Esophageal tissue and / or organoid compositions and methods for their preparation

Abstract

The present disclosure relates to methods for converting mammalian definitive endoderm (DE) cells into specific tissues or organs by directed differentiation. In particular, the present disclosure relates to the formation of esophageal tissue and / or organoids formed from differentiated definitive endoderm.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the priority and benefit of U.S. Provisional Application 62 / 570,182, filed Oct. 10, 2017, by James Wells, the content of which is incorporated in its entirety for all purposes.

[0003] Statement Regarding Federally Sponsored Research

[0004] This invention was made with government support under NIH grant number P01HD093363. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0005] The esophagus actively promotes the passage of food from the oral cavity and pharynx into the stomach. It consists of a stratified squamous epithelium, a muscular layer, and an enteric nervous system that senses distension and controls peristalsis. Congenital disorders (e.g., esophageal atresia) are caused by genetic mutations that result in a narrowed or discontinuous lumen. Other diseases affect the esophagus later in life, such as esophageal cancer, eosinophilic esophagitis, achalasia, and other motility disorders. Tracheal and esophageal diseases are common in humans and difficult to accurately model in mice. Despite the prevalence of the above disease states and because of the substantial differences in tissue architecture between the mouse and human esophagus, there is a need in the art for human esophageal tissue models for research. The present disclosure addresses one or more of the foregoing needs in the art. SUMMARY OF THE INVENTION

[0006] The present disclosure relates to methods for converting mammalian definitive endoderm (DE) cells into specific tissues or organs by directed differentiation. In particular, the present disclosure relates to the formation of esophageal tissue and / or organoids formed from differentiated definitive endoderm. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Those skilled in the art will understand that the drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of the teachings in any way.

[0008] Figures 1A - 1KSpecification of anterior foregut fate by modulating Wnt and retinoic acid signaling during foregut spheroid development. (1A) Experimental protocol for patterning foregut spheroids along the anterior-posterior axis by manipulating the duration of Wnt activation (chiron-chr). (1B-1C) qPCR analysis of the patterning of foregut spheroids with different durations of chiron treatment, as measured by (1B) the foregut marker SOX2 and the mid / hindgut marker CDX2, and (1C) the anterior foregut (“AFG”) marker HNF1B, and the posterior foregut markers PROX1 and HNF6. (D-E) Whole-mount immunofluorescence (“IF”) analysis of HNF1B, SOX2, and CTNNB1 in neonatal spheroids (day 6) treated with chiron for 1 day (1D) and 3 days (1E). (1F) Experimental protocol for patterning foregut spheroids along the anterior-posterior axis using retinoic acid (RA). (1G) Effect of altering the duration of RA treatment on 3-day-old foregut spheroids, as measured by SOX2, TP63 (ΔN isoform), GATA4, and PDX1. (1J-1K) IF analysis of the early esophageal markers SOX2 and p63 in untreated spheroids (1I) and spheroids treated with RA for 1 day (1J) or 4 days (1K). 1l-1, 1J-1, and 1K-1 show p63 staining alone. (1H) Quantification of the percentage of SOX2+ and p63+ epithelial cells in each spheroid. Scale bar = 25 μm. See the Quantification and Statistical Analysis section for details. See also Figure 8 and Figure 9A -R.

[0009] Figures 2A - 2I Anterior foregut spheroids have esophageal respiratory capacity. (2A) Schematic diagram depicting the experimental protocol for patterning AFG spheroids along the dorsal-ventral axis. (2B) Current simplified model of cues guiding the dorsal-ventral patterning of the AFG in mouse and frog embryos. (2C-2G) qPCR analysis of 3-day-old spheroids (day 9) treated with Noggin for 3 days, untreated (-ctrl), or chiron and BMP4 (10 ng / mL), using the dorsal markers SOX2 and MNX1 (2C+2E), the respiratory marker NKX2-1 (2D), the ΔN splice variant of TP63 (2F), and the stratified squamous epithelial marker KRT4 (2G). (2H-2I) IF staining of SOX2, NKX2-1, CDH1, and nuclei (DAPI) in spheroids treated with Noggin (2H) compared to chiron + BMP4 (2I). Scale bar = 25 μm. See the Quantification and Statistical Analysis section for details. See also Figures 10A - 10J 。

[0010] Figures 3A - 3Y。The dorsal anterior foregut spheres form organoids that contain a stratified squamous epithelium expressing esophageal markers. (3A) Schematic diagram depicting the differentiation of DE into human esophageal organoids (HEO). (3B - 3F) Bright - field images depicting the growth of nascent spheres into HEO. (G - R) IF analysis by the transcription factors Sox2 and p63 (3G - 3I), the epithelial marker Krt8 compared to Krt14 (3J - 3O), and the suprabasal marker Krt13 (3P - 3R), comparison of E17.5 esophagus (G, J, M, K) with 1 - month - old and 2 - month - old HEO (3H - 3I, 3K - 3L, 3N - 3O, 3Q - 3R). (3S - 3V) qPCR analysis of the identity and maturation of 1 - month - old and 2 - month - old esophageal organoids by the stratified squamous epithelium markers p63, KRT5, KRT13, IVL, CRNN, compared to human gastric and intestinal organoids (HGO and HIO) and pediatric esophageal biopsies. (3W) Unsupervised hierarchical clustering of 2 - month - old HEO compared to various biopsies of the gastrointestinal tract. (3X) Principal component analysis of 1 - month - old HIO, HGO, and HEO. (3Y) Heatmap of log2 - transformed normalized TPM values of selected genes (esophagus, stomach, intestine) averaged across replicates. SSE = stratified squamous epithelium; b = basal; sb = suprabasal; scale bar = 500 μm (3B - 3F), 50 μm (3G - 3L), 100 μm (3O - 3R), and 25 μm (3O - 1 - 3R - 1). See the Quantitative and Statistical Analysis section for details. Also see Figures 11A - 11CC 。

[0011] Figures 4A - 4BB。The HEO contains progenitor cells that give rise to differentiated stratified squamous epithelium. (4A - 4B) H&E staining was used to compare 7-week-old HEO with organotypic rafts generated using HEO derived from keratinocytes. (4C - 4N) 7-week-old HEO was compared with organotypic rafts by IF analysis of the transcription factors SOX2 and p63 (4C - 4D), the basal marker KRT14 (4E - 4F), the suprabasal keratins KRT4 (4G - 4H) and KRT13 (4I - 4J), and the differentiation markers IVL, CRNN, and FLG (4K - 4N). (4O - 4U) Esophageal biopsies, 7-week-old HEO, keratinocyte-derived HEO, and organotypic rafts were subjected to qPCR analysis for SOX2 and TP63 (4O), KRT5 (4P), KRT14 (4Q), KRT4 and KRT13 (4R), IVL (4S), CRNN (4T), and the esophageal-specific marker TMPRSS11A / D (4U). (4V) Protocol for the EdU pulse-chase labeling experiment in HEO. (4W - 4Z) IF images of HEO at various time points after labeling. (4AA - 4BB) Analysis of the IF images using 2D histograms of P63 intensity versus EdU intensity (4AA) and 1D histograms of the percentage of total EdU-labeled cells versus distance from the epithelial basal (4BB). b = basal; sb = suprabasal. Scale bars = 50 μm (C - N), 100 μm (4A - 4B, 4S - 4V). See the Quantitative and Statistical Analysis section for details. See also Figures 12A - 12R 。

[0012] Figures 5A - 5L 。Early endodermal deletion of Sox2 results in esophageal hypoplasia in mice. (5A - 5d) IF analysis of Sox2 and Nkx2-1 in control embryos (Sox2 fl / fl ) and Sox2 conditional endodermal knockout embryos (Sox2-DE-LOF, FoxA2 CreER ; Sox2 fl / fl ) from pregnant dams gavaged with tamoxifen at 6.5 dpc. Embryonic sections at E9.5 (5A - 5B) and whole-mount IF at E11.5 (5C - 5D), where overlaid images highlight the endoderm. (5E - 5F) IF images of sections with relative sections indicated in the whole-mount images (5C - 5D) for Nkx2-1 (5E) and p63 (5F). Insets show only the Sox2 channel (left) and the green / right (Nkx2-1 or p63) channel. (5G - 51H) By E10.5 Sox2 cKO from pregnant dams gavaged at 8.5 dpc (Sox2 CreER / fl) Cleaved Caspase 3 staining in embryos for analysis of cell death. The boxed area is magnified and shown in (5G-1-5H-1), with the endoderm outlined in white and only cleaved Caspase 3 shown. (5I-5L) IF analysis of E11.5 mouse control and Sox2 cKO embryos (Sox2 CreER / fl ) from pregnant dams gavaged at 9.5 dpc. (5I and 5J) Whole mount IF of Nkx2-1 and Foxa2 in the foregut from lateral and frontal projections. (5K and 5L) Sections of the E11.5 foregut corresponding to their relative positions in the whole mount IF projections (5I-5J), stained for Nkx2-1 (5K) and p63 (5J), with yellow arrows pointing to the mutant esophagus. Scale bars are 50 μm in all IF sections and 100 μm in all IF whole mount projections. See the Quantitative and Statistical Analysis section for details. fg = foregut, dfg = dorsal foregut, vfg = ventral foregut, eso = esophagus, tr = trachea, br = bronchus, st = stomach. Also see Figure 13 A-13F.

[0013] Figures 6A - 6T . Sox2 inhibits respiratory fate and promotes dorsal (esophageal) lineage. (6A-6F) In situ hybridization of nkx2-1 in control (6A, 6C, 6E) or Sox2 MO-injected (6B, 6D, 6F) Xenopus endoderm explants analyzed at stage NF35 treated with Bio (GSK3β inhibitor) and Bio + BMP4. (6G) Schematic diagram depicting the experimental protocol for generating human dorsal (Noggin) and ventral (BMP) AFG cultures. +SOX2 indicates tet-inducible SOX2, while -SOX2 indicates SOX2 CRISPRi. (6H-6N) Analysis of day 9 AFG cultures patterned along the dorsal-ventral axis using Dox-inducible CRISPRi at days 3-9, with or without SOX2 knockdown in dorsal cultures; (6H-6K) IF staining of the cultures for SOX2 and NKX2-1 and quantification in (6L). (6M-6N) qPCR analysis of SOX2 and NKX2-1 in response to these patterning conditions. (6O-6T) Doxycycline-induced exogenous SOX2 expression in day 8 abdominal cultures and analysis at day 9. (6O-6R) IF staining of the cultures for NKX2-1 and HA-SOX2; and (6S-6T) qPCR analysis of SOX2 and NKX2-1 in response to patterning conditions. Scale bars are 50 μm for IF images and 200 μm for Xenopus explant images. See the Quantitative and Statistical Analysis section for details.

[0014] Figures 7A - 7L. Sox2 regulates the expression of secreted Wnt antagonists and Wnt signaling activity in the dorsal foregut endoderm. (7A) Clustered heatmap of differentially expressed genes from RNA sequencing of day 9 dorsal (+Noggin) or ventral (+BMP4) AFG cultures, with (+dox) and without SOX2 CRISPR interference (CRISPRi). (7B) Venn diagram analysis of genes upregulated in dorsal and ventral cultures compared to genes that are increased or decreased after knockdown of SOX2 by CRISPRi. (7C) Gene ontology (GO) term analysis of biological processes for genes positively regulated by SOX2. (7D) Number of genes enriched in dorsal and ventral cultures and whether their expression is SOX2-dependent. (7E) Gene set enrichment analysis of the gene ontology term "regulation of Wnt signaling pathway", with red indicating higher expression and blue indicating lower expression. (7F-7G) In situ hybridization of the Wnt-responsive gene Axin2 to the E9.5 mouse anterior foregut of (7F) control (Sox2 fl / fl ) and (7G) Sox2-DE-LOF (FoxA2 CreER ; Sox2 fl / fl ) embryos. (7H-7I) In situ hybridization of Axin2 to the E10.5 mouse embryonic foregut of (7H) control (Sox2 fl / + ) and (7I) Sox2 cKO (Sox2 CreER / fl ) embryos obtained from dams gavaged at 8.5 dpc. The number of embryos analyzed is shown in the upper left. The boxed regions (7F-7I) highlight the dorsal foregut region. (7J) qPCR analysis of AXIN2 in day 9 dorsal and ventral foregut cultures with or without exogenous expression of SOX2. (7K) Plotted TPM values of the Wnt antagonists SFRP1, SFRP2, and DKK1 from RNA-seq of AFG cultures. (7L) Proposed model for the role of Sox2 in the dorsal-ventral patterning of the anterior foregut. Scale bar = 100 μm. See Materials and Methods and the Quantitative and Statistical Analysis sections for details. Also see Figures 14A - 14D .

[0015] Figures 8A - 8VModulate the duration of Wnt and retinoic acid signaling to coordinate foregut patterning along the anteroposterior axis. (8A) Schematic diagram depicting the experimental protocol for patterning foregut spheroids along the anteroposterior axis using CHIR99021 (chiron, or chr) and retinoic acid (RA). (8B-8G) qPCR analysis of day 6 spheroids generated by varying the duration of chiron treatment, with or without retinoic acid treatment, for (B) anterior and posterior foregut markers HNF1B, (8C-8D) posterior foregut markers PROX1 and HNF6, (8E) hindgut marker CDX2, and (8F-8G) pharyngeal markers PAX9 and OTX2. (8H-8N) Comparison of chiron versus Wnt3a treatment of endoderm by qPCR analysis of (8H) foregut marker SOX2, (8I) HNF1B, PROX1, HNF6, and CDX2, (8j) Wnt target genes AXIN2, LEF1, and TCF1, and (8K) epithelial and neural markers CDH1 and NESTIN. Spheroids generated with a one-day chiron treatment had the same gene expression profile as those generated with two days of Wnt3a. (8L-8N) Bright-field imaging of nascent spheroids generated by chiron versus Wnt3a treatment showed that the efficiency of spheroid generation was not affected under different conditions. (8O-8R) Analysis of day 9 spheroids generated by varying the duration of retinoic acid treatment starting on day 5. (8O) qPCR analysis of retinoic acid targets HOXA1, HOXB1, CYP26C1. (8S) Schematic diagram depicting the experimental protocol for modulating retinoic acid signaling using the synthetic inhibitor DEAB. (8T-8U) (8T) Foregut markers at day 6, (8U) dorsal anterior foregut markers SOX2 and TP63 (ΔN isoform) at day 9, and (8V) RA targets HOXA1 and HOXB1 at day 9 by qPCR analysis. Scale bars are 500 μm in (8L-8N) and 50 μm in (8P-8R). Error bars represent SD. For two-tailed t-tests, *p < 0.05, **p < 0.01, and ***p < 0.001.

[0016] Figures 9A - 9R。Early regulation of Wnt and retinoic acid signaling affects subsequent differentiation into human esophageal organoids. (9A) Schematic depicting the experimental protocol for generating organoids starting from foregut spheroids treated with chiron for 1 or 3 days. (9B–9C) qPCR analysis of organoids at day 35 (1 month old) for (9B) the stratified squamous epithelium markers KRT5, KRT13, and KRT13, and (9C) the posterior foregut markers GATA4 and PDX1. (9D–9O) Analysis of day 35 (1 month old) organoids resulting from altering the duration of retinoic acid treatment starting at day 5. (9D) qPCR analysis of the anterior foregut basal transcription factors SOX2 and the ΔN isoform of TP63, (9E) the antral stomach and pancreas marker PDX1, and (9F) the stratified squamous epithelium markers KRT5, KRT13, and IVL. Immunofluorescence analysis of (9G–9I) SOX2 and p63, (9J–9L) KRT13, and (9M–9O) PDX1 in 1-month-old organoids. (9P) Schematic describing the experimental protocol for modulating retinoic acid signaling using the synthetic inhibitor DEAB. (9Q–9R) qPCR analysis of (9Q) the esophageal basal markers SOX2 and TP63 and (9R) the stratified squamous epithelium markers KRT5, KRT13, and IVL in day 35 organoids. These data represent two independent experiments with n = 3 wells per experiment. Scale bar = 100 μm. Error bars represent SD. For two-tailed t tests, *p < 0.05 and **p < 0.01.

[0017] Figures 10A - 10J 。Pattern formation of the foregut into anterior foregut spheroids under these culture conditions does not require TGFβ inhibition. (10A) Schematic depicting the experimental protocol for testing the requirement for TGFβ signaling in anterior–posterior patterning of the foregut. (10B–10F) qPCR analysis of day 6 anterior foregut spheroids treated with and without Wnt3a and the TGFβ inhibitor (SB431542, 10 μM) for (10B–10C) the foregut marker SOX2 and the hindgut marker CDX2, (10D) the foregut marker HNF1B, and (10E–10F) the posterior foregut markers PROX1 and HNF6. (10G) Schematic depicting the experimental protocol for testing the ability of anterior foregut spheroids treated with and without Wnt3a or the TGFβ inhibitor SB431542 to respond to respiratory induction. (H–J) NKX2-1 in day 9 spheroids analyzed by (10H–10I) immunofluorescence and (10J) qPCR. Scale bar = 50 μm. Error bars represent SD. For two-tailed t tests, *p < 0.05 and **p < 0.01.

[0018] Figures 11A - 11CCRobust growth of human esophageal organoids and comparison with mouse embryonic esophageal development. Improved sphere outgrowth efficiency of organoids treated with FGF10 from day 6 - 13, as shown by (A - D) bright - field imaging and (11E) quantitative analysis of images. (11F - 11Q) Comparative analysis by immunofluorescence staining of mouse embryonic esophagus at E12.5 (11F, 11J, 11N) and E14.5 (11G, 11K, 11O) and human esophageal organoids (HEO) at week 2 (11H, 11L, 11P) and week 3 (11I, 11M, 11Q). (R11) Gene expression of stratified squamous epithelial markers over time in mouse esophagus; public data obtained from GEO dataset GSE34728 (Chen et al., 2012). (11S) qPCR analysis of stratified squamous epithelial markers at various time points during the differentiation of definitive endoderm into human esophageal organoids. (11T) Quantification of the percentage of area of KRT5 - and KRT13 - positive epithelium in HEO at day 62. Each point is an individual organoid, and sub - panels “a” and “b” are representative images of different organoids depicted in this figure. (11U) Quantification of the percentage of nuclei of SOX2 - and p63 - positive epithelium in HEO at day 62. Each point is an individual organoid. (11V - 11CC) Immunofluorescence analysis of 1 - month - old HEO in different cell lines tested, examining esophageal - enriched markers SOX2 and p63 (11V - 11Y) and KRT13 (11Z - 11CC). Scale bars are 500 μm in (11A - 11D), 50 μm in (11F - 11Q, 11V - 11CC), and 100 μm in (11T - 11U). Error bars represent SD. For two - tailed t - tests, *p < 0.05, **p < 0.01, and ***p < 0.001.

[0019] Figures 12A - 12RAlternative methods for human esophageal organoid maturation and expansion. (12A - 12F) Analysis of HEOs grown for 2 months in the renal capsule of immunodeficient mice by immunofluorescence images of early (KRT8) and differentiated (KRT13, KRT14, and IVL) esophageal - specific markers (12A - 12E) and H&E (12F). (12G - 12R) Analysis of HEOs mechanically passaged (dissociated and recultured) twice. (12G - 12N) IF images of the transcription factors SOX2 and p63, (12I - 12J) immature (KRT8) and basal markers (KRT14), (12K - 12L) basal (KRT5) and suprabasal (KRT13) markers, and (12M - 12N) suprabasal differentiation markers KRT4, CRNN, and IVL in passaged organoids. (12O - 12R) qPCR analysis comparing the transcriptional markers SOX2 and TP63, (12P) stratified squamous markers KRT5, KRT13, IVL, and (12Q - 12R) patterning markers of the lung (NKX2 - 1), stomach (GATA4), and intestine (GATA4 and CDX2) in passaged HEOs versus normal HEOs and gastric organoids (hAGO). For two - tailed t - tests, *p < 0.05, **p < 0.01, and ***p < 0.001.

[0020] Figure 13 A - 13V. Endoderm or widespread Sox2 knockout after gastrulation led to a similar phenotype of esophageal hypoplasia. (13A - 13B) Whole - mount immunofluorescence (IF) analysis of the dorsal marker Sox2 (red) and the respiratory marker Nkx2 - 1 (green) in E9.5 control (Sox2 fl / fl ) and Sox2 - DE - LOF (FoxA2 CreER ; Sox2 fl / fl ) embryos. (13C - 13D) Whole - mount IF analysis of Sox2 and Nkx2 - 1 in E10.5 control (Sox2 fl / fl ) and Sox2 - DE - LOF (FoxA2 CreER ; Sox2 fl / fl ) embryos. White arrows highlight normal versus ectopic Nkx2 - 1 expression. (13E - 13F) IF analysis of the apical marker aPKC (green) in foregut sections of E11.5 embryos. (G - H) E11.5 control (Sox2 fl / + ) and Sox2 - cKO (Sox2 CreER / fl)Whole-mount IF analysis of Nkx2-1 in embryos. (13I-13L) Immunofluorescence analysis of E11.5 embryos (similar to those in 13E-13F) for (13I-13J) Sox2 and Nkx2-1 and (13K-13L) Sox2 and p63. (13M-13T) IF analysis of E10.5 control (Sox2 fl / + ) and Sox2-cKO (Sox2 CreER / fl ) embryos, for epithelial morphology from the anterior axis (13M, 13N) to the posterior axis (13S, 13T). (13S, 13T) Quantification of (13U) cleaved Caspase 3 and (13V) Ki67 IF staining for cell death in the anterior foregut of E10.5 mouse embryos during dissection of the dorsal and ventral foregut in control (Cre-, Sox2 fl / + ) and Sox2 cKO (Cre+, Sox2 CreER / fl ) embryos from dams gavaged at 8.5 dpc. Scale bars are 100 μm in (13A-13D, 13G-13H), 50 μm in (13I-13T), and 25 μm in (13E-13F). For Sox2-DE-LOF embryos, n = 3 embryos per genotype at E9.5, and n = 2 embryos per genotype per analysis at E11.5 (minimum of 2 litters harvested per analysis and time point). For Sox2-driven Sox2 cKO embryos, n = 3 embryos per genotype. Error bars represent SD. *For two-tailed t-test, *p ≤ 0.05. fg = foregut, dfg = dorsal foregut, vfg = ventral foregut, ph = pharyngeal endoderm, eso = esophagus, r = respiratory progenitor, thy = thyroid, tr = trachea, br = bronchus, st = stomach.

