Generates dorsal foregut and anterior domain endoderm cells

Abstract

Provided herein are compositions, systems, kits, and methods for producing dorsal foregut endoderm (DFE) cells by contacting pluripotent stem cells with retinoic acid signaling pathway agonists and bone morphogenesis (BMP) pathway inhibitors without exposing stem cells to transforming growth factor β (TGFβ) pathway agonists. In certain embodiments, DFE cells are contacted with retinoic acid signaling pathway agonists and FGFR pathway inhibitors to produce pancreatic endoderm (PE) cells (e.g., with dorsal features (dorsal identity)). In other embodiments, PE cells are contacted with ALK5 inhibitors and Notch inhibitors to produce endocrine cells (e.g., endocrine cells expressing insulin). In certain embodiments, compositions, systems, kits, and methods for producing anterior domain endoderm (ADE) cells are provided.

Description

[0001] This application claims priority to U.S. Provisional Application Serial No. 62 / 851,348, filed on May 22, 2019, which is incorporated herein by reference in its entirety. Technical Field

[0002] Provided herein are compositions, systems, kits, and methods for producing dorsal foregut endoderm (DFE) cells by contacting pluripotent stem cells with retinoic acid signaling pathway agonists and bone morphogenesis (BMP) pathway inhibitors without exposing stem cells to transforming growth factor β (TGFβ) pathway agonists. In certain embodiments, DFE cells are contacted with retinoic acid signaling pathway agonists and FGFR pathway inhibitors to produce pancreatic endoderm (PE) cells (e.g., with dorsal features (dorsal identity)). In other embodiments, PE cells are contacted with ALK5 inhibitors and Notch inhibitors to produce endocrine cells (e.g., cells expressing insulin). In certain embodiments, compositions, systems, kits, and methods for producing anterior domain endoderm (ADE) cells are provided. Technical Background

[0003] Endoderm is the germ layer that produces most of the cells in most internal organ systems (such as lungs, stomach, pancreas, liver and intestines). Almost all efforts to induce endoderm from pluripotent cells rely on the use of TGFβ pathway agonists (most commonly activin A (AA)) as node mimics to promote pluripotent cells through in vitro gastrulation events (D'Amour et al., 2005, Gadue et al., 2006). This has led to endoderm populations that can be used to produce multiple offspring fates including intestine (Spence et al., 2011), pancreas (Kroon et al., 2008, Rezania et al., 2014, Pagliuca et al., 2014) and liver (Sampaziotis et al., 2015). By providing patterning inputs at subsequent stages, the generation of more anterior endoderm fates such as lungs has been achieved (Green et al., 2011). However, recent studies have suggested that the initial patterning of definitive endoderm may occur during its generation (Matsuno et al., 2016; Loh et al., 2014).

[0004] The pancreas is particularly meaningful for cell-based therapies of diabetes characterized by insulin-producing cell defects. The pancreas is formed by two spatially different primordia, which appear in the dorsal and ventral sides of the original intestinal tube, which fuses subsequently. Although both pancreatic buds can produce all lineages of adult pancreas (Matsuura et al., 2009), the initial induction of the pancreatic domain (pancreatic domain) on the opposite side of the intestinal tube is controlled by different transcriptional programs. In mice, the ventral pancreatic bud is first formed by the endoderm region with the bipotential ability of pancreas and liver at about embryonic day 8.5 (E8.5) (Angelo et al., 2012, Deutsch et al., 2001, Tremblay and Zaret, 2005). This early ventral endoderm region is composed of a progenitor cell group that transiently co-expresses Pdx1 / Sox17, and the progenitor cell group is divided by E9.5 to form the ventral pancreas and the hepatobiliary system (Spence et al., 2009). The specification of the ventral pancreas depends on the expression of HHex, in which the gene ablation model exhibits complete ventral dysplasia without affecting the formation of the dorsal pancreatic bud (Bort et al., 2004). In contrast, the dorsal pancreatic bud of mice appears at approximately embryonic day 9.0 and grows from a process at the tail of the gastric antrum region. Studies on mice have also identified factors that are involved in the specification of the dorsal pancreas but have no effect on ventral organogenesis. The Mnx1 (Hlxb9) knockout model shows that dorsal dysplasia occurs without a ventral phenotype (Li et al., 1999). Mnx1 expression is observed in the ventral region, but only after Pdx1 expression, while in the dorsal region, Mnx1 is expressed before Pdx1. The Raldh2 knockout model results in dorsal-specific dysplasia, which is attributed to the absence of Pdx1 and Prox1 expression in the dorsal bud (Martin et al., 2005, Molotkov et al., 2005). Furthermore, studies in chicks have shown that initial budding of the dorsal pancreas depends on selective repression of SHH in the dorsal midgut (Hebrok et al., 1998). Although it is unclear whether the murine system is conserved between species, a recent study using laser capture followed by deep sequencing analysis described some fundamental differences between the ventral and dorsal pancreas during human development (Jennings et al., 2017).

[0005] Despite the presence of different pathway utilization and distinct cell-intrinsic factors, the dorsal and ventral pancreatic programs have much in common. HNF1β (Tcf2) is required for pancreatic specification in both pancreatic buds and is critical during pancreatic development. Tcf2 knockout mice fail to generate a ventral pancreas and have greatly reduced dorsal buds that fail to differentiate or proliferate (Haumaitre et al., 2005). HNF1β is expressed in the anterior pancreatic foregut, where the gene acts at the apex of a sequential transcriptional cascade leading to activation of Hnf6 (Oc1) and then Pdx1 (Poll et al., 2006). Conditional inactivation of HNF1β results in loss of Glis3 and Ngn3 expression and leads to loss of the pancreas and anterior endocrine field characterized by a cystic duct (De Vas et al., 2015). In human development, the importance of HNF1β is highlighted by the emergence of the "maturity-onset diabetes of the young type 5" (MODY5) syndrome, a disorder attributed to mutations in the HNF1β gene. Although heterozygous mutations in HNF1β show no phenotype in mouse studies, in humans, heterozygous mutations in HNF1β have been shown to be associated with MODY5 or complete pancreatic dysgenesis, suggesting that the role of HNF1β in human pancreatic development is more important than that in mice (Body-Bechou et al., 2014). Summary of the invention

[0006] Provided herein are compositions, systems, kits, and methods for producing dorsal foregut endoderm (DFE) cells by contacting pluripotent stem cells with retinoic acid signaling pathway agonists and bone morphogenesis (BMP) pathway inhibitors without exposing stem cells to transforming growth factor β (TGFβ) pathway agonists. In certain embodiments, DFE cells are contacted with retinoic acid signaling pathway agonists and FGFR pathway inhibitors to produce pancreatic endoderm (PE) cells (e.g., with dorsal features (dorsal identity)). In other embodiments, PE cells are contacted with ALK5 inhibitors and Notch inhibitors to produce endocrine cells (e.g., endocrine cells expressing insulin). In certain embodiments, compositions, systems, kits, and methods for producing anterior domain endoderm (ADE) cells are provided.

[0007] In some embodiments, provided herein is a method for producing dorsal foregut endoderm cells, comprising: a) contacting a population of pluripotent stem cells (e.g., iPSCs or embryonic stem cells) with a retinoic acid signaling pathway agonist and a bone morphogenesis (BMP) pathway inhibitor; and b) culturing at least a portion of the pluripotent stem cell population to produce a dorsal foregut endoderm (DFE) cell population; wherein the stem cells are not exposed to a transforming growth factor β pathway (TGFβ) agonist during the culturing or contacting period.

[0008] In certain embodiments, provided herein is a composition comprising: a cell, wherein the cell comprises: i) an exogenous retinoic acid signaling pathway agonist, and ii) an exogenous bone morphogenesis (BMP) pathway inhibitor, and wherein the cell: i) does not contain any exogenous transforming growth factor β (TGFβ) pathway agonist, and ii) is a pluripotent stem cell (e.g., an iPSC or an embryonic stem cell) or a dorsal foregut endoderm cell.

[0009] In specific embodiments, the compositions provided herein include: a cell culture medium, wherein the cell culture medium includes: i) a retinoic acid signaling pathway agonist compound, and ii) a bone morphogenesis (BMP) pathway inhibitor; and wherein the culture medium does not contain or detectably does not contain any transforming growth factor β (TGFβ) pathway agonist. In specific embodiments, the cell culture medium includes a suitable energy source and a compound that regulates the cell cycle. In certain embodiments, the cell culture medium includes most or all of the following: amino acids, vitamins, inorganic salts, glucose, and serum as sources of growth factors, hormones, and attachment factors. In some embodiments, the composition further includes: a cell population, wherein the cells are pluripotent stem cells (e.g., iPSC or embryonic stem cells) or dorsal foregut endoderm (DFE) cells. In other embodiments, the cells are pluripotent stem cells, and wherein the retinoic acid signaling pathway agonist and the BMP pathway inhibitor are present in the culture medium at a concentration that causes at least a portion of the pluripotent stem cells to become DFE when they are cultured in the culture medium for at least 2 days.

[0010] In other embodiments, provided herein are kits and systems comprising, consisting essentially of, or consisting of: a) a population of pluripotent stem cells (e.g., iPSCs or embryonic stem cells) present in a cell culture vessel; b) a retinoic acid signaling pathway agonist compound present in a first vessel; and c) a bone morphogenesis (BMP) pathway inhibitor present in a second vessel; and wherein the cell culture vessel is free of any or any detectable exogenous transforming growth factor β (TGFβ) pathway agonist, and wherein the kits and systems are free of any transforming growth factor β (TGFβ) pathway agonist in the first or second vessel or in the third vessel. In additional embodiments, the kits and systems further include a culture medium within the culture vessel.

[0011] In other embodiments, the BMP pathway inhibitor is a BMP4 pathway inhibitor. In certain embodiments, the BMP pathway inhibitor comprises LDN193189. In additional embodiments, the BMP pathway inhibitor is selected from the group consisting of: DMH1, DMH2, Dorsopmorphin, K02288, LDN214117, LDN212854, folistain, ML347, Noggin. In some embodiments, the retinoic acid signaling pathway agonist is selected from the group consisting of: i) retinoid compounds, ii) retinoid X receptor (RXR) agonists, and iii) retinoic acid receptor (RAR) agonists. In specific embodiments, the retinoic acid signaling pathway agonist is selected from the group consisting of: retinoic acid, Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxo retinoic acid and all-trans retinoic acid (ATRA). In specific embodiments, culturing is performed for 1-5 days (eg, 1 day, 2 days, 3 days, 4 days, or 5 days) or 2-4 days.

