Patent Application
20110053166
The present invention relates to nucleic acid sequences comprising regulatory sequences that direct expression of tag sequences in stem cells. The invention furthermore provides nucleic acid sequences that direct expression in embryonic or induced pluripotent stem cells.
Embryonic stem cell-like pluripotent stem cells, called induced Pluripotent Stem (iPS) cells, can be induced by introducing 1 to 4 genes into somatic cells using retroviral vectors in vitro (see, for example Okita et al., 2007). This ‘reprogramming’ of iPS cells is inefficient, and optimization may be desirable. The unique morphology of mouse and human iPS cells can be used to isolate reprogrammed cells, however distinguishing of iPS morphology requires substantial experience of stem cell culture to allow for ease of identification (Takahashi et al 2007). More consistent methods of identification of iPS cells are needed.
In order to facilitate identification of reprogrammed cells, they may be screened for the expression of markers expressed at the embryonic stage, for example SSEA-1 (stage-specific embryonic antigen-1) for mouse iPS cells; SSEA-3 (stage-specific embryonic antigen-3), SSEA-4 (stage-specific embryonic antigen-4), TAR-1-60 or TAR-1-81 for human iPS. Those surface markers may not directly reflect the reprogrammed nuclear state, since there is no functional link between surface markers and pluripotency. Use of an antibody to interact with the surface marker may adversely affect the cell, and, depending on the details of the method used, the tested cells may not be viable. Since the efficiency of reprogramming is very low, a method to enrich a population of cells for reprogrammed cells when sorting may be useful.
Viral vectors, such as retroviral vectors, represent efficient vehicles for introduction of foreign nucleic acid into iPS cells. Retroviral transgene expression after integration, however, tends to be silenced or attenuated in pluripotent stem cells, such as embryonic stem cells (ES), embryonic carcinoma cells (EC) and iPS cells (see, for example, Yao et al., 2004; Okita, supra; Wernig et al., 2007; Meissner et al., 2007), thus conveying a marker that is intended to be expressed only in ES or iPS cells in such a vehicle may be counterproductive.
Stem cells are cells that retain the ability to self-renew (undergo multiple cycles of cell division while maintaining an undifferentiated state), and are capable of differentiation into other cell lineages or specialized cell types (potency). Embryonic stem cells (ES cells, or ES) are stem cells found at the blastocyst stage of embryonic development. ES cells generally have the potential to differentiate into any or all of the specialized embryonic tissues in any of the three primary germ layers—endoderm, ectoderm, and mesoderm.
A pluripotent stem cell is capable of giving rise to any or all of the various cell types that make up the body, but cannot normally differentiate into extraembryonic tissues.
Both human ES cells (hES cells, or hES) and mouse or murine ES cells (mES cells, or mES) are the subject of research—both have key stem cell characteristics of pluripotency and self-renewal. The growth conditions and markers required for each differ however—for example, mES may be grown on a layer of gelatin, and require the presence of LIF (leukemia inhibitory factor) in the culture medium, while hES generally require a feeder layer of mouse embryonic fibroblasts (MEF), and FGF-2 (fibroblast growth factor-2) in the culture medium. Thus, experimental manipulations that are demonstrated to work in mES do not always transfer to a human system—the outcome may be unpredictable.
Human ES or mES, when injected directly into a subject, will differentiate into a variety of cell types, and form a generally disorganized mass referred to as a teratoma. In order for hES or mES to be used in therapeutic applications, or even as a consistent source of experimental material, differentiation must be controlled to provide for useable cells. Residual undifferentiated cells must be killed or otherwise removed to prevent teratoma formation after transplantation into a subject.
A variety of protocols for differentiating ES into specific cell types are known, and the selection of a suitable protocol may depend on the source of the ES (e.g. human or mouse, or other species), the desired tissue, cell type or developmental stage that the ES is to be differentiated into, or the desired end use of the differentiated cell. See for example, Current Protocols in Stem Cell Biology (Wiley Interscience)
An iPS is a pluripotent stem cell artificially derived from an adult somatic cell, through introduction of specific transcription factors. Methods of inducing pluripotent stem cells from mouse and human fibroblasts are described in, for example Takahashi, supra; and Takahashi and Yamanaka, 2006, both of which are herein incorporated by reference. These methods involve introduction of pluripotency factors into human or murine fibroblasts. Pluripotency factors include transcription factors that, when expressed in a somatic cell, result in the reprogramming of the cell and induce it to develop into a pluripotent state.
A vehicle for introducing nucleic acid sequences to be expressed specifically in ES or iPS cells is desired.
The present invention relates to nucleic acid sequences comprising regulatory sequences that direct expression of tag sequences in stem cells. The invention furthermore provides nucleic acid sequences that direct expression in embryonic or induced pluripotent stem cells.
In accordance with one aspect of the invention, there is provided a nucleic acid comprising a pluripotent stem cell specific promoter, operatively linked to a tag sequence. The pluripotent stem cell-specific promoter may be an ETn promoter sequence (SEQ ID NO: 1), an ETn poly A mutated (pAMu) promoter sequence (SEQ ID NO: 2), or other pluripotent stem-cell specific promoter.
In accordance with another aspect of the invention, the nucleic acid may further comprise one or more than one pluripotent stem cell specific enhancer sequence. A pluripotent stem cell specific enhancer sequence is an enhancer sequence active in a pluripotent stem cell. Each of the pluripotent stem cell specific enhancer sequences is operatively linked to the pluripotent stem cell specific promoter and may be in a forward (positive or “+”) or reverse (negative or “−”) orientation. The one or more than one pluripotent stem cell specific enhancer sequence may be operatively linked 5′ or 3′ relative to the promoter, or the tag sequence, or both the promoter and tag sequence. The pluripotent stem cell specific enhancer sequence may be CR4, SRR2, a combination of CR4 and SRR2, or may be selected from the group comprising SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.
In accordance with another aspect of the invention, there is provided a nucleic acid comprising an ETn poly A mutated (pAMu) promoter sequence (SEQ ID NO: 2) operatively linked to a tag sequence and a first sequence, the first sequence comprising an enhancer motif active in a pluripotent stem cell. The enhancer motif may be selected from the group consisting of two or more than two SRR2 enhancer sequences, and one or more than one CR4 enhancer sequence. The enhancer motif may be located upstream of the pAMu promoter sequence.
In accordance with another aspect of the invention, the one or more than one CR4 enhancer sequence is selected from the group consisting of SEQ ID NOS: 3 and 5.
In accordance with another aspect of the invention, the two or more than two SRR2 enhancer sequence is selected from the group consisting of SEQ ID NOS: 4 and 6.
In accordance with another aspect of the invention, the tag sequence may encode an amino acid sequence permitting antibiotic selection, drug selection, color selection, negative selection, cell-surface selection or fluorescence selection. The tag sequence may alternately encode a pluripotency factor, or a differentiation factor to drive differentiation into specific cell lineages (for example MyoD directs differentiation into muscle).
In accordance with another aspect of the invention, there is provided a cell comprising a nucleic acid, the nucleic acid comprising a pluripotent stem cell specific promoter, operatively linked to a tag sequence. The pluripotent stem cell-specific promoter may be an ETn promoter sequence (SEQ ID NO: 1), an ETn poly A mutated (pAMu) promoter sequence (SEQ ID NO: 2), or other pluripotent stem-cell specific promoter.
In accordance with another aspect of the invention, the cell may be an adult somatic cell, such as, but not limited to a fibroblast, a keratinocyte, a cynoviocyte, a mesenchymal stem cell, a neural stem/progenitor cell, a skin progenitor cell, a hepatocyte, a gastric epithelial cell, a pluripotent stem cell, or an induced pluripotent stem cell.
In accordance with another aspect of the invention, there is provided a vector comprising a nucleic acid, the nucleic acid comprising a pluripotent stem cell specific promoter, operatively linked to a tag sequence. The pluripotent stem cell-specific promoter may be an ETn promoter sequence (SEQ ID NO: 1), an ETn poly A mutated (pAMu) promoter sequence (SEQ ID NO: 2), or other pluripotent stem-cell specific promoter.
In accordance with another aspect of the invention, the vector may be a viral vector, such as a retroviral or lentiviral vector. The vector may further be a self-inactivating vector.
