Human embryonic lung epithelial tips are multipotent progenitors that can be expanded in vitro as long-term self-renewing organoids

Abstract

The embryonic mouse lung is a widely used substitute for human lung development. For example, attempts to differentiate human pluripotent stem cells to lung epithelium rely on passing through progenitor states that have only been described in mouse. The tip epithelium of the branching mouse lung is a multipotent progenitor pool that self-renews and produces differentiating descendants. We hypothesized that the human distal tip epithelium is an analogous progenitor population and tested this by examining morphology, gene expression and in vitro self-renewal and differentiation capacity of human tips. These experiments confirm that human and mouse tips are analogous and identify signalling pathways that are sufficient for long-term self-renewal of human tips as differentiation-competent organoids. Moreover, we identify mouse-human differences, including markers that define progenitor states and signalling requirements for long-term self-renewal. Our organoid system provides a genetically-tractable tool that will allow these human-specific features of lung development to be investigated.

Keywords: alveolar differentiation; branching; developmental biology; human; human biology; lung development; medicine; stem cell; stem cells; tip progenitor.

Figure 1.. Evolution of alveolar and bronchiolar marker gene expression during human embryonic lung development.

Sections of human embryonic and adult lungs. (A, B) 11 and 20 pcw. Green: HTII-280; red: SOX9 (tips); white: ECAD (epithelial cells). Arrow = HTII-280 positive terminal airway. (C) Adult. Green: HTII-280 (AT2 cells); red: pro-SFTPC (AT2 cells). Arrow = selected AT2 cells. (D–F) 11, 17 and 20 pcw. Green: HTII-280; red: pro-SFTPC. (G–H) 11 and 20 pcw. Green: HTII-280; red: HOPX; white: SOX2 (bronchiolar cells). Dotted bracket = terminal airway cells co-expressing SOX2 and HTII-280. Arrowheads = differentiating AT1 cells. (I, J) 11 and 20 pcw. Green: HTI-56; red: HOPX. K. Adult. Red: HOPX (AT1 cells). Arrowheads = HOPX⁺ nuclei. (L) 7 pcw. Green: TP63; red: SOX2. Arrowheads = TP63/SOX2 dual-positive cells in the more proximal airway region. Boxed region is shown as an inset in L’ with channels separated. (M) Adult. Red: TP63 (basal cells). Arrowheads = TP63⁺ cells in an intra-lobar bronchiole. (N) Adult. Green: FOXJ1 (ciliated cells); red: SCGB1A1 (secretory cells). (O, P) 11 pcw proximal and distal airway from the same lung. Green: TP63; red: SOX2. Arrowheads = TP63/SOX2 dual-positive cells in the more distal airway. (Q–S) 16 pcw proximal and distal airway from the same individual. Green: KRT5; red: TP63. Arrows = developing sub-mucosal glands. Arrowheads = KRT5⁺, TP63^- cells. Dashed lines = patches of KRT5⁺, TP63⁺ cells. (T) 16 pcw proximal airway. Green: MUC5AC. Arrows = developing sub-mucosal gland. Arrowheads = mucous cells. Blue: DAPI (nuclei). Bars = 100 μm (A,B,D,E,F,G,H,I,J,Q.R); 50 μm (B’,C, F’,H’,H’’,J’,K,L,L’,M,N,O,O’,P,P’,S,T); 25 μm (insets in C,F’,I,L’). DOI: http://dx.doi.org/10.7554/eLife.26575.003

Figure 1—figure supplement 1.. Representative morphology of human embryonic lung samples.

Wholemount images of human embryonic lungs. (A) CS15 (~5 pcw). (B) 6 pcw. (C) 8 pcw. (D) 10 pcw. (E) 16 pcw. Bars = 2.5 mm (A, B); 5 mm (C, D); 3 cm (E). DOI: http://dx.doi.org/10.7554/eLife.26575.004

Figure 1—figure supplement 2.. The evolution of alveolar marker gene expression in the human embryonic lung.

