阅读说明：本技术 食道组织和/或类器官组合物及其制备方法 (Esophageal tissue and/or organoid compositions and methods of making same ) 是由 J·M·威尔斯 S·特里斯诺 于 2018-10-05 设计创作，主要内容包括：本公开涉及通过定向分化将哺乳动物定形内胚层(DE)细胞转化为特定组织或器官的方法。特别地,本公开涉及从分化的定形内胚层形成的食道组织和/或类器官的形成。(The present disclosure relates to methods for transforming mammalian Definitive Endoderm (DE) cells to 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 shaped endosymbiods.)

1. A method of preparing an Esophageal Organoid (EO), comprising:

a. contacting definitive endoderm with a BMP inhibitor, a Wnt activator, a FGF activator, and Retinoic Acid (RA) for a first period of time to form a foregut culture, wherein the foregut culture expresses SOX2 and HNF1B, wherein the foregut culture does not substantially express PROX1 and HNF 6;

b. contacting the foregut culture with a BMP inhibitor (Noggin) and an EGF activator for a second period of time sufficient to form a dorsal foregut ("dAFG") sphere, wherein the dAFG expresses SOX2 and TP63 but does not express PDX1, PAX9, or NKX 2.1;

c. culturing said dAFG for a third period of time sufficient to allow formation of Esophageal Organoids (EO), wherein said culturing is in the presence of EGF, further optionally comprising an FGF signaling pathway activator, preferably FGF 10.

2. The method of claim 1, wherein the BMP signaling pathway inhibitor is selected from Noggin, Dorsomorphin, LDN189DMH-1, and combinations thereof, preferably wherein the BMP signaling pathway inhibitor is Noggin.

3. The method of claim 1 or 2, wherein the BMP inhibitor is present in a concentration of about 50 to about 1500 ng/ml.

4. The method according to any of the preceding claims, wherein the WNT activator is selected from one or more molecules selected from the group consisting of: wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, GSK β inhibitors (e.g. CHIR99021, i.e. "CHIRON"), BIO, LY2090314, SB-216763, lithium, porcine inhibitor IWP, LGK974, C59, SFRP inhibitor WAY-316606, β -catenin activator DCA.

5. The method according to any of the preceding claims, wherein the concentration of the Wnt pathway activator is at a concentration of about 50 to about 1500 ng/ml.

6. The method of any one of the preceding claims, wherein the FGF activator is selected from one or more molecules selected from the group consisting of: 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.

7. The method of any one of the preceding claims, wherein the concentration of the FGF pathway activator is a concentration of about 50 to about 1500 ng/ml.

8. The method of any one of the preceding claims, wherein the first period of time is about three days ± 24 hours.

9. The method of any one of the preceding claims, wherein the second period of time is three days ± 24 hours

10. The method of any one of the preceding claims, wherein the third period of time is about 28 days ± 48 hours, or about 21 days to about 90 days, or about 30 days to about 60 days.

11. The method according to any of the preceding claims, wherein the definitive endoderm is derived from a precursor cell selected from the group consisting of embryonic stem cells, embryonic germ cells, induced pluripotent stem cells, mesodermal cells, definitive endoderm cells, posterior endoderm cells and posterior intestinal cells, preferably derived from the definitive endoderm of pluripotent stem cells, more preferably derived from the definitive endoderm of pluripotent stem cells selected from the group consisting of embryonic stem cells, adult stem cells or induced pluripotent stem cells.

12. The method of any of the preceding claims, wherein the definitive endoderm is obtained by contacting a pluripotent stem cell with a subgroup of BMPs selected from the TGF- β superfamily of activins, growth factors; nodal, Activin A, Activin B, BMP4, Wnt3a, and combinations thereof. The method of any one of the preceding claims, wherein the DE is a DE monolayer wherein greater than 90% of the cells in the DE monolayer co-express FOXA2 and SOX 17.

13. The method of any one of the preceding claims, wherein the BMP inhibitor is selected from Noggin, Dorsomorphin, LDN189, DMH-1, and combinations thereof.

14. The method according to any one of the preceding claims, wherein the retinoic acid of step a is contacted with the DE for a period of time from about 12 hours to about 48 hours, or from about 20 hours to about 40 hours, or about 24 hours, or until treatment results in loss of PDX expression and p63 expression.

15. The method of any one of the preceding claims, wherein step c is performed for a period of time sufficient to form a stratified epithelium lacking KRT 8.

16. The method according to any one of the preceding claims, wherein said step c is carried out for a period of time sufficient to form stratified squamous epithelium expressing regional keratin.

17. The method of any preceding claim wherein step c is performed for a period of time sufficient for the HEO to express INV.

18. The method of any one of the preceding claims, wherein steps a-c are performed in vitro.

19. The method of any one of the preceding claims, further comprising contacting the forepart foregut culture of step a) or the spheres of step b) with a matrix selected from collagen, basement membrane matrix (matrigel), or a combination thereof.

20. A composition comprising esophageal tissue produced according to any of the preceding claims, wherein the esophageal tissue is characterized by an absence of innervation and/or blood vessels.

21. A Human Esophageal Organoid (HEO) composition, wherein said HEO composition is substantially free of one or more of submucosal glands, transition regions, vasculature, immune cells, or submucosa.

22. An esophageal progenitor cell capable of being organized into an organotypic culture, wherein the esophageal cell is derived from the method of any one of claims 1-19.

23. A method of making stratified squamous epithelium comprising the steps of:

a. enzymatically dissociating the HEO of any one of claims 1-19 to release progenitor cells, wherein the HEO is from about 3 weeks to about 10 weeks of age, or from about 4 weeks to about 8 weeks of age, or about 5 weeks of age;

b. expanding the progenitor cells into a monolayer; and

c. redifferentiating the dissociated HEO on a collagen-coated membrane into a stratified squamous epithelium for a period of time sufficient to produce the stratified squamous epithelium, wherein the stratified squamous epithelium expresses keratin and one or more markers selected from the group consisting of IVL, CRNN, and FLG.

24. The stratified squamous epithelium as claimed in claim 23, wherein the stratified squamous epithelium comprises esophageal cells substantially organized in the form of lamellae.

25. A method of treating an esophageal disorder in an individual in need thereof, wherein the disorder is selected from the group consisting of congenital disorders (atresia), functional disorders (achalasia and other dyskinesias), immunological disorders (eosinophilic esophagitis), pathological disorders (barrett's esophagus and esophageal cancer), and combinations thereof, comprising the step of contacting an esophageal composition according to claim 1(HEO) or claim _ (esophageal sheet) with the individual.

26. The method of claim 25, wherein the disease comprises an ulcer, and wherein the esophageal composition comprises esophageal cells substantially organized in a sheet form, and further comprising the step of contacting the sheet with the ulcer.

27. A method of identifying a treatment for eosinophilic esophagitis comprising contacting a potential therapeutic agent of interest with the organoid of any of the foregoing claims, detecting a measure of eosinophilic esophagitis activity, and determining whether the potential therapeutic agent of interest improves the measure of eosinophilic esophagitis activity.

28. A method of making a fanconi anemia model comprising the steps of any one of claims 1 to 19, wherein the DE is obtained from FANCA deficient precursor cells.

29. A method of making a model of barrett's metaplastic disease comprising the step of inducing CDX2 and activating BMP in the HEO of any one of claims 1-19.

30. A method of making an eosinophilic esophagitis disease model wherein the HEO of any one of claims 1 to 19 is contacted 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.

31. A method of identifying an active agent capable of treating an esophageal disease state, comprising the steps of: contacting a test agent with the disease model of any one of claims 28 to 30 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 increased esophageal gene expression such as SOX2, p63, KRT13, CRNN, IVL, and loss of Barrett's intestinal genes.