In accordance with another aspect of the invention, there is provided a stem cell expression vector comprising, an ETn pAMu promoter sequence operatively linked to a tag sequence, and one or more enhancer sequences, the one or more than one enhancer sequence selected from the group comprising CR4, SRR2, and a combination of CR4 and SRR2.
In accordance with another aspect of the invention, there is provided a method of producing an induced pluripotent stem cell comprising, inducing pluripotency to a cell with one or more than one pluripotency factor to produce a pluripotent cell, transfecting the pluripotent cell with a nucleic acid comprising a pluripotent stem cell specific promoter operatively linked to a tag sequence, to produce a transfected cell, growing the transfected cell, and selecting for the induced pluripotent stem cell.
A cell may be induced to become pluripotent by transfection with one or more than one pluripotency factor, or by transfection with one or more than one vector encoding the one or more than one pluripotency factor. A cell may be induced to become pluripotent by exposure to one or more than one pluripotency factor in a culture medium.
In accordance with another aspect of the invention, there is provided a method of producing an induced pluripotent stem cell comprising, transfecting a cell with a nucleic acid, the nucleic acid comprising a pluripotent stem cell specific promoter operatively linked to a tag sequence to produce a transfected cell, inducing pluripotency to the transfected cell with one or more than one pluripotency factor to produce the induced pluripotent stem cell, and growing the induced pluripotent stem cell.
In accordance with another aspect of the invention, there is provided a method of producing an induced pluripotent stem cell comprising either: Ai) reprogramming a cell to induce pluripotency producing a pluripotent cell; Aii) transfecting the pluripotent cell with the nucleic acid of claim 1 to produce a transfected pluripotent cell; or Bi) transfecting a cell with the nucleic acid of claim 1 to produce a transfected cell; Bii) reprogramming a cell to induce pluripotency to produce a transfected pluripotent cell; iii) growing the transfected pluripotent cell; and iv) selecting for an induced pluripotent stem cell.
According to some embodiments of the invention, the step of reprogramming may comprise: transfecting a cell with one or more than one pluripotency factors; adding one or more than one chemical, cytokine, or hormone into culture medium of a cell; transfecting a cell with one or more than one pluripotency factors and adding one or more than one chemical, cytokine, or hormone into culture medium of a cell; nuclear transfer of a cell into a pluripotent stem cell or an oocyte; or cell-cell fusion of a cell with a pluripotent stem cell.
In accordance with another aspect of the invention, there is provided a method of identifying a pluripotent stem cell comprising, providing a population of pluripotent stem cells, transfecting the population of pluripotent stem cells with a nucleic acid comprising a pluripotent stem cell specific promoter, operatively linked to a tag sequence, expressing the nucleic acid and selecting for an amino acid sequence of interest encoded by a tag sequence.
In accordance with another aspect of the invention, there is provided a method of overcoming silencing of one or more than one gene or nucleotide sequence following retroviral transfection, the method comprising, transfecting an adult fibroblast or an embryonic stem cell with a vector comprising a nucleic acid, the nucleic acid comprising a pluripotent stem cell specific promoter operatively linked to a tag sequence, and expressing the nucleic acid thereby overcoming silencing of the one or more gene or nucleotide sequence.
In accordance with another aspect of the invention, there is provided a stem cell expression cassette comprising, a nucleic acid, the nucleic acid comprising a pluripotent stem cell specific promoter, operatively linked to a tag sequence.
In accordance with another aspect of the invention, there is provided a method of maintaining a pluripotent stem cell in a pluripotent state comprising, providing a population of pluripotent stem cells comprising a nucleic acid, the nucleic acid comprising a pluripotent stem cell specific promoter operatively linked to a tag sequence, and expressing the nucleic acid thereby maintaining the pluripotent stem cell in the pluripotent state.
In accordance with another aspect of the invention, there is provided a method of purging one or more than one undifferentiated pluripotent stem cell from a population of differentiated stem cells during directed differentiation comprising, providing a population of pluripotent stem cells each comprising a nucleic acid, the nucleic acid comprising a pluripotent stem cell specific promoter operatively linked to a tag sequence, expressing the nucleic acid and differentiating the population of pluripotent stem cells, and killing any pluripotent stem cells that continue to express an amino acid sequence of interest encoded by the tag sequence of the nucleic acid thereby purging the one or more than one undifferentiated pluripotent stem cell from the population of differentiated stem cells.
In accordance with another aspect of the invention, there is provided a method for identifying a potential pluripotency factor comprising, providing a population of cells comprising a nucleic acid, the nucleic acid comprising a pluripotent stem cell specific promoter operatively linked to a tag sequence, exposing the population of cells to media comprising the potential pluripotency factor, expressing the nucleic acid and selecting for a pluripotent cell expressing an amino acid sequence of interest encoded by the tag sequence of the nucleic acid, whereby selection of the pluripotent cell expressing the tag sequence is indicative of the occurrence of the potential pluripotency factor in the media.
In accordance with another aspect of the invention, there is provided a kit for identification, production, or both identification and production, of a pluripotent stem cell or an embryonic stem cell, the kit comprising a nucleic acid comprising an ETn pAMu promoter sequence operatively linked to a tag sequence and instructions for its use. The kit may further comprise one or more than one pluripotency factor, media, other agents useful in selecting a pluripotent stem cell, or a combination thereof. The kit may further provide one or more than one nucleic acid comprising a sequence encoding one or more than one pluripotency factor. The kit may further comprise one or more than one transfection reagent for transfecting a cell.
This summary of the invention does not necessarily describe all features of the invention. Other aspects, features and advantages of the present invention will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the invention.
These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:
The present invention relates to nucleic acid sequences comprising regulatory sequences that direct expression of tag sequences in stem cells. The invention furthermore provides nucleic acid sequences that direct expression in embryonic or induced pluripotent stem cells.
The following description is of a preferred embodiment.
The present invention provides a nucleic acid construct comprising an pluripotent stem cell specific promoter sequence operatively linked to a tag sequence. Non-limiting examples of a pluripotent stem cell specific promoter sequence include a murine ETn (early transposon) element (SEQ ID NO: 1), or an ETn pAMu promoter (SEQ ID NO:2). The Etn pAMu promoter comprises a point mutation at position 183 of the ETn promoter, where an A is substituted for a T (this mutation is designated as: A183T). Other examples of pluripotent stem cell specific promoter sequences include promoter sequences from genes expressed in pluripotent stem cells, examples of such genes including, but not limited to, Oct-4 (Okumura-Nakanishi et al 2005), Nanog (Kuroda et al., 2005; Rodda et al., 2005), Sox2 (Tomioka et al., 2002), FGF-4 (Ambrosetti et al., 2000), Fbx15 (Tokuzawa et al., 2003), Utf1 (Nishimoto et al, 1999), Lefty1 (Nakatake et al, 2006), and Zfp206 (Wang et al., 2007), and Lin28.
An advantage of using a nucleic acid construct comprising, for example but not limited to, an ETn promoter sequence (SEQ ID NO: 1), or an ETn pAMu promoter sequence (SEQ ID NO: 2), operatively linked to a tag sequence to mark or select for iPS, is that standard protocols may be used, and similarly, the protocol is cell type neutral. Transfection of the construct may be carried out by any convenient or suitable method, and is the procedure is not dependent upon the transformation vector.
The nucleic acid construct according to some embodiments of the present invention may further comprise one or more than one enhancer sequence.
