Research ArticleCELL BIOLOGY

Directed differentiation of human pluripotent stem cells to blood-brain barrier endothelial cells

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Science Advances  08 Nov 2017:
Vol. 3, no. 11, e1701679
DOI: 10.1126/sciadv.1701679
  • Fig. 1 Schematic of BMEC differentiation protocol.

    (A) Singularized hPSCs are seeded on six-well plates coated with Matrigel, vitronectin, or Synthemax and expanded for 3 days in mTeSR1. Differentiation to primitive streak is initiated by 24-hour treatment with 6 μM CHIR99021 in DeSR1. Cells progress to intermediate mesoderm and endothelial progenitors during culture in serum-free defined DeSR2 medium. At day 6, BMEC specification is induced by culture in hESFM supplemented with 2% B27, 10 μM RA, and bFGF (20 ng/ml) (hECSR1) for 2 days. After replating on Matrigel or fibronectin/collagen IV substrates, BMECs are obtained. (B to D) The pluripotent state of expanded hPSCs was verified before differentiation by immunofluorescence for OCT4 (B), NANOG (C), and TRA-1-60 (D). DAPI, 4′,6-diamidino-2-phenylindole. (E and F) The expression of the primitive streak marker brachyury was assessed by immunofluorescence (E) and flow cytometry (F) 24 hours after CHIR99021 treatment. IgG, immunoglobulin G; FSC, forward scatter. (G and H) On day 4 of differentiation, the expression of the intermediate mesoderm marker PAX2 was quantified. (I and J) On day 5, the expression of the endothelial progenitor marker VEGFR2 was analyzed. Scale bars, 100 μm.

  • Fig. 2 hPSC-derived BMECs express key BMEC proteins and have gene expression profiles similar to those of primary human BMECs.

    (A to I) At day 10, BMECs differentiated as shown in Fig. 1A were characterized by immunocytofluorescence (A) and flow cytometry (B to I) for key endothelial and BMEC markers. Scale bars, 100 μm. (J) Hierarchical clustering of whole transcripts counted by RNA sequencing was plotted using GENE-E. Hierarchical clustering analysis was performed on log2-transformed gene counts of RNA sequencing expression data of undifferentiated hPSCs (7678); hPSC-derived endoderm (Endo) (76), ectoderm (Ecto) (77), and mesoderm (Mes) (78); BMECs differentiated under defined conditions as illustrated in Fig. 1A (D-BMEC1, D-BMEC2, and D-BMEC3: IMR90-4–derived BMECs at day 10 generated from three independent differentiations); BMECs differentiated in UM (UM-BMECs); and human primary BMECs (hBMECs). Distances were computed using one minus Pearson correlation with average linkage. (K) A set of 506 tight junction and transporter genes (table S1) was used to investigate gene expression similarity between primary human BMECs, hPSC-derived BMECs differentiated under defined conditions, and hPSC-derived BMECs differentiated in UM (32). The gene set included 20 tight junction–related genes (1, 5356), all 25 CLDN genes, all 407 solute carrier (SLC) transporters, and all 53 ATP-binding cassette (ABC) transporters. CLDN, SLC, and ABC genes were included without previous knowledge of BBB association. A threshold of >1 fragment per kilobase million (FPKM) was used to define expressed versus nonexpressed transcripts.

  • Fig. 3 hPSC-derived BMECs exhibit key BBB phenotypes.

    hPSC-derived BMECs were differentiated as illustrated in Fig. 1A. (A) Immunofluorescence images of vWF (red) and DAPI staining (blue) in hPSC-derived BMECs at day 10. (B) hPSC-derived BMECs were dissociated with Accutase and replated at 1 × 105 cells/cm2 on Matrigel. Images were taken after 24 hours in hECSR2 with VEGF (50 ng/ml). (C) hPSC-derived BMECs at day 10 were analyzed with an LDL Uptake Assay kit. LDL is shown in red on a merged bright-field image. (D to F) ICAM-1 induction in hPSC-derived BMECs. hPSC-derived BMECs at day 10 were treated with TNF-α (10 ng/ml) for 16 hours. Immunofluorescence images for ICAM-1 were acquired before (D) and after (E) TNF-α treatment. (F) Cells were dissociated with Accutase, and ICAM-1 expression was quantified by flow cytometry before and after TNF-α treatment. Efflux transporter activities were measured by the intracellular accumulation of (G) rhodamine 123, (H) Hoechst, and (I) DCFDA, substrates for Pgp, BCRP, and MRP, respectively. CsA, Ko143, and MK571 were used as specific inhibitors of Pgp, BCRP, and MRP, respectively. (J) The polarization of Pgp was measured by rhodamine 123 transport across the BMEC monolayer from the apical to basolateral side (A-B) and vice versa (B-A). Inhibitor-treated samples were independently normalized to each respective non–inhibitor-treated control sample. (K) TEER was measured in hPSC-derived BMECs cocultured with astrocytes, neurons, and pericytes. Data were collected from three independent replicates and are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 100 μm.

  • Fig. 4 Initial seeding density is critical for BMEC differentiation.

