Methods and compositions for small molecule-based pancreatic beta cell induction

Abstract

Disclosed is a method and composition for producing human pancreatic beta cells and their progenitor cells from human pluripotent stem cells using chemically defined medium.This method allows FOXA2+ HNF1b+ dorsal foregut endoderm cells (DFEC) to be produced first, and according to the provided method, this FOXA2+ HNF1b+ dorsal foregut endoderm cells (DFEC) can be further differentiated into human PTF1A+ PDX1+ pancreatic progenitor cells (PPC) and human INS+ PDX+ pancreatic beta cells (PBC).Also provided are medium, isolated cell population, and kit.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 400,349, filed August 23, 2022. The entire contents of U.S. Provisional Patent Application No. 63 / 400,349 are incorporated herein by reference. [Background technology]

[0002] Background of the Invention The successful application of tissue engineered medical products (TEMPs) depends heavily on the generation of highly specialized cell types. Diseases that could potentially be treated or cured using TEMPs include diabetes, heart disease, and neurodegenerative diseases. A common strategy relies on instructing stem cells to differentiate into desired cell types through a directed differentiation process. Conceptually, such attempts seek to deliver cellular signals designed to mimic natural developmental cues, with the differentiation process occurring through discrete steps. Currently, such methods are developed through tedious testing, in which the combinatorial space of plausible signaling inputs is inadequately explored, a process that rarely takes into account the cost of consumables / reagents. For example, protein-type agonists are commonly used to mimic natural developmental cues, but they are 100–1000 times more expensive than small molecule agonists or antagonists. Proteins also degrade over time, thereby reducing overall potency and necessitating greater QC requirements for the production process. The high cost is further amplified with each intermediate step that occurs and the overall duration of the differentiation protocol. Many of the directed differentiation protocols that have been developed are also impaired by insufficient robustness, with only a small percentage of cultures acquiring the desired phenotype.

[0003] Current pancreatic beta cell (PBC) protocols use six separate steps and can take as long as 34 days to generate highly variable, functional beta cells (Pagliuca et al., 2014; Rezania et al., 2014; and Velazco et al., 2020). Despite the above, PBC development holds great potential for future medical applications. Specifically, TEMP (i.e., stem cell-derived insulin-producing cells) derived from PBC protocols are currently in clinical trials. For example, Vertex acquired SEMMA, the originator of TEMP, and received Phase 1 / 2 accelerated approval (NCT04786262) (17 patients enrolled) to initiate a stem cell-based therapy for metabolically unstable type 1 diabetes.

[0004] Thus, although some progress has been made, there remains a need for efficient and robust methods and compositions for generating human pancreatic beta cells and their progenitors from human pluripotent stem cells. Summary of the Invention

[0005] The present disclosure provides a method for generating human pancreatic beta cells (PBCs), including human dorsal foregut endoderm cells (DFECs) and human pancreatic progenitor cells (PPCs), using a three-step protocol that can be completed in just 16 days. The method uses a chemically defined medium that allows for the generation of DFECs within three days of culture, PPCs within six days of culture, and PBCs within 16 days of culture. The defined medium used to obtain different types of progenitor cells contains small molecule agents that stimulate or attenuate specific signaling pathways in pluripotent stem cells to promote differentiation along the endodermal lineage, resulting in cell maturation and the expression of PBC-associated biomarkers. The disclosed method uses different components from those used in previous protocols and uses a medium for differentiation that avoids the use of certain components required by other protocols. The disclosed method also has the advantage that the use of small molecule agents in the medium allows for precise control of culture components, resulting in fewer differentiation steps and a shorter differentiation time to PBCs compared to prior art protocols.

[0006] Accordingly, in one aspect, the present disclosure relates to a method for producing human dorsal foregut endoderm cells (DFECs), comprising culturing human pluripotent stem cells (PSCs) in a medium comprising a BMP pathway antagonist, an RA pathway agonist, a TGF-β pathway antagonist, and a MEK pathway antagonist on days 0 to 3 to obtain human DFECs.

[0007] The method may further include culturing the human DFECs in a medium containing a BMP pathway antagonist, an RA pathway agonist, a TGF-β pathway antagonist, a TAK1 pathway antagonist, a bFGF mimetic, and an Akt pathway antagonist on days 4 to 6 to obtain human pancreatic progenitor cells (PPCs).

[0008] The method may then further include culturing the human pancreatic beta cells (PPCs) in a medium containing a Notch pathway inhibitor (e.g., a gamma-secretase inhibitor), a TGF-β pathway antagonist, and a flavonoid on days 7 to 16 to obtain human pancreatic beta cells (PBCs).

[0009] In one embodiment, the human pluripotent stem cells are induced pluripotent stem cells (iPSCs). In another embodiment, the human pluripotent stem cells are embryonic stem cells. In another embodiment, the human pluripotent stem cells are attached to vitronectin-coated plates during culture.

[0010] In another embodiment, the BMP pathway antagonist is selected from the group consisting of LDN193189, DMH1, DMH2, dorsomorphin, K02288, LDN214117, LDN212854, follistatin, ML347, noggin, and combinations thereof. In one embodiment, the BMP pathway antagonist is present in the medium at a concentration of 100 to 400 nM. In another embodiment, the BMP pathway antagonist is LDN193189, and the LDN193189 is present in the medium at a concentration of 250 nM.

[0011] In another embodiment, the RA pathway antagonist is selected from the group consisting of retinoic acid (RA), TTNPB, AM580, CD1530, CD2314, Ch55, BMS753, tazarotene, isotretinoin, AC261066, Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxo retinoic acid, and all-trans retinoic acid (ATRA). In one embodiment, the RA pathway antagonist is present in the medium at a concentration ranging from 0.5 to 4 nM. In another embodiment, the RA pathway antagonist is RA, and the RA is present in the medium at a concentration of 2 μM.

[0012] In another embodiment, the TGF-β pathway antagonist is selected from the group consisting of A8301, SB-431542, GW788388, SB525334, TP0427736, Repsox, SD-208, and combinations thereof. In one embodiment, the TGF-β pathway antagonist is present in the medium at a concentration of 200-1000 nM. In another embodiment, the TGF-β pathway antagonist is A8301, and the A8301 is present in the medium at a concentration of 500 nM.

[0013] In another embodiment, the MEK pathway antagonist is selected from the group consisting of PD0325901, binimetinib (MEK162), cobimetinib (XL518), selumetinib, trametinib (GSK1120212), CI-1040 (PD-184352), refametinib, ARRY-142886 (AZD-6244), PD98059, U0126, BI-847325, RO5126766, and combinations thereof. In one embodiment, the MEK pathway antagonist is present in the medium at a concentration ranging from 200 to 1000 nM. In another embodiment, the MEK pathway antagonist is PD0325901, and PD0325901 is present in the medium at a concentration of 250 nM.

[0014] In another embodiment, the TAK1 pathway antagonist is selected from the group consisting of Taki ((5Z)-7-oxozeaenol), takinib, dehydroabietic acid, NG25, sarsasapogenin, and combinations thereof. In one embodiment, the TAK1 pathway antagonist is present in the medium at a concentration in the range of 200 to 1000 nM. In another embodiment, the TAK1 pathway antagonist is Taki ((5Z)-7-oxozeaenol), and the Taki ((5Z)-7-oxozeaenol) is present in the medium at a concentration of 500 nM.

[0015] In another embodiment, the bFGF mimetic is present in the medium at a concentration in the range of 150 to 600 nM. In one embodiment, the bFGF mimetic is SUN11602, and SUN11602 is present in the medium at a concentration of 300 nM.

[0016] In another embodiment, the AKT pathway antagonist is selected from the group consisting of AT7867, MK2206, GSK690693, perifosine (KRX-0401), ipatasertib (GDC-0068), capivasertib (AZD5363), PF-04691502, triciribine (NSC154020), ARQ751, milansertib (ab235550), volsertib, cerisertib, and combinations thereof. In one embodiment, the AKT pathway antagonist is present in the medium at a concentration ranging from 100 to 400 nM. In another embodiment, the AKT pathway antagonist is AT7867, and AT7867 is present in the medium at a concentration of 250 nM.

[0017] In another embodiment, the Notch pathway antagonist is selected from the group consisting of GSI-XX, RO4929097, semagacestat, dibenzazepine, LY411575, crenigacestat, IMR-1, IMR-1A, FLI-06, DAPT, valproic acid, YO-01027, CB-103, tangeretin, BMS-906024, avagacestat, bruceine D, and combinations thereof. In one embodiment, the Notch pathway antagonist is present in the medium at a concentration ranging from 50 to 200 nM. In another embodiment, the Notch pathway antagonist is GSI-XX, and the GSI-XX is present in the medium at a concentration of 100 nM.

[0018] In another embodiment, the flavonoid is selected from the group consisting of quercetin, a quercetin analog (e.g., dihydroquercetin, 6,2',4',5'-pentahydroxyflavone, quercetin-3-O-propionate (Q-pr), quercetin-3-O-butyrate (Q-bu), quercetin-3-O-valerate, or 3,4'-di-O-methylquercetin), genistein, anthocyanins, catechins, gallocatechins (e.g., epigallocatechin-3-gallate (EGCG)), anthocyanidins, apigenin, luteolin, kaempferol, curcumin, myricetin, daidzein, naringin, rutin, and hesperitin. The flavonoid is present in the medium at a concentration ranging from 3 to 50 μM. In another embodiment, the flavonoid is quercetin, and the quercetin is present in the medium at a concentration of 15 μM.

[0019] In another aspect, the present disclosure provides a method for producing human DFE cells, the method comprising culturing human pluripotent stem cells in a medium containing a BMP pathway antagonist, an RA pathway agonist, a TGF-β pathway antagonist, and a MEK pathway antagonist on days 0 to 3 to obtain human DFE cells.

[0020] In another embodiment, the BMP pathway antagonist is LDN193189, the RA pathway agonist is retinoic acid, the TGF-β pathway antagonist is A8301, and the MEK pathway antagonist is PD0325901.

[0021] In another embodiment, LDN193189 is present in the medium at a concentration in the range of 100 to 400 nM, retinoic acid is present in the medium at a concentration in the range of 0.4 to 4 μM, A8301 is present in the medium at a concentration in the range of 200 to 1000 nM, and PD0325901 is present in the medium at a concentration in the range of 100 to 400 nM.

[0022] In another embodiment, LDN193189 is present in the medium at a concentration of 250 nM, retinoic acid is present in the medium at a concentration of 2 μM, A8301 is present in the medium at a concentration of 500 nM, and PD0325901 is present in the medium at a concentration of 250 nM.

[0023] In another aspect, the present disclosure provides a method of generating human pancreatic progenitor cells (PPCs), comprising: (a) culturing human pluripotent stem cells in a medium containing a BMP pathway antagonist, an RA pathway agonist, a TGF-β pathway antagonist, and a MEK pathway antagonist on days 0 to 3 to obtain human dorsal foregut endoderm cells (DFECs); and (b) further culturing the DFECs in a medium containing a BMP pathway antagonist, an RA pathway agonist, a TGF-β pathway antagonist, a TAK1 pathway antagonist, a bFGF mimetic, and an Akt pathway antagonist on days 4 to 6 to obtain human pancreatic progenitor cells (PPCs); The present invention provides a method comprising:

[0024] In one embodiment, the BMP pathway antagonist is LDN193189, the RA pathway agonist is retinoic acid, the TGF-β pathway antagonist is A8301, the MEK pathway antagonist is PD0325901, the TAK1 pathway antagonist is Taki ((5Z)-7-oxozeaenol), the bFGF mimetic is SUN11602, and the Akt pathway antagonist is AT7867.

[0025] In another embodiment, LDN193189 is present in the medium at a concentration in the range of 100 to 400 nM, retinoic acid is present in the medium at a concentration in the range of 0.5 to 4 μM, A8301 is present in the medium at a concentration in the range of 200 to 1000 nM, PD0325901 is present in the medium in step (a) at a concentration in the range of 100 to 400 nM, Taki ((5Z)-7-oxozeaenol) is present in the medium at a concentration in the range of 200 to 1000 nM, SUN11602 is present in the medium at a concentration in the range of 150 to 600 nM, and AT7867 is present in the medium in step (a) at a concentration in the range of 100 to 400 nM.

[0026] In another embodiment, LDN193189 is present in the culture medium at a concentration of 250 nM, retinoic acid is present in the culture medium at a concentration of 2 μM, A8301 is present in the culture medium at a concentration of 500 nM, PD0325901 is present in the culture medium at a concentration of 250 nM, Taki ((5Z)-7-oxozeaenol) is present in the culture medium at a concentration of 500 nM, SUN11602 is present in the culture medium at a concentration of 300 nM, and AT7867 is present in the culture medium at a concentration of 250 nM in step (a).

[0027] In yet another aspect, the present disclosure provides a method of producing human pancreatic beta cells (PBCs), comprising: (a) culturing human pluripotent stem cells (hPSCs) in a medium containing a BMP pathway antagonist, an RA pathway agonist, a TGF-β pathway antagonist, and a MEK pathway antagonist on days 0 to 3 to obtain human dorsal foregut endoderm cells (DFECs); (b) further culturing the DFECs in a medium containing a BMP pathway antagonist, an RA pathway agonist, a TGF-β pathway antagonist, a TAK1 pathway antagonist, a bFGF mimetic, and an Akt pathway antagonist on days 4 to 6 to obtain pancreatic progenitor cells (PPCs); and (c) further culturing the human pancreatic beta cells (PPCs) in a medium containing a Notch pathway antagonist, a TGF-β pathway antagonist, and a flavonoid on days 7 to 16 to obtain human pancreatic beta cells (PBCs); The present invention provides a method comprising:

[0028] In one embodiment, the BMP pathway antagonist is LDN193189, the RA pathway agonist is retinoic acid, the TGF-β pathway antagonist is A8301, the MEK pathway antagonist is PD0325901, the TAK1 pathway antagonist is Taki ((5Z)-7-oxozeaenol), the bFGF mimetic is SUN11602, the Akt pathway antagonist is AT7867, and the Notch pathway antagonist is gamma-secretase inhibitor XX (GSI-XX).

