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CELL B IOLOGY

Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA.*Corresponding author. Email: sppalecek@wisc.edu (S.P.P.); eshusta@wisc.edu(E.V.S.)

Qian et al., Sci. Adv. 2017;3 : e1701679 8 November 2017

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Directed differentiation of human pluripotent stem cellsto blood-brain barrier endothelial cellsTongcheng Qian, Shaenah E. Maguire, Scott G. Canfield, Xiaoping Bao, William R. Olson,Eric V. Shusta,* Sean P. Palecek*

The blood-brain barrier (BBB) is composed of specialized endothelial cells that are critical to neurological health. Akey tool for understanding human BBB development and its role in neurological disease is a reliable and scalablesource of functional brain microvascular endothelial cells (BMECs). Human pluripotent stem cells (hPSCs) can the-oretically generate unlimited quantities of any cell lineage in vitro, including BMECs, for disease modeling, drugscreening, and cell-based therapies. We demonstrate a facile, chemically defined method to differentiate hPSCsto BMECs in a developmentally relevant progression via small-molecule activation of key signaling pathways.hPSCs are first induced to mesoderm commitment by activating canonical Wnt signaling. Next, these mesodermprecursors progress to endothelial progenitors, and treatment with retinoic acid leads to acquisition of BBB-specificmarkers and phenotypes. hPSC-derived BMECs generated via this protocol exhibit endothelial properties, includ-ing tube formation and low-density lipoprotein uptake, as well as efflux transporter activities characteristic ofBMECs. Notably, these cells exhibit high transendothelial electrical resistance above 3000 ohm·cm2. These hPSC-derived BMECs serve as a robust human in vitro BBB model that can be used to study brain disease and informtherapeutic development.

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INTRODUCTIONAn intact blood-brain barrier (BBB) serves as a key interface betweenthe blood circulation and the central nervous system (CNS). The pri-mary anatomical component of the BBB is provided by brain micro-vascular endothelial cells (BMECs) (1, 2) that work in concert withsupporting cells, such as astrocytes, pericytes, and neurons, to formthe neurovascular unit (1, 3, 4). BMECs are connected by tight junc-tions and display low levels of vesicular traffic, leading to extremelylow vascular permeability. BMECs also express molecular influx andefflux transporters, which regulate the delivery of nutrients from theblood to the brain and removal of compounds from the brain, respec-tively. A functional BBB prevents most of the small-molecule drugsand nearly all large-molecule biologics from entering the brain (5).Thus, the BBB is a highly efficient barrier that protects the brainand limits CNS drug delivery (6). Moreover, BBB dysfunction hasbeen associated with many CNS disorders, including stroke (7–9),Alzheimer’s disease (10, 11), multiple sclerosis (12), Parkinson’s dis-ease (13), traumatic brain injury (14, 15), and HIV (16–18).

Although the BBB has been extensively studied in animal models(19–21), in vitro models based on primary human BMECs (22, 23),and immortalized human brain EC lines (2, 24, 25), these models lackkey attributes of the human BBB. Animal models cannot fully repre-sent the human BBB because of species differences, particularly intransporter expression and function (26). Human primary BMECsare difficult to obtain in sufficient quantities for drug screening anddiseasemodels and cannot be readily expanded in high-fidelity cultures.Immortalized cell lines exhibit a loss of BMEC-specific properties, in-cluding loss of tight junctions yielding subphysiologic transendothelialelectrical resistance (TEER) (27). These limitations have preventedour full understanding of humanBBBdevelopment, function, and dis-ease (28).

Humanpluripotent stemcells (hPSCs) have the potential to generatelarge quantities of specialized human cells for studying developmentand modeling disease (29–31). Previously, we reported the generationof pure populations of hPSC-derived BMECs via co-differentiation ofhPSCs to neural and endothelial progenitors, followed by selective pu-rification of the BMECs (32). In addition, we demonstrated that retinoicacid (RA) addition during BMEC differentiation enhanced barrierproperties to physiologic levels (33). Presumably, the neural progenitorsin this co-differentiation platform induce the endothelial progenitors toacquire BMEC-specific traits, which are then enhanced by RA treat-ment.However, the undefined nature of this co-differentiation platformcomplicates the investigation ofmechanisms that specify BMEC fates inthe hPSC-derived ECs. In addition, this undefined protocol can result inline-to-line and batch-to-batch variability in BMEC yield and pheno-types (32, 34–36), despite recent efforts that have explored the use ofdefined medium to accelerate portions of the differentiation process(36). Other studies have also shown that human BMEC-like cells canbe generated from alternative stem and progenitor cell sources, includ-ing hematopoietic stem cells (37), endothelial progenitors (38), andhPSC-derived ECs cocultured with hPSC-derived pericytes, astrocytes,and neurons (39) or C6 glioma cells (40). Nevertheless, none of theseprevious studies report a chemically defined, robust process for gener-ating human BMECs exhibiting physiologic BBB phenotypes.

