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

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics 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. https://doi.org/10.7554/eLife.26575.001 eLife digest Degenerative lung disease occurs when the structure of the lungs breaks down, which makes it harder to get enough oxygen into the bloodstream. Most, but not all, cases occur in smokers and ex-smokers or people who have been exposed to a lot of air pollution. Currently, there is no way to reverse the damage, and even slowing the progress of the disease is extremely difficult. Some researchers are looking for ways to treat patients with degenerative lung diseases by regenerating the surface of their lungs. However, it is still not clear what the most effective route towards this long-term goal will be. One approach to lung regeneration is to use findings from developmental biology to understand how embryos normally build the gas exchange surfaces in the lungs. This knowledge may allow scientists to trigger a similar process in an adult lung to renew or replace any diseased tissue. Alternatively, cells could be collected from patients, reprogrammed and then coaxed into becoming a gas exchange surface in the laboratory. Such a “lung-in-a-dish” could be used to understand how degenerative diseases develop, to discover and test new drugs, or even to treat the patient directly via a transplant. To date, the embryonic development of lungs has mostly been studied using mouse lungs as a model system. However, it was not clear if human lungs actually develop in similar ways to mouse lungs, and whether using mice is a valid research strategy. Nikolić et al. compared embryonic lungs from humans and mice and showed that they are indeed very similar in terms of the cell types that they contain and how they mature. However, some key differences were identified that can only be explored in human cells and tissue. Nikolić et al. went on to identify conditions that allowed them to grow cells from human embryonic lungs indefinitely in a dish. These cells can now be used to investigate the aspects of lung development that are specific to humans. Together these findings provide a useful guide to allow scientists to coax human cells growing in a laboratory to become lung cells. Further improvements to this process will make the lungs-in-a-dish more true to the real organs, meaning that they could be used to better understand lung disease and identify new medicines. In the longer term, Nikolić et al. hope to gain enough insight from the human lung-in-a-dish model to eventually be able to regenerate the lungs of patients with degenerative lung disease. However, this possibility is still many years away. https://doi.org/10.7554/eLife.26575.002 Introduction During mouse lung development the distal tip epithelial cells are SOX9+, ID2+ and function as multipotent progenitors producing first bronchiolar, and then alveolar, descendants (Alanis et al., 2014; Rawlins et al., 2009). Between ~E10–15 cells that exit the distal tip turn off SOX9, upregulate SOX2 and differentiate along bronchiolar lineages. Whereas, ~E16–18 cells exiting the tip turn off SOX9 and co-express markers of alveolar type 1 (AT1) and alveolar type 2 (AT2) fate. As morphogenesis proceeds these bipotent cells line developing alveolar sacs and differentiate as mature AT1 or AT2 cells (Desai et al., 2014; Treutlein et al., 2014). Many factors controlling self-renewal and differentiation in the developing mouse lung epithelium have been identified. By contrast, relatively little is known about human lung development. This is largely due to practical considerations about tissue availability and culture system limitations (Benlhabib et al., 2015; Haitchi et al., 2009; Rajatapiti et al., 2010). A small number of human studies show the detailed expression of specific genes (Al Alam et al., 2015; Gonzalez et al., 1996; Khoor et al., 1993, Khoor et al., 1994; Laresgoiti et al., 2016; Stahlman et al., 2007; Zhang et al., 2012). Whereas, transcriptomics has provided a genome-wide view of human lung developmental transitions, but currently lacks cellular resolution (Feng et al., 2014; Kho et al., 2010, 2016). Improved in vitro models of human disease are needed to complement available mouse models. One recent approach to disease modelling is to use self-renewing human organoids which recapitulate aspects of organ morphogenesis/physiology (Dekkers et al., 2013; Ettayebi et al., 2016; Huch et al., 2015). Human