A tag sequence comprises one or more than one nucleic acid sequence encoding an amino acid sequence of interest. The amino acid sequence of interest may be a marker for color or fluorescence selection, for example, but not limited to GFP (Green fluorescent protein), EGFP (enhanced green fluorescent protein), Beta-galactosidase, luciferase, GUS, or the like (see, for example GUS Protocols: Using the GUS Gene as a Reporter of Gene Expression, S. R. Gallagher, Ed., Academic Press, Inc. (1992); Bronstein, I., et al. 1994. Anal. Biochem. 219:169-181; Alam, J. and Cook, J. L. 1990. Anal. Biochem. 188:245-254; WO 1997/042320; Nordeen, S. K. 1988. BioTechniques 6:454-457). The amino acid sequence of interest may be an antibiotic, drug or toxin resistance gene product, for example but not limited to puromycin-N-acetyl-transferase (confers resistance to puromycin; Vara et al. Nucl. Acids Res. 1986; 14: 4617-4624), aminoglycoside 3′ phosphotransferase (produced by the neo gene of Tn5, and confers resistance to G418 and neomycin; U.S. Pat. No. 4,784,949, or other antibiotic or toxin-resistance gene product, or the like, that allows cells expressing the amino acid sequence to survive and grow in the presence of the antibiotic, drug or toxin. SEQ ID NO's: 23 and 24 provide non-limiting examples of tag sequences. The amino acid sequence of interest may be an enzyme which catalyses the conversion of a non-toxic prodrug into a toxic form (so called suicide gene), for example but not limited to thymidine kinase gene, cytosine deaminase, cytochrome P450, nitroreductase, carboxypeptidase G2, purine nucleoside phosphorylase or the like (see, for example “Suicide genes for cancer therapy.” Portsmouth D. et al., Mol Aspects Med. 2007; 28:4-41), that allows cells expressing the amino acid sequence to kill or stop growing in the presence of the drug (“negative selection”). The amino acid sequence of interest may be a cell-surface antigen which can be recognized by a specific antibody (“cell-surface selection”)
A tag sequence may be assayed, for example, using colorimetric assays, drug selection assay, FACS analysis (Shapiro, H. M. 1988. Practical Flow Cytometry, 2nd ed Wiley-Liss, New York), CELISA, ELISA (Lequin R, 2005. Clin. Chem. 51: 2415-8), western blot (Burnette, W. N. 1981. Anal. Biochem. 112:195-203), Northern blot, Southern blot, PCR (Saiki, R. K. et al. 1988. Science 239:487-491), RT-PCR (Frohman, M. A., Dush, M. K., and Martin, G. R. 1988. Proc. Natl. Acad. Sci. U.S.A. 85:8998-9002), or the like. The nucleic acid transcript of the tag sequence may also be assayed by RT-PCR, northern blotting (Alwine et al 1977. Proc. Natl. Acad. Sci 74:5350), Southern blotting (Southern, E M. 1975. J. Mol Biol. 98:503), 5′ RACE, 3′RACE, sequencing (Sanger F, et al. Proc Natl Acad Sci USA. 1977. 74:5463-7; Maxam A M, Gilbert W., Proc Natl Acad Sci USA. 1977. 74:560-4), or other sequence-based assays.
Tag sequences comprising more than one nucleic acid sequence encoding an amino acid sequence of interest may further comprise a nucleotide sequence to facilitate expression of more than one nucleic acid sequence, for example, a nucleotide sequence comprising an internal ribosome entry site (IRES). Examples of IRES sequences are taught in, for example, Baird S D et al., RNA. 12:1755-85 2006). Another example of such a tag sequence may comprise a sequence encoding enhanced green fluorescent protein (EGFP) operatively linked to an IRES and a sequence encoding an amino acid sequence that confers resistance to antibiotics, such as puromycin (PuroR). When the nucleic acid is transcribed, all three sequences are produced as a single RNA transcript. When the RNA transcript is translated, ribosomes recognize both the 5′ cap (a translation initiation signal) and the IRES, and translation proceeds in a normal manner. A cell that comprises and expresses such a construct is therefore identifiable both by fluorescence, and by growth on culture medium comprising puromycin (“puro”). Other examples of such nucleic acids may comprise neomycin phosphotransferase (confers neomycin resistance) or hygromycin phosphotransferase (confers hygromycin resistance) in place of puromycin phosphotransferase (confers puromycin resistance). Other examples of such nucleic acids may comprise beta-galactosidase in place of the EGFP
Another example of a tag sequence according to some embodiments of the invention includes a nucleic acid comprising a nucleotide sequence encoding a fusion protein, such as beta-geo, HygroTK or NeoEGFP. A nucleotide sequence encoding beta-geo comprises a sequence encoding beta-galactosidase fused in-frame with a sequence encoding a protein for neomycin phosphotransferase. A nucleotide sequence encoding HygroTK comprises a sequence encoding a protein for hygromycin phosphotransferase fused in-frame with a sequence encoding thymidine kinase. A nucleotide sequence encoding NeoEGFP comprises a sequence encoding a protein for neomycin phosphotransferase fused in-frame with a sequence encoding enhanced green fluorescent protein. Another example of a tag sequence may include a nucleic acid comprising two or more nucleotide sequences encoding amino acid sequences of interest and connected by a nucleotide sequence encoding a cleavage peptide, such as a Picornavirus 2A peptide, or a 2A-like peptide (Szymczak A L et al., Nat Biotechnol. 2004). Such tag sequences may be expressed from a single promoter of a vector comprising the nucleic acid, and the translated polypeptide self-cleaved into the individual amino acid sequences of interest. Examples of such amino acid sequences include, but are not limited to, pluripotency factors, beta galactosidase, hygromycin phosphotransferase, neomycin phosphotransferase, thymidine kinase, green fluorescent proteins and
A tag sequence may also express other factors in a cell, for example one or more than one pluripotency factor, or one or more than one factor for expression in a developmental-stage specific manner. Examples of such pluripotency factors include, but are not limited to Oct-4, Nanog, Sox2, FGF-4, Fbx15, Utf1, Lefty1, Klf-4, c-Myc, Lin28 or Zfp206 (see references supra.). Pluripotency factors may also include various compounds, agents, proteins, peptides or other molecules that may be transfected into, or added to the culture medium of a cell to be induced to pluripotency, or to maintain pluripotency in a cell as would be known to one of skill in the art.
By “operatively linked” it is meant that the particular sequences, for example a promoter or enhancer, and a coding region of interest, interact either directly or indirectly to carry out an intended function, such as mediation or modulation of gene expression. The interaction of operatively linked sequences may, for example, be mediated by proteins that interact with the operatively linked sequences. Additionally, an IRES may be operatively linked to a nucleic acid sequence facilitating translation of the nucleic acid.
The relative position of any two or more operatively linked elements may be described as “in cis” or “in trans”. Elements that are in cis are found on the same molecule, for example encoded by the same nucleic acid, e.g. a vector. Elements that are in trans are found on two or more separate molecules. The relative position of two or more elements in cis may also be described as being upstream (5′) or downstream (3′) from an element. Elements in cis may be adjacent, or may be separated by one or more other elements, for example a tag sequence, or a regulatory element such as a promoter, another enhancer, a termination signal or the like.
An enhancer sequence (may also be referred to as a motif) is a nucleic acid sequence or region of DNA that aids in the transcription of a gene or nucleotide sequence. An enhancer sequence may be located at a distance from the nucleotide sequence being transcribed, for example, it may be located on a separate chromosome, or on a separate nucleic acid molecule. Enhancer sequences may be located 5′ or 3′ to the nucleotide sequence being transcribed, and may function in either ‘orientation’ (either positive “+”, or negative “−” orientation). Enhancer sequences may also be 5′ or 3′ relative to the promoter directing transcription of a coding sequence of the nucleotide sequence being transcribed. These variants may be combined in a single construct to modify transcription of the coding sequence.
An “enhancer unit” may comprise one or more than one enhancer sequences, or motifs. Each of the one or more than one enhancer sequence may function constitutively, function in a developmental stage or tissue specific manner, or function constitutively and in a developmental stage or tissue-specific manner. An enhancer sequence, or an enhancer unit, selective for, for example, an embryonic stem cell stage, may be combined with the ETn promoter to obtain an increase in expression of a tag sequence. An increase in expression of the tag sequence can be determined by comparing the level of expression of the tag sequence (or coding sequence product) that is modified by an enhancer sequence or an enhancer unit, to the level of expression of the tag sequence (or coding sequence product) obtained in the absence of the enhancer sequence or enhancer unit. Enhancer sequences may be obtained from genes expressing in ES cells. Examples of such genes include, but are not limited to, Oct-4 (Okumura-Nakanishi et al 2005), Nanog (Kuroda et al., 2005; Rodda et al., 2005), Sox2 (Tomioka et al., 2002), FGF-4 (Ambrosetti et al., 2000), Fbx15 (Tokuzawa et al., 2003), Utf1(Nishimoto et al, 1999), Lefty1 (Nakatake et al, 2006), and Zfp206 (Wang et al., 2007).
