WO2010115052A2 - Induced pluripotent stem cells - Google Patents
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- WO2010115052A2 WO2010115052A2 PCT/US2010/029704 US2010029704W WO2010115052A2 WO 2010115052 A2 WO2010115052 A2 WO 2010115052A2 US 2010029704 W US2010029704 W US 2010029704W WO 2010115052 A2 WO2010115052 A2 WO 2010115052A2
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- C12N2501/606—Transcription factors c-Myc
Definitions
- the present invention relates to the production and use of pluripotent cells.
- a pluripotent stem cell has the potential to differentiate into any one of the three different types of germ layers: (1) endoderm cells which constitute the interior stomach lining, gastrointestinal tract, and the lungs; (2) mesoderm cells, which constitute muscle, bone, blood, urogenital cells; and (3) ectoderm cells, which constitute epidermal tissues and nervous system cells. Because of these properties, pluripotent stem cells offer a powerful way to remake or replenish cells in various cell replacement therapies. Pluripotent stem cells thus provide a unique way to obtain a renewable source of healthy cells and tissues, which can be useful for treating any of a number of diseases.
- Pluripotent stem cells are typically isolated from human embryos that are a few days old. Cells from these embryos can be used to create pluripotent stem cell lines that can be grown indefinitely in the laboratory. Multipotent stem cell lines have also been developed from fetal tissue obtained from fetal tissue (older than 8 weeks of development). However, even though pluripotent stem cells can give rise to any fetal or adult cell types, they cannot by themselves divide and develop into a fetal or adult animal because pluripotent stem cells lack the potential to contribute to extraembryonic tissue, such as the placenta. Nevertheless, under the right circumstances, a pluripotent stem cell that is isolated from an embryo can produce almost all of the cells in the body.
- iPS induced pluripotent stem cells
- Induced pluripotent stem cells therefore are a type of pluripotent stem cell that is artificially derived from a differentiated cell, such as an adult somatic cell, by forcing the expression of certain genes in that differentiated cell, which effectively resets the genotype of the cell to that of a pluripotent state.
- iPS cells are believed to have many features in common with natural pluripotent stem cells, such as embryonic stem cells, with regard to the expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, viable chimera formation, and potency and differentiability.
- iPS cells are typically derived by transfection of certain stem cell-associated genes (reprogramming factors; "RPFs") into non-pluripotent cells, such as adult fibroblasts. Transfection is typically achieved through viral vectors, such as retroviruses. Trans fected genes include the master transcriptional regulators Oct-3/4 (Pouf51) and Sox2, which are described herein as nuclear reprogramming factors. After about a month, small numbers of transfected cells begin to become morphologically and biochemically similar to pluripotent stem cells, and are typically isolated through morphological selection, doubling time, or through a reporter gene and antibiotic selection.
- RPFs stem cell-associated genes
- iPS cells Oct-3/4, SOX2, c-Myc, and Klf4.
- a preferred marker that is used to detect iPS is "Nanog" a gene that is important in embryonic stem cells, and a major determinant of cellular pluripotency.
- these reprogramming factors are oncogenic and/or tumor- related.
- the viral transfection systems used to insert the genes at random locations in the host's genome can also lead to undesirable oncogenic and tumorogenic growths because the integration of expression cassettes abnormally disrupts the host cell's genome.
- the current methods for reprogramming differentiated cells require the use of viral vectors to express gene sequences that encode the transcription factors important for reprogramming. This is undesirable and problematic because viral vectors are known to integrate into host cell genome, which is harmful to the cell and the organism. Accordingly, the present invention provides a different, non-dangerous, and non-oncogenic method of producing iPS cells without use of ant DNA or viral vectors.
- One aspect of the present invention is a method for producing a pluripotent stem cell, comprising contacting a differentiated cell (e.g., a somatic cell) with at least one reprogramming factor protein wherein the reprogramming factor protein(s) causes the differentiated cell to dedifferentiate.
- a differentiated cell e.g., a somatic cell
- the reprogramming factor protein(s) causes the differentiated cell to dedifferentiate.
- the reprogramming factor is selected from the group consisting of Oct4, Sox2, c-Myc, Klf4, Nanog, and Lin28.
- the method further comprises contacting the differentiated cells, which have been exposed to a reprogramming factor, with any protein that has reprogramming activity or reprogramming-enhancing activity.
- the method further comprises contacting the differentiated cells with at least one of an inhibitor of p53, pl6(Ink4a), and pl9(Arf), ERas, ECAT15-2, Tell, and beta-catenin, ECATl, Esgl, Dnmt3L, ECAT8, GdO, Soxl5, ECAT15-1, Fthll7, Sall4, Rexl, UTFl, Stella, Stat3, and Grb2.
- the reprogramming factor is linked to a cell penetrating peptide.
- the reprogramming factor is chemically conjugated to the cell penetrating peptide or is recombinantly linked to the cell penetrating peptide as a fusion protein.
- the cell penetrating peptide is a peptide that comprises (a) at least nine contiguous lysine amino acid resides, (b) at least nine contiguous arginine amino acid residues; (c) at least nine residues of a mixture of lysine and arginine amino acids, or (d) the HIV-TAT protein or a fragment thereof.
- the differentiated cell is contacted with the at least one reprogramming factor either in vitro or in vivo.
- the method comprises contacting said differentiated cell with an Oct4 reprogramming factor protein, wherein the Oct4 reprogramming factor protein comprises at least nine contiguous lysine resides or at least nine contiguous arginine residues, and wherein the presence of the Oct4 reprogramming factor protein in the differentiated cell causes the differentiated cell to become a pluripotent stem cell (i.e, and induced pluripotent cell; "iPS").
- iPS pluripotent stem cell
- the method comprises contacting said differentiated cell with a Sox2 reprogramming factor protein, wherein the Sox2 reprogramming factor protein comprises at least nine contiguous lysine resides or at least nine contiguous arginine residues, and wherein the presence of the Sox2 reprogramming factor protein in the differentiated cell causes the differentiated cell to become a pluripotent stem cell (i.e, and induced pluripotent cell; "iPS").
- a pluripotent stem cell i.e, and induced pluripotent cell; "iPS"
- the method comprises contacting said differentiated cell with a c-Myc reprogramming factor protein, wherein the c-Myc protein comprises at least nine contiguous lysine resides or at least nine contiguous arginine residues, and wherein the presence of the c-Myc reprogramming factor protein in the differentiated cell causes the differentiated cell to become a pluripotent stem cell (i.e, and induced pluripotent cell; "iPS").
- a pluripotent stem cell i.e, and induced pluripotent cell; "iPS"
- the method comprises contacting said differentiated cell with a Klf4 reprogramming factor protein, wherein the Klf4 reprogramming factor protein comprises at least nine contiguous lysine resides or at least nine contiguous arginine residues, and wherein the presence of the Klf4 reprogramming factor protein in the differentiated cell causes the differentiated cell to become a pluripotent stem cell (i.e, and induced pluripotent cell; "iPS").
- a pluripotent stem cell i.e, and induced pluripotent cell; "iPS"
- the method comprises contacting said differentiated cell with an Oct4 conjugate, a Sox2 conjugate, a c-Myc conjugate, and a Klf4 conjugate, wherein each of said conjugates comprises a cell penetrating peptide, wherein said conjugates cause said cell to become a pluripotent stem cell (i.e, and induced pluripotent cell; "iPS").
- a pluripotent stem cell i.e, and induced pluripotent cell; "iPS"
- the cell penetrating peptide comprises a plurality of substituents selected from amines, guanidines, amidines, N-containing heterocycles, or combinations thereof.
- the cell penetrating peptide comprises a plurality of reactive units selected from the group consisting of alpha-amino acids, beta-amino acids, gamma- amino acids, cationically functionalized monosaccharides, cationically functionalized ethylene glycols, ethylene imines, substituted ethylene imines, N-substituted spermine, N- substituted spermidine, and combinations thereof.
- the cell penetrating peptide is an oligomer selected from the group consisting of oligopeptide, oligoamide, cationically functionalized oligoether, cationically functionalized oligosaccharide, oligoamine, oligoethyleneimine, and combinations thereof.
- the oligomer is an oligopeptide.
- substantially all of the amino acid residues of the oligopeptide are capable of forming positive charges.
- the oligopeptide comprises 5 to 15 amino acids.
- the oligopeptide comprises 5 to 10 amino acids.
- the oligopeptide comprises at least nine contiguous lysine resides, at least nine contiguous arginine residues; or combinations thereof.
- the differentiated cells is contacted with the reprogramming factor conjugate protein either in vitro or in vivo.
- a reprogramming factor protein comprising a cell penetrating peptide linked to a reprogramming factor selected from the group consisting of Oct4, Sox2, c-Myc, Lin28, Nanog, and Klf4.
- the present invention is not limited to only these particular reprogramming factors; other reprogramming factors may be used in accordance with the methods and techniques disclosed herein for producing iPS cells.
- the cationic conjugate comprises a plurality of substituents selected from amines, guanidines, amidines, N-containing heterocycles, or combinations thereof.
- the cationic conjugate comprises a plurality of reactive units selected from the group consisting of alpha-amino acids, beta-amino acids, gamma-amino acids, cationically functionalized monosaccharides, cationically functionalized ethylene glycols, ethylene imines, substituted ethylene imines, N-substituted spermine, N-substituted spermidine, and combinations thereof.
- the cationic conjugate is an oligomer selected from the group consisting of oligopeptide, oligoamide, cationically functionalized oligoether, cationically functionalized oligosaccharide, oligoamine, oligoethyleneimine, and combinations thereof.
- the oligomer is an oligopeptide. In one embodiment, substantially all of the amino acid residues of said oligopeptide are cationic. In another embodiment, the oligopeptide comprises 5 to 15 amino acids. In another embodiment, the oligopeptide comprises 5 to 10 amino acids. In one embodiment, the oligopeptide comprises at least nine contiguous lysine resides, at least nine contiguous arginine residues; or combinations thereof.
