Influence of Mesenchymal and Biophysical Components on Distal Lung Organoid Differentiation

Influence of Mesenchymal and Biophysical Components on Distal Lung Organoid Differentiation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Influence of Mesenchymal and Biophysical Components on Distal Lung Organoid Differentiation Olivia Goltsis, Claudia Bilodeau, Jinxia Wang, Daochun Luo, Meisam Asgari, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4510238/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Sep, 2024 Read the published version in Stem Cell Research & Therapy → Version 1 posted 5 You are reading this latest preprint version Abstract Background Chronic lung disease of prematurity, called bronchopulmonary dysplasia (BPD), lacks effective therapies, stressing the need for preclinical testing systems that reflect human pathology for identifying causal pathways and testing novel compounds. Alveolar organoids derived from human pluripotent stem cells (hPSC) are promising test platforms for studying distal airway diseases like BPD, but current protocols do not accurately replicate the distal niche environment of the native lung. Herein, we investigated the contributions of cellular constituents of the alveolus and fetal respiratory movements on hPSC-derived alveolar organoid formation. Methods Human PSCs were differentiated in 2D culture into lung progenitor cells (LPC) which were then further differentiated into alveolar organoids before and after removal of co-developing mesodermal cells. LPCs were also differentiated in Transwell® co-cultures with and without human fetal lung fibroblast. Forming organoids were subjected to phasic mechanical strain using a Flexcell® system. Differentiation within organoids and Transwell® cultures was assessed by flow cytometry, immunofluorescence, and qPCR for lung epithelial and alveolar markers of differentiation including GATA Binding Protein 6 (GATA 6), E-Cadherin (CDH1), NK2 Homeobox 1 (NKX2-1), HT2-280, Surfactant Proteins B (SFTPB) and C (SFTPC). Results We observed that co-developing mesenchymal progenitors promote alveolar epithelial type 2 cell (AEC2) differentiation within hPSC-derived lung organoids. This mesenchymal effect on AEC2 differentiation was corroborated by co-culturing hPSC-NKX2-1 + lung progenitors with human embryonic lung fibroblasts. The stimulatory effect did not require direct contact between fibroblasts and NKX2-1 + lung progenitors. Additionally, we demonstrate that episodic mechanical deformation of hPSC-derived lung organoids, mimicking in situ fetal respiratory movements, increased AEC2 differentiation without affecting proximal epithelial differentiation. Conclusion Our data suggest that biophysical and mesenchymal components promote AEC2 differentiation within hPSC-derived distal organoids in vitro . alveolar organoids lung fibroblasts pluripotent stem cells mechanical strain Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION One of the leading morbidities in preterm infants is bronchopulmonary dysplasia (BPD). This disease has many causes, and the incidence has not decreased in over 4 decades. To lessen the burden of disease we need robust preclinical platforms to mimic lung pathology and to test novel compounds. Human alveolar organoids created by directed differentiation of human pluripotent stem cells (hPSC) are promising test platforms to investigate distal lung diseases like BPD. Ample studies have demonstrated differentiation of hPSC (ESC and iPSC) towards alveolar progeny in 2D and 3D culture systems ( 1 – 4 ). In general, published protocols recapitulate chemical cues present during development in a stepwise fashion. Expression of the homeodomain transcription factor NKX2-1 has been used to monitor the efficiency of differentiation from PSCs into lung progenitors( 1 , 2 , 5 – 8 ). The yield of NKX2-1 + lung progenitors vary widely between protocols and hPSC lines ( 1 – 4 , 9 , 10 ). Alveolar epithelial cells (AEC) are derived from NKX2-1 + lung progenitors ( 1 , 7 ). While several protocols have been explored for their differentiation ( 1 , 9 , 11 ), the most effective method involves sorting the progenitor cells into a homogenous population and culturing them in Matrigel ( 1 , 4 , 9 ). In this hydrogel substratum, NKX2-1 + lung progenitors self-assemble into 3-dimensional (3D) structures called organoids ( 12 ). These organoids, with appropriate chemical cues, promote the differentiation of alveolar epithelial type II cells (AEC2). The efficiency varies widely, ranging from 3–85%, depending on the specific protocol, the hPSC line used, as well as the seeding density ( 1 , 2 , 4 , 9 , 11 , 13 ). In vivo , epithelial-mesenchymal crosstalk is pivotal for proper lung development ( 14 ). Mesenchymal cells located near epithelial cells lining the distal air sacs in the fetal lung are essential for AEC2 differentiation ( 15 ) and fate determination ( 16 ). Similarly, in the alveolar niche of the adult lung, a comparable population of mesenchymal cells adjacent to AEC2 influences their cellular behavior( 17 , 18 ). In vitro studies have shown that primary AEC2 require mesenchymal support to form organotypic alveolospheres ( 17 , 19 ). In organotypic aggregates, embryonic lung mesenchyme induces hPSC-derived endoderm differentiation into AEC2 ( 20 ). In alveolar organoids from hPSC-derived lung progenitors, differentiation into AEC2 is increased when progenitors are directly cultured with human embryonic lung fibroblasts ( 1 , 4 ). Several studies have shown that lung organoids derived from unsorted hPSC-endodermal cells undergoing directed differentiation to AEC2 contain cells expressing mesenchymal markers ( 10 , 11 , 13 , 21 , 22 ). Recently, mouse PSCs have been differentiated into lung-specific mesenchymal cells that, in co-culture experiments, stimulate mPSC-derived NKX2-1 + lung epithelial differentiation ( 23 ). However, whether the emerging mesenchymal population during directed differentiation of unsorted hPSC-derived endodermal cells into AEC2 using organoids has a similar effect on epithelial differentiation has not been investigated. Most published protocols for generating alveolar organoids have ignored biophysical cues. During development, the epithelium secretes fluid, which is circulated by the contraction of embryonic airway smooth muscles, creating a phasic hydrostatic distension of the developing airways ( 24 ). Additionally, episodic fetal breathing movements (FBM) begin in the first trimester of pregnancy and change the distal lung surface area ( 25 , 26 ). Drainage of lung fluid volume ( 27 ) or abolition of FBM ( 28 ) in experimental animal models leads to lung hypoplasia, underscoring the importance of physical forces in lung development. In vitro studies have demonstrated that stretch regimens reproducing FBM increase fetal AEC2 differentiation ( 29 , 30 ). Thus, cyclic mechanical deformation may represent a crucial biophysical cue for optimal AEC2 differentiation within organoids from hPSC-derived progenitors in vitro . In the present study, we investigated how the seeding density of NKX2-1 + lung progenitors affect AEC2 differentiation and whether co-developing mesenchymal cells within hPSC-derived lung organoids enhance the differentiation of human lung progenitors into AEC2. Additionally, we investigated the impact of cyclic mechanical deformation, mimicking respiratory movements of the fetal lung, on AEC2 differentiation within hPSC-derived lung organoids. We observed that both biophysical and mesenchymal cues stimulated AEC2 differentiation. METHODS Maintenance of human pluripotent cells The human pluripotent cell lines were utilized in accordance with the guidelines provided by the Stem Cell Oversight Committee of The Canadian Institute of Health Research. Human CA1 ES and NCRM1 iPS cells were cultured in feeder-free conditions on Matrigel-coated (Matrigel®GFR; Corning, #354230) plates in mTeSR™ Plus media (StemCell Technologies, #05825) with 1% (v/v) penicillin/streptomycin (Gibco, #15070-063). Cells were passaged weekly using ReLeSR™ (StemCell Technologies, #05825) and Gentle Cell Dissociation Reagent (GCDR, Stemcell, #07174), respectively. Cultures were maintained at 5% CO 2 in ambient air (21% O 2 ) at 37°C. Lentiviral infection of ES and iPS cells with hSFTPC GFP reporter is detailed in the online supplement. Differentiation into NKX2-1 + lung progenitor cells The differentiation protocol (Fig. 1 A) was adapted with minor modifications from Yamamoto et al., 2017, 2020 and is described in detail in the online supplement. Briefly, human ES and/or iPS cells were seeded at a density of 1.5 x 10 6 cells/well onto 6-well ultra-low attachment plates and differentiated into definitive endoderm in RPMI1640 (Gibco, #118755-119) supplemented with 2% B27 without vitamin A (Gibco, #12587-010), 100 ng/mL Activin A (Stem Cell Technologies, #78132), 1 µM CHIR99021 (Tocris, #4423) and 1% (v/v) penicillin/streptomycin (Gibco, #15140-122). On Day 6 of differentiation, cells were harvested with TrypLE™ Express (Gibco, #12605028) and analysed by flow cytometry for definitive endoderm (DE) markers c-KIT and CRCX4 ( Fig. S1 B ). Efficiency of DE induction (% c-KIT + /CXCR4 + cells) was consistently greater than 85%. DE cells were further differentiated to ventral anterior foregut endoderm (VAFE) by seeding unsorted cells onto 6-well Matrigel-coated plates (1.0x10 6 cells/well) in serum-free differentiation media (SFDM) containing 0.5µM dorsomorphin homologue 1 (DMH1) (StemCell Technologies, #73634) and 10µM SB431542 (Tocris, #1614) for anterior foregut (AFE) patterning. SFDM consisted of 1:1 (v/v) DMEM/F12 with 2% B27, 1% N2 (Gibco, #17502-048), 0.05mg/mL L-ascorbic acid (Sigma, #A92902), 1% (v/v) Glutamax (Gibco, #35050-061), 0.4mM 1-thioglycerol (Sigma, #M6145) 1% (v/v) penicillin/streptomycin. On Day 11 of differentiation, cells were analysed by flow cytometry for the AFE surface markers CD56 and CD271 ( Fig. S1 B ). Efficiency of AFE induction (% CD56 + /CD271 + cells) was consistently greater than 65%. At day 11, medium was changed to SFDM supplemented with BMP4 (R&D system, #314-BP), 0.5µM retinoic acid (Sigma, #R2625) and 3.5µM CHIR99021 for ventral anterior foregut endoderm (VAFE) induction. On Day 15, cells were harvested with TrypLE™ Express, analysed for NKX2-1 expression by flow cytometry, re-seeded on Matrigel-coated plates (1.0x10 6 cells/well) and incubated for 7 days in SFDM without N2, supplemented with 10ng/mL FGF7 (StemCell Technologies, #78046.1), 10ng/mL FGF10 (StemCell Technologies, #78037.1) and 3µM CHIR99021 for lung progenitor cell (LPC) formation. Medium was changed every other day. For Notch-inhibited cultures, 20µM DAPT (Stem Cell Technologies) was added. All differentiations were performed at 37°C in a 5% CO 2 and 5% O 2 environment. On Day 22 of differentiation, cells were dissociated with TrypLE™ Express in single cells, passed through a 40-µm pore size cell strainer, and analysed by flow cytometry for CD26/CD47, CPM and NKX2-1 expression - markers of LPC ( Fig. S1 B ). For source and dilution of antibodies see Table 1 in the online supplement. Culture and irradiation of human embryonic lung fibroblasts Human embryonic lung fibroblasts (hELF, 17.5 weeks of gestation, DV Biologics) were cultured in DMEM (Gibco, #11965118) containing 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin. Cells were passaged maximally 5 times. At 80–90% confluency, hELF were irradiated (Gammacell 40 SN 264) with 31,000 rad and then frozen in 40% DMEM, 50% FBS and 10% DMSO. Four days before experimentation, irradiated fibroblasts (ihELF) were thawed, seeded onto 12-well plates (3 x 10 5 cells/well) in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin. Based on VivaFix 410/450 (Bio-Rad, #1351112) viability measurements > 80% of the ihELF cells remained viable for at least 3 weeks in culture. Differentiation of NKX2-1 + lung progenitor cells into AEC2 cells Only differentiations that yielded > 50% NKX2-1 + progenitors were used. The cells (2.5 x 10 4 or 10 x 10 4 ) were resuspended in a 400µL 1:1 mixture of Matrigel® GFR and distalizing medium consisting of Ham’s F12-based SFDM containing 100µM 8-Br-cAMP (Sigma-Aldrich, #B7880), 100µM 3-Isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich, #I5879), 50nM dexamethasone (Sigma, #D4902), 50-100ng/mL FGF7, 10µM SB431542, 3µM CHIR99021, 0.25% (v/v) bovine albumin (Gibco, #15260-037), 15mM HEPES (Sigma, #H-0891), 0.8mM CaCl2 (Sigma-Aldrich, #449709), 0.1% (v/v) ITS Premix (Sigma, #13146) and 1% (v/v) penicillin/streptomycin. The cell suspension was loaded onto a Transwell® insert, placed in a well of a 12-well plate and 0.5 mL of AEC2 induction medium was added to the well and changed every other day (Fig. 1 C). For mechanical stretching, lung progenitor (> 50% NKX2-1 + ) cells (2.5 x 10 5 /mL) were resuspended in a mixture of AEC2 induction medium and 80% Matrigel® GFR. One mL of cell suspension was then seeded into each well of a 6-well tissue train® circular foam culture plate (FlexCell, TTCF-5001U) and incubated at 37°C for 2 hours to facilitate gelation. Three mL of induction medium was added which was changed every 2–3 days. Mechanical stretching was implemented 3 days post-seeding, the tissue train circular foam culture plate was connected to the FX-4000 Tension System (FlexCell). Settings were set to mimic fetal breathing movements, i,e, 5% elongation, frequencies of 1 Hz, square waveform, continuous cycle of 15 minutes ON and 45 minutes OFF ( 31 ). Organoids were subjected to this phasic strain regimen for 21 days. All cultures were maintained at 37°C in a 5% CO 2 and 5% O 2 environment. Sorting CPM + lung progenitor cells On Day 22 of differentiation, cells were harvested with TrypLE™ Express and passed through a 40-µm pore size cell strainer. Cells were stained with unconjugated mouse anti-human CPM monoclonal antibody for 30 minutes at 4°C and subsequently incubated with Alexa Fluor 647 donkey anti-mouse IgG and VivaFix 410/450 viability dye for 30 minutes at 4°C. A MoFlo XDP U/VBR (Beckman Coulter) sorter was used for isolating viable CPM + cells. For source and dilution of antibodies see Table 1 in the online supplement. Flow Cytometry and Immunofluorescence analysis Flow cytometry and immunofluorescence was conducted as previously described ( 32 , 33 ). The procedure of both methods is detailed in the online supplement. Antibodies and their concentrations are described in Table 1 in the online supplement. Electron Microscopy Samples processing and microscopy was performed as previously reported ( 34 ). Details are described in the online Supplement. RNA isolation, cDNA preparation and real-time PCR Total RNA was isolated from cells and organoids using the PureLink RNA Micro kit (Thermo Fisher Scientific, #12183016) and reverse transcribed with the SuperScript™ IV SuperScript IV First-Strand Synthesis System (Invitrogen, #18091050). PCR amplification was conducted using the SYBR Select Master Mix (Applied Biosystems, #34472908) and probes targeting various genes of interest are listed in Table 2 in the online supplement. Gene expression was normalized to beta-actin (ACTB) and expressed relative to selected appropriate positive or negative controls. Measuring organoid diameter Organoid diameter was measured using the built-in scale bar function on the EVOS M5000 imaging system (ThermoFisher Scientific). To obtain the mean of each biological replicate, more than 150 organoids from each condition were imaged and measured. Matrigel stiffness The mechanical properties of 50 versus 80% Matrigel were assessed by atomic force microscopy as detailed in the online supplement. Statistical analysis All data are presented as mean ± Standard Error of Mean (SEM). Statistical analysis was performed using GraphPad Prism Version 7.0.1 software. For multiple groups, one-way ANOVA followed by Tukey’s Multiple Comparisons Test was used. For less than three groups, a paired or unpaired Student t-test was used if both distributions were normal while a Mann-Whitney test was used if normality was not confirmed. Single asterisks (*) indicate statistical significance of p < 0.05. RESULTS Higher density seeding of lung progenitor cells improves AEC2 differentiation in organoids. Using an adapted protocol ( 4 , 35 ), we differentiated human CA1 ES cells to NKX2-1 + lung progenitor (LPC) cells (Figs. 1 A, S1). In contrast to the published protocol, we did not observe any benefit from adding DAPT, a γ-secretase inhibitor of the NOTCH pathway, to the media for differentiating ventral anterior foregut endoderm (VAFE) to LPCs ( Fig. S2 ). We introduced DAPT at different time points and durations during the VAFE to LPC differentiation period ( Fig. S2 A ) and validated its inhibition of NOTCH ( Fig. S2 B ) but did not find any effect on NKX2-1 + LPC induction ( Fig. S2 C ). However, extending the original duration of differentiation of AFE to LPC from D11-15 ( 4 , 35 ) to D11-22 doubled the induction efficiency of NKX2.1 + cells from 21.79 ± 4.21% at D15 (n = 14 biological replicates) to 44.35 ± 3.32% at D22 (n = 25 biological replicates; a.k.a. 25 separate differentiations) (Fig. 1 B). Similar NKX2-1 induction efficiencies (45–55%) at D22 of differentiation were observed with CA1 ES and NCRM1 iPS cells that both were stably transfected with a hSFTPC GFP reporter ( Fig. S3B ). The flow cytometry data was corroborated by nuclear immunostaining of the cells for NKX2-1 (Figs. 1 B, S3B). Flow cytometry and real-time PCR analysis also indicated a greater commitment of LPCs to SOX9 + than SOX2 + progenitor lineages ( Fig. S3C ). When more than 50% of the LPC population at D22 was positive for NKX2-1 ( 22 ), cells were embedded in a 1:1 diluted (50%) Matrigel for organoid formation and then incubated in a distalizing medium promoting AEC2 induction (Fig. 1 C). Organoid size and number, but not morphology, were dependent on the seeding density of CA1 LPCs (Fig. 1 D,E). Cultures started with 2.5x10 4 LPCs yielded less organoids than a 4-fold higher concentration of LPCs. Moreover, organoids in cultures started with 2.5x10 4 LPCs were on average double the size of those found in the higher density seeded cultures (215.95 ± 11.45µm vs 129.12 ± 5.66µm diameter