[0021] Figures 14A - 14DAnalysis of loss or gain of function of Sox2 in human cultures. (14A) Principal component analysis of transcriptomes generated from day 9 anterior foregut cultures (with and without SOX2) patterned along the dorsal-ventral axis (with or without Dox treatment of the CRISPR interference construct activated). Dorsal compared to ventral anterior foregut (dAFG compared to vAFG) is represented as chr+Nog and chr+BMP4 respectively. Knockdown is indicated by +Dox. (14B) TPM values of SOX2 and NKX2-1. (14C) qPCR analysis of SFRP2 in day 9 anterior foregut cultures patterned along the dorsal (dAFG) and ventral (vAFG) axes, including induction of exogenous HA-tagged SOX2 in ventral cultures by Dox treatment at day 8. (14D) Genomic browser view of Sox2 peaks at the SFRP2 locus in hPSC-derived endoderm (GSM1505764) and mesendoderm (GSM1505767) from the GEO dataset GSE61475 (Tsankov et al., 2015). Scale bar = 500 μm. Error bars represent SD. *p≤0.05 for two-tailed t-test.

[0022] Figures 15A - 15E Role of Sox2 in esophageal development after anterior foregut isolation. (15A) Schematic of mouse breeding and tamoxifen administration protocol. (15B) Confocal immunofluorescence (IF) images of esophageal sections at E14.5 (left), E17.5 (middle), and P7 (right), pregnant dams gavaged at 11.5 dpc (left), 14.5 dpc (middle), pups gavaged at P1 (right). Sections were stained for E-cadherin to visualize the epithelium, Sox2 and Nkx2-1 for respiratory identity. (15C) IF images of various markers in the E17.5 esophagus from pregnant dams gavaged at 11.5 dpc: p63 and Sox2 to confirm esophageal identity, basal marker Krt14, suprabasal marker Krt13, immature or columnar marker Krt8, proliferation marker Ki67. The green arrow in the bottom middle right panel highlights suprabasal Ki67 staining. (15D) High-magnification IF image of E-cadherin in the E17.5 esophagus from pregnant dams gavaged at 11.5 dpc. (15E) IF images of patterned markers in the E17.5 esophagus from pregnant dams gavaged at 11.5 dpc: intestinal marker Cdx2, gastric / intestinal markers Gata4 and Pdx1, respiratory marker Nkx2-1, and smooth muscle marker Desmin. Yellow arrows highlight rare Nkx2-1 positive cells in mutant esophagi. Scale bars are 100 μm in (15B, 15C, 15E) and 25 μm in (15D).

[0023] Figures 16A - 16GModeling the effect of Fanconi anemia (FANCA loss) in HEO. (16A) Schematic diagram depicting the experimental protocol for generating HEO with (+dox) or without FANCA. **Note: Hydroxyurea (HU) was used for Western blot analysis in (16E). (16B) IF images of AFG monolayers on day 6 stained for the foregut marker SOX2 (green) and the hindgut marker CDX2 (red). (16C) Bright-field images of HEO with or without doxycycline treatment at week 0 (day 6), week 2 (day 20), and week 4 (day 35) of organoid growth. (16D) IF images of SOX2 and the proliferation marker KI67 in HEO. (16E) Western blot analysis of FANCA and FANCD2 to confirm dox-induced expression and function of FANCA protein. (16F) Size quantification of 2-week-old HEO from bright-field images. (16G) Quantification of proliferating (KI67+) epithelial cells in 1-month-old (day 36) HEO. Scale bars are 100 μm in (16B, 16D) and 250 μm in (16C). For Mann-Whitney nonparametric test, *p ≤ 0.05 and **p ≤ 0.01. DE = definitive endoderm; AFG = anterior foregut; HEO = human esophageal organoid.

[0024] Figures 17A - 17LInduction of CDX2 in human foregut and HEO cultures. (17A) Schematic diagram depicting the experimental protocol for inducing CDX2 in foregut and HEO cultures. (17B) Schematic diagram of the transduced lentiviral vector for inducing CDX2 after doxycycline administration. (17C) IF analysis of the foregut marker SOX2 and the hindgut marker CDX2 in the anterior foregut monolayer on day 6 treated with different levels of doxycycline (20, 100, and 500 ng / mL). (17D + 17E) Quantification of the IF images (as in 17C) by scatter plot of SOX2 intensity compared to CDX2 intensity. The vertical line in the scatter plot is the "gate" defining CDX2+ versus CDX2− cells. (17E) Bar graph of the percentage of CDX2+ cells. (17F) IF analysis of the stratified squamous markers SOX2 and p63 and the hindgut (induced) marker CDX2 in 1-month-old HEOs treated with or without doxycycline. (17G - 17L) qPCR analysis of the hindgut markers (17G) CDX1, (17H) CDX2, (17I) CDH17, (17J) MUC2 and the foregut / stratified squamous markers (17K) SOX2 and (17L) p63. Scale bar = 100 μm. For Student's t-test with two-tailed distribution assuming unequal variances, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001. DE = definitive endoderm; FG = foregut; AFG = anterior foregut; HEO = human esophageal organoids.

[0025] Figures 18A - 18I. Late induction of CDX2 in HEOs leads to repression of esophageal transcription factors SOX2 and p63. 18A. Schematic illustration of the experimental protocol for inducing CDX2 in more mature HEOs (6 - 7 weeks old). 18B. IF images of the stratified squamous markers SOX2 and p63 and the hindgut marker CDX2 in day 58 HEOs treated with (+CDX2) or without doxycycline. (18C - 18E) qPCR analysis of (18C) CDX2, (18D) SOX2, and (18E) p63 in day 58 HEOs. (18F) Scatter plot of CDX2 versus p63 intensity in basal epithelial cells of HEOs, analysis of IF images (e.g., 18B). The vertical line separates p63 - negative (left) and p63 - positive (right) cells. The horizontal line separates CDX2 - negative (bottom), CDX2 - low (middle), and CDX2 - high (top) cells. (18G - 18I) Quantification of IF analysis (see 18B and 18F) of (18G) all CDX2 - high and CDX2 - low basal cells, (18H) CDX2 - high and CDX2 - low basal cells that are SOX2 +, and (18I) CDX2 - high and CDX2 - low basal cells that are p63 +. Scale bar = 100 μm. For Student t - test with two - tailed distribution not assuming equal variance, ***p ≤ 0.0001. DE = definitive endoderm; AFG = anterior foregut; dAFG = dorsal anterior foregut; HEO = human esophageal organoids.

[0026] Figures 19A - 19H . Loss of esophageal differentiation in CDX2 - induced HEOs. (19A) Schematic illustration of the experimental protocol for inducing CDX2 with or without Notch inhibition in HEOs. (19B - 19G) qPCR analysis of the stratified squamous markers (19B) KRT5, (19C) KRT13, and (19D) IVL, (19E) the intestinal epithelial marker CDH17, (19F) the Notch target HES5, and (19G) the BMP target ID1 in d58 HEOs. (19H) IF images of CDH17, KRT5, and KRT13 (upper row); and the differentiated stratified squamous markers CRNN and IVL (lower row) in HEOs treated with doxycycline and the γ - secretase (Notch) inhibitor DAPT. Scale bar = 100 μm. For Student t - test with two - tailed distribution not assuming equal variance, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. DE = definitive endoderm; AFG = anterior foregut; dAFG = dorsal anterior foregut; HEO = human esophageal organoids.

[0027] Figures 20A - 20HHEO treated with IL-13 upregulates IL-13 target genes and increases proliferation. (20A) Schematic diagram depicting the experimental protocol for treating IL-13 in late HEO. (20B–20E) qPCR analysis of known IL-13 target genes (20B) CCL26, (20C) CDS26, (20D) CAPN14, and (20E) SERPINB4 in 62-day HEO treated with IL-13 for 2 days before harvest. (20F) Western blot analysis of SERPINB13, CDH26, and housekeeping protein GAPDS in 50-day HEO treated with IL-13 (100 ng / mL) for 1 week before harvest. (20G) IF images of 62-day HEO treated with IL-13 (100 ng / mL) for 2 weeks and EdU (10 μM) for 2 days before harvest. (20H) Quantification of (20G) the percentage of labeled EdU in all the most basal (basal-most) p63 cells. For qPCR data, *p ≤ 0.05 and **p ≤ 0.01 for two-tailed Student t test with unequal variance assumed. For organoid EdU incorporation quantification, *p < 0.05 for Mann–Whitney nonparametric test. Scale bar = 100 μm. DE = definitive endoderm; AFG = anterior foregut; HEO = human esophageal organoid.

[0028] Figure 21A-L. Impaired differentiation and altered morphology of HEOs treated with IL-13. (21A) IF analysis of 62-day HEOs treated with IL-13 (100 ng / mL) for 2 weeks before harvest. (21B) Western blot analysis of structural and differentiation proteins of 56-day HEOs treated with IL-13 (100 ng / mL) for 1 week before harvest. (21C-21F) qPCR analysis of (21C) IVL, (21D) CRNN, and BMP antagonists (21E) NOG and (21F) FST in 62-day HEOs treated with IL-13 (100 ng / mL) for 2 weeks before harvest. (21G) Structural analysis of 62-day HEOs treated with IL-13 (100 ng / mL) for 2 weeks before fixation by H&E staining (left column) and electron micrographs (right column). (21H-21L) qPCR analysis of (21H) SOX2, (21I) CCL26, (21J) CDH26, (21K) hedgehog target PTCH1, and (21L) BMP target ID3 in 62-day HEOs treated with IL-13 (100 ng / mL) and BMP4 (100 ng / mL). For IF and H&E images, scale bar = 100 μm, and for electron micrographs, scale bar = 6 μm. For Student t-tests with two-tailed distributions not assuming equal variances, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

[0029] Figures 22A - 22H . Induced upregulation of stratified squamous markers by SOX2 in human intestinal organoids (HIOs). (22A) Schematic diagram depicting the experimental protocol for inducing SOX2 in HIO. (22B) Schematic diagram of a transcriptional lentiviral vector that induces HA-tagged SOX2 after doxycycline administration. (22C) IF images of (top) foregut marker SOX2 and hindgut marker CDX2, (middle) anterior foregut marker p63, gastric / intestinal marker PDX1, and HA tag; (bottom) intestinal marker CDH17 and gastric marker CLDN18 in 36-day HIOs treated with or without doxycycline. (22D-22H) qPCR analysis of various regional markers (22D) SOX2, (22E) p63, (22F) PDX1, (22G) CDX2, and (22H) upper basal stratified squamous marker KRT13. Scale bar = 100 μm. For Student t-tests with two-tailed distributions not assuming equal variances, *p ≤ 0.05, **p ≤ 0.01, and ****p ≤ 0.0001. DE = definitive endoderm; HG = hindgut; HIO = human intestinal organoid.

[0030] Figures 23A - 23JActivation of BMP in HEO results in loss of proliferation and differentiation. (23A) Schematic depicting the experimental protocol for activation of BMP signaling in late-stage HEO. (23B) IF images of (left) pSMAD1 / 5 / 9 and SOX2, (middle) p63 and EdU, (right) IVL and CRNN in day 62 HEO treated with or without BMP4 (100 ng / mL). (23C) Quantification of the percentage of labeled EdU in all the most basal epithelial cells in HEO with or without BMP4. (23D-23J) qPCR analysis of various markers in day 62 HEO: (D) SOX2, (23E) p63, (23F) ID3, (23G) KRT5, (23H) KRT13, (23I) IVL, and (23J) CRNN. Scale bar = 100 μm. For qPCR data, *p ≤ 0.05, ****p ≤ 0.0001 for two-tailed Student t test with unequal variance assumed. For organoid EdU incorporation quantification (23C), **p ≤ 0.01 for Mann-Whitney non-parametric test. DE = definitive endoderm; AFG = anterior foregut; dAFG = dorsal anterior foregut; HEO = human esophageal organoid. Detailed implementation

[0031] Definitions

[0032] Unless otherwise specified, those of ordinary skill in the relevant art should understand the terms according to their ordinary usage. In case of conflict, this document including the definitions shall prevail. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. All published documents, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0033] As used herein and in the appended claims, the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes a plurality of such methods, reference to "a dose" includes reference to one or more doses known to those skilled in the art and their equivalents, and so on.

[0034] The terms "about" or "approximately" mean within an acceptable error range of a particular value, as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measuring system. For example, in accordance with the practice in the art, "about" can mean within one or more standard deviations. Alternatively, "about" can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Or, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of a numerical value, preferably within 5-fold, more preferably within 2-fold. Where a particular value is described in the present application and claims, unless otherwise stated, the term "about" should be assumed to mean that the particular value is within an acceptable error range.

[0035] The terms "individual", "host", "subject" and "patient" are used interchangeably and refer to an animal that is the subject of treatment, observation, and / or experimentation. Generally, the term refers to a human patient, however the methods and compositions are equally applicable to non-human subjects, such as other mammals. In some embodiments, the term refers to a human. In other embodiments, the term can refer to a child.

[0036] As used herein, the term "definitive endoderm (DE) cell" refers to one of the three primary germ layers generated through the process of gastrulation.

[0037] As used herein, the term "wnt signaling pathway" refers to the wnt / β-catenin pathway and is a signaling pathway mediated by Wnt ligands and Frizzled cell surface receptors that act through β-catenin.

[0038] As used herein, the term "activator" with respect to a pathway such as the "wnt pathway" refers to a substance that activates the Wnt / β-catenin pathway such that the Wnt / β-catenin target is increased.

[0039] As used herein, the term "FGF signaling pathway activator" refers to a substance that activates the FGF pathway such that the FGF target is increased.

[0040] As used herein, the term "BMP signaling pathway inhibitor" is a substance that interferes with the BMP pathway and reduces the BMP target.

[0041] As used herein, the term "growth factor" refers to a substance that is capable of stimulating cellular processes including but not limited to growth, proliferation, morphogenesis or differentiation.

[0042] As used herein, the term "stable expression" of a marker refers to an expression that does not change upon a change in the growth environment.

[0043] As used herein, the term "totipotent stem cell" (also referred to as "omnipotent stem cell") is a stem cell that can differentiate into embryonic and extraembryonic cell types. Such cells can build a complete, viable organism. These cells are derived from the fusion of an egg and a sperm cell. Cells generated by the first few divisions of a fertilized egg are also totipotent.

[0044] As used herein, the term "pluripotent stem cell (PSC)", often also referred to as a PS cell, includes any cell that can differentiate into almost all cell types, i.e., cells derived from any of the three germ layers (germ epithelium), including the endoderm (inner lining of the stomach, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital), and ectoderm (epidermal tissue and nervous system). A PSC can be a descendant of a totipotent cell, derived from an embryo (including embryonic germ cells), or obtained by forcing the expression of certain genes in a non-pluripotent cell (such as a somatic cell).

[0045] As used herein, the term "induced pluripotent stem cell (iPSC)", often also abbreviated as an iPS cell, refers to a type of pluripotent stem cell that is artificially derived from a normal non-pluripotent cell such as a somatic cell by inducing the "forced" expression of certain genes.

[0046] As used herein, the term "precursor cell" encompasses any cell that can be used in the methods described herein, and one or more precursor cells acquire the ability to self-renew or differentiate into one or more specialized cell types. In some embodiments, the precursor cell is pluripotent or has the ability to become pluripotent. In some embodiments, the precursor cell is treated with external factors (e.g., growth factors) to obtain pluripotency. In some embodiments, the precursor cell can be a totipotent stem cell; a pluripotent stem cell (induced or non-induced); a multipotent stem cell; and a unipotent stem cell. In some embodiments, the precursor cell can be from an embryo, infant, child, or adult. In some embodiments, the precursor cell can be a somatic cell that is treated such that pluripotency is conferred by genetic manipulation or protein / peptide treatment.

[0047] In developmental biology, cell differentiation is the process by which less specialized cells become more specialized cell types. As used herein, the term "directed differentiation" describes the process by which less specialized cells become a specific specialized target cell type. The specificity of the specialized target cell type can be determined by any suitable method that can be used to define or alter the initial cell fate. Exemplary methods include, but are not limited to, genetic manipulation, chemical treatment, protein treatment, and nucleic acid treatment.

[0048] As used herein, the term "cellular component" refers to individual genes, proteins, mRNA-expressed genes, and / or any other variable cellular component or protein activity, such as the degree of protein modification (e.g., phosphorylation), which is typically measured by those skilled in the art in biological experiments (e.g., by microarray or immunohistochemistry). Significant discoveries related to the complex network of biological processes underlying living systems, common human diseases, and gene discovery and structure determination can now be attributed to the application of cellular component abundance data as part of the research process. Cellular component abundance data can help identify biomarkers, distinguish disease subtypes, and identify toxicity mechanisms.

[0049] Pluripotent Stem Cells Derived from Embryonic Cells

[0050] In some embodiments, an important step is to obtain pluripotent or inducible pluripotent stem cells. In some embodiments, pluripotent stem cells are derived from embryonic stem cells, which in turn are derived from the totipotent cells of an early mammalian embryo and are capable of unlimited, undifferentiated proliferation in vitro. Embryonic stem cells are pluripotent stem cells derived from the inner cell mass (early embryo) of a blastocyst. Methods for deriving embryonic stem cells from blastocysts are well known in the art. The human embryonic stem cell line H9 (H9-hESC) is used in the exemplary embodiments described in this application, but those skilled in the art will understand that the methods and systems described herein can be applied to any stem cell.

[0051] Additional stem cells that can be used in embodiments according to the present invention include, but are not limited to, those provided or described in databases hosted by the National Stem Cell Bank (NSCB), the Human Embryonic Stem Cell Research Center at the University of California, San Francisco (UCSF); the WiCell Research Institute's WiSC Cell Bank; the University of Wisconsin Stem Cell and Regenerative Medicine Center (UW-SCRMC); Novocell, Inc. (San Diego, California); Cellartis AB (Gothenburg, Sweden); ES Cell International Pte Ltd (Singapore); the Technion at the Israel Institute of Technology (Haifa, Israel); and stem cell databases hosted by Princeton University and the University of Pennsylvania. Exemplary embryonic stem cells that can be used in embodiments according to the present invention include, but are not limited to, SA01 (SA001); SA02 (SA002); ES01 (HES-1); ES02 (HES-2); ES03 (HES-3); ES04 (HES-4); ES05 (HES-5); ES06 (HES-6); BG01 (BGN-01); BG02 (BGN-02); BG03 (BGN-03); TE03 (13); TE04 (14); TE06 (16); UC01 (HSF1); UC06 (HSF6); WA01 (H1); WA07 (H7); WA09 (H9); WA13 (H13); WA14 (H14).

[0052] More details regarding embryonic stem cells can be found, for example, in Thomson et al., 1998, “Embryonic Stem Cell Lines Derived from Human Blastocysts,” Science 282(5391):1145-1147; Andrews et al., 2005, “Embryonic stem (ES) cells and embryonal carcinoma (EC) cells: opposite sides of the same coin,” Biochem Soc Trans 33:1526-1530; Martin 1980, “Teratocarcinomas and mammalian embryogenesis,” Science 209(4458):768-776; Evans and Kaufman, 1981, “Establishment in culture of pluripotent cells from mouse embryos,” Nature 292(5819):154-156; Klimanskaya et al., 2005, “Human embryonic stem cells derived without feeder cells,” Lancet 365(9471):1636-1641; the respective full texts of which are incorporated herein by reference.

[0053] Induced Pluripotent Stem Cells (iPSC)

[0054] In some embodiments, iPSCs are derived by transfecting certain stem cell-related genes into non-pluripotent cells (e.g., adult fibroblasts). Transfection is typically achieved through viral vectors (e.g., retroviruses). The genes transfected include the master transcriptional regulators Oct-3 / 4 (Pouf51) and Sox2, although other genes have been proposed to enhance the induction efficiency. After 3-4 weeks, a small number of transfected cells begin to become morphologically and biochemically similar to pluripotent stem cells and are typically isolated by morphological selection, doubling time, or by reporter genes and antibiotic selection. As used herein, iPSCs include, but are not limited to, first-generation iPSCs, second-generation iPSCs, and human induced pluripotent stem cells in mice. In some embodiments, a retroviral system is used to convert human fibroblasts into pluripotent stem cells using four key genes: Oct3 / 4, Sox2, Klf4, and c-Myc. In alternative embodiments, a lentiviral system is used to convert somatic cells with OCT4, SOX2, NANOG, and LIN28. Genes whose expression is induced in iPSCs include, but are not limited to, Oct-3 / 4 (e.g., Pou5f1); certain members of the Sox gene family (e.g., Sox1, Sox2, Sox3, and Sox15); certain members of the Klf family (e.g., Klf1, Klf2, Klf4, and Klf5), certain members of the Myc family (e.g., C-myc, L-myc, and N-myc), Nanog, and LIN28.

[0055] In some embodiments, non-viral-based techniques are employed to generate iPSCs. In some embodiments, adenoviruses can be used to deliver the four essential genes into the DNA of mouse skin and liver cells, generating cells identical to embryonic stem cells. Since adenoviruses do not incorporate any of their own genes into the targeted host, the risk of tumor formation is eliminated. In some embodiments, although with very low efficiency, reprogramming can be accomplished with plasmids that do not have any viral transfection system at all. In other embodiments, direct delivery of proteins is used to generate iPSCs, thus eliminating the need for viral or genetic modification. In some embodiments, it is possible to generate mouse iPSCs using a similar methodology: repeated treatment of cells with certain proteins delivered into the cells via polyarginine anchors is sufficient to induce pluripotency. In some embodiments, the expression of pluripotency-inducing genes can also be increased by treating somatic cells with FGF2 under hypoxic conditions.

[0056] More details regarding embryonic stem cells can be found, for example, in Kaji et al., 2009, “Virus free induction of pluripotency and subsequent excision of reprogramming factors,” Nature 458:771-775; Woltjen et al., 2009, “piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells,” Nature 458:766-770; Okita et al., 2008, “Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors,” Science 322(5903):949-953; Stadtfeld et al., 2008, “Induced Pluripotent Stem Cells Generated without Viral Integration,” Science 322(5903):945-949; and Zhou et al., 2009, “Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins,” Cell Stem Cell 4(5):381-384; the entire texts of each are incorporated herein by reference.

[0057] In some embodiments, exemplary iPS cell lines include, but are not limited to, iPS-DF19-9; iPS-DF19-9; iPS-DF4-3; iPS-DF6-9; iPS (Foreskin); iPS (IMR90); and iPS (IMR90).

[0058] For more details on the function of signaling pathways involved in DE development, see, for example, Zorn and Wells, 2009, “Vertebrate endoderm development and organ formation,” Annu Rev Cell Dev Biol 25:221-251; Dessimoz et al., 2006, “FGF signaling is necessary for establishing gut tube domains along the anterior-posterior axis in vivo,” Mech Dev 123:42-55; McLin et al., 2007, “Repression of Wnt / β-catenin signaling in the anterior endoderm is essential for liver and pancreas development. Development,” 134:2207-2217; Wells and Melton, 2000, Development 127:1563-1572; de Santa Barbara et al., 2003, “Development and differentiation of the intestinal epithelium,” Cell Mol Life Sci 60(7):1322-1332; the respective full texts of which are incorporated herein by reference.

