Mammalian livestock pluripotent stem cells derived from delayed embryos

Abstract

This invention provides a method for deriving pluripotent stem cell lines from mammals and livestock. [Solution] A method is provided comprising (a) culturing a mammalian livestock embryo that is at least 7 days and within 21 days after fertilization in ex vivo for a culture period of at least 4 days and within 21 days after fertilization to obtain an embryo containing epiblast cells and / or late pluripotent stem cells; (b) isolating epiblast cells and / or late pluripotent stem cells from the embryo; and (c) culturing the epiblast cells and / or late pluripotent stem cells under conditions suitable for the proliferation of undifferentiated mammalian livestock pluripotent stem cells to obtain a population of mammalian livestock pluripotent stem cells and deriving a mammalian livestock pluripotent stem cell line.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 047,375, filed 2 July 2020, the entire contents of which are incorporated herein by reference as constituting an integral part of this specification.

[0002] Sequence listing The 40,960-byte ASCII file "87134Sequence Listing.txt", created on July 1, 2021, and filed concurrently with the filing of this application, is incorporated herein by reference as constituting a part of this specification. [Background technology]

[0003] In some embodiments, the present invention relates to isolated mammalian livestock pluripotent stem cells and methods for producing the same, and more specifically to cultured mammalian livestock (e.g., cattle) pluripotent stem cells and cells differentiated therefrom, but is not limited thereto.

[0004] Embryonic development begins immediately after fertilization with the division, proliferation, and differentiation of blastomeres. In developing mammalian embryos, blastomeres are totipotent until the morula contraction stage. In the contracting embryo, blastomeres begin to polarize, resulting in two distinct cell populations: the inner cell mass (ICM) that contributes to the embryo proper, and the ectotrophectoderm that develops into the extraembryonic layer. Immediately after implantation, the ICM separates into a layer of primitive endoderm that gives rise to the extraembryonic endoderm and another layer of primitive ectoderm that gives rise to the embryo and several extraembryonic derivatives [Gardner 1982]. After implantation and gastrulation, the cells gradually become limited to specific lineages, losing their pluripotency and being considered multipotent progenitor cells. Therefore, it should be noted that pluripotent embryonic stem cells proliferate and replicate only for a limited period in intact embryos.

[0005] Embryonic stem cell (ESC) lines are pluripotent strains derived from mammalian embryos at the blastocyst stage. Human ESCs have been isolated and characterized [Thomson et al. 1998, Reubinoff et al. 2000], but the pluripotency of uncultured human post-implantation embryonic cells between implantation and gastrulation has not been studied.

[0006] While the ability to culture human embryos in vitro up to day 9 has been reported, demonstrating proliferative and healthy ICMs [Edwards and Surani, 1978], these reports failed to answer several important questions, such as whether pluripotent stem cells still exist in implanted embryos and whether isolation and continuous culture are appropriate to enable their characterization.

[0007] International Publication No. 2006 / 040763 discloses isolated primate embryonic cells characterized by brachyury expression and the ability to differentiate into derivatives of endoderm, mesoderm, and ectoderm tissues. Human blastocysts were cultured as whole embryos on MEFs for 9–14 days post-fertilization until large cysts developed, and isolated cells were prepared.

[0008] The success rate of extracting bovine embryonic stem cells (ESCs) has been reported to be low (Mitalipova et al, 2001), and only a few studies have reported on the extraction of characterized bovine ESCs.

[0009] Recently, Bogliotti YS, et al., 2018 (PNAS, 115: 2090-2095) described the extraction of stable prime pluripotent embryonic stem cells from bovine blastocysts using TeSR1-basic medium supplemented with FGF2 and WNT signaling inhibitor (IWR1), with an extraction rate of 44-58%. The resulting bovine ESCs were then treated with SOX2. + / OCT4 + / CDX2 - / GATA6 -While expression signatures were shown, the clearly defined colony boundaries characteristic of human ESCs and mouse EpiSCs were not.

[0010] However, there are no previous reports of PSCs at the epiblast stage or late embryonic stage being derived from mammalian livestock such as cattle. [Overview of the project]

[0011] In one embodiment of several embodiments of the present invention, a method for deriving a mammalian livestock pluripotent stem cell line, (a) Mammalian livestock embryos at least 7 days after fertilization are cultured ex vivo for a period of at least 4 days and no more than 21 days after fertilization to obtain embryos containing epiblast cells and / or late pluripotent stem cells. (b) Isolate epiblast cells and / or late pluripotent stem cells from the embryo, (c) Epiblast cells and / or late pluripotent stem cells are cultured under conditions suitable for the proliferation of undifferentiated mammalian livestock pluripotent stem cells to obtain a population of mammalian livestock pluripotent stem cells. A method is provided that includes deriving a mammalian livestock pluripotent stem cell line.

[0012] In one embodiment of several embodiments of the present invention, there is provided an isolated mammalian livestock pluripotent stem cell prepared by a method of several embodiments of the present invention, wherein the isolated mammalian livestock pluripotent stem cell is capable of differentiating into the ectoderm, mesoderm, and embryonic germ layer of the ectoderm, and is capable of spontaneously differentiating into adipogenic cells when cultured in a dexamethasone-free medium.

[0013] In one embodiment of several embodiments of the present invention, a method for producing adipocytes is provided, comprising culturing isolated mammalian livestock pluripotent stem cells of several embodiments of the present invention, or a population of mammalian livestock pluripotent stem cells obtained by the method of several embodiments of the present invention, in a culture medium without chemical or hormonal induction to adipogenesis for a period of at least 10 days and no more than 60 days without subculturing.

[0014] In one aspect of some embodiments of the present invention, there is provided a method for producing a food product, comprising introducing adipocytes produced by the method of some embodiments of the present invention into the food product to produce the food product.

[0015] In one aspect of some embodiments of the present invention, there is provided a food product comprising adipocytes produced by the method of some embodiments of the present invention.

[0016] According to some embodiments of the present invention, mammalian livestock pluripotent stem cells can spontaneously differentiate into adipocytes in the absence of an adipogenic differentiation inducer.

[0017] According to some embodiments of the present invention, mammalian livestock pluripotent stem cells can spontaneously differentiate into adipocytes when cultured in a medium not containing dexamethasone.

[0018] According to some embodiments of the present invention, isolation is performed when the embryo develops a cyst characterized by a diameter of about 0.4 millimeters (mm) to about 1 mm.

[0019] According to some embodiments of the present invention, epiblast cells and / or late pluripotent stem cells are contained in the embryonic disc-like structure, and the isolation further comprises removing trophectoderm cells or cells differentiated from trophectoderm cells surrounding the disc-like structure.

[0020] According to some embodiments of the present invention, the method further comprises removing the zona pellucida of the mammalian livestock embryo before culturing the mammalian livestock embryo.

[0021] According to some embodiments of the present invention, mammalian livestock embryo culture further comprises replating the mammalian livestock embryo on a fresh feeder cell layer or on a fresh extracellular matrix during the culture period.

[0022] According to some embodiments of the present invention, the method further comprises removing peripheral fibroblasts from the mammalian livestock embryo before replating.

[0023] According to some embodiments of the present invention, epiblast cells and / or late pluripotent stem cells are characterized by a large nucleus-to-cytoplasm ratio.

[0024] According to some embodiments of the present invention, the method further includes mechanically passaging a population of mammalian domestic animal pluripotent stem cells for at least two passages to obtain a population rich in mammalian domestic animal pluripotent stem cells.

[0025] According to some embodiments of the present invention, the method further includes mechanically passaging a population of mammalian domestic animal pluripotent stem cells for at least about 4 to 6 passages to obtain a population rich in mammalian domestic animal pluripotent stem cells.

[0026] According to some embodiments of the present invention, the passage of the population rich in mammalian domestic animal pluripotent stem cells is performed every 5 to 10 days.

[0027] According to some embodiments of the present invention, the passage of the population rich in mammalian domestic animal pluripotent stem cells is performed by enzymatic passage.

[0028] According to some embodiments of the present invention, the passage of the population rich in mammalian domestic animal pluripotent stem cells is performed by mechanical passage.

[0029] According to some embodiments of the present invention, the culture of mammalian domestic animal embryos is performed in a two-dimensional culture system.

[0030] According to some embodiments of the present invention, the culture of mammalian domestic animal embryos is performed on feeder cells.

[0031] According to some embodiments of the present invention, the culture of epiblast cells and / or late pluripotent stem cells is performed on a two-dimensional culture system.

[0032] According to some embodiments of the present invention, the two-dimensional culture system includes a matrix without feeder cells.

[0033] According to some embodiments of the present invention, the feeder cell-free matrix is ​​selected from the group consisting of Matrigel® matrix, fibronectin matrix, laminin matrix, and vivonectin matrix.

[0034] According to some embodiments of the present invention, the isolation of epiblast cells and / or late pluripotent stem cells is performed using a hypodermic needle under stereoscopic guidance.

[0035] According to some embodiments of the present invention, mammalian livestock embryos are cultured in a culture medium containing a limited amount of fetal mammalian livestock serum.

[0036] According to some embodiments of the present invention, the culture medium comprises a basic medium selected from the group consisting of DMEM / F12, KO-DMEM, and DMEM.

[0037] According to some embodiments of the present invention, mammalian livestock embryos are cultured in a culture medium containing an IL6RIL6 chimera.

[0038] According to some embodiments of the present invention, epiblast cells and / or late pluripotent stem cells are cultured in a culture medium containing an IL6RIL6 chimera.

[0039] According to some embodiments of the present invention, the culture medium further comprises basic fibroblast growth factor (bFGF).

[0040] According to some embodiments of the present invention, the culture medium further comprises a serum substitute.

[0041] According to some embodiments of the present invention, mammalian livestock embryos are cultured in a culture medium containing the Wnt3a polypeptide.

[0042] According to some embodiments of the present invention, epiblast cells and / or late pluripotent stem cells are cultured in a culture medium containing Wnt3a polypeptide.

[0043] According to some embodiments of the present invention, the culture medium further comprises basic fibroblast growth factor (bFGF) and leukemia suppressor factor (LIF).

[0044] According to some embodiments of the present invention, the culture medium further comprises a serum substitute.

[0045] According to some embodiments of the present invention, mammalian livestock embryos are obtained by in vitro fertilization of mammalian livestock oocytes.

[0046] According to some embodiments of the present invention, mammalian livestock embryos are obtained by nuclear transfer (NT) of mammalian livestock cells.

[0047] According to some embodiments of the present invention, mammalian livestock embryos are obtained by parthenogenesis.

[0048] According to some embodiments of the present invention, bovine embryos are obtained by in vitro fertilization of bovine oocytes.

[0049] According to some embodiments of the present invention, bovine embryos are obtained by nuclear transfer (NT) of bovine cells.

[0050] According to some embodiments of the present invention, bovine embryos are obtained by parthenogenesis.

[0051] According to some embodiments of the present invention, mammalian livestock embryos are placed in a two-dimensional culture system using a 27g needle or a stretched Pasteur pipette.

[0052] According to some embodiments of the present invention, mammalian livestock embryos are placed on feeder cells using a 27g needle or a stretched Pasteur pipette.

[0053] According to some embodiments of the present invention, a mammalian livestock embryo is covered with a drop of extracellular matrix before culturing.

[0054] According to some embodiments of the present invention, cells from a population of mammalian livestock pluripotent stem cells are capable of differentiating into the embryonic germ layers of the endoderm, mesoderm, and ectoderm.

[0055] According to some embodiments of the present invention, cells from a population of mammalian livestock pluripotent stem cells are capable of differentiating into embryoid bodies.

[0056] According to some embodiments of the present invention, cells from a population of mammalian livestock pluripotent stem cells spontaneously differentiate into adipogenic cell lines by passage in culture medium for approximately 14 to 21 days.

[0057] According to some embodiments of the present invention, the culture medium includes serum.

[0058] According to some embodiments of the present invention, the culture medium contains an IL6RIL6 chimera.

[0059] According to some embodiments of the present invention, the culture medium does not contain dexamethasone.

[0060] According to some embodiments of the present invention, the culture medium includes serum.

[0061] According to some embodiments of the present invention, the culture medium contains an IL6RIL6 chimera.

[0062] According to some embodiments of the present invention, the mammalian livestock is a ruminant mammalian livestock.

[0063] According to some embodiments of the present invention, the mammalian livestock is a ruminant mammalian livestock.

[0064] According to some embodiments of the present invention, the ruminant mammal domestic is selected from the group consisting of the Bovidae subfamily, sheep, goats, deer, and camels.

[0065] According to some embodiments of the present invention, domesticated ruminant mammals of the Bovidae subfamily are cattle or yaks.

[0066] According to some embodiments of the present invention, the domesticated ruminant mammal of the Bovidae subfamily is the cattle.

[0067] According to some embodiments of the present invention, the livestock is a buffalo, a bison, or a dairy cow (cattle).

[0068] According to some embodiments of the present invention, the mammalian livestock is a dairy cow (cattle).

[0069] According to some embodiments of the present invention, the livestock is a dairy cow (cattle).

[0070] According to some embodiments of the present invention, the ruminant mammalian livestock is selected from a group consisting of pigs, rabbits, and horses.

[0071] According to some embodiments of the present invention, the ruminant mammal domestic is a horse.

[0072] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as those commonly understood by a person of ordinary skill in the art to which this invention belongs. Similar or equivalent methods and materials to those described herein may be used in carrying out or testing embodiments of the invention, but exemplary methods and / or materials are described below. In case of any inconsistency, including definitions, the prevailing view shall prevail. Furthermore, materials, methods, and examples are illustrative and not necessarily intended to be limiting.

[0073] Some embodiments of the present invention will be described here, with reference to the accompanying drawings, as merely examples. While the drawings will be given in particular detail, it is important to emphasize that the details presented here are illustrative and intended for specific consideration of embodiments of the present invention. In this regard, the description provided in conjunction with the drawings will make it clear to those skilled in the art how embodiments of the present invention can be carried out. [Brief explanation of the drawing]