Sections of human embryonic and adult lungs. (A) 14 and 17 pcw. Green: HTII; red: SOX9 (tips); white: ECAD (epithelial cells). (B) 14 and 17 pcw. Green: HTII-280; red: HOPX; white: SOX2 (bronchiolar cells). (C) 20 pcw Green: HTII-280; red: HOPX; white: SOX2 (bronchiolar cells). Dotted line indicates SOX2⁺ cells that do not express the alveolar markers. Arrow heads = triple-positive (SOX2⁺, HTII-280⁺, HOPX⁺) cells. Green/white and red/white channels also shown separately for clarity. (D) 11, 14 and 17 pcw. Green: HTI-56; red: SOX2. (E) 16 pcw. Green: LPCAT1; red: SOX2. (F) Adult. Green: LPCAT1 (AT2 cells); red: ECAD (epithelial cells). (F’) Adult. Green: ABCA3 (AT2 cells); red: ECAD (epithelial cells). Blue: DAPI (nuclei). Bars = 50 μm all panels. DOI: http://dx.doi.org/10.7554/eLife.26575.005

Figure 1—figure supplement 3.. Epithelial PDPN expression is not specific to AT1 cells during human lung development.

Sections of human and adult embryonic lungs. (A, A’) nine pcw. Green: NKX2-1; red: PDPN. Arrowheads = low levels of PDPN in the developing airways. Arrows = PDPN in lymphatic endothelial cells. (B) 14 pcw. Green: ECAD (epithelial cells); red: PDPN. Arrowheads = PDPN is stronger in the more distal epithelium than the developing airways (arrow). (C) 17 pcw. Green: SOX9 (epithelial tips); red: PDPN; white: ECAD (epithelial cells). Low levels of PDPN can be seen in the tips, likely due to the angle of the section. (D) 17 pcw. Green: pro-SFTPC; red: PDPN; white: HTII-280. Arrowheads = PDPN and HTII-280 are largely co-expressed in cells that have already exited the tips. (E) 20 pcw. Green: SOX9 (epithelial tips); red: PDPN. Arrowheads = PDPN is observed in cells that have exited the epithelial tips. (F) 17 pcw. Green: pro-SFTPC; red: PDPN; white: HTII-280. The majority of cells lining the developing alveolar sacs are PDPN⁺, HTII-280⁺ dual-positive, although levels of each protein vary (seen clearly in F’ and F’’). (G) 20 pcw. Green: NKX2-1; red: PDPN. Epithelial cells lining the alveolar sacs are mostly PDPN⁺, low levels of PDPN are still observed in the developing bronchioles (arrow). (H, H’) 20 pcw. Green: AQP5; red: PDPN. Line = rows of continuous epithelial cells lining parts of the developing alveolar sacs are AQP5⁺, PDPN⁺ dual-positive. Arrows = distinct, individual, cells are AQP5⁺, PDPN⁺ dual-positive and express a higher level of AQP5 (likely developing AT1 cells). Arrowhead = individual cells that are AQP5^-, PDPN^- dual-negative (likely developing AT2 cells) can also be distinguished. (I) Adult lung. Green: pro-SFTPC; red: PDPN. Arrowheads = adult AT2 cells are pro-SFTPC⁺ and PDPN^-. All panels: blue = DAPI (nuclei). Bars = 200 μm A; 50 μm B, C, D, E, F, F’, F’’, G, H, H’’, I; 25 μm inset in D. DOI: http://dx.doi.org/10.7554/eLife.26575.006

Figure 1—figure supplement 4.. Epithelial AQP5 expression is not specific to AT1 cells during human lung development.