An enhancer sequence may include a CR4 element from Oct-4 (SEQ ID NO: 3), an SRR2 element from Sox2 (SEQ ID NO: 4), a CR4 element in reverse orientation (SEQ ID NO: 5), a SRR2 element in reverse orientation (SEQ ID NO: 6), or a combination thereof.
An enhancer unit may comprise one or more than one enhancer sequence. An enhancer sequence or motif may be 5′ to a promoter, or 3′ to a promoter. For example, a nucleic acid may comprise a promoter operatively linked to a tag sequence, and have an enhancer motif located 5′ relative to the promoter, 3′ relative to the promoter and 5′ relative to the tag sequence, 3′ relative to both the promoter and tag sequence, or a combination thereof. Sequences comprising an enhancer unit may be all in a positive orientation, all in a negative orientation, or a combination of both positive and negative orientation. If the enhancer unit comprises two or more enhancer sequences of both positive and negative orientation, the most-upstream sequence may have a positive or negative orientation. Orientation of the enhancer sequence does not need to relate or correspond to the order or position of the enhancer sequence within the enhancer motif.
SEQ ID NOS: 7, 8, 11 and 12 are non-limiting examples of nucleic acid constructs comprising one enhancer sequence (SRR2 or CR4) located 5′ to a promoter sequence (ETn pAMu) in a forward (SEQ ID NOS: 7 and 11) or reverse (SEQ ID NOS: 8, 12) orientation.
SEQ ID NOS: 9, 10, 13 and 14 are non-limiting examples of nucleic acid constructs comprising more than one enhancer sequence (SRR or CR4) located 5′ to a promoter sequence (ETn pAMu) in a forward (SEQ ID NOS: 9, 13 and 14) or reverse (SEQ ID NO: 10) orientation.
SEQ ID NOS: 15-18 are non-limiting examples of nucleic acid constructs comprising one enhancer sequence (CR4 or SRR2) located 3′ to a nucleic acid sequence encoding a tag sequence (EGFP), in the forward (SEQ ID NOS: 15, 17) or reverse (SEQ ID NOS: 16, 18) orientation.
SEQ ID NOS: 19-22 are non-limiting examples of nucleic acid constructs comprising one or more than one enhancer sequence located 5′ to a promoter sequence (ETn pAMu) and located 3′ to a tag sequence (EGFP). The enhancer sequences may be in the forward or reverse orientation.
Therefore, the present invention provides for a nucleic acid comprising an ETn poly A mutated (pAMu) promoter sequence (SEQ ID NO: 2) operatively linked to a tag sequence and one or more than one enhancer sequence, the one or more than one enhancer sequence active in a pluripotent stem cell. The one or more than one enhancer sequence may be selected from one or more than one CR4 enhancer sequences, two or more than two SRR2 enhancer sequences, or a combination thereof. The enhancer motif may be located upstream of the pAMu promoter sequence.
In some embodiments of the invention, the one or more than one CR4 enhancer sequence may be selected from SEQ ID NOS: 3 and 5. In some embodiments of the invention, the one or more than one SRR2 enhancer sequence may be selected from SEQ ID NOS: 4 and 6.
Therefore, the invention provides a nucleic acid construct comprising an ETn pAMu promoter sequence and one or more than one enhancer sequence. The one or more than one enhancer sequence may be in a forward or reverse orientation, located 5′ to the promoter, 3′ to the promoter, 5′ and, 3′ to the promoter, or a combination thereof. The one or more than one enhancer sequence in the nucleic acid construct may be the same, or may be different. Non-limiting examples of such constructs may be found with reference to
The present invention also provides for a nucleic acid comprising an ETn poly A mutated (pAMu) promoter sequence (SEQ ID NO: 2) operatively linked to a tag sequence and operatively linked to one, two, three, four or more SRR2 enhancer sequences, the enhancer sequences may be located 5′ to the ETn pAMu promoter.
The present invention also provides for a nucleic acid comprising an ETn poly A mutated (pAMu) promoter sequence (SEQ ID NO: 2) operatively linked to a tag sequence, and operatively linked to one, two, three, four or more CR4 enhancer sequences in a positive orientation and may be located 5′ to the pAMu promoter.
The present invention also provides for a nucleic acid comprising an ETn poly A mutated (pAMu) promoter sequence (SEQ ID NO: 2) operatively linked to a tag sequence, and operatively linked to one, two, three, four or more CR4 enhancer sequences in a negative orientation, and located 5′ to the pAMu promoter.
The present invention also provides a method of identifying an embryonic stem cell (ES). To identify an ES cell, the nucleic acid comprising an ETn sequence operatively linked to a tag sequence is transfected into a cell or population of cells. The cells are grown in suitable medium and assayed for expression of the tag sequence, or the presence or expression of the amino acid sequence encoded by the tag sequence, where the detected of the tag sequence or amino acid sequence encoded by the tag protein identifies the ES cell.
The present invention also provides a method of identifying an induced pluripotent stem cell (iPS). To identify an iPS cell, a population of cells comprising the iPS is transfected with a nucleic acid comprising an ETn or ETn pAMu promoter or an ETn pAMu promoter operatively linked to one or more than one enhancer sequence, and directing expression of a tag sequence. The transfected cells are grown under suitable conditions and the cells expressing the tag sequence are selected.
The method of selection of a tag sequence in the above methods, will be dependent on the tag sequence used. For example, if the tag sequence provides for expression of EGFP, the iPS cells may be selected by FACS analysis (see, for example, Shapiro, H. M. 1988. Practical Flow Cytometry, 2nd ed Wiley-Liss, New York). Use of a fluorescent marker such as EGFP (thus enabling use of FACS) provides an additional advantage of separating out the iPS from the remainder of the population of cells, enriching for iPS. Positive selection may also be used. As an example, the tag sequence may encode an amino acid sequence of interest that provides resistance (for example a puromycin resistance enzyme) to an agent in the culture medium (for example puromycin). Cells that express the enzyme will continue to grow in the presence of puromycin, while those that do not express the enzyme (those that are not at a developmental stage where the tag sequence is expressed) will die.
The present invention also provides a method of identifying or killing a residual undifferentiated cell following induced differentiation. To identify a residual undifferentiated cell, a nucleic acid comprising an ETn or ETn pAMu sequence operatively linked to a tag sequence is transfected into a cell or population of cells. The cells are differentiated into suitable cell types and assayed for expression of the tag sequence to identify or to negatively select an undifferentiated cell.
The tag sequence as described herein may be one or more negative selection markers, for example a suicide genes which catalyse the conversion of a non-toxic prodrug into a toxic form, for example but not limited to thymidine kinase (or a nucleic acid comprising a nucleotide sequence encoding thymidine kinase), cytosine deaminase, or cytochrome P450, or the like (see for example Portsmouth D. et al., Mol Aspects Med. 2007; 28:4-41; herein incorporated by reference), that allows cells expressing the amino acid sequence to kill or to stop growing in the presence of a pharmaceutical agent drug (such as ganciclovir, or acyclovir). An advantage to performing such a negative selection may include the reduction or removal of potential teratoma-forming cells in the population.
Examples of such directed differentiation methods or procedures include using a defined set of growth factors added to the cell media to induce differentiation into specific cell lineages such as neural, cardiac, or pancreatic cells. Chemicals, cytokines or hormones such as Retinoic Acid, or the introduction of lineage-specifying master genes (for example MyoD to induce muscle) that direct differentiation into a specific lineage or cell type may also be used.
Negative selection may also be used in combination with other selection criteria to identify selected cells. For example, tag sequences encoding both a drug resistance enzyme (e.g. a sequence encoding a puromycin resistance enzyme) and a negative selection element (e.g. a sequence encoding thymidine kinase) may be transfected into the cells. Reprogrammed cells may be selected for by growing in puromycin. During a subsequent directed differentiation method or procedure, gancyclovir may be added to the growth medium to select against cells expressing thymidine kinase (e.g. those that did not undergo subsequent directed differentiation). An advantage to performing such a multi-step selection may include reduction or removal of potential teratoma-forming cells in the population, and be of particular interest if the iPS cells are to be used in a subject.