at D36) (Fig. 1 F). Independent of seeding density, cells within the organoids were highly proliferative based on Ki-67 expression (Fig. 1 G). In both cultures, we observed a slight but non-significant decrease in Ki-67 + cells at D36 versus D29, suggesting a small reduction in proliferation as differentiation progressed. Thus, seeding density of LPCs affects the size of the organoids, especially on D36 of differentiation. We next investigated whether seeding density (i.e., size of the organoid) impacted AEC2 differentiation. Flow cytometry for HT2-280, a human AEC2 surface marker ( 36 ), revealed an increase in HT2-280 expression over time under both seeding conditions (Fig. 2 A,B). The percentage of HT2-280 + cells at D43 of differentiation was significantly greater in high-density versus low-density seeded ( i.e. , smaller versus larger) organoid cultures (20.15 ± 6.05% vs. 7.20 ± 1.20%, p < 0.05). However, the percentage of CDH1 (E-cadherin) positive epithelial cells was significantly lower in high-density compared to low-density seeded organoid cultures. The efficiency in AEC2 differentiation was validated with CA1 and NCRM1 cells transduced with a hSFTPC GFP reporter ( Fig. S4 ). The hSFTPC promoter has previously been validated to label murine AEC2 in situ ( 37 , 38 ). Immunostaining of D43 CA1-SFTPC GFP organoid cells ( Fig. S4A ) revealed that GFP + cells, representing SFTPC expression, were positive for CDH1 (see inlet ), and that 16.81 ± 1.22% (n = 3) of the organoid cells stained positive for HT2-280 ( Fig. S4C ). Flow cytometry for GFP ( Fig.S4A -right panel) of D43 organoids formed from 10 5 CA1-hSFTPC GFP or NCRM1-hSFTPC GFP LPCs demonstrated that 20.42 ± 2.76% and 21.06 ± 4.40%, respectively, of the organoid cells expressed GFP ( Fig. S4B ), matching the differentiation efficiency of LPCs into AEC2 when using HT2-280 as readout (Fig. 2 B) and was in line with the immunostaining findings. We then assessed whether time in culture increased AEC2 differentiation. We dissociated the organoids and passaged them into fresh Matrigel at D14 after the initial start of organoid culture from LPCs, extending the duration of organoid differentiation by another week (D50 instead of D43). This did not result in a further increase hSFTPC-driven GFP expression ( Fig. S4D ). Real-time PCR analysis of GATA6 , a transcription factor essential for distal lung epithelial differentiation ( 39 , 40 ), and AEC2 markers SFTPB and SFTPC substantiated the HT2-280 and GFP cytometry findings. In both low- and high-density seeded organoid cultures, GATA6 , SFTPB , and SFTPC mRNA expression increased compared to either ESC/iPSC ( Fig. S5 ) or D22 LPCs (Fig. 2 C), with high-density seeded organoids exhibiting the highest expression. To visualise the diversity of cell types within D43 organoid cultures, we conducted immunofluorescence confocal microscopy for distal and proximal epithelial lung markers. Consistent with the gene expression findings, we identified pro-SFTPB + and HT-280 + cells ( Fig. S6A -bottom panels) within the CDH1 + /NKX2-1 + ( Fig. S6A -top panels) organoids at D43. Positive immunostaining for mature SFTPB (mSFTB, see inlets of pro-SFTB images) indicated the presence of cells capable of processing pro-SFTPB into its mature form. HTII-280 staining was apical within the lumen of the organoids, in line with an apical-in orientation. Immunostaining for SCGB1A1 (club cell marker) and TUBB4A (ciliated cell marker) was negative, aligning with the reduction in proximal gene expression Fig. S6A- middle panels). Ultrastructural electron microscopy (EM) analysis of the D43 organoids revealed cell junctions between epithelial cells with microvilli at the apical surface (Fig. S6B- left panels ) . Immuno-gold EM visualized mature SFTPB within cytoplasmic pre-lamellar multivesicular structures ( Fig. S6B- left panels), consistent with previous reports for fetal AEC2 in situ ( 41 ). Removal of mesenchymal progenitor cells limits AEC2 differentiation within organoids. We next enriched the CA1 NKX2-1 + expressing population before organoid formation using carboxypeptidase M (CPM), an enzyme present on the surface of lung progenitor cells ( 1 ). Sorting for CPM + cells at D22 (43.43 ± 3.48% of total cells were CPM + at D22, n = 8 biological replicates; Fig. 3 A) resulted in an enrichment of the NKX2-1 expressing LPCs from 44.35 ± 3.32% to 80.54 ± 4.05% (Fig. 3 B). NKX2-1 immunostaining further confirmed the enrichment in the number of NKX2-1 + LPCs (Fig. 3 B). As depicted in Fig. 3C , 10 5 CPM + -sorted LPCs formed organoids within a few days of seeding ( 1 , 42 ) that were similar in shape and size to organoids formed from unsorted (> 50% NKX-2-1 + ) LPCs (Fig. 1 D- 3 C). Surprisingly, the percentage of HT2-280 + cells in D43 organoids established with CPM + -sorted LPCs was significantly lower compared to organoids formed with unsorted LPCs (Fig. 3 D), while the percentage of CDH1 + epithelial cells remained unchanged. We hypothesized that the reduction of HT2-280 + cells in the organoids formed from CPM + -sorted LPCs occurred due to the loss of mesodermal cells, which are known to be essential for distal epithelial differentiation ( 16 – 18 , 43 ). In our differentiation protocol (Fig. 1 A), we did not sort for a pure definitive endoderm population as ≥ 85% of the cells at D6 of differentiation were double positive for c-KIT and CXCR4 ( Figs. S1B, S3A ). Therefore, we started our differentiation towards AEC2 with a definitive endoderm population that was contaminated by other lineages, including mesoderm ( 44 , 45 ). By sorting the D22 cell population for CPM + cells, we likely removed most of the mesodermal progenitor cells that had emerged during the differentiation protocol ( 10 , 11 , 13 , 21 ). To verify, we compared the expression of mesenchymal markers vimentin (VIM) and actin alpha 2 (ACTA2) in our CPM + -sorted versus unsorted organoid cultures ( 17 , 46 , 47 ). Immunofluorescence confocal microscopy revealed the presence of VIM + cells in the unsorted organoid cultures, while VIM + cells were sparse in the CPM + -sorted organoid cultures (Fig. 4 A). Reduced VIM and ACTA2 expression in the CPM + -sorted compared to unsorted organoid cultures corroborated the immunofluorescence findings (Fig. 4 B). Together, the findings suggest that organoids formed with CPM + -sorted LPCs contain fewer mesenchymal progenitor cells. The loss in VIM + cells could also be due to less epithelial-mesenchymal transition in the sorted LPC organoid cultures, but the similar percentages of CDH1 + epithelial cells in the unsorted and sorted LPC organoid cultures (Fig. 3 D) argue against epithelial dedifferentiation. Mesenchymal support for LPC differentiation into AEC2 was corroborated by incubating LPCs in the absence and presence of human embryonic lung fibroblasts (hELF). Human ELFs were mitotically inactivated by irradiation (ihELF) to prevent overgrowth during co-culturing without influencing their secretome ( 48 , 49 ). D22 NKX2-1 + LPCs were grown on Transwell® inserts pre-coated with either Matrigel, Laminin, or Collagen type IV (the latter two matrices are the main components of Matrigel). The inserts were cultured in distalizing medium with and without ihELFs seeded in the bottom wells of the Transwell® plates. The percentage of CDH1 + cells did not differ between LPCs cultured for 22 days with or without ihELFs (Fig. 5 A). However, independent of the coated matrix, co-culture with ihELFs increased the percentage of HT2-280 + cells (Fig. 5 A), confirming the importance of mesenchymal-epithelial crosstalk for fetal AEC2 differentiation ( 15 , 16 , 43 ). Direct contact between ihELF and LPCs within organoids did not further improve HT-280 induction above indirect co-culturing (Fig. 5 B). Biophysical forces stimulate AEC2 differentiation within organoids. To examine whether mechanical strain affects the differentiation of LPC into AEC2 within organoids, we recapitulated fetal breathing movements in vitro using a 3D stretching device (Fig. 6 A). Within 3 days of culture of D22 unsorted (> 50% NKX2-1 + ) LPCs in 80% Matrigel (concentration required for applying strain to organoids using the FlexCell® system), organoid structures began to form. No apparent differences were observed in the morphology of organoids in static compared to stretched conditions (Fig. 6 B ) . The size of the organoids did not change with periodic stretching compared to static controls (128.10 ± 7.54 vs 134.02 ± 22.12 mm, mean ± range, n = 3 biological repeats, static versus stretch at D42). Flow cytometry for Ki-67 and CDH1 in episodically stretched organoids and paired static controls demonstrated no significant differences in proliferation and epithelial lineage expression, respectively (Figs. 6 C, S7). However, flow cytometry for HT2-280 revealed that episodically stretched organoids had significantly more HT2-280 + cells than organoids from static cultures (Figs. 6 D, S 7). Increased gene expression of SFTPC in the stretched organoids corroborated these HT2-280 findings (Fig. 6 D). Gene expression of FOXJ1 and SCGB1A1 was similar between static and stretched organoids (Fig. 6 D), suggesting that episodic stretching does not contribute to the commitment of LPCs to proximal epithelial lung lineages. To exclude that LPC to AEC2 differentiation varied with the stiffness of the Matrigel, we determined the elastic moduli by atomic force microscopy (AFM) micro-indentation. The 80% Matrigel used in the episodic stretch experiments had a similar elastic modulus at room temperature as 50% Matrigel used in all other experiments (150 ± 13 Pa vs 125 ± 12 Pa, n ≥ 17 measurements). Also, organoid size (128.10 ± 7.54 vs 129.12 ± 5.66µm diameter) and SFTPC induction (Fig. 6 D vs . Figure 2 C) under static conditions did not differ between 80 and 50% Matrigel. DISCUSSION Replicating the alveolar microenvironment for lung regenerative purposes has been challenging. Cellular and chemical cues of the distal niche have been used to create complex human alveolar organoids using either fetal lung bud tip progenitors ( 50 , 51 ) or adult AEC2 cells ( 12 , 52 ). The impact of cellular and biophysical cues on AEC2 differentiation within hPSC-derived organoids is not well understood. Here, we demonstrate that seeding density, mesenchymal presence and mechanical strain promote AEC2 differentiation within hPSC-derived organoids. In the present study, our adapted directed differentiation protocol yielded 44–55% NKX2-1 + lung progenitor cells without sorting. This was significantly lower than the 85% reported by Yamamoto et al. ( 4 ), but it matched or exceeded what has been reported for other human cell lines ( 1 , 2 ). The variation in induction efficiency could be attributed to factors such as the cell line used, the composition of the media and growth factors, the type of extracellular matrix coating, or the methodology for evaluating NKX2-1 + expression. Immunostaining for NKX2-1 + consistently yielded higher percentages ( 1 , 4 ) compared to NKX2-1 GFP reporters ( 2 ). We observed significant oscillation in the percentage of NKX2-1 + progenitors between experiments, consistent with previous reports ( 2 ). This inter-experiment variability was independent of media composition, hPSC passage number, efficiency of differentiation into definitive endoderm, or cell seeding density. The induction efficiencies of NKX2-1 + LPCs at D22 were validated using CD26 low /CD47 high and CPM + flow cytometry. Herein, we demonstrated that the concentration of Matrigel did not affect organoid formation from LPCs. While Hawkins et al. ( 2 ) used pure Matrigel, we utilized 50% and 80% Matrigel to enrobe the cells. Despite this difference, we did not observe any significant variations in organoid formation between 50 and 80% Matrigel. Previous studies have shown that lineage differentiation of human lung progenitors can be influenced by matrix stiffness ( 53 ). To investigate this further, we used AFM micro-indentation to measure the mechanical properties of 50% and 80% Matrigel at room temperature. Interestingly, we found that the elastic moduli of both concentrations were very similar, measuring around 120–150 Pa. These values closely align with those reported for pure Matrigel (~ 120 Pa) taken with AFM ( 54 ). Thus, it is unlikely that variations in LPC differentiation towards AEC2 between the various published reports are due to differences in Matrigel stiffness. However, it’s worth noting that these measured values are significantly lower than the elastic moduli (~ 4.5–10 kPa) reported for an alveolar wall ( 55 ) and decellularized alveolar ECM ( 56 , 57 ). This suggests that stiffer matrices than Matrigel may be required to accurately replicate lineage differentiation in the in situ distal niche. In this study, we evaluated the efficiency of AEC2 differentiation using HT2-280 protein expression. HT2-280 is present on the surface of both fetal and adult human AEC2 ( 36 , 58 , 59 ), and its expression aligns with that of SFTPC ( 17 ). Several studies have used HT2-280 to sort for human AEC2s for the generation of alveolar organoid cultures ( 12 , 17 , 60 ). While a recent study reported challenges in sorting AEC2 with HT2-280 from hiPSC-derived alveolar organoids ( 58 ), it’s important to note that this difficulty may be due to certain PSC lines, as other studies have successfully identified AEC2s using HT2-280 in hPSC-derived alveolar organoids ( 21 ). To validate our findings, we compared the HT2-280 flow cytometry results with those obtained from the GFP expression in organoids established with hPSCs that were stably transduced with a hSFTPC GFP reporter. Both flow cytometric readouts, HT2-280 and hSFTPC-GFP, consistently identified comparable viable AEC2 populations within the organoids, which were further confirmed through immunostaining for HT2-280. Our induction efficiency of 20–30% HT2-280 + cells falls within reported values ranging from 4–50% using SFTPC reporters ( 1 , 4 , 61 ). Our starting population consisted of 50–60% NKX2-1 + LPCs. Surprisingly, enriching the NKX2-1 + LPC population to more than 80% with CPM + sorting resulted in a decrease in HT2-280 + AEC2 induction within the organoids. The absence of any enrichment in SFTPB and SFTPC gene expression corroborated that CPM sorting did not lead to improved AEC2 differentiation (not shown). By contrast, previous studies have reported that enriching the NKX2-1 population resulted in a higher yield of SFTPC + cells within the organoids ( 1 , 4 , 9 ). However, these yield gains were always in comparison to the NKX2-1 negative populations ( 1 , 2 , 4 , 9 ), while we compared our findings to the unsorted > 50% NKX2-1 + LPC population. To complicate straight comparisons even further, some studies included direct co-cultures with fetal lung fibroblast cells ( 1 , 4 ). We also observed that our CA1 organoids comprised heterogenous cell types. Approximately one-third of the CDH1 + epithelial population in our organotypic cultures were positive for HT2-280, which raises questions about the identity of the remaining 65% CDH1 + cells. Some studies have reported positive staining for AEC1 markers in their hPSC-derived organoids ( 4 , 13 ), while others showed negative staining ( 9 ). Recently, Kanagaki and colleagues reported that presence of mesenchymal cells was crucial for AEC1 differentiation in organoids ( 62 ). Our CA1 organoid cultures contained hPSC-derived mesenchymal VIM + cells but stained negative for AEC1 markers PDPN and AQP5 (not shown). Also, unlike in other studies, no TUBB4 + ciliated and SCGB1A1 + secretory club cells were detected, ( 4 , 13 ). We speculate that a portion of the CDH + cells in the organoids represent non-lung endodermal lineages ( 63 ). Ultrastructural and immuno-gold EM analysis revealed cell junctions between epithelial cells within the organoids and presence of SFTPB-positive pre-lamellar multivesicular bodies, but no lamellar bodies, suggesting an immature AEC2 phenotype in HT2-280 + cells at D43 within the CA1 organoids. It has been reported that 14 days post-NKX2-1 enrichment, the NKX2-1 + population within lung organoids fell to 60–70% ( 9 ), a level comparable to our starting population, which raises questions about the advantage of enriching the NKX2-1 + population for distal differentiation within organoids. The heterogenicity in organoid cell composition among various studies is likely due to variability in differentiation protocols, cellular plasticity, contaminating cell populations, and hPSC lines used, highlighting the need for standardized protocols to facilitate better comparisons. One major difference between protocols is whether the epithelial LPCs are co-cultured with mesenchyme. Similar to other studies ( 1 , 4 ), our organoids and Transwell cultures showed significantly more HT2-280 + cells when mesenchymal cells were present. We highlighted the importance of mesenchymal cells for AEC2 differentiation within organoids by removing co-developed VIM + mesodermal cells from our LPC population using CPM sorting. This resulted in less HT2-280 + cells within the organoids during distal differentiation. Co-culturing LPCs with irradiated embryonic lung fibroblasts (iELF) in Transwell plates had the opposite effect and resulted in more HT2-280 + cells. The stimulatory effect on HT2-280 induction was independent of the substratum used and did not require direct contact between LPCs and iELF. The ELF-secretome is unaffected by irradiation ( 48 , 49 ) and contains various factors, including FGFs and WNTs, that are key to alveolar organoid formation ( 18 , 20 , 64 , 65 ). The cocktail of ELF factors stimulating optimal AEC differentiation remains yet unresolved. Finally, we demonstrated the importance of mechanical strain in alveolar development. In utero , the lung is exposed to mechanical forces generated by fetal breathing movements (FBM) that stimulate epithelial lung growth and differentiation ( 31 , 66 , 67 ). In a recent study using a postnatal lung organoid model of CD326 + epithelial cells and fibroblasts, it was shown that static stretch increased cell proliferation, while cycle stretch promoted mesenchymal lineage gene expression ( 68 ). In our study, we did not observe an