Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
  • A01K67/027—New or modified breeds of vertebrates
  • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
  • A01K67/027—New or modified breeds of vertebrates
  • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
  • A01K67/0276—Knock-out vertebrates
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
  • A01K67/027—New or modified breeds of vertebrates
  • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/48—Reproductive organs
  • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
  • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
  • C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
  • C07K16/3092—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated mucins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0603—Embryonic cells ; Embryoid bodies
  • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2227/00—Animals characterised by species
  • A01K2227/10—Mammal
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2227/00—Animals characterised by species
  • A01K2227/10—Mammal
  • A01K2227/105—Murine
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2267/00—Animals characterised by purpose
  • A01K2267/02—Animal zootechnically ameliorated
  • A01K2267/025—Animal producing cells or organs for transplantation

Definitions

• the present application relates to methods of creating human tissue in non-human animals.
• the present application also relates to testing for drugs on the non-human animal as it relates to human tissue present in the non-human animal.
• the present application also relates to methods of treating patients with the human tissue or organs obtained from the non- human animal.
• the present application also relates to methods of generating transgenic animals that would enable incorporation of human cells and tissues.
• Ethical issues may impede the generation of non-human primate-human chimeras. Although more distant mammal-human chimeras may overcome certain ethical issues, attempts to generate non-primate species-human chimeras have thus far failed.
• the present invention solves the problem by providing a method for generating human stem cells in the naive state for the generation of non-human-human chimeric animals and also for providing an environment suitable for the growth of pluripotent human stem cells in a non-human environment.
• the present invention solves the problem by providing a method for "humanizing" host animals by genetically altering them such that they express human factors that enable growth and expansion of the human cells in the non-human host animal.
• the present invention is directed to a method of testing for efficacy or toxicity of a potential drug agent in a chimeric animal that expresses some human DNA or some human tissues.
• an animal that expresses some human DNA or tissues is generated by introducing human naive state stem cells into a non-human cell or cells.
• the non-human cell is an egg, in another aspect it is a fertilized egg, in another aspect, the cells are a morula, blastocyst or embryo.
• the agent that maintains stem cells in the naive state or reverts primed stem cells to the naive state may be an NME protein, 2i, 5i, or other cocktails of inhibitors, chemicals, or nucleic acids.
• the NME protein may be NME1 dimer, NME7 monomer, NME7-AB, NME7-X1, NME6 dimer, or bacterial NME.
• the non-human mammal may be a rodent, such as a mouse or rat, primate, including macaque, rhesus monkey, ape, chimp, bonobo and the like, or a domestic animal including pig, sheep, bovine, and the like.
• the chimeric animal may have a genetic disorder, have an induced disease, or a cancer that may be spontaneously generated or implanted from cells derived from a human being.
• the non-human animal may be transgenic, wherein the animal expresses human MUC1 or MUC1* or NME protein in the germ cells or somatic cells, wherein the germ cells and somatic cells contain a recombinant human MUC1 or MUC1* or NME gene sequence introduced into said animal.
• the gene expressing the human MUC1 or MUC1* or NME protein may be under control of an inducible promoter.
• the promoter may be inducibly responsive to a naturally occurring protein in the non-human animal or an agent that can be administered to the animal before, after or during development.
• the non-human animal may be transgenic, wherein the animal expresses its native sequence MUC1 or MUC1* or NME protein in the germ cells or somatic cells, wherein the germ cells and somatic cells contain a recombinant native species MUC1 or MUC1* or NME gene sequence introduced into said mammal.
• the NME species can be NME7, NME7-X1, NME1, NME6 or a bacterial NME.
• the agent that maintains stem cells in the naive state or reverts primed stem cells to the naive state may be an NME protein, 2i, 5i, or other cocktails of inhibitors, chemicals, or nucleic acids.
• the NME protein may be NME1 dimer, NME7 monomer, NME7-AB, NME7-X1, NME6 dimer, or bacterial NME.
• the agent may suppress expression of MBD3, CHD4, BRD4 or JMJD6.
• the agent may be siRNA made against MBD3, CHD4, BRD4 or JMJD6, or siRNA made against any gene that encodes a protein that upregulates expression of MBD3, CHD4, BRD4 or JMJD6.
• the cancer stem cell may be characterized by increased expression of CXCR4 or E-cadherin (CDH1) compared with cancer cells or normal cells.
• the invention is directed to a method for generating tissue from xenograft in a non-human mammal, comprising: (i) generating a transgenic non-human mammal, wherein the mammal expresses human MUC1 or MUC1* or NME protein in the germ cells and somatic cells, wherein the germ cells and somatic cells contain a recombinant human MUC1 or MUC1* or NME gene sequence introduced into said mammal, wherein the expression of the gene sequence may be under control of an inducible and repressible regulatory sequence; (ii) transferring stem cells or progenitor cells that are xenogeneic in origin to the non-human mammal such that the gene may be induced to be expressed so as to multiply the number of stem or progenitor cells; and (iii) repressing the gene expression so as to generate tissue from the xenografted stem cells.
• the gene expression repression may be carried out by contacting the stem cells with a tissue differentiation factor, or in step (iii) the gene expression repression may be carried out naturally in the mammal in response to naturally produced host tissue differentiation factor.
• the transferred cells may be human.
• the tissue may be an organ.
• the NME protein may be NME7, NME7-AB, NME7-X1, NME1, NME6, or bacterial NME.
• the animal may be a mammal, a rodent, a primate or domesticated animal such as a pig, sheep, or bovine species.
• the present invention is directed to making animals having at least some human cells or cells in which at least some of the DNA is of human origin.
• Such animals would grow human tissue, tissue containing some human cells or cells containing some human DNA for the generation of human or human-like tissue.
• Such animals would grow organs comprising at least some human cells.
• such animals would grow organs comprised entirely of human cells.
• host animals can be genetically or molecularly manipulated even after development to grow human limbs.
• Limbs, nerves, blood vessels, tissues, organs, or factors made in them, or secreted from them, would then be harvested from the animals and used for a multitude of purposes including but not limited to: 1) transplant into humans; 2) administration into humans for medicinal benefit, including anti-aging; and 3) scientific experiments including drug testing and disease modeling.
• the invention is directed to a method for generating human tissues in a non-human animal comprising: (i) generating human naive state stem cells and injecting them into a fertilized egg, morula, blastocyst or embryo of a non-human animal such that a chimeric animal is generated; (ii) harvesting human tissues, organs, cells or factors secreted by or made in the human tissues or cells from the chimeric animal; and (iii) transplanting or administering the harvested material into a human.
• the naive state stem cells may be generated using NME7, NME7-AB, NME7-X1 or dimeric NME1.
• the naive stem cells may be iPS cells that have been reprogrammed in a medium containing NME7, NME7-AB, NME7-X1 or dimeric NME1.
• the naive stem cells may be embryonic stem cells that have been cultured in a medium containing NME7, NME7-AB, NME7-X1 or dimeric NME1.
• the non-human cells of the blastocyst or embryo may have been genetically altered. And the genetic alteration may result in the host animal being unable to generate a certain tissue or organ.
• the genetic alteration may be to make the non-human animal express human molecules that facilitate or enhance the incorporation or growth of human stem or progenitor cells in the non-human host animal.
• the agent that maintains stem cells in the naive state or reverts primed stem cells to the naive state may be an NME protein, 2i, 5i, chemical, or nucleic acid.
• the NME protein may be NME1 dimer, NME7 monomer, NME7-AB, NME6 dimer, or bacterial NME, or NME7-X1.
• the non-human animal may be a rodent, mouse, rat, pig, sheep, non-human primate, macaque, chimpanzee, bonobo, gorilla or any non-human mammal.
• the non-human animal is chosen for its high sequence homology to human NME protein, especially human NME7-AB or NME7-X1 or high sequence homology to human MUC1* extracellular domain.
• the NME protein may be present in serum-free media as the single growth factor.
• a chimeric animal is if stem cells from a first species are able to incorporate into the inner cell mass (ICM) of a second species. Chimeric animals are more readily generated when the two different species are closely related, for example two rodents.
• human naive state stem cells that had been generated in human NME7-AB, then cultured in human were injected into a mouse morula 2.5 days after fertilization of the egg. This is before the inner cell mass forms. Forty-eight (48) hours later, the morula was analyzed and such analysis showed that the human stem cells had incorporated into the inner cell mass, indicating that a chimeric animal will develop.
• Figure 1 shows a graph of RT-PCR measurements of the expression of master pluripotency regulator genes Oct4 and Nanog in somatic fibroblast, 'fbb' cells that were cultured in either normal fibroblast growth media, or a serum free minimal media to which was added a human recombinant NM23, also called NME1 dimers, NME7-AB, or HSP593 bacterial NME1 dimers.
• '+ ROCi' refers to Rho kinase inhibitor that was added to some cells to make them adhere to the surface.
• Figure 2 shows a graph of RT-PCR measurements of the expression of genes that code for chromatin rearrangement factors BRD4, JMJD6, MBD3 and CHD4, in somatic fibroblast, 'fbb' cells that were cultured in either normal fibroblast growth media, or a serum free minimal media to which was added a human recombinant NM23, also called NME1 dimers, NME7-AB, or HSP593 bacterial NME1 dimers.
• '+ ROCi' refers to Rho kinase inhibitor that was added to some cells to make them adhere to the surface.
• Figure 3 shows a graph of RT-PCR measurements of the expression of pluripotency genes OCT4 and NANOG, chromatin rearrangement factors BRD4, JMJD6, MBD3 and CHD4, and NME1 and NME7 in somatic fibroblast, 'fbb' cells that were cultured in either normal fibroblast growth media, or a serum free minimal media to which was added a human recombinant NM23, also called NME1 dimers, NME7-AB, or HSP593 bacterial NME1 dimers.
• '+ ROCi' refers to Rho kinase inhibitor that was added to some cells to make them adhere to the surface.
• Figure 4A-4B shows photographs of human embryonic stem cells with pluripotent morphology wherein the stem cells have been cultured in a serum-free minimal media with recombinant human NME1 dimers as the only growth factor added.
• Fig. 4 A shows photograph taken at 4X magnification
• Fig. 4B shows photograph taken at 20X magnification.
• Figure 5A-5C shows photographs of human embryonic stem cells with pluripotent morphology wherein the stem cells have been cultured in a serum-free minimal media with recombinant human NME7-AB as the only growth factor added.
• Fig. 5A shows photograph taken at 4X magnification
• Fig. 5B shows photograph taken at 10X magnification
• Fig. 5C shows photograph taken at 20X magnification.
• Figure 6A-6B shows graph of HRP signal from ELISA sandwich assay showing NME7-AB dimerizes MUCl* extra cellular domain peptide.
• Fig. 6A shows an amount of NME7-AB that bound to a surface coated with the MUCl* extracellular domain peptide PSMGFR, and
• Fig. 6B shows a second MUCl* peptide binding to a second site on the bound NME7-AB.
• Figure 7 shows a graph of RT-PCR measurement of the expression levels of transcription factors BRD4 and co-factor JMJD6 in the earliest stage naive human stem cells compared to the later stage primed stem cells.
• Figure 8 shows photographs of human fibroblast cells after 18 days in culture in a serum-free media containing human NME1 in dimer form at 4X magnification.
• Figure 9 shows photographs of human fibroblast cells after 18 days in culture in a serum-free media containing human NME1 in dimer form at 20X magnification.
• Figure 10 shows photographs of human fibroblast cells after 18 days in culture in a serum-free media containing human NME7-AB at 4X magnification.
• Figure 11 shows photographs of human fibroblast cells after 18 days in culture in a serum-free media containing human NME7-AB at 20X magnification.
• Figure 12 shows photographs of human fibroblast cells after 18 days in standard media without NME protein at 4X magnification.
• Figure 13 shows photographs of human fibroblast cells after 18 days in standard media without NME protein at 20X magnification.
• Figure 14A-14C shows a cartoon of mechanistic model of inventors' discovery of how human stem cells limit their self-replication.
• Fig. 14A shows that NME7-AB is secreted from naive stem cells and how NME7-AB dimerizes MUCl* growth factor receptor as a monomer, but is later suppressed by BRD4 while co-factor JMJD6 upregulates NME1
• Fig. 14B shows that NME1 is secreted by a later naive state stem cell, but only binds to MUCl* as a dimer
• Fig. 14C shows that as number of stem cells increases so does concentration of NME1 causing it to form hexamers that do not bind to MUC1* and they induce differentiation.
