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. 2025 Dec 12;27:89. doi: 10.1186/s12931-025-03441-0

Modulation of an aberrant basal cell program in human alveolar epithelial spheroids and lung slices

Yong Li 1,5, Jack H Wellmerling 1, Lorena Rosas 4, Patrick A Link 6, Daniela Chow 1,3, Joshua Alvarez 4, Kyoung M Choi 1, Ana M Diaz Espinosa 1, Rachel M Gilbert 1, Nicholas P Goplen 1, Donghao Li 1,3, Andrew J Haak 1,3, Y S Prakash 1,2, Mauricio Rojas 4, Daniel J Tschumperlin 1,✉
PMCID: PMC12918232  PMID: 41388287

Abstract

Rationale

Idiopathic Pulmonary Fibrosis (IPF) is a progressive scarring disease marked by the accumulation of aberrant basal cells that are thought to promote disease progression. The cellular origin and disease-relevant cues that lead to aberrant basal cells remain poorly understood.

Objectives

We sought to identify the signals regulating formation and maintenance of aberrant basal cells from human alveolar type II (ATII) cells using 3D spheroids, and test whether inhibition of select pathways could reduce aberrant basal signatures in human precision-cut lung slices (PCLS) from diseased lungs.

Methods

We characterized aberrant basal cell signatures in human ATII spheroids in response to TGFβ1 and hypoxia mimic dimethyloxalylglycine (DMOG) treatment alone or in combination. We tested whether a Notch inhibitor (LY-411575) could inhibit/reverse these signatures in human ATII spheroids and IPF PCLS. Readouts included immunofluorescence analysis, western blotting, and quantitative PCR of aberrant basal signature genes.

Main results

We found that human ATII spheroids acquire aberrant basal cell signatures upon TGFβ1 and DMOG treatment, with the combination most effectively programming cells to an aberrant basal state. LY-411575 was able to significantly inhibit or reverse a subset of the aberrant basal cell signatures in 3D spheroids and IPF PCLS.

Conclusions

TGFβ1 and hypoxia are disease relevant signals capable of driving ATII cells to acquire aberrant basal cell signatures found in IPF. Notch inhibition may provide a tractable approach to normalize these programs in the fibrotic human lung.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12931-025-03441-0.

Introduction

Idiopathic Pulmonary Fibrosis is increasingly recognized to be driven by aberrant cellular programs that emerge under the combined effects of aging, genetic susceptibility, and environmental exposures [1]. Single cell RNA sequencing has identified novel cell populations unique to the IPF lung, including the ectopic presence of basal epithelial cells, termed aberrant basal cells [2–5]. Spatial transcriptomics has further confirmed that aberrant basal cells occupy a distal location in the fibrotic lung closely associated with fibroblasts and macrophages that also exhibit disease-associated programs [6–8]. Detailed characterization of aberrant epithelial cells from human and animal models has suggested they take on intermediate nonregenerative states [1, 3], occupy pathologic niches [9], and release mediators regulating pathogenic macrophage [10] and fibroblast [11–13] accumulation and activation. Aberrant basal epithelial cells are thus increasingly recognized as likely drivers of fibrosis in the lungs, necessitating a better understanding of their origin and identification of strategies to modulate their pathogenic behaviors.

Considerable ambiguity remains regarding the origin of ectopic aberrant basal cells in human IPF [1, 3, 6, 14–17], with evidence supporting their potential emergence from alveolar type II (ATII) cells [6, 8, 10, 11, 17, 18], as well as airway secretory [3, 4, 19] and basal cells [13, 20, 21]. Consensus markers of aberrant basal cells include the expression of both airway epithelial and mesenchymal markers, genes related to senescence and IPF, and diminished or low expression of traditional alveolar epithelial markers [2, 8, 9]. Mouse genetic models have provided insight into the formation and function of aberrant epithelial cells. Transgenic models including SftpcC121G, ATII conditional loss of Sin3a, and Notch intracellular domain 1 overexpression promote lung fibrosis and accumulation of aberrant basal cells [11, 22, 23], while lung fibrosis and aberrant basal cell accumulation are ameliorated in Krt8 KO, Il-11 KO or Hif1a/2a KO mice [4, 10, 24–26]. Compounds including X203 (IL-11 inhibitor), saracatinib (SRC kinase inhibitor), dasatinib plus quercetin (senolytic drug cocktail) and PT2385 (HIF2a inhibitor) have exhibited some capacity to modulate aberrant basal cell abundance and programs [4, 13, 23, 25, 27]. The effects of these compounds and mouse knockouts support a role for hypoxia and Notch signaling as drivers of the aberrant basal cells state. However, there is less evidence supporting compounds that perform a similar function in human cells due to the challenges in studying this cell population in human systems. Differences in human and mouse lung architecture and cellular composition [28–30] as well as limitations of mouse models to fully recapitulate the complexity of human IPF [7, 8, 28, 30, 31] emphasize the need to develop and test tractable models of human aberrant basal cell formation.

Current models used to study human IPF diseased cell states include three-dimensional (3D) human models such as iPSC-derived alveolar organoids [32] as well as precision cut lung slices (PCLS) [29, 33]. These models have identified a fibrosis cocktails [29, 33–36] that combine pro-fibrotic and pro-inflammatory signals which act to drive epithelial cells toward aberrant basal states. These studies align with prior efforts to delineate signals promoting aberrant basal cell states, which have supported potential interactions among pro-fibrotic and pro-inflammatory signals, hypoxia, and Notch signaling [17, 18, 22, 26, 37]. To date these developed human models appear to only partially recapitulate the broad signatures of aberrant basal cells leaving gaps in our understanding of the aberrant basal cell disease state. Better understanding of the relevant cues that induce ATII aberrant reprogramming into aberrant basal cells would allow for development of interventions that can reverse the aberrant differentiation processes.

