Wnt signaling mediates acquisition of blood–brain barrier properties in naïve endothelium derived from human pluripotent stem cells

  1. Benjamin D Gastfriend
  2. Hideaki Nishihara
  3. Scott G Canfield
  4. Koji L Foreman
  5. Britta Engelhardt
  6. Sean P Palecek  Is a corresponding author
  7. Eric V Shusta  Is a corresponding author
  1. Department of Chemical and Biological Engineering, University of Wisconsin–Madison, United States
  2. Theodor Kocher Institute, University of Bern, Switzerland
  3. Department of Neurological Surgery, University of Wisconsin–Madison, United States

Abstract

Endothelial cells (ECs) in the central nervous system (CNS) acquire their specialized blood–brain barrier (BBB) properties in response to extrinsic signals, with Wnt/β-catenin signaling coordinating multiple aspects of this process. Our knowledge of CNS EC development has been advanced largely by animal models, and human pluripotent stem cells (hPSCs) offer the opportunity to examine BBB development in an in vitro human system. Here, we show that activation of Wnt signaling in hPSC-derived naïve endothelial progenitors, but not in matured ECs, leads to robust acquisition of canonical BBB phenotypes including expression of GLUT-1, increased claudin-5, decreased PLVAP, and decreased permeability. RNA-seq revealed a transcriptome profile resembling ECs with CNS-like characteristics, including Wnt-upregulated expression of LEF1, APCDD1, and ZIC3. Together, our work defines effects of Wnt activation in naïve ECs and establishes an improved hPSC-based model for interrogation of CNS barriergenesis.

Editor's evaluation

This work provides a considerable novelty to the view on barrier induction in endothelial cells in the central nervous system by the Wnt/β-catenin pathway. By recapitulating the endothelial differentiations events towards a blood–brain barrier phenotype, the presented results offer novel ways to establish cell culture systems that faithfully recapitulate the blood–brain barrier ex vivo, and thereby supporting basic as well as translational research.

https://doi.org/10.7554/eLife.70992.sa0

eLife digest

The cells that line the inside of blood vessels are called endothelial cells. In the blood vessels of the brain, these cells form a structure called the ‘blood-brain barrier’, which allows nutrients to pass from the blood into the brain, while at the same time preventing harmful substances like toxins from crossing. Faults in the blood-brain barrier can contribute to neurological diseases, but the blood-brain barrier can also restrict drugs from accessing the brain, making it difficult to treat certain conditions. Understanding how the endothelial cells that form the blood-brain barrier develop may offer insight into new treatments for neurological diseases.

During the development of the embryo, endothelial cells develop from stem cells. They can also be generated in the laboratory from human pluripotent stem cells or ‘hPSCs’, which are cells that can produce more cells like themselves, or differentiate into any cell type in the body. Scientists can treat hPSCs with specific molecules to make them differentiate into endothelial cells, or to modify their properties. This allows researchers to monitor how different types of endothelial cells form.

Endothelial cells at the blood-brain barrier are one of these types. During their development, these cells gain distinct features, including the production of proteins called GLUT-1, claudin-5 and LSR. GLUT-1 transports glucose across endothelial cells’ membranes, while claudin-5 and LSR tightly join adjacent cells together, preventing molecules from leaking into the brain through the space between cells. In mouse endothelial cells, a signaling protein called Wnt is responsible for turning on the genes that code for these proteins. But how does Wnt signaling impact human endothelial cells?

Gastfriend et al. probed the effects of Wnt signaling on human endothelial cells grown in the lab as they differentiate from hPSCs. They found that human endothelial cells developed distinct blood-brain barrier features when Wnt signaling was activated, producing GLUT-1, claudin-5 and LSR. Gastfriend et al. also found that human endothelial cells were more responsive to Wnt signaling earlier in their development. Additionally, they identified the genes that became activated in human endothelial cells when Wnt signaling was triggered.

These findings provide insight into the development and features of the endothelial cells that form the human blood-brain barrier. The results are a first step towards a better understanding of how this structure works in humans. This information may also allow researchers to develop new ways to deliver drugs into the brain.

Introduction

In the central nervous system (CNS), vascular endothelial cells (ECs) are highly specialized, with complex tight junctions, expression of a spectrum of nutrient and efflux transporters, low rates of vesicle trafficking, no fenestrae, and low expression of immune cell adhesion molecules (Reese and Karnovsky, 1967; Obermeier et al., 2013). ECs bearing these attributes, often referred to as the blood–brain barrier (BBB), work in concert with the other brain barriers to facilitate the tight regulation of the CNS microenvironment required for proper neuronal function (Daneman and Engelhardt, 2017; Profaci et al., 2020). During development, the Wnt/β-catenin signaling pathway drives both CNS angiogenesis, during which vascular sprouts originating from the perineural vascular plexus invade the developing neural tube, and the coupled process of barriergenesis by which resulting ECs begin to acquire BBB properties (Liebner et al., 2008; Stenman et al., 2008; Daneman et al., 2009; Engelhardt and Liebner, 2014; Umans et al., 2017). Specifically, neural progenitor-derived Wnt7a and Wnt7b ligands signal through Frizzled receptors and the obligate co-receptors RECK and GPR124 (ADGRA2) on ECs (Kuhnert et al., 2010; Cullen et al., 2011; Vanhollebeke et al., 2015; Cho et al., 2017; Eubelen et al., 2018; Vallon et al., 2018). Other ligands function analogously in other regions of the CNS, including Norrin in the retina and cerebellum (Ye et al., 2009; Wang et al., 2012) and potentially Wnt3a in the dorsal neural tube (Daneman et al., 2009). Furthermore, Wnt/β-catenin signaling is required for maintenance of CNS EC barrier properties in adulthood (Tran et al., 2016), with astrocytes as a major source of Wnt7 ligands (He et al., 2018; Vanlandewijck et al., 2018; Guérit et al., 2021).

Molecular hallmarks of Wnt-mediated CNS EC barriergenesis are (i) acquisition of glucose transporter GLUT-1 expression, (ii) loss of plasmalemma vesicle-associated protein (PLVAP), and (iii) upregulation of claudin-5 (Daneman et al., 2009; Kuhnert et al., 2010; Cho et al., 2017; Umans et al., 2017; Wang et al., 2019). Notably, the Wnt-mediated switch between the ‘leaky’ EC phenotype (GLUT-1 PLVAP+ claudin-5low) and the barrier EC phenotype (GLUT-1+ PLVAP claudin-5high) correlates with reduced permeability to molecular tracers (Wang et al., 2012; Cho et al., 2017) and is conserved in multiple contexts. For instance, medulloblastomas that produce Wnt-inhibitory factors have leaky vessels (Phoenix et al., 2016). Moreover, vasculature perfusing circumventricular organs is leaky due to low levels of Wnt signaling (Benz et al., 2019; Wang et al., 2019). Notably, ectopic activation of Wnt in ECs of circumventricular organs induces GLUT-1 and suppresses PLVAP (Benz et al., 2019; Wang et al., 2019). However, similar ectopic activation of Wnt in liver and lung ECs produces only very minor barriergenic effects (Munji et al., 2019), and Wnt activation in cultured primary mouse brain ECs does not prevent culture-induced loss of barrier-associated gene expression (Sabbagh and Nathans, 2020). The reasons for the apparent context-dependent impacts of Wnt activation in ECs remain unclear and motivate systematic examination of this process in a simplified model system. Further, given species differences in brain EC transporter expression (Uchida et al., 2011), drug permeability (Syvänen et al., 2009), and gene expression (Song et al., 2020), this process warrants investigation in human cells to complement mouse in vivo studies.

Prior studies have evaluated the impact of Wnt activation in immortalized human brain ECs and observed only modest effects on barrier phenotype (Paolinelli et al., 2013; Laksitorini et al., 2019). Combined with the aforementioned deficits observed in primary adult mouse brain ECs that are not rescued by ectopic Wnt activation (Sabbagh and Nathans, 2020), one possibility is that mature, adult endothelium is largely refractory to Wnt activation, and that Wnt responsiveness is a property of immature ECs analogous to those in the perineural vascular plexus. Human pluripotent stem cells (hPSCs) offer an in vitro human model system for systematic investigation of molecular mechanisms of BBB phenotype acquisition, especially given their ability to model early stages of endothelial specification and differentiation. However, currently available hPSC-based models of CNS endothelial-like cells are not well suited for modeling the BBB developmental progression as they do not follow a developmentally relevant differentiation trajectory, lack definitive endothelial identity, or have been incompletely characterized with respect to the role of developmental signaling pathways (Lippmann et al., 2020; Workman and Svendsen, 2020; Lu et al., 2021). As a potential alternative, hPSCs can also be used to generate immature, naïve endothelial progenitors (Lian et al., 2014) that could be used to better explore the induction of BBB phenotypes. For example, we recently reported that extended culture of such hPSC-derived endothelial progenitors in a minimal medium yielded ECs with improved BBB tight junction protein expression and localization, which led to improved paracellular barrier properties (Nishihara et al., 2020). However, as shown below, these cells exhibit high expression of PLVAP and little expression of GLUT-1, indicating the need for additional cues to drive CNS EC specification.

In this work, we aimed to define the effects of activating Wnt/β-catenin signaling in hPSC-derived, naïve endothelial progenitors and assess the extent to which this strategy would drive development of a CNS EC-like phenotype. We found that many aspects of the CNS EC phenotype, including the canonical GLUT-1, claudin-5, and PLVAP expression effects, were regulated by CHIR 99021, a small molecule agonist of Wnt/β-catenin signaling. CHIR treatment in matured ECs produced a more limited response. Whole-transcriptome analysis revealed definitive endothelial identity of the resulting cells and CHIR-upregulated expression of known CNS EC transcripts, including LEF1, APCDD1, AXIN2, SLC2A1, CLDN5, LSR, ABCG2, SOX7, and ZIC3. We also observed an unexpected CHIR-mediated upregulation of caveolin-1, which did not, however, correlate with increased uptake of a dextran tracer. Thus, we provide evidence that Wnt activation in hPSC-derived naïve endothelial progenitors is sufficient to induce many aspects of the CNS barrier EC phenotype, and we establish a model system for further systematic investigation of putative barriergenic cues.

Results

Wnt activation in hPSC-derived endothelial progenitors

We adapted an existing protocol to produce endothelial progenitor cells (EPCs) from hPSCs (Lian et al., 2014; Bao et al., 2016; Figure 1A). To achieve mesoderm specification, this method employs an initial activation of Wnt/β-catenin signaling with CHIR 99021 (CHIR), a small molecule inhibitor of glycogen synthase kinase-3 (GSK-3), which results in inhibition of GSK-3β-mediated β-catenin degradation. After 5 days of expansion, the resulting cultures contained a mixed population of CD34+CD31+ EPCs and CD34CD31 non-EPCs (Figure 1B and C). We used magnetic-activated cell sorting (MACS) to isolate CD31+ cells from this mixed culture and plated these cells on collagen IV-coated plates in a minimal EC medium termed hECSR (Nishihara et al., 2020). We first asked whether Wnt3a, a ligand widely used to activate canonical Wnt/β-catenin signaling (Kim et al., 2005; Kim et al., 2008; Liebner et al., 2008; Cecchelli et al., 2014; Praça et al., 2019), could induce GLUT-1 expression in the resulting ECs. After 6 days of treatment, we observed a significant increase in the fraction of GLUT-1+ ECs in Wnt3a-treated cultures compared to controls (Figure 1D and E). Consistent with previous observations (Nishihara et al., 2020), we also detected a population of calponin+ smooth muscle protein 22-⍺+ putative smooth muscle-like cells (SMLCs) outside the endothelial colonies (Figure 1—figure supplement 1), and these SMLCs expressed GLUT-1 in both control and Wnt3a-treated conditions (Figure 1D).

Figure 1 with 1 supplement see all
Human pluripotent stem cell (hPSC)-derived endothelial progenitors as a model for studying Wnt-mediated barriergenesis.

(A) Overview of the endothelial differentiation and Wnt treatment protocol. (B) Immunocytochemistry analysis of CD34 and CD31 expression in D5 endothelial progenitor cells (EPCs) prior to magnetic-activated cell sorting (MACS). Hoechst nuclear counterstain is overlaid in the merged image. Scale bars: 200 μm. (C) Flow cytometry analysis of CD34 and CD31 expression in D5 EPCs prior to MACS. (D) Immunocytochemistry analysis of β-catenin and GLUT-1 expression in Passage 1 ECs treated with Wnt3a or control. Hoechst nuclear counterstain is overlaid. Arrowheads indicate smooth muscle-like cells (SMLCs). Scale bars: 200 μm. (E) Quantification of the percentage of GLUT-1+ ECs in control- and Wnt3a-treated conditions. Points represent replicate wells from two independent differentiations of the IMR90-4 line, each differentiation indicated with a different color. Bars indicate mean values. p-value: two-way ANOVA.

Based on these promising results with Wnt3a, we next tested a low concentration (4 µM) of the GSK-3 inhibitor CHIR because of its ability to activate Wnt signaling in a receptor/co-receptor-independent manner. In addition to GLUT-1, we evaluated expression of two other key proteins: claudin-5, which is known to be upregulated in CNS ECs in response to Wnt (Benz et al., 2019), and caveolin-1, given the low rate of caveolin-mediated transcytosis in CNS compared to non-CNS ECs (Reese and Karnovsky, 1967; Andreone et al., 2017; Figure 2A). 4 µM CHIR robustly induced GLUT-1 expression in approximately 90% of ECs while increasing EC number and increasing EC purity to nearly 100% (Figure 2B). Furthermore, CHIR led to an approximately 1.5-fold increase in average claudin-5 abundance and a 10- to 30-fold increase in GLUT-1 abundance, but also a 2- to 4-fold increase in caveolin-1 (Figure 2B). We therefore titrated CHIR to determine an optimal concentration for EC expansion, purity, GLUT-1 induction, and claudin-5 upregulation while limiting the undesirable non-CNS-like increase in caveolin-1 abundance. Although 2 µM CHIR did not lead to increased caveolin-1 expression compared to vehicle control (DMSO), it also did not elevate claudin-5 or GLUT-1 expression compared to control and was less effective in increasing EC number and EC purity than 4 µM CHIR (Figure 2—figure supplement 1). On the other hand, 6 µM CHIR further increased GLUT-1 abundance but also further increased caveolin-1 abundance and did not improve EC number, EC purity, or claudin-5 expression (Figure 2—figure supplement 1). Therefore, we conducted further experiments using 4 µM CHIR. We confirmed that the CHIR-mediated increases in EC purity, EC number, and caveolin-1 and GLUT-1 expression were conserved in an additional hPSC line, although claudin-5 upregulation was not apparent (Figure 2—figure supplement 2). We also used two hPSC lines with doxycycline-inducible expression of short hairpin RNAs targeting CTNNB1 (β-catenin) to confirm that CHIR-mediated upregulation of GLUT-1 in ECs was β-catenin-dependent. Indeed, doxycycline treatment in combination with CHIR significantly reduced GLUT-1 abundance in ECs derived from these hPSC lines (Figure 2—figure supplement 3). Finally, we confirmed that increased EC number was the result of increased EC proliferation in CHIR-treated cultures (Figure 2—figure supplement 4). Together, these results suggest that activation of the Wnt/β-catenin pathway is capable of inducing CNS-like phenotypes in hPSC-derived endothelial progenitors.

Figure 2 with 4 supplements see all
Effect of CHIR on endothelial properties.

(A) Immunocytochemistry analysis of claudin-5, caveolin-1, and GLUT-1 expression in Passage 1 endothelial cells (ECs) treated with DMSO or 4 µM CHIR. Hoechst nuclear counterstain is overlaid in the merged images. Dashed lines indicate the border between an EC colony and smooth muscle-like cells (SMLCs) in the DMSO condition. Scale bars: 200 μm. (B) Quantification of images from the conditions described in (A) for number of ECs per 20× field, percentage of ECs (claudin-5+ cells relative to total nuclei), percentage of GLUT-1+ ECs (relative to total claudin-5+ ECs), and mean fluorescence intensity of claudin-5, caveolin-1, and GLUT-1 normalized to Hoechst mean fluorescence intensity within the area of claudin-5+ ECs only. Points represent replicate wells from 2 to 6 independent differentiations of the IMR90-4 line, each differentiation indicated with a different color. Bars indicate mean values. For the fluorescence intensity plots, values were normalized within each differentiation such that the mean of the DMSO condition equals 1. p-values: two-way ANOVA on unnormalized data.

Effects of CHIR-mediated Wnt activation in endothelial progenitors

Since CHIR elicited a robust Wnt-mediated response, we next asked whether other aspects of the CNS EC barrier phenotype were CHIR-regulated. PLVAP, a protein that forms bridges across both caveolae and fenestrae (Herrnberger et al., 2012), is one such canonically Wnt-downregulated protein. We therefore first evaluated PLVAP expression in Passage 1 control (DMSO) or CHIR-treated ECs using confocal microscopy (Figure 3A). We observed numerous PLVAP+ punctate vesicle-like structures in both conditions, with CHIR treatment reducing PLVAP abundance by approximately 20% (Figure 3A and B). This effect was not apparent in western blots of Passage 1 ECs, likely due to the relatively modest effect (Figure 4A and B). However, after two more passages (Figure 1A), Passage 3 ECs demonstrated a robust downregulation of PLVAP in CHIR-treated cells compared to controls (Figure 4C and D). We also used western blotting to confirm CHIR-mediated upregulation of GLUT-1 and claudin-5 both at Passage 1 and Passage 3 (Figure 4A–D). We next evaluated expression of the tricellular tight junction protein LSR (angulin-1) because of its enrichment in CNS versus non-CNS ECs, and the temporal similarity between LSR induction and the early stage of Wnt-mediated CNS barriergenesis (Sohet et al., 2015). We found that CHIR treatment led to a strong increase in LSR expression in both Passage 1 and Passage 3 ECs (Figure 4A–D), suggesting that Wnt signaling upregulates multiple necessary components of the CNS EC bicellular and tricellular junctions.

