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. 2024 Dec 12;20(1):102376. doi: 10.1016/j.stemcr.2024.11.006

Nascent matrix deposition supports alveolar organoid formation from aggregates in synthetic hydrogels

Madeline K Eiken 1, Charlie J Childs 2, Lindy K Brastrom 2, Tristan Frum 2, Eleanor M Plaster 1, Donia W Ahmed 1, Ryan C Spencer 2, Orren Shachaf 3, Suzanne Pfeiffer 1, Justin E Levine 1, Konstantinos-Dionysios Alysandratos 4,5, Darrell N Kotton 4,5, Jason R Spence 1,2,6,7, Claudia Loebel 1,8,9,∗
PMCID: PMC11784465  PMID: 39672155

Summary

Human induced pluripotent stem cell (iPSC)-derived alveolar organoids have emerged as a system to model the alveolar epithelium in homeostasis and disease. However, alveolar organoids are typically grown in Matrigel, a mouse sarcoma-derived basement membrane matrix that offers poor control over matrix properties, prompting the development of synthetic hydrogels as a Matrigel alternative. Here, we develop a two-step culture method that involves pre-aggregation of organoids in hydrogel-based microwells followed by embedding in a synthetic hydrogel that supports alveolar organoid growth, while also offering considerable control over organoid and hydrogel properties. We find that the aggregated organoids secrete their own nascent extracellular matrix (ECM) both in the microwells and upon embedding in synthetic hydrogels, which supports their growth. Thus, the synthetic hydrogels described here allow us to de-couple exogenous and nascent ECM to interrogate the role of ECM in organoid formation.

Keywords: organoids, alveolospheres, hydrogels, extracellular matrix, lung

Graphical abstract

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Highlights

  • •

    A two-step method for alveolar organoid culture in hyaluronic acid (HA) hydrogels

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    The two-step method offers control over alveolar size, density, and growth

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    Alveolar organoids maintain their AT2 identity in HA hydrogels

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    Alveolar organoids secrete nascent extracellular matrix supporting organoid growth

Eiken et al. develop a two-step method for alveolar organoid culture in synthetic hydrogels that maintains organoid identity and provides experimental control over organoid size and density. Alveolar aggregates and organoids secrete nascent extracellular matrix before and upon embedding in synthetic hydrogels, which supports their growth in the absence of Matrigel.

Introduction

Organoids are three-dimensional (3D) in vitro organ-like structures that recapitulate some elements of in vivo organs, including cellular diversity, spatial organization, and function (Childs et al., 2022; Clevers, 2016; Frum and Spence, 2021). Organoids representative of diverse organ systems have become powerful tools for studying a range of biological questions, which include interrogating physiologic and pathologic states to understand cell-cell and cell-matrix interactions in vitro (Nikolić and Rawlins, 2017; Yi et al., 2021).

Induced pluripotent stem cell (iPSC)-derived alveolar type II (AT2) organoids have emerged as a powerful tool to study alveolar homeostasis and disease such as severe acute respiratory syndrome coronavirus 2 (Huang et al., 2020; Hurley et al., 2020; Jacob et al., 2017; Mirabelli et al., 2021). Alveolar organoids are traditionally grown in Matrigel, a commercially available complex extracellular matrix (ECM) that supports proliferation, 3D growth, and viability (Aisenbrey and Murphy, 2020; Jacob et al., 2017). However, Matrigel and other commercially available ECMs are derived from non-human sources or cancer cell lines, resulting in a high biological complexity, high batch-to-batch variation, low tunability, and limited control over proteins or growth factors found within the ECM (Hughes et al., 2010; Kozlowski et al., 2021). To address these limitations, synthetic hydrogels have emerged as an alternative to commercially available complex ECMs for organoid culture (Gan et al., 2023; Hofer and Lutolf, 2021; Magno et al., 2020). Synthetic hydrogels are typically based on a polymeric backbone modified with functional moieties that provide control over the mechanical moduli (e.g., elasticity and viscosity), degradation, and the presentation of ligands to direct biological interactions (e.g., cell adhesion) (Kratochvil et al., 2019).

Several synthetic hydrogel systems have been developed for organoid culture, including hydrogel-based microwells that include an array of microcavities for aggregation and growth of single cells into organoids (Brandenberg et al., 2020; Chen et al., 2021; Choi et al., 2010; Decembrini et al., 2020; Gracz et al., 2015; Karp et al., 2007; Luan et al., 2022; Moeller et al., 2008; Wiedenmann et al., 2021). Building upon this concept, we recently developed a hydrogel-based microwell system that supports the formation and culture of alveolar organoids (Loebel et al., 2022). Although this microwell system enables the formation of alveolar organoids with defined shape and size, it is limited for studies that aim to understand the mechanisms of organoid-hydrogel and organoid-ECM interactions.