[0059] Any method for generating definitive endoderm from pluripotent cells (e.g., iPSC or ESC) is applicable to the methods described herein. In some embodiments, the pluripotent cells are derived from a morula. In some embodiments, the pluripotent stem cells are stem cells. The stem cells used in these methods can include, but are not limited to, embryonic stem cells. Embryonic stem cells can be derived from the inner cell mass of an embryo or from the embryonic genital ridge. Embryonic stem cells or germ cells can be derived from a variety of animal species, including, but not limited to, a variety of mammalian species, including humans. In some embodiments, human embryonic stem cells are used to generate definitive endoderm. In some embodiments, human embryonic germ cells are used to generate definitive endoderm. In some embodiments, iPSCs are used to generate definitive endoderm.

[0060] Tracheal and esophageal diseases are prevalent in humans and are difficult to accurately model in mice. Therefore, the applicant established a three-dimensional organoid model of esophageal development through the directed differentiation of human pluripotent stem cells. Sequential manipulation of the BMP, WNT, and RA signaling pathways allowed patterning of definitive endoderm into foregut, anterior foregut (AFG), and dorsal AFG spheres. Dorsal AFG spheres grown in 3D matrix formed human esophageal organoids (HEOs), and HEO cells could be converted to two-dimensional cultures and grown into esophageal organotypic rafts. In both configurations, esophageal tissue had proliferative basal progenitor cells and differentiated stratified squamous epithelium. Using HEO cultures to model human esophageal birth defects, the applicant determined that Sox2 promoted esophageal specification, in part by inhibiting Wnt signaling and promoting survival in dorsal AFG. Consistently, Sox2 ablation caused esophageal hypoplasia in mice. Thus, HEOs provide a powerful platform for modeling human pathology and tissue engineering.

[0061] Human tissue organoids differentiated from pluripotent stem cells (PSCs) or obtained directly from organs have proven to be excellent models of tissue physiology and pathology (McCauley and Wells, 2017). Generally, the process of converting PSCs into organ cell types relies on recapitulating the stepwise differentiation of organogenesis, including the formation of definitive endoderm (DE), anteroposterior patterning into foregut, midgut, and hindgut, organ specification, and differentiation into organ-specific lineages. This approach has been used to generate human anterior and posterior endoderm organoids, including the respiratory tract, stomach, small intestine, and colon (Chen et al., 2017; Dye et al., 2015, 2016; McCracken et al., 2014, 2017; Múnera et al., 2017; Spence et al., 2011). However, human PSC-derived esophageal tissue has not been reported. Dual inhibition of BMP and TGFβ after DE induction produced anterior foregut (AFG); however, this produced a mixture of tissues including pharynx, esophagus, and respiratory endoderm (Green et al., 2011; Kearns et al., 2013; Longmire et al., 2012). This suggests the need for a more refined patterning approach based on the pathways controlling esophageal development to direct the specific differentiation of PSCs into the esophagus.

[0062] Multiple signaling pathways direct the differentiation and morphogenesis of the developing esophagus. The esophageal epithelium is derived from definitive endoderm (DE), a two-dimensional sheet of cells that forms during gastrulation (Zorn and Wells, 2007). The DE is then patterned along the anterior-posterior axis via Wnt, BMP, and FGF signaling and forms the primitive gut tube, which is roughly divided into the foregut, midgut, and hindgut (Dessimoz et al., 2006; McLin et al., 2007; Stevens et al., 2017; Zorn and Wells, 2009). The foregut is further patterned into the posterior foregut by retinoic acid (RA) (Bayha et al., 2009; Niederruth et al., 1999; Wang et al., 2006). The anterior foregut (AFG) gives rise to the esophagus and respiratory tract. Respiratory specification in response to Wnt and BMP activation leads to the expression of the transcription factor Nkx2-1, while BMP inhibition in the dorsal foregut promotes the development of the esophageal epithelium expressing Sox2 (Domyan et al., 2011; Goss et al., 2009; Harris-Johnson et al., 2009; Que et al., 2006; Rankin et al., 2016). The esophagus begins as simple cuboidal epithelium but develops into a stratified squamous epithelium expressing multiple keratins and a basal layer expressing Sox2 and p63 (Rosekrans et al., 2015; Zhang et al., 2016).

[0063] Disclosed herein is the temporal manipulation of the above signaling pathways to differentiate human PSCs into esophageal organoids. After DE formation, the applicant determined that precise temporal manipulation of the BMP, WNT, and RA pathways directs the formation of AFG spheres. Consistent with in vivo data, AFG spheres acquire a respiratory fate by activating the WNT and BMP pathways, while BMP inhibition promotes the formation of dorsal foregut spheres, which form human esophageal organoids (HEOs) after 1-2 months of continuous growth. HEOs contain a stratified squamous epithelium with distinct basal and luminal cell layers and harbor proliferative esophageal progenitors that can be expanded and differentiated into esophageal epithelium in organotypic raft cultures. HEOs used in parallel with mouse embryos can be used to identify molecular pathways affected by loss of SOX2 function, which is one of the causes of esophageal atresia in humans and mice (Domyan et al., 2011; Que et al., 2007). While reduced Sox2 function results in esophageal atresia in mice, complete loss of Sox2 in the mouse foregut endoderm results in esophageal agenesis. Loss of SOX2 function and transcriptional profiling of the human and mouse foregut determined that SOX2 regulates the dorsal expression of Wnt antagonists such as SFRP2, indicating that SOX2 inhibits the ability of Wnt to induce a respiratory fate in the dorsal foregut. Next, the applicant has found that the disclosed HEOs provide a complementary platform to study human esophageal organogenesis, congenital defects, and diseases.

[0064] In one aspect, a method of manufacturing esophageal organoids (EO) is disclosed. The method may include the step of contacting definitive endoderm with a BMP inhibitor, a Wnt activator, an FGF activator, and retinoic acid (RA). The contacting step may continue for a first time period sufficient to form an anterior foregut culture. In one aspect, the anterior foregut culture expresses SOX2 and HNF1B after such first time period and substantially does not express PROX1 and HNF6. The method may further include contacting the anterior foregut culture with a BMP inhibitor (Noggin) and an EGF activator for a second time period sufficient to form a dorsal anterior foregut ("dAFG") sphere, wherein the dAFG may express SOX2 and TP63 but does not express PDX1, PAX9 or NKX2.1. The method may further include the step of culturing the dAFG for a third time period sufficient to allow the formation of esophageal organoids (EO), wherein the culturing is carried out in the presence of EGF and further optionally includes an FGF signaling pathway activator, preferably FGF10. In one aspect, the EO is a human esophageal organoid (HEO).

[0065] Exemplary gene (or mRNA when the gene is not available) accession numbers are provided as follows: SOX2 (NG_009080.1); HNF1B (NG_013019.2), PROX1 (NC_000001.11); HNF6 (NM_214659.1); TP63 (NG_007550.1); PDX1 (NG_008183.1), PAX9 (NG_013357.1); and NKX2.1 (NG_013365.1). It should be noted that the gene names listed above (i.e., SOX2, HNF1B, etc.) are sufficient for a person of ordinary skill in the art to identify the genes described. The genes mentioned are intended to encompass variations of the genes and are not intended to be limited to the nomenclature of the exemplary accession numbers provided. That is, the accession numbers provided are not intended to limit the scope of the genes and / or claims, but are one of many identifiers of these genes / mRNAs / proteins and are essentially only exemplary. That is, the identifier may only refer to a particular isoform / variant that may be one of many. A person of ordinary skill in the art will readily understand this distinction, and a person of ordinary skill in the art will understand that the genes described encompass variants and genes having sequences different from the sequences associated with the accession numbers above.

[0066] In one aspect, definitive endoderm can be derived from precursor cells selected from embryonic stem cells, embryonic germ cells, induced pluripotent stem cells, mesoderm cells, definitive endoderm cells, posterior endoderm cells, posterior endoderm cells and hindgut cells. In one aspect, definitive endoderm can be derived from pluripotent stem cells. In one aspect, definitive endoderm can be derived from pluripotent stem cells selected from embryonic stem cells, adult stem cells or induced pluripotent stem cells. In one aspect, the DE can be a DE monolayer, wherein greater than 90% of the cells in the DE monolayer co-express FOXA2 and SOX17.

[0067] In one aspect, definitive endoderm can be derived by contacting pluripotent stem cells with one or more molecules selected from Activin, BMP subgroup of the TGF-β superfamily of growth factors; Nodal, Activin A, Activin B, BMP4, Wnt3a and combinations thereof.

[0068] In one aspect, inhibitors of the BMP signaling pathway can be selected from Noggin, Dorsomorphin, LDN189, DMH-1 and combinations thereof. In one aspect, the inhibitor of the BMP signaling pathway is Noggin. The BMP inhibitor can be present at a concentration of about 50 to about 1500 ng / ml.

[0069] In one aspect, the WNT activator can be selected from one or more molecules selected from: Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, GSKβ inhibitors (such as CHIR99021, i.e., "CHIRON"), BIO, LY2090314, SB-216763, lithium, porcupine inhibitor IWP, LGK974, C59, SFRP inhibitor WAY-316606, β-catenin activator DCA. The concentration of the Wnt pathway activator can, for example, be used at a concentration of about 50 to about 1500 ng / ml. There are many ways to activate the Wnt / β-catenin pathway (see http: / / web.stanford.edu / group / nusselab / cgi-bin / wnt / ). Some suitable existing wnt signaling pathway activators include, but are not limited to, protein-based activators, which can include Wnt ligands, which include, but are not limited to, Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt8, etc.; modifiers of Wnt ligand activity, including, but not limited to, activated Wnt frizzled receptors, (LRP) co-receptors, R-spondin proteins, Dkk proteins, regulators of Wnt ligand secretion and transport (Wntless, Porcupine), inhibition of β-catenin degradation APC and GSK3β inhibition, activated β-catenin, constitutively active TCF / Lef proteins, and chemical activators, which can include more than 28 known chemicals that activate or inhibit Wnt / β-catenin signaling. Some activators include, but are not limited to, the GSK3-β inhibitor CHIR99021 (CHIRON), BIO, LY2090314, SB-216763, lithium, porcupine inhibitor IWP, LGK974, C59, SFRP inhibitor WAY-316606, β-catenin activator DCA.

[0070] In one aspect, the FGF activator can be one or more molecules selected from: FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, and combinations thereof, preferably FGF4 or FGF10, or combinations thereof. In one aspect, the concentration of the FGF pathway activator can be used at a concentration of about 50 to about 1500 ng / ml. Proteins and chemicals that stimulate FGF receptors and downstream signaling components of the receptor, including MAPK, MEK, ERK proteins, and chemicals that regulate their activities. FGF signaling can be activated by inhibitors that inhibit the FGF signaling pathway, including but not limited to members of the Sprouty protein family.

[0071] In one aspect, the retinoic acid of step a can be contacted with DE for a period of about 12 hours to about 48 hours, or about 20 hours to about 40 hours, or a period of about 24 hours, or until the treatment results in the expression of PDX and the loss of P63 expression.

[0072] In one aspect, step c can be carried out for a period sufficient to form a stratified epithelium lacking KRT8. In one aspect, step c can be carried out for a period sufficient to form a stratified squamous epithelium expressing regional keratin. In one aspect, step c can be carried out for a period sufficient to cause the HEO to express INV.

[0073] In one aspect, the first time period can be a time period of about three days ± 24 hours. In one aspect, the second time period can be a time period of about three days ± 24 hours. In one aspect, the third time period can be a time period of about 28 days ± 48 hours, or about 21 days to about 90 days, or about 30 days to about 60 days. In one aspect, steps a to c can be carried out in vitro.

[0074] In one aspect, the method can further comprise the step of contacting the anterior foregut culture of step a) or the spheroid of step b) with a matrix selected from collagen, basement membrane matrix (Matrigel), or combinations thereof.

[0075] In one aspect, the esophageal composition described herein can be characterized by being devoid of innervation and / or vasculature. In one aspect, the composition is a human esophageal organoid (HEO) composition, wherein the HEO composition is substantially free of one or more of submucosal glands, transitional zones, vasculature, immune cells, or the submucosa.

[0076] In one aspect, an esophageal progenitor cell capable of organizing into an organotypic culture is disclosed. The esophageal progenitor cell can be derived from the method disclosed herein.

[0077] In one aspect, a method of manufacturing stratified squamous epithelium is disclosed. The method may include enzymatically dissociating the herein-described HEO to release progenitor cells, wherein the HEO is from about 3 weeks to about 10 weeks old, or from about 4 weeks to about 8 weeks old, or about 5 weeks old; expanding the progenitor cells into a monolayer; and redifferentiating the dissociated HEO into stratified squamous epithelium on a collagen-coated membrane for a period of time sufficient to produce non-keratinized stratified squamous epithelium, wherein the non-keratinized stratified squamous epithelium expresses keratin and one or more markers selected from IVL, CRNN, and FLG. In one aspect, the stratified squamous epithelium may comprise esophageal cells organized substantially in the form of a sheet.

[0078] In one aspect, a method of treating esophageal diseases in an individual in need thereof is disclosed. The diseases may be selected from congenital diseases (atresia), functional diseases (achalasia and other motility disorders), immune diseases (eosinophilic esophagitis), pathological diseases (Barrett's esophagus and esophageal cancer), and combinations thereof, and the method includes the step of contacting the individual with an esophageal composition (such as HEO or esophageal sheet) disclosed herein.

[0079] In one aspect, the disease may include an ulcer or ulcerated tissue of the esophagus, and the disclosed esophageal composition may be used to contact and repair the ulcerated tissue. For example, in one aspect, the esophageal composition may comprise esophageal cells organized substantially in the form of a sheet, which may be contacted with the patient.

[0080] In one aspect, a method of identifying a treatment for eosinophilic esophagitis is disclosed. In this aspect, the method may include contacting a potential therapeutic agent of interest with an organoid or esophageal tissue described herein, detecting a measure of eosinophilic esophagitis activity, and determining whether the potential therapeutic agent of interest improves the measure of eosinophilic esophagitis activity.

[0081] In one aspect, a method of manufacturing a Fanconi anemia disease model is disclosed. In this aspect, the method of preparing HEO or esophageal sheet disclosed herein is carried out, wherein the DE is obtained from FANCA-deficient progenitor cells.

[0082] In one aspect, a method of manufacturing a Barrett's metaplasia disease model is disclosed. In this aspect, the method may include the step of inducing CDX2 and activating BMP in HEO or esophageal sheet prepared according to the method disclosed herein.

[0083] In one aspect, a method of making an eosinophilic esophagitis disease model is disclosed. In this aspect, the method can include contacting HEO or esophageal slices prepared according to the methods disclosed herein with IL-13 for a period of time sufficient to increase the expression of CCL26 and CAPN14 and decrease the expression of CRNN and IVL.

[0084] In one aspect, a method of identifying an active agent capable of treating an esophageal disease state is disclosed, which includes the steps of: contacting a test agent with HEO or esophageal slices made according to the methods disclosed herein for a period of time sufficient to cause a physiological change in the disease model; and detecting a decrease in the expression of CCL26 and CAPN14 and an increase in the expression of CRNN and IVL in EoE; or detecting an increase in esophageal gene expression such as SOX2, p63, KRT13, CRNN, IVL, and the loss of Barrett's intestinal genes.

[0085] Examples

[0086] The following non-limiting examples are provided to further illustrate the embodiments of the invention disclosed herein. Those skilled in the art should understand that the techniques disclosed in the following examples represent methods that have been found to function well in the practice of the invention and thus can be considered as examples of a mode for its practice. However, based on the present disclosure, those skilled in the art should understand that many changes can be made in the specific embodiments disclosed and still obtain similar or comparable results without departing from the spirit and scope of the invention.

[0087] Wnt and Retinoic Acid Signaling Control Anterior Foregut Fate Relative to Posterior Foregut

[0088] To generate foregut derivatives, hPSCs are first induced into DE as previously described (Figure 1), followed by three-dimensional (3D) SOX2-expressing foregut spheroids (D'Amour et al., 2005; Dye et al., 2016; McCracken et al., 2014, 2017). When attempting to generate esophageal organoids, the major challenge is to generate foregut tissue with the correct regional identity. Endoderm patterning is regulated by differential BMP, WNT, and RA signaling, where the highest activation levels of these pathways promote midgut and hindgut fates and lower levels promote foregut fate (Bayha et al., 2009; Davenport et al., 2016; Matt et al., 2003; McLin et al., 2007; Tiso et al., 2002; Wang et al., 2006). Based on our previous studies showing that the duration of signaling is important for differentiation, the applicants tested the effect of the duration of Wnt activation during foregut spheroid formation on anterior-posterior identity (Spence et al., 2011). The applicants found that a shorter duration of activation of the canonical Wnt pathway agonist chiron (by GSK3β inhibition), or Wnt3a treatment after DE formation, led to the formation of anterior foregut (AFG) spheroids expressing HNF1β and SOX2, with low levels of the posterior foregut markers PROX1 and HNF6 ( Figures 1A - 1E , 8A-8G). HNF1β is not expressed in pharyngeal endoderm, indicating that the AFG spheroids are not pharyngeal ( Figures 1C - 1E , 8B). The mid / hindgut marker CDX2 is not expressed ( Figure 1B , 1D, 1E). Thus, the applicants concluded that the regional identity of these foregut spheroids (HNF1B+ / SOX2+, PROX1- / HNF6-) is closer to the posterior foregut and away from the pharynx.

[0089] It is also known that four days of RA treatment posteriorizes foregut spheroids (McCracken et al., 2014), and loss of RA signaling results in abnormal development of posterior foregut organs (Bayha et al., 2009; Wang et al., 2006). Therefore, the applicants investigated whether shortening the duration of RA signaling in foregut cultures would promote a more anterior fate. Foregut cultures treated with RA for 4 days expressed the posterior foregut markers GATA4 and PDX1, while treatment with RA for 1 day led to spheroids expressing TP63, a marker expressed in the developing esophagus ( Figure 1F -K, 8O-8R). Cultures lacking RA or containing the aldehyde dehydrogenase inhibitor DEAB (blocking RA synthesis) produced spheroids with very low TP63 expression and increased levels of the pharyngeal markers PAX9 and OTX2 ( Figures 1G - 1K, 8F - 8G, 8S - 8V). Together, these data indicate that transient activation of RA promotes foregut regional identity consistent with the presumptive esophageal domain.

[0090] Anterior Foregut Spheres Have the Capacity to Form Esophageal or Respiratory Lineages

[0091] The presumptive esophageal / respiratory region of the foregut is patterned along the dorsal - ventral (D - V) axis, leading to the specification of esophageal and respiratory fates, respectively. The Applicant predicts that AFG spheres will respond to D - V patterning cues to acquire esophageal or respiratory fates. Studies in vertebrate embryos have demonstrated that BMP and Wnt signaling promote respiratory fate (NKX2 - 1+SOX2 - ), while Noggin - mediated inhibition of BMP signaling in the dorsal foregut tube is required for esophageal development (Domyan et al., 2011; Fausett et al., 2014; Goss et al., 2009; Harris - Johnson et al., 2009; Que et al., 2006). Thus, the Applicant treated day 6 AFG spheres with 3 days of chiron and BMP4 or alternatively with Noggin (to inhibit BMP signaling) ( Figures 2A - 2B ). Treatment with chiron + BMP4 led to the induction of NKX2 - 1 and the inhibition of SOX2, while treatment with Noggin dorsalized the spheres, as marked by elevated levels of SOX2, MNX1, KRT4, and TP63 ( Figure 2C -I) (Daniely et al., 2004; Sherwood et al., 2009). Overall, inhibition of BMP signaling in cultures of AFG spheres promoted dorsal anterior foregut identity.

[0092] Formation of Esophageal Organoids with Stratified Squamous Epithelium

[0093] To determine whether dorsal - patterned AFG spheres are capable of growing into esophageal organoids, the Applicant cultured them suspended in Matrigel with EGF alone or in the context of manipulation of other pathways predicted to promote esophageal development, including Wnt activation (chiron), long - term BMP inhibition (Noggin), activation of Hedgehog (SAG, a smoothened agonist), and FGF10. Although most of these manipulations had no effect on growth (data not shown), the Applicant found that addition of FGF10 to the cultures from day 6 to day 13 led to an improved efficiency of sphere growth into organoids ( Figures 11A - 11E ), and indeed did not affect patterning and differentiation into esophageal organoids (data not shown).

[0094] Next, the applicant compared the morphological and molecular development of putative human esophageal organoids (HEO) with the normal development of the embryonic mouse esophagus. During embryonic growth and development, the esophagus transitions from a simple cuboidal epithelium at E12.5 to a multi-layered / stratified epithelium between E14.5 and E17.5 ( Figure 3G , 3J, 3M, 3P, 11F-11O, 11R) (Chen et al., 2012). Similarly, over the course of one month, the size of HEO expands from approximately 50 μm in diameter to 200 - 400 μm ( Figures 3B - 3F ). Moreover, the organoid epithelium transitions from a simple epithelium that is predominantly SOX2, p63 double-positive to a multi-layered epithelium that expresses markers of esophageal stratified squamous epithelium ( Figures 11H - 11Q , 11S). At one month, HEO express the basal markers p63 and KRT14 as well as the suprabasal marker KRT13 ( Figure 3H , 3K, 3N, 3Q). This expression pattern is similar to that of the E17.5 esophagus, which contains a multi-layered epithelium ( Figure 3G , 3J, 3M, 3P).

[0095] One-month HEO remain relatively immature, as demonstrated by widespread SOX2 epithelial expression and the expression of the immature esophageal marker KRT8 ( Figure 3J , 11L-11M). Therefore, the applicant extended the culture period to 2 months, which led to additional growth and formation of a stratified epithelium lacking KRT8. The applicant observed robust expression of KRT14 throughout the basal layer and KRT13 and IVL throughout the differentiated suprabasal layer, demonstrating the presence of a stratified squamous epithelium ( Figure 3I , 3L, 3O, 3R). Histologically, the basal layer and squamous cells of 2-month HEO are more suprabasally defined, without any signs of keratinization ( Figure 4A ). The epithelial morphology of HEO is readily distinguishable from other organoids, including gastric (HGO), intestinal (HIO), or colon organoids, each of which has an epithelial morphology unique to that organ type (McCracken et al., 2014, 2017; Múnera et al., 2017; Spence et al., 2011).

