WO2012112603A1 - Génération de cellules ips et procédés associés - Google Patents

Génération de cellules ips et procédés associés Download PDF

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Publication number
WO2012112603A1
WO2012112603A1 PCT/US2012/025117 US2012025117W WO2012112603A1 WO 2012112603 A1 WO2012112603 A1 WO 2012112603A1 US 2012025117 W US2012025117 W US 2012025117W WO 2012112603 A1 WO2012112603 A1 WO 2012112603A1
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cells
reprogramming
cell
construct
promoter
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PCT/US2012/025117
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English (en)
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Stefan M. Pulst
Sharan PAUL
Warunee DANSITHONG
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University Of Utah Research Foundations
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Priority to EP12719479.3A priority Critical patent/EP2675903A1/fr
Publication of WO2012112603A1 publication Critical patent/WO2012112603A1/fr
Priority to US13/975,004 priority patent/US9228204B2/en
Priority to US14/984,861 priority patent/US9920333B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/602—Sox-2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/603—Oct-3/4
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/604—Klf-4
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/606—Transcription factors c-Myc
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/20—Vector systems having a special element relevant for transcription transcription of more than one cistron

Definitions

  • iPS induced pluripotent stem
  • Lenti- or Retro- or Adeno-viruses requires multiple viral vectors for gene delivery. Lenti- or Retro- viruses can also result in insertional mutagenesis and can present significant 20 barriers to research, clinical, and therapeutic application of iPS cells.
  • feeder cells such as inactivated mouse embryonic fibroblasts
  • Feeder 25 cells provide the essential support and nutrients to allow ES/iPS cells to grow, attach, and proliferate. The risk of contamination of viruses or other macromolecules from the mouse cells limits the use of such iPS cells for therapeutic purposes.
  • a reprogramming cell and loading viral particles In one aspect, for example, a
  • transformation construct for generating iPS cells can include a vector backbone having a plurality of reprogramming factors, each reprogramming factor being under control of a separate promoter, wherein the vector backbone is contained in an adenovirus particle.
  • the vector backbone has a size of at least 8 kb. In another aspect, the vector backbone has a size of at least 10 kb.
  • the plurality of reprogramming factors includes OCT3/4, SOX2, and at least one member selected from KLF4, c-Myc, NANOG, or LI 28.
  • the plurality of reprogramming factors includes OCT3/4, SOX2, KLF4, and c-Myc.
  • the plurality of reprogramming factors consists of OCT3/4, SOX2, and KLF4.
  • the vector backbone has a sequence that is at least 80% homologous to SEQ ID 1. In another aspect, the vector backbone has a sequence that is at least 80% homologous to SEQ ID 4. In some aspects, each reprogramming factor is under the control of a CMV promoter.
  • the present invention also provides a method of generating iPS cells.
  • Such a method can include separately cloning reprogramming factors including OCT3/4, SOX2, and at least one member selected from the group consisting of KLF4, c-Myc, NANOG, or LIN28 into separate vectors, each reprogramming factor being controlled by a promoter, and consecutively cloning each of the reprogramming factors including each promoter into a single shuttle vector.
  • the method can also include linearizing the shuttle vector and recombining in bacterial cells to create a recombinant adenoviral plasmid, linearizing the recombinant adenoviral plasmid and transfecting the recombinant adenoviral plasmid into an adenovirus packaging cell line.
  • the adenoviral plasmids can then be harvested from the adenovirus packaging cell line.
  • the method can additionally include infecting transformable cells with the adenoviral plasmids and growing the transformable cells for a period of time to generate iPS cells.
  • the reprogramming factors are cloned into separate vectors using blunt end ligation.
  • the period of time can be from about 2 days to about 10 days. In another aspect, the period of time can be from about 2 days to about 6 days.
  • the iPS cells are generated in the absence of feeder cells. In a further aspect, the iPS cells are generated in the absence of a matrigel matrix. In yet a further aspect, the iPS cells are generated in the absence of feeder cells and a matrigel matrix.
  • an iPS cell is provided that is generated according to the methods disclosed herein.
  • the present disclosure provides a differentiated cell derived from an iPS cell as disclosed herein. A variety of differentiated cells are contemplated, including without limitation, endoderm, ectoderm, mesoderm, and the like. In another aspect, the differentiated cell can be a neuron.
  • FIG. 1 is a schematic view of a vector system in accordance with one aspect of the present disclosure
  • FIG. 2A is a schematic view of a vector system in accordance with another aspect of the present disclosure.
  • FIG. 2B shows data demonstrating protein expression of multiple reprogramming factors in accordance with another aspect of the present disclosure
  • FIG. 3 A is a schematic view of an adenoviral construct in accordance with yet another aspect of the present disclosure.
  • FIG. 3B shows iPS cells generated with adenoviral constructs without feeder cells, IMR90 cells transduced with Ad-GFP, and iPS cell colonies in IMR90 cells transduced with Ad-SOK in accordance with another aspect of the present disclosure
  • FIG. 4A shows IMR90 cells transduced with adenoviruses, either Ad-GFP (a) or
  • Ad-SOK (b) in accordance with a further aspect of the present disclosure.
  • FIG. 4B shows RT-PCR data of EX cell marker genesin accordance with yet a further aspect of the present disclosure
  • FIG. 5A shows a timeline for transformation in accordance with another aspect of the present disclosure
  • FIG. 5B shows cells tested for ALP staining in accordance with another aspect of the present disclosure
  • FIG. 5C shows a Western blot analyses in accordance with another aspect of the present disclosure
  • FIG. 6A shows real-time PCR and Western blot data in accordance with another aspect of the present disclosure
  • FIG. 6B shows real-time PCR and Western blot data in accordance with another aspect of the present disclosure
  • FIG. 7A shows data demonstrating expression of markers in iPS cells in accordance with another aspect of the present disclosure
  • FIG. 7B shows data demonstrating expression of markers in iPS cells in accordance with another aspect of the present disclosure
  • FIG. 8A shows data representing isolated RNA from iPS cells that demonstrate high expression of undifferentiated ES cell-marker genes in accordance with another aspect of the present disclosure
  • FIG. 8B shows data representing isolated RNA from iPS cells that demonstrate high expression of undifferentiated ES cell-marker genes in accordance with another aspect of the present disclosure
  • FIG. 8C shows data representing isolated RNA from iPS cells that demonstrate high expression of undifferentiated ES cell-marker genes in accordance with another aspect of the present disclosure
  • FIG. 8D shows data representing isolated RNA from iPS cells that demonstrate high expression of undifferentiated ES cell-marker genes in accordance with another aspect of the present disclosure
  • FIG. 8E shows data representing isolated RNA from iPS cells that demonstrate high expression of undifferentiated ES cell-marker genes in accordance with another aspect of the present disclosure
  • FIG. 8F shows data representing isolated RNA from iPS cells that demonstrate high expression of undifferentiated ES cell-marker genes in accordance with another aspect of the present disclosure
  • FIG. 9 shows images of cells undergoing morphological changes in accordance with another aspect of the present disclosure.
