Research ArticleNEUROSCIENCE

Human pluripotent stem cell–derived brain pericyte–like cells induce blood-brain barrier properties

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Science Advances  13 Mar 2019:
Vol. 5, no. 3, eaau7375
DOI: 10.1126/sciadv.aau7375
  • Fig. 1 Generation of multipotent NCSC populations.

    (A) NCSC differentiation timeline. Small-molecule activation of canonical WNT signaling and small-molecule inhibition of activin/nodal/TGFβ/BMP signaling in minimal medium produce H9-derived NCSCs over a 15-day treatment window. NCSCs are then magnetically sorted and replated for subsequent mural cell differentiation. (B) Immunocytochemistry images of H9 hESCs differentiated in E6-CSFD probed for the presence of HNK1 and p75-NGFR at D15. NCSCs are HNK1+/p75-NGFR+ cells. Hoechst nuclear counterstain (blue) is also included. Scale bars, 100 μm. (C) AP-2 immunocytochemistry images for H9-derived NCSCs at D15. Hoechst nuclear counterstain (blue) is also included. Scale bar, 100 μm. (D) Temporal polymerase chain reaction (PCR) analysis of pluripotency (NANOG and POU5F1) and NCSC (TFAP2A, B3GAT1, NGFR, SOX9, and SOX10) transcripts. (E) Quantification of NCSC expansion in population doublings over the 15 days of NCSC differentiation. Plotted are the means ± SD of three technical replicates of a representative differentiation. (F) Flow cytometry analysis of H9-derived NCSCs. Panels include isotype controls (i), NCSC (HNK1+/p75-NGFR+) purity before magnetic-activated cell sorting (MACS) (ii), and NCSC purity following MACS (iii). Inset percentages are included in each quadrant. Quantitation is shown in (J). (G) Immunocytochemistry analysis of D16 NCSCs following MACS and replating. NCSCs maintained HNK1 and p75-NGFR expression. Hoechst nuclear counterstain (blue) is also included. Scale bars, 100 μm. (H) Immunocytochemistry analysis of H9-derived NCSCs subsequently differentiated in peripheral neuron medium. Resultant cells were positive for βIII-tubulin and peripherin expression. Hoechst nuclear counterstain (blue) is also included. Scale bar, 200 μm. (I) H9-derived NCSCs could be differentiated into mesenchymal derivatives, including Oil Red O–stained adipocytes (i, red), Alizarin red–stained osteocytes (ii, red), and Alcian blue–stained chondrocytes (ii, blue). Scale bars, 200 μm. (J) NCSC and pericyte-like cell differentiation efficiencies for three hPSC lines.

  • Fig. 2 Serum treatment directs H9-derived NCSCs toward mural cells.

    (A) Differentiation timeline for mural cell differentiation. Replated NCSCs are differentiated to mural cells in E6 medium + 10% FBS for 9 days. (B) PDGFRβ and NG2 immunocytochemistry of cells obtained after treating replated H9-derived NCSCs for 6 days in E6, E6 + TGFβ1 + PDGF-BB, or E6 + 10% FBS on uncoated tissue culture polystyrene or E6 + 10% FBS on gelatin-coated tissue culture polystyrene. Scale bars, 200 μm. (C) Temporal flow cytometry analysis for PDGFRβ+ and NG2+ cells in H9-derived NCSCs treated with E6 + 10% FBS. Depicted are the means ± SEM of at least two independent differentiations at each time point, *P < 0.05 versus D15 NCSCs using analysis of variance (ANOVA) followed by Dunnett’s test. (D) Representative PDGFRβ and NG2 flow cytometry plots for H9-derived NCSCs treated for 9 days with E6 + 10% FBS medium. Quantitative data can be found in Fig. 1J. (E) Temporal PCR analysis of mural and pericyte transcripts for the differentiating H9 hESCs. (F) PDGFRβ and NG2 immunocytochemistry of H9-derived NCSCs (D16), mural cells (D22), and primary pericytes. Hoechst nuclear counterstain (blue) is also included. Scale bars, 200 μm. (G) Calponin and SM22α immunocytochemistry of H9-derived NCSCs (D16), mural cells (D22), and primary pericytes. Hoechst nuclear counterstain (blue) is also included. Scale bars, 200 μm. (H) α-SMA immunocytochemistry of H9-derived NCSCs (D16), mural cells (D22), and primary pericytes. Hoechst nuclear counterstain (blue) is also included. Scale bars, 200 μm. (I) CD13 immunocytochemistry of H9-derived mural cells (D22). Hoechst nuclear counterstain (blue) is also included. Scale bar, 200 μm. (J) Desmin immunocytochemistry of H9-derived mural cells (D22). Hoechst nuclear counterstain (blue) is also included. Scale bar, 200 μm.