    (A) hPSCs were seeded at the indicated densities (from 8800 to 140,000 cells/cm2) and differentiated to BMECs, as illustrated in Fig. 1A. TEER was measured 2 days after replating on Transwell membranes at 106 cells/cm2. (B) TEERs of hPSC-derived BMECs were measured daily for 7 days after replating on Transwell membranes. Data were collected from three independent replicates and are means ± SEM. ***P < 0.001. (C to E) The percentage of claudin-5–positive cells and expression levels of claudin-5 were quantified by flow cytometry at day 8 for cells differentiated at the indicated seeding density (cells/cm2). (F) The localization of claudin-5 in cells differentiated at different seeding densities was investigated by immunofluorescence. White arrows indicate sample areas lacking claudin-5 expression, and red arrows indicate discontinuous claudin-5. (G to I) The percentage of occludin-positive cells and expression levels of occludin were quantified by flow cytometry at day 8 for cells differentiated at the indicated seeding density (cells/cm2). (J) The localization of occludin in cells differentiated at different seeding densities was investigated by immunofluorescence. White arrows indicate sample areas lacking occludin expression, and red arrows indicate discontinuous occludin. Flow cytometry plots are representative of three independent experiments. Insets indicate the mean percentage of cells in the immunopositive gated region ± SEM. Scale bars, 100 μm.

  • Fig. 5 RA induces acquisition of key BMEC phenotypes in EC progenitors.

    BMECs were differentiated as shown in Fig. 1A in the presence or absence of RA, as indicated. (A) At day 8, the expression of tight junction and transporter genes was assessed by qPCR. (B) Flow cytometry for CD31 expression was performed at days 6 (D6) and 8 (D8). (C) Pgp expression was quantified by flow cytometry at day 10. At day 6, the medium was switched to hESFM containing or lacking RA, as indicated. (D) At day 8, cells were replated onto Matrigel-coated Transwell membranes at 106 cells/cm2 in the presence or absence of 10 μM Y-27632. TEER was measured at day 10, 2 days after replating. (E to G) Occludin (E and G) and ZO-1 (F and G) expression and localization were assessed by flow cytometry and immunofluorescence at day 10. Red arrows indicate discontinuous occludin or ZO-1 localization. (H and I) At day 10, the expression levels of claudin-5 in BMECs differentiated in the presence or absence of RA were assessed by flow cytometry. (J) The localization and expression of claudin-5 of cells differentiated in the absence of RA were determined via immunofluorescence (white arrows indicate cells lacking claudin-5, and red arrows indicate discontinuous claudin-5). Images, bar graphs, and flow cytometry plots are representative of three independent experiments. Data from three independent replicates are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Insets denote either percentage of positively labeled cells or percentage increase in marker expression upon RA treatment. Scale bars, 100 μm.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/11/e1701679/DC1

    fig. S1. Gene expression during hPSC differentiation to BMECs.

    fig. S2. BMECs differentiated from H9 hESCs and 19-9-11 iPSCs express EC- and BMEC-related proteins.

    fig. S3. BMECs differentiated on Synthemax and vitronectin express EC- and BMEC-related proteins and have efflux transporter activities.

    fig. S4. BMECs differentiated from hPSCs in defined and undefined protocols exhibit similar Pgp activities.

    fig. S5. BMECs differentiated at different seeding densities express VEGFR2 and CD31.

    fig. S6. BMECs differentiated at different seeding densities express Pgp.

    fig. S7. BMECs differentiated at different densities express BMEC proteins but can exhibit aberrant protein localization.

    fig. S8. TEER of BMECs differentiated from different hPSC lines.

    fig. S9. TEER in BMECs differentiated from hPSCs at different seeding densities.

    fig. S10. BMECs differentiated in the absence of RA exhibit low expression and mislocalization of EC and BMEC proteins.

    fig. S11. RA enhances TEER for cells differentiated on Matrigel-, vitronectin-, and Synthemax-coated surfaces.

    table S1. Expression of tight junction and transporter genes in iPSC-derived and primary human BMECs.

    table S2. Antibodies used in this study.

    table S3. qPCR primers used in this study.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Gene expression during hPSC differentiation to BMECs.
    • fig. S2. BMECs differentiated from H9 hESCs and 19-9-11 iPSCs express EC- and BMEC-related proteins.
    • fig. S3. BMECs differentiated on Synthemax and vitronectin express EC- and BMEC-related proteins and have efflux transporter activities.
    • fig. S4. BMECs differentiated from hPSCs in defined and undefined protocols exhibit similar Pgp activities.
    • fig. S5. BMECs differentiated at different seeding densities express VEGFR2 and CD31.
    • fig. S6. BMECs differentiated at different seeding densities express Pgp.
    • fig. S7. BMECs differentiated at different densities express BMEC proteins but can exhibit aberrant protein localization.
    • fig. S8. TEER of BMECs differentiated from different hPSC lines.
    • fig. S9. TEER in BMECs differentiated from hPSCs at different seeding densities.
    • fig. S10. BMECs differentiated in the absence of RA exhibit low expression and mislocalization of EC and BMEC proteins.
    • fig. S11. RA enhances TEER for cells differentiated on Matrigel-, vitronectin-, and Synthemax-coated surfaces.
    • Legend for table S1
    • table S2. Antibodies used in this study.
    • table S3. qPCR primers used in this study.

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    Other Supplementary Material for this manuscript includes the following:

    • table S1 (Microsoft Excel format). Expression of tight junction and transporter genes in iPSC-derived and primary human BMECs.

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