Germline competent rabbit embryonic stem cells

Abstract

Provided herein are compositions comprising germline-competent rabbit embryonic stem cells (rbESCs) and compositions and methods for their generation, maintenance, and use.

Description

Docket No. UM-43841.601GERMLINE COMPETENT RABBIT EMBRYONIC STEM CELLSRELATED APPLICATION INFORMATION

[0001] The present application claims priority to United States Provisional Patent Application Serial Number 63 / 748,241, filed January 22, 2025 and 63 / 807,121, filed May 16, 2025, the disclosure of which are herein incorporated by reference in their entireties.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under OD023245 awarded by the National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0003] Provided herein are compositions comprising germline-competent rabbit embryonic stem cells (rbESCs) and compositions and methods for their generation, maintenance, and use.BACKGROUND

[0004] Embryonic stem cells (ESCs) are a cornerstone of biomedical research due to their remarkable ability to self-renew indefinitely and differentiate into nearly any cell type in the body. These unique properties have positioned ESCs as invaluable tools for studying developmental biology7, modeling human diseases, and advancing regenerative medicine. Among the most extensively used models for ESC research are mouse models, which have significantly contributed to our understanding of gene function, cellular processes, and therapeutic strategies.

[0005] Mouse ESCs, derived from rodent species, have been instrumental in scientific breakthroughs, including the creation of genetically modified models for disease studies. However, these systems are not without limitations. Rodents differ significantly from humans in terms of developmental biology, physiology, and lifespan, which can limit the applicability of findings to human health. Furthermore, their small size presents practical challenges for certain experimental procedures, such as surgical inter entions.

[0006] To address these challenges, there is an urgent need for models that better approximate human embryology and stem cell biology. Such models should possess a closer resemblance to human developmental processes, allow for practical surgical interventions, and have a lifespan long enough to permit clinically relevant research over time. By expanding the scopeDocket No. UM-43841.601of ESC research beyond rodent models, scientists can improve the efficiency and relevance of biomedical studies, bridging the gap between laboratory findings and real-world medical applications.SUMMARY

[0007] Provided herein are compositions comprising germline-competent rabbit embry onic stem cells (rbESCs) and compositions and methods for their generation, maintenance, and use.

[0008] Embodiments of the present disclosure include methods for generating germline-competent rabbit embryonic stem cells (rbESCs). In some embodiments, the methods comprise culturing precursor cells in the presence of one or more agents. In some embodiments, the one or more agents comprise a plurality' of inhibitors of differentiation (IDs). In some embodiments the methods comprise one or more or each of: (a) obtaining a blastocyst from a suitable biological source; (b) isolating an inner cell mass (ICM) from the blastocyst and culturing (e.g., in the presence of feeder cells) in a chemically defined medium comprising the plurality of IDs for a suitable time period (e.g., 3-6 days) under conditions sufficient to suppress differentiation and retain pluripotency; (c) removing cells into suspension, pelleting, resuspending, and culturing (e.g., until at least 80% confluence); and (d) passaging, storing and / or utilizing as desired. In some embodiments, rbESCs are isolated or selected based on the expression of pluripotency markers.

[0009] In some embodiments, the plurality of IDs modulate the STAT3 pathway by acting on STAT3 or upstream and / or downstream targets. In some embodiments, the plurality7of inhibitors of differentiation (IDs) is selected from the group consisting of: cytokines and growth factors (e.g., leukemia inhibitory factor (LIF), IL-6, IL-10. Oncostatin M (OSM)). JAK kinases (e.g., JAK1, JAK2, JAK3, TYK2), erythropoietin (EPO), Src kinases, granulocyte colonystimulating factor (G-CSF), the PI3K / AKT pathway, Bcl-2, Mcl-1, c-Myc, Cyclin DI, vascular endothelial growth factor (VEGF), suppressor of cytokine signaling 3 (S0CS3), Fas ligand (FasL), matrix metalloproteinases (MMPs), c-Myb, hypoxia-inducible factor 1-alpha (HIF-la), GSK-3 (e.g.. GSK-3a and / or GSK-3(3) inhibitors or WNT activators (e.g.. CHIR99021), platelet-derived growth factor receptor (PDGFR) inhibitors (e.g., CP673451), GLI1, and / or Twist. In some embodiments, the plurality of IDs comprises: at least one leukemia inhibitory factor (LIF) receptor regulator, at least one GSK-3 (e.g., GSK-3a and / or GSK-3( )Docket No. UM-43841.601inhibitor / WNT activator, and at least one PDGFR inhibitor. In some embodiments, the plurality of IDs comprises: LIF. CHIR99021, and CP673451 or analogs or derivatives thereof.