  • Fig. 3 RNA-seq of pericyte-like cells and related cell types.

    (A) Hierarchical clustering based on all transcripts of undifferentiated H9 hESCs; H9-derived NCSCs at D15 and after an additional 40 days in E6-CSFD (D55); H9-derived pericyte-like cells at D19, D22, and D25 (three independent differentiations at the D25 time point, indicated as “H9-A,” “H9-B,” and “H9-C”); H9-derived pericyte-like cells maintained for an additional 20 days in E6 + 10% FBS (D45); CS03n2- and IMR90C4-derived pericyte-like cells at D25; and primary brain pericytes (from two distinct cultures of the same cell source, indicated as “Primary-A” and “Primary-B”). (B) Expression (FPKM) of selected transcripts in H9 hPSCs (day “0”), NCSCs (“15”), and during the differentiation of pericyte-like cells (“19,” “22,” “25,” and “45”). The mean transcript expression in all D25 hPSC-derived pericyte-like cells (H9-A to H9-C, CS03n2, and IMR90C4; “H”) and in primary brain pericytes (“P”) is also shown. Error bars represent SEM of five independent differentiations (“H”) or of two primary pericyte samples (“P”). (C) Top 10 GO terms sorted by enrichment score [ES = −log10(FDR)] for hPSC-derived pericyte-like cells. Genes included in the dataset were enriched in pericyte-like cells (average of all D25 samples) compared to NCSCs (average of D15 and D55 samples) (FPKMpericyte-like cells/FPKMNCSC ≥ 10) and were expressed at ≥1 FPKM in pericyte-like cells. (D) Expression (≥1 FPKM) of murine pericyte-enriched transcripts [46 transcripts (40)] in hPSC-derived pericyte-like cells (29 transcripts) and primary brain pericytes (26 transcripts). A detailed listing of genes and FPKM values can be found in table S2.

  • Fig. 4 hPSC-derived pericyte-like cell assembly with endothelial cells.

    (A) Self-assembly schematic. hPSC-derived pericyte-like cells self-assemble with HUVECs to form vascular cords. (B) Confocal immunocytochemistry images of primary pericytes and H9-derived pericyte-like cells (NG2) aligning with and extending processes along HUVEC cords (CD31). Hoechst nuclear counterstain (blue) is also included. Scale bars, 50 μm. (C) Immunocytochemistry images of HUVECs alone or cultured with HEK293 fibroblasts (+HEK293), primary human brain pericytes (+Primary pericytes), CS03n2-derived pericyte-like cells (+CS03n2), H9-derived pericyte-like cells (+H9), or IMR90C4-derived pericyte-like cells (+IMR90C4). Hoechst nuclear counterstain (blue) is also included. Scale bars, 100 μm. (D) Representative bright-field images of HUVECs alone or cultured with the various cell types. Scale bars, 300 μm. (E) Quantification of the average segment lengths from bright-field images in (D). Plotted are means ± SEM of three independent pericyte-like cell differentiations. *P < 0.05 versus HUVEC monoculture, ANOVA followed by Dunnett’s test. (F) Quantification of the number of segments per field normalized to HUVEC monoculture from bright-field images in (D). Plotted are means ± SEM of three independent pericyte-like cell differentiations. *P < 0.05 versus HUVEC monoculture, ANOVA followed by Dunnett’s test.

  • Fig. 5 Measurement of the effects of hPSC-derived pericyte-like cells on BBB phenotypes.