[0029] In another embodiment, LDN193189 is present in the medium in steps (a) and (b) at a concentration in the range of 100 to 400 nM, retinoic acid is present in the medium in steps (a) and (b) at a concentration in the range of 0.5 to 2 μM, A8301 is present in the medium in steps (a) to (c) at a concentration in the range of 200 to 1000 nM, PD0325901 is present in the medium in step (a) at a concentration in the range of 100 to 400 nM, and Taki ((5Z)-7-oxozeaenol) is The medium in step (b) is present at a concentration in the range of 200 to 1000 nM, SUN11602 is present at a concentration in the range of 150 to 600 nM, AT7867 is present at a concentration in the range of 100 to 400 nM, γ-secretase inhibitor XX (GSI-XX) is present at a concentration in the range of 50 to 200 nM, and quercetin is present at a concentration in the range of 5 to 30 μM.

[0030] In another embodiment, LDN193189 is present in the culture medium in steps (a) and (b) at a concentration of 250 nM, retinoic acid is present in the culture medium in steps (a) and (b) at a concentration of 2 μM, A8301 is present in the culture medium in steps (a) to (c) at a concentration of 500 nM, PD0325901 is present in the culture medium in step (a) at a concentration of 250 nM, Taki ((5Z)-7-oxozeaenol) is present in the culture medium in step (b) at a concentration of 500 nM, SUN11602 is present in the culture medium in step (b) at a concentration of 300 nM, AT7867 is present in the culture medium in step (b) at a concentration of 250 nM, GSI-XX is present in the culture medium in step (c) at a concentration of 100 nM, and quercetin is present in the culture medium in step (c) at a concentration of 15 μM.

[0031] The disclosed methods and compositions are useful in generating pancreatic β cells or their precursor cells for use in clinical therapy, research, development, and commercial purposes. For clinical use, the in vitro generated PBCs of the present invention can be administered directly to a subject or systemically to treat or prevent type 1 diabetes, type 2 diabetes, prediabetes, conditions resulting from significant trauma (i.e., damage to the pancreas or loss of islet β cells or damage to islet β cells), or other metabolic diseases or disorders associated with a deficiency in the number of β cells (e.g., a reduced number of pancreatic cells), an insufficient level of β cell biological activity (e.g., a deficiency in glucose-stimulated insulin secretion, or a deficiency in insulin production).

[0032] Other features and advantages of the invention will be apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0033] [Figure 1]1 is a schematic diagram of a representative culture method of the present disclosure using a three-step protocol for generating pancreatic beta cells (PBCs) from human induced pluripotent stem cells (hiPSCs). The method first allows for the generation of FOXA2+ HNF1b+ dorsal foregut endoderm cells (DFECs), which are further cultured to generate human PTF1A+ PDX1+ pancreatic progenitor cells (PPCs), and then human INS+ PDX+ pancreatic beta cells (PBCs). [Figure 2] Optimization of HNF1B activation from the iPSC line CR01. Cultures of the iPSC cell line Cr01 were exposed to an HD-DoE perturbation matrix consisting of compounds modulating the BMP, retinoid, TGF-β, and FGF pathways. This analysis utilized pathways previously shown to be involved in dorsal foregut endoderm differentiation. MODDE software was used to predict optimal conditions for maximizing HNF1b activation. The predicted factor additions for this optimization are shown, along with the relative expression of the measured genes under these conditions. [Figure 3] Optimization of FOXA2 expression from the iPSC line CR01. Cultures of the iPSC cell line Cr01 were exposed to an HD-DoE perturbation matrix consisting of compounds modulating the BMP, retinoid, TGF-β, and FGF pathways, pathways previously shown to be involved in dorsal foregut endoderm differentiation. MODDE software was used to predict optimal conditions for maximizing FOXA2 activation. The predicted factor additions for this optimization are shown, along with the relative expression of all measured genes under these conditions. [Figure 4]Dual optimization of FOXA2 and HNF1B expression leads to dorsal foregut endoderm (DFE). Cultures of the iPSC cell line Cr01 were exposed to an HD-DoE perturbation matrix consisting of compounds modulating the BMP, retinoid, TGF-β, and FGF pathways. Pathways previously shown to be involved in dorsal foregut endoderm differentiation were evaluated. MODDE software was used to predict optimal conditions for maximizing FOXA2 activation. The predicted factor additions for this optimization and the relative expression of all measured genes under these conditions are shown. [Figure 5] Dynamic profiling demonstrates that MEK and TGF-β inhibition are required for FOXA2 induction. Cultures of the iPSC cell line Cr01 were exposed to HD-DoE perturbation matrices consisting of compounds modulating BMP, retinoid, TGF-β, and FGF pathways. Use of MODDE software to visualize dynamic profiling indicates the relative contribution of key effectors. [Figure 6] Coefficient plots show the main effectors required for DFE differentiation. Cultures of the iPSC cell line Cr01 were exposed to an HD-DoE perturbation matrix consisting of compounds modulating the BMP, retinoid, TGF-β, and FGF pathways. Coefficient plots are shown for the FOXA2 and HNF1B genes. The size and direction of the bars indicate the overall contribution of the factor to gene activation or repression. Here, positive bars indicate contributions to activation, and negative bars indicate contributions to inhibition. [Figure 7]The heat map identifies the critical input components required for DFE generation. Duplicate samples of iPSC (CR01) cultures were exposed to media containing different combinations. Here, retinoic acid, LDN193189, A8301, PD0325, and / or Wnt3a were added to the cultures. The combinations used are indicated at the top of the heat map. Samples were subjected to RNA purification and sent for sequencing. Genes representing different endoderm regions were monitored, and the relative levels of these transcripts are shown. A number of genes previously identified as contributing to neural fate repression are indicated at the bottom of the heat map. The tissue regions corresponding to the genes are indicated to the left of the heat map. [Figure 8] DFE is induced directly without passing through either the primitive streak or the definitive endoderm intermediate. Duplicate samples of iPSC(CR01) cultures were exposed to medium containing different combinations of the additives retinoic acid, LDN193189, A8301, PD0325, and / or Wnt3a. The combinations used are indicated above the heatmap. Samples were subjected to RNA purification and sent for sequencing. The relative expression levels of genes representing the primitive streak or generalized definitive endoderm state are indicated to the left of the heatmap. [Figure 9] Achieving optimal FOXA2 expression. IPSC (CR01) cultures were exposed to medium containing different combinations of the additives retinoic acid, LDN193189, A8301, and / or PD0325, as indicated on the left side of the staining. After 3 days, cultures were fixed and subjected to immunohistochemical staining for FOXA2 and HNF1b protein antigens. [Figure 10] Dynamic profiling demonstrates the potential for pancreatic targeting. Dynamic profiling of gene combinations known to indicate the location of the future pancreatic field was demonstrated. This was performed using the HD-Doe design shown in Examples 1-3. [Figure 11]Modeling for maximal PDX1 induction. Cultures of the iPSC cell line CR01 were differentiated to the DFE state and then subjected to an HD-DoE perturbation matrix consisting of the compounds shown in the figure. MODDE software was used to predict optimal conditions for maximizing PDX1 activation. The predicted factor additions for this optimization are shown, along with the relative expression of the measured genes under these conditions. [Figure 12] The coefficient plot shows the main effectors for generating pancreatic progenitor cells from DFE progenitor cells. The coefficients for the HD-DoE design shown in Example 9 are shown for PDX1 and PTF1A. Co-expression of these two genes indicates the pancreatic field. [Figure 13] Optimization for PDX1 induction. iPSC cultures were differentiated into DFE cultures and then subjected to two opposing PDX1 optimizers. ESC CPPopt refers to previously identified conditions (Bukys et al., 2020) and are the conditions specified in Example 10. Cultures were further differentiated in these conditions for 3 days before being formalin-fixed and stained with antibodies recognizing the protein transcription factors PDX1 and NKX6.1. Co-expression of PDX1 and NKX6.1 indicates pro-endocrine pancreatic progenitor cells. [Figure 14] Dynamic profiling to explore endocrine cell subtypes. iPSC cultures were differentiated into DFE cultures and then subjected to HD-DoE matrices as outlined in Example 10. MODDE software was used to display dynamic profiles of GCG, INS, and SST, which are indicative of distinct pancreatic endocrine subtypes corresponding to α-, β-, and δ-cells, respectively. [Figure 15]Modeling for Maximum Insulin Expression. Cultures of the iPSC cell line CR01 were differentiated to the DFE state and then subjected to an HD-DoE perturbation matrix consisting of the compounds shown in the figure. The cultures were then incubated for an additional 5 days in the presence of γ-secretase inhibitor XX (GSI-XX) and A8301, compounds known to induce endocrine commitment from pancreatic progenitor cells. MODDE software was used to predict optimal conditions for maximizing PDX1 activation. The predicted factor additions for this optimization are shown, along with the relative expression of the measured genes under these conditions. [Figure 16] Dynamic profiling of endocrine cell subtypes. Cultures of the iPSC cell line CR01 were differentiated to the DFE state and then subjected to an HD-DoE perturbation matrix consisting of the compounds shown in the figure. These cultures were then incubated for an additional 5 days in the presence of GSI-XX and A8301, compounds known to induce endocrine commitment from pancreatic progenitor cells. MODDE software was used to display dynamic profiles of GCG, INS, and SST, which represent distinct pancreatic endocrine subtypes corresponding to α, β, and δ cells, respectively. [Figure 17] Immunohistochemical validation of new β cells. Cultures of the iPSC cell line CR01 were differentiated to the DFE state for 3 days. These cultures were then incubated for an additional 3 days in the presence of stage 2 medium, followed by an additional 10 days in the presence of medium containing GSI-XX and A8301. Cultures were then formalin-fixed and stained with the antibodies used, which recognize the proteins PDX1 / insulin or PDX1 / C-peptide, as shown in Figure 17. [Figure 18]HD-DoE screening for optimal de novo β-cell induction. Cultures of the iPSC cell line CR01 were differentiated to the DFE state for three days. These cultures were then incubated for an additional three days in the presence of Stage 2 medium. These cultures were then incubated for an additional five days in the presence of GSI-XX and A8301, followed by three days in the presence of a perturbation matrix composed of the compounds shown in the different panels. This was performed three separate times (one for each of the three panels). These HD-DoE experiments were then analyzed using MODDE software for maximum insulin induction. The predicted results are shown. [Figure 19] Heat map showing descending factor contributions to INS induction. Three HD-DoE experiments shown in Example 17 were used to model the maximum expression of several other genes, including GCG, SST, PDX1, PAX4, MAFA, and NKX2.2. GCG, SST, PDX1, PAX4, MAFA, and NKX2.2 represent endocrine cell types, focusing on pancreatic β cells as shown at the top of the heat map. The relative factor contributions for activating these different genes were then used to create a secondary heat map representing gene induction logic, which was then ranked according to the ability of compounds to induce INS. For the activation of the indicated genes, dark blue indicates high induction potential, while dark red indicates high repression potential. [Figure 20] HD-DoE screening for optimal de novo β-cell induction. Cultures of the iPSC cell line CR01 were differentiated to the DFE state for three days. These cultures were then incubated in the presence of Stage 2 medium for an additional three days. These cultures were then incubated in the presence of GSI-XX and A8301 for an additional five days, followed by incubation in the presence of a perturbation matrix composed of the compounds shown in the different panels for three days. This was performed four separate times (one for each of the four panels). These HD-DoE experiments were then analyzed using MODDE software for maximum insulin induction. The predicted results are shown. [Figure 21] Heat map showing descending factor contributions for INS induction. The four HD-DoE experiments described in Example 19 were used to model the maximal expression of several other genes. These genes were GCG, SST, PDX1, PAX4, MAFA, and NKX2.2. All of these genes represent endocrine cell types, with a focus on beta cells, as shown at the top of the heat map. The relative factor contributions for activating these different genes were then used to create a secondary heat map representing these gene induction logics, which were then ranked according to the ability of compounds to induce INS. For the activation of the indicated genes, dark blue indicates a high induction potential, while dark red indicates a high inhibitory effect. [Figure 22] Dynamic Profiling of Endocrine Subtype Regulation. One of the HD-DoE experiments shown in Example 19 was analyzed using MODDE software to demonstrate the dynamic profiling of the endocrine products GCG, SST, and INS. [Figure 23] Dynamic Profiling of Endocrine Subtype Regulation. One of the HD-DoE experiments shown in Example 19 was analyzed using MODDE software to demonstrate the dynamic profiling of the endocrine products GCG, SST, and INS. [Figure 24] Dynamic Profiling of Endocrine Subtype Regulation. One of the HD-DoE experiments shown in Example 19 was analyzed using MODDE software to demonstrate the dynamic profiling of the endocrine products GCG, SST, and INS. [Figure 25] Dynamic Profiling of Endocrine Subtype Regulation. One of the HD-DoE experiments shown in Example 19 was analyzed using MODDE software to demonstrate the dynamic profiling of the endocrine products GCG, SST, and INS. [Figure 26]Immunohistochemical verification of new β-cell generation. Cultures of the iPSC cell line CR01 were differentiated to the DFE state for 3 days. These cultures were then incubated in the presence of Stage 2 medium for an additional 3 days. These cultures were then incubated in the presence of quercetin, GSI-XX, and A8301 for an additional 10 days. Cultures were then fixed with formalin and stained with antibodies recognizing the proteins PDX1 and CPEP. [Figure 27] Immunohistochemical verification of new beta cell generation. Cultures of the iPSC cell line CR01 were differentiated to the DFE state for 3 days. These cultures were then incubated in the presence of Stage 2 medium for an additional 3 days, followed by incubation in the presence of quercetin, GSI-XX, and A8301 for an additional 10 days. Cultures were then fixed with formalin and stained with antibodies recognizing the proteins CGC / CPEP, PDX1 / CPEP, or PDX1 / SOX9, as indicated. [Figure 28] Functional analysis of β cells. Cultures of the iPSC cell line CR01 were differentiated to the DFE state for 3 days. These cultures were then incubated in the presence of stage 2 medium for an additional 3 days, followed by quercetin, GSI-XX, and A8301 for an additional 10 days. The cultures were washed with PBS and then incubated in medium containing 3 mM glucose (basal medium) for 15 minutes. The respective medium was then replaced with medium containing either 17.5 mM glucose or 30 mM KCl, as indicated on the x-axis. Samples were run in triplicate. Quantification was performed using a C-peptide ELISA. Samples were normalized to the C-peptide levels detected in the basal medium samples. [Figure 29] The directed differentiation β-cell protocol is applicable to suspension culture systems. The differentiation protocol was carried out in a PBS vertical wheel bioreactor as shown in the figure. Photographs of aggregates from different stages are shown. [Figure 30]Proliferation and expansion occurs throughout the differentiation process. Samples were taken daily from the bioreactor shown in Figure 29. Samples were quantified on a Cell Countess and graphed using Graphpad prism software. [Figure 31] Differentiation in a suspension system results in a similar induction of beta cells. At the end of each stage, samples were taken from the bioreactor shown in Figure 29 and subjected to RNA isolation. Technical replicates were then subjected to cDNA conversion, and relative levels were quantified on a QuantStudio using custom-designed chips. Relative expression levels of key genes are shown compared to those obtained from control stage 3, which consisted of cells differentiated using the same protocol in adherent culture. [Figure 32] De novo insulin production in bioreactors. Quantitation of samples from stage 3 bioreactors is shown in Figure 29. Potassium-mediated release was performed on cell culture inserts, transferring the inserts from basal medium to a 30 mM KCl solution. Samples were removed from the bioreactor and the number of aggregates per ml was calculated. This was then centrifuged and a sample of the supernatant was used to quantify the conditioned medium. The pellet was dissolved in TPER and then used to quantify the C-peptide content per aggregate. All quantitation was performed using C-peptide ELISA. [Figure 33] Figures 33A-33B. Bioreactor runs consistently yield insulin-producing cells. Figure 33A shows a schematic diagram referencing different stages assessed by immunohistochemistry (IHC) analysis in panel B. Figure 33B shows a brightfield image of iPSC aggregates. [Figure 34] Endocrine cells produced by bioreactors have similar expression patterns compared to human islets. For comparative IHC analysis, aggregates generated within bioreactor runs and primary human islets were seeded onto Matrigel. The gene expression patterns of the cells were assessed, demonstrating similar expression patterns. [Figure 35]Determination of the average insulin content per cell. Bar graph shows the results in pg insulin / cell. [Figure 36] Insulin production continues for up to two weeks after initial induction. The graph shows the results of bioreactor cultures monitored and sampled over the course of stage 3 induction. [Figure 37] Glucose-stimulated insulin secretion is not maintained after cryopreservation. The results in the graph show that iPSC derivatives exhibit varying degrees of functionality when exposed to glucose influx. [Figure 38] Bioreactor-based production runs consistently produce insulin-producing cells at levels comparable to human islets. RNA sequencing results are from three different bioreactor runs of stage 3 cells performed to assess expression patterns representative of different aspects of the differentiation process. [Figure 39] The function of insulin-secreting cells produced by bioreactors is mediated by cAMP agonism. The bar graph shows the results of assaying the insulin secretion mechanism of iPSC derivatives. This shows that cAMP agonists can increase insulin secretion when used in conjunction with the GSIS assay. [Figure 40] The mature glucose-stimulated insulin secretion mechanism is underdeveloped in immature iPSC derivatives. RNA sequencing results for the expression of the indicated genes were presented. This demonstrated that expression of the incretin receptor GLPR1 was very low in iPSC derivatives, glucokinase (GCK) expression was similar between iPSC derivatives and primary β-cells, and GLUT2 transporter expression was lower in iPSC derivatives. DETAILED DESCRIPTION OF THE INVENTION