• Figure 15A-15B shows photographs of human blastocysts that were stained with an antibody the inventors developed that recognizes NME7-AB and NME7-X1.
• Fig. 15 A shows a Day 3 blastocyst wherein every cell stains positive for presence of NME7-AB or NME7-X1
• Fig. 15B shows a Day 5 blastocyst wherein only the naive cells of the inner cell mass stain positive for presence of NME7-AB or NME7-X1.
• Figure 16A-16B shows photographs of Western blot gels from a co- immunoprecipitation experiment in which human naive state induced pluripotent stem (iPS) cells and embryonic stem (ES) cells were lysed and an antibody against the cytoplasmic tail of MUC1 (Ab5) was used to co-immunoprecipitate species that bind to MUC1. The immunoprecipitates were then assayed by Western blot.
• iPS human naive state induced pluripotent stem
• ES embryonic stem
• FIG. 16A shows a photograph of the Western blot that was probed with an anti-NME7 antibody and shows two NME7 species, one with molecular weight of 30 kDa and the other 33 kDa, bound to MUC1, whereas full- length NME7 in crude cell lysate has molecular weight of 42 kDa
• Fig. 16B shows a photograph of the Western blot wherein the gel of Fig. 16A was stripped and re-probed with an anti-MUCl* extracellular domain antibody, showing that NME7-AB or NME7-X1 bound to the cleaved form of MUC1 called MUC1* that runs with a molecular weight of 17-25 kDa, depending on glycosylation.
• Figure 17 shows a cartoon of the inventive method for growing human stem cell in the naive state using NME7-AB as the only growth factor on an adhesion surface of MNC3 anti-MUCl* extracellular domain antibody.
• a synthetic MUC1* extracellular domain peptide is added to bind up all the NME7-AB.
• Figure 18A-18C shows heat maps from an RNA SEQ experiment in which human embryonic stem (ES) cells that were derived in FGF, and thus in the primed state, were reverted to a less mature naive state by culturing in NME7-AB or in NME1 dimers.
• Fig. 18A shows heat map of parent FGF-cultured ES cells
• Fig. 18B shows heat map of parent cells cultured for 10 passages in NME7-AB
• Fig. 18C shows heat map of parent cells cultured for 10 passages in NME1 dimers.
• FIG. 19A-19B shows photographs of human embryonic stem (ES) cells stained with an antibody that binds to tri-methylated Lysine 27 on Histone 3, wherein the presence of red foci indicates that the second X chromosome of female source stem cells has been inactivated, XaXi, which is a sign that the cells are primed state and have initiated early differentiation and have made some cell fate decisions ; the presence of a red cloud or absence of red staining indicates that both X chromosomes are still active, XaXa, and thus no differentiation decisions have been made yet, so are naive.
• Fig. 19A shows photographs of female source stem cells that have been cultured for at least 10 passages in FGF media
• Fig. 19B shows photographs of the same cells that have been cultured for 10 passages in NME7-AB media.
• Figure 20A-20B shows photographs of human pluripotent stem cells in culture in NME7-AB media over a MNC3 anti-MUCl* antibody surface at two time points showing a 10-20-fold expansion rate over a period of four days which is a much faster growth rate than primed state stem cells and is another indication that stem cells cultured in NME7-AB are in naive state.
• Fig. 20A shows the stem cells at Day 4, and
• Fig.20B shows stem cells at Day 4.
• Figure 21A-21C shows photographs of fibroblasts being reprogrammed to become pluripotent stem cells using Yamanaka master reprogramming factors OCT4, NANOG, KLF4 and c-Myc delivered using Sendai viral transduction wherein newly reprogrammed induced pluripotent stem cells are visualized via staining with alkaline phosphatase.
• Fig. 21 A shows reprogramming being done in FGF media over MEF feeder cells
• Fig. 2 IB shows reprogramming being done in mTeSR media over Matrigel
• Fig. 21C shows reprogramming being done in NME7-AB media over MNC3 anti-MUCl* antibody surface.
• Figure 22A-22D shows photographs of human iPS cells that carry a fluorescent marker, yellow cells, which have been derived and cultured in NME7-AB, injected into a mouse blastocyst at Day 2.5 then imaged 48 hours later at Day 4.5 and show that human iPS cells cultured in NME7-AB have ability to integrate into the inner cell mass of mouse embryo.
• Fig. 22A shows fluorescent microscopy photographs of Clone E human iPS cells (yellow) that have found their way into the mouse embryo's inner cell mass that contains the naive stem cells, Fig.
• FIG. 22B is the same photograph but with another fluorescent channel opened to allow imaging of DAPI, blue, to show all the cells and showing that the NME7-AB cells have only integrated into inner cell mass plus a whiff in the trophectoderm that will develop into placenta
• Fig. 22C shows fluorescent microscopy photographs of Clone R human iPS cells (yellow) that have found their way into the mouse embryo's inner cell mass that contains the naive stem cells
• Fig. 22D is the same photograph but with another fluorescent channel opened to allow imaging of DAPI to show all the cells and showing that the NME7-AB cells have only integrated into inner cell mass plus a whiff in the trophectoderm that will develop into placenta.
• Figure 23A-23J shows confocal images of NME7-AB generated human iPS cells, clone E, that were injected into a Day 2.5 mouse morula. Staining for human Tra 1-81 (red) 48 hours later shows incorporation of the NME7-AB human stem cells into the inner cell mass of the mouse morula.
• Figures 23A, 23C, 23E, 23G, and 231 are fluorescent images.
• Figures 23B, 23D, 23F, 23H, and 23J are bright field images.
• Figure 24A-24J shows confocal images of NME7-AB generated human iPS cells, clone R, that were injected into a Day 2.5 mouse morula. Staining for human Tra 1-81 (red) 48 hours later shows incorporation of the NME7-AB human stem cells into the inner cell mass of the mouse morula.
• Figures 24A, 24C, 24E, 24G, and 241 are fluorescent images. Arrows point to incorporation of naive state NME7-AB human stem cells into the mouse morula, indicating forming of a chimeric animal.
• Figures 24B, 24D, 24F, 24H, and 24J are bright field images.
• Figure 25A-25D shows confocal images of FGF generated human iPS cells that were injected into a Day 2.5 mouse morula. Staining for human Tra 1-81 (red) and staining for trophectoderm (green) 48 hours later shows some scattered human stem cells but no incorporation of the primed state human stem cells into the inner cell mass of the mouse morula, indicating failure to start to form a chimeric animal.
• Figures 25A and 25C are fluorescent images, and Figures 25B and 25D are bright field images.
• Figure 26A-26D shows confocal images of human iPS cells that were cultured in 50% NME7-AB media and 50% KSOM, Potassium Simplex Optimized Medium, then injected into a Day 2.5 mouse morula. Fluorescent as well as bright field images were taken 48 hours later. Staining for human Tra 1-81 (red) and staining for trophectoderm (green) 48 hours later shows minimal number of human cells, if any, integrating and no incorporation of the primed state human stem cells into the inner cell mass of the mouse morula, indicating failure to start to form a chimeric animal.
• Figures 26A, 26C, 26 E and 26G are fluorescent images, and figures 26B, 26D, 26F and 26H include DAP staining.
• Figure 27A-27F shows confocal fluorescent images of NME7-AB generated human iPS cells, clone E, that were transfected with a fluorescent label, tdtomato, so they would self-fluoresce red. They were injected into a Day 2.5 mouse morula. Images were taken 48 hours later and show integration of the NME7-AB human cells into the inner cell mass of the mouse morula.
• Figures 27 A, 27B, 27C show NME7-AB human cells in red, the trophectoderm of the mouse cells in green and Figures 27D, 27E, and 27F show NME7-AB human cells in red, the trophectoderm of the mouse cells in green and all cell nuclei in blue from DAPI staining.
• Figure 28A-28J shows confocal fluorescent images of NME7-AB generated human iPS cells, clone E, that were transfected with a fluorescent label, tdtomato, so they would self-fluoresce red. They were injected into a Day 2.5 mouse morula andimages were taken 48 hours later.
• FIGS. 28A, 28C, 28E, 28G, and 281 show NME7- AB human cells in red, the trophectoderm of the mouse cells in green
• Figures 28B, 28D, 28F, and 28H show NME7-AB human cells in red, the trophectoderm of the mouse cells in green and all cell nuclei in blue from DAPI staining.
• Figure 29A-29J shows confocal fluorescent images of NME7-AB generated human iPS cells, clone R, that were transfected with a fluorescent label, tdtomato, so they would self-fluoresce red. They were injected into a Day 2.5 mouse morula and images were taken 48 hours later. The photographs show integration of the NME7-AB human cells into the inner cell mass of the mouse morula.
• Figures 29A, 29C, 29E, 29G, 291 show NME7-AB human cells in red, the trophectoderm of the mouse cells in green and Figures 29B, 29D, 29F, and 29H show NME7-AB human cells in red, the trophectoderm of the mouse cells in green and all cell nuclei in blue from DAPI staining.
• FIG. 30A-30J shows confocal fluorescent images of NME7-AB generated human iPS cells, clone R, that were transfected with a fluorescent label, tdtomato, so they would self-fluoresce red. They were injected into a Day 2.5 mouse morula. Images were taken 48 hours later. The photographs show integration of the NME7-AB human cells into the inner cell mass of the mouse morula. Arrows indicate incorporation of the human cells into the mouse inner cell mass.
• Figures 30A, 30C, 30E, 30G, and 301 show NME7-AB human cells in red, the trophectoderm of the mouse cells in green and Figures 30B, 30D, 30F, and 30H show NME7-AB human cells in red, the trophectoderm of the mouse cells in green and all cell nuclei in blue from DAPI staining.
• Figure 31A-31F shows photographs of control plates for experiment demonstrating generation of non-human primate induced pluripotent stem cells.
• fibroblasts from crab-eating macaques were cultured but the core pluripotency genes were not transduced. Images show the morphology of fibroblasts not of stem cells.
• Figures 31A and 3 ID were photographed at 4X magnification
• Figures 3 IB and 3 IE were photographed at 10X magnification
• Figures 31C and 3 IF were photographed at 20X magnification
• Figure 32A32-C shows Day 6 photographs of fibroblasts from crab-eating macaques that have been reprogrammed into induced pluripotent stem (iPS) cells by culturing in an NME7 media, in this case NME7-AB, over a surface of anti-MUCl* antibody, in this case MN-C3 and by transducing the cells with core pluripotency genes, in this case Oct4, Sox2, Klf4, and c-Myc.
• NME7 media in this case NME7-AB
• anti-MUCl* antibody in this case MN-C3
• core pluripotency genes in this case Oct4, Sox2, Klf4, and c-Myc.
• Figure 32A was photographed at 4X magnification
• Figure 32B was photographed at 10X magnification
• Figure 32C was photographed at 20X magnification.
• Figure 33A-33F shows Day 6 photographs of fibroblasts from crab-eating macaques that have been reprogrammed into induced pluripotent stem (iPS) cells by culturing in an NME7 media, in this case NME7-AB, over a surface of anti-MUCl* antibody, in this case MN-C3 and by transducing the cells with core pluripotency genes, in this case Oct4, Sox2, Klf4, and c-Myc. 100,000 fibroblasts from crab-eating macaques were plated per well of a 6-well plate. Emerging colonies with stem-like morphology are circled. Figures 33A and 33D were photographed at 4X magnification, Figures 33B and 33E were photographed at 10X magnification, and Figures 33C and 33F were photographed at 20X magnification.
• NME7 media in this case NME7-AB
• MN-C3 anti-MUCl* antibody
• core pluripotency genes in this case Oct4, Sox2, Kl
• Figure 34A-34F shows Day 14 photographs of fibroblasts from crab-eating macaques that have been reprogrammed into induced pluripotent stem (iPS) cells by culturing in an NME7 media, in this case NME7-AB, over a surface of anti-MUCl* antibody, in this case MN-C3 and by transducing the cells with core pluripotency genes, in this case Oct4, Sox2, Klf4, and c-Myc. Stem cell colonies are clearly visible.
• NME7 media in this case NME7-AB
• MN-C3 anti-MUCl* antibody
• core pluripotency genes in this case Oct4, Sox2, Klf4, and c-Myc. Stem cell colonies are clearly visible.