[0074] [Figure 1] Figures 1A-C show images illustrating the derivation of bovine pluripotent stem cell lines (bPSC lines) from delayed blastocysts. Figure 1A: Bovine bud cells with a prominent inner cell mass (ICM) 8 days post-fertilization. Figure 1B: Whole embryo plated with mouse embryonic fibroblasts (MEFs) 11 days post-fertilization. Prominent cysts have developed. Figure 1C: The same embryo 14 days post-fertilization. The cysts have proliferated further, and secondary cysts have developed. Size bars: Figure 1A is 1 mm, Figure 1B is 1 mm, Figure 1C is 1 mm. [Figure 2-1] Figures 2A-D show images illustrating the morphology of bPSC colonies cultured under different culture conditions. Figure 2A: bPSC colonies cultured on MEF in the presence of serum-containing culture medium ("Culture X"). Figure 2B: bPSC colonies cultured on Matrigel® matrix in the presence of serum-free culture medium (IL6RIL6 chimera). Figure 2C: bPSC colonies cultured on MEF in the presence of culture medium supplemented with serum substitute (IL6RIL6 chimera). Figure 2D: Magnified image of Figure 2A. In Figure 2A (and more clearly, the magnified Figure 2D) and Figure 2C, gaps are visible between cells within the colonies, and the cells have a large nucleus-to-cytoplasmic ratio, a typical characteristic of pluripotent stem cells (PSCs). Size bars: Figure 2A is 1 mm, Figure 2B is 1 mm, Figure 2C is 1 mm, Figure 2D is 1 mm. [Figure 2-2] Same as above [Figure 3] Figures 3A and 3B show images of immunofluorescence staining for OCT4, a key pluripotency marker. Figure 3A: DAPI staining of the same field as Figure 3B. Figure 3B: Positive staining for Oct4 (red). Size bars: Figure 3A is 100 μm (micrometers), Figure 3B is 100 μm. [Figure 4]Figures 4A-C show the morphology of 3rd passage bPSCs that spontaneously differentiated when cultured in a culture medium such as DMEM enriched with 10-20% v / v FBS. Figures 4A and 4B show examples of bPSC colonies consisting of differentiated cells. Figure 4C: Cystic EB formed by bPSCs cultured in a serum-containing culture medium ("Medium X"). Size bars: 100 μm for Figure 4A, 50 μm for Figure 4B, and 100 μm for Figure 4C. [Figure 5] Figures 5A-D show images of immunofluorescence staining of key differentiation markers following spontaneous differentiation of bPSCs in culture, representing representative cells from the three embryonic germ layers. Figure 5A: Positive staining of alpha-fetoprotein (indicating endodermal germ layer). Figure 5B: The same staining as in Figure 5A overlaid with DAPI (nuclear) staining. Figures 5C-D: Figure 5D shows co-staining of EMOS (red, indicating mesodermal germ layer) and 3-beta-tubulin (green, indicating ectoderm germ layer) with DAPI (blue, nuclear staining). Figure 5C shows only DAPI nuclear staining of the same microscopic field as shown in Figure 5D. Size bars: Figure 5A is 100 μm, Figure 5B is 100 μm, Figure 5C is 50 μm, Figure 5D is 50 μm. [Figure 6] Figures 6A and 6B show images illustrating the spontaneous differentiation of bovine pluripotent cells into adipocytes. Bovine PSCs were cultured in serum-supplemented medium (medium X) without subculturing for at least 14 days and spontaneously differentiated into adipocytes exhibiting lipid droplets. The intracellular lipid droplets (white arrows) in Figures 6A and 6B were positively stained with oil red. Size bars: Figure 6A is 20 μm, and Figure 6B is 50 μm. [Figure 7] Figures 7A and 7B show images illustrating the derivation of the bovine pluripotent stem cell (bPSC) line BVN6 from delayed bovine blastocysts. Figure 7A: Bovine bud cells, 8 days post-fertilization; Figure 7B: Whole embryo plated with MEF, 16 days post-fertilization. Significant cyst formation occurred (white arrow in Figure 7B). The culture medium used to derive the bovine delayed blastocyst cell line was medium X. Scale bars: 50 μm in Figure 7A, 200 μm in Figure 7B. [Figure 8]Figures 8A-D show images illustrating the derivation of equine PSC lines from delayed blastocysts. Figure 8A: Elongated equine blastocyst with prominent inner cell mass (ICM, white arrow), 8 days post-fertilization. Figure 8B: Whole equine embryo plated with mouse embryonic fibroblasts (MEF), 16 days post-fertilization. Prominent cysts have developed (arrows). The microscope focus is on the cysts. Figure 8C: Same field of view as Figure 8B, but the microscope focus is on the cells. Figure 8D: Cell colonies derived from the second passage, cultured in medium X. Scale bars: Figure 8A is 200 μm, Figure 8B is 100 μm, Figure 8C is -100 μm, and Figure 8D is 50 μm. [Figure 9] Figures 9A-D show images illustrating the morphology of bovine pluripotent stem cell (bPSC) colonies. Figure 9A: bPSC strain BVN1 at passage 30 (p30), overall view of the colony; Figure 9B: bPSC strain BVN1 at p30, magnified to show the cell nuclei within the colony; Figure 9C: bPSC strain BVN2 at p8; Figure 9D: bPSC strain BVN5 at p9. All cell lines were derived using medium X. Scale bars: Figure 9A is 100 μm, Figure 9B is 50 μm, Figure 9C is 50 μm, Figure 9D is 100 μm. [Figure 10] Figures 10A-D show immunofluorescence staining of TRA1-60 (red) and TRA1-81 (green), key pluripotency markers, in the 8th passage (p8) bPSC strain BVN5. Figure 10A: DAPI (nuclear counterstaining) of the cells shown in Figure 10B, Figure 10B: Positive staining for TRA1-60, Figure 10C: DAPI of the cells shown in Figure 10D, Figure 10D: Positive staining for TRA1-81. Scale bars: Figure 10A is 50 μm, Figure 10B is 50 μm, Figure 10C is 100 μm, Figure 10D is 100 μm. [Modes for carrying out the invention]

[0075] In some embodiments, the present invention relates to isolated mammalian livestock pluripotent stem cells and methods for producing them, and more specifically to cultured mammalian livestock (e.g., cattle, horses) pluripotent stem cells and cells differentiated therefrom, but is not limited thereto.

[0076] Before describing in detail at least one embodiment of the present invention, it should be understood that the present invention is not necessarily limited in its application to the details shown in the following description or illustrated in the examples. The present invention can enable other embodiments or be carried out or performed in a variety of ways.

[0077] The derivation of bovine embryonic stem cells has been reported to have a low success rate (Mitalipova et al, 2001), and only a few studies have reported on the derivation of characteristic bovine ESCs.

[0078] Recently, Bogliotti YS, et al., 2018 (PNAS, 115: 2090-2095) described the extraction of stable prime pluripotent embryonic stem cells from bovine blastocysts using TeSR1-basic medium supplemented with FGF2 and WNT signaling inhibitor (IWR1), with an extraction rate of 44-58%. The resulting bovine ESCs were then treated with SOX2. + / OCT4 + / CDX2 - / GATA6 - While expression signatures were shown, the clearly defined colony boundaries characteristic of human ESCs and mouse EpiSCs (epiblast stem cells) were not observed.

[0079] However, there are no previous reports of PSCs at the epiblast stage or late embryonic stage being derived from mammalian livestock such as cattle or horses.

[0080] The inventors have surprisingly found that mammalian livestock embryos (bovine or horse embryos) cultured ex vivo for at least 4 days beyond the blastocyst stage (7 days post-fertilization) and within 21 days post-fertilization can be isolated. Example 1 in the Examples section below shows the derivation of several bovine pluripotent stem cell lines from various bovine embryos 7 days post-fertilization, which are considered to be within 7 days of in-vivo fertilization. Normally, fertilization occurs in vivo (in the uterus) within 0 to 24 hours after insemination in female mammalian livestock (e.g., dairy cows or horses). Embryos 7 days post-fertilization are in the early blastocyst or blastocyst stage. Embryos were removed from the uterus of dairy cows by washing and then cultured ex vivo for 6 to 13 days (thus becoming embryos 13 to 20 days post-fertilization). Embryos 13–20 days post-fertilization were observed under a microscope to evaluate the formation of disc-like structures containing epiblasts and late pluripotent stem cells. The disc-like structures were removed from each embryo, and the isolated cells contained within the disc-like structures were cultured in vitro, continuously passaged every 4–10 days, to obtain bovine pluripotent stem cell populations. The bovine pluripotent stem cell lines were named "BVN1," "BVN2," "BVN5," and "BVN6." It should be noted that BVN1 embryos were cultured ex vivo for 7 days after insemination, BVN2 embryos for 12 days after insemination, BVN5 embryos for 13 days after insemination, and BVN6 embryos for 11 days after insemination. Subsequently, the disc-like structure (containing the epiblast and late pluripotent stem cells) was removed, and the epiblast and late pluripotent stem cells were cultured in vitro while being continuously passaged every 4 to 10 days.

[0081] The examples described later demonstrate that bovine PSCs were cultured on a feeder cell layer (Figure 2A, Figure 2C, and Figure 2D) or matrix (e.g., Matrigel®, Figure 2B) and maintained pluripotency, as demonstrated by OCT4 (Figure 3A-B), TRA1-60, and TRA1-81 (Figure 10A-D).

[0082] Example 2 in the section on examples described later demonstrates the derivation of a horse pluripotent stem cell line from an 8-day post-fertilization female horse embryo (therefore, a 16-day post-fertilization embryo) cultured ex vivo for 8 days. Subsequently, the disc-like structure containing the epiblast and late pluripotent stem cells was removed from the embryo, and the isolated cells were cultured in vitro while continuously passaged every 5-10 days to obtain a population of horse pluripotent stem cells. The horse pluripotent stem cell line was named "HRS1".

[0083] The examples described later further demonstrate that, upon removal from the feeder cell layer or its supporting matrix, and in the presence of serum-containing medium (e.g., "Medium X"), bPSCs (bovine pluripotent stem cells) spontaneously differentiate into embryoid bodies (Figure 4A-C), which contain cells differentiated into all three embryonic germ layers: mesoderm, ectoderm, and endoderm (Figure 5A-D).

[0084] In addition, when bPSCs were left in a two-dimensional culture system for 14-21 days without subculturing in a medium that did not contain adipogenic differentiation agents (such as dexamethasone), the cells spontaneously differentiated into adipocytes that stained positively with oil red (Figure 6A-B).

[0085] According to one aspect of several embodiments of the present invention, a method for deriving a mammalian livestock pluripotent stem cell line, (a) Mammalian livestock embryos at least 7 days after fertilization are cultured ex vivo for a period of at least 4 days and no more than 21 days after fertilization to obtain embryos containing epiblast cells and / or late pluripotent stem cells. (b) Isolate epiblast cells and / or late pluripotent stem cells from the embryo, (c) Epiblast cells and / or late pluripotent stem cells are cultured under conditions suitable for the proliferation of undifferentiated mammalian livestock pluripotent stem cells to obtain a population of mammalian livestock pluripotent stem cells. A method is provided that includes deriving a mammalian livestock pluripotent stem cell line.

[0086] As used in this application, the term "stem cell" refers to cells that remain undifferentiated for a long period during culture (e.g., totipotent, pluripotent, or multipotent stem cells) until they are induced to evolve into other types of cells with specific, characteristic functions (e.g., fully differentiated cells).

[0087] The term "pluripotent stem cell" refers to a cell that can differentiate into all three types of embryonic germ layers (i.e., endoderm, ectoderm, and mesoderm), or that can remain undifferentiated.

[0088] In this application, the term "derive" in relation to "mammalian livestock pluripotent stem cell lines" means creating a population of mammalian livestock pluripotent stem cells from at least one type of stem cell (e.g., epiblast cells or late pluripotent stem cells) isolated from a single mammalian livestock embryo (e.g., an ex-vivo cultured bovine embryo).

[0089] According to the methods of some embodiments of the present invention, mammalian livestock embryos at least 7 days post-fertilization are cultured ex vivo. It should be noted that mammalian livestock embryos at 7 days post-fertilization are at the blastocyst stage and are characterized by the presence of an inner cell mass (ICM), trophoblast layer, and cysts.

[0090] According to some embodiments of the present invention, mammalian livestock embryos are obtained before the embryo implants in the uterus.

[0091] According to some embodiments of the present invention, mammalian livestock embryos are obtained by in vitro fertilization of mammalian livestock oocytes.

[0092] According to some embodiments of the present invention, mammalian livestock embryos are obtained by nuclear transfer (NT) of mammalian livestock cells. Methods of nuclear transfer are known in the art and are described, for example, in Steven L. Stice, et al., 1996 ("Pluripotent Bovine Embryonic Cell Lines Direct Embryonic Development Following Nuclear Transfer"; BIOLOGY OF REPRODUCTION 54, 100-110), the entirety of which is incorporated herein by reference. Such methods include, for example, nuclear transfer into oocytes or nuclear transfer into zygotes when recipient cells have stopped at mitosis.

[0093] According to some embodiments of the present invention, mammalian livestock embryos are obtained by parthenogenesis, which is performed by stimulating unfertilized eggs (parthenogenetic organisms), for example, as described in Kitai Kim et al., 2007 ("Histocompatible Embryonic Stem Cells by Parthenogenesis"; SCIENCE, VOL 315; pages: 482-486), the entirety of which is incorporated herein by reference.

[0094] According to some embodiments of the present invention, mammalian livestock embryos are placed on a two-dimensional culture system or feeder cells using a 27g needle or a stretched Pasteur pipette.

[0095] According to some embodiments of the present invention, a mammalian livestock embryo is covered with a drop of extracellular matrix before culturing.

[0096] The extracellular matrix may consist of components derived from the basement membrane and / or extracellular matrix components that form part of the receptor-ligand coupling of adhesion molecules. Matrigel® (Becton Dickinson, USA) is an example of a commercially available matrix suitable for use in the present invention. Matrigel® is a soluble preparation derived from Engelbreth-Holm-Swarm tumor cells that gels at room temperature to reconstitute the basement membrane, and Matrigel® is also available as a growth inhibitor preparation. Other extracellular matrix components and component mixtures suitable for use in the present invention include foreskin matrix, laminin matrix, fibronectin matrix, proteoglycan matrix, entactin matrix, heparan sulfate matrix, collagen matrix, etc., each individually or in various combinations thereof.

[0097] According to some embodiments of the present invention, the matrix is ​​xeno-free.

[0098] The term "xeno" is a prefix based on the Greek word "Xenos," meaning "other." As used herein, "xenofree" means free from any components / impurities of a different (xenos) (i.e., non-same) species. These components may be impurities such as pathogens associated with a different species (e.g., infections), cellular components of a different species, or non-cellular components of a different species (e.g., bodily fluids).

[0099] When completely xeno-free culture conditions are desired, the matrix is ​​preferably derived from the same raw materials as the embryo, for example, from mammalian livestock (e.g., cattle), or synthesized using recombinant technology. Examples of such matrices include recombinant fibronectin, recombinant laminin, synthetic fibronectin matrix, vitronectin matrix, and / or collagen matrix. Synthetic fibronectin matrix is ​​available from Sigma in St. Louis, Missouri, USA.

[0100] According to some embodiments of the present invention, the method further comprises removing the zona pellucida of mammalian livestock embryos before culturing them ex vivo.

[0101] Methods for removing the zona pellucida include, but are not limited to, chemical digestion (e.g., with Tyrode's acidic solution), enzymatic digestion (e.g., using trypsin-like enzymes or collagenase), or mechanical methods using, for example, a micropipette or micromanipulator (e.g., using a laser).

[0102] According to some embodiments of the present invention, the zona pellucida is removed by chemical digestion with Tyrode's acidic solution.

[0103] According to some embodiments of the present invention, ex-vivo culture of mammalian livestock embryos is performed in a two-dimensional culture system.

[0104] According to some embodiments of the present invention, ex-vivo culture of mammalian livestock embryos is performed on feeder cells.

[0105] Once placed on a two-dimensional culture system or feeder cell layer, mammalian livestock embryos spontaneously attach to the surface of the two-dimensional culture system or feeder cell layer, continue to proliferate ex vivo, and develop.

[0106] According to methods of some embodiments of the present invention, mammalian livestock embryos are cultured ex vivo outside the mammalian livestock uterus under conditions that allow for further development, so as to obtain embryos containing epiblast cells and / or late pluripotent stem cells.

[0107] According to some embodiments of the present invention, conditions that enable further development outside the mammalian uterus include a culture system (e.g., a feeder cell layer or matrix) and a suitable culture medium that allows for the undifferentiated proliferation of epiblast cells and late pluripotent stem cells contained within the mammalian embryo.

[0108] As described above, the methods in some embodiments of the present invention include ex-vivo culturing mammalian livestock embryos in a culture medium for at least 4 days.

[0109] According to some embodiments of the present invention, the culture medium used for ex-vivo culture of mammalian livestock embryos comprises a basic medium and serum.

[0110] According to some embodiments of the present invention, ex-vivo culture of mammalian livestock embryos is performed in a culture medium containing a limited amount of fetal bovine serum.

[0111] According to some embodiments of the present invention, the culture medium comprises a basic medium selected from the group consisting of DMEM / F12, KO-DMEM, and DMEM.

[0112] According to some embodiments of the present invention, the culture medium used for ex-vivo culture of mammalian livestock embryos is serum-free.

[0113] As used herein, the term "serum-free" refers to a product that does not contain human or animal serum.

[0114] It should be noted that the function of serum in culture protocols is to provide cultured cells with an environment similar to that which exists in vivo (i.e., the environment in the organism from which the cells originate, e.g., the blastocyst of an embryo). However, the use of animal-derived serum (e.g., mammalian livestock serum (e.g., cattle)) or human-derived serum (human serum) is limited by the significant variability in serum components between individuals and the risk of contamination by heterologous contaminants (if serum from other species is used).

[0115] According to some embodiments of the present invention, the serum-free culture medium does not contain serum or any part thereof.

[0116] According to some embodiments of the present invention, ex-vivo culture of mammalian livestock embryos is performed in a culture medium containing an IL6RIL6 chimera.

[0117] According to some embodiments of the present invention, the culture medium for ex-vivo culture of mammalian livestock embryos contains IL6RIL6 chimeras at a concentration of about 100 pg / m to about 300 pg / ml (e.g., about 100 pg / ml).

[0118] According to some embodiments of the present invention, the culture medium for ex-vivo culture of mammalian livestock embryos contains IL6RIL6 chimera at a concentration of about 100 ng / ml to about 300 ng / ml (e.g., about 100 ng / ml).

[0119] According to some embodiments of the present invention, the culture medium containing the IL6RIL6 chimera further contains basic fibroblast growth factor (bFGF).

[0120] According to some embodiments of the present invention, a culture medium containing an IL6RIL6 chimera for ex-vivo culture of mammalian livestock embryos further contains bFGF at a concentration of about 20 ng / ml to about 100 ng / ml (e.g., about 50 ng / ml, e.g., about 100 ng / ml).

