How to reprogram human cells

Abstract

Methods for generating induced trophoblast stem cells (iTSCs) from human cells are provided. Thus, methods are provided that include expressing exogenous GATA3 and OCT4 transcription factors in human cells under conditions that allow for the generation of iTSCs from the cells. Methods for rejuvenating and / or dedifferentiating human cells are also provided. Nucleic acid constructs, protein formulations, isolated human cells, human iTSCs, rejuvenated cells, and dedifferentiated cells are also provided.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to U.S. Patent Application No. 63 / 210,030, filed June 13, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing An ASCII file entitled 92637SequenceListing.txt, created on June 9, 2022 and submitted contemporaneously with the filing of this application, containing 114,688 bytes, is hereby incorporated by reference. [Background technology]

[0003] FIELD AND BACKGROUND OF THE PRESENT ART The present invention, in some embodiments thereof, relates to methods for reprogramming human cells, and more particularly, but not exclusively, to methods for reprogramming human cells into induced trophoblast stem cells (iTSCs) or rejuvenated cells.

[0004] Regenerative medicine is a new and expanding field that aims to replace lost or damaged cells, tissues, or organs in the human body through cell transplantation. Embryonic stem cells (ESCs) are pluripotent cells capable of long-term growth and self-renewal and can give rise to all cells, tissues, and organs of the fetal body. Thus, ESCs have great potential for cell therapy as a source of differentiated diverse cell types. Few major obstacles to realizing such potential are the risk of teratoma formation, alloimmune rejection of ESC-derived cells by the recipient, and ethical issues. The discovery of induced pluripotent stem cells (iPSCs) and direct transformation approaches have opened an attractive avenue to solve these challenges.

[0005] Master regulators are the most potent transcription factors that determine cell identity. Each cell type expresses a specific combination of master regulators, which together regulate the gene expression program of the cell. Along with the master regulators, there are thousands of transcription factors, cofactors, and chromatin modifiers whose expression in the cell is important to maintain a stable cellular state. The transcriptome of each cell type is tightly controlled by these factors, which allows the cell to perform its functions properly. The first report demonstrating how powerful master regulators are in regulating cell identity was in the 1980s, when Davis et al. showed that ectopic expression of MyoD in fibroblasts could convert fibroblasts into myocyte-like cells [Davis et al., Cell (1987) 51, pp. 987-1000]. Nearly two decades later, Xie et al. demonstrated that forced expression of C / EBPα / β could convert differentiated B cells into macrophage-like cells [Xie et al., Cell (2004) 117, 663-676]. These two studies demonstrated how fragile and delicate the balance between cell identity and cell plasticity is, and suggested that key regulators can influence cell fate when overexpressed.

[0006] In 2006, the way we use to think about cellular plasticity was transformed when two Japanese scientists, Takahashi and Yamanaka, showed that fibroblasts could be reprogrammed into functional embryonic stem cell-like cells (also named induced pluripotent stem cells (iPSCs)) by the introduction of four transcription factors, namely Oct4, Sox2, Klf4, and Myc (OSKM) [Takahashi, K. and Yamanaka, S., Cell (2006) 126, 663-676]. The idea that as few as four factors are sufficient to reset a cell's epigenome opened new avenues for scientists to attempt to convert various adult cells into other somatic cell types derived from ontogenetically distinct lineages by using a specific subset of key regulators and by circumventing the pluripotent state. Using this approach, several subsets of cell types, such as hematopoietic cells, various neuronal cells, cardiomyocytes, hepatocytes, embryonic Sertoli cells, endothelial cells, and RPE, have been converted from a variety of somatic cells.

[0007] In mammals, specialized cell types in the placenta mediate the physiological exchange between fetus and mother during pregnancy. The progenitors of these differentiated cells are trophoblast stem cells (TSCs). In the preimplantation embryo, trophoblast cells are the first differentiated cells that can differentiate from the pluripotent inner cell mass and form the outermost layer of the blastocyst [Roberts, RM and Fisher, SJ, Biology of reproduction (2011) 84, 412-421]. Lineage trophoblast cells are the source of the most fundamental cell types for the major structural and functional components of the placenta. Thus, TSCs have great biomedical relevance, as one-third of all human pregnancies are affected by placenta-related disorders [James et al., Placenta (2014) 35, 77-84].

[0008] In mice, TSCs can be isolated and cultured from the blastocyst polar trophectoderm (TE) or from outgrowths of the extraembryonic ectoderm (ExE) that originate from the polar TE after implantation [e.g., Latos and Hemberger, (2014) Placenta. 35 Suppl: S81-5]. All attempts to isolate and grow human TSCs (hTSCs) in vitro have failed for a long time. Very recently, hTSCs were successfully cultured for the first time [Okae et al., Cell stem cell (2018) 22, 50-63 e56]. These hTSCs give rise to all major trophoblast cell types following differentiation, display similar transcriptional and epigenetic signatures as primary placental cells, and give rise to a pathology of trophoblast cells when injected into NOD / SCID mice, suggesting that they are fully functional hTSCs (Okae et al., 2018).

[0009] The generation of induced TSC-like cells (iTSCs) from embryonic stem cells (ESCs) and somatic cells, such as fibroblasts, has been described previously (Cambuli et al., 2014; Kuckenberg et al., 2010; Lu et al., 2008; Ng et al., 2008; Nishioka et al., 2009; Niwa et al., 2000; Niwa et al., 2005; Ralston et al., 2010; and 15-17). However, in all models, lineage conversion remained incomplete and failed to confer a stable true TSC phenotype. Recently, it has been shown that transient ectopic expression of four mouse key trophectoderm (TE) genes, namely GATA3, Eomes, Tfap2c, and Myc (GETM), reprograms fibroblasts into stable and fully functional mouse induced trophoblast stem cells (miTSCs) [Benchetrit et al., Cell stem cell (2015) 17, 543-556].