Sections of human and adult embryonic lungs. (A–D) 11, 14, 15 and 20 pcw. Green: AQP5; red: ECAD (epithelial cells). Patches of diffuse, apical AQP5 staining can be observed from 11 pcw. Arrowheads = elongated cells with more intense AQP5 staining observed from 20 pcw. However, AQP5 expression is widespread and also observed in cells with a more columnar epithelial appearance (arrows). (E) 20 pcw. Green: SOX9 (epithelial tips); red: AQP5; white: ECAD (epithelium). AQP5 extends to the epithelial tips. (F) Adult alveolar region. Red: AQP5; white ECAD (epithelial cells). Arrowheads = AT1 staining. (G) Adult terminal bronchiole. Green: TP63 (basal cells); red: AQP5; white: ACT (ciliated cells). Arrowheads = AQP5⁺ secretory cells. All panels: blue = DAPI (nuclei). Bars = 50 μm all panels. DOI: http://dx.doi.org/10.7554/eLife.26575.007

Figure 1—figure supplement 5.. NKX2-1 and FOXA2 are expressed in all human lung epithelial cells up to 20 pcw.

Sections of human embryonic lungs. (A–D). 15, 17, 20 and 21 pcw. Green: NKX2-1; red: FOXA2; white: HTII-280. Arrowheads in D’ = NKX2-1^- cells apparently lining the developing alveolar sacs. E-G. 20 pcw. (E) Green: NKX2-1; red: PDPN. Arrowheads = apparently PDPN⁺, NKX2-1^- cells lining the developing alveolar sacs. (F) Green: NKX2-1; red: PDPN; white: ECAD (epithelial cells). Arrowheads = NKX2-1^- cells are also ECAD^- and therefore not epithelial. (G) Green: NKX2-1; red: VECAD (vascular endothelium). Mesenchymal cells can appear to line the alveolar sacs in thin sections. Arrowheads = NKX2-1^-, VECAD⁺ endothelial cells apparently lining the developing alveolar sacs. All panels: blue = DAPI (nuclei). Bars = 100 μm A; 50 μm B, C, D’, E, F, G; 200 μm D. DOI: http://dx.doi.org/10.7554/eLife.26575.008

Figure 2.. The tip and stalk epithelial cell populations are clearly demarcated in branching human, pseudoglandular stage, lungs.

(A–E) Sections of human embryonic lungs. (A) 11 pcw. Green: SOX9 (tip); red: SOX2 (stalk); white: α-SMA (smooth muscle). (B) 8 pcw. Green: SOX9 (tip); red: SOX2 (stalk); white: KI67 (proliferating cells). (C, D) 17 pcw. (E) 20 pcw. Green: SOX9 (tip); red: SOX2 (stalk). Arrowheads = SOX9⁺, SOX2^- co-expressing tips. Arrows = SOX9⁺, SOX2^- tips. Blue: DAPI (nuclei). (F) Experimental schematic for tip versus stalk RNAseq. (G) Venn diagram showing common and differentially-expressed transcripts based on a fold-change of at least 2. (H) Unsupervised hierarchical clustering of tip, stalk and published foetal lungs based on the differentially-expressed genes. (I) Chart to show the percentage of the gene ontology classes represented in the differential expression data. (J) List of transcription factors enriched at least two-fold in the tips. Number in brackets indicates fold-change over the stalk. * indicates reported mouse tip expression, see Supplementary file 2. (K) List of differentiation markers enriched in the stalk. (L) List of transcription factors enriched in stalks that were previously reported as expressed in the mesenchyme . Scale bars = 50 μm (A, C, D); 100 μm (B, E); 2 mm (F). DOI: http://dx.doi.org/10.7554/eLife.26575.009

Figure 2—figure supplement 1.. Pseudoglandular stage human lung tips co-express SOX9 and SOX2.

(A) E13.5 mouse lung staining illustrating absence of SOX2 in SOX9⁺ tip cells. Green: SOX9; red: SOX2; white: ECAD. (B) Whole-mount staining of a 5 pcw human embryonic lung showing the primary branches and SOX2/SOX9 co-expression in the tips (boxed area in B is magnified in B’). Green: SOX9; red: SOX2; white: ECAD. (C) Confocal image of a 8 pcw human embryonic lung illustrating SOX2 expression in SOX9⁺ tip cells. Green: SOX9; red: SOX2. (D) qRT-PCR of microdissected tip and stalk compared to whole lung: SOX9 and SOX2 data confirm immunostainings. TBX4 is a mesenchymal marker. ID2 is another well-characterised mouse tip gene. Scale bars = 50 μm (A, C); 200 μm (B); 100 μm (B’). DOI: http://dx.doi.org/10.7554/eLife.26575.010

Figure 2—figure supplement 2.. Human lung tips down-regulate SOX2 during the canalicular stage.