Effect of CHIR on endothelial PLVAP expression.

(A) Confocal immunocytochemistry analysis of VE-cadherin and PLVAP expression in Passage 1 endothelial cells (ECs) treated with DMSO or CHIR. Hoechst nuclear counterstain is overlaid. Eight serial confocal Z-slices with 1 μm spacing are shown. Scale bars: 50 μm. (B) Quantification of PLVAP and VE-cadherin area under the curve (AUC) of mean fluorescence intensity versus Z-position normalized to Hoechst AUC. Points represent replicate wells from three independent differentiations of the IMR90-4 line, each differentiation indicated with a different color. Bars indicate mean values, with values normalized within each differentiation such that the mean of the DMSO condition equals 1. p-values: two-way ANOVA on unnormalized data.

Effect of CHIR on protein expression in Passage 1 and Passage 3 endothelial cells (ECs).

(A) Western blots of Passage 1 ECs treated with DMSO or CHIR probed for GLUT-1, claudin-5, PLVAP, VE-cadherin, LSR, and β-actin. (B) Quantification of western blots of Passage 1 ECs. GLUT-1, claudin-5, PLVAP, VE-cadherin, and LSR band intensities was normalized to β-actin band intensity. Points represent replicate wells from 2 to 3 independent differentiations of the IMR90-4 line, each differentiation indicated with a different color. Bars indicate mean values, with values normalized within each differentiation such that the mean of the DMSO condition equals 1. p-values: two-way ANOVA on unnormalized data. (C) Western blots of Passage 3 ECs treated with DMSO or CHIR probed for GLUT-1, claudin-5, PLVAP, VE-cadherin, LSR, and β-actin. (D) Quantification of western blots of Passage 3 ECs. GLUT-1, claudin-5, PLVAP, VE-cadherin, and LSR band intensities was normalized to β-actin band intensity. Points represent replicate wells from three independent differentiations of the IMR90-4 line, each differentiation indicated with a different color. Bars indicate mean values, with values normalized within each differentiation such that the mean of the DMSO condition equals 1. p-values: two-way ANOVA on unnormalized data.

CHIR treatment produced two apparently competing changes in ECs related to vesicular transport: an expected downregulation of PLVAP and an unexpected upregulation of caveolin-1. We therefore asked whether the rate of total fluid-phase endocytosis differed between CHIR-treated and control ECs using a fluorescently labeled 10 kDa dextran as a tracer. After incubating Passage 1 cultures with dextran for 2 hr at 37°C, we used flow cytometry to gate CD31+ ECs and assess total dextran accumulation (Figure 5A and B). In ECs incubated at 37°C, CHIR treatment did not change the geometric mean dextran signal compared to DMSO (Figure 5B and C), but did cause a broadening of the distribution of dextran intensities as quantified by the coefficient of variation (CV), indicative of subpopulations of cells with decreased and increased dextran uptake (Figure 5B and D). We confirmed that the dextran signal measured by this assay was endocytosis-dependent by carrying out the assay at 4°C and with inhibitors of specific endocytic pathways (Figure 5—figure supplement 1A–C). Compared to vehicle control, chlorpromazine (inhibitor of clathrin-mediated endocytosis) and rottlerin (inhibitor of macropinocytosis) both decreased dextran uptake, while nystatin (inhibitor of caveolin-mediated endocytosis) did not significantly affect uptake (Figure 5—figure supplement 1B and C), consistent with the very small number of dextran+ caveolin-1+ puncta observed by confocal imaging (Figure 5—figure supplement 1D). Thus, despite the generally uniform elevation of caveolin-1 and decrease of PLVAP observed by immunocytochemistry in CHIR-treated ECs, our functional assay suggests neither an overall increase nor decrease in total fluid-phase endocytosis. Instead, it indicates that CHIR increases the heterogeneity of the EC population with respect to the rate of endocytosis.

Figure 5 with 1 supplement see all
Functional properties of CHIR- and DMSO-treated endothelial cells (ECs).

(A) Flow cytometry analysis of CD31 expression in Passage 1 ECs following the dextran internalization assay. CD31+ cells were gated for further analysis. (B) Flow cytometry analysis of 10 kDa dextran-Alexa Fluor 488 (AF488) abundance in CD31+ cells. Cells were treated with DMSO or CHIR for 6 days prior to the assay. Representative plots from cells incubated with dextran for 2 hr at 37°C are shown. (C) Quantification of 10 kDa dextran-AF488 geometric mean fluorescence intensity in CD31+ cells. Treatment and assay conditions were as described in (B). Points represent replicate wells from three independent differentiations of the IMR90-4 line, each differentiation indicated with a different color. Bars indicate mean values. p-value: two-way ANOVA. (D) Quantification of the coefficient of variation (CV) of 10 kDa dextran-AF488 fluorescence intensity in CD31+ cells. Points represent replicate wells from three independent differentiations of the IMR90-4 line, each differentiation indicated with a different color. Bars indicate mean values. p-value: two-way ANOVA. (E) Transendothelial electrical resistance (TEER) of Passage 3 ECs. The x-axis indicates the number of days after seeding cells on Transwell inserts. Points represent replicate wells from three independent differentiations of the IMR90-4 line, each differentiation indicated with a different shape. p-value: two-way ANOVA. (F) Permeability of Passage 3 ECs to sodium fluorescein. Points represent replicate wells from two independent differentiations of the IMR90-4 line, each differentiation indicated with a different color. Bars indicate mean values. p-value: two-way ANOVA.

We also compared the paracellular barrier properties of DMSO- and CHIR-treated ECs. Because Passage 1 cultures contain SMLCs that preclude formation of a confluent endothelial monolayer, we evaluated paracellular barrier properties of Passage 3 ECs that had undergone selective dissociation and replating (see Materials and methods), a strategy that effectively purifies the cultures (Nishihara et al., 2020). CHIR-treated Passage 3 ECs had elevated transendothelial electrical resistance (TEER) (Figure 5E) and decreased permeability to the small molecule tracer sodium fluorescein (Figure 5F). Together, these results are consistent with CHIR-mediated increases to tight junction protein expression (e.g., claudin-5 and LSR) and suggest that Wnt activation leads to functional improvements to paracellular barrier in this system.

Given the relatively weak responses to Wnt activation in adult mouse liver ECs in vivo (Munji et al., 2019) and adult mouse brain ECs cultured in vitro (Sabbagh and Nathans, 2020), we sought to determine whether the immature, potentially more plastic state of hPSC-derived endothelial progenitors contributed to the relatively robust CHIR-mediated response we observed. To test this hypothesis, we matured hPSC-derived ECs in vitro for four passages (until approximately day 30) prior to initiating CHIR treatment for 6 days and compared the resulting cells to differentiation-matched samples treated with CHIR immediately after MACS (Figure 6A). Both Passage 1 DMSO-treated ECs and Passage 5 DMSO-treated ECs, which are analogous to EECM-BMEC-like cells we previously reported (Nishihara et al., 2020), did not have detectable GLUT-1 expression (Figure 6B). Compared to DMSO controls, the CHIR-treated Passage 5 ECs exhibited no increase in GLUT-1 abundance (Figure 6B–D), which contrasts with the marked increase observed when CHIR treatment was initiated immediately after MACS (Figure 6B–D). Furthermore, CHIR treatment in matured ECs did not increase claudin-5 expression and did not increase EC number (Figure 6B–D), in contrast to the increases observed in both properties when treatment was initiated immediately after MACS (Figure 6B–D). We observed a similar lack of robust GLUT-1 induction in an additional differentiation and an additional hPSC line in which CHIR treatment was carried out at Passage 4 (Figure 6—figure supplement 1). Together, these data suggest that early, naïve endothelial progenitors are more responsive to Wnt activation than more mature ECs derived by the same differentiation protocol.

Figure 6 with 1 supplement see all
Effect of CHIR treatment in endothelial progenitor cells (EPCs) and matured endothelium.

(A) Overview of the endothelial differentiation, extended culture, and CHIR treatment protocols. (B) Immunocytochemistry analysis of claudin-5 and GLUT-1 expression in endothelial cells (ECs) treated with DMSO or CHIR as outlined in (A). Images from the IMR90-4 line are shown. Hoechst nuclear counterstain is overlaid. Scale bars: 100 μm. (C) Quantification of images from the conditions described in (B) for number of ECs per 30× field. Points represent replicate wells from one differentiation of the IMR90-4 line. Bars indicate mean values. p-values: ANOVA followed by Tukey’s honest significant difference (HSD) test. (D) Quantification of images from the conditions described in (B) for GLUT-1 and claudin-5 mean fluorescence intensity normalized to Hoechst mean fluorescence intensity within the area of claudin-5+ ECs only. Points represent replicate wells from one differentiation of the IMR90-4 line. Bars indicate mean values, with values normalized such that the mean of the DMSO condition equals 1. p-values: ANOVA followed by Tukey’s HSD test.

Comprehensive profiling of the Wnt-regulated endothelial transcriptome

We turned next to RNA-sequencing as an unbiased method to assess the impacts of Wnt activation on the EC transcriptome. We performed four independent differentiations and analyzed Passage 1 ECs treated with DMSO or CHIR using fluorescence-activated cell sorting (FACS) to isolate CD31+ ECs from the mixed EC/SMLC cultures. We also sequenced the SMLCs from DMSO-treated cultures at Passage 1 from two of these differentiations. DMSO- and CHIR-treated ECs at Passage 3 from three of these differentiations were also sequenced. Principal component analysis of the resulting whole-transcriptome profiles revealed that the two cell types (ECs and SMLCs) segregated along principal component (PC) 1, which explained 57% of the variance. In ECs, the effects of passage number and treatment were reflected in PC 2, which explained 21% of the variance (Figure 7A). We next validated the endothelial identity of our cells; we observed that canonical endothelial marker genes (including CDH5, CD34, PECAM1, CLDN5, ERG, and FLI1) were enriched in ECs compared to SMLCs and had high absolute abundance, on the order of 100–1000 transcripts per million (TPM) (Figure 7B, Supplementary file 1). SMLCs expressed mesenchymal (mural/fibroblast)-related transcripts (including PDGFRB, CSPG4, PDGFRA, TBX2, CNN1, and COL1A1), which ECs generally lacked, although we did observe slight enrichment of some of these genes in Passage 1 DMSO-treated ECs, likely reflective of a small amount of SMLC contamination despite CD31 FACS (Figure 7B). SMLCs also expressed SLC2A1 (Supplementary file 1) consistent with protein-level observations (Figure 1D). We also observed little to no expression of the epithelial genes CDH1, EPCAM, CLDN1, CLDN3 (Castro Dias et al., 2019), CLDN4, and CLDN6, reflecting the definitive endothelial nature of the cells (Figure 7B, Supplementary file 1).

Figure 7 with 6 supplements see all
RNA-seq of DMSO- and CHIR-treated endothelial cells (ECs).

(A) Principal component (PC) analysis of EC and smooth muscle-like cell (SMLC) whole-transcriptome data subject to variance stabilizing transformation by DESeq2. Points from Passage 1 ECs represent cells from four independent differentiations of the IMR90-4 line, points from Passage 3 ECs represent cells from three independent differentiations of the IMR90-4 line, and points from SMLCs represent two independent differentiations of the IMR90-4 line. Points are colored based on treatment: DMSO (black), CHIR (red). Data are plotted in the space of the first two PCs, with the percentage of variance explained by PC1 and PC2 shown in axis labels. Dashed lines indicate points from Passage 1 and Passage 3 ECs, and are not confidence ellipses. (B) Heatmap of transcript abundance [log2(TPM + 1)] for endothelial, mesenchymal, and epithelial genes across all samples. Abundance data for all transcripts are provided in Supplementary file 1. (C) Differential expression analysis of Passage 1 CHIR-treated ECs compared to Passage 1 DMSO-treated ECs. Differentially expressed genes (adjusted p-values<0.05, DESeq2 Wald test with Benjamini–Hochberg correction) are highlighted in green (upregulated) and red (downregulated). The number of upregulated, downregulated, and nonsignificant (ns) genes is shown in the legend. Complete results of differential expression analysis are provided in Supplementary file 2. (D) Transcript abundance (TPM) of Wnt-regulated, barrier-related genes in Passage 1 DMSO- and CHIR-treated ECs. Points represent cells from four independent differentiations of the IMR90-4 line and lines connect points from matched differentiations. All genes shown were differentially expressed (adjusted p-values<0.05, DESeq2 Wald test with Benjamini–Hochberg correction). p-values are provided in Supplementary file 2. (E) Venn diagram illustrating the number of genes identified as upregulated or downregulated (adjusted p-values<0.05, DESeq2 Wald test with Benjamini–Hochberg correction) in ECs treated with CHIR versus DMSO at Passage 1 compared to Passage 3. Gene lists are provided in Supplementary file 2, and selected genes are shown in Figure 7—figure supplement 1 and Figure 7—figure supplement 2.

First comparing CHIR- and DMSO-treated ECs at Passage 1, we identified 1369 significantly upregulated genes and 2037 significantly downregulated genes (Figure 7C, Supplementary file 2). CHIR-upregulated genes included SLC2A1, CLDN5, LSR, and CAV1, consistent with protein-level assays. PLVAP was downregulated, as were a number of mesenchymal genes (TAGLN, COL1A1), again reflective of slight contamination of SMLC transcripts in the DMSO-treated EC samples (Figure 7C and D). Additionally, important downstream effectors of Wnt signaling were upregulated, including the transcription factors LEF1 and TCF7, the negative regulator AXIN2, and the negative regulator APCDD1, which is known to modulate Wnt-regulated barriergenesis in retinal endothelium (Mazzoni et al., 2017; Figure 7C and D). We also identified upregulated transcription factors: ZIC3, which is highly enriched in brain and retinal ECs in vivo and downstream of Frizzled4 signaling (Wang et al., 2012; Sabbagh et al., 2018), and SOX7, which acts cooperatively with SOX17 and SOX18 in retinal angiogenesis (Zhou et al., 2015), were upregulated by CHIR in our system (Figure 7D). MSX1 and EBF1, which are expressed by murine brain ECs in vivo (Vanlandewijck et al., 2018), were also CHIR-upregulated (Figure 7D). Additional CHIR-upregulated genes included ABCG2 (encoding the efflux transporter breast cancer resistance protein [BCRP]), APLN, a tip cell marker enriched in postnatal day 7 murine brain ECs compared to those of other organs, and subsequently downregulated in adulthood (Sabbagh et al., 2018; Sabbagh and Nathans, 2020), and FLVCR2, a disease-associated gene with a recently identified role in brain angiogenesis (Santander et al., 2020; Figure 7C and D). Finally, we detected CHIR-mediated downregulation of the fatty acid-binding protein-encoding FABP4, which is depleted in brain ECs compared to those of peripheral organs (Sabbagh et al., 2018). We also observed similar downregulation of SMAD6, which is depleted in brain ECs compared to lung ECs and is a putative negative regulator of BMP-mediated angiogenesis (Mouillesseaux et al., 2016; Vanlandewijck et al., 2018; Figure 7D).

In Passage 3 ECs, many of the CHIR-mediated gene expression changes observed at Passage 1 persisted, including SLC2A1, LSR, LEF1, AXIN2, APCDD1, ZIC3, EBF1, FLVCR2, and ABCG2 upregulation and PLVAP downregulation (Figure 7E, Figure 7—figure supplement 1). Additional concordantly CHIR-upregulated genes encoding secreted factors, transcription factors, and transmembrane proteins are shown in Figure 7—figure supplement 2 and include REEP1, a gene enriched in brain versus non-brain ECs (Sabbagh et al., 2018; Vanlandewijck et al., 2018) that encodes a regulator of endoplasmic reticulum function and the Notch ligand-encoding gene JAG2. On the other hand, at Passage 3, CLDN5 was not upregulated in CHIR-treated cells compared to DMSO-treated cells, but was highly expressed (~2500 TPM). Similarly, CAV1 abundance remained high, but was not CHIR-upregulated in Passage 3 cells (Figure 7—figure supplement 1). Conversely, JAM2, which encodes junctional adhesion molecule 2, a component of EC tight junctions (Aurrand-Lions et al., 2001; Tietz and Engelhardt, 2015), was upregulated by CHIR at Passage 3, but not at Passage 1, as was the retinol-binding protein-encoding gene RBP1 (Figure 7—figure supplement 1).

We used weighted gene correlation network analysis (WGCNA) (Zhang and Horvath, 2005; Langfelder and Horvath, 2008) to identify modules containing genes with highly correlated expression across the 14 EC samples (Figure 7—figure supplement 3A, Supplementary file 3). One such module (the green module, containing 441 genes) had a representative gene expression profile (module eigengene) with a strong, positive correlation with CHIR treatment (Figure 7—figure supplement 3B). Importantly, genes central to this module included canonical transcriptional targets of Wnt/β-catenin signaling, including AXIN2 and APCDD1, further supporting the key role of β-catenin signaling in transcriptional changes observed in CHIR-treated ECs. Additional central (highly correlated) genes within the green module included SLC2A1, ZIC3, and FLVCR2, consistent with pairwise differential expression analysis, transcription factors (CASZ1, PRRX1), and genes with putative roles in vesicle trafficking (SNX4, ARL8B, AP1AR, VTI1A, VPS41) and lipid metabolism (AGPAT5, ASAH1) (Figure 7—figure supplement 3C).

