Patent Application
20180235193
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.
Currently, there is considerable research in the area of making chimeric animals by inserting stem cells into a developing embryo or blastocyst. The end goal is to be able to generate human organs or tissue in an animal such as a cow, sheep or pig, wherein the resultant organs or tissues would be used for transplant into humans. The donor stem cells could be from a patient in need of a new liver, heart or the like. If successful, the technology would replace the need for an organ donor; currently most patients die before a suitable organ donor is identified.
Technology now exists for: 1) making a knockout animal that will not develop a heart, a pancreas, lungs or other organ or tissue; 2) making a chimeric animal by injecting donor stem cells into a morula or blastocyst of another; 3) making a chimeric animal wherein the fertilized or unfertilized egg has had gene(s) knocked out such that they no longer have the capacity to form a particular organ or tissue, but where the donor stem cells do have the capacity to form that particular organ or tissue, so chimeric animal may have up to 100% DNA of the donor stem cells in the targeted organ. These techniques have been shown to work for across species applications such as mouse to rat and rat to mouse. The size of the organ generated depends on the size of the recipient animal. However, rat and mouse are close species.
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.
One problem is that, at least in the rodent examples, only naïve state stem cells were able to incorporate into the morula or inner cell mass and form chimeras. Primed state stem cells were not able to.
The present invention solves the problem by providing a method for generating human stem cells in the naïve 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.
I. In one aspect, 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. In this method, an animal that expresses some human DNA or tissues is generated by introducing human naïve state stem cells into a non-human cell or cells. In one aspect 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. For ethical concerns or other reasons, it may be advantageous to generate chimeric animals wherein the integrating naïve state stem cells are also non-human, but of a different species than the recipient cell, cells, morula, blastocyst or embryo. In the method above, the agent that maintains stem cells in the naïve state or reverts primed stem cells to the naïve 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.
In the method above, 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. Alternatively, 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.
In the method above, the agent that maintains stem cells in the naïve state or reverts primed stem cells to the naïve 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.
In this method, 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.
In another aspect, 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.
In this method, in step (iii), 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.
II. In other aspects, 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. In other cases such animals would grow organs comprising at least some human cells. In other cases such animals would grow organs comprised entirely of human cells. In yet other cases, 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.
In one aspect, the invention is directed to a method for generating human tissues in a non-human animal comprising: (i) generating human naïve 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 naïve state stem cells may be generated using NME7, NME7-AB, NME7-X1 or dimeric NME1. The naïve stem cells may be iPS cells that have been reprogrammed in a medium containing NME7, NME7-AB, NME7-X1 or dimeric NME1. Or, the naïve 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. Further, the agent that maintains stem cells in the naïve state or reverts primed stem cells to the naïve 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. In one aspect of the invention, 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. In some cases, the NME protein may be present in serum-free media as the single growth factor.
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. We injected human naïve state stem cells into a mouse morula and showed they incorporated into the inner cell mass. In a specific example, human naïve 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 invention will become more fully understood from the detailed description given herein below, and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein;
FIG. 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-MUC1* antibody, in this case MN-C3 and by transducing the cells with core pluripotency genes, in this case Oct4, Sox2, Klf4, and c-Myc. 50,000 fibroblasts from crab-eating macaques were plated per well of a 6-well plate. Emerging colonies with stem-like morphology are circled.
As used herein, the “MUC1*” extra cellular domain is defined primarily by the PSMGFR sequence (GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:6)). Because the exact site of MUC1 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 MUC1* extra cellular domain may vary at the N-terminus.
As used herein, the term “PSMGFR” is an acronym for Primary Sequence of MUC1 Growth Factor Receptor as set forth as GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:6). In this regard, the “N-number” as in “N-10 PSMGFR”, “N-15 PSMGFR”, or “N-20 PSMGFR” refers to the number of amino acid residues that have been deleted at the N-terminal end of PSMGFR. Likewise “C-number” as in “C-10 PSMGFR”, “C-15 PSMGFR”, or “C-20 PSMGFR” refers to the number of amino acid residues that have been deleted at the C-terminal end of PSMGFR.
As used herein, the “extracellular domain of MUC1*” refers to the extracellular portion of a MUC1 protein that is devoid of the tandem repeat domain. In most cases, MUC1* is a cleavage product wherein the MUC1* portion consists of a short extracellular domain devoid of tandem repeats, a transmembrane domain and a cytoplasmic tail. The precise location of cleavage of MUC1 is not known perhaps because it appears that it can be cleaved by more than one enzyme. The extracellular domain of MUC1* will include most of the PSMGFR sequence but may have an additional 10-20 N-terminal amino acids.
As used herein, “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 H1, H2 and so on. Herein, the terms NM23 and NME are interchangeable. Herein, terms NME1, 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. coli or are more soluble than the native sequence protein. For example, 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. “NME1” 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 NME1 dimers.
Various artificially created NME1 dimers are disclosed in PCT/US2012/036975, filed May 8, 2012, titled “Genetically Engineered Growth Factor Variants”, the contents of which are incorporated by reference herein as regards the dimeric growth factors that are disclosed therein.
