Patent Application
20150320840
1. Field of the Invention
The present application relates to the field of inducing pluripotency in cells.
2. General Background and State of the Art:
It has been demonstrated, in mouse and human, that somatic cells can be reprogrammed by ectopic expression of transcription factors (Lowry et al., 2008; Maherali et al., 2007; Nakagawa et al., 2008; Okita et al., 2007; Park et al., 2008; Takahashi et al., 2006; Takahashi and Yamanaka, 2006; Wernig et al., 2007; Yu et al., 2006) to become pluripotent. The generation of induced pluripotent stem (iPS) cells holds great promise for the realization of truly personalized regenerative medicine (Yamanaka, 2007; Jaenish and Young, 2008) because stem cells derived from a patient's own skin cell can be used to generate cells and tissues to repair damage caused by disease or aging. Forced expression of combinations of the transcription factors, Oct4, Sox2, Klf4 and c-Myc or Oct4, Sox2, Nanog and Lin28 have been shown to cause mature cells to revert to the pluripotent state.
In earlier studies, the transcription factors were expressed using multiple viral vectors (Takahashi and Yamanaka, 2006; Okita et al., 2007; Maherali et al., 2007; Wernig et al., 2007; Takahashi et al., 2006; Yu et al., 2006; Park et al., 2008). The use of multiple vectors presented a problem because of multiple integration events, which could lead to increased risk of oncogenicity (Takahashi and Yamanaka, 2006; Aoi et al., 2008). Researchers have tried to overcome this problem by using single vector systems (Sommer et al., 2009), excisable vectors (Kaji et al., 2009; Soldner et al., 2009; Woltjen et al., 2009), non-integrating vectors (Stadtfeld et al., 2009; Yu et al., 2009) and transient transfections (Okita et al., 2009). However, these methods are extremely inefficient at achieving epigenetic reprogramming.
Methods for inducing pluripotency include transfection of the oncogene c-Myc, which is undesirable because of its potential to cause cancer. iPS cells can be generated without transfecting c-Myc (Nakagawa et al., 2008; Wernig et al., 2008). However, the efficiency of reprogramming was greatly decreased. Similarly, Klf4 can induce dysplasia (Foster et al., 2005).
Because of the problems associated with multiple viral vector integration and undesirable side effects of some of the genes that induce pluripotency, there is a need to replace the use of some or all of the pluripotency-inducing genes with the protein gene products and proteins that regulate their expression or whose expression is regulated by the pluripotency-inducing genes or small molecules that regulate the expression of genes or proteins that induce pluripotency. To this end, it has been reported that the introduction of the gene products, rather than the genes, also induced pluripotency (Zhou et al., 2009). Recombinant Oct4, Sox2, Klf4 and c-Myc, tagged with poly-arginine to facilitate entry into the cell, reprogrammed mouse somatic cells. Others have used small molecules to replace the need for one of the genes of the core set. A small molecule that upregulated Nanog eliminated the need for the Klf4 gene, which also upregulates Nanog (Lyssiotis et al., 2009). In another study, a small molecule HDAC inhibitor removed the requirement for both Klf4 and c-Myc (Huangfu et al., 2008, a&b). These studies show that: 1) the protein gene products can replace the need for the genes; 2) small molecules that upregulate genes can replace the need for the genes; and 3) genes (or gene products) in the same regulatory pathway can substitute for one another.
Despite these achievements, a major problem that remains is that these methods suffer from low efficiency of reprogramming Current rates of inducing pluripotency in somatic cells are so low that they make therapeutic uses of iPS cells impractical. Therefore, what is needed is to identify proteins and small molecules that either alone or in addition to those already identified, induce pluripotency or improve the efficiency of the induction of pluripotency in cells.
In one aspect, the present invention is directed to a method for generating less mature cells from starting cells comprising inducing the starting cells to revert to a less mature state, comprising contacting the starting cells with a biological or chemical agent that
(i) increases amount of MUC1 or NME protein in the cell;
(ii) increases expression MUC1 or NME protein in the cell; or
(iii) increases activity of MUC1 or NME protein.
The method may include transfecting the starting cells with a nucleic acid that directly or indirectly causes an increase in the amount, expression or activity of the MUC1 or NME protein. NME protein may be NME7 or a NME7 variant thereof. Or, the NME protein may be NME1 or a NME1 variant thereof. The MUC1 may be MUC1*.
In another aspect, the method above may include transfecting the starting cells with a nucleic acid that directly or indirectly causes an increase in the amount, expression or activity of MUC1 cleavage enzyme. The cleavage enzyme may be MMP-16, MMP-14 or ADAM-17.
In another aspect, the method above may further include transfecting the starting cell with a nucleic acid that directly or indirectly causes an increase in the amount, expression or activity of Oct4, Sox2, Klf4, c-Myc, Lin28, or Nanog.
The above method may further include contacting the starting cells with the peptide or protein that directly or indirectly causes an increase in the amount, expression or activity of Oct4, Sox2, Klf4, c-Myc, Lin28, or Nanog.
The biological species may be a peptide or protein. The peptide or protein may be modified with a moiety or sequence that enhances its ability to enter a cell. The peptide or protein may be an NME protein or variant thereof. The NME protein may be NME7 or NME1. The peptide or protein may also be MUC1, or a portion of MUC1. And the above method may further include contacting the starting cells with the peptide or protein that directly or indirectly causes an increase in the amount, expression or activity of Oct4, Sox2, Klf4, c-Myc, Lin28, or Nanog. The above method may also include transfecting the starting cells with a nucleic acid that directly or indirectly causes an increase in the amount, expression or activity of Oct4, Sox2, Klf4, c-Myc, Lin28, or Nanog.
In the above-described method, the chemical species may directly or indirectly cause an increase in the amount, expression or activity of NME7, NME1, MUC1, MUC1*, MMP16, MMP14 or ADAM17. And this method may further include contacting the starting cells with chemical species that directly or indirectly cause an increase in the amount, expression or activity of Oct4, Sox2, Klf4, c-Myc, Lin28 or Nanog. The less mature state may be characterized by an increase in expression of at least one of OCT4, SOX2, KLF4, KLF2, NANOG, LIN28, MUC1, NME1 or NME7. The less mature state may be a pluripotent state.
In the method above, the protein may be a MUC1* ligand. The ligand may dimerize MUC1*. The ligand may be an NME family member, such as NME1, NME6 or NME7. NME1 and NME6 may be in dimeric form and NME7 in monomeric form.
In another aspect, the ligand may be an antibody that recognizes the PSMGFR sequence of MUC1*.
In the method above, the chemical agent may be a small molecule that enhances the transcription of MUC1, transcription of MUC1 cleavage enzyme, or transcription of an NME family member. The cleavage enzyme may be MMP-16, MMP-14 or ADAM-17. The small molecule may enhance cleavage of MUC1, such as phorbol ester.
In the methods described above, the nucleic acid used may encode MUC1, such as MUC1*. Or, the nucleic acid may encode a ligand of MUC1*, and the ligand may be MUC1* antibody or an NME protein.
In the methods described above, the method may further include contacting the starting cell with a molecule that increases expression of gene products that induce pluripotency. Such gene product may include OCT4, SOX2, NANOG, KLF4 or LIN28. In particular, OCT4, and SOX2.
The cells in the invention may be mammalian, including human.
In still another aspect, the present invention is directed to a method of generating less mature cells from starting cells comprising inducing the starting cells to revert to a less mature state, comprising contacting the starting cells with a biological or chemical agent that increases the amount of MUC1 or NME in the cells, and further contacting the starting cells with a biological or chemical species that increases the amount of one or more of OCT4, SOX2, NANOG, KLF4 or LIN28. The biological species that increases the amount of MUC1 or NME may be the nucleic acid that encodes MUC1, MUC1*, NME1 or NME7 or variants thereof.
The starting cells may be pluripotent stem cells, multipotent stem cells or terminally differentiated cells. The pluripotent stem cell may be in the primed state. The multipotent stem cell may be a hematopoietic cell, a bone marrow cell or a neuronal cell. The terminally differentiated cell may be a fibroblast, a dermablast, a blood cell, or a neuronal cell.
The generated cells may be then differentiated, in vitro or in vivo.
The present invention is directed to a method of administering the generated cells, which includes differentiating the generated cells, and administering the differentiated cells to a patient in need thereof. The present invention is directed to a method of administering the generated cells, and administering the generated cells to a patient in need thereof. The generated cell may be from the patient or a donor.
In still another aspect, the invention is directed to a method of differentiating stem cells including
(i) inducing starting cells to revert to a less mature state or to maintain it in the less mature state comprising carrying out the method described above; and
(ii) causing the less mature cells to differentiate.
The cells maybe differentiated into ectodermal, mesodermal or endodermal cells.
In yet another aspect, the invention is directed to a method of healing a disease or wound that would be healed by the induction or maintenance of stem cells at the site of injury, comprising administering to a person in need thereof an effective amount of an pluripotency inducing agent. The pluripotency inducing agent may be a MUC1* activator. The agent may be NME. The generated cells may be administered to the subject by injection, transplantation or topical application.
In still another aspect, the invention is directed to a method of rescuing degraded stem cells comprising contacting the cells with NME.
In yet another aspect, the invention is directed to a method of increasing efficiency of induction of pluripotency of a cell comprising contacting the cells with NME.
In another aspect, the invention is directed to a method of increasing expression of MUC1 or MUC1* in a cell comprising contacting the cell with NME. The cell may be stem cell.
In another aspect, the invention is directed to a method of generating a less mature cell, including induced pluripotent cell comprising contacting starting cells with NME in the absence of bFGF.
These and other objects of the invention will be more fully understood from the following description of the invention, the referenced drawings attached hereto and the claims appended hereto.
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;
TABLE 2 shows how many stem-like colonies resulted by Day 19 from induction of pluripotency of human fibroblasts under a variety of conditions and calculates induction efficiency, which is the number of cells required for generating a single colony, and induction rate, which is the inverse of that number.
In the present application, “a” and “an” are used to refer to both single and a plurality of objects.
As used herein, “increasing MUC1* activity” refers to directly or indirectly increasing MUC1* signaling, and includes without limitation the dimerization of MUC1* receptor and also increased production of MUC1* by cleavage of the MUC1 receptor. MUC1* activity may be also increased by higher transcriptional expression of MUC1 receptor, which is further cleaved and dimerized. Therefore, in one aspect, MUC1* activity may be increased by a higher activity of the effector molecule that dimerizes MUC1*, or the higher activity of the cleavage molecule that cleaves MUC1 so that MUC1* is formed, or increased expression of the MUC1. Therefore, any chemical or biological species that is able to increase the activity of the MUC1* dimerizing ligand, MUC1 cleavage enzyme to form MUC1*, or any transcriptional activator that enhances expression of MUC1, is encompassed as a species that “increases MUC1* activity”.