  • FIG. 10A shows data from SkMC-derived iPS cells in accordance with another aspect of the present disclosure
  • FIG. 10B shows data from SkMC-derived iPS cells in accordance with another aspect of the present disclosure
  • FIG. I OC shows data from SkMC-derived iPS cells in accordance with another aspect of the present disclosure
  • FIG. 10D shows data from SkMC-derived iPS cells in accordance with another aspect of the present disclosure
  • FIG. 10E shows data from SkMC-derived iPS cells in accordance with another aspect of the present disclosure
  • FIG. 1 1 A shows data from SCA2 skin fibroblast-derived iPS cells in accordance with another aspect of the present disclosure
  • FIG. 1 IB shows data from SCA2 skin fibroblast-derived iPS cells in accordance with another aspect of the present disclosure
  • FIG. 1 1C shows data from SCA2 skin fibroblast-derived iPS cells in accordance with another aspect of the present disclosure
  • FIG. 1 ID shows data from SCA2 skin fibroblast-derived iPS cells in accordance with another aspect of the present disclosure
  • FIG. 1 IE shows data from SCA2 skin fibroblast-derived iPS cells in accordance with another aspect of the present disclosure
  • FIG. 12 shows immunohistochemistry data from differentiated iPS cells in accordance with another aspect of the present disclosure
  • FIG. 13 shows histological data revealing development of muscle and adipose tissues in accordance with another aspect of the present disclosure
  • FIG. 14 shows an illustration of an experimental time line in accordance with another aspect of the present disclosure.
  • FIG. 15A shows a heat-map of a gene expression profile in accordance with another aspect of the present disclosure
  • FIG. 15B shows a heat-map of a gene expression profile in accordance with another aspect of the present disclosure
  • FIG. 16 shows the construction of a CMV weak promoter in accordance with another aspect of the present disclosure
  • FIG. 17 shows the validation of the CMV weak promoter in accordance with another aspect of the present disclosure.
  • FIGs. 18A-C shows the generation of iPS cells using the CMV weak promoter in accordance with another aspect of the present disclosure.
  • the term “substantially” refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result.
  • an object that is “substantially” enclosed would mean that the object is either completely enclosed or nearly completely enclosed.
  • the exact allowable degree of deviation from absolute completeness may in some cases depend on the specific context. However, generally speaking die nearness of completion will be so as to have the same overall result as if absolute and total completion were obtained.
  • the use of “substantially” is equally applicable when used in a negative connotation to refer to the complete or near complete lack of an action, characteristic, property, state, structure, item, or result.
  • compositions that is "substantially free of particles would either completely lack particles, or so nearly completely lack particles that the effect would be the same as if it completely lacked particles.
  • a composition that is "substantially free of an ingredient or element may still actually contain such item as long as there is no measurable effect thereof.
  • the term "about” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “a little above” or “a little below” the endpoint.
  • the inventors have developed a non-integrating vector system where multiple reprogramming factors are cloned in a single cassette in an adenoviral vector.
  • all defined reprogramming factors that are sufficient for the generation of iPS cells are cloned in a single cassette in an adenoviral vector.
  • each gene corresponding to each reprogramming factor is expressed under the control of its own independent promoter that allows the balanced expression of all genes in one cell.
  • cells are reprogramed into iPS cells in about 2- 10 days with greater than about 80% efficiency without the need for feeder cells. These iPS cells show human ES cell morphology, express ES cell surface markers and pluripotent cell-specific genes.
  • the iPS cells can also be differentiated into cells of the three germ layers. iPS cells can be generated from a variety of cells, including, without limitation, human skeletal muscle cells and skin fibroblasts.
  • iPS cells generated by the present invention in as little as 2-3 days without feeder cells meet all the reported criteria seen in iPS cells generated by other methods.
  • Cells reprogrammed using the present techniques display features typical of human ES cells, including the presence of an unmethylated NANOG promoter, early initiation of mesenchymal to epithelial transition, expression of ES cell-marker genes and cell surface markers, as well as differentiation into germ layers in vitro and in vivo, including neurons.
  • promoters are contemplated, and any promoter that can be utilized as described herein is considered to be within the present scope.
  • the promoter can be a CMV promoter. Additionally, in some aspects each
  • each reprogramming factor can have a separate promoter of the same promoter type, e.g. each reprogramming factor can have a separate CMV promoter. In other aspects, each reprogramming factor can have a separate promoter, but the promoters may not be of the same promoter type. Thus, in some cases different promoters can be utilized to affect the balance of expression of the reprogramming factors in a cell. It is also noted that a promoter can be modified to increase or decrease expression of a reprogramming factor if desired.
  • a promoter-driven reporter can be included in the cassette to track the recombinant adenovirus and transgene expression. Any reporter that can be loaded into the cassette with the reprogramming factors is considered to be within the present scope. In one specific example, the reporter is a promoter driven GFP marker.
  • the methods for plasmid construction, gene expressions of recombinant adenoviral vectors, generation of iPS cells from 1MR90 fibroblast cells without using feeder cells, and- the like are shown as non-limiting examples in the following discussion.
  • the techniques described herein can be utilized in a variety of contemplated transformation systems, and should not be seen as being limited to the iPS transformation system disclosed herein. Variation in the number of reprogramming factors included in the cassette and the specific types of reprogramming factors can vary, both within the iPS system and in other transformation systems.
  • the present disclosure provides a transformation construct for generating iPS cells that can include a vector backbone having a plurality of reprogramming factors, where each reprogramming factor is under control of a separate promoter.
  • the vector backbone can be contained in a suitable delivery package such as, for example, an adenovirus particle.
  • the vector backbone can have a size of at least 8 kb.
  • the vector backbone can have a size of at least 10 kb.
  • such large size plasmids can be introduced into an adenoviral vector. It is believed that such a large plasmid represents at least a 60% increase in plasmid size loaded into such viruses as compared to those outside of the present methods.
  • iPS human induced pluripotent stem
  • adenoviral construct allows balanced expression of all reprogramming factors in single cell, and greatly speeds up the reprogramming efficiency of cells in a short period of time over conventional iPS cell generation methods.
  • traditional iPS cell generation methods can take about 30-45 days, whereas the present methods can generate iPS cells in 10 days or less.
  • iPS cells can be generated in from about 2 to about 10 days.
  • iPS cells can be generated in from about 2 to about 6 days.
  • iPS cells can be generated in from about 2 to about 3 days.
  • the time period for the generation of iPS cells is measured from the time of transfection with a viral cassette until the observable appearance of stem celllike colonies.
  • the current methods allow iPS cells to be generated without the use of feeder cells and/or matrigel systems. While not intending to be bound to any scientific theory, this may be due to a more rapid transformation of the cells into iPS cells. Because the multiple reprogramming factors are introduced simultaneously into recipient cells under the control of separate promoters, this may allow a more rapid transformation into iPS cells that more quickly form colonies of cells that have increased surface area compared to, for example, adhered fibroblasts. Such increased surface area also results in increased access to nutrients in the culture medium, thus rendering feeder cells unnecessary. Furthermore, the simultaneous or near simultaneous transformation into iPS cells may promote survival of iPS cells through secreted factors.
  • cells that are just about to transform into iPS cells, or that have just undergone this transition begin to migrate towards each other to form colonies. This also suggests the presence of secretable factors to indicate location and initiate locomotion toward iPS cell colonies.
  • secretable factors include, without limitation, peptides, proteins, lipoproteins, glycoproteins, glycolipids, and the like, and may be co-expressed with receptor molecules at the cell surface.