To address this, we describe a two-step culture method that leverages the advantages of microwells to control the aggregation of AT2 cells and 3D hydrogels to study organoid-hydrogel and organoid-ECM interactions. First, we demonstrated that microwell-aggregated alveolar organoids can be successfully cultured in norbornene-modified hyaluronic acid (HA) hydrogels. The HA hydrogel consists of a HA backbone modified with norbornenes, which enable crosslinking via a thiol-ene reaction of norbornenes with dithiols and modification with thiol moieties such as the cell-adhesive ligand RGD (Plaster et al., 2023). Using a two-step culture method, cells were first pre-aggregated in hydrogel-based microwells, then the aggregates were embedded in HA hydrogels. We demonstrated that this method offers tunability across a range of hydrogel moduli, organoid size, and density and supports high organoid viability and formation efficiency, identity maintenance, and growth. Next, we interrogated the mechanism of organoid formation and found that aggregates secrete their own matrix, including the basement membrane proteins laminin and collagen IV, within the microwells and after embedding in HA hydrogels, which supports organoid formation without Matrigel, highlighting the capacity of synthetic hydrogels to de-couple the role of exogenous and nascent ECM in organoid formation.

Results

Formation of alveolar organoids in HA hydrogels

First, we sought to test whether iPSC-derived alveolar progenitor cells form organoids within HA hydrogels fabricated with a modulus (i.e., Young’s modulus, stiffness) from 1.5 to 20 kPa, reflecting the Young’s moduli of alveolar regions within healthy and diseased lungs (Hinz, 2012). Mature alveolar progenitors are commonly passaged as organoids in Matrigel for up to 1 year, which allows for multiple subsequent studies (Jacob et al., 2017). Thus, we first compared the traditional approach of embedding single alveolar progenitor cells (400 cells/μL) into Matrigel or into HA hydrogels with initial Young’s moduli of 1.5 (soft), 4 (medium), or 20 kPa (stiff) (Figure 1A) (Jacob et al., 2017, 2019). The alveolar progenitor cells used here contain a tdTomato reporter targeted to the endogenous surfactant protein C (SFTPC) loci as an indicator of AT2 identity (Jacob et al., 2017, 2019). Within 10 days, single cells gave rise to alveolar organoids in Matrigel, whereas no organoids were observed within HA hydrogels (Figures 1B and S1A). Quantification of organoid formation efficiency, defined as the number of multicellular structures present at day 14 divided by the number of aggregates at day 1, showed significant reduction for single alveolar progenitor cells in HA hydrogels (average formation efficiency: 1.3% ± 1.6%) compared to Matrigel (formation efficiency: 4.1% ± 1.1%) (Figure 1C). Although single alveolar progenitor cells did not give rise to organoids in HA hydrogels, single cells remained viable throughout 14 days (average viability: 62.0% ± 19.2%), although viability was significantly lower than Matrigel (97.7% ± 3.5%) (Figure 1D). These data indicate that single alveolar progenitor cells cannot form organoids within HA hydrogels.

Figure 1.

Figure 1

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Embedding of alveolar progenitor cell aggregates enables viable alveolar organoid formation in synthetic HA hydrogels of various moduli

(A) Schematic overview of experimental approach using single alveolar progenitor cells: alveolar progenitors are isolated from alveolar organoids in Matrigel and resuspended into single cells prior to embedding into HA hydrogels.

(B) Representative bright-field and fluorescence images showing SFTPC-tdTomato (red) expression in Matrigel and HA hydrogels with Young’s moduli of 1.5 (“soft”), 4 (“medium”), and 20 kPa (‘stiff') at day 10 (d10). Scale bar 300 μm.

(C) Quantification of organoid formation efficiency in Matrigel and HA hydrogels at day 14, n ≥ 8 individual hydrogels from 3 independent experiments.

(D) Viability of embedded single cells in Matrigel and HA hydrogels at day 10. n ≥ 10 regions of interest (ROIs) from 3 independent experiments.

(E) Schematic overview of experimental approach using aggregated alveolar progenitor cells: alveolar progenitor cells are isolated from alveolar organoids in Matrigel, resuspended into single cells, and aggregated within HA hydrogel-based microwells for 3 days prior embedding into HA hydrogels.

(F) Representative bright-field and fluorescence images of aggregates in Matrigel and HA hydrogels at day 10. Scale bar 300 μm.

(G) Quantification of organoid formation efficiency of aggregates in Matrigel and HA hydrogels at day 14. n ≥ 8 individual hydrogels from 3 independent experiments.

(H) Quantification of projected tdTomato area per live cell area of aggregates at day 10. n ≥ 6 ROI from 3 independent experiments. (C, D, G, and H) ∗∗∗p < 0.001, ∗∗p < 0.01, ns: no significant difference by ANOVA with Tukey’s multiple comparisons test.