[0121] According to some embodiments of the present invention, a culture medium containing an IL6RIL6 chimera further comprises a serum substitute.

[0122] According to some embodiments of the present invention, the culture medium containing the IL6RIL6 chimera further comprises basic fibroblast growth factor (bFGF) and a serum substitute.

[0123] According to some embodiments of the present invention, ex-vivo culture of mammalian livestock embryos is performed in a culture medium containing Wnt3a polypeptide.

[0124] According to some embodiments of the present invention, the culture medium for ex-vivo culture of mammalian livestock embryos contains WNT3A polypeptide at a concentration of about 10 ng / ml to about 50 ng / ml (e.g., about 10 ng / ml).

[0125] According to some embodiments of the present invention, the culture medium containing the Wnt3a polypeptide further contains basic fibroblast growth factor (bFGF).

[0126] According to some embodiments of the present invention, a culture medium containing WNT3A polypeptide for ex-vivo culture of mammalian livestock embryos contains bFGF at a concentration of about 20 ng / ml to about 100 ng / ml (e.g., about 50 ng / ml).

[0127] According to some embodiments of the present invention, the culture medium containing the Wnt3a polypeptide further contains a leukemia suppressor (LIF).

[0128] According to some embodiments of the present invention, a culture medium containing WNT3A polypeptide for ex-vivo culture of mammalian livestock embryos contains LIF at a concentration of about 1000 U / ml (units per milliliter) to about 3000 U / ml (e.g., about 3000 U / ml).

[0129] According to some embodiments of the present invention, the culture medium comprising the Wnt3a polypeptide further comprises basic fibroblast growth factor (bFGF) and leukemia suppressor factor (LIF).

[0130] According to some embodiments of the present invention, the culture medium for ex-vivo culture of mammalian livestock embryos comprises Wnt3a polypeptide in a concentration range of 5 to 50 ng / ml, bFGF in a concentration range of 20 to 100 ng / ml, and LIF in a concentration range of 1000 to 3000 U / ml.

[0131] As used in this application, the term "culture medium" means a liquid substance that supports cell proliferation. In some embodiments, the culture medium used in the present invention may be an aqueous medium containing a combination of substances such as salts, nutrients, minerals, vitamins, amino acids, nucleic acids, and proteins (e.g., cytokines, growth factors, and hormones), all of which are necessary for cell proliferation and / or differentiation.

[0132] For example, the culture medium in one aspect of several embodiments of the present invention may be a synthetic tissue culture medium obtained by adding essential additives, as described in detail below, to a basic medium such as Dulbecco's Modified Eagle Medium (DMEM, e.g., available from Gibco-Invitrogen Corporation products, Grand Island, New York, USA), DMEM / F12 (e.g., available from Biological Industries, Beyemeck, Israel), MEM Alpha (e.g., available from Biological Industries, Beyemeck, Israel), Ham's F-12 (e.g., available from Invitrogen / Thermo Fisher Scientific), Ko-DMEM (e.g., available from Gibco-Invitrogen Corporation products, Grand Island, New York, USA), or Eagle Minimum Essential Medium (EMEM, e.g., available from Gibco-Invitrogen Corporation products, Grand Island, New York, USA). The concentration of the basic medium depends on the concentration of other medium ingredients, such as serum substitutes, as described below.

[0133] According to some embodiments of the present invention, the culture medium is a limited culture medium.

[0134] A "limited" culture medium is a chemically restricted culture medium manufactured from known components at specific concentrations. For example, a limited culture medium is a non-conditional culture medium.

[0135] Conditional culture media are growth media for monolayer cell cultures (i.e., feeder cells) present after a specific culture period. Conditional culture media contain growth factors and cytokines secreted by the monolayer cells in the culture.

[0136] Conditional culture media can be recovered from various cells that form a monolayer in the culture. Examples include mouse embryonic fibroblast (MEF) conditional culture, foreskin conditional culture, human embryonic fibroblast conditional culture, and human fallopian tube epithelial cell conditional culture.

[0137] It should be noted that after a certain period of culture, the feeder cells or matrix should be replaced with a layer of fresh feeder cells or fresh matrix of the same type to support the proliferation and development of ex-vivo mammalian livestock embryos.

[0138] According to some embodiments of the present invention, ex-vivo culture of mammalian livestock (e.g., bovine) embryos further comprises replating the mammalian livestock embryos onto a fresh feeder cell layer or a fresh extracellular matrix during the culture period.

[0139] According to some embodiments of the present invention, the method further comprises removing peripheral fibroblasts from mammalian livestock embryos before replating onto a fresh feeder cell layer or a fresh extracellular matrix.

[0140] According to some embodiments of the present invention, the ex-vivo culture period of mammalian livestock embryos is at least 4 days of culture, for example, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, or at least 13 days, and is a period during which the embryo does not reach 21 days after fertilization.

[0141] According to some embodiments of the present invention, the ex-vivo culture period of mammalian livestock embryos is at least 4 days of culture, for example, at least 5 days, at least 6 days, at least 7 days, at least 8 days, during which the embryo has not reached 21 days post-fertilization, 20 days post-fertilization, 19 days post-fertilization, 18 days post-fertilization, or 17 days post-fertilization.

[0142] The inventors have found that when mammalian livestock embryos cultured ex vivo develop cysts of a specific size (e.g., as shown in Figures 1B-C), epiblast cells or late pluripotent stem cells contained in the embryos can be isolated and cultured in vitro for the purpose of deriving pluripotent stem cell lines.

[0143] According to some embodiments of the present invention, once cysts of ex-vivo cultured mammalian livestock embryos are characterized by a diameter of approximately 0.4 mm to approximately 1 mm, epiblast cells and / or late pluripotent stem cells can be isolated and cultured in vitro.

[0144] According to some embodiments of the present invention, isolation is performed when the embryo develops a cyst with a diameter of approximately 0.6 to 1 mm.

[0145] The term "epiblast cells" as used in this application refers to cells of the embryonic epiblast. These cells are pluripotent and therefore capable of differentiating into all three types of embryonic germ layers.

[0146] The term "late pluripotent stem cells" as used in this application refers to cells derived from the late epiblast stage up to gastrulation. These cells are pluripotent and therefore capable of differentiating into all three types of embryonic germ layers.

[0147] According to some embodiments of the present invention, epiblast cells and / or late pluripotent stem cells are characterized by a large nucleus-to-cytoplasmic ratio.

[0148] According to some embodiments of the present invention, in ex-vivo cultured mammalian livestock embryos, epiblast cells and / or late pluripotent stem cells are contained within the embryonic disc-like structure.

[0149] The isolation of epiblast cells or late pluripotent stem cells can be performed by removing the disc-like structure from an ex-vivo cultured embryo and transferring the cells contained within the disc-like structure to a fresh culture dish covered with a matrix or feeder cell layer.

[0150] The isolation of epiblast cells or late pluripotent stem cells from ex-vivo cultured mammalian livestock embryos can be carried out using various techniques, preferably with the use of a microscope or stereoscope.

[0151] For example, epiblast cells or late-stage pluripotent stem cells can be captured with a needle under stereoscopic guidance.

[0152] According to some embodiments of the present invention, the process further includes removing trophectoderm cells, or cells differentiated from trophectoderm cells, that surround the disc-like structure before culturing the disc-like structure cells in a culture dish (or culture vessel).

[0153] Next, epiblast cells or late pluripotent stem cells can be cultured on a feeder cell layer or matrix within a two-dimensional culture system in the presence of a suitable culture medium that maintains the cells in a pluripotent and undifferentiated state.

[0154] According to some embodiments of the present invention, epiblast cells and / or late pluripotent stem cells are cultured on a two-dimensional culture system.

[0155] According to some embodiments of the present invention, the two-dimensional culture system includes a matrix without feeder cells.

[0156] As described above, once epiblast cells and / or late pluripotent stem cells are isolated from mammalian livestock embryos, these isolated cells are further cultured in vitro in the presence of culture medium.

[0157] According to some embodiments of the present invention, epiblast cells and / or late pluripotent stem cells are cultured in a culture medium containing an IL6RIL6 chimera.

[0158] According to some embodiments of the present invention, the culture medium for culturing epiblast cells and / or late mammalian livestock pluripotent stem cells contains an IL6RIL6 chimera in a concentration range of 50 to 300 pg / ml (e.g., a concentration of about 100 pg / ml).

[0159] According to some embodiments of the present invention, a culture medium for culturing epiblast cells and / or late mammalian livestock pluripotent stem cells contains an IL6RIL6 chimera in a concentration range of 50 to 300 ng / ml (e.g., a concentration of about 100 ng / ml).

[0160] According to some embodiments of the present invention, epiblast cells and / or late pluripotent stem cells are cultured in a culture medium containing an IL6RIL6 chimera, basic fibroblast growth factor (bFGF), and a serum substitute.

[0161] According to some embodiments of the present invention, a culture medium for culturing epiblast cells and / or late mammalian livestock pluripotent stem cells comprises an IL6RIL6 chimera in a concentration range of 50–300 pg / ml (e.g., a concentration of about 100 pg / ml), bFGF in a concentration range of 20–100 ng / ml (e.g., a concentration of about 50 ng / ml), and a serum substitute in a concentration range of 10–20% v / v (e.g., about 15% v / v).

[0162] According to some embodiments of the present invention, a culture medium for culturing epiblast cells and / or late mammalian livestock pluripotent stem cells comprises an IL6RIL6 chimera in a concentration range of 50–300 ng / ml (e.g., a concentration of about 100 ng / ml), bFGF in a concentration range of 20–100 ng / ml (e.g., a concentration of about 50 ng / ml), and a serum substitute in a concentration range of 10–20% v / v (e.g., about 15% v / v).

[0163] According to some embodiments of the present invention, epiblast cells and / or late pluripotent stem cells are cultured in a culture medium containing Wnt3a polypeptide.

[0164] According to some embodiments of the present invention, the culture medium for culturing epiblast cells and / or late mammalian livestock pluripotent stem cells comprises Wnt3a polypeptide in a concentration range of 5 to 50 ng / ml (e.g., a concentration of about 10 ng / ml).

[0165] According to some embodiments of the present invention, epiblast cells and / or late pluripotent stem cells are cultured in a culture medium containing Wnt3a polypeptide and basic fibroblast growth factor (bFGF).

[0166] According to some embodiments of the present invention, a culture medium for culturing epiblast cells and / or late mammalian livestock pluripotent stem cells comprises Wnt3a polypeptide in a concentration range of 5 to 50 ng / ml (e.g., a concentration of about 10 ng / ml) and bFGF in a concentration range of 20 to 100 ng / ml (e.g., a concentration of about 50 ng / ml).

[0167] According to some embodiments of the present invention, epiblast cells and / or late pluripotent stem cells are cultured in a culture medium containing Wnt3a polypeptide and leukemia suppressor (LIF).

[0168] According to some embodiments of the present invention, the culture medium for culturing epiblast cells and / or late mammalian livestock pluripotent stem cells comprises Wnt3a polypeptide in a concentration range of 5 to 50 ng / ml (e.g., a concentration of about 10 ng / ml) and LIF in a concentration range of 1000 to 3000 u / ml (e.g., a concentration of about 3000 u / ml). According to some embodiments of the present invention, the culture of epiblast cells and / or late pluripotent stem cells is carried out in a culture medium comprising Wnt3a polypeptide, basic fibroblast growth factor (bFGF), and leukemia suppressor factor (LIF).

[0169] According to some embodiments of the present invention, a culture medium for culturing epiblast cells and / or late mammalian livestock pluripotent stem cells comprises Wnt3a polypeptide in a concentration range of 5 to 50 ng / ml (e.g., a concentration of about 10 ng / ml), bFGF in a concentration range of 20 to 100 ng / ml (e.g., a concentration of about 50 ng / ml), and LIF in a concentration range of 1000 to 3000 u / ml (e.g., a concentration of about 3000 u / ml).

[0170] According to some embodiments of the present invention, the culture medium used for culturing epiblast cells and / or late pluripotent stem cells further comprises a serum substitute.

[0171] As used herein, the term "serum substitute" refers to a specified preparation that substitutes for serum function by providing pluripotent stem cells with components necessary for their proliferation and survival.

[0172] Various serum substitute formulations are well known in the industry and are commercially available.

[0173] For example, GIBCO® Knockout® Serum Replacement (manufactured by Gibco-Invitrogen Corporation, Grand Island, New York, USA, catalog number 10828028) is a standard serum-free formulation optimized for growing and maintaining undifferentiated ES cells in culture. It should be noted that GIBCO® Knockout® Serum Replacement formulations contain animal-derived Albumax (lipid-rich bovine serum albumin) (International Publication No. 98 / 30679 by Price, PJ et al). However, a recent publication by Crook et al., 2007 (Crook JM., et al., 2007, Cell Stem Cell, 1: 490-494) describes six clinical-grade hESC lines prepared in Knockout® Serum Replacement (Invitrogen Corporation, USA, catalog number 04-0095), manufactured according to cGMP using FDA-approved clinical-grade preputial fibroblasts.

[0174] According to some embodiments of the present invention, the concentration of GIBCO® Knockout® serum substitute in the culture medium is in the range of approximately 1% [volume / volume (v / v)] to approximately 50% (v / v), for example, approximately 5% (v / v) to approximately 40% (v / v), for example, approximately 5% (v / v) to approximately 30% (v / v), for example, approximately 10% (v / v) to approximately 30% (v / v), for example, approximately 10% (v / v) to approximately 25% (v / v), for example, approximately 10% (v / v) to approximately 20% (v / v), for example, approximately 10% (v / v), for example, approximately 15% (v / v), for example, approximately 20% (v / v), for example, approximately 30% (v / v).

[0175] Another commercially available serum substitute is a vitamin A-free B27 supplement, available from Gibco-Invitrogen Corporation, Grand Island, New York, USA, catalog number 12587-010. The B27 supplement is a serum-free preparation containing d-biotin, fatty acid-free fraction V of bovine serum albumin (BSA), catalase, L-carnitine HCl, corticosterone, ethanolamine HCl, D-galactose (anhydrous), glutathione (reduced), recombinant human insulin, linoleic acid, linolenic acid, progesterone, putrescine-2-HCl, sodium selenite, superoxide dismutase, T-3 / albumin complex, DL-alpha-tocopherol, and DL-alpha-tocopherol acetate.

[0176] According to some embodiments of the present invention, the serum substitute is xenofree.

[0177] For example, xenofree serum substitutes may include a combination of insulin, transferrin, and selenium.

[0178] A non-limiting example of a commercially available xenofree serum substitute composition is the ITS (insulin, transferrin, and selenium) premix (ITS, Invitrogen, catalog number 51500-056) available from Invitrogen Corporation.

[0179] According to some embodiments of the present invention, the ITS (Invitrogen Corporation) or SR3 (Sigma) xenofree serum substitute formulation is diluted in a 1:100 ratio to achieve a 1× active concentration.

[0180] According to some embodiments of the present invention, a suitable culture medium for culturing mammalian livestock pluripotent stem cells in an undifferentiated state is a basic medium such as DMEM / F12 or KO-DMEM (e.g., about 80% v / v) supplemented with serum (e.g., limited fetal bovine serum (FBS), e.g., about 20% v / v). According to some embodiments of the present invention, the culture medium further comprises 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, and 1% v / v non-essential amino acid stock. It should be noted that this culture medium can support the undifferentiated proliferation of bovine PSCs cultured on feeder cells such as MEFs, with passage every 5-10 days. However, when bovine PSCs are cultured at high density (e.g., without passage for at least 14 days) in a culture system without MEFs or feeders, at least 25% of the bovine PSCs spontaneously differentiate into adipogenic cell lines. For example, when bovine PSCs are cultured at high density (e.g., without passage for at least 21 days) in a culture system without an MEF or feeder, at least 50% of the bovine PSCs spontaneously differentiate into adipogenic cell lines.

[0181] According to some embodiments of the present invention, a suitable culture medium for culturing mammalian livestock pluripotent stem cells in an undifferentiated state is a basic medium such as DMEM / F12 or KO-DMEM (e.g., about 85% v / v) supplemented with ko-serum substitute (about 15% v / v), IL6RIL6 chimera (concentration range of 50-150 pg / ml, e.g., about 100 pg / ml), and bFGF (concentration range of 40-60 ng / ml, e.g., about 50 ng / ml). According to some embodiments of the present invention, the culture medium further comprises 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, and 1% v / v non-essential amino acid stock.