[0012] In some embodiments, the method further comprises: c) contacting at least a portion of the DFE cell population with a retinoic acid signaling pathway agonist and a FGFR pathway inhibitor; and d) culturing at least a portion of the DFE cell population to produce a pancreatic endoderm (PE) cell population. In certain embodiments, the DFE cells are not exposed to a transforming growth factor β (TGFβ) pathway agonist during the culture period of step d) or the contact period of step c). In some embodiments, the culture in step d is performed for about 1-5 days (e.g., 1 day, 2 days, 3 days, 4 days, or 5 days) or 2-4 days. In certain embodiments, the PE cells have dorsal pancreatic features. In some embodiments, the FGFR pathway inhibitor is selected from the group consisting of: PD0325901, Arctigenin, PD184352, PD198306, PD334581, SL 327, U0126, MEK inhibitors, FGFR inhibitors, and MAPK inhibitors. In certain embodiments, the contacting in step c) further comprises contacting the DFE cell population with a sonic hedhog (SHH) pathway inhibitor.

[0013] In certain embodiments, the method further comprises: e) contacting at least a portion of the PE cell population with a Notch pathway inhibitor and an ALK5 inhibitor; and f) culturing at least a portion of the PE cell population to produce an endocrine cell population. In some embodiments, the endocrine cells are cells that express insulin. In other embodiments, the endocrine cells are fetal β cells. In certain embodiments, the Notch pathway inhibitor includes a gamma secretase inhibitor XX, also known as DBZ. In other embodiments, the Notch pathway inhibitor is selected from the group consisting of: DAPT, MRK-003, MRK-0752, z-Ile-leu-CHO, a gamma secretase inhibitor, L-685,485, LY411575, Compound E, F-03084014, RO4929097, and BMS-906024. In certain embodiments, the ALK5 inhibitor comprises A8301. In another embodiment, the ALK5 inhibitor is selected from the group consisting of A7701, A83-01, SB505124, SB431542, Alantolactone and LY2157299. In a further embodiment, the culturing in step f is performed for 5-15 days (e.g., 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or 15 days).

[0014] In some embodiments, cells (e.g., endocrine cells) produced by the methods herein are transplanted into a subject to provide therapeutic benefit. In some embodiments, when the subject suffers from diabetes, the transplanted cells are cells that express insulin.

[0015] In certain embodiments, the methods herein further comprise: c) contacting at least a portion of the DFE cell population with EGF; and d) culturing at least a portion of the DFE cell population to generate a gastric endoderm (SE) cell population.

[0016] In other embodiments, the methods herein further comprise: c) contacting at least a portion of the DFE cell population with BMP4 or a BMP pathway agonist (eg, sb4); and d) culturing at least a portion of the DFE cell population to produce a hepatic endoderm (LE) cell population.

[0017] In other embodiments, the methods herein are used to generate anterior definitive endoderm (ADE). Anterior definitive endoderm is located more anterior to DFE and DE and encompasses corresponding cells of pharyngeal endoderm. The ADE expresses specific marker genes, such as TBX1, PAX9 and OSR1, and represents the progenitor cells of the cell lineage of the pharyngeal region (including but not limited to the lung, thymus and thyroid).

[0018] In some embodiments, provided herein is a method for producing anterior domain endoderm cells, comprising: a) contacting a pluripotent stem cell population with: i) Nodal protein and BMP protein or BMP pathway agonist, or one or more vectors encoding the Nodal protein and the BMP protein or BMP pathway agonist (if it is a protein), and ii) an ALK5 inhibitor; and b) culturing at least a portion of the pluripotent stem cell population to produce anterior domain endoderm (ADE) cell population. In certain embodiments, during the culturing or the contacting, the pluripotent stem cells are not exposed to: i) a BMP inhibitor, and / or ii) a transforming growth factor β (TGFβ) pathway agonist.

[0019] In certain embodiments, compositions are provided herein comprising: a) Nodal protein or a vector encoding Nodal protein; b) BMP protein or BMP pathway agonist or a vector encoding BMP protein or BMP agonist (if it is a protein), and c) an ALK5 inhibitor. In some embodiments, the composition does not contain: a BMP inhibitor, and / or ii) a transforming growth factor β (TGFβ) pathway agonist. In certain embodiments, a single vector encodes a Nodal protein and a BMP protein (or a BMP pathway agonist, if it is a protein), while in other embodiments, a separate vector is used. In other embodiments, the composition further comprises: a cell population, wherein the cells are pluripotent stem cells or anterior domain endoderm (ADE) cells.

[0020] In some embodiments, provided herein is a kit or system consisting essentially of or consisting of: a) a population of cells present in a cell culture container; b) a Nodal protein or a vector encoding a Nodal protein present in a first container; c) a BMP protein or a BMP pathway agonist or a vector encoding a BMP protein or a BMP agonist (if a protein) present in a second container; and d) an ALK5 inhibitor present in a third container.

[0021] In certain embodiments, the cell culture container does not contain any or any detectable exogenous BMP inhibitor and / or exogenous transforming growth factor β (TGFβ), and wherein the kit and system do not contain any detectable exogenous BMP inhibitor and / or exogenous transforming growth factor β (TGFβ) in the first, second, third container or in the fourth container. In other embodiments, the cells are pluripotent stem cells or anterior domain endoderm (ADE) cells. In further embodiments, the ALK5 inhibitor is selected from the group consisting of: A7701, A-83-01, SB505124, SB431542, scutellariae lactone and LY2157299. In additional embodiments, the BMP protein is a BMP4 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The patent or application file contains at least one drawing printed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0023] Figure 1 An exemplary method for generating dorsal foregut endoderm (DFE) cells is shown.

[0024] Figure 2 shows the results of effective endoderm induction in the absence of AA / WNT. A: Schematic diagram of endoderm genes induced by activin A induction of the anterior primitive streak (APS) and optimized HNF1β induction. B: Predicted conditions that meet the conditions for APS gene induction (top circles) and HNF1β induction (bottom circles). C: Validation of induction of endoderm markers using two separate protocols. D: Coefficient plot for selecting endoderm genes (main effectors only).

[0025] Figure 3 Figure 3. Non-APS-derived endoderm is shown to be severely activated by retinoic acid and BMP inhibition and to display dorsal foregut characteristics. A: Schematic diagram of the experiments performed. B: Graph showing the relative expression of several endoderm genes that respond to retinoic acid and BMP inhibition. Genes were normalized to the average expression of endogenous levels of YWHAZ, GAPDH, and TBP: DE - definitive endoderm, RA - retinoic acid, LDN - LDN3189 (a BMP inhibitor), and WIN - Win XXX (an ALDH inhibitor). C: Heatmap containing key endoderm genes from pluripotent cultures that were either subjected to classical definitive endoderm differentiation conditions or differentiated using conditions predicted in the HNF1β optimizer.

[0026] Figure 4 shows that DFE can generate pancreatic endoderm with highly dorsalized properties. A: Schematic diagram showing the optimization modeling of gastric genes SOX2, OSR1 and pancreatic gene PDX1. B: The corresponding optimizers for the predicted maximum induction of SOX2, OSR1 and PDX1, respectively. C: Dynamic map of the effectors most responsible for the activation of the corresponding genes. D: PDX1 Opt Representative IHC of A. E: KeyGenes predictions of the corresponding DE- and DFE-derived pancreatic endoderm. F: Heatmap evaluating the differential expression of several pancreatic and dorsal-specific pancreatic genes between the two protocols.

[0027] Figure 5 Results are shown for the efficient and rapid induction of pancreatic endocrine fate from pluripotency via DFE endoderm using small molecules. A: Schematic showing the responses performed to alter pancreatic induction. B: C-peptide levels detected across different experimental conditions as determined by ELISA. C: QuantStudio-based expression levels of several key pancreatic and endocrine-specific genes normalized to the average expression of GAPDH, TBP, and YWHAZ.

[0028] Figure 6 Results showing that DFE-derived endocrine cells have an immature phenotype are shown. A: Schematic diagram showing the directed differentiation protocol used to generate the endocrine domain. B: IHC evaluation of endpoint cultures of various endocrine specific factors. C: Endocrine induction was measured daily for 14 days by ELISA for c-peptide production. D: Retention of dithizone by multipotent derivatives grown as aggregates. E: Glucose stimulated insulin release assay (GSIS) assessment of endocrine function of endocrine cultures. F: Microfluidic analysis of internal calcium release in response to glucose and potassium challenge.

[0029] Figure 7 The results of the bioequivalence test of the small molecule-based pancreatic endocrine induction protocol are shown. A: A schematic diagram showing the reactions performed, where HNF1β Opt and PDX1 Opt Only the factors with the highest predictor contribution from the modeling experiments (retinoic acid and LDN3189 or retinoic acid and PD0325, respectively) were replaced. B: QuantStudio-based transcriptional analysis of the four corresponding reaction conditions performed at the PE stage (in light grey) or the endocrine stage (in dark grey) as normalized to the average expression of GAPDH, TBP and YWHAZ is shown.

[0030] Figure 8 The kinetics of DFE induction are shown. A: shows the kinetics of DFE induction using HNF1βopt A: Graph of the relative percentage of FOXA2, HNF1β, HLXB9 and OCT3 / 4 positive cells during the 4-day incubation period. B: Representative IHC of HNF1β, FOXA2 and HLXB9 at day 3 of the differentiation protocol.

[0031] Fig. 9 Protocol input showing DFE does not alter patterning of DE. A: Representative IHC showing differential expression of TGFβ or retinoic acid responsive genes.

[0032] Fig.10 Showing that the patterning ability of DFE is different from that of DE-derived endoderm A: Schematic diagram showing how HNF1β-optimized cultures or cultures differentiated into definitive endoderm respond to stimulation for differentiation toward gastric, pancreatic, or liver fates. B: Relative expression of selection markers for genes indicated between different protocols. Blue bars represent cultures that were provided conditions designed to differentiate toward gastric differentiation. Yellow bars are cultures that were provided conditions designed to generate pancreas. Red bars represent cultures that were provided conditions designed to generate liver. C: Schematic diagram of subsequent experiments in which organoids were derived from HNF1β stimulated with gastric inducing conditions. opt D: DFE-derived organoids that have been differentiated and grown for 23 days. E: Representative organoid staining for gene markers of PSC-derived putative antral PDX1+ / SOX2+ / OSR1+ stomach.

