Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Technical Report
  • Published:

Efficient derivation of microglia-like cells from human pluripotent stem cells

Nature Medicine volume 22, pages 1358–1367 (2016)Cite this article

Subjects

Abstract

Microglia, the only lifelong resident immune cells of the central nervous system (CNS), are highly specialized macrophages that have been recognized to have a crucial role in neurodegenerative diseases such as Alzheimer's, Parkinson's and adrenoleukodystrophy (ALD). However, in contrast to other cell types of the human CNS, bona fide microglia have not yet been derived from cultured human pluripotent stem cells. Here we establish a robust and efficient protocol for the rapid production of microglia-like cells from human (h) embryonic stem (ES) and induced pluripotent stem (iPS) cells that uses defined serum-free culture conditions. These in vitro pluripotent stem cell–derived microglia-like cells (termed pMGLs) faithfully recapitulate the expected ontogeny and characteristics of their in vivo counterparts, and they resemble primary fetal human and mouse microglia. We generated these cells from multiple disease-specific cell lines and find that pMGLs derived from an hES model of Rett syndrome are smaller than their isogenic controls. We further describe a platform to study the integration and live behavior of pMGLs in organotypic 3D cultures. This modular differentiation system allows for the study of microglia in highly defined conditions as they mature in response to developmentally relevant cues, and it provides a framework in which to study the long-term interactions of microglia residing in a tissue-like environment.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Induction of primitive myelogenesis from human pluripotent stem cells.
Figure 2: Characterization of phagocytes delaminating from cystic YS-EBs.
Figure 3: pMGLs adopt ramified morphologies over time and express specific markers of microglia.
Figure 4: pMGLs cytokine profiles in response to endotoxin challenge.
Figure 5: pMGLs recapitulate the consensus signature distinguishing primary microglia from other macrophages.
Figure 6: Neural co-cultures enhance the microglial signature of pMGLs.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

Referenced accessions

Gene Expression Omnibus

References

  1. Tremblay, M.E. et al. The role of microglia in the healthy brain. J. Neurosci. 31, 16064–16069 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Alliot, F., Godin, I. & Pessac, B. Microglia derive from progenitors, originating from the yolk sac, and which proliferate in the brain. Brain Res. Dev. Brain Res. 117, 145–152 (1999).

    Article  CAS  PubMed  Google Scholar 

  3. Vitry, S., Bertrand, J.Y., Cumano, A. & Dubois-Dalcq, M. Primordial hematopoietic stem cells generate microglia but not myelin-forming cells in a neural environment. J. Neurosci. 23, 10724–10731 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Ginhoux, F. et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science 330, 841–845 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hong, S., Dissing-Olesen, L. & Stevens, B. New insights on the role of microglia in synaptic pruning in health and disease. Curr. Opin. Neurobiol. 36, 128–134 (2016).

    Article  CAS  PubMed  Google Scholar 

  6. Smith, A.M., Gibbons, H.M., Lill, C., Faull, R.L. & Dragunow, M. Isolation and culture of adult human microglia within mixed glial cultures for functional experimentation and high-content analysis. Methods Mol. Biol. 1041, 41–51 (2013).

    Article  CAS  PubMed  Google Scholar 

  7. Hu, B.Y., Du, Z.W., Li, X.J., Ayala, M. & Zhang, S.C. Human oligodendrocytes from embryonic stem cells: conserved SHH signaling networks and divergent FGF effects. Development 136, 1443–1452 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Hu, B.Y. & Zhang, S.C. Differentiation of spinal motor neurons from pluripotent human stem cells. Nat. Protoc. 4, 1295–1304 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Grigoriadis, A.E. et al. Directed differentiation of hematopoietic precursors and functional osteoclasts from human ES and iPS cells. Blood 115, 2769–2776 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Bennett, M.L. et al. New tools for studying microglia in the mouse and human CNS. Proc. Natl. Acad. Sci. USA 113, E1738–E1746 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Hickman, S.E. et al. The microglial sensome revealed by direct RNA sequencing. Nat. Neurosci. 16, 1896–1905 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Ginhoux, F. & Prinz, M. Origin of microglia: current concepts and past controversies. Cold Spring Harb. Perspect. Biol. 7, a020537 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Butovsky, O., Bukshpan, S., Kunis, G., Jung, S. & Schwartz, M. Microglia can be induced by IFN-gamma or IL-4 to express neural or dendritic-like markers. Mol. Cell. Neurosci. 35, 490–500 (2007).

    Article  CAS  PubMed  Google Scholar 

  14. Chen, Y. et al. NS21: re-defined and modified supplement B27 for neuronal cultures. J. Neurosci. Methods 171, 239–247 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Zhou, J. et al. High-efficiency induction of neural conversion in human ESCs and human induced pluripotent stem cells with a single chemical inhibitor of transforming growth factor beta superfamily receptors. Stem Cells 28, 1741–1750 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Wei, S. et al. Functional overlap but differential expression of CSF-1 and IL-34 in their CSF-1 receptor-mediated regulation of myeloid cells. J. Leukoc. Biol. 88, 495–505 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Wang, Y. et al. IL-34 is a tissue-restricted ligand of CSF1R required for the development of Langerhans cells and microglia. Nat. Immunol. 13, 753–760 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Palis, J., McGrath, K.E. & Kingsley, P.D. Initiation of hematopoiesis and vasculogenesis in murine yolk sac explants. Blood 86, 156–163 (1995).

