WO2012098260A1

WO2012098260A1 - A non-viral system for the generation of induced pluripotent stem (ips) cells

Info

Publication number: WO2012098260A1
Application number: PCT/EP2012/050984
Authority: WO
Inventors: Amir KERIC; Andreas Ehlich; Michael W. HOFMANN; Olga RUBENCHIK; Noushin EHLICH; Heribert Bohlen; Eugen Kolossov
Original assignee: Axiogenesis Ag
Priority date: 2011-01-21
Filing date: 2012-01-23
Publication date: 2012-07-26

Abstract

Provided is a method using non-viral expression plasmids for the generation of induced pluripotent stem (iPS) cells from somatic cells. Furthermore, iPS cells or differentiated cells derived by the method of the present invention are provided suitable for use in tissue regeneration, therapeutic and as well non-therapeutic applications such as drug or toxicological screening as well.

Description

A non-viral system for the generation of induced pluripotent stem (iPS) cells

Field of the invention

The present invention is concerned generally with a method for the generation of induced pluripotent stem (iPS) cells from somatic cells. Furthermore, the present invention is concerned with iPS cells and redifferentiated cells derived by the method of the present invention, suitable for use in tissue regeneration, therapeutic and as well as non-therapeutic applications such as drug or toxicological screening.

Background of the invention

Stem cells of various kinds have become an extremely attractive modality in regenerative medicine. They can be proliferated in culture, and then differentiated in vitro or in situ into the cell types needed for therapy (Yu and Thomson, Genes Dev. 22 (2008), 1987-1997; Yamanaka, Cell Stem Cell. 1 (2007), 39-49; Lengner, Ann. N. Y. Acad. Sci., 1192 (2010); 38-44). Comparable to organ transplantation however, if established from human embryos, stem cell transplantations pose the problem of causing possible rejections. In addition to these technical, ethical and legal problems arise since human embryos have to be destructed in the way to provide stem cells.

To overcome such problems, methods for dedifferentiation, or reprogramming of animal and even more of human adult somatic cells into pluripotent and proliferating cells similar to the ES cells are highly desirable. Traditionally, four methods are used for reprogramming somatic cells into pluripotent cells: nuclear transfer, cell fusion, provision of cell extracts and direct reprogramming by introduction of a selection of reprogramming/transcription factors into somatic cells (see for review, e.g., Patel and Yang, Stem Cell Rev. 6 (2010), 367-380).

Direct reprogramming of somatic cells into such induced pluripotent stem (iPS) cells has been achieved using retroviral and lentiviral delivery of Oct4, Sox2, Klf4 and c-Myc reprogramming factors first in mice (Takahashi and Yamanaka, Cell 126 (2006), 663-676) and later in human cells as well (Park et. al, Nature 451 (2008), 141-146). Because of viral integration, these methods introduce also unacceptable risks due to the resulting genomic alteration and possible reactivation of viral transgenes. Therefore, new methods are required combining reliable and efficient reprogramming strategies with minimized risk by using non-viral and possibly non-integrating carriers for the reprogramming factors. Several plasmid based systems for reprogramming have been used, e.g., by, using episomal vectors derived from the Epstein-Barr virus (Yu et al, Science 324 (2009), 797-801). However, this technique requires three individual plasmids carrying a total of seven factors, including the oncogene SV40 large T gene, and has not been shown to successfully reprogram cells from adult donors, a more clinically relevant target population. Furthermore, expression of the EBNA1 protein, as was required for this technique, may increase immune cell recognition of transfected cells, thus potentially limiting clinical application if the transgene is not completely removed (Jia et al, Nat. Methods 7 (2010), 197- 199).

Jia et al, used mini circle DNA for reprogramming of somatic cells (Jia et al, Nat. Methods 7 (2010), 197-199). Since reprograming needs a continuous dose of the mini circle DNA (for the generation of the transcription factors), the mini circle DNAs have to be delivered several times, which may have caused the observed low reprogramming efficiency in comparison to the viral-based methods (minicircle 0.005%; viral-based 0.01% reprogramming efficiency).

In another approach a piggyBac (PB) transposon gene delivery system was used to enhance transfection efficiency (Kaji et al, Nature 458 (2009), 771-775). Albeit PB is a non-viral transposon system, it shows an integration pattern resembling that of integrating viral vectors raising thereby similar concerns due to possible insertional mutagenesis and unpredictable genetic dysfunction effects because of the genomic integration itself and its pattern as well; see also Wilson et al, Molecular Therapy 15 (2007), 139-145. Furthermore, due to the existence of PB-like elements dispersed on different human chromosomes it cannot be excluded that an endogenous PB-like transposase could excise and integrate a transposon flanked with PB termini or, conversely, that an exogenous source of PB transposase may mobilize endogenous human PB-like elements; see Feschotte, Proc. Natl. Acad. Sci. USA 103 (2006), 14981-14982.

Finally, mouse somatic cells have been successfully reprogrammed into iPS cells using conventional plasmids (Okita et al, Science 322 (2008), 949-53). In this context, international application WO 2009/133971 with two authors of Okita et al. (2008) as the named inventors describes experiments reporting generation of human iPS cells, particularly cells with an ES cell-like morphology from an HDF cell line derived from a six years old Japanese female, when allowing the cells to express the Slc7al gene and transfecting the cells with six kinds of genes. However, except ES cell-like morphology there is no evidence that those cells are indeed pluripotent and capable of differentiating into any cell type of the three germinal layers, the lack of evidence probably also explaining the corresponding successful experiments have not been published in literature yet. Indeed, because of low reprogramming efficiency though, there were concerns questioning the feasibility of this method towards human cells (Yu et al, Science 324 (2009), 797-801) confirmed at first by publications reporting that corresponding efforts failed, i.e. failure of generation of iPS cells using a regular plasmid vector (Jia et al, Nat. Methods. 7 (2010), 197-199).

In conclusion, availability of a simplified and reliable, non-viral introduction system is desirable for broader applications to alter cellular states of potency, i.e. reprogramming of human somatic cells into human iPS cells.

Summary of the invention

The present invention relates to a method of producing an induced pluripotent stem cell, wherein somatic cells, preferably of human origin transfected with preferably no more than 4 kinds of reprogramming factors in the presence of at least one small molecule chemical promoting the reprogramming efficiency for at least more than 7 days, wherein transfection of the reprogramming factors and subjecting the culture to the small chemical molecules is preferably repeated over time. In particular, the method of the present invention preferably comprises the steps of:

(i) introducing at least one non-viral expression vector, preferably plasmid vector comprising at least one DNA sequence encoding at least one reprogramming factor into somatic cells;

(ii) cultivating the cells for a time period sufficient for the introduced reprogramming factor being expressed and capable of reprogramming the somatic cells into pluripotent cells, characterized in that during the cultivating step at least one histone deacetylase (HDAC) inhibitor and/or at least one p53 inhibitor are added to the cell culture; and

(iii) selection of pluripotent cells.

In a preferred embodiment of the present invention, the reprogramming factor comprises one or more factors selected from the group consisting of the protein families OCT, SOX, KLF and MYC, wherein in one preferred embodiment of the present invention the DNA-sequences encoding said factors are introduced incorporated within two vectors into the somatic cells.

In another preferred embodiment of the present invention the particular reprogramming factors OCT3/4, SOX2, KLF-4 and c-MYC are selected from said protein families and their respective encoding DNA-sequences are preferably simultaneously introduced into the somatic cells in the order Oct3/4-JRES-Sox2 and/or Klf4-JRES-c-Myc from the 5' to the 3' end on preferably two vectors. Here, the intervening IRES sequence is an internal ribosome entry site allowing polycistronic expression of the respective flanking DNA sequences, preferably under the control of the CMV early enhancer and the chicken β-actin promoter.

According to one embodiment of the present invention the introduction of the vectors is at least repeated once, preferably at least two times every second day after the first introduction, most preferably at least three times every third day after the first introduction of the vectors into the cells.

In one preferred embodiment of the present invention the HDAC inhibitor is 2- propylpentanoic acid (Valproic Acid/VPA) and/or the p53 inhibitor is pifithrin-alpha (PFTalpha).

The method of the present invention can be advantageously applied to adult somatic cells, preferably fibroblasts, more preferably dermal fibroblasts, most preferably derived from adult skin. In one particular preferred embodiment of the present invention the cells are human cells.

Furthermore, in one embodiment of the present invention the somatic cells are derived from a diseased cell, tissue or organ, preferably wherein the disease is selected from the group consisting of:

(i) cardiovascular diseases such as cardiomyopathy, cardiac (or ventricular) hypertrophy, atherosclerosis, hypertension, congenital heart disease (CHD), coronary heart disease, ischemia, heart failure, inflammatory heart disease, Brugada syndrome;

(ii) neurological diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, multiple sclerosis; (iii) metabolic diseases such as diabetes mellitus type II, metabolic syndrom, obesity, lysosomal storage disease;

(iv) autoimmune and inflammatory diseases such as diabetes mellitus type I, rheumatoid arthritis, psoriasis, inflammatory bowel, ulcerative colitis, Crohn's disease, celiac (or coeliac) disease, multiple sclerosis, asthma, allergy; and

(v) monogenetic diseases such as Brugada syndrome, Huntington's disease, cystic fibrosis/mucoviscidosis, Duchenne muscular dystrophy, phenylketonuria, lysosomal storage diseases. In a further aspect, the present invention also relates to induced pluripotent (iPS) cells obtainable by the method of the present invention and to in vitro differentiated cells derived from these iPS cells.

In a still further aspect, the present invention relates to kits comprising one or more vector(s) and/or inhibitors promoting the reprogramming efficiency, and optionally cell culture reagents for use in the method of the present invention are provided.

In a further embodiment of the present invention the iPS cells and the in vitro differentiated cells of the present invention are provided for use in toxicity screening, drug development, transplantation therapy or drug target validation.

Other embodiments of the invention will be apparent from the description that follows

Brief description of the drawings

Figure 1: Plasmid maps of pOIS (A) and pKIM (B). Under control of the constitutive chicken beta-actin promoter the four reprogramming factors OCT3/4 with SOX2 and KLF4 with c-MYC, both linked via IRES-2 sequence, are expressed from bicistronic messages to induce reprogramming of adult somatic cells. Some of the sequences specific for the vectors and their starting and end coordinates in base pairs in relation to a chosen starting base pair are indicated by arrows at the schematic vector maps, for example the Ampicilin resistance gene (AmpR), the Origin of replication (ColEl), the early SV40 polyadenylation signal (SV40 early polyA signal) and the above mentioned chicken beta-actin promoter and the four reprogramming factors Oct3/4 with Sox2 and Klf4 with c-Myc. Right to the plasmid maps schematic figures of the respective expression cassettes comprised in the vectors are indicated.

Figure 2: Outline of the iPS cells generation by transfection of the plasmids pOIS and ρΚΓΜ, in the absence or presence of small molecules, e.g. like pifithrin-alpha (PFTalpha) and valproic acid (VP A). The iPS generation process may be subdivided into three phases: (A) the nucleofection phase, starting here at day 0, ending at day 6. The upper bar indicates that fibroblast medium was used during this phase, the bar below indicates that the cells were grown on a surface coated with 0, 1 % gelatin. (B) the reprogramming phase from day 6 until day 31 with an indicated period from day 8 until day 24 of addition of small molecule chemicals pifithrin-alpha and valproic acid to the cell culture. The cells were grown in hES medium on inactivated mouse embryonic fibroblasts (MEF) and (C) the subsequent colony picking phase, wherein an arrow indicates a possible time point of picking a colony at day 35.

Figure 3: Sequences of PCR-Primers used for detection of the transfected plasmids and therein included expression cassettes containing different reprogramming factors within DNA extracts of iPS cell clones, (f) indicates a forward, (r) a reverse primer.

