Abstract
Direct reprogramming is a breakthrough technology that can alter the fate of cells without the passage of stem cells. However, direct reprogramming of somatic cells into pulmonary alveolar epithelial cells has not yet been achieved. Here, we report the direct reprogramming of mouse tail tips and embryonic fibroblasts into induced pulmonary alveolar epithelial-like cells (iPULs) using four transcription factor-coding genes (Nkx2-1, Foxa1, Foxa2, and Gata6) and three-dimensional culture. The iPULs showed lamellar body-like structures and displayed key properties of pulmonary alveolar epithelial cells. Although the potential for iPULs to morphologically differentiate into alveolar epithelial type 1 cells was limited in vitro, the intratracheal administration of iPULs in a bleomycin-induced mouse model of pulmonary fibrosis led to their integration into the alveolar surface, where they formed both alveolar epithelial type 1 and type 2-like cells. Thus, reprogrammed fibroblasts may represent a new source of pulmonary alveolar epithelial cells for regenerative medicine.
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Introduction
Various respiratory diseases, such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD), are intractable and irreversible, and lack effective treatment1,2. Despite its potential as a curative treatment, the use of lung transplantation is limited by patient age and dearth of donors, resulting in the death of ~20% patients before transplantation3. Additionally, postoperative rejection remains an unresolved problem. Therefore, novel therapies involving regeneration of lung tissue and restoration of normal function in intractable lung diseases are urgently required. Alveolar epithelial type 2 (AT2) cells act as tissue stem cells that maintain the alveolar epithelium and regenerate the alveoli after injury4. Research on AT2 cell maintenance and differentiation has advanced remarkably in recent years5,6,7. Moreover, as AT2 cell injury and senescence contribute significantly to the pathogenesis of IPF and COPD8,9, establishing a method for AT2 cell regeneration is of considerable clinical importance10.
Despite the discovery of embryonic stem cells (ESCs)11,12, the use of regenerative medicine has been hindered by ethical issues. However, the advent of the induced pluripotent stem cell (iPSC) technology in 2006 led to a significant breakthrough13. Recent advancements in iPSC technology, combined with gene-editing technology and three-dimensional (3D) organoid culture, have led to the creation of diverse research and development infrastructures14,15,16. Lung epithelial cells induced from iPSCs and their long-term culture have been applied to elucidate the pathological mechanisms of diseases and screen for the toxicity of new drugs17,18,19,20. However, complex culture conditions must be varied at each stage of the differentiation process to generate cells of lung epithelial lineage from stem cells. Therefore, appropriate iPSC clones must be selected17, as cells initiate fate changes once differentiated, which might bias the subsequent differentiation tropism or lead to instability in the genome21,22. This selection is labor-intensive and expensive, and contamination with undifferentiated iPSCs during cell therapy can lead to tumor formation23.
Earlier reports showed that direct reprogramming by overexpressing specific transcription factors (TFs) has been reported to directly convert differentiated cells into other cell types such as cardiomyocytes24, neurons25, and hepatocytes26,27 without entering the pluripotent cell state28,29. This method is less time-consuming and is associated with low risk of carcinogenesis. However, direct reprogramming of fibroblasts into lung alveolar epithelial cells has not been reported. This technology is anticipated to accelerate research in the distal lung region and expand treatment options for refractory lung disease.
Here, we report the direct induction of mouse fibroblasts into pulmonary alveolar epithelial-like cells using defined TFs and 3D organoid culture techniques. These induced pulmonary alveolar epithelial-like cells (iPULs) present an AT2-like transcriptome and remain viable in mice without disrupting pre-existing alveolar structures. Our observations represent a departure from the results of conventional lung regeneration research and have potential in various future applications.
Results
Selection of Nkx2-1, Foxa1, Foxa2, and Gata6 in a screen of fibroblast reprogramming
To screen for pulmonary alveolar epithelial fate-inducing factors, we selected 14 candidate genes associated with pulmonary alveolar epithelial cell differentiation during lung development: Nkx2-1, Foxa1, Foxa2, Foxj1, Tcf21, Hoxa5, Sox17, Gata6, Tbx4, Gata5, Foxf1, Foxl1, Gli2, and Gli330,31,32. A mixture of retrovirus vectors carrying each of the 14 genes (hereafter referred to as “14 factors”) was used to transduce mouse tail-tip fibroblasts (TTFs), which were subsequently cultured in two-dimensional (2D) dishes for 7 d. Transduction of all 14 factors into fibroblasts strongly induced the expression of the AT2 cell marker, surfactant protein-C (Sftpc). To screen for reprogramming factors, Sftpc expression was assessed by systematically eliminating one factor at a time from the pool of 14 factors. Importantly, removal of Nkx2-1 from the pool of 14 factors drastically reduced Sftpc expression (Supplementary Fig. 1a). Next, we examined the cooperative effects of Nkx2-1 and the other factors. Nkx2-1 elicited high Sftpc expression in combination with Foxa1, Foxa2, and Gata6; in particular, combined expression of Nkx2-1/Foxa1/Foxa2/Gata6 increased Sftpc expression the most (Supplementary Fig. 1b, c). We confirmed that one to three colonies of 1 × 105 TTFs (the reprogramming efficiency was 0.002 ± 0.0004% [mean ± standard error of the mean (SEM)]) transduced with the four defined TFs (4TFs: Nkx2-1, Foxa1, Foxa2, and Gata6) were Sftpc-positive cells (Supplementary Fig. 1d).
Improving reprogramming efficiency using 3D organoid culture and fluorescence-activated cell sorting (FACS)
To improve reprogramming efficacy, we modified the provisional protocol; we used 3D organoid cultures instead of 2D cultures and mouse embryonic fibroblasts (MEFs) collected from Sftpc-GFP mice instead of neonatal TTFs, and supplemented the serum-free medium with various factors, including Wnt pathway activators33, several growth factors6,34, and Smad inhibitors35, important factors associated with alveolar cultures. Fluorescence microscopy confirmed the presence of the red fluorescent dye, DsRed, and flow cytometry showed that ~80% of the cells were DsRed-positive 3 d after introduction into a 3D organoid culture of MEFs using this method (Supplementary Fig. 1e, f). Figure 1a shows a schematic illustration of the 3D culture experiment with MEFs isolated from Sftpc-GFP mice transduced with the 4TFs. The transduced MEFs gradually lost their fibroblast marker, Vim, but showed increased Sftpc expression, resulting in the formation of Sftpc-GFP-emitting organoids in the 3D culture system (Fig. 1b and Supplementary Fig. 1g, h). To isolate MEF-derived AT2-like cells, we sorted Sftpc-GFP-positive, Thy1.2-negative (fibroblast marker), and EpCAM-positive (epithelial cell marker) cells using FACS. The number of Sftpc-GFP+ Thy1.2– cells (originating from the initial Sftpc-GFP– MEFs) increased with time; ~34% of the cells were Sftpc-GFP+ Thy1.2– cells at day 7 post-transduction. Notably, 10.1% of the Sftpc-GFP+ Thy1.2– cells were EpCAM+, whereas the remaining were EpCAM– (Fig. 1c). Sftpc-GFP+ Thy1.2– EpCAM– cells were categorized as cells that were not completely reprogrammed and could not be epithelialized. We named the Sftpc-GFP+ Thy1.2– EpCAM+ AT2-like cells isolated from MEFs transduced with the 4TFs as iPULs, which accounted for 2–3% of all sorted cells on day 7 (Fig. 1d). AT2 cells were isolated from adult mouse lungs under the same Sftpc-GFP+ Thy1.2– EpCAM+ conditions, and most cells maintained similar characteristics after several passages (Fig. 1e). Remarkably, purified iPULs sorted using FACS formed organoids resembling those formed by AT2 cells (Fig. 1f). For the purified iPULs, which were stabilized after several passages, the percentage of Sftpc-GFP+ Thy1.2- EpCAM+ AT2-like cells was comparable to that of AT2 cells (Fig. 1g, h).
a Schematic of the experimental design. b Low-power bright-field, fluorescence, and overlay images of MEF organoids and unpurified iPULs. Unpurified iPULs were observed 7 d after transduction. Scale bar: 1 mm. c Representative two-parameter contour plots of unpurified iPULs (Sftpc-GFP on the x-axis (i) and Thy1.2-PECy7 on the x-axis (ii)) at the indicated time points. d Percentage of GFP+ Thy1.2- EpCAM+ cells in unpurified iPULs at each time point. Data are presented as mean values ± SEM; n = 4 biologically independent samples. e Representative two-parameter contour plots of AT2 cells at passage 5 (P5). f Bright-field and fluorescence images of organoids containing purified iPULs and AT2 cells. Organoids were observed on day 8 in P2 (purified iPULs) and P3 (AT2 cells) after the initiation of 3D culture. Scale bar: 1 mm (low power) and 100 µm (high power). g Representative two-parameter dot plots of the indicated samples. h Percentage of GFP+ Thy1.2- EpCAM+ cells in each sample. Data are presented as mean values ± SEM; n = 3 biologically independent samples.
At this stage, we reviewed the factors that could be excluded or added to the 4TFs again. First, we excluded Gata6, which had the most unstable and exhibited the smallest increase in Sftpc expression level in combination with Nkx2-1 in the 2D culture screening. In the 3TFs without Gata6, fewer Sftpc-GFP-emitting organoids were formed at day 7 (Supplementary Fig. 2a), and FACS also revealed a decrease in the percentage of AT2-like cells (Supplementary Fig. 2b, c). The Sftpc-GFP– Thy1.2– EpCAM+ cells that acquired epithelial features but did not develop an AT2-like pattern failed to express Sftpc, even after sorting and continued culture (Supplementary Fig. 2d). Second, based on the results of a literature survey, three new factors—Sox9, Id2, and Etv536,37,38—in addition to the 14 factors, were considered candidates for further analysis. The effect of the combinations of these three individual factors or their association with the 4TFs was assessed; however, Sftpc expression and the formation of Sftpc-GFP-emitting organoids were the strongest with 4TFs alone, even in 3D organoid culture (Supplementary Fig. 2e, f). Therefore, we used Nkx2-1, Foxa1, Foxa2, and Gata6 in further experiments.