NME7 as referred to herein is intended to mean native NME7 having a molecular weight of about 42 kDa, a cleaved form having a molecular weight between 25 and 33 kDa, 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.
As used herein, “NME1 dimers” also known as “NM23-H1 dimers” can be two NME1 proteins that are non-covalently bound to each other, two NME1 proteins covalently linked to each other or two NME1 proteins that are genetically fused together, including via a linker. NME1 dimers can be genetically engineered by making a DNA construct comprised of two NME1 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. Other 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.
As used herein, an “an agent that maintains stem cells in the naïve state or reverts primed stem cells to the naïve state” refers to a protein, small molecule or nucleic acid that alone or in combination maintains stem cells in the naïve 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.
As used herein, 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 50 Da and 2000 Da, more preferably between 150 Da and 1000 Da, still more preferably between 200 Da and 750 Da.
As used herein, in reference to an agent being referred to as a “natural product”, it may be chemical molecule or a biological molecule, so long as the molecule exists in nature.
As used herein, “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.
As used herein, FGF, FGF-2 or bFGF refer to fibroblast growth factor.
As used herein, “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.
As used herein, the term “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.
Sequence Listing Free Text
As regards the use of 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.
describes full-length human MUC1 Receptor (Mucin 1 precursor, Genbank Accession number: P15941).
SEQ ID NOS:2, 3 and 4 describe N-terminal MUC-1 signaling sequence for directing MUC1 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).
tgtcagtgccgccgaaagaactacgggcagctggacatctttccagcccgggatacctaccatcctatgagcgagta ccccacctaccacacccatgggcgctatgtgccccctagcagtaccgatcgtagcccctatgagaaggtttctgcaggtaacggtggc agcagcctctcttacacaaacccagcagtggcagccgcttctgccaacttg (SEQ ID NO:10) describes human MUC1 cytoplasmic domain nucleotide sequence.
CQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVS AGNGGSSLSYTNPAVAAASANL (SEQ ID NO:11) describes human MUC1 cytoplasmic domain amino acid sequence.
gagatcctgagacaatgaatcatagtgaaagattcgttttcattgcagagtggtatgatccaaatgcttcacttcttcgac gttatgagcttttattttacccaggggatggatctgttgaaatgcatgatgtaaagaatcatcgcacctttttaaagcggaccaaatatgata acctgcacttggaagatttatttataggcaacaaagtgaatgtcttttctcgacaactggtattaattgactatggggatcaatatacagctc gccagctgggcagtaggaaagaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataa acaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagacc ctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactg ctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcag cgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaa atttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggtatgttgaatacactatattcagtacattttgttaataggagag caatgtttattttcttgatgtactttatgtatagaaaataa (SEQ ID NO:12) describes human NME7 nucleotide sequence (NME7: GENBANK ACCESSION AB209049).
DPETMNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRT FLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAI SKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEIL RDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELF FPSSGGCGPANTAKFTNCTCCIVKPHAVSEGMLNTLYSVHFVNRRAMFIFLMYFMY RK (SEQ ID NO:13) describes human NME7 amino acid sequence (NME7: GENBANK ACCESSION AB209049).
atggtgctactgtctactttagggatcgtctttcaaggcgaggggcctcctatctcaagctgtgatacaggaaccatgg ccaactgtgagcgtaccttcattgcgatcaaaccagatggggtccagcggggtcttgtgggagagattatcaagcgttttgagcagaaa ggattccgccttgttggtctgaaattcatgcaagcttccgaagatcttctcaaggaacactacgttgacctgaaggaccgtccattctttgc cggcctggtgaaatacatgcactcagggccggtagttgccatggtctgggaggggctgaatgtggtgaagacgggccgagtcatgct cggggagaccaaccctgcagactccaagcctgggaccatccgtggagacttctgcatacaagttggcaggaacattatacatggcag tgattctgtggagagtgcagagaaggagatcggcttgtggtttcaccctgaggaactggtagattacacgagctgtgctcagaactgga tctatgaatga (SEQ ID NO:14) describes human NM23-H1 also known as NME1 nucleotide sequence (NM23-H1: GENBANK ACCESSION AF487339).
MVLLSTLGIVFQGEGPPISSCDTGTMANCERTFIAIKPDGVQRGLVGEIIKR FEQKGFRLVGLKFMQASEDLLKEHYVDLKDRPFFAGLVKYMHSGPVVAMVWEGL NVVKTGRVMLGETNPADSKPGTIRGDFCIQVGRNIIHGSDSVESAEKEIGLWFHPEEL VDYTSCAQNWIYE (SEQ ID NO:15) NM23-H1 describes amino acid sequence (NM23-H1 also known as NME1: GENBANK ACCESSION AF487339).
