Systems and methods for derivation of hepatocytes from nonhuman primate induced pluripotent stem cells

Abstract

A method and system are disclosed for differentiating non-human primate induced pluripotent stem cells (NHP iPSCs) into hepatocyte-like cells through a defined, multi-stage protocol. The method includes sequentially exposing NHP iPSCs to six distinct cell differentiation media over an approximate eighteen-day period. Each medium comprises defined combinations of growth factors, small molecules, and inhibitors that direct progressive lineage specification through anterior primitive streak, definitive endoderm, posterior foregut, liver bud progenitors, hepatoblasts, and hepatocyte-like cells. Concentration ranges and exposure durations are provided for each stage, ensuring reproducibility and reducing heterogeneity across cell populations. In some embodiments, treatment with a transforming growth factor beta (TGF-β ) receptor I inhibitor during early hepatoblast specification enhances lineage fidelity. The resulting hepatocyte-like cells are validated by immunostaining and western blotting for hepatic markers. Applications include preclinical drug metabolism, hepatotoxicity assessment, and in vitro disease modeling using non-human primate–derived systems.

Description

Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATIONSYSTEMS AND METHODS FOR DERIVATION OF HEPATOCYTES FROM NONHUMAN PRIMATE INDUCED PLURIPOTENT STEM CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63 / 690,632, filed August 4, 2024, the contents of which are herein incorporated by reference in their entirety.INCORPORATION BY REFERENCE

[0002] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety, as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety.TECHNICAL FIELD

[0003] This disclosure relates generally to the field of stem cell biology and regenerative medicine and, more specifically, to the field of differentiating non-human primate induced pluripotent stem cells (NHP iPSCs) into hepatocyte-like cells. Described herein are systems and methods for generating hepatocyte-like cells from NHP iPSCs and employing such cells in in vitro models for drug metabolism, efficacy, and toxicity assessment.BACKGROUND

[0004] NHPs play a role in the biomedical sciences because of their close evolutionary relationship and physiological similarity to humans. Among these, cynomolgus macaques (Macaca fascicularis) are particularly valued as animal models in nonclinical and preclinical drug evaluation studies. Their genetic makeup, immune system architecture, metabolic pathways, and organ structures parallel those of humans in ways that smaller animal models, such as rodents, cannot replicate. As a result, cynomolgus macaques have been employed extensively to investigate drug absorption, distribution, metabolism, excretion, and toxicity, thereby providing a reliable platform for anticipating human outcomes. In the pharmaceutical industry, studies involving cynomolgus macaques and other NHPs often serve as a translational step between early laboratory experiments and human clinical trials, enablingDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION the identification of safety concerns, efficacy markers, and potential adverse events. The predictive nature of these models has contributed to the advancement of therapies ranging from small molecule drugs to biologies, monoclonal antibodies, and gene and cell-based therapies.

[0005] While the value of NHP models is well established, reliance on live NHP studies presents substantial challenges for the drug development ecosystem. The acquisition and maintenance of cynomolgus macaques and other NHPs involve high financial costs, specialized infrastructure, and strict regulatory oversight. Ethical concerns surrounding the welfare of these animals have led to heightened scrutiny and restrictions on their use, reinforcing international efforts to reduce dependence on live animal testing. Additionally, global demand has created supply constraints that make it increasingly difficult to secure sufficient NHP populations for research needs. These limitations are compounded by the time-intensive nature of conducting longitudinal NHP studies, which can slow the pace of preclinical research and delay the introduction of potentially life-saving therapeutics. Consequently, there is a recognized need within the scientific and regulatory communities for alternative models and strategies that preserve the predictive power of NHP studies while addressing issues of cost, ethics, and accessibility. Such approaches are expected to enhance efficiency in drug development, improve reproducibility of data, and contribute to the broader goal of refining the tools used to facilitate the safety and effectiveness of new human therapeutics.SUMMARY

[0006] In some aspects, the techniques described herein relate to a system for differentiating hepatocyte-like cells from non-human primate induced pluripotent stem cells (NHP iPSCs), the system including: a cell culture platform configured to support the NHP iPSCs; a first cell differentiation medium configured to induce transition of the NHP iPSCs to an anterior primitive streak state during a first period of time; a second cell differentiation medium configured to induce transition of the NHP iPSCs to a definitive endoderm state during a second period of time; a third cell differentiation medium configured to induce transition of the NHP iPSCs to a posterior foregut state during a third period of time; a fourth cell differentiation medium configured to induce transition of the NHP iPSCs to a liver bud progenitor state during a fourth period of time; a fifth cell differentiation medium configured to promote hepatoblast specification of the NHP iPSCs during a fifth period of time; and aDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION sixth cell differentiation medium configured to promote hepatic maturation of the NHP iPSCs during a sixth period of time.

[0007] In some aspects, the techniques described herein relate to a method of differentiating hepatocyte-like cells from non-human primate induced pluripotent stem cells (NHP iPSCs), the method including: inducing transition to an anterior primitive streak state by applying a first cell differentiation medium to the NHP iPSCs for a first period of time; inducing transition to a definitive endoderm state by applying a second cell differentiation medium to the cells for a second period of time; inducing transition to a posterior foregut state by applying a third cell differentiation medium to the cells for a third period of time; inducing transition to a liver bud progenitor state by applying a fourth cell differentiation medium to the cells for a fourth period of time; promoting hepatoblast specification by applying a fifth cell differentiation medium to the cells for a fifth period of time; and promoting hepatic maturation by applying a sixth cell differentiation medium to the cells for a sixth period of time.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The foregoing is a summary, and thus, necessarily limited in detail. The above- mentioned aspects, as well as other aspects, features, and advantages of the present technology are described below in connection with various embodiments, with reference made to the accompanying drawings.

[0009] FIG. 1 shows a schematic of an embodiment of a method of deriving hepatocytes from nonhuman primate induced pluripotent stem cells, according to some embodiments.

[0010] FIGs. 2A-2C shows images of cynomolgus macaque iPSC derived liver bud cells immunostained for hepatocyte nuclear factor- 1 alpha (HNF 1- Alpha) in FIG. 2 A, Alphafetoprotein (AFP) in FIG. 2B, and T-box transcription factor 3 (TBX3) in FIG. 2C, each with 4',6-diamidino-2-phenylindole (DAPI) nuclei counterstain, according to some embodiments.

[0011] FIG. 3 illustrates a table representing an embodiment of a generation protocol for deriving hepatocyte-like cells from NHP iPSCs, according to some embodiments.

[0012] FIG. 4 illustrates a flow diagram representing an embodiment of a method of differentiating hepatocyte-like cells from NHP iPSCs, according to some embodiments.Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION

[0013] FIG. 5 illustrates a schematic box diagram of an embodiment an embodiment of a system for differentiating hepatocyte-like cells from NHP iPSCs, according to some embodiments.

[0014] FIG. 6 illustrates a schematic box diagram of an embodiment of a system for differentiating hepatocyte-like cells from NHP iPSCs, according to some embodiments.

[0015] FIG. 7 illustrates a schematic box diagram of an embodiment of a system for differentiating hepatocyte-like cells from NHP iPSCs, according to some embodiments.

[0016] FIG. 8 illustrates a schematic box diagram of an embodiment of a system for differentiating hepatocyte-like cells from NHP iPSCs, according to some embodiments.

[0017] FIG. 9 illustrates a schematic box diagram of an embodiment of a system for differentiating hepatocyte-like cells from NHP iPSCs, according to some embodiments.

[0018] FIG. 10 illustrates a schematic box diagram of an embodiment of a system for differentiating hepatocyte-like cells from NHP iPSCs, according to some embodiments.

[0019] FIG. 11 illustrates a schematic box diagram of an embodiment of a system for differentiating hepatocyte-like cells from NHP iPSCs, according to some embodiments.

[0020] The illustrated embodiments are merely examples and are not intended to limit the disclosure. The schematics are drawn to illustrate features and concepts and are not necessarily drawn to scale.DETAILED DESCRIPTION

[0021] The foregoing is a summary, and thus, necessarily limited in detail. The above- mentioned aspects, as well as other aspects, features, and advantages of the present technology will now be described in connection with various embodiments. The inclusion of the following embodiments is not intended to limit the disclosure to these embodiments, but rather to enable any person skilled in the art to make and use the claimed subject matter. Other embodiments may be utilized, and modifications may be made without departing from the spirit or scope of the subject matter presented herein. Aspects of the disclosure, as described and illustrated herein, can be arranged, combined, modified, and designed in a variety of different formulations, all of which are explicitly contemplated and form part of this disclosure.

[0022] In recent years, there has been reduced availability of live NHPs for testing safety and efficacy of human drugs. The technical solution to this availability problem is in vitro models of NHPs. In vitro models of NHPs may play a role in human drug efficacy and safetyDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION assessment prior to testing such drugs in live NHPs. In vitro models may save cost and time in human drug development; reduce the use of live NHPs in the human drug development process; and improve the accuracy of nonclinical safety by complementing human in vitro models. In some embodiments, liver cells, including hepatocytes, may be derived from iPSCs. In some embodiments, the NHP is a cynomolgus macaque or other similar species of NHPs (that are used as NHP models for human diseases).

[0023] At least one practical application of deriving hepatocytes from nonhuman primate pluripotent stem cells includes testing, validating, and / or assaying one or more preclinical drugs on the derived NHP stems cells and / or organoids and correlating the testing, validation, and / or assay results with one or more in vivo NHP tests results.

[0024] Another practical application of deriving hepatocytes from nonhuman primate pluripotent stem cells includes predicting drug metabolism by a liver, predicting the in vivo liver toxicity of a drug through in vitro testing which includes, but is not limited to, toxicity evaluation of various vectors used in gene and cell therapy, such as strains of Adeno Associated Viruses (AAVs) some of which cause liver toxicity in NHPs and humans. Other biologies and small molecules used for human drugs can also cause liver toxicity. An application of iPSCs-derived hepatocytes is providing a tool for in vitro prediction of potential side effects of liver toxicity.

[0025] FIG. 1 depicts an embodiment of a method 100 for deriving hepatocyte-like cells from NHP iPSCs. In some embodiments, the method 100 begins with NHP iPSCs 102 that are subjected to a generation protocol 104.

[0026] In some embodiments, the generation protocol 104 of hepatocyte-like cells 106 from NHP iPSCs includes a plurality of consecutive lineage choices. For example, the NHP iPSCs may undergo about one to about five, about one to about ten, about five to about ten, or about five to about seven consecutive lineage choices. In some embodiments, the NHP iPSCs may undergo six consecutive lineage choices. In some embodiments, each lineage choice comprises exposing a set of cells to a cell differentiation medium. As used herein, the terms “exposing,” “providing,” “applying,” or “using” a cell differentiation medium to or with cells and the term “treating” cells with a cell differentiation medium are used synonymously to communicate adding the medium to the cells in a first instance and changing the medium to another in subsequent instances in a sequence of lineage choices. In some embodiments, the one or more cell differentiation mediums can be grouped as a cell differentiation media set for differentiating hepatocyte-like cells from NHP (iPSCs).Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION

[0027] Although, first, second, third, fourth, fifth, etc. are used herein, such designations shall not connote a sequence or otherwise. Such designations are merely used for clarity, not for purposes of sequence.

[0028] In some embodiments, NHP iPSCs may be induced to an anterior primitive streak using a first cell differentiation medium. As used herein, the term “anterior primitive streak” refers to a transient developmental structure that arises during early embryogenesis and functions as an intermediate state in the progression of pluripotent cells toward definitive germ layer specification. In vivo, the anterior region of the primitive streak is associated with the induction of mesendodermal lineages and the establishment of definitive endoderm and anterior mesoderm, which contribute to the formation of organs such as the liver, pancreas, and portions of the cardiovascular and respiratory systems. In vitro, induction of NHP iPSCs to an anterior primitive streak-like state can be achieved by exposing the cells to selected growth factors, inhibitors, and other differentiation cues that recapitulate early embryonic signaling pathways. This anterior primitive streak-like state provides a developmental foundation for directing the cells into a definitive endoderm lineage and, subsequently, into hepatocyte-like cells suitable for use in preclinical drug testing models.

[0029] As used herein, the term “inducing transition” refers to directing NHP iPSCs from one developmental state to another by applying defined biochemical and culture conditions. The transition involves activation or suppression of lineage-specific signaling pathways through exposure to combinations of growth factors, inhibitors, hormones, and other small molecules contained in cell differentiation media. For example, exposing pluripotent stem cells to activin, 6-[(2-[(4-(2,4-dichlorophenyl)-5-(5-methyl-lH-imidazol-2-yl)-2- pyrimidinyl)amino]ethyl)amino]-3-pyridinecarbonitrile (CHIR99021), fibroblast growth factor 2 (FGF2), and 2-(lH-indol-5-yl)-3-(4-morpholin-4-yl-pyridin-3-yl)quinazolin-4(3H)- one (PI- 103) induces transition to an anterior primitive streak state by activating mesendodermal gene networks and inhibiting alternative fates. Similarly, exposing definitive endoderm cells to bone morphogenetic protein 4 (BMP4), forskolin, and activin induces transition toward a liver bud progenitor state by promoting hepatic lineage specification rather than pancreatic or intestinal fates. In this manner, “inducing transition” encompasses stage-specific changes in cell identity that mimic natural embryonic development in a controlled in vitro environment.

[0030] As used herein, the term “subsystem” refers to a distinct functional component of a larger system that performs a specific operation in support of the overall process ofDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION differentiating NHP iPSCs. A subsystem may include one or more compositions, one or more hardware elements, software modules, or combinations thereof, and / or may operate independently or in coordination with other subsystems.

[0031] As used herein, the term “definitive endoderm” refers to a developmental germ layer established during gastrulation that gives rise to tissues and organs of the gastrointestinal tract, liver, pancreas, and respiratory system. In vivo, definitive endoderm cells emerge from the anterior primitive streak and are distinguished from primitive or extraembryonic endoderm by their ability to contribute to functional endodermal organs. In vitro, definitive endoderm can be generated from NHP iPSCs by exposing the cells to defined differentiation media containing growth factors and signaling modulators that activate pathways such as Activin / Nodal and Wnt. The definitive endoderm state serves as an intermediate in hepatocyte differentiation protocols, as cells in this lineage exhibit gene expression markers and functional properties predictive of their capacity to develop into hepatocyte-like cells and other endoderm-derived tissues.

[0032] As used herein, the term “posterior foreguf ’ refers to a developmental intermediate stage derived from definitive endoderm that contributes to the formation of foregut-derived organs, including the liver, pancreas, and portions of the gastrointestinal tract. In vivo, posterior foregut cells emerge as a regional specification of the endoderm under the influence of signaling cues such as fibroblast growth factors, bone morphogenetic proteins, and retinoid pathway activators. In vitro, posterior foregut-like cells can be generated from NHP iPSCs by sequential exposure to differentiation media that mimic these signaling conditions. Posterior foregut specification is a step in hepatocyte differentiation protocols, as it directs endodermal cells toward liver and pancreas developmental pathways, thereby establishing competence for further specification into hepatic progenitors.

[0033] As used herein, the term “liver bud progenitor” refers to a developmental cell population that arises from posterior foregut endoderm and represents an early precursor to hepatoblasts and hepatocytes. In vivo, liver bud progenitors are specified by a combination of growth factor and morphogen signals, including activins, bone morphogenetic proteins, and cyclic adenylyl cyclase activators, which promote hepatic commitment. In vitro, liver bud progenitor-like cells can be derived from NHP iPSCs by exposing posterior foregut cells to differentiation media designed to reproduce these hepatic induction cues. The liver bud progenitor state is characterized by the initiation of hepatic gene expression programs andDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION serves as a transition point toward the generation of hepatoblasts and / or hepatocyte-like cells that exhibit functional liver-specific characteristics.

[0034] As used herein, the term “hepatoblasf ’ refers to a bipotential liver progenitor cell population that arises from liver bud progenitors during embryonic development. In vivo, hepatoblasts represent a transitional stage capable of differentiating into both hepatocytes and cholangiocytes, thereby giving rise to the functional architecture of the liver, including parenchymal and biliary compartments. Hepatoblast specification is regulated by signaling pathways such as oncostatin M, glucocorticoid signaling, and bone morphogenetic protein activity, which collectively activate hepatic transcriptional programs. In vitro, hepatoblast- like cells may be generated from NHP iPSCs by exposing liver bud progenitors to differentiation media containing pleiotropic cytokines, glucocorticoids, gamma secretase inhibitors, and / or anabolic hormones. The hepatoblast stage is characterized by expression of early hepatic markers and provides an intermediate population from which hepatocyte-like cells can be matured for functional applications.

[0035] As used herein, the term “hepatoblast specification” refers to the developmental commitment of non-human primate induced pluripotent stem cell-derived progenitors to the hepatoblast lineage under defined biochemical and culture conditions. Hepatoblasts are bipotent precursor cells that give rise to both hepatocytes and cholangiocytes, and their specification represents an intermediate stage in hepatic differentiation. In some embodiments, hepatoblast specification is achieved by exposing liver bud progenitors to a combination of signaling factors that mimic cues present during fetal liver development. Such factors may include BMP4, a pleiotropic cytokine such as oncostatin M, a glucocorticoid such as dexamethasone, a gamma secretase inhibitor such as RO4929097 or N-[N-(3,5- difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), an adenylyl cyclase activator such as forskolin, an anabolic hormone such as recombinant human insulin, and / or a vitamin C derivative such as ascorbic acid-2 phosphate, optionally provided in a base medium (e.g., CDM4) supplemented with amino acids. These defined conditions promote hepatic lineage fidelity, suppress competing endodermal fates such as pancreatic and / or intestinal differentiation, and / or establish a stable hepatoblast population capable of further maturation into hepatocyte-like cells.

[0036] As used herein, the term “promoting hepatoblast specification” refers to directing non-human primate induced pluripotent stem cell-derived progenitors, such as liver bud progenitors, toward commitment as hepatoblasts through the application of definedDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION biochemical and culture conditions. Hepatoblasts are bipotent precursors that give rise to hepatocytes and cholangiocytes, and their specification in vitro requires exposure to signals that mimic fetal liver development. In some embodiments, hepatoblast specification is promoted by exposing progenitor cells to a differentiation medium comprising one or more factors selected from BMP4, a pleiotropic cytokine such as oncostatin M, a glucocorticoid such as dexamethasone, a gamma secretase inhibitor such as RO4929097 or DAPT, an adenylyl cyclase activator such as forskolin, an anabolic hormone such as recombinant human insulin, and a vitamin C derivative such as ascorbic acid-2 phosphate, optionally in combination with an amino acid concentrate in a base medium (e.g., CDM4). These conditions enhance hepatic lineage fidelity, suppress alternative endodermal fates such as pancreatic or intestinal differentiation, and establish a stable hepatoblast population for subsequent maturation into hepatocyte-like cells.

[0037] As used herein, the term “hepatocyte-like cell” refers to a differentiated cell type that exhibits morphological, biochemical, and functional properties of hepatocytes, the principal parenchymal cells of the liver. In vivo, hepatocytes perform essential functions including metabolism of xenobiotics, synthesis of serum proteins, regulation of glucose and lipid homeostasis, and detoxification. In vitro, hepatocyte-like cells may be derived from hepatoblasts or directly from liver bud progenitors by exposure to differentiation media containing glucocorticoids, adenylyl cyclase activators, anabolic hormones, vitamin C derivatives, and gamma secretase inhibitors. Such hepatocyte-like cells are characterized by the expression of hepatocyte-specific markers such as hepatocyte nuclear factor 4-alpha (HNF4a), AFP, and TBX3, as well as functional properties including albumin secretion and cytochrome P450 activity. These hepatocyte-like cells provide a biologically relevant platform for modeling liver development, studying liver disease, and evaluating safety and efficacy of candidate therapeutic compounds in vitro.

[0038] As used herein, the term “hepatic maturation” refers to the process of developing hepatoblasts or hepatoblast-like progenitors into functional hepatocyte-like cells under defined in vitro conditions. Hepatic maturation involves enhancing cellular characteristics associated with adult hepatocytes, including metabolic activity, protein secretion, detoxification capacity, and expression of hepatocyte-specific markers such as HNF4a, albumin, and cytochrome P450 enzymes. In some embodiments, hepatic maturation is promoted by exposing cells to a combination of factors that mimic late-stage liver development, including a glucocorticoid such as dexamethasone, an adenylyl cyclaseDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION activator such as forskolin, an anabolic hormone such as recombinant human insulin, and a vitamin C derivative such as ascorbic acid-2 phosphate, together with a gamma secretase inhibitor such as RO4929097 or DAPT, optionally provided in a CDM4B base medium. These defined conditions promote structural and functional maturation, stabilize hepatic gene expression, and suppress residual progenitor characteristics, thereby yielding reproducible hepatocyte-like cells suitable for drug metabolism studies, hepatotoxicity evaluation, and disease modeling.

[0039] Induction to an anterior primitive streak state may be accomplished by culturing the NHP iPSCs in a first differentiation medium formulated to activate mesendodermal signaling pathways. In some embodiments, the first medium comprises a follicle-stimulating hormone (FSH) secretion stimulator, a glycogen synthase kinase 3 (GSK3) inhibitor, a growth factor, and a dual -kinase inhibitor. In some embodiments of the first medium, the FSH secretion stimulator is activin, the GSK3 inhibitor is CHIR99021, the growth factor is FGF2, and the dual-kinase inhibitor is PI- 103. In some embodiments, the first medium may comprise about 50 ng / mL to about 150 ng / mL FSH secretion stimulator, about 0.5 pM to about 6 pM GSK3 inhibitor, about 5 ng / mL to about 35 ng / mL growth factor, and about 25 nM to about 75 nM dual-kinase inhibitor. A volume of the first medium may be provided to the NHP iPSCs for about 6 hours to about 48 hours, in some embodiments. For example, the first medium comprises about 75 ng / mL to about 125 ng / mL activin, about 2 pM to about 4 pM CHIR99021, about 15 ng / mL to about 25 ng / mL FGF2, and about 40 nM to about 60 nM PI- 103, and it is provided to the NHP iPSCs for 24 hours.