To determine the effects of extended culture, we next compared control (DMSO-treated) ECs at Passage 3 versus Passage 1 (Figure 7—figure supplement 4, Supplementary file 2). Extended culture to Passage 3 in the absence of exogeneous Wnt activation led to 1521 upregulated genes, including CLDN5 and CAV1, consistent with previously reported protein-level observations in EECM-BMEC-like cells (Nishihara et al., 2020), which are analogous to Passage 3 DMSO-treated cells. We also observed 1625 downregulated genes, including marked downregulation of PLVAP (Figure 7—figure supplement 4). SLC2A1, however, was not upregulated at Passage 3 (Figure 7—figure supplement 4), concordant with absence of GLUT-1 protein expression in the control ECs (Figure 6B), nor was LSR. Further, despite some similarly regulated genes between the passage number and CHIR treatment comparisons (e.g., CLDN5, CAV1, PLVAP), the transcriptional responses to these two experimental variables were globally distinct as assessed by gene correlation network analysis (Figure 7—figure supplement 3B). We also evaluated transcript-level expression of components of the Wnt signaling pathway in Passage 3 control (DMSO-treated) ECs as a first step towards understanding the relative lack of responsiveness observed when CHIR treatment was initiated in matured (Passage 4) ECs (Figure 7—figure supplement 4). While CTNNB1, GSK3B, and genes encoding components of the destruction complex were not significantly different between Passage 3 and Passage 1, LEF1 and TCF7 were strongly downregulated in Passage 3 cells (Figure 7—figure supplement 4).

Finally, to further understand the strengths and limitations of this model system both as a readout of early developmental changes in CNS ECs (Passage 1 cells) or as a source of CNS-like ECs for use in downstream modeling applications, we evaluated absolute transcript abundance and effects of treatment or passage number on 53 characteristic CNS EC genes encompassing tight junction components, vesicle trafficking machinery, solute carriers, and ATP-binding cassette (ABC) efflux transporters selected based on high expression in human brain ECs from a meta-analysis of single-cell RNA-seq data (Gastfriend et al., 2021; Figure 7—figure supplement 5). While ECs expressed CLDN5, TJP1, TJP2, OLCN, and LSR, they lacked MARVELD2 (encoding tricellulin) under all conditions. ECs under all conditions also lacked MFSD2A and, despite CHIR-mediated downregulation of PLVAP, retained high absolute expression of this and other caveolae-associated genes. Finally, while many solute carriers and ABC transporters were expressed (SLC2A1, SLC3A2, SLC16A1, SLC38A2, ABCG2), others expressed at the in vivo human BBB were not (SLC5A3, SLC7A11, SLC38A3, SLCO1A2, ABCB1) (Figure 7—figure supplement 5). Thus, while CHIR treatment yields ECs with certain elements of CNS-like character, additional molecular signals are likely necessary to impart other aspects of the in vivo CNS EC phenotype.

The Wnt-regulated endothelial transcriptome in multiple contexts

To globally assess whether CHIR-mediated gene expression changes in our system are characteristic of the responses observed in ECs in vivo and similar to those observed in other in vitro contexts, we compared our RNA-seq dataset to those of studies that employed a genetic strategy for β-catenin stabilization (the Ctnnb1flex3 allele) in adult mouse ECs in several contexts: (i) pituitary ECs, which acquire some BBB-like properties upon β-catenin stabilization Wang et al., 2019; (ii) liver ECs, which exhibit little to no barriergenic response to β-catenin stabilization (Munji et al., 2019); (iii) brain ECs briefly cultured in vitro, which rapidly lose their BBB-specific gene expression profile even with β-catenin stabilization (Sabbagh and Nathans, 2020), and offer the most direct comparison to our in vitro model system. Upon recombination, the Ctnnb1flex3 allele produces a dominant mutant β-catenin lacking residues that are phosphorylated by GSK-3β to target β-catenin for degradation (Harada et al., 1999); as such, this strategy for ligand- and receptor-independent Wnt activation by β-catenin stabilization is similar to CHIR treatment, although GSK-3 phosphorylates targets other than β-catenin (discussed below).

We first used literature RNA-seq data from postnatal day 7 murine brain, liver, lung, and kidney ECs (Sabbagh et al., 2018) to define core sets of genes in brain ECs that are differentially expressed compared to all three of the other organs (Figure 8A and B). Using the resulting sets of 1094 brain-enriched and 506 brain-depleted genes, we asked how many genes in our Passage 1 ECs were concordantly regulated by CHIR: 130 of the brain-enriched genes were CHIR-upregulated and 116 of the brain-depleted genes were CHIR-downregulated (Figure 8C). At Passage 3, 61 genes were concordantly upregulated and 46 downregulated (Figure 8—figure supplement 1). In pituitary ECs with β-catenin stabilization, 102 of the brain-enriched genes were upregulated and 48 of the brain-depleted genes were downregulated (Figure 8D). Compared with the pituitary ECs, there were far fewer concordantly regulated genes in liver ECs with β-catenin stabilization, with 25 upregulated and 1 downregulated (Figure 8E). Finally, cultured primary mouse brain ECs with β-catenin stabilization exhibited 72 concordantly upregulated and 16 downregulated genes (Figure 8F). The only gene concordantly regulated in all four comparisons was the canonical Wnt target AXIN2. Several additional genes were concordantly upregulated in three of four, including TCF7, FAM107A, NKD1, TNFRSF19, GLUL, SLC30A1, and ABCB1, which was the only gene concordantly regulated in all comparisons except the hPSC-derived ECs (Figure 8G). Several canonical target genes were shared by the hPSC-derived EC and pituitary EC systems, including APCDD1, LEF1, CLDN5, and SLC2A1; also in this category were LSR, the zinc/manganese transporter SLC39A8, and 12 additional genes (Figure 8G). Notably, the caveolae inhibitor MFSD2A was robustly upregulated by β-catenin in pituitary ECs, but not in any other context (Figure 8C–F), suggesting that other brain-derived factors may cooperate with Wnt to regulate expression of this important inhibitor of caveolin-mediated transcytosis. Complete gene lists from this comparative analysis are provided in Supplementary file 4. In sum, the data suggest that the hPSC-derived ECs responded to Wnt activation in a fashion that led to modest induction of CNS transcriptional programs and that the response was most similar to the pituitary β-catenin stabilization model. Importantly, this analysis also supports the hypothesis that immature endothelium is highly responsive to Wnt activation where mature (adult) endothelium is largely refractory except in regions proximal to barrier-forming regions.

Figure 8 with 2 supplements see all
Identification of concordantly Wnt-regulated central nervous system (CNS) endothelial cell (EC)-associated genes in RNA-seq data.

(A) Differential expression analysis of P7 murine brain ECs compared to liver, lung, or kidney ECs (Sabbagh et al., 2018). Differentially expressed genes (adjusted p-values<0.05, DESeq2 Wald test with Benjamini–Hochberg correction) are highlighted in green (up, brain-enriched) and red (down, brain-depleted). The number of up, down, and nonsignificant (ns) genes is shown in the legends. (B) Venn diagrams illustrating the number of genes identified as brain EC-enriched (left) or brain EC-depleted (right) versus liver, lung, or kidney ECs (adjusted p-values<0.05, DESeq2 Wald test with Benjamini–Hochberg correction). The 1094 genes enriched in brain ECs compared to each other organ, and the 506 genes depleted in brain ECs compared to each other organ, were used for subsequent analysis of the effects of Wnt activation in the various experimental contexts. (C–F) In each plot, the x-axis indicates average log2(fold change) of gene expression in brain ECs compared to liver, lung, and kidney ECs for the 1094 brain EC-enriched genes and 506 brain EC-depleted genes described in (B) with known mouse-human homology. Homologous human gene names are shown. The y-axes indicate differential expression [log2(fold change)] in Passage 1 CHIR-treated ECs compared to Passage 1 DMSO-treated ECs (C), in adult mouse pituitary ECs with stabilized β-catenin (gain-of-function, GOF) compared to controls (Wang et al., 2019) (D), in adult mouse liver ECs with stabilized β-catenin compared to controls (Munji et al., 2019) (E), or in cultured adult mouse brain ECs with stabilized β-catenin compared to controls (Sabbagh and Nathans, 2020) (F). Points are highlighted in blue if concordantly regulated (upregulated in both comparisons or downregulated in both comparisons). The number of concordantly upregulated and concordantly downregulated genes is shown. Genes were identified as upregulated or downregulated based on adjusted p-values<0.05, DESeq2 Wald test with Benjamini–Hochberg correction. (G) Venn diagrams illustrating the number of brain EC-enriched genes concordantly upregulated with β-catenin GOF (top) and the number of brain EC-depleted genes concordantly downregulated with β-catenin GOF (bottom) for the four comparisons shown in (C–F). Complete results of this analysis are provided in Supplementary file 4.

Last, because GSK-3 is a component of numerous signaling pathways in addition to Wnt/β-catenin (Eto et al., 2005; Beurel et al., 2015; Hermida et al., 2017), we used RNA-seq data to infer pathways that might be differentially regulated by the two strategies for activating Wnt/β-catenin signaling employed in the experiments above: CHIR treatment, which increases β-catenin stability by inhibiting GSK-3, or direct stabilization of β-catenin. We tested lists of upregulated genes in (i) our Passage 1 ECs treated with CHIR versus DMSO, (ii) Passage 3 ECs treated with CHIR versus DMSO, and (iii) pituitary ECs with β-catenin stabilization versus controls (Wang et al., 2019), against the Hallmark gene set collection (Liberzon et al., 2015; Figure 8—figure supplement 2, Supplementary file 5). In all three comparisons, the Wnt/β-catenin signaling gene set was significantly enriched (Figure 8—figure supplement 2A). Similarly, the Notch signaling, TNFα signaling via NF-κB, KRAS signaling up, and several additional gene sets were consistently enriched in all three comparisons (Figure 8—figure supplement 2A and B, Supplementary file 5), suggesting similar regulation by GSK-3 inhibition and direct β-catenin stabilization. In contrast, the PI3K AKT mTOR signaling gene set was enriched in Passage 1 ECs, but not in Passage 3 ECs or pituitary ECs. Similarly, the gene set mTORC1 signaling was enriched in Passage 1 ECs and pituitary ECs, but genes driving this enrichment were distinct (Figure 8—figure supplement 2C), and this gene set was not enriched in Passage 3 ECs. Thus, given the known, bidirectional interactions of GSK-3 and AKT/mTOR pathway components (Hermida et al., 2017), these results suggest that CHIR-mediated inhibition of GSK-3 may transiently activate this pathway in Passage 1 ECs. Conversely, the gene set TGF-β signaling was enriched only in pituitary ECs with β-catenin stabilization (Figure 8—figure supplement 2, Supplementary file 5). Taken together, these results, coupled with those of our CTNNB1 knockdown experiments and gene correlation network analysis, suggest a central role for β-catenin as a key effector of CHIR-mediated signaling, but also highlight some potential differences in the pathways activated in response to CHIR treatment versus β-catenin stabilization. Differences in other aspects of these two experimental paradigms (in vitro versus in vivo, naïve versus CNS-proximal, human versus mouse), however, caution against overinterpretation of these results.

Discussion

The Wnt/β-catenin signaling pathway plays a central role in CNS angiogenesis and in establishing the unique properties of CNS ECs (Liebner et al., 2008; Stenman et al., 2008; Daneman et al., 2009; Kuhnert et al., 2010; Cullen et al., 2011; Vanhollebeke et al., 2015; Cho et al., 2017). In this work, we investigated the role of Wnt/β-catenin signaling in induction of BBB properties in a human EC model using naïve endothelial progenitors derived from hPSCs. We reasoned that these immature EPCs (Lian et al., 2014) would be similar to the immature endothelium in the perineural vascular plexus and thus competent to acquire CNS EC phenotypes in response to Wnt activation. To activate Wnt signaling, we evaluated the widely used ligand Wnt3a (Liebner et al., 2008) and the GSK-3 inhibitor CHIR.

We found that CHIR treatment robustly induced several canonical CNS EC molecular phenotypes, including a marked induction of GLUT-1, upregulation of claudin-5, and downregulation of PLVAP, which correlated with differential gene expression in RNA-seq data. We also observed a functional decrease in paracellular permeability. Further, using RNA-seq and western blotting, we identified LSR (angulin-1) as CHIR-induced in this system, supporting the notion that this highly CNS EC-enriched tricellular tight junction protein (Daneman et al., 2010a; Sohet et al., 2015) is Wnt-regulated. In RNA-seq data, we observed differential expression of known CNS EC-enriched/depleted and Wnt-regulated genes including upregulated LEF1, AXIN2, APCDD1, ABCG2, SOX7, ZIC3, FLVCR2, JAM2, and RBP1, and downregulated PLVAP, FABP4, SMAD6, and SLIT2. These RNA-seq data should therefore be useful in generating hypotheses of BBB-associated genes regulated by Wnt activation in ECs for future functional studies. Our work also defines an important set of phenotypes for which Wnt activation in ECs is not sufficient in our system: in the context of vesicle trafficking, we observed caveolin-1 (CAV1) upregulation, no change in mean functional endocytosis, virtually no expression of MFSD2A, and high absolute PLVAP abundance in RNA-seq data despite CHIR-mediated downregulation. Given roles of brain pericytes in regulating PLVAP, MFSD2A, and functional transcytosis (Armulik et al., 2010; Daneman et al., 2010b; Ben-Zvi et al., 2014; Stebbins et al., 2019), and the observation that MFSD2A is Wnt-regulated in pituitary ECs in vivo (Wang et al., 2019), where pericytes are present, it is plausible that pericyte-derived cues are necessary in addition to Wnts to achieve the characteristically low rate of CNS EC pinocytosis. Next, while ABCG2 (BCRP) was Wnt-induced in our system, other hallmark efflux transporters were not Wnt-regulated and either expressed at low levels (e.g., ABCC4, encoding MRP-4) or not expressed (e.g., ABCB1, encoding P-glycoprotein). Notably, however, Abcb1a was Wnt-regulated in the three other β-catenin stabilization experiments from the literature that we evaluated (Munji et al., 2019; Wang et al., 2019; Sabbagh and Nathans, 2020). Thus, pericyte-derived cues, astrocyte-derived cues, and/or activation of the pregnane X or other nuclear receptors may be important for complete acquisition of the complement of CNS EC efflux transporters (Bauer et al., 2004; Berezowski et al., 2004; Praça et al., 2019).

CHIR is widely used to activate Wnt/β-catenin signaling in cell culture (Lian et al., 2012; Lian et al., 2014; Patsch et al., 2015; Sakaguchi et al., 2015; Gomez et al., 2019; Pellegrini et al., 2020; Guo et al., 2021). It remains unknown, however, to what extent CHIR-mediated inhibition of GSK-3 in ECs mimics the effects of Wnt ligand-induced inhibition of GSK-3 or direct stabilization of β-catenin. In our system, although the GLUT-1-inductive effect of CHIR was partially inhibited by β-catenin knockdown and our RNA-seq data revealed a transcriptional response characteristic of canonical Wnt signaling, it is possible that CHIR affects other signaling pathways, as suggested by pathway enrichment analysis. Thus, employing ligand-based strategies to activate Wnt signaling will be an important next step. Our RNA-seq data suggest that the receptors and coreceptors necessary to transduce Wnt7 and Norrin signaling (e.g., FZD4, LRP6, RECK, ADGRA2 [GPR124], TSPAN12, DVL2) are expressed by hPSC-derived ECs (Figure 7—figure supplement 6). Given evidence that Wnt ligands have poor solubility (Janda et al., 2012) and our preliminary data suggesting that supplementation of culture medium with Wnt7a and Wnt7b is largely ineffective in activating Wnt/β-catenin signaling in this system, special emphasis should be placed on strategies that present Wnt7a, Wnt7b, and/or Norrin in a manner that concentrates ligands at the cell surface, for example, by using direct cocultures of endogenously Wnt-producing cells (neural progenitors or astrocytes) or Wnt-overexpressing cells. Importantly, neural progenitor cells and astrocytes likely would also contribute other yet-unidentified ligands important for acquisition of CNS EC phenotype. Finally, it would also be informative to directly compare CHIR and/or Wnt ligand treatment to direct stabilization of β-catenin in this system, for example, by generating an hPSC line with inducible expression of a dominant active β-catenin.

We also directly addressed the hypothesis that immature ECs are more plastic, that is, more competent to acquire BBB properties upon Wnt activation than mature ECs. This hypothesis is supported by existing observations that ectopic expression of Wnt7a is sufficient to induce GLUT-1 expression in non-CNS regions of the mouse embryo (Stenman et al., 2008), but β-catenin stabilization in adult mouse liver and lung ECs produces only a slight effect (Munji et al., 2019). We repeated our CHIR treatment paradigm in hPSC-derived ECs after an extended period of in vitro culture (Passage 4 ECs) and observed much weaker induction of GLUT-1 and no pro-proliferative effect. Thus, our results support this hypothesis and suggest that the loss of BBB developmental plasticity in ECs is an intrinsic, temporally controlled process rather than a result of the peripheral organ environment. The molecular mechanisms underlying this loss of plasticity remain poorly understood. While previous studies have demonstrated that the level of Wnt/β-catenin signaling in CNS ECs peaks early in development and subsequently declines (Corada et al., 2019; Hübner et al., 2018), this finding does not address mechanisms underlying the competence of ECs (CNS and non-CNS) to respond to Wnt signals. In RNA-seq data of Passage 3 control (DMSO-treated) ECs, LEF1 and TCF7 were strongly downregulated compared to Passage 1 cells. This result suggests that low baseline expression of these transcription factors, which form a complex with nuclear β-catenin to regulate Wnt target genes, may partially explain the poor efficacy of CHIR in matured ECs, although additional work is necessary to assess the functional relevance of these differences. Interestingly, ECs in non-BBB-forming regions of the CNS (i.e., circumventricular organs), and in the anterior pituitary, which is directly proximal to the CNS, retain some of their plasticity in adulthood (Wang et al., 2019), possibly as the result of a delicate balance between Wnt ligands and Wnt-inhibitory factors in these regions. Our model should facilitate additional systematic examination of factors that may enhance or attenuate EC Wnt responsiveness.

