1. Author Response:

    Reviewer #1 (Public Review):

    Gastfriend et al present an analysis of the properties and gene expression patterns elicited by pharmacologically activating the Wnt signaling pathway in human pluripotent stem cells (hPSCs) that have been cultured under conditions that favor their differentiation to naïve endothelial cell (EC) progenitors. The result of Wnt activation is partial induction of blood-brain barrier-like properties, as judged by immunostaining for several well characterized marker proteins, trans-epithelial resistance, and RNAseq. Most of the experiments, and the largest effects, were obtained with CHIR99021, a small molecular weight inhibitor of GSK3-beta, the kinase that phosphorylates beta-catenin, leading to its ubiquitinylation and proteosomal degradation. Interestingly, low-passage ("naïve") hPSC-derived ECs were more susceptible to CHIR99021-induced BBB-like conversion than higher passage ("mature") hPSC-derived ECs. The experimental data is of uniformly high quality.

    By way of context, several publications describe the use of hPSC or other starting cell sources for the generation of ECs with BBB-like properties, and several have described the BBB-enhancing effects of activating different signaling pathways (retinoic acid, TGF-beta, Wnt). The effect on BBB properties of manipulations that would be predicted to increase Wnt signaling varied among published studies - it was detectable in some studies (e.g. Paolinelli et al., 2013, Laksitorini et al., 2019) and it was undetectable in another (Sabbagh and Nathans, 2020). The present work adds to this literature and presents additional information on this attractive experimental system for dissecting Wnt signaling and potentially other signaling systems that affect the BBB-like differentiation of CNS ECs.

    One potential question raised by the present study is the interpretation of the response to CHIR99021. GSK3-beta has many substrates, not just beta-catenin. Could the bioactivity of CHIR99021 for the BBBlike conversion reflect a combination of beta-catenin stabilization and reduced phosphorylation of other GSK3-beta substrates?

    We thank the reviewer for this important comment and have addressed this possibility with expanded analysis of RNA-seq data and expanded discussion.

    A minor point on page 8, lines 180-185. It might be appropriate to note that neural-rosette-conditioned and astrocyte-conditioned media may contain factors in addition to Wnt7a.

    Because we have removed the neural rosette- and astrocyte-CM approaches from the Results section of the manuscript, we have instead included the following statement in the Discussion:

    “Importantly, neural progenitor cells and astrocytes likely would also contribute other yet-unidentified ligands important for acquisition of CNS EC phenotype.”

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  2. Reviewer #4 (Public Review):

    This work provides a new method to generate blood-brain barrier (BBB) endothelial cells (ECs) from human pluripotent stem cells (hPSCs), by activating the Wnt/β-catenin pathway at an early, susceptible time point when EC are still at a naïve state during differentiation. Although physiological inducers of Wnt/β-catenin signaling have been investigated, the work shows best performance for the small molecular GSK3 inhibitor CHIR99021 (CHIR) in the employed in vitro setting. Wnt/β-catenin hPSCs upregulated important BBB signature genes and promoted tightening of an EC monolayer. The experimental model could evolve into a more general technique typically used in BBB research, so it may allow rigorous testing of basic research questions at the BBB in vitro as well as compounds crossing the BBB for drug development.

    Strengths:

    The work deciphers in detail the schedule of EC differentiation from hPSCs and identified the correct time point for BBB-EC generation by Wnt/β-catenin activation. Hence, beside offering a tool for BBB and drug delivery research, the work opens the possibility to generate human BBB models from human pluripotent stem cells for personalized medicine.

    Weaknesses:

    Although the paper does have strengths in principle, the weaknesses of the paper are insufficient and in some case inappropriate analyses are performed to fully support the key claims in the manuscript by the data presented. In particular:

    It would be important to 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). The authors do not address in sufficient depth the cellular mechanisms of how naïve cells are susceptible for Wnt/β-catenin activation.