During embryonic development, mesoderm-derived ECs form avascular plexus covering the developing neural tube (41, 42). Asnascent blood vessels enter the developing CNS, canonical Wntsignaling is necessary to induce BMEC barrier properties (43–45).RA has also been shown to regulate BMEC specification. DuringBBB development, radial glial cells supply the CNS with RA (46),and this RA signaling induces barrier formation and BBB-specificgene expression (33, 46, 47). In addition toWnt regulation of BBB in-duction in vivo, previous studies have demonstrated that activation ofcanonicalWnt signaling can also direct hPSCs tomesodermal lineagesin vitro (31, 48–50). Thus, we hypothesized that appropriate differenti-ation stage–specific modulation of canonical Wnt signaling would in-ducemesodermal and endothelial commitment in hPSCs, and combine

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with subsequent RA signaling to drive the acquisition of BMECmarkersand phenotypes.

Here, we report a chemically definedmethod to differentiate hPSCsto BMECs via sequentialWnt and RA pathway activation. During thisdifferentiation process, hPSCs progress through stages resemblingprimitive streak and intermediate mesoderm to vascular endothelialgrowth factor receptor 2–positive (VEGFR2+) endothelial progenitorsthat become virtually pure populations of CD31+ ECs displaying keyBMECphenotypes including tight junctions, low passive permeability,and polarized efflux transporters. The resultant, developmentally rele-vant BMEC differentiation strategy is defined, robust, and facile.

Qian et al., Sci. Adv. 2017;3 : e1701679 8 November 2017

RESULTShPSCs progress from intermediate mesoderm to BMECsGiven the roles of canonical Wnt signaling in both mesoderm specifi-cation and BBBdevelopment, we first treated hPSCswithCHIR99021,a glycogen synthase kinase 3b (GSK-3b) inhibitor and canonical Wntpathway agonist, to direct hPSCs to mesoderm-derived endothelialprogenitors. Before treatment, IMR90-4 induced pluripotent stem cells(iPSCs) were seeded on a Matrigel-coated six-well plate at a density of35 × 103 cells/cm2 and expanded in an undifferentiated state for 3 daysin mTeSR1 (Fig. 1A). Previously, we showed that 6 mM CHIR99021treatment induced hPSC differentiation to a primitive streak–like stage

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Fig. 1. Schematic of BMEC differentiation protocol. (A) Singularized hPSCs are seeded on six-well plates coated with Matrigel, vitronectin, or Synthemax andexpanded for 3 days in mTeSR1. Differentiation to primitive streak is initiated by 24-hour treatment with 6 mM CHIR99021 in DeSR1. Cells progress to intermediatemesoderm and endothelial progenitors during culture in serum-free defined DeSR2 medium. At day 6, BMEC specification is induced by culture in hESFM supplementedwith 2% B27, 10 mM 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) Thepluripotent 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 afterCHIR99021 treatment. IgG, immunoglobulin G; FSC, forward scatter. (G and H) On day 4 of differentiation, the expression of the intermediate mesoderm markerPAX2 was quantified. (I and J) On day 5, the expression of the endothelial progenitor marker VEGFR2 was analyzed. Scale bars, 100 mm.

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in a serum- and albumin-free medium (51). Hence, at day 0, 6 mMCHIR99021 was added to DeSR1 [unconditioned medium (UM)lacking KnockOut Serum Replacement: DMEM/F12, 1%minimum es-sential medium (MEM)–nonessential amino acids (NEAA), 0.5%GlutaMAX, and 0.1 mM b-mercaptoethanol (32)] to initiate differenti-ation. After 24 hours, the medium was removed and cells were transi-tioned to DeSR2 (DeSR1 plus B27 supplement) for another 5 days withdaily medium changes. At day 0, pluripotency was verified by OCT4,NANOG, and TRA-1-60 immunofluorescence (Fig. 1, B to D). After24 hours of CHIR99021 treatment, almost 100% of the cells expressedthe primitive streakmarker brachyury, as assessed by immunolabeling(Fig. 1E) and flow cytometry (Fig. 1F). In concert with brachyury ex-pression, primitive streak genesT andMIXL1 (52) peaked at day 2 andthenmarkedly decreased (fig. S1). At day 4, more than 90% of the cellsexpressed the intermediatemesodermmarker PAX2 (Fig. 1, G andH),andPAX2 expression peaked at day 6 (fig. S1). Nearly 100% of the cellsexpressed the endothelial progenitor marker VEGFR2 at day 5 (Fig. 1,I and J), whereas the expression level of the endothelial progenitormarker CD34 gradually increased and then diminished after day 6(fig. S1).

At day 6, cells were switched to hECSR1 medium [human endo-thelial serum-free medium (hESFM) supplemented with basic fibro-blast growth factor (bFGF, 20 ng/ml), 10 mMRA, and B27] to induceRA signaling in the hPSC-derived endothelial progenitors in an at-tempt to drive the specification to BMECs. Cells were maintained inthis medium for 2 days. At day 8, cells were replated onto aMatrigel-coated substrate in hECSR1, and at day 9, the medium was switchedto hECSR2 (hECSR1 lacking RA and bFGF). The expression of CDH5[vascular endothelial cadherin (VE-cadherin)]was substantially inducedafter RA treatment (fig. S1). The expression of the tight junction–relatedgenes TJP1, CLDN5, and OCLN and the efflux transporter ABCB1 alsoincreased during differentiation (fig. S1). The resultant day 10 BMEC-like cells were a pure population expressing endothelial markers (CD31and VE-cadherin), BBB glucose transporter (GLUT-1), tight junctionproteins (ZO-1, claudin-5, andoccludin), and efflux transporters (BCRP,MRP1, and Pgp) (Fig. 2, A to I). Thus, sequential treatment of hPSCswith CHIR99021 and RA directed hPSCs through endothelial pro-genitors to ECs that expressed BMECmarkers. We next tested whetherthe differentiation protocol illustrated in Fig. 1A generated cells ex-pressing BMEC markers in additional hPSC lines, including H9 hu-man embryonic stem cells (hESCs) and 19-9-11 iPSCs. These linesalso produced cells expressing endothelial and BMEC markers, in-cluding CD31, GLUT-1, ZO-1, claudin-5, occludin, MRP1, BCRP1,and Pgp, at day 10 (fig. S2).