Here we report that stimulation of human ATII spheroids with a hypoxia mimic (DMOG) or TGFβ1 alone incompletely promotes aberrant basal signatures, whereas combined stimulation with both is more effective. Removal of both stimuli incompletely reverts aberrant basal cell signatures. A Notch inhibitor (LY-411575) partially reverses aberrant basal cell signatures even in the presence of continued hypoxia and TGFb1 signaling, and when applied to human IPF PCLS significantly decreases aberrant basal cell signatures, suggesting potential therapeutic utility of targeting Notch signaling to reduce aberrant basal cells in IPF.

Methods

Human lung tissue and distal lung cell isolation and organoid formation

The collection of human lung tissue samples for ATII spheroid culture was approved by the Mayo Institutional Review Board (IRB) under protocol no. 16–009655. Standard informed consent for research was obtained in writing from all patients who contributed to this study before tissue procurement and all experiments followed relevant guidelines and regulations. Human lung dissociation was performed as described previously [38]. Briefly, peripheral lung tissues were obtained from discarded surgical tissue from lobectomies (Table S1). Human lung tissue (approximately 2 g) was washed with PBS containing 1% Antibiotic-Antimycotic and minced into small pieces. Samples were digested with 10 mL of enzyme mixture (Collagenase type IV: 2 mg/mL, DNase I: 2 mg/mL) at 37 °C for 1 h with rotation. The digested tissued suspension was sequentially sheared using 10- and 5-mL plastic pipettes. The suspension was filtered through a 100 μm cell strainer and rinsed with 15 mL Advanced DMEM/F12 + 2% FBS media through the strainer. The supernatant was removed after centrifugation at 450g for 10 min and the cell pellet was resuspended in 10 mL red blood cell lysis buffer for 90 s at room temperature, washed with 10 mL PBS. For distal lung organoid culture, cell clusters were centrifuged at 450g for 5 min, the pellet was resuspended in Matrigel and 50µL mixture (10 to 20e6/ml) seeded into each well of 24-well plate. The Serum Free Feeder Free media (SFFF) was changed every other day. Distal lung organoids were generated through self-organization and harvested after 7–10 days of culture.

The collection of human lung tissue samples for PCLS was conducted by The Ohio State University (OSU) Comprehensive Transplant Center (CTC) Human Tissue Biorepository, which adheres to ISBER Best Practices for Biorepositories. IPF patients were consented to the Total Transplant Care Protocol (IRB protocol # 2017H0309) and donor research authorization was obtained by our local organ procurement agency, Lifeline of Ohio (Table S2). Samples were distributed to our laboratory by the CTC Biorepository via an Honest Broker process (IRB protocol # 20170310) under a secondary research IRB protocol #2021H0180.

Distal lung organoid digestion and alveolar epithelial isolation

For distal lung organoid digestion, we added 1 mL gentle dissociation media (STEMCELL TECH, Cat# 100–0485) into the each well of 24-well plate, incubated for 1 min at room temperature, mechanically sheared mixtures using 1 mL pipette, centrifuge and add 10 mL trypLE for 10 min at 37 °C, remove the supernatant after centrifuge, and add 10 mL 0.25% trypsin for 5–10 min at 37 °C, centrifuge after adding 1 mL FBS and the suspension was filtered through a 70 μm cell strainer.

Human ATIIs were isolated by Fluorescence-Activated Cell Sorting (FACS) based protocols [39]. The above approximately 10–20 × 106 cells were resuspended in Advanced DMEM/F12 buffer + 2% FBS containing DNase I and incubated with APC-CD326 (EpCAM) (5uL/e6 cells), epithelial cell surface marker, and HTII-280 (1:50 dilution) antibodies, ATII cells surface marker, for 30 min at 4 °C, with gentle shaking once every 10 min. The cells were washed twice with Advanced DMEM/F12 buffer + 2% FBS containing DNase I and centrifuged at 150g for 12 min at 4 °C. After removal of the supernatant cells were suspended with secondary Alexa Fluor 488 anti-mouse IgM (1:100) in Advanced DMEM/F12 buffer + 2% FBS + DNase I for 30 min at 4 °C. The cells were washed twice with Advanced DMEM/F12 buffer + 2% FBS containing DNase I. Sorting was performed using a FACS Vantage SE.

ATII spheroid culture

CD326 + HTII-280 positive human ATIIs (5000–8000/well) were resuspended in SFFF media and mixed with an equal volume of matrigel. 50µL of this mixture was seeded into each well of a 24-well plate. After 10 to 15 min media was added to the matrigel domes. SFFF was used for the first week, after which ATII spheroids were cultured in human Alveolar II Maintenance Medium (hATIIM). The media was changed every other day. Spheroids were passaged every 10–14 days. For detailed media compositions see Table S3.

ATII spheroid aberrant differentiation

For ATII spheroid aberrant differentiation, a 50µL mixture containing 1:1 matrigel and human ATIIs was seeded into each well of 96-well plate cultured in SFFF media for 7–10 days, and then switched to hATIIM and cultured for an additional 7–10 days, and then switch to hATIIM without SB431542 (hATIIM-SB) plus treatment compounds (TGFβ1, DMOG and LY-411575) for another 7 to 10 days. The hATIIM-SB media was changed every other day.

