Differentiation of human intestinal organoids with endogenous vascular endothelial cells

Background

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.

 

 

References:

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

doi: https://doi.org/10.1242/prelights.20257

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