[0010] Further background art includes the following: U.S. Patent No. 7,642,091; U.S. Patent No. 6,630,349; U.S. Patent Application Publication No. 20050191742; International Application Publication No. WO 2006052646; Canadian Patent Application Publication No. CA 2588088; International Application Publication No. WO2016 / 005985; and Fogarty et al., Nature (2017) 550(7674): 67-73. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] U.S. Patent No. 7,642,091 [Patent Document 2] U.S. Patent No. 6,630,349 [Patent Document 3] U.S. Patent Application Publication No. 20050191742 [Patent Document 4] International Application Publication No. WO 2006052646 [Patent Document 5] Canadian Patent Application Publication No. CA 2588088 [Patent Document 6] International Application Publication No. WO2016 / 005985 [Patent Document 7] International Application Publication No. WO 2013049389 [Patent Document 8] U.S. Patent No. 8,557,972 [Patent Document 9] International Patent Application No. WO 2014085593 [Patent Document 10] International Patent Application No. WO 2009071334 [Patent Document 11] International Patent Application No. WO 2011146121 [Patent Document 12] U.S. Patent No. 8,771,945 [Patent Document 13] U.S. Patent No. 8,586,526 [Patent Document 14] U.S. Patent No. 6,774,279 [Patent Document 15] U.S. Patent Application Publication No. 20030232410 [Patent Document 16] U.S. Patent Application Publication No. 20050026157 [Patent Document 17] U.S. Patent Application Publication No. 20060014264 [Patent Document 18] U.S. Patent No. 5,464,764 [Patent Document 19] U.S. Patent No. 5,487,992 [Patent Document 20] U.S. Patent No. 4,666,828 [Patent Document 21] U.S. Patent No. 4,683,202 [Patent Document 22] U.S. Patent No. 4,801,531 [Patent Document 23] U.S. Patent No. 5,192,659 [Patent Document 24] U.S. Patent No. 5,272,057 [Patent Document 25] U.S. Patent No. 3,791,932 [Patent Document 26] U.S. Patent No. 3,839,153 [Patent Document 27] U.S. Patent No. 3,850,752 [Patent Document 28] U.S. Patent No. 3,850,578 [Patent Document 29] U.S. Patent No. 3,853,987 [Patent Document 30] U.S. Patent No. 3,867,517 [Patent Document 31] U.S. Patent No. 3,879,262 [Patent Document 32] U.S. Patent No. 3,901,654 [Patent Document 33] U.S. Patent No. 3,935,074 [Patent Document 34] U.S. Patent No. 3,984,533 [Patent Document 35] U.S. Patent No. 3,996,345

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Non-licensed literature

[0012] [Non-licensed document 1] Davis, Cell (1987) 51, 987-1000

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[0013] According to an aspect of some embodiments of the present invention, there is provided a method of generating induced trophoblast stem cells (iTSCs) from human cells, the method comprising expressing exogenous GATA3 and OCT4 transcription factors in the cells under conditions that allow for generation of iTSCs from the cells, thereby generating iTSCs from the cells.

[0014] According to an aspect of some embodiments of the present invention, there is provided a method of generating induced trophoblast stem cells (iTSCs) from human cells, the method comprising expressing exogenous GATA3, OCT4, and KLF transcription factors in the cells under conditions that allow for generation of iTSCs from the cells, thereby generating iTSCs from the cells.

[0015] According to an aspect of some embodiments of the present invention there is provided a method of rejuvenating and / or dedifferentiating a human cell, the method comprising expressing exogenous GATA3 and OCT4 transcription factors in the cell under conditions allowing for the rejuvenation and / or dedifferentiation of the cell, thereby generating a rejuvenated and / or dedifferentiated cell.

[0016] According to an aspect of some embodiments of the present invention there is provided a method of rejuvenating and / or de-differentiating a human cell, the method comprising expressing exogenous GATA3, OCT4, and KLF transcription factors in the cell under conditions allowing for rejuvenation and / or de-differentiation of the cell, thereby generating a rejuvenated and / or de-differentiated cell.

[0017] According to some embodiments of the invention, the expressing step comprises transiently expressing.

[0018] According to some embodiments of the invention, the method further comprises expressing an exogenous c-MYC transcription factor in the cell.

[0019] According to some embodiments of the invention, the method further comprises expressing an exogenous KLF4 transcription factor in the cell.

[0020] According to some embodiments of the invention, the method further comprises expressing an exogenous KLF transcription factor in the cell.

[0021] According to some embodiments of the invention, the conditions are such that the expressing step is for at least 14 days following introduction of the exogenous transcription factor into the cell.

[0022] According to some embodiments of the invention, the conditions are such that the expressing step lasts for no more than 30 days after introduction of the exogenous transcription factor into the cell.

[0023] According to some embodiments of the invention, the conditions are such that the expressing step is for at least one day after introduction of the exogenous transcription factor into the cell.

[0024] According to some embodiments of the invention, the conditions are such that the expressing step is for less than 25 days following introduction of the exogenous transcription factor into the cell.

[0025] According to some embodiments of the invention, the iTSCs do not express exogenous transcription factors as determined by at least one of PCR, Western blot, and / or flow cytometry.

[0026] According to some embodiments of the invention, the rejuvenated and / or de-differentiated cells do not express exogenous transcription factors as determined by at least one of PCR, Western blot, and / or flow cytometry.

[0027] According to some embodiments of the invention, the expressing step comprises introducing a polynucleotide encoding the transcription factor into the cell.

[0028] According to some embodiments of the invention, the polynucleotide is DNA.

[0029] According to some embodiments of the invention, the polynucleotide is RNA.

[0030] According to some embodiments of the invention, the method includes isolating iTSCs from non-iTSCs.

[0031] According to some embodiments of the invention, the method includes assaying the generation of iTSCs.

[0032] According to some embodiments of the invention, the method comprises isolating the rejuvenated cells from non-rejuvenated cells.

[0033] According to some embodiments of the invention, the method includes assaying for rejuvenation.