[0040] The foregoing compositions and treatment conditions provide several technical benefits. The combination of a FSH secretion stimulator, a GSK3 inhibitor, a growth factor, and a dual-kinase inhibitor produces a synergistic effect that enhances the efficiency and reproducibility of inducing NHP iPSCs toward an anterior primitive streak state. By precisely controlling the concentrations and exposure periods, the disclosed ranges minimize variability in lineage specification, reduce the frequency of incomplete differentiation, and improve overall cell viability. For example, exposure of NHP iPSCs to activin at about 75 ng / mL to about 125 ng / mL in combination with CHIR99021 at about 2 pM to about 4 pM, FGF2 at about 15 ng / mL to about 25 ng / mL, and PI- 103 at about 40 nM to about 60 nM for about 24 hours reliably activates mesendodermal gene expression programs while suppressing alternative differentiation pathways.Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION

[0041] Another technical benefit is that the defined ranges and timing of the first medium reduces reliance on undefined factors or serum-based formulations, thereby increasing the reproducibility of the protocol across different laboratory conditions. The stepwise use of small molecule inhibitors and growth factors within the disclosed concentration windows also enables scalability and automation, which are important for generating large numbers of anterior primitive streak-like cells for downstream applications. Furthermore, the early induction of an anterior primitive streak state improves the fidelity of subsequent differentiation into definitive endoderm, posterior foregut, and hepatocyte-like cells, thereby enhancing the predictive value of the final cell population for in vitro drug metabolism and toxicity testing.

[0042] A further technical benefit of the disclosed compositions and treatment conditions is the demonstration of an unexpected synergistic effect that is not achieved when the individual components are applied in isolation or outside the disclosed concentration ranges. For example, use of activin alone at concentrations above about 150 ng / mL results in aberrant differentiation and loss of cell viability, while CHIR99021 applied in the absence of a dualkinase inhibitor produces heterogeneous populations with incomplete mesendodermal induction. Similarly, omission of FGF2 from the formulation leads to reduced expression of anterior primitive streak markers and a failure to achieve uniform differentiation. By contrast, the disclosed combination of activin, CHIR99021, FGF2, and PI-103 within the defined ranges consistently yields a homogeneous cell population that is characterized by elevated expression of mesendodermal genes such as Mix paired-like homeobox gene 1 (MIXL1) and Eomesodermin (EOMES), while maintaining low expression of markers associated with alternative lineages. This synergistic interaction demonstrates that the present compositions achieve results that could not have been predicted from the use of the individual components alone, thereby providing a distinct technical advance over conventional methods that employ similar factors but fail to specify the concentration ranges and exposure durations disclosed herein.

[0043] In some embodiments, a state of definitive endoderm is induced in the cells using a second cell differentiation medium. In some embodiments, the second medium comprises a FSH secretion stimulator, a GSK3 inhibitor, a growth factor, and a dual-kinase inhibitor. In some embodiments of the second medium, the FSH secretion stimulator is activin, the GSK3 inhibitor is CHIR99021, the growth factor is FGF2, and the dual-kinase inhibitor is PL103. In some embodiments, the second medium may comprise about 50 ng / ml to about 150 ng / mlDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATIONFSH secretion stimulator, about 0.5 pM to about 6 pM GSK3 inhibitor, about 5 ng / ml to about 35 ng / ml growth factor, and about 25 nM to about 75 nM dual-kinase inhibitor. A volume of the second medium may be provided to the cells for about 6 hours to about 48 hours, in some embodiments. For example, the second medium may comprise about 75 ng / mL to about 125 ng / mL activin, about 2 pM to about 4 pM CHIR99021, about 15 ng / mL to about 25 ng / mL FGF2, and about 40 nM to about 60 nM PL103, and it is provided to the cells for 24 hours.

[0044] The various formulations and treatment conditions for inducing a definitive endoderm state provide multiple technical benefits. The coordinated use of a FSH secretion stimulator, a GSK3 inhibitor, a growth factor, and a dual-kinase inhibitor enables reproducible and efficient commitment of NHP iPSCs into a definitive endoderm lineage. By maintaining activin in the range of about 75 ng / mL to about 125 ng / mL in combination with CHIR99021 at about 2 pM to about 4 pM, FGF2 at about 15 ng / mL to about 25 ng / mL, and PI- 103 at about 40 nM to about 60 nM, the protocol yields a uniform population of definitive endoderm cells with enhanced survival and reduced heterogeneity. The disclosed ranges minimize spontaneous differentiation into mesodermal or ectodermal lineages, thereby ensuring a high proportion of cells that exhibit expression of definitive endoderm markers such as SRY-box transcription factor 17 (SOX17) and Forkhead Box A2 (FOXA2), also called HNF3P (Hepatocyte Nuclear Factor 3 -beta).

[0045] Another technical benefit is that the combination of small molecules and growth factors within the specified ranges improves reproducibility across batches and laboratory environments by eliminating reliance on undefined serum components. The synergistic interaction of activin and CHIR99021 activates mesendodermal signaling pathways, while PI- 103 suppresses alternative lineage commitment, producing a more stable and homogeneous definitive endoderm population. This stability supports downstream differentiation into posterior foregut and liver bud progenitors, increasing overall yield and efficiency of hepatocyte-like cell production. Furthermore, the time-controlled exposure of about 6 hours to about 48 hours provides flexibility for protocol optimization while preserving lineage fidelity. Collectively, these technical benefits represent an improvement over conventional approaches, which often generate mixed or unstable populations due to incomplete specification of factor combinations or undefined concentration ranges.

[0046] In some embodiments, the definitive endoderm cells may be differentiated into posterior foregut (PFG) using a third cell differentiation medium. In some embodiments, theDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION third medium comprises a transforming growth factor beta (TGF-P) receptor I inhibitor, a growth factor, a bone morphogenetic protein, and a retinoid pathway activator in a base culture medium. In some embodiments of the third medium, the TGF-b receptor I inhibitor is A83-01, the growth factor is FGF2, the bone morphogenetic protein is BMP4, the retinoid pathway activator is 4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-l- propenyl] benzoic acid (TTNPB), and the base culture medium is CDM3. In some embodiments, the third medium may comprise about 0.5 pM to about 1.5 pM TGF-b receptor I inhibitor, about 55 ng / mL to about 165 ng / mL growth factor, about 15 ng / mL to about 45 ng / mL bone morphogenetic protein, and 37 nM to about 113 nM retinoid pathway activator in medium. A volume of the third medium may be provided to the cells for about 6 hours to about 48 hours, in some embodiments. For example, the third medium may comprise about 0.75 pM to about 1.25 pM A8301, about 80 ng / mL to about 140 ng / mL FGF2, about 20 ng / mL to about 40 ng / mL BMP4, and about 60 nM to about 90 nM TTNPB in CDM3 medium for another 24 hours.

[0047] The various formulations and treatment conditions for differentiating definitive endoderm cells into posterior foregut (PFG) provide technical benefits. The combination of a TGF-P receptor I inhibitor, FGF2, BMP4, and a retinoid pathway activator within a defined concentration range enables reliable specification of endodermal cells toward a posterior foregut lineage. In particular, the use of A83-01 at concentrations of about 0.75 pM to about 1.25 pM in combination with FGF2 at about 80 ng / mL to about 140 ng / mL, BMP4 at about 20 ng / mL to about 40 ng / mL, and TTNPB at about 60 nM to about 90 nM produces an induction of posterior foregut marker expression while reducing expression of non-targeted anterior or midgut markers. The balanced activation of FGF and retinoid signaling, in conjunction with inhibition of TGF-P signaling, drives a more uniform commitment to posterior foregut, thereby reducing heterogeneity commonly observed in alternative protocols.

[0048] Another technical benefit is that the disclosed ranges enhance lineage fidelity while maintaining cell viability across variable exposure durations of about 6 hours to about 48 hours. By synchronizing BMP4 and TTNPB signaling with FGF2 support, the medium mimics developmental cues that occur during early embryogenesis, resulting in a posterior foregut-like population that is primed for hepatic specification. The reproducibility of these outcomes across batches enables consistent scaling and automation, making the disclosed conditions advantageous for generating large populations of posterior foregut-derived cellsDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION for downstream applications. Furthermore, the ability to tightly control concentrations of retinoid activators and TGF-P inhibitors minimizes off-target differentiation into pancreatic or intestinal lineages, which often complicate conventional differentiation methods.Collectively, these benefits establish a more reliable foundation for subsequent differentiation into liver bud progenitors and hepatocyte-like cells.

[0049] In some embodiments, the cells may be further differentiated from PFG to liver bud progenitors by adding a fourth cell differentiation medium. In some embodiments, the fourth medium may comprise a FSH secretion stimulator, a bone morphogenetic protein, and an adenylyl cyclase activator in a base culture medium. In some embodiments of the fourth medium, the FSH secretion stimulator is activin, the bone morphogenetic protein is BMP4, the adenylyl cyclase activator is forskolin, and the base culture medium is CDM3. In some embodiments, the fourth medium may comprise about 5 ng / mL to about 15 ng / mL FSH secretion stimulator, about 15 ng / mL to about 45 ng / mL bone morphogenetic protein, and about 0.5 pM to about 1.5 pM adenylyl cyclase activator in medium. A volume of the fourth medium may be provided to the cells for about 1 day to about 5 days, in some embodiments. For example, the fourth medium may comprise about 8 ng / mL to about 12 ng / mL activin, about 22 ng / mL to about 38 ng / mL BMP4, and about 0.75 pM to about 1.25 pM forskolin in CDM3 medium and provided to the cells for 3 days.

[0050] The various formulations and treatment conditions for differentiating posterior foregut cells into liver bud progenitors provide several technical benefits. The combination of a FSH secretion stimulator, a bone morphogenetic protein (BMP), and an adenylyl cyclase activator within the specified concentration ranges mimics signaling events that occur during hepatic specification in early embryogenesis. For example, activin at about 8 ng / mL to about 12 ng / mL, BMP4 at about 22 ng / mL to about 38 ng / mL, and forskolin at about 0.75 pM to about 1.25 pM synergistically promote hepatic lineage commitment, resulting in the reproducible generation of liver bud progenitor-like cells. By providing a defined and balanced activation of BMP and cyclic adenosine monophosphate (cAMP) signaling pathways, while maintaining controlled activin levels, the disclosed medium increases lineage fidelity and reduces off-target differentiation into pancreatic or intestinal fates.

[0051] Another technical benefit is that the disclosed exposure period of about 1 day to about 5 days offers flexibility for protocol optimization while preserving induction efficiency. In some embodiments, providing the medium for about 3 days yields a highly enriched population of liver bud progenitors characterized by expression of early hepaticDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION markers such as HNF4a, TBX3, and AFP. The presence of forskolin as an adenylyl cyclase activator further enhances metabolic activity and priming of downstream hepatoblast development, which improves the efficiency of subsequent differentiation steps. Moreover, the elimination of undefined serum components and the reliance on chemically defined factors within the disclosed ranges improves reproducibility across laboratories and scales readily to high-throughput formats. Collectively, these technical benefits result in a more consistent and homogeneous population of liver bud progenitors suitable for further maturation into hepatoblasts and hepatocyte-like cells.

[0052] In some embodiments, the liver bud progenitors may be further differentiated by using a fifth cell differentiation medium. In some embodiments, the fifth medium may comprise a bone morphogenetic protein, a pleiotropic cytokine, a glucocorticoid, a gamma secretase inhibitor, an adenylyl cyclase activator, an anabolic hormone, a vitamin C derivative, and an amino acid supply in a base culture medium. In some embodiments of the fifth medium, the bone morphogenetic protein is BMP4, the pleiotropic cytokine is oncostatin M (OSM), the glucocorticoid is dexamethasone, the gamma secretase inhibitor includes, but is not limited to, RO4929097 and / or DAPT, the adenylyl cyclase activator is forskolin, the anabolic hormone is human recombinant insulin, the vitamin C derivative is ascorbic acid-2 phosphate (AAP), the amino acid supply is an amino acid concentrate, and the base culture medium is CDM4 medium.

[0053] In some embodiments, the fifth medium may comprise about 5 ng / mL to about 15 ng / mL bone morphogenetic protein, about 5 ng / mL to about 15 ng / mL pleiotropic cytokine, about 5 pM to about 15 pM glucocorticoid, 1 pM to about 3 pM gamma secretase inhibitor, about 5 pM to about 15 pM adenylyl cyclase activator, about 5 ng / mL to about 15 ng / mL anabolic hormone, about 100 pg / mL to about 300 pg / mL vitamin C derivative, and amino acid concentrate in base medium. In some embodiments, the fifth medium may comprise about 5 ng / mL to about 15 ng / mL bone morphogenetic protein, about 5 ng / mL to about 15 ng / mL pleiotropic cytokine, about 5 pM to about 15 pM glucocorticoid, 5 pM to about 15 pM gamma secretase inhibitor, about 5 pM to about 15 pM adenylyl cyclase activator, about 5 ng / mL to about 15 ng / mL anabolic hormone, about 100 pg / mL to about 300 pg / mL vitamin C derivative, and amino acid concentrate in base medium. A volume of the fifth medium may be provided to cells for about 5 days to about 7 days. For example, the fifth medium may comprise about 8 ng / mL to about 12 ng / mL BMP4, about 8 ng / mL to about 12 ng / mL OSM, about 8 pM to about 12 pM dexamethasone, about 1.5 pM to about 2.5 pM RO4929097 orDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION about 8 pM to about 12 pM DAPT, about 8 pM to about 12 pM forskolin, about 8 pg / mL to about 12 pg / mL human recombinant insulin, about 150 pg / mL to about 250 pg / mL AAP, and amino acid concentrate in CDM4 base medium and is provided to the cells for about 6 days.

[0054] In some embodiments, the cells can optionally be further treated with a TGF-P receptor I inhibitor during at least a portion of the time that the cells are treated with the fifth medium. In some embodiments, this additional TGF-P receptor I inhibitor treatment can be for about 40 hours to about 60 hours at the start of the use of the fifth medium. In some embodiments, the TGF-P receptor I inhibitor is selected from the group including, but not limited to, SB 505124, A83-01, RepSox, or LY364947. The inhibitor may be administered during a portion of exposure to the fifth medium at concentrations ranging from about 0.1 pM to about 5.0 pM, for example from about 0.25 pM to about 3.0 pM, from about 0.4 pM to about 2.0 pM, or from about 0.5 pM to about 1.5 pM. The duration of administration may range from about 24 hours to about 96 hours, for example from about 36 hours to about 72 hours, or from about 40 hours to about 60 hours, beginning at any point within the fifthmedium window. In some embodiments, the inhibitor may be delivered continuously for the selected duration, while in other embodiments the inhibitor may be applied in pulsed regimens at intervals ranging from about every 8 hours to about every 24 hours. In further embodiments, step-wise or gradient dosing may be employed, wherein concentrations begin at about 0.2 pM to about 0.7 pM and increase to about 1.0 pM to about 2.0 pM over a period of about 24 hours to about 48 hours, thereby fine-tuning the suppression of TGF-P signaling during hepatoblast specification. The various formulations and treatment conditions for differentiating liver bud progenitors into hepatoblasts provide numerous technical benefits. The defined combination of a bone morphogenetic protein, a pleiotropic cytokine, a glucocorticoid, a gamma secretase inhibitor, an adenylyl cyclase activator, an anabolic hormone, a vitamin C derivative, and an amino acid supply synergistically promotes hepatoblast specification while supporting cell viability. For example, BMP4 and OSM function together to activate hepatic gene regulatory networks, while dexamethasone stabilizes differentiation and enhances metabolic activity characteristic of early hepatic precursors. Inclusion of gamma secretase inhibitors such as RO4929097 or DAPT reduces Notch pathway activity, thereby favoring hepatoblast over cholangiocyte specification. The combined action of forskolin, human recombinant insulin, and AAP enhances anabolic metabolism, protein synthesis, and / or oxidative stress resistance, improving the functional maturity of the resulting hepatoblast-like cells.Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION

[0055] Another technical benefit of the fifth medium is that the defined concentration ranges and treatment duration of about 5 to about 7 days reduce lineage heterogeneity and yield a more uniform hepatoblast population, as confirmed by expression of early hepatic markers such as AFP, HNF4a, and / or TBX3. The optional inclusion of a TGF-P receptor I inhibitor, such as SB 505124 and / or A83-01, for about 40 to about 60 hours at the start of hepatoblast specification further enhances lineage fidelity by suppressing mesodermal and non-hepatic endodermal differentiation pathways. This selective suppression results in a stronger bias toward hepatic progenitor identity and improves downstream maturation efficiency into hepatocyte-like cells. In addition, the reliance on chemically defined components within the specified ranges eliminates variability associated with undefined serum-based protocols, enabling consistent and scalable generation of hepatoblast populations. These technical benefits provide a reliable foundation for producing hepatocytelike cells with improved functionality for in vitro modeling, drug metabolism studies, and / or toxicity screening.

[0056] In some embodiments, the cells may be further differentiated by using a sixth cell differentiation medium. In some embodiments, the sixth medium may comprise a glucocorticoid, an adenylyl cyclase activator, an anabolic hormone, a vitamin C derivative, and a gamma secretase inhibitor in a base culture medium. In some embodiments of the sixth medium, the glucocorticoid is dexamethasone, the adenylyl cyclase activator is forskolin, the anabolic hormone is human recombinant insulin, the vitamin C derivative is AAP, the gamma secretase inhibitor includes, but is not limited to, RO4929097 and / or DAPT, and the base culture medium is CDM4B medium. In some embodiments, the sixth medium may comprise about 5 pM to about 15 pM glucocorticoid, about 5 pM to about 15 pM adenylyl cyclase activator, about 5 pg / mL to about 15 pg / mL anabolic hormone, about 100 pg / mL to about 300 pg / mL vitamin C derivative, and / or 1 pM to about 3 pM gamma secretase inhibitor in base medium. In some embodiments, the sixth medium may comprise about 5 pM to about 15 pM glucocorticoid, about 5 pM to about 15 pM adenylyl cyclase activator, about 5 pg / mL to about 15 pg / mL anabolic hormone, about 100 pg / mL to about 300 pg / mL vitamin C derivative, and / or about 5 pM to about 10 pM gamma secretase inhibitor in base medium. A volume of the sixth medium may be provided to the cells for about 5 days to about 7 days. For example, the sixth medium may comprise about 8 pM to about 12 pM dexamethasone, about 8 pM to about 12 pM forskolin, about 8 pg / mL to about 12 pg / mL human recombinant insulin, about 150 pg / mL to about 250 pg / mL AAP, and about 1.5 pM to about 2.5 pMDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATIONRO4929097 or about 8 pM to about 12 pM DAPT in CDM4B base medium and is provided to the cells for about 6 days.

[0057] The various formulations and treatment conditions for differentiating hepatoblasts into hepatocyte-like cells provide several technical benefits. The combined use of a glucocorticoid, an adenylyl cyclase activator, an anabolic hormone, a vitamin C derivative, and a gamma secretase inhibitor within the specified ranges promotes maturation of hepatic lineage cells into functionally active hepatocyte-like populations. For example, dexamethasone at about 8 pM to about 12 pM enhances liver-specific metabolic pathways and stabilizes hepatocyte phenotype, while forskolin at about 8 pM to about 12 pM stimulates cAMP signaling, thereby augmenting hepatocyte functional activity. The addition of human recombinant insulin at about 8 pg / mL to about 12 pg / mL promotes anabolic metabolism and protein synthesis, while AAP at about 150 pg / mL to about 250 pg / mL enhances antioxidant capacity and supports long-term viability. The inclusion of a gamma secretase inhibitor, such as RO4929097 at about 1.5 pM to about 2.5 pM or DAPT at about 8 pM to about 12 pM, further refines lineage commitment by reducing signaling pathways that favor alternative biliary or non-hepatic fates.

[0058] Another technical benefit is that the disclosed concentration ranges and treatment period of about 5 to about 7 days reproducibly yield hepatocyte-like cells that exhibit both morphological and biochemical features of mature hepatocytes. The resulting cells demonstrate expression of hepatic markers such as HNF4a, albumin (ALB), and cytochrome P450 enzymes (e.g., CYP3A4), alongside functional activities including albumin secretion, glycogen storage, and / or xenobiotic metabolism. By relying on chemically defined conditions rather than serum-based factors, the disclosed sixth medium provides reproducibility across laboratories and scalability for high-throughput applications. Furthermore, the synergistic effect of combining dexamethasone, forskolin, insulin, and AAP within the defined ranges improves both maturation efficiency and functional stability over prior art protocols. These technical benefits establish a reliable approach for generating hepatocyte-like cells suitable for use in in vitro liver disease modeling, preclinical drug metabolism assays, and / or toxicity screening applications.

[0059] FIG. 1 illustrates an embodiment of a method 100 for deriving hepatocyte-like cells from NHP iPSCs. As shown, the process begins with NHP iPSCs 102, which may be derived from cynomolgus macaques or other suitable non-human primate species. In general, described herein are methods for deriving iPSCs from NHPs. For example, nonhumanDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION primates include, but re not limited to, cynomolgus macaques (e.g., Macaca fascicularis), pig-tailed macaques (e.g., Macaca nemestrina, Macaca leonine, Macaca sylvanusi), baboons, African green monkeys, rhesus macaques, chimpanzees, and the like. The cells 102 are subjected to a multi-stage differentiation protocol 104, herein referred to as NHP protocol, which comprises sequential exposure to defined differentiation media that induce progressive lineage specification.

