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Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells

Nature Biotechnology volume 30, pages 783–791 (2012)Cite this article

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Abstract

The blood-brain barrier (BBB) is crucial to the health of the brain and is often compromised in neurological disease. Moreover, because of its barrier properties, this endothelial interface restricts uptake of neurotherapeutics. Thus, a renewable source of human BBB endothelium could spur brain research and pharmaceutical development. Here we show that endothelial cells derived from human pluripotent stem cells (hPSCs) acquire BBB properties when co-differentiated with neural cells that provide relevant cues, including those involved in Wnt/β-catenin signaling. The resulting endothelial cells have many BBB attributes, including well-organized tight junctions, appropriate expression of nutrient transporters and polarized efflux transporter activity. Notably, they respond to astrocytes, acquiring substantial barrier properties as measured by transendothelial electrical resistance (1,450 ± 140 Ω cm2), and they possess molecular permeability that correlates well with in vivo rodent blood-brain transfer coefficients.

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Figure 1: Differentiation of hPSCs into BMECs.
Figure 2: Wnt-β-catenin signaling involvement in BBB specification from hPSCs.
Figure 3: Purification of hiPSC-derived BMECs on collagen-fibronectin matrix.
Figure 4: Functional barrier properties and BBB characteristics of purified hiPSC-derived BMECs.

References

  1. Butt, A.M., Jones, H.C. & Abbott, N.J. Electrical resistance across the blood-brain barrier in anaesthetized rats: a developmental study. J. Physiol. (Lond.) 429, 47–62 (1990).

    CAS  PubMed Central  Google Scholar 

  2. Pardridge, W.M. Blood-brain barrier drug targeting: the future of brain drug development. Mol. Interv. 3, 90–105, 151 (2003).

    CAS  PubMed  Google Scholar 

  3. Weiss, N., Miller, F., Cazaubon, S. & Couraud, P.O. The blood-brain barrier in brain homeostasis and neurological diseases. Biochim. Biophys. Acta 1788, 842–857 (2009).

    CAS  PubMed  Google Scholar 

  4. Deli, M.A., Abraham, C.S., Kataoka, Y. & Niwa, M. Permeability studies on in vitro blood-brain barrier models: physiology, pathology, and pharmacology. Cell. Mol. Neurobiol. 25, 59–127 (2005).

    PubMed  Google Scholar 

  5. Syvanen, S. et al. Species differences in blood-brain barrier transport of three positron emission tomography radioligands with emphasis on P-glycoprotein transport. Drug Metab. Dispos. 37, 635–643 (2009).

    PubMed  Google Scholar 

  6. Warren, M.S. et al. Comparative gene expression profiles of ABC transporters in brain microvessel endothelial cells and brain in five species including human. Pharmacol. Res. 59, 404–413 (2009).

    CAS  PubMed  Google Scholar 

  7. Cecchelli, R. et al. Modelling of the blood-brain barrier in drug discovery and development. Nat. Rev. Drug Discov. 6, 650–661 (2007).

    CAS  PubMed  Google Scholar 

  8. Weksler, B.B. et al. Blood-brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J. 19, 1872–1874 (2005).

    CAS  PubMed  Google Scholar 

  9. Forster, C. et al. Differential effects of hydrocortisone and TNF-α on tight junction proteins in an in vitro model of the human blood-brain barrier. J. Physiol. (Lond.) 586, 1937–1949 (2008).

    Google Scholar 

  10. Man, S. et al. Human brain microvascular endothelial cells and umbilical vein endothelial cells differentially facilitate leukocyte recruitment and utilize chemokines for T cell migration. Clin. Dev. Immunol. 2008, 384982 (2008).

    PubMed  PubMed Central  Google Scholar 

  11. Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).

    CAS  PubMed  Google Scholar 

  12. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    CAS  PubMed  Google Scholar 

  13. Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

    CAS  PubMed  Google Scholar 

  14. Goldman, O. et al. A boost of BMP4 accelerates the commitment of human embryonic stem cells to the endothelial lineage. Stem Cells 27, 1750–1759 (2009).

    CAS  PubMed  Google Scholar 

  15. James, D. et al. Expansion and maintenance of human embryonic stem cell-derived endothelial cells by TGF-β inhibition is Id1 dependent. Nat. Biotechnol. 28, 161–166 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Levenberg, S., Golub, J.S., Amit, M., Itskovitz-Eldor, J. & Langer, R. Endothelial cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 99, 4391–4396 (2002).

    CAS  PubMed  Google Scholar 

  17. Nakahara, M. et al. High-efficiency production of subculturable vascular endothelial cells from feeder-free human embryonic stem cells without cell-sorting technique. Cloning Stem Cells 11, 509–522 (2009).

    CAS  PubMed  Google Scholar 

  18. Wang, L. et al. Endothelial and hematopoietic cell fate of human embryonic stem cells originates from primitive endothelium with hemangioblastic properties. Immunity 21, 31–41 (2004).