Figure 4: Immunostaining for pluripotency markers Nanog, OCT3/4, SSEA-4, Tra-1-60 and Tra- 1-81 for exemplary clone 06-08. The first column shows a phase contrast, the second a DAPI-staining of cell nuclei and the third the respective immunostainings revealing the respective expression and distribution of the pluripotency markers as indicated.

Figure 5: Embryoid bodies (EB) formation in Aggrewells™ from cells of exemplary clone

06-08.

Figure 6: First indication of differentiation of cells of the exemplary hiPS cell clone 06-08.

Development of Embryoid Bodies (EB) and potential ectodermal, i.e. neuronal structures (NS) at day 22.

Figure 7: Further differentiated structures of cells of the exemplary hiPS cell clone 06-08.

Development of mesodermal structures, i.e. beating cardiomyocyte areas at day

23.

Figure 8: Proceeding differentiation of cells of the exemplary clone 06-08. Several beating cardiomyocyte areas at day 31.

Figure 9: PCR analysis outcome of the induced exemplary hiPS clones and positive

(plasmid DNA) and negative controls (Water, mouse ES cell line DNA). A generated product is indicated by a (+), lack of a product by a (-) sign. Primer pairs (Primer) used and the product length (PCR Product) are indicated in both top rows Definitions

For the purposes of this description, the term "stem cell" can refer to either a stem cell or a germ cell, for example embryonic stem (ES) and germ (EG) cell, respectively, but also including adult stem cells and induced pluripotent stem (iPS) cells. Minimally, a stem cell has the ability to proliferate and form cells of more than one different phenotype, and is also capable of self renewal either as part of the same culture, or when cultured under different conditions. Embryonic stem cells are also typically telomerase positive and OCT-4 positive. Telomerase activity can be determined using TRAP activity assay (Kim et al, Science 266 (1997), 2011), using a commercially available kit (TRAPeze(R) XK Telomerase Detection Kit, Cat. s7707; Intergen Co., Purchase N.Y.; or TeloTAGGG(TM) Telomerase PCR ELISAplus, Cat. 2,013,89; Roche Diagnostics, Indianapolis). hTERT expression can also be evaluated at the mRNA level by RT-PCR. The LightCycler TeloTAGGG(TM) hTERT quantification kit (Cat. 3,012,344; Roche Diagnostics) is available commercially for research purposes. In accordance with the present invention, the term "embryonic stem (ES) cell" includes cells that are able to self-renew indefinitely. These cells are generally collected from the inner cell mass of a 5 day old blastocyst. They are unspecialized cells that have the ability to differentiate into several different cell types that are derivatives of all of the three germinal layers (endoderm, mesoderm, and ectoderm), according to a standard art-accepted test, such as the ability to form a teratoma in 8-12 week old SCID mice.

"Embryonic germ cells" or "EG cells" are cells derived from primordial germ cells. The term "embryonic germ cell" is used to describe cells of the present invention that exhibit an embryonic pluripotent cell phenotype. The terms "human embryonic germ cell (EG)" or "embryonic germ cell" can be used interchangeably herein to describe mammalian, preferably human cells, or cell lines thereof, of the present invention that exhibit a pluripotent embryonic stem cell phenotype as defined herein. Thus, EG cells are capable of differentiation into cells of ectodermal, endodermal, and mesodermal germ layers. EG cells can also be characterized by the presence or absence of markers associated with specific epitope sites identified by the binding of particular antibodies and the absence of certain markers as identified by the lack of binding of certain antibodies.

"Pluripotent" refers to cells that retain the developmental potential to differentiate into a wide range of cell lineages including the germ line. The terms "embryonic stem cell phenotype," "embryonic stem-like cell," "induced pluripotent stem cell" and "induced pluripotent cell" also are used interchangeably herein to describe cells that are undifferentiated and thus are pluripotent cells and that preferably are capable of being visually distinguished from other adult cells of the same animal.

Included in the definition of ES cells (ESC) are embryonic cells of various types, exemplified by human embryonic stem cells, described by Thomson et al. (Science 282 (1998), 1145); embryonic stem cells from other primates, such as Rhesus stem cells (Thomson et al, Proc. Natl. Acad. Sci. USA 92 (1995), 7844), marmoset stem cells (Thomson et al, Biol. Reprod. 55 (1996), 254) and human embryonic germ (hEG) cells (Shamblott et al, Proc. Natl. Acad. Sci. USA 95 (1998), 13726). Any cells of mammal origin that are capable of producing progeny that are derivatives of all three germinal layers are included, regardless of whether they were derived from embryonic tissue, fetal tissue, or other sources except sources such as adult somatic cells, which derivates are defined below.

Cells which are substantially genetically identical to their respective differentiated somatic cell of origin and display characteristics similar to cells of higher potency, or pluripotent cells, such as ES cells, as described herein, are interchangeably referred to as "induced pluripotent cells," "induced pluripotent stem cells," "iPS cells," (iPSC's) or respective "hiPS cells" (hiPSC's) if derived from human cells. For example, iPS cells exhibit morphological features (i.e., colony formation with round-flattened shape, large nuclei and scant cytoplasm) akin to ES cells. In addition, iPS cells express pluripotent cellspecific markers (e.g., OCT3/4, SOX2, NANOG, growth and differentiation factor 3 (GDF3), reduced expression 1 (REXl), fibroblast growth factor 4 (FGF4), embryonic cell-specific gene 1 (ESG1), developmental pluripotency-associated 2 (DPPA2), DPPA4, and telomerase reverse transcriptase (hTERT), stage-specific ambryonic antigen SSEA-3, SSEA-4, tumor-related antigen Tra-1-60, Tra-1-81, but not SSEA-1; Takashi et al, Cell 131 (2007), 861-872). Further, iPS cells can be transmitted to the germ line (Okita et al, Nature 448 (2007), 313-317) and give rise to viable mice by tetraploid complementation assays (Boland et al, Nature 461 (2009), 91-94; Kang et al, Cell Stem Cell. 5 (2009), 135-138 and Zhao et al, Nature 461 (2009), 86-90).

The stem cells employed in accordance with the present invention are preferably (but not always necessarily) karyotypically normal. However, it is preferred not to use stem cells that are derived from a malignant source.

The terms "nuclear reprogramming factor" and "reprogramming factor" are used interchangeably in this disclosure to refer to DNA-sequences encoding proteins and the encoded proteins, i.e. defined transcription factors which have the capability to reprogram or transform somatic cells into pluripotent, ESC-like cells, or restore one or more of their pluripotency associated characteristics, as already described above concerning the ES cells, such as in vitro differentiation into cell types of different germ layers, teratoma formation, contribution to chimeras, germline transmission and tetraploid complementation (Maherali and Hochedlinger, Cell Stem Cell 3 (2008), 595-605; Woltjen et al, Nature 458 (2009), 766- 70). The particular DNA-sequences and the encoded corresponding proteins may be identified by screening methods as described in the international application WO2005/80598. Examples of the gene and corresponding protein families which may be identified by this method or a method modified from the above described are the members of the group consisting of the Oct family genes, the Klf family genes, the Sox family genes, the Myc family genes, the Lin family genes and the Nanog gene which are utilized in different combinations for reprogramming the somatic cells, such as are described by way of example in the international applications WO2007/069666, WO2008/118820 and in Yu et al, Science 318 (2007), 1917-1920.

"Feeder cells" or "feeders" are terms used to describe cells of one type that are co-cultured with cells of another type, to provide an environment in which the cells of the second type can grow. The feeder cells are optionally from a different species as the cells they are supporting. For example, certain types of ES cells or iPS cells can be supported by primary mouse embryonic fibroblasts, immortalized mouse embryonic fibroblasts (such as murine STO cells, e.g., Martin and Evans, Proc. Natl. Acad. Sci. USA 72 (1975), 1441-1445), or human fibroblast-like cells differentiated from human ES cells. The term "STO cell" refers to embryonic fibroblast mouse cells such as are commercially available and include those deposited as ATCC CRL 1503. The term "embryoid bodies" (EBs) is a term of art synonymous with "aggregate bodies". The terms refer to aggregates of differentiated and undifferentiated cells that appear when ES cells or iPS cells overgrow in monolayer cultures, or are maintained in suspension cultures. Embryoid bodies are a mixture of different cell types, typically from several germ layers, distinguishable by morphological criteria; see also infra. As used herein, "embryoid body", "EB" or "EB cells" typically refers to a morphological structure comprised of a population of cells, the majority of which are derived from iPS cells that have undergone differentiation. Under culture conditions suitable for EB formation {e.g., the removal of Leukemia inhibitory factor (LIF) for mouse and of bFGF for human ES cells, or of other, similar blocking factors), ES cells or iPS cells proliferate and form a small mass of cells that begin to differentiate. In the first phase of differentiation, usually corresponding to about days 1-4 of differentiation for humans, the small mass of cells forms a layer of endodermal cells on the outer layer, and is considered a "simple embryoid body". In the second phase, usually corresponding to about days 3-20 post-differentiation for humans, "complex embryoid bodies" are formed, which are characterized by extensive differentiation of ectodermal and mesodermal cells and derivative tissues. As used herein, the term "embryoid body" or "EB" encompasses both simple and complex embryoid bodies unless otherwise required by context. The determination of when embryoid bodies have formed in a culture of iPS cells or ES cells is routinely made by persons of skill in the art by, for example, visual inspection of the morphology. Floating masses of about 20 cells or more are considered to be embryoid bodies; see, e.g., Schmitt et al, Genes Dev. 5 (1991), 728-740; Doetschman et al, J. Embryol. Exp. Morph. 87 (1985), 27-45. It is also understood that the term "embryoid body", "EB", or "EB cells" as used herein encompasses a population of cells, the majority of which are pluripotent cells capable of developing into different cellular lineages when cultured under appropriate conditions.

The terms "polynucleotide" and "nucleic acid molecule" refer to a polymer of nucleotides of any length. Included are genes and gene fragments, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA and RNA, nucleic acid probes, and primers. As used in this disclosure, the term polynucleotides refer interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention that is a polynucleotide encompasses both a double-stranded form, and each of the two complementary single-stranded forms known or predicted to make up the double-stranded form. Included are nucleic acid analogs such as phosporamidates and thiophosporamidates.

A cell is said to be "genetically altered", "transfected", or "genetically transformed" when a polynucleotide has been transferred into the cell by any suitable means of artificial manipulation, or where the cell is a progeny of the originally altered cell that has inherited the polynucleotide. The polynucleotide will often comprise a transcribable sequence encoding a protein of interest, which enables the cell to express the protein at an elevated level. The genetic alteration is said to be "inheritable" if progeny of the altered cell have the same alteration.

A "regulatory sequence" or "control sequence" is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, such as replication, duplication, transcription, splicing, polyadenylation, translation, or degradation of the polynucleotide. Transcriptional control elements include promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, splice junctions, and the like, which collectively provide for the replication, transcription, post-transcriptional processing and translation of a coding sequence in a recipient cell.

Particular gene sequences referred to as promoters, like the "chicken beta-actin", "alpha- MHC" or "collagen" promoter, are polynucleotide sequences derived from the gene referred to that promote transcription of an operatively linked gene expression product in a manner that is similar to the transcription of the polynucleotide molecule that is normally associated with the promoter. It is recognized that various portions of the upstream and intron untranslated gene sequence may in some instances contribute to promoter activity, and that all or any subset of these portions may be present in the genetically engineered construct referred to. Particular gene sequence referred to as enhancer, like the CMV (cytomegalovirus) immediate-early enhancer (Boshart et al, Cell 41 (1985), 521-530), may be operably linked to any of the above mentioned promoters {e.g., chicken beta-actin promoter; see also Niwa et al, Gene 108 (1991), 193-199), effectively conferring increased transcription activity relative to the transcription activity resulting from a promoter in the absence of the enhancer domain. Chosen combination of the promoter and enhancer control elements may lead to constitutively active {e.g., combination of the CMV immediate early enhancer with the chicken beta-actin promoter of the present invention) or under defined circumstances inducible transcription of the operatively linked genes.