    Article  CAS  PubMed  Google Scholar 

  19. Hoeffel, G. & Ginhoux, F. Ontogeny of tissue-resident macrophages. Front. Immunol. 6, 486 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Sturgeon, C.M., Ditadi, A., Awong, G., Kennedy, M. & Keller, G. Wnt signaling controls the specification of definitive and primitive hematopoiesis from human pluripotent stem cells. Nat. Biotechnol. 32, 554–561 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. McKercher, S.R. et al. Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. EMBO J. 15, 5647–5658 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Smith, A.M. et al. The transcription factor PU.1 is critical for viability and function of human brain microglia. Glia 61, 929–942 (2013).

    Article  PubMed  Google Scholar 

  23. Feng, R. et al. PU.1 and C/EBPα/β convert fibroblasts into macrophage-like cells. Proc. Natl. Acad. Sci. USA 105, 6057–6062 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Ito, D. et al. Microglia-specific localisation of a novel calcium binding protein, Iba1. Brain Res. Mol. Brain Res. 57, 1–9 (1998).

    Article  CAS  PubMed  Google Scholar 

  25. Murray, P.J. & Wynn, T.A. Protective and pathogenic functions of macrophage subsets. Nat. Rev. Immunol. 11, 723–737 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Li, Y. et al. Global transcriptional and translational repression in human-embryonic-stem-cell-derived Rett syndrome neurons. Cell Stem Cell 13, 446–458 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Derecki, N.C., Cronk, J.C. & Kipnis, J. The role of microglia in brain maintenance: implications for Rett syndrome. Trends Immunol. 34, 144–150 (2013).

    Article  CAS  PubMed  Google Scholar 

  28. Derecki, N.C. et al. Wild-type microglia arrest pathology in a mouse model of Rett syndrome. Nature 484, 105–109 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Butovsky, O. et al. Identification of a unique TGF-β-dependent molecular and functional signature in microglia. Nat. Neurosci. 17, 131–143 (2014).

    Article  CAS  PubMed  Google Scholar 

  30. Haynes, S.E. et al. The P2Y12 receptor regulates microglial activation by extracellular nucleotides. Nat. Neurosci. 9, 1512–1519 (2006).

    Article  CAS  PubMed  Google Scholar 

  31. Zhang, Y. et al. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J. Neurosci. 34, 11929–11947 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Vitner, E.B., Futerman, A.H. & Platt, N. Innate immune responses in the brain of sphingolipid lysosomal storage diseases. Biol. Chem. 396, 659–667 (2015).

    Article  CAS  PubMed  Google Scholar 

  33. Beutner, C., Roy, K., Linnartz, B., Napoli, I. & Neumann, H. Generation of microglial cells from mouse embryonic stem cells. Nat. Protoc. 5, 1481–1494 (2010).

    Article  CAS  PubMed  Google Scholar 

  34. Kierdorf, K. et al. Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways. Nat. Neurosci. 16, 273–280 (2013).

    Article  CAS  PubMed  Google Scholar 

  35. Schulz, C. et al. A lineage of myeloid cells independent of Myb and hematopoietic stem cells. Science 336, 86–90 (2012).

    Article  CAS  PubMed  Google Scholar 

  36. Hoeffel, G. et al. C-Myb+ erythro-myeloid progenitor-derived fetal monocytes give rise to adult tissue-resident macrophages. Immunity 42, 665–678 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ginhoux, F. & Guilliams, M. Tissue-resident macrophage ontogeny and homeostasis. Immunity 44, 439–449 (2016).

    Article  CAS  PubMed  Google Scholar 

  38. Nimmerjahn, A., Kirchhoff, F. & Helmchen, F. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308, 1314–1318 (2005).

    Article  CAS  PubMed  Google Scholar 

  39. Wu, Y., Dissing-Olesen, L., MacVicar, B.A. & Stevens, B. Microglia: dynamic mediators of synapse development and plasticity. Trends Immunol. 36, 605–613 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Aubourg, P. Cerebral adrenoleukodystrophy: a demyelinating disease that leaves the door wide open. Brain 138, 3133–3136 (2015).

    Article  PubMed  Google Scholar 

  41. Ransohoff, R.M. & El Khoury, J. Microglia in health and disease. Cold Spring Harb. Perspect. Biol. 8, a020560 (2015).

    Article  PubMed  Google Scholar 

  42. Cartier, N. et al. Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science 326, 818–823 (2009).

    Article  CAS  PubMed  Google Scholar 

  43. Yirmiya, R., Rimmerman, N. & Reshef, R. Depression as a microglial disease. Trends Neurosci. 38, 637–658 (2015).

    Article  CAS  PubMed  Google Scholar 

  44. Lengner, C.J. et al. Derivation of pre-X inactivation human embryonic stem cells under physiological oxygen concentrations. Cell 141, 872–883 (2010).