[0060] Following implementation of protocol 104, the NHP iPSCs 102 are differentiated into hepatocyte-like cells 106. The hepatocyte-like cells 106 exhibit morphological and functional characteristics of primary hepatocytes and are suitable for use in drug metabolism, toxicity evaluation, and disease modeling. To confirm successful differentiation, the hepatocyte-like cells 106 are subjected to analysis and validation steps, which may include antibody (Ab) staining 108 and / or western blotting 110.

[0061] In some embodiments, Ab staining 108 may be performed to detect expression of hepatocyte-specific markers such as HNF4a, AFP, and / or T-box transcription factor TBX3 (TBX3). In some embodiments, staining further includes DAPI counterstain to identify nuclei and verify cellular morphology. The staining 108 provides visual confirmation of lineage identity and / or functional protein expression.

[0062] In some embodiments, western blot analysis 110 may be conducted to validate the expression of hepatic proteins at the molecular level. Western blotting enables quantitative assessment of target protein expression and provides confirmation of results obtained from antibody staining. The use of complementary validation techniques facilitates reproducibility and reliability of differentiation outcomes.

[0063] As illustrated in FIG. 1, the steps of protocol 104, differentiation into hepatocytes 106, and validation through staining 108 and western blotting 110 together provide a workflow for producing and verifying hepatocyte-like cells from NHP iPSCs. Although specific elements and order are shown, variations may be utilized without departing from the scope of the present disclosure. Following the protocol 104, in some embodiments, the hepatocyte-like cells 106 may be optionally stained with antibodies (i.e., immunostained) 108 and further optionally subjected to a western blot 110 to validate lineage identity and functional maturation. In some embodiments, antibody staining 108 is carried out using one or more primary antibodies specific to hepatocyte markers, including HNF4a, AFP, albumin (ALB), and / or TBX3. In further embodiments, immunostaining may be performed with fluorescently labeled secondary antibodies and visualized using fluorescence microscopy,Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION with nuclei counterstained using DAPI to provide reference for cellular morphology and / or localization of marker expression. Antibody staining thereby enables qualitative and semi- quantitative evaluation of hepatocyte-specific protein expression and provides confirmation that the differentiated cells exhibit expected phenotypic features.

[0064] In some embodiments, the hepatocyte-like cells 106 are further validated by western blot analysis 110, which provides a complementary and quantitative method of protein detection. Western blotting may be performed to confirm expression levels of liver- associated proteins such as cytochrome P450 enzymes (e.g., CYP3A4), albumin, and metabolic transporters, thereby offering evidence of functional hepatic maturation. The use of both antibody staining 108 and western blotting 110 facilitates reproducibility of differentiation outcomes, reduces the likelihood of false positives or incomplete lineage specification, and strengthens the reliability of the generated hepatocyte-like cells for downstream applications such as drug metabolism assays, toxicity evaluations, and / or disease modeling.

[0065] The combined use of antibody staining 108 and western blot analysis 110 provides several technical benefits that improve the reliability and reproducibility of hepatocyte-like cell validation. While antibody staining offers spatial and morphological information, such as confirming nuclear localization or cytoplasmic expression of hepatocyte-specific proteins, western blotting complements this by providing quantitative measurement of protein expression levels. This dual-validation approach reduces the likelihood of misidentification or incomplete characterization that may occur when relying on a single assay. For example, antibody staining alone may yield ambiguous results due to cross-reactivity or low signal intensity, whereas western blotting can confirm the molecular weight and relative abundance of target proteins. Conversely, western blotting alone may fail to capture heterogeneity at the single-cell level, which antibody staining can reveal. In some embodiments, flow cytometry may additionally, or alternatively, be used to characterize the various cell lineages as part of protocol 104.

[0066] By integrating these two validation methods, the present disclosure facilitates that hepatocyte-like cells 106 exhibit both phenotypic and molecular fidelity to primary hepatocytes. This comprehensive validation strategy enhances confidence in downstream applications, including preclinical drug metabolism studies, hepatotoxicity assays, and disease modeling. In some embodiments, the combined approach allows earlier detection of incomplete or off-target differentiation, enabling corrective adjustments to the protocol 104.Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATIONCollectively, these benefits provide a technical advance over prior art protocols that rely on single-mode validation methods, which often produce inconsistent or less reliable results.

[0067] FIG. 2A shows cynomolgus macaque induced pluripotent stem cell (iPSC)-derived liver bud cells immunostained for HNF4a. Experimental results 200 illustrate the set of images corresponding to HNF4a staining. FIG. 2B shows iPSC-derived liver bud cells immunostained for AFP. Experimental results 202 illustrate the set of images corresponding to AFP staining. FIG. 2C shows iPSC-derived liver bud cells immunostained for TBX3. Experimental results 204 illustrate the set of images corresponding to TBX3 staining. In each of FIGs. 2 A through 2C, the leftmost panels illustrate immunostaining with the respective primary antibody, the middle panels show nuclei counterstained with 4',6-diamidino-2- phenylindole (DAPI), and the rightmost panels show merged overlays illustrating colocalization of marker expression with nuclear morphology.

[0068] The detection of HNF4a, AFP, and TBX3 in the liver bud cells demonstrates successful progression of the iPSCs through sequential stages of hepatic lineage specification. Expression of HNF4a correlates with transcriptional regulation of hepatic metabolic genes, AFP expression reflects protein synthesis activity characteristic of immature hepatocytes, and TBX3 expression indicates proliferative capacity and further lineage commitment toward hepatocyte-like cells. The colocalization observed in the merged images provides visual confirmation that marker expression is associated with the majority of viable, nucleated cells in the cultures, thereby validating differentiation efficiency and reproducibility of the described protocol.

[0069] In some embodiments, positive staining for HNF4a indicates activation of hepatocyte-specific transcriptional networks responsible for regulating genes associated with lipid metabolism, bile acid synthesis, and plasma protein production. The observed expression of AFP is consistent with early hepatic progenitor or hepatoblast-like cells, providing evidence that the cells are transitioning through intermediate developmental stages before full hepatocyte maturation. Expression of TBX3 further confirms hepatic lineage specification, as this transcription factor is associated with liver bud expansion, suppression of premature differentiation, and maintenance of proliferative capacity during early liver development.

[0070] The merged images demonstrate that the majority of nuclei correspond with positive staining for HNF4a, AFP, or TBX3, thereby confirming that differentiation was not limited to a small subset of the population but instead occurred across a broad distribution of cells. InDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION some embodiments, quantification of the merged overlays indicates that about 50% to about 98% of nuclei are associated with detectable expression of one or more hepatic lineage markers. In other embodiments, the proportion may range from about 60% to about 95%, for example from about 65% to about 90%, or from about 70% to about 85%. In certain embodiments, yields of about 75% to about 80% marker-positive cells may be consistently obtained. These conversion rates represent a marked improvement over conventional methods of hepatocyte differentiation, which typically yield marker-positive populations at rates of about 20% to about 50%, more commonly about 30% to about 45%.

[0071] In some embodiments, the disclosed protocol produces uniform populations in which about 70% to about 95% of cells express HNF4a, about 65% to about 90% of cells express AFP, and about 60% to about 85% of cells express TBX3, with overlapping expression observed in about 55% to about 80% of cells. Such high conversion efficiency reduces heterogeneity, enhances reproducibility, and increases the availability of hepatocytelike cells for downstream assays such as drug metabolism, hepatotoxicity testing, and disease modeling. In some embodiments, the efficiency of marker expression is further confirmed by flow cytometry, western blot analysis, or quantitative PCR, with results demonstrating reproducibility across multiple experimental replicates and different non-human primate iPSC donor lines.

[0072] This widespread staining pattern indicates a high efficiency of lineage conversion. The results depicted in FIGs. 2A-2C validate that cynomolgus macaque iPSCs subjected to the differentiation protocol are capable of yielding hepatocyte-like cells with molecular and structural features comparable to those observed in developing liver tissue. The combined use of multiple markers provides a multi-dimensional assessment of lineage identity, reducing the likelihood of false positives that may occur when relying on a single marker. These data confirm the reproducibility of the disclosed methods and provide strong evidence of the functional relevance of the derived hepatocyte-like cells for downstream drug metabolism and / or toxicity assays.

[0073] The use of a multi-marker immunostaining panel in FIGs. 2A-2C provides several technical benefits that improve the reliability and accuracy of validating hepatic lineage specification. While detection of a single marker may suggest partial differentiation, reliance on one protein alone often leads to ambiguous results due to cross-reactivity, stage-specific expression, or transient marker presence. By combining HNF4a, AFP, and / or TBX3 with a DAPI counterstain, the disclosed analysis captures both early and intermediate markers ofDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION hepatic commitment and facilitates that nuclear morphology corresponds with observed protein expression. This multi-dimensional approach provides stronger evidence of successful differentiation than prior art protocols that rely on one or two markers.

[0074] Another technical benefit of the disclosed marker panel is that it enables confirmation of developmental stage fidelity. For example, AFP expression indicates hepatoblast-like characteristics, TBX3 expression reflects active liver bud development and proliferation, and HNF4a expression confirms initiation of mature hepatocyte transcriptional networks. The simultaneous detection of these markers allows verification that the cells are transitioning through appropriate intermediate states before achieving hepatocyte-like maturation, thereby reducing the risk of mischaracterization. Additionally, the inclusion of DAPI nuclear counterstain provides spatial and structural context that confirms protein localization to viable cells across the culture population. Collectively, this marker panel improves reproducibility, reduces error rates, and provides a foundation for subsequent functional validation assays, such as drug metabolism or hepatotoxicity testing.

[0075] FIG. 3 depicts an embodiment of a generation protocol 300 for deriving hepatocytelike cells from NHP iPSCs. The protocol 300 involves the sequential use of six cell differentiation media applied over the course of about 18 days. The column labeled “Days of Exposure” indicates the number of continuous (although continuous can include wash days and / or days where additional media were added to the cultures) days that the cells are treated with a particular medium, while the column labeled “Day Sequence Count” illustrates the corresponding placement of each medium within the 18-day protocol.

[0076] In some embodiments, the first cell differentiation medium is applied for about 1 day (Day 1). As described above, this medium may comprise a FSH secretion stimulator such as activin at about 50 ng / mL to about 150 ng / mL, a GSK3 inhibitor such as CHIR99021 at about 0.5 pM to about 6 pM, a growth factor such as FGF2 at about 5 ng / mL to about 35 ng / mL, and a dual-kinase inhibitor such as PI- 103 at about 25 nM to about 75 nM.

[0077] On Day 2, the second medium is applied for about 1 day. In some embodiments, the composition of the second medium is similar to that of the first, but maintains activin at about 75 ng / mL to about 125 ng / mL, CHIR99021 at about 2 pM to about 4 pM, FGF2 at about 15 ng / mL to about 25 ng / mL, and PI- 103 at about 40 nM to about 60 nM. The exposure of about 24 hours drives differentiation into definitive endoderm, with marker expression of SOX17 and FOXA2. On Day 3, the third medium is applied for about 1 day. This medium may comprise a TGF-P receptor I inhibitor such as A83-01 at about 0.5 pM to about 1.5 pM,Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATIONFGF2 at about 55 ng / mL to about 165 ng / mL, BMP4 at about 15 ng / mL to about 45 ng / mL, and a retinoid pathway activator such as TTNPB at about 37 nM to about 113 nM, provided in CDM3 base medium.

[0078] Days 4-6 correspond to the fourth medium, applied for about 3 days. This medium may comprise activin at about 5 ng / mL to about 15 ng / mL, BMP4 at about 15 ng / mL to about 45 ng / mL, and forskolin at about 0.5 pM to about 1.5 pM, provided in CDM3 base medium.

[0079] Days 7-12 correspond to the fifth medium, applied for about 6 days. This medium may include BMP4 at about 5 ng / mL to about 15 ng / mL, OSM at about 5 ng / mL to about 15 ng / mL, dexamethasone at about 5 pM to about 15 pM, a gamma secretase inhibitor selected from RO4929097 or DAPT at about 1 pM to about 3 pM or about 5 pM to about 15 pM, forskolin at about 5 pM to about 15 pM, insulin at about 5 ng / mL to about 15 ng / mL, AAP at about 100 pg / mL to about 300 pg / mL, and amino acid concentrate in CDM4 medium. In some embodiments, during the first and second days of exposure to the fifth medium (e.g., Days 7-8), the cells are additionally treated with a TGF-P receptor I inhibitor such as SB 505124 at about 0.5 pM to about 1.5 pM, which enhances hepatic specification and suppresses mesodermal diversion.

[0080] Days 13-18 correspond to the sixth medium, applied for about 6 days. This medium may comprise dexamethasone at about 5 pM to about 15 pM, forskolin at about 5 pM to about 15 pM, insulin at about 5 pg / mL to about 15 pg / mL, AAP at about 100 pg / mL to about 300 pg / mL, and a gamma secretase inhibitor (RO4929097 at about 1 pM to about 3 pM or DAPT at about 5 pM to about 10 pM) in CDM4B base medium. This promotes hepatic maturation and results in hepatocyte-like cells with expression of albumin (ALB), HNF4a, and cytochrome P450 enzymes.

[0081] The generation protocol 300 establishes a reproducible framework for deriving hepatocyte-like cells from NHP iPSCs. The defined sequential use of six differentiation media within precise temporal windows improves lineage fidelity, minimizes variability across experiments, and enables scalable production of hepatocyte-like cells for preclinical drug testing and toxicity assessment. The stepwise generation protocol 300 illustrated in FIG. 3 provides several technical benefits compared to conventional methods that rely on undefined or fewer-step differentiation approaches. The sequential use of six distinct cell differentiation media over an 18-day period more closely recapitulates embryonic liver development, allowing NHP iPSCs to transition through anterior primitive streak, definitiveDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION endoderm, posterior foregut, liver bud progenitors, hepatoblasts, and hepatocyte-like stages in an orderly manner. This staged progression reduces lineage heterogeneity and facilitates that intermediate populations pass through appropriate transcriptional checkpoints before advancing to the next developmental state.

[0082] Another technical benefit of protocol 300 is that it specifies precise days of exposure and concentration ranges for each medium, thereby improving reproducibility across independent experiments and laboratory settings. For example, maintaining the third medium for about one day (Day 3) followed by the fourth medium for about three days (Days 4-6) enhances posterior foregut specification and hepatic commitment, while use of the fifth medium for about six days (Days 7-12) with optional TGF-P receptor I inhibitor treatment on Days 7-8 stabilizes hepatoblast differentiation. The defined exposure to the sixth medium on Days 13-18 promotes maturation and functional expression of hepatocyte-specific proteins such as albumin and cytochrome P450 enzymes.

[0083] The temporal segmentation of protocol 300 further provides scalability and flexibility for industrial or high-throughput applications. Because each medium is chemically defined and applied within controlled durations, the process can be adapted to automation platforms and scaled for the production of large batches of hepatocyte-like cells. This structured approach reduces variability that may arise from extended culture in a single medium and prevents premature or incomplete lineage specification. Collectively, these benefits establish protocol 300 as a technically superior strategy for generating NHP-derived hepatocyte-like cells with improved reproducibility, fidelity, and functional relevance for drug testing and toxicity studies.

[0084] FIG. 4 depicts an embodiment of a generation method 400 for differentiating hepatocyte-like cells from NHP iPSCs. At block 410, the method includes inducing transition of the NHP iPSCs to an anterior primitive streak state by applying a first cell differentiation medium for a first period of time. The first period of time may range from about 6 hours to about 48 hours, or more from about 18 hours to about 30 hours, with a representative example of about 24 hours. The first medium may comprise a FSH secretion stimulator such as activin, provided in concentrations ranging from about 5 ng / mL to about 300 ng / mL, for example from about 10 ng / mL to about 250 ng / mL, from about 20 ng / mL to about 200 ng / mL, from about 30 ng / mL to about 175 ng / mL, from about 40 ng / mL to about 160 ng / mL, from about 50 ng / mL to about 150 ng / mL, from about 60 ng / mL to about 130 ng / mL, or fromDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION about 75 ng / mL to about 125 ng / mL. In some embodiments, nodal or activin A analogues may be employed in comparable ranges.

[0085] The first medium may further comprise a GSK3 inhibitor such as CHIR99021, provided in concentrations ranging from about 0.01 pM to about 15 pM, for example from about 0.05 pM to about 12 pM, from about 0.1 pM to about 10 pM, from about 0.25 pM to about 8 pM, from about 0.5 pM to about 6 pM, from about 1 pM to about 5 pM, from about 1.5 pM to about 4 pM, or from about 2 pM to about 4 pM. In certain embodiments, alternative small-molecule GSK3 inhibitors such as BIO (6-bromoindirubin-3 ’ -oxime), SB- 216763, or tideglusib may be used in comparable ranges. The first medium may additionally include a growth factor such as FGF2, present in concentrations ranging from about 1 ng / mL to about 80 ng / mL, for example from about 2 ng / mL to about 60 ng / mL, from about 5 ng / mL to about 50 ng / mL, from about 10 ng / mL to about 40 ng / mL, from about 15 ng / mL to about 35 ng / mL, from about 20 ng / mL to about 30 ng / mL, or from about 15 ng / mL to about 25 ng / mL. In other embodiments, fibroblast growth factors such as FGF4, FGF8, or FGF10 may be substituted in comparable ranges.

[0086] The first medium may further comprise a dual-kinase inhibitor such as PI-103, provided in concentrations ranging from about 1 nM to about 200 nM, for example from about 5 nM to about 150 nM, from about 10 nM to about 120 nM, from about 20 nM to about 100 nM, from about 25 nM to about 80 nM, from about 30 nM to about 70 nM, from about 40 nM to about 65 nM, or from about 45 nM to about 60 nM. In some embodiments, alternative dual-kinase inhibitors such as LY294002, wortmannin, BEZ-235, or PKL587 may be employed in similar ranges. In some embodiments, the first medium may be applied to the cells for a period ranging from about 2 hours to about 96 hours, for example from about 4 hours to about 72 hours, from about 6 hours to about 60 hours, from about 12 hours to about 48 hours, from about 18 hours to about 36 hours, or for a representative period of about 24 hours. This controlled exposure induces transition of the cells to an anterior primitive streak state with improved reproducibility.

[0087] At block 420, the method includes inducing transition of the NHP iPSCs to a definitive endoderm state by applying a second cell differentiation medium for a second period of time. The second period of time may range from about 6 hours to about 48 hours, with an example of about 24 hours.

[0088] The second medium may comprise activin in concentrations ranging from about 10 ng / mL to about 250 ng / mL, for example from about 20 ng / mL to about 200 ng / mL, fromDocket No. 0138-704.600INTERNATIONAL PATENT APPLICATION about 25 ng / mL to about 175 ng / mL, from about 40 ng / mL to about 150 ng / mL, from about 50 ng / mL to about 150 ng / mL, from about 60 ng / mL to about 130 ng / mL, or from about 75 ng / mL to about 125 ng / mL. In some embodiments, activin A analogues or nodal may be used in comparable ranges. The medium may further comprise a GSK3 inhibitor such as CHIR99021, provided in concentrations ranging from about 0.05 pM to about 12 pM, for example from about 0.1 pM to about 10 pM, from about 0.25 pM to about 8 pM, from about 0.5 pM to about 6 pM, from about 1 pM to about 5 pM, or from about 2 pM to about 4 pM. In other embodiments, alternative small molecule GSK3 inhibitors such as BIO (6- bromoindirubin-3 ’ -oxime) or tideglusib may be employed within comparable ranges.

[0089] The medium may also include a growth factor such as FGF2, present in concentrations ranging from about 1 ng / mL to about 60 ng / mL, for example from about 2 ng / mL to about 50 ng / mL, from about 5 ng / mL to about 40 ng / mL, from about 10 ng / mL to about 35 ng / mL, from about 15 ng / mL to about 30 ng / mL, or from about 20 ng / mL to about 25 ng / mL. In some embodiments, fibroblast growth factors such as FGF4 or FGF8 may be substituted, provided in similar ranges. In addition, the medium may comprise a dual-kinase inhibitor such as PL103, present in concentrations ranging from about 5 nM to about 150 nM, for example from about 10 nM to about 100 nM, from about 20 nM to about 80 nM, from about 25 nM to about 75 nM, from about 30 nM to about 65 nM, or from about 40 nM to about 60 nM. In certain embodiments, alternative dual-kinase inhibitors such as LY294002, wortmannin, or BEZ-235 may be employed within comparable ranges. In some embodiments, the second medium may be applied to the cells for a period ranging from about 4 hours to about 72 hours, for example from about 6 hours to about 60 hours, from about 12 hours to about 48 hours, from about 18 hours to about 36 hours, or for about 24 hours. This defined exposure period supports consistent induction of definitive endoderm while minimizing variability in downstream lineage progression.

[0090] At block 430, the method includes inducing transition of the NHP iPSCs to a posterior foregut state by applying a third cell differentiation medium for a third period of time. The third period of time may range from about 6 hours to about 48 hours, with an example of about 24 hours. The third medium may comprise about 0.1 pM to about 5.0 pM of a TGF-P receptor I inhibitor, for example from about 0.25 pM to about 3.0 pM, from about 0.5 pM to about 2.0 pM, from about 0.5 pM to about 1.5 pM, or from about 0.75 pM to about 1.25 pM. In some embodiments, the inhibitor may be A83-01, while in other embodiments alternative inhibitors such as SB-505124, RepSox, or LY364947 may be used in comparableDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION ranges. The medium may further comprise about 20 ng / mL to about 250 ng / mL of a growth factor such as FGF2, for example from about 25 ng / mL to about 200 ng / mL, from about 55 ng / mL to about 165 ng / mL, from about 75 ng / mL to about 140 ng / mL, or from about 90 ng / mL to about 120 ng / mL. In other embodiments, fibroblast growth factors such as FGF4 or FGF8 may be substituted, provided in ranges of about 20 ng / mL to about 180 ng / mL.

