Compositions and methods for promoting the generation of pharyngeal foregut endoderm and its derivatives

Abstract

The disclosure herein provides, in example embodiments, methods to generate human pharyngeal foregut endoderm, e.g., from pluripotent stem cells, as well as methods and compositions for treatment of defects, e.g., developmental defects affecting the pharyngeal endoderm and its derivatives.

Description

COMPOSITIONS AND METHODS FOR PROMOTING THE GENERATION OF PHARYNGEAL FOREGUT ENDODERM AND ITS DERIVATIVESRELATED APPLICATION(S)

[0001] This application claims the benefit of U.S. Patent Application No. 63 / 538,801, filed on September 16, 2023. The entire teachings of the above application(s) are incorporated herein by reference.GOVERNMENT SUPPORT

[0002] This invention was made with government support under DK104218, DK089572, All 14525, and AI132963 from National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Developmental defects affecting the pharynx and derived tissues are found in a variety of syndromes. The associated malformations and dysfunctions of pharynx-derived tissues precipitate phenotypes ranging from malformed features and endocrine disorders to severe immunodeficiency, some of which are associated with high morbidity or elaborate treatment and health care costs.

[0004] While some of the developmental defects have been linked to mutations or singlenucleotide polymorphisms in transcription factors (TFs) or cis-regulatory elements (CREs), the origins of most pharynx-specific birth defects remain poorly understood, and the underlying genetic causes as well as the epigenetic context remain elusive. Unfortunately, restricted human tissue access, as well as discordance of genetic factor requirement and phenotypic penetrance of disease-associated human mutations when modeled in mice, have prohibited detailed insights on the molecular basis of these mentioned human diseases. Accordingly, there is a need for improved treatment of defects, including developmental defects affecting the pharyngeal endoderm and its derivatives.SUMMARY

[0005] There is a critical need to develop methods, compositions, and therapeutic agents for treating disorders including defects (e.g., developmental defects) affecting the pharynxand derived tissues and the syndromes that result therefrom. The disclosure provides such methods and compositions.

[0006] The disclosure provided herein is based, in part, on an efficient step-wise differentiation method to generate human pharyngeal foregut endoderm (PFE) cells from pluripotent stem cells, as well as a method to generate organoids, e.g., thymic organoids from the PFE cells. Accordingly, in some embodiments, the disclosure relates to compositions (e.g., a medium for differentiating a pluripotent stem cell, an organoid comprising differentiated cells), methods, and therapeutic agents that are useful for treating defects of the pharynx, pharynx-derived tissue, and related syndromes.

[0007] In some aspects, the invention provides a method for producing anterior foregut endoderm (AFE) cells, the method comprising the step of culturing definitive endoderm (DE) cells in a medium, the medium comprising about 100 nM retinoic acid, thereby producing AFE cells.

[0008] In some embodiments: the medium further comprises an ALK / TGF-PRI inhibitor, an ALK / BMP inhibitor, a WNT inhibitor, or a combination thereof; culturing the DE cells in the medium occurs on about days 4-8 of differentiation and / or for a period of about five days; or both of the foregoing.

[0009] In some embodiments, the medium comprises: about 1 pM of ALK / TGF-PRI inhibitor, about 250 nM of ALK / BMP inhibitor, about 250 nM of WNT inhibitor, or a combination of the foregoing.

[0010] In some embodiments, the medium further comprises about 100 nM retinoic acid, about 1 pM of ALK / TGF-PRI inhibitor A83-01, about 250 nM of ALK / BMP inhibitor LDN193189, about 250 nM of WNT inhibitor IWR-1, or a combination of the foregoing.

[0011] In some embodiments, the medium comprises: about 100 nM retinoic acid, about 1 pM of ALK / TGF-PRI inhibitor A83-01, about 250 nM of ALK / BMP inhibitor LDN193189, and about 250 nM of WNT inhibitor IWR-1.

[0012] In some embodiments, the method further comprises the step of culturing the DE cells in a medium, the medium comprising about 0 nM retinoic acid prior to culturing the DE cells in the medium comprising about 100 nM retinoic acid.

[0013] In some embodiments, the method further comprises singularizing and plating the DE cells prior to culturing the DE cells in the medium comprising about 0 nM retinoic acid, optionally wherein singularizing and plating the DE cells occur on about day 3 of differentiation; the medium comprising about 0 nM retinoic acid further comprises anALK / TGF-PRI inhibitor, an ALK / BMP inhibitor, a ROCK inhibitor, or a combination thereof; culturing the DE cells in the medium comprising about 0 nM retinoic acid occurs on about day 3 of differentiation and / or for a period of about one day; or a combination thereof.

[0014] In some embodiments, the medium comprising about 0 nM retinoic acid comprises about 1 pM of ALK / TGF-PRI inhibitor, about 250 nM of ALK / BMP inhibitor, about 10 pM of ROCK inhibitor, or a combination of the foregoing.

[0015] In some embodiments, the medium comprising about 0 nM retinoic acid comprises about 1 pM of ALK / TGF-PRI inhibitor A83-01, about 250 nM of ALK / BMP inhibitor LDN193189, about 10 pM of ROCK inhibitor Y-27632, or a combination of the foregoing.

[0016] In some embodiments, the medium comprising about 0 nM retinoic acid further comprises: about 1 pM of ALK / TGF-PRI inhibitor A83-01, about 250 nM of ALK / BMP inhibitor LDN193189, and about 10 pM of ROCK inhibitor Y-27632.

[0017] In some embodiments, the AFE cells: functionally express VGLL2, EYA1, SIX1, or a combination thereof; do not functionally express POU5F1, SOX17, or a combination thereof; or both of the foregoing.

[0018] In some embodiments, the method further comprises the step of producing the DE cells prior to culturing the DE cells, by culturing pluripotent stem cells (PSCs) in a medium on about day 0 of differentiation and / or for a period of about 1 day, the PSCs comprising induced PSCs (iPSCs) or embryonic stem cells (ESCs), and the medium comprising: a Roswell Park Memorial Institute 1640 cell culture medium (RPMI 1640), about 0.2% HYCLONE® Defined fetal bovine serum (FBS), about 1% of L-glutamine supplement GLUTAMAX® (200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl), about 1% 5,000 IU penicillin-5,000 pg / mL streptomycin (P / S), about 100 ng / mL Activin A, about 3 pM of GSK3 inhibitor CHIR99021, and about 50 nM of PI3K / mT0R inhibitor PI- 103; and culturing the PSCs in a medium on about days 1-2 of differentiation and / or for a period of about 2 days, the medium comprising: RPMI 1640, about 0.2% HYCLONE® Defined FBS, about 1% GLUTAMAX® (200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl), about 1% P / S, about 100 ng / mL Activin A, and about 250 nM of ALK / BMP inhibitor LDN193189; thereby producing DE cells; optionally wherein the PSCs are human PSCs or murine PSCs.

[0019] In some embodiments, the DE cells functionally express SOX17, GATA4, GATA6, EOMES, LEFTY1, CER1, or a combination thereof; do not functionally expressP0U5F1, VGLL2, EYA1, PAX9, PAX1, F0XE1, PBX1, or a combination thereof (z.e., do not functionally express one or more of POU5F1, VGLL2, EYA1, PAX9, PAX1, FOXE1, PBX1); or both of the foregoing.

[0020] In some aspects, the invention provides a method for producing pharyngeal foregut endoderm (PFE) cells, the method comprising the step of culturing anterior foregut endoderm (AFE) cells in a medium, the medium comprising: retinoic acid, optionally wherein the retinoic acid is at a concentration of about 100 nM; a bone morphogenic protein (BMP), optionally wherein the BMP comprises BMP4, and / or optionally wherein the BMP is at a concentration of about 50 ng / mL; a fibroblast growth factor (FGF), optionally wherein the FGF comprises FGF10, and / or optionally wherein the FGF is at a concentration of about 50 ng / mL; a ROCK inhibitor, optionally wherein the ROCK inhibitor is Y-27632, and / or optionally wherein the ROCK inhibitor is at a concentration of about 10 pM; or a combination of the foregoing, thereby producing PFE cells.

[0021] In some embodiments, the AFE cells are produced by a method disclosed herein.

[0022] In some embodiments, the method further comprises singularizing and plating the AFE cells prior to culturing the AFE cells in the medium, optionally wherein singularizing and plating the AFE cells occur on about day 9 of differentiation.

[0023] In some embodiments, the AFE cells are cultured in a medium comprising retinoic acid, BMP, FGF, and a ROCK inhibitor. In some embodiments, culturing the AFE cells occurs for a first period of time and a second period of time, and the AFE cells are cultured for a first period of time in the medium comprising retinoic acid, BMP, FGF, and a ROCK inhibitor; and / or the AFE cells are cultured for a second period of time in the medium comprising retinoic acid, BMP, and FGF.

[0024] In some embodiments: the first period of time occurs on about day 9 of differentiation and / or for a period of about one day; and / or the second period of time occurs on about days 10-14 of differentiation and / or for a period of about five days.

[0025] In some embodiments, the PFE cells: functionally express FOXA2, SOX2, PAX9, PAX1, FOXE1, PBX1 or a combination thereof; do not functionally express POU5F1, SOX17, VGLL2, or a combination thereof; or both of the foregoing.

[0026] In some embodiments, the PFE cells functionally express at least about two-fold the level of PAX9 functionally expressed by the AFE cells.

[0027] In some aspects, the invention provides a method for producing thymic epithelial progenitor (TEP) cells, the method comprising the step of culturing pharyngeal foregutendoderm (PFE) cells in a medium, the medium comprising: retinoic acid, optionally wherein the retinoic acid is at a concentration of about 100 nM; a fibroblast growth factor (FGF), optionally wherein the FGF comprises FGF10, and / or optionally wherein the FGF is at a concentration of about 50 ng / mL; an ALK / TGF-PRI inhibitor, optionally wherein the ALK / TGF-PRI inhibitor comprises A83-01, and / or optionally wherein the ALK / TGF-PRI inhibitor is at a concentration of about 1 pM; a ROCK inhibitor, optionally wherein the ROCK inhibitor comprises Y-27632, and / or optionally wherein the ROCK inhibitor is at a concentration of about 10 pM; or a combination of the foregoing, thereby producing thymic epithelial progenitor (TEP) cells.

[0028] In some embodiments, the PFE cells are produced by a method disclosed herein.

[0029] In some embodiments, culturing the PFE cells occurs for a first period of time and a second period of time. In some embodiments, the PFE cells are cultured for a first period of time in the medium comprising retinoic acid and FGF; and / or the PFE cells are cultured for a first period of time in the medium comprising ALK / TGF-PRI inhibitor.

[0030] In some embodiments: the first period of time occurs on about days 15-21 of differentiation and / or for a period of about 7 days; and / or the second period of time occurs on about days 22-28 of differentiation and / or for a period of about 7 days.

[0031] In some embodiments, the TEP cells: functionally express PAX1, PAX9, FOXN1, GATA3, or a combination thereof; do not functionally express VGLL2, SOX17, CER1, POU5F1, NANOG, or a combination thereof; or both of the foregoing.

[0032] In some aspects, the invention provides a method for producing an organoid comprising thymic epithelial cells (TECs), the method comprising the steps of: seeding thymic epithelial progenitor (TEP) cells onto a scaffold, thereby initiating an organoid; and culturing the organoid in a medium, the medium comprising: an ALK / TGF-PRI inhibitor, optionally wherein the ALK / TGF-PRI inhibitor is A83-01, and / or optionally wherein the ALK / TGF-PRI inhibitor is at a concentration of about IpM; a ROCK inhibitor, optionally wherein the ROCK inhibitor is Y-27632, and / or optionally wherein the ROCK inhibitor is at a concentration of about 10 pM; or both of the foregoing, thereby producing an organoid comprising TECs.

[0033] In some embodiments, the TEP cells are produced by a method disclosed herein.

[0034] In some embodiments: the method further comprises singularizing the TEP cells prior to seeding the TEP cells onto a scaffold; singularizing and seeding the TEP cells onto a scaffold occur on about day 29 of differentiation; the scaffold is contained in a plate,comprises a decellularized thymic lobe or a cell culture filter insert, is coated with a basement membrane or extracellular matrix, or a combination thereof, optionally wherein the decellularized thymic lobe is from a mouse, optionally wherein the mouse is of about 4-6 weeks of age, is of an ICR strain, or both; culturing the organoid in the medium occurs on about days 29-45 of differentiation and / or for a period of about 17 days; or a combination thereof.

[0035] In some aspects, the invention provides a method for producing thymic epithelial cells (TECs), the method comprising the steps of producing an organoid comprising thymic epithelial cells (TECs) according to a method disclosed herein; and isolating the TECs from the organoid comprising TECs, thereby producing the TECs.

[0036] In some embodiments, the TECs: functionally express GCM2, FOXN1, CCL25, PAX1, PAX9, GATA3, MAFB, or a combination thereof; do not functionally express VGLL2, SOX17, CER1, POU5F1, NANOG, or a combination thereof; or both of the foregoing.

[0037] In some embodiments, singularizing comprises: dissociating with a proteolytic enzyme, wherein the proteolytic enzyme is capable of detaching and / or dissociating adherent cells in culture, optionally wherein the proteolytic enzyme, or a solution containing the proteolytic enzyme, comprises trypsin, TRYPLE® recombinant dissociation enzyme, ACCUTASE® cell detachment solution, DISPASE® dissociating enzyme, or a combination thereof; scraping and then triturating; or both of the foregoing.

[0038] In some embodiments, culturing comprises growing cells on a basement membrane or extracellular matrix, optionally wherein the basement membrane or extracellular matrix comprises 804G supernatant, MATRIGEL® basement membrane matrix, MATRIGEL® Growth Factor Reduced (GFR) basement membrane matrix, or a combination thereof.

[0039] In some embodiments, the medium further comprises: a Roswell Park Memorial Institute 1640 cell culture medium (RPMI 1640); a neuronal cell culture supplement, optionally wherein the neuronal cell culture supplement comprises B-27® minus vitamin A, and / or optionally wherein the neuronal cell culture supplement is at a concentration of about 0.5x; an L-glutamine supplement, optionally wherein the L-glutamine supplement comprises GLUTAMAX® (200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl), and / or optionally wherein the L-glutamine supplement comprises about 1% of the medium; an antibiotic, optionally wherein the antibiotic comprises 5,000 IU penicillin-5,000 pg / mLstreptomycin (P / S), and / or optionally wherein the antibiotic comprises about 1% of the medium; optionally, a ROCK inhibitor, optionally wherein the ROCK inhibitor is Y-27632, and / or optionally wherein the ROCK inhibitor is at a concentration of about 10 pM; or a combination of the foregoing.

[0040] In some aspects, the invention provides an anterior foregut endoderm (AFE) cell produced by a method disclosed herein.

[0041] In some aspects, the invention provides a pharyngeal foregut endoderm (PFE) cell produced by a method disclosed herein.

[0042] In some aspects, the invention provides a thymic epithelial progenitor (TEP) cell produced by a method disclosed herein.

[0043] In some aspects, the invention provides a thymic epithelial cell (TEC) produced by a method disclosed herein.

[0044] In some aspects, the invention provides an organoid comprising thymic epithelial cells (TECs) produced by a method disclosed herein.

[0045] In some aspects, the invention provides a pharmaceutical composition comprising the pharyngeal foregut endoderm (PFE) cell disclosed herein, the thymic epithelial progenitor (TEP) cell disclosed herein, the thymic epithelial cell (TEC) disclosed herein, or the organoid comprising TECs disclosed herein.

[0046] In some aspects, the invention provides a method of recovering or restoring impaired tissue in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharyngeal foregut endoderm (PFE) cell disclosed herein, the thymic epithelial progenitor (TEP) cell disclosed herein, the thymic epithelial cell (TEC) disclosed herein, or a population or organoid comprising such a cell.

[0047] In some aspects, the invention provides a method of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount the pharyngeal foregut endoderm (PFE) cell disclosed herein, the thymic epithelial progenitor (TEP) cell disclosed herein, the thymic epithelial cell (TEC) disclosed herein, or a population or organoid comprising such a cell.

[0048] In some embodiments, the subject has a thymus, parathyroid, and / or thyroid tissue disorder, and wherein definitive endoderm (DE) cells are differentiated from pluripotent stem cells (PSCs) obtained from a biological sample from the subject.

[0049] In some aspects, the invention provides a method of evaluating a biological response to a compound or an environmental stimulus, the method comprising: producing afirst and a second population of pluripotent stem cells (PSCs); exposing the second population of PSCs to the compound or environmental stimulus; producing a first population of definitive endoderm (DE) cells by culturing the first population of PSCs and a second population of DE cells by culturing the second population of PSCs according to a method disclosed herein; producing a first population of anterior foregut endoderm (AFE) cells from the first population of DE cells and a second population of AFE cells from the second population of DE cells according to a method disclosed herein; producing a first population of pharyngeal foregut endoderm (PFE) cells from the first population of AFE cells and a second population of PFE cells from the second population of AFE cells according to a method disclosed herein; and comparing the first and second populations of PFE cells, thereby evaluating the biological response.

[0050] In some embodiments, the method further comprises: producing a first population of thymic epithelial progenitor (TEP) cells from the first population of PFE cells and a second population of TEP cells from the second population of PFE cells according to a method disclosed herein; producing a first population of thymic epithelial cells (TECs) from the first population of TEP cells and a second population of TECs from the second population of TEP cells according to a method disclosed herein; and comparing the first and the second population of TECs, thereby evaluating the biological response.

[0051] In some embodiments: the first population of PSCs is obtained from a subject or produced from cells obtained from the subject prior to exposing the subject to the compound or environmental stimulus; exposing the second population of PSCs to the compound or environmental stimulus comprises exposing the subject to the compound or environmental stimulus; and the second population of PSCs is obtained from the subject or produced from cells obtained from the subject after exposing the subject to the compound or environmental stimulus.

[0052] In some aspects, the invention provides a composition for identifying a therapeutic compound for treatment of a thymus, parathyroid, and / or thyroid tissue dysfunction, the composition comprising the pharyngeal foregut endoderm (PFE) cell disclosed herein, the thymic epithelial progenitor (TEP) cell disclosed herein, the thymic epithelial cell (TEC) disclosed herein, or a population or organoid comprising such a cell.

[0053] In some aspects, the invention provides a method of increasing pharyngeal foregut endoderm (PFE) cells, thymic epithelial progenitor (TEP) cells, and / or thymic epithelial cells (TECs) in a subject in need thereof, the method comprising administeringretinoic acid to the subject in an amount sufficient to increase PFE cells, TEP cells, and / or TECs.

[0054] In some aspects, the invention provides a medium for producing anterior foregut endoderm (AFE) cells, the medium comprising: an ALK / TGF-PRI inhibitor, optionally wherein the ALK / TGF-PRI inhibitor is A83-01, and / or optionally wherein the ALK / TGF- PRI inhibitor is at a concentration of about IpM; an ALK / BMP inhibitor, optionally wherein the ALK / BMP inhibitor comprises LDN193189, and / or optionally wherein the ALK / BMP inhibitor is at a concentration of about 250 nM; and optionally, a ROCK inhibitor, optionally wherein the ROCK inhibitor comprises Y-27632, and / or optionally wherein the ROCK inhibitor is at a concentration of about 10 pM.

[0055] In some aspects, the invention provides a medium for producing anterior foregut endoderm (AFE) cells, the medium comprising: about 100 nM retinoic acid, an ALK / TGF- PRI inhibitor, optionally wherein the ALK / TGF-PRI inhibitor is A83-01, and / or optionally wherein the ALK / TGF-PRI inhibitor is at a concentration of about IpM; an ALK / BMP inhibitor, optionally wherein the ALK / BMP inhibitor comprises LDN193189, and / or optionally wherein the ALK / BMP inhibitor is at a concentration of about 250 nM; and a WNT inhibitor, optionally wherein the WNT inhibitor comprises IWR-1, and / or optionally wherein the WNT inhibitor is at a concentration of about 250 nM.

[0056] In some aspects, the invention provides a medium for producing pharyngeal foregut endoderm (PFE) cells, the medium comprising: retinoic acid, optionally wherein the retinoic acid is at a concentration of about 100 nM; a bone morphogenic protein (BMP), optionally wherein the BMP comprises BMP4, and / or optionally wherein the BMP is at a concentration of about 50 ng / mL; a fibroblast growth factor (FGF), optionally wherein the FGF comprises FGF10, and / or optionally wherein the FGF is at a concentration of about 50 ng / mL; and optionally, a ROCK inhibitor, optionally wherein the ROCK inhibitor is Y- 27632, and / or optionally wherein the ROCK inhibitor is at a concentration of about 10 pM.

[0057] In some aspects, the invention provides a medium for producing pharyngeal foregut endoderm (PFE) cells, the medium comprising: retinoic acid, optionally wherein the retinoic acid is at a concentration of about 100 nM; a bone morphogenic protein (BMP), optionally wherein the BMP comprises BMP4, and / or optionally wherein the BMP is at a concentration of about 50 ng / mL; and a fibroblast growth factor (FGF), optionally wherein the FGF comprises FGF10, and / or optionally wherein the FGF is at a concentration of about 50 ng / mL.

[0058] In some aspects, the invention provides a medium for producing thymic epithelial progenitor (TEP) cells, the medium comprising: retinoic acid, optionally wherein the retinoic acid is at a concentration of about 100 nM; and a fibroblast growth factor (FGF), optionally wherein the FGF comprises FGF10, and / or optionally wherein the FGF is at a concentration of about 50 ng / mL.

[0059] In some aspects, the invention provides a medium for producing thymic epithelial progenitor (TEP) cells, the medium comprising: an ALK / TGF-PRI inhibitor, optionally wherein the ALK / TGF-PRI inhibitor comprises A83-01, and / or optionally wherein the ALK / TGF-PRI inhibitor is at a concentration of about IpM.

[0060] In some aspects, the invention provides a medium for producing thymic epithelial cells (TECs) and / or an organoid comprising TECs, the medium comprising: an ALK / TGF- PRI inhibitor, optionally wherein the ALK / TGF-PRI inhibitor is A83-01, and / or optionally wherein the ALK / TGF-PRI inhibitor is at a concentration of about IpM; and optionally, a ROCK inhibitor, optionally wherein the ROCK inhibitor is Y-27632, and / or optionally wherein the ROCK inhibitor is at a concentration of about 10 pM.

[0061] In some embodiments, a medium disclosed herein further comprises: a Roswell Park Memorial Institute 1640 cell culture medium (RPMI 1640); a neuronal cell culture supplement, optionally wherein the neuronal cell culture supplement comprises B-27® minus vitamin A, and / or optionally wherein the neuronal cell culture supplement is at a concentration of about 0.5x; an L-glutamine supplement, optionally wherein the L-glutamine supplement comprises GLUTAMAX® (200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl), and / or optionally wherein the L-glutamine supplement comprises about 1% of the medium; and an antibiotic, optionally wherein the antibiotic comprises 5,000 IU penicillin- 5,000 pg / mL streptomycin (P / S), and / or optionally wherein the antibiotic comprises about 1% of the medium.BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.

[0063] FIG. 1A shows a schematic of an example small molecule-directed protocol utilized to generate definitive endoderm and anterior foregut cells from human pluripotent stem cells. Pluripotent stem cells (PSC), definitive endoderm (DE) and anterior foregut endoderm (AFE) differentiation stages, small molecules and timeline are described. FIG. IB shows representative intracellular flow cytometry of day 0 (PSC), day 3 (DE), and day 9 (AFE) cultures for SOX2 and FOXA2. Percentages of populations are indicated. FIG. 1C shows representative immunofluorescence staining of cultures on day 0 (PSC), day 3 (DE) and day 9 (AFE) of differentiation for FOXA2 and SOX2 with Hoechst stain indicating nuclei. Scale bar = 100pm. FIG. ID shows quantitative gene expression analysis of POU5F1, NANOG, 80X17, GA TA 6, 0X2 and OTX2 in differentiation cultures at day 0 (PSC), day 3 (DE), and day 9 (AFE). Expression levels are relative to ACTB. N = 3 independent experiments. Error bars show + / - standard deviation (S.D.). FIG. IE shows quantitative gene expression analysis of PAX9, NKX2.6, PAX1, HOXA3, 81X1, VGI.I.2, HHEX, EYA 1, and OTX2 at day 9 (AFE) treated with the indicated concentrations of retinoic acid (RA), or I M of the retinoic acid receptor inverse agonist BMS493 (BMS). Expression levels are relative to day 3 (DE). N = 3 independent experiments. Error bars show + / - S.D. An asterisk (*) denotes p<0.01 relative to the no retinoic acid (RA) condition. FIG. IF shows UMAP (uniform manifold approximation and projection) visualization of single cell transcriptomic profiles at AFE in the presence of varying concentrations of RA added at DE with cells colored by RA condition. FIG. 1G is a column-standardized heatmap displaying expression of anterior, posterior and mid / hindgut markers from literature at each of the four retinoic acid conditions profiled by scRNA sequencing.

[0064] FIG. 2A shows UMAP visualization of single cell transcriptomic profiles at AFE in the presence of varying concentrations of RA added at DE with cells shaded by unsupervised leiden cluster assignment. Each dot represents a single cell in the global transcriptomic space. FIG. 2B shows pie plots indicating the percentage of cells in each RA condition for each of the four leiden clusters (FIG. 2C and FIG. 2D). FIG. 2C shows UMAP visualization from FIG. 2A with cells shaded by an RA response signature gene score curated from literature (Table S2 of Lopez-Perez et cd., 2021). FIG. 2D shows transcripts associated with anterior foregut endoderm (80X2, OTX2), pharyngeal endoderm (PAX1, PAX ) and posterior foregut endoderm (PDXP) identity. FIG. 2E is a dot plot displaying expression patterns of the top 20 differentially expressed transcripts ranked by increasing adjusted p- value (log2foldchange > 1, adjusted p-value < 0.05, fraction of cells expressing the genewithin the group > 0.25, fraction of cells expressing the gene outside the group < 0.5). Transcripts upregulated in more than 1 condition were displayed according to the condition with the least adjusted p-value. The size of the dot indicates the fraction of cells per RA condition expressing a given gene, and the shading indicates mean expression in that condition. FIG. 2F provides a curated list of terms from an EnrichR analysis of the differentially upregulated genes per RA condition with shade indicating the scaled z-score enrichment of the term for the genes in that condition. FIG. 2G shows UMAP visualization with cells shaded by the inferred cell-cycle phase Gl, G2M or S. Variable radius pie plots indicating the mean percentage of cells in each phase + / - standard error across the two replicates per RA condition. Radii are proportional to the mean percentage of cells in that phase.

[0065] FIG. 3A shows quantitative gene expression analysis of PAX9 in differentiation cultures at day 15 (PFE) at the indicated time window of RA treatment. Expression levels are displayed relative to the no RA condition. N = 3 independent experiments. Error bars show + / - S.D. An asterisk (*) denotes p<0.01 relative to the no RA condition. FIG. 3B shows representative immunofluorescence staining of day 15 (PFE) cultures generated with no RA, lOOnM RA and lOuM RA from days 4-15 for PAX9 and EPCAM. Hoechst stain indicates nuclei. Scale bars = 100pm. FIG. 3C shows immunofluorescence staining of transplanted PFE following 10 weeks of maturation in vivo for FOXN1 co-stained with PAX9, HLA- DR / DP / DQ, K5, K8, UEA1 and CLDN4. Scale bars = 100pm, final right panel, scale bar = 50pm. FIG. 3D shows quantitative gene expression analysis of PAX9, PAX1, H0XA3, FOXG1, FOXN1, and PSMB11, of day 0 (PSC), day 3 (DE), day 9 (AFE), day 15 (PFE) and 10-week transplanted cells (Graft). Expression levels are relative to transplanted cells. N = 3 independent experiments. Error bars show + / - S.D. RA = retinoic acid.

[0066] FIG. 4A shows immunofluorescence microscopy series over the entire time course of pharyngeal foregut endoderm differentiation using Hl embryonic stem cells, capturing the expression of SOX17, POU5F1, FOXA2, SOX2, H0XA3 and EPCAM across PSC, DE, AFE and PFE stages. Nuclei were stained with Hoechst. Scale bar = 100pm. FIG. 4B shows quantitative gene expression analysis of FOXA2, SOX2, TBX1, TP63, HLA-DRA1, DLL4, IL7, SCF, CXCL12 and CCL25 on day 0 (PSC), day 3 (DE), day 9 (AFE), day 15 (PFE) and 10-week transplanted cells (Graft). Expression levels are relative to transplanted cells. N = 3 independent experiments. Error bars show + / - S.D. FIG. 4C shows karyotyping analysis showing that the CBOOl-iPSCs have a normal karyotype. FIG. 4D showsquantitative gene expression analysis of S0X17, F0XA2, S0X2 and PAX9 of CBOOl-iPSCs at PSC, DE, AFE and PFE stages. Expression levels are relative to ACTB. N = 2 independent experiments. Error bars show + / - S.D. FIG. 4E shows differentiation of the cord blood- derived induced pluripotent stem cell line (CBOOl-iPSC) into human pharyngeal foregut endoderm. Immunofluorescence microscopy series capturing the expression of critical genes SOX 17, P0U5F1, F0XA2, S0X2, H0XA3 and EPCAM across PSC, DE, AFE and PFE stages. Nuclei were stained with Hoechst. Scale bar = 100pm.

[0067] FIG. 5A shows a schematic showing an example of the approach: a scRNAseq directed differentiation time course sampled at 4 stages; two published scRNAseq mouse in vivo endoderm development datasets and a novel approach “CellMatch” for classifying a clustered query dataset onto a clustered reference based on a correlation distance metric. CellMatch compares neighboring reference-query matches and outputs a per-gene evaluation for the best match. FIG. 5B shows UMAP visualization of single cell transcriptomic profiles of the directed differentiation time course from PSC to PFE (N =3571 cells) shaded by the differentiation stage of the cells and unsupervised leiden clusters labelled by the stage of the cells within the cluster. Each dot represents a single cell in the global transcriptomic space. FIG. 5C shows a column-standardized heatmap displaying expression of markers from literature at each of the timepoints. FIG. 5D shows UMAP visualization of single cell transcriptomic profiles of known transcripts at PSC (POU5FPR DE (CER1, SOX 17}, AFE (VGLL2, EYA1),- DE to PFE (FOXA2),- PSC, AFE and PFE (50X2); and PFE (PAX9, PAX1, F0XE1, PBX1). FIG. 5E shows a heatmap of the normalized gene expression values plotted along increasing pseudotime with a bar showing the Wanderlust computed pseudotime. Heat is calculated by averaging over 100 cells, followed by row wise standard scaling. Time series plots of the probability of finding a cell in each leiden cluster is shown at the bottom. Confidence intervals, as determined from the 1% and 99% quantiles from bootstrapping for 1000 iterations, are shown as shaded regions around the calculated probability. Transcripts in the heatmap and clusters in the probability plots are ordered in increasing order of the pseudotime at which they attain their maximum value. FIG. 5F shows force directed graph layout for integrated mouse embryonic endoderm (E3.5-E8.75) (Nowotschin et al., 2019) and mouse embryonic PFE (E9.5-E12.5) (Magaletta et al, 2022). Each dot represents a cell in the global transcriptomic space (N = 76,894 cells). Non-endodermal cells are excluded. Relevant cell types are labelled, namely inner cell mass (ICM), Epiblast (EPI), Definitive Endoderm (DE), Gut Tube, Liver, Pancreas, Thyroid, Early and Late Pharyngeal Foregut Endoderm(PFE), Parathyroid, Thymic Epithelial Cells (TECs), Ultimobranchial Body (Ubb) and Esophagus and Oropharynx. FIG. 5G shows force directed graph layout of integrated mouse embryonic endoderm as in FIG. 5F. One dot is shown for each in vivo cell. Clusters with no stem cell equivalents are light gray. Clusters with stem cell equivalents are shaded according to the stage at which their equivalents are present. Labels denote in vitro to in vivo classification results, namely PSCs to Epiblast, DE to DE, AFE to early PFE, and PFE to late PFE. Early PFE denotes an E9.5 dominated PFE population while late PFE denotes an El 1.5 dominated PFE population. FIG. 5H shows a bar plot indicating the proportion of cells shaded by embryonic day from the four in vivo clusters to which the in vitro cells are classified: namely, Epiblast (EPI), Definitive Endoderm (DE), Early Pharyngeal Foregut Endoderm Cluster 16 (Early PFE(C16)) and Late Pharyngeal Foregut Endoderm Cluster 0 (Late PFE(CO)).

