protocol described in materials and methods. B) Characteristics of the defined and quick protocol. C) Histogram represents RFP expression by PSC-MDM differentiated from PSC transduced with EF1a-RFP-IRES-PuromycinR (black line) or control PSC-MDM (shaded gray).CC BY · PubMed Central
Figure 2 Monocytopoiesis characterization in differentiation cultures. A) Time-course analysis of cell surfaces marker expressed during monocytopoiesis. Adherent EBs were harvested at 12, 21 and 33 days after EB formation and stained for various cell surface markers. Immuno-fluorescence dot plot analysis of CD38 and CD34; Thy-1 and CD34; CD45 and CD14. Gates were determined by using the relevant isotype control antibodies. B) Forward Scatter (FSC) and Side Scatter (SSC) dot plot of harvested PSC-MC showing a gate around the homogenous cell population. C) Non-adherent PSC-MC were harvested from the supernatant of differentiation cultures and counted using a cell counter (Chemometec). The media were replaced for repeated PSC-MC harvests over a period of 52 week. Data represent the cumulative number of viable PSC-MC from 6 wells.
Figure 2 Monocytopoiesis characterization in differentiation cultures. A) Time-course analysis of cell surfaces marker expressed during monocytopoiesis. Adherent EBs were harvested at 12, 21 and 33 days after EB formation and stained for various cell surface markers. Immuno-fluorescence dot plot analysis of CD38 and CD34; Thy-1 and CD34; CD45 and CD14. Gates were determined by using the relevant isotype control antibodies. B) Forward Scatter (FSC) and Side Scatter (SSC) dot plot of harvested PSC-MC showing a gate around the homogenous cell population. C) Non-adherent PSC-MC were harvested from the supernatant of differentiation cultures and counted using a cell counter (Chemometec). The media were replaced for repeated PSC-MC harvests over a period of 52 week. Data represent the cumulative number of viable PSC-MC from 6 wells.CC BY · PubMed Central
Figure 3 PSC-MC characterization compared to b-MC. A) Morphology of MC. From left to right: Eosin-methylene blue staining of MC from cytospins, Scanning Electron Microscopy, and Transmission Electron Microscopy. Scale bar, 10 µM. D) Diameter of PSC-MC (n = 7) and b-MC (n = 3) were determined by an AO-DAPI stain (Chemometec). Bars represent the mean diameter ± SEM. C) Phenotype of MC. Surface expression of CD14, CD16, CD163, CD86 and MHC II were measured by flow cytometry. Histograms represent surface staining (black line) compared to the isotype control (shaded gray). D) The symbols reflect the relative ratio of the geometric mean fluorescence intensity (MFI) over the isotype control of independent experiments.
Figure 3 PSC-MC characterization compared to b-MC. A) Morphology of MC. From left to right: Eosin-methylene blue staining of MC from cytospins, Scanning Electron Microscopy, and Transmission Electron Microscopy. Scale bar, 10 µM. D) Diameter of PSC-MC (n = 7) and b-MC (n = 3) were determined by an AO-DAPI stain (Chemometec). Bars represent the mean diameter ± SEM. C) Phenotype of MC. Surface expression of CD14, CD16, CD163, CD86 and MHC II were measured by flow cytometry. Histograms represent surface staining (black line) compared to the isotype control (shaded gray). D) The symbols reflect the relative ratio of the geometric mean fluorescence intensity (MFI) over the isotype control of independent experiments.CC BY · PubMed Central
Figure 4 PSC-MDM characterization compared to b-MC. A) Morphology of MDM. From left to right: Representative brightfield image (scale bare 200 µM), Scanning Electron Microscopy and Transmission Electron Microscopy (scale bar, 10 µM) D) Diameter of PSC-MDM (n = 3) and b-MDM (n = 6) were determined by an AO-DAPI stain (Chemometec). Bars represent the mean diameter ± SEM. C) Phenotype of MC. Surface expression of CD14, CD16, CD163, CD86 and MHC II were measured by flow cytometry. Histograms represent surface staining (black line) compared to the isotype control (shaded gray). D) The symbols reflect the relative ratio of the geometric mean fluorescence intensity (MFI) over the isotype control of independent experiments.
Figure 4 PSC-MDM characterization compared to b-MC. A) Morphology of MDM. From left to right: Representative brightfield image (scale bare 200 µM), Scanning Electron Microscopy and Transmission Electron Microscopy (scale bar, 10 µM) D) Diameter of PSC-MDM (n = 3) and b-MDM (n = 6) were determined by an AO-DAPI stain (Chemometec). Bars represent the mean diameter ± SEM. C) Phenotype of MC. Surface expression of CD14, CD16, CD163, CD86 and MHC II were measured by flow cytometry. Histograms represent surface staining (black line) compared to the isotype control (shaded gray). D) The symbols reflect the relative ratio of the geometric mean fluorescence intensity (MFI) over the isotype control of independent experiments.CC BY · PubMed Central
Figure 5 PSC-MDM activation. A) Cytokine production and surface expression of CD206 from resting (top), classically activated (middle) and alternatively activated (bottom) PSC-MDM. Histograms represent surface staining (black line) compared to the isotype control (shaded gray).
Figure 5 PSC-MDM activation. A) Cytokine production and surface expression of CD206 from resting (top), classically activated (middle) and alternatively activated (bottom) PSC-MDM. Histograms represent surface staining (black line) compared to the isotype control (shaded gray).CC BY · PubMed Central
Figure 6 PSC-MDM pathogen interactions. A) Phagocytosis of fluorescent zymosan particles. Representative histrogram showing percentage of zymosan-postive cells. B) Surface expression of the HIV-1 (co-) receptors CD4, CCR5 and CXCR4 were measured by flow cytometry. Histograms represent surface staining (black line) compared to the isotype control (shaded gray). C) Reverse transcription of HIV-1 was measured by detecting late (pol) products by q-PCR after 30 h of infection. Symbols represent the relative mean number of copies of HIV-1 DNA ± SEM of independent experiments (n = 3), normalised to the number of cells using a β-actin control. D) HIV-1 replication was measured by p24 ELISA. Symbols represent the mean p24 concentration at different time points ± SEM of independent experiments (n = 7).