[0010] In some embodiments, provided herein is a method of generating germline-competent rabbit embryonic stem cells (rbESCs). comprising contacting a rabbit precursor cell, in culture, with a STAT3 pathway regulator. In some embodiments, the rabbit precursor cell comprises an isolated inner cell mass. In some embodiments, the isolated inner cell mass is obtained from a blastocyst. In some embodiments, the STAT3 pathway regulator comprises a leukemia inhibitory factor (LIF) receptor regulator. In some embodiments, the LIF receptor regulator comprises leukemia inhibitory factor. In some embodiments, the method further comprises contacting the rabbit precursor cell with a glycogen synthase kinase-3 (GSK-3) inhibitor. In some embodiments, the GSK-3 inhibitor is selective for GSK-3a,GSK-3(3 or both isoforms. In some embodiments, the GSK-3 inhibitor is CHIR99021. In some embodiments, the method further comprises contacting said rabbit precursor cell with a platelet-derived growth factor receptors (PDGFR) inhibitor. In some embodiments, the PDGFR inhibitor is CP673451. In some embodiments, the method further comprises contacting said rabbit precursor cell with a LATS1 / 2 inhibitor. In some embodiments, the LATS1 / 2 inhibitor is TRULI and / or TDI-011536 (TDI). In some embodiments, the method further comprises the step of isolating generated rbESCs. In some embodiments, the method further comprises the step of genetically modifying a cell. In some embodiments, the rabbit precursor cells and / or the rbESCs is genetically modified).

[0011] In some embodiments, the rbESCs are collected and stored (e.g., frozen). In some embodiments, the rbESCs are utilized in research, diagnostic, or therapeutic applications. The cells may be used directly or tissues, organs, or animals containing the cells, or progeny thereof, or derived from the cells may be used. For example, in some embodiments, the cells are used to generate a chimeric animal or to generate offspring. In some embodiments, the cells are used to study how diseases develop, how healthy development occurs, or to find new treatments. In some embodiments gene editing techniques are used to create genetically modified cells to study how specific genes contribute to disease or development. In some embodiments, the cells are used for drug testing. For example, in some embodiments, the cells are used to screen for drug candidates or to test drug safety or efficacy. In some embodiments, the cells are used in regenerative medicine (e.g., to repair or replace damaged or diseased organs or tissue).

[0012] For example, in some embodiments, provided herein are methods of introducing an rbESC into an embryo or animal; methods of generating offspring from an rbESC; methods ofDocket No. UM-43841.601analyzing an rbESC, or a tissue, organ, or animal containing or generated therefrom, (e.g., determining an effect of agent on said rbESC, tissue, organ, or animal).

[0013] Embodiments of the present disclosure include cells, or compositions comprising thereof, generated through the methods described above or elsewhere herein. In some embodiments, an isolated rbESC, or a population thereof, made by any of the methods is provided. In some embodiments, the cells are provided in a kit (e.g.. container). Frozen cells may be stored in a preservation media (e.g., glycerol). Frozen cells may be stored in one or more vials in a low temperature container (e.g., liquid nitrogen-containing container).DEFINITIONS

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0015] The terms “comprise(s),’' “include(s),'’ “having,’" “has,’" “can,’" “contain(s),"’ and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0016] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0017] As used herein the term “stem cell” (“SC”) refers to cells that can self-renew and differentiate into multiple lineages. A stem cell is a developmentally pluripotent or multipotent cell. A stem cell can divide to produce two daughter stem cells, or one daughter stem cell and one progenitor (“transit”) cell, which then proliferates into the tissue's mature, fully formed cells. Stem cells may be derived, for example, from embryonic sources (“embryonic stemDocket No. UM-43841.601cells”) or derived from adult sources. For example, U.S. Pat. No. 5,843,780 to Thompson describes the production of stem cell lines from human embryos. PCT publications WO 00 / 52145 and WO 01 / 00650 describe the use of cells from adult humans in a nuclear transfer procedure to produce stem cell lines.

