A three-dimensional model of human lung development and disease from pluripotent stem cells

Journal name:
Nature Cell Biology
Volume:
19,
Pages:
542–549
Year published:
DOI:
doi:10.1038/ncb3510
Received
Accepted
Published online

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

Figures

  1. Generation of lung bud organoids.
    Figure 1: Generation of lung bud organoids.

    (a) Development of adherent structures during ventralization of AFE between day 6 and day 8 (see protocol Supplementary Fig. 1b), which could be expanded in suspension culture (day 10, day 20). Representative of >50 independent experiments (ESCs and iPSCs). Scale bars, 250μm. (b) Cellular expansion during the generation of LBOs (mean ± s.e.m., n = 3 independent experiments in RUES2 ESCs). The source data can be found in Supplementary Table 4. (c) Expression of EPCAM, KRT8, NKX2.1, FOXA1, and P63 in day 25 LBOs. Representative of >10 independent experiments in ESCs and iPSCs. Scale bars, 100μm. (d) Staining of day 25 LBO for ECAD and PDGFRA. Representative of 3 independent experiments in RUES2 ESCs. Scale bar, 250μm. (e) Expression of endodermal and mesodermal markers in the EPCAM+ and EPCAM fraction of day 25 LBOs determined by RNAseq (3 independent biological replicates, RUES2 ESCs).

  2. In vivo potential of LBOs.
    Figure 2: In vivo potential of LBOs.

    (a) Macroscopic aspect of growths 1.5 months after transplantation of 106 LBO cells embedded in Matrigel under the kidney capsule of NSG mice. Scale bar, 1cm. (b) Haematoxylin–eosin (HE) stain of LBO-derived growth 1.5 months after transplantation. Scale bar, 500μm. (c) Immunofluorescence for indicated markers in LBO-derived growths 1.5 months after transplantation. Scale bars, 100μm. (d) HE staining of LBO-derived growths 5 months after transplantation. Scale bars, 250μm. (e) Immunofluorescence for indicated markers in LBO-derived growths 5 months after transplantation. Scale bars, 250μm. (f) Dot blots for proteins marked on the left in aspirates from tubules in LBO-derived growths 5 months after transplantation. (g) HE staining and immunofluorescence for indicated markers in LBO-derived growths 7 months after transplantation. Scale bars, 100μm. All panels used RUES2 ESCs, representative of 4 independent experiments.

  3. LBO differentiation in Matrigel at day 70.
    Figure 3: LBO differentiation in Matrigel at day 70.

    (a) Bright-field images of the development of an LBO into a branching structure after plating in Matrigel. RUES2 ESCs. Arrows indicate branching distal tip. Representative of >50 independent experiments. Scale bars, 500μm. (b) Immunofluorescence staining for indicated markers in day 70 RUES2-derived LBOs plated in Matrigel at day 25. Representative of 4 independent experiments. Scale bars, 250μm.

  4. Long-term development of LBOs in vitro.
    Figure 4: Long-term development of LBOs in vitro.

    (a) Macroscopic appearance of day 170 RUES2 LBOs embedded in Matrigel at day 25. Representative of >50 independent experiments. Scale bar, 5mm. (b) Bright-field images of day 170 RUES2 and C12 LBOs embedded in Matrigel at day 25. Representative of >50 independent experiments. Scale bars, 500μm. (c) Immunofluorescence for indicated markers in day 170 RUES2 LBOs embedded in Matrigel at day 25. Representative of 3 independent experiments. Scale bars for MUC1 + SFTPB and HT2-280, 100μm. Scale bar for SFTPC, 10μm. (d) Electron microscopy of day 170 LBOs embedded in Matrigel at day 25 in RUES2 ESCs and HDF SV iPSCs. Arrows indicate LBs. Representative of 3 independent experiments. (e) Uptake of SFTPB-BODIPY (green) in day 170 LBOs embedded in Matrigel at day 25. Representative of 4 independent experiments. Scale bars, 100μm. (f) Time-course of uptake of SFTPB-BODIPY in day 170 LBOs embedded in Matrigel at day 25 (mean ± s.e.m., n = 4 independent experiments in RUES2 ESCs). The source data can be found in Supplementary Table 4. (g) Comparison of genome-wide expression in day 170 LBOs derived from hESCs and hiPSCs (12 biologically independent samples) with the KeyGenes database, showing the best match with second trimester human lung.

  5. Potential application of LBOs in modelling human diseases.
    Figure 5: Potential application of LBOs in modelling human diseases.