Next, RNA sequencing was used to compare global gene expres-sion profiles in the hPSC-derived BMECs differentiated, as shownin Fig. 1A, with BMECs generated from our previously reported co-differentiation system [UM (32)] and primary human BMECs. Asexpected, hPSC-derived BMECs from three independent differentia-tions clustered closely and were similar to those generated from theundefinedUMplatform.Moreover, the hPSC-derivedBMECs clusteredwith primary human BMECs and were distinct from undifferentiatedhPSCs andhPSC-derived ectoderm, endoderm, andmesoderm (Fig. 2J).The Pearson correlation coefficient between defined BMECs andprimary human BMECs was 0.77 (P < 0.001), which indicates a strongpositive association between these two groups. We next analyzed theexpression of a subset of genes that regulate key BBB attributes, includ-ing tight junctions and molecular transporters. The gene set comprises20 tight junction–related genes (1, 53–56) and an unbiased list of all

Qian et al., Sci. Adv. 2017;3 : e1701679 8 November 2017

25 CLDN genes, all 407 solute carrier (SLC) transporters, and all 53adenosine 5′-triphosphate (ATP)–binding cassette (ABC) transport-ers regardless of previous knowledge of BBB association (table S1).Primary human BMECs expressed 234 of these genes. BMECs differ-entiated from hPSCs via the definedmethod expressedmany of thesesame genes [206 of 234 (88%)], as did BMECs differentiated via theUMmethod [208 of 234 (89%); Fig. 2K], indicating a close similaritybetween hPSC-derived BMECs and primary human BMECs with re-spect to transcripts having likely relevance to BBB function.

Initially, differentiation was performed onMatrigel, which has beenshown to support BMEC generation from hPSCs (32). However, to re-move the lot-to-lot variability inherent to Matrigel and to fully definethe differentiation platform, we explored differentiation on Synthemaxand recombinant human vitronectin coatings. UndifferentiatedIMR90-4 iPSCs were expanded on either Synthemax- or vitronectin-coated surfaces for 3 days and then subjected to the differentiationprocess shown in Fig. 1A. Cells were replated onto a human placenta–derived collagen IV/human plasma–derived fibronectin-coated sur-face at day 8. Immunofluorescence at day 10 demonstrated theexpression of key BMEC proteins in cells differentiated on definedmatrices (fig. S3, A and B).

hPSC-derived BMECs exhibit BBB phenotypesIn addition to the examination of BMEC gene and protein expression,we also evaluated endothelial and BMEC phenotypes. After 8 days ofdifferentiation, cells were replated onto a Matrigel-coated surface at1 million cells/cm2 and maintained in hECSR1 medium. At day 9,culture medium was switched to hECSR2. Day 10 hPSC-derivedBMECs exhibited EC properties, including the expression of vonWillebrand factor (vWF) (Fig. 3A), formation of tube-like structureson Matrigel in the presence of VEGF (Fig. 3B), uptake of acetylatedlow-density lipoprotein (LDL) (Fig. 3C), and up-regulation of inter-cellular adhesion molecule 1 (ICAM-1) expression after treatmentwith tumor necrosis factor–a (TNF-a) (Fig. 3, D to F). BMEC effluxtransporter activities were also measured at day 10. Efflux transport-er accumulation assays were performed by quantifying intracellularaccumulation of fluorescent substrates, including the Pgp substraterhodamine 123, the MRP family substrate 2′,7′-dichlorofluoresceindiacetate (DCFDA), and the BCRP family substrate Hoechst. In thepresence of the transporter-specific inhibitors cyclosporine A (CsA;Pgp), MK571 (MRP), and Ko143 (BCRP), the intracellular accumu-lation of fluorescent substrates increased between 150 and 220%, in-dicating the activity of each class of transporters in hPSC-derivedBMECs (Fig. 3, G to I). In addition, BMECs differentiated on definedvitronectin or Synthemax substrates also exhibited Pgp, MRP, andBCRP transporter activities similar to those differentiated on Matrigel(fig. S3C). Next, polarization of Pgp activity was demonstrated bymeasuring rhodamine 123 flux across theBMECmonolayer in the pres-ence and absence of the Pgp-specific inhibitor CsA and in both the ap-ical to basolateral (A-B) and basolateral to apical (B-A) directions. Asshown in Fig. 3J, CsA treatment increased rhodamine 123 transportacross the BMEC monolayer by 160% in the A-B direction. In con-trast, CsA inhibition resulted in a 23% decrease in rhodamine 123crossing the barrier in the B-A direction (indicated in Fig. 1A), indi-cating Pgp efflux function polarized in the B-A direction. Finally,BMECs differentiated via the defined protocol exhibited Pgp accu-mulation and transport similar to those differentiated via our previ-ously reported undefined co-differentiation protocol (fig. S4, UMprotocol).