Similar changes were observed in mouse dermal fibroblasts (MDFs) derived from adult mouse tissue (Supplementary Fig. 3a–c), where Sftpc expression did not differ with culture dimension (Supplementary Fig. 3d). We also experimented with adult lung fibroblasts (ALFs) using the same protocol. The qPCR results showed that Sftpc expression was ~1000-fold on day 7 (Supplementary Fig. 3e). However, unlike with MEFs and MDFs, only a few GFP+ Thy1.2− EpCAM+ cells were detected by FACS, which made passaging difficult.
Characteristics of the purified iPULs
Purified iPULs were capable of long-term fibroblast-free expansion beyond 180 days, with sub-passages every 7–10 days, although the expression of some AT2 cell markers decreased gradually (Fig. 2a and Supplementary Fig. 4a, b). Moreover, the expanded iPULs could be cryopreserved for a long time (100 days) and the thawed cells reformed their organoids (Supplementary Fig. 4c).
a Schematic showing the passaging and cryopreservation of iPULs. b mRNA expression levels of the indicated genes in MEFs, unpurified iPULs (8 days after transduction), purified iPUL (P6–7) organoids, AT2 organoids (P3–6), primary AT2 cells isolated from Sftpc-GFP mice (8–18 weeks old), whole adult lungs (12–18 weeks old), and fetal lungs (E18.5). Data are presented as mean values ± SEM; n = 3 biologically and technically independent samples. c Representative hematoxylin and eosin (H & E)-stained images, GFP luminescence (green), and immunofluorescence staining for pro-Sftpc (AT2 cell marker, red) and Ager (AT1 cell marker, gray) in iPUL organoids. Scale bar: 50 µm (H & E) and 100 µm (others). d Transmission electron microscopy images of iPUL organoids (P6) containing lamellar bodies and microvilli (white arrows). Scale bar: left = 5 µm and right = 500 nm. e Live cell imaging of iPUL organoids at the indicated time points after the addition of LysoTracker Red DND-99 (100 nM) to the medium. Scale bar: 100 µm. f Principal component analysis (i), row-normalized heatmap data, and hierarchical clustering (ii) based on the RNA-seq analysis of MEFs, unpurified iPULs (8 days after transduction), purified iPULs (P6–7), AT2 organoids (P3–6), primary AT2 cells isolated from Sftpc-GFP mice (8–18 weeks old), adult lungs (12–18 weeks old), and fetal lungs (E18.5).
The expression of lung epithelial cell markers and the 4TFs was compared across various cell types, including MEFs and cultured or isolated primary AT2 cells from Sftpc-GFP mice, and in healthy adult and fetal lungs. The expression of the AT2 cell markers (Sftpc and Sftpb) increased in the purified iPULs, reaching values equal to or greater than those observed in AT2 organoids (Fig. 2b). Among alveolar epithelial type 1 (AT1) cell markers, Ager expression was comparable among unpurified iPULs, purified iPULs, and organoid-cultured AT2 cells; however, Aqp5 expression was lower in the purified iPULs (Fig. 2b). Although the expression levels of Scgb1a1 (a club cell marker), Foxj1 (a ciliated cell marker), and Krt5 (a basal cell marker), were slightly elevated compared to those in MEFs, they did not exceed those in organoid and primary-isolated AT2 cells from Sftpc-GFP mice. Compared with that in MEFs, the expression of Vim (a mesenchymal cell marker) was reduced in iPULs (Supplementary Fig. 4d). The expression levels of the 4TFs in the purified iPULs were higher than those in the MEFs and AT2 organoids, even after several passages (Supplementary Fig. 4e). Furthermore, normal karyotypes were maintained even under forced TF expression conditions (Supplementary Fig. 4f). To examine whether retroviral transgene expression was still detectable in the purified iPULs, quantitative polymerase chain reaction (qPCR) and electrophoresis were performed using primers specific for the four defined endogenous or exogenous TFs. The four exogenous TFs continued to be expressed in the iPULs after 10 passages (Supplementary Fig. 4g, 5). We conclude that exogenous 4TFs are stable and predominant in iPULs, therefore continuing to express Sftpc in the long term, similar to AT2 cells.
Immunofluorescence staining using the alveolosphere, which grew into a hollow organoid, revealed that the iPULs expressed the AT2 cell markers, pro-Sftpc, Sftpb, Abca3, and E-cadherin (an epithelial cell marker; Fig. 2c and Supplementary Fig. 6a); in contrast, Vimentin was enriched in MEFs, whereas it was not detected in the purified iPULs (Supplementary Fig. 6a, b). The AT2 organoids also expressed pro-Sftpc and Abca3 (Supplementary Fig. 6c). These results indicated that MEFs infected with the 4TFs transformed into AT2-like cells and lost their fibroblast-like characteristics. Ager was also expressed in few iPULs and AT2 organoids, while Aqp5 expression was slightly stronger in AT2 organoids than in iPULs, which was in agreement with the results of our qPCR analysis (Fig. 2c and Supplementary Fig. 6a, c). However, both iPULs and AT2 cells showed low expression of the common AT2 cell sorting marker, MHC class II39 (Supplementary Fig. 7a).
Transmission electron microscopy analyses further revealed that iPULs contained lamellar bodies, which are lysosome-related organelles specific to AT2 cells40, and surface microvilli (Fig. 2d), which are characteristic features of AT2 cells41. Live cell imaging showed that the lamellar body dye, lysotracker42, accumulated on iPUL organoids in 30 min (Fig. 2e).
We further performed bulk RNA-sequencing (RNA-seq) to examine the global gene expression profiles (Fig. 2f). Our principal component analysis included 16,745 genes, and the purified iPULs were plotted close to organoid AT2 cells. The unpurified iPULs were located between the purified iPULs and MEFs, whereas the primary-isolated AT2 cells were located between organoid AT2 cells and whole lungs (adult and fetal lungs). Previously reported major markers for lung epithelial cells and fibroblasts were selected for clustering analysis18,43,44. The purified iPULs clustered close to the organoid AT2 cells and were separated from MEFs. Consistent with the results of FACS analysis, the expression of MHC class II-related genes was lower in cultured cells than in isolated primary AT2 cells and adult lungs (Supplementary Fig. 7b).
Single-cell RNA profiling of the cell components of MEFs reprogrammed into unpurified and purified iPULs
We examined the heterogeneity of MEF-derived iPULs using single-cell RNA-sequencing (scRNA-seq). scRNA-seq was performed on organoids of 4TF-transduced MEFs cultured for 7 days (unpurified iPUL), organoids that were FACS-sorted and passaged several times (purified iPUL), and organoid AT2 cells from Sftpc-GFP mice that were moderately passaged (Fig. 3a). The agreement was satisfactory in the uniform manifold approximation and projection (UMAP) plot, in which the samples were integrated and delineated in the analytical phase, and the comprehensive transcriptomes showed similarities (Fig. 3b). In total, 25,946 cells were analyzed, and cell clustering using the Louvain algorithm resulted in six distinct clusters. We analyzed the characteristics of each cluster by referring to the violin plot of key marker expression, differentially expressed genes (DEGs), Gene Ontology (GO), and pathway analyses (Fig. 3c, Supplementary Fig. 8a–c, and Supplementary Fig. 9a–d). “Cluster 1”, “cluster 4”, and “cluster 5” expressed Sftpc (AT2 cell marker), Scgb1a1 (Club cell marker), and several stromal cell markers particularly well, respectively (Fig. 3d and Supplementary Fig. 8b, c). Pathway analysis also yielded terms associated with the cell type expressing each marker (Supplementary Fig. 9a–d). Genes and terms related to cell division and proliferation were prominent in “cluster 3”. Although “cluster 2” was defined as that in which the expression of some AT2 cell markers was slightly weaker than that in “cluster 1”, the other signatures were similar. The proportion of cluster cells comprising each sample differed; however, purification allowed the purified iPULs to exclude “cluster 5” with the lowest percentage of EpCAM+ cells and “cluster 6” with relatively high expression of stromal cell markers (Fig. 3e and Supplementary Table 1). Immunofluorescence staining of purified iPUL organoids showed that Krt8 (a pre-AT1 transitional cell state marker) and Mki67 (proliferating cell marker) signals partially overlapped with the GFP emission region (Fig. 3f). The DEGs among the cells classified into each cluster are listed in Supplementary Table 2.
a Schematic showing the experimental design of scRNA-seq of unpurified iPULs, purified iPULs, and AT2 organoids. Clustering of transcriptomes using UMAP plots. Cells are colored based on the cell origin (b) and Louvain algorithm (c). d Violin plots of AT2 cell marker expression at the single-cell level for each cluster. e Breakdown of the clustered cells per sample. f GFP luminescence (green) and immunofluorescence staining for Mki67 (proliferating cell marker, red) and Krt8 (transient cell marker, gray) in iPUL organoids. Scale bar: 100 µm. g Heatmap data of Pearson correlation coefficients for each cell type in the scMCA45. Z-scores were calculated for each column. “Dividing cells” represent the AT2-dividing cells. h RNA velocity analysis of the three samples.