increase in progenitor cell proliferation in organoids subjected to a stretch regimen mimicking FBMs. This is not surprising, as more than 70% of the progenitors in static organoid cultures are proliferating. Despite no change in proliferation, we observed a marked increase in the number of HT2-280 + cells and SFTPC expression within the organoids. This aligns with previous studies demonstrating that a similar stretch regimen increased the differentiation of primary fetal epithelial cells into AEC2 based on surfactant phospholipid ( 30 , 69 ) and SFTPC ( 29 ) production. The mechanotransduction pathways stimulating AEC2 differentiation are unknown. Recently, it has been reported that ROCK-Yap/Taz signaling is essential to regulate AEC1 differentiation in response to mechanical loading (stretching) of the fetal lung ( 70 ). The role of this mechanotransduction pathway in episodic stretch stimulated AEC2 differentiation remains to be elucidated. Our study had several strengths, including the utilization of both iPSC and ESC lines, as well as the incorporation of SFTPC-GFP reporter lines. The exploration of mechanical strain in a 3D culture system, rather than a 2D system, is a novel approach with clinical relevance. Future applications could involve investigations in the impact of injurious strain (high VT ventilation) on AEC2 proliferation and differentiation in 3D organoid culture. However, there were also several limitations of our findings. One limitation is our reliance on HT2-280 + AEC2 marker expression. Single-cell transcriptomics might have provided a more comprehensive understanding of the distal epithelial population within our organoids and identified non-lung cell populations within them ( 63 ). Additionally, a longer duration of culture might be necessary to improve alveolar differentiation efficiency, although we observed that passaging of the organoid cells did not further enhance AEC2 induction. CONCLUSIONS Our study highlights the critical factors influencing efficient and validated AEC2 differentiation from hPSCs, including lung progenitor concentration, mesenchymal population, and mechanical strain. While these factors increase the yield of AECs, further investigation is needed to understand the mechanisms driving AEC1 generation in hPSC-derived organoids to achieve a more accurate modeling of the human lung in situ. List Of Abbreviations ACTA2 : Actin alpha 2 AEC : Alveolar epithelial cell AEC2 : Alveolar epithelial type 2 cell AEC1 : Alveolar epithelial type 1 cell AFE : Anterior foregut endoderm AFM : Atomic force microscopy AQP5 : Aquaporin 5 BPD : Bronchopulmonary dysplasia BMP4 : Bone morphogenic protein 4 CDH1 : E-cadherin CPM : Carboxypeptidase M c-Kit : KIT proto-oncogene, receptor tyrosine kinase CXCR4 : C-X-C chemokine receptor type 4 DMH1 : Dorsomorphin homologue 1 ECM : Extracellular matrix EM : Electron microscopy ESC : Embryonic stem cell FBM : Fetal breathing movements FOXJ1 : Forkhead box J1 GATA6 : Gata binding protein 6 GFP : Green fluorescent protein hELF : Human embryonic lung fibroblasts hPSC : Human pluripotent stem cell ihELF ; Irradiated human embryonic lung fibroblast LPC : Lung progenitor cells NKX2-1 : NK2 homeobox 1 PDPN : Podoplanin PSC : Pluripotent stem cells iPSC : Induced pluripotent stem cell PCR : Polymerase chain reaction qPCR : Quantitative PCR SCGB1A1 : Secretoglobin family 1A member 1 SFDM : Serum free differentiation medium SOX2 : SRY-Box Transcription Factor 2 SOX9 : SRY-Box Transcription Factor 9 STFPB : Surfactant protein B SFTPC : Surfactant protein C TUBB4A : Tubulin Beta 4A Class IVa VAFE : Ventral anterior foregut endoderm VIM : Vimentin Declarations Ethics approval and consent of participants: This study does not involve animal experiments or human participants.Use of human pluripotent cell lines was approved by the Stem Cell Oversight Committee of The Canadian Institute of Health Research (Patient-specific alveolar type II (ATII) cells from surfactant protein-B deficient induced pluripotent stem cells) in March 2014. The Research Ethical Board of the Hospital for Sick Children confirmed no need for additional ethical approval. Not applicable Consent for publication: Not applicable. Competing interests: The authors declare that they have no competing interests. Artificial intelligence: The authors declare that artificial intelligence is not used in this study. Availability of data and materials: The data that support the findings of this study are available from the corresponding author upon reasonable request. Funding: This research was supported by Canadian Institutes of Health Research (FND-143309 to MP). Authors contribution: OG and CB: Conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing. JW, DL, MA, LB, and AP: Design, Collection and/or assembly of data. SLL: manuscript writing. MP: Conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript. Acknowledgements: We like to thank Dr. Andras Nagy, University of Toronto, and the Center for Commercialization of Regenerative Medicine, Toronto, for their generous gifts of CA1 ES and NCRM1 iPS cells, respectively. The hSFTPC promoter construct was a generous gift of Dr. Jeffrey Whistsett, Cincinatti, OH. Claudia Bilodeau was supported by a RESTRACOMP scholarship award from the Hospital for Sick Children. References Gotoh S, Ito I, Nagasaki T, Yamamoto Y, Konishi S, Korogi Y, et al. Generation of alveolar epithelial spheroids via isolated progenitor cells from human pluripotent stem cells. Stem Cell Rep. 2014;3(3):394–403. Hawkins F, Kramer P, Jacob A, Driver I, Thomas DC, McCauley KB, et al. Prospective isolation of NKX2-1-expressing human lung progenitors derived from pluripotent stem cells. J Clin Invest. 2017;127(6):2277–94. Huang SX, Islam MN, O'Neill J, Hu Z, Yang YG, Chen YW, et al. Efficient generation of lung and airway epithelial cells from human pluripotent stem cells. Nat Biotechnol. 2014;32(1):84–91. Yamamoto Y, Gotoh S, Korogi Y, Seki M, Konishi S, Ikeo S, et al. Long-term expansion of alveolar stem cells derived from human iPS cells in organoids. Nat Methods. 2017;14(11):1097–106. Green MD, Chen A, Nostro MC, d'Souza SL, Schaniel C, Lemischka IR, et al. Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells. Nat Biotechnol. 2011;29(3):267–72. Huang SX, Green MD, de Carvalho AT, Mumau M, Chen YW, D'Souza SL, et al. The in vitro generation of lung and airway progenitor cells from human pluripotent stem cells. Nat Protoc. 2015;10(3):413–25. Longmire TA, Ikonomou L, Hawkins F, Christodoulou C, Cao Y, Jean JC, et al. Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells. Cell Stem Cell. 2012;10(4):398–411. Mou H, Zhao R, Sherwood R, Ahfeldt T, Lapey A, Wain J, et al. Generation of multipotent lung and airway progenitors from mouse ESCs and patient-specific cystic fibrosis iPSCs. Cell Stem Cell. 2012;10(4):385–97. Jacob A, Morley M, Hawkins F, McCauley KB, Jean JC, Heins H, et al. Differentiation of Human Pluripotent Stem Cells into Functional Lung Alveolar Epithelial Cells. Cell Stem Cell. 2017;21(4):472–88. e10. Leibel SL, McVicar RN, Winquist AM, Snyder EY. Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease. J Vis Exp. 2021(170). Dye BR, Hill DR, Ferguson MA, Tsai YH, Nagy MS, Dyal R et al. In vitro generation of human pluripotent stem cell derived lung organoids. Elife. 2015;4. Barkauskas CE, Chung MI, Fioret B, Gao X, Katsura H, Hogan BL. Lung organoids: current uses and future promise. Development. 2017;144(6):986–97. Leibel SL, Winquist A, Tseu I, Wang J, Luo D, Shojaie S, et al. Reversal of Surfactant Protein B Deficiency in Patient Specific Human Induced Pluripotent Stem Cell Derived Lung Organoids by Gene Therapy. Sci Rep. 2019;9(1):13450. Morrisey EE, Hogan BL. Preparing for the first breath: genetic and cellular mechanisms in lung development. Dev Cell. 2010;18(1):8–23. Caniggia I, Tseu I, Han RN, Smith BT, Tanswell K, Post M. Spatial and temporal differences in fibroblast behavior in fetal rat lung. Am J Physiol. 1991;261(6 Pt 1):L424–33. Deimling J, Thompson K, Tseu I, Wang J, Keijzer R, Tanswell AK, et al. Mesenchymal maintenance of distal epithelial cell phenotype during late fetal lung development. Am J Physiol Lung Cell Mol Physiol. 2007;292(3):L725–41. Barkauskas CE, Cronce MJ, Rackley CR, Bowie EJ, Keene DR, Stripp BR, et al. Type 2 alveolar cells are stem cells in adult lung. J Clin Invest. 2013;123(7):3025–36. Zepp JA, Zacharias WJ, Frank DB, Cavanaugh CA, Zhou S, Morley MP, et al. Distinct Mesenchymal Lineages and Niches Promote Epithelial Self-Renewal and Myofibrogenesis in the Lung. Cell. 2017;170(6):1134–e4810. Lee JH, Bhang DH, Beede A, Huang TL, Stripp BR, Bloch KD, et al. Lung stem cell differentiation in mice directed by endothelial cells via a BMP4-NFATc1-thrombospondin-1 axis. Cell. 2014;156(3):440–55. Fox E, Shojaie S, Wang J, Tseu I, Ackerley C, Bilodeau M, et al. Three-dimensional culture and FGF signaling drive differentiation of murine pluripotent cells to distal lung epithelial cells. Stem Cells Dev. 2015;24(1):21–35. Chen YW, Huang SX, de Carvalho A, Ho SH, Islam MN, Volpi S, et al. A three-dimensional model of human lung development and disease from pluripotent stem cells. Nat Cell Biol. 2017;19(5):542–9. Leibel SL, McVicar RN, Winquist AM, Niles WD, Snyder EY. Generation of Complete Multi-Cell Type Lung Organoids From Human Embryonic and Patient-Specific Induced Pluripotent Stem Cells for Infectious Disease Modeling and Therapeutics Validation. Curr Protoc Stem Cell Biol. 2020;54(1):e118. Alber AB, Marquez HA, Ma L, Kwong G, Thapa BR, Villacorta-Martin C, et al. Directed differentiation of mouse pluripotent stem cells into functional lung-specific mesenchyme. Nat Commun. 2023;14(1):3488. Jesudason EC. Airway smooth muscle: an architect of the lung? Thorax. 2009;64(6):541–5. Harding R. Fetal pulmonary development: the role of respiratory movements. Equine Vet J Suppl. 1997(24):32–9. Kitterman JA. The effects of mechanical forces on fetal lung growth. Clin Perinatol. 1996;23(4):727–40. Moessinger AC, Harding R, Adamson TM, Singh M, Kiu GT. Role of lung fluid volume in growth and maturation of the fetal sheep lung. J Clin Invest. 1990;86(4):1270–7. Wigglesworth JS, Desai R. Effect on lung growth of cervical cord section in the rabbit fetus. Early Hum Dev. 1979;3(1):51–65. Nakamura T, Liu M, Mourgeon E, Slutsky A, Post M. Mechanical strain and dexamethasone selectively increase surfactant protein C and tropoelastin gene expression. Am J Physiol Lung Cell Mol Physiol. 2000;278(5):L974–80. Sanchez-Esteban J, Cicchiello LA, Wang Y, Tsai SW, Williams LK, Torday JS, et al. Mechanical stretch promotes alveolar epithelial type II cell differentiation. J Appl Physiol (1985). 2001;91(2):589–95. Liu M, Skinner SJ, Xu J, Han RN, Tanswell AK, Post M. Stimulation of fetal rat lung cell proliferation in vitro by mechanical stretch. Am J Physiol. 1992;263(3 Pt 1):L376–83. Shojaie S, Ermini L, Ackerley C, Wang J, Chin S, Yeganeh B, et al. Acellular lung scaffolds direct differentiation of endoderm to functional airway epithelial cells: requirement of matrix-bound HS proteoglycans. Stem Cell Rep. 2015;4(3):419–30. Litvack ML, Wigle TJ, Lee J, Wang J, Ackerley C, Grunebaum E, et al. Alveolar-like Stem Cell-derived Myb(-) Macrophages Promote Recovery and Survival in Airway Disease. Am J Respir Crit Care Med. 2016;193(11):1219–29. Bilodeau C, Shojaie S, Goltsis O, Wang J, Luo D, Ackerley C, et al. TP63 basal cells are indispensable during endoderm differentiation into proximal airway cells on acellular lung scaffolds. NPJ Regen Med. 2021;6(1):12. Yamamoto Y, Korogi Y, Hirai T, Gotoh S. A method of generating alveolar organoids using human pluripotent stem cells. Methods Cell Biol. 2020;159:115–41. Gonzalez RF, Allen L, Gonzales L, Ballard PL, Dobbs LG. HTII-280, a biomarker specific to the apical plasma membrane of human lung alveolar type II cells. J Histochem Cytochem. 2010;58(10):891–901. van Tuyl M, Groenman F, Kuliszewski M, Ridsdale R, Wang J, Tibboel D, et al. Overexpression of lunatic fringe does not affect epithelial cell differentiation in the developing mouse lung. Am J Physiol Lung Cell Mol Physiol. 2005;288(4):L672–82. Tibboel J, Groenman FA, Selvaratnam J, Wang J, Tseu I, Huang Z, et al. Hypoxia-inducible factor-1 stimulates postnatal lung development but does not prevent O2-induced alveolar injury. Am J Respir Cell Mol Biol. 2015;52(4):448–58. Keijzer R, van Tuyl M, Meijers C, Post M, Tibboel D, Grosveld F, et al. The transcription factor GATA6 is essential for branching morphogenesis and epithelial cell differentiation during fetal pulmonary development. Development. 2001;128(4):503–11. Yang H, Lu MM, Zhang L, Whitsett JA, Morrisey EE. GATA6 regulates differentiation of distal lung epithelium. Development. 2002;129(9):2233–46. Ridsdale R, Post M. Surfactant lipid synthesis and lamellar body formation in glycogen-laden type II cells. Am J Physiol Lung Cell Mol Physiol. 2004;287(4):L743–51. McCauley KB, Hawkins F, Serra M, Thomas DC, Jacob A, Kotton DN. Efficient Derivation of Functional Human Airway Epithelium from Pluripotent Stem Cells via Temporal Regulation of Wnt Signaling. Cell Stem Cell. 2017;20(6):844–57. e6. Shannon JM. Induction of alveolar type II cell differentiation in fetal tracheal epithelium by grafted distal lung mesenchyme. Dev Biol. 1994;166(2):600–14. Murry CE, Keller G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell. 2008;132(4):661–80. Cheng X, Ying L, Lu L, Galvao AM, Mills JA, Lin HC, et al. Self-renewing endodermal progenitor lines generated from human pluripotent stem cells. Cell Stem Cell. 2012;10(4):371–84. Farahani RM, Xaymardan M. Platelet-Derived Growth Factor Receptor Alpha as a Marker of Mesenchymal Stem Cells in Development and Stem Cell Biology. Stem Cells Int. 2015;2015:362753. Broers JL, de Leij L, Rot MK, ter Haar A, Lane EB, Leigh IM, et al. Expression of intermediate filament proteins in fetal and adult human lung tissues. Differentiation. 1989;40(2):119–28. Eiselleova L, Peterkova I, Neradil J, Slaninova I, Hampl A, Dvorak P. Comparative study of mouse and human feeder cells for human embryonic stem cells. Int J Dev Biol. 2008;52(4):353–63. Liu X, Krawczyk E, Suprynowicz FA, Palechor-Ceron N, Yuan H, Dakic A, et al. Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens. Nat Protoc. 2017;12(2):439–51. Miller AJ, Hill DR, Nagy MS, Aoki Y, Dye BR, Chin AM, et al. In Vitro Induction and In Vivo Engraftment of Lung Bud Tip Progenitor Cells Derived from Human Pluripotent Stem Cells. Stem Cell Rep. 2018;10(1):101–19. Lim K, Donovan APA, Tang W, Sun D, He P, Pett JP, et al. Organoid modeling of human fetal lung alveolar development reveals mechanisms of cell fate patterning and neonatal respiratory disease. Cell Stem Cell. 2023;30(1):20–e379. Liao D, Li H. Dissecting the Niche for Alveolar Type II Cells With Alveolar Organoids. Front Cell Dev Biology. 2020;8:419. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell. 2006;126(4):677–89. Alcaraz J, Xu R, Mori H, Nelson CM, Mroue R, Spencer VA, et al. Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia. EMBO J. 2008;27(21):2829–38. Bou Jawde S, Takahashi A, Bates JHT, Suki B. An Analytical Model for Estimating Alveolar Wall Elastic Moduli From Lung Tissue Uniaxial Stress-Strain Curves. Front Physiol. 2020;11:121. Luque T, Melo E, Garreta E, Cortiella J, Nichols J, Farre R, et al. Local micromechanical properties of decellularized lung scaffolds measured with atomic force microscopy. Acta Biomater. 2013;9(6):6852–9. Jorba I, Beltran G, Falcones B, Suki B, Farre R, Garcia-Aznar JM, et al. Nonlinear elasticity of the lung extracellular microenvironment is regulated by macroscale tissue strain. Acta Biomater. 2019;92:265–76. Korogi Y, Gotoh S, Ikeo S, Yamamoto Y, Sone N, Tamai K, et al. In Vitro Disease Modeling of Hermansky-Pudlak Syndrome Type 2 Using Human Induced Pluripotent Stem Cell-Derived Alveolar Organoids. Stem Cell Rep. 2019;12(3):431–40. Zacharias WJ, Frank DB, Zepp JA, Morley MP, Alkhaleel FA, Kong J, et al. Regeneration of the lung alveolus by an evolutionarily conserved epithelial progenitor. Nature. 2018;555(7695):251–5. Konda B, Mulay A, Yao C, Beil S, Israely E, Stripp BR. Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture. J Vis Exp. 2020(161). Ghaedi M, Calle EA, Mendez JJ, Gard AL, Balestrini J, Booth A, et al. Human iPS cell-derived alveolar epithelium repopulates lung extracellular matrix. J Clin Invest. 2013;123(11):4950–62. Kanagaki S, Ikeo S, Suezawa T, Yamamoto Y, Seki M, Hirai T, et al. Directed induction of alveolar type I cells derived from pluripotent stem cells via Wnt signaling inhibition. Stem Cells. 2021;39(2):156–69. Hurley K, Ding J, Villacorta-Martin C, Herriges MJ, Jacob A, Vedaie M, et al. Reconstructed Single-Cell Fate Trajectories Define Lineage Plasticity Windows during Differentiation of Human PSC-Derived Distal Lung Progenitors. Cell Stem Cell. 2020;26(4):593–e6088. Shiraishi K, Shichino S, Ueha S, Nakajima T, Hashimoto S, Yamazaki S, et al. Mesenchymal-Epithelial Interactome Analysis Reveals Essential Factors Required for Fibroblast-Free Alveolosphere Formation. iScience. 2019;11:318–33. Nabhan AN, Brownfield DG, Harbury PB, Krasnow MA, Desai TJ. Single-cell Wnt signaling niches maintain stemness of alveolar type 2 cells. Science. 2018;359(6380):1118–23. Li J, Wang Z, Chu Q, Jiang K, Li J, Tang N. The Strength of Mechanical Forces Determines the Differentiation of Alveolar Epithelial Cells. Dev Cell. 2018;44(3):297–312. e5. Liu M, Tanswell AK, Post M. Mechanical force-induced signal transduction in lung cells. Am J Physiol. 1999;277(4):L667–83. Joshi R, Batie MR, Fan Q, Varisco BM. Mouse lung organoid responses to reduced, increased, and cyclic stretch. Am J Physiol Lung Cell Mol Physiol. 2022;322(1):L162–73. Sanchez-Esteban J, Wang Y, Gruppuso PA, Rubin LP. Mechanical stretch induces fetal type II cell differentiation via an epidermal growth factor receptor-extracellular-regulated protein kinase signaling pathway. Am J Respir Cell Mol Biol. 2004;30(1):76–83. Nguyen TM, van der Merwe J, Elowsson Rendin L, Larsson-Callerfelt AK, Deprest J, Westergren-Thorsson G, et al. Stretch increases alveolar type 1 cell number in fetal lungs through ROCK-Yap/Taz pathway. Am J Physiol Lung Cell Mol Physiol. 