(A–G) Sections of human embryonic lungs at 11, 14, 17 and 20 pcw stained for green: SOX9; red: SOX2. Slides were stained simultaneously and imaged using the same microscope settings such that protein levels can be compared between lungs. Each row shows representative images from a different individual lung. SOX2 is also shown as a separate channel. SOX2 is significantly down-regulated in the epithelial tips by 17 pcw, although still present in some tips. It is absent in the tips by 20pcw. Arrows indicate SOX9⁺ tips. (A) 11 pcw. (B, C) 14 pcw. (D–F) 17 pcw. G. 20 pcw. Blue = DAPI (nuclei). Bar = 50 μm in all panels. DOI: http://dx.doi.org/10.7554/eLife.26575.011

Figure 2—figure supplement 3.. Transcriptional differences and similarities between human pseudoglandular stage tip and stalk populations.

(A) Chart to show the percentage of the expressed genes in the major gene ontology classes represented in the data. (Note: tip and stalk data are reproduced from Figure 2I). (B–E) Human embryonic lung sections. (B) 11 pcw. Green: NKX2.1 (lung epithelium); red: FOXA2 (lung epithelium); white: ECAD (epithelial cells). (C) 11 pcw. Green: β-III-Tubulin (differentiating neurons, including neuroendocrine cells); red: SOX2; white: PECAM (endothelial cells). (D) 12 pcw. Green: β-III-Tubulin (differentiating neurons, including neuroendocrine cells); red: SOX2. Arrowheads = β-III-Tubulin⁺, SOX2⁺ neuroendocrine cells. (E) 10 pcw. Green: SPRY2; red: SOX2; white: ECAD (epithelial cells). (F, G) 8 pcw. Green: FGFR2; red: SOX2; white: ECAD (epithelial cells). Scale bars = 100 μm (A, B, C, E, F, G); 50 μm (D). DOI: http://dx.doi.org/10.7554/eLife.26575.012

Figure 2—figure supplement 4.. Specific transcription factors are enriched at the protein level in human distal epithelial lung tips.

(A–D) Sections of 8 pcw human embryonic lung. (A) Green: ETV5; red: SOX2; white: ECAD (epithelial cells). (B) Green: HMGA2; red: SOX2; white: ECAD (epithelial cells). (C) Green: HNF1B; red: SOX2; white: ECAD (epithelial cells). (D) Green: ID2; red: HMGA1; white: ECAD (epithelial cells). Arrows = distal tips. Blue = DAPI (nuclei). Bars = 50 μm all panels. DOI: http://dx.doi.org/10.7554/eLife.26575.013

Figure 2—figure supplement 5.. Tip gene expression is highly conserved between mouse and human.

(A) Pie chart showing over-lap of orthologous human-mouse genes called as present in 6–7 pcw human tips and E11.5 mouse tips. (B) Scatter plot to estimate the relative levels of expression of orthologous genes between mouse and human tips. We reasoned that the microarray signal saturates and therefore generated a scatter plot of mean microarray signal, versus mean log-transformed RPKM for each orthologous gene identified. r² = 0.423; p<10⁻¹⁶. DOI: http://dx.doi.org/10.7554/eLife.26575.014

Figure 3.. Long-term, self-renewing organoid culture of human lung epithelial tip cells with a initial colony forming efficiency of 100%.