[0018] Examples of adult stem cells include, but are not limited to, hematopoietic stem cells, neural stem cells, mesenchymal stem cells, and bone marrow stromal cells. These stem cells have demonstrated the ability to differentiate into a variety of cell types including adipocytes, chondrocytes, osteocytes, myocytes, bone marrow stromal cells, and thymic stroma (mesenchymal stem cells); hepatocytes, vascular cells, and muscle cells (hematopoietic stem cells); myocytes, hepatocytes, and glial cells (bone marrow stromal cells) and, indeed, cells from all three germ layers (adult neural stem cells).

[0019] As used herein, the term "‘totipotent cell” refers to a cell that is able to form a complete embryo (e.g., a blastocyst).

[0020] As used herein, the term “pluripotent cell” or “pluripotent stem cell” refers to a cell that has complete differentiation versatility, e.g., the capacity to grow into any of the mammalian body's approximately 260 cell types. A pluripotent cell can be self-renewing and can remain dormant or quiescent within a tissue. Unlike a totipotent cell (e.g., a fertilized, diploid egg cell), a pluripotent cell, even a pluripotent embryonic stem cell, cannot usually form anew blastocyst.

[0021] As used herein, the term “induced pluripotent stem cells” (“iPSCs”) refers to a stem cell induced from a somatic cell, e.g.. a differentiated somatic cell, and that has a higher potency than said somatic cell. iPS cells are capable of self-renewal and differentiation into mature cells.

[0022] As used herein, the term “multipotent cell” refers to a cell that has the capacity to grow into a subset of the mammalian body's approximately 260 cell types. Unlike a pluripotent cell, a multipotent cell does not have the capacity to form all of the cell types.

[0023] As used herein, the term “progenitor cell” refers to a cell that is committed to differentiate into a specific t pe of cell or to form a specific type of tissue.

[0024] As used herein, the term “embryonic stem cell” (“ES cell” or ESC”) refers to a pluripotent cell that is derived from the inner cell mass of a blastocyst (e.g., a 4- to 5-day-old human embryo) and has the ability to yield many or all of the cell types present in a mature animal.

[0025] As used herein the term “feeder cells” refers to cells used as a growth support in some tissue culture systems. Feeder cells may be embryonic striatum cells or stromal cells.Docket No. UM-43841.601

[0026] As used herein, the term “chemically defined medium"’ refers to a growth medium used for in vitro cell culture that contains known chemical components

[0027] As used herein, the term “serum-free media” refers to culture media that is devoid of serum, but not necessarily of other undefined components.

[0028] As used herein, the terms “germline cells” or “germline-competent cells” refer to cells that have the potential to contribute to the germline of an organism, including the formation of eggs or sperm, and are capable of being passed on to subsequent generations. Germline-competent cells, such as germline-competent embryonic stem cells (ESCs), are pluripotent cells that retain the ability to differentiate into all cell types, including those that form the reproductive tissues, and are capable of transmitting genetic information to offspring.

[0029] A cell has been “genetically modified,” “transformed,” or “transfected” by exogenous DNA, e.g., a recombinant expression vector, when such DNA has been introduced inside the cell. The presence of the exogenous DNA results in permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. For example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones that comprise a population of daughter cells containing the transforming DNA.

[0030] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:Docket No. UM-43841.601

[0032] FIG. 1 demonstrates the derivation of rabbit embry onic stem cells (rbESCs). (A) A representative image of dome shaped rbESCs colonies. (B) Representative images of single cell derived rbESCs culture after lentivirus mediated transfection of Green Fluorescent Protein (GFP). Left: bright field images of the rbESCs clones. Right: fluorescent images of rbESCs clones. Green color indicate successful gene transfection.

[0033] FIG.2 demonstrates ESC-positive rbESCs contribution to the inner cell mass (ICM) in the injected embryos. Left: fluorescent image of the ESC-injected embryos. Green signals indicate the presence of cells derived from GFP-positive ESCs. Right: bright field image of the ESC-injected embryos.