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Finally, previous studies have shown that coculturingBMECs, includ-ing those that are iPSC-derived, with neural progenitor cells, astrocytes,and pericytes can enhance BBB properties such as TEER (33, 57–60).Day 8 iPSC-derived BMECs seeded on Transwells were either main-

Qian et al., Sci. Adv. 2017;3 : e1701679 8 November 2017

tained as a monoculture or cocultured with primary human pericytesfor the first 24 hours, followed by coculture with hPSC EZ sphere–derived astrocytes and neurons (1:3) (61) for additional 3 days. Max-imum TEER for the monoculture system was ~3000 ohm·cm2, and

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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, BMECsdifferentiated 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 mm. (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 (76–78); 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 threeindependent differentiations); BMECs differentiated in UM (UM-BMECs); and human primary BMECs (hBMECs). Distances were computed using one minus Pearson correlationwith 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, 53–56), all25 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 knowl-edge of BBB association. A threshold of >1 fragment per kilobase million (FPKM) was used to define expressed versus nonexpressed transcripts.

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coculture further elevated TEER by 30% at day 2 and helpedmaintainan elevated TEER throughout the duration of the experiment com-pared to the monoculture control (Fig. 3K).

Cell density is crucial for BMEC differentiationCell density has been shown to be crucial for efficient hPSC differen-tiation to a variety of lineages, including BMECs (35, 62–64). Thus, in

Qian et al., Sci. Adv. 2017;3 : e1701679 8 November 2017

addition to the optimal initial day −3 cell seeding density used above(35 × 103 cells/cm2), we tested a range of seeding densities, from 8.8 ×103 to 140 × 103 cells/cm2, to explore how density affects BMEC yieldand phenotype. As shown in Fig. 4A, TEER was a strong function ofseeding density, with only 35 × 103 cells/cm2 yielding BMECs withsubstantial barrier function at day 2 after transfer onto Transwells.The BMEC TEER peaked 2 days after replating and plateaued above

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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 amerged bright-field image. (D to F) ICAM-1 induction in hPSC-derived BMECs. hPSC-derived BMECs at day 10 were treated with TNF-a (10 ng/ml) for 16 hours. Immuno-fluorescence images for ICAM-1 were acquired before (D) and after (E) TNF-a treatment. (F) Cells were dissociated with Accutase, and ICAM-1 expression was quantified byflow cytometry before and after TNF-a 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 ofPgp 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 wereindependently normalized to each respective non–inhibitor-treated control sample. (K) TEER was measured in hPSC-derived BMECs cocultured with astrocytes, neurons, andpericytes. Data were collected from three independent replicates and are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 100 mm.

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2000 ohm·cm2 through day 7 (Fig. 4B). At non-optimum seeding den-sities, TEER gradually increased through 6 days after replating, reach-ing approximately 1000 ohm·cm2. We next assessed the expression ofendothelial markers to investigate the endothelial specification pro-

Qian et al., Sci. Adv. 2017;3 : e1701679 8 November 2017

cess. Cells differentiated at all densities tested yielded EC populationswith nearly 100% VEGFR2+ cells at day 5 and more than 90% CD31+

cells at day 10 (fig. S5). This suggested that deficits in barrier functionmay be a result of poor BMEC specification. Thus, we assessed BMEC

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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 differ-entiated 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 weremeasured 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) Thepercentage 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 sampleareas lacking claudin-5 expression, and red arrows indicate discontinuous claudin-5. (G to I) The percentage of occludin-positive cells and expression levels of occludinwere 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 atdifferent seeding densities was investigated by immunofluorescence. White arrows indicate sample areas lacking occludin expression, and red arrows indicate dis-continuous occludin. Flow cytometry plots are representative of three independent experiments. Insets indicate the mean percentage of cells in the immunopositivegated region ± SEM. Scale bars, 100 mm.

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marker expression in populations differentiated at different seedingdensities. Nearly 100% of cells expressed Pgp after the differentiationprocess at either day 8 or day 10 (fig. S6). However, only cells differ-entiated at the optimal seeding density of 35 × 103 cells/cm2 yielded apure claudin-5–expressing population with maximal claudin-5 expres-sion (Fig. 4, C to E). In addition, a subpopulation of cells differentiatedfrom non-optimal starting densities also lacked junctional claudin-5(Fig. 4F, white arrows) or exhibited discontinuous claudin-5 localizationat cell junctions (Fig. 4F, red arrows). Similarly, only cells differen-tiated from seeding densities of at least 35 × 103 cells/cm2 yielded anearly pure population of occludin-expressing cells (Fig. 4, G to I).Immunofluorescence analysis of occludin also showed that a largefraction of cells differentiated from initial densities less than 35 ×103 cells/cm2 lacked occludin expression (Fig. 4J, white arrows). Un-like claudin-5 and occludin, seeding density did not have a significanteffect on ZO-1 expression, but cells differentiated at initial densities of8.8 × 103 cells/cm2 showed discontinuous ZO-1 localization (fig. S7, redarrows). Immunofluorescence for additional BMEC markers also indi-cated poor localization of CD31, MRP1, and BCRP in cells differen-tiated at non-optimum initial cell densities (fig. S7, compare 35k toother densities). H9 hESCs and 19-9-11 iPSCs yielded BMECs exhibit-ing TEER at or above 2000 ohm·cm2 at an optimal initial seeding den-sity of 35 × 103 cells/cm2 (fig. S8, A and B). We also compared thedifferentiation reproducibility with that of the previously reportedUM protocol (33). Although both methods produce BMECs capableof substantial barrier formation from multiple hPSC lines, BMECsdifferentiated from H9 hESCs and 19-9-11 iPSCs using the definedmethod exhibited higher TEERs and lower batch-to-batch variation(fig. S8A). Finally, 35 × 103 cells/cm2 was also found to be the optimalseeding density for differentiation on Synthemax and vitronectinsubstrates, with vitronectin substrates performing more closely toMatrigel than Synthemax substrates in TEER assays (fig. S9).