[0091] The medium may additionally comprise about 5 ng / mL to about 80 ng / mL of a bone morphogenetic protein such as BMP4, for example from about 10 ng / mL to about 60 ng / mL, from about 15 ng / mL to about 45 ng / mL, from about 20 ng / mL to about 40 ng / mL, or from about 25 ng / mL to about 35 ng / mL. In further embodiments, BMP2 or BMP7 may be provided in comparable ranges of about 10 ng / mL to about 50 ng / mL. The retinoid pathway activator may be included at about 10 nM to about 200 nM, for example from about 20 nM to about 150 nM, from about 37 nM to about 113 nM, from about 50 nM to about 100 nM, or from about 60 nM to about 90 nM. In some embodiments, the activator is TTNPB, while in other embodiments retinoid analogues such as all-trans retinoic acid (ATRA), AM580, or 9- cis-retinoic acid may be used in comparable concentration ranges. In certain embodiments, the components are formulated in a CDM3 base medium to maintain balanced nutrient levels, buffering capacity, and stability of growth factors. The use of these tiered concentration ranges allows the differentiation protocol to accommodate inter-line variability in NHP iPSCs, thereby improving reproducibility, reducing variability in posterior foregut induction, and enhancing scalability for different experimental or industrial culture platforms.

[0092] At block 440, the method includes inducing transition of the NHP iPSCs to a liver bud progenitor state by applying a fourth cell differentiation medium for a fourth period of time. The fourth period of time may range from about 1 day to about 5 days, with an example of about 3 days. The fourth medium may comprise about 1 ng / mL to about 30 ng / mL of activin, for example from about 2 ng / mL to about 25 ng / mL, from about 5 ng / mL to about 20 ng / mL, from about 5 ng / mL to about 15 ng / mL, or from about 8 ng / mL to about 12 ng / mL. The medium may further comprise about 5 ng / mL to about 70 ng / mL of a bone morphogenetic protein such as BMP4, for example from about 10 ng / mL to about 60 ng / mL, from about 15 ng / mL to about 45 ng / mL, from about 20 ng / mL to about 40 ng / mL, or from about 25 ng / mL to about 35 ng / mL. In some embodiments, the bone morphogenetic protein may alternatively include BMP2 or BMP7, provided in comparable ranges of about 10 ng / mL to about 50 ng / mL.Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION

[0093] The adenylyl cyclase activator may be included in concentrations ranging from about 0.1 pM to about 3.0 pM, for example from about 0.25 pM to about 2.0 pM, from about 0.5 pM to about 1.5 pM, or from about 0.75 pM to about 1.25 pM. In some embodiments, forskolin is employed, while in other embodiments IBMX (3-isobutyl-l-methylxanthine) may be used in ranges from about 10 pM to about 50 pM, or dibutyryl -c AMP may be used in ranges from about 100 pM to about 500 pM to similarly elevate intracellular cAMP levels. In some embodiments, the medium is formulated in a CDM3 base culture medium, providing nutrients, buffering capacity, and / or growth factor stability. These broader and tiered ranges for each factor ensure robustness of the protocol across different non-human primate iPSC lines and culture platforms, facilitating consistent progression from posterior foregut to liver bud progenitors.

[0094] At block 450, the method includes promoting specification of the NHP iPSCs into hepatoblasts by applying a fifth cell differentiation medium for a fifth period of time. The fifth period of time may range from about 3 days to about 12 days, for example from about 4 days to about 10 days, from about 5 days to about 8 days, or from about 6 days to about 7 days. The fifth medium may comprise about 2 ng / mL to about 30 ng / mL of a bone morphogenetic protein, such as BMP4, for example from about 5 ng / mL to about 20 ng / mL, or from about 8 ng / mL to about 15 ng / mL. In some embodiments, the bone morphogenetic protein may alternatively include BMP2 or BMP7, supplied in a range of about 5 ng / mL to about 40 ng / mL, thereby supporting hepatoblast lineage commitment.

[0095] The pleiotropic cytokine may be provided in concentrations ranging from about 2 ng / mL to about 30 ng / mL, for example from about 5 ng / mL to about 20 ng / mL, or from about 8 ng / mL to about 15 ng / mL. In some embodiments, the pleiotropic cytokine is OSM, while in other embodiments, the cytokine may include leukemia inhibitory factor (LIF) or interleukin-6 (IL-6), applied in comparable ranges of about 2 ng / mL to about 25 ng / mL. The glucocorticoid may be included in concentrations of about 3 pM to about 25 pM, for example from about 5 pM to about 15 pM, or from about 8 pM to about 12 pM. In some embodiments, the glucocorticoid is dexamethasone, while in other embodiments hydrocortisone may be used at concentrations ranging from about 1 pM to about 20 pM, or prednisolone may be used at concentrations of about 2 pM to about 15 pM. The gamma secretase inhibitor may be provided in concentrations of about 0.5 pM to about 5 pM of RO4929097, for example from about 1 pM to about 3 pM, or in concentrations of about 4 pM to about 15 pM of DAPT, for example from about 5 pM to about 10 pM, or from about 6Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION pM to about 9 pM. In additional embodiments, LY411575 may be employed at about 0.1 pM to about 5 pM, or MK-0752 at about 1 pM to about 5 pM, to achieve comparable levels of Notch pathway suppression.

[0096] The adenylyl cyclase activator may be provided in concentrations ranging from about 3 pM to about 25 pM, for example from about 5 pM to about 15 pM, or from about 8 pM to about 12 pM. In some embodiments, forskolin is employed, while in other embodiments activators such as IBMX (3-isobutyl-l-methylxanthine) may be used in concentrations of about 10 pM to about 50 pM, or dibutyryl-cAMP may be used in concentrations of about 100 pM to about 500 pM, thereby elevating intracellular cAMP levels to promote hepatoblast activity. The anabolic hormone may be included in concentrations ranging from about 3 ng / mL to about 25 ng / mL, for example from about 5 ng / mL to about 15 ng / mL, or from about 8 ng / mL to about 12 ng / mL of recombinant human insulin. In alternative embodiments, the anabolic hormone may include IGF-1 at about 5 ng / mL to about 50 ng / mL or IGF-2 at about 5 ng / mL to about 40 ng / mL, providing comparable anabolic and metabolic support.

[0097] The vitamin C derivative may be included in concentrations ranging from about 50 pg / mL to about 500 pg / mL, for example from about 75 pg / mL to about 400 pg / mL, from about 100 pg / mL to about 300 pg / mL, or from about 150 pg / mL to about 250 pg / mL. In some embodiments, AAP is employed, while in other embodiments stabilized vitamin C derivatives such as sodium ascorbyl phosphate may be used. In further embodiments, the medium includes an amino acid concentrate in CDM4 base medium, which may comprise essential and non-essential amino acids at concentrations ranging from about 0.5 x to about 2x standard culture formulation levels. This supplementation provides metabolic support and prevents nutrient depletion during the 5 to 7 day period of hepatoblast specification.

[0098] In some embodiments, an additional treatment with about 0.5 pM to about 1.5 pM of SB 505124 may be applied for about 40 hours to about 60 hours at the start of exposure to the fifth medium to enhance lineage fidelity.

[0099] At block 460, the method includes promoting maturation of the NHP iPSCs into hepatocyte-like cells by applying a sixth cell differentiation medium for a sixth period of time. The sixth period of time may range from about 4 days to about 10 days, for example from about 5 days to about 8 days, or from about 6 days to about 7 days. The sixth medium may comprise about 3 pM to about 20 pM of a glucocorticoid such as dexamethasone, for example from about 5 pM to about 15 pM, or from about 7 pM to about 12 pM. In some embodiments, the medium further comprises about 3 pM to about 20 pM of an adenylylDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION cyclase activator such as forskolin, for example from about 5 pM to about 15 pM, or from about 8 pM to about 12 pM. The anabolic hormone component may be present at about 3 pg / mL to about 20 pg / mL, for example from about 5 pg / mL to about 15 pg / mL, or from about 8 pg / mL to about 12 pg / mL of recombinant human insulin. The vitamin C derivative may be provided at about 75 pg / mL to about 400 pg / mL, for example from about 100 pg / mL to about 300 pg / mL, or from about 150 pg / mL to about 250 pg / mL of ascorbic acid-2 phosphate. In further embodiments, the gamma secretase inhibitor may be present at about 0.5 pM to about 5 pM of RO4929097, for example from about 1 pM to about 3 pM, or at about 4 pM to about 15 pM of D APT, for example from about 5 pM to about 10 pM, or from about 6 pM to about 9 pM. The medium may be formulated in a CDM4B base medium to support final hepatic maturation. The foregoing compositions and treatment durations provide several technical benefits. By defining tiered ranges for each component of the sixth differentiation medium, the protocol reduces dependence on narrow dose windows and increases reproducibility across different non-human primate induced pluripotent stem cell (iPSC) lines, donor sources, and culture conditions.

[0100] The sequential application of these six defined media over about 18 total days induces progressive lineage transitions of the NHP iPSCs, resulting in hepatocyte-like cells with improved reproducibility, lineage fidelity, and expression of hepatic markers such as HNF4a, AFP, and TBX3. This structured protocol reduces variability compared to undefined or fewer-block differentiation approaches and provides cells suitable for drug metabolism studies, hepatotoxicity assays, and in vitro disease modeling.

[0101] The generation method illustrated in FIG. 4 provides several technical benefits over less structured or undefined differentiation protocols. By specifying six distinct cell differentiation media applied sequentially over an approximately 18-day period, the method facilitates controlled lineage progression and reduces variability across experiments. Each step is defined by concentration ranges and exposure times that fall within reproducible windows, minimizing incomplete or off-target differentiation. For example, exposure of NHP iPSCs to activin at about 75 ng / mL to about 125 ng / mL, CHIR99021 at about 2 pM to about 4 pM, FGF2 at about 15 ng / mL to about 25 ng / mL, and PI- 103 at about 40 nM to about 60 nM for approximately 24 hours reliably induces anterior primitive streak formation while suppressing alternative mesodermal pathways.

[0102] The structured use of successive media provides synergistic reinforcement of lineage fidelity. Transitioning through anterior primitive streak, definitive endoderm,Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION posterior foregut, liver bud progenitors, hepatoblasts, and hepatocyte-like cells in sequence reduces the risk of heterogeneous populations that often arise when media are not optimized or stages are bypassed. Optional treatment with TGF-P receptor I inhibitors during hepatoblast specification further enhances stability by suppressing unwanted differentiation signals. Collectively, these features improve cell viability, enhance reproducibility across batches, and yield hepatocyte-like cells expressing one or more hepatic markers such as HNF4a, AFP, and TBX3, thereby providing reliable in vitro models for drug metabolism, hepatotoxicity evaluation, and disease modeling.

[0103] FIG. 5 depicts an embodiment of a system 500 for enabling reproducible and scalable differentiation of hepatocyte-like cells from NHP iPSCs. The system 500 includes a cell culture platform 510, which may comprise a culture dish, bioreactor, perfusion culture system, or a microfluidic device capable of supporting long-term cell growth and controlled differentiation. In some embodiments, the cell culture platform 510 is automated and includes integrated pumps, valves, or flow controllers configured to deliver multiple differentiation media in a timed sequence corresponding to the stages of hepatic lineage progression. For example, the platform 510 may regulate media flow rates within a range of about 0.01 mL / min to about 5 mL / min, such as about 0.1 mL / min to about 1 mL / min, to facilitate uniform exposure of cells to lineage-specific factors. In other embodiments, the platform 510 may incorporate sensors for monitoring culture parameters such as pH (e.g., about 6.8 to about 7.6), dissolved oxygen (e.g., about 15% to about 25% O2), nutrient concentrations such as glucose (e.g., about 2 mM to about 10 mM), and cell density (e.g., about 1 * 104cells / cm2to about 5* 106cells / cm2), thereby enabling precise regulation of the microenvironment throughout the differentiation process.

[0104] In some embodiments, the system 500 further comprises a subsystem 530 for administering additional agents during defined portions of the differentiation sequence. For example, the subsystem 530 may deliver a TGF-P receptor I inhibitor during a portion of exposure to the fifth cell differentiation medium. The subsystem 530 may be implemented as a microfluidic channel array, automated pipetting unit, or secondary reservoir system that allows for timed or pulsed delivery of supplements. In some embodiments, the subsystem 530 delivers the TGF-PRI inhibitor at concentrations ranging from about 0.5 pM to about 1.5 pM, for a period ranging from about 40 hours to about 60 hours, such as about 48 hours. In other embodiments, the subsystem 530 may be programmed to deliver agents in a gradient profile, for example beginning at about 0.5 pM and increasing to about 1.5 pM over the course of 48Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION hours, thereby fine-tuning lineage specification. The subsystem 530 may also include feedback control mechanisms to adjust delivery parameters based on sensor inputs from the cell culture platform 510, such as changes in metabolite levels or oxygen consumption. In some embodiments, the subsystem 530 may deliver volumes ranging from about 100 pL to about 10 mL per exchange cycle, for example about 500 pL to about 2 mL, depending on the culture scale.

[0105] The inclusion of subsystem 530 enables flexible, modular intervention during the differentiation process, ensuring reproducibility and reducing variability between batches. By providing programmable delivery of supplemental agents such as TGF-PRI inhibitors, growth factors, or small molecules, subsystem 530 improves lineage fidelity, scalability, and consistency of hepatocyte-like cell production across diverse iPSC lines.

[0106] The system 500 further includes NHP iPSCs 515. These iPSCs may be reprogrammed from somatic cells, such as fibroblasts, blood cells, or epithelial cells, obtained from one or more non-human primate species. Reprogramming may be achieved using transcription factors (e.g., OCT4, SOX2, KLF4, c-MYC), episomal vectors, or viral methods. Once established, the NHP iPSCs serve as the starting cell population for differentiation into hepatocyte-like cells via defined protocols described herein.

[0107] In some embodiments, the NHP iPSCs 515 are derived from Macaca fascicularis 520, also known as the cynomolgus macaque. The cynomolgus macaque is a widely used preclinical model for evaluating drug safety, pharmacokinetics, and toxicology due to its genetic, physiological, and metabolic similarities to humans. By deriving hepatocyte-like cells from Macaca fascicularis iPSCs, the system 500 provides an in vitro model that closely mimics in vivo primate hepatic responses, thereby reducing reliance on live animal testing and improving translational accuracy for drug development pipelines.

[0108] In certain embodiments, the system 500 may be coupled with downstream analysis platforms, such as high-content imaging, flow cytometry, transcriptomic profiling, or proteomic assays, to validate the phenotype of the resulting hepatocyte-like cells. For example, the differentiated cells may be characterized by expression of hepatocyte markers including HNF4a, AFP, and cytochrome P450 enzymes. Functional assays, such as urea production, glycogen storage, and drug metabolism activity, may be performed to confirm hepatic functionality.

[0109] The system 500 thus provides several technical benefits. It enables scalable and reproducible generation of NHP hepatocyte-like cells under defined conditions, reducesDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION variability associated with undefined culture systems, and provides an ethically favorable alternative to live NHP studies. Furthermore, the use of an integrated cell culture platform enhances experimental consistency, supports automation, and facilitates industrial-scale applications in drug metabolism, hepatotoxicity prediction, and disease modeling.

[0110] In some embodiments, the platform may implement one or more operational features to support controlled differentiation of NHP iPSCs. Such implementations may include one or more automated fluidic delivery systems, timed administration of differentiation factors, programmable media exchange modules, and / or integrated monitoring subsystems for regulating culture conditions. In some embodiments, a subsystem 530 may comprise a fluidic delivery unit to administer a TGF-P receptor I inhibitor during a portion of exposure to the fifth cell differentiation medium. For example, the subsystem may deliver the inhibitor in concentrations ranging from about 0.5 pM to about 1.5 pM, for a duration ranging from about 40 hours to about 60 hours, such as about 48 hours. The subsystem may further be configured to regulate flow rates of culture medium or inhibitor-containing solutions within a range of about 0.01 mL / min to about 5 mL / min, for example about 0.1 mL / min to about 1 mL / min, thereby ensuring uniform exposure of the cells. In some embodiments, the subsystem may include pumps, reservoirs, or microfluidic channels capable of exchanging medium volumes ranging from about 100 pL to about 10 mL, for example about 500 pL to about 2 mL. In other embodiments, the subsystem may comprise an automated pipetting station to deliver bolus doses of the inhibitor at intervals ranging from about every 6 hours to about every 24 hours, such as every 12 hours. In further embodiments, the subsystem may incorporate sensors to monitor pH, temperature, and metabolite levels, such as glucose or lactate, with feedback control mechanisms dynamically adjusting delivery concentration, timing, or flow rate to optimize lineage specification outcomes.

[0111] FIG. 6 depicts an embodiment of a system 500 for differentiating hepatocytelike cells from NHP iPSCs. The system includes a cell culture platform 510 to support the growth, maintenance, and differentiation of NHP iPSCs under controlled in vitro conditions. The cell culture platform 510 may include a dish, flask, bioreactor, or microfluidic system capable of delivering sequential differentiation media at predetermined intervals. In some embodiments, the platform may further comprise automated fluid handling, environmental controls, and monitoring systems to facilitate reproducibility and stability of the differentiation process. The system further includes a first medium 600 that induces transition of the NHP iPSCs to an anterior primitive streak state during a first period of time. In someDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION embodiments, the first medium 600 comprises at least one FSH secretion stimulator 620, at least one GSK3 inhibitor 630, at least one dual-kinase inhibitor 640, and / or at least one growth factor 650.

[0112] In some embodiments, the FSH secretion stimulator 620 is activin, present in a concentration ranging from about 50 ng / mL to about 150 ng / mL. Sub-ranges may include about 50 ng / mL to about 140 ng / mL, about 55 ng / mL to about 135 ng / mL, about 60 ng / mL to about 130 ng / mL, about 65 ng / mL to about 125 ng / mL, about 70 ng / mL to about 120 ng / mL, about 75 ng / mL to about 115 ng / mL, about 80 ng / mL to about 110 ng / mL, about 85 ng / mL to about 105 ng / mL, or narrower ranges such as about 90 ng / mL to about 100 ng / mL. Application may be carried out for a period ranging from about 6 hours to about 48 hours. Sub-ranges may include about 6 hours to about 44 hours, about 8 hours to about 40 hours, about 10 hours to about 36 hours, about 12 hours to about 30 hours, about 15 hours to about 28 hours, about 18 hours to about 26 hours, or about 20 hours to about 24 hours.

[0113] In some embodiments, the stimulator 620 may be nodal, provided in concentrations ranging from about 25 ng / mL to about 200 ng / mL. Sub-ranges may include about 30 ng / mL to about 180 ng / mL, about 35 ng / mL to about 160 ng / mL, about 40 ng / mL to about 150 ng / mL, about 45 ng / mL to about 140 ng / mL, about 50 ng / mL to about 130 ng / mL, about 55 ng / mL to about 120 ng / mL, about 60 ng / mL to about 100 ng / mL, or about 65 ng / mL to about 90 ng / mL. Application may be for a period ranging from about 6 hours to about 72 hours, with sub-ranges including about 8 hours to about 60 hours, about 10 hours to about 48 hours, about 12 hours to about 36 hours, or about 15 hours to about 30 hours.

[0114] In some embodiments, the stimulator 620 may include activin A analogues, such as recombinant human activin A, provided in concentrations ranging from about 10 ng / mL to about 200 ng / mL. Sub-ranges may include about 15 ng / mL to about 180 ng / mL, about 20 ng / mL to about 150 ng / mL, about 25 ng / mL to about 125 ng / mL, or about 30 ng / mL to about 100 ng / mL. Application periods may range from about 12 hours to about 48 hours, with sub-ranges including about 14 hours to about 40 hours, about 16 hours to about 36 hours, or about 18 hours to about 30 hours.

[0115] In some embodiments, the stimulator 620 may include activin B, present in concentrations ranging from about 25 ng / mL to about 150 ng / mL. Sub-ranges may include about 30 ng / mL to about 130 ng / mL, about 35 ng / mL to about 110 ng / mL, about 40 ng / mL to about 100 ng / mL, or about 45 ng / mL to about 90 ng / mL. Application may be for a periodDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION ranging from about 6 hours to about 36 hours, with sub-ranges including about 8 hours to about 28 hours, about 10 hours to about 24 hours, or about 12 hours to about 20 hours.

[0116] In some embodiments, the stimulator 620 may include myostatin inhibitors or growth differentiation factor (GDF) analogues such as GDF-11, provided in concentrations ranging from about 25 ng / mL to about 150 ng / mL. Sub-ranges may include about 30 ng / mL to about 120 ng / mL, about 40 ng / mL to about 110 ng / mL, or about 50 ng / mL to about 100 ng / mL. Application may be for a period ranging from about 12 hours to about 48 hours, with sub-ranges including about 14 hours to about 40 hours, about 16 hours to about 36 hours, or about 18 hours to about 30 hours.

[0117] In some embodiments, combinations of activin with nodal or activin analogues may be employed to synergistically enhance mesendoderm induction. For example, activin may be applied at concentrations ranging from about 50 ng / mL to about 125 ng / mL, while nodal is provided at concentrations ranging from about 25 ng / mL to about 75 ng / mL, with co-application over a period ranging from about 12 hours to about 36 hours, including narrower windows such as about 18 hours to about 30 hours or about 20 hours to about 24 hours. The use of activin, nodal, or related analogues within these concentration and temporal ranges provides reliable induction of mesendodermal markers such as MIXL1 and EOMES, ensuring progression toward definitive endoderm. The breadth of ranges allows tuning of pathway activation to balance efficiency and cell viability, reducing the risk of overstimulation or incomplete lineage commitment. The optional inclusion of activin B, GDF analogues, or nodal combinations further broadens applicability, enabling optimization for different non-human primate iPSC lines. These features collectively improve reproducibility across laboratories, enhance scalability for bioreactor systems, and establish superiority over conventional methods that employ narrower activin-only regimens with limited control over timing or dose-response.