• Figures 34A, 34B, and 34C were photographed at 10X magnification
• Figures 34D, 34E, and 34F were photographed at 20X magnification
• Figures 34A and 34D show results from 24,000 macaque fibroblasts being plated
• Figures 34B and 34E show results from 6,000 macaque fibroblasts being plated
• Figures 34C and 34F show results from 12,000 macaque fibroblasts being plated.
• Figure 35A-35D shows photographs of rhesus macaque embryonic stem (ES) cells being proliferated in a serum-free media containing NME7-AB as the only growth factor on Day 1 of the second passage in NME7-AB media on an anti-MUCl* antibody surface. Embryonic stem cell colonies are clearly visible.
• ES rhesus macaque embryonic stem
• Figures 35A and 35B were photographed at 4X magnification, and Figures 35C and 35D were photographed at 10X magnification, [0031]
• Figure 36A-36D shows photographs of rhesus macaque embryonic stem (ES) cells being proliferated in a serum-free media containing NME7-AB as the only growth factor on Day 3 of the second passage in NME7-AB media on an anti-MUCl* antibody surface. Embryonic stem cell colonies are clearly visible.
• Figures 36A and 36B were photographed at 4X magnification, and Figures 36C and 36D were photographed at 10X magnification.
• Figure 37A-37B shows photographs of rhesus macaque embryonic stem (ES) cells being proliferated in a serum-free media containing NME7-AB as the only growth factor on Day 1, passage 3.
• Figure 37A shows scale bar at lOOOum and
• Figure 37B shows scale bar at 400um.
• Figure 38A-38H shows photographs of rhesus macaque embryonic stem (ES) cells being proliferated in a serum-free media containing NME7-AB as the only growth factor on Day 4, passage 3.
• Figures 38A, 38C, 38F were photographed at 4X
• Figures 38B, 38D, 38G were photographed at 10X
• Figures 38E and 38H were photographed at 20X.
• Figure 39A-39C shows Day 14 photographs of fibroblasts from rhesus macaques that have been reprogrammed into induced pluripotent stem (iPS) cells by culturing in an NME7 media, in this case NME7-AB, over a surface of anti-MUCl* antibody, in this case MN-C3 and by transducing the cells with core pluripotency genes, in this case Oct4, Sox2, Klf4, and c-Myc on Day 14 post transduction of pluripotency genes.
• Figure 39A was photographed at 4X
• Figure 39B was photographed at 10X
• Figure 39C was photographed at 20X.
• Figure 40A-40C shows photographs of serial passaging of fibroblasts from rhesus macaques that have been reprogrammed into induced pluripotent stem (iPS) cells by culturing in an NME7 media, in this case NME7-AB, over a surface of anti-MUCl* antibody, in this case MN-C3 and by transducing the cells with core pluripotency genes, in this case Oct4, Sox2, Klf4, and c-Myc.
• Figure 40A was photographed at 4X
• Figure 40B was photographed at 10X
• Figure 40C was photographed at 20X.
• Figure 41A-41D shows photographs of Day 16 derivation of macaque iPS cells in NME7-AB media by transducing the cells with core pluripotency genes, in this case Oct4, Sox2, Klf4, and c-Myc under identical conditions except that in one case the macaque fibroblasts were plated onto MNC3 anti-MUCl* antibody surface and in the other case onto mouse embryonic feeder cells, MEFs. It is clearly observed that iPS derivation of non-human primates is more efficient when plated in the absence of mouse feeder cells.
• Figures 41 A and 41B were plated onto MNC3 antibody surface and Figures 41C and 41D were plated onto mouse feeder cells.
• Figure 42A-42B shows Day 14 photographs of a fibroblasts from rhesus macaques that have been reprogrammed into induced pluripotent stem (iPS) cells by culturing in an NME7 media, in this case NME7-AB, over a surface of MEFs and by transducing the cells with core pluripotency genes, in this case Oct4, Sox2, Klf4, and c-Myc.
• Figure 42 A was photographed at 10X magnification
• Figure 42B was photographed at 20X magnification.
• Figure 43A-43E shows photographs of NME7-AB induced pluripotent stem cells from rhesus macaques over a surface of anti-MUCl* antibody, in this case MN-C3 on Day 3, passage 1.
• Figure 43 A was photographed at 4X magnification
• Figure 43B was photographed at 10X magnification
• Figure 43C was photographed at 20X magnification
• Figures 43D and 43E were photographed at 40X magnification.
• Figure 44A-44F shows photographs of serial passaging of NME7-AB induced pluripotent stem cells from rhesus macaques over a surface of anti-MUCl* antibody, in this case MN-C3 on Day 1, passage 2. As is clearly visible, stem cell colonies are present.
• Figures 44A and 44D were photographed at 10X magnification
• figures 44B and 44E were photographed at 20X magnification
• figures 44C and 44F were photographed at 40X magnification.
• Figure 45A-45H shows photographs of serial passaging of NME7-AB induced pluripotent stem cells derived from rhesus macaque fibroblasts over a surface of mouse feeder cells, MEFs, on Day 2 of passage 2.
• Figures 45A and 45E were photographed at 4X magnification
• figures 45B and 45F were photographed at 10X magnification
• figures 45C and 45G were photographed at 20X magnification
• figures 45D and 45H were photographed at 40X magnification.
• Figure 46A-46H shows photographs of serial passaging of NME7-AB induced pluripotent stem cells from rhesus macaques over mouse feeder cells, MEFs, on Day 3 of passage 2.
• Figures 46A and 46E were photographed at 4X magnification
• figures 46B and 46F were photographed at 10X magnification
• figures 46C and 46G were photographed at 20X magnification
• figures 46D and 46H were photographed at 40X magnification.
• Figure 47A-47G shows photographs of serial passaging of NME7-AB induced pluripotent stem cells from rhesus macaques over mouse feeder cells, MEFs, on Day 1 and on Day 2 of passage 3.
• Figures 47A, 47B, 47C and 47D were photographed on Day 1 of passage 3
• figures 47E, 47F, 47G were photographed on Day 2 of passage 3
• figures 47 A and 47E were photographed at 4X magnification
• figures 47B and 47F were photographed at 10X magnification
• figures 47C and 47G were photographed at 20X magnification
• figure 47D was photographed at 40X magnification.
• Figure 48A-48D shows photographs of serial passaging of NME7-AB induced pluripotent stem cells from rhesus macaques mouse feeder cells, MEFs, on Day 3, passage 4.
• Figure 48A was photographed at 4X magnification
• Figures 48B and 48C were photographed at 10X magnification
• Figure 48D was photographed at 20X magnification.
• the "MUCl*" extra cellular domain is defined primarily by the PSMGFR sequence (GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:6)). Because the exact site of MUCl cleavage depends on the enzyme that clips it, and that the cleavage enzyme varies depending on cell type, tissue type or the time in the evolution of the cell, the exact sequence of the MUCl* extra cellular domain may vary at the N-terminus.
• PSMGFR Primary Sequence of MUCl Growth Factor Receptor as set forth as
• N-10 PSMGFR N-10 PSMGFR
• N-15 PSMGFR N-20 PSMGFR
• C-number refers to the number of amino acid residues that have been deleted at the C- terminal end of PSMGFR.
• extracellular domain of MUCl* refers to the extracellular portion of a MUCl protein that is devoid of the tandem repeat domain.
• MUCl* is a cleavage product wherein the MUCl* portion consists of a short extracellular domain devoid of tandem repeats, a transmembrane domain and a cytoplasmic tail.
• the precise location of cleavage of MUCl is not known perhaps because it appears that it can be cleaved by more than one enzyme.
• the extracellular domain of MUCl* will include most of the PSMGFR sequence but may have an additional 10-20 N-terminal amino acids.
• NME family proteins or “NME family member proteins”, numbered 1-10, are proteins grouped together because they all have at least one NDPK (nucleotide diphosphate kinase) domain. In some cases, the NDPK domain is not functional in terms of being able to catalyze the conversion of ATP to ADP.
• NME proteins were formally known as NM23 proteins, numbered HI, H2 and so on. Herein, the terms NM23 and NME are interchangeable.
• NMEl, NME2, NME6 and NME7 are used to refer to the native protein as well as NME variants. In some cases these variants are more soluble, express better in E.
• NME7 as used in the specification can mean the native protein or a variant, such as NME7-AB that has superior commercial applicability because variations allow high yield expression of the soluble, properly folded protein in E. coli.
• NME as referred to herein is interchangeable with "NM23-H1". It is also intended that the invention not be limited by the exact sequence of the NME proteins.
• the mutant NME1-S120G, also called NM23-S120G, are used interchangeably throughout the application. The S120G mutants and the P96S mutant are preferred because of their preference for dimer formation, but may be referred to herein as NM23 dimers or NMEl dimers.
• NME7 as referred to herein is intended to mean native NME7 having a molecular weight of about 42kDa, a cleaved form having a molecular weight between 25 and 33kDa, a variant devoid of the DM10 leader sequence, NME7-AB or a recombinant NME7 protein, or variants thereof whose sequence may be altered to allow for efficient expression or that increase yield, solubility or other characteristics that make the NME7 more effective or commercially more viable.
• NMEl dimers also known as “NM23-H1 dimers” can be two NMEl proteins that are non-covalently bound to each other, two NMEl proteins covalently linked to each other or two NMEl proteins that are genetically fused together, including via a linker.
• NMEl dimers can be genetically engineered by making a DNA construct comprised of two NMEl proteins, which can be separated by a flexible linker. The two NME proteins need not be the full protein or the native sequence. For example, C-terminal deletions that promote dimer formation and stability may be used. Mutations such as S120G and/or P96S that promote dimer formation and stability may be used.
• NME family member dimers such as NME2 or NME6 dimers, are similarly two NME proteins that are non-covalently bound to each other, two NME proteins covalently linked to each other or two NME proteins that are genetically fused together, including via a linker.
• an "an agent that maintains stem cells in the naive state or reverts primed stem cells to the naive state” refers to a protein, small molecule or nucleic acid that alone or in combination maintains stem cells in the naive state, resembling cells of the inner cell mass of an embryo. Examples include but are not limited to NME1 dimers, human or bacterial, NME7, NME7-AB, 2i, 5i, nucleic acids such as siRNA that suppress expression of MBD3, CHD4, BRD4, or JMJD6.
• small molecule in reference to an agent being referred to as a "small molecule”, it may be a synthetic chemical or chemically based molecule having a molecular weight between 50Da and 2000Da, more preferably between 150 Da and 1000 Da, still more preferably between 200Da and 750Da.
• 2i inhibitor refers to small molecule inhibitors of GSK3-beta and MEK of the MAP kinase signaling pathway.
• the name 2i was coined in a research article (Silva J et al 2008), however herein “2i” refers to any inhibitor of either GSK3-beta or MEK, as there are many small molecules or biological agents that if they inhibit these targets, have the same effect on pluripotency or tumorigenesis.
• FGF fibroblast growth factor
• Rho associated kinase inhibitors may be small molecules, peptides or proteins (Rath N, et al, 2012). Rho kinase inhibitors are abbreviated here and elsewhere as ROCi or ROCKi, or Ri. The use of specific rho kinase inhibitors are meant to be exemplary and can be substituted for any other rho kinase inhibitor.
• stem-like refers to a state in which cells acquire characteristics of stem cells or progenitor cells, share important elements of the gene expression profile of stem cells progenitor cells.
• Stem-like cells may be somatic cells undergoing induction to a less mature state, such as increasing expression of pluripotency genes.
• Stem-like cells also refers to cells that have undergone some de-differentiation or are in a meta-stable state from which they can alter their terminal differentiation.
• nucleotide symbols other than a, g, c, t they follow the convention set forth in WIPO Standard ST.25, Appendix 2, Table 1, wherein k represents t or g; n represents a, c, t or g; m represents a or c; r represents a or g; s represents c or g; w represents a or t and y represents c or t.
• SEQ ID NOS:2, 3 and 4 describe N-terminal MUC-1 signaling sequence for directing MUCl receptor and truncated isoforms to cell membrane surface. Up to 3 amino acid residues may be absent at C-terminal end as indicated by variants in SEQ ID NOS:2, 3 and 4.
• GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGW GIALLVLVCVLVALAIVYLIALAVCQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHG RYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAAASANL (SEQ ID NO:5) describes a truncated MUC1 receptor isoform having nat- PSMGFR at its N-terminus and including the transmembrane and cytoplasmic sequences of a human full-length MUC1 receptor.
• GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:6) describes the extracellular domain of Native Primary Sequence of the human MUC1 Growth Factor Receptor (nat-PSMGFR - an example of "PSMGFR"):
• TINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:7) describes the extracellular domain of Native Primary Sequence of the human MUC1 Growth Factor Receptor (nat-PSMGFR - An example of "PSMGFR"), having a single amino acid deletion at the N-terminus of SEQ ID NO:6).
• GTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 8) describes the extracellular domain of "SPY” functional variant of the native Primary Sequence of the MUC1 Growth Factor Receptor having enhanced stability (var- PSMGFR - An example of "PSMGFR").
• TINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:9) describes the extracellular domain of "SPY” functional variant of the native Primary Sequence of the MUC1 Growth Factor Receptor having enhanced stability (var- PSMGFR - An example of "PSMGFR"), having a single amino acid deletion at the C-terminus of SEQ ID NO:8).
• CQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVS AGNGGSSLSYTNPAVAAASANL (SEQ ID NO: 11) describes human MUC1 cytoplasmic domain amino acid sequence.
• NM23-H1 describes amino acid sequence (NM23-H1 also known as NMEl: GENBANK ACCESSION AF487339).
• MANLERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVAMKFLRASEEHLKQ HYIDLKDRPFFPGLVKYMNSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPGTIR GDFCIQVGRNIIHGSDSVKSAEKEISLWFKPEELVDYKSCAHDWVYE (SEQ ID NO: 19) describes NM23-H2 amino acid sequence (NM23-H2: GENBANK ACCESSION AK313448).
• RNAAHGPDSFASAAREMELFF- (SEQ ID NO:43)
• nucleoside diphosphate kinase 7 isoform a [Homo sapiens] (Hu_7)
• Human NME7 isoform a represented by SEQ ID NO:84 has 90.4% sequence identity with Human NME7 isoform b represented by SEQ ID NO:21 in 376 amino acid overlap region.
• Mouse MUC1* extracellular domain PSMGFR is 51.1% identical to human in a 45 amino acid overlap region
• GTFSASDVKSQLIQHKKEA-DDYNLTISEVKVNEMQFPPSAQSRP (SEQ ID NO: 87)
• nucleoside diphosphate kinase 7 isoform 1 Mus musculus (Mo_7)
• Mouse isoform 2 NME7 is 88.4% identical to human NME7 isoform a in a 253 amino acid overlap region
• nucleoside diphosphate kinase 7 isoform 2 [Mus musculus] (Mo2-7)
• Pig MUC1* PSMGFR is 52.2% identical to human in a 46 amino acid overlap
• Pig NME7 is 65.6% identical to human NME7 isoform a in a 453 amino acid overlap region
• nucleoside diphosphate kinase 7 [Sus scrofa, Pig] (Pi_7)
• Sheep NME7 is 88.4% identical to human NME7 isoform a in a 395 amino acid overlap region
• nucleoside diphosphate kinase 7 isoform XI [Ovis aries, Sheep] (Sh_7)
• Crab-eating Macque NME7 is 98% identical to human NME7 isoform a in a 251 amino acid overlap
• Rhesus Macaque NME7 is 98.4% identical to human NME7 isoform a in a 376 amino acid overlap region
• Bonobo MUC1* extracellular domain PSMGFR is 100% identical to human.
• Bonobo PREDICTED nucleoside diphosphate kinase 7 [Pan paniscus, Bonobo] (Bo_7)
• Chimpanzee NME7 is 99.7% identical to human NME7 isoform a in a 376 amino acid overlap region
• nucleoside diphosphate kinase 7 [Pan troglodytes, Chimp] (CH_7)
• Gorilla NME7 is 78.2% identical to human NME7 isoform a in a 376 amino acid overlap region
• NME7 [ENSGGOP00000002464], Gorilla gorilla (Go_7)
• NNATKTFREFCGPADP SEQ ID NO: 1028
• nucleoside diphosphate kinase 7 [Mus musculus] (Mo_7)
• Mouse NME7 is 87.8% identical to human NME7 isoform a in a 378 amino acid overlap region
• nucleoside diphosphate kinase 7 isoform XI [Mus musculus] (MoXl-7)
• Mouse NME7 is 79.8% identical to human NME7 isoform a in a 376 amino acid overlap region
• Macaca NME7 is 98.0% identical to human NME7 isoform a in a 251 amino acid overlap region
• Macaca NME7 is 98.4% identical to human NME7 isoform a in a 376 amino acid overlap region
• nucleoside diphosphate kinase 7 b [Pan troglodytes, Chimp] (CHb_7)
• Chimp NME7 is 90.2% identical to human NME7 isoform a in a 376 amino acid overlap region
• Sheep NME7 is 92.6% identical to human NME7 isoform a in a 377 amino acid overlap region
• nucleoside diphosphate kinase 7 isoform X3 [Ovis aries, Sheep] (Shx3_7)
• Sheep NME7 is 83.6% identical to human NME7 isoform a in a 377 amino acid overlap region
• Macaca mulatta rhesus macaque MUC1
• DIFPARDAYHPMSEYPTYHTHGRYVPAGGTNRSPYE (SEQ ID NO: 117)
• Macaca fascicularis_MUCl (isoform XI)
• VSAGNGGSSLSYTNPAVAATSANL (SEQ ID NO: 118)
• the present invention is directed to a method of testing for efficacy or toxicity of a potential drug agent in a chimeric animal that expresses some human DNA or some human tissues.
• an animal that expresses some human DNA or tissues is generated by introducing human naive state stem cells into a non-human cell or cells.
• the non-human cell is an egg, in another aspect it is a fertilized egg, in another aspect, the cells are a morula, blastocyst or embryo.
• the agent that maintains stem cells in the naive state or reverts primed stem cells to the naive state may be an NME protein, 2i, 5i, or other cocktails of inhibitors, chemicals, or nucleic acids.
• the NME protein may be NME1 dimer, NME7 monomer, NME7-AB, NME7-X1, NME6 dimer, or bacterial NME.
• the non-human mammal may be a rodent, such as a mouse or rat, primate, including macaque, rhesus monkey, ape, chimp, bonobo and the like, or a domestic animal including pig, sheep, bovine, and the like.
• the chimeric animal may have a genetic disorder, have an induced disease, or a cancer that may be spontaneously generated or implanted from cells derived from a human being.
• the non-human animal may be transgenic, wherein the animal expresses human MUCl or MUCl* or NME protein in the germ cells or somatic cells, wherein the germ cells and somatic cells contain a recombinant human MUCl or MUCl* or NME gene sequence introduced into said animal.
• the gene expressing the human MUCl or MUCl* or NME protein may be under control of an inducible promoter.
• the promoter may be inducibly responsive to a naturally occurring protein in the non-human animal or an agent that can be administered to the animal before, after or during development.
• the non-human animal may be transgenic, wherein the animal expresses its native sequence MUC1 or MUC1* or NME protein in the germ cells or somatic cells, wherein the germ cells and somatic cells contain a recombinant native species MUC1 or MUC1* or NME gene sequence introduced into said mammal.
• the NME species can be NME7, NME7-X1, NME1, NME6 or a bacterial NME.
• the agent that maintains stem cells in the naive state or reverts primed stem cells to the naive state may be an NME protein, 2i, 5i, or other cocktails of inhibitors, chemicals, or nucleic acids.
• the NME protein may be NME1 dimer, NME7 monomer, NME7-AB, NME7-X1, NME6 dimer, or bacterial NME.
• the agent may suppress expression of MBD3, CHD4, BRD4 or JMJD6.
• the agent may be siRNA made against MBD3, CHD4, BRD4 or JMJD6, or siRNA made against any gene that encodes a protein that upregulates expression of MBD3, CHD4, BRD4 or JMJD6.
• the cancer stem cell may be characterized by increased expression of CXCR4 or E-cadherin (CDH1) compared with cancer cells or normal cells.
• the invention is directed to a method for generating tissue from xenograft in a non-human mammal, comprising: (i) generating a transgenic non-human mammal, wherein the mammal expresses human MUC1 or MUC1* or NME protein in the germ cells and somatic cells, wherein the germ cells and somatic cells contain a recombinant human MUC1 or MUC1* or NME gene sequence introduced into said mammal, wherein the expression of the gene sequence may be under control of an inducible and repressible regulatory sequence; (ii) transferring stem cells or progenitor cells that are xenogeneic in origin to the non-human mammal such that the gene may be induced to be expressed so as to multiply the number of stem or progenitor cells; and (iii) repressing the gene expression so as to generate tissue from the xenografted stem cells.
• the gene expression repression may be carried out by contacting the stem cells with a tissue differentiation factor, or in step (iii) the gene expression repression may be carried out naturally in the mammal in response to naturally produced host tissue differentiation factor.
• the transferred cells may be human.
• the tissue may be an organ.
• the NME protein may be NME7, NME7-AB, NME7-X1, NME1, NME6, or bacterial NME.
• the animal may be a mammal, a rodent, a primate or domesticated animal such as a pig, sheep, or bovine species. [00351] II.
• the present invention is directed to making animals having at least some human cells or cells in which at least some of the DNA is of human origin.
• Such animals would grow human tissue, tissue containing some human cells or cells containing some human DNA for the generation of human or human-like tissue.
• Such animals would grow organs comprising at least some human cells.
• such animals would grow organs comprised entirely of human cells.
• host animals can be genetically or molecularly manipulated even after development to grow human limbs.
• Limbs, nerves, blood vessels, tissues, organs, or factors made in them, or secreted from them, would then be harvested from the animals and used for a multitude of purposes including but not limited to: 1) transplant into humans; 2) administration into humans for medicinal benefit, including anti-aging; and 3) scientific experiments including drug testing and disease modeling.
• the invention is directed to a method for generating human tissues in a non-human animal comprising: (i) generating human naive state stem cells and injecting them into a fertilized egg, morula, blastocyst or embryo of a non-human animal such that a chimeric animal is generated; (ii) harvesting human tissues, organs, cells or factors secreted by or made in the human tissues or cells from the chimeric animal; and (iii) transplanting or administering the harvested material into a human.
• the naive state stem cells may be generated using NME7, NME7-AB, NME7-X1 or dimeric NME1.
• the naive stem cells may be iPS cells that have been reprogrammed in a medium containing NME7, NME7-AB, NME7-X1 or dimeric NME1.
• the naive stem cells may be embryonic stem cells that have been cultured in a medium containing NME7, NME7-AB, NME7-X1 or dimeric NME1.
• the non-human cells of the blastocyst or embryo may have been genetically altered. And the genetic alteration may result in the host animal being unable to generate a certain tissue or organ.
• the genetic alteration may be to make the non-human animal express human molecules that facilitate or enhance the incorporation or growth of human stem or progenitor cells in the non-human host animal.
• the agent that maintains stem cells in the naive state or reverts primed stem cells to the naive state may be an NME protein, 2i, 5i, chemical, or nucleic acid.
• the NME protein may be NME1 dimer, NME7 monomer, NME7-AB, NME6 dimer, or bacterial NME, or NME7-X1.
• the non-human animal may be a rodent, mouse, rat, pig, sheep, non-human primate, macaque, chimpanzee, bonobo, gorilla or any non-human mammal.
• the non-human animal is chosen for its high sequence homology to human NME protein, especially human NME7-AB or NME7-X1 or high sequence homology to human MUC1* extracellular domain.
• the NME protein may be present in serum-free media as the single growth factor.
• a chimeric animal One test of whether or not a chimeric animal can be generated is if stem cells from a first species are able to incorporate into the inner cell mass (ICM) of a second species. Chimeric animals are more readily generated when the two different species are closely related, for example two rodents.
• human naive state stem cells that had been generated in human NME7-AB, then cultured in human were injected into a mouse morula 2.5 days after fertilization of the egg. This is before the inner cell mass forms. Forty-eight (48) hours later, the morula was analyzed and such analysis showed that the human stem cells had incorporated into the inner cell mass, indicating that a chimeric animal will develop.
