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Definitions

• the present disclosure relates to bovine stem cells and more specifically to naive bovine embryonic stem cells, associated methods, and compositions.
• Naive embryonic stem cells can differentiate into all types of cells in the body, including extraembryonic cells such as trophoblast stem cells and extraembryonic endodermal lineage cells.
• Mouse blastocyst-like structures also called iblastoids
• EPS Expanded Pluripotent Stem
• iPS induced Pluripotent Stem cells
• bovine naive stem cells may facilitate the efficient multiplication of embryos with desirable characteristics. Desirable genetic characteristics in embryos may arise as a result of processes such as meiosis, mutation, and the immigration of genes, which can occur naturally, may be generated using assisted reproductive technologies such as Smith et al., WO 2020/168422, or by genetic modification.
• naive bovine embryonic stem cells and associated methods for iblastoid production can realize a broad array of benefits from naive stem cell technology by providing an efficient platform for producing genetically modified animals at scale, delivering important constituent technologies for in vitro breeding programs, and enabling the development and delivery of advanced veterinary medical biologies and therapeutics.
• naive bovine embryonic stem cells and associated methods for iblastoid production can realize a broad array of benefits from naive stem cell technology by providing an efficient platform for producing genetically modified animals at scale, delivering important constituent technologies for in vitro breeding programs, and enabling the development and delivery of advanced veterinary medical biologies and therapeutics.
• Described herein are materials and methods useful for achieving attachment and outgrowth formation using bovine embryos, such as morula and blastocyst stage embryos, for establishing bovine naive embryonic stem cells.
• bovine embryos such as morula and blastocyst stage embryos
• ZP-free bovine embryos plated on layered ECM-coated substrates provide greater attachment rates and outgrowth formation compared to embryos plated on conventional ECM-coated substrates.
• outgrowth media compositions and associated methods which support embryo attachment and outgrowth formation, as well as the propagation of the inner cell mass (ICM) cells from such outgrowths, allowing for the derivation of naive embryonic stem cells from the ICM.
• the embodiments described herein are therefore useful for deriving naive bovine embryonic stem cells and optionally for use in breeding programs such as for generating iblastoid structures, multiplying preimplantation embryos having desirable genetic characteristics, deriving primordial germ cells and/or gametes for in vitro breeding programs, and/or developing and delivering veterinary medical biologicals and therapeutics.
• the method comprises: providing a Zona Pellucida (ZP)-free bovine embryo comprising naive bovine embryonic stem cells; contacting the ZP-free bovine embryo with an extracellular matrix (ECM)-coated substrate, wherein the ECM-coated substrate comprises a substrate comprising a negatively charged substrate surface adjacent to a positively charged biocompatible polymer layer, and a negatively charged ECM layer adjacent to the positively charged biocompatible polymer layer; and culturing the ZP-free bovine embryo in the presence of outgrowth medium to induce attachment of the ZP-free bovine embryo to the ECM-coated substrate and outgrowth of an inner cell mass (ICM) comprising derived naive bovine embryonic stem cells.
• ZP Zona Pellucida
• ECM extracellular matrix
• the bovine embryo is genetically modified.
• the ZP-free bovine embryo is obtained from a reconstructed diploid embryo.
• the biocompatible polymer is negatively charged at physiological pH.
• the biocompatible polymer is type A gelatin.
• the ECM comprises EHS-ECM.
• the substrate comprises polystyrene.
• the outgrowth medium comprises a base medium, and one or more components described herein useful for inducing attachment of the ZP-free embryo to the ECM coated substrate and outgrowth of the ICM in a feeder-free culture system.
• the outgrowth medium comprises one or more of: a 1 :1 mixture of DMEM/F12 and Neurobasal medium; an N2B27 component; a Wnt activator component; a Wnt inhibitor component; a MEK/ERK inhibitor component; a ROCK inhibitor component; a LIF component; a PKC inhibitor component; and an insulin component.
• the outgrowth medium further com prises an Activin A component.
• the outgrowth medium comprises: the N2B27 component; the Wnt activator component; the Wnt inhibitor component; the MEK/ERK inhibitor component; the ROCK inhibitor component; the LIF component; the Activin A component; the PKC inhibitor; and the insulin component.
• the N2B27 component comprises B27 supplement and N2 supplement, optionally about 1 % B27 supplement and about 0.5% N2 supplement;
• the Wnt activator component comprises CHIR99021 , BIO, CHIR-98014, LY2090314, and/or IM-12;
• the Wnt inhibitor component comprises XAV939, IWR-1 , and/or IWP -2;
• the MEK/ERK inhibitor component comprises PD0325901 , Ravoxertinib, GSK1120212, MEK162, PD184352, Trametinib, LY3214996, and/or Ulixertinib;
• the ROCK inhibitor component comprises Y27632, Thiazovivin, and/or Blebbistatin;
• the LIF component comprises human LIF;
• the Activin A comprises human Activin A;
• the PKC inhibitor comprises G66983, G66976, LY317615, LY333531 , PKC412, GSK690693,
• the ZP-free bovine embryo is a morula (stage 4); a blastocyst (stage 5); an expanding blastocyst (stage 6); an expanded blastocyst (stage 7); a hatching blastocyst (stage 8) or a hatched blastocyst (stage 9).
• the ZP-free bovine embryo is obtained by enzyme- assisted ZP removal.
• the method comprises obtaining the ZP-free bovine embryo by enzyme-assisted ZP removal.
• Also provided herein is a method of enzyme-assisted ZP removal comprising the steps of: a) providing an embryo; b) contacting the embryo with a protease solution; c) incubating the embryo in the protease solution to partially digest the ZP and obtain a ZP-thinned embryo; d) contacting the ZP-thinned embryo with a protease inactivation medium to inactivate the protease; e) rupturing the ZP; and f) manipulating the embryo to separate the ZP from the embryo.
• the concentration of protease in step c) is about 0.1 % to about 0.5%, about 0.2% to 0.3%, or about 0.25%.
• the embryo and protease solution are incubated for between about 30-60 seconds in step c), optionally for about 45 seconds.
• the ZP is ruptured in step e) using a microblade.
• manipulating the embryo in step f) comprises pipetting.