- Another aspect of the present invention provides induced pluripotent stem cells produced from any of the methods disclosed herein.
- the differentiated cells are produced by (A) producing embryonic bodies from the induced pluripotent stem cells made using any of the methods described herein, and (2) incubating the embryonic bodies on ITSFn media, wherein the embryonic bodies differentiate into the cellular morphology of at least one germ layer selected from the group consisting of endoderm germ later cells, mesoderm germ layer cells, and ectoderm germ layer cells.
- Another aspect of the present invention is a method for making differentiated cells, comprising (A) producing embryonic bodies from the induced pluripotent stem cells made using any of the methods described herein, and (2) incubating the embryonic bodies on ITSFn media, wherein the embryonic bodies differentiate into the cellular morphology of at least one germ layer selected from the group consisting of endoderm germ later cells, mesoderm germ layer cells, and ectoderm germ layer cells.
- the differentiated cells are selected from the group consisting of neural cells (e.g., neurons, astrocytes, oligodendrocytes, or Schwann cells), epidermal cells, striated muscle cells, adipose cells, epithelial cells (e.g., respiratory epithelial cells or skin epithelial cells), fibroblasts (e.g., skin fibroblasts), and cornea-like epithelial cells.
- neural cells e.g., neurons, astrocytes, oligodendrocytes, or Schwann cells
- epidermal cells e.g., striated muscle cells
- adipose cells e.g., epithelial cells (e.g., respiratory epithelial cells or skin epithelial cells), fibroblasts (e.g., skin fibroblasts), and cornea-like epithelial cells.
- epithelial cells e.g., respiratory epithelial cells or skin epithelial cells
- Another aspect of the present invention is a method for treating an individual with a disease or injury, comprising (A) producing pluripotent stem cells by contacting differentiated cells from an individual with at least one reprogramming factor protein selected from the group consisting of Oct4, Sox2, c-Myc, and Klf4, wherein the presence of the reprogramming factor protein(s) in the individual's differentiated cells induces development of a pluripotent stem cell; (B) producing differentiated germ layer cells from the induced pluripotent stem cells; and (C) administering the differentiated germ layer cells to the individual, wherein the differentiated germ layer cells are disease-free and useful for treating the individual's disease or injury.
- A producing pluripotent stem cells by contacting differentiated cells from an individual with at least one reprogramming factor protein selected from the group consisting of Oct4, Sox2, c-Myc, and Klf4, wherein the presence of the reprogramming factor protein(s) in the individual's differentiated cells induces development of a pluri
- the disease or injury is a neurodegenerative diseases, leukemia, lymphoma, type 1 diabetes, a traumatic brain and/or spinal cord injury, degenerative spinal cord injuries and diseases, an injury to cardiac muscle, an injury to skeletal muscle, or an injury to skin.
- the neurodegenerative disease is Parkinson's disease, Huntington's disease, or Alzheimer's disease; the degenerative spinal cord injury or disease is amyotrophic lateral sclerosis; the injury to the cardiac muscle is ischemia damage; and the injury to skin are lacerations, chemical burns, and thermal burns.
- Another aspect of the present invention is a method for treating an individual with a disease or injury, comprising administering to said individual pluripotent stem cells made by any of the methods disclosed herein.
- the individual has a neurodegenerative diseases, leukemia, lymphoma, type 1 diabetes, a traumatic brain and/or spinal cord injury, degenerative spinal cord injuries and diseases, an injury to cardiac muscle, an injury to skeletal muscle, or an injury to skin.
- the neurodegenerative disease is Parkinson's disease, Huntington's disease, or Alzheimer's disease; the degenerative spinal cord injury or disease is amyotrophic lateral sclerosis; the injury to the cardiac muscle is ischemia damage; and the injury to skin are lacerations, chemical burns, and thermal burns.
- An aspect of the present invention is a pharmaceutical composition, comprising pluripotent stem cells made by any of the methods disclosed herein.
- a "pluripotent stem cell” is an undifferentiated cell that is capable of differentiating into various cell types of all three germ layers and is capable of in vitro self-replication, or self-renewal, for multiple generations and possibly an indefinite period of time, wherein the resultant daughter cells retain the undifferentiated characteristics of the parent cell.
- Pluripotent cells include embryonic stem cells but, other examples of pluripotent cells include induced pluripotent cells (see, for example, Takahashi et al., Cell, 126: 663-676, 2006; Cell, 131 : 861-872, 2007; and Nakagawa et al., Nat. Biotechnol. 26: 101-106, 2008), pluripotent cells derived by nuclear transfer.
- Figure 1 Cellular uptake of reprogramming proteins fused with 9 repeat arginine as a cell-penetrating peptide: (a) Schematic representation of the mammalian expression vector of reprogramming factors. Each of KLF4, c-MYC, OCT4, and SOX2 cDNA was connected with the 9 repeat arginine as a cell-penetrating peptide and myc tagging peptide at C- terminus; (b) Human newborn fibroblast (HNF) cells were incubated with cell extracts from HEK 293 cells expressing each reprogramming protein at 37°C for 8 hours and were subjected to immunocytochemistry using myc antibodies. Nuclei were counterstained with DAPI.
- HNF Human newborn fibroblast
- FIG. 2 Stable expression of reprogramming factors in HEK293 cells.
- HEK293 cells were transfected with pCMV hKLF4-9xArg-myc, pCMV hOCT4-9xArg-myc, pCMV hSOX2-9xArg-myc, and pCMV hc-MYC-9xArg-myc vectors, respectively and were grown in the presence of G-418 to select the stably transformed cells.
- 50 ⁇ g of cell lystaes protein were subjected to SDS-PAGE followed by western blotting using an anti-myc antibodies.
- FIG. 3 Fluorescence microscopy analysis of COS-7 and HNF cells treated with dsRED and dsRED-9R proteins, a, COS-7 cells and b, HNF cells were treated with the extract of HEK 293 cells expressing dsRED (upper panel) and dsRED fused with 9 repeat arginine (dsRED-9R; lower panel) for 8 hours and were shown in 594nm fluorescence.
- Figure 4 Various protocols for induction of p-iPS cells from human newborn fibroblasts.
- Figure 5 Generation and characterization of rv-hiPSOl cell line derived from human newborn fibroblasts (HNFs) using retroviral factors
- a Immunofluorescence staining of rv- hiPSOl clone shows expression of human ES makers including TRA- 1-60, Oct-4, and SSEA- 4.
- b Embryoid body (EB) -mediated differentiation of rv-hiPS 01 cells.
- EBs are made by suspension culture of rv- iPS cells at day 8. Phase contrast images show all three germ cells differentiated from EBs at day 24 including neural cells (ectodermal), endothelial- like cells (mesodermal), and endoderm-like cells (endoderm).
- Figure 6 Induction and characterization of p-hiPS (01 and 02) cell lines derived from human newborn fibroblasts (HNFs) using a novel protocol for p-iPS cell generation, a, The protocol shows a repeated process for generation of p-iPS cells from HNFs (first image of top panel), b, Protein treated-HNFs were changed in their morphology from 3 cycle repeats (second image of top panel) and repeated processes induce more colony formations after 6 cycles (third image of top panel). These colonies are clearly stained with alkaline phosphatase (AP) (fourth image of top panel).
- AP alkaline phosphatase
- AP -positive colonies form two independent human ES cell lines with similar ES-like morphology on the mouse embryonic fibroblast (MEF).; AP staining of formed iPS cell-like colonies (fourth image of top panel), the early morphology of p-iPS colonies derived from AP-stained iPS cell-like colonies at high magnification (fifth image of top panel), typical morphology of established p-iPS cell line at passage number 10 (second image of top panel).
- Immunofluorescence staining of p-hiPSOl clone (second panel) and p-hiPS02 clone (bottom panel) show expression of human ES makers including AP staining, SSEA-3, SSEA-4, Oct-4, Nanog, and TRA-1-60. Nuclei were stained with 4,6-
- Diamidino-2- phenylindole (blue at second and third row panel), c, Efficiency of iPS- like colony formation.
- FIG. 7 Gene expression and epigenetic status in protein-induced human iPS cells
- a Quantitative RT-PCR was performed to asses the expression of c-MYC, GDF3, KLF4, NANOG, OCT4, REXl, SOX2, and hTERT, in protein-induced human iPS, H9, and HNF cells. Fold change represents the relative gene expression in HNF cells normalized to ⁇ -actin expression. This experiment has been repeated twice in triplicate using independently prepared cDNAs.
- b Bisulfite sequencing analysis of the promoter region of NANOG and OCT4 in protein-induced iPS, H9, and HNF cells. Open and closed circles indicate unmethylated and methylated, respectively. Numbers in the top show CpG location relative to the transcription start site. Percentages of CpG methylation (%Me) were shown in the right of each panel.
- Figure 8 Expression of hES marker genes in HNF, H9, protein-induced iPS (p- hiPSOl and p-hiPS02), and retrovirus-induced iPS (rv-hiPSOl) cells.
- RT-PCR was performed using different primers to discriminate the relative expression of total, endogenous, and retrovirally expressed genes.
- ⁇ -Actin is used for an amplification control.
- Genomic DNAs were prepared from protein- induced iPS, retrovirus-induced iPS, H9, and HNF cells. Genomic PCR was performed using the primer sets containing a highly variable of tandem repeats (Table 3). Amplified PCR products were subjected to 7% acrylamide gel electrophoresis.
- FIG 10 Embryoid body (EB) -mediated differentiation of p- hiPS (01 and 02) cells in vitro and in vivo differentiation
- a EBs are made by suspension culture of two p- iPS cell lines at day 8 (Left at top row panels).
- Phase contrast images (Top row panels) and immunostaining images (second and third row panels) show all three germ cells differentiated from EBs at day 24 including neural cells (ectodermal), muscle (mesodermal) endothelial- like cells (mesodermal), and endoderm-like cells (endoderm).
