Differentiation of human intestinal organoids with endogenous vascular endothelial cells

Emily M. Holloway, Joshua H. Wu, Michael Czerwinkski, Caden W. Sweet, Angeline Wu, Yu-Hwai Tsai, Sha Huang, Amy E. Stoddard, Meghan M. Capeling, Ian Glass, Jason R. Spence

Preprint posted on March 15, 2020

Differentiating cells find the spot: precise replication of organ-specific endothelial cells uncovered in human organoid culture.

Selected by Nozomu Takata


The creation of complex tissues in vitro is one of the current attractive themes in biology. However, most of the established directed differentiation cultures lack several cell types found in the native organ. Vascular endothelial cells (ECs) are such an example, which build the vasculature that is responsible for tissue communication and delivering nutrients and gas to the organs. There are several approaches that have pioneered the assembly of stem cell-derived tissues with or without exogenous ECs for in vitro platforms, for example, vascularized liver, brain and nephron organoids (Takebe et al., 2013) (Cakir et al., 2019; Czerniecki et al., 2018; Low et al., 2019).


3D human small intestinal organoids (HIOs) have led to a useful experimental platform to understand human intestinal development and physiology. However, HIOs have not yet fully recapitulated the complexity of the native human intestine because of the lack of cellular components such as the gut vasculature. ECs are known to possess organ-specific gene expression, morphology, and function. Previously, it has been shown that ECs in each organ have a distinct molecular signature based on data from single-cell RNA sequencing (Kalucka et al., 2020).  Although we now know the molecular identity of each organ-derived EC, the possibility of making the gut ECs in a human 3D culture platform hasn’t yet been explored.


Key findings

The main focus of this study was to understand how the EC progenitors in the gut become differentiated and distinct from other ECs. The authors here have tackled the challenge with a human gut organoid culture where they can trace embryonic cell lineages and molecular identities in a temporal manner.

One of the main findings is capturing a transient population of the gut ECs present via single-cell RNA sequencing when they are differentiating in culture. The number of the EC progenitors in the conventional method, however, is not abundant, based on the time-course analysis.  Therefore, they have explored the possibility of enhancing the co-differentiation of ECs within HIOs to push the number of the ECs up. They succeeded by timely modification of key growth factors known to help EC differentiation and maintenance. The improved culture gave them a rich EC population defined by a gene set expected to be present in regular ECs, including CDH5, KDR, FLT1, and ESAM.

Next, they took primary ECs from different human organs using the markers CD31+/CD144+ in flow cytometric analysis. Surprisingly, the ECs they co-differentiated were not just regular ECs. It turns out that gut-enriched ECs from the HIOs grown in vitro share the highest similarity with native intestinal ECs relative to the ones in kidney and lung. The authors also confirmed with multiplexed FISH that the gut ECs express gut EC transcripts but they don’t express detectable amount of lung and kidney signature genes. These evidence highlight the clear molecular identities in the gut ECs produced in vitro. The authors showed that taking advantage of primary human intestinal, lung, and kidney EC data sets would serve as a useful guideline for in vitro EC formation in organ differentiation.

Maximum intensity projection of a wholemount confocal z-series staining for the EC marker


What you like about this preprint/why you think the work is important; 

The group has previously proposed in vitro platforms of human gut culture (Spence et al., 2011). As a continuation of their previous research, they have sought to identify the key developmental time window and molecular pathways of the gut ECs, wisely using 3D culture and single-cell RNA sequencing. A strength of this paper is their ability to uncover a transient cell population using ways previously never explored. They have led us to think about how to approach such problems better, and provided us with useful molecular maps for the research in human ECs. In the future clinical setting, their findings would become essential to succeed in assembling ECs within the human body, including the nervous, circulatory, and internal organ systems.

Box-and-whiskers plot of individual data points (the organ-specific endothelial cell type scoring)


Future directions and questions for the authors;

A critical aspect of this paper is the formation of the human gut with endogenous endothelial cells in vitro. Although they demonstrated the molecular similarity, the question now becomes how similar the cellular and molecular processes of EC formation are in the gut during human development. It would be interesting to compare the stages, cell population, and morphology in vivo during  human development. Also, they have shown several key genes, including MEOX1, NKX2-3, FABP4. How important are those genes during gut EC formation? Could those genes have the power to transform progenitors or even other ECs into gut-specific ECs? When the candidate genes are deleted, will the gut ECs  loose their cell fate? If those problems are solved clearly, then what could be the main factor that drives EC identities in the gut. We hope to understand those remaining questions thoroughly, and one day we might be able to accurately control gut EC formation in disease and tissue repair.