[0034] FIG.3 demonstrates Polymerase Chain Reaction (PCR) confirmation of GFP-positive cells in different organs of animal #R644-4. Left: A representative electrophoresis gel image showing the PCR product after amplifying the GFP specific sequence in the liver, kidney and ovary7tissue samples collected from a wildtype (i.e., negative control) animal (Lanes 3, 7, and 11), R644-4 (Lanes 4, 8, and 12), R644-2 (Lanes 5, 9, and 13), and R644-5 (Lanes 6, 10, and 14). GFP positive (+); GFP negative (-). Right: ID information for different lanes.

[0035] FIG.4 A western blot confirmation of GFP-positive cells in different organs of animal R644-4.

[0036] FIG. 5 Representative immunofluorescence (IF) staining of GFP in frozen sections of the ovarian tissues from a wild-ty pe control (i.e., GFP-negative; top row), and R644-4 (644-4) animals (i.e., GFP-positive; bottom row).

[0037] FIG. 6 Representative fluorescence imaging of embryos produced by R644-2 (GFP-negative; top row) and R644-4 (GFP-positive; bottom row).

[0038] FIG. 7 PCR confirmation of GFP-positive cells in different organs of two wild-ty pe animals (CLN00126520 and CLN00137965; GFP-negative). and the R644-4‘s offspring (CLN00135500; GFP-positive). Left: A representative electrophoresis gel image showing the PCR product after the GFP specific sequence was amplified in the skin, heart, lung, liver, kidney and ovary' tissues (lanes 1 to 6, respectively). Right: ID information for different lanes.TYR: internal control band showing the presence of the Tyrosinase (TYR) gene in all tissues of all animals tested.DETAILED DESCRIPTION

[0039] Provided herein are compositions comprising germline-competent rabbit embry onic stem cells (rbESCs) and compositions and methods for their generation, maintenance, and use.Docket No. UM-43841.601

[0040] Embodiments of the present disclosure include methods of treating cells (e.g., rabbit precursor cells) with a plurality of inhibitors of differentiation (IDs). In some embodiments, the cells are derived from a blastocyst from a suitable biological source (e.g., rabbit). In some embodiments, the cells are derived from an inner cell mass (ICM) isolated from the blastocyst and cultured. In some embodiments, the ICM is cultured in the presence of feeder cells. In some embodiments, the IDs are provided within a chemically defined medium. The ICMs are then cultured under conditions sufficient to suppress differentiation and retain pluripotency. In some embodiments, such conditions comprise 3-6 days of culturing in the chemically defined medium, although longer or shorter times may be employed. Next, cells are removed into suspension, pelleted, and resuspended in culture medium. The cells are transferred to a culture system (e.g., a culture plate comprising feeder cells) and cultured (e.g., until at least 80% confluence) and then passaged or used as desired.

[0041] In some embodiments, one or more of the plurality of IDs modulate the STAT3 pathway by regulating STAT3 or acting on upstream and downstream targets. In some embodiments, the plurality of inhibitors of differentiation (IDs) is selected from the group consisting of: cytokines and growth factors (e.g., leukemia inhi bi tory factor (LIF), IL-6, IL- 10, Oncostatin M (OSM)), JAK kinases (e.g., JAK1, JAK2. JAK3, TYK2), erythropoietin (EPO). Src kinases, granulocyte colony-stimulating factor (G-CSF), the PI3K / AKT pathway, Bcl-2, Mcl-1, c-Myc, Cyclin DI, vascular endothelial growth factor (VEGF), suppressor of cytokine signaling 3 (S0CS3), Fas ligand (FasL), matrix metalloproteinases (MMPs), c-Myb, hypoxia-inducible factor 1-alpha (HIF-la), GSK-3 (e.g., GSK-3oc and / or GSK-3P) inhibitors or WNT activators (e.g.. CHIR99021). platelet-derived growth factor receptor (PDGFR) inhibitors (e.g., CP673451), GLI1, and / or Twist. In some embodiments, the plurality of IDs comprises: at least one cytokine, at least one GSK-3 (e.g., GSK-3a and / or GSK-3(3) inhibitor / WNT activator, and at least one PDGFR inhibitor. In some embodiments, the plurality of IDs comprises: LIF, CHIR99021, and CP673451 or analogs or derivatives thereof.