- b Hematoxylin and eosin staining of teratoma derived from p-hiPS cells (01 and 02).
- the tumors are well-developed from a single injection site after cells were transplanted under the kidney capsules of SCID mice.
- the resulting teratomas demonstrate the following features representing ectoderm, mesoderm and endoderm differentiation (fourth row panel (p-hiPSOl) and bottom row panel (p-hiPS02).
- Ectoderm pigmented retinal cells and neural tissue; mesoderm: muscle and adipocyte, endoderm: respiratory epithelium and gut-like epithelium.
- Figure 11 Scheme 1 illustrates exemplary reprogramming factor protein linked with cationic conjugate at C or N terminus of the protein.
- the polypeptides comprise alpha amino acids, e.g. glycine, alanine, lysine, arginine or histidine, or beta amino acids, e.g. beta-alanine.
- Figure 12 Scheme 2: illustrates exemplary reprogramming factor protein linked with cationic conjugate via the amino acid side chain of the protein with or without linkers.
- the exemplary conjugated reprogramming factor proteins coupled with a cationic conjugate (a polypeptide or a polyamine) from its amino acid side chain directly or indirectly via a disulfide or urea linkage.
- the present invention provides compositions of pluripotent stem cells derived from somatic cells, including biopsy fibroblasts, and methods for producing the same.
- the pluripotent stem cells are provided as relatively substantially homogeneous populations having defined characteristics.
- a substantially homogenous cell population is a population or sample of cells which contain a majority (i.e., >50%) of cells having the desired phenotype (i.e., trait(s) of interest).
- substantially homogenous populations contain at least 60%, at least 70%, at least 80%, at least 90%, or more of the cells having the desired phenotype.
- the reprogrammed pluripotent cell of the present invention can then be cultured to develop and differentiate into a particularly desirable cell type under appropriate culture conditions.
- Methods for cell culturing, developing, and differentiating pluripotent stem cells may be carried out with reference to standard literature in the field. Suitable techniques are described by Wiles et al., Meth. Enzymol. 225:900, 1993; and in Embryonic Stem Cells (Turksen ed., Humana Press, 2002). Established protocols for generation, passaging and preservation of rodent and human pluripotent stem cells are described by, for example, Iannaccone et al. (Dev. Biol.
- culturing step entails the use of a feeder cell layer such as a fibroblast feeder cell layer to culture the iPS cells.
- the present invention also provides a method for preparing clinically feasible cell sources for treatment of human diseases, such a neurodegenerative diseases, in an individual by administering to them a composition or formulation containing iPS cells generated by the methods of the present invention.
- Diseases and disorders amenable to treatment using the therapeutic compositions and cells of the invention include, for example, neurodegenerative diseases (e.g., Parkinson's disease, Huntington's disease, and Alzheimer's disease), leukemia, lymphoma, type 1 diabetes, traumatic brain and/or spinal cord injury, degenerative spinal cord injuries and diseases (e.g., amyotrophic lateral sclerosis), injuries to the cardiac muscle (e.g., following ischemia damage such as a myocardial infarction, or traumatic injury), injuries to the skeletal muscle, and injuries to the skin (e.g., lacerations, chemical burns, and thermal burns).
- neurodegenerative diseases e.g., Parkinson's disease, Huntington's disease, and Alzheimer's disease
- the cells contained in the therapeutic composition are encapsulated. See the subsection below on Genetic Diseases where this embodiment of the present invention is explained in more detail.
- the therapeutic compositions may be administered to the patient by any appropriate, known route.
- cells derived from an iPS cell refers to cells that are either pluripotent or terminally differentiated as a result of the in vitro culturing or in vivo transplantation of iPS cells.
- somatic and differentiated cells to be reprogrammed according to the present invention to become iPS cells are not particularly limited, and any kinds of somatic cells may be used.
- matured somatic cells may be used, as well as somatic cells of an embryonic period, as well as fibroblasts, hepatocytes, and gastric mucous cells.
- any cell types from (1) endoderm cells, such as cells of the interior stomach lining, gastrointestinal tract, and the lungs; (2) mesoderm cells of muscle, bone, blood, urogenital cells; and (3) ectoderm cells of epidermal tissues and nervous system cells may be manipulated according to the present invention and exposed to the reprogramming proteins disclosed herein in order to produce iPS cells.
- the differentiated cell may be any mammalian cell, for example a mouse, human, rat, bovine, ovine, horse, hamster, dog, guinea pig, or ape cell.
- mammalian cell for example a mouse, human, rat, bovine, ovine, horse, hamster, dog, guinea pig, or ape cell.
- somatic cells For example, direct reprogramming of such somatic cells provides an opportunity to generate individual- or disease-specific pluripotent stem cells.
- Mouse iPS cells are virtually indistinguishable from ES cells in morphology, proliferation, gene expression, and teratoma formation.
- mouse iPS cells when transplanted into blastocysts, mouse iPS cells can give rise to adult chimeras, which are competent for germline transmission (Maherali et al., Cell Stem Cell 1 :55-70, 2007; Okita et al., Nature 448:313-17, 2007; Wemig et al., Nature 448:318-324, 2007).
- Human iPS cells are also expandable and virtually indistinguishable from human embryonic stem (ES) cells in morphology and proliferation. Furthermore, these cells can differentiate into cell types of the three germ layers in vitro and in teratomas.
- informed and express consent from a human individual from whom cells are to be obtained directly may be required, such as by needle aspiration withdrawal of tissue and cells, or from any biological sample, such as a blood sample, tissue sample, saliva sample, hair sample, or semen sample, or any bodily fluid sample. See Aalto- Setala, et al., PLoS Biology, vol. 7(2), pp.:204-208 (February 2009), on issues concerning informed consent.
- human somatic cells may be obtained from a biological depository such as the ATCC and cultured according to the accompanying conditions provided with the deposit.
- the reprogramming factors that may be delivered directly to and into cells of the present invention include, but are not limited to families of proteins expressed from genes of the Oct4 family, Sox2 family, Klf4 family, and c-myc family. See Yamanaka, "Strategies and New Developments in the Generation of Patient-Specific Pluripotent Stem Cells," Cell Stem Cell 1 :39-49 (July 2007), which is incorporated herein by reference. Briefly, Oct4 is a transcription factor belonging to the Oct family and is specifically expressed in EC cells, early embryos, and germ cells. The Oct transcription factors contain a POU domain, which is about 150 amino acid region, that binds to the octamer sequence ATTA/TGCAT.
- Oct4 plays an important role in promoting cellular differentiation, and particularly so in neural and cardiac cell differentiation in mice.
- Sox2 is a Sox (SRY-related HMG box) protein which contains a high mobility group (HMG) domain that binds to DNA by recognizing the binding motif A/TA/TCAAA/TG. Sox2 is expressed in the ICM, epiblast, and germ cells and is also expressed by the multipotential cells of the extraembryonic ectoderm. It is associated with uncommitted dividing stem and precursor cells of the central nervous system. For clinical purposes of iPS cells, it is desirable to use human Sox2 cDNA sequences as a fused form with CPP and/or other sequences such as histidine repeat for purification purposes.
- Klf4 is a Kruppel-like factor, zinc-finger protein that is highly expressed in differentiated, postmitotic epithelial cells of the skin and gastrointestinal tract. Klf4 is also expressed in fibroblasts and in undifferentiated mouse ES cells. K14 functions as a tumor suppressor and Oncogene. For clinical purposes of iPS cells, it is desirable to use human Klf4 cDNA sequences as a fused form with CPP and/or other sequences such as histidine repeat for purification purposes.
- c-Myc binds, via its N-terminus, to several proteins, particularly to histone complex components.
- the C-terminus of c-Myc contains a basic region/helix-loop-helix/leucine zipper domain which binds c-Myc to its partner protein Max.
- the c-Myc-Max dimers bind to DNA sequences with the motif CAC A/GTG, which exists throughout the human genome. Binding of the c-Myc-Max dimer can therefore modify chromatin structure and regulate gene expression.
- a problem with c-Myc is that it is known to induce oncogenesis.
- c-Myc cDNA sequences For clinical purposes of iPS cells, it is desirable to use human c-Myc cDNA sequences as a fused form with CPP and/or other sequences such as histidine repeat for purification purposes.
- Each of such nuclear reprogramming proteins may be used alone or in combination with other nuclear reprogramming proteins as disclosed herein.
- a reprogramming protein of the present invention may be used in conjunction with small molecules, compounds, or other agents in order to obtain iPS cells.
- proteins can be co-delivered to the somatic or differentiated cell to help promote or enhance the induced pluripotent stem cell genotype, such as but not limited to Lin28, Nanog, ERas, ECAT15-2, Tell, and .beta.- catenin. Nanog is particularly useful for promoting pluripotency.
- the following proteins also can be delivered to the differentiated cell in conjunction with one or more of the reprogramming factor proteins (e.g., Oct4 protein, Sox2 protein, Klf4 protein, and c-myc protein): ECATl, Esgl, Dnmt3L, ECAT8, GdO, Soxl5, ECAT15-1, Fthll7, Sall4, Rexl, UTFl, Stella, Stat3, and Grb2.
- reprogramming factor proteins e.g., Oct4 protein, Sox2 protein, Klf4 protein, and c-myc protein
- reprogramming factor proteins e.g., Oct4 protein, Sox2 protein, Klf4 protein, and c-myc protein
- ECATl Esgl, Dnmt3L, ECAT8, GdO, Soxl5, ECAT15-1, Fthll7, Sall4, Rexl, UTFl, Stella, StaO, and Grb2.
- another embodiment of the present invention therefore includes the use of any protein with reprogramming activity or reprogramming-enhancing activity to help enhance or promote creation of a pluripotent stem cell.
- any protein with reprogramming activity or reprogramming-enhancing activity to help enhance or promote creation of a pluripotent stem cell.