organoids are typically derived from adult stem cells limiting their use for studying paediatric disease and disease progression. An alternative is to derive the organ of interest from pluripotent stem cells by directed differentiation (Dye et al., 2016; Merkle and Eggan, 2013). The ability to in vitro self-renew and differentiate bona fide human lung tip progenitors could provide a genetic system for fundamental developmental biology and paediatric disease modelling. Moreover, an improved understanding of human lung progenitor states and human fate specification would facilitate strategies for directed differentiation of pluripotent cells. We have extensively characterized human epithelial tip progenitors, and the early stages of fate specification, revealing mouse-human differences in the expression of key marker genes including SOX2 and pro-SFTPC. We developed methods to grow human tip epithelium as long-term self-renewing, branching, organoids and therefore investigate tip signalling requirements and differentiation capacity. Our human tip organoids can be directed to differentiate towards alveolar or bronchiolar fate in vitro and can engraft into the adult or developing mouse lung. These experiments confirm that human and mouse tips are analogous in function and validate our organoid conditions. These conditions are sufficient to convert differentiating human embryonic lung epithelial stalks to tip fate, illustrating the plasticity of the developing lung. However, they do not support the long-term self-renewal of mouse tips, highlighting species-specific regulatory differences. Our organoid system thus provides a genetically-tractable in vitro model to accelerate studies of human lung development. Results Initiation of alveolar and bronchiolar differentiation in human embryonic lungs We characterized the evolution of marker gene expression in human embryonic lungs 6–21 pcw (post-conception weeks; Figure 1—figure supplement 1). This period covers the pseudoglandular stage (~5–16 pcw) when the bronchiolar tree is established and the early canalicular stage (~16–21 pcw) when alveolar sac formation begins (Burri, 1984; Rackley and Stripp, 2012). Throughout the time-course, we observed SOX9 (SRY-box 9) localized to the distal epithelial tips (Figure 1A,B). In the adult human lung, pro-SFTPC (Surfactant Protein C, or SPC) and a monoclonal antibody, HTII-280, are markers of AT2 cells (Figure 1C) (Barkauskas et al., 2013; Gonzalez et al., 2010). We first detected low levels of HTII-280 in SOX9- epithelium at 11 pcw, adjacent to the distal tip (Figure 1A). Similarly at 14 and 17 pcw (Figure 1—figure supplement 2A). At 20 pcw, HTII-280 was more heterogeneous in the alveolar sacs with cells having either high, or undetectable, levels (Figure 1B). We confirmed that HTII-280 was not in the larger airways (Figure 1B, arrowhead), but it was ubiquitous in the columnar epithelium of the terminal bronchioles (Figure 1B, arrow). By contrast, pro-SFTPC was first detected at very low levels at 17 pcw, particularly in tip epithelium (Figure 1—figure supplement 3D), and more robustly in distal squamous cells by 20 pcw (Figure 1D–F). At 20 pcw, pro-SFTPC was mostly co-expressed with HTII-280 (Figure 1F’), although we also observed a small number of pro-SFTPC+, HTII-280- cells and many more pro-SFTPC-, HTII-280+ cells. This human embryonic pro-SFTPC staining differs from that observed in mouse development in which pro-SFTPC is expressed throughout the epithelium from ~E10, increases in canalicular stage tips and is further up-regulated in differentiating AT2 cells (Laresgoiti et al., 2016; Wuenschell et al., 1996). However, our human data are consistent with previous reports of pro-SFTPC staining in human embryos (Khoor et al., 1994). We previously used LPCAT1 (Lysophosphatidylcholine acyltransferase 1) as a specific marker of alveolar/AT2 fate in mouse development (Laresgoiti et al., 2016). However, it was expressed widely throughout the epithelium of the developing human lung from six pcw, although specific to AT2 cells in the adult (Figure 1—figure supplement 2E,F). ABCA3 (ATP binding cassette subfamily A member three) is present on the surface of lamellar bodies in mature AT2 cells, but has not been detected in human embryonic lungs prior to 28 weeks gestation (Stahlman et al., 2007). Consistent with this, we could not detect ABCA3 in any of the embryonic lungs we stained (6–21 pcw), but reproducibly saw expression in adult AT2 cells (Figure 1—figure supplement 2F). This