Alternately, negative selection may be employed in vivo post-transplantation. A subject may be administered a population of cells, or tissue comprising such cells, the cells having previously been transfected with a nucleic acid comprising a negative selection element, directed to differentiate to the desired cell or tissue type. Once the cells or tissue are transplanted or administered to the subject, the subject may be administered a pharmaceutical agent or drug to select against any undifferentiated cells. For example, if the negative selection element is a nucleic acid encoding thymidine kinase operatively linked to a promoter sequence that is selectively expressed in an iPS cell, any undifferentiated iPS cells will be killed following administration of a course of, for example, gancyclovir, or acyclovir to the subject.
Transfection refers generally to the introduction of foreign material, frequently nucleic acid, into a cell, such as a mammalian cell. Transfection of a cell frequently results in a change in one or more properties of the cell, for example, expression of a foreign transcript or protein, alteration in growth pattern, or the like. Cells may be transfected by any of several methods known in the art, for example use of calcium phosphate (Graham F L, van der Eb A J, Virology. 1973 52(2):456-467); use of dendrimers to bind the nucleic acid and enhance uptake; liposomal transfection (Sells, M. A., Li., J., and Chernoff, J. 1995. BioTechniques 19:72-78); transfection using cationic polymers such as DEAE-dextran, polyethylenimine or poly-L-ornithine (Scangus G and Ruddle F H. 1981 Gene 14:1-10); ‘gene gun’ or biolistic particle delivery (U.S. Pat. No. 4,956,050, U.S. Pat. No. 5,204,253, U.S. Pat. No. 6,194,389); nucleofection (Aluigi M et al 2006. Stem cells 24:454-461); electroporation; heat shock; magnetofection (Plank C et al 2003. Biol. Chem 384:737-47; U.S. Pat. No. 5,547,932); or transfection using viral vectors, such as retroviral or lentiviral vectors (Wilson et al., 1990. PNAS 87:439-443, Kasid et al., 1990). Protocols for such methods and techniques may be found in, for example, Current Protocols in Molecular Biology (Ausubel et al., Editors. Wiley Interscience 2008).
Cells may be stably or transiently transfected. If the transfected nucleic acid is to persist in daughter cells following mitosis or meiosis, stable transfection is preferable. The transfected nucleic acid may be co-transfected with another gene that provides a selective advantage, such as resistance to a drug or agent (where the drug or agent is added to the cell culture medium following transfection), or ability to survive in the absence of a particular metabolite. The transfected nucleic acid may also be co-transfected with another gene that increase the transfection efficiency (such as a DNA binding protein which has a cell-permeabilization signal), increase the chromosomal integration efficiency (such as an integrase or a transposase), increase the targeting into a specific locus of chromosome (such as a zinc-finger protein which bind to a specific DNA sequence, or a site-specific endonuclease), facilitate homologous recombination, or maintain nucleic acid as an episome (such as SV40 large T antigen or Epstein-Barr Virus nuclear antigen 1). Examples for such methods and techniques may be found in, for example, Palazzoli F. et al., Current Gene Therapy, 2008. “Transduction”, “infection” (in reference to transfection using a viral vector, such as a retroviral vector), “infection by transformation” are other terms that may be used interchangeably with transfection, in reference to the introduction of foreign material such as nucleic acid into a cell, and the systems that facilitate such introduction.
Viral vectors, such as retroviral vectors, for example but not limited to gammaretroviral or lentiviral vectors, are one option available for introducing foreign nucleic acid into cells, in particular primary fibroblasts, ES or iPS cells. Other examples of viral vectors include, but are not limited to adenovirus vectors, parvovirus vectors, herpesvirus vectors, adeno-associated virus vector, poxivirus vectors, or the like. Nucleic acids according to some embodiments of the invention may be incorporated in a retroviral vector for delivery to the cells. The silencing or attenuation of nucleotide sequences may be observed following retroviral vector transfection. This silencing may include tag sequences. As shown in the examples, silencing may be overcome through the use of an ETn or ETn pAMu promoter, or an ETn pAMu promoter operatively linked to one or more than one enhancer sequence, in the vector to direct transcription of the tag sequence. Thus, a method to overcome silencing of genes or nucleotide sequences following retroviral transfection is provided. The method comprises transfecting an adult fibroblast or embryonic stem cell with a vector comprising an ETn or an ETn pAMu promoter operatively linked to a tag sequence, and expressing the tag sequence.
A retroviral or lentiviral vector that is self-inactivating (SIN) may also be suitable for introducing foreign nucleic acid into cells. An example of a self-inactivating retroviral vector is HSC-1 (Osborne et al., 1999). HSC1 retroviral vector has a self-inactivating deletion in 3′LTR U3 and do not contain any known ES-specific silencer binding sites. After reverse transcription and integration, the self-inactivating (SIN) deletion is copied into 5′LTR so that EGFP is only expressed from an internal promoter.
Transposon vectors may also be used to introduce foreign nucleic acid into cells, including primary fibroblasts, ES or iPS cells. Examples of transposon vectors include piggyBac (Cary, L. C. et al., 1989. Virology 172: 156-169; Fraser, M. J., L. et al., 1995. Virology 211:397-407) or the Sleeping Beauty Transposon™ System (SBTS) (U.S. Pat. No. 6,489,458).
The present invention, further provides for a method of identifying an iPS comprising transfecting a cell with a viral vector comprising one or more pluripotency factors, transfecting the cell with a nucleic acid comprising an ETn promoter sequence (SEQ ID NO: 1) or an ETn pAMu promoter sequence (SEQ ID NO: 2), growing the cell, and selecting for the iPS.
Also provided by the present invention is a cell comprising a nucleic acid comprising an ETn promoter sequence (SEQ ID NO: 1) or an ETn pAMu promoter sequence (SEQ ID NO: 2) operatively linked to a tag sequence. The cell may be an iPS, or may be an ES from a subject.
Also provided by the present invention is a method of producing an induced pluripotent stem cell. This method comprises transfecting a cell with one or more than one pluripotency factors to produce a pluripotent cell, transfecting the pluripotent cell with a nucleic acid comprising one or more than one regulatory sequence that direct expression of tag sequences in a stem cell to produce a transfected cell, growing the transfected cell, and selecting for an induced pluripotent stem cell.
The present invention also provides another method of producing an induced pluripotent stem cell. This method comprises transfecting a cell with a nucleic acid comprising regulatory sequences that direct expression of tag sequences in a stem cell to produce a transfected cell; transfecting the transfected cell with one or more than one pluripotency factor to produce a pluripotent cell, growing the pluripotent cell, and selecting for an induced pluripotent stem cell.
The methods of Takahashi et al., 2006, Okita et al., 2007 or Takahashi et al., 2007 may be employed, for example, to induce pluripotent stem cells from fibroblast cultures. Alternately, the methods of Okita et al., 2008 or Stadtfeld et al., 2008 may be employed to induce pluripotent stem cells from differentiated, or partially differentiated cell cultures.
Other methods of introducing pluripotency factors to a cell to induce reprogramming to an iPS state may also be used. For example, pluripotency factors may be introduced using vectors other than viral vectors (e.g. episomal nucleic acid), or by transfection of the pluripotency factors themselves directly into the cell. The pluripotency factors may further comprise amino acid motifs or domains that facilitate entry of a protein into a cell, for example, protein transduction domains. Protein transduction domains may be fused, bound or coupled to a pluripotency factor. Examples of protein transduction domains include HIV TAT, cell-penetrating peptides, antennapedia protein transduction domain, polyarginine oligomers, polylysine oligomers, KALA, MAP, transportan, PTD-5 or the like (see, for example, Kabouridis 2003. Trends in Biotechnology 21:498-503). Other methods may further include chemical induction of pluripotency (e.g. “chemical reprogramming”) by addition of small molecules that mimic pluripotency factors, activate pluripotency factors, or enhance reprogramming efficiency to the culture medium.
For example, a cell reprogrammed to an iPS state may further have nucleic acids according to some embodiments of the invention, or vectors comprising such nucleic acids delivered concurrently with, a vector or vectors for reprogramming, or subsequent to the reprogramming. Alternately, reprogramming may be facilitated by any method, including chemical reprogramming (e.g. addition of small molecules that mimic pluripotency factors or enhance reprogramming efficiency directly to the culture medium) or transient methods of delivering pluripotency factors to the cells (e.g. using adenovirus vectors, plasmid vectors, episomes, tat-fusion proteins or the like). The resulting iPS cells may be used, for example, to generate new lung, heart or bone tissue for patient-specific personalized regenerative medicine. For example, US Patent Publications 2004/0072343, 2007/00207759, 2007/0025973, 2003/0211603, 2007/0196918 disclose various methods of reprogramming differentiated, or partially differentiated cells.