(A) Frames from Video 1 showing bright field images of a single microdissected tip taken every 24 hr for 12 days. Representative bright field images of tip organoid cultures from P0, P6 and P15. A typical organoid after matrigel removal is shown and after further microdissection of branched structures (inset). (B) Confocal images of tip organoids at P0, P6 and P15. Green: SOX9; red: SOX2; white: ECAD. (C) Bright field images of stalk organoids cultured in self-renewing medium at P0, P1, P5. (D) Confocal images of stalk organoids at P3. Green: SOX9; red: SOX2; white: ECAD. (E) Multidimensional scaling plot showing the distribution of fresh tip and stalk transcriptomes and cultured organoids. (F) Heat map of selected tip, stalk and differentiation markers. Bars = 1 mm (A, C); 50 μm (B, D). DOI: http://dx.doi.org/10.7554/eLife.26575.015

Figure 3—figure supplement 1.. Organoids passaged in self-renewing medium retain tip-specific transcription factor proteins.

(A–F) Cryosections of self-renewing tip organoids between passage 5 and 7. (A) Green: SOX9; red: SOX2; white: ECAD (epithelial cells). (B) Green: HMGA2; red: SOX2; white: ECAD (epithelial cells). (C) Green: NKX2-1; red: SOX2; white: ECAD (epithelial cells). (D) Green: ETV5; red: SOX2; white: ECAD (epithelial cells). (E) Green: HNF1B; red: SOX2; white: ECAD (epithelial cells). (F) Green: ID2; red: HMGA1; white: ECAD (epithelial cells). Bar = 50 μm all panels. DOI: http://dx.doi.org/10.7554/eLife.26575.016

Figure 3—figure supplement 2.. Organoids passaged in self-renewing medium retain a normal karyotype.

(A–G) Karypotype of 7 organoid lines between passage 4 and 8. 7/7 organoid lines tested retained a normal karypotype. DOI: http://dx.doi.org/10.7554/eLife.26575.017

Figure 3—figure supplement 3.. SOX2^-, SOX9⁺ canalicular stage human embryonic tips can be grown as SOX2⁺, SOX9⁺self-renewing organoids.

(A–D) Bright field images of 19 pcw tips growing as self-renewing organoids. (E) 19 pcw tip organoids co-express SOX2 and SOX9. Green: SOX9; red: SOX2; white: ECAD. Blue: DAPI. Scale bars = 2 mm (A–D); 100 μm (E). DOI: http://dx.doi.org/10.7554/eLife.26575.018

Figure 3—figure supplement 4.. Box plots of selected tip and stalk specific genes showing transcript levels in fresh tissue and cultured organoids.

Normalised RNAseq data was used to generate box plots of selected genes. DOI: http://dx.doi.org/10.7554/eLife.26575.019

Figure 3—figure supplement 5.. E12.5 mouse tips do not long-term self-renew in the growth medium developed for human tips.

(A, C) Bright-field images of mouse E12.5 tip epithelium growing as organoids in self-renewal medium +/- SB431542. (Factors adjusted to be mouse-specific). (B, D) Confocal images of mouse organoids. These were initially SOX9⁺, SOX2^- similar to the in vivo tissue, but they decreased SOX9 expression and turned on SOX2 over time. Green: SOX9; red: SOX2; white: ECAD. Blue: DAPI. Scale bars = 2 mm (A, C); 100 μm (B, D). DOI: http://dx.doi.org/10.7554/eLife.26575.020

Figure 4.. All factors added to the medium are required for culture establishment.

(A) Whole-mount staining for DAPI and ECAD was performed to estimate the fraction of mesenchyme in a microdissected tip (dotted area) and stalk (dashed box). (B) Quantitation of percentage of mesenchyme in four microdissected tips and stalks. (C) Presence of mesenchyme was assessed in six organoid lines over multiple passages by staining for ECAD (arrowhead = mesenchymal cells). (D) Bright field and confocal images of P0 Day 13 organoids cultured in self-renewing medium, or without the indicated factors. Green: SOX9; red: SOX2; white: ECAD. (E). Bright field and confocal images of organoids at P1 Day 6 cultured in self-renewing medium, or without FGF10, or Noggin. Green: SOX9; red: SOX2; white: ECAD. Boxed area is magnified in inset. (F) Quantitation of organoid forming efficiency and size with, or without, TGFβ inhibition. Bars = SEM. Three biological replicates were analysed; 61 tip cultures without TGFβ inhibition, 38 tip cultures with TGFβ inhibition. (G) Established organoid lines were grown for 3 days in self-renewing, or indicated test medium, and levels of SOX2 and SOX9 assessed by qRT-PCR (values normalized to one for self-renewing controls). Three independent tip organoid lines at P9, 15 and 21 were used. Bars = SEM. * = p-value<0.05. Bars = 50 μm (A, C; D, E confocal images); 1 mm (D, E bright field). DOI: http://dx.doi.org/10.7554/eLife.26575.023