suggests that mature lamellar body-containing AT2 cells are not present in human embryonic lungs by 21 pcw consistent with previous analysis (Oulton et al., 1980). Figure 1 with 5 supplements see all Download asset Open asset 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’). https://doi.org/10.7554/eLife.26575.003 In mouse development the AT1 marker HOPX (Hop Homeobox) is first detected ubiquitously in cells that have exited the canalicular stage distal tip, subsequently becoming heterogeneous and marking future AT1 cells (Jain et al., 2015; Laresgoiti et al., 2016). Similarly, we detected HOPX in cells that had exited the distal tip at 11 pcw, although there was also co-expression of HOPX and SOX2 (SRY-Box 2) in the smaller airways (Figure 1G). HOPX and HTII-280 were frequently co-expressed between 11 and 17 pcw (Figure 1—figure supplement 2B). By 20 pcw, a mixture of single and co-expressing distal epithelial cells were observed lining the developing alveolar sacs (Figure 1H). Interestingly, at 20 pcw cells in the terminal bronchioles co-expressed SOX2, HTII-280 and HOPX (Figure 1H’ and Figure 1—figure supplement 2C). A monoclonal antibody, HTI-56, has been reported as human AT1 cell specific (Dobbs et al., 1999; Gonzales et al., 2015). Consistent with this, we observed the highest levels of expression in HOPX+ cells adjacent to the distal tip from 11 pcw (Figure 1I). By 20 pcw HTI-56 was becoming heterogeneous, whilst HOPX remained ubiquitous (Figure 1J,J’). However, we also observed a low level of HTI-56 expression throughout the SOX2+ airways, beyond the terminal bronchioles, making it less useful as an AT1 marker in vitro (Figure 1—figure supplement 2C). In adult human alveoli a relatively small number of HOPX+ nuclei can be observed whilst the entire alveolar surface is covered by HOPX+ cell membranes extending from the AT1 cells (Figure 1K). PDPN (Podoplanin, also known as T1α) protein is detected in mouse lung epithelium from the time of alveolar specification at ~E16.5 and becomes restricted to differentiating AT1 cells by E18.5 (Laresgoiti et al., 2016). It is also a marker of airway basal cells and lymphatic endothelium (Breiteneder-Geleff et al., 1999; Farr et al., 1992). We detected low levels of apical PDPN expression in columnar (not basal) cells of the developing human bronchioles from 9 pcw (Figure 1—figure supplement 3A). At later stages expression is stronger in the more distal regions where PDPN and HTII-280 are co-expressed ubiquitously on the developing alveolar surface at 17 pcw, but PDPN is absent from the distal tips (Figure 1—figure supplement 3B–G). By 20 pcw PDPN can be observed to be co-expressed with AQP5 (Aquaporin 5) in distinct, individual cells, rather than running continuously through the alveolar duct (Figure 1—figure supplement 3H). It is strongly expressed in adult AT1 cells as expected (Figure 1—figure supplement 3H). AQP5 has been described in mouse as specifically detected in differentiating AT1 cells and not expressed in the bipotent alveolar progenitors which co-express AT1 and 2 markers (Desai et al., 2014). It is therefore a putative marker of differentiating AT1 cells. We first detect AQP5 in distal regions of the lung from 11 pcw (Figure 1—figure supplement 4A–C). By 20 pcw it can be observed to be expressed ubiquitously in the low columnar epithelium that lines the developing alveolar ducts and extends to the distal tips. It is expressed at higher levels in elongating cells with very short membrane extensions (Figure 1—figure supplement 4D,E; these are similar in appearance to the HOPX+ cells marked with arrowheads in Figure 1H’’ and are likely to be differentiating AT1 cells that are beginning to extend their membranes). In the adult alveoli AQP5 is highly expressed in AT1 cells, but it can also be observed on the surface of non-ciliated cells in the lower airways (Figure 1—figure supplement 4F,G). NKX2-1 is ubiquitously expressed in lung epithelial cells from the time of lung specification in the foregut (Lazzaro et al., 1991). NKX2-1 levels have been reported to be heterogeneous in mature alveolar cells in the adult mouse and rat (Desai et al., 2014; Liebler et al., 2016). We observed that NKX2-1 was co-expressed with FOXA2 in all human embryonic lung epithelial cells from 9 to 21 pcw, albeit at higher levels in alveolar-fated than bronchiolar-fated cells (Figure 1—figure supplement 5A–C). NKX2-1-,FOXA2- cells which apparently lined the more mature alveolar ducts were identified, but closer inspection