2021;321(5):L814–26. Supplementary Files SupplementaryFiguresStemCellResearchandTherapy2024.pdf SupplementaryMaterialsStemCellResearchandTherapy2024.pdf Cite Share Download PDF Status: Published Journal Publication published 02 Sep, 2024 Read the published version in Stem Cell Research & Therapy → Version 1 posted Reviewers agreed at journal 21 Jun, 2024 Reviewers invited by journal 21 Jun, 2024 Editor assigned by journal 12 Jun, 2024 First submitted to journal 12 Jun, 2024 Editorial decision: Major Revision 10 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4510238","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":317324843,"identity":"dc43e1de-de1d-4364-9fc9-d29a5535dd7f","order_by":0,"name":"Olivia Goltsis","email":"","orcid":"","institution":"The Hospital for Sick Children","correspondingAuthor":false,"prefix":"","firstName":"Olivia","middleName":"","lastName":"Goltsis","suffix":""},{"id":317324844,"identity":"96da3abd-7e07-4a0c-b331-77b96b918cd3","order_by":1,"name":"Claudia Bilodeau","email":"","orcid":"","institution":"The Hospital for Sick Children","correspondingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Bilodeau","suffix":""},{"id":317324845,"identity":"f1bcbc35-cd0f-4139-b5c2-12ab2e403a39","order_by":2,"name":"Jinxia Wang","email":"","orcid":"","institution":"The Hospital for Sick Children","correspondingAuthor":false,"prefix":"","firstName":"Jinxia","middleName":"","lastName":"Wang","suffix":""},{"id":317324846,"identity":"76be4780-e5e6-458a-bc29-63e4a57df5da","order_by":3,"name":"Daochun Luo","email":"","orcid":"","institution":"The Hospital for Sick Children","correspondingAuthor":false,"prefix":"","firstName":"Daochun","middleName":"","lastName":"Luo","suffix":""},{"id":317324847,"identity":"c48317e3-cce3-4960-8942-b5263d06d15d","order_by":4,"name":"Meisam Asgari","email":"","orcid":"","institution":"McGill University","correspondingAuthor":false,"prefix":"","firstName":"Meisam","middleName":"","lastName":"Asgari","suffix":""},{"id":317324848,"identity":"6a91ec64-d09d-4db9-a12a-e3ad48a3cdc4","order_by":5,"name":"Laurent Bozec","email":"","orcid":"","institution":"University of Toronto Faculty of Dentistry","correspondingAuthor":false,"prefix":"","firstName":"Laurent","middleName":"","lastName":"Bozec","suffix":""},{"id":317324849,"identity":"84f3d363-8e42-4b2a-8ad7-d670e6420c8b","order_by":6,"name":"Ante Petterson","email":"","orcid":"","institution":"The Hospital for Sick Children","correspondingAuthor":false,"prefix":"","firstName":"Ante","middleName":"","lastName":"Petterson","suffix":""},{"id":317324850,"identity":"9b466f90-2ceb-4175-990b-4df65c185f9c","order_by":7,"name":"Sandra Leibel","email":"","orcid":"","institution":"Rady Children's Hospital: Rady Children's Hospital San Diego","correspondingAuthor":false,"prefix":"","firstName":"Sandra","middleName":"","lastName":"Leibel","suffix":""},{"id":317324851,"identity":"c0001f29-8be8-43fb-b952-058de70b3d66","order_by":8,"name":"Martin Post","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYLCCBAjF+IDBgMFAghlIEgCMDRAtzMwGxGuB0MxsEkDSAEjg16Lbfvb5g4c7GPL4+c8fq/xRYGcs2c68+QNDjR1OLWZn0g0bEs8wFEvOSGa7zWOQbCbNzFYmwXAsGbeWA2mMDYltDIkbbjCz3WYwOGAjx8xjBnQsM24t559BtZw/zFb4A6LF+ANjQz1uLTdgthxIZmPgMTgAdBiPgQRjw2E8Wp4xzkhskwD5xVga6BdjyWagXxKOHcfjsDSGjz/bbIAhdvDhxx9/7AxnnD+8+cOHmmqcWqBAIgGVn4BNERogRs0oGAWjYBSMVAAAQBNN1MAsD8gAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-4258-9303","institution":"The Hospital for Sick Children","correspondingAuthor":true,"prefix":"","firstName":"Martin","middleName":"","lastName":"Post","suffix":""}],"badges":[],"createdAt":"2024-05-31 16:36:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4510238/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4510238/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13287-024-03890-2","type":"published","date":"2024-09-02T16:05:40+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60141710,"identity":"432d4b8b-bd0e-4f6f-a3da-07d66a023e45","added_by":"auto","created_at":"2024-07-12 09:09:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1479524,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOrganoid size depends on lung progenitor cell seeding density. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Directed differentiation schematic for derivation of NKX2-1\u003csup\u003e+ \u003c/sup\u003elung progenitor (LPC) cells. (\u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eEfficiency of NKX2-1 specification at day 22 (n=25 biological replicates; a.k.a. separate differentiations) analyzed via flow cytometry and immunofluorescence. A threshold of ≥ 50% NKX2-1\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;cells was used for experiments involving further differentiation to alveolar epithelial type II (AEC2) cells. (\u003cstrong\u003eC\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eSchematic depicting the directed differentiation of D22 LPC to HT2-280\u003csup\u003e+\u003c/sup\u003e/SFTPC\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;AEC2 cells in organoid culture. (\u003cstrong\u003eD\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eRepresentative brightfield images of organoid cultures throughout days 28 to 42 of differentiation from different seeding densities. (\u003cstrong\u003eE\u003c/strong\u003e) Representative H\u0026amp;E-stained organoid sections derived from different seeding densities at day 42 showing hollow structure. (\u003cstrong\u003eF\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eAverage organoid diameter in day 29 and day 36 organoid cultures from 2.5 x 10\u003csup\u003e4\u003c/sup\u003e\u0026nbsp;or 10 x 10\u003csup\u003e4\u003c/sup\u003e\u0026nbsp;LPCs. Calculations are based on the measurement of ≥ 180 organoids per condition for three biological replicates. (\u003cstrong\u003eG\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003ePercentage of Ki-67\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;cells, as measured by flow cytometry, in day 29 and day 36 organoids from different seeding densities (mean ± SEM, n=3 biological replicates. *p \u0026lt; 0.05 by one-way ANOVA).\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4510238/v1/2d1037ff05c93c57130e7305.png"},{"id":60141703,"identity":"765ed66a-b0f8-4fad-b2f7-2cea1313389a","added_by":"auto","created_at":"2024-07-12 09:09:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":450648,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigher density seeding of lung progenitor cells improves AEC2 differentiation in organoids. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003ePercentage of HT2-280\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;and CDH1\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;cells from different seeding densities in D29, D36 and D43 organoids assessed by flow cytometry (mean ± SEM, n≥3 biological replicates, *p \u0026lt; 0.05 by one-way ANOVA). Inviable cells were identified and removed from flow cytometric analysis using the VivaFix 410/450 viability stain. (\u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eRepresentative flow cytometry density plots of HT2-280\u003csup\u003e+ \u003c/sup\u003ecells in D43 organoid cultures from 2.5 x 10\u003csup\u003e4 \u003c/sup\u003eand 10 x 10\u003csup\u003e4\u003c/sup\u003e\u0026nbsp;LPCs. Left plot: control density plots – PE-conjugated IgG\u003csub\u003e1 \u003c/sub\u003eisotype only. (\u003cstrong\u003eC\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eGene expression measured by real-time PCR of distal (AEC2: \u003cem\u003eSFTPB\u003c/em\u003e, \u003cem\u003eSFTPC\u003c/em\u003e) and proximal (\u003cem\u003eFOXJ1\u003c/em\u003e: ciliated cells; \u003cem\u003eMUC5AC\u003c/em\u003e: goblet cells; \u003cem\u003eSCGB1A1\u003c/em\u003e: club cells) lung cell markers in day 43 organoids from different seeding densities relative to day 22 LPCs (mean ± SEM, n≥3 biological replicates).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4510238/v1/b4f3757df10c8476062e6836.png"},{"id":60141706,"identity":"36e78302-7eec-4b3a-a710-36214cd071f7","added_by":"auto","created_at":"2024-07-12 09:09:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":739062,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnriching the NKX2-1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e\u0026nbsp;lung progenitor population does not increase HT-280\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e AEC2 differentiation in organoids. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Gating strategy to sort for CPM\u003csup\u003e+ \u003c/sup\u003elung progenitor cells on day 22 (D22) of differentiation. (\u003cstrong\u003eB) Left panel\u003c/strong\u003e: Percentage of NKX2-1 positive cells of CPM\u003csup\u003e+\u003c/sup\u003e-\u003csup\u003e \u003c/sup\u003esorted and unsorted D22 LPCs (n=4 biological replicates).\u003cstrong\u003e Right panel\u003c/strong\u003e:\u003cstrong\u003e \u003c/strong\u003eRepresentative immunofluorescence staining for NKX2-1 of CPM\u003csup\u003e+\u003c/sup\u003e-sorted and unsorted D22 LPCs.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eC\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eRepresentative brightfield images of organoid formation by 10\u003csup\u003e5\u003c/sup\u003e\u0026nbsp;CPM\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;sorted LPCs at days 28-39. (\u003cstrong\u003eD\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003ePercentage of HT2-280\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;and CDH1\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;cells in D43 organoids formed with 10\u003csup\u003e5\u003c/sup\u003e\u0026nbsp;CPM\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;sorted \u003cem\u003evs\u003c/em\u003e unsorted LPCs. Unviable cells were identified and removed from flow cytometric analysis using the VivaFix 410/450 viability stain (n=4 biological replicates, *p \u0026lt; 0.05 by two-tailed unpaired t-test).\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4510238/v1/24e26ef1efd43a28a1ec1187.png"},{"id":60141709,"identity":"07a84791-0f12-4d12-8f5f-512645830b23","added_by":"auto","created_at":"2024-07-12 09:09:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":761435,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMesenchymal cells are reduced in organoids formed with CPM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e\u0026nbsp;(NKX2-1 enriched) sorted lung progenitor cells. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eRepresentative immunofluorescence images of epithelial (CDH1) and mesenchymal (VIM) cell markers in day 43 organoids formed with CPM\u003csup\u003e+\u003c/sup\u003e-sorted \u003cem\u003evs\u003c/em\u003e unsorted LPCs. (\u003cstrong\u003eB\u003c/strong\u003e) Gene expression measured by real-time PCR of VIM and ACTA2 in day 43 organoids from CPM\u003csup\u003e+\u003c/sup\u003e-sorted \u003cem\u003evs\u003c/em\u003e unsorted LPCs relative to day 22 lung progenitors (mean ± SEM, n=4 biological replicates. *p\u003cem\u003e \u0026lt; \u003c/em\u003e0.05 by paired t-test).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4510238/v1/2e47456f3353fc26a24cad52.png"},{"id":60141711,"identity":"e30fccad-ef48-44bf-b53a-ad30cd036938","added_by":"auto","created_at":"2024-07-12 09:09:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":184279,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEmbryonic lung fibroblasts stimulate the differentiation of lung progenitors into HT-280\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eAEC2 cells.\u003c/strong\u003e (\u003cstrong\u003eA) Top\u003c/strong\u003e \u003cstrong\u003epanel:\u003c/strong\u003e Schematic of the indirect Transwell® co-culture system to determine the effect of irradiated human embryonic lung fibroblasts (ihELF) on the differentiation of D22 NKX2-1\u003csup\u003e+\u003c/sup\u003e LPCs into AEC2. \u003cstrong\u003eBottom\u003c/strong\u003e \u003cstrong\u003epanel:\u003c/strong\u003e Flow cytometry analysis for CDH1 and HT2-280 of organoids grown in different matrices. Data are expressed as mean ± SEM, n=4 biological replicates. Dashed lines indicate mean % of HT2-280 positive cells, respectively, after combining the matrix data in both groups. *p\u0026lt;0.05 by Mann-Whitney U test, no ihELF \u003cem\u003evs\u003c/em\u003e ihELF. (\u003cstrong\u003eB) Left panel\u003c/strong\u003e: Schematic of experimental design to determine the effect of direct \u003cem\u003eversus\u003c/em\u003e indirect contact of LPCs with ihELF on the differentiation of D22 NKX2-1\u003csup\u003e+\u003c/sup\u003e LPCs to AEC2.\u003cstrong\u003e Right panel:\u003c/strong\u003e Flow cytometry analysis for HT2-280 of organoids grown directly \u003cem\u003eversus\u003c/em\u003e indirectly with ihELF after 20 days of culture. Data are expressed as mean ± SEM, n ≥ 10 biological replicates.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4510238/v1/e382c08658c1dd64a3293264.png"},{"id":60142381,"identity":"e9aa6ee6-297d-498f-b47b-c64e884f65e9","added_by":"auto","created_at":"2024-07-12 09:17:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":539721,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhasic mechanical stretch improves AEC2 differentiation in organoids. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Schematic outlining the parameters for episodic mechanical stretch from day 22 to 43. (\u003cstrong\u003eB\u003c/strong\u003e) Representative brightfield images of organoids formed under stretched and static conditions from day 25 to 42. (\u003cstrong\u003eC\u003c/strong\u003e) Flow cytometry analysis of Ki-67 and CDH1 in D43 stretched \u003cem\u003evs\u003c/em\u003e static organoid cultures. (\u003cstrong\u003eD) Top panels\u003c/strong\u003e: Flow cytometry and gene expression of AEC2 markers HT2-280 and \u003cem\u003eSFTPC\u003c/em\u003e, respectively, in D43 stretched and static organoid cultures. Flow data for Ki-67 and HT2-280 data are presented as a ratio compared to paired static controls. Data are mean ± SEM, n=4 biological replicates, *p\u0026lt;0.05 by paired t-test. \u003cstrong\u003eBottom panels\u003c/strong\u003e: Gene expression of proximal ciliated (\u003cem\u003eFOXJ1\u003c/em\u003e) and club (\u003cem\u003eSCGB1A1\u003c/em\u003e) cell markers in D43 stretched and static organoid cultures (mean ± SEM, n=4 biological replicates).\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4510238/v1/36554f8b236fbddae610be2b.png"},{"id":64185853,"identity":"fae71395-302c-4e10-a32f-6c95d50f3219","added_by":"auto","created_at":"2024-09-09 16:22:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5034371,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4510238/v1/49db8dce-1f78-4361-a397-5c7c728c3ac0.pdf"},{"id":60141708,"identity":"578c0d2c-2a90-44de-b318-38cd5b4f70f8","added_by":"auto","created_at":"2024-07-12 09:09:55","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4901896,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiguresStemCellResearchandTherapy2024.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4510238/v1/32e5f43c3f45ba19b5e055e8.pdf"},{"id":60142382,"identity":"cb0d6460-d01b-427d-95fd-654f2391c825","added_by":"auto","created_at":"2024-07-12 09:17:55","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":286592,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialsStemCellResearchandTherapy2024.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4510238/v1/9dead16e239fac6865abffbb.pdf"}],"financialInterests":"","formattedTitle":"Influence of Mesenchymal and Biophysical Components on Distal Lung Organoid Differentiation","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eOne of the leading morbidities in preterm infants is bronchopulmonary dysplasia (BPD). This disease has many causes, and the incidence has not decreased in over 4 decades. To lessen the burden of disease we need robust preclinical platforms to mimic lung pathology and to test novel compounds. Human alveolar organoids created by directed differentiation of human pluripotent stem cells (hPSC) are promising test platforms to investigate distal lung diseases like BPD. Ample studies have demonstrated differentiation of hPSC (ESC and iPSC) towards alveolar progeny in 2D and 3D culture systems (\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). In general, published protocols recapitulate chemical cues present during development in a stepwise fashion. Expression of the homeodomain transcription factor NKX2-1 has been used to monitor the efficiency of differentiation from PSCs into lung progenitors(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). The yield of NKX2-1\u003csup\u003e+\u003c/sup\u003e lung progenitors vary widely between protocols and hPSC lines (\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Alveolar epithelial cells (AEC) are derived from NKX2-1\u003csup\u003e+\u003c/sup\u003e lung progenitors (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). While several protocols have been explored for their differentiation (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), the most effective method involves sorting the progenitor cells into a homogenous population and culturing them in Matrigel (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). In this hydrogel substratum, NKX2-1\u003csup\u003e+\u003c/sup\u003e lung progenitors self-assemble into 3-dimensional (3D) structures called organoids (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). These organoids, with appropriate chemical cues, promote the differentiation of alveolar epithelial type II cells (AEC2). The efficiency varies widely, ranging from 3\u0026ndash;85%, depending on the specific protocol, the hPSC line used, as well as the seeding density (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vivo\u003c/em\u003e, epithelial-mesenchymal crosstalk is pivotal for proper lung development (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Mesenchymal cells located near epithelial cells lining the distal air sacs in the fetal lung are essential for AEC2 differentiation (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) and fate determination (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Similarly, in the alveolar niche of the adult lung, a comparable population of mesenchymal cells adjacent to AEC2 influences their cellular behavior(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). \u003cem\u003eIn vitro\u003c/em\u003e studies have shown that primary AEC2 require mesenchymal support to form organotypic alveolospheres (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). In organotypic aggregates, embryonic lung mesenchyme induces hPSC-derived endoderm differentiation into AEC2 (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). In alveolar organoids from hPSC-derived lung progenitors, differentiation into AEC2 is increased when progenitors are directly cultured with human embryonic lung fibroblasts (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Several studies have shown that lung organoids derived from unsorted hPSC-endodermal cells undergoing directed differentiation to AEC2 contain cells expressing mesenchymal markers (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Recently, mouse PSCs have been differentiated into lung-specific mesenchymal cells that, in co-culture experiments, stimulate mPSC-derived NKX2-1\u003csup\u003e+\u003c/sup\u003e lung epithelial differentiation (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). However, whether the emerging mesenchymal population during directed differentiation of unsorted hPSC-derived endodermal cells into AEC2 using organoids has a similar effect on epithelial differentiation has not been investigated.