• the method further comprises performing genetic testing to determine one or more genotypes of the ZP-free bovine embryo for one or more biomarkers.
• the method further comprises selecting the ZP-free bovine embryo based on genetic testing for one or more biomarkers.
• the method further comprises performing genetic testing to determine one or more genotypes of the derived naive bovine embryonic stem cells.
• the ZP-free bovine embryo is obtained from a fresh embryo, optionally a fresh biopsied embryo.
• the embryo of step a) is a genetically modified embryo.
• the ZP-free bovine embryo is obtained from a frozen embryo, optionally a biopsied-frozen embryo.
• the method comprises thawing the frozen embryo and contacting the embryo with a recovery medium.
• the ZP-free bovine embryo is obtained by a method comprising: thawing the frozen embryo; contacting the frozen embryo with the recovery medium; manipulating the embryo to separate the ZP from the embryo in the recovery medium; and incubating the ZP-free bovine embryo in the recovery medium.
• the recovery medium comprises a glycogen synthase kinase 3 (GSK-3) inhibitor, a MEK/ERK kinase inhibitor and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor.
• GSK-3 glycogen synthase kinase 3
• MEK/ERK kinase inhibitor a MEK/ERK kinase inhibitor
• ROCK Rho-associated, coiled-coil containing protein kinase
• the recovery medium comprises CHIR99021 , PD0325901 and Y27632.
• the method further comprises incubating the ZP-free embryo with an adaptation medium, wherein the adaptation medium comprises a combination of recovery medium and outgrowth medium.
• the adaptation medium comprises a combination of recovery medium and outgrowth medium at a ratio of between about 0.5:1 and 1.5:1 optionally about 1 :1.
• a naive bovine stem cell derived using the methods described herein includes use of a naive bovine stem cell derived using the methods described herein in a breeding scheme or genetic improvement program, and for multiplying preimplantation embryos having desirable genetic characteristics, deriving primordial germ cells and/or gametes for in vitro breeding programs, and/or developing and delivering veterinary medical biologicals and therapeutics.
• a method of preparing an extracellular matrix (ECM)-coated substrate comprising: providing a substrate comprising a negatively charged surface; contacting the negatively charged surface with a first solution comprising a biocompatible polymer, wherein the biocompatible polymer is positively charged; incubating the substrate in contact with the first solution such that a layer of the positively charged biocompatible polymer is deposited on the negatively charged surface of the substrate; removing the first solution and optionally washing the substrate; contacting the substrate with a second solution comprising an extracellular matrix (ECM), wherein the ECM is negatively charged; and incubating the substrate in contact with the second solution such that a layer of the negatively charged ECM is deposited on the layer of the positively charged biocompatible polymer.
• ECM extracellular matrix
• the biocompatible polymer comprises type A gelatin.
• the ECM comprises EHS-ECM.
• the substrate comprises polystyrene.
• An aspect includes an ECM-coated substrate produced according to the methods described herein.
• a further aspect includes an ECM-coated substrate comprising: a substrate comprising a positively charged surface; a layer of a positively charged biocompatible polymer in contact with the negatively charged surface of the substrate; a layer of a negatively charged ECM in contact with the layer of the positively charged biocompatible polymer.
• the biocompatible polymer comprises type A gelatin.
• the ECM comprises EHS-ECM.
• the substrate comprises polystyrene.
• An aspect of the disclosure includes use of an ECM-coated substrate described herein for culturing an embryo, optionally to induce ICM outgrowth and/or the derivation of naive embryonic stem cells.
• the embryo is a bovine embryo, optionally a bovine embryo between day 5 and day 7.
• the bovine embryo is a reconstructed diploid embryo.
• the embryo is a genetically modified embryo.
• a media composition comprising base media and one or more components for culturing an embryo, optionally to induce ICM outgrowth and/or the derivation of naive embryonic stem cells.
• the media composition comprises one or more of: an N2B27 component; a Wnt activator component; a Wnt inhibitor component; a MEK/ERK inhibitor component; a ROCK inhibitor component; a LIF component; a PKC inhibitor; and an insulin component.
• the media composition further comprises an Activin A component.
• the N2B27 component comprises about 1 % B27 supplement and about 0.5% N2 supplement;
• the Wnt activator component comprises CHIR99021 , BIO, CHIR-98014, LY2090314, or IM-12;
• the Wnt inhibitor component comprises XAV939, IWR-1 , or IWP-2;
• the MEK/ERK inhibitor component comprises PD0325901 , Ravoxertinib, GSK1120212, MEK162, PD184352, Trametinib, LY3214996, or Ulixertinib;
• the ROCK inhibitor component comprises Y27632, Thiazovivin, or Blebbistatin;
• the LIF component comprises human LIF;
• the Activin A component comprises human Activin A;
• the PKC inhibitor comprises G66983, G66976, LY317615, LY333531 , PKC412, GSK690693, Sotrastaurin, Staurosporine, or Bisindoly
• a further aspect includes use of a media composition described herein for culturing an embryo to induce ICM outgrowth formation, optionally wherein the embryo is a bovine embryo, a reconstructed diploid bovine embryo, and/or the embryo is a genetically modified embryo.
• a further aspect includes a recovery medium comprising: a glycogen synthase kinase 3 (GSK-3) inhibitor, optionally CHIR99021 , a MEK/ERK kinase inhibitor, optionally PD0325901 , and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, optionally Y27632.
• GSK-3 glycogen synthase kinase 3
• CHIR99021 a glycogen synthase kinase 3
• MEK/ERK kinase inhibitor optionally PD0325901
• a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor optionally Y27632.
• FIG. 1 shows results of experiments to determine the attachment and outgrowth rates of embryos plated on different coating materials.
• Fig. 2 shows an image of a fabricated dish surface with Geltrex and 30um pore cell strainer (magnification of image; x100).
• FIG. 3 shows a schematic of the chemical structure of a plasma treated Poly styrene dish surface.
• Fig. 4 shows an illustration of Layer-by-Layer (LbL) coating.
• Fig. 5 shows improved derivation efficiency using a Layer-by-Layer ECM- coated substrate.
