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. 2020 Dec 12;12(9):717–733. doi: 10.1007/s13238-020-00811-w

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Generation of human airway and alveolar organoids from hESCs. (A) Schematic of differentiation protocol and stages from hESCs to human airway organoids (hAWOs) and human alveolar organoids (hALOs). (B) Representative images at the indicated differentiation stages. Scale bar, 500 μm. (C) Fold change of lineage marker genes from day 0 (D0) to D41 over undifferentiated hESCs by quantitative RT-PCR (2^−ΔΔCt). D0–D21, hLOs early stage. D21–D41, organoids split into two groups with different differentiated medium (hAWOs and hALOs). POU5F1, embryonic stem cell marker, SOX17, definitive endoderm marker, SOX2, embryonic stem cell and proximal airway cell marker, SOX9, distal alveolar progenitor cell marker, FOXA2 and NKX2.1, lung progenitor lineage marker, P63, basal cell marker, SCGB1A1 (CC10), club cell marker, MUC5AC, goblet cell marker, SPC, AT2 cell marker. Normalized to GAPDH. Bars represent mean ± SD, n = 3. (D–F) Cell lineage marker expression in human lung progenitor organoids (hLOs), human airway organoids (hAWOs), and human alveolar organoids (hALOs). Immunofluorescence images of NKX2.1 and E-Cadherin (epithelial cells) expression in D21 hLOs (D), of P63, SOX2, CC10, Ki67 (proliferation cells) and acetylated tubulin (ciliated cells), SOX9, MUC5AC, E-Cadherin protein expression in D35 hAWOs (E), and of SPC, AQP5 (AT1) and PDPN (AT1) expression in D35 hALOs (F). Nuclei were counterstained with DAPI. Scale bar, 100 μm (left panel); 20 μm (right panel). Boxes represent zoom views. (G) Fold change of ACE2 and TMPRSS2 gene expression from D0 to D41 over undifferentiated hESCs by quantitative RT-PCR (2^−ΔΔCt). Normalized to GAPDH. Bars represent mean ± SD, n = 3