- recent studies showed that dominant negative form of p53 or an inhibitor of p53 increases the reprogramming efficiency (Hong et al, 2009, Nature, 460, 1132; Utikal et al, 2009, Nature, 460, 1145).
- one embodiment comprises exposing cells, such as differentiated cells, to a dominant negative form of p53 or to an inhibitor of p53.
- Another embodiment comprises coadministering one of the reprogramming factors disclosed herein with a dominant negative form of p53 or with an inhibitor of p53, such a short hairpin RNA molecule (Hong et al.).
- Primary cell populations with low endogenous levels of active Trp53 (i.e., p53), pl6(Ink4a), and pl9(Arf) may also be useful for producing iPS cells at high efficiency (Utikal et al.)
- one embodiment of the present invention contemplates exposing cells to combinations of (1) at least one of Oct4, Sox2, c-Myc, Klf4, Nanog, and Lin28, and (2) a dominant negative form of p53 or an inhibitor of p53, or in conjunction with any protein reprogramming activity or reprogramming-enhancing activity, as described herein.
- Reprogramming factor proteins of the present invention may be extracted, isolated, and purified from tissue samples or cell culture samples.
- Klf4 for instance, is highly expressed in differentiated, postmitotic epithelial cells of the skin and gastrointestinal tract. Hence, it is reasonable to obtain skin and gastrointestinal tract tissue samples from which Klf4 could be extracted and purified.
- the Sox2 is expressed in the ICM, epiblast, and germ cells and multipotential cells of the extraembryonic ectoderm. Hence, these tissue types provide a source of Sox2 proteins.
- Oct4 proteins may be isolated from neural and cardiac cells. There are various standard techniques for isolating proteins from cells, tissues, and cell extracts.
- a reprogramming factor of the present invention may be produced recombinantly by expressing the gene sequence, or a functional part thereof, in a cell.
- An cell expression system may be used to express a nucleotide encoding a functional factor, such as a human cell expression system, chicken hamster ovary cell expression system (CHO), or in insect cells or bacterial cells, for example. It could be desirable to use a mammalian cell expression system to express a reprogramming factor or another enhancing/promoting protein, e.g., nanog, to ensure the resultant expressed protein is properly or authentically folded and formed so as to maximize the activity of the expressed protein.
- a mammalian cell expression system to express a reprogramming factor or another enhancing/promoting protein, e.g., nanog, to ensure the resultant expressed protein is properly or authentically folded and formed so as to maximize the activity of the expressed protein.
- an expression cassette of the present invention may comprise a polynucleotide sequence encoding any one of an Oct4, Sox2, c-Myc, Nanog, Lin28, and Klf4, operably linked to a promoter and appropriate polyadenylation and/or termination signal sequences.
- an expression cassette of the present invention comprises a polynucleotide encoding Oct4, or a functional part thereof, operably linked to suitable regulatory elements that facilitate expression of Oct4 in a cell.
- an expression cassette of the present invention comprises a polynucleotide encoding Sox2, or a functional part thereof, operably linked to suitable regulatory elements that facilitate expression of Sox2 in a cell.
- an expression cassette of the present invention comprises a polynucleotide encoding c-Myc, or a functional part thereof, operably linked to suitable regulatory elements that facilitate expression of c-Myc in a cell.
- an expression cassette of the present invention comprises a polynucleotide encoding Nanog, or a functional part thereof, operably linked to suitable regulatory elements that facilitate expression of Nanog in a cell.
- an expression cassette of the present invention comprises a polynucleotide encoding Klf4, or a functional part thereof, operably linked to suitable regulatory elements that facilitate expression of KlO in a cell.
- an expression cassette of the present invention comprises a polynucleotide encoding Lin28, or a functional part thereof, operably linked to suitable regulatory elements that facilitate expression of Lin28 in a cell.
- a nucleotide sequence that encodes a "functional part" of one of these reprogramming transcription factors is one that encodes that part of the protein sequence responsible for interacting with genomic DNA or with other transcription factors, such as those parts of the factor that recognize particular DNA binding domains or are responsible for homo- or hetero- dimerization.
- another embodiment of the present invention entails making an expression cassette in which a polynucleotide sequence that encodes a functional part of any one of Oct4, Sox2, c-Myc, Nanog, and Kf4, that is operably linked to regulatory sequences, such as to a promoter and appropriate polyadenylation and/or termination signal sequences.
- an expression vector may comprise two or more expression cassettes, each of which expresses a different reprogramming factor. Accordingly, one transformation event may express two or more different reprogramming transcription factors.
- a recombinantly expressed factor of the present invention may then be purified from the extract of the cell expression system according to standard techniques.
- the reprogramming factor protein may be fused chemically or recombinantly, or otherwise associated with a cationic conjugate, such as a cationic lysine-rich or arginine-rich peptide, or with other cationic conjugates as described below.
- a cationic conjugate such as a cationic lysine-rich or arginine-rich peptide, or with other cationic conjugates as described below.
- the cationic conjugate may linked to the protein directly or via an appropriate linkers under suitable conditions.
- the present invention is the creation of a fusion protein, for instance by chemical conjugation or recombinantly, between a reprogramming factor and a cationic conjugate.
- Cationic conjugate herewith comprises a plurality of residues selected from amines, guanidines, amidines, N-containing heterocycles, or combinations thereof.
- the cationic conjugate may comprise a plurality of reactive units selected from the group consisting of alpha-amino acids, beta-amino acids, gamma-amino acids, cationically functionalized monosaccharides, cationically functionalized ethylene glycols, ethylene imines, substituted ethylene imines, N-substituted spermine, N-substituted spermidine, and combinations thereof.
- the cationic conjuage is an oligomer selected from the group consisting of oligopeptide, oligoamide, cationically functionalized oligoether, cationically functionalized oligosaccharide, oligoamine, oligoethyleneimine, and the like, as well as combinations thereof.
- the oligomers may be oligopeptides where amino acid residues of the oligopeptide are capable of forming positive charges.
- the oligopeptides may comprise 5 to 25 amino acids; preferably 5 to 15 amino acids; more preferably 5 to 10 amino acids.
- the present invention provides fusion proteins comprising oligopeptides (polypeptides) where amino acid residues of the oligopeptide are capable of forming positive charges.
- cationic polypeptide such as, but not limited to Oct4-polylysine, Oct4-polyarginine, Sox2-polylysine, Sox2-polyarginine, c-Myc-polylysine, c-Myc- polyarginine, Klf4-polylysine, Klf4-polyarginine, and Nanog-polylysine and Nanog- polyarginine.
- oligopeptides comprising any natural or unnatural amino acid residues capable of forming positive charge, e.g. histidine, ornithine, homoarginine and the like.
- positive charge e.g. histidine, ornithine, homoarginine and the like.
- the present invention also provides fusion proteins that have a reprogramming factor linked to a hybrid cell penetrating peptide made up of both lysine and arginine amino acids and the like.
- the present invention also provides fusion proteins such as Oct4- polylysine/polyarginine, Sox2-polylysine/polyarginine, c-Myc-polylysine/polyarginine, Klf4- polylysine/polyarginine, and Nanog-polylysine/polyarginine.
- a cationic oligopeptide or polypeptide of the present invention includes, but is not limited to, a contiguous string of lysine amino acids, or arginine amino acids, or a mixture of lysine and arginine amino acids, or the like.
- the present invention provides homopolymeric lysine peptides, homopolymeric arginine peptides, heteropolymeric lysine/arginine peptides, and the like, which may be fused, conjugated, ligated, or otherwise joined to a reprogramming factor protein.
- the present invention provides the fusion of the same or different cell penetrating peptide at one or both ends of the reprogramming factor protein.
- the present invention provides the fusion of, for example, a 6-lysine peptide at the N-terminus of a reprogramming factor and a 9-lysine peptide at its C-terminus.
- the length of a cationic lysine or arginine peptide of the present invention may be 5 amino acids in length, 6 amino acids in length, 7 amino acids in length, 8 amino acids in length, 9 amino acids in length, 10 amino acids in length, 11 amino acids in length, 12 amino acids in length, 13 amino acids in length, 14 amino acids in length, 15 amino acids in length, 16 amino acids in length, 17 amino acids in length, 18 amino acids in length, 19 amino acids in length, 20 amino acids in length, or more than 20 amino acids in length.
- the peptide may be between 5 and 10 amino acids in length.
- the cell penetrating peptide comprises 9 residues, i.e., 9 contiguous lysine residues or 9 contiguous arginine residues.
- one or more lysines or arginines may reside within the longer arginine-rich or lysine-rich peptide, respectively. That is, a predominantly lysine-rich peptide may comprise one or more arginine residues; likewise, a predominantly arginine-rich peptide may comprise one or more lysine residues.
- a hetero-cell penetrating peptide of the present invention may comprises a string of contiguous lysine residues adjacent to a string of contiguous arginine residues.
- a hetero-cell penetrating peptide of the present invention may contain 9 lysine residues joined to 9 arginine residues.
- the present invention is not limited to any particular embodiment of cell penetrating peptide composition but may comprises various permutations and arrangements that help facilitate the delivery of a protein to which they are attached across a cell membrane and into the cell environment.
- a reprogramming factor/lysine-or-arginine cell penetrating peptide fusion protein of the present invention can be made in different ways, such as by chemical conjugation and recombinant expression methods.
- a reprogramming factor of the present invention may be chemically conjugated to a cationic oligopeptide of the present invention.
- Standard techniques for conjugating one peptide to another are well known. See, for instance Kennerly S. Patrick, Chemistry of Peptide Synthesis, CRC; 1 edition (August 12, 2005).
- the present invention provides the conjugation of a cell penetrating peptide at the N- terminus of a reprogramming factor or at its C-terminus. Furthermore, the present invention provides the conjugation of the same or different cell penetrating peptide at both N- and C- termini of a reprogramming factor. See Figures 18 and 19 for exemplary embodiments of chemically-conjugated cell penetrating peptides and reprogramming factors.
