Figure 2. Adult stem cell-derived lung organoids are propagatable models with both proximal and distal airway components.

(A) Schematic lists the various markers used here for qPCR and immunofluorescence to confirm the presence of all cell types in the 3D lung organoids here and in 2D monolayers later (in Figure 3). (B–H) Bar graphs display the relative abundance of various cell-type markers (normalized to 18S) in adult lung organoids (ALO), compared to the airway ( normal human bronchial epithelial cell [NHBE]) and/or alveolar (AT2) control cells, as appropriate. p-values were analyzed by one-way ANOVA. Error bars denote SEM; n = 3–6 datasets from three independent ALOs and representing early and late passages. See also Figure 2—figure supplement 2 for individual ALOs. (I, J). H&E-stained cell blocks were prepared using HistoGel (I). Slides were stained for the indicated markers and visualized by confocal immunofluorescence microscopy. Representative images are shown in (J). Scale bar = 50 µm. (K) 3D organoids grown in 8-well chamber slides were fixed, immunostained, and visualized by confocal microscopy as in (J). Scale bar = 50 µm. See also Figure 2—figure supplement 2. Top row (ACE2/KRT5-stained organoids) displays the single and merged panels as max projections of z-stacks (top) and a single optical section (bottom) of a selected area. For the remaining rows, the single (red/green) channel images are max projections of z-stacks; however, merged panels are optical sections to visualize the centers of the organoids. All immunofluorescence images showcased in this figure were obtained from ALO lines within passage #3–6. See also Figure 2—figure supplements 3–5 for additional evidence of mixed cellularity of ALO models, their similarity to lung tissue of origin, and stability of cellular composition during early (#1–8) and late (#8–15) passages, as determined by qPCR and flow cytometry.

Figure 2—figure supplement 1. Lung organoids are reproducibly established from three different donors and propagated in each case over 10 passages.

(A) Schematic displaying the key demographics of the patients who served as donors of the lung tissue as a source of adult stem cells for the generation of organoids. Three organoid lines were generated, ALO1-3. ALO, adult lung organoids. (B–D) Bright-field microscopy of organoids in 3D culture grown in different media/conditions (B), imaged serially over days (C), and at different passages (D). Scale bar = 100 µm. (E) Serial cuts of HistoGel-embedded organoids were analyzed by H&E staining. Scale bar = 50 µm.

Figure 2—figure supplement 2. Adult stem cell-derived lung organoids are propagatable models with both proximal and distal airway components.

(A) Schematic lists the various markers used here for qPCR and immunofluorescence to confirm the presence of all cell types in the 3D lung organoids here and in 2D monolayers later (in Figure 3). (B–H) Bar graphs display the relative abundance of various cell-type markers (normalized to 18S) in adult lung organoids (ALO), compared to the airway ( normal human bronchial epithelial cell [NHBE]) and/or alveolar (AT2) control cells, as appropriate. p-values were analyzed by one-way ANOVA. Error bars denote SEM; n = 3–6 datasets. (I) 3D organoids grown in 8-well chamber slides were fixed, immunostained, and visualized by confocal microscopy, as in Figure 2K. Scale bar = 50 µm.

Figure 2—figure supplement 3. Adult stem cell-derived lung organoids (ALO) generally recapitulate cell-type-specific gene expression patterns observed in the adult lung tissue (ALT) from which they originate.

(A, B) Schematics depict the study goal in this figure, that is, analysis of cell-type-specific transcripts in ALO vs. ALT. (C–L) Bar graphs display the relative abundance of various cell-type markers (normalized to 18S) in adult lung organoids from early passage (ALO), compared to the adult lung tissue (ALT) from which they were derived. p-values were analyzed by one-way ANOVA. Error bars denote SEM; n = 3–6 datasets. Statistically significant differences were not noted in any of the transcripts analyzed.

Figure 2—figure supplement 4. Adult stem cell-derived lung organoids (ALO) generally maintain their cellular composition from early (E) to late (L) passages, as determined by cell-type-specific gene expression by qPCR.

(A, B) Schematics depict the study goal in this figure, that is, analysis of cell-type-specific transcripts in early (E) vs. late (L) passages of ALO1-3 lines. (C–K) Bar graphs display the relative abundance of various cell-type markers (normalized to 18S) in adult lung organoids from either early (E) or late (L) passages of ALO lines 1–3. p-values were analyzed by one-way ANOVA. Error bars denote SEM; n = 3–6 datasets. Statistically significant differences were not noted in any of the transcripts analyzed.

Figure 2—figure supplement 5. Adult stem cell-derived lung organoids (ALO) comprised both proximal and distal airway epithelial population and generally maintain such diversity from early (E) to late (L) passages, as determined by FACS.

Lung monolayers were dissociated into single cells and analyzed using flow cytometry. Gating strategy depicted in (A), isotype controls in (B) and (C) show various lung cell types. Numbers denote %.Table in (D) lists marker-positive cell fractions in ALO1-3, presented either as averaged over both early and late passages combined (column 2), or separated into early (column 3) or late (column 4) passages. These findings are consistent with others’ findings by multichannel FACS (Bonser et al., 2021) showing that although many of these markers are highly expressed in a certain cell type, they are shared at lower levels among other cell types.