Background

The esophagus actively promotes the passage of food from the mouth and pharynx to the stomach. It consists of stratified squamous epithelium, muscle layers and the enteric nervous system that senses stretch and controls peristalsis. Congenital diseases (e.g., esophageal blockage) are caused by mutations in genes that result in narrowing or discontinuity of the lumen. Other diseases affect the esophagus later in life, such as esophageal cancer, eosinophilic esophagitis, achalasia and other movement disorders. Tracheal and esophageal diseases are common in humans and difficult to model accurately in mice. Despite the prevalence of the disease states described above, and because of the substantial differences in tissue structure between 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.

Disclosure of Invention

The present disclosure relates to methods for transforming mammalian Definitive Endoderm (DE) cells to 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 shaped endosymbiods.

Drawings

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

FIGS. 1A-1K. The anterior foregut fate is regulated by modulating Wnt and retinoic acid signaling during foregut spheroid development. (1A) Experimental protocol for modeling the foregut sphere along the antero-posterior axis by manipulating the duration of Wnt activation (chiron-chr). (1B-1C) patterned qPCR analysis of foregut spheroids for different chiron treatment durations as measured by (1B) foregut marker SOX2 and mid/hindgut marker CDX2, and (1C) foregut ("AFG") marker HNF1B, and hindgut markers PROX1 and HNF 6. (D-E) Whole pack immunofluorescence ("IF") analysis of newborn spheres (day 6) treated with chiron for 1 day (1D) and 3 days (1E) HNF1B, SOX2 and CTNNB 1. (1F) Protocol for modeling foregut spheroids along the antero-posterior axis using Retinoic Acid (RA). (1G) Effect of varying duration of RA treatment on 3 days old enteric spheroids as measured by SOX2, TP63 (subtype Δ N), GATA4 and PDX 1. (1J-1K) IF analysis of early esophageal markers SOX2 and p63 in untreated spheres (1I) and spheres treated with RA for 1 day (1J) or 4 days (1K). 1l-1, 1J-1 and 1K-1 showed individual staining of p 63. (1H) Quantification of the percentage of SOX2+ and p63+ epithelial cells in each sphere. Scale bar 25 μm. For details see the quantitative and statistical analysis sections. See also fig. 8 and fig. 9A-R.

FIGS. 2A-2I. The anterior foregut sphere has esophageal breathing ability. (2A) Schematic depicting an experimental protocol for modeling AFG spheres along the dorsoventral axis. (2B) Current simplified models that guide cues for dorsal-ventral patterns of AFG of mouse and frog embryos. (2C-2G) qPCR analysis of 3 day old spheres (day 9) treated with Noggin for 3 days, untreated (-ctrl), or chiron and BMP4(10ng/mL) using dorsal markers SOX2 and MNX1(2C +2E), respiratory marker NKX2-1(2D), the Δ N splice variant of TP63 (2F), and the stratified squamous cell marker KRT4 (2G). (2H-2I) IF staining of Noggin (2H) compared to chiron + BMP4(2I) treated spheres of SOX2, NKX2-1, CDH1 and nuclei (DAPI). Scale bar 25 μm. For details see the quantitative and statistical analysis sections. See also fig. 10A-10J.

FIGS. 3A-3Y. The dorsal anterior foregut spheroid forms an organoid comprising stratified squamous epithelium expressing esophageal markers. (3A) Schematic representation depicting DE differentiation into Human Esophageal Organoids (HEO). (3B-3F) brightfield image depicting the growth of nascent spheres to HEO. (G-R) comparison of E17.5 esophagus (G, J, M, K) with HEO (3H-3I, 3K-3L, 3N-3O, 3Q-3R) at 1 and 2 months of age by IF analysis of transcription factors Sox2 and P63(3G-3I), epithelial marker Krt8 compared to Krt14(3J-3O), and suprabasal marker Krt13 (3P-3R). (3S-3V) by qPCR analysis of the stratified squamous epithelial markers p63, KRT5, KRT13, IVL, CRNN, identity and maturation of 1-and 2-month-old esophageal organoids 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) Log2 of selected genes (esophagus, stomach, intestine) averaged over the replicates transformed a heatmap normalizing the TPM values. SSE ═ stratified squamous epithelium; b is a substrate; sb ═ upper substrate; scale bar 500 μm (3B-3F), 50 μm (3G-3L), 100 μm (3O-3R), and 25 μm (3O-1-3R-1). For details see the quantitative and statistical analysis sections. See also fig. 11A-11 CC.

FIGS. 4A-4 BB. HEO contains progenitor cells that produce differentiated stratified squamous epithelium. (4A-4B) H & E staining comparing 7 week old HEO with organotypic rafts (raft) generated using HEO derived from keratinocytes. (4C-4N) 7-week-old HEO was compared to organotypic rafts by IF analysis of transcription factors SOX2 and p63(4C-4D), basal markers KRT14(4E-4F), upper basal keratins KRT4(4G-4H) and KRT13(4I-4J) and differentiation markers IVL, CRNN, and FLG (4K-4N). (4O-4U) esophageal biopsy, 7-week-old HEO, keratinocyte-derived HEO, and qPCR analysis of organotypic rafts 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 EdU pulse chase labeling experiments in HEO. (4W-4Z) IF images of HEO at various time points after labeling. (4AA-4BB) analysis of IF images using a 2D histogram of P63 intensity versus EdU intensity (4AA) and a 1D histogram of the percentage of total EdU labeled cells versus distance from the epithelial substrate (4 BB). b is the base; sb ═ upper substrate. Scale bar 50 μm (C-N), 100 μm (4A-4B, 4S-4V). For details see the quantitative and statistical analysis sections. See also fig. 12A-12R.

FIGS. 5A-5L. Early endoderm depletion of Sox2 resulted in mouse esophagusIncomplete development. (5A-5d) control embryos from pregnant dams gavage with tamoxifen at 6.5dpc (Sox 2)^fl/fl) And Sox2 conditional endoderm knockout embryos (Sox2-DE-LOF, FoxA2^CreER；Sox2^fl/fl) IF analysis of Sox2 and Nkx2-1 in (1). Embryonic sections at E9.5(5A-5B) and complete IF at E11.5(5C-5D), with the images masked, the endoderm highlighted. (5E-5F) IF images of sections with relative sections indicated in the package images (5C-5D) for Nkx2-1(5E) and p63 (5F). The inset shows only the Sox2 channel (left) and the green/right (Nkx2-1 or p63) channel. (5G-51H) by E10.5 Sox2cKO (Sox 2) from pregnant dam gavaged at 8.5dpc^CreER/fl) Cleavage Caspase3 staining in embryos, analysis of cell death. The boxed area is enlarged and shown in (5G-1-5H-1), the endoderm is outlined in white and only cleaved Caspase3 is shown. (5I-5L) E11.5 mouse controls and Sox2cKO embryos from pregnant dams gavage at 9.5dpc (Sox 2)^CreER/fl) IF analysis of (3). (5I and 5J) complete IF from Nkx2-1 and Foxa2 of the lateral and frontal projections of the foregut. Sections of (5K and 5L) E11.5 foregut, corresponding to their relative position in the whole mount IF projection (5I-5J), stained Nkx2-1(5K) and p63(5J), with yellow arrows pointing to the mutated esophagus. The scale bar is 50 μm in all IF slices and 100 μm in all IF integer projections. For details see the quantitative and statistical analysis sections. fg, dfg, dorsal, vfg, ventral, eso, esophagus, tr, trachea, br, and st, stomach. See also fig. 13A-13F.