We used a previously reported lung cell dataset from the single-cell Mouse Cell Atlas (scMCA)45 to validate our findings. Assuming that the TF-transduced fibroblasts did not differentiate into hematopoietic cells, the lung cell dataset, excluding hematopoietic cell clusters, was used for this analysis. We calculated the Pearson correlation coefficients between our scRNA-seq data and each cell type in the lung cell dataset (Fig. 3g). Ten clusters with differential expression of representative gene markers were identified based on the scMCA annotation (Supplementary Fig. 10a). In particular, purification of iPULs eliminated “Stromal cell” (24% to 0%) and reduced the “unclassified” (42% to 24%) category while significantly increasing the percentage of “AT2 cell” (26% to 58%). Regarding the distribution of AT2 organoids, “AT2 cell,” “Club cell,” and “Dividing cell” accounted for approximately a third of the cells each (Supplementary Fig. 10b, c and Supplementary Table 3). Although the majority of “cluster 1” through “cluster 6” were also strongly assigned in scMCA predictions to cell types characterized by markers highly expressed in each cluster, “cluster 2” of unpurified iPULs contained 66% of “unclassified” category (Supplementary Fig. 10d). Sorting and passaging of the EpCAM- GFP+ population in unpurified iPULs revealed the presence of cells that highly express EpCAM over time; it is possible that these populations were included in the “unclassified” category, and that purification and passage may have reduced their proportion (Supplementary Fig. 10e, f). RNA velocity analysis revealed that unpurified iPULs have a higher ability to resist reprogramming than purified iPULs and return to MEFs (stromal cells; Fig. 3h).
Contribution of media components to cell maintenance and proliferation
iPULs did not express genes such as Sox2 (a proximal lung progenitor cell marker), Pax8/Foxe1 (thyroid cell markers), and Emx2/Pax6 (forebrain markers), confirming the lack of differentiation toward these cells (Supplementary Fig. 11a). In contrast, Sox9, the distal lung epithelial progenitor cell marker, was expressed. Suppression of the glucocorticoid pathway owing to the removal of 8-bromoadenosine 3ʹ,5ʹ-cyclic monophosphate (8-Br-cAMP) and 3-isobutyl-1-methylxanthine (IBMX) from the culture medium did not result in significant differences in the gene expression levels of Sftpc, Sox2, and Sox9, as well as the protein levels of Sox2 and Sox9 (Supplementary Fig. 11b–d). In agreement with the fragments per kilobase of exon per million mapped reads (FPKM) values in RNA-seq analysis, the low expression of Sox2 negated the airway fate, while the higher expression of Sox9 in iPULs than that in AT2 cells indicated that the cells acquired a distal alveolar epithelial fate (Supplementary Fig. 11e). Hence, iPULs were cultured in strictly controlled media supplemented with various factors previously reported to enable efficient induction and maintenance of AT2 cells from lung specimens, iPSCs, and/or ESCs9,18,19,33,46,47. Gene set enrichment analysis indicated that purified iPULs and AT2 organoids differed with respect to proliferation, differentiation, and response to stimuli (Fig. 4a). Hence, we first examined differences in iPUL and AT2 cell growth rates under complete medium culture conditions. The doubling time during the logarithmic growth phase (days 2–5) was 24.3 h for iPULs and 16.4 h for AT2 cells (Fig. 4b, c). Next, to assess the importance of each factor in iPUL formation and growth compared with their necessity for AT2 cell proliferation in culture, each factor was successively excluded from the culture medium, and the subsequent effect on the number of cells was observed. Activation of fibroblast growth factors (FGFs; FGF2, FGF7, and FGF10), Wnt signaling pathway members (Afamin-Wnt/R-spondin), and epidermal growth factor (EGF), and inhibition of Smad signaling (A83-01) were required for iPUL self-renewal; however, vascular endothelial growth factor (VEGF) activation was not essential. Despite some differences in responsiveness to the bone morphogenetic protein (BMP) signaling pathway, the percentage of cells under each condition tended to be similar in iPULs and AT2 cells (Fig. 4d, e). This suggested that iPULs closely resembled AT2 cells in terms of their niche dependence in the context of cell proliferation within the microenvironment.
a Gene set enrichment analysis of purified iPULs and AT2 organoids. The identified, activated, or suppressed gene groups in iPULs were compared to those in AT2 organoids (biological process). b Growth curves of iPULs (P5–7, red solid line) and AT2 cells (P5–7, blue dotted line) with 5000 cells seeded per well. c Average doubling times for iPULs (P5–7) and AT2 cells (P5–7) during the logarithmic growth phase (d 2–5). d iPUL and AT2 organoid growth in iPUL basal medium (iPUL medium) or without niche factors (d 7, P8 for iPUL; day 4, P6 for AT2). Scale bar: 1 mm. e Total cell count per well in conditioned medium (iPUL: day 7, P5–7; AT2: day 5, P6–7). Data are presented as mean values ± SEM; n = 3 biologically independent samples. f mRNA levels of AT2 and AT1 markers in organoids under various conditions (iPUL: day 7, P5–7; AT2: day 5, P5–7). Data are presented as mean values ± SEM; n = 3 biologically and technically independent samples. g Organoid growth in iPUL medium or without FGFs (iPUL: day 6, P8; AT2: day 4, P7). Scale bar: 1 mm. h Total cell count without FGFs (iPUL: day 6–7, P5–8; AT2: day 5–7, P5–6). Data are presented as mean values ± SEM; n = 3 biologically independent samples. i mRNA levels of AT2 and AT1 markers without FGFs (iPUL: day 7, P5–6; AT2: day 5–7, P5–6). Data are presented as mean values ± SEM; n = 3 biologically and technically independent samples. j GFP luminescence (green) and immunofluorescence staining for Ager (AT1 cell marker, red) and Krt8 (transient cell marker, gray) in iPUL organoids. Scale bar: 100 µm. GFP luminescence and immunofluorescence staining for Ager (red) and Krt8 (gray) in iPULs and AT2 cells (P7) (k) and percentage of cells positive for each marker (l) before and after 7-day culture in AT2 differentiation medium. Scale bar: 100 µm. Data are presented as mean values ± SEM; n = 3 biologically independent samples.
The expression of key alveolar epithelial markers in iPULs remained generally unchanged under all conditions, possibly because of their robustness following the forced expression of 4TFs. In contrast, the levels of both Sftpc (AT2 marker) and Ager (AT1 marker) were elevated in AT2 cells in the absence of FGFs or EGF activation, suggesting a higher sensitivity to these factors (Fig. 4f and Supplementary Fig. 11f). We subsequently used the same approach to determine which of the three types of FGFs exerted the strongest effect on the medium. Lack of FGF7 activation inhibited the proliferation of both iPULs and AT2 cells (Fig. 4g, h). Expression of the AT1 markers tended to be high in iPULs, whereas the AT2 cells showed elevated levels of both Sftpc and Ager, as was observed with FGFs (Fig. 4i).
While evaluating differentiation ability, we confirmed that normal cultures in iPUL medium expressed Krt8, whereas Ager expression was negligible (Fig. 4j). iPULs and AT2 cells were cultured under conditions that possibly induced differentiation into AT1 cells48,49. First, we evaluated differentiation under organoid culture conditions that mimicked the in vivo 3D environment without Wnt signaling33. We used a differentiation medium (AT2 differentiation medium50) that did not contain factors involved in the Wnt pathway or FGF7, the importance of which was demonstrated in our niche-dependent experiments. After 7 day of incubation, the percentage of Ager+ and Krt8+ cells increased even in iPULs, thereby indicating a tendency to differentiate into AT1 cells (Fig. 4k, l).
After 7 day of 2D culture, the iPULs did not show any clear morphological differences from the pre-culture in bright-field image analysis. In contrast, most AT2 cells showed flat thin cytoplasm characteristic of AT1 cells, within a few days. Immunofluorescence images showed that most cells had lost GFP luminescence; however, Aqp5 was more universally expressed in AT2 cells than in iPULs (Supplementary Fig. 11g).
Regenerative potential of iPULs in vivo
To assess whether iPULs can be engrafted into the lungs, we used a mouse model of bleomycin-induced pulmonary fibrosis. iPULs were labeled with mCherry via lentivirus-mediated transduction, and mCherry+ iPULs (GFP+ Thy1.2– EpCAM+ mCherry+) were sorted using FACS immediately before transplantation (Fig. 5a and Supplementary Fig. 12a, b). As a control, AT2 cells prepared using the same procedure were administered intratracheally into the lungs of immunocompromised CB17/Icr-Prkdcscid/CrlCrlj (SCID) mice, and the cell type could be evaluated through fluorescent immunostaining (Supplementary Fig. 12c). To assess their long-term safety in vivo, iPULs were first transplanted subcutaneously into SCID mice. Control SCID mice transplanted with human and mouse lung cancer cell lines showed tumor development; however, iPULs did not form tumors even after more than 70 d (Supplementary Fig. 12d).
a Schematic showing the mCherry-expressing iPUL production process. b Schematic showing the experimental procedure. c Immunofluorescence staining of lung tissue 14 d after transplantation with mCherry-expressing iPULs. Scale bar: 100 µm. d Percentage of positive areas for each cell marker in mCherry-positive transplanted iPULs 14 day after transplantation. Data are presented as mean values ± SEM; n = 4 biologically independent samples. e Two-parameter dot plots of recipient lung epithelium 42 days after transplantation. f Percentage of transplanted iPULs in the recipient lung epithelium 42 days after transplantation. Data are presented as mean values ± SEM; n = 5 biologically independent samples. g The percentage of each fluorophore expression pattern in iPULs was re-assessed 42 days after transplantation. h Immunofluorescence staining of lung tissue 42 days after the transplantation of mCherry-expressing iPULs. Scale bar: 100 µm. i Percentage of positive areas for each cell marker in mCherry-positive iPULs 42 days after transplantation. Data are presented as mean values ± SEM; n = 3 biologically independent samples.
Next, 10 days after the intratracheal administration of bleomycin to SCID mice, the sorted iPULs were intratracheally administered and analyzed for engraftment and differentiation status in three phases: early (after another 14 days), mid (42 days), and late (100 days; Fig. 5b). We observed that mCherry+ iPULs engrafted into the lungs early after transplantation were localized only in the alveolar regions and not in the airway regions, which harbored the club cells (Scgb1a1-positive; Supplementary Fig. 12e). The transplanted iPULs coexpressed pro-Sftpc, Ager, and Krt8, suggesting that iPULs tended to differentiate into AT1-like cells in vivo, similar to that observed in vitro (Fig. 5c, d). In the mid-phase, iPULs represented ~0.5% of the recipient lung epithelium. These presumably included cells that lost mCherry expression due to gene silencing and those that lost GFP expression because of differentiation (Fig. 5e–g). Immunofluorescence staining revealed similar percentages of positive areas for cell markers in the early and mid-phases (Fig. 5h, i). iPULs remained visible as grafts in the late phase (Supplementary Fig. 12f).