revealed that these were ECAD- non-epithelial cells their presence at the alveolar surface likely being an artefact due to the angle of sectioning (Figure 1—figure supplement 5D–G). Our results show that the broad picture of mouse alveolar development (low-level co-expression of AT1 and 2 markers in cells that exit the canalicular stage distal tip, followed by lineage-specific expression as morphogenesis proceeds) is conserved in human. However, even in this limited analysis there are striking mouse-human differences. The timing of alveolar marker gene onset in human is ~11 pcw, preceding the canalicular stage by approximately five weeks. In addition, the relative timing of expression of specific markers can differ (pro-SFTPC, LPCAT1, AQP5). Moreover, the developing human terminal bronchioles are SOX2+, but co-express alveolar markers which to our knowledge has never been observed in mouse (Laresgoiti et al., 2016). The first signs of cellular heterogeneity/differentiation in the bronchioles were observed from 7 pcw where we saw that a sub-set of SOX2+ cells co-expressed low levels of TP63 (Tumour Protein P63) in the larger airways (Figure 1L, arrowheads). These are likely to be differentiating basal cells which are TP63+ in the adult (Figure 1M, arrowheads) (Rock et al., 2009). FOXJ1 and SCGB1A1 were also readily detected in adult airways (Figure 1N). By 11 pcw, TP63 staining was much stronger and localised to a sub-set of basally-located airway nuclei (Figure 1O). TP63 was also stronger in more proximal, versus distal, bronchioles within the same lungs (Figure 1O,P). We first detected KRT5 (Keratin 5) at 16 pcw in the larger airways, particularly in invaginating submucosal gland buds (Figure 1Q–S). KRT5 was also seen in patches of TP63+ airway cells, likely differentiating basal cells (Figure 1—figure supplement 1S, dotted lines). KRT5+, TP63- cells were also observed, frequently in a non-basal position (Figure 1—figure supplement 1S, arrowheads). The first signs of columnar cell differentiation were detected at 16 pcw when rare MUC5AC+ (Mucin 5AC) cells were identified in the proximal airways (Figure 1T). This is consistent with a proximal-distal pattern of airway differentiation as in other species (Plopper et al., 1992; Toskala et al., 2005). Characterization of human embryonic lung tip progenitors In the pseudoglandular stage mouse lung, there is a clear demarcation between SOX9+ tip and SOX2+ stalk (differentiating bronchiole) cells and multiple signalling mechanisms regulate the boundary between the two populations (Hrycaj et al., 2015; Mahoney et al., 2014; Wang et al., 2013). We observed a tip-stalk boundary in the pseudoglandular stage human lungs with SOX9 restricted to the tip and SOX2 expressed highly in the stalk. Differentiating αSMA+ (α-Smooth Muscle Actin) smooth muscle was observed around the SOX2+ future airways and SOX9 was present throughout the mesenchyme at low levels (Figure 2A). We noted that human tip epithelium was more proliferative than stalk (Figure 2B), consistent with mouse results (Okubo et al., 2005). However, in contrast to the mouse, low levels of SOX2 were co-expressed with SOX9 in the distal tip epithelium throughout the pseudoglandular stage (Figure 2B; Figure 2—figure supplement 1A–C). SOX2, SOX9 co-expression at the tip was confirmed by qRT-PCR in microdissected tip and stalk cells (Figure 2—figure supplement 1D). Further examination of our time-course revealed that SOX2 gradually decreased over time and disappeared from the tip epithelium at the transition to the canalicular stage of development. This happened heterogeneously throughout the lung. For example, at 17 pcw we observed a mixture of SOX2+ and SOX2- distal tips within individual lungs (Figure 2C,D; Figure 2—figure Supplement 2). However, by 20 pcw all distal tips were SOX2- (Figure 2E; Figure 2—figure Supplement 2). Moreover, there was a SOX2-, SOX9- zone adjacent to the 20 pcw distal tips which corresponds to the developing saccules where markers of alveolar differentiation are expressed (compare Figure 2E with Figure 1H). Figure 2 with 5 supplements see all Download asset Open asset 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 at least in the tips. in fold-change over the stalk. reported mouse tip see 2. List of differentiation markers in the stalk. (L) List of transcription factors in stalks that were previously reported as expressed in the mesenchyme = 50 μm (A, C, 100 μm 2 To tip-stalk gene expression we microdissected tip and stalk