FIGS. 6A-6T. Sox2 inhibited respiratory fate and promoted the dorsal (esophageal) lineage. (6A-6F) in situ hybridization of nkx2-1 of either controls (6A, 6C, 6E) or Xenopus endoderm explants injected with Sox2 MO (6B, 6D, 6F) analyzed by NF35 at the stage of treatment with Bio (GSK3 β inhibitor) and Bio + BMP 4. (6G) Schematic diagram depicting experimental protocols for generating human dorsal (Noggin) and ventral (BMP) AFG cultures. + SOX2 indicates tet-induced SOX2, and-SOX 2 indicates SOX2 CRISPRi. (6H-6N) analysis of day 9 AFG cultures modelled along the dorsal-ventral axis using Dox-inducible CRISPR on days 3-9, with or without SOX2 knockdown in dorsal cultures; (6H-6K) IF staining of cultures against SOX2 and NKX2-1 and quantification in (6L). (6M-6N) qPCR analysis for SOX2 and NKX2-1 in response to these patterned conditions. (6O-6T) exogenous SOX2 expression induced by doxycycline in abdominal cultures on day 8 and analysis on day 9. IF staining of (6O-6R) cultures against NKX2-1 and HA-SOX 2; and (6S-6T) qPCR analysis for SOX2 and NKX2-1 in response to patterned conditions. The scale bar is 50 μm for IF images and 200 μm for Xenopus explant images. For details see the quantitative and statistical analysis sections.

FIGS. 7A-7L. Sox2 regulates the expression and Wnt signaling activity of secreted Wnt antagonists in dorsal foregut endoderm. (7A) Cluster heatmaps 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) Wien plot analysis of genes up-regulated in dorsal and ventral cultures compared to genes that were elevated or lowered after knock-down of SOX2 by CRISPRi. (7C) Gene Ontology (GO) terminology analysis of biological processes of genes positively regulated by SOX 2. (7D) The number of genes enriched in dorsal and ventral cultures, and whether their expression is SOX2 dependent. (7E) Gene ontology the term "regulation of the Wnt signaling pathway" gene set enrichment analysis, red indicates higher expression and blue indicates lower expression. (7F-7G) Wnt responsive Gene Axin2 vs (7F) control (Sox 2)^fl/fl) And (7G) Sox2-DE-LOF (FoxA 2)^CreER；Sox2^fl/fl) In situ hybridization of the anterior foregut of E9.5 mice in embryos. (7H-7I) control (7H) obtained from dam gavage at 8.5dpc (Sox 2)^fl/+) And (7I) Sox2cKO (Sox 2)^CreER/fl) Embryonic E10.5 in situ hybridization of Axin2 in the embryonic foregut of mice. The number of embryos analyzed is shown at the top left. The boxed area (7F-7I) highlights the dorsal foregut region. (7J) qPCR analysis of AXIN2 in dorsal and ventral foregut cultures on day 9, with or without exogenously expressed SOX 2. (7K) TPM values plotted for Wnt antagonists SFRP1, SFRP2 and DKK1 from RNA-seq of AFG cultures. (7L) proposed model for the role of Sox2 in dorsal abdominal patterning of the anterior foregut. Scale bar 100 μm. For details see materials and methods and quantitative and statistical analysis sections. See also fig. 14A-14D.

FIGS. 8A-8V. The duration of Wnt and retinoic acid signaling is modulated to coordinate foregut patterning in the anterior-posterior axis. (8A) Schematic depicting an experimental protocol for modeling the foregut sphere along the antero-posterior axis using CHIR99021(chiron, or chr) and Retinoic Acid (RA). (8B-8G) qPCR analysis of day 6 spheres 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) posterior gut marker CDX2, and (8F-8G) pharyngeal markers PAX9 and OTX 2. (8H-8N) comparison of treatment of endoderm compared to Wnt3a by qPCR analysis of (8H) foregut markers SOX2, (8I) HNF1B, PROX1, HNF6, and CDX2, (8j) Wnt target genes AXIN2, LEF1 and TCF1, and (8K) epithelial and neurological markers CDH1 and NESTIN, respectively. Treatment with a one day chiron produced spheres with the same gene expression profile as spheres produced with 2 day Wnt3 a. (8L-8N) brightfield imaging of nascent spheres from chiron compared to Wnt3a treatment showed that the efficiency of sphere production was not affected under different conditions. (8O-8R) analysis of day 9 spheres resulting from varying the duration of retinoic acid treatment from day 5 onwards. (8O) qPCR analysis of retinoic acid targets HOXA1, HOXB1, CYP26C 1. (8S) schematic, depicting an experimental protocol for modulating retinoic acid signaling using the synthetic inhibitor DEAB. (8T-8U) (8T) day 6 foregut markers, (8U) day 9 dorsal foregut markers SOX2 and TP63 (subtype Δ N), and (8V) qPCR analysis of day 9 RA targets HOXA1 and HOXB 1. The scale bar is 500 μm in (8L-8N) and 50 μm in (8P-8R). Error bars represent SD. For the two-tailed t-test, p <0.05, p <0.01 and p < 0.001.

FIGS. 9A-9R. Early regulation of Wnt and retinoic acid signaling affects subsequent differentiation into human esophageal organoids. (9A) Schematic, depicting experimental protocol for generation of organoids starting with foregut spheroids treated with chiron for 1 or 3 days. (9B-9C) qPCR analysis of organoids at day 35 (1 month old) for (9B) stratified squamous epithelial markers KRT5, KRT13 and KRT13, and (9C) posterior foregut markers GATA4 and PDX 1. (9D-9O) analysis of day 35 (1 month old) organoids by varying the duration of retinoic acid treatment from day 5 onwards. (9D) Δ N subtypes of the anterior foregut basal transcription factors SOX2 and TP63, (9E) gastric sinus and pancreatic markers PDX1, and (9F) qPCR analysis of stratified squamous epithelial markers KRT5, KRT13 and IVL. Immunofluorescence analysis of one month old organoids (9G-9I) SOX2 and p63, (9J-9L) KRT13, and (9M-9O) PDX 1. (9P) schematic, depicting an experimental protocol for modulating retinoic acid signaling using the synthetic inhibitor DEAB. (9Q-9R) qPCR analysis of (9Q) esophageal basal markers SOX2 and TP63, of organoids on day 35, (9R) stratified squamous epithelial markers KRT5, KRT13 and IVL. These data represent 2 independent experiments with n-3 wells per experiment. Scale bar 100 μm. Error bars represent SD. For the two-tailed t-test, p <0.05 and p < 0.01.

FIGS. 10A-10J. Modeling the foregut to foregut spheroids under these culture conditions does not require TGF β inhibition. (10A) Schematic depicting an experimental protocol testing the need for TGF signaling in anterior-posterior modeling of the foregut. (10B-10F) qPCR analysis of day 6 anterior foregut spheres treated with and without Wnt3a and a TGF β inhibitor (SB431542, 10 μ M) against (10B-10C) foregut marker SOX2 and hindgut marker CDX2, (10D) foregut marker HNF1B, and (10E-10F) hindgut marker PROX1 and HNF 6. (10G) Schematic depicting experimental protocol for the ability of anterior segment foregut spheroids tested and not treated with Wnt3a or TGF β inhibitor SB431542 to respond to respiratory induction. (H-J) the spheres at day 9 were analyzed for NKX2-1 by (10H-10I) immunofluorescence and (10J) qPCR. Scale bar 50 μm. Error bars represent SD. For the two-tailed t-test, p <0.05 and p < 0.01.