\u003c/p\u003e \u003cp\u003eMost published protocols for generating alveolar organoids have ignored biophysical cues. During development, the epithelium secretes fluid, which is circulated by the contraction of embryonic airway smooth muscles, creating a phasic hydrostatic distension of the developing airways (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Additionally, episodic fetal breathing movements (FBM) begin in the first trimester of pregnancy and change the distal lung surface area (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Drainage of lung fluid volume (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) or abolition of FBM (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) in experimental animal models leads to lung hypoplasia, underscoring the importance of physical forces in lung development. \u003cem\u003eIn vitro\u003c/em\u003e studies have demonstrated that stretch regimens reproducing FBM increase fetal AEC2 differentiation (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Thus, cyclic mechanical deformation may represent a crucial biophysical cue for optimal AEC2 differentiation within organoids from hPSC-derived progenitors \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn the present study, we investigated how the seeding density of NKX2-1\u003csup\u003e+\u003c/sup\u003e lung progenitors affect AEC2 differentiation and whether co-developing mesenchymal cells within hPSC-derived lung organoids enhance the differentiation of human lung progenitors into AEC2. Additionally, we investigated the impact of cyclic mechanical deformation, mimicking respiratory movements of the fetal lung, on AEC2 differentiation within hPSC-derived lung organoids. We observed that both biophysical and mesenchymal cues stimulated AEC2 differentiation.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaintenance of human pluripotent cells\u003c/h2\u003e \u003cp\u003eThe human pluripotent cell lines were utilized in accordance with the guidelines provided by the Stem Cell Oversight Committee of The Canadian Institute of Health Research. Human CA1 ES and NCRM1 iPS cells were cultured in feeder-free conditions on Matrigel-coated (Matrigel\u0026reg;GFR; Corning, #354230) plates in mTeSR\u0026trade; Plus media (StemCell Technologies, #05825) with 1% (v/v) penicillin/streptomycin (Gibco, #15070-063). Cells were passaged weekly using ReLeSR\u0026trade; (StemCell Technologies, #05825) and Gentle Cell Dissociation Reagent (GCDR, Stemcell, #07174), respectively. Cultures were maintained at 5% CO\u003csub\u003e2\u003c/sub\u003e in ambient air (21% O\u003csub\u003e2\u003c/sub\u003e) at 37\u0026deg;C. Lentiviral infection of ES and iPS cells with hSFTPC\u003csup\u003eGFP\u003c/sup\u003e reporter is detailed in the online supplement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDifferentiation into NKX2-1\u003csup\u003e+\u003c/sup\u003e lung progenitor cells\u003c/h2\u003e \u003cp\u003eThe differentiation protocol (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) was adapted with minor modifications from Yamamoto et al., 2017, 2020 and is described in detail in the online supplement. Briefly, human ES and/or iPS cells were seeded at a density of 1.5 x 10\u003csup\u003e6\u003c/sup\u003e cells/well onto 6-well ultra-low attachment plates and differentiated into definitive endoderm in RPMI1640 (Gibco, #118755-119) supplemented with 2% B27 without vitamin A (Gibco, #12587-010), 100 ng/mL Activin A (Stem Cell Technologies, #78132), 1 \u0026micro;M CHIR99021 (Tocris, #4423) and 1% (v/v) penicillin/streptomycin (Gibco, #15140-122). On Day 6 of differentiation, cells were harvested with TrypLE\u0026trade; Express (Gibco, #12605028) and analysed by flow cytometry for definitive endoderm (DE) markers c-KIT and CRCX4 (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB\u003c/b\u003e). Efficiency of DE induction (% c-KIT\u003csup\u003e+\u003c/sup\u003e/CXCR4\u003csup\u003e+\u003c/sup\u003e cells) was consistently greater than 85%. DE cells were further differentiated to ventral anterior foregut endoderm (VAFE) by seeding unsorted cells onto 6-well Matrigel-coated plates (1.0x10\u003csup\u003e6\u003c/sup\u003e cells/well) in serum-free differentiation media (SFDM) containing 0.5\u0026micro;M dorsomorphin homologue 1 (DMH1) (StemCell Technologies, #73634) and 10\u0026micro;M SB431542 (Tocris, #1614) for anterior foregut (AFE) patterning. SFDM consisted of 1:1 (v/v) DMEM/F12 with 2% B27, 1% N2 (Gibco, #17502-048), 0.05mg/mL L-ascorbic acid (Sigma, #A92902), 1% (v/v) Glutamax (Gibco, #35050-061), 0.4mM 1-thioglycerol (Sigma, #M6145) 1% (v/v) penicillin/streptomycin. On Day 11 of differentiation, cells were analysed by flow cytometry for the AFE surface markers CD56 and CD271 (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB\u003c/b\u003e). Efficiency of AFE induction (% CD56\u003csup\u003e+\u003c/sup\u003e/CD271\u003csup\u003e+\u003c/sup\u003e cells) was consistently greater than 65%. At day 11, medium was changed to SFDM supplemented with BMP4 (R\u0026amp;D system, #314-BP), 0.5\u0026micro;M retinoic acid (Sigma, #R2625) and 3.5\u0026micro;M CHIR99021 for ventral anterior foregut endoderm (VAFE) induction. On Day 15, cells were harvested with TrypLE\u0026trade; Express, analysed for NKX2-1 expression by flow cytometry, re-seeded on Matrigel-coated plates (1.0x10\u003csup\u003e6\u003c/sup\u003e cells/well) and incubated for 7 days in SFDM without N2, supplemented with 10ng/mL FGF7 (StemCell Technologies, #78046.1), 10ng/mL FGF10 (StemCell Technologies, #78037.1) and 3\u0026micro;M CHIR99021 for lung progenitor cell (LPC) formation. Medium was changed every other day. For Notch-inhibited cultures, 20\u0026micro;M DAPT (Stem Cell Technologies) was added. All differentiations were performed at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e and 5% O\u003csub\u003e2\u003c/sub\u003e environment. On Day 22 of differentiation, cells were dissociated with TrypLE\u0026trade; Express in single cells, passed through a 40-\u0026micro;m pore size cell strainer, and analysed by flow cytometry for CD26/CD47, CPM and NKX2-1 expression - markers of LPC (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB\u003c/b\u003e). For source and dilution of antibodies see Table\u0026nbsp;1 in the online supplement.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCulture and irradiation of human embryonic lung fibroblasts\u003c/h2\u003e \u003cp\u003eHuman embryonic lung fibroblasts (hELF, 17.5 weeks of gestation, DV Biologics) were cultured in DMEM (Gibco, #11965118) containing 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin. Cells were passaged maximally 5 times. At 80\u0026ndash;90% confluency, hELF were irradiated (Gammacell 40 SN 264) with 31,000 rad and then frozen in 40% DMEM, 50% FBS and 10% DMSO. Four days before experimentation, irradiated fibroblasts (ihELF) were thawed, seeded onto 12-well plates (3 x 10\u003csup\u003e5\u003c/sup\u003e cells/well) in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin. Based on VivaFix 410/450 (Bio-Rad, #1351112) viability measurements\u0026thinsp;\u0026gt;\u0026thinsp;80% of the ihELF cells remained viable for at least 3 weeks in culture.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDifferentiation of NKX2-1\u003csup\u003e+\u003c/sup\u003e lung progenitor cells into AEC2 cells\u003c/h2\u003e \u003cp\u003eOnly differentiations that yielded\u0026thinsp;\u0026gt;\u0026thinsp;50% NKX2-1\u003csup\u003e+\u003c/sup\u003e progenitors were used. The cells (2.5 x 10\u003csup\u003e4\u003c/sup\u003e or 10 x 10\u003csup\u003e4\u003c/sup\u003e) were resuspended in a 400\u0026micro;L 1:1 mixture of Matrigel\u0026reg; GFR and distalizing medium consisting of Ham\u0026rsquo;s F12-based SFDM containing 100\u0026micro;M 8-Br-cAMP (Sigma-Aldrich, #B7880), 100\u0026micro;M 3-Isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich, #I5879), 50nM dexamethasone (Sigma, #D4902), 50-100ng/mL FGF7, 10\u0026micro;M SB431542, 3\u0026micro;M CHIR99021, 0.25% (v/v) bovine albumin (Gibco, #15260-037), 15mM HEPES (Sigma, #H-0891), 0.8mM CaCl2 (Sigma-Aldrich, #449709), 0.1% (v/v) ITS Premix (Sigma, #13146) and 1% (v/v) penicillin/streptomycin. The cell suspension was loaded onto a Transwell\u0026reg; insert, placed in a well of a 12-well plate and 0.5 mL of AEC2 induction medium was added to the well and changed every other day (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eFor mechanical stretching, lung progenitor (\u0026gt;\u0026thinsp;50% NKX2-1\u003csup\u003e+\u003c/sup\u003e) cells (2.5 x 10\u003csup\u003e5\u003c/sup\u003e/mL) were resuspended in a mixture of AEC2 induction medium and 80% Matrigel\u0026reg; GFR. One mL of cell suspension was then seeded into each well of a 6-well tissue train\u0026reg; circular foam culture plate (FlexCell, TTCF-5001U) and incubated at 37\u0026deg;C for 2 hours to facilitate gelation. Three mL of induction medium was added which was changed every 2\u0026ndash;3 days. Mechanical stretching was implemented 3 days post-seeding, the tissue train circular foam culture plate was connected to the FX-4000 Tension System (FlexCell). Settings were set to mimic fetal breathing movements, i,e, 5% elongation, frequencies of 1 Hz, square waveform, continuous cycle of 15 minutes ON and 45 minutes OFF (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Organoids were subjected to this phasic strain regimen for 21 days. All cultures were maintained at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e and 5% O\u003csub\u003e2\u003c/sub\u003e environment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSorting CPM\u003csup\u003e+\u003c/sup\u003e lung progenitor cells\u003c/h2\u003e \u003cp\u003eOn Day 22 of differentiation, cells were harvested with TrypLE\u0026trade; Express and passed through a 40-\u0026micro;m pore size cell strainer. Cells were stained with unconjugated mouse anti-human CPM monoclonal antibody for 30 minutes at 4\u0026deg;C and subsequently incubated with Alexa Fluor 647 donkey anti-mouse IgG and VivaFix 410/450 viability dye for 30 minutes at 4\u0026deg;C. A MoFlo XDP U/VBR (Beckman Coulter) sorter was used for isolating viable CPM\u003csup\u003e+\u003c/sup\u003e cells. For source and dilution of antibodies see Table\u0026nbsp;1 in the online supplement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFlow Cytometry and Immunofluorescence analysis\u003c/h2\u003e \u003cp\u003eFlow cytometry and immunofluorescence was conducted as previously described (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). The procedure of both methods is detailed in the online supplement. Antibodies and their concentrations are described in Table\u0026nbsp;1 in the online supplement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eElectron Microscopy\u003c/h2\u003e \u003cp\u003eSamples processing and microscopy was performed as previously reported (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Details are described in the online Supplement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation, cDNA preparation and real-time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from cells and organoids using the PureLink RNA Micro kit (Thermo Fisher Scientific, #12183016) and reverse transcribed with the SuperScript\u0026trade; IV SuperScript IV First-Strand Synthesis System (Invitrogen, #18091050). PCR amplification was conducted using the SYBR Select Master Mix (Applied Biosystems, #34472908) and probes targeting various genes of interest are listed in Table\u0026nbsp;2 in the online supplement. Gene expression was normalized to beta-actin (ACTB) and expressed relative to selected appropriate positive or negative controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMeasuring organoid diameter\u003c/h2\u003e \u003cp\u003eOrganoid diameter was measured using the built-in scale bar function on the EVOS M5000 imaging system (ThermoFisher Scientific). To obtain the mean of each biological replicate, more than 150 organoids from each condition were imaged and measured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMatrigel stiffness\u003c/h2\u003e \u003cp\u003eThe mechanical properties of 50 \u003cem\u003eversus\u003c/em\u003e 80% Matrigel were assessed by atomic force microscopy as detailed in the online supplement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard Error of Mean (SEM). Statistical analysis was performed using GraphPad Prism Version 7.0.1 software. For multiple groups, one-way ANOVA followed by Tukey\u0026rsquo;s Multiple Comparisons Test was used. For less than three groups, a paired or unpaired Student t-test was used if both distributions were normal while a Mann-Whitney test was used if normality was not confirmed. Single asterisks (*) indicate statistical significance of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eHigher density seeding of lung progenitor cells improves AEC2 differentiation in organoids.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eUsing an adapted protocol (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), we differentiated human CA1 ES cells to NKX2-1\u003csup\u003e+\u003c/sup\u003e lung progenitor (LPC) cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, S1). In contrast to the published protocol, we did not observe any benefit from adding DAPT, a γ-secretase inhibitor of the NOTCH pathway, to the media for differentiating ventral anterior foregut endoderm (VAFE) to LPCs (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). We introduced DAPT at different time points and durations during the VAFE to LPC differentiation period (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA\u003c/b\u003e) and validated its inhibition of NOTCH (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB\u003c/b\u003e) but did not find any effect on NKX2-1\u003csup\u003e+\u003c/sup\u003e LPC induction (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC\u003c/b\u003e). However, extending the original duration of differentiation of AFE to LPC from D11-15 (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e) to D11-22 doubled the induction efficiency of NKX2.1\u003csup\u003e+\u003c/sup\u003e cells from 21.79\u0026thinsp;\u0026plusmn;\u0026thinsp;4.21% at D15 (n\u0026thinsp;=\u0026thinsp;14 biological replicates) to 44.35\u0026thinsp;\u0026plusmn;\u0026thinsp;3.32% at D22 (n\u0026thinsp;=\u0026thinsp;25 biological replicates; a.k.a. 25 separate differentiations) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Similar NKX2-1 induction efficiencies (45\u0026ndash;55%) at D22 of differentiation were observed with CA1 ES and NCRM1 iPS cells that both were stably transfected with a hSFTPC\u003csup\u003eGFP\u003c/sup\u003e reporter (\u003cb\u003eFig. S3B\u003c/b\u003e). The flow cytometry data was corroborated by nuclear immunostaining of the cells for NKX2-1 (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, S3B). Flow cytometry and real-time PCR analysis also indicated a greater commitment of LPCs to \u003cem\u003eSOX9\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e than \u003cem\u003eSOX2\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e progenitor lineages (\u003cb\u003eFig. S3C\u003c/b\u003e). When more than 50% of the LPC population at D22 was positive for NKX2-1 (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), cells were embedded in a 1:1 diluted (50%) Matrigel for organoid formation and then incubated in a distalizing medium promoting AEC2 induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eOrganoid size and number, but not morphology, were dependent on the seeding density of CA1 LPCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD,E). Cultures started with 2.5x10\u003csup\u003e4\u003c/sup\u003e LPCs yielded less organoids than a 4-fold higher concentration of LPCs. Moreover, organoids in cultures started with 2.5x10\u003csup\u003e4\u003c/sup\u003e LPCs were on average double the size of those found in the higher density seeded cultures (215.95\u0026thinsp;\u0026plusmn;\u0026thinsp;11.45\u0026micro;m \u003cem\u003evs\u003c/em\u003e 129.12\u0026thinsp;\u0026plusmn;\u0026thinsp;5.66\u0026micro;m diameter at D36) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Independent of seeding density, cells within the organoids were highly proliferative based on Ki-67 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). In both cultures, we observed a slight but non-significant decrease in Ki-67\u003csup\u003e+\u003c/sup\u003e cells at D36 \u003cem\u003eversus\u003c/em\u003e D29, suggesting a small reduction in proliferation as differentiation progressed. Thus, seeding density of LPCs affects the size of the organoids, especially on D36 of differentiation.