• the LbL ECM-coated substrate exhibited a high and stable attachment rate, TE (trophectodermal cell) and ICM (inner cell mass) growth compared to control protocols using EHS-ECM only.
• Fig. 6 shows (Left) a schematic representation of the layout of a protease treatment dish and (Right) an image of protease-treated poor-quality embryos where the ZP was fully removed by the enzymatic treatment.
• Fig. 7 shows ZP-free day 6 fresh embryos. All embryos appear healthy after ZP removal.
• Fig. 8 shows (Top Left) a schematic representation of the layout of a postthawing recovery dish, (Top right) and images showing the effect of 2iY media on the recovery of post-thaw embryos.
• (Bottom) 2iY treated embryos has shown faster recovery than control embryo culture media and higher derivation efficiency as observed by the faster re-expansion of the embryos.
• 2iY treated embryos has shown bigger size and clear ICM than control group (dashed circle).
• Fig. 9 is a graph showing the effect of 2iY on the quality of post-thaw embryos. 2iY treated embryos exhibit more advanced stage of development after postthawing recovery.
• Fig. 10 shows a schematic representation of the layout of the adaptation medium dish #1 and dish #2. Bottom is a graph showing the effect of adaptation time on the derivation of outgrowths.
• Fig. 11 is a graph showing derivation results with various types of serum sources.
• FBS fetal bovine serum
• KOSR knock-out serum replacement
• SR serum replacement
• N2B27 N2 supplement + B27 supplement (see Examples).
• Fig. 12 is a schematic showing various Wnt signaling pathway and targets.
• Fig. 13 is a graph showing the effect of Forskolin and the dual kinase Wnt pathway modification on the naive stem cell derivation.
• Fig. 14 is a graph showing the effect of IL-6 and SRC inhibitor on the derivation of outgrowths. Bottom is schematic showing various pathways downstream of SRC.
• Fig. 15 shows the effect of a MEK inhibitor on the formation of naive outgrowth.
• Fig. 16 is a graph showing the derivation efficiency between naive stem cell media (left) and an image showing an outgrowth colony derived by t2iLG6Y media.
• Fig. 17 shows (Left) Endodermal differentiation of outgrowth. ICM colony (arrow) is covered by undifferentiated (arrowhead) and differentiated (star) endodermal cells. (Right) ICM cell colony (chunk of cells which have bright edge) after 1 st passaging following insulin addition.
• Fig. 18 shows a schematic of the steps involved in deriving cell lines for subsequently generating primordial germ cell lines and gametes and generating multiple genetically identical embryos (also called iblastoids) based on the derivation of naive bovine embryonic stem cells.
• the phrase "at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
• This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.
• the inventors have demonstrated naive bovine stem cell outgrowth formation in a feeder- free culture system comprising a layered ECM-coated substrate and a specially formulated outgrowth media.
• the layered ECM-coated substrate and outgrowth media support ex vivo or in vitro attachment and growth of inner cell mass (ICM) cells derived from embryos, such as morula or blastocyst stage (e.g. bovine day 6 or day 7) embryos.
• ICM inner cell mass
• the materials and methods described herein are therefore useful for deriving and maintaining naive bovine embryonic stem cells and optionally for use in breeding programs such as for the multiplication of preimplantation embryos with desirable genetic characteristics and/or the production of iblastoid structures, deriving primordial germ cells and/or gametes for in vitro breeding programs, and developing and delivering veterinary medical biologicals and therapeutics. Desirable genetic characteristics can arise via natural processes or by genetic modification.
• the method comprises: providing a Zona Pellucida (ZP)-free bovine embryo comprising naive bovine embryonic stem cells; contacting the ZP-free bovine embryo with an extracellular matrix (ECM)-coated substrate, wherein the ECM-coated substrate comprises a substrate comprising a negatively charged substrate surface adjacent to a positively charged biocompatible polymer layer, and a negatively charged ECM layer adjacent to the positively charged biocompatible polymer layer; and culturing the ZP-free bovine embryo in the presence of outgrowth medium to induce attachment of the ZP-free bovine embryo to the ECM-coated substrate and outgrowth of an inner cell mass (ICM) comprising derived naive bovine embryonic stem cells.
• ZP Zona Pellucida
• ECM extracellular matrix
• naive embryonic stem cell is used to refer to embryonic stem cells which substantially retain the molecular characteristics of cells of a morula stage embryo, such as a day-5 or day-6 bovine embryo, where the cells are still in an undifferentiated state.
• Naive embryonic stem cells may be found within the inner cell mass (ICM) of a blastocyst (such as a day-7 embryo).
• ICM inner cell mass
• blastocyst such as a day-7 embryo
• Naive embryonic stem cells may be capable of developing into a complete organism and/or may retain the capacity to give rise to the full complement of adult tissues and/or cell types.
• Naive embryonic stem cells are capable of being derived and maintained in an undifferentiated state of self-renewal without the need for exogenously expressed pluripotency factors as opposed to induced Pluripotent Stem Cells (iPSC).
• iPSC induced Pluripotent Stem Cells
• bovine embryonic stem cell refers to a naive embryonic stem cell of bovine origin.
• Naive embryonic stem cells such as naive bovine embryonic stem cells
• naive bovine embryonic stem cells may be derived from sufficiently undifferentiated tissues, such as for example the embryonic cells of an embryo, for example a morula (stage 4); a blastocyst (stage 5); an expanding blastocyst (stage 6); an expanded blastocyst (stage 7); a hatching blastocyst (stage 8) or a hatched blastocyst (stage 9).
• naive bovine embryonic stem cells may be derived from a 3- to 8-day bovine embryo, optionally a 3-, 4-, 5-, 6-, 7- , or 8-day bovine embryo, or a 5- to 7- day bovine embryo.
• the bovine embryo is a morula (stage 4); a blastocyst (stage 5); an expanding blastocyst (stage 6); an expanded blastocyst (stage 7); a hatching blastocyst (stage 8) ora hatched blastocyst (stage 9).
• the bovine embryo is a 3- to 7-day embryo, optionally a 5- to 7- day embryo or 6- or 7-day embryo.
• the embryo is a preimplantation embryo.
• the embryo is an embryo that has previously been frozen and/or biopsied.
• the embryo is genetically modified.