FIGS. 11A-11 CC. Robust growth of human esophageal organoids and comparison to mouse embryonic esophageal development. (11A-11E) improved sphere outgrowth efficiency of organoids treated with FGF10 on days 6-13, as determined by (a-D) brightfield imaging and (11E) quantitative analysis of the 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 weeks 2 (11H, 11L, 11P) and 3 (11I, 11M, 11Q). (R11) gene expression of stratified squamous epithelial markers in mouse esophagus over time; public data obtained from the GEO data set GSE34728(Chen et al, 2012). (11S) qPCR analysis of stratified squamous epithelial markers at various time points during differentiation of definitive endoderm into adult esophageal organoids. (11T) quantification of% area of KRT5 and KRT13 positive epithelium by HEO on day 62. Each point is an individual organoid, and the subgraphs "a" and "b" are representative images of the different organoids depicted in this figure. (11U) quantification of SOX2 and p 63-positive% of epithelial nuclei by HEO at day 62. Each point is an individual organoid. (11V-11CC) immunofluorescence analysis of 1 month old HEO in the different cell lines tested, examining the esophageal enrichment markers SOX2 and p63(11V-11Y) and KRT13(11Z-11 CC). The scale bar is 500 μm in (11A-11D), 50 μm in (11F-11Q, 11V-11CC), and 100 μm in (11T-11U). Error bars represent SD. For the two-tailed t-test, p <0.05, p <0.01 and p < 0.001.

FIGS. 12A-12R. Alternative to human esophageal organoids maturation and expansion. (12A-12F) analysis of HEO growth 2 months after transplantation into kidney capsule of immunodeficient mice by immunofluorescence imaging of early (KRT8) and differentiation (KRT13, KRT14 and IVL) esophageal specific markers (12A-12E) and (12F) H & E. (12G-12R) analysis of HEO from mechanical passages (dissociation and re-culture) twice. (12G-12N) IF images of (12G-12H) transcription factors SOX2 and p63, (12I-12J) immature (KRT8) and basal markers (KRT14), (12K-12L) basal (KRT5) and superior basal (KRT13) markers, and (12M-12N) superior basal differentiation markers KRT4, CRNN and IVL of passaged organoids. (12O-12R) qPCR analysis of patterned markers of passage HEO compared to normal HEO and the (12O) transcriptional markers SOX2 and TP63 of gastric organoids (hAGO), (12P) stratified squamous markers KRT5, KRT13, IVL and (12Q-12R) lung (NKX2-1), stomach (GATA4) and intestine (GATA4 and CDX 2). For the two-tailed t-test, p <0.05, p <0.01 and p < 0.001.

FIGS. 13A-13V. Gastrulation posterior endoderm or extensive Sox2 knockouts lead to a similar phenotype of esophageal hypoplasia. (13A-13B) control at E9.5 (Sox 2)^fl/fl) And Sox2-DE-LOF (FoxA 2)^CreER；Sox2^fl/fl) Whole-pack Immunofluorescence (IF) analysis of dorsal marker Sox2 (red) and respiratory marker Nkx2-1 (green) in embryos. (13C-13D) at E10.5 control(Sox2^fl/fl) And Sox2-DE-LOF (FoxA 2)^CreER；Sox2^fl/fl) Ready-packaged IF analysis of Sox2, Nkx2-1 in embryos. White arrows highlight normal versus ectopic Nkx2-1 expression. (13E-13F) IF analysis of foregut sections from E11.5 embryos against apical marker aPKC (green). (G-H) E11.5 control (Sox 2) from dam gavage at 8.5dpc^fl/+) And Sox2-cKO (Sox 2)^CreER/fl) Whole pack IF analysis of embryos Nkx 2-1. Immunofluorescence analysis of (13I-13L) E11.5 embryos (analogous to that in 13E-13F) against (13I-13J) Sox2 and Nkx2-1 and (13K-13L) Sox2 and p 63. (13M-13T) E10.5 control (Sox 2) from dam gavage at 8.5dpc^fl/+) And Sox2-cKO (Sox 2)^CreER/fl) IF analysis of embryos for epithelial morphology across the anterior axis (13M, 13N) to the posterior axis (13S, 13T). (13S, 13T) in a control (Cre-, Sox2) isolated from dam gavage at 8.5dpc^fl/+) And Sox2cKO (Cre +, Sox2)^CreER/fl) Quantification of cell death (13U) cleaved Caspase3 and (13V) Ki67 IF staining in the foregut of E10.5 mouse embryos at dorsal and ventral foregut in embryos. The scale bar is 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 2 embryos per analysis per genotype at E11.5 (minimal 2 litters were collected per analysis and time point). . For Sox2cKO embryos driven by Sox2, each genotype was n-3 embryos. Error bars represent SD. For two-tailed t-test, p ≦ 0.05. fg, dfg, dorsal, vfg, ventral, ph pharyngeal endoderm, eso, esophagus, r, respiratory progenitor, thy, tr, trachea, br, bronchi, st, stomach.

FIGS. 14A-14D. Analysis of loss of function or gain of Sox2 in human cultures. (14A) Principal component analysis of transcriptomes generated from day 9 anterior foregut cultures (with and without SOX2) modelled along the dorsal-ventral axis (no or with Dox treatment activating CRISPR interfering constructs). Dorsal versus ventral anterior foregut (dAFG versus vAFG) are indicated as chr + Nog and chr + BMP4, respectively. The knockdown is represented by + Dox. (14B) TPM values for SOX2 and NKX 2-1. (14C) qPCR analysis of SFRP2 of day 9 anterior foregut cultures patterned along the dorsal (dAFG) and ventral (vAFG) axes, including induction of the exogenous HA marker SOX2 in ventral cultures by Dox treatment on day 8. (14D) Genome browser view of Sox2 peak at the SFRP2 locus in hPSC-derived endoderm (GSM1505764) and mesendoderm (GSM1505767) from GEO dataset GSE61475(Tsankov et al, 2015). Scale bar 500 μm. Error bars represent SD. For the two-tailed t-test, p ≦ 0.05.

FIGS. 15A-15E. Role of Sox2 in esophageal development after separation of the anterior foregut. (15A) Schematic representation of mouse breeding and tamoxifen administration regimen. (15B) Confocal Immunofluorescence (IF) images of E14.5 (left), E17.5 (center), and P7 (right) esophageal sections, pregnant dams were gavaged at 11.5dpc (left), 14.5dpc (center), and young pups were gavaged at P1 (right). Sections were stained for E-cadherin to visualize the epithelium, Sox2 and Nkx2-1 were directed to respiratory properties. (15C) IF images of various markers from E17.5 esophagus of pregnant dams gavage at 11.5 dpc: p63 and Sox2 confirming the identity of esophagus, basal marker Krt14, suprabasal marker Krt13, immature or columnar marker Krt8, proliferation marker Ki 67. The green arrow in the bottom middle right panel highlights the upper base Ki67 staining. (15D) High magnification IF images of E-cadherin from E17.5 esophagus of pregnant dams gavage at 11.5 dpc. (15E) IF images of patterned markers from E17.5 esophagus of pregnant dams gavaged at 11.5 dpc: intestinal markers Cdx2, gastric/intestinal markers Gata4 and Pdx1, respiratory marker Nkx2-1, and smooth muscle marker Desmin (Desmin). Yellow arrows highlight rare Nkx2-1 positive cells in the mutant esophagus. The scale bar is 100 μm in (15B, 15C, 15E) and 25 μm in (15D).

FIGS. 16A-16G. The effect of fanconi anemia (loss of FANCA) was modeled in HEO. (16A) Schematic diagram depicting experimental protocols for generating HEO with (+ dox) or without FANCA. Note: hydroxyurea (HU) was used for western blot analysis in (16E). (16B) IF images of day 6 AFG monolayers 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) SOX2 from HEO and IF images of proliferation marker KI 67. (16E) Western blot analysis of FANCA and FANCD2 confirming expression and function of dox-induced FANCA protein. (16F) Size quantification of 2 week old HEO from bright field images. (16G) Quantification of proliferative (KI67+) epithelial cells in HEO at 1 month of age (day 36). The scale bar is 100 μm in (16B, 16D) and 250 μm in (16C). For the Mann-Whitney non-parametric test, p.ltoreq.0.05 and p.ltoreq.0.01. DE ═ definitive endoderm; AFG ═ anterior foregut; HEO is a human esophageal organoid.