- amino acids include the (D) and (L) stereoisomers of such amino acids when the structure of the amino acid admits of stereoisomeric forms.
- the configuration of the amino acids and amino acid residues herein are designated by the appropriate symbols (D), (L) or (DL), furthermore when the configuration is not designated the amino acid or residue can have the configuration (D), (L) or (DL).
- D amino acids
- L amino acid residues
- the structure of some of the compounds of this invention includes asymmetric carbon atoms. It is to be understood accordingly that the isomers arising from such asymmetry are included within the scope of this invention. Such isomers can be obtained in substantially pure form by classical separation techniques and by sterically controlled synthesis.
- a named amino acid shall be construed to embrace both the (D) and (L) stereoisomers.
- the term "cationically functional monosaccharides” may include any amine-containing monosaccharide such as glucosamine, galactosamine and 2-amino-sialic acid. It may also include any natural or unnatural derivatized monosaccharides containing one or more functional groups that can form positive charge, e.g. amine and phosphorus containing groups.
- the term “cationically functionalized oligosaccharide” is an oligosaccharide comprising one or more "cationically functional monosaccharides.”
- the term "cationically functionalized ethylene glycols” may include any substituted ethylene glycols where the substituents comprise functional groups that can form positive charge, e.g. amine and phosphorus containing groups.
- the term “cationically functionalized oligoether” may include any substituted oligoether where the substituents comprise functional groups that can form positive charge, e.g. amine and phosphorus containing groups.
- the linker may be selected from the group consisting of a disulfide linkage, a protected disulfide linkage, an ether linkage, a thioether linkage, a sulfoxide linkage, an amine linkage, a hydrazone linkage, a sulfonamide linkage, an urea linkage, a sulfonate linkage, an ester linkage, an amide linkage, a carbamate linkage, a dithiocarbamate linkage, and the like, as well as combinations thereof.
- the linkage can be prepared in a variety of ways, e.g. by functional group conversion at C or N terminus of a reprogramming factor protein (e.g.
- linkage an amine linkage, a sulfonamide linkage, an ester linkage, an amide likage
- linkage of the side chain to appropriate linkers (e.g. a thioether linkage, a sulfoxide linkage, linkage, an ether linkage, an amine linkage, an ester linkage).
- linkers used in the present invention are selected based on the desired length of the linkers, the chemical property of the linkers and the chemistry employed for derivatization. Linkers with more than one possible orientation for attachment to reprogramming factor protein should be understood to embrace all possible orientations for attachment.
- an ester linkage can be linked via hydroxy (-QC(O)-) or via oxo (- C(O)O-) moiety; a sulfonate linkage may be linked via hydroxy (-OS(O) 2 -) or via mercapto (- S(O) 2 O-) moiety; a thiocarbamate linkage may be linked via hydroxy (-OC(S)NH-) or via amino (-NHC(S)O-) moiety.
- Other suitable linkers for attachment of each cationic conjugate can be readily identified.
- CPPs Cell penetrating peptides
- One function of CPPs is to deliver the cargo into cells, a process that commonly occurs through endocytosis, with the cargo delivered to the endosomes of living mammalian cells.
- a "cargo” can be anything from small chemical molecules to nanosize particles and large fragments of DNA and proteins. Any such cargo can be co-joined to a CPP peptide either through chemical linkage via covalent bonds or through non-covalent interactions. Or a nucleotide sequence that encodes a CPP peptide can be engineered into or subcloned adjacent to a polynucleotide that encodes a cargo protein or nucleic acid sequence of interest. Thus expression of the entire sequence could produce, for example, a fusion protein comprising the cargo protein fused to the CPP peptide. Further below is a discussion of the use of CPPs for delivering oligonucleotide cargos to cells.
- CPPs typically have an amino acid composition containing either a high relative abundance of positively charged amino acids such as lysine or arginine, or have sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids.
- HAV-TAT human immunodeficiency virus transactivator of transcription
- a CPP employed in accordance with one aspect of the invention may include 3 to 35 amino acids, preferably 5 to 25 amino acids, more preferably 10 to 25 amino acids, or even more preferably 15 to 25 amino acids.
- the present invention also contemplates encoding nucleotide sequences that encode these peptidic lengths of CPPs.
- a CPP may also be chemically modified, such as prenylated near the C-terminus of the CPP.
- Prenylation is a post-translation modification resulting in the addition of a 15 (farneysyl) or 20 (geranylgeranyl) carbon isoprenoid chain on the peptide.
- a chemically modified CPP can be even shorter and still possess the cell penetrating property.
- a CPP pursuant to another aspect of the invention, is a chemically modified CPP with 2 to 35 amino acids, preferably 5 to 25 amino acids, more preferably 10 to 25 amino acids, or even more preferably 15 to 25 amino acids.
- a CPP suitable for carrying out one aspect of the invention may include at least one basic amino acid such as arginine, lysine and histidine.
- arginine may be encoded by the sequences CGU, CGC, CGA, CGG, AGA, and AGG; lysine may be encoded by the sequences AAA and AAG; and histidine encoded by the sequences CAU and CAC. Accordingly, combinations of these codons can be designed to create particular or desirable CPPs as described next.
- the CPP may include more, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such basic amino acids, or alternatively about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50% of the amino acids are basic amino acids.
- the CPP contains at least two consecutive basic amino acids, or alternatively at least three, or at least five consecutive basic amino acids.
- the CPP includes at least two, three, four, or five consecutive arginine.
- the CPP includes more arginine than lysine or histidine, or preferably includes more arginine than lysine and histidine combined.
- CPPs may include acidic amino acids but the number of acidic amino acids should be smaller than the number of basic amino acids.
- the CPP includes at most one acidic amino acid.
- the CPP does not include acidic amino acid.
- a suitable CPP is the HIV-TAT peptide.
- CPPs can be linked to a protein recombinantly, covalently or non-covalently.
- a recombinant protein having a CPP peptide can be prepared in bacteria, such as E. coli, a mammalian cell such as a human HEK293 cell, or any cell suitable for protein expression.
- Covalent and non-covalent methods have also been developed to form CPP/protein complexes.
- a CPP, Pep-1 has been shown to form a protein complex and proven effective for delivery (Kameyama et al., 2006 Bioconjugate Chem. 17:597-602).
- CPPs also include cationic conjugates which also may be used to facilitate delivery of the proteins into the progenitor or stem cell.
- Cationic conjugates may include a plurality of residues including amines, guanidines, amidines, N-containing heterocycles, or combinations thereof.
- the cationic conjugate may comprise a plurality of reactive units selected from the group consisting of alpha-amino acids, beta-amino acids, gamma- amino acids, cationically functionalized monosaccharides, cationically functionalized ethylene glycols, ethylene imines, substituted ethylene imines, N-substituted spermine, N- substituted spermidine, and combinations thereof.
- the cationic conjugate also may be an oligomer including an oligopeptide, oligoamide, cationically functionalized oligoether, cationically functionalized oligosaccharide, oligoamine, oligoethyleneimine, and the like, as well as combinations thereof.
- the oligomers may be oligopeptides where amino acid residues of the oligopeptide are capable of forming positive charges.
- the oligopeptides may contain 5 to 25 amino acids; preferably 5 to 15 amino acids; more preferably 5 to 10 cationic amino acids or other cationic subunits.
- Recombinant proteins anchoring CPP to the proteins can be generated to be used for delivery to neural progenitor cells or stem cells to prepare mature and functional DA neurons.
- the invention provides a method for producing a neural cell from neural progenitor cells or stem cells by contacting a neural progenitor cell or neural stem cell with at least one protein of the Wntl-Lmxla signaling pathway selected from the group consisting of Wntl, Lmxlb, Lmxlb, Otx2 and Pitx3 and at least one protein of the SHH-FoxA2 signaling pathway selected from the group consisting of SHH, FoxA2 and
- each of the proteins is attached to a CPP.
- the proteins comprise FoxA2, Lmxla and/or Otx2, or alternatively include Nurrl, Pitx3 and/or Lmxla, or alternatively include Nurrl, Pitx3, Lmxla, FoxA2 and/or Otx2.
- the neural cells can be further in contact with one or more of EnI, En2 and/or Ngn2, which can be optionally attached to a CPP.
- the invention provides modified polypeptides useful for producing neural cells from progenitor cells.
- the polypeptides include the various Wntl-Lmxla signaling pathway members (e.g., Wntl, Lmxla, Lmxlb, Otx2 and Pitx3) and the various SHH-FoxA2 signaling pathway members (e.g., SHH, FoxA2 and Nurrl), fused to CPP.
- the CPP is fused to the C-terminus of the proteins either directly or through a linker (e.g., an amino acid or polymer linker).
- Suitable CPPs include, for example, the HIV TAT protein or any polycationic polypeptide or polymer (e.g., at least five consecutive arginine residues).
- One, two, three, four, five, or more of these polypeptides may be incorporated into a pharmaceutical formulation which itself may be administered to a patient, in a therapeutically effective amount, for the treatment or prevention of Parkinson's Disease.
- one embodiment of the present invention concerns providing to a cell a reprogramming factor selected from the group consisting of Oct4, Sox2, c-Myc, Klf4, Nanog, and Lin28, that is linked to a CPP peptide as described herein.
- the present invention contemplates conjugates such as Oct4-CPP, Sox2-CPP, c-Myc-CPP, Klf4-CPP, Nanog-CPP, and Lin28-CPP.
- trans-activating transcriptional activator from human immunodeficiency virus 1 (HIV-I) is a CPP, which is able to deliver different proteins, such as horseradish peroxidase and RNase A across cell membrane into the cytoplasm in different cell lines. Wadia et al. (2004) Nat. Med 10:310-15. Accordingly, in one aspect, a protein, such as Lmxlb, can be delivered to a neural precursor cell using TAT as a vehicle to increase the biological activity of Lmxlb in the cell.