atggtgctactgtctactttagggatcgtctttcaaggcgaggggcctcctatctcaagctgtgatacaggaaccatgg ccaactgtgagcgtaccttcattgcgatcaaaccagatggggtccagcggggtcttgtgggagagattatcaagcgttttgagcagaaa ggattccgccttgttggtctgaaattcatgcaagchccgaagatcttctcaaggaacactacgttgacctgaaggaccgtccattctttgc cggcctggtgaaatacatgcactcagggccggtagttgccatggtctgggaggggctgaatgtggtgaagacgggccgagtcatgct cggggagaccaaccctgcagactccaagcctgggaccatccgtggagacttctgcatacaagttggcaggaacattatacatggcgg tgattctgtggagagtgcagagaaggagatcggcttgtggtttcaccctgaggaactggtagattacacgagctgtgctcagaactgga tctatgaatga (SEQ ID NO:16) describes human NM23-H1 S120G mutant nucleotide sequence (NM23-H1: GENBANK ACCESSION AF487339).
MVLLSTLGIVFQGEGPPISSCDTGTMANCERTFIAIKPDGVQRGLVGEIIKR FEQKGFRLVGLKFMQASEDLLKEHYVDLKDRPFFAGLVKYMHSGPVVAMVWEGL NVVKTGRVMLGETNPADSKPGTIRGDFCIQVGRNIIHGGDSVESAEKEIGLWFHPEEL VDYTSCAQNWIYE (SEQ ID NO:17) describes NM23-H1 S120G mutant amino acid sequence (NM23-H1: GENBANK ACCESSION AF487339).
atggccaacctggagcgcaccttcatcgccatcaagccggacggcgtgcagcgcggcctggtgggcgagatcatc aagcgcttcgagcagaagggattccgcctcgtggccatgaagttcctccgggcctctgaagaacacctgaagcagcactacattgac ctgaaagaccgaccattcttccctgggctggtgaagtacatgaactcagggccggttgtggccatggtctgggaggggctgaacgtg gtgaagacaggccgagtgatgcttggggagaccaatccagcagattcaaagccaggcaccattcgtggggacttctgcattcaggtt ggcaggaacatcattcatggcagtgattcagtaaaaagtgctgaaaaagaaatcagcctatggtttaagcctgaagaactggttgacta caagtcttgtgctcatgactgggtctatgaataa (SEQ ID NO:18) describes human NM23-H2 nucleotide sequence (NM23-H2: GENBANK ACCESSION AK313448).
MANLERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVAMKFLRASEEHLKQ HYIDLKDRPFFPGLVKYMNSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPGTIR GDFCIQVGRNIIHGSDSVKSAEKEISLWFKPEELVDYKSCAHDWVYE (SEQ ID NO:19) describes NM23-H2 amino acid sequence (NM23-H2: GENBANK ACCESSION AK313448).
Human NM23-H7-2 isoform b sequence optimized for E. coli expression:
Human NME7-A:
Human NME7-A1:
Human NME7-A2:
Human NME7-A3:
Human NME7-B:
Human NME7-B1:
Human NME7-B2:
Human NME7-B3:
Human NME7-AB:
Human NME7-AB1:
Human NME7-A sequence optimized for E. coli expression:
Human NME7-A1 sequence optimized for E. coli expression:
Human NME7-A2 sequence optimized for E. coli expression:
Human NME7-A3 sequence optimized for E. coli expression:
Human NME7-B sequence optimized for E. coli expression:
Human NME7-B1 sequence optimized for E. coli expression:
Human NME7-B2 sequence optimized for E. coli expression:
Human NME7-B3 sequence optimized for E. coli expression:
Human NME7-AB sequence optimized for E. coli expression:
Human NME7-AB1 sequence optimized for E. coli expression:
Mouse NME6
Human NME6:
Human NME6 1:
Human NME6 2:
Human NME6 3:
Human NME6 sequence optimized for E. coli expression:
Human NME6 1 sequence optimized for E. coli expression:
Human NME6 2 sequence optimized for E. coli expression:
Human NME6 3 sequence optimized for E. coli expression:
Histidine Tag
Strept II Tag
PSMGFR N-10 peptide:
PSMGFR C-10 peptide
Human NME7
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.
Human NME7-X1
Mouse MUC1 and NME7
Mouse MUC1* extracellular domain PSMGFR is 51.1% identical to human in a 45 amino acid overlap region
Mouse isoform 1 NME7 84.3% identical to human NME7 isoform a in 395 amino acid overlap region
nucleoside diphosphate kinase 7 (mouse NME7) 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 Sus scrofa MUC1 and NME7
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
PREDICTED: nucleoside diphosphate kinase 7 [Sus scrofa, Pig] (Pi_7)
Sheep MUC1 and NME7
Sheep MUC1* extracellular domain PSMGFR is 46.8% identical to human
Sheep NME7 is 88.4% identical to human NME7 isoform a in a 395 amino acid overlap region
PREDICTED: nucleoside diphosphate kinase 7 isoform X1 [Ovis aries, Sheep] (Sh_7)
Crab-eating Macque Macaca fascicularis MUC1 and NME7
Crab-eating macaque MUC1* extracellular domain PSMGFR is 88.9% identical to human
Crab-eating Macque NME7 is 98% identical to human NME7 isoform a in a 251 amino acid overlap
unnamed protein product [Macaca fascicularis] (Ma_7) (sequence incomplete, only NME7A)
Rhesus macaque MUC1 and NME7
Rhesus macque MUC1* PSMGFR
Rhesus macaque MUC1* extracellular domain PSMGFR is 88.9% identical to human
Rhesus Macaque NME7 is 98.4% identical to human NME7 isoform a in a 376 amino acid overlap region
Macaca mulatta (Rhesus macaque) (Mm_7)
Bonobo MUC1 and NME7
Bonobo Pan paniscus MUC1
Bonobo MUC1* extracellular domain PSMGFR is 100% identical to human.