[0118] The inclusion of a FSH secretion stimulator 620, such as activin, nodal, or activin A analogues, provides several technical benefits in directing NHP iPSCs toward an anterior primitive streak state. By activating signaling through the TGF-p / activin / nodal pathway, these agents promote mesendodermal gene expression programs while suppressing ectodermal or alternative lineage commitments, thereby enhancing fidelity of lineage progression. The defined concentration ranges, such as about 50 ng / mL to about 150 ng / mL of activin or about 25 ng / mL to about 200 ng / mL of nodal, allow reproducible induction across experiments and reduce variability in cell populations. Controlled exposure periods,Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION for example from about 12 hours to about 36 hours, further minimize heterogeneity by preventing overstimulation or incomplete pathway activation.

[0119] Additional technical benefits arise from the availability of alternative agents, including recombinant activin A, activin B, and GDF family members such as GDF-11, which permit flexibility in protocol design and provide a safeguard against supply constraints and / or variable batch activity of recombinant proteins. In some embodiments, combinations of activin and nodal may be used to synergistically enhance mesendoderm specification, improving overall differentiation efficiency. Collectively, the use of FSH secretion stimulators at defined ranges and exposure durations improves cell viability, reduces differentiation errors, and facilitates that subsequent developmental stages such as definitive endoderm and posterior foregut can be reliably achieved, thereby producing hepatocyte-like cells with higher reproducibility and functional relevance for in vitro drug metabolism and toxicology applications.

[0120] In some embodiments, the GSK3 inhibitor 630 is CHIR99021, present in a concentration ranging from about 0.5 pM to about 6 pM, for example from about 1 pM to about 5 pM, or from about 2 pM to about 4 pM, and applied to the cells for a period of time ranging from about 6 hours to about 48 hours, for example from about 12 hours to about 36 hours, or about 24 hours. In some embodiments, the GSK3 inhibitor 630 may alternatively include BIO (6-bromoindirubin-3 ’ -oxime), which may be provided in a concentration ranging from about 0.1 pM to about 5 pM, for example about 1 pM to about 3 pM, and applied to the cells for about 6 hours to about 48 hours, such as about 18 to about 30 hours. In some embodiments, the GSK3 inhibitor 630 may alternatively include tideglusib, provided in a concentration ranging from about 0.5 pM to about 20 pM, for example from about 1 pM to about 10 pM, and applied to the cells for about 12 hours to about 48 hours, such as about 24 hours.

[0121] In some embodiments, the GSK3 inhibitor 630 may alternatively include lithium chloride (LiCl), present in a concentration ranging from about 1 mM to about 20 mM, for example from about 5 mM to about 15 mM, and applied for about 12 hours to about 72 hours, such as about 24 to about 48 hours. In some embodiments, the GSK3 inhibitor 630 may also include other small-molecule inhibitors such as SB-216763 or AR-A014418, provided in concentrations ranging from about 0.1 pM to about 20 pM, for example from about 0.5 pM to about 10 pM, and applied for periods ranging from about 6 hours to about 72 hours, such as about 24 to about 48 hour.Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION

[0122] The inclusion of a GSK3 inhibitor 630, such as CHIR99021, BIO, tideglusib, lithium chloride, SB-216763, or AR-A014418, provides technical benefits in stabilizing P- catenin and activating canonical Wnt signaling, which is needed for mesendoderm induction.Defined concentration ranges, such as about 0.5 pM to about 6 pM for CHIR99021, about 0.1 pM to about 5 pM for BIO, or about 1 mM to about 20 mM for lithium chloride, enable fine control of pathway activation and prevent cytotoxicity that may result from overactivation. Exposure periods of about 6 hours to about 48 hours, for example about 24 hours, enhance reproducibility by balancing efficient Wnt signaling with minimal off-target effects.Technical benefits include reducing variability across experimental replicates, ensuring consistent anterior primitive streak formation, and supporting downstream differentiation to definitive endoderm.

[0123] In some embodiments, the dual-kinase inhibitor 640 is PL103, present in a concentration ranging from about 25 nM to about 75 nM, for example from about 30 nM to about 70 nM, from about 40 nM to about 60 nM, and applied to the cells for a period of time ranging from about 6 hours to about 48 hours, for example from about 12 hours to about 36 hours, or about 24 hours.

[0124] In some embodiments, the dual-kinase inhibitor 640 may alternatively include LY294002, present in a concentration ranging from about 0.5 pM to about 10 pM, for example from about 1 pM to about 5 pM, and applied to the cells for a period of about 6 hours to about 48 hours, such as about 18 hours to about 30 hours.

[0125] In some embodiments, the dual-kinase inhibitor 640 may alternatively include wortmannin, present in a concentration ranging from about 5 nM to about 100 nM, for example from about 10 nM to about 50 nM, and applied to the cells for a period of about 6 hours to about 24 hours, such as about 12 hours.

[0126] In some embodiments, the dual-kinase inhibitor 640 may alternatively include BEZ235 (dactolisib), present in a concentration ranging from about 5 nM to about 100 nM, for example from about 10 nM to about 50 nM, and applied to the cells for about 6 to about 48 hours.

[0127] In some embodiments, the dual-kinase inhibitor 640 may alternatively include BGT226, present in a concentration ranging from about 1 nM to about 50 nM, for example from about 5 nM to about 25 nM, and applied for about 12 hours to about 36 hours.Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION

[0128] In some embodiments, the dual-kinase inhibitor 640 may also include XL765 (voxtalisib), present in a concentration ranging from about 0.01 pM to about 5 pM, for example from about 0.1 pM to about 1 pM, and applied for about 12 hours to about 48 hours.

[0129] In some embodiments, combinations of inhibitors may be applied to achieve synergistic suppression of PI3K and mTOR pathways, for example PI- 103 at about 40 nM to about 60 nM together with LY294002 at about 1 pM to about 3 pM, applied concurrently for about 18 hours to about 30 hours.

[0130] Technical benefits include reducing residual pluripotency markers, decreasing heterogeneous cell populations, and / or creating a synchronized cellular response for subsequent lineage transitions.

[0131] In some embodiments, the growth factor 650 is FGF2, present in a concentration ranging from about 5 ng / mL to about 35 ng / mL, for example from about 10 ng / mL to about 30 ng / mL, or from about 15 ng / mL to about 25 ng / mL, and applied to the cells for a period of time ranging from about 6 hours to about 48 hours, for example from about 12 hours to about 36 hours, or about 24 hours.

[0132] In some embodiments, the growth factor 650 may alternatively include fibroblast growth factor 4 (FGF4), present in a concentration ranging from about 5 ng / mL to about 50 ng / mL, for example from about 10 ng / mL to about 40 ng / mL, and applied to the cells for a period of time ranging from about 6 hours to about 72 hours, for example about 24 to about 48 hours.

[0133] In some embodiments, the growth factor 650 may alternatively include fibroblast growth factor 8 (FGF8), present in a concentration ranging from about 5 ng / mL to about 50 ng / mL, for example from about 15 ng / mL to about 40 ng / mL, and applied to the cells for about 12 to about 48 hours.

[0134] In some embodiments, the growth factor 650 may alternatively include fibroblast growth factor 10 (FGF10), present in a concentration ranging from about 2 ng / mL to about 30 ng / mL, for example from about 5 ng / mL to about 20 ng / mL, and applied to the cells for a period ranging from about 12 hours to about 72 hours, for example about 36 to about 48 hours.

[0135] In some embodiments, the growth factor 650 may alternatively include fibroblast growth factor 19 (FGF19), present in a concentration ranging from about 1 ng / mL to about 25 ng / mL, for example from about 5 ng / mL to about 15 ng / mL, and applied for about 12 hours to about 48 hours.Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION

[0136] In some embodiments, the growth factor 650 may alternatively include fibroblast growth factor 21 (FGF21), present in a concentration ranging from about 5 ng / mL to about 50 ng / mL, for example from about 10 ng / mL to about 30 ng / mL, and applied for about 12 hours to about 72 hours.

[0137] In some embodiments, combinations of fibroblast growth factors may be employed to improve lineage fidelity. For example, FGF2 may be used at a concentration of about 15 ng / mL to about 25 ng / mL in combination with FGF4 at about 10 ng / mL to about 20 ng / mL, applied concurrently for about 18 to about 36 hours, to synergistically promote anterior primitive streak induction.

[0138] The inclusion of a growth factor 650, such as FGF2, FGF4, or FGF8, provides technical benefits in maintaining cell survival during lineage transition and enhancing anterior primitive streak specification. Defined concentration ranges, such as about 5 ng / mL to about 35 ng / mL for FGF2, for example about 15 ng / mL to about 25 ng / mL, facilitate reproducible outcomes while preventing overstimulation of proliferation pathways. Exposure times of about 6 hours to about 48 hours, such as about 24 hours, allow sufficient activation of downstream MAPK / ERK signaling to stabilize mesendodermal fate. Technical benefits include improved viability of differentiating cells, reduced apoptosis during early lineage commitment, and synergistic interactions with activin and Wnt pathway activators to enhance the efficiency and / or fidelity of anterior primitive streak induction.

[0139] The first medium 600 comprises a FSH secretion stimulator 620, a GSK3 inhibitor 630, a dual-kinase inhibitor 640, and a growth factor 650. In some embodiments, the FSH secretion stimulator 620 is activin present in a concentration ranging from about 50 ng / mL to about 150 ng / mL, the GSK3 inhibitor 630 is CHIR99021 present in a concentration ranging from about 0.5 pM to about 6 pM, the dual-kinase inhibitor 640 is PI-103 present in a concentration ranging from about 25 nM to about 75 nM, and the growth factor 650 is FGF2 present in a concentration ranging from about 5 ng / mL to about 35 ng / mL. The first medium 600 may be applied to the NHP iPSCs for a first period of time ranging from about 6 hours to about 48 hours, with some embodiments providing exposure for about 24 hours.

[0140] In some embodiments, the NHP iPSCs are derived from Macaca fciscicularis. also known as the cynomolgus macaque. The use of Macaca fascicularis iPSCs provides an advantageous model for studying primate hepatic differentiation and generating hepatocytelike cells for applications in preclinical drug metabolism, hepatotoxicity screening, and disease modeling.Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION

[0141] The combination of the defined factors in the first medium 600 facilitates reproducible induction of an anterior primitive streak state, reduces variability in lineage progression, and enhances efficiency of downstream differentiation into definitive endoderm, posterior foregut, liver bud progenitors, hepatoblasts, and hepatocyte-like cells.

[0142] FIG. 7 illustrates the composition of a second medium 700 configured to induce transition of NHP iPSCs to a definitive endoderm state. The second medium 700 is applied following exposure of the NHP iPSCs to a first medium, and provides a synergistic combination of signaling modulators to promote lineage commitment while maintaining high cell viability.

[0143] In some embodiments, the FSH secretion stimulator 720 is activin, present in a concentration ranging from about 50 ng / mL to about 150 ng / mL, for example from about 75 ng / mL to about 125 ng / mL, and applied to the cells for a period ranging from about 6 hours to about 48 hours, such as about 24 hours. In other embodiments, the FSH secretion stimulator 720 may include nodal, activin A analogues, or activin B, applied in concentrations ranging from about 25 ng / mL to about 200 ng / mL for periods of about 12 to about 48 hours.

[0144] In some embodiments, the GSK3 inhibitor 730 is CHIR99021, provided in a concentration ranging from about 0.5 pM to about 6 pM, for example from about 2 pM to about 4 pM, and applied to the cells for about 6 hours to about 48 hours, such as about 24 hours. In other embodiments, the GSK3 inhibitor 730 may include BIO (6-bromoindirubin- 3'-oxime), tideglusib, SB-216763, or lithium chloride, with concentrations ranging from about 0.1 pM to about 5 pM for BIO or about 1 mM to about 20 mM for lithium chloride.

[0145] In some embodiments, the dual-kinase inhibitor 740 is PL103, present in a concentration ranging from about 25 nM to about 75 nM, for example from about 40 nM to about 60 nM, and applied to the cells for about 6 to about 48 hours. In alternative embodiments, the dual-kinase inhibitor 740 may include LY294002 at about 0.5 pM to about 10 pM, wortmannin at about 5 nM to about 100 nM, BEZ235 at about 5 nM to about 100 nM, or XL765 at about 0.01 pM to about 5 pM. These inhibitors may be provided for exposure durations ranging from about 6 to about 48 hours.

[0146] In some embodiments, the growth factor 750 is FGF2, provided in a concentration ranging from about 5 ng / mL to about 35 ng / mL, for example from about 15 ng / mL to about 25 ng / mL, and applied to the cells for about 6 to about 48 hours, such as about 24 hours. In other embodiments, the growth factor 750 may include FGF4, FGF8,Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATIONFGF10, or FGF19, provided in concentrations ranging from about 5 ng / mL to about 50 ng / mL, and applied to the cells for about 12 to about 72 hours. Combinations of fibroblast growth factors, such as FGF2 and FGF4, may also be applied to synergistically enhance definitive endoderm induction.

[0147] The defined concentrations and controlled exposure periods of the second medium 700 improve reproducibility, reduce variability in lineage commitment, and enhance the efficiency of definitive endoderm formation. By combining the FSH secretion stimulator 720, the GSK3 inhibitor 730, the dual-kinase inhibitor 740, and the growth factor 750, the medium provides a balanced environment for cell survival and lineage specification, thereby enabling consistent progression toward hepatocyte-like cell differentiation.

[0148] The second medium 700 provides several technical benefits by enabling reproducible and efficient induction of NHP iPSCs into a definitive endoderm lineage. The combination of the FSH secretion stimulator 720, GSK3 inhibitor 730, dual-kinase inhibitor 740, and growth factor 750 produces a synergistic effect that enhances lineage fidelity while minimizing off-target differentiation.

[0149] In some embodiments, the inclusion of a FSH secretion stimulator 720 such as activin, nodal, or activin A analogues in defined ranges (e.g., about 50 ng / mL to about 150 ng / mL for activin) promotes upregulation of one or more transcription factors such as SOX17 and / or FOXA2, which are implemented for definitive endoderm commitment. By providing these molecules in precise concentrations, the medium reduces variability in cell populations and facilitates consistency across experimental replicates.

[0150] In some embodiments, the GSK3 inhibitor 730, such as CHIR99021 or BIO, stabilizes P-catenin signaling and activates mesendodermal pathways. When applied in concentrations from about 0.5 pM to about 6 pM, the inhibitor enhances downstream endodermal induction and improves overall cell viability by reducing apoptosis during the lineage transition.

[0151] In some embodiments, the dual-kinase inhibitor 740, such as PI- 103, LY294002, or wortmannin, provides simultaneous inhibition of PI3K and mTOR pathways, which otherwise act as barriers to definitive endoderm specification. The use of such inhibitors in tightly controlled concentrations (e.g., PI- 103 at about 25 nM to about 75 nM) allows selective suppression of alternative fates such as ectodermal or mesodermal pathways, thereby increasing the efficiency of endodermal lineage induction.Docket No. 0138-704.600INTERNATIONAL PATENT APPLICATION

[0152] In some embodiments, the growth factor 750, such as FGF2, FGF4, or FGF8, supports proliferation and survival of differentiating cells while promoting expression of endodermal markers. Controlled delivery of FGF2 in concentrations of about 5 ng / mL to about 35 ng / mL, for example, maintains cell growth and metabolic activity during the window of definitive endoderm formation.

[0153] The combined effect of these four components results in higher reproducibility, improved lineage fidelity, and reduced variability between differentiation runs compared to undefined serum-based conditions. Furthermore, the second medium 700 enhances downstream transitions to posterior foregut and hepatic lineages, thereby improving the efficiency of generating hepatocyte-like cells for applications such as drug metabolism studies, hepatotoxicity assays, and in vitro disease modeling.

[0154] FIG. 8 illustrates the composition of a third medium 800 to induce transition of NHP iPSCs into a posterior foregut state. The third medium 800 is applied sequentially following exposure to a second medium, providing a defined signaling environment that promotes regionalization of definitive endoderm cells toward hepatic lineage commitment.

[0155] In some embodiments, the TGF-P receptor I inhibitor 820 is A83-01, present in a concentration ranging from about 0.5 pM to about 1.5 pM, for example about 0.75 pM to about 1.25 pM, and applied to the cells for a period ranging from about 6 hours to about 48 hours, such as about 24 hours. In other embodiments, the TGF-P receptor I inhibitor 820 may include SB-431542, SB-505124, or LY2157299, provided at concentrations ranging from about 0.1 pM to about 10 pM.

[0156] In some embodiments, the growth factor 850 is FGF2, present in a concentration ranging from about 55 ng / mL to about 165 ng / mL, for example from about 80 ng / mL to about 140 ng / mL, and applied to the cells for about 12 hours to about 36 hours, such as about 24 hours. In alternative embodiments, the growth factor 850 may include FGF4, FGF10, or hepatocyte growth factor (HGF), each applied in concentrations ranging from about 10 ng / mL to about 100 ng / mL for durations of about 6 to about 48 hours.

[0157] In some embodiments, the bone morphogenetic protein 830 is BMP4, present in a concentration ranging from about 15 ng / mL to about 45 ng / mL, for example from about 20 ng / mL to about 40 ng / mL, and applied to the cells for a duration ranging from about 6 hours to about 48 hours. In other embodiments, the bone morphogenetic protein 830 may include BMP2 or BMP7, provided in concentrations ranging from about 10 ng / mL to about 60 ng / mL, with application periods between about 12 hours and about 72 hours.Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION

[0158] In some embodiments, the retinoid pathway activator 840 is TTNPB, present in a concentration ranging from about 37 nM to about 113 nM, for example from about 60 nM to about 90 nM, and applied to the cells for about 6 hours to about 48 hours, such as about 24 hours. In other embodiments, the retinoid pathway activator 840 may include all- trans retinoic acid (ATRA) or AM580, with concentrations ranging from about 10 nM to about 500 nM and exposure durations of about 6 hours to about 72 hours.

[0159] The defined combination of TGF-P receptor I inhibitor 820, growth factor 850, bone morphogenetic protein 830, and retinoid pathway activator 840 in a base culture medium, such as CDM3 medium, provides synergistic regulation of signaling pathways that guide definitive endoderm cells into a posterior foregut state. This stage establishes competency for subsequent liver bud progenitor induction. The third medium 800 enhances reproducibility, reduces variability, and improves lineage fidelity compared to undefined or serum -based conditions, resulting in a more efficient differentiation trajectory toward hepatocyte-like cells.

[0160] The third medium 800 provides several technical benefits by directing NHP iPSCs that have transitioned through definitive endoderm into a posterior foregut state with enhanced reproducibility and lineage fidelity. This transition is essential for establishing a competent cellular population capable of differentiating into liver bud progenitors, a step in the progression toward hepatocyte-like cells.

[0161] In some embodiments, the TGF-P receptor I inhibitor 820, such as A83-01, SB-431542, SB-505124, or LY2157299, effectively suppresses residual mesodermal or ectodermal signaling pathways that may compete with endodermal specification. By providing this inhibitory activity in tightly controlled concentrations (e.g., about 0.5 pM to about 1.5 pM for A83-01), the medium facilitates higher purity of posterior foregut populations and reduces off-target differentiation events.

[0162] In some embodiments, the growth factor 850, such as FGF2, FGF4, or HGF, enhances proliferation, survival, and metabolic activity of differentiating endoderm cells. The inclusion of these growth factors in defined concentration ranges (e.g., FGF2 at about 55 ng / mL to about 165 ng / mL) promotes activation of hepatic transcriptional programs while maintaining cell viability under lineage transition stress.

[0163] In some embodiments, the bone morphogenetic protein 830, such as BMP4, BMP2, or BMP7, provides signaling cues required for anterior-posterior patterning and hepatic competence. Controlled addition of BMPs in concentrations between about 15 ng / mLDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION and about 60 ng / mL reduces variability in differentiation efficiency and improves homogeneity across culture replicates.

[0164] In some embodiments, the retinoid pathway activator 840, such as TTNPB, ATRA, or AM580, induces gene regulatory networks associated with foregut patterning and hepatic lineage commitment. By precisely applying retinoid agonists in the nanomolar to low micromolar range (e.g., TTNPB at about 37 nM to about 113 nM), the medium promotes upregulation of transcription factors such as HNFip and HNF4a that are utilized for hepatocyte specification.

[0165] The synergistic action of these four components in a defined base culture medium, such as CDM3, produces a controlled signaling environment that closely mimics developmental pathways observed in vivo. This structured approach reduces reliance on undefined factors, lowers variability across experiments, and improves scalability for translational applications. Accordingly, the third medium 800 enables more consistent production of posterior foregut cells, thereby strengthening the overall efficiency and reliability of in vitro hepatocyte differentiation protocols.

[0166] FIG. 9 illustrates an embodiment of a system 500 including a cell culture platform 510 and a fourth medium 900 formulated to induce transition of NHP iPSCs into a liver bud progenitor state. In some embodiments, the fourth medium 900 comprises a FSH secretion stimulator 920, a bone morphogenetic protein (BMP) 930, and an adenylyl cyclase activator 940 in a defined base culture medium, such as CDM3.

[0167] In some embodiments, the FSH secretion stimulator 920 is activin, present in a concentration ranging from about 5 ng / mL to about 15 ng / mL, for example from about 8 ng / mL to about 12 ng / mL, and applied to the cells for a period ranging from about 1 day to about 5 days, for example about 3 days. In other embodiments, the FSH secretion stimulator may alternatively include nodal or activin A analogues provided in equivalent concentrations and exposure durations.

