Successful Establishment of Primary Type 2 Alveolar Epithelium with 3D Organotypic Co-Culture

Successful Establishment of Primary Type 2 Alveolar Epithelium with 3D Organotypic Co-Culture

Running title: 3D Co-Culture Establishes Primary AT2 cells

Key words: AT2 cell, epithelial-mesenchymal interactions, organotypic co-culture, lung model

ABSTRACT

 

Alveolar type 2 (AT2) epithelial cells are uniquely specialized to produce surfactant in the lung and act as progenitor cells in the process of repair after lung injury. AT2 cell injury has been implicated in several lung diseases, including idiopathic pulmonary fibrosis and bronchopulmonary dysplasia. The inability to maintain primary AT2 cells in culture has been a significant barrier in the investigation of pulmonary biology. We have addressed this knowledge gap by developing a 3-dimensional organotypic co-culture using primary human fetal AT2 cells and pulmonary fibroblasts. Grown on top of matrix-embedded fibroblasts, the primary human AT2 cells establish a monolayer and have direct contact with the underlying pulmonary fibroblasts. Unlike conventional 2D culture, the structural and functional phenotype of the AT2 cells in our 3D organotypic culture was preserved over 7 days of culture, as evidenced by the presence of lamellar bodies and by production of surfactant proteins B and C. Importantly, the AT2 cells in 3D co-cultures maintained the ability to replicate, with approximately 60% of  AT2 cells staining positive for the proliferation marker Ki67, while no such proliferation is evident in 2D cultures of the same primary AT2 cells. This organotypic culture system enables interrogation of AT2 epithelial biology by providing a reductionist in vitro model in which to investigate the response of AT2 epithelial cells and AT2-fibroblast interactions during lung injury and repair.

INTRODUCTION

 

The lung is comprised of more than 40 resident cell types, each with specific physiologic functions(1). Within the alveolus, the alveolar type 2 (AT2) epithelial cell is particularly important as it has the specialized function of surfactant production and also serves as a resident stem cell which proliferates in response to injury and subsequently differentiates into alveolar type 1 (AT1) epithelial cells(2-4). While conventional 2D culture methods have forged major advances in our understanding of the biosynthetic and regenerative capacity of AT2 cells, our knowledge of AT2 cell biology, including the response to injury, has been hindered by the difficulty in culturing AT2 cells in vitro(5). With conventional 2D culture methods the distinct pulmonary epithelial phenotype of AT2 cells is lost within 3-5 days of culture as evidenced by the loss of  their characteristic cuboidal shape and diminished surfactant production, including the loss of lamellar bodies (6). To date, the barrier to culturing primary AT2 cells has been circumvented by using stable cell lines that approximate AT2 function, but these immortalized cells do not fully recapitulate the biology of primary AT2 cell(5, 6).

Major advances in bioengineering have attempted to replicate the microenvironment of the lung, modeling lung development and respiratory disease using human primary or induced pluripotent stem cells(7, 8). These new technologies, which include organoid culture, alveolospheres, and lung-on-a-chip, have advanced the field of lung biology and have demonstrated the importance of culturing cells in 3-dimensions (3D)(4, 9, 10). Prior work with rat AT2 cells co-cultured with fibroblasts and collagen (11, 12) and more recent alveolosphere experiments with co-cultured human epithelial cells and mesenchyme (13-15) illustrate the important contribution that mesenchymal cells make to the maintenance of AT2 cell survival and function.  Other groups have generated complex lung bud organoids with human induced pluripotent stem cells (hiPSCs), and have demonstrated the spatial organization of many cell types in the developing lung(16). While these methods are improvements over conventional 2D culture, they have proven difficult to standardize, are not readily scaled for higher throughput, and allow for only limited access to the different cellular compartments, thereby restricting the experimental manipulation and interrogation of the culture system.

Building upon prior work in other organ systems(17), we have developed a system of 3D organotypic co-culture that can be used with primary human AT2 cells and pulmonary fibroblasts. Whereas 2D cultures of primary AT2 cells lose many of their defining characteristics, we demonstrate that primary lung epithelial cells cultured in this 3D organotypic co-culture retain their AT2 physiology and molecular characteristics. This novel method enables routine and standardizable long-term culture of AT2 cells and has the potential for use in modeling their contribution to the emergence and resolution of diseases of the human lung.