• the embryo has been selected based on genetic testing for one or more biomarkers.
• a “genetically modified embryo” refers to an embryo where genomic DNA of the cells in the embryo have been manipulated to express one or more exogenous genes and/or to introduce mutation(s) within endogenous genes or intergenic regions which affects expression or functional activity of one or more endogenous genes or gene products. Examples of successful genetic modifications in bovine embryos have included the introduction of transgenes by microinjection (U.S.
• a “genetically modified cell” refers to a cell where the genomic DNA of the cell has been manipulated to express one or more exogenous genes and/or to introduce mutation(s) within endogenous genes or intergenic regions which affects expression or functional activity of one or more endogenous genes or gene products.
• the Zona Pellucida prevents attachment of embryonic cells to culture substrates. Accordingly, in an embodiment, the ZP of the embryo is removed prior to contact with the layered ECM-coated substrate and/or outgrowth media. ZP-free embryos can be provided or obtained using any suitable method.
• the ZP may be thinned and/or ruptured using enzymatic, chemical, and/or mechanical means, and subsequently separated from the embryo by mechanical manipulation to obtain a ZP-free embryo.
• Suitable enzymatic or chemical means for thinning and/or rupturing the ZP include for example the use of proteases such as pronase or acidified Tyrode’s solution.
• Suitable mechanical methods for rupturing the ZP include for example the use of a microblade, micropipette, microneedle, or laser.
• the ruptured ZP may be separated from the embryo for example by agitation such as pipetting, vortexing, or direct manipulation using a micropipette or microneedle.
• the ZP-free embryo is obtained by enzyme-assisted ZP removal.
• protease treatment of a morula (6-day) bovine embryo, followed by mechanical rupture and separation of the ZP results in a ZP-free embryo suitable for deriving naive embryonic stem cells as described herein.
• the ZP-free embryo is obtained from a biopsied embryo.
• Desirable genetic characteristics in embryos may arise as a result of processes such as meiosis, mutation, and the immigration of genes, which can occur naturally or by genetic modification. Accordingly, in an embodiment the ZP-free embryo is obtained from a genetically modified embryo.
• Embryos from which the ZP-free bovine embryo is obtained may be fresh or previously frozen, and optionally may be obtained from biopsied-frozen embryos. In an embodiment the embryo is a genetically tested embryo.
• the bovine embryo is a reconstructed diploid embryo. Reconstructed diploid embryos are described, for example in Smith et al., WO 2020/168422, the contents of which is incorporated by reference herein in its entirety.
• Diploid embryos with predetermined genomes can be generated in vitro by reconstructing biparental embryos using screened and selected androgenetic and parthenogenetic embryonic haploid cells (Smith et al., WO 2020/168422).
• Genomes for the reconstructed diploid embryos can be produced to contain a unique combination of alleles, haplotypes, or traits meeting stringent genetic criteria for a large complement of genetic or genomic characteristics such as production traits (e.g. milk, fat, protein, fat%, protein%, milk protein variant composition. g. A2A2 milk), meat quality traits, growth traits, health traits (e.g. somatic cell score, mastitis resistance, immune response, livability, disease resistance), reproductive traits (e.g.
• calving traits e.g. calving ease, calving to first insemination, stillbirths
• conformation traits e.g. polled traits, udder and teat traits, feet and leg traits, body traits, dimension traits
• efficiency traits e.g. feed efficiency traits, workability, longevity, productive life
• novel traits e.g. robotic milking traits, heat tolerance, activity traits and behavior traits
• composite index traits e.g. LPI (Life Production Index) , TPI (Total Production Index)
• LPI Life Production Index
• TPI Total Production Index
• stromal derived cells Most cells in culture (except stromal derived cells) require a supporting layer to attach and proliferate in vitro such as in a tissue culture dish.
• This supporting layer can be made from stromal cells (directly attached on the dish), commonly known as a feeder cell system.
• stromal cells directly attached on the dish
• MEF mouse embryonic fibroblast
• a feeder system is frequently used for the culture of embryonic stem cells.
• MEF mouse embryonic fibroblast
• a feeder system is the potential of cross-species contamination when cells, such as embryonic stem cells, from a species different than the mouse is cultured on MEF.
• Another option is to use a protein matrix as a supporting layer, also known as feeder-free system.
• an ECM-coated substrate generated using a Layer-by-Layer (LbL) protocol with a positively charged biocompatible polymer binding layer exhibited higher and more stable attachment rates as well as TE (trophectodermal cell) and ICM (inner cell mass) growth compared to a control substrate using ECM extracted from Engelbreth-Holm-Swarm murine sarcoma cells, such as Matrigel (from Corning) or Geltrex (from Invitrogen).
• LbL Layer-by-Layer
• a substrate comprising a positively charged surface; a layer of a positively charged biocompatible polymer in contact with the negatively charged surface of the substrate; and a layer of a negatively charged ECM in contact with the layer of the positively charged biocompatible polymer.
• the method comprises: a) providing a substrate comprising a negatively charged surface, b) contacting the negatively charged surface with a first solution comprising a biocompatible polymer, wherein the biocompatible polymer is positively charged; c) incubating the substrate in contact with the first solution such that a layer of the positively charged biocompatible polymer is deposited on the negatively charged surface of the substrate; d) removing the first solution and optionally washing the substrate; e) contacting the substrate with a second solution comprising an extracellular matrix (ECM), wherein the ECM is negatively charged; and f) incubating the substrate in contact with the second solution such that a layer of the negatively charged ECM is deposited on the layer of the positively charged biocompatible polymer.
• ECM extracellular matrix
• the term “substrate” generally means a physical surface onto which layer(s) of materials are deposited or adhered.
• the substrate may be rigid or flexible and may be made of any suitable material, for example a plastic such as polystyrene.
• the substrate may be treated to render it hydrophilic and/or impart a charge such as a negative charge to the surface.
• the substrate is plasma-treated.
• the substrate is plasma-treated polystyrene (also known as tissue culture plastic).
• biocompatible polymer generally means a polymer that is compatible with living tissues or cells, for example a polymer which is non- toxic and does not elicit undesirable effects on for example the survival, growth, proliferation and/or other biological activities of cells.