We hypothesized that pre-aggregating alveolar progenitors within our recently developed hydrogel-based microwells improves formation efficiency of alveolar organoids upon embedding into HA hydrogels (Loebel et al., 2022). Dissociated alveolar progenitor cells were first seeded into microwells and allowed to aggregate for 3 days, and then embedded in Matrigel or HA hydrogels of various Young’s moduli (soft, medium, and stiff) (Figure 1E). Within 10 days after embedding, aggregates formed into alveolar organoids in both Matrigel and HA hydrogels independent of the initial Young’s modulus (Figure 1F) and continued to increase in size over 14 days (Figure S1B). Accordingly, quantification of the organoid formation efficiency showed no significant difference between aggregates embedded in Matrigel (77.4% ± 12.4%) and HA hydrogels (average: 76.8% ± 14.9%) (Figure 1G). Cell viability was maintained above 88% for aggregates embedded in Matrigel and HA hydrogels throughout the entire culture period of 14 days post embedding (Figure S1C). Notably, SFTPC-tdTomato reporter expression of embedded aggregates showed no significant differences between Matrigel and HA hydrogels at day 14 (Figure 1H) and throughout the entire culture period (Figure S1D). Maintenance of SFPTC-tdTomato reporter expression was further confirmed by flow cytometry, demonstrating no significant difference in the percent of cells positive for the SFTPC-tdTomato reporter between Matrigel and HA hydrogels (Figure S1E). Taken together, these data show that pre-aggregation of alveolar progenitor cells within hydrogel-based microwells enhances viable alveolar organoid formation in HA hydrogels and that HA hydrogels maintain SFTPC-tdTomato reporter expression at comparable levels to Matrigel.

Alveolar organoids maintain AT2 identity in HA hydrogels

After showing viable alveolar organoids in HA hydrogels, we next aimed to characterize their identity and function. We selected 4 kPa HA hydrogels (medium) as it reflects the stiffness of the healthy lung (Hinz, 2012). Given that SFTPC-tdTomato reporter expression has limited sensitivity in detecting differences in SFTPC expression (Sun et al., 2021), we first aimed to confirm that alveolar organoids maintain the expression of AT2 identity markers upon embedding. Thus, we compared the transcriptional and protein expression of common AT2 cell identity markers in Matrigel and HA hydrogels. We first assessed the expression of canonical AT2 genes in alveolar organoids in Matrigel and HA hydrogels using quantitative reverse-transcription polymerase chain reaction (RT-qPCR) (Frum et al., 2023; Herriges and Morrisey, 2014). Culture in microwells did not significantly impact the expression of canonical AT2 genes when compared to Matrigel (Figure S2A). No significant differences were observed for the expression of the lamellar body marker LAMP3 and surfactant protein B (SFTPB) (Figure 2A). In contrast, expression of surfactant protein A (SFTPA), a marker of AT2 maturity, and the surfactant processing protein Napsin A (NAPSA) was significantly lower for alveolar organoids in HA hydrogels compared to Matrigel (Figure 2B). These observations suggest that AT2s are less mature in HA hydrogels. Notably, SFTPC expression was almost 3-fold higher in HA hydrogels when compared to Matrigel (Figure 2C), which we also previously observed in HA microwells (Loebel et al., 2022). Given that SFTPC is expressed in nascent AT2 cells, these data further support the notion that HA hydrogels may prevent AT2 maturation. This is further supported by lower cell numbers and smaller lumen of alveolar organoids in HA hydrogels compared to Matrigel (Figure S2B). To assess whether HA hydrogels promote the expression of non-alveolar lung markers in the organoids, we measured expression of lung development markers, including SOX9, SOX2, and TP63, which were lower for alveolar organoids in HA hydrogels (Figure S2C), indicating that alveolar identity is maintained in HA hydrogels. In addition, expression of the lung epithelial cell marker NKX2.1 showed lower mRNA levels in HA hydrogels; however, similar protein expression in Matrigel and HA hydrogel argues against a functional difference (Figure S2D). Taken together, these transcriptional differences in the expression of AT2 markers suggest that Matrigel maintains alveolar progenitor organoids at a more mature state than HA hydrogels.

Figure 2.

Figure 2

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HA hydrogels maintain expression of AT2 cell identity markers of alveolar organoids

(A–C) Quantification of bulk gene expression of AT2 cell identity markers, including LAMP3, SFTPB, SFTPA, NAPSA, and SFPTC for embedded alveolar progenitor cell aggregates in Matrigel and HA hydrogel (medium, 4 kPa) at day 14. n = 9 repeated qPCR measurements from 3 independent experiments. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗p < 0.05, ns: no significant difference by unpaired Student’s t test.

(D) Representative fluorescent images of alveolar progenitor cell aggregates embedded in Matrigel and HA hydrogel and immunostained for SFTPC, SFTPB, and SFTPA and Hoechst at day 14.

(E) Representative fluorescent images of alveolar progenitor cell aggregates in Matrigel and HA hydrogels immunostained for MUC1 at day 14.

(F) Representative fluorescent images of alveolar organoids in Matrigel and HA hydrogels immunostained for epithelial cadherins (ECAD) and zonula occludens-1 (ZO1) at day 14. (D–F) Scale bars 50 μm (inset 25 μm).

(G) Representative TEM images of organoids in Matrigel and HA hydrogels showing lamellar bodies and tight junctions (arrowheads) at day 14. Scale bars 400 nm.