  • reprogramming factors can be cloned separately into separate vectors, where each reprogramming factor is under the control of a separate promoter, such as, for example, a cytomegalovirus (CMV) promoter.
  • a separate promoter such as, for example, a cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • pAdTrack-CMV cytomegalovirus
  • the present scope includes any promoter that is capable of being separately associated with a set of reprogramming factors and loaded into a viral cassette.
  • Non-limiting examples can include the CAG- promoter, a combination of CMV early enhancer elements, chicken beta-actin promoter, and the like.
  • a mutated promoter such as a mutated CMV promoter, can be utilized to alter the expression of the associated reprogramming factor.
  • reprogramming factors along with the associated promoters are consecutively subcloned into a shuttle vector.
  • a shuttle vector can be using blunt end ligation. Any number of reprogramming factors can be subcloned into the shuttle vector. In one aspect, at least two reprogramming factors can be subcloned therein. In another aspect, at least three reprogramming factors can be subcloned therein. In another aspect, at least four reprogramming factors can be subcloned therein.
  • Non-liming examples of reprogramming factors include OCT3/4, SOX2, KLF4, c-Myc, NANOG, LIN28, and the like, including combinations thereof.
  • the reprogramming factors can include OCT3/4, SOX2 and at least one factor selected from KLF4, c-Myc, NANOG, or LIN28.
  • the reprogramming factors can include OCT3/4, SOX2, and at least two factors selected from, KLF4, c-Myc, NANOG, or LIN28.
  • the reprogramming factors include OCT3/4, SOX2, KLF4, and c-Myc.
  • one example of an OCT3/4, SOX2, KLF4, and c-Myc construct is at least 80% homologous to SEQ ID 1.
  • the construct can be at least 95% homologous, or 100% homologous to SEQ ID 1.
  • the reprogramming factors include or consist of OCT3/4, SOX2, and KLF4.
  • SOX2, and KLF4 construct is at least 80% homologous to SEQ ID 4.
  • the construct can be at least 95% homologous, or 100% homologous to SEQ ID 4.
  • the reprogramming factors include or consist of OCT3/4, SOX2, NANOG, and LIN28.
  • the reprogramming factors include OCT3/4, SOX2, NANOG, and LI 28.
  • other members of the OCT, SOC, NANOG, and LIN families can be utilized.
  • At least one reprogramming factor can be selected from AIRE, CBFA2T3, CEBPE, CRABP2, EGR4, HIC2, IRX4, IRF7, KCNH2, KLF3, KLF4, KLF9, LIN28B, LHX6, LHX1 , NFATC 1 , NFATC2, PEG3, POLR3G, PAX8, RAX2, RUNX3, SFRS 17A, SOX8, TAF4B, ZNF57, and the like.
  • At least one reprogramming factor can be selected from ETS 1 , FOXM 1 , HEY 1 , HOXA4, HOXA3, KLF6, KLF2, LZTS 1 , LBX2, MYBL 1 , MYBL2, MITF, POGK, RUNX 1 , SALL2, SOX6, SP140, SMAD6, SMAD9, TCF 19, TOP2A, VGLL3, ZNF641 , ZNF671 , ZNF70, and the like.
  • reprogramming factors can include on or more selected from DBP, ZNF33B, CREB3L2, ZSCAN 16, AHR, ZNF 138, HSF4, HMX2, HEY1 , ZNF 192, PITX2, MAX, CIR l , PBX3, ZNF3, PRDM2, HOXC9, NFKB2, NRL, BATF3, SOX4, BARHL 1 , TSC22D3, MEF2D, GATAD2B, ZNF33A, KLF7, NR 1 D2, AHR, ZNF639, ETV6, IKZF4, NR1D2, ZNF217, HOXC8, GLIS3, HOXC6, ZNF397, AR1D4A, ZNF496, MLLTIO, ZFP36L1, NRL, PKNOXl, MTA3, PAX7, DMTF1, MZF1, RUNX1, HOXA2, ⁇ , MLLTIO, NFE2L1, PBX3,
  • SCAND2 HOXA6, LM04, SNAPC5, FOXC1, PCGF6, TAF5L, HOXB4, ETV6, HOXA4, ZNF256, ZNF449, ZNF193, RUNX1, ZBTB17, MYOG, NFIC, TBX5, HOXA5, CUX1, GLI3, CNOT7, TCF25, CNOT7, NPAT, SP4, MSC, IRF2, TBX5, RUNX 1 , ZBTB38, CREM, ZNF397, NR2F1, ZNF217, KLF5, RFXAP, HMGB2, CBL, ZNF93, ZSCA 12, MYST2, EGR2, SATB1, E2F1, PLAG1, PFDN1, E2F3, ZNF18, ENST00000300681, HLX, E2F2, SALL2, L3MBTL1, RCAN1, ARNT2, RERE, GTF2I, HIF1A, RU X1, SMAD9, ZNF211,
  • reprogramming factors can include on or more selected from SLC30A9, ZFP36L2, ELK4, ZHX3, TCFL5, GABPBl, NKX3-1, BLZF1, BLZFl, ZSCAN2, ZNF134, AFF1, NFYA, NCOR1, TRPS1,
  • HOXB9 HES6, MESP1, LBX-1, STAT4, DUX4, NFX1, NR2F6, HES6, HES1, HMX1, PPARA, ZNF445, DUX4L4, SOX11, EVX1, PBX4, ZXDC, ZNF131, LMOl, ZNF3, KDM5B, STAT5A, DUX4, PHF5A, REL, ZNF446, MLL4, ZNF157, IRF5, HOXA9, TAF10, HSFI, ZNF133, TRERF1, NR1I3, ISL2, LMX1B, SIM1, SCAND2, MYNN, ARX, TBX6, VSX 1 , TBX 10, NR5A2, GATA6, PAX6, TFDP2, DM5B, SNAPC5, HAND 1 , PAX4, DUX4, NFX1, ZNF277, SNAPC5, ZBTB48, POU5F1, ESRRG, HOXD9, CBFA2
  • any one or more of the above reprogramming factors can be utilized with any other reprogramming factor described herein or in any combination with any other reprogramming factor described herein.
  • FIG.1 a schematic outline of one non-limiting example of an expression system (e.g. AdEasy-1) is provided.
  • Reprogramming factors of interest such as, and without limitation, OCT3/4, SOX2, LF4, and c-Myc, can be first cloned separately into a vector under separate promoters 102 (e.g. pAdTrack-CMV). Then, each reprogramming factor along with the promoter can be consecutively subcloned into a shuttle vector 104 (e.g. apAdTrack) using a technique such as, for example, blunt end ligation.
  • a shuttle vector 104 e.g. apAdTrack
  • the resultant plasmid can be linearized by digesting with a restriction endonuclease such as Pme ⁇ , and recombination can be carried out using high competence bacterial cells, such as E. coli BJ5 183 cells, by homologous recombination. In some cases, high competence bacterial cells can allow for more efficient recombination.
  • the recombinant adenoviral plasmid 106 e.g. pSOKcM-AdEasy-1
  • an enzyme such as Pad
  • transfected into an adenovirus packaging cell line for virus production is HEK 293A.
  • adenoviral backbone vector 106 represent the regions mediating homologous recombination between the shuttle vector 104 and the adenoviral backbone vector 106.
  • the recombination can be confirmed by multiple restriction endonuclease analyses, and the production of recombinant adenoviruses can be followed by GFP expression.