[0034] Detailed Description of the Invention Described herein are methods and compositions that enable the generation of pancreatic beta cells (PBCs) and their progenitor cells from human pluripotent stem cells under chemically defined culture conditions using a small molecule-based approach. The disclosed method generates PBCs and their progenitor cells using a three-step protocol. In this protocol, FOXA2+ HNF1b+ dorsal foregut endoderm (DFE) is generated in 3 days, followed by PDX1+ PTF1A+ NKX6.1+ pancreatic progenitor cells (PPCs) by day 6 of culture, and then pancreatic beta cells (PBCs) are generated by day 11 of culture. Thus, the present disclosure enables the generation of PBCs using chemically defined culture conditions in a significantly shorter time than prior art protocols.

[0035] Various aspects of the invention are described in further detail in the following subsections.

[0036] I. cell The starting cells in the culture are human pluripotent stem cells. As used herein, the term "human pluripotent stem cells" ("hPSCs") refers to human stem cells that have the ability to differentiate into a variety of different cell types. As used herein, the term "pluripotent" refers to cells that have the ability to differentiate under various conditions into cell types characteristic of all three germ layers (endoderm, mesoderm, and ectoderm). Pluripotent cells are primarily characterized by their ability to differentiate into all three germ layers, for example, using nude mice and teratoma formation assays. While pluripotency can be demonstrated by the expression of embryonic stem (ES) cell markers, the preferred test for pluripotency is the demonstration of the ability to differentiate into cells of each of the three germ layers.

[0037] hPSCs include, for example, induced pluripotent stem cells (iPSCs) and human embryonic stem cells, such as ES cell lines. Non-limiting examples of induced pluripotent stem cells (iPSCs) include 19-11-1, 19-9-7, or 6-9-9 cells (e.g., as described in Yu, J. et al. (2009) Science 324:797-801). Non-limiting examples of human embryonic stem cell lines include ES03 cells (WiCell Research Institute) and H9 cells (Thomson, JA et al. (1998) Science 282:1145-1147). Human pluripotent stem cells (PSCs) express cell markers that can be used to determine whether a cell is a PSC. Non-limiting examples of pluripotent stem cell markers include TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG, and / or SOX2. Because the disclosed methods of generating progenitor cell populations are during differentiation from a starting pluripotent stem cell population, in various embodiments, the progenitor cell populations generated by the disclosed methods lack expression of one or more stem cell markers, including the stem cell markers described in the Examples.

[0038] Common practices and methods for coating TC plates or passaging iPSCs may be used. In an exemplary embodiment, PSC cultures, such as the CR01 iPSC line, can be maintained and expanded on vitronectin-coated 6-well tissue culture (TC) plates. E8 medium may be used for general maintenance of these cells. PSC cultures are typically passaged every 3–4 days using EDTA to disrupt cell-to-cell adhesion. This can be accomplished by removing the E8 medium and washing each well of the TC plate with 2 ml of PBS. A 3-minute incubation in the presence of 5 mM EDTA can then be performed at 37°C. The wells are then aspirated, and the cells are washed off the plate and seeded in fresh E8 medium supplemented with 1x RevitaCell. Typically, each passaged well is seeded into six wells of a newly vitronectin-coated TC plate for 1–6 expansion of the iPSC line.

[0039] Passaging of PSCs may be performed using collagenase, accutase, trypsin, TyrPLE, or other digestive enzymes instead of EDTA. PSCs can also be maintained and grown on supports other than vitronectin. Commonly used supports include gelatin, Matrigel, Geltrex, or other ECMs or charged surface coatings. Additionally, other ROCK inhibitors may be used in place of the ROCK inhibitor found in RevitaCell supplements. A commonly used inhibitor is Y27632.

[0040] Pluripotent stem cells are subjected to culture conditions that induce cell differentiation, as described herein. As used herein, the term "differentiation" refers to the development of cells from a more primitive stage into more mature (i.e., less primitive) cells that typically exhibit phenotypic characteristics of commitment to a particular cell lineage.

[0041] In some embodiments, the cells produced by the method of the present disclosure are dorsal foregut endoderm cells (DFEC).As used herein, " dorsal foregut endoderm cells " or " DFEC " refers to cells that are more differentiated than pluripotent stem cells in that they are committed to endodermal lineage, but still have the ability to differentiate into different types of cells along the same lineage.DFEC expresses biomarkers FOXA2 and HNF1b.

[0042] DFECs may also express additional biomarkers, including, but not limited to, endodermal region markers PROM1, CPB1, ONECUT1, and CXCL4; dorsal endoderm markers SFRP5, MNX1, PTCH1, and PAX6; and midgut markers FOXA2, HNF1b, HOXA3, and ONECUT2. In contrast, DFECs exhibit very low or undetectable expression of ventral markers HHEX and NR5A2 and classically defined definitive endoderm markers SOX17, GSC, MIXL1, and CER.

[0043] In some embodiments, the cells produced by the methods of the present disclosure are pancreatic progenitor cells (PPCs), which are more differentiated (mature) than DFECs and are committed to specific cell types within the endodermal lineage. The PPCs of the present invention express the biomarkers PTF1A and PDX1.

[0044] Committed PPCs generated by the methods of the present disclosure can be further cultured in vitro to generate mature human pancreatic beta cells (PBCs). As used herein, "pancreatic beta cells" or "PBCs" refer to stem cell-derived pancreatic beta cells that express the biomarkers INS and PDX.

[0045] In some embodiments, cells can be identified and characterized based on the expression of one or more biomarkers that are specific or characteristic of DFEC, PPC or PBC.A " positive " biomarker is a biomarker that is expressed on the surface of cells of interest, whereas a " negative " biomarker is a biomarker that is not expressed on the surface of cells of interest.In the embodiments described herein, DFEC is FOXA2+ HNF1b+, PPC is PTF1A+ PDX1+, and PBC is INS+ PDX+.

[0046] As used herein, a very "low" level of cellular expression of a biomarker of interest is intended to refer to a level that is at most 20% above background levels, more preferably less than 20%, less than 15%, less than 10%, or less than 5% (background levels correspond, for example, to the expression level of a negative control marker that is considered not to be expressed by the cells).

[0047] II. Medium components The disclosed methods for generating DFECs, PPCs, and PBCs involve culturing human pluripotent stem cells in a medium containing specific agonists and / or antagonists of cell signaling pathways, which in some embodiments lack serum, lack exogenously added growth factors, lack animal products, are serum-free, xeno-free, and / or are feeder-layer free.

[0048] As shown in the exemplary embodiment depicted in Figure 1, medium containing a BMP pathway antagonist (e.g., LDN193189), an RA pathway agonist (e.g., RA), a TGF-β pathway antagonist (e.g., A8301), and a MEK pathway antagonist (e.g., PD032901) was sufficient to generate foregut endoderm cells (DFECs) in as little as three days (referred to herein as "stage 1" of the differentiation protocol). In some embodiments, pluripotent cells may be cultured for up to five days in stage 1 of the differentiation protocol.

[0049] Further differentiation of DFECs into pancreatic progenitor cells (PPCs) can be achieved after another 3 days by culturing DFECs in a medium containing a BMP pathway antagonist, such as LDN193189; an RA pathway agonist, such as retinoic acid (RA); a TGF-β pathway antagonist, such as A8301; a TAK1 pathway antagonist, such as Taki; a bFGF mimetic, such as SUN11602; and an Akt pathway antagonist, such as AT7867 (referred to herein as "stage 2"). In some embodiments, DFECs can be cultured for up to 5 days in stage 2 of the differentiation protocol.

[0050] Further differentiation of PPCs into pancreatic beta cells (PBCs) can be achieved in as little as 5 days (referred to as "stage 3") by culturing PPCs in medium containing a Notch pathway antagonist, e.g., gamma-secretase inhibitor XX (GSI-XX); a TGF-β pathway antagonist, e.g., A8301; and a flavonoid, e.g., quercetin or a quercetin analog thereof. In some embodiments, DFECs can be cultured in stage 3 medium of the differentiation protocol for up to 20 days.

[0051] As used herein, an "agonist" of a cell signaling pathway is used in reference to an agent that stimulates (upregulates) that cell signaling pathway. In some embodiments, stimulation of a cell signaling pathway can be initiated extracellularly, e.g., by using an agonist that activates a cell surface receptor involved in the signaling pathway (e.g., the agonist can be a receptor ligand). Additionally or alternatively, stimulation of cell signaling can be initiated intracellularly, e.g., by using a small molecule agonist that interacts intracellularly with one or more components of the signaling pathway.

[0052] As used herein, an "antagonist" of a cell signaling pathway refers to an agent that inhibits (downregulates) a cell signaling pathway. In some embodiments, inhibition of a cell signaling pathway can be initiated extracellularly, for example, by using an antagonist that blocks a cell surface receptor involved in the signaling pathway. Additionally or alternatively, inhibition of cell signaling can be initiated intracellularly, for example, by using a small molecule antagonist that interacts intracellularly with one or more components of the signaling pathway.

[0053] The agonist and antagonist used in the method of the present disclosure are known and / or commercially available.They are used in medium at effective concentrations to achieve desired outcome, for example, the production of DFE, PPC or PBC, each of which is characterized by specific corresponding markers.The non-limiting examples of suitable agonist and antagonist acting substance and effective concentration range will be further described below.