• Biocompatible polymers may be inert with respect to such activities, and/or may support desired activities.
• the biocompatible polymer may be a naturally occurring polymer, may be prepared from a naturally occurring polymer, or may be a synthetic polymer with the desired properties. Suitable biocompatible polymers have properties so as to result in deposition and/or adherence of the polymer onto the surface of the substrate under conditions used for coating the substrate with the polymer.
• suitable properties of the biocompatible polymer may include for example a charge, such as a positive charge, at the pH of the solution used for coating.
• a charge such as a positive charge
• the interaction between polymer and substrate should be maintained under conditions (e.g. pH) used for subsequent washing and ECM coating steps, as well as conditions used for cell culture (e.g. physiological pH).
• Suitable polymers include gelatin type A (such as that derived from acid-cured tissue). Accordingly, in an embodiment, the biocompatible polymer is gelatin type A, optionally porcine gelatin type A.
• incubate or “incubating” means to maintain for example a substance, material, composition, etc. at a particular temperature, or within a temperature range, for a period of time.
• physiological pH means a pH of about 7.1 to about 7.6, optionally about 7.15 to about 7.45, about 7.2 to about 7.4, about 7.25 to about 7.35, or about 7.3.
• extracellular matrix or “ECM” as used herein generally means a biocompatible matrix comprising one or more macromolecule components, such as for example proteins, glycosaminoglycans (GAGs), and/or proteoglycans, which provides attachment and support for the growth and proliferation of cells, such as for example cells grown ex vivo or in vitro.
• macromolecule components such as for example proteins, glycosaminoglycans (GAGs), and/or proteoglycans, which provides attachment and support for the growth and proliferation of cells, such as for example cells grown ex vivo or in vitro.
• Common ECM components may include, without limitation, one or more of laminin, collagen (e.g. collagen l-XIV), fibronectin, vitronectin, entactin/nidogen, heparan sulfate proteoglycans, and/or one or more functional variants thereof.
• ECM commonly includes basement membrane extracts such as those isolated from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, and hereinafter referred to as “EHS-ECM”, (for example sold under trade names MatrigelTM (Corning) and GeltrexTM (Thermo Fisher)).
• EHS-ECM Engelbreth-Holm-Swarm
• MatrigelTM Corening
• GeltrexTM GeltrexTM
• the EHS-ECM may comprise for example laminin, collagen IV, entactin/nidogen, and heparan sulfate proteoglycans.
• other sources of ECM with different compositions and/or purified components e.g. FN
• synthetic ECM-like substrates e.g.
• ECM comprising RGD peptides
• RGD peptides could also be used in the coating methods if desired.
• ECM component(s) will depend on a number of factors including without limitation, cell type, stage of differentiation, and other experimental parameters.
• EHS-ECM is demonstrated herein to be suitable for culture (e.g. attachment and outgrowth) of bovine embryos. Accordingly, in an embodiment, the ECM is EHS-ECM, optionally Matrigel or Geltrex.
• the attachment and outgrowth formation from bovine embryos is influenced by the composition of the outgrowth medium in which the embryos are cultured.
• the outgrowth medium may comprise for example a base medium, and one or more small molecules, growth factors, and/or nutrients.
• Suitable base media can be readily determined by the skilled person and includes without limitation DMEM/F12, advanced DMEM/F12 and Neurobasal medium.
• Suitable supplements can be readily determined by the skilled person and include without limitation MEM non-essential amino acids, L-glutamine, Glutamax, ascorbic acid, insulin, BSA (fraction V), betamercaptoethanol and penicillin/streptomycin.
• outgrowth media components useful for deriving and maintaining naive embryonic stem cells including the attachment and outgrowth of the ICM.
• the outgrowth media comprises a base medium and/or supplements as well as one or more outgrowth medium components.
• the outgrowth medium comprises one or more of an N2B27 component comprising B27 supplement and N2 supplement, optionally comprising about 1 % B27 supplement and about 0.5% N2 supplement; a Wnt activator component, optionally CHIR99021 , BIO, CHIR-98014, LY2090314, or IM-12; a Wnt inhibitor component, optionally XAV939, IWR-1 , or lWP-2; a MEK/ERK inhibitor component, optionally PD0325901 , Ravoxertinib, GSK1120212, MEK162, PD184352, Trametinib, LY3214996, or Ulixertinib; a ROCK inhibitor component, optionally Y27632, Thiazovivin, or Blebbistatin; a LIF component, optionally human LIF; a PKC inhibitor, optionally G66983, G66976, LY317615, LY333531 , PKC412, GSK690693, So
• the outgrowth medium may comprise one or more of N2B27, CHIR99021 (a Wnt activator), XAV939 (a Wnt inhibitor), PD0325901 (a MEK/ERK inhibitor), Ravoxertinib (ERK specific inhibitor) G66983 (PKC inhibitor), Y27362 (ROCK inhibitor), and/or LIF.
• the outgrowth media further comprises an Activin A component, optionally human Activin A.
• an ECM-coated substrate comprising a substrate comprising a negatively charged substrate surface adjacent to a positively charged biocompatible polymer layer and a negatively charged ECM layer adjacent to the positively charged biocompatible polymer layer.
• the biocompatible polymer is type A gelatin.
• the ECM comprises EHS-ECM.
• the substrate is a plastic substrate suitable for cell culture such as polystyrene.
• the outgrowth medium comprises a base medium and one or more components identified herein.
• the outgrowth medium comprises one or more of an N2B27 component, a Wnt activator component, a Wnt inhibitor component, a MEK/ERK inhibitor component, a ROCK inhibitor component, a LIF component, a PKC inhibitor, and an insulin component.
• the outgrowth medium comprises an N2B27 component, a Wnt activator component, a Wnt inhibitor component, a MEK/ERK inhibitor component, a ROCK inhibitor component, a LIF component, a PKC inhibitor, and an insulin component.
• the N2B27 component comprises B27 supplement and N2 supplement, optionally about 1 % B27 supplement and about 0.5% N2 supplement.
• the Wnt activator component comprises CHIR99021 , BIO, CHIR-98014, LY2090314, or IM-12.