As used herein, “MUC1 Growth Factor Receptor” (MGFR) is a functional definition meaning that portion of the MUC1 receptor that interacts with an activating ligand, such as a growth factor or a modifying enzyme such as a cleavage enzyme. The MGFR region of MUC1 is that extracellular portion that is closest to the cell surface and is defined by most or all of the PSMGFR, as defined below. The MGFR is inclusive of both unmodified peptides and peptides that have undergone enzyme modifications, such as, for example, phosphorylation, glycosylation and so forth.
As used herein, “Primary Sequence of the MUC1 Growth Factor Receptor” (PSMGFR) refers to peptide sequence that defines most or all of the MGFR in some cases, and functional variants and fragments of the peptide sequence. The PSMGFR is defined as SEQ ID NO:6, and all functional variants and fragments thereof having any integer value of amino acid substitutions up to 20 (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) and/or any integer value of amino acid additions or deletions up to 20 at its N-terminus and/or C-terminus A “functional variant or fragment” in the above context refers to such variant or fragment having the ability to specifically bind to, or otherways specifically interact with, ligands that specifically bind to, or otherwise specifically interact with, the peptide of SEQ ID NO:6, while not binding strongly to identical regions of other peptide molecules identical to themselves, such that the peptide molecules would have the ability to aggregate (i.e. self-aggregate) with other identical peptide molecules. One example of a PSMGFR that is a functional variant of the PSMGFR peptide of SEQ NO:6 is SEQ ID NO:8, which differs from SEQ ID NO:6 by including an -SPY- sequence instead of the -SRY-.
As used herein, “MUC1*” refers to the MUC1 protein with the N-terminus truncated such that the extracellular domain is essentially comprised of the PSMGFR (SEQ ID NO:5).
As used herein “MUC1* associated factors” refers to agents that modify, activate, modulate the activity of, or modulate the expression of MUC1*. MUC1* associated factors include, without limitation, agents that affect dimerization of MUC1* receptor, increased production of MUC1*, induce cleavage of the MUC1 receptor, agents that increase MUC1* activity by higher transcriptional expression of MUC1 receptor, which is further cleaved and dimerized.
As used herein, “effective amount” is an amount sufficient to effect beneficial or desired clinical or biochemical results. An effective amount can be administered one or more times. For purposes of this invention, an effective amount of an inhibitor compound is an amount that is sufficient to induce or maintain pluripotency of a cell or activate MUC1*
As used herein, “fragments” or “functional derivatives” refers to biologically active amino acid sequence variants and fragments of the native ligands or receptors of the present invention, as well as covalent modifications, including derivatives obtained by reaction with organic derivatizing agents, post-translational modifications, derivatives with nonproteinaceous polymers, and immunoadhesins.
As used herein, “immature” cells refers to cells that can undergo at least one more step of differentiation and expresses markers of a particular cell type that is known to be able to undergo at least one more step of differentiation.
As used herein, “cell having less mature state than starting cell” refers to a cell that has de-differentiated so that it has an increased ability to differentiate into a different cell type than the starting cell or has an increased ability to differentiate into more cell types than the starting cell. A cell in a less mature state can be identified by measuring an increase in the expression of pluripotency markers, by a determination that the expression levels of pluripotency markers are closer to those of pluripotent stem cells or by measuring markers of a less mature state than the starting cells. For example, hematopoietic stem cells that can differentiate into any blood cell type are characterized by the expression of CD34 and the absence of CD38. As these cells differentiate, they go from CD34+/CD38− to CD34+/CD38− then to CD34−/CD38+. If one were to induce the CD34−/CD38+ cells to revert to a less mature state, the cells would regain expression of CD34. The technique of transdifferentiation involves reverting starting cells to a less mature state wherein the cells become unstable and can be directed to differentiate into a differentiate cell type than the starting cell, even if the starting cell was at the same relative level of differentiation as the resultant cell (Iede et al 2010; Efe et al 2011). For example, cardio fibroblasts have been reverted to a less mature state by brief ectopic expression of OCT4, SOX2, KLF4 and c-MYC, then from this unstable state, differentiated into cardiomyocytes.
As used herein, “ligand” refers to any molecule or agent, or compound that specifically binds covalently or transiently to a molecule such as a polypeptide. When used in certain context, ligand may include antibody. In other context, “ligand” may refer to a molecule sought to be bound by another molecule with high affinity, such as but not limited to a natural or unnatural ligand for MUC1* or a cleaving enzyme binding to MUC1 or MUC1* or a dimerizing ligand for MUC1*.
As used herein, “Naïve stem cells” are those that resemble and share quantifiable characteristics with cells of the inner mass of a blastocyst. Naïve stem cells have quantifiable differences in expression of certain genes compared to primed stem cells, which resemble and share traits and characteristics of cells from the epiblast portion of a blastocyst. Notably, naïve stem cells of a female source have two active X chromosomes, referred to as XaXa, whereas the later primed stem cells of a female source have one of the X chromosomes inactivated.
As used herein, “NME” family proteins is a family of ten (10) proteins, some of which have been recently discovered, wherein they are categorized by their shared sequence homology to nucleoside diphosphate kinase (NDPK) domains, even though many of the NME family members are incapable of kinase activity. NME proteins were previously known as NM23-H1 and NM23-H2 then NM23-H3 through NM23-10 as they were being discovered. The different NME proteins function differently. Herein, NME1 and NME6 bind to and dimerize the MUC1* receptor (wherein its extra cellular domain is comprised essentially of the PSMGFR sequence) when they are in dimer form; NME7 has two (2) binding sites for MUC1* receptor extra cellular domain and also dimerizes the receptor. NME1 dimers, NME6 dimers and NME7 are the preferred NME family members for use as MUC1* ligands to induce or maintain cells in a less mature state than the starting cells. Other NME family members that are able to bind to and dimerize the MUC1* receptor are also contemplated for use as MUC1* ligands to induce or maintain cells in a less mature state than the starting cells.
As used herein, “pluripotency markers” are those genes and proteins whose expression is increased when cells revert to a less mature state than the starting cells. Pluripotency markers include OCT4, SOX2, NANOG, KLF4, KLF2, Tra 1-60, Tra 1-81, SSEA4, and REX-1 as well as others previously described and those currently being discovered. For example, fibroblast cells express no detectable or low levels of these pluripotency markers, but express a fibroblast differentiation marker called CD13. To determine if a cell is becoming less mature than the starting cells, one could measure a difference in the expression levels of the pluripotency markers between the starting cells and the resultant cells.
As used herein, “primed stem cells” are cells that resemble and share traits and characteristics of cells from the epiblast portion of a blastocyst.
As used herein, the term “specifically binds” refers to a non-random binding reaction between two molecules, for example between an antibody molecule immunoreacting with an antigen, or a non-antibody ligand reacting with another polypeptide, such as NM23 specifically binding with MUC1* or an antibody binding to MUC1* or a cleaving enzyme binding to MUC1 or MUC1*.
As used herein, “pluripotent” stem cell refers to stem cells that can differentiate to all three germlines, endoderm, ectoderm and mesoderm, to differentiate into any cell type in the body, but cannot give rise to a complete organism. A totipotent stem cell is one that can differentiate or mature into a complete organism such as a human being. With reference to embryonic pluripotent stem cells, they are cells derived from the inner cell mass of a blastocyst. Typical markers of pluripotency are OCT4, KLF4, NANOG, Tra 1-60, Tra 1-81 and SSEA4.
As used herein, “multipotent” stem cells refer to stem cells that can differentiate into other cell types wherein the number of different cell types is limited.
As used herein, “semi-pluripotent” or “pre-iPS state” refers to a cell that has some or all of the morphological characteristics of a pluripotent stem cell, but its level of expression of the pluripotency markers or its ability to differentiate to all three germlines is less than that of a pluripotent stem cell.
As used herein, “stem-like” morphology refers to a morphology that resembles that of a stem cell, a level of expression of one or more of the pluripotency genes, or an ability to differentiate into multiple cell types. Stem-like morphology is when the cells have a rounded shape, and are rather small compared to the size of their nucleus, which is often has a large nucleus to cytoplasm ratio, which is characteristic of pluripotent stem cells. By contrast, fibroblast morphology is when cells have a long, spindly shape and do not have a large nucleus to cytoplasm ratio. Additionally, pluripotent stem cells are non-adherent, whereas other cell types, such as fibroblasts, are adherent.
As used herein, “vector”, “polynucleotide vector”, “construct” and “polynucleotide construct” are used interchangeably herein. A polynucleotide vector of this invention may be in any of several forms, including, but not limited to, RNA, DNA, RNA encapsulated in a retroviral coat, DNA encapsulated in an adenovirus coat, DNA packaged in another viral or viral-like form (such as herpes simplex, and adeno-associated virus (AAV)), DNA encapsulated in liposomes, DNA complexed with polylysine, complexed with synthetic polycationic molecules, complexed with compounds such as polyethylene glycol (PEG) to immunologically “mask” the molecule and/or increase half-life, or conjugated to a non-viral protein. Preferably, the polynucleotide is DNA. As used herein, “DNA” includes not only bases A, T, C, and G, but also includes any of their analogs or modified forms of these bases, such as methylated nucleotides, internucleotide modifications such as uncharged linkages and thioates, use of sugar analogs, and modified and/or alternative backbone structures, such as polyamides.
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 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.
describes a truncated MUC1 receptor isoform having nat-PSMGFR at its N-terminus and including the transmembrane and cytoplasmic sequences of a full-length MUC1 receptor.
describes Native Primary Sequence of the MUC1 Growth Factor Receptor (nat-PSMGFR—an example of “PSMGFR”):
describes Native Primary Sequence of the 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).
describes “SPY” functional variant of the native Primary Sequence of the MUC1 Growth Factor Receptor having enhanced stability (var-PSMGFR—An example of “PSMGFR”).
describes “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).
describes MUC1 cytoplasmic domain nucleotide sequence.
describes MUC1 cytoplasmic domain amino acid sequence.
describes NME7 nucleotide sequence (NME7: GENBANK ACCESSION AB209049).
describes NME7 amino acid sequence (NME7: GENBANK ACCESSION AB209049).
describes NM23-H1 nucleotide sequence (NM23-H1: GENBANK ACCESSION AF487339).