[0068] FIG. 6A shows schematic showing essentials of the CellMatch approach. Given a clustered reference dataset and a clustered single-cell RNA query dataset, CellMatch robustly selects an optimal matching model, then shows a gene-by-gene rationale for that model and a comparison to neighboring models. FIG. 6B shows performance on PBMC cross-platform benchmark data following Abdelaal et al. 2019. N / A means that a comparison was not attempted either because it was not cross-platform (diagonal entries) or because the indicated cell type was not present in one or both platforms (remaining entries). FIG. 6C shows model comparison results for classification of 10X v3 PBMC data using 10X v2 as the reference set. Starting from the optimal model, this compares all models within an edit distance of 1. The horizontal axis shows which query cluster had its match edited. The vertical axis specifies the match after the edit. Black dots show the best-fitting model, from which edits were made. FIG. 6D shows a cross-species benchmark task was selected following Abdelaal et al. 2019 using Allen Brain Atlas data from mouse (Tasic et al. 2018) and human (Hodge et al. 2019). Brain icons and data embedding icons are from the original papers. FIG. 6E shows Allen Brain Atlas benchmark results. Shading shows median Fl score across cell types. Scores for SingleCellNet (Tan and Cahan, 2019), ACTINN (Ma and Pellegrini, 2019), and SVM, are transcribed from Abdelaal et al. 2019. Scores for cellmatch use the ground-truth partition of the test data, and scores for cellmatch recluster use unsupervised clustering. FIG. 6F shows a subset of genes supporting CellMatch classification of human MTG data using mouse VI as the reference set. Shading shows expression in counts per million, rescaled to a maximum of 1 within each gene. Sidebar shows correlation distance by gene. FIG. 6G shows a bimodaldistribution of correlation distances across all genes used in CellMatch classification of human MTG data with mouse VI as the reference set.

[0069] FIG. 7A shows a heatmap illustrating the pairwise Pearson correlation between every pair of samples across the differentiation timecourse. FIG. 7B shows UMAP visualization of single cell transcriptomic profiles of the directed differentiation time course from PSC to PFE with cells shaded by replicate. FIG. 7C shows cell number proportions for each of the populations across the embryonic days. FIG. 7D shows a force directed graph layout of integrated mouse embryonic endoderm shaded by embryonic day. FIG. 7E shows a heatmap showing the column scaled cell-type averaged normalized scRNA expression for known literature markers. Cell-types are annotated with the prefix ‘P:’ followed by the annotation from the manuscript denoting cells from Nowotschin et cd.. 2019. Populations are annotated as Early, Mid and Late based on whether most cells in the population were at E9.5, E10.5 or El 1.5 respectively and numbered CO to C27 as described previously (in Supplementary Figure 3a of Magaletta et al., 2022). FIG. 7F shows model comparison results for staging of stem cell data using the combined mouse endoderm reference. Starting from the optimal model, this compares all models within an edit distance of 1. The vertical axis shows which query cluster had its match edited. The horizontal axis specifies the new match after the edit. Black dots show the best-fitting model, from which edits were made.Heat is the correlation distance for the corresponding reference-query model match. FIG. 7G shows genes supporting the CellMatch classification of human in vitro differentiation data using the mouse atlas as the reference set. Shading shows the normalized gene expression on the in vitro query dataset rescaled to a maximum of 1 within each gene. Sidebar shows correlation distance by gene. FIG. 7H shows transcript signature scores for the four sets of genes supporting the classification of PSC to Epiblast, DE to DE, AFE to Early PFE (Cl 6) and PFE to Late PFE (CO), respectively, computed on the mouse integrated atlas and visualized on the force directed layout embedding.

[0070] FIG. 8A shows principal component analysis of the eight samples (PSC, DE, AFE, PFE with 2 replicates each) profiled showing similarity between replicates and dissimilarity across stages. FIG. 8B shows pairwise distance between samples across all expressed transcripts. Samples are clustered using correlation distances. FIG. 8C shows a heatmap showing curated literature markers highlighting transcriptomic changes across the differentiation time course. FIGs. 8D-8G show volcano plots of pairwise differentially expressed genes with known literature markers labelled based on -logio adjusted p-value andlog2foldchange cutoffs as follows: “p-value and log2FC” (-logiopadj > 2, log2foldchange > 1), “p-value” ( -logiopadj > 2, log2foldchange < 1), “log2FC” ( -logiopadj < 2, log2foldchange > 1), and “NS” (not significant; -logiopadj < 2, log2foldchange < 1).

[0071] FIG. 9A shows a heatmap showing z-scored rlog normalized transcript expression of differentially expressed genes (number of genes = 6161) (adjusted p-val < 0.1, log2- foldchange > 0.5) from RNA comparisons between every pair across the four timepoints profiled, namely PSC, DE, AFE, and PFE with the two replicates indicated. Genes are grouped into 10 clusters using unsupervised k-means clustering on the centered and scaled normalized expression across the timepoints. Literature markers from each of the 10 clusters are shown. FIG. 9B shows immunofluorescence microscopy series over the entire time course of pharyngeal foregut endoderm differentiation, capturing PSC, DE, AFE, and PFE stages with antibodies against PBX1, FOXE1, GATA3, and PAX9. FIG. 9C shows curated list of terms from an EnrichR analysis of the 10 gene clusters in FIG. 9A with heat indicating the scaled z-score enrichment of the term for the genes in the cluster.

[0072] FIG. 10A shows a Venn diagram indicating overlap between the consensus set of ATAC regions across the four stem cell differentiation timepoints profiled: PSC, DE, AFE, and PFE. FIG. 10B shows pairwise correlation distance between samples across the consensus set of 202,709 ATAC peaks with a dendrogram indicating clustered sample distances. FIG. 10C shows principal component analysis of the ATAC samples on the consensus peak set from FIG. 10B showing similarity between replicates and dissimilarity across differentiation stages. FIG. 10D shows pairwise correlation distance between PSC, DE, AFE and PFE sample histone marks (H3K27me3, H3K4me2 and H3K27ac) with a dendrogram indicating clustered sample distances. FIG. 10E shows a heatmap of the top 40,163 regions with varying accessibility (coefficient of variation across the time course > 1) from normalized ATAC signal averaged over replicates across the four timepoints profiled, namely PSC, DE, AFE, and PFE. Regions are grouped into four clusters using unsupervised k-means clustering on the centered and scaled normalized signal.

[0073] FIGs. 11A-11D show browser tracks showing the ATAC, H3K27ac, H3K27me3, and H3K4me2 signal at SOX17 (FIG. 11 A), GATA6 (FIG. 11C), FOXA2 (FIG. 1 IB), and PAX1 (FIG. 1 ID) loci. FIG. HE shows per base averaged PhastCon conservation score across 100 vertebrates as a function of distance from peak center over the set VP of 40,163 variable ATAC peaks from FIG. 10E and on a background. FIG. HF shows plot of mean peak PhastCon conservation score by quantile on proximal and distal regions of arepresentative ENCODE DNAse Hypersensitivity peak set as well as the variable peak set from our differentiation, along with a random background set. FIGs. 11G-11J show heatmaps of transcription factor (TF) regulators split by the timepoint (FIG. 11G, PSC; FIG. 11H, DE; FIG. 1 II, AFE; FIG. 11 J, PFE) on which they have the maximum effect. Regulators were selected based on their motif enrichment in variable regions (VP) from (B), correlation (> 0.7) of ATAC motif enrichment and RNA expression across the TF family and correlation (>0.5) between RNA expression and motif enrichment in at least one of the three profiled histone marks on peaks in VP. Motif enrichment in VP regions is shown on ATAC, H3K27ac, H3K27me3 and H3K4me2 along with scaled TPM normalized RNA expression (cividis). In case of multiple TFs with the same motif enrichment correlation, the top two having the maximum expression variability across the time course were displayed.

[0074] FIG. 12A shows a heatmap showing the row normalized PageRank score of the top 150 TF drivers across all stages of the differentiation with importance determined by their PageRank score. Drivers are visualized as PSC, DE, AFE, or PFE based on the timepoint at which they have the most importance. FIG. 12B shows a gene regulatory network of the top TFs at PFE from the heatmap in FIG. 12A and their high weighted edge connectivity (weight > 10). Nodes are shaded based on their PageRank and directed edges indicate a putative enhancer binding event between a TF and its target.

[0075] FIG. 13 shows an experiment workflow to test maturation of PFE in a three- dimensional (3-D or 3D) environment in vitro. An Hl-FoxNl-luciferace-GFP (Hl-FNl-luc- GFP) embryonic stem cell line was used to generate PFE cells. PFE cells matured, z.e., are differentiated into, thymic epithelial progenitor (TEP) cells. TEP cells were subsequently used to generate organoids of thymic epithelial cells (TECs) on a scaffold (decellularized mouse thymus; ECM) or as a reaggregated organ culture (ROC). After reaching the TEP stage, cells were either continuously cultured in TEP differentiation medium without splitting (Untreated), split and seeded into a fresh monolayer culture (Mono), split and seeded onto a decellularized mouse thymus scaffold (ECM Recell), or split and seeded onto prepared tissue culture insert (TEP ROC).

[0076] FIGs. 14A-14C show that 3D culture induces an increased percentage of FOXN1- expressing (FoxNl+) and HLA-expressing (HLA+) human stem cell-derived TECs. FIG.14A shows flow cytometry results measuring the expression of epithelial cell adhesion molecule (EpCam) and HLA-DR in TEPs grown in a monolayer on extracellular matrix / basement membrane (Monolayer), TEPs seeded onto a decellularized mouse thymusscaffold and cultured (ECM Recell), and TEPs grown as a reaggregated organ culture (TEP ROC). Gates capture epithelial cells (EpCam+ only), TEPs (EpCam+ and FoxNl+), TECs (EpCam+, FoxNl+, and HLA+), and non-epithelial cells (EpCam-). FIG. 14B shows changes in the proportion of cells expressing EpCam, FoxNl (GFP), and HLA in ECM and TEP ROC 3D cultures relative to TEPs grown in Monolayer. FIG. 14C shows respective yields of TECs, TEPs, epithelial cells, and non-epithelial cells in ECM and TEP ROC cultures. The ECM and TEP ROC cultures were derived from an original plating of 2e+06 Hl-FNl-luc-GFP cells per well.

[0077] FIGs. 15A-15B show a high level of expression of FoxNl protein in ECM and TEP ROC cryosections after 17 days of organoid culture. Colocalization of Pax9 and FoxNl staining indicates a pharyngeal cell lineage. Staining of FoxNl detects FoxNl protein, not the associated GFP reporter. FIG. 15A depicts immunofluorescence detection of robust Pax9 and FoxNl expression in ECM culture. First column shows merge. Bottom row shows inset. Nuclei were stained with DAPI. FIG. 15B depicts immunofluorescence detection of robust Pax9 and FoxNl expression in TEP ROC culture. First column shows merge. Bottom row shows inset. Nuclei were stained with DAPI.

[0078] FIGs. 16A-B show HLA expression detected on FoxNl -expressing cells in both ECM and TEP ROC cultures. Staining of FoxNl detects FoxNl protein, not the associated GFP reporter. Colocalization of FoxNl and GFP staining demonstrates reporter fidelity. FIG. 16A depicts immunofluorescence detection of FoxNl, aGFP, and HLA in ECM culture. First column shows merge of FoxNl, aGFP, and DAPI. Bottom row shows inset. Nuclei were stained with DAPI. Last column (inset only) shows merge of FoxNl and HLA. FIG. 16B depicts immunofluorescence detection of FoxNl, aGFP, and HLA in TEP ROC culture. First column shows merge of FoxNl, aGFP, and DAPI. Bottom row shows inset. Nuclei were stained with DAPI. Last column (inset only) shows merge of FoxNl and HLA.

[0079] FIGs. 17A-17B show that 3D culture systems such as ECM and TEP ROC induce expression of cortical TEC maturation markers critical for thymus function and T-cell interaction. Expression of markers is shown in Day 0 scTEPs (e.g., single-cell TEPs prior to organoid culture), ECM, TEP ROC, TEPs grown in monolayer, and human fetal thymus. FIG. 17A depicts expression of cortical TEC markers PRSS16 and Psmbl 1 and general maturation markers FoxNl and HLA-DRA. FIG. 17B depicts expression of medullary TEC markers TNFRSF1 la and AIRE and general maturation markers DLL4 and IL-7.

[0080] FIGs. 18A-18B show enrichment of maturation markers in FoxNl-GFP positive, EpCam positive flow cytometry cell sorted populations in TEP ROC cultures. FIG. 18A depicts the gates used to sort cell populations within a TEP ROC culture. The upper left gate captures EpCam+ FoxNl / GFP+ cells. The upper right gate captures EpCam+ FoxNl / GFP- cells. The lower gate captures EpCam- FoxNl / GFP- cells. FIG. 18B depicts the fold-change in expression of maturation markers HLA-DR, DLL4, and IL-7 in different cell cultures and populations compared to expression in human fetal thymus. TEP ROC cultures were sorted according to FIG. 18 A, then qPCR was performed. In each graph, the first column shows Untreated (no split) cells, the second column shows TEP ROC FoxNl / GFP+ EpCam+ cells (upper right gate of FIG. 18 A), the third column shows Monolayer (split / re-plated) cells, the fourth column shows TEP ROC FoxNl / GFP- EpCam+ cells (upper left gate of FIG. 18 A), the fifth column shows bulk TEP ROC cells (unsorted), and the sixth column shows FoxNl / GFP- EpCam- cells (lower gate of FIG. 18A). Note scale bar is normalized to B-actin, then fold expression relative to human fetal thymus.

[0081] FIGs. 19A-19D show novel 3D-culture benefits in vitro human embryonic stem cell (hESC) differentiation toward pharyngeal endoderm derivatives, including thymic and parathyroid epithelium progenitors by single cell RNA-seq evaluation. FIG. 19A depicts the differentiation workflow and experimental groups (split / singularized monolayer, monolayer (no split), and organoid) that underwent single-cell RNA sequencing. FIG. 19B shows a UMAP visualization based on transcriptomic profiles of split monolayer, monolayer, and organoid cultures. FIG. 19C shows a heatmap of the mean expression of various markers (left) at the different stages of differentiation (top; the shaded label at the top of each column corresponds to, from left to right, ESC, DE, AFE, PFE1, PFE2, split monolayer, monolayer, and organoid as in FIG. 19A). FIG. 19D shows UMAP visualizations of expression of various differentiation and maturation markers in the cells shown in FIG. 19B.

[0082] FIGs. 20A-20C show novel 3D-culture benefits in vitro human embryonic stem cell (hESC) differentiation toward pharyngeal endoderm derivatives, including thymic and parathyroid epithelium progenitors by single cell RNA-seq evaluation. FIG. 20A depicts the differentiation workflow resulting in TEP ROC culture, which then underwent single cell RNA sequencing as in FIGs. 19A-D. FIG. 20B shows the normalized expression of various markers (left) at the different stages of differentiation (labeled at top). FIG. 20C shows an image of a TEP ROC organoid (top row, left), unsupervised leiden cluster assignment (toprow, first from left, “clusters”), and UMAP visualizations of expression of various differentiation and maturation markers.

[0083] FIGs. 21A-21C show computational classification of huma stem cell-derived organoid cells that confirms thymic and parathyroid signatures. FIG. 21A shows the computational classification workflow. FIG. 21B depicts the organoid culture leiden clusters from FIG. 20C (left) overlay ed on a roadmap projection (right). Reference test data classification (from works referenced herein) suggest that classifications correlate with the organoid clusters from FIG. 20C and FIG. 21B. FIG. 21C shows agreement between the transcriptional signatures of organoid clusters (top panel) and cell / tissue identity classifications (bottom panel). Labeled signature rows in the top panel correspond to signatures marked with an asterisk (*) in the bottom panel.DETAILED DESCRIPTION

[0084] A description of example embodiments follows.Several aspects of the disclosure are described below, with reference to examples for illustrative purposes only. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the disclosure. One having ordinary skill in the relevant art, however, will readily recognize that the disclosure can be practiced without one or more of the specific details or practiced with other methods, protocols, reagents, cell lines and animals. The present disclosure is not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and / or concurrently with other acts or events. Furthermore, not all illustrated acts, steps or events are required to implement a methodology in accordance with the present disclosure.Introduction

[0085] Presented herein, in some embodiments, is an efficient step-wise differentiation method to generate human pharyngeal foregut endoderm (PFE) cells from pluripotent stem cells, as well as methods to generate thymic organoids from the PFE cells. Dose and temporal requirements of signaling pathway engagement for optimized differentiation were determined and the differentiation products were characterized on cellular and integrated molecular levels.

[0086] In addition, presented herein are a computational classification tool, “CellMatch”, and a transcriptomic classification of differentiation products on an integrated mouse singlecell RNA sequencing (scRNAseq) developmental roadmap confirms cellular maturation. Integrated transcriptomic and chromatin analyses infer differentiation stage-specific gene regulatory networks.

[0087] The disclosure herein also provides methods and an integrated multi-omic resource for the investigation of disease-relevant loci and gene regulatory networks, and their role in developmental defects affecting the pharyngeal endoderm and its derivatives.

[0088] The developing pharynx is a critical developmental intermediate for proper organ formation. Notably, the pharyngeal endoderm has been pinpointed as contributing to various epithelia that are essential for organ functions involved in immune system and metabolic homeostasis. Various birth defects and organ failures have been linked to transcription factor dysfunctions in pharyngeal endoderm and its derivatives but have remained largely inaccessible for study due to scarce tissue access.

[0089] Approaches to study human pharyngeal foregut endoderm, a developmental intermediate that is linked to various human syndromes involving pharynx development and organogenesis of tissues such as thymus, parathyroid and thyroid, have been hampered by scarcity of tissue access and cellular models.

[0090] Human pluripotent stem cells (hPSCs) could provide a genetically tractable model to decipher the molecular logic of normal and impaired pharynx development. Previous attempts to develop directed differentiation protocols to generate pharyngeal endoderm from hPSCs have met with mixed efficacies (Green et aL, 2011; Kearns et aL, 2013). In addition, success in generating pharyngeal endoderm derivatives such as thymic, parathyroid and thyroid epithelial cells has been reported (Kurmann et al, 2015; Lawton et al., 2020; Parent et aL, 2013; Soh et aL, 2014; M. Su et al., 2015; X. Sun et al., 2013; Zeleniak et al., 2022). However, the pharyngeal endoderm intermediate remains not as well defined or is heterogeneous, the reported pharyngeal derivatives exhibit molecular inaccuracies, and protein expression of disease-associated TFs remains largely elusive Accordingly, there is a need for a well-defined hPSC differentiation protocol to generate pharyngeal endoderm.

[0091] A well-defined hPSC differentiation protocol to pharyngeal endoderm is not only the basis to understanding molecular mechanisms guiding cell fate transitions in this important progenitor population, but will also benefit generation of molecularly and functionally faithful pharyngeal derivatives.

[0092] To this end, through optimization of dose and temporal activation of culture conditions, provided herein, in an example embodiment, is a differentiation protocol to generate human pharyngeal endoderm from hPSCs in vitro at high efficacy. The novel differentiation product is benchmarked on a multi-omic level and dynamic gene regulatory principles of the differentiation intermediates are synthesized as an extended resource.

[0093] In some embodiments, the present disclosure presents a differentiation protocol to generate pharyngeal endoderm from human pluripotent stem cells in vitro at high efficacy. Differentiation to pharyngeal foregut endoderm (PFE) was achieved by optimizing temporal and dose-dependent retinoic acid (RA)-mediated patterning of anterior foregut endoderm (AFE) to PFE. While various studies have focused on generating pharyngeal endoderm derivatives (Kurmann et al., 2015; Lawton et al., 2020; Parent et al., 2Q ,' Soh et al., 2014; M. Su et al., 2015; X. Sun et al., 2013; Zeleniak et al., 2022), these studies have not fully captured the PFE intermediate and the temporal- and dose-dependent role of RA in this step, including the role of RA in promoting pharyngeal foregut identity and the posteriorizing effects of high RA concentrations.

[0094] The present disclosure also demonstrates example requirements for RA to engage expression of pharyngeal endoderm signature genes effectively at transcript and protein levels, distinguishing it from anteriorization platforms without RA (Green et al., 2011; K. M. Loh et al., 2014).

[0095] One consideration in the development of pharyngeal endoderm differentiation protocols is the detection of protein expression of known pharyngeal endoderm-specific transcription factors (TFs), which is a prerequisite to the study of functional and defective gene regulation by disease associated TFs. Notably, while PAX9 expression is detected at our AFE stage consistent with previous reports (Green et al., 2011), the additional differentiation step to PFE generates robust and widespread PAX9 protein expression at an unprecedented level. Caudal pharyngeal pouch associated H0XA3 is also detected at protein level starting at AFE and more ubiquitously at PFE. In addition, expression of additional TFs, such as PAX1, GATA3, TBX1, EYA1, SIX1 and FOXE1, that have been linked to developmental defects and pharyngeal-derivative organ dysfunction in humans, was confirmed (Chang et al., 2004; Clifton-Bligh et al., 1998; Muroya et al., 2001; Ruf et al., 2004; Yagi et al., 2003; Yamazaki et al., 2020).

[0096] Robustness of embodiments of the protocol described herein is demonstrated by its applicability for differentiation of the cord blood-derived hiPSCs. Accordingly, thepresently disclosed protocol has potential for use in modeling patient-specific pharyngeal- derivative organ dysfunction and syndromes.

[0097] Further maturation was achieved upon transplantation in immuno-deficient NSG mice, with spontaneous generation of third pharyngeal pouch derivative thymic epithelial cells, demonstrating developmental competence of the differentiation culture and induction of FOXN1 protein expression, a TF itself linked to functional defects of pharyngeal derivatives (Du et al.. 2019; Gallo et al.. 2017).

[0098] While it is significant and impactful to detect known pharyngeal markers, it is also important to place the differentiated cellular state in the context of in vivo developmental state in more comprehensive ways. To this end, the present disclosure not only presents an integrated developmental cell census including pluripotent states as well as broader endodermal gut tube and pharyngeal endoderm derivatives, it also develops a novel computational label transfer tool, CellMatch, which classifies query single-cell RNA-seq datasets based on such a reference dataset. This method is shown to be robust across platforms or species, performs well in comparisons with state-of-the-art classification approaches, as exhibited with multiple datasets, allows for classification of hPSC differentiation products. The scRNAseq analyses of the hPSC differentiation process not only revealed a highly efficient and homogeneous differentiation process to PFE, but also provided further support of curated gene list- and CellMatch-based characterization of the differentiation products by tracking the developmental cell state progression. Thus, the novel single cell RNA-seq (scRNAseq) compatible label transfer tool described herein allows for characterization of the differentiation intermediates in the context of in vivo development.

[0099] Further, the disclosure characterizes the differentiation intermediates at a multi- omic level and reveals transcriptomic and chromatin dynamics across the differentiation process. These data are integrated and synthesized to gene regulatory networks, highlighting predicted molecular logic of human pharyngeal endoderm development at unprecedented level.

[0100] Overall, the present disclosure not only presents insights on RA-dose and temporal response requirements for PFE differentiation, resulting in a novel and optimized protocol to generate human pharyngeal endoderm, e.g., for study, but also characterizes the resource on molecular, protein, and maturation levels. Further, the present disclosure provides CellMatch as a novel tool for cell classification and synthesize the molecular logic of PFE lineage differentiation, which provides context for some disease associatedtranscription factors. The present disclosure provides for a resource that will be a cornerstone for molecular and functional approaches toward studying the human pharyngeal endoderm lineage and its derivatives, which will impact current efforts on dissecting developmental defects and work toward cell replacement therapies of pharyngeal endoderm derivatives, such as thymus, parathyroid or thyroid.Definitions

[0101] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. It will be further understood that terms, such as those defined in commonly-used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and / or as otherwise defined herein.

[0102] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0103] As used herein, the indefinite articles “a,” “an” and “the” should be understood to include plural reference unless the context clearly indicates otherwise.

[0104] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of, e.g., a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integer or step. When used herein, the term “comprising” can be substituted with the term “containing” or “including.”

[0105] “About” means within an acceptable error range for the particular value, as determined by one of ordinary skill in the art. Typically, an acceptable error range for a particular value depends, at least in part, on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of ± 20%, e.g., ± 10%, ± 5% or ± 1% of a given value. It is to be understood that the term “about” can precede any particular value specified herein, except for particular valuesused in the Exemplification. When “about” precedes a range, as in “about 24-96 hours,” the term “about” should be read as applying to both of the given values of the range, such that “about 24-96 hours” means about 24 hours to about 96 hours. As used herein, the symbol “±” denotes a range, that is, where a given value is “N±X” a range from N-X to N+X, wherein the range includes the values of N-X and N+X.

[0106] As used herein, “consisting of’ excludes any element, step, or ingredient not specified in the claim element. When used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any of the terms “comprising,” “containing,” “including,” and “having,” whenever used herein in the context of an aspect or embodiment of the invention, can in some embodiments, be replaced with the term “consisting of,” or “consisting essentially of’ to vary scopes of the disclosure.

[0107] As used herein, the conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and / or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and, therefore, satisfy the requirement of the term “and / or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and, therefore, satisfy the requirement of the term “and / or.”

[0108] When a list is presented, unless stated otherwise, it is to be understood that each individual element of that list, and every combination of that list, is a separate embodiment. For example, a list of embodiments presented as “A, B, or C” is to be interpreted as including the embodiments, “A,” “B,” “C,” “A or B,” “A or C,” “B or C,” or “A, B, or C.”

[0109] When introducing elements disclosed herein, the articles “a,” “an,” “the,” and “said” are intended to mean that there are one or more of the elements. Further, the one or more elements may be the same or different. Thus, for example, unless the context clearly indicates otherwise, “an agent” includes a single agent, and two or more agents. Further the two or more agents can be the same or different as, for example, in embodiments wherein a first agent comprises a polynucleotide of a first sequence and a second agent comprises a polynucleotide of a second sequence.

[0110] The terms “subject” and “patient” are used interchangeably herein. The term “patient” refers to a human, while the term “subject” may refer to a human or a non-human animal.

[0001] As used herein, a “subject” is a vertebrate, including any member of the class mammalia.

[0002] As used herein, a “mammal” refers to any mammal including but not limited to human, mouse, rat, sheep, monkey, goat, rabbit, hamster, horse, cow or pig.

[0003] A “non-human mammal”, as used herein, refers to any mammal that is not a human.

[0111] In some embodiments, the subject is a human.

[0112] In some embodiments, the subject has one X chromosome and one Y chromosome. In some embodiments, the subject has two X chromosomes. In some embodiments, the subject has two X chromosomes and one Y chromosome. In some embodiments, the subject has one X chromosome and two Y chromosomes. In some embodiments, the subject is a human male. In some embodiments the subject is human female.

[0113] In some embodiments, the subject is at least about 1 month of age, for example, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18 or 21 months of age, or at least about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 years of age. In some embodiments, the subject is about: 1-100, 1-80, 1-60, 1-30, 1-24, 1-20, 1-18, 1-12, 1- 10, 1-8, 1-6, 2-100, 2-80, 2-60, 2-30, 2-24, 2-20, 2-18, 2-12, 2-10, 2-8, 2-6, 3-100, 3-80, 3-60,3-30, 3-24, 3-20, 3-18, 3-12, 3-10, 3-8, 3-6, 4-100, 4-80, 4-60, 4-30, 4-24, 4-20, 4-18, 4-12,4-10, 4-8, 4-6, 5-100, 5-80, 5-60, 5-30, 5-24, 5-20, 5-18, 5-12, 5-10, 5-8, 6-100, 6-80, 6-60, 6-30, 6-24, 6-20, 6-18, 6-12, 6-10, 8-100, 8-80, 8-60, 8-30, 8-24, 8-20, 8-18, 8-12, 10-100, 10-80, 10-60, 10-30, 10-24, 10-20, 10-18, 12-100, 12-80, 12-38, 12-60, 12-50, 12-40, 12-30, 12-24, 12-20, 12-18, 18-100, 18-80, 18-60, 18-50, 18-40, 18-30, 18-24, 20-100, 20-80, 20-60, 20-50, 20-40, 20-30, 20-25, 30-100, 30-80, 30-60, 30-55, 30-50, 30-45, 30-40, 40-100, 40-80, 40-60, 40-55 or 40-50 years of age. In some embodiments, the subject is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80 or 100 years of age. In some embodiments, the subject is about 12-38 years of age. In other embodiments, the subject is a fetus. In some embodiments, the subject is a neonatal subject.

[0114] In some embodiments, the subject is 18 years of age or older, e.g., 18 to less than 40 years of age, 18 to less than 45 years of age, 18 to less than 50 years of age, 18 to less than55 years of age, 18 to less than 60 years of age, 18 to less than 65 years of age, 18 to less than70 years of age, 18 to less than 75 years of age, 40 to less than 75 years of age, 45 to less than75 years of age, 50 to less than 75 years of age, 55 to less than 75 years of age, 60 to less than75 years of age, 65 to less than 75 years of age, 60 to less than 75 years of age, 40 years of age or older, 45 years of age or older, 50 years of age or older, 55 years of age or older, 60 years of age or older, 65 years of age or older, 70 years of age or older, 75 years of age or older or 90 years of age or older. In some embodiments, the subject is 50 years of age or older. In some embodiments, the subject is a child. In some embodiments, the subject is 18 years of age or younger, e.g., 0-18 years of age, 0-12 years of age, 0-16 years of age, 0-17 years of age, 2-12 years of age, 2-16 years of age, 2-17 years of age, 2-18 years of age, 3-12 years of age, 3-16 years of age, 3-17 years of age, 3-18 years of age, 4-12 years of age, 4-16 years of age, 4-17 years of age, 4-18 years of age, 6-12 years of age, 6-16 years of age, 6-17 years of age, 6-18 years of age, 9-12 years of age, 9-16 years of age, 9-17 years of age, 9-18 years of age, 12-16 years of age, 12-17 years of age or 12-18 years of age.

[0115] In some embodiments, the subject is about 2-11, 4-17, 12-18, 18-50, 18-90 or 50- 90 years of age.

[0116] The phrase “pharmaceutically acceptable” means that the substance or composition the phrase modifies is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio.

[0117] As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of mammals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, the relevant teachings of which are incorporated herein by reference in their entirety. Pharmaceutically acceptable salts of the compounds described herein include salts derived from suitable inorganic and organic acids, and suitable inorganic and organic bases.

[0118] Examples of salts derived from suitable acids include salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid,sulfuric acid and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts derived from suitable acids include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cinnamate, citrate, cyclopentanepropionate, di gluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, glutarate, glycolate, hemisulfate, heptanoate, hexanoate, hydroiodide, hydroxybenzoate, 2-hydroxy-ethanesulfonate, hydroxymaleate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 2-phenoxybenzoate, phenyl acetate, 3 -phenylpropionate, phosphate, pivalate, propionate, pyruvate, salicylate, stearate, succinate, sulfate, tartrate, thiocyanate, p- toluenesulfonate, undecanoate, valerate salts, and the like.

[0119] Either the mono-, di- or tri-acid salts can be formed, and such salts can exist in either a hydrated, solvated or substantially anhydrous form.

[0120] Salts derived from appropriate bases include salts derived from inorganic bases, such as alkali metal, alkaline earth metal, and ammonium bases, and salts derived from aliphatic, alicyclic or aromatic organic amines, such as methylamine, trimethylamine and picoline, or N+((Ci-C4)alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, barium and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxyl, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.

[0121] “Therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of a therapeutic or a combination of therapeutics to elicit a desired response in the individual.

[0122] In some embodiments, an agent disclosed herein (e.g., retinoic acid) is in a form of a pharmaceutical composition, or a pharmaceutically acceptable salt thereof. A “pharmaceutical composition” refers to a formulation of one or more therapeutic agents and a medium generally accepted in the art for delivery of a biologically active agent to subjects, e.g., humans. In some embodiments, a pharmaceutical composition may include one or morepharmaceutically acceptable excipients, diluents, or carriers. “Pharmaceutically acceptable carrier, diluent, or excipient” includes any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.

[0123] In some embodiments, a pharmaceutical composition disclosed herein is formulated as a solution.

[0124] “Pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In some embodiments, the carrier may be a diluent, adjuvant, excipient, or vehicle with which the agent (e.g., polynucleotide) is administered. Such vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc. The concentration of the agent in such pharmaceutical formulation may vary widely, z.e., from less than about 0.5%, to at least about 1%, or to as much as 15% or 20%, 25%, 30%, 35%, 40%, 45% or 50% by weight. The concentration will be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the mode of administration.Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington: The Science and Practice of Pharmacy, 21stEdition, Troy, D.B. ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing: 691-1092 (e.g, pages 958-89).

[0125] In some embodiments, a pharmaceutical composition suitable for use in methods disclosed herein further comprises one or more pharmaceutically acceptable carriers. The term “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject and should not interfere with the efficacy of the active ingredient. A pharmaceutically acceptable carrierincludes, but is not limited to, such as those widely employed in the art of drug manufacturing. The carrier may be a diluent, adjuvant, excipient, or vehicle with which the agent is administered. Such vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4% saline and 0.3% glycine may be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc. The concentration of the agent in such pharmaceutical formulation may vary widely, e.g., from less than about 0.5%, usually to at least about 1% to as much as 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% by weight. The concentration will be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the particular mode of administration selected. Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in e.g., Remington: The Science and Practice of Pharmacy, 21stEdition, Troy, D. B. ed., Lipincott Williams and Wilkins, Philadelphia, Pa. 2006, Part 5, Pharmaceutical Manufacturing pp 691- 1092, see especially pp. 958-89.

