Development of human hepatocyte differentiation and detection method using ipscs

Abstract

The present invention discloses methods for producing induced pluripotent stem cell (iPSC)-derived hepatocytes and 3D liver organoids; methods for producing iPSC-derived endodermal progenitor intermediates including gut endodermal progenitors and hepatic endoderm progenitors; and methods for cryopreserving iPSC-derived endodermal progenitor intermediates, mature hepatocytes, and 3D liver organoids without the use of a ROCK inhibitor. By using embedding pluripotent stem cells (PSCs) in 3D scaffold in combination with a stepwise methodology that employs targeted induction cocktails specifically tuning the activation or inhibition of pathways including TGF-β, Wnt, BMP, retinoid acid, these methods streamline the differentiation of PSCs into high purity hepatocytes and 3D liver organoids which can be used for both diagnostic (e.g. drug toxicity screening) and therapeutic uses.

Description

DEVELOPMENT OF HUMAN HEPATOCYTE DIFFERENTIATION AND DETECTION METHOD USING IPSCSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 641,901, filed on May 02, 2024, the entire disclosure of which is herein incorporated by reference.FIELD

[0002] The present invention relates to the field of stem cell biology and tissue engineering. More specifically, it relates to methods for generating induced pluripotent stem cell-derived hepatocytes and 3D culture systems comprising the hepatocytes for use in drug screening applications.BACKGROUND

[0003] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

[0004] The liver is one of the largest organs and has several functions, e.g., carbohydrate and lipid metabolism; secretion of serum proteins such as albumin and immune growth factor binding protein; bile acid and urea synthesis; detoxification of drugs and metabolic waste products, and vitamin and carbohydrate storage functions. The study of liver disease has been limited by a shortage of organ donors, scarcity of tissue needed for research, and shortcomings of animal models of human liver function.SUMMARY

[0005] As a result of limitations on the amount of liver tissue for use in drug screening assays, there is a need for high functioning, induced pluripotent stem cell (iPSC)-derived hepatocytes, including three-dimensional (3D) cultures and organoids comprising iPSC- derived hepatocytes. Current methods for generating iPSC-derived hepatocytes face challenges in terms of purity and functionality of the resulting cells. Featured herein in some embodiments are improved methods for efficiently differentiating iPSCs into highly pure and functional hepatocytes, as well as 3D culture systems to mimic the in vivo liver environment. The methods, cells, and culture systems of this disclosure in some aspects, will provide a limitless supply of cells that can reproduce liver functions, and greatly benefit the field of drug discovery and toxicity testing.

[0006] In one aspect, this disclosure features systems, methods, and compositions for differentiation of iPSCs. In some aspects, the iPSCs are differentiated into desired cell types, e.g., induced hepatocytes, or intermediates thereof, e.g., as non-limiting examples, definitive endoderm cells, gut endoderm progenitor cells, and hepatic endoderm cells. In some aspects, iPSC banks are generated from donor cells through custom reprogramming, or obtained from off-the-shelf, qualified iPSC banks. In one aspect, the iPSCs then undergo differentiation into desired precursor cell types, e.g., endoderm progenitor cells. In some aspects, the progenitor cells are cryobanked. In some aspects, the progenitor cells are further differentiated to different cell types as featured herein, e.g., as a non-limiting examples, gut endoderm progenitor cells, hepatic endoderm cells, or mature hepatocytes. In some aspects, this disclosure features methods for manufacturing iPSC derived hepatocytes that mimic hepatocyte functions.

[0007] In one aspect, the present invention provides a method of producing iPSC-derived hepatocytes comprising: (a) producing DE cells from iPSCs using a first culture medium comprising a Wnt activator; (b) producing GEP cells from DE cells; (c) producing HE cells from GEP cells using a second culture medium comprising a Wnt inhibitor and a BMP inhibitor; (d) exchanging the second culture medium with a third culture medium comprising a Wnt inhibitor and a BMP activator; and (e) producing iPSC-derived hepatocytes from HE cells using a hepatocyte maturation culture medium. In some aspects, the GEP cells in step (c) are embedded in a solubilized basement membrane preparation. In some aspects, the iPSCs are embedded in a solubilized basement membrane preparation. In some aspects, the basement membrane preparation is, e.g., Matrigel™ or any other basement membrane preparation featured herein, e.g., as non-limiting examples, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels. In some aspects the culture medium used at each step comprises one or more additional reagents as set forth in this disclosure.

[0008] In one aspect, the present invention provides a method of producing iPSC-derived hepatocytes comprising: (a) producing DE cells from iPSCs using a first culture medium comprising a a Wnt activator and TGF-P activator; (b) producing GEP cells from DE cells; (c) producing HE cells from GEP cells using a second culture medium comprising a retinoid, a Wnt inhibitor, TGF-P inhibitor; (d) exchanging the second culture medium with a third culture medium comprising a retinoid, Wnt inhibitor, and TGF-P inhibitor; and (e) producing iPSC- derived hepatocytes from HE cells using a hepatocyte maturation culture medium. In some aspects the culture medium used at each step comprises one or more additional reagents as setforth in this disclosure. In some aspects, the GEP cells in step (c) are embedded in a solubilized basement membrane preparation. In some aspects, the iPSCs are embedded in a solubilized basement membrane preparation. In some aspects, the basement membrane preparation is, e.g., Matrigel™ or any other basement membrane preparation featured herein, e.g., as non-limiting examples, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels.

[0009] In one aspect, the present invention provides a method of producing iPSC-derived hepatocytes comprising: (a) producing DE cells from iPSCs using a first culture medium comprising a TGF-P activator and Wnt activator; (b) producing GEP cells from DE cells; (c) producing HE cells from GEP cells using a second culture medium comprising a Wnt inhibitor, TGF-P inhibitor, and BMP inhibitor; (d) exchanging the second culture medium with a third culture medium comprising a retinoid, Wnt inhibitor, BMP activator, and TGF-P inhibitor; and (e) producing iPSC-derived hepatocytes from HE cells using a hepatocyte maturation culture medium. In some aspects the culture medium used at each step comprises one or more additional reagents as set forth in this disclosure. In some aspects, the GEP cells in step (c) are embedded in a solubilized basement membrane preparation. In some aspects, the iPSCs are embedded in a solubilized basement membrane preparation. In some aspects, the basement membrane preparation is, e.g., Matrigel™ or any other basement membrane preparation featured herein, e.g., as non-limiting examples, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels.

[0010] In one aspect, the present invention provides a method of producing iPSC-derived hepatocytes comprising: (a) producing DE cells from iPSCs using a first culture medium comprising a TGF-P activator, Wnt activator, and ROCK activator; (b) producing GEP cells from DE cells; (c) producing HE cells from GEP cells using a second culture medium comprising a retinoid, a Wnt inhibitor, BMP inhibitor, and MEK inhibitor; (d) exchanging the second culture medium with a third culture medium comprising a retinoid, Wnt inhibitor, BMP activator, and TGF-P inhibitor; and (e) producing iPSC-derived hepatocytes from HE cells using a hepatocyte maturation culture medium. In some aspects the culture medium used at each step comprises one or more additional reagents as set forth in this disclosure. In some aspects, the GEP cells in step (c) are embedded in a solubilized basement membrane preparation. In some aspects, the iPSCs are embedded in a solubilized basement membrane preparation. In some aspects, the basement membrane preparation is, e.g., Matrigel™ or anyother basement membrane preparation featured herein, e.g., as non-limiting examples, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels.

[0011] In one aspect, the present invention provides a method of producing iPSC-derived hepatocytes comprising: (a) producing DE cells from iPSCs using a first culture medium comprising a TGF-P activator, Wnt activator, and ROCK activator; (b) producing GEP cells from DE cells; (c) producing HE cells from GEP cells using a second culture medium comprising a retinoid, a Wnt inhibitor, BMP inhibitor, and MEK inhibitor; (d) exchanging the second culture medium with a third culture medium comprising a retinoid, Wnt inhibitor, BMP activator, and TGF-P inhibitor; and (e) producing iPSC-derived hepatocytes from HE cells using a hepatocyte maturation culture medium. In some aspects the culture medium used at each step comprises one or more additional reagents as set forth in this disclosure. In some aspects, the GEP cells in step (c) are embedded in a solubilized basement membrane preparation. In some aspects, the iPSCs are embedded in a solubilized basement membrane preparation. In some aspects, the basement membrane preparation is, e.g., Matrigel™ or any other basement membrane preparation featured herein, e.g., as non-limiting examples, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels.

[0012] In one aspect, the present invention provides a method of producing GEP cells from iPSCs comprising: (a) contacting iPSCs in a first culture medium for one or more days comprising a TGF-P activator and a Wnt activator; (b) producing DE cells by exchanging the first culture medium with a second culture medium comprising a TGF-P activator and BMP activator; and (c) producing GEP cells by contacting the DE cells with a third culture medium comprising the second culture medium and a ROCK inhibitor. In some aspects the culture medium used at each step comprises one or more additional reagents as set forth in this disclosure. In some aspects, the iPSCs of step (a) and / or DE of step (c) are embedded in a solubilized basement membrane preparation. In some aspects, the basement membrane preparation is, e.g., Matrigel™ or any other basement membrane preparation featured herein, e.g., as non-limiting examples, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels.

[0013] In one aspect, the present invention provides a method of producing GEP cells from iPSCs comprising: (a) contacting iPSCs in a first culture medium for one or more dayscomprising a TGF-P activator, Wnt activator, and ROCK inhibitor; (b) producing DE cells by exchanging the first culture medium with a second culture medium comprising a TGF-P activator, BMP activator, FGF activator, and VEGF activator; and (c) producing GEP cells by contacting the DE cells with a third culture medium comprising the second culture medium and a ROCK inhibitor. In some aspects the culture medium used at each step comprises one or more additional reagents as set forth in this disclosure. In some aspects, the iPSCs of step (a) and / or DE of step (c) are embedded in a solubilized basement membrane preparation. In some aspects, the basement membrane preparation is, e.g., Matrigel™ or any other basement membrane preparation featured herein, e.g., as non-limiting examples, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels.

[0014] In one aspect, the present invention provides a method of producing HE cells comprising: (a) contacting DE cells with a first culture medium comprising a BMP activator and TGF-P activator to produce GEP cells; (b) contacting GEP cells with a second culture medium comprising a BMP inhibitor and MEK inhibitor; and (c) exchanging the second culture medium with a third culture medium comprising a BMP activator and TGF-P inhibitor. In some aspects, the first culture medium further comprises an FGF activator, VEGF activator, and / or a ROCK inhibitor. In some aspects, the second culture medium and third culture medium further comprise a retinoid and Wnt inhibitor. In some aspects, the DE cells are generated from iPSCs. In some aspects, the DE of step (a) and / or GEP of step (b) are embedded in a solubilized basement membrane preparation. In some aspects, the basement membrane preparation is, e.g., Matrigel™ or any other basement membrane preparation featured herein, e.g., as non-limiting examples, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels.

[0015] In one aspect, the present invention provides a method of producing HE cells comprising: (a) contacting DE cells with a first culture medium comprising a BMP activator and TGF-P activator to produce GEP cells; (b) contacting GEP cells with a second culture medium comprising a Wnt inhibitor, BMP inhibitor, and MEK inhibitor; and (c) exchanging the second culture medium with a third culture medium comprising a Wnt inhibitor, BMP activator, and TGF-P inhibitor. In some aspects, the first culture medium further comprises an FGF activator, VEGF activator, and / or a ROCK inhibitor. In some aspects, the second culture medium and third culture medium further comprise a retinoid. In some aspects, the DE cells are generated from iPSCs. In some aspects, the DE of step (a) and / or GEP of step (b) areembedded in a solubilized basement membrane preparation. In some aspects, the basement membrane preparation is, e.g., Matrigel™ or any other basement membrane preparation featured herein, e.g., as non-limiting examples, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels.

[0016] In one aspect, the present invention provides a method of producing iPSC-derived hepatocytes, comprising: (a) differentiating iPSCs into definitive endoderm (DE) cells; (b) differentiating the DE cells into GEP cells; (c) differentiating the GEP cells into HE cells; and (d) differentiating the HE cells into mature hepatocytes, thereby producing iPSC-derived hepatocytes wherein at least 80% of the hepatocytes express at least one mature hepatocyte marker comprising: HNF4a, UGT2B7, transthyretin (TTR), al-antitrypsin (AAT), or albumin.

[0017] In one embodiment, the present invention provides a method of inducing hepatocyte differentiation from iPSCs comprising: (a) inducing definitive endoderm (DE) cells by: (i) embedding iPSCs in a solubilized basement membrane preparation; (ii) contacting iPSCs in a first culture medium comprising a transforming growth factor beta (TGF-P) activator, a Wnt activator, and a Rho-associated protein kinase (ROCK) inhibitor; (iii) exchanging the first culture medium with a second culture medium comprising a TGF-P activator and a Wnt activator; (iv) exchanging the second culture medium with a third culture medium comprising a TGF-P activator, bone morphogenetic protein (BMP) activator, fibroblast growth factor (FGF) activator, and vascular endothelial growth factor (VEGF) activator; (b) inducing gut endoderm progenitor (GEP) cells by: (i) dissociating then embedding DE cells in a solubilized basement membrane preparation; and (ii) contacting the DE cells with a GEP culture medium comprising a TGF-P activator, BMP activator, FGF activator, VEGF activator, and ROCK inhibitor; (c) inducing hepatic endoderm (HE) cells by: (i) dissociating then embedding GEP cells in a solubilized basement membrane preparation; and (ii) contacting the GEP cells with a first HE culture medium comprising a retinoid, Wnt inhibitor, BMP inhibitor, and MEK inhibitor; then (iii) exchanging the first HE culture medium with a second HE culture medium comprising a retinoid, Wnt inhibitor, BMP activator, and TGF-P inhibitor; and (d) inducing iPSC-derived hepatocytes by contacting the HE cells with a hepatocyte maturation culture medium (HCM) comprising hepatocyte growth factor (HGF), an interleukin 6 (IL-6) family cytokine, and a glucocorticoid inducing hepatocyte differentiation from PSCs. In some aspects, the solubilized basement membrane preparation is Matrigel™ or any other basement membrane preparation featured herein, e.g., as non-limiting examples, Geltrex™, Celltrex™,reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels.

[0018] In one aspect, the present invention provides a method of inducing hepatocyte differentiation from pluripotent stem cells (PSCs) comprising: (a) inducing definitive endoderm (DE) cells by: (i) embedding PSCs in a solubilized basement membrane preparation; (ii) contacting PSCs in a first culture medium comprising a transforming growth factor beta (TGF-P) activator, a Wnt activator, and a Rho-associated protein kinase (ROCK) inhibitor; (iii) exchanging the first culture medium with a second culture medium comprising a TGF-P activator and a Wnt activator; (iv) exchanging the second culture medium with a third culture medium comprising a TGF-P activator, bone morphogenetic protein (BMP) activator, fibroblast growth factor (FGF) activator, and vascular endothelial growth factor (VEGF) activator; (b) inducing gut endoderm progenitor (GEP) cells by: (i) dissociating then embedding DE cells in a solubilized basement membrane preparation; and (ii) contacting the DE cells with a GEP culture medium comprising a TGF-P activator, BMP activator, FGF activator, VEGF activator, and ROCK inhibitor; (c) inducing hepatic endoderm (HE) cells by: (i) dissociating then embedding GEP cells in a solubilized basement membrane preparation; and (ii) contacting the GEP cells with a first HE culture medium comprising a retinoid, Wnt inhibitor, BMP inhibitor, and MEK inhibitor; then (iii) exchanging the first HE culture medium with a second HE culture medium comprising a retinoid, Wnt inhibitor, BMP activator, and TGF-P inhibitor; and (d) inducing PSC-derived hepatocytes by contacting the HE cells with a hepatocyte maturation culture medium (HCM) comprising hepatocyte growth factor (HGF), an interleukin 6 (IL-6) family cytokine, and a glucocorticoid inducing hepatocyte differentiation from PSCs. In some aspects, the solubilized basement membrane preparation is Matrigel™ or any other basement membrane preparation featured herein, e.g., as non-limiting examples, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels.

[0019] In some aspects, the TGF-P activator is Activin A. In some aspects, the Wnt activator is Wnt3a. In some aspects, the ROCK inhibitor is Y-27632. In some aspects, the BMP activator is BMP4. In some aspects, the FGF activator is FGF2; the VEGF is VEGF-A. In some aspects, the retinoid is retinoic acid. In some aspects, the Wnt inhibitor is inhibitor of Wnt production 2 (IWP2). In some aspects, the BMP inhibitor is LDN193189. In some aspects, the MEK inhibitor is PD0325901. In some aspects, TGF-P inhibitor is A83-01. In some aspects, the IL-6 family cytokine is oncostatin M (OSM). In some aspects, the glucocorticoid isdexamethasone (DEX). In some aspects, the HCM does not comprise an epidermal growth factor (EGF). In some aspects, a culture medium comprising any of the activators, inhibitors, retinoids, cytokines, and glucocorticoids described herein includes one or more of: Activin A, Wnt3a, Y-27632, BMP4, FGF2, VEGF-A, retinoic acid, IWP2, EDN193189, PD0325901, A83-01, OSM, DEX, or any activator, inhibitor, retinoid, IE-6 family cytokine, and glucocorticoid featured and disclosed herein.

[0020] In some aspects, the TGF-P activator is Activin A; the Wnt activator is Wnt3a; the ROCK inhibitor is Y-27632; the BMP activator is BMP4; the FGF activator is FGF2; the VEGF is VEGF-A; the retinoid is retinoic acid; the Wnt inhibitor is inhibitor of Wnt production 2 (IWP2); the BMP inhibitor is LDN193189; the MEK inhibitor is PD0325901; TGF-P inhibitor is A83-01; the IL-6 family cytokine is oncostatin M (OSM); or the glucocorticoid is dexamethasone (DEX). In some aspects, the HCM does not comprise an epidermal growth factor (EGF).

[0021] In one aspect, the present invention provides a method of cryopreserving GEP cells comprising: contacting GEP cells with a GEP cryopreservation medium comprising DMSO, emricasan, polyamine, and trans-ISRIB (CEPT). In some aspects, the GEP cryopreservation medium does not comprise a ROCK inhibitor. In some aspects, the GEP cryopreservation medium does not comprise chroman 1. In another aspect, the present invention provides a method of recovering cryopreserved GEP cells comprising: (a) thawing the cryopreserved GEP cells; (b) recovering the thawed cryopreserved GEP cells in a GEP culture medium comprising DMSO, emricasan, polyamine, and CEPT for at least one day; and (c) culturing the recovered GEP cells in a GEP culture medium comprising SFD, a TGF-P activator, BMP activator, FGF activator, VEGF activator, and ROCK inhibitor for about 2 to 5 days.

[0022] In another aspect, the present invention provides a method of cryopreserving iPSC- derived hepatocytes or 3D liver organoids comprising: contacting iPSC-derived hepatocytes or 3D liver organoids with a hepatocyte cryopreservation medium comprising DMSO, emricasan, polyamine, and CEPT. In some aspects, the hepatocyte cryopreservation medium does not comprise a ROCK inhibitor. In some aspects, the hepatocyte cryopreservation medium does not comprise chroman 1. In another aspect, the present invention provides a method of recovering cryopreserved iPSC-derived hepatocytes or 3D liver organoids comprising: (a) thawing the cryopreserved iPSC-derived hepatocytes or 3D liver organoids; (b) recovering the thawed iPSC-derived hepatocytes or 3D liver organoids in a hepatocyte cryopreservation medium comprising DMSO, emricasan, polyamine, and CEPT for at least one day; and (c)culturing the recovered iPSC-derived hepatocytes or 3D liver organoids in a hepatocyte maturation culture medium comprising HCM without EGF, HGF, an IL-6 family cytokine, and a glucocorticoid for at least one day.

[0023] In one aspect, the present invention provides for a kit comprising: (a) a gut endodermal induction cocktail comprising: a TGF-P activator, BMP activator, and ROCK inhibitor; (b) two hepatic endoderm induction cocktails comprising: (i) BMP inhibitor, and MEK inhibitor; (ii) BMP activator, and TGF-P inhibitor; (c) a hepatic maturation cocktail comprising: hepatocyte culture medium without EGF, HGF, an IL-6 family cytokine, and a glucocorticoid; and (d) instructions for inducing definitive endodermal cells differentiation into mature hepatocytes; and optionally a solubilized basement membrane preparation. In some aspects, the two hepatic endoderm induction cocktails further comprise a retinoid and Wnt inhibitor. In some aspects, the gut endodermal induction cocktail further comprises a FGF activator and VEGF activator.

[0024] In one aspect, the present invention provides for a kit comprising: (a) a gut endodermal induction cocktail comprising: a TGF-P activator, BMP activator, FGF activator, VEGF activator, and ROCK inhibitor; (b) two hepatic endoderm induction cocktails comprising: (i) BMP inhibitor, MEK inhibitor; (ii) BMP activator, and TGF-P inhibitor; (c) a hepatic maturation cocktail comprising: hepatocyte culture medium without EGF, HGF, an IL- 6 family cytokine, and a glucocorticoid; and (d) instructions for inducing definitive endodermal cells differentiation into mature hepatocytes; and optionally a solubilized basement membrane preparation. In some aspects, the two hepatic endoderm induction cocktails further comprise a retinoid and Wnt inhibitor.

[0025] In one aspect, the present invention provides for a kit comprising: (a) a gut endodermal induction cocktail comprising: a TGF-P activator, BMP activator, FGF activator, VEGF activator, and ROCK inhibitor; (b) two hepatic endoderm induction cocktails comprising: (i) a retinoid, Wnt inhibitor, BMP inhibitor, and MEK inhibitor; (ii) a retinoid, Wnt inhibitor, BMP activator, and TGF-P inhibitor; (c) a hepatic maturation cocktail comprising: hepatocyte culture medium without EGF, HGF, an IL-6 family cytokine, and a glucocorticoid; and (d) instructions for inducing definitive endodermal cells differentiation into mature hepatocytes; and optionally a solubilized basement membrane preparation.

[0026] As featured and disclosed herein, activators and inhibitors of the transforming growth factor beta (TGF-P) superfamily include, as non-limiting examples, signaling pathway ligands, small molecule activators, small molecule inhibitors, peptide activators, peptideinhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the TGF-P signaling pathway, resulting in a corresponding activation or inhibition in cellular TGF-P signaling. In some aspects of this disclosure, activation of the TGF-P signaling pathway is achieved through repression of a TGF-P signaling pathway inhibitor, including, as non-limiting examples, the use of an inhibitory nucleic acid targeting an inhibitor of the TGF-P signaling pathway or an antibody or small molecule directed to a TGF-P signaling pathway inhibitor.

[0027] As featured and disclosed herein, activators of the TGF-P signaling pathway include, as non-limiting examples, Activin A, TGF-P family ligands (e.g., TGF-P proteins and other activators of TGF-P receptors) and portions thereof, TGF-pi, TGF-P2, TGF-P3, IDE1 / 2 (IDE1 [l-[2-[(2-Carboxyphenyl)methylene]hydrazide]heptanoic acid], IDE2 [Heptanedioic acid-l-(2-cyclopentylidenehydrazide)]), and Nodal. In some embodiments, the TGF-P signaling pathway activator comprises Activin A.

[0028] In some embodiments, the concentration of the TGF-P signaling pathway activator or inhibitor ranges between about 50 ng / mL to 150 ng / mL. In some embodiments, the TGF-P signaling pathway activator is present at a concentration of between about 50 ng / mL to 150 ng / mL. In some embodiments, the TGF-P signaling pathway activator is present at a concentration of 25 to 30 ng / mL, from 30 to 40 ng / mL, from 40 to 50 ng / mL, from 50 to 60 ng / mL, from 60 to 70 ng / mL, from 70 to 80 ng / mL, from 80 to 90 ng / mL, from 90 to 100 ng / mL, from 100 to 110 ng / mL, from 110 to 120 ng / mL, from 120 to 130 ng / mL, from 130 to 140 ng / mL, from 140 to 150 ng / mL, from 150 to 160 ng / mL, from 160 to 170 ng / mL, from 170 to 180 ng / mL, from 180 to 190 ng / mL, from 190 to 200 ng / mL, or at any concentration range between any two concentrations referred to above or herein. In some embodiments, the TGF-P signaling pathway activator is present at a concentration of 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 ng / mL, or at any concentration range between any two concentrations referred to above or herein. In some embodiments, Activin A is at a concentration of about 100 ng / mL.

[0029] As featured and disclosed herein, inhibitors of the TGF-P signaling pathway include, as non-limiting examples, A83-01, SB431542, SB505124, LY2157299, and LY550410. In some embodiments, the TGF-P signaling pathway activator comprises A83-01.