NM23-H1 describes amino acid sequence (NM23-H1: GENBANK ACCESSION AF487339).
describes NM23-H1 S120G mutant nucleotide sequence (NM23-H1: GENBANK ACCESSION AF487339).
describes NM23-H1 S120G mutant amino acid sequence (NM23-H1: GENBANK ACCESSION AF487339).
describes human SOX2 nucleotide sequence (SOX2:GENBANK ACCESSION NM—003106).
describes human SOX2 amino acid sequence (SOX2:GENBANK ACCESSION NM—003106).
describes Human OCT4 nucleotide sequence.
describes Human OCT4 amino acid sequence.
describes NM23-H2 nucleotide sequence (NM23-H2: GENBANK ACCESSION AK313448).
describes NM23-H2 amino acid sequence (NM23-H2: GENBANK ACCESSION AK313448).
describes KLF4 nucleotide sequence (KLF4: GENBANK ACCESSION AF022184).
describes KLF4 amino acid sequence (KLF4: GENBANK ACCESSION AF022184).
describes c-Myc nucleotide sequence (c-Myc:GENBANK ACCESSION BC000917).
describes c-Myc amino acid sequence (c-Myc: GENBANK ACCESSION BC000917).
describes LIN28 nucleotide sequence (LIN28: GENBANK ACCESSION AF521099).
describes LIN28 amino acid sequence (LIN28: GENBANK ACCESSION AF521099).
describes MMP14 nucleotide sequence (MMP14: GENBANK ACCESSION BC064803).
describes MMP14 amino acid sequence (MMP14: GENBANK ACCESSION BC064803).
describes MMP16 nucleotide sequence (MMP16:GENBANK ACCESSION AB009303).
describes MMP 16 amino acid sequence (MMP16:GENBANK ACCESSION AB009303)
Human NME7-AB sequence optimized for E. coli expression:
(DNA)
describes NME7-AB nucleotide sequence
(Amino Acids)
describes NME7-AB amino acid sequence
describes membrane proximal portion of human MUC1 receptor.
describes “N-10,” which is missing ten amino acids at the N-terminus of PSMGFR.
describes amino sequence encompassing N-terminal adjacent portion of the amino acid sequence of SEQ ID NO:37.
describes self-aggregation domain of MUC1.
describes an irrelevant peptide.
Induction of Cells to Less Mature State
It was recently discovered that somatic cells can be reprogrammed to revert to the pluripotent state. The genes that code for transcription factors OCT4, SOX2, KLF4, NANOG, c-MYC and LIN28 or the proteins themselves can be introduced into somatic cells and cause a reversion to the pluripotent state. Many of these pluripotency factors were previously thought of as oncogenes. C-Myc is a well known oncogene and similarly, Klf4 has been shown to induce dysplasia (Foster et al., 2005). OCT4 was once thought of as the gold standard for identifying pluripotent stem cells. The presence of OCT4 in the nucleus was thought to indicate that the cell is pluripotent and its absence indicates that the cell has entered the differentiation process and is no longer able to differentiate into any cell type. Recently, it became known that OCT4 is also present in the nucleus of many cancer cells, but not in normal mature cells. The present inventors recently discovered that a cleaved form of the MUC1 transmembrane protein (SEQ ID NO:1), MUC1*, is a powerful growth factor receptor that is expressed on an estimated 75% of solid tumor cancers (Raina et al., 2009) and that it is also expressed in this “tumorigenic” form on all pluripotent stem cells, including embryonic stem (ES) cells as well as induced pluripotent stem (iPS) cells (Hikita et al., 2008; Smagghe et al, 2013). The present invention relates to MUC1* and MUC1* associated factors as well as methods employing them for the induction or maintenance of pluripotency or to enhance the efficiency of inducing pluripotency.
MUC1* is a primal growth factor receptor that mediates growth and pluripotency of stem cells and cancer cells. Introducing MUC1*-associated factors induces cells to revert to a less mature state than that of the starting cells. NM23 in a bivalent or dimeric form is a ligand of MUC1* growth factor receptor. Interruption of the NM23-MUC1* interaction induces expression of microRNA-145 (miR-145) which is a microRNA that signals pluripotent stem cells to exit from pluripotency and initiate a maturation process.
Treating somatic cells with a protein belonging to NME family of proteins such as dimeric NM23, a bivalent NM23, or NME7 in the presence or absence of pluripotency genes, caused mature cells to revert to a less mature state than the starting state of the cell. Fibroblasts that were transfected with two or more of the pluripotency genes, Oct4, Sox2, Klf4 and c-Myc, were induced to become pluripotent stem cells at a much faster rate and with enhanced efficiency of induction when they were cultured in a medium that contained dimeric NM23. Cells that were not transfected with any of the pluripotency genes but cultured in dimeric NM23 expressed Oct4 and reverted to a stem-like morphology within days. Additionally, somatic cells treated with a bivalent antibody that recognizes the PSMGFR portion of MUC1*, also reverted to a less differentiated state than the starting cells. Thus, bivalent MUC1* ligands, such as dimeric NME family proteins, in particular NME7 which has two binding sites for MUC1* and thus dimerizes MUC1*, NME family members in dimeric form or antibodies against the PSMGFR region, promote growth of undifferentiated stem cells, as well as inducing cells to revert to a less mature state, wherein the cells that can be reverted to a less mature state are chosen from the group comprising totipotent stem cells, pluripotent stem cells, multipotent stem cells as well as differentiated cells.
In addition to making mature cells revert to a less mature state or further to a pluripotent state, we have also demonstrated that treating stem cells with a MUC1 ligand such as NM23 dimers or NME7 causes the stem cells to revert to a less mature state, i.e. a more pluripotent state.
Stem Cell Rescue
Stem cell lines often “go bad” as evidenced by their inability to differentiate properly. Researchers may use a stem cell line for months that can be directed to differentiate into cardiomyocytes, for example, then efficiency of differentiation begins to decline and eventually, the cell line just stops working. When the cell lines are assayed for the presence of typical pluripotency markers, the reduction in the expression of convenient surface markers like Tra 1-60, SSEA4 or Rex-1 is slight. However, if these cells are then assayed for the presence of MUC1*, we found that there was minimal expression of MUC1*. Treatment of these “pluripotent” stem cells (iPS or ES) with NM23 dimers or NME7 caused a dramatic increase n the expression of MUC1* that coincided with increased expression of the pluripotency markers. Some experiments that studied this aspect of the invention are detailed in Example 14,
Because stem cells differentiate into mature adult cells in stages, it is not necessary to bring cells all the way back to a pluripotent stem cell state before having them differentiate into mature cells. Cells can be differentiated to a desired cell type from an interim state. Therefore, cells can merely be induced to revert to a less mature state from which they are able to differentiate into the desired cell or tissue type. Some refer to this interim, less mature state as a pre-iPS state. The technique is referred to as “transdifferentiation” and sometimes “direct differentiation.” For example, somatic cells or mature cells such as fibroblasts or dermablasts can be induced to become somewhat pluripotent, and then directed to differentiate or be allowed to differentiate into some desired cell type (Iede et al 2010; Efe et al 2011). For example, cardio fibroblasts can be brought to a less mature state and then differentiated into beating cardiomyocytes. In other cases, it is advantageous to start with cells from the same lineage as the desired final cell type. In this way, cells are only reverted to a less mature state, which is earlier than some decision point, and then directed to differentiate into the desired cell type.
Methods for directing differentiation into cardiomyocytes, neuronal cells, islet cells and the like are known by those skilled in the art. The hurdles that need to be overcome include very low efficiency of directed differentiation and the use of plasmids and viral vectors to introduce agents to induce pluripotency. Protein agents that induce cells to revert to a less mature state, increase efficiency of that induction and/or increase the efficiency of directed differentiation would solve these problems that have thus far prevented clinical application of stem cell therapies. NM23 and/or other MUC1* associated factors can be introduced to cells to induce them to become less mature or more stem-like. NM23 can be used alone or in conjunction with other factors to induce cells to become stem-like. In one embodiment, NM23 in the dimeric form or in a bivalent form is provided to cells to induce cells to become less mature. In other embodiments, NM23 (H1 or H6 dimers or NME7) is added along with other genes, proteins, or small molecules that induce cells to become more stem-like. From this stem-like or semi-pluripotent state, the induced cells can be allowed to differentiate into a desired cell type by merely placing the cells in an environment of cells of the desired cell type or in an environment of factors that will influence the induced cells to differentiate into the desired cell or tissue type.
In one embodiment, the cells that are induced to become less mature are cells present in a host animal or human. In some cases, the factor(s) that induce the cells to become less mature are added systemically. In other cases, the factor(s) that induce the cells to become less mature are added locally. To facilitate the local introduction of these factor(s), the inducing factors can be impregnated into or attached to a dressing, for example, to expedite wound healing. Alternatively, they can be injected locally, alone, or in a carrier material, which could be a hydrogel or other material. The inducing factor(s) could also be attached to a biocompatible material that could be topically applied, surgically inserted or ingested. Cartilage repair could be facilitated by introducing factor(s) that induce the cells to become less mature into a joint. Persons suffering from neurodegenerative diseases such as Alzheimer's or Parkinson's diseases could be treated by inducing local brain cells to revert to a less mature state from which they would be able to differentiate into functional brain cells.
The invention also includes attaching factors that induce cells to become less mature to substrates that perform an unrelated function, such as stents for blood vessel repair, tape-like materials to hold two pieces of substance together while encouraging cellular regeneration in the gap, scaffolds to shape the formation of tissues either over or within the structure, substrates that are patterned for example for the formation of nerves and other biological structures. The invention further includes attaching factors that induce a semi-pluripotent state to substrates for the generation of structured cells and tissues such as those that make up the eye.
In some cases, factors that direct the pre-iPS cells to differentiate are added either concurrently or at a later time to the site of the cells that were induced to become less mature. In other cases, no factors that direct differentiation are added. Instead, factors secreted by the local environment are relied upon to direct the induced cells to differentiate into the desired cell type or tissue type.
In a preferred embodiment, the factors that induce cells to become less mature are MUC1*-associated factors, including but not limited to bivalent anti-MUC1* antibodies and antibody-like proteins, enzymes or agents that increase MUC1 cleavage, as well as introduction of genes that increase expression of MUC1 or NM23 (H1 or H6 dimers or NME7). The invention includes introducing a nucleic acid that codes for a MUC1 cleavage product whose extra cellular domain is comprised essentially of the PSMGFR sequence which is the approximately 45 amino acids that are membrane proximal. In an especially preferred embodiment, the MUC1* associated factor is NM23 in a bivalent or dimeric form, except when NME7 is used as it is a natural “dimer” having two binding sites for MUC1* and able to dimerize it. MUC1* associated factors that induce cells to become less mature can be added alone or together with other pluripotency inducing factors including but not limited to OCT4, SOX2, KLF4, NANOG, c-MYC and/or LIN28.
Sequence Homology of MUC1 Among Mammals
The portions of MUC1 that are membrane proximal are highly conserved among mammals. The membrane proximal portion of human MUC1 is:
RYNLTISDVSVSDV
PFPFSAQSGAC.
We previously showed that the MUC1* activating ligand NM23 binds to the portion of MUC1* that contains the sequence QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:37), also referred to in our previous patent applications as “N-10,” which is missing ten amino acids at the N-terminus of PSMGFR. This portion of MUC1 is 72% homologous between human and mouse with 58% identity. The N-terminal adjacent portion that contains the sequence
GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDV
is 71% homologous between human and mouse with 47% sequence identity. The portion of MUC1 that we previously showed is a self-aggregation domain, GGFLGLSNIKFRPGSVVVQLTLAFRE (SEQ ID NO:39), is 85% homologous between human and mouse with 69% identity.