[0033] Fig.11 Showing the enhanced pancreatic competence of DFE-derived endoderm compared to DE-derived endoderm, A: Schematic showing four different early endoderm genes that were optimized by computer modeling and subsequently stimulated for pancreatic competence. B: IHC validation of four corresponding cultures for the general endoderm gene FOXA2. C: IHC validation of four corresponding cultures for the pancreatic-specific gene PDX1.

[0034] Fig.12 Showing the results of robust and uniform induction of pancreatic fate from DFE using iPSCs and female hESCs A, A: HNF1β opt A: IHC validation of the protocol between pluripotent cell lines in which the corresponding expression levels of three different key pancreatic factors were upregulated. B: IHC validation of pancreatic induction from DFE between pluripotent cell lines in which the corresponding expression levels of three different key pancreatic factors were upregulated.

[0035] Fig.13Hierarchical clustering comparing pluripotent derived cells to several primary tissues is shown. Hierarchical clustering was performed on the KeyGenes dataset in R. Since all pluripotent derived samples belonged to the same cluster containing human fetal stomach, fetal pancreas, and adult islet samples, only this cluster region is shown.

[0036] Fig.14 It is shown that retinoic acid metabolites are effective in activating dorsal foregut endoderm. A: Schematic diagram of the experiments performed. B: HNF1β used in the experiments described in panel A Opt C: Graph showing the differential effects of different retinoids on activation of selected endoderm genes. D: Comparison of original HNF1β Opt Corresponding IHC validation of protein levels between conditions predicted in the current experimental design. E: for HNF1β called old and new Opt The additive compound of the reaction conditions.

[0037] Fig.15 All trans-retinoic acids are shown to activate Nodal. All trans-retinoic acids and retinoic acid metabolites have differential ability to activate the primitive streak marker Nodal. A: Schematic diagram of the experiments performed. B: Relative expression of selected endoderm genes compared to retinoid-based induction. C: Schematic diagram of the effects of retinoid-based induction on NODAL. opt Schematic diagram of the experiments performed for the conditions.

[0038] Fig.16 Shown is the patterning of the endoderm fate space by initiation of gastrulation. A: PSC cultures were exposed to a matrix of DoE-defined compounds known to initiate or inhibit gastrulation. B: tSNE plots of all optimizers obtained for the assayed endoderm genes. C: Corresponding heatmaps showing which genes segregate into each of the four defined populations shown as defined in the tSNE plots.

[0039] Fig.17 Contributions of Activin A, BMP4 and Retinoic Acid to patterning endoderm during gastrulation are shown. The relevant factor contributions obtained from DoE-based modeling experiments determining the role of Activin A, BMP4 and Retinoic Acid during in vitro gastrulation events are shown in the table.

[0040] Fig.18.Quantification of DFE protocols. A: Representative IHCs from four different DFE protocols as described in the schematic above the images. A': Corresponding quantifications of the different protocols from panel A. B: Representative IHCs from four different DFE protocols further differentiated to pancreatic progenitor cells as detailed in the schematic above the images. B': Corresponding quantifications of the different protocols from panel B. C: Representative IHCs from four different DFE protocols further differentiated to pancreatic progenitor cells using only the key process parameters as defined by the differentiation model and detailed in the schematic above the images. C': Corresponding quantifications of the different protocols from panel B.

[0041] Fig.19 . Relative effector contributions defining representative patterned endoderm populations. A: Ridge plot showing the relative levels of different effectors used throughout the experiment. Different plots are re-directed to optimizer clustering and colors match different clusters. B: For ease of reference, the original clusters are shown again and the corresponding cluster numbers (0-3) are provided.

[0042] Fig. 20 .Evaluation of expression patterns within endoderm clusters. A: For ease of reference, the original clusters are shown again and the corresponding cluster numbers (0-3) are provided. B: Cluster 0 consists of dorsal foregut endoderm (DFE). C: Cluster 1 consists of anterior midgut endoderm cluster (ADE). D: Expression patterns of primitive streak markers Nodal and ALDH1A2.

[0043] Fig.21 Anterior and midgut genes are induced in the absence of AA. Violin plots define the relative expression levels in the different clusters of select endoderm genes.

[0044] Fig. 22 Anterior endoderm genes are responsive to BMP4 and Nodal. Twenty independent optimizers are shown for each gene annotated in a single figure, focusing on the predicted concentrations of the TGFβ family ligands AA, Nodal, and BMP4 involved in activation of the corresponding gene.

[0045] Fig.23 AA, BMP, Nodal, and retinoic acid all have unique responsive gene networks. The effector contributions from all corresponding optimizers for the measured endoderm genes were normalized and ranked from the most responsive genes to the least responsive genes.

[0046] Fig.24AA-stimulated pluripotent cells activate several TGFβ family ligands. A: RNA Seq data were used to determine the expression patterns of TGFβ family ligands activated under DE and DFE conditions. Genes were ranked from most to least expressed transcripts in the DE population, and undetected levels were removed from the heatmap. B: IHC confirmed the expression of Nodal in AA-stimulated pluripotent cells.

[0047] Fig.25 . Anterior and midgut patterning occurs in the absence of AA signaling. Representative optimizers of master regulators of anterior and midgut genes. DETAILED DESCRIPTION

[0048] Provided herein are compositions, systems, kits, and methods for producing dorsal foregut endoderm (DFE) cells by contacting pluripotent stem cells with retinoic acid signaling pathway agonists and bone morphogenesis (BMP) pathway inhibitors without exposing stem cells to transforming growth factor β (TGFβ) pathway agonists. In certain embodiments, DFE cells are contacted with retinoic acid signaling pathway agonists and FGFR pathway inhibitors to produce pancreatic endoderm (PE) cells (e.g., with dorsal features (dorsal identity)). In other embodiments, PE cells are contacted with ALK5 inhibitors and Notch inhibitors to produce endocrine cells (e.g., endocrine cells expressing insulin). In certain embodiments, systems, kits, and methods for producing anterior domain endoderm (ADE) cells are provided.

[0049] The derivation of endoderm and offspring organs (such as pancreas, liver and intestine) is affecting disease modeling and regenerative medicine. The common method for inducing definitive endoderm from pluripotent cells always involves the use of TGFβ signaling to excite, the most common being activin A. Work performed during the development of the embodiments described herein determined that activin A is not necessary, but BMP antagonism and retinoid input can be successfully used to cause the induction of dorsal foregut endoderm (DFE), and the endoderm induced by activin causes cells showing ventral heterogeneous characteristics. This shows that pancreatic features can be induced quickly and strongly from DFE, and such cells have dorsal pancreatic features. In this work, the DFE group is very capable of differentiating into gastric organoids and pancreatic tissue types, and can effectively produce fetal β cells by two subsequent differentiations based on a small molecule process. The generation of such pancreatic insulin-producing cells can be used, for example, for cell-based therapies for patients with diabetes.

[0050] The work carried out in the development process of the embodiments herein has proved that the patterned endoderm of effective and regionalization can be directly and powerfully differentiated from pluripotent cells without using TGFβ agonism, and pancreas and other endoderm derivatives can be produced from such cells. The embodiments herein provide a robust scheme for inducing the specialized human endoderm representing the dorsal foregut region of the intestinal tube. The cells of such generation can be effectively converted into dorsal pancreatic progenitor cells, which can then adopt the endocrine fate including the generation of fetal-like β cells.

[0051] The present disclosure is not limited by the type of culture medium or culture system adopted. Any suitable culture medium and culture system can be used. For example, it is understood by those skilled in the art that a variety of methods for cultivating and expanding pluripotent stem cells (using and not using mesenchymal feeder cells, and using and not using conditions determined by complete chemical composition, such as culture in the presence of serum or other complex additives that are not completely determined) are available. In addition, a variety of flexible growth medium bases (growth media base) are available, examples of which have mixed culture media (e.g., culture medium mixtures, such as CDM2 used therein), and specific culture media (such as Essential-8, Essential-6, TesR culture media), and various supplements (such as KOSR), which are used to support the expansion of pluripotent stem cell states, regardless of cell source (such as ESC or iPSC). In certain embodiments, a series of continuous, essentially continuous specific culture media can be used, all of which have potential basal culture media, which can be flexibly explored from widely used cell culture media such as RPMI1640, DMEM, F2 and other culture media. Such media are then provided with a range of metabolic support molecules, such as pyruvate, anaplerotic agents such as glutamine, chemically defined lipids, fuel sources such as glucose, and in some cases chemically defined growth support agents, such as B27, as is routinely performed by those skilled in the art. In certain embodiments, a variety of basal media are used along with metabolic support molecules.

[0052] In certain embodiments, the cells produced by the methods and compositions herein are endocrine pancreatic insulin-producing cells. In a specific embodiment, such cells are macroencapsulated as known in the art and then transplanted into patients with diabetes (see, e.g., Hwa et al., Curr Diab Rep. June 16, 2018; 18(8):50, which is incorporated herein by reference). In other embodiments, insulin-producing cells are transplanted into the liver of a subject (e.g., a subject with diabetes). In other embodiments, such cells are subcutaneously transplanted into a protective device, as described in clinical trial NCT03163511. In yet other embodiments, such cells are transplanted into microencapsulated spheres, as described in clinical trial NCT01739829. In other embodiments, such cells are implanted into an oxygen supply device, as described in clinical trial NCT02064309. In other embodiments, such cells can be transplanted (e.g., subcutaneously, intraperitoneally, intravenously) to a suitable site without physical protection but in the presence of an immunomodulator. In other embodiments, such cells can be transplanted using a retrievable polymer nanoporous wire coated with alginate and implanted intraperitoneally as described in An et al., Proc Natl Acad Sci US A. January 9, 2018; 115(2): E263-E272 (incorporated herein by reference). In some embodiments, the diabetic patient can be a type II diabetic patient without autoimmune signs. In some embodiments, the cell can be derived from a pluripotent stem cell reprogramming process and has a genetic composition similar to that of the recipient.