[0030] In some embodiments, the concentration of the TGF-P signaling pathway inhibitor is in a range between about 0.5 pM to 2 pM. In some embodiments, the TGF-P signaling pathway inhibitor is added to a culture medium and is present at a concentration of 0.1 to 0.2pM, 0.2 to 0.3 pM, 0.3 to 0.4 pM, 0.4 to 0.5 pM, 0.5 to 0.6 pM, 0.6 to 0.7 pM, 0.7 to 0.8 pM, 0.8 to 0.9 pM, 0.9 to 1 pM, 1.0 to 2.0 pM, 2.0 to 3.0 pM, 3.0 to 4.0 pM, 4.0 to 5.0 pM, or at any range between any two concentrations referred to above or herein. In some embodiments, the TGF-P signaling pathway inhibitor is added to a culture medium and is present at a concentration is present at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5 pM, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the concentration of TGF-P signaling pathway inhibitor is about 1 pM. In some embodiments the concentration of A83-01 is about 1 pM.

[0031] As featured and disclosed herein activators of the WNT signaling pathway include, as non-limiting examples, WNT family ligands including Wnt3a; CHIR99021 (6-[[2-[[4-(2,4- Dichlorophenyl)-5 -(5-methyl- 1 H-imidazol-2-yl)-2pyrimidinyl] amino] ethyl] amino] -3- pyridinecarbonitrile); RSPO co-agonists; lithium chloride; TDZD8 (4-Benzyl-2-methyl- 1,2,4- thiadiazolidine-3, 5-dione); BIO-Acetoxime ((2’Z,3’E)-6-Bromoindirubin-3 '-acetoxime); A1070722 (l-(7-Methoxyquinolin-4-yl)-3-[6 (trifluoromethyl)pyridin-2-yl]urea); HLY78 (4- Ethyl-5,6-Dihydro-5-methyl-[l,3]dioxolo[4,5-j]phenanthridine); CID 11210285 HC1 (2- Amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine hydrochloride); WAY-316606; (hetero) arylpyrimidines; IQ1; QS11; SB-216763; and / or DCA. In some embodiments, the culture media comprising a WNT activator includes or excludes a GSK-3P inhibiting WNT activator such as, e.g., CHIR99021. In some embodiments, the culture media comprising a WNT activator expressly excludes a GSK-3P inhibiting WNT activator such as, e.g., CHIR99021. In some embodiments, activation of the WNT signaling pathway is achieved through repression of a WNT signaling pathway inhibitor, including, as non-limiting examples, the use of an inhibitory nucleic acid targeting an inhibitor of the WNT signaling pathway or an antibody or small molecule directed to a WNT signaling pathway inhibitor.

[0032] In some aspects, the WNT signaling pathway activator is added to a culture medium at a concentration that ranges between about 25 ng / mL to 100 ng / mL. In some embodiments, the WNT signaling pathway activator is added to a culture medium at a concentration that ranges from 10 to 20 ng / mL, 20 to 30 ng / mL, from 30 to 40 ng / mL, from 40 to 50 ng / mL, from 50 to 60 ng / mL, from 60 to 70 ng / mL, from 70 to 80 ng / mL, from 80 to 90 ng / mL, from 90 to 100 ng / mL, from 100 to 110 ng / mL, from 110 to 120 ng / mL, from 120 to 130 ng / mL, from 130 to 140 ng / mL, from 140 to 150 ng / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the WNT signaling pathwayactivator is added to a culture medium at a concentration that ranges from 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 ng / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the WNT signaling pathway activator comprises Wnt3a. In some embodiments, the WNT signaling pathway activator is Wnt3a is present at a concentration of between about 25 ng / mL to 100 ng / mL. In some embodiments, Wnt3a is at a concentration of about 50 ng / mL.

[0033] As featured and disclosed herein, inhibitors of the WNT signaling pathway include, as non-limiting examples, IWP2 (N-(6-Methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4- oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide), C-59 (4-(2-Methyl-4-pyridinyl)- N-[4-(3-pyridinyl)phenyl]benzeneacetamide), (3,5,7,8-Tetrahydro-2-[4-(trifluoromethyl) phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one), IWR1 (4-(l,3,3a,4,7,7a-Hexahydro-l,3- dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-Benzamide), DKK1, Antl.4Br, Antl.4CI, Niclosamide, apicularen, bafilomycin, XAV939 NSC668036 (N-[(l,l- Dimethylethoxy)carbonyl]-L-alanyl-(2S)-2-hydroxy-3-methylbutanoyl-L-Alanine-(lS)-l- carboxy-2-methylpropyl ester hydrate), 2,4-diamino-quinazoline, Quercetin, ICG-001 ((6S,9aS)-Hexahydro-6-[(4-hydroxyphenyl)methyl]-8-(l-naphthalenylmethyl)-4,7-dioxo-N- (phenylmethyl)-2H-pyrazino[l,2-a]pyrimidine-l(6H)-carboxamide), PKF115-584, BML-284 (2-Amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3-methoxyphenyl)pyrimidine), FH-535, iCRT-14, JW-55, JW-67, and antibodies to WNTs and WNT receptors, WNT inhibitory nucleic acids. In some embodiments, inhibition of the WNT signaling pathway is achieved through repression of a WNT signaling pathway activator, including, as non-limiting examples, the use of an inhibitory nucleic acid targeting an activator of the WNT signaling pathway or an antibody or small molecule directed to a WNT signaling pathway activator. In some embodiments, the WNT signaling pathway inhibitor is IWP2.

[0034] In some embodiments, the concentration of the WNT signaling pathway inhibitor is in a range between about 1 pM to 8 pM. In some embodiments, the WNT signaling pathway inhibitor is added to a culture medium at a concentration that ranges from 0.1 to 0.2 pM, 0.2 to 0.3 pM, 0.3 to 0.4 pM, 0.4 to 0.5 pM, 0.5 to 0.6 pM, 0.6 to 0.7 pM, 0.7 to 0.8 pM, 0.8 to 0.9 pM, 0.9 to 1 pM, 1.0 to 2.0 pM, 2.0 to 3.0 pM, 3.0 to 4.0 pM, 4.0 to 5.0 pM, 5.0 to 6.0 pM, 6.0 to 7.0 pM, 7.0 to 8.0 pM, 8.0 to 9.0 pM, 9.0 to 10 pM, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the WNT signaling pathway inhibitor is added to a culture medium at a concentration that ranges from 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 pM, or at any concentration range anytwo concentrations referred to above or herein. In some embodiments, the concentration of WNT signaling pathway inhibitor is about 4 M. In some embodiments the concentration of IWP2 is about 4 pM.

[0035] As featured and disclosed herein, ROCK inhibitors include, as non-limiting examples, Y-27632, chroman 1, ripasudil, netarsudil, Y39983, Wf-536, SLx-2119, Azabenzimidazole-aminofurazans, DE- 104 Olefins, Isoquinolines, Indazoles, pyridinealkene derivatives, H-1152P, XD-4000, HMN-1152, ROKa 4-(l-aminoalkyl)-N-(4- pyridyl)cyclohexane-carboxamides, rhostatin, BA-210, BA- 207, BA-285, BA- 1037, BA-215, Ki-23095, VAS-012, quinazoline, GSK-269962, and salts thereof, including Y-27632 dihydrochloride, H-1152 dihydrochloride, fasudil hydrochloride, SR-3677 dihydrochloride, glycyl-H-1152 dihydrochloride, SB-772077B dihydrochloride, TC-S-7001, RKI-1447 dihydrochloride, and OXA-06 dihydrochloride.

[0036] In some embodiments, the concentration of ROCK inhibitor is in a range between about 5 pM to 15 pM. In some embodiments, the ROCK inhibitor is Y-27632 at concentration of about 10 pM. In some embodiments, the ROCK inhibitor is at a concentration that ranges from 1.0 to 2.0 pM, from 2.0 to 3.0 pM, from 3.0 to 4.0 pM, from 4.0 to 5.0 pM, from 5.0 to 6.0 pM, from 6.0 to 7.0 pM, from 7.0 to 8.0 pM, from 8.0 to 9.0 pM, from 9.0 to 10 pM, from 10 to 11 pM, from 11 to 12 pM, from 12 to 13 pM, from 13 to 14 pM, from 14 to 15 pM, from 15 to 16 pM, from 16 to 17 pM, from 17 to 18 pM, from 18 to 19 pM, from 19 to 20 pM, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the ROCK inhibitor is added to a culture medium at a concentration that ranges from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 pM, or at any concentration range any two concentrations referred to above or herein. In some embodiments, Y-27632 is at a concentration of about 10 pM.

[0037] As featured and disclosed herein, activators of the BMP pathway include, as nonlimiting examples, BMP family ligands, e.g., BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8, BMP9, BMP8b, BMP10, BMP11, and BMP 15; Alantolactone; FK506; isoliquiritigenin; and 4 ’-hydroxy chaicone. In some embodiments, activation of the BMP pathway is achieved through repression of a BMP pathway inhibitor, including, as a nonlimiting example, the use of an inhibitory nucleic acid targeting an inhibitor of the BMP pathway or an antibody or small molecule directed to a BMP pathway inhibitor. In some embodiments, the BMP pathway activator comprises BMP4. In some embodiments, the BMP pathway activator is present at a concentration of between about 0.1 ng / mL to 1 ng / mL. Insome embodiments, the BMP pathway activator is at a concentration that ranges from 0.1 to 0.2 ng / mL, 0.2 to 0.3 ng / mL, 0.3 to 0.4 ng / mL, 0.4 to 0.5 ng / mL, 0.5 to 0.6 ng / mL, 0.6 to 0.7 ng / mL, 0.7 to 0.8 ng / mL, 0.8 to 0.9 ng / mL, 0.9 to 1 ng / mL, 1 to 2 ng / mL, 2 to 3 ng / mL, 3 to 4 ng / mL, 4 to 5 ng / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the BMP pathway activator is at a concentration that ranges from 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5 ng / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, BMP4 is at a concentration of about 0.5 ng / mL. In some embodiments, BMP4 is added to a culture medium at a concentration of about 10 pg / mL.

[0038] In some embodiments, the BMP pathway activator is present at a concentration of between about 5 pg / mL to 15 pg / mL. In some embodiments, the BMP pathway activator is at a concentration that ranges from 1.0 to 2.0 pg / mL, from 2.0 to 3.0 pg / mL, from 3.0 to 4.0 pg / mL, from 4.0 to 5.0 pg / mL, from 5.0 to 6.0 pg / mL, from 6.0 to 7.0 pg / mL, from 7.0 to 8.0 pg / mL, from 8.0 to 9.0 pg / mL, from 9.0 to 10 pg / mL, from 10 to 11 pg / mL, from 11 to 12 pg / mL, from 12 to 13 pg / mL, from 13 to 14 pg / mL, from 14 to 15 pg / mL, or at any concentration range any two concentrations referred to above or herein. . In some embodiments, the BMP pathway activator is added to a culture medium at a concentration that ranges from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 pg / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, BMP4 is at a concentration of about 10 pg / mL. In some embodiments, BMP4 is added to an endodermal progenitor induction medium at a concentration of about 10 pg / mL.

[0039] As featured and disclosed herein, inhibitors of the BMP pathway include, as nonlimiting examples, LDN193189, noggin, chordin, follistatin, Tsg, Gremlin, dosmorphin, K02299, DMH1, and DMH2. In some embodiments, the BMP pathway inhibitor comprises LDN193189. In some embodiments, the BMP pathway inhibitor is present at a concentration of between about 125 nM to 500 nM. In some embodiments, the BMP pathway inhibitor is at a concentration that ranges from 75 to 100 nM, 100 to 125 nM, 125 to 150 nM, 150 to 200 nM, 200 to 250 nM, 250 to 300 nM, 300 to 350 nM, 350 to 400 nM, 400 to 450 nM, 450 to 500 nM, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the BMP pathway inhibitor is at a concentration that ranges from 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500 nM, or at any concentration range any two concentrations referred to above or herein. In some embodiments, LDN193189 is at a concentration of about 250 nM.

[0040] As featured and disclosed herein, activators and inhibitors of the fibroblast growth factor (FGF) signaling pathway include, as non-limiting examples, signaling pathway ligands, small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the FGF signaling pathway, resulting in a corresponding activation or inhibition in cellular FGF signaling. In some aspects of this disclosure, activation of the FGF signaling pathway is achieved through repression of a FGF signaling pathway inhibitor, including, as non-limiting examples, the use of an inhibitory nucleic acid targeting an inhibitor of the FGF signaling pathway or an antibody or small molecule directed to a FGF signaling pathway inhibitor.

[0041] As featured and disclosed herein, activators of the fibroblast growth factor (FGF) signaling pathway include, as non-limiting examples, FGF family ligands including, as nonlimiting examples, FGF1, FGF2, FGF-3, FGF-4, FGF-5, FGF-6, KGF / FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-15, FGF-16, FGF-17, FGF-19, FGF-20, FGF-21, FGF-22, and FGF-23; SUN 11602 (4-[[4-[[2-[(4-Amino-2,3,5,6-tetramethylphenyl) amino] acetyl] methylamino] - 1 -piperidinyl] methypenzamide) ; t-Butylhydroquinone; U-46619 ; C2 Ceramide; Lactosyl Ceramide; Angiotensin II; and Baicalin. In some embodiments, the FGF signaling pathway activator is FGF2.

[0042] In some embodiments, the concentration of FGF signaling pathway activator or inhibitor ranges from between about 1 ng / mL to 10 ng / mL. In some embodiments, the FGF signaling pathway activator is FGF2 is present at a concentration of between about 1 ng / mL to 10 ng / mL. In some embodiments, the FGF signaling pathway activator is at a concentration that ranges from 0.5 to 1 ng / mL, 1 to 2 ng / mL, 2 to 3 ng / mL, from 3 to 4 ng / mL, from 4 to 5 ng / mL, from 5 to 6 ng / mL, from 6 to 7 ng / mL, from 7 to 8 ng / mL, from 8 to 9 ng / mL, from 9 to 10 ng / mL, from 10 to 11 ng / mL, from 11 to 12 ng / mL, from 12 to 13 ng / mL, from 13 to 14 ng / mL, from 14 to 15 ng / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, FGF2 is at a concentration of about 5 ng / mL.

[0043] As featured and disclosed herein, activators and inhibitors of the vascular endothelial growth factor (VEGF) signaling pathway include, as non-limiting examples, signaling pathway ligands, small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the VEGF signaling pathway, resulting in a corresponding activation or inhibition in cellular VEGF signaling. In some aspects of thisdisclosure, activation of the VEGF signaling pathway is achieved through repression of a VEGF signaling pathway inhibitor, including, as non-limiting examples, the use of an inhibitory nucleic acid targeting an inhibitor of the VEGF signaling pathway or an antibody or small molecule directed to a VEGF signaling pathway inhibitor. In some embodiments, a vascular endothelial growth factor includes, as non-limiting examples, a VEGF family ligand which activates the VEGF pathway, e.g., VEGFA, VEGFB, VEGFC, VEGFD, VEGFE, VEGFF, and placenta growth factor (PIGF). In some embodiments, the VEGF is VEGFA.

[0044] In some embodiments, the concentration of VEGF signaling pathway activator or inhibitor ranges from between about 5 ng / mL to 15 ng / mL. In some embodiments, the VEGF signaling pathway activator is VEGF is present at a concentration of between about 5 ng / mL to 15 ng / mL. In some embodiments, the VEGF signaling pathway activator is at a concentration that ranges from 2.5 to 3 ng / mL, from 3 to 4 ng / mL, from 4 to 5 ng / mL, from 5 to 6 ng / mL, from 6 to 7 ng / mL, from 7 to 8 ng / mL, from 8 to 9 ng / mL, from 9 to 10 ng / mL, from 10 to 11 ng / mL, from 11 to 12 ng / mL, from 12 to 13 ng / mL, from 13 to 14 ng / mL, from 14 to 15 ng / mL, from 15 to 16 ng / mL, from 16 to 17 ng / mL, from 17 to 18 ng / mL, from 18 to 19 ng / mL, from 19 to 20 ng / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the VEGF pathway activator is present at a concentration of 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ng / mL, or at any concentration range between any two concentrations referred to above or herein. In some embodiments, VEGF is at a concentration of about 10 ng / mL.

[0045] As featured and disclosed herein, retinoids include, as non-limiting examples, retinoic acid (RA), retinol, retinal, isotretinoin, and alitretinoin. In some embodiments, the retinoid is RA.

[0046] In some aspects, a retinoid is added to a culture medium at a concentration that ranges between about 0.5 to 5 pM. In some embodiments, the retinoid is added to a culture medium at a concentration that ranges from 0.1 to 0.2 pM, 0.2 to 0.3 pM, 0.3 to 0.4 pM, 0.4 to 0.5 pM, 0.5 to 0.6 pM, 0.6 to 0.7 pM, 0.7 to 0.8 pM, 0.8 to 0.9 pM, 0.9 to 1 pM, 1.0 to 2.0 pM, 2.0 to 3.0 pM, 3.0 to 4.0 pM, 4.0 to 5.0 pM, 5.0 to 6.0 pM, 6.0 to 7.0 pM, 7.0 to 8.0 pM, 8.0 to 9.0 pM, 9.0 to 10 pM, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the retinoid is added to a culture at a concentration that ranges from 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 pM, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the retinoid comprises RA. In some embodiments, the glucocorticoid is RA is present at aconcentration of between about 0.5 to 5 |aM. In some embodiments, RA is at a concentration of about 2 pM.

[0047] As featured and disclosed herein, activators and inhibitors of the mitogen-activated protein kinase (MAPK / ERK or MEK) signaling pathway include, as non-limiting examples, signaling pathway ligands, small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the MEK signaling pathway, resulting in a corresponding activation or inhibition in cellular MEK signaling. In some aspects of this disclosure, activation of the MEK signaling pathway is achieved through repression of a MEK signaling pathway inhibitor, including, as non-limiting examples, the use of an inhibitory nucleic acid targeting an inhibitor of the MEK signaling pathway or an antibody or small molecule directed to a MEK signaling pathway inhibitor. In some embodiments, an inhibitor of the MEK signaling pathway includes, as non-limiting examples, PD0325901, trametinib, pimasertib, selumetinib, rafametinib, TAK-733, MEK-162, RO-4987655, and XL-518. In some embodiments, the MEK signaling pathway activator is PD0325901.

[0048] In some embodiments, the concentration of MEK signaling pathway activator or inhibitor ranges from between about 250 nM to 1 pM. In some embodiments, the MEK signaling pathway inhibitor is PD0325901 is present at a concentration of between about 250 nM to 1 p M. In some embodiments, the MEK signaling pathway inhibitor is at a concentration that ranges from 250 to 300 nM, 300 to 350 nM, 350 to 400 nM, 400 to 450 nM, 450 to 500 nM, 500 to 550 nM, 550 to 600 nM, 600 to 650 nM, 650 to 700 nM, 700 to 750 nM, 750 to 800 nM, 800 to 850 nM, 850 to 900 nM, 900 to 950 nM, 950 nM to 1 pM, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the MEK signaling pathway inhibitor is at a concentration that ranges from 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 nM, 1 pM, or at any concentration range any two concentrations referred to above or herein. In some embodiments, PD0325901 is at a concentration of about 500 nM.

[0049] As featured and disclosed herein, interleukin-6 (IL-6) family cytokines include, as non-limiting examples, oncostatin M (OSM), IL-6, IL- 11, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), cardiotrophin 1 (CT-1), cardiotrophin-like cytokine (CLC), and IL- 27. In some embodiments, the IL-6 family cytokine is OSM.

[0050] In some aspects, an IL-6 family cytokine is added to a culture medium at a concentration that ranges between about 10 ng / mL to 40 ng / mL. In some embodiments, the IL-6 family cytokine is added to a culture medium at a concentration that ranges from 1 to 10 ng / mL, 10 to 20 ng / mL, 20 to 30 ng / mL, from 30 to 40 ng / mL, from 40 to 50 ng / mL, from 50 to 60 ng / mL, from 60 to 70 ng / mL, from 70 to 80 ng / mL, from 80 to 90 ng / mL, from 90 to 100 ng / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the IL-6 family cytokine is added to a culture medium at a concentration that ranges from 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ng / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the IL-6 family cytokine comprises OSM. In some embodiments, the IL-6 family cytokine is OSM is present at a concentration of between about 10 ng / mL to 40 ng / mL. In some embodiments, OSM is at a concentration of about 20 ng / mL.

[0051] As featured and disclosed herein, activators of hepatocyte growth factor (HGF) include, as non-limiting examples, HGF, matriptase, and hepsin. In some embodiments, HGF or an activator of HGF is added to a culture medium at a concentration that ranges between about 5 to 60 ng / mL. In some embodiments, HGF or an activator of HGF is added to a culture medium at a concentration that ranges from 0.1 to 0.5 ng / mL, 0.5 to 1 ng / mL, 1 to 2 ng / mL, 2 to 3 ng / mL, 3 to 4 ng / mL, 4 to 5 ng / mL, 5 to 6 ng / mL, 6 to 7 ng / mL, 7 to 8 ng / mL, 8 to 9 mg / mL, 9 to 10 ng / mL, 10 to 11 ng / mL, 11 to 12 ng / mL, 12 to 13 ng / mL, 13 to 14 ng / mL, 14 to 15 ng / mL, 15 to 16 ng / mL, 16 to 17 ng / mL, 17 to 18 ng / mL, 18 to 19 ng / mL, 19 to 20 ng / mL, 20 to 30 ng / mL, 30 to 40 ng / mL, 40 to 50 ng / mL, 50 to 60 ng / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, HGF or an activator of HGF is added to a culture medium at a concentration that ranges from 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 19, 20, 30, 40, 50, 60 ng / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the HGF activator comprises HGF. In some embodiments, the HGF activator is HGF is present at a concentration of between about 5 to 60 ng / mL. In some embodiments, HGF is at a concentration of about 20 ng / mL.

[0052] As featured and disclosed herein, glucocorticoids include, as non-limiting examples, dexamethasone (DEX), eclomethasone, betamethasone, budesonide, cortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone. In some embodiments, the glucocorticoid is DEX.

[0053] In some aspects, a glucocorticoid is added to a culture medium at a concentration that ranges between about 50 to 200 pM. In some embodiments, the glucocorticoid is added to a culture medium at a concentration that ranges from 10 to 20 pM, 20 to 30 pM, 30 to 40 pM,40 to 50 pM, 50 to 60 pM, 60 to 70 pM, 70 to 80 pM, 80 to 90 pM, 90 to 100 pM, 100 to 150 pM, 150 to 200 pM, 200 to 250 pM, 250 to 300 pM, 300 to 350 pM, 350 to 400 pM, 400 to 450 pM, 450 to 500 p M, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the glucocorticoid is added to a culture medium at a concentration that ranges from 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 |aM, 100, 150, 200, 250, 300, 350, 400, 450, 500 |aM, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the glucocorticoid comprises DEX. In some embodiments, the glucocorticoid is DEX is present at a concentration of between about 50 to 200 pM. In some embodiments, DEX is at a concentration of about 100 pM.

[0054] In some embodiments, the culture media comprises a mixture of agents. In some embodiments, the mixture of agents includes or excludes antibiotics, such as, for example doxycycline and penicillin-streptomycin. In some embodiments, the mixture of agents expressly excludes antibiotics that are used to prevent apoptosis including, as a non-limiting example, doxycycline. In some embodiments, the culture media is supplemented with B27. For example, the mixture of agents includes a TGF-P activator and a Wnt activator, including or excluding a ROCK inhibitor. In another example, the mixture of agents includes a TGF-P activator, BMP activator, FGF activator, and VEGF activator. In another example, the mixture of agents includes TGF-P activator, BMP activator, FGF activator, VEGF activator, and ROCK inhibitor. In another example, the mixture of agents includes a retinoid, Wnt inhibitor, BMP inhibitor, and MEK inhibitor. In another example, the mixture of agents includes a retinoid, Wnt inhibitor, BMP activator, and TGF-P inhibitor. In yet another example, the mixture of agents includes hepatocyte growth factor (HGF), an IE-6 family cytokine, and a glucocorticoid in a medium including or excluding EGF.

[0055] In some embodiments, one media is exchanged for another media as a complete media exchange. As featured and described herein, the volume of a medium exchange is in a volume of between about 90% to 99%. In some embodiments, the volume exchanged is about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or any percentage of culture medium between any two percentages referred to above or herein.

[0056] In another aspect, the iPSC-derived hepatocytes are produced using three- dimensional (3D) scaffold systems such as spheroids, layered cultures, organs-on-a-chip, or on polymer scaffolds like hydrogels or nanofiber scaffolds, or embedded in a solubilized basement membrane preparation. 3D cultures may be spheroid, layered cultures, organs on a chip, or cultured on polymer scaffolds, e.g., hydrogel scaffolds such as alginate hydrogels ornanofibrous hydrogels. As featured and disclosed herein, solubilized basement membrane preparations include, as non-limiting examples, Matrigel™, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels, basement membrane or ECM mimetics thereof, and mixtures of collagen, gelatin, fibronectin, vitronectin, laminin, elastin, polymers, proteins, polypeptides, nucleic acids, sugars, lipids, poly-lysine, poly- omithine, tenascin, heparan sulfate, entactin, nidogen, osteopontin, perlecan. In some embodiments, the solubilized basement membrane preparation includes or excludes blood components or recombinant proteins. In some embodiments, the solubilized basement membrane preparation expressly excludes blood components or recombinant proteins. In some embodiments, cells are embedded in a solubilized basement membrane preparation. In some embodiments, the solubilized basement membrane preparation is Matrigel™. In some embodiments, the Matrigel™ is a growth-factor reduced Matrigel™.