METHODS

 

Primary epithelial cell isolation

Second trimester human lung tissues were isolated as previously published(18, 19) in accordance with protocols approved by the Vanderbilt University Institutional Review Board, with details in the Supplement.

Isolation of primary lung fibroblasts

Using the same second trimester fetal lung tissues described above, fetal lung fibroblasts were isolated and cells between passages 5 and 15 were used for experiments, with details of cell culture in the Supplement.

Isolation of primary mouse lung epithelium

All mouse experiments were approved by the Vanderbilt Institutional Animal Care and Use Committee. Lungs were isolated from C57BL/6 mice, aged 8-12 weeks, from both males and females, and AT2 cells were isolated using protocol detailed in the Supplement.

Mouse lung epithelium (MLE-15) Cell line

MLE-15 cells were obtained from Dr. Jeffrey Whitsett (Cincinnati Children’s Hospital, Cincinnati, OH)(20), with additional culture details found in the Supplement.

Assembly of 3D organotypic co-cultures

3D organotypic co-cultures were assembled on 24 mm transwells with 3 m pore polycarbonate membrane insert (Corning 3414) and placed into deep-well culture plates (Corning 355467) (Figure 1A, B). Co-cultures were assembled in two layers, a 3D matrix with human fetal lung fibroblasts and a layer of AT2 cells (primary human fetal AT2 cells, mouse AT2 cells, or MLE-15), with complete details of organotypic co-culture assembly and processing for analysis found in the Supplement. Full details of control experiments with matrix-free co-culture and cell-free AT2-matrix culture are also in the Supplement.

2D Culture

AT2 cells were isolated from second trimester fetal lung as described above and cultured on Matrigel-coated glass coverslips in serum-free Waymouth’s medium with DCI added as previously described(19). Cells were cultured for 7 days, changing medium every 24 hrs. For the comparison with 3D organotypic co-cultures, AT2 cells from the same lung were used. For experiments with added growth factors, keratinocyte growth factor (KGF) at 10 ng/ml, and hepatocyte growth factor (HGF) at 20 ng/ml were used, in accordance with previously published experiments using these growth factors and AT2 cells (21).

RNA isolation and real-time quantitative PCR (qPCR)

Total RNA was isolated and reverse transcribed by standard methods using the Qiagen RNEasy kit (Qiagen, Hilden, Germany) and SuperScript VILO cDNA synthesis kit (Invitrogen), with additional details in the Supplement.

Immunofluorescence

Immunofluorescence (IF) was performed as described previously(10, 22, 23), with additional details about specific antibodies, cyclic staining, and microscopy in the Supplement.

Western Blot Analysis

AT2 cells were isolated from fetal lungs that had been cultured as explants in DCI medium for 96 hrs and were grown in 3D organotypic co-cultures as described above. Human fetal lung explants grown in DCI medium for 6 days were used as positive controls. Full details of protein isolation and immunoblot preparation can be found in the Supplement.

Transmission Electron Microscopy and Scanning Electron Microscopy

Specimens were processed for transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and imaged in the Vanderbilt Cell Imaging Shared Resource Electron Microscopy facility.  Details of the fixation and microscopy protocols used for TEM and SEM can be found in the Supplement.

Statistics

Experiments were performed a minimum of three times, with triplicate samples used in qPCR experiments. Quantitative microscopy was performed with a minimum of 25 high-powered fields counted per sample. All values are reported as mean values ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism (version 7). Two-tailed students’s t-test was used  for two-group comparisons, with Shapiro-Wilk test for normality and correction for multiple comparisons using the Holm-Sidak method. One-way analysis of variance was used for comparisons between multiple groups. For analysis of variance, the Tukey method was used to correct for multiple comparisons. Adjusted P values less than 0.05 were considered to be statistically significant.

RESULTS

3-dimensional organotypic co-cultures support a monolayer of epithelial cells and allow for direct contact between epithelial cells and fibroblasts.

Assembly of the lung epithelium atop matrix-embedded fibroblasts suspended on a transwell insert generates a 3D organotypic co-culture that maintains AT2 cells in close proximity to lung fibroblasts (Figure 1B, C, D). This models the interaction that exists between these two cell types in lung tissue (Figure 1E, F, G). By hematoxylin and eosin staining, the primary fetal AT2 cells established a continuous monolayer on the 3D fibroblast matrix (Figure 1C) as did primary AT2 cells isolated from adult mice (Figure 1D). The co-culture of these two cell types in close proximity facilitates paracrine signaling as is found in vivo. Further, the primary AT2 cells appear to have direct contact with the fibroblast cells in a manner that resembles the direct interaction between epithelial cells and fibroblasts in developing fetal lung (Figure 1F) and term infant lung (Figure 1G). To enable direct comparison between our new organotypic model and existing research using established lines, we implemented the model with MLE-15 cells, a mouse cell line, which closely resembles human AT2 cells and exhibits surfactant protein production (20). When seeded onto the 3D fibroblast matrix, the MLE-15 cells established a monolayer (Supplemental Figure E1A) that emulated the morphology of the human fetal AT2 cells.

The type 2 alveolar epithelial cells grown in this 3D organotypic co-culture system exhibit ultrastructural features characteristic of AT2 cells.

We next examined whether the structural features that define AT2 cells (2) were preserved in our 3D organotypic cultures. Using SEM, we confirmed that AT2 cells in this system exhibited the characteristic cuboidal shape with apical microvilli (Figures 2A, B). This is in contrast to 2D culture where the AT2 cells flatten after 24-48 hrs(3). TEM further revealed the presence of lamellar bodies in the AT2 cells (Figure 2C, D), a cardinal feature of fully-differentiated AT2 cells(5). In addition, TEM demonstrated the direct interaction between AT2 cells and fibroblasts (Figure 2E), a novel feature of our system compared to the separation by an artificial membrane that occurs in traditional transwell cultures.

Epithelial cells and fibroblasts exist in the co-culture system as distinct geographically defined populations, retaining expression of cell-type specific genes.

Immunofluorescence demonstrated the presence of distinct populations of epithelial cells (that stained positive for epithelial markers pan-cytokeratin and Ep-CAM) and fibroblasts (expressing the mesenchymal marker vimentin) that spatially exist in separate regions of the organotypic co-culture (Figure 3A, B). In other culture models using fibroblasts as a feeder layer for epithelial cells, induction of cell-cycle arrest is often required to prevent fibroblasts from taking over the culture(24). In this organotypic system, however, the 3D matrix allows for physiological levels of proliferation in the fibroblast population and direct contact with epithelial cells, but prevents their overgrowth in the co-culture. Cells expressing both pro-surfactant protein C (SP-C) and pancytokeratin, a generic marker of epithelial cells, resided only in the top epithelial layer (Figure 3A, C), and this region was negative for the mesenchymal marker vimentin (Figure 3A, C). The top layer of cells was also for surfactant protein B (SP-B) in addition to pro-SP-C reflecting expression of both surfactant genes SFTPB and SFTPC by qPCR (Figure 3D). Expression of vimentin a fibroblast marker, was only seen in the lower layer of the organotypic cultures (Figure 3D). As expected, we also found evidence of expression of VIM (vimentin) by qPCR (Figure 3D). RT-qPCR of 3D cultures showed nearly no expression of  AT1 markers AQP5 (aquaporin 5), HOPX and PDPN (podoplanin), with cycle threshold (CT) values >30 (Figure 3D). As club cells have also been shown to express SP-B, we tested for expression of club cell specific marker SCGB1A1 and found very little mRNA (CT>35) (Figure 3D). When compared with human fetal AT2 cells cultured in 2D conditions for 7 days, we found significant differences in the pattern of expression of AT2 and AT1 cell markers. After 7 days in culture, expression of SFTPB was approximately 10-fold greater in the 3D organotypic co-cultures relative to the 2D culture, with significantly increased SFTPC expression as well (Figure 3D). Interestingly, the 2D AT2 monocultures exhibited significantly increased expression of AT1 markers PDPN and HOPX when compared to 3D organotypic co-cultures. Increased expression of the fibroblast marker vimentin in the 3D organotypic cultures was expected since the organotypic culture contained both AT2 cells and fibroblasts, whereas the 2D cultures contained AT2 depleted of fibroblasts. Expression and full proteolytic processing of proSP-B from a 42 kDa proprotein to an 8 kDa mature protein is a cardinal feature of AT2 cells that is not demonstrated by club cells(25).  Western immunoblotting of 3D co-cultures demonstrated the presence of mature and immature forms of SP-B as did explants from second trimester human fetal lung grown for 6 days in DCI (Figure 3E). 3D co-cultures using primary mouse AT2 cells and human fetal lung fibroblasts demonstrated similar results to human fetal AT2 cells in co-culture.  Specifically, primary mouse cells demonstrated spatial segregation of  SP-C-positive cells in the top layer of cells and vimentin-positive fibroblasts in the bottom layer of cells within the matrix (Figure 3F), and maintenance of expression of Sftpb and Sftpc by RT-qPCR after 7 d in co-cultures (Figure 3G). Similar results were found in the co-cultures that combined MLE-15 cells and human fibroblasts (Figure E1B, C).

AT2 cells in 3D co-culture have higher rates of proliferation when compared to AT2 cells grown with traditional 2D culture methods.

As mentioned previously, 2D cultures of AT2 cells often require serum-free conditions to minimize fibroblast overgrowth which also results in suppression of AT2 proliferation.  Despite using serum-free culture media for both 3D co-cultures and 2D cultures, we observed that 30.4% (+/-2.4%) of the total cells in in the 3D organotypic co-culture culture immunostained positive for Ki-67, a marker of cellular proliferation, whereas only 1.8% (+/-0.8%) of cells were Ki67-positive under 2D conditions (Figure 3H, I, p<0.0001). Co-staining with NKX2-1, a marker specific to AT2 cells, revealed that 62.9%(+/6.0%) of Ki67 positive cells were positive for NKX2-1 in the 3D co-culture. This was in striking contrast to AT2 cells grown in 2D for 7 days in DCI medium, where none of the Ki67 cells were positive for NKX2-1 (Figure 3H, I). Instead, all of the Ki67-positive cells in the 2D cultures were also vimentin-positive, whereas fewer than 30% of the Ki67-positive cells in 3D co-culture were vimentin-positive (Figure 3H). The 1.8% of Ki67-positive vimentin-positive cells in the AT2 2D culture likely represent contaminating fibroblasts. Thus, 3D organotypic co-cultures preserve the capacity of AT2 cells to proliferate, a critical characteristic of AT2 cells in vivo (26).

To understand the importance of the complex structure of the 3D co-culture to the preservation of at AT2 phenotype, we assessed proliferation and apoptosis in AT2 cultures missing key features of the 3D co-cultures and in 2D cultures supplemented with growth factors.  Human fetal AT2 cells cultured on the complex Matrigel-collagen matrix without any fibroblasts for 7 days demonstrated few surviving AT2 cells, and very little RNA remained.  TUNEL staining showed a very high rate of AT2 apoptosis in the absence of matrix-embedded fibroblasts (Supplement E2A-E). In contrast, <5% TUNEL-positive staining was evident in organotypic cultures constructed using human fetal lung AT2, mouse primary AT2, or MLE-15 cells (p<0.0001).  AT2 cells cultured on the upper surface of transwell membranes with fibroblasts growing on the bottom surface of the well exhibited increased rates of proliferation indicated by Ki67 immunostaining, but all of the Ki67-positive cells were vimentin-positive fibroblasts, and none were Nkx2-1-positive AT2 (Supplement E3A). Epithelial cell proliferation in 2D culture did not achieve the levels seen in 3D organotypic cultures even when KGF, HGF, or both were added to 2D cultures for 7 days, demonstrating no cells immunopositive for both Ki67 and the AT2 marker Nkx2-1 (Supplement E4 A, C, E). Under matrix-free co-culture or added growth factor conditions, we observed no detectable SFPTC expression, decreased SFTPB expression, and increased expression of AT1 markers relative to AT2 cells grown in 3D organotypic co-culture by qPCR (Supplement E3C and E4 B, D, F).

Organotypic co-cultures can be used to model injury and to examine cell-cell interactions

Hyperoxia has been used in in vivo and in vitro systems to model lung injury, particularly injury occurring after pre-term birth(27-29). We exposed our 3D organotypic co-cultures to 48 hrs of 70% oxygen and found that many of the previously reported transcriptomic features were replicated, including increased expression of COL1A1 (Collagen 1), and ACTA2 (a-SMA) relative to the organotypic co-cultures grown in normoxia (Figure 3J). Prior work has demonstrated that in AT2 cells increase surfactant production in response to hyperoxia (30). We found a greater increase in the expression of SFTPB and SFTPC in the hyperoxia-exposed 3D organotypic cultures than in the hyperoxia-exposed AT2 cells growing in traditional 2D conditions (Figure 3J).

DISCUSSION

There is significant evidence that epithelial-mesenchymal interactions are key drivers of normal lung development(31-33) and of the pathology of many forms of lung injury and fibrotic lung disease (34-36). Our ability to understand the complex biology and behavior of AT2 cells has been limited by the difficulty of culturing AT2 cells and maintaining the relevant features of their phenotype(5). The development of a 3D organotypic co-culture system that preserves direct contact between AT2 cells and mesenchymal cells is therefore a critical step towards developing in vitro models of these diseases.  The presented model allows for the interrogation of essential drivers of epithelial-mesenchymal cell interactions by allowing access to each cell type in the co-culture for up to 7 days, and enabling the manipulation of the growth medium of each compartment. In addition, this system allows for epithelial-mesenchymal interactions to be investigated over at least a 7-day time course, a significant improvement from traditional 2D methods, where primary AT2 cells begin to lose their characteristic structure and surfactant production within 3-5 days(24, 37).

The lung epithelial cells maintained in the 3D organotypic culture retain many of the specialized functions of AT2 cells. These include lamellar body production and full processing of surfactant proteins, which are considered metrics of fully-differentiated AT2 cells in vivo(38). Considering the role of AT2 cells in lung repair, proliferation is essential for AT2 cells to replenish the AT2 cell population and provide progenitor cells that can differentiate into AT1 cells(26). While significant evidence of differentiation from type 2 to type 1 cells occurs in traditional culture systems after 3-5 days, we did not observe such type 1 cell differentiation in our 3D system. This is not entirely unexpected because the secreted factors from the fibroblasts that maintain the AT2 phenotype most likely prevent this AT2-to-AT1 differentiation. We found that the maintenance of SP-B and SP-C expression and cellular proliferation were critically dependent on the direct interaction with fibroblasts growing in the 3D matrix. AT2 cells grown in 2D culture (with and without KGF and HGF), cell-free matrix, and matrix-free transwell co-cultures all had significantly less surfactant protein expression and AT2 proliferation when compared with cells growing in 3D organotypic co-culture. While the AT2 cells cultured in matrix-free transwells and 2D culture with growth factors showed some evidence of proliferation, all of the dividing cells were negative for AT2 marker Nkx2-1. We have not elucidated the precise mechanism by which the organotypic co-culture system maintains the AT2 phenotype and promotes AT2 cell proliferation, but we speculate that the fibroblasts growing in the 3D matrix secrete growth factors and cytokines that support the physiology of AT2 cells in a manner that reproduces the in vivo condition.  Indeed, one advantage of this system is that it allows for the maintenance of primary AT2 cells without the addition of exogenous growth factors, which might have unintended effects on the lung biology and pathology being modeled. Based on the work of other groups in culturing AT2 cells(39), it is possible that transcriptomic changes and epigenetic shifts occur during the cell isolation and the culture process, and we did not investigate these changes in our system in this study. Future work to examine the features of each cell type in our organotypic culture system and how these features evolve over time will be important in understanding the features and limitations of this system and how this system might be used to study lung development and lung diseases that result from AT2 cell injury.

As with all reductionist models, this system has several limitations. It does not measure or replicate all of the functions of AT2 cells; specifically, it does not study fluid transport, vectorial ion transport, or the properties of tight junctions, which have been previously modeled in transwell cultures(40-42). While we have demonstrated lamellar body production by EM, expression of SFTPB and SFTPC by qPCR, and processing of SP-B by Western blot, we have not isolated mature surfactant proteins or demonstrated surfactant arranged in a tubular myelin structure from the secreted media, as other models using alveolospheres in Matrigel have done(43).  In addition, this system includes only two cell types, and does not include endothelial cells or circulating immune cells, both of which have also been implicated as key drivers of lung disease. Future work to expand this model and incorporate these cell types may produce a more complex model that has even greater fidelity with human lung. Indeed, recent studies of complex alveolospheres and lung buds incorporating multiple cell types represent major advances in in vitro modeling of lung development and disease(4, 16). One benefit of our more simplified system is that the media of each cell type can be accessed and manipulated separately and that the ratio of AT2 cells to fibroblasts can be carefully controlled, in contrast to the more stochastic assembly of alveolosphere-based co-cultures.

Currently, the longevity of our co-culture is limited by contraction of the matrix layer which prevents culture of the system beyond 17-21 total days (10-14 days after adding AT2 cells). As the matrix pulls away from the sides of the transwell insert, separation of the two media compartments is lost, and the surface of the epithelial side of the matrix deforms. Additional studies that change the stiffness and composition of the matrix may provide a solution to this barrier to long-term culture.

Previous studies have demonstrated the utility of 3D organotypic co-culture between fibroblasts and epithelial cells in other organ systems, and in transformed cancer cell lines from the lung(17, 44). Our experimental method differs from these methods in the composition of the mesenchymal matrix and in the use of primary lung fibroblasts to create a tissue culture matrix that is hospitable to fragile primary AT2 cells, creating an opportunity to study their intrinsic vulnerability to injury and characterize their basic biologic features. Understanding the specialized AT2 cell and its critical contribution to lung development, injury, and repair is of paramount importance in understanding the pathobiology of multiple human lung diseases and developing new therapeutic strategies. This system of 3D organotypic co-culture not only enables the study of the epithelial cells themselves, but also allows for the investigation of interaction between the epithelium and the underlying stroma, a critical communication that is central to repair after lung injury. We have demonstrated that at least a portion of the transcriptomic response of AT2 cells and fibroblasts to hyperoxia can be replicated in this model, and in fact the AT2 response to hyperoxia appears to be related to the epithelial-mesenchymal interactions afforded by the 3D model. Future development of this model will allow us to model disease using human primary cells as well as mouse primary cells from genetically modified animals. By replicating the epithelial-mesenchymal proximity found natively in the alveolus, we have created a system that allows for the organotypic cultivation and manipulation of AT2 cells and fibroblasts, thus creating a path for broadening our understanding of the biology of the distal lung in development and disease. Importantly, the ease-of-use and scalability of this model system will enable high-throughput discovery and preclinical screening of therapeutic agents that may lead to novel interventional strategies.

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FIGURE LEGENDS

Figure 1: 3D Organotypic Co-Cultures Mimic the Relationship Between Epithelial and Mesenchymal Cells. A: Flowchart depicting the assembly of 3D organotypic co-cultures. Fibroblasts are cultured in a 3D matrix, plated on a transwell membrane and matured for 7 days. AT2 cells are then added and co-cultured for 7 days. B: Schematic overview of 3D organotypic co-cultures.   C: Hematoxylin and eosin (H&E) stain of 3D organotypic co-culture with fetal lung fibroblasts in matrix and primary human fetal AT2 cells on top layer. 3D culture has been embedded in paraffin on-edge and sectioned. D: H&E stain of 3D organotypic co-culture with human fetal lung fibroblasts in matrix and primary mouse AT2 cells in top layer. E: Schematic of human lung alveolus, demonstrating the relationship between AT2 cells and fibroblasts. F: H&E staining of 18-week human fetal lung. G: H&E staining of 40-week term infant lung. Scale bar = 25 m.

Figure 2: Primary AT2 Cells Grown in 3D Organotypic Co-Culture Have Preserved Ultrastructural Features. A: Scanning electron microscopy (SEM) of surface of 3D organotypic co-culture, demonstrating the cuboidal shape of the primary AT2 cells. Scale bar = 5 m B: SEM of the 3D organotypic co-culture showing the presence of microvilli of the AT2 cells. Scale bar = 5 m. C, D: Transmission electron microscopy (TEM) shows the presence of lamellar bodies in AT2 cells, LB=lamellar body, Nuc=nucleus, M=mitochondria. Panels on right are enlargements of areas indicated by red boxes.  Scale bar C = 2 m, with inset scale bar = 660 nm scale bar D = 500 nm, with inset scale bar=1.4 m  E: TEM shows direct contact between AT2 cells and fetal lung fibroblasts (FLF). Scale bar = 2m.