[0096] To show that HEO are human esophageal epithelium, the applicant compared 1- and 2-month HEO with human esophageal biopsies and 1-month HGO and HIO by qPCR. The key stratified squamous epithelium markers p63, KRT5, KRT13, IVL, and CRNN were expressed highest in human esophageal biopsies and 2-month HEO, while HGO and HIO expressed these transcripts negligibly ( Figures 3S - 3V)。In addition, the applicant compared the entire transcriptome of HEO with the transcriptomes of other human epithelial tissues isolated from the esophagus, lung, skin, stomach, small intestine, colon, HGO, and HIO. Clustering analysis of the RNA sequencing data revealed that, in contrast to the stomach, small intestine, and colon, HEO was most closely related to the human esophagus and the esophageal keratinocyte cell line EPC2( Figure 3W )。The applicant used principal component analysis to compare HEO with HIO and HGO and found that even 1-month-old HEO was completely different from other gastrointestinal organoids( Figure 3X )。Comparison of markers of the esophagus, skin, stomach, and colon revealed that HEO was highly similar to the human esophagus, again affirming the qPCR analysis. Although there was significant overlap between the skin and the esophagus, there were also distinct differences, including KRT1 in the skin and KRT4 and 13 in the esophagus. Notably, HEO did not express the gastric or intestinal markers TFF2, CLDN18, GATA4, PDX1, CDX2, and CDH17( Figure 3Y )。

[0097] Given that in vivo growth of PSC-derived organoids has been shown to promote further maturation and function, the applicant studied HEO in vivo using three different transplantation-based methods. The applicant first transplanted 1-month-old HEO into the renal capsules of immunodeficient (NSG) mice and allowed them to grow for 8 weeks, which led to the maturation of a subset of the transplanted organoids (2 / 5)( Figures 12A - 12F )。The applicant used two other unsuccessful transplantation methods: inoculating HEO onto biodegradable PEG scaffolds and then transplanting them into the fat pads of mice; or implanting HEO into the forestomach of NSG mice (data not shown) (Dye et al., 2016). Overall, growth of spheres and organoids outgrowth was robust in various ES and iPS lines, generating the vast majority of organoids expressing stratified squamous markers in each generation (Figure T-11CC). Together, these data demonstrate that PSC-derived dorsal foregut spheroids form esophageal organoids with well-differentiated, non-keratinized stratified squamous epithelium.

[0098] HEO Contains Progenitor Cells Capable of Reconstituting Stratified Squamous Epithelium

[0099] The esophagus contains basal progenitor cells, which can give rise to all the differentiated stratified layers (DeWard et al., 2014; Doupe et al., 2012; Kalabis et al., 2008). This property allows esophageal cells to be isolated, expanded in culture, and then redifferentiated into stratified squamous epithelium. The applicant first tested whether HEO contains esophageal progenitor cells by enzymatically dissociating 5-week-old HEO into single cells, expanding them in monolayer culture, and then testing their ability to redifferentiate into stratified squamous epithelium using the organotypic raft culture method (Hoskins et al., 2009). After 14 days in organotypic culture, HEO-derived keratinocytes generated unkeratinized stratified squamous epithelium, which expressed the appropriate keratins and the differentiation markers IVL, CRNN, FLG( Figures 4A - 4N ). HEO, HEO-derived keratinocytes, and organotypic raft cultures all expressed high levels of the basal markers p63, KRT5, and KRT14( Figure 4O -Q), while the organotypic rafts were the most differentiated, expressing CRNN, IVL, KRT13, TMPRSS11A, and D at levels comparable to human esophageal biopsies( Figures 4R - 4U ). However, efforts to expand these progenitor cells long-term in 3D organoid cultures were not successful. Dissociated organoids replated in 3D Matrigel grew for several weeks, maintained patterning (SOX2+p63+), and were passaged multiple times (re-dissociated)( Figures 12G - 12H , 12O, 12Q-12R). However, the passaging efficiency decreased over time, and the applicant was unable to induce differentiation or stratification in these passaged organoids( Figure 12I -N, 12P). This result was similar to esophageal progenitor cells derived from the human esophagus, demonstrating that these cells generally cannot be cultured long-term (Kasagi et al., 2018).

[0100] Another approach to studying basal progenitor cell differentiation is to pulse-chase label proliferating basal progenitor cells and track the labeled cells as they differentiate into stratified layers over time. The applicant labeled proliferating cells in 40-day-old HEO by a 1-day EdU treatment and analyzed them immediately (day 0), or after chase periods of 2, 6, and 13 days after labeling( Figures 4V - 4BB ). EdU-labeled cells initially appeared in basal cells expressing p63, but over time, these cells moved into the upper basal compartment, lost p63 expression, and ultimately sloughed into the lumen 13 days after labeling( Figures 4W - 4BB ). Thus, it is believed that HEO contains basal progenitor cells that differentiate and migrate into stratified layers, similar to the esophageal epithelium.

[0101] Using HEO and Mouse Genetics to Determine the Mechanisms of Esophageal Development

[0102] While the establishment of the PSC-derived HEO model system is an important advance, it is necessary to demonstrate that HEO can be used to study human development and disease. The applicant chose to use HEO in parallel with two well-established vertebrate model systems (mouse and Xenopus laevis) to model esophageal birth defects by studying loss of function of SOX2. First, the applicant determined the consequences of complete loss of Sox2, since partial loss of SOX2 function in humans and mice results in partial loss of the esophagus (atresia) (OMIM206900, Fantes et al., 2003; Que et al., 2007; Williamson et al., 2006). The applicant generated two mouse models to inducibly delete Sox2 in the foregut endoderm before the onset of esophageal development (FoxA2 CreER ; Sox2 fl / fl and Sox2 CreER / fl )(Arnold et al., 2011; Park et al., 2008; Shaham et al., 2009). In both models, early deletion of Sox2 in the foregut results in complete esophageal agenesis, with the foregut region between the pharynx and stomach remaining as a tube lacking p63 and widely expressing the respiratory marker Nkx2-1( Figures 5C - 5F , 13A-13D). Lung buds are largely similar to control embryos, and cell polarity is not affected( Figure 13E -F). Deletion of Sox2 after the onset of esophageal development results in partial loss of esophageal tissue, with the esophageal region severely hypoplastic at E11.5. In some regions, the esophagus is only 2-3 cells wide, remains simple cuboidal epithelium, lacks p63 expression, and expresses Nkx2-1 in some cells( Figures 5I - 5L ). These data indicate that Sox2 function is required to initiate esophageal development, and loss of Sox2 one day later results in reduced esophageal tissue and identity.

[0103] To study whether esophageal agenesis is caused by changes in cell death and proliferation or only due to the absence of partitioning from the common foregut, the applicant analyzed E10.5 embryos at the time of partitioning. Sox2 CreER / f1 embryos have increased cleaved Caspase3 staining in the dorsal foregut at the level where partitioning would normally occur, indicating that cells in the presumptive esophagus undergo cell death( Figures 5G - 5H , 13U). Proliferation marked by Ki67+ cells is unchanged( Figure 13V ). In both control and Sox2 knockout foreguts, there appears to be a point where the epithelium narrows midway along the dorsoventral axis, indicating that the epithelium attempts to divide into two tubes regardless of the presence or absence of Sox2 protein( Figure 6M -T). The results in mice demonstrate that Sox2 is required for esophageal development, survival, and restriction of Nkx2-1 to the ventral / respiratory domain in the foregut.

[0104] The BMP - Independent Role of Sox2 in Suppressing Nkx2 - 1 Expression

[0105] The applicant next wanted to take advantage of the in vitro advantages of human and Xenopus foregut cultures to mechanically explore how Sox2 initiates esophageal development. From previous studies, Sox2 is believed to inhibit respiratory (ventral) development and promote esophageal (dorsal) development. To promote ventral identity, BMP signaling is believed to inhibit Sox2 in the ventral foregut, thereby allowing Wnt-mediated induction of Nkx2-1 expression (Domyan et al., 2011). The applicant tested whether the only function of BMP was to inhibit Sox2 by suppressing Sox2 and then activating Wnt, which was predicted to be sufficient to activate Nkx2-1 in the absence of BMP. Using morpholino injection to knockdown sox2 and activating canonical Wnt signaling with Bio in Xenopus endoderm explants did not activate nkx2-1 expression in the absence of BMP4 ( Figure 6A -B, 6E-6F). However, treatment with Bio and BMP4, as in the mouse Sox2 knockout, expanded the nkx2-1 domain after sox2 knockdown ( Figures 6C - 6D ). These data suggest two things: one, BMP signaling is required for Nkx2-1 expression independent of Sox2 inhibition; two, Sox2 is required to inhibit ectopic Nkx2-1 expression outside the respiratory domain.

[0106] To determine whether human SOX2 is required to prevent ectopic expression of NKX2-1, the applicant used an iPSC line to inducibly express a repressor form of the CRISPR protein, which inhibits transcription at the SOX2 locus (CRISPRi-SOX2) (Mandegar et al., 2016). Knockdown of SOX2 in human dorsal anterior foregut cultures (dAFG) led to ectopic expression of NKX2-1 mRNA and protein ( Figures 6G - 6J , 6L-6M). Optimal induction of NKX2-1 in ventral anterior foregut (vAFG) still depended on the presence of BMP ( Figures 6K - 6N ). To determine whether SOX2 expression was sufficient to inhibit NKX2-1, the applicant generated a stable tet-inducible hPSC line that expressed HA-tagged SOX2 in the ventral foregut during respiratory induction. Expression of SOX2 in the ventral foregut led to a significant downregulation of NKX2-1 mRNA and protein ( Figures 6Q - 6T ). Together, these data suggest that BMP has an additional function for respiratory induction and confirm that Wnt signaling is required for NKX2-1 expression in all cases. In addition, SOX2 expression is sufficient to inhibit NKX2-1 expression by an unknown mechanism.

[0107] Sox2 Regulates the Expression of Wnt Antagonists during Dorsal - Ventral Patterning

[0108] The ease of manipulation and scalability of foregut cultures are ideally suited to "omics" approaches. Accordingly, the applicant employed an RNA-sequencing-based approach to identify genes regulated by SOX2 and / or BMP signaling during dorsal-ventral (esophagus-respiratory) patterning. Principal component analysis (PCA) determined that the largest groups of regulated genes were for dorsal-ventral patterning (±BMP4 or Noggin) and for SOX2-regulated genes (±dox-SOX2 CRISPRi)( Figure 14A ). Additionally, SOX2 regulates different groups of genes in dorsal (Noggin) cultures compared to ventral (BMP4) cultures, as shown along principal component axis 1 in the cluster heatmap and PCA ( Figure 7A , 14A). The use of BMP4 or Noggin resulted in the expected changes in dorsal-ventral patterning markers, such as upregulation of NKX2-1 and inhibition of SOX2, MNX1, KRT4, PAX9 in ventral (BMP-high) cultures. Loss of SOX2, however, led to many transcriptional changes in the dorsal foregut and relatively fewer changes in the ventral foregut, including increased NKX2-1 expression and decreased FOXE1, NTN1, and GDNF expression ( Figures 7A - 7B , 7D, 14B).

[0109] Among dorsal and ventral genes, there are transcripts that change (increase or decrease) in response to CRISPRi-SOX2 alteration and genes that do not (SOX2-independent). For example, among 542 genes enriched in the dorsal side, 75.6% (410 genes) are SOX2-dependent. 17.2% (93 genes) in the dorsal foregut are downregulated after SOX2 knockdown, which the applicant termed "positively regulated by SOX2". There are 39 transcripts that increase in response to SOX2 knockdown, indicating that they are "negatively regulated by SOX2". In ventral cultures, 374 genes were upregulated by BMP treatment, of which 38 decreased and 5 increased in response to loss of SOX2 ( Figure 7D ). Not surprisingly, more genes are regulated by SOX2 in the dorsal foregut, as ventral SOX2 expression is already significantly downregulated in response to BMP. The applicant used cross-analysis to identify genes that BMP might regulate by inhibiting SOX2 and found that 46 genes (12.3%) were upregulated by both BMP treatment and SOX2 knockdown ("genes negatively regulated by SOX2"). The applicant also found 81 genes (14.9%) that were downregulated by both BMP treatment and SOX2 knockdown ("genes positively regulated by SOX2") ( Figure 7B ). Additionally, >80% of BMP-regulated transcripts did not change in response to SOX2 knockdown, which is consistent with the conclusion that BMP has a role independent of SOX2 inhibition in ventral foregut specification.

[0110] Gene ontology analysis of all 404 unique genes whose expression was reduced in dAFG in response to SOX2 knockdown yielded numerous important gene ontology terms, including two terms related to the Wnt signaling pathway ( Figure 7C ). Gene set enrichment analysis also found that several Wnt signaling components were significantly altered in response to SOX2 knockdown ( Figure 7E ). The secreted canonical Wnt signaling inhibitors SFRP1, SFRP2, and DKK1 were all downregulated upon loss of SOX2 ( Figure 7E , 7K). In addition, overexpression of SOX2 in ventral cultures upregulated SFRP2, which is consistent with published ChIP-seq data in hPSC-derived mesendoderm and endoderm, showing SOX2 binding peaks at the SFRP2 locus ( Figures 14C - 14D ) (Tsankov et al., 2015).

[0111] Since SOX2 positively regulates the expression of Wnt antagonists, the applicant hypothesized that SOX2 could inhibit canonical Wnt signaling in the dorsal foregut. To investigate this, the applicant used two genetic models, Foxa2 CreER ; Sox2 fl / fl and Sox2 CreER / fl to delete Sox2 from the mouse foregut and measured canonical Wnt / β-catenin activity by analyzing the expression of the Wnt target gene Axin2 (Jho et al., 2002; Lustig et al., 2002). In situ hybridization revealed high levels of Axin2 mRNA in the ventral foregut endoderm and low levels in the dorsal foregut endoderm of control embryos. In contrast, when Sox2 was deleted, the dorsal foregut had increased Axin2 staining ( Figures 7F - 7I ). Similarly, in human ventral foregut cultures with exogenous expression of SOX2, AXIN2 transcript levels were reduced ( Figure 6G , 7J). In addition to increasing the expression of canonical Wnt genes, the Nkx2-1 expression domain also expanded into the dorsal foregut ( Figure 5E , 13C-13D). Together with the data from human foregut cultures, the applicant proposed a model in which Sox2 positively regulates the expression of secreted Wnt antagonists in the dorsal foregut, which inhibits canonical Wnt signaling in the dorsal foregut and restricts the expression of Nkx2-1 to the ventral foregut.

[0112] Discussion

[0113] The generation of HEO and organotypic raft cultures from primary esophageal cells and cell lines has been described (Andl et al., 2003; Kalabis et al., 2012; Kasagi et al., 2018). In addition, human PSC-derived anterior foregut (AFG) endoderm cultures generate a heterogeneous mixture of multiple AFG derivatives (Green et al., 2011; Kearns et al., 2013; Longmire et al., 2012). To enrich esophageal endoderm by this method, cell sorting and subsequent culture must be relied upon, as achieved by Zhang et al. Alternatively, directed differentiation into specific foregut derivatives, such as the esophagus, benefits from a more nuanced recapitulation of early organ development. Here, the applicant has specifically differentiated human PSCs into HEO using a stepwise approach approximating DE formation, foregut patterning and morphogenesis, AFG patterning into a presumptive esophagus-respiratory domain, and finally dorsal foregut patterning. This method progressively restricts endoderm differentiation potential, leaving behind dorsal AFG endoderm that grows into esophageal organoids.

[0114] One challenge is to find conditions that generate the respiratory-esophageal anterior region of the foregut but not the most anterior pharyngeal region. BMP inhibition is necessary for foregut specification, and the applicant found that transient Wnt and RA activation patterns the foregut into esophagus-respiratory endoderm rather than pharyngeal endoderm. Moreover, the applicant found that 1 day of RA promoted the expression of TP63 and KRT4 but not the posterior foregut markers GATA4 and PDX1. Without being bound by theory, this effect of RA may be direct, as RA promotes the expression of KRT4 and TP63 in keratinocytes (Bamberger et al., 2002). Due to the lack of specific esophageal markers, the applicant relied on the presence or absence of regional expression markers to determine early anterior foregut endoderm identity and exclude pharyngeal, respiratory, liver, pancreatic, and gastric endoderm.

[0115] The applicant also used functional assays to show that the esophageal-respiratory region of the foregut had been generated. This anterior-posterior level of the foregut should have the ability to generate esophageal and respiratory lineages. The applicant showed that AFG spheres could respond to respiratory induction signals (BMP4 and Wnt activation by chiron) by upregulating NKX2-1. Conversely, inhibition of BMP signaling dorsalized the spheres based on the expression of SOX2, TP63, and MNX1. Interestingly, under the applicant's culture conditions, in contrast to other protocols, addition of a TGFβ inhibitor during foregut induction led to an increase in posterior foregut markers and a decrease in the upregulation of NKX2-1 ( Figure 10A -J), which exemplifies how timing and combinatorial signaling pathway manipulation can lead to different outcomes.

[0116] The ultimate evidence for esophageal lineage commitment from dorsal foregut spheroids is that they grow into three-dimensional HEOs with a stratified squamous epithelium expressing regional keratin. After long-term culture or in vivo transplantation, the HEOs show a significant increase in maturity both morphologically and by analysis of late esophageal markers (IVL, CRNN, FLG). In addition, HEOs can be dissociated in organotypic raft cultures, expanded into keratinocytes, and differentiated into stratified squamous epithelium, demonstrating that HEOs have basal progenitors similar to the human esophagus (Doupe et al., 2012; Kalabis et al., 2008). Indeed, the expression levels of differentiation markers in organotypic raft cultures are close to those in the human esophagus. Generating fully differentiated and mature cell types from human PSCs is a challenge across organ systems, and our data indicate that PSC-derived esophageal epithelium is among the most highly differentiated tissues derived to date.

[0117] HEOs will undoubtedly facilitate research on human esophageal diseases. As an example, the applicant demonstrated how HEOs can model human esophageal birth defects. Since SOX2 mutations can cause esophageal atresia in mice and humans, the applicant used HEOs to determine how SOX2 controls human esophageal development, as its mechanism of action is not well understood (Fantes et al., 2003; Que et al., 2007; Williamson et al., 2006). The applicant first determined the transcriptional changes that occur upon loss of SOX2. Current models suggest that the main role of BMP signaling is to inhibit Sox2, which in turn inhibits Nkx2-1. However, the applicant has determined that BMP signaling regulates many transcriptional changes independently of SOX2, indicating that the current model is overly simplistic (Domyan et al., 2011; Rankin et al., 2012)( Figure 2B ). In addition, the data indicate that SOX2 inhibits canonical Wnt signaling and promotes dorsal endoderm survival. In other contexts, Sox2 can inhibit and promote Wnt signaling through diverse mechanisms, including direct binding to TCF / LEF and regulation of secreted Wnt antagonists (Chen et al., 2008; Kormish et al., 2010; Li et al., 2016; Sinner et al., 2007; Zhou et al., 2016). The use of Barx1 knockout mice has shown the role of the secreted Wnt antagonists Sfrp1 and Sfrp2 in tracheoesophageal septation (Woo et al., 2011). In human foregut cultures, SOX2 also regulates the transcriptional levels of SFRP2, and loss of SOX2 leads to increased Wnt activity in the dorsal foregut. The applicant concludes that SOX2 restricts the respiratory lineage from the dorsal foregut endoderm, possibly by inhibiting canonical Wnt signaling.

[0118] In summary, the applicant has developed a method for generating human PSC-derived HEO based on the temporal manipulation of signals that pattern the early endoderm and foregut. HEO development is highly similar to mouse esophageal development and results in patterned stratified squamous epithelium. The applicant used human foregut cultures and genetic approaches in mice and frogs to identify molecular pathways regulated by Sox2 during dorsoventral patterning and esophageal specification. The applicant determined that SOX2 inhibits Wnt activity in both humans and mice, and failure to do so leads to inappropriate dorsal activation of the respiratory program. Thus, HEO provides a powerful model for studying esophageal development and disease.

[0119] Experimental Models and Subject Details

[0120] Animal

[0121] According to the Guide for the Care and Use of Laboratory Animals of the NIH, all mice and frogs were housed in the animal facilities of Cincinnati Children's Hospital Medical Center (CCHMC). The animals were maintained on a 12-hour light / dark cycle with free access to water and standard chow. Wild-type and mutant mice and Xenopus laevis were used for studies of foregut and esophageal embryonic development. The sex of the embryos was not determined. Male immunodeficient NSG (NOD.Cg-Prkdc -scid Il2rg tm1Wjl / SzJ) mice at 8-16 weeks of age were used for transplantation experiments. Healthy animals were used for all experiments. All experiments were conducted with the approval of the Institutional Animal Care and Use Committee of CCHMC (protocols IACUC2016-0004 and IACUC2016-0059).

[0122] Human ESC / IPSC

[0123] The human embryonic stem cell (ESC) line H1 (WA01) was purchased from WiCell. Unmodified iPSC lines 65.8, 72.3, and 263.10 were generated and obtained from the CCHMC Pluripotent Stem Cell Facility and approved by the Institutional Review Board of CCHMC. The CRISPR interference iPSC line (WTC11 genetic background) was generated and obtained from the Conklin laboratory at the University of California, San Francisco (Mandegar et al., 2016). The H1 line is male, the iPSC65.8 line is female, the iPSC72.3 line is male, the iPSC263.10 line is male, and the SOX2 CRISPR interference line (CRISPRi-SOX2) is male. All iPSC lines were examined and determined to have normal karyotypes, and iPSC65.8 and iPSC72.3 have been tested using the in vivo teratoma assay.

[0124] Human biopsy tissues

[0125] During endoscopy, human esophageal tissues were collected from pediatric patients (all male, aged 3 to 13 years) who consented to provide esophageal biopsy specimens for research purposes. This study was approved by the Institutional Review Board of Cincinnati Children's Hospital Medical Center (Protocol 2008-0090). Samples were used as positive controls for esophageal tissue identity by RNA quantification.

[0126] Method Details:

[0127] Experimental Design

[0128] Pluripotent stem cell lines and maintenance

[0129] Both human embryonic stem cells and induced pluripotent stem cells (hESC and hiPSC) were maintained in feeder-free cultures. Cells were plated on hESC-qualified Matrigel (BD Biosciences, San Jose, California) and maintained at 37 °C, 5% CO2 with daily medium change of mTeSR1 medium (STEMCELL, Vancouver, Canada); cells were routinely passaged every 4 days using Dispase (STEMCELL Technologies). Lentivirus was generated with the help of the Viral Vector Core Laboratory at CCHMC by cloning the human SOX2 ORF into pINDUCER20 (Addgene #44012, Meerbrey et al., 2011), and hESC were transduced with 2 μL of virus to generate the H1 HA-tagged SOX2 dox-inducible line; the line was maintained during selection using mTeSR1 and G418 (500 μg mL-1, ThermoFisher Scientific).