\u003c/p\u003e \u003cp\u003eWe next investigated whether seeding density (i.e., size of the organoid) impacted AEC2 differentiation. Flow cytometry for HT2-280, a human AEC2 surface marker (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), revealed an increase in HT2-280 expression over time under both seeding conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,B). The percentage of HT2-280\u003csup\u003e+\u003c/sup\u003e cells at D43 of differentiation was significantly greater in high-density \u003cem\u003eversus\u003c/em\u003e low-density seeded (\u003cem\u003ei.e.\u003c/em\u003e, smaller \u003cem\u003eversus\u003c/em\u003e larger) organoid cultures (20.15\u0026thinsp;\u0026plusmn;\u0026thinsp;6.05% \u003cem\u003evs.\u003c/em\u003e 7.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, the percentage of CDH1 (E-cadherin) positive epithelial cells was significantly lower in high-density compared to low-density seeded organoid cultures. The efficiency in AEC2 differentiation was validated with CA1 and NCRM1 cells transduced with a hSFTPC\u003csup\u003eGFP\u003c/sup\u003e reporter (\u003cb\u003eFig. S4\u003c/b\u003e). The hSFTPC promoter has previously been validated to label murine AEC2 \u003cem\u003ein situ\u003c/em\u003e (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Immunostaining of D43 CA1-SFTPC\u003csup\u003eGFP\u003c/sup\u003e organoid cells (\u003cb\u003eFig. S4A\u003c/b\u003e) revealed that GFP\u003csup\u003e+\u003c/sup\u003e cells, representing SFTPC expression, were positive for CDH1 (see \u003cb\u003einlet\u003c/b\u003e), and that 16.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22% (n\u0026thinsp;=\u0026thinsp;3) of the organoid cells stained positive for HT2-280 (\u003cb\u003eFig. S4C\u003c/b\u003e). Flow cytometry for GFP (\u003cb\u003eFig.S4A\u003c/b\u003e-right panel) of D43 organoids formed from 10\u003csup\u003e5\u003c/sup\u003e CA1-hSFTPC\u003csup\u003eGFP\u003c/sup\u003e or NCRM1-hSFTPC\u003csup\u003eGFP\u003c/sup\u003e LPCs demonstrated that 20.42\u0026thinsp;\u0026plusmn;\u0026thinsp;2.76% and 21.06\u0026thinsp;\u0026plusmn;\u0026thinsp;4.40%, respectively, of the organoid cells expressed GFP (\u003cb\u003eFig. S4B\u003c/b\u003e), matching the differentiation efficiency of LPCs into AEC2 when using HT2-280 as readout (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and was in line with the immunostaining findings.\u003c/p\u003e \u003cp\u003eWe then assessed whether time in culture increased AEC2 differentiation. We dissociated the organoids and passaged them into fresh Matrigel at D14 after the initial start of organoid culture from LPCs, extending the duration of organoid differentiation by another week (D50 instead of D43). This did not result in a further increase hSFTPC-driven GFP expression (\u003cb\u003eFig. S4D\u003c/b\u003e). Real-time PCR analysis of \u003cem\u003eGATA6\u003c/em\u003e, a transcription factor essential for distal lung epithelial differentiation (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), and AEC2 markers \u003cem\u003eSFTPB\u003c/em\u003e and \u003cem\u003eSFTPC\u003c/em\u003e substantiated the HT2-280 and GFP cytometry findings. In both low- and high-density seeded organoid cultures, \u003cem\u003eGATA6\u003c/em\u003e, \u003cem\u003eSFTPB\u003c/em\u003e, and \u003cem\u003eSFTPC\u003c/em\u003e mRNA expression increased compared to either ESC/iPSC (\u003cb\u003eFig. S5\u003c/b\u003e) or D22 LPCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), with high-density seeded organoids exhibiting the highest expression.\u003c/p\u003e \u003cp\u003eTo visualise the diversity of cell types within D43 organoid cultures, we conducted immunofluorescence confocal microscopy for distal and proximal epithelial lung markers. Consistent with the gene expression findings, we identified pro-SFTPB\u003csup\u003e+\u003c/sup\u003e and HT-280\u003csup\u003e+\u003c/sup\u003e cells (\u003cb\u003eFig. S6A\u003c/b\u003e-bottom panels) within the CDH1\u003csup\u003e+\u003c/sup\u003e/NKX2-1\u003csup\u003e+\u003c/sup\u003e (\u003cb\u003eFig. S6A\u003c/b\u003e-top panels) organoids at D43. Positive immunostaining for mature SFTPB (mSFTB, see inlets of pro-SFTB images) indicated the presence of cells capable of processing pro-SFTPB into its mature form. HTII-280 staining was apical within the lumen of the organoids, in line with an apical-in orientation. Immunostaining for SCGB1A1 (club cell marker) and TUBB4A (ciliated cell marker) was negative, aligning with the reduction in proximal gene expression \u003cb\u003eFig. S6A-\u003c/b\u003emiddle panels). Ultrastructural electron microscopy (EM) analysis of the D43 organoids revealed cell junctions between epithelial cells with microvilli at the apical surface \u003cb\u003e(Fig. S6B-\u003c/b\u003eleft panels\u003cb\u003e)\u003c/b\u003e. Immuno-gold EM visualized mature SFTPB within cytoplasmic pre-lamellar multivesicular structures (\u003cb\u003eFig. S6B-\u003c/b\u003eleft panels), consistent with previous reports for fetal AEC2 \u003cem\u003ein situ\u003c/em\u003e (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eRemoval of mesenchymal progenitor cells limits AEC2 differentiation within organoids.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe next enriched the CA1 NKX2-1\u003csup\u003e+\u003c/sup\u003e expressing population before organoid formation using carboxypeptidase M (CPM), an enzyme present on the surface of lung progenitor cells (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Sorting for CPM\u003csup\u003e+\u003c/sup\u003e cells at D22 (43.43\u0026thinsp;\u0026plusmn;\u0026thinsp;3.48% of total cells were CPM\u003csup\u003e+\u003c/sup\u003e at D22, n\u0026thinsp;=\u0026thinsp;8 biological replicates; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) resulted in an enrichment of the NKX2-1 expressing LPCs from 44.35\u0026thinsp;\u0026plusmn;\u0026thinsp;3.32% to 80.54\u0026thinsp;\u0026plusmn;\u0026thinsp;4.05% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). NKX2-1 immunostaining further confirmed the enrichment in the number of NKX2-1\u003csup\u003e+\u003c/sup\u003e LPCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). As depicted in \u003cb\u003eFig.\u0026nbsp;3C\u003c/b\u003e, 10\u003csup\u003e5\u003c/sup\u003e CPM\u003csup\u003e+\u003c/sup\u003e-sorted LPCs formed organoids within a few days of seeding (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) that were similar in shape and size to organoids formed from unsorted (\u0026gt;\u0026thinsp;50% NKX-2-1\u003csup\u003e+\u003c/sup\u003e) LPCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Surprisingly, the percentage of HT2-280\u003csup\u003e+\u003c/sup\u003e cells in D43 organoids established with CPM\u003csup\u003e+\u003c/sup\u003e-sorted LPCs was significantly lower compared to organoids formed with unsorted LPCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), while the percentage of CDH1\u003csup\u003e+\u003c/sup\u003e epithelial cells remained unchanged. We hypothesized that the reduction of HT2-280\u003csup\u003e+\u003c/sup\u003e cells in the organoids formed from CPM\u003csup\u003e+\u003c/sup\u003e-sorted LPCs occurred due to the loss of mesodermal cells, which are known to be essential for distal epithelial differentiation (\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). In our differentiation protocol (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), we did not sort for a pure definitive endoderm population as \u0026ge;\u0026thinsp;85% of the cells at D6 of differentiation were double positive for c-KIT and CXCR4 (\u003cb\u003eFigs. S1B, S3A\u003c/b\u003e). Therefore, we started our differentiation towards AEC2 with a definitive endoderm population that was contaminated by other lineages, including mesoderm (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). By sorting the D22 cell population for CPM\u003csup\u003e+\u003c/sup\u003e cells, we likely removed most of the mesodermal progenitor cells that had emerged during the differentiation protocol (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). To verify, we compared the expression of mesenchymal markers vimentin (VIM) and actin alpha 2 (ACTA2) in our CPM\u003csup\u003e+\u003c/sup\u003e-sorted \u003cem\u003eversus\u003c/em\u003e unsorted organoid cultures (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Immunofluorescence confocal microscopy revealed the presence of VIM\u003csup\u003e+\u003c/sup\u003e cells in the unsorted organoid cultures, while VIM\u003csup\u003e+\u003c/sup\u003e cells were sparse in the CPM\u003csup\u003e+\u003c/sup\u003e-sorted organoid cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Reduced \u003cem\u003eVIM\u003c/em\u003e and \u003cem\u003eACTA2\u003c/em\u003e expression in the CPM\u003csup\u003e+\u003c/sup\u003e-sorted compared to unsorted organoid cultures corroborated the immunofluorescence findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Together, the findings suggest that organoids formed with CPM\u003csup\u003e+\u003c/sup\u003e-sorted LPCs contain fewer mesenchymal progenitor cells. The loss in VIM\u003csup\u003e+\u003c/sup\u003e cells could also be due to less epithelial-mesenchymal transition in the sorted LPC organoid cultures, but the similar percentages of CDH1\u003csup\u003e+\u003c/sup\u003e epithelial cells in the unsorted and sorted LPC organoid cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) argue against epithelial dedifferentiation. Mesenchymal support for LPC differentiation into AEC2 was corroborated by incubating LPCs in the absence and presence of human embryonic lung fibroblasts (hELF). Human ELFs were mitotically inactivated by irradiation (ihELF) to prevent overgrowth during co-culturing without influencing their secretome (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). D22 NKX2-1\u003csup\u003e+\u003c/sup\u003e LPCs were grown on Transwell\u0026reg; inserts pre-coated with either Matrigel, Laminin, or Collagen type IV (the latter two matrices are the main components of Matrigel). The inserts were cultured in distalizing medium with and without ihELFs seeded in the bottom wells of the Transwell\u0026reg; plates. The percentage of CDH1\u003csup\u003e+\u003c/sup\u003e cells did not differ between LPCs cultured for 22 days with or without ihELFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). However, independent of the coated matrix, co-culture with ihELFs increased the percentage of HT2-280\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), confirming the importance of mesenchymal-epithelial crosstalk for fetal AEC2 differentiation (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Direct contact between ihELF and LPCs within organoids did not further improve HT-280 induction above indirect co-culturing (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e \u003cb\u003eBiophysical forces stimulate AEC2 differentiation within organoids.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo examine whether mechanical strain affects the differentiation of LPC into AEC2 within organoids, we recapitulated fetal breathing movements \u003cem\u003ein vitro\u003c/em\u003e using a 3D stretching device (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Within 3 days of culture of D22 unsorted (\u0026gt;\u0026thinsp;50% NKX2-1\u003csup\u003e+\u003c/sup\u003e) LPCs in 80% Matrigel (concentration required for applying strain to organoids using the FlexCell\u0026reg; system), organoid structures began to form. No apparent differences were observed in the morphology of organoids in static compared to stretched conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. The size of the organoids did not change with periodic stretching compared to static controls (128.10\u0026thinsp;\u0026plusmn;\u0026thinsp;7.54 vs 134.02\u0026thinsp;\u0026plusmn;\u0026thinsp;22.12 mm, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;range, n\u0026thinsp;=\u0026thinsp;3 biological repeats, static \u003cem\u003eversus\u003c/em\u003e stretch at D42). Flow cytometry for Ki-67 and CDH1 in episodically stretched organoids and paired static controls demonstrated no significant differences in proliferation and epithelial lineage expression, respectively (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, S7). However, flow cytometry for HT2-280 revealed that episodically stretched organoids had significantly more HT2-280\u003csup\u003e+\u003c/sup\u003e cells than organoids from static cultures (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, S 7). Increased gene expression of \u003cem\u003eSFTPC\u003c/em\u003e in the stretched organoids corroborated these HT2-280 findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Gene expression of \u003cem\u003eFOXJ1\u003c/em\u003e and \u003cem\u003eSCGB1A1\u003c/em\u003e was similar between static and stretched organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), suggesting that episodic stretching does not contribute to the commitment of LPCs to proximal epithelial lung lineages. To exclude that LPC to AEC2 differentiation varied with the stiffness of the Matrigel, we determined the elastic moduli by atomic force microscopy (AFM) micro-indentation. The 80% Matrigel used in the episodic stretch experiments had a similar elastic modulus at room temperature as 50% Matrigel used in all other experiments (150\u0026thinsp;\u0026plusmn;\u0026thinsp;13 Pa vs 125\u0026thinsp;\u0026plusmn;\u0026thinsp;12 Pa, n\u0026thinsp;\u0026ge;\u0026thinsp;17 measurements). Also, organoid size (128.10\u0026thinsp;\u0026plusmn;\u0026thinsp;7.54 vs 129.12\u0026thinsp;\u0026plusmn;\u0026thinsp;5.66\u0026micro;m diameter) and \u003cem\u003eSFTPC\u003c/em\u003e induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD \u003cem\u003evs\u003c/em\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) under static conditions did not differ between 80 and 50% Matrigel.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eReplicating the alveolar microenvironment for lung regenerative purposes has been challenging. Cellular and chemical cues of the distal niche have been used to create complex human alveolar organoids using either fetal lung bud tip progenitors (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e) or adult AEC2 cells (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). The impact of cellular and biophysical cues on AEC2 differentiation within hPSC-derived organoids is not well understood. Here, we demonstrate that seeding density, mesenchymal presence and mechanical strain promote AEC2 differentiation within hPSC-derived organoids.\u003c/p\u003e \u003cp\u003eIn the present study, our adapted directed differentiation protocol yielded 44\u0026ndash;55% NKX2-1\u003csup\u003e+\u003c/sup\u003e lung progenitor cells without sorting. This was significantly lower than the 85% reported by Yamamoto et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), but it matched or exceeded what has been reported for other human cell lines (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The variation in induction efficiency could be attributed to factors such as the cell line used, the composition of the media and growth factors, the type of extracellular matrix coating, or the methodology for evaluating NKX2-1\u003csup\u003e+\u003c/sup\u003e expression. Immunostaining for NKX2-1\u003csup\u003e+\u003c/sup\u003e consistently yielded higher percentages (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) compared to NKX2-1\u003csup\u003eGFP\u003c/sup\u003e reporters (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). We observed significant oscillation in the percentage of NKX2-1\u003csup\u003e+\u003c/sup\u003e progenitors between experiments, consistent with previous reports (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). This inter-experiment variability was independent of media composition, hPSC passage number, efficiency of differentiation into definitive endoderm, or cell seeding density. The induction efficiencies of NKX2-1\u003csup\u003e+\u003c/sup\u003e LPCs at D22 were validated using CD26\u003csup\u003elow\u003c/sup\u003e/CD47\u003csup\u003ehigh\u003c/sup\u003e and CPM\u003csup\u003e+\u003c/sup\u003e flow cytometry.\u003c/p\u003e \u003cp\u003eHerein, we demonstrated that the concentration of Matrigel did not affect organoid formation from LPCs. While Hawkins et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) used pure Matrigel, we utilized 50% and 80% Matrigel to enrobe the cells. Despite this difference, we did not observe any significant variations in organoid formation between 50 and 80% Matrigel. Previous studies have shown that lineage differentiation of human lung progenitors can be influenced by matrix stiffness (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). To investigate this further, we used AFM micro-indentation to measure the mechanical properties of 50% and 80% Matrigel at room temperature. Interestingly, we found that the elastic moduli of both concentrations were very similar, measuring around 120\u0026ndash;150 Pa. These values closely align with those reported for pure Matrigel (~\u0026thinsp;120 Pa) taken with AFM (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). Thus, it is unlikely that variations in LPC differentiation towards AEC2 between the various published reports are due to differences in Matrigel stiffness. However, it\u0026rsquo;s worth noting that these measured values are significantly lower than the elastic moduli (~\u0026thinsp;4.5\u0026ndash;10 kPa) reported for an alveolar wall (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e) and decellularized alveolar ECM (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). This suggests that stiffer matrices than Matrigel may be required to accurately replicate lineage differentiation in the \u003cem\u003ein situ\u003c/em\u003e distal niche.