Mitrofanova, O. | bioRxiv Science

bioRxiv2023

An integrated transcriptomic cell atlas of human endoderm-derived organoids

Human stem cells can generate complex, multicellular epithelial tissues of endodermal origin in vitro that recapitulate aspects of developing and adult human physiology. These tissues, also called organoids, can be derived from pluripotent stem cells or tissue-resident fetal and adult stem cells. However, it has remained difficult to understand the precision and accuracy of organoid cell states through comparison with primary counterparts, and to comprehensively assess the similarity and differences between organoid protocols. Advances in computational single-cell biology now allow the integration of datasets with high technical variability. Here, we integrate single-cell transcriptomes from 218 samples covering organoids of diverse endoderm-derived tissues including lung, pancreas, intestine, liver, biliary system, stomach, and prostate to establish an initial version of a human endoderm organoid cell atlas (HEOCA). The integration includes nearly one million cells across diverse conditions, data sources and protocols. We align and compare cell types and states between organoid models, and harmonize cell type annotations by mapping the atlas to primary tissue counterparts. To demonstrate utility of the atlas, we focus on intestine and lung, and clarify ontogenic cell states that can be modeled in vitro. We further provide examples of mapping novel data from new organoid protocols to expand the atlas, and showcase how integrating organoid models of disease into the HEOCA identifies altered cell proportions and states between healthy and disease conditions. The atlas makes diverse datasets centrally available, and will be valuable to assess organoid fidelity, characterize perturbed and diseased states, and streamline protocol development.

cell biology↗

bioRxiv2023

Bioengineered human colon organoids with in vivo-like complexity and function

Organoids and microphysiological systems, such as organs-on-a-chip, have emerged as powerful tools for modeling human gut physiology and disease in vitro. However, although physiologically relevant, these systems often lack the environmental milieu, spatial organization, cell-type diversity, and maturity necessary for mimicking adult human intestinal mucosa. To instead generate models closely resembling the in vivo cell-type composition and spatial compartmentalization, we herein integrated organoid and organ-on-a-chip technology to develop a primary human stem-cell-derived organoid model, called mini-colons. The luminal access and flow in human mini-colons removes shed cells to greatly enhance tissue longevity and differentiation over physically inaccessible human intestinal organoids that accumulate trapped cellular debris and waste. By establishing a gradient of growth factors, we replicated and sustained in vivo-like cell fate patterning and concurrent differentiation to secretory cell types and colonocytes. These long-lived human mini-colons contain abundant mucus-producing Goblet cells that lubricate the colonic epithelial lining. The stem and proliferative progenitor cells are also realistically confined to the crypts, facilitating stable homeostatic tissue turnover and preserving tissue integrity for several weeks. Also signifying mini-colon in vivo-like maturation, single-cell RNA sequencing showed emerging mature colonocytes and absorptive BEST4+ colonocytes. This methodology could be expanded to generate microtissues derived from the small intestine and incorporate additional microenvironmental components, thus emulating the intricate complexity of the native gut in an in vitro setting. Our bioengineered human organoids provide a highly accurate, long-lived, functional platform to systematically study human gut physiology and pathology, and for the development of novel therapeutic strategies.

bioengineering↗