[0053] In certain embodiments, certain cells replace pluripotent stem cells as the original cell group to be processed. Such cells include, but are not limited to, EP (endodermal progenitor cells, such as described by Cheng et al., Cell Stem Cell. April 6, 2012; 10(4): 371-84 (which is incorporated herein by reference)); EPIBLAST stem cells, such as described by Tesar Proc Natl Acad Sci USA. June 7, 2005; 102(23): 8239-44 (which is incorporated herein by reference); or embryonic germ cells, such as described by Kimura et al., Stem Cells. October 2014; 32(10): 2668-78 (which is incorporated herein by reference). The present disclosure is not limited by the species from which the pluripotent stem cells or other cells described above are derived. All vertebrate species produce similarly patterned endoderm, including the formation of those described therein, located in the front of the derivatives, the production of anterior endoderm descending tissue (anterior endodermal descending tissue), all vertebrate species produce cells of this endoderm, the cells produce insulin molecules that control glucose homeostasis. In some embodiments, the production of pancreatic endoderm, pancreatic insulin-producing cells from non-human species, when such as combined with microencapsulation devices, large encapsulation devices or immunosuppressive drug regimens, or alternatively, when genetically modified to achieve the loss of immune detection, can be advantageously used to provide a means of deriving cell-based therapies. In addition, regardless of the species source, such cells can be used as the basis for drug discovery of insulin-producing cells derived in vitro, and thus lead to the identification of novel drugs that affect diabetic states.

[0054] In certain embodiments, insulin-producing cells produced by the methods and compositions described herein are used with the technology of any of the following companies: 1) Seraxis (cell replacement therapy for type 1 diabetes); 2) Unicyte AG (cell therapy for diabetes); 3) ViaCyte (insulin for diabetes); 4) Sernova (stem cells for the treatment of diabetes); 5) Betalin Therapeutics (insulin production); 6) AltuCell (stem cell therapy for type 2 diabetes); 7) NextCellPharma AB (stem cell therapy for type 1 diabetes); 8) Osiris Therapeutics (type 1 diabetes control); 9) Mesoblast (type 2 diabetes treatment); 10) Evotec and Sanofi (stem cells for diabetes); 11) Orgenesis (insulin-producing cells); 12) Semma Therapeutics (type 1 diabetes therapy); 13) NovoNordisk (treatment for type 1 diabetes); 14) Beta-O2 (artificial pancreas for diabetes); 15) Eli Lilly andSigilon (stem cells for diabetes).

[0055] Example

[0056] Example 1

[0057] Robust and rapid induction conditions for dorsal pancreatic endoderm from pluripotent cells

[0058] This example describes the generation of dorsal foregut endoderm (DFE) cells from pluripotent stem cells. This example also describes the generation of dorsal pancreatic endoderm (DFE) cells from DFE cells.

[0059] result

[0060] There are two separate and distinct pathways for endoderm induction

[0061] We initially set out to predict the conditions required to define the definitive endoderm population of the progeny of the primitive streak, as suggested by the literature. This was achieved by modeling maximal expression of MESP1, EOMES, and BRACHYURY / T while minimizing EVX1 (a marker of the posterior primitive streak) to define the anterior primitive streak (APS) population (Loh et al., 2014). By ISPA, the conditions predicted to generate this differentiation event consisted of low tolerance to Wnt inhibition (FC = 18.65) and high Activin A levels (FC = 18.85) ( Figure 2A and Figure 2B). These conditions are consistent with current protocols for generating definitive endoderm (DE) (D'Amour et al., 2005), which rely on Nodal signaling. In fact, when differentiating pluripotent cultures using these conditions, the majority of cultures converted to a FOXA2+ / SOX17+ population within 3 days ( Figure 2C By ISPA, many other genes were predicted to be highly expressed under these conditions (carrier genes), including COL6A1, HHEX, MESP2, SOX17) ( Figure 2D and data not shown). Notably, POU5F1 (OCT4) remained high, consistent with a node-like state. These genes are known to be elevated in direct descendants of the mouse after gastrulation. However, through ISPA, we noticed that not all genes known to be expressed in the early endoderm were uniquely maximized by the APS condition. Examining the known space from the above experiments yielded a completely different set of solutions focused on HNF1β (TCF2) expression, also known to be expressed in the definitive endoderm. Maximizing HNF1β expression resulted in expression of concomitant genes such as FOXA2, HNF4a, MNX1, CXCR4, MTF1 (all of which are known markers of endoderm), while expression of HHEX and SOX17 remained low (ISPA results not shown). We continued to characterize these unique states, and the requirements for their induction.

[0062] Retinoic acid and Bmp inhibition synergistically induce an endodermal program that is mutually exclusive with activin-induced endoderm.

[0063] Using ISPA, we extracted the underlying logic governing endoderm gene induction. For APS-derived DE, it was clear that many early endoderm genes were under the direct control of TGFβ signaling, showing strong positive coefficient terms from AA in their complex regulatory model. These genes include, but are not limited to, SOX17, CXCR4, LEFTY1, MIXL1, and HHEX ( Figure 2D ). This is not the case for a variety of other known endoderm markers. A subset of endoderm genes is unresponsive to activin A stimulation but is directly dependent on retinoic acid signaling and, to a lesser extent, requires inhibition of the BMP pathway. These genes include FOXA2, EPCAM, ONECUT1, CDX2, and MNX1 ( Figure 2D ), and these genes are predicted to be directly controlled by the synergistic effects of retinoic acid and BMP inhibition, with high factor contributions including FOXA2 (FC=24.8 in RA, BMPi=25.3), HNF1β (FC=30.1 in RA, BMPi=31.7), MNX1 (FC=22.7 in RA, BMPi=30.5) ( Figure 2B, data not shown). Notably, Activin A was not predicted to contribute to activation of these genes, whereas inhibition of the TGFβ pathway was predicted to favor expression of these genes, with Alk5i contributing a factor of 7.59 to HNF1β, 8.88 to FOXA2, and 18.98 to MNX1 ( Figure 2B The predicted HNF1β optimization (HNF1β) was tested on differentiated pluripotent cells. Opt ) conditions to validate based on protein expression, where HNF1β, MNX1, and FOXA2 can all be activated and cells can be efficiently transformed. Opt Application of these conditions resulted in the majority of cells being HNF1β+ ( Figure 8 ), while APS differentiated cells showed only weak HNF1β expression ( Figure 2C ).

[0064] The two pathways of endoderm activation are fundamentally different and based on conflicting input logics, suggesting that the pathways are mutually exclusive. We tested this by creating hybrid protocols that measured the effects of RA and BMPi in the presence of activin A ( Figure 3 A). Inclusion of retinoic acid in the APS-based DE generation protocol (D'Amour et al., 2005) was shown to only modestly increase gene expression of retinoic acid-responsive genes, with only CDX2 (data not shown) and OSR1 ( Figure 3 In contrast, when retinoic acid was included in the APS-type DE generation response including HHEX, SOX17 and GSC, known TGFβ-responsive genes showed significant downregulation ( Figure 3 B and Supplementary Fig. 2). This suggests that the presence of either key program driver (AA vs. RA) inhibits the other. Furthermore, as also predicted by ISPA, genes upregulated in the presence of retinoic acid and LDN3189 were activated more efficiently when activin A was excluded from these responses. Importantly, HNF1β OPT Two key conditions: input of RA and LDN3189 is sufficient to effectively initiate differentiation ( Figure 3 B).

[0065] Retinoic acid / BMP inhibition induces endoderm with dorsal foregut characteristics

[0066] To better understand the different nature of the endoderm populations, we performed RNA sequencing on the cultures. Common endoderm genes expressed in both populations included CXCR4, FOXA2, EPCAM, GATA4, and GATA6 ( Figure 3C) However, significant differences were observed in genes associated with patterning, suggesting that HNF1β Opt The induced endoderm was enriched for genes known to be specific to dorsal (MNX1 and PAX6) and foregut endoderm HOXA1, HOXA3, HNF4A, and HNF1β ( Figure 3 C). In contrast, APS-type DE showed enrichment for genes representing the ventral endoderm NR5A2, HHEX, and SOX17, as well as more posterior endoderm (SOX17 and AFP, but not CDX1) ( Figure 3 C) Due to HNF1β Opt The cultures appeared to have a more dorsal foregut endoderm (DFE) phenotype, so we stimulated the ability of this culture to differentiate into stomach, pancreas, and liver (tissues derived from the posterior foregut) using conditions previously shown to induce these fates in APS-derived DE (Supplementary Figure 3 A) and compared with APS-derived DE. Significant differences in potency were observed when DFE cohorts were compared with APS-DE cohorts. In all cases, APS-DE cultures activated liver genes (APOB, HHEX, and EVX1 (Supplementary Figure 2) more efficiently than DFE. Figure 3 However, gastric (OSR1) and pancreatic (PDX1) were more efficiently activated in DFE cultures. When DFE cultures were stimulated to generate gastric organoids ( Fig.10 C and Fig.10 D, (McCracken et al., 2014)), co-expression of OSR1 and PDX1, as well as co-expression of SOX2 and PDX1, was observed, indicating that gastric organoids preferentially transformed into the antral-type, most posterior stomach ( Fig.10 E).

[0067] Mechanisms of organ territory specification derived from the dorsal foregut endoderm

[0068] When stimulated for pancreatic induction, DFE-patterned cultures had a stronger ability to activate PDX1 compared to conventional DE induction methods ( Fig.10 A). We therefore returned to ISPA to investigate pancreatic induction by optimizing different early endoderm markers at stage 1 to determine which markers are best suited for pancreatic transformation. We used ISPA to identify and compare independent conditions that maximized expression of HNF1β or OC2 as representative genes under the control of retinoic acid, or optimized expression of NR5A2 or SOX17 as representative genes under the control of TGFβ signaling ( Fig.11A). By first exposing pluripotent cultures to induction conditions for each of the four genes (3 days), we then induced pancreatogenesis using previously defined conditions (Mfopou et al., 2010). Cultures optimized for either HNF1β or OC2 showed much more uniform induction of the pancreatic markers PDX1 and FOXA2 ( Fig.11 B and Fig.11 C) Since we observed that DFE cultures were capable of both gastric and pancreatic induction, we next evaluated this bipotential property through sequential DoE modeling experiments performed on DFE cultures.