[0054] Antagonists of the BMP (bone morphogenetic protein) pathway include agents, molecules, compounds, or substances that can inhibit (downregulate) the BMP signaling pathway, which is biologically activated by the binding of BMP to a BMP receptor that is an activin receptor-like kinase (ALK) (e.g., type I BMP receptor, including, but not limited to, ALK2 and ALK3). In one embodiment, the BMP pathway antagonist is selected from the group consisting of LDN193189, DMH1, DMH2, dorsomorphin, K02288, LDN214117, LDN212854, follistatin, ML347, noggin, and combinations thereof. In one embodiment, the BMP pathway antagonist is present in the culture medium at a concentration within the range of 100-500 nM, 100-400 nM, 150-350 nM, or 200-300 nM. In one embodiment, the BMP pathway antagonist is LDN193189. In another embodiment, the BMP pathway antagonist is LDN193189, and the LDN193189 is present in the medium at a concentration within the range of 100-500 nM, 100-400 nM, 150-350 nM, or 200-300 nM. In another embodiment, the BMP pathway antagonist is LDN193189, and the LDN193189 is present in the medium at a concentration of 250 nM in steps (a) and (b) (i.e., stages 1 and 2) of the method.

[0055] Agonists of retinoic acid (RA) pathway include agents, molecules, compounds, or substances that can activate (upregulate) RA signaling pathway. In one embodiment, the RA pathway agonist is selected from the group consisting of retinoic acid (RA), TTNPB, AM580, CD1530, CD2314, CD437, Ch55, BMS753, BMS961, tazarotene, tamibarotene, isotretinoin, tretinoin, AC261066, AC55649, Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxoretinoic acid, and all-trans retinoic acid (ATRA), AY9944 dihydrochloride, ciliobrevin A, cyclopamine, or combinations thereof. In one embodiment, the RA pathway agonist is present in the medium at a concentration ranging from 0.05 to 5 μM, 0.5 to 5 μM, or 1 to 3 μM. In another embodiment, the RA pathway agonist is RA, and the RA is present in the culture at a concentration ranging from 0.2 to 5 μM, 0.5 to 4 μM, or 1 to 3 μM. In another embodiment, the RA pathway agonist is RA, and the RA is present in the medium in steps (a) and (b) (i.e., stages 1 and 2) of the method at a concentration of 2 μM.

[0056] TGFβ (transforming growth factor β) pathway antagonists include agents, molecules, compounds, or substances that can inhibit (downregulate) signaling through members of the TGFβ receptor family, a family of serine / threonine kinase receptors. In one embodiment, the TGFβ pathway antagonist is selected from the group consisting of A8301, SB-431542, GW788388, SB525334, TP0427736, Repsox, SD-208, and combinations thereof. In one embodiment, the TGFβ pathway antagonist is present in the culture medium at a concentration within the range of 200-1000 nM, 250-750 nM, 300-650 nM, or 400-600 nM. In one embodiment, the TGFβ pathway antagonist is A8301. In another embodiment, the TGFβ pathway antagonist is A8301, and A8301 is present in the medium at a concentration of 200-1000 nM, 250-750 nM, 300-650 nM, or 400-600 nM. In another embodiment, the TGFβ pathway antagonist is A8301, and A8301 is present in the medium in steps (a)-(c) (i.e., stages 1-3) of the method at a concentration of 500 nM.

[0057] Antagonists of the MEK pathway include agents, molecules, compounds, or substances that can inhibit (downregulate) one or more signaling pathways of components of the MAPK / ERK pathway (also known as the Ras-Raf-MEK-ERK pathway). In one embodiment, the MEK pathway antagonist is selected from the group consisting of PD0325901, binimetinib (MEK162), cobimetinib (XL518), selumetinib, trametinib (GSK1120212), CI-1040 (PD-184352), refametinib, ARRY-142886 (AZD-6244), PD98059, U0126, BI-847325, RO5126766, and combinations thereof. In one embodiment, the MEK pathway antagonist is present in the medium at a concentration within the range of 25-1000 nM, 50-750 nM, 75-500 nM, 100-400 nM, or 150-300 nM. In one embodiment, the MEK pathway antagonist is PD0325901. In another embodiment, the MEK pathway antagonist is PD0325901, and PD0325901 is present in the medium at a concentration within the range of 25-300 nM, 50-150 nM, 50-250 nM, or 150-300 nM. In another embodiment, the MEK pathway antagonist is PD0325901, and PD0325901 is present in the medium in step (a) (i.e., stage 1) of the method at a concentration of 250 nM.

[0058] Antagonists of the TAK1 (also known as MAP3K7) pathway include agents, molecules, compounds, or substances that can inhibit (downregulate) signaling through TAK1 (MAP3K7). In one embodiment, the TAK1 pathway antagonist is selected from the group consisting of Taki ((5Z)-7-oxozeaenol), takinib, dehydroabietic acid, NG25, sarsasapogenin, and combinations thereof. In one embodiment, the TAK1 pathway antagonist is present in the medium at a concentration within the range of 200-1000 nM, 250-750 nM, 300-650 nM, or 400-600 nM. In another embodiment, the TAK1 pathway antagonist is Taki, and Taki is present in the medium at a concentration of 300-800 nM, 250-750 nM, 300-650 nM, or 400-600 nM. In another embodiment, the TAK1 pathway antagonist is Taki, and Taki is present in the medium in step (b) (i.e., stage 2) of the method at a concentration of 500 nM.

[0059] bFGF mimetics include agents, molecules, compounds, or substances that can activate (upregulate) signal transduction through the fibroblast growth factor 2 (FGF2) signaling pathway. In one embodiment, the bFGF mimetic is SUN11602.

[0060] Antagonists of the AKT pathway include agents, molecules, compounds, or substances that can inhibit (downregulate) the signaling pathways of one or more of the serine / threonine kinase AKT family members, including AKT1 (also known as PKB or RacPK), AKT2 (also known as PKBβ or RacPK-β), and AKT3 (also known as PKBγ or thyoma viral proto-oncogene 3). In one embodiment, the AKT pathway antagonist is selected from the group consisting of AT7867, MK2206, GSK690693, perifosine (KRX-0401), ipatasertib (GDC-0068), capivasertib (AZD5363), PF-04691502, triciribine (NSC154020), ARQ751, milansertib (ab235550), volsertib, cerisertib, and combinations thereof. In one embodiment, the AKT pathway antagonist is present in the medium at a concentration within the range of 25-1000 nM, 50-750 nM, 75-500 nM, 100-400 nM, or 150-300 nM. In one embodiment, the AKT pathway antagonist is AT7867. In another embodiment, the AKT pathway antagonist is AT7867, and AT7867 is present in the medium at a concentration within the range of 25-300 nM, 50-150 nM, 50-250 nM, or 150-300 nM. In another embodiment, the AKT pathway antagonist is AT7867, and AT7867 is present in the medium in step (b) (i.e., stage 2) of the method at a concentration of 250 nM.

[0061] Notch pathway antagonists include agents, molecules, compounds, or substances that can inhibit (downregulate) signaling through or activity of Notch transcription factors, including gamma-secretase inhibitors (GSIs). In one embodiment, the Notch pathway antagonist is selected from the group consisting of GSI-XX, GSI-X, RO4929097, semagacestat, avagacestat, dibenzazepine, LY411575, LY450149, DAPT, crenigacestat, MK0752, BMS-708163, BMS-906024, CB-103, AL101, Compound E, Compound X(CX), IMR-1, IMR-1A, FLI-06, valproic acid, YO-01027, tangeretin, brucein D, and combinations thereof. In one embodiment, the Notch pathway antagonist is present in the medium at a concentration ranging from 25 to 200 nM, 50 to 150 nM, or 75 to 125 nM. In another embodiment, the Notch pathway antagonist is GSI-XX. In another embodiment, the Notch pathway antagonist is GSI-XX, and the GSI-XX is present in the medium at a concentration of 25 to 200 nM, 50 to 150 nM, or 75 to 125 nM. In another embodiment, the Notch pathway antagonist is GSI-XX, and the GSI-XX is present in the medium in step (c) (i.e., stage 3) of the method at a concentration of 100 nM.

[0062] Flavonoids include agents, molecules, compounds, or substances selected from the group consisting of quercetin, quercetin analogs (e.g., dihydroquercetin, 6,2',4',5'-pentahydroxyflavone, quercetin-3-O-propionate (Q-pr), quercetin-3-O-butyrate (Q-bu), quercetin-3-O-valerate, or 3,4'-di-O-methylquercetin), genistein, anthocyanins, catechins, gallocatechins (e.g., epigallocatechin-3-gallate (EGCG)), anthocyanidins, apigenin, luteolin, kaempferol, curcumin, myricetin, daidzein, naringin, rutin, and hesperitin. In one embodiment, the flavonoid is quercetin. In one embodiment, the flavonoid is present in the medium at a concentration within the range of 3 to 50 μM, 5 to 30 μM, or 10 to 20 μM. In another embodiment, the flavonoid is quercetin, and the quercetin is present in the medium at a concentration within the range of 3 to 50 μM, 5 to 30 μM, or 10 to 20 μM. In another embodiment, the flavonoid is quercetin, and the quercetin is present in the medium at a concentration of 15 μM.

[0063] When an agonist or antagonist is used in multiple steps of the method, the agent may be the same or different for one or more of the steps in which it is present in the medium. Furthermore, when an agonist or antagonist is used in multiple steps of the method, the concentration of the same agonist or antagonist may be the same or different for each step in which it is present in the medium.

[0064] III. Culture conditions In combination with the chemically defined and optimized media described in subsection II above, the methods for producing DFE, PP, and PBC of the present disclosure use standard culture conditions established in the art for cell culture. For example, cells can be cultured at 37°C under 5% O2 and 5% CO2 conditions. In one embodiment, cells can be cultured in a standard culture vessel or plate, such as a 96-well plate. In certain embodiments, the starting pluripotent stem cells are attached to a plate, preferably a plate coated with an extracellular matrix material such as vitronectin. In one embodiment, the stem cells are cultured on a vitronectin-coated culture surface (e.g., a vitronectin-coated 96-well plate).

[0065] Pluripotent stem cells can be cultured in commercially available media prior to differentiation. For example, stem cells can be cultured in Essential 8 Flex medium (Thermo Fisher # A2858501) for at least one day before the differentiation protocol begins. In a non-limiting exemplary embodiment, stem cells are cultured at a density of 150,000 cells / cm. 2 The cells were passaged onto vitronectin (Thermo Fisher # A14700) coated 96-well plates at a cell density of 1000 and cultured in Essential 8 Flex medium for 1 day before differentiation.

[0066] To initiate the differentiation protocol, the medium in which the stem cells are cultured is exchanged for a basal differentiation medium supplemented with signaling pathway agonists and / or antagonists as described above in Subsection II. The basal differentiation medium may comprise, for example, a commercially available base supplemented with additional standard media components necessary to maintain cell survival and proliferation, but lacking serum (basal differentiation medium is serum-free medium) or any other exogenously added growth factors, such as bFGF (FGF2), PDGF, or HGF. In a non-limiting exemplary embodiment, the basal differentiation medium contains 1x IMDM (Thermo Fisher #12440046), 1x F12 (Thermo Fisher #11765047), 1 mg / ml poly(vinyl alcohol) (Sigma #p8136), 1% chemically defined lipid concentrate (Thermo Fisher #11905031), 450 μM 1-thioglycerol (Sigma #M6145), 0.7 μg / ml insulin (Sigma #11376497001), and 15 μg / ml transferrin (Sigma #10652202001) (also referred to herein as "CDM2" medium, as used in the exemplary differentiation protocol set forth herein and illustrated in FIG. 1). The medium is typically replaced periodically with fresh medium. For example, in one embodiment, the medium is replaced every 24 hours.

[0067] To generate DFECs, PPCs, and PBCs, starting stem cells are cultured in optimized medium for a time sufficient for cell differentiation and expression of committed DFE, PPC, or PBC markers. As described in the Examples, culturing pluripotent stem cells in a three-stage method—one stage optimized for generating DFECs, a second stage optimized for generating PPCs, and a third stage optimized for generating PBCs—was found to generate PBCs in as little as 16 days of culture. The culture period for the first stage (to generate DFECs) corresponds to days 1–3, the culture period for the second stage (to generate PPCs) corresponds to days 4–6, and the culture period for the third stage (to generate PBCs) corresponds to days 7–16.

[0068] Thus, in the first stage of the method for generating DFEC, also referred to herein as "step (a)" or "stage 1," pluripotent stem cells are cultured in stage 1 optimized medium from days 0 to 3, or starting from day 0 and continuing through day 3, or for 72 hours (3 days), or for at least 60 hours, or at least 64 hours, or at least 68 hours, or at least 70 hours, or 60 hours, or 64 hours, or 68 hours, or 70 hours, or 72 hours. In some embodiments, pluripotent cells may be cultured for up to 5 days in stage 1 of the differentiation protocol. Thus, in certain embodiments, pluripotent stem cells may be cultured in Stage 1 optimized medium for 72 hours, followed by at least an additional 8 hours, at least an additional 8 hours, at least an additional 16 hours, at least an additional 24 hours, at least an additional 32 hours, at least an additional 40 hours, or may be cultured for 72 hours, followed by an additional 8 hours, at least an additional 16 hours, at least an additional 24 hours, at least an additional 32 hours, at least an additional 40 hours, or at least an additional 48 hours (i.e., up to a total of 5 days).