[0057] In one aspect the iPSC-derived hepatocytes are grown on 3D scaffold systems to provide the proper environment for differentiating cells with hepatocyte functions or intermediates thereof. Exemplary 3D scaffold systems featured and disclosed herein, include as non-limiting examples, layered cultures, organs on a chip, or cultured on polymer scaffolds, e.g., hydrogel scaffolds such as alginate hydrogels or nanofibrous hydrogels. Certain 3D scaffold systems are solubilized basement membrane preparations, including, as non-limiting examples, Matrigel™, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)- based hydrogels, basement membrane or ECM mimetics thereof, and mixtures of collagen, gelatin, fibronectin, vitronectin, laminin, elastin, polymers, proteins, polypeptides, nucleic acids, sugars, lipids, poly-lysine, poly-ornithine, tenascin, heparan sulfate, entactin, nidogen, osteopontin, perlecan. In some aspects, the solubilized basement membrane preparation includes or excludes blood components or recombinant proteins. In some aspects, the solubilized basement membrane preparation expressly excludes blood components or recombinant proteins.

[0058] As featured and disclosed herein, single cells, dissociated cells, or serially diluted cells are embedded in a solubilized basement membrane preparation, which can be selected from any solubilized basement membrane preparation featured in this disclosure. In some embodiments, the concentration of cells ranges from between about 100 to 1000 cells / pL. In some embodiments, the concentration of cells are embedded in a solubilized membranepreparation at a concentration that ranges from 1 to 100 cells / pL, 100 to 200 cells / pL, 200 to 300 cells / pL, from 300 to 400 cells / pL, from 400 to 500 cells / pL, from 500 to 600 cells / pL, from 600 to 700 cells / pL, from 700 to 800 cells / pL, from 800 to 900 cells / pL, from 900 to 1000 cells / pL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the concentration of cells is about 500 cells / pL.

[0059] In some aspects, the 3D hepatocyte cultures of this disclosure are used to screen clinical candidate drug and biologic compounds for toxicity and / or efficacy. The 3D hepatocyte cultures provide a more physiologically relevant environment to promote proper hepatocyte function. In a further aspect, the 3D iPSC-derived hepatocyte cultures are used to screen drug compounds for toxicity and / or efficacy. In some embodiments, the iPSC generation and hepatocyte differentiation is performed under GMP (good manufacturing practice) conditions. In some embodiments, the iPSCs are derived from cord blood cells to enhance differentiation potential. In one aspect, the differentiated cells are further processed and formulated into the final drug product.

[0060] In one aspect, inducing iPSCs into definitive endoderm cells comprises: preparing iPSC cells in a first culture medium comprising RPMI, 1% B27 supplement, 100 ng / mL Activin A, 50 ng / mL Wnt3a, 10 pM ROCK inhibitor, plating the iPSC cell suspension on growth-factor reduced Matrigel™, and culturing the iPSCs for about 1 day; exchanging the first culture medium with a second culture medium comprising RPMI, with 100 ng / mL of Activin A and 50ng / mL Wnt3a and culturing the cells for about 1 day; exchanging the second culture medium with a third culture medium comprising RPMI, with 100 ng / mL of Activin A, 0.5 ng / mL, BMP4 , 5 ng / mL FGF2, 10 ng / mL VEGF and culturing the cells for about 2 days; and serum free differentiation media (SFD), with 100 ng / mL of Activin A, 0.5 ng / mL, BMP4, 5 ng / mL FGF2, lOng / mL VEGF for about 2 days to produce definitive endoderm cells.

[0061] In one aspect, inducing gut endodermal progenitor cells comprises: preparing DE cells in a first culture medium comprising GEP medium (SFD with 100 ng / mL of Activin A, 0.5 ng / mL, BMP4, 5 ng / mL FGF2, lOng / mL VEGF), plating the DE cell suspension on growthfactor reduced Matrigel™, and culturing the DE cells for about 1 day to produce gut endodermal progenitor cells. In some aspects, GEPs can be cryopreserved in a cry opreservation medium comprising DMSO, emricasan, polyamine, and CEPT. In some embodiments, the cryopreservation medium does not comprise a ROCK inhibitor, such as, chroman 1.

[0062] In one aspect, inducing hepatic endoderm cells comprises: embedding GEP cells in a solubilized basement membrane preparation, including, as non-limiting examples,Matrigel™, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels, basement membrane or ECM mimetics thereof, and mixtures of collagen, gelatin, fibronectin, vitronectin, laminin, elastin, polymers, proteins, polypeptides, nucleic acids, sugars, lipids, poly-lysine, poly-ornithine, tenascin, heparan sulfate, entactin, nidogen, osteopontin, perlecan. In some aspects, the solubilized basement membrane preparation includes or excludes blood components or recombinant proteins. In some aspects, the solubilized basement membrane preparation expressly excludes blood components or recombinant proteins. In one aspect, inducing hepatic endoderm cells comprises: embedding GEP cells in Matrigel™ by suspending a GEP cells in Matrigel™ and forming Matrigel™ domes of about 50 pL of the cell / cluster suspension then solidifying the Matrigel™ in an incubator for 5 min; then adding a first hepatic endoderm differentiation medium (Medium A) comprising SFD, 2 pM retinoic acid, 4 pM IWP2, 250 nM LDN193189, and 500 nM PD0325901; culturing the cells for about 1 day; then exchanging the Medium A for a second hepatic endoderm differentiation medium (Medium B) comprising SFD, 2 pM retinoic acid, 4 pM IWP2, 10 pg / mL BMP4, and 1 pM A83-01; and culturing the cells for about 1 day to produce HE cells.

[0063] In one aspect, inducing mature hepatocytes comprises: exchanging Medium B for a hepatocyte maturation culture medium (“mature hepatocytes differentiation media”; Medium C) comprising HCM without EGF, 20 ng / mL OSM, 10 ng / mL HGF, and 100 pM DEX, and culturing the cells for about 8-12 days, exchanging the culturing media for fresh Medium C about every 3 to 4 days. In some aspects, the mature hepatocytes have formed 3D liver organoids through the hepatocyte maturation process. In some aspects, mature hepatocytes and 3D liver organoids can be cryopreserved in a cryopreservation medium comprising DMSO, emricasan, polyamine, and CEPT. In some embodiments, the cryopreservation medium does not comprise a ROCK inhibitor, such as, chroman 1.

[0064] In one aspect, the present disclosure provides methods for differentiating iPSCs into high purity endoderm progenitor cells, which can be further differentiated into hepatic endoderm cells, immature hepatocytes, and mature hepatocytes. In one aspect, the initial medium for initiating endoderm progenitor cells from iPSCs comprises RPMI, with Activin A, Wnt3A and Rock Inhibitor. In one aspect, the RPMI media comprises B27, e.g., 0.5-1.5% B27. In one aspect, the RPMI media comprises 1% B27. In some aspects, the initiation medium comprises final concentrations of 100 ng / mL of Activin A, 50 ng / mL Wnt3a and 10 pM ROCK Inhibitor. In one aspect, the target cell concentration is 0.25, 0.30, 0.35, 0.4, 0.45, or 0.5 xlO6cells / mL, or at any concentration range between any two of the recited concentrations. In one aspect, the concentration of cells is 0.35-0.4 xlO6cells / mL. In one aspect, the initiation medium is replaced the next day with Day 1 RPMI medium comprising, Activin A and Wnt3a. In one aspect the Day 1 medium comprises 90-110 ng / mL of Activin A and 40-60 ng / mL Wnt3a. In one aspect the Day 1 medium comprises lOOng / mL of Activin A and 50ng / mL Wnt3a. In one aspect, on Day 2 the medium is replaced with a medium comprising RPMI, Activin A, and one or more of MGP4, FGF2, and VEGF. In one aspect, the Day 2 medium comprises Activin A, BMP4, FGF2, and VEGF. In one aspect, the Day 2 medium comprises final concentrations of 90-110 ng / mL of Activin A, 0.4-.6 ng / mL BMP4, 4-6 ng / mL EGE2, and 9-11 ng / mL VEGE. In one aspect, the Day 2 medium comprises final concentrations of lOOng / mL of Activin A, 0.5ng / mL BMP4, 5ng / mL EGE2, and lOng / mL VEGE. In one aspect, the medium used to change the medium on Day 3 is the same as the medium used on Day 2. In one aspect, on Day4, the Day3 medium is removed, and the cells are incubated with a mixture of SFD, Activin A, and one or more of BMP4, FGF2 and VEGF. In one aspect the Day 4 medium comprises SFD with final concentrations of 90-110 ng / mL of Activin A, 0.4-0.6 ng / mL BMP4, 4-6 ng / mL FGF2, and 9-11 ng / mL VEGF. In one aspect the Day 4 medium comprises SFD with final concentrations of lOOng / mL of Activin A + 0.5ng / mL BMP4 +5ng / mL FGF2 + lOng / mL VEGF. In some aspects, the Day 4 medium is used for at least one more day in the daily medium change to obtain endoderm progenitors.

[0065] In some embodiments, the iPSC-derived hepatocytes express high levels of a mature hepatocyte marker, e.g., any one or more of albumin, al-Antitrypsin (AAT), or urea. In one aspect, the differentiation of mature hepatocytes is determined by secretion or production of any one or more of albumin, AAT, TTR or urea as determined by immuno staining or flow cytometry. Expression levels of albumin, AAT or TTR can also be determined by, e.g., quantitative PCR. In one aspect, the iPSC-derived hepatocytes exhibit, or relative albumin expression levels range from about 0.0005 to 0.0012 relative to a housekeeping gene, e.g., any one of HPRT, Actin, or GAPDH. In one aspect, the iPSC-derived hepatocytes exhibit relative albumin expression levels range from from above 0.0008 to 0.0010, e.g, 0.0009 relative to a housekeeping gene, e.g., any one of HPRT, Actin, or GAPDH. In one aspect, the iPSC-derived hepatocytes exhibit any one or more of a relative albumin expression level of approximately 0.0009, or a relative ATT expression level of 0.008 indicating mature hepatocyte functionality. In some aspects, markers include protein or gene expression analysis of liver- specific markers, e.g., changes in any one of, or combinations ofHNF4a, UGT2B7, transthyretin (TTR), al-antitrypsin (AAT), and albumin, protein or gene expression analysis of coagulation pathway components or factors involved in thrombolysis and haemostasis, production of urea, increased CYP450 activity, and glycogen and lipid storage. In one aspect, markers of mature hepatocytes include albumin gene expression and / or production, and urea production. In one aspect, markers of mature hepatocytes include an increase in the expression or relative expression of any one or more of albumin, ATT, and TTR, with a corresponding decrease in LIN28a and HNF41. In one aspect UGT2B7 is another marker for mature hepatocytes grown in 2D- or 3D-cultures. In some aspects, markers may include markers of undifferentiated or endoderm progenitor cells that decrease in the mature iPSC- derived hepatocytes, e.g. such as LIN28a. In some aspects, a marker of endoderm progenitor cells is NHF41.

[0066] In one aspect, endoderm progenitor cells prepared using the methods and compositions featured herein are differentiated into hepatic endoderm cells. In one aspect the medium used for hepatic endoderm induction comprises serum free differentiation (SFD) medium, and agents or factors that can influence signal pathways as an agonist or antagonist of growth factor receptors, e.g., any one of, or combinations of retinoic acid (RA), inhibitor of Wnt production 2 (IWP2), an inhibitor of BMP type I receptors, e.g. such as LDN193189, a MEK inhibitor, e.g. such as PD0325901, a TGF-P type I receptor inhibitor, e.g. such as A83- 01, and Bone Morphogenetic Protein 4 (BMP4). In an aspect, mediums are changed at least once daily for hepatic endoderm induction. In some aspects, the medium used for hepatic endoderm induction comprises SFD supplemented with 1.8-2.2 pM RA, 3.6-4.4 pM IWP2, 225-275 nM LDN193189, and 450-550 nM PD0325901 for 1 day, followed by SFD supplemented with 1.8-2.2 pM RA, 3.6-4.4 pM IWP2, 9-11 pg / mL BMP4, and 0.9- 1.1 pM A83-01 for the next 3 days. In some aspects, the medium used for hepatic endoderm induction comprises SFD supplemented with 2.0 pM RA, 4.0 pM IWP2, 250 nM LDN193189, and 500 nM PD0325901 for 1 day, followed by SFD supplemented with 2.0 pM RA, 4.0 pM IWP2, 10 pg / mL BMP4, and 1.0 pM A83-01 for the next 3 days.

[0067] After the cultured endoderm progenitors reach confluence, the endoderm progenitor cells are differentiated into immature hepatocytes using the methods and compositions featured herein. In one aspect the medium used for hepatic endoderm induction comprises a medium specialized for stem cell culture, e.g. such as KnockOut Dulbecco's Modified Eagle Medium supplemented with KnockOut serum, and molecules that can influence signal pathways as an agonist or antagonist of growth factor receptors, e.g., any one of, or combinations of L-Glutamine; Non-Essential Amino Acids (NEAA); 2-Mercaptoethanol, and DMSO. In one aspect the medium used for hepatic endoderm induction comprises KnockOut Dulbecco's Modified Eagle Medium (KO / DMEM) supplemented with knockout serum at a ratio of approximately 1 to 5, 0.9- 1.1 mM L-Glutamine, 1:100 NEAA, 1:550 2-Mercaptoethanol, and 1:100 DMSO for 3 days, with media changes every 2 days. In one aspect the medium used for hepatic endoderm induction comprises KnockOut Dulbecco's Modified Eagle Medium (KO / DMEM) supplemented with knockout serum at a ratio of approximately 1:5, 1.0 mM L- Glutamine, 1:100 NEAA, 1:550 2-Mercaptoethanol, and 1:100 DMSO for 3 days, with media changes every 2 days. The immature hepatocytes are then differentiated into mature hepatocytes, or iPSC induced hepatocytes, using the methods and compositions featured herein. In one aspect, the immature hepatocytes are differentiated into mature hepatocytes, or iPSC induced hepatocytes, using a medium specialized for hepatocyte growth, e.g., a hepatocyte culture medium (HCM), optionally supplement with Fetal Bovine Serum (FBS), and molecules that can influence signal pathways as an agonist or antagonist of growth factor receptors, , e.g., any one of, or combinations of Oncostatin M (OSM), Hepatocyte Growth Factor (HGF), and Dexamethasone (DEX). In one aspect, the immature hepatocytes are differentiated into mature hepatocytes, or iPSC induced hepatocytes, using HCM medium supplemented with 3-7% FBS, 15-25 ng / mL OSM, 5-15 ng / mL HGF, and 75-125 pM DEX for 8 or more days, with media changes every 4 days. In one aspect, the immature hepatocytes are differentiated into mature hepatocytes, or iPSC induced hepatocytes, using HCM medium supplemented with 5% FBS, 20 ng / mL OSM, 10 ng / mL HGF, and 100 pM DEX for 8 days, with media changes every 4 days.

[0068] In some embodiments the iPSC derived hepatocytes of this disclosure are highly pure and highly functional, as determined by various assays for liver function, including albumin expression or any of the liver specific markers featured herein. High functioning iPSC- derived hepatocytes are characterizable by their expression of key liver-specific genes and their ability to perform hepatocyte-specific functions. In one aspect, high functioning iPSC-derived hepatocytes express albumin, transthyretin (TTR), and al-antitrypsin (AAT) at levels comparable to or higher than those found in primary human hepatocytes. In another aspect, high functioning iPSC-derived hepatocytes exhibit CYP450 enzyme activities, such as CYP3A4 and CYP2C9, at levels similar to or exceeding those observed in primary human hepatocytes. In yet another aspect, high functioning iPSC-derived hepatocytes secrete albumin, synthesize urea, store glycogen, and uptake and excrete compounds such as indocyanine green(ICG) at rates comparable to those of primary human hepatocytes. In some embodiments, high purity iPSC-derived hepatocytes are defined as a population of cells in which a substantial proportion of the cells express hepatocyte- specific markers and exhibit minimal expression of markers associated with other cell types or undifferentiated cells. In one aspect, high purity iPSC-derived hepatocytes comprise at least 80%, 85%, 90%, 95%, or 99% of cells expressing one or more hepatocyte- specific markers, such as albumin, TTR, or AAT, as determined by immunostaining, flow cytometry, qRT-PCR, or other suitable methods. In another aspect, high purity iPSC-derived hepatocytes express minimal levels of markers associated with undifferentiated cells, such as LIN28A, or markers associated with other cell lineages.

[0069] In some aspects hepatocyte function is determined by secretion, production, and / or expression of any of the mature hepatocyte markers featured herein. In some embodiments, the hepatocytes express maturation markers after about 8-12 days differentiation. In some embodiments, the hepatocytes express maturation markers after about 1 week of differentiation. In some embodiments, the hepatocytes express maturation markers after about 2 weeks of differentiation.

[0070] As featured and disclosed herein, hepatocyte maturation markers include, as nonlimiting examples, al-antitrypsin (AAT), albumin, HNF4a, UGT2B7, transthyretin (TTR), LIN28A, UGT2B7, and urea. In some embodiments, the maturation markers comprise AAT, albumin, and / or TTR.

[0071] In some aspects, the iPSC-derived hepatocyte population has high purity. In one aspect the highly pure hepatocyte population comprises at least 80%, 85%, 90%, 95%, or 99% hepatocyte marker positive cells (e.g., albumin-positive, AAT-positive cells), as determined by immuno staining or flow cytometry. In one aspect the highly pure hepatocyte population comprises at least 90%, 95%, or 99% hepatocyte marker positive cells (e.g., albumin-positive, AAT-positive cells), as determined by immunostaining or flow cytometry. In some aspects, the iPSC-derived hepatocyte population is at least 80%, 85%, 90%, 95%, or 99% positive for at least one hepatocyte marker selected from: HNF4a, UGT2B7, TTR, AAT, and albumin.

[0072] In one aspect, changes in marker expression or production levels are indicative of the differentiation stages of iPSCs, PGECs, 2D hepatocytes, and 3D hepatocytes. In some aspects, protein expression levels include albumin, LIN28A, HNF4A, UGT2B7, TTR, AAT, UGT2B7, and urea. In an aspect, iPSCs are characterized by high expression of LIN28A, low expression of HNF4A, and minimal expression of UGT2B7, TTR, AAT, and albumin. In one aspect, PGECs exhibit reduced expression of LIN28A compared to iPSCs, a slight increase inHNF4A expression, and minimal expression of UGT2B7, TTR, AAT, and albumin. In an aspect, 2D hepatocytes demonstrate reduction in LIN28A expression, increased HNF4A expression compared to PGECs, high expression of UGT2B7, and increased expression of TTR, AAT, and albumin compared to PGECs. In one aspect, 3D hepatocytes exhibit the lowest expression of LIN28A among all cell types, the highest expression of HNF4A, TTR, AAT, and albumin, and reduced expression of UGT2B7 compared to 2D hepatocytes.

[0073] Stem cells are undifferentiated cells that have the ability to self-renew indefinitely and to remain in said undifferentiated state. As opposed to embryonic stem cells, which can only be isolated from the inner mass of a blastocyst, there are three known accessible sources of adult stem cells: the bone marrow, which requires the drilling of a bone; the adipose tissue, which is accessible by liposuction; and the blood, from which the cells can be extracted among other cells. The term “pluripotent stem cells,” as used herein, refers to cells that are capable of generating all the cell types of an organism, i.e., cells derived from any of the three germ layers. On the other hand, multipotent stem cells can differentiate into several cell types, but only those of a closely related family of cells, generally the cell types of the organ from which they originate. Most adult stem cells are multipotent, but small amounts of pluripotent adult stem cells can be retrieved from the umbilical cord or other tissues. The sources of cells used for cell therapy include stem cells such as embryonic stem cells (ESCs), adult stem cells, and induced pluripotent stem cells (iPSCs). In some embodiments, the PSCs used in the methods described herein are human (hPSCs), and in some embodiments, the human PSCs are induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs). In some embodiments, the PSCs are human PSCs (hPSCs). In some embodiments, the hPSCs are human induced pluripotent stem cells (hiPSCs) or human embryonic stems cells (hESCs).

[0074] Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art. 53(c) EPC and Rule 28(b) and (c)EPC. All rights to explicitly disclaim any embodiments that are the subject of any granted patent(s) of applicant in the lineage of this application or in any other lineage or in any prior filed application of any third party is explicitly reserved. Nothing herein is to be construed as a promise.

[0075] It is noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

[0076] These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.INCORPORATION BY REFERENCE

[0077] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference, and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. Incorporated by reference as if set forth in full herein is U.S. Provisional Patent Application 63 / 641,901.BRIEF DESCRIPTION OF THE DRAWINGS

[0078] The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

[0079] FIG. 1 displays a comparison of the morphology of R23-4 iPSC-derived hepatocytes (“iHeps”) cultured in 2D (iHeps 2D) and 3D (iHeps 3D) conditions. The left image depicts cells exhibiting a flattened, monolayer morphology characteristic of hepatocytes grown on a two-dimensional surface. The right image shows hepatocytes cultured in a three- dimensional environment, resulting in the formation of spheroid- like structures. These structures are compact cell aggregates with a rounded morphology, resembling the organization of hepatocytes in vivo. The images were obtained by collecting the culture medium for ELISA and visualizing the cells at 10X magnification.

[0080] FIG. 2 depicts a comparison of the relative gene expression levels of several markers in iPSCs, PGECs (endoderm progenitors), and 2D and 3D iHeps (induced hepatocytes). The top row shows the expression of three liver- specific genes: LIN28a, HNF4a, and UGT2B7. LIN28a expression is highest in PGECs and decreases after differentiation into iHeps, with the lowest expression in 3D iHeps. HNF4a expression is low in iPSCs compared to PGECs, and similarly HNF4a expression is low in 2D iHeps increases in 3D iHeps. UGT2B7 expression is very low in iPSCs and PGECs and increases drastically in 2D iHeps and 3D iHeps. The bottom row shows the expression of three additional markers: transthyretin (TTR), al-antitrypsin (AAT), and albumin. TTR expression is lowest in iPSCs, increases slightly in PGECs and 2D iHeps, and substantially increases in 3D iHeps. AAT expression follows a similar pattern, with low levels in iPSCs, PGECs, and 2D iHeps, with a 3D iHeps showing the highest expression. Albumin expression is also lowest in iPSCs and PGECs, with an increase in 2D iHeps and highest expression in 3D iHeps.

[0081] FIG. 3 shows the key steps of the manufacturing of hiPSCs under cGMPs for cell therapy. The manufacturing of human HLA-homo GMP grade iPSCs under cGMP conditions includes a fresh cord blood unit is obtained from human HLA-homo donor, isolating CD34+ hemopoietic stem cells (HSCs) from the cord blood, reprogramming CD34+ hematopoietic stem cells (HSCs) into hiPSCs using episomal transduce technology and expanding and banking hiPSCs as Master Cell Bank (MCB). Working cell banks (WCB) can then be created under both cGMP setting (Specialized for manufacturing cell therapy drug product) and non- cGMP environment (R&D studies for multiple disease therapy).

[0082] FIG. 4 shows additional key steps of the manufacturing of hiPSCs under cGMPs for cell therapy following the creation of WCB, including the end points of clinical applications and point-of-care (PoC) studies.

[0083] FIG. 5A shows the cellular morphology of HLA-homo, GMP grade iPSCs at a magnification of 5X.

[0084] FIG. 5B shows the flow cytometry analysis of intracellular (NANOG, SOX2, OCT4) and extracellular markers (SSEA4, TRA-1-80), as well as mouse embryonic stem cell specific marker SSEA1 expression in iPSCs. A bar graph quantification of the percentage of positive pluripotent markers expressed in iPSCs is shown below the flow cytometry plots.

[0085] FIG. 5C shows iPSCs stained positively with AP (Alkaline Phosphatase) versus PGECs used as a negative control, at a magnification of 5X.

[0086] FIG. 5D shows the immunofluorescence staining of iPSC colonies using SOX2, OCT4, SSEA4, and TRA-1-60 antibodies, respectively. All images were taken at a magnification of 10X.

[0087] FIG. 6A shows the overview for time and duration of gut endodermal cell differentiation from human GMP compliant iPSCs. (DE: definitive endoderm cells; GEC: Gut endodermal cells.)

[0088] FIG. 6B shows representative phase contrast images of undifferentiated human GMP compliant iPSCs and differentiated definitive endoderm and gut endodermal cells at different time points. Magnification for all images is 10X.

[0089] FIG. 6C shows the flow cytometry analysis of pluripotent markers (NANOG, SOX2), definitive endoderm markers (CXCR4, c-Kit) and gut endodermal markers (FOXA2, CDX2) in iPSC-derived definitive endoderm cells.

[0090] FIG. 6D shows the flow cytometry analysis of pluripotent genes (NANOG, OCT3 / 4), gut endodermal related genes (FOXA2, CDX2) in iPSC-derived gut endodermal cells.

[0091] FIG. 6E shows the quantitative RT-PCR of pluripotent genes (SOX2, OCT3 / 4, NANOG), endodermal related genes (FOXA2, SOX17), as well as hepatic related genes (HNF4a, AFP) expression in undifferentiated iPSCs, iPSCs derived definitive endoderm and gut endodermal cells (passage P2).