The promoter may be based on the gene sequence of any species having the gene, unless explicitly restricted, and may incorporate any additions, substitutions or deletions desirable, as long as the ability to promote transcription in the target tissue. Genetic constructs designed for treatment of humans typically comprise a segment that is at least 90 % identical to a promoter sequence of a human gene or are generally selected to be active in the relevant host cell. According to the present invention, the term "cell- and/or development-dependent promoter" is intended to mean a promoter which displays its promoter activity only in particular cell types and/or only in particular stages of cellular development, in cell cultures (embryoid bodies), tissues, organs and in transgenic non-human mammals derived from the iPS cells according to the invention. In addition, any other known cell-specific promoter can be employed, e.g., for nerve cells, heart cells, neurons, glia cells, hematopoietic cells, endothelial cells, smooth muscle cells, skeletal muscle cells, cartilage cells, fibroblasts and epithelial cells.

Genetic elements are said to be "operatively linked" if they are in a structural relationship permitting them to operate in a manner according to their expected function. For instance, if a promoter helps initiate transcription of the coding sequence, the coding sequence can be referred to as operatively linked to (or under control of) the promoter. There may be intervening sequence between the promoter and coding region so long as this functional relationship is maintained. DNA-sequences which are "operatively linked" in a way as described above, that means which are capable of directing transcription are also described with the term "expression construct" or "expression cassette". An expression construct includes, at the least, a promoter or a structure functionally equivalent to a promoter. Additional elements, such as an enhancer, and/or a transcription termination signal, may also be included.

In the context of encoding sequences, promoters, and other genetic elements, the term "heterologous" indicates that the element is derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared. For example, a promoter or gene introduced by genetic engineering techniques into an animal of a different species is said to be a heterologous polynucleotide. An "endogenous" genetic element is an element that is in the same place in the chromosome where it occurs in nature, although other elements may be artificially introduced into a neighboring position. The terms "polypeptide", "peptide" and "protein" are used interchangeably in this disclosure to refer to polymers of amino acids of any length. The polymer may comprise modified amino acids, it may be linear or branched, and it may be interrupted by non-amino acids.

The slow kinetics and low efficiency of reprogramming methods to generate human induced pluripotent stem cells impose major limitations on their utility in biomedical applications. Therefore, additional chemical substances are used which improve the efficiency of iPS cell generation. The terms "small molecule chemicals," "chemicals," "small molecules" or "inhibitors" have been used interchangeably in this disclosure to refer to said substances. In particular small molecule chemicals may be used which promote reprogramming efficiency basing on different effects on the cells, such as inhibition of p53, inhibition of DNA- methylation and inhibition of histone deacetylase (HDAC). However, the mechanisms by which the small molecule chemicals of the different classes are promoting generation of induced pluripotent stem cells are not clarified completely. For example, there are indications that different HDAC inhibitors may have different potency or range of HDAC inhibition or some additional activities beyond HDAC inhibition because not all of them can be used for generating pluripotent cells (Ware et al, Cell Stem Cell 4 (2009), 359-369). The same is true in view of the mechanism of action of p53 inhibitors, where no clear consensus about their reprogramming mechanism has been found until now (Puzio-Kuter and Levine, Nat. Biotechnol. 27 (2009), 914-915). For this reason, reference is made to different small molecule chemicals found in different screens as described in Xu et al, Nature 453 (2008), 338-344, Feng et al, Cell Stem Cell 4 (2009), 301-312 and Hoangfu et al, Nat. Biotechnol. 26 (2008), 795-797. Exemplary small molecule chemicals as they are used in the present invention are indicated below without any intention to be bound by the presented selection of molecules.

Valproic acid (VP A) is an exemplary HDAC inhibitor and enables efficient induction of pluripotent stem cells even if one of the reprogramming factors, like c-Myc, is missing (Huangfu D., et al. Nature Biotechnol. 26 (2008), 1269-1275). Pifithrin-α (Pifithrin-alpha, PFT-α or PFTa) is an exemplary reversible inhibitor of p53. The reduction of p53 activity has been shown to enhance reprogramming efficiency of human and mouse somatic cells (Kawamura et al, Nature 460 (2009), 1140-1144). 5-aza-cytidine (5-AzaC) is an exemplary inhibitor of DNA methyltransferase which has been shown to improve the reprogramming efficiency of somatic cells by reversing silencing of genes by methylation (Huangfu et al, Nat. Biotech. 26 (2008), 795-797)

If not stated otherwise the term "drug," "medicine," or "medicament" are used interchangeably herein and shall include but are not limited to all (A) articles, medicines and preparations for internal or external use, and any substance or mixture of substances intended to be used for diagnosis, cure, mitigation, treatment, or prevention of disease of either man or other animals; and (B) articles, medicines and preparations (other than food) intended to affect the structure or any function of the body of man or other animals; and (C) articles intended for use as a component of any article specified in clause (A) and (B). The term "drug," "medicine," or "medicament" shall include the complete formula of the preparation intended for use in either man or other animals containing one or more "compounds", "substances" or "(chemical) compositions" as an active agent and in some other context also other pharmaceutically inactive excipients as fillers, disintegrants, lubricants, glidants, binders or ensuring easy transport, disintegration, disaggregation, dissolution and biological availability of the "drug," "medicine," or "medicament" at an intended target location within the body of man or other animals, e.g., at the skin, in the stomach or the intestine.

Differentiation is the process whereby relatively unspecialized cells {e.g., stem cells) acquire specialized structural and/or functional features characteristic of mature cells. Similarly, "differentiate" refers to this process. Typically, during differentiation, cellular structure alters and tissue-specific proteins appear.

Detailed description of the invention

Pluripotent stem cells of various kinds have become an extremely attractive modality in regenerative medicine. However, the derivation of human embryonic stem cells posses many ethical problems concerning the provision of the cells from inner cell mass in 5 day old blastocyst, which have to be destroyed during this process. Generation of induced pluripotent stem cells based on viral gene delivery carries risk of integration during the reprogramming procedure into the genome of the stem cells. Therefore, new methods are required combining reliable and efficient reprogramming strategies with minimized risk by using non-viral and possibly non-integrating carriers for the factors reprogramming somatic into pluripotent, ES- like cells.

The present invention relates to a method of producing an induced pluripotent stem (iPS) cell, the method comprising the steps of (i) introducing at least one non-viral expression vector comprising at least one DNA sequence encoding at least one reprogramming factor into somatic cells; (ii) cultivating the cells for a time period sufficient for the introduced reprogramming factor being expressed and capable of reprogramming the somatic cells into pluripotent cells, characterized in that during the cultivating step at least one hi stone deacetylase (HDAC) inhibitor and/or at least one p53 inhibitor are added to the cell culture; and (iii) selection of pluripotent cells. The present invention is based on the surprising observation that pluripotent cells can be generated from somatic cells, in particular human somatic cells transfected with plasmid-based vectors encoding reprogramming factors of the families Oct, Sox, Klf and Myc without the need of additional reprogramming factors or tumor antigens as taught in the prior art. In particular, a method could be developed as illustrated in the Examples, wherein repeated transfection of plasmids encoding the reprogramming factors into somatic cells and concomitant subjection of the cultured cells to compounds which counteract silencing of transcriptional activity in the cell, for example due to histon deacetylase (HDAC) and senescent signals in the cells such as apoptosis induced by p53 or other effects induced by p53. In this context, experiments performed within the scope of the present invention revealed that continuous application of such compounds to the cell culture during a given period of time is sufficient and necessary to generate cells which are pluripotent. The iPS cells generated in accordance with the method of the present invention are preferably characterized by expressing the endogenous Oct3/4 gene at mRNA and at protein level, the endogenous Nanog protein, the pluripotent surface stem cell markers Tra-1- 60, Tra-1-81, and/or SSEA4. Furthermore, in accordance with the present invention it could be demonstrated that the iPS cells produced by the method of the present invention are capable of forming Embryoid Bodies and can be differentiated into the cells of all three germ layers in accordance with the common definition of pluripotentcy. In particular, it could be shown that iPS cells generated in accordance with the present invention from somatic cells can be differentiated into cardiomyocytes as well as into neural and neuronal cells.

This observation is quite surprising since in the experiments performed in accordance with the present invention human adult fibroblasts have been used rather than somatic cells from children such as proposed in international application WO 2009/133971, which may still be considered to keep at least some differentiation properties.

Thus, the method of the present invention for the first time enables the generation of iPS cells from somatic cells obtained from adults including elderly people, who have been exposed to the environment for a long time and have acquired a determined status including epigenetic factors over time. This is particular advantageous since somatic cells of children who are still in a maturing process may not adequately reflect somatic cells of an adult which are fully differentiated and terminated. Accordingly, in particular in view to drug development and use of iPS cells in transplantation therapy the present invention provides an important contribution to the art in that iPS cells can be generated from somatic cells of subjects which suffer from a disorder or disease, for example cardiomyopathy or other heart diseases, or which have a predisposition for such disorder and diseases. While the present invention is illustrated in the Examples with an HDAC inhibitor and an inhibitor of p53, the person skilled in the art would understand that equivalent compounds may be used, in particular in case the culture conditions as used in the Examples, i.e. repeated transfection of the plasmids encoding the reprogramming factors and/or continuous supply of those compounds over at least one, preferably more than two weeks is followed. In this context, the use of only one of such compounds, for example the HDAC inhibitor may be sufficient to arrive at at least one colony of iPS cells or for example if the culture period with that compound is extended to over more than 16 days as performed in the Examples.

Regarding possible non-viral expression vectors which are used according to the method of the present invention, preferably plasmid vectors are employed. Examples of the plasmid vector include, but are not limited to, Escherichia coli-derived plasmids (ColE-series plasmids such as pBR322, pUC18, pUC19, pUC118, pUC119, and pBluescript, and the like), Actinomyces-derived plasmids (pIJ486 and the like), Bacillus subtilis-derived plasmids {e.g., pUBHO, pSH19 and others), yeast-derived plasmids (YEpl3, YEp 24, YcpSO and the like) and the like, as well as artificial plasmid vectors and the like.

Examples of easily available non-viral expression vectors include, but are not limited to, pCAGGS and its derivatives such as pCX-GFP (Niwa et. al, Gene 108 (1991), 193-199; Okabe et al, FEBS Lett. 407 (1997), 313-319), pIRES2-EGFP (Clontech), pCMV6-XL3 (OriGene Technologies Inc.), pEGFP-Cl (Clontech), pGBT-9 (Clontech), pcDNAI (FUNAKOSHI), pcDM8 (FI AKOSHI), pAGE107 (Miyaji et al., Cytotechnology 3 (1990), 133-140), pCDM8 (Seed, Nature 329 (1987), 840-842), pcDNAI/AmP (Invitrogen), pREP4 (Invitrogen), pAGE103, pAGE210 (both: Mizukami and Itoh, J. Biochem., 101 (1987), 1307- 1310,) and the like.

For a better selection of transfected cells, the expression vector may also contain a selectable marker or reporter gene. Examples for markers are visible markers, or selectable markers, i.e. genes permitting the capability to the cells to survive under particular selective conditions. For example, selective markers are resistance genes permitting resistance to different antibiotics. Examples for these are nucleoside and aminoglycoside-antibiotic-resistance genes, e.g. puromycin (puromycin-N-acetyltransferase), streptomycin, neomycin, gentamycin or hygromycin. Further examples for resistance genes are dehydrofolate-reductase, which confers a resistance against aminopterine and methotrexate, as well as multi drug resistance genes, which confer a resistance against a number of antibiotics, e.g. against vinblastin, doxorubicin and actinomycin D. Alternatively the reporter may be a gene and its associated product which confers an observable or measurable phenotype to the cells, such as fluorescent proteins. According to the present invention, the green fluorescent protein (GFP) from the Aequorea victoria (described in WO95/07463, W096/27675 and W095/21191) and its derivates "Blue GFP" (Heim et al, Curr. Biol. 6 (1996), 178-182) and "Redshift GFP" (Muldoon et al, Biotechniques 22 (1997), 162-167) can be used. Particularly preferred is the Enhanced Green Fluorescent Protein (EGFP). Further embodiments are the Enhanced Yellow and Cyan Fluorescent Proteins (EYFP and ECFP, respectively) and Red Fluorescent proteins (DsRed, HcRed). Further fluorescent proteins are known to the person skilled in the art and can be used according to the invention as long as they do not damage the cells.