[0182] According to some embodiments of the present invention, a suitable culture medium for culturing mammalian livestock pluripotent stem cells in an undifferentiated state is a basic medium such as DMEM / F12 or KO-DMEM (e.g., about 85% v / v) supplemented with ko-serum substitute (about 15% v / v), IL6RIL6 chimera (concentration range of 50-150 pg / ml, e.g., about 100 pg / ml), and bFGF (concentration range of 40-60 ng / ml, e.g., about 50 ng / ml). According to some embodiments of the present invention, the culture medium further comprises 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, and 1% v / v non-essential amino acid stock.

[0183] According to some embodiments of the present invention, a suitable culture medium for culturing mammalian livestock pluripotent stem cells in an undifferentiated state is a basic medium such as DMEM / F12 or KO-DMEM (e.g., about 85% v / v) supplemented with ko-serum substitute (at a concentration of about 15% v / v), WNT3A (in a concentration range of 5-50 ng / ml, e.g., about 10 ng / ml), bFGF (in a concentration range of 20-100 ng / ml, e.g., about 50 ng / ml), and leukemia suppressor factor (LIF) (in a concentration range of 1000-3000 U / ml, e.g., about 3000 U / ml). According to some embodiments of the present invention, the culture medium further comprises 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, and a 1% v / v non-essential amino acid stock.

[0184] During culture, epiblast cells or late-stage pluripotent stem cells can be passaged to obtain a population of mammalian livestock pluripotent stem cells.

[0185] As used in this application, the terms “subculturing” or “to subculture” mean dividing the cells in a culture vessel into two or more culture vessels, and typically include the addition of fresh culture medium. Subculturing is typically performed when the culture reaches a certain density.

[0186] According to some embodiments of the present invention, subculturing is performed by mechanical subculturing.

[0187] As used herein, the term "mechanical dissociation" refers to the separation of pluripotent stem cell aggregates into single cells by using physical force rather than enzymatic activity.

[0188] For mechanical dissociation, a pellet of pluripotent stem cells (obtained by cell centrifugation) or isolated pluripotent stem cell aggregates can be dissociated by pipetting the cells up and down in a small amount (e.g., 0.2–1 ml) of culture medium. For example, pipetting can be performed several times (e.g., 3–20 times) using a 200 μl or 1000 μl pipette tip.

[0189] Alternatively, or in addition to the above, mechanical dissociation of large pluripotent stem cell masses can be performed using a device designed to break the mass into predetermined sizes. Such devices are available from CellArtis in Goetheborg, Sweden. Alternatively, or in addition to the above, mechanical dissociation can be performed manually using a needle, for example, a 27g needle (BD Microlance, Drogheda, Ireland), while observing the mass under an inverted microscope.

[0190] According to some embodiments of the present invention, the subculturing is carried out under conditions without enzymatic dissociation.

[0191] According to some embodiments of the present invention, the method further comprises at least 2 to 6, for example, at least 2 to 5, for example, at least 2 to 4, mechanical passaging of a population of mammalian livestock pluripotent stem cells to obtain a population of mammalian livestock pluripotent stem cells.

[0192] According to some embodiments of the present invention, subculturing is performed by enzymatic dissociation of the cell aggregate.

[0193] Enzymatic digestion of pluripotent stem cell aggregates can be carried out by subjecting the aggregates or colonies to enzymes such as type IV collagenase (Worthington Biochemical Corporation, Lakewood, New Jersey, USA) and / or dispase (Invitrogen Corporation products, Grand Island, New York, USA). The incubation time with the enzyme depends on the size of the cell aggregates and colonies present in the cell culture medium. Typically, if pluripotent stem cell aggregates in culture are dissociated every 5–7 days, incubation with 1.5 mg / ml type IV collagenase for 20–60 minutes will yield small cell aggregates that can be further cultured in an undifferentiated state. Alternatively, pluripotent stem cell aggregates can be incubated with 1.5 mg / ml type IV collagenase for approximately 25 minutes, followed by incubation with 1 mg / ml dispase for 5 minutes.

[0194] According to some embodiments of the present invention, the method further comprises enzymatic passage of a population of mammalian livestock pluripotent stem cells at least 2 to 6 times, for example, at least 2 to 5 times, for example, at least 2 to 4 times, in order to obtain a population of mammalian livestock pluripotent stem cells.

[0195] The phrase "population rich in mammalian livestock pluripotent stem cells" as used in this application means a cell population containing at least 70% mammalian livestock pluripotent stem cells.

[0196] According to some embodiments of the present invention, a population rich in mammalian livestock pluripotent stem cells comprises at least 71% undifferentiated and pluripotent mammalian livestock stem cells, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more undifferentiated and pluripotent mammalian livestock stem cells.

[0197] Once obtained, a population rich in mammalian livestock pluripotent stem cells can be cultured continuously through successive subgenerations.

[0198] According to some embodiments of the present invention, a population of pluripotent stem cells is proliferated in an undifferentiated state over an extended period of time through continuous passage.

[0199] According to some embodiments of the present invention, the extended period is at least two weeks, for example, at least one month, for example, at least three, four, five, six, seven months or longer of cultivation.

[0200] According to some embodiments of the present invention, once obtained, a population rich in mammalian livestock pluripotent stem cells can be frozen in liquid nitrogen using a freezing solution such as a solution consisting of 10% v / v dimethyl sulfoxide (DMSO) (e.g., available from Sigma, St. Louis, Missouri, USA), 10% v / v fetal bovine serum (FBS) (e.g., available from Hyclone, Utah, USA), and 80% v / v DMEM / F12 (e.g., available from Biological Industries, Israel).

[0201] According to some embodiments of the present invention, successive passages of a population enriched with mammalian livestock pluripotent stem cells are performed every 4 to 10 days, for example, every 5 to 7 days.

[0202] According to some embodiments of the present invention, the passage of a population enriched with mammalian livestock pluripotent stem cells is carried out by enzymatic passage (e.g., using type IV collagenase, dispase, and TryPLE trypsin).

[0203] According to some embodiments of the present invention, the subculturing of a population enriched with mammalian livestock pluripotent stem cells is performed by mechanical subculturing.

[0204] According to some embodiments of the present invention, the passage of a population enriched with mammalian livestock pluripotent stem cells is performed by mechanical passage without enzymatic passage.

[0205] Therefore, the methods of some embodiments of the present invention result in mammalian livestock pluripotent stem cell lines, which include populations rich in mammalian livestock pluripotent stem cells.

[0206] According to some embodiments of the present invention, cells from a population of mammalian livestock pluripotent stem cells are capable of differentiating into the embryonic germ layers of the endoderm, mesoderm, and ectoderm.

[0207] In some embodiments of the present invention, differentiation of mammalian livestock pluripotent stem cells into the endoderm, mesoderm, and ectoderm embryonic germ layer can be carried out by direct differentiation in cell ploidy, differentiation into embryoid bodies, and / or teratoma formation.

[0208] According to some embodiments of the present invention, cells from a population of mammalian livestock pluripotent stem cells are capable of differentiating into embryoid bodies.

[0209] As used in this application, the term "embryoid body" (EB) refers to a three-dimensional multicellular aggregate of differentiated and undifferentiated cell derivatives of the embryonic germline.

[0210] Embryoid bodies are formed when pluripotent stem cells are removed from conditions that maintain an undifferentiated state, such as a feeder layer, a culture system without feeder cells, or a culture medium capable of maintaining cells in an undifferentiated and pluripotent state. Removal of pluripotent stem cells from a matrix with or without feeder cells can be carried out by treatment with type IV collagenase for a limited time. Following dissociation from the culture surface, the cells are transferred to a tissue culture plate containing culture medium supplemented with serum and amino acids.

[0211] During the culture period, EB cells are monitored for their differentiation status. Cell differentiation can be determined by examining cell or tissue-specific markers known to indicate differentiation. For example, EB-induced differentiated cells may express 68KD neuronal filaments, a marker characteristic of ectoderm cell lines.

[0212] The differentiation level of EB cells can be monitored by tracking the loss of OCT-4 expression, as well as the increased expression of other markers such as α-fetoprotein, NF-68kDa, α-cardiac markers, and albumin. Methods useful for monitoring the expression levels of specific genes are widely known in the field and include RT-PCR, semi-quantitative RT-PCR, Northern blotting, RNA in situ hybridization, Western blotting analysis, and immunohistochemistry.

[0213] Teratoma The pluripotency of pluripotent stem cells in some embodiments of the present invention can also be confirmed by injecting the cells into SCID mice [Evans MJ and Kaufman M (1983) Pluripotential cells grown directly from normal mouse embryos. Cancer Surv. 2: 185-208], which induces teratomas. The teratomas are fixed with 4% v / v paraformaldehyde and histologically examined for three embryonic germ layers (i.e., endoderm, mesoderm, and ectoderm).

[0214] In addition to monitoring the differentiation state, stem cells are often monitored for karyotype to confirm their cytological euploidy, that is, the presence of all chromosomes and no detectable changes during culture. The karyotype of cultured stem cells can be determined by standard Giemsa staining and comparison with the published karyotype of the corresponding species.

[0215] It is widely known in the field that pluripotent stem cells can be induced to differentiate into adipogenic lines by direct induction in the presence of an effective amount of adipogenic differentiation-inducing agent. For example, direct differentiation can be achieved by culturing pluripotent stem cells in the presence of bone morphogenetic protein 4 (BMP4), as substantially described in Qi-Qun Tang, 2004 [Proc. Natl. Acad. Sci. USA 101(26): 9607-9611 “Commitment of C3H10T1 / 2 pluripotent stem cells to the adipocyte lineage”]. In addition, or alternatively, pluripotent stem cells can also be differentiated into adipogenic cells by embryoid body (EB) differentiation. For example, 10-day-old EB cells can be plated onto a gelatin-coated plate containing 20% ​​v / v KSR (knockout serum substitute) (e.g., DMEM / F12), and after another 10 days, the derivatives can be cultured in a medium containing DMEM / F12 and 10% v / v KSR supplemented with IBMX (1-methyl-3-isobutylxanthine, e.g., 0.5 mM), dexamethasone (e.g., 0.25 μM), T3 (e.g., 0.2 nM), insulin (e.g., 1 μg / ml), and rosiglitazone (e.g., 1 μM), as described in Tala Mohsen-Kanson et al., 2014 (Stem Cells, 32: 1459-1467), which largely incorporates this reference.

[0216] As used in this application, the term "adipogenic differentiation inducer" means a substance, such as a hormone and / or chemical reagent, that, when added to pluripotent stem cells in culture in vitro, induces the differentiation of cells into adipogenic cell lines, ultimately resulting in the production of adipocytes.

[0217] According to some embodiments of the present invention, adipogenic differentiation inducers induce the differentiation of pluripotent stem cells cultured in a two-dimensional culture system (e.g., on a matrix or on a feeder cell layer) into adipogenic lines.

[0218] Non-limiting examples of known adipogenic differentiation inducers include, but are not limited to, IBMX (1-methyl-3-isobutylxanthine or 3-isobutyl-1-methylxanthine, as used substituted in this application), hydrocortisone, dexamethasone, BMP (bone morphogenetic protein), T3 (triiodothyronine), indomethacin, and fatty acids or branched fatty acids such as monounsaturated omega-5 (e.g., myristoleic acid), monounsaturated omega-7 (e.g., palmitoleic acid), and monounsaturated omega-9 (e.g., erucic acid, elaidic acid, oleic acid) (e.g., phytanic acid and pristanic acid). These are substantially the same as those described in F. Mehta et al 2019 Sissel Beate Ronning (ed.), Myogenesis: Methods and Protocols, Methods in Molecular Biology, vol. 1889, Springer Science+Business Media, LLC, part of Springer Nature 2019.

[0219] The following are exemplary effective concentration ranges suitable for inducing adipogenic differentiation of pluripotent stem cells such as human ESCs or iPSCs. The adipogenic differentiation medium may contain 0.01–1 mM of3-isobutyl-1-methylxanthine, 0.1–10 μM hydrocortisone, 0.01–1 mM indomethacin, 0.4–0.6 mM IBMX, 0.2–0.3 μM dexamethasone, 0.15–0.3 nM T3, 1–2 μg / ml insulin, and 1–2 μM rosiglitazone.

[0220] Compared to already identified pluripotent stem cells, mammalian livestock PSCs (e.g., bovine PSCs) of some embodiments of the present invention can spontaneously differentiate into adipocytes without the addition of any adipogenic differentiation inducers (e.g., hormones or chemicals) that induce differentiation into adipogenic lines.

[0221] Examples 1 in the section on examples described later, and Figures 6A and 6B, demonstrate that bovine pluripotent stem cells in several embodiments of the present invention can spontaneously differentiate into adipocytes without the addition of any adipogenic differentiation inducer (e.g., hormones or chemicals). The presence of adipocytes can be confirmed by visualization of oil droplets stained positively with oil red staining.

[0222] Mammalian livestock PSCs of some embodiments of the present invention can spontaneously differentiate into adipocytes as background differentiation or when cultured on feeder cells (e.g., MEF feeder layer) in the presence of culture media that do not contain adipogenic differentiation inducers, such as "Culture Medium X" or IL6RIL6 chimeric medium (a serum-free medium), without passage.

[0223] Therefore, mammalian livestock PSCs of some embodiments of the present invention can differentiate into adipocytes in the absence of serum, i.e., in serum-free culture medium.

[0224] According to some embodiments of the present invention, mammalian livestock pluripotent stem cells can spontaneously differentiate into adipocytes in the absence of adipogenic differentiation inducers.

[0225] According to some embodiments of the present invention, mammalian livestock pluripotent stem cells can spontaneously differentiate into adipocytes in the absence of adipogenic differentiation inducers and in serum-free culture media.

[0226] In this application, the phrase "in the absence of" used with respect to the adipogenic differentiation inducer means that an effective amount of the aforementioned adipogenic agent is not included.

[0227] It should be noted that culture media without adipogenic differentiation inducers do not contain an effective amount, and therefore may contain a trace amount of adipogenic differentiation inducer that does not cause differentiation into adipocytes when added to human embryonic stem cells or human embryoid bodies cultured for approximately 14-21 days without passage.

[0228] According to some embodiments of the present invention, mammalian livestock pluripotent stem cells can spontaneously differentiate into adipocytes when cultured for at least 10 days, for example, 14 days or longer, without passage in a dexamethasone-free medium.

[0229] According to some embodiments of the present invention, mammalian livestock pluripotent stem cells can spontaneously differentiate into adipocytes when cultured for at least 10 days, for example, 14 days or longer, without subculturing in a medium that does not contain IBMX (1-methyl-3-isobutylxanthine).

[0230] According to some embodiments of the present invention, mammalian livestock pluripotent stem cells can spontaneously differentiate into adipocytes when cultured for at least 10 days, for example, 14 days or longer, without passage in a BMP-free medium.

[0231] According to some embodiments of the present invention, mammalian livestock pluripotent stem cells can spontaneously differentiate into adipocytes when cultured for at least 10 days, for example, 14 days or longer, without passage in a T3-free medium.

[0232] According to some embodiments of the present invention, when cells from a population of mammalian livestock pluripotent stem cells are cultured in culture medium for about 10 to 14 days without passage, they spontaneously differentiate into adipogenic cell lines.

[0233] According to some embodiments of the present invention, the culture medium used for spontaneous differentiation into adipogenic lines includes serum.

[0234] According to some embodiments of the present invention, the culture medium used for spontaneous differentiation into adipogenic lines contains an IL6RIL6 chimera.

[0235] According to one aspect of several embodiments of the present invention, there is a description of isolated mammalian livestock pluripotent stem cells prepared by a method of several embodiments of the present invention, wherein the isolated mammalian livestock pluripotent stem cells are capable of differentiating into ectoderm, mesoderm and ectoderm-embryonic germ layer, and are capable of spontaneously differentiating into adipogenic cells when cultured in a medium that does not contain adipogenic differentiation inducers.

[0236] According to some embodiments of the present invention, isolated mammalian livestock pluripotent stem cells are characterized by positive expression of OCT4 (a pluripotent stem cell marker).

[0237] According to one aspect of several embodiments of the present invention, a method for producing adipocytes is provided, comprising culturing isolated mammalian livestock pluripotent stem cells of several embodiments of the present invention or a population of mammalian livestock pluripotent stem cells obtained by the method of several embodiments of the present invention in a culture medium that does not contain an adipogenic differentiation inducer, for a period of at least 4 days and no more than 60 days without subculturing, for example, for a period of at least 10 days and no more than 60 days without subculturing, for example, for a period of at least 14 days and no more than 50 days without subculturing, for example, for a period of at least 14 days and no more than 40 days without subculturing, for example, for a period of at least 14 days and no more than 30 days without subculturing, for example, for a period of at least 14 days and no more than 25 days without subculturing.