Other methods of reprogramming to convert a cell into a pluripotent state may also be used. For example, nuclear transfer from a somatic cell into a pluripotent cell or into an oocyte (Yang X. et al., nature Genetics, 2007), or cell-cell fusion of a somatic cell and a pluripotent stem cell (Cowan et al., Science, 2005), where the somatic cell comprising one or more than one regulatory sequence that direct expression of tag sequences in a stem cell to produce a tranfected cell.
The nucleic acid may furthermore comprise sequences that direct expression in embryonic or induced pluripotent stem cells, examples of such sequences include ETn (SEQ ID NO: 1) and ETn pAMu (SEQ ID NO: 2). The nucleic acid may further comprise enhancer sequences, such as those selected from the group comprising SEQ ID NO: 3, 4, 5 or 6; the tag sequence may encode an amino acid sequence of interest.
These methods may be applied to any adult cell that may be reprogrammed to an ES-like stage, for example, an iPS. Examples of adult cells include, but are not limited to, fibroblasts, cynoviocytes (Takahashi et al., Cell, 2007), mesenchymal stem cells (Park et al., Nature, 2007), hepatocytes, keratinocytes, neural stem cell or neural progenitor cell, skin progenitor cell, epiblast derived stem cell, or gastric epithelial cells (Aoi et al., Science, 2008).
By the term “subject”, it is meant an organism, from whom cells may be isolated, or to whom cells according to some embodiments of the invention, may be administered. Examples of a subject include, but are not limited to, humans, primates, birds, swine, sheep, horse, dogs, cats, livestock, rabbits, mice, rats, guinea pigs or other rodents, and the like. Such target organisms are exemplary, and are not to be considered limiting to the applications and uses of the present invention.
Cells that have been selected on the basis of the tag sequence may be further characterized to determine the insertion point of the transgenes, or other characteristics as may be suitable for the desired application of the cells, including modeling human disease states or for therapeutic applications. For example, iPS cells may be generated from cells obtained from a subject or animal model of a disease or disorder having one or more genetic components, and these iPS cells used to generate a renewable source of cells or tissue demonstrating a particular characteristic or phenotype found in cell or tissue of the affected subjects or animal models. Such a renewable source of cells or tissue may be used to study the defects that underlie the particular disease or disorder and for evaluating the role of various genes in this process, for example, via rescue experiments or drug screenings. Examples of such diseases or disorders include any genetic disorder or congenital defect, such as, but not limited to, various neural defects having a genetic component (e.g. autism, Rett syndrome, schizophrenia), cystic fibrosis, various cardiac defects having a genetic component (e.g. Hypertrophic Cardiomyopathy (HCM), Marfan Syndrome, Long QT Syndrome, DiGeorge Syndrome), various musculoskeletal disorders having a genetic component (e.g. Muscular Dystrophy, Marfan Syndrome), Progeria, various cancers, or the like.
In addition cells according to some embodiments of the invention may be used to repair, regenerate or replace damaged tissue, such as lung, heart, or bone tissue for subject-specific regenerative medicine.
The invention also provides for a kit for identifying a pluripotent stem cell or an embryonic stem cell, comprising a nucleic acid comprising an ETn pAMu promoter sequence operatively linked to a tag sequence, and instructions for its use. The kit may further comprise one or more than one pluripotency factor, media, one or more than one other agents useful in selecting a pluripotent stem cell, or a combination thereof. The kit may further provide nucleic acids comprising a sequence encoding one or more than one pluripotency factor, such as Oct4, Sox2, Klf4, c-Myc or a combination thereof, and may further comprise transfection reagents for transfecting a cell. Instructions for use of the nucleic acids as described herein, transfection reagents or instructions for transfecting a cell, as well as instructions for screening for iPS cells as described herein may also be provided in such a kit.
The ability of EOS vectors to mark, enrich and maintain ES and iPS cell lines makes such vectors useful as reporters to aid in increasing the efficiency of isolating reprogrammed iPS cell lines from transgenic animals or from patient biopsies to model disease in vitro. Further, EOS vectors may be used to optimize reprogramming technologies by aiding in quantification and/or isolation of induced cells at the appropriate developmental stage.
The nucleic acid constructs and vectors provided by the present invention further allow for constant selection to maintain and expand iPS cells in a pluripotent state. It has been previously demonstrated that, in the context of in vitro iPS cell applications, retroviral or lentiviral integrations do not hinder disease-specific iPS cell line generation, nor do they influence phenotyping of affected cell types (Park et al., 2008; Dimos et al., 2008). The ability of EOS vectors to be imaged for EGFP expression or selected for puromycin resistance may be useful and valuable attributes for optimizing novel reprogramming technologies employing transient factor delivery methods or using high-throughput screens of small molecules. Directed differentiation procedures may need to be optimized for each disease-specific iPS cell line generated, and EOS vector expression may be used to monitor the numbers of responding, or non-responding pluripotent stem cells in this context.
Sequences according to various embodiments of the invention are described in Tables 1 and 2 and in the figures and accompanying text of the specification.
For all of SEQ ID NOS: 7-14 (Table 1), the ETn pAMu sequence may be operatively linked 5′ to a tag sequence (e.g. EGFP, NeoR-IRES-EGFP (“NIE”) or EGFP-IRES-PuroR (“EiP”), or another tag sequence as described herein). For SEQ ID NOS: 15-22, the EGFP tag sequence may be substituted by another tag sequence, e.g. NeoR-IRES-EGFP (“NIE”) or EGFP-IRES-PuroR (“EiP”) or another tag sequence as described herein.
SEQ ID NOS: 7-14 may be operatively linked 5′ to a tag sequence. Examples of tag sequences include a sequence encoding EGFP, sequences encoding a gene product for puromycin resistance, a sequence encoding a gene product for neomycin or G418 resistance, or a sequence encoding EGFP operatively linked to a sequence encoding a gene product for puromycin resistance and further comprising an operatively-linked IRES (SEQ ID NO: 23), or a sequence encoding a gene product for neomycin resistance operatively linked to a sequence encoding EGFP and further comprising an operatively linked IRES (SEQ ID NO: 24).
Plasmid Vector Constructions
The HSC-1 retrovirus (Osborne et al., J. Virol., 1999) and PL (self-inactivating) lentivirus vector backbones (Buzina et al, 2008 PLOS Genetics in press) have been previously described. The mouse PGK promoter was derived from SM-2 vector, ETnII LTR#6 promoter is described previously (Maksakova et al., 2005) but introduced a single nucleotide mutation in poly A signal by 2 step PCR method using primers ETn-pA-Mu-s, ETn-pA-Mu-a, RVP3(Promega) and GLP2(Promega) (Table 2). Human Nanog promoter was PCR amplified from BAC RP11-277J24 (AC006517) containing human chromosome 12 using following primers: Nanog-NcoI and Nanog-BamHI. Mouse Oct-4 promoter was derived from the 2.7 kb HindIII fragment of GOF-18 GFP (Yeom et al., 1996). Mouse Oct-4 enhancer CR4 (Okumura-Nakanishi, supra) and Sox enhancer SRR2 (Tomioka, supra) were PCR amplified from genomic DNA of J1 ES cells (strain 129S4/Jae) using primers mOct4-CR4-s(EcoRI), mOct4-CR4-a(XhoI), mSox2-SRR2-s(EcoRI) and mSox2-SRR2-a(XhoI) (Table 2).
All promoters and enhancers were confirmed by DNA sequencing.
Cell Culture
J1 mouse ES cells were cultured on gelatin-coated dishes using mouse ES medium (DMEM with 15% FBS supplement with 4 mM L-glutamin, 0.1 mM MEM non-essential amino acids, 1 mM sodium pyruvate, 0.55 mM 2-mercaptoethanol, and LIF), unless specified. Plat-E cells (Morita et al., 2000) were maintained in DMEM with 10% FBS containing blasticidin (10 μg/ml) and puromycin (1 μg/ml). 293T, NIH3T3 and MEF cells were cultured in DMEM with 10% FBS supplement with 4 mM L-glutamine. MEFs were isolated from E15.5-E17.5 CD-1 mouse embryos.