Because of the great sequence conservation, ligands that recognize human MUC1* receptor, also recognize murine MUC1* receptor. For example, human NM23 in dimeric or bivalent form binds to MUC1 on mouse embryonic stem cells and enables growth while maintaining as well as inducing pluripotency. The addition of human NM23 dimers in minimal stem cell media (MM) completely abolished the need for LIF. Further, growth of mouse ES cells in LIF increased the percentage of cells that expressed pluripotency markers and also increased expression of MUC1, which is itself a pluripotency factor. Therefore, human or mouse NM23 in dimer or bivalent form promotes growth and pluripotency of mouse stem cells, embryonic or hematopoietic, in the absence of any other growth factors. In addition, human or mouse NM23 induces pluripotency in mouse somatic cells. In addition to the natural ligand, NM23, bivalent antibodies that recognize the membrane proximal portion of MUC1 promote and maintain as well as induce pluripotency in murine cells. Because of the great sequence conservation in the membrane proximal regions of MUC1, the invention includes the use of NM23 as well as bivalent antibodies, which recognize the approximately 50 membrane proximal amino acids, for the growth, maintenance and induction of pluripotency in mammalian cells and in mammals in general.
The present invention also encompasses using MUC1* associated factors, which include protein factors, genes that encode them, or small molecules that affect their expression, to induce or improve the efficiency of generating iPS cells. We have shown that a cleaved form of the MUC1 transmembrane protein—MUC1*—is a primal growth factor receptor that mediates the growth of both cancer and pluripotent stem cells. Complete disruption of the interaction between MUC1* extracellular domain and its activating ligand, NM23, is lethal to pluripotent stem cells (Hikita et al., 2008), while treatment with lower concentrations of these inhibitors induced differentiation. The interaction between MUC1* and NM23 was specifically interrupted by treating with an anti-MUC1* Fab to block NM23-induced dimerization of the MUC1* receptor or by adding a synthetic peptide having the same sequence as the MUC1* extra cellular domain so that it would competitively inhibit the interaction between NM23 and its target MUC1* extra cellular domain. These findings indicate that the MUC1-NM23 pathway is critical for pluripotency. NM23 is a ligand that activates MUC1* (Mahanta et al., 2008, Hikita et al, 2008; Smagghe et al, 2013) (SEQ ID NOS:12-17, 22-23, and 34-35). In addition to its ability to stimulate pluripotent stem cell growth, while inhibiting differentiation, NM23 has been reported to induce transcription of c-Myc (Dexheimer at al., 2009), which is a known pluripotency factor. In addition, stimulation of MUC1*, by either NM23 or a bivalent anti-MUC1* antibody, activates the MAP kinase proliferation pathway, which increases cell survival (Mahanta et al., 2008). NANOG expression induces pluripotency; the tumor suppressor p53 suppresses Nanog expression (Lin et al., 2007). Therefore, the need for NANOG for inducing pluripotency is reduced or eliminated by suppressing p53. An ectopically expressed 72-amino acid fragment of the MUC1 cytoplasmic tail (MUC1-CT) has been shown to be present in the nucleus of cancer cells where it binds to the p53 promoter (Wei et al., 2007). The approximately 72 amino acid fragment of MUC1-CD such as shown in SEQ ID NO:11 can be used in combination with other pluripotency-inducing factors to induce or enhance iPS cell generation. However, this peptide does not correspond to a naturally occurring MUC1 species, and therefore may produce undesired effects. The present inventors disclose that MUC1* translocates to the nucleus (Examples 1 and 9, and
The present invention discloses novel reagents and methods, involving MUC1* and its ligands, for inducing cells to revert to a less mature state and even to a pluripotent state. These reagents and methods are used to induce pluripotency in somatic or mature cells. Alternatively, they can be used to induce cells to a less mature state wherein the starting cells are mature cells, progenitor cells or cells that are partially differentiated. In another aspect of the invention, they are used to increase the efficiency of inducing pluripotency in mature cells. In yet another aspect of the invention they are used to maintain immature cells in an immature state. In another aspect of the invention they are used to inhibit differentiation. In another aspect of the invention, these reagents and methods are used for maintaining stem cells in the pluripotent state.
The invention involves reversing differentiation or maintaining stem-like characteristics by introducing to mature cells, or somewhat differentiated cells, genes or gene products that affect the expression of MUC1* and its associated factors. MUC1* is the cleaved form of the transmembrane protein MUC1. MUC1* associated factors include, but are not limited to, enzymes that cleave MUC1, MUC1* activating ligands and also transcription factors that affect the expression of MUC1 or MUC1*. The invention is also drawn to the introduction of the genes or gene products for MUC1* or MUC1* associated factors to mature cells or somewhat differentiated cells, which will induce pluripotency or stem-ness in those cells or their progeny. The present application describes their use for maintaining pluripotency in stem cells. Agents that affect expression of MUC1* or MUC1* associated factors, such as NM23-H1 dimers or NM23-H7 monomers, can be added in combination with, or to replace one or more genes or gene products that are already known to induce pluripotency including OCT4, SOX2, KLF4, NANOG, c-MYC and LIN28.
Forced expression of combinations of the transcription factors, Oct4, Sox2, Klf4 and c-Myc or Oct4, Sox2, Nanog and Lin28 have been shown to cause mature cells to revert to the pluripotent state (Takahashi and Yamanaka, 2006). Each of the transcription factors that induce pluripotency regulates the transcription of about a dozen genes. Among these were several that the inventor has identified as being MUC1-associated factors. OCT4 and SOX2 bind to the MUC1 promoter itself. SOX2 and NANOG bind to the NM23 (NME7) promoter. NM23 (also known as NME) was previously identified, by the present inventor, as the activating ligand of MUC1* (Mahanta et al., 2008). NM23-H1 (also called NME1) binds to MUC1* extra cellular domain and induces dimerization of MUC1* if the NM23-H1 is in dimer form; at higher concentrations, or without mutations that make NM23 prefer dimer formation, NM23 wild type is a hexamer, which does not bind to MUC1* or dimerize it. Therefore, it is only NM23-H1 in dimeric form that induces pluripotency in cells. NM23-H6, also called NME6 can also be dimeric and as such binds to and dimerizes MUC1* growth factor receptor which induces pluripotency. NME7 is also an activating ligand of MUC1*. However, NME7 as a monomer has two binding sites for the MUC1* extra cellular domain and dimerizes MUC1*, thus inducing and maintaining pluripotency. OCT4 and SOX2 both bind to the promoter for MMP16 which we disclose herein is a cleavage enzyme of MUC1. OCT4, SOX2 or NANOG also bind to promoter sites for cleavage enzymes MMP2, MMP9, MMP10, ADAM TSL-1, ADAM TS-4, ADAM-17 (a MUC1 cleavage enzyme), ADAM-TS 16, ADAM-19 and ADAM-28. Some or all of these cleavage enzyme may be upregulated to enhance the cleavage of MUC1 to the MUC1* form to induce pluripotency or maintain it (Boyer et al, 2005).
Our previous work with embryonic stem cells, which only express the cleaved form of MUC1, MUC1*, showed that dimerization of its extracellular domain stimulate growth and inhibit differentiation (Hikita et al., 2008). These effects were achieved by dimerizing the MUC1* extracellular domain using either a bivalent anti-MUC1* antibody, recombinant NM23, or a mutant NM23 (S120G) that preferentially forms dimers (Kim et al., 2003) Inhibition of MUC1* extracellular domain using the monovalent anti-MUC1* Fab was lethal within hours. These findings indicate that MUC1* is a significant factor in maintaining stem cells or causing reversion of cells to a less mature state. In addition, OCT4 and SOX2 bind to the MUC1 gene promoter and also to the promoter of its cleavage enzymes. SOX2 and NANOG bind to the NM23 (NME7) promoter. Since blocking the extracellular domain of MUC1* are lethal to hESCs, it follows that the pluripotency genes, OCT4, SOX2, and NANOG, all induce expression of MUC1, its cleavage enzyme and its activating ligand. One or more of the genes or gene products that have already been shown to induce pluripotency can be replaced by transfecting the gene or introducing the gene product, for MUC1* alone or in addition to its cleavage enzymes and/or activating ligands, NME7, NME1, NME2, NME6 or an antibody that dimerizes the PSMGFR epitope of MUC1 or MUC1*.
As those who are skilled in the art are familiar, nucleic acids that encode the pluripotency genes or the proteins or peptides themselves can be modified with moieties or sequences that enhance their entry into the cell. Similarly, signal sequences can direct the localization of the transfected gene or gene product. Examples of signal sequences are given as SEQ ID NOS:2-4. The invention contemplates the use of gene and protein modifications to any of the pluripotency genes described above to enhance cellular entry of nucleic acids encoding the proteins or the proteins themselves, wherein the proteins include MUC1, MUC1*, NME7, NME1, NME6 and variants thereof. MUC1* is generally described as a truncated form of the transmembrane receptor MUC1, wherein most of the extra cellular domain is not present and the remaining extra cellular domain contains most or all of the PSMGFR sequence. However, MUC1 may be cleaved by different enzymes depending on tissue type or cell type. For example, in stem cells, MUC1 is cleaved to MUC1* by MMP14, MMP16 and ADAM17, whereas in breast cancer T47D cells, MUC1 cleavage is dominated by MMP16 and in DU145 prostate cancer cells it is cleaved by MMP14. Therefore, MUC1* extra cellular domain essentially consists of the PSMGFR sequence, but may be further extended at the N-terminus to comprise additional amino acids. The invention contemplates that the N-terminal domain of MUC1* may be truncated or extended by up to nine (9) amino acids without substantially altering its activity. MUC1* exemplified as SEQ ID NO:5 and variants whose extracellular domain is essentially comprised of the sequences given in SEQ ID NOS: 6, 7, 8 and 9 are preferred.
MUC1, MUC1*, or associated factors, including those listed above, can substitute for one or more of the genes or gene products that induce pluripotency and may be used to induce pluripotency or transition to a less mature state or to maintain that state.