- CPPs examples therefore include the HIV-I TAT sequence, e.g., Tat-(48-60) GRKKRRQRRRPPQ, and the homeodomain of the Drosophila homeoprotein Antennapaedia (penetratin residues 43-58) RQIKIWFQNRRMKWKK; and R8 (octo-arginine) RRRRRRRR. See Howl et al, Biochemical Society Transactions (2007), vol. 35, part 4, pp.767-769, which is incorporated herein by reference. Howl reports that there are various chimeric CPP peptides that can be made which are capable of traversing the cell membrane, which may provide improved translocation properties compared to non-chimeric CPPs.
- chimeric CPPs may include peptides associated with tumor-homing peptides, nuclear localization sequences, protease-cleavable sites, integrin-binding RGD (Arg-Gly-Asp) sequences, sugars, lipids, and membrane-active peptides.
- integrin-binding RGD Arg-Gly-Asp sequences
- sugars lipids
- membrane-active peptides as associated with tumor-homing peptides
- Such chimeric CPPs may be useful for better targeting particular cell types, disease sites, and organelles.
- CPPs can also be used to deliver oligonucleotide cargoes to cells. Howl et al. (supra).
- nucleic acids that can benefit from attachment to a CPP include but are not limited to neutral peptide nucleic acids (PNAs), phosphorodiamidate morpholino oligomers (PMOs), and double-stranded RNA molecules, such as hairpin RNAs and duplexes used in RNA interference or as short interfering RNAs.
- Arginine-rich CPPs and R6-penetratin are useful for delivering PNAs and PMOs.
- a CPP peptide may be non- covalently linked to RNA molecules to improve cellular uptake of siRNA molecules.
- a fusion protein of the present invention also may be made recombinantly.
- An expression cassette that expresses a reprogramming factor, as described in the preceding passages, may be further engineered to comprise a sequence that encodes one or more cationic lysine or arginine peptides. Codons that encode lysine include AAA and AAG. Codons that encode arginine include CGT, CGC, CGA, CGG, AGA, and AGG. Accordingly, permutations of these codons can be engineered into a polynucleotide sequence so that a particular cell penetrating peptide is expressed recombinantly.
- the present invention provides the integration of a polynucleotide that comprises a string of AAA codons, or AAG codons, or combinations of both AAA and AAG codons, into an expression cassette operably linked to the polynucleotide sequence encoding a particular reprogramming factor.
- the lysine polynucleotide sequence may be positioned at the 5 '-end of the reprogramming factor sequence or at the 3 '-end of the reprogramming factor sequence, or a lysine polynucleotide may be positioned at both ends of the factor sequence.
- an arginine-encoding polynucleotide may be made up of one or more CGT, CGC, CGA, CGG, AGA, and AGG codons or permutations thereof; and likewise positioned at the 5 '-end or 3 '-end of a reprogramming factor sequence in the expression cassette.
- Hybrid hetero-lysine/arginine-encoding polynucleotide sequences can be made by combining different codons for each residue, and incorporating that sequence into the expression cassette.
- an expression cassette of the present invention may comprise a polynucleotide sequence that, when expressed, produces a fusion protein comprising at least one cell penetrating peptide and a reprogramming factor.
- the expression cassette may for instance comprise a promoter operably linked to an insert that comprises a polynucleotide sequence encoding a homo- or heter-cell penetrating peptide which is operably linked to a sequence encoding a reprogramming factor, which is operably linked to appropriate termination and polyadenylation sequences to facilitate timely and appropriate expression of that insert.
- the recombinant expression cassette can be expressed in any of a number of available expression systems, such as in human cells, e.g., HEK cells, Chinese Hamster Ovary cells (CHO), insect expression systems, bacterial cell expression systems, or yeast expression systems.
- human cells e.g., HEK cells, Chinese Hamster Ovary cells (CHO), insect expression systems, bacterial cell expression systems, or yeast expression systems.
- the present invention is not limited to the creation of lysine- or arginine-specific fusion proteins with reprogramming factors.
- Other cell penetrating peptides may be chemically conjugated or recombinantly engineered onto a reprogramming factor of the present invention.
- Such other cell penetrating peptides includes, but is not limited to TAT peptides, Penetratin, VP22, and Buforin II.
- one or more reprogramming factor proteins are added to cells which are to become iPS pluripotent cells.
- the reprogramming factor proteins can be isolated and purified from a cell expression system and those purified proteins added to the target cells; or the cellular extract of cells expressing the reprogramming factor proteins can be obtained (by lysing or otherwise breaking open the expression system cells).
- a reprogramming factor of the present invention may be covalently, non-covalently, or recombinantly linked to, joined to, fused to, or associated with any cell penetrating peptide (CPP), as described herein.
- CPP cell penetrating peptide
- either the purified protein or protein extract can be added directly to the target cells.
- These techniques are well known. See for instance Example 3 below. Basically, to prepare cell protein extracts, cells are lysed in a lysis buffer, resuspended, and sonicated. The centrifuged sonicated material consists of a pellet of coarse cellular material and the supernatant the protein extract, which can then be removed and frozen until needed.
- the target cells e.g., differentiated somatic cells
- the target cells can then be incubated with purified individual or multiple reprogramming factor proteins, or protein cellular extract(s) for a first incubation period of a number of hours per week for a number of weeks in a suitable medium, such as ES 1 medium described herein (see Example 4).
- ESl medium is similar to regular media of MEF, but contains a little higher FBS and other ingredients and is useful for facilitating fibroblast-like cells to grow.
- the target cells can be incubated with the reprogramming factor protein(s) for about 5 hours per week, about 6 hours per week, about 7 hours per week, about 8 hours per week, about 9 hours per week, about 10 hours per week, about 11 hours per week, about 12 hours per week, or more than about 12 hours per week; and this cycle can be repeated for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks, or more than about 12 weeks.
- the target cells can be incubated with the reprogramming factor protein(s) for about 0.1 hours per day, about 0.5 hours per day, about 1.0 hours per day, about 2.0 hours per day, about 3.0 hours per day about 4.0 hours per day, about 5.0 hours per day, about 6.0 hours per day, or more than about 6.0 hours per day; and this cycle can be repeated for about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, or about 16 days, or more than about 16 days.
- the cells can then be cultured on ES 2 medium (a mix of ESl and ES3 media for culturing intermediate cells between fibroblast and iPS), for one, two, three, four, five, six, seven days, or more than seven days.
- ES 2 medium a mix of ESl and ES3 media for culturing intermediate cells between fibroblast and iPS
- the cells are incubated for a week in ES 2 medium.
- the cells can be cultured with
- ES 3 Medium for final establishment of iPS cells ; same as normal ES media) for a period of time until iPS formation can be observed. See Example 4.
- Pluripotent stem cell development induced by introduction of one or more reprogramming factor proteins into a differentiated cell can be identified and tracked in a number of ways.
- the iPS cell can be analyzed for the presence of certain marker genes that are expressed in pluripotent cells. See Example 5 and the representative marker genes shown in Table 1.
- the pluripotent marker genes can be identified by RT-PCR of total RNA extracted from sample cells that have been exposed to the reprogramming factor protein(s). See Example 5.
- other standard molecular biology techniques such as Northern blot and probe hybridization identification techniques can be used to determine whether a pluripotent-specific gene is expressed in the post-factor-treated cells of the present invention.
- Treated cells of the present invention also can be assessed for pluripotent stem cell status based on bisulfite genomic sequencing. See Example 6. This technique measures methylation patterns associated with regulatory and promoter regions of a gene. The Nanog and Oct4 genes of pluripotent stem cells have a distinct methylation pattern and thus the identification of these methylation patterns in what used to be a differentiated cell is indicative of the change of the differentiated cell to a pluripotent cell.
- Cells may also be fingerprinted to create a DNA fingerprint profile, such as by the amplification of tandem repeats and microsatellites, and use of standard fingerprinting techniques such as random amplified polymorphic DNAs and restriction fragment length polymorphisms, which produce a DNA pattern based upon cDNAs made from the RNAs expressed in the cell before and after exposure to the reprogramming factor protein(s) of the present invention. See Example 7.
- protein profiles of cells treated with the reprogramming factor protein(s) of the present invention can be generated using Western Blotting techniques (Example 8) and antibody-based immunocytochemistry (Example 9) to identify the presence of pluripotent- specific proteins in cells exposed to the reprogramming factor(s).
- the present invention is not limited to the use of only these particular methods of identifying iPS cells, and one or more of these methods can be used collectively to confirm the change of a differentiated or somatic cell to the status of a pluripotent stem cell.
- Pluripotent stem cells that have been created according to the present invention can then be cultured and grown into a specific type of cell.
- iPS cells can be dissociated and embryonic bodies (EBs) allowed to form for four days after plating of iPS cells in bacterial dishes in the ES medium without bFGF (which makes cells stop proliferating and start differentiating).
- EBs were allowed one day to attach to tissue culture dishes and neuronal precursor were then selected for by incubation in serum-free ITSFn medium for a month.
- ITSFn medium is DMEM/F12 (1 :1) supplemented with insulin (5ug/ml) transferring (50ug/ml), selenium chloride (3OnM), and fibronectin (5ug/ml). (Okabe et al., (1996) Mech Dev 59:89-102). Serum-free ITSFn medium is selectively good for neural precursor cells. Thereafter, various differentiated cell types are seen from EBs.
- EBs are spherical multi-cellular aggregates that comprise a variety of cell populations.
- HNF hepatocyte nuclear factor
- MSCs Mesenchymal stem cells
- MSCs Mesenchymal stem cells
- they can differentiate into at least three lineages (osteogenic, chondrogenic, and adipogenic) when cultured under defined in vitro conditions.