Alveolar spheroid fixation and sectioning

Alveolar spheroids were fixed with 4% paraformaldehyde (PFA) at room temperature for 20 min. For OCT frozen blocks, samples were washed with PBS, embedded in OCT and cryosectioned at 8–10 μm thickness. For paraffin blocks, samples were dehydrated, embedded in paraffin and sectioned at 5 μm thickness.

Immunostaining alveolar spheroids and in situ whole-mount spheroids

Paraffin sections were first dewaxed and rehydrated before antigen retrieval. OCT sections were defrosted and washed with PBS. Antigen retrieval was performed using 10mM sodium citrate buffer in water bath (95 °C for 10 min). Sections were washed with PBS, permeabilized in PBST (0.1% Triton X-100 in PBS), and incubated with 3% BSA and 0.1% Triton X-100 in PBS for 1 h at room temperature followed by primary antibodies at 4 °C overnight. Sections were then washed 3 times in PBST, incubated with secondary antibodies in blocking buffer for 1 h at room temperature, washed with PBST 3 times, and mounted with mounting media containing nuclear counterstain DAPI (Vector Lab). Primary antibodies were as follows (Table S4): Surfactant Protein C (ms, 1:100), KRT17 (rb, 1:250). For spheroid whole-mount staining, spheroids in the 96-well plate were fixed with 4% PFA overnight, spheroids were washed with PBS, permeabilized in PBST, and incubated with 3% BSA and 0.1% Triton X-100 in PBS for 4 °C overnight followed by primary antibodies KRT 17 (rb, 1:250) and SFTPC (ms, 1:100) at 4 °C overnight. After overnight incubation and washing, spheroids were incubated with secondary Alexa-488 and Alexa-555 conjugated secondary antibody (1:500, Molecular Probes) and counterstained with DAPI (Vector Lab).

Spheroid-forming efficiency assays

Spheroid-forming efficiency was determined by quantification of spheroid numbers and size [40]. In brief, 200 sorted HTII-280 positive cells seeded into 20 wells (~ 10 cells/well) out of 96-well plate, a 50µL mixture containing 1:1 matrigel and cultured in SFFF media. Spheroid-forming efficiency (SFE) on day 21 post culture by counting the number of spheres >45 μm in diameter.

Image acquisition, processing and quantification

All confocal images were collected using an Olympus Confocal Microscope FV3000 equipped with 10×, 20× and 40× objectives using Fluoview FV1200. Experiments were performed on at least three biological replicates unless stated otherwise and representative images are shown. All other phase contrast images were taken using a 20× or a 40× objective using a Zeiss Axiovert 200 microscope using Zen 2012 Blue Edition software. For quantification of cells in each spheroid were manually counted using the Cell Counter plugin in ImageJ and quantified as a percentage of DAPI + cells.

RNA isolation and qRT-PCR

Media cultured with spheroids was completely removed, and the spheroids were harvested with 350uL RLT lysis buffer containing 1% 2-Mercaptoethanol from each well of 96-well plate. The total RNA was isolated by RNeasy Micro kit (Qiagen). For lung PCLS, the protocol following Direct-zol™ RNA Miniprep plus Cat No. R2070. cDNA was synthesized with SuperScript™ IV Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions with DNase I treatment. Gene expression levels were quantified by qRT-PCR on the Lightcycler 96 Real-Time PCR System (Roche) according to the manufacturer’s instruction. qRT-PCR was performed by incubating at 95 °C for 10 min and then cycling 45 times at 95 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s. Ct values within each experiment were normalized against GUSB. If a CT value wasn’t detected, it was considered to be 45. Primers are listed in Table S5.

Protein extraction and Western blot

Proteins were extracted using RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA). Protein concentration was determined using the Pierce BCA Protein Assay (Thermo Fisher Scientific, Waltham, MA). Equal amount of protein from each sample or equal volume of conditioned media were loaded on a 4–15% polyacrylamide Mini-PROTEAN precast gel (Bio-Rad, Hercules, CA, USA) and separated by electrophoresis. The proteins were then transferred onto PVDF membrane, blocked with 5% Bovine Serum Albumin (Sigma, A9647), and incubated with primary antibodies overnight at 4 °C. Western blotting analysis of cell lysates was performed using the following antibodies against: SFTPC (ms, 1:500), MMP7 (rb, 1:2000), Beta-actin (ms, 1:5000), KRT17 (rb, 1:1000), CDH2 (rb, 1:1000), COL1A1 (rb, 1000). Blots were then washed and incubated with appropriate IgG-HRP conjugated antibodies for 1 h at room temperature. Bands were visualized by using Super Signal West Pico Plus or Super Signal West Femto or Super Signal West Atto (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Western blot images were quantified using Image Lab 6.0.1 (Bio-Rad, Hercules, CA, USA).

Human precision-cut lung slices (PCLS) Preparation

PCLS from IPF explant lungs were prepared as described previously [41]. Briefly, approximately one cm3 of lung tissue was filled with warm 3% of UltraPure™ Low Melting Point Agarose (16520100, Thermo Fisher) in a sterile media (DMEM, Gibco) via a visible bronchus. Lung segments were transferred into a tube with phosphate-buffered saline (PBS) and cooled on ice for 30 min to allow gelling of the agarose, embedded, and sliced at 400 μm of thickness using a vibratome (0.10–0.30 mm/s; Leica VT 1200). The slices were incubated at 37 °C in a tissue incubator with 5% CO2 and then washed in a sterile media (DMEM/F-12, Gibco) three times to remove agarose. Followed by overnight incubation in DMEM/F-12 media (Gibco) with 1% fetal bovine serum (FBS, Gibco) and 1% Antibiotic-Antimycotic mixed solution (Gibco). PCLS came from IPF-Upper lobe and were subdivided into two groups: (1) PCLS + vehicle (0.1% DMSO, control group) and (2) PCLS + LY-411575 at 10 µM up to 72 h. After treatment, PCLS were collected at 24 h, 48 h and 72 h by snap freezing in liquid nitrogen and stored at −80 °C before analyses.