The invention contemplates using any mature cell, including without limitation, somatic cells, which include without limitation, fibroblasts, dermablasts, blood cells, hematopoietic progenitors, nerve cells and their precursors and virtually any kind of cell that is more differentiated than a pluripotent stem cell. In one case, somatic cells such as fibroblasts, dermal fibroblasts, blood cells or nerve cells are transfected with a gene that encodes the MUC1 protein, which aids in inducing stem cell-like features and in some cases induces progeny to become pluripotent stem cells. In another aspect of the invention, a gene for MUC1* is transfected into cells to induce a reversion to a less mature or stem cell-like state and in some cases induce generation of actual pluripotent stem cells. Each of the MUC1 or MUC1* genes may be introduced to the cell alone or in combination with other genes that aid in inducing pluripotency or stem cell-like characteristics. For example, DNA encoding MUC1 or preferentially MUC1* is introduced to the cell along with one or more of the genes that encode OCT4, SOX2, NANOG, LIN28, KLF4, and/or c-MYC. DNA encoding a truncated form of MUC1, preferentially MUC1*, is transfected into fibroblasts along with one or more of the genes encoding OCT4, SOX2, NANOG, and LIN28 (Yu et al., 2007). In another embodiment, DNA encoding a truncated form of MUC1, preferentially MUC1*, is transfected into somatic cells, fibroblasts, or other cells, along with genes encoding OCT4, SOX2, KLF4, and c-Myc (Takahashi et al., 2007). Similarly, DNA encoding MUC1* and/or its activating ligand, NM23 is transfected into cells to induce reversion to a less mature state. In a preferred embodiment, the NM23 family member is NME1 or the S120G mutant of NME1 that prefers dimer formation, NME6 in dimer form, or NME7. In a preferred embodiment, the NME family member is added to the cell culture medium. In a more preferred embodiment, the NME family member is NME7 which may optionally consist of subunits A and B, devoid of the “M” leader sequence: NME7-AB (SEQ ID NOS: 34-35) MUC1* and/or NM23 may be introduced to cells along with other genes such as OCT4, SOX2, NANOG, LIN28, KLF4, and/or c-MYC to induce pluripotency or stem cell-like characteristics. DNA encoding antibodies that recognize MUC1* or MUC1 may also be transfected into cells alone or with other genes to induce stem cell characteristics in the cells or their progeny. If secreted, anti-MUC1* antibodies will dimerize and thus activate the MUC1* receptor, which will function to promote or maintain stem-like characteristics. Alternatively, an anti-MUC1* antibody is exogenously added to cells undergoing induction to a less mature state. In a preferred embodiment, the MUC1* antibody is attached to a surface upon which cells are attached.
Similarly, factors such as nucleic acids, proteins, modified proteins or small molecules that affect the expression of MUC1, MUC1* or their associated factors are introduced to cells to induce characteristics of stem cells or to induce a return to pluripotency. For example, genes or gene products for MUC1 cleavage enzymes, MMP14, MMP16, MMP2, MMP9, MMP10, ADAM TSL-1, ADAM TS-4 ADAM-17 (a MUC1 cleavage enzyme), ADAM-TS16, ADAM-19 and ADAM-28 are introduced to cells to induce pluripotency or similar characteristics.
In another embodiment, non-protein agents are added to cells to induce or enhance the induction of pluripotency. For example the phorbol ester phorbol 12-myristate 13-acetate (PMA) is a small molecule that increases the cleavage of MUC1 to MUC1* (Thathiah et al., 2003). In one aspect of the invention, phorbol ester is added to cells undergoing conversion to pluripotency to induce or increase the efficiency of iPS generation.
In another example, ligands that interact with MUC1 or MUC1* are added to somatic cells, dermal fibroblasts, fibroblasts, or somewhat differentiated cells to induce pluripotency either alone or in combination with other genes to induce or maintain stem-like features or pluripotency. For example, one or more of the genes encoding OCT4, SOX2, NANOG, LIN28, KLF4, and/or c-MYC are transfected into fibroblasts or other cells and then are cultured in the presence of ligands that activate MUC1 or MUC1*. Dimeric, protein ligands of MUC1* are preferred. In a preferred embodiment, a bivalent anti-MUC1* antibody is added to cells that have been transfected with genes that influence cells or their progeny to become pluripotent stem cells.
In a preferred embodiment, NM23 (NM23-H1, NM23-H2, NME6, or variants thereof that are able to dimerize the MUC1* extra cellular domain, NME7 or NME7-AB) is introduced to cells, as the gene that encodes it, as the protein itself or as a protein bearing a leader sequence such as a poly-arginine tract, to facilitate entry into the cell, to aid in the induction or maintenance of pluripotency. The inventors recently showed that when NM23 is secreted by pluripotent stem cells (and cancer cells), it is an activating ligand of the cleaved form of MUC1-MUC1*—and triggers the MAP kinase proliferation pathway. NM23 stimulation of MUC1* was shown to promote the growth of pluripotent hESCs and inhibited their differentiation (Hikita et al., 2008). NM23 also induces the transcription of c-Myc (Dexheimer at al., 2009) and replaces the need for c-MYC. NM23 is added exogenously either in its native state to activate the MUC1* growth factor receptor or with a poly arginine tract to facilitate entry into the cell and nucleus where it induces c-MYC expression. NM23 (NME) may be added as the encoding nucleic acid, or as the expressed protein with or without a modification that facilitates entry into the cell. NME-H1 or -H6 can be used in their native state or in mutant forms that favor the dimeric state, such as the S120G mutation. NME7 is used as the monomeric protein, optionally as a human recombinant protein that is expressed from a construct that encodes the A and B domains but is devoid of the M leader sequence, which we call NME7-AB (SEQ ID NOS:34-35).
In another aspect of the invention, a bivalent antibody that binds to the extracellular domain of MUC1* (PSMGFR) or a dimeric MUC1* ligand, such as NM23, or genes encoding them are added to MUC1*-expressing cells to induce pluripotency, increase the efficiency of the induction of pluripotency, to maintain pluripotency or to inhibit differentiation. The cells to which these MUC1 or MUC1* interacting proteins are added may be naturally occurring cells or those into which genes to induce stem cell-like characteristics have been added, or have already entered the differentiation process or may be stem cells.
Genes for inducing pluripotency may be introduced on the same or different plasmids, which may be lenti viral vector driven or adenovirus vectors or any integrating or non-integrating viral or non-viral vector, or any other system that facilitates introduction of these genes into the desired cells.
In many cases, it is preferential to achieve the effects of pluripotency-inducing proteins by introducing the proteins themselves rather than the nucleic acids or genes that encode them. The invention encompasses genes disclosed herein for the induction of stem-like characteristics or pluripotency that can be replaced by the gene products, the proteins, either in their native state or modified with leader sequences such as poly-arginine tracts to allow entry into the cells. The products of these genes, i.e. proteins, or other proteins which interact with one or more of the products of the transfected genes are introduced to cells to induce or maintain pluripotency or other stem-cell like characteristics.
In other cases, it may be beneficial to introduce synthetic agents, such as small molecules, to induce stem-ness in mature or differentiated cells (Lyssiotis et al. 2009). In one aspect of the invention, small molecules are added to cells that either directly or indirectly induce the transcription of genes that induce pluripotency. In other cases, small molecules that directly or indirectly increase the production of MUC1* are added. In one instance, these small molecules increase cleavage of MUC1 to the MUC1* form, which is a characteristic of stem cells. Phorbol ester, for example, is a small molecule that increases cleavage of MUC1 to MUC1*, so when added to cells, it promotes induction or maintenance of pluripotent state by generating MUC1*.
Use of P53 Inhibitor
P53, which is also known as a tumor suppressor, promotes apoptosis. It would therefore be advantageous to inhibit p53 when culturing stem cells or inducing pluripotency in somatic or other cells. The present invention anticipates using p53 suppressors along with other reagents and methods of the invention to maintain stem-ness or induce stem-like or pluripotent characteristics. P53 can be suppressed by a number of methods. Small molecules such as Pifithrin-μ inhibits the pro-apoptotic effects of p53 (Strom, et al., 2006 September; Komarov, et al., 1999) and thus are optionally added to cells to increase efficiency of induction of pluripotency or to maintain stem-ness. In a preferred embodiment, p53 inhibitors are used along with genes or gene products that up-regulate MUC1 or MUC1*, including but not limited to the MUC1 or MUC1* genes or gene products, their activating ligands and their cleavage enzymes.
Another method for suppressing p53 activity to increase the efficiency of inducing pluripotency or maintaining stem-ness is by the introduction of the MUC1* protein to cell cultures. The MUC1* protein or portions thereof, such as the cytoplasmic domain alone, can be modified by adding on a poly-arginine tract to facilitate entry into the cell. It has been reported that the overexpression of the cytoplasmic tail, alone, of MUC1 (MUC1-CD) resulted in its translocation to the nucleus where it was found to bind to the p53 promoter. These studies could not determine whether MUC1-CD down or up-regulated p53. The present invention is also drawn to the repression of p53 by the ectopic expression of MUC1*, to increase the efficiency of inducing pluripotency or other stem-like characteristics. MUC1* can be introduced by inserting the gene into the cell, by adding the protein itself exogenously or by adding the MUC1* protein that is optionally modified with a poly-arginine tract.
In one aspect of the invention, a MUC1* ligand is added into cell culture media; cells, which may be somatic, differentiated or somewhat differentiated are contacted with the media over the course of several days to several weeks until cells have reverted to the desired state which is a less mature state than the starting cells. In a preferred embodiment, the MUC1* ligand is NME1 in dimeric form. In a more preferred embodiment, the MUC1* ligand is monomeric NME7, which may be devoid of the “M” leader sequence (NME7-AB). Contacting cells with a MUC1* ligand alone is sufficient to make cells revert to a less mature state. In a preferred embodiment, cells to be reverted to a less mature state are contacted by two different types of MUC1* ligand: one that enables attachment of the cells to a surface, such as an anti-MUC1* antibody, and the other a ligand free in solution or media, such as dimeric NM23-H1 or NME7. Optionally a rho kinase inhibitor can also be added to the cell culture media. Evidence of cells reverting to a less mature state by contacting the cells with a MUC1* ligand can be seen in
NME Causes Expression of MUC1 and MUC1*
Culturing cells in NM23-H1 dimers or NME7 causes expression of MUC1 and MUC1* in particular to be increased. Increased expression or activity of MUC1 or MUC1* makes cells revert to a less mature state, which can be a pluripotent state. Evidence of this is documented by detecting a concomitant increase in markers of the pluripotent stem cell state, such as OCT4, SSEA4, Tra 1-60, REX-1, NANOG, KLF4 and others known to those skilled in the art. RT-PCR measurements show that cells cultured in NM23 media have increased expression of MUC1; because PCR measures the RNA transcript, it cannot tell whether or not the protein will be post-translationally modified, such as cleaved to produce MUC1*. However, immunocytochemistry experiments, clearly show that culturing cells or contacting cells with NM23-H1 dimers or with NME7 causes a dramatic increase in the amount of MUC1* expressed. For example, when human fibroblasts were transfected with three (3) or four (4) of the pluripotency inducing genes, also called the “Yamanaka factors” (Oct4, Sox2, Klf4 and c-Myc) and cultured either by the standard method in FGF media or in NM23 media (dimeric form of NM23-H1) then assayed by RT-PCR to quantify expression levels of pluripotency markers as well as MUC1 and MUC1* ligand, NME7, it was observed that as the cells increased expression of the pluripotency markers, there was an associated increase in the expression of MUC1 and the MUC1* ligand NME7. A representative graph of several RT-PCR experiments that showed this effect can be seen in
In another aspect of the invention, cells are reverted to a less mature state and even further to a pluripotent state by contacting the cells with a MUC1* ligand, such as NM23-H1 dimers, NME7, NME7-AB and/or an anti-MUC1* antibody, while also being contacted with other biological or chemical agents that induce pluripotency. In a preferred embodiment, the agents that induce pluripotency are the genes or nucleic acids that encode them, or the proteins themselves, selected from the group comprising OCT4, SOX2, KLF4, c-MYC, NANOG and LIN28. It is known that ectopic expression of two or more of the pluripotency genes selected from the group above will cause cells to revert to the pluripotent state. The state of the art for inducing pluripotency in a more mature cell is to cause the cells to express one or more of the pluripotency genes, while in culture in a medium containing bFGF and sometimes bFGF and TGF-beta. The efficiency of inducing pluripotency (making induced pluripotent stem (iPS) cells) is very low.