[0034] According to some embodiments of the invention, the method comprises isolating the de-differentiated cells from non-de-differentiated cells.

[0035] According to some embodiments of the invention, the method includes assaying dedifferentiation.

[0036] According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct or system comprising at least one polynucleotide comprising a nucleic acid sequence encoding GATA3 and OCT4 transcription factors.

[0037] According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct or system comprising at least one polynucleotide comprising a nucleic acid sequence encoding a GATA3, OCT4, or KLF transcription factor.

[0038] According to some embodiments of the invention, the at least one polynucleotide further comprises a nucleic acid sequence encoding a c-MYC transcription factor.

[0039] According to some embodiments of the invention, the at least one polynucleotide further comprises a nucleic acid sequence encoding a KLF4 transcription factor.

[0040] According to some embodiments of the invention, the at least one polynucleotide further comprises a nucleic acid sequence encoding a KLF transcription factor.

[0041] According to some embodiments of the invention, at least one polynucleotide is RNA.

[0042] According to an aspect of some embodiments of the invention there is provided a protein preparation comprising GATA3 and OCT4 transcription factor polypeptides at a purity level of at least 20%.

[0043] According to an aspect of some embodiments of the invention there is provided a protein preparation comprising GATA3, OCT4, and KLF transcription factor polypeptides at a purity level of at least 20%.

[0044] According to some embodiments of the invention, the protein formulation further comprises a c-MYC transcription factor polypeptide.

[0045] According to some embodiments of the invention, the protein formulation further comprises a KLF4 transcription factor polypeptide.

[0046] According to some embodiments of the invention, the protein formulation further comprises a KLF transcription factor polypeptide.

[0047] According to an aspect of some embodiments of the present invention there is provided an isolated human cell expressing exogenous GATA3 and OCT4 transcription factors.

[0048] According to an aspect of some embodiments of the present invention there is provided an isolated human cell expressing exogenous GATA3, OCT4, and KLF transcription factors.

[0049] According to some embodiments of the invention, the isolated cells further express an exogenous c-MYC transcription factor.

[0050] According to some embodiments of the invention, the isolated cells further express an exogenous KLF4 transcription factor.

[0051] According to some embodiments of the invention, the isolated cells further express an exogenous KLF transcription factor.

[0052] According to some embodiments of the invention, the cell comprises a DNA molecule encoding a transcription factor.

[0053] According to some embodiments of the invention, the cell comprises an RNA molecule encoding a transcription factor.

[0054] According to some embodiments of the invention, the RNA is modified RNA.

[0055] According to some embodiments of the invention, the cell comprises a protein molecule of a transcription factor.

[0056] According to some embodiments of the invention, the expressing is not at the native location and / or expression level of the native gene of the transcription factor.

[0057] According to some embodiments of the invention, the cell is a somatic cell.

[0058] According to some embodiments of the invention, the cells are fibroblasts.

[0059] According to some embodiments of the invention, the cell is selected from the group consisting of a keratinocyte, a hematopoietic cell, a retinal cell, a fibroblast, a hepatic cell, a cardiac cell, a renal cell, a pancreatic cell, and a neuron.

[0060] According to some embodiments of the invention, the cells are hematopoietic cells or mesenchymal stem cells.

[0061] According to an aspect of some embodiments of the present invention there is provided an isolated induced trophoblast stem cell (iTSC) obtainable according to the method.

[0062] According to an aspect of some embodiments of the invention there is provided an isolated rejuvenated and / or de-differentiated cell obtainable according to the method.

[0063] According to an aspect of some embodiments of the present invention there is provided an isolated human induced trophoblast stem cell (iTSC) comprising ectopic DNA of GATA3 and OCT4 transcription factors integrated into its genome.

[0064] According to an aspect of some embodiments of the present invention, there is provided an isolated human induced trophoblast stem cell (iTSC) comprising ectopic DNA for GATA3, OCT4, and KLF transcription factors integrated into its genome.

[0065] According to some embodiments of the invention, the cell further comprises ectopic DNA of a c-MYC transcription factor integrated into the genome.

[0066] According to some embodiments of the invention, the cell further comprises ectopic DNA of a KLF4 transcription factor integrated into the genome.

[0067] According to some embodiments of the invention, the cell further comprises ectopic DNA of a KLF transcription factor integrated into the genome.

[0068] According to some embodiments of the invention, the isolated iTSCs maintain a level of differentiation of trophoblast stem cells for at least 20 passages in culture.

[0069] According to some embodiments of the present invention, the iTSC comprises: (i) TSC morphology; (ii) TSC markers determined by immunocytochemistry and / or PCR assays; (iii) the absence of fibroblast-specific markers as determined by immunocytochemistry and / or PCR assays; (iv) a transcriptome similar to that of blastocyst-derived TSCs, as determined by RNA sequencing assays; (v) genomic stability similar to blastocyst-derived TSCs, as determined by chromosomal microarray analysis; (vi) a methylation pattern similar to blastocyst-derived TSCs, as determined by bisulfate assay; (vii) in vitro differentiation following culture in medium lacking factors that support an undifferentiated state or in medium that promotes directed differentiation, as determined by morphology, flow cytometry, and / or PCR assays; (viii) in vitro and / or in vivo differentiation into trophectoderm lineage derivatives as determined by morphology, immunocytochemistry, immunocytochemistry, flow cytometry, and / or PCR assays; (ix) the ability to form three-dimensional organoid cultures as determined by morphology, immunocytochemistry, immunocytochemistry, and / or PCR assays; (x) in vivo formation of trophoblast cell lesions as determined by histological evaluation; (xi) no change in the level of differentiation for at least 20 passages in culture as determined by at least one of the assays (i) to (x). is characterized by at least one of the following:

[0070] According to some embodiments of the invention, the methylation pattern comprises hypomethylation of the ELF5 promoter region and / or hypermethylation of the Nanog promoter compared to somatic cells and / or ESC cells.