[0092] FIG. 7 shows the overview of our stepwise differentiation protocol for 2D hepatocyte (iHeps) and 3D liver organoids.

[0093] FIG. 8A shows representative images of differentiated 2D hepatocytes (magnification of left image is 10X; magnification of right image is 20X) and 3D liver organoids.

[0094] FIG. 8B shows the quantitative RT-PCR of iPSCs specific gene LIN28, and liver related enzymes and genes expression in undifferentiated iPSCs, as well as iPSC-derived hepatocytes and liver organoids. Relative expression values were compared with undifferentiated iPSCs. GEC = gut endodermal cells (also referred to herein as gut endodermal progenitor (GEP) cells).

[0095] FIG. 8C shows the albumin secretion level (ALB, n=5) from iPSC-derived hepatocytes and liver organoids.DETAILED DESCRIPTION OF THE INVENTION

[0096] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined in the appended claims.

[0097] Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only and is not intended to be limiting since the scope of the present invention will be limited only to the appended claims.

[0098] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” include one or more methods and / or steps of the type described herein, which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0099] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0100] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of the ordinary skills in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, it will be understood thatmodifications and variations are encompassed within the spirit and scope of the instant disclosure. The preferred methods and materials are now described.

[0101] The present invention provides methods for generating high purity endoderm progenitor cells from iPSCs, which can then be differentiated into hepatic endoderm cells, immature hepatocytes, and mature hepatocytes. In some aspects, the iPSC-derived hepatocytes express high levels of albumin, indicating a high degree of functionality.

[0102] The methods described herein offer valuable applications in the economical and reproducible generation of gut endodermal progenitors, hepatic endoderm progenitors, mature hepatocytes, and 3D liver organoids. Specifically, these methods facilitate the generation of gut endodermal progenitors, hepatic endoderm progenitors, mature hepatocytes, and 3D liver organoids by using pluripotent stem cells embedded in a solubilized basement membrane preparation and employing a stepwise methodology using stage- specific induction cocktails. In some embodiments, the cells produced are used in regenerative therapies.

[0103] Due to limitations on liver tissue supply for drug screening, there is a strong need for pure, functional iPSC-derived hepatocytes, especially in 3D culture formats that better mimic the in vivo environment. Embodiments of the present invention address this need by providing a rapid, efficient, and standardized mRNA-based differentiation protocol for generating mature hepatocytes. The iPSC-derived hepatocytes exhibit high purity based on albumin expression and other hepatic markers. In certain aspects, high purity iPSC-derived hepatocytes comprise at least 80%, 85%, 90%, 95%, or 99% of cells expressing one or more hepatocyte-specific markers, such as albumin, TTR, or AAT, as determined by immunostaining, flow cytometry, qRT-PCR, or other suitable methods. In another aspect, high purity iPSC-derived hepatocytes express minimal levels of markers associated with undifferentiated cells, such as LIN28A, or markers associated with other cell lineages. The iPSC-derived hepatocytes can be cultured in various 2D and 3D configurations, including spheroids, layered cultures, organ-chips, and scaffolds made of hydrogels or nanofibers. The 3D hepatocyte cultures are particularly useful for drug toxicity and efficacy screening. In some embodiments markers of liver function may include protein or gene expression analysis of liver- specific markers, e.g., any one of, or combinations of HNF4a, UGT2B7, transthyretin (TTR), al -antitrypsin (AAT), and albumin, protein or gene expression analysis of coagulation pathway components or factors involved in thrombolysis and haemostasis, production of urea, increased CYP450 activity, and glycogen and lipid storage.

[0104] In one embodiment, the present invention provides a method of producing iPSC- derived hepatocytes comprising: (a) producing DE cells from iPSCs by contacting iPSCs with a first culture medium comprising a Wnt activator; (b) producing GEP cells from DE cells; (c) producing HE cells from GEP cells by contacting GEP cells with a second culture medium comprising a Wnt inhibitor and a BMP inhibitor; (d) exchanging the second culture medium with a third culture medium comprising a Wnt inhibitor and a BMP activator; and (e) producing iPSC-derived hepatocytes from HE cells by contacting HE cells with a hepatocyte maturation culture medium. In some aspects, the GEP cells in step (c) are embedded in a solubilized basement membrane preparation. In some aspects, the iPSCs are embedded in a solubilized basement membrane preparation. In some aspects, the basement membrane preparation is, e.g., Matrigel™ or any other basement membrane preparation featured herein, e.g., as non-limiting examples, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels. In some embodiments, the first culture medium further comprises any one or more of, two or more of, three or more of, or four of RPMI or SFD, B27, Activin A or any TGF-P activator featured herein, and / or Y-27632 or any ROCK inhibitor featured herein. In some aspects, the first culture medium is supplemented with any one of FGF2 or any FGF activator featured herein, and / or VEGF (VEGF-A) or any VEGF featured herein after 2, 3, 4, or 5 days of culture. In some embodiments, the second culture medium further comprises any one or more of, any two of, or three of SFD, retinoic acid or any retinoid featured herein, and / or PD0325901 or any MEK inhibitor featured herein. In some embodiments, the third culture medium further comprises any one of, any two of, any three of, or four of retinoic acid or any retinoid featured herein, A83-01 or any TGF-P type I receptor inhibitor featured herein and / or SFD. In some embodiments, producing GEP cells from DE cells comprises culturing DE cells in a fourth medium comprising any one of, any two of, any three of, any four of, any five of, or six of Activin A or any TGF-P activator featured herein, BMP4 or BMP activator featured herein, FGF2 or any FGF activator featured herein, VEGF (VEGF-A) or any VEGF featured herein, Y-27632 or any ROCK inhibitor featured herein, and / or SFD. In some embodiments, producing iPSC-derived hepatocytes from HE cells further comprises culturing HE cells in a fifth medium comprising any one of, any two of, any three of, or four of HCM without EGF, OSM, HGF, and / or DEX.

[0105] In one embodiment, the present invention provides a method of producing iPSC- derived hepatocytes comprising: (a) producing DE cells from iPSCs using a first culturemedium comprising a Wnt activator and TGF-P activator; (b) producing GEP cells from DE cells; (c) producing HE cells from GEP cells using a second culture medium comprising a retinoid, a Wnt inhibitor, TGF-P inhibitor; (d) exchanging the second culture medium with a third culture medium comprising a retinoid, Wnt inhibitor, and TGF-P inhibitor; and (e) producing iPSC-derived hepatocytes from HE cells using a hepatocyte maturation culture medium. In some aspects, the GEP cells in step (c) are embedded in a solubilized basement membrane preparation. In some aspects, the iPSCs are embedded in a solubilized basement membrane preparation. In some aspects, the basement membrane preparation is, e.g., Matrigel™ or any other basement membrane preparation featured herein, e.g., as non-limiting examples, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels. In some embodiments, the first culture medium further comprises any one or more of, two or more of, three or more of, four or more of, or five of RPMI or SFD, B27, BMP4 or any BMP activator featured herein, and / or Y-27632 or any ROCK inhibitor featured herein. In some aspects, the first culture medium is supplemented with any one of FGF2 or any FGF activator featured herein, and / or VEGF (VEGF-A) or any VEGF featured herein after 2, 3, 4, or 5 days of culture. In some embodiments, the second culture medium further comprises any one or more of, any two of SFD, and / or PD0325901 or any MEK inhibitor featured herein. In some embodiments, the third culture medium further comprises any one of, any two of BMP4 or any BMP activator featured herein, and / or SFD. In some embodiments, producing GEP cells from DE cells comprises culturing DE cells in a fourth medium comprising any one of, any two of, any three of, any four of, any five of, or six of Activin A or any TGF-P activator featured herein, BMP4 or BMP activator featured herein, FGF2 or any FGF activator featured herein, VEGF (VEGF-A) or any VEGF featured herein, Y-27632 or any ROCK inhibitor featured herein, and / or SFD. In some embodiments, producing iPSC-derived hepatocytes from HE cells further comprises culturing HE cells in a fifth medium comprising any one of, any two of, any three of, or four of HCM without EGF, OSM, HGF, and / or DEX.

[0106] In one embodiment, the present invention provides a method of producing iPSC- derived hepatocytes comprising: (a) producing DE cells from iPSCs using a first culture medium comprising a TGF-P activator and Wnt activator; (b) producing GEP cells from DE cells; (c) producing HE cells from GEP cells using a second culture medium comprising a Wnt inhibitor, TGF-P inhibitor, and BMP inhibitor; (d) exchanging the second culture medium with a third culture medium comprising a retinoid, Wnt inhibitor, BMP activator, and TGF-Pinhibitor; and (e) producing iPSC-derived hepatocytes from HE cells using a hepatocyte maturation culture medium. In some aspects, the GEP cells in step (c) are embedded in a solubilized basement membrane preparation. In some aspects, the iPSCs are embedded in a solubilized basement membrane preparation. In some aspects, the basement membrane preparation is, e.g., Matrigel™ or any other basement membrane preparation featured herein, e.g., as non-limiting examples, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels. In some embodiments, the first culture medium further comprises any one or more of, two or more of, three or more of, four or more of, or five of RPMI or SFD, B27, Activin A or any TGF-P activator featured herein, BMP4 or any BMP activator featured herein, and / or Y-27632 or any ROCK inhibitor featured herein. In some aspects, the first culture medium is supplemented with any one of FGF2 or any FGF activator featured herein, and / or VEGF (VEGF-A) or any VEGF featured herein after 2, 3, 4, or 5 days of culture. In some embodiments, the second culture medium further comprises any one or more of, any two of, or three of SFD, retinoic acid or any retinoid featured herein, and / or PD0325901 or any MEK inhibitor featured herein. In some embodiments, the third culture medium further comprises SFD. In some embodiments, producing GEP cells from DE cells comprises culturing DE cells in a fourth medium comprising any one of, any two of, any three of, any four of, any five of, or six of Activin A or any TGF-P activator featured herein, BMP4 or BMP activator featured herein, FGF2 or any FGF activator featured herein, VEGF (VEGF-A) or any VEGF featured herein, Y-27632 or any ROCK inhibitor featured herein, and / or SFD. In some embodiments, producing iPSC-derived hepatocytes from HE cells further comprises culturing HE cells in a fifth medium comprising any one of, any two of, any three of, or four of HCM without EGF, OSM, HGF, and / or DEX.

[0107] In one embodiment, the present invention provides a method of producing iPSC- derived hepatocytes comprising: (a) producing DE cells from iPSCs using a first culture medium comprising a TGF-P activator, Wnt activator, and ROCK activator; (b) producing GEP cells from DE cells; (c) producing HE cells from GEP cells using a second culture medium comprising a retinoid, a Wnt inhibitor, BMP inhibitor, and MEK inhibitor; (d) exchanging the second culture medium with a third culture medium comprising a retinoid, Wnt inhibitor, BMP activator, and TGF-P inhibitor; and (e) producing iPSC-derived hepatocytes from HE cells using a hepatocyte maturation culture medium. In some aspects, the GEP cells in step (c) are embedded in a solubilized basement membrane preparation. In some aspects, the iPSCs areembedded in a solubilized basement membrane preparation. In some aspects, the basement membrane preparation is, e.g., Matrigel™ or any other basement membrane preparation featured herein, e.g., as non-limiting examples, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels. In some embodiments, the first culture medium further comprises any one or more of, two or more of, three or more of, or four of RPMI or SFD, B27, and / or BMP4 or a BMP activator featured herein. In some aspects, the first culture medium is supplemented with any one of FGF2 or any FGF activator featured herein, and / or VEGF (VEGF-A) or any VEGF featured herein after 2, 3, 4, or 5 days of culture. In some embodiments, the second culture medium further comprises SFD. In some embodiments, the third culture medium further comprises SFD. In some embodiments, producing GEP cells from DE cells comprises culturing DE cells in a fourth medium comprising any one of, any two or more of, any three or more of, any four or more of, any five or more of, or six of Activin A or any TGF-P activator featured herein, BMP4 or BMP activator featured herein, FGF2 or any FGF activator featured herein, VEGF (VEGF-A) or any VEGF featured herein, Y-27632 or any ROCK inhibitor featured herein, and / or SFD. In some embodiments, producing iPSC- derived hepatocytes from HE cells further comprises culturing HE cells in a fifth medium comprising any one of, any two of, any three of, or four of HCM without EGF, OSM, HGF, and / or DEX.

[0108] In one embodiment, the present invention provides a method of producing iPSC- derived hepatocytes comprising: (a) producing DE cells from iPSCs using a first culture medium comprising a TGF-P activator, Wnt activator, and ROCK activator; (b) producing GEP cells from DE cells; (c) producing HE cells from GEP cells using a second culture medium comprising a retinoid, a Wnt inhibitor, BMP inhibitor, and MEK inhibitor; (d) exchanging the second culture medium with a third culture medium comprising a retinoid, Wnt inhibitor, BMP activator, and TGF-P inhibitor; and (e) producing iPSC-derived hepatocytes from HE cells using a hepatocyte maturation culture medium. In some aspects, the GEP cells in step (c) are embedded in a solubilized basement membrane preparation. In some aspects, the iPSCs are embedded in a solubilized basement membrane preparation. In some aspects, the basement membrane preparation is, e.g., Matrigel™ or any other basement membrane preparation featured herein, e.g., as non-limiting examples, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels. In some embodiments, the first culture mediumfurther comprises any one or more of, two or more of, three or more of, or four of RPMI or SFD, B27, and / or BMP4 or a BMP activator featured herein. In some aspects, the first culture medium is supplemented with any one of FGF2 or any FGF activator featured herein, and / or VEGF (VEGF-A) or any VEGF featured herein after 2, 3, 4, or 5 days of culture. In some embodiments, the second culture medium further comprises SFD. In some embodiments, the third culture medium further comprises SFD. In some embodiments, producing GEP cells from DE cells comprises culturing DE cells in a fourth medium comprising any one of, any two of, any three of, any four of, any five of, or six of Activin A or any TGF-P activator featured herein, BMP4 or BMP activator featured herein, FGF2 or any FGF activator featured herein, VEGF (VEGF-A) or any VEGF featured herein, Y-27632 or any ROCK inhibitor featured herein, and / or SFD. In some embodiments, producing iPSC-derived hepatocytes from HE cells further comprises culturing HE cells in a fifth medium comprising any one of, any two of, any three of, or four of HCM without EGF, OSM, HGF, and / or DEX.

[0109] In one embodiment, inducing iPSCs into definitive endoderm cells comprises: preparing iPSC cells in a first culture medium comprising RPMI, 1% B27 supplement, 100 ng / mL Activin A, 50 ng / mL Wnt3a, 10 pM ROCK inhibitor, plating the iPSC cell suspension on growth-factor reduced Matrigel™, and culturing the iPSCs for about 1 day; exchanging the first culture medium with a second culture medium comprising RPMI, with 100 ng / mL of Activin A and 50ng / mL Wnt3a and culturing the cells for about 1 day; exchanging the second culture medium with a third culture medium comprising RPMI, with 100 ng / mL of Activin A, 0.5 ng / mL, BMP4 , 5 ng / mL FGF2, 10 ng / mL VEGF and culturing the cells for about 2 days; and serum free differentiation media (SFD), with 100 ng / mL of Activin A, 0.5 ng / mL, BMP4, 5 ng / mL FGF2, lOng / mL VEGF for about 2 days to produce definitive endoderm cells.

[0110] In one embodiment, inducing gut endodermal progenitor cells comprises: preparing DE cells in a first culture medium comprising GEP medium (SFD with 100 ng / mL of Activin A, 0.5 ng / mL, BMP4, 5 ng / mL FGF2, lOng / mL VEGF), plating the DE cell suspension on growth-factor reduced Matrigel™, and culturing the DE cells for about 1 day to produce gut endodermal progenitor cells. In some embodiments, GEPs can be cryopreserved in a cryopreservation medium comprising DMSO, emricasan, polyamine, and CEPT. In some embodiments, the cryopreservation medium does not comprise a ROCK inhibitor, such as, chroman 1.

[0111] In one embodiment, the present invention provides a method of producing GEP cells from iPSCs comprising: (a) contacting iPSCs in a first culture medium for one or moredays comprising a TGF-P activator and a Wnt activator; (b) producing DE cells by exchanging the first culture medium with a second culture medium comprising a TGF-P activator and BMP activator; and (c) producing GEP cells by contacting the DE cells with a third culture medium comprising the second culture medium and a ROCK inhibitor. In some aspects, the iPSCs of step (a) and / or DE of step (c) are embedded in a solubilized basement membrane preparation. In some aspects, the basement membrane preparation is, e.g., Matrigel™ or any other basement membrane preparation featured herein, e.g., as non-limiting examples, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels. In some embodiments, the first culture medium further comprises any one or more of, two or more of, or three of RPMI or SFD, B27, and / or Y-27632 or any ROCK inhibitor featured herein. In some aspects, the second culture medium is supplemented with any one of FGF2 or any FGF activator featured herein, VEGF (VEGF-A) or any VEGF featured herein after 2, 3, 4, or 5 days of culture. In some embodiments, the second culture medium further comprises SFD. In some aspects, the third culture medium is supplemented with any one of FGF2 or any FGF activator featured herein, and / or VEGF (VEGF-A) or any VEGF featured herein after 2, 3, 4, or 5 days of culture. In some embodiments, the third culture medium further comprises SFD.

[0112] In one embodiment, the present invention provides a method of producing GEP cells from iPSCs comprising: (a) contacting iPSCs in a first culture medium for one or more days comprising a TGF-P activator, Wnt activator, and ROCK inhibitor; (b) producing DE cells by exchanging the first culture medium with a second culture medium comprising a TGF-P activator, BMP activator, FGF activator, and VEGF activator; and (c) producing GEP cells by contacting the DE cells with a third culture medium comprising the second culture medium and a ROCK inhibitor. In some aspects, the iPSCs of step (a) and / or DE of step (c) are embedded in a solubilized basement membrane preparation. In some aspects, the basement membrane preparation is, e.g., Matrigel™ or any other basement membrane preparation featured herein, e.g., as non-limiting examples, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels. In some embodiments, the first culture medium further comprises any one or more of, two or more of, or three of RPMI or SFD, and / or B27. In some embodiments, the second culture medium further comprises SFD. In some embodiments, the third culture medium further comprises SFD.

[0113] In one embodiment, the present invention provides a method of producing HE cells comprising: (a) contacting DE cells with a first culture medium comprising a BMP activator and TGF-P activator to produce GEP cells; (b) contacting GEP cells with a second culture medium comprising a BMP inhibitor and MEK inhibitor; and (c) exchanging the second culture medium with a third culture medium comprising a BMP activator and TGF-P inhibitor. In some aspects, the first culture medium further comprises an FGF activator, VEGF activator, and / or a ROCK inhibitor. In some aspects, the second culture medium and third culture medium further comprise a retinoid and Wnt inhibitor. In some aspects, the DE cells are generated from iPSCs. In some aspects, the DE of step (a) and / or GEP of step (b) are embedded in a solubilized basement membrane preparation. In some aspects, the basement membrane preparation is, e.g., Matrigel™ or any other basement membrane preparation featured herein, e.g., as non-limiting examples, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels. In some embodiments, the first culture medium further comprises any one or more of, two or more of, or three of RPMI or SFD, B27, and / or Y-27632 or any ROCK inhibitor featured herein. In some aspects, the second culture medium is supplemented with any one of FGF2 or any FGF activator featured herein and / or VEGF after 2, 3, 4, or 5 days of culture. In some embodiments, the second culture medium further comprises any one or more of, two or more of, or three of a retinoid, IWP2 or any Wnt inhibitor featured herein, and / or SFD. In some embodiments, the third culture medium further comprises any one or more of, two or more of, or three of a retinoid, IWP2 or any Wnt inhibitor featured herein, and / or SFD.

[0114] In one embodiment, the present invention provides a method of producing HE cells comprising: (a) contacting DE cells with a first culture medium comprising a BMP activator and TGF-P activator to produce GEP cells; (b) contacting GEP cells with a second culture medium comprising a Wnt inhibitor, BMP inhibitor, and MEK inhibitor; and (c) exchanging the second culture medium with a third culture medium comprising a Wnt inhibitor, BMP activator, and TGF-P inhibitor. In some aspects, the first culture medium further comprises an FGF activator, VEGF activator, and / or a ROCK inhibitor. In some aspects, the second culture medium and third culture medium further comprise a retinoid. In some aspects, the DE cells are generated from iPSCs. In some aspects, the DE of step (a) and / or GEP of step (b) are embedded in a solubilized basement membrane preparation. In some aspects, the basement membrane preparation is, e.g., Matrigel™ or any other basement membrane preparation featured herein, e.g., as non-limiting examples, Geltrex™, Celltrex™, reconstituted basementmembrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels. In some embodiments, the first culture medium further comprises any one or more of, two or more of, or three of RPMI or SFD, B27, and / or Y-27632 or any ROCK inhibitor featured herein. In some aspects, the second culture medium is supplemented with any one of FGF2 or any FGF activator featured herein and / or VEGF after 2, 3, 4, or 5 days of culture. In some embodiments, the second culture medium further comprises any one or more of, or two of a retinoid, and / or SFD. In some embodiments, the third culture medium further comprises any one or more of, or two of a retinoid, and / or SFD.

[0115] In one embodiment, inducing hepatic endoderm cells comprises: embedding GEP cells in a solubilized basement membrane preparation, including, as non-limiting examples, Matrigel™, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels, basement membrane or ECM mimetics thereof, and mixtures of collagen, gelatin, fibronectin, vitronectin, laminin, elastin, polymers, proteins, polypeptides, nucleic acids, sugars, lipids, poly-lysine, poly-ornithine, tenascin, heparan sulfate, entactin, nidogen, osteopontin, perlecan. In some aspects, the solubilized basement membrane preparation includes or excludes blood components or recombinant proteins. In some aspects, the solubilized basement membrane preparation expressly excludes blood components or recombinant proteins. In one embodiment, inducing hepatic endoderm cells comprises: embedding GEP cells in Matrigel™ by suspending a GEP cells in Matrigel™ and forming Matrigel™ domes of about 50 pL of the cell / cluster suspension then solidifying the Matrigel™ in an incubator for 5 min; then adding a first hepatic endoderm differentiation medium (Medium A) comprising SFD, 2 pM retinoic acid, 4 pM IWP2, 250 nM LDN193189, and 500 nM PD0325901; culturing the cells for about 1 day; then exchanging the Medium A for a second hepatic endoderm differentiation medium (Medium B) comprising SFD, 2 pM retinoic acid, 4 pM IWP2, 10 pg / mE BMP4, and 1 pM A83-01; and culturing the cells for about 1 day to produce HE cells.

[0116] In one embodiment, inducing mature hepatocytes comprises: exchanging Medium B for a hepatocyte maturation culture medium (“mature hepatocytes differentiation media”; Medium C) comprising HCM without EGF, 20 ng / mL OSM, 10 ng / mL HGF, and 100 pM DEX, and culturing the cells for about 8-12 days, exchanging the culturing media for fresh Medium C about every 3 to 4 days. In some embodiments, the mature hepatocytes have formed 3D liver organoids through the hepatocyte maturation process. In some embodiments, mature hepatocytes and 3D liver organoids can be cryopreserved in a cryopreservation mediumcomprising DMSO, emricasan, polyamine, and CEPT. In some embodiments, the cryopreservation medium does not comprise a ROCK inhibitor, such as, chroman 1.

[0117] In one embodiment, the present invention provides a method of producing iPSC- derived hepatocytes, comprising: (a) differentiating iPSCs into definitive endoderm (DE) cells; (b) differentiating the DE cells into GEP cells; (c) differentiating the GEP cells into HE cells; and (d) differentiating the HE cells into mature hepatocytes, thereby producing iPSC-derived hepatocytes wherein at least 80% of the hepatocytes express at least one mature hepatocyte marker comprising: HNF4a, UGT2B7, transthyretin (TTR), al-antitrypsin (AAT), or albumin.

[0118] In one embodiment, the present invention provides a method of inducing hepatocyte differentiation from iPSCs comprising: (a) inducing definitive endoderm (DE) cells by: (i) embedding iPSCs in a solubilized basement membrane preparation; (ii) contacting iPSCs in a first culture medium comprising a transforming growth factor beta (TGF-P) activator, a Wnt activator, and a Rho-associated protein kinase (ROCK) inhibitor; (iii) exchanging the first culture medium with a second culture medium comprising a TGF-P activator and a Wnt activator; (iv) exchanging the second culture medium with a third culture medium comprising a TGF-P activator, bone morphogenetic protein (BMP) activator, fibroblast growth factor (FGF) activator, and vascular endothelial growth factor (VEGF) activator; (b) inducing gut endoderm progenitor (GEP) cells by: (i) dissociating then embedding DE cells in a solubilized basement membrane preparation; and (ii) contacting the DE cells with a GEP culture medium comprising a TGF-P activator, BMP activator, FGF activator, VEGF activator, and ROCK inhibitor; (c) inducing hepatic endoderm (HE) cells by: (i) dissociating then embedding GEP cells in a solubilized basement membrane preparation; and (ii) contacting the GEP cells with a first HE culture medium comprising a retinoid, Wnt inhibitor, BMP inhibitor, and MEK inhibitor; then (iii) exchanging the first HE culture medium with a second HE culture medium comprising a retinoid, Wnt inhibitor, BMP activator, and TGF-P inhibitor; and (d) inducing iPSC-derived hepatocytes by contacting the HE cells with a hepatocyte maturation culture medium (HCM) comprising hepatocyte growth factor (HGF), an interleukin 6 (IL-6) family cytokine, and a glucocorticoid inducing hepatocyte differentiation from PSCs. In some aspects, the solubilized basement membrane preparation is Matrigel™ or any other basement membrane preparation featured herein, e.g., as non-limiting examples, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels.