• the Wnt activator component comprises CHIR99021 , optionally at a concentration of about 0.1 uM to about 5 uM, optionally about 1 uM, to about 3 uM, optionally about 1 uM, about 2 uM, or about 3 uM.
• the Wnt inhibitor component comprises XAV939, IWR- 1 , or IWP-2.
• the Wnt inhibitor component comprises XAV939, optionally at a concentration of about 0.2uM to about 10uM, optionally about 1 uM to about 5uM, optionally about 2uM.
• the Wnt inhibitor component comprises IWR-1 , optionally at a concentration of about 0.25 uM to about 10 uM, optionally about 1 uM to about 5uM, optionally about 2.5uM.
• the MEK/ERK inhibitor component comprises PD0325901 , Ravoxertinib, GSK1120212, MEK162, PD184352, Trametinib, LY3214996, or Ulixertinib.
• the MEK/ERK inhibitor component comprises PD0325901 or Ravoxertinib.
• the MEK/ERK inhibitor component comprises PD0325901 at a concentration of about 0.05 uM to about 5 uM, optionally about 0.1 uM to about 2 uM, optionally about 1 uM.
• the MEK/ERK inhibitor component comprises Ravoxertinib at a concentration of about 0.25 uM to about 10 uM, optionally about 1 uM to about 5uM, optionally about 2.5uM.
• the ROCK inhibitor component comprises Y27632 Thiazovivin, or Blebbistatin.
• the ROCK inhibitor component comprises Y27632, optionally at a concentration of about 0.5 uM to about 20 uM, optionally about 5 uM to about 10 uM, optionally about 5 uM or about 10uM.
• the LIF component comprises human LIF, optionally at a concentration of about 1 ng/ml to about 1000 ng/ml, optionally about 5 ng/ml to about 100 ng/ml, optionally about 5 ng/ml, about 10 ng/ml, about 20 ng/ml, or about 100 ng/ml.
• the Activin A comprises human Activin A, optionally at a concentration of about 1 ng/ml to about 50 ng/ml, optionally about 5 ng/ml to about 50 ng/ml, optionally about 10 ng/ml or about 20 ng/ml.
• the PKC inhibitor comprises G66983, G66976, LY317615, LY333531 , PKC412, GSK690693, Sotrastaurin, Staurosporine, or Bisindolylmaleimide.
• the PKC inhibitor comprises G66983, optionally at a concentration of about 0.2 uM to about 25 uM, optionally about 2 uM to about 2.5 uM, optionally about 2 uM or about 2.5 uM.
• the insulin component comprises insulin peptide, optionally at a concentration of about 2 ug/ml to about 200 ug/ml, optionally about 20 ug/ml.
• formulations suitable for the post-thawing recovery of embryos As shown in Figures 8 and 9, embryos treated with a recovery media formulated as described herein exhibited a faster recovery and a higher derivation efficiency as observed by the faster re-expansion of the embryos relative to controls. Embryos treated with recovery media also appeared to be larger and had a clearer ICM relative to controls.
• the recovery media comprises a glycogen synthase kinase 3 (GSK-3) inhibitor, a MEK/ERK kinase inhibitor, and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor.
• GSK-3 inhibitors include for example CHIR99021.
• MEK/ERK inhibitors include for example PD0325901.
• Suitable ROCK inhibitors include for example Y27632.
• the recovery medium comprises CHIR99021 , optionally 0.1 uM to about 5 uM, optionally about 1 uM, to about 3 uM, optionally about 1 uM, about 2 uM, or about 3uM; PD0325901 , optionally about 0.05 uM to about 5 uM, optionally about 0.1 uM to about 2 uM, or about 1 uM; and Y27632, optionally about 0.5 uM to about 20 uM, optionally about 5 uM to about 10 uM, or about 10uM.
• kits comprising one or more of an ECM-coated substrate, an outgrowth media and/or a recovery media as described herein.
• the ECM-coated substrate, outgrowth media and/or recovery media are packaged in separate containers.
• An aspect includes a naive bovine stem cell derived using the methods described herein.
• the naive bovine stem cell is a genetically modified stem cell.
• an ECM-coated substrate as described herein for culturing embryos and/or cells derived from embryos.
• the ECM-coated substrate is useful for promoting the attachment and/or outgrowth of embryos as well as the derivation of naive embryonic stem cells, optionally naive bovine embryonic stem cells.
• an outgrowth media as described herein for culturing embryos and/or cells derived from embryos.
• the outgrowth medium is useful for promoting for the attachment and/or outgrowth of embryos, as well as the derivation of naive embryonic stem cells, optionally naive bovine embryonic stem cells.
• the outgrowth media is for use in combination with an ECM-coated substrate as described herein.
• a recovery media or adaptation media as described herein for treating embryos, optionally bovine embryos and optionally reconstructed diploid bovine embryos.
• the recovery media is useful for treating fresh embryos, or embryos that have previously been biopsied and/or frozen. Recovery media is also useful for processes in which embryos can benefit from recovery media such as, for example, ZP removal, thawing, biopsy, gene editing, or cell derivation.
• a further aspect includes use of a naive bovine stem cell derived using the methods described herein in a breeding scheme or genetic improvement program, or for multiplying preimplantation embryos having desirable genetic characteristics, deriving primordial germ cells and/or gametes for in vitro breeding programs, and/or developing and delivering veterinary medical biologicals and therapeutics.
• Coated substrates were incubated in a humidified 5% CO2 incubator at 38.5°C for 1 hour.
• First attachment rate was determined at 48 hours of outgrowth culture and first media change was done right after first attachment determination.
• Cell strainer membrane (30 urn pore size) was cut into 1 cm 2 size.
• Cut membrane was placed on a 35 mm 2 dish.
• Coating material was allowed to polymerize in the humidified incubator at 38.5°C for 1 hour and then allowed to dry on the bench (under the laminar flow hood) at room temperature for 30 min.
• a grooved dish surface can easily be made with this protocol. However, the grooves collapsed rapidly following the addition of culture media.
• Example 3 ECM-coated substrates generated using a Layer-by-layer (LbL) protocol
• the NuncTM delta (plasma treated) polystyrene dishes used in Examples 1 and 2 have a negatively charged surface (see e.g. Figs. 3 and 4).