  • FIG. 2A shows a schematic representation of an adenoviral vector containing multi-reprogramming factors in a single cassette, pSOKcM-AdEAsy-1 102.
  • FIG. 2B shows SH5Y cells that were transiently transfected with pSOKcM-AdEasy- 1 and pSOKcM-AdShuttle constructs. Protein extracts from harvested cells at 40-54 hrs post-transfection were probed by Western blot analysis using the antibodies indicated. Blots were re-probed for Actin as an internal loading control.
  • iPS cells expresses all proteins from the adenoviral vector in cells tested.
  • iPS cells can thus be generated using such an adenoviral vector containing multi- reprogramming factors.
  • iPS cells can be generated from a variety of transfectable cell types, and any type of cell capable of transfection is considered to be within the present scope.
  • One specific example of such a transfectable cell type includes IMR90 human fetal fibroblasts.
  • iPS cells can be generated with such an adenoviral vector without feeder cells or a matrigel matrix.
  • AdEasy-1 Ad-GFP or Ad-SOK are shown.
  • FIG. 3B a timeline of experimental design is shown. IMR90 cells were transduced with adenoviruses, Ad-GFP or Ad-SOK on day 2. Culture medium was changed every day with regular cell culture medium. Colonies appeared at days 4-7 in culture dishes. The top of FIG. 3B shows photomicrographs of IMR90 cells transduced with Ad-GFP on day 7; phase contrast in the top left image and GFP expression in the top right image.
  • FIG. 3B middle and bottom images show iPS cell-like colonies appearing in IMR90 cells transduced with Ad-SOK on days 4-7, as shown by phase contrast (FIG. 3B middle and bottom left). GFP expression in fluorescence microscopy of the same colony is shown in FIG. 3B bottom right. Thus by days 4-7, several colonies showing ES cell-like morphology emerged and all colonies looked identical. The resultant colonies (iPS cells) can be further expanded or subjected to characterization.
  • iPS cells can be further characterized.
  • the undifferentiated state of human ES cells/iPS cells express high levels of membrane alkaline phosphatase (AP), and AP staining can be used to characterize such stem cells.
  • AP staining iPS cells are generated from iMR90 cells in 24 well plates using the methods as described. At day 7, iPS cells are fixed with 4% paraformaldehyde for 2 minutes, followed by 15- minute incubation with staining solution (Alkaline Phosphatase Detection Kit; Millipore).
  • staining solution Alkaline Phosphatase Detection Kit; Millipore.
  • AP staining data demonstrate the positive staining for iPS cells, as shown in FIG. 4A.
  • IMR90 cells are transduced with adenoviruses, Ad-GFP or Ad-SOK for 7 days.
  • Human iPS cells generated from Ad-SOK are positive for alkaline phosphatase (AP) staining.
  • FIG. 4B shows RT-PCR analyses of ES cell marker genes. IMR90 cells are transduced with adenoviruses, Ad-GFP or Ad-SOK for 7 days. Total RNA is isolated from harvested cells and synthesized cDNAs (150 ng) are used for RT-PCR analyses.
  • Human iPS cells express many undifferentiated ES cell marker genes including telomerase reverse transcriptase (hTERT) and growth and differentiation factor 3 (GDF3).
  • FIG. 4B shows an expression profile by RT-PCR analyses, demonstrating that iPS cells derived from IMR90 cells highly express the hTERT and GDF3 genes.
  • somatic cell reprogramming was tested using the adenovirus containing OCT3/4, SOX2, KLF4, and c-Myc (Ad-SOcMK) shown in FIG. 1.
  • IMR90 cells were transduced with the adenovirus, and the timeline for transformation is shown in FIG. 5A. Briefly, the IMR90 cells were transduced with Ad- SOcMK or Ad-GFP for 12 or 24 hrs, after which the medium was replaced with human ES cell medium. Within 1 day, Ad-SOcMK-transduced cells took on a different appearance and began to form small cell clusters. By day 2 or 3, several colonies of cells showing ES cell-like morphology emerged in the dish (FIG.
  • I B top right and middle right.
  • Cells were also tested for ALP staining, as is shown in FIG. 5B, bottom right.
  • the ALP assay reveals strong staining of IMR90-derived iPS cells indicating pluripotency, while no ALP staining is observed in the GFP-transduced cells (FIG. 5B, lower left).
  • the expression of exogenous individual protein factors in protein extracts from harvested cells was also investigated by Western blot analyses, as is shown in FIG. 5C. The results demonstrate that all RFs in the adenovirus are highly expressed in transduced IMR90 cells but not in Ad-GFP-transduced cells.
  • mesenchymal-to-epithelial transition is a key regulatory event during reprogramming of somatic cells to the pluripotent state.
  • Expression of exogenous reprogramming factors effectively activate the epithelial program and shut down key mesenchymal genes to favor the MET transition of somatic cells toward induced pluripotency.
  • THY l mesenchymal marker
  • CDH I 22-24 upregulation of the epithelial marker
  • RT PCR and Western blot analyses reveals upregulation of CDHI and concomitant reduction of THY l in iPSCs when compared with control (See FIG. 6A).
  • THY l is exclusively expressed in fibroblasts and fibroblast cells dramatically switched the state. in a short period of time, the expression level of THY l by real-time PCR can be determined as a function of reprogramming efficiency.
  • Real-time PCR and Western blot data reveals a decrease in levels of THYl by -80% in Ad-SOcMK transduced cells as cells are reprogrammed (See FIG. 6A lower panel, and FIG. 6B).
  • iPS cells generated with Ad-SOcMK express human ES cell-marker genes such as NANOG, Telomerase reverse transcriptase (TERT), LIN28, stage specific embryonic. antigens (SSEA- 1 , -3, and -4), and tumor-related antigens (TRA 1 -60 and -81 ).
  • ES cell-marker genes such as NANOG, Telomerase reverse transcriptase (TERT), LIN28, stage specific embryonic. antigens (SSEA- 1 , -3, and -4), and tumor-related antigens (TRA 1 -60 and -81 ).
  • SSEA- 1 , -3, and -4 stage specific embryonic. antigens
  • TRA 1 -60 and -81 tumor-related antigens
  • Real-time RT-PCR, semi-quantitative PCR, and Western blot analyses can be performed.
  • Real-time and semi-quantitative PCR analysis of isolated RNA from iPS cells demonstrate high expression of undifferentiated ES cell-marker genes, including NANOG, TERT, L1N28, ALPL, growth and differentiation factor 3 (GDF3), fibroblast growth factor 4 (FGF4), developmental pluripotency-associated 5 (DPPA5), interferon induced transmembrane protein 1 (LFIT 1), galanin prepropeptide (GAL), gamma-aminobutyric acid (GAB A) A receptor, beta 3 (GABRB3), teratocarcinoma-derived growth factor 1 (TDGF 1), Nodal homolog (NODAL) and podocalyxin-like 2 (PODXL2) (See FIGs. 8A-F).
  • Ad-SOcMK transduced cells undergo progressive epithelial-like morphological changes from elongated fibroblasts (FIG. 9, panels a, b) to packed clusters of rounded cells as visualized by phase contrast microscopy (FIG. 9, panels d, f, h). Morphological changes occur in close association with expression of ALP. ALP-positive cells appeared as early as day 1 in Ad-SOcMK transduced cells and ALP positive cells progressively increased as reprogramming time increased (FIG. 9, panels 1, n, p). Cells transduced with Ad-GFP showed neither morphological changes (FIG.