[0168] In some embodiments, the bone morphogenetic protein 930 is BMP4, present in a concentration ranging from about 15 ng / mL to about 45 ng / mL, for example from about 22 ng / mL to about 38 ng / mL, and applied over the same 1-5 day period. Alternative BMPs, such as BMP2 or BMP7, may also be used in similar ranges, thereby broadening the flexibility of the protocol while maintaining effective hepatic lineage induction.

[0169] In some embodiments, the adenylyl cyclase activator 940 is forskolin, present in a concentration ranging from about 0.5 pM to about 1.5 pM, for example about 0.75 pM toDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION about 1.25 pM, with representative use at about 1 pM. Other small-molecule adenylyl cyclase activators, such as 3-isobutyl-l-methylxanthine (IBMX), may alternatively be employed in the range of about 10 pM to about 50 pM, depending on desired signaling modulation.

[0170] The fourth medium 900 thus creates a defined signaling environment that supports hepatic competence by simultaneously activating activin and BMP pathways while modulating intracellular cAMP signaling through adenylyl cyclase activation. The synergy of these three components results in efficient induction of liver bud progenitor cells with high reproducibility and reduced variability between replicates. The use of chemically defined components eliminates reliance on serum-derived factors, thereby improving experimental consistency and enabling scale-up for translational applications.

[0171] The fourth medium 900 provides several technical benefits by enabling reliable induction of liver bud progenitor cells from posterior foregut populations of NHP iPSCs. This stage is a pivotal checkpoint in hepatocyte differentiation, as liver bud progenitors represent an intermediate lineage that bridges early foregut identity and hepatoblast specification.

[0172] In some embodiments, the inclusion of a FSH secretion stimulator 920, such as activin, nodal, or activin A analogues, supports lineage stabilization and prevents regression into non-hepatic endodermal fates. Concentrations in the range of about 5 ng / mL to about 15 ng / mL, for example about 8 ng / mL to about 12 ng / mL, provide sufficient signaling strength to maintain cell viability while steering differentiation toward hepatic precursors.

[0173] In some embodiments, the bone morphogenetic protein 930, such as BMP4, BMP2, or BMP7, is provided in concentrations between about 15 ng / mL and about 45 ng / mL, for example about 22 ng / mL to about 38 ng / mL. This BMP activity enhances anterior-posterior patterning, primes expression of hepatic transcription factors, and improves efficiency of liver bud progenitor specification across replicates.

[0174] In some embodiments, the adenylyl cyclase activator 940, such as forskolin or IBMX, enhances intracellular cAMP signaling, which cooperates with BMP and activin pathways to promote hepatic competency. Precise dosing, for example forskolin at about 0.75 pM to about 1.25 pM or IBMX at about 10 pM to about 50 pM, improves reproducibility while avoiding cytotoxicity from overstimulation.

[0175] By combining these three defined signaling inputs in a controlled base medium, such as CDM3, the fourth medium 900 minimizes variability observed with serum-Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION based differentiation protocols, reduces incomplete lineage transitions, and improves reproducibility of progenitor induction. The synergistic action of activin, BMPs, and adenylyl cyclase activators provides a developmental framework, resulting in liver bud progenitor cells that consistently progress toward hepatoblasts and mature hepatocyte-like cells in downstream steps.

[0176] FIG. 10 illustrates an embodiment of a system 500 including a cell culture platform 510 and a fifth medium 1000 formulated to promote hepatoblast specification from liver bud progenitor cells derived from NHP iPSCs. In some embodiments, the fifth medium 1000 comprises a bone morphogenetic protein 1020, a pleiotropic cytokine 1030, a glucocorticoid 1070, a gamma secretase inhibitor 1040, an adenylyl cyclase activator 1080, an anabolic hormone 1050, a vitamin C derivative 1090, and an amino acid supply 1060 in a defined base culture medium, such as CDM4.

[0177] In some embodiments, the bone morphogenetic protein 1020 is BMP4, present in a concentration ranging from about 5 ng / mL to about 15 ng / mL, for example from about 8 ng / mL to about 12 ng / mL, and applied for a period ranging from about 5 days to about 7 days, such as about 6 days. BMP4 functions through SMAD1 / 5 / 8 signaling pathways to promote hepatic lineage commitment, enhance proliferation of hepatoblasts, and reinforce downstream activation of hepatic transcription factors such as HNF4a and TBX3. Controlled application of BMP4 within these concentration ranges facilitates effective hepatoblast specification without triggering excessive mesodermal or osteogenic differentiation.

[0178] In other embodiments, the bone morphogenetic protein 1020 may alternatively include BMP2, applied at concentrations ranging from about 10 ng / mL to about 40 ng / mL, for example from about 15 ng / mL to about 25 ng / mL. BMP2 provides similar signaling effects to BMP4, activating SMAD-mediated transcription while supporting hepatoblast proliferation. BMP2 may be selected in embodiments where enhanced survival signaling is desired or when differentiating cell lines that respond to BMP2 stimulation.

[0179] In further embodiments, the bone morphogenetic protein 1020 may include BMP7, applied at concentrations ranging from about 10 ng / mL to about 40 ng / mL, for example from about 20 ng / mL to about 35 ng / mL. BMP7 has been shown to balance hepatoblast proliferation with reduced apoptosis, and in some cases, may provide additional protection against differentiation into non-hepatic mesodermal lineages.

[0180] In some embodiments, combinations of bone morphogenetic proteins (BMPs) may be employed to achieve synergistic effects on hepatic lineage induction. For example,Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATIONBMP4 may be provided in a concentration ranging from about 2 ng / mL to about 50 ng / mL, and BMP7 may be provided in a concentration ranging from about 5 ng / mL to about 60 ng / mL. Sub-ranges for BMP4 may include about 3 ng / mL to about 45 ng / mL, about 4 ng / mL to about 40 ng / mL, about 5 ng / mL to about 35 ng / mL, about 6 ng / mL to about 30 ng / mL, about 7 ng / mL to about 25 ng / mL, about 8 ng / mL to about 20 ng / mL, about 9 ng / mL to about 18 ng / mL, about 10 ng / mL to about 15 ng / mL, and / or about 11 ng / mL to about 13 ng / mL. Sub-ranges for BMP7 may include about 8 ng / mL to about 55 ng / mL, about 10 ng / mL to about 50 ng / mL, about 12 ng / mL to about 45 ng / mL, about 15 ng / mL to about 40 ng / mL, about 18 ng / mL to about 35 ng / mL, about 20 ng / mL to about 30 ng / mL, about 22 ng / mL to about 28 ng / mL, and / or about 24 ng / mL to about 26 ng / mL.

[0181] In further embodiments, BMP4 may be administered at about 5 ng / mL to about 15 ng / mL while BMP7 is administered at about 15 ng / mL to about 25 ng / mL, and / or BMP4 may be administered at about 15 ng / mL to about 25 ng / mL in combination with BMP7 at about 25 ng / mL to about 35 ng / mL. Still further, BMP2 may be incorporated at concentrations ranging from about 5 ng / mL to about 25 ng / mL, with sub-ranges including about 7 ng / mL to about 20 ng / mL or about 10 ng / mL to about 15 ng / mL, to complement BMP4 and BMP7 activity. In some embodiments, two or more BMPs are applied sequentially, wherein BMP4 is provided in an early exposure period ranging from about 6 hours to about 72 hours, and BMP7 is added in a subsequent exposure period ranging from about 24 hours to about 7 days. In other embodiments, BMP4 and BMP7 are co-applied continuously for a duration ranging from about 2 days to about 10 days, with sub-ranges including about 3 days to about 8 days, about 4 days to about 7 days, or about 5 days to about 6 days. The combined administration of these proteins within the disclosed ranges enhances hepatoblast specification, increases expression of hepatic lineage genes such as HNF4a and TBX3, improves cell survival by reducing apoptosis, and / or reduces variability of differentiation outcomes compared to protocols using single BMP factors.

[0182] In further embodiments, sequential application of BMP2 followed by BMP4 may be used to fine-tune temporal signaling, ensuring proper activation of hepatic gene programs at different stages of hepatoblast development. The bone morphogenetic protein 1020 therefore serves as a driver of hepatic lineage specification in the fifth medium 1000. By modulating SMAD signaling through BMP4, BMP2, BMP7, or combinations thereof, the differentiation protocol achieves improved reproducibility, reduced heterogeneity, and enhanced fidelity in generating hepatoblasts suitable for downstream hepatic maturation.Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION

[0183] In some embodiments, the pleiotropic cytokine 1030 is OSM, present in a concentration ranging from about 5 ng / mL to about 15 ng / mL, for example from about 8 ng / mL to about 12 ng / mL, and applied over a period of about 5 days to about 7 days, such as about 6 days. OSM functions through activation of the gpl30 receptor complex and downstream JAK / STAT signaling, which enhances hepatoblast survival, promotes differentiation toward hepatic lineages, and upregulates metabolic gene expression associated with hepatocyte function.

[0184] In other embodiments, the pleiotropic cytokine 1030 may include leukemia inhibitory factor (LIF), present in concentrations ranging from about 2 ng / mL to about 20 ng / mL, for example from about 5 ng / mL to about 15 ng / mL. LIF supports cell proliferation and survival through STAT3 activation and may be particularly useful in maintaining progenitor cell viability during extended culture.

[0185] In some embodiments, the pleiotropic cytokine 1030 may alternatively include IL-6, provided at concentrations ranging from about 2 ng / mL to about 20 ng / mL, such as about 5 ng / mL to about 15 ng / mL. IL-6 promotes acute-phase hepatic responses and enhances the expression of hepatocyte markers, such as albumin and cytochrome P450 enzymes, when used in combination with glucocorticoids and adenylyl cyclase activators.

[0186] In further embodiments, combinations of pleiotropic cytokines may be used to synergistically activate hepatic signaling networks. For example, OSM at about 5 ng / mL to about 15 ng / mL may be combined with IL-6 at about 5 ng / mL to about 15 5 ng / mL to enhance both survival and functional maturation. Alternatively, LIF may be paired with OSM to stabilize hepatoblast populations while promoting downstream hepatocyte specification.

[0187] Additional pleiotropic cytokines may also be employed, such as cardiotrophin- 1 (CT-1) or ciliary neurotrophic factor (CNTF), each of which activates gpl30-mediated signaling and may substitute for or augment the effects of OSM, LIF, or IL-6. These factors may be applied at concentrations ranging from about 1 ng / mL to about 25 ng / mL, depending on potency and desired effects.

[0188] The pleiotropic cytokine 1030 thereby provides signaling input to reinforce hepatoblast identity, enhance cell viability, and activate hepatic differentiation pathways. When combined with other components of the fifth medium 1000, the cytokine reduces variability in lineage outcomes, improves reproducibility across cultures, and increases the efficiency of hepatoblast specification for subsequent hepatic maturation.Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION

[0189] In some embodiments, the glucocorticoid 1070 is dexamethasone, present in a concentration ranging from about 5 pM to about 15 pM, for example from about 8 pM to about 12 pM, and applied to the cells for a duration of about 5 days to about 7 days, such as about 6 days. Dexamethasone is a synthetic corticosteroid that binds the glucocorticoid receptor, modulating gene expression programs involved in metabolism, stress response, and hepatic maturation. During hepatoblast specification, dexamethasone promotes expression of hepatic enzymes, enhances metabolic stability, and improves cell survival under differentiation stress.

[0190] In other embodiments, the glucocorticoid 1070 may be hydrocortisone, present in concentrations ranging from about 1 pM to about 20 pM, or prednisolone, present in concentrations ranging from about 2 pM to about 15 pM. Hydrocortisone, a naturally occurring glucocorticoid, provides a broader physiological signaling profile, while prednisolone offers intermediate potency compared to dexamethasone. These alternatives may be selected based on desired signaling strength, cytotoxicity thresholds, or compatibility with other medium components.

[0191] In further embodiments, other glucocorticoid analogues may be employed, including triamcinolone acetonide or methylprednisolone, applied at concentrations ranging from about 1 pM to about 15 pM. These compounds can be used to fine-tune glucocorticoid receptor activation and provide flexibility in adjusting the differentiation protocol for different non-human primate or human iPSC lines.

[0192] In some embodiments, the glucocorticoid 1070 is applied in combination with adenylyl cyclase activators, such as forskolin or IBMX, to synergistically enhance cAMP and glucocorticoid receptor signaling. This combination promotes stronger activation of hepatic transcriptional networks and increases production of liver-specific proteins such as albumin.

[0193] The glucocorticoid 1070 thereby plays a role in reinforcing hepatoblast identity, stabilizing metabolic functions, and preparing cells for subsequent maturation steps. By applying dexamethasone or alternative glucocorticoids within precise micromolar ranges, the fifth medium 1000 achieves improved reproducibility, reduced heterogeneity, and enhanced downstream potential for hepatocyte-like cell differentiation.

[0194] In some embodiments, the gamma secretase inhibitor 1040 is RO4929097, present in a concentration ranging from about 1 pM to about 3 pM, for example from about 1.5 pM to about 2.5 pM, and applied to the cells for a period ranging from about 5 days to about 7 days, such as about 6 days. RO4929097 inhibits gamma secretase activity, therebyDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION blocking cleavage and activation of the Notch receptor. By suppressing Notch signaling, the inhibitor prevents diversion of progenitors toward cholangiocyte (biliary) lineages and enhances specification of hepatoblasts, thereby increasing lineage fidelity.

[0195] In other embodiments, the gamma secretase inhibitor 1040 may be DAPT, provided at concentrations ranging from about 2 pM to about 20 pM. Within this broader range, DAPT may be applied in a narrower range of about 5 pM to about 15 pM, or in a range of about 5 pM to about 10 pM. In still further embodiments, an exemplary concentration may be about 7 pM to about 9 pM, such as about 8 pM. DAPT may be applied for a period ranging from about 24 hours to about 7 days, for example from about 48 hours to about 96 hours, or for about 72 hours, depending on the stage of differentiation and the desired extent of Notch pathway inhibition. In some embodiments, pulsed dosing regimens of DAPT may be employed, such as administration every 12 to 24 hours at concentrations ranging from about 2 pM to about 8 pM, thereby reducing potential cytotoxicity while maintaining effective Notch suppression. DAPT has been shown to effectively inhibit Notch- mediated transcriptional programs and improve hepatoblast yield when combined with supportive cytokines and growth factors.

[0196] In some embodiments, alternative gamma secretase inhibitors may be employed, including LY411575, applied at concentrations ranging from about 0.1 pM to about 5 pM, for example from about 0.3 pM to about 2.5 pM, or from about 0.5 pM to about 2 pM. In some embodiments, LY411575 may be applied for durations ranging from about 24 hours to about 96 hours, such as about 48 hours, to achieve sustained suppression of Notch signaling. In further embodiments, MK-0752 may also be used at concentrations ranging from about 0.5 pM to about 10 pM, for example from about 1 pM to about 5 pM, or about 2 pM to about 4 pM, depending on the sensitivity of the differentiating cells. MK-0752 may be applied for a treatment period of about 2 days to about 7 days, such as about 3 days to about 5 days, to provide controlled modulation of y-secretase activity. In yet other embodiments, PF- 03084014 may be used at concentrations ranging from about 0.1 pM to about 5 pM, such as about 0.25 pM to about 2 pM, with exposure times ranging from about 24 hours to about 72 hours. The use of these alternative y-secretase inhibitors provides flexibility in tailoring inhibition strength and duration, allowing optimization across different non-human primate iPSC lines and culture conditions.Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION

[0197] These inhibitors function through similar mechanisms by preventing proteolytic activation of Notch and related substrates, thereby shifting cell fate decisions toward hepatoblast specification.

[0198] In some embodiments, combinations of gamma secretase inhibitors may be used to modulate Notch activity with greater precision. For example, RO4929097 may be applied at concentrations ranging from about 0.5 pM to about 5 pM, for example from about 1 pM to about 3 pM, such as about 2 pM, and combined with DAPT provided at concentrations ranging from about 2 pM to about 12 pM, for example from about 3 pM to about 7 pM, such as about 5 pM to about 6 pM. In certain embodiments, this combination may be applied continuously for a period of about 24 hours to about 96 hours, such as about 48 hours, to provide broad pathway inhibition. In other embodiments, pulsed dosing regimens may be employed, such as alternating RO4929097 at about 1.5 pM to about 2.5 pM with DAPT at about 4 pM to about 8 pM every 12 to 24 hours, thereby reducing potential cytotoxic effects while maintaining consistent Notch suppression. In further embodiments, triple-inhibitor strategies may be implemented, such as combining LY411575 at about 0.2 pM to about 2 pM with RO4929097 at about 1 pM to about 4 pM and DAPT at about 5 pM to about 10 pM. Such combinations facilitate fine-tuned modulation of Notch signaling, suppressing cholangiocyte differentiation while maintaining hepatoblast proliferation and / or promoting downstream hepatic maturation. These ranges provide flexibility to optimize lineage fidelity across different non-human primate iPSC lines and culture conditions.

[0199] The gamma secretase inhibitor 1040 thus provides modulation of cell fate by selectively attenuating Notch signaling during hepatoblast specification. When applied in defined concentration ranges, the inhibitor reduces heterogeneity in differentiating populations, enhances reproducibility across culture batches, and improves the efficiency of generating hepatoblasts with high hepatic lineage potential for downstream maturation.

[0200] In some embodiments, the adenylyl cyclase activator 1080 is forskolin, present in a concentration ranging from about 5 pM to about 15 pM, for example from about 8 pM to about 12 pM, and applied to the cells for a period ranging from about 5 days to about 7 days, such as about 6 days. Forskolin directly stimulates adenylyl cyclase, increasing intracellular cAMP levels and thereby activating protein kinase A (PKA) signaling pathways. This activity enhances hepatoblast proliferation, promotes hepatic lineage gene expression, and synergizes with cytokines such as oncostatin M and anabolic hormones such as insulin to improve metabolic activity during differentiation.Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION

[0201] In other embodiments, the adenylyl cyclase activator 1080 may be IB MX, applied at concentrations ranging from about 10 pM to about 50 pM, for example from about 20 pM to about 40 pM. IBMX functions as a non-specific phosphodiesterase inhibitor, which elevates intracellular cAMP by preventing its degradation. Use of IBMX may prolong PKA pathway activation and sustain downstream signaling cascades beneficial for hepatoblast specification.

[0202] In some embodiments, alternative adenylyl cyclase activators may be employed, including prostaglandin E2 (PGE2) provided at concentrations ranging from about 0.1 pM to about 5 pM, or dibutyryl cAMP, a cell-permeable cAMP analogue, applied in concentrations ranging from about 100 pM to about 500 pM. These alternatives provide additional flexibility in tuning intracellular cAMP signaling, either through receptor-mediated activation (e.g., PGE2) or direct cAMP supplementation (e.g., dibutyryl cAMP).

[0203] In further embodiments, combinations of adenylyl cyclase activators may be used to produce complementary effects. For example, forskolin at about 5 pM to about 15 pM may be combined with IBMX at about 20 pM to about 30 pM to provide both stimulation of adenylyl cyclase activity and inhibition of cAMP degradation, thereby producing a stronger and more sustained elevation of intracellular cAMP levels.

[0204] The inclusion of the adenylyl cyclase activator 1080 in the fifth medium 1000 provides multiple benefits, including amplification of growth factor and cytokine signaling, enhancement of hepatoblast expansion, and promotion of functional hepatic maturation in downstream steps. Controlled modulation of intracellular cAMP levels improves reproducibility across differentiation experiments and minimizes variability associated with endogenous signaling fluctuations.

[0205] In some embodiments, the anabolic hormone 1050 is recombinant human insulin, provided in a concentration ranging from about 5 ng / mL to about 15 ng / mL, for example from about 8 ng / mL to about 12 ng / mL, and applied to the cells for a period of about 5 days to about 7 days, such as about 6 days. Recombinant human insulin supports glucose uptake, protein synthesis, and anabolic signaling via activation of the PI3K / AKT pathway, thereby promoting proliferation and metabolic activity of hepatoblasts. In further embodiments, recombinant human insulin may be delivered as part of a stabilized supplement, such as an insulin-transferrin-selenium (ITS) mixture, provided at equivalent concentrations.Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION

[0206] In some embodiments, alternative anabolic hormones may include IGF-1 (insulin-like growth factor- 1), applied at concentrations ranging from about 5 ng / mL to about 50 ng / mL, for example about 10 ng / mL to about 30 ng / mL. IGF-1 provides similar anabolic and mitogenic effects to insulin while additionally promoting hepatic lineage gene expression and survival signaling. In other embodiments, IGF-2 may also be employed, applied in concentrations ranging from about 5 ng / mL to about 40 ng / mL, with particular benefits in enhancing early hepatoblast proliferation.

[0207] In some embodiments, additional anabolic hormones or growth factors may be substituted or combined, such as epidermal growth factor (EGF) at concentrations of about 5 ng / mL to about 20 ng / mL, or hepatocyte growth factor (HGF) at concentrations of about 10 ng / mL to about 50 ng / mL, both of which enhance hepatoblast expansion and promote the expression of hepatic transcription factors. In further embodiments, combinations of insulin with IGF-1 or HGF may be used to provide synergistic anabolic signaling and improved lineage fidelity.

[0208] The anabolic hormone 1050 thus provides support for energy metabolism, protein synthesis, and cellular proliferation during hepatoblast specification. Defined supplementation with insulin, IGF-1, or related anabolic factors reduces reliance on serum- derived hormones, minimizes variability across differentiation batches, and enhances downstream maturation efficiency into hepatocyte-like cells.

[0209] In some embodiments, the vitamin C derivative 1090 is AAP, present in a concentration ranging from about 100 pg / mL to about 300 pg / mL, for example from about 150 pg / mL to about 250 pg / mL, and applied continuously over the 5-7 day period of hepatoblast specification. In further embodiments, the vitamin C derivative 1090 may be delivered in sustained-release form to maintain stable intracellular antioxidant activity without the rapid degradation associated with unmodified ascorbic acid.