[0130] Differentiation of anterior foregut cultures and spheres

[0131] Confluent hPSC cultures were treated with Acutase (STEMCELL Technologies) to dissociate into single cells and resuspended in mTeSR1 and Y-27632 (10 μM, Tocris) and plated onto Matrigel. The next day, differentiation into definitive endoderm was performed as previously described (McCracken et al., 2014). Briefly, on day 1, cells were treated with Activin A (100 ng mL-1, R&D systems, Minneapolis, MN) and BMP4 (50 ng mL-1, R&D systems) in RPMI 1640 medium (Life Technologies). Cells on the subsequent two days were treated with Activin A (100 ng mL-1) only in RPMI 1640, with a stepwise increase in the concentration of HyClone defined fetal bovine serum (dFBS, GE Healthcare Life Sciences) from 0.2% to 2%.

[0132] For anterior foregut monolayer cultures, cells were treated with Noggin (200 ng mL-1) in RPMI 1640 containing 2% dFBS for 3 days, and on day 3 with all-trans retinoic acid (2 μM, Sigma, St. Louis, MO).

[0133] Alternatively, to generate anterior foregut spheres from definitive endoderm, cells were treated with FGF4 (500 ng mL-1, R&D systems), Noggin (200 ng mL-1) in RPMI 1640 containing 2% dFBS for 3 days. Additional factors were tested during this period (described in the results), such as CHIR99021 (“chiron” or “chr”, 2 μM, Tocris), Wnt3a (500 ng mL-1, R&D systems), SB431542 (10 μM, Tocris), DEAB (10 μM, Sigma), and retinoic acid (2 μM).

[0134] Three-dimensional cultures and anterior foregut spheres differentiate into human esophageal organoids

[0135] Transfer the anterior foregut spheroids into Matrigel in 50 μL droplets and culture them in Advanced DMEM / F12 (ThermoFisher Scientific) basal (“Gut”) medium supplemented with B27 supplement (1X, ThermoFisher Scientific), N2 supplement (1X, ThermoFisher Scientific), HEPES (13 mM, ThermoFisher Scientific), L-glutamine (2 mM, ThermoFisher Scientific), penicillin / streptomycin (1X, ThermoFisher Scientific), and EGF (100 ng mL -1 , R&D systems) for 3 - 58 days. In addition to this basal medium, Noggin (200 ng mL -1 ), FGF10 (50 ng mL -1 ), and CultureOne supplement (1X, ThermoFisher Scientific) were supplemented for the first three days. The supplementation with FGF10 and CultureOne was continued until the end of the first week in the three-dimensional culture. The medium was changed every 3 - 4 days. For EdU labeling, EdU (10 μM, Invitrogen) was supplemented into the medium for a defined period of time and removed by washing twice with sterile PBS before changing to medium without EdU.

[0136] Keratinocytes and organotypic raft cultures

[0137] Dissociate the HEO on day 41 at 37 °C using TrypLE Select (Gibco) for 30 - 40 minutes, during which they are triturated with a 22 1 / 2 & 27 1 / 2 gauge needle. After dissociation, the cells are reconstituted in complete keratinocyte serum-free medium (K-SFM, Gibco) supplemented with Y-27632 (10 μM), EGF (10 ng mL-1), and penicillin / streptomycin (1X), and then plated at 1.5×10 4 cells cm -2 onto collagen IV (Sigma)-coated plates (1.5 μg cm -2 ). After reaching 90% confluence, the HEO-derived keratinocytes are dissociated into single cells using TrypLE Select and transferred into organotypic raft cultures. Slightly modified organotypic rafts are generated as previously described (Hoskins et al., 2009). Briefly, 1.2×10 6Individual HEO-derived keratinocytes were plated on 24-mm collagen matrices (rat tail, EMD Millipore) that were seeded with embedded mouse fibroblasts (J2-3T3 cells). The rafts were first cultured for 4 days with the addition of Y-27632 (10 μM) prior to exposure to the air-liquid interface to generate stratified epithelia. After 14 days, the rafts were fixed in 4% PFA and embedded in paraffin. Sections were stained with H&E and examined for histopathology by conventional microscopy.

[0138] Mouse model

[0139] All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Cincinnati Children's Hospital Medical Center (CCHMC). FoxA2 CreER Mice were obtained from Anne Moon's laboratory (Park et al., 2008), Sox2 fl / fl Mice were obtained from Richard Lang's laboratory (Shaham et al., 2009), and Sox2 CreER (Stock No. 017593, Arnold et al., 2011) were obtained from The Jackson Laboratory. Mice were housed in the CCHMC animal facility and embryos were obtained using timed matings at relevant stages. Pregnant dams were gavaged with tamoxifen at 0.12 mg / g mouse at different stages to activate CreER at the appropriate stage. Specifically, FOXA2 CreER Pregnant dams in the experiment were gavaged at 6.5 dpc to achieve efficient recombination. In Sox2 CreER In the experiment, pregnant dams were gavaged at 8.5 dpc before tracheoesophageal septation to knockout Sox2, and at 9.5 dpc during / after tracheoesophageal septation to knockout Sox2.

[0140] Xenopus laevis experiments

[0141] Adult Xenopus laevis were purchased from Nasco (Fort Atkinson, Wisconsin) and housed according to the CCMCC IACUC protocol. Ovulation, in vitro fertilization, and dejellification of embryos were performed as described (Sive et al., 2000). A mixture of morpholinos (MOs; VanRaay et al., 2005) targeting the 5′UTR (Sox2-UTR MO) and the ATG start codon (Sox2-ATG MO) of Sox2, which had been previously validated, was injected into each vegetal blastomere at the 8-cell stage (2 ng total MO per blastomere, 8 ng total per embryo) to target the endoderm. The MOs were synthesized and purchased from GeneTools. Equal amounts of control MO were used in control injections.

[0142] For Xenopus explant studies, NF20 stage foregut endoderm tissue was microdissected in 1X MBS (modified Barth's saline; Sive et al., 2000) + 50 μg / mL gentamicin sulfate (MP Biochemicals), + 4% Ficoll-400 (Sigma) and the explants were then cultured in 0.5X MBS + 0.1% fatty acid-free BSA (Fisher) + 50 μg / mL gentamicin sulfate, with or without the following concentrations of small molecules or recombinant proteins from NF25-NF38 stage (approx. 48 hours): 3.5 μM Bio (Tocris), 50 ng / mL recombinant human BMP4 (R&D systems).

[0143] In situ hybridization

[0144] Mouse sections were subjected to in situ hybridization by generating DIG-labeled probes from linearized mouse cDNA plasmids. The probes were hybridized overnight at 65 °C. The next day, the probes were washed thoroughly and then incubated overnight at 4 °C with a 1:5,000 dilution of anti-DIG alkaline phosphatase antibody (Sigma) in MAB buffer (maleic acid buffer, 100 mM maleic acid, 150 mM NaCl, pH 7.5) + 10% heat-inactivated lamb serum (Gibco) + 2% blocking reagent (Sigma). Multiple washes were performed before developing the slides with BM purple. In situ hybridization of Xenopus explants was mainly performed as described in (Sive et al., 2000). Briefly, embryos and explants were fixed overnight at 40 °C in MEMFA (0.1 M MOPS, 2 mM EGTA, 1 mM MgSO4, 3.7% formaldehyde), dehydrated directly in 100% ethanol, and stored at -20 °C. The following minor modifications to the in situ protocol were used: protease K (ThermoFisher) was used at 2 μg / mL for 10 minutes on day 1 for the explants; the RNAse A step was omitted on day 2; and finally, a 1:5,000 dilution of anti-DIG alkaline phosphatase antibody in MAB buffer + 10% heat-inactivated lamb serum + 2% blocking reagent was used on days 2 / 3.

[0145] In situ hybridization of whole mount Xenopus embryos was performed by generating an antisense DIG-labeled nkx2-1 in situ probe according to the manufacturer's instructions. The probe was generated using a linearized plasmid full-length nkx2-1 cDNA template (Small et al., 2000; XbaI for linearization, T7 for antisense RNA synthesis) with 10X DIG RNA labeling mix (Sigma).

[0146] Immunofluorescence analysis

[0147] The tissue cultures were fixed with 4% paraformaldehyde for 15 minutes at room temperature, for cryosections fixed with 4% paraformaldehyde for 2 hours at 4°C, or for paraffin-embedded / sectioned tissues and mouse embryos fixed with 4% paraformaldehyde overnight at 4°C. For paraffin embedding and sectioning, after fixation, the tissues were dehydrated and embedded in paraffin blocks. Then, the slides of paraffin sections were deparaffinized and antigen retrieval was performed in 10 mM sodium citrate for 30 minutes before staining. For cryosections, the tissues were thoroughly washed in PBS, placed overnight in 30% sucrose, embedded in OCT compound (VWR), and sectioned at a thickness of 8 μm. Usually, then the slides were permeabilized with a PBS solution of 0.5% Triton X-100 for 10 minutes, blocked with 5% normal donkey serum (Jackson ImmunoResearch) for 1 hour, and then incubated overnight at 4°C in the primary antibody. The next day, the slides were thoroughly washed in PBS, incubated in the secondary antibody (1:500) for 1 hour, and then washed thoroughly again. For EdU visualization, the applicant used the Click-iT EdU Alexa Fluor 488 Imaging Kit (Invitrogen) before blocking. For whole-mount immunofluorescence staining, the embryos were immediately placed in 100% methanol after fixation. The embryos were then permeabilized with Dent’s Bleach (4:1:1 MeOH:DMSO:30% H2O2) for 2 hours at room temperature, rehydrated with methanol washes, and blocked for several hours at room temperature to overnight at 4°C. The primary antibody was applied and the embryos were incubated overnight at 4°C. Then the embryos were thoroughly washed in a PBS solution of 0.1% Triton X-100, and then incubated overnight at 4°C in the secondary antibody. Finally, the embryos were washed again, dehydrated with methanol washes, and cleared with Murray's Clear (2:1 benzyl benzoate:benzyl alcohol, Sigma) for at least 15 minutes before imaging. A list of the antibodies and dilutions used is shown in Table 2.

[0148] RNA Isolation and qPCR

[0149] Spheres and organoids were harvested whole, including the embedded Matrigel. The collagen plugs from the organotypic raft cultures were first removed from the transwells on day 14 and then harvested whole. Total RNA was isolated using the NucleoSpin RNA kit (Macherey-Nagel) and reverse transcribed into cDNA using the SuperScript VILO cDNA Synthesis kit (ThermoFisher Scientific). For qRT-PCR, the applicant used the Quantitect SYBR-Green premix (Qiagen) and ran the reactions on a QuantStudio 6 machine (ThermoFisher Scientific). A list of the primers used is shown in Table 1.

[0150] Table 1: List of primers for qPCR analysis. Related to Figures 1-7.

[0151]

[0152]

[0153]

[0154] Table 2: List of antibodies for immunofluorescence staining. Related to Figures 1-7.

[0155]

[0156]

[0157] Source table

[0158]

[0159]

[0160]

[0161] RNA sequencing and analysis

[0162] Total transcriptome RNA sequencing of anterior foregut cultures and HEO (n = 3 for each condition or time point) was performed on Poly(A) and TruSeq libraries generated from isolated total RNA by the DNA Sequencing and Genotyping Core Facility on the Illumina Hi-Seq 2500 platform. RNA sequencing parameters were 75bp single-end sequencing with a depth of 10M reads per sample. Fastq read files for each sample were obtained and then aligned using Computational Suite for Bioinformaticians and Biologists version 2.1 (CSBB-v2.1, https: / / sourceforge.net / projects / csbb-v2-1 / ). Raw transcript counts and normalized transcripts per million (TPM) values were obtained, and differential expression and gene set enrichment analysis (GSEA, Subramanian et al., 2005) were performed using CSBB-v2.1. For differential expression, statistical and biological significance was set at P < 0.05, FDR < 0.05, log fold change > 1, and at least 3 transcript counts in 3 out of 6 samples. For heatmap visualization and hierarchical clustering analysis, Morpheus (https: / / software.broadinstitute.org / morpheus / ) was used.

[0163] Using HOMER, the anterior foregut transcriptome was cross-referenced with SOX2 and SMAD1 ChIP-seq peaks from the GEO datasets (GSE61475, Tsankov et al., 2015; GSE47058, Watanabe et al., 2014) to obtain a list of genes whose expression is potentially regulated by these transcription factors. The peak cut-off distance from the transcription start site of any given gene was set at 50 kb.

[0164] The HEO analysis was compared to previously published RNA-seq samples of in vitro-generated organoids (intestine and stomach), EPC2 cultures, and biopsies from the ENCODE Roadmap project, which included the following tissues: skin, esophagus, small intestine, stomach, colon, and lung. To compare internal data with public data, the applicant used Upper Quantile [normalization between lanes] from EDASeq [http: / / bioconductor.org / packages / release / bioc / vignettes / EDASeq / inst / doc / EDASeq.pdf]. The applicant used the UpperQuantile module of CSBB's [Computational Suite for Bioinformaticians and Biologists] version 3.0 [https: / / github.com / csbbcompbio / CSBB-v3.0]. The applicant generated an expression matrix spanning genes in internal and public samples and used the UpperQuantile module of CSBB-v3.0 for quantile normalization. The applicant then log2-transformed the quantile-normalized matrix in R.Log2.

[0165] The transformed matrix was used for all downstream analyses.

[0166] The applicant also used SVA [https: / / bioconductor.org / packages / release / bioc / vignettes / sva / inst / doc / sva.pdf] on the log2-transformed quantile-normalized matrix to check for any potential variables / surrogate variables to correct for. The applicant did not find any surrogate variables to correct for. This approach gave the applicant confidence that UpperQuantile normalization and subsequent Log2 transformation were robust enough to remove batch and sequencing effects from the data.

[0167] Quantitative and Statistical Analyses

[0168] For experiments involving sphere patterning, organoid growth, and raft assays, "n" represents the number of replicates performed in each experiment (in Matrigel cultures, 3 - 7 organoids or 30 - 50 spheres were collected per well for each replicate, and all samples from 1 well of an organotypic raft culture were considered one replicate). For animal experiments, "n" represents the number of embryos analyzed. All data quantifications are presented as mean ± SD. To compare the various conditions tested in sphere patterning and organoid growth, a two-tailed t-test with unequal (i.e., not equal) variances was used in Microsoft Excel, where *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

[0169] Details of the quantification and statistical analysis of Figures 1 - 7

[0170] Figure 1: For 1H, at least 20 spheres from two experiments were evaluated. For all qPCR results, the data represent at least 2 independent experiments for each experiment, with n = 3 wells (50 - 100 spheres per well). The RA experiments were repeated in both the H1 and iPS263.10 cell lines.

[0171] Figure 2: The data represent 2 independent experiments with n = 3 wells (an average of 30 - 50 spheres per well) in each experiment using the H1 hESC line.

[0172] Figure 3: Organoid generation represents >40 experiments across 4 hES and iPS cell lines: H1, iPS65.8, iPS72.3, iPS263.10. The qPCR data represent 2 independent experiments with n = 3 wells (3 - 12 organoids per well) and were compared to n = 5 patient biopsy samples.

[0173] Figure 4: For the HEO to organotypic raft culture experiment, n = 2 - 4 wells; for the EdU experiment, n = 6 - 10 organoids per time point. n = 5 patient esophageal biopsy samples. The experiments were performed in the H1 hESC line.

[0174] Figure 5: For Sox2-DE-LOF embryos, n = 3 embryos of each genotype at E9.5 and n = 2 embryos of each genotype of each analysis type at E11.5. For Sox2-driven Sox2 cKO embryos, the various stages of tamoxifen administration and the corresponding stage-harvested IF were analyzed in n = 3 embryos.

[0175] Figure 6: All data from human PSC-derived cultures represent 3 separate experiments with n = 3 wells per condition per experiment.

[0176] Data and software availability

[0177] The accession number for the data generated is Gene Expression Omnibus (GEO): GSE112886. This includes the organoid growth comparison (1 month versus 2 months of human esophageal organoids, Figure 3), as well as the SOX2 knockdown experiments in dorsal and ventral anterior foregut cultures (Figures 7 and Figures 14A - 14D ).

[0178] ChIP-seq data for SOX2 and SMAD1 ChIP-seq peaks were downloaded from the public database GEO with accession numbers GSE61475 (Tsankov et al., 2015) and GSE47058 (Watanabe et al., 2014), respectively. RNA-seq data for biopsies and EPC2 cultures were downloaded from the public database GEO. The accession numbers for the samples were: GSM1120313 and GSM1120314 (small intestine), GSM1010946 and GSM1120308 (lung), GSM1120307 and GSM11010960 (stomach), GSM1120315 and GSM1010974 (large intestine), GSM1010956 and GSM1120303 (esophagus), GSM2343841 and GSM234564 (calf skin), and GSM1592609 - GSM1592611 (EPC2 day 0 cultures). The complete RNA-seq processing pipeline was completed using Computational Suite for Bioinformaticians and Biologists version 2.1 (CSBB-v2.1), available from https: / / sourceforge.net / projects / csbb-v2-1 / .

[0179] Disease state

[0180] With the emergence of the human esophageal organoid model described herein, the applicant sought to apply this system to the study of various diseases affecting the esophagus. The applicant examined the role of Sox2 in the patterning and segregation of the anterior foregut and esophageal formation. Although esophageal atresia mainly focuses on the proper resolution of the esophagus and respiratory tract, its sequelae in human patients far exceed this main problem. This is because the treatment for esophageal atresia is a surgical operation to repair the anatomical defect. However, the underlying mechanisms leading to the main defect and how they impair the normal development of the esophagus (and respiratory tract) have not been resolved. Among the many genes associated with esophageal atresia, the roles of two genes, Sox2 and Fanconi anemia gene (FANCA), in esophageal development are discussed herein.

[0181] Previous studies have examined the role of Sox2 in esophageal development and homeostasis. In Sox2 hypomorphic embryos, the esophagus of mutant embryos in which foregut separation occurs has altered properties, including lack of expression of the stratified squamous epithelial markers p63 and Krt14, and expression of gastric and intestinal markers (Que et al., 2007). However, it has been challenging to interpret these results in terms of the role of Sox2 in early versus late esophageal development, as these changes could be the result of early mispatterning in the mutant foregut rather than a continued requirement for Sox2 to drive esophageal growth and maturation. Studies of the adult esophagus do suggest that Sox2 continues to play a role after the developmental stage. Cells expressing Sox2 in the esophagus are essential for maintaining homeostasis, and upon ablation, this results in a complete loss of resident basal and atypical cells in the esophagus (Arnold et al., 2011). In addition to acting as a marker for basal cells in the adult esophagus, overexpression of Sox2 in the esophagus also results in an expansion of the basal compartment and reduced differentiation of the upper basal layer (Liu et al., 2013). In addition to the esophagus, Sox2 also has a homeostatic role in other endodermal organs. In the trachea, loss of Sox2 after birth results in reduced proliferation and a decreased proportion of basal (less p63+), ciliated, and Clara cells (Que et al., 2009). However, in the adult stomach, although Sox2 can have a tumor suppressor role, it appears to be dispensable for tissue homeostasis (Sarkar et al., 2016). Nevertheless, the role of Sox2 in the developing esophagus (after foregut separation) remains to be carefully examined, which could provide insights into other problems in patients with esophageal atresia.

[0182] Fanconi anemia is a recessive disorder (for any particular FANC complementation group gene), and is associated with esophageal atresia among other GI defects, although these GI problems occur only in a subset of patients (i.e., low penetrance) (Fausett and Klingensmith, 2012). Some patients do not have severe congenital defects, although many patients have defects across multiple organ systems. Among various patients, in addition to hematological problems, the defects that occur are also similar to the symptoms of the VACTERL association, including vertebral, anal, cardiac, tracheo-esophageal, renal, and limb defects (Auerbach, 2009). Among all the FANC complementation group genes that cause Fanconi anemia, the most common are FANCA, FANCC, and FANCG (Auerbach, 2009; Nebert et al., 2016). Based on the similarity of the symptoms of Shh mutants and Fanconi anemia, it has been suggested that the Shh signaling pathway may be involved in Fanconi anemia (Lubinsky, 2015). However, in some cases of Fanconi anemia, little is known about how esophageal atresia develops. One obstacle to understanding how esophageal atresia develops in patients with Fanconi anemia is that mouse models of Fanconi anemia do not recapitulate most of the developmental abnormalities (Bakker et al., 2013). Therefore, using human organ development models can provide a breakthrough in understanding the pathogenesis of various GI malformations in Fanconi anemia.

[0183] In addition to the congenital disorder esophageal atresia, the applicant is also interested in later diseases that affect the esophagus, such as Barrett's esophagus. Barrett's esophagus (or Barrett's metaplasia) is a condition in which the stratified squamous epithelium of the esophagus is transformed into columnar (intestinal-like) epithelium, which makes the patient prone to esophageal adenocarcinoma (DeJonge et al., 2014). The underlying mechanisms of this transformation are being actively studied, and there are multiple hypotheses regarding the origin cells of this ectopic columnar epithelium. Among the more likely models include the transformation of esophageal cells into intestinal-like cells, the transformation of transitional epithelium at the gastroesophageal junction, and the transformation of submucosal glands (Jiang et al., 2017; Leedham et al., 2008; Wang et al., 2010).

[0184] Studies that previously tested this hypothesis have found that acid and bile salts may upregulate Cdx1 and Cdx2 in cultured esophageal cells by modulating the Cdx2 promoter (Huo et al., 2010; Kazumori et al., 2006, 2009; Liu et al., 2007). In addition, in culture, acid and bile salts result in downregulation of stratified squamous epithelial genes (Ghatak et al., 2013). However, Cdx2 induction alone does not appear to fully transform esophageal cells, although it can cause mild downregulation of some stratified squamous epithelial genes, upregulation of some intestinal genes (Muc2, Villin), and a mild change in epithelial morphology (Kong et al., 2011; Liu et al., 2007). BMP activation alone or in combination with Cdx2 appears to have a stronger effect on downregulating stratified squamous epithelial genes in mouse esophagus and cultured human esophageal cells, although these changes are far from complete transformation into columnar epithelium by morphology or gene expression (Mari et al., 2014). Other signaling pathways, such as Wnt and Notch, can be altered in esophageal cells exposed to bile acids (Chen et al., 2012). Notch inhibition results in some changes in cultured esophageal cells, including downregulation of stratified squamous epithelial genes, upregulation of columnar epithelial genes, and an increase in the intercellular space in the basal compartment (Kasagi et al., 2018; Vega et al., 2014). However, from these studies, it is unclear which signaling pathways contribute to the pathogenesis of Barrett's metaplasia, rather than just being altered as a result of all the other morphological changes. Thus, HEO can allow for rapid combinatorial screening of these suspect signaling pathways in a biologically more relevant model.