Human ES cell line CA1 was maintained on feeders in Knockout DMEM (Invitrogen) supplemented with 15% Serum Replacement (Invitrogen), 2 mM Glutamax (Invitrogen), penicillin/streptomycin, 0.1 mM non-essential amino acids, 0.5 mM mercaptoethanol, and 10 ng/mL recombinant FGF2 (Peprotech). Human embryonic stem cells were grown on matrigel in the presence of MEF-conditioned medium as previously reported (Bendall et al., 2007). Human dermal fibroblasts (HDFs) were isolated skin biopsy from 8-years old male by distal humerus osteotomy. Feeder cells for CA-1 and iPS cultures were isolated from E15.5 embryo of Tg(DR4)1Jae/J mice (Stock No. 003208, Jackson Laboratory) for puromycin resistance.
Virus Production and Infection
Production of retroviral and lentiviral vectors were as described previously (Buzina et al., 2008; Hotta et al., 2006). Briefly, Plat-E cells were plated at a density of 1×105 cells/cm2. Next day, the cells were transfected with the appropriate plasmids using 1 μl/1×105 cells of Lipofectamine 2000 (Invitrogen).
For lentiviral EOS vector production, 293T cells were plated at a density of 8×106 in T-75 flasks. The following day, the cells were transfected using Lipofectamine 2000 (Invitrogen) with 10 μg HPV275 (gag/pol expression plasmid), 10 μg P633 (rev expression plasmid), 10 μg HPV17 (tat expression plasmid), 5 μg pVSV-G (VSV-G expression plasmid) and 15 μg of EOS lentiviral plasmid which is derived from the PL.SIN.EF1a-EGFP backbone (Buzina et al, 2008). The lentiviruses were collected in 20 mL media after 48 hours, filtered through 0.45 μM filters to remove cell debris. If necessary, viruses were concentrated by ultracentrifugation at 4° C., 2 hours, 30,000 rpm with T-865 rotor (Sorvall). The viral pellet was resuspended in 40 μl Hanks' balanced salt solution (Invitrogen) overnight at 4° C. Titer for PL-EOS-C(3+)-EiP lentiviral vectors was approximately 1×107 IU/ml assayed on J1 mouse ES cells, and the titer was used to estimate the MOI of fibroblast infections.
One day before infection, target cells were seeded at 5×104 cells (for NIH3T3 and MEFs) or 1×104 cells (for J1) per wells of a 24-well plate. For infection, virus was added to the target cells with several dilutions in the presence of 8 microgram/ml polybrene (hexadimethrine bromide, Sigma). Twenty four hours post infection, virus was removed and transgene expression was analyzed 2 to 3 days post infection.
Surface Marker Staining
Cells were trypsinized into single cell suspension and incubated with Mouse IgM anti SSEA-1 antibody (MC-480, Hybridoma Bank) for 30 min on ice. After washing with PBS, cells were incubated with PE-Cy5.5 conjugated anti mouse IgM antibody (35-5790, eBioscience) for 30 min on ice.
Flow Cytometry
Trypsinized cells were suspended in PBS with 5% FBS. Single cell suspensions were filtered through 70 μm pore nylon membrane and analyzed by a FACScan (Becton Dickinson) flow cytometry using CellQuest software. Before each experiment, the machine was calibrated using calibration beads (FL-2056-2, Spherotech). Cell debris was excluded from analysis by using forward- and side-scatter gating. In each cell type, mock-infected or non-infected cells were used as a negative control to adjust FL1 gain to detect EGFP fluorescence. Obtained data were analyzed by FlowJo software (Tree Star Inc.).
Immunocytochemistry
Cells were fixed with 4% formaldehyde in PBS for 20 min, permeabilized with 0.2% NP-40 for 5 min, blocked with 0.5% BSA and 6% normal goat serum for 1-2 hours, and incubated with primary antibodies with 0.25% BSA and 3% normal goat serum in PBS overnight. After washing 3 times with PBS, cells were incubated with secondary antibodies for 45 minutes. Immunostaining images were taken with a Zeiss Axiovert 200M microscope equipped with AxioCam HRm camera and AxioVision software. Antibodies used in this study are listed in Table 3.
Microscopy Imaging
Live cell images were captured using a Leica DM IL inverted contrasting microscope equipped with Leica DC500 digital color camera by OpenLab software. Acquired images are copied onto Microsoft PowerPoint software and phase-contrast images were converted to gray scale. For EGFP fluorescence, band-pass 450-490 nm filter was used for excitation and low-pass 520 nm filter was used for detection of fluorescence.
Mouse ES Cell Differentiation
J1 ES cell colonies cultured on gelatin-coated dishes were loosely detached by trypsin-EDTA treatment and suspended in mouse ES medium without LIF. The J1 ES colonies were cultured as suspension in non-treated Petri dishes for 4 days to make embryoid bodies (EB). The cells were treated with 5 μM all trans retinoic acid (RA, Sigma) for 24 hours and cultured further as EBs for 3 days. The EBs were trypsinized to suspend into single cells and plated onto tissue culture grade dishes for an additional 3-5 days.
Alkaline Phosphatase Staining
Cells were fixed by 4% formaldehyde and stained by 1 mg/ml Fast Red TR hemi (zinc chloride) salt (F8764, Sigma) and 0.4 mg/ml Naphthol phosphate disodium salt (N7255, Sigma) in 0.1M Tris-HCl (pH=8.6) for 10 min at room temperature. Wild type J1 ES cells were used for staining control and NIH3T3 or MEF (mouse embryonic fibroblast) cells were used for negative control.
Mouse iPS Cell Induction
The induction of iPS cells was performed based on the Yamanaka protocol (Nakagawa et al., Nature Biotechnology, 2007; Takahashi et al., Nature Protocols, 2007). In brief, retrovirus vectors encoding Oct-4, Sox2, Klf4, and c-Myc were produced using Plat-E cells by plasmid transfection of either pMXs-Oct4, pMXs-Sox2, pMXs-K1f4, or pMXs-c-Myc (Addgene plasmid 13366, 13367, 13370, and 13375, respectively). One million cells per 10 cm dish of MEFs (isolated from wild type strain CD-1 or MeCP2 mutant mice [Stock No. 005439 Jackson Laboratory]) were infected with 2.5 ml each of unconcentrated retrovirus vector in the presence of 8 μg/ml polybrene. One day after infection, the cells were trypsinized and 6×105 cells were transferred onto feeder cells in a 10 cm dish in mouse ES media. Colonies were picked and dissociated by trypsinization. All EOS infected iPS cell lines were maintained in mouse ES media containing 1 μg/ml puromycin on feeders.
Human iPS Cell Induction
Human BJ fibroblasts (ATCC, CRL-2522) or Rett Syndrome patient fibroblasts (Coriell, GM11270) were infected with pLenti6/UbC/mS1c7a1 lentiviral vector (Addgene, 17224) expressing the mouse S1c7a1 gene and selected with blasticidin prior to reprogramming experiments. Cells were seeded at 8×105 cells per 10 cm dish and transduced twice with pMXs retroviral vectors encoding hOCT4, hSOX2, hKLF4, and hc-MYC (Addgene 17217, 17218, 17219, and 17220, respectively) (Lowry et al., 2008), together with pMXs-mRFP1 (monomeric Red Fluorescence Protein 1) retrovirus for monitoring infectivity and viral silencing. One week after transduction, cells were trypsinized and seeded onto 10 cm feeder dish in human ES cell media. Emerged colonies were picked and mechanically dissociated at initial passages up to the 6-well plate, then adapted to collagenase treatment. All EOS infected iPS cell lines are maintained in human ES media containing 1 μg/ml puromycin on feeders.
Teratoma Formation
Mouse iPS cells were suspended in PBS with 5% FBS and injected into the testes of NOD/SCID mice. Four to five weeks after injection, tumors were weighed. Human iPS cells were suspended in a mixture of KO-DMEM, Matrigel and collagen to inject intramuscularly into NOD/SCID mice, as previously described (Park et al., 2008). Tumors were harvested 9 weeks after injection. Fixed tumors were embedded in paraffin, sectioned and stained with hematoxylin and eosin for pathological analysis. Mouse and human ES cells were used as positive control for teratoma formation. Parental fibroblasts for iPS derivation did not form teratomas.