The detection of fluorescent proteins takes place through per se known fluorescence detection methods; see, e.g., Kolossov et al, J. Cell Biol. 143 (1998), 2045-2056. Alternatively to the fluorescent proteins, particularly in in vivo applications, other detectable proteins, particularly epitopes of those proteins, can also be used. Also protein epitopes, selected as not to damage the cells, can be used; see also the international application WO2002/051987.

For reprogramming of somatic into pluripotent cells an introduced vector has to comprise genes encoding proteins capable of performing such a transformation. Overexpression of a single transcription factor in somatic cells was found to activate genes that are typical of other somatic cell types, first m Drosophila (Gehring, Genes Cells 1 (1996), 11-15; Schneuwly et al, Nature 325(1987), 816-818) than in mammals as well. Though, due to many failed attempts to identify factors that can induce reprogramming of vertebratae somatic into pluripotent cells, until about 2006 the view prevailed that this process might require the cooperation of up to 100 factors (Yamanaka and Blau, Nature 465 (2010), 704-712). Then, members of the group consisting of the Oct family genes, the Klf family genes, the Sox family genes, the Myc family genes, the Lin family genes and the Nanog gene were identified as reprogramming factors by screening methods as described in the international application No. WO 2005/080598. In particular potency-determining factors that can reprogram somatic cells include, but are not limited to, factors such as Oct-4, Sox2, FoxD3, UTF1, Stella, Rexl, ZNF206, Soxl5, Mybl2, Lin28, Nanog, DPPA2, ESG 1, Otx2, SV40 large antigen or combinations thereof. They may be utilized in different combinations for reprogramming the somatic cells, such as are described by way of example in the international application WO2007/069666, WO2008/118820 or in Yu et al, Science 318 (2007), 1917-1920 and Patel and Yang, Stem Cell Rev. 6 (2010), 367-380. The introduction of the reprogramming factors into somatic cells may be obtained by the introduction of one or more expression cassettes on one or more vectors and using different combinations of said reprogramming factors as described in the references above.

In one embodiment of the present invention the reprogramming factor comprises one or more factors selected from the group consisting of the protein families OCT, SOX, KLF and MYC, preferably wherein at least two vectors, preferably plasmids are introduced into the somatic cells which comprise different expression cassettes encoding particular combinations of the reprogramming factors. The reprogramming factors from the above mentioned families may be introduced in several combinatorial mixtures from one, two, three or four genes encoding members of the respective protein families on one or more vectors, at once or subsequently into the somatic cells. In one preferred embodiment of the present invention the factors are introduced on two different vectors into a somatic cell, wherein a first vector comprises a DNA sequence encoding reprogramming factors of the OCT and SOX family and a second vector comprises a DNA sequence encoding reprogramming factors of the KLF and MYC family, preferably wherein the reprogramming factors are OCT3/4, SOX2, KLF-4 and c- MYC, as shown, e.g., in Example 1 of the present invention.

As mentioned supra, factors identified as capable of reprogramming somatic into pluripotent cells comprise members of the LIN protein family, the NANOG protein and/or the SV40 large antigen. However, it is a particular achievement of the present invention to provide a method wherein the reprogramming factor does not include any of this factors, i.e. not the NANOG protein, neither member of the LIN protein family, nor the SV40 large antigen.

The vectors may be introduced subsequently into the somatic cells, however in one further embodiment of the method of the present invention the vectors are introduced simultaneously into the somatic cells. Any suitable expression vector for this purpose can be used. Suitable vector systems for producing stem cells altered according to this invention can be prepared using commercially available mammalian expression plasmids. The introduction of the vector construct or constructs into the somatic as well as the iPS cells occurs in a known manner, e.g., by transfection, electroporation, lipofection or nucleofection(TM). Exemplary is the formulation Lipofectamine 2000(TM), available from Gibco/Life Technologies. Another exemplary reagent is FuGENE(TM) 6 Transfection Reagent, a blend of lipids in non- liposomal form and other compounds in 80 % ethanol, obtainable from Roche Diagnostics Corporation. A further example is the The Nucleofector™ technology, wherein the transfection kits and devices (Nucleofector(TM)) are available from Lonza (Lonza GmbH, Cologne, Germany and Lonza Group Ltd, Basel, Switzerland)

The introduction of the one or more vectors comprising reprogramming factors into the somatic cells may occur once or several times according to the method of the present invention. The repeated introduction may be also performed at different time schedules. For example, the transfection may be performed four times at every subsequent or more preferably at every second day, because of the destructive effects of some of the transfection methods on the cells. Alternatively, the transfection may be performed three times every subsequent, or more preferably every second, every third or every fourth day (see also Table 2 and Example 1). In one particular preferred embodiment of the present invention the introduction of the vectors is at least repeated once, preferably at least two times every second day after the first introduction, most preferably at least three times every third day after the first introduction of the vectors into the cells.

In certain embodiments of the invention, internal ribosome entry sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning modus of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, Nature. 334 (1988), 320-325; Pelletier and Sonenberg, J Virol. 63 (1989), 441-444). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, as above), and may be used also to induce translation from eukaryotic RNA (Macejak and Sarnow, Nature 353 (1991), 90-94). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (see U.S. Patent Nos. 5,925,565 and 5,935,819, both incorporated herein by reference).

In one preferred embodiment of the present invention the DNA sequences encoding the reprogramming factors are separated by an intervening sequence allowing polycistronic expression of the DNA sequences, most preferably wherein the intervening sequence is an internal ribosome entry site (IRES).

In a particular preferred embodiment of the invention, a vector or more vectors are used according to the method of the present invention, wherein the order of the DNA sequences is Oct3/4- ES-Sox2 and/or Kl/4-JRES-c-Myc from the 5' to the 3' end. Further, in another aspect of the present invention the DNA sequence encoding the reprogramming factor is operably linked to expression control sequences, preferably wherein the expression control sequence comprises a promoter and preferably an enhancer.

Non-limiting examples of promoters include early or late viral promoters, such as, SV40 early or late promoters, cytomegalovirus (CMV) immediate early promoters, Rous Sarcoma Virus (RSV) early promoters; eukaryotic cell promoters, such as, e. g., beta actin promoter (Ng, S. Y, Nuc. Acid Res. 17 (1989), 601-615; Quitsche et at., J. Biol. Chem. 264 (1989), 9539- 9545), GADPH promoter (Alexander et al, Proc. Nat. Acad. Sci. USA 85 (1988), 5092- 5096; Ercolani et al, J. Biol. Chem. 263 (1988), 15335-15341), metallothionein promoter (Karin et al. Cell 36 (1989), 371-379; Richards et al, Cell 37 (1984), 263-272,).

An enhancer which may be used along with a promoter is, e.g., the CMV (cytomegalovirus) immediate-early enhancer (Boshart et al, Cell 41 (1985), 521-530), which may be operably linked to any of the above mentioned promoters, e.g., the chicken beta-actin promoter; see also Niwa et al, Gene 108 (1991), 193-199.

In one embodiment of the present invention the promoter is a constitutive promoter, preferably the chicken β-actin promoter and/or the enhancer sequence is the CMV early enhancer.

Small molecule chemicals which may be used according to the methods of the present invention can be provided in different combinations, concentrations and schedules to the cell culture. For example, small molecule chemicals of three different classes may be added, i.e. inhibitors of methylation, p53 and of histone deacetylase (FID AC). Alternatively inhibitors of only one or two of the above mentioned processes may be added to the cell culture; see also Table 2 and Example 1. The person skilled in the art will understand that equivalent compounds to the above-mentioned small molecule chemicals may be found when performing experiments orientated towards the examples of the present application.

In a particular preferred embodiment of the invention the HDAC inhibitor is 2- propylpentanoic acid (Valproic Acid/VPA) and/or the p53 inhibitor is pifithrin-alpha (PFTalpha).

Furthermore, the small chemicals may be used at different concentrations and may be added for different time periods to the culture. For example, the p53 inhibitor 5-AzaC may be present in the culture in a concentration of about 0,1 to 10 μΜ or preferably in a concentration of about 1 to 5 μΜ. In an alternative or additional embodiment of the present invention the HE) AC inhibitor is present in the culture in a concentration of about 0, 1 mM to 10 mM, preferably at concentration of about 1 to 5 mM In a further alternative or additional embodiment the p53 inhibitor is present in the cell culture in a concentration of about 1 μΜ to 100 μΜ, preferably in a concentration of about 5 to 20 μΜ. The inhibitors may be also added for different time periods to the cell culture of between 5 and 20 days. In a preferred embodiment of the invention the cells are cultured in the presence of the inhibitors for a time period of 7 to 16 days, most preferably for at least 16 days. Addition of the inhibitors to the cell culture may start at different time points. Preferably the addition of the inhibitors to the cell culture starts after the last introduction of the reprogramming factors to the cell culture. In a preferred embodiment of the present invention the inhibitors are added to the cell culture 1 to 5 days, preferably 2 days after the last introduction of the reprogramming factor to the cell culture.

Usually said induced pluripotent stem cell (iPSC) or stem cells are derived from somatic or differentiated cells, preferably wherein the somatic cells are fibroblasts, preferably dermal fibroblasts, most preferably derived from adult skin from mouse or rat, or in particular from human. Therefore, in one preferred embodiment of the invention, the somatic cells are adult somatic cells.

The invention can be practiced using stem cells of any vertebrate species. Included are somatic cells from humans; as well as non- human primates, domestic animals, livestock, and other non-human mammals. Amongst the stem cells suitable for use in this invention are primate pluripotent stem cells derived from tissue originating from all three germ layers, derived from individuals of every age, i.e. new born, young, adolescent, adult or aged individuals, wherein in one preferred embodiment of the invention the somatic cells are adult somatic cells. In another preferred embodiment of the invention the somatic cells are fibroblasts, preferably dermal fibroblasts, most preferably derived from adult skin. Further, in another preferred embodiment of the present invention the somatic cells are human cells, i.e. the somatic cells used and reprogrammed by the methods of the present invention are of human origin.

The somatic cells may be derived from healthy individuals, however, in one embodiment of the present invention the somatic cells are derived from a diseased cell, tissue or organ.

In a further preferred embodiment of the present invention the disease is selected from the group consisting of: (i) cardiovascular diseases such as cardiomyopathy, cardiac hypertrophy, atherosclerosis, hypertension, congenital heart disease, coronary heart disease, ischemia, heart failure, inflammatory heart disease, Brugada syndrome; (ii) neurological diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, multiple sclerosis; (iii) metabolic diseases such as diabetes mellitus type II, metabolic syndrome, obesity, lysosomal storage disease; (iv) autoimmune and inflammatory diseases such as diabetes mellitus type I, rheumatoid arthritis, psoriasis, inflammatory bowel, ulcerative colitis, Crohn's disease, celiac disease, multiple sclerosis, asthma, allergy; and (v) monogenetic diseases such as Brugada syndrome, Huntington's disease, cystic fibrosis/mucoviscidosis, Duchenne muscular dystrophy, phenylketonuria, lysosomal storage diseases. Derived from a healthy as well as from a diseased cell, tissue or organ, either iPS cells of the present invention may also be used in therapeutic approaches on the field of the regenerative medicine to produce endoderm-derived tissues or organs such as the liver and pancreas. In one preferred embodiment of the invention an induced pluripotent iPS cell obtainable by the methods of the invention is provided. Because of their ES cell like pluripotency the iPS cells, in particular human iPS cells of the present invention may be used in a way similar to human embryonic stem cells and their use for preparing different cell and tissue types as they are also described in Reprod. Biomed. Online 4 (2002), 58-63 or in Kiessling and Anderson, Harvard Medical School, in Human Embryonic Stem Cells: An Introduction to the Science and Therapeutic Potential; (2003) Jones and Bartlett Publishers; ISBN: 076372341X.

