Human apical-out nasal organoids reveal an essential role of matrix metalloproteinases in airway epithelial differentiation

Abstract

Extracellular matrix (ECM) assembly/disassembly is a critical regulator for airway epithelial development and remodeling. Airway organoid is widely used in respiratory research, yet there is limited study to indicate the roles and mechanisms of ECM organization in epithelial growth and differentiation by using in vitro organoid system. Moreover, most of current Matrigel-based airway organoids are in basal-out orientation where accessing the apical surface is challenging. We present a human apical-out airway organoid using a biochemically defined hybrid hydrogel system. During human nasal epithelial progenitor cells (hNEPCs) differentiation, the gel gradually degrade, leading to the organoid apical surfaces facing outward. The expression and activity of ECM-degrading enzymes, matrix metalloproteinases (MMP7, MMP9, MMP10 and MMP13) increases during organoid differentiation, where inhibition of MMPs significantly suppresses the normal ciliation, resulting in increased goblet cell proportion. Moreover, a decrease of MMPs is found in goblet cell hyperplastic epithelium in inflammatory mucosa. This system reveals essential roles of epithelial-derived MMPs on epithelial cell fate determination, and provides an applicable platform enabling further study for ECM in regulating airway development in health and diseases.

Fig. 1. Characterization of hANOs during epithelial differentiation.

A, B Representative pictures of hANOs morphology (by bright field microscopy, at 100x and 200x magnification, respectively) and candidate markers (p63, βIV-tubulin, MUC5AC, FOXJ1, and ZO-1) of hANO development (by multiphoton microscopy) at different stages of hANO differentiation (D10, D17, and D24). Time lapse microscopy videos for hANOs in different differentiation stages shown in Supplementary Movie 1 to 3; Cilia beating patterns of hANOs shown in Supplementary Movie 4. C Gel degradation and F-CHP at different stages of hANO differentiation (D10, D17, and D24). D Collagen degradation was analyzed by quantifying the ratio of F-CFP/Collagen gel signals; the Mann–Whitney test was used in comparison analysis; data present the median with interquartile range, n = 8 experiments per condition; the experiment was independently performed in three organoid lines from three different donors; ns, not significant.

Fig. 2. Molecular characteristics of hANO development.

A, B PCA plots show distinct molecular profiles during hANOs differentiation. C Volcano plots show the differentially expressed genes in differentiating (D17) or differentiated (D24) cells versus proliferated cells (D10). Differentially expressed genes in different stages of hANOs shown in Supplementary Data 1. D Biological process, KEGG analysis, and GSEA analysis demonstrate the relevant functional terms in different differentiation stages of hANOs. E Heatmap demonstrates key genes associated with airway epithelial proliferation and differentiation based on RNAseq analysis. The values in heatmaps are the log2-scaled RNA-seq read counts. n = 3 experiment per condition; the experiment was independently performed in three organoid lines from three different donors.

Fig. 3. Expression and activity of MMPs during hANO development.

A Heatmap shows expression levels of MMP7, MMP9, MMP10 and MMP13 based on RNA-seq data. B mRNA levels of MMP7, MMP9, MMP10, and MMP13 were analyzed in hANOs at D10, D17 and D24 by qPCR assay; the experiment was independently performed in five organoid lines from five different donors. C Protein levels of MMP7, MMP9, MMP10, and MMP13 were analyzed in hANOs at D10, D17 and D24 by Luminex assay; the experiment was independently performed in four organoid lines from four different donors. D Pan-MMP activity was analyzed in hANOs at D10, D17, and D24, Pan-MMP activity was analyzed in hANOs at D10, D17, and D24; NC-1 and NC-2 were negative controls in which the organoids treated with culture medium and assay buffer, respectively; while PC was the positive control in which the organoids treated with APMA pre-treated MMP9 recombinant protein; RFU relative fluorescence units; data present the mean with SEM, n = 5 experiments per condition; the experiment was independently performed in five organoid lines from five different donors. The Friedman test was used in comparison analysis in (B) and (C); the 2way ANOVA with Tukey’s multiple comparison test was performed in (D).