Bonobo NME7 100% identical to human NME7 isoform a in a 376 amino acid overlap region
Bonobo PREDICTED: nucleoside diphosphate kinase 7 [Pan paniscus, Bonobo] (Bo_7)
Chimpanzee MUC1 and NME7
Pan troglodytes MUC1
Chimpanzee MUC1* extracellular domain PSMGFR is 100% identical to human
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 MUC1 and NME7
Gorilla gorilla MUC1
Gorilla MUC1* PSMGFR is 100% identical to human
Gorilla NME7 is 78.2% identical to human NME7 isoform a in a 376 amino acid overlap region
NME7 [ENSGGOP00000002464], Gorilla gorilla (Go_7)
Mouse
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 X1 [Mus musculus] (MoX1-7)
Mouse NME7 is 79.8% identical to human NME7 isoform a in a 376 amino acid overlap region
Macaca fascicularis
unnamed protein product [Macaca fascicularis] (Ma_7) (sequence incomplete, only NME7A)
Macaca NME7 is 98.0% identical to human NME7 isoform a in a 251 amino acid overlap region
Macaca mulatta (Rhesus macaque) (Mm_7)
Macaca NME7 is 98.4% identical to human NME7 isoform a in a 376 amino acid overlap region
Chimp
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
Ovis aries
PREDICTED: nucleoside diphosphate kinase 7 isoform X2 (Shx2_7) [Ovis aries, Sheep]
Sheep NME7 is 92.6% identical to human NME7 isoform a in a 377 amino acid overlap region
PREDICTED: 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
Mouse
Mouse MUC1
Macaca mulatta (rhesus macaque) MUC1, partial
Macaca fascicularis_MUC1 (isoform X1)
Sus scrofa MUC1, partial
Ovis aries MUC1 (Sheep)
I. In one aspect, 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. In this method, an animal that expresses some human DNA or tissues is generated by introducing human naïve state stem cells into a non-human cell or cells. In one aspect 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. For ethical concerns or other reasons, it may be advantageous to generate chimeric animals wherein the integrating naïve state stem cells are also non-human, but of a different species than the recipient cell, cells, morula, blastocyst or embryo. In the method above, the agent that maintains stem cells in the naïve state or reverts primed stem cells to the naïve 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.
In the method above, 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. Alternatively, 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.
In the method above, the agent that maintains stem cells in the naïve state or reverts primed stem cells to the naïve 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.
In this method, 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.
In another aspect, 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.
In this method, in step (iii), 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.
II. In other aspects, 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. In other cases such animals would grow organs comprising at least some human cells. In other cases such animals would grow organs comprised entirely of human cells. In yet other cases, 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.
In one aspect, the invention is directed to a method for generating human tissues in a non-human animal comprising: (i) generating human naïve 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 naïve state stem cells may be generated using NME7, NME7-AB, NME7-X1 or dimeric NME1. The naïve stem cells may be iPS cells that have been reprogrammed in a medium containing NME7, NME7-AB, NME7-X1 or dimeric NME1. Or, the naïve 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. Further, the agent that maintains stem cells in the naïve state or reverts primed stem cells to the naïve 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. In one aspect of the invention, 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. In some cases, the NME protein may be present in serum-free media as the single growth factor.
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. We injected human naïve state stem cells into a mouse morula and showed they incorporated into the inner cell mass. In a specific example, human naïve 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.
III. In one aspect, the present is directed to a method for generating human tissues or organ in a non-human animal host comprising: (i) generating human naïve 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 naïve state stem cells are generated using NME7, NME7-AB, NME7-X1, NME6 or dimeric NME1. The naïve stem cells are iPS cells that have been reprogrammed in a medium containing NME7, NME7-AB, NME6, NME7-X1 or dimeric NME1. The naïve stem cells are embryonic stem cells that have been cultured in a medium containing NME7, NME7-AB, NME6, NME7-X1 or dimeric NME1. 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 naïve state or reverts primed stem cells to the naïve state is an NME protein, 2i, 5i, chemical, or nucleic acid. The NME protein is NME1 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 NME1 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.