Since the iPS cells obtainable by the methods of the present invention are found to resemble many of the known characteristics of embryonic stem (ES) cells, they may be also cultivated by the use of the same methods as used for the culture of said ES cells. It is referred to the literature of Anderson et al, Nat. Med. 7 (2001), 393-395; Gage, Science 287 (2000), 433-438, and Prockop, Science 276 (1997), 71-74, wherein the extraction and culture of ES cells is described, which culturing methods are also applicable for the iPS cells provided by the present invention. Media for isolating and propagating stem cells can have any of several different formulas, as long as the cells obtained have the desired characteristics, and can be propagated further. Suitable sources include Iscove's modified Dulbecco's medium (IMDM), Gibco, #12440-053; Dulbecco's modified Eagles medium (DMEM), Gibco #11965-092; Knockout Dulbecco's modified Eagles medium (KO DMEM), Gibco #10829-018; 200 mM L- glutamine, Gibco # 15039-027; non-essential amino acid solution, Gibco # 11140-050; [beta]- mercaptoethanol, Sigma # M7522; human recombinant basic fibroblast growth factor (bFGF), Gibco # 13256-029. Exemplary serum-containing ES medium and conditions for culturing stem cells are known, and can be optimized appropriately according to the cell type. Media and culture techniques for particular cell types referred to in the previous section are provided in the references cited herein. Further media types and other reagents and materials are described and enlisted in the Examples below.

Stem cells can be propagated continuously in culture, using a combination of culture conditions that promote proliferation without promoting differentiation. Traditionally, embryonic stem cells are cultured on a layer of feeder cells, typically fibroblast type cells, often derived from embryonic or fetal tissue. The cell lines are plated to near confluence, usually irradiated to prevent proliferation, and then used to support when cultured in medium conditioned by certain cells (e.g. Koopman and Cotton, Exp. Cell 154 (1984), 233-242; Smith and Hooper, Devel. Biol. 121 (1987), 1-91), or by the exogenous addition of leukemia inhibitory factor (LIF), or basic fibroblast growth factor (bFGF). Such cells can be grown relatively indefinitely using the appropriate culture conditions without differentiation.

In the absence of feeder cells, exogenous leukemia inhibitory factor (LIF)/ basic fibroblast growth factor (bFGF), or conditioned medium, iPS, ES or EG cells spontaneously differentiate into a wide variety of cell types, including cells found in each of the endoderm, mesoderm, and ectoderm germ layers. With the appropriate combinations of growth and differentiation factors, however, cell differentiation can be controlled. For example, mouse ES and EG cells can generate cells of the hematopoietic lineage in vitro (Keller et al, Mol. Cell. Biol. 13 (1993), 473-486; Palacios et al, Proc. Natl. Acad. Sci USA 92 (1995), 7530-7534; Rich, Blood 86 (1995), 463-472). Additionally, mouse ES cells have been used to generate in vitro cultures of neurons (Bain et a/., Developmental Biology 168 (1995), 342-357; Fraichard et al, J. Cell Science 108 (1995), 3161-3188), cardiomyocytes (heart muscle cells) (Klug et al, Am. J. Physiol. 269 (1995), H1913-H1921), skeletal muscle cells (Rohwedel et al, Dev. Biol. 164 (1994), 87-101), vascular cells (Wang et al, Development 114 (1992), 303-316), US patent US-A-5, 773, 255 relates to glucose-responsive insulin secreting pancreatic beta cell lines, US patent US-A-5, 789,246 relates to hepatocyte precursor cells. Hepatic differentiation of murine embryonic stem cells is also described in Jones et al, Exp. Cell Res. 272 (2002), 15-22. Other progenitors of interest include but are not limited to chondrocytes, osteoblasts, retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal duct epithelial cells, smooth and skeletal muscle cells, testicular progenitors, and vascular endothelial cells. Embryonic stem cell differentiation models for cardiogenesis, myogenesis, neurogenesis, epithelial and vascular smooth muscle cell differentiation in vitro have been generally described in Guan et al, Cytotechnology 30 (1999), 211-226. For a review of differentiation potential of ES cells to clinically relevant cell populations see also Muny and Keller, Cell 132 (2008), 661-680.

In vitro differentiated cardiomyocytes, neural cells, hepatocytes, adipocytes, skeletal muscle cells, vascular endothelial cells and osteoblasts are described in US patent application US2002/142457. The preparation of cells of the cardiomyocyte lineage produced from human pluripotent stem cells is described in international application WO03/006950; see also references cited therein. A method for the generation of in vitro differentiated cardiomyocytes from particular stem cells called spoc cells is described in international application WO03/035838. The production of cardiomyocyte-enriched cellular populations, and methods and materials for obtaining the same are also described in international application WO01/68814.

Induced pluripotent stem cells (iPSC's) may also be differentiated into different cell populations, though there are reports saying that differentiation of the iPS'c may be less efficient than differentiation of human embryonic stem cells and that the iPS'c may be less predictable than the ES cells showing need for improving differentiation potency (Hu et al, Proc. Natl. Acad. Sci USA 107 (2010), 4335-4340).

However, several studies have demonstrated iPSC's ability to generate cells of the cardiovascular and hematopoietic lineages (Choi et al, Stem Cells 27 (2009), 559-567; Schenke-Layland et al, Stem Cells 26 (2008), 1537-1546) insulin-secreting islet like structures (Tateishi et al, J. Biol.Chem. 283 (2008), 31601-31607), functional cardiomyocytes (Zhang et al, Circ. Res. 104 (2009), e30-41), cells of the neural lineages (Chambers et al, Nat. Biotechnol. 27 (2009), 275-280), cells of the adipose lineage (Taura et al, FEBS Lett. 583 (2009), 1029-1033) and retinal cells (Hirami et al, Neurosci. Lett. 458 (2009), 126-131). Because of their resemblance of many characteristics of ES cells, the above described differentiation methods concerning ES cells may be also used or adapted for iPS cells.

For example, in certain embodiments of the invention, differentiation is promoted by withdrawing one or more medium component(s) that promote(s) growth of undifferentiated cells, or act(s) as an inhibitor of differentiation. Examples of such components include certain growth factors, mitogens, leukocyte inhibitory factor (LIF), and basic fibroblast growth factor (bFGF). Differentiation may also be promoted by adding a medium component that promotes differentiation towards the desired cell lineage, or inhibits the growth of cells with undesired characteristics. iPSC's provided by the methods of the present invention are well capable to redifferentiate as exemplary shown by the development of Embryoid Bodies (EB) and the differentiation of hiPSC's of the present invention into cells of different germ layers, i.e. neural cells and cardiomyocytes; see also Example 1 and 2 and Figures 6 to 9. In one preferred embodiment of the present invention such an in vitro differentiated cell derived from the iPS cell obtainable by the methods of the present invention is provided.

It is known that every tissue consists of a main specific cell type which determines its functional role along with supporting cell types {e.g. fibroblasts, stromal, endothelial, glial cells, etc.), which can be important for maintaining of three-dimensional architectonic structure of tissue, its trophic function and interconnections with other tissue systems of the whole organism. Therefore, in one embodiment of the method of the present invention an in vitro differentiated cell of one cell type is cocultured with at least one cell of a second cell type, and/or comprised in tissue or tissue-like structures comprising at least one second cell type such as any one of those described hereinbefore. Said second cell type may be for example an embryonic second cell type. Preferably, the in vitro differentiated cell in said tissue or tissue-like structure is obtained by culturing an induced pluripotent stem (iPS) cell derived first cell type in the presence of at least one embryonic second cell type; and allowing integration and alignment of said at least two cell types into tissue or tissue-like structures. Said at least second cell type may also be generated as the first cell type, i.e. by in vitro differentiation of ES cells which have been genetically engineered with corresponding marker genes; see also supra for appropriate methods and materials. A corresponding method for providing a variety of tissue or tissue-like structures and like in vitro differentiated cells and tissue is described in detail in co-pending European patent application "Tissue modeling in embryonic stem (ES) cell system", application number EP 03 013 980.2, filed on June 20, 2003 and its subsequent application, the disclosure content of which is incorporated herein by reference.

Accordingly, the term "in vitro differentiated cell" is also meant to include a plurality of in vitro differentiated cells of the same or different cell types as well as in vitro differentiated tissue and organs, and cocultures of in vitro differentiated cells with other cell types such as of embryonic origin. Thus, the term "in vitro differentiated cell" does not necessarily exclude the presence of a cell or cell type other than that which the original stem cell has been differentiated to. However, in most embodiments the use of a substantially pure culture of in vitro differentiated cells is preferred or the use of even a single cell.

In one embodiment, wherein said in vitro differentiated cell is a cardiomyocyte said at least second cell type preferably corresponds to an endothelial cell and/or fibroblast. For example, it has been reported that bradykinin blocks angiotensin II-induced hypertrophy in the presence of endothelial cells; see Ritchie et al., Hypertension 31 (1998), 39-44. In those experiments effects of bradykinin on isolated ventricular cardiomyocytes from adult and neonatal rat hearts have been determined and the extent to which bradykinin blocks hypertrophy in vitro. Bradykinin was found to be a hypertrophic agonist, as defined by increased protein synthesis and atrial natriuretic peptide secretion and expression. However, in cardiomyocytes cocultured with endothelial cells, bradykinin did not increase protein synthesis. In conclusion, bradykinin has a direct hypertrophic effect on ventricular myocytes. The presence of endothelial cells is required for the antihypertrophic effects of bradykinin.

Thus, depending on the nature of the disease and the type of diseased tissue or organ to be investigated the use of cocultures of differentiated cells or in vitro differentiated tissue in the method of the present invention may be taken into account.

The in vitro differentiated cell is obtained by a method which is preferably performed such that it allows self-assembly of the different cell types, for example into the desired tissue or tissue-like structures that should reflect the tissue or organ of a mammal, preferably human. The induced pluripotent stem cells are in a preferred embodiment of the invention available in form of aggregates that are known as embryoid bodies (EBs). International application No. WO 02/051987 describes a protocol to obtain embryoid bodies. The manufacturing may take place with the "hanging drop" method or by methylcellulose culture (International application No. WO 2002/051987 and Wobus et al, Differentiation 48 (1991), 172-182). Alternatively Embryoid Bodies may be prepared according a recent developed "mass culture" system described in detail in the international application No. WO2005/005621. In one embodiment of the present invention the manufacturing of EBs is performed by other methods, such as the method described in the international application No. WO2008/106771 using a microwell device with a high density of microwells for the production of cell aggregates which is sold as Aggrewell(TM) (see also Example 1 and Figure 5 of the present invention), or similar methods as described in the international application WO2010/142755.

Alternatively to this, spinner flasks (stirring cultures) can be used as culture method. Therefore, the undifferentiated iPS cells are introduced into stirring cultures and are mixed permanently according to an established procedure. Therefore, 10 million iPS cells are introduced into 150 ml medium with 20 % FBS and are stirred constantly with the rate of 20 rpm., wherein the direction of the stirring motion is changed regularly. 24 hours after introduction of the iPS cells an extra 100 ml medium with serum is added and thereupon 100 - 150 ml of the medium is exchanged every day (Wartenberg et al, FASEB J. 15 (2001), 995-1005). Under these culture conditions large amounts of iPS cell-derived cells, i.e. cardiomyocytes, endothelial cells, neurons etc. depending on the composition of the medium can be obtained. The cells are selected by means of the resistance gene either still within the stirring culture or after plating, respectively.