• the present is directed to a method for generating human tissues or organ in a non-human animal host comprising: (i) generating human naive state stem cells and injecting them into a fertilized egg, morula, blastocyst, embryo or developing fetus of the non-human animal host such that a chimeric animal is generated; (ii) harvesting human tissues, organs, cells or factors secreted by or made in the human tissues or cells from the chimeric animal; (iii) transplanting or administering the harvested material into a human resulting in generation of human tissues.
• the naive state stem cells are generated using NME7, NME7-AB, NME7-X1, NME6 or dimeric NMEl.
• the naive stem cells are iPS cells that have been reprogrammed in a medium containing NME7, NME7-AB, NME6, NME7-X1 or dimeric NMEl.
• the naive stem cells are embryonic stem cells that have been cultured in a medium containing NME7, NME7-AB, NME6, NME7-X1 or dimeric NMEl.
• Non-human cells of the blastocyst or embryo have been genetically altered. The genetic alteration results in the host animal being unable to generate a certain tissue or organ.
• the agent that maintains stem cells in the naive state or reverts primed stem cells to the naive state is an NME protein, 2i, 5i, chemical, or nucleic acid.
• the NME protein is NMEl dimer, NME7 monomer, NME7- AB, NME6 dimer, or bacterial NME.
• the non-human animal is a rodent, pig bovine, sheep or primate. The rodent is a mouse or rat.
• the NME protein is present in serum free media as the single growth factor.
• the non-human animal host expresses NME protein having a sequence that is homologous to the native sequence of the species of the stem cells to be generated.
• the NME protein is NME7, NME7-AB, NME7-X1, or dimeric NMEl or NME6.
• the NME protein is NME7.
• the NME protein is at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% homologous to the native NME protein sequence of the species of the stem cells to be generated.
• the NME protein is at least 60% homologous to the native sequence of the species of the stem cells to be generated.
• the NME protein is at least 70% homologous to the native sequence of the species of the stem cells to be generated.
• an NME protein having a sequence at least 75% homologous to native mouse NME protein an NME protein having a sequence at least 75% homologous to native rat NME protein, an NME protein having a sequence at least 75% homologous to native pig NME protein, an NME protein having a sequence at least 75% homologous to native sheep NME protein, an NME protein having a sequence at least 75% homologous to native bovine NME protein, an NME protein having a sequence at least 75% homologous to native crab-eating macaque NME protein, an NME protein having a sequence at least 75% homologous to native rhesus monkey NME protein, an NME protein having a sequence at least 75% homologous to native chimpanzee NME protein, an NME protein having a sequence at least 75% homologous to native bonobo NME protein, an NME protein having a sequence at least 75% homologous to native gorilla NME protein
• the invention is directed to a method for generating stem cells, inducing pluripotency in somatic cells or culturing stem cells comprising the steps of contacting cells with an NME protein and/or an anti-MUCl* antibody wherein the NME protein is at least 75% homologous to the sequence of the donor cells and the anti-MUCl* antibody binds to a peptide comprising the sequence of a MUC1* extracellular domain wherein the sequence is at least 75% homologous to the native sequence of the species that donated the cells.
• the invention is directed to a method of treating a person in need of generated tissue or organ, comprising carrying out the steps described above.
• the invention is directe to a method of generating a first non-human mammal that comprises DNA, molecules, cells, tissue or organ specifically originating from a second mammal that does or does not belong to the same species or genus as the first non-human mammal, comprising introducing cells from the second mammal into the first non-human mammal.
• the cells from the second mammal are progenitor cells, stem cells or naive state stem cells.
• the naive state stem cells are generated by culturing cells in a media that contains NME.
• the NME is dimeric NMEl, dimeric NME6, NME7-X1 or NME7-AB.
• the NME has sequence endogenous to the second mammal.
• the second mammal is human.
• the first non-human mammal is a rodent, a domesticated mammal, pig, bovine, or a non-human primate.
• the progenitor cells, stem cells or naive stem cells are introduced into the fertilized egg, morula, blastocyst, embryo or developing fetus of the first non-human mammal.
• further steps include: allowing the first non- human mammal to develop and harvesting from the first non-human mammal molecules, cells, tissues or organs that have incorporated some second mammalian DNA; and administering to the second mammal in need thereof the molecules, cells, tissues or organs for the treatment or prevention of a disease or condition.
• the progenitor cells, stem cells or naive stem cells are iPS cells.
• the somatic cells from which the iPS cells are generated are from the second mammal to which the obtained molecules, cells, tissues or organs for the treatment or prevention of a disease or condition is administered.
• further steps include: determining an organ developmental time period and endogenous genes involved in the development of the organ; and knocking out or knocking down the endogenous gene during the developmental time period of the organ in the first non-human mammal, wherein the organ is caused to be produced from the cells from the second mammal.
• the first non-human mammal is close to the second mammal with global sequence identity that is greater than 70%, 75%, 80%, 85%, 90%, or 95% or NME sequence identity that is greater than 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
• further steps include: determining an organ developmental time period and endogenous genes involved in the development of the organ; and genetically altering the fertilized egg, cells of morula, cells of the blastocyst, or cells of the embryo or developing fetus of the first non-human mammal such that second mammalian NME7-AB or NME1 is expressed from an inducible or repressable promoter such that the second mammalian cells are timely expanded in response to the non-mammalian NME7-AB or NME1.
• Further steps include injecting the second mammalian stem cells into embryo at a later stage of development at the location where the desired organ or tissue would normally develop.
• Further steps include expanding the mammalian stem cells by inducing expression of either first non-human mammalian or second mammalian NME7 or NME1 at that location. Further steps include expanding the mammalian stem cells by inducing expression of either first non-human mammalian or second mammalian NME1 at that location.
• a second mammalian promoter is linked to an endogenous first non-human mammalian protein and is expressed at a desired time and location, then introducing an agent that directs the development of the desired tissue.
• the endogenous first non-human mammalian protein is a protein that induces expression of NME1 or NME7, preferably NME1.
• the invention is directed to a method of testing for efficacy or toxicity of a potential drug agent in a chimeric animal that expresses some second mammalian DNA or some second mammalian tissue, comprising: (i) generating a first non- human mammal that comprises DNA, molecules, cells, tissue or organ specifically originating from a second mammal that does or does not belong to the same species or genus as the first non-human mammal, comprising introducing cells from the second mammal into the first non-human mammal; and (ii) administering a test drug to the first non-human mammal for the effect on the tissue or organ originating from the second mammal.
• NME is expressed in the first non-human mammal that enhances proliferation of the cells originating from the second mammal.
• the invention is directed to a method discovering a potential drug agent in a chimeric animal that expresses some second mammal DNA or some second mammal tissues, comprising: (i) generating a first non-human mammal that comprises DNA, molecules, cells, tissue or organ specifically originating from a second mammal that does or does not belong to the same species or genus as the first non-human mammal, comprising introducing cells from the second mammal into the first non-human mammal; and (ii) administering a compound to the first non-human mammal for the effect on the tissue or organ originating from the second mammal, wherein efficacious effects indicate that the present of a potential drug.
• NME is expressed in the first non-human mammal that enhances proliferation of the cells originating from the second mammal.
• MUC1*/NME in Stem Cell Proliferation and Induction [00366] We discovered that human stem cells overexpress MUC1* which is a potent growth factor receptor.
• the ligand of MUC1*, NM23-H1, also called NME1 in dimeric form is alone sufficient to make human stem cells grow in the pluripotent state, without the need for feeder cells, conditioned media, or any other growth factors or cytokines (Mahanta S et al 2008; Hikita S et al 2008).
• NME1 dimers are ligands of the MUC1* growth factor receptor, wherein MUC1* is the remaining transmembrane portion after most of the extra cellular domain has been cleaved and shed from the cell surface. The remaining portion of the extra cellular domain is comprised essentially of the PSMGFR sequence. NME1 dimers bind to and dimerize the MUC1* receptor. Competitive inhibition of the NME-MUC1* interaction, using a synthetic PSMGFR peptide, induced differentiation of pluripotent stem cells, which shows that pluripotent stem cell growth is mediated by the interaction between NME1 dimers and MUC1* growth factor receptor.
• Human stem cells secrete NME1 where, after dimerization, it binds to the MUC1* receptor and stimulates growth and pluripotency of human stem cells.
• Competitive inhibition of the interaction by either addition of the PSMGFR peptide or by adding the Fab of an anti-MUCl* (anti- PSMGFR) antibody induced cell death and differentiation (Hikita et al 2008) in vitro.
• NME7 a new growth factor of the NME family, NME7, that makes human stem cells grow and inhibits their differentiation, in the absence of FGF or any other growth factor.
• NME7-AB a truncated, recombinant human NME7 that we call NME7-AB or rhMNE7-AB. It is devoid of the N-terminal DM10 domain and has a molecular weight of approximately 33kDa.
• NME7-AB rhMNE7-AB
• NME7-AB rhMNE7-AB
• An alternative splice variant of NME7 called NME7-X1 was theorized.
• NME7-X1 does exist in nature and is also secreted by human stem cells and is 30kDa.
• NME7-X1 does exist in nature and is also secreted by human stem cells and is 30kDa.
• some bacterial NME1 proteins that have high sequence homology to human NME1 act the same as human NME1. They bind to and dimerize the MUC1* extracellular domain and induce pluripotency.
• One such bacterial NME1 protein that we showed supports human stem cell growth and induces pluripotency is Halomonas Sp. 593, also known as HSP593. Fibroblasts are somatic cells, not stem cells.
• NME7-AB, NME7-X1, NME1 dimers and HSP593 dimers are able to induce somatic cells to revert to a less mature state.
• Figure 1 shows that simply culturing human fibroblasts in NME7-AB, NME1 dimers or HSP593 NME1 dimers causes upregulation of stem cell markers OCT4 and NANOG.
• Figure 2 shows that these NME proteins suppress expression of MBD3 and CHD4; suppression of these genes was previously shown to make human stem cells revert to a more naive state (Rais Y et al, 2013).
• BRD4 has been shown to suppress expression of NME7 and its co-factor JMJD6 upregulates NMEl.
• the PCR graph of Figure 3 shows that NME7-AB or NMEl dimers induce pluripotency by upregulating pluripotency genes and suppressing those that others have reported are suppressed in naive state stem cells.
• Figures 4A and 4B show that culturing human stem cells in NMEl dimers as the only added growth factor fully supports pluripotent stem cell growth.
• Figures 5A-5C show that culturing human stem cells in NME7-AB monomers as the only added growth factor fully supports pluripotent stem cell growth.
• NMEl must be a dimer in order to bind to and dimerize the MUC1* growth factor receptor, monomeric NME7-AB and NME7-X1 have two binding sites for MUC1*.
• Figure 6A and 6B show that in a sandwich ELISA assay, NME7-AB is able to simultaneously bind to two MUC1* extracellular domain peptides, also referred to herein as PSMGFR peptides (JHK SEQ ID?). BRD4 and co-factor JMJD6 are suppressed in naive state stem cells as is shown in Figure 2 and Figure 7.
• Figures 8-11 show that NMEl dimers and NME7-AB induce human fibroblasts to revert to a less mature, stem-like state as can be seen by their dramatic change in morphology that resembles stem cell morphology and is without question unlike fibroblast morphology which is shown in Figures 12-13.
• NME7-AB and NME7- XI are secreted which allows them to bind to and dimerize the extracellular domain of MUC1* growth factor receptor.
• NME7-AB stabilizes a first naive state.
• BRD4 suppresses NME7 and its co-factor JMJD6 upregulates expression of NMEl that must be a dimer to bind to and dimerize the MUC1* growth factor receptor.
• NMEl dimers stabilize a second naive-like state.
• Figure 15B shows that by Day 5 the morula has developed into a blastocyst and at this stage of development, the NME7-positive cells are restricted to the inner cell mass, which are known to be in naive state.
• naive state human stem cells express and secrete two truncated forms of NME7 that are both devoid of the N-terminal DM10 domain. These truncated NME7 species bind to the extracellular domain of MUC1* growth factor receptor.
• One NME7 form that we call NME7-AB has undergone post-translational cleavage to produce an NME7 species that runs with an apparent molecular weight of ⁇ 33kDa.
• NME7-X1 The other truncated form is an alternative splice isoform called NME7-X1 that is -30 kDa. This is in contrast to full-length NME7 that has a calculated molecular weight of ⁇ 42kDa and which appears to be restricted to the cytoplasm.