Figure 5.. Disscoiated self-renewing organoids are competent to differentiate under the kidney capsule in the presence of embryonic mouse lung cells.

(A) Experimental schematic. Dissociated organoids were mixed with dissociated E13.5 mouse lungs (either MF1 outbred strain for grafts harvested at 3 and 7 weeks, or Rosa26R-mT-mG strain for grafts harvested at 12 weeks.) Animals culled at 12 weeks received 3x dexamethasone injections 1 week before culling. The 7 and 12 week grafts looked identical and thus no effects on the grafts were observed from the dexamethasone injections. Three independent organoid lines were used and three mice (one per organoid line) culled at each time point. Grafts were clearly visible growing beneath the kidney capsule in all nine kidneys harvested. (B) Chimeric mouse/human lung structures were found in all kidneys. Green: ECAD (epithelium); red: HuNu (human nuclei). (B’) It can clearly be seen that mouse and human cells tend to segregate within the grafts, possibly due to their differing size or surface properties. Wide-spread regions of mouse pro-SFTPC⁺ cells were always visible. Green: ECAD; red: HuNu; white: pro-SFTPC. (C) Rare human pro-SFTPC⁺ cells (arrow) were identified in all samples. Green: ECAD; red: HuNu; white: pro-SFTPC. (D) Human cells arranged into airway-like structures were strongly SOX9⁺, SOX2⁺ at 3 weeks, although patches of cells which were down-regulating SOX9 were visible (bracket). By 7 and 12 weeks SOX9 was expressed at very low levels in some human cells. Green: SOX9; red: HuNu; white: SOX2. (E, F) Rare patches of differentiated human airway cells were identified in 2/3 organoid lines in kidneys harvested at 12 weeks. These were lined with basal, goblet and ciliated cells similar to the in vivo human airways. The remaining human airway-like structures contained goblet cells only and were found in every graft harvested (e.g. arrows in E and F). (E) Green: KRT5 (basal cells); red: Td-Tomato (mouse cells); white: MUC5AC (goblet cells). (F) Green: KRT5 (basal cells); red: Td-Tomato (mouse cells); white: ACT (cilia). Bars = 50 μm (C, D, E’, F’); 2 mm (A); 0.5 mm (B); 200 μm (B’); 100 μm (E, F); 20 μm (E’’, F’’). DOI: http://dx.doi.org/10.7554/eLife.26575.027

Figure 5—figure supplement 1.. Cells isolated from self-renewing organoids are competent to engraft into adult mouse lungs.

(A) Oropharyngeal administration of 1 mg/ml bleomycin at a dose of 1 μl per gram body weight is sufficient to injure adult male NSG mouse lungs. Representative haematoxylin and eosin stained lung sections. B. Experimental schematic: 6 × 10⁵ cells were administered 2 days post-bleomycin. C. Grafts were only detected in animals that received both injury and cells. Bars = SEM. D. Grafted human cells (arrow or bracket) identified at day 2 and day 8 post-cell administration. Green: ECAD (epithelial cells); red: HuNu (Human nuclear marker). E. Graph showing number of cells per graft counted at day 2 and day 8. F-I. Sections of day eight grafts. F. Green: NKX2-1; red: HuNu (Human nuclear marker); white: FOXA2. Arrow = NKX2-1⁺ grafted cell; although the majority of the graft is NKX2-1^-,FOXA2⁺. G. Green: SOX9; red: HuNu (Human nuclear marker); white: SOX2. H. Green: MUC5AC (goblet cells); red: TRP63 (basal cells); white: ECAD (epithelial cells). I. Green: KRT5 (basal cells); red: HuNu (Human nuclear marker); white: ECAD (epithelial cells). Arrows = KRT5⁺ graft cells. Bars = 1 mm (A); 50 μm (D,F–I). DOI: http://dx.doi.org/10.7554/eLife.26575.028