Finally, our work establishes an improved hPSC-based model for investigating mechanisms of BBB development in naïve ECs. hPSCs are an attractive model system to complement in vivo animal studies because they (i) are human, (ii) permit investigation of developmental processes in contrast to primary or immortalized cells, (iii) are highly scalable, (iv) can be derived from patients to facilitate disease modeling and autologous coculture systems, and (v) are genetically tractable. While widely used hPSC-based BBB models are useful for measuring molecular permeabilities and have been employed to understand genetic contributions to barrier dysfunction (Vatine et al., 2017; Vatine et al., 2019; Lim et al., 2017), they have not been shown to proceed through a definitive endothelial progenitor intermediate (Lippmann et al., 2012; Lu et al., 2021) and express epithelial-associated genes (Qian et al., 2017; Delsing et al., 2018; Vatine et al., 2019; Lu et al., 2021). Thus, new models with developmentally relevant differentiation trajectories and definitive endothelial phenotype are needed for improved understanding of developmental mechanisms. Motivated in part by prior use of ECs derived from hematopoietic progenitors in human cord blood to generate BBB models (Boyer-Di Ponio et al., 2014; Cecchelli et al., 2014), we and others recently showed that hPSC-derived naïve endothelial progenitors or ECs are good candidates for such a system (Praça et al., 2019; Nishihara et al., 2020; Roudnicky et al., 2020a; Roudnicky et al., 2020b). For example, Praça et al. showed that a combination of VEGF, Wnt3a, and retinoic acid directed EPCs to brain capillary-like ECs with moderate TEER similar in order of magnitude to that reported here. We previously showed that BBB-like paracellular barrier characteristics are induced in hPSC-EPC-derived ECs after extended culture in a minimal medium. These so-called EECM-BMEC-like cells had TEER and small molecule permeability similar to primary human brain ECs, well-developed tight junctions, and an immune cell adhesion molecule profile similar to brain ECs in vivo (Nishihara et al., 2020). In this study, we showed it was possible to use the small molecule Wnt agonist CHIR to induce additional hallmarks of CNS EC phenotype in hPSC-EPC-derived ECs, including canonical GLUT-1, claudin-5, and PLVAP effects (both Passage 1 and 3 CHIR-treated ECs). However, it is important to note that despite the improvements in CNS EC character with CHIR treatment, further improvements to functional endocytosis, and efflux transporter and solute carrier phenotype should be targets of future study and may be facilitated by cocultures and/or additional molecular factors. Along these lines, the Passage 1 CHIR-treated CNS-like ECs would be at a differentiation stage well suited to investigate cues subsequent to Wnt signaling that may be key for the induction of additional CNS EC properties. Alternatively, the Passage 3 CHIR-treated CNS-like ECs may be suitable for other BBB modeling applications. In summary, our work has defined the EC response to Wnt activation in a simplified, human system and established a new hPSC-derived in vitro model that will facilitate improved understanding of endothelial barriergenesis.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (human)iPSC: IMR90-4Available from WiCell; Yu et al., 2007RRID:CVCL_C437
Cell line (human)iPSC: WTC11Available from Gladstone Institutes; Kreitzer et al., 2013RRID:CVCL_Y803
Cell line (human)iPSC: 19-9-11-7TGP-ishcat3Laboratory stock
Cell line (human)hESC: H9-7TGP-ishcat2Laboratory stock
Lian et al., 2013
Cell line (human)hESC: H9-CDH5-eGFPLaboratory stock
Bao et al., 2017
AntibodyAnti-CD31-FITC (mouse monoclonal IgG1, clone AC128)Miltenyi BiotecCat# 130-117-390;RRID:AB_2733637
AntibodyAnti-CD31-APC (mouse monoclonal IgG1, clone AC128)Miltenyi BiotecCat# 130-119-891;RRID:AB_2784124
AntibodyAnti-CD34-FITC (mouse monoclonal IgG2a, clone AC136)Miltenyi BiotecCat# 130-113-178;RRID:AB_2726005
AntibodyAnti-β-catenin-Alexa Fluor 488 (mouse monoclonal IgG1, clone 14)BD BiosciencesCat# 562505;RRID:AB_11154224(1:100, ICC)
AntibodyAnti-GLUT-1 (mouse monoclonal IgG2a, clone SPM498)InvitrogenCat# MA5-11315;RRID:AB_10979643(1:100, ICC)(1:500, WB)
AntibodyAnti-calponin (mouse monoclonal IgG1, clone hCP)Sigma-AldrichCat# C2687;RRID:AB_476840(1:15,000, ICC)
AntibodyAnti-SM22⍺ (rabbit polyclonal)AbcamCat# ab14106;RRID:AB_443021(1:1000, ICC)
AntibodyAnti-claudin-5 (mouse monoclonal IgG1, clone 4C3C2)InvitrogenCat# 35-2500;RRID:AB_2533200(1:100, ICC)(1:500, WB)
AntibodyAnti-caveolin-1 (rabbit polyclonal)Cell Signaling TechnologyCat# 3238;RRID:AB_2072166(1:500, ICC)
AntibodyAnti-CD31 (rabbit polyclonal)Lab VisionCat# RB-10333-P;RRID:AB_720502(1:100, ICC)
AntibodyAnti-Ki67 (mouse monoclonal IgG1, clone B56)BD BiosciencesCat# 550609;RRID:AB_393778(1:100, ICC)
AntibodyAnti-VE-cadherin (mouse monoclonal IgG2a, clone BV9)Santa Cruz BiotechnologyCat# sc-52751;RRID:AB_628919(1:100, ICC)(1:250, WB)
AntibodyAnti-β-actin (rabbit monoclonal IgG, clone 13E5)Cell Signaling TechnologyCat# 4970;RRID:AB_2223172(1:1000, WB)
AntibodyAnti-PLVAP (rabbit polyclonal)Prestige AntibodiesCat# HPA002279;RRID:AB_1079636(1:200, ICC)(1:250, WB)
AntibodyAnti-LSR (rabbit polyclonal)Prestige AntibodiesCat# HPA007270;RRID:AB_1079253(1:250, WB)
AntibodyAlexa Fluor 488 goat anti-mouse IgG (goat polyclonal)InvitrogenCat# A-11001;RRID:AB_2534069(1:200, ICC)
AntibodyAlexa Fluor 647 goat anti-rabbit IgG (goat polyclonal)InvitrogenCat# A-21245;RRID:AB_2535813(1:200, ICC)
AntibodyAlexa Fluor 488 goat anti-mouse IgG1 (goat polyclonal)InvitrogenCat# A-21121;RRID:AB_2535764(1:200, ICC)
AntibodyAlexa Fluor 647 goat anti-mouse IgG2a (goat polyclonal)InvitrogenCat# A-21241;RRID:AB_2535810(1:200, ICC)
AntibodyAlexa Fluor 555 goat anti-rabbit IgG (goat polyclonal)InvitrogenCat# A-21428;RRID:AB_2535849(1:200, ICC)
AntibodyIRDye 800CW goat anti-mouse IgG (goat polyclonal)LI-COR BiosciencesCat# 926-32210;RRID:AB_621842(1:5000, WB)
AntibodyIRDye 800CW goat anti-rabbit IgG (goat polyclonal)LI-COR BiosciencesCat# 926-32211;RRID:AB_621843(1:5000, WB)
AntibodyIRDye 680RD goat anti-rabbit IgG (goat polyclonal)LI-COR BiosciencesCat# 926-68071;RRID:AB_10956166(1:5000, WB)
Commercial assay or kitRNeasy Plus Micro KitQiagenCat# 74034
Chemical compound or drugCHIR 99021TocrisCat# 4423
Chemical compound or drugVybrant DyeCycle Green StainInvitrogenCat# V35004
Chemical compound or drugDextran, Alexa Fluor 488; 10,000 MW, Anionic, FixableInvitrogenCat# D22910
Software or algorithmRSEMLi and Dewey, 2011RRID:SCR_013027v1.3.3
Software or algorithmBowtie2Langmead and Salzberg, 2012RRID:SCR_016368v2.4.2
Software or algorithmRR FoundationRRID:SCR_001905v3.6.3
Software or algorithmDESeq2Love et al., 2014RRID:SCR_015687v1.26.0
Software or algorithmbiomaRtDurinck et al., 2009RRID:SCR_019214v2.42.1
Software or algorithmWGCNALangfelder and Horvath, 2008RRID:SCR_003302v1.70-3
Software or algorithmCytoscapeShannon et al., 2003RRID:SCR_003032v3.8.2
Software or algorithmFIJI/ImageJSchindelin et al., 2012RRID:SCR_002285v2.0.0-rc-68
Software or algorithmImage StudioLI-COR BiosciencesRRID:SCR_015795v5.2
Software or algorithmFlowJoBD BiosciencesRRID:SCR_008520v10.7.1
Software or algorithmJMP ProSAS InstituteRRID:SCR_014242v15.0.0
Software or algorithmPrismGraphPad SoftwareRRID:SCR_002798v5.0.1

hPSC maintenance

Request a detailed protocol

Tissue culture plates were coated with Matrigel, Growth Factor Reduced (Corning, Glendale, AZ). A 2.5 mg aliquot of Matrigel was thawed and resuspended in 30 mL DMEM/F-12 (Life Technologies, Carlsbad, CA), and the resulting solution used to coat plates at 8.7 µg/cm2 (1 mL per well for 6-well plates; 0.5 mL per well for 12-well plates). Plates were incubated at 37°C for at least 1 hr prior to use. hPSCs were maintained on Matrigel-coated plates in E8 medium (STEMCELL Technologies, Vancouver, Canada) at 37°C, 5% CO2. hPSC lines used were IMR90-4 iPSC, WTC11 iPSC, H9-CDH5-eGFP hESC, H9-7TGP-ishcat2 hESC, and 19-9-11-7TGP-ishcat3 iPSC. Medium was changed daily. When hPSC colonies began to touch, typically at approximately 70–80% confluence, cells were passaged using Versene (Life Technologies). Briefly, cells were washed once with Versene, then incubated with Versene for 7 min at 37°C. Versene was removed and cells were dissociated into colonies by gentle spraying with E8 medium. Cells were transferred at a split ratio of 1:12 to a new Matrigel-coated plate containing E8 medium. hPSC cultures were routinely tested for mycoplasma contamination using a PCR-based assay performed by the WiCell Research Institute (Madison, WI).

Endothelial progenitor cell differentiation

Request a detailed protocol

EPCs were differentiated according to previously published protocols (Lian et al., 2014; Bao et al., 2016; Nishihara et al., 2020) with slight modifications. On day –3 (D-3), hPSCs were treated with Accutase (Innovative Cell Technologies, San Diego, CA) for 7 min at 37°C. The resulting single-cell suspension was transferred to 4× volume of DMEM/F-12 (Life Technologies) and centrifuged for 5 min, 200× g. Cell number was quantified using a hemocytometer. Cells were resuspended in E8 medium supplemented with 10 µM ROCK inhibitor Y-27632 dihydrochloride (Tocris, Bristol, UK) and seeded on Matrigel-coated 12-well plates at a density of (1.5–2.5) × 104 cells/cm2, 1 mL per well. Cells were maintained at 37°C, 5% CO2. On the following two days (D-2 and D-1), the medium was replaced with E8 medium. The following day (D0), differentiation was initiated by changing the medium to LaSR medium (Advanced DMEM/F-12 [Life Technologies], 2.5 mM GlutaMAX [Life Technologies], and 60 µg/mL l-ascorbic acid 2-phosphate magnesium [Sigma-Aldrich, St. Louis, MO]) supplemented with 7–8 µM CHIR 99021 (Tocris), 2 mL per well. The following day (D1), medium was replaced with LaSR medium supplemented with 7–8 µM CHIR 99021, 2 mL per well. On the following three days (D2, D3, and D4), the medium was replaced with pre-warmed LaSR medium (without CHIR), 2 mL per well.

On D5, EPCs were isolated using CD31 MACS. Cells were treated with Accutase for 15–20 min at 37°C. The resulting cell suspension was passed through a 40 µm cell strainer into an equal volume of DMEM (Life Technologies) supplemented with 10% FBS (Peak Serum, Wellington, CO) and centrifuged for 5 min, 200× g. Cell number was quantified using a hemocytometer. Cells were resuspended in MACS buffer (Dulbecco’s phosphate buffered saline without Ca and Mg [DPBS; Life Technologies] supplemented with 0.5% bovine serum albumin [Sigma-Aldrich] and 2 mM EDTA [Sigma-Aldrich]) at a concentration of 107 cells per 100 µL. The CD31-FITC antibody (Miltenyi Biotec, Auburn, CA) was added to the cell suspension at a dilution of 1:50. The cell suspension was incubated for 30 min at room temperature (RT), protected from light. The cell suspension was brought to a volume of 15 mL with MACS buffer and centrifuged for 5 min, 200× g. The supernatant was aspirated and the pellet resuspended in MACS buffer at a concentration of 107 cells per 100 µL. The FITC Selection Cocktail from the EasySep Human FITC Positive Selection Kit (STEMCELL Technologies) was added at a dilution of 1:10, and the cell suspension was incubated for 20 min at RT, protected from light. The Dextran RapidSpheres (magnetic particles) solution from the Selection Kit was added at a dilution of 1:20, and the cell suspension was incubated for an additional 15 min at RT.

The cell suspension was brought to a total volume of 2.5 mL with MACS buffer (for total cell number less than 2 × 108, the approximate maximum yield from two 12-well plates; for a larger number of plates/cells, a total volume of 5 mL was used). 2.5 mL of cell suspension was transferred to a sterile 5 mL round-bottom flow cytometry tube and placed in the EasySep magnet (STEMCELL Technologies) for 5 min. The magnet was inverted to pour off the supernatant, the flow tube removed, the retained cells resuspended in 2.5 mL of MACS buffer, and the flow tube placed back in the magnet for 5 min. This step was repeated three times, and the resulting cell suspension transferred to a centrifuge tube, and centrifuged for 5 min, 200× g. Cell number was quantified using a hemocytometer. Resulting EPCs were used directly for experiments as described below or cryopreserved in hECSR medium supplemented with 30% FBS and 10% DMSO for later use. hECSR medium is Human endothelial serum-free medium (Life Technologies) supplemented with 1× B-27 supplement (Life Technologies) and 20 ng/mL FGF2 (Waisman Biomanufacturing, Madison, WI).

Endothelial cell culture and treatment

Request a detailed protocol

Collagen IV (Sigma-Aldrich) was dissolved in 0.5 mg/mL acetic acid to a final concentration of 1 mg/mL. Collagen IV-coated plates were prepared by diluting a volume of this stock solution 1:100 in water, adding the resulting solution to tissue culture plates, or #1.5 glass-bottom plates (Cellvis, Sunnyvale, CA) for cells intended for confocal imaging (1 mL per well for 6-well plates, 0.5 mL per well for 12-well plates, 0.25 mL per well for 24-well plates), and incubating the plates for 1 hr at RT. Collagen IV coating solution was removed, and EPCs obtained as described above were suspended in hECSR medium and plated at approximately 3 × 104 cells/cm2. In some experiments, ligands and small molecules were added to hECSR medium: CHIR 99021 (Tocris) was used at 4 µM except where indicated; DMSO (Sigma-Aldrich) was used as a vehicle control for CHIR; Wnt3a (R&D Systems) was used at 20 ng/mL; doxycycline was used at 1, 2, or 4 µg/mL. The hECSR medium, including any ligands or small molecules, was replaced every other day until confluent (typically 6 days). We denote this time point as ‘Passage 1.’

For extended culture, ECs were selectively dissociated and replated as previously described (Nishihara et al., 2020). Cells were incubated with Accutase until ECs appeared round, typically 2–3 min at 37°C. The plate was tapped to release the ECs while SMLCs remained attached, and the EC-enriched cell suspension transferred to 4× volume of DMEM/F-12 and centrifuged for 5 min, 200× g. Cells were resuspended in hECSR medium and seeded on a new collagen IV-coated plate at approximately 3 × 104 cells/cm2. hECSR medium was replaced every other day until confluent (typically 6 days). The selective dissociation and seeding described above was repeated, and hECSR medium was again replaced every other day until confluent (typically 6 days). We denote this time point as ‘Passage 3.’ In one experiment, these steps were repeated for another two passages. Except where indicated, CHIR 99021 or vehicle (DMSO) was included in the hECSR medium for the entire duration of culture.

RNA-seq

Request a detailed protocol

RNA-seq was performed on ECs and SMLCs from the IMR90-4 hPSC line. Four independent differentiations were performed, with DMSO- and CHIR-treated ECs at Passage 1 analyzed from all four differentiations. DMSO- and CHIR-treated ECs at Passage 3 were analyzed from three of the four differentiations. DMSO-treated SMLCs at Passage 1 were analyzed from two of the four differentiations. FACS was used to isolate CD31+ ECs and CD31 SMLCs from mixed Passage 1 cultures. Cells were incubated with Accutase for 10 min at 37°C, passed through 40 µm cell strainers into 4× volume of DMEM/F-12, and centrifuged for 5 min, 200× g. Cells were resuspended in MACS buffer and incubated with CD31-APC antibody (Miltenyi Biotec) for 30 min at 4°C, protected from light. The cell suspension was brought to a volume of 15 mL with MACS buffer and centrifuged at 4°C for 5 min, 200× g. Cells were resuspended in MACS buffer containing 2 µg/mL 4′,6-diamidino-2-phenylindole (DAPI; Life Technologies). A BD FACSAria III Cell Sorter (BD Biosciences, San Jose, CA) was used to isolate DAPICD31+ cells (live ECs) and DAPICD31 cells (live SMLCs). The resulting cell suspensions were centrifuged at 4°C for 5 min, 200× g, and cell pellets immediately processed for RNA extraction as described below.