  • somatic cells Such reprogramming of somatic cells is also accompanied by significant epigenetic changes.
  • the NANOG promoter changes from a highly methylated state in somatic cells to being unmethylated and active in iPS cells.
  • bisulfite genomic sequence analysis can be used to evaluate the methylation status of cytosine guanine dinucleotides (CpGs) in the NANOG promoter. CpGs are highly unmethylated in iPS cells when compared with the highly methylated CpGs in parent
  • IMR90 cells This indicates that the NANOG promoter is active in iPS cells derived from IMR90 cells resulting in increased steady-state levels (FIG. 8B, lower panel).
  • Southern blot analysis can be performed by digesting genomic DNA from iPS cells generated with Ad- SOcMK with BamHI and Ascl for KLF4 and c-MYC probes, respectively. Notably, Southern blot analyses does not detect genomic integration of the adenoviral transgene into iPS cells derived from IMR90 cells (data not shown).
  • chromosomal G- band analyses showed that iPS .cells generated with Ad-SOcMK had a normal karyotype of 46XX (data not shown).
  • a variety of cell types can be utilized to generate iPS cells according to aspects of the present disclosure, and any such capable cell is considered to be within the present scope.
  • human skeletal muscle cells (SkMCs) and spinocerebellar ataxia 2 (SCA2) patient skin fibroblasts were used.
  • SkMCs and SCA2 skin fibroblasts are transduced with Ad-SOcMK, several iPS cell colonies resembling ES cell-like morphology emerge in the dishes as early as day 3.
  • the SkMC and SCA2 skin fibroblast-derived iPS cells positively stain for ALP, and
  • iPS cells One of the useful characteristics of pluripotency is the ability of iPS cells to differentiate into all three germ layers.
  • the following non-limiting example is provided to show such differentiation.
  • freshly prepared iPS cells with Ad-SOcMK as have been described were cultured in ES cell medium without basic fibroblast growth factor (bFGF) for 8-9 days.
  • the resultant embryoid bodies (EBs) in suspension cultures are allowed to differentiate further in chamber slides. After 9-10 days in adherent culture, attached cells show various types of morphologies.
  • Immunocytochemistry reveals the detection of Nestin (ectoderm, FIG. 12, panel d), smooth muscle actin (SMA) (mesoderm, FIG.
  • iPS cells are seeded on inactivated MEF cells and cultured for 22-25 days. Morphological and immunostaining data revealed that the iPS cells were differentiated into neurons with a subpopulation of neurons staining with the dopaminergic marker tyrosine hydroxylase (TH) (FIG. 12, panels g, h). To examine developmental potential in vivo, iPS cells generated with Ad-SOcMK are injected into NOD/SCID mice subcutaneously.
  • TH dopaminergic marker tyrosine hydroxylase
  • iPS cells generated according to aspects of the present 5 disclosure show pluripotency with the potential of differentiating into germ layers in vitro and in vivo.
  • RNAs are isolated from Ad-SOcMK and Ad-GFP transduced IMR90 cells at 0, 24, 48 and 72 hrs post-transduction and queried for global gene
  • Differential expression analyses shows changes in 6,852 genes for 0/24 hr, 12,945 for 0/48 hr, and 14,158 for 0/72 hr (data not shown).
  • R A expression is analyzed at 6 hr intervals for 84 hrs after0 Ad-SOcMK transduction.
  • FIG. 14 shows an illustration of the experimental time line. To identify temporal waves of gene expression across time points, the entire data set is analyzed, including Ad-GFP-transduced control cells (> 1.5-fold differential expression) by using singular value decomposition (SVD)25.
  • SVD singular value decomposition
  • FIG. 15A shows a heat-map of the gene expression profile for this data set5 including lincRNAs (21 ,372 genes).
  • lincRNAs 21 ,372 genes.
  • a large class of RNAs is highly expressed in IMR90 cells with rapid reduction in the following 12-24 hrs.
  • a second class of RNAs shows little change initially, and then exhibits increased expression with a return to or below initial levels by 72-84 hrs.
  • genes in a third group have low expression in the first 24-48 hrs, but then become highly expressed from that0 time on. Similar clustering of lincRNA expression can be observed (>four-fold
  • an altered promoter can be utilized to alter the expression of a particular reprogramming factor.
  • CMV W p a weakened CMV promoter
  • CMV W p CMV W p
  • CMV W p a weakened CMV promoter
  • 589bp has previously been used in mammalian system to express a protein in order to study protein functionality. Decreasing the expression of a reprogramming factor can be benficial in the reprogramming process. In some case, overexpression of a protein may actually hamper the reprogramming process. As such, in some cases factors can be tuned to more beneficial rates of expression.
  • CMVwp has been developed to, among other reasons, to regulate protein expression and allow a higher amount of genetic material to be cloned into a single cassette.
  • the inventors have constructed a series of mutant CMV promoters by deleting 200 or 322 bp from original CMV promoter sequence of pEGFPNl (Clontech Inc., USA) plasmid using either PCR or restriction digestion methods.
  • the resultant mutant CMV promoters are tested for promoter activity by Western blot analyses expressing in HEK293 or SH-SY5Y cells.
  • CMV A('/ ? / . 4 ⁇ -GFP constmct designated as CMV weak promoter-GFP (CMVn> / >-GFP) results in the significant reduction of the GFP protein expression by >60% when compared with or CMV A( i.2oo)bp-GF?.
  • CMV weak promoter-GFP CMV weak promoter-GFP
  • CMV promoter variants construction of CMV promoter variants is shown. 200 bp deleted from the 5' end through PCR or 322 bp deleted by Aatll digestion from the CMV promoter region of pEGFPNl is shown in the upper panel of FIG. 16. Validation of promoter activity is shown in the lower panel of FIG. 16. Protein extracts from HEK293 or SH-SY5Y cells transfected with CMV promoter variants were subjected to Western blot analyses using the antibodies indicated. The blots were re-probed for Actin as an internal loading control. promoter [CMV weak promoter ⁇ CMV wp)] results in significant reduction of GFP protein expression.
  • iPS cells are then generated using the CMVjjp in the viral cassette as has previously been disclosed for the CMV promoter.
  • 1MR90 cells are plated at a density of 1.5-2.5 x 10 6 cells per 10 cm tissue culture dish without feeder cells.
  • TMR90 cells are about 60-70% confluent and the cells are transduced with medium (DMEM, 5%FBS, 1 % NEAA, 0.5% penicillin-streptomycin) containing adenoviruses, Ad-GFP (control) or Ad(CMV
  • the culture medium is replaced with human ES cell medium including
  • DMEM/F 12 20% Knockout Serum Replacement (KSR), I X nonessential amino acids, I X sodium pyruvate, IX L-glutamine, 0.1 mM b-mercaptoethanol, 25 ng/ml basic fibroblast growth factor (bFGF), and 0.5% penicillin-streptomycin.
  • KSR Knockout Serum Replacement
  • I X nonessential amino acids I X sodium pyruvate
  • IX L-glutamine 0.1 mM b-mercaptoethanol
  • 25 ng/ml basic fibroblast growth factor (bFGF) 25 ng/ml basic fibroblast growth factor (bFGF), and 0.5% penicillin-streptomycin.