FIGS. 17A-17L. Induction of CDX2 in human foregut and HEO cultures. (17A) Schematic diagram depicting experimental protocols for induction of CDX2 in foregut and HEO cultures. (17B) Schematic representation of transduced lentiviral vectors that induced CDX2 following doxycycline administration. (17C) IF analysis of foregut marker SOX2 and hindgut marker CDX2 of foregut monolayers on day 6 treated with different levels of doxycycline (20, 100 and 500 ng/mL). (17D +17E) quantification of IF images (as in 17C) by scattergrams of (17D) SOX2 versus CDX2 intensities. The vertical lines in the scatter plot define the "gate" of CDX2+ versus CDX 2-cells. (17E) Histogram of percent CDX2+ cells. (17F) IF analysis of stratified squamous markers SOX2 and p63 and the hindgut (induced) marker CDX2 of 1 month old HEO with or without doxycycline treatment. qPCR analysis of (17G-17L) hindgut marker (17G) CDX1, (17H) CDX2, (17I) CDH17, (17J) MUC2 and foregut/stratified squamous markers (17K) SOX2 and (17L) p 63. Scale bar 100 μm. For the Student's t-test with two-tailed distribution of not assumed equal variance, p ≦ 0.01, p ≦ 0.001, and p ≦ 0.0001. DE ═ definitive endoderm; FG — foregut; AFG ═ anterior foregut; HEO is a human esophageal organoid.

FIGS. 18A-18I. 18A) Late induction of CDX2 in HEO resulted in the inhibition of the esophageal transcription factors SOX2 and p 63. Schematic, experimental protocol for induction of CDX2 in more mature HEO (6-7 weeks old) is described. IF images of the stratified squamous markers SOX2 and p63 and the hindgut marker CDX2 of HEO on day 58 treated with (+ CDX2) or without doxycycline. qPCR analysis of (18C) CDX2, (18D) SOX2 and (18E) p63 of HEO at day 58 (18C-18E). (18F) Analysis of IF images (e.g. 18B) by CDX2 versus p63 intensity scattergrams of HEO basal epithelial cells. Vertical lines separated p63 negative (left) and p63 positive (right) cells. Horizontal lines separated CDX 2-negative (bottom), CDX 2-low (middle) and CDX 2-high (top) cells. (18G-18I) (18G) quantification of IF analysis of all CDX 2-high and CDX 2-low basal cells, (18H) CDX 2-high and CDX 2-low basal cells which are SOX2+, (18I) CDX 2-high and CDX 2-low basal cells which are p63+ (see 18B and 18F). Scale bar 100 μm. For Student's t-test with two-tailed distribution of not assumed equal variance, p ≦ 0.0001. DE ═ definitive endoderm; AFG ═ anterior foregut; dAFG ═ dorsal anterior foregut; HEO is a human esophageal organoid.

FIGS. 19A-19H. CDX 2-induced loss of esophageal differentiation in HEO. (19A) Schematic of experimental protocol for induction of CDX2 with or without Notch inhibition in HEO. (19B-19G) stratified squamous markers of D58 HEO (19B) KRT5, (19C) KRT13 and (19D) IVL, (19E) intestinal epithelial marker CDH17, (19F) Notch target HES5 and (19G) qPCR analysis of BMP target ID 1. (19H) CDH17, KRT5 and KRT13 (upper panel) of HEO treated with doxycycline and the gamma-secretase (Notch) inhibitor DAPT; and IF images of the differentiated stratified squamous markers CRNN and IVL (lower row). Scale bar 100 μm. For the Student's t-test with two-tailed distribution of non-assumed equal variance, p ≦ 0.05, p ≦ 0.01, and p ≦ 0.001. DE ═ definitive endoderm; AFG ═ anterior foregut; dAFG ═ dorsal anterior foregut; HEO is a human esophageal organoid.

FIGS. 20A-20H. IL-13 treated HEO up-regulates IL-13 target genes and increases proliferation. (20A) Schematic, depicting an experimental protocol for the treatment of IL-13 in late HEO. (20B-20E) qPCR analysis of known IL-13 target genes (20B) CCL26, (20C) CDS26, (20D) CAPN14 and (20E) SERPINB4 of 62-day HEO treated with IL-13 for 2 days prior to harvest. (20F) Western blot analysis of HEO on day 50 treated with IL-13(100ng/mL) for 1 week prior to harvest, SERPINB13, CDH26, and housekeeping protein GAPDS. (20G) IF images of day 62 HEO treated 2 weeks with IL-13(100ng/mL) and 2 days with EdU (10 μ M) before harvest. (20H) Quantification of the percentage of labelled EdU (of 20G) of all basal (basal-most) p63 cells. For the qPCR data, for the Student's t-test with two-tailed distribution that did not assume equal variance, p ≦ 0.05 and p ≦ 0.01. Quantification was introduced for organoid EdU, p <0.05 for Mann-Whitney nonparametric test. Scale bar 100 μm. DE ═ definitive endoderm; AFG ═ anterior foregut; HEO is a human esophageal organoid.

FIGS. 21A-L. Impaired differentiation and altered morphology of HEO treated with IL-13. (21A) IF analysis of 62-day HEO treated with IL-13(100ng/mL) for 2 weeks prior to harvest. (21B) Western blot analysis of structural and differentiation proteins of HEO on day 56 treated with IL-13(100ng/mL) for 1 week prior to harvest. (21C-21F) qPCR analysis of (21C) IVL, (21D) CRNN, and BMP antagonists (21E) NOG and (21F) FST of day 62 HEO treated with IL-13(100ng/mL) for 2 weeks prior to harvest. (21G) Structural analysis by H & E staining (left column) and electron micrographs (right column) of HEO at day 62 treated with IL-13(100ng/mL) for 2 weeks prior to fixation. (21H-21L) qPCR analysis of (21H) SOX2, (21I) CCL26, (21J) CDH26, (21K) hedgehog target PTCH1 and (21L) BMP target ID3 of day 62 HEO treated with IL-13(100ng/mL) and BMP4(100 ng/mL). For the IF and H & E images, the scale bar is 100 μm, and for the electron micrographs, the scale bar is 6 μm. For the Student's t-test with two-tailed distribution that did not assume equal variance, p ≦ 0.05, p ≦ 0.01, p ≦ 0.001, and p ≦ 0.0001.

FIGS. 22A-22H. SOX2 in Human Intestinal Organoids (HIOs) induces upregulation of stratified squamous markers. (22A) Schematic diagram depicting experimental protocol for induction of SOX2 in HIO. (22B) Schematic representation of a retroviral vector that induces HA-tagged SOX2 following doxycycline administration. (22C) (top) foregut marker SOX2 and hindgut marker CDX2, (middle) foregut marker p63, stomach/gut marker PDX1, and HA-tag of day 36 HIO with or without doxycycline treatment; IF images of the (bottom) intestinal marker CDH17 and the gastric marker CLDN 18. (22D-22H) qPCR analysis of the various regional markers (22D) SOX2, (22E) p63, (22F) PDX1 and (22G) CDX2 and the basal stratified squamous marker KRT13 on (22H). Scale bar 100 μm. For the Student's t-test with two-tailed distributions that do not assume equal variance, p ≦ 0.05, p ≦ 0.01, and p ≦ 0.0001. DE ═ definitive endoderm; HG ═ hindgut; HIO is a human intestinal organoid.