We also demonstrated that the transplanted iPULs may contribute to the regeneration of injured lungs. The organoid culture of iPULs re-collected in the mid-phase using FACS showed adequate proliferation of mCherry+ and/or GFP+ cells (Fig. 5b and Supplementary Fig. 13a). Next, a second dose of bleomycin was administered 21 days after iPUL transplantation to induce re-injury and analyze whether the previously engrafted iPULs proliferated in vivo (Supplementary Fig. 13b). The transplanted iPULs expressed Mki67 at a higher rate than endogenous AT2 cells, indicating that they proliferated well in the injured lung (Supplementary Fig. 13c, d).
Discussion
In the present study, we showed that mouse fibroblasts can be directly reprogrammed into AT2-like cells (iPULs) using a combination of 4TFs (Nkx2-1, Foxa1, Foxa2, and Gata6). The reprogrammed cells can be transplanted into the alveolar region of immunocompromised mice, and they resemble AT2 organoids in the following aspects. First, purification of a defined cell population (GFP+ Thy1.2– EpCAM+ cells) at the initial stage supported long-term expanded culture in vitro while maintaining the expression of Sftpc, the AT2 cell marker. Second, the cells were cuboidal in shape with microvilli and intracellular lamellar body-like structures, which store and secrete surfactant phosoholipids40. Third, bulk and scRNA-seq profiling revealed a transcriptome similar to that of cells of the AT2 lineage. Fourth, the niches of the AT2 cells and iPULs were similar in terms of cell proliferation. Fifth, under specific conditions, iPULs showed the potential to differentiate into AT1-like cells both in vitro and in vivo4.
The results of this study are in agreement with those of previous studies on the alveolar epithelial region represented by iPSC-derived AT2-like cells, highlighting commonalities that reinforce their findings. Recent studies using AT2 cells and iPSC/ESC-derived AT2-like cells have used organoids, a 3D tissue platform that closely mimics the native tissue microenvironment in vitro51,52,53. We successfully developed an organoid culture system that considerably improved the induction efficiency of iPULs and maintained tissue stem cell properties over time. Moreover, based on recent insights into the largely uncharacterized niche factors of AT2 cells, iPULs can be cultured under feeder-free conditions after purification by adjusting the medium composition. However, precise replication of the in vivo environment under in vitro conditions is challenging, and the effect of culture on the transcriptomic program cannot be eliminated in alveolar epithelial cells48. In fact, the results of our qPCR and RNA-seq analyses indicated that the RNA expression profiles of iPULs were more similar to those of organoid AT2 cells than those of isolated primary AT2 cells. We also observed that compared to the primary AT2 cells, the iPULs showed lower expression of genes related to MHC class II54. This feature is similar to that of iPSC-derived AT2-like cells, albeit not specific to engineered cells52. Unlike mature AT2 cells, iPULs may have to be refined further to induce cells that can attain full functional maturity, as indicated by their expression of Sox9, a marker of distal progenitors.
We identified 4TFs, the expression of which was critical in generating iPULs that maximized Sftpc expression. We performed iPUL niche searches focusing on cell proliferation and the expression of representative AT1/AT2 cell markers. We concluded that these TFs play important roles in the direct reprogramming of iPULs. Nkx2-1 is selectively expressed in the lungs, thyroid gland, and central nervous system55,56. Nkx2-1 has been reported to control the expression of surfactant proteins essential for lung stability and pulmonary host defense, including Sftpc57. Foxa1 and Foxa2 are closely related members of the Foxa gene family that are coexpressed in subsets of pulmonary epithelial cells to perform complementary functions during lung morphogenesis58,59. Both genes activate Nkx2-1 transcription in pulmonary epithelial cells60. Gata6 is a member of the zinc-finger transcriptional regulator family and the only known Gata factor expressed in the distal epithelium during lung development61,62. Gata6 and Nkx2-1 directly interact with and regulate the expression of Sftpc63. Based on these reports, we hypothesized that these 4TFs facilitate Sftpc expression.
A series of feeder-free culture systems and exploratory experiments following the introduction of the 4TFs provided interesting insights regarding the iPUL niche. Feeder-free cultures have recently replaced co-culture with fibroblasts as the mainstream method because of ease in cell maintenance and elimination of the step where unwanted cells had to be removed prior to in vivo administration47. In this study, we added Wnt pathway activators, growth factors (FGFs, EGF, and VEGF), and dual Smad signaling (TGF-β and BMP) inhibitors to the culture medium. Activation of the Wnt pathway potentiates the self-renewal of AT2 cells and inhibits their differentiation into AT1 cells9,33,46. The addition of FGF7 (KGF), an important mediator of lung organogenesis and primary AT2 cell maturation and proliferation, has been shown to promote alveolosphere growth47,64,65. FGF2 and FGF10 are key molecules responsible for the efficient generation of lung and airway epithelial cells34,66. These factors can increase the efficiency of organoid formation67 or lead to the formation of larger organoids51 in vitro. However, the importance of FGFR2 as a ligand of FGF7 and FGF10 in adult AT2 cell homeostasis remains controversial6,68,69,70. Although AT2 cells and iPULs exhibited a similar niche in terms of cell proliferation, iPULs appeared to depend more on FGF7 than AT2 cells. In terms of TGF-β/BMP signaling, AT2 cells may be inhibited in the context of fibroblast-epithelial interactions35,47.
This study offers novel preclinical proof of concept for cell therapies for refractory lung diseases. Although our lung transplantation study demonstrated the regenerative potential of AT2 cells as stem cells in damaged lungs, their practical application in clinical settings was hindered by the difficulty in obtaining large quantities of normal AT2 cells or iPSCs. Several reports have demonstrated the successful transplantation of iPSCs/ESC-derived lung epithelial cells or their progenitor cells into a mouse model of lung injury and their successful engraftment in the alveolar region52,71,72. These studies uncovered the potential for both indirect fibrosis alleviation via paracrine signaling from the induced cells and direct replacement and regeneration of lung epithelial tissue. Our direct reprogramming protocol has the advantages of easy induction, short turnaround time, and potential for use in vivo reprogramming, potentially replacing iPSC-derived AT2 cells in lung regeneration.
This study has some limitations. First, TF selection may have been biased as the expression of only Sftpc was considered among the AT2 cell markers. Fibroblasts that successfully switched fate to iPULs following forced expression of the 4TFs were strongly oriented toward differentiation into AT2 cells. Our in vitro iPUL culture showed limited AT2 cell features, such as the ability to morphologically differentiate into AT1 cells. Although direct reprogramming of fibroblasts into alveolar epithelial-like cells was demonstrated, we also detected a population of cells that gradually acquired epithelial characteristics through several passages after direct reprogramming. We have also confirmed that the same 4TF transfection method used with mouse cells cannot induce AT2-like cells in human cells. Hence, more efficient and applicable methods have to be developed. A growing body of research on cell types that can be successfully and directly reprogrammed includes the transition from mouse models to human applications28 and investigations regarding in vivo direct reprogramming using reliable delivery methods28,73,74. Second, the therapeutic effects of iPULs were not assessed in vivo owing to the low percentages of transplanted cells, which hindered the determination of the exact effect of replacing the damaged alveolar epithelium with iPULs. More efficient engraftment would enable comparisons of the iPUL transcriptome pre- and post-administration. Detailed study of the expression of classical AT1 markers, Ager, Aqp5, and Hopx, as well as that of new markers such as Gprc5a and Rtkn275, may help in revealing the mechanism underlying the repair process after lung injury and maturation over time.
In conclusion, we demonstrated that the 4TFs induced functional pulmonary epithelial cell-like cells in mice. Further studies, including those on human cells, are required to adapt this technology to clinical practice, particularly for regenerative therapy.
Methods
Animals
This study was conducted in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals, the National Institutes of Health. All animal experiments were approved by the Animal Use Committee of the Keio University School of Medicine (protocol No. A2022-268 and A2022-276). Pregnant C57BL/6 female mice were purchased from CLEA Japan, and neonatal C57BL/6 mice were used for TTF culture. SFTPC-GFP mice with a CBA/Ca×C57BL/6J mixed genetic background (kindly provided by Prof. Brigid Horgan, Duke University) were bred in-house and were used to prepare MEFs and control samples. Fetal lungs (E18.5), healthy adult lungs (male and female, 12–16 weeks old), and AT2 cells (male, 8 weeks old) were isolated from mice euthanized by intraperitoneal administration of combined triad anesthesia of medetomidine, midazolam, and butorphanol (0.3, 4.0, and 5.0 mg/kg). For the in vivo experiments, C.B-17 SCID mice (male, 8–10 weeks old) were obtained from Charles River Laboratories Japan, Inc. (Yokohama, Japan). All mice were maintained under specific pathogen-free conditions in a facility supervised by the Laboratory Animal Center at Keio University School of Medicine.
Primary cell cultures
To prepare TTFs, neonatal or adult (8 weeks old) C57BL/6 mice were anesthetized by intraperitoneal injection of a mixture of ketamine and xylazine (100 and 20 mg/kg), then their tails were cut off and washed with phosphate-buffered saline (PBS). The tails were minced into 2-mm pieces and placed in a 0.1% gelatin-coated plate with Iscove’s modified Dulbecco’s medium containing 20% fetal bovine serum (FBS), 50 U/mL penicillin/streptomycin (P/S), and 2.5 μg/mL amphotericin B (all from Thermo Fisher Scientific, Waltham, MA, USA). After incubation at 37 °C for 5 d in an atmosphere of 5% CO2, the fibroblasts migrated out of the tails and were cultured until they reached confluence. Thereafter, the cells were passaged using 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA; Thermo Fisher Scientific).