[0071] According to some embodiments of the invention, the rejuvenated cells are (i) The morphology of cells of the same type and developmental stage that have not been subjected to the method; (ii) markers for cells of the same type and developmental stage that were not subjected to the method, as determined by immunostaining, Western blot, and / or PCR assays; (iii) a transcriptome with the exception of age-associated genes, similar to cells of the same type and developmental stage that were not subjected to the method, with the exception of genes determined by RNA sequencing assays; (iv) a methylation pattern, as determined by a bisulfate assay, that is distinct from cells of the same type and developmental stage that have not been subjected to the method. It is characterized by at least one of the following:

[0072] According to an aspect of some embodiments of the present invention there is provided a cell culture comprising isolated cells and a culture medium.

[0073] According to an aspect of some embodiments of the present invention there is provided a cell culture comprising isolated cells and a culture medium.

[0074] According to some embodiments of the invention, the culture medium comprises a composition of components shown to support the culture of human TSCs.

[0075] According to some embodiments of the invention, the isolated cell is a cell line.

[0076] According to an aspect of some embodiments of the present invention there is provided a cell line of the isolated cells.

[0077] According to an aspect of some embodiments of the present invention there is provided an isolated population of cells, wherein at least 80% of the cells are iTSCs.

[0078] According to an aspect of some embodiments of the present invention there is provided an isolated population of cells, wherein at least 80% of the cells are rejuvenated and / or de-differentiated cells.

[0079] According to an aspect of some embodiments of the present invention there is provided an isolated population of cells, wherein at least 80% of the cells are cells disclosed herein.

[0080] According to an aspect of some embodiments of the invention there is provided a pharmaceutical composition comprising iTSCs or a population of cells and a pharma- ceutically acceptable carrier or diluent.

[0081] According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising the construct or system or the protein formulation and a pharma- ceutically acceptable carrier or diluent.

[0082] According to an aspect of some embodiments of the present invention there is provided a cosmetic composition comprising the construct or system or protein formulation and a cosmetic carrier or diluent.

[0083] According to some embodiments of the present invention, the cosmetic product is formulated as a cream, facial mask, scrub, soap, wash, or gel.

[0084] According to an aspect of some embodiments of the invention there is provided an isolated assembly, organoid, placenta, developing embryo, or synthetic embryo comprising an iTSC, construct or system, or protein preparation.

[0085] According to an aspect of some embodiments of the invention there is provided a method of enhancing a placenta, a developing embryo, or a synthetic embryo comprising introducing an iTSC, construct or system, or a protein preparation into the placenta, the developing embryo, or the synthetic embryo.

[0086] According to an aspect of some embodiments of the invention there is provided a method of generating an aggregate or organoid comprising trophoblast cells, the method comprising introducing an iTSC, construct or system, or protein formulation into a scaffold or matrix.

[0087] According to an aspect of some embodiments of the present invention, there is provided a method of treating and / or preventing a disorder associated with trophoblast cell development and / or activity in a subject in need thereof, the method comprising administering a therapeutically effective amount of an iTSC or cell population, pharmaceutical composition, construct or system, or protein formulation to the subject, thereby treating and / or preventing a disorder associated with trophoblast cell development and / or activity in the subject.

[0088] According to an aspect of some embodiments of the present invention there is provided a method of treating and / or preventing an age-related disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a cell or population of cells, pharmaceutical composition, construct or system, or protein formulation, thereby treating and / or preventing the disease in the subject.

[0089] According to some embodiments of the invention, the disease is a vision-related disease.

[0090] According to some embodiments of the invention, the disease is selected from the group consisting of glaucoma, cataracts, high myopia, retinitis pigmentosa, cone dystrophy, cone-rod dystrophy, Usher syndrome, Stargardt's disease, Bardet-Biedl syndrome, Best disease, and hereditary macular degeneration.

[0091] According to some embodiments of the invention, the disease is selected from the group consisting of myelodysplastic syndrome (MDS), cancer, graft rejection, graft versus host disease (GVHD), infectious disease, cytokine storm, radiation injury, neurodegenerative disease, and wound.

[0092] According to an aspect of some embodiments of the present invention there is provided a method of performing cosmetic care in a subject in need thereof, the method comprising applying a therapeutically effective amount of the construct or system, protein formulation, or cosmetic method composition to the skin of the subject, thereby performing the cosmetic care.

[0093] According to some embodiments of the invention, the KLF transcription factor is selected from the group consisting of KLF4, KLF5, KLF6, and KLF15.

[0094] According to some embodiments of the invention, the KLF transcription factor is selected from the group consisting of KLF4 and KLF5.

[0095] According to some embodiments of the invention, the KLF transcription factor comprises at least two distinct KLF transcription factors.

[0096] According to some embodiments of the invention, the KLF transcription factors include at least KLF4 and KLF5.

[0097] According to an aspect of some embodiments of the present invention there is provided a method for identifying an agent capable of modulating trophoblast cell development and / or activity, the method comprising: (i) contacting an isolated iTSC or cell population, aggregate, organoid, or placenta with a candidate agent; and (ii) comparing the development and / or activity of the isolated iTSCs, cell populations, aggregates, organoids, or placenta following contact with the agent to the development and / or activity of the isolated iTSCs, cell populations, aggregates, organoids, or placenta in the absence of the agent. Including, An effect of the agent on the development and / or activity of the isolated iTSCs, cell population, aggregate, organoid, or placenta that is greater than a predetermined level compared to the development and / or activity of the isolated iTSCs, cell population, aggregate, organoid, or placenta in the absence of the agent indicates that the agent modulates the development and / or activity of trophoblast cells.

[0098] According to an aspect of some embodiments of the present invention there is provided a method of obtaining a compound produced by a trophoblast cell, the method comprising culturing isolated iTSCs, a population of cells, or a cell culture and isolating a compound secreted by the cells from the culture medium, thereby obtaining the compound produced by the trophoblast cell.