[0069] In the second stage of the method for generating PPCs, also referred to herein as "step (b)" or "stage 2," the DFECs generated in step (a) are further cultured in stage 2 optimized medium on days 4-6, or starting on day 4 and continuing through day 6, or starting on day 4 and continuing for 72 hours (3 days), or starting on day 4 and continuing for at least 60 hours, or at least 64 hours, or at least 68 hours, or at least 70 hours, or at least 72 hours, or starting on day 4 and continuing for 60 hours, or 64 hours, or 68 hours, or 70 hours, or 72 hours, starting on day 4. In some embodiments, DFECs may be cultured for up to 5 days in stage 2 of the differentiation protocol. Thus, in certain embodiments, DFEC may be cultured in Stage 2 optimized medium for 72 hours, followed by at least an additional 8 hours, at least an additional 16 hours, at least an additional 24 hours, at least an additional 32 hours, at least an additional 40 hours, or may be cultured for 72 hours, followed by an additional 8 hours, an additional 16 hours, an additional 24 hours, an additional 32 hours, an additional 40 hours, or an additional 48 hours (i.e., up to a total of 5 days).

[0070] In the third stage of the method for generating PPCs, also referred to herein as "step (c)" or "stage 3," the PPCs generated in step (b) are further cultured in stage 3 optimized medium from days 7 to 16, or starting on day 7 and continuing through day 16, or starting on day 7 and continuing for 240 hours (10 days), or starting on day 7 and continuing for at least 216 hours, or starting on day 7 and continuing for at least 220 hours, or starting on day 7 and continuing for at least 224 hours, or starting on day 7 and continuing for at least 228 hours, or starting on day 7 and continuing for 216 hours, or 220 hours, or 224 hours, or 228, or 232 hours. In some embodiments, PPCs may be cultured for up to 20 days in stage 3 of the differentiation protocol. Thus, in certain embodiments, PPCs may be cultured in Stage 3 optimized medium for 240 hours (10 days) followed by at least 1 additional day, at least 2 additional days, at least 3 additional days, at least 4 additional days, at least 5 additional days, at least 6 additional days, at least 7 additional days, at least 8 additional days, or at least 9 additional days, or may be cultured for 240 hours (10 days) followed by 1 additional day, 2 additional days, 3 additional days, 4 additional days, 5 additional days, 6 additional days, 7 additional days, 8 additional days, 9 additional days, or 10 additional days (i.e., up to 20 days total).

[0071] In an exemplary embodiment, TC plates are plated in Essential 8 (E8) medium at 62,500 cells / cm. 2Cells were seeded at an initial cell density of 1000 kJ / well and grown overnight. The next day, the growth medium was replaced with stage 1 medium (CDM2), which consisted of basal medium supplemented with 250 nM LDN193189, 2 μM retinoic acid, 500 nM A8301, and 250 nM PD0325901. CDM2 medium can be used for all stages of the different protocols and is described by Loh et al. (2014). Cultures were maintained in stage 1 medium for three consecutive days. On day 3, the differentiation medium was replaced with stage 2 medium, which consisted of basal medium CDM2 supplemented with 250 nM LDN193189, 500 nM (5Z)-7-oxozeanol, 2 μM retinoic acid, 300 nM SUN11602, 250 nM AT7867, and 500 nM A8301. Cultures are incubated in Stage 2 medium for 3 consecutive days until Stage 2 medium is replaced with Stage 3 medium, which consists of CDM2 supplemented with 100 nM γ-secretase, 500 nM A8301, and 15 µM quercetin. Cultures are incubated in the presence of Stage 3 medium for 5-10 consecutive days.

[0072] The differentiation protocol described above has been shown to be highly reproducible, resulting in consistent differentiation regardless of the adherence system used, and has been successfully used in most TC formats, including 96-well, 24-well, 6-well, and T75 flasks. This protocol has also been shown to work in 3D suspension culture, as described below. Throughout all stages, differentiated PSC cultures are supplied with fresh medium daily via medium changes.

[0073] To ensure that the PSCs do not settle or remain in suspension, some cell suspension embodiments use a shaker flask expansion method, which involves filling a flask approximately one-third full with growth medium, adding the PSCs, and placing the entire flask on a shaker. The medium solution is shaken so that the liquid is constantly circulating without splashing. This method was originally developed for the growth of bacteria or yeast, adapted for the growth of mammalian cells, and later for the growth of PSC aggregates. Other cell suspension embodiments may use spinner flasks. Spinner flasks have a propeller attached to a rod that reaches into the flask for agitation. The propeller is horizontal and designed to be moved by a magnetic stir plate on which the flask rests.

[0074] In some embodiments, a bioreactor system is used to culture the cells. An exemplary bioreactor system is the PBS Vertical Wheel Bioreactor System (https: / / www.pbsbiotech.com / uploads / 1 / 7 / 9 / 9 / 17996975 / 2021_borys_et_al._-_overcoming_bioprocess_bottlenecks.pdf). The PBS VW bioreactor comprises an enclosed vesicle that can accommodate various sizes of suspension cultures, including 100 ml, 500 ml, 3,000 ml, and 15,000 ml. The culture vessel sits on a magnetized vertical wheel with a corresponding motorized unit that rotates it to continuously agitate the suspension culture. In one embodiment, StemScale medium is used for general expansion of PSCs in the bioreactor.

[0075] In an exemplary embodiment, initial seeding of the VW bioreactor occurs from an adherent culture, but subsequent passaging events can be accomplished between bioreactors. On day 3–4, multiple wells of a 6-well plate are washed with PBS and then incubated in the presence of TryPLE for 3 minutes to remove cells from the plate. Three to four of these wells are then washed with basal medium to remove any cells that have not completely detached from the plate. Cells are counted and seeded at a concentration of 150,000 cells / ml into a PBS VW bioreactor containing StemScale medium supplemented with 10 μM Y27632. The following day, and every two days thereafter, a demi-depletion is performed on the bioreactor. This is accomplished by removing the bioreactor from its base and placing it in a hood for 5 minutes to allow aggregates to settle to the bottom of the bioreactor. At this time, half of the bioreactor volume is removed without disturbing the bottom of the reactor. This volume is then replaced with fresh StemScale medium. Bioreactors are passaged every 4–5 days. All media containing aggregates is removed from the bioreactor and placed in a centrifuge tube. The sample is spun at 400 × g for 4 minutes, after which the supernatant media is aspirated, leaving behind a pellet of cell aggregates. This pellet is then incubated in the presence of Accutase for 10 minutes to disaggregate the aggregates into small fragments and individual cells. The Accutase is then diluted so that it can be aspirated from the sample, and the sample is centrifuged. Passaging within the bioreactor contents can be performed using a scale-up model or a continuous culture model. Scale-up occurs when all biomaterial is placed into a larger bioreactor for subculture, such as the next size bioreactor for passage of a 100 ml sample into a 500 ml bioreactor. A continuous model may be used when only a portion of the sample is subcultured in a subsequent bioreactor, for example, when a sample is processed from a 100 ml run and only a portion of it is returned to inoculate the 100 ml bioreactor.

[0076] IV. use The disclosed methods and compositions for producing DFECs, PPCs, and PBCs enable efficient and robust access to these cell populations for a variety of uses in clinical therapy, research, development, and commercial purposes. For therapeutic purposes, the in vitro-produced PBCs of the present invention may be administered systemically or directly to a subject to treat or prevent type 1 diabetes, type 2 diabetes, prediabetes, conditions resulting from significant trauma (i.e., damage to the pancreas or loss of islet β cells or damage to islet β cells), or other metabolic diseases or disorders associated with a deficiency in the number of β cells (e.g., reduced pancreatic cell numbers), insufficient levels of β cell biological activity (e.g., deficiency in glucose-stimulated insulin secretion, deficiency in insulin production).

[0077] In one embodiment, the PBC cells of the present invention are directly injected into an organ of interest (e.g., the pancreas). Alternatively, a composition comprising the β-like cells of the present invention is indirectly provided to the organ of interest, for example, by administration to the circulatory system (e.g., the pancreatic vasculature). To increase the production of cells capable of producing insulin in vitro or in vivo, augmentation and differentiation agents may be provided before, during, or after cell administration. In some embodiments, the PBCs of the present application may be genetically modified to enhance their therapeutic and / or safety profile before administration. The cells may be dissolved in any physiologically acceptable vehicle and administered, typically intravascularly, but may also be introduced into another convenient site where the cells may find a suitable site for regeneration and differentiation.

[0078] In one embodiment, at least 100,000, 250,000, or 500,000 cells are injected. In other embodiments, 750,000 or 1,000,000 cells are injected. In other embodiments, at least 1 x 10 cells are injected. 5 cells, at least approximately 1 x 10 6 At least about 1 x 10 pieces 7 pieces, or 1 x 10 8 ~1×10 10Many cells, or even more, may be administered. The dosage can be easily adjusted by one skilled in the art (e.g., a decrease in purity may require an increased dosage). PBC cells may be introduced by catheter administration, systemic injection, local injection, including intravenous injection, or parenteral administration. When administered, the therapeutic compositions of the present invention (e.g., pharmaceutical compositions containing selected cells) are generally formulated in a unit-dose injectable form (solution, suspension, emulsion).

[0079] In other aspects, the PBC cells of the present disclosure can be used to screen potential drugs for treating diseases or disorders associated with PBC dysfunction or to develop new cell therapies for treating diseases or disorders associated with PBC dysfunction.

[0080] In other aspects, the methods and compositions can be used in studying the development and biology of pancreatic beta cell progenitors, including their differentiation into PBCs, to aid in the understanding and potential treatment of diseases and disorders associated with abnormal pancreatic beta cell function, such as diabetes.

[0081] In certain embodiments, DFEC, PPC, or PBC produced using the methods of the present disclosure can be further purified according to methods established in the art using agents that bind to surface markers expressed on the cells. Thus, in one embodiment, the present disclosure provides a method for isolating DFEC, PPC, or PBC, comprising contacting DFEC, PPC, or PBC produced by the methods of the present disclosure with one or more binding agents (e.g., monoclonal antibodies (mAbs)) that bind to one or more cell surface markers expressed on the DFEC, PPC, or PBC, and isolating cells that bind to the binding agents to facilitate isolation of the DFEC, PPC, or PBC. Cells that bind to the antibodies can be isolated by methods known in the art, including, but not limited to, fluorescent-activated cell-sorting (FACS) and magnetic-activated cell sorting (MACS).

[0082] V. composition In other aspects, the disclosure provides compositions related to methods of producing DFECs, PPCs, and PBCs, including media and cell cultures, and isolated progenitor cells and cell populations thereof.

[0083] In one aspect, the present disclosure provides a medium for obtaining human DFE, comprising a medium containing a BMP pathway antagonist, an RA pathway agonist, a TGF-β pathway antagonist, and a MEK pathway antagonist. Suitable agents and concentrations thereof include those described in subsection II.

[0084] In another aspect, the present disclosure provides a medium for obtaining human PPCs, comprising a BMP pathway antagonist, an RA pathway agonist, a TGF-β pathway antagonist, a TAK1 pathway antagonist, a bFGF mimetic, and an Akt pathway antagonist. Suitable agents and concentrations thereof include those described in subsection II.

[0085] In another aspect, the present disclosure provides a medium for obtaining human pancreatic beta cells (PBCs), comprising a Notch pathway antagonist, e.g., a gamma-secretase inhibitor, a TGF-beta pathway antagonist, and a flavonoid, e.g., quercetin or a quercetin analog. Suitable agents and concentrations thereof include those described in subsection II.

[0086] In another aspect, the present disclosure provides an isolated cell culture of human DFEC, comprising human DFEC cultured in a medium comprising a BMP pathway antagonist, an RA pathway agonist, a TGF-β pathway antagonist, and a MEK pathway antagonist. Suitable agents and concentrations thereof include those described in subsection II.

[0087] In another aspect, the present disclosure provides an isolated cell culture of human PPCs, the cell culture comprising human PPCs cultured in a medium comprising a BMP pathway antagonist, an RA pathway agonist, a TGF-β pathway antagonist, a TAK1 pathway antagonist, a bFGF mimetic, and an Akt pathway antagonist. Suitable agents and concentrations thereof include those described in subsection II.

[0088] In another aspect, the present disclosure provides an isolated cell culture of human pancreatic beta cells (PBCs), comprising human PBCs cultured in a medium comprising a Notch pathway antagonist, e.g., a gamma-secretase inhibitor, a TGF-beta pathway antagonist, and a flavonoid, e.g., quercetin or a quercetin analog. Suitable agents and concentrations thereof include those described in Subsection II.

[0089] In another aspect, the present disclosure provides human DFEC produced by the methods of the present disclosure (ie, step (a) or stage 1 of the culture protocol).

[0090] In another aspect, the present disclosure provides human PPCs produced by the methods of the present disclosure (ie, steps (a) and (b), or stages 1 and 2, of the culture protocol).

[0091] In another aspect, the present disclosure provides human PBCs produced by the methods of the present disclosure (ie, steps (a), (b) and (c), or stages 1, 2 and 3, of the culture protocol).

[0092] The present invention is further illustrated by the following examples, which should not be construed as further limiting. The figures, as well as the contents of all references, patents, and published patent applications cited throughout this application, are hereby expressly incorporated by reference. [Example]

[0093] Examples 1 and 2 : Optimization of HNF1B from iPSC line CR01 Dorsal foregut endoderm (DFE) is defined as a pluripotent-derived endoderm population that patterns the region of the early developing embryo that arises dorsal to the midgut of the primitive digestive tract. Expression patterns within this population are indicated by the dorsal endoderm markers SFRP5, MNX1, PTCH1, and PAX6, and robust expression of the midgut markers FOXA2, HNF1b, HOXA3, and ONECUT2. High expression of these markers contrasts with their absence in ventral, more posterior endoderm regions. DFE was found to have very low or absent expression of the ventral markers HHEX and NR5A2, as well as the classically defined definitive endoderm markers SOX17, GSC, MIXL1, and CER. It has previously been demonstrated that the DFE state can be achieved by dual optimization of HNF1b and FOXA2, and that this induction can be achieved by retinoid signaling, which occurs in the absence of BMP signaling. Previous studies on the DFE population used one iPSC line and embryonic stem cells.