MDFs were isolated from adult (8 weeks old) Sftpc-GFP mice as described previously76, with slight modifications. Briefly, the ears were cut using scissors such that their radius was ~7–8 mm and the pieces were incubated in 40 mL of 70% ethanol in sterile 50 mL conical tubes for 5 min. Hair was removed after air-drying and the ears were cut into <3 mm-thick sections. Next, the sections were transferred into 1.8 mL cryotube vials, filled with the liberase (Roche, Basel, Switzerland)/dispase (Sigma-Aldrich, St. Louis, MO, USA) enzyme mixture, and incubated horizontally on a shaker at 200 rpm for 90 min at 37 °C. Next, 10 mL of high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Wako Chemicals, Neuss, Germany) containing 10% FBS and 50 U/mL P/S was added to a 10-cm cell culture dish. A 70-µm cell strainer was placed over it, in which digested tissue was ground using a 10-mL syringe plunger for 5 min. The resulting cell suspension was centrifuged at 580 × g for 7 min; the supernatant was removed, and resuspended in 10 mL high-glucose DMEM containing 10% FBS, 50 U/mL P/S, and 10 μL amphotericin B solution (stock solution: 250 µg/mL), and incubated in a 10-cm dish at 37 °C in an atmosphere of 5% CO2.
Female Sftpc-GFP mice were sacrificed 13.5 d post-coitum by intraperitoneal administration of combined triad anesthesia to prepare MEFs. The embryos were separated from the placenta and surrounding membranes. Limbs isolated from the embryos were transferred to a clean dish and minced finely. The minced fragments were placed in tubes containing PBS and centrifuged at 300 × g for 6 min. The supernatant was discarded, the pellet was suspended in 0.05% trypsin/EDTA, and the cells were dissociated into single cells via pipetting and repeated filtering through a 70-μm cell strainer (Falcon). Single cells were plated onto a dish and cultured in high-glucose DMEM supplemented with 10% FBS and 50 U/mL P/S. Upon reaching confluence, the cells were passaged using 0.25% trypsin/EDTA.
To prepare AT2 cells from Sftpc-GFP mice, lung digestion was performed as described previously77. Briefly, the lung lobes were collected and finely minced using scissors. These samples were subsequently incubated in the liberase/dispase enzyme mixture for 10 min at 37 °C. The solution was passed through a 21G needle (four times) and filtered through a 100-μM mesh. DNase (Worthington, Columbus, OH, USA) was added to the mesh to release the entangled cells. The cells were sorted using a cell sorter to obtain GFP+ CD31− CD45− Thy1.2− EpCAM+ cells (AT2 cells). When the cells were plated onto a dish prior to sorting and cultured under the same conditions as MEFs, the cells that adhered to the bottom of the dish and proliferated were collected as ALFs.
Molecular cloning and retroviral production
To prepare the pMXs retroviral vectors, the open reading frame of each candidate gene (Nkx2-1, Foxa1, Foxa2, Foxj1, Tcf21, Hoxa5, Sox17, Gata6, Tbx4, Gata5, Foxf1, Foxl1, Gli2, and Gli3) was amplified using PCR and AccuPrime Pfx DNA polymerase (Thermo Fisher Scientific), followed by subcloning into the pMXs retroviral vector (RTV-010 Cell Biolabs, San Diego, CA, USA) using the Gateway cloning kit, according to the manufacturer’s protocol (Thermo Fisher Scientific).
Platinum-E (Plat-E) cells (Cell Biolabs, San Diego, CA, USA) were subcultured in 10 mL fibroblast-platinum (FP) medium supplemented with 1 μg puromycin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and 10 μg blasticidin S (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The FP medium consisted of high-glucose DMEM supplemented with 10% FBS, 50 U/mLP/S, 1 mM sodium pyruvate (Sigma-Aldrich), 1% MEM nonessential amino acid solution (Sigma-Aldrich), and 1% GlutaMAX (Thermo Fisher Scientific).
For retroviral production, Plat-E cells were seeded at the density of 3.6 × 106 cells/10 mL FP medium in gelatin-coated 100-mm culture dishes. The cells were incubated overnight at 37 °C in an atmosphere of 5% CO2. The next day, 300 μL Opti-MEM1 (Thermo Fisher Scientific) was transferred to a 1.5-mL tube, followed by mixing with 27 μL FuGENE 6 (Promega) transfection reagent and incubation for 5 min at 20–25 °C. Next, 9 μg of each pMXs plasmid DNA was added to a FuGENE 6/Opti-MEM-containing tube and incubated for 15 min at 20–25 °C. The DNA/FuGENE 6 complex was gently added to the Plat-E dish, which was incubated overnight at 37 °C in the presence of 5% CO2. The transfection reagent-containing medium was aspirated, and 10 mL of fresh FP medium was added to the dish.
Retroviral infection
Twenty-four hours after changing the medium, the retrovirus-containing medium in the Plat-E dish was collected and filtered through a 0.45-μm pore-size sterile syringe filter (Corning, NY, USA). Freshly generated retroviruses were used for each experiment to maximize reprogramming efficiency, with a multiplicity of infection of pMX-Nkx2-1/Foxa1/Foxa2/Gata6 of ~10 (8, 13, and 10 for three independent experiments). Polybrene solution (40 µg, Millipore, Burlington, MA, USA) was added to 10 mL of the filtered virus-containing medium; the mixture of the virus-containing medium was prepared based on the gene-induction plan. Approximately 5–10 × 105 fibroblasts were seeded onto a 10-cm dish 1 day before infection. The culture medium was aspirated, and 10 mL of polybrene/virus-containing medium was added to the fibroblast dish for retroviral infection. For 2D cultures, the medium was removed from the fibroblast dish and 10 mL of fresh FP medium was added after 24 h of incubation. The medium was changed every other day during the culture period. For 3D cultures, MEFs transduced with retroviruses encoding the 4TFs were collected from a 100-mm dish 24 h after infection and cultured in the 3D organoid system.
3D organoid culture
MEFs transduced with the 4TFs or DsRed, purified iPULs, or AT2 cells (1–2 × 104 cells each) were embedded in 20–25 µL Matrigel (Corning), plated onto each well of a 48-well plate (Corning), and cultured in iPUL medium, which contained advanced DMEM/F12 supplemented with 10 mM HEPES, 2 mM GlutaMAX, 100 U/mL penicillin, 100 mg/mL streptomycin, 1 × B27 (Thermo Fisher Scientific), 1 mM N-acetylcysteine (Sigma-Aldrich), 0.1 mM 8-Br-cAMP (Biolog Life Science Institute, Bremen, Germany), 0.1 mM IBMX (Wako Chemicals), and niche factors, including 5% Afamin-Wnt-3A serum-free conditioned medium78, 50 ng/mL mouse recombinant EGF (Thermo Fisher Scientific), 10 ng/mL FGF2 (BioLegend, San Diego, CA, USA), 100 ng/mL mouse recombinant FGF7 (R&D Systems, Minneapolis, MN, USA), 100 ng/mL FGF10 (BioLegend), 5 ng/mL VEGF (Sigma-Aldrich), 20 ng/mL mouse recombinant Noggin (Peprotech, Cranbury, NJ, USA), 2% R-spondin-1 conditioned medium (R&D Systems)79, and 500 nM A83-01 (Tocris, Bristol, UK). To prevent anoikis, 10 µM Y-27632 (FUJIFILM Wako Pure Chemical) was added to the culture medium for the first 2 d. To investigate niche factor dependency, 10 ng/mL TGF-β (R&D Systems) and 100 ng/mL BMP4 (Peprotech) were used. The medium was changed every other day. Fluorescence imaging with GFP was performed using a BZ-X810 digital microscope (Keyence, Osaka, Japan).
To prepare single-cell suspensions for flow cytometry, passaging, and cryopreservation, iPULs and AT2 organoids were dissociated via gentle pipetting with TrypLE Express (Thermo Fisher Scientific). For immunofluorescence staining and electron microscopy analysis, organoids were released from the Matrigel using a Cell Recovery Solution (Corning).
2D culture for differentiation
Approximately 3–5 × 105 iPULs or AT2 cells were sorted as GFP/Sftpc+ EpCAM+ using FACS immediately before seeding onto a 35-mm glass-bottom dish (Corning) pre-coated with PureCol (Advanced BioMatrix, Carlsbad, CA, USA). Previously reported DCIR medium5 was used with slight modifications: advanced DMEM/F12 was supplemented with 10 mM HEPES, 100 U/mL penicillin, 100 mg/mL streptomycin, 1 × B27, 1 mM N-acetylcysteine, 0.25% bovine serum albumin (Sigma), 50 mM dexamethasone (Sigma), 100 µM 8-Br-cAMP, 100 µM 3-isobutyl-1-methylxanthine, 10 ng/mL mouse recombinant FGF7, and 0.1% insulin-transferrin-selenium (Thermo Fisher Scientific) to promote differentiation. Y-27632 (10 mM) was added for the first 2 d.
Lentiviral infection
Two wells of iPUL organoids (~4–5 × 105 cells) were dissociated into a single-cell state using TrypLE Express and centrifuged at 400 × g for 3 min to obtain a visible cell pellet. Next, the pellets were suspended in a mixture of 1 mL lentivirus concentrate encoding mCherry, 10 µg polybrene solution, 10 µM Y-27632, and 30 µL Matrigel and seeded into a 24-well clear flat bottom ultra-low attachment multiple-well plate (Corning). The plate was centrifuged at 600 × g and 32 °C for 1 h and then incubated for 6 h at 37 °C in the presence of 5% CO2. Once the mixture and iPULs were collected in a 15-mL conical tube, the cells were dissociated using TrypLE Express and cultured as 3D organoids for normal passages. Target iPULs expressing mCherry were sorted using flow cytometry as described below.