FIGS. 23A-23J. BMP activation in HEO results in loss of proliferation and differentiation. (23A) Schematic drawing depicting an experimental protocol for activating BMP signaling in late-phase HEO. (23B) IF images of (left) pSMAD1/5/9 and SOX2, (center) p63 and EdU, (right) IVL and CRNN of day 62 HEO with or without BMP4(100 ng/mL). (23C) Quantification of percent labeled EdU of all basal-most epithelial cells in HEO with or without BMP 4. (23D-23J) qPCR analysis of various markers of HEO at day 62: (D) SOX2, (23E) p63, (23F) ID3, (23G) KRT5, (23H) KRT13, (23I) IVL and (23J) CRNN. Scale bar 100 μm. For the qPCR data, for the Student's t-test with two-tailed distribution that did not assume equal variance, p ≦ 0.05, p ≦ 0.0001. Quantification was introduced for organoid EdU (23C), for Mann-Whitney non-parametric test, p ≦ 0.01. DE ═ definitive endoderm; AFG ═ anterior foregut; dAFG ═ dorsal anterior foregut; HEO is a human esophageal organoid.

Detailed Description

Definition of

Unless otherwise indicated, terms should be understood by those of ordinary skill in the relevant art based on conventional usage. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

As used herein and in the appended claims, the singular forms "a", "an", 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 and equivalents thereof known to those skilled in the art, and so forth.

The term "about" or "approximately" means within an acceptable error range for a particular value, as determined by one of ordinary skill in the art, which error will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within 1 or greater than 1 standard deviation, according to practice in the art. Alternatively, "about" may represent a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5-fold, more preferably within 2-fold, of the value. Where a particular value is described in the application and claims, unless otherwise stated, the term "about" shall be assumed to indicate that the particular value is within an acceptable error range.

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

As used herein, the term "Definitive Endoderm (DE) cell" refers to one of the three major germ layers produced by the process of gastrulation.

As used herein, the term "Wnt signaling pathway" refers to the Wnt/β -catenin (catenin) pathway and is a signaling pathway mediated by Wnt ligands and frizzled cell surface receptors that function through β -catenin.

As used herein, the term "activator" with respect to a pathway such as the "Wnt pathway" refers to an agent that activates the Wnt/β -catenin pathway such that the Wnt/β -catenin target is increased.

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.

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

As used herein, the term "growth factor" refers to a substance capable of stimulating a cellular process including, but not limited to, growth, proliferation, morphogenesis or differentiation.

As used herein, the term "stable expression" of a marker refers to expression that does not change after a change in the growth environment.

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 extra-embryonic cell types. Such cells can construct whole, viable organisms. These cells are generated from the fusion of egg and sperm cells. The cells produced by the first few divisions of the zygote are also totipotent.

As used herein, the term "Pluripotent Stem Cell (PSC)", also commonly referred to as PS cell, includes any cell that can differentiate into almost all cells, i.e., cells derived from any of the three germ layers (germ epithelium), including endoderm (gastric mucosa, gastrointestinal tract, lung), mesoderm (muscle, bone, blood, urogenital), and ectoderm (epidermal tissue and nervous system). PSCs can be progeny of totipotent cells, derived from embryos (including embryonic germ cells), or obtained by inducing non-pluripotent cells (e.g., adult cells) by forcing expression of certain genes.

As used herein, the term "Induced Pluripotent Stem Cell (iPSC)", also commonly abbreviated as iPS cell, refers to a type of pluripotent stem cell that is artificially derived from normal non-pluripotent cells, such as adult cells, by inducing "forced" expression of certain genes.

As used herein, the term "precursor cell" encompasses any cell useful in the methods described herein by which 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 cells are pluripotent or have the ability to become pluripotent. In some embodiments, the precursor cells are treated with an external agent (e.g., a growth factor) to achieve pluripotency. In some embodiments, the precursor cell may be a totipotent stem cell; pluripotent (pluripotent) stem cells (induced or non-induced); pluripotent (multipotent) stem cells; and unipotent stem cells. In some embodiments, the precursor cells may be from an embryo, infant, child or adult. In some embodiments, the precursor cells may be somatic cells that are treated so as to be rendered pluripotent by gene manipulation or protein/peptide treatment.

In developmental biology, cell differentiation is the process by which less specialized cells become more specialized cell types. As used herein, the term "committed differentiation" describes the process by which a less specialized cell becomes a specific specialized target cell type. The specificity of a particular 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, gene manipulation, chemical treatment, protein treatment, and nucleic acid treatment.

As used herein, the term "cellular component" is an individual gene, protein, mRNA expressed gene, and/or any other variable cellular component or protein activity, such as the degree of protein modification (e.g., phosphorylation), for example, which is typically measured by one of skill in the art in biological experiments (e.g., by microarray or immunohistochemistry). Significant findings associated with the complex network of living systems, common human diseases, and gene discovery and structural determination of underlying biochemical processes can now be attributed to the use of cellular component abundance data as part of the research process. The cellular constituent abundance data can help identify biomarkers, differentiate disease subtypes, and identify toxic mechanisms.

Pluripotent stem cells derived from embryonic cells

In some embodiments, an important step is to obtain stem cells that are or can be induced to be pluripotent. In some embodiments, the pluripotent stem cells are derived from embryonic stem cells, which in turn are derived from totipotent cells of early mammalian embryos and are capable of unlimited, undifferentiated proliferation in vitro. Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of embryonic cells (early embryo). Methods for deriving embryonic stem cells from embryonic cells are well known in the art. Human embryonic stem cells H9(H9-hESC) are used in the exemplary embodiments described in this application, but one skilled in the art will appreciate that the methods and systems described herein can be applied to any stem cell.

Additional stem cells that may be used in embodiments according to the present invention include, but are not limited to, the human embryonic stem cell research center by National Stem Cell Bank (NSCB), university of California, san Francisco (UCSF); wi Cell institute WISC Cell bank; university of Wisconsin Stem cells and regenerative medicine center (UW-SCRMC); novocell, Inc (san diego, california); cellartis AB (goldburg, sweden); ES Cell International Pte Ltd (singapore); a database hosted by Technion (israel sea) of israel institute of technology; and those provided or described in the stem cell databases hosted by university of princeton and 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(SA 001); SA02(SA 002); 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(HSF 1); UC06(HSF 6); WA01 (H1); WA07 (H7); WA09 (H9); WA13 (H13); WA14 (H14).

More details on Embryonic stem cells can be found, for example, in Thomson et al, 1998, "Embryonic StemShell Lines Derived from humanBlastocysts," Science282(5391): 1145-1147; andrews et al, 2005, "Embryonic Stem (ES) cells and Embryonic Carcinoma (EC) cells: opposissides of the same coin, Biochem Soc Trans 33: 1526-; martin 1980, "Teratoccarinomas and mammalian embryo genetics,". Science 209(4458): 768-776; evans and Kaufman,1981, "achievement in culture of pluripotent cells from mice emulsions," Nature 292(5819): 154-; klimanskaya et al, 2005, "Human anatomical cells derived with feeder cells," Lancet 365(9471): 1636-; each of which is incorporated herein in its entirety.

Induced Pluripotent Stem Cells (iPSC)

In some embodiments, ipscs are derived by transfecting certain stem cell-associated genes into non-pluripotent cells (e.g., adult fibroblasts). Transfection is typically accomplished by viral vectors (e.g., retroviruses). The transfected genes included the major transcriptional regulators Oct-3/4(Pouf51) and Sox2, although other genes were proposed to enhance 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 usually isolated by morphological selection, doubling time, or by reporter gene and antibiotic selection. As used herein, ipscs include, but are not limited to, first generation ipscs, second generation ipscs in mice, and human induced pluripotent stem cells. In some embodiments, four key genes are used: oct3/4, Sox2, Klf4, and c-Myc, a retroviral system used to convert human fibroblasts into pluripotent stem cells. In an alternative embodiment, a lentiviral system is used to transform the somatic cells with OCT4, SOX2, NANOG and LIN 28. Genes whose expression is induced in ipscs include, but are not limited to, Oct-3/4 (e.g., Pou5f 1); certain members of the Sox gene family (e.g., Sox1, Sox2, Sox3, and Sox 15); 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 LIN 28.