  • the medium is changed every day and incubated for 7-8 days. By days 4-7, several colonies showing ES cell-like morphology emerge and all colonies look identical, as shown in FIG. 18B.
  • the resultant colonies (iPSCs) can be further expanded or subjected to
  • SEQ ID 2, SEQ ID 3, and SEQ ID 4 are examples of adenovirus cassettes utilizing CMV W p- Additionally, as has been described, the undifferentiated state of human ES/iPS cells express high levels of membrane alkaline phosphatase (ALP) and ALP staining can be used to characterize the stem cells.
  • ALP staining iPS cells are generated from iMR90 cells in 12 wells plate using the methods described. An experimental timeline is shown in FIG. 18A. At day 4, iPS cells are fixed with 4% paraformaldehyde for 2 minutes, followed by 15-minute incubation with staining solution (Alkaline Phosphatase Detection Kit; Millipore).
  • ALP staining data demonstrates the positive staining for iPS cells, as is shown in FIG. 1 8C.
  • the CMVWP promoter can be utilized to generate iPS cells according to the methods and techniques described herein.
  • Reprogramming factors OCT3/4, SOX2, GKLF4 and c-Myc. Plasmids containing the reprogramming factors (pEP4 E02S ET2K, pCEP4-M2L, pEP4 E02S EN2K, pEP4 E02S CK2M EN2L) are purchased from Addgene Inc., USA.
  • Each of the reprogramming factors was PCR amplified from the plasmids with Nhel restriction sites. The authenticity of each gene was verified by Nhel restriction digestion analyses and DNA sequencing.
  • Plasmid, pEGP-Nl (4.7 kb) is purchased from Clontech Inc., USA.
  • Adenoviral plasmid (pAdEasy- 1 , 33.4 kb), Shuttle vectors (pAdTrack and pAdTrack-CMV), Competent cells (AdEasier cells: E coli BJ5183 containing pAdEasy- 1 backbone), and Packaging cells (HEK 293A) were generous gift from CoraliePoizet, Larry Kedes Lab, University of Southern California, Los Angeles, California, USA.
  • Human embryonic fibroblast IMR90 cells were obtained from the American Type Culture Collection (ATCC), Catalog No. CCL- 186. IMR90 cells were maintained in DMEM medium containing 10% fetal bovine serum (FBS).
  • ATCC American Type Culture Collection
  • FBS fetal bovine serum
  • SkMCs Human skeletal muscle cells
  • SCA2 skin fibroblasts containing (CAG)57 were obtained from Coriell Cell Repositories, USA Catalog No. # GM04319. SCA2 skin fibroblasts were cultured in MEM medium containing 15% FBS.
  • KO serum replacement Fetal bovine serum, FBS (Hyclone, Thermo Scientific) KO serum replacement (KOSR; Invitrogen, cat. no. 10828-028)
  • Nonessential amino acid solution (NEAA) (Invitrogen Inc.,)
  • bFGF Basic fibroblast growth factor
  • Penicillin/streptomycin, 100X (Invitrogen Inc.,)
  • Collagenase type IV (Invitrogen, cat. no. 17104-019)
  • Biosafety cabinet with aspirator for tissue culture fitted for stereomicroscope Tissue culture centrifuge, Sorvall, Legend XI Centrifuge, Thermo Scientific. Tissue culture dishes and Flasks, 100mm, 150 mm and T-25
  • Plastic disposable transfer pipettes 1 , 5, 10 and 25 ml
  • Disposable sterile filter system (0.22 ⁇ , 250 ml and 500 ml)
  • Disposable syringes 60, 30, 10 and 1 ml
  • Freezing container (Nalgene Labware, cat. no. 5100) Cell lifter (Corning, cat. no. 3008) REAGENT SETUP .
  • CM- 1 Culture medium 1 (CM- 1): DMEM, 10% FBS, and 1% penicillin-streptomycin
  • CM-2 culture medium 2 (CM-2): DMEM, 5%FBS, 1% NEAA, and 0.5% penicillin-streptomycin.
  • CM-3 Culture medium 3 (CM-3): DMEM, 10%FBS, 1 % NEAA, and 0.5% penicillin- streptomycin.
  • Mouse embryo fibroblast (MEF) medium DMEM, 10%FBS, 1% NEAA, and 0.5% penicillin-streptomycin.
  • hiPS cell medium DMEM/F 12 containing 20% KOSR (vol/vol), 50 ng/ml bFGF, IX L-Gln, IX NEAA, IX Sodium Pyruvate, 100 ⁇ 2-mercaptoethanol, 50 U/ml penicillin, and 50 mg/ml streptomycin.
  • 2X cell-freezing medium DMEM, 20% DMSO (vol/vol), 40% FBS (vol/vol), and 1% penicillin-streptomycin
  • 2X iPS cell-freezing medium DMEM/F 12, 20% DMSO (vol/vol), 60% FBS (vol/vol), and 20% hiPS medium (vol/vol).
  • pEGFP-Nl plasmid (4.7 kb; purchased from Clontech Inc., USA.) is digested with Bglll and NotI to remove the GFP open reading frame (ORF) from the plasmid backbone.
  • the digestion reaction mix is as follows: pEGFP-N l Plasmid DNA (1 ⁇ ) 10 ⁇
  • the digestion reaction mix is incubated at 37° C for 3-4 hrs. Heat inactivation is performed at 65° C for 30 min.
  • the digested product is then electrophoresed on a 0.8% agarose gel and the plasmid back bone (3.9 kb) is purified using a gel extraction kit (Qiagen).
  • Each of the reprogramming factors (OCT3/4, SOX2, GKLF4 and c-Myc) are PCR amplified from pEP4 E02S ET2K or pCEP4-M2L or pEP4 E02S EN2K or pEP4 E02S CK2M EN2L plasmids (Addgene) with Nhel restriction sites.
  • the PCR products are cloned into pEGFP Nl (GFP deletion) at Nhel site from the above digestion reaction.
  • the ligation reaction mix is as follows:
  • the ligation reaction mix is incubated at 16°C for 18-24 hrs.
  • the DNA is mixed with DH5a competent cells (New England Biolabs Inc.) and the transformation is performed.
  • the cell suspension is inoculated onto 10 cm petri dishes containing LB-agar plus 50 ⁇ g/ml of kanamycin.
  • the agar plates are incubated at 37°C for 20-24 hrs.
  • the positive clones of each gene are verified by Nhel restriction digestion analyses and DNA sequencing.
  • each cassette from the above reaction is consecutively subcloned into the shuttle vector (Sox2 cassette at Hindlll site, OCT3/4 cassette at EcoRV site, KLF4 cassette at Sail site,and c-Myc cassette at Notl site), designated as pAdSOcMK shuttle vector, as is shown in FIG. 1 .
  • the ligation reaction mix is as follows: pShuttle Vector DNA ( 10 ng/ ⁇ ) 1 ⁇
  • the ligation reaction mix is incubated at 16°C for 18-24 hrs.
  • the DNA is mixed with DH5a competent cells and the transformation isperformed.
  • the cell suspension is inoculated onto 10 cm petri dishes containing LB-agar plus 50 ⁇ g/ml of kanamycin.
  • the agar plates are incubated at 37°C for 20-24 hrs.