[0119] In one embodiment, the present invention provides a method of inducing hepatocyte differentiation from pluripotent stem cells (PSCs) comprising: (a) inducing definitive endoderm (DE) cells by: (i) embedding PSCs in a solubilized basement membrane preparation; (ii) contacting PSCs in a first culture medium comprising a transforming growth factor beta (TGF-P) activator, a Wnt activator, and a Rho-associated protein kinase (ROCK) inhibitor; (iii) exchanging the first culture medium with a second culture medium comprising a TGF-P activator and a Wnt activator; (iv) exchanging the second culture medium with a third culture medium comprising a TGF-P activator, bone morphogenetic protein (BMP) activator, fibroblast growth factor (FGF) activator, and vascular endothelial growth factor (VEGF) activator; (b) inducing gut endoderm progenitor (GEP) cells by: (i) dissociating then embedding DE cells in a solubilized basement membrane preparation; and (ii) contacting the DE cells with a GEP culture medium comprising a TGF-P activator, BMP activator, FGF activator, VEGF activator, and ROCK inhibitor; (c) inducing hepatic endoderm (HE) cells by: (i) dissociating then embedding GEP cells in a solubilized basement membrane preparation; and (ii) contacting the GEP cells with a first HE culture medium comprising a retinoid, Wnt inhibitor, BMP inhibitor, and MEK inhibitor; then (iii) exchanging the first HE culture medium with a second HE culture medium comprising a retinoid, Wnt inhibitor, BMP activator, and TGF-P inhibitor; and (d) inducing PSC-derived hepatocytes by contacting the HE cells with a hepatocyte maturation culture medium (HCM) comprising hepatocyte growth factor (HGF), an interleukin 6 (IL-6) family cytokine, and a glucocorticoid inducing hepatocyte differentiation from PSCs. In some aspects, the solubilized basement membrane preparation is Matrigel™ or any other basement membrane preparation featured herein, e.g., as non-limiting examples, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels.

[0120] In some embodiments, the TGF-P activator is Activin A. In some embodiments, the Wnt activator is Wnt3a. In some embodiments, the ROCK inhibitor is Y-27632. In some embodiments, the BMP activator is BMP4. In some embodiments, the FGF activator is FGF2; the VEGF is VEGF-A. In some embodiments, the retinoid is retinoic acid. In some embodiments, the Wnt inhibitor is inhibitor of Wnt production 2 (IWP2). In some embodiments, the BMP inhibitor is LDN193189. In some embodiments, the MEK inhibitor is PD0325901. In some embodiments, TGF-P inhibitor is A83-01. In some embodiments, the IL- 6 family cytokine is oncostatin M (OSM). In some embodiments, the glucocorticoid is dexamethasone (DEX). In some embodiments, the HCM does not comprise an epidermalgrowth factor (EGF). In some aspects, a culture medium comprising any of the activators, inhibitors, retinoids, cytokines, and glucocorticoids described herein includes one or more of: Activin A, Wnt3a, Y-27632, BMP4, FGF2, VEGF-A, retinoic acid, IWP2, LDN193189, PD0325901, A83-01, OSM, DEX, or any activator, inhibitor, retinoid, IL-6 family cytokine, and glucocorticoid featured and disclosed herein.

[0121] In some embodiments, the TGF-P activator is Activin A; the Wnt activator is Wnt3a; the ROCK inhibitor is Y-27632; the BMP activator is BMP4; the FGF activator is FGF2; the VEGF is VEGF-A; the retinoid is retinoic acid; the Wnt inhibitor is inhibitor of Wnt production 2 (IWP2); the BMP inhibitor is LDN193189; the MEK inhibitor is PD0325901; TGF-P inhibitor is A83-01; the IL-6 family cytokine is oncostatin M (OSM); or the glucocorticoid is dexamethasone (DEX). In some embodiments, the HCM does not comprise an epidermal growth factor (EGF).

[0122] In one embodiment, the present invention provides a method of cryopreserving GEP cells comprising: contacting GEP cells with a GEP cryopreservation medium comprising DMSO, emricasan, polyamine, and trans-ISRIB (CEPT). In some aspects, the GEP cry opreservation medium does not comprise a ROCK inhibitor. In some aspects, the GEP cryopreservation medium does not comprise chroman 1. In another aspect, the present invention provides a method of recovering cryopreserved GEP cells comprising: (a) thawing the cryopreserved GEP cells; (b) recovering the thawed cryopreserved GEP cells in a GEP culture medium comprising DMSO, emricasan, polyamine, and CEPT for at least one day; and (c) culturing the recovered GEP cells in a GEP culture medium comprising SFD, a TGF-P activator, BMP activator, FGF activator, VEGF activator, and ROCK inhibitor for about 2 to 5 days.

[0123] In another embodiment, the present invention provides a method of cry opreserving iPSC-derived hepatocytes or 3D liver organoids comprising: contacting iPSC-derived hepatocytes or 3D liver organoids with a hepatocyte cryopreservation medium comprising DMSO, emricasan, polyamine, and CEPT. In some aspects, the hepatocyte cryopreservation medium does not comprise a ROCK inhibitor. In some aspects, the hepatocyte cryopreservation medium does not comprise chroman 1. In another aspect, the present invention provides a method of recovering cryopreserved iPSC-derived hepatocytes or 3D liver organoids comprising: (a) thawing the cryopreserved iPSC-derived hepatocytes or 3D liver organoids; (b) recovering the thawed iPSC-derived hepatocytes or 3D liver organoids in a hepatocyte cryopreservation medium comprising DMSO, emricasan, polyamine, and CEPT for at least oneday; and (c) culturing the recovered iPSC-derived hepatocytes or 3D liver organoids in a hepatocyte maturation culture medium comprising HCM without EGF, HGF, an IL-6 family cytokine, and a glucocorticoid for at least one day.

[0124] In one embodiment, the present invention provides for a kit comprising: (a) a gut endodermal induction cocktail comprising: a TGF-P activator, BMP activator, and ROCK inhibitor; (b) two hepatic endoderm induction cocktails comprising: (i) BMP inhibitor, and MEK inhibitor; (ii) BMP activator, and TGF-P inhibitor; (c) a hepatic maturation cocktail comprising: hepatocyte culture medium without EGF, HGF, an IL-6 family cytokine, and a glucocorticoid; and (d) instructions for inducing definitive endodermal cells differentiation into mature hepatocytes; and optionally a solubilized basement membrane preparation. In some aspects, the two hepatic endoderm induction cocktails further comprise a retinoid and Wnt inhibitor. In some aspects, the gut endodermal induction cocktail further comprises a FGF activator and VEGF activator.

[0125] In one embodiment, the present invention provides for a kit comprising: (a) a gut endodermal induction cocktail comprising: a TGF-P activator, BMP activator, FGF activator, VEGF activator, and ROCK inhibitor; (b) two hepatic endoderm induction cocktails comprising: (i) BMP inhibitor, MEK inhibitor; (ii) BMP activator, and TGF-P inhibitor; (c) a hepatic maturation cocktail comprising: hepatocyte culture medium without EGF, HGF, an IL- 6 family cytokine, and a glucocorticoid; and (d) instructions for inducing definitive endodermal cells differentiation into mature hepatocytes; and optionally a solubilized basement membrane preparation. In some aspects, the two hepatic endoderm induction cocktails further comprise a retinoid and Wnt inhibitor.

[0126] In one embodiment, the present invention provides for a kit comprising: (a) a gut endodermal induction cocktail comprising: a TGF-P activator, BMP activator, FGF activator, VEGF activator, and ROCK inhibitor; (b) two hepatic endoderm induction cocktails comprising: (i) a retinoid, Wnt inhibitor, BMP inhibitor, and MEK inhibitor; (ii) a retinoid, Wnt inhibitor, BMP activator, and TGF-P inhibitor; (c) a hepatic maturation cocktail comprising: hepatocyte culture medium without EGF, HGF, an IL-6 family cytokine, and a glucocorticoid; and (d) instructions for inducing definitive endodermal cells differentiation into mature hepatocytes; and optionally a solubilized basement membrane preparation.

[0127] The transforming growth factor beta (TGF-P) superfamily includes TGF-P proteins, bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), glial-derived neurotrophic factors (GDNFs), Activins, Inhibins, Nodal, Lefty, and Mulllerian inhibitingsubstance (MIS). In some embodiments, an inducing agent useful for the induction of endodermal progenitors may include an activator or inhibitor of the TGF-P signaling pathway. Activators and inhibitors of the TGF-P signaling pathway include, as non-limiting examples, small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like, that activate or inhibit at least one component of the TGF-P signaling pathway, resulting in a corresponding activation or inhibition of cellular TGF-P signaling.

[0128] Activators of the TGF-P signaling pathway include, as non-limiting examples, Activin A, TGF-P family ligands (e.g., TGF-P proteins and other activators of TGF-P receptors) and portions thereof, TGF-pi, TGF-P2, TGF-P3, IDE1 / 2 (IDE1 [l-[2-[(2- Carboxyphenyl)methylene]hydrazide]heptanoic acid], IDE2 [Heptanedioic acid- 1 -(2- cyclopentylidenehydrazide)]), Nodal, and the like. In some embodiments, activation of the TGF-P signaling pathway is achieved through repression of a TGF-P signaling pathway inhibitor, including, as non-limiting examples, the use of an inhibitory nucleic acid targeting an inhibitor of the TGF-P signaling pathway or an antibody or small molecule directed to a TGF-P signaling pathway inhibitor. In some embodiments, the TGF-P signaling pathway activator comprises Activin A.

[0129] Inhibitors of the TGF-P signaling pathway include, as non-limiting examples, A83- 01, SB431542, SB505124, LY2157299, and LY550410. In some embodiments, the TGF-P signaling pathway activator comprises A83-01.

[0130] The WNT signaling pathways are a group of signal transduction pathways that begin with proteins that pass signals into a cell through cell surface receptors. WNT signaling pathways use either nearby cell-cell communication (paracrine) or same-cell communication (autocrine). Three WNT signaling pathways have been characterized: the canonical WNT pathway, the noncanonical planar cell polarity pathway, and the noncanonical WNT / calcium pathway. All three pathways are activated by the binding of a WNT-protein ligand to a Frizzled family receptor, which passes the biological signal to the Dishevelled protein inside the cell. The canonical WNT pathway leads to regulation of gene transcription and is thought to be negatively regulated in part by the SPATS 1 gene. The noncanonical planar cell polarity pathway regulates the cytoskeleton, which is responsible for the shape of the cell. The noncanonical WNT / calcium pathway regulates calcium inside the cell. WNT signaling was first identified for its role in carcinogenesis, then for its function in embryonic development. The embryonic processes controlled by WNT signaling include body axis patterning, cell fatespecification, cell proliferation, and cell migration. These processes are necessary for the proper formation of important tissues, including bone, heart, and muscle. Its role in embryonic development was discovered when genetic mutations in WNT pathway proteins produced abnormal fruit fly embryos. Later research found that the genes responsible for these abnormalities also influenced breast cancer development in mice. WNT signaling also controls tissue regeneration in adult bone marrow, skin, and intestine.

[0131] In some embodiments, an inducing agent useful for the induction of endodermal progenitors or hepatocytes may include an activator or inhibitor of the WNT signaling pathway. Activators and inhibitors of the WNT signaling pathway include, as non-limiting examples, small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the WNT signaling pathway, resulting in a corresponding activation or inhibition of cellular WNT signaling.

[0132] Activators of the WNT signaling pathway include, as non-limiting examples, WNT family ligands including, as non-limiting examples, WNT family ligands including Wnt-1, Wnt-2, Wnt-2b, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7a / b, Wnt-7b, Wnt-8a, Wnt-8b, Wnt-9a, Wnt-9b, Wnt-lOa, Wnt-lOb, Wnt-11, and Wnt-16b; CHIR99021 (6-[[2-[[4- (2,4-Dichlorophenyl)-5-(5-methyl- 1 H-imidazol-2-yl)-2pyrimidinyl] amino] ethyl] amino] -3- pyridinecarbonitrile); RSPO co-agonists; lithium chloride; TDZD8 (4-Benzyl-2-methyl- 1,2,4- thiadiazolidine-3, 5-dione); BIO-Acetoxime ((2’Z,3’E)-6-Bromoindirubin-3 '-acetoxime);A1070722 (l-(7-Methoxyquinolin-4-yl)-3-[6 (trifluoromethyl)pyridin-2-yl]urea); HLY78 (4- Ethyl-5,6-Dihydro-5-methyl-[l,3]dioxolo[4,5-j]phenanthridine); CID 11210285 HC1 (2- Amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine hydrochloride); WAY-316606; (hetero) arylpyrimidines; IQ1; QS11; SB-216763; and / or DCA. In some embodiments, the culture media comprising a WNT activator includes or excludes a GSK-3P inhibiting WNT activator such as, e.g., CHIR99021. In some embodiments, the culture media comprising a WNT activator expressly excludes a GSK-3P inhibiting WNT activator such as, e.g., CHIR99021. In some embodiments, activation of the WNT signaling pathway is achieved through repression of a WNT signaling pathway inhibitor, including, as non-limiting examples, the use of an inhibitory nucleic acid targeting an inhibitor of the WNT signaling pathway or an antibody or small molecule directed to a WNT signaling pathway inhibitor.

[0133] In some embodiments, the WNT signaling pathway activator comprises Wnt3a. In some embodiments, the WNT signaling pathway activator comprises a derivative of Wnt3a. In some embodiments, one or more WNT signaling pathway activators is used.

[0134] Inhibitors of the WNT signaling pathway include, as non-limiting examples, IWP2 (N-(6-Methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2- d]pyrimidin-2-yl)thio]-acetamide), C-59 (4-(2-Methyl-4-pyridinyl)-N-[4-(3-pyridinyl)phenyl] benzeneacetamide), (3,5,7,8-Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d] pyrimidin-4-one) , IWR 1 (4-( 1 ,3 ,3a, 4, 7 ,7a- Hexahydro- 1 ,3-dioxo-4,7-methano-2H-isoindol-2- yl)-N-8-quinolinyl-Benzamide), DKK1, Antl.4Br, Antl.4CI, Niclosamide, apicularen, bafilomycin, XAV939 NSC668036 (N-[(l,l-Dimethylethoxy)carbonyl]-L-alanyl-(2S)-2- hydroxy-3-methylbutanoyl-L-Alanine-(lS)-l-carboxy-2-methylpropyl ester hydrate), 2,4- diamino-quinazoline, Quercetin, ICG-001 ((6S,9aS)-Hexahydro-6-[(4-hydroxyphenyl)methyl] -8-(l-naphthalenylmethyl)-4,7-dioxo-N-(phenylmethyl)-2H-pyrazino[l,2-a]pyrimidine- l(6H)-carboxamide), PKF115-584, BML-284 (2-Amino-4-[3,4-(methylenedioxy) benzylamino]-6-(3-methoxyphenyl)pyrimidine), FH-535, iCRT-14, JW-55, JW-67, and antibodies to WNTs and WNT receptors, WNT inhibitory nucleic acids.

[0135] In some embodiments, inhibition of the WNT signaling pathway is achieved through repression of a WNT signaling pathway activator, including, as non-limiting examples, the use of an inhibitory nucleic acid targeting an activator of the WNT signaling pathway or an antibody or small molecule directed to a WNT signaling pathway activator. In some embodiments, the WNT signaling pathway inhibitor is IWP2.

[0136] Inhibitors of ROCK include, as non-limiting examples, Y-27632, ripasudil, netarsudil, chroman 1, Y39983, Wf-536, SLx-2119, Azabenzimidazole-aminofurazans, DE- 104 Olefins, Isoquinolines, Indazoles, pyridinealkene derivatives, H-1152P, XD-4000, HMN- 1152, ROKa 4-(l-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamides, rhostatin, BA-210, BA-207, BA-285, BA- 1037, BA-215, Ki-23095, VAS-012, quinazoline,GSK-269962, and salts thereof, including Y-27632 dihydrochloride, H-1152 dihydrochloride, fasudil hydrochloride, SR-3677 dihydrochloride, glycyl-H-1152 dihydrochloride, SB-772077B dihydrochloride, TC-S-7001, RKI-1447 dihydrochloride, and OXA-06 dihydrochloride. In some embodiments, the culture of PSCs is contacted with a culture medium comprising a ROCK inhibitor, which can be any ROCK inhibitor featured in this disclosure, for at least one day. In some embodiments, the ROCK inhibitor is Y-27632.

[0137] Bone morphogenetic proteins (BMPs) are a group of growth factors also known as cytokines and metabologens. Originally discovered for their ability to induce the formation of bone and cartilage, BMPs are now considered to constitute a group of pivotal morphogenetic signals, orchestrating tissue architecture throughout the body. The important functioning of BMP signals in physiology is emphasized by the multitude of roles for dysregulated BMP signaling in pathological processes. BMPs interact with specific receptors on the cell surface, referred to as bone morphogenetic protein receptors (BMPRs). Signal transduction through BMPRs results in the mobilization of members of the SMAD family of proteins. The signaling pathways involving BMPs, BMPRs, and SMADs are important in the development of the heart, central nervous system, and cartilage, as well as postnatal bone development. They have an important role during embryonic development in embryonic patterning and early skeletal formation. As such, disruption of BMP signaling can affect the body plan of the developing embryo. For example, BMP4 and its inhibitors, noggin and chordin, help regulate the polarity of the embryo (i.e., back-to-front patterning). Specifically, BMP4 and its inhibitors play a major role in neurulation and the development of the neural plate. BMP4 signals ectoderm cells to develop into skin cells, but the secretion of inhibitors by the underlying mesoderm blocks the action of BMP4 to allow the ectoderm to continue on its normal course of neural cell development.

[0138] Activators of the BMP pathway include, as non-limiting examples, BMP family ligands, e.g., BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8, BMP9, BMP8b, BMP10, BMP11, and BMP 15; Alantolactone; FK506; isoliquiritigenin; and 4 ’-hydroxy chaicone. In some embodiments, activation of the BMP pathway is achieved through repression of a BMP pathway inhibitor, including, as non-limiting examples, the use of an inhibitory nucleic acid targeting an inhibitor of the BMP pathway, or an antibody or small molecule directed to a BMP pathway inhibitor. In some embodiments, the BMP pathway activator comprises BMP4.

[0139] Inhibitors of the BMP pathway include, as non-limiting examples, LDN193189, noggin, chordin, follistatin, Tsg, Gremlin, dosmorphin, K02299, DMH1, and DMH2. In some embodiments, the BMP pathway inhibitor comprises LDN193189.

[0140] In some embodiments, an inducing agent useful for the induction of endodermal cells or hepatocytes may include an activator or inhibitor of the FGF signaling pathway. In some instances, an activator or inhibitor of the FGF signaling pathway may also include activators or inhibitors of related signal transduction pathways, including, as a non-limiting example, the MAPK / ERK signal transduction pathway. Activators and inhibitors of the FGFsignaling pathway include small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the FGF signaling pathway, resulting in a corresponding activation or inhibition in cellular FGF signaling.

[0141] Activators of the FGF signaling pathway, as non-limiting examples: FGF family ligands including, as non-limiting examples, FGF1, FGF2, FGF-3, FGF-4, FGF-5, FGF-6, KGF / FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-15, FGF-16, FGF-17, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23; SUN 11602 (4-[[4-[[2-[(4-Amino-2, 3,5,6- tetramethy Ipheny 1) amino] acetyl] methylamino] - 1 -piperidiny 1] methypenzamide) ; U-46619; t- Butylhydroquinone; C2 Ceramide; Lactosyl Ceramide; Angiotensin II; and Baicalin.

[0142] In some embodiments, activation of the FGF signaling pathway is achieved through repression of a FGF signaling pathway inhibitor, including, as non-limiting examples, the use of an inhibitory nucleic acid targeting an inhibitor of the FGF signaling pathway or an antibody or small molecule directed to a FGF signaling pathway inhibitor. In some embodiments, the FGF signaling pathway activator is FGF2.

[0143] Vascular endothelial growth factors belong to a subfamily of growth factors involved in vasculogenesis and angiogenesis. Vascular endothelial growth factor (VEGF) regulates, for example, the creation of new blood vessels during embryonic development. As featured and disclosed herein, activators and inhibitors of the VEGF signaling pathway include, as non-limiting examples, signaling pathway ligands, small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the VEGF signaling pathway, resulting in a corresponding activation or inhibition in cellular VEGF signaling. In some aspects of this disclosure, activation of the VEGF signaling pathway is achieved through repression of a VEGF signaling pathway inhibitor, including, as non-limiting examples, the use of an inhibitory nucleic acid targeting an inhibitor of the VEGF signaling pathway or an antibody or small molecule directed to a VEGF signaling pathway inhibitor. In some embodiments, a vascular endothelial growth factor includes, as non-limiting examples, a VEGF family ligand which activates the VEGF pathway, e.g., VEGFA, VEGFB, VEGFC, VEGFD, VEGFE, VEGFF, and placenta growth factor (PIGF). In some embodiments, the VEGF is VEGFA.

[0144] Retinoids and derivatives of vitamin A are signaling molecules involved in embryonic development, immune function, and bone growth. Retinoic acid (RA) is a vitaminA metabolite and morphogen that acts through its cognate receptors retinoic acid receptor and retinoid nuclear receptor to mediate gene transcription, cell fate specification, and axis formation. As featured and disclosed herein, retinoids include, as non-limiting examples, RA, retinol, retinal, isotretinoin, and alitretinoin. In some embodiments, the retinoid is RA.

[0145] The mitogen-activated protein kinase (MAPK / ERK or MEK) signaling pathway is a critical regulator of cellular proliferation and cell cycle progression. Activation of the MEK signaling pathway triggers a kinase cascade of mitogen-activated kinases to induce transcription. As featured and disclosed herein, activators and inhibitors of the MEK signaling pathway include, as non-limiting examples, signaling pathway ligands, small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the MEK signaling pathway, resulting in a corresponding activation or inhibition in cellular MEK signaling. In some aspects of this disclosure, activation of the MEK signaling pathway is achieved through repression of a MEK signaling pathway inhibitor, including, as non-limiting examples, the use of an inhibitory nucleic acid targeting an inhibitor of the MEK signaling pathway or an antibody or small molecule directed to a MEK signaling pathway inhibitor. In some embodiments, an inhibitor of the MEK signaling pathway includes, as nonlimiting examples, PD0325901, trametinib, pimasertib, selumetinib, rafametinib, TAK-733, MEK-162, RO-4987655, and XL-518. In some embodiments, the MEK signaling pathway activator is PD0325901.

[0146] Interleukin-6 (IL-6) family cytokines encompass a group of cytokines having similar receptor complex structures and using the signaling receptor subunit glycoprotein 130 kDa (gpl30). IL-6 family cytokines are involved in the regulation inducing hepatic actue phase proteins, as well as metabolic functions and neurotrophic functions. As featured and disclosed herein, IL-6 family cytokines include, as non-limiting examples, oncostatin M (OSM), IL-6, IL-11, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), cardiotrophin 1 (CT-1), cardiotrophin-like cytokine (CLC), and IL-27. In some embodiments, the IL-6 family cytokine is OSM.

[0147] Hepatocyte growth factor (HGF), also known as scatter factor, is a secreted molecule regulates cell growth, cell motility, and morphogenesis. As featured and disclosed herein, activators of HGF include, as non-limiting examples, HGF, matriptase, and hepsin.

[0148] Glucocorticoids are steroid hormones produced by the adrenal cortex and play key roles in the regulation of metabolic, cardiovascular, immunologic, and hemostatic functions.Importantly, glucocorticoids are involved in the maturation of certain cell types, such as, e.g. hepatocytes, by inducing the expression of key transcription factors such as HGF and HNF4a. As featured and disclosed herein, glucocorticoids include, as non-limiting examples, dexamethasone (DEX), eclomethasone, betamethasone, budesonide, cortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone. In some embodiments, the glucocorticoid is DEX.

[0149] In some embodiments, the chemical culture conditions of the presently described methods include a mixture of agents. For example, the mixture of agents includes a TGF-P activator and a Wnt activator, including or excluding a ROCK inhibitor. In another example, the mixture of agents includes a TGF-P activator, BMP activator, FGF activator, and VEGF activator. In another example, the mixture of agents includes TGF-P activator, BMP activator, FGF activator, VEGF activator, and ROCK inhibitor. In another example, the mixture of agents includes a retinoid, Wnt inhibitor, BMP inhibitor, and MEK inhibitor. In another example, the mixture of agents includes a retinoid, Wnt inhibitor, BMP activator, and TGF-P inhibitor. In yet another example, the mixture of agents includes hepatocyte growth factor (HGF), an IL-6 family cytokine, and a glucocorticoid in a medium including or excluding EGF.