[0099] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0100] Some embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: Reference will now be made in detail to the drawings, stressing that the particulars presented are by way of example and for the purposes of explanatory discussion of embodiments of the present invention, and in this regard, it will become apparent to those skilled in the art how to practice embodiments of the present invention, taking the description together with the drawings. [Brief description of the drawings]

[0101] [Figure 1] Figure 1A-E demonstrates that human fibroblasts are converted into trophoblast stem cell-like cells by ectopic expression of GATA3, OCT4, KLF4, and MYC (GOKM). (A) Schematic representation of the protocol to reprogram human foreskin fibroblasts (HFF, KEN or PCS201) into human induced trophoblast stem cells (hiTSC). M2rtTA-containing HFFs (passages 7-14) were infected with lentiviral vectors encoding the indicated transcription factors. Infected HFFs were exposed to doxycycline (dox) for 28 days while changing the relevant medium as illustrated in the scheme. 7-10 days after dox withdrawal, stable epithelial colonies were picked and plated onto feeder-containing plates. Colonies were passaged until fully stabilized. (B) Brightfield images of two TSC lines derived from human blastocysts, hbdTSC#2 and hbdTSC#9, and four representative hiTSC colonies, HFF lines of KEN origin hiTSC#1 and hiTSC34, or PCS201 origin hiTSC#11 and hiTSC#12. (C-D) qPCR analysis of mRNA levels for TSC-specific TFAP2C, TP63, KRT7, and endogenous GATA3 (C), and mesenchymal-specific genes, THY1, ZEB1, VIM, and ACTA2 (D), in four hiTSC colonies, two hbdTSC lines, two HFF lines, hESCs, and iPSCs. Results are shown as relative values ​​to the maximum expressing sample and normalized to the mRNA levels of the housekeeping control gene GAPDH. Bars indicate standard deviation between technical duplicates. A typical experiment from three independent experiments is shown. (E) Immunofluorescence staining of PFA-fixed hbdTSC line hbdTSC#9 and representative hiTSC clone hiTSC#1 for TSC markers GATA2, GATA3, KRT7, epithelial markers KRT18 and CDH1, and mesenchymal marker VIM. The experiment was repeated with two hbdTSC lines and two hiTSC lines with similar results. [Diagram 2]2A-C show the results of RNAseq analysis showing that hiTSCs and hbdTSCs have highly similar transcriptomes. (A-C) Plots representing RNA-seq data-based comparisons of the whole transcriptomes of HFFs, hESCs, hiPSCs, two hbdTSC lines, namely hbdTSC#2 and hbdTSC#9, and two biological duplicates of three hiTSC clones, namely hiTSC#1, hiTSC#4, and hiTSC#7. Principal component analysis (PCA) plots (A), correlation heatmaps (B), and scatter plots (C) of bulk RNA showing the transcriptional similarity between hbdTSCs and hiTSCs and their distance from pluripotent stem cells (PSCs) and HFFs. Hierarchical clustering in (B) was generated using the Spearman correlation coefficient of log2-CPM expression data values. Note that hiTSC lines cluster closer to hbdTSCs than hbdTSC lines cluster with each other. Pairwise scatterplot comparison of global gene expression profiles of hbdTSC#9 with hESC, HFF, hbdTSC#2, and three hiTSC colonies (C) shows high correlations between different colonies of hbdTSC and only between different colonies of hbdTSC and hiTSC. Representative genes expressed in ESC (NANOG, OCT4), fibroblasts (VIM, ZEB1), and hTSC (GATA3, TP63, TEAD4, TFAP2C) are shown. [Diagram 3]Figure 3A-D shows RRBS analysis demonstrating trophoblast cell specific changes in hiTSC methylation. Methylation analysis of HFFs, hESCs, two hbdTSC lines, namely hbdTSC#2 and hbdTSC#9, and three biological duplicates of four hiTSC clones, namely hiTSC#1, hiTSC#2, hiTSC#4, and hiTSC#11, assessed by RRBS. CpG methylation rate analysis was calculated based on 100 bp tiles with a sequencing depth of at least 10 reads per tile. (A, left) Heatmap showing 4676 differentially methylated regions (DMRs) of 100 bp that were hypomethylated in HFFs and hypermethylated in hbdTSCs. The methylation difference is more than 50%. It is shown that hiTSCs successfully acquired virtually all methylation patterns similar to hbdTSCs. (A, right) Box plot showing the average methylation for each biological sample. (B, left) Heatmap showing 24205 DMRs of 100 bp that are hypermethylated in HFFs and hypomethylated in hdTSCs. The difference in methylation is more than 50%. hiTSCs are shown to have successfully acquired the majority methylation pattern similar to hbdTSCs. (B, right) Box plot showing the average methylation for each biological sample. (C) Genome browser capture of the methylation levels of different tiles assessed by RRBS at the ELF5 locus. Note the trophoblast cell-specific hypomethylation upstream of the ELF5 gene. (D) Genome browser capture of the methylation levels of different tiles assessed by RRBS at the NANOG locus. Note the PSC-specific hypomethylation upstream of the NANOG gene compared to hbdTSC and hiTSC samples. Black boxes indicate 1000 bp tiles. [Figure 4]Figure 4A-E demonstrates the differentiation of hiTSCs into multinucleated ST cells. (A) Flow cytometry analysis of propidium iodide (PI) nuclear stained cells at 0, 4, and 8 days after changing to basal differentiation medium (BDM) consisting of DMEM supplemented with 10% FBS, showing spontaneous differentiation and formation of multinucleated syncytia. The experiment was repeated with two hbdTSC and two hiTSC lines with similar results. (B) Brightfield images of hbdTSC#2 and hiTSC#4 after 6 days in medium for directed differentiation of TSCs into syncytial trophoblast cells (STM) (Okae et al., 2018). (C) qPCR analysis of relative mRNA levels of ST-specific markers, namely CSH1, GCM1, SDC1, and CGB, for the indicated samples at 0, 2, and 6 days in STM. Results are presented as fold change relative to the day of maximum expression for each colony and normalized to the housekeeping control gene GAPDH. (D and E) Immunofluorescence staining of PFA-fixed undifferentiated hbdTSC#2 and hiTSC#4 and their ST derivatives after 6 days of ST differentiation. Cells were stained for DAPI (blue), epithelial-specific protein CDH1 (green), and pan-trophoblast cell marker KRT7 (red, D), as well as CSH1 and SDC1 (green) as ST-specific markers (E). White arrows indicate areas of spontaneous ST differentiation in undifferentiated TSCs, and yellow arrows indicate undifferentiated cells that are CDH1 positive in the ST differentiation plate. [Diagram 5]Figure 5A-C demonstrates that hiTSCs differentiate into HLA-G positive EVT cells. (A) Bright field images of hbdTSC#2, hiTSC#4, and hiTSC#2 and their EVT derivatives after 6 days of directed differentiation. (B) qPCR analysis of relative mRNA levels of EVT specific markers, namely HLA-G, MMP2, ITGA5, and ITGA1, at 0, 6, and 14 days of directed differentiation into extravillous trophoblast cells. Results are presented as fold change relative to the day of maximum expression of each colony and normalized to the housekeeping control gene GAPDH. (C) Immunofluorescence staining of PFA-fixed undifferentiated hbdTSC#2 and hiTSC#4 and their EVT derivatives after 14 days of EVT differentiation. Cells were stained for