[0094] The NCRM1 cell line was selected to assay the efficacy of potentially clinical-grade iPSC lines. HD-DoE-based optimization was used to validate HNF1b and FOXA2 optimizers on this cell line. The HD-DOE method was applied with the intent of discovering conditions for inducing endodermally expressed genes directly from a pluripotent stem cell state. In this example, we use a method previously described by Bukys et al. (2020) Iscience 23:101346. This method uses computerized design geometry to simultaneously test multiple process inputs and provides mathematical modeling of deep effector / response space. This method allows for the discovery of combinatorial signaling inputs that control complex processes, such as those during cell differentiation. This method allows for the testing of multiple plausible key process parameters as they affect output responses, such as gene expression. Because gene expression is a hallmark of phenotype, e.g., of human cells, the methods can be applied to identify and understand which signaling pathways control cell fate.

[0095] To develop cell culture recipes for each stage, we tested and modeled the effects of agonists and antagonists (referred to herein as effectors) of multiple signaling pathways on the expression of preselected genes. The influence of each effector on gene expression levels is revealed by a parameter called the factor contribution, which is calculated for each effector during modeling. These effectors are small molecules or proteins commonly used during stepwise stem cell differentiation toward specific fates. The selection of effectors was based on the latest literature on stem cell differentiation into endodermal progenitor cells.

[0096] In both optimizations, BMP pathway inhibition was the most important component of the optimization, with factor contributions of LDN193189 of 31.26 and 31.11 for HNF1b (Figure 2) and FOXA2 (Figure 3), respectively. MEK pathway inhibition with PD0325901 was important for HNF1b and FOXA2 (factor contributions of 15.01 and 29.01, respectively), but FOXA2 activation was more dependent on MEK inhibition. Two other pathways determined to be critical for regulating DFE activation were retinoid signaling and TGF-β pathway inhibition. These two pathways had different effects on FOXA2 and HNF1b. Inhibition of the TGF-β pathway with A8301 was critical for optimal FOXA2 activation, with a positive correlation and a significant FC of 17.72 (Figure 3). In contrast, the use of A8301 had little effect on HNF1b activation (Figure 2, negative correlation and negligible FC of 3.02). Retinoid signaling was significant in HNF1b activation with a FC of 14.43 (Figure 2), but not significantly involved in FOXA2 activation with a negative correlation and FC of 5.44 (Figure 3).

[0097] Example 3 : Dual optimization of FOXA2 and HNF1B leads to dorsal foregut endoderm (DFE) Performing a dual optimizer for the combined expression of FOXA2 and HNF1b further demonstrated the importance of simultaneously regulating these four pathways, as the optimizer predicted that active retinoid signaling was required in the presence of BMP, TGF-β, and MEK pathway inhibition (Figure 4). We concluded that while both retinoid signaling and BMP pathway inhibition are still required for DFE generation, the CR01 iPSC cell line significantly requires active inhibition of both TGF-β and MEK signaling.

[0098] Example 4 Dynamic profiling demonstrates that MEK and TGF-β inhibition are required for FOXA2 induction As shown in Figure 5, dynamic profiling further confirmed the need to simultaneously control all four pathways. The most important process parameter to control in DFE production was the inhibition of BMP signaling by LDN193189. This was crucial for both HNF1b and FOXA2 expression, whereas other components present in the HNF1B / FOXA2 optimizer had different effects on these two fate-driving genes. Retinoid signaling was shown to significantly increase HNF1b activation while having a slight inhibitory effect on FOXA2 expression, but this inhibitory effect was negligible. Similarly, inhibiting both the MEK and TGF-β pathways was highly beneficial for FOXA2 activation but had little or no effect on HNF1b expression.

[0099] Example 5 : Coefficient plot shows the main effectors required for DFE differentiation As shown in Figure 6, the coefficient plots confirmed that BMP pathway inhibition was fundamentally important for the activation of HNF1b (top) and FOXA2 (bottom). Inhibition of the TGF-β and MEK pathways was determined to be more directly involved in FOXA2 activation.

[0100] Example 6 Heatmap identifies key input components required for DFE creation By using previously identified key process parameters, retinoic acid and LDN193189, in the presence or absence of A8301 and PD0325901, we found robust FOXA2 and HNF1b expression dependent on the addition of either of these compounds (Figure 7). Supplying this differentiation medium with active Wnt signaling (an HNF1b optimizer identified in embryonic stem cells) separated HNF1b co-expression from FOXA2 co-expression and significantly reduced endoderm genes overall, suggesting differences between pluripotent starting materials. By actively inhibiting both the TGF-β and MEK pathways, we observed an increase in genes representative of the dorsal foregut and a generalized endoderm fate (Figure 7). Furthermore, greater pancreatic fate was observed in this progenitor population. All conditions demonstrated evidence of genes known to suppress neural fates.

[0101] Example 7 : DFE is directly induced without passing through the primitive streak or definitive endoderm intermediate As shown in the heatmap in Figure 8, genes classically defined as primitive streak and definitive endoderm markers were not activated during DFE induction, further demonstrating that iPSCs directly convert to a patterned endoderm fate. None of the genes classically defined as primitive streak markers, including NODAL, FGF8, WNT3, TBXT, LHX1, and FGF4, showed any level of activation. Similarly, with the exception of the DE markers FOXA2 and CXCR4, which are associated with DFE patterning, the definitive endoderm markers SOX17, GSC, MIXL1, and CER1 were not expressed.

[0102] Example 8 : Achieving optimal FOXA2 expression As shown in Figure 9, immunohistochemical analysis confirmed the significant contribution of MEK (PD0325901) and TGF-β (A8301) pathway inhibition to FOXA2 activation. Notably, the inclusion of either PD0325901 or A8301 increased FOXA2 activation compared with RA alone and LDN alone. However, this increase was even greater when both PD0325901 and A8301 were used together.

[0103] Example 9 Dynamic profiling demonstrates potential for the pancreas To assess the ability of DFE cultures to generate pancreatic fields, genes associated with this endodermal region were evaluated for expression during pancreatic bud formation. These genes include PROM1, CPB1, and ONECUT1. As shown in the dynamic profiling diagram in Figure 10, these genes were shown to be similarly regulated, and all were activated in DFE cultures. The combined effects of BMP pathway inhibition and active retinoid signaling increased ONECUT1 and CPB1 activation. PROM1 activation did not depend on BMP inhibition, but showed a stronger response to the combined effects of retinoid signaling in the presence of TGF-β and MEK inhibition. Collectively, the combined expression of HNF1B, FOXA2, PROM1, CPB1, and ONECUT1 indicates an endodermal population capable of inducing pancreatic fields.

[0104] Example 10 : Modeling for maximum PDX1 induction To determine the best approach to induce pancreatic fate from DFE progenitors, we performed a subsequent HD-DoE analysis. We identified optimal conditions for PDX1 induction by exposing DFE cultures to a perturbation matrix consisting of all previously identified effectors shown to have a positive effect on PDX1 activation. As shown in Figure 11, inhibition of retinoid signaling (retinoic acid) and the Akt / PKA pathway (AT7867) had the strongest effect on PDX1 activation, with a factor contribution of 19.2 and 15.5, respectively. Furthermore, inhibition of the TAK1 pathway ((5Z)-7-oxozeanol), inhibition of the SHH pathway (SANT1), and simultaneous stimulation of the FGF pathway (SUN11602) had a diminishing effect on PDX1 activation, with a factor contribution of 8.2, 5.0, and 4.7, respectively.

[0105] Example 11 : Coefficient plot shows the main effectors for generating pancreatic progenitor cells from DFE progenitor cells Coefficient plots comparing the input logic of the pancreas-specific genes PTF1A and PDX1 revealed that the combined effect of active retinoid signaling (retinoic acid) in the presence of MEK pathway inhibition (PD0325901) are the two most important pathways controlling pancreatic field activation from the regionalized dorsal foregut endoderm field (Figure 12). Combinatorial expression of PTF1A and PDX1 occurs exclusively within the early pancreatic field of the developing embryo, and not in any other tissue type.

[0106] Example 12 : Optimization for PDX1 induction As shown in Figure 13, immunohistochemical analysis demonstrated that retinoic acid and PD0325901 are the main factors required for PDX1 activation, but inclusion of other indicated pathways also significantly increased PDX1 activation. As shown in Figure 16 in Example 15, inhibition of both the BMP (LDN) and SHH (sant1) pathways while providing PKC agonism increased PDX1 activation.

[0107] Example 13 : Dynamic profiling to discover endocrine cell subtypes Comparison of PDX1 optimizers with markers of pancreatic endocrine fate revealed that the predicted conditions were compatible with the creation of an endocrine fate (Figure 14). Notably, only retinoic acid had a strong inhibitory effect on the activation of SST (a δ-cell marker), and the FGF agonist SUN11602 had a slight negative effect on the activation of INS (a β-cell marker). These findings suggest that this predicted PDX1 induction represents a pre-endocrine area.

[0108] Example 14 : Modeling for maximum insulin expression Because PDX1+ cells can give rise to all cell types in the pancreas and gallbladder, as well as gastric and intestinal cells, we sought optimal conditions for β-cell induction. This was accomplished by parallel experiments in which cells were exposed to the same perturbation matrix and then differentiated for an additional 5 days in the presence of a Notch pathway inhibitor (γ-secretase inhibitor (GSI)-XX) and a TGF-β pathway inhibitor (A8301). It is well established that inhibition of these pathways induces β-cell fate from pre-endocrine progenitor cells. Optimizing these cultures for INS predicted similar culture conditions to the original PDX1 optimizer. Notably, as shown in Figure 15, both the retinoid agonist and the Akt / PKA pathway inhibitor increased the factor contributions by 33.3 and 25.5, respectively. Furthermore, while inhibition of both the BMP pathway (LDN) and the TGF-β pathway (A8301) was required, active inhibition of the SHH pathway was no longer required.

[0109] Example 15 : Dynamic profiling of endocrine cell subtypes Comparing the regulatory inputs of the effectors used in the insulin optimizer with PDX1 activation revealed nearly identical input logics for the two genes. As shown in Figure 16, the strongest regulatory inputs for activating both PDX1 and INS are active retinoid signaling and Akt / PKA pathway inhibition. GCG (an α-cell marker) showed an input logic identical to that of INS, whereas SST (a δ-cell marker) showed an input logic opposite to that of PDX1 / INS / GCG. This suggests a significantly different regulatory system for δ cells.

[0110] Example 16 : Immunohistochemical verification of novel β cells As shown in Figure 17, immunohistochemical analysis confirmed the ability of PDX1-optimized cultures to activate endocrine genes. PDX1-expressing cultures were subjected to the TGF-β pathway inhibitor A8301 and the Notch pathway inhibitor gamma-secretase inhibitor XX (GSI-XX), and then assayed for INS expression. The results of this analysis demonstrated that the resulting PDX1 cells were proendocrine. Furthermore, C-peptide staining confirmed that the INS detected originated within the cultures and was not absorbed from the growth medium used to maintain the cultures (Figure 17). C-peptide is produced when proinsulin is processed into a functional insulin molecule, and C-peptide is only present when de novo insulin is produced.

[0111] Example 17 : HD-DoE screening for optimal de novo β-cell induction To clarify the factors that contribute to INS activation, we performed a series of HD-DoE perturbations using factors selected to target different aspects of β-cell biology. As shown in Figure 18, the compounds that demonstrated the greatest contribution to INS activation targeted inhibition of the PI3K / AKT pathway (quercetin) and inhibition of α2-adrenergic agonism (clonidine), with factor contributions of 15.6 and 23.4, respectively. Other compounds that increased INS induction to a lesser extent, with factor contributions of 5–10, were SEMA3, GH, and rapamycin.

[0112] Example 18 : Heatmap showing the descending factor contributions for INS induction To determine which factors may potentially favor β cells over other endocrine cell types, optimizers for markers of other endocrine cell types and β cell-specific markers were compared. The relative factor contributions from the various optimizers were compared. As shown in Figure 19, the compounds shown to be beneficial for all genes optimized in this way were quercetin, a PI3K / AKT pathway inhibitor; rapamycin, an mTOR pathway inhibitor; and IBMX, a cAMP pathway activator.

[0113] Example 19 : HD-DoE screening for optimal de novo β-cell induction To identify factors contributing to INS activation, we performed a series of HD-DoE perturbations using factors selected to target different aspects of β-cell biology. Factors tested that were determined to have potent INS activation capabilities included the Axl inhibitor R248, the SHH inhibitor SANT1, the retinoid agonist TTNPB, the TPH1 inhibitor LP533401, and the amino acids tryptamine and glutamine (Figure 20). Their respective factor contributions were 22.9, 15.0, 12.7, 10.7, 21.2, and 10.4, respectively. Other compounds that had positive effects on INS induction included the hormone obestatin, the GLP-1 activator exendrin4, the RET pathway inhibitor SPP86, the thiazolidinedione rosiglitazone, and the short-chain fatty acid propionate. The factor contributions of these compounds for INS induction are 9.6, 6.0, 4.8, 6.0, and 5.3, respectively (FIG. 20).

[0114] Example 20 : Heatmap showing the descending factor contributions for INS induction We generated optimizers for key pancreatic endocrine hormones and β-cell-specific transcription factors to compare their contributions to this set of HD-DoEs. In this manner, we identified compounds that may selectively favor β-cell differentiation relative to α- and δ-cell differentiation. Notably, the retinoid agonist TTNPB potently inhibited both α- and δ-cell fates while potently activating all β-cell-specific transcription factors assayed in this manner (Figure 21). Propionate and glutamax had similar, albeit smaller, effects preferentially favoring β-cell fate. Serotonin had little effect on INS induction, but potently activated GCG induction and inhibited SST induction (Figure 21).