- Previously attempts at differentiation of mature hepatocytes from adult BM including human MSCs have been reported. See Camper and Tilghman, Biotechnology 16, 81-87, 1991; Nahon, Biochimie. 69, 445-459, 1987; and Medvinsky and Smith, Nature 422, 823-825, 2003); and United States Patent 7,332,336, all of which are incorporated herein by reference.
- the reprogramming factor fusion proteins of the present invention may be useful for administering to individuals to correct or treat certain genetic diseases.
- Genetic diseases such as adenosine deaminase deficiency-related severe combined immunodeficiency (ADA- SCID), Shwachman-Bodian-Diamond syndrome (SBDS), Gaucher disease (GD) type III, Duchenne (DMD) and Becker muscular dystrophy (BMD), Parkinson disease (PD), Huntington disease (HD), juvenile-onset, type 1 diabetes mellitus (JDM), Down syndrome (DSytrisomy 21, and the carrier state of Lesch-Nyhan syndrome, may be treated by reprogramming those cells expressing the mutant phenotype into a undifferentiated state.
- the resultant disease-specific stem cells may prove helpful in obtaining normal and pathologic human tissue formation in vitro, thereby enabling disease investigation and drug development. See Park et al., Cell, 134, 877 (2008).
- iPS cells from patient have fundamental dysfunction
- iPS cells or cells derived thereof can be further improved by genetic engineering or other methods so that they can be functional after transplantation. For example, see the paper by Jaenish (Science, 318: 1920- 1923)
- iPS cells from an individual permits the large-scale production of the cell types affected by that individual's disease. These cells could in turn be used for disease modeling, drug discovery, and eventually autologous cell replacement therapies. See Dimos et al, Science, 321, 1218 (2008).
- the present invention provides a "personalized medicine" approach to reprogramming an individual's genetically-diseased cells that can be re-differentiated into a desirable, and genetically- "correct,” genotype.
- the present invention permits the administration of fusion proteins of the present invention directly to an individual, where cells in vivo are reprogrammed; as well as the extraction of cells from an individual to which one or more fusion proteins of the present invention are delivered directly in vitro, whereupon the resultant undifferentiated cells are placed back into the individual, or are further treated and cultured in vitro to differentiate into a new cell type. That new cell type can then be reintroduced into the individual.
- a permanent cell line could be made from the individual's cells and treated with one or more fusion proteins as described herein, and then that undifferentiated cell line used in screening assays to determine the effect of certain drugs, chemicals, compounds, and genetic expression studies to evaluate particular treatment schemes.
- Such regenerative treatments are therefore particularly amenable to the use of the iPS cells produced according to the present invention, such as regenerative treatments for neuropathy and cardiopathy.
- somatic cells involved in diseases can be used to generate iPS cells by adding the reprogramming factor proteins disclosed herein to the somatic cells such that a healthy and replenishable source of new undifferentiated cells can be cultured into a "disease- free" cell line and reintroduced into the individual.
- a method for selecting induced pluripotent stem cells that appear in a medium according to the method of the present invention is not particularly limited, and a well-known means may be suitably employed, for example, a drug resistance gene or the like can be used as a marker gene to isolate induced pluripotent stem cells using drug resistance as an index.
- induced pluripotent stem cells can be efficiently isolated by using a combination of appropriate media. Differentiation and proliferation abilities of isolated induced pluripotent stem cells can be easily confirmed by using confirmation means widely applied to ES cells. See US 2009/0047263, which is incorporated herein by reference.
- the iPS cells of the present invention which are created from the reprogramming effects of certain proteins on differentiated cells, are very useful as drug discovery tools.
- the iPS cells can be used in high-content screening (HCS) assays and used to study and identify cellular events and phenotypes resultant from exposure of the iPS cells to certain compounds and substances.
- HCS high-content screening
- a "compound” or drug which is added to an iPS cell of the present invention can be a chemical, a drug, a therapeutic substance, a compound, a protein, a peptide, or a nucleic acid.
- an iPS cell can be engineered to express a DNA or RNA molecule and used as a host to identify genetic interactions that may prove to be useful targets for the design of particular therapeutics.
- an iPS cell can be useful as a host for sense RNA, antisense RNA, or RNA interference (RNAi) assays, to identify the phenotypic and molecular effect(s) of suppression of expression of one or more particular endogenous genes.
- RNAi RNA interference
- the effectuating nucleic acid which brings about the silencing or downregulation of a gene in the iPS cell may therefore be a candidate for a gene based therapy.
- any compound, drug, or therapeutic substance can be administered to an iPS cell of the present invention and then the iPS cell monitored for any particular effects of the compound, drug, or substance on the phenotype or biochemical or molecular properties of the iPS cell.
- a differentiated cell made from an iPS of the present invention also can be likewise treated with one or more various substances to determine the effect of that substance on the differentiated cell phenotype.
- the iPS cells generated by the methods of the present invention can be useful for, but are not limited to, ADME/Tox assays, cytotoxicity assays, cell authentication screens, studies of apoptosis, cell signaling studies, determination of cell viability, imaging and immunological detection, kinase assays, cytokine assays, and protease assays.
- iPS cells generated by the methods of the present invention are their use in screening for anti-cancer drugs.
- One key mechanism in cancer concerns the abnormal activation or mutation of kinases.
- Kinase inhibitor drugs are an important class of targeted therapeutics with the clinical success of several kinase inhibitors including Gleevec, Sutent, and Sprycell.
- an iPS cell of the present invention can be used to identify kinase inhibitors.
- other classes of proteins and substances can be identified as therapeutic inhibitors of cancers and other diseases, as mentioned in the preceding subsection.
- iPS cells generated by the methods of the present invention can be useful for biological, medical, and cosmetic purposes.
- stem cells are useful in improving cosmetic and therapeutic cosmetic applications.
- iPS cells of the present invention cell- conditioned media and extracts can be used to treat wrinkles, rejuvenate and whiten skin, grow cartilage, and bone, thus making them useful tools in surgery, reconstructive procedures, and cosmetic surgery in procedures such as anti-aging or anti-wrinkle applications and various implantations, such as in breast implant surgery.
- the iPS cells of the present invention are particularly useful in regenerating skin from a patient.
- One beneficial application is therefore the use of the iPS cells created by the present invention for creating skin grafts that can be applied to burn victims, treatment of skin inflammation, and for healing or repairing wounds. See also U.S. Patent No. 7,479,279, which is incorporated herein by reference.
- iPS cells of the present invention are a useful and more patient-friendly alternative to certain existing cosmetic procedures currently available, such as painful Botox injections.
- iPS cells or extracts or isolates e.g., proteins, RNAs, and other isolated cellular components
- cytokines can be isolated from the iPS cells of the present invention. Cytokines are useful signaling molecules that, generally-speaking, have autocrine, paracrine, and endocrine functions, which regulate immunity, inflammation, and hematopoiesis. The largest group of cytokines stimulates immune cell proliferation and differentiation.
- This group includes Interleukin 1 (IL-I), which activates T cells; IL-2, which stimulates proliferation of antigen-activated T and B cells; IL-4, IL-5, and IL-6, which stimulate proliferation and differentiation of B cells; Interferon gamma (IFNg), which activates macrophages; and IL-3, IL-7 and Granulocyte Monocyte Colony-Stimulating Factor (GM-CSF), which stimulate hematopoiesis.
- IL-I Interleukin 1
- IL-2 which stimulates proliferation of antigen-activated T and B cells
- IL-4, IL-5, and IL-6 which stimulate proliferation and differentiation of B cells
- IFNg Interferon gamma
- IL-3, IL-7 Granulocyte Monocyte Colony-Stimulating Factor
- GM-CSF Granulocyte Monocyte Colony-Stimulating Factor
- the invention provides cosmetic preparations (e.g., skin creams, lotions, and solutions) which contain cell culture media in which the iPS cells of the present invention have been grown, or extracts or filtrates thereof ("conditioned cell media").
- cosmetic preparations e.g., skin creams, lotions, and solutions
- cell culture media in which the iPS cells of the present invention have been grown, or extracts or filtrates thereof
- HNF Human newborn fibroblasts
- ATCC CCL-117
- HNF Human newborn fibroblasts
- DMEM Dulbecco's modified Minimal Essential Medium
- Invitrogen Carlsbad, CA
- 2mM L-glutamine Invitrogen
- ImM ⁇ -mercaptoethanol, Ix non-essential amino acids NEAA
- FBS fetal bovine serum
- penicillin 100 ⁇ g/ml streptomycin
- iPS cells were generated and maintained in human ES 3 medium (DMEM (Invitrogen, Carlsbad, CA), supplemented with 2mM L-glutamine (Invitrogen).
- DMEM Human ES 3 medium
- Human ES cells and iPS cells were maintained on feeder layers of mitomycin C (lO ⁇ g/ml media, Sigma- Aldrich)-treated MEF cells.
- mitomycin C lO ⁇ g/ml media, Sigma- Aldrich
- human iPS cells were washed once with ES medium and then mechanically handpicked (up to passage 35) or incubated with 0.1 % collagenase type IV solution for 10 min. An appropriate volume of the medium was added, and the contents were transferred to a new dish onto MEF feeder cells. The split ratio was 1 :1 (until passage 3) and after that, routinely 1 :5.
- the plate was coated with gelatin (StemCell Tech.).
- EXAMPLE 2 PLASMID CONSTRUCTION
- Human cDNAs for OCT4, SOX2, KLF4, and c-MYC were amplified by RT-PCR from human ES poly(A + )RNA using the primers 5 '-GGA TCC GAA TTC ATG GCG GGA CAC CTG GCT TCGG-3' (SEQ ID NO.: 1) and 5'-AAA AAA GTC GAC gcg gcg tct gcg tct gcg gcg tct gcg GTT TGA ATG CAT GGG AGA GCC-3 ' (SEQ ID NO.
- HEK 293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum containing 100 units/ml penicillin and 100 ⁇ g/ml streptomycin.