[0042] In some embodiments, the plurality of IDs comprises: at least one leukemia inhibitory factor (LIF) receptor regulator, at least one GSK-3 (e.g., GSK-3a and / or GSK-30) inhibitor / WNT activator, and at least one PDGFR inhibitor.

[0043] In some embodiments, the plurality of IDs comprises: LIF (e.g., hLIF (e.g., human LIF, amino acids (Ser23-Phe202) (Accession# NP_002300.1)) (Peprotech, catalog #300-05)), CHIR99021 (e.g., Selleckchem, catalog #1263), and CP673451 (e.g., Selleckchem, catalogDocket No. UM-43841.601#1536) or analogs or derivatives thereof (see e g., US Pat. No. 6,417,185 and WO 2001040217 herein incorporated by reference in their entireties).

[0044] In some embodiments, the method further comprises contacting the rabbit precursor cell with a LATS 1 / 2 inhibitor. LATS 1 and LATS2 are key components of the Hippo signaling pathway and function as downstream kinases, receiving signals from upstream regulators and passing them on by phosphorylating downstream effectors. The Hippo pathway is an important regulator of cell proliferation, apoptosis, organ size, and stem cell behavior, acting as a growth-suppressive mechanism that helps maintain tissue homeostasis by preventing uncontrolled cell division. Upstream kinases MST1 / 2 activate the downstream kinases LATS 1 / 2 through phosphorylation. Once activated, LATS 1 / 2 phosphorylate the transcriptional co-activators YAP and TAZ, leading to their retention in the cytoplasm and / or targeting for degradation, thereby preventing their transcriptional activity in the nucleus. When the Hippo pathway is inactive, YAP / TAZ translocate into the nucleus, where they bind to TEAD transcription factors and drive the expression of genes involved in cell growth, survival, and pluripotency.

[0045] In some embodiments, the inhibition of the Hippo pathway can enhance YAP / TAZ activity7, thereby promoting self-renewal, proliferation, and tissue regeneration. In some embodiments, the Hippo pathway may be inhibited by a small molecule. In some embodiments, the small molecule inhibitor function by inhibiting LATS1 and / or LATS2 activity7. For example, non-limiting examples of small molecule inhibitors of LATS1 / 2, include but are not limited to, GA-017, NIBR-LTSi, GA-002 or Merlin. In some embodiments, the small molecule inhibitors of LATS 1 / 2 include TRULI and its derivative, TDI-011536 (TDI). In exemplary embodiments. LATS1 / 2 are inhibited through genetic approaches used to reduce and / or eliminate LATS 1 / 2 function. In exemplary embodiments, LATS 1 / 2 are inhibited through CRISPR / Cas9 knockout directly knocking out the LAT1 and / or LATS2 genes, preventing protein production. In exemplary7embodiments, LATS 1 / 2 are inhibited through RNA interference (RNAi) using siRNA or shRNA to reduce LATS 1 / 2 mRNA levels, decreasing protein expression. In exemplary embodiments, LATS 1 / 2 are inhibited through dominant-negative mutants wherein modified versions of LATS 1 / 2 block normal LATS 1 / 2 activity7when overexpressed.

[0046] In exemplary7embodiments, LATS1 / 2 inhibition is achieved indirectly by7targeting upstream activators of the Hippo pathway. In exemplary7embodiments, the upstream activators is MST1 / 2. In exemplary embodiments, the upstream activators are scaffoldingDocket No. UM-43841.601proteins SAV1 and M0B1. In exemplary' embodiments, 14-3-3 proteins are blocked and / or disrupted from binding to phosphory lated sites on their partner proteins. In exemplary embodiments, mechanical and / or environmental cues are modulated to influence Hippo pathway activity. In exemplary' embodiments, the YAP and TAZ are modulated directly.