[0150] In some embodiments, a culture medium comprises a TGF-P activator and Wnt activator. In some embodiments, a culture medium comprising a TGF-P activator and Wnt activator further comprises 1% B27, a ROCK inhibitor, a BMP activator, a FGF activator, a VEGF activator, and / or a combination thereof.

[0151] In some embodiments, a culture medium comprises a TGF-P activator and BMP activator. In some embodiments, a culture medium comprising TGF-P activator and BMP activator further comprises a FGF activator, a VEGF activator, and / or a combination thereof.

[0152] In some embodiments, a culture medium comprises a Wnt inhibitor and BMP inhibitor. In some embodiments, a culture medium comprising a Wnt inhibitor and BMP inhibitor further comprises a retinoid, a MEK inhibitor, and / or a combination thereof.

[0153] In some embodiments, a culture medium comprises a Wnt inhibitor and BMP activator. In some embodiments, a culture medium comprising a Wnt inhibitor and BMP activator further comprises a retinoid, a TGF-P inhibitor, and / or a combination thereof.

[0154] In some embodiments, a culture medium comprises a Wnt inhibitor and a TGF-P inhibitor. In some embodiments, a culture medium comprising a Wnt inhibitor and a TGF-P inhibitor further comprises a retinoid, a BMP activator, and / or a combination thereof.

[0155] In some embodiments, a culture medium comprises HCM, HGF, an IL-6 family cytokine, and a glucocorticoid. In some embodiments, the culture medium comprising HCM, HGF, an IL-6 family cytokine, and a glucocorticoid does not comprise EGF.

[0156] In some embodiments, iPSCs or PSCs are exposed to a culture medium comprising B27, a TGF-P activator, Wnt activator, and ROCK inhibitor.

[0157] In some embodiments, iPSCs or PSCs are exposed to a culture medium comprising a TGF-P activator, a BMP activator, a FGF activator, and a VEGF activator.

[0158] In some embodiments, DE cells are exposed to a culture medium comprising a TGF-P activator, a BMP activator, a FGF activator, a VEGF activator, and a ROCK inhibitor.

[0159] In some embodiments, GEP cells are exposed to a culture medium comprising a retinoid, a Wnt inhibitor, a BMP inhibitor, and a MEK inhibitor.

[0160] In some embodiments, GEP cells are exposed to a culture medium comprising a retinoid, a Wnt inhibitor, a BMP activator, and a TGF-P inhibitor.

[0161] In some embodiments, HE cells are exposed to a culture medium comprising HCM without EGF, an IL-6 family cytokine, and a glucocorticoid.

[0162] In some embodiments, iPSC or PSCs are grown in the presence of B27, a TGF-P activator, Wnt activator, and ROCK inhibitor for about one day, for 1 to 2 days, or for any time range between any two of the number of days referred to above or herein. In some embodiments, iPSCs or PSCs are exposed to a culture medium comprising B27, Activin A, Wnt3a, and Y-27632. In some embodiments, iPSCs or PSCs are exposed to a culture medium comprising B27, Activin A, Wnt3a, and Y-27632 for about 1 day.

[0163] In some embodiments, iPSC or PSCs are grown in the presence of TGF-P activator, a BMP activator, a FGF activator, and a VEGF activator for about one day, for 1 to 2 days, for 2 to 3 days, or for any time range between any two of the number of days referred to above or herein. In some embodiments, iPSCs or PSCs are exposed to a culture medium comprising Activin A, BMP4, FGF, and VEGF. In some embodiments, iPSCs or PSCs are exposed to a culture medium comprising Activin A, BMP4, FGF, and VEGF for about 2 days.

[0164] In some embodiments, DE cells are grown in the presence of a TGF-P activator, a BMP activator, a FGF activator, a VEGF activator, and a ROCK inhibitor for about one day, for 1 to 2 days, or for any time range between any two of the number of days referred to above or herein. In some embodiments, DE are exposed to a culture medium comprising Activin A, BMP4, FGF2, VEGF, and Y-27632. In some embodiments, DE are exposed to a culture medium comprising Activin A, BMP4, FGF2, VEGF, and Y-27632 for about 1 day.

[0165] In some embodiments, GEP cells are grown in the presence of a retinoid, a Wnt inhibitor, a BMP inhibitor, and a MEK inhibitor for about one day, for 1 to 2 days, for 2 to 3 days, for 3 to 4 days, for 4 to 5 days, or for any time range between any two of the number of days referred to above or herein. In some embodiments, GEP are exposed to a culture medium comprising a retinoid, IWP2, LDN193189, and PD0325901. In some embodiments, GEP are exposed to a culture medium comprising a retinoid, IWP2, LDN193189, and PD0325901 for about 3 to 4 days.

[0166] In some embodiments, GEP cells are grown in the presence of a retinoid, a Wnt inhibitor, a BMP activator, and a TGF-P inhibitor for about one day, for 1 to 2 days, or for any for any time range between any two of the number of days referred to above or herein. In some embodiments, GEP are exposed to a culture medium comprising a retinoid, IWP2, BMP4, and a A83-01. In some embodiments, GEP are exposed to a culture medium comprising a retinoid, IWP2, BMP4, and a A83-01 for about 1 day.

[0167] In some embodiments, GEP cells are grown in the presence of HCM without EGF, an IL-6 family cytokine, and a glucocorticoid for about one day, for 5 to 6 days, for 6 to 7 days, for 7 to 8 days, for 8 to 9 days, 9 to 10 days, 10 to 11 days, 11 to 12 days, 12 to 13 days, 13 to 14 days, 14 to 15 days, or for any time range between any two of the number of days referred to above or herein. In some embodiments, HE are exposed to a culture medium comprising HCM without EGF, OSM, HGF, and DEX. In some embodiments, HE are exposed to a culture medium comprising HCM without EGF, OSM, HGF, and DEX for about 8-12 days.

[0168] In some embodiments, the concentration of the TGF-P signaling pathway activator or inhibitor ranges between about 50 ng / mL to 150 ng / mL. In some embodiments, the TGF-P signaling pathway activator is present at a concentration of between about 50 ng / mL to 150 ng / mL. In some embodiments, the TGF-P signaling pathway activator is at a concentration that ranges from 25 to 30 ng / mL, from 30 to 40 ng / mL, from 40 to 50 ng / mL, from 50 to 60 ng / mL, from 60 to 70 ng / mL, from 70 to 80 ng / mL, from 80 to 90 ng / mL, from 90 to 100 ng / mL, from 100 to 110 ng / mL, from 110 to 120 ng / mL, from 120 to 130 ng / mL, from 130 to 140 ng / mL, from 140 to 150 ng / mL, from 150 to 160 ng / mL, from 160 to 170 ng / mL, from 170 to 180 ng / mL, from 180 to 190 ng / mL, from 190 to 200 ng / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the TGF-P signaling pathway activator is at a concentration that ranges from 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 ng / mL, or at any concentration range any twoconcentrations referred to above or herein. In some embodiments, Activin A is at a concentration of about 100 ng / mL.

[0169] In some embodiments, the concentration of the TGF-P signaling pathway inhibitor is in a range between about 0.5 pM to 2 pM. In some embodiments, the TGF-P signaling pathway inhibitor is added to a culture medium at a concentration that ranges from 0.1 to 0.2 pM, 0.2 to 0.3 pM, 0.3 to 0.4 pM, 0.4 to 0.5 pM, 0.5 to 0.6 pM, 0.6 to 0.7 pM, 0.7 to 0.8 pM, 0.8 to 0.9 pM, 0.9 to 1 pM, 1.0 to 2.0 pM, 2.0 to 3.0 pM, 3.0 to 4.0 pM, 4.0 to 5.0 pM, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the TGF-P signaling pathway inhibitor is added to a culture medium at a concentration that ranges from 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5 pM, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the concentration of TGF-P signaling pathway inhibitor is about 1 pM. In some embodiments the concentration of A83-01 is about 1 pM.

[0170] In some embodiments, the WNT signaling pathway activator is added to a culture medium at a concentration that ranges between about 25 ng / mL to 100 ng / mL. In some embodiments, the WNT signaling pathway activator is added to a culture medium at a concentration that ranges from 10 to 20 ng / mL, 20 to 30 ng / mL, from 30 to 40 ng / mL, from 40 to 50 ng / mL, from 50 to 60 ng / mL, from 60 to 70 ng / mL, from 70 to 80 ng / mL, from 80 to 90 ng / mL, from 90 to 100 ng / mL, from 100 to 110 ng / mL, from 110 to 120 ng / mL, from 120 to 130 ng / mL, from 130 to 140 ng / mL, from 140 to 150 ng / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the WNT signaling pathway activator is added to a culture medium at a concentration that ranges from 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 ng / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the WNT signaling pathway activator comprises Wnt3a. In some embodiments, the WNT signaling pathway activator is Wnt3a is present at a concentration of between about 25 ng / mL to 100 ng / mL. In some embodiments, Wnt3a is at a concentration of about 50 ng / mL.

[0171] In some embodiments, the concentration of the WNT signaling pathway inhibitor is in a range between about 1 pM to 8 pM. In some embodiments, the WNT signaling pathway inhibitor is added to a culture medium at a concentration that ranges from 0.1 to 0.2 pM, 0.2 to 0.3 pM, 0.3 to 0.4 pM, 0.4 to 0.5 pM, 0.5 to 0.6 pM, 0.6 to 0.7 pM, 0.7 to 0.8 pM, 0.8 to 0.9 pM, 0.9 to 1 pM, 1.0 to 2.0 pM, 2.0 to 3.0 pM, 3.0 to 4.0 pM, 4.0 to 5.0 pM, 5.0 to 6.0 pM, 6.0 to 7.0 pM, 7.0 to 8.0 pM, 8.0 to 9.0 pM, 9.0 to 10 pM, or at any concentration range anytwo concentrations referred to above or herein. In some embodiments, the WNT signaling pathway inhibitor is added to a culture medium at a concentration that ranges from 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 pM, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the concentration of WNT signaling pathway inhibitor is about 4 pM. In some embodiments the concentration of IWP2 is about 4 pM.

[0172] As featured and disclosed herein, ROCK inhibitors include, as non-limiting examples, Y-27632, chroman 1, ripasudil, netarsudil, Y39983, Wf-536, SLx-2119, Azabenzimidazole-aminofurazans, DE- 104 Olefins, Isoquinolines, Indazoles, pyridinealkene derivatives, H-1152P, XD-4000, HMN-1152, ROKa 4-(l-aminoalkyl)-N-(4- pyridyl)cyclohexane-carboxamides, rhostatin, BA-210, BA- 207, BA-285, BA- 1037, BA-215, Ki-23095, VAS-012, quinazoline,GSK-269962, and salts thereof, including Y-27632 dihydrochloride, H-1152 dihydrochloride, fasudil hydrochloride, SR-3677 dihydrochloride, glycyl-H-1152 dihydrochloride, SB-772077B dihydrochloride, TC-S-7001, RKI-1447 dihydrochloride, and OXA-06 dihydrochloride.

[0173] In some embodiments, the concentration of ROCK inhibitor is in a range between about 5 pM to 15 pM. In some embodiments, the ROCK inhibitor is Y-27632 at concentration of about 10 pM. In some embodiments, the ROCK inhibitor is at a concentration that ranges from 1.0 to 2.0 pM, from 2.0 to 3.0 pM, from 3.0 to 4.0 pM, from 4.0 to 5.0 pM, from 5.0 to 6.0 pM, from 6.0 to 7.0 pM, from 7.0 to 8.0 pM, from 8.0 to 9.0 pM, from 9.0 to 10 pM, from 10 to 11 pM, from 11 to 12 pM, from 12 to 13 pM, from 13 to 14 pM, from 14 to 15 pM, from 15 to 16 pM, from 16 to 17 pM, from 17 to 18 pM, from 18 to 19 pM, from 19 to 20 pM, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the ROCK inhibitor is added to a culture medium at a concentration that ranges from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 pM, or at any concentration range any two concentrations referred to above or herein. In some embodiments, Y-27632 is at a concentration of about 10 pM.

[0174] As featured and disclosed herein, activators of the BMP pathway include, as nonlimiting examples, BMP family ligands, e.g., BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8, BMP9, BMP8b, BMP10, BMP11, and BMP 15; Alantolactone; FK506; isoliquiritigenin; and 4 ’-hydroxy chaicone. In some embodiments, activation of the BMP pathway is achieved through repression of a BMP pathway inhibitor, including, as a nonlimiting example, the use of an inhibitory nucleic acid targeting an inhibitor of the BMPpathway or an antibody or small molecule directed to a BMP pathway inhibitor. In some embodiments, the BMP pathway activator comprises BMP4. In some embodiments, the BMP pathway activator is present at a concentration of between about 0.1 ng / mL to 1 ng / mL. In some embodiments, the BMP pathway activator is at a concentration that ranges from 0.1 to 0.2 ng / mL, 0.2 to 0.3 ng / mL, 0.3 to 0.4 ng / mL, 0.4 to 0.5 ng / mL, 0.5 to 0.6 ng / mL, 0.6 to 0.7 ng / mL, 0.7 to 0.8 ng / mL, 0.8 to 0.9 ng / mL, 0.9 to 1 ng / mL, 1 to 2 ng / mL, 2 to 3 ng / mL, 3 to 4 ng / mL, 4 to 5 ng / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the BMP pathway activator is at a concentration that ranges from 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5 ng / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, BMP4 is at a concentration of about 0.5 ng / mL. In some embodiments, BMP4 is at a concentration of about 0.5 ng / mL. In some embodiments, BMP4 is added to a culture medium at a concentration of about 10 pg / mL.

[0175] In some embodiments, the BMP pathway activator is present at a concentration of between about 5 pg / mL to 15 pg / mL. In some embodiments, the BMP pathway activator is at a concentration that ranges from 1.0 to 2.0 pg / mL, from 2.0 to 3.0 pg / mL, from 3.0 to 4.0 pg / mL, from 4.0 to 5.0 pg / mL, from 5.0 to 6.0 pg / mL, from 6.0 to 7.0 pg / mL, from 7.0 to 8.0 pg / mL, from 8.0 to 9.0 pg / mL, from 9.0 to 10 pg / mL, from 10 to 11 pg / mL, from 11 to 12 pg / mL, from 12 to 13 pg / mL, from 13 to 14 pg / mL, from 14 to 15 pg / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the BMP pathway activator is added to a culture medium at a concentration that ranges from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 pg / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, BMP4 is at a concentration of about 10 pg / mL. In some embodiments, BMP4 is added to an endodermal progenitor induction medium at a concentration of about 10 pg / mL.

[0176] In some embodiments, the BMP pathway inhibitor is present at a concentration of between about 125 nM to 500 nM. In some embodiments, the BMP pathway inhibitor is at a concentration that ranges from 75 to 100 nM, 100 to 125 nM, 125 to 150 nM, 150 to 200 nM, 200 to 250 nM, 250 to 300 nM, 300 to 350 nM, 350 to 400 nM, 400 to 450 nM, 450 to 500 nM, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the BMP pathway inhibitor is at a concentration that ranges from 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500 nM, or at any concentration range any twoconcentrations referred to above or herein. In some embodiments, LDN193189 is at a concentration of about 250 nM.

[0177] As featured and disclosed herein, activators of the fibroblast growth factor (FGF) signaling pathway include, as non-limiting examples, FGF family ligands including, as nonlimiting examples, FGF1, FGF2, FGF-3, FGF-4, FGF-5, FGF-6, KGF / FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-15, FGF-16, FGF-17, FGF-19, FGF-20, FGF-21, FGF-22, and FGF-23; SUN 11602 (4-[[4-[[2-[(4-Amino-2,3,5,6-tetramethylphenyl)amino] acetyl]methylamino]-l-piperidinyl]methypenzamide); t-Butylhydroquinone; U-46619; C2 Ceramide; Lactosyl Ceramide; Angiotensin II; and Baicalin. In some embodiments, the FGF signaling pathway activator is FGF2.

[0178] In some embodiments, the concentration of FGF signaling pathway activator or inhibitor ranges from between about 1 ng / mL to 10 ng / mL. In some embodiments, the FGF signaling pathway activator is FGF2 is present at a concentration of between about 1 ng / mL to 10 ng / mL. In some embodiments, the FGF signaling pathway activator is at a concentration that ranges from 0.5 to 1 ng / mL, 1 to 2 ng / mL, 2 to 3 ng / mL, from 3 to 4 ng / mL, from 4 to 5 ng / mL, from 5 to 6 ng / mL, from 6 to 7 ng / mL, from 7 to 8 ng / mL, from 8 to 9 ng / mL, from 9 to 10 ng / mL, from 10 to 11 ng / mL, from 11 to 12 ng / mL, from 12 to 13 ng / mL, from 13 to 14 ng / mL, from 14 to 15 ng / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, FGF2 is at a concentration of about 5 ng / mL.

[0179] In some embodiments, the concentration of VEGF signaling pathway activator or inhibitor ranges from between about 5 ng / mL to 15 ng / mL. In some embodiments, the VEGF signaling pathway activator is VEGF is present at a concentration of between about 5 ng / mL to 15 ng / mL. In some embodiments, the VEGF signaling pathway activator is at a concentration that ranges from 2.5 to 3 ng / mL, from 3 to 4 ng / mL, from 4 to 5 ng / mL, from 5 to 6 ng / mL, from 6 to 7 ng / mL, from 7 to 8 ng / mL, from 8 to 9 ng / mL, from 9 to 10 ng / mL, from 10 to 11 ng / mL, from 11 to 12 ng / mL, from 12 to 13 ng / mL, from 13 to 14 ng / mL, from 14 to 15 ng / mL, from 15 to 16 ng / mL, from 16 to 17 ng / mL, from 17 to 18 ng / mL, from 18 to 19 ng / mL, from 19 to 20 ng / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the VEGF pathway activator is at a concentration that ranges from 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ng / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, VEGF is at a concentration of about 10 ng / mL.

[0180] In some embodiments, a retinoid is added to a culture medium at a concentration that ranges between about 0.5 to 5 |aM. In some embodiments, the retinoid is added to a culture medium at a concentration that ranges from 0.1 to 0.2 |jM, 0.2 to 0.3 |aM, 0.3 to 0.4 |jM, 0.4 to 0.5 |jM, 0.5 to 0.6 |jM, 0.6 to 0.7 |jM, 0.7 to 0.8 |aM, 0.8 to 0.9 |aM, 0.9 to 1 |aM, 1.0 to 2.0 |jM, 2.0 to 3.0 |jM, 3.0 to 4.0 |aM, 4.0 to 5.0 |aM, 5.0 to 6.0 |aM, 6.0 to 7.0 |aM, 7.0 to 8.0 |aM, 8.0 to 9.0 |jM, 9.0 to 10 |aM, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the retinoid is added to culture medium at a concentration that ranges from 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 |jM, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the retinoid comprises RA. In some embodiments, the glucocorticoid is RA is present at a concentration of between about 0.5 to 5 pM. In some embodiments, RA is at a concentration of about 2 pM.

[0181] In some embodiments, the concentration of MEK signaling pathway activator or inhibitor ranges from between about 250 nM to 1 pM. In some embodiments, the MEK signaling pathway inhibitor is PD0325901 is present at a concentration of between about 250 nM to 1 pM. In some embodiments, the MEK signaling pathway inhibitor is at a concentration that ranges from 250 to 300 nM, 300 to 350 nM, 350 to 400 nM, 400 to 450 nM, 450 to 500 nM, 500 to 550 nM, 550 to 600 nM, 600 to 650 nM, 650 to 700 nM, 700 to 750 nM, 750 to 800 nM, 800 to 850 nM, 850 to 900 nM, 900 to 950 nM, 950 nM to 1 pM, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the MEK signaling pathway inhibitor is at a concentration that ranges from 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 nM, 1 pM, or at any concentration range any two concentrations referred to above or herein. In some embodiments, PD0325901 is at a concentration of about 500 nM.

[0182] In some embodiments, an IL-6 family cytokine is added to a culture medium at a concentration that ranges between about 10 ng / mL to 40 ng / mL. In some embodiments, the IL- 6 family cytokine is added to a culture medium at a concentration that ranges from 1 to 10 ng / mL, 10 to 20 ng / mL, 20 to 30 ng / mL, from 30 to 40 ng / mL, from 40 to 50 ng / mL, from 50 to 60 ng / mL, from 60 to 70 ng / mL, from 70 to 80 ng / mL, from 80 to 90 ng / mL, from 90 to 100 ng / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the IL-6 family cytokine is added to a culture medium at a concentration that ranges from 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ng / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the IL-6 familycytokine comprises OSM. In some embodiments, the IL-6 family cytokine is OSM is present at a concentration of between about 10 ng / mL to 40 ng / mL. In some embodiments, OSM is at a concentration of about 20 ng / mL.

[0183] In some embodiments, HGF or an activator of HGF is added to a culture medium at a concentration that ranges between about 5 to 60 ng / mL. In some embodiments, HGF or an activator of HGF is added to a culture medium at a concentration that ranges from 0.1 to 0.5 ng / mL, 0.5 to 1 ng / mL, 1 to 2 ng / mL, 2 to 3 ng / mL, 3 to 4 ng / mL, 4 to 5 ng / mL, 5 to 6 ng / mL, 6 to 7 ng / mL, 7 to 8 ng / mL, 8 to 9 mg / mL, 9 to 10 ng / mL, 10 to 11 ng / mL, 11 to 12 ng / mL, 12 to 13 ng / mL, 13 to 14 ng / mL, 14 to 15 ng / mL, 15 to 16 ng / mL, 16 to 17 ng / mL, 17 to 18 ng / mL, 18 to 19 ng / mL, 19 to 20 ng / mL, 20 to 30 ng / mL, 30 to 40 ng / mL, 40 to 50 ng / mL, 50 to 60 ng / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, HGF or an activator of HGF is added to a culture medium at a concentration that ranges from 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 19, 20, 30, 40, 50, 60 ng / mL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the HGF activator comprises HGF. In some embodiments, the HGF activator is HGF is present at a concentration of between about 5 to 60 ng / mL. In some embodiments, HGF is at a concentration of about 20 ng / mL.

[0184] In some embodiments, a glucocorticoid is added to a culture medium at a concentration that ranges between about 50 to 200 pM. In some embodiments, the glucocorticoid is added to a culture medium at a concentration that ranges from 10 to 20 pM, 20 to 30 pM, 30 to 40 pM, 40 to 50 pM, 50 to 60 pM, 60 to 70 pM, 70 to 80 pM, 80 to 90 pM, 90 to 100 pM, 100 to 150 pM, 150 to 200 pM, 200 to 250 pM, 250 to 300 pM, 300 to 350 pM, 350 to 400 pM, 400 to 450 pM, 450 to 500 pM, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the glucocorticoid is added to a culture medium at a concentration that ranges from 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 pM, 100, 150, 200, 250, 300, 350, 400, 450, 500 pM, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the glucocorticoid comprises DEX. In some embodiments, the glucocorticoid is DEX is present at a concentration of between about 50 to 200 pM. In some embodiments, DEX is at a concentration of about 100 pM.

[0185] In one aspect the iPSC-derived hepatocytes are grown on 3D scaffold systems to provide the proper environment for hepatocyte functions. In some embodiments, the 3D hepatocyte cultures of this disclosure are used to screen clinical candidate drug and biologic compounds for toxicity and / or efficacy. The 3D hepatocyte cultures provide a morephysiologically relevant environment to promote proper hepatocyte function. In a further aspect, the 3D iPSC-derived hepatocyte cultures are used to screen drug compounds for toxicity and / or efficacy. In some embodiments, the iPSC generation and hepatocyte differentiation is performed under GMP (good manufacturing practice) conditions. In some embodiments, the iPSCs are derived from cord blood cells to enhance differentiation potential. In one aspect, the differentiated cells are further processed and formulated into the final drug product.

[0186] Media exchange is replacing one media used in a culture for new media and is also known as a “complete media change” or “complete media exchange.” As featured and described herein, the volume of a medium exchange is in a volume of between about 90% to 99%. In some embodiments, the volume exchanged is about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or any percentage of culture medium between any two percentages referred to above or herein.

[0187] Physical culture conditions include, as non-limiting examples, the gas concentration in the incubator (e.g., CO2 concentration, 02 concentration), the pH of the culture medium, the temperature, and the culture environment of the cell (e.g., adherent versus suspension culture, or in two-dimensional versus in three-dimensional culture systems). Physical culture conditions include the gas concentration in the incubator. Incubation of cell cultures is typically performed in a normal atmosphere with 15-22% oxygen and 5% CO2 for expansion and seeding. In some embodiments, the PSCs are grown in a humidified atmosphere with about 5% CO2 concentration and normoxic conditions (non-hypoxic 02 concentration). While hypoxic culture conditions are thought to support stem cell performance in general, in the present methods, the PSCs are cultured under conditions that are not hypoxic. As used herein, “normoxic” conditions refer to culture conditions including atmospheric 02 concentration (e.g., about 15-25% 02 concentration). As used herein, hypoxic conditions are characterized by a lower oxygen concentration as compared to the oxygen concentration of ambient air (approximately 15%-25% oxygen).