Cakir, B., Xiang, Y., Tanaka, Y., Kural, M.H., Parent, M., Kang, Y.J., Chapeton, K., Patterson, B., Yuan, Y., He, C.S., et al. (2019). Engineering of human brain organoids with a functional vascular-like system. Nat Methods 16, 1169-1175.

Czerniecki, S.M., Cruz, N.M., Harder, J.L., Menon, R., Annis, J., Otto, E.A., Gulieva, R.E., Islas, L.V., Kim, Y.K., Tran, L.M., et al. (2018). High-Throughput Screening Enhances Kidney Organoid Differentiation from Human Pluripotent Stem Cells and Enables Automated Multidimensional Phenotyping. Cell Stem Cell 22, 929-940 e924.

Kalucka, J., de Rooij, L., Goveia, J., Rohlenova, K., Dumas, S.J., Meta, E., Conchinha, N.V., Taverna, F., Teuwen, L.A., Veys, K., et al. (2020). Single-Cell Transcriptome Atlas of Murine Endothelial Cells. Cell 180, 764-779 e720.

Low, J.H., Li, P., Chew, E.G.Y., Zhou, B., Suzuki, K., Zhang, T., Lian, M.M., Liu, M., Aizawa, E., Rodriguez Esteban, C., et al. (2019). Generation of Human PSC-Derived Kidney Organoids with Patterned Nephron Segments and a De Novo Vascular Network. Cell Stem Cell 25, 373-387 e379.

Spence, J.R., Mayhew, C.N., Rankin, S.A., Kuhar, M.F., Vallance, J.E., Tolle, K., Hoskins, E.E., Kalinichenko, V.V., Wells, S.I., Zorn, A.M., et al. (2011). Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470, 105-U120.

Takebe, T., Sekine, K., Enomura, M., Koike, H., Kimura, M., Ogaeri, T., Zhang, R.R., Ueno, Y., Zheng, Y.W., Koike, N., et al. (2013). Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 499, 481-484.

Tags: human intestinal organoids, single cell rna sequencing, the gut specific endothelial cells

Posted on: 8th May 2020 , updated on: 11th May 2020


Read preprint (No Ratings Yet)

  • Sign up to customise the site to your preferences and to receive alerts

    Register here

    Also in the developmental biology category:

    Planar Cell Polarity – PCP

    This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.


    List by Ana Dorrego-Rivas

    Cell Polarity

    Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.


    List by Yamini Ravichandran

    TAGC 2020

    Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20


    List by Maiko Kitaoka, Madhuja Samaddar, Miguel V. Almeida, Sejal Davla, Jennifer Ann Black, Gautam Dey

    3D Gastruloids

    A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells)


    List by Paul Gerald L. Sanchez and Stefano Vianello

    ASCB EMBO Annual Meeting 2019

    A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)


    List by Madhuja Samaddar, Ramona Jühlen, Amanda Haage, Laura McCormick, Maiko Kitaoka

    EDBC Alicante 2019

    Preprints presented at the European Developmental Biology Congress (EDBC) in Alicante, October 23-26 2019.


    List by Sergio Menchero, Jesus Victorino, Teresa Rayon, Irepan Salvador-Martinez

    EMBL Seeing is Believing – Imaging the Molecular Processes of Life

    Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019


    List by Gautam Dey

    SDB 78th Annual Meeting 2019

    A curation of the preprints presented at the SDB meeting in Boston, July 26-30 2019. The preList will be updated throughout the duration of the meeting.


    List by Alex Eve

    Lung Disease and Regeneration

    This preprint list compiles highlights from the field of lung biology.


    List by Rob Hynds

    Young Embryologist Network Conference 2019

    Preprints presented at the Young Embryologist Network 2019 conference, 13 May, The Francis Crick Institute, London


    List by Alex Eve

    Pattern formation during development

    The aim of this preList is to integrate results about the mechanisms that govern patterning during development, from genes implicated in the processes to theoritical models of pattern formation in nature.


    List by Alexa Sadier

    BSCB/BSDB Annual Meeting 2019

    Preprints presented at the BSCB/BSDB Annual Meeting 2019


    List by Gautam Dey

    Zebrafish immunology

    A compilation of cutting-edge research that uses the zebrafish as a model system to elucidate novel immunological mechanisms in health and disease.


    List by Shikha Nayar