[0126] Non-limiting examples of pharmaceutically acceptable carriers are solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible, such as salts, buffers, antioxidants, saccharides, aqueous or non-aqueous carriers, preservatives, wetting agents, surfactants or emulsifying agents, or combinations thereof.

[0127] Non-limiting examples of buffers that may be used are acetic acid, citric acid, formic acid, succinic acid, phosphoric acid, carbonic acid, malic acid, aspartic acid, histidine, boric acid, Tris buffers, HEPPSO and HEPES.

[0128] Non-limiting examples of antioxidants that may be used are ascorbic acid, methionine, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, lecithin, citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol and tartaric acid.

[0129] Non-limiting examples of amino acids that may be used are histidine, isoleucine, methionine, glycine, arginine, lysine, L-leucine, tri-leucine, alanine, glutamic acid, L- threonine, and 2-phenylamine.

[0130] Non-limiting examples of surfactants that may be used are polysorbates (e.g., polysorbate-20 or polysorbate-80); polyoxamers (e.g., poloxamer 188); Triton; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl- dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and the MONAQUA™ series (Mona Industries, Inc., Paterson, N.J.), poly ethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., PLURONICS™, PF68, etc.).

[0131] Non-limiting examples of preservatives that may be used are phenol, m-cresol, p- cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride, alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof.

[0132] Non-limiting examples of saccharides that may be used are monosaccharides, di saccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, nonreducing sugars such as glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, dextran, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, mellibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, glucitol, maltitol, lactitol or iso-maltulose.

[0133] Non-limiting examples of salts that may be used are acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N’ -dibenzylethylenediamine, N-m ethyl glucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like. In some embodiments, the salt is sodium chloride (NaCl).

[0134] Agents (e.g., retinoic acid, a PFE cell, a TEP cell, and / or a TEC produced by the disclosed method, or a population or organoid comprising such a cell) disclosed herein may be prepared in accordance with standard procedures and are administered at dosages that areselected to reduce, prevent, or eliminate, or to slow or halt progression of, a condition being treated (See, e.g., Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, and Goodman and Gilman’s The Pharmaceutical Basis of Therapeutics, McGraw-Hill, New York, N.Y., the contents of which are incorporated herein by reference, for a general description of methods for administering various agents for human therapy).

[0135] In some embodiments, an agent disclosed herein (e.g., retinoic acid, a PFE cell, a TEP cell, and / or a TEC produced by the disclosed method, or a population or organoid comprising such a cell) is delivered using controlled or sustained-release delivery systems (e.g., capsules, biodegradable matrices). Example delayed-release delivery systems for drug delivery that would be suitable for administration of a composition described herein are described in U.S. Patent Nos. US 5,990,092 (issued to Walsh); 5,039,660 (issued to Leonard); 4,452,775 (issued to Kent); and 3,854,480 (issued to Zaffaroni), the entire teachings of which are incorporated herein by reference. Example biodegradable matrices are discussed in Li et al. (Li, C., Guo, C., Fitzpatrick, V. et al. Design of biodegradable, implantable devices towards clinical translation. Nat Rev Mater 5, 61-81 (2020). https: / / doi.org / 10.1038 / s41578- 019-0150-z).

[0136] In some embodiments, an agent disclosed herein (e.g., a PFE cell, a TEP cell, and / or a TEC produced by the disclosed method, or a population or organoid comprising such a cell) is delivered using a decellularized scaffold, z.e., an organ, tissue, or part thereof, which is collected from a living or non-living human (allogenic) or non-human animal (xenogenic), decellularized, recellularized with a PFE cell, a TEP cell, and / or a TEC produced by the disclosed method, or a population comprising such a cell, and implanted into the subject. For a discussion of allogenic decellularized scaffolds, see, for example, Yu et al. (Yu Y, Alkhawaji A, Ding Y, Mei J. Decellularized scaffolds in regenerative medicine. Oncotarget. 2016 Sep 6;7(36):58671-58683. Doi: 10.18632 / oncotarget.10945. PMID: 27486772; PMCID: PMC5295461). For a discussion including xenogenic decellularized scaffolds, see, for example, Mazloomnejad et al. (Mazloomnejad R, Babajani A, Kasravi M, Ahmadi A, Shariatzadeh S, Bahrami S, Niknejad H. Angiogenesis and Re-endothelialization in decellularized scaffolds: Recent advances and current challenges in tissue engineering. Front Bioeng Biotechnol. 2023 Feb 16; 11 : 1103727. doi: 10.3389 / fbioe.2023.1103727. PMID: 36873356; PMCID: PMC9978201).

[0137] For oral administration, an agent such as retinoic acid may be in the form of, for example, a tablet, capsule, suspension or liquid. An agent such as retinoic acid is preferablymade in the form of a dosage unit containing a therapeutically effective amount of an active ingredient. Examples of such dosage units are tablets and capsules. For therapeutic purposes, tablets and capsules can contain, in addition to an active ingredient, conventional carriers such as binding agents, for example, acacia gum, gelatin, polyvinylpyrrolidone, sorbitol, or tragacanth; fillers, for example, calcium phosphate, glycine, lactose, maize-starch, sorbitol, or sucrose; lubricants, for example, magnesium stearate, polyethylene glycol, silica, or talc; disintegrants, for example potato starch, flavoring or coloring agents, or acceptable wetting agents. Oral liquid preparations generally in the form of aqueous or oily solutions, suspensions, emulsions, syrups or elixirs may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous agents, preservatives, coloring agents and flavoring agents. Examples of additives for liquid preparations include acacia, almond oil, ethyl alcohol, fractionated coconut oil, gelatin, glucose syrup, glycerin, hydrogenated edible fats, lecithin, methyl cellulose, methyl or propyl para-hydroxybenzoate, propylene glycol, sorbitol, or sorbic acid.

[0138] Administration of the agent to the subject can be by parenteral or non-parenteral means. In some embodiments, an agent disclosed herein (e.g., retinoic acid, a PFE cell, a TEP cell, and / or a TEC produced by the disclosed method, or a population or organoid comprising such a cell) is administered intravenously, intra-arterially, intrathecally, intraventricularly, intramuscularly, intradermally, subcutaneously, intracranially, or spinally. “Administering” or “administration” as used herein, refers to taking steps to deliver an agent to a subject, such as a mammal, in need thereof. Administering can be performed, for example, once, a plurality of times, and / or over one or more extended periods. Administration includes both direct administration, including self-administration, and indirect administration, including an act of prescribing a drug or directing a subject to consume an agent. For example, as used herein, one (e.g., a physician) who instructs a subject (e.g., a patient) to self-administer an agent (e.g., a drug), or to have an agent administered by another and / or who provides a patient with a prescription for a drug is administering an agent to a subject. Administration of an agent can be once in a day or more than once in a day (e.g., twice a day or more). Administration of the agent can be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months or longer. Repeated courses of treatment are also possible, as is chronic administration. The repeated administration may be at the same dose or at a different dose.

[0139] In some embodiments, an agent disclosed herein e.g., retinoic acid, a PFE cell, a TEP cell, and / or a TEC produced by the disclosed method, or a population or organoid comprising such a cell) is delivered systemically, such as via intravenous or subcutaneous injection. In some embodiments, the agent (e.g., retinoic acid, a PFE cell, a TEP cell, and / or a TEC produced by the disclosed method, or a population or organoid comprising such a cell) is delivered using an approach that enhances bioavailability in the target tissue or organ after administration. These approaches can include modification of the sugars or phosphate linkages, delivering as a duplex with a ligand-conjugated RNA molecule, formulation into an artificial exosome, liposome, polymer nanoparticle or lipid nanoparticle, or conjugation to lipids, antibodies, peptides, sugars, neuroactive molecules, or other moieties that enhance delivery to the target tissue or organ. Other methods of enhancing bioavailability in the target tissue or organ after administration will be known to one skilled in the art.

[0140] In some embodiments, a method disclosed herein comprises administering to the subject two or more agents, for example, 2, 3, 4, or 5 or more agents. In some embodiments, the two or more agents are administered together. In other embodiments, the two or more agents are administered separately, e.g., sequentially. In some embodiments, two or more agents are administered in the same composition. In some embodiments, two or more agents are administered in different compositions.

[0141] As used herein, an “antibody” is any immunoglobulin polypeptide, or fragment thereof, having immunogen binding ability.

[0142] As used herein, a “fragment” is a portion of a polypeptide or nucleic acid molecule. This portion contains in example embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0143] As used herein, an “agonist” is an agent that causes an increase in the expression or activity of a target gene or protein, respectively. An agonist can bind to and activate its cognate receptor in some fashion, which directly or indirectly brings about this physiological effect on the target gene or protein.

[0144] As used herein, an “inhibitor” is an agent that causes a decrease in the expression or activity of a target gene or protein, respectively. An “antagonist” can be an inhibitor, but is more specifically an agent that binds to a receptor, and which in turn decreases or eliminates binding by other molecules.

[0145] As used herein, the term “stem cell” refers to a multipotent cell having the capacity to self-renew and to differentiate into multiple cell lineages.

[0146] As used herein, the term “differentiation” is used interchangeably to describe: the process of a cell differentiating into a cell lineage; the process of a person of skill in the art differentiating a cell into a cell lineage; a state of a cell in the process of differentiating into a cell lineage or having differentiated into a cell lineage; a protocol for the differentiation of a cell into a cell lineage; or a combination thereof. In some embodiments, the term “day” denotes a day of a differentiation, z.e., day 3 of differentiation. In some embodiments, the term “day” denotes a day of a differentiation protocol, e.g., day 3 of a differentiation protocol. In some embodiments, a given day of differentiation means a given day of a differentiation protocol.

[0147] As used herein, the term “epithelial stem cell” refers to a multipotent cell which has the potential to become committed to multiple cell lineages, including cell lineages resulting in epithelial cells.

[0148] As used herein, the term “progenitor cell” refers to a lineage-restricted cell derived from a stem cell.

[0149] As used herein, the term “epithelial progenitor cell” refers to a multipotent cell which has the potential to become restricted to cell lineages resulting in epithelial cells.

[0150] As used herein, the term “self-renewal” refers to the process by which a stem cell divides to generate one (asymmetric division) or two (symmetric division) daughter cells with development potentials that are indistinguishable from those of the mother cell. Self-renewal involves both proliferation and the maintenance of an undifferentiated state.

[0151] As used herein, the term “graft,” “engraft,” or “engraftmenf ’ refers to the process of stem or progenitor cell incorporation into a tissue of interest in vivo through contact with existing cells of the tissue.

[0152] As used herein, the term “isolated” refers to a material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings.

[0153] As used herein, a “population” of cells is any number of cells greater than 1, such as at least 1X103cells, at least 1X104cells, at least at least 1X105cells, at least 1X106cells, at least 1X107cells, at least 1X108cells, at least 1X109cells, or at least 1X1010cells.

[0154] As used herein, the term “organoid” or “epithelial organoid” refers to a cell cluster or aggregate that resembles an organ, or part of an organ, and possesses cell types relevant tothat particular organ. Alternatively, “organoid,” “3D culture,” “re-aggregate,” “reaggregated organ culture (ROC),” or “droplet organ culture” refers to a model of an organ or tissue, e.g., a simplified and / or miniaturized version of the organ or tissue that recapitulates characteristics of the organ or tissue in a state that is undeveloped, developing, fully developed, mature, aging, healthy, injured, diseased, and / or at risk for disease. An organoid can be a model of any stage organ development. The terms “organoid,” “3D culture,” “reaggregate,” “reaggregated organ culture (ROC),” and “droplet organ culture” may be used interchangeably herein.

[0155] The terms “media” and “medium” may be used interchangeably herein when in reference to cell culture.

[0156] As used herein, a “subject” is a vertebrate, including any member of the class mammalia.

[0157] As used herein, a “mammal” refers to any mammal including but not limited to human, mouse, rat, sheep, monkey, goat, rabbit, hamster, horse, cow or pig.

[0158] A “non-human mammal”, as used herein, refers to any mammal that is not a human.

[0159] As used herein, “increases” also means increases by at least 1-fold, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000-fold or more, for example, as compared to the level of a as compared to the level of a reference standard.

[0160] As used herein, “decreasing” refers to decreasing by at least 5%, for example, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%, for example, as compared to the level of reference.

[0161] As used herein, “decreases” also means decreases by at least 1-fold, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000-fold or more, for example, as compared to the level of a reference.

[0162] As used herein, the term “reference” means a standard or control condition (e.g., untreated with a test agent or combination of test agents). Alternatively, “reference” may refer to a resource, such as an annotated genome, transcriptome, or the like, that is used to assemble, analyze, and / or interpret data.

[0163] As used herein, the term “eliminate” means to decrease to a level that is undetectable.

[0164] As used herein, the terms “treat,” “treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and / or symptoms associated therewith. It will beappreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

[0165] As used herein, “functionally express” refers to the ability of a cell to express an RNA and / or protein at a level, or within a range, that is known in the art to correlate with an identity and / or function of a cell. In some embodiments, functional expression is of a transcript and / or a protein. In some embodiments, functional expression, or the lack thereof, is detected by quantitative polymerase chain reaction (qPCR), RNA sequencing (RNA-seq), flow cytometry, immunofluorescence (IF), immunohistochemistry (IHC), immunocytochemistry (ICC), or similar methodology. In some embodiments, detection of RNA is an acceptable surrogate for protein detection where protein detection has been validated and / or a reasonable expectation of successful translation exists.

[0166] As used herein, the terms “HLA,” “HLA-DR,” and “HLA-DRA” are used interchangeably.

[0167] As used herein, gene, transcript, and protein nomenclature uses lowercase and uppercase letters interchangeably. As used herein, gene and transcript nomenclature uses italicized and non-italicized names and symbols interchangeably.

[0168] Other definitions appear in context throughout this disclosure.Example Embodiments

[0169] There is a critical need to develop methods, compositions, and therapeutic agents for treating disorders including defects (e.g., developmental defects) affecting the pharynx and derived tissues and the syndromes that result therefrom. The disclosure provides such methods and compositions.

[0170] The disclosure provided herein is based, in part, on an efficient step-wise differentiation method to generate human pharyngeal foregut endoderm (PFE) cells from pluripotent stem cells, as well as a method to generate organoids, e.g., thymic organoids from the PFE cells. Accordingly, the disclosure generally relates to compositions (e.g., a medium for differentiating a pluripotent stem cell, an organoid comprising differentiated cells), methods, and therapeutic agents that are useful for treating defects of the pharynx, pharynx- derived tissue, and related syndromes.

[0171] In one aspect, the invention provides a method for producing pharyngeal foregut endoderm (PFE) cells, the method comprising a) differentiating definitive endoderm (DE) cells into anterior foregut endoderm (AFE) cells, wherein differentiating comprises i)culturing the DE cells in a medium comprising about OnM retinoic acid and ii) further culturing the DE cells in a medium comprising about lOOnM retinoic acid, thereby producing AFE cells; and b) further differentiating the AFE cells into PFE cells, wherein differentiating comprises culturing the AFE cells in media comprising about lOOnM retinoic acid, thereby producing the PFE cells.

[0172] In one aspect, the invention provides a method for producing pharyngeal foregut endoderm (PFE) cells by obtaining definitive endoderm (DE) cells, differentiating the DE cells into anterior foregut endoderm (AFE) cells, and further differentiating the AFE cells into PFE cells. In this aspect, differentiating comprises culturing the DE, AFE, and PFE cells in a medium comprising about lOOnM retinoic acid.

[0173] In one aspect, the invention provides a method for producing pharyngeal foregut endoderm (PFE) cells, the method comprising differentiating definitive endoderm (DE) cells into anterior foregut endoderm (AFE) cells, and further differentiating the AFE cells into PFE cells, wherein differentiating comprises: culturing the DE cells in a medium comprising about OnM retinoic acid, further culturing the DE cells in a medium comprising about lOOnM retinoic acid, and culturing the AFE cells in media comprising about lOOnM retinoic acid.

[0174] In some embodiments, the medium used to produce PFE cells (i.e., the first, fourth, fifth, and sixth medium) comprises RPMI 1640, a neuronal cell culture supplement, an L-glutamine supplement, an antibiotic, or a combination thereof. In some embodiments, the neuronal cell culture supplement is B-27® minus vitamin A, the L-glutamine supplement is GLUTAMAX®, the antibiotic is P / S, or a combination thereof. In some embodiments, the medium comprises about 0.5x B-27® minus vitamin A neuronal cell culture supplement, 1% GLUTAMAX®, 1% P / S, or a combination thereof.Example Differentiation of Pluripotent Stem Cells into Definitive Endoderm Cells

[0175] In some embodiments, the invention provides a method of obtaining DE cells by differentiating pluripotent stem cells (PSCs). In some embodiments, the PSCs are human induced PSCs (iPSCs). In other embodiments, the PSCs are human embryonic stem cells (hESCs).

[0176] In some embodiments, differentiating the PSCs comprises culturing the PSCs on a basement membrane or extracellular matrix. In some embodiments, the basement membrane or extracellular matrix is MATRIGEL® Growth Factor Reduced (GFR) basement membranematrix. In other embodiments, a different basement membrane or extracellular matrix may be used.

[0177] In some embodiments, differentiating the PSCs comprises culturing the PSCs in a first medium comprising Roswell Park Memorial Institute 1640 cell culture medium (RPMI 1640), fetal bovine serum (FBS), an L-glutamine supplement, an antibiotic, Activin A, a GSK3 inhibitor, a PI3K / mTOR inhibitor, or a combination thereof. In some embodiments, culturing the PSCs in the first medium occurs on about day 0 of differentiation. In some embodiments, culturing the PSCs in the first medium occurs for a period of about 1 day. In some embodiments, the FBS is HYCLONE® Defined FBS, the L-glutamine supplement is GLUTAMAX® (200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl), the antibiotic is 5,000 IU penicillin-5,000 pg / mL streptomycin (P / S), the GSK3 inhibitor is CHIR99021, the PI3K / mTOR inhibitor is PI-103, or a combination thereof. In some embodiments, the first medium comprises about 0.2% HYCLONE® Defined FBS, 1% GLUTAMAX®, 1% P / S, lOOng / mL Activin A, 3pM CHIR99021, 50nM PL103, or a combination thereof.

[0178] In some embodiments, differentiating the PSCs further comprises culturing the PSCs in a second medium comprising RPMI 1640, FBS, an L-glutamine supplement, an antibiotic, Activin A, an ALK / BMP inhibitor, or a combination thereof. In some embodiments, culturing the PSCs in the second medium occurs on about days 1-2 of differentiation. In some embodiments, culturing the PSCs in the second medium occurs for a period of about 2 days. In some embodiments, the FBS is HYCLONE® Defined FBS, the L- glutamine supplement is GLUTAMAX®, the antibiotic is P / S, the ALK / BMP inhibitor is LDN193189, or a combination thereof. In some embodiments, the second medium comprises about 0.2% HYCLONE® Defined FBS, 1% GLUTAMAX®, 1% P / S, lOOng / mL Activin A, 250 nM LDN193189, or a combination thereof.

[0179] In some embodiments, the DE cells obtained by differentiating PSCs functionally express SOX17, GATA4, GATA6, EOMES, LEFTY1, CER1, or a combination thereof; and / or do not functionally express POU5F1, VGLL2, EYA1, PAX9, PAX1, FOXE1, PBX1, or a combination thereof.Example Differentiation of Definitive Endoderm Cells into Anterior Foregut Endoderm Cells

[0180] In some embodiments, differentiating DE cells into AFE cells comprises singularizing the DE cells, plating the DE cells, culturing the DE cells in a third medium, and culturing the DE cells in a fourth medium comprising about lOOnM retinoic acid.

[0181] In some embodiments, singularizing the DE cells and plating the DE cells occur on about day 3 of differentiation. In some embodiments, singularizing comprises dissociating the DE cells with a proteolytic enzyme, scraping the DE cells and then triturating the DE cells (e.g., triturating by pipette), or a combination thereof. In some embodiments, the proteolytic enzyme is capable of detaching and / or dissociating adherent cells in culture. In some embodiments, the proteolytic enzyme, or a solution containing the proteolytic enzyme, comprises trypsin, TRYPLE® recombinant dissociation enzyme, ACCUTASE® cell detachment solution, DISEASE® dissociating enzyme, or a combination thereof.

[0182] In some embodiments, culturing the DE cells comprises culturing the DE cells on a basement membrane or extracellular matrix. In some embodiments, the basement membrane or extracellular matrix comprises 804G supernatant, MATRIGEL® basement membrane matrix, MATRIGEL® Growth Factor Reduced (GFR) basement membrane matrix, or a combination thereof.

[0183] In some embodiments, culturing the DE cells in the third medium occurs on about day 3 of differentiation. In some embodiments, culturing the DE cells in the third medium occurs for a period of about one day. In some embodiments, the third medium comprises an ALK / TGF-PRI inhibitor, an ALK / BMP inhibitor, a ROCK inhibitor, or a combination thereof. In some embodiments, the ALK / TGF-PRI inhibitor is A83-01, the ALK / BMP inhibitor is LDN193189, the ROCK inhibitor is Y-27632, or a combination thereof. In some embodiments, the third medium comprises about: IpM A83-01, 250nM LDN193189, lOpM Y-27632, or a combination thereof.

[0184] In some embodiments, culturing the DE cells in the fourth medium occurs on about days 4-8 of differentiation. In some embodiments, culturing the DE cells in the fourth medium occurs for a period of about five days. In some embodiments, the fourth medium comprises an ALK / TGF-PRI inhibitor, an ALK / BMP inhibitor, a WNT inhibitor, retinoic acid, or a combination thereof. In some embodiments, the ALK / TGF-PRI inhibitor is A83-01, the ALK / BMP inhibitor is LDN193189, the WNT inhibitor is IWR-1, or a combination thereof. In some embodiments, the fourth medium comprises about IpM A83-01, 250nM LDN193189, 250nM IWR-1, lOOnM retinoic acid, or a combination thereof.

[0185] In some embodiments, the AFE cells produced by differentiating DE cells functionally express VGLL2, EYA1, SIX1, or a combination thereof; and / or do not functionally express POU5F1, SOX17, or a combination thereof.Example Differentiation of Anterior Foregut Endoderm Cells into Pharyngeal Foregut Endoderm Cells

[0186] In some embodiments, differentiating AFE cells into PFE cells comprises singularizing the AFE cells, plating the AFE cells, and culturing the AFE cells in a fifth and a sixth medium comprising about lOOnM retinoic acid.

[0187] In some embodiments, singularizing the AFE cells and plating the AFE cells occur on about day 9 of differentiation. In some embodiments, wherein singularizing comprises dissociating the AFE cells with a proteolytic enzyme, scraping the AFE cells and then triturating the AFE cells by pipette, or a combination thereof. In some embodiments, the proteolytic enzyme is capable of detaching and / or dissociating adherent cells in culture. In some embodiments, the proteolytic enzyme, or a solution containing the proteolytic enzyme, comprises trypsin, TRYPLE® recombinant dissociation enzyme, ACCUTASE® cell detachment solution, DISPASE® dissociating enzyme, or a combination thereof.

[0188] In some embodiments, culturing the AFE cells comprises culturing the AFE cells on a basement membrane or extracellular matrix. In some embodiments, the basement membrane or extracellular matrix comprises 804G supernatant, MATRIGEL® basement membrane matrix, MATRIGEL® Growth Factor Reduced (GFR) basement membrane matrix, or a combination thereof.

[0189] In some embodiments, culturing the AFE cells in the fifth medium occurs on about day 9 of differentiation. In some embodiments, culturing the AFE cells in the fifth medium occurs for a period of about one day. In some embodiments, the fifth medium comprises a bone morphogenic protein 4 (BMP4), a fibroblast growth factor 10 (FGF10), a retinoic acid, a ROCK inhibitor, or a combination thereof. In some embodiments, the ROCK inhibitor is Y-27632. In some embodiments, the fifth medium comprises about 50 ng / mL BMP4, 50 ng / mL FGF10, 100 nM retinoic acid, lOpM Y-27632, or a combination thereof.

[0190] In some embodiments, culturing the AFE cells in the sixth medium occurs on about days 10-14 of differentiation. In some embodiments, culturing the AFE cells in the sixth medium occurs for a period of about five days. In some embodiments, the sixth medium comprises a bone morphogenic protein 4 (BMP4), a fibroblast growth factor 10 (FGF10), a retinoic acid, or a combination thereof. In some embodiments, the sixth medium comprises about 50 ng / mL BMP4, 50 ng / mL FGF10, 100 nM retinoic acid, or a combination thereof.

[0191] In some embodiments, the PFE cells produced by differentiating AFE cells functionally express FOXA2, SOX2, PAX9, PAX1, FOXE1, PBX1 or a combination thereof; and / or do not functionally express POU5F1, SOX17, VGLL2, or a combination thereof.Example Differentiation of Pharyngeal Foregut Endoderm Cells into an Organoid

[0192] In another aspect, the invention provides a method for producing thymic epithelial cells (TECs), the method comprising the following steps in order: obtaining PFE cells produced by the disclosed method; culturing the PFE cells in a sixth medium, thereby producing thymic epithelial progenitor (TEP) cells; singularizing the TEP cells; seeding the TEP cells onto a scaffold, thereby initiating an organoid; and culturing the TEP organoid in a seventh and an eighth medium, thereby producing TECs and an organoid comprising TECs.

[0193] In some embodiments, the sixth, seventh, and eighth medium comprise RPMI 1640, a neuronal cell culture supplement, an L-glutamine supplement, an antibiotic, or a combination thereof. In some embodiments, the neuronal cell culture supplement is B-27® minus vitamin A, the L-glutamine supplement is GLUTAMAX®, the antibiotic is P / S, or a combination thereof. In some embodiments, the sixth, seventh, and eighth medium comprise about 0.5x B-27® minus vitamin A neuronal cell culture supplement, 1% GLUTAMAX®, 1% P / S, or a combination thereof.

[0194] In some embodiments, culturing the PFE cells in the sixth medium occurs on about days 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 of differentiation, or a combination thereof. In some embodiments, culturing the PFE cells in the sixth medium occurs for a period of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, the sixth medium comprises a bone morphogenic protein 4 (BMP4), a fibroblast growth factor 10 (FGF10), a retinoic acid, or a combination thereof. In some embodiments, the sixth medium comprises about 50 ng / mL BMP4, 50 ng / mL FGF10, retinoic acid, or a combination thereof.

[0195] In some embodiments, the TEP cells produced from the PFE cells functionally express PAX1, PAX9, FOXN1, GATA3, or a combination thereof; and / or do not functionally express VGLL2, SOX17, CER1, POU5F1, NANOG, or a combination thereof.

[0196] In some embodiments, singularizing the TEP cells comprises: dissociating the TEP cells with a proteolytic enzyme, scraping the TEP cells and then triturating the TEP cells by pipette, or a combination thereof. In some embodiments, the proteolytic enzyme is capable of detaching and / or dissociating adherent cells in culture. In some embodiments, the proteolytic enzyme, or a solution containing the proteolytic enzyme, comprises trypsin,TRYPLE® recombinant dissociation enzyme, ACCUTASE® cell detachment solution, DISEASE® dissociating enzyme, or a combination thereof.

[0197] In some embodiments, the scaffold onto which the TEP cells are seeded is contained in a plate. In some embodiments, the scaffold is a decellularized thymic lobe or a cell culture filter insert. In some embodiments, the decellularized thymic lobe is from a mouse. In some embodiments, the mouse is 4-6 weeks of age. In some embodiments, the mouse is of an ICR strain.

[0198] In some embodiments, culturing the TEP organoid in the seventh medium occurs on about day 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, or 41 of differentiation, or a combination thereof. In some embodiments, culturing the TEP organoid in the seventh medium occurs for a period of about 1 day. In some embodiments, culturing the TEP organoid in the seventh medium occurs for a period of about 1 or more days. In some embodiments, culturing the TEP organoid in the seventh medium occurs for a period of about 1-16 days. In some embodiments, the seventh medium comprises an ALK / TGF-PRI inhibitor, a ROCK inhibitor, or a combination thereof. In some embodiments, the ALK / TGF-PRI inhibitor is A83-01, the ROCK inhibitor is Y-27632, or a combination thereof. In some embodiments, the seventh medium comprises about IpM A83- 01, lOpM Y-27632, or a combination thereof.

[0199] In some embodiments, culturing the TEP organoid in the eighth medium occurs on about days 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, or 41 of differentiation, or a combination thereof. In some embodiments, culturing the TEP organoid in the eighth medium occurs for a period of about 16 days. In some embodiments, the eighth medium comprises an ALK / TGF-PRI inhibitor. In some embodiments, the ALK / TGF-PRI inhibitor is A83-01. In some embodiments, the eighth medium comprises about IpM A83-01.

[0200] In some embodiments, the TECs produced by the disclosed method functionally express GCM2, FOXN1, CCL25, PAX1, PAX9, GATA3, MAFB, or a combination thereof; and / or do not functionally express VGLL2, SOX17, CER1, POU5F1, NANOG, or a combination thereof.

[0201] In some embodiments, the invention provides a method for producing thymic epithelial progenitor (TEP) cells, the method comprising the following steps in order: obtaining pharyngeal foregut endoderm (PFE) cells produced by the disclosed method; and culturing the PFE cells in a medium, thereby producing thymic epithelial progenitor (TEP)cells. In some embodiments, the medium comprises: RPMI 1640, a neuronal cell culture supplement, an L-glutamine supplement, an antibiotic, or a combination thereof. In some embodiments, the neuronal cell culture supplement is B-27® minus vitamin A, the L- glutamine supplement is GLUTAMAX® (200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl), the antibiotic is 5,000 IU penicillin-5,000 pg / mL streptomycin (P / S), or a combination thereof. In some embodiments, the medium comprises: about 0.5x B-27® minus vitamin A neuronal cell culture supplement, about 1% GLUTAMAX® (200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl), about 1% P / S, or a combination thereof. In some embodiments, the medium comprises: a bone morphogenic protein 4 (BMP4), a fibroblast growth factor 10 (FGF10), a retinoic acid, or a combination thereof. In some embodiments, the medium comprises: about 50 ng / mL BMP4, about 50 ng / mL FGF10, about lOOnM retinoic acid, or a combination thereof. In some embodiments, the TEP cells: functionally express PAX1, PAX9, FOXN1, GATA3, or a combination thereof; do not functionally express VGLL2, SOX17, CER1, POU5F1, NANOG, or a combination thereof; or both.

[0202] In some embodiments, the disclosure provides a method for producing thymic epithelial cells (TECs), the method comprising the following steps in order: obtaining pharyngeal foregut endoderm (PFE) cells produced by the disclosed method; culturing the PFE cells in a first medium, and culturing the PFE cells in a second medium, thereby producing thymic epithelial progenitor (TEP) cells; singularizing the TEP cells; seeding the TEP cells onto a scaffold, thereby initiating a TEP organoid; and culturing the TEP organoid in a third medium, thereby producing TECs and an organoid comprising TECs. In some embodiments, the first, second, and third medium each comprise RPMI 1640, a neuronal cell culture supplement, an L-glutamine supplement, an antibiotic, or a combination thereof. In some embodiments, the the neuronal cell culture supplement is B-27® minus vitamin A, the L-glutamine supplement is GLUTAMAX® (200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl), the antibiotic is 5,000 IU penicillin-5,000 pg / mL streptomycin (P / S), or a combination thereof. In some embodiments, the first, second, and third medium each comprise about 0.5x B-27® minus vitamin A neuronal cell culture supplement, about 1% GLUTAMAX® (200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl), about 1% P / S, or a combination thereof. In some embodiments, the first medium comprises an ALK / TGF-PRI inhibitor, a fibroblast growth factor 10 (FGF10), a retinoic acid, or a combination thereof. In some embodiments, the first medium comprises about IpM A83-01, about 50 ng / mL FGF10, about lOOnM retinoic acid, or a combination thereof. In some embodiments, the secondmedium comprises an ALK / TGF-PRI inhibitor. In some embodiments, the ALK / TGF-PRI inhibitor is A83-01. In some embodiments, the second medium comprises about IpM A83- 01. In some embodiments, the third medium comprises an ALK / TGF-PRI inhibitor, a ROCK inhibitor, or both. In some embodiments, the ALK / TGF-PRI inhibitor is A83-01, the ROCK inhibitor is Y-27632, or both. In some embodiments, the third medium comprises about IpM A83-01, about lOpM Y-27632, or both. In some embodiments, culturing the PFE cells in the first medium occurs on about days 15-21 of differentiation. In some embodiments, culturing the PFE cells in the first medium occurs for a period of about 7 days. In some embodiments, culturing the PFE cells in the second medium occurs on about days 22-28 of differentiation. In some embodiments, culturing the PFE cells in the second medium occurs for a period of about 7 days. In some embodiments, culturing the TEP organoid in the third medium occurs on about days 29-45 of differentiation. In some embodiments, culturing the TEP organoid in the third medium occurs for a period of about 17 days.Example Differentiation Media

[0203] In another aspect, the invention provides a composition for a medium for the differentiation of DE cells into AFE cells, wherein the medium comprises about IpM A83- 01, 250nM LDN193189, lOpM Y-27632, or a combination thereof. In some embodiments, the medium also comprises RPMI 1640, 0.5x B-27® minus vitamin A neuronal cell culture supplement, 1% GLUTAMAX®, 1% P / S, or a combination thereof. In some embodiments, the composition for the medium, z.e., the medium, comprises about IpM A83-01, about 250nM LDN193189, and about lOpM Y-27632. In some embodiments, the medium comprises about IpM A83-01, about 250nM LDN193189, about lOpM Y-27632, RPMI 1640, about 0.5x B-27® minus vitamin A neuronal cell culture supplement, about 1% GLUTAMAX®, and about 1% P / S. In some embodiments, the medium comprises about IpM A83-01 and about 250nM LDN193189. In some embodiments, the medium comprises about IpM A83-01, about 250nM LDN193189, RPMI 1640, about 0.5x B-27® minus vitamin A neuronal cell culture supplement, about 1% GLUTAMAX®, and about 1% P / S.