In another alternative method, the EBs differentiated in the hanging drop might be not plated, but kept simply in suspension. Even under these conditions a progression of a differentiation could be observed experimentally. The washing off of the non-desired cell types can be done with mechanical mixing alone and addition of low concentration of enzyme {e.g., collagenase, trypsin); a single cell suspension is achieved with easy washing off of the non-desired cell types. As mentioned before, embryoid bodies represent a complex group of cells differentiating into different tissues. In one embodiment, the cells within an embryoid body are substantially synchronized for their differentiation. Accordingly, at known intervals, the majority of the synchronized cells differentiate into the three embryonic germ layers and further differentiate into multiple tissue types, such as cartilage, bone, smooth and striated muscle, and neural tissue, including embryonic ganglia; see also Snodgrass et al, "Embryonic Stem Cells: Research and Clinical Potentials" in Smith and Sacher, eds. Peripheral Blood Stem Cells American Association of Blood Banks, Bethesda MD (1993). Thus, the cells within embryoid bodies provide a much closer model to the complexity of whole organisms than do traditional single cell or yeast assays, while still avoiding the cost and difficulties associated with the use of mice and larger mammals. Moreover, the recent availability of human embryoid bodies improves the predictive abilities of the invention by providing a vehicle for modeling toxicity and identification of drugs useful for the treatment of heart disorders in human organ systems, and in humans.

Typically, non-viral plasmids are lost over time after a period sufficient to induce cells into a pluripotent or a desired cell state. Therefore, an inherent feature of these methods produces progeny cells essentially free of exogenous genetic elements and a negative selection may facilitate the process. These methods enable isolation of iPS cells or any desired cell types essentially free of vector elements by altering differentiation status. However, many of the iPS cell lines generated after transient transfection may contain integrated plasmids though (Okita K, et al, Science 322 (2008), 949-953), suggesting that stable integration of exogenous sequences can occur maybe because of the selection pressure for iPS cell induction. In one embodiment of the present invention, within the iPS cells provided by the methods of the present invention one or more DNA sequences encoding reprogramming factors are integrated into the cellular genome in the form of a plasmid (see, e.g., Example 3 and Figure 9 of the present invention).

In a further embodiment, an in vitro differentiated cell is provided, derived from an iPS cell obtainable by the methods of the present invention, wherein the in vitro differentiated cell is characterized by the lack of expression of said at least one reprogramming factor.

In another embodiment of the present invention the non-lethal marker and also other parts of the introduced vectors can be constructed to enable their subsequent removal using any of a variety of art-recognized techniques, such as removal via Cre-mediated, site-specific gene excision. For example, it may become desirable to delete the marker gene after the pluripotent cell population is obtained, to avoid interference by the marker gene product in the experiment or process to be performed with the cells. The same is true, if parts or whole plasmids which were used for the introduction of the reprogramming factors integrate into the cells genome, introducing possible safety risk by a potential reactivation of the reprogramming factors at any time point after the redifferentiation and possible therapeutic use of the iPS cells. Their removal, e.g., removal of introduced sequences encoding the reprogramming factors, may be achieved by targeted deletions which can be accomplished by providing structure(s) near a given gene sequence that permits its ready excision. That is, a Cre/Lox genetic element can be used. The Lox sites can be built into the vectors and subsequently cells. If it is desired to remove the introduced gene sequences from the pluripotent or redifferentiated cells, the Cre agent can be added to the cells. Other similar systems also can be used.

The present invention further relates to a kit comprising one or more vector(s) and/or inhibitors as defined supra, and optionally cell culture reagents for use in the method of the present invention. This kit may be used for reliable dedifferentiation of somatic cells of different kind into iPS cells and accordingly of the generation of redifferentiated cells thereof.

The present invention also relates to the use of the iPS cell and in vitro (re)differentiated cells of the present invention in toxicity screening, drug development, transplantation therapy or drug target validation as described in detail in the international patent application WO2005/108598, for example.

These and other embodiments are disclosed and encompassed by the description and examples of the present invention. Further literature concerning any one of the materials, methods, uses and compounds to be employed in accordance with the present invention may be retrieved from public libraries and databases, using for example electronic devices. For example the public database "Medline" may be utilized, which is hosted by the National Center for Biotechnology Information and/or the National Library of Medicine at the National Institutes of Health. Further databases and web addresses, such as those of the European Bioinformatics Institute (EBI), which is part of the European Molecular Biology Laboratory (EMBL) are known to the person skilled in the art and can also be obtained using internet search engines. An overview of patent information in biotechnology and a survey of relevant sources of patent information useful for retrospective searching and for current awareness is given in Berks, TIBTECH 12 (1994), 352-364. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples and figures which are provided herein for purposes of illustration only and are not intended to limit the scope of the invention. The contents of all cited references (including literature references, issued patents, published patent applications as cited throughout this application and manufacturer's specifications, instructions, etc) are hereby expressly incorporated by reference; however, there is no admission that any document cited is indeed prior art as to the present invention.

EXAMPLES

The examples which follow further illustrate the invention, but should not be construed to limit the scope of the invention in any way. Detailed descriptions of conventional methods, such as those employed herein can be found in the cited literature; see also "The Merck Manual of Diagnosis and Therapy" Seventeenth Ed. ed by Beers and Berkow (Merck & Co., Inc. 2003).

General methods of the invention

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art.

For further elaboration of general techniques concerning stem cell technology, the practitioner can refer to standard textbooks and reviews, for example Teratocarcinomas and embryonic stem cells: A practical approach (E. J. Robertson, ed., IRL Press Ltd. 1987); Guide to Techniques in Mouse Development (P. M. Wasserman et al, eds., Academic Press 1993); Embryonic Stem Cell Differentiation in Vitro (Wiles, Meth. Enzymol. 225 (1993), 900,); Properties and uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy (Rathjen et al, Reprod. Fertil. Dev. 10 (1998), 31,). Differentiation of stem cells is reviewed in Robertson, Meth. Cell Biol. 75 (1997), 173; and Pedersen, Reprod. Fertil. Dev. 10 (1998), 31. Besides the sources for stem cells described already above further references are provided; see Evans and Kaufman, Nature 292 (1981), 154-156; Handyside et al, Roux's Arch. Dev. Biol, 196 (1987), 185-190; Flechon et al, J. Reprod. Fertil. Abstract Series 6 (1990), 25; Doetschman et al, Dev. Biol. 127 (1988), 224-227; Evans et al, Theriogenology 33 (1990), 125-128; Notarianni et al, J. Reprod. Fertil. Suppl, 43 (1991), 255-260; Giles et al, Biol. Reprod. 44 (Suppl. 1) (1991), 57; Strelchenko et al, Theriogenology 35 (1991), 274; Sukoyan et al, Mol. Reprod. Dev. 93 (1992), 418-431; Iannaccone et al., Dev. Biol. 163 (1994), 288-292.

General methods in molecular and cellular biochemistry, and genetic engineering can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al, Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); DNA Cloning, Volumes I and II (Glover ed., 1985); Oligonucleotide Synthesis (Gait ed., 1984); Nucleic Acid Hybridization (Hames and Higgins eds. 1984); Transcription And Translation (Hames and Higgins eds. 1984); Culture Of Animal Cells (Freshney and Alan, Liss, Inc., 1987); Gene Transfer Vectors for Mammalian Cells (Miller and Calos, eds.); Current Protocols in Molecular Biology and Short Protocols in Molecular Biology, 3rd Edition (Ausubel et al, eds.); and Recombinant DNA Methodology (Wu, ed., Academic Press). Gene Transfer Vectors For Mammalian Cells (Miller and Calos, eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al, eds.); Immobilized Cells And Enzymes (IRL Press, 1986); Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (Weir and Blackwell, eds., 1986). Protein Methods (Bollag et al, John Wiley & Sons 1996); Non- viral Vectors for Gene Therapy (Wagner et al eds., Academic Press 1999); Viral Vectors (Kaplitt & Loewy eds., Academic Press 1995); Immunology Methods Manual (Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998). Reagents, cloning vectors and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech. General techniques in cell culture and media collection are outlined in Large Scale Mammalian Cell Culture (Hu et al, Curr. Opin. Biotechnol. 8 (1997), 148); Serum-free Media (Kitano, Biotechnology 17 (1991), 73); Large Scale Mammalian Cell Culture (Curr. Opin. Biotechnol. 2 (1991), 375); and Suspension Culture of Mammalian Cells (Birch et al., Bioprocess Technol. 19 (1990), 251); Extracting information from cDNA arrays, Herzel et al, CHAOS 11 (2001), 98-107. REAGENTS

Compounds or equipment Supplier

Invitrogen, Carlsbad, U.S.A., Cat. No. 41965-

Dulbecco's Modified Eagle's Medium (DMEM)

039

Invitrogen, Carlsbad, U.S.A., Cat. No. 10829-

Knockout (KO) DMEM

018

Invitrogen, Carlsbad, U.S.A., Cat. No. 21980-

Iscove's Modified Dulbecco's Medium (IMDM)

065

Invitrogen, Carlsbad, U.S.A., Cat. No. 10828-

KO serum replacement (KOSR)

028

Biochrom AG, Berlin, Germany, Cat. No. S-

Heat-inactivated Fetal Bovine Serum (FBS)

0115

Dulbecco's Phosphate buffered saline (PBS) lx, PAA Laboratories GmbH,

without Ca/Mg Pasching, Austria, Cat. No. H 15-002

Minimum Essential Medium (MEM) Invitrogen, Carlsbad, U.S.A., Cat. No. 11140- Non-Essential Amino Acids 068

Invitrogen, Carlsbad, U.S.A., Cat. No. 35050-

GlutaMAX(TM)

038

Invitrogen, Carlsbad, U.S.A., Cat. No. 11360-

Sodium Pyruvate

088

Invitrogen, Carlsbad, U.S.A., Cat. No. 12587-

B27 supplement w/o vitamin A

010

Invitrogen, Carlsbad, U.S.A., Cat. No. 12563-

TrypLE™ Select 12563

011

Invitrogen, Carlsbad, U.S.A., Cat. No. 31350-

2-Mercaptoethanol

010

Sigma-Aldrich, St. Louis, U.S.A., Cat. No.

DMSO

D2650

Stemgent, San Diego, U.S.A., Cat. No. 04-

Pifitrin-alpha

0038

Sigma-Aldrich, St. Louis, U.S.A., Cat. No.

Valproic Acid (2-PROPYLPENTANOIC ACID SODIUM)

P4543

Sigma-Aldrich, St. Louis, U.S.A., Cat. No.

5-AzaC (5-AZA-2'-DEOXYCYTIDINE

A3656

Ultrapure Water with 0.1% Gelatin Millipore, Billerica, U.S.A., Cat. No. ES-006-B

Invitrogen, Carlsbad, U.S.A., Cat. No. 13256-

Basic fibroblasts growth factor (bFGF)

029

Amaxa NHDF Nucleofector Kit Lonza, Basel, Switzerland, Cat. No. VPD-1001

Sigma-Aldrich, St. Louis, U.S.A., Cat. No.

Y-27632 ROCK inhibitor

Y0503

Nalgene Labware, Rochester, U.S.A., Cat. No.

Freezing container

5100

nexttec GmbH Biotechnologie,

nexttec™ Genomic DNA Isolation Kit for Tissue and Cells

Hilgertshausen, Germany, Cat. No. 10.250

Solis BioDyne, Tartu, Estonia, Cat. No. 01-01-

FIREPol® DNA Polymerase

02000

5X Green GoTaq® Flexi Reaction Buffer Promega, Madison, U.S.A., Cat No. M8911

25mM Deoxynucleotide Triphosphates (dNTPs) dilutet Fermentas GmbH, St. Leon-Rot, Germany, from lOOmM stock solutions Cat. No. R0181

Invitrogen, Carlsbad, U.S.A., Cat. No.13256-

Basic fibroblasts growth factor (bFGF)

029

STEMCELL Technologies, Grenoble, France,

AggreWell(TM) plates 400

Cat. No. 27845

STEMCELL Technologies, Grenoble, France, mTeSR® medium Kit

Cat. No. 05850

STEMCELL Technologies, Grenoble, France,

ACCUTASE®

Cat. No. 07920 GFL GmbH, Burgwedel, Germany, Cat. No. rocking platform GFL 3006

3006

Becton Dickinson and Company, Franklin

10 cm petri dishes

Lakes, U.S.A., Cat. No. 351029

Becton Dickinson and Company, Franklin

10 cm Falcon™ tissue culture dishes

Lakes, U.S.A., Cat. No. 353003

Normal human dermal fibroblasts (adult skin) Lonza, Basel, Switzerland, Cat. No. CC-2511

Fibronectin from bovine plasma Sigma- Aldrich, Cat. No. F1141

Table 1: List of compounds and technical equipments with according suppliers.