RA enhances BMEC phenotypesPreviously, we have shown that RA induces BBB properties in hPSC-derived BMECs (33). Other studies have also demonstrated that RAsignaling regulates BBB formation and induces BBB phenotypes(46, 47). To determine the role of RA in specifying BMEC differen-tiation and enhancing the BMEC phenotypes described in Figs. 2and 3, we compared differentiation in the presence and absence ofRA using the protocol illustrated in Fig. 1A. From day 6 to day 8, cellswere maintained either in hECSR1 or in hECSR1 lacking RA. Quan-titative polymerase chain reaction (qPCR) showed that the expres-sion of the tight junction–related genes TJP1, CLDN5, and OCLNand the efflux transporters ABCG2, ABCC1, and ABCB1 was greater(3- to 20-fold) in cells exposed to RA (Fig. 5A). Nearly 100% of cellsexpressed CD31 at day 6, and this expression was preserved in thepresence of RA induction at day 8 (Fig. 5B). Immunofluorescence forCD31 and other BMECmarkers for cells differentiated in the absenceof RA, including VE-cadherin, GLUT-1, and MRP1, is shown in fig.S10A. Nearly 100% of cells differentiated in the absence and presenceof RA expressed Pgp, but RA-treated cells expressed more Pgp thannontreated cells (Fig. 5C). To evaluate the barrier formation potentialof the differentiated BMECs and to assess the effects of RA treatment,we replated day 8 BMECs onto Transwells andmeasured TEER at day10. As shown in Fig. 5D, cells differentiated in the presence of RA ex-hibited physiologically relevant TEER (~4000 ohm·cm2), whereasthose differentiated in the absence of RA exhibited significantly re-duced barrier properties. Similarly, BMECs differentiated on vitronec-

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tin or Synthemax required RA supplementation to achieve substantialbarrier properties (fig. S11). We then investigated the expression andlocalization of tight junction proteins. Both occludin and ZO-1 wereexpressed in nearly all cells at day 10 regardless of RA treatment; how-ever, RA treatment significantly increased the expression levels ofoccludin and ZO-1 (Fig. 5, E and F). Although occludin and ZO-1 ex-pression was lower in the absence of RA, immunofluorescence resultsindicate that nearly all the cells differentiated in the absence of RA stillexpressed occludin and ZO-1; however, the junctional distributionwas nonuniform (Fig. 5G, indicated with red arrows and compareto Fig. 2A). In contrast to the results with occludin and ZO-1, in theabsence of RA, only around 60% of the ECs expressed claudin-5compared to 100% of RA-treated cells expressing claudin-5 (Fig. 5,H and I). In addition, claudin-5 expression was also substantiallygreater in RA-treated cells (Fig. 5I). Immunofluorescence indicatedthat in the absence of RA, many of the cells did not express claudin-5(Fig. 5J, white arrows), and those that did exhibited nonuniform junc-tional distribution of claudin-5 similar to that observed with occludinand ZO-1 (Fig. 5J, red arrows). Together, these results suggest that RAis not necessary for hPSC differentiation to ECs but enhances keyBMECphenotypes in the hPSC-derived ECs, including the expressionand localization of tight junction proteins that promote barrier func-tion as measured by TEER.

DISCUSSIONHere, we demonstrate a robust and efficient process to differentiatehPSCs to BMECs in a defined manner. The cells progress as a homo-geneous population from a pluripotent state to primitive streak–likeand intermediate mesoderm–like stages, to endothelial progenitors,and eventually to ECs that express BMEC markers and exhibit BBBbarrier and efflux transporter properties. This differentiation methoduses a completely defined platform, including culture medium andsubstrates. Defined reagents exhibit less lot-to-lot variability, leadingto more robust and efficient differentiation and allowing differentia-tion results to bemore reliable and reproducible.We have tested threedifferent hPSC lines with this differentiation protocol, and all of theselines were able to differentiate into pure populations of BMEC, withdefinitive BMEC properties at the same optimum cell density.