Lactate dehydrogenase (LDH) activity in PCLS supernatant

The viability of cultured PCLS was evaluated by quantifying lactate dehydrogenase (LDH) activity in the culture supernatant. Beginning 24 h after tissue preparation, LDH activity was measured at 24-hour intervals over a 72-hour cultivation period. Measurements were performed using the LDH-Glo™ Cytotoxicity Assay Kit (J2380, Promega), in accordance with the manufacturer’s instructions. Prior to analysis, supernatants were diluted 1:100 in culture medium. Standards and diluted samples were plated in triplicate into white 96-well plate (50 µl per well), followed by the addition of the LDH detection reagent at a 1:1 volume ratio. Then, the plate was incubated in the dark for 1 h at room temperature, and luminescence was detected using a BioTek Synergy H1 microplate reader (Agilent). LDH activity was expressed as a percentage of LDH activity, with 100% activity defined by treatment of PCLS with 10% Triton X-100 for 15 min prior to sample collection.

LIVE/DEAD staining in PCLS

Cell viability in PCLS tissue was evaluated using the LIVE/DEAD® Viability/Cytotoxicity Kit (L3224, Thermo Fisher Scientific), according to the manufacturer’s protocol. PCLS were washed three times with 1x PBS, then incubated with 2 µM Calcein AM and 2 µM ethidium homodimer-1 in 1 mL of 1x PBS for 45 min at room temperature. After incubation, the PCLS were washed twice with 1x PBS. Imaging was performed immediately, and whole-slice tiled images were acquired at 10x magnification using a Keyence BZ-X800 fluorescence microscope.

Statistical analysis

Statistical methods relevant to each figure are outlined in the figure legend. Data is presented as means with standard error of the mean (SEM) to indicate the variation within each experiment. The quantified values were averaged and plotted using GraphPad Prism 9.4.1 (GraphPad Software). Statistical analysis was performed using one-way ANOVA (nonparametric) among groups and unpaired, two-tailed, Student’s t-test between groups. p values less than 0.05 were accepted as significant.

Results

ATII spheroids acquire select aberrant basal cell signatures in response to hypoxia mimic and TGFβ1

To test the various factors that are able to promote signatures of IPF-associated aberrant epithelial cell states, we first expanded epithelial population in 3D organoids. These organoids were created from mixed population of freshly dissociated human distal lung tissue cultured in matrigel. The organoids were matured for 7 days in SFFF media containing a Wnt agonist (CHIR99021), TGFbR inhibitor (SB431542), p38 MAPK inhibitor (BIRB796), FGF10 and EGF to promote growth of ATII cells [42]. The resulting organoids were then dissociated, and ATII cells positive for EpCAM and HTII-280 were sorted and re-seeded in matrigel. ATII spheroids were enriched and allowed to mature in SFFF and human ATII Maintenance media (hATIIM) (Fig. 1A, Table S3) [43]. To assess epithelial cell status we developed a panel of transcripts based on prior human single cell RNAseq studies [2, 3]. From these studies we selected canonical markers of alveolar Type I (AGER) and Type II (SFTPC) epithelium, a marker of aberrant basaloid state (KRT17), markers of IPF (MMP7), epithelial-mesenchymal transition (EMT) markers (COL1A1, CDH2), and senescence markers (CDKN1A, GDF15). Together these markers broadly characterize ATII, ATI and aberrant basal cells [2, 3].

Fig. 1.

Fig. 1

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The individual effect of TGFβ1 and DMOG on aberrant basal signatures. A Schematic representation of the time-course for generation of human ATII spheroids and stimulation of ATII spheroids with TGFβ1 or DMOG. SB: SB431542 (TGFbR inhibitor). B and D Transcript expression of ATII spheroids treated with TGFβ1 or DMOG at day0, day3, day5, day7. Experiments were performed with ATII spheroids formed from four independent donor lungs for TGFβ1 treatment and three for DMOG treatment. Values in graphs represent mean ± SEM. (*P < 0.05; ** P < 0.01). C and E Western blots of KRT17, MMP7 and SFTPC protein expression in ATII spheroids treated with TGFβ1 or DMOG. F-K SFTPC and KRT17 immunostaining for ATII spheroids treated with TGFβ1 or DMOG at day 0, day 3 and day 7. Scale bar: 100 μm

We first tested the impact of TGFβ1 on aberrant basal markers. TGFβ is a well-known driver of IPF that is enriched in the myofibroblast-rich niches adjacent to aberrant basal cells in IPF lung tissue [44]. Because the spheroid culture media hATIIM includes an inhibitor of TGFβ signaling, this inhibitor was removed from the base media and ATII spheroids were exposed to 1 ng/mL TGFβ1 for one week. Cells were harvested at several time points and assayed for the genes of interest, normalized to transcript levels at day 0. Analysis of transcripts by qRT-PCR showed that KRT17, COL1A1, CDH2, MMP7 and AGER significantly increased whereas GDF15 significantly decreased over time in response to TGFb1 (Fig. 1B). Western blotting from a representative sample confirmed KRT17 and MMP7 increased (Figs. 1C) in response to TGFb1. Immunostaining mirrored what was seen by western blotting (Fig. 1F and H). However, TGFβ alone failed to increase the epithelial stress and senescence markers CDKN1A and GDF15, characteristic of aberrant epithelial cells states. Moreover, TGFβ increased expression of ATI marker AGER, consistent with prior work [45]. Thus, TGFβ alone could induce a subset of aberrant epithelial signatures in ATII spheroids but also promoted the transition toward an ATI fate.