Plasmids

Vector pCX-EGFP (Okabe et al, FEBS Lett. 407 (1997), 313-319; derived from pCAGGS, which was described in Niwa et al, Gene 108 (1991), 193-199) containing the CMV immediate early enhancer operably linked to the chicken β-actin promoter was used as a starting vector for the construction of the vectors pOIS and pKJJVI.

To generate the pKIM vector sequences encoding KLF4 and c-MYC were derived from clones IRATp970H0848D respective IRATp970F0881D (Source BioScience imaGenes, Berlin, Germany) and cloned together with an intervening IRES-sequence of the encephalomyocarditis virus (ECMV) (Jackson et al, Trends Biochem. Sci.15 (1990), 477- 483; Jang et al, J. Virol. 62 (1990),2636-2643) from pIRES2-EGFP vector (Clontech, Mountain View, U.S.A., Cat. No. #6029-1) to replace EGFP encoding sequence within said pCX- Vector.

Similary, pOIS was constructed by exchanging EGFP encoding sequence in pCX-EGFP by sequences encoding OCT3/4 and SOX3 derived from clones 40125986 (OpenBiosystem products / Thermo Fisher Scientific, Huntsville, U.S.A., Cat. No. MHS4426-99239368) respective IRAUp969A0546D (Source BioScience imaGenes, Berlin, Germany) with an intervening IRES-sequence as mentioned above from pIRES2-EGFP vector (Clontech, Mountain View, U.S.A., Cat. No. #6029-1).

cDNA and protein sequences of the respective reprogramming factors may be also derived from the NIH GenBank® using following database accession numbers: KLF4 (GenBank: BC029923), c-MYC (GenBank: BC058901), OCT3/4 (GenBank: BC117435) and SOX3 (GenBank: BC013923) Culture medium compositions and setup

hFiB medium: consists of DMEM containing 10% FBS (vol/vol), 2mM GlutaMAX, ΙΟΟμΜ MEM-NEAA, 1% sodium pyruvate. To prepare 500ml of the medium, 50 ml FBS, 5 ml GlutaMAX and 5 ml sodium pyruvate are mixed and then filled up to 500 ml with DMEM. hES cell medium: consists of Knockout-DMEM containing 20% KOSR (vol/vol), lOng/ml bFGF, 2mM GlutaMAX, ΙΟΟμΜ nonessential amino acids, O. lmM 2-Mercaptoethanol, B27 supplement (IX) are mixed and filled up to 500 ml with Knockout-DMEM. lOng/ml bFGF are added freshly to the media.

2 X iPS cell-freezing medium: consists of 20%DMSO (vol/vol) and 80%FBS (vol/vol).

Example 1: Reprogramming of human adult somatic cells to induced pluripotent stem cells (hiPSC)

It was the aim of the present invention to provide a protocol to reprogram human somatic cells into induced pluripotent stem cells in a reliable way, lowering at the same time the perils connected with the well-established methods using virus based vectors. To do so, sequences encoding reprogramming factors were introduced into normal plasmid vectors pOIS and pKFM and several reprogramming experimental strategies were tested, as outlined in Figure 2 and indicated in Table 2 below. From four different setups tested, experimental setup 4 provides in the most reliable way hiPSC and is described in detail below.

Table 2: Comparison of different reprogramming experimental setups and protocols provided by the present invention.

Transfection of the somatic cells

The two plasmids pOIS and pKIM containing expression cassettes encoding the four reprogramming factors Oct3/4, Sox2, Klf4 and c-Myc are cotransfected ^g DNA of each plasmid respectively) into human dermal fibroblasts (approximately 1.0 x 10⁶ cells per reaction; e.g., from LONZA). For transfection, the Amaxa NHDF Nucleofector™ Kit (Lonza, Basel, Switzerland; VPD-1001; with U-23 program) is used with the Nucleofector™ device (Lonza, Basel, Switzerland; cat. No. AAD-1001) for high transfection efficiency according to the description of the manufacturer. The person skilled in the art will know how to use said kits and devices, further information may be found at the homepage of the manufacturer (http://www.lonzabio.com/cell-biology/transfection/nucleofectiontrade-products/ and http://www.lonzabio.com) and in the patent specifications concerning Nucleofector™ kits and devices (see also the European Application EP 1 607 484 and international patent applications WO 02/00871, WO 02/086134, WO 02/055721, WO 02/086129, WO 03/070875, WO 2004/027015, WO 2005/039692, WO 2005/090547, WO 2007/006487, WO 2008/031598, WO 2008/101697). The transfection reaction is performed three times subsequently every third day. The transfected fibroblasts are plated then to a 10 cm gelatin- coated dish in human fibroblast medium, which medium is exchanged every day. After the final transfection the cells are plated on inactivated mouse embryonic fibroblasts (MEF) in human ES cell culture medium, which starts the reprogramming phase of the experiment.

Culture and reprogramming of the transfected cells on mouse embryonic feeder cells

Transfected cells are cultured on 10cm Falcon™ tissue culture dishes (Becton Dickinson) at a density of lxlO⁶ in hES medium containing lOng/ml of bFGF on a layer of feeder cells (inactivated mouse embryonic fibroblasts, prepared according standard protocols; see also description of the invention above). Cells were incubated at 37°C, 7% C0₂ and 95% humidity. Medium change is perfomed on a daily basis.

Two days after the last transfection, the reprogramming is supported by the addition of the small molecules pifithrin-alpha (PFTalpha, 10μΜ) and valporic acid (2mM) continuously for 16 days. The culture medium supplemented with PFTalpha and VP A has to be exchanged every day in this process (from Monday to Friday at least). The reprogramming phase is completed after 60 days. hiPS cells aggregation and preparation of Embryoid Bodies (EBs)

After the reprogramming phase (see Figure 2 for an overview of the cell culture schedule) hES medium is aspirated from the hiPSCs culture plate(s), and the cells are rinsed once with 2 ml of PBS (without Ca and Mg). After subsequent removal of PBS, 2.5 ml of ACCUTASE^® (STEMCELL Technologies, Grenoble, France) per 100 mm dish are added to cover the cells followed by incubation at 37°C and 7% C0₂ until cells detach easily from the plate with gentle shaking (5-10 min). The cell suspension is pipetted 2-3 times with a serological pipette to ensure any remaining clumps are fully dissociated and to dislodge any cells that are still attached to the surface of the dish. The cells are transferred to a 15 ml or 50 ml conical tube and centrifuged at 300x g for 5 min at room temperature (15-25°C). The supernatant is removed and the pellet is resuspended in a small volume of mTeSR medium (STEMCELL Technologies, Grenoble, France) supplemented with 10 μΜ Rho-associated kinase (ROCK) inhibitor (Sigma, Germany) to a density of 0.5-1.0xl0⁷ cells/ml. The single cell suspension is added to the AggreWell plates, centrifuged to distribute the cells evenly among the microwells, and then cultured for a minimum of 24 hours to allow aggregation of the cells and EB formation within each microwell. After said 24 hours the plates may be inspected microscopically for EB formation in which case the EBs are washed-out in mTeSR medium and plated on petri dishes (10 cm in diameter) in hES medium for further six days. Afterwards the EBs are transferred on fibronectin-coated dishes (19 cm in diameter) for two days. At day 8 the medium is exchanged to EVIDM + 20% FCS to allow differentiation. All cell culture incubation steps occur at 37°C, 7% C0₂ and 95% humidity. Alternatively EBs formation may be also provided by the following protocol, leading however to higher heterogeneity in size and shape of the EBs formed. hiPS cells from one or more petri dishes are trypsinised to obtain a single cell suspension and collected by centrifugation (800g for 5 min). Cells are resuspended to a density of 2xl0⁶cells/ml in Iscove's Modified Dulbecco's Medium (IMDM, Invitrogen) supplemented with 20% (v/v) fetal bovine serum (FBS, Invitrogen, batch controlled). 4 ml of this suspension are incubated per 6 cm petri dish (bacterial grade; Greiner Bio-One GmbH, Frickenhausen, Germany) on a rocking table (model 3006, GFL GmbH, Burgwedel, Germany) at 50 rpm, 37°C, 5% C0₂ and 95% humidity for 6h. After this time, the suspension is diluted 1 : 10 (e.g. 2ml of suspension added to 18ml IMDM 20% FCS) in several T25 tissue culture flasks (Falcon, Becton Dickinson) and incubated under the same conditions for additional 18h. After this time, hiPS cell aggregates ("embryoid bodies", EBs) are formed, typically around 500 per ml of suspension.

Example 2: Characterization of generated human induced pluripotent stem cells

(hiPSC's)

Multiple markers may be used to analyze the state of the reprogrammed adult cells and to distinguish between non-reprogrammed, partially-reprogrammed and fully-reprogrammed human iPS cells (Chan et al., Nat. Biotechnol. 27 (2009), 1033-1037).

For example, human iPS cells may be characterized by ES cell-like morphology at first and have then their iPS status confirmed more reliably by the expression of pluripotency markers, such as OCT4, Nanog, SSEA4, TRA-1-60 and TRA-1-81 (Takeshi et al, Cell 131 (2007), 861-872). As to the endogenous OCT4, its promoter sequence may also be checked for demethylation demonstrating reactivated transcription due to the induced pluripotent state of the cells. Further, expression of genes related to pluripotency including OCT-3/4, SOX2, NANOG, GDF3, REXl, FGF4, ESGl, DPPA2, DPPA4, and hTERT and to the cell-origin specific genes may be analyzed on RNA level (qRT-PCR). Additionally, in vitro differentiation by EB formation or by redifferentiation to cells of all germ layers is used to measure the developmental potential of the human iPS cells.

Immunocytochemistry for pluripotency markers:

The pluripotency markers Oct4, Nanog, Tra-1-81 and SSEA-4 are characteristic for reprogrammed hiPS cells, while Tra-1-60 is exclusively found only on fully-reprogrammed iPS cells (Park et al, Nature Protocols 3 (2008), 1180-1186). The presence of the markers was analyzed by immunocytochemistry with antibodies as indicated in Table 3 below and the following staining procedure

First antibody

Antigen (human) Antigen Isotype Supplier Cat. No. Working

dilution

Nanog Rabbit IgG Stemgent, San

09-0020 1 : 100

Diego, U.S.A.

Oct3/4 Mouse IgG Santa Cruz

sc-9081 1 :200

Biotechnology, Inc.,

Santa Cruz, U.S.A.

SSEA-4 Mouse IgG Millipore, Billerica,

MAB 4304 1 : 100

U.S.A.

Tra-1-60 Mouse IgG Millipore, Billerica,

MAB 4360 1 : 100

U.S.A. Tra-1-81 Mouse IgG Millipore, Billerica,

MAB 4381 1 : 100

U.S.A.

Secondary antibody

Antigen Fluorescent dye Supplier Working

dilution

Goat-anti-mouse IgG Cy3 Dianova,

115-165-003 1 :400

Hamburg, Germany

Goat-anti-rabbit IgG Alexa 488 Invitrogen,

A11008 1 :200

Carlsbad, U.S.A.

DAPI Sigma-Aldrich, St.

D9564 1 :4000

(4',6-Diamidino- Louis, U.S.A.