Figure 5—figure supplement 2.. Mouse regions of chimeric human-mouse kidney capsule grafts differentiate efficiently.

The extent of differentiation of the mouse cells was indistinguishable at 3, 7 and 12 weeks post-grafting and a selection of representative images from the various times are shown. Regions of mouse alveoli are found throughout the grafts illustrated by pro-SFTPC staining (A) and HOPX staining (B). Mouse airway epithelial cells can be KRT5⁺, MUC5AC⁺ (C), SCGB1A1⁺ (D) and ACT⁺ (E). Human airway epithelial cells very occasionally have a low level of SCGB1A1 staining (arrow in D). F. Very rare human cells express pro-SFTPC. These are always at the periphery of a tubular structure (arrow). Green: ECAD (epithelium); red: HuNu (Human Nuclei); white: pro-SFTPC. HOPX⁺ human cells were not identified. Bars = 100 μm (A, B, D); 50 μm (C, E, F). DOI: http://dx.doi.org/10.7554/eLife.26575.029

Figure 6.. In vitro differentiation of self-renewing organoids towards bronchiolar and alveolar lineages.

(A) Experimental schematic for 3 weeks organoid differentiation in Pneumacult-ALI medium. (B) In low passage number organoids, mesenchyme expanded (arrows), basally-located TP63⁺ basal cells differentiated and rare ACT⁺ ciliated cells were seen. Dashed arrow = ciliated cell. Arrow heads = basal cells. Arrow = mesenchyme. Green: ACT (cilia); red: TP63 (basal cells); white: SOX2. (C) Differentiated organoids were predominantly composed of MUC5AC⁺ goblet cells, although low levels of SCGB1A1 were observed in some cells. Cells retained SOX9 suggesting that differentiation was not complete. Green: MUC5AC (mucous); red: SOX9; white: SCGB1A1. (D) Experimental schematic for 3 week alveolar differentiation experiment. (E) All organoid cells retained a columnar appearance and expressed relatively low levels of apical pro-SFTPC. Red: pro-SFTPC; white: ECAD. (F) Experimental schematic for 3 week alveolar differentiation in the presence of freshly-isolated 19 pcw human mesenchyme. (G) Mesenchymal cells were observed in the cultures (arrowheads), moreover cells expressing higher levels of pro-SFTPC with a more squamous appearance were also obtained. Red: pro-SFTPC; white: ECAD. (H) Experimental schematic for 3 week alveolar differentiation of 19 pcw organoid in the presence of expanded human 20 pcw mesenchymal cells. DCI = dexamethasone, cAMP, IBMX. (I) Organoid epithelium took on a more squamous appearance and was surrounded by mesenchymal cells (arrowheads). White: ECAD. (J) AT2 markers were expressed robustly. Green: HTII-280; red: pro-SFTPC. (K) AT1 and AT2 markers were co-expressed. Green: HOPX; red: pro-SFTPC. Blue: DAPI. Bars = 1 mm (A, H); 100 μm (B, C); 20 μm (B’, E’); 10 μm (C’, G’); 50 μm (E, G, I, J, K). DOI: http://dx.doi.org/10.7554/eLife.26575.030

Figure 6—figure supplement 1.. Exposure of self-renewing organoids to Pneumacult^TM medium leads to efficient goblet cell differentiation with rare patches of KRT5⁺ basal cells.