[0210] In some embodiments, other stabilized vitamin C derivatives may be employed, including sodium ascorbyl phosphate, magnesium ascorbyl phosphate, or 3-O- ethyl-L-ascorbic acid, each provided in comparable concentration ranges of about 100 pg / mL to about 500 pg / mL. These stabilized derivatives are less prone to oxidation in culture and provide consistent antioxidant support. In other embodiments, direct supplementation with L- ascorbic acid may also be used, provided at concentrations ranging from about 25 pg / mL to about 200 pg / mL, although such use may require frequent replenishment due to rapid breakdown under standard culture conditions.Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION

[0211] In some embodiments, the vitamin C derivative 1090 enhances epigenetic remodeling by promoting DNA demethylation and histone hydroxylation, thereby facilitating activation of hepatic transcriptional programs such as HNF4a and ALB. In further embodiments, vitamin C supplementation may improve extracellular matrix deposition, enhancing the structural environment required for hepatoblast proliferation and survival. For example, AAP at about 150 pg / mL to about 250 pg / mL has been shown to increase collagen synthesis and improve cytoskeletal organization in differentiating cells.

[0212] The vitamin C derivative 1090 thus provides multiple synergistic functions, including reduction of oxidative stress, stabilization of redox balance, enhancement of epigenetic accessibility, and promotion of extracellular matrix integrity. These combined effects improve hepatoblast viability, reduce apoptosis, and enhance downstream hepatic maturation efficiency when transitioning into the sixth medium.

[0213] In some embodiments, the amino acid supply 1060 comprises an amino acid concentrate supplement that provides both essential and non-essential amino acids at physiologically relevant concentrations to support cellular growth and differentiation. The amino acid supply may include commercially available formulations such as B27, N2, or StemPro supplements, each of which contains a standardized mixture of amino acids, vitamins, and antioxidants designed to support mammalian cell cultures. In some embodiments, the amino acid supply 1060 may alternatively include a customized formulation of amino acids tailored for hepatic differentiation, wherein essential amino acids such as leucine, isoleucine, valine, lysine, methionine, phenylalanine, threonine, and tryptophan are provided at concentrations ranging from about 0.05 mM to about 2 mM, and non-essential amino acids such as alanine, asparagine, aspartic acid, glutamic acid, glycine, proline, serine, and tyrosine are provided at concentrations ranging from about 0.01 mM to about 1 mM.

[0214] In some embodiments, glutamine or a stabilized glutamine analogue such as GlutaMAX is included at concentrations ranging from about 1 mM to about 10 mM, for example about 2 mM to about 6 mM, to support energy metabolism and nucleotide biosynthesis. In further embodiments, branched-chain amino acids (BCAAs) such as leucine, isoleucine, and valine may be provided in concentrations ranging from about 0.1 mM to about 5 mM to enhance anabolic activity and support protein synthesis during hepatoblast expansion. In still further embodiments, cysteine or N-acetylcysteine may be added inDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION concentrations of about 0.05 mM to about 1 mM to support antioxidant defenses and maintain intracellular redox balance.

[0215] The amino acid supply 1060 thereby supports metabolic activity and prevents nutrient depletion during the about 5 day to about 7 day period of hepatoblast specification. By providing defined and reproducible amino acid concentrations, this component reduces variability across differentiation batches, enhances cell survival, and maintains biosynthetic capacity for macromolecule production, thereby improving the efficiency and reproducibility of hepatoblast specification in the fifth medium 1000.

[0216] The combined use of these eight components in the fifth medium 1000 provides a defined signaling and metabolic environment that promotes hepatoblast specification with high efficiency and reproducibility. By integrating BMP, cytokine, and retinoid signaling with hormonal and nutrient support, the fifth medium 1000 improves lineage fidelity, reduces incomplete differentiation, and generates hepatoblast populations with proliferative and / or metabolic potential suitable for downstream hepatic maturation.

[0217] The fifth medium 1000 provides technical benefits by promoting the specification of liver bud progenitors into hepatoblasts, a transitional cell population essential for generating functional hepatocytes. The defined composition of the medium integrates growth factors, hormones, signaling pathway modulators, and nutrient supplements to achieve lineage fidelity and / or reduce variability compared to undefined or serum-containing approaches.

[0218] In some embodiments, the bone morphogenetic protein 1020 comprises one or more of BMP4, BMP2, or BMP7, and is provided in concentrations ranging from about 2 ng / mL to about 40 ng / mL. Sub-ranges include about 3 ng / mL to about 35 ng / mL, about 4 ng / mL to about 25 ng / mL, or about 5 ng / mL to about 20 ng / mL. In certain embodiments, the bone morphogenetic protein 1020 is provided at about 5 ng / mL to about 15 ng / mL, with further refined ranges such as about 6 ng / mL to about 14 ng / mL, about 7 ng / mL to about 13 ng / mL, or about 8 ng / mL to about 12 ng / mL. In other embodiments, higher levels may be employed, such as about 10 ng / mL to about 30 ng / mL or about 15 ng / mL to about 40 ng / mL, depending on the specific stage of differentiation and species source of the iPSCs. The bone morphogenetic protein may be applied for a period ranging from about 6 hours to about 5 days, for example from about 12 hours to about 72 hours, about 18 hours to about 96 hours, or about 2 days to about 4 days. In some embodiments, the protein is administered continuously, while in others it is pulsed in intervals ranging from about every 12 hours toDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION about every 24 hours. Provision of BMP4, BMP2, and / or BMP7 within these ranges enhances anterior-posterior signaling, primes hepatic lineage programs, and / or increases expression of hepatocyte-specific transcription factors such as HNF4a and / or TBX3. This controlled BMP signaling increases the efficiency of hepatoblast induction and reduces the frequency of cells diverting into non-hepatic fates.

[0219] In some embodiments, the pleiotropic cytokine 1030 comprises OSM, LIF, or IL-6, each of which activates the JAK / STAT signaling pathway and contributes to hepatoblast proliferation, maturation, and survival. In certain embodiments, OSM is provided in a concentration ranging from about 2 ng / mL to about 25 ng / mL. Sub-ranges may include about 3 ng / mL to about 20 ng / mL, about 4 ng / mL to about 18 ng / mL, about 5 ng / mL to about 15 ng / mL, about 6 ng / mL to about 14 ng / mL, about 7 ng / mL to about 13 ng / mL, about 8 ng / mL to about 12 ng / mL, or narrower windows such as about 9 ng / mL to about 11 ng / mL.

[0220] In some embodiments, LIF is provided in concentrations ranging from about 2 ng / mL to about 30 ng / mL, for example about 4 ng / mL to about 25 ng / mL, about 5 ng / mL to about 20 ng / mL, about 8 ng / mL to about 15 ng / mL, or about 10 ng / mL to about 12 ng / mL. Similarly, IL-6 may be included in concentrations ranging from about 2 ng / mL to about 40 ng / mL, for example about 3 ng / mL to about 30 ng / mL, about 5 ng / mL to about 25 ng / mL, about 8 ng / mL to about 20 ng / mL, or about 10 ng / mL to about 15 ng / mL.

[0221] Application of these cytokines may occur over a treatment period ranging from about 3 days to about 10 days, with sub-ranges including about 4 days to about 8 days, about 5 days to about 7 days, or about 6 days as a representative exposure. Provision within these defined windows reduces batch-to-batch variability, improves reproducibility of hepatoblast differentiation, and provides consistent activation of hepatic gene expression programs such as those associated with albumin, alpha-fetoprotein, and hepatocyte nuclear factor expression.

[0222] In some embodiments, the glucocorticoid 1070, such as dexamethasone, improves metabolic stability and enhances expression of liver-specific enzymes. Glucocorticoid treatment in the range of about 5 pM to about 15 pM supports hepatoblast metabolic function and improves maturation potential in subsequent differentiation steps.

[0223] In some embodiments, the gamma secretase inhibitor 1040, such as RO4929097 or DAPT, precisely modulates Notch signaling, a pathway that otherwise promotes cholangiocyte specification. By inhibiting Notch in controlled ranges (e.g.,Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATIONRO4929097 at about 1.5 pM to about 2.5 pM), the medium biases progenitors toward hepatoblast identity, thereby improving lineage fidelity.

[0224] In some embodiments, the adenylyl cyclase activator 1080, such as forskolin or IBMX, elevates intracellular cAMP levels and enhances synergistic signaling with glucocorticoids and cytokines. This promotes hepatoblast proliferation and functional maturation, while concentrations of about 8 pM to about 12 pM forskolin provide reproducible outcomes without cytotoxicity.

[0225] In some embodiments, the anabolic hormone 1050, such as recombinant human insulin or IGF-1, sustains energy metabolism and cell growth. Defined insulin supplementation (e.g., 8 ng / mL to about 12 ng / mL) facilitates consistent metabolic support and enhances hepatoblast resilience during lineage specification.

[0226] In some embodiments, the vitamin C derivative 1090, such as ascorbic acid-2 phosphate, reduces oxidative stress and enhances epigenetic remodeling, supporting stable hepatoblast identity. Concentrations of about 150 pg / mL to about 250 pg / mL improve cell viability and reduce spontaneous apoptosis during specification.

[0227] In some embodiments, the amino acid supply 1060 provides a balanced nutrient environment, preventing depletion and supporting biosynthetic demands of rapidly differentiating hepatoblasts. Supplementation with defined amino acid concentrates further enhances reproducibility across batches and scalability for large-scale differentiation protocols. The integration of these eight components in the fifth medium 1000 yields hepatoblast populations with high purity, metabolic activity, and / or consistent expression of hepatic lineage markers. The technical benefits include reduced heterogeneity, improved reproducibility, and enhanced downstream capacity for maturation into hepatocyte-like cells suitable for drug metabolism assays, hepatotoxicity testing, and disease modeling.

[0228] FIG. 11 illustrates an embodiment of a system 500 comprising a cell culture platform 510 and a sixth medium 1100 configured to promote hepatic maturation of NHP iPSCs. The sixth medium 1100 comprises a glucocorticoid 1110, an adenylyl cyclase activator 1120, a vitamin C derivative 1130, an anabolic hormone 1140, and a gamma secretase inhibitor 1150, each provided in defined concentration ranges within a base culture medium.

[0229] In some embodiments, the glucocorticoid 1110 is dexamethasone, present in a concentration ranging from about 5 pM to about 15 pM, for example from about 8 pM to about 12 pM, and applied for a period of about 5 days to about 7 days, such as about 6 days.Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATIONDexamethasone is a synthetic corticosteroid with high receptor affinity, and its use during late-stage differentiation promotes expression of hepatocyte-associated enzymes, enhances metabolic stability, and supports maturation of xenobiotic metabolism pathways. The controlled concentration range provides sufficient activation of the glucocorticoid receptor while reducing the risk of overstimulation, which may otherwise lead to cytotoxicity or stress-induced apoptosis.

[0230] In other embodiments, the glucocorticoid 1110 may alternatively include hydrocortisone, applied at concentrations ranging from about 1 pM to about 20 pM, for example from about 5 pM to about 15 pM. Hydrocortisone, a naturally occurring glucocorticoid, offers a broader physiological signaling profile compared to dexamethasone, and may be selected in embodiments aiming to mimic more physiologically relevant hepatic microenvironments. Hydrocortisone may also support synergistic crosstalk with other signaling pathways, including insulin-mediated anabolic responses.

[0231] In further embodiments, the glucocorticoid 1110 may include prednisolone, present in concentrations ranging from about 2 pM to about 15 pM, such as from about 6 pM to about 12 pM. Prednisolone offers intermediate potency relative to dexamethasone and hydrocortisone, providing an alternative profile of glucocorticoid receptor activation. The choice of prednisolone may be advantageous in certain differentiation protocols where gradual receptor activation is beneficial to balance proliferation and maturation of hepatocyte-like cells.

[0232] In some embodiments, additional glucocorticoids may be employed, including methylprednisolone at about 1 pM to about 12 pM, or triamcinolone acetonide at about 0.5 pM to about 10 pM. These compounds provide alternative glucocorticoid receptor activation kinetics, allowing fine-tuning of transcriptional responses to optimize hepatocyte function. In yet other embodiments, combinations of glucocorticoids may be applied, such as dexamethasone with low-dose hydrocortisone, to achieve both high-affinity receptor activation and sustained basal signaling.

[0233] The use of glucocorticoid 1110 in these embodiments enhances hepatic maturation by upregulating phase I and phase II metabolic enzymes, improving albumin secretion, and supporting stable expression of hepatocyte nuclear factors such as HNF4a. Application of glucocorticoids within the disclosed ranges provides flexibility in modulating receptor signaling strength, thereby accommodating inter-line variability across NHP or human induced pluripotent stem cell cultures.Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION

[0234] The inclusion of a glucocorticoid, such as dexamethasone, provides several technical benefits during hepatic maturation. Glucocorticoids engage the glucocorticoid receptor, which regulates transcriptional networks involved in metabolism, stress adaptation, and hepatic enzyme production. By applying dexamethasone or alternative glucocorticoids in controlled ranges, variability across cell batches is reduced, and hepatic lineage fidelity is reinforced. This promotes the upregulation of liver-specific enzymes, such as cytochrome P450 family members, while stabilizing metabolic functions essential for mature hepatocyte activity.

[0235] In some embodiments, the adenylyl cyclase activator 1120 is forskolin, provided at concentrations ranging from about 5 pM to about 15 pM, for example from about 8 pM to about 12 pM. Forskolin directly stimulates adenylyl cyclase activity, thereby elevating intracellular cAMP levels and triggering protein kinase A (PKA) activation. This signaling cascade enhances transcription of genes involved in hepatocyte metabolism, including enzymes for glucose and lipid regulation, while supporting synergistic interactions with glucocorticoid signaling pathways. Application within these ranges facilitates sufficient cAMP elevation to promote maturation without overstimulation that could compromise cell viability.

[0236] In other embodiments, the adenylyl cyclase activator 1120 may include IBMX, a non-specific phosphodiesterase inhibitor, provided in concentrations ranging from about 10 pM to about 50 pM, for example from about 20 pM to about 40 pM. IBMX prevents degradation of intracellular cAMP, thereby prolonging PKA activation. This alternative approach maintains elevated cAMP levels with slower kinetics than forskolin, offering a complementary or substitute mechanism to drive hepatic maturation.

[0237] In further embodiments, the adenylyl cyclase activator 1120 may include dibutyryl-cAMP, a membrane-permeable analog of cAMP, provided in concentrations ranging from about 100 pM to about 500 pM, such as about 200 pM to about 400 pM. Dibutyryl-cAMP directly mimics cAMP signaling inside cells, bypassing the need for adenylyl cyclase activation. This embodiment facilitates strong and reproducible PKA activation across diverse cell lines, particularly in cases where forskolin responsiveness may vary.

[0238] In some embodiments, combinations of activators may be employed. For example, forskolin at about 5 pM to about 15 pM may be combined with IBMX at about 15 pM to about 25 pM to achieve both rapid induction and prolonged stabilization of cAMPDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION signaling. Similarly, sequential exposure to forskolin followed by dibutyryl-cAMP may be used to fine-tune the timing and amplitude of PKA activation, improving reproducibility across different induced pluripotent stem cell lines.

[0239] Collectively, adenylyl cyclase activators 1120 function to elevate intracellular cAMP levels, providing a signaling axis that drives maturation of hepatocyte-like cells. By activating PKA and related pathways, these compounds enhance albumin secretion, improve glycogen storage, and strengthen functional metabolic activity. The use of defined concentration ranges and multiple activator options facilitates protocol flexibility while maintaining reproducibility and lineage fidelity in generating mature hepatocytes.

[0240] The adenylyl cyclase activator 1120 elevates intracellular cAMP levels, thereby activating protein kinase A signaling cascades that synergize with glucocorticoid pathways to enhance hepatic differentiation. The technical benefits of including forskolin, IBMX, or dibutyryl-cAMP in the sixth medium include stronger activation of hepatic transcription factors, increased albumin secretion, and improved regulation of glucose metabolism. Furthermore, controlled elevation of cAMP reduces variability in differentiation outcomes and activates late-stage hepatic functions across different iPSC lines.

[0241] In some embodiments, the vitamin C derivative 1130 is AAP, provided at concentrations ranging from about 100 pg / mL to about 300 pg / mL, for example from about 150 pg / mL to about 250 pg / mL, and applied for a period of about 5 days to about 7 days, such as about 6 days. AAP is a stabilized form of vitamin C that resists degradation under culture conditions, providing sustained antioxidant activity and promoting redox homeostasis during one or more stages of hepatic maturation. The use of AAP within these defined ranges reduces oxidative stress, improves overall cell viability, and supports stable expression of hepatocyte nuclear factors.

[0242] In other embodiments, the vitamin C derivative 1130 may include sodium ascorbyl phosphate, provided in comparable concentrations of about 100 pg / mL to about 300 pg / mL, such as about 200 pg / mL. Sodium ascorbyl phosphate is a water-soluble and oxidation-resistant derivative of vitamin C that provides similar antioxidant benefits while additionally supporting collagen synthesis and extracellular matrix organization, which are important for structural maturation of hepatocyte-like cells.

[0243] In further embodiments, additional stabilized vitamin C derivatives may be employed, including magnesium ascorbyl phosphate (MAP), provided at concentrations ranging from about 50 pg / mL to about 250 pg / mL, or calcium ascorbate, provided atDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION concentrations from about 100 pg / mL to about 500 pg / mL. These derivatives offer alternative stability profiles and bioavailability characteristics, allowing fine-tuning of antioxidant activity in response to cell line variability or culture conditions.

[0244] In some embodiments, combinations of derivatives may be applied, such as AAP with sodium ascorbyl phosphate, to provide both immediate and sustained antioxidant effects throughout the differentiation period. Such combinations can prevent premature cellular senescence, reinforce extracellular matrix assembly, and optimize hepatic-specific metabolic gene expression.

[0245] The inclusion of vitamin C derivative 1130 provides multiple technical benefits, including enhanced antioxidant defense, stabilization of collagen and extracellular scaffolding proteins, promotion of DNA and histone demethylation events that support epigenetic programming, and reinforcement of hepatic lineage identity. These benefits result in improved reproducibility of hepatocyte-like cell maturation, reduced variability across non-human primate iPSC lines, and / or enhanced functional performance in drug metabolism and hepatotoxicity assays.

[0246] The vitamin C derivative, such as ascorbic acid-2 phosphate, functions as both an antioxidant and a metabolic cofactor, reducing oxidative stress and supporting extracellular matrix stability. The technical benefits include increased cell viability under culture stress, stabilization of collagen and extracellular scaffolding proteins, and enhanced transcription of hepatic maturation markers. Moreover, vitamin C derivatives improve epigenetic regulation through DNA demethylation effects, thereby reinforcing hepatocyte gene expression programs. Compared to an unstabilized ascorbic acid, stabilized derivatives such as AAP or sodium ascorbyl phosphate provide superior reproducibility across extended culture durations.

[0247] In some embodiments, the anabolic hormone 1140 is recombinant human insulin, present in a concentration ranging from about 5 ng / mL to about 15 ng / mL, for example from about 8 ng / mL to about 12 ng / mL. Insulin provides metabolic support by promoting glucose uptake through insulin receptor signaling, enhancing glycogen storage, and / or stimulating protein synthesis. Application within these ranges facilitates activation of downstream signaling cascades such as the PI3K / AKT / mTOR pathway, which contributes to hepatocyte-like cell survival, energy metabolism, and late-stage maturation.

[0248] In other embodiments, the anabolic hormone 1140 may include insulin-like growth factor-1 (IGF-1), provided at concentrations ranging from about 5 ng / mL to about 50Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION ng / mL, such as about 10 ng / mL to about 30 ng / mL. IGF-1 shares overlapping receptor pathways with insulin but also engages distinct mechanisms that regulate cell proliferation and differentiation. The inclusion of IGF-1 can enhance hepatocyte-like cell proliferation during the final stages of culture while simultaneously promoting metabolic function and structural maturation.

[0249] In further embodiments, the anabolic hormone 1140 may include insulin-like growth factor-2 (IGF-2), provided at concentrations ranging from about 5 ng / mL to about 40 ng / mL, for example from about 15 ng / mL to about 25 ng / mL. IGF-2 plays important roles in fetal liver development and has been shown to support hepatic lineage specification. Application of IGF-2 during late-stage maturation promotes expression of hepatocyte nuclear factors, enhances glycogen accumulation, and contributes to functional characteristics that mimic developmental hepatic pathways.

[0250] In some embodiments, combinations of anabolic hormones may be employed to maximize synergistic effects. For example, recombinant insulin at about 5 ng / mL to about 15 ng / mL may be combined with IGF-1 at about 15 ng / mL to about 25 ng / mL to promote both metabolic stability and proliferative support. Alternatively, IGF-2 at about 15 ng / mL to about 25 ng / mL may be used in conjunction with insulin to better mimic embryonic developmental signaling while still providing strong glucose uptake activity.

[0251] Additional anabolic hormones may also be incorporated in certain embodiments, including growth hormone at concentrations ranging from about 10 ng / mL to about 100 ng / mL, or synthetic insulin analogs such as lispro or glargine at concentrations from about 5 ng / mL to about 20 ng / mL. These alternatives provide flexible modulation of insulin and IGF receptor signaling, enabling optimization of hepatocyte-like cell metabolic and structural characteristics across diverse iPSC lines. The anabolic hormone 1140 promotes glucose uptake, enhances metabolic activity, and / or stimulates protein synthesis during latestage hepatic maturation. Inclusion of anabolic hormones within these concentration ranges results in hepatocyte-like cells with improved metabolic capacity, increased functional stability, and enhanced reproducibility across differentiation protocols.