Given that gene and protein expression are not always correlated (Cote et al., 2016), we next sought to confirm the maintenance of AT2 identity at the protein level (Beers and Moodley, 2017; Frum et al., 2023; Jacob et al., 2017; Sucre et al., 2018; Wang et al., 2007). First, we immunostained embedded alveolar organoids for SFTPC, SFTPB, and SFTPA and observed the expected punctate staining patterns in both Matrigel and HA hydrogels (Figure 2D). In addition, AT2 cell surface markers, HTII-280 and MUC1, were examined with immunofluorescence staining and showed apical expression in alveolar organoids in both Matrigel and HA hydrogels (Figures 2E and S2E) (Gonzalez et al., 2010; Jarrare et al., 1998), indicating that cells are polarized. As expected, the apical markers were within the lumen of the organoids in Matrigel, indicating an “apical in” orientation. In contrast, apical markers were on the membrane furthest from the lumen in HA hydrogels indicating an “apical out” orientation. We further interrogated organoid orientation through the presence of junctional proteins, such as epithelial cadherin (ECAD) and the tight junction protein zonula occludens-1 (ZO1). ECAD was expressed at the basolateral side of the cells, whereas ZO1 was expressed as punctate spots on the apical side of cells within organoids (Figure 2F). Similar to the expression of AT2 cell surface markers, within Matrigel, ZO1 was mostly expressed in the lumen of the organoid, indicating an “apical-in” orientation, whereas within HA hydrogels, ZO1 was expressed on the outside of the organoid, indicating an “apical-out” orientation, confirming flipped epithelial cell polarity (Cereijido et al., 2008) in HA hydrogels. Finally, we confirmed AT2 identity through the presence of lamellar bodies in both Matrigel and HA hydrogels using transmission electron microscopy (TEM) (Jacob et al., 2017; Loebel et al., 2022; Schmitz and Muller, 1991). We observed no significant difference in the number of lamellar bodies per cell in Matrigel and HA hydrogels (Figure S2F). TEM imaging also showed the expression of zipper-like structures between adjacent cells, confirming the formation of tight junctions (Figure 2G) (Buckley and Turner, 2018). Taken together, these data indicate that alveolar organoids in HA hydrogels maintain their identity as AT2 cells.

Initial size and density of alveolar aggregates influence organoid growth

We next sought to determine whether aggregate size or embedding density directs alveolar organoid growth. Our previous work demonstrated that the number of seeded cells per microwells correlates with alveolar organoid size (Loebel et al., 2022). Thus, we hypothesized that, by controlling the initial size of alveolar progenitor cell aggregates, we can control the final alveolar organoid size (Figure 3A). We altered the seeding density in the microwells to 10 cells/microwell (small), 40 cells/microwell (medium), or 120 cells/microwell (large), which all formed aggregates within 3 days. After embedding, small aggregates showed limited formation of cystic structures whereas medium and large aggregates increased in size at day 14 (Figure 3B), which was consistent throughout the culture period (Figure S3A). Aggregate size impacted the growth rate of the aggregates with an average increase of 220 (small), 345 (medium), and 700 (large) μm2/organoid/day. To assess the role of aggregate size on proliferation, we incubated cells for the first (days 0–7) or second half (days 7–14) of the culture period with 5-ethynyl 2′-deoxyuridine (EdU). Between days 0 and 7, the number of EdU-positive nuclei increased as a function of aggregate size (Figure 3C). Interestingly, no significant difference was observed between different-sized aggregates when comparing EdU expression between day 7 and 14 (Figure S3B). This indicates that modulating the size of embedded alveolar progenitor cell aggregates controls the ultimate size of organoids, likely through differences in growth rate during the initial 7 days of culture.

Figure 3.

Figure 3

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Alveolar progenitor cell aggregation enables control over alveolar organoid size and density

(A) Schematic of experimental approach: the relationship between cell density in the microwell array (i.e., aggregate size) prior to embedding and final size.

(B) Representative bright-field and fluorescent images and quantification of projected organoid size of alveolar organoids formed from small, medium, and large aggregates embedded in HA hydrogels at day 14. Scale bar 300 μm. n = 2 independent experiments. Shaded error bars represent standard error of the mean (SEM).

(C) Representative fluorescent images and quantification of EdU incorporation from days 0–7 of alveolar organoids formed from small, medium, and large aggregates embedded in HA hydrogels at day 7. Scale bar 50 μm. n ≥ 49 alveolar organoids from 3 independent experiments.

(D) Schematic of experimental approach: the relationship between the number of embedded alveolar progenitor aggregates (medium size) and aggregate growth in HA hydrogels.

(E) Representative bright-field and fluorescent images of SFTPC-tdTomato (red) expression and quantification of projected organoid size of alveolar organoids formed from low, medium, and high-density aggregates embedded in HA hydrogels at day 14. Scale bar 300 μm. n = 2 independent experiments. Shaded error bars represent SEM.

(F) Representative fluorescent images and quantification of EdU incorporation from days 0–7 of alveolar organoids formed from low, medium, and high-density aggregates embedded in HA hydrogels at day 7. Scale bar 50 μm. n ≥ 34 alveolar organoids from 3 independent experiments. (C and F) ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ns: no significant difference by one-way ANOVA with Tukey’s multiple comparisons test.