We demonstrated that substituting NM23 media for bFGF media vastly improves the efficiency of inducing pluripotency. In addition, the use of feeder cells can be substituted for a layer of extra cellular matrix proteins, or fragments thereof, or a layer of a MUC1* ligand. In a preferred embodiment, cells undergoing induction to a less mature state are plated over a layer of an anti-MUC1* antibody that recognizes MUC1* on stem cells. Table 2 (see Drawings section) shows that substitution of bFGF for NM23 dimers in the media resulted in as much as a 100-fold increase in the efficiency of iPS generation wherein efficiency is calculated by the number colonies generated with stem-like morphology divided by the number of cells required to produce that number of colonies, which is also referred to as an induction rate.
In yet another aspect of the invention, cells are reverted to a less mature or pluripotent state by contacting the cells with a biological or chemical agent that increase expression of MUC1 or MUC1*. Cells transfected with pluripotency genes OCT4, SOX2, KLF4 and c-MYC and cultured in fibroblast serum-containing media then FGF media as is the standard practice for making iPS cells, causes an increase in MUC1 expression that coincides with the expected increases in expression of pluripotency markers such as OCT4, Tra 1-60 and the like. An even greater increase in MUC1 expression is obtained when pluripotency genes are caused to be expressed and cells are contacted with a MUC1* ligand in a media or attached to a surface. However, the invention contemplates culturing cells in suspension or on other surfaces including surfaces coated with extra cellular matrix proteins, fragments of ECM proteins such a fibronectin fragments, vitronectin, feeder cells, cancer cells and the like.
Human fibroblasts were subjected to the standard method for inducing pluripotency, wherein one or more of the genes encoding the Yamanaka factors OCT4, SOX2, KLF4 and c-MYC were used. However, to assess methods of the invention for their ability to increase the efficiency of iPS generation or to induce pluripotency on their own, we used a culture medium that contained NM23 dimers or NME7 instead of the standard bFGF. In one condition of the experiment, no genes were transfected, but the fibroblasts were cultured in a serum-free media with NM23-S120G in dimeric form or NME7 as the only exogenously added growth factor. Some or all of the pluripotency genes were transfected in another arm of the experiment. Another variable of the experiment was that NM23 media was introduced either from the onset of the experiment or at Day 7, when according to the standard protocol, fibroblast medium (FM) would be exchanged for a serum-free medium containing 4 ng/mL of bFGF. At this timepoint, according to the standard protocol, the cells would be moved to fibroblast feeder cells. This was done, but in addition, NM23 cultured cells were moved to a plastic culture plate that had been coated with an mouse monoclonal anti-MUC1* antibody called MN-C3 that the inventors developed for attaching human stem cells to surfaces. “MN-C3” (short hand “C3”) and “MN-C8” (short hand “C8”) are mouse monoclonal antibodies developed by the inventors to specifically bind to MUC1* as it appears on human stem cells. When surfaces are coated with either of these antibodies, it enables human stem cell adhesion, whereas pluripotent human stem cells are non-adherent cells.
Resultant cells were characterized by photographs, RT-PCR quantification of the pluripotency genes, immunocytochemistry and FACS to assess the presence of pluripotency markers; characterization was performed on cells taken between Day 4 and Day 30.
Fibroblasts cultured in either NM23 (dimers) in serum-free media without any genes transfected revert to a less mature state as evidenced by a dramatic change in their morphology, going from fibroblast morphology to a stem cell-like morphology within days. By Day 20, there were no visible differences between the mock transfection cells and actual pluripotent stem cells. Measurement of pluripotency markers indicated that the cells expressed increased levels of pluripotency markers. Mock transfectants cultured in FGF media showed no changes in morphology or in the measurement of pluripotency markers. Cells transfected with some or all of the pluripotency genes Oct4, Sox2, Klf4, c-Myc, Lin28, or Nanog, that were cultured in NM23-S120G dimers consistently expressed pluripotency markers before comparable cells cultured in FGF media, and had a much higher efficiency of inducing pluripotency than the standard FGF method. Several experiments were performed and representative data are described below.
RT-PCR (real time PCR) was also performed at various timepoints for the cells pictured in the figures described above in order to quantify expression levels of key pluripotency genes, such as OCT4, NANOG, KLF4, and sometimes SOX2. Human fibroblasts were transfected with either three (3) or four (4) of the pluripotency genes Oct4, Sox2, Klf4 and c-Myc. It can be seen that by Day 4 post transfection cells cultured in NM23-H1 dimers, which are also a MUC1* ligand that dimerizes the MUC1* extra cellular domain, expressed increased amounts of the pluripotency markers OCT4, NANOG and KLF4, whereas the same cells cultured in fibroblast media showed only a modest increase in OCT4 by Day 4. Therefore, it is concluded that MUC1* ligand NM23 induces pluripotency or reverts the cells to a less mature state over and above that which is due to transfection of the pluripotency genes alone. At the same time, cells induced to revert to a less mature state also have increased expression of MUC1 and NME7, a MUC1* ligand. Recall that RT-PCR detects the nucleic acid so that this assay cannot differentiate MUC1 from MUC1*, since MUC1 is post-translationally cleaved to yield MUC1*. By Day 20, the cells undergoing the standard method for inducing pluripotency wherein FGF media replaces fibroblast media (serum-containing) on Day 7, show increased expression of pluripotency markers OCT4, NANOG and KLF4 as well as a dramatic increase in the expression of MUC1 and NME7.
These results show that the pluripotency genes Oct4, Sox2, Klf4 and c-Myc induce expression of MUC1 and a MUC1* ligand. From our earlier work (Hikita et al) and
Transfectants cultured in fibroblast media showed less than a 2-fold increase in expression of Oct4, Nanog and Klf4 by Day 4, whereas cells transfected with OSK and cultured in NM23 media showed significant increases in the expression of the key pluripotency genes. OCT4 expression increased by 70-fold higher than cells transfected with OSKM but cultured in fibroblast media. NANOG increased 7-fold, and KLF4 increased 4.5-fold over the same cells transfected with all four pluripotency genes but cultured in fibroblast media Importantly, we note that contacting cells with the MUC1* ligand, NM23 caused a 4.5 increase in the expression of MUC1.
MUC1* ligands induce pluripotency and expression of MUC1*. In the induction of pluripotency experiments described above, it was observed that whenever there was an increase in the expression of pluripotency markers, there was an associated increase in the expression of MUC1, MUC1* and MUC1* ligand NME7.
Immunocytochemistry experiments were performed so that MUC1* could be measured directly. iPS and ES cells that were previously cultured in FGF over a layer of mouse feeder cells (MEFs) were switched to culture in a serum-free media containing either NME7 or NM23 dimers as the single growth factor; no other growth factors or cytokines were added. Subsequent analysis by immunocytochemistry of pluripotency markers as well as MUC1* showed that human embryonic stem (hES) and induced pluripotent stem (hiPS) that had been cultured long term in FGF media, which drives human pluripotent stem cells from the naïve state to the primed state (Hanna et al. a, 2010 and Hanna et al. b, 2010)), showed minimal expression of MUC1* that was dramatically increased after being cultured in either NM23 dimer media or NME7 (
Consistent results were obtained that were essentially that: 1) culturing cells in NM23-H1 dimers or in NME7 increased the efficiency of iPS generation wherein two or more of the pluripotency genes were ectopically expressed; 2) culturing cells in NM23-H1 dimers or NME7 caused fibroblasts to revert to a stem-like state without ectopic expression of pluripotency genes; 3) culturing cells in NM23-H1 dimers or NME7 caused an increase in the amount of MUC1 or MUC1* that the cells expressed; and 4) forced expression of pluripotency genes Oct4, Sox2, Klf4 and/or c-Myc caused an increase in the amount of MUC1 or MUC1* that the cells expressed.
Variations in Number of the Yamanaka Pluripotency Genes Used
In this set of experiments, human fibroblasts were transfected with either all four pluripotency genes OSKM, or Three (3) OSK or OSM and cultured in the standard media or in NM23-MM-A or NM23-MM-R. To further characterize the molecular makeup of the cells induced to become pluripotent, cells were transferred from plastic to chamber slides at Day 5, grown until Day 10, then stained for the presence of pluripotency marker Tra 1-60 and for nuclei using DAPI.
Cells and Source of Cells
The invention is not meant to be limited by the type of cell or the source of the cell. We have demonstrated that contacting a cell with NM23-H1 dimers, NM23 mutants or variants that induce dimerization of MUC1, or NME7 induces cells to revert to a less mature state and showed that the progression to a less mature state or a fully pluripotent state occurs over a period of time. In addition to contacting cells with an NME family member, we showed that contacting the cells with a biological or chemical agent that induces expression of one or more pluripotency gene increases the efficiency of reverting the cells to a less mature state. We have demonstrated these discoveries using embryonic stem cells and iPS cells, which are fully pluripotent cells. We have also demonstrated these discoveries using fibroblasts. This choice of demonstration cell types, thus covers the range from the most primitive pluripotent cell to a mature cell. The invention can be used to make virtually any type of cell revert to a less mature state or a completely pluripotent state. Starting cell types include but are not limited to somatic cells, cells from cord blood, bone marrow cells, peripheral blood cells, mobilized blood cells, hematopoietic stem cells, dermablasts, fibroblasts, neuronal cells, nerve cells, hair follicules, mesenchymal stem cells and cells from cerebrospinal fluid.
Cells to be used with methods of the invention may be derived from any one of a number of sources. Cells may be obtained from a patient for autologous uses, or from donors.
In a preferred embodiment, the cells are human. However, we have demonstrated that we can grow mouse stem cells in human NM23-H1 dimers or in NME7 and it alone is sufficient to maintain mouse stem cells in the pluripotent state, without the use of the conventionally “required” LIF. Conversely, the NM23 proteins need not be human because of the large degree of conservation among species. However, human NM23-H1, NME6 and NME7 are preferred. Mutant NM23 proteins, such as the S120G mutation in NM23-H1, that favor dimerization are preferred.
Uses of Stem Cells
Methods of the invention are envisioned to be used in a number of in vitro, in vivo and ex vivo applications. In one aspect, methods of the invention are used to make induced pluripotent stem (iPS) cells in vitro, which can then be used as is or differentiated for any number of applications, including research, drug testing, toxicology, or therapeutic uses.