RNA was isolated using the RNeasy Plus Micro Kit (Qiagen, Germantown, MD). Buffer RLT Plus supplemented with 1% β-mercaptoethanol was used to lyse cells (pellets from FACS of Passage 1 cells, or directly on plates for Passage 3 ECs). Lysates were passed through gDNA Eliminator spin columns, loaded onto RNeasy MinElute spin columns, washed with provided buffers according to the manufacturer’s instructions, and eluted with RNase-free water. Sample concentrations were determined using a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA) and RNA quality assayed using an Agilent 2100 Bioanalyzer with Agilent RNA 6000 Pico Kit (Agilent, Santa Clara, CA). First-strand cDNA synthesis was performed using the SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio, Mountain View, CA) with 5 ng input RNA followed by nine cycles of PCR amplification and library preparation using the Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA). Sequencing was performed on a NovaSeq 6000 (Illumina), with approximately 40–60 million 150 bp paired-end reads obtained for each sample.

FASTQ files were aligned to the human genome (hg38) and transcript abundances quantified using RSEM (v1.3.3) (Li and Dewey, 2011) calling bowtie2 (v2.4.2) (Langmead and Salzberg, 2012). Estimated counts from RSEM were input to DESeq2 (v1.26.0) (Love et al., 2014) implemented in R (v3.6.3) for differential expression analysis. Elsewhere, transcript abundances are presented as TPM. Differentiation pairing as described above was included in the DESeq2 designs. The Wald test with Benjamini–Hochberg correction was used to generate adjusted p-values. Principal component analysis was performed on counts after the DESeq2 variance stabilizing transformation. Transcription factor annotations were based on the list available at http://humantfs.ccbr.utoronto.ca/ (Lambert et al., 2018); secreted and transmembrane annotations were based on the UniProt database (UniProt Consortium, 2021). WGCNA (v1.70–3) (Zhang and Horvath, 2005; Langfelder and Horvath, 2008) was performed on the 14 EC datasets. Genes with an average of fewer than 50 estimated counts across these datasets were excluded, and the DESeq2 variance stabilizing transformation was used to generate the expression matrix for input to WGCNA. The topological overlap matrix (TOM) was constructed using the signed network type and a power of 20. Hierarchical clustering was performed on dissimilarity (1 – TOM) with average linkage. Gene modules were detected by a constant height (0.99) cut of the hierarchical clustering dendrogram with a minimum module size of 30 genes. Module eigengenes (the first principal component of the expression matrix for genes in each module) were computed as described, and the Pearson correlation between module eigengenes and experimental variables (CHIR vs. DMSO: CHIR = 1, DMSO = 0; Passage 3 vs. Passage 1: Passage 3 = 1, Passage 1 = 0) was used to identify modules of interest. Cytoscape (v3.8.3) (Shannon et al., 2003) was used to visualize the 30 genes in the green module (strong positive correlation with CHIR treatment) with the highest intramodular connectivity. The list of genes, corresponding modules, and correlations to experimental variables and module eigengenes is provided in Supplementary file 3.

Bulk RNA-seq data from the literature (FASTQ files; see ‘Previously published datasets used’) were obtained from the Gene Expression Omnibus (GEO). These FASTQ files were aligned to the mouse genome (mm10) and transcript abundances quantified as described above. DESeq2 was used for differential expression analysis as described above. For direct comparison of human and mouse data, the biomaRt package (v2.42.1) (Durinck et al., 2009) and Ensembl database (Yates et al., 2020) were used to map human gene names to mouse homologs. Venn diagrams were generated using the tool available at http://bioinformatics.psb.ugent.be/webtools/Venn/. To identify solute carrier and efflux transporter genes highly expressed at the human BBB in vivo, we used five human brain scRNA-seq datasets (Han et al., 2020; Hodge et al., 2019; La Manno et al., 2016; Polioudakis et al., 2019; Zhong et al., 2020; see ‘Previously published datasets used’) integrated in a previous meta-analysis (Gastfriend et al., 2021). SLC and ABC genes with average expression greater than 100 TPM in ECs across the five independent datasets were selected. For pathway enrichment analysis, lists of upregulated genes (log2(fold change) > 0, adjusted p-value<0.05, DESeq2 Wald test with Benjamini–Hochberg correction) were tested against the Hallmark gene sets collection (Liberzon et al., 2015) using the tool available at http://www.gsea-msigdb.org/gsea/msigdb/annotate.jsp.

Immunocytochemistry

Request a detailed protocol

Immunocytochemistry was performed in 24-well plates. Cells were washed once with 500 µL DPBS and fixed with 500 µL cold (–20°C) methanol for 5 min, except cells intended for calponin/SM22a and CD31/Ki67 detection, which were fixed with 500 µL of 4% paraformaldehyde for 15 min. Cells were washed three times with 500 µL DPBS and blocked in 150 µL DPBS supplemented with 10% goat serum (Life Technologies) for 1 hr at RT, except cells intended for calponin/SM22⍺ detection, which were blocked and permeabilized in DPBS supplemented with 3% BSA and 0.1% Triton X-100, or cells intended for CD31/Ki67 detection, which were blocked and permeabilized in DPBS supplemented with 5% non-fat dry milk and 0.4% Triton X-100. Primary antibodies diluted in 150 µL of the above blocking solutions (see Key resources table for antibody information) were added to cells and incubated overnight at 4°C on a rocking platform. Cells were washed three times with 500 µL DPBS. Secondary antibodies diluted in 150 µL of the above blocking solutions (see Key resources table for antibody information) were added to cells and incubated for 1 hr at RT on a rocking platform, protected from light. Cells were washed three times with 500 µL DPBS, followed by 5 min incubation with 500 µL DPBS plus 4 µM Hoechst 33342 (Life Technologies). Images were acquired using an Eclipse Ti2-E epifluorescence microscope (Nikon, Tokyo, Japan) with a 20× or 30× objective or an A1R-Si+ confocal microscope (Nikon) with a 100× oil objective. Confocal images were acquired with 1 µm slice spacing.

Images were analyzed using FIJI (ImageJ) software. For epifluorescence images, five fields (20× or 30×) were analyzed per well, with 3–4 wells per treatment condition. For quantification of cell number, EC colonies were manually outlined, and the Analyze Particles function was used to estimate the number of nuclei within the EC colonies. Nuclei outside the EC colonies were manually counted. EC purity (% EC) was calculated as the number of nuclei within EC colonies relative to total nuclei. To estimate % GLUT-1+ ECs, cells within the EC colonies with membrane-localized GLUT-1 immunoreactivity were manually counted. To estimate % Ki67+ ECs, cells within the EC colonies with at least one nuclear-localized Ki67 punctum were manually counted. For quantification of fluorescence intensity in epifluorescence images, EC colonies were manually outlined, and the Measure function was used to obtain the mean fluorescence intensity for each image channel (fluorophore). A cell-free area of the plate was similarly quantified for background subtraction. Following background subtraction, the mean fluorescence intensity of each protein of interest was normalized to the mean fluorescence intensity of Hoechst to correct for effects of cell density. For confocal images, 3–4 fields (100×) containing only VE-cadherin+ ECs were analyzed per well, with four wells per treatment condition. The first slice with visible nuclei (closest to glass) was defined as Z = 0, and the Measure function was used to obtain the mean fluorescence intensity for each image channel (fluorophore) in each slice from Z = 0 to Z = 7 µm. A cell-free area of the plate was similarly quantified for background subtraction. After background subtraction, to approximate total abundance (area under the fluorescence versus Z curve [AUC]) for each channel, mean fluorescence intensities were summed across all slices. AUCs for the proteins of interest were normalized to Hoechst AUC.

Cell cycle analysis

Request a detailed protocol

Passage 1 cultures were dissociated by treatment with Accutase for 10 min at 37°C. Cell suspensions were passed through 40 µm cell strainers into 4× volume of DMEM/F-12 and centrifuged for 5 min, 200× g. Approximately 5 × 105 cells per replicate were resuspended in MACS buffer and incubated with the CD31-APC antibody (Miltenyi Biotec) for 30 min at 4°C, protected from light. Cell suspensions were brought to a volume of 5 mL with MACS buffer and centrifuged at 4°C for 5 min, 200× g. Cells were resuspended in 500 µL MACS buffer containing 2 µg/mL DAPI and 0.5 µL Vybrant DyeCycle Green Stain (Invitrogen) and incubated at RT for 1 hr, protected from light. Cells were analyzed on an Attune NxT flow cytometer (Invitrogen). FlowJo software (BD Biosciences) was used to gate CD31+ cells and quantify the percentage of S/G2/M phase cells.

Western blotting

Request a detailed protocol

To enrich samples from Passage 1 cultures for ECs, the Accutase-based selective dissociation method described above was employed. Dissociated cells were centrifuged for 5 min, 200× g, and resulting cell pellets were lysed in RIPA buffer (Rockland Immunochemicals, Pottstown, PA) supplemented with 1× Halt Protease Inhibitor Cocktail (Thermo Scientific). Passage 3 cells were lysed with the above buffer directly on plates. Lysates were centrifuged at 4°C for 5 min, 14,000× g, and protein concentration in supernatants quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific). Equal amounts of protein were diluted to equal volume with water, mixed with sample buffer, and heated at 95°C for 5 min, except lysates intended for GLUT-1 western blotting, which were not heated. Samples were resolved on 4–12% Tris-Glycine gels and transferred to nitrocellulose membranes. Membranes were blocked for 1 hr in Tris-buffered saline plus 0.1% Tween-20 (TBST) supplemented with 5% non-fat dry milk. Primary antibodies (see Key resources table for antibody information) diluted in TBST plus 5% non-fat dry milk were added to membranes and incubated overnight at 4°C on a rocking platform. Membranes were washed five times with TBST. Secondary antibodies (see Key resources table for antibody information) diluted in TBST were added to membranes and incubated for 1 hr at RT on a rocking platform, protected from light. Membranes were washed five times with TBST and imaged using an Odyssey 9120 (LI-COR, Lincoln, NE). Band intensities were quantified using Image Studio software (LI-COR).

Dextran accumulation assay

Request a detailed protocol

A fixable, Alexa Fluor 488-conjugated dextran with an average molecular weight of 10 kDa (Invitrogen) was used as a tracer to estimate total fluid-phase endocytosis. Dextran was added at 10 µM to the medium of Passage 1 cultures. Plates were incubated on rotating platforms at 37 or 4°C for 2 hr. For inhibitor experiments, 20 µM chlorpromazine (Sigma), 100 U/mL nystatin (Sigma), or 2 µM rottlerin (Tocris) were added to the medium 30 min prior to addition of dextran. Medium was removed and cells were washed once with DPBS, and then incubated with Accutase for 10 min at 37°C. Cell suspensions were passed through 40 µm cell strainers into 4× volume of DMEM/F-12 and centrifuged for 5 min, 200× g. Cells were resuspended in MACS buffer and incubated with the CD31-APC antibody (Miltenyi Biotec) for 30 min at 4°C, protected from light. Cell suspensions were brought to a volume of 5 mL with MACS buffer and centrifuged at 4°C for 5 min, 200× g. Pellets were resuspended in DPBS supplemented with 4% paraformaldehyde and incubated for 15 min at RT, protected from light. Cells were centrifuged for 5 min, 200× g. Pellets were resuspended in MACS buffer and analyzed on a BD FACSCalibur flow cytometer (BD Biosciences). FlowJo software was used to gate CD31+ cells and quantify geometric mean fluorescence intensity and CV of dextran. For imaging, the dextran accumulation assay was performed on cells cultured on #1.5 glass-bottom plates. After 2 hr of dextran treatment, medium was removed and cells washed with DPBS. Cells were fixed with 4% paraformaldehyde for 15 min. Cells were washed three times with 500 µL DPBS and blocked and permeabilized with DPBS supplemented with 10% goat serum and 0.1% Triton X-100 for 1 hr at RT. Cells were stained with the caveolin-1 primary antibody and imaged on a confocal microscope as described above.

Transendothelial electrical resistance and sodium fluorescein permeability

Request a detailed protocol

Transwell inserts (6.5 mm diameter with 0.4 µm pore polyester filters) (Corning) were coated with 50 µL of a solution of collagen IV (400 µg/mL) and fibronectin (100 µg/mL) in water for 4 hr at 37°C. Passage 3 DMSO- and CHIR-treated ECs were seeded on Transwell inserts at 105 cells/cm2 in hECSR medium supplemented with DMSO or CHIR. Medium volumes were 200 µL for the apical chamber and 800 µL for the basolateral chamber. Beginning the day after seeding, TEER was measured daily for 6 days using an EVOM2 epithelial voltohmmeter with STX2 chopstick electrodes (World Precision Instruments, Sarasota, FL). Medium was replaced every other day. TEER values were corrected by subtracting the resistance of a collagen IV/fibronectin-coated Transwell insert without cells and multiplying by the filter surface area of 0.33 cm2. Permeability of endothelial monolayers to sodium fluorescein was assessed 6 days after seeding cells on Transwell inserts. Medium in both apical and basolateral chambers was replaced and cells returned to the incubator for 1 hr. Medium in apical chambers, including the apical chamber of a collagen IV/fibronectin-coated Transwell insert without cells, was then replaced with medium supplemented with 10 µM sodium fluorescein (Sigma-Aldrich), and plates placed on an orbital platform in an incubator. At 15, 30, 45, and 60 min, an 80 µL sample of the basolateral chamber medium was withdrawn from each Transwell, transferred to a 96-well plate, and 80 µL fresh medium replaced in the basolateral chamber of each Transwell. At 60 min, an 80 µL sample of apical chamber medium was also withdrawn from each Transwell and transferred to the 96-well plate. 80 µL of medium lacking sodium fluorescein was also transferred to the 96-well plate for background subtraction. Fluorescence intensity of all samples was measured using an Infinite M1000 PRO plate reader (Tecan, Männedorf, Switzerland) with 485 nm excitation and 530 nm emission wavelengths. Background-subtracted fluorescence intensity values at the 30, 45, and 60 min timepoints were corrected for sampling-induced dilution as previously described (Stebbins et al., 2016). The endothelial permeability coefficient (Pe), which is a concentration-independent parameter corrected for the permeability of a cell-free Transwell insert, was calculated as previously described (Stebbins et al., 2016).

Statistics

Individual wells of cultured cells that underwent identical experimental treatments are defined as replicates, and all key experiments were repeated using multiple independent hPSC differentiations. Detailed information about replication strategy is provided in figure legends. Student’s t test was used for comparison of means from two experimental groups. One-way analysis of variance (ANOVA) was used for comparison of means from three or more experimental groups, followed by Dunnett’s post-hoc test for comparison of multiple treatments to a single control, or Tukey’s honest significant difference (HSD) post-hoc test for multiple pairwise comparisons. When data from multiple differentiations were combined, two-way ANOVA (one factor being the experimental treatment and one factor being the differentiation) was used for comparison of means to achieve blocking of differentiation-based variability, followed by post-hoc tests as described above if more than two experimental treatments were compared. For fluorescence intensities (a.u.), two-way ANOVA was performed prior to normalization of these values to the control group within each differentiation (for visualization in plots). Statistical tests were performed in JMP Pro (v15.0.0). For RNA-seq differential expression analysis, the DESeq2 Wald test with Benjamini–Hochberg correction was used to calculate p-values. Descriptions of the statistical tests used are provided in figure legends.

Data availability

RNA-seq data have been deposited in GEO under accession number GSE173206.

The following data sets were generated
    1. Gastfriend BD
    2. Palecek SP
    3. Shusta EV
    (2021) NCBI Gene Expression Omnibus
    ID GSE173206. RNA-sequencing of human pluripotent stem-cell derived endothelial cells under control and Wnt-activating conditions.
The following previously published data sets were used
    1. Sabbagh MF
    2. Heng J
    3. Luo C
    4. Castanon RG
    5. Nery JR
    6. Rattner A
    7. Goff LA
    8. Ecker JR
    9. Nathans J
    (2018) NCBI Gene Expression Omnibus
    ID GSM3040844 GSM3040845 GSM3040852 GSM3040853 GSM3040858 GSM3040859 GSM3040864 GSM3040865. Transcriptional and Epigenomic Landscapes of CNS and non-CNS Vascular Endothelial Cells.
    1. Munji R
    2. Daneman R
    (2018) NCBI Gene Expression Omnibus
    ID GSM2498580 GSM2498581 GSM2498582 GSM2498583 GSM2498584 GSM2498585 GSM2498586 GSM2498587. Gene expression profiles of liver and lung endothelial cells during normal and upregulation of Wnt/beta-catenin signaling.
    1. Wang Y
    2. Sabbagh MF
    3. Gu X
    4. Rattner A
    5. Williams J
    6. Nathans J
    (2019) NCBI Gene Expression Omnibus
    ID GSM3455653 GSM3455654 GSM3455657 GSM3455658 GSM3455661 GSM3455662 GSM3455665 SM3455666. The role of beta-catenin signaling in regulating barrier vs. non-barrier gene expression programs in circumventricular organ and ocular vasculatures.
    1. Sabbagh MF
    2. Nathans J
    (2020) NCBI Gene Expression Omnibus
    ID GSM4160534 GSM4160535 GSM4160536 GSM4160537 GSM4160538 GSM4160539 GSM4160540 GSM4160541 GSM4160542 GSM4160543. A genome-wide view of the de-differentiation of central nervous system endothelial cells in culture.
    1. Allen Institute
    (2019) Allen Brain Map
    ID human-multiple-cortical-areas-smart-seq. Human Multiple Cortical Areas SMART-seq.
    1. Polioudakis D
    (2019) UCLA
    ID CoDex. Cortical Development Expression.
    1. Guo G
    2. Han X
    3. Zhou Z
    4. Fei L
    5. Sun H
    6. Wang R
    7. Wang J
    8. Chen H
    (2020) NCBI Gene Expression Omnibus
    ID GSM3980129 GSM4008656 GSM4008657 GSM4008658. Construction of A Human Cell Landscape by Single-cell mRNA-seq.
    1. LaManno G
    2. Gyllborg D
    3. Arenas E
    4. Linnarsson S
    (2016) NCBI Gene Expression Omnibus
    ID GSE76381. Single Cell RNA-seq Study of Midbrain and Dopaminergic Neuron Development in Mouse, Human, and Stem Cells.
    1. Zhong S
    2. Wu Q
    3. Wang X
    (2019) NCBI Gene Expression Omnibus
    ID GSE119212. Characterization of the transcriptional landscape of human developing hippocampus by scRNA-seq.