[0185] Another disease that affects the pediatric and adult esophagus is eosinophilic esophagitis, which is a chronic immune-mediated disease that causes feeding difficulties or food impaction, vomiting, and abdominal pain. The diseased esophagus undergoes several changes, including esophageal stricture due to fibrosis and thickening of the muscular wall, basal cell hyperplasia, and expansion of the intercellular space in the epithelium, as well as the hallmark finding of high levels of eosinophils in the esophageal epithelium (Furuta and Katzka, 2015). Traditionally, it has been thought that a damaged barrier and exposure to certain antigens initiate and then maintain the disease because the inflamed esophagus recruits immune cells and further maintains the damaged barrier (Caldwell et al., 2017; Furuta and Katzka, 2015).

[0186] The recruited T helper 2 cells (and other immune cells) secrete a variety of cytokines, which cause extensive changes in the esophageal epithelium and surrounding layers. In the epithelium, IL-13 upregulates eotaxin-3 (or CCL26), which is a chemokine that attracts eosinophils. In addition to CCL26 and other target inflammatory genes, IL-13 exposure also leads to basal cell hyperplasia in the mouse esophagus and downregulates markers of differentiated stratified squamous epithelium (such as FLG, IVL, SPRR protein family) (Blanchard et al., 2010; Jiang et al., 2015; KC et al., 2015; Rochman et al., 2017). Air-liquid interface cultures of esophageal keratinocytes treated with IL-13 also showed reduced transepithelial electrical resistance (TEER), demonstrating barrier dysfunction (D'Mello et al., 2016; Davis et al., 2016; Wu et al., 2018).

[0187] Results

[0188] The role of Sox2 in late esophageal development

[0189] To examine the role of Sox2 in esophageal development and maturation (after the esophagus has separated from the respiratory tract), the applicant conditionally knocked out Sox2 in Sox2-expressing cells using the same mouse model as in the previous chapter. The applicant mated female Sox2 fl / f mice with male Sox2 CreERT2 / + mice and gavaged the dams with tamoxifen at E11.5 and E14.5, or injected the pups with tamoxifen at P1, and then harvested them a few days after gavage to study the effects of Sox2 loss in the esophagus at various developmental stages ( Figure 1A ). Early loss of Sox2 (gavage at E11.5) led to delayed stratification at E14.5, as confirmed by the simplest columnar epithelium in the esophagus of the conditional knockout compared to the wild type, which had stratified into 2 layers at this stage ( Figure 1B ). Late knockout of Sox2 (gavage at E14.5) led to a slightly smaller esophagus, although the stratification appeared normal. Finally, early postnatal knockout of Sox2 (P1) did not appear to cause obvious changes in the esophagus ( Figure 1B ). In these embryos with Sox2 knocked out after separation of the anterior foregut, Nkx2-1 was not immediately re-expressed in the esophagus, although some cells in the esophagus of embryos gavaged at E11.5 had Nkx2-1 expression at E17.5 ( Figure 1B , 1E).

[0190] Because the most obvious changes occurred only in the gavaged E11.5 embryos, the applicant more closely examined various patterning and differentiation markers in these esophagi at E17.5. Overall, the esophageal epithelium appeared less folded (in itself) and had a larger average lumen diameter. Except for the confirmed loss of Sox2, the basal layer of the Sox2 knockout esophagus appeared normal, expressing p63 and Krt14 and lacking Krt8 expression ( Figure 1C ). However, the upper basal layer was significantly altered: Krt13 was absent while Krt8 was robustly expressed. There were proliferative (Ki67+) positive cells that were absent in the wild-type esophagus; and, the epithelial morphology remained columnar epithelium ( Figure 1C -D). However, the applicant found no evidence of Muc2 or Muc5ac production, although further future analysis (such as Alcian blue staining) may reveal mucus production (data not shown). Except for a few cells expressing Nkx2-1 in the Sox2 knockout esophagus, other key intestinal and gastric markers were not expressed in either wild-type or Sox2 knockout esophagi ( Figure 1E ). In addition to the key role of Sox2 in early patterning, these data also directly demonstrated the necessity of Sox2 for proper esophageal development and maturation after the separation of the esophagus from the respiratory tract.

[0191] Fanconi anemia and early esophageal development

[0192] In addition to Sox2, a variety of other genes are associated with esophageal atresia, and the mechanisms of foregut defects caused by mutations in some of these genes are not well understood. Loss of Fanconi anemia complementation group genes can lead to multiple problems with variable penetrance, including GI problems such as atresia (esophagus, duodenum, anus), CNS defects, kidney defects, and growth abnormalities (Auerbach, 2009; De Jong et al., 2010). To begin to understand how these genes contribute to esophageal atresia, the applicant sought to use HEOs to model the role of FANCA deficiency in esophageal development.

[0193] The applicant used an iPSC line generated from a patient lacking FANCA, which could be maintained / rescued using a dox-inducible FANCA construct. This line could successfully generate HEOs with or without doxycycline treatment as described herein ( Figure 2A ). In low or absent expression of FANCA, foregut patterning did not appear to be altered ( Figure 2B ). When dorsal anterior foregut spheres grew out (2 - 4 weeks) into HEOs, FANCA-deficient organoids consistently appeared smaller than "control" / rescued (+dox) organoids ( Figure 2C , 2F). However, at 1 month, FANCA-deficient HEOs had an increased number of KI67+ cells compared to control HEOsFigure 2D , 2G). In both cases, cell death (stained by cleaved Caspase 3) appeared to be no different (data not shown). Additionally, the applicant did not find a significant difference in the expression of stratified squamous epithelial markers between the two cases (data not shown). To confirm that the organoids respond to doxycycline treatment and express FANCA, when FANCA is present, the applicant probed the response of FANCA and FANCD2 to hydroxyurea treatment. Western blot clearly showed the knockout and rescue of FANCA with or without doxycycline treatment respectively, and the change in the size of FANCD2 protein after doxycycline and hydroxyurea treatment ( Figure 2E ).

[0194] Modeling Barrett's esophagus using HEOs

[0195] In addition to studying early defects in foregut and esophageal development, the applicant also wanted to apply HEOs to model late-stage diseases affecting the esophagus, such as Barrett's metaplasia. Considerable work has been done to try to identify the origin cells and understand the mechanisms that lead to the transformation of stratified squamous epithelium into intestinal (columnar) epithelium. However, due to the challenges in precisely modeling human pathological processes in mice, it is believed that using the HEO model can be a complementary approach to understanding how Barrett's esophagus develops.

[0196] The applicant began by focusing on early development of the foregut and esophagus. The applicant used a dox-inducible CDX2 construct stably transduced in hPSCs, which was then used to generate HEOs ( Figures 3A - 3B ). First, anterior foregut (AFG) cultures were treated with doxycycline at different doses for 1 day to measure the concentration required to induce CDX2 in most cells, which appeared to saturate at approximately 100 - 500 ng / mL ( Figures 3C - 3E ). Interestingly, SOX2 expression was not inhibited by the transient (24-hour) induction of CDX2 in anterior foregut cells, indicating that CDX2 alone does not directly inhibit SOX2 transcription ( Figures 3C - 3D ).

[0197] Continuous doxycycline treatment during the first month of HEO growth led to the upregulation of some intestinal markers in HEOs, such as CDX1 and CDH17, while MUC2 remained unchanged ( Figures 3G - 3J ). The stratified squamous marker p63 was inhibited and SOX2 showed a moderate downregulation, which was consistent with the early results of anterior foregut cultures on day 6 ( Figure 3D, 3K-3L). Additionally, the stratified squamous markers KRT5 and KRT13 were not altered by CDX2 induction in developing HEOs (data not shown). To further examine the regulatory interactions between these two major transcription factors, SOX2 in the foregut and CDX2 in the hindgut, the applicant induced SOX2 in human intestinal organoids (HIOs)( Figures 8A - 8B ). In these cultures, the applicant found that treating HIOs with SOX2 led to an (albeit mild) upregulation of the stratified squamous markers p63 and KRT13, and a downregulation of CDH17 and CDX2( Figure 8C -E, 8G-8H). In SOX2-induced HIOs, there was also an upregulation of CLDN18, a marker expressed in the stomach, although it is important to note some variability in control HIOs( Figure 8C , data not shown). PDX1, a distal stomach or proximal intestine marker, tended to be downregulated by SOX2 induction, although its expression was also often variable in control HIOs( Figure 8F ). Together, these data indicate that although SOX2 plays a strong role in suppressing mid / hindgut fates, CDX2 alone cannot robustly suppress foregut / esophageal fates during early development.

[0198] To determine whether the regulatory role of CDX2 persists during later development (or in the HEO protocol) when plasticity is classically thought to become increasingly restricted, the applicant grew HEOs to 6 weeks of age and treated them with doxycycline for 8 days( Figure 4A ). In this context, SOX2 and p63 were downregulated in response to CDX2 induction, although some HEOs responded poorly and / or had low CDX2 induction( Figures 4B - 4E , data not shown). Due to the variability of the CDX2-inducible system, the applicant chose an analysis at the cellular resolution. The applicant counted and classified cells into 3 categories: uninduced, CDX2 low (induced), and CDX2 high (induced)( Figures 4F - 4G ). Restricting the analysis to basal cells, both SOX2 and p63 were strongly inhibited in CDX2-high basal cells, while many cells could co-express SOX2 or p63 with CDX2 in CDX2-low basal cells( Figures 4H - 4I ). This indicates that these key esophageal transcription factors are effectively inhibited if all cells express CDX2 at high levels.

[0199] To further examine the role of CDX2 in suppressing esophageal (stratified squamous) epithelial identity, the applicant examined esophageal differentiation markers with CDX2 induction and in combination with the Notch (γ-secretase) inhibitor DAPT( Figure 5A ). The Notch signaling target gene HES5 in the esophagus was downregulated upon addition of DAPT(Figure 5F )。KRT5 was downregulated by CDX2 induction and further downregulated by DAPT addition, as demonstrated by some organoids that completely lost KRT5 expression ( Figure 5B , 5H). KRT13 was also downregulated, although it did not appear to synergize with DAPT treatment ( Figure 5C ). More differentiation markers IVL and CRNN were downregulated in CDX2-induced HEOs with or without DAPT treatment ( Figure 5D , 5H). Finally, CDH17 appeared to be moderately upregulated by CDX2 induction, although few organoids with visible CDH17 protein expression were found ( Figure 5E , 5H). This indicates that CDX2 can inhibit stratified squamous epithelial markers, and Notch inhibition rarely enhances this effect.

[0200] Since BMP signaling has been associated with the transformation of the esophagus into intestinal cells, the applicant also examined the role of BMP signaling in HEOs ( Figure 9A )(Mari et al., 2014). Treating 6-week-old HEOs with BMP4 for an additional 2 weeks resulted in the loss of stratification and expression of differentiation markers ( Figure 9B , 9G-9J). The remaining basal cells had active BMP signaling (pSMAD1 / 5 / 9+) and expressed p63, but lost SOX2 expression ( Figure 9B , 9D-9E). Additionally, very little EdU incorporation was introduced into BMP4-treated HEOs, meaning that the cell renewal rate was significantly slowed ( Figures 9B - 9C ). Although CDX2 induction led to the downregulation of the BMP target ID1, CDX2 expression did not change in BMP4-treated HEOs ( Figure 5G , data not shown). Together, this indicates that BMP signaling can enhance the downregulation of stratified squamous epithelium by counteracting the attenuation of BMP signaling upon CDX2 induction.

[0201] Modeling eosinophilic esophagitis using HEOs

[0202] Finally, the applicant tested whether HEOs could be used to model eosinophilic esophagitis (an inflammatory disease affecting the esophagus). The applicant focused on the epithelial response to the cytokine IL-13, which is known to cause extensive changes in the esophagus, to validate HEOs in modeling eosinophilic esophagitis. The applicant treated 6- to 8-week-old HEOs with IL-13 for different durations and examined various properties of the HEOs ( Figure 6A ). First, the applicant verified the response of the target genes CCL26, CDH26, CAPN14, and SERPINB4 to short-duration IL-13 treatment ( Figures 6B - 6E)。Long-term treatment with IL-13 maintained the upregulation of certain target genes, SERPINB13 and CDH26 ( Figure 6F )。In addition to the upregulation of these target genes, EdU incorporation increased in the most basal p63+ cells, indicating that basal cells were more proliferative upon long-term exposure to IL-13 ( Figures 6G - 6H )。

[0203] The applicant then observed the morphology and differentiation of the stratified squamous epithelium after treatment with IL-13. Treatment of HEOs with IL-13 downregulated the late differentiation and structural proteins DSG1, IVL, and CRNN, although control organoids had significant variability in expressing these differentiation markers by RNA ( Figures 7A - 7D )。In addition, the epithelium had dilated intercellular spaces basally, and upon electron microscopy examination, the spaces between individual cells increased and cell-cell contacts decreased ( Figure 7A , 7G). These data indicate that, compared to other model systems, in most cases, HEOs responded to IL-13 treatment as expected.

[0204] Finally, since BMP signaling has also been associated with eosinophilic esophagitis, particularly with the downregulation of the BMP antagonist follistatin in diseased esophagus, the applicant examined whether BMP activation could reverse certain aspects of the disease process (Jiang et al., 2015). However, unlike IL-13-induced mouse esophagus, HEOs did not upregulate the BMP antagonists NOG and FST ( Figure 7E -F) (Jiang et al., 2015). This was reflected by a modest increase in the BMP target gene ID3 in response to IL-13 (and BMP4) treatment ( Figure 7L )。Nonetheless, compared to IL-13 alone, the expression of SOX2 and PTCH1 normalized back to control after treatment of HEOs with IL-13 and BMP4 ( Figure 7H , 7K). The IL-13 targets CCL26 and CDH26 were also downregulated upon addition of BMP4 ( Figures 7I - 7J )。However, the stratified squamous markers KRT5, KRT13, IVL, and CRNN were not consistently altered upon addition of BMP4 and IL-13 compared to IL-13 treatment alone (data not shown). Thus, BMP signaling activation can reverse or counteract certain aspects of the disease process induced by IL-13.

[0205] This result shares some similarities with previously published experiments. In contrast to the hypomorphic Sox2 model, which has lower Sox2 levels throughout embryonic development, the applicant was able to examine closely when and for which processes Sox2 is necessary in esophageal development. Thus, the applicant was able to distinguish that the basal layer of the esophagus appears intact in mid-gestation embryos (E11.5) with a Sox2 knockout by the expression of Krt14 and p63, while the esophagus in earlier knockouts (E8.5) or in hypomorphic Sox2 embryos has reduced expression / absence of expression of these proteins (Que et al., 2007). This result could be due to the establishment of p63 expression in the esophagus by the time of the late Sox2 knockout, and then p63 alone may be able to maintain basal development. Another difference between the global Sox2 hypomorph and the mid-gestation Sox2 knockout is that the later Sox2 knockout esophagus does not appear to acquire a different tissue identity (intestinal, gastric, respiratory), indicating that esophageal fate is determined by E11.5. Interestingly, this result also shows that loss of Sox2 at E11.5 leads to loss of basal layer differentiation and increased suprabasal proliferation, which is similar to how Sox2 can inhibit proliferation during gastric development (Hagey et al., 2018). Late embryonic or early postnatal knockout of Sox2 appears to have little effect on the esophagus, indicating that Sox2 may not play a major role in the adult esophagus and is more similar to the stomach compared to the trachea (Que et al., 2009; Sarkar et al., 2016). Thus, although esophageal fate can be set by mid-gestation embryonic development, it turns out that persistent Sox2 expression after the initial patterning of the anterior foregut is required for proper esophageal differentiation and maturation. However, long-term studies on more mice and injury models may uncover later roles of Sox2 in the late embryonic and postnatal esophagus.

[0206] For other diseases: Fanconi anemia, Barrett's esophagus, and eosinophilic esophagitis, the applicant has attempted to use HEOs to model changes in the esophagus. When attempting to model esophageal atresia in Fanconi anemia with HEOs, the major obstacle was that only a small number of Fanconi anemia patients have GI malformations. The specific patient used to generate the iPS lines in this study did not have esophageal atresia, although FANCA mutations have been associated with esophageal atresia (Feng et al., 2018). Since loss of FANCA leads to increased sensitivity to DNA damage, the applicant examined cell death (by cleaved Caspase 3 staining), but found no differences (or rather, no significant amount of cell death in either case), which could be expected since the applicant did not add any chemotherapeutic agents. Interestingly, despite the smaller size of FANCA-deficient HEOs, there were more proliferative cells compared to control HEOs, similar to what was found in FANCA-deficient skin keratinocytes in culture (Hoskins et al., 2009). Apart from this difference, the applicant did not find changes in differentiation. One caveat to using HEOs to model esophageal atresia is that culture conditions can compensate for or supplement the defect, which could bypass most of the disease process. Additionally, the analysis at the 1-month time point may be too late since the defect may have most of its impact at an earlier stage of the protocol.

[0207] The applicant next sought to use HEOs to model Barrett's esophagus. In older HEOs, the applicant found that CDX2 induction led to downregulation of some stratified squamous markers, which contrasted with the minimal changes produced in similar experiments conducted in an esophageal keratinocyte line (EPC2) (Mari et al., 2014). The applicant's results are consistent with CDX2 induction not robustly upregulating intestinal genes, suggesting the need for additional factors, as shown by using DNA-methyltransferase inhibitors in other models (Kong et al., 2009, 2011; Mari et al., 2014). Different from previous studies using organotypic raft cultures (Vega et al., 2014), Notch inhibition appeared to only slightly (or negligibly) increase this transformation in HEOs. The applicant similarly found that BMP activation also downregulated stratified squamous genes, although in our case, this could be mediated by cell cycle arrest of the dry / progenitor cells (Mari et al., 2014).

[0208] The applicant also induced CDX2 and SOX2 in the HEO or HIO differentiation protocols, respectively, to examine the transcriptional network that regulates this switch between the alternative fates. Consistent with studies in mice, SOX2 induction in HIO downregulated CDX2 and upregulated genes of a variety of foregut lineages: p63, KRT13, and CLDN18, although the epithelium remained predominantly columnar (Kuzmichev et al., 2012). Interestingly, transient CDX2 induction in early foregut cultures did not directly regulate / suppress SOX2 expression, and some SOX2+CDX2+ double-positive cells have been shown to exist in the early endoderm (Sherwood et al., 2009). However, extending the culture and CDX2 induction to early (or late) HEO led to the suppression of SOX2 and p63, suggesting that CDX2 can alter downstream targets, which then regulate SOX2 and p63. Moreover, unlike later CDX2 induction, early onset of CDX2 induction led to a significant upregulation of CDH17, indicating that some of the early plasticity is lost upon organoid maturation. Thus, this suggests that multiple changes are required for the complete conversion of esophageal epithelium to intestinal epithelium and vice versa. Alternatively, the cells that give rise to metaplasia in Barrett's esophagus may not be stratified squamous epithelium.

[0209] Finally, HEO can be used to model the epithelial changes in eosinophilic esophagitis. Treatment of HEO with IL-13 led to responses similar to those of air-liquid interface (ALI) esophageal cultures treated with IL-13, such as downregulation of genes of differentiated stratified squamous epithelium (Blanchard et al., 2010; KC et al., 2015). In addition, increased proliferation in IL-13-treated HEO may indicate the onset of basal cell hyperplasia, although in HEO, the actual thickening of the basal layer is difficult to assess and may not occur because organoids suspended in three-dimensional cultures are able to expand freely. Similar to other studies, although barrier function was not directly assessed by measuring transepithelial electrical resistance (TEER) as in ALI cultures, signs of barrier dysfunction, including increased intercellular spaces and downregulation of DSG1, could be observed (D'Mello et al., 2016; Davis et al., 2016; Kasagi et al., 2018). Thus, it appears that HEO responds as expected to IL-13 treatment.

[0210] A variety of signaling pathways that may be misregulated in eosinophilic esophagitis were investigated. In human biopsies of eosinophilic esophagitis and experiments where IL-13 was abnormally expressed in the mouse esophagus, BMP signaling was reduced and the BMP antagonist follistatin was upregulated (Jiang et al., 2015). However, the applicant found that in HEO treated with IL-13, there were slight but opposite changes in BMP antagonist and BMP activation. Interestingly, however, BMP activation in IL-13-treated HEO reversed some of the changes that occurred in HEO treated with IL-13 alone, as demonstrated by SOX2 downregulation and certain IL-13 target genes. In addition, using PTCH1 as a readout of hedgehog signaling activity, IL-13 treatment upregulated PTCH1 (Robbins et al., 2012). This potential increase in hedgehog signaling may be associated with increased epithelial proliferation and decreased differentiation, similar to studies on the esophagus of Ptch1 mutant mice and esophageal squamous cell carcinoma (van Dop et al., 2012). By modeling these selected esophageal pathologies, the applicant has demonstrated that HEO can be used as a complementary model to better understand these and, hopefully, the mechanisms of other diseases affecting the esophagus.

[0211] Materials and Methods

[0212] mouse

[0213] Wild-type and mutant mice were used for studies of foregut and esophageal development. Sox2 fl / fl Mice were obtained from the laboratory of Richard Land (Shaham et al., 2009), and Sox2 CreER (stock number #017593, Arnold et al., 2011) were obtained from The Jackson Laboratory. Pregnant dams were gavaged with tamoxifen at 0.12 mg / g mouse at different stages to activate CreER at the appropriate stage. Mice were housed in the animal facilities of Cincinnati Children's Hospital Medical Center (CCHMC) according to the National Institutes of Health guidelines for the care and use of laboratory animals. Animals were maintained on a 12-hour light-dark cycle with free access to water and standard chow. Healthy animals were used for all experiments. All experiments were conducted with the approval of the Institutional Animal Care and Use Committee of CCHMC (protocol IACUC2016-0004).

[0214] Human ESC / IPSC and maintenance

[0215] The human embryonic stem cell (hESC) line H1 (WA01, male) was purchased from WiCell. The iPSC line iPS106 was generated in-house and approved by the Institutional Review Board of CCHMC. All hPSCs were maintained on feeder-free media: cells were plated on hESC-qualified Matrigel (BD Biosciences, San Jose, California) and maintained at 37 °C, 5% CO2, with daily medium change of mTeSR1 medium (STEMCELL Technologies, Vancouver, Canada); cells were routinely passaged every 4 days using Dispase (STEMCELL Technologies). HA-tagged SOX2, CDX2, or FANCA dox-inducible lines were generated by first cloning the human SOX2 ORF, CDX2 ORF, or FANCA ORF (respectively) into pINDUCER20 (Addgene #44012, Meerbrey et al., 2011). Next, lentiviruses were produced with the help of the Viral Vector Core Facility at CCHMC. The lentiviruses for HA-SOX2 and CDX2 were transduced into hESCs together with 2 μL of virus; these lines were maintained in selection with mTeSR1 and G418 (500 μg mL -1 , ThermoFisher Scientific). The lentivirus for FANCA was transduced prior to the generation of the iPS106 line, as FANCA is required to maintain hPSC characteristics. The hESC H1 line (and transduced derivatives) was used for all experiments except for Fanconi anemia modeling in HEO, which used the iPS106 line.

