Recapitulation of lung development from human pluripotent stem cells (hPSCs) in three dimensions (3D) would allow deeper insight into human development, as well as the development of innovative strategies for disease modelling, drug discovery and regenerative medicine1. We report here the generation from hPSCs of lung bud organoids (LBOs) that contain mesoderm and pulmonary endoderm and develop into branching airway and early alveolar structures after xenotransplantation and in Matrigel 3D culture. Expression analysis and structural features indicated that the branching structures reached the second trimester of human gestation. Infection in vitro with respiratory syncytial virus, which causes small airway obstruction and bronchiolitis in infants2, led to swelling, detachment and shedding of infected cells into the organoid lumens, similar to what has been observed in human lungs3. Introduction of mutation in HPS1, which causes an early-onset form of intractable pulmonary fibrosis4, 5, led to accumulation of extracellular matrix and mesenchymal cells, suggesting the potential use of this model to recapitulate fibrotic lung disease in vitro. LBOs therefore recapitulate lung development and may provide a useful tool to model lung disease.
At a glance
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- Supplementary Figure 1: Characterization of lung bud organoids. (343 KB)
(a)Published 2D directed differentiation protocol for the generation of lung and airway epithelial cells1, 2. (b) Schematic overview of the protocol for generating and differentiating LBOs. (c) Unsupervised clustering of RNAseq data generated from EPCAM+ and EPCAM− cells isolated from d25 RUES2 LBOs (3 independent biological replicates). (d) Expression SHH and of its transcriptional targets, GLI1, PTCH and HHIP, of genes expressed in AFE, in lung and airway, and in other AFE-derived lineages in d25LBOs (extracted from the RNAseq data shown in Supplementary Fig. 1c; mean ± s.e.m.,n = 3 independent experiments in RUES2 ESCs).The source data can be found in Supplementary Table 4. (e) ISH for SHH in LBOs at d15, d20 and d25. Representative of 3 independent experiments, RUES2 ESCs. Scale bars 250 μm.
- Supplementary Figure 2: Potential of LBOs in vivo. (1,659 KB)
(a) Staining for human nuclei of RUES2 ESC LBO-derived growths 1.5 months after transplantation under the kidney capsule of NSG mice. Scale bars 500 μm. (b) Staining of LBO-derived growths 5 months after transplantation for murine CD31 (mCD31). Scale bars 50 μm. (c) Staining of LBO-derived growths 5 months after transplantation for SMA and EPCAM. Scale bars 500 μm. (d) Hematoxyline-eosine stain of LBO-derived growths showing ciliated cells 5 months after transplantation under the kidney capsule of NSG mice. Scale bars 25 μm. (e) Hematoxyline-eosine stain of LBO-derived growths showing submucosal glands 5 months after transplantation under the kidney capsule of NSG mice. Scale bars 100 μm. All panels used RUES2 ESCs, representative of 4 independent experiments.
- Supplementary Figure 3: Branching in iPSC and ESC-derived LBOs and mesoderm requirement for branching. (975 KB)
(a) Branching colonies in d70 cultures of LBOs derived from RUES2 and three different iPS lines plated at d25 in Matrigel 3D culture in the presence of CHIR99021, BMP4, FGF7, FGF10, and RA. Representative of >10 independent experiments. Scale bar 100 μm. (b) Branching colonies 90 days after plating RUES2 LBOs in Matrigel at 1 (top) or 4 LBOs (bottom) per 6.4 μm well. Scale bars 2.5 μm. All images are representative of >10 independent experiments. (c) Fraction of EPCAM− cells in LBOs (mean ± s.e.m.,n = 3 independent experiments, RUES2 ESCs). The source data can be found in Supplementary Table 4. (d) Colonies from single EPCAM+ and EPCAM− cells isolated from LBOs. Representative of 5 experiments. Scale bar 500 μm. (e) IF of colonies generated from single cells derived from LBOs in Matrigel 3D culture. Representative of 5 experiments. Scale bars 500 μm, 25 μM for SFTPB and SFTPC.
- Supplementary Figure 4: LBO maturation in Matrigel at d170. (1,524 KB)
(a) Morphology of d170 cultures of LBOs derived from three iPS lines plated at d25 in Matrigel in the presence of CHIR99021, BMP4, FGF7, FGF10, and RA. Representative of >10 independent experiments. Scale bar 250 μm. (b) Low-magnification tile scan immunofluorescence images after staining for indicated markers. Staining performed on serial sections of a d170 culture of LBOs derived from C12 iPS line plated at d25 in Matrigel in the presence of CHIR99021, BMP4, FGF7, FGF10, and RA. Representative of 4 independent experiments. Scale bars 1 μm. (c) Electron microscopy of d170 LBOs embedded in Matrigel at d25 in HDF mRNA iPSCs. Arrows indicate LBs. Representative of 3 independent experiments. Scale bar 1 μm. (d) Hematoxylin-Eosin stain (left) and expression of SOX2 and SOX9 in week 14 distal human fetal lung (HFL). Note tubes that co-express SOX2 and SOX9 (arrows). Representative of 3 independent experiments. Scale bar 250 μm. (e) Hierarchical clustering of the genome-wide expression profile in d170 LBOs with genome-wide expression profiles of 2nd trimester human organs and tissues from the KeyGenes database3.