This change in the growth rate of different aggregate sizes may indicate that the relative proximity of alveolar progenitor cells impacts proliferation within aggregates. To test this, we modulated aggregate density in the HA hydrogels by varying the number of medium-sized aggregates per HA hydrogel (Figure 3D). Embedding aggregates at low, medium, and high densities showed little differences in alveolar organoid structure and size at day 14; however, the density of aggregates controlled growth kinetics (Figures 3E and S3C). Alveolar organoids embedded at high and medium densities showed higher growth rates between day 0 and 7 of culture (700 and 800 μm2/day), while low densities showed reduced growth rates (350 μm2/day). Incorporation of EdU confirmed these findings with an almost 300% increase in EdU expression for alveolar organoids formed from aggregates in medium and high densities (31% ± 28% and 33% ± 29%) when compared to low densities (10% ± 12%) (Figure 3F). No significant differences in EdU incorporation were observed between day 7 and 14 (Figure S3D). When embedded into Matrigel, both size and density of aggregates had minimal effects on alveolar organoid growth and proliferation (Figure S3E). These data indicate that the proximity of alveolar progenitor cells controls alveolar organoid growth within and between embedded aggregates. Taken together, our two-step culture method enables control over the size and density of embedded aggregates and thus provides a useful tool to study the role of cell signaling within and between organoids.

Aggregates secrete nascent ECM in microwells and upon embedding in HA hydrogels

After showing that cellular interactions are important for organoid growth in HA hydrogels, we next sought to investigate the interactions between cells and their surrounding ECM. Building upon previous work showing ECM deposition by epithelial organoids (Blatchley et al., 2022), we hypothesized that aggregates start to secrete components of the ECM within the microwells (Figure 4A). To interrogate nascent ECM secretion, we leveraged a glycan labeling approach that is based on the incorporation of a galactosamine analog (Ac4GalNAz) into nascent glycans. Upon secretion, Ac4GalNAz-containing glycans are modified with a fluorophore using click chemistry (Dube et al., 2006; Laughlin et al., 2008; Zhang and Zhang, 2013). In addition, we stained for the basement membrane proteins, laminin and collagen IV (Figure 4B), which are secreted by lung epithelial cells (Nguyen et al., 2002; Pierce et al., 1998; Rosmark et al., 2023). Nascent glycan expression was largely confined to the cell membranes, likely labeling O-linked mucins or glycosphingolipids in the glycocalyx on the cell surface (Aich and Yarema, 2008). In contrast, aggregates were surrounded by laminin, whereas collagen IV was deposited in the center of the aggregate. Given that collagen IV but not laminin contains galactosamine in the globular domain (Weber et al., 1984), collagen IV staining overlapped with nascent glycan staining (Figure S4A). Importantly, no exogenous laminin or collagen IV was added to the cultures. Thus, these findings show that alveolar progenitor cells secrete nascent ECM that forms a shell around aggregates. Based on this observation, we next hypothesized that organoids continue to secrete nascent ECM upon embedding in the HA hydrogels (Figure 4C). To test this, we continued nascent ECM labeling after embedding aggregates into HA hydrogels and throughout the 14 days of culture (Figure S4B). Nascent glycans showed a significant increase in overall glycan thickness on day 14 (Figures 4D and S4C); however, expression of lamininβ1 (LAMB1) and collagen IVα5 (COL4A5) showed no significant differences between HA hydrogels and Matrigel (Figure S4D). These observations suggest that alveolar organoids continue to secrete nascent ECM upon embedding within HA hydrogels without difference in ECM gene expression. Previous work has shown that nascent ECM deposition within synthetic hydrogels plays a synergistic role in directing cell function (Ferreira et al., 2018; Loebel et al., 2019). Thus, we next assessed whether nascent glycans influence the ability of organoids to directly interact with the HA hydrogel. To quantify the organoid-hydrogel distance, we co-embedded fluorescent microbeads (0.5 μm) to visualize the hydrogel (Loebel et al., 2020) (Figure S4E). Similar to nascent glycan thickness, organoid-hydrogel distance, as measured by the distance between the cell membrane and the microbeads, increased over 14 days of culture (Figure 4E). In addition, no fluorescent microbeads were shown within the nascent glycans (Figure S4F), suggesting that the nascent ECM forms an interface between organoids and HA hydrogels. Staining for laminin and collagen IV at day 14 showed constant (laminin) or decreasing (collagen IV) thickness when normalized to organoid size, likely due to organoid growth (Figures 4F and S4G). Co-staining of nascent glycans, laminin, and collagen IV confirmed the formation of a near-complete shell around alveolar organoids at day 14 (Figure 4G). Electron microscopy showed the presence of extracellular fibrils adjacent to the cell membranes, further confirming nascent ECM proteins within HA hydrogels (Figure 4H). Taken together, these data indicate that nascent ECM forms an interface between cells and the hydrogel that supports organoid function.

Figure 4.

Figure 4

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Alveolar progenitor cell aggregates deposit ECM in the microwells and upon embedding in HA hydrogels

(A) Schematic of experimental approach: ECM deposition by aggregates in microwells was visualized.