epithelium from individual pcw lungs and (Figure 2F). We detected a of with the expressed in tip and stalk (Figure This level of was expected the stalk cells are the descendants of the tip a in expression level genes that were in stalk or tip were also identified. clustering of the based on these transcripts revealed a clear into tip and stalk populations (Figure Moreover, these genes were expressed at similar levels in published human foetal lung (Figure et al., 2010). analysis of the expressed transcripts to cell transcription and developmental expected for embryonic progenitors (Figure These were also a of genes that were co-expressed in tip and stalk (Figure 2—figure supplement 3A). and which we detect as ubiquitous throughout the developing human lung epithelium (Figure 2—figure supplement were not in tip or stalk. The human data genes as transcription factors of which had previously been characterized as expressed in mouse tips (Figure 1 and 2). These and SOX9 which have in mouse et al., 2013; et al., 2013; et al., 2014; Zhang et al., Moreover, we were able to confirm human of and at the protein level (Figure 2—figure supplement the tip transcription we identified only which is likely to be expressed et al., et al., a very low level of A of our human tip with a previously published mouse tip (Laresgoiti et al., that of genes that were expressed in human tips were also present in mouse (Figure 2—figure supplement was also a between levels of mouse-human gene expression (Figure 2—figure supplement We detect the first signs of cellular in the stalk by 7 pcw (Figure we were able to identify transcripts that are characterized as expressed in differentiating airway cells (Figure These and cell markers et al., et al., et al., 2015; and 2009; et al., 2009). Consistent with this, we observed cells in pseudoglandular stage airways (Figure 2—figure supplement we the stalk transcripts as transcription factors in more we that had published mouse airway expression However, of these were expressed in mouse rather than epithelium (Figure a level of in the stalk the we identified of the and signalling pathways 1). We noted that signalling were in tip and and were more likely to be tip or stalk signalling is to mouse lung branching morphogenesis and and protein expression of was confirmed by staining (Figure 2—figure supplement were differences in tip signalling pathways between mouse and human. For example, and were highly in human tips compared with in the mouse et al., and in human where mouse has et al., This analysis suggests that the human tip epithelium is analogous to the mouse population with a highly conserved and similar signalling However, we also differences that are likely to be of in vitro self-renewing culture conditions for human epithelial tips The mouse distal tip population is a progenitor that self-renews extensively throughout lung development. Moreover, our recent experiments that is largely by (Laresgoiti et al., 2016). We therefore that we be able to tip self-renewing in vitro by the of This would be analogous to the long-term self-renewal of cell as cells. We microdissected human epithelial tips from 5 to 9 pcw lungs in Figure and them in in the presence of 7 a and a was based on the conditions used to grow adult foregut as the on mouse lung development and our analysis et al., et al., 2009; and 2015; et al., 2014). In these conditions human lung epithelial tips organoids with = tips from within for and then by culture 14 the organoids a of tips (Figure 1). We the organoids 2 weeks by into smaller and in a similar in later although the appearance of the more heterogeneous on the of during (Figure 3A). organoids SOX2 and SOX9 expression over multiple (Figure Moreover, they expressed the transcription NKX2-1 and the marker that we have (Figure supplement 1). a and were of a single epithelial 2) similar to the in of the tip epithelium (Figure 2—figure supplement tip organoid line we to grow in the same way for at least 9 over = 11 organoid lines were for in morphology, or SOX2 and SOX9 some have been for to 9 were also (Figure supplement 2). We that of and and of and are sufficient to grow human epithelial tips as self-renewing organoids with an of Figure with 5 supplements see all Download asset Open asset self-renewing organoid culture of human lung epithelial tip cells with a of (A) from 1 showing of a single microdissected tip for of tip organoid from and A organoid is shown and further of (B) of tip organoids at and Green: red: white: (C) of