[0216] Differentiation of anterior foregut cultures and spheres

[0217] Confluent hPSC cultures were treated with Acutase (STEMCELL Technologies) to be resuspended as single cells in mTeSR1 and Y-27632 (10 μM, Tocris) and plated on Matrigel. The next day, differentiation into definitive endoderm was performed as previously described (McCracken et al., 2014). Briefly, cells were treated on day 1 with Activin A (100 ng mL-1, R&D systems, Minneapolis, Minnesota) and BMP4 (50 ng mL-1, R&D systems) in RPMI 1640 medium (Life Technologies). Cells for the subsequent two days were treated only with Activin A (100 ng mL-1) in RPMI 1640, with incremental concentrations of 0.2% and 2% HyClone defined fetal bovine serum (dFBS, GE Healthcare Life Sciences).

[0218] For the generation of anterior foregut spheroids from definitive endoderm, cells were treated with Wnt3a (500 ng mL-1, R&D systems) for 2 days and with FGF4 (500 ng mL-1, R&D systems), Noggin (200 ng mL-1) in RPMI 1640 containing 2% dFBS for 3 days. Three-dimensional cultures of anterior foregut spheroids were obtained as described above and differentiated into human esophageal organoids.

[0219] Immunofluorescence analysis

[0220] Tissue cultures were fixed with 4% paraformaldehyde for 15 minutes at room temperature (for monolayer cultures) or overnight at 4 °C (for organoids). Tissues were thoroughly washed in PBS and stained for monolayer cultures, while organoids were thoroughly washed, then placed overnight in 30% sucrose, embedded in OCT compound (VWR), and sectioned to a thickness of 8 μm. The slides were then permeabilized with a PBS solution of 0.5% Triton X-100 for 10 minutes, blocked in 5% normal donkey serum (Jackson ImmunoResearch) for at least 1 hour, and incubated overnight at 4 °C in the primary antibody. The next day, the slides were thoroughly washed in PBS, incubated in the secondary antibody (1:500) for 1 hour, and then washed thoroughly again. For EdU visualization, the Click-iT EdU Alexa Fluor 488 Imaging Kit (Invitrogen) was used before blocking. Table 1 shows the antibodies and dilutions used.

[0221] RNA isolation and qPCR

[0222] Monolayer cultures and organoids were harvested en masse, including plating / embedding in Matrigel. Total RNA was isolated using the NucleoSpin RNA kit (Macherey-Nagel) and reverse transcribed into cDNA using the SuperScript VILO cDNA Synthesis kit (ThermoFisher Scientific). For qRT-PCR, the applicant used the Quantitect SYBR-Green premix (Qiagen), and the reaction was run on a QuantStudio 6 machine (ThermoFisher Scientific). The primers used are shown in Table 2.

[0223] References

[0224] Andl, C.D., Mizushima, T., Nakagawa, H., Oyama, K., Harada, H., Chruma, K., Herlyn, M., and Rustgi, A.K. (2003). Epidermal growth factor receptor mediates increased cell proliferation, migration, and aggregation in esophageal keratinocytes in vitro and in vivo. Journal of Biological Chemistry, 278(3), 1824–1830. https: / / doi.org / 10.1074 / jbc.M209148200

[0225] Arnold, K., Sarkar, A., Yram, M.A., Polo, J.M., Bronson, R., Sengupta, S., Seandel, M., Geijsen, N., and Hochedlinger, K. (2011). Sox2+ Adult Stem and Progenitor Cells Are Important for Tissue Regeneration and Survival of Mice. Cell Stem Cell, 9(4), 317–329. https: / / doi.org / 10.1016 / j.stem.2011.09.001Bamberger, C., Pollet, D., and Schmale, H. (2002). Retinoic acid inhibits downregulation of DeltaNp63alpha expression during terminal differentiation of human primary keratinocytes. The Journal of Investigative Dermatology, 118(1), 133–8. https: / / doi.org / 10.1046 / j.0022-202x.2001.01649.x

[0226] Bayha, E., M.C., Serup, P., and Grapin-Botton, A. (2009). Retinoic acid signaling organizes endodermal organ specification along the entire antero-posterior axis. PloS One, 4(6), e5845. https: / / doi.org / 10.1371 / journal.pone.0005845

[0227] Chen, H., Li, J., Li, H., Hu, Y., Tevebaugh, W., Yamamoto, M., Que, J., and Chen, X. (2012). Transcript profiling identifies dynamic gene expression patterns and an important role for Nrf2 / Keap1 pathway in the developing mouse esophagus. PloS One, 7(5), e36504. https: / / doi.org / 10.1371 / journal.pone.0036504

[0228] Chen, Y.-W., Huang, S.X., de Carvalho, A.L.R.T., Ho, S.-H., Islam, M.N., Volpi, S., Notarangelo, L.D., Ciancanelli, M., Casanova, J.-L., Bhattacharya, J., Liang, A.F., et al. (2017). A three-dimensional model of human lung development and disease from pluripotent stem cells. Nature Cell Biology, 19(5), 542–549. https: / / doi.org / 10.1038 / ncb3510

[0229] Chen, Y., Shi, L., Zhang, L., Li, R., Liang, J., Yu, W., Sun, L., Yang, X., Wang, Y., Zhang, Y., and Shang, Y. (2008). The Molecular Mechanism Governing the Oncogenic Potential of SOX2 in Breast Cancer. Journal of Biological Chemistry, 283(26), 17969–17978. https: / / doi.org / 10.1074 / jbc.M802917200

[0230] D’Amour, K. a, Agulnick, A. D., Eliazer, S., Kelly, O. G., Kroon, E., and Baetge, E. E. (2005). Efficient differentiation of human embryonic stem cells to definitive endoderm. Nature Biotechnology, 23(12), 1534–1541. https: / / doi.org / 10.1038 / nbt1163

[0231] Daniely, Y., Liao, G., Dixon, D., Linnoila, R. I., Lori, A., Randell, S. H., Oren, M., and Jetten, A. M. (2004). Critical role of p63 in the development of a normal esophageal and tracheobronchial epithelium. American Journal of Physiology. Cell Physiology, 287(1), C171–C181. https: / / doi.org / 10.1152 / ajpcell.00226.2003 Davenport, C., Diekmann, U., Budde, I., Detering, N., and Naujok, O. (2016). The Anterior-Posterior Patterning of Definitive Endoderm Generated from Human Embryonic Stem Cells Depends on the Differential Signaling of Retinoic Acid, Wnt-and BMP-Signaling. Stem Cells, 34(11), 2635–2647. https: / / doi.org / 10.1002 / stem.2428

[0232] Dessimoz, J., Opoka, R., Kordich, J. J., Grapin-Botton, A., and Wells, J. M. (2006). FGF signaling is necessary for establishing gut tube domains along the anterior-posterior axis in vivo. Mechanisms of Development, 123(1), 42–55. https: / / doi.org / 10.1016 / j.mod.2005.10.001

[0233] DeWard, A.D., Cramer, J., and Lagasse, E. (2014). Cellular heterogeneity in the mouse esophagus implicates the presence of a nonquiescent epithelial stem cell population. Cell Reports, 9(2), 701–11. https: / / doi.org / 10.1016 / j.celrep.2014.09.027

[0234] Domyan, E.T., Ferretti, E., Throckmorton, K., Mishina, Y., Nicolis, S.K., and Sun, X. (2011). Signaling through BMP receptors promotes respiratory identity in the foregut via repression of Sox2. Development (Cambridge, England), 138(5), 971–981. https: / / doi.org / 10.1242 / dev.053694

[0235] Doupe, D.P., Alcolea, M.P., Roshan, A., Zhang, G., Klein, a.M., Simons, B.D., and Jones, P.H. (2012). A Single Progenitor Population Switches Behavior to Maintain and Repair Esophageal Epithelium. Science, 337(6098), 1091–1093. https: / / doi.org / 10.1126 / science.1218835

[0236] Dye, B.R., Dedhia, P.H., Miller, A.J., Nagy, M.S., White, E.S., Shea, L.D., and Spence, J.R. (2016). A bioengineered niche promotes in vivo engraftment and maturation of pluripotent stem cell derived human lung organoids. eLife, 5 (September 2016). https: / / doi.org / 10.7554 / eLife.19732

[0237] Dye, B.R., Hill, D.R., Ferguson, M.A., Tsai, Y.-H., Nagy, M.S., Dyal, R., Wells, J.M., Mayhew, C.N., Nattiv, R., Klein, O.D., White, E.S., et al. (2015). In vitro generation of human pluripotent stem cell derived lung organoids. eLife, 4, e05098. https: / / doi.org / 10.7554 / eLife.05098

[0238] Fantes, J., Ragge, N.K., Lynch, S.-A., McGill, N.I., Collin, J.R.O., Howard-Peebles, P.N., Hayward, C., Vivian, A.J., Williamson, K., van Heyningen, V., and FitzPatrick, D.R. (2003). Mutations in SOX2 cause anophthalmia. Nature Genetics, 33(4), 461–463. https: / / doi.org / 10.1038 / ng1120

[0239] Fausett, S.R., Brunet, L.J., and Klingensmith, J. (2014). BMP antagonism by Noggin is required in presumptive notochord cells for mammalian foregut morphogenesis. Developmental Biology, 391(1), 111–24. https: / / doi.org / 10.1016 / j.ydbio.2014.02.008

[0240] Goss, A.M., Tian, Y., Tsukiyama, T., Cohen, E.D., Zhou, D., Lu, M.M., Yamaguchi, T.P., and Morrisey, E.E. (2009). Wnt2 / 2b and beta-catenin signaling are necessary and sufficient to specify lung progenitors in the foregut. Developmental Cell, 17(2), 290–8. https: / / doi.org / 10.1016 / j.devcel.2009.06.005

[0241] Green, M.D., Chen, A., Nostro, M.-C., d’Souza, S.L., Schaniel, C., Lemischka, I.R., Gouon-Evans, V., Keller, G., and Snoeck, H.-W. (2011). Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells. Nature Biotechnology, 29(3), 267–72. https: / / doi.org / 10.1038 / nbt.1788 Harris-Johnson, K.S., Domyan, E.T., Vezina, C.M., and Sun, X. (2009). beta-Catenin promotes respiratory progenitor identity in mouse foregut. Proceedings of the National Academy of Sciences of the United States of America, 106(38), 16287–92. https: / / doi.org / 10.1073 / pnas.0902274106

[0242] Hoskins, E.E., Morris, T.A., Higginbotham, J.M., Spardy, N., Cha, E., Kelly, P., Williams, D.A., Wikenheiser-Brokamp, K.A., Duensing, S., and Wells, S.I. (2009). Fanconi anemia deficiency stimulates HPV-associated hyperplastic growth in organotypic epithelial raft culture. Oncogene, 28(5), 674–685. https: / / doi.org / 10.1038 / onc.2008.416

[0243] Jho,E.,Zhang,T.,Domon,C.,Joo,C.-K.,Freund,J.-N.,and Costantini,F.(2002).Wnt / beta-catenin / Tcf signaling induces the transcription of Axin2,anegative regulator of the signaling pathway.Molecular and Cellular Biology,22(4),1172–83.https: / / doi.org / 10.1128 / MCB.22.4.1172

[0244] Kalabis,J.,Oyama,K.,Okawa,T.,Nakagawa,H.,Michaylira,C.Z.,Stairs,D.B.,Figueiredo,J.,Mahmood,U.,Diehl,J.A.,Herlyn,M.,and Rustgi,A.K.(2008).Asubpopulation of mouse esophageal basal cells has properties of stem cellswith the capacity for self-renewal and lineage specification.Journal ofClinical Investigation,(118),3860–3869.https: / / doi.org / 10.1172 / JCI35012

[0245] Kalabis,J.,Wong,G.S.,Vega,M.E.,Natsuizaka,M.,Robertson,E.S.,Herlyn,M.,Nakagawa,H.,and Rustgi,A.K.(2012).Isolation and characterization of mouseand human esophageal epithelial cells in 3D organotypic culture.NatureProtocols,7(2),235–246.https: / / doi.org / 10.1038 / nprot.2011.437

[0246] Kasagi, Y., Chandramouleeswaran, P. M., Whelan, K. A., Tanaka, K., Giroux, V., Sharma, M., Wang, J., Benitez, A. J., DeMarshall, M., Tobias, J. W., Hamilton, K. E., et al. (2018). The Esophageal Organoid System Reveals Functional Interplay BetweenNotch and Cytokines in Reactive Epithelial Changes. Cmgh, 5(3), 333–352. https: / / doi.org / 10.1016 / j.jcmgh.2017.12.013

[0247] Kearns, N. a, Genga, R. M. J., Ziller, M., Kapinas, K., Peters, H., Brehm, M. a, Meissner, A., and Maehr, R. (2013). Generation of organized anterior foregutepithelia from pluripotent stem cells using small molecules. Stem CellResearch, 11(3), 1003–12. https: / / doi.org / 10.1016 / j.scr.2013.06.007

[0248] Kormish, J.D., Sinner, D., and Zorn, A.M. (2010). Interactions between SOX factors and Wnt / beta-catenin signaling in development and disease. Developmental Dynamics: An Official Publication of the American Association of Anatomists, 239 (August 2009), 56–68. https: / / doi.org / 10.1002 / dvdy.22046 Li, H., Zhou, H., Fu, X., and Xiao, R. (2016). Directed differentiation of human embryonic stem cells into keratinocyte progenitors in vitro: an attempt with promise of clinical use. In vitro Cellular & Developmental Biology - Animal, 52(8), 885–893. https: / / doi.org / 10.1007 / s11626-016-0024-2

[0249] Longmire, T.a, Ikonomou, L., Hawkins, F., Christodoulou, C., Cao, Y., Jean, J.C., Kwok, L.W., Mou, H., Rajagopal, J., Shen, S.S., Dowton, A.a, et al. (2012). Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells. Cell Stem Cell, 10(4), 398–411. https: / / doi.org / 10.1016 / j.stem.2012.01.019

[0250] Lustig,B.,Jerchow,B.,Sachs,M.,Weiler,S.,Pietsch,T.,Karsten,U.,van de Wetering,M.,Clevers,H.,Schlag,P.M.,Birchmeier,W.,and Behrens,J.(2002).Negative feedback loop of Wnt signaling through upregulation of conductin / axin2 in colorectal and liver tumors.Molecular and Cellular Biology,22(4),1184–93.https: / / doi.org / 10.1128 / MCB.22.4.1184

[0251] Mandegar,M.A.,Huebsch,N.,Frolov,E.B.,Shin,E.,Truong,A.,Olvera,M.P.,Chan,A.H.,Miyaoka,Y.,Holmes,K.,Spencer,C.I.,Judge,L.M.,et al.(2016).CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs.Cell Stem Cell,18(4),541–553.https: / / doi.org / 10.1016 / j.stem.2016.01.022

[0252] Matt,N.,Ghyselinck,N.B.,Wendling,O.,Chambon,P.,and Mark,M.(2003).Retinoic acid-induced developmental defects are mediated by RARβ / RXR heterodimers in the pharyngeal endoderm.Development,130(10),2083–2093.https: / / doi.org / 10.1242 / dev.00428

[0253] McCauley, H. A., and Wells, J. M. (2017). Pluripotent stem cell-derived organoids: using principles of developmental biology to grow human tissues in a dish. Development, 144(6), 958–962. https: / / doi.org / 10.1242 / dev.140731

[0254] McCracken, K. W., Aihara, E., Martin, B., Crawford, C. M., Broda, T., Treguier, J., Zhang, X., Shannon, J. M., Montrose, M. H., and Wells, J. M. (2017). Wnt / β-catenin promotes gastric fundus specification in mice and humans. Nature, 541(7636), 182–187. https: / / doi.org / 10.1038 / nature21021

[0255] McCracken, K. W., Catá, E. M., Crawford, C. M., Sinagoga, K. L., Schumacher, M., Rockich, B. E., Tsai, Y.-H., Mayhew, C. N., Spence, J. R., Zavros, Y., and Wells, J. M. (2014). Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature, 516(7531), 400–404. https: / / doi.org / 10.1038 / nature13863

[0256] McLin, V. a, Rankin, S. a, and Zorn, A. M. (2007). Repression of Wnt / beta-catenin signaling in the anterior endoderm is essential for liver and pancreas development. Development (Cambridge, England), 134(12), 2207–17. https: / / doi.org / 10.1242 / dev.001230

[0257] Meerbrey, K. L., Hu, G., Kessler, J. D., Roarty, K., Li, M. Z., Fang, J. E., Herschkowitz, J. I., Burrows, A.

[0258] Ciccia, A., Sun, T., Schmitt, E. M., et al. (2011). The pINDUCER lentiviral toolkit for inducible RNA interference in vitro and in vivo. Proceedings of the National Academy of Sciences of the United States of America, 108(9), 3665–3670. https: / / doi.org / 10.1073 / pnas.1019736108

[0259] Múnera, J. O., Sundaram, N., Rankin, S. A., Hill, D., Watson, C., Mahe, M., Vallance, J. E., Shroyer, N. Sinagoga, K. L., Zarzoso-Lacoste, A., Hudson, J. R., et al. (2017). Differentiation of Human Pluripotent Stem Cells into Colonic Organoids via Transient Activation of BMP Signaling. Cell Stem Cell, 21(1), 51–64.e6. https: / / doi.org / 10.1016 / j.stem.2017.05.020

[0260] Niederreither, K., Subbarayan, V., Dolle, P., and Chambon, P. (1999). Embryonic retinoic acid synthesis is essential for early mouse post-implantation development. Nature Genetics, 21(4), 444–448. https: / / doi.org / 10.1038 / 7788

[0261] Park, E. J., Sun, X., Nichol, P., Saijoh, Y., Martin, J. F., and Moon, A. M. (2008). System for tamoxifen-inducible expression of Cre-recombinase from the Foxa2 locus in mice. Developmental Dynamics, 237(2), 447–453. https: / / doi.org / 10.1002 / dvdy.21415

[0262] Que, J., Choi, M., Ziel, J. W., Klingensmith, J., and Hogan, B. L. M. (2006). Morphogenesis of the trachea and esophagus: current players and new roles for noggin and Bmps. Differentiation, 74(7), 422–437. https: / / doi.org / 10.1111 / j.1432-0436.2006.00096.x

[0263] Que, J., Okubo, T., Goldenring, J. R., Nam, K.-T., Kurotani, R., Morrisey, E. E., Taranova, O., Pevny, L. H., and Hogan, B. L. M. (2007). Multiple dose-dependent roles for Sox2 in the patterning and differentiation of anterior foregut endoderm. Development (Cambridge, England), 134(13), 2521–31. https: / / doi.org / 10.1242 / dev.003855

[0264] Rankin, S. A., Han, L., McCracken, K. W., Kenny, A. P., Anglin, C. T., Grigg, E. A., Crawford, C. M., Wells, J. M., Shannon, J. M., and Zorn, A. M. (2016). A Retinoic Acid-Hedgehog Cascade Coordinates Mesoderm-Inducing Signals and Endoderm Competence during Lung Specification. Cell Reports, 16(1), 66–78. https: / / doi.org / 10.1016 / J.CELREP.2016.05.060

[0265] Rankin, S. a, Gallas, A. L., Neto, A., Gómez-Skarmeta, J. L., and Zorn, A. M. (2012). Suppression of Bmp4 signaling by the zinc-finger repressors Osr1 and Osr2 is required for Wnt / β-catenin-mediated lung specification in Xenopus. Development (Cambridge, England), 139(16), 3010–20. https: / / doi.org / 10.1242 / dev.078220

[0266] Rosekrans, S.L., Baan, B., Muncan, V., and van den Brink, G.R. (2015). Esophageal development and epithelial homeostasis. American Journal of Physiology - Gastrointestinal and Liver Physiology, 309(4), G216 - 228. https: / / doi.org / 10.1152 / ajpgi.00088.2015

[0267] Shaham, O., Smith, A.N., Robinson, M.L., Taketo, M.M., Lang, R.a, and Ashery - Padan, R. (2009). Pax6 is essential for lens fiber cell differentiation. Development (Cambridge, England), 136(15), 2567–2578. https: / / doi.org / 10.1242 / dev.032888

[0268] Sherwood, R.I., Chen, T.-Y.A., and Melton, D. (2009). Transcriptional dynamics of endodermal organ formation. Developmental Dynamics: An Official Publication of the American Association of Anatomists, 238(1), 29–42. https: / / doi.org / 10.1002 / dvdy.21810

[0269] Sinner, D., Kordich, J. J., Spence, J. R., Opoka, R., Rankin, S., Lin, S. J., Jonatan, D., Zorn, A. M., and Wells, J. M. (2007). Sox17 and Sox4 differentially regulate beta-catenin / T-cell factor activity and proliferation of colon carcinoma cells. Molecular and Cellular Biology, 27(22), 7802–7815. https: / / doi.org / 10.1128 / MCB.02179-06

[0270] Sive, H., Grainger, R., and Harland, R. (2000). Early Development of Xenopus laevis: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

[0271] Spence, J. R., Mayhew, C. N., Rankin, S. a, Kuhar, M. F., Vallance, J. E., Tolle, K., Hoskins, E. E., Kalinichenko, V. V, Wells, S. I., Zorn, A. M., Shroyer, N. F., and Wells, J. M. (2011). Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature, 470(7332), 105–109. https: / / doi.org / 10.1038 / nature09691

[0272] Stevens, M. L., Chaturvedi, P., Rankin, S. A., Macdonald, M., Jagannathan, S., Yukawa, M., Barski, A., and Zorn, A. M. (2017). Genomic integration of Wnt / β-catenin and BMP / Smad1 signaling coordinates foregut and hindgut transcriptional programs. Development, 144(7), 1283–1295. https: / / doi.org / 10.1242 / dev.145789

[0273] Subramanian, A., Tamayo, P., Mootha, V. K., Mukherjee, S., Ebert, B. L., Gillette, M. A., Paulovich, A., Pomeroy, S. L., Golub, T. R., Lander, E. S., and Mesirov, J. P. (2005). Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles. Proceedings of the National Academy of Sciences, 102(43), 15545–15550. https: / / doi.org / 10.1073 / pnas.0506580102