(A) Experimental schematic. (B) Every organoid examined had many MUC5AC⁺ goblet cells and little or no SOX9. Green: MUC5AC (mucous); red: SOX9. (C) More rarely, some organoids contained patches of KRT5+ basal cells. Green: MUC5AC (mucous); red: KRT5 (basal cells). Blue: DAPI. Bar = 50 μm (B, C). DOI: http://dx.doi.org/10.7554/eLife.26575.031

Figure 6—figure supplement 2.. Testing media conditions for ability to promote human organoid alveolar differentiation.

(A) Experimental schematic. (B) Alveolar differentiation media tested. DCI = dexamethasone, cAMP, IBMX. (C-D) Self-renewing controls co-express SOX2 and SOX9, whereas SOX2 is retained and SOX9 is lost following 2 weeks in most media tested. Green: SOX9; red: SOX2; white: ECAD. (F) Green: HTII-280; red: pro-SFTPC cannot be detected in self-renewing control conditions. (G–M) The majority of media tested resulted in patchy loss of SOX2 and upregulation of pro-SFTPC. Green: SOX2; red: pro-SFTPC; white: ECAD. (N) HTII-280 expression was very rarely observed in any differentiation condition. Green: HTII-280; red: pro-SFTPC. Scale bars = 1 mm (A); 50 μm (C–N); 10 μm (insets). DOI: http://dx.doi.org/10.7554/eLife.26575.032

Figure 6—figure supplement 3.. A combination of canalicular stage lung embryonic mesenchyme and alveolar differentiation medium together promote the most efficient organoid alveolar differentiation.

(A) Experimental schematic for 3 week alveolar differentiation of psedoglandular stage-derived organoids in the presence of freshly-isolated 19 pcw human mesenchyme. DCI = dexamethasone, cAMP, IBMX. (B, C) Regions of organoid that have turned off SOX2 and turned on pro-SFTPC can readily be distinguished. Green: SOX2; red: pro-SFTPC; white: ECAD. DOI: http://dx.doi.org/10.7554/eLife.26575.033

Figure 6—figure supplement 4.. Expanded canalicular stage mesenchyme and alveolar medium can promote organoid alveolar differentiation.

(A) Experimental schematic for 3 week alveolar differentiation of psedoglandular and canalicular stage-derived organoids in the presence of expanded 19 or 20 pcw human mesenchyme. (B–D) Pseudoglandular stage organoids took on a squamous appearance and expressed pro-SFTPC and, more rarely, HTII-280. The AT1 marker, HOPX was also expressed. (E–I) Canalicular stage organoids also took on a squamous appearance and expressed higher levels of AT2 markers, in addition to HOPX and PDPN. Cells retained co-expression of AT1 and AT2 markers. Results shown are from two independent 19 pcw organoid experiments are shown. B, C, E, F. Green: HTII-280; red: pro-SFTPC. (D, G) Green: HOPX; red: HTII-280. (H, I) Green: NKX2-1; red: PDPN; white: HTII-280. Blue = DAPI. Bars = 50 μm all panels. DOI: http://dx.doi.org/10.7554/eLife.26575.034

Figure 6—figure supplement 5.. Expanded fibroblasts used for organoid co-cultures are a heterogeneous population expressing various lung embryonic fibroblast markers.

(A–D) 20 pcw lung sections stained for mesenchymal markers. (A) Green: PDGFRA; red: PDPN (alveolar epithelium and lymphatic endothelium); white: SMA. (B) Green: PDGFRB; red: PDPN (alveolar epithelium and lymphatic endothelium). (C) Green: CD90; red: PDPN (alveolar epithelium and lymphatic endothelium). (D) Green: VECAD (vascular endothelium); red: PDPN (alveolar epithelium and lymphatic endothelium). (E–F) Passage three fibroblasts expanded from 19 pcw human embryonic lung. (E) Green: PDGFRA; red: SMA. (F). Green: PDGFRB; red: CD90. Expanded fibroblasts are all PDGFRB⁺, but co-express a variety of other markers. All panels: blue = DAPI (nuclei). Bars = 50 μm all panels. DOI: http://dx.doi.org/10.7554/eLife.26575.035