Live cell imaging
LysoTracker Red DND-99 (100 nM; Thermo Fisher Scientific) was added to the medium of iPUL organoids grown on a 35-mm glass-bottom dish. Images were captured using a TCS SP5 (Leica, Wetzlar, Germany) every minute for 30 min.
Gene expression analysis
Total RNA was isolated from TTFs, MEFs, iPULs, AT2 organoids, and whole lungs using the RNeasy mini kit (Qiagen, Hilden, Germany). cDNA was synthesized via reverse transcription of RNA using the ReverTra Ace qPCR RT master mix (TOYOBO, Osaka, Japan) and TaKaRa thermal cycler Dice Touch (Takara, Kusatsu, Japan). RNA was quantified using NanoDrop One (Thermo Fisher Scientific) and the amount of RNA input for cDNA synthesis was standardized within each experiment. Expression levels were determined using the Quantstudio 5 real-time qPCR system (Applied Biosystems, Foster City, CA, USA) and the KAPA SYBR FAST quantitative PCR (qPCR) kit (KAPA Biosystems, Wilmington, MA, USA) or THUNDERBIRD Next SYBR qPCR mix (TOYOBO). Beta-actin was used for normalization in these studies.
According to a previous report80, the DNA of endogenous or exogenous 4TFs (Nkx2-1, Foxa1, Foxa2, Gata6) were amplified using reverse transcription (RT)-PCR. The PCR samples were stained using 5 × GelPilot DNA loading dye (Qiagen) and separated via agarose gel electrophoresis at 100 V using Mupid-exU (Takara) filled with 1 × Tris-acetic acid-EDTA buffer (Nippon Gene, Tokyo, Japan). The target area of the agarose gel containing 2% Midori Green Xtra (Nippon Genetics, Tokyo, Japan) was imaged using FAS-IV (Nippon Genetics) and aligned with the position of the molecular weight marker. The primer sequences for each target gene are listed in Supplementary Table 4.
Immunofluorescence staining
To stain the 2D-cultured TTFs, cells were cultured on cell chamber slides (Falcon) for 14 days and fixed using 4% paraformaldehyde for 15 min, after which the slides were washed twice with PBS and blocked with goat serum (Vector Laboratories, Newark, CA, USA) for 30 min. The primary rabbit anti-SFTPC antibody (1:400, #ABC99, Millipore) was diluted in protein blocking reagent and incubated overnight at 4 °C. Alexa Fluor-conjugated secondary antibodies (Thermo Fisher Scientific) were used for double staining. Nuclei were counterstained using 4′,6-diamidino-2-phenylindole (DAPI; #H-1200, Vector Laboratories).
To stain iPULs and AT2 organoids, the organoids released from the Matrigel were fixed using 4% paraformaldehyde for 20 min at 20–25 °C. The organoids were washed twice using PBS and further blocked using Power Block Universal blocking reagent (BioGenex, Fremont, CA, USA) for 10 min at 20–25 °C, followed by membrane permeabilization using 0.2% Triton X-100 in PBS and incubation with the primary antibody overnight at 4 °C with gentle rocking. The organoids were subsequently washed thrice with PBS and stained with secondary antibodies for 30 min at 20–25 °C with gentle rocking in the dark. Nuclear counterstaining was performed simultaneously using Hoechst 33342 (1:1000, #H3570, Thermo Fisher Scientific). The stained organoids were suspended in one drop of ProLong Diamond antifade mountant (Thermo Fisher Scientific) and mounted onto a 35-mm glass-bottom dish. For cells in 2D culture, a series of protocols was performed on a glass-bottom dish. Images were captured using a TCS SP5 microscope (Leica). The antibodies used for imaging analysis were rabbit anti-proSP-C (1:400, #AB3786, Millipore), mouse anti-Sftpb (1:500, #sc-133143, Santa Cruz Biotechnology), rabbit anti-Abca3 (1:400, #WRAB-70565, Seven Hills Bioreagents, Cincinnati, OH, USA), rabbit anti-Aqp5 (1:1000, #ab78486, Abcam, Cambridge, UK), goat anti-Rage/Ager (1:400, #AF1145, R&D Systems), rabbit anti-vimentin (1:250, #5741, Cell Signaling Technology), rat anti-vimentin (1:200, #MAB2105, R&D Systems), Alexa Fluor 647 mouse anti-E-cadherin (1:200, #560062, BD Biosciences, Franklin Lakes, NJ, USA), rat anti-cytokeratin 8 (1:500, #TROMA-I, Developmental Studies Hybridoma Bank, Iowa City, IA, USA), and rabbit anti-Ki67 (1:500, #ab16667, Abcam). Subsequently, the appropriate Alexa Fluor-coupled secondary antibodies (1:250, Thermo Fisher Scientific, or Abcam) were used as required.
Lung tissues were fixed using 4% paraformaldehyde, embedded in paraffin, and sectioned before staining. The lung sections were deparaffinized in xylene and rehydrated using a graded ethanol series, and antigen retrieval was performed by heating the samples in sodium citrate buffer at 95 °C for 20 min. A non-serum protein block (Dako, Golstrup, Denmark) was applied for 30 min. The rabbit anti-proSP-C (1:200, #AB3786, Millipore), rabbit anti-RFP (1:200, #PM005, MBL Life Science, Tokyo, Japan), mouse anti-DsRed (1:200, #sc-390909, Santa Cruz Biotechnology), goat anti-Rage/Ager (1:400, #AF1145, R&D Systems), rat anti-cytokeratin 8 (1:50, #TROMA-I, Developmental Studies Hybridoma Bank), rabbit anti-Ki67 (1:200, #ab16667, Abcam), and/or goat anti-CCSP/CC10/Scgb1a1 (1:200, #sc-9772, Santa Cruz Biotechnology) primary antibodies were diluted in a protein block and incubated overnight at 4 °C. Appropriate Alexa Fluor-coupled secondary antibodies (1:250, Thermo Fisher Scientific or Abcam) were used to double- and triple-stain the sections. Nuclei were counterstained using DAPI (#H-1200, Vector Laboratories).
Flow cytometry
Single-cell suspensions of iPULs and lung cells were prepared as described above. Single cells were washed in FACS buffer (PBS containing 2% FBS and 1 mg/mL sodium azide), transferred to test tubes with cell strainers (Corning), and incubated with an anti-CD16/CD32 monoclonal antibody (1:100, #14-0161-82, Thermo Fisher Scientific/eBioscience) for 10 min, followed by staining for 20 min with the following antibodies conjugated with the indicated fluorophores: APC anti-mouse CD326 (EpCAM; 1:200, #118214), PE/Cy7 anti-mouse CD90.2 (Thy1.2; 1:200, #140310), PE/Cy7 anti-mouse CD31 (1:200, #102418), PE/Cy7 anti-mouse CD45 (1:200, #103114), and APC-Cy7 anti-mouse I-A/I-E (1:200, #107627; all from BioLegend). The cells were subsequently washed twice with FACS buffer and stained using the live/dead Fixable near-IR dead cell stain kit (1:1000, #L34975, Thermo Fisher Scientific) or propidium iodide (1:1000, #556463, BD Biosciences). The stained cells were processed using a MoFlo XDP cell sorter (Beckman Coulter, Brea, CA, USA), and the data were analyzed using the FlowJo software v. 10.6.2 (Tree Star, Inc., Ashland, OR, USA).
RNA-seq analysis
RNA-seq libraries were constructed from polyA-selected RNA using TruSeq Stranded mRNA library preparation kit (Illumina, San Diego, CA, USA), and 100-bp single-end reads were generated on an Illumina NovaSeq 6000 system. After removing the reads aligned to ribosomal RNA using Bowtie 2.2.481, the remaining reads were aligned to the mouse reference genome (mm 10) using STAR 2.6.0a82. The number of reads aligned to each gene was counted using featureCounts v. 1.6.283 and RefSeq84, whereas the reads per kilobase per million mapped reads (RPKM) values for each gene were calculated based on the read count.
scRNA-seq analysis
The library for scRNA-seq was prepared using Chromium single cell 3’ reagent kits v3 (10X Genomics, Pleasanton, CA, USA). The scRNA-seq library of 28- and 98-bp paired-end sequencing reads was constructed using the NovaSeq6000 platform and one lane of the S1 flow cell. The sequenced data were processed using the CellRanger 3.1.0 pipeline. Briefly, the sequences were aligned with the mouse reference genome, the reads were separated using cell barcodes, the reads aligned to each gene were counted, and the PCR duplicates were normalized using a unique molecular identifier. The Seurat 4.4.0 and velocyto. R 0.6 software were used for single-cell analysis, following the guided clustering tutorial85. Single-cell data from samples were used when the number of detected genes was between 2000 and 8000 and the percentage of aligned reads to mitochondrial genes was ≤20%. Dimension reduction was performed using UMAP and clustering was performed using the first 30 principal components. For the classification of the predicted cell types, the Pearson correlation coefficients between our scRNA-seq data and each cell type in the scMCA were calculated45. When a single cell’s highest coefficient was ≥0.3 with a particular cell type, it was classified as that cell type. The single cells for which the highest correlation coefficient with any cell type was <0.3 were defined as “unclassified.”