Hypoxia is a sentinel feature of IPF [46] and aberrant basaloid signatures are strongly associated with hypoxia-induced signaling via hypoxia-inducible factors (HIFs) [4]. To test the effects of hypoxia signaling in ATII spheroids, we selected a hypoxia mimic (DMOG). Under normoxic condition, HIF is rapidly degraded by HIF prolyl hydroxylase. DMOG mimics the effects of hypoxia by competitively inhibiting HIF prolyl hydroxylase, causing the stabilization of HIFs and mimicking the transcriptional effects of hypoxia [47]. Human ATII spheroids were exposed to 2.5 mM DMOG [48] for one week with cells harvested at several time points. For consistency with the above study, the TGFβR inhibitor was again omitted during the test phase. Analysis of transcripts by qRT-PCR showed that KRT17, CDH2, and GDF15 significantly increased, and SFTPC significantly decreased in response to DMOG (Fig. 1D). These results were confirmed at the protein level in a representative sample using western blotting (Fig. 1E). To verify the effects of DMOG as a hypoxia mimic, we assayed for SLC2A1, encoding glucose transporter protein type 1 (GLUT1), which is a known hypoxia target gene [49]. SLC2A1 was significantly increased in response to DMOG, confirming the expected response to a hypoxia mimic. Immunostaining mirrored the KRT17 expression increase observed by western blotting (Fig. 1I and K). The hypoxia mimic and TGFβ exhibited overlapping effects on some key transcripts (KRT17, CDH2), but complementary effects on others (MMP7, GDF15), with each individual treatment only partially recapitulating the full aberrant basal signature, suggesting that combined effect of TGFβ and DMOG may more fully recapitulate aberrant basal signatures in ATII spheroids.

As above, ATII spheroids were thus cultured in 3D matrigel in hATIIM minus the TGFβR inhibitor, with spheroids stimulated with TGFβ1 alone, DMOG alone, or both in combination for 7 days (Fig. 2A). Analysis of transcripts by qRT-PCR showed that in response to the combined stimulation with TGFβ1 and DMOG, both ATII (SFPTC), and ATI (AGER) markers significantly decreased, whereas aberrant basal (KRT17), EMT (COL1A1, CDH2), and senescence (CDKN1A) markers all increased. In only two transcripts, GDF15 and MMP7, was the combination of stimuli less effective than single stimulation with TGFβ1 or DMOG (Fig. 2B). Of interest, a Notch pathway target gene, HES4, was significantly increased by the combined stimulation (Fig. 2B). Western blotting confirmed that SFTPC decreased and KRT17 increased in response to combined hypoxia mimic and TGFb1 stimulation (Fig. 2C, Fig S1). Immunostaining mirrored the KRT17 expression by western blotting (Fig. 2D and G). Thus, combined stimulation of human ATII spheroids for seven days with TGFβ1 and DMOG was broadly effective, and more effective than either alone, at producing aberrant epithelial signatures characteristic of IPF.

Fig. 2.

Fig. 2

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ATII aberrant basal signatures under combined TGFβ1 and DMOG stimulation. A Schematic representation of the conditions used to stimulate ATII spheroids with TGFβ1 and DMOG alone and in combination. SB: SB431542 (TGFbR inhibitor). B Transcript expression of ATII spheroids treated with TGFβ1, DMOG and both combination for 7 days. Experiments were performed with ATII spheroids formed from five independent lung tissues. Values in graphs represent mean ± SEM. (*P < 0.05; ** P < 0.01). C Representative western blots of KRT17, MMP7 and SFTPC protein expression in ATII spheroids treated with TGFβ1, DMOG or both. Experiments were performed with four independent normal donor lungs. D-G SFTPC and KRT17 immunostaining for ATII spheroids treated with TGFβ1 or DMOG at day 7. Scale bar: 100 μm

ATII cells have variable capacity for aberrant differentiation

One possible explanation for emergence of the aberrant basaloid signature in our ATII spheroid experiments is the potential presence of rare contaminating basal or other cell types that may have expanded in response to the selected stimuli. It is known that HTII-280 positive cells are not entirely exclusive to SFTPC positive ATII cells [50], and that sorting strategies are not 100% effective, which may also contribute to the variability in baseline expression of epithelial markers (e.g. MMP7) across experiments. To address this concern, we prepared single cell suspensions of HTII-280 positive cells from distal lung organoids as described above, and reseeded the cells at very low density (~ 10 cells per well) in matrigel in 96-well plates (Fig. 3A). We cultured cells in SFFF media and observed spheroid formation efficiency of ~ 20% after 3 weeks culture (Fig S2A and S2B). Irregular edge and multiple layer ATII spheroids were formed (Fig S2C). ATII spheroids that formed from single cells were then treated in the presence or absence of combined TGFβ1 and DMOG for five days to assess capacity to express aberrant epithelial signatures (Fig. 3A). Immunostaining of untreated spheroids identified 2.7% KRT17 positive and 83% SFPTC positive cells, consistent with high ATII purity and relatively rare spontaneous expression of an aberrant epithelial marker (Fig. 3B). In response to TGFβ1 and DMOG stimulation, 37.1% of spheroids were KRT17 positive and 77% were SFTPC positive (Fig. 3B and F). However, substantial variation was observed, with the single positive SFTPC + KRT17- population diminishing and the SFTPC-KRT17 + population increasing, and with the emergence of a double positive SFTPC + KRT17 + population in TGFb1 and DMOG stimulated spheroids (Fig. 3G). The dramatic overall increase in KRT17 + cells within 5 days of combined stimulation is consistent with ATII spheroids acquiring an aberrant basal signature as the predominant response. However, these results also highlight the variability in the capacity of human distal lung epithelial cells to express aberrant markers in response to TGFβ1 and DMOG, suggesting potential heterogeneity in ATII populations that merits further study.