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  3. Evaluation Summary:

    The manuscript by Gastfriend et al. establishes a novel protocol for the differentiation of blood-brain barrier endothelial cells by activating Wnt/β-catenin signaling in human pluripotent stem cells at a critical stage of naïve endothelial progenitors. The characterization of naïve endothelial progenitors and the novel model has potential impact on basic research approaches as well as on the use of in vitro models in drug development and pharmacology. The strength of the study is the comprehensive analysis of the differentiated blood-brain barrier endothelial cells, whereas weaknesses are present in some experimental setups and data conclusion, requiring additional experimental support.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

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  4. Reviewer #1 (Public Review):

    Gastfriend et al present an analysis of the properties and gene expression patterns elicited by pharmacologically activating the Wnt signaling pathway in human pluripotent stem cells (hPSCs) that have been cultured under conditions that favor their differentiation to naïve endothelial cell (EC) progenitors. The result of Wnt activation is partial induction of blood-brain barrier-like properties, as judged by immunostaining for several well characterized marker proteins, trans-epithelial resistance, and RNAseq. Most of the experiments, and the largest effects, were obtained with CHIR99021, a small molecular weight inhibitor of GSK3-beta, the kinase that phosphorylates beta-catenin, leading to its ubiquitinylation and proteosomal degradation. Interestingly, low-passage ("naïve") hPSC-derived ECs were more susceptible to CHIR99021-induced BBB-like conversion than higher passage ("mature") hPSC-derived ECs. The experimental data is of uniformly high quality.

    By way of context, several publications describe the use of hPSC or other starting cell sources for the generation of ECs with BBB-like properties, and several have described the BBB-enhancing effects of activating different signaling pathways (retinoic acid, TGF-beta, Wnt). The effect on BBB properties of manipulations that would be predicted to increase Wnt signaling varied among published studies - it was detectable in some studies (e.g. Paolinelli et al., 2013, Laksitorini et al., 2019) and it was undetectable in another (Sabbagh and Nathans, 2020). The present work adds to this literature and presents additional information on this attractive experimental system for dissecting Wnt signaling and potentially other signaling systems that affect the BBB-like differentiation of CNS ECs.

    One potential question raised by the present study is the interpretation of the response to CHIR99021. GSK3-beta has many substrates, not just beta-catenin. Could the bioactivity of CHIR99021 for the BBB-like conversion reflect a combination of beta-catenin stabilization and reduced phosphorylation of other GSK3-beta substrates?

    A minor point on page 8, lines 180-185. It might be appropriate to note that neural-rosette-conditioned and astrocyte-conditioned media may contain factors in addition to Wnt7a.

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  5. Reviewer #2 (Public Review):

    The authors optimized the generation of endothelial progenitor cells from hPSCs and describe the effects of different Wnt-pathway activations on establishment of blood-brain barrier (BBB) characteristics.

    Specifically they show, that Wnt pathway activation by Wnt 7a/b, neural rosette medium or most strongly the GSK-3 inhibitor CHIR is capable of inducing CNS-like phenotypes measured by expression of glut-1, claudin5 and caveolin-1. They further analyzed the barrier phenotype in CHIR treated cells and show an increase in tight junction protein expression. They also analyzed the timing and found that naïve endothelial progenitors are more responsive to Wnt activation than more mature ECs.

    Using RNA sequencing for transcriptome analysis they show that many BBB EC-properties can be induced by Wnt signaling activation through CHIR, however other characteristic gene expressions are still lacking, indicating that other signaling pathways or stimuli are needed for developing an in vivo equivalent of BBB endothelial cells.

    The authors succeed in the analysis of the model represented, however the impact for the community is limited by the lack of novelty and the (expected and existing) variation between the different differentiation protocols.

    In general many of the claims (e.g. comparing between different effects of WNT ligands) are not supported by statistical analysis, as all conditions were only significant compared to the control.

    The use of the different quantification methods (eg, number of Glut-1 expressing cells versus Glut1/Hoechst intensity of expression) is not always optimally adapted to the question, and therefore the drawn conclusions have to be observed with caution.

    Overall this represents more a methods paper and will be valuable to the community as such.

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  6. Reviewer #3 (Public Review):

    Benjamin D. Gastfriend et al. used endothelial progenitors derived from human pluripotent stem cells to decipher the effects of activation of the canonical Wnt pathway in naïve endothelial cells. This is an interesting approach to better understand the role of Wnt pathway in endothelial barrier genesis.

    Strength: The experiments are well designed, and the findings are supported by data. A massive amount of data has been generated, notably using RNA sequencing and these data have been well exploited, notably by cross comparison with other datasets to compare the CHIR-regulated expression of genes in ECs to CNS-like ECs characteristics in other dataset. In addition, the dataset has been made available and could be used for further investigations in the field.

    Weakness: This is a data-rich manuscript which sometimes could be simplified to facilitate understanding by readers.

    Nevertheless, the conclusions of this paper are well supported by data and provide interesting insight in the contribution of Wnt pathway BBB formation.

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