• NME1 dimers, NME6 dimers, NME7-AB and NME7-X1 function as a growth factors for human stem cells. They promote growth, pluripotency and induction of naive state by binding to and dimerizing the MUC1* growth factor receptor. However, NME7-AB and NME7-X1 do so as monomers as they have two binding sites for the extracellular domain of MUC1*.
• Figure 16A-16B shows photographs of Western blot gels from a co-immunoprecipitation experiment in which human naive state induced pluripotent stem (iPS) cells and embryonic stem (ES) cells were lysed and an antibody against the cytoplasmic tail of MUC1 (Ab5) was used to co-immunoprecipitate species that bind to MUC1. The immunoprecipitates were then assayed by Western blot.
• iPS human naive state induced pluripotent stem
• ES embryonic stem
• FIG. 16A shows a photograph of the Western blot that was probed with an anti-NME7 antibody and shows two NME7 species, one with molecular weight of 30 kDa and the other 33 kDa, bound to MUC1, whereas full-length NME7 in crude cell lysate has molecular weight of 42 kDa
• Fig. 16B shows a photograph of the Western blot wherein the gel of Fig. 16A was stripped and re-probed with an anti-MUCl* extracellular domain antibody, showing that NME7-AB or NME7-X1 bound to the cleaved form of MUC1 called MUC1* that runs with a molecular weight of 17-25 kDa, depending on glycosylation.
• Figure 17 depicts the inventive method for growing stem cells in the earliest naive state.
• NME7-AB or NME7-X1 is added to a serum-free media at low nanomolar concentrations, with the range being between 1 ⁇ -60 ⁇ , with 2nM-32nM more preferred, 2nM-10nM more preferred and 4nM most preferred.
• Feeder cells and extracellular matrix proteins and mixtures contain growth factors and other biological molecules that deliver signals, so it is desirable to avoid their use when inducing or stabilizing naive state.
• an anti-MUCl* antibody such as MN-C3, MN-C8 or humanized versions or fragments of MN-C3 or MN-C8, or an NME protein as the surface coating that makes stem cells adhere to the surface.
• the cells could be cultured in suspension to avoid to need for an adhesive layer.
• a peptide comprising most or all of the PSMGFR peptide is added. Adding a peptide having the sequence of most or all of the extracellular domain of MUC1* growth factor receptor acts as a ligand sink and binds up all the NME growth factor so that differentiation is synchronized and more complete.
• This peptide also ensures that all the OCT4 positive cells are induced to differentiate which minimizes or eliminates the risk of teratoma formation.
• These methods are used to produce stem cells for research, drug discovery, therapeutic use, or can be implanted into a fertilized egg, morula, blastocyst, embryo or fetus for the generation of non-human animals that have some human DNA, molecules, cells, tissues or organs. Such molecules, cells, tissues or organs that contain at least some human DNA can then be used for research, drug discovery, drug testing, or administered to a human for the treatment or prevention of a disease or condition.
• the NME7-AB or NME7-X1 should have the human sequence or a sequence at least 80% identical to the native human sequence.
• a non-human primate-non-primate chimera could be created to avoid ethical concerns.
• the sequence of the NME protein could be human or the sequence of the non-human primate because of the high sequence homology.
• the NME7 sequence should be that of the mammal whose stem cells are to be inserted into the host morula or blastocyst.
• stem cells that are not in the earliest naive state but in a later naive state.
• NME1 dimers would be used in to culture the stem cells in the methods described above.
• Figure 18 shows a heat map generated from RNA-SEQ experiments. Human embryonic stem cells that had been derived and grown in FGF so they were in the primed state, were then transferred to the culture system described above wherein the added growth factor was either NME7-AB or NME1 dimers.
• the heat map of gene expression shows that FGF grown primed state cells have a completely different gene expression signature from that of NME7-AB grown cell or NME1 grown cells, indicating that they are different from primed cells and are naive.
• NME7 and NME1 gorwn cells are in the naive state is that iPS cell generation in NME7-AB, NME1 dimers or NME7-X1 is much more efficient than iPS generation in FGF based media.
• Figure 19A-19C shows that reprogramming somatic cells to become induced pluripotent stem cells (iPS cells) in FGF-based media has very low efficiency. Stem cell colonies are visualized by staining with alkaline phosphatase. FGF-based media used with mouse embryonic fibroblasts (MEFs) looks relatively efficient at this early stage but only about 15% of the picked colonies proceed to become bona fide stem cell lines (Fig. 19A).
• the mouse feeder layer introduces non-human non-quantifiable and non- human species into the method which is frowned up for eventual therapeutic use of the cells or their progeny in humans.
• mTeSR is an FGF-based media that can be used feeder-free by plating cells onto Matrigel but this method is very inefficient as can be seen by the sparse and small colonies of Figure 19B.
• iPS reprogramming in NME7-AB over a layer of anti-MUCl* antibody is highly efficient with an abundance of stem cell colonies that arise quicker than FGF-based methods and grow faster (Fig. 19C).
• Fig. 19C shows that over 86% of colonies picked from NME7 -generated iPS colonies go on to become bona fide naive state iPS stem cell lines.
• Another indicator of stem cells being in the naive state is if the second X chromosome of female source cells is still active.
• One of the earliest differentiation decisions that stem cells make is which X chromosome will be turned off in female cells.
• Lysine 27 of histone 3 is tri-methylated.
• Figure 20A shows female source embryonic stem cells derived and grown in FGF media. The focal red dot in the nucleus of each cell is a fluorescent antibody binding to the tri-methylated Lysine 27 in histone 3 showing that in primed state stem cells the second X chromosome has been turned off, called XaXi.
• Figure 20B shows that after culturing these same cells in NME7-AB, the second X is reactivated (XaXa) resulting in the disappearance of the focal red dot. The same results were obtained after culture in NME1 dimers.
• XaXa reactivated resulting in the disappearance of the focal red dot.
• Figure 21A-21D shows fluorescent images of our human NME7-AB stem cells incorporating into the inner cell mass of a mouse blastocyst. Yet another indicator of naive state stem cells is if they grow without spontaneous differentiation and if they grow faster than primed state cells.
• Figure 20A-20B shows that human NME7-AB grown stem cells have a much faster growth rate than primed stem cells. They undergo a 10-20-fold expansion in 4 days, which is 2-3-times faster than primed state cells.
• NME proteins promote growth and pluripotency of embryonic and iPS cells as well as inducing cells to revert to a stem-like state or a naive state.
• the NME family member protein is NME1 or an NME protein having greater than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% sequence identity to NME1, wherein said protein is a dimer.
• the NME family member protein is NME7 or an NME protein having greater than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% sequence identity to at least one of the NME7 domains A or B and able to dimerize the MUC1* growth factor receptor.
• NME1 in dimer form NME7-AB or NME7-X1 were able to: a) fully support human ES or iPS growth and pluripotency, while inhibiting differentiation; b) revert somatic cells to a more stem-like or naive-like state; and c) produce naive state human stem cells that were able to integrate into blastocysts of other species.
• NME7-AB naive human stem cells were injected into a mouse morula at Day 2.5.
• Figure 22A-22D shows that 48 hours later when the morula has developed into a blastocyst, the human cells (yellow) are within the inner cell mass (circled) where the mouse naive cells reside. Such incorporation into the inner cell mass is required for formation of a chimeric animal.
• Figures 23A-23J and Figures 24A-24J show confocal images of other mouse blastocysts into which human NME7- nai ' ve cells were injected at Day 2.5 and also show integration into inner cell mass.
• Figures 25A-25D and Figure 26A- 26H show that primed state, FGF grown human stem cells do not incorporate into the inner cell mass of mouse blastocysts under the same conditions.
• Figures 27A-27F, Figures 28A-26J, Figures 29A-29J, and Figures 30A-30J show human NME7-AB grown naive stem cells injected at Day 2.5 into a mouse morula, are found concentrated in the inner cell mass of the blastocyst 48 hours later at Day 4.5.
• NME is a universal stem cell growth factor
• NME7 and truncated forms are universal stem cell and pluripotency growth factors across many species. For example, we have been able to proliferate rhesus macaque and crab-eating macaque stem cells, including embryonic and iPS stem cells using human NME7-AB. We have also succeeded in generating iPS cells in rhesus and crab-eating macaque using NME7-AB along with reprogramming factors of Yamanaka or Thomson, in the absence of feeder cells using an anti-MUCl* antibody, MN-C3, to facilitate surface attachment. The sequence of both macaque NME7 is 98% identical to human NME7 and its target growth factor receptor, MUC1* extracellular domain is 90% identical to human PSMGFR.
• Figures 31A-31F show photograph of control plates containing fibroblasts from crab-eating macaques in culture in NME7-AB for 6 days but without transduction of Yamanaka pluripotency factors OCT4, SOX2, NANOG and c-Myc.
• Figures 32A-32C, Figures 33A-33F, and Figures 34A-34F show fibroblasts from crab-eating macaques being reprogrammed by Yamanaka factors and NME7-AB. After Day6, emerging iPS colonies are transferred to an anti-MUCl* antibody surface, where they continue to expand until about Day 14-17 when individual clones are picked and expanded.
• embryonic or iPS stem cells are proliferated, maintained or generated in other non-human species by culturing fertilized eggs or embryos or reprogramming cells using a media that contains an NME protein.
• the NME protein may be NME1 dimers or NME6 dimers, NME7, NME7-AB or NME7-X1.
• the sequence of the NME protein may be the human sequence.
• the sequence of the NME protein is the sequence of the non-human species that the stem cells, embryo or cells for reprogramming came from.
• the sequence of NME7 in the non-human species is too diverse from that of human NME7, then better results are obtained using an NME protein having a sequence that has higher sequence identity to the non-human target species or an NME protein having the sequence of the native NME protein of that species.
• the NME protein is NME1 or NME6 dimers, NME7, NME7-AB or NME7-X1.
• a method for determining which species a particular NME protein will work in as a pluripotency growth factor is to test whether or not the NME protein binds to a PSMGFR peptide having the sequence of the target species. If the NME protein binds to the PSMGFR peptide of the target species, then the NME protein will work as a stem cell growth factor or pluripotency factor.
• non-human stem cells are proliferated or maintained by culturing the cells in a media containing an NME protein of that non-human species.
• non-human stem cells are proliferated or maintained by culturing the cells in a media containing an NME protein having a sequence that is at least 40% homologous to that of the non-human species stem cells.
• non-human stem cells are proliferated or maintained by culturing the cells in a media containing an NME protein that is able to bind to a peptide having the sequence of that species' MUC1* extracellular domain also called the PSMGFR peptide.
• the non-human stem cells are proliferated or maintained by culturing the cells in a media containing a human sequence NME protein.
• the NME protein may be NME1 or NME6 dimers, NME7, NME7-AB or NME7-X1.
• non-human stem cells are generated using reprogramming technology such as introducing combinations of the genes or gene products that include Oct4, Sox2, Klf4, c-Myc, Nanog, Lin28, in the presence of a media containing an NME protein of that non-human species.
• non-human stem cells are generated using reprogramming technology in a media containing an NME protein having a sequence that is at least 40% homologous to that of the non-human species stem cells.
• non-human stem cells are generated using reprogramming technology in a media containing an NME protein that is able to bind to a peptide having the sequence of that species' MUC1* extracellular domain also called the PSMGFR peptide.
• the non-human stem cells are generated using reprogramming technology in a media containing a human sequence NME protein.
• the NME protein may be NME1 or NME6 dimers, NME7, NME7-AB or NME7-X1.
• non-human embryonic stem cells are generated by culturing fertilized eggs or embryos in a media containing an NME protein of that non- human species.
• non-human embryonic stem cells are generated by culturing the fertilized eggs or embryos in a media containing an NME protein having a sequence that is at least 40% homologous to that of the non-human species stem cells.
• non-human embryonic stem cells are generated by culturing fertilized eggs or embryos in a media containing an NME protein that is able to bind to a peptide having the sequence of that species' MUC1* extracellular domain also called the PSMGFR peptide.
• the non-human embryonic stem cells are generated by culturing fertilized eggs or embryos in a media containing a human sequence NME protein.
• the NME protein may be NME1 dimers, or NME6 dimers, NME7, NME7-AB or NME7-X1.
• the non-human species when it is desired to have human stem cells incorporate into the developing blastocyst or embryo of a non-human species whose NME protein or MUC1* protein have low sequence identity to human, the non-human species is humanized.