    Article  CAS  PubMed  Google Scholar 

  45. Harrington, M.G. et al. Cerebrospinal fluid sodium rhythms. Cerebrospinal Fluid Res. 7, 3 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Bardy, C. et al. Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro. Proc. Natl. Acad. Sci. USA 112, E2725–E2734 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Hogins, J., Crawford, D.C., Zorumski, C.F. & Mennerick, S. Excitotoxicity triggered by Neurobasal culture medium. PLoS One 6, e25633 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Nakamichi, N. et al. Protection by exogenous pyruvate through a mechanism related to monocarboxylate transporters against cell death induced by hydrogen peroxide in cultured rat cortical neurons. J. Neurochem. 93, 84–93 (2005).

    Article  CAS  PubMed  Google Scholar 

  49. Jourdain, P. et al. l-Lactate protects neurons against excitotoxicity: implication of an ATP-mediated signaling cascade. Sci. Rep. 6, 21250 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Soldner, F. et al. Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations. Cell 146, 318–331 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors thank L. Freland-Gobaille, D. Fu, R. Alagappan, T. Lungjangwa and S. Elmsaouri for technical support, and all members of the Jaenisch lab for helpful discussions. We thank P. Thiru and G. Bell at WIBR's Bioinformatics and Research Computing group for help and advice with sequencing-data analysis. Confocal microscopy and wound assays were performed at the Keck Facility, with the precious help of W. Salmon. We thank P. Wisniewski and C. Zollo for help with cell sorting. We thank T. Volkert, J. Love and S. Gupta at the WIGTC for help with library preparation and sequencing. J.M. received funding from the European Leukodystrophy Association and a NARSAD Young Investigator Grant from the Brain & Behavior Research Foundation. Y.L. received funding from a Simons postdoctoral fellowship, an International Rett Syndrome Foundation (IRSF) postdoctoral fellowship and a NARSAD Young Investigator Grant from the Brain & Behavior Research Foundation. S.C. was the recipient of a Simons undergraduate scholarship and an Amgen Scholarship. G.B. was supported by H.H.M.I. Work for this project was supported by a grant from the Simons Foundation (SFARI 204106 R.J.), NIH grants HD 045022, R37-CA084198 and NS088538, the ELA foundation, the Emerald foundation and Biogen (to R.J.) and NIH 1RF1 AG042978 to L.-H.T.

Author information

Author notes

  1. Julien Muffat and Yun Li: These authors contributed equally to this work.

Authors and Affiliations

  1. Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA

    Julien Muffat, Yun Li, Bingbing Yuan, Maisam Mitalipova, Attya Omer, Sean Corcoran, Grisilda Bakiasi & Rudolf Jaenisch

  2. University of Paris-Sud, Institut National de la Santé et de la Recherche Médicale U1169, France

    Attya Omer & Patrick Aubourg

  3. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Sean Corcoran & Rudolf Jaenisch

  4. Bryn Mawr College, Bryn Mawr, Pennsylvania, USA

    Grisilda Bakiasi

  5. Department of Brain and Cognitive Sciences, Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Li-Huei Tsai

  6. GTDesign, Kremlin-Bicetre, France

    Patrick Aubourg

  7. Biogen, Cambridge, Massachusetts, USA

    Richard M Ransohoff

Authors
  1. Julien Muffat
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yun Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bingbing Yuan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Maisam Mitalipova
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Attya Omer
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sean Corcoran
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Grisilda Bakiasi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Li-Huei Tsai
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Patrick Aubourg
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Richard M Ransohoff
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Rudolf Jaenisch
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.M., Y.L. and R.J. conceived the project, designed and supervised the experiments, interpreted results and wrote the paper with input from R.M.R., and all other authors. P.A. provided primary AMN and ALD fibroblast cultures and helped with study inception and manuscript revision. L.-H.T. provided iPS-wt5 and input on study design and manuscript revision. J.M., Y.L. and R.J. designed the differentiation method and growth conditions for pMGLs. B.Y. performed transcriptome profile analyses and comparisons. M.M. provided additional hES and hiPS lines for the study and performed their pMGL differentiation. J.M. and Y.L. performed and analyzed all other experiments. A.O., G.B. and S.C. assisted with cell culture, sample preparation and data analysis.

Corresponding author

Correspondence to Rudolf Jaenisch.

Ethics declarations

Competing interests

R. Jaenisch is an adviser to Stemgent, and cofounder of Fate Therapeutics and Fulcrum Therapeutics. R. Ransohoff is Senior Research Fellow with Biogen. P. Aubourg is cofounder of GTDesign.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Muffat, J., Li, Y., Yuan, B. et al. Efficient derivation of microglia-like cells from human pluripotent stem cells. Nat Med 22, 1358–1367 (2016). https://doi.org/10.1038/nm.4189

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.4189

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing