Higher density seeding of lung progenitor cells improves AEC2 differentiation in organoids.
Using an adapted protocol (4, 35), we differentiated human CA1 ES cells to NKX2-1+ lung progenitor (LPC) cells (Figs. 1A, S1). In contrast to the published protocol, we did not observe any benefit from adding DAPT, a γ-secretase inhibitor of the NOTCH pathway, to the media for differentiating ventral anterior foregut endoderm (VAFE) to LPCs (Fig. S2). We introduced DAPT at different time points and durations during the VAFE to LPC differentiation period (Fig. S2A) and validated its inhibition of NOTCH (Fig. S2B) but did not find any effect on NKX2-1+ LPC induction (Fig. S2C). However, extending the original duration of differentiation of AFE to LPC from D11-15 (4, 35) to D11-22 doubled the induction efficiency of NKX2.1+ cells from 21.79 ± 4.21% at D15 (n = 14 biological replicates) to 44.35 ± 3.32% at D22 (n = 25 biological replicates; a.k.a. 25 separate differentiations) (Fig. 1B). Similar NKX2-1 induction efficiencies (45–55%) at D22 of differentiation were observed with CA1 ES and NCRM1 iPS cells that both were stably transfected with a hSFTPCGFP reporter (Fig. S3B). The flow cytometry data was corroborated by nuclear immunostaining of the cells for NKX2-1 (Figs. 1B, S3B). Flow cytometry and real-time PCR analysis also indicated a greater commitment of LPCs to SOX9+ than SOX2+ progenitor lineages (Fig. S3C). When more than 50% of the LPC population at D22 was positive for NKX2-1 (22), cells were embedded in a 1:1 diluted (50%) Matrigel for organoid formation and then incubated in a distalizing medium promoting AEC2 induction (Fig. 1C).
Organoid size and number, but not morphology, were dependent on the seeding density of CA1 LPCs (Fig. 1D,E). Cultures started with 2.5x104 LPCs yielded less organoids than a 4-fold higher concentration of LPCs. Moreover, organoids in cultures started with 2.5x104 LPCs were on average double the size of those found in the higher density seeded cultures (215.95 ± 11.45µm vs 129.12 ± 5.66µm diameter at D36) (Fig. 1F). Independent of seeding density, cells within the organoids were highly proliferative based on Ki-67 expression (Fig. 1G). In both cultures, we observed a slight but non-significant decrease in Ki-67+ cells at D36 versus D29, suggesting a small reduction in proliferation as differentiation progressed. Thus, seeding density of LPCs affects the size of the organoids, especially on D36 of differentiation.
We next investigated whether seeding density (i.e., size of the organoid) impacted AEC2 differentiation. Flow cytometry for HT2-280, a human AEC2 surface marker (36), revealed an increase in HT2-280 expression over time under both seeding conditions (Fig. 2A,B). The percentage of HT2-280+ cells at D43 of differentiation was significantly greater in high-density versus low-density seeded (i.e., smaller versus larger) organoid cultures (20.15 ± 6.05% vs. 7.20 ± 1.20%, p < 0.05). However, the percentage of CDH1 (E-cadherin) positive epithelial cells was significantly lower in high-density compared to low-density seeded organoid cultures. The efficiency in AEC2 differentiation was validated with CA1 and NCRM1 cells transduced with a hSFTPCGFP reporter (Fig. S4). The hSFTPC promoter has previously been validated to label murine AEC2 in situ (37, 38). Immunostaining of D43 CA1-SFTPCGFP organoid cells (Fig. S4A) revealed that GFP+ cells, representing SFTPC expression, were positive for CDH1 (see inlet), and that 16.81 ± 1.22% (n = 3) of the organoid cells stained positive for HT2-280 (Fig. S4C). Flow cytometry for GFP (Fig.S4A-right panel) of D43 organoids formed from 105 CA1-hSFTPCGFP or NCRM1-hSFTPCGFP LPCs demonstrated that 20.42 ± 2.76% and 21.06 ± 4.40%, respectively, of the organoid cells expressed GFP (Fig. S4B), matching the differentiation efficiency of LPCs into AEC2 when using HT2-280 as readout (Fig. 2B) and was in line with the immunostaining findings.
We then assessed whether time in culture increased AEC2 differentiation. We dissociated the organoids and passaged them into fresh Matrigel at D14 after the initial start of organoid culture from LPCs, extending the duration of organoid differentiation by another week (D50 instead of D43). This did not result in a further increase hSFTPC-driven GFP expression (Fig. S4D). Real-time PCR analysis of GATA6, a transcription factor essential for distal lung epithelial differentiation (39, 40), and AEC2 markers SFTPB and SFTPC substantiated the HT2-280 and GFP cytometry findings. In both low- and high-density seeded organoid cultures, GATA6, SFTPB, and SFTPC mRNA expression increased compared to either ESC/iPSC (Fig. S5) or D22 LPCs (Fig. 2C), with high-density seeded organoids exhibiting the highest expression.
To visualise the diversity of cell types within D43 organoid cultures, we conducted immunofluorescence confocal microscopy for distal and proximal epithelial lung markers. Consistent with the gene expression findings, we identified pro-SFTPB+ and HT-280+ cells (Fig. S6A-bottom panels) within the CDH1+/NKX2-1+ (Fig. S6A-top panels) organoids at D43. Positive immunostaining for mature SFTPB (mSFTB, see inlets of pro-SFTB images) indicated the presence of cells capable of processing pro-SFTPB into its mature form. HTII-280 staining was apical within the lumen of the organoids, in line with an apical-in orientation. Immunostaining for SCGB1A1 (club cell marker) and TUBB4A (ciliated cell marker) was negative, aligning with the reduction in proximal gene expression Fig. S6A-middle panels). Ultrastructural electron microscopy (EM) analysis of the D43 organoids revealed cell junctions between epithelial cells with microvilli at the apical surface (Fig. S6B-left panels). Immuno-gold EM visualized mature SFTPB within cytoplasmic pre-lamellar multivesicular structures (Fig. S6B-left panels), consistent with previous reports for fetal AEC2 in situ (41).
Removal of mesenchymal progenitor cells limits AEC2 differentiation within organoids.
We next enriched the CA1 NKX2-1+ expressing population before organoid formation using carboxypeptidase M (CPM), an enzyme present on the surface of lung progenitor cells (1). Sorting for CPM+ cells at D22 (43.43 ± 3.48% of total cells were CPM+ at D22, n = 8 biological replicates; Fig. 3A) resulted in an enrichment of the NKX2-1 expressing LPCs from 44.35 ± 3.32% to 80.54 ± 4.05% (Fig. 3B). NKX2-1 immunostaining further confirmed the enrichment in the number of NKX2-1+ LPCs (Fig. 3B). As depicted in Fig. 3C, 105 CPM+-sorted LPCs formed organoids within a few days of seeding (1, 42) that were similar in shape and size to organoids formed from unsorted (> 50% NKX-2-1+) LPCs (Fig. 1D-3C). Surprisingly, the percentage of HT2-280+ cells in D43 organoids established with CPM+-sorted LPCs was significantly lower compared to organoids formed with unsorted LPCs (Fig. 3D), while the percentage of CDH1+ epithelial cells remained unchanged. We hypothesized that the reduction of HT2-280+ cells in the organoids formed from CPM+-sorted LPCs occurred due to the loss of mesodermal cells, which are known to be essential for distal epithelial differentiation (16–18, 43). In our differentiation protocol (Fig. 1A), we did not sort for a pure definitive endoderm population as ≥ 85% of the cells at D6 of differentiation were double positive for c-KIT and CXCR4 (Figs. S1B, S3A). Therefore, we started our differentiation towards AEC2 with a definitive endoderm population that was contaminated by other lineages, including mesoderm (44, 45). By sorting the D22 cell population for CPM+ cells, we likely removed most of the mesodermal progenitor cells that had emerged during the differentiation protocol (10, 11, 13, 21). To verify, we compared the expression of mesenchymal markers vimentin (VIM) and actin alpha 2 (ACTA2) in our CPM+-sorted versus