• the fertilized or unfertilized egg, or stem cells of the non-human species are transduced with vectors that enable the expression of human NME protein and or human MUC1* protein, wherein their expression may be inducible or repressible.
• NME1 dimers or NME7 can be introduced into an animal by a variety of methods. It can be mixed in with the tumor cells prior to implantation, or it can be injected into the animal bearing the tumor.
• a transgenic animal is generated that expresses human NME7 or a fragment thereof.
• the NME7 may be carried on an inducible promoter so that the animal can develop naturally, but expression of the NME7 or the NME7 fragment can be turned on during implantation of human tumors or for the evaluation of drug efficacies or toxicities.
• the NME7 species that is introduced to the test animal is NME7-AB.
• a transgenic animal can be made wherein the animal expresses human MUC1, MUC1*, NME7 and/or NME1 or NME2, a variant of NME1 or NME2 that prefers dimer formation, single chain constructs or other variants that form dimers.
• NME proteins and MUC1 are parts of a feedback loop in humans, wherein expression of one can cause upregulation of the other, it could be advantageous to generate transgenic animals that express human NME protein(s) and MUC1 or the cleaved form MUC1*.
• a natural or an engineered NME species can be introduced into animals, such as mice, by any of a variety of methods, including generating a transgenic animal, injecting the animal with natural or recombinant NME protein or a variant of an NME protein, wherein NMEl, NME6 and NME7 proteins or variants are preferred and NMEl, NME6 and NME7 proteins or variants that are able to dimerize MUC1*, specifically the PSMGFR peptide, are especially preferred.
• the NME species is a truncated form of NME7 having an approximate molecular weight of 33kDa.
• the NME7 species is devoid of the DM10 domain of its N-terminus.
• the NME7 species is human.
• NME family proteins especially NMEl, NME6 and NME7 are expressed in human cancers where they function as growth factors that promote the growth and metastasis of human cancers. Therefore, human NME protein or active forms of NME protein should be present for proper growth, evolution and evaluation of human cancers and for determining their response to compounds, biologicals or drugs.
• the protein expression may be linked to an inducible genetic element such as a regulatable promoter.
• the NME protein that is introduced into an animal to increase engraftment of human stem cells or cancer cells is human NME7.
• the NME7 protein is a fragment that is ⁇ 33kDa.
• the NME protein is human NME7-AB.
• chromatin re-arrangement factors MBD3 and CHD4 were recently reported to block the induction of pluripotency (Rais Y et al, 2013).
• siRNA suppression of the chromatin re- arrangement factors MBD3 and CHD4 were shown to be key components of a method for reverting human primed stem cells to the naive state.
• Transcription factors BRD4 and co-factor JMJD6 reportedly suppress NME7 and up-regulate NMEl (Lui W et al, 2013).
• NMEl dimers also called NM23-H1, bacterial NMEl, NME6, NME7-X1 and NME7-AB promote the growth of embryonic stem cells and induced pluripotent stem cells, inhibit their differentiation and maintain them in a naive state as evidenced by global genetic analysis, having both X chromosomes in the active state if stem cell donor is human and by having the ability to form teratomas in a host animal.
• a transgenic animal that expresses human NME7 or NME7-AB is generated. Because NMEl, human or bacterial, and NME7 inhibit differentiation of stem cells, it may be advantageous to use technology in which the timing of expression of the NME protein, preferably NME7 or NME7-antibody, in the transgenic animal can be controlled. It would be advantageous to have the human NME7 on an inducible promoter, for example to avoid potential problems of NME7 expression during development of the animal. Methods for making the expression of foreign genes inducible in the host animal are known to those skilled in the art. Expression of NME7 or NME7-AB can be inducible using any one of many methods for controlling expression of transgenes that are known in the art.
• the expression or timing of expression, of NME7 may be controlled by the expression of another gene which may be naturally expressed by the mammal.
• the NME7 or NME7 variant may be expressed in a certain tissue, such as the heart.
• the gene for NME7 is then operably linked to the expression of a protein expressed in the heart such as MHC.
• MHC protein expressed in the heart
• the expression of NME7 is turned off when and where the MHC gene product is expressed.
• human NME6 or NME7 in a non- human mammal can be controlled by genes expressed in mammary tissues.
• human NME6 or human NME7 is expressed from the prolactin promoter, or a similar gene. In this way, it would be possible to induce or repress expression of the human NME protein in a site specific manner.
• an animal model for the development of cancer metastasis is generated by making a transgenic animal that expresses human NME7 or more preferably NME7-AB.
• the NME7 is on an inducible promoter to allow the animal to correctly develop.
• a metastatic animal model preferably rodent, is made by making a transgenic animal that expresses human NME or human NME7 or NME7-AB.
• the animal is a transgenic animal in which the kinases inhibited by 2i or 5i are suppressed via inducible promoters or agents to suppress the kinases are administered to the test animal.
• Metastatic animal models are then used to study the basic science of the development or progression of cancers as well as to test the effects of compounds, biologicals, drugs and the like on the development of cancers.
• an animal transgenic for human NME1, bacterial NME1 or human NME7, preferable NME7-AB is implanted or engrafted with human cells which may be stem cells or progenitor cells or incipient mesodermal cells.
• human cells which may be stem cells or progenitor cells or incipient mesodermal cells.
• an animal such as a mouse, pig, sheep, bovine animals and primates, that will grow human tissue in its heart, liver, skin or other organ.
• One method for doing so is to generate a kind of chimeric animal by implanting human stem cells into an animal that has been made to express human NME7 or human NME7-AB.
• the human stem cells or progenitor cells can be implanted at various stages of the animal's development, including in vitro and in vivo, at the blastocyst, embryo or fetus stage of development. Because NME7 inhibits differentiation, the NME7 or NME7-AB transgene would be linked to a method by which the timing of its expression is controllable.
• NME7 inducible or repressible
• Methods are known to those skilled in the art which could be used such that expression of the human NME7 or NME7-AB is turned off or decreased at times or locations where it is desirable to have differentiation or maturation occur.
• One method for making the transgene, preferably NME7, inducible or repressible is to link its expression or repression to the expression of a gene that is only expressed later in development. In such cases, one would make a transgenic animal in which expression of NME7 or NME7-AB is linked to the expression of a later gene expressed in heart or in heart progenitor cells.
• the expression or timing of expression, of NME7 is controlled by the expression of another gene which may be naturally expressed by the mammal.
• the NME7 or NME7 variant may be expressed in a certain tissue, such as the heart.
• the gene for the NME7 is then operably linked to the expression of a protein expressed in the heart such as MHC.
• the expression of NME7 is decreased or turned off when and where the MHC gene product is expressed.
• the expression of human NME1 or NME7 in a non-human mammal can be controlled by genes expressed in mammary tissues.
• human NME1 or human NME7 can be expressed or repressed by the prolactin promoter, or a similar gene.
• an animal transgenic for human NME7 or NME7-AB can be allowed to grow to a point, then implanted with human stem or progenitor cells, where they proliferate because of contact with human NME protein.
• the expression of the human NME is then turned off such that a specific organ or part of an organ in the animal would develop as a human tissue.
• the invention contemplates many applications of animals transgenic for human NME1, bacterial NME1 or human NME7, or NME7-AB.
• human stem or progenitor cells are implanted in the NME transgenic animal or germ cells of what will be a transgenic animal. Expression of the NME may be inducible or repressible. Depending on the site and timing of the implantation of the stem or progenitor cells, the resulted animal can be made to express human heart, liver, neuronal cells or skin.
• human tissues can be generated in a transgenic non-human mammal, wherein the mammal expresses human MUC1 or MUC1* or NME protein in the germ cells or somatic cells, wherein the germ cells or somatic cells contain a recombinant human MUC1 or MUC1* or NME gene sequence introduced into said mammal, wherein the expression of the gene sequence can be induced or repressed either by introduction of an external composition or by linking its expression or repression to the expression or repression of a naturally occurring gene of the host animal.
• Stem cells or progenitor cells that are xenogeneic in origin to the non-human mammal are transferred to the transgenic animal such that the gene is induced to be expressed so as to multiply the stem or progenitor cells and then repressing the gene expression so as to generate tissue from the xenografted stem cells.
• One method by which repression of the transgene is carried out is by contacting the stem cell or progenitor cells with a tissue differentiation factor. Transgene repression is also carried out naturally in the mammal in response to naturally produced host tissue differentiation factors.
• These animals can be used for drug discovery. They can also be used for toxicity testing, to use an animal to determine the effects of a compound, biological or drug on human tissue or on the development of human tissue.
• the transgenic animal implanted with human stem or progenitor cells is used to grow human tissue for transplant into a human patient.
• the stem or progenitor cells that are implanted are from a patient who will be the recipient of the human tissue harvested from the transgenic animal.
• the MUC1, MUC1* or NME protein expression may be induced until the amount of transferred stem or progenitor cells are sufficiently large.
• the MUC1, MUC1* or NME protein expression may then be shut down by injecting the host mammal with a substance that represses the expression of MUC1, MUC1* or NME protein.
• the population of stem or progenitor cells may be induced to differentiate by either natural methods such as by the expression in the mouse of a differentiation inducing factor for a particular tissue or organ type, or chemical or protein substances may be injected into the host at the site of stem or progenitor cell transference to cause differentiation to desired tissue type.
• differentiation/transformation agents for endoderm cell tissue may include without limitation the following agents: hepatocyte growth factor, oncostatin-M, epidermal growth factor, fibroblast growth factor-4, basic-fibroblast growth factor, insulin, transferrin, selenius acid, BSA, linoleic acid, ascorbate 2-phosphate, VEGF, and dexamethasone, for the following cell types: liver, lung, pancreas, thyroid, and intestine cells.
• differentiation/transformation agents for mesoderm tissue include without limitation the following agents: insulin, transferrin, selenous acid, BSA, linoleic acid, TGF- ⁇ , TGF- 3, ascorbate 2-phosphate, dexamethasone, ⁇ -glycerophosphate, ascorbate 2- phosphate, BMP, and indomethacine, for the following cell types: cartilage, bone, adipose, muscle, and blood cells.
• differentiation/transformation agents for ectoderm tissue include without limitation the following agents: dibutyryl cyclin AMP, isobutyl methylxanthine, human epidermal growth factor, basic fibroblast growth factor, fibroblast growth factor- 8, brain-derived neurotrophic factor, and/or other neurotrophic growth factor, for the following cell types: neural, skin, brain, and eye cells.
• Regulators of NME protein or downstream effectors of NME protein can substitute for the NME protein
• MUC1* MUC1 transmembrane protein
• NME proteins exert their effects is by being transported to the nucleus where they function directly or indirectly to stimulate or suppress other genes. It has been previously reported (Boyer et al, 2005) that OCT4 and SOX2 bind to the promoter sites of MUC1 and its cleavage enzyme MMP16. The same study reported that SOX2 and NANOG bind to the promoter site of NME7. We conclude, on the basis of our experiments that these 'Yamanaka' pluripotency factors (Takahashi and Yamanaka, 2006) up-regulate MUC1, its cleavage enzyme MMP16 and its activating ligand NME7.
• NME7 suppresses NME7, while its co-factor JMJD6 up-regulates NMEl (Thompson et al), which we have demonstrated is a self-regulating stem cell growth factor that is expressed later than NME7 in embryogenesis.
• siRNA suppression of Mbd3 or Chd4 greatly reduced resistance to iPS generation (Rais Y et al 2013 et al.) and was able to maintain stem cells in the naive state.
• Evidence presented here shows that there is a reciprocal feedback loop wherein NME7 suppresses BRD4 and JMJD6, while also suppressing inhibitors of pluripotency Mbd3 and CHD4.
• NME7 up-regulates SOX2 (>150X), NANOG ( ⁇ 10X), OCT4 ( ⁇ 50X), KLF4 (4X) and MUC1 (10X).
• NME7 up-regulates cancer stem cell markers including CXCR4 (-200X) and E-cadherin (CDH1).
• CXCR4 -200X
• E-cadherin CDH1
• the invention contemplates substituting downstream effectors of NME7 for NME7.
• agents that suppress MBD3, CHD4, BRD4 or JMJD6 can be substituted in any of the methods described herein, for NME7, which we have shown suppresses MBD3, CHD4, BRD4 or JMJD6.