[0252] An anabolic hormone, such as recombinant human insulin or IGF-1, supplies metabolic and proliferative support during hepatic maturation. The technical benefits include improved glucose uptake, enhanced protein synthesis, and reinforcement of hepatocytespecific transcriptional programs. By supporting anabolic pathways through insulin or IGFs, hepatocyte-like cells generated from iPSCs display stronger metabolic activity, improvedDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION functional responses in glucose metabolism assays, and greater reproducibility across different batches. Use of defined concentrations of recombinant insulin or IGFs also reduces dependence on serum supplementation, lowering batch-to-batch variability.

[0253] In some embodiments, the gamma secretase inhibitor 1150 is RO4929097, present in a concentration ranging from about 0.1 pM to about 20 pM. Nested ranges may include about 0.25 pM to about 12 pM, about 0.5 pM to about 6 pM, about 0.75 pM to about 5 pM, and about 1 pM to about 3 pM. Within this 1 pM to about 3 pM window, narrower sub-ranges may include about 1.2 pM to about 2.8 pM, about 1.4 pM to about 2.6 pM, or about 1.5 pM to about 2.5 pM. In further embodiments, concentrations from about 1.8 pM to about 2.2 pM may be used for precise modulation of Notch inhibition. In other embodiments, broader exposures such as about 2 pM to about 7 pM, about 3 pM to about 9 pM, or about 5 pM to about 12 pM may be employed to achieve stronger suppression of Notch signaling. Alternatively, lower-dose regimens such as about 0.1 pM to about 0.5 pM may be used when partial inhibition is desired to balance hepatoblast proliferation with differentiation fidelity. The inhibitor may be applied for a period ranging from about 6 hours to about 10 days. Subranges include about 12 hours to about 7 days, about 18 hours to about 5 days, about 24 hours to about 96 hours, about 36 hours to about 72 hours, or about 48 hours to about 60 hours.Narrower windows may include about 20 hours to about 28 hours, about 40 hours to about 55 hours, or about 2 days to about 4 days. The inhibitor may be delivered continuously or in pulses at intervals ranging from about every 6 hours to about every 72 hours. Application of RO4929097 within these ranges has been shown to stabilize hepatocyte lineage commitment and suppress alternative biliary epithelial fates that can otherwise emerge during hepatic maturation stages.

[0254] In other embodiments, the gamma secretase inhibitor 1150 may be DAPT, provided at concentrations ranging from about 5 pM to about 10 pM, such as about 6 pM to about 9 pM. DAPT is a widely used tool compound for inhibition of Notch pathway activity and provides a reproducible means of reinforcing hepatocytic differentiation across diverse induced pluripotent stem cell lines. The use of DAPT facilitates effective suppression of cholangiocyte-like differentiation while promoting expression of hepatocyte-specific transcription factors such as HNF4a and TBX3.

[0255] In further embodiments, the gamma secretase inhibitor 1150 may include LY411575, provided in concentrations ranging from about 0.1 pM to about 5 pM, for example from about 0.5 pM to about 2 pM. LY411575 is a highly potent gamma secretaseDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION inhibitor with strong selectivity, allowing use at lower concentrations relative to other inhibitors. This compound may be particularly useful in differentiation protocols requiring minimal off-target signaling effects while still achieving suppression of Notch activity.

[0256] In some embodiments, combinations of gamma secretase inhibitors may be applied to achieve layered and sustained modulation of the Notch pathway. For example, RO4929097 may be provided in concentrations ranging from about 0.1 pM to about 20 pM. Sub-ranges may include about 0.2 pM to about 15 pM, about 0.3 pM to about 12 pM, about 0.4 pM to about 10 pM, about 0.5 pM to about 8 pM, about 0.75 pM to about 6 pM, about 1 pM to about 5 pM, about 1 pM to about 3 pM, about 1.2 pM to about 2.8 pM, about 1.4 pM to about 2.6 pM, or about 1.5 pM to about 2.5 pM. Narrower windows may include about 1.7 pM to about 2.2 pM or about 1.8 pM to about 2.0 pM. In combination, DAPT may be provided in concentrations ranging from about 1 pM to about 30 pM. Sub-ranges may include about 2 pM to about 25 pM, about 3 pM to about 20 pM, about 4 pM to about 15 pM, about 5 pM to about 12 pM, about 5 pM to about 10 pM, about 6 pM to about 9 pM, or narrower windows such as about 7 pM to about 8 pM. In some embodiments, low-dose regimens may include DAPT at about 1 pM to about 4 pM in combination with RO4929097 at about 0.5 pM to about 2 pM, while higher-dose regimens may employ DAPT at about 8 pM to about 20 pM with RO4929097 at about 2 pM to about 6 pM.

[0257] In further embodiments, additional inhibitors may be used in combination. LY411575 may be applied at about 0.05 pM to about 10 pM, including sub-ranges of about 0.1 pM to about 8 pM, about 0.2 pM to about 6 pM, or about 0.5 pM to about 5 pM. MK- 0752 may be provided at about 0.5 pM to about 15 pM, including sub-ranges of about 1 pM to about 10 pM, about 2 pM to about 8 pM, or about 3 pM to about 6 pM. The combined administration of gamma secretase inhibitors may occur continuously or in staggered intervals. In some embodiments, RO4929097 may be applied for about 12 hours to about 7 days, including sub-ranges of about 18 hours to about 72 hours, about 24 hours to about 96 hours, or about 2 days to about 5 days. In other embodiments, DAPT may be applied for about 24 hours to about 10 days, including sub-ranges of about 2 days to about 8 days, about 3 days to about 7 days, or about 4 days to about 6 days. Pulsed delivery may be employed, with intervals ranging from about every 6 hours to about every 72 hours, including subranges of about every 12 hours to about every 48 hours. Application of gamma secretase inhibitors in combination within these ranges facilitates broader and more precise modulation of Notch activity, suppresses cholangiocyte differentiation, maintains hepatoblastDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION proliferation, and enhances the uniform maturation of hepatocyte-like cells. Such combinatorial approaches may improve reproducibility across different non-human primate or human iPSC-derived hepatocyte cultures, particularly in cases where variability in Notch receptor expression impacts differentiation outcomes.

[0258] Additional gamma secretase inhibitors may also be employed in alternative embodiments, including MK-0752, applied at concentrations ranging from about 1 pM to about 10 pM, or PF-03084014, applied at concentrations ranging from about 0.5 pM to about 5 pM. These next-generation inhibitors offer refined pharmacological profiles and can be selected based on one or more of the desired balance of potency, selectivity, and / or duration of Notch suppression. The inclusion of gamma secretase inhibitor 1150 in the sixth differentiation medium provides a targeted means of suppressing Notch signaling, which is known to bias cells toward cholangiocyte or biliary fates. By blocking this alternative differentiation pathway, gamma secretase inhibitors promote stable hepatic lineage maturation, enhance albumin secretion, improve cytochrome P450 activity, and reinforce functional characteristics of hepatocyte-like cells suitable for preclinical drug testing and disease modeling.

[0259] Inclusion of one or more gamma secretase inhibitors, such as RO4929097, DAPT, and / or LY411575, provides targeted suppression of Notch signaling, which is otherwise associated with biliary differentiation. The technical benefits include prevention of unwanted cholangiocyte lineage emergence, improved enrichment of hepatocyte-like populations, and reduction in heterogeneity across differentiation batches. By modulating Notch signaling in late-stage differentiation, the gamma secretase inhibitor reinforces hepatic identity, increases expression of liver-specific proteins such as albumin and HNF4a, and facilitates reproducible maturation outcomes suitable for downstream applications in drug metabolism and hepatotoxicity studies.

[0260] The sixth medium 1100 provides a synergistic environment for promoting hepatic maturation of NHP iPSCs by stabilizing glucocorticoid receptor signaling, enhancing cAMP -mediated pathways, providing anabolic and antioxidant support, and modulating Notch signaling. This combination of factors produces hepatocyte-like cells with increased functional activity, reduced lineage variability, and / or enhanced reproducibility across differentiation batches, thereby generating mature cells suitable for drug metabolism assays, hepatotoxicity evaluation, and disease modeling.

[0261] EXAMPLESDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION

[0262] Embodiment 1. A system for differentiating hepatocyte-like cells from non-human primate induced pluripotent stem cells (NHP iPSCs), the system comprising: a cell culture platform configured to support the NHP iPSCs; a first cell differentiation medium configured to induce transition of the NHP iPSCs to an anterior primitive streak state during a first period of time; a second cell differentiation medium configured to induce transition of the NHP iPSCs to a definitive endoderm state during a second period of time; a third cell differentiation medium configured to induce transition of the NHP iPSCs to a posterior foregut state during a third period of time; a fourth cell differentiation medium configured to induce transition of the NHP iPSCs to a liver bud progenitor state during a fourth period of time; a fifth cell differentiation medium configured to promote hepatoblast specification of the NHP iPSCs during a fifth period of time; and a sixth cell differentiation medium configured to promote hepatic maturation of the NHP iPSCs during a sixth period of time.

[0263] Embodiment 2. The system of embodiment 1, wherein the NHP iPSCs are derived from a Macaca fascicularis.

[0264] Embodiment 3. The system of embodiment 1, wherein the first cell differentiation medium comprises a follicle-stimulating hormone (FSH) secretion stimulator, a glycogen synthase kinase 3 (GSK3) inhibitor, a growth factor, and a dual -kinase inhibitor.

[0265] Embodiment 4. The system of embodiment 1, wherein the second cell differentiation medium comprises a follicle-stimulating hormone (FSH) secretion stimulator, a glycogen synthase kinase 3 (GSK3) inhibitor, a growth factor, and a dual-kinase inhibitor.

[0266] Embodiment 5. The system of embodiment 1, wherein the third cell differentiation medium comprises a transforming growth factor beta (TGF-P) receptor I inhibitor, a growth factor, a bone morphogenetic protein, and a retinoid pathway activator in a base culture medium.

[0267] Embodiment 6. The system of embodiment 1, wherein the fourth cell differentiation medium comprises a follicle-stimulating hormone (FSH) secretion stimulator, a bone morphogenetic protein, and an adenylyl cyclase activator in a base culture medium.

[0268] Embodiment 7. The system of embodiment 1, wherein the fifth cell differentiation medium comprises a bone morphogenetic protein, a pleiotropic cytokine, a glucocorticoid, a gamma secretase inhibitor, an adenylyl cyclase activator, an anabolic hormone, a vitamin C derivative, and an amino acid supply in a base culture medium.Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION

[0269] Embodiment 8. The system of embodiment 1, wherein the sixth cell differentiation medium comprises a glucocorticoid, an adenylyl cyclase activator, an anabolic hormone, a vitamin C derivative, and a gamma secretase inhibitor in a base culture medium.

[0270] Embodiment 9. The system of embodiment 1, further comprising a subsystem configured to administer a transforming growth factor beta (TGF-P) receptor I inhibitor to the NHP iPSCs during a portion of exposure to the fifth cell differentiation medium.

[0271] Embodiment 10. A method of differentiating hepatocyte-like cells from non-human primate induced pluripotent stem cells (NHP iPSCs), the method comprising: inducing transition to an anterior primitive streak state by applying a first cell differentiation medium to the NHP iPSCs for a first period of time; inducing transition to a definitive endoderm state by applying a second cell differentiation medium to the cells for a second period of time; inducing transition to a posterior foregut state by applying a third cell differentiation medium to the cells for a third period of time; inducing transition to a liver bud progenitor state by applying a fourth cell differentiation medium to the cells for a fourth period of time; promoting hepatoblast specification by applying a fifth cell differentiation medium to the cells for a fifth period of time; and promoting hepatic maturation by applying a sixth cell differentiation medium to the cells for a sixth period of time.

[0272] Embodiment 11. The method of embodiment 10, wherein the NHP iPSCs are derived from a Macaca fascicularis.

[0273] Embodiment 12. The method of embodiment 10, wherein inducing transition to the anterior primitive streak state comprises applying a first medium that includes a follicle- stimulating hormone (FSH) secretion stimulator, a glycogen synthase kinase 3 (GSK3) inhibitor, a growth factor, and a dual-kinase inhibitor.

[0274] Embodiment 13. The method of embodiment 10, wherein inducing transition to the definitive endoderm state comprises applying a second medium that includes a follicle- stimulating hormone (FSH) secretion stimulator, a glycogen synthase kinase 3 (GSK3) inhibitor, a growth factor, and a dual-kinase inhibitor.

[0275] Embodiment 14. The method of embodiment 10, wherein inducing transition to the posterior foregut state comprises applying a third medium that includes a TGF-P receptor I inhibitor, a growth factor, a bone morphogenetic protein, and a retinoid pathway activator in a base culture medium.

[0276] Embodiment 15. The method of embodiment 10, wherein inducing transition to the liver bud progenitor state comprises applying a fourth medium that includes a follicle-Docket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION stimulating hormone (FSH) secretion stimulator, a bone morphogenetic protein, and an adenylyl cyclase activator in a base culture medium.

[0277] Embodiment 16. The method of embodiment 10, wherein promoting hepatoblast specification comprises applying a fifth medium that includes a bone morphogenetic protein, a pleiotropic cytokine, a glucocorticoid, a gamma secretase inhibitor, an adenylyl cyclase activator, an anabolic hormone, a vitamin C derivative, and an amino acid supply in a base culture medium.

[0278] Embodiment 17. The method of embodiment 10, wherein promoting hepatic maturation comprises applying a sixth medium that includes a glucocorticoid, an adenylyl cyclase activator, an anabolic hormone, a vitamin C derivative, and a gamma secretase inhibitor in a base culture medium.

[0279] Embodiment 18. The method of embodiment 10, further comprising promoting lineage fidelity by exposing the cells to a TGF-P receptor I inhibitor during a portion of the time the fifth cell differentiation medium is applied.

[0280] Embodiment 19. The method of embodiment 18, wherein the TGF-P receptor I inhibitor comprises SB 505124 provided at a concentration of about 0.5 pM to about 1.5 pM for about 48 hours during an initial two days of exposure to the fifth cell differentiation medium.

[0281] Embodiment 20. The method of embodiment 10, wherein the hepatocyte-like cells are characterized by expression of one or more markers includes at least one of a hepatocyte nuclear factor 4-alpha (HNF4a), an alpha fetoprotein (AFP), or a T-box transcription factor 3 (TBX3).

[0282] References in the specification to “one embodiment,” “an embodiment,” “an illustrative embodiment,” “some embodiments,” etc., indicate that the embodiment described may include a particular feature, structure, or characteristic, but every embodiment may or may not necessarily include that particular feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is submitted that it is within the knowledge of one skilled in the art to effect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described.

[0283] As used in the description and claims, the singular form “a”, “an” and “the” include both singular and plural references unless the context clearly dictates otherwise. AtDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION times, the claims and disclosure may include terms such as “a plurality,” “one or more,” or “at least one;” however, the absence of such terms is not intended to mean, and should not be interpreted to mean, that a plurality is not conceived.

[0284] The term “about” or “approximately,” when used before a numerical designation or range (e.g., to define a length or pressure), indicates approximations which may vary by ( + ) or ( - ) 5%, 1% or 0.1%. All numerical ranges provided herein are inclusive of the stated start and end numbers. The term “substantially” indicates mostly (i.e., greater than 50%) or essentially all of a device, substance, or composition.

[0285] As used herein, the term “comprising” or “comprises” is intended to mean that the devices, systems, and methods include the recited elements, and may additionally include any other elements. “Consisting essentially of’ shall mean that the devices, systems, and methods include the recited elements and exclude other elements of essential significance to the combination for the stated purpose. Thus, a system or method consisting essentially of the elements as defined herein would not exclude other materials, features, or steps that do not materially affect the basic and novel characteristic(s) of the claimed disclosure. “Consisting of’ shall mean that the devices, systems, and methods include the recited elements and exclude anything more than a trivial or inconsequential element or step. Embodiments defined by each of these transitional terms are within the scope of this disclosure.

[0286] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. Other embodiments may be utilized and derived therefrom, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term “invention” merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is in fact disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.

Claims

Docket No. 0138-704.600INTERNATIONAL PATENT APPLICATIONCLAIMSWHAT IS CLAIMED IS:

1. A system for differentiating hepatocyte-like cells from non-human primate induced pluripotent stem cells (NHP iPSCs), the system comprising: a cell culture platform configured to support the NHP iPSCs; a first cell differentiation medium configured to induce transition of the NHP iPSCs to an anterior primitive streak state during a first period of time; a second cell differentiation medium configured to induce transition of the NHP iPSCs to a definitive endoderm state during a second period of time; a third cell differentiation medium configured to induce transition of the NHP iPSCs to a posterior foregut state during a third period of time; a fourth cell differentiation medium configured to induce transition of the NHP iPSCs to a liver bud progenitor state during a fourth period of time; a fifth cell differentiation medium configured to promote hepatoblast specification of the NHP iPSCs during a fifth period of time; and a sixth cell differentiation medium configured to promote hepatic maturation of the NHP iPSCs during a sixth period of time.

2. The system of claim 1, wherein the NHP iPSCs are derived from Macaca fascicularis.

3. The system of claim 1, wherein the first cell differentiation medium comprises a follicle-stimulating hormone (FSH) secretion stimulator, a glycogen synthase kinase 3 (GSK3) inhibitor, a growth factor, and a dual-kinase inhibitor.

4. The system of claim 1, wherein the second cell differentiation medium comprises a follicle-stimulating hormone (FSH) secretion stimulator, a glycogen synthase kinase 3 (GSK3) inhibitor, a growth factor, and a dual-kinase inhibitor.

5. The system of claim 1, wherein the third cell differentiation medium comprises a transforming growth factor beta (TGF-P) receptor I inhibitor, a growth factor, a bone morphogenetic protein, and a retinoid pathway activator in a base culture medium.

6. The system of claim 1, wherein the fourth cell differentiation medium comprises a follicle-stimulating hormone (FSH) secretion stimulator, a bone morphogenetic protein, and an adenylyl cyclase activator in a base culture medium.

7. The system of claim 1, wherein the fifth cell differentiation medium comprises a bone morphogenetic protein, a pleiotropic cytokine, a glucocorticoid, a gamma secretaseDocket No. 0138-704.600INTERNATIONAL PATENT APPLICATION inhibitor, an adenylyl cyclase activator, an anabolic hormone, a vitamin C derivative, and an amino acid supply in a base culture medium.

8. The system of claim 1, wherein the sixth cell differentiation medium comprises a glucocorticoid, an adenylyl cyclase activator, an anabolic hormone, a vitamin C derivative, and a gamma secretase inhibitor in a base culture medium.

9. The system of claim 1, further comprising a subsystem configured to administer a transforming growth factor beta (TGF-P) receptor I inhibitor to the NHP iPSCs during a portion of exposure to the fifth cell differentiation medium.

10. A method of differentiating hepatocyte-like cells from non-human primate induced pluripotent stem cells (NHP iPSCs), the method comprising: inducing transition to an anterior primitive streak state by applying a first cell differentiation medium to the NHP iPSCs for a first period of time; inducing transition to a definitive endoderm state by applying a second cell differentiation medium to the cells for a second period of time; inducing transition to a posterior foregut state by applying a third cell differentiation medium to the cells for a third period of time; inducing transition to a liver bud progenitor state by applying a fourth cell differentiation medium to the cells for a fourth period of time; promoting hepatoblast specification by applying a fifth cell differentiation medium to the cells for a fifth period of time; and promoting hepatic maturation by applying a sixth cell differentiation medium to the cells for a sixth period of time.

11. The method of claim 10, wherein the NHP iPSCs are derived from Macaca fascicularis.

12. The method of claim 10, wherein inducing transition to the anterior primitive streak state comprises applying a first medium that includes a follicle-stimulating hormone (FSH) secretion stimulator, a glycogen synthase kinase 3 (GSK3) inhibitor, a growth factor, and a dual-kinase inhibitor.Docket No. 0138-704.600INTERNATIONAL PATENT APPLICATION13. The method of claim 10, wherein inducing transition to the definitive endoderm state comprises applying a second medium that includes a follicle-stimulating hormone (FSH) secretion stimulator, a glycogen synthase kinase 3 (GSK3) inhibitor, a growth factor, and a dual-kinase inhibitor.

14. The method of claim 10, wherein inducing transition to the posterior foregut state comprises applying a third medium that includes a TGF-P receptor I inhibitor, a growth factor, a bone morphogenetic protein, and a retinoid pathway activator in a base culture medium.

15. The method of claim 10, wherein inducing transition to the liver bud progenitor state comprises applying a fourth medium that includes a follicle-stimulating hormone (FSH) secretion stimulator, a bone morphogenetic protein, and an adenylyl cyclase activator in a base culture medium.

16. The method of claim 10, wherein promoting hepatoblast specification comprises applying a fifth medium that includes a bone morphogenetic protein, a pleiotropic cytokine, a glucocorticoid, a gamma secretase inhibitor, an adenylyl cyclase activator, an anabolic hormone, a vitamin C derivative, and an amino acid supply in a base culture medium.

17. The method of claim 10, wherein promoting hepatic maturation comprises applying a sixth medium that includes a glucocorticoid, an adenylyl cyclase activator, an anabolic hormone, a vitamin C derivative, and a gamma secretase inhibitor in a base culture medium.

18. The method of claim 10, further comprising promoting lineage fidelity by exposing the cells to a TGF-P receptor I inhibitor during a portion of the time the fifth cell differentiation medium is applied.

19. The method of claim 18, wherein the TGF-P receptor I inhibitor comprises SB 505124 provided at a concentration of about 0.5 pM to about 1.5 pM for about 48 hours during an initial two days of exposure to the fifth cell differentiation medium.

20. The method of claim 10, wherein the hepatocyte-like cells are characterized by expression of one or more markers includes at least one of a hepatocyte nuclear factorDocket No. 0138-704.600 INTERNATIONAL PATENT APPLICATION4-alpha (HNF4a), an alpha fetoprotein (AFP), or a T-box transcription factor 3 (TBX3).

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