[0204] In another aspect, the invention provides a composition for a medium for the differentiation of DE cells into AFE cells, wherein the medium comprises about IpM A83- 01, 250nM LDN193189, 250nM IWR-1, lOOnM retinoic acid, or a combination thereof. In some embodiments, the medium also comprises RPMI 1640, 0.5x B-27® minus vitamin A neuronal cell culture supplement, 1% GLUTAMAX®, 1% P / S, or a combination thereof. Insome embodiments, the composition for the medium, i.e., the medium, comprises about IpM A83-01, about 250nM LDN193189, about 250nM IWR-1, and about lOOnM retinoic acid. In some embodiments, the composition for the medium, i.e., the medium, comprises about IpM A83-01, about 250nM LDN193189, about 250nM IWR-1, about lOOnM retinoic acid, RPMI 1640, about 0.5x B-27® minus vitamin A neuronal cell culture supplement, about 1% GLUTAMAX®, and about 1% P / S.

[0205] In another aspect, the invention provides a composition for a medium for the differentiation of AFE cells into PFE cells, wherein the medium comprises about 50 ng / mL BMP4, 50 ng / mL FGF10, 100 nM retinoic acid, lOpM Y-27632, or a combination thereof. In some embodiments, the medium also comprises RPMI 1640, 0.5x B-27® minus vitamin A neuronal cell culture supplement, 1% GLUTAMAX®, 1% P / S, or a combination thereof. In some embodiments, the composition for the medium, i.e., the medium, comprises about 50 ng / mL BMP4, about 50 ng / mL FGF10, about 100 nM retinoic acid, and about lOpM Y- 27632. In some embodiments, the medium comprises about 50 ng / mL BMP4, about 50 ng / mL FGF10, about 100 nM retinoic acid, about lOpM Y-27632, RPMI 1640, about 0.5x B- 27® minus vitamin A neuronal cell culture supplement, about 1% GLUTAMAX®, and about 1% P / S.

[0206] In another aspect, the invention provides a composition for a medium for the differentiation of AFE cells into PFE cells, wherein the medium comprises about 50 ng / mL BMP4, 50 ng / mL FGF10, 100 nM retinoic acid, or a combination thereof. In some embodiments, the medium also comprises RPMI 1640, 0.5x B-27® minus vitamin A neuronal cell culture supplement, 1% GLUTAMAX®, 1% P / S, or a combination thereof. In some embodiments, the composition for the medium, i.e., the medium, comprises about 50 ng / mL BMP4, about 50 ng / mL FGF10, and about 100 nM retinoic acid. In some embodiments, the composition for the medium, i.e., the medium, comprises about 50 ng / mL BMP4, about 50 ng / mL FGF10, about 100 nM retinoic acid, RPMI 1640, about 0.5x B-27® minus vitamin A neuronal cell culture supplement, about 1% GLUTAMAX®, and about 1% P / S.

[0207] In another aspect, the disclosure provides a medium for the differentiation of pharyngeal foregut endoderm (PFE) cells into thymic epithelial progenitor (TEP) cells, wherein the medium comprises about IpM A83-01, about 50 ng / mL FGF10, about lOOnM retinoic acid, or a combination thereof. In some embodiments, the composition for the medium, i.e., the medium, comprises about IpM A83-01, about 50 ng / mL FGF10, and aboutlOOnM retinoic acid. In some embodiments, the composition for the medium, i.e., the medium, comprises about IpM A83-01, about 50 ng / mL FGF10, about lOOnM retinoic acid, RPMI 1640, about 0.5x B-27® minus vitamin A neuronal cell culture supplement, about 1% GLUTAMAX®, and about 1% P / S. In some embodiments, the composition for the medium, i.e., the medium, comprises about 50 ng / mL FGF10 and about lOOnM retinoic acid. In some embodiments, the composition for the medium, i.e., the medium, comprises about 50 ng / mL FGF10, about lOOnM retinoic acid, RPMI 1640, about 0.5x B-27® minus vitamin A neuronal cell culture supplement, about 1% GLUTAMAX®, and about 1% P / S.

[0208] In another aspect, the disclosure provides a medium for the differentiation of pharyngeal foregut endoderm (PFE) cells into thymic epithelial progenitor (TEP) cells, wherein the medium comprises about IpM A83-01. In some embodiments, the composition for the medium, i.e., the medium, comprises about IpM A83-01, RPMI 1640, about 0.5x B- 27® minus vitamin A neuronal cell culture supplement, about 1% GLUTAMAX®, and about 1% P / S.

[0209] In another aspect, the disclosure provides a medium for the differentiation of thymic epithelial progenitor (TEP) cells into thymic epithelial cells (TECs), wherein the medium comprises IpM A83-01 and lOpM Y-27632. In some embodiments, the composition for the medium, i.e., the medium, comprises about IpM A83-01, about lOpM Y-27632, RPMI 1640, about 0.5x B-27® minus vitamin A neuronal cell culture supplement, about 1% GLUTAMAX®, and about 1% P / S.

[0210] In some embodiments, a medium for the differentiation of pharyngeal foregut endoderm (PFE) cells into thymic epithelial progenitor (TEP) cells also comprises RPMI 1640, 0.5x B-27® minus vitamin A neuronal cell culture supplement, 1% GLUTAMAX® (200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl), 1% P / S, or a combination thereof.

[0211] In some embodiments, the disclosure provides a kit comprising one or more medium disclosed herein. In some embodiments, the disclosure provides a kit comprising components of one or more medium disclosed herein.Example Differentiation Products

[0212] In another aspect, the invention provides a PFE cell produced (i.e., obtained or differentiated) by a method disclosed herein.

[0213] In another aspect, the invention provides a TEP cell produced by a method disclosed herein.

[0214] In another aspect, the invention provides a TEC produced by a method disclosed herein.

[0215] In another aspect, the invention provides an organoid comprising the TEP cell or the TEC produced by a method disclosed herein.

[0216] In another aspect, the invention provides compositions, e.g., populations (e.g., isolated populations or organoids) comprising a cell produced by a method disclosed herein.

[0217] In another aspect, the invention provides an organoid or population comprising a PFE cell or a TEC produced by a method disclosed herein. In another aspect, the invention provides a population comprising a TEP cell or a TEC produced by a method disclosed herein.

[0218] In another aspect, the invention provides a kit comprising means to produce a PFE cell, TEP cell, TEC, population of any such cell, or organoid comprising any such cell, according to a method disclosed herein.Example Compositions

[0219] In another aspect, the invention provides a composition (e.g., a pharmaceutical composition) comprising the pharyngeal foregut endoderm (PFE) cell, thymic epithelial progenitor (TEP) cell, thymic epithelial cell (TEC), or organoid comprising TECs produced according to a method disclosed herein.

[0220] In another aspect, the invention provides a composition (e.g., a pharmaceutical composition) comprising the pharyngeal foregut endoderm (PFE) cell produced according to a method disclosed herein.

[0221] In another aspect, the invention provides a composition (e.g., a pharmaceutical composition) comprising the thymic epithelial progenitor (TEP) cell produced according to a method disclosed herein.

[0222] In another aspect, the invention provides a composition (e.g., a pharmaceutical composition) comprising the thymic epithelial cell (TEC) produced according to a method disclosed herein.

[0223] In another aspect, the invention provides a composition (e.g., a pharmaceutical composition) comprising the organoid comprising TECs produced according to a method disclosed herein.Example Applications

[0224] In another aspect, the invention provides a method of recovering or restoring impaired tissue in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the PFE cell, the TEP cell, and / or the TEC produced by the disclosed method, or a population or organoid comprising such a cell.

[0225] In another aspect, the invention provides a method of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the PFE cell, the TEP cell, and / or the TEC produced by the disclosed method, or a population or organoid comprising such a cell.

[0226] In some embodiments, the subject has a thymus, parathyroid, and / or thyroid tissue disorder, and wherein the DE cells are differentiated from PSCs obtained from a biological sample from the subject.

[0227] In another aspect, the invention provides a method of evaluating a biological response to a compound or an environmental stimulus, the method comprising producing a first and a second population of PSCs, exposing the second population of PSCs to the compound or environmental stimulus, differentiating the first and the second population of PSCs into a first and a second population of PFE cells, and comparing the first and second populations of PFE cells, thereby evaluating the biological response. In some embodiments, the method further comprises differentiating the first and the second population of PFE cells into a first and a second population of TEP cells, producing a first and a second population of TECs from the first and the second population of TEP cells, and comparing the first and the second population of TEP cells, and / or the first and the second population of TECs, thereby evaluating the biological response.

[0228] In some embodiments, the first and second population of PSCs are obtained from a subject. In some embodiments, the first and second population of PSCs are produced from cells (e.g., non-pluripotent cells) obtained from a subject. In some embodiments, the first and second population of PSCs are obtained from the same subject. In some embodiments, the first and second population of PSCs are produced from cells (e.g., non-pluripotent cells) obtained from the same subject. In some embodiments, the first population of PSCs is obtained from a subject before the subject is exposed to a compound or environmental stimulus. In some embodiments, the first population of PSCs is produced from cells obtained from a subject before the subject is exposed to a compound or environmental stimulus. In some embodiments, the second population of PSCs is obtained from a subject after the subject is exposed to a compound or environmental stimulus. In some embodiments, thesecond population of PSCs is produced from cells obtained from a subject after the subject is exposed to a compound or environmental stimulus, z.e., from cells that were exposed to the compound or environmental stimulus.

[0229] In some embodiments, a composition or compound comprises, for example, a growth factor or cytokine. In some embodiments, and environmental stimulus comprises, for example, an extracellular matrix.

[0230] In another aspect, the invention provides a method of using the PFE cell, the TEP cell, and / or the TEC produced by the disclosed method, or a population or organoid comprising such a cell, in identifying a therapeutic compound for treatment of a thymus, parathyroid, and / or thyroid tissue dysfunction.

[0231] In another aspect, the invention provides a method of increasing PFE cells, TEP cells, and / or TECs in a subject in need thereof, the method comprising administering retinoic acid to the subject in an amount sufficient to increase PFE cells, TEP cells, and / or TECs.Additional Example Embodiments

[0232] In some embodiments, a method for producing pharyngeal foregut endoderm (PFE) cells comprises a) differentiating definitive endoderm (DE) cells into anterior foregut endoderm (AFE) cells, wherein differentiating comprises i) culturing the DE cells in a medium comprising about OnM retinoic acid and ii) further culturing the DE cells in a medium comprising about lOOnM retinoic acid, thereby producing AFE cells; and b) further differentiating the AFE cells into PFE cells, wherein differentiating comprises culturing the AFE cells in media comprising about lOOnM retinoic acid, thereby producing the PFE cells.

[0233] In some embodiments, a method for producing pharyngeal foregut endoderm (PFE) cells comprises obtaining definitive endoderm (DE) cells, differentiating the DE cells into anterior foregut endoderm (AFE) cells, and further differentiating the AFE cells into PFE cells.

[0234] As used herein, “differentiating” refers to culturing a multipotent cell in such a way that its potential to become committed to multiple cell lineages is reduced, z.e., such that the multipotent cell exhibits, to varying degrees, a more differentiated cell identity.

[0235] In some embodiments, differentiating comprises culturing the DE, AFE, and PFE cells in a medium comprising about lOOnM retinoic acid. In some embodiments, such a medium comprises about lOnM, 20nM, 30nM, 40nM, 50nM, 60nM, 70nM, 80nM, 90nM, orlOOnM retinoic acid. In some embodiments, a medium comprises OnM retinoic acid. In some embodiments, a medium comprises lOOnM retinoic acid.

[0236] In some embodiments, a medium comprises one or more supplements. In some embodiments, a supplement comprises a small molecule, an amino acid, a protein, a serum, a nutrient mixture, and the like, or a combination thereof. In some embodiments, a disclosed supplement may be replaced by a “suitable substitute.” In some embodiments, a suitable substitute comprises a supplement or combination of supplements that is different from but equivalent to the disclosed supplement, i.e. a supplement that exhibits the same or a similar biological effect as the disclosed supplement.

[0237] In some embodiments, a method of obtaining DE cells comprises differentiating pluripotent stem cells (PSCs). In some embodiments, the PSCs are human induced PSCs (iPSCs). In other embodiments, the PSCs are human embryonic stem cells (hESCs). In some embodiments, cells from a subject or patient are cultured to become iPSCs.

[0238] In some embodiments, the subject is a patient. In some embodiments, the subject is a human. In some embodiments, the subject or patient has, or is predisposed to have, one or more defects, e.g., developmental defects affecting the pharynx and derived tissues, e.g., defects found in a variety of syndromes, including but not limited to, immunodeficiency, DiGeorge / 22ql l.2 deletion, branchiootorenal (BOR) syndrome, Bamforth-Lazarus syndrome, CHARGE syndrome, and otofaciocervical syndromes. CHARGE is an abbreviation for several features common in the syndrome, including coloboma, heart defects, atresia choanae (choanal atresia), growth retardation, genital abnormalities, and ear abnormalities. In some embodiments, the patient suffers from thymus, parathyroid, and / or thyroid tissue dysfunction. In some embodiments, the patient suffers from organ failure.

[0239] In some embodiments, differentiating the PSCs comprises culturing the PSCs on a basement membrane or extracellular matrix. In some embodiments, plates (e.g. bottoms of wells of tissue culture plates) are coated with a basement membrane or extracellular matrix (ECM). In some embodiments, a basement membrane or extracellular matrix comprises CORNING® MATRIGEL® hESC-Qualified Matrix (Corning, Corning, NY, Cat. No. 354277). “MATRIGEL®” refers to a basement membrane matrix produced by Engelbreth- Holm- Swarm mouse sarcomas and is commonly used to support the culture of multipotent cells, cells in the process of differentiation, and 3D / organoid cultures. In some embodiments, the matrix is growth factor reduced, for example, growth factor reduced MATRIGEL® (MATRIGEL® (GFR). MATRIGEL® (GFR) differs from regular MATRIGEL® by anadditional treatment with ammonium sulfate, resulting in the reduction of growth factors bound to the extracellular matrix components contained within the MATRIGEL®.

[0240] In some embodiments, a different basement membrane or extracellular matrix may be used, non-limiting examples of which include: Matrigengel Matrix Growth Factor Reduced basement membrane extracts (ACRO Biosystems, Newark, DE, Cat. No. AC- M082701), CULTREX® Reduced Growth Factor Basement Membrane Extract (R&D Systems via Bio-Techne, Minneapolis, MN, Cat. No. 3433-005-01), Vitronectin XF™ defined xeno-free cell culture matrix (STEMCELL Technologies, Vancouver, Canada, Cat. No. 07180), GELTREX® LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Gibco via Thermo Fisher Scientific, Waltham, MA, Cat. No. A1413201), and the like.

[0241] In some embodiments, differentiating the PSCs comprises culturing the PSCs in a first medium comprising Roswell Park Memorial Institute 1640 cell culture medium (RPMI 1640), fetal bovine serum (FBS), an L-glutamine supplement, an antibiotic, Activin A (z.e., Activin beta-A chain, Inhibin beta-1; e.g. recombinant human / mouse / rat Activin A protein; GenBank Accession No. P08476), a GSK3 inhibitor, a PI3K / mTOR inhibitor, or a combination thereof.

[0242] In some embodiments, an RPMI 1640 comprises glucose, a pH indicator, buffering salts, amino acids, and vitamins. In some embodiments, a pH indicator comprises phenol red. In some embodiments, buffering salts comprise sodium chloride, sodium bicarbonate, disodium phosphate, potassium chloride, magnesium sulfate, calcium nitrate, and the like, or a combination thereof. In some embodiments, an RPMI 1640 comprises glutathione, biotin, vitamin B12, PABA, inositol, choline, or a combination thereof.

[0243] In some embodiments, culturing the PSCs in the first medium occurs on about day 0 of differentiation. In some embodiments, culturing the PSCs in the first medium occurs for a period of about 1 day.

[0244] In some embodiments, a “day” means a period of about 24 hours. In some embodiments, a “day” means a period of about 24±12 hours. In some embodiments, a “day” means a period of time that spans two calendar days, i.e. a period of time that begins on a given calendar day and ends on the next calendar day. In some embodiments, “day N of differentiation,” where “N” is an integer, indicates the length of time that has passed since the initiation of the differentiation protocol or method. In some embodiments, N=0, indicating that the associated method or parts thereof comprise the initiation of the differentiationprotocol or method. In some embodiments, “about” with respect to a day N of differentiation means a day N±l, N±2, or N±3 of differentiation.

[0245] In some embodiments, the FBS is HYCLONE® Defined FBS, the L-glutamine supplement is GLUTAMAX® (200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl), the antibiotic is 5,000 IU penicillin-5,000 pg / mL streptomycin (P / S), the GSK3 inhibitor is CHIR99021 (i.e. 6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-U / -imidazol-2-yl)-2- pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile, CT99021; CAS No. 252917-06-9), the PI3K / mT0R inhibitor is PI-103 i.e. 3-(4-(4-Morpholinyl)pyrido[3',2':4,5]furo[3,2- d]pyrimidin-2-yl)phenol, mTOR Inhibitor V, PI 3-K Inhibitor V; CAS No. 371935-74-9), or a combination thereof.

[0246] In some embodiments, the first medium comprises about 0.2% HYCLONE® Defined FBS, 1% GLUTAMAX® (200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl), 1% P / S, lOOng / mL Activin A, 3pM CHIR99021, 50nM PL103, or a combination thereof.

[0247] In some embodiments, the first medium comprises about 0.2% HYCLONE® Defined FBS, 1% GLUTAMAX® (200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl), 1% P / S, lOOng / mL Activin A, 3pM CHIR99021, and 50nM PI-103 in RPMI 1640.

[0248] In some embodiments, RPMI 1640 may be substituted with an alternative cell culture medium, non-limiting examples of which include: Dulbecco’s Modification of Eagle’s Medium (DMEM), Minimum Essential Medium (MEM), Ham’s F-12 Nutrient Mix, or the like. In some embodiments, substituting RPMI 1640 with an alternative cell culture medium requires the addition of supplements such as biotin, vitamin B12, and PABA i.e., 4- Aminobenzoic acid, para-Aminobenzoic acid, Vitamin Bx, Vitamin Hi, CAS No. 150-13-0), to function as an equivalent to RPMI 1640.

[0249] In some embodiments, an FBS is not heat inactivated or heat inactivated. In some embodiments, an FBS is defined or undefined. In some embodiments, an FBS is an FBS replacement or equivalent, non-limiting examples of which include KnockOut™ Serum Replacement (Gibco via Thermo Fisher Scientific, Waltham, MA, Cat. No. 10828010), FETALGRO® EX Bovine Growth Serum Premium FBS Replacement (Rocky Mountain Biologicals, Missoula, MT, Cat. No. FGX-BBT), or the like. FBS is also known as fetal calf serum (FCS).

[0250] In some embodiments, an antibiotic is ampicillin, gentamicin, kanamycin, neomycin, penicillin, streptomycin, or a combination thereof. In some embodiments, anantibiotic comprises one or more different antibiotics suitable for use in cell culture. In some embodiments, an antibiotic further comprises an antimycotic.

[0251] In some embodiments, a GSK3 inhibitor is a WNT agonist, a GSK-3P inhibitor, or both. In some embodiments, a GSK inhibitor may inhibit additional pathways and / or proteins. WNT agonists comprising GSK-3P inhibitors are well known in the art and include, but are not limited to, 1, 6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-lH-imidazol-2-yl)-2- pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile “CHIR99021” (Ring et cd., 2003), LiCl (Klein et al., 1996), BlO-acetoxime ((2'Z,3'E)-6-Bromoindirubin-3 '-oxime) (Meijer et al., 2003), N6-[2-[[4-(2,4-Dichlorophenyl)-5-(lH-imidazol-2-yl)-2- pyrimidinyl]amino]ethyl]-3-nitro-2,6-pyridinediamine “CHIR98014” (Ring et al., 2003), 3- (2, 4-Dichlorophenyl)-4-(l-methyl-lH-indol-3-yl)-lH-pyrrole-2, 5-dione “SB 216763” also known as GSK-3 Inhibitor IV (Coghlan etal., 2000), 3-[(3-Chloro-4-hydroxyphenyl)amino]- 4-(2-nitrophenyl)-lH-pyrrol-2, 5-dione “SB 415286” (Coghlan et al., 2000), 5-ethyl-7,8- dimethoxy-lH-pyrrolo[3,4-c]-isoquinoline-l,3-(2H)-dione “3F8” (Zhong et al., 2009), 9- Bromo-7,12-dihydro-indolo[3,2-d][l]benzazepin-6(5H)-one “Kenpaullone” (Schultz et al. ,1999; Zaharevitz etal., 1999), 9-Bromo-7,12-dihydro-pyrido[3',2':2,3]azepino[4,5- b]indol-6(5H)-one “1-Azakenpaullone” (Schultz et al., 1999; Zaharevitz et al., 1999), N-(3- Chloro-4-methylphenyl)-5-(4-ni_,trophenyl)-l,3,4-oxadiazol-2-amine “TC-G 24” (Khanfar et al., 2010), 2-Methyl-5-[3-[4-(methylsulfinyl)phenyl]-5-benzofuranyl]-l,3,4-oxadiazole “TCS 2002” (Saitoh etal., 2009), N-[(4-Methoxyphenyl)methyl]-N'-(5-nitro-2-thiazolyl)urea “AR- A 014418” (Bhat et al., 2003), 3-[5-[4-(2-Hydroxy-2-methyl-l-oxopropyl)-l-piperazinyl]-2- (trifluoromethyl)phenyl]-4-(U / -indol-3-yl)-U / -pyrrole-2, 5-dione “TCS 21311” (Thoma et al., 2011), 3-[[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]-phenol “TWS 119” (Ding et al., 2003), ((2'Z,3'E)-6-Bromoindirubin-3 '-acetoxime) “BlO-acetoxime” also known as GSK-3 Inhibitor IX (Meijer et al., 2003), 4-(2-Amino-4-oxo-2-imidazolin-5-ylidene)-2- bromo-4,5,6,7-tetrahydropyrrolo[2,3-c]azepin-8-one “lOZ-Hymenial disine” (Breton etal., 1997), 2-[(3-iodophenyl)methylsulfanyl]-5-pyridin-4-yl-l,3,4-oxadiazole, also known as GSK-3P Inhibitor II (Wada, 2009), 4-Benzyl-2-methyl-l, 2, 4-thiadiazolidine-3, 5-dione, also known as GSK-3 P Inhibitor I (Wada, 2009), 3-Amino-6-(4-((4-methylpiperazin-l- yl)sulfonyl)phenyl)-N-(pyridin-3-yl)pyrazine-2-carboxamide, HC1 , also known as GSK-3P Inhibitor XXVII (US Patent Pub. No. 2006 / 0173014), 4,5-bis(l-Methyl-lH-indol-3-yl)-l,2- dihydropyrazol-3-one, also known as GSK-3P Inhibitor XXVI (Chen et al, 2011), FRATtide peptide (Bax etal., 2001), 3-Amino-lH-pyrazolo[3,4-b]quinoxaline “Cdkl / 5 Inhibitor”(Andreani et aL, 1996, 2000; Katoh et aL, 2011) and 4-((5-Bromo-2-pyridinyl)amino)-4- oxobutanoic acid “Bikinin” (De Rybel et al., 2009).

[0252] In some embodiments, a PI3K / mTOR inhibitor is a PI3K inhibitor, an mTOR inhibitor, a DNA-PK inhibitor, an AKT inhibitor, or a combination thereof. In some embodiments, a PI3K / mTOR inhibitor may inhibit additional pathways and / or proteins. In some embodiments, a PI3K / mTOR inhibitor is PI-103, PI-103 hydrochloride (3-[4-(4- Morpholinylpyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl]phenol hydrochloride, CAS No. 371935-79-4), Z STK474 (2-(2-Difluoromethylbenzimidazol- 1 -yl)-4, 6-dimorpholino- 1,3,5- triazine, 2-(Difluoromethyl)-l-(4,6-di-4-morpholinyl-l,3,5-triazin-2-yl)-lH-benzimidazole, 4,4'-(6-(2-(Difluoromethyl)-lH-benzo[d]imidazol-l-yl)-l,3,5-triazine-2,4-diyl)dimorpholine, ZSTK 474, ZSTK-474, 2-(2-Difluoromethylbenzoimidazol-l-yl)-4, 6-dimorpholino- 1, 3, 5- triazine, CAS No. 475110-96-4), and the like, or a combination thereof.

[0253] In some embodiments, differentiating the PSCs further comprises culturing the PSCs in a second medium comprising RPMI 1640, FBS, an L-glutamine supplement, an antibiotic, Activin A, an ALK / BMP inhibitor, or a combination thereof. In some embodiments, culturing the PSCs in the second medium occurs on about days 1-2 of differentiation. In some embodiments, culturing the PSCs in the second medium occurs for a period of about 2 days. In some embodiments, the FBS is HYCLONE® Defined FBS, the L- glutamine supplement is GLUTAMAX® (200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl), the antibiotic is 5,000 IU penicillin-5,000 pg / mL streptomycin (P / S), the ALK / BMP inhibitor is LDN193189 (i.e. DM3189, 4-[6-[4-(l-Piperazinyl)phenyl]pyrazolo[l,5- a]pyrimidin-3-yl]quinoline dihydrochloride; CAS No. 1062368-24-4), or a combination thereof. In some embodiments, the second medium comprises about 0.2% HYCLONE® Defined FBS, 1% GLUTAMAX® (200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl), 1% P / S, lOOng / mL Activin A, 250 nM LDN193189, or a combination thereof.

[0254] In some embodiments, an ALK / BMP inhibitor comprises a BMP inhibitor, a BMP pathway inhibitor, an ALK1 inhibitor, an ALK2 inhibitor, and ALK3 inhibitor, an ALK6 inhibitor, an AMPK pathway inhibitor, or a combination thereof. In some embodiments, an ALK / BMP inhibitor may inhibit additional pathways and / or proteins. In some embodiments, an ALK / BMP inhibitor comprises LDN193189, dorsomorphin (z.e., dorsomorphin dihydrochloride, 6-[4-[2-(l-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[l,5- a]pyrimidine dihydrochloride, Compound C, BML-275, CAS No. 1219168-18-9), or the like, or a combination thereof. Bone Morphogenic Proteins (BMPs) are members of the TGF-betasuperfamily and comprise metalloproteases implicated in embryonic patterning among diverse species as well as post-embryonic cell signaling. Inhibitors of BMPs include, for example, agents that bind to a BMP molecule to form a complex wherein the BMP activity is decreased or eliminated, for example by preventing or inhibiting the binding of the BMP molecule to a BMP receptor. Alternatively, the inhibitor is an agent that acts as an antagonist or reverse agonist. This type of inhibitor binds to a BMP receptor and prevents binding of a BMP to the receptor. An example of a latter agent is an antibody that binds a BMP receptor and prevents binding of BMP to the antibody -bound receptor. Inhibitors of BMPs are well known in the art (Rider et al., 2010) and can include, but are not limited to, Noggin, Chordin, Follistatin (Schneyer et al., 1994), DAN, proteins comprising a DAN cysteine-knot domain (including Cerberus and Gremlin), Sclerostin, Twisted Gastrulation, Uterine Sensitivity- Associated Gene-1, Connective-Tissue Growth Factor (Abreu et al., 2002), Inhibin (Wiater and Vale, 2003), BMP-3 (Gamer et al., 2005), Dorsomorphin (Yu et al., 2008) and derivatives, including DMH1 (Hao et al., 2010) and LDN-193189 (Cuny et al., 2008).

[0255] In some embodiments, differentiation of PSCs into DE cells can comprise culturing the PSCs in a defined medium such as STEMDIFF® Definitive Endoderm Kit (STEMCELL Technologies Cat. No. 05115) or the like.

[0256] In some embodiments, the DE cells obtained by differentiating PSCs functionally express SOX17, GATA4, GATA6, EOMES, LEFTY1, CER1, or a combination thereof; and / or do not functionally express POU5F1, VGLL2, EYA1, PAX9, PAX1, FOXE1, PBX1, or a combination thereof.

[0257] In some embodiments, functional expression or the lack thereof is of an RNA (transcript). In some embodiments, functional expression or the lack thereof is of a polypeptide (protein).

[0258] In some embodiments, the medium used to produce PFE cells (i.e. the third, fourth, fifth, and sixth medium) comprises RPMI 1640, a neuronal cell culture supplement, an L-glutamine supplement, an antibiotic, suitable substitutes for the foregoing, or a combination thereof. In some embodiments, the neuronal cell culture supplement is B-27® minus vitamin A, the L-glutamine supplement is GLUTAMAX® (200 mM L-alanyl-L- glutamine dipeptide in 0.85% NaCl), the antibiotic is 5,000 IU penicillin-5,000 pg / mL streptomycin (P / S), or a combination thereof. In some embodiments, the medium comprises about 0.5x B-27® minus vitamin A neuronal cell culture supplement, 1% GLUTAMAX® (200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl), 1% P / S, or a combination thereof.In some embodiments, B-27® minus vitamin A neuronal cell culture supplement comprises progesterone (CAS No. 57-83-0).

[0259] In some embodiments, a neuronal cell culture supplement may be replaced with an equivalent supplement. In some embodiments, a neuronal cell culture supplement or equivalent lacks vitamin A. In some embodiments, a neuronal cell culture supplement or equivalent comprises B-27® minus vitamin A, MACS® NEUROBREW®-21 without Vitamin A neural supplements for propagation of neural stem cells of primary, embryonic stem, or induced pluripotent stem cell origin (Miltenyi Biotec, Cat. No. 130-097-263), NEUROCULT™ SMI without Vitamin A supplement for the culture of neural progenitor cells (STEMCELL Technologies, Cat. No. 05731), or the like, or a combination thereof.

[0260] In some embodiments, differentiating DE cells into AFE cells comprises singularizing the DE cells, plating the DE cells, culturing the DE cells in a third medium, and culturing the DE cells in a fourth medium comprising about lOOnM retinoic acid.

[0261] “Singularizing” refers to the process of dissociating cell aggregates, i.e. adherent cells (e.g., cells attached to other cells and / or cells attached to a growth surface or matrix), into a suspension comprising cells that are primarily singularized, i.e. not attached to other cells or not attached to many other cells.

[0262] “Plating” refers to the action of dispensing cells (i.e., singularized cells or small aggregates of cells) onto / into a plate. As used herein, “seeding” refers to plating a number of cells with the intention of culturing said cells to divide and produce a larger number of cells.

[0263] In some embodiments, singularizing the DE cells and plating the DE cells occur on about day 3 of differentiation. In some embodiments, singularizing comprises dissociating the DE cells with a proteolytic enzyme, scraping the DE cells and then triturating the DE cells by pipette, or a combination thereof. In some embodiments, the proteolytic enzyme is capable of detaching and / or dissociating adherent cells in culture.

[0264] In some embodiments, the proteolytic enzyme, or a solution containing the proteolytic enzyme, comprises any enzyme capable of dissociating adherent cells. In some embodiments, the proteolytic enzyme, or a solution containing the proteolytic enzyme, is trypsin (i.e., Cocoonase, Tryptar, Tryptase; CAS No. 9002-07-7), TRYPLE® recombinant dissociation enzyme, ACCUTASE® cell detachment solution, DISPASE® dissociating enzyme, or a combination thereof.

[0265] In some embodiments, culturing the DE cells comprises culturing the DE cells on a basement membrane or extracellular matrix. In some embodiments, the basementmembrane or extracellular matrix comprises 804G supernatant, MATRIGEL® basement membrane matrix, MATRIGEL® Growth Factor Reduced (GFR) basement membrane matrix, or a combination thereof.