• the present invention discloses methods for generating, maintaining or proliferating human stem cells in a naive state and using the resultant cells in a non-human host animal or a fertilized egg, blastocyst or embryo of a non-human animal in order to generate chimeric organisms or animals that are comprised of DNA of the non-human host and DNA of the human donor stem cells.
• Limbs, nerves, blood vessels, tissues, organs, or factors made in them, or secreted from them, of a chimeric species that contains some human DNA can be harvested for several uses including transplant into humans, administration into humans for medicinal benefit, including anti-aging, and scientific experiments including drug testing and disease modeling.
• human naive state stem cells are generated, maintained or proliferated by contacting human primed state stem cells with an NME family protein or an agent that dimerizes the MUC1* growth factor receptor.
• human naive state stem cells are generated, maintained or proliferated by inducing somatic cells to revert to a less mature state, such as through the use of iPS technologies, wherein cells are reprogrammed in the presence of an NME family protein or an agent that dimerizes the MUC1* growth factor receptor.
• human naive state stem cells are generated, maintained or proliferated by culturing cells obtained from a human embryo, blastocyst or fertilized egg in the presence of an NME family protein or an agent that dimerizes the MUC1* growth factor receptor.
• NME proteins or agents that dimerize MUC1* convert human primed state stem cells to a naive state.
• Said NME proteins or agents that dimerize MUC1* also support the derivation of naive state embryonic stem cell lines from cells taken from a human embryo.
• Said NME proteins or agents that dimerize MUC1* support the generation of naive state induced pluripotent stem cells lines, wherein differentiated cells are reprogrammed to a stem cell state.
• the NME family protein is NME7.
• the NME family member is NME7-AB or NME7-X1 or other isoform or truncation of NME7.
• the NME family member is dimeric NM23, aka NME1.
• the NME family member is NME6.
• the agent that dimerizes MUC1* is an antibody that binds to the PSMGFR peptide of the MUC1* extracellular domain.
• naive state human stem cells generated by a method that includes contacting human cells with NME1 dimers, NME6 dimers, NME7, NME7-AB or NME7-X1, then inserting or injecting said cells into a morula, blastocyst, embryo or fetus of a non-human animal.
• Chimeric animals are generated that have some tissues, organs or other body parts that are at least in part of human origin and emanated from the human stem cells inserted into the blastocyst or embryo. The tissues, organs or other body parts are harvested from the host animal when completely developed or at any earlier stage of development.
• the tissues, organs, or body parts or factors generated by these human body parts are then transplanted into or administered to a human recipient in need of a new organ or in need of regenerative properties of factors secreted by the human tissues or organs of the host non-human.
• the cells of the non-human animal have been genetically altered or treated for example with biochemical inhibitors such that the developing non-human animal is not able to generate certain tissues or organs.
• the chimeric animal would generate certain tissues or organs that emanate from, or have significant contribution from, the human donor stem cells and will be partially or entirely human.
• the donor stem cells are from a donor in need of the tissue or organ that is generated in the non-human animal and at some stage of development or after the animal is mature, the tissue or organ is harvested and transplanted into the donor human.
• the donor stem cells are from a donor who is not the intended recipient of the tissues, organs or other material generated in the chimeric species.
• the donor stem cells are iPS cells and in another aspect the stem cells are embryonic stem cells.
• stem cells are cultured in a media containing an NME protein.
• the NME protein can be dimeric NME1, dimeric NME6, NME7, dimeric B domain of NME7, NME7-X1 or NME7-AB.
• the NME protein is dimeric NME1.
• the NME protein is NME7-X1.
• the NME protein is NME7-AB.
• stem cell attachment to a surface is facilitated by coating said surface with an anti-MUCl* antibody wherein the antibody has the ability to bind to a peptide comprising at least 15 contiguous amino acids of the PSMGFR sequence.
• stem cell attachment to a surface is facilitated by coating said surface with an NME protein, which in some cases may be histidine-tagged and coated onto a surface presenting a metal-chelate-metal moiety such as nitrile-tri-acetic acid- Nickel, aka NTA-Ni++.
• NME protein which in some cases may be histidine-tagged and coated onto a surface presenting a metal-chelate-metal moiety such as nitrile-tri-acetic acid- Nickel, aka NTA-Ni++.
• stem cell attachment to a surface is facilitated by coating said surface with an integrin or integrin fragment wherein the integrin is vitronectin, fibrinectin, collagen and the like.
• stem cell attachment to a surface is facilitated by coating said surface with peptides, small molecules or polymers.
• a Rho I kinase inhibitor is added to the culture media to further enhance surface attachment.
• the generation, induction or maintenance of stem cells is achieved according to parts or all of the methods described above.
• an NME protein whose sequence is that of native pig NME6, NME1, NME7, NME7-X1 or NME7- AB.
• the surface may be advantageous to coat the surface with an antibody selected for its ability to bind to a peptide comprising at least 15 contiguous amino acids of the PSMGFR region of the MUC1* extracellular domain, wherein the sequence of the peptide is the native sequence of pig MUC1* extracellular domain.
• Serum-free Minimal Media 500 mL includes the following components:
• NME media Culturing Stem Cells in NME media.
• Stem cells can be grown in suspension in an NME media or on cell culture plates. If stem cells are being cultured on cell culture plates coated with an anti-MUCl* antibody such as MN-C3, then a Rho Kinase inhibitor was added such as Y-26732 to a final concentration of lOuM.
• Cell culture plates were prepared at least 1 day before stem cell plating.
• Cell culture plates were coated with a solution containing -12.5 ug/mL of MN-C3 anti-MUCl* antibody. The coated plates were incubated overnight at 4 degrees before stem cells were plated onto to them, and without a pre-wash step.
• Stem cells were plated at densities broadly corresponding to the density obtained when 100,000 cells to 300,000 cells are plated per well of a 6-well plate.
• Cells are suspended in NME Media to which was added a Rho kinase inhibitor.
• Cells were incubated undisturbed in a 5%C02/5%02 incubator for 48 hours. Thereafter, media was changed every 24 or 48 hours until cells reached -80% confluency (Fig. 20B).
• Cells were dissociated to single cells using Trypsin/EDTA or TryplE. Repeat process from beginning to expand.
• Fibroblast Media was changed to NME Media (Example 2).
• Fibroblasts were transduced with master ⁇ programming factors, such as Yamanaka factors or Thomson factors, according to standard protocol. Any method of introducing nucleic acids that will cause expression of OCT4, SOX2, NANOG or KLF4 and c-Myc if desired will suffice.
• Non-integrating viral delivery systems such as lenti virus, Sendai virus, gamma retrovirus or transposons such as Sleeping Beauty.
• Day 3 Change media with 2mL to 4 mL per well of NME Media, without Rho kinase inhibitor.
• Day 5 Change media with 2mL to 4 mi_ per well of NME Media, without Rho nase inhibitor.
• Cell culture plates were prepared with an anti-MUCl* antibody such as MN-C3 coated onto cell culture plates at a concentration of 3.25ug/mL to 24ug/mL with about 12.5ug/mL preferred and incubate the antibody coated plates at 4°C overnight.
• an anti-MUCl* antibody such as MN-C3 coated onto cell culture plates at a concentration of 3.25ug/mL to 24ug/mL with about 12.5ug/mL preferred and incubate the antibody coated plates at 4°C overnight.
• reprogrammed cells were then plated at 5x10 - 1x10 " per well. From this point onward, cells are cultured in an NME media with a Rho kinase inhibitor such as lOuM Y-26732 added and incubated for first 48 hours undisturbed in 5% C0 2 /5%0 2 - Day 9 and onward: Change media daily using the same NME media throughout plus Rho kinase inhibitor such as lOuM Y-26732.
• Rho kinase inhibitor such as lOuM Y-26732
• IPS cells were also generated from blood using same process as Day 1 and onward.
• fibroblast cells were cultured in Minimal Media to which was added recombinant human NME1/NM23 dimers, bacterial HSP593 NMEl dimers or human recombinant NME7-AB.
• the fibroblasts were cultured in their normal media, which is for 500 mL, 445 mL DMEM high glucose base media, 5 mL GlutaMAX and 50 mL of fetal bovine serum (FBS).
• FBS fetal bovine serum
• FIG. 2 shows a graph of RT-PCR measurements of the expression of genes that code for the chromatin rearrangement factors BRD4, JMJD6, MBD3 and CHD4.
• Figure 3 shows a graph of RT-PCR measurements of the expression of pluripotency genes, genes that code for chromatin rearrangement factors BRD4, JMJD6, MBD3 and CHD4 and NME proteins.
• 'minus ROCi' refers to cells that became nonadherent and floated off the surface. The morphology of the cells also completely changed so that they no longer were recognizable as fibroblasts and look like stem cells (Figs. 8-11).
• NME7-AB cultured human stem cells incorporate into inner cell mass of mouse morula.
• Mouse eggs were fertilized in vitro.
• 10 human stem cells that had been generated in and cultured in NME7-AB were separately injected into the fertilized eggs.
• Day 2.5 is before the inner cell mass has formed.
• morulas were stained with a fluorescent antibody that stains human Tra 1-81.
• arrows point to human naive NME7-AB cells that have incorporated into the inner cell mass, which indicates the development of a chimeric animal.
• FIGS 22A-22D, Figures 23A-23J and Figures 24A-24J which show confocal images of other mouse blastocysts into which human NME7-nai ' ve cells were injected at Day 2.5 and also show integration into inner cell mass of blastocyst at Day 4.5.
• Figures 27A-27F, Figures 28A-26J, Figures 29A-29J, and Figures 30A-30J which also show human NME7-AB grown naive stem cells injected at Day 2.5 into a mouse morula, are found concentrated in the inner cell mass of the blastocyst 48 hours later at Day 4.5.
• FGF- grown primed state human stem cells were injected into fertilized eggs at Day 2.5 and at Day 4.5 stained with anti-human Tra 1-81 and also with CDX2 which stains non- inner cell mass region called the Trophoectoderm.
• Figures 25A-25D and 26A-26H show that these primed state cells did not incorporate into the inner cell mass but are in the trophectoderm.
• NME7-AB cultured human stem cells were transfected with a red fluorophore called tomato red or TDtomato. These fluorescent human naive cells were then injected into Day 2.5 fertilized mouse eggs and imaged at Day 4.5. The morulas were also stained with DAPI and a fluorescent antibody that stains the trophectederm. See Figures 27A-27F, Figures 28A-26J, Figures 29A-29J, and Figures 30A-30J show human NME7-AB grown naive stem cells injected at Day 2.5 into a mouse morula, are found concentrated in the inner cell mass of the blastocyst 48 hours later at Day 4.5. Arrows indicate where the human cells incorporated into the inner cell mass, indicating formation of a chimeric animal.
• Example 7 Non-human primate species stem cells were generated in NME7-AB media in the absence of bFGF. Rhesus macaque and crab-eating macaque fibroblasts were transfected with core pluripotency factors in a media containing NME7-AB and in the absence of bFGF or feeder cells. In this case the Yamanaka factors Oct 4, Sox2, Klf4, and c-Myc were used but could be substituted for other pluripotency factors, either genes or gene products. Between Day 5 and 7 post gene transfection, when cells began to detach from the surface, cells were re-plated onto a surface coated with an anti-MUCl* antibody, in this case MN-C3.
• an anti-MUCl* antibody in this case MN-C3.
• Figures 31A-31F show photograph of control plates containing fibroblasts from crab- eating macaques in culture in NME7-AB for 6 days but without transduction of Yamanaka pluripotency factors OCT4, SOX2, NANOG and c-Myc.
• Figures 32A-32C, Figures 33A-33F, and Figures 34A-34F show fibroblasts from crab-eating macaques being reprogrammed by Yamanaka factors and NME7-AB. After Day6, emerging iPS colonies are transferred to an anti-MUCl* antibody surface, where they continue to expand until about Day 14-17 when individual clones are picked and expanded.
• Klf2 is an essential factor that sustains ground state pluripotency. Cell Stem Cell. Jun 5;14(6):864-72.
• MUCl* is a determinant of
• trastuzumab Herceptin resistance in breast cancer cells
• CXCR4 axis cell trafficking in the cancer stem cell niche of head and neck squamous cell carcinoma. Oncol. Rep. 2013 Jun;29(6):2325-31.
• nucleoside diphosphate kinase A a metastasis suppressor. Proteins. 2002, 46:340- 342.

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