    (a) Confocal images of whole mount day 170 LBOs 1 and 2 days after infection with RSV and stained using anti-RSV (all antigens) antibody. Arrows: infected cells in the lumen. Representative of 3 independent experiments. Scale bars, 100μm. (b) Bright-field images of day 50 LBO-derived Matrigel colonies from RUES2 and RUES2-HPS1 cells. Representative of six independent experiments. Scale bars, 500μm. (c) Fraction of EPCAM+ and EPCAM cells in day 50 LBO-derived colonies in 3D Matrigel cultures of RUES2 and RUES2-HPS1 cells. (n = 6, mean ± s.e.m. of 3 technical replicates from two experiments; P < 0.0001; two-tailed Students t-test). The source data can be found in Supplementary Table 4. (d) Immunofluorescence staining for mesenchymal markers and ECM components in 3D Matrigel cultures of RUES2 and RUES2-HPS1 cells. Representative of 3 independent experiments. Scale bars, 500μm.

  6. Characterization of lung bud organoids.
    Supplementary Fig. 1: Characterization of lung bud organoids.

    (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 250μm.

  7. Potential of LBOs in vivo.
    Supplementary Fig. 2: Potential of LBOs in vivo.

    (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 500μm. (b) Staining of LBO-derived growths 5 months after transplantation for murine CD31 (mCD31). Scale bars 50μm. (c) Staining of LBO-derived growths 5months after transplantation for SMA and EPCAM. Scale bars 500μm. (d) Hematoxyline-eosine stain of LBO-derived growths showing ciliated cells 5 months after transplantation under the kidney capsule of NSG mice. Scale bars 25μm. (e) Hematoxyline-eosine stain of LBO-derived growths showing submucosal glands 5 months after transplantation under the kidney capsule of NSG mice. Scale bars 100μm. All panels used RUES2 ESCs, representative of 4 independent experiments.

  8. Branching in iPSC and ESC-derived LBOs and mesoderm requirement for branching.
    Supplementary Fig. 3: Branching in iPSC and ESC-derived LBOs and mesoderm requirement for branching.

    (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 100μm. (b) Branching colonies 90 days after plating RUES2 LBOs in Matrigel at 1 (top) or 4 LBOs (bottom) per 6.4μm well. Scale bars 2.5μm. 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 500μm. (e) IF of colonies generated from single cells derived from LBOs in Matrigel 3D culture. Representative of 5 experiments. Scale bars 500μm, 25μM for SFTPB and SFTPC.

  9. LBO maturation in Matrigel at d170.
    Supplementary Fig. 4: LBO maturation in Matrigel at d170.

    (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 250μm. (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 1μm. (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 1μm. (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 250μm. (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.

  10. Modeling of HPS-associate interstitial pneumonia (HPSIP).
    Supplementary Fig. 5: Modeling of HPS-associate interstitial pneumonia (HPSIP).

    (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 1μm. (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 Students 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 Students 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 Students t-test after normalizing RUES2 controls to 1 in each experiment). The source data can be found in Supplementary Table 4. (hj) 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.

Videos

  1. Beating cilia in the LBO-derived growth.
    Video 1: Beating cilia in the LBO-derived growth.
    Bright field movie showing beating cilia in LBO-derived growth (RUES2) 5 months after engraftment. Section was made using a vibratome. Scale bar 75μm.
  2. Morphology of d170 LBOs.
    Video 2: Morphology of d170 LBOs.
    Bright field movie showing the connected dilated saccules and structures resembling pulmonary acini in C12 LBOs. Scale bar 100μm.
  3. Uptake of SPB-BODIPY in d170 LBOs.
    Video 3: Uptake of SPB-BODIPY in d170 LBOs.
    Time lapse movie of several distal buds of different d170 LBOs (RUES2) taken at 2min intervals. The movie shows uptake of SPB-BODIPY over time in the cells and secretion into the lumen. Green: SPB-BODIPY. Scale bar 50μm.
  4. Modeling respiratory syncytial virus infection in d170 LBOs.
    Video 4: Modeling respiratory syncytial virus infection in d170 LBOs.
    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 50μm.

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Author information

Affiliations

  1. Columbia Center for Human Development, Columbia University Medical Center, New York, New York 10032, USA

    • Ya-Wen Chen,
    • Sarah Xuelian Huang,
    • Ana Luisa Rodrigues Toste de Carvalho &
    • Hans-Willem Snoeck
  2. Columbia Center for Translational Immunology, Columbia University Medical Center, New York, New York 10032, USA

    • Ya-Wen Chen,
    • Sarah Xuelian Huang,
    • Ana Luisa Rodrigues Toste de Carvalho,
    • Siu-Hong Ho &
    • Hans-Willem Snoeck
  3. Department of Medicine, Columbia University Medical Center, New York, New York 10032, USA

    • Ya-Wen Chen,
    • Sarah Xuelian Huang,
    • Ana Luisa Rodrigues Toste de Carvalho,
    • Siu-Hong Ho,
    • Mohammad Naimul Islam,
    • Jahar Bhattacharya &
    • Hans-Willem Snoeck
  4. Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, 4710-057 Braga, Portugal