Trans-Differentiation
In another aspect, methods of the invention are used to make cells revert to a less mature state and then differentiated such that they develop into a desired cells type. Methods of the invention can therefore be used in trans-differentiation techniques, which are also called direct differentiation techniques, wherein cells are only partially reverted to a pluripotent state. Current techniques for trans-differentiation involve inducing expression of two or more of the pluripotency genes (Oct4, Sox2, Klf4, c-Myc, Lin28 and Nanog) for shorter periods of time than is required to make cells fully pluripotent, then introducing biological or chemical agents that direct differentiation to a particular cell lineage or cell type. For example, methods of the invention can be used to induce cells to revert to a less mature state, whereupon the cells are differentiated to a desired state. This can be carried out in vitro, in vivo, or ex vivo. In one aspect of the invention, the cells are in vivo, for example in a patient, where they are reverted to a less mature state and then induced to differentiate to a desired state. For example, about half of the heart cells are fibroblasts. Therefore a treatment for heart conditions that could be ameliorated by increasing the number of healthy cardiomyocytes, is to cause the cardio fibroblasts in situ to revert to a less mature state using methods of the invention, and then contacted them with other agents to induce them to differentiate into cardiomyocytes.
Techniques of the invention need not be carried out completely in vitro or in vivo. In another aspect, cells can be harvested from the cerebrospinal fluid, or from another source, reverted to a less mature state using methods of the invention, then differentiated into some other desired cell type or lineage, such as neuronal cells and then introduced into patient, for example into the spinal fluid, which has access to the brain. The source cells may be obtained from the patient and then re-introduced in a differentiated or semi-differentiated state. Alternatively, cells could be harvested from a patient or donor, which may be derived from the desired lineage and reverted to a less mature state, then introduced to site where they are influenced to become the desired cell type. In this way cells can be harvested from a patient or donor, reverted to a less mature state and then introduced to the part of the patient in need of therapeutic cells, wherein the local environment or the addition of exogenous agents would make the cells differentiate into the desired cell type.
Wound Healing
In yet another aspect, methods of the invention can be used in settings suitable for the promotion of wound healing. In this aspect, agents of the invention are in a medium or immobilized on a support, which may be a bandage, a stent, a scaffold, a scaffold for tissue regeneration, a scaffold or support for spinal cord regeneration and the like. In one example, a bandage coated with a MUC1* ligand such as NM23-H1 dimers or NME7 would revert cells, proximal to an injury, to a stem-like state whereupon they would accelerate healing.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. The following examples are offered by way of illustration of the present invention, and not by way of limitation.
The following examples show that: 1) MUC1 associated factors induce pluripotency in a cell that is not pluripotent; 2) MUC1 associated factors increase the efficiency of iPS generation; 3) MUC1 associated factors replace the requirement for one or more genes that are currently thought to be required for inducing pluripotency. NM23 is a ligand of MUC1* and is a MUC1* associated factor.
MUC1* promotes growth and cell death resistance.
MUC1* promotes clonogenic growth (colony expansion) of fibroblasts. Single cell clones of 3Y1 cells transfected with either full-length MUC1 (SEQ ID NO:1), MUC1*1110 (SEQ ID NO:5) or empty vector were plated at 1000 cells per 60 mm dish in DMEM media containing 10% fetal bovine serum, penicillin/streptomycin and G418 (600 μg/ml). Cells were grown for 9 days and then fixed in 4% paraformaldehyde for 15 minutes at room temperature. Dishes were washed with water and then stained with 1% crystal violet in 70% methanol for 20 minutes at room temperature. Dishes were washed three times with water and allowed to dry overnight at room temperature and photographed.
Anti-MUC1* Fab blocks resistance to cell death by TAXOL® in trastuzumab (HERCEPTIN®)-resistant cells (made resistant by culture in 1 ug/ml HERCEPTIN®). Fessler et al., 2009 reported that HERCEPTIN® resistant cells are also resistant to TAXOL®, doxorubicin and cyclophosphamide. As reported, these drug resistant cancer cells achieve resistance by overexpressing MUC1*. The following experiment showed that blocking the PSMGFR portion of the MUC1* extracellular domain reversed acquired drug resistance in cancer cells. Parental (BT474) or resistant (BTRes1) cells were plated at a density of 10,000 cells/well in 96 well plates, 4 wells/condition. The following day, Anti-MUC1* Fab, control Fab, or no Fab were added to cells in the presence or absence of TAXOL® (Paclitaxel Sigma T7191). Two days later, cells were resuspended in 50 μl trypsin, and counted in the presence of trypan blue. Percent cell death was calculated as percent trypan blue uptake. BT474 cells underwent cell death in response to TAXOL® under each condition, and BTRes1 cells only underwent cell death in the presence of MUC1* antibody (
MUC1* acts as a growth factor receptor, and is activated by dimerization of its extracellular domain using an artificial (anti-MUC1* antibody) or its natural ligand, NM23 (NME). MUC1*-positive ZR-75-30 cells, 6000/well, or control (MUC1-negative) HEK293 cells 4000/well, were plated in 96 well plates. The following day, zero hour cell counts were taken, and different concentrations of anti-MUC1* antibody or Fab were added in medium with low (0.1%) serum every 24 or 48 hours. After several days of incubation, cells were resuspended in trypsin and counted, and percent normalized growth was calculated. Stimulation of ZR-75-30 cells, shown as a bell-shaped curve, as is demonstrated for ligand-induced growth stimulation, but not HEK293 cells (
NM23 binds specifically to the PSMGFR peptide which is comprised essentially of the extracellular domain of MUC1*. Binding was measured by Surface Plasmon Resonance, using a Biacore3000 instrument and BiaEvaluation software. Histidine-tagged MUC1*1110-ecd (SEQ ID NO:5) or irrelevant peptide (HHHHHH-SSSSGSSSSGSSSSGGRGDSGRGDS—SEQ ID NO:40) were immobilized on separate flow channels of 5.7% NTA-Ni++ SAM-coated SPR chips, prepared in our lab as described in Mahanta et al. 2008. 35 μL plugs of NM23, purified bovine or recombinant human, were injected into a constant flow stream of 5 uL/minute and sensograms were recorded. NM23 purified from bovine liver (Sigma N-2635) was diluted in PBS alone. Affinities were measured over a wide range of concentrations using a 1:1 Langmuir model. Actual affinities may vary as first order kinetics cannot adequately describe this system. (
MUC1* Growth Factor Receptor and its ligand NM23 are on undifferentiated hESC, but not differentiated hESC. Human embryonic stem cells in the undifferentiated (pluripotent) state or in the newly differentiating state were analyzed by immunocytochemistry (ICC). Human embryonic stem cells (hESCs) were manually dissected and plated in 8-well chamber slides (Nunc) that had been pre-coated with matrigel. For undifferentiated cells, cells were fixed 5-7 days after plating. For differentiated cells, bFGF was removed from the culture medium 5-7 days after plating and cells were allowed to differentiate for 14 days before fixation. Cells were washed with PBS prior to fixation with 4% paraformaldehyde in 0.1M cacodylate buffer for 15 minutes at 4° C. Cells were blocked for 1 hour with 1% BSA and 1% donkey or goat serum in PBS. 0.1% NP-40 was used with antibodies against intracellular antigens. Primary antibodies were diluted in block and incubated with cells for 1 hour at 4° C. The primary antibodies for the following proteins were used: OCT4 (Santa Cruz, Clone Clones H-134 and C-10, 1:100 dilution), full-length MUC1 (VU4H5, Santa Cruz Biotechnology, 1:50 dilution), MUC1* (Minerva, 1:250 dilution), or NM23 (Santa Cruz, Clone NM301, 1:100 dilution)). Cells were washed 3 times in PBS for 5 minutes prior to incubation for 30 minutes with secondary antibodies: AlexaFluor 488 Goat anti-rabbit IgG, AlexaFluor 555 Goat anti-mouse IgG, AlexaFluor 350 Goat anti-rabbit IgG (Invitrogen, 1:200); Goat anti-mouse IgM-TR (Santa Cruz, 1:100). Cells were washed 3 times in PBS for 5 minutes prior to coverslip mounting using an anti-fade mounting medium (Biomeda). Nuclei were visualized by DAPI staining (1 g/ml) for 5 minutes. Immunostained cells were visualized on an Olympus BX-51 epifluorescent microscope. Results of these experiments show that MUC1* is on the surface of undifferentiated cells (pluripotent stem cells) (
MUC1* mediates growth of pluripotent stem cells.
The following experiment was performed to determine the effect of stimulating MUC1*, using a bivalent anti-MUC1*, on pluripotent stem cells. The results show that adding a MUC1* dimerizing ligand stimulates pluripotent (OCT4-positive) stem cell growth and also enables their growth in the absence of feeder cells, their extracts or bFGF.
Long term growth of pluripotent (OCT4-positive) hESC is mediated by MUC1* stimulation. hESCs were trypsin-dissociated and seeded in 8-well chamber slides pre-coated with matrigel at 4×104 cells/well. Media was changed and antibodies added every other day at a final concentration of 1 μg/ml for bivalent anti-MUC1* until discrete colonies were visible. Culture conditions include ‘minimal stem cell medium’ (hESC media without feeder-conditioned medium) and Hs27-conditioned medium, with and without bFGF supplementation. For each condition, cells were grown in quadruplicate. Cells were washed with PBS and fixed, and OCT4 immunostaining was conducted as described above.
The effect of stimulating MUC1* to enhance the growth of pluripotent stem cells was directly measured in a quantitative Calcein assay. Human embryonic stem cells (hESCs) were manually dissected and grown on matrigel-coated wells of a 96 well plate at a density of 1.9×104 cells/well. Culture media contained hESC media supplemented with 30% Hs27-conditioned medium and 4 ng/ml bFGF. Antibodies were added at a final concentration of 1 μg/ml for bivalent anti-MUC1* and 100 μg/ml for monovalent anti-MUC1*. Experiments were performed in triplicate. 41 hours-post antibody treatment, live and dead cells were quantified with the LIVE/DEAD viability/cytotoxicity kit (Molecular Probes), following manufacturer's instructions. Fluorescence was measured using a Victor3V plate reader (Perkin Elmer). The bar graph of
A long-term stem cell growth experiment was done to compare the effects of stimulating the growth of stem cells using a bivalent anti-MUC1* antibody, NM23, NM23-mutant, or bFGF. hESCs were dissociated with trypsin and seeded in 8-well chamber slides pre-coated with Matrigel at a cell density of 8.2×104 cells/well. Media was changed and antibodies or wild type or mutant NM23 proteins were added every other day at final concentrations of 80 ng/ml for Anti-MUC1* antibody, 1 nM for wild type recombinant NM23 or mutant (S120G) NM23, or recombinant bFGF at a final concentration of 4 ng/ml in ‘minimal stem cell medium’ (hESC media without feeder-conditioned medium). Cells were also grown as a control in minimal stem cell medium with 30% conditioned medium from Hs27 fibroblasts and 4 ng/ml recombinant bFGF (Peprotech #100-18B). Results of this experiment show that MUC1* ligands do a better job of stimulating growth in minimal media of pluripotent colonies than does conditioned media plus bFGF, the ‘normal’ growth medium of these cells on Matrigel. Table 1 details the results.