Figure 3: Primary AT2 Cells in Organotypic Culture Express Surfactant and Retain Ability to Replicate. A: Cyclic immunofluorescence of large section of organotypic co-culture, with staining for epithelial marker pan-cytokeratin (red), type 1 collagen (green), nuclei (DAPI), pro-surfactant protein C (aqua) and nuclear marker histone H3 (red). Scale bar = 100 m. B: Immunofluorescence (IF) of 3D organotypic co-culture with human fetal AT2 cells for Ep-CAM (green) and vimentin (red). Scale bar = 25 m. C: Immunofluorescence of 3D organotypic co-culture with primary human fetal AT2 cells for SP-B and pro-SP-C (green) and vimentin (red). Scale bar = 25m. D: RT-qPCR expression of SFTPC, SFTPB, AQP5, HOPX, PDPN, SCGB1A1, and VIM in 3D co-cultures with primary fetal AT2 cells. E: Western blot with an antibody that binds immature and mature forms of SP-B, with the mature form measuring 8kDa. OTC= organotypic co-culture. HFL= human fetal lung explants. F: Immunofluorescence of 3D organotypic co-culture with primary mouse AT2 cells and human fetal lung fibroblasts for SP-C (green) and vimentin (red). Scale bar = 25m. G: RT-qPCR expression of Sftpb and Sftpc in 3D co-cultures with primary mouse AT2 cells. H: Immunofluorescence of human fetal AT2 cells cultured in 2D and 3D for proliferation marker Ki67 (green) and AT2 marker Nkx2-1 (red) and Ki67 (green) and fibroblast marker vimentin (red). Scale bar = 25 m. I: Quantification of 1,000 cells per sample showed significant increase in the percentage of Ki67 positive cells in 3D culture when compared with the same cells cultured in 2D Matrigel (p<0.0001). There was a significant increase in the percentage of NKX2-1 cells that were also positive for Ki67 in 3D culture when compared with 2D culture (p<0.0001). J: Exposure of 3D organotypic co-culture (OTC) to hyperoxia replicated many of the transcriptomic features described in bronchopulmonary dysplasia (OTC), with significant increases in COL1A1 and ACTA2 expression. Comparing AT2 cells grown in 2D monoculture or 3D OTC, there was a greater response to hyperoxia, as demonstrated by the increase in expression of SP-B and SP-C in OTC.

Supplemental Figure E1: A: Hematoxylin and eosin (H&E) stain of 3D organotypic co-culture with fetal lung fibroblasts in matrix and MLE-15 cells on top layer.  Scale bar = 25 m. B: Immunofluorescence of 3D organotypic co-culture with MLE-15 cells and human fetal lung fibroblasts for SP-B (green) and vimentin (red). C: RT-qPCR expression of Sftpb and Sftpc in 3D co-cultures with MLE-15 cells.

Supplemental Figure E2: A-C: TUNEL stain (green) of 3D organotypic co-culture with fetal lung fibroblasts in the matrix and human fetal AT2 (A), mouse AT2 (B), and MLE-15 (C) epithelial cells on top. Counterstain with SP-C (red). D: Human fetal AT2 cells cultured on top of cell-free Matrigel and collagen I matrix, with TUNEL staining (green) and SP-C counter stain (red). E: Quantification of TUNEL positive cells shows significant difference between the amount of apoptosis seen in the cell-free matrix cultures and the other 3 organotypic co-cultures, ****p<0.0001. Scale bar = 25 m.

Supplemental Figure E3: A: IF of human fetal AT2 cells in matrix free transwell co-cultures for proliferation marker Ki67 (green) and fibroblast marker vimentin (red) and Ki67 (green) and AT2 marker Nkx2-1 (red). Scale bar = 25 m. B: All of the Ki67 cells were positive for vimentin, and there were no Nkx2-1 positve cells that were Ki67 positive. C: qPCR for expression of markers of AT2, AT1, and fibroblasts in matrix free transwell co-cultures, showing no detectable SP-C expression, and a 10-fold decrease in SP-B expression when compared with 3D OTC. There was detectable expression of AT1 markers PDPN and HOPX as well as vimentin.

Supplemental Figure E4: A, C, E:  IF of human fetal AT2 cells in 2D cultures with added keratinocyte growth factor (KGF), hepatocyte growth factor (HGF) or both for proliferation marker Ki67 (green) and AT2 marker Nkx2-1 (red) and Ki67 (green) and fibroblast marker vimentin (red).Scale bar = 25 m. B, D, F: qPCR for expression of markers of AT2, AT1, and fibroblasts in matrix free transwell co-cultures, showing no detectable SP-C expression, and a 10-fold decrease in SP-B expression when compared with 3D OTC. There was detectable expression of AT1 markers PDPN and HOPX as well as vimentin, suggesting some AT2 cell differentiation in 2D culture, even in the setting of KGF and