    • Ana Luisa Rodrigues Toste de Carvalho
  5. ICVS/3Bs, PT Government Associate Laboratory, 4710-057 Braga/Guimarães, Portugal

    • Ana Luisa Rodrigues Toste de Carvalho
  6. Division of Immunology and Manton Center for Orphan Disease Research, Childrens Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Stefano Volpi &
    • Luigi D. Notarangelo
  7. U.O. Pediatria 2, Istituto Giannina Gaslini, Genoa 16148, Italy

    • Stefano Volpi
  8. St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, New York 10065, USA

    • Michael Ciancanelli &
    • Jean-Laurent Casanova
  9. Department of Physiology & Cellular Biophysics, Columbia University Medical Center, New York, New York 10032, USA

    • Jahar Bhattacharya &
    • Anne Moscona
  10. OCS Microscopy Core, New York University Langone Medical Center, New York, New York 10016, USA

    • Alice F. Liang
  11. Department of Pediatrics, Columbia University Medical Center, New York, New York 10032, USA

    • Laura M. Palermo,
    • Matteo Porotto &
    • Anne Moscona
  12. Center for Host-Pathogen Interaction, Columbia University Medical Center, New York, New York 10032, USA

    • Laura M. Palermo,
    • Matteo Porotto &
    • Anne Moscona
  13. Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York 10032, USA

    • Anne Moscona &
    • Hans-Willem Snoeck

Contributions

Y.-W.C. designed and performed most experiments, contributed to the concept, and co-wrote the manuscript with H.-W.S. S.X.H. and A.L.R.T.d.C. assisted Y.-W.C. A.F.L. performed transmission electron microscopy. S.-H.H. provided assistance with flow cytometry. J.-L.C. and M.C. provided patient material. L.D.N. and S.V. generated the C12 IRF7-deficient iPS line. M.N.I. and J.B. provided SFTPB-BODIPY. A.M., M.P. and L.M.P. generated and provided virology reagents, and provided design and instruction for experiments involving RSV. H.-W.S. provided concept and guidance, and co-wrote with Y.-W.C.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

Supplementary Figures

  1. 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 250μm.

  2. 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 500μm. (b) Staining of LBO-derived growths 5 months after transplantation for murine CD31 (mCD31). Scale bars 50μm. (c) Staining of LBO-derived growths 5months after transplantation for SMA and EPCAM. Scale bars 500μm. (d) Hematoxyline-eosine stain of LBO-derived growths showing ciliated cells 5 months after transplantation under the kidney capsule of NSG mice. Scale bars 25μm. (e) Hematoxyline-eosine stain of LBO-derived growths showing submucosal glands 5 months after transplantation under the kidney capsule of NSG mice. Scale bars 100μm. All panels used RUES2 ESCs, representative of 4 independent experiments.

  3. 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 100μm. (b) Branching colonies 90 days after plating RUES2 LBOs in Matrigel at 1 (top) or 4 LBOs (bottom) per 6.4μm well. Scale bars 2.5μm. 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 500μm. (e) IF of colonies generated from single cells derived from LBOs in Matrigel 3D culture. Representative of 5 experiments. Scale bars 500μm, 25μM for SFTPB and SFTPC.

  4. 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 250μm. (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 1μm. (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 1μm. (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 250μm. (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.

  5. 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 1μm. (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 Students 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 Students 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 Students t-test after normalizing RUES2 controls to 1 in each experiment). The source data can be found in Supplementary Table 4. (hj) 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

  1. Video 1: Beating cilia in the LBO-derived growth. (28.59 MB, Download)
    Bright field movie showing beating cilia in LBO-derived growth (RUES2) 5 months after engraftment. Section was made using a vibratome. Scale bar 75μm.
  2. Video 2: Morphology of d170 LBOs. (45.83 MB, Download)
    Bright field movie showing the connected dilated saccules and structures resembling pulmonary acini in C12 LBOs. Scale bar 100μm.
  3. Video 3: Uptake of SPB-BODIPY in d170 LBOs. (17.62 MB, Download)
    Time lapse movie of several distal buds of different d170 LBOs (RUES2) taken at 2min intervals. The movie shows uptake of SPB-BODIPY over time in the cells and secretion into the lumen. Green: SPB-BODIPY. Scale bar 50μm.
  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 50μm.

PDF files

  1. Supplementary Information (127 MB)

    Supplementary Information

Excel files

  1. Supplementary Table 1 (13 KB)

    Supplementary Information

  2. Supplementary Table 2 (10 KB)

    Supplementary Information

  3. Supplementary Table 3 (13 KB)

    Supplementary Information

  4. Supplementary Table 4 (634 KB)

    Supplementary Information

Additional data