References

    1. Kreitzer FR
    2. Salomonis N
    3. Sheehan A
    4. Huang M
    5. Park JS
    6. Spindler MJ
    7. Lizarraga P
    8. Weiss WA
    9. So P-L
    10. Conklin BR
    (2013)
    A robust method to derive functional neural crest cells from human pluripotent stem cells
    American Journal of Stem Cells 2:119–131.

Decision letter

  1. Stefan Liebner
    Reviewing Editor; University Hospital, Goethe University Frankfurt, Germany
  2. Didier YR Stainier
    Senior Editor; Max Planck Institute for Heart and Lung Research, Germany
  3. Maxime Culot
    Reviewer; Artois University, France

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Wnt signaling mediates acquisition of blood-brain barrier properties in naïve endothelium derived from human pluripotent stem cells" for consideration by eLife. Your article has been reviewed by 4 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Didier Stainier as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Maxime Culot (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) The authors need to explore in more depth the differences in the expression profile between naïve endothelial progenitors and (partially) differentiated ECs by RNA-Seq.

2) Moreover, the authors should investigate other pathways induced by CHIR (GSK3 substrates; Akt, IκB-α, presenilin etc.) in comparison with dominant-active β-catenin.

3) Given that the mechanistic insight in the discrepancies between the stimulations with CHIR and Wnt7a/b, as well as with conditioned medium from neurospheres and astrocytes is limited, the authors should omit these data and add this approach to the discussion.

4) The authors should clearly delineate the aim of the study that, as perceived by the reviewers, appears to be the characterization of the Wnt pathway effects on directing human pluripotent stem cells toward the differentiation to a BBB endothelium.

Reviewer #1 (Recommendations for the authors):

See public review.

Reviewer #2 (Recommendations for the authors):

While this paper may be a worthwhile method development paper (as published in PLoS One, Science Reports or Microvascular research; eg. Cecchelli 2014 doi:10.1371/journal.pone.0099733; Linville 2020 doi: 10.1016/j.mvr.2020.104042), it lacks scientific novelty and depth required for a publication in eLife.

Numerous papers have identified Wnt3a for mesoderm induction and Wnt7a as the major brain EC ligand, and the use of a chemical GSK-3 inhibitor for more powerful Wnt signaling pathway activation is not novel or surprising. Indeed many other models and Wnt-pathway response papers have been published, numerous in the last two years (e.g. Laksitorini et al., Sci Rep 2019 9(1):19718. "Modulation of Wnt/β-catenin signaling promotes blood-brain barrier phenotype in cultured brain endothelial cells").

In addition one major claim of archievement is the analysis of EC maturity linked to Wnt-responsiveness. However, it has already been shown that there is a crucial timing difference in the responsiveness to Wnt signals both in vivo and in vitro (including, but not at all limited to the following examples: Hübner et al., Nat commun 2018 Nov 19;9(1):4860 "Wnt/β-catenin signaling regulates VE-cadherin-mediated anastomosis of brain capillaries by counteracting S1pr1 signaling"; Corada et al., Circ Res 2019 124(4):511-525 "Fine-Tuning of Sox17 and Canonical Wnt Coordinates the Permeability Properties of the Blood-Brain Barrier").

Additionally many of the mentioned examples not only analyze the contribution of WNT signaling, but also link other signaling pathways, whereas this paper identifies other but unknown signaling contributions for establishing ECs with full BBB properties.

Given the lack of novelty and the following mentioned difficulties of biological sample variations, I do not recommend the paper for publication in eLife.

Other comments:

Figure 1 shows more than factor2 differences between different differentiation samples (in terms of glut-1 positive cells), making this a less robust model than indicated.

The overall percentage of Glut-1 positive cells in Figure 1 and 2 is very low, making the comparison between the effectiveness of the different ligands while statistically significant compared to the control, most likely not significant compared between the ligands. Therefore the claims of e.g. Wnt7a being more effective is based on one of 3 samples of the same differentiation. All these comaparative claims will have to be either supported by much more data or taken out. The only clear result is that CHIR can stimulate expression of Glut-1 in around 90% of the cells, whereas the offered Wnt ligands affect 2-4%. To me this suggests that soluble WNT ligands in the culture medium are least effective in stimulating a response. It would have been most interesting to explore if co-cultures and hence the supply in e.g. Cytonems or other vesicle or membrane-bound delivery systems would make a huge difference. However this is not the scope of the paper.The analysis of mean fluorescence intensity of claudin-5, caveolin-1, and GLUT-1 normalized to Hoechst mean fluorescence intensity within the area of claudin-5+ ECs only is less informative, as one highly expressing cell will outweigh many low expressing cells and there is no published link of an endothelial cell resembling a BBB endothelial cells more closely in correlation with their Glut-1 expression intensity.

Unfortunately, when analysing the effects of conditioned media in Figure3 only those relative quantifications were used and again the spread between the two different differentiation rounds was so high, that a comparative analysis is hindered and not represented by statistical analysis compared to control only. For example the Caveolin/Hoest values of the orange dataset were increased in the CHIR treatment, but unchanged in Control and NR-CM, but in contrast in the green dataset the NR-CM resulted in a higher index compared to CHIR or the control. Again were no statistical comparison was done between samples, the drawn conclusions can not indicate there are any.

Figure 5 Anal;yses the effect of passage number in combination with CHIR treatment. However the individual differentiation samples again behave partially opposite: the green sample Glut1/ß-actin is only weakle elevated in passage 1 and upregulated after passage3, the purple sample is high in passage 1 and becomes downregulated after passage 3.

Figure 7 the effects in the mature EC should be directly compared to the effects in more naïve from the same differentiation.

Reviewer #3 (Recommendations for the authors):

Although I have a positive opinion on the manuscript, I personally think that the manuscript could be slightly simplified (and shorten). Therefore, I have listed below several points that I think should be addressed (and possibly used to shorten the manuscript) by the authors before publication of the manuscript.

– Page 8 Line 173- 192 : "In the CNS, neural progenitors ….. ….. the Wnt ligand signal (analyzed further below)”.

This part and associated figure 3 compare the effects of neural rosette-conditioned medium and astrocyte-conditioned medium to the one of WNT7A and CHIR based on the fact that both cell types were suggested to express WNT7A (Vatine et al., 2016; Shang et al., 2018)..

Although the data are interesting per se., these data in my opinion, do not really have an added value to the main topic of the study (i.e deciphering the effects of wnt signaling in human endothelial cell progenitor with a focus on barrier formation).

In addition, the reported effects of neural rosette and astrocyte-conditioned media on endothelial progenitors were weaker than the one of CHIR which the authors suggest could be due to the potency or concentration of ligands in conditioned media. Indeed, there can be numerous hypothesis made here as there can be numerous secreted factors in those conditioned media.

Therefore, instead of including these data (i.e Figure 3) in this manuscript, I would rather suggest the authors to use it as the basis of a study on the contribition of wnt ligands in the effects of neural rosette and/or astrocyte-conditioned media-effects on ECs derived from hPSC. They could make use of their hPSC lines with doxycycline-inducible expression of short hairpin RNAs targeting CTNNB1 to confirm that neural rosette and/or astrocyte-conditioned media-effects in ECs (notably on GLUT-1, Claudin 5, caveolin-1) are β-catenin dependent.

In such experiment a comparison of the effects of wnt7A, neural rosette and astrocyte-conditioned media (+ brain pericyte conditioned medium or even coculture) on hPSC-derived endothelial progenitors (including from doxycycline-inducible expression of short hairpin RNAs targeting CTNNB1) would probably clarify the role of wnt ligand in the effects of neural rosette and astrocyte-conditioned media (or even coculture).

– Page 10-Line 217 (and associated Figure 6C): "We first confirmed that the process of dextran internalization required the membrane fluidity of an endocytosis-dependent process by carrying out the assay at 4C”

Although I get the idea of comparing fluorescent dextran internalization with or without CHIR treatment, I miss the point of the necessity of comparing the 4C and 37C. I assume 4C would not only affect membrane fluidity but also would reduce any active processes taking place in the cells as well as reduce thermal molecular agitation within the assay.

Therefore, I would suggest to remove this experiment at 4{degree sign}C which I find confusing and possibly replace it by experiment performed with inhibitors of the caveolin pathway (e.g Filipin, Genistein, Nystatin,.;)

– Page 21 – Line 483:

Although I agree with the statement that Praça et al., reported moderate improvement in barrier function using a combination of VEGF, Wnt3a, and retinoic acid, in my opinion TEER values should be used intra assay (e.g as in the manuscript to compare the effects of CHIR on TEER) not from one study to the next as several parameters could influence TEER (e.g Temperature, buffer capacity, surface of the insert,…).

Reviewer #4 (Recommendations for the authors):

The manuscript by Gastfriend et al., describes a novel approach to generate a blood-brain barrier (BBB) in vitro model from human pluripotent stem cells (hPSCs) by inducing BBB characteristics in yet undifferentiated, naïve endothelial precursor cells with Wnt/β-catenin activating agents. Specifically, the authors identified the inhibitor of glycogen synthase kinase 3β (GSK3β) CHIR99021 (CHIR) to consistently induce crucial BBB-relevant genes such as GLUT-1, Cldn5, LEF1, APCDD1 and ZIC3. At the same time GSK3β inhibition lead to reduced expression of PLVAP. Interestingly, the authors also observed, along with the up-regulation of BBB genes, an up-regulation of caveolin-1 (CAV1) that however, did not result in increased transendothelial permeability. Further the authors compared the treatment with CHIR to the stimulation with recombinant Wnt7a, Wnt7b and combinations thereof, as well as to the treatment with conditioned medium (CM) from astrocytes and neuroblast rosettes. Wnt7a and the combination of Wnt7a and Wnt7b showed a pronounced effect on endothelial purity during IPSC differentiation, but considerably lower effects on the expression of barrier genes than CHIR. Neuroblast and astrocyte CM showed effects on endothelial purity and neuroblast CM also some effect on GLUT-1 expression.

The authors also investigated the barrier characteristics at different stages of endothelial progenitor differentiation, i.e. at passage 1 and passage 3, as well as at a higher maturity at passages 4 and 5. The presented data suggest that at passage 1 and 3 CHIR is able to induce expression of GLUT-1, claudin-5, LSR and barrier properties measured by TEER, whereas at later passages 4 and 5 these effects were considerably lower or absent.

Consequently, the author interpret this finding as a a result of endothelial differentiation, suggesting that naïve progenitors are susceptible to Wnt induction, whereas terminally differentiated cells are not or at lower level.

Finally, the authors performed RNA-Seq of CHIR and DMSO treated progenitor cells at passages 1 and 3, as well as comparing Wnt7a and DMSO. Expression profiling demonstrated that CHIR induces crucial BBB genes which essentially overlapped with the inducing effects of Wnt7a. Interestingly, only Wnt7a resulted in the induction of Sox17, which has been shown to be up-regulated in the maturing BBB.

The concept of a time-dependent induction of BBB characteristics by Wnt/β-catenin induction as a “window of opportunity" is interesting and innovative, although others have already proposed this.

The presented data are solid and the manuscript is well written with high standard figures. However, some experimental details and interpretation of the data require the authors attention to augment the scientific merit of the manuscript:

It would be important that the authors clearly claim if the present manuscript describes a novel in vitro model or a basic stem cell biological finding.

The following critical points are related to this first issue because the that

The authors show that the treatment with Wnt7a and in particular with Wnt7b only show little or even no effects on Wnt pathway activation (Figure 8—figure supplement 1 B; no AXIN2 induction by Wnt7a/7b!!) and induction of BBB characteristics. It would be important to further investigate this topic to clearly demonstrate that Wnt7a/7b indeed are functional as recombinant proteins in the described experimental setting. Wnt7 has been shown to be only little diffusible, requiring the direct contact of the sending cell to the receiving cells (Eubelen, M. et al., Science (New York, NY) 361, eaat1178, 2018). Therefore, the authors should elaborate on an experimental setting to stimulate the naïve endothelial progenitors with Wnt7a/7b in a paracrine manner. This would help to clarify the question regarding the discrepancy between the CHIR and the Wnt7a treatment.

Essentially, the same critique can be raised concerning the treatment with conditioned medium from neuroblast rosettes and astrocytes. Although both may express Wnt7a/7b, specifically these Wnts may not accumulate in the CM and hence, the effects may be minor.

Page 16, line 359-363: The assumption made in the sentence “Upon recombination, the Ctnnb1 flex3 allele produces a dominant mutant β-catenin lacking residues that are phosphorylated by GSK-3β to target β-catenin for degradation (Harada et al., 1999); as such, this strategy for ligand- and receptor-independent Wnt activation by β-catenin stabilization is directly analogous to CHIR treatment." might be wrong, as blocking GSK3beta potentially has many other effects than “just" activating β-catenin-mediated transcription!! The authors should discuss this topic in more depth.

One of the highlight of the present manuscript is the inducibility of naïve endothelial progenitors by CHIR compared to differentiated endothelial cells. Hence it would be important to compare the expression pattern by RNA-Seq of these two conditions.

https://doi.org/10.7554/eLife.70992.sa1

Author response

Essential revisions:

1) The authors need to explore in more depth the differences in the expression profile between naïve endothelial progenitors and (partially) differentiated ECs by RNA-Seq.

We agree with the reviewers that a more comprehensive analysis and discussion of transcriptomics data is warranted. We have greatly expanded the analysis of our RNA-seq data, focusing on the attributes of the partially differentiated ECs resulting from CHIR treatment. We have also included new analyses that deploy weighted gene correlation network analyses to distinguish the unique features of CHIR treatment compared with those acquired through extended culture. Finally, we have performed enrichment analyses to compare and contrast CHIR effects to those elicited by direct stabilization of β-catenin. We have substantially revised the corresponding sections of the Results and Discussion to highlight these additional analyses, but given the number of figures that have been edited and added, have not duplicated the revised figures in the response document. In detail:

– We have highlighted additional genes of interest induced by CHIR in Passage 1 ECs, including additional transcription factors also expressed by brain ECs in vivo (MSX1, EBF1), and FLVCR2, a gene with a known role in brain angiogenesis (Figure 7).

From Results: “We also identified upregulated transcription factors: ZIC3, which is highly enriched in brain and retinal ECs in vivo and downstream of Frizzled4 signaling (Wang et al., 2012; Sabbagh et al., 2018), and SOX7, which acts cooperatively with SOX17 and SOX18 in retinal angiogenesis (Zhou et al., 2015), were upregulated by CHIR in our system (Figure 7D). MSX1 and EBF1, which are expressed by murine brain ECs in vivo (Vanlandewijck et al., 2018) were also CHIR-upregulated (Figure 7D). Additional CHIR-upregulated genes included ABCG2 (encoding the efflux transporter Breast Cancer Resistance Protein, BCRP), APLN, a tip cell marker enriched in postnatal day 7 murine brain ECs compared to those of other organs, and subsequently downregulated in adulthood (Sabbagh et al., 2018; Sabbagh and Nathans, 2020), and FLVCR2, a disease-associated gene with a recently-identified role in brain angiogenesis (Santander et al., 2020) (Figure 7C-D).”

– We have better enumerated genes induced by CHIR in Passage 3 ECs: In Figure 7—figure supplement 1, we have highlighted genes up- and down-regulated by CHIR in a format analogous to Figure 7; In Figure 7—figure supplement 2, we have identified genes encoding transcription factors, secreted proteins, and transmembrane proteins that are consistently CHIR-upregulated at Passage 1 and Passage 3.

From Results: “In Passage 3 ECs, many of the CHIR-mediated gene expression changes observed at Passage 1 persisted, including SLC2A1, LSR, LEF1, AXIN2, APCDD1, ZIC3, EBF1, FLVCR2 and ABCG2 upregulation and PLVAP downregulation (Figure 7E; Figure 7—figure supplement 1). […] Conversely, JAM2, which encodes junctional adhesion molecule 2, a component of EC tight junctions (Aurrand-Lions et al., 2001; Tietz and Engelhardt, 2015), was upregulated by CHIR at Passage 3, but not at Passage 1, as was the retinol-binding protein-encoding gene RBP1 (Figure 7—figure supplement 1).”

– We used Weighted Gene Correlation Network Analysis to identify highly correlated gene modules with EC passage number and CHIR treatment (Figure 7—figure supplement 3). Importantly, this analysis complements gene-by-gene differential expression analysis by (a) demonstrating that genes central to the module that strongly correlates with CHIR treatment are canonical targets of Wnt/β-catenin signaling, (b) identifying novel genes that strongly correlate with these known targets, and (c) demonstrating that CHIR treatment and passage number have distinct effects on the transcriptome as a whole despite some concordantly regulated genes (e.g., CLDN5 and PLVAP).

From Results: “We used Weighted Gene Correlation Network Analysis (WGCNA) (Zhang and Horvath, 2005; Langfelder and Horvath, 2008) to identify modules containing genes with highly correlated expression across the 14 EC samples (Figure 7—figure supplement 3A; Supplementary file 3). […] Further, despite some similarly-regulated genes between the passage number and CHIR treatment comparisons (e.g., CLDN5, CAV1, PLVAP), the transcriptional responses to these two experimental variables were globally distinct as assessed by gene correlation network analysis (Figure 7—figure supplement 3B).”

– We have performed enrichment analysis to identify pathways that may be upstream of observed transcriptional changes upon CHIR treatment at passage 1 and 3. We have also performed this analysis on literature data from pituitary ECs to investigate how pathways induced by CHIR-mediated inhibition of GSK-3 might differ from pathways induced by direct stabilization of β-catenin (described in additional detail below under point 2.) These data are in Figure 8—figure supplement 2 and Supplementary file 5.