[0115] Example 21 : Dynamic profiling of endocrine subtype regulation Dynamic profiling of the critical beta cell-specific markers INS and PDX1 was used to compare the regulatory profile of beta cells with that of alpha and delta cells (Figure 22). Within this HD-DoE matrix, tryptamine had the strongest INS-inducing effect, selectively increasing INS induction while having no effect on any other transcripts measured. Other compounds that contributed to INS induction were LP533401, obestatin, clonidine, and serotonin, but these had smaller effects and were not specific for beta cell induction.

[0116] Example 22 : Dynamic profiling of endocrine subtype regulation Dynamic profiling of the crucial beta cell-specific markers INS and PDX1 was used to compare the regulatory profile of beta cells with that of alpha and delta cells (Figure 23). The strongest inducers of beta cell status were the Axl inhibitor R428 and the retinoid agonist TTNPB. TTNPB was shown to be highly selective for beta cell status because it is inhibitory to the divergent endocrine fates of alpha and delta cells. Exendin-4 showed a similar pattern of preferentially driving beta cell fates from pre-endocrine cultures, although INS induction by this compound was less potent. The FGF pathway agonist FGF19 also contributed to overall INS induction, but to a much smaller extent than the other mentioned effectors.

[0117] Example 23 : Dynamic profiling of endocrine subtype regulation Dynamic profiling of the critical beta cell-specific markers INS and PDX1 was used to compare the regulatory profile of beta cells with that of alpha and delta cells. As shown in Figure 24, the JNK pathway inhibitor SP600125 and the amino acid Glutamax were both shown to be positive inducers of endocrine fate. However, both increased GCG and SST levels throughout the culture, and were therefore not beta cell specific. Cardiotropin and diazoxide both positively increased INS induction, but not significantly.

[0118] Example 24 : Dynamic profiling of endocrine subtype regulation Dynamic profiling of the crucial beta cell-specific markers INS and PDX1 was used to compare the regulatory profile of beta cells with that of alpha and delta cells. As shown in Figure 25, the SHH pathway inhibitors SANT1 and Glutamax both increased the induction of endocrine fate without overall specificity to beta cells. Although the effects of SANT1 and Glutamax on INS induction were more potent, increased induction of GCG and SST was observed. The compounds rosiglitazone and propionate significantly increased INS levels and inhibited both GCG and SST induction, suggesting specificity for the beta cell state. Other compounds that had positive effects on INS induction but much smaller contributions to overall INS levels were SPP86, A8301, and dorsomorphin.

[0119] Example 25 : Immunohistochemical verification of new β-cell generation Addition of quercetin to stage 3 medium increased overall endocrine conversion and induced greater overall new β-cell generation, as shown by immunohistochemical analysis of day 16 cultures shown in Figure 26. C-peptide staining was performed to distinguish between new β-cell generation and possible insulin absorption from the medium.

[0120] Example 26 : Immunohistochemical verification of new β-cell generation As shown in Figure 27, the cultures were highly homogenous for endocrine cells, as reflected by wide-field expression of CGA, a common endocrine marker. C-PEP co-staining was consistent with cells possessing the β-cell subtype. Areas interspersed with non-endocrine cell types were shown to be proendocrine trunk progenitor cells, as indicated by SOX9 expression.

[0121] Example 27 : Functional analysis of β cells To assess the functional maturity of the generated new β-cells, a glucose-stimulated insulin secretion assay was performed. The growth medium was removed from the assay samples, and the assay samples were washed once with PBS to remove any residual medium that may have been previously conditioned by secreted C-PEP. All samples were then incubated at 37°C for 15 minutes in the presence of basal medium. The basal medium consisted of RPMI containing 3 mM glucose. This medium was then replaced with medium supplemented with 17.5 mM glucose or 30 mM KCl and incubated for an additional 15 minutes. As shown in Figure 28, samples exposed to 17.5 mM glucose medium exhibited a two-fold increase in secreted C-peptide levels. In contrast, samples exposed to 30 mM KCl produced approximately four-fold increases in C-peptide levels compared to basal C-peptide secretion levels. These results demonstrate that the response of new β-cells to glucose exposure does not reach the cells' maximum secretory capacity, suggesting a limited functional capacity that reflects a more immature cellular state.

[0122] Example 28 : Directed differentiation of β cells is applicable to suspension culture systems To apply the iPSC-derived DFE protocol to a PBS vertical wheel bioreactor, we chose the bioreactor system illustrated in Figure 29. This culture system was ideal due to its scalability, with bioreactors available in sizes from 0.1 to 80 liters. Initial efforts in adapting the protocol focused on efficiently progressing the differentiation process.

[0123] Example 29 : Proliferation and expansion occur throughout the differentiation process Cultures exhibited an initial proliferation phase that disappeared within 4–5 days, after which the number of cells surviving subsequent steps in the protocol decreased (Figure 30). The observed decrease in viability and overall cessation of differentiation events was attributed to increasing aggregate size. As aggregate size increases, nutrient availability becomes limited due to limited diffusivity, a known issue in bioreactors. To overcome these limitations, we incorporated periodic disruption of aggregates between separate protocol steps. This maintained small aggregate size, ensured nutrient availability, and allowed for a continuous proliferation state. A decrease in proliferation rate was observed throughout the differentiation process. Progression from DFE progenitors to PP progenitors in the PBS vertical wheel bioreactor showed a 5-fold and 3-fold increase, respectively. This was expected, as progenitor cells are highly proliferative, whereas terminally differentiated cells are quiescent.

[0124] Example 30 Comparable phenotypes were observed between the bioreactor and the newborn protocol. Continued growth in the bioreactor significantly increased the biomass produced, with an estimated 20- to 50-fold expansion potential for iPSC cultures. To initially establish the bioreactor-based protocol, biomass reduction throughout the process was used to maintain optimal cell density within the bioreactor. To confirm a similar phenotype between cells differentiated in the bioreactor and those differentiated using the newborn protocol, transcript levels of several key endocrine genes were compared. The endocrine products INS, representing pancreatic alpha cells, GCG, representing pancreatic beta cells, and SST, representing pancreatic delta cells, were measured throughout different stages of the bioreactor run. As expected, these hormones were undetectable in the early stages but increased significantly by stage 3. Two highly specific beta-specific genes, NEUROD and NKX2.2, were also measured, showing similar expression patterns. No significant differences in transcript levels were observed when bioreactor runs were compared with control cultures differentiated under adherent TC conditions.

[0125] Example 31 : Novel insulin production in bioreactors Stage 3 bioreactor runs consistently produced an average density of 100 aggregates / ml, with each aggregate consisting of approximately 500 cells. C-peptide was detected both in the stage 3 medium used to differentiate the cell aggregates and within the cell aggregates themselves (Figure 32). Basal insulin secretion is consistent in the immature β-cell phenotype observed by many other groups. C-peptide was detected at levels of 6.5 ng / L in the medium used in the bioreactor. C-peptide is produced when proinsulin is processed into a functional insulin molecule, and C-peptide is only present when de novo insulin is produced. The C-peptide detection method used is highly specific for human C-peptide, and the differentiation medium used contains only recombinant insulin lacking this region of the peptide. To further confirm that the C-peptide present in the conditioned medium originated from the aggregates, control medium samples not exposed to the aggregates during differentiation were analyzed in parallel. The control medium samples showed no detectable levels (Figure 32). Furthermore, cell lysates from stage 3 aggregates were assayed for the presence of C-peptide. A cellular content of 250–500 pg of C-peptide per aggregate was detected, demonstrating that iPSCs differentiated in bioreactors both produce and process insulin.

[0126] Example 32 : Further characterization of insulin-producing cells In this example, additional experiments were performed to characterize the insulin-producing cells.

[0127] As demonstrated in Figures 33A-B, the bioreactor run consistently yielded insulin-producing cells. iPSC cultures were grown as aggregates to an average size of 150 μm in diameter. After stage 1 medium induction of dorsal foregut endoderm, samples of the aggregates were treated with Accutase and plated on vitronectin. This was followed by staining for the markers FOXA2 and HNF1B to confirm DFE induction in the bioreactor. The DFE cultures were then exposed to stage 2 medium, after which samples were treated with Accutase and plated on vitronectin for IHC imaging. The cultures were stained for the pancreatic progenitor marker PDX1. These PP cultures were then exposed to stage 3 medium and plated on Matrigel to maintain the aggregates. The following day, they were stained for the beta cell-specific markers CPEP and PDX1.

[0128] As shown in Figure 34, bioreactor-produced endocrine cells have similar gene expression patterns compared to human islets. For comparison, IHC analysis of aggregates generated within the bioreactor run and primary human islets was performed on Matrigel. The expression patterns of PDX1 and CPEP in the cells were assessed, demonstrating similar expression patterns. It should be noted that PDX1 expression in iPSC derivatives was not restricted to CPEP+ cells, demonstrating the continued generation of pancreatic progenitor cells within the cultures. Examination of endocrine subpopulations revealed the presence of all major endocrine subtypes within the iPSC derivatives. Within the same cells, cells expressing CPEP (β cells), GCG (α cells), and SST (δ cells) were observed, as well as cells expressing multiple endocrine products. These represent immature, uncommitted endocrine cells.

[0129] The average insulin content per cell was determined. These results are shown in Figure 35. The aggregates were lysed using TPER, and CPEP content was assessed by ELISA. This was then divided by the estimated number of cells in the aggregate sample and compared to the theoretical estimate of insulin content in human islets. The aggregates produced throughout the bioreactor run were found to contain approximately 50% of the expected insulin levels in human islets. Note that the conversion between measured CPEP content and measured INS content is a 1:1 ratio between CPEP and INS, because they are initially synthesized as a single polypeptide, then processed and stored together in vesicles.

[0130] As shown in Figure 36, insulin production continued for up to two weeks after initial induction. Bioreactor cultures were monitored and sampled over the course of stage 3 induction. CPEP content was observed to increase until day 14, when CPEP levels plateaued. This indicates that increasing the length of time aggregates are maintained in stage 3 medium increases the overall efficiency of the differentiation event.

[0131] As shown in Figure 37, glucose-stimulated insulin secretion was not maintained after cryopreservation. iPSC derivatives exhibit varying degrees of function when exposed to glucose influx. Results showed that the function and overall insulin content of iPSC derivatives decreased upon cryopreservation, decreasing from a stimulation index (SI) of 2.7 before freezing to an SI of 1.3 after recovery.

[0132] As shown in Figure 38, bioreactor-based production runs consistently produced insulin-producing cells at levels comparable to human islets. Using three different bioreactor runs of stage 3 cells, RNA sequencing was performed to assess expression patterns representing different aspects of the differentiation process. Genes representing dorsal foregut endoderm were found to be more highly expressed in the iPSC derivatives than in the primary islets, indicating retention of the dorsal phenotype. Notably, SOX2, NODAL, and FOXA2 expression remained elevated in the iPSC derivatives. Genes representing the exocrine component of the pancreas were also shown to be expressed at higher levels in the primary human islets. This is consistent with low levels of exocrine tissue carrying over into the extraction process during islet purification, and also indicates the absence of exocrine tissue in the bioreactor-based iPSC derivative production. Several endocrine-specific genes were assessed. Notably, iPSC derivatives consistently showed high levels of CHGA (a pan-endocrine marker), indicative of high levels of general endocrine cells, but low levels of actual endocrine products, as observed by low levels of INS, GCG, and SST expression. High expression of the early α-cell marker ARX and low expression of the mature α-cell marker MAFB were observed in iPSC derivatives, indicating an early, immature α-cell phenotype in iPSC derivatives. A similar lack of mature phenotype markers was observed in iPSC-derived insulin-producing cells, with low expression of NKX2.2, MNX1, INSM1, and ESR2 when compared to human islets.

[0133] As shown in Figure 39, the overall function of insulin-secreting cells produced in a bioreactor occurs via cAMP agonism. Assaying the insulin secretion mechanism of iPSC derivatives revealed that cAMP agonists could increase insulin secretion when used in conjunction with the GSIS assay. This increase in insulin release due to cAMP agonism did not occur when the cAMP agonists used were GLP1 or exendin 4 (EX4). Fully matured β cells respond to incretins such as GLP1 via a cAMP-mediated response. iPSC derivatives do not respond to GLP1 or the commonly used GLP1 pathway agonist EX4. Expression of the receptor for these incretins (GLPR1) in iPSC derivatives was shown to be very low (Figure 40). The rate of insulin secretion is also directly related to the rate of mitochondrial respiration; fully matured β cells lack LDHA expression and are therefore only able to metabolize glucose via mitochondrial respiration. Although LDHA expression in iPSC derivatives was similar to primary islets, anabolic factors that directly nourish mitochondria, which typically increase overall mitochondrial respiration, had little effect on increasing insulin secretion (Figure 39), suggesting that iPSC derivatives lack the mitochondrial mass found in fully functional β-cells.

[0134] The results of RNA sequencing of gene expression are shown in Figure 40. Fully functional β-cells contain a specific, constitutively functional GLUT2 glucose transporter, which allows intracellular glucose concentrations to be regulated by serum glucose levels. Fully functional β-cells also contain a highly specific hexokinase enzyme that is responsible for phosphorylating intracellular glucose and forcing it into glycolysis. This hexokinase, glucokinase (GCK), has a low binding affinity, Km, which allows intracellular glucose concentrations to be regulated to ideal serum levels. While GCK expression is similar between iPSC derivatives and primary β-cells, expression of GLUT2 transporters is lower. This limits the ability of iPSC derivatives to match their intracellular glucose concentrations to those of the basal medium and, as a result, reduces their ability to respond appropriately to glucose fluctuations.

[0135] Furthermore, iPSC derivatives have continued expression of HK1, HK2, and SLC16A1. Downregulation of these three genes has been shown to be crucial for achieving a fully functional phenotype. iPSC derivatives also show reduced expression of other crucial maturation markers, FFAR1 and KIR6-2. Taken together, this demonstrates that iPSC derivatives are not fully functional and have a limited response to glucose.