Fig. 4. Characterization of hANOs treated with MMP inhibitor.

A MMP activity was measured in actinonin treated and untreated hANOs, data present the mean with SEM, n = 4 experiments per condition; the experiment was independently performed in three organoid lines from three different donors. B Representative pictures of candidate cellular markers (βIV-tubulin and MUC5AC) of hANO (by multiphoton microscopy and immunofluorescent microscopy) in differentiated hANOs (D24) treated with actinonin versus untreated organoids. C, D βIV-tubulin⁺ cells, MUC5AC⁺ cells and mRNA expression of FOXJ1 and MUC5AC were compared in actinonin-treated to untreated hANOs; for positive cell analysis, data present the median with interquartile, n = 9 experiments per condition; for qPCR analysis, data present the mean with SD, n = 6 experiments per condition; the above two experiments were independently performed in three organoid lines from three different donors. E, F Gel degradation and F-CHP in differentiating (D17) and differentiated (D24) hANOs treated with actinonin versus untreated organoids. G Collagen degradation was analyzed by quantifying the ratio of F-CFP/Collagen gel signals, data present the median with interquartile, n = 9 experiments per condition; the experiment was independently performed in three organoid lines from three different donors. The 2way ANOVA with Tukey’s multiple comparison test was performed in (A); the Mann–Whitney test was used in comparison analysis in (C), (D) and (G); ns not significant.

Fig. 5. Molecular characteristics of hANOs treated with actinonin.

A, B PCA plots and Heatmap show distinct molecular profiles in actinonin-treated versus untreated hANOs; the values in heatmaps are the log2-scaled RNA-seq read counts. C Volcano plots show the differentially expressed genes in actinonin-treated versus untreated hANOs. Differentially expressed genes in actinonin-treated versus untreated hANOs shown in Supplementary Data 2. D Biological process and GSEA analysis demonstrates the changes of key relevant functional terms in actinonin-treated versus untreated hANOs. E Heatmap demonstrats key genes associated with ciliated cells and goblet cells in hANOs treated with and without actinonin during epithelial differentiation. n = 3 experiments per condition; the experiment was independently performed in three organoid lines from three different donors.

Fig. 6. Evaluation of basement membrane and MMP expression in normal epithelium and epithelium with goblet cell hyperplasia in nasal mucosa.

Representative pictures of MMP7, MMP9, MMP10, and MMP13 in normal and remodeled nasal epithelium. Thickness of basement membrane and MMP protein levels were compared between normal and goblet cell hyperplastic epithelium. Data present the median with interquartile, n = 14 experiments per group; the experiment was independently performed in tissues from five CRS patients and five control subjects, respectively. The Mann–Whitney test was used in comparison analysis. ns not significant.

Fig. 7. Characterization of hANOs treated with MM9 or MMP13 specific inhibitor, and neutralizing antibody of MMP7 or MMP10.

A–D Representative pictures of gel degradation and candidate cellular markers (βIV-tubulin and MUC5AC) of hANO (by multiphoton microscopy and immunofluorescent microscopy) in differentiated hANOs (D24) treated with MMP9 or MMP13 specific inhibitor, and MMP7 or MMP10 neutralizing antibody versus untreated organoids. E, F βIV-tubulin⁺ cells, MUC5AC⁺ cells, and mRNA expression of FOXJ1 and MUC5AC were compared in hANOs treated with MMP9 or MMP13 specific inhibitor, and MMP7 or MMP10 neutralizing antibody versus untreated hANOs, for positive cell analysis, data present the median with interquartile, n = 9 experiments per condition; for qPCR analysis, data present the mean with SD, n = 5 experiments per condition. The experiment was independently performed in three organoid lines from three different donors. The Mann–Whitney test was performed in (E); the unpaired t test was used in comparison analysis in (F). ns not significant.

Fig. 8. Diagram of MMP mechanism on ECM degradation associated with epithelial ciliation and polarity by using current hANO system.

Schematic diagram shows the comparison between CAH gel based hANO and Matrigel based airway organoids, and the roles of MMPs in airway organoid development.