Fig. 5 |. Expected outcomes of the protocol.

a, Bright-field image of foregut spheroids on the day of collection, cultured in a Matrigel droplet. Scale bar, 200 µm. b,c, The overwhelming majority of foregut spheroid cells are (b) NKX2.1⁺ and (c) SOX2⁺. Scale bars, 50 µm. d–j, Expected outcomes of the human lung organoid protocol. d, Bright-field images showing expected growth patterns for human lung organoids. Scale bars, 500 µm. e, Human lung organoids have persisting epithelial structures and surrounding mesenchyme, as shown by H&E staining at day 100 of culture. Scale bar, 500 µm. f, Epithelial structures contain P63⁺ basal-like cells and an organized airway-like epithelium. Scale bar, 50 µm. g, Surrounding mesenchyme is positive for smooth muscle marker smooth muscle actin (SMA), and epithelial structures contain P63⁺ cells on the basolateral surface, with FOXJ1⁺ cells on the luminal side of the epithelium. P63⁺ cells constituted, on average, roughly 40% of all cells within organoids. Though FOXJ1⁺ cells composed ~5% of the total cells in the HLOs, mature ACTUB structures were not seen at day 65 and were rare at 85 d (data not shown). Scale bar, 50 µm. h–j, Scale bars, 50 µm. h, Cells surrounding day-65 HLO ECAD⁺ epithelial structures were positive for multiple mesenchymal-cell-type markers, including PDGFRα and vimentin (VIM). i, Rare cells within HLOs express SOX9 and markers of the AECII marker surfactant protein C (SFTPC). These cells make up roughly 5% of all cells within an organoid. j, Rare cells within HLOs express SOX9 and the AECI marker HOPX. These cells make up roughly 5% of all cells within an organoid. k–m, Expected outcomes of the bud tip progenitor organoid protocol if organoids are maintained by regular needle passaging. k, Epithelial structures form and can be isolated by day 12 in culture. Scale bar, 500 µm. l, By day 14 of culture, over 95% of epithelial cells are NKX2.1⁺SOX2⁺. Scale bars, 50 µm. m, Needle-passaged bud tip progenitor organoids have a simple epithelial structure composed of SOX9⁺SOX2⁺ cells similar to that observed in the native human lung bud tip progenitors before week 16 of gestation. Scale bars, 50 µm. n–r, Expected outcomes of the bud tip progenitor organoid protocol if organoids are not maintained by needle passage and are instead allowed to grow intact into complex epithelial structures. n, Expected growth patterns over time of bud tip progenitor organoids. Epithelial structures predictably begin to fold at ~3 weeks, and form branch-like structures at 5–6 weeks. Scale bar, 200 µm; all images taken at the same magnification. o, Bright-field images over 5 d in culture, showing bifurcation of an epithelial bud structure. Bifurcated bud tips are marked with an asterisk. Scale bar, 200 µm; all images taken at the same magnification. p, Cartoon representation of the interior and bud tip regions of non-needle-passaged bud tip progenitor organoids. q, In the interior regions of these structures, roughly 5% of cells exhibit SCGB1A1 staining, and roughly 2% of cells stain positive for goblet cell marker MUC5AC. Scale bar, 50 µm. r, The bud tip regions of these structures maintain the expression of bud tip progenitor markers SOX9 and pro-surfactant protein C (SFTPC). Scale bars, 50 µm. Detailed information about antibodies used for staining can be found in Table 3. g–j, Reproduced with permission from ref. ¹⁵, eLife Sciences Publications (licensed under CC BY-NC-ND 4.0, https://creativecommons.org/licenses/by/4.0/). l–r, reproduced with permission from ref. ¹⁷, Elsevier (licensed under CC BY-NC-ND 4.0, https://creativecommons.org/licenses/by/4.0/).