Other aspects of the invention are directed to: 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, an antibody that binds to a peptide comprising the sequence of the extracellular domain of MUC1*, wherein the sequence is non-human, an antibody that binds to a peptide comprising the sequence of the extracellular domain of MUC1*, wherein the sequence is primate, an antibody that binds to a peptide comprising the sequence of the extracellular domain of MUC1*, wherein the sequence is macaque, chimpanzee, ape, bonobo, or gorilla, an antibody that binds to a peptide comprising the sequence of the extracellular domain of MUC1*, wherein the sequence is non-primate, an antibody that binds to a peptide comprising the sequence of the extracellular domain of MUC1*, wherein the sequence is rodent, an antibody that binds to a peptide comprising the sequence of the extracellular domain of MUC1*, wherein the sequence is mouse or rat, an antibody that binds to a peptide comprising the sequence of the extracellular domain of MUC1*, wherein the sequence is mammalian, an antibody that binds to a peptide comprising the sequence of the extracellular domain of MUC1*, wherein the sequence is pig, bovine, or sheep.
In another aspect, 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-MUC1* antibody wherein the NME protein is at least 75% homologous to the sequence of the donor cells and the anti-MUC1* 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.
In another aspect, 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.
In yet another aspect, 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 naïve state stem cells. The naïve state stem cells are generated by culturing cells in a media that contains NME. The NME is dimeric NME1, 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 naïve stem cells are introduced into the fertilized egg, morula, blastocyst, embryo or developing fetus of the first non-human mammal.
In the above described methods, 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 naïve 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.
In the above described methods, 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.
In the above methods, 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%.
In the above described methods, 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.
In another aspect, 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.
In another aspect, 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
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). We previously showed that 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-MUC1* (anti-PSMGFR) antibody induced cell death and differentiation (Hikita et al 2008) in vitro.
We discovered 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. We made 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 33 kDa. A naturally occurring NME7 cleavage product that is secreted from stem cells appears to be essentially the same as our recombinant NME7-AB. An alternative splice variant of NME7 called NME7-X1 was theorized. We showed by PCR that NME7-X1 does exist in nature and is also secreted by human stem cells and is 30 kDa. We have also made a human recombinant NME7-X1. In addition, we showed that 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. However, we found that NME7-AB, NME7-X1, NME1 dimers and HSP593 dimers are able to induce somatic cells to revert to a less mature state.
We theorized that in the very earliest human stem cells, NME7-AB and NME7-X1 are secreted which allows them to bind to and dimerize the extracellular domain of MUC1* growth factor receptor. Thus, NME7-AB stabilizes a first naïve state. At a later stage, BRD4 suppresses NME7 and its co-factor JMJD6 upregulates expression of NME1 that must be a dimer to bind to and dimerize the MUC1* growth factor receptor. Thus, NME1 dimers stabilize a second naïve-like state. As the stem cells of a developing morula or blastocyst multiply, the amount of NME1 that is secreted increases and the dimers become hexamers. Hexameric NME1 does not bind to MUC1* and induces differentiation (Smagghe et al 2013).
For the generation of a truly chimeric animal, the stem cells to be injected into the fertilized egg, morula, or blastocyst must be in a naïve state.
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 naïve state. In a preferred embodiment 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. In a more preferred embodiment, 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.
Here, we report that 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 naïve state human stem cells that were able to integrate into blastocysts of other species. NME7-AB naïve human stem cells were injected into a mouse morula at Day 2.5.
We made recombinant human NME1, dimers that bear the 5120G mutation that stabilizes dimers. We previously reported that human NME1 dimers bind to the PSMGFR portion of the extracellular domain of the MUC1* receptor (Smagghe et al. 2013). We also made recombinant human NME7-AB and NME7-X1 that, as monomers, bind to and dimerize the PSMGFR portion of the extracellular domain of the MUC1* receptor on pluripotent stem cells, which stimulates growth and pluripotency and induces stem cells to revert to the earliest naïve state.
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-MUC1* 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.
In another aspect of the invention, 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. In some cases, the sequence of the NME protein may be the human sequence. In other cases 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. If 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. In one aspect of the invention 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.
In one aspect of the invention, non-human stem cells are proliferated or maintained by culturing the cells in a media containing an NME protein of that non-human species. In another aspect of the invention, 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. In another aspect of the invention, 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. In another aspect of the invention, 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.
In another aspect of the invention, 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. In another aspect of the invention, 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. In another aspect of the invention, 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. In another aspect of the invention, 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.
In another aspect of the invention, 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. In another aspect of the invention, 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. In another aspect of the invention, 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. In another aspect of the invention, 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.
In another aspect of the invention, 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.
Testing for Potential Drug Agent in Chimeric Animal
The current practice for testing cancer drugs in mice and other animals is to inject human cancer cells into the animal and either immediately or after several days or weeks of engraftment, inject the animal with the test drug. However, this approach is fundamentally flawed because the host does not naturally produce the growth factors that the human cancer cells need to grow or engraft. Additionally, because the host does not produce the growth factor or the same levels of the growth factor or the human form of the growth factor, drugs being tested in the animals will not have the same effect as they would in humans. Mouse NME7 is only 84% homologous to human NME7 and is not expressed in the adult. Therefore, current xenograft methods for anti-cancer drug testing often fall short in predicting human response to those drugs. This problem could be solved by introducing NME1 dimers or NME7 into the mice so that the human tumor cells have their cognate growth factor to feed the tumor. 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.