    CAS  PubMed  Google Scholar 

  19. Choi, K.D. et al. Hematopoietic and endothelial differentiation of human induced pluripotent stem cells. Stem Cells 27, 559–567 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Vodyanik, M.A., Bork, J.A., Thomson, J.A. & Slukvin, I.I. Human embryonic stem cell-derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential. Blood 105, 617–626 (2005).

    CAS  PubMed  Google Scholar 

  21. Cleaver, O. & Melton, D.A. Endothelial signaling during development. Nat. Med. 9, 661–668 (2003).

    CAS  PubMed  Google Scholar 

  22. Weidenfeller, C., Svendsen, C.N. & Shusta, E.V. Differentiating embryonic neural progenitor cells induce blood-brain barrier properties. J. Neurochem. 101, 555–565 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Bauer, H.C. et al. Neovascularization and the appearance of morphological characteristics of the blood-brain barrier in the embryonic mouse central nervous system. Brain Res. Dev. Brain Res. 75, 269–278 (1993).

    CAS  PubMed  Google Scholar 

  24. Daneman, R., Zhou, L., Kebede, A.A. & Barres, B.A. Pericytes are required for blood-brain barrier integrity during embryogenesis. Nature 468, 562–566 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Stewart, P.A. & Hayakawa, K. Early ultrastructural changes in blood-brain barrier vessels of the rat embryo. Brain Res. Dev. Brain Res. 78, 25–34 (1994).

    CAS  PubMed  Google Scholar 

  26. Daneman, R. et al. Wnt/β-catenin signaling is required for CNS, but not non-CNS, angiogenesis. Proc. Natl. Acad. Sci. USA 106, 641–646 (2009).

    CAS  PubMed  Google Scholar 

  27. Stenman, J.M. et al. Canonical Wnt signaling regulates organ-specific assembly and differentiation of CNS vasculature. Science 322, 1247–1250 (2008).

    CAS  PubMed  Google Scholar 

  28. Ying, Q.L., Stavridis, M., Griffiths, D., Li, M. & Smith, A. Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat. Biotechnol. 21, 183–186 (2003).

    CAS  PubMed  Google Scholar 

  29. Kane, N.M. et al. Derivation of endothelial cells from human embryonic stem cells by directed differentiation: analysis of microRNA and angiogenesis in vitro and in vivo. Arterioscler. Thromb. Vasc. Biol. 30, 1389–1397 (2010).

    CAS  PubMed  Google Scholar 

  30. Wang, Z.Z. et al. Endothelial cells derived from human embryonic stem cells form durable blood vessels in vivo. Nat. Biotechnol. 25, 317–318 (2007).

    CAS  PubMed  Google Scholar 

  31. Daneman, R. et al. The mouse blood-brain barrier transcriptome: a new resource for understanding the development and function of brain endothelial cells. PLoS ONE 5, e13741 (2010).

    PubMed  PubMed Central  Google Scholar 

  32. Calabria, A.R., Weidenfeller, C., Jones, A.R., de Vries, H.E. & Shusta, E.V. Puromycin-purified rat brain microvascular endothelial cell cultures exhibit improved barrier properties in response to glucocorticoid induction. J. Neurochem. 97, 922–933 (2006).

    CAS  PubMed  Google Scholar 

  33. Yu, J. et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797–801 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Liebner, S. et al. Wnt/β-catenin signaling controls development of the blood-brain barrier. J. Cell Biol. 183, 409–417 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Xu, Q. et al. Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair. Cell 116, 883–895 (2004).

    CAS  PubMed  Google Scholar 

  36. Ye, X. et al. Norrin, frizzled-4, and Lrp5 signaling in endothelial cells controls a genetic program for retinal vascularization. Cell 139, 285–298 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Robitaille, J. et al. Mutant frizzled-4 disrupts retinal angiogenesis in familial exudative vitreoretinopathy. Nat. Genet. 32, 326–330 (2002).

    CAS  PubMed  Google Scholar 

  38. Kawaguchi, R. et al. A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A. Science 315, 820–825 (2007).

    CAS  PubMed  Google Scholar 

  39. Szeto, W. et al. Overexpression of the retinoic acid-responsive gene Stra6 in human cancers and its synergistic induction by Wnt-1 and retinoic acid. Cancer Res. 61, 4197–4205 (2001).

    CAS  PubMed  Google Scholar 

  40. Shimomura, Y. et al. APCDD1 is a novel Wnt inhibitor mutated in hereditary hypotrichosis simplex. Nature 464, 1043–1047 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Anderson, K.D. et al. Angiogenic sprouting into neural tissue requires Gpr124, an orphan G protein-coupled receptor. Proc. Natl. Acad. Sci. USA 108, 2807–2812 (2011).

    CAS  PubMed  Google Scholar 

  42. Cullen, M. et al. GPR124, an orphan G protein-coupled receptor, is required for CNS-specific vascularization and establishment of the blood-brain barrier. Proc. Natl. Acad. Sci. USA 108, 5759–5764 (2011).