DAPI (blue), epithelial specific protein EPCAM (green), and EVT specific marker HLA-G (red). [Figure 6]6A-C demonstrate that hiTSCs transplanted into NOD-SCID mice form trophoblast cell lesions. hiTSCs can be used to establish three-dimensional organoid cultures. (A, left) Lesions extracted from NOD-SCID mice after subcutaneous injection of 4×106 cells of hbdTSC#2 or hiTSC#3 lines. Lesions were harvested 9 days after injection. (A, right) Stained sections of trophoblast cell lesions extracted from NOD-SCID mice. Hematoxylin and eosin staining and KRT7 immunohistochemical staining with hematoxylin counter staining are shown. (B, left) A commercially available pregnancy test that detects the presence of hCG. All hTSC cultures show positive results, but HFFs and PSCs show negative results. (B, right) qPCR analysis of mRNA levels of the CGB gene, which encodes the beta subunit of the trophoblast cell-specific hormone hCG. Results are presented as fold change relative to the maximum expressing sample and normalized to the housekeeping control gene GAPDH. (C, left) Brightfield images of hbdTSC#2 and hiTSC#4 at days 1 and 10 of the organoid formation protocol. (C, right) Spinning disk confocal imaging of formed organoids after immunofluorescence staining for DAPI, the pan-trophoblast cell marker KRT7, and the proliferative cell marker Ki-67. White arrows indicate areas of differentiation that are KRT7 positive but KI-67 negative. [Figure 7]Figure 7A-G demonstrate that hiTSCs reprogrammed by forced expression of GOKM undergo MET and express trophoblast cell markers. (A) qPCR analysis of the indicated transgenes in the depicted hiTSC colonies and control HFF(KEN), HFF(PCS), and hbdTSC#2. Transgene integration was assessed by designing a forward primer for the last exon of the transgene and a reverse primer matching the sequence of the FUW-tetO plasmid (see Table 1). Results are shown for the largest sample and normalized to the intronic region of the GAPDH gene. Bars indicate the standard deviation between two duplicates. (B) qPCR analysis of the mRNA levels of the indicated transgenes in infected HFFs 3 days after dox exposure. Three independent infections are shown compared to uninfected HFFs. (C) qPCR analysis of the mRNA levels of trophoblast cell markers GATA2 and TFAP2A. Note that TFAP2A and to some extent GATA2 are expressed in fibroblasts. (D) qPCR analysis of mRNA levels of HLA class I gene HLA-A normalized to the housekeeping control gene GAPDH. Results are presented as fold change with the maximum sample set as 1. (E) qPCR analysis of mRNA levels of epithelial markers KRT18, CDH1, OCLN, and EPCAM in the indicated hiTSC colonies and control HFF(KEN), HFF(PCS), ESC, and iPSC#1. Results are shown relative to the maximum expressing sample for each gene and normalized to the mRNA of the GAPDH gene. Bars indicate standard deviation between technical duplicates in a typical experiment. (F, top) Immunofluorescence staining for DAPI and TSC-specific marker TEAP2C in PFA-fixed hbdTSC#9, hiTSC#1, and HFF control. (F, bottom) Immunofluorescence staining for DAPI, GATA3, KRT7, KRT18, CDH1, and VIM in PFA-fixed HFFs. (G) Histograms showing flow cytometry analysis of classical HLA class I protein expression (HLA-A / B / C) using the well-characterized W6 / 32 antibody in the indicated hiTSC colonies, hbdTSC#2, and HFFs.HFFs and hbdTSC#2 were stained with secondary antibodies only to control for non-specific staining. [Figure 8] 8A-C show RNA-seq analysis showing that hiTSCs and hbdTSCs have transcriptomes enriched for gene ontology terms related to placental development. (A and B) Gene expression differences between hTSCs (hbdTSCs or hiTSCs) and hESCs and HFFs revealed significant enrichment of gene ontology terms related to placental and embryo-placental morphogenesis and development according to the Human Gene Atlas. (C) Network analysis showing associations between gene ontology terms and gene expression differences in (B). [Figure 9] Figure 1 shows karyotype analysis of hiTSC and hbdTSC. Two hbdTSC lines, hbdTSC#2 and hbdTSC#9, and four hiTSC clones, hiTSC#1, hiTSC#2, hiTSC#4, and hiTSC#11, were subjected to karyotype analysis using Affymetrix CytoScan 750K arrays. 50% of the hbdTSC lines and 50% of the hiTSC lines have intact karyotypes. The other 50% of the colonies showed few abnormalities in a small fraction of cells. The specific abnormalities and the associated affected fraction of cells are noted below each plot. [Figure 10]10A-F show that hiTSCs differentiate into ST-like and EVT-like cells. (A) qPCR analysis of relative mRNA levels of ST markers, namely ERVFRD-1, CSH1, SDC1, CGB, and PSG1, and EVT markers, namely NOTCH1, HLA-G, and MMP2, over 5 days in BDM. Results are presented as fold change relative to the day of maximum expression of each colony and normalized to the housekeeping control gene GAPDH. Bars indicate standard deviation among technical duplicates in a typical experiment. (B) Flow cytometry analysis of propidium iodide (PI) nuclear stained cells at 0, 4, and 8 days after change to BDM in hiTSC#1, showing spontaneous differentiation and formation of multinucleated syncytia. (C) Brightfield image of hiTSC#2 after 6 days in medium for directed differentiation of TSCs into syncytial trophoblast cells (STM). (D) qPCR analysis of relative mRNA levels of ST marker genes, PSG1, CHSY1, and ERVFRD-1, at 0, 2, and 6 days in STM. Results are presented as fold change relative to the day of maximum expression of each colony and normalized to the mRNA of the GAPDH gene. (E) Immunofluorescence staining for DAPI, epithelial-specific protein CDH1, and pan-trophoblast cell marker KRT7 in PFA-fixed undifferentiated hiTSC#2 cells after 6 days of ST differentiation in STM. (F) Bright field image and immunofluorescence staining for ST markers CSH1 and SDC1 in PFA-fixed ST cells originating from hiTSC#2 after 6 days of differentiation in STM. [Figure 11] Figure 1 demonstrates that hiTSCs transplanted into NOD-SCID mice form trophoblast cell lesions. (Left) Lesions extracted from NOD-SCID mice after subcutaneous injection of hiTSC cells. For each lesion, approximately 4x106 cells were injected subcutaneously into NOD-SCID mice. Lesions were harvested 9 days after injection. (Right) Stained sections of trophoblast cell lesions extracted from NOD-SCID mice. Hematoxylin and eosin staining (middle), KRT7 immunohistochemistry staining with hematoxylin counter staining (right). [Figure 12]Figure 1 demonstrates that ectopic expression of GATA3, OCT4, KLF4, KLF5, and MYC converts human fibroblasts into trophoblast stem cell-like cells. Senescent fibroblasts were transduced with GATA3, OCT4, KLF4, KLF5, and MYC and reprogrammed for 28 days, followed by dox withdrawal for 10 days. Representative images are shown demonstrating the morphology of parental fibroblasts (left) and the various hiTSC colonies that emerged following the reprogramming process (right). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0102] The present invention, in some embodiments thereof, relates to methods for reprogramming human cells, and more particularly, but not exclusively, to methods for reprogramming human cells into induced trophoblast stem cells (iTSCs) or rejuvenated cells.