In a preferred embodiment, 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. In a preferred embodiment, the NME7 species that is introduced to the test animal is NME7-AB.
Alternatively, 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. Because 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 NME1, NME6 and NME7 proteins or variants are preferred and NME1, NME6 and NME7 proteins or variants that are able to dimerize MUC1*, specifically the PSMGFR peptide, are especially preferred. In a preferred embodiment, the NME species is a truncated form of NME7 having an approximate molecular weight of 33 kDa. In a more preferred embodiment, the NME7 species is devoid of the DM10 domain of its N-terminus. In a still more preferred embodiment, the NME7 species is human.
NME family proteins, especially NME1, 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.
Humanized Animals
In some instances, it is desirable to be able to control the timing of the expression of the NME protein. In these cases, the protein expression may be linked to an inducible genetic element such as a regulatable promoter. In a preferred embodiment, the NME protein that is introduced into an animal to increase engraftment of human stem cells or cancer cells is human NME7. In a yet more preferred embodiment, the NME7 protein is a fragment that is ˜33 kDa. In a still more preferred embodiment, the NME protein is human NME7-AB.
Others have reported that inhibitors called ‘2i’ (Silva J et al 2008) and ‘5i’ (Theunissen T W et al 2014) are able to maintain stem cells in naïve-like state. Treatment with the ‘2i’ inhibitors or ‘5i’ inhibitors caused stem cells to revert to a more naïve state. 2i refers to inhibitors of the MAP kinase pathway and GSK3 inhibitors such as PD0325901 and CHIR99021. However, these and other methods that depend on the use of biochemical inhibitors have not satisfied the criteria for being naïve, such as being able to integrate into inner cell mass of other species and in addition they report that they cannot propagate the stem cells for 10 or more passages without either rampant spontaneous differentiation or abnormal karyotype or both.
Naïve State.
Other agents have been reported to maintain stem cells in the naïve state or revert primed stem cells to the naïve state. Chromatin re-arrangement factors MBD3 and CHD4 were recently reported to block the induction of pluripotency (Rais Y et al, 2013). For example, 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 naïve state. Transcription factors BRD4 and co-factor JMJD6 reportedly suppress NME7 and up-regulate NME1 (Lui W et al, 2013). We found that these factors were expressed at lower levels in naïve stem cells than they were in the later stage primed stem cells. We observed that these four (4) genes, MBD3, CHD4, BRD4 and JMJD6, are naturally suppressed in cancer cells that were cultured in NME1 dimers, NME7 or NME7-AB or NME7-X1 (
We have demonstrated that human NME1 dimers, also called NM23-H1, bacterial NME1, 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 naïve 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.
Several examples have been presented here that indicate that contacting cells with an agent or agents that are able to revert stem cells from the primed state to the less mature naïve state are also able to revert a wide variety of cell types to a less mature state: somatic cells to stem or progenitor cells and stem cells back to naïve state.
In a preferred embodiment, a transgenic animal that expresses human NME7 or NME7-AB is generated. Because NME1, 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.
Alternatively, 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. For example, it may be desirable for the NME7 or NME7 variant to 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. In this instance, the expression of NME7 is turned off when and where the MHC gene product is expressed. Similarly, one may want to have the expression of human NME1, NME6 or NME7 turn on or off in the prostate such that the location and timing of its expression is controlled by the expression of for example, a prostate specific protein. Similarly, the expression of human NME6 or NME7 in a non-human mammal can be controlled by genes expressed in mammary tissues. For example, in a transgenic mouse, 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
Animals xenografted with human tumors and also injected with human NME7 developed metastatic cancers. Therefore, 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. Optimally the NME7 is on an inducible promoter to allow the animal to correctly develop. Alternatively, a metastatic animal model, preferably rodent, is made by making a transgenic animal that expresses human NME or human NME7 or NME7-AB. Alternatively, 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.
Generation of Animals that Express Human Tissue
Other applications are envisioned wherein 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. For example in some cases it is desirable to generate 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. 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. Thus, the expression or timing of expression, of NME7 is controlled by the expression of another gene which may be naturally expressed by the mammal. For example, it may be desirable for the NME7 or NME7 variant to 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. In this instance, the expression of NME7 is decreased or turned off when and where the MHC gene product is expressed. Similarly, one may want to have the expression of human NME1, NME6 or NME7 turn on in the prostate such that the location and timing of its expression is controlled by the expression of for example, a prostate specific protein. Similarly, the expression of human NME1 or NME7 in a non-human mammal can be controlled by genes expressed in mammary tissues. For example, in a transgenic mouse, human NME1 or human NME7 can be expressed or repressed by the prolactin promoter, or a similar gene.
In this way, 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.
As an aside, it is also contemplated that primates or any animal which shares close global sequence identity to humans may not be a good host animal candidate as cross-species interaction may occur and thus ethical issues may arise.