2) Moreover, the authors should investigate other pathways induced by CHIR (GSK3 substrates; Akt, IκB-α, presenilin etc.) in comparison with dominant-active β-catenin.

We thank the reviewers for the suggestion to compare pathways induced by CHIR (in our system) versus stabilized β-catenin (literature data). For stabilized β-catenin, we focused on the RNA-seq data from the mouse pituitary EC system, as this system demonstrated the strongest transcriptional response to β-catenin stabilization (compared to mouse liver ECs and cultured mouse brain ECs). We tested lists of CHIR- and β-catenin-upregulated genes against the Hallmark gene set collection, a curated database of high-confidence links between genes and potential upstream biological processes and signaling pathways. Complete results are shown in Figure 8—figure supplement 2 and Supplementary file 5, and are summarized in the Results section. Briefly, we found that multiple signaling pathways (including Wnt/β-catenin signaling, Notch signaling, TNFα signaling via NF-κB, KRAS signaling up) were enriched in both CHIR-treated and β-catenin-stabilized systems. A key potential difference is enrichment of the PI3K AKT mTOR signaling gene set in Passage 1 CHIR-treated ECs; such enrichment is not present in pituitary ECs with β-catenin-stabilization, or Passage 3 CHIR-treated ECs. This result may suggest that CHIR transiently activates the AKT/mTOR pathway. Overall, however, the strong and persistent enrichment of the Wnt/β-catenin signaling gene set in CHIR-treated ECs at both Passage 1 and 3 (along with upregulation of canonical transcriptional targets of β-catenin, the centrality of such canonical transcriptional targets in gene correlation network analysis, and the attenuation of GLUT-1 induction with CTNNB1 knockdown), support the notion that Wnt/β-catenin signaling is a central mediator of our observed phenotypes. We agree that other GSK-3 targets may participate, and believe a direct comparison of CHIR treatment and β-catenin stabilization in the hPSC-derived EC system could be informative in the future. Finally, we note that β-catenin stabilization is also an imperfect mimic of Wnt ligand-induced signaling, as ligand-induced signaling also involves GSK-3 inhibition; this further motivates future work using Wnt ligands and the endogenous Wnt receptors and intracellular transduction machinery.

From Results: “Last, because GSK-3 is a component of numerous signaling pathways in addition to Wnt/β-catenin (Eto et al., 2005; Beurel et al., 2015; Hermida et al., 2017), we used RNA-seq data to infer pathways that might be differentially regulated by the two strategies for activating Wnt/β-catenin signaling employed in the experiments above: CHIR treatment, which increases β-catenin stability by inhibiting GSK-3, or direct stabilization of β-catenin. […] Differences in other aspects of these two experimental paradigms (in vitro versus in vivo, naïve versus CNS-proximal, human versus mouse), however, caution against over-interpretation of these results.”

From Discussion: “CHIR is widely used to activate Wnt/β-catenin signaling in cell culture (Lian et al., 2012, 2014; Patsch et al., 2015; Sakaguchi et al., 2015; Gomez et al., 2019; Pellegrini et al., 2020; Guo et al., 2021). It remains unknown, however, to what extent CHIR-mediated inhibition of GSK-3 in ECs mimics the effects of Wnt ligand-induced inhibition of GSK-3 or direct stabilization of β-catenin. In our system, although the GLUT-1-inductive effect of CHIR was partially inhibited by β-catenin knockdown and our RNA-seq data revealed a transcriptional response characteristic of canonical Wnt signaling, it is possible that CHIR affects other signaling pathways, as suggested by pathway enrichment analysis. Thus, employing ligand-based strategies to activate Wnt signaling will be an important next step. […] Finally, it would also be informative to directly compare CHIR and/or Wnt ligand treatment to direct stabilization of β-catenin in this system, for example, by generating a hPSC line with inducible expression of a dominant active β-catenin.”

3) Given that the mechanistic insight in the discrepancies between the stimulations with CHIR and Wnt7a/b, as well as with conditioned medium from neurospheres and astrocytes is limited, the authors should omit these data and add this approach to the discussion.

As suggested, we have now removed these data from the manuscript and instead added a paragraph summarizing these approaches to the Discussion. While Wnt7a/b and conditioned media treatment are rational and important approaches, we agree that there are many potential explanations for their lack of efficacy (lack of ligand bioactivity, lack of necessary cell-cell contact, lack of ligand accumulation in the medium, lack of necessary receptor machinery on endothelial progenitors). We also agree that further mechanistic examination of this phenomena should be the subject of future work.

From Discussion: “CHIR is widely used to activate Wnt/β-catenin signaling in cell culture (Lian et al., 2012, 2014; Patsch et al., 2015; Sakaguchi et al., 2015; Gomez et al., 2019; Pellegrini et al., 2020; Guo et al., 2021). […] Importantly, neural progenitor cells and astrocytes likely would also contribute other yet-unidentified ligands important for acquisition of CNS EC phenotype.”

4) The authors should clearly delineate the aim of the study that, as perceived by the reviewers, appears to be the characterization of the Wnt pathway effects on directing human pluripotent stem cells toward the differentiation to a BBB endothelium.

We appreciate the suggestion to clarify the aim of the study. We have reworded the thesis statement in the final paragraph of the introduction to better convey the goal of the work:

From Introduction: “In this work, we aimed to define the effects of activating Wnt/β-catenin signaling in hPSC-derived, naïve endothelial progenitors and assess the extent to which this strategy would drive development of a CNS EC-like phenotype.”

Statements in the Abstract and Discussion also emphasize this overarching aim:

From Abstract: “Together, our work defines effects of Wnt activation in naïve ECs and establishes an improved hPSC-based model for interrogation of CNS barriergenesis.”

From Discussion: “In this work, we investigated the role of Wnt/β-catenin signaling on induction of BBB properties in a human EC model, using naïve endothelial progenitors derived from hPSCs.”

Reviewer #2 (Recommendations for the authors):

While this paper may be a worthwhile method development paper (as published in PLoS One, Science Reports or Microvascular research; eg. Cecchelli 2014 doi:10.1371/journal.pone.0099733; Linville 2020 doi: 10.1016/j.mvr.2020.104042), it lacks scientific novelty and depth required for a publication in eLife.

Numerous papers have identified Wnt3a for mesoderm induction and Wnt7a as the major brain EC ligand, and the use of a chemical GSK-3 inhibitor for more powerful Wnt signaling pathway activation is not novel or surprising. Indeed many other models and Wnt-pathway response papers have been published, numerous in the last two years (e.g. Laksitorini et al., Sci Rep 2019 9(1):19718. "Modulation of Wnt/β-catenin signaling promotes blood-brain barrier phenotype in cultured brain endothelial cells").

We agree that a substantial body of previous research has demonstrated the ability of Wnt signaling to both induce mesoderm differentiation and to induce EC BBB properties, and that many previously reported methods have used GSK-3 inhibition as a Wnt activation strategy. We wish, however, to emphasize the novelty of our work in applying Wnt activation to achieve a BBB-like phenotype in hPSC-derived definitive ECs, and in comprehensively profiling the resulting transcriptome and other aspects of cellular phenotype.

Several previous papers have examined the effects of Wnt activation in immortalized ECs (Laksitorini et al.,) or hematopoietic stem cell-derived ECs (Cecchelli et al.,). In contrast to these cell sources, hPSCs offer the opportunity to track EC differentiation along the entire developmental trajectory, and probe temporal aspects of Wnt activation (discussed further below). We have clarified this point in the Introduction:

From Introduction: “Human pluripotent stem cells (hPSCs) offer an in vitro human model system for systematic investigation of molecular mechanisms of BBB phenotype acquisition, especially given their ability to model early stages of endothelial specification and differentiation.”

The few papers that have attempted Wnt activation in hPSC-derived ECs have not isolated the effects of Wnt and/or performed comprehensive analysis of Wnt-mediated changes. For example, Linville et al., evaluated Wnt7a only in combination with VEGF, and then only on “sprouting” and not on BBB phenotype. Praça et al., demonstrated a slight impact of Wnt3a on permeability, but analyzed protein/gene expression changes with Wnt3a in combination with VEGF or VEGF and retinoic acid. Further, none of the above works have used RNA-sequencing to comprehensively profile the Wnt-regulated EC transcriptome. Other hPSC-derived BBB models for which RNA-seq data are available do not address the impact of Wnt signaling on the transcriptome, do not proceed through definitive endothelial progenitors, and have an underlying epithelial gene signature (Vatine et al., 2017, Cell Stem Cell; Qian et al., 2017, Sci Adv).

To the best of our knowledge, our work represents the first report that activation of Wnt signaling in hPSC-derived ECs can partially achieve BBB phenotype. We further suggest that our transcriptome data defining Wnt-regulated genes, and importantly, BBB-associated genes not regulated by Wnt, will be an important addition to the literature for those studying human BBB development and those wishing to continue to improve our ability to recapitulate the BBB in the dish.

In addition one major claim of archievement is the analysis of EC maturity linked to Wnt-responsiveness. However, it has already been shown that there is a crucial timing difference in the responsiveness to Wnt signals both in vivo and in vitro (including, but not at all limited to the following examples: Hübner et al., Nat commun 2018 Nov 19;9(1):4860 "Wnt/β-catenin signaling regulates VE-cadherin-mediated anastomosis of brain capillaries by counteracting S1pr1 signaling"; Corada et al., Circ Res 2019 124(4):511-525 "Fine-Tuning of Sox17 and Canonical Wnt Coordinates the Permeability Properties of the Blood-Brain Barrier").

Additionally many of the mentioned examples not only analyze the contribution of WNT signaling, but also link other signaling pathways, whereas this paper identifies other but unknown signaling contributions for establishing ECs with full BBB properties.

We thank the reviewer for this comment and we have now expanded the Discussion to incorporate these concepts and citations. We note that these reports have primarily characterized the endogenous temporal profile of Wnt/β-catenin signaling in the context of CNS angiogenesis and barriergenesis, and used loss of function approaches (e.g., dominant negative TCF, IWR-1 treatment) to identify timepoints during normal CNS vascular development at which Wnt/β-catenin is necessary for specific phenotypes. A major conclusion is that the level of Wnt/β-catenin in CNS ECs peaks early in development and subsequently declines, but this result does not directly address the competence of ECs to respond to Wnt signals, which varies across different contexts, including time (embryo versus adult), organ (CNS versus CNS-proximal versus non-CNS), and in vitro versus in vivo (e.g., Munji et al., 2019, Nat Neurosci; Wang et al., 2019, eLife; Sabbagh et al., 2020, eLife). To address this gap in understanding of the role of Wnt/β-catenin in imparting BBB properties to ECs, we performed gain of function experiments by activating Wnt/β-catenin with CHIR at different timepoints during the hPSC differentiation to ECs, and found that early ECs were more responsive than those after extended culture. As discussed above, hPSCs are particularly well suited to this type of longitudinal analysis; we further contend that this treatment paradigm is novel and that the results will be a useful contribution to the literature.

From Discussion: “Thus, our results support this hypothesis and suggest that the loss of BBB developmental plasticity in ECs is an intrinsic, temporally-controlled process rather than a result of the peripheral organ environment. The molecular mechanisms underlying this loss of plasticity remain poorly understood. While previous studies have demonstrated that the level of Wnt/β-catenin signaling in CNS ECs peaks early in development and subsequently declines (Corada et al., 2018; Hübner et al., 2018), this finding does not address mechanisms underlying the competence of ECs (CNS and non-CNS) to respond to Wnt signals. In RNA-seq data of Passage 3 control (DMSO-treated) ECs, LEF1 and TCF7 were strongly downregulated compared to Passage 1 cells. This result suggests that low baseline expression of these transcription factors, which form a complex with nuclear β-catenin to regulate Wnt target genes, may partially explain the poor efficacy of CHIR in matured ECs, although additional work is necessary to assess functional relevance of these differences.”

Given the lack of novelty and the following mentioned difficulties of biological sample variations, I do not recommend the paper for publication in eLife.

Please see the above description of novelty and impact.

Other comments:

Figure 1 shows more than factor2 differences between different differentiation samples (in terms of glut-1 positive cells), making this a less robust model than indicated.

We agree that the percentage of GLUT-1+ ECs resulting from Wnt3a treatment appears to differ between the two differentiations performed; in each case, however, Wnt3a significantly increased the percentage of GLUT-1+ ECs over control (Differentiation 1, control: 1.3±0.4%, Wnt3a: 9.1±1.0%, P = 0.00028, Student’s t test; Differentiation 2, control: 0.5±0.3%, Wnt3a: 2.9±0.4%, P = 0.00096, Student’s t test). We feel it is important to present both differentiations to provide a realistic picture of expected variability, which, unlike with immortalized cell lines, is an inherent aspect of hPSC differentiation. We also believe that this variability, as well as the small proportion of GLUT-1+ ECs elicited by Wnt3a treatment, motivates our subsequent use of CHIR.

The overall percentage of Glut-1 positive cells in Figure 1 and 2 is very low, making the comparison between the effectiveness of the different ligands while statistically significant compared to the control, most likely not significant compared between the ligands. Therefore the claims of e.g. Wnt7a being more effective is based on one of 3 samples of the same differentiation. All these comaparative claims will have to be either supported by much more data or taken out. The only clear result is that CHIR can stimulate expression of Glut-1 in around 90% of the cells, whereas the offered Wnt ligands affect 2-4%. To me this suggests that soluble WNT ligands in the culture medium are least effective in stimulating a response. It would have been most interesting to explore if co-cultures and hence the supply in e.g. Cytonems or other vesicle or membrane-bound delivery systems would make a huge difference. However this is not the scope of the paper.The analysis of mean fluorescence intensity of claudin-5, caveolin-1, and GLUT-1 normalized to Hoechst mean fluorescence intensity within the area of claudin-5+ ECs only is less informative, as one highly expressing cell will outweigh many low expressing cells and there is no published link of an endothelial cell resembling a BBB endothelial cells more closely in correlation with their Glut-1 expression intensity.

As discussed under “Essential Revisions” point 3, we have removed data on Wnt7a/b from the manuscript and instead focus now in Figure 2 on the effect of CHIR. Because CHIR induces GLUT-1 expression in ~90% of ECs, and images do not indicate substantial heterogeneity in claudin-5 or caveolin-1 immunofluorescence across ECs, we believe mean fluorescence intensity within EC colonies is a reasonable metric for comparison of the DMSO and CHIR conditions. We have added replicates from several additional differentiations to Figure 2 to accurately convey robustness of these effects. Moreover, the protein level increases are also quantified in Figure 4 and are concordant with those determined by quantitative image analysis.

Unfortunately, when analysing the effects of conditioned media in Figure3 only those relative quantifications were used and again the spread between the two different differentiation rounds was so high, that a comparative analysis is hindered and not represented by statistical analysis compared to control only. For example the Caveolin/Hoest values of the orange dataset were increased in the CHIR treatment, but unchanged in Control and NR-CM, but in contrast in the green dataset the NR-CM resulted in a higher index compared to CHIR or the control. Again were no statistical comparison was done between samples, the drawn conclusions can not indicate there are any.

We agree that the discrepancy between two differentiations in the conditioned medium-elicited changes in caveolin-1 (and claudin-5) fluorescence intensity motivates additional replication of these treatments. As discussed under “Essential Revisions” point 3, we have removed the conditioned medium data from this manuscript and will instead address this in future work.

Figure 5 Anal;yses the effect of passage number in combination with CHIR treatment. However the individual differentiation samples again behave partially opposite: the green sample Glut1/ß-actin is only weakle elevated in passage 1 and upregulated after passage3, the purple sample is high in passage 1 and becomes downregulated after passage 3.

The data shown in panels B and D are not from matched differentiations. We recognize that the color scheme may have implied this, and we have adjusted the colors accordingly. Thus, quantitative comparison of data in panels B and D is not possible. We have also added data from one additional differentiation to Figure 4D to better estimate the expected GLUT-1 induction at Passage 3.

Figure 7 the effects in the mature EC should be directly compared to the effects in more naïve from the same differentiation.

We thank the reviewer for suggesting this key experiment, which we have now performed and the results of which we have included as a revised Figure 6 (formerly Figure 7). In a direct comparison of GLUT-1 fluorescence intensity in ECs from the same differentiation, CHIR treatment initiated at the early timepoint (D5; “Passage 0”) led to a ~70-fold increase, while no statistically significant increase was observed when treatment was carried out at Passage 4. Similarly, CHIR-mediated increases in EC number and claudin-5 abundance were observed when treatment was carried out at D5, but not when treatment was carried out at Passage 4. These results strengthen the conclusion that EC responsiveness to Wnt activation decreases with extended culture.

From Results: “To test this hypothesis, we matured hPSC-derived ECs in vitro for 4 passages (until approximately day 30) prior to initiating CHIR treatment for 6 days, and compared the resulting cells to differentiation-matched samples treated with CHIR immediately after MACS (Figure 6A). Both Passage 1 DMSO-treated ECs and Passage 5 DMSO-treated ECs, which are analogous to EECM-BMEC-like cells we previously reported (Nishihara et al., 2020), did not have detectable GLUT-1 expression (Figure 6B). Compared to DMSO controls, the CHIR-treated Passage 5 ECs exhibited no increase in GLUT-1 abundance (Figure 6B-D), which contrasts with the marked increase observed when CHIR treatment was initiated immediately after MACS (Figure 6B-D). Furthermore, CHIR treatment in matured ECs did not increase claudin-5 expression and did not increase EC number (Figure 6B-D), in contrast to the increases observed in both properties when treatment was initiated immediately after MACS (Figure 6B-D).”

Reviewer #3 (Recommendations for the authors):

Although I have a positive opinion on the manuscript, I personally think that the manuscript could be slightly simplified (and shorten). Therefore, I have listed below several points that I think should be addressed (and possibly used to shorten the manuscript) by the authors before publication of the manuscript.