In some embodiments, a non-virus based technique is employed to generate ipscs. In some embodiments, the adenovirus can be used to transfer the necessary four genes into the DNA of the skin and liver cells of the mouse, resulting in the same cells as embryonic stem cells. Since adenovirus does not bind any of its own genes to the targeted host, the risk of developing tumors is eliminated. In some embodiments, reprogramming can be accomplished by a plasmid that does not have any viral transfection system at all, although the efficiency is very low. In other embodiments, direct delivery of the protein 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 similar methodologies: repeated treatment of cells with certain proteins delivered into the cells via poly-arginine anchors is sufficient to induce pluripotency. In some embodiments, expression of the pluripotency-inducing gene can also be increased by treating somatic cells with FGF2 under hypoxic conditions.

More details on embryonic stem cells can be found, for example, in Kaji et al, 2009, "Virus free induced pluripotent and subcoquest exclusion of developmental factors," Nature458: 771-775; woltjen et al, 2009, "piggyBac translation programs identified by public sources cells," Nature458: 766-; okita et al, 2008, "Generation of mouse Induced Pluripotent Stem Cells with out Viral Vectors," Science 322(5903): 949-; stadtfeld et al, 2008, "Induced Pluripotent Stem Cells Generation with visual Integration," Science 322(5903):945 949; and Zhou et al, 2009, "Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins," Cell Stem Cells 4(5): 381-384; each of which is incorporated herein in its entirety.

In some embodiments, exemplary iPS cell lines include, but are not limited to iPS-DF 19-9; iPS-DF 19-9; iPS-DF 4-3; iPS-DF 6-9; ips (freskin); iPS (IMR 90); and iPS (IMR 90).

More details regarding the function of signaling pathways involved in DE development can be found, for example, in Zorn and Wells,2009, "Vertebrate endberm degradation and organ format," Annu Revcell Dev Biol 25: 221-; dessimoz et al, 2006, "FGF signaling is a scientific for evaluating gum tube domains along the antigen-poreror axis in vivo," MechDev 123: 42-55; McLin et al, 2007, "reproduction of Wnt/β -catenin signaling in the antisense end expression for lever and amplification, development," 134: 2207-; wells and Melton,2000, Development 127: 1563-1572; de Santa Barbara et al, 2003, "Development and differentiation of the interactive epiphyllum," Cell Mol Life Sci 60(7): 1322-1332; each of which is incorporated herein in its entirety.

Any method of producing definitive endoderm from pluripotent cells (e.g., ipscs or ESCs) is suitable for use in the methods described herein. In some embodiments, the pluripotent cells are derived from morula. In some embodiments, the pluripotent stem cells are stem cells. The stem cells used in these methods may include, but are not limited to, embryonic stem cells. Embryonic stem cells may be derived from the inner cell mass of an embryo or from the gonadal ridges of an embryo. Embryonic stem or germ cells can be derived from a wide variety of animal species, including but not limited to a wide variety of mammalian species, including humans. In some embodiments, human embryonic stem cells are used to produce definitive endoderm. In some embodiments, human embryonic germ cells are used to produce definitive endoderm. In some embodiments, ipscs are used to produce definitive endoderm.

Tracheal and esophageal diseases are ubiquitous in humans and difficult to model accurately in mice. Therefore, the applicant established a three-dimensional organoid model of esophageal development by directed differentiation of human pluripotent stem cells. Sequential manipulation of BMP, WNT and RA signaling pathways allowed modeling of definitive endoderm as foregut, Anterior Foregut (AFG) and dorsal AFG spheres. Dorsal AFG spheres grown in 3D matrices form Human Esophageal Organoids (HEOs) and HEO cells can be transformed into two-dimensional cultures and grown as esophageal organorafts. In both configurations, esophageal tissue has proliferative basal progenitor cells and differentiated stratified squamous epithelium. Using HEO cultures to model human esophageal birth defects, applicants determined that Sox2 promotes esophageal specification (specification) in part by inhibiting Wnt signaling and promoting survival in dorsal AFG. Consistently, Sox2 ablation caused esophageal hypoplasia in mice. Thus, HEO provides a powerful platform for modeling human pathology and tissue engineering.

Human tissue organoids differentiated from Pluripotent Stem Cells (PSC) 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 to organ cell types relies on a reiteration of 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 method has been used to generate human anterior and posterior endodermal embryonic layer organoids including respiratory tract, stomach, small intestine and colon (Chen et al, 2017; Dye et al, 2015, 2016; McCracken et al, 2014, 2017; M-nera et al, 2017; Spence et al, 2011). However, human PSC-derived esophageal tissue has not been reported. Dual inhibition of BMP and TGF β following DE induction produced anterior segment foregut (AFG); however, this produces a mixture of tissues including the pharynx, esophagus and respiratory endoderm (Green et al, 2011; Kearns et al, 2013; Longmire et al, 2012). This suggests that more elaborate patterning methods based on pathways controlling esophageal development are needed to direct PSC to differentiate specifically into the esophagus.

A variety of 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 formed during gastrulation (Zorn and Wells, 2007). DE then patterns and forms primitive gut tubes, roughly divided into foregut, midgut and hindgut, along the anteroposterior axis by Wnt, BMP and FGF signaling (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) creates the esophagus and respiratory tract. Respiratory norms in response to Wnt and BMP activation result in the expression of the transcription factor Nkx2-1, whereas inhibition of BMP in the dorsal foregut promotes the development of esophagal epithelium expressing Sox2 (domean et al, 2011; Goss et al, 2009; Harris-Johnson et al, 2009; Que et al, 2006; rankine et al, 2016). The esophagus starts in simple cubic epithelium, but develops into a stratified squamous epithelium expressing a variety of keratins and a basal layer expressing Sox2 and p63 (Rosekrans et al, 2015; Zhang et al, 2016).

Disclosed herein are manipulations of the above-mentioned signaling pathways in time to differentiate human PSCs into esophageal organoids. After DE formation, applicants determined that precise time manipulation of BMP, WNT and RA pathways guided AFG sphere formation. Consistent with in vivo data, AFG spheroids acquire a respiratory fate by activating WNT and BMP pathways, while BMP inhibition promotes the formation of dorsal foregut spheroids, which form Human Esophageal Organoids (HEO) after 1-2 months of sustained growth. HEO contains stratified squamous epithelium with distinct basal and luminal cell layers with proliferative esophageal progenitor cells that can expand in organotypic raft cultures and differentiate into esophageal epithelium. HEO used in parallel with mouse embryos can be used to identify molecular pathways affected by SOX2 loss of function, which is one of the causes of esophageal atresia in humans and mice (domean et al, 2011; quee et al, 2007). Although reduced Sox2 function resulted in mouse esophageal atresia, complete loss of Sox2 in mouse foregut endoderm resulted in esophageal hypoplasia. Loss of SOX2 function and analysis of transcription profiles of the human and mouse foregut determined that SOX2 modulates dorsal expression of Wnt antagonists (such as SFRP2), suggesting the ability of SOX2 to inhibit Wnt-induced respiratory fate in the dorsal foregut. Applicants have next discovered that the disclosed HEOs provide a complementary platform to study human esophageal organogenesis, congenital defects and disease.