• Common ECM components for example basement membrane extracts, are also negatively charged under conditions used for coating and/or at physiological pH.
• Geltrex A14133, Thermo fisher
• pH 7.2 which is the pH of the diluent buffer used for coating (isoelectric point of EHS-ECM: 4-5).
• the molecules of a protein matrix can be adhered with plasma treated polystyrene surfaces by hydrogen binding with the -OH residue of the surface (Lerman et al., 2018).
• the dish was incubated in a humidified 5% CO2 incubator at 38.5°C for 1 hour.
• EHS-ECM solution was removed, and 50 ul of media was applied; drops were covered with 4 ml of mineral oil, and 50 uL of additional media was added into each drop.
• the media was allowed to equilibrate in 5% CO2 incubator for 2 hours.
• the embryo also called blastocyst
• the embryo is surrounded by a thick membrane of glycoprotein which forms the zona pellucida (ZP).
• ZP zona pellucida
• the blastocyst will start to expand and ultimately “hatch”, which will allow the blastocyst to get out of that “shell”. It is possible to obtain hatching and hatched blastocysts generally around day-8 of in vitro culture.
• bovine in vitro embryo culture media can efficiently support the development until day-7, after which the embryo needs to be either transferred into a recipient or frozen. From preliminary experiments, the ZP is found to block the attachment of embryo, so it should be removed prior to outgrowth culture. Therefore, investigations were performed to try and improve ZP removal from either fresh day-6/day-7 and/or frozen/thawed day-7 embryos.
• Enzyme-based approach is a frequently used technique for ZP removal.
• a concentration of Protease from Streptomyces griseus, Pronase, Sigma P8811 ) between 0.05 to 0.5 % is commonly used for that protocol.
• protease is not a glycoprotein specific enzyme, it can also damage the embryo once the ZP has been completely digested.
• a protease treatment dish was prepared (see Fig. 6).
• the extended exposure to protease can cause dissociation of the embryo itself and damage the cells (as illustrated by dark and nontransparent cells).
• ZP digestion protocol was as follows:
• a protease dish was prepared with 0.25% protease (e.g. as shown in Fig. 6). Protease drop was prepared a maximum of 2 hours before use.
• One or more embryos were transferred from embryo culture medium to drop 1 made with embryo handling media.
• Enzyme assisted ZP removing protocol can be used to produce ZP-free morulas as shown in Fig. 7.
• Example 5 Compositions to improve post-thaw quality of biopsied frozen embryos
• biopsied-frozen embryos are typically genetically identified (e.g. screened for biomarkers), allowing practitioners to select those have desired genetic characteristics. Even though it is possible to biopsy and freeze day-7 embryos, those embryos are exposed to more stress compared to fresh embryos. Biopsied-frozen embryos have opened ZP (because of the biopsy procedure), therefore ZP can be easily removed by gentle pipetting. However, biopsied-frozen-thawed embryo quality is generally inferior to fresh embryos.
• a post-thawing recovery medium (called “2iY” media or “recovery media” herein) is described, which includes three inhibitors: CHIR99021 , PD0325901 and Y27632.
• CHIR99021 and PD0325901 are well characterized inhibitors, also known as “2i” in the stem cell field. Those two inhibitors modulate two important pathways involved in transcription factor activity of naive embryonic stem cells (CHIR99021 : Wnt pathway activator though inhibition of GSK3, PD0325901 : MEK pathway inhibition).
• Y27632 which is a ROCK inhibitor, is an actin filament stabilizer. The ability of these three inhibitors to protect the cells from the postthawing stress and to promote a faster cell recovery by modulating key sternness pathways was tested. The experiments shown herein demonstrate that this protocol significantly improves the quality of the embryos post-thaw, which allows for more efficient derivation of out-growths.
• a thawing dish was prepared using 2iY media (see Fig. 8) or control embryo culture media. 3. One or more embryos were thawed according to protocol.
• the 2iY treated group exhibited higher attachment and outgrowth forming rate than the control group when plated for 2 days and 4-5 days, respectively, on LbL-ECM coated dishes.
• 2iY treated embryos demonstrate faster recovery than control embryo culture media and higher derivation efficiency as observed by the faster re-expansion of the embryos.
• 2iY treated embryos are larger and show a clear ICM relative to the control group (dashed circles in Fig. 8) after 4 hours of recovery.
• 2iY treated group exhibits more advanced stage and improved quality of embryos 4 hours after thawing from cryopreservation (see Fig. 9).
• Adaptation dishes were prepared as shown in Fig. 10. 3. After post-thaw recovery, embryos were washed twice in adaptation media (1 :1 ratio of thawing media (e.g. embryo handling media and outgrowth media (e.g. DMEM)) ) before being put in the adaptation drop in the Dish #1 (Fig. 10).
• adaptation media (1 :1 ratio of thawing media (e.g. embryo handling media and outgrowth media (e.g. DMEM))
• Embryos were then moved to the outgrowth media (DMEM) for plating.
• DMEM outgrowth media
• Table 1 Measurements of pH and osmolarity (Osm) for various media. Measurements were obtained in duplicate.
• Cell culture media contain various components to support general maintenance of cells such as metabolism, survival and proliferation. Additional growth factors or inhibitors may also be added to promote differentiation, self-renewal or simply boost cell growth. To derive naive embryonic stem cells and maintain them in an undifferentiated state, requires a combination of growth factors and inhibitors in the culture media. Improper combinations of additives or different concentration of those molecules can induce irreversible differentiation of stem cells.
• Fig. 11 Type of serum/serum replacement [00164] As shown in Fig. 11 , five different serum sources/concentrations conditions were tested. FBS (10091 , GibcoTM), Knock-out Serum Replacement (10828-10, GibcoTM), Serum Replacement (S0638, Sigma), B27 supplement (17504-044, GibcoTM), and N2 supplement (17502-048, GibcoTM) were used for this test.