- Supplementary Figure 5: Modeling of HPS-associate interstitial pneumonia (HPSIP). (760 KB)
(a) Schematic representation of the HPS1 gene, and location of sequence complementary to the gRNA (upper). Nucleotide sequence of wild type alleles in RUES2 and of both targeted alleles in RUES2-HPS1 cells in the region targeted by the gRNA (middle). Nucleotide and amino acid sequence of exons 15 and 16 of HPS1, showing deletions and premature stop codons in the targeted alleles of RUES2-HPS1 cells (lower). (b) Representative example (of two biological replicates each consisting of three technical replicates) of flow cytometric analysis of EPCAM+ and EPCAM− cells in d50 LBO-derived colonies in 3D Matrigel cultures of RUES2 and RUES2-HPS1 cells. (c) Tile scan images of immunofluorescence staining for EPCAM and PDGFRA of LBO-derived branching colonies in 3D Matrigel cultures generated from parental RUES2 cells and from RUES2-HPS1 cells. Representative of five independent experiments. Scale bars 1 μm. (d) Representative example of the expression of the proliferation antigen Ki67 in EPCAM+ and EPCAM− cells from d40 LBOs derived from parental RUES2 and mutant RUES2-HPS1 cells. Representative of three independent experiments. (e) Fraction of Ki67+ (proliferating) cells in EPCAM+ and EPCAM− cells from LBO in suspension (d15, d25) and in 3D Matrigel cultures (d40) of RUES2 and RUES2-HPS1 cells (mean ± s.e.m., n = 4 for d15 and d25, n = 3 for d40 independent experiments; ∗P < 0.0001 compared to RUES2; two-tailed Student’s t-test). The source data can be found in Supplementary Table 4. (f) Hydroxyproline content in LBO-derived colonies in 3D Matrigel cultures of RUES2 and RUES2-HPS1 cells (mean ± s.e.m.,n = 3 independent experiments; ∗P < 0.05; two-tailed Student’s t-test). The source data can be found in Supplementary Table 4. (g) Quantification of collagens I and III and fibronectin in 3D Matrigel cultures of RUES2 and RUES2-HPS1 cells using immunofluorescence intensity relative to DAPI (mean ± s.e.m., n = 3 independent experiments; ∗P < 0.05 for fibronectin, ∗P < 0.01 for collagens; two-tailed Student’s t-test after normalizing RUES2 controls to 1 in each experiment). The source data can be found in Supplementary Table 4. (h–j) Mixing experiments suggest that epithelial cells drive the accumulation of mesenchymal cells. It is still debated to what extent the pathogenetic origin IPF is epithelial, mesenchymal or both4, 5, 6, although most evidence points towards a prime role for epithelial injury, in particular ATII injury7. We devised a mixing strategy to address this issue without disrupting the LBO structures. When definitive endoderm cells derived from ZsGreen- or mCherry-expressing RUES2 cells were mixed at d4 of the protocol (see Supplementary Fig. 1b), after dissociation of embryoid bodies and before replating in 2D to generate anterior foregut cells, the resulting LBOs contained homogenously distributed red and green cells (h, left panel). However, when red and green LBOs were first generated separately and then grown together in suspension from d10 on, fusion between LBOs occurred such that large sections were either green or red (h, right panel). When ZsGreen+ RUES2 cells were mixed with non-fluorescent mutant RUES2-HPS1 cells at d4, the fraction of both wild type and mutant mesenchymal cells was increased (i, blue bars). As both genotypes were equally affected, these data indicate that a primary mesenchymal cause is unlikely. When LBOs were fused at d10, only sections of the resulting branching colonies were ZsGreen+ after plating in Matrigel, as expected (j). In these cultures, only accumulation of mutant mesenchymal cells could be demonstrated (i, orange bars). These data are consistent with the notion that fibrosis in HPS1 is driven by mutant epithelial cells7, 8, and suggest that the interaction between mutant epithelial cells and mesenchymal cells occurs over a short range. Mean ± s.e.m., n = 3 independent experiments; ∗P < 0.01 compared to parental RUES2 and to parental RUES2 mixed at d10 with RUES2-HPS1; one way ANOVA. The source data can be found in Supplementary Table 4.
- Video 4: Modeling respiratory syncytial virus infection in d170 LBOs. (2.89 MB, Download)
- Confocal microscopy movie of a distal bud of a d170 LBO (RUES2) showing infected cells in the lumen (RSV antigen, green; DAPI, blue). Scale bar 50 μm.
- Supplementary Information (127 MB)