[0266] In some embodiments, culturing the DE cells in the third medium occurs on about day 3 of differentiation. In some embodiments, culturing the DE cells in the third medium occurs for a period of about one day. In some embodiments, the third medium comprises an ALK / TGF-PRI inhibitor, an ALK / BMP inhibitor, a ROCK inhibitor, or a combination thereof. In some embodiments, the ALK / TGF-PRI inhibitor is A83-01 (i.e. 3-(6-Methyl-2- pyridinyl)-N-phenyl-4-(4-quinolinyl)-lH-pyrazole-l-carbothioamide; CAS No. 909910-43- 6), the ALK / BMP inhibitor is LDN193189, the ROCK inhibitor is Y-27632 (i.e. Y-27632 2HC1, Y-27632 dihydrochloride, y27632, ROCK1 inhibitor, trans-4-[(lR)-l-Aminoethyl]-N- 4-pyridinylcyclohexanecarboxamide dihydrochloride; CAS No. 129830-38-2), or a combination thereof. In some embodiments, the third medium comprises about: IpM A83- 01, 250nM LDN193189, lOpM Y-27632, or a combination thereof.

[0267] In some embodiments, an ALK / TGF-PRI inhibitor is an ALK inhibitor, a TGF inhibitor, a TGF-P inhibitor, a TGF-PRI inhibitor, an ALK5-TD inhibitor, an ALK4-TD inhibitor, an ALK7-TD inhibitor, or a combination thereof. In some embodiments, an ALK / TGF-PRI inhibitor may inhibit additional pathways and / or proteins. In some embodiments, an ALK / TGF-PRI inhibitor comprises A83-01. In some embodiments, an ALK / TGF-PRI inhibitor comprises SD-208 (2-(5-Chloro-2-fluorophenyl)-4-[(4- pyridyl)amino]pteridine, CAS No. 627536-09-8), SB525334 (6-[2-(l,l-Dimethylethyl)-5-(6- methyl-2-pyridinyl)-lH-imidazol-4-yl]quinoxaline , CAS No. 356559-20-1), E-616452 (2-(3- (6-Methylpyridine-2-yl)-lH-pyrazol -4-yl)- 1,5-naphthyri dine, RepSox, SIN 2511, ALK5 Inhibitor II, CAS No. 446859-33-2), LY2157299 (4-[5,6-Dihydro-2-(6-methyl-2-pyridinyl)- 4H-pyrrolo[l,2-b]pyrazol-3-yl]-6-quinolinecarboxamide, galunisertib, CAS No. 700874-72- 2), LY2109761 (4-[5,6-Dihydro-2-(2-pyridinyl)-4H-pyrrolo[l,2-b]pyrazol-3-yl]-7-[2-(4- morpholinyl)ethoxy] quinoline, CAS No. 700874-71-1), or the like, or a combination thereof.

[0268] In some embodiments, a ROCK inhibitor comprises a Rho kinase inhibitor, a Rho- associated protein kinase inhibitor, a ROCK1 inhibitor, a ROCK 2 inhibitor, or a combination thereof. In some embodiments, a ROCK inhibitor may additionally inhibit other pathways and / or proteins. In some embodiments, a ROCK inhibitor comprises Y-27632 (trans-4-[(lR)~ l-Aminoethyl]-A-4-pyridinylcyclohexanecarboxamide, CAS No. 146986-50-7), Y-27632 2HC1 ( / raw -4-[(lA)-l-Aminoethyl]-7V-4-pyridinylcyclohexanecarboxamide dihydrochloride,y27632, Y-276322HC1, ROCK inhibitor, R0CK1 inhibitor, CAS No. 129830-38-2), GSK429286A (4-[4-(Trifluoromethyl)phenyl]-A-(6-Fluoro-lZ7-indazol-5-yl)-2-methyl-6- oxo-l,4,5,6-tetrahydro-3-pyridinecarboxamide, GSK 429286, CAS No. 864082-47-3), RKI- 1447 (A-[(3-Hydroxyphenyl)methyl]-7V’ -[4-(4-pyridinyl)-2-thiazolyl]urea dihydrochloride, RKI 1447 dihydrochloride, CAS No. 1782109-09-4), Chroman 1 ((3S)-A-[2-[2- (Dimethylamino)ethoxy]-4-(l / / -pyrazol -4-yl)phenyl]-3,4-dihydro-6-methoxy -2 / 7-1 - benzopyran-3 -carboxamide dihydrochloride, CAS No. 1273579-40-0), a CEPT cocktail comprising Chroman 1, Emricasan (A-[2-(l,l-Dimethylethyl)phenyl]-2-oxoglycyl-A-[(15)-l- (carboxymethyl)-2-oxo-3-(2,3,5,6-tetrafluorophenoxy)propyl]-L-alaninamide, CAS No. 254750-02-2), Putrescine (1,4-Butanediamine dihydrochloride, 1,4-Diaminobutane dihydrochloride, Tetramethylenediamine dihydrochloride, Putrescine dihydrochloride, CAS No. 333-93-7), Spermine (N, N"-Bis(3 -aminopropyl)- 1,4-butanediamine tetrahydrochloride, Spermine tetrahydrochloride, CAS No. 306-67-2), Spermidine (l,8-Diamino-4-azaoctane, N- (3 -Aminopropyl)- 1,4-diaminobutane, CAS No. 124-20-9), and trans-ISRIB N,N’-trans-l,4- cyclohexanediylbis[2-(4-chlorophenoxy)acetamide], CAS No. 1597403-47-8); or the like, or a combination thereof.

[0269] In some embodiments, culturing the DE cells in the fourth medium occurs on about days 4-8 of differentiation. In some embodiments, culturing the DE cells in the fourth medium occurs for a period of about five days. In some embodiments, the fourth medium comprises an ALK / TGF-PRI inhibitor, an ALK / BMP inhibitor, a WNT inhibitor, retinoic acid, or a combination thereof. In some embodiments, the ALK / TGF-PRI inhibitor is A83-01, the ALK / BMP inhibitor is LDN193189, the WNT inhibitor is IWR-1 (z.e., e / rab-IWR 1, rel- 4-[(3a / ?,4k,7 / ?,7ak)-l,3,3a,4,7,7a-Hexahydro- l,3-dioxo-4,7-methano-2 / / -isoindol-2-yl]-N-8- quinolinylbenzamide; CAS No. 1127442-82-3), or a combination thereof. In some embodiments, the fourth medium comprises about IpM A83-01, 250nM LDN193189, 250nM IWR-1, lOOnM retinoic acid, or a combination thereof.

[0270] In some embodiments, a WNT inhibitor may be a P-catenin inhibitor, PORCN inhibitor. In some embodiments, a WNT inhibitor may inhibit one or more additional proteins and / or pathways. In some embodiments, a WNT inhibitor comprises IWR-1. In some embodiments, a WNT inhibitor comprises IWP 2 (z.e., A-(6-Methyl-2-benzothiazolyl)-2- [(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-J]pyrimidin-2-yl)thio]-acetamide, CAS No. 686770-61-6), XAV 939 (z.e., 3,5,7,8-Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4Z7-thiopyrano[4,3-J]pyrimidin-4-one, CAS No. 284028-89-3), or the like, or a combination thereof.

[0271] In some embodiments, the AFE cells produced by differentiating DE cells functionally express VGLL2, EYA1, SIX1, or a combination thereof; and / or do not functionally express POU5F1, SOX17, or a combination thereof.

[0272] In some embodiments, differentiating AFE cells into PFE cells comprises singularizing the AFE cells, plating the AFE cells, and culturing the AFE cells in a fifth and a sixth medium comprising about lOOnM retinoic acid.

[0273] In some embodiments, singularizing the AFE cells and plating the AFE cells occur on about day 9 of differentiation. In some embodiments, wherein singularizing comprises dissociating the AFE cells with a proteolytic enzyme, scraping the AFE cells and then triturating the AFE cells by pipette, or a combination thereof. In some embodiments, the proteolytic enzyme is capable of detaching and / or dissociating adherent cells in culture. In some embodiments, the proteolytic enzyme, or a solution containing the proteolytic enzyme, comprises trypsin, TRYPLE® recombinant dissociation enzyme, ACCUTASE® cell detachment solution, DISPASE® dissociating enzyme, or a combination thereof.

[0274] In some embodiments, culturing the AFE cells comprises culturing the AFE cells on a basement membrane or extracellular matrix. In some embodiments, the basement membrane or extracellular matrix comprises 804G supernatant, MATRIGEL® basement membrane matrix, MATRIGEL® Growth Factor Reduced (GFR) basement membrane matrix, or a combination thereof.

[0275] In some embodiments, culturing the AFE cells in the fifth medium occurs on about day 9 of differentiation. In some embodiments, culturing the AFE cells in the fifth medium occurs for a period of about one day. In some embodiments, the fifth medium comprises a bone morphogenic protein 4 (BMP4), a fibroblast growth factor 10 (FGF10), a retinoic acid, a ROCK inhibitor, or a combination thereof. In some embodiments, the ROCK inhibitor is Y-27632. In some embodiments, the fifth medium comprises about 50 ng / mL BMP4, 50 ng / mL FGF10, 100 nM retinoic acid, lOpM Y-27632, or a combination thereof.

[0276] In some embodiments, culturing the AFE cells in the sixth medium occurs on about days 10-14 of differentiation. In some embodiments, culturing the AFE cells in the sixth medium occurs for a period of about five days. In some embodiments, the sixth medium comprises a bone morphogenic protein 4 (BMP4), a fibroblast growth factor 10 (FGF10), aretinoic acid, or a combination thereof. In some embodiments, the sixth medium comprises about 50 ng / mL BMP4, 50 ng / mL FGF10, 100 nM retinoic acid, or a combination thereof.

[0277] In some embodiments, the PFE cells produced by differentiating AFE cells functionally express FOXA2, SOX2, PAX9, PAX1, FOXE1, PBX1 or a combination thereof; and / or do not functionally express POU5F1, SOX17, VGLL2, or a combination thereof. In some embodiments, the PFE cells functionally express PAX9 at least about two-fold the level of PAX9 functionally expressed by the AFE cells.

[0278] In some embodiments, producing PFE cells comprises culturing DE and AFE cells in media continuously comprising retinoic acid. In some embodiments, producing PFE cells comprises culturing DE and AFE cells in media continuously comprising lOOnM retinoic acid.

[0279] In some embodiments, producing PFE cells comprises culturing DE and AFE cells in media comprising retinoic acid beginning after the initialization of DE to AFE differentiation. In some embodiments, producing PFE cells comprises culturing DE and AFE cells in media comprising lOOnM retinoic acid beginning after the initialization of DE to AFE differentiation.

[0280] In some embodiments, producing PFE cells comprises culturing DE and AFE cells in media comprising retinoic acid beginning about 1 day after the initialization of DE to AFE differentiation. In some embodiments, producing PFE cells comprises culturing DE and AFE cells in media comprising lOOnM retinoic acid beginning about 1 day after the initialization of DE to AFE differentiation.

[0281] In another embodiment, the invention provides a composition for a medium for the differentiation of DE cells into AFE cells, wherein the medium comprises about IpM A83-01, 250nM LDN193189, lOpM Y-27632, or a combination thereof. In some embodiments, the medium also comprises RPMI 1640, 0.5x B-27® minus vitamin A neuronal cell culture supplement, 1% GLUTAMAX® (200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl), 1% P / S, or a combination thereof.

[0282] In another embodiment, the invention provides a composition for a medium for the differentiation of DE cells into AFE cells, wherein the medium comprises about IpM A83-01, 250nM LDN193189, 250nM IWR-1, lOOnM retinoic acid, or a combination thereof. In some embodiments, the medium also comprises RPMI 1640, 0.5x B-27® minus vitamin A neuronal cell culture supplement, 1% GLUTAMAX® (200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl), 1% P / S, or a combination thereof.

[0283] In another embodiment, the invention provides a composition for a medium for the differentiation of AFE cells into PFE cells, wherein the medium comprises about 50 ng / mL BMP4, 50 ng / mL FGF10, 100 nM retinoic acid, lOpM Y-27632, or a combination thereof. In some embodiments, the medium also comprises RPMI 1640, 0.5x B-27® minus vitamin A neuronal cell culture supplement, 1% GLUTAMAX® (200 mM L-alanyl-L- glutamine dipeptide in 0.85% NaCl), 1% P / S, or a combination thereof.

[0284] In another embodiment, the invention provides a composition for a medium for the differentiation of AFE cells into PFE cells, wherein the medium comprises about 50 ng / mL BMP4, 50 ng / mL FGF10, 100 nM retinoic acid, or a combination thereof. In some embodiments, the medium also comprises RPMI 1640, 0.5x B-27® minus vitamin A neuronal cell culture supplement, 1% GLUTAMAX® (200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl), 1% P / S, or a combination thereof.

[0285] In some embodiments, the invention provides a PFE cell obtained by the disclosed method. In another embodiment, the invention provides an organoid or population comprising a PFE cell obtained by the disclosed method.

[0286] In some embodiments, the invention provides a method for producing thymic epithelial cells (TECs), the method comprising obtaining PFE cells produced by the disclosed method; culturing the PFE cells in a sixth medium, thereby producing thymic epithelial progenitor (TEP) cells; singularizing the TEP cells; seeding the TEP cells onto a scaffold, thereby initiating an organoid; and culturing the TEP organoid in a seventh and an eighth medium, thereby producing TECs and an organoid comprising TECs. In some embodiments, a scaffold is coated in an extracellular matrix or basement membrane. In some embodiments, a scaffold is not coated in an extracellular matrix or basement membrane.

[0287] A “scaffold” refers to a biomaterial or composition of biomaterials that provides structural support for cells in culture, thereby enabling differentiation and / or development of organoids that would otherwise be poorly supported by conventional two-dimensional (2D) tissue culture. A scaffold may be prepared from an organism, such as a non-human animal, by decellularizing an existing organ or part thereof.

[0288] In some embodiments, the sixth, seventh, and eighth medium comprise RPMI 1640, a neuronal cell culture supplement, an L-glutamine supplement, an antibiotic, or a combination thereof. In some embodiments, the neuronal cell culture supplement is B-27® minus vitamin A, the L-glutamine supplement is GLUTAMAX® (200 mM L-alanyl-L- glutamine dipeptide in 0.85% NaCl), the antibiotic is 5,000 IU penicillin-5,000 pg / mLstreptomycin (P / S), or a combination thereof. In some embodiments, the sixth, seventh, and eighth medium comprise about 0.5x B-27® minus vitamin A neuronal cell culture supplement, 1% GLUTAMAX® (200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl), 1% P / S, or a combination thereof.

[0289] In some embodiments, culturing the PFE cells in the sixth medium occurs on about days 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 of differentiation, or a combination thereof. In some embodiments, culturing the PFE cells in the sixth medium occurs for a period of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, the sixth medium comprises a bone morphogenic protein 4 (BMP4), a fibroblast growth factor 10 (FGF10), a retinoic acid, or a combination thereof. In some embodiments, the sixth medium comprises about 50 ng / mL BMP4, 50 ng / mL FGF10, retinoic acid, or a combination thereof.

[0290] In some embodiments, the TEP cells produced from the PFE cells functionally express PAX1, PAX9, FOXN1, GATA3, or a combination thereof; and / or do not functionally express VGLL2, SOX17, CER1, POU5F1, NANOG, or a combination thereof.

[0291] In some embodiments, singularizing the TEP cells comprises dissociating the TEP cells with a proteolytic enzyme, scraping the TEP cells and then triturating the TEP cells by pipette, or a combination thereof. In some embodiments, the proteolytic enzyme is capable of detaching and / or dissociating adherent cells in culture. In some embodiments, the proteolytic enzyme, or a solution containing the proteolytic enzyme, comprises trypsin, TRYPLE® recombinant dissociation enzyme, ACCUTASE® cell detachment solution, DISPASE® dissociating enzyme, or a combination thereof.

[0292] In some embodiments, the scaffold onto which the TEP cells are seeded is contained in a plate. In some embodiments, the scaffold is a decellularized thymic lobe, lobule, or part thereof, or a cell culture filter insert.

[0293] In some embodiments, the lobe or lobule is from a human. In some embodiments, the lobe or lobule is from a non-human animal.

[0294] In some embodiments, the decellularized thymic lobe is from a mouse. In some embodiments, the mouse is a young mouse. In some embodiments, the mouse is 4-6 weeks of age. In some embodiments, the mouse is of an ICR strain (e.g. JAX Strain No. 009122 “ICR / HaJ”, Taconic Strain No. IcrTacTCR “ICR”).

[0295] In some embodiments, culturing the TEP organoid in the seventh medium occurs on about day 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41 of differentiation, or a combination thereof. In some embodiments,culturing the TEP organoid in the seventh medium occurs for a period of about 1 day. In some embodiments, culturing the TEP organoid in the seventh medium occurs for a period of about 1 or more days. In some embodiments, culturing the TEP organoid in the seventh medium occurs for a period of about 1-16 days. In some embodiments, the seventh medium comprises an ALK / TGF-PRI inhibitor, a ROCK inhibitor, or a combination thereof. In some embodiments, the ALK / TGF-PRI inhibitor is A83-01, the ROCK inhibitor is Y-27632, or a combination thereof. In some embodiments, the seventh medium comprises about IpM A83- 01, lOpM Y-27632, or a combination thereof.

[0296] In some embodiments, culturing the TEP organoid in the eighth medium occurs on about days 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41 of differentiation, or a combination thereof. In some embodiments, culturing the TEP organoid in the eighth medium occurs for a period of about 16 days. In some embodiments, the eighth medium comprises an ALK / TGF-PRI inhibitor. In some embodiments, the ALK / TGF-PRI inhibitor is A83-01. In some embodiments, the seventh medium comprises about IpM A83-01.

[0297] In some embodiments, a medium comprises one or more suitable substitutes for one or more of the disclosed components thereof.

[0298] In some embodiments, the TECs produced by the disclosed method functionally express GCM2, FOXN1, CCL25, PAX1, PAX9, GATA3, MAFB, or a combination thereof; and / or do not functionally express VGLL2, SOX17, CER1, POU5F1, NANOG, or a combination thereof.

[0299] In another embodiment, the invention provides a TEP cell produced by the disclosed method.

[0300] In another embodiment, the invention provides a TEC produced by the disclosed method.

[0301] In another embodiment, the invention provides an organoid or a population comprising a cell, e.g., a TEP cell or a TEC, produced by a method disclosed herein.

[0302] In another embodiment, the invention provides a method of recovering or restoring impaired tissue in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the PFE cell, the TEP cell, and / or the TEC produced by the disclosed method, or a population or organoid comprising such a cell.

[0303] In another embodiment, the invention provides a method of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of aPFE cell, the TEP cell, and / or the TEC produced by a disclosed method disclosed herein, and / or a population or organoid comprising such a cell.

[0304] In some embodiments, the subject has a thymus, parathyroid, and / or thyroid tissue disorder, and, e.g., the DE cells are differentiated from PSCs obtained from a biological sample from the subject.

[0305] In another embodiment, the invention provides a method of evaluating a biological response to a compound or an environmental stimulus, the method comprising producing a first and a second population of PSCs, exposing the second population of PSCs to the compound or environmental stimulus, differentiating the first and the second population of PSCs into a first and a second population of PFE cells, and comparing the first and second populations of PFE cells, thereby evaluating the biological response. In some embodiments, the method further comprises differentiating the first and the second population of PFE cells into a first and a second population of TEP cells, producing a first and a second population of TECs from the first and the second population of TEP cells, and comparing the first and the second population of TEP cells, and / or the first and the second population of TECs, thereby evaluating the biological response.

[0306] In another aspect, the invention provides a method of using the PFE cell, the TEP cell, and / or the TEC produced by the disclosed method, or a population or organoid comprising such a cell, in identifying a therapeutic compound for treatment of a thymus, parathyroid, and / or thyroid tissue dysfunction.

[0307] In another embodiment, the invention provides a method of increasing PFE cells, TEP cells, and / or TECs in a subject in need thereof, the method comprising administering retinoic acid to the subject in an amount sufficient to increase PFE cells, TEP cells, and / or TECs.

[0308] In another embodiment, the following table shows a non-limiting example of a method of producing PFE cells, TEP cells, TECs, and / or an organoid comprising such a cell.aAbout the day number(s) of protocol on which steps occurbAbout the duration of time, in days, associated with feeding cells a given mediacAs referenced with respect to each stage of differentiationEXEMPLIFICATIONEXAMPLE 1 : MATERIALS AND METHODSHuman embryonic stem cell culture andAFE differentiation

[0309] Hl hESCs were maintained on CORNING® MATRIGEL® hESC-Qualifted Matrix (BD Biosciences, Franklin Lakes, NI, Cat. No. 354277) in MTESR®1 (STEMCELL Technologies, Vancouver, Canada, Cat. No. 05850). Cells were fed daily and split every 3-4 days with DISPASE® (STEMCELL Technologies, Cat. No. 07923). For initiation of differentiation, maintenance cultures were singularized with TRYPLE® Express (Gibco via Thermo Fisher Scientific, Waltham, MA, Cat. No. 12604) and plated at 2xl05cells / cm2onto MATRIGEL® (GFR)-coated (BD Biosciences, Cat. No. 356231) 6-well plates in MTESR®1 supplemented with 10 pM Y-27632 (Selleck Chemicals, Houston, TX, Cat. No. S1049). Forty-eight hours later (day 0, z.e., on day 0 of a differentiation protocol, z.e., on day 0 of differentiation) DE differentiation was initiated by replacing the media with DE differentiation media (RPMI 1640 (Gibco via Thermo Fisher Scientific, Cat. No. 21870-092), 0.2% HYCLONE® FBS (Cytiva via Fisher Scientific, Waltham, MA, Cat. No. SH30070.03), 1% GLUTAMAX® (Gibco via Thermo Fisher Scientific, Cat. No. 35050079), and 1% penicillin-streptomycin (Corning, Cat. No. 30-001 -CI)) supplemented with 3uM CHIR99021 (Tocris Bioscience via Bio-Techne, Minneapolis, MN, Cat. No. 4423), 50nM PI-103 (Selleck Chemicals, Cat. No. SI 038) and lOOng / ml Activin A (R&D Systems via Bio-Techne, Minneapolis, MN, Cat. No. 338-AC). On days 1 and 2 cells were fed with DE differentiationmedia supplemented with lOOng / ml Activin A (R&D Systems via Bio-Techne, Cat. No. 338- AC) and 250nM LDN193189 (Selleck Chemicals, Cat. No. S2618).

[0310] To generate AFE, on day 3, DE cultures were singularized using TRYPLE® Express (Gibco via Thermo Fisher Scientific, Cat. No. 12604) and plated onto 804G- supematant coated plates at 3xl05cells / cm2in B27 differentiation media (RPMI 1640 (Gibco via Thermo Fisher Scientific, Cat. No. 21870-092), 0.5x B27 minus vitamin A (Gibco via Thermo Fisher Scientific, Cat. No. 12587-010), 1% GLUTAMAX® (Gibco via Thermo Fisher Scientific, Cat. No. 35050079), and 1% penicillin-streptomycin (Corning, Cat. No. 30- 001-CI)) supplemented with lOpM Y-27632, IpM A83-01 (Tocris Bioscience via Bio- Techne, Cat. No. 2939) and 250nM LDN193189. On days 4-8, cells were fed with B27 differentiation media supplemented with IpM A83-01, 250nM LDN193189, 250nM IWR-1 (Tocris Bioscience via Bio-Techne, Cat. No. 3532).

[0311] To determine the effect of retinoic acid signaling on pharyngeal endoderm marker expression by qPCR, differentiation cultures were treated with either IpM BMS493 (Tocris Bioscience via Bio-Techne, Cat. No. 3509) or varying concentrations of retinoic acid (Sigma- Aldrich via MilliporeSigma, Burlington, MA, Cat. No. R2625) as indicated. To further dissect the transcriptomic effects of retinoic acid at single-cell resolution, differentiation cultures generated with either OnM, lOOnM, IpM, or lOpM of retinoic acid were collected and processed for single cell transcriptomic analysis. 804G supernatant was prepared and used as described (Bosco et al., 2000; Genga et al., 2019).PFE differentiation

[0312] To generate PFE, DE was generated as described above. On day 3 DE cultures were fed with B27 differentiation media supplemented with IpM A83-01 and 250nM LDN193189. On days 4-8 cells were fed with B27 differentiation media supplemented with luM A83-01, 250nM LDN193189, 250nM IWR-1, and lOOnM retinoic acid (Sigma-Aldrich via MilliporeSigma, Cat. No. R2625). On day 9, cells were singularized using TRYPLE® Express (Gibco via Thermo Fisher Scientific, Cat. No., 12604) and plated onto 804G- supematant coated plates at 3xl05cells / cm2in B27 differentiation media supplemented with lOpM Y-27632, 50ng / ml bone morphogenetic protein 4 (BMP4) (R&D Systems via Bio- Techne, Cat. No. 314-BP), lOOnM retinoic acid and 50ng / ml FGF10 (R&D Systems via Bio-Techne, Cat. No. 345-FG). On days 10-14, cells continued to be fed with B27 differentiation media supplemented with 50ng / ml BMP4, lOOnM retinoic acid and 50ng / ml FGF10.Generation and differentiation of human cord-blood derived induced pluripotent stem cells

[0313] Human umbilical cord blood (UCB) was obtained in accordance with the Committee for the Protection of Human Subjects in Research guidelines of the University of Massachusetts Chan Medical School. UCB was provided by the medical staff of the University of Massachusetts Memorial Umbilical Cord Blood Donation Program. Mononuclear cells were isolated from human umbilical cord blood (HuCBOOl) using HISTOPAQUE®-1077 (Sigma-Aldrich via MilliporeSigma, Cat. No. 10771). Briefly, l lmL of whole blood was layered onto 1 ImL of HISTOPAQUE®-1077 and centrifuged at 400xg for 30min at room temperature. After centrifugation, monocytic cells were isolated at the HISTOPAQUE®-1077 / plasma interface. On the day of transduction, 3xl05monocytic cells were transduced with premixed sendai viral factors and spinfected at 900xg for 90min. After centrifugation, cells were incubated at 37°C overnight. On the next day, cells were harvested with TRYPLE®, centrifuged at 500xg for 5min, and resuspended with fresh STEMSPAN® SFEM media (STEMCELL Technologies, Cat. No. 09650). 72 hours after sendai virus transduction, cells were harvested again and 3.3xl05cells were seeded into 1 well of 6-well plate pre-coated with hESC MATRIGEL® in STEMSPAN® SFEM media. Two days later, media was changed to ReproTESR® Medium for Reprogramming (STEMCELL Technologies, Cat. No. 05926) and cells were fed for another four days before being maintained in MTESR®1 media. Cord blood cells and iPSCs were analyzed for HLA isoform to confirm source:iPSC relationship.

[0314] The cord blood-derived human induced pluripotent stem cells (CBOOl-iPSC) were then differentiated into DE, AFE and PFE cells. Cells were harvested with TRYPLE® and plated at 2xl05cells / cm2onto MATRIGEL® (GFR)-coated 6-well plates in MTESR®1 supplemented with 10 pM Y-27632. To generate definitive endoderm, cells on day 0 were fed with DE differentiation media supplemented with 3uM CHIR99021, 50nM PI- 103 and lOOng / ml Activin A, followed by a supplementation with lOOng / ml Activin A and 250nM LDN193189 on day 1 and day 2. To generate AFE, on day 3 DE cultures were replated onto 804G-supernatant coated plates at 3xl05cells / cm2in B27 differentiation media supplemented with lOpM Y-27632, IpM A83-01 and 250nM LDN193189. On days 4-8 cells were fed withB27 differentiation media supplemented with IpM A83-01, 250nM LDN193189, 250nM IWR-1, and lOOnM retinoic acid. To generate PFE, cells were singularized using TRYPLE® and plated onto 804G-supernatant coated plates at 3xl05cells / cm2in B27 differentiation media supplemented with lOpM Y-27632, 50ng / ml BMP4, lOOnM retinoic acid and 50ng / ml FGF10. On days 10-14, cells continued to be fed with B27 differentiation media supplemented with 50ng / ml BMP4, lOOnM retinoic acid and 50ng / ml FGF10.RNA extraction, cDNA preparation and quantitative PCT analysis

[0315] RNA was isolated using TRIZOL® (Invitrogen via Thermo Fisher Scientific, Waltham, MA, Cat. No. 15596-018) according to the manufacturer’s instructions and Ipg of RNA was reverse transcribed to cDNA using SUPERSCRIPT® III (Invitrogen via Thermo Fisher Scientific, Cat. No. 18080-044). 30ng cDNA was analyzed using TAQMAN® Master Mix (Applied Biosystems via Thermo Fisher Scientific, Waltham, MA, Cat. No. 4440040) and gene specific TAQMAN® assays according to the manufacturer’s instructions. Alternatively, 30ng cDNA was analyzed using SYBR® FAST (KAPA Biosystems via Roche Diagnostics, Indianapolis, IN, Cat. No. KK4600) with gene specific primers (Integrate DNA Technologies, Coralville, IA). Targets were amplified and measured over the course of 35 cycles. Relative gene expression was calculated using either the AACT method to the indicated control condition; or by using the ACT method. Significance testing was performed in GraphPad Prism (Dotmatics, Boston, MA) by ANOVA with Dunnett’s multiple testing correction against a control condition. All genes were normalized to beta-actin. TAQMAN® assay kits (Life Technologies Cat. No. 4331182) and primer sequences used are listed in the following tables.Immunofluorescence analysis

[0316] Cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature, washed with PBS, washed with PBS + 0.2% Triton X-100 (PBST), and then incubated with 5% donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA, Cat. No. 017- 000-121) in PBST for 1 hour at room temperature. Primary antibodies were diluted in blocking buffer and incubated overnight at 4C. Cells were washed three times in PBST and incubated with appropriate Alexa dye-conjugated secondary antibodies (Invitrogen via Thermo Fisher Scientific) at room temperature for 2 hours. Cells were washed three times in PBST. Nuclei were stained with Hoechst (Invitrogen via Thermo Fisher Scientific, Cat. No. H3570). Images were visualized using a Nikon Eclipse Ti microscope (Nikon, Melville, NY) or a Leica TCS SP5 II confocal microscope (Leica Microsystems, Deerfield, IL). Primary and secondary antibodies with their dilutions are listed in the table below.Intracellular flow cytometry

[0317] Cells were dissociated with TRYPLE® Express (Gibco via Thermo Fisher Scientific, Cat. No., 12604) and stained with fixable LIVE / DEAD (Thermo Fisher Scientific, Cat. No. L23102) in PBS supplemented with 2% FBS on ice. Cells were washed in PBS then fixed in 2% PFA for 30 min at 4C. Cells were blocked and permeabilized with blocking buffer (5% donkey serum, 0.25% saponin in PBS) for 30 min at 4C. Primary antibodies were diluted in blocking buffer at the indicated concentration and incubated for 1 hour 4C. Cells were washed three times in wash buffer (0.25% saponin in PBS), and incubated with appropriate Alexa dye-conjugated secondary antibodies (Invitrogen via Thermo Fisher Scientific) diluted in blocking buffer at for 1 hour at room temperature. Cells were washed three times in wash buffer (0.25% saponin in PBS). Flow cytometry was performed using a BD Accuri C6 flow cytometer and all data was analyzed using FlowJo (Tree Star, Inc., Ashland, OR). Dead cells were excluded based on fixable LIVE / DEAD staining. Primary and secondary antibodies with their dilutions are listed in the following table.Transplantation assay

[0318] 3xl06PFE cells were resuspended in 20pl of blood from mouse of same strain and transplanted under the renal capsules of NOD-.scvt / I 2rgnuil(NSG) mice (The Jackson Laboratory, Bar Harbor, ME). After 10 weeks, transplants were recovered and fixed in 4% paraformaldehyde at room temperature, then incubated in 30% sucrose overnight at 4C. Tissues were equilibrated in OCT and then frozen in OCT for cryosectioning. Slides were immunostained for markers of pharyngeal pouch derivatives.Single cell RNA (scRNA) library preparation and sequencingRA titration AFE cell isolation. Hashtagging, and IPX scRNAseq library preparation

[0319] Cells were washed once with PBS, singularized using TRYPLE® and resuspended in PBS + 0.5% BSA (FACS buffer) at the concentration of IxlO6cells / ml. Approximately 500,000 cells for each sample were pelleted at 500 x g for 5 min at 4°C and resuspended in 100 pL FACS buffer supplemented with 500 ng of one of four Hashtag Antibodies: A0251 anti-human Hashtag 1 Antibody (BioLegend, Cat. No. 394601) for 0 nM RA, A0252 anti-human Hashtag 2 Antibody (BioLegend, Cat. No. 394603) for 100 nM RA, A0253 anti-human Hashtag 3 Antibody (BioLegend, Cat. No. 394605) for 1 pM RA, A0254 anti-human Hashtag 4 Antibody (BioLegend, Cat. No. 394607) for 10 pM RA. Samples were incubated on ice for 30 min and then washed twice with 1 mL of FACS buffer. Cells were pelleted as before and resuspended in 250 pL FACS Buffer. Cells were filtered through 40pM cell strainers (Fisher Scientific, Cat. No. 22363547) and counted on a hemocytometer. Equal number of cells for each of the four treatments were combined and cells were recounted using a hemocytometer. 9,900 cells were used to generate 10X scRNAseq library using Chromium Next GEM Single Cell 3’ v3.1 reagents according to manufacturer’s instructions (lOx Genomics, Pleasanton, CA, Cat. No. CG000206 Rev D). Hashtag libraries were prepared according to the manufacturer’s protocol (BioLegend). scRNAseq libraries were indexed using 10X SI-GA-B10 and SI-GA-B11 indices and HTO libraries were indexed using D701 (CGAGTAAT) and D702 (TCTCCGGA) indices. Libraries were sequenced on a Nextseq 550 using a High Output v2.5 75-cycle kit (Illumina, San Diego, CA. Cat. No. 20024906) to a mean read depth of 57,698 and 59,121 reads per cell for the two RNA replicates and 567 and 770 reads per cell for the respective hashtag libraries.Drop-seq and library preparation

[0320] Drop-seq was performed as described before (Genga et al., 2019) following the Drop-seq Laboratory Protocol version 3.1 (http: / / mccarrolllab.com / dropseq / ). Briefly, cells were harvested with TRYPLE® and resuspended in PBS + 0.01% BSA at the concentration of IxlO5cells / ml. The single cell suspension was then diluted and loaded onto a co-flow microfluidic device (Nanoshift, Richmond, CA) together with barcoded beads (ChemGenes, Wilmington, MA, Cat. No. MACOSKO-2011-10) at 4mL / hr. The flow rate of droplet generation oil was set at 15mL / hr. Subsequently, droplets were broken and beads were isolated to generate cDNA libraries. Tagmentation was performed using Nextera XT DNA Library Preparation kit (Illumina, Cat. No. FC-131-1024) and size selection was done using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, Cat. No. A63881). Final sequencing libraries were analyzed on an Agilent High Sensitivity DNA bioanalyzer chip (Agilent Technologies, Santa Clara, CA) to determine average library size and final concentrations with a QUBIT® High Sensitivity DNA assay kit (Invitrogen via Thermo Fisher Scientific, Cat. No. Q32854). The final Drop-seq libraries were paired-end sequenced using the Nextseq500 / 550 High Output v2 75-cycle kit (Illumina, Cat. No. FC-404-2005) on a NextSeq500. scRNAseq data processing and analysisRA titration scRNAseq data processing and analysis at AFE

[0321] To generate demultiplexed count matrices from fastq files, cellranger multi was used with GRCh38 genome version 2020-A as reference. Using the scanpy interface, the sample_feature_bc_matrix.h5 for each concentration in a single replicate were concatenated. Default parameters were used for everything unless otherwise specified. Scrublet (Wolock et al., 2019) was run on each replicate to compute doublet scores and remove obvious doublets. Using scanpy’ s calculate qc metrics, basic QC metrics were calculated including percent ribosomal RNA, percent mitochondrial RNA, number of genes, and number of counts. An additional layer of filtering to remove empty droplets and dead cells included forcing the percent of mitochondrial RNA to be between 1 and 10, and the loglp(number of genes) to be greater than 7.5. This filtered 3,824 cells to 3,250 cells in replicate 1 and 3,554 to 2,971 cells in replicate 2. Replicates 1 and 2 were concatenated into a single scanpy object for further study. In order to avoid the effects of cell cycle, stress, number of genes, the total counts of all cells were normalized to have the same number of acceptable genes. Acceptable genes are defined as all genes aside from mitochondrial, ribosomal, and cell cycle genes. Cell cycle genes were obtained from previous work (Tirosh et al., 2016). The scanpy object was then loglp transformed and phases were computed using scanpy’ s score genes cell cycle function. Next, highly variable genes were computed and filtered to only acceptable genes. Pea, neighbors, umap, and leiden (with resolution 0.25) were computed. 3 cycles of removing nuisance effects from the embedding were carried out. The first removed effects due to replicates, the second removed cell cycle and count effects, and the third filtered doublets. To remove the batch effects, ridge regression was fit to the data using the scipy function Ridge (with fit_intercept=False, and alpha=l) with 1 hot encodings of the two replicates. This provided coefficients to compute approximations of true gene expression based on the explanatory variables. The gene expression due to the nuisance explanatory variables’ effects on gene expression (replicate) was subtracted from the gene expression matrix and pea, neighbors, umap, and leiden (resolution=0.25) were recomputed. This new embedding captured the variation due to RA, but also the variation due to cell cycle and count effects. Ridge regression was run again to get explanation of gene expression due to 1 hot encodings of phase, binned gene counts (4 bins representing quantiles), and leiden clustering. The variation due to phase and binned gene counts was subtracted from the transformed gene expression matrix and pea, neighbors, umap, and leiden clustering were run again to remove the effects of phase and gene counts from the embedding. In the new embedding, a small group of cells with high doublet scores containing cells from all concentrations was observed.At leiden clustering with resolution of 2 (resulting in 16 clusters), the median doublet score of each cluster was computed as the representative doublet score. Any cluster with representative doublet score more than 2 times the standard deviation of the representative doublet scores above the mean representative doublet score was considered to be doublets and removed (resulting in the removal of the 1 small cluster representing doublets). Pea, neighbors, umap, and leiden were recomputed. All subsequent analysis and figures were computed from this final object’s clustering and embedding.