[0047] In some embodiments, chemically defined media is employed. For animal ESCs, chemically defined media (CDM) can include basal media containing essential nutrients such as salts, vitamins, glucose, and amino acids. Media include DMEM / F12 or Neurobasal medium, both of which can be customized with additives to optimize stem cell culture. For example, to prepare N2B27 medium, which finds use for maintaining the pluripotency of ESCs, a combination of DMEM / F12-N2 medium and Neurobasal / B27 medium is mixed in a 1 : 1 ratio. To the N2B27 medium, 0-mercaptoethanol is added at a final concentration of 0.1 rnM to help reduce oxidative stress, supporting cell growth and survival.

[0048] rbESCs, once generated, can be further passaged, used, or stored, as desired. In some embodiments, cells are cryopreserved for storage. In some embodiments, cells are cooled at a controlled rate (e.g., 1-2°C per minute) and then stored frozen in liquid nitrogen.

[0049] Rabbit embryonic stem cells (rbESCs) find use in a variety of research, diagnostic, and therapeutic applications. In some embodiments, cultured cells are used. In some embodiments, cells are transplanted into an embry o or animal. In some embodiments, cells are developed into gametes and are used to generate animals.

[0050] In some embodiments, cells are employed to screen for factors or agents (e.g., small molecules, peptides, antibodies, nucleic acids, gene therapies, environmental conditions) that influence stem cell maintenance, differentiation, or reprogramming. Screening applications may include identifying compounds that enhance or inhibit signaling pathways or testing drugs for cytotoxicity. The effects of compounds on cell morphology', marker expression, or metabolic activity' can be assessed and correlated with functional outcomes. High-throughput screening (HTS) platforms using cells allows for the rapid evaluation of large compound libraries to detect potential therapeutic or adverse effects. These methods support the development of targeted therapies, optimization of culture conditions, and investigation of signaling pathways in animal models.

[0051] Particular screening applications involve the testing of pharmaceutical compounds in drug research. Assessing the activity of candidate pharmaceutical compounds generally involves combining the cells with the candidate compound, determining any changes in cellDocket No. UM-43841.601morphology, marker phenotype, or metabolic activity attributable to the compound (compared with untreated cells or those treated with an inert compound), and correlating these effects with observed changes. Screening can be performed to evaluate pharmacological effects, unintended side effects, or to identity compounds in a library for desired effects. Two or more drugs may be tested in combination (either simultaneously or sequentially) to detect possible drug-drug interaction effects. In some applications, compounds are also screened for cytotoxicity.EXAMPLES

[0052] The present disclosure has multiple aspects, illustrated by the non-limiting examples as described herein.

[0053] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents.Example 1 :Materials and Methods

[0054] N2B27 medium. Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 supplemented with N2. (DMEM / F12-N2) medium: To 100 ml of DMEM / F12. add 1 ml of N2(Gibco) 100*stock solution. Neurobasal / B27 medium: To 100 ml of neurobasal medium, add 2 ml of B27(Gibco), and 0.5 ml of 200 mM 1-glutamine. N2B27 medium: Mix DMEM / F12-N2 medium with neurobasal / B27 medium at a ratio of 1:1. To 200 ml of N2B27 medium, add 200 pl ofO.l M (3-mercaptoethanol. The final concentration ofp-mercaptoethanol in N2B27 medium is 0.1 mM.

[0055] Rabbit Embryonic Stem Cell (ESC) medium. Add 1 pM CP673451 (Selleckchem Cat# 1536), 3 pM CHIR-99021 (Selleckchem Cat# 1263)), and 20 ng / ml human Leukemia Inhibitory' Factor (LIF) (Peprotech Cat#300-05) to the N2B27 medium.

[0056] DF medium. DMEM medium with 10% Fetal bovine serum (FBS).

[0057] Feeder Cell Pre-treatment Plate. Pre-treat the culture plates with 0.1% gelatin for 1 hour. Thaw previously irradiated mouse embryonic fibroblast (MEF) cells, and transfer the cells to the gelatin-coated culture plates. Culture the cells in DF medium overnight until they reach approximately 40% confluence.Example 2:Derivation of Rabbit ES CellsDocket No. UM-43841.601

[0058] Rabbit embryos at the pronuclear stage were collected following standard superovulation, breeding and embryo flushing protocols. The embryos were cultured in vitro until they reach the blastocyst stage following a protocol that is previously described.