In vivo, ECs that form the BBB originate frommesoderm progeni-tors located outside the CNS (65). In contrast to previous BMEC dif-ferentiation protocols (32, 37) that rely on coculture of endothelialprogenitors with pericytes, astrocytes, or differentiating neural cells,this differentiation strategy instead relies on sequential Wnt and RAsignaling activation to first specify ECs and then enhance BMECprop-erties, respectively. First, activation of canonical Wnt signaling byCHIR99021 addition directs hPSCs to brachyury-positive primitivestreak–like cells that then differentiate to PAX2-positive intermediatemesoderm and EC progenitors when differentiated at the appropriatedensity in DeSR2 medium. Next, RA treatment for 2 days drives theseendothelial progenitor cells to express key BMECmarkers and exhibitBMEC-specific properties, including high TEER and efflux transporteractivity. Our experiments showed that although RA was not necessaryto obtain ECs, RA treatment significantly increasedBBBproperties suchas TEER. The TEER enhancement correlated with increased expressionand improved localization of the tight junction proteins occludin andclaudin-5. These findings are similar to those results observed after RAtreatment of hPSC-derivedBMECs generated by co-differentiationwithneural cells using our previously reported undefined protocol (33), in

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addition to those studies that have explored the barrier-enhancingeffects of astrocyte or neuron coculture with hPSC-derived BMECs(33, 60). However, even in the absence of RA treatment, the previouslyreported undefined protocol still yielded pure populations of claudin-5–expressing BMECs, whereas RA was critical in the defined protocol forgenerating pure claudin-5–expressing populations. Thus, the effects ofdefined differentiation do not exactly mimic those provided by cocul-tured neural cells, yet the resultant BMECs were quite similar on awhole-transcriptome level to BMECs generated using the undefined co-culture approach and to primary human BMECs.

Previously, we have shown that cell seeding density can affectBMEC differentiation from hPSCs using the neural co-differentiationprotocol (35). Other studies have also demonstrated that cell seedingdensity is crucial for efficient hPSC differentiation to various lineages

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(66–68). An initial cell seeding density of 35 × 103 cells/cm2 at day −3is necessary to yield homogeneous populations of BMECs with highexpression and proper localization of key BBB proteins, in turnleading to optimal barrier properties. In addition, this optimumseeding density translated to multiple hPSC lines and to differentia-tion on defined matrices. Cells differentiated at non-optimal seedingdensities expressed BMEC markers but exhibited a reduced TEER,likely resulting from diminished claudin-5 and occludin expressionand improper junctional localization. Thus, RA signaling and celldensity similarly regulate the capability for the endothelial progenitorsto acquire BMEC properties. Although we determined that seedingdensity is a crucial regulator of hPSC differentiation to BMECs, wedo not yet know the mechanism by which density affects differentia-tion efficiency.

IsotypeNO RA-D10RA-D10

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NO RA-D1095.1 ± 0.6% RA-D10 99.1 ± 0.5%191% increase

NO RA-D1099.1 ± 0.3% RA-D10 99.4 ± 0.1%328% increase

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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, asindicated. (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 mM Y-27632. TEER was measured at day 10, 2 days afterreplating. (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 indicatediscontinuous 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 byflow cytometry. (J) The localization and expression of claudin-5 of cells differentiated in the absence of RA were determined via immunofluorescence (white arrows indicatecells 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 increasein marker expression upon RA treatment. Scale bars, 100 mm.

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Coculturing hPSC-derived BMECs with pericytes, astrocytes, andneurons further elevated TEER, consistent with previous studies thatshowed that coculturing BMECs with these neural cells can enhanceBBBproperties (57, 58, 69–71). These data suggest that it will be possibleto integrate these defined hPSC-derived BMECs with other cells of theneurovascular unit to create an isogenic patient-derived model that canbe used to study the role of neurovascular unit in human neurologicaldiseases (60). In addition, this defined BMEC differentiation methodhas the potential to be a powerful and robust tool for preclinical studiesof pharmaceutical transport through the BBB.

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MATERIALS AND METHODShPSC culture and differentiationHuman iPSCs [iPS(IMR90)-4 (72), iPS-DF 19-9-11T (73), and hESCs(H9) (29)] were maintained onMatrigel (Corning)–coated surfaces inmTeSR1 (STEMCELL Technologies), as previously described (74).Before differentiation, hPSCs were singularized with Accutase (Inno-vative Cell Technologies) and plated onto Matrigel-coated plates at adensity between 25 × 103 and 50 × 103 cells/cm2 in mTeSR1 supple-mented with 10 mM Rho-associated protein kinase (ROCK) inhibitorY-27632 (Selleckchem). hPSCs were expanded in mTeSR1 for 3 days.For differentiation on defined substrates, singularized hPSCs wereplated onto the vitronectin-coated (Thermo Fisher Scientific) orSynthemax-coated (Corning) surface at a density between 25 × 103