The invention contemplates many applications of animals transgenic for human NME1, bacterial NME1 or human NME7, or NME7-AB. In one aspect of the invention, 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.
Thus 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. Alternatively, the transgenic animal implanted with human stem or progenitor cells is used to grow human tissue for transplant into a human patient. In some cases, 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.
In one aspect, 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.
Induction, 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.
Induction, differentiation/transformation agents for mesoderm tissue include without limitation the following agents: insulin, transferrin, selenous acid, BSA, linoleic acid, TGF-β1, TGF-β3, ascorbate 2-phosphate, dexamethasone, β-glycerophosphate, ascorbate 2-phosphate, BMP, and indomethacin, for the following cell types: cartilage, bone, adipose, muscle, and blood cells.
Induction, 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
These studies have shown that one way in which NME proteins function to promote stem-like or cancer-like growth is by binding to a clipped form of the MUC1 transmembrane protein, herein referred to as MUC1*, which consists primarily of the PSMGFR sequence. Dimerization of the MUC1* extracellular domain stimulates growth and de-differentiation of somatic cells, stem cells and cancer cells, making them more metastatic.
Another way that 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. It has also been previously reported that BRD4 suppresses NME7, while its co-factor JMJD6 up-regulates NME1 (Thompson et al), which we have demonstrated is a self-regulating stem cell growth factor that is expressed later than NME7 in embryogenesis. Still others recently reported that 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 naïve 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. We note that in naïve human stem cells, these four factors BRD4, JMJD6, Mbd3 and CHD4 are suppressed compared to their expression in later stage ‘primed’ stem cells. We also note that the 2i inhibitors (inhibitors of Gsk3β and MEK) that revert mouse primed stem cells to the naïve state, also down regulated the same four factors BRD4, JMJD6, Mbd3 and CHD4.
We have also discovered that NME7 up-regulates SOX2 (>150×), NANOG (˜10×), OCT4 (˜50×), KLF4 (4×) and MUC1 (10×). Importantly, we have shown that NME7 up-regulates cancer stem cell markers including CXCR4 (˜200×) and E-cadherin (CDH1). Taken together these multiple lines of evidence point to the conclusion that NME7 is the most primitive stem cell growth and pluripotency mediator and that it is a powerful factor in the transformation of somatic cells to a cancerous state as well as transforming cancer cells to the more metastatic cancer stem cells.
Therefore, the present invention contemplates substituting genes and gene products that increase expression of NME7 for NME7. Similarly, the invention contemplates substituting downstream effectors of NME7 for NME7. For example, alone or in combination, 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.
Stem Cell-Based Organ and Tissue Generation
The present invention discloses methods for generating, maintaining or proliferating human stem cells in a naïve 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.
In a first method, human naïve 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.
In a second method, human naïve 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.
In a third method, human naïve 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.
Said NME proteins or agents that dimerize MUC1* convert human primed state stem cells to a naïve state. Said NME proteins or agents that dimerize MUC1* also support the derivation of naïve state embryonic stem cell lines from cells taken from a human embryo. Said NME proteins or agents that dimerize MUC1* support the generation of naïve state induced pluripotent stem cells lines, wherein differentiated cells are reprogrammed to a stem cell state.
In a preferred embodiment, the NME family protein is NME7. In a still more preferred embodiment the NME family member is NME7-AB or NME7-X1 or other isoform or truncation of NME7. In another embodiment the NME family member is dimeric NM23, aka NME1. In yet another embodiment, the NME family member is NME6. In a preferred embodiment the agent that dimerizes MUC1* is an antibody that binds to the PSMGFR peptide of the MUC1* extracellular domain.
In one aspect of the invention, naïve 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.
In one aspect of the invention, 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. In this aspect, 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.
In one aspect of the invention, 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. In another aspect of the invention, 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. In one aspect the donor stem cells are iPS cells and in another aspect the stem cells are embryonic stem cells.
In one aspect of the invention, 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. In a preferred embodiment the NME protein is dimeric NME1.
In a more preferred embodiment the NME protein is NME7-X1. In a still more preferred embodiment, the NME protein is NME7-AB. In some cases, stem cell attachment to a surface is facilitated by coating said surface with an anti-MUC1* antibody wherein the antibody has the ability to bind to a peptide comprising at least 15 contiguous amino acids of the PSMGFR sequence. In another case, 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++. In other instances, 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. In other instances, stem cell attachment to a surface is facilitated by coating said surface with peptides, small molecules or polymers. In some cases 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. However, for the generation, induction or maintenance of non-human stem cells, it may be advantageous to contact the cells with an NME protein whose sequence is that of the non-human species. For example, for generating, inducing pluripotency or maintaining pig stem cells, it may be advantageous to use an NME protein whose sequence is that of native pig NME6, NME1, NME7, NME7-X1 or NME7-AB. Facilitate surface attachment, it 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.
Minimal Media
Serum-free Minimal Media 500 mL includes the following components:
0.9 mL β-mercaptoethanol, 55 mM stock.