[0059] Rabbit blastocysts were placed in N2B27 medium. A 3 ml syringe with a needle (23G X 1, 0.6mm X 25mm) was used to strip and remove the zona pellucida from the embry os. The same size syringe and needle were then used to strip the zona-free blastocysts and isolate the inner cell mass.

[0060] The inner cell mass (ICM) cells were transferred onto a culture plate pre-seeded with feeders and cultured in rabbit ESC medium (REM). A cocktail of inhibitors of differentiation (IDs) which include human leukemia inhibitory factor (hLIF), CHIR99021, and CP673451 are supplied in the REM to facilitate the development of rbESCs.

[0061] The ICM cells were maintained at 5% CO2 and 38°C for 3 to 6 days. After this period, the REM was removed, and 0.025% trypsin / EDTA was added, followed by' incubation at 37°C for 2 minutes. To neutralize the digestion, DF medium was added at twice the volume of the trypsin / EDTA. The cells were then pipetted gently to form a single-cell suspension, transferred to a 15 ml centrifuge tube, and centrifuged at 300g for 3 minutes. The supernatant was discarded, and the cell pellet was resuspended in REM. The cells were then transferred onto a feeder-seeded culture plate and cultured in REM until they reached 80% confluence, at which point they were passaged.Example 3:Propagation of Rabbit ES Cells

[0062] When the cells reached 80% confluence, they were treated with 0.025% trypsin / EDTA at 37°C for 2-3 minutes. Once all cells were detached, as observed under the microscope, DF medium was added at twice the volume of the 0.025% trypsin / EDTA used. The cells were then pipetted to form a single-cell suspension, transferred into a 15 ml centrifuge tube, and centrifuged at 300g for 3 minutes. The supernatant was discarded and the cell pellet was resuspended in REM. Cells were then transferred onto a culture plate pre-seeded with feeders and culture them in REM. The cells were then maintained at 5% CO2 and 38°C.Example 4:The rbESCs can be cultured for prolonged time and amenable for genetic engineering

[0063] The derived rbESCs were passaged in vitro for prolonged time up to 40 passages (and counting), yet the rbESCs still maintained their dome shaped stem cell morphology which was considered an indicator of germline transmission status (FIG. 1 A).Docket No. UM-43841.601

[0064] Importantly, these rbESCs were amenable to genetic engineering. In one example, rbESCs were subjected to lentivirus mediated integration of green florescence protein (GFP) expressing vectors (FIG. IB).Example 5:The rbESCs support the production of chimeric animals after ESC injection to host embryos

[0065] To evaluate if the rbESCs supported the production of chimeric animals. ESC injections were conducted. In the well-established mouse ESC validations, ESCs are injected into blastocyst stage host embryos, followed by embryo transfer of these ESC-injected embryos to recipient female. The newborn mouse pups would be evaluated if any ESCs contributed to the embryo and ultimately the pup development, and if so such pups are called "‘chimeric7; Detection of ESC origin, often marked by the presence of a certain transgene such as GFP, in the pup is used for determination of ’‘chimeric animal”.

[0066] GFP positive rbESCs were injected into GFP negative, i.e., wild-type (WT) host embryos. Approximately 8-15 rbESCs were injected to one 8-cell stage rabbit embryo. ESC-injected embryos were cultured in vitro until they reached blastocyst stage and evaluated for any GFP-positive ESCs contributed to the formation of ICM. Fluorescence microscopy images demonstrated that GFP-positive ESCs successfully contributed to the formation of ICM (FIG.2).

[0067] In parallel, ESC-injected embryos (n=25) were transferred immediately to one prepregnant female (R644) rabbit. After gestation, five kits (i.e., newborn rabbits, R644-1 to R644-5) were produced, three of which are female (R644-2, R644-4, and R644-5).

[0068] These three female ESC-injected embryos derived animals were maintained to adulthood. Prior to euthanasia, the animals were super ovulated and bred to produce fertilized embryos followed by embryo transfer to test germline transmission.