(B) Representative fluorescent images of ECM secreted by the aggregates in the microwells (day 3). Scale bar 50 μm. White arrowhead shows collagen IV in the middle of an aggregate.

(C) Schematic of experimental approach: ECM deposition by aggregates/organoids upon embedding into HA hydrogels was visualized and quantified.

(D) Quantification of projected nascent glycan thickness beyond the cell membrane across the culture period. n ≥ 20 alveolar organoids from 3 independent experiments.

(E) Quantification of the organoid-hydrogel distance (i.e., the area between the aggregate/organoid and hydrogel) across the culture period. n ≥ 20 alveolar organoids from 3 independent experiments.

(F) Quantification of projected laminin and collagen IV per aggregate/organoid area. n ≥ 28 aggregates/organoids from 3 independent experiments.

(G) Representative max-project images of alveolar organoids in HA hydrogels co-stained for nascent glycans, laminin, and collagen IV at day 14. Scale bar 50 μm.

(H) Representative transmission electron microscopy images of alveolar organoids in HA hydrogels at day 14. Arrowheads show extracellular fibrillar structures. Scale bar 100 nm.

(I) Schematic of experimental approach: laminin was co-embedded in the HA hydrogels with aggregates, and subsequent ECM deposition was assessed.

(J) Quantification of collagen IV per cell area in HA hydrogels without laminin (blue bars) and with added laminin (green bars) at day 1 and day 14. n ≥ 31 ROI across 3 independent experiments. Scale bars 50 μm.

(K) Quantification of projected nascent glycan thickness in HA hydrogels with no additional proteins and HA hydrogels with laminin. n ≥ 13 ROI across 2 independent experiments. (D and F) ∗∗∗∗p < 0.0001, ∗p < 0.05, ns: no significant difference by one-way ANOVA with Tukey’s multiple comparisons test. (E, J, and K) ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns: no significant difference by unpaired Student’s t test.

Prior studies showed that organoid formation from single cells within synthetic hydrogels requires the addition of exogenous ECM proteins (Broguiere et al., 2018; Gjorevski et al., 2016; Loebel et al., 2022). Thus, we next aimed to determine if the lack of exogenous ECM in our system promotes nascent ECM deposition (Figure 4I). To test this, we compared nascent glycan and laminin deposition of alveolar organoids within HA hydrogels without or with laminin/entactin (2 mg/mL) mixed into the hydrogel. We observed a significant decrease in the expression of collagen IV in alveolar organoids cultured within HA hydrogels with added laminin (HA hydrogel + laminin) when compared to HA hydrogels without additional laminin (Figures 4J and S4H). Although not significant, collagen IV mRNA was also reduced for alveolar organoids in HA/laminin hydrogels (Figure S4I). There was no significant difference in nascent glycan thickness in the HA hydrogels with or without laminin (Figures 4K and S4J). This suggests that exogenous ECM proteins reduce alveolar progenitor cell-secreted ECM in HA hydrogels. In summary, microwells support the formation of aggregates and nascent ECM deposition within synthetic hydrogels.

Aggregation and nascent ECM deposition are required for organoid formation in HA hydrogels

Given that aggregates deposit nascent ECM, we investigated how the initial aggregation phase regulates the formation and growth of organoids in HA hydrogels. Culture in microwells leads to the formation of cell-cell contacts and ECM deposition, which may support aggregate growth upon embedding in HA hydrogels. To investigate how cell-cell contact and nascent ECM regulate organoid growth, we performed several perturbation studies. First, we cultured individual cells in suspension (“suspension”) to test if individual cells secrete nascent ECM that supports organoid formation. Next, we allowed cells to aggregate for short time (“pre-aggregates”) to prevent nascent ECM secretion prior embedding. In addition, we embedded aggregates upon enzymatic digestion of nascent ECM (“digested”) (Figure 5A). Perturbations did not affect cell viability, with >95% viability for all conditions (Figure S5A). After 3 days in microwells, aggregates were surrounded by a shell of secreted laminin, which enabled alveolar organoid growth over 14 days in HA hydrogels (Figure 5B). In both suspension and pre-aggregates groups, little laminin and collagen IV was detectable (Figure 5C) with a significant decrease in ECM area per cell area (Figure S5B). After 14 days of culture, control aggregates showed a 5-fold increase in projected area. In contrast, suspension and pre-aggregates groups showed minimal increase in alveolar organoid area after 14 days of culture in HA hydrogels (Figure 5D). As such, the initiation of cell-to-cell contact and nascent ECM deposition during aggregate formation in microwells is required for alveolar organoid formation within HA hydrogels.

Figure 5.

Figure 5

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Aggregation and nascent ECM deposition support alveolar organoid formation and growth in HA hydrogels

(A) Schematic of experimental approach and perturbation studies. Aggregates are formed from individual cells seeded within microwells and cultured for 3 days, which enables formation of cell-to-cell contacts and deposition of nascent ECM. Aggregates are then embedded into HA hydrogels to grow and form organoids. Perturbation studies include embedding of (i) individual cells cultured in suspension for 3 days, (ii) immature aggregates after 15 min within microwells, and (iii) aggregates with digested nascent ECM.