[0188] There are two basic systems for growing cells in culture: monolayers on an artificial substrate (i.e., adherent culture) or free-floating in the culture medium (suspension culture). The majority of the cells derived from vertebrates, with the exception of hematopoietic cell lines and a few others, are anchorage-dependent and have to be cultured on a suitable substrate that is specifically treated to allow cell adhesion and spreading (i.e., tissue-culture-treated). However, many cell lines can also be adapted for suspension culture. In some aspects, the cells are grown in a two-dimensional culture system.

[0189] Another system for growing cells in culture involves three-dimensional (3D) systems such as spheroids, layered cultures, organs-on-a-chip, or on polymer scaffolds like hydrogels or nano fiber scaffolds. Typically for a 3D culturing system, cells are seeded onto scaffolds or matrices, or embedded in a scaffold such as for example, Matrigel™. 3D cultures may be spheroid, layered cultures, organs on a chip, or cultured on polymer scaffolds, e.g., hydrogel scaffolds such as alginate hydrogels or nanofibrous hydrogels, layered cultures. In some embodiments, the iPSC-derived hepatocytes are grown on 3D scaffold systems to provide the proper environment for hepatocyte functions. In some embodiments, the 3D hepatocyte cultures of this disclosure are used to screen clinical candidate drug and biologic compounds for toxicity and / or efficacy. The 3D hepatocyte cultures provide a more physiologically relevant environment to promote proper hepatocyte function.

[0190] Solubilized basement membrane preparations include, as non-limiting examples, Matrigel™, Geltrex™, Celltrex™, reconstituted basement membrane mixtures, extracellular cellular matrices, basement membrane matrices, extracellular matrix (ECM)-based hydrogels, basement membrane or ECM mimetics thereof, and mixtures of collagen, gelatin, fibronectin, vitronectin, laminin, elastin, polymers, proteins, polypeptides, nucleic acids, sugars, lipids, poly-lysine, poly-ornithine, tenascin, heparan sulfate, entactin, nidogen, osteopontin, perlecan. In some embodiments, the solubilized basement membrane preparation includes or excludes blood components or recombinant proteins. In some embodiments, the solubilized basement membrane preparation expressly excludes blood components or recombinant proteins. In some embodiments, cells are embedded in a solubilized basement membrane preparation. In some embodiments, the solubilized basement membrane preparation is Matrigel™. In some embodiments, the Matrigel™ is a growth-factor reduced Matrigel™.

[0191] In another aspect, the iPSC-derived hepatocytes are produced using three- dimensional (3D) systems such as spheroids, layered cultures, organs-on-a-chip, or on polymer scaffolds like hydrogels or nanofiber scaffolds, or embedded in a solubilized basement membrane preparation. 3D cultures may be spheroid, layered cultures, organs on a chip, layered cultures, or cultured on polymer scaffolds, e.g., hydrogel scaffolds such as alginate hydrogels or nanofibrous hydrogels. In some embodiments, the solubilized basement membrane preparation comprises a hydrogel, which is typically hydrophilic polymer networks filled with water. Hydrogels have the advantage of selectively triggering polymer swelling. Depending on the composition of the polymer network, swelling of the microparticle may be triggered by a variety of stimuli, including pH, ionic strength, thermal, electrical, ultrasound, and enzymeactivities. Non-limiting examples of polymers useful in hydrogel compositions include, among others, those formed from polymers of poly (lactide-co-glycolide); poly (N- isopropylacrylamide); poly (methacrylic acid-g-poly ethylene glycol); poly aery lie acid and poly (oxypropylene-co-oxyethylene) glycol; and natural compounds such as chrondroitan sulfate, chitosan, gelatin, fibrinogen, or mixtures of synthetic and natural polymers, for example, chitosan-poly (ethylene oxide). The polymers may be cross-linked reversibly or irreversibly to form gels adaptable for forming three-dimensional tissues. In certain aspects, the cells are embedded in a solubilized basement membrane preparation such as Matrigel™. In some embodiments, the solubilized basement membrane preparation includes or excludes blood components or recombinant proteins. In some embodiments, the solubilized basement membrane preparation expressly excludes blood components or recombinant proteins.

[0192] As featured and disclosed herein, single cells, dissociated cells, or serially diluted cells are embedded in a solubilized membrane preparation. In some embodiments, the concentration of cells ranges from between about 100 to 1000 cells / pL. In some embodiments, the concentration of cells are embedded in a solubilized membrane preparation at a concentration that ranges from 1 to 100 cells / pL, 100 to 200 cells / pL, 200 to 300 cells / pL, from 300 to 400 cells / pL, from 400 to 500 cells / pL, from 500 to 600 cells / pL, from 600 to 700 cells / pL, from 700 to 800 cells / pL, from 800 to 900 cells / pL, from 900 to 1000 cells / pL, or at any concentration range any two concentrations referred to above or herein. In some embodiments, the concentration of cells is about 500 cells / pL.

[0193] In some aspects, hepatocyte function is determined by secretion, production, and / or expression of any of the mature hepatocyte markers featured herein. In some embodiments, the hepatocytes express maturation markers after about 8-12 days differentiation. In certain aspects, the hepatocytes express maturation markers after about 1 week of differentiation. In some certain, the hepatocytes express maturation markers after about 2 weeks of differentiation.

[0194] As featured and disclosed herein, hepatocyte maturation markers include, as nonlimiting examples, al-antitrypsin (AAT), albumin, HNF4a, UGT2B7, transthyretin (TTR), LIN28A, UGT2B7, and urea. In some embodiments, the maturation markers comprise AAT, albumin, and / or TTR.

[0195] In some aspects, the iPSC-derived hepatocyte population has high purity. In one aspect the highly pure hepatocyte population comprises at least 80%, 85%, 90%, 95%, or 99% hepatocyte marker positive cells (e.g., albumin-positive, AAT-positive cells), as determined byimmuno staining or flow cytometry. In some aspects, the highly pure hepatocyte population comprises at least 90%, 95%, or 99% hepatocyte marker positive cells (e.g., albumin-positive, AAT-positive cells), as determined by immunostaining or flow cytometry. In some aspects, the iPSC-derived hepatocyte population is at least 80%, 85%, 90%, 95%, or 99% positive for at least one hepatocyte marker selected from: HNF4a, UGT2B7, TTR, AAT, and albumin.

[0196] In some aspects, changes in marker expression or production levels are indicative of the differentiation stages of iPSCs, PGECs, 2D hepatocytes, and 3D hepatocytes. In some aspects, protein expression levels include albumin, LIN28A, HNF4A, UGT2B7, TTR, AAT, UGT2B7, and urea. In an aspect, iPSCs are characterized by high expression of LIN28A, low expression of HNF4A, and minimal expression of UGT2B7, TTR, AAT, and albumin. In one aspect, PGECs exhibit reduced expression of LIN28A compared to iPSCs, a slight increase in HNF4A expression, and minimal expression of UGT2B7, TTR, AAT, and albumin. In an aspect, 2D hepatocytes demonstrate reduction in LIN28A expression, increased HNF4A expression compared to PGECs, high expression of UGT2B7, and increased expression of TTR, AAT, and albumin compared to PGECs. In one aspect, 3D hepatocytes exhibit the lowest expression of LIN28A among all cell types, the highest expression of HNF4A, TTR, AAT, and albumin, and reduced expression of UGT2B7 compared to 2D hepatocytes.

[0197] Stem cells are undifferentiated cells that have the ability to self-renew indefinitely and to remain in said undifferentiated state. In contrast to embryonic stem cells, which can only be isolated from the inner mass of a blastocyst, there are three known accessible sources of adult stem cells: the bone marrow, which requires the drilling of a bone; the adipose tissue, which is accessible by liposuction; and the blood, from which the cells can be extracted among other cells. The term “pluripotent stem cells,” as used herein, refers to cells that are capable of generating all the cell types of an organism, i.e., cells derived from any of the three germ layers. On the other hand, multipotent stem cells can differentiate into several cell types, but only those of a closely related family of cells, generally the cell types of the organ from which they originate. Most adult stem cells are multipotent, but small amounts of pluripotent adult stem cells can be retrieved from the umbilical cord or other tissues. The sources of cells used for cell therapy include stem cells such as embryonic stem cells (ESCs), adult stem cells, and induced pluripotent stem cells (iPSCs). In some embodiments, the PSCs used in the methods described herein are human (hPSCs), and in some embodiments, the human PSCs are induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs). In some embodiments, the PSCsare human PSCs (hPSCs). In some embodiments, the hPSCs are human induced pluripotent stem cells (hiPSCs) or human embryonic stems cells (hESCs).

[0198] Unless otherwise specified, pluripotent stem cells are maintained in a stem cell medium suitable for the culture and propagation of pluripotent stem cells. Non-limiting examples of such suitable media include, as a non-limiting example, StemMACS iPS-Brew XF.

[0199] As used herein, the term “basal medium” generally refers to a base medium without any additives added (for example, a basal medium refers to a medium as commercially available). Those generally include water, nutrients, salts, and amino acids, but no additives or supplements. In some embodiments, the basal medium is a chemically defined medium including, as non-limiting examples, RPMI, DMEM, and DMEM-F12. In some embodiments, the culture medium is DMEM-F12, RPMI, serum free differentiation media (SFD), hepatocyte culturing media (HCM), Knockout-DMEM, IMDM, or StemMACS iPS-Brew XF. In some embodiments, the culture medium is DMEM-F12. In some embodiments, the culture medium is RPMI. In some embodiments, the culture medium is serum free differentiation media (SFD).

[0200] A basal medium can be supplemented with general additives to obtain a “supplemented basal medium.” In some embodiments, the culture medium is a preparation of DMEM-F12, RPMI, serum free differentiation media (SFD), hepatocyte culturing media (HCM), Knockout-DMEM, IMDM, StemMACS iPS-Brew XF, or related base medium supplemented with growth factors, accessory factors, cytokines, activators, or inhibitors. In some embodiments, the culture medium is a basal medium, supplemented basal medium, defined medium, final medium, complete medium, or cell-specific medium. Non-limiting examples of supplements include, as non-limiting examples, insulin or ascorbate. A basal medium can also be completed with specific signaling molecules, such as those identified by a user as necessary to achieve a particular goal with the cell culture, such as, for example, driving the differentiation of a cell type of interest into a target cell type. A non-limiting example of a basal medium supplemented with a specific signaling molecule includes a ROCK inhibitor medium containing Y-27632. Such a complete basal medium can be referred to as a “final,” “complete,” or “cell- specific” medium. Such supplemented basal medium used for endodermal progenitor generation, e.g., generating gut endodermal progenitors or hepatic endoderm cells, uses small molecules of interest as needed. Similarly, such supplemented basal medium used for hepatocyte maturation for hepatocyte generation uses small molecules of interest as needed. For hepatocyte maturation, the endodermal progenitor cells are switched to SFD.

[0201] Chemical culture conditions include, as non-limiting examples, the agents or molecules that are added to the culture medium to achieve the desired effects sought after (i.e., differentiation of PSCs into hepatocytes). The terms “agent” and “molecule” are used interchangeably and include, as non-limiting examples, small molecules (including small molecules that do not have optimal cell permeability), lipids, nucleosides, nucleotides, nucleic acids, polynucleotides, oligonucleotides, antibodies, toxins, negatively charged polymers, and other polymers, for example proteins, peptides, hormones, carbohydrates, or polyamines.

[0202] In some embodiments, the culture media comprises a mixture of agents. In some embodiments, the mixture of agents includes or excludes antibiotics, such as, for example doxycycline and penicillin-streptomycin. In some embodiments, the mixture of agents expressly excludes antibiotics that are used to prevent apoptosis including, as a non-limiting example, doxycycline. In some embodiments, the culture media is supplemented with B27. For example, the mixture of agents includes a TGF-P activator and a Wnt activator, including or excluding a ROCK inhibitor. In another example, the mixture of agents includes a TGF-P activator, BMP activator, FGF activator, and VEGF activator. In another example, the mixture of agents includes TGF-P activator, BMP activator, FGF activator, VEGF activator, and ROCK inhibitor. In another example, the mixture of agents includes a retinoid, Wnt inhibitor, BMP inhibitor, and MEK inhibitor. In another example, the mixture of agents includes a retinoid, Wnt inhibitor, BMP activator, and TGF-P inhibitor. In yet another example, the mixture of agents includes hepatocyte growth factor (HGF), an IL-6 family cytokine, and a glucocorticoid in a medium including or excluding EGF.

[0203] In some aspects, one media is exchanged for another media as a complete media exchange. As featured and described herein, the volume of a medium exchange is in a volume of between about 90% to 99%. In some embodiments, the volume exchanged is about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or any percentage of culture medium between any two percentages referred to above or herein.

[0204] In certain aspects, iPSCs are first differentiated into endoderm progenitor cells using a specific protocol and timeline. In some aspects, the differentiation involves culturing the iPSCs with various factors such as Activin A, Wnt3a, BMP4, FGF2, and VEGF at defined timepoints. In certain embodiments, the resulting endoderm progenitors can be passaged and expanded multiple times. In certain embodiments, the resulting endoderm progenitors are not passaged. In certain embodiments, the resulting endoderm progenitors are not expanded, canbe passaged and expanded multiple times. In certain aspects, the progenitors are cultured in media with OSM, HGF and dexamethasone to induce hepatocyte differentiation.

[0205] In some aspects, iPSCs are seeded at an appropriate density on substrates and in media that promote loss of pluripotency and to initiate differentiation. In certain aspects, mesendoderm specification is stimulated by factors like Activin A and Wnt3a. In some aspects, multiple transfection doses are given over several days to act synergistically with the media conditions and to generate a highly pure endoderm population.

[0206] In some aspects, the endoderm progenitors are differentiated into hepatic endoderm cells by culturing to confluence and exposing to a specified hepatic endoderm medium. In other aspects, the differentiation into immature hepatocytes is achieved through additional media changes. In certain aspects, the immature hepatocytes are matured into functional hepatocytes by culturing in a defined maturation medium. In some aspects, the endoderm cells are cultured in media containing BMP4 and FGF2 to promote hepatic lineage specification.

[0207] In some aspects, the iPSC-derived hepatocytes are cultured in 3D systems to better mimic the native liver environment and promote hepatocyte function. In certain aspects, the 3D cultures can take the form of spheroids, organoids, layered cultures, organs-on-a-chip, or cells grown on polymer scaffolds such as hydrogels or nanofiber matrices. In certain aspects, iPSC generation and hepatocyte differentiation are performed under GMP conditions for clinical applications.

[0208] The 3D iPSC-derived hepatocyte cultures have applications in drug screening assays to assess toxicity and efficacy of compounds. In one aspect of the methods featured herein, the entire process from iPSC generation to hepatocyte differentiation is carried out under GMP conditions for increased clinical relevance. In one aspect, the use of cord blood- derived iPSCs may provide enhanced differentiation potential in some aspects.

[0209] Certain Exemplary Definitions

[0210] By ‘ ‘contacting,” “exposing”, or “culturing,” it is meant that the cells are cultured with one or more agents of interest, added to the defined basal or supplemented medium. That is, the cells are cultured in their regular culture basal or supplemented medium, in which a desired concentration of one or more agents of interest is added.

[0211] A “signaling pathway activator,” as used herein, refers to any molecule that is capable of activating, enhancing, or inducing a signaling pathway of interest. A signaling pathway is a series of chemical reactions in which a group of molecules in a cell work together to control a cell function, such as cell differentiation. A cell receives signals from itsenvironment when a molecule, such as a hormone or growth factor, binds to a specific protein receptor on or in the cell. After the first molecule in the pathway receives a signal, it activates another molecule. This process is repeated through the entire signaling pathway until the last molecule is activated and cell function is carried out. Abnormal activation of signaling pathways or inhibition of a signaling pathway may lead to diseases or, in the case of pluripotent cells, to an alteration of the pluripotent state and therefore to differentiation. The term “molecule” includes, as non-limiting examples, small molecules (including small molecules that do not have optimal cell permeability), lipids, nucleosides, nucleotides, nucleic acids, polynucleotides, oligonucleotides, antibodies, toxins, negatively charged polymers, and other polymers, for example proteins, peptides, hormones, carbohydrates, or polyamines. Nonlimiting examples of polynucleotides include short interfering nucleic acid (siNA), antisense, enzymatic nucleic acid molecules, 2’, 5 ’-oligoadenylate, triplex forming oligonucleotides, aptamers, and decoys. Biologically active molecules include antibodies (e.g., monoclonal, chimeric, humanized, etc.), cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, allozymes, aptamers, decoys, and analogs thereof, and small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double- stranded RNA (dsRNA), micro-RNA (miRNA), antagomirs, and short hairpin RNA (shRNA) molecules.

[0212] ‘ ‘Induced pluripotent stem cells (iPSCs)” refer to pluripotent stem cells generated from adult somatic cells that have been reprogrammed back into an embryonic-like pluripotent state.

[0213] ‘ ‘Endoderm progenitors” are multipotent cells derived from the endodermal germ layer that can give rise to various endoderm-derived tissues, such as liver, pancreas, lung, etc. Examples of endoderm progenitors include definitive endoderm cells, gut endodermal progenitor cells, and hepatic endoderm cells.

[0214] ‘ ‘Definitive Endodermal (DE) Cells” are early precursor cells that give rise to the epithelial lining of the gut and associated organs.

[0215] “Gut Endodermal Progenitor (GEP) Cells” or “Gut Endoderm Cells (GEC)” are an intermediate precursor cells more differentiated than DE cells and not yet committed to hepatic specification.

[0216] “Hepatic Endoderm (HE) Cells” are the earliest precursors in liver development, located in the anterior part of the definitive endoderm, receiving signals from mesodermal tissues to initiate hepatic specification. The HE cells differ from both hepatic progenitor cells (which are bipotential cells that can differentiate into both hepatocytes and biliary epithelial cells) and immature hepatocyte-like cells, which are cells that are differentiated towards a hepatocyte fate but do not fully possess mature hepatocyte characteristics.

[0217] “Hepatocytes” are the main parenchymal cells of the liver that carry out the majority of liver functions, including protein synthesis, detoxification, metabolism, and more.

[0218] “Progenitor cell” as used herein includes liver cells, as well as cells that have attributes and characteristics of liver cells, such as the expression of markers associated with liver progenitor cells.

[0219] “Pluripotent stem cells (PSCs)” as used herein refer to stem cells with the ability to differentiate into many cell types, and includes both embryonic stem cells (ESCs) and iPSCs.

[0220] The present invention is also directed to kits for the induction, propagation and / or isolation of liver progenitor cells. The present invention is also directed toward kits for the transplantation of liver progenitor cells to liver damaged models. Each kit is an assemblage of materials or components. The exact nature of the components configured in each inventive kit depends on its intended purpose. For example, some embodiments are configured for the purpose of inducing and / or propagating hepatocyte precursors. In some embodiments, instructions for use are included in the kit. In some embodiments, instructions for use are not included in the kit. “Instructions for use” typically include a tangible expression describing the technique to be employed in using the components of the kit for a desired purpose, such as for induction, propagation, and / or isolation of liver progenitor cells.

[0221] Optionally, the kits also contain other useful components, such as those described herein, or buffers (e.g., PBS), growth media, tissue culture plates, multiple well plates, flasks, chamber slides, differentiation media, stem cell media, goat serum, fetal bovine serum, basic fibroblast growth factor, epidermal growth factor, diluents, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, or other useful paraphernalia as will be readily recognized by those of skill in the art.

[0222] The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. In some aspects, the components are in dissolved, dehydrated, or lyophilized forms. In some aspects,the components are provided at room, refrigerated, or frozen temperatures. The components are typically contained in suitable packaging material(s).

[0223] In some embodiments a culture medium can comprise one or more of the reagents exemplified in Table 1. In some embodiments, a culture medium comprises one or more of the reagents at any concentration featured in this disclosure, e.g., a concentration within the recited range of concentration, e.g., as a non-limiting example, at the final exemplary concentration.

[0224] Table 1. Reagent concentration ranges.

[0225] In some embodiments, iPSCs are cultured in a culture medium comprising a TGF- P activator, Wnt activator, and ROCK inhibitor. In some embodiments, iPSCs are cultured in a culture medium comprising a TGF-P activator, BMP activator, FGF activator, and VEGF activator. In some embodiments, iPSCs are cultured in a definitive endoderm medium comprising TGF-P activator, BMP activator, FGF activator, and VEGF activator. Exemplary concentrations for the iPSC culture media and DE medium are described in Table 2. In some embodiments, a culture medium comprises one or more of the reagents at any concentration featured in this disclosure, e.g., a concentration within the recited range of concentration, e.g., as a non-limiting example, at the final exemplary concentration.

[0226] Table 2. Exemplary reagents used in culture media to induce definitive endoderm differentiation.

[0227] In some embodiments, a gut endodermal progenitor medium comprises a TGF-P activator, BMP activator, FGF activator, VEGF activator, and ROCK inhibitor. In some embodiments, DE cells are cultured in a gut endodermal progenitor medium comprising a TGF- P activator, BMP activator, FGF activator, VEGF activator, and ROCK inhibitor. Exemplary concentrations for the GEP medium are described in Table 3. In some embodiments, a culture medium comprises one or more of the reagents at any concentration featured in this disclosure, e.g., a concentration within the recited range of concentration, e.g., as a non-limiting example, at the final exemplary concentration.

[0228] Table 3. Exemplary reagents used in culture media to induce gut endodermal progenitor differentiation.

[0229] In some embodiments, a hepatic endoderm medium comprises a retinoid, Wnt inhibitor, BMP inhibitor, and MEK inhibitor. In some embodiments, a hepatic endoderm medium comprises a retinoid, a Wnt inhibitor, BMP activator, and TGF-P inhibitor. In some embodiments, GEP cells are cultured in a first hepatic endoderm medium comprising a retinoid, Wnt inhibitor, BMP inhibitor, and MEK inhibitor, then the first hepatic endoderm medium is exchanged for a second hepatic endoderm medium comprising a retinoid, a Wnt inhibitor, BMP activator, and TGF-P inhibitor. Exemplary concentrations for the HE media are described in Table 4. In some embodiments, a culture medium comprises one or more of the reagents at any concentration featured in this disclosure, e.g., a concentration within the recited range of concentration, e.g., as a non-limiting example, at the final exemplary concentration.

[0230] Table 4. Exemplary reagents used in culture media to induce hepatic endoderm differentiation.

[0231] In some embodiments, a hepatocyte maturation medium comprises an IL-6 family cytokine, HGF, and glucocorticoid. In some embodiments, a hepatocyte maturation medium does not comprise EGF. In some embodiments, HE cells are cultured in a hepatocyte maturation medium comprising an IL-6 family cytokine, HGF, and glucocorticoid, and does not comprise EGF. Exemplary concentrations for the hepatocyte maturation medium are described in Table 5. In some embodiments, a culture medium comprises one or more of the reagents at any concentration featured in this disclosure, e.g., a concentration within the recited range of concentration, e.g., as a non-limiting example, at the final exemplary concentration.

[0232] Table 5. Exemplary reagents used in culture media to induce hepatic endoderm differentiation.

[0233] The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way.EXAMPLES

[0234] The invention is further illustrated by the following non-limiting examples:

[0235] Example 1: Generation of endoderm progenitors from iPSCs

[0236] iPSCs were differentiated into endoderm progenitors (PGECs) following Method 1 1, Step A. The expression of the pluripotency marker LIN28a increased during the differentiation process from iPSCs to PGECs from iPSCs to PGECs and decreased from PGECs to iHeps (FIG. 2), indicating a loss of pluripotency as the cells differentiated towards the mature hepatic lineage.

[0237] Method 1:

[0238] A. Initiation Endoderm Progenitors from Human Induced Pluripotent Stem Cells

[0239] The following steps (1-11) provide an exemplary protocol starting from preparing iPSCs for endoderm differentiation.1. Prepare iPSC colonies (3~4 days after passage) in 100mm dish. Full 100mm dish will serve 5-7 xl06 cells.2. Rinse the 100-mm dish with 5 mL sterile Ca+ / Mg+-free PBS and discard.3. Add 1 mL Accutase to the dish; incubate at 37 °C and 5% CO2 for 4 ~5 min.4. If the cells are lifting away and floating from the bottom of the dish, neutralize and harvest cells with 9 mL of fresh pure DMEM-F12 without cytokines, extract 50pl for cell count.5. Spin 90g, 5min.6. Aspirate the supernatant and re-suspend the cell pellet with RPMI with final lOOng / mL of Activin A, 50ng / mL Wnt3a + final 10 pM Rock Inhibitor, target cell concentration = 0.35-0.4 xl06 cells / mL.7. Aspirate the DMEM-F12 from the Matrigel™ (growth-factor reduced)-coated 6-well plate, and transfer 2mL of the above cell suspension. (=0.7-0.8 xl06 cells / well), as dayO.8. Incubate at 37 °C and 5% CO2 overnight.9. Next day (Day 1 post plating), aspirate the medium, replace with RPMI, with final lOOng / mL of Activin A + 50ng / mL Wnt3a.10. Day2, aspirate the medium. Replace with RPMI, with final lOOng / mL of Activin A + 0.5ng / mL BMP4 +5ng / mL FGF2 + lOng / mL VEGF x 2 day, daily medium change.11. Day4, aspirate the media, incubate with final lOOng / mL of Activin A + 0.5ng / mL BMP4 +5ng / mL FGF2 + lOng / mL VEGF x for 2 days with daily medium to get the endoderm progenitors.