[0103] Before describing at least one embodiment of the present invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by way of examples, as the invention is capable of other embodiments or of being practiced or carried out in various ways.

[0104] Regenerative medicine is a new and expanding field that aims to replace lost or damaged cells, tissues, or organs in the human body through cell transplantation. Induced stem cell generation and direct conversion approaches provide a valuable resource of cells for regenerative medicine and disease modeling. Here, direct conversion approaches refer to both somatic cell dedifferentiation and stem cell reprogramming. In mammals, specialized cell types in the placenta mediate the physiological exchange between fetus and mother during pregnancy. The precursors of these differentiated cells are trophoblast stem cells (TSCs), and thus TSCs have great biomedical relevance.

[0105] While putting embodiments of the present invention into practice, the inventors discovered that transient ectopic expression of key regulators of TSC in human cells results in the formation of stable, transgene-independent iTSCs that resemble endogenous TSCs in their transcriptome, methylome, and function.

[0106] As described hereafter and in the Examples section below, we have shown that transient ectopic expression of factors including GATA3 and OCT4 in human fibroblasts initiates a reprogramming process that leads to the formation of stable, transgene-independent induced trophoblast stem cells (iTSCs) (Examples 1 and 7, Figures 1A-1E, 7A-7G, and 12). The induced TSCs can be cultured for multiple passages (>20 passages) independent of exogenous factors and resemble blastocyst-derived TSCs in their morphology, genomic integrity, expression of TSC-specific markers, non-expression of ESC-specific and fibroblast-specific markers, and transcriptome and methylation status (Example 2, Figures 2A-2C, 3A-3D, 8A-8C, and 9). The inventors further demonstrated that the generated iTSCs could differentiate into syncytial trophoblast cells (ST) and extravillous trophoblast cells (EVT) to form trophoblast cell lesions in NOD / SCID mice and form functional organoids in Matrigel (Examples 3-5, Figures 4A-6C, and Figures 10A-11), suggesting that iTSCs acquire all the attributes of TSCs.

[0107] Thus, certain embodiments suggest the use of GATA3, OCT4, and optionally KLF4, KLF5, and / or c-MYC for the generation of iTSCs from human somatic cells and for their further use in, for example, regenerative medicine, disease modeling, drug screening, and placenta enhancement. Moreover, this is the first time that isolated human iTSCs have been generated that maintain their differentiation level in culture over the long term (more than 20 passages) without expressing the exogenous transcription factors (i.e., GATA3, OCT4, KLF4, and c-MYC) used to reprogram the parental cells.

[0108] Thus, according to one aspect of the present invention, there is provided an isolated human induced trophoblast stem cell (iTSC) comprising ectopic DNA of GATA3 and OCT4 transcription factors integrated into its genome.

[0109] According to certain embodiments, the isolated iTSCs contain ectopic DNA of the c-MYC transcription factor integrated into the genome.

[0110] According to certain embodiments, the isolated iTSCs contain ectopic DNA of a KLF transcription factor integrated into the genome.