\u003c/p\u003e \u003cp\u003eIn this study, we evaluated the efficiency of AEC2 differentiation using HT2-280 protein expression. HT2-280 is present on the surface of both fetal and adult human AEC2 (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e), and its expression aligns with that of SFTPC (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Several studies have used HT2-280 to sort for human AEC2s for the generation of alveolar organoid cultures (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). While a recent study reported challenges in sorting AEC2 with HT2-280 from hiPSC-derived alveolar organoids (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e), it\u0026rsquo;s important to note that this difficulty may be due to certain PSC lines, as other studies have successfully identified AEC2s using HT2-280 in hPSC-derived alveolar organoids (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). To validate our findings, we compared the HT2-280 flow cytometry results with those obtained from the GFP expression in organoids established with hPSCs that were stably transduced with a hSFTPC\u003csup\u003eGFP\u003c/sup\u003e reporter. Both flow cytometric readouts, HT2-280 and hSFTPC-GFP, consistently identified comparable viable AEC2 populations within the organoids, which were further confirmed through immunostaining for HT2-280. Our induction efficiency of 20\u0026ndash;30% HT2-280\u003csup\u003e+\u003c/sup\u003e cells falls within reported values ranging from 4\u0026ndash;50% using SFTPC reporters (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). Our starting population consisted of 50\u0026ndash;60% NKX2-1\u003csup\u003e+\u003c/sup\u003e LPCs. Surprisingly, enriching the NKX2-1\u003csup\u003e+\u003c/sup\u003e LPC population to more than 80% with CPM\u003csup\u003e+\u003c/sup\u003e sorting resulted in a decrease in HT2-280\u003csup\u003e+\u003c/sup\u003e AEC2 induction within the organoids. The absence of any enrichment in \u003cem\u003eSFTPB\u003c/em\u003e and \u003cem\u003eSFTPC\u003c/em\u003e gene expression corroborated that CPM sorting did not lead to improved AEC2 differentiation (not shown). By contrast, previous studies have reported that enriching the NKX2-1 population resulted in a higher yield of SFTPC\u003csup\u003e+\u003c/sup\u003e cells within the organoids (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). However, these yield gains were always in comparison to the NKX2-1 negative populations (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), while we compared our findings to the unsorted\u0026thinsp;\u0026gt;\u0026thinsp;50% NKX2-1\u003csup\u003e+\u003c/sup\u003e LPC population. To complicate straight comparisons even further, some studies included direct co-cultures with fetal lung fibroblast cells (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe also observed that our CA1 organoids comprised heterogenous cell types. Approximately one-third of the CDH1\u003csup\u003e+\u003c/sup\u003e epithelial population in our organotypic cultures were positive for HT2-280, which raises questions about the identity of the remaining 65% CDH1\u003csup\u003e+\u003c/sup\u003e cells. Some studies have reported positive staining for AEC1 markers in their hPSC-derived organoids (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), while others showed negative staining (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Recently, Kanagaki and colleagues reported that presence of mesenchymal cells was crucial for AEC1 differentiation in organoids (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). Our CA1 organoid cultures contained hPSC-derived mesenchymal VIM\u003csup\u003e+\u003c/sup\u003e cells but stained negative for AEC1 markers PDPN and AQP5 (not shown). Also, unlike in other studies, no TUBB4\u003csup\u003e+\u003c/sup\u003e ciliated and SCGB1A1\u003csup\u003e+\u003c/sup\u003e secretory club cells were detected, (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). We speculate that a portion of the CDH\u003csup\u003e+\u003c/sup\u003e cells in the organoids represent non-lung endodermal lineages (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). Ultrastructural and immuno-gold EM analysis revealed cell junctions between epithelial cells within the organoids and presence of SFTPB-positive pre-lamellar multivesicular bodies, but no lamellar bodies, suggesting an immature AEC2 phenotype in HT2-280\u003csup\u003e+\u003c/sup\u003e cells at D43 within the CA1 organoids. It has been reported that 14 days post-NKX2-1 enrichment, the NKX2-1\u003csup\u003e+\u003c/sup\u003e population within lung organoids fell to 60\u0026ndash;70% (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), a level comparable to our starting population, which raises questions about the advantage of enriching the NKX2-1\u003csup\u003e+\u003c/sup\u003e population for distal differentiation within organoids. The heterogenicity in organoid cell composition among various studies is likely due to variability in differentiation protocols, cellular plasticity, contaminating cell populations, and hPSC lines used, highlighting the need for standardized protocols to facilitate better comparisons.\u003c/p\u003e \u003cp\u003eOne major difference between protocols is whether the epithelial LPCs are co-cultured with mesenchyme. Similar to other studies (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), our organoids and Transwell cultures showed significantly more HT2-280\u003csup\u003e+\u003c/sup\u003e cells when mesenchymal cells were present. We highlighted the importance of mesenchymal cells for AEC2 differentiation within organoids by removing co-developed VIM\u003csup\u003e+\u003c/sup\u003e mesodermal cells from our LPC population using CPM sorting. This resulted in less HT2-280\u003csup\u003e+\u003c/sup\u003e cells within the organoids during distal differentiation. Co-culturing LPCs with irradiated embryonic lung fibroblasts (iELF) in Transwell plates had the opposite effect and resulted in more HT2-280\u003csup\u003e+\u003c/sup\u003e cells. The stimulatory effect on HT2-280 induction was independent of the substratum used and did not require direct contact between LPCs and iELF. The ELF-secretome is unaffected by irradiation (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e) and contains various factors, including FGFs and WNTs, that are key to alveolar organoid formation (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). The cocktail of ELF factors stimulating optimal AEC differentiation remains yet unresolved.\u003c/p\u003e \u003cp\u003eFinally, we demonstrated the importance of mechanical strain in alveolar development. \u003cem\u003eIn utero\u003c/em\u003e, the lung is exposed to mechanical forces generated by fetal breathing movements (FBM) that stimulate epithelial lung growth and differentiation (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e). In a recent study using a postnatal lung organoid model of CD326\u003csup\u003e+\u003c/sup\u003e epithelial cells and fibroblasts, it was shown that static stretch increased cell proliferation, while cycle stretch promoted mesenchymal lineage gene expression (\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e). In our study, we did not observe an increase in progenitor cell proliferation in organoids subjected to a stretch regimen mimicking FBMs. This is not surprising, as more than 70% of the progenitors in static organoid cultures are proliferating. Despite no change in proliferation, we observed a marked increase in the number of HT2-280\u003csup\u003e+\u003c/sup\u003e cells and \u003cem\u003eSFTPC\u003c/em\u003e expression within the organoids. This aligns with previous studies demonstrating that a similar stretch regimen increased the differentiation of primary fetal epithelial cells into AEC2 based on surfactant phospholipid (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e) and SFTPC (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) production. The mechanotransduction pathways stimulating AEC2 differentiation are unknown. Recently, it has been reported that ROCK-Yap/Taz signaling is essential to regulate AEC1 differentiation in response to mechanical loading (stretching) of the fetal lung (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e). The role of this mechanotransduction pathway in episodic stretch stimulated AEC2 differentiation remains to be elucidated.\u003c/p\u003e \u003cp\u003eOur study had several strengths, including the utilization of both iPSC and ESC lines, as well as the incorporation of SFTPC-GFP reporter lines. The exploration of mechanical strain in a 3D culture system, rather than a 2D system, is a novel approach with clinical relevance. Future applications could involve investigations in the impact of injurious strain (high VT ventilation) on AEC2 proliferation and differentiation in 3D organoid culture. However, there were also several limitations of our findings. One limitation is our reliance on HT2-280\u003csup\u003e+\u003c/sup\u003e AEC2 marker expression. Single-cell transcriptomics might have provided a more comprehensive understanding of the distal epithelial population within our organoids and identified non-lung cell populations within them (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). Additionally, a longer duration of culture might be necessary to improve alveolar differentiation efficiency, although we observed that passaging of the organoid cells did not further enhance AEC2 induction.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eOur study highlights the critical factors influencing efficient and validated AEC2 differentiation from hPSCs, including lung progenitor concentration, mesenchymal population, and mechanical strain. While these factors increase the yield of AECs, further investigation is needed to understand the mechanisms driving AEC1 generation in hPSC-derived organoids to achieve a more accurate modeling of the human lung \u003cem\u003ein situ.\u003c/em\u003e\u003c/p\u003e "},{"header":"List Of Abbreviations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eACTA2\u003c/em\u003e\u003c/strong\u003e: Actin alpha 2\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAEC\u003c/em\u003e\u003c/strong\u003e: Alveolar epithelial cell\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAEC2\u003c/em\u003e\u003c/strong\u003e: Alveolar epithelial type 2 cell\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAEC1\u003c/em\u003e\u003c/strong\u003e: Alveolar epithelial type 1 cell\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAFE\u003c/em\u003e\u003c/strong\u003e:\u0026nbsp;Anterior foregut endoderm\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAFM\u003c/em\u003e\u003c/strong\u003e: Atomic force microscopy\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAQP5\u003c/strong\u003e: Aquaporin 5\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBPD\u003c/em\u003e\u003c/strong\u003e: Bronchopulmonary dysplasia\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBMP4\u003c/em\u003e\u003c/strong\u003e: Bone morphogenic protein 4\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCDH1\u003c/em\u003e\u003c/strong\u003e: E-cadherin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCPM\u003c/em\u003e\u003c/strong\u003e: Carboxypeptidase M\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ec-Kit\u003c/em\u003e\u003c/strong\u003e: KIT proto-oncogene, receptor tyrosine kinase\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCXCR4\u003c/em\u003e\u003c/strong\u003e: C-X-C chemokine receptor type 4\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDMH1\u003c/em\u003e\u003c/strong\u003e: Dorsomorphin homologue 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eECM\u003c/em\u003e\u003c/strong\u003e: Extracellular matrix\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEM\u003c/em\u003e\u003c/strong\u003e: Electron microscopy\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eESC\u003c/em\u003e\u003c/strong\u003e: Embryonic stem cell\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFBM\u003c/em\u003e\u003c/strong\u003e: Fetal breathing movements\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFOXJ1\u003c/em\u003e\u003c/strong\u003e:\u0026nbsp;Forkhead box J1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGATA6\u003c/em\u003e\u003c/strong\u003e: Gata binding protein 6\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGFP\u003c/em\u003e\u003c/strong\u003e: Green fluorescent protein\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ehELF\u003c/em\u003e\u003c/strong\u003e: Human embryonic lung fibroblasts\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ehPSC\u003c/em\u003e\u003c/strong\u003e: Human pluripotent stem cell\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eihELF\u003c/em\u003e\u003c/strong\u003e; Irradiated human embryonic lung fibroblast\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLPC\u003c/em\u003e\u003c/strong\u003e: Lung progenitor cells\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eNKX2-1\u003c/em\u003e\u003c/strong\u003e: NK2 homeobox 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePDPN\u003c/em\u003e\u003c/strong\u003e: Podoplanin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePSC\u003c/em\u003e\u003c/strong\u003e: Pluripotent stem cells\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eiPSC\u003c/em\u003e\u003c/strong\u003e: Induced pluripotent stem cell \u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePCR\u003c/em\u003e\u003c/strong\u003e: Polymerase chain reaction \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eqPCR\u003c/em\u003e\u003c/strong\u003e: Quantitative PCR\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSCGB1A1\u003c/em\u003e\u003c/strong\u003e:\u0026nbsp;Secretoglobin family 1A member 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSFDM\u003c/em\u003e\u003c/strong\u003e: Serum free differentiation medium\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSOX2\u003c/em\u003e\u003c/strong\u003e:\u0026nbsp;SRY-Box Transcription Factor 2\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSOX9\u003c/em\u003e\u003c/strong\u003e: SRY-Box Transcription Factor 9\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSTFPB\u003c/em\u003e\u003c/strong\u003e: Surfactant protein B\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSFTPC\u003c/em\u003e\u003c/strong\u003e: Surfactant protein C\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTUBB4A\u003c/em\u003e\u003c/strong\u003e: Tubulin Beta 4A Class IVa\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eVAFE\u003c/em\u003e\u003c/strong\u003e: Ventral anterior foregut endoderm \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eVIM\u003c/em\u003e\u003c/strong\u003e: Vimentin\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent of participants:\u0026nbsp;\u003c/strong\u003eThis study does not involve animal experiments or human participants.Use of human pluripotent cell lines was approved by the Stem Cell Oversight Committee of The Canadian Institute of Health Research (Patient-specific alveolar type II (ATII) cells from surfactant protein-B deficient induced pluripotent stem cells) in March 2014. The Research Ethical Board of the Hospital for Sick Children\u0026nbsp;confirmed no need for additional ethical approval.\u003c/p\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eArtificial intelligence:\u0026nbsp;\u003c/strong\u003eThe authors declare that artificial intelligence is not used in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research was supported by Canadian Institutes of Health Research (FND-143309 to MP).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contribution:\u0026nbsp;\u003c/strong\u003eOG and CB: Conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing. \u0026nbsp;JW, DL, MA, LB, and AP: Design, Collection and/or assembly of data. \u0026nbsp;SLL: manuscript writing. \u0026nbsp;MP: Conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eWe like to thank Dr. Andras Nagy, University of Toronto, and the Center for Commercialization of Regenerative Medicine, Toronto, for their generous gifts of CA1 ES and NCRM1 iPS cells, respectively. The hSFTPC promoter construct was a generous gift of Dr. Jeffrey Whistsett, Cincinatti, OH. Claudia Bilodeau was supported by a RESTRACOMP scholarship award from the Hospital for Sick Children.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGotoh S, Ito I, Nagasaki T, Yamamoto Y, Konishi S, Korogi Y, et al. Generation of alveolar epithelial spheroids via isolated progenitor cells from human pluripotent stem cells. Stem Cell Rep. 2014;3(3):394\u0026ndash;403.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHawkins F, Kramer P, Jacob A, Driver I, Thomas DC, McCauley KB, et al. Prospective isolation of NKX2-1-expressing human lung progenitors derived from pluripotent stem cells. J Clin Invest. 2017;127(6):2277\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang SX, Islam MN, O'Neill J, Hu Z, Yang YG, Chen YW, et al. Efficient generation of lung and airway epithelial cells from human pluripotent stem cells. Nat Biotechnol. 2014;32(1):84\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamamoto Y, Gotoh S, Korogi Y, Seki M, Konishi S, Ikeo S, et al. Long-term expansion of alveolar stem cells derived from human iPS cells in organoids. Nat Methods. 2017;14(11):1097\u0026ndash;106.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGreen MD, Chen A, Nostro MC, d'Souza SL, Schaniel C, Lemischka IR, et al. Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells. Nat Biotechnol. 2011;29(3):267\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang SX, Green MD, de Carvalho AT, Mumau M, Chen YW, D'Souza SL, et al. The in vitro generation of lung and airway progenitor cells from human pluripotent stem cells. Nat Protoc. 2015;10(3):413\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLongmire TA, Ikonomou L, Hawkins F, Christodoulou C, Cao Y, Jean JC, et al. Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells. Cell Stem Cell. 2012;10(4):398\u0026ndash;411.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMou H, Zhao R, Sherwood R, Ahfeldt T, Lapey A, Wain J, et al. Generation of multipotent lung and airway progenitors from mouse ESCs and patient-specific cystic fibrosis iPSCs. Cell Stem Cell. 2012;10(4):385\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJacob A, Morley M, Hawkins F, McCauley KB, Jean JC, Heins H, et al. Differentiation of Human Pluripotent Stem Cells into Functional Lung Alveolar Epithelial Cells. Cell Stem Cell. 2017;21(4):472\u0026ndash;88. e10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeibel SL, McVicar RN, Winquist AM, Snyder EY. Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease. J Vis Exp. 2021(170).