(B and C) Representative bright-field image and fluorescent image of structures at day 14 in HA hydrogels. Scale bar 300 μm. Inset: representative immunofluorescence images of ECM secreted prior embedding into HA hydrogels. Scale bar 50 μm.

(D) Quantification of the fold change of average organoid size in HA hydrogels at day 14 compared to day 1 post embedding. n ≥ 4 individual hydrogels across 3 independent experiments. ∗∗p < 0.01, ns: no significant difference by one-way ANOVA with Tukey’s multiple comparisons test.

Discussion

Here, we described a two-step culture method for the formation of alveolar organoids. Our method includes the pre-aggregation of dissociated cells in microwells, followed by embedding in an HA hydrogel, which supports high viability and growth of AT2 organoids as well as maintenance. Importantly, the microwells provide precise control and tunability over aggregate size when compared to the traditional method of forming organoids in Matrigel, which results in high heterogeneity. The ability to control the seeding density in HA hydrogels provides means to control cell-cell and organoid-organoid signaling, which are critical factors in directing organoid growth. Our findings further show that single cells are not able to form viable organoids when directly embedded in hydrogels. In contrast, pre-aggregated cells deposit their own ECM, supporting organoid formation and growth upon embedding in HA hydrogels. This two-step method also enables the culture of alveolar organoids in HA hydrogels of various moduli, which may provide a useful tool to study how matrix stiffness and organoid-matrix interactions direct alveolar progenitor cell function (Ahmed et al., 2023; Liu and Tschumperlin, 2011).

Interestingly, our study showed that organoids grow with an apical-out orientation in HA hydrogels in contrast to the typical apical-in orientation within Matrigel as shown by HTII280, MUC1, and ZO1 staining (Figures 2E, 2F, and S2C). Such flipped orientations have been observed in other organoids in the absence of Matrigel (Capeling et al., 2019; 2022; Co et al., 2019; 2021; Fiorotto et al., 2023; Kakni et al., 2022) and are thought to be due to the basement membrane proteins present in Matrigel that direct polarity through integrins. Given that cell polarity is largely directed by integrin binding to the ECM (Streuli, 2009), the presence of laminin and collagen IV in Matrigel likely guides cellular orientation and the apical-in polarization with the apical side of the organoids facing away from the Matrigel. While laminin is well known to direct epithelial cell polarity (Matlin et al., 2017), nascent laminin had little effect on apical-out orientations upon HA hydrogel embedding (Figure 4B). Indeed, other studies have shown that laminin alone is not sufficient in re-orienting organoid polarity (Martins-Costa et al., 2023). Similarly, we previously observed an apical-out orientation of organoids after long-term culture in microwells (Loebel et al., 2022). Thus, it is possible that nascent collagen IV in the center of the aggregates may guide their orientation (Figure 4B). It has also been suggested that once polarity is established, cells may not be able to re-orient themselves (Wijesekara et al., 2022). Re-orientation in response to nascent ECM secretion is also limited by the capacity of cells to remodel the relatively stiff and non-degradable HA hydrogels. In summary, our two-step culture method provides a useful platform to further study the role of the nascent ECM in guiding organoid polarity.

Another critical component of this study is the introduction of click chemistry to label nascent glycans that are produced by aggregates and organoids. Nascent glycan labeling has been used extensively in glycobiology (Dube et al., 2006; Saxon and Bertozzi, 2000; Zhang and Zhang, 2013), and the galactosamine analog has been used to label nascent glycans in developing zebrafish (Laughlin et al., 2008). Specifically, click chemistry approaches have been used to visualize the cell-hydrogel interface of single cells embedded in synthetic hydrogels (Günay et al., 2023; Loebel et al., 2019). Here, we employ this technique to study the cell-hydrogel interface of organoids.

It is well established that cells remodel their environment both in vivo and in vitro. Multiple cell types have been shown to deposit ECM in synthetic hydrogels, including stromal cells (Ferreira et al., 2018; Hezaveh et al., 2018; Loebel et al., 2019), fibroblasts (Ivarsson et al., 1997; Mochitate et al., 1991; Zhou et al., 2014), endothelial-fibroblast co-culture (Friend et al., 2023; Moon et al., 2010), chondrocytes (Bryant and Anseth, 2002; Kisiday et al., 2002; Richardson et al., 2019), and oocytes (Tomaszewski et al., 2021). We recently reported that nascent ECM directs cellular mechanosensing of synthetic hydrogels (Loebel et al., 2019). Thus, nascent ECM secretion may also explain why hydrogel stiffness has little influence on organoid formation (Figure 1G). While it is known that epithelial cells secrete ECM (Qureshi et al., 2017; Rosmark et al., 2023; Van Der Velden et al., 2018), the concept of nascent ECM in organoid formation has just recently been explored (Below et al., 2022; Fiorotto et al., 2023; Güney et al., 2021; Hushka et al., 2024). Here, we show that pre-aggregation is critical for the secretion of a nascent ECM layer that supports organoid growth in synthetic hydrogels (Figure 5). Overall, this study uses a two-step culture method to support Matrigel-free alveolar organoid culture, highlighting the importance of both cell-cell and cell-ECM interactions in organoid formation and growth in synthetic hydrogels.