MUC1* translocates to nucleus of cells. Anti-MUC1* monoclonal Ab was labeled in vitro with Alexa 555 dye, and bound at 4° C. to HCT-116 cells (MUC1-negative) transfected with MUC1*, that had been washed in cold PBS, at 4° C. After 20 min, cells were washed twice in cold PBS, and cells were either fixed in 4% paraformaldehyde, or incubated with pre-warmed growth medium. Cells were washed after 40 minutes, and fixed with 4% paraformaldehyde for 5 minutes, then blocked and permeabilized with 2.5% BSA, 2.5% FBS and 0.1% NP-40 in PBS. Endosomes were stained using an anti-EEA1 antibody (Cell Signaling Technologies, 2411S) and Alexa 488 (Invitrogen 1:200) (
In the examples described below, cells are cultured in either standard fibroblast media (FM), bFGF-based media (FGF-MM) or NM23−S120G-dimer in a minimal stem cell media (NM23-MM) or NME7 devoid of its M leader sequence such that it only contains its A and B domains (referred to as simply NME7 herein or NME7-AB).
Fibroblast Media, “FM”:
for 500 mLs: 435 mL DMEM (ATCC #30-2002 Manassas, Va.), 50 mL fetal bovine serum (FBS, #35-011-cv, Mediatech, Manassas, Va.), 5 mL of 100× stock Glutamax, (#35050061, LifeTechnologies, Carlsbad, Calif.), 5 mL 100× non-essential amino acids (#11140050, LifeTechnologies), 5 mL 100× penicillin/streptomycin (#17-602E, Lonza, Allendale, N.J.)
bFGF Media, “FGF-MM”
for 500 mLs: 400 mL DMEM/F12 (#10565-042), 100 mL Knock Out Serum Replacement, “KOSR” (#10828-028), 5 mL 100× non-essential amino acids (#11140050), 0.9 mL 100× stock 2-mercaptoethanol (#21985-023), all from LifeTechnologies and 2 ug bFGF (#100-18B, Peprotech, Rocky Hill, N.J.).
NM23 Media, “NM23-MM”
for 500 mLs: 400 mL DMEM/F12 (#10565-042), 100 mL Knock Out Serum Replacement, “KOSR” (#10828-028), 5 mL 100× non-essential amino acids (#11140050), 0.9 mL 100× stock 2-mercaptoethanol (#21985-023), all from LifeTechnologies and 8 nM NM23dimer (Minerva, Waltham, Mass.).
OSKM: Oct4, Sox2, Klf4, c-Myc are the pluripotency genes, combinations of which were used in these experiments to induce pluripotency. Several experiments were performed with results being consistent across all experiments. In some cases a lenti viral system was used to cause ectopic expression of the pluripotency genes, while in other cases nucleic acids were used. In still other cases, the proteins themselves were used rather than the genes that encode them.
NM23-MM-R or NM23-R: NM23-MM Replaces the fibroblast media (FM) on Day 7 instead of the standard method of replacing the FM with bFGF-based media (bFGF-M).
NM23-MM-A or NM23-A: NM23-MM is Always present from the onset of the experiment.
TC-MUC1* Ab: Fibroblasts are plated onto a cell culture plate (often tissue culture treated, but not necessarily) that has been coated with an anti-MUC1* antibody (mAb MN-C3 and MN-C8 at 12.5 ug/mL especially preferred here) instead of plastic, then transferring to a layer of fibroblast feeder cells at Day 5.
Vita™-MUC1* Ab: Fibroblasts are plated onto a cell culture plate (Vita™: ThermoFisher) that has been coated with an anti-MUC1* antibody (mAb MN-C3 and MN-C8 at 12.5 ug/mL especially preferred here) instead of plastic, then transferring to a layer of fibroblast feeder cells at Day 5.
HS27: human foreskin fibroblast feeder cells (inactivated)
MEFs: mouse embryonic fibroblast feeder cells (inactivated)
The effect of using NM23 or NME7 on inducing cells to revert to a less mature or pluripotent state.
The procedure for iPS generation was performed wherein all four genes (OSKM) were transfected using a lenti virus system. In this experiment, human fibroblasts were transfected with the four (4) pluripotency genes (ref Yamanaka): Oct4, Sox2, Klf4 and c-Myc, hereafter referred to as OSKM. The standard protocol is to first plate dermablasts or fibroblasts (human fibroblasts “hFFn”: #PC501A-hFF, System Biosciences, Mountain View, Calif.) on plastic and culture them in fibroblast media (FM), changed every 24 hours. After 5 days, the cells are transferred to a surface coated with inactivated fibroblast feeder cells, which can be mouse (MEFs) or human (HS27). For the next 2 days, cells remain in FM. On Day 7 the media is changed to bFGF-M, described above, and media is changed every 24 hours. ˜2-4 weeks post initial plating, colonies (clones) that have embryonic stem (ES) cell-like morphology are selected and individually plated into new wells coated with inactivated feeder cells (MEFs, mouse or HS27 human fibroblasts) and sequentially passaged every 3-4 days. Wells that continue to grow as ES-like cells were propagated and tested for the presence of pluripotency markers.
Contrary to the standard protocol, we tested the effect of NM23 media added Always (NM23-MM-A) or at Day 7 (NM23-MM-R) to replace the fibroblast media (FM) after cells had been transferred onto a layer of fibroblast feeder cells. As controls, the transfection reagent was added but the genes were omitted, “mock transfection” or neither the genes nor the transfection reagents was added, “untransfected,” or the cells were treated according to standard protocol as described above.
The results of the experiment and the rates of iPS induction are shown in Table 2. As can be seen in Table 2, fibroblasts that were not transfected with any genes, but cultured in NM23-S120G in dimer form in a media devoid of serum or other growth factors or cytokines produced colonies with stem-like morphology at a rate at least double that of cells transfected with Oct4, Sox2, Klf4 and c-Myc (OSKM) and cultured according to standard methods, which includes culture in FGF media after Day 7. Fibroblasts transfected with three (3) or four (4) of the pluripotency genes and cultured in NM23-S120G in dimer form in a media devoid of serum or other growth factors or cytokines produced colonies with stem-like morphology at a rate of up to 100-times that of the standard method which uses FGF media. Thus, the efficiency of generating induced pluripotent stem cells or cells that are in a less mature state than the starting cells is far greater when cells are contacted by a MUC1* ligand, wherein NM23-H1 in dimeric form or NME7 are preferred. Induction rate is calculated as the number of colonies with stem-like morphology divided by the number of starting cells.
The effect of NM23 on iPS generation wherein only three (3) of the pluripotency genes were transfected using a lenti virus system. In this experiment, we tested the effect of omitting one of the pluripotency genes. Human fibroblasts (hFFn) were transfected with either the four (4) pluripotency genes, Oct4, Sox2, Klf4 and c-Myc, “OSKM”, or three (3), OSK, or OSM. The gene expression levels of certain genes that are indicative of pluripotency were assessed on Day 4 and on Day 20 using RT-PCR techniques and immunocytochemistry. Note that the primers used in these experiments are designed such that they will not amplify the genes that are being ectopically expressed. RT-PCR was used to quantify the expression level of genes Oct4, Nanog, Klf4, which are indicators of pluripotency. Expression of MUC1 was also measured.
The results of the RT-PCR experiments are summarized in the graphs of
The use of NM23 media enabled the elimination of at least 1 of the 4 pluripotency genes.
In this set of experiments, human fibroblasts were transfected with either all four pluripotency genes OSKM, or three (3) OSK or OSM and cultured in the standard media or in NM23-MM-A or NM23-MM-R. To further characterize the molecular makeup of the cells induced to become pluripotent, cells were transferred from plastic to chamber slides at Day 5, grown until Day 10, then stained for the presence of pluripotency marker Tra 1-60 and for nuclei using DAPI.
FACS was done on populations of all the cells transfected with 4 or 3 genes. Sorting was done to identify cells that were positive for pluripotency markers Tra 1-60 and SSEA4 but negative for the fibroblast marker CD13. The results, shown in Table 3 below show that cells transfected with OSK and not c-Myc and cultured in NM23-MM-A had the highest number of pluripotent stem cells.
In addition, cells induced to be pluripotent by transfecting genes OSKM, OSK, or OSM when cultured in NM23-MM or bFGF-MM were analyzed by RT-PCR to quantify the amount of the pluripotency marker OCT4 they expressed on Day 4 then again at Day 20. As the graphs of
Because of the great conservation of MUC1 proximal regions among mammals, we compared growth of mouse stem cells in the standard media with LIF as the growth factor to growth using NM23 as the growth factor. Mouse ES cells grew as well or better in NM23-MM than mouse ES cells cultured in the standard media containing LIF as the growth factor.
The resultant cells of Examples 10-12 described above, which were repeated four (4) times, were also subjected to FACS analysis to determine the percentage of cells subjected to the pluripotency induction methods that expressed surface markers of pluripotency. FACS analysis should indicate human fibroblast cells induced to revert to a less mature state and to a pluripotent state. The standard method for generating iPS cells takes about 3-4 weeks to develop clones that are pluripotent as evidenced by the expression of pluripotency markers familiar to those skilled in the art. For example, the starting cells in these experiments were fibroblasts. CD13 is a fibroblast marker, whereas cells that have effectively begun to revert to a pluripotent state are CD13-negative. It is known in the art that cells that are pluripotent are positive for pluripotency markers OCT4, KLF4, NANOG, REX-1 and others. Cytoplasmic or nuclear markers are often detected by RT-PCR as described above, while other surface markers are convenient for detecting or sorting live cells by fluorescence activated cell sorting (FACS). At Day 18 and Day 19 after fibroblasts were transfected with some or all of the four “Yamanaka” pluripotency genes, resultant cells were analyzed by FACS for the presence of pluripotency surface proteins SSEA4 and Tra 1-60; cells were also stained for CD13 so that gates could be chosen to exclude any cell that remained positive for the fibroblast marker. Results are shown in
MUC1* ligands increase expression of MUC1*. Experiments were performed with human commercially available human induced pluripotent stem (iPS) cells as well as with human embryonic stem (ES) cells in which we tested the ability of MUC1* ligands to increase the expression or activity of MUC1* as well as their ability to induce cells to revert to a less mature (more pluripotent) state. SC101A-iPSC human iPS cells from Systems Biosciences Inc. were cultured in FGF (4-8 ng/mL) Media over MEFs or in NME7 (a MUC1* ligand—8-16 nM) Media over a surface coated with anti-MUC1* antibody MN-C3 (also a MUC1* ligand-12.5 ug/mL coated onto a Vita™ plate). Except for the growth factor, FGF or NME7, the base media was identical and is described above as “Minimal Media”. Resultant cells were assayed by immunocytochemistry to determine the extent of cells that stained positive for pluripotency markers. The results showed that contacting the cells with MUC1* ligand NME7 caused an increase in the expression of MUC1*, which coincided with an increase in the percentage of cells that stained positive for pluripotency markers. Nuclear stain, DAPI, shows many nuclei that are not also stained by the markers for pluripotency when the cells are cultured in FGF.
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.
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| 61675264 | Jul 2012 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US13/50563 | Jul 2013 | US |
| Child | 14596051 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US12/60684 | Oct 2012 | US |
| Child | PCT/US13/50563 | US |