[0136] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the scope of the following claims.

Claims

1. 1. A method for generating human FOXA2+ HNF1b+ dorsal foregut endoderm cells (DFECs), comprising: The method comprises culturing human pluripotent stem cells (PSCs) in a medium containing a BMP pathway antagonist, an RA pathway agonist, a TGF-β pathway antagonist, and a MEK pathway antagonist to obtain human FOXA2+ HNF1b+ human DFECs.

2. The method of claim 1, further comprising culturing the DFEC in a medium containing a BMP pathway antagonist, an RA pathway agonist, a TGF-β pathway antagonist, a TAK1 pathway antagonist, a bFGF mimetic, and an Akt pathway antagonist to obtain human PTF1A+ PDX1+ pancreatic progenitor cells (PPCs).

3. The method of claim 2, wherein the PPCs are further cultured in a medium containing a gamma-secretase inhibitor, a TGF-β pathway antagonist, and a flavonoid to obtain human INS+ PDX+ pancreatic beta cells (PBCs).

4. The method of any one of claims 1 to 3, wherein the human pluripotent stem cells are induced pluripotent stem cells (iPSCs).

5. The method of any one of claims 1 to 3, wherein the human pluripotent stem cells are embryonic stem cells.

6. The method of any one of claims 1 to 5, wherein the human pluripotent stem cells are attached to a vitronectin-coated plate during culture.

7. The method of any one of claims 1 to 6, wherein the BMP pathway antagonist is selected from the group consisting of LDN193189, DMH1, DMH2, dorsomorphin, K02288, LDN214117, LDN212854, follistatin, ML347, noggin, and combinations thereof.

8. 8. The method of claim 7, wherein the BMP pathway antagonist is present in the medium at a concentration in the range of 100 to 400 nM.

9. 8. The method of claim 7, wherein the BMP pathway antagonist is LDN193189, and the LDN193189 is present in the culture medium at a concentration of 250 nM.

10. 7. The method of any one of claims 1 to 6, wherein the RA pathway agonist is selected from the group consisting of retinoic acid (RA), TTNPB, AM580, CD1530, CD2314, Ch55, BMS753, tazarotene, isotretinoin, AC261066, Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxo retinoic acid, all-trans retinoic acid (ATRA), and combinations thereof.

11. 11. The method of claim 10, wherein the RA pathway agonist is present in the medium at a concentration in the range of 0.5 to 4 nM.

12. The method of claim 10, wherein the RA pathway agonist is RA, and the RA is present in the medium at a concentration of 2 nM.

13. 7. The method of any one of claims 1 to 6, wherein the TGF-β pathway antagonist is selected from the group consisting of A8301, SB-431542, GW788388, SB525334, TP0427736, Repsox, SD-208, and combinations thereof.

14. 14. The method of claim 13, wherein the TGF-β pathway antagonist is present in the medium at a concentration in the range of 200 to 1000 nM.

15. 14. The method of claim 13, wherein the TGF-β pathway antagonist is A8301, and the A8301 is present in the culture medium at a concentration of 500 nM.

16. 7. The method of any one of claims 1 to 6, wherein the MEK pathway antagonist is selected from the group consisting of PD0325901, binimetinib (MEK162), cobimetinib (XL518), selumetinib, trametinib (GSK1120212), CI-1040 (PD-184352), refametinib, ARRY-142886 (AZD-6244), PD98059, U0126, BI-847325, RO5126766, and combinations thereof.

17. 17. The method of claim 16, wherein the MEK pathway antagonist is present in the medium at a concentration in the range of 100 to 400 nM.

18. 17. The method of claim 16, wherein the MEK pathway antagonist is PD0325901, and the PD0325901 is present in the medium at a concentration of 250 nM.

19. The method of any one of claims 2 to 6, wherein the TAK1 pathway antagonist is selected from the group consisting of Taki ((5Z)-7-oxozeaenol), takinib, dehydroabietic acid, NG25, sarsasapogenin, and combinations thereof.

20. 20. The method of claim 19, wherein the TAK1 pathway antagonist is present in the medium at a concentration in the range of 200 to 1000 nM.

21. The method of claim 19, wherein the TAK1 pathway antagonist is Taki ((5Z)-7-oxozeaenol), and the Taki ((5Z)-7-oxozeaenol) is present in the culture medium at a concentration of 500 nM.

22. The method of any one of claims 2 to 6, wherein the bFGF mimetic is present in the culture medium at a concentration in the range of 150 to 600 nM.

23. 23. The method of claim 22, wherein the bFGF mimetic is SUN11602.

24. 24. The method of claim 23, wherein SUN11602 is present in the medium at a concentration of 300 nM.

25. 7. The method of any one of claims 2 to 6, wherein the AKT pathway antagonist is selected from the group consisting of AT7867, Sc79, demethyl-coclaurine, LM22B-10, YS-49, YS-49 monohydrate, demethylasteriquinone B1, resilisib, N-oleiolglycine, NSC45586 sodium, periplosin, CHPG sodium salt, bilobalide, 6-hydroxyflavone, musk ketone, SEW2871, 8-prenylnaringenin, razuprotafib, and combinations thereof.

26. 26. The method of claim 25, wherein the AKT pathway antagonist is present in the culture medium at a concentration in the range of 100 to 400 nM.

27. 26. The method of claim 25, wherein the AKT pathway antagonist is AT7867, and the AT7867 is present in the culture medium at a concentration of 250 nM.

28. 7. The method of any one of claims 3 to 6, wherein the Notch pathway antagonist is selected from the group consisting of GSI-XX, GSI-X, RO4929097, semagacestat, avagacestat, dibenzazepine, LY411575, LY450149, DAPT, crenigacestat, MK0752, BMS-708163, BMS-906024, CB-103, AL101, Compound E, Compound X(CX), IMR-1, IMR-1A, FLI-06, valproic acid, YO-01027, tangeretin, brucein D, and combinations thereof, and combinations thereof.

29. 29. The method of claim 28, wherein the Notch pathway antagonist is a gamma secretase inhibitor, and the gamma secretase inhibitor is present in the medium at a concentration in the range of 25 to 200 nM.

30. 29. The method of claim 28, wherein the Notch pathway antagonist is gamma secretase inhibitor XX, and the gamma secretase inhibitor XX is present in the medium at a concentration of 100 nM.

31. 7. The method of any one of claims 3 to 6, wherein the flavonoid is quercetin.

32. 32. The method of claim 31, wherein quercetin is present in the medium at a concentration in the range of 5 to 30 μM.

33. 32. The method of claim 31, wherein quercetin is present in the medium at a concentration of 15 μM.

34. 1. A method for generating human FOXA2+ HNF1b+ dorsal foregut endoderm cells (DFECs), comprising: The method comprises culturing human pluripotent stem cells in a medium containing a BMP pathway antagonist, an RA pathway agonist, a TGF-β pathway antagonist, and a MEK pathway antagonist to obtain the human DFEC.

35. 1. A method for generating human PTF1A+ PDX1+ pancreatic progenitor cells (PPCs), comprising: (a) culturing human pluripotent stem cells in a medium containing a BMP pathway antagonist, an RA pathway agonist, a TGF-β pathway antagonist, and a MEK pathway antagonist to obtain human FOXA2+ HNF1b+ DFECs; and (b) further culturing the DFECs in a medium containing a BMP pathway antagonist, an RA pathway agonist, a TGF-β pathway antagonist, a TAK1 pathway antagonist, a bFGF mimetic, and an Akt pathway antagonist to obtain human PTF1A+ PDX1+ PPCs; The method comprises:

36. The method of claim 35, wherein the BMP pathway antagonist is LDN193189, the RA pathway agonist is retinoic acid, the TGF-β pathway antagonist is A8301, the MEK pathway antagonist is PD0325901, the TAK1 pathway antagonist is Taki ((5Z)-7-oxozeaenol), the bFGF mimetic is SUN11602, and the AKT pathway antagonist is AT7867.

37. 37. The method of claim 36, wherein LDN193189 is present in the medium at a concentration in the range of 100 to 400 nM, retinoic acid is present in the medium at a concentration in the range of 0.5 to 2 μM, A8301 is present in the medium at a concentration in the range of 200 to 1000 nM, PD0325901 is present in the medium in step (a) at a concentration in the range of 100 to 400 nM, Taki ((5Z)-7-oxozeaenol) is present in the medium at a concentration in the range of 200 to 1000 nM, SUN11602 is present in the medium at a concentration in the range of 150 to 600 nM, and AT7867 is present in the medium in step (a) at a concentration in the range of 100 to 400 nM.

38. 37. The method of claim 36, wherein LDN193189 is present in the medium at a concentration of 250 nM, retinoic acid is present in the medium at a concentration of 2 μM, A8301 is present in the medium at a concentration of 500 nM, PD0325901 is present in the medium at a concentration of 250 nM, Taki ((5Z)-7-oxozeaenol) is present in the medium at a concentration of 500 nM, SUN11602 is present in the medium at a concentration of 300 nM, and AT7867 is present in the medium in step (a) at a concentration of 250 nM.

39. 1. A method for producing human pancreatic beta cells (PBCs), comprising: (a) culturing human pluripotent stem cells in a medium containing a BMP pathway antagonist, an RA pathway agonist, a TGF-β pathway antagonist, and a MEK pathway antagonist to obtain human dorsal foregut endoderm cells (DFECs); (b) further culturing the DFECs in a medium containing a BMP pathway antagonist, an RA pathway agonist, a TGF-β pathway antagonist, a TAK1 pathway antagonist, a bFGF mimetic, and an Akt pathway antagonist to obtain human pancreatic progenitor cells (PPCs); and (c) further culturing the human pancreatic beta cells (PPCs) in a medium containing a Notch pathway antagonist, a TGF-β pathway antagonist, and a flavonoid to obtain human pancreatic beta cells (PBCs). The method comprises:

40. The method of claim 39, wherein the BMP pathway antagonist is LDN193189, the RA pathway agonist is retinoic acid, the TGF-β pathway antagonist is A8301, the MEK pathway antagonist is PD0325901, the TAK1 pathway antagonist is Taki ((5Z)-7-oxozeaenol), the bFGF mimetic is SUN11602, the AKT pathway antagonist is AT7867, the Notch pathway antagonist is gamma-secretase inhibitor XX (GSI-XX), and the flavonoid is quercetin.

41. LDN193189 is present in the medium in steps (a) and (b) at a concentration in the range of 100 to 400 nM, retinoic acid is present in the medium in steps (a) and (b) at a concentration in the range of 0.5 to 2 μM, A8301 is present in the medium in steps (a) to (c) at a concentration in the range of 200 to 1000 nM, PD0325901 is present in the medium in step (a) at a concentration in the range of 100 to 400 nM, and Taki ((5Z)-7-oxozeaenol) is 41. The method of claim 40, wherein SUN11602 is present in the medium in step (b) at a concentration in the range of 150 to 600 nM, AT7867 is present in the medium in step (b) at a concentration in the range of 100 to 400 nM, GSI-XX is present in the medium in step (c) at a concentration in the range of 50 to 200 nM, and quercetin is present in the medium in step (c) at a concentration in the range of 5 to 30 μM.

42. LDN193189 was present in the medium in steps (a) and (b) at a concentration of 250 nM, retinoic acid was present in the medium in steps (a) and (b) at a concentration of 2 μM, A8301 was present in the medium in steps (a) to (c) at a concentration of 500 nM, PD0325901 was present in the medium in step (a) at a concentration of 250 nM, and Taki ((5Z)-7-oxozeaenol) 41. The method of claim 40, wherein SUN11602 is present in the culture medium in step (b) at a concentration of 300 nM, AT7867 is present in the culture medium in step (b) at a concentration within the range of 250 nM, GSI-XX is present in the culture medium in step (c) at a concentration of 100 nM, and quercetin is present in the culture medium in step (c) at a concentration of 15 μM.

43. A medium for obtaining human dorsal foregut endoderm cells (DFEC), comprising a medium containing a BMP pathway antagonist, an RA pathway agonist, a TGF-β pathway antagonist, and a MEK pathway antagonist.

44. A medium for obtaining human pancreatic progenitor cells (PPCs), comprising a BMP pathway antagonist, an RA pathway agonist, a TGF-β pathway antagonist, a TAK1 pathway antagonist, a bFGF mimetic, and an Akt pathway antagonist.

45. A medium for obtaining human pancreatic beta cells (PBCs), comprising a Notch pathway antagonist, a TGF-beta pathway antagonist, and a flavonoid.

46. 1. An isolated cell culture of human FOXA2+ HNF1b+ dorsal foregut endoderm cells (DFEC), comprising: The cell culture comprises human DFECs cultured in a medium comprising a BMP pathway antagonist, an RA pathway agonist, a TGF-β pathway antagonist, and a MEK pathway antagonist.

47. 1. An isolated cell culture of human PTF1A+ PDX1+ pancreatic progenitor cells (PPCs), comprising: The cell culture comprises human PPCs cultured in a medium comprising a BMP pathway antagonist, an RA pathway agonist, a TGF-β pathway antagonist, a TAK1 pathway antagonist, a bFGF mimetic, and an Akt pathway antagonist.

48. 1. An isolated cell culture of human INS+ PDX+ pancreatic beta cells (PBC), comprising: The cell culture comprises human PBCs cultured in a medium comprising a Notch pathway antagonist, a TGF-β pathway antagonist, and a flavonoid.

49. 10. Human FOXA2+ HNF1b+ dorsal foregut endoderm cells (DFECs) produced by the method of claim 1.

50. 3. A human PTF1A+ PDX1+ pancreatic progenitor cell (PPC) produced by the method of claim 2.

51. Human INS+ PDX+ pancreatic beta cells (PBCs) produced by the method of claim 3.

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