Figure 6 PSC-MDM pathogen interactions. A) Phagocytosis of fluorescent zymosan particles. Representative histrogram showing percentage of zymosan-postive cells. B) Surface expression of the HIV-1 (co-) receptors CD4, CCR5 and CXCR4 were measured by flow cytometry. Histograms represent surface staining (black line) compared to the isotype control (shaded gray). C) Reverse transcription of HIV-1 was measured by detecting late (pol) products by q-PCR after 30 h of infection. Symbols represent the relative mean number of copies of HIV-1 DNA ± SEM of independent experiments (n = 3), normalised to the number of cells using a β-actin control. D) HIV-1 replication was measured by p24 ELISA. Symbols represent the mean p24 concentration at different time points ± SEM of independent experiments (n = 7).CC BY · PubMed Central
Figure 7 Phenotype of human Pluripotent Stem Cell lines. A) PluriTest analysis of Illumina HT12v4 transcriptome array data shows iPS-OX1-18, iPS-OX1-19 and iPS-OX1-23 to cluster with pluripotent stem cells (red cloud) and not with partly- or differentiated cells (blue clouds). Each circle represents one hiPSC line. B) Expression of human pluripotent stem cell markers. Surface expression of TRA-1-60 and SSEA-4, as well as total expression of Nanog and Oct 3/4, were measured by flow cytometry. Histograms represent surface staining (black line) compared to the isotype control (shaded gray).
Figure 7 Phenotype of human Pluripotent Stem Cell lines. A) PluriTest analysis of Illumina HT12v4 transcriptome array data shows iPS-OX1-18, iPS-OX1-19 and iPS-OX1-23 to cluster with pluripotent stem cells (red cloud) and not with partly- or differentiated cells (blue clouds). Each circle represents one hiPSC line. B) Expression of human pluripotent stem cell markers. Surface expression of TRA-1-60 and SSEA-4, as well as total expression of Nanog and Oct 3/4, were measured by flow cytometry. Histograms represent surface staining (black line) compared to the isotype control (shaded gray).CC BY · PubMed Central
Figure 8 Further phenotype of human Pluripotent Stem Cell lines. Pluripotent stem cells were harvested after adaptation to matrigel/mTeSR™ and after undirected differentiation for 16d as embryoid bodies (10,000 cells per EB) and expression of transcripts was assessed by qPCR. A) Silencing of transgenes in iPS cell lines (normalised to NHDF transduced with Yamanaka reprogramming retroviruses 5d before harvest, ‘NHDF+Y’, relative to actin). B) Expression of endogenous version of reprogramming genes. C) Expression of additional endogenous pluripotency-associated genes. D) Expression of Endoderm-associated genes. E) Expression of mesoderm-associated genes (BRACHYURY and MSX1) and Ectoderm-associated genes (PAX6 and MAP2). B–E normalised to corresponding HUES2 transcripts (relative to actin).
Figure 8 Further phenotype of human Pluripotent Stem Cell lines. Pluripotent stem cells were harvested after adaptation to matrigel/mTeSR™ and after undirected differentiation for 16d as embryoid bodies (10,000 cells per EB) and expression of transcripts was assessed by qPCR. A) Silencing of transgenes in iPS cell lines (normalised to NHDF transduced with Yamanaka reprogramming retroviruses 5d before harvest, ‘NHDF+Y’, relative to actin). B) Expression of endogenous version of reprogramming genes. C) Expression of additional endogenous pluripotency-associated genes. D) Expression of Endoderm-associated genes. E) Expression of mesoderm-associated genes (BRACHYURY and MSX1) and Ectoderm-associated genes (PAX6 and MAP2). B–E normalised to corresponding HUES2 transcripts (relative to actin).CC BY · PubMed Central
Figure 9 CytoSNP interrogation of human Pluripotent Stem Cell lines. KaryoStudio Detected Regions for NHDF-OX1 (parental fibroblasts) and derived hiPSC lines iPS-OX1-18, iPS-OX1-19 and iPS-OX1-23. The chromosomes which contained regions detected by KaryoStudio as deviating from expected (indicated in ‘Found Region’ column by green for amplification, orange for deletion) are shown, as are the X and Y chromosomes (which, being single copy, are Called despite being the expected copy number). Where an amplification has been detected, the SNP B allele frequency can be seen to show a corresponding small triploid region and the Smoothed Log R Ratio shows a small spike. Chromosomes that are not shown here were those (the majority) that had no regions detected by KaryoStudio as deviating from expected, and so are considered karyotypically normal. Note that the small changes detected here are well below the level of detection in other karyotype methods. The genes that these affect are listed in Table 1.
Figure 9 CytoSNP interrogation of human Pluripotent Stem Cell lines. KaryoStudio Detected Regions for NHDF-OX1 (parental fibroblasts) and derived hiPSC lines iPS-OX1-18, iPS-OX1-19 and iPS-OX1-23. The chromosomes which contained regions detected by KaryoStudio as deviating from expected (indicated in ‘Found Region’ column by green for amplification, orange for deletion) are shown, as are the X and Y chromosomes (which, being single copy, are Called despite being the expected copy number). Where an amplification has been detected, the SNP B allele frequency can be seen to show a corresponding small triploid region and the Smoothed Log R Ratio shows a small spike. Chromosomes that are not shown here were those (the majority) that had no regions detected by KaryoStudio as deviating from expected, and so are considered karyotypically normal. Note that the small changes detected here are well below the level of detection in other karyotype methods. The genes that these affect are listed in Table 1.CC BY · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Show all 44 references