[0111] According to certain embodiments, the isolated iTSCs contain ectopic DNA of the KLF4 transcription factor integrated into the genome.

[0112] According to certain embodiments, the isolated iTSCs contain ectopic DNA of the KLF5 transcription factor integrated into the genome.

[0113] According to certain embodiments, the isolated iTSCs comprise ectopic DNA of at least one of KLF4 and KLF5 transcription factors integrated into the genome. According to certain embodiments, the isolated iTSCs comprise ectopic DNA of KLF4 and KLF5 transcription factors integrated into the genome.

[0114] As used herein, the term "isolated" means at least partially separated from its natural environment, e.g., from a mammalian (e.g., human) embryo or mammalian (e.g., human) body, or from other cells in culture.

[0115] According to certain embodiments, isolation may be performed to produce a pure population, e.g., greater than 80%, greater than 85%, greater than 90%, greater than 95%, or 100% iTSCs, rejuvenated cells, or de-differentiated cells.

[0116] As used herein, the term "induced trophoblast stem cells (iTSCs)" refers to cells obtained by cell dedifferentiation or cell reprogramming. iTSCs thus produced acquire pluripotency and can differentiate into trophoblast cell lineages in this case. According to certain embodiments, such cells are obtained from differentiated cells (e.g. somatic cells such as fibroblasts) and undergo dedifferentiation by genetic manipulation that reprograms the cells to acquire the characteristics of trophoblast stem cells (TSCs). According to certain embodiments, iTSCs can differentiate into three types of trophoblast lineage cells in placental tissue: villous cytotrophoblasts, syncytiotrophoblasts, and extravillous trophoblasts. Villous cytotrophoblasts are specialized placental epithelial cells that differentiate, proliferate, and invade the uterine wall to form villi. Cytotrophoblasts reside in the anchoring villi and can fuse to form syncytiotrophoblasts or form columnar structures of extravillous trophoblasts (Cohen S. et al., 2003. J. Pathol. 200: 47-52).

[0117] According to certain embodiments, the iTSCs are human cells.

[0118] iTSCs typically resemble TSCs derived from mammalian embryonic placenta, for example, in morphology, expression of specific markers, transcriptome, methylation patterns, and function, as further described below.

[0119] According to certain embodiments, the iTSCs comprise: (i) TSC morphology, for example by microscopic evaluation (bright field or H&E staining, electron microscopy) (according to certain embodiments, TSC morphology is characterized by flat, dense colonies with high edges). (ii) TSC markers determined by immunocytochemistry and / or PCR assays; (iii) the absence of fibroblast-specific markers as determined by immunocytochemistry and / or PCR assays; (iv) a transcriptome similar to that of blastocyst-derived TSCs, as determined by RNA sequencing assays; (v) genomic stability similar to blastocyst-derived TSCs, as determined by chromosomal microarray analysis; (vi) a methylation pattern similar to blastocyst-derived TSCs, as determined by bisulfate assay; (vii) in vitro differentiation following culture in medium lacking factors that support an undifferentiated state (e.g., when cultured in DMEM medium containing 10% FBS) or in medium that promotes directed differentiation (e.g., Okae et al., Cell Stem Cell. 2018 Jan 4;22(1):50-63, or Haider et al., Stem Cell Reports. 2018 Aug 14;11(2):537-551, the contents of which are incorporated herein by reference in their entireties), as determined by morphology, flow cytometry, and / or PCR assays; (viii) in vitro and / or in vivo differentiation into trophectoderm lineage derivatives as determined by morphology, immunocytochemistry, immunocytochemistry, flow cytometry, and / or PCR assays; (ix) the ability to form three-dimensional organoid cultures as determined by morphology, immunocytochemistry, immunocytochemistry, and / or PCR assays (e.g., as described in Haider et al., Stem Cell Reports. 2018 Aug 14;11(2):537-551, the contents of which are incorporated herein by reference in their entirety); (x) in vivo formation of trophoblast cell lesions (e.g., in NOD / SCID mice) as determined by histological evaluation; (xi) no change in the level of differentiation for at least 20 passages in culture as determined by at least one of the assays (i) to (x). is characterized by at least one of the following:

[0120] According to certain embodiments, the TSC marker is selected from the group consisting of KRT7, GATA2, GATA3, TFAP2A, TFAP2C, TP63.

[0121] According to certain embodiments, the ESC-specific marker is selected from the group consisting of NANOG, OCT4, SOX2.

[0122] According to certain embodiments, the mesenchymal marker is selected from the group consisting of THY1, ZEB1, VIM, ACTA2.

[0123] According to certain embodiments, the methylation pattern comprises hypomethylation of the ELF5 promoter region and / or hypermethylation of the Nanog promoter compared to the non-reprogrammed parental cell and / or the ESC cell.

[0124] According to certain embodiments, iTSCs are characterized by the absence of embryonic stem cell (ESC) specific markers (eg, NANOG, OCT4, and SOX2) as determined by immunocytochemistry and / or PCR assays.

[0125] According to certain embodiments, the iTSCs maintain the differentiation level of TSCs for at least 20 passages, at least 30 passages, or at least 50 passages in culture.

[0126] According to certain embodiments, the iTSCs maintain their level of differentiation of TSCs for at least 20 passages.

[0127] According to other specific embodiments, the iTSCs maintain the differentiation level of TSCs without expression of exogenous transcription factors, for example as determined by a PCR assay.

[0128] According to certain embodiments, the iTSCs do not express exogenous transcription factors as determined by PCR, Western blot, and / or flow cytometry.

[0129] According to certain embodiments, the iTSCs do not express exogenous GATA3, OCT4, KLF, and c-MYC transcription factors as determined by PCR, Western blot, and / or flow cytometry.

[0130] According to certain embodiments, the iTSCs do not express exogenous GATA3, OCT4, KLF4, and c-MYC transcription factors as determined by PCR, Western blot, and / or flow cytometry.

[0131