    (A) Schematic of Transwell setup for coculture assays. (B) Maximum TEER achieved by IMR90C4-derived BMEC monoculture or coculture with 3T3 mouse fibroblasts, primary human brain pericytes, H9-derived pericyte-like cells, CS03n2-derived pericyte-like cells, or IMR90C4-derived pericyte-like cells. Plotted are the means ± SEM of at least three independent differentiations per condition. *P < 0.05 versus monoculture, ANOVA followed by Dunnett’s test. (C) Sodium fluorescein permeability for IMR90C4-derived BMECs in monoculture or coculture with cell types as described in (B). Plotted are the means ± SEM of at least three independent differentiations per condition. *P < 0.05 versus monoculture, ANOVA followed by Dunnett’s test. (D) Representative images of occludin immunocytochemistry of BMECs cultured for 48 hours in EC medium (Mono) or EC medium conditioned by the cell types described in (B). Enlarged example of a frayed junction is inset in the monoculture panel. Scale bar, 25 μm. (E) Quantification of occludin area fraction index for the samples described in (D). Plotted are the means ± SEM of three independent differentiations. No significant difference by ANOVA. (F) Quantification of frayed junctions visualized by occludin immunocytochemistry for the samples described in (D). Plotted are the means ± SEM of three independent differentiations. *P < 0.05 versus monoculture, ANOVA followed by Dunnett’s test. (G) Accumulation of Alexa 488–tagged 10-kDa dextran in IMR90C4-derived BMECs following 48 hours of coculture with cell types as described in (B). All results are normalized to BMEC monoculture control. Plotted are the means ± SD of three Transwells. Results are representative of three independent differentiations. *P < 0.05 versus monoculture, ANOVA followed by Dunnett’s test. (H and I) Transcytosis of Alexa 488–tagged 10-kDa dextran at 37°C (H) or 4°C (I) across IMR90C4-derived BMECs following 48 hours of coculture with the cell types as described in (B). All results are normalized to BMEC monoculture control. Plotted are the means ± SD from three Transwells. Results are representative of three independent differentiations. *P < 0.05 versus monoculture, ANOVA followed by Dunnett’s test. No significant differences at 4°C by ANOVA.

  • Fig. 6 IMR90C4-derived pericyte-like cells integrate into a complete isogenic NVU model.

    (A) Schematic of IMR90C4-derived BMEC coculture set up with IMR90C4-derived NVU cell types. (B) Maximum TEER achieved in IMR90C4-derived BMECs following monoculture or coculture. Plotted are the means ± SD from three Transwells. Results are representative of three independent differentiations. *P < 0.05 versus monoculture, #P < 0.05 versus pericyte-like cell coculture, and %P < 0.05 versus astrocyte/neuron coculture, ANOVA followed by Tukey’s honestly significant difference (HSD) test. (C) Sodium fluorescein permeability in IMR90C4-derived BMECs following 48 hours of monoculture or coculture. Plotted are the means ± SD from three Transwells. Results are representative of three independent differentiations. *P < 0.05 versus monoculture, ANOVA followed by Tukey’s HSD test.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/3/eaau7375/DC1

    Fig. S1. Generation of multipotent NCSC populations from multiple hPSC lines.

    Fig. S2. Serum treatment directs iPSC-derived NCSCs toward mural cells.

    Fig. S3. Supplemental analysis of hPSC-derived pericyte-like cells.

    Fig. S4. Supplemental analysis of BMEC/hPSC-derived pericyte-like cell cocultures.

    Fig. S5. Measurement of the effects of hPSC-derived pericyte-like cells on primary rat BMEC phenotypes.

    Fig. S6. NCSCs maintained in E6-CSFD retain neural crest marker expression and do not develop pericyte marker expression.

    Fig. S7. hPSC-derived pericyte-like cell assembly with brain endothelial cells.

    Table S1. Antibody staining conditions and DNA primer sequences and running conditions.

    Table S2. Pericyte-enriched genes identified by single-cell RNA-seq in mouse (40) with human homologs.

    Table S3. RNA-seq FPKM data for all samples.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Generation of multipotent NCSC populations from multiple hPSC lines.
    • Fig. S2. Serum treatment directs iPSC-derived NCSCs toward mural cells.
    • Fig. S3. Supplemental analysis of hPSC-derived pericyte-like cells.
    • Fig. S4. Supplemental analysis of BMEC/hPSC-derived pericyte-like cell cocultures.
    • Fig. S5. Measurement of the effects of hPSC-derived pericyte-like cells on primary rat BMEC phenotypes.
    • Fig. S6. NCSCs maintained in E6-CSFD retain neural crest marker expression and do not develop pericyte marker expression.
    • Fig. S7. hPSC-derived pericyte-like cell assembly with brain endothelial cells.
    • Table S1. Antibody staining conditions and DNA primer sequences and running conditions.
    • Table S2. Pericyte-enriched genes identified by single-cell RNA-seq in mouse (40) with human homologs.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S3 (Microsoft Excel format). RNA-seq FPKM data for all samples.

    Files in this Data Supplement:

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