[0322] Differential gene expression was performed over retinoic acid concentration using scanpy rank genes groups with method set to wilcoxon followed by filter rank gene groups with default parameters. Dotplots show the top 20 genes with smallest adjusted p value up to a maximum of 0.05. If a gene is upregulated in multiple groups, it is shown in the heatmap corresponding to the group with the lower p value. EnrichR analysis on the differentially upregulated genes in each condition was performed using gget (Luebbert and Pachter, 2022) with several databases. A list of GO terms was curated and the enrichR z-scores were read into anndata (Virshup et cd., 2021) to create a Scanpy (vl.8.2) object. Term scores defined as the standardized (subtracted by the minimum and divided by the maximum) enrichR z-scores were plotted using scanpy. pl. heatmap with standard_scale='var'. ). Cell cycle phase was computed similar to (Satija et al., 2015) using G2M and S phase gene lists from (Macosko et al., 2015) and scanpy. tl.score genes cell cycle function. Variable radii pie plots were made with custom code included in our repository. scRNAseq timecourse processing and quality control

[0323] Sequenced Drop-seq scRNAseq data were processed as described (Genga et al., 2019). Ensembl release 106 GRCh38 gtf and release 108 GRCh38 primary assembly genome were used for annotation and alignment. The gtf was filtered with Cellanger (v6.1.2) using the command cellranger mkgtf with allowed attributes for gene biotype being protein coding, IncRNA, antisense, IG LV gene, IG V gene, IG_V_pseudogene, IG D gene, IG_J_pseudogene, IG C gene, IG_C_pseudogene, TR V gene, TR_V_pseudogene, TR D gene, TR J gene, TR_J_pseudogene, and TR C gene. Transcript quantification was done using commands as in the table below with STAR aligner (v2.7.10), Picard tools (v2.26.10), and Drop-seq tools (v2.5.1).scRNAseq timecourse analysis

[0324] The Scanpy vl.9.1 python package (Wolf et cd.. 2018) was used for data analyses. The 9 experiments were integrated into an anndata object containing 38,525 hESC, 26,907 hDE, 22,251 hAFE, and 14,377 hPFE droplets, “scanpy. pp.filter_cells” was used to filter cells with less than 100 reads by setting the min counts parameter to 100.“scanpy. external. pp.scrublet” was used separately on each batch to filter potential doublets, “scanpy. pp.calculate qc metrics” with the loglp parameter set to True was used to get histograms of loglp n genes by counts to generate sample specific minimum loglp(gene count) cutoffs which were chosen as 7, 7, 8, 8, 8, 7.3, 8, 8 for hESC_repl, hESC_rep2, hESC_rep3, hDE repl, hDE_rep2, hAFE repl, hAFE_rep2 and hPFE respectively. Next, the mitochondrial contents of each sample were plotted. Additionally, an outlier population of 44 cells with low mitochondrial content (<=0.5%), which were likely empty cells, were removed from hESC repl. For all samples, an upper bound of 10% mitochondrial content was set as it is indicative of the cells being dead. This left 897 hESC cells, 339 hDE cells, 1481 hAFE cells, and 509 hPFE cells. The data were normalized using the “scanpy. pp.normalize total” followed by “scanpy. pp. loglp” functions. Feature selection was done using “scanpy. pp. highly variable genes” and mitochondrial, ribosomal, and cell cycle genes as previously defined (Tirosh et al., 2016) were excluded from the highly variable gene list. Dimensionality reduction was done with “scanpy. pp. pea” with “use highly variable” set to true and “n comps” set to 20. Batch integration was completed using“scanpy. external. pp. harmony integrate” and neighbors were computed with “scanpy. pp. neighbors” on the Harmony(Korsunsky etal., 2019) corrected PCA representation with default parameters. Leiden clustering and umap embeddings were obtained with the default parameters using “scanpy .tl.leiden” and “scanpy. tl. umap”. Two leiden clusters with less than 20 cells each contained a mix of cells from multipledifferentiation stages and were removed as they most likely represent noise. Highly variable genes, principal components, data integration, leiden and umap were recomputed.

[0325] In order to compute pseudo time on the AFE-PFE cells, the AnnData object was subset to cells within the AFE or PFE experiments. Highly variable genes were computed using scanpy’s “highly_variable_genes” function with “n_top_genes” set to 1000. Mitochondrial, ribosomal, and cell cycle genes were removed from the top genes selected. Phase effect was removed using combat and, 150 principal components were computed, and harmony was run on the principal components to remove effects from experiment. Neighbors was run on the harmony integrated pea with the method set to “gauss” and n neighbors set to 10. Diffmap was run with 15 components. In order to remove components that corresponded to count effects, any embedding dimension which has a pearsonsr > 0.5 with pval < 0.01 was removed, calculated using scipy. stats’s pearsonr function. A neighborhood graph was recomputed on the diffmap using neighbors with n neighbors = 10 and method- ’gauss”. To compute pseudo times, Wanderlust (Bendall etal., 2014) was run using scanpy. external. tl.wishbone (Setty et al., 2016). It run on the embedding from diffusion map components 0 and 1 and the starting cell was set to the cell with highest value for diffusion map component 1.

[0326] Pseudo time heatmaps were made by sorting the cells by pseudo time and averaging the gene’s normalized expression value with a window size of 100 cells. The leiden probabilities correspond to the number of cells within that leiden cluster divided by 100. The leiden probability confidence intervals were obtained using the 1’st and 99’th percentiles after bootstrapping sets of cells for 1000 iterations.Mouse embryonic development atlasEarly endoderm data processing

[0327] Early endoderm data (Nowotschin et al., 2019) were downloaded as FASTQ files from GEO (accession: GSE123046). Filtered count matrices were produced using 10X Cellranger v3.0.1 with reference version mml0-3.0.0. Chemistry was specified as SC3Pv2 and otherwise default parameters were used. Software and reference were downloaded from https: / / support.10xgenomics.com / single-cell-gene-expression / software / downloads. Cell metadata and the h5ad file containing cells were downloaded from https: / / endoderm-explorer.com / . 6 samples with only yolk sac and parietal endoderm were omitted, but samples including visceral endoderm were retained.

[0328] Cellranger filtered count matrices comprising 122,520 cells and 31,053 transcripts across all the samples from embryonic day (E) 3.5 to 8.75 were processed with scanpy (vl.6.0). 30885 cells excluded from the analysis in the original publication (according to the h5ad file) were removed. Non-endodermal cells annotated as 'Midline', 'Mes', 'Endothelial', 'exVE', 'ParE', 'YsE', 'ExE', 'Blood', 'TE' or 'GermCells' in the original manuscript were also removed, leaving 42856 cells. Parietal endoderm clusters labeled as 'E3.5 :0', 'E4.5:0', 'E4.5 : 1', and 'E3.5 :4', visceral endoderm annotated as 'emVE and 'VE' and an E7.5 cluster ‘E7.5: 13’ annotated as Epiblast in the original manuscript were removed. Finally extra-embryonic derived endoderm from GFP+ samples Libl l-2_E8.5_GFP+’, ‘Libi 1-1 E8.5 GFP+’, ‘Lib5- 3 E7.5 GFP+', 'Lib5-2_E7.5_GFP+' and 'Lib5-1_E7.5_GFP+' were removed leaving 22850 cells.Pharyngeal endoderm atlas and data integration

[0329] The 54,044 cells from our previously described pharyngeal endoderm atlas (Magaletta et cd., 2022) from were integrated with the early endoderm atlas (Nowotschin et al., 2019). Briefly, the pharyngeal endoderm dataset was concatenated with the early embryo and endoderm dataset based on the Ensembl IDs, and transcripts missing from either dataset were omitted. 5,608 genes expressed in less than 3 cells were filtered out for a final object with 76,894 cells and 21,906 genes. Cells were annotated using the original publications. For the pharyngeal endoderm dataset, Cluster 0 to 27 (from Supplementary Figure 3a in Magaletta et al., 2022) were labeled as CO to C27 with prefixes for cell type annotations (from Figure 1c in Magaletta et al., 2022) for Thyroid, Respiratory, Pouchl, Pouch2, UBB, Parathyroid, Esophagus and Oropharynx, Ctec and Mtec. Cluster 9 was annotated as Thymic progenitors. The remainder of the immature pharynx populations (from Figure 1c in Magaletta et al., 2022) were annotated as Early, Mid and Late based on whether they were dominated by cells from embryonic day 9.5, 10.5 or 11.5 respectively. For the early embryo and endoderm dataset (Nowotschin et al. , 2019), annotations from the CellType metadata field were prefixed with a ‘P:’. Further, clusters E3.5: l, E3.5:3 were called ‘P:ICM’, denoting inner cell mass, and clusters ‘E3.5:2', 'E4.5:2’ were called P:Epi, denoting Epiblast cells, in accordance with the manuscript (Nowotschin et al., 2019).

[0330] Downstream processing was performed in Scanpy. The raw UMI counts were normalized using scanpy normalize total with total counts = 10000 and the resulting log- transformed pseudo-counts were obtained using the scanpy log Ip function. For feature selection, ribosomal (Rps and Rpl), mitochondrial(mt-), mitochondrial-ribosomal (Mtrpl and Mtrps) transcripts were excluded. Genes associated with cell cycle (from Table S8 in Park et al., 2020) were also excluded and the highly variable genes were obtained using highly variable genes with min_mean=0.0125, max_mean=3, and min_disp=0.5. The top 1000 principal components were computed on this set of highly variable genes using the pea function. Harmony (Nowotschin et al. , 2019) was used to obtain an augmented affinity matrix with additional connections between consecutive time-points. 100 principal components were used, and random number generators were seeded for repeatability (np. random. seed(O)). The affinity matrix was used as input for a force-directed graph layout using ‘force directed layout’ from harmony. Finally this was plotted with scanpy. pl. embedding.

[0331] To obtain the normalized expression of transcripts for downstream analysis and classification, size factors were computed on raw UMI counts using compute SumFactors in the Scran package (Lun et al., 2016) with min. mean = 0.1 and clusters from the “Clusters” and “louvain” metadata in (Nowotschin et al. , 2019) and (Magaletta et al. , 2022) respectively. Counts were normalized using these size factors and log-transformed with scanpy log Ip function. These normalized counts were used to plot the expression of markers from literature and for the classification.CellMatch classification tool

[0332] Input for CellMatch consists of 1) a reference single-cell RNA sequencing (scRNAseq) dataset with cell type annotations, and 2) a query scRNAseq dataset with unannotated clusters. CellMatch works by averaging data within clusters; selecting a set of informative genes from reference data; pairing clusters using a robust penalty function (correlation distance); and creating graphics to assess alternate models and explain individual matches. These steps are described in greater detail below.Gene selection

[0333] CellMatch is designed to be used after selection of informative genes (2,000 by default). Gene selection for CellMatch is done via a simple overdispersion estimates, implemented in the SelectlnformativeGenes function. For the reference dataset X, SelectlnformativeGenes removes genes below a cutoff (default: 1). A pseudocount (default: 0.01) is added and the mean, and the coefficient of variation (CV) is computed for each gene. A nonparametric regression is fitted to predict CV based on mean expression (via gam from the R package mgcv). Overdispersion is measured as the CV minus the predicted CV. By default, the top 2000 genes are retained, but for less complex datasets, better results may be obtained by selecting fewer genes. The default pseudocount and expression cutoff are intended for use when RNA quantities are measured in counts per million, and in other situations they may need to be adjusted. In lieu of this step, CellMatch also accepts user- specified genesets.Model selection

[0334] At the core of CellMatch is the concept of a matching model. Consider a set of reference RNA profiles represented as a matrix X e ]RR><C', where R is the number of profiles and G is the number of transcripts measured. Typically, this is a normalized expression matrix whose values are measured in counts per million, and there will be one profile per cell type, computed by averaging cells. Consider also a matrix of Q query profiles Y e ]R<3><G, measured on the same genes. Let the query profiles be named QltQ2, ... and the reference profiles be named d R1, R2< ■■■ Then a matching model M is a set of Q ordered pairs■ ■■ that associates each query profile with a reference profile. A matching model can also be represented as a pair of matrices Y,Z where Y is the original query matrix and Z contains the corresponding columns of the reference XM1,XMz, ...XM(Z has the same dimensions as Y, and Z may contain repeated columns.)

[0335] In CellMatch, user-specified matching models can be evaluated using a carefully selected metric for goodness of fit. By directly optimizing this metric, CellMatch can also select a matching model. The default metric is the correlation distancewhere p(Zg, Yg) is the Pearson correlation between the query and the corresponding reference values and G is the set of genes selected. (In a slight abuse of notation, subscripting heredenotes rows (genes) even though it previously denoted columns (profiles)). The motivation for the factor of 2 comes from the Law of Cosines, but using a multiple of this distance instead of the distance itself would not affect the results. The sum ranges over a set of genes that can be user-defined or selected using a built-in utility function, which is described above.

[0336] This metric was selected due to its strong empirical performance on pilot tests unrelated to this manuscript, but we note it also has two appealing robustness properties. First, it is invariant to gene-specific changes in location and scale: pa + bYg). Biologically, this allows any gene to be globally higher expressed (or more easily detected) in either dataset, without altering the results. Second, the maximum penalty per gene is capped at 2, so results are based on consensus of many genes, and they cannot be arbitrarily affected by a small number of "corrupted" genes that do not behave as expected.

[0337] CellMatch also offers other penalty functions, including the following, which are compared against the default correlation distance on the cross-species brain benchmark. A subscript k denotes all expression values for a given cluster, K denotes the set of clusters, the Pearson correlation is denoted p, and the Spearman rank correlation is denoted ps.

[0338] Sample-to-sample correlation distance:

[0339] Sample-to-sample rank-based correlation distance:

[0340] Absolute deviation:

[0341] In CellMatch, models are selected by minimizing the selected penalty via a simple coordinate descent routine. Five random initializations are used, and the lowest-distance matching model among the results is retained. Most parameters, including the distance function itself, can be overridden by the user, but it is strongly suggested to try the default settings first.Transparency and model comparison

[0342] CellMatch aims not only to classify cells, but also to summarize uncertainty and evidence for each model. Given a matching model, CellMatch provides two types of output for this purpose. For the first type of output, CellMatch compares the selected model to all neighboring models in terms of the penalty function of choice. The resulting heatmaps provide an intuitive picture of which alternative matching models are also supported by thedata. These features are implemented in the functions MutateModel and PlotNeighboringModels.

[0343] In the second type of output, CellMatch heatmaps a set of “shared specific genes.” By default, a gene is included when it has a two-fold increase in any query cluster relative to the rest and a two-fold increase in the corresponding reference cluster relative to the rest. To show deficiencies of the matching model, remaining genes (any gene present in the selection step but not in the “shared specific” list) are grouped by expression pattern and presented to the user via additional heatmaps. These features are implemented in the function EvaluateByGene. In addition to heatmaps, output from CellMatch includes tables of genes for each cluster, allowing users to evaluate matches by looking for well-known genes or using annotation tools such as Enrichr (Chen et aL, 2013; Xie et al., 2021). Since a query cluster might not be present in the reference, CellMatch optionally warns users when the number of genes supporting a reference - query match is less than one percent of the size of the gene list used for classification. Users are encouraged to inspect gene lists to ensure that the results of CellMatch are biologically meaningful.Benchmark on PBMC data

[0344] PBMC evaluations were run using training and test sets that were previously published (Abdelaal etal., 2019). This included PBMC datasets generated across seven scRNAseq technologies and manually annotated using consistent nomenclature. Training and test datasets for cross-platform comparisons follow as published example (see Fig. 3 in Abdelaal et al., 2019): in particular, when datasets were split (e.g. “PbmcBench pbmcl, 10Xv2b” and “PbmcBench pbmc2.10Xv2b”), the same splits were used. CellMatch was run with default parameters: counts per million were averaged within each cluster; 2000 highly-variable genes were selected; and clusters were matched by optimizing the correlation distance summed over all 2000 selected genes. Test set cells were aggregated by the same labels used as the ground truth. Five random initializations were used for coordinate descent. Correct matches and incorrect matches are shown by technology and by cell type. Matches that were impossible (cell type was not present in either dataset) or not attempted (within- platform comparisons) are shown as N / A.Benchmark on Brain atlas data

[0345] Brain atlas evaluations were run using the data and evaluation framework downloaded from Abdelaal et al., 2019. The evaluation included all pairs of datasets from Hodge et al. (human MTG) (Hodge et al. , 2019) and Tasic et al. (mouse VI and ALM) (Tasic etal., 2016) (Hodge etal., 2019) plus a comparison using both mouse datasets as a combined reference. Their chosen metric, Fl score, was used as the measure of performance. Performance of SingleCellNet, ACTINN, and SVM was transcribed directly (Fig. 4b from Abdelaal et al., 2019).

[0346] For results labeled cellmatch, test set cells were aggregated by the same labels used as the ground truth, but for results labeled cellmatch reclustered, test set cells were aggregated by unsupervised clustering. Unsupervised clustering was carried out on dataset separately using the R package Seurat (3.1.1) (Stuart et al., 2019). Default parameters were used except as described: briefly, data were scaled to 10,000 counts per cell and log- transformed with a pseudocount of 1. Informative genes were selected via FindVariableFeatures and centered / scaled with ScaleData. PCA was run on the scaled data and principal components with eigenvalues exceeding the Marchenko-Pastur upper bound were retained. Retained components were used as input for UMAP (uniform manifold approximation and projection), shared-nearest-neighbor graph construction, and Leiden clustering (with resolution parameter 1).

[0347] CellMatch was run using default parameters (2000 genes, 5 random initializations), but final cell classifications were made using a model averaging strategy. For model averaging, each cell was classified with 50% probability according to either the optimal model or another high-scoring model. This alternate model was obtained from the function GetReasonableAlternative, which begins with the selected model and, for each query cluster, searches over all reference clusters to find the one producing the second-best correlation distance. This second-best match is returned unless it is “substantially worse,” in which case the best match is returned. “Substantially worse” is defined as follows. The relative difference between the best and second-best scores: g =Sbest Ssecondwas computed sbest(note that this quantity is non-positive, and higher values (less negative) indicate higher- quality second-best matches). For any coordinate where g is above its mean, GetReasonableAlternative returns the second best match; otherwise, the best match is returned. In formulas, GetReasonableAlternative returns the second best match if:Ej e Q 9j 9iIQIwhere i is a query cluster and Q is the set of all query clusters.

[0348] Example output showing model comparison, supporting genes, and distribution of the correlation distance was generated using the human MTG data as the query and the mouse VI data as the reference. The histogram in FIG. 6F uses default cellmatch output, which includes correlation distance contributions for each gene given the selected model.Classification with CellMatch on hESC differentiation time course

[0349] The human in-vitro stem cell differentiation scRNA raw counts on the quality- controlled cells obtained as described earlier were read into scanpy and a standard processing workflow was followed. 2741 transcripts having no counts in any cell were excluded. Counts per million (CPM) normalized pseudo-counts were log transformed using normalize_per_cell and log Ip functions. The top 30 principal components (pea, random_state=0, svd solver- arpack') were used to obtain a nearest neighbor graph and perform Louvain clustering at resolution = 0.5 with the top 2000 variable genes. Size factors were computed using compute SumF actors in the scran package (Lun et al.. 2016) with min. mean = 0.1 and using the Louvain clusters obtained above. Raw UMI counts were normalized using these size factors and log transformed.

[0350] In order to obtain a consensus set of genes between human and mouse, the set of Orthologous Mouse Genes were downloaded from Ensembl (Cunningham et al., 2022) biomart with human genome version GRCh38.pl3. The orthologs files used have been shared in our code repository. In case of duplicate mouse or human ortholog matches, only the first was retained and the subset of transcripts expressed in our mouse integrated atlas and human differentiation was retained. The scran normalized counts obtained as described above for human and mouse were input to CellMatch.

[0351] CellMatch was run with number of genes = 2000, number of initializations = 10 and 20 iterations for gradient descent. Clusters for the mouse atlas were used as defined above. For the human stem cell differentiation, clusters were defined as described earlier.

[0352] For the display of CellMatch results on the mouse endoderm atlas, clusters with no stem cell equivalents are colored gray. Clusters with stem cell equivalents are colored according to the stage at which their equivalents are present. Clusters with more than one stem cell equivalent, have a fraction of their cells randomly chosen and colored by each equivalent.

[0353] The standard CellMatch output representing the transcripts responsible for classification are visualized on the stem cell differentiation time course with their correlation scores. The transcripts responsible for classification to each of the 4 stages, namely Epiblast, Definitive Endoderm, Early PFE (Cl 6) and Late PFE (CO) were scored in Scanpy using the score genes function and visualized on the mouse atlas embedding.Bulk RNA-SeqLibrary preparation

[0354] Total RNA was extracted from differentiation intermediates using TRIZOL® Reagent (Thermo Fisher Scientific, Cat. No. 15596018) following the manufacturer’s instructions. Strand-specific RNA-seq libraries were generated utilizing dUTP second-strand marking following the published protocol (Z. Zhang et al., 2012). Briefly, 4 pg of total RNA from each sample was depleted of ribosomal RNA using the RIBO-ZERO® rRNA Removal Kit (Illumina, Cat. No. MRZH116). rRNA-depleted samples were purified with the RNA Clean and Concentrator-5 Kit (Zymo Research, Irvine, CA, Cat. No. R1015) and treated with TURBO™ DNase (Ambion via Thermo Fisher Scientific, Waltham, MA, Cat. No. AM2239) to remove contaminating DNA. RNA samples were randomly fragmented in 5x first strand buffer at 94C and then reversed transcribed into cDNA using SUPERSCRIPT® III Reverse Transcriptase (Invitrogen via Thermo Fisher Scientific, Cat. No. 18080093). dUTP (BioBasic, Amherst, NY, Cat. No. DM1244) was incorporated during second-strand cDNA synthesis and subsequently degraded with uracil-DNA glycosylase (UDG) (New England Biolabs, Ipswich, MA, Cat. No. M0280S) during library preparation to preserve strand information. Final strand-specific RNA-seq libraries were quantified using the QUBIT® High Sensitivity DNA kit (Invitrogen via Thermo Fisher Scientific, Cat. No. Q32854) and the size distribution of each library was determined through bioanalysis with an Agilent High Sensitivity DNA chip. Paired end sequencing was performed on the final libraries with Hi- Seq2000.RNA-seq data processing and quality control

[0355] RNA-seq sequencing data were processed using the ENCODE RNA-seq pipeline version 1.2.3 (https: / / github.com / ENCODE-DCC / rna-seq-pipeline) using default settings with the GRCh38 V29 reference genome. Standard ENCODE RNA-seq reference files wereused as found at https: / / github.eom / ENCODE-DCC / rna-seq- pipeline / blob / 1.2.3 / docs / reference.md#genome-reference-files.

[0356] RSEM (Li and Dewey, 2011) counts from the ENCODE pipeline were loaded into R using the tximeta package {Love:2020em}. A DeSeq2 (Love et al., 2014) object was constructed from the matrix of counts, timepoint and replicate information. Genes with at least 3 counts in at least 2 samples were retained. The matrix was rlog transformed to estimate the between sample correlation using the dist function in R. Clustering and visualization were done using the pheatmap package in R. Principal component analysis was performed using the plotPCA function from the DeSeq2 package on the rlog normalized counts.RNA-seq differentiatial gene expression analysis

[0357] Differential gene expression analysis was performed using the default DeSeq2 workflow with the DeSeq function. The results for the pairwise differential gene expression analysis were obtained using the results function from DeSeq2 between the 6 possible contrasts: DE and ES, AFE and DE, AFE and ES, PFE and AFE, PFE and DE and DE and ES. The significance cutoff for independent filtering of genes was set at 0.1. For every pairwise contrast, genes with log2-foldchange > 0.5 and the adjusted p-value < 0.1 were retained leaving 6161 genes across the 6 contrasts. Transcripts were annotated using the EnsDb.Hsapiens.v86 database from the ensembldb package (Rainer etal., 2019). The rlog normalized counts were centered, scaled and visualized using the ComplexHeatmap package(Gu et al., 2016) in R. Unsupervised k-means clustering was performed on the genes to obtain 10 gene clusters by setting row km = 10 in the Heatmap function. EnrichR analysis on the 10 gene clusters was performed using gget (Luebbert and Pachter, 2022) with several databases. A list of GO terms was curated and the enrichR z-scores were read into anndata (Virshup et al., 2021) to create a Scanpy (vl.8.2) object. Term scores defined as the standardized (subtracted by the minimum and divided by the maximum) enrichR z-scores were plotted using scanpy. pl. heatmap with standard_scale='var'.

[0358] The rlog normalized and scaled gene expression was averaged across the replicates and visualized in R for a curated list of markers with the Heatmap function from the ComplexHeatmap package (Gu et al., 2016) with cluster_rows=TRUE to group the transcripts based on their expression pattern across the 4 stages The results of the pairwisedifferential expression were visualized with volcano plots using the enhanced volcano package (https: / / github.com / kevinblighe / EnhancedVolcano) with adjusted pvalue cutoff = 0.01 and log2foldchange cutoff = 1.ATAC-seqATAC-seq library preparation and sequencing

[0359] Samples were processed as described before (Genga et al., 2019). Briefly, 50,000 cells were pelleted at 750 x g for 15 minutes at 4°C. Cells were lysed with cold lysis buffer (10 mM Tris-Cl pH 7.4, 10 mM NaCl, 3 mM MgC12, and 0.1% IGEPAL CA-630).Immediately following, nuclei were pelleted at 750 x g for 15 minutes at 4°C. The supernatant was discarded, and transposition reaction mix was added. The transposase reaction was performed at 37°C for 30 minutes. Following purification, the tagmented DNA was amplified for 9-10 cycles. To perform size selection, 0.55X volume of AMPure XP beads (Beckman Coulter, Cat. No. A63881) were added and incubated for 5 minutes at room temperature. The supernatant was taken to a new tube and IX volume of AMPure beads was added and incubated for 5 minutes at room temperature. The beads were washed twice with 75% ethanol and air-dried. The final library was eluted EB buffer. Sequencing was performed on an Illumina NextSeq 500 platform using the paired-end, 75-75 configuration.ATAC-seq data processing and analysis

[0360] ATAC-seq sequencing data were processed using the ENCODE ATAC-seq pipeline version 1.9.1 (https: / / github.com / ENCODE-DCC / atac-seq-pipeline) using default settings with the hg38 genome. Standard ENCODE ATAC-seq reference files were used as found in https: / / storage.googleapis.com / encode-pipeline-genome- data / genome_tsv / v4 / hg38. tsv .

[0361] Narrowpeaks from each replicate of ESC, DE, AFE, and PFE replicate 1 output by the Encode pipeline were filtered by excluding peaks with signal <= 3 or -log 10 qvalue <= 3. These were read into R and the standard DiffBind (Ross-Innes et al., 2012) workflow was followed. In brief, per time point consensus intervals were obtained by merging overlapping peaks in both replicates. Peaks with no overlap across replicates (where applicable) were excluded. Further a single set of unique genomic intervals across the entire dataset (C) was obtained by merging the per time point consensus interval sets. The Venn diagram for C wasplotted using the dba.plotVenn function in diffbind. The dba.count function was used to obtain the final consensus set of peaks (P) and the count matrix across the time course for each peak in P. In brief, summits were obtained for each interval in C based on the read pileups at each base for each sample the peak was called in. Peaks intervals were respecified to the default 200bp + / - the summit. Finally, reads overlapping the intervals were counted. Peaks having RPKM <1 in all the samples were excluded leaving 202,708 peaks with FrIP = 0.15, 0.23, 0.16, 0.30, 0.32, 0.22 and 0.14 across hESC repl, hESC_rep2, hDE repl, hDE_rep2, hAFE repl, hAFE_rep2 and hPFE repl respectively. The correlation and PCA plots were plotted with DiffBind functions plot and plotPCA respectively on the count matrix. Peaks were annotated using annotatePeaks.pl from Homer (Heinz et cd.. 2010) with hg38 and the default parameters. EdgeR trimmed mean of M values (TMM) normalization was performed using dba.normalize in diffbind with normalize=DBA_NORM_TMM and the count matrix (counts tmm) was extracted with dba.peakset. Quantile normalization was performed in R using normalize. quantiles from preprocessCore (https: / / github.com / bmbolstad / preprocessCore, preprocessCore: A collection of preprocessing functions. R package version 1.60.0). Resulting quantile normalized TMM counts were averaged across replicates, where applicable to obtain “counts tmm norm”. To obtain peaks that have varying signal across the differentiation time course, the coefficient of variation (CV) was defined for each peak as the ratio of standard deviation to mean on counts tmm norm. Peaks with CV greater than one standard deviation above the median CV were retained in a variable peak set (peak var) comprising 40,163 peaks.“Counts tmm norm” was visualized on the “peak var” set of peaks using the ComplexHeatmap package (Gu etal., 2016) in R. Unsupervised k-means clustering was performed on the peaks. The number of clusters=4 was set using the elbow plot of the total within clusters sum of squares to the number of clusters. This was obtained using the kmeans function in R with number of clusters (or centers) varying from 1 to 50, iter.max = 15 and nstart = 50.