• t2iLG6Y media (1 :1 mixture of DMEM/F12 and neurobasal medium + 1 % MEM non-essential amino acid + 2m M Glutamax + 50 pg/ml BSA, fraction V + 100 pM beta-mercaptoethanol + 100 IU penicillin/streptomycin + growth factors/inhibitors including 1 pM CHIR99021 , 1 pM PD0325901 , 10 pM Y27632, 2.5 pM G66983 and 10 ng/ml LIF) was used for the testing of N2B27 group.
• the reason for that different combination is because the 2 i L medium has a much simpler formulation (compared to t2iLGoY medium), which makes it more suitable for basic serum sources such as FBS; KOSR or SR.
• KOSR Knock-Out Serum Replacement
• Serum replacement which contains bovine origin components, has shown much better efficiency compared to KOSR.
• N2B27 media which includes 1 % of B27 supplement and 0.5% N2 supplement, exhibited the best result for bovine embryo outgrowth derivation (TE and ICM growth) compared to the other components tested.
• n Attach % TE % ICM % [00169] A positive attachment rate was obtained, but outgrowth forming rate was low with only 19% of ICM expansion and 50% TE expansion.
• Naive Human Stem cell Media - NHSM (Gafni et al., 2013): Media composition: DMEM/F12:Neurobasal media (1 :1 mixture) + N2B27 serum + 8 ng/ml FGF + 1 ng/ml TGF-b + 20 ng/ml LIF + 3 uM CHIR99021 + 1 uM PD0325901 + 10 uM SP600125 + 10 uM + SB203580
• Forskolin is an adenylyl cyclase stimulator and increases cAMP level in the cells which is secondary messenger involved in many signaling pathways.
• the combination of forskolin and 2iL media revealed good efficiency in the derivation of human naive stem cells (Hanna et al., 2010) and the reprogramming of bovine naive-like pluripotent stem cell (Kawaguchi et al., 2015).
• Wnt is known to be a key player for naive stem cell signaling pathways.
• the combination of Wnt activator CHIR99021 and Wnt inhibitor such as IWR-1 or XAV939 (both inhibitor of the same protein complex, but each are targeting a different unit) cause cytoplasmic accumulation of beta-catenin and can promote self-renewal of mouse pluripotent stem cells through stabilization of E-cadherin which is key component of adherent junctions (Kim et al. 2013).
• the ability of the dual modulation of Wnt to overcome the poor expansion observed with 2i LFk media was tested.
• Dual Wnt media 1 .5 uM CHIR99021 + 2.5 uM IWR-1 + 1 uM PD0325901 + 100 ng/ml LIF + 10 uM Forskolin.
• Bovine IL-6 (superfamily of LIF) and SRCi:
• Bovine IL-6 (superfamily of LIF) and SRC inhibitor were tested as a replacement for hLIF and PD0325901 , respectively, used in t2iLG6Y media, which has been used for human naive stem cell derivation (titrated 2i/LIF/G66983/Y27632, Guo et al., 2016).
• SRC inhibitor which is an RTK inhibitor, is involved in most of the signaling pathways induced by growth factors (Theunissen et al., 2014). Knowing that the endpoint of SRC inhibition would ultimately target the ERK/MEK pathway, a SRC inhibitor was tested as an alternative and novel strategy to the MEK pathway inhibition (though the MEKi PD0325901 ).
• t2iLG6Y media DMEM/F12:Neurobasal media (1 :1 mixture) + N2B27 serum + 1 uM CHIR99021 + 1 uM PD0325901 + 10 ng/ml human LIF + 2.5 uM G66983 + 10 uM Y27632.
• N2B27 G66983 2.5 pM 67% 50% 0% inhibitor Neurobasal embryo
• t2iLGoY medium showed similar ICM growth rate but much higher rate in attachment and outgrowth formation, especially TE growth.
• ICM cell morphology was not maintained after first passage using various media compositions described above. ICM cells quickly differentiated into extraembryonic endoderm cells (hypoblast) during outgrowth culture and after passaging.
• Khan et al. (2021) used high-throughput chemical screening to identify the best culture conditions for human naive stem cells, and inhibition of ERK pathway was identified as a key factor to maintain stable human naive stem cells culture (Khan et al., 2021).
• naive stem cells are generally derived and maintained on feeder cells, and naive stem cell media is technically designed for such culture systems. According to Cosin-Roger et al., 2019 and Talbot et al., 2012, feeder cells secrete several important growth factors and more importantly Wnt ligands.
• the Wnt pathway is not only inhibited but also completely depleted from Wnt ligands, which are normally secreted from feeder cells.
• the effects of Wnt inhibition may therefore not be the same using feeder-free conditions compared with cells maintained on feeder cells, especially after several passages of culture.
• t2iLGdY may therefore be more suitable for bovine naive cell derivation using feeder-free conditions.
• Neurobasal medium Y27632 10 pM hLIF : 20 ng/ml
• Stable bovine naive stem cells generated using the high efficiency protocols described in the preceding Examples are used to reconstruct iBIastoid structures using an approach similar to what was recently published by Liu et al. (2021 ), Yu et al. (2021 ), and Yanagida et al. (2021 ). Protocol
• iBIastoid Media 1 will allow the differentiation of naive stem cell into trophoblast stem cells, to form the outer layer (TE) of the blastocyst.
• iBIastoid media 1 is Advanced-DMEM/F12 + 0.5 % Serum replacement + 1 % MEM NEAA + 1 % GlutamaxTM + 0.1 mM beta-mercaptoethanol + Gentamycin + 2 pM CHIR99021 + 5 pM Y27632 + 0.5 mM Valproic acid + 1 pM A83-01 + 50 ng/ml EGF.
• Formulation of iBIastoid media 2 is 1 :1 mixture of DMEM/F12 and neurobasal medium + 1 % MEM NEAA + 1 % GlutamaxTM + 0.1 mM beta- mercaptoethanol + 100 lll/ml Pen/Strep + 100 ng/ml Activin A + 3 pM CHIR99021 + 10 ng/ml LIF.
• Lentiviral vectors are they the future of animal transgenesis? Physiol Genomics. 31 : 159-173.
• Zhao L Gao X, Zheng Y, Wang Z, Zhao G, Ren J, Zhang J, Wu J, Wu B, Chen Y et al. (2021) Establishment of bovine expanded potential stem cells. Proceedings of the National Academy of Sciences of the United States of America 118.

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