Fig. 3.

Fig. 3

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ATII spheroids formed from single ATII cell exhibit heterogeneous capacity for aberrant differentiation. A Schematic representation of ATII spheroids formed from single ATII cells and treated with TGFβ1 and DMOG. SB: SB431542 (TGFbR inhibitor). B-C In Situ whole mount immunostaining for SFTPC and KRT17 in ATII spheroids in the absence or presence TGFβ1 and DMOG. Scale bar: 50µm. E-G Quantification of KRT17 and SFTPC for cells out of ATII spheroids in the absence (12 spheroids) or presence (33 spheroids) of TGFβ1 and DMOG from three independent lung tissues. Values in graphs represent mean ± SEM. (** P<0.01)

Notch inhibition can partially prevent and reverse aberrant basal signatures

As noted above, combined stimulation of human ATII spheroids with TGFβ1 and DMOG resulted in increased transcript levels of HES4, a Notch responsive gene (Fig. 2B). Prior work has implicated the Notch pathway in lung epithelial differentiation and pulmonary fibrosis [18, 21, 22, 26, 51]. We thus tested the effect of a Notch signaling inhibitor, LY-411575, on aberrant basal signatures in ATII spheroids. We first tested ATII spheroids treated simultaneously with TGFβ1 plus DMOG in the presence or absence LY-411575 for seven days (Fig. 4A). Transcripts analyzed by qRT-PCR and protein by western blotting demonstrated that LY-411575 inhibited KRT17 and MMP7 expression (Fig. 4B and C). Immunostaining mirrored the KRT17 expression response observed by western blotting (Fig. 4D and F). However, LY-411575 had modest but non-significant effects on other aberrant epithelial transcripts (Fig. 4B and F), suggesting a limited role of Notch inhibition in preventing the emergence of an aberrant epithelial state [18, 37].

Fig. 4.

Fig. 4

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Notch inhibitor LY-411575 can partially prevent acquisition of aberrant basal signatures. A Schematic representation of ATII spheroids treated with hATII-SB, TGFβ1 + DMOG and TGFβ1 + DMOG + LY-411575 for one week. SB: SB431542 (TGFbR inhibitor), LY-411: LY-411575 (Notch signaling inhibitor). B Transcript expression of ATII spheroids treated with TGFβ1 + DMOG and TGFβ1 + DMOG + LY-411575. Experiments were performed with ATII spheroids formed from five independent lungs. Values in graphs represent mean ± SEM. (*P < 0.05; ** P < 0.01). C Western blots of KRT17, MMP7 and SFTPC protein expression in ATII spheroids treated with TGFβ1 + DMOG and TGFβ1 + DMOG + LY-411575. D-F SFTPC and KRT17 immunostaining for ATII spheroids treated with hATIIM-SB, TGFβ1 + DMOG and TGFβ1 + DMOG + LY-411575 at day 7. Scale bar: 100 μm

Next, we wanted to test the transient nature of the aberrant basal signature. Therefore, we tested if cells would spontaneously revert to a healthy state upon removal of the initiating stimuli, TGFβ1 and DMOG. We additionally wanted to test whether LY-411575 could reduce aberrant signatures even in the continued presence of TGFβ1 and DMOG. We first stimulated ATII spheroids with combined TGFβ1 and DMOG for seven days, then maintained stimulation for the next three days, added LY-411575 to the ongoing TGFb1 and DMOG treatment, or removed both TGFβ1 and DMOG (Fig. 5A). Overall, we observed relatively low reversibility of aberrant signatures upon removal of TGFβ1 and DMOG over this limited time course. Promisingly, analysis of transcripts by qRT-PCR demonstrated that LY-411575 significantly reduced KRT17, MMP7 and COL1A1 expression even in the continued presence of TGFβ1 and DMOG (Fig. 5B), in many cases as effectively as removing both stimuli. Western blotting from a representative subject confirmed KRT17 and MMP7 protein expression were reduced by LY-411575 (Fig. 5C). Immunostaining mirrored the KRT17 expression effect observed by western blotting (Fig. 5D and F). These results demonstrate that Notch inhibition can partially reverse aberrant epithelial signatures in a spheroid model of aberrant ATII differentiation driven by TGFβ1 and hypoxia mimetic, motivating us to expand our work into human PCLS.

Fig. 5.