2-phenylindole

dihydrochloride,

dilactate)

Antibodies and DNA labeling agents used for detection and validation by immunocytochemistry of pluripotency markers

The staining procedure is performed as described in the following steps (i) to (xii)

(i) Fixation: lx 20min in PBS with 4% paraformaldehyd, pH7.4, at room temperature (RT)

(ii) Washing: 3x 5 min with PBS (without (w/o) Ca2⁺ and Mg²⁺) at RT

For intracellular staining:

(iii) Permeabilization: lx 15 min with 0.5% Triton/PBS (w/o Ca²⁺ and Mg²⁺)

(iv) Washing: 2x with PBS (w/o Ca²⁺&Mg²⁺ ions)

(v) Blocking: lx 60 min with PBS +10% FBS at RT

(vi) Incubation with first antibody: overnight in PBS +5% FBS at 4°C (antibody

concentration as indicated in Table 1 above)

(vii) Washing: 3x 5 min in PB S (w/o Ca²⁺ and Mg²⁺) at RT

(viii) Incubation with second antibody: lx 60 min in PBS +5% FBS at RT (antibody

concentration as indicated in Table 1 above)

(ix) Washing: 3x 5 min with PBS (w/o Ca²⁺ and Mg²⁺) at RT

(x) DAPI staining: lx 15 min with DAPI in PBS (w/o Ca²⁺ and Mg²⁺) at RT

(xi) Washing: 3x 5 min PBS (w/o Ca²⁺ and Mg²⁺) at RT

(xii) Covering with aluminum foil and storage at 4°C before fluorescent microscopy Five of the generated twelve hiPS cell clones were exemplary analyzed by the above- mentioned immunocytochemical method and pluripotency markers could be detected in all of them; see Figure 5 for the exemplary hiPS cell clone 06-08.

In vitro hiPS cell differentiation

hiPS cells raised by, e.g., the method using AggreWell(TM) plates described in Example 1 can be differentiated to different cell types demonstrating their pluripotency and applicability in provision of redifferentiated cells for further usage according to the present invention.

After said 24 hours the plates may be inspected microscopically for EB formation in which case the EBs are washed-out in mTeSR medium and plated on petri dishes (10 cm in diameter) in hES medium for further six days. Afterwards the EBs are transferred on fibronectin-coated dishes (19 cm in diameter) for 2 days. At day 8 the medium is exchanged to IMDM + 20% FCS to allow differentiation. All cell culture incubation steps occur at 37°C, 7% C0₂ and 95% humidity. After 23 days, structures belonging to different germ layers occur, such as potential neuronal (ectoderm) and first beating cardiomyocytes (mesoderm) appear in the EBs and may be visualized using an appropriate microscope, giving proof to the assumed pluripotency of the hiPS cells provided by the present invention.

Example 3: Analysis of genomic integration by genomic PCR

After introduction into somatic cells non-viral expression vectors, such as plasmid vectors are typically not integrated into the genome. Under selection pressure however, and by usage of a transfection method introducing DNA molecules efficiently into the nucleus non-viral expression vectors may be as well stable integrated into chromosomal DNA. To analyze this possibility iPS cells which are generated by the method of the present invention may be examined by genomic PCR as described below for exemplary human iPS cell clones obtained in an experiment as described in Example 1.

Genomic DNA preparation

Genomic DNA is extracted and purified from cell culture cells with the nexttec™ Genomic DNA Isolation Kit for Tissue and Cells (nexttec GmbH Biotechnologie, Hilgertshausen, Germany) according to the description of the manufacturer, but may be as well obtained by conventional methods as described in Wu et al, Nucleic Acids Res. 23 (1995), 5087-5088. First, the nextec cleanColumns have to be equilibrated. For this purpose, 350μ1 of Prep Buffer are added of to a nexttec cleanColumn. After a subsequent incubation with the column for 5 min at RT, the column is centrifuged at 2300 rpm for 1 min in a table centrifuge. The supernatant has to be removed and the nexttec cleanColumn to be positioned into a new DNA collection tube. Thus prepared, the cleanColumn may be stored for a week at +2 to +8°C.

Lysis protocol:

Cells (1-2 x 10⁶) or tissue (5-30 mg) is provided into a small reaction tube (1.5-2 ml volume size, e.g., from Eppendorf, Germany) and centrifuged for 2 min at 2300 rpm. After removal of the supernatant and two subsequent washing steps with 500 μΐ PBS w/o MgCl₂ each, the probes are centrifuged again at 2300rpm for lmin. Supernatant is removed again and 265μ1 of Gl, 10 μΐ of G2 and 25 μΐ of the G3 buffer added (optionally with 3 μΐ DTT in addition). The samples are incubated in a thermomixer at 60°C, 1200 rpm for at least 30 min or over night (o/n).

Genomic DNA purification

120 μΐ of the lysate obtained by the above described protocol are provided to an equilibrated nexttec cleanColumn and incubated for 3 min at RT. The nexttec CleanColumn is centrifuged then at 3300 rpm for 1 min, the flow-through containing purified genomic DNA collected and stored at 4°C until it is required for analysis by PCR.

PCR on genomic DNA

Purified DNA of the previous step is analysed by the PCR-protocol as indicated in Table 4 below for presence of specific DNA sequences. Appropriate forward and reverse primers are selected from within the primers indicated in Figure 4 allowing amplification of sequence fragments of the introduced plasmids.

Table 4: PCR protocol: Composition of a respective reaction mixture in left part (A), reaction course in the right part (B) of the table.

Genomic DNA from all twelve exemplary hiPS cell lines was analyzed. Since the primer pairs used were designed to amplify regions of the chicken beta-actin promoter and the expression cassettes encoding the transformation factors with the intervening IRES- sequences, at least the integration of this vector parts was found in all twelve hiPS cell lines; see also the table in Figure 9 where (+) shows that an corresponding PCR product was generated which indicates plasmid integration in all generated exemplary hiPS cell lines.

Example 4: Usage of hiPS cells in pharmacological compound screening

The hiPS cells and the redifferentiated cells of the present invention may be used in pharmacological compound screenings in a way comparable to ES cells and their redifferentiated descendents. For example, hiPS cells of the present invention differentiate to beating cardiomyocytes as described supra. Adding of compounds interfering with normal cardiac physiology {e.g. Nifedipine, 4-aminopyridine) alters beating frequence and/or intensity. Therefore, said redifferentiated cardiomyocytes and this method can be used in pharmacological compound screening as described in detail in the international application WO 2005/108598.

Additionally, the method of said application WO 2005/108598 can be used to detect embryotoxic compounds in a high-throughput in vitro system. Therefore, the EBs used by said method can be manufactured using hiPS cells with an appropriate reporter gene {e.g. a fluorescent reporter like GFP) driven by a tissue specific promoter {e.g., alpha-MHC for cardiomyocytes). After plating into 96 well plates (flat bottom, black; Falcon, Becton Dickinson), the EBs are challenged with the test compounds at different concentrations or with the diluents as control. Half of the medium is replaced with fresh medium and compound twice a week. After differentiation towards cardiomyocytes appears in the control EBs, the fluorescence in all EBs is measured using a fluorescence spectrophotometer (Tecan). The embryotoxic effect of the test compounds is calculated as percent of the controls, which are defined as 100%.

Claims

A method of producing an induced pluripotent stem cell, the method comprising the steps of:

(i) introducing at least one non-viral expression vector comprising at least one DNA sequence encoding at least one reprogramming factor into somatic cells, preferably wherein the vector is a plasmid vector;

(ii) cultivating the cells for a time period sufficient for the introduced reprogramming factor being expressed and capable of reprogramming the somatic cells into pluripotent cells, characterized in that during the cultivating step at least one histone deacetylase (HDAC) inhibitor and at least one p53 inhibitor are added to the cell culture; and

(iii) selection of pluripotent cells.

The method of claim 1, wherein the reprogramming factor comprises one or more factors selected from the group consisting of the protein families OCT, SOX, KLF and MYC.

The method of claim 1 or 2, wherein at least two vectors are introduced into the somatic cells, preferably wherein a first vector comprises a DNA sequence encoding reprogramming factors of the OCT and SOX family and a second vector comprises a DNA sequence encoding reprogramming factors of the KLF and MYC family, preferably wherein the reprogramming factors are OCT3/4, SOX2, KLF-4 and c-MYC and most preferably wherein the vectors are introduced simultaneously into the somatic cells.

The method of any one of claims 1 to 3, wherein the DNA sequences encoding the reprogramming factors are separated by an intervening sequence allowing polycistronic expression of the DNA sequences, preferably wherein the intervening sequence is an internal ribosome entry site (IRES) most preferably wherein the order of the DNA sequences is Oct3/4-JRES-Sox2 and/or Klf4-JRES-c-Myc from the 5' to the 3' end.

The method of any one of claims 1 to 4, wherein the DNA sequence encoding the reprogramming factor is operably linked to expression control sequences, preferably wherein the expression control sequence comprises a promoter and preferably an enhancer, most preferably wherein the promoter is a constitutive promoter, preferably the chicken β-actin promoter and/or the enhancer sequence is the CMV early enhancer.

The method of any one of claims 1 to 5, wherein the introduction of the vectors is at least repeated once, preferably at least two times every second day after the first introduction, most preferably at least three times every third day after the first introduction of the vectors into the cells.

The method of any one of claims 1 to 6, wherein the HDAC inhibitor is 2- propylpentanoic acid (Valproic Acid/VPA), preferably wherein the HDAC inhibitor is present in the culture in a concentration of about 0,1 mM to 10 mM, preferably at concentration of about 1 to 5 mM and/or the p53 inhibitor is pifithrin-alpha (PFTalpha), preferably wherein the p53 inhibitor is present in the cell culture in a concentration of about 1 μΜ to 100 μΜ, preferably in a concentration of about 5 to 20 μΜ.

The method of any one of claims 1 to 7, wherein the cells are cultured in the presence of the inhibitors for a time period of 7 to 16 days, most preferably for at least 16 days, preferably wherein the inhibitors are added to the cell culture 1 to 5 days, most preferably 2 days after the last introduction of the reprogramming factor to the cell culture.

The method of any one of claims 1 to 8, wherein the somatic cells are adult somatic cells, preferably derived from human, preferably wherein the somatic cells are fibroblasts, preferably dermal fibroblasts, most preferably derived from adult skin.

10. The method of any one of claims 1 to 9, wherein the somatic cells are derived from a diseased cell, tissue or organ, preferably wherein the disease is selected from the group consisting of:

(i) cardiovascular diseases such as cardiomyopathy, cardiac hypertrophy, atherosclerosis, hypertension, congenital heart disease, coronary heart disease, ischemia, heart failure, inflammatory heart disease, Brugada syndrome;

(ii) neurological diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, multiple sclerosis;

(iii) metabolic diseases such as diabetes mellitus type II, metabolic syndrome, obesity, lysosomal storage disease;

(iv) autoimmune and inflammatory diseases such as diabetes mellitus type I, rheumatoid arthritis, psoriasis, inflammatory bowel, ulcerative colitis, Crohn's disease, celiac disease, multiple sclerosis, asthma, allergy; and

(v) monogenetic diseases such as Brugada syndrome, Huntington's disease, cystic fibrosis/mucoviscidosis, Duchenne muscular dystrophy, phenylketonuria, lysosomal storage diseases.

11. An induced pluripotent (iPS) cell obtainable by the method of any one of claims 1 to 10.

12. The iPS cell of claim 11, wherein one or more DNA sequences encoding reprogramming factors are integrated into the cellular genome in the form of a plasmid.

13. An in vitro differentiated cell derived from the iPS cell of any one of claims, preferably wherein said in vitro differentiated cell is characterized by the lack of expression of said at least one reprogramming factor.

14. A kit comprising one or more vector(s) and/or inhibitors as defined in any one of the preceding claims, and optionally cell culture reagents for use in the method of any one of claims 1 to 10.

15. Use of the iPS cell of the claim 11 or 12 or the in vitro differentiated cell of claim 13 in toxicity screening, drug development, transplantation therapy or drug target validation.