- DMEM Dulbecco's modified Eagle's medium
- a 2 ⁇ g plasmid DNA was transfected to approximately 4 x 10 5 cells by LipofectamineTM (Invitrogen).
- 1/1000 of cells 48 hours after transfection were seeded and further cultured in the presence of 500 ⁇ g/ml neomycin (G418 Sulfate, Clontech, Palo Alto, CA). An individual colony was isolated from neomycin-resistant colonies. The expression of 4 factors from neomycin-resistant colonies was determined by Western blot analysis ( Figure 2).
- cell extracts were washed in PBS and in cell lysis buffer (100 mM HEPES, pH 8.2, 50 mM NaCl, 5 mM MgCl 2 , 1 mM dithiothreitol, and protease inhibitors), sedimented at 40Og, resuspended in 1 volume of cold cell lysis buffer, and incubated for 30- 45 min on ice.
- Cells were sonicated on ice using a Labsonic-M pulse sonicator fitted with a 3-mm-diameter probe until all cells and nuclei were lysed, as judged by microscopy.
- the lysate was sedimented at 15,00Og for 15 min at 4°C to pellet the coarse material.
- the supernatant was filtered with 0.2 ⁇ m membrane, aliquoted, frozen quickly in a dry ice, and stored at -80 0 C.
- EXAMPLE 4 RETROVIRAL INFECTION. PROTEIN TRANSDUCTION AND IPS GENERATION
- HEF cells cultured in vitro were incubated with the viral supernatant containing polybrene (hexadimethrine bromide; 1 ⁇ g/ml; Sigma) for 2-4 h. After infection, the cells were incubated five more days in normal culture media (DMEM with 15% defmed-FBS). At six days of infection, cells were replated on gelatin-coated plate. Then virus-infected HEF cells are cultured DMEM with 15% defmed-FBS for 24 hrs.
- polybrene hexadimethrine bromide
- HEF cells cultured in vitro were incubated with the 4 protein factors (each 12 ⁇ g/ ⁇ l) for 8 hrs per week up to 6 weeks with ES 1 medium (DMEM supplemented with 2mM L-glutamine, ImM ⁇ -mercaptoethanol, Ix non-essential amino acids, 20% FBS, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin and 1500 U/ml LIF).
- ES 1 medium DMEM supplemented with 2mM L-glutamine, ImM ⁇ -mercaptoethanol, Ix non-essential amino acids, 20% FBS, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin and 1500 U/ml LIF.
- ES 2 medium mixed medium (ratio; 1 (ESl medium) :3 (ES 3 medium)
- ES 3 medium DMEM supplemented with 2mM L- glutamine, ImM ⁇ -mercaptoethanol, Ix non-essential amino acids, 20% KSR, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin
- ES 3 medium DMEM supplemented with 2mM L- glutamine, ImM ⁇ -mercaptoethanol, Ix non-essential amino acids, 20% KSR, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin
- Genomic DNA from cells was performed with the DNeasy Tissue Kit (Qiagen). Bisulfite treatment was done using the EpiTect Kit (Qiagen) following the manufacturer's instruction.
- the promoter regions of the human NANOG and OCT4 were amplified by PCR using primers published previously (Deb-Rinker et al., 2005 JBC 280:6257-6260; Freberg et al, 2007 MCB 18:1543-1553; Table 2).
- the resulting amplified PCR products were gel- purified, subcloned into the pGEM-T Easy vector (Promega) and sequenced.
- VNTR highly variable numbers of tandem repeats
- the cells were lysed with RIPA buffer containing of 5OmM Tris (pH7.5), 15OmM NaCl, 1% NP-40, 0.5% deoxycholic acid, and 0.1% SDS), supplemented with protease inhibitor cocktail (Roche) and mixed with an equal volume of a sodium dodecyl sulfate (SDS)-sample buffer consisting of 125mM Tris (pH 6.8), 2% SDS, 15% glycerol, 5% ⁇ -mercaptoethanol, 0.05% bromophenol blue. Samples were subjected to SDS- polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose membrane (Hybond-ECL, Amersham).
- SDS sodium dodecyl sulfate
- the membrane was incubated with 1 :3000- dilution of an anti-myc antibody (Roche), followed by reaction with 1 :300-dilution of a horseradish peroxidase-conjugated anti-mouse immunoglobulin G (IgG) antibody (Amersham). Detection was achieved using an enhanced-chemiluminescent substrate (Amersham).
- Alkaline phosphatase (AP) staining was performed using the Alkaline phosphatase staining kit II (Vector Vector Laboratories, Burlingame, CA).
- iPS cells were dissociated and EBs were allowed to form for four days after plating of iPS cells in bacterial dishes in the ES medium without nFGF. EBs were allowed one day to attach to tissue culture dishes and neuronal precursor were then selected for by incubation in serum-free ITSFn medium for a month. Thereafter, various differentiated cell types are seen from EBs.
- Protein-derived Human iPS cells (clone 1 and 2) were suspended in DMEM containing 10% FBS. Nude mice were anesthetized with diethyl ether. We injected the cell suspension under the kidney capsule. Four weeks after the injection, tumors were surgically dissected from the mice. Samples were weighed, fixed in PBS containing 4% formaldehyde, and embedded in paraffin. Sections were stained with hematoxylin and eosin.
- EXAMPLE 12 DIRECT DELIVERY OF RECOMBINANT REPROGRAMMING PROTEINS TO HUMAN SOMATIC CELLS
- HNF human newborn fibroblasts
- RFP-9R red fluorescent protein
- Figure 3 Next stable HEK293 cell lines were generated that can express each of the four human reprogramming factors (Oct4, Sox2, Klf4, and c-myc) fused with 9R and the myc tag ( Figure 1).
- HNF cells (5x10 5 ) were treated with combined total extracts of four stable HEK 293 cell lines for 16 hours. See Protocol 1 in Figure 4. After washing, cells were incubated for 6 days in ES Media 1 (DMEM supplemented with 2mM L-glutamine, ImM ⁇ - mercaptoethanol, Ix non-essential amino acids, 20% FBS, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin and 1500 U/ml LIF). Those cells were then transferred onto mouse embryonic feeders (MEF). These transferred cells were incubated with ES Media 2 (mixed medium (ratio; 1 (ESl medium) :3 (ES 3 medium)) for up to 4 weeks but no reprogrammed colonies were identified.
- ES Media 1 DMEM supplemented with 2mM L-glutamine, ImM ⁇ - mercaptoethanol, Ix non-essential amino acids, 20% FBS, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin and
- ImM ⁇ -mercaptoethanol Ix non-essential amino acids (NEAA; Invitrogen, Carlsbad, CA), 20% knock-out serum replacement (KSR, Invitrogen, Carlsbad, CA), 100 U/ml penicillin, 100 ⁇ g/ml streptomycin (Invitrogen)), for 7 days each.
- NEAA Invitrogen, Carlsbad, CA
- KSR knock-out serum replacement
- 100 U/ml 100 U/ml penicillin
- 100 ⁇ g/ml streptomycin Invitrogen
- EXAMPLE 14 CHARACTERIZATION OF PROTEIN-INDUCED hiPS CELL LINES
- Human iPS lines were also generated from the same HNFs using the retroviral vectors expressing the same four reprogramming factors (Takahashi et al., 2007 Cell 131 :861; Yu et al, 2007 Science 318:1917; Park et al., 2008 Nature 451 :141), which satisfied the same criteria of iPS cells as described in this study ( Figure 8) One of them (rv-hiPSOl) was used as a control ( Figure 9). Since hES H9 and rv-hiPS01 lines were used as control lines, it was important to rule out the possibility that the p-hiPS cells were derived from these contaminating cells.
- RT-PCR analyses detected chromosomal integrations of the transgenes in rv-hiPSOl cells, but not in p-hiPSOl and p-hiPS02 cell lines (Figure 9). Furthermore, DNA fingerprinting analysis demonstrated that the patterns of both p-hiPS lines and rv-hiPSOl cells are identical to parental FINF cells, but are different from those of hES H9 cells, thus confirming that both p-hiPS lines are derived from HNF cells (ATCC cat no. SCRC1071). Furthermore, both p-hiPS lines exhibited the normal karyotype of 46XX at least for 35 passages (Figure 10).
- Embryoid body (EB) formation was then next induced from these p-hiPS cells by suspension culture ( Figure 10). After 8 days, well-formed EB structures were generated from both p-iPS clones. When these EB-like structures were incubated on gelatin-coated tissue culture plates in ITSFn media for 15 to 25 days, they differentiated to various cell morphologies of all three germ layers, including neural cells, muscle cells, and endoderm-like cells ( Figure 10).
- teratomas contained tissues of all three germ layers including neural tissues (ectoderm), epidermal tissues (ectoderm), striated muscle (mesoderm), adipose tissue (mesoderm), respiratory epithelium (endoderm) and cornea-like epithelial tissues (endoderm) (Figure 10), showing that both p-hiPS clones exhibit pluripotency both in vitro and in vivo.
- neural tissues ectoderm
- epidermal tissues ectoderm
- striated muscle mesoderm
- adipose tissue meoderm
- respiratory epithelium endoderm
- cornea-like epithelial tissues endoderm
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- 2010-04-01 KR KR1020117026184A patent/KR20110134939A/en not_active Application Discontinuation
- 2010-04-01 EP EP10759439.2A patent/EP2414510A4/en not_active Withdrawn
- 2010-04-01 WO PCT/US2010/029704 patent/WO2010115052A2/en active Application Filing
- 2010-04-01 US US13/260,897 patent/US20120128655A1/en not_active Abandoned
Non-Patent Citations (1)
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Also Published As
Publication number | Publication date |
---|---|
EP2414510A4 (en) | 2013-04-17 |
EP2414510A2 (en) | 2012-02-08 |
WO2010115052A3 (en) | 2011-08-04 |
KR20110134939A (en) | 2011-12-15 |
US20120128655A1 (en) | 2012-05-24 |
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