[0238] As used in this application, the term "mammalian livestock" means domesticated mammalian animals, typically those used as food ingredients such as meat and / or milk.

[0239] According to some embodiments of the present invention, the mammalian livestock is a ruminant mammalian livestock.

[0240] According to some embodiments of the present invention, the mammalian livestock is a ruminant mammalian livestock.

[0241] According to some embodiments of the present invention, the ruminant mammal domestic is selected from the group consisting of the Bovidae subfamily, sheep, goats, deer, and camels.

[0242] According to some embodiments of the present invention, domesticated ruminant mammals of the Bovidae subfamily are cattle or yaks.

[0243] According to some embodiments of the present invention, the domesticated ruminant mammal of the Bovidae subfamily is the cattle.

[0244] According to some embodiments of the present invention, the livestock is a buffalo, a bison, or a dairy cow (cattle).

[0245] According to some embodiments of the present invention, the mammalian livestock is a dairy cow (cattle).

[0246] According to some embodiments of the present invention, the livestock is a dairy cow (cattle).

[0247] According to some embodiments of the present invention, the ruminant mammalian livestock is selected from a group consisting of pigs, rabbits, and horses.

[0248] According to some embodiments of the present invention, the mammalian livestock is a horse.

[0249] According to one aspect of several embodiments of the present invention, a method for producing food is provided, which includes introducing adipocytes prepared by the method of several embodiments of the present invention into food and producing food.

[0250] According to some embodiments of the present invention, the food comprises cultured meat or cultured cells that are combined with other substances to obtain cultured meat.

[0251] As used in this application, the term "cultured meat" refers to animal cells cultured in vitro that have been processed to impart the sensory receptivity and texture of meat.

[0252] Cultured meat products may contain various cells, including, but are not limited to, adipocytes, muscle cells, blood cells, chondrocytes, osteocytes, connective tissue cells, fibroblasts, and / or cardiomyocytes.

[0253] According to some embodiments of the present invention, the animal cells cultured in vitro are mammalian livestock cells.

[0254] According to some embodiments of the present invention, the animal cells cultured in vitro are bovine cells (however, other cells, such as fish, pigs, and birds, may be included).

[0255] According to some embodiments of the present invention, the animal cells cultured in vitro are horse cells (however, other cells, such as fish, pigs, and birds, may be included). According to some embodiments of the present invention, the animal cells cultured in vitro are adipocytes obtained by the spontaneous differentiation of mammalian livestock pluripotent stem cells according to some embodiments of the present invention.

[0256] According to some embodiments of the present invention, cultured meat is substantially free from contamination by harmful microorganisms or parasites.

[0257] As described above, the cultured meat contains adipocytes spontaneously differentiated from mammalian livestock (e.g., cattle) pluripotent stem cells of several embodiments of the present invention.

[0258] While meat with more fat is generally considered tastier, it's important to note that a higher fat content carries a greater risk of undesirable health problems, such as heart disease.

[0259] According to some embodiments of the present invention, cultured meat has a muscle cell / adipocyte ratio that is adjustable to produce meat products with optimal taste and health benefits. For example, such a ratio can be adjusted by the desired cells initially implanted in the culture medium, or by controlling the differentiation of mammalian livestock pluripotent stem cells into muscle cells, chondrocytes, hematopoietic cells, or adipocytes.

[0260] Differentiation may occur on a support layer that provides support for the structure and / or texture of the cultured meat.

[0261] According to some embodiments of the present invention, aseptic techniques may be used in cell culture to obtain meat products that are substantially free of harmful microorganisms such as bacteria, fungi, viruses, prions, protozoa, or any combination thereof. Examples of harmful microorganisms include pathogenic microorganisms such as Salmonella, Campylobacter, and Escherichia coli O156:H7. Aseptic techniques can also be used when meat products are removed from the biological production line and packaged. Such quality assurance may be monitored with standard assays for microorganisms or drugs known in the industry. "Substantially free" means that the concentration of microorganisms or parasites is below a clinically significant level of contamination, i.e., below a level that would cause disease or an undesirable health condition through ingestion.

[0262] According to some embodiments of the present invention, the nutritional value of meat may be increased by adding other nutrients, such as vitamins, that are usually lacking in meat products obtained from animal bodies. This can be achieved by directly adding nutrients to the growth medium or by genetic engineering techniques. For example, one or more genes for enzymes responsible for the biosynthesis of specific vitamins, such as vitamin D, A, or complexes of different vitamin B groups, may be transfected into cultured muscle cells to produce specific vitamins.

[0263] According to some embodiments of the present invention, the meat product derived from in vitro cultured cells may include various meat product derivatives. Such derivatives can be produced, for example, by grinding or shaving in vitro grown tissue and mixing it with appropriate seasonings for meatballs, fish balls, hamburger patties, etc. Alternatively, the derivatives can be cut from layers of tissue and seasoned with spices for beef jerky, Bologna sausage, salami, etc. Thus, the meat product of the present invention may be used in the production of any food product made from animal meat.

[0264] According to one aspect of several embodiments of the present invention, a food containing adipocytes prepared by the method of several embodiments of the present invention is provided.

[0265] As described above, mammalian pluripotent stem cells of some embodiments of the present invention can be induced to differentiate into various cell lineages and cell types. Next, non-limiting methods for differentiating mammalian pluripotent stem cells of some embodiments of the present invention are shown.

[0266] Differentiation into erythrocytes: Pluripotent stem cells can be induced to differentiate into hematopoietic cells such as erythrocytes using various protocols.

[0267] For example, differentiation into hematopoietic cells can be achieved by differentiating pluripotent stem cells into embryoid bodies (EBs).

[0268] Pluripotent stem cells can be induced to differentiate into hematopoietic cells through spontaneous differentiation into embryoid bodies (EBs), as substantially described in H. Lapillonne, et al., 2010 [haematologica, 95(10): 1651-1659; “Red blood cell generation from human induced pluripotent stem cells: perspectives for transfusion medicine”], which is incorporated entirely by reference here. In summary, differentiation into EB cells is performed in the presence of a culture medium such as Iscove's modified Dulbecco's medium-glutamax containing human plasma, and in the presence of stem cell factors (SCF, e.g., approximately 100 ng / mL), thrombopoietin (TPO, e.g., approximately 100 ng / mL), FLT3 ligand (e.g., approximately 100 ng / mL), recombinant human bone morphogenetic protein 4 (BMP4, e.g., approximately 10 ng / mL), recombinant human vascular endothelial growth factor (VEGF-A165, e.g., approximately 5 ng / mL), interleukin-3 (IL-3, e.g., approximately 5 ng / mL), interleukin-6 (IL-6, e.g., approximately 5 ng / mL), and erythropoietin (Epo, e.g., approximately 3 U / mL). Embryoid bodies obtained after approximately 20 days of culture contain cells with early erythrocyte commitment. EB cells are then dissociated into single cells and further cultured in a culture medium containing plasma (e.g., approximately 10% v / v), insulin (e.g., approximately 10 μg / ml), and heparin (e.g., approximately 3 U / mL), along with additional factors such as SCF (e.g., approximately 100 ng / mL), IL-3 (e.g., approximately 5 ng / mL), and Epo (e.g., approximately 3 U / mL). Following 8 days of culture, the medium is replaced with a culture medium supplemented with SCF (e.g., approximately 100 ng / mL) and Epo (e.g., approximately 3 U / mL), and the cells are cultured for a further 3 days. From days 11 to 25, the cells can be cultured in a medium supplemented with Epo (3 U / mL). This protocol can yield final erythrocytes capable of maturing into enucleated erythrocytes containing functional tetrameric fetal hemoglobin.

[0269] Alternatively, pluripotent stem cells can also be directly differentiated into definitive erythroblasts, as substantially described in Bin Mao et al. (2016, Stem Cell Reports, Vol. 7, pp 869-883), which is incorporated herein by reference in its entirety. In summary, differentiation into the hematopoietic lineage can be induced by changing the culture medium of pluripotent stem cells cultured on a two-dimensional matrix or feeder cells from an hPSC maintenance medium to a hematopoietic development-inducing medium. For example, the hematopoietic development-inducing medium can be Iscove's Modified Dulbecco's Medium (IMDM) supplemented with fetal bovine serum (FBS, e.g., about 10% v / v) (e.g., Hyclone), 1% v / v non-essential amino acids, ascorbic acid (e.g., about 50 mg / mL), and VEGF (vascular endothelial growth factor, e.g., about 20 ng / mL). The culture can be for a culture period of about 1-12 days, and hematopoietic precursors and erythroid precursors are obtained. The co-culture is harvested on days 10-12 and transferred to an ultra-low attachment plate containing a serum-free growth medium supplemented with stem cell factor (SCF, e.g., about 100 ng / mL), interleukin-6 (IL-6, e.g., about 100 ng / mL), interleukin-3 (IL-3, e.g., about 5 ng / mL), fetal liver (e.g., about 10 ng / mL), thrombopoietin (TPO, e.g., about 10 ng / mL), erythropoietin (EPO, e.g., about 4 IU / mL), and VEGF (e.g., about 20 ng / mL) for 6 days, and then further cultured for 7-8 days in a serum-free medium supplemented with stem cell factor, interleukin-3 (IL-3), and erythropoietin. Finally, as substantially described in Giarratana, M.C., 2005 (Nat. Biotechnol. 23, 69-74), which is incorporated herein by reference in its entirety, the cells are cultured for about 1-2 weeks in a serum-free RBC medium supplemented with erythropoietin (EPO) for the maturation of erythroblasts. Mature erythroblasts (derived from pluripotent stem cells) exhibit higher levels of beta-globin expression and a progressive loss of mesodermal and endothelial properties, identified by GPA+CD36 low / + Note that it can also be identified by CD36 suppressed at the end.

[0270] <00008In addition, or alternatively, as described in Kenichi Miharada et al., 2006 ("Efficient Enucleation of Erythroblasts Differentiated in Vitro From Hematopoietic Stem and Progenitor Cells"; Nat. Biotechnol. 24(10):1255-6), whose entire content is incorporated herein by reference, once CD34+ cells are obtained or isolated, enucleated erythrocytes can be obtained under culture conditions without feeder cells. In summary, CD34+ cells are cultured for the first passage in a culture medium containing stem cell factor (SCF), erythropoietin (EPO), interleukin-3 (IL-3), vascular endothelial growth factor (VEGF), and insulin-like growth factor II (IGF-II), and then the second and third passages are cultured in a medium supplemented only with SCF and EPO to obtain approximately 77% nucleated erythrocytes.

[0271] Differentiation into cardiomyocytes: For pluripotent stem cells, refer to the following references: PW Burridge et al., (2014; Nat Methods. 11: 855-860; “Chemically defined generation of human cardiomycytes”), I. Batalov et al., (2015; Biomarker Insights 2015:10(S1); “Differentiation of Cardiomycytes from Human Pluripotent Stem Cells Using Monolayer Culture”), and PW Burridge et al. 2013 (Chapter 12 In: Methods in Molecular Biology 997; Uma Lakshmipathy and Mohan C. Vemuri Editors; Pluripotent Stem Cells, Methods and Protocols; “Highly Efficient Directed Differentiation of Human Induced Pluripotent Stem Cells into Differentiation into cardiomyocytes can be induced using various known methods described in "Cardiomyocytes." For example, to differentiate into cardiomyocytes, pluripotent stem cells are cultured in a medium that allows for the formation of embryoid bodies (EBs), and then exposed to a serum-containing medium (e.g., fetal bovine serum) for cell adhesion and the formation of contractile cardiomyocytes.

[0272] Differentiation into smooth muscle cells: Pluripotent stem cells can be induced to differentiate into smooth muscle cells using various known methods. For example, using pluripotent vasculogenic pericytes that successfully differentiate into smooth muscle cells, differentiation into smooth muscle cells can be induced using the method substantially described in Dar A., ​​et al., 2012 (Circulation. 125: 87-99; “Multipotent Vasculogenic Pericytes From Human Pluripotent Stem Cells Promote Recovery of Murine Ischemic Limb”), the entire content of which is referenced here. In summary, pluripotent stem cells spontaneously differentiate into EB cells and CD105 + / CD90 + / CD73 + / CD31 - When EB cells, which are pluripotent, clonal mesodermal precursors, are isolated using MACS microbeads, pericytes develop, which can then proliferate further and differentiate into smooth muscle cells.

[0273] In addition, or alternatively, as substantially described in ELLIOT W. SWARTZ, et al., 2016 ("A Novel Protocol for Directed Differentiation of C9orf72-AssociatedHumanInduced Pluripotent Stem Cells Into Contractile Skeletal Myotubes"; STEM CELLS TRANSLATIONAL MEDICINE 2016;5:1461-1472), whose entire contents are incorporated by this reference, culturing pluripotent stem cells in a chemically restricted culture medium containing a phosphoinositide 3-kinase (PI3K) inhibitor and a glycogen synthase kinase (GSK3b) inhibitor, and adding bone morphogenesis protein 4 (BMP4) and fibroblast growth factor 2 (FGF2), converts approximately 60% or less of the cells into the myogenic program shown by the MYOG+ cell population by day 36.

[0274] Further appropriate methods for inducing the differentiation of pluripotent stem cells into muscle cells are described in Jerome Chal et al., 2016 ("Generation of human muscle fibers and satellite-like cells from human pluripotent stem cells in vitro"; Nature protocols; VOL.11: 1833-1850) and Nunnapas Jiwlawat et al., 2018 ("Current Progress and Challenges for Skeletal Muscle Differentiation from Human Pluripotent Stem Cells Using Transgene-Free Approaches"; Stem Cells International, Volume 2018, pp: 1-18), the entire contents of which are referenced here.

[0275] Differentiation into chondrocytes: Pluripotent stem cells can be differentiated into chondrocytes via embryoid body formation, as substantially described in Sergey P. Medvedev et al., 2011 ("Human Induced Pluripotent Stem Cells Derived from Fetal Neural Stem Cells Successfully Undergo Directed Differentiation into Cartilage"; STEM CELLS AND DEVELOPMENT, Volume 20, Number 6: 1099-1112), for example, the entire content of which will be referenced here. In summary, pluripotent stem cells are spontaneously differentiated into embryoid bodies for 8-15 days. To differentiate directly into cartilage, embryoid bodies are cultured for a further 21 days in chondroforming medium containing DMEM and supplemented with bovine serum (e.g., approximately 5% v / v), dexamethasone (e.g., approximately 10 nM), ascorbic acid (e.g., approximately 50 μg / mL), L-proline (e.g., approximately 40 μg / mL), transforming growth factor b3 (TGFβ3; e.g., approximately 10 ng / mL), and bone morphogenetic protein-2 (BMP2; e.g., approximately 10 ng / mL). For further cartilage self-assembly, EB is deaggregated (e.g., using trypsin) and cultured in a 96-well plate (e.g., coated with agarose) with 10 units per well. 5 The cells can be further transferred based on their density and cultured in the same medium.

[0276] In addition, or alternatively, pluripotent stem cells can also be directly differentiated into chondrocytes by plating cells onto a matrix in the presence of chondrogenesis-inducing culture medium, using various protocols such as those described in Michal Lach et al., 2014. Journal of Tissue Engineering Volume 5: 1-9, the entire content of which is incorporated herein by reference. For example, pluripotent stem cells can be cultured on a matrix in a medium supplemented with various growth factors such as WNT-3a, activin, follistatin, BMP4, fibroblast growth factor 2 (FGF2), growth / differentiation factor 5 (GDF5), and neurotrophin 4 (NT4), as substantially described in Oldershaw RA, et al. 2010 ("Directed differentiation of human embryonic stem cells toward chondrocytes"; Nat Biotechnol 28(11): 1187-1194), the entire content of which is incorporated herein by reference.

[0277] In addition, or alternatively, pluripotent stem cells can be cultured in a medium containing only six growth factors: WNT-3a, activin, follistatin, BMP4, fibroblast growth factor 2 (FGF2), and growth / differentiation factor 5 (GDF5), as substantially described in Yang SL, et al. 2012 ("Compound screening platform using human induced pluripotent stem cells to identify small molecules that promote chondrogenesis". Protein Cell, 3(12): 934-942), the entire contents of which are referenced here. These protocols can differentiate chondrocyte-like cells that exhibit high expression of COL2A1 (type II collagen alpha 1) and SRY (sex-determining region Y)-box 9 (SOX9), as well as low expression of pluripotency markers, compared to control cell lines.