– Page 8 Line 173- 192 : "In the CNS, neural progenitors ….. ….. the Wnt ligand signal (analyzed further below)”.

This part and associated figure 3 compare the effects of neural rosette-conditioned medium and astrocyte-conditioned medium to the one of WNT7A and CHIR based on the fact that both cell types were suggested to express WNT7A (Vatine et al., 2016; Shang et al., 2018)..

Although the data are interesting per se., these data in my opinion, do not really have an added value to the main topic of the study (i.e deciphering the effects of wnt signaling in human endothelial cell progenitor with a focus on barrier formation).

In addition, the reported effects of neural rosette and astrocyte-conditioned media on endothelial progenitors were weaker than the one of CHIR which the authors suggest could be due to the potency or concentration of ligands in conditioned media. Indeed, there can be numerous hypothesis made here as there can be numerous secreted factors in those conditioned media.

Therefore, instead of including these data (i.e Figure 3) in this manuscript, I would rather suggest the authors to use it as the basis of a study on the contribition of wnt ligands in the effects of neural rosette and/or astrocyte-conditioned media-effects on ECs derived from hPSC. They could make use of their hPSC lines with doxycycline-inducible expression of short hairpin RNAs targeting CTNNB1 to confirm that neural rosette and/or astrocyte-conditioned media-effects in ECs (notably on GLUT-1, Claudin 5, caveolin-1) are β-catenin dependent.

In such experiment a comparison of the effects of wnt7A, neural rosette and astrocyte-conditioned media (+ brain pericyte conditioned medium or even coculture) on hPSC-derived endothelial progenitors (including from doxycycline-inducible expression of short hairpin RNAs targeting CTNNB1) would probably clarify the role of wnt ligand in the effects of neural rosette and astrocyte-conditioned media (or even coculture).

We thank the reviewer for the suggestion to simplify the manuscript and focus here on the effects of CHIR. We agree that the data on Wnt7 and conditioned media treatments would best serve as the basis for a future study where their ability to activate Wnt/β-catenin signaling, and the β-catenin dependence of observed responses, could be unambiguously confirmed. As discussed above under “Essential Revisions” point 3, we have therefore removed these data from the present manuscript, focus on the effects of CHIR in the Results, and mention future avenues for research involving Wnt ligands and conditioned media in the Discussion.

– Page 10-Line 217 (and associated Figure 6C): "We first confirmed that the process of dextran internalization required the membrane fluidity of an endocytosis-dependent process by carrying out the assay at 4C”

Although I get the idea of comparing fluorescent dextran internalization with or without CHIR treatment, I miss the point of the necessity of comparing the 4C and 37C. I assume 4C would not only affect membrane fluidity but also would reduce any active processes taking place in the cells as well as reduce thermal molecular agitation within the assay.

Therefore, I would suggest to remove this experiment at 4{degree sign}C which I find confusing and possibly replace it by experiment performed with inhibitors of the caveolin pathway (e.g Filipin, Genistein, Nystatin,.;)

While 4°C indeed affects membrane fluidity and thereby reduces/eliminates endocytosis processes, we better clarified the processes by which dextran is internalized by these cells as requested by the reviewer. We repeated the internalization assay in the presence of nystatin (inhibitor of caveolin-mediated endocytosis), chlorpromazine (inhibitor of clathrin-mediated endocytosis), or rottlerin (inhibitor of macropinocytosis). We also used confocal microscopy to visualize dextran and identify whether dextran colocalized with caveolin-1+ puncta. In general, these assays suggest that clathrin-mediated endocytosis and macropinocytosis are the predominant pathways of dextran internalization under the conditions of this assay, although a small number of dextran+ caveolin-1+ puncta were apparent. We have included these data in Figure 5—figure supplement 1, and have also moved the 4°C control to Figure 5—figure supplement 1 to simplify the main figure.

From Results: “We confirmed that the dextran signal measured by this assay was endocytosis-dependent by carrying out the assay at 4°C and with inhibitors of specific endocytic pathways (Figure 5—figure supplement 1A-C). Compared to vehicle control, chlorpromazine (inhibitor of clathrin-mediated endocytosis) and rottlerin (inhibitor of macropinocytosis) both decreased dextran uptake, while nystatin (inhibitor of caveolin-mediated endocytosis) did not significantly affect uptake (Figure 5—figure supplement 1B,C), consistent with the very small number of dextran+ caveolin-1+ puncta observed by confocal imaging (Figure 5—figure supplement 1D).”

– Page 21 – Line 483:

Although I agree with the statement that Praça et al., reported moderate improvement in barrier function using a combination of VEGF, Wnt3a, and retinoic acid, in my opinion TEER values should be used intra assay (e.g as in the manuscript to compare the effects of CHIR on TEER) not from one study to the next as several parameters could influence TEER (e.g Temperature, buffer capacity, surface of the insert,…).

We included the TEER value of ~60 Ω×cm2 reported by Praça et al., to draw attention to the similarity in order of magnitude to our results, and agree completely that TEER values obtained in two different assays/laboratories are not directly comparable. We have therefore reworded this sentence to clarify this point:

From Discussion: “For example, Praça et al., showed that a combination of VEGF, Wnt3a, and retinoic acid directed EPCs to brain capillary-like ECs with moderate transendothelial electrical resistance (TEER) similar in order of magnitude to that reported here.”

Reviewer #4 (Recommendations for the authors):

[…]

The presented data are solid and the manuscript is well written with high standard figures. However, some experimental details and interpretation of the data require the authors attention to augment the scientific merit of the manuscript:

It would be important that the authors clearly claim if the present manuscript describes a novel in vitro model or a basic stem cell biological finding.

Our primary goal was to provide insight into the cellular phenotype elicited by activation of Wnt/β-catenin signaling in naïve hPSC-derived endothelial cells, particularly as it relates to gain of CNS EC phenotype. As discussed above under “Essential Revisions” point 4, we have clarified this goal in the Introduction. We do not believe the two goals described by the reviewer are mutually exclusive, however, and we would suggest that others consider this hPSC-based definitive EC in vitro model system for future studies of CNS EC barriergenesis.

From Introduction: “In this work, we aimed to define the effects of activating Wnt/β-catenin signaling in hPSC-derived, naïve endothelial progenitors and assess the extent to which this strategy would drive development of a CNS EC-like phenotype.”

The following critical points are related to this first issue because the that

The authors show that the treatment with Wnt7a and in particular with Wnt7b only show little or even no effects on Wnt pathway activation (Figure 8—figure supplement 1 B; no AXIN2 induction by Wnt7a/7b!!) and induction of BBB characteristics. It would be important to further investigate this topic to clearly demonstrate that Wnt7a/7b indeed are functional as recombinant proteins in the described experimental setting. Wnt7 has been shown to be only little diffusible, requiring the direct contact of the sending cell to the receiving cells (Eubelen, M. et al., Science (New York, NY) 361, eaat1178, 2018). Therefore, the authors should elaborate on an experimental setting to stimulate the naïve endothelial progenitors with Wnt7a/7b in a paracrine manner. This would help to clarify the question regarding the discrepancy between the CHIR and the Wnt7a treatment.

Essentially, the same critique can be raised concerning the treatment with conditioned medium from neuroblast rosettes and astrocytes. Although both may express Wnt7a/7b, specifically these Wnts may not accumulate in the CM and hence, the effects may be minor.

We agree that the lack of upregulation of canonical transcriptional targets (e.g., AXIN2, LEF1) in the RNA-seq data of Wnt7a/b-treated ECs is strong evidence that these ligands are not effective in activating Wnt/β-catenin signaling in this system. As described under “Essential Revisions” point 3, we have thus removed these data from the paper. In the Discussion, we have also added suggestions of coculture-based strategies that may mitigate the poor solubility/diffusability of Wnt ligands:

From Discussion: “Given evidence that Wnt ligands have poor solubility (Janda et al., 2012) and our preliminary data suggesting that supplementation of culture medium with Wnt7a and Wnt7b is largely ineffective in activating Wnt/β-catenin signaling in this system, special emphasis should be placed on strategies that present Wnt7a, Wnt7b, and/or Norrin in a manner that concentrates ligands at the cell surface, for example, by using direct cocultures of endogenously Wnt-producing cells (neural progenitors or astrocytes) or Wnt-overexpressing cells.”

Page 16, line 359-363: The assumption made in the sentence “Upon recombination, the Ctnnb1 flex3 allele produces a dominant mutant β-catenin lacking residues that are phosphorylated by GSK-3β to target β-catenin for degradation (Harada et al., 1999); as such, this strategy for ligand- and receptor-independent Wnt activation by β-catenin stabilization is directly analogous to CHIR treatment." might be wrong, as blocking GSK3beta potentially has many other effects than “just" activating β-catenin-mediated transcription!! The authors should discuss this topic in more depth.

We have reworded the sentence to clarify this important difference between the two approaches, and added additional data analysis and discussion of this topic to both the Results and Discussion sections (as discussed above under “Essential Revisions” point 2).

From Results: “this strategy for ligand- and receptor-independent Wnt activation by β-catenin stabilization is similar to CHIR treatment, although GSK-3 phosphorylates targets other than β-catenin (discussed below).”

One of the highlight of the present manuscript is the inducibility of naïve endothelial progenitors by CHIR compared to differentiated endothelial cells. Hence it would be important to compare the expression pattern by RNA-Seq of these two conditions.

We agree that comparing the endothelial progenitors (day 5 cells) to matured ECs (Passage 4) would provide valuable insight into to their differential responsiveness to CHIR. Although we do not have RNA-seq data perfectly matched to these two time points, we can use our RNA-seq data of Passage 1 and Passage 3 control (DMSO-treated) ECs as a surrogate for this comparison. We found marked downregulation of LEF1 and TCF7 at Passage 3, and suggest this as a possible explanation for the attenuated CHIR-responsiveness of matured ECs. We have added these data to Figure 7—figure supplement 4 and to the Results section. We also added this observation to the Discussion section and clearly highlighted the imperfect nature of the comparison and the need for additional follow-up studies:

From Results: “We also evaluated transcript-level expression of components of the Wnt signaling pathway in Passage 3 control (DMSO-treated) ECs as a first step towards understanding the relative lack of responsiveness observed when CHIR treatment was initiated in matured (Passage 4) ECs (Figure 7—figure supplement 4). While CTNNB1, GSK3B, and genes encoding components of the destruction complex were not significantly different between Passage 3 and Passage 1, LEF1 and TCF7 were strongly downregulated in Passage 3 cells (Figure 7—figure supplement 4B).”

From Discussion: “In RNA-seq data of Passage 3 control (DMSO-treated) ECs, LEF1 and TCF7 were strongly downregulated compared to Passage 1 cells. This result suggests that low baseline expression of these transcription factors, which form a complex with nuclear β-catenin to regulate Wnt target genes, may partially explain the poor efficacy of CHIR in matured ECs, although additional work is necessary to assess the functional relevance of these differences.”

https://doi.org/10.7554/eLife.70992.sa2

Article and author information

Author details

  1. Benjamin D Gastfriend

    Department of Chemical and Biological Engineering, University of Wisconsin–Madison, Madison, United States
    Contribution
    Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft
    Competing interests
    Inventor on a provisional US patent application (63/185815) related to this work
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4677-1455
  2. Hideaki Nishihara

    Theodor Kocher Institute, University of Bern, Bern, Switzerland
    Contribution
    Conceptualization, Investigation, Methodology, Validation, Writing – review and editing
    Competing interests
    Inventor on a provisional US patent application (63/185815) related to this work
  3. Scott G Canfield

    Department of Chemical and Biological Engineering, University of Wisconsin–Madison, Madison, United States
    Present address
    Department of Anatomy, Cell Biology, and Physiology, Indiana University School of Medicine, Terre Haute, United States
    Contribution
    Conceptualization, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
  4. Koji L Foreman

    Department of Chemical and Biological Engineering, University of Wisconsin–Madison, Madison, United States
    Contribution
    Conceptualization, Investigation, Writing – review and editing
    Competing interests
    No competing interests declared
  5. Britta Engelhardt

    Theodor Kocher Institute, University of Bern, Bern, Switzerland
    Contribution
    Conceptualization, Funding acquisition, Supervision, Writing – review and editing
    Competing interests
    Inventor on a provisional US patent application (63/185815) related to this work
  6. Sean P Palecek

    Department of Chemical and Biological Engineering, University of Wisconsin–Madison, Madison, United States
    Contribution
    Conceptualization, Funding acquisition, Supervision, Writing – review and editing
    For correspondence
    sppalecek@wisc.edu
    Competing interests
    Inventor on a provisional US patent application (63/185815) related to this work
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4917-5584
  7. Eric V Shusta

    1. Department of Chemical and Biological Engineering, University of Wisconsin–Madison, Madison, United States
    2. Department of Neurological Surgery, University of Wisconsin–Madison, Madison, United States
    Contribution
    Conceptualization, Funding acquisition, Supervision, Writing – review and editing
    For correspondence
    eshusta@wisc.edu
    Competing interests
    Inventor on a provisional US patent application (63/185815) related to this work
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4297-0158

Funding

National Institutes of Health (R01 NS103844)

  • Sean P Palecek
  • Eric V Shusta

National Institutes of Health (R01 NS107461)

  • Sean P Palecek
  • Eric V Shusta

National Institutes of Health (T32 GM008349)

  • Benjamin D Gastfriend

National Science Foundation (1747503)

  • Benjamin D Gastfriend

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (310030_189080)

  • Britta Engelhardt

Bern Center for Precision Medicine

  • Britta Engelhardt

Japan Society for the Promotion of Science (Overseas Research Fellowship)

  • Hideaki Nishihara

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We acknowledge the University of Wisconsin–Madison Biotechnology Center Gene Expression Center and DNA Sequencing Facility for providing library preparation and next-generation sequencing services. We acknowledge the University of Wisconsin–Madison Biochemistry Optical Core for use of a confocal microscope. We acknowledge the University of Wisconsin Carbone Cancer Center Flow Cytometry Laboratory (supported by NIH Cancer Center Support Grant P30 CA014520) for use of a flow cytometer and for performing FACS. We thank Richard Daneman for advice related to this work.

Senior Editor

  1. Didier YR Stainier, Max Planck Institute for Heart and Lung Research, Germany

Reviewing Editor

  1. Stefan Liebner, University Hospital, Goethe University Frankfurt, Germany

Reviewer

  1. Maxime Culot, Artois University, France

Publication history

  1. Preprint posted: May 14, 2021 (view preprint)
  2. Received: June 4, 2021
  3. Accepted: November 9, 2021
  4. Accepted Manuscript published: November 10, 2021 (version 1)
  5. Accepted Manuscript updated: November 12, 2021 (version 2)
  6. Version of Record published: December 10, 2021 (version 3)

Copyright

© 2021, Gastfriend et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,520
    Page views
  • 211
    Downloads
  • 1
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Benjamin D Gastfriend
  2. Hideaki Nishihara
  3. Scott G Canfield
  4. Koji L Foreman
  5. Britta Engelhardt
  6. Sean P Palecek
  7. Eric V Shusta
(2021)
Wnt signaling mediates acquisition of blood–brain barrier properties in naïve endothelium derived from human pluripotent stem cells
eLife 10:e70992.
https://doi.org/10.7554/eLife.70992

Further reading

    1. Developmental Biology
    2. Neuroscience
    Eleni Chrysostomou et al.
    Research Article

    Neurogenesis is the generation of neurons from stem cells, a process that is regulated by SoxB transcription factors (TFs) in many animals. Although the roles of these TFs are well understood in bilaterians, how their neural function evolved is unclear. Here, we use Hydractinia symbiolongicarpus, a member of the early-branching phylum Cnidaria, to provide insight into this question. Using a combination of mRNA in situ hybridization, transgenesis, gene knockdown, transcriptomics, and in-vivo imaging, we provide a comprehensive molecular and cellular analysis of neurogenesis during embryogenesis, homeostasis, and regeneration in this animal. We show that SoxB genes act sequentially at least in some cases. Stem cells expressing Piwi1 and Soxb1, which have a broad developmental potential, become neural progenitors that express Soxb2 before differentiating into mature neural cells. Knockdown of SoxB genes resulted in complex defects in embryonic neurogenesis. Hydractinia neural cells differentiate while migrating from the aboral to the oral end of the animal, but it is unclear whether migration per se or exposure to different microenvironments is the main driver of their fate determination. Our data constitute a rich resource for studies aiming at addressing this question, which is at the heart of understanding the origin and development of animal nervous systems.

    1. Developmental Biology
    Noah P Mitchell et al.
    Research Article

    Organ architecture is often composed of multiple laminar tissues arranged in concentric layers. During morphogenesis, the initial geometry of visceral organs undergoes a sequence of folding, adopting a complex shape that is vital for function. Genetic signals are known to impact form, yet the dynamic and mechanical interplay of tissue layers giving rise to organs' complex shapes remains elusive. Here, we trace the dynamics and mechanical interactions of a developing visceral organ across tissue layers, from sub-cellular to organ scale in vivo. Combining deep tissue light-sheet microscopy for in toto live visualization with a novel computational framework for multilayer analysis of evolving complex shapes, we find a dynamic mechanism for organ folding using the embryonic midgut of Drosophila as a model visceral organ. Hox genes, known regulators of organ shape, control the emergence of high-frequency calcium pulses. Spatiotemporally patterned calciumpulses triggermuscle contractions via myosin light chain kinase. Muscle contractions, in turn, induce cell shape change in the adjacent tissue layer. This cell shape change collectively drives a convergent extension pattern. Through tissue incompressibility and initial organ geometry, this in-plane shape change is linked to out-of-plane organ folding. Our analysis follows tissue dynamics during organ shape change in vivo, tracing organ-scale folding to a high-frequency molecular mechanism. These findings offer a mechanical route for gene expression to induce organ shape change: genetic patterning in one layer triggers a physical process in the adjacent layer - revealing post-translational mechanisms that govern shape change.