  • the positive clones of each gene are verified by restriction digestion analyses and DNA sequencing.
  • High competence bacterial cells (E coli BJ5183) are utilized in the following methods to achieve efficient recombination.
  • Recombinant pAdShuttle plasmid clones containing the reprogramming factors (pAdSOcMK) from Example 1 are grown in 4.0 ml LB/kanamycin in a 5-ml conical tube, and shaken overnight in a 37°C orbital shaker.
  • the plasmid DNA is purified by an alkaline lysis procedure. It has been found that efficient homologous recombination in
  • AdEasiercells is improved by maintaining the integrity of the shuttle vector DNAs.
  • Plasmids purified with commercial DNA minipreparation kits can contain significant numbers of nicked DNA molecules that may be detrimental to efficient and faithful recombination.
  • the conventional alkaline lysis procedure can provide consistent and reliable results.
  • the recombinant shuttle vector plasmid is linearized by digesting with the restriction endonuclease Pmel, and purified using a gel extraction kit (Qiagen).
  • the digestion reaction mix is as follows:
  • Recombinant shuttle vector DNA ( 1 ⁇ g/ ⁇ l) 10 ⁇
  • the digestion reaction is incubated at 37°C for 3-4 hrs. Heat inactivation is performed at 65°C for 30 min.
  • the digested product is electrophoresed on a 0.8% agarose gel and the plasmid back bone is purifiedusing a gel extraction kit (Qiagen).
  • 100 ⁇ of the cell suspension is inoculated onto each of three 10 cm petri dishes containing LB-agar plus 50 ⁇ g/ml of kanamycin.
  • the agar plates are incubated at 24- 30°C for 2-3 days until colonies appear.
  • Each colony is isolated and grown in 4 ml LB medium containing 50 ⁇ g/ml of kanamycin at 24-30°C for 2 days in an orbital shaker.
  • Plasmid DNA is isolated using the conventional alkaline lysis method. Pad restriction digestion is performed on candidate clones. Correct recombinants usually yield a large fragment ( ⁇ 30 kb) and a smaller fragment of 4.5 kb.1 -3 ⁇ of correct recombinant plasmids (pAdSOcMK adenoviral vector) are retransformed into DHIOB competent cells. The correct clones are subjected to restriction enzyme and/or PCR analysis to verify authenticity. The plasmids are purified with Pure Link Maxi Kit (invitrogen Inc.,) in order to transfect into the packaging cells (HEK 293A cells) for virus production.
  • Example 3 Adenovirus production in packaging cells (HEK 293A)
  • HEK 293A cells E 1 -transformed human embryonic kidney cells
  • HEK 293A cells E 1 -transformed human embryonic kidney cells
  • cell culture medium containing DMEM, 10% FBS, and 1 % penicillin-streptomycin.
  • the cells are incubated at 37°C, 5% C0 2 for 24 hr.
  • the recombinant adenoviral plasmids (pAdSOcMK) are digested with Pad (often 5 ⁇ g DNA is needed for one transfection).
  • the digested plasmids are ethanol precipitated and resuspended in 25-30 ⁇ of sterile H2O.
  • a standard lipofectaminetransfection is performed according to manufacturer's protocol (Invitrogen Inc.).
  • DMEM fetal calf serum
  • Opti-Mem I is added to a T-25 flask containing the cells. Incubate the cells (37°C, 5% C02) for 10-15 min. The lipofectamine-DNA mix is added to the flasks with the cells and returned to the incubator for 5-6 hrs. The lipofectamine/DNA medium is removed and 5-7 ml of fresh cell culture medium is added, and the cells are incubated at 37°C, 5% C02.
  • the cells are scraped off the flask with a scrapper at 20-30 days post-transfection and the medium with the cells is collected in 15/50 ml conical tubes.
  • the tubes are spun in a benchtop centrifuge, and supernatant (sup 1) is collected and the pellet is resuspended in 2-3 ml sterile PBS.
  • the cells are frozen in a dry ice/methanol bath or a -80°C freezer, thawed in a 37°C water bath, and vortexed vigorously. This procedure of freeze/thaw/vortex is repeated for 3-4 more cycles.
  • adenovirus particles The adenovirus particles are filtered with a 0.45 ⁇ syringe filter and stored at -20/-80oC until use.
  • Example 4 Amplification of adenoviruses
  • HEK 293A cells Two 50-70% confluent T-25 flasks of HEK 293A cells are infected using 40-50% of the viral supernatant containing the adenovirus particles from Example 3 for each flask. Cytopathic effect (CPE) or cell lysis should appear at 7-10 days post infection. Effective production of adenoviruses can be monitored by GFP expression. When >90% of the cells die, the cells are scraped off and adenoviral supernatant is prepared as described in Example 3. Authenticity of recombinant adenovirus can be confirmed by infecting the viral supernatant to any infectable cells and Western blot and/or PCR analyses of target genes. Multiple rounds of infection cycles in HEK 293 A cells can be carried out to harvest adenoviral particles.
  • Example 5 Generation of iPS cells by adenoviral vector containing multi- reprogramming factors from IMR90 human fetal fibroblasts without using feeder cells
  • Human embryonic fibroblast IMR90 cells are purchased from the American Type
  • IMR90 cells are cultured and maintained in culture medium 1 (CM-1 ) containing DMEM, 10% FBS, and 1% penicillin-streptomycin according to manufacturer's protocol.
  • CM-1 culture medium 1
  • the fibroblasts 1MR90 are thawed as follows:
  • a vial of frozen fibroblasts is removed from the liquid nitrogen tank and placed into a 37°C water bath until most (but not all) cells are thawed.
  • the tube is centrifuged at 1 100 rpm for 3 minutes, and the supernatant is discarded.
  • the cells are re-suspended in 10 ml of CM-1 , and transferred to a 100 mm dish (0.5-1 x 105 cells/dish). The cells are incubated in a 37°C, 5% C02 incubator until the cells become 80-90% confluent. The medium is changed every other day.
  • the fibroblasts are passaged as follows:
  • the medium is discarded the cells are washed once with PBS.
  • the PBS is aspirated, and 1.5 ml per dish of 0.05% trypsin/0.53 mM EDTA is added, and the cells are incubated for 1 -2 minutes at 37°C.
  • CM-1 CM-1
  • Adenoviral transduction is accomplished as follows:
  • IMR90 cells are plated at a density of 1 .5-2.5 x 10 6 cells per 10 cm tissue culture dish in CM- 1 without feeder cells and incubated at 37°C, 5% C0 2 for 24 hr.
  • CM-2 culture medium 2
  • DMEM fetal calf serum
  • 5%FBS fetal calf serum
  • NEAA fetal calf serum
  • NEAA fetal calf serum
  • adenovirus particles as generated in either of Examples 3 and 4, Ad-GFP or Ad-SOK or Ad-SOcMK at 100- 500 pfu/cell.
  • the cells are incubated at 37°C, 5% C02 for 24 hr.
  • CM-3 culture medium 3
  • DMEM fetal calf serum
  • NEAA fetal calf serum
  • penicillin- streptomycin 0.5% penicillin- streptomycin.
  • the cells are incubated at 37°C, 5% C02 for 24 hr.
  • Day 4 Incubation continues. The medium is changed every day with CM-3 and incubated for more 3-4 days.