[0278] Neural progenitor cells To differentiate EBs into neural precursors according to some embodiments of the present invention, 4-day-old EBs are cultured for 5-12 days in a tissue culture dish containing DMEM / F-12 medium (TSFn medium, Okabe, S. et al., 1996, Mech. Dev. 59: 89-102) containing 5 mg / ml insulin, 50 mg / ml transferrin, 30 nM selenium chloride, and 5 mg / ml fibronectin. The resulting neural precursors can be further transplanted to generate neurons in vivo (Brustle, O. et al., 1997. In vitro-generated neural precursors participate in mammalian brain development. Proc. Natl. Acad. Sci. USA. 94: 14809-14814). Prior to transplantation, the neural precursors are trypsinized and ground in the presence of 0.1% DNase to obtain a single-cell suspension.

[0279] Oligodendrocytes and myelinated cells EBs of some embodiments of the present invention can be differentiated into oligodendrocytes and myelinated cells by culturing in a modified SATO medium, namely DMEM containing bovine serum albumin (BSA), pyruvic acid, progesterone, putrescine, thyroxine, triiodothyronine, insulin, transferrin, sodium selenite, amino acids, neurotrophin 3, ciliary neurotrophic factor and Hepes (Bottenstein, J. E. & Sato, G. H., 1979, Proc. Natl. Acad. Sci. USA 76, 514-517, Raff, M. C., Miller, R. H., & Noble, M., 1983, Nature 303: 390-396). Summarizing, the EBs are dissociated using 0.25% v / v trypsin / EDTA (for 5 minutes at 37°C) and triturated to form a single cell suspension. The suspended cells are seeded into a flask containing SATO medium supplemented with 5% v / v horse serum and 5% v / v fetal calf serum (FCS). After 4 days of culture, the flask is gently agitated to suspend the weakly adherent cells (primary oligodendrocytes), leaving the astrocytes attached to the flask, and conditioned medium is further produced. The primary oligodendrocytes are transferred to a new flask containing SATO medium for an additional 2 days. After a total of 6 days of culture, the oligospheres are either partially dissociated and resuspended in SATO medium or completely dissociated for seeding into the oligosphere conditioned medium derived from the previous shaking step for cell transplantation [Liu, S. et al., (2000). Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation. Proc. Natl. Acad. Sci. USA. 97: 6126-6131].

[0280] Mast cell For mast cell differentiation, 2-week-old EBs of several embodiments of the present invention are transferred to a tissue culture dish containing DMEM medium supplemented with 10% v / v FCS, 2 mM L-glutamine, 100 units / ml penicillin, 100 mg / ml streptomycin, 20% (v / v) WEHI-3 cell condition medium, and 50 ng / ml recombinant rat stem cell factor (rrSCF, Tsai, M. et al., 2000. In vivo immunological function of mast cells derived from embryonic stem cells: An approach for the rapid analysis of even embryonic lethal mutations in adult mice in vivo. Proc Natl Acad Sci USA. 97: 9186-9190). The cells are transferred to a new flask weekly, and half of the culture medium is replaced to grow the culture.

[0281] Blood lymphoid cells To generate hemato-lymphoid cells from embryoid bodies (EBs) of some embodiments of the present invention, an incubator capable of adjusting the amount of oxygen is used, and 2- to 3-day-old EBs are transferred to a gas-permeable culture dish in the presence of 7.5% CO2 and 5% O2. Following 15 days of differentiation, the cells are harvested and dissociated by mild digestion with both collagenase (0.1 units / mg) and dispase (0.8 units / mg), both available from F. Hoffman-La Roche Ltd, Basel, Switzerland. CD45-positive cells are isolated using the anti-CD45 monoclonal antibody (mAb) M1 / 9.3.4.HL.2 and goat anti-rat immunoglobulin-conjugated paramagnetic microbeads (Miltenyi) as described by Potocnik, A.J. et al., (Immunology Hemato-lymphoid in vivo reconstitution potential of subpopulations derived from in vitro differentiated embryonic stem cells. Proc. Natl. Acad. Sci. USA. 1997, 94: 10295-10300). The isolated CD45-positive cells can be further enriched by passing them once through a MACS column (Miltenyi).

[0282] Note that because EBs have a complex structure, differentiation of EBs into specific differentiated cells, tissues or organs requires isolation of lineage-specific cells from the EBs.

[0283] Such isolation may be performed by sorting of EB cells by fluorescence-activated cell sorter (FACS) or by mechanical separation of cells, tissues and / or tissue-like structures contained in the EBs.

[0284] Methods for isolating EB-derived differentiated cells by FACS analysis are known in the industry. One method involves deaggregating EB cells using a solution of trypsin and EDTA (0.025% v / v and 0.01% v / v, respectively), washing with 5% v / v fetal bovine serum (FBS) in phosphorylated saline (PBS), and incubating on ice for 30 minutes with a fluorescently labeled antibody against a cell surface antigen characteristic of a specific cell lineage. For example, endothelial cells are isolated by conjugating them with an antibody against platelet endothelial cell adhesion molecule 1 (PECAM1), such as the fluorescently labeled PECAM1 antibody (30884X) available from PharMingen (Becton Dickinson Bio Sciences, California, USA), as described in Levenberg, S. et al., (Endothelial cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. USA. 2002. 99: 4391-4396). Hematopoietic cells are isolated using fluorescently labeled antibodies such as CD34-FITC, CD45-PE, CD31-PE, CD38-PE, CD90-FITC, CD117-PE, CD15-FITC, and Class I-FITC, all of which are available from PharMingen as IgG1, or CD133 / 1-PE(IgG1) (available from Miltenyi Biotec, Auburn, California) and glycophorin A-PE(IgG1) (available from Immunotech, Miami, Florida). Live cells (i.e., unfixed) are analyzed using FACScan (Becton Dickinson Bio Sciences) with either PC-LYSIS or CELLQUEST software, after removing dead cells using propidium iodide.The isolated cells can be further enriched using magnetically labeled secondary antibodies and magnetic separation columns (MACS, Miltenyi), as described in Kaufman, DS et al., (Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. USA. 2001, 98: 10716-10721).

[0285] An example of mechanical isolation of pulsating cardiomyocytes from EB is disclosed in U.S. Patent Application Publication No. 20030022367 by Xu et al. In summary, EB of several embodiments of the present invention at 4 days old are transferred to a gelatin-coated plate or chamber slide, where they adhere and differentiate. Cells that spontaneously contract, observed from day 8 of differentiation, are mechanically separated and collected in a 15 mL tube containing low-calcium medium or PBS. Depending on collagenase activity, the cells are dissociated by collagenase B digestion at 37°C for 60–120 minutes. The dissociated cells are resuspended in differentiation KB medium (85 mM KCI, 30 mM K2HPO4, 5 mM MgSO4, 1 mM EGTA, 5 mM creatine, 20 mM glucose, 2 mM Na2ATP, 5 mM pyruvate, and 20 mM taurine, buffered to pH 7.2, Maltsev et al., Circ. Res. 75:233, 1994) and incubated at 37°C for 15-30 minutes. Following dissociation, the cells are implanted into chamber slides and cultured in differentiation medium to produce pulsatile single cardiomyocytes.

[0286] Appropriate culture conditions for the differentiation and proliferation of isolated lineage-specific cells include various tissue culture media, growth factors, antibiotics, amino acids, etc. It should be noted that determining the conditions to be adapted for the proliferation and differentiation of a particular cell type and / or cell lineage is within the scope of the skills of those skilled in the art [reported in Fijnvandraat AC, et al., Cardiovasc Res. 2003; 58: 303-12, Sachinidis A, et al., Cardiovasc Res. 2003; 58: 278-91, and Stavridis MP and Smith AG, 2003; Biochem Soc Trans. 31(Pt 1): 45-9].

[0287] Cell lines of some embodiments of the present invention can be produced by immortalizing EB-derived cells by methods known in the art, such as expressing the telomerase gene in cells (Wei, W. et al., 2003. Mol Cell Biol. 23: 2859-2870), or co-culturing cells with NIH 3T3 hph-HOX11 retrovirus-producing cells (Hawley, RG et al., 1994. Oncogene 9: 1-12).

[0288] The following are non-limiting examples of appropriate culture conditions for differentiated and / or proliferating lineage-specific cells derived from pluripotent stem cells (e.g., ESCs and iPS cells).

[0289] CD73-positive and SSEA-4-negative mesenchymal stem cells can be generated from pluripotent stem cells by mechanically increasing the proportion of fibroblast-like differentiated cells formed in the pluripotent stem cell culture, as substantially described in Trivedi P and Hematti P. Exp Hematol. 2008, 36(3):350-9. In summary, extending the interval between culture media changes to 3-5 days to induce differentiation of pluripotent stem cells causes the cells around the ESC colony to resemble cone-shaped fibroblasts. Under these conditions, after 9-10 days, when approximately 40-50% of the cultured cells have acquired a fibroblast-like appearance, the undifferentiated portion of the pluripotent stem cell colony is physically removed, and the remaining differentiated cells are subcultured in a new culture dish under the same conditions.

[0290] To induce differentiation of pluripotent stem cells into dopaminergic (DA) neurons, cells can be co-cultured with mouse stromal cell lines PA6 or MS5, as substantially described in Vazin T, et al., PLoS One. 2009 Aug 12; 4(8):e6606 and Elkabetz Y., et al., Genes Dev. 2008 January 15; 22: 152-165, or cultured with a combination of stromal cell-derived factor 1 (SDF-1 / CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), and ephyrin B1 (EFNB1).

[0291] To generate midbrain dopamine (mesDA) neurons, pluripotent stem cells can be genetically modified to express the transcription factor Lmx1a, as substantially described in Friling S., et al., Proc Natl Acad Sci US A. 2009, 106: 7613-7618 (e.g., using a lentiviral vector containing the PGK promoter and Lmx1a).

[0292] To generate lung epithelium (type II lung cells) from pluripotent stem cells, pluripotent stem cells can be cultured in the presence of commercially available cell culture medium (Small Airway Growth Medium, College Park, Maryland, Cambrex), as described in Rippon HJ., et al., Proc Am Thorac Soc. 2008; 5: 717-722, or alternatively, in the presence of a condition medium recovered from a lung cell line (e.g., A549 human lung adenocarcinoma cell line).

[0293] To induce differentiation of pluripotent stem cells into neurons, pluripotent stem cells can be cultured for approximately 5 days in serum substitute medium supplemented with a TGF-β inhibitor (SB431542, Tocris, e.g., 10 nM) and noggin (R&D, e.g., 500 ng / ml), as substantially described in Chambers SM., et al., Nat Biotechnol. 2009, 27: 275-280, followed by culture in increasing amounts (e.g., 25%, 50%, 75%, changed every 2 days) of N2 medium (Li XJ., et al., Nat Biotechnol. 2005, 23:215-21) in the presence of 500 ng / mL of noggin.

[0294] In addition to strain-specific primary culture, the EB of the present invention can be used to create strain-specific cell lines that can grow without restriction during culture.

[0295] Cell lines of some embodiments of the present invention can be produced by immortalizing EB-derived cells by methods known in the art, such as expressing the telomerase gene in cells (Wei, W. et al., 2003. Mol Cell Biol. 23: 2859-2870), or co-culturing cells with NIH 3T3 hph-HOX11 retrovirus-producing cells (Hawley, RG et al., 1994. Oncogene 9: 1-12).

[0296] As used herein, the term "IL6RIL6 chimera" refers to a chimeric polypeptide comprising a soluble portion of the interleukin-6 receptor (IL-6-R, e.g., GenBank accession number AAH89410, human IL-6-R described in SEQ ID NO: 1) (e.g., amino acids 112-355 of GenBank accession number AAH89410, part of the soluble IL6 receptor described in SEQ ID NO: 2) and interleukin-6 (IL-6) (e.g., GenBank accession number CAG29292, human IL-6 described in SEQ ID NO: 3) or its bioactive fraction (e.g., receptor-binding domain). The IL6RIL6 chimera used by the method according to this aspect of the present invention is preferably capable of supporting the undifferentiated proliferation of human pluripotent stem cells while maintaining their pluripotency. When constructing an IL6RIL6 chimera, it should be understood that the two functional parts (i.e., IL6 and its receptor) can be directly fused to each other (e.g., by binding or translational fusion, i.e., so that they are encoded by a single open reading frame) or conjugated via a suitable linker (e.g., a polypeptide linker). Preferably, the IL6RIL6 chimeric polypeptide exhibits glycosylation in amounts and patterns similar to those of naturally occurring IL6 and its receptor. For example, a suitable IL6RIL6 chimera is shown in SEQ ID NO: 4 and in Figure 11 of International Publication No. 99 / 02552 by Revel M., et al., which is fully incorporated herein by reference.

[0297] As used herein, the term "WNT3A" refers to a member of the WNT gene family. The WNT gene family consists of structurally related genes that encode secretory signaling proteins. These proteins are known to be involved in oncogenicity as well as in several developmental processes, including the regulation of cell fate and pattern formation during embryonic development.

[0298] The WNT3A mRNA (GenBank accession number NM_033131.3, SEQ ID NO: 5) encodes the WNT3A polypeptide (GenBank accession number NP_149122.1, SEQ ID NO: 6). The WNT3A polypeptide is available from various manufacturers, including R&D SYSTEMS (e.g., catalog number 5036-WN-010).

[0299] As used herein, the term "basic fibroblast growth factor (bFGF)" refers to polypeptides of the fibroblast growth factor (FGF) family that bind to heparin and possess broad mitogenic and angiogenic activity. The bFGF gene mRNA has multiple polyadenylation sites and is translated alternately from non-AUG (CUG) start codons and AUG start codons, resulting in five distinct isoforms with individual characteristics. The CUG-initiated isoform is localized to the nucleus and is responsible for endocrine effects, while the majority of the AUG-initiated forms are cytoplasmic and are responsible for the paracrine and autocrine effects of this FGF.

[0300] bFGF polypeptide is available under GenBank accession number NP_001997 (SEQ ID NO: 7) and can be obtained from various manufacturers such as Peprotech, R&D Systems (e.g., catalog number 233-FB), and Millipore.

[0301] The bovine bFGF polypeptide is provided by GenBank accession number NP_776481.2 (SEQ ID NO: 11) and is encoded by SEQ ID NO: 12 (GenBank accession number NM_174056.4). Bovine bFGF is available from R&D systems, for example, as bovine FGFbasic / FGF2 / bFGF (derived from bovine brain, catalog number 133-FB-025) or recombinant bovine FGFbasic / FGF2 / bFGF (derived from E. coli, catalog number 2099-FB-025). As used herein, the term "leukemia inhibitory factor (LIF)" refers to a multifunctional cytokine that is involved in the induction of hematopoietic differentiation, the induction of neuronal differentiation, and the regulation of the conversion of mesenchymal cells to epithelial cells during kidney development, and may also play a role in immune tolerance at the maternal-fetal interface.

[0302] The LIF used in the culture medium of some embodiments of the present invention may be a purified, synthetic, or recombinantly expressed LIF protein [e.g., human LIF polypeptide, GenBank accession number NP_002300.1 (SEQ ID NO: 8), human LIF polynucleotide, GenBank accession number NM_002309.4 (SEQ ID NO: 9), bovine LIF polypeptide encoded by GenBank accession number NM_173931.1 (SEQ ID NO: 11), GenBank accession number NP_776356.1 (SEQ ID NO: 10)]. It should be noted that for the preparation of xeno-free culture medium, recombinantly expressed LIF is preferably used. Recombinant human LIF can be obtained from various suppliers such as Chemicon (catalog number LIF10100) in the United States and AbD Serotec (MorphoSys US Inc., Raleigh, North Carolina 27604, USA). Mouse LIF ESGRO® (LIF) can be obtained from Millipore (catalog number ESG1107) in the United States.

[0303] According to some embodiments of the present invention, the concentration of LIF in the culture medium is about 1,000 units / ml to about 10,000 units / ml, for example, about 2,000 units / ml to about 10,000 units / ml, about 2,000 units / ml to about 8,000 units / ml, for example, about 2,000 units / ml to about 6,000 units / ml, for example, about 2,000 units / ml to about 5,000 units / ml, for example, about 2,000 units / ml to about 4,000 units / ml, for example, about 2,500 units / ml to about 3,500 units / ml, for example, about 2,800 units / ml to about 3,200 units / ml, for example, about 2,900 units / ml to about 3,100 units / ml, for example, about 3,000 units / ml.