Fig. 5

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A Schematic representation of ATII spheroids stimulated with TGFb1 + DMOG to establish aberrant signatures, followed by continued stimulation with TGFβ1 + DMOG, TGFβ1 + DMOG + LY-411575, or removal of both TGFβ1 and DMOG for three days. SB: SB431542 (TGFbR inhibitor), LY-411: LY-411575 (Notch signaling inhibitor). B Transcript expression pattern in spheroids treated with continuing TGFβ1 + DMOG, switch to TGFβ1 + DMOG + LY-411575, or removal of both TGFβ1 and DMOG for three days. Experiments were performed with ATII spheroids formed from four independent lungs. Values in graphs represent mean ± SEM. (*P < 0.05; ** P < 0.01). C Western blots of KRT17, MMP7 and SFTPC protein expression. D-F SFTPC and KRT17 immunostaining for ATII-derived aberrant basal cells treated with TGFβ1 + DMOG, TGFβ1 + DMOG + LY-411575 and hATIIM-SB at day 3

Notch inhibition can partially reduce aberrant basal signatures in IPF-PCLS

Human PCLS spatially retain the cellular diversity and architecture of the native lung [29, 33]. We thus tested whether LY-411575 treatment of PCLS established from IPF subjects (IPF-PCLS) would beneficially impact aberrant basal and profibrotic signatures. IPF-PCLS were cultured with DMEM/F-12 media plus 1% fetal bovine serum in the absence or presence LY-411575 up to three days (Fig. 6A). Analysis of transcripts by qRT-PCR demonstrated that LY-411575 significantly reduced KRT17, MMP7, CDH2 and COL1A1 expression (Fig. 6B). Western blotting confirmed KRT17, MMP7, CDH2 and COL1A1 expression were significantly reduced by LY-411575. However, SFTPC expression was very dim or not detectable throughout (Fig. 6C). To rule out problems with PCLS viability we measured live/dead cell fractions and LDH levels in PCLS, both of which confirmed robust viability (Fig S3), consistent with the absence of SFTPC reflecting loss of ATII cells in IPF, not lack of cell viability [52]. Together these results imply that Notch inhibition can partially reverse aberrant epithelial signatures in IPF-PCLS, though with limited capacity to elevate an ATII marker in the three-day time span tested here.

Fig. 6.

Fig. 6

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Notch inhibitor LY-411575 can partially reduce aberrant basal signatures in IPF-PCLS. A Schematic representation of IPF-PCLS treated in the absence or presence LY-411575. LY-411: LY-411575 (Notch signaling inhibitor). B Transcript expression pattern in IPF-PCLS treated in the absence or presence LY-411575 (three independent IPF patients including two 2 days LY-411575 treatment and 1 day LY-411575 treatment). Values in graphs represent mean ± SEM. (*P < 0.05; ** P < 0.01). C Western blots of KRT17, MMP7, COL1A1, CDH2 and SFTPC protein expression in IPF-PCLS treated in the absence or presence LY-411575 for three days, along with quantification from three independent IPF-PCLS (*P < 0.05; ** P < 0.01)

Discussion

Model systems that recapitulate the formation and maintenance of aberrant basal cell signatures in human lung epithelium offer potential for basic mechanism studies as well as identification of therapeutic targets and potential translational strategies. Although the origins of aberrant basal cells in IPF are still debated, multiple lines of evidence point toward the formation of KRT17+ aberrant basal cells as functional disease drivers for IPF [6–13]. Our work demonstrates that ATII spheroids can acquire key aberrant basal cell signatures under combined treatment with TGFβ1 and the hypoxia mimic DMOG, suggesting these disease-relevant cues are sufficient to reprogram human ATII cells toward aberrant basal states (Fig S4). ATII spheroids derived from single ATII cells are heterogeneously responsive to these stimuli and exhibit varying acquisition of KRT17 positivity by immunostaining. Once acquired, aberrant basal signatures exhibited only limited spontaneous reversal within the first three days of removal of TGFb1 and DMOG. Inhibition of Notch signaling with LY-411575 partially prevented and reversed aberrant basal signatures in spheroid cultures and reduced key signatures in human IPF-PCLS, identifying Notch as a key target for reducing aberrant signatures in human cells and diseased tissue.

Previous investigations have implicated both TGFβ and hypoxia as central players in epithelial differentiation in IPF [53]. TGFβ1 has been shown to be a key component for modeling fibrotic cellular responses in a variety of in vitro and ex vivo fibrosis models [29, 33, 34, 36], and participates in various differentiation process including ATII differentiation to ATI cells, formation of transitional cell states, as well as aberrant basal cell formation. However, there appear to be differences in study conclusions based on species and methods of study [45, 54, 55]. Our data confirmed TGFβ1 enhanced the ATI signature in ATII spheroids, while also promoting markers associated with EMT, expression of the aberrant basal marker KRT17 and expression of the disease marker MMP7 [56–59]. Likewise, prior work has implicated HIF signaling in aberrant basal signatures and HIF2 inhibition in preventing emergence of aberrant intermediate cells from lung spheroids [4, 60]. In our studies, DMOG reduced ATII marker SFTPC without enhancing ATI marker AGER, and increased aberrant basal signatures including KRT17, GDF15 and CDH2, but exhibited no effect on COL1A1, MMP7 and CDKN1A. This partial response prompted us to explore cooperative actions of TGFβ and hypoxia signaling, two pathways previously shown to interact in pulmonary fibrosis [61–63]. Our data revealed that combined stimulation with both TGFb1 and DMOG was more effective than either alone in stimulating most aberrant basal cell signatures, providing an important step toward modeling formation and maintenance of human aberrant basal cells from ATII spheroids.

Prior work has identified variations in cell subpopulations that emerge in lung injury, with three distinct ATII populations emerging based on hypoxia signaling in single-cell RNA-sequencing (scRNA-seq) analysis of primary human lung epithelial cells from normal and fibrotic lung [26]. Our data indicate that ATII cells under TGFb1 and DMOG treatment also exhibit three distinct ATII populations based on SFTPC and KRT17 expression. As the origin of human cell subpopulations under maladaptive repair states may arise from several different sources, including resident ATII cells, secretory ATII cells [64], airway secretory cells [65–67], and club cells [68], the different degree of airway basal cell response we identity under the stimulation of TGFb1 and DMOG may indicate that distinct cell populations in the distal lung possess higher airway differentiation potential [64].