[0274] Tiso, N., Filippi, A., Pauls, S., Bortolussi, M., and Argenton, F. (2002). BMP signalling regulates anteroposterior endoderm patterning in zebrafish. Mechanisms of Development, 118(1–2), 29–37. Retrieved from http: / / www.ncbi.nlm.nih.gov / pubmed / 12351167

[0275] Tsankov, A.M., Gu, H., Akopian, V., Ziller, M.J., Donaghey, J., Amit, I., Gnirke, A., and Meissner, A. (2015). Transcription factor binding dynamics during human ES cell differentiation. Nature, 518(7539), 344–9. https: / / doi.org / 10.1038 / nature14233

[0276] Van Raay, T.J., Moore, K.B., Iordanova, I., Steele, M., Jamrich, M., Harris, W.A., and Vetter, M.L. (2005). Frizzled 5 signaling governs the neural potential of progenitors in the developing Xenopus retina. Neuron, 46(1), 23–36. https: / / doi.org / 10.1016 / j.neuron.2005.02.023

[0277] Wang, Z., Dollé, P., Cardoso, W.V., and Niederreither, K. (2006). Retinoic acid regulates morphogenesis and patterning of posterior foregut derivatives. Developmental Biology, 297(2), 433–445. https: / / doi.org / 10.1016 / j.ydbio.2006.05.019

[0278] Watanabe, H., Ma, Q., Peng, S., Adelmant, G., Swain, D., Song, W., Fox, C., Francis, J. M., Pedamallu, C. S., DeLuca, D. S., Brooks, A. N., et al. (2014). SOX2 and p63 colocalize at genetic loci in squamous cell carcinomas. Journal of Clinical Investigation, 124(4), 1636–1645. https: / / doi.org / 10.1172 / JCI71545 Williamson, K. a., Hever, A. M., Rainger, J., Rogers, R. C., Magee, A., Fiedler, Z., Keng, W. T., Sharkey, F. H., McGill, N., Hill, C. J., Schneider, A., et al. (2006). Mutations in SOX2 cause anophthalmia-esophageal-genital (AEG) syndrome. Human Molecular Genetics, 15(9), 1413–1422. https: / / doi.org / 10.1093 / hmg / ddl064

[0279] Woo, J., Miletich, I., Kim, B.-M., Sharpe, P. T., and Shivdasani, R. a. (2011). Barx1-mediated inhibition of Wnt signaling in the mouse thoracic foregut controls tracheo-esophageal septation and epithelial differentiation. PloSOne, 6(7), e22493. https: / / doi.org / 10.1371 / journal.pone.0022493

[0280] Zhang, Y., Jiang, M., Kim, E., Lin, S., Liu, K., Lan, X., and Que, J. (2016). Development and Stem Cells of the Esophagus. Seminars in Cell & Developmental Biology, 66, 25–35. https: / / doi.org / 10.1016 / j.semcdb.2016.12.008

[0281] Zhou, C., Yang, X., Sun, Y., Yu, H., Zhang, Y., and Jin, Y. (2016). Comprehensive profiling reveals mechanisms of SOX2-mediated cell fate specification in human ESCs and NPCs. Cell Research, 26(2), 171–189. https: / / doi.org / 10.1038 / cr.2016.15

[0282] Zorn, A. M., and Wells, J. M. (2007). Molecular Basis of Vertebrate Endoderm Development. In International Review of Cytology (Vol. 259, pp. 49–111). https: / / doi.org / 10.1016 / S0074-7696(06)59002-3 Zorn, A. M., and Wells, J. M. (2009). Vertebrate Endoderm Development and Organ Formation. Annual Review of Cell and Developmental Biology, 25(1), 221–251. https: / / doi.org / 10.1146 / annurev.cellbio.042308.113344

[0283] Arnold, K., Sarkar, A., Yram, M. A., Polo, J. M., Bronson, R., Sengupta, S., Seandel, M., Geijsen, N., and Hochedlinger, K. (2011). Sox2+ Adult Stem and Progenitor Cells Are Important for Tissue Regeneration and Survival of Mice. Cell Stem Cell, 9(4), 317–329. https: / / doi.org / 10.1016 / j.stem.2011.09.001

[0284] Auerbach, A. D. (2009). Fanconi anemia and its diagnosis. Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, 668(1–2), 4–10. https: / / doi.org / 10.1016 / j.mrfmmm.2009.01.013

[0285] Bakker, S. T., de Winter, J. P., and Riele, H. t. (2013). Learning from a paradox: recent insights into Fanconi anaemia through studying mouse models. Disease Models & Mechanisms, 6(1), 40–47. https: / / doi.org / 10.1242 / dmm.009795

[0286] Blanchard,C.,Stucke,E.M.,Burwinkel,K.,Caldwell,J.M.,Collins,M.H.,Ahrens,A.,Buckmeier,B.K.,Jameson,S.C.,Greenberg,A.,Kaul,A.,Franciosi,J.P.,etal.(2010).Coordinate Interaction between IL-13 and Epithelial DifferentiationCluster Genes in Eosinophilic Esophagitis.The Journal of Immunology,184(7),4033–4041.https: / / doi.org / 10.4049 / jimmunol.0903069

[0287] Caldwell,J.M.,Paul,M.,and Rothenberg,M.E.(2017).Novel immunologicmechanisms in eosinophilic esophagitis.Current Opinion in Immunology,48,114–121.https: / / doi.org / 10.1016 / j.coi.2017.08.006

[0288] Chen,X.,Jiang,K.,Fan,Z.,Liu,Z.,Zhang,P.,Zheng,L.,Peng,N.,Tong,J.,andJi,G.(2012).Aberrant expression of Wnt and Notch signal pathways in Barrett’sesophagus.Clinics and Research in Hepatology and Gastroenterology,36(5),473–483.https: / / doi.org / 10.1016 / j.clinre.2012.06.001

[0289] D’Mello, R.J., Caldwell, J.M., Azouz, N.P., Wen, T., Sherrill, J.D., Hogan, S.P., and Rothenberg, M.E. (2016). LRRC31 is induced by IL-13 and regulates kallikrein expression and barrier function in the esophageal epithelium. Mucosal Immunology, 9(3), 744–756. https: / / doi.org / 10.1038 / mi.2015.98

[0290] Davis, B.P., Stucke, E.M., Khorki, M.E., Litosh, V.A., Rymer, J.K., Rochman, M., Travers, J., Kottyan, L.C., and Rothenberg, M.E. (2016). Eosinophilic esophagitis–linked calpain 14 is an IL-13–induced protease that mediates esophageal epithelial barrier impairment. JCI Insight, 1(4). https: / / doi.org / 10.1172 / jci.insight.86355

[0291] De Jong, E.M., Felix, J.F., De Klein, A., and Tibboel, D. (2010). Etiology of esophageal atresia and tracheoesophageal fistula: “Mind the gap.” Current Gastroenterology Reports, 12(3), 215–222. https: / / doi.org / 10.1007 / s11894-010-0108-1

[0292] De Jonge, P. J. F., Van Blankenstein, M., Grady, W. M., and Kuipers, E. J. (2014). Barrett’s oesophagus: Epidemiology, cancer risk and implications for management. Gut, 63(1), 191–202. https: / / doi.org / 10.1136 / gutjnl-2013-305490

[0293] Fausett, S. R., and Klingensmith, J. (2012). Compartmentalization of the foregut tube: developmental origins of the trachea and esophagus. Wiley Interdisciplinary Reviews. Developmental Biology, 1(2), 184–202. https: / / doi.org / 10.1002 / wdev.12

[0294] Feng, Y., Chen, R., Da, M., Qian, B., and Mo, X. (2018). Identification of rare heterozygous missense mutations in FANCA in esophageal atresia patients using next generation sequencing. Gene, 661(January), 182–188. https: / / doi.org / 10.1016 / j.gene.2018.03.097

[0295] Furuta, G. T., and Katzka, D. A. (2015). Eosinophilic Esophagitis. New England Journal of Medicine, 373(17), 1640–1648. https: / / doi.org / 10.1056 / NEJMra1502863

[0296] Ghatak, S., Reveiller, M., Toia, L., Ivanov, A., Godfrey, T. E., and Peters, J. H. (2013). Bile acid at low pH reduces squamous differentiation and activates EGFR signaling in esophageal squamous cells in 3-D culture. Journal of Gastrointestinal Surgery: Official Journal of the Society for Surgery of the Alimentary Tract, 17(10), 1723–31. https: / / doi.org / 10.1007 / s11605-013-2287-1

[0297] Hagey, D. W., Klum, S., Kurtsdotter, I., Zaouter, C., Topcic, D., Andersson, O., Bergsland, M., and Muhr, J. (2018). SOX2 regulates common and specific stem cell features in the CNS and endoderm derived organs. PLOS Genetics, 14(2), e1007224. https: / / doi.org / 10.1371 / journal.pgen.1007224

[0298] Huo, X., Zhang, H. Y., Zhang, X. I., Lynch, J. P., Strauch, E. D., Wang, J. Y., Melton, S. D., Genta, R. M., Wang, D. H., Spechler, S. J., and Souza, R. F. (2010). Acid and Bile Salt-Induced CDX2 Expression Differs in Esophageal Squamous Cells From Patients With and Without Barrett’s Esophagus. Gastroenterology, 139(1), 194–203.e1. https: / / doi.org / 10.1053 / j.gastro.2010.03.035

[0299] Jiang, M., Ku, W., Zhou, Z., Dellon, E. S., Falk, G. W., Nakagawa, H., Wang, M., Liu, K., Wang, J., Katzka, D. a, Peters, J. H., et al. (2015). BMP-driven NRF2 activation in esophageal basal cell differentiation and eosinophilic esophagitis. The Journal of Clinical Investigation, 125(14), 1–12. https: / / doi.org / 10.1172 / JCI78850DS1

[0300] Jiang, M., Li, H., Zhang, Y., Yang, Y., Lu, R., Liu, K., Lin, S., Lan, X., Wang, H., Wu, H., Zhu, J., et al. (2017). Transitional basal cells at the squamous-columnar junction generate Barrett’s oesophagus. Nature, 550(7677), 529–533. https: / / doi.org / 10.1038 / nature24269

[0301] Kasagi, Y., Chandramouleeswaran, P. M., Whelan, K. A., Tanaka, K., Giroux, V., Sharma, Wang, J., Benitez, A. J., DeMarshall, M., Tobias, J. W., Hamilton, K. E., et al. (2018). The Esophageal Organoid System Reveals Functional Interplay BetweenNotch and Cytokines in Reactive Epithelial Changes. Cmgh, 5(3), 333–352. https: / / doi.org / 10.1016 / j.jcmgh.2017.12.013

[0302] Kazumori, H., Ishihara, S., and Kinoshita, Y. (2009). Roles of caudal-related homeobox gene Cdx1 in oesophageal epithelial cells in Barrett’sepithelium development. Gut, 58(5), 620–628. https: / / doi.org / 10.1136 / gut.2008.152975

[0303] Kazumori, H., Ishihara, S., Rumi, M. A. K., Kadowaki, Y., and Kinoshita, Y. (2006). Bile acids directly augment caudal related homeobox gene Cdx2expression in oesophageal keratinocytesinBarrett’sepithelium. Gut, 55(1), 16–25. https: / / doi.org / 10.1136 / gut.2005.066209

[0304] KC, K., Rothenberg, M. E., and Sherrill, J. D. (2015). In vitro model for studying esophageal epithelial differentiation and allergic inflammatory responses identifies keratin involvement in eosinophilic esophagitis. PloS One, 10(6), e0127755. https: / / doi.org / 10.1371 / journal.pone.0127755

[0305] Kong, J., Crissey, M. A., Funakoshi, S., Kreindler, J. L., and Lynch, J. P. (2011). Ectopic Cdx2 expression in murine esophagus models an intermediate stage in the emergence of Barrett’s esophagus. PLoS ONE, 6(4), 1–12. https: / / doi.org / 10.1371 / journal.pone.0018280

[0306] Kong, J., Nakagawa, H., Isariyawongse, B. K., Funakoshi, S., Silberg, D. G., Rustgi, A. and Lynch, J. P. (2009). Induction of intestinalization in human esophageal keratinocytes is a multistep process. Carcinogenesis, 30(1), 122–130. https: / / doi.org / 10.1093 / carcin / bgn227

[0307] Kuzmichev, A.N., Kim, S.K., D’Alessio, A.C., Chenoweth, J.G., Wittko, I.M., Campanati, and McKay, R.D. (2012). Sox2 acts through Sox21 to regulate transcription in pluripotent and differentiated cells. Current Biology, 22(18), 1705–1710. https: / / doi.org / 10.1016 / j.cub.2012.07.013

[0308] Leedham, S.J., Preston, S.L., McDonald, S.A.C., Elia, G., Bhandari, P., Poller, D., Harrison, R., Novelli, M.R., Jankowski, J.A., and Wright, N.A. (2008). Individual crypt genetic heterogeneity and the origin of metaplastic glandular epithelium in human Barrett’s oesophagus. Gut, 57(8), 1041–1048. https: / / doi.org / 10.1136 / gut.2007.143339

[0309] Liu, K., Jiang, M., Lu, Y., Chen, H., Sun, J., Wu, S., Ku, W.-Y., Nakagawa, H., Kita, Y., Natsugoe, S., Peters, J.H., et al. (2013). Sox2 Cooperates with Inflammation-Mediated Stat3 Activation in the Malignant Transformation of Foregut Basal Progenitor Cells. Cell Stem Cell, 12(3), 304–315. https: / / doi.org / 10.1016 / j.stem.2013.01.007

[0310] Liu, T., Zhang, X., So, C. K., Wang, S., Wang, P., Yan, L., Myers, R., Chen, Z., Patterson, A. P., Yang, C. S., and Chen, X. (2007). Regulation of Cdx2 expression by promoter methylation, and effects of Cdx2 transfection on morphology and gene expression of human esophageal epithelial cells. Carcinogenesis, 28(2), 488–496. https: / / doi.org / 10.1093 / carcin / bgl176

[0311] Lubinsky, M. (2015). Sonic Hedgehog, VACTERL, and Fanconi anemia: Pathogenetic connections and therapeutic implications. American Journal of Medical Genetics, Part A, 167(11), 2594–2598. https: / / doi.org / 10.1002 / ajmg.a.37257

[0312] Mari, L., Milano, F., Parikh, K., Straub, D., Everts, V., Hoeben, K. K., Fockens, P., Buttar, N. S., and Krishnadath, K. K. (2014). A pSMAD / CDX2 complex is essential for the intestinalization of epithelial metaplasia. Cell Reports, 7(4), 1197–1210. https: / / doi.org / 10.1016 / j.celrep.2014.03.074

[0313] Meerbrey, K. L., Hu, G., Kessler, J. D., Roarty, K., Li, M. Z., Fang, J. E., Herschkowitz, J. I., Burrows, A. E., Ciccia, A., Sun, T., Schmitt, E. M., et al. (2011). The pINDUCER lentiviral toolkit for inducible RNA interference in vitro and in vivo. Proceedings of the National Academy of Sciences, 108(9), 3665–3670.

[0314] https: / / doi.org / 10.1073 / pnas.1019736108

[0315] Nebert, D. W., Dong, H., Bruford, E. A., Thompson, D. C., Joenje, H., and Vasiliou, V. (2016). Letter to the editor for “Update of the human and mouse Fanconi anemia genes.” Human Genomics, 10(1), 25. https: / / doi.org / 10.1186 / s40246-016-0081-3

[0316] Que, J., Luo, X., Schwartz, R. J., and Hogan, B. L. M. (2009). Multiple roles for Sox2 in the developing and adult mouse trachea. Development (Cambridge, England), 136(11), 1899–1907. https: / / doi.org / 10.1242 / dev.034629

[0317] Robbins, D. J., Fei, D. L., and Riobo, N. A. (2012). The Hedgehog Signal Transduction Network. Science Signaling, 5(246), re6-re6. https: / / doi.org / 10.1126 / scisignal.2002906

[0318] Rochman, M., Travers, J., Miracle, C. E., Bedard, M. C., Wen, T., Azouz, N. P., Caldwell, J. M., KC, K., Sherrill, J. D., Davis, B. P., Rymer, J. K., et al. (2017). Profound loss of esophageal tissue differentiation in patients with eosinophilic esophagitis. Journal of Allergy and Clinical Immunology, 140(3), 738–749.e3.

[0319] https: / / doi.org / 10.1016 / j.jaci.2016.11.042

[0320] Sarkar, A., Huebner, A. J., Sulahian, R., Anselmo, A., Xu, X., Flattery, K., Desai, N., Sebastian, C., Yram, M. A., Arnold, K., Rivera, M., et al. (2016). Sox2 Suppresses Gastric Tumorigenesis in Mice. Cell Reports, 16(7), 1929–1941.

[0321] https: / / doi.org / 10.1016 / j.celrep.2016.07.034

[0322] van Dop, W.a., Rosekrans, S.L., Uhmann, a., Jaks, V., Offerhaus, G.J.a., vanden Bergh Weerman, M.a., Kasper, M., Heijmans, J., Hardwick, J.C.H., Verspaget, H.W., Hommes, D.W., et al. (2012). Hedgehog signalling stimulates precursor cell accumulation and impairs epithelial maturation in the murine oesophagus. Gut, 62(3), 348–57. https: / / doi.org / 10.1136 / gutjnl-2011-301141

[0323] Vega, M.E., Giroux, V.V., Natsuizaka, M., Liu, M., Klein-Szanto, A.J., Stairs, D.B., Nakagawa, H., Wang, K.K., Wang, T.C., Lynch, J.P., and Rustgi, A.K. (2014). Inhibition of notch signaling enhances transdifferentiation of the esophageal squamous epithelium towards a Barrett’s-like metaplasia via KLF4. Cell Cycle, 13(24), 3857–3866. https: / / doi.org / 10.4161 / 15384101.2014.972875

[0324] Wang, D. H., Clemons, N. J., Miyashita, T., Dupuy, A. J., Zhang, W., Szczepny, A., Corcoran-Schwartz, I. M., Wilburn, D. L., Montgomery, E. A., Wang, J. S., Jenkins, N. A., et al. (2010). Aberrant Epithelial-Mesenchymal Hedgehog Signaling Characterizes Barrett’s Metaplasia. Gastroenterology, 138(5), 1810–1822.e2.

[0325] https: / / doi.org / 10.1053 / j.gastro.2010.01.048

[0326] Wu, L., Oshima, T., Li, M., Tomita, T., Fukui, H., Watari, J., and Miwa, H. (2018). Filaggrin and tight junction proteins are crucial for IL-13-mediated esophageal barrier dysfunction. American Journal of Physiology-Gastrointestinal and Liver Physiology, ajpgi.00404.2017. https: / / doi.org / 10.1152 / ajpgi.00404.2017

[0327] Unless otherwise specified, all percentages and ratios are by weight.

[0328] Unless otherwise specified, all percentages and ratios are based on the total composition.

[0329] It should be understood that every maximum numerical limitation given at any point in this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given at any point in this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given at any point in this specification will include every narrower numerical range that falls within the broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0330] The dimensions and values disclosed herein should not be construed as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to represent the recited value and a functionally equivalent range around that value. For example, a dimension disclosed as "20 mm" is intended to represent "about 20 mm".

[0331] Unless expressly excluded or otherwise limited, each document cited herein, including any cross-referenced or related patent or application, is incorporated herein by reference in its entirety. Citing any document does not imply admission that it is prior art to any invention disclosed or claimed herein, or that it teaches, suggests, or discloses any such invention, alone or in combination with any other reference. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

[0332] Although specific embodiments of the invention have been shown and described, it will be apparent to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. Accordingly, all such changes and modifications are intended to be covered by the appended claims within the scope of the invention.

Claims

1. A method for preparing esophageal organoids in vitro, comprising: a. Activating the Wnt signaling pathway and the FGF signaling pathway and inhibiting the BMP signaling pathway in a culture medium containing 2 μM retinoic acid for a first period of time to form an anterior foregut culture, wherein the definitive endoderm is derived from induced pluripotent stem cells, wherein the anterior foregut culture expresses SOX2 and HNF1B, and wherein the first period of time is three days ± 24 hours; b. Inhibiting the BMP signaling pathway of the anterior foregut culture and activating the EGF signaling pathway for a second period of time to form dorsal anterior foregut spheres, wherein the dorsal anterior foregut expresses SOX2 and TP63 but does not express PDX1, PAX9 or NKX2.1, and wherein the second period of time is three days ± 24 hours; and c. Culturing the dorsal anterior foregut in the presence of EGF for a third period of time to allow the formation of esophageal organoids, wherein the third period of time is from 21 days to 90 days; wherein the Wnt signaling pathway is activated by a Wnt signaling pathway activator Wnt3a, and wherein the Wnt signaling pathway activator is at a concentration of 50 to 1500 ng / ml; wherein the retinoic acid contacts the definitive endoderm for a period of 12 hours to 48 hours; wherein the FGF signaling pathway is activated by an FGF signaling pathway activator of at least one of FGF4, FGF10 or a combination thereof, and wherein the FGF signaling pathway activator is present at a concentration of 50 to 1500 ng / ml; wherein the BMP signaling pathway is inhibited using a BMP signaling pathway inhibitor Noggin at a concentration of 50 to 1500 ng / ml; wherein the EGF signaling pathway is activated using an EGF signaling pathway activator EGF, and wherein the EGF is present at a concentration of 100 ng / ml.

2. The method according to claim 1, wherein culturing the dorsal anterior foregut further comprises activating the FGF signaling pathway, wherein the FGF signaling pathway is activated by an FGF signaling pathway activator, wherein the FGF signaling pathway activator is at least one of FGF4, FGF10 or a combination thereof, and wherein the FGF signaling pathway activator is at a concentration of 50 to 1500 ng / ml.

3. The method according to claim 1, wherein the definitive endoderm is derived from iPSCs by contacting pluripotent stem cells with one or more molecules selected from the group consisting of activin, members of the BMP subgroup of the TGF-β superfamily of growth factors, Nodal, Activin A, Activin B, BMP4, Wnt3a and combinations thereof.

4. The method according to claim 1, wherein the definitive endoderm is a definitive endoderm monolayer, and wherein more than 90% of the cells in the definitive endoderm monolayer co-express FOXA2 and SOX17.

5. The method according to claim 1, wherein the third time period is 60 to 90 days, and wherein the esophageal organoid is characterized by forming a stratified epithelium lacking KRT8 expression and / or a stratified epithelium expressing KRT13, KRT14, and IVL.

6. The method according to claim 1, further comprising: contacting the anterior foregut culture or the dorsal anterior foregut with a matrix selected from collagen, basement membrane matrix, or a combination thereof.

7. The method according to claim 1, wherein in step c, the dorsal anterior foregut is cultured in the presence of EGF at a concentration of 100 ng / ml.

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