[0069] The results from ISPA maximization of SOX2, OSR1, or PDX1 expression were compared ( Figure 4A and Figure 4B ). Dynamic graph analysis ( Figure 4C ) showed that OSR1 (stomach) and PDX1 (pancreas) were under similar control mechanisms. Both genes responded strongly to RA, with FC=30.68 and FC=31.38, respectively ( Figure 4B and Figure 4C ). However, differential responses to FGF signaling were predicted, with FGF4 predicted to be important for OSR1(stomach) activation with an FC of 13.45, while MEK pathway inhibition was shown to contribute substantially to PDX1 activation, with the small molecule inhibitor PD0325901 having a substantial FC of 29.62 ( Figure 4B and Figure 4C ). Thus, the dual potential of gastric / pancreatic fate is resolved by FGF / FGFi input, respectively. Notably, the expression of SOX2 is strongly decreased by RA and highly regulated by SHH, with corresponding FCs of -28.59 and 29.58 ( Figure 4B and Figure 4C ). SOX2 is a persistent marker of the esophageal and fundic stomachs and is therefore located just anterior to the antral region expressing PDX1 in rodents (McCracken et al., 2014). Our data suggest that retinoic acid protects the posterior antral region and that active inhibition of SHH promotes downregulation of SOX2 expression, allowing the switch from the gastric to the pancreatic region. This observation is supported by developmental studies in the chick (Hebrok et al., 1998).

[0070] DFE-derived pancreas has dorsal features

[0071] Use the PDX1 optimizer conditions defined by ISPA (such as Figure 4B), demonstrating that DFE-derived PE cultures expressing PDX1 co-expressed several known pancreatic progenitor markers including FOXA2, NR5A2, GATA4, and SOX9, indicating that a bona fide PE state was rapidly induced ( Figure 4D This DFE-derived pancreatic induction was shown to be reproducible in the H9 female embryonic stem cell line as well as in iPSC cultures ( Fig.12 RNASeq-based KeyGenes analysis (Roost et al., 2015) was used to validate that this DFE-derived PDX1 induction was a bona fide pancreatic fate ( Figure 4E We compared pancreatic endoderm (PE) derived from DFE with stage 4 PE derived from APS-type DE as previously published (Xie et al., 2013). Interestingly, both starting populations, DE and DFE cultures, initially displayed limited brain features that were lost at successive stages in both protocols ( Figure 4E This may be attributed to the emergency expression of neuronal fate repressor genes (Jennings et al., 2017), which are activated at one or more successive stages in both populations ( Figure 4F ). The published APS-derived DE population showed similarity to the lung, whereas the DFE population did not ( Figure 4E ). This is presumably because the lung buds are derived exclusively from the ventral endoderm. Hierarchical clustering comparisons between DFE, DE, and their derived PE populations showed that the DFE and DE populations were so similar that the DFE population clustered between triplicate DE sample groups ( Fig.13 ), demonstrating that both ventral and dorsal programs acquire a basic endoderm program. However, when PE derived from DFE or DE were compared to genes highly enriched in the dorsal pancreas during human development (Jennings et al., 2017), we found that the DFE population already expressed several of these genes, including DLL1, CNR1 FRZB, HOXA1, and ARMC3, while the APS-DE population showed only low expression of CNR1 and FRZB. In addition, expression of MNX1 (Hlxb9), a previously described dorsal marker, was expressed throughout DFE culture, while DE-derived PE only began to have detectable MNX1 expression at the PFG stage ( Figure 4F ). Subsequently, PE derived from DFE continues to express most dorsal-specific genes ( Figure 4F). When compared to current pancreatic DD protocols (Xie et al., 2013), we conclude that DE-derived PE adopt a pancreatic phenotype at the published ‘PGT’ stage as demonstrated by expression of HHEX, HNF1b, RFX6, HNF4A, PDX1, PROM1, and PROX1, but have no discernible dorsal features until cultures are exposed to retinoic acid at the ‘PFG’ stage. Considering the results of ISPA as previously described, although the present invention is not limited to any particular mechanism, and understanding of the mechanism is not necessary to understand or practice the present invention, this implies that the dorsal phenotype previously attributed to these protocols is the result of redirecting the population toward a dorsal fate later in the protocol.

[0072] The effective induction of endocrine pancreas by PE derived from DFE was time-dependent.

[0073] To further validate the pancreatic progenitor cell identity, we characterized the forward differentiation potential of DFE-derived PEs toward terminal pancreatic fate using pancreatic lineage-specific markers. The importance of NOTCH pathway inhibition for terminal differentiation of endocrine cells is well known (Jensen et al., 2000, Afelik et al., 2012), so we used Figure 5 The three media inputs outlined in A (all three media inputs contained a NOTCH pathway inhibitor (g-secretase inhibitor XX)) evaluated the temporal effects of NOTCH inhibition on endocrine commitment and evaluated single SMAD inhibition (Alk5 inhibitor) and dual SMAD inhibition (Alk5i / LDN). Regardless of the conditions, these media were able to induce endocrine differentiation. Single SMAD inhibition appeared to be superior to dual SMAD inhibition, and the absence of SMAD inhibition resulted in a slight increase in acinar differentiation ( Figure 5 B to Figure 5 C). Because multiple developmental systems (e.g., somitogenesis, neural development) depend on the total duration of NOTCH signaling, we varied the length-of-stay during pancreatic induction, i.e., the time at which the NOTCH pathway begins to become active. Strikingly, prolonging the PE induction period reduced endocrine competence. These data reveal that, following PDX1 activation, cultures progress through a competence window that, if prolonged, results in loss of endocrine fate even under permissive endocrine induction conditions ( Figure 5C). Notably, increasing the duration of the PE stage resulted in an increased capacity towards acinar and finally ductal differentiation. When PE cultures remained able to engage NOTCH signaling for up to 5 days before being subjected to NOTCH inhibition, an increase in acinar capacity as measured by expression of CPB1 and MIST1 (BHLHA15) was observed. Figure 5 C). This implies that endocrine lineage competence is temporally regulated first before the acinar lineage and, subsequently, ultimately towards the ductal lineage ( Fig.14 A and Fig.14 C). Ductal differentiation, as observed by expression of F3, HNF1β, and PROM1, increases as the PE population is allowed to remain in an active NOTCH PE state ( Figure 5 C), which is consistent with NOTCH being permissive for ductal fate (Afelik et al., 2012).

[0074] Dorsal-type PE-derived β cells are fetal-like.

[0075] Given that conditions for forward differentiation toward an endocrine fate rely primarily on inhibition of NOTCH signaling and have not been optimized using a QbD approach, we examined the resulting endocrine output and also determined the maturation state. Figure 6 A), we found that PDX1+ / NKX6.1+ co-expression (a cell state that represents TrPC pro-endocrine cells and is required for the generation of functional β cells) occurred throughout the culture ( Figure 6 B). In humans, but not in mice, NKX2.2 is β-cell specific and is not expressed by MPCs. NKX2.2 was co-expressed with insulin C-peptide within plaques throughout culture. Furthermore, induction of multiple endocrine products was observed with cells co-expressing INS+ / GCG+ or INS+ / SST+ ( Figure 6 B) C-peptide production increased as cultures were exposed to Notch inhibition, with peak levels occurring after 10 days and then leveling off ( Figure 7 C) Cell aggregates exhibit dithizone retention and thus Zn 2+ load( Figure 6 D). KCl induced a 4-fold increase in detectable C-peptide levels when insulin secretion was stimulated, but glucose stimulation (2 mM > 20 mM) did not induce C-peptide secretion ( Figure 6 E). Microfluidic evaluation of endocrine-derived cultures showed a detectable calcium influx of approximately 6% ( Figure 6F); again no glucose response was observed. The results from the classical GSIS and microfluidic analysis assays are consistent and demonstrate that immature β-cell physiology is capable of synthesizing and storing insulin but has limited functional profile in response to glucose fluctuations, a characteristic of the fetal-like β-cell state previously observed in β-cells derived from APS-DE (Hrvatin et al., 2014).

[0076] A small molecule approach for inducing fetal-like beta cells

[0077] PDX1 Opt Examination of the conditions identified that retinoic acid and PD0325 had the highest factor contributions, 31.38 and 29.62, respectively ( Figure 7 A), while all other tested effectors had low factor contributions < 10. In order to seek a minimally complex approach to achieve pancreatic endocrine cell induction, we combined these components (RA and LDN (R / L)) for induction of DFE with PDX1 Opt The combination of RA and PD0325 (R / P) was finally transformed into an endocrine fate by NOTCH and ALK5 inhibition. opt After induction of DFE followed by R / P, a significant increase in measured pancreatic markers was observed, and this increase was amplified when endocrine induction was subsequently performed ( Figure 7 B) When HNF1β is replaced using the (R / L) step Opt A further increase in pancreatic genes was observed when the R / L step was used, followed by Pdx1 opt Conditions produced the highest levels of pancreatic genes INS, GCG, NKX6.1, CHGA, GLP1R, and PDX1 ( Figure 7 B) However, when HNF1β Opt and PDX1 Opt Comparable changes were observed when both R / L and R / P were substituted. A slight decrease in the expression of INS, GCG, NKX6.1, CHGA, GLP1R, and PDX1 was observed under these conditions, whereas a modest compensatory increase was observed in the expression of NEUROD1, NGN3, PAX4, MAFA, and SST. We conclude that directed differentiation of pluripotent cells to fetal β cells can be rapidly achieved through three sequential stages using five small molecules: RA, LDN3189, PD0325901, γ-XX, and A8301.

[0078] We have demonstrated a novel protocol that enables the rapid conversion of pluripotent cells into a compartmentalized endoderm population (DFE) that is competent to form dorsal pancreatic progenitors and undergo endocrine conversion via a three-stage protocol that relies on the use of only five small molecules.

[0079] RNA-Seq data suggest that DFE is a dorsal signature based on multiple genes. Jennings et al. investigated the dorsal versus ventral signature of the pancreas during human development and demonstrated that a clear set of 13 genes distinguishes the dorsal pancreas from the ventral pancreas. As the basis for our interpretation, the DFE population is consistent with a dorsal fate. This dorsal aspect is instilled into APS-DE by administering retinoic acid at a later stage. For DFE, dorsal signatures continue into the pancreas, and this state allows for the induction of all pancreatic lineages. Previous studies have shown that DEs have a hepatovagal fate (Mfopou et al., 2010), whereas DFE-derived pancreas do not, although we note that it has a dual potential for antral induction. This is expected because the ventral pancreas has a dual potential for liver induction (Angelo et al., 2012, Deutsch et al., 2001, Tremblay and Zaret, 2005, Bort et al., 2004). Early on, lateral plate-derived BMPs direct this precursor to a liver fate (Chung et al., 2008), and PE treated with BMP4 also induces liver genes. In contrast, ISPA analysis suggests that SHH and retinoic acid are important process inputs affecting bipotential pancreatic / gastric progenitors. These findings are consistent with previous studies on dorsal pancreas specification in the chick (Hebrok et al., 1998). Regardless of the dorsal / ventral origin of the endoderm population, both DFE and APS-DE readily generate pancreatic endoderm capable of generating endocrine cells. As observed for APS-DE-derived cells, DFE-derived endocrine cells are functionally more similar to fetal β cells than to fully mature glucose-responsive β cells.