Experimental procedures

Cell culture

At day 3, aggregates were embedded into HA hydrogels of varying stiffness using different concentrations of peptide crosslinker and cultured for up to 14 days. For nascent ECM perturbation studies, alveolar progenitor cells were embedded as single cells after suspension culture or as aggregates following nascent ECM digestion with dispase.

Cell analyses

Cell viability was measured using CalceinAM (live) and NucRed Dead or Sytox staining and quantified as live per total cell area using ImageJ. Flow cytometry was performed on a Sony MA900 after retrieving organoids from HA hydrogels using 2 wt % hyaluronidase. For gene expression, RNA was isolated from pelleted and flash-frozen retrieved organoids, followed by cDNA synthesis and RT-qPCR using QuantiTect SYBR Green PCR Kit. Gene expression was calculated relative to RN18S and reported as arbitrary units (Table S1). Immunofluorescence was performed on fixated whole mount or paraffin-embedded organoids, after permeabilization, staining with primary and secondary antibodies (Table S2), and imaging on a Leica THUNDER microscope. For nascent ECM analysis, cell culture media was supplemented with azide-modified galactosamine followed by click labeling with dibenzocyclooctyne 488 prior fixation. Quantification of nascent ECM thickness was performed using BoneJ (Loebel et al., 2020) TEM was performed by the University of Michigan BRCF Microscopy and Image Analysis Laboratory.

Statistical analysis

Statistical comparisons were performed using unpaired Student’s t tests and one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. All experiments were repeated as described in the text.

Resource availability

Lead contact

Further information and request for materials and reagents should be directed to and will be fulfilled by the lead contact, Claudia Loebel (loebelcl@umich.edu).

Materials availability

Requests for materials including silicone molds, norbornene-modified HA hydrogels, and iPSC-derived alveolar progenitor cells should be directed to the lead contact and will be shared based on material transfer agreements.

Data and code availability

All data reported in this paper will be shared by the lead contact upon request. No large datasets were generated in this study. Any additional information required to re-analyze the raw data reported in this paper is available from the lead contact upon request.

Acknowledgments

C.L. is supported by the National Heart, Lung, and Blood Institute (NHLBI; R00-HL151670), the American Lung Association (IA-939940), and David and Lucile Packard Foundation. J.R.S. is supported by the Cystic Fibrosis Foundation Epithelial Stem Cell Consortium; the Chan Zuckerberg Initiative, an advised fund of the Silicon Valley Community Foundation (CZF2019-002440); and the National Heart, Lung, and Blood Institute (NHLBI; R01-HL166139). M.K.E. is supported by the National Science Foundation Graduate Research Fellowship (DGE-2241144). Human iPSC-derived AT2 cells were received from the Center of Regenerative Medicine of Boston University and Boston Medical Center, NIH/NHLBI grants N01: 75N92020C00005 and R01HL095993 to D.N.K. Any opinion, findings, and conclusions or recommendations expressed in this manuscript are those of the authors. The authors are grateful for support from the University of Michigan BRCF Microscopy and Image Analysis Laboratory, the University of Michigan’s BRCF Flow Cytometry Core, and Dr. Minli Xing from the University of Michigan BioNMR (U-M BioNMR) Core Facility (supported by the U-M College of Literature, Sciences, and Arts, the Life Sciences Institute, the College of Pharmacy, and the U-M Biosciences Initiative).

Author contributions

M.K.E., C.L., and J.R.S. designed the experiments. T.F. provided critical expertise and experimental input. M.K.E., C.J.C., L.K.B., E.M.P., S.P., R.C.S., and D.W.A. performed the experiments and collected the data. M.K.E., C.J.C., O.S., and J.E.L. analyzed the data. D.N.K. and K.-D.A. provided critical materials (alveolar organoids). M.K.E., C.L., and J.R.S. interpreted the data and wrote and prepared the manuscript. All authors contributed to manuscript review and editing.

Declaration of interests

D.N.K. holds intellectual property relating to alveolar organoids. J.R.S. and T.F. hold intellectual property related to lung organoid technologies.

Published: December 12, 2024

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.stemcr.2024.11.006.

Supplemental information

Document S1. Figures S1–S5, Tables S1 and S2, and supplemental experimental procedures
mmc1.pdf (11.2MB, pdf)
Document S2. Article plus supplemental information
mmc2.pdf (16.5MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S5, Tables S1 and S2, and supplemental experimental procedures
mmc1.pdf (11.2MB, pdf)
Document S2. Article plus supplemental information
mmc2.pdf (16.5MB, pdf)

Data Availability Statement

All data reported in this paper will be shared by the lead contact upon request. No large datasets were generated in this study. Any additional information required to re-analyze the raw data reported in this paper is available from the lead contact upon request.

Articles from Stem Cell Reports are provided here courtesy of Elsevier

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