Genotyping of MeCP2 Mutation
For mouse RTT-iPS cells, PCR on genomic DNA yielded an amplicon of 396 by for wild-type Mecp2 and 318 by for the truncated Mecp2308 allele using the following primers: (see Table 2 for sequences) IMR3912 (common forward primer) (SEQ ID NO: 35), IMR3913 (WT allele specific reverse primer) (SEQ ID NO: 36), IMR3914 (MUT allele specific reverse primer) (SEQ ID NO: 37). For human RTT-iPS cells, genomic DNA was extracted from R306C hiPS cells and PCR was performed using the following primers: RTT-Fwd (SEQ ID NO: 38) and RTT-Rev (SEQ ID NO: 39). The PCR amplicon was isolated and DNA sequencing was performed using the RTT-Fwd Primer.
To characterize the transduction efficiency of ES cells by gammaretroviral vectors, we inserted several ES-specific or ubiquitous promoters into HSC1 vector backbone, as an internal promoter (
For ES-specific expression, we tested Nanog (Nanog-EP, 1.5 kb; Nanog-P, 490 bp) and Oct4 (Oct4-EOP, 2.1 kb; Oct4-OP, 475 bp) promoters.
Both Nanog and Oct4 promoters express to low levels in ES cells. Since Nanog and Oct4 are both transcriptional factors, ES cells may not need to express those proteins to such a high level as metabolic enzymes, like PGK. A vector without promoter (LTR promoter is self-inactivation and no internal promoter) was used as a negative control to estimate the background expression of EGFP (HSC1-Non-EGFP, referred as “Non” in
Given the fact that Nanog and Oct4 are not expressed in viral producer cells (293T based Plat-E) (data not shown), those promoters may work as a transcriptional repressor of 5′LTR and may be preventing virus production. Interestingly, EGFP expression from the 5′ LTR promoter was suppressed by introduction of Nanog and Oct4 promoters in the retrovirus producer cells (
As an alternative of Nanog and Oct4 promoter, the ETn LTR promoter was tested. The ETn is an LTR-type retrotransposon and highly transcribed in pluripotent stem cells, such as ES and EC cells. Among several subfamilies of ETn promoter, we used the type II #6 LTR promoter. Surprisingly, ETn promoter has higher titer and EGFP expression compared with Nanog and Oct4 promoters in ES cells (
To investigate whether expression from the ETn promoter can be increased by one or more ES-specific enhancer elements, we cloned Oct4 core enhancer element (CR4) or Sox2 core enhancer element (SRR2), or a combination of CR4 and SRR2, into the HSC1-pAMu-EGFP vector (
Introduction of one or more copies of CR4 (SEQ ID NO: 3 for forward orientation; SEQ ID NO: 5 for reverse orientation) or SRR2 (SEQ ID NO: 4 for forward orientation; SEQ ID NO: 6 for reverse orientation) enhancer sequences in forward or reverse orientation, or a combination thereof, upstream of the ETn pAMU promoter, EGFP expression was increased in ES cells (
Next, to test the expression pattern of EOS cassette further, EOS constructs EOS-C(3+) and EOS-S(4+) were transferred into a self-inactivating lentiviral vector (
To test the specificity of the expression of the EOS cassette in the pluripotent state, we performed differentiation experiments of mouse ES cells. First, lentiviral vectors were infected into J1 cells (cultured on gelatin) and spread onto duplicate plates. One plate was maintained as an undifferentiated ES culture, and another plate was differentiated as described in the Materials and Methods. As expected, EGFP expression from the EOS cassettes diminished and was almost indistinguishable from mock-infected negative control by flow cytometry (
Among several differentiation experiments, some residual GFP positive cells were observed in ES-like or EB-like colonies alter differentiation, most likely due to insufficient dissociation of EBs (
Six day differentiated EBs express the pluripotent marker SSEA-1 (stage-specific embryonic antigen-1) in 50-75% of the cells (
We also examined EOS expression specificity in human CA-1 ES cell lines (Peerani et al., 2007). CA-1 cells on feeders were infected with concentrated lentiviral vectors and EGFP expression from lentiviral vectors were examined 3 days after infection by fluorescence microscopy (
To determine whether EOS lentivirus expression is specific for pluripotent stem cells, primary human dermal fibroblasts were infected. Flow cytometry and fluorescence microscopy demonstrate that the ubiquitous PGK and Ef1α promoter vectors express in the primary fibroblasts whereas the Oct4, Nanog and EOS vectors do not (
Another application of the EOS vectors is the selective growth of pluripotent stem cells expressing antibiotic resistance. A viral vector which expresses the neomycin (G418) resistance gene under the control of EOS-C(3+) promoter was constructed (
An EOS-C(3+) lentivirus vector containing an EGFP-ires-Puro cassette was constructed to mark iPS cells generated by infection of MEFs using the Yamanaka reprogramming retrovirus vectors (
These data further indicate that that EOS is an effective marker of reprogrammed colonies and pluripotency, and enriches for the isolation and maintenance of iPS cell lines.
The pluripotency of established iPS clones was examined by in vitro differentiation.
After EB mediated in vitro differentiation, dissociated cells were stained for the three germ layer markers, beta-III tubulin (ectoderm), alpha-actinin (mesoderm) and alpha-fetoprotein (endoderm). Most of the lines (9 out of 10) differentiated into the three germ layers, indicating those iPS clones are pluripotent.
Three mouse iPS lines —EOS3 #24, #28 and #29—were selected for further study. To assess their in vivo pluripotency, we injected the cells into NOD/SCID mice for teratoma formation. Four to five weeks after injection, injected mice developed teratomas that contain the 3 germ layers (
We also investigated EOS expression during differentiation of mouse iPS cell lines using the same protocol used for mouse ES cells. As expected, the EOS-EGFP expression was extinguished upon differentiation (
To test whether residual EOS-EGFP expression marked persisting undifferentiated cells, we injected the differentiated cells into testes of NOD/SCID mice for teratoma formation. After 5 weeks of injection, the differentiated EOS3#24 cells (EOS-GFPpositive) formed significantly larger teratomas with a wide variety of tissue types, whereas the EOS3 #28 and #29 (EOS-GFP-negative) cells had no aggressive tumorigenicity nor obvious teratoma pathology (
To assess the effect of EOS vector selection on reprogramming of human somatic cells, human fibroblasts expressing the mS1c7a1 (ecotropic gammaretrovirus receptor) were infected with EOS lentiviral vectors encoding EGFP-IRES-Puro, prior to infection with MoMLV-based retroviral vectors (pMXs) encoding the four human Yamanaka factors (
These selected iPS cell lines were isolated and continued to express EOS-EGFP coincident with the endogenous pluripotent markers NANOG, TRA-1-81, SSEA-4, and TRA-1-60 (
As a proof-of-principle for EOS selection reproducibility in a disease context, we generated Rett Syndrome-specific iPS cell lines. Heterozygous MEFs from Mecp2308 mice (Shahbazian et al., 2002) were isolated and genotyped for reprogramming experiments (
These results demonstrate that EOS selection can be used to establish disease-specific iPS cell lines from a subject—such as a patient, or a mouse model of a disease such as a knockout mouse.
These data further illustrate that EOS lentiviral vectors direct pluripotent stem cells specific expression and resist vector silencing while under puromycin selection.
Negative selection may also be used in combination with other selection means to identify selected cells. A nucleic acid comprising a nucleotide sequence encoding a fusion protein of hygromycin phosphotransferase and thymidine kinase (SEQ ID NO: 40, 41) may be transfected into a population of cells that is to be, or has been induced to pluripotency as described. Successful transformants are first selected by growing in hygromycin-containing medium as described. During, or after a subsequent directed differentiation procedure, gancyclovir (or a similar reagent) is added to the growth medium to select against cells expressing thymidine kinase (e.g. those that did not undergo subsequent directed differentiation), providing a doubly-selected population of differentiated cells.
All citations are herein incorporated by reference.
One or more currently preferred embodiments have been described by way of example. It will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2,621,155 | Mar 2008 | CA | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA2009/000230 | 2/27/2009 | WO | 00 | 10/22/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 61064366 | Feb 2008 | US |