[0080] Materials and methods

[0081] Cell culture

[0082] Pluripotent cultures were adapted to Essential 8 media (Gibco A15169-01) and grown on vitronectin (Gibco A14700)-coated plates (Corning Inc. 3598). Differentiation experiments were performed at 75,000 cells / cm 2The cells were plated and maintained in E8 medium for 48 hours until the cultures reached approximately 90% confluence before initiating differentiation. All growth factors and small molecules were reconstituted according to the supplier's recommendations and aliquots were stored at -80°C for up to one year. The growth medium was changed daily, and the basal medium used in all differentiation experiments except the DE protocol was CDM2 (Loh et al., 2014). APS Opt It is composed of 250nM LDN3189 (Sellekchem S2618), 50ng / ml Activin A (Peprotech 120-14), 20ng / ml bFGF (Gibco 13256029), and 100nM Sant1 (Sellekchem S7092). HNF1β Opt Supplemented with 250nM LDN3189, 500nM A8301 (Biogems 4463325), 12.6ng / ml Wnt3a (R&D 5036WN / CF), 25ng / ml bFGF, 250nM PD0325 (Selleckchme S1036), 40ng / ml SHH (Peprotech 100-45) and 2uM retinoic acid Sigma Aldrich R2625). DE was generated using previously described conditions (Rezania et al., 2014, D'Amour et al., 2005). PDX1 Opt It consisted of 500nM A8301, 250nM LDN3189, 100nM Ant1, 250nM PD0325, 2uM retinoic acid and 1% B27 supplement (Gibco 12587) and was used for 3 consecutive days on differentiated cells unless otherwise stated in the text. Endocrine push medium was supplemented with 100nM γ-secretase inhibitor XX (EMD Millipore 565789) and 500nM A8301. Gastric organoids were generated using a modified protocol (McCracken et al., 2014). Briefly, HNF1β OptThe culture was incubated for three days with stage 2 medium supplemented with 500ng / ml Wnt3a, 500ng / ml FGF4 (R&D 7460-F4), 250nM LDN3189 and 2uM retinoic acid (RA only on the first day). The culture was passaged 1:6 (where the plating was performed in a 1:4 matrigel (FisherScientific354230) / CDM2 mixture) and incubated at 37°C for 30 minutes, followed by 3 days of stage 3 medium application, which consisted of 2uM retinoic acid, 250nM LDN3189 and 100ng / ml EGF (R&D 236-EG). The culture was then supplemented with stage 4 medium supplemented with 100ng / ml EGF for 23 days, and the organoids were then recovered and fixed for slicing.

[0083] Computer modeling

[0084] All DoE experimental designs were computer generated using D-optimal interactive design in MODDE software (Sartorius Stedim Data Analytical Solutions, SSDAS). Perturbation matrices were generated (96 independent experimental runs) on a Freedom Evo150 liquid handling robot (TECAN, CH). Each DoE differentiation experiment used a 3-day perturbation matrix application with daily media changes. All cell culture, including manual pre-amplification and seeding and any robotic manipulation, was contained in a modular X-Vivo system (Biospherix, NY, USA), which provides process analysis technology (PAT) for cell culture conditions (providing N2%, O2%, CO2% and temperature control and monitoring at any point during the experiment). Endpoint data collection was performed using an open array RT-QPCR method (QuantStudio, Life Technologies, 53 genes / sample / card). QuantStudio gene expression data were normalized, standardized within Expression SuiteTM (Life Tech), and exported to MODDE. Mathematical modeling of the differential space was performed using PLS to maximize the explained variance in MODDE, and gene-specific models were subsequently adjusted by Q2 maximization. Fitted model optimization conditions that maximized or minimized responses were extracted using the software prediction tools "Optimizer" and "Dynamic Profile" analysis. Virtual experiments were performed in the known space covered by the experimental design, resolving the relative contribution of pathways to the desired parameters.

[0085] RNA and cDNA preparation

[0086] RNA extraction for modeling analysis was performed using MagMax-96 total RNA isolation kit (Life Technologies AM1830) and performed according to the manufacturer's protocol. Reverse transcription of RNA samples was performed using the reaction conditions provided with a high-capacity cDNA RT kit (Life Technologies 4368814). RNA extraction for RNASeq and QRT-PCR validation was performed in quadruplicate, using a Trizol (Life Technologies 15596018)-based method for RNA extraction. For open array validation (non-modeling), cDNA was then subjected to QuantStudio analysis. The resulting data was then normalized to housekeeping genes and graphed using GraphPad Prism 5.02 software.

[0087] QRT-PCR for modeling analysis.

[0088] Samples were loaded onto custom-designed Quant Studio cards using the OpenArray AccuFill System (Life Technologies 4471021) and run on the QuantStudio 12k Flex Real-Time PCR System (LifeTechnologies 4471090). QuantStudio Open Array analysis provided expression information for 53 selected genes for each open array configuration. The resulting data were QC evaluated in ExpressionSuite to remove any genes that failed to amplify and were normalized to three internal standards (internal standards were changed between designs). The initial design included genes that responded to all major sub-lineage fates downstream of pluripotent cells, while later designs focused more on the endoderm lineage, with an emphasis on pancreatic fate. For all open array designs, the primer / TaqMan probe sets used for each platform provided typical confidence amplification (Cq Conf.) scores of 0.975 and above. Each modeling experiment generated approximately 10,000 individual gene expression response data points, and MODDE optimization was based on these data points.

[0089] Immunohistochemical analysis of cultures

[0090] Histological characterization was performed by treating cell cultures with 4% paraformaldehyde (EMD 30525-89-4) solution at ambient temperature for 15 minutes. The samples were then blocked and infiltrated for 1 hour using 0.1M Tris / HCl (Promega H5123) pH 7.5 supplemented with 0.5% blocking agent (Perkin Elmer FP1012) and 0.1% TrinTonx-100 (Fisher Scientific BP151). All antibody solutions were diluted in 0.1M Tris / HCl (Promega H5123) at pH 7.5, and the primary antibody was incubated overnight, while the secondary antibody was incubated for 1 hour. All incubations were performed at ambient temperature. The cultures were treated with Vectashield mounting medium (Vector Laboratories H-1200) diluted 1:2. The specific antibodies and dilutions used are provided in Table 1.

[0091] Table 1

[0092] Primary Antibody

[0093]

[0094]

[0095] Secondary Antibodies

[0096]

[0097]

[0098] Cultures were generally stained within the TC culture vesicles in which they differentiated, whereas aggregates or organoids were fixed and embedded in OCT (Sakura 4583) and then cryosectioned prior to staining.

[0099] Characterization of endocrine cells

[0100] GSIS assays were performed by incubating cells in Krebs-Ringer buffer supplemented with 2 mM glucose for 30 minutes to determine basal C-peptide levels. The reaction buffer was then changed to Krebs-Ringer buffer supplemented with 20 mM glucose or 30 mM KCl and incubated for another 30 minutes. Buffer samples were collected and C-peptide levels were quantified using ELISA (Mercodia 10-1141-01) according to the manufacturer's protocol. Microfluidics assays were performed as previously described (Adewola et al., 2010, Wang et al., 2012).

[0101] RNA Seq and key gene analysis

[0102] RNA samples were sequenced by the University of Chicago Genomics Core. FASTQ files were aligned to reference hg19 using HISAT2 version 2.0.5 software. Gene counts of aligned reads were generated using RefSeq annotation files by HTSeq version 0.8.0. Differential expression analysis was performed using edgeR version 3.18.1, with a cutoff of 4 counts / million. KeyGenes analysis was performed using the provided training set according to the published protocol (Roost et al., 2015). Hierarchical clustering of 87 KeyGenes classifier genes was performed using RStudio software with R packages gplots and ggplot2, with only the portion containing all hESC derivatives shown. Differential expression was determined using the EdgeR package, and heat maps were generated using Microsoft Excel software.

[0103] Example 2

[0104] Generation of DFE cells using retinoid compounds

[0105] This example describes the generation of dorsal foregut endoderm cells from pluripotent stem cells using retinoids with known biological activity. This example also describes how endoderm is patterned during gastrulation by the synergistic activities of TGFb agonism, TGFb inhibition, and retinoid agonism.

[0106] result

[0107] Retinoic acid is produced in the body through a series of sequential enzymatic oxidation reactions that convert retinol to retinal and finally to retinoic acid. It is generally accepted that all-trans retinoic acid is the biologically relevant form of the molecule, however, due to its long conjugated backbone, retinoic acid exists in many isomeric forms. Three prevalent isoforms are observed in the circulation; 7-cis retinoic acid, 13-cis retinoic acid, and all-trans retinoic acid, and the serum ratios of these different isomers have been shown to be consistent over time. Experiments using liver lysates have shown that conversions between these different isomers are enzymatically regulated, however, the enzymes responsible for these conversions have not been reliably identified. Studies have shown that these isoforms have differential binding affinities for RARs and RXRs. When retinoic acid is produced in or enters cells, it induces the production of Cyp26a, an enzyme that converts retinoic acid to 4-oxo-retinoic acid. Although the production of 4-oxo-retinoic acid was initially thought to be an inactivator, several studies have shown that these 4-oxo-retinoic acids have differential binding affinities for several retinoic acid targets. Altogether, this suggests that different retinoic acid isomers may have unique biological activities.

[0108] When retinoic acid is used in directed differentiation protocols, the selected form used is all-trans retinoic acid, however this molecule is highly unstable and rapidly degrades over time or when exposed to light. Both time and light can produce a mixture of different isoforms and produce epoxy and keto forms of retinoic acid. These epoxy and keto retinoic acid derivatives have been shown to have biological activity and affinity for retinoic acid binding sites. In short, when retinoic acid is used in in vitro experiments, this produces several potential bioactive molecules with different differentiation potentials. In order to determine which retinoic acid isoforms and derivatives can induce dorsal foregut endoderm, the ISPA approach based on DoE was used.