In one aspect, a method of manufacturing an Esophageal Organoid (EO) is disclosed. The method may comprise the step of contacting definitive endoderm with a BMP inhibitor, a Wnt activator, a FGF activator and Retinoic Acid (RA). The contacting step may be continued for a first period of time sufficient to form an anterior foregut culture. In one aspect, the anterior foregut culture expresses SOX2 and HNF1B after such a first period of time, and does not substantially express PROX1 and HNF 6. The method can further comprise contacting the foregut culture with a BMP inhibitor (Noggin) and an EGF activator for a second period of time sufficient to form a dorsal foregut ("dAFG") sphere, wherein the dAFG can express SOX2 and TP63, but does not express PDX1, PAX9, or NKX 2.1. The method may further comprise the steps of: culturing dAFG for a third period of time sufficient to allow formation of Esophageal Organoids (EO), wherein said culturing is in the presence of EGF, further optionally including an FGF signaling pathway activator, preferably FGF 10. In one aspect, EO is Human Esophageal Organoid (HEO).

Exemplary gene (or mRNA when a gene is not available) accession numbers are provided below: 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 is noted that the above-listed gene names (i.e., SOX2, HNF1B, etc.) are sufficient for one of ordinary skill in the art to identify the recited genes. Reference to a gene is intended to encompass variations of the gene and is not intended to be limited by 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 merely exemplary in nature. That is, an identifier may refer to only a particular isoform/variant that may be one of many. One of ordinary skill in the art will readily appreciate this distinction, and one of ordinary skill in the art will appreciate that the genes described encompass variants and genes having sequences that differ from the sequences associated with the above accession numbers.

In one aspect, definitive endoderm may be derived from a precursor cell selected from the group consisting of embryonic stem cells, embryonic germ cells, induced pluripotent stem cells, mesodermal cells, definitive endoderm cells, posterior endoderm cells, and posterior intestinal cells. In one aspect, definitive endoderm may be derived from pluripotent stem cells. In one aspect, definitive endoderm may be derived from pluripotent stem cells selected from embryonic stem cells, adult stem cells or induced pluripotent stem cells. In one aspect, the DE may be a DE monolayer, wherein greater than 90% of the cells in the DE monolayer co-express FOXA2 and SOX 17.

In one aspect, definitive endoderm may be obtained by contacting pluripotent stem cells with a subgroup of BMPs selected from Activin, the TGF- β superfamily of growth factors; nodal, Activin A, Activin B, BMP4, Wnt3a, and combinations thereof.

In one aspect, the BMP signaling pathway inhibitor may be selected from the group consisting of Noggin, Dorsomorphin, LDN189, DMH-1, and combinations thereof. In one aspect, the BMP signaling pathway inhibitor is Noggin. The BMP inhibitor may be present in a concentration of from about 50 to about 1500 ng/ml.

In one aspect, WNT activators may be selected from one or more molecules selected from the group consisting of: wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, GSK β inhibitors (e.g. CHIR99021, i.e. "CHIRON"), BIO, LY2090314, SB-216763, lithium, swine inhibitor (porcinhibitor) IWP, LGK974, C59, SFRP inhibitors WAY-316606, β -catenin activator DCA. The concentration of Wnt pathway activator may be used, for example, 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 /). Suitable some existing Wnt signaling pathway activators include, but are not limited to, protein-based activators, which may include Wnt ligands, including, but not limited to, Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt8, and the like; alterations in Wnt ligand activity, including but not limited to, activated Wnt frizzled receptor, (LRP) co-receptor, R-spondin protein, Dkk protein, modulators of Wnt ligand secretion and transport (wnless, Porcupine), inhibition of β -catenin degradation APC and GSK3 β inhibition, activated β -catenin, constitutively active TCF/Lef protein, and chemical activators, which may include more than 28 chemicals known to activate or inhibit Wnt/β -catenin signaling. Some activators include, but are not limited to, GSK 3-beta inhibitor CHIR99021(CHIRON), BIO, LY2090314, SB-216763, lithium, porcupine inhibitor IWP, LGK974, C59, SFRP inhibitor WAY-316606, beta-catenin activator DCA.

In one aspect, the FGF activator can be one or more molecules selected from the group consisting of: 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 signaling components downstream of the receptors, including MAPK, MEK, ERK proteins and chemicals that modulate their activity. FGF signaling can be activated by inhibiting inhibitors of the FGF signaling pathway, including but not limited to, members of the Sprouty family of proteins.

In one aspect, the retinoic acid of step a may be contacted with DE for a period of time from about 12 hours to about 48 hours, or from about 20 hours to about 40 hours, or about 24 hours, or until the treatment results in loss of PDX expression as well as P63 expression.

In one aspect, said step c may be performed for a period of time sufficient to form a stratified epithelium lacking KRT 8. In one aspect, step c can be performed for a period of time sufficient to form stratified squamous epithelium expressing the regional keratin. In one aspect, step c can be performed for a period of time sufficient for the HEO to express INV.

In one aspect, the first time period may be a period of about three days ± 24 hours. In one aspect, the second time period can be a period of about three days ± 24 hours. In one aspect, the third time period may 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 through c may be performed in vitro.

In one aspect, the method may further comprise the step of contacting the forepart foregut culture of step a) or the spheres of step b) with a matrix selected from collagen, basement membrane matrix (Matrigel), or a combination thereof.

In one aspect, the esophageal compositions described herein can be characterized by the absence of innervation and/or blood vessels. 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, transition regions, vasculature, immune cells, or submucosa.

In one aspect, an esophageal progenitor cell capable of being organized into an organotypic culture is disclosed. The esophageal progenitor cells can be derived from the methods disclosed herein.

In one aspect, a method of making a stratified squamous epithelium is disclosed. The method may comprise enzymatically dissociating HEO as described herein to release progenitor cells, wherein the HEO is between about 3 weeks and about 10 weeks of age, or between about 4 weeks and about 8 weeks of age, or about 5 weeks of age; expanding the progenitor cells into a monolayer; redifferentiating the dissociated HEO on a collagen-coated membrane into stratified squamous epithelium 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 the group consisting of IVL, CRNN and FLG. In one aspect, stratified squamous epithelium may comprise esophageal cells organized substantially in the form of lamellae.

In one aspect, a method of treating esophageal disease in an individual in need thereof is disclosed. The disease may be selected from congenital diseases (atresia), functional diseases (achalasia and other movement disorders), immunological diseases (eosinophilic esophagitis), pathological diseases (barrett's esophagus and esophageal cancer), and combinations thereof, the method comprising the step of contacting an esophageal composition (e.g., HEO or esophageal sheet) disclosed herein with the subject.

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

In one aspect, a method of identifying a treatment for eosinophilic esophagitis is disclosed. In this aspect, the method can include contacting a potential therapeutic agent of interest with an organoid or esophageal tissue as 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.

In one aspect, a method of making a model of fanconi anemia disease is disclosed. In this aspect, the disclosed method of making a HEO or esophageal sheet is performed as described herein, wherein the DE is obtained from a FANCA deficient precursor cell.

In one aspect, a method of making a model of barrett's metaplastic disease is disclosed. In this aspect, the method can include the step of inducing CDX2 and activating BMP in a HEO or esophageal sheet prepared according to the methods disclosed herein.

In one aspect, a method of making an eosinophilic esophagitis disease model is disclosed. In this aspect, the method may comprise contacting HEO or esophageal flakes 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.

In one aspect, a method of identifying an active agent capable of treating an esophageal disease state is disclosed, comprising the steps of: contacting a test agent with a HEO or esophageal sheet 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 increased esophageal gene expression such as SOX2, p63, KRT13, CRNN, IVL, and loss of Barrett's intestinal genes.