Our results indicate that inhibition of TGFb1 and hypoxia signaling may provide paths toward modulating aberrant basal cell signatures, but we observed limited reversal of signatures when these were removed, and therapeutic targeting of both remains challenging. We thus sought to identify a common mediator that may be more amenable to intervention. Notch signaling plays a major role during lung development [69, 70] and the differentiation of epithelial-specific lineages in the airway epithelium [71–73], alveolar cell fate decisions [68, 69, 74] and maintenance of dysplastic alveolar repair [21]. TGFβ-mediated effects are attenuated by Notch inhibition in retinal fibrosis [75], whereas Notch is activated by hypoxia [18, 26] and contributes to maladaptive repair [22]. We thus reasoned that Notch inhibition may be effective in counteracting the combined influence of TGFβ and hypoxia signaling in alveolar epithelial cells. Our data revealed that Notch inhibition can partially reverse aberrant cell phenotypes in spheroids even when TGFb1 and DMOG remain present. These results were then tested in human IPF-PCLS samples. However, whereas others identified partial restoration of alveolar signatures [22, 64] with Notch inhibition, we did not observe such changes in the three-day time frame of our experiments. Nevertheless, our results provide further evidence that Notch inhibition could be a promising therapeutic target for reversing pathologic epithelial states in IPF [76].

We acknowledge several limitations in our work. First, Notch inhibition only partially reversed the aberrant basal cell signatures and could not restore ATII status. In addition, pan-Notch inhibitors have serious Notch-associated adverse events [77]. Thus, deciphering specific ligand-receptor interactions and selective targeting of these interactions or their downstream components may be necessary for in vivo applications. Our results suggest potential involvement of Notch1, consistent with prior work [22], but confirmation will require specific genetic or pharmacologic ablation of specific Notch signals. In addition, we have largely relied on bulk analysis of ATII, ATI, and aberrant epithelial signatures. We acknowledge that a continuum of epithelial states likely exists between and within ATII, ATI, and aberrant basaloid populations; future work using scRNA-seq approaches will be needed to more fully characterize these populations in spheroid models. This will allow us to better define transcriptional identity, map transitional trajectories, and identify markers that distinguish these populations. Beyond these specific limitations, we acknowledge that although both 3D models used here are useful for disease modeling and drug testing, they still lack other physiological features such as immune cell recruitment and mechanical cues. While both models benefit from utilizing primary human lung cells and tissues, because of donor-specific genetic and epigenetic differences, potential differences in the cellular composition of starting material, and intrinsic variability in organoid culture systems, we observed substantial variability across samples, highlighting the need for expanded banks of human primary cells, spheroids, and PCLS for testing.

We limited our investigation to spheroids from nonfibrotic lungs and PCLS from fibrotic lungs. Additional work will be needed to develop and characterize IPF-derived spheroids and to investigate mechanisms of aberrant epithelial formation in PCLS derived from nonfibrotic lung tissue, as well as the functional roles of ATII derived aberrant basaloid cells in the IPF phenotype formation. Finally, we have not investigated other potential cellular sources of aberrant epithelial cells in our spheroid model and have not evaluated whether the signals investigated here are similarly responsible for their emergence from other potential cellular origins.

In conclusion, we report that combined stimulation with TGFβ and a hypoxia mimic broadly recapitulates aberrant basal cell signatures in human ATII spheroids. Human ATII cells exhibit a high but variable capacity to acquire aberrant basal signatures, and the inhibition of Notch partially reverses aberrant basal cell signatures in both spheroid and PCLS models. The establishment of tractable models for studying the formation and maintenance of aberrant epithelial cell states should support further investigation of these cells, the signals that drive their emergence and maintenance, their paracrine effects on other lung cell populations, and identification and testing of strategies to redirect aberrant epithelium toward states that facilitate lung repair.

Supplementary Information

Supplementary Material 1. (3.2MB, docx)
Supplementary Material 2. (206.5KB, docx)
Supplementary Material 3. (191.6KB, docx)
Supplementary Material 4. (180.6KB, docx)
Supplementary Material 5. (298.6KB, docx)
Supplementary Material 6. (356KB, docx)

Acknowledgements

We thank Dr. Yunzhou Dong and Dr. Mihael A Thompson for assistance in hypoxia culture and procuring human lung tissue samples, Alex Anwar and Duane Deal for the help in confocal microscopy.

Authors’ contributions

Y.L., Y.S.P., and D.T. conceived and designed research; Y.L. performed experiments; J.H.W, L.R, P.A.L, D.C, J.A, K.M.C., A.D. E, R.G, N.P.G, D.H.L, A.J.W, M.R. provided technical support. Y.L., and D.T. analyzed data; Y.L. and D.T. interpreted results of experiments; Y.L. prepared figures; Y.L. drafted manuscript; and D.T, Y.L, R.G, J.H.W, A.D.E, edited and revised manuscript; D.T. approved final version of manuscript.

Funding

This work was supported by NIH R01 HL092961 and U01 HL152967.

Data availability

Data in the manuscript: available.

Ethics approval and consent to participate

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

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Supplementary Materials

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Supplementary Material 6. (356KB, docx)

Data Availability Statement

Data in the manuscript: available.

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