[0362] The consensus set of peaks were scanned for motif footprints using gimmemotifs (Bruse and van Heeringen, 2018; van Heeringen and Veenstra, 2011) scan on hg38 with the options -b, -gc and -z which calculates the z-score normalized logodds motif binding score using a background with similar GC content. Further, the per sample motif footprint differential enrichment was computed on the peak var subset using maelstrom fromgimmemotifs on the time point averaged quantile and TMM normalized scores obtained above with the -no-filter option.Evolutionary conservation

[0363] PhastCon scores (Siepel et al., 2005) for 100 vertebrates were downloaded from http: / / hgdownload.cse.ucsc.edu / goldenpath / hg38 / phastConsl00way / . The evolutionary conservation score of a peak was considered as the average signal of hg38.phastConsl00way.bw over the peak using bigWigAverageOverBed. The background peak set was made using bedtools random with default parameters and the ATAC peaks were obtained from the variable peak set (peak var) defined above. ENCODE representative DNAse hypersensitivity site peak set was downloaded from https: / / www.encodeproject.org / files / ENCFF088UEJ / and used as the reference. Peaks were annotated using annotatePeaks.pl from Homer(Heinz etal., 2010) with hg38 and the default parameters. Proximal regions were defined as promoter-TSS labeled peaks. Peaks not in promoter-TSS, exon, intron, TTS, 3’ or 5’ were annotated as distal.

[0364] To look at the base conservation rate bedtools (A. R. Quinlan and Hall, 2010) and pyBigWig from deepTools2 (Ramirez et al., 2016) were used, bedtools slop -n 10000 was used on the consensus set of ATAC peaks and the background peak set. For each base, the conservation across all peaks using pyBigWig was averaged and plotted where the average nucleotide conservation as a function of distance from the peak center.ChlP-seqChlP-seq library preparation and sequencing

[0365] Approximately 3-5 million cells were used for each histone ChlP-seq experiment. Cells were cross-linked with 1% formaldehyde for 10 minutes, followed by quenching with 125mM glycine for 4-5 minutes and washed for twice with PBS. The cell pellet was lysed in cell lysis buffer (20mM Tris-HCl, pH8, 85mM KC1, 0.5% NP-40) supplemented with lx protease inhibitor cocktail (Roche Diagnostics, Indeanapolis, IN, Cat. No. 11836170001) on ice for 20 minutes then pelleted at 5000rpm for 10 minutes. The pellet was resuspended in sonication buffer (lOmM Tris pH7.5, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS) with lx protease inhibitor cocktail and incubated for 10 minutes at 4oC. In order to achieve 200-700nt DNA fragmentation range, nuclei were sonicated (Bronson sonifier model 250with the following conditions: Amplitude = 15%, Time interval = 3 min (total of 8-12min) and pulse ON / OFF = 0.7sec / 1.3sec). Chromatin was pre-cleared with DYNABEADS® Protein A (Invitrogen via Thermo Fisher Scientific, Cat. No. 10002D) for 1 hour and incubated with antibody overnight at 4C. Antibodies included: anti-H3K27ac (5 pg; Diagenode, Denville, NJ, Cat. No. Cl 5410196), anti-H3K27me3 (5 pg; Sigma- Aldrich via MilliporeSigma, Cat. No. 07-449) and anti-H3K4me2 (5 pg; Active motif 39141). On the following day, 30-40pL of DYNABEADS® Protein A were added to the chromatin and antibody mix for 2-3 hours and then the captured immuno-complexes were washed as follows - lx low-salt buffer, lx High-salt buffer, lx LiCl salt buffer and lx TE. And the washed immuno-complexes were eluted in ChlP-DNA elution buffer (lOmM TriHcl pH8, lOOmM NaCl, 20mM EDTA and 1% SDS) for 20 minutes, reverse cross-linked overnight at 65C, followed by proteinase K treatment (Invitrogen via Thermo Fisher Scientific, Cat. No. 25530- 049), RNase A (Thermo Fisher Scientific, Cat. No. ENO531) treatment andPhenol: Chloroform: isoamyl alcohol extraction. The Illumina library construction steps were carried out with 5-1 Ong of purified ChlP’ed DNA. During the library construction, PCR purification was performed after every step using a QIAquick PCR purification kit (Qiagen, Germantown, MD, Cat. No. 28104) or QIAquick gel extraction kit (Qiagen, Cat. No. 28706). The library reaction steps with ChlP’ed DNA as follows: 1.) End-repair - T4 PNK (New England Biolabs, Cat. No. M0201L), KI enow fragment (New England Biolabs, Cat. No. M0210S), T4 DNA Polymerase (New England Biolabs, Cat. No. M0203L) and dNTP mix (Thermo Fisher Scientific, Cat. No. 18427013) incubated at RT for 45min, 2.) 3’ end A-base addition - 3’ -> 5’ exo- Klenow fragment (New England Biolabs, Cat. No. M0212L) and dATP (New England Biolabs, Cat. No. N0440S) incubated at 37°C for 30min, 3.) Adapter ligation - T4 DNA ligase (New England Biolabs, Cat. No. M0202M) and Illumina adapters incubated at 16°C overnight and 4.) PCR amplification - 2x PfU Ultra Hotstart master mix (Agilent Technologies, Cat. No. 600850-51) for 10-14 cycles. The amplified libraries were size selected for 200-450nt on 2% Agarose E-gel (Thermo Fisher Scientific, Cat. No. G402002). Sequencing was performed on Illumina NextSeq 500 or Hi-Seq2000.ChlP-seq data processing and quality control

[0366] ChlP-seq sequencing data were processed using the ENCODE ChlP-seq pipeline version 1.6.1 (https: / / github.com / ENCODEDCC / chip-seq-pipeline2) using default settingswith the hg38 genome. Standard ENCODE ChlP-seq reference files were used as found in https: / / storage.googleapis.com / encode-pipeline-genome-data / genome_tsv / v4 / hg38.tsv. The peak caller option was set to MACS2 for all data.

[0367] All peaks and bam files were read into R with the standard DiffBind (Ross-Innes et al., 2012) workflow similar to the ATAC-seq processing described above to obtain the sample correlation plot.ChlP-seq downstream analysis

[0368] For each histone mark, the peaks and bam files were read into R with the standard DiffBind (Ross-Innes etal., 2012) workflow described above for ATAC-seq processing and a count matrix was obtained. The consensus ATAC peakset (202,708 peaks) was imported and TMM normalized counts for the histone data in the ATAC-seq peak regions were obtained with dba. count with options summits=0, score=DBA_NORM_TMM. These were quantile normalized and averaged between replicates like in the ATAC-seq processing workflow and sites with any signal in the ATAC peak var set were retained for further processing. Per sample motif footprint differential enrichment was then computed using maelstrom from gimmemotifs with the -no-filter option.Signal track generation

[0369] Bigwigs from the Encode pipeline were used for samples with a single replicate. In case of samples with two replicates, bigwigCompare from deepTools(Ramirez et al., 2016) was used to take the mean over the per sample bigwigs output by encode with options — operation mean, — skipZeroOverZero, — outFileFormat bigwig, — numberofProcessors max.Data integration

[0370] The aggregated z-score (Zagg) from the combination of methods output by gimme maelstrom for ATAC and histone marks (H3K4me2, H3K27ac, H3K27me3) in ATAC variable peak regions was standardized (subtracted by the minimum and divided each by its maximum) and visualized. Replicate averaged TPM normalized and standardized RNA expression obtained as described above was also plotted. TFs were split into PSC, DE, AFE or PFE drivers based on the time point at which they had maximum Zagg.Matching motifs to Transcription Factors (TFs)

[0371] The default motifs were obtained from gimmemotifs using the default_motifs() function. For each, the predicted direct and indirect transcription factors were obtained using custom code modified from CellOracle (Kamimoto etal., 2020) thus obtaining a set of motif- TF pairs. TFs from the set of 6161 variable transcripts in Fig 4 A were retained thus keeping only transcripts varying along the differentiation. TFs were matched to their family by excluding special and terminal numeric characters. Pearson correlation (atac corr) between TPM normalized TF expression and Zagg for every motif-TF pair within the same family was computed using pearsonr from the stats in python. Similarly, correlation was computed between Zagg for each of the three histone marks and the TPM normalized TF expression obtaining h3k27ac_corr, h3k4me2_corr and h3k27me3_corr. Motif-TF pairs with atac corr <= 0.7 were excluded. Additionally, motif-TF pairs with h3k27ac_corr, h3k4me2_corr or h3k27me3_corr <= 0.5 were excluded. Additionally, pairs were excluded if they had aggregated z-scores (Zagg) <= 2 at all the time points. For each TF, the motif(s) with the maximum atac corr were kept. The TF variability defined as the ratio of standard deviation to mean across the 4 samples was computed and the top 2 TFs (based on TF variability) for each motif were displayed.Gene regulatory network analysis

[0372] To infer the GRN, the consensus set of AT AC peaks, scanned for footprints as described earlier, was used. Transcription start sites (TSS) overlapping any ATAC peak were determined. Genes without ATAC signal at the TSS are considered off and ignored. Any ATAC peak from the consensus set within 100,000 nucleotides of a peak overlapping the TSS was considered a putative regulatory region for that gene.

[0373] Motifs were associated with transcription factors using the Pearson correlation between every pair within the TF family as described above. However, unlike above, any motif with a positive correlation between Zagg on ATAC and its associated TF is considered a binding site for that TF and no additional exclusion criteria were applied. Any transcription factors or their downstream genes that are not up-regulated in some sample compared (as defined as having an adjusted p-value < 0.1 and log fold change > 0.5 from differential analysis on RNA-seq described earlier) to some other sample are excluded for all analysis. The expression of RNA in a cell type is considered loglp(TPM) averaged over replicates.

[0374] To identify the importance of each TF in each cell type, PageRank was used. To get the weight associated with the link between the TF and downstream gene, 2 factors were used: uncertainty in the association and RNA amount. To get the factor associated with uncertainty, the uncertainty in the association between the motif and TF and the motif and target was examined. Equal probability that a gene is regulated by each of its putative regulatory motifs was assigned. It was also assigned equal probability that a motif is bound by each of the potential TFs. To get the total uncertainty weight for a TF target interaction, the probability that is equal to randomly selecting a putative motif as a regulator for the gene followed by randomly selecting a TF associated with that motif was used. To get the weight associated with RNA, the RNA of the TF was multiplied with the target. To get the total directed edge weight from a target to a TF, the uncertainty weight was multiplied with the RNA weight.

[0375] This graph was constructed using networkx (Hagberg et cd.. 2008). “networkx.pagerank” was called to predict which transcription factors have the largest effect on overall RNA amounts in each cell type with default parameters on the weighted digraph. The personalization factor of a gene was given by exp(z-scored over cell types(expression of RNA)).

[0376] The hPFE adjacency graph was obtained from the TF source-target gene interactions as described above. For the GRN visualization, only the top 40 TF drivers of PFE obtained from the PageRank score analysis described earlier were considered and edges with weight > 10 were retained. A directed graph was built with igraph (Csardi and Nepusz, 2006) using graph. data. frame with directed=TRUE. The Fruchterman-Reingold layout to visualize the graph was obtained using layout with fr. This directed graph was plotted with ggplot with nodes colored by their PageRank score.Organoid Generation

[0377] Recellularized mouse thymic lobes are used to generate organoids. A murine thymic lobe is harvested from a 4-6 week old ICR mouse and the lobe is decellularized by 3X freeze thaw cycles followed by incubation with 0.1% sodium dodecyl sulfate detergent until lobes appear translucent. Lobes are washed three time with PBS 1% triton X-100. Lobes are then washed three times in PBS with pen / strep. TEP cells are dissociated to single cells and pelleted to achieve a cell slurry at about 2.5E+05 cells / pL. Decellularized lobes are placed ona 6-well plate cell culture insert filter with B27 differentiation media supplemented with A83- 01 and Y-27623. A volume of IpL of cell slurry is injected into interior of the decellularized lobe. The 6-well plate is placed in a humidified container and incubated at 37°C with 5% CO2 for 17 days, replenishing fresh media every 3-4 days. Organoids are also generated by droplet thymic organ culture. TEP cells are dissociated to single cells and pelleted to achieve a cell slurry at about 2.5E+05 cells / pL. A 6-well culture plate is prepared with cell culture insert filter with B27 differentiation media supplemented with A83-01 and Y-27623. Droplets IpL in volume are added to cell culture filter inserts to initiate TEP TOC culture. The 6-well plate is placed in a humidified container and incubated at 37 degrees with 5% CO2 for 17 days, replenishing fresh media every 3-4 days.EXAMPLE 2Retinoic acid induces human anterior foregut endoderm patterning in a dose- and timingdependent manner

[0378] To generate pharyngeal endoderm from human pluripotent stem cells (PSC), a previously described protocol for definitive endoderm (DE) differentiation (Genga etal., 2019) was extended toward anterior foregut endoderm (AFE). Consistent with previous reports (Green et al., 2011; Huang et al., 2015; Mou et al., 2012), temporal inhibition of bone morphogenetic protein (BMP), Transforming growth factor beta (TGFP), and Wnt with small molecules to transition DE toward AFE was included (FIG. 1 A).

[0379] Hl human embryonic stem cells (hESCs) effectively responded with a cell fate transition as monitored by flow cytometry and immunofluorescence for SOX2 and FOXA2 protein expression (PSC: SOX2+FOXA2-, DE: SOX2-FOXA2+, AFE: SOX2+FOXA2+, FIG. IB and FIG. 1C). This transition was further supported by stage-specific detection of transcripts associated with PSC (POU5F1, NANOG, SOX2), DE (GATA6, SOX17, 07X2), and AFE (SOX2+ but POU5F 1- and SOX17-negative) states (FIG. ID).

[0380] Given the involvement of the retinoid pathway in mammalian pharyngeal endoderm pouch development (Kopinke et al., 2006; R. Quinlan et al., 2002; Wendling et al., 2000), the response of the differentiation culture to different retinoic acid (RA) levels was explored. While previous efforts on generating pharyngeal endoderm derivatives, such as thymus or parathyroid, include RA treatment (e.g. (Lawton et al., 2020; Parent et al., 2013)), a careful analysis of dose- and timing-responses remains elusive. Titration of RA levels between lOuM and 300pM, in addition to treatments including a carrier or RA-pathwayinhibitor (BMS493), revealed RA dosage-dependent expression of embryonic anterior- and pharyngeal-foregut associated transcripts, such as PAX9, NKX2.6, PAX1, H0XA3, SIX1, VGLL2, HHEX, EYA1, and 0TX2 (Bogue et al., 2000; Farley et al., 2013; Hou et al., 2007; Johnson et al., 2011; Magaletta et al., 2022; Neubiiser et al., 1995; Tanaka et al, 2000; Xu et al., 2002; 1997) (FIG. IE, Table 1). An RA concentration of lOOnM offers a balance of expression of multiple markers. Notably, H0XA3, a transcription factor linked to caudal pouch identity (Gordon, 2018; Manley and Capecchi, 1998), was highly enriched with high RA concentrations but only partially overlapped with optimized expression of pharyngeal pouch associated transcripts such as PAX9, NKX2.6, and PAX1 as found at lower RA levels. Thus, optimization for H0XA3 levels alone at this stage would have been an insufficient readout.ANOVA with Dunnett's multiple comparisons test

[0381] To discriminate further the molecular states of the AFE products in the presence of different RA concentrations, single cell transcriptomic profiling of AFE from cultures treated with four separate concentrations of RA was performed: no RA as described previously (K. M. Loh et al, 2014); 100 nM, as selected for this differentiation protocol; IpM, as used before in endoderm to pharyngeal patterning (X. Sun et al., 2013); and lOpM. After sample demultiplexing and quality control, 1885, 1838, 1184, and 1271 cells were obtained from no RA, lOOnM, IpM and lOpM conditions across two replicates expressing 10412, 10223, 9969, and 10362 median genes, respectively. Highly variable gene selection, principal component analysis and visualization on a UMAP (uniform manifold approximation and projection) embedding revealed a clear separation in transcriptomic space between cultures treated with different RA concentrations, with IpM and lOpM being closer than other pairs of concentrations (FIG. IF). Unsupervised leiden clustering resulted in four clusters, each almost completely capturing cells of a single RA concentration (FIG. 2A and FIG. 2B), supporting a largely culture-wide consistent response. To verify the downstream activation of RA response genes on the data, a RA response signature from literature was curated (Lopez-Perez et al., 2021) and a per-cell RA signature gene score was obtained. Consistent with the dose titration, highest RA response signature was observed at lOpM followed by IpM then lOOnM, supporting an analog, culture-wide dosage response rather than threshold-stimulated responses in subsets of the cells (FIG. 2C).

[0382] Next the expression of gut endoderm region-specific genes was examined (K. M. Loh et aL, 2014) (Nowotschin et aL, 2019) across the four RA concentrations (FIG. 1G and FIG. 2D). OTX2 and ISL1, transcripts associated with anterior foregut identity were highest at no RA treatment but also expressed at lOOnM. However, PFE-associated transcripts, including EYA1, IRX1 / 3 / 5, NKX2-3, RIPPI.Y3, SIX1, SOX2 and TBX1, were highest at lOOnM and lower at no RA conditions. AFE-and PFE-associated transcripts were largely absent at higher RA concentrations, throughout. Importantly, PAX9 and PAX1, two PFE- specific transcripts were largely undetected or low at all concentrations except lOOnM, further supporting the optimal RA concentration required for pharyngeal identity.

[0383] Surprisingly, despite the presence of TGFbeta- and BMP-inhibitor containing anteriorizing media, higher RA concentrations (IpM and lOpM) provoked posterior foregut and hindgut-associated transcripts including, AFP, FOXA3, GATA4, HNF1B, HNF4A, ONECUT1, ON ECUT2, TTR, FABP1 and OSRE Moreover, PDX1, CDX2 and HHEX expression further supports diversion toward alternative, more posterior gut signatures of the endoderm (FIG. 1G and FIG. 2D, (Sherwood et al., 2009)).

[0384] To further characterize phenotypes associated with the different concentrations of RA, differential expression across RA concentrations was performed and followed by an enrichment analysis of the upregulated genes (log2foldchange > 1, p-value < 0.05) and curated a list of terms (FIG. 2E and FIG. 2F). Consistent with the expression of endoderm region-associated transcripts described above, the pathway enrichment analysis identified terms associated with digestive tract, intestinal epithelial cell, type B pancreatic cell and enteroendocrine cell differentiation which were enriched at higher RA concentrations 1 pM and lOpM, while anterior associated terms including the branchial arch and cranium phenotypes were triggered by PFE-associated transcripts most enriched at lOOnM (FIG.2F). Differences in cell cycle distributions between RA concentrations during the scRNAseq data processing were noted (FIG. 2G). Analysis of G2M, S or G1 phase predictions suggests that high concentrations of RA reduce cell proportions in S phase compared to control treated AFE (FIG. 2G). Notably, lOOnM RA did not seem to have this effect, which could suggest that the different concentrations of RA not only affect gut tube regionalized gene expression, but also impact cell division.

[0385] It was concluded that an optimal RA concentration of lOOnM is favorable for engaging and fine-tuning pharyngeal foregut endoderm (PFE)-like gene expression signatures, whereas higher RA concentrations seem to pattern the endoderm toward theposterior, mid and hindgut regions, and the absence of RA fails to promote pharyngeal endoderm identity.EXAMPLE 3Directed differentiation of highly pure human pharyngeal foregut from embryonic stem cells

[0386] Given the RA-dose dependency of pharyngeal gene expression, next the temporal effect of the treatment on AFE patterning was evaluated. As a target of optimization, PAX9 expression as a highly pharyngeal endoderm- and pouch-specific transcript was used (Farley et al., 2013; Magaletta et al., 2022; Neubiiser et al., 1995). In addition to using lOOnM RA, the culture after the AFE stage was supplemented with BMP4 and FGF10 to engage the BMP and fibroblast growth factor (FGF) pathways that have been implicated in maturation of pharyngeal endoderm to pharyngeal derivatives such as thymic epithelial cells (Gardiner et al., 2012; Gordon et al., 2010; Neves et al., 2012; Patel et al., 2006; Soza-Ried et al., 2008). The time course revealed a strong RA treatment timing-dependency of high PAX9 expression, with highest induction of PAX9 with RA treatments prior to the AFE stage (FIG. 3 A). The increased PAX9 transcript levels in PFE cultures that receive RA from day 4 of differentiation was also RA dose dependent and translated to PAX9 protein positive EPCAM+ cells (FIG. 3B).

[0387] To confirm the stage-specific expression of transcription factors on the protein level, differentiation cultures were surveyed for protein level expression of POU5F1 (PSC- stage specific), SOX17 (DE-stage specific), FOXA2 (DE- and AFE-lineage specific), SOX2 (PSC-state and AFE-lineage specific) and H0XA3 (caudal pharyngeal region marker). Consistent with progression of the cellular differentiation, these transcription factors were tightly regulated in the differentiation culture, and not only appropriately upregulated, but also downregulated, dependent on differentiation stage (FIG. 4A).

[0388] To assess whether the optimized PFE culture could mature into pharyngeal derivatives, the PFE cultures were transplanted under the kidney capsule of immuno-deficient NOD-.scid l / .2rg‘ul1(NSG) mice. 10 weeks after transplantation, the transplants were evaluated for expression of pharyngeal endoderm and thymic epithelial cell-related proteins. Small clusters of keratin positive, and PAX9 and FOXN1 expressing cells were detected (FIG. 3C). These FOXN1 -positive cells were reactive to lectins and antibodies detecting additional thymic epithelial cell-related molecules such as UEA-1, CLDN4, and HLA-DR / DP / DQ (Gras-Pena et aL, 2022; Rouse et aL, 1982; S. Sun et aL, 2022). Concordantly, quantitative PCR analysis revealed upregulation of additional thymic epithelial progenitorspecific transcripts in the transplants, such as FOXG1, PSMB11, IL7 and DLL4 (Apavaloaei et al., 2019; Itoi et al., 2007; Ribeiro et al., 2013; Wei and Condie, 2011), compared to the in vitro differentiation intermediates (FIG. 3D and FIG. 4B).

[0389] The protocol development was pioneered in human embryonic stem cells (hESCs). To verify that the approach is robust and could be transferred to human induced pluripotent stem cells (hiPSCs), e.g., for future applications in personalized disease models, hiPSCs from human cord blood were generated and then differentiated to pharyngeal endoderm (FIG. 4C, FIG. 4D, and FIG. 4E). Similar to Hl hESCs (FIG. 3D and FIG. 4B), a clear transition based on evaluation of differentiation state-specific transcripts SOX17, FOXA2, SOX2, and PAX9 was found (FIG. 4D). Importantly, regulation of transcript was reflected in corresponding protein expression (FIG. 4E), suggesting that transcriptional changes translate into functional transcription factors on an induced pluripotent stem cell (iPSC) differentiation culture-wide cellular level.

[0390] Thus, it was concluded that the in vitro generated PFE is dependent on timing of RA and exhibits the developmental competence to mature further in transplant settings. Further, the presently described protocol has high efficacy, is robust, and is applicable to personalized iPSC disease model studies.EXAMPLE 4Human pluripotent stem cell-derived pharyngeal endoderm exhibits molecular accuracy and developmental competence

[0391] To evaluate further the molecular state of the PFE product, its heterogeneity as well as its global transcriptomic similarity to in vivo development, an unbiased cell classification approach was developed. First, single cell transcriptomic profiling of the PSC to PFE differentiation intermediates was performed. Next, a single cell mouse roadmap of the developing pharyngeal endoderm from the inner cell mass through endodermal gut tube maturation to pharyngeal development and derivative specification by integrating data from Nowostschin et al. was compiled (Nowotschin et al. , 2019) and a previous study on mouse pharyngeal endoderm development (Magaletta et al., 2022). These resources contain the four hPSC differentiation intermediates and an almost daily sampling of the embryonic pharyngeal endoderm lineage differentiation intermediates from the pluripotent state to pharyngeal endoderm derived organ domains (FIG. 5A). Finally, a computational tool,termed CellMatch, was developed for identification of hPSC differentiation intermediates by classifying them on high-dimensional transcriptomic space to in vivo populations (FIG. 5A).

[0392] CellMatch works by matching all the query cell populations or clusters to reference clusters via a robust correlation-based distance and selecting the match with the minimum distance across all reference-query cluster pairs. Further, CellMatch evaluates neighboring matches, z.e., matches created by editing a single reference query pair. Finally, the per gene correlation scores were obtained across the reference-query cluster matches to identify genes supporting the predicted classification thereby allowing the biologist to evaluate the results based on known literature markers. Importantly, because of the simultaneous matching of all query-to-reference profiles and the use of a robust distance metric, the approach works well for classification across platform, species or system. To evaluate the performance of CellMatch on studies independent of this dataset, its performance on two benchmarks comprising a cross-platform study and a cross-species study was validated, as well as compared its performance to other classification tools (see Methods and Materials and FIGs. 6A-6G).

[0393] After quality control, the scRNAseq dataset yields a resource of 886, 336, 1465, and 503 cells at PSC, DE, AFE, and PFE, respectively (median of 3319.5 unique genes per cell) with excellent reproducibility across replicates (average Pearson correlation between replicates = 0.95) (FIG. 7A and FIG. 7B). Identification of highly variable genes, dimensionality reduction, data integration and unsupervised leiden clustering identifies 10 clusters among the cell states, each containing differentially upregulated genes (log2foldchange > 1, p-value < 0.05) (FIG. 5B). Consistent with bulk qPCR and immunofluorescence analyses, clear separation of the differentiation stages on a UMAP (uniform manifold approximation and projection) embedding was found, although some differentiation stage-dependent sub-clustering can be achieved. The AFE and PFE cultures are closest in transcriptomic space with some interspersed PFE cells in the AFE clusters. Evaluation of stage-specific transcripts further extends insights on culture stage appropriate expression such as POU5Fl at PSC (Y.-H. Loh et al., 2006), CER1 and SOX17 at DE (Kanai-Azuma et cd., 2002), with downregulation at later stages. Anterior patterning markers EYA1 (Xu et al., 2002) and VGLL2 (Mielcarek et al., 2002) are most highly expressed at AFE, while SOX2 is absent at DE but present elsewhere, and FOXA2 is expressed from DE onward. Importantly, pharyngeal endoderm-specific transcripts, such as PAX9 (Peters et al., 1998), PAX1 (D. Su et al., 2001), FOXE1 (Dathan et al., 2002; Zannini et al., 1997), andPBX1 (Manley et aL, 2004) are most highly expressed in PFE (FIG. 5C, FIG. 5D), although some PBX1 expression was also noted in PSCs. To assess the heterogeneity at AFE, pseudotime analysis was run on the transition from AFE to PFE. The analysis supports the progression from AFE to PFE through timepoint specific markers, such as EYA1 (Xu et al.. 2002) and VGLL2 (Mielcarek et al., 2002) appearing earlier than PAX9 (Peters et al., 1998) despite all of them being widely expressed at AFE (FIG. 5E).

[0394] The integrated mouse in vivo developmental roadmap captures 76,894 endodermal lineage cells from the inner cell mass (ICM), epiblast, definitive endoderm, gut tube, liver, pancreas, small intestine, as well the developing PFE and patterning into the pharyngeal pouches and derived organs including the early respiratory system, eustachian tube, thyroid, thymus (cortex and medulla), parathyroid, ultimobranchial body (UBB), oropharynx, and esophagus (FIG. 5E). Consistent with the endodermal origin and described patterning of PFE, the integration shows a continuous progression from pluripotent cells of the ICM and epiblast to pharyngeal endoderm derivatives. Notably, embryonic PFE from E9.5 to E12.5 displays increasing diversity with increase in embryonic day (FIG. 7C, FIG. 7D). Curated literature transcripts validate stage and cell type specificity of markers (FIG. 7E).

[0395] Next, CellMatch was applied to stage PSC derivatives from the differentiation with respect to the comprehensive endoderm reference roadmap. Concordant with the step- wise differentiation approach, matched embryonic cell types fall in order on the integrated reference with human PSC classifying to the E3.5-E6.5 epiblast population dominated by E5.5 cells, DE cells to embryonic definitive endoderm at E7.5, AFE to an E9.5 dominated early developing pharyngeal endoderm population and human PFE to an El 1.5 dominated late developing pharyngeal endoderm population (FIG. 5F, FIG. 5G). Importantly, all subclusters at the same stage classified to the same mouse population suggesting stage specific homogeneity within the differentiation. While the identity of PSC populations is unambiguous, DE exhibits high scoring matches (low correlation distance) with developing gut-tube populations suggesting some patterning even at DE (FIG. 7F). AFE clusters have high-scoring matches with other early pharyngeal populations suggesting differentiation potential to multiple pharyngeal lineages. The PFE cluster has high scoring matches with several mid and late pharyngeal populations including ones annotated as Esophagus and Oropharynx (C6) and Mid_C2 which was annotated as a third pouch progenitor previously (Magaletta et al., 2022) (FIG. 7D). This range of matches suggests maturation along the pharyngeal endoderm lineage with possible developmental potential toward severalderivative lineages including the esophagus and oropharynx and pouch 3 derived organs such as the thymus and parathyroid. The match of the in vitro intermediates with the mouse in vivo roadmap is supported by a high number of markers at PSC (206, 216 and 204 transcripts for the 3 PSC populations), DE (144 transcripts) and PFE (98 transcripts) with relatively fewer markers for the five AFE populations (12, 28, 44, 40, 30) indicating slightly less confidence in the classification of AFE. The 647 unique transcripts identified in support of the classification exhibit stage specificity on the in vitro differentiation as well as the mouse roadmap (FIG. 7G, FIG. 7H). CellMatch identifies pluripotency associated transcripts such as NANOG and F0XD3 in support of the match at PSC. The match at DE has endoderm specific CER1, SOX17, GATA6 as the top three transcripts (lowest correlation distance or best model fit). The AFE match included anterior foregut associated transcripts such as VGLL2, NKX2-5, NKX2-6, EYA1, PRRX2, SIX2, F0XG1, PAX1, BMP3 as well as HOX genes associated with regional patterning of the gut tube into pharyngeal pouches, including H0XA2, H0XA3, HOXA4, H0XB1, HOXB3, H0XB4, H0XB5. However, the correlation distance of H0XA3 and H0XB3, transcripts associated with caudal pouch identity, was low (< 1) suggesting that they are more responsible for the classification than the other HOX genes. Finally, the best-fit genes for PFE include PAX9, FOXE1, BARX2, DMRT2, SHH, GRHI.3, MEIS2, HOXB3, and F0XP2 with previously described roles in pharyngeal development. Overall, the list of transcripts supporting the classification by CellMatch contains several known markers associated with the respective cell type identity, suggesting that the in vitro populations correspond to biologically relevant types present in the reference data. The exception is AFE cl, which is ambiguous since it had too few transcripts (12) supporting the classification.

[0396] In conclusion, the in vitro human directed differentiation toward PFE has been characterized on single cell transcriptomic level, benchmarked based on previous knowledge, and curated signature from comparing it to an integrated mouse in vivo developmental reference roadmap. Overall, the intermediate differentiation stages sampled appeared highly homogeneous, although pseudotime analyses suggest some maturation intermediates at least at the AFE stage. Further, the in vitro cell clusters were classified with a novel staging tool, CellMatch, which was independently validated on cross platform and cross species benchmark datasets. Staging with CellMatch suggests that PSC, DE, AFE, and PFE stages correspond well with in vivo Epiblast, DE, Early PFE, and Late PFE respectively and provides a set of markers supporting the classification predictions.EXAMPLE 5Global transcriptomic profiling reveals stage-specific and dynamic expression changes during pharyngeal endoderm differentiation

[0397] To characterize the differentiation culture more comprehensively, bulk RNA sequencing was performed at all four stages of differentiation: PSC (Day 0), DE (Day 3), AFE (Day 9), and PFE (Day 15). Principal component analysis confirmed scRNAseq results, highlighting that AFE is considerably closer to PFE as compared to PSC and DE (FIG. 8A). This relationship is further supported by high correlation between samples (FIG. 8B). A curated list of transcripts from literature confirms stage and time dependent expression of markers across the time course (FIG. 8C).