unsorted organoid cultures (17, 46, 47). Immunofluorescence confocal microscopy revealed the presence of VIM+ cells in the unsorted organoid cultures, while VIM+ cells were sparse in the CPM+-sorted organoid cultures (Fig. 4A). Reduced VIM and ACTA2 expression in the CPM+-sorted compared to unsorted organoid cultures corroborated the immunofluorescence findings (Fig. 4B). Together, the findings suggest that organoids formed with CPM+-sorted LPCs contain fewer mesenchymal progenitor cells. The loss in VIM+ cells could also be due to less epithelial-mesenchymal transition in the sorted LPC organoid cultures, but the similar percentages of CDH1+ epithelial cells in the unsorted and sorted LPC organoid cultures (Fig. 3D) argue against epithelial dedifferentiation. Mesenchymal support for LPC differentiation into AEC2 was corroborated by incubating LPCs in the absence and presence of human embryonic lung fibroblasts (hELF). Human ELFs were mitotically inactivated by irradiation (ihELF) to prevent overgrowth during co-culturing without influencing their secretome (48, 49). D22 NKX2-1+ LPCs were grown on Transwell® inserts pre-coated with either Matrigel, Laminin, or Collagen type IV (the latter two matrices are the main components of Matrigel). The inserts were cultured in distalizing medium with and without ihELFs seeded in the bottom wells of the Transwell® plates. The percentage of CDH1+ cells did not differ between LPCs cultured for 22 days with or without ihELFs (Fig. 5A). However, independent of the coated matrix, co-culture with ihELFs increased the percentage of HT2-280+ cells (Fig. 5A), confirming the importance of mesenchymal-epithelial crosstalk for fetal AEC2 differentiation (15, 16, 43). Direct contact between ihELF and LPCs within organoids did not further improve HT-280 induction above indirect co-culturing (Fig. 5B).
Biophysical forces stimulate AEC2 differentiation within organoids.
To examine whether mechanical strain affects the differentiation of LPC into AEC2 within organoids, we recapitulated fetal breathing movements in vitro using a 3D stretching device (Fig. 6A). Within 3 days of culture of D22 unsorted (> 50% NKX2-1+) LPCs in 80% Matrigel (concentration required for applying strain to organoids using the FlexCell® system), organoid structures began to form. No apparent differences were observed in the morphology of organoids in static compared to stretched conditions (Fig. 6B). The size of the organoids did not change with periodic stretching compared to static controls (128.10 ± 7.54 vs 134.02 ± 22.12 mm, mean ± range, n = 3 biological repeats, static versus stretch at D42). Flow cytometry for Ki-67 and CDH1 in episodically stretched organoids and paired static controls demonstrated no significant differences in proliferation and epithelial lineage expression, respectively (Figs. 6C, S7). However, flow cytometry for HT2-280 revealed that episodically stretched organoids had significantly more HT2-280+ cells than organoids from static cultures (Figs. 6D, S 7). Increased gene expression of SFTPC in the stretched organoids corroborated these HT2-280 findings (Fig. 6D). Gene expression of FOXJ1 and SCGB1A1 was similar between static and stretched organoids (Fig. 6D), suggesting that episodic stretching does not contribute to the commitment of LPCs to proximal epithelial lung lineages. To exclude that LPC to AEC2 differentiation varied with the stiffness of the Matrigel, we determined the elastic moduli by atomic force microscopy (AFM) micro-indentation. The 80% Matrigel used in the episodic stretch experiments had a similar elastic modulus at room temperature as 50% Matrigel used in all other experiments (150 ± 13 Pa vs 125 ± 12 Pa, n ≥ 17 measurements). Also, organoid size (128.10 ± 7.54 vs 129.12 ± 5.66µm diameter) and SFTPC induction (Fig. 6D vs. Figure 2C) under static conditions did not differ between 80 and 50% Matrigel.