and 50 × 103 cells/cm2 in mTeSR1 supplemented with 10 mM ROCKinhibitor Y-27632 (Selleckchem). To initiate differentiation at day 0,cells were treated with 6 mM CHIR99021 (Selleckchem) in DeSR1:DMEM/Ham’s F12 (Thermo Fisher Scientific), 1× MEM-NEAA(Thermo Fisher Scientific), 0.5× GlutaMAX (Thermo Fisher Scientific),and 0.1 mM b-mercaptoethanol (Sigma). After 24 hours, the mediumwas changed toDeSR2: DeSR1 plus 1× B27 (Thermo Fisher Scientific)every day for another 5 days. At day 6, the medium was switched tohECSR1: hESFM (Thermo Fisher Scientific) supplemented with bFGF(20 ng/ml), 10 mMRA, and 1× B27. After 2 days of culture in hECSR1medium, day 8 cells were dissociated with Accutase and plated at1 × 106 cells/cm2 in hECSR1 onto 48-well tissue culture plates or1.12-cm2 Transwell-Clear permeable inserts (0.4 mmpore size) coatedwith Matrigel (100 mg/ml). For cells differentiated on vitronectin orSynthemax substrates, cells were dissociated with Accutase and platedat 1 × 106 cells/cm2 in hECSR1 onto 48-well tissue culture plates or1.12-cm2 Transwell-Clear permeable inserts (0.4 mmpore size) coatedwith human placenta–derived collagen IV (400 mg/ml)/human plasma–derived fibronectin (100 mg/ml). At day 9, themediumwas changed tohECSR2 (hECSR1 without RA or bFGF) for longer-term mainte-nance. Y-27632 was added to increase the attachment (75) of cells dif-ferentiated in the absence of RAandwas permitted to ensure confluentmonolayer formation and confirm that the reduction in TEERwas notonly a result of incomplete monolayer formation.

ImmunochemistryCells were rinsed with ice-cold phosphate-buffered saline (PBS) onceand fixed in either ice-cold methanol or 4% paraformaldehyde (PFA)for 15 min. Cells were then blocked with 10% goat serum in PBScontaining 0.3%TritonX-100 for 30min (10%PBSGT). Primary anti-bodies were diluted in 10% PBSGT, and cells were incubated in theantibody solutions at 4°C overnight or at room temperature for 2 hours.After three PBSwashes, cellswere incubatedwith secondary antibodiesin 10% PBSGT (goat anti-rabbit Alexa Fluor 594 and goat anti-mouse

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Alexa Fluor 488; 1:200) for 1 hour at room temperature. Cells werethen washed with PBS three times, followed by treatment with DAPIFluoromount-G (Southern Biotech), and were visualized. A detailedlist of antibodies is shown in table S2.

Flow cytometryCells were dissociated with Accutase, fixed in 1% PFA for 15 min atroom temperature, and thenwashedwith 0.5% bovine serum albumin(BSA) (Bio-Rad) plus 0.1% Triton X-100 three times. Cells were stainedwith primary and secondary antibodies diluted in 0.5% BSA plus0.1% Triton X-100, as previously described (31). Data were collectedon a FACSCalibur flow cytometer (Becton Dickinson) and analyzedusing FlowJo. Corresponding isotype antibodies were used as FACS(fluorescence-activated cell sorting) gating control. Details about anti-body source and usage are provided in table S2.

Quantitative real-time PCRTotal RNAwas extractedwith theRNeasyMiniKit (Qiagen) and treatedwith deoxyribonuclease (Qiagen). One microgram of total RNA wasreverse-transcribed into complementary DNA using an oligo(dT)primer with SuperScript III Reverse Transcriptase (Invitrogen).Quantitative real-time PCRwas done in triplicatewith iQ SYBRGreenSuperMix (Bio-Rad). Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) was used as an endogenous housekeeping control. Primersequences are provided in table S3.

LDL uptake assayDifferentiated BMECs at day 10 were analyzed with the LDL UptakeAssay Kit (Abcam). Culture medium was aspirated and replaced withLDL-DyLight 550 working solution. Cells were then incubated for3 hours at 37°C, followed by three washes with PBS, and then visualizedvia fluorescentmicroscopy with excitation and emission wavelengths of540 and 570 nm, respectively. After visualization, cells were fixed with acell-based fixative solution for 10min. Cells were then washedwith tris-buffered saline plus 0.1% Triton X-100 for 5 min, followed by 30-minblocking with Cell-Based Assay Blocking Solution. Cells were thenstained with rabbit anti-LDL receptor primary antibody and DyLight488–conjugated secondary antibody. Images were taken with a fluores-cent microscope with excitation and emission wavelengths of 485 and535 nm, respectively.

Efflux transporter accumulation and transport assayPgp, BCRP, andMRP functionality was assessed by intracellular accu-mulation of fluorescent transporter substrates and transport of fluo-rescent substrates across BMECmonolayers. Rhodamine 123 (10 mM;Sigma), Hoechst (20 mM; Thermo Fisher Scientific), and DCFDA(10 mM; Life Technologies) were used as the specific substrates forPgp, BCRP1, andMRP1, respectively. BMECs at day 10were pretreatedfor 1 hour with or without specific transporter inhibitors [10 mMCsA(Pgp inhibitor), 10 mM Ko143 (BCRP inhibitor) (Sigma), and 1 mMMK571 (MRP inhibitor) (Sigma) in Hanks’ balanced salt solution(HBSS)]. Cells were then treated with transporter substrates in HBSSand incubated for 1 hour at 37°C on an orbital shaker. Cells werewashed with PBS three times and then lysed with radioimmunoprecip-itation assay buffer (Pierce Biotechnology). Fluorescence intensity wasmeasured on a plate reader (485-nm excitation and 530-nm emissionfor rhodamine 123 and DCFDA and 360-nm excitation and 497-nmemission for Hoechst). Fluorescence intensity was subsequently nor-malized to cell number determined using a hemocytometer.