[0304] According to some embodiments of the present invention, the concentration of LIF in the culture medium is at least about 1000 units / ml, for example, at least about 2000 units / ml, for example, at least about 2100 units / ml, for example, at least about 2200 units / ml, for example, at least about 2300 units / ml, for example, at least about 2400 units / ml, for example, at least about 2500 units / ml, for example, at least about 2600 units / ml, for example, at least about 2700 units / ml, for example, at least about 2800 units / ml, for example, at least about 2900 units / ml, for example, at least about 2950 units / ml, for example, at least about 3000 units / ml.

[0305] As described above, any protein factors used in the culture medium of some embodiments of the present invention (e.g., bFGF, IL6RIL6 chimera, WNT3a, LIF) may be recombinantly expressed or biochemically synthesized. In addition, naturally occurring protein factors such as bFGF, WNT3a, and LIF can be purified from biological samples (e.g., human serum, cultured cells) by known methods. It should be noted that recombinantly expressed protein factors are preferable for the preparation of xeno-free culture medium.

[0306] The biochemical synthesis of protein factors can be carried out using standard solid-phase techniques. These methods include exclusive solid-phase synthesis, partial solid-phase synthesis, fragment condensation, and classical solution synthesis.

[0307] Recombinant expression of the protein factor of the present invention is described in Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680, Brogli et al., (1984) Science 224:838-843, Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press. They can be prepared using the recombinant techniques described in NY, Section VIII, pp 421-463. Specifically, IL6RIL6 chimeras can be prepared as described in International Publication No. 99 / 02552 of Revel M., et al. and Chebath J, et al., 1997, which are fully incorporated herein by reference.

[0308] As described above, some embodiments of the present invention utilize the culture of mammalian livestock (e.g., bovine) embryos or stem cells on or without a feeder cell layer.

[0309] The following is an illustrative and non-exclusive description of the feeder cell layer.

[0310] Mouse feeder layer: The most common method for culturing pluripotent stem cells is based on a feeder cell layer of mouse embryonic fibroblasts (MEFs) supplemented with tissue culture medium containing serum or leukemia inhibitory factor (LIF) that supports the proliferation and pluripotency of pluripotent stem cells [Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. (1998). Embryonic stem cell lines derived from human blastocysts. Science 282: 1145-7, Reubinoff BE, Pera MF, Fong C, Trounson A, Bongso A. (2000). Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat. Biotechnol. 18: 399-404]. MEF cells are induced from 12-13 day old mouse embryos in a culture medium supplemented with fetal bovine serum. Under these conditions, mouse ES cells can be maintained as pluripotent stem cells that preserve phenotypic and functional characteristics. It should be noted that the use of feeder cells substantially increases production costs. In addition, since feeder cells are metabolically inactive and do not proliferate beyond stem cells, fresh feeder cells are required each time the pluripotent stem cell culture is divided.

[0311] Pluripotent stem cells can also be cultured on MEFs under serum-free conditions using a serum substitute supplemented with basic fibroblast growth factor (bFGF) [Amit M, Carpenter MK, Inokuma MS, Chiu CP, Harris CP, Waknitz MA, Itskovitz-Eldor J, Thomson JA. (2000). Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol. 227: 271-8]. Under these conditions, the cloning efficiency of ES cells is four times higher than in the presence of fetal bovine serum. In addition, even after 6 months of culture in the presence of the serum substitute, ES cells still retain their pluripotency, as demonstrated by their ability to form teratomas containing all three types of embryonic germ layers. Although this system uses more restricted culture conditions, the presence of mouse cells in culture limits its use in cell therapy because it may expose pluripotent stem cell cultures to mouse pathogens.

[0312] Human embryonic fibroblasts or adult fallopian tube epithelial cells as feeder cell layers: Embryonic stem cells can be proliferated and maintained using human embryonic fibroblasts or adult fallopian tube epithelial cells. When grown on these human feeder cells, embryonic stem cells exhibit a normal karyotype, exert alkaline phosphatase activity, express Oct-4 and other embryonic cell surface markers (including SSEA-3, SSEA-4, TRA-1-60, and GCTM-2), form teratomas in vivo, and maintain all key morphological features [Richards M, Fong CY, Chan WK, Wong PC, Bongso A. (2002). Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat. Biotechnol. 20: 933-6].

[0313] Foreskin Feeder Layer: Embryonic stem cells can be cultured in the human foreskin feeder layer disclosed in U.S. Patent Application Publication No. 10 / 368,045. The foreskin-derived feeder cell layer provides a completely animal-free environment suitable for culturing embryonic stem cells. In addition, foreskin cells can be maintained in culture for up to 42 passages from their derivation, providing a relatively constant environment for embryonic stem cells. Under these conditions, embryonic stem cells were found to be functionally indistinguishable from cells grown using alternative protocols (e.g., MEF). Following differentiation, embryonic stem cells expressed genes related to all three embryonic germ layers in vitro and formed teratomas in vivo consisting of tissues derived from all three embryonic germ layers. Furthermore, unlike human fallopian tube epithelial cells or human embryonic fibroblasts, human embryonic stem cells cultured on the foreskin feeder layer were maintained in a pluripotent and undifferentiated state for at least 87 passages during culture. However, even if foreskin cells are maintained in culture for a long period (i.e., 42 passages), the foreskin culture system is not precisely defined due to differences between individual batches. In addition, culture systems based on human feeder layers still require the simultaneous proliferation of both the feeder layer and hES cells. Therefore, a culture system without feeder cells has been developed.

[0314] The following is an illustrative and non-limiting description of a culture system without feeder cells.

[0315] Stem cells can grow on solid surfaces such as extracellular matrix (e.g., Matrigel® or laminin) in the presence of culture medium. Unlike feeder-based cultures, which require the simultaneous growth of feeder cells and stem cells and can result in mixed cell populations, stem cells grown in feeder-free systems are easily separated from the surface. For stem cell proliferation, culture media containing factors that effectively inhibit differentiation and promote proliferation, such as MEF-conditioned medium or bFGF, are used. However, commonly used feeder-free culture systems utilize animal-based matrices (e.g., Matrigel®) supplemented with mouse or bovine serum or MEF-conditioned medium [Xu C, et al. (2001). Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol. 19: 971-4], which carries the risk of cross-transfer of animal pathogens to human ES cells, hindering future clinical use.

[0316] According to some embodiments of the present invention, the feeder cell matrix is ​​selected from the group consisting of Matrigel® matrix, fibronectin matrix, laminin matrix, and vivonectin matrix.

[0317] Pluripotent stem cells of some embodiments of the present invention, or cells differentiated therefrom (e.g., adipocytes, muscle cells, blood cells, chondrocytes, osteocytes, connective tissue cells, fibroblasts, and / or cardiomyocytes), can be identified using various expression markers that characterize these cells. Expression markers can be identified at the RNA or protein level.

[0318] Methods for detecting RNA expression levels include, but are not limited to, Northern blot analysis, RT-PCR analysis, RNA in situ hybridization staining, in situ RT-PCR staining, DNA microarrays / DNA chips, and oligonucleotide microarrays.

[0319] Methods for detecting protein expression and / or activity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), fluorescence-activated cell sorting (FACS) for sorting adipocytes, muscle cells, blood cells, chondrocytes, osteocytes, connective tissue cells, fibroblasts, and / or cardiomyocytes, immunohistochemical analysis, and in-situ activity analysis.

[0320] As used in this specification, the term "approximately" refers to ±10%.

[0321] According to some embodiments of the present invention, the term “about” means ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.1%.

[0322] The terms "comprises," "comprising," "includes," "including," and "having," along with their conjugations, all mean "to include (but not limited to)."

[0323] The term "consisting of" means "including and limited to."

[0324] The term "consisting essentially of" means that a composition, method, or structure may include additional components, steps, and / or parts, provided that such additional components, steps, and / or parts do not materially alter the fundamental and novel features of the composition, method, or structure described in the claims.

[0325] As used herein, the singular forms "a," "an," and "the" include multiple references unless the context otherwise explicitly states. For example, the terms "a compound" or "at least one compound" may include multiple compounds (including mixtures thereof).

[0326] Throughout this application, various embodiments of the present invention can be presented in scope form. It should be understood that the scope form is merely for convenience and brevity and should not be interpreted as an irrevocable limitation of the scope of the present invention. Therefore, a scope description should be considered to specifically disclose not only all possible subranges but also the individual numerical values ​​within that range. For example, a scope description such as 1 to 6 should be considered to specifically disclose not only subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, and 3 to 6, but also the individual numerical values ​​within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0327] Where a numerical range is indicated herein, it shall always include any cited numerical value (fraction or integer) within that range. The phrases “range between” the first and second indicators, and furthermore, “range from” the first indicator to the second indicator, are used synonymously herein and shall include the first and second indicators and all fractions and integers between them.

[0328] As used herein, the term “method” means a method, means, technique, and procedure for accomplishing a given task, and includes, but is not limited to, methods, means, techniques, and procedures known to those skilled in the art in the fields of chemistry, pharmacy, biology, biochemistry, and medicine, or methods, means, techniques, and procedures that can be readily developed from methods, means, techniques, and procedures known to those skilled in the art.

[0329] As used herein, the term “treat” means to neutralize, substantially inhibit, slow down, or reverse the progression of a disease, including substantial improvement of the clinical or aesthetic symptoms associated with the disease, or substantial prevention of the appearance of the clinical or aesthetic symptoms associated with the disease.

[0330] When referring to a specific sequence listing, such reference includes sequences that substantially correspond to its complementary sequence, even those with minor sequence changes resulting from sequencing errors, cloning errors, or other modifications that lead to base substitutions, base deletions, or base additions, provided that the frequency of such changes is less than 1 per 50 nucleotides, or less than 1 per 100 nucleotides, or less than 1 per 200 nucleotides, or less than 1 per 500 nucleotides, or less than 1 per 1000 nucleotides, or less than 1 per 5000 nucleotides, or less than 1 per 10000 nucleotides.

[0331] It should be understood that the sequence numbers (SEQ ID NOs) disclosed in this application may represent either DNA or RNA sequences, depending on the context in which the SEQ ID NO is described, even if the sequence is expressed only in the form of a DNA sequence or only in the form of an RNA sequence. For example, SEQ ID NO: 13 is expressed in the form of a DNA sequence (e.g., describing T for thymine), but it may represent a DNA sequence corresponding to a bovine bFGF nucleic acid sequence, or an RNA sequence which is the nucleic acid sequence of an RNA molecule. Similarly, some sequences are expressed in the form of an RNA sequence (e.g., describing U for uracil), but depending on the actual type of molecule described, it may represent a sequence of an RNA molecule containing dsRNA, or a sequence of a DNA molecule corresponding to the RNA sequence shown therein. In any case, both DNA and RNA molecules with sequences containing any substitutions are envisioned.

[0332] Features of the present invention described as separate embodiments for clarity should be understood to be able to be combined and provided as a single embodiment. Conversely, various features of the present invention described as a single embodiment for brevity can also be provided individually, in any suitable partial combination, or in a suitable combination with other embodiments described herein. Features described in relation to various embodiments are not considered essential requirements of those embodiments unless the embodiment is inoperable without those features.

[0333] The various embodiments and aspects of the invention, as described in detail above and claimed within the scope of the claims below, are experimentally supported by the following examples. [Examples]

[0334] The following examples, along with the above description, illustrate some embodiments of the present invention without limiting them.

[0335] The nomenclature and experimental procedures used herein typically include molecular techniques, biochemical techniques, microbiological techniques, and recombinant DNA techniques. Such techniques are well described in the literature. For example, "Molecular Cloning: A Laboratory Manual" Sambrook et al., (1989), "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994), Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989), Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988), Watson et al., "Recombinant DNA", Scientific American Books, New York, Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998), methods described in U.S. Patent Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659, and 5,272,057, "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, JE, ed. (1994), "Current Protocols in Immunology", Volumes I-III Coligan, JE, ed. (1994), Stites et al.See (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994), and Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", WH Freeman and Co., New York (1980). Available immunoassays are widely described in patents and scientific literature, for example, U.S. Patent Nos. 3,791,932, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, 3,879,262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, 4,098,876, 4,879,219, 5,011,771, and 5,281,521, "Oligonucleotide Synthesis" Gait, MJ, ed. (1984), “Nucleic Acid Hybridization” Hames, BD, and Higgins SJ, eds. (1985), “Transcription and Translation” Hames, BD, and Higgins SJ, Eds. (1984), “Animal Cell Culture” Freshney, RI, ed. (1986), “Immobilized Cells and Enzymes” IRL Press, (1986), "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol.See 1-317, Academic Press, "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990), and Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual", CSHL Press (1996). All of the above references are fully incorporated herein by reference. Other general references are provided herein. The procedures described therein are considered well known in the art and are provided for the convenience of the reader. All information contained therein is incorporated herein by reference.

[0336] General materials and experimental methods One difference between delayed blastocysts in cattle and horses is that in cattle, embryos (tightly packed embryos or early blastocysts) are obtained from cattle 7 days after insemination, while in horses, embryos (blastocysts or proliferating blastocysts, usually only one embryo) are obtained from 8 days after insemination.

[0337] Culture of bovine blastocysts: Seven-day-old embryos were obtained by washing the uterus of Holstein cattle undergoing uterine fertilization. The embryos were washed and maintained in Holding and Transfer Medium (BioLife, product number C15C, USA) for up to 1 hour before being transferred to culture conditions.

[0338] Blastocysts can also be obtained from the following raw materials. - Commercially available products (Israel, Ha'fetz Ha'im, Sion) - IVF, in vitro oocyte fertilization - Nuclear transfer (NT) of bovine cells - unisexual reproduction

[0339] Derivation of bovine PSC strains: After digesting the zona pellucida with Tyrode's acidic solution (Sigma Aldrich, St. Louis, Missouri, USA), the exposed blastocysts were plated. Two different plating methods were used. (i) A feeder layer of mitotically inactive mouse embryonic fibroblasts (MEFs) or mitotically inactive prepectineal fibroblasts, (ii) A suitable matrix (Matrigel® matrix, fibronectin, laminin, vitronectin, or commercially available cell matrix).

[0340] The embryo was attached to the surface using one of the following techniques. (i) Use of 27g needles, (ii) Use of an extended Pasteur pipette, (iii) Covering the embryo with a drop of suitable matrix, or (iv) Leave the embryos until they spontaneously attach to the surface.

[0341] Attached bovine blastocysts were cultured as whole embryos on MEF for 7–21 days post-fertilization until large cysts developed (e.g., 14 days post-fertilization as shown in Figure 1C). If necessary due to the quality of the MEF or matrix, the entire embryo was transferred to a new MEF-coated plate using a 27-gauge needle, leaving a small number of peripheral fibroblasts. Once the embryo developed cysts, the disc-like structures were isolated and individually plated on fresh MEF or matrix-coated plates. Cells with stem cell morphology (small cells with large nuclei) were mechanically passaged. After several passages (4–6 passages), once a population rich in bovine pluripotent stem cells was established, the cells were regularly passaged every 5–10 days using 1 mg / ml type IV collagenase (Gibco Invitrogen Corporation products, San Diego, California, USA).

[0342] Culture medium: Candidate 1: Medium X, consisting of 80% v / v DMEM / F12 or KO-DMEM supplemented with 20% v / v limited fetal bovine serum (FBS) (HyClone, Utah, USA), 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, and 1% v / v non-essential amino acid stock (all products of Gibco Invitrogen Corporation, San Diego, California, USA).

[0343] Medium X can support the undifferentiated proliferation of bovine PSCs cultured on feeder cells such as MEFs. However, when bovine PSCs are cultured in this medium at high density (e.g., without passage for at least 14 days) on MEFs, the bovine PSCs spontaneously differentiate. In addition, when bovine PSCs are cultured in this medium in a culture system without feeder cells, the bovine PSCs spontaneously differentiate.

[0344] Candidate 2: Cells were cultured in a medium consisting of IL6RIL6 chimeric medium, 85% v / v DMEM / F12 (or KO-DMEM), supplemented with 15% v / v KO-serum substitute, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1% non-essential amino acid stock, 100 pg / ml IL6-IL6 receptor chimera (chimera, biotest), and 50 ng / ml basic fibroblast growth factor (bFGF) (all products except the IL6-IL6 receptor chimera are Gibco Invitrogen Corporation products from San Diego, California, USA). The cells were frozen in liquid nitrogen using a freezing solution consisting of 10% v / v DMSO (Sigma, St. Louis, Missouri, USA), 10% v / v FBS (HyClone, Utah, USA), and 80% v / v DMEM / F12.