[0109] BMP inhibition in the presence of 4-oxo and 9-cis retinoic acid induces DFE.

[0110] like Fig.14 As shown in A, a DoE design covering several known bioactive retinoids was created. When performing ISPA-based optimization ( Figure 4B ), several of the retinoids tested were predicted to contribute to HNF1β induction. Both 13-cis retinoic acid (significant at FC=24.1609) and all-trans retinoic acid (low significance at FC=2.77281) were predicted to contribute negatively to HNF1β induction, while the enzymatically degraded form of retinoic acid, 4-oxo-retinoic acid (FC=13.21), had the highest factor contribution of all the retinoic acids tested ( Figure 4B To validate ISPA, transcript analysis of several key DFE genes was performed. The results showed that the new optimizer, composed of the two retinoids predicted to have the highest contribution factors in the presence of LDN193189, activated HNF1β to levels comparable to those of native HNF1β. Opt The same level ( Fig.14 C and Fig.14 D) This novel HNF1β was further confirmed by activation of several previously described DFE markers including MNX1, SFRP5, OSR1, ONECUT1, MYT1, FOXA2, and FRZB. Opt The phenotype ( Fig.14 C and Fig.14 D). Replacing 4-oxo-retinoic acid with freshly reconstituted retinoic acid was able to activate several DFE genes, but at much lower levels. When LDN193189 was removed from the differentiation medium formulation, the results showed that most DFE gene markers were not activated, however, several endoderm genes (OSR1, ONECUT1, MYT1, CDX2, and data not shown) were activated by the use of retinoids including the gastrulation gene NODAL ( Figure 4C and 4D), suggesting that differentially patterned endoderm is being induced.

[0111] All isomers of retinoic acid can induce NODAL signaling.

[0112] Most directed differentiation protocols use Activin A as a Nodal mimetic to generate endoderm to induce gastrulation events in vitro, however, during normal embryogenesis, primitive streak formation is initiated by Nodal, not Activin A. Previous studies have shown that Nodal and Activin A signaling are not equivalent and that definitive endoderm generated by Nodal signaling is different from that generated by Activin A. Since our ISPA model of retinoid signaling predicts that retinoic acid can activate NODAL in the absence of BMP-inhibition, we next evaluated NODAL-induced endoderm formation versus retinoic acid induction of NODAL. The responses using differentiation medium supplemented with NODAL and CRIPTO (a binding partner of NODAL) in the presence of BMP4, Activin A, or retinoic acid were compared with our ISPA-based NODAL induction assay. Opt For comparison ( Fig.15 A). Note that all tested responses were able to induce the early and mid-gastrulation markers NODAL and ALDH1A2, respectively, but that exposure to NODAL Opt The lowest levels were observed in cultures with Fig.15 B) Consistent with previous observations, the ventral marker HHEX was induced in all responses to retinoic acid deprivation, whereas the highest induction of the classical definitive endoderm marker SOX17 occurred in response to exposure to retinoic acid-based NODAL Opt All differentiation responses supplemented with retinoic acid showed the highest induction of the DFE markers HNF1β, MNX1, SFRP5, and OSR1, but the levels obtained in the absence of BMP inhibition were an order of magnitude lower ( Fig.15 B).

[0113] To better understand how the in vitro generated endoderm populations under study are patterned, we next performed subsequent DoE-based gastrulation modeling experiments ( Fig.16 A). Cluster analysis of ISPA optimization for all measured individual endoderm genes was then performed using the Seurat R package ( Fig.16 B). This analysis separated all optimizers into four distinct groups consisting of a single cluster containing DFEs as patterned by retinoic acid and LDN193189 ( Fig.16 C and Fig.17The other cluster is patterned via Nodal / Bmp4 and consists of endoderm patterned primarily toward anterior and midgut fates, although the hindgut marker CDX2 is elevated in this cluster ( Fig.16 C and Fig.17 The cluster patterned by Nodal / Activin A consists of endoderm patterned toward ventral and posterior fates ( Fig.16 C and Fig.17 When considering the contribution of the individual genes measured throughout this experiment, it is noted that anterior and dorsal (except for GSC) genes are generally not activated in the presence of activin A ( Fig.17 ). Although some of these anterior or dorsal endoderm genes are predicted to be activated by the coordinated action of Nodal / retinoic acid or Nodal / BMP, it is important to note that the gene markers DFE, HNF1b, MNX1, HNF6, OSR1, and SFRP5 are best induced using retinoic acid in the absence of Nodal.

[0114] in conclusion

[0115] Although NODAL Opt There are strong contributions to all biologically relevant isomers of retinoic acid (9-cis, 13-cis, and all-trans), but it also has a significant contribution to the CYP26A1 inhibitor talaroazole, ensuring that no catalytic conversion to their respective 4-oxo forms occurs ( Fig.15 A) This is consistent with HNF1β in which both the 13-cis and all-trans forms of retinoic acid are not tolerated, but the 4-oxo metabolite and the 9-cis form are required. Opt on the contrary( Fig.14 B) Taken together, this suggests that, although retinoic acid can induce the DFE phenotype in a Nodal-dependent manner when using retinoic acid isomers or in a Nodal-independent manner when using 4-oxoretinoic acid, optimal induction of DFE occurs after retinoic acid is enzymatically degraded to 4-oxoretinoic acid. This implies that the initial HNF1β Opt This may occur through unnecessary transient NODAL induction, however once retinoic acid induces cyp26a1, any Nodal production ceases. Altogether, this suggests that optimal DFE induction occurs in the absence of Nodal signaling.

[0116] Example 3

[0117] Validation of the DFE optimizer conditions using immunohistochemistry.

[0118] To further validate the induction conditions for the DFE markers, immunostaining was performed followed by high-content imaging to assess conversion efficiency. Testing alternative agonists of the retinoic acid pathway here, we demonstrated that the use of a mixture of 4-oxo-retinoic acid and 9-cis-retinoic acid in combination with complete HNF1β Opt Furthermore, the photostable RA agonist EC23 was comparable to the RA and 4-oxo / 9-cis conditions.

[0119] Example 4

[0120] We evaluate important process parameters associated with endoderm induction and identify multiple endoderm fates derived from pluripotent cells based on the relative inputs of nodal, BMP, and retinoic acid.

[0121] HD-DoE designed with activin A (AA), BMP4, NODAL, retinoic acid, and corresponding inhibitors were used in pluripotent cultures and key endoderm genes ( Fig.16 The resulting data were entered into analysis software and a computer model predicting the optimal culture conditions for activation of all genes monitored was clustered ( Fig.19 B). Four unique endoderm populations were predicted, all of which are tolerant to NODAL. The corresponding ridge plots ( Fig.19 A) shows that retinoic acid signaling contributes to the induction of endoderm populations defined by cluster 0 and cluster 1 (red and green clusters, respectively). Cluster 0 is the dorsal foregut endoderm previously characterized with retinoic acid and BMP-inhibited CPPs, and cluster 1 is the anterior endoderm population described below, which is generated and patterned by the combined effects of Nodal, BMP, and retinoic acid. Only cluster 2 (blue cluster) was found to be dependent on AA, as the concentration of AA was highly variable in both clusters 1 and 3 (green and purple clusters), and the Alk4 / 5 / 7 inhibitor A8301, which specifically targets AA signaling, was present at high levels in clusters 0, 1, and 3. A8301 is largely absent in the proposed AA-driven cluster 2 (blue cluster), allowing uninhibited signaling. A similar pattern was observed for BMP4 signaling, with only cluster 1 (green cluster) having high levels of BMP4 ( Fig.19). The ALK1 / 2 / 6 inhibitor K02288 was tolerated in cluster 1, suggesting that BMP4 signaling occurs through the ALK3 receptor in this endoderm population. Nodal has been shown to be present in all clusters, with the two strongest contributions being cluster 2 (blue cluster) and cluster 1 (green cluster), which are the proposed AA and BMP4 driven clusters, respectively. This suggests that Nodal can signal through the Smad 2 / 3 mediated AA pathway classically attributed to Nodal signaling, and potentially plays a role in the Smad1 / 5 / 8 BMP driven signaling pathway, which Nodal has recently been shown to activate. Also considering that both clusters 0 and 3 show significant contributions to the effectors AA, BMP4 and Nodal and their corresponding inhibitors, this implies that endoderm patterning may have a strong contribution from non-classical TGF-β signaling.

[0122] Example 5

[0123] Identification of anterior domain endoderm populations by analysis of region-specific genes.

[0124] We attempt to further clarify Fig.16 and Fig.19 Phenotypes of specific clusters identified in B. Fig. 20 The location of specific genes within four major clusters was revealed. First, we demonstrated that cluster 0 represents the previously identified DFE. Fig. 20 B and Fig.21 As can be seen in the Figure 3, the ventral marker HHEX is largely absent and the dorsal marker MNX1 is only expressed in cluster 0. Both SFRP5 and HNF1β are more broadly expressed outside of cluster 0 and are also present in cluster 1. This is because SFRP5 and HNF1β are not restricted to the dorsal region of the gut tube, but are also expressed along the entire anterior region and throughout the midgut. Cluster 1 represents a unique endoderm population distinct from DFE and standard literature-induced DE (represented by cluster 2). Due to the specific marker expression, we determined that cluster 1 represents an endoderm population representing the anterior / midgut region of the gut tube due to the expression pattern of the anterior genes TBX1, PAX9, and OSR1 ( Fig. 20 C and Fig.21 ). We refer to this population here as the anterior domain endoderm (ADE). During development, TBX1 and PAX9 are both expressed in the anterior pharyngeal region, whereas OSR1 is expressed throughout the pharynx and is expressed in the midgut ( Fig. 20 C and Fig.21 ).

[0125] Check the important process inputs of ADE through ridge plot analysis ( Fig.19A). Induction of ADE in the absence of BMP inhibition depends primarily on the provision of Nodal, BMP, and ALK5 inhibition. It is tolerant to RA signaling inputs. This input set reveals a unique BMP-dependency signature not present in other endoderm types. While the ridge plots show clear contributions of retinoic acid and Nodal to the induction of ADE ( Fig.19 A), but the RA-generating enzyme ALDH1A2 is not expressed, and Nodal is variably expressed throughout cluster 1. Since retinoic acid production begins later in mid-gastrulation, the lack of ALDH1A2 is consistent with this population representing a more anterior region. Therefore, any involvement in the patterning of the anterior endoderm may be achieved through the diffusion of retinoids in the embryo. The lack of Nodal expression can be attributed to the fact that the most anterior region of the primitive streak is considered to exit gastrulation first, meaning that this population may have exited the primitive streak phenotype but retained the response to Nodal. In summary, we defined a novel endoderm type and the inductive inputs behind its generation.