[0069] To check the chimeric status of these animals, PCR, western blots and immunofluorescence (IF) staining was used to check the presence of GFP-positive cells in different organs after the animals were humanly euthanized. The data consistently suggested that R644-4 was a positive chimeric animal (i.e., GFP-positive rbESCs contributed to the embryo development) (FIG. 3, FIG. 4 and FIG. 5). In contrast, R644-2 and R644-5 were negative for chimeric animal test. The successful rate of chimeric animal production calculated based on the number of embryos used was one out of twenty-five (4%); if calculated based on the number of tested animals, it was one out of three (33%).Example 6:Docket No. UM-43841.601The rbESCs are capable for germline transmission

[0070] After R644-2, R644-4 and R644-5 reached sexual maturation, the animals were super ovulated, bred with GFP-negative wildtype male animals. The embryos from each animal were separately collected and immediately checked under fluorescence microscopy to check for GFP signals. Only embry os produced by R644-4 had GFP-positive signals, again confirming the chimeric positive status of R644-4 animal, and the chimeric negative status of R644-2 (FIG.6).

[0071] Next, twenty GFP-positive embryos produced were transferred by R644-4 to a wildtype prepregnant female animal and successfully produced five ESC-derived Fl generation animals. These animals were GFP-positive in all tissues examined (FIG. 6).

[0072] These results especially evidenced by the results from animal R644-4 indicated that the rbESCs were capable for germline development. The injected GFP-positive ESCs not only contributed to the development of ICM cells (FIG. 2), but they also further made contributions to the development of different organs (FIG. 3, FIG. 4 and FIG. 5), and ultimately to the germ cells (oocyte in female), the derived embryos (FIG. 6), and the production of live animals (FIG.7).Example 7:LATS1 / 2 inhibitors enhance rbESCs maintenance

[0073] Screening of additional small molecules identified the LATS1 / 2 inhibitor TRULI and its optimized derivative TDI-011536 (TDI) as effective agents for improving rbESC stability and maintenance.

Claims

Docket No. UM-43841.601CLAIMSWhat is claimed is:

1. A method of generating germline-competent rabbit embryonic stem cells (rbESCs), comprising: contacting a rabbit precursor cell, in culture, with a STAT3 pathway regulator.

2. The method of claim 1, wherein the rabbit precursor cell comprises an isolated inner cell mass.

3. The method of claim 2, wherein the isolated inner cell mass is obtained from a blastocyst.

4. The method of any one of claims 1 through 3, wherein said STAT3 pathway regulator comprises a leukemia inhibitory factor (LIF) receptor regulator.

5. The method of claim 4, wherein the LIF receptor regulator comprises leukemia inhibitory factor.

6. The method of any one of claims 1 through 5, further comprising contacting said rabbit precursor cell with a glycogen synthase kinase-3 (GSK-3) inhibitor.

7. The method of claim 6, wherein the GSK-3 inhibitor is CHIR99021.

8. The method of any one of claims 1 through 7, further comprising contacting said rabbit precursor cell with a platelet-derived growth factor receptors (PDGFR) inhibitor.

9. The method of claim 8, wherein the PDGFR inhibitor is CP673451.

10. The method of any one of claims 1 through 9, further comprising contacting said rabbit precursor cell with a LATS1 / 2 inhibitor.

11. The method of claim 10, wherein the LATS1 / 2 inhibitor is TRULI and TDI-011536 (TDI).

12. The method of any one of claims 1 through 11. wherein said contacting comprises culturing for a period of 3-6 days.

13. The method of any one of claims 1 through 12, further comprising the step of isolating generated rbESCs.

14. The method of any one of claims 1 through 13, further comprising the step of genetically modifying a cell.Docket No. UM-43841.60115. The method of claim 14, wherein said rabbit precursor cell is genetically modified.

16. The method of claim 14, wherein said rbESC is genetically modified.

17. An isolated rbESC made by the method of any of claims 1 through 16.

18. A method comprising: passaging, in culture, an rbESC of claim 17.

19. A method comprising: introducing an rbESC of claim 17 into an embryo or an animal.

20. A method comprising: generating offspring from an rbESC of claim 17.

21. A method comprising: analyzing an rbESC of claim 17, or a tissue, an organ, or an animal containing or generated therefrom.

22. The method of claim 21, wherein said analyzing comprises determining an effect of agent on said rbESC, tissue, organ, or animal.

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