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Endothelial cell tube formationEach well of a 24-well tissue culture plate was coated with 300 ml ofMatrigel (10 mg/liter). BMECs at day 10 were dissociated with Ac-cutase and plated in hECSM1 plus VEGF (50 ng/ml) without RA orbFGF at 2 × 105 cells per well. Phase-contrast images were acquiredafter 24 hours.

RNA sequencing and data analysisTotal RNA of day 10 IMR90-4 iPSC–derived BMECs with eitherdefined protocol or UM protocol and primary human brain micro-vascular ECs (ACBRI 376, Cell Systems) were prepared with theDirect-zol RNA MiniPrep Plus Kit (Zymo Research) according tothe manufacturer’s instructions. Samples were sequenced on IlluminaHiSeq 2500 at the University of Wisconsin-Madison BiotechnologyCenter. The resulting sequence reads were mapped to the human ge-nome (hg19) usingHISAT49, and the RefSeq transcript levels (FPKMs)were quantified using the Python script rpkmforgenes.py50. A hierar-chical clustering of whole transcripts was performed using GENE-Eon the log2-transformed gene counts. Distances were computed usingone minus Pearson correlation with average linkage. FASTQ files ofhPSCs (76–78) and hPSC-derived ectoderm (77), endoderm (76), andmesoderm (78) were downloaded from theGeneExpressionOmnibus(GEO) or ArrayExpress (www.ebi.ac.uk/arrayexpress/) database. Theexpression of a subset of genes that regulate key BBB attributes, in-cluding tight junctions and molecular transporters, was analyzed. Thegene set comprises 20 tight junction–related genes (1, 53–56) and anunbiased list of all 25 CLDN genes, all 407 solute carrier (SLC) trans-porters, and all 53ATP-binding cassette (ABC) transporters regardlessof previous knowledge of BBB association (table S1). Transcript levels(FPKMs)were set at a threshold of >1 FPKM,which indicatesmoderateexpression (79). Primary human BMECs were used to screen out theBBB-related genes from the gene list with the threshold of >1 FPKM.

Coculture of hPSC-derived BMECs with pericytes, neurons,and astrocyteshPSC-derived BMECs at day 8 were replated onto 12-well Transwellinserts at a density of 1 × 106 cells/cm2 and cocultured with primaryhumanpericytes residing on the bottomof the 12-well plates (1×105 cellsperwell) for 24hours in hECSR1medium. Following coculturewith peri-cytes, BMECs were cocultured with EZ sphere–derived neurons and as-trocytes (a total of 1 × 105 cells per well; 1:3) in hECSR2 for the remainderof the experiment. TEER was measured as a function of time followinginitiation of coculture.

StatisticsData are presented as means ± SEM. Statistical significance was de-termined by Student’s t test (two-tailed) between two groups. Threeor more groups were analyzed by one-way analysis of variance(ANOVA) followed by Tukey’s post hoc tests. P < 0.05 was consideredstatistically significant.

SUPPLEMENTARY MATERIALSSupplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/11/e1701679/DC1fig. S1. Gene expression during hPSC differentiation to BMECs.fig. S2. BMECs differentiated from H9 hESCs and 19-9-11 iPSCs express EC- and BMEC-relatedproteins.fig. S3. BMECs differentiated on Synthemax and vitronectin express EC- and BMEC-relatedproteins and have efflux transporter activities.

Qian et al., Sci. Adv. 2017;3 : e1701679 8 November 2017

fig. S4. BMECs differentiated from hPSCs in defined and undefined protocols exhibit similarPgp 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 exhibitaberrant 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 mislocalizationof 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 primaryhuman BMECs.table S2. Antibodies used in this study.table S3. qPCR primers used in this study.

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Acknowledgments: We acknowledge the use of the Illumina RNA Seq Services of theUniversity of Wisconsin-Madison Biotechnology Center. Funding: This work was supportedby the NIH (grants R21 NS085351, R01 NS083688, and R01 EB007534) and the TakedaPharmaceuticals New Frontier Science Program. Author contributions: The project wasconceived by T.Q., E.V.S., and S.P.P. The experiments were designed by T.Q., E.V.S., and S.P.P.and were carried out by T.Q., S.E.M., S.G.C., X.B., and W.R.O. The manuscript was writtenby T.Q., E.V.S., and S.P.P. Competing interests: T.Q., E.V.S., and S.P.P. are authors on a patentapplication related to this work (U.S. patent application no. 15/478,463, filed 4 April 2017,published 5 October 2017). All other authors declare that they have no competing interests.Data and materials availability: The final analyzed data and raw FASTQ files weresubmitted to GEO with the accession number GSE97575 (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE97575). All data needed to evaluate the conclusions in the paper are presentin the paper and/or the Supplementary Materials. Additional data related to this papermay be requested from the authors.

Submitted 19 May 2017Accepted 11 October 2017Published 8 November 201710.1126/sciadv.1701679

Citation: T. Qian, S. E. Maguire, S. G. Canfield, X. Bao, W. R. Olson, E. V. Shusta, S. P. Palecek,Directed differentiation of human pluripotent stem cells to blood-brain barrier endothelialcells. Sci. Adv. 3, e1701679 (2017).

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

PalecekTongcheng Qian, Shaenah E. Maguire, Scott G. Canfield, Xiaoping Bao, William R. Olson, Eric V. Shusta and Sean P.

DOI: 10.1126/sciadv.1701679 (11), e1701679.3Sci Adv 

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