[0240] B. Expansion and. Maintenance of Endoderm Progenitors

[0241] The following steps (1 and 2) provide an exemplary protocol for expanding and maintaining endoderm progenitors.1. Passage P01.1. Prepare the induced endoderm progenitor cells in 60mm dish. Full 60mm dish will serve 3-4 xlO6cells.1.2. Rinse the 600-mm dish with 3 mL sterile Ca+ / Mg+-free PBS and discard.1.3. Add 1 mL Accutase to the dish; incubate at 37°C and 5% CO2 for 4 -5 min.1.4. If the cells are lifting away and floating from the bottom of the dish, neutralize and harvest cells with 9 mL of fresh pure DMEM-F12 without cytokines, extract 50 pl forcell count.1.5. Spin 80~90g, 5min.1.6. Aspirate the supernatant and re-suspend the cell pellet with final 10|iM Rock Inhibitor, target cell concentration = 25 xlO4cells / mL.1.7. Aspirate the DMEM-F12 from the Matrigel™ (growth-factor reduced) (1:30 dilution)-coated 6-well plate, and transfer 2mL of the above cell suspension. (=5 xlO5cells / well)1.8. Incubate at 37°C and 5% CO2 overnight.1.9. Next day (Day 1 post plating), aspirate the medium, wash with sterile PBS and remove the PBS. Detach the cells for later passage or cell stock. Those cells were named as EP-PO.2. Passage more than Pl2.1. Prepare EP-PO or EP (passage > 0) cell, dilute the cells with final 10qM Rock Inhibitor, target cell concentration = 5 xlO4cells / mL.2.2. Aspirate the DMEM-F12 from the Matrigel™ (growth-factor reduced) (1:30 dilution)-coated 6-well plate, and transfer 2mL of the above cell suspension. (=5 xlO5cells / well)2.3. Incubate at 37°C and 5% CO2 overnight.2.4. Next day (Day 1 post plating), aspirate the medium, Replace with EPM for cell expansion, change medium every 2 days until cell confluent.2.5. For long-term expansion and maintenance, the endoderm progenitors, repeat the steps.

[0242] C. Hepatic Endoderm Induction

[0243] After the cultured endoderm progenitors got confluent, remove the media, wash with sterile PBS and remove, change media to specified hepatic endoderm differentiation media (A) described in Table 1 for 1 day. Subsequent media changes from media A to media B, then to the immature hepatocytes differentiation media, and finally to the mature hepatocytes maturation media are performed as full media exchanges and after at least one day of culturing in each respective media. Hepatocyte maturation can be performed using the maturehepatocytes maturation media over at least one week, or about 2 weeks, or more than 2 weeks with media exchanges for fresh mature hepatocytes maturation media every 3 to 4 days.

[0244] The media used at each stage of Method 1 is described below in Tables 6-9.

[0245] Table 6. Hepatic Endoderm differentiation media (A)

[0246] Table 7. Hepatic Endoderm differentiation media (B)

[0247] Table 8. Immature Hepatocytes differentiation media

[0248] Table 9. Mature Hepatocytes differentiation media

[0249] Example 2: Differentiation of endoderm progenitors into hepatocytes.

[0250] The endoderm progenitors (PGECs) generated in Example 1 were further differentiated into hepatocytes (iHeps) using a multi-step protocol involving the generation ofhepatic endoderm cells and immature hepatocytes, followed by maturation into fully functional hepatocytes as outlined in Method 1, Steps B and C.

[0251] The iHeps were cultured in both 2D and 3D conditions, and their morphology and gene expression were compared. In 2D culture, the iHeps exhibited a flattened, polygonal morphology characteristic of hepatocytes grown on a two-dimensional surface (FIG. 1, left). In contrast, 3D culture resulted in the formation of compact, spheroid-like structures more closely resembling the organization of hepatocytes in vivo (FIG. 1, right).

[0252] Gene expression analysis of the expression of liver- specific markers, including HNF4a, UGT2B7, transthyretin (TTR), al-antitrypsin (AAT), and albumin is shown in FIG. 2. The top row of FIG. 2 shows the expression of three liver- specific genes: LIN28a, HNF4a, and UGT2B7. LIN28a expression is highest in PGECs and decreases after differentiation into iHeps, with the lowest expression in 3D iHeps. HNF4a expression is low in iPSCs compared to PGECs, and similarly HNF4a expression is low in 2D iHeps increases in 3D iHeps. UGT2B7 expression is very low in iPSCs and PGECs and increases drastically in 2D iHeps and 3D iHeps. The top row of FIG. 2 shows expression of transthyretin (TTR), al-antitrypsin (AAT), and albumin. TTR expression is lowest in iPSCs, increases slightly in PGECs and 2D iHeps, and substantially increases in 3D iHeps. AAT expression follows a similar pattern, with low levels in iPSCs, PGECs, and 2D iHeps, with a 3D iHeps showing the highest expression. Albumin expression is also lowest in iPSCs and PGECs, with an increase in 2D iHeps and highest expression in 3D iHeps. Notably, 3D iHeps showed the highest expression levels of UGT2B7, TTR, AAT, and Albumin, suggesting that the 3D culture system promotes a more mature hepatocyte phenotype compared to 2D culture.Example 3: Drug screening using 3D iPSC-derived hepatocyte cultures.

[0253] 3D cultures of iPSC-derived hepatocytes (3D iHeps) were exposed to various compounds to test for hepatotoxicity and effects on CYP450 metabolic activity. Compared to primary hepatocytes or hepatic cell lines, the 3D iHeps exhibited higher sensitivity in detecting drug-induced toxicity and more accurately predicted effects on liver enzyme function. The enhanced functionality of 3D iHeps can be attributed to their more physiologically relevant organization, as demonstrated by the formation of spheroid-like structures (FIG. 1), and increased expression of key hepatocyte markers (FIG. 2). These results highlight the utility of the iPSC-derived hepatocyte 3D culture platform for preclinical drug development and safety assessment.

[0254] Example 4: Improved Method for generating iPSC-derived hepatocytes and liver organoids

[0255] Method 2 advantageously streamlines Method 1 presented in Examples 1-3 by condensing the stages of endoderm differentiation and simplifying the media used for each stage of differentiation. Immature hepatocytes are not generated in the improved Method 2, but rather a hepatic lineage endoderm (hepatic endoderm (HE) cells) differentiated from definitive endoderm cells are generated. An intermediate endodermal cell type, gut endodermal cells (also referred to as gut endodermal progenitor cells; GEC or GEP cells) are induced from DE and are then cultured in a stepwise order using targeted induction culture media (also referred to as induction cocktails) to produce HE cells. The induction cocktails used specifically tune the activation or inhibition of critical pathways to hepatic differentiation including the TGF-P, Wnt, BMP, retinoid acid signaling pathways. The HE cells are then matured directly, using a hepatocyte maturation media (Media C) described in Table 7 below. Further, the use of serum is eliminated in, for example, Media C, removing excess growth factors not necessary for producing high purity mature hepatocytes and 3D liver organoids.

[0256] Notably, Method 2 also advantageously uses a solubilized basement membrane preparation (e.g., Matrigel™) for each stage: for the induction of DE, GEP cells, and HE cells, mimicking the scaffolding and microenvironment that hepatic cells are matured in, resulting in improved morphology, mature hepatocyte marker positivity, and hepatocyte functionality (FIG. 7 and FIGs. 8A-8C). Altogether, the aforementioned improvements streamline the differentiation of PSCs into high purity hepatocytes and 3D liver organoids which can be used for both diagnostic (e.g. drug toxicity screening) and therapeutic uses.

[0257] Method 2:

[0258] The following steps (1-29) provide an exemplary protocol starting from preparing iPSCs for endoderm differentiation.

[0259] Induction of Definitive Endoderm (DE) cells from Human iPSCs [5 days]1. DayO, prepare iPSC colonies (3~4 days after passage) in 100mm dish.2. Rinse the 100-mm dish with 5 mL sterile Ca+ / Mg+-free PBS and discard.3. Add 1 mL Accutase to the dish; incubate at 37 °C and 5% CO2 for 4 ~5 min.4. If the cells are lifting away and floating from the bottom of the dish, neutralize and harvest cells with 9 mL of fresh pure DMEM-F12 without cytokines, extract 50 pl for cell count.5. Spin 90g, 5min.6. Aspirate the supernatant and re-suspend the cell pellet with RPMI (1% B27), with final lOOng / mL of Activin A, 50ng / mL Wnt3a + final 10 pM Rock Inhibitor, target cell concentration = 0.35-0.4 xlO6cells / mL.7. Aspirate the DMEM-F12 from the Matrigel™ (growth-factor reduced)-coated 6-well plate, and transfer 2mL of the above cell suspension. (=0.7-0.8 xlO6cells / well), as dayO.8. Incubate at 37°C and 5% CO2.9. Day 1, aspirate the medium, replace with RPMI, with final lOOng / mL of Activin A + 50ng / mL Wnt3a.10. Day2-day3, aspirate the medium. Replace with RPMI, with final 100 ng / mL of Activin A + 0.5ng / mL BMP4 +5ng / mL FGF2 + lOng / mL VEGF x 2 days.11. Day4-day5, aspirate the media, incubate with a mixture of SFD, with final lOOng / mL of Activin A + 0.5ng / mL BMP4 +5ng / mL FGF2 + lOng / mL VEGF x for 2 days to get the definitive endoderm cells.

[0260] Induction of Gut Endodermal Progenitor (GEP) cells [1 day]12. Day 6, prepare the induced definitive endoderm cells in 60mm dish. Full 60mm dish will serve 3-4 xl06 cells.13. Rinse the 600-mm dish with 3 mL sterile Ca+ / Mg+-free PBS and discard.14. Add 1 mL Trypsin / EDTA to the dish; incubate at 37°C and 5% CO2 for 4 -5 min.15. If the cells are lifting away and floating from the bottom of the dish, neutralize and harvest cells with 9 mL of fresh pure DMEM-F12 without cytokines, extract 50 pl for cell count.16. Spin 80~90g, 5min.17. Aspirate the supernatant and re- suspend the cell pellet with GEP medium + final 10 pM Rock Inhibitor, target cell concentration = 25 xl04 cells / mL.18. Aspirate the DMEM-F12 from the Matrigel™ (growth-factor reduced) (1:30 dilution)- coated 6-well plate, and transfer 2mL of the above cell suspension. (=5 xl05 cells / well)19. Incubate at 37 °C and 5% CO2 overnight.20. Day7, aspirate the medium, wash with sterile PBS and remove the PBS. Detach the cellsfor later cell embedding or cell stock. Those cells were named gut endodermal progenitor cells (GEP).

[0261] Hepatic Endoderm (HE) Induction [4 days]21. Day 7, detach the cells from above step, centrifuge for 3min at 300g, at RT.22. Completely aspirate the supernatant from the cell pellet.23. Using a P1000 pipet, add 1000 pL of Matrigel™ to the cell pellet in 15mL conical tube from one well of a six well plate and gently and slowly pipet.24. To each well of a 6 well plate, drop 50 pL of the cell / cluster suspension to the well, forming a dome-shape, plate six drops in one well of a six well plate.25. Gently place the plate in a 37 °C incubator for 5min to solidify the Matrigel™.26. Add 4 mL of Medium A to each well of a 6 well plate and place back in the incubator.27. Day 9-day 10, gently aspirate the Medium A from each well, taking care to not disturb the Matrigel™ drops and adding 4 mL fresh Medium B to each well.

[0262] Mature Hepatocytes Induction [8-12 days]28. Day 11, aspirate Medium B and add 4 mL of Medium C to each well of a 6 well plate.29. Every 3-4 days the media exchange by aspirating the media in the well and adding 4 mL fresh Medium C.

[0263] The media used at each stage of Method 2 is described below in Tables 10-12.

[0264] Table 10. Hepatic Endoderm differentiation media (Medium A)

[0265] Table 11. Hepatic Endoderm differentiation media (Medium B)

[0266] Table 12. Hepatocyte Maturation media (Medium C)

[0267] Example 5: Methods for cry opreserving endodermal progenitors and iPSC- derived hepatocytes and liver organoids

[0268] As part of Method 2, cryopreservation of endodermal progenitors including DE, GEP, HE, and mature hepatocytes and liver organoids was also improved upon. Unlike other methods which utilize a ROCK inhibitor, an effective cryopreservation medium was developed without a ROCK inhibitor, using a combination of DMSO, emricasan, polyamine, and trans- ISRIB (CEPT).

[0269] Method 3:

[0270] A. If cryopreserving endodermal progenitors or non-organoid mature hepatocytes:1. Dissociate cells using an appropriate reagent (e.g., Accutase, Trypsin, TryplE, or similar).2. Neutralize and harvest cells using a media without cytokines and pellet the cells (for example, spin 80-90g for 5 min). Optionally wash the pellet in Ca+ / Mg+-free PBS or media and pellet the cells.3. Aspirate the supernatant and re-suspend the cell pellet in cryopreservation media (DMSO, emricasan, polyamine, and trans-ISRIB (CEPT)) and transfer to -80C freezer. Optionally transfer the frozen cells from the -80C freezer after about 24 hours to liquid nitrogen for longer term storage.

[0271] B. If cryopreserving 3D liver organoids:1. Centrifuge the plate of solubilized basement membrane domes at 1100 rpm for 5 minutes.2. Carefully aspirate the solubilized basement membrane from the organoid pellet.3. Wash organoids with media or PBS. Centrifuge plate and aspirate the wash from the organoid pellet.4. Add cryopreservation media (DMSO, emricasan, polyamine, and trans-ISRIB (CEPT)) and transfer to -80C freezer. Optionally transfer the suspended organoids (i.e., suspended in cry opreservation media) to a cryofreezing tubes then transfer the organoids to the -80Cfreezer, and after about 24 hours, optionally transfer the organoids to liquid nitrogen for longer term storage.

[0272] Example 6: Methods for recovering cryopreserved endodermal progenitors and iPSC-derived hepatocytes and liver organoids

[0273] Method 4 below is an exemplary method for thawing cryopreserved endodermal progenitors including DE, GEP, HE, and mature hepatocytes and liver organoids.

[0274] Method 4:1. Transfer liquid nitrogen-retrieved tubes or -80C-frozen tubes onto ice.2. Quickly thaw tubes in a 37C water bath then spin down the cells or organoids (for example, 80-90g for 5 min, or 1 lOOrpm for 5 min) and gently aspirate the medium.3. Resuspend the cells in an appropriate medium that is cold; for example, if thawing GEP cells, resuspend the cells in GEP media. If thawing organoids, gently resuspend the organoids in hepatocyte maturation medium. Optionally add cryopreservation media (DMSO, emricasan, polyamine, and trans-ISRIB (CEPT)) to the resuspension media.4. If cyropreservation media was added to the resuspension media incubate the cells or organoids for about 24 hours, then exchange the media for fresh media excluding cry opreservation reagents.* * *

[0275] All documents cited or referenced herein (“herein cited documents”), together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0276] Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

Claims

CLAIMSWhat is claimed is:

1. A method of producing induced pluripotent stem cell (iPSC)-derived hepatocytes comprising:(a) producing definitive endoderm (DE) cells from iPSCs by contacting iPSCs with a first culture medium comprising a TGF-P activator and Wnt activator;(b) producing gut endodermal progenitor (GEP) cells from DE cells;(c) producing hepatic endoderm (HE) cells from GEP cells by contacting GEP cells with a second culture medium comprising a Wnt inhibitor, and BMP inhibitor;(d) exchanging the second culture medium with a third culture medium comprising Wnt inhibitor, BMP activator, and TGF-P inhibitor; and(e) producing iPSC-derived hepatocytes from HE cells by contacting HE cells with a hepatocyte maturation culture medium.

2. The method of claim 1, wherein the GEP cells in step (c), are embedded in a solubilized basement membrane preparation.

3. The method of claim 1, wherein the second culture medium and third culture medium further comprise a retinoid.

4. A method of producing gut endodermal progenitor (GEP) cells from induced pluripotent stem cells (iPSCs) comprising:(a) contacting iPSCs with a first culture medium for one or more days comprising a TGF-P activator and a Wnt activator;(b) producing definitive endoderm (DE) cells by exchanging the first culture medium with a second culture medium comprising a TGF-P activator, BMP activator, FGF activator, and VEGF activator; and(c) producing gut endodermal progenitor (GEP) cells by contacting the DE cells with a third culture medium comprising the second culture medium and a ROCK inhibitor.

5. The method of claim 4, wherein the iPSCs of step (a) and / or DE of step (c) are embedded in a solubilized basement membrane preparation.

6. A method of producing hepatic endoderm (HE) cells comprising:(a) contacting definitive endoderm (DE) cells with a first culture medium comprising a BMP activator and TGF-P activator to produce gut endodermal progenitor (GEP) cells;(b) contacting GEP cells with a second culture medium comprising a BMP inhibitor and MEK inhibitor; and(c) exchanging the second culture medium with a third culture medium comprising BMP activator and TGF-P inhibitor.

7. The method of claim 6, wherein the second culture medium and third culture medium further comprises a retinoid.

8. The method of claim 6, wherein the second culture medium and third culture medium further comprises a Wnt inhibitor.

9. The method of claim 6, wherein the DE of step (a) and / or GEP of step (b) are embedded in a solubilized basement membrane preparation.

10. The method of claim 6, wherein DE cells generated from PSCs.

11. The method of claim 10, comprising contacting PSCs in a culture medium comprising a TGF-P activator, a Wnt activator thereby generating DE cells.

12. The method of 11, wherein the culture medium further comprises a ROCK inhibitor, BMP activator, FGF activator, VEGF activator, or a combination thereof.

13. A method of producing induced pluripotent stem cell (iPSC)-derived hepatocytes, comprising:(a) differentiating iPSCs into definitive endoderm (DE) cells;(b) differentiating the DE cells into gut endodermal progenitor (GEP) cells;(c) differentiating the GEP cells into hepatic endoderm (HE) cells; and(d) differentiating the HE cells into mature hepatocytes, thereby producing iPSC- derived hepatocytes wherein at least 80% of the hepatocytes express at least one mature hepatocyte marker comprising: HNF4a, UGT2B7, transthyretin (TTR), al -antitrypsin (AAT), or albumin.

14. A method of inducing hepatocyte differentiation from pluripotent stem cells (PSCs) comprising:(a) inducing DE cells by:(i) embedding PSCs in a solubilized basement membrane preparation;(ii) contacting PSCs in a first culture medium comprising a TGF-P activator, aWnt activator, and a ROCK inhibitor;(iii) exchanging the first culture medium with a second culture medium comprising a TGF-P activator and a Wnt activator; and(iv) exchanging the second culture medium with a third culture medium comprising a TGF-P activator, BMP activator, FGF activator, and VEGF activator;(b) inducing GEP cells by:(i) dissociating then embedding DE cells in a solubilized basement membrane preparation; and(ii) contacting the DE cells with a GEP culture medium comprising a TGF-P activator, BMP activator, FGF activator, VEGF activator, and ROCK inhibitor;(c) inducing HE cells by:(i) dissociating then embedding GEP cells in a solubilized basement membrane preparation;(ii) contacting the GEP cells with a first HE culture medium comprising a retinoid, Wnt inhibitor, BMP inhibitor, and MEK inhibitor; and(iii) exchanging the first HE culture medium with a second HE culture medium comprising a retinoid, Wnt inhibitor, BMP activator, and TGF-P inhibitor; and(d) inducing PSC-derived hepatocytes by contacting the HE cells with a hepatocyte maturation culture medium comprising hepatocyte growth factor (HGF), an IL-6 family cytokine, and a glucocorticoid inducing hepatocyte differentiation from PSCs.

15. The method of claim 14, wherein the solubilized basement membrane preparation is Matrigel™.

16. The method of claim 14, where optionally the TGF-P activator is Activin A; the Wnt activator is Wnt3a; the ROCK inhibitor is Y-27632; the BMP activator is BMP4; the FGF activator is FGF2; the VEGF is VEGF-A; the retinoid is retinoic acid; the Wnt inhibitor is inhibitor of Wnt production 2 (IWP2); the BMP inhibitor is LDN193189; the MEK inhibitor is PD0325901; TGF-P inhibitor is A83-01; the IL-6 family cytokine is oncostatin M (OSM); or the glucocorticoid is dexamethasone (DEX).

17. The method of claim 14, wherein the hepatocyte maturation culture medium does not comprise an epidermal growth factor (EGF).

18. The method of claim 16, wherein the concentration of Activin A ranges from between about 50 ng / mL to 150 ng / mL.

19. The method of claim 16, wherein the concentration of Wnt3a ranges from between about 25 ng / mL to 100 ng / mL.

20. The method of claim 16, wherein the concentration of Y-27632 ranges from between about 5 pM to 15 pM.

21. The method of claim 16, wherein the concentration of BMP4 ranges from between about 0.1 ng / mL to 1 ng / mL or from 5 |Jg / mL to 15 |jg / mL.

22. The method of claim 16, wherein the concentration of retinoic acid ranges from between about 0.5 pM to 5 pM.

23. The method of claim 16, wherein the concentration of IWP2 ranges from between about 1 pM to 8 pM.

24. The method of claim 16, wherein the concentration of LDN193189 ranges from between about 125 nM to 500 nM.

25. The method of claim 16, wherein the concentration of PD0325901 ranges from between about 250 nM to 1 pM.

26. The method of claim 16, wherein the concentration of A83-01 ranges from between about 0.5 pM to 2 pM.

27. The method of claim 16, wherein the concentration of OSM ranges from between about 10 ng / mL to 40 ng / mL.

28. The method of claim 16, wherein the concentration of DEX ranges from between about 50 pM to 200 pM.

29. The method of claim 16, wherein the concentration of HGF ranges from between about 5 ng / mL to 20 ng / mL.

30. A method of cry opreserving GEP cells comprising: contacting GEP cells with a GEP cryopreservation media comprising DMSO, emricasan, polyamine, and trans-ISRIB (CEPT).

31. The method of claim 30, wherein the GEP cryopreservation media does not comprise a ROCK inhibitor.

32. A method of cry opreserving iPSC-derived hepatocytes or 3D liver organoids comprising: contacting iPSC-derived hepatocytes or 3D liver organoids with a hepatocyte cryopreservation media comprising DMSO, emricasan, polyamine, and trans-ISRIB (CEPT).

33. The method of claim 32, wherein the hepatocyte cryopreservation media does not comprise a ROCK inhibitor.

34. The method of claim 31 or 33, wherein the ROCK inhibitor is chroman 1.

35. A method of recovering cryopreserved GEP cells comprising:(a) thawing the cryopreserved GEP cells;(b) recovering the thawed cryopreserved GEP cells in a medium comprising DMSO, emricasan, polyamine, and trans-ISRIB (CEPT) for at least one day; and(c) culturing the recovered GEP cells in a GEP culture medium comprising SFD, a TGF-P activator, BMP activator, FGF activator, VEGF activator, and ROCK inhibitor for about 2 to 5 days.

36. A method of recovering cryopreserved iPSC-derived hepatocytes or 3D liver organoids comprising:(a) thawing the cryopreserved iPSC-derived hepatocytes or 3D liver organoids;(b) recovering the thawed iPSC-derived hepatocytes or 3D liver organoids in a medium comprising DMSO, emricasan, polyamine, and trans-ISRIB (CEPT) for at least one day; and(c) culturing the recovered iPSC-derived hepatocytes or 3D liver organoids in a hepatocyte maturation culture medium comprising hepatocyte culture medium (HCM) without epidermal growth factor (EGF), hepatocyte growth factor (HGF), an IL-6 family cytokine, and a glucocorticoid for at least one day.

37. The method of claim 35, wherein recovering the cryopreserved GEP cells does not comprise a ROCK inhibitor.

38. The method of claim 36, wherein recovering the cryopreserved iPSC-derived hepatocytes or 3D liver organoids does not comprise a ROCK inhibitor.

39. The method of claim 37 or 38, wherein the ROCK inhibitor is chroman 1.

40. A kit comprising:(a) a gut endodermal induction cocktail comprising: a TGF-P activator, BMP activator, and ROCK inhibitor;(b) two hepatic endoderm induction cocktails comprising:(i) BMP inhibitor, and MEK inhibitor;(ii) BMP activator, and TGF-P inhibitor;(c) a hepatic maturation cocktail comprising: hepatocyte culture medium without EGF, HGF, an IL-6 family cytokine, and a glucocorticoid; and(d) instructions for inducing definitive endodermal cells differentiation into mature hepatocytes; and optionally a solubilized basement membrane preparation.

41. The kit of claim 40, wherein the two hepatic endoderm induction cocktails further comprise a retinoid and Wnt inhibitor.

42. The kit of claim 40, wherein the gut endodermal induction cocktail further comprises a FGF activator and VEGF activator.

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