  • Example 6 Adenovirus transduction and iPSC generation
  • 1MR90 cells (1.0-1.5 X 106) are cultured overnight on 100 mm dishes without feeder cells. On the following day, cells are transduced with Ad-SOcMK or Ad-GFP (control). Adenoviruses are removed at 24 hrs post-transduction (day 1), and replaced with human ES cell medium consisting of DMEM/F 12 (#1 1330-32, Invitrogen Inc., USA), 20% Knockout Serum Replacement (KSR) (#10828-028, Invitrogen Inc., USA), IX nonessential amino acids, IX sodium pyruvate, IX L-glutamine, 0.1 mM ⁇ - mercaptoethanol, 25 ng/ml basic fibroblast growth factor (bFGF) (#PHG0263, Invitrogen Inc., USA), and 0.5% penicillin-streptomycin. The medium is changed every day and by days 2-3, several colonies showing ES cell-like morphology emerged on the dish. The same protocol is used to generate iPSCs
  • Protein extracts are resolved by SDS-PAGE and transferred to Hybond P membranes (Amersham Bioscience Inc., USA). After blocking with 5% skim milk in 0.1% Tween 20 PBS, the membranes are incubated with primary antibodies in 5% skim milk in 0.1% Tween 20/PBS for 2 hrs at room temperature or overnight at 4°C. After several washes with 0.1 % Tween 20/PBS, the membranes are incubated with the corresponding secondary antibodies conjugated with HRP in 5% skim milk in 0.1 % Tween 20/PBS for 2 hrs at room temperature. Following three additional washes with 0.1% Tween 20 PBS, signals are detected by using the Immobilon Western
  • SMA Smooth Muscle Actin
  • AFP Alpha Feto Protein
  • NANOG 1 3000 1 : 500 USA 3580 THY1 1 : 4000 9798 Nestin 1 :500 Millipore Inc., USA AB5922 mAb conjugated with HRP:
  • Beta-Actin (AC- 5) Sigma Inc., USA A3854
  • ALP staining was performed using the Alkaline Phosphatase Detection Kit (#SCR004, Millipore Inc., USA). Briefly, iPS cells are fixed with 4%
  • Total RNA is prepared from harvested cells using the RNAeasy Kit (Qiagen Inc., USA). cDNA is synthesized from 5 ⁇ g of total RNA using MMLV reverse transcriptase and random hexanucleotide primers (New England Biolab Inc., USA) according to the manufacuirer's protocol. To study gene expression of iPS cells, cDNAs (150 ng for semiquantitative and 5 ng for real-time PCR) derived from the total RNA is subjected to PCR analysis. In regular PCR, the PCR products are cloned and verified by sequencing. Primer sequences used for semi-quantitative and real-time PCR are listed in Tables 3 and 4.
  • genomic DNA is purified from 1MR90 cells transduced with Ad-GFP or Ad-SOcMK using the DNeasy Kit (Qiagen Inc., USA). Purified genomic DNA (1 ⁇ g) is used to convert unmethylated cytosines (C) to uracil (U) using EZ DNA methylation kit
  • Treated DNA is purified with QIAquick column (Qiagen Inc., USA) and purified DNA ( 150 ng) from each sample is subjected to PCR analyses for the promoter region of NANOG using the following primers: forward 5 ' -CACC ATGCGTGGCTA ATTTTTGT A-3 ' , reverse 5 '- TTAAAATCCTGG AGTCTCTAG ATTT-3 ' .
  • the resulting PCR products are subcloned into the pCR2.1 -TOPO vector (Invitrogen Inc., USA). Ten clones of each sample are verified by sequencing.
  • Example 1 In Vitro Differentiation
  • Embryoid bodies To determine the differentiation ability of iPS cells in vitro, the floating culture method is used to form Embryoid bodies (EBs). Briefly, IMR90 cells are transduced with Ad-SOcMK. On day 3, the resultant iPS cells are mechanically dissociated and cultured in ES cell medium (without bFGF) in non-coated T25 flasks. The medium is changed every other day. After 7 days in floating culture, ball-shaped structures typical for EBs are formed. EBs are then transferred to 0. 1 % gelatin-coated chamber slides using the same medium. The medium is changed every other day once EBs are attached to the slide. Differentiated cells are fixed after 8 days in adherent culture and stained with antibodies recognizing marker proteins for each germ layer.
  • Example 12 Teratoma Formation
  • IMR90 cells are transduced with Ad-SOcMK.
  • the resultant iPSCs are injected subcutaneously to 4 of 6-week-old male nonobese diabetic severe combined immunodeficient (NOD/SCID) mice (Charles River Laboratories) (3X 106 iPSCs for each mouse).
  • NOD/SCID mice Charles River Laboratories
  • IMR90 cells 3X 106 cells
  • tumors are dissected and fixed in 4% paraformaldehyde.
  • Teratoma experiments are conducted in Comparative Oncology Resource Core at the University of Utah.
  • 1MR90 cells are transduced with Ad-SOcMK or Ad-GFP.
  • Adenoviruses are removed at 12 hrs post-transduction and cells are sampled at every 6 hrs.
  • Total RNA is prepared from each sample using Qiagen RNeasy kit according to manufacturer's protocol.
  • LincRNA targets are used for microarray hybridization to examine the global gene expression. Approximately 1 ⁇ g of RNA from each sample is labeled using Agilent Two-Color Quick Amp Labeling Kit following manufacturer's instructions. All arrays are hybridized at 65°C for 17 hrs and scanned using an Agilent scanner G2505C. The gene expression raw data is extracted using Agilent Feature Extraction Software version 10.5. Quality control is done on the basis of Agilent quality control metrics. Singular value decomposition (SVD) of the qualified data, with gene expression centered at its time average, identified several "eigengenes," i.e., significant patterns of expression variation across time. Sorting the data according to the two most significant eigengenes gives a global picture of the dynamics of gene expression, in which individual genes appear to be classified into groups of similar regulation and function25. Array experiments are performed in Microarray Core Facility at the University of Utah.

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Abstract

La présente invention concerne des systèmes, des constructions géniques, et des procédés de reprogrammation de cellules et de charge de particules virales. Selon un aspect, par exemple, une construction génique de transformation destinée à générer des cellules iPS peut comprendre un squelette de vecteur ayant une pluralité de facteurs de reprogrammation, chaque facteur de reprogrammation étant sous contrôle d'un promoteur séparé, le squelette de vecteur étant contenu dans une particule adénovirale. Selon un aspect, le squelette de vecteur a une taille d'au moins 8 kb. Selon un autre aspect, le squelette de vecteur a une taille d'au moins 10 kb.
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AU2017225350B2 (en) * 2016-03-01 2023-07-20 Oxford Genetics Limited Promoter
WO2018020269A1 (fr) * 2016-07-28 2018-02-01 Oxford University Innovation Limited Cellules souches et cancer
CN110846343A (zh) * 2019-11-09 2020-02-28 河南理工大学 一种表达Sox4基因重组腺病毒的制备及纯化方法
CN110804651A (zh) * 2019-11-15 2020-02-18 南方医科大学珠江医院 一种cited2与cmva的相关性的体外实验法
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CN112175909B (zh) * 2020-09-11 2023-04-28 中山大学中山眼科中心 一株vsx2绿色荧光报告基因人诱导多能干细胞系及其构建方法

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