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDye BR, Hill DR, Ferguson MA, Tsai YH, Nagy MS, Dyal R et al. In vitro generation of human pluripotent stem cell derived lung organoids. Elife. 2015;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarkauskas CE, Chung MI, Fioret B, Gao X, Katsura H, Hogan BL. Lung organoids: current uses and future promise. Development. 2017;144(6):986\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeibel SL, Winquist A, Tseu I, Wang J, Luo D, Shojaie S, et al. Reversal of Surfactant Protein B Deficiency in Patient Specific Human Induced Pluripotent Stem Cell Derived Lung Organoids by Gene Therapy. Sci Rep. 2019;9(1):13450.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorrisey EE, Hogan BL. Preparing for the first breath: genetic and cellular mechanisms in lung development. Dev Cell. 2010;18(1):8\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaniggia I, Tseu I, Han RN, Smith BT, Tanswell K, Post M. Spatial and temporal differences in fibroblast behavior in fetal rat lung. Am J Physiol. 1991;261(6 Pt 1):L424\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeimling J, Thompson K, Tseu I, Wang J, Keijzer R, Tanswell AK, et al. Mesenchymal maintenance of distal epithelial cell phenotype during late fetal lung development. Am J Physiol Lung Cell Mol Physiol. 2007;292(3):L725\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarkauskas CE, Cronce MJ, Rackley CR, Bowie EJ, Keene DR, Stripp BR, et al. Type 2 alveolar cells are stem cells in adult lung. J Clin Invest. 2013;123(7):3025\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZepp JA, Zacharias WJ, Frank DB, Cavanaugh CA, Zhou S, Morley MP, et al. Distinct Mesenchymal Lineages and Niches Promote Epithelial Self-Renewal and Myofibrogenesis in the Lung. Cell. 2017;170(6):1134\u0026ndash;e4810.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee JH, Bhang DH, Beede A, Huang TL, Stripp BR, Bloch KD, et al. Lung stem cell differentiation in mice directed by endothelial cells via a BMP4-NFATc1-thrombospondin-1 axis. Cell. 2014;156(3):440\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFox E, Shojaie S, Wang J, Tseu I, Ackerley C, Bilodeau M, et al. Three-dimensional culture and FGF signaling drive differentiation of murine pluripotent cells to distal lung epithelial cells. Stem Cells Dev. 2015;24(1):21\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen YW, Huang SX, de Carvalho A, Ho SH, Islam MN, Volpi S, et al. A three-dimensional model of human lung development and disease from pluripotent stem cells. Nat Cell Biol. 2017;19(5):542\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeibel SL, McVicar RN, Winquist AM, Niles WD, Snyder EY. Generation of Complete Multi-Cell Type Lung Organoids From Human Embryonic and Patient-Specific Induced Pluripotent Stem Cells for Infectious Disease Modeling and Therapeutics Validation. Curr Protoc Stem Cell Biol. 2020;54(1):e118.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlber AB, Marquez HA, Ma L, Kwong G, Thapa BR, Villacorta-Martin C, et al. Directed differentiation of mouse pluripotent stem cells into functional lung-specific mesenchyme. Nat Commun. 2023;14(1):3488.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJesudason EC. Airway smooth muscle: an architect of the lung? Thorax. 2009;64(6):541\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarding R. Fetal pulmonary development: the role of respiratory movements. Equine Vet J Suppl. 1997(24):32\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKitterman JA. The effects of mechanical forces on fetal lung growth. Clin Perinatol. 1996;23(4):727\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoessinger AC, Harding R, Adamson TM, Singh M, Kiu GT. Role of lung fluid volume in growth and maturation of the fetal sheep lung. J Clin Invest. 1990;86(4):1270\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWigglesworth JS, Desai R. Effect on lung growth of cervical cord section in the rabbit fetus. Early Hum Dev. 1979;3(1):51\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakamura T, Liu M, Mourgeon E, Slutsky A, Post M. Mechanical strain and dexamethasone selectively increase surfactant protein C and tropoelastin gene expression. Am J Physiol Lung Cell Mol Physiol. 2000;278(5):L974\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanchez-Esteban J, Cicchiello LA, Wang Y, Tsai SW, Williams LK, Torday JS, et al. Mechanical stretch promotes alveolar epithelial type II cell differentiation. J Appl Physiol (1985). 2001;91(2):589\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu M, Skinner SJ, Xu J, Han RN, Tanswell AK, Post M. Stimulation of fetal rat lung cell proliferation in vitro by mechanical stretch. Am J Physiol. 1992;263(3 Pt 1):L376\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShojaie S, Ermini L, Ackerley C, Wang J, Chin S, Yeganeh B, et al. Acellular lung scaffolds direct differentiation of endoderm to functional airway epithelial cells: requirement of matrix-bound HS proteoglycans. Stem Cell Rep. 2015;4(3):419\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLitvack ML, Wigle TJ, Lee J, Wang J, Ackerley C, Grunebaum E, et al. Alveolar-like Stem Cell-derived Myb(-) Macrophages Promote Recovery and Survival in Airway Disease. Am J Respir Crit Care Med. 2016;193(11):1219\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBilodeau C, Shojaie S, Goltsis O, Wang J, Luo D, Ackerley C, et al. TP63 basal cells are indispensable during endoderm differentiation into proximal airway cells on acellular lung scaffolds. NPJ Regen Med. 2021;6(1):12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamamoto Y, Korogi Y, Hirai T, Gotoh S. A method of generating alveolar organoids using human pluripotent stem cells. Methods Cell Biol. 2020;159:115\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGonzalez RF, Allen L, Gonzales L, Ballard PL, Dobbs LG. HTII-280, a biomarker specific to the apical plasma membrane of human lung alveolar type II cells. J Histochem Cytochem. 2010;58(10):891\u0026ndash;901.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Tuyl M, Groenman F, Kuliszewski M, Ridsdale R, Wang J, Tibboel D, et al. Overexpression of lunatic fringe does not affect epithelial cell differentiation in the developing mouse lung. Am J Physiol Lung Cell Mol Physiol. 2005;288(4):L672\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTibboel J, Groenman FA, Selvaratnam J, Wang J, Tseu I, Huang Z, et al. Hypoxia-inducible factor-1 stimulates postnatal lung development but does not prevent O2-induced alveolar injury. Am J Respir Cell Mol Biol. 2015;52(4):448\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKeijzer R, van Tuyl M, Meijers C, Post M, Tibboel D, Grosveld F, et al. The transcription factor GATA6 is essential for branching morphogenesis and epithelial cell differentiation during fetal pulmonary development. Development. 2001;128(4):503\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang H, Lu MM, Zhang L, Whitsett JA, Morrisey EE. GATA6 regulates differentiation of distal lung epithelium. Development. 2002;129(9):2233\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRidsdale R, Post M. Surfactant lipid synthesis and lamellar body formation in glycogen-laden type II cells. Am J Physiol Lung Cell Mol Physiol. 2004;287(4):L743\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcCauley KB, Hawkins F, Serra M, Thomas DC, Jacob A, Kotton DN. Efficient Derivation of Functional Human Airway Epithelium from Pluripotent Stem Cells via Temporal Regulation of Wnt Signaling. Cell Stem Cell. 2017;20(6):844\u0026ndash;57. e6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShannon JM. Induction of alveolar type II cell differentiation in fetal tracheal epithelium by grafted distal lung mesenchyme. Dev Biol. 1994;166(2):600\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurry CE, Keller G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell. 2008;132(4):661\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng X, Ying L, Lu L, Galvao AM, Mills JA, Lin HC, et al. Self-renewing endodermal progenitor lines generated from human pluripotent stem cells. Cell Stem Cell. 2012;10(4):371\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFarahani RM, Xaymardan M. Platelet-Derived Growth Factor Receptor Alpha as a Marker of Mesenchymal Stem Cells in Development and Stem Cell Biology. Stem Cells Int. 2015;2015:362753.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBroers JL, de Leij L, Rot MK, ter Haar A, Lane EB, Leigh IM, et al. Expression of intermediate filament proteins in fetal and adult human lung tissues. Differentiation. 1989;40(2):119\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEiselleova L, Peterkova I, Neradil J, Slaninova I, Hampl A, Dvorak P. Comparative study of mouse and human feeder cells for human embryonic stem cells. Int J Dev Biol. 2008;52(4):353\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu X, Krawczyk E, Suprynowicz FA, Palechor-Ceron N, Yuan H, Dakic A, et al. Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens. Nat Protoc. 2017;12(2):439\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiller AJ, Hill DR, Nagy MS, Aoki Y, Dye BR, Chin AM, et al. In Vitro Induction and In Vivo Engraftment of Lung Bud Tip Progenitor Cells Derived from Human Pluripotent Stem Cells. Stem Cell Rep. 2018;10(1):101\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLim K, Donovan APA, Tang W, Sun D, He P, Pett JP, et al. Organoid modeling of human fetal lung alveolar development reveals mechanisms of cell fate patterning and neonatal respiratory disease. Cell Stem Cell. 2023;30(1):20\u0026ndash;e379.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiao D, Li H. Dissecting the Niche for Alveolar Type II Cells With Alveolar Organoids. Front Cell Dev Biology. 2020;8:419.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEngler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell. 2006;126(4):677\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlcaraz J, Xu R, Mori H, Nelson CM, Mroue R, Spencer VA, et al. Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia. EMBO J. 2008;27(21):2829\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBou Jawde S, Takahashi A, Bates JHT, Suki B. An Analytical Model for Estimating Alveolar Wall Elastic Moduli From Lung Tissue Uniaxial Stress-Strain Curves. Front Physiol. 2020;11:121.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuque T, Melo E, Garreta E, Cortiella J, Nichols J, Farre R, et al. Local micromechanical properties of decellularized lung scaffolds measured with atomic force microscopy. Acta Biomater. 2013;9(6):6852\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJorba I, Beltran G, Falcones B, Suki B, Farre R, Garcia-Aznar JM, et al. Nonlinear elasticity of the lung extracellular microenvironment is regulated by macroscale tissue strain. Acta Biomater. 2019;92:265\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKorogi Y, Gotoh S, Ikeo S, Yamamoto Y, Sone N, Tamai K, et al. In Vitro Disease Modeling of Hermansky-Pudlak Syndrome Type 2 Using Human Induced Pluripotent Stem Cell-Derived Alveolar Organoids. Stem Cell Rep. 2019;12(3):431\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZacharias WJ, Frank DB, Zepp JA, Morley MP, Alkhaleel FA, Kong J, et al. Regeneration of the lung alveolus by an evolutionarily conserved epithelial progenitor. Nature. 2018;555(7695):251\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKonda B, Mulay A, Yao C, Beil S, Israely E, Stripp BR. Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture. J Vis Exp. 2020(161).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhaedi M, Calle EA, Mendez JJ, Gard AL, Balestrini J, Booth A, et al. Human iPS cell-derived alveolar epithelium repopulates lung extracellular matrix. J Clin Invest. 2013;123(11):4950\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKanagaki S, Ikeo S, Suezawa T, Yamamoto Y, Seki M, Hirai T, et al. Directed induction of alveolar type I cells derived from pluripotent stem cells via Wnt signaling inhibition. Stem Cells. 2021;39(2):156\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHurley K, Ding J, Villacorta-Martin C, Herriges MJ, Jacob A, Vedaie M, et al. Reconstructed Single-Cell Fate Trajectories Define Lineage Plasticity Windows during Differentiation of Human PSC-Derived Distal Lung Progenitors. Cell Stem Cell. 2020;26(4):593\u0026ndash;e6088.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShiraishi K, Shichino S, Ueha S, Nakajima T, Hashimoto S, Yamazaki S, et al. Mesenchymal-Epithelial Interactome Analysis Reveals Essential Factors Required for Fibroblast-Free Alveolosphere Formation. iScience. 2019;11:318\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNabhan AN, Brownfield DG, Harbury PB, Krasnow MA, Desai TJ. Single-cell Wnt signaling niches maintain stemness of alveolar type 2 cells. Science. 2018;359(6380):1118\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, Wang Z, Chu Q, Jiang K, Li J, Tang N. The Strength of Mechanical Forces Determines the Differentiation of Alveolar Epithelial Cells. Dev Cell. 2018;44(3):297\u0026ndash;312. e5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu M, Tanswell AK, Post M. Mechanical force-induced signal transduction in lung cells. Am J Physiol. 1999;277(4):L667\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJoshi R, Batie MR, Fan Q, Varisco BM. Mouse lung organoid responses to reduced, increased, and cyclic stretch. Am J Physiol Lung Cell Mol Physiol. 2022;322(1):L162\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanchez-Esteban J, Wang Y, Gruppuso PA, Rubin LP. Mechanical stretch induces fetal type II cell differentiation via an epidermal growth factor receptor-extracellular-regulated protein kinase signaling pathway. Am J Respir Cell Mol Biol. 2004;30(1):76\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen TM, van der Merwe J, Elowsson Rendin L, Larsson-Callerfelt AK, Deprest J, Westergren-Thorsson G, et al. Stretch increases alveolar type 1 cell number in fetal lungs through ROCK-Yap/Taz pathway. Am J Physiol Lung Cell Mol Physiol. 2021;321(5):L814\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"alveolar organoids, lung fibroblasts, pluripotent stem cells, mechanical strain","lastPublishedDoi":"10.21203/rs.3.rs-4510238/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4510238/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eChronic lung disease of prematurity, called bronchopulmonary dysplasia (BPD), lacks effective therapies, stressing the need for preclinical testing systems that reflect human pathology for identifying causal pathways and testing novel compounds. Alveolar organoids derived from human pluripotent stem cells (hPSC) are promising test platforms for studying distal airway diseases like BPD, but current protocols do not accurately replicate the distal niche environment of the native lung. Herein, we investigated the contributions of cellular constituents of the alveolus and fetal respiratory movements on hPSC-derived alveolar organoid formation.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eHuman PSCs were differentiated in 2D culture into lung progenitor cells (LPC) which were then further differentiated into alveolar organoids before and after removal of co-developing mesodermal cells. LPCs were also differentiated in Transwell\u0026reg; co-cultures with and without human fetal lung fibroblast. Forming organoids were subjected to phasic mechanical strain using a Flexcell\u0026reg; system. Differentiation within organoids and Transwell\u0026reg; cultures was assessed by flow cytometry, immunofluorescence, and qPCR for lung epithelial and alveolar markers of differentiation including GATA Binding Protein 6 (GATA 6), E-Cadherin (CDH1), NK2 Homeobox 1 (NKX2-1), HT2-280, Surfactant Proteins B (SFTPB) and C (SFTPC).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe observed that co-developing mesenchymal progenitors promote alveolar epithelial type 2 cell (AEC2) differentiation within hPSC-derived lung organoids. This mesenchymal effect on AEC2 differentiation was corroborated by co-culturing hPSC-NKX2-1\u003csup\u003e+\u003c/sup\u003e lung progenitors with human embryonic lung fibroblasts. The stimulatory effect did not require direct contact between fibroblasts and NKX2-1\u003csup\u003e+\u003c/sup\u003e lung progenitors. Additionally, we demonstrate that episodic mechanical deformation of hPSC-derived lung organoids, mimicking \u003cem\u003ein situ\u003c/em\u003e fetal respiratory movements, increased AEC2 differentiation without affecting proximal epithelial differentiation.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur data suggest that biophysical and mesenchymal components promote AEC2 differentiation within hPSC-derived distal organoids \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Influence of Mesenchymal and Biophysical Components on Distal Lung Organoid Differentiation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-12 09:09:50","doi":"10.21203/rs.3.rs-4510238/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-06-21T12:38:47+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-21T12:37:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-13T02:16:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Stem Cell Research \u0026 Therapy","date":"2024-06-12T12:53:14+00:00","index":"","fulltext":""},{"type":"decision","content":"Major Revision","date":"2024-06-10T07:27:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5705bcf7-6fc7-40c2-a368-4b6c7ad436f1","owner":[],"postedDate":"July 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-09T16:13:49+00:00","versionOfRecord":{"articleIdentity":"rs-4510238","link":"https://doi.org/10.1186/s13287-024-03890-2","journal":{"identity":"stem-cell-research-and-therapy","isVorOnly":false,"title":"Stem Cell Research \u0026 Therapy"},"publishedOn":"2024-09-02 16:05:40","publishedOnDateReadable":"September 2nd, 2024"},"versionCreatedAt":"2024-07-12 09:09:50","video":"","vorDoi":"10.1186/s13287-024-03890-2","vorDoiUrl":"https://doi.org/10.1186/s13287-024-03890-2","workflowStages":[]},"version":"v1","identity":"rs-4510238","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4510238","identity":"rs-4510238","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}