    CAS  PubMed  Google Scholar 

  43. Kuhnert, F. et al. Essential regulation of CNS angiogenesis by the orphan G protein-coupled receptor GPR124. Science 330, 985–989 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Vittet, D. et al. Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps. Blood 88, 3424–3431 (1996).

    CAS  PubMed  Google Scholar 

  45. Rubin, L.L. et al. A cell culture model of the blood-brain barrier. J. Cell Biol. 115, 1725–1735 (1991).

    CAS  PubMed  Google Scholar 

  46. Liebner, S., Kniesel, U., Kalbacher, H. & Wolburg, H. Correlation of tight junction morphology with the expression of tight junction proteins in blood-brain barrier endothelial cells. Eur. J. Cell Biol. 79, 707–717 (2000).

    CAS  PubMed  Google Scholar 

  47. Roberts, L.M. et al. Expression of the thyroid hormone transporters monocarboxylate transporter-8 (SLC16A2) and organic ion transporter-14 (SLCO1C1) at the blood-brain barrier. Endocrinology 149, 6251–6261 (2008).

    CAS  PubMed  Google Scholar 

  48. Uchida, Y. et al. Quantitative targeted absolute proteomics of human blood-brain barrier transporters and receptors. J. Neurochem. 117, 333–345 (2011).

    CAS  PubMed  Google Scholar 

  49. Perriere, N. et al. A functional in vitro model of rat blood-brain barrier for molecular analysis of efflux transporters. Brain Res. 1150, 1–13 (2007).

    CAS  PubMed  Google Scholar 

  50. Cohen-Kashi Malina, K., Cooper, I. & Teichberg, V.I. Closing the gap between the in vivo and in vitro blood-brain barrier tightness. Brain Res. 1284, 12–21 (2009).

    CAS  PubMed  Google Scholar 

  51. Ludwig, T.E. et al. Feeder-independent culture of human embryonic stem cells. Nat. Methods 3, 637–646 (2006).

    CAS  PubMed  Google Scholar 

  52. de Planell-Saguer, M., Rodicio, M.C. & Mourelatos, Z. Rapid in situ codetection of noncoding RNAs and proteins in cells and formalin-fixed paraffin-embedded tissue sections without protease treatment. Nat. Protoc. 5, 1061–1073 (2010).

    CAS  PubMed  Google Scholar 

  53. Huang, S.M. et al. Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling. Nature 461, 614–620 (2009).

    CAS  PubMed  Google Scholar 

  54. Dravid, G. et al. Defining the role of Wnt/β-catenin signaling in the survival, proliferation, and self-renewal of human embryonic stem cells. Stem Cells 23, 1489–1501 (2005).

    CAS  PubMed  Google Scholar 

  55. Tuomi, J.M., Voorbraak, F., Jones, D.L. & Ruijter, J.M. Bias in the Cq value observed with hydrolysis probe based quantitative PCR can be corrected with the estimated PCR efficiency value. Methods 50, 313–322 (2010).

    CAS  PubMed  Google Scholar 

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Acknowledgements

This work was funded in part by US National Institutes of Health (NIH) grants NS056249 (E.V.S.), AA020476 (E.V.S.) and EB007534 (S.P.P.) and US National Science Foundation (NSF) grant EFRI-0735903 (S.P.P.). E.S.L. is a recipient of a NIH Chemistry Biology Interface traineeship (T32 GM008505) and S.M.A. is the recipient of a NSF Graduate Research fellowship. We thank the WiCell Research Institute for providing research support and W.M. Pardridge (University of California–Los Angeles) for the gift of GLUT-1 antiserum.

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  1. Ethan S Lippmann and Samira M Azarin: These authors contributed equally to the work.

  2. palecek@engr.wisc.edu

Authors and Affiliations

  1. Department of Chemical and Biological Engineering, University of Wisconsin–Madison, Madison, Wisconsin, USA

    Ethan S Lippmann, Samira M Azarin, Jennifer E Kay, Hannah K Wilson, Abraham Al-Ahmad, Sean P Palecek & Eric V Shusta

  2. Central Microscopy Research Facility, University of Iowa, Iowa City, Iowa, USA

    Randy A Nessler

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Contributions

E.S.L., S.M.A., S.P.P. and E.V.S. conceived the hPSC-derived BMEC strategy, designed all experiments, analyzed all data and wrote the paper. E.S.L. and S.M.A. did the majority of experiments. J.E.K. did RT-PCR experiments, R.A.N. carried out freeze-fracture microscopy and contributed to its interpretation, H.K.W. did fluorescence in situ hybridization experiments and A.A.-A. contributed to characterization of the BMECs.

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Correspondence to Sean P Palecek or Eric V Shusta.

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The authors declare no competing financial interests.

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Lippmann, E., Azarin, S., Kay, J. et al. Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells. Nat Biotechnol 30, 783–791 (2012). https://doi.org/10.1038/nbt.2247

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