[0398] To investigate transcriptional changes over the course of PFE differentiation, a differential analysis was performed between every pair of stages across 21,672 transcripts and identified 6161 transcripts that were differentially expressed across the time course (absolute log2foldchange > 0.5, adjusted p-value < 0.1). There were comparable number of differential transcripts between all pairs except the PFE:AFE comparison (9%), which is consistent with the observed similarity between AFE and PFE. Volcano plots for the DE over PSC differential expression analysis reveal known upregulated (LEFTY2, BMP2, NODAL, KOMIN CER1, FGF17, SOX17, GATA4, GATA3, GATA6, LEFTY1, OTX2) and downregulated transcripts (80X2, POU5FL) (FIGs. 8D-8G). Similarly, transcripts associated with Anterior Foregut patterning such as A / A7A7, MEIS2, 0X2, HES1, EYA1, ISL1, SEXI, VGLL2, PBX1 are upregulated in AFE:DE. Anterior HOX genes H0XA2, H0XB2, H0XB3, H0XB4 are differentially upregulated at AFE:DE while more posterior HOX genes are missing from the differential analysis as expected from anterior foregut identity (Nowotschin et al., 2019). Of note, NR2F1, NR2F2, RARB, and MEIS2 associated with retinoic acid signaling are upregulated at AFE:DE. Further, GATA4, and GATA6, markers associated with DE are downregulated at AFE while GATA3 with known role in pharyngeal endoderm development (Grigorieva et al., 2010) is upregulated. PAX9 is differentially upregulated in AFE:DE consistent with previous conclusions of early pharyngeal endoderm behavior at AFE (Green et al., 2011; Kearns et al, 2013). Finally, PFE:DE differential analysis yields an even broader set of pharyngeal markers including CDH1, FN1, BMP5, TP63, SHH, BMP1, HEY1 and F0XE1 suggesting further maturation toward the pharyngeal endoderm lineage in thePFE cultures (FIGs. 8D-8G). The pharyngeal marker, PAX1, increases over course of the differentiation and consequently is captured in a differential analysis of PFE over PSC (FIG. 8C).

[0399] To identify groups of transcripts with similar behavior and their associated pathways from the combined list of 6161 pairwise differentially expressed transcripts, 10 clusters were characterized based on their dynamic expression throughout the differentiation time course (FIG. 9A). Pluripotency state-specific transcripts, such as POU5F1, NANOG, and F0XD3 fall into cluster 7 and 8, clusters that capture downregulated transcripts upon differentiation of PSCs to DE. In turn, transcripts associated with a definitive endoderm state, including SOX17, GATA4, GATA6, EOMES, and LEFTY 1, are found in clusters 9 and 10, with transient high expression in definitive endoderm. Further, the sequential differentiation signature toward the pharyngeal endoderm state is captured by clusters 1, 3 and 4, where transcripts are upregulated and continue to be expressed in the subsequent differentiation process. Notable examples are initiation of pioneer-factor FOXA2 expression at DE, SEXI, EYA 1, and PAX9 expression at AFE, and F0XE1 and PAX1 expression at PFE. Specificity combined with observed dynamics support the maturation along the pharyngeal endoderm lineage.

[0400] Importantly, upregulated transcripts along the differentiation axis include transcripts from loci associated with various pharyngeal malformations and pharynx derivative-dependent diseases were detected. Specifically, upregulation of Hypoparathyroidism, sensorineural deafness and renal (HDR) disease-associated GATA3 (Muroya etal., 2001), DiGeorge syndrome and thymic hypoplasia-associated TBX1 (Yagi et al., 2003), Otofaciocervical syndrome-2 with T-cell deficiency (OTFCS2)-associated PAX1 (Yamazaki et al., 2020), Branchiootorenal (BOR) syndrome-associated EYA1 and SIX1 (Chang et al., 2004; Ruf et al, 2004), and Bamforth-Lazarus syndrome and thyroid aplasia associated F0XE1 were observed (Clifton-Bligh et al., 1998).

[0401] To determine whether changes in gene expression were reflected in protein expression changes of stage-specific transcription factors, the differentiation cultures for protein level expression of markers were surveyed. PFE-specific, protein level, nuclear expression of PAX9 and F0XE1, two pharyngeal TFs that so far have remained elusive in such context in previous efforts to generate pharyngeal endoderm and its derivatives from PSCs, were detected. Notably, dynamic protein expression of PBX1 and GATA3, two TFsimplicated in pharyngeal pouch development and hypoparathyroidism, were confirmed (Grigorieva et al., 2010; Manley et al., 2004; Muroya et al., 2001) (FIG. 9B).

[0402] To uncover biological pathways and phenotypes in the gene clusters, gene set enrichment analysis and curated cluster specific enriched Gene expression Ontology (GO) terms were performed. This substantiated the anticipated differentiation process and revealed GO-term enrichment of “endoderm development”, “regulation of gastrulation” and “morphogenesis of an epithelium” for DE state-specific clusters 9 and 10, whereas GO-terms such as “pharyngeal system development”, embryonic organ development”, “gland development” and “extracellular matrix organization” were enriched in clusters that captured specific signatures of the later differentiation stages (FIG. 9C).EXAMPLE 6Dynamic global chromatin activity and epigenetic changes reveal molecular basis and putative drivers of pharyngeal endoderm differentiation

[0403] Given the observation of dynamic pharyngeal-lineage specific TF protein expression, chromatin dynamics that accompany transcriptomic changes were elucidated. To this end, open chromatin (ATAC-seq) and histone modifications (ChlP-seq) were profiled. H3K4me2 was measured for active or poised chromatin (Bernstein et al., 2005; Orford et al., 2008), H3K27ac for active chromatin (Heintzman et al., 2009; 2007) and H3K27me3 to determine regions of poly comb repression (Schuettengruber et al., 2007).

[0404] The chromatin and histone data showed reproducibility and identified 20,108, 29,701, 26,593, and 49,811 consensus open chromatin regions specific to PSC, DE, AFE, and PFE respectively and 29,456 regions common to all four stages (FIG. 10A, FIG. 10B, FIG. 10C, FIG. 10D).

[0405] ATAC and histone modification signals reveal strong concordance of chromatin accessibility and modifications over the loci of genes linked to pharyngeal endoderm function and development. Importantly, putative cell-type-specific regulatory elements were captured, as exemplified in the distal regions of genes each of which performs critical functions in development of the endoderm (SOX17, GATA6) and the endodermal lineage (FOXA2). Critically, histone modification data support PFE as a separate cellular state than AFE, shown by a marked increase in H3K27ac and H3K4me2 and loss of H3K27me3 over the PAX1 gene and distal elements. These data suggest a pattern of polycomb repression of this locus that is maintained through the PSC, DE, and AFE stages, but lost prior to PFE (FIGs. 11 A-l ID).

[0406] To identify the set of open chromatin regions which vary across the differentiation, peaks with high coefficient of variation in their normalized peak signal across the 4 stages were selected to obtain a set of 40,163 highly variable peaks. To find sets of open variable regions with similar behavior, optimal set of four clusters were grouped using the normalized and replicate averaged signal and found that clusters with 4889 (cluster4), 12,949 (clusters), 17,860 (cluster2) and 4465 (clusterl) peaks were specific to PSC, DE, AFE, and PFE respectively (FIG. 10E). The base conservation in this variable set of chromatin- accessible peaks was high among vertebrate species compared to a background, suggesting a high level of conservation among vertebrates of the regions putatively driving pharyngeal endoderm development (FIG. 1 IE). Expectedly, the conservation among proximal peaks in this variable set is higher than distal. Of note, the distribution of conservation of both proximal and distal regions responsible for pharyngeal development, is similar to the corresponding distribution from representative DNase hypersensitivity sites from ENCODE (ENCODE Project Consortium et aL, 2020) (FIG. 1 IF).

[0407] To determine the putative drivers of differentiation states, chromatin accessibility and histone modification signal in the variable regions, predicted TF footprint binding, and determined stage specific enrichment of the binding motif using the Stouffer’s method in gimmemotifs were obtained (Bruse and van Heeringen, 2018; van Heeringen and Veenstra, 2011) to combine z-score enrichment from multiple approaches. Further, motifs were paired with candidate regulatory TFs using motif databases and correlation between motif enrichment and RNA expression within the TF family. Thus a total of 247 TF-motif pairs were identified with 21, 12, 25 and 55 unique motifs at PSC, DE, AFE and PFE respectively. For these pairs, the median correlation of TF expression with TF motif enrichment in open chromatin (ATAC), active or poised (H3K4me2), active (H3K27ac), and polycomb repressive (H3K27me3) regions is 0.93, 0.86, 0.79 and -0.50 respectively. The correlation of TF expression with TF motif enrichment in active (H3K27ac) chromatin regions was used to identify putative activators of each stage, such as POU5F1 and NANOG at PSC; GA TA4, GATA6, EOMES, SOX / 7, OTX2, and LHX1 at DE; FOXA2, SIX2, NKX2-5, HOXA1, RARB, NR2F1, and TBX1 at AFE; and PAX1, PAX9, GATA3, BACH2, HOXB2 3 4, HOXA2, NFKB1, FOXA1, PBX1, SMAD 3 / 6 / 779, NR2F2, STAT3, MEIS 1 2 3, TP63 73, GRHL1 / 2 / 3, and TEAD1 / 3 at PFE. Similarly, the correlation of TF expression with TF motif enrichment in polycomb repressor (H3K4me3) chromatin regions was used to identify putative repressorssuch as PKNOX1, SIX1, 1X)XI3, STAT3, SIX4, TFDP2, NFIB, E2F8, FOSB, ATF4, HMGA2, RFX3, LHX1, REST, SIX2, PAX9, and SNAI2. (FIGs. 11G-11 J).

[0408] In conclusion, for each of the four stages of our differentiation protocol, chromatin with ATAC-seq as well as active, poised, and repressive regulatory regions was profiled with H3K27ac, H3K4me2 and H3K27me3 ChlP-seq respectively. Known and putative novel regulatory regions associated with key drivers of pharyngeal development were found and it was demonstrated that these regions are conserved across vertebrates, and integrated ATAC, RNA, and histone data to identify putative drivers of each differentiation stage.EXAMPLE 7Gene regulatory network inference prioritizes transcription factor regulators during human pharyngeal endoderm differentiation

[0409] To gain additional insights on key drivers of the PFE lineage differentiation intermediates, gene regulatory networks were inferred by identifying putative regulatory regions and associated TF footprints for each gene from the accessible peaks within + / - lOOkb of the TSS. Further, a strategy to infer the extent of an interaction between a TF and target was developed by combining RNA signal of the source and target, uncertainty of transcription factor binding, and uncertainty over motif regulation of the target. Finally, PageRank (Hagberg et al., 2008) was applied to rank TFs within a differentiation intermediate and score them to determine relevance across all the stages. In this manner, the top 150 TFs driving the differentiation and grouped them by the stage on which they had the highest score were identified (FIG. 12A). Consistent with their central role in pluripotency, POU5F1, NANOG, and SOX2 were retrieved among the top ranked TFs at the PSC state. TFs previously associated with developmental defects in endoderm development, such as GATA6 (Tiyaboonchai et al. , 2017), SOX17 (Genga et al., 2019; Kanai-Azuma et al., 2002), and EOMES (Teo et al., 2011), are highly ranked and specific in DE. F0XA2 is highly ranked in AFE as well as DE and PFE, consistent with its role as a pioneer factor at definitive and anterior foregut endoderm (Genga et al., 2019; Wang et al., 2015). Additional TFs that are top ranked in AFE include PBX1, RARB, H0XA1, ZBTB16, and MEIS2, all which have recently been associated with RA responsiveness through conserved RA response elements (RAREs) (Berenguer et al., 2020). TFs previously determined to be critical for mouse pharyngeal endoderm or epithelia development, namely PAX9, F0XE1, and ISL1, wereprominent. Additionally, human disease relevant PAX1 and GATA3 are highly ranked in the network analysis.

[0410] To uncover the important regulatory nodes and edges of the PFE gene regulatory network, the network comprising the top TF regulators was extracted and retained high confidence putative enhancer interactions by preserving edges with weight > 10. The resulting network was visualized using a force-directed layout with high confidence interactions grouping toward the center of the embedding and lower confidence interactions branching out toward the periphery (FIG. 12B). This gene regulatory network analysis uncovers putative high confidence targets of key pharyngeal markers including PAX9 with predicted targets including SMAD3 / 7, HES1, TP63, FOXE1, TEAD3, BACH 2. NKX2.5, all of which have been implicated in pharyngeal development (FIG. 12B).

[0411] Despite performing scRNAseq in the presence of various RA conditions as well as on the differentiation time course, the present disclosure did not pinpoint major heterogeneity in the differentiation product intermediates. While it is believed to suggest homogeneity in the populations, it is possible that heterogeneity is not captured due to inherent limitations with single cell assays including dropout and the inability to confidently capture smaller populations.

[0412] Overall, the present multi-omic analysis distills the data resource to accessible gene regulatory networks that provide context of known and novel players in human pharyngeal endoderm development, including embedding of disease relevant transcription factors in gene regulatory context.References

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Claims

CLAIMSWhat is claimed is:

1. A method for producing anterior foregut endoderm (AFE) cells, the method comprising the step of culturing definitive endoderm (DE) cells in a medium, the medium comprising about 100 nM retinoic acid, thereby producing AFE cells.

2. The method of claim 1, wherein: the medium further comprises an ALK / TGF-PRI inhibitor, an ALK / BMP inhibitor, a WNT inhibitor, or a combination thereof; culturing the DE cells in the medium occurs on about days 4-8 of differentiation and / or for a period of about five days; or both of the foregoing.

3. The method of claim 2, wherein the medium comprises: about 1 pM of ALK / TGF-PRI inhibitor, about 250 nM of ALK / BMP inhibitor, about 250 nM of WNT inhibitor, or a combination of the foregoing.

4. The method of any one of claims 1-3, wherein the medium further comprises: about 1 pM of ALK / TGF-PRI inhibitor A83-01, about 250 nM of ALK / BMP inhibitor LDN193189, about 250 nM of WNT inhibitor IWR-1, or a combination of the foregoing.

5. The method of any one of claims 1-4, wherein the medium comprises: about 100 nM retinoic acid, about 1 pM of ALK / TGF-PRI inhibitor A83-01, about 250 nM of ALK / BMP inhibitor LDN193189, and about 250 nM of WNT inhibitor IWR-1.

6. The method of any one of claims 1-5, further comprising the step of culturing the DE cells in a medium, the medium comprising about 0 nM retinoic acid prior to culturing the DE cells in the medium comprising about 100 nM retinoic acid.

7. The method of claim 6, wherein: the method further comprises singularizing and plating the DE cells prior to culturing the DE cells in the medium comprising about 0 nM retinoic acid, optionally wherein singularizing and plating the DE cells occur on about day 3 of differentiation; the medium comprising about 0 nM retinoic acid further comprises an ALK / TGF-PRI inhibitor, an ALK / BMP inhibitor, a ROCK inhibitor, or a combination thereof; culturing the DE cells in the medium comprising about 0 nM retinoic acid occurs on about day 3 of differentiation and / or for a period of about one day; or a combination thereof.

8. The method of claim 6 or 7, wherein the medium comprising about 0 nM retinoic acid comprises: about 1 pM of ALK / TGF-PRI inhibitor, about 250 nM of ALK / BMP inhibitor, about 10 pM of ROCK inhibitor, or a combination of the foregoing.

9. The method of any one of claims 6-8, wherein the medium comprising about 0 nM retinoic acid further comprises: about 1 pM of ALK / TGF-PRI inhibitor A83-01, about 250 nM of ALK / BMP inhibitor LDN193189, about 10 pM of ROCK inhibitor Y-27632, or a combination of the foregoing.

10. The method of any one of claims 6-9, wherein the medium comprising about 0 nM retinoic acid further comprises: about 1 pM of ALK / TGF-PRI inhibitor A83-01, about 250 nM of ALK / BMP inhibitor LDN193189, and about 10 pM of ROCK inhibitor Y-27632.

11. The method of any one of claims 1-10, wherein the AFE cells: functionally express VGLL2, EYA1, SIX1, or a combination thereof; do not functionally express POU5F1, SOX17, or a combination thereof; orboth of the foregoing.

12. The method of any one of claims 1-11, further comprising the step of producing the DE cells prior to culturing the DE cells, by: culturing pluripotent stem cells (PSCs) in a medium on about day 0 of differentiation and / or for a period of about 1 day, the PSCs comprising induced PSCs (iPSCs) or embryonic stem cells (ESCs), and the medium comprising: a Roswell Park Memorial Institute 1640 cell culture medium (RPMI 1640), about 0.2% HYCLONE® Defined fetal bovine serum (FBS), about 1% of L-glutamine supplement GLUTAMAX® (200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl), about 1% 5,000 IU penicillin-5,000 pg / mL streptomycin (P / S), about 100 ng / mL Activin A, about 3 pM of GSK3 inhibitor CHIR99021, and about 50 nM of PI3K / mT0R inhibitor PI- 103; and culturing the PSCs in a medium on about days 1-2 of differentiation and / or for a period of about 2 days, the medium comprising: RPMI 1640, about 0.2% HYCLONE® Defined FBS, about 1% GLUTAMAX® (200 mM L-alanyl-L- glutamine dipeptide in 0.85% NaCl), about 1% P / S, about 100 ng / mL Activin A, and about 250 nM of ALK / BMP inhibitor LDN193189, thereby producing DE cells; optionally wherein the PSCs are human PSCs or murine PSCs.

13. The method of any one of claims 1-12, wherein the DE cells: functionally express SOX17, GATA4, GATA6, EOMES, LEFTY1, CER1, or a combination thereof; do not functionally express POU5F1, VGLL2, EYA1, PAX9, PAX1, FOXE1, PBX1, or a combination thereof; or both of the foregoing.

14. A method for producing pharyngeal foregut endoderm (PFE) cells, the method comprising the step of culturing anterior foregut endoderm (AFE) cells in a medium, the medium comprising: retinoic acid,optionally wherein the retinoic acid is at a concentration of about 100 nM; a bone morphogenic protein (BMP), optionally wherein the BMP comprises BMP4, and / or optionally wherein the BMP is at a concentration of about 50 ng / mL; a fibroblast growth factor (FGF), optionally wherein the FGF comprises FGF10, and / or optionally wherein the FGF is at a concentration of about 50 ng / mL; a ROCK inhibitor, optionally wherein the ROCK inhibitor is Y-27632, and / or optionally wherein the ROCK inhibitor is at a concentration of about 10 pM; or a combination of the foregoing, thereby producing PFE cells.

15. The method of claim 14, wherein the AFE cells are produced by the method of any one of claims 1-13.

16. The method of claim 14 or 15, the method further comprising singularizing and plating the AFE cells prior to culturing the AFE cells in the medium, optionally wherein singularizing and plating the AFE cells occur on about day 9 of differentiation.

17. The method of any one of claims 14-16, wherein culturing the AFE cells occurs for a first period of time and a second period of time, and wherein: the AFE cells are cultured for a first period of time in the medium comprising retinoic acid, BMP, FGF, and a ROCK inhibitor; and / or the AFE cells are cultured for a second period of time in the medium comprising retinoic acid, BMP, and FGF.

18. The method of claim 17, wherein: the first period of time occurs on about day 9 of differentiation and / or for a period of about one day; and / orthe second period of time occurs on about days 10-14 of differentiation and / or for a period of about five days.

19. The method of any one of claims 14-18, wherein the PFE cells: functionally express FOXA2, SOX2, PAX9, PAX1, FOXE1, PBX1 or a combination thereof; do not functionally express POU5F1, SOX17, VGLL2, or a combination thereof; or both of the foregoing.

20. The method of any one of claims 14-19, wherein the PFE cells functionally express at least about two-fold the level of PAX9 functionally expressed by the AFE cells.

21. A method for producing thymic epithelial progenitor (TEP) cells, the method comprising the step of culturing pharyngeal foregut endoderm (PFE) cells in a medium, the medium comprising: retinoic acid, optionally wherein the retinoic acid is at a concentration of about 100 nM; a fibroblast growth factor (FGF), optionally wherein the FGF comprises FGF10, and / or optionally wherein the FGF is at a concentration of about 50 ng / mL; an ALK / TGF-PRI inhibitor, optionally wherein the ALK / TGF-PRI inhibitor comprises A83-01, and / or optionally wherein the ALK / TGF-PRI inhibitor is at a concentration of about IpM; a ROCK inhibitor, optionally wherein the ROCK inhibitor comprises Y-27632, and / or optionally wherein the ROCK inhibitor is at a concentration of about10 pM; or a combination of the foregoing, thereby producing thymic epithelial progenitor (TEP) cells.

22. The method of claim 21, wherein the PFE cells are produced by the method of any one of claims 14-20.

23. The method of claim 21 or 22, wherein culturing the PFE cells occurs for a first period of time and a second period of time, and wherein: the PFE cells are cultured for a first period of time in the medium comprising retinoic acid and FGF; and / or the PFE cells are cultured for a first period of time in the medium comprising ALK / TGF-PRI inhibitor.

24. The method of claim 23, wherein: the first period of time occurs on about days 15-21 of differentiation and / or for a period of about 7 days; and / or the second period of time occurs on about days 22-28 of differentiation and / or for a period of about 7 days.

25. The method of any one of claims 21-24, wherein the TEP cells: functionally express PAX1, PAX9, FOXN1, GATA3, or a combination thereof; do not functionally express VGLL2, SOX17, CER1, POU5F1, NANOG, or a combination thereof; or both of the foregoing.

26. A method for producing an organoid comprising thymic epithelial cells (TECs), the method comprising the steps of: seeding thymic epithelial progenitor (TEP) cells onto a scaffold, thereby initiating an organoid; and culturing the organoid in a medium, the medium comprising: an ALK / TGF-PRI inhibitor, optionally wherein the ALK / TGF-PRI inhibitor is A83-01, and / or optionally wherein the ALK / TGF-PRI inhibitor is at a concentration of about 1 pM; a ROCK inhibitor,optionally wherein the ROCK inhibitor is Y-27632, and / or optionally wherein the ROCK inhibitor is at a concentration of about 10 pM; or both of the foregoing, thereby producing an organoid comprising TECs.

27. The method of claim 14, wherein the TEP cells are produced by the method of any one of claims 21-25.

28. The method of claim 26 or 27, wherein: the method further comprises singularizing the TEP cells prior to seeding the TEP cells onto a scaffold; singularizing and seeding the TEP cells onto a scaffold occur on about day 29 of differentiation; the scaffold is contained in a plate, comprises a decellularized thymic lobe or a cell culture filter insert, is coated with a basement membrane or extracellular matrix, or a combination thereof, optionally wherein the decellularized thymic lobe is from a mouse, optionally wherein the mouse is of about 4-6 weeks of age, is of an ICR strain, or both; culturing the organoid in the medium occurs on about days 29-45 of differentiation and / or for a period of about 17 days; or a combination thereof.

29. A method for producing thymic epithelial cells (TECs), the method comprising the steps of: producing an organoid comprising thymic epithelial cells (TECs) according to the method of any one of claims 26-28; and isolating the TECs from the organoid comprising TECs, thereby producing the TECs.

30. The method of any one of claims 26-29, wherein the TECs: functionally express GCM2, FOXN1, CCL25, PAX1, PAX9, GATA3, MAFB, or a combination thereof;do not functionally express VGLL2, SOX17, CER1, P0U5F1, NANOG, or a combination thereof; or both of the foregoing.

31. The method of any one of claims 7, 16, and 28, wherein singularizing comprises: dissociating with a proteolytic enzyme, wherein the proteolytic enzyme is capable of detaching and / or dissociating adherent cells in culture, optionally wherein the proteolytic enzyme, or a solution containing the proteolytic enzyme, comprises trypsin, TRYPLE® recombinant dissociation enzyme, ACCUTASE® cell detachment solution, DISPASE® dissociating enzyme, or a combination thereof; scraping and then triturating; or both of the foregoing.

32. The method of any one of claims 1-31, wherein culturing comprises growing cells on a basement membrane or extracellular matrix, optionally wherein the basement membrane or extracellular matrix comprises 804G supernatant, MATRIGEL® basement membrane matrix, MATRIGEL® Growth Factor Reduced (GFR) basement membrane matrix, or a combination thereof.

33. The method of any one of claims 1-11 and 13-32, wherein the medium further comprises: a Roswell Park Memorial Institute 1640 cell culture medium (RPMI 1640); a neuronal cell culture supplement, optionally wherein the neuronal cell culture supplement comprises B- 27® minus vitamin A, and / or optionally wherein the neuronal cell culture supplement is at a concentration of about 0.5x; an L-glutamine supplement, optionally wherein the L-glutamine supplement comprises GLUTAMAX® (200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl), and / or optionally wherein the L-glutamine supplement comprises about 1% of the medium; an antibiotic,optionally wherein the antibiotic comprises 5,000 IU penicillin-5,000 pg / mL streptomycin (P / S), and / or optionally wherein the antibiotic comprises about 1% of the medium; optionally, a ROCK inhibitor, optionally wherein the ROCK inhibitor is Y-27632, and / or optionally wherein the ROCK inhibitor is at a concentration of about 10 pM; or a combination of the foregoing.

34. An anterior foregut endoderm (AFE) cell produced by the method of any one of claims 1-13.

35. A pharyngeal foregut endoderm (PFE) cell produced by the method of any one of claims 14-20.

36. A thymic epithelial progenitor (TEP) cell produced by the method of any one of claims 21-25.

37. A thymic epithelial cell (TEC) produced by the method of claim 29.

38. An organoid comprising thymic epithelial cells (TECs) produced by the method of any one of claims 26-28.

39. A pharmaceutical composition comprising the pharyngeal foregut endoderm (PFE) cell of claim 35, the thymic epithelial progenitor (TEP) cell of claim 36, the thymic epithelial cell (TEC) of claim 37, or the organoid comprising TECs of claim 38.

40. A method of recovering or restoring impaired tissue in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharyngeal foregut endoderm (PFE) cell of claim 35, the thymic epithelial progenitor (TEP) cell of claim 36, the thymic epithelial cell (TEC) of claim 37, or a population or organoid comprising such a cell.

41. A method of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount the pharyngeal foregut endoderm (PFE) cellof claim 35, the thymic epithelial progenitor (TEP) cell of claim 36, the thymic epithelial cell (TEC) of claim 37, or a population or organoid comprising such a cell.

42. The method of claim 40 or 41, wherein the subject has a thymus, parathyroid, and / or thyroid tissue disorder, and wherein definitive endoderm (DE) cells are differentiated from pluripotent stem cells (PSCs) obtained from a biological sample from the subject.

43. A method of evaluating a biological response to a compound or an environmental stimulus, the method comprising: producing a first and a second population of pluripotent stem cells (PSCs); exposing the second population of PSCs to the compound or environmental stimulus; producing a first population of definitive endoderm (DE) cells by culturing the first population of PSCs and a second population of DE cells by culturing the second population of PSCs according to claim 12; producing a first population of anterior foregut endoderm (AFE) cells from the first population of DE cells and a second population of AFE cells from the second population of DE cells according to any one of claims 1-11 and 13; producing a first population of pharyngeal foregut endoderm (PFE) cells from the first population of AFE cells and a second population of PFE cells from the second population of AFE cells according to any one of claims 14-20; and comparing the first and second populations of PFE cells, thereby evaluating the biological response.

44. The method of claim 43, wherein the method further comprises: producing a first population of thymic epithelial progenitor (TEP) cells from the first population of PFE cells and a second population of TEP cells from the second population of PFE cells according to any one of claims 21-25; producing a first population of thymic epithelial cells (TECs) from the first population of TEP cells and a second population of TECs from the second population of TEP cells according to claim 29; and comparing the first and the second population of TECs, thereby evaluating the biological response.

45. The method of claim 43 or 44, wherein: the first population of PSCs is obtained from a subject or produced from cells obtained from the subject prior to exposing the subject to the compound or environmental stimulus; exposing the second population of PSCs to the compound or environmental stimulus comprises exposing the subject to the compound or environmental stimulus; and the second population of PSCs is obtained from the subject or produced from cells obtained from the subject after exposing the subject to the compound or environmental stimulus.

46. A composition for identifying a therapeutic compound for treatment of a thymus, parathyroid, and / or thyroid tissue dysfunction, the composition comprising the pharyngeal foregut endoderm (PFE) cell of claim 35, the thymic epithelial progenitor (TEP) cell of claim 36, the thymic epithelial cell (TEC) of claim 37, or a population or organoid comprising such a cell.

47. A method of increasing pharyngeal foregut endoderm (PFE) cells, thymic epithelial progenitor (TEP) cells, and / or thymic epithelial cells (TECs) in a subject in need thereof, the method comprising administering retinoic acid to the subject in an amount sufficient to increase PFE cells, TEP cells, and / or TECs.

48. A medium for producing anterior foregut endoderm (AFE) cells, the medium comprising: an ALK / TGF-PRI inhibitor, optionally wherein the ALK / TGF-PRI inhibitor is A83-01, and / or optionally wherein the ALK / TGF-PRI inhibitor is at a concentration of about IpM; an ALK / BMP inhibitor, optionally wherein the ALK / BMP inhibitor comprises LDN193189, and / or optionally wherein the ALK / BMP inhibitor is at a concentration of about 250 nM; and optionally, a ROCK inhibitor,optionally wherein the ROCK inhibitor comprises Y-27632, and / or optionally wherein the ROCK inhibitor is at a concentration of about10 pM.

49. A medium for producing anterior foregut endoderm (AFE) cells, the medium comprising: about 100 nM retinoic acid, an ALK / TGF-PRI inhibitor, optionally wherein the ALK / TGF-PRI inhibitor is A83-01, and / or optionally wherein the ALK / TGF-PRI inhibitor is at a concentration of about IpM; an ALK / BMP inhibitor, optionally wherein the ALK / BMP inhibitor comprises LDN193189, and / or optionally wherein the ALK / BMP inhibitor is at a concentration of about 250 nM; and a WNT inhibitor, optionally wherein the WNT inhibitor comprises IWR-1, and / or optionally wherein the WNT inhibitor is at a concentration of about 250 nM.

50. A medium for producing pharyngeal foregut endoderm (PFE) cells, the medium comprising: retinoic acid, optionally wherein the retinoic acid is at a concentration of about 100 nM; a bone morphogenic protein (BMP), optionally wherein the BMP comprises BMP4, and / or optionally wherein the BMP is at a concentration of about 50 ng / mL; a fibroblast growth factor (FGF), optionally wherein the FGF comprises FGF10, and / or optionally wherein the FGF is at a concentration of about 50 ng / mL; and optionally, a ROCK inhibitor,optionally wherein the ROCK inhibitor is Y-27632, and / or optionally wherein the ROCK inhibitor is at a concentration of about 10 pM.

51. A medium for producing pharyngeal foregut endoderm (PFE) cells, the medium comprising: retinoic acid, optionally wherein the retinoic acid is at a concentration of about 100 nM; a bone morphogenic protein (BMP), optionally wherein the BMP comprises BMP4, and / or optionally wherein the BMP is at a concentration of about 50 ng / mL; and a fibroblast growth factor (FGF), optionally wherein the FGF comprises FGF10, and / or optionally wherein the FGF is at a concentration of about 50 ng / mL.

52. A medium for producing thymic epithelial progenitor (TEP) cells, the medium comprising: retinoic acid, optionally wherein the retinoic acid is at a concentration of about 100 nM; and a fibroblast growth factor (FGF), optionally wherein the FGF comprises FGF10, and / or optionally wherein the FGF is at a concentration of about 50 ng / mL.

53. A medium for producing thymic epithelial progenitor (TEP) cells, the medium comprising: an ALK / TGF-PRI inhibitor, optionally wherein the ALK / TGF-PRI inhibitor comprises A83-01, and / or optionally wherein the ALK / TGF-PRI inhibitor is at a concentration of about IpM.

54. A medium for producing thymic epithelial cells (TECs) and / or an organoid comprising TECs, the medium comprising: an ALK / TGF-PRI inhibitor, optionally wherein the ALK / TGF-PRI inhibitor is A83-01, and / or optionally wherein the ALK / TGF-PRI inhibitor is at a concentration of about 1 pM; and optionally, a ROCK inhibitor, optionally wherein the ROCK inhibitor is Y-27632, and / or optionally wherein the ROCK inhibitor is at a concentration of about 10 pM.

55. The medium of any one of claims 48-54, the medium further comprising: a Roswell Park Memorial Institute 1640 cell culture medium (RPMI 1640); a neuronal cell culture supplement, optionally wherein the neuronal cell culture supplement comprises B- 27® minus vitamin A, and / or optionally wherein the neuronal cell culture supplement is at a concentration of about 0.5x; an L-glutamine supplement, optionally wherein the L-glutamine supplement comprises GLUTAMAX® (200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl), and / or optionally wherein the L-glutamine supplement comprises about 1% of the medium; and an antibiotic, optionally wherein the antibiotic comprises 5,000 IU penicillin-5,000 pg / mL streptomycin (P / S), and / or optionally wherein the antibiotic comprises about 1% of the medium.

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