Research works printed but not linked: 1

  1. K Karlsson, 2008, Homogeneous monocytes and macrophages from human embryonic stem cells following coculture-free differentiation in M-CSF and IL-3, Experimental hematology, 36, 1167, 10.1016/j.exphem.2008.04.009 · DOI · Paper page

Cited by

Sorted by citations

See all 360

Related

Metadata

Authors
Wilgenburg, B. V., Browne, C., Vowles, J., & Cowley, S. A.
Affiliations
University of Oxford (GB)
Corresponding author
Cowley, Sally A.
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2013-08-12
Volume
8
Issue
8
Pages
e71098
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0071098
PMC
PMC3741356
OpenAlex
W2121999038
Cited by
353
References
44
Citations by year
2026: 34 · 2025: 30 · 2024: 50 · 2023: 37 · 2022: 39 · 2021: 36 · 2020: 35 · 2019: 19 · 2018: 27 · 2017: 18 · 2016: 13 · 2015: 10 · 2014: 3 · 2013: 2
FWCI
4.18
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • Wellcome Trust (91070, WT082260, G090074791070, G0900747, LC0910-004, 090532, 090532/Z/09/Z, 090532/Z/09/, 090532/Z)
  • Parkinson's UK (J-0901)
  • Medical Research Council (G0900747 91070, Hub grant G0900747 91070, 090532/Z09/Z)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Mesenchymal stem cell research
  • Health Sciences › Medicine › Hematology › Hematopoietic Stem Cell Transplantation
Keywords
monocyte-derived macrophages, human pluripotent stem cells, human embryonic stem cells, human induced pluripotent stem cells, defined culture conditions, serum-free culture, HIV-1 infection, lentiviral transduction, transgene expression, macrophage phagocytosis, inflammatory cytokines, macrophage infection model
MeSH terms
Cell Differentiation, Cell Line, Culture Media, Gene Expression Regulation, Humans, Macrophages, Monocytes, Phagocytosis, Phenotype, HIV-1, Macrophage Colony-Stimulating Factor, Cell Culture Techniques, Polymorphism, Single Nucleotide, Pluripotent Stem Cells, Embryonic Stem Cells, Induced Pluripotent Stem Cells

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review