[0126] Example 6

[0127] Comparison of important process parameters associated with genes expressed in specific endoderm

[0128] To better understand the importance of Nodal and BMP4 signaling in the generation of anterior / midgut endoderm populations, we compiled all prediction models optimized for TBX1, PAX9, and OSR1 ( Fig. 22 ). Nodal and BMP4 both showed equal average cumulative contributions to activation of the anterior endoderm, as shown by the TBX1 and PAX9 optimizers. Although BMP4 still has an important role, the more ubiquitously expressed OSR1 (anterior midgut) showed a much stronger response to Nodal signaling. This means that the further down gut tube patterning occurs, the less contribution BMP signaling makes. It is important to note that AA made little to no contribution in inducing TBX1, PAX9, or OSR1 ( Fig. 22 ), as all previous efforts to generate pharyngeal or gastric progeny relied on the initial use of AA. In turn, AA was noted to have increased effector contributions in selected genes representing midgut, posterior, and ventral endoderm fates, as shown by the exemplary optimizers selected for APOB, CDX2, and SOX17, respectively ( Fig. 22 ). This suggests that the AA inputs often encountered in the literature contribute to the establishment of more posterior endoderm regions.

[0129] To directly determine which measured endoderm genes were responsive to the corresponding TGFβ agonists, we next performed ISPA on all measured key endoderm genes that define discrete regions along the gut tube. The corresponding effector contributions were then normalized for AA, Nodal, BMP4, and RA and ranked from the most responsive genes to the least responsive genes. Fig.23 Our previous finding that RA plays a central role in the generation of DFE was reaffirmed as HNF6, HNF1β, SFRP5, and MNX1 were all shown to be highly regulated by RA and inhibited by AA ( Fig.23 ). Consistent with the conditions used to generate ADE, Nodal, BMP4, and RA were all shown to be involved in the activation of TBX1, PAX9, and OSR1, with AA having highly negative contributions to the activation of TBX1, PAX9, and OSR1, with normalized factor contributions of -0.59, -0.85, and -1.0, respectively. Consistent with the current hypothesis that AA and Nodal have redundant functions, a number of genes with overlapping input logics were observed between the two effectors. These included the primitive streak genes GSC and NODAL (NODAL was not expressed in Fig.23 shown in Fig.21 ), the midgut genes NR5A2, CDX2, and PROX1, and the ventrally localized genes BMP4 and SOX17.

[0130] Since our results showed that both BMP4 and NODAL were activated by AA, we next used RNA Seq data to assess which TGFβ family ligands were activated in both the classical DE and novel DFE protocols. Incubation of pluripotent cultures with AA has been shown to induce endogenous expression of several TGFβ family members, including TDGF1, LEFTY1 / 2, NODAL, BMP1 / 2 / 4 / 7, and TGFβ1, which may explain previous findings attributing broader effects to endoderm induction with AA ( Fig.24 A). This high level of Nodal induction through the production of DE was confirmed by IHC-based protein detection ( Fig.24 B). RA / LDN treatment of pluripotent cells leads to selective activation of BMP1 / 2 / 4 / 7, GDF11, AMH and TGFβ2 ( Fig.24 ). This retinoic acid-mediated induction of BMP explains the key role of LDN in dorsalization of the DFE protocol. In summary, we demonstrate that NODAL and BMP pattern the endoderm along the anterior part of the midgut tube and that AA-responsive genes are more representative of the primitive streak, ventral midgut, and more posterior regions. The optimizer for selective activation of several Nodal / BMP / RA-responsive genes that are unresponsive to AA is presented as an example in Fig.25 Given in.

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[0184] All publications and patents mentioned in this specification and / or listed below are incorporated herein by reference. Various improvements and variations of the described method and system of the present invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in conjunction with specific embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications to the described mode for performing the present invention that are obvious to those skilled in the relevant art are expected to be within the scope described herein.

Claims

1. A method for producing endoderm cells, wherein: A) the endoderm cell is a dorsal foregut endoderm cell, and wherein the method comprises: a) contacting a population of pluripotent stem cells with a retinoic acid signaling pathway agonist and a bone morphogenetic (BMP) pathway inhibitor; and b) culturing at least a portion of the pluripotent stem cell population to generate a dorsal foregut endoderm (DFE) cell population; wherein said stem cells are not exposed to a transforming growth factor beta (TGFβ) pathway agonist during said culturing or during said contacting.

2. The method of claim 1, wherein in A), a) The BMP pathway inhibitor i) is a BMP4 pathway inhibitor; ii) including LDN193189; or iii) selected from the group consisting of: DMH1, DMH2, Dorsopmorphin, K02288, LDN214117, ML347 and Noggin; or b) the culturing is performed for 1-5 days or 2-4 days.

3. The method of claim 1, wherein in A), the method further comprises: c) contacting at least a portion of the DFE cell population with a retinoic acid signaling pathway agonist and a FGFR pathway inhibitor; as well as d) culturing at least a portion of the DFE cell population to generate a pancreatic endoderm (PE) cell population.

4. The method of claim 3, wherein i) during said culturing in step d) or during said contacting in step c), said DFE cells are not exposed to a transforming growth factor β (TGFβ) pathway agonist; ii) the culturing in step d) is carried out for 1-5 days or 2-4 days; iii) the PE cells have dorsal pancreatic characteristics; iv) the FGFR pathway inhibitor is selected from the group consisting of PD0325901, arctigenin, PD184352, PD198306, PD334581, SL 327, U0126, MEK162, GSK1120212, PD325901, CI-1040, TAK-733, selumetinib and XL518; or v) said contacting in step c) further comprises contacting said DFE cell population with a sonic hedgehog (SHH) pathway inhibitor.

5. The method of claim 3, further comprising: e) contacting at least a portion of the PE cell population with a Notch pathway inhibitor and an ALK5 inhibitor; as well as f) culturing at least a portion of the PE cell population to produce an endocrine cell population.

6. The method of claim 5, wherein i) the endocrine cell is an insulin-expressing cell or a fetal β cell; ii) the Notch pathway inhibitor comprises a gamma secretase inhibitor XX, or is selected from the group consisting of: DAPT, MRK-003, MRK-0752, z-Ile-leu-CHO, a gamma secretase inhibitor, L-685,485, LY411575, RO4929097, BMS-906024, Dapt, FLI-06, YO-01027, LY450139, Agastogram, Begacestat, BMS299897, Flurizan, L-685,458 and MRK560; iii) the ALK5 inhibitor comprises A8301, or is selected from the group consisting of A7701, A83-01, SB505124, SB431542, scutellariae lactone, LY2157299, RepSox, R268712, SM16, IN1130, BI4659, SD-208, GW788388, TP0427736HCL, TEW-7197, DMH1, LDN212854, D4476 and SB525334; or iv) the culturing in step f) is carried out for 5-15 days.

7. The method of claim 1, wherein in A), the method further comprises: c) contacting at least a portion of the DFE cell population with EGF; as well as d) culturing at least a portion of the DFE cell population to generate a gastric endoderm (SE) cell population; or c) contacting at least a portion of the DFE cell population with a BMP pathway agonist or BMP4; as well as d) culturing at least a portion of the DFE cell population to generate a hepatic endoderm (LE) cell population.

8. A composition comprising: A) pluripotent stem cells, wherein the pluripotent stem cells comprise: i) exogenous retinoic acid signaling pathway agonist, and ii) exogenous bone morphogenetic (BMP) pathway inhibitors, and The pluripotent stem cells: do not contain any exogenous transforming growth factor β (TGFβ) pathway agonist; or B) a cell culture medium, wherein the cell culture medium comprises: i) a retinoic acid signaling pathway agonist compound, and ii) bone morphogenetic (BMP) pathway inhibitors, and The culture medium does not contain or detectably does not contain any transforming growth factor β (TGFβ) pathway agonist.

9. The composition of claim 8, wherein in A) a) the exogenous BMP pathway inhibitor comprises LDN193189; or b) the cell is a DFE cell, and wherein the DFE cell further comprises an exogenous FGFR pathway inhibitor.

10. The composition of claim 9, wherein the FGFR pathway inhibitor is selected from the group consisting of PD0325901, arctigenin, PD184352, PD198306, PD334581, SL 327, U0126, MEK162, GSK1120212, PD325901, CI-1040, TAK-733, selumetinib and XL518.

11. The composition of claim 8, in B), further comprising: a cell population, wherein the cells are pluripotent stem cells or dorsal foregut endoderm (DFE) cells.

12. The composition of claim 11, wherein the cells are pluripotent stem cells, and wherein the retinoic acid signaling pathway agonist and the BMP pathway inhibitor are present in the culture medium at concentrations that cause at least a portion of the pluripotent stem cells to become DFEs when the pluripotent stem cells are cultured in the culture medium for at least 2 days.

13. A kit comprising or consisting of: A) a) a population of pluripotent stem cells present in a cell culture vessel, optionally including a culture medium in said culture vessel; b) a retinoic acid signaling pathway agonist compound present in the first container; and c) a bone morphogenetic (BMP) pathway inhibitor present in a second container; and wherein the cell culture vessel does not contain any or any detectable exogenous transforming growth factor β (TGFβ) pathway agonist, and wherein the kit does not contain any transforming growth factor β (TGFβ) pathway agonist in the first container or the second container.

14. The composition of claim 8 or the kit of claim 13, wherein in A) or B) of claim 8 or in A) of claim 13, the BMP pathway inhibitor is selected from the group consisting of DMH1, DMH2, Dorsopmorphin, K02288, LDN214117, ML347, Noggin, LDN193189, LDN212854 and follistatin.

15. The method of claim 1, the composition of claim 8, or the kit of claim 13, wherein in A) of claim 1, A) and B) of claim 8, or A) of claim 13, the retinoic acid signaling pathway agonist is selected from the group consisting of a retinoid compound, a retinoid X receptor (RXR) agonist, a retinoic acid receptor (RAR) agonist, retinoic acid, Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxo retinoic acid, and all-trans retinoic acid (ATRA).

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