Electron microscopy
The iPUL organoids were prepared as described previously. The organoids were fixed overnight using 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4 °C. Subsequently, the organoids were washed thrice using 0.1 M cacodylate buffer (pH 7.4) and post-fixed using 1% OsO4 in 0.1 M cacodylate buffer (pH 7.4) at 4 °C for 2 h. The organoids were electronically stained with 1% uranyl acetate (UA) at 4 °C for 30 min, dehydrated in graded ethanol solutions, and infiltrated twice with QY-1 (n-butylglycidyl ether; Nisshin EM Co., Ltd., Tokyo, Japan) for 20 min each, followed by incubation in 3:1, 1:1, and 1:3 mixtures of QY-1 and epoxy resin (Epok-812; Oken-shoji Co., Ltd., Tokyo, Japan) for 1 h each. Then, they were transferred to fresh 100% resin and infiltrated overnight. After two changes with fresh resin, the organoids were embedded in the resin and polymerized at 60 °C for 72 h. The polymerized resins were ultra-thin-sectioned at 80 nm using a diamond knife mounted onto an ultramicrotome (UC7; Leica). The sections were mounted on copper grids and subsequently stained with UA at 20–25 °C for 10 min, washed with distilled water, and secondarily stained with a lead staining solution at 20–25 °C for 10 min. The grids were observed using a transmission electron microscope (JEM-1400Plus, JEOL Ltd., Tokyo, Japan) at an acceleration voltage of 100 kV. Digital images (2048 × 2048 pixels) were captured using a CCD camera (EM-Z12162TTKDC; JEOL Ltd.).
Karyotype assay
Demecolcine (Sigma-Aldrich) was added to the iPUL medium of 70–90% confluent iPUL organoids to a final concentration of 0.01 μg/mL. After 3.5 h, a 100-fold diluted chromosome resolution additive (Genial Genetic Solutions, Chester, UK) was added to the 1% medium and placed in an incubator. After another 1.5 h, the iPUL organoids were dissociated into a single-cell suspension using TrypLE Express (Thermo Fisher Scientific) and collected in iPUL medium before centrifugation. The remaining cell pellet was resuspended in 0.075 M KCl and treated with a hypotonic solution at 20–25 °C for 15 min. An equal volume of Carnoy’s solution (methanol: glacial acetic acid 3:1) and KCl was added and mixed via pipetting. The cell pellets were centrifuged at 1500 × g for 5 min and pipetted twice in Carnoy’s solution to ensure adequate fixation. The cells were sent to Trans Chromosomics, Inc. (Tottori, Japan) for G-band analysis.
Bleomycin-induced mouse model of pulmonary fibrosis
Bleomycin (2.5 U/kg in 0.9% sterile saline; Sigma-Aldrich, B8416) was administered intratracheally to C.B-17 SCID mice anesthetized by intraperitoneal injection of a mixture of ketamine and xylazine. 10 d after bleomycin injection, the mice were injected via the same route with 1 × 106 in vitro-expanded mCherry+ iPULs (GFP+ Thy1.2– EpCAM+ cells), which were sorted on the day of administration and resuspended in 50 μL PBS. Some mice received another bleomycin injection after 21 days. The lungs collected from mice euthanized by intraperitoneal administration of combined triad anesthesia 14 (early), 42 (middle), and 100 (late) days after cell transplantation were evaluated. Independent experiments were performed with three mice per group.
Tumorigenicity and teratogenicity assay
In total, 1 × 105 3D organoid-cultured iPULs in 100 µL iPUL medium and A549 cells or Lewis lung carcinoma (3LL) cells cultured under 2D conditions in high-glucose DMEM supplemented with 10% FBS and 50 U/mL P/S were mixed with equal amount of Matrigel and injected subcutaneously into the backs of SCID mice. The mice were monitored weekly and evaluated for tumorigenicity 26 or 75 days after injection. We have complied with all relevant ethical regulations in the handling of A549 cells, including the Declaration of Helsinki.
Quantification and statistical analysis
Data are presented as mean ± SEM. Graphs were generated and statistical analyses were performed using the R software or GraphPad Prism 8 (GraphPad Software). Quantification of cell numbers and areas was performed using the ImageJ 1.54f software.
Data availability
The accession numbers for the RNA-seq and scRNA-seq data reported in this paper are DDBJ Sequence Read Archive (DRA): PRJDB17433.
References
Mora, A. L., Rojas, M., Pardo, A. & Selman, M. Emerging therapies for idiopathic pulmonary fibrosis, a progressive age-related disease. Nat. Rev. Drug Discov. 16, 755–772 (2017).
Agustí, A. & Hogg, J. C. Update on the pathogenesis of chronic obstructive pulmonary disease. N. Engl. J. Med. 381, 1248–1256 (2019).
Valapour, M. et al. OPTN/SRTR 2021 annual data report: lung. Am. J. Transplant. 23, S379–S442 (2023).
Barkauskas, C. E. et al. Type 2 alveolar cells are stem cells in adult lung. J. Clin. Investig. 123, 3025–3036 (2013).
Leiby, K. L. et al. Rational engineering of lung alveolar epithelium. NPJ Regen. Med. 8, 22 (2023).
Brownfield, D. G. et al. Alveolar cell fate selection and lifelong maintenance of AT2 cells by FGF signaling. Nat. Commun. 13, 7137 (2022).
Juul, N. H., Stockman, C. A. & Desai, T. J. Niche cells and signals that regulate lung alveolar stem cells in vivo. Cold Spring Harb. Perspect. Biol. 12. https://doi.org/10.1101/cshperspect.a035717 (2020).
Evans, K. V. & Lee, J. H. Alveolar wars: the rise of in vitro models to understand human lung alveolar maintenance, regeneration, and disease. Stem Cells Transl. Med. 9, 867–881 (2020).
Basil, M. C. et al. The cellular and physiological basis for lung repair and regeneration: past, present, and future. Cell Stem Cell 26, 482–502 (2020).
Nadkarni, R. R., Abed, S. & Draper, J. S. Stem cells in pulmonary disease and regeneration. Chest 153, 994–1003 (2018).
Evans, M. J. & Kaufman, M. H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981).
Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).
Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).
Matano, M. et al. Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids. Nat. Med. 21, 256–262 (2015).
Sato, T. & Clevers, H. Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science 340, 1190–1194 (2013).
Shi, Y., Inoue, H., Wu, J. C. & Yamanaka, S. Induced pluripotent stem cell technology: a decade of progress. Nat. Rev. Drug Discov. 16, 115–130 (2017).
Gotoh, S. et al. Generation of alveolar epithelial spheroids via isolated progenitor cells from human pluripotent stem cells. Stem Cell Rep. 3, 394–403 (2014).
Yamamoto, Y. et al. Long-term expansion of alveolar stem cells derived from human iPS cells in organoids. Nat. Methods 14, 1097–1106 (2017).
Jacob, A. et al. Differentiation of human pluripotent stem cells into functional lung alveolar epithelial cells. Cell Stem Cell 21, 472–488.e410 (2017).
Masui, A., Hirai, T. & Gotoh, S. Perspectives of future lung toxicology studies using human pluripotent stem cells. Arch. Toxicol. 96, 389–402 (2022).
Lee, J. H. et al. Somatic transcriptome priming gates lineage-specific differentiation potential of human-induced pluripotent stem cell states. Nat. Commun. 5, 5605 (2014).
Bock, C. et al. Reference Maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines. Cell 144, 439–452 (2011).
Nori, S. et al. Long-term safety issues of iPSC-based cell therapy in a spinal cord injury model: oncogenic transformation with epithelial-mesenchymal transition. Stem Cell Rep. 4, 360–373 (2015).
Ieda, M. et al. Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell 142, 375–386 (2010).
Vierbuchen, T. et al. Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463, 1035–1041 (2010).
Sekiya, S. & Suzuki, A. Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors. Nature 475, 390–393 (2011).
Huang, P. et al. Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors. Nature 475, 386–389 (2011).
Wang, H., Yang, Y., Liu, J. & Qian, L. Direct cell reprogramming: approaches, mechanisms and progress. Nat. Rev. Mol. Cell Biol. 22, 410–424 (2021).
Xu, J., Du, Y. & Deng, H. Direct lineage reprogramming: strategies, mechanisms, and applications. Cell Stem Cell 16, 119–134 (2015).
Costa, R. H., Kalinichenko, V. V. & Lim, L. Transcription factors in mouse lung development and function. Am. J. Physiol. Lung Cell Mol. Physiol. 280, L823–L838 (2001).
Lange, A. W., Keiser, A. R., Wells, J. M., Zorn, A. M. & Whitsett, J. A. Sox17 promotes cell cycle progression and inhibits TGF-beta/Smad3 signaling to initiate progenitor cell behavior in the respiratory epithelium. PLoS One 4, e5711 (2009).
Mendelson, C. R. Role of transcription factors in fetal lung development and surfactant protein gene expression. Annu. Rev. Physiol. 62, 875–915 (2000).
Nabhan, A. N., Brownfield, D. G., Harbury, P. B., Krasnow, M. A. & Desai, T. J. Single-cell Wnt signaling niches maintain stemness of alveolar type 2 cells. Science 359, 1118–1123 (2018).
Fox, E. et al. Three-dimensional culture and FGF signaling drive differentiation of murine pluripotent cells to distal lung epithelial cells. Stem Cells Dev. 24, 21–35 (2015).
Chung, M. I., Bujnis, M., Barkauskas, C. E., Kobayashi, Y. & Hogan, B. L. M. Niche-mediated BMP/SMAD signaling regulates lung alveolar stem cell proliferation and differentiation. Development 145. https://doi.org/10.1242/dev.163014 (2018).
Rockich, B. E. et al. Sox9 plays multiple roles in the lung epithelium during branching morphogenesis. Proc. Natl. Acad. Sci. USA110, E4456–E4464 (2013).
Rawlins, E. L., Clark, C. P., Xue, Y. & Hogan, B. L. The Id2+ distal tip lung epithelium contains individual multipotent embryonic progenitor cells. Development 136, 3741–3745 (2009).
Zhang, Z. et al. Transcription factor Etv5 is essential for the maintenance of alveolar type II cells. Proc. Natl. Acad. Sci. USA114, 3903–3908 (2017).
Hasegawa, K. et al. Fraction of MHCII and EpCAM expression characterizes distal lung epithelial cells for alveolar type 2 cell isolation. Respir. Res. 18, 150 (2017).