When a rho kinase inhibitor, “Ri” or “ROCi”, was added it was Y27632 from Stemgent (Cambridge, Mass.), added immediately before use to a final concentration of 10 uM.
Culturing Stem Cells in NME Media
To serum-free Minimal Media, described in Example 1, add one of the following NME proteins: 8 nM (final concentration) dimeric rhNME1 (aka NM23) preferably having the S120G mutation to ensure stable dimers, 8 nM dimeric NME6, 8 nM bacterial HSP593 recombinant NME1, 4 nM NME7AB or 4 nM NME7-X1. 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-MUC1* antibody such as MN-C3, then a Rho Kinase inhibitor was added such as Y-26732 to a final concentration of 10 uM.
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-MUC1* 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% CO2/5%02 incubator for 48 hours. Thereafter, media was changed every 24 or 48 hours until cells reached ˜80% confluency (
iPS Generation in NME Media
Day −2 (48 hours before reprogramming): Fibroblasts were seeded onto standard tissue culture-treated 6-well plates in 2 mLs per well of Fibroblast Medium (DMEM High Glucose with glutamine, 10% FBS), at 25,000-100,000 cells per well. Culture for 48 hours in 5% CO2.
Day 0: Fibroblast Media was changed to NME Media (Example Fibroblasts were transduced with master reprogramming 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. Common methods use non-integrating viral delivery systems such as lend virus, Sendai virus, gamma retrovirus or transposons such as Sleeping Beauty.
Day 1: Cells were washed with Minimal Media to remove virus and cell debris, then replaced with 2 mL-4 mL per well of NME Media, without Rho kinase inhibitor.
Day 3: Change media with 2 mL to 4 mL per well of NME Media, without Rho kinase inhibitor.
Day 5: Change media with 2 mL to 4 mL per well of NME Media, without Rho kinase inhibitor.
Day 6: Cell culture plates were prepared with an anti-MUC1* antibody such as MN-C3 coated onto cell culture plates at a concentration of 3.25 ug/mL to 24 ug/mL with about 12.5 ug/mL preferred and incubate the antibody coated plates at 4° C. overnight.
Day 7: Transduced cells, which were changing morphology to that of stem cells by this time, were dissociated with Trypsin/EDTA, passed through a cell strainer, and seeded onto MN-C3 coated plates in NME Media plus a Rho kinase inhibitor (such as 10 uM Y-26732). The reprogrammed cells were then plated at 5×104-1×105 per well. From this point onward, cells are cultured in an NME media with a Rho kinase inhibitor such as 10 uM Y-26732 added and incubated for first 48 hours undisturbed in 5% CO2/5% O2.
Day 9 and onward: Change media daily using the same NME media throughout plus Rho kinase inhibitor such as 10 uM Y-26732.
Day 16-Day 21: Colonies were picked and each clone was cultured on MN-C3 coated surfaces, first in 96-well plates, then 24-well, then 12-well, then 6-well and larger formats after stem cells were characterized and found to express all normal pluripotency genes, naïve genes, formed teratomas when implanted into animals and had normal karyotype.
iPS cells were also generated from blood using same process as Day 1 and onward.
In the cells shown in
Reprogramming Capability of NME Proteins in the Absence of Master Pluripotency Regulators OCT4, SOX2, KLF4 or c-Myc
In this example, fibroblast cells were cultured in Minimal Media to which was added recombinant human NME1/NM23 dimers, bacterial HSP593 NME1 dimers or human recombinant NME7-AB. As a control, 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). After 15-20 days in culture in Minimal Media with either NME1/NM23 dimers, bacterial HSP593 NME1 dimers or NME7-AB, RT-PCR showed that the resultant cells greatly increased expression of stem cell marker genes OCT4 and NANOG, see
NME7-AB cultured human stem cells incorporate into inner cell mass of mouse morula. Mouse eggs were fertilized in vitro. At Day 2.5 post fertilization, 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. 48 hours later, at Day 4.5, morulas were stained with a fluorescent antibody that stains human Tra 1-81. in some of the figures, arrows point to human naïve NME7-AB cells that have incorporated into the inner cell mass, which indicates the development of a chimeric animal See
NME7-AB cultured human stem cells were transfected with a red fluorophore called tomato red or TDtomato. These fluorescent human naïve 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
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-MUC1* antibody, in this case MN-C3. Beginning on Day 6 for crab-eating macaques and Day 14 for rhesus macaques, colonies began to appear.
Primate species embryonic stem cells proliferate and are maintained in NME7-AB media. After colonies were picked, they were replated onto MN-C3 antibody coated surfaces or onto MEFs and could be serially passaged indefinitely in a serum-free media containing NME7-AB at a concentration between 2 nM and 32 nM wherein 4 nM worked best. See
All of the references cited herein are incorporated by reference in their entirety.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention specifically described herein. Such equivalents are intended to be encompassed in the scope of the claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US16/40880 | 7/1/2016 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62187638 | Jul 2015 | US | |
| 62234035 | Sep 2015 | US |