[0345] Candidate 3: Cells were cultured in Wnt3a medium, 85% v / v DMEM / F12 (or KO-DMEM), supplemented with 15% v / v ko-serum substitute, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1% v / v non-essential amino acid stock, 10 ng / ml Wnt3a (Biotest), 100 ng / ml basic fibroblast growth factor (bFGF), and 3000 U / ml leukemia suppressor factor (LIF) (unless otherwise specified, all products are from Gibco Invitrogen Corporation, San Diego, California, USA). Cells were frozen in liquid nitrogen using a freezing solution consisting of 10% v / v DMSO (Sigma, St. Louis, Missouri, USA), 10% v / v FBS (HyClone, Utah, USA), and 80% v / v DMEM / F12.

[0346] Culture of horse blastocysts: Eight-day-old embryos were obtained by washing the uterus of horses undergoing uterine fertilization. The embryos were washed and maintained in Holding and Transfer Medium (BioLife, product number C15C) until ready for culture (up to 1 hour).

[0347] Blastocysts can also be obtained from the following raw materials. - Commercially available products - In vitro fertilization (IVF) - In vitro fertilization of oocytes - Nuclear transfer (NT) of horse cells - unisexual reproduction

[0348] Derivation of the horse PSC strain: After digesting the zona pellucida with Tyrode's acidic solution (Sigma Aldrich, St. Louis, Missouri, USA), the exposed blastocysts were plated. Two possible plating methods were used: (i) a feeder layer such as mitotically inactive mouse embryonic fibroblasts (MEF) or mitotically inactive prepectinate fibroblasts, and (ii) a suitable matrix (Matrigel® matrix, fibronectin, laminin, vitronectin, commercially available cell matrix). Embryos were attached to the surface by covering them with a 27g needle, a stretched Pasteur pipette, or a drop of suitable matrix, or by leaving the embryos to adhere to the surface spontaneously. The attached blastocysts were cultured as whole embryos on MEFs for 8–21 days post-fertilization (e.g., a specific HRS1 embryo was cultured 16 days post-fertilization as shown in Figure 8B) until large cysts developed. If necessary due to the quality of the MEF or matrix, the entire embryo is transferred to a new MEF-coated plate using a 27-gauge needle, leaving a small number of peripheral fibroblasts. Once the embryo develops cysts, the disc-like structures are isolated from them and individually plated onto fresh MEF or matrix-coated plates. Cells with stem cell morphology (small cells with large nuclei) are mechanically passaged. After several passages (3-6 passages), once homogeneous culture is achieved, the cells are regularly passaged every 5-10 days using 1 mg / ml type IV collagenase (Gibco Invitrogen Corporation products, San Diego, California, USA).

[0349] Culture medium: The culture medium X mentioned above.

[0350] Formation of EB: To form embryoid bodies (EBs), two of the four confluent wells in a four-well plate were used. Cells were left undivided for 14 days to allow EBs to form spontaneously. Some remained attached to the culture surface, while others were suspended EBs (Figure 3A-B). The EBs were grown using medium X.

[0351] Immunostaining: The cells were fixed at room temperature and exposed to the primary antibody. Next, the cells were incubated with the secondary antibody. The reaction conditions and antibodies are summarized in Table 1 below.

[0352] [Table 1]

[0353] Table 1 provides immunohistochemical staining conditions, the antibodies used, and the antigens identified by the antibodies. It also provides characteristics of cells exhibiting positive antigen expression. For example, OCT4 is an undifferentiated pluripotent stem cell marker. "Host serum" refers to serum derived from the same species as the host animal from which the antibody was produced. "NA" indicates no applicable term.

[0354] Spontaneous differentiation into adipocytes: Bovine PSCs were cultured in "Culture Medium X" (without dexamethasone) for 14-21 days without cell passaging, and then fixed with paraformaldehyde for evaluation of lipid droplets by oil red staining.

[0355] Oil red dye: The cells were fixed with 4% v / v paraformaldehyde (PFA) at room temperature (RT) for 20 minutes. After washing off the PFA with phosphate-buffered saline (PBS), the cells were incubated with oil red O solution (Sigma) at RT for 10 minutes. The culture was washed with water and visualized using a phase-contrast microscope.

[0356] Example 1 Derivation of bovine pluripotent stem cells from elongated blastocysts Experimental results Using the derivation method described in "General Materials and Experimental Methods" above, the inventors demonstrated the derivation of four bovine cell lines (BVN1, BVN2, BVN5, and BVN6).

[0357] Derivation of the bovine pluripotent stem cell line BVN1: The inventors used two types of bovine embryos 7 days post-fertilization: one consisting of incomplete pseudoblast cells and the other a normal blastocyst. In vitro on MEF, the normal bovine embryo proliferated until day 13 post-fertilization, but the incomplete embryo did not continue development in vitro. Therefore, the experiment was continued with the normal blastocyst.

[0358] Derivation of bovine pluripotent stem cell lines from elongated blastocysts: Bovine embryos 7 days post-fertilization were cultured whole on MEFs in the presence of medium X until 13 days post-fertilization (i.e., 6 days in vitro on MEFs) until large cysts developed (Figure 1A-C).

[0359] After the embryos developed cysts, disc-like structures were isolated from the embryos and individually plated on fresh MEF or matrix-coated plates (Figure 1A-C). During passage, two colonies were transferred to IL6RIL6 chimeric medium and Matrigel®. Cells with stem cell morphology (small cells with large nuclei) were mechanically passaged. After several passages (4-6 passages), once a population rich in cultured bovine pluripotent stem cells was achieved, the cells were regularly passaged every 5-10 days using 1 mg / ml type IV collagenase (Gibco Invitrogen Corporation products, San Diego, California, USA).

[0360] The resulting bovine pluripotent stem cells, named "bPSC," were the first bovine pluripotent stem cells obtained using elongated blastocyst culture technology, and the initial strain was designated "BVN1."

[0361] Bovine pluripotent stem cells exhibit a morphology similar to that of human ESCs: As shown in Figures 1A-C, light microscopy revealed that colonies formed by bPSCs exhibit a morphology similar to that of hESCs, such as round colonies with intercellular spaces, relatively large nuclei, and clearly defined nuclear structures.

[0362] Bovine pluripotent stem cells (bPSCs) can be maintained on feeder cell layers in various culture media: As shown in Figures 2A-C, bPSCs were cultured under different conditions and maintained the morphology of the bPSC colonies. For example, bPSCs were cultured on feeder cells such as MEFs in the presence of a serum-containing culture medium ("Culture X") (Figure 2A).

[0363] In addition, bPSCs were successfully cultured on MEFs even in the presence of serum-free culture media such as IL6RIL6 chimeric culture medium (Figure 2C).

[0364] As shown in Figures 2A and 2C, bPSCs cultured on feeder cells exhibited typical intercellular spaces within the colonies, and the cells showed a high nucleus-to-cytoplasmic ratio typical of pluripotent stem cells (PSCs).

[0365] Bovine pluripotent stem cells (bPSCs) can be maintained in serum-free culture medium in a feeder cell-free culture system: When cultured on a Matrigel™ matrix in the presence of serum-free culture medium (IL6RIL6 chimera), bPSCs were successfully cultured under feeder cell-free conditions.

[0366] It should be noted that bovine PSCs cultured in either Wnt3a-containing medium or IL6RIL6 chimeric medium while being passed through remained in an undifferentiated state (Figures 2A-C, and data are not shown).

[0367] Bovine pluripotent stem cells derived from elongated blastocysts exhibit a pluripotent cell phenotype: Immunostaining of bPSCs with the embryonic pluripotency marker Oct4 showed positive staining (Figure 3A-B).

[0368] Bovine pluripotent stem cells derived from elongated blastocysts are capable of differentiating into embryoid bodies: bPSCs were transferred to a 4-well plate in the presence of medium X (consisting of 80% v / v DMEM / F12 or KO-DMEM supplemented with 20% v / v restricted fetal bovine serum). The cells were left undivided for 14 days until confluent culture was achieved, allowing for spontaneous formation of embryoid bodies (EBs). Some remained attached to the culture surface, while others were suspended EBs (Figure 4A-C).

[0369] Embryoid bodies derived from bovine pluripotent stem cells contain differentiated cells representing all three types of embryonic germ layers: Immunostaining of differentiation markers revealed the ability to differentiate into cells representing all three types of embryonic germ layers (Figure 5A-D).

[0370] Bovine pluripotent stem cells (PSCs) can spontaneously differentiate into adipocytes: When bovine PSCs were cultured on an MEF feeder layer in the presence of medium X or IL6RIL6 chimeric medium, they spontaneously differentiated into adipocytes as background differentiation or after being left for more than 14 days without passage. Spontaneous differentiation was recognized by staining of oil droplets with oil red staining. Note that no forced induction into the adipogenic lineage was performed. The medium used for spontaneous differentiation did not contain dexamethasone, which is known as an inducer of stem cells into the adipogenic lineage.

[0371] In stark contrast to the bovine PSCs described in this application, the human delayed blastocyst cells described in International Publication No. 2006 / 040763 did not spontaneously differentiate into adipocytes without induction by EB formation or adipocyte differentiation medium and removal of the MEF feeder layer. As shown in Figures 6A-B, the spontaneous differentiation of bovine pluripotent cells into adipocytes was revealed by intracellular lipid droplets stained with oil red. These results demonstrate the ability of bPSCs to spontaneously differentiate into adipocytes without the use of chemical or hormonal induction.

[0372] Derivation of bovine pluripotent stem cells from the delayed bovine blastocyst line BVN6: Bovine blastocysts were obtained 8 days post-fertilization, and the entire embryo was plated onto feeder cells (mouse embryonic fibroblasts) until 16 days post-fertilization (Figure 7A-B). As shown in Figure 7B, prominent cysts developed at 16 days post-fertilization, and the disc-like structures were subsequently isolated from the embryos and further plated individually onto fresh MEF. To derive the bovine delayed blastocyst cell line, the cells were further cultured in culture medium (medium X).

[0373] Morphological characterization of bovine pluripotent stem cell (bPSC) colonies of BVN1, BVN2, and BVN5 strains: As shown in Figures 9A–D, cells of bPSC strains BVN1, BVN2, and BVN5 maintained small cells with large nuclei, each exhibiting typical pluripotent stem cell morphology, at various passage numbers (8, 9, and 30 passages) in culture media such as medium X (e.g., up to 15 passages in medium X). Long-term culture and passage were performed using culture media containing the IL6RIL6 chimera.

[0374] Expression of pluripotency markers TRA1-60 and TRA1-81 in the BVN5 bovine cell line derived from delayed bovine blastocysts: Figures 10A-D show that the BVN5 strain of bPSCs maintained an undifferentiated and pluripotent state after at least 8 passages in the presence of medium X, as evidenced by positive staining for TRA1-60 (red) and TRA1-81 (green).

[0375] Example 2 Derivation of equine pluripotent stem cells from elongated blastocysts Experimental results Using the derivation method described in "General Materials and Experimental Methods" above, the inventors demonstrated the derivation of one type of horse cell line (HRS1).

[0376] Derivation of Equine Delayed Blastocyst Cell Lines: The inventors used equine embryos 8 days post-fertilization. As shown in Figure 8A, elongated equine blastocysts with prominent inner cell masses (ICM, white arrows) were observed 8 days post-fertilization. The entire equine embryo was plated with mouse embryonic fibroblasts (MEFs) and cultured in vitro in the presence of medium X until 16 days post-fertilization, when prominent cysts developed (Figure 8B). Figures 8B and 8C show the same embryo at 16 days post-fertilization at different microscopic focuses. On 16 days post-fertilization, the disc-like structures were isolated from the embryos and individually plated further on fresh MEFs using medium X. As shown in Figure 8D, the derived cells formed colonies of pluripotent stem cells characterized by small cells with large nuclei.

[0377] While the present invention has been described in relation to its specific embodiments, numerous changes, modifications, and variations will be apparent to those skilled in the art. Therefore, all such changes, modifications, and variations are intended to be included within the spirit and broader scope of the appended claims.

[0378] All publications, patents, and patent applications referenced herein are incorporated herein by reference in the same manner as each individual publication, patent, and patent application is incorporated herein by reference in specific and individual terms. In addition, any citation or specification of references herein should not be construed as acceptance of such references being available as prior art of the present invention. Nor should the titles of each section be construed as necessarily limiting to the extent in which they are used. Furthermore, the documents relating to the basic application of this application are also fully incorporated herein by reference in their entirety.

[0379] References (Further references are cited within the text.) Bogliotti YS, Wu J, Vilarino M, Okamur et al. Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts. PNAS, 115 (9): 2090-2095, 2018. Edwards R.G., Surani M.A.H. (1978). The primate blastocyst and its environment. Upsala Journal of Medical Sciences 22: 39-50. Gardner RL. Investigation of cell lineage and differentiation in the extraembryonic endoderm of the mouse embryo. J. Embryological Experiment and Morphology 1982; Mitalipova M, Beyham Z, and First N. Pluripotency of Bovine Embryonic Cell Line Derived from Precompacting Embryos Cloning, 3 (2):59-, 2001. Reubinoff, B.E., Pera, M.F., Fong, C. Trounson, A., Bongso, A. (2000). Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat. Biotechnol. 18, 399-404. Thomson, JA, Itskovitz-Eldor, J., Shapiro, SS, Waknitz, MA, Swiergiel, JJ, Marshall, VS, Jones, JM (1998). Embryonic stem cell lines derived from human blastocysts. Science 282, 1145-1147 [erratum in Science (1998) 282, 1827]. [Sequence Listing Free Text]

[0380] Sequence ID 4: Amino acid sequence of the IL6RIL6 chimera

Claims

1. A method for deriving mammalian livestock pluripotent stem cell lines, (a) Mammalian livestock embryos that are at least 7 days and within 21 days after fertilization at the start of culture in ex-vivo are cultured in ex-vivo for a culture period of at least 4 days and within 21 days after fertilization to obtain embryos containing epiblast cells and / or late pluripotent stem cells. (b) Isolate the epiblast cells and / or late pluripotent stem cells from the embryo, (c) The epiblast cells and / or late pluripotent stem cells are cultured under conditions suitable for the proliferation of undifferentiated mammalian livestock pluripotent stem cells to obtain a population of mammalian livestock pluripotent stem cells. However, one of the above cultures is performed in a culture medium containing the IL6RIL6 chimera. A method comprising deriving the aforementioned mammalian livestock pluripotent stem cell line.

2. The method according to claim 1, wherein the mammalian livestock pluripotent stem cells are capable of spontaneously differentiating into adipocytes in the absence of an adipogenic differentiation inducer.

3. The method according to claim 2, wherein the mammalian livestock pluripotent stem cells are capable of spontaneously differentiating into adipocytes when cultured in a medium that does not contain dexamethasone.

4. The method according to claim 1, wherein the culture in step (a) is at least 4 days and 14 days or less.

5. The method according to claim 1, further comprising mechanically passage the population of mammalian livestock pluripotent stem cells for at least two generations to obtain a population rich in the mammalian livestock pluripotent stem cells.

6. The method according to claim 1, wherein the culture of the mammalian livestock embryo is performed in a two-dimensional culture system.

7. The method according to claim 1, wherein the culture of the mammalian livestock embryo is performed on feeder cells.

8. The method according to claim 1, wherein the culture of the epiblast cells and / or late pluripotent stem cells is performed on a two-dimensional culture system, and optionally the two-dimensional culture system includes a matrix without feeder cells.

9. The method according to claim 1, wherein the culture of the mammalian livestock embryo, or the culture of the epiblast cells and / or late pluripotent stem cells, is carried out in a culture medium containing Wnt3a polypeptide.

10. The method according to claim 1, wherein, before the culture, the mammalian livestock embryo is covered with a drop of extracellular matrix.

11. The method according to claim 1, wherein the cells of the population of mammalian livestock pluripotent stem cells spontaneously differentiate into adipogenic cell lines by passage in culture medium for a period of 14 to 21 days ± 10%.

12. The method according to claim 1, further comprising culturing the population of mammalian livestock pluripotent stem cells in a culture medium without chemical or hormonal induction to adipogenesis for at least 10 days and no more than 60 days without subculturing to produce adipocytes.

13. The method according to claim 12, wherein the culture medium does not contain dexamethasone.

14. A method for producing food, comprising introducing adipocytes prepared by the method of claim 12 into food and producing food.

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