Weaver, T. E., Na, C. L. & Stahlman, M. Biogenesis of lamellar bodies, lysosome-related organelles involved in storage and secretion of pulmonary surfactant. Semin. Cell Dev. Biol. 13, 263–270 (2002).
Kemp, S. J. et al. Immortalization of human alveolar epithelial cells to investigate nanoparticle uptake. Am. J. Respir. Cell Mol. Biol. 39, 591–597 (2008).
Van der Velden, J. L., Bertoncello, I. & McQualter, J. L. LysoTracker is a marker of differentiated alveolar type II cells. Respir. Res. 14, 123 (2013).
Treutlein, B. et al. Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq. Nature 509, 371–375 (2014).
Kobayashi, Y. et al. Persistence of a regeneration-associated, transitional alveolar epithelial cell state in pulmonary fibrosis. Nat. Cell Biol. 22, 934–946 (2020).
Han, X. et al. Mapping the mouse cell atlas by microwell-seq. Cell 172, 1091–1107.e1017 (2018).
Liao, D. & Li, H. Dissecting the niche for alveolar type II cells with alveolar organoids. Front. Cell Dev. Biol. 8, 419 (2020).
Shiraishi, K. et al. Mesenchymal-epithelial interactome analysis reveals essential factors required for fibroblast-free alveolosphere formation. iScience 11, 318–333 (2019).
Alysandratos, K. D. et al. Culture impact on the transcriptomic programs of primary and iPSC-derived human alveolar type 2 cells. JCI Insight 8. https://doi.org/10.1172/jci.insight.158937 (2023).
Chen, Q. & Liu, Y. Isolation and culture of mouse alveolar type II cells to study type II to type I cell differentiation. STAR Protoc. 2, 100241 (2021).
Katsura, H. et al. Human lung stem cell-based alveolospheres provide insights into SARS-CoV-2-mediated interferon responses and pneumocyte dysfunction. Cell Stem Cell 27, 890–904.e898 (2020).
Weiner, A. I. et al. Mesenchyme-free expansion and transplantation of adult alveolar progenitor cells: steps toward cell-based regenerative therapies. NPJ Regen. Med. 4, 17 (2019).
Herriges, M. J. et al. Durable alveolar engraftment of PSC-derived lung epithelial cells into immunocompetent mice. Cell Stem Cell. https://doi.org/10.1016/j.stem.2023.07.016 (2023).
Suezawa, T. et al. Disease modeling of pulmonary fibrosis using human pluripotent stem cell-derived alveolar organoids. Stem Cell Rep. 16, 2973–2987 (2021).
Toulmin, S. A. et al. Type II alveolar cell MHCII improves respiratory viral disease outcomes while exhibiting limited antigen presentation. Nat. Commun. 12, 3993 (2021).
DeFelice, M. et al. TTF-1 phosphorylation is required for peripheral lung morphogenesis, perinatal survival, and tissue-specific gene expression. J. Biol. Chem. 278, 35574–35583 (2003).
Hawkins, F. & Kotton, D. N. Embryonic and induced pluripotent stem cells for lung regeneration. Ann. Am. Thorac. Soc. 12 (Suppl 1), S50–S53 (2015).
Boggaram, V. Thyroid transcription factor-1 (TTF-1/Nkx2.1/TITF1) gene regulation in the lung. Clin. Sci.116, 27–35 (2009).
Whitsett, J. A. & Matsuzaki, Y. Transcriptional regulation of perinatal lung maturation. Pediatr. Clin. North Am. 53, 873–887 (2006).
Wan, H. et al. Compensatory roles of Foxa1 and Foxa2 during lung morphogenesis. J. Biol. Chem. 280, 13809–13816 (2005).
Ikeda, K., Shaw-White, J. R., Wert, S. E. & Whitsett, J. A. Hepatocyte nuclear factor 3 activates transcription of thyroid transcription factor 1 in respiratory epithelial cells. Mol. Cell. Biol. 16, 3626–3636 (1996).
Yang, H., Lu, M. M., Zhang, L., Whitsett, J. A. & Morrisey, E. E. GATA6 regulates differentiation of distal lung epithelium. Development 129, 2233–2246 (2002).
Liao, C. M. et al. GATA6 suppression enhances lung specification from human pluripotent stem cells. J. Clin. Investig. 128, 2944–2950 (2018).
Liu, C., Glasser, S. W., Wan, H. & Whitsett, J. A. GATA-6 and thyroid transcription factor-1 directly interact and regulate surfactant protein-C gene expression. J. Biol. Chem. 277, 4519–4525 (2002).
Yano, T. et al. KGF regulates pulmonary epithelial proliferation and surfactant protein gene expression in adult rat lung. Am. J. Physiol. Lung Cell Mol. Physiol. 279, L1146–L1158 (2000).
Zepp, J. A. et al. Distinct mesenchymal lineages and niches promote epithelial self-renewal and myofibrogenesis in the lung. Cell 170, 1134–1148.e1110 (2017).
Huang, S. X. et al. Efficient generation of lung and airway epithelial cells from human pluripotent stem cells. Nat. Biotechnol. 32, 84–91 (2014).
Rabata, A., Fedr, R., Soucek, K., Hampl, A. & Koledova, Z. 3D Cell culture models demonstrate a role for FGF and WNT signaling in regulation of lung epithelial cell fate and morphogenesis. Front. Cell Dev. Biol. 8, 574 (2020).
Liberti, D. C. et al. Alveolar epithelial cell fate is maintained in a spatially restricted manner to promote lung regeneration after acute injury. Cell Rep. 35, 109092 (2021).
MacKenzie, B. et al. Attenuating endogenous Fgfr2b ligands during bleomycin-induced lung fibrosis does not compromise murine lung repair. Am. J. Physiol. Lung Cell Mol. Physiol. 308, L1014–L1024 (2015).
Ahmadvand, N. et al. Fgfr2b signaling is essential for the maintenance of the alveolar epithelial type 2 lineage during lung homeostasis in mice. Cell. Mol. Life Sci. 79, 302 (2022).
Louie, S. M. et al. Progenitor potential of lung epithelial organoid cells in a transplantation model. Cell Rep. 39, 110662 (2022).
Nichane, M. et al. Isolation and 3D expansion of multipotent Sox9(+) mouse lung progenitors. Nat. Methods 14, 1205–1212 (2017).
Qian, L. et al. In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes. Nature 485, 593–598 (2012).
Song, G. et al. Direct reprogramming of hepatic myofibroblasts into hepatocytes in vivo attenuates liver fibrosis. Cell Stem Cell 18, 797–808 (2016).
Horie, M. et al. Integrated single-cell RNA-sequencing analysis of aquaporin 5-expressing mouse lung epithelial cells identifies GPRC5A as a novel validated type I cell surface marker. Cells 9. https://doi.org/10.3390/cells9112460 (2020).
Khan, M. & Gasser, S. Generating primary fibroblast cultures from mouse ear and tail tissues. J. Vis. Exp. https://doi.org/10.3791/53565 (2016).
Hegab, A. E. et al. Mimicking the niche of lung epithelial stem cells and characterization of several effectors of their in vitro behavior. Stem Cell Res. 15, 109–121 (2015).
Mihara, E. et al. Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/α-albumin. Elife 5. https://doi.org/10.7554/eLife.11621 (2016).
Ootani, A. et al. Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nat. Med. 15, 701–706 (2009).
Miura, S. & Suzuki, A. Generation of mouse and human organoid-forming intestinal progenitor cells by direct lineage reprogramming. Cell Stem Cell 21, 456–471.e455 (2017).
Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).
O’Leary, N. A. et al. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res. 44, D733–D745 (2016).
Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol. 36, 411–420 (2018).
Acknowledgements
We are grateful to Dr. Akinori Hashiguchi and Ms. Nobuko Moritoki (Electron Microscope Laboratory, Keio University School of Medicine) for their assistance with the electron microscopy analysis; Ms. Miyuki Yamamoto, Dr. Shizuko Kagawa, Dr. Haiyue Zhang, Ms. Mikiko Shibuya, Ms. Chinatsu Yonekawa, Ms. Yoko Inamori, Dr. Mari Ozaki, and Mr. Tomoya Tamaki (Keio University School of Medicine) for their research assistance; Mr. Akira Sonoda and Ms. Mari Fujiwara (Core Facility, Collaborative Research Resources, Keio University School of Medicine) for their skilled technical assistance; and Ms. Terumi Horiuchi, Ms. Erina Ishikawa, Ms. Kazumi Abe, and Ms. Kiyomi Imamura (The University of Tokyo) for sequencing analysis. This study was funded by JSPS KAKENHI Grant Numbers JP24K02113, JP24K23468, JP22KJ2672, JP21J21344, JP21H02926, JP19K17682, JP18K19566, JP18H02821, JP15K19945, and AMED Grant Numbers JP21bm0404053, JP23wm0325031, JP24ym0126807, and JP25ek0109788.
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A.M., M. Ishii, T.A., M.Y., A.E.H., T.O., T.S. and M. Ieda designed the experiments. A.M., M. Ishii, M. Y., A.E.H., T.K., Y.N., M.O., F.S., J.H. and S.S. performed the in vitro experiments. A.M., T.A., M.Y., A.E.H., H.K., H.N., and S.O. performed the in vivo experiments. A.M., M. Ishii, T.A., M.Y., A.E.H., T.K., N.H., Y.N., M.O., T.E., H.Y., Y.K., M.S., Y.S., H.A., H.W. and M.K. analyzed the data. N.H., M. Ieda, and K.F. supervised the study. A.M., M. Ishii, T.A., M.Y., T.O. and A.E.H. wrote the manuscript. All authors have edited, reviewed, and approved the final manuscript.
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Morita, A., Ishii, M., Asakura, T. et al. Direct reprogramming of mouse fibroblasts into self-renewable alveolar epithelial-like cells. npj Regen Med 10, 30 (2025). https://doi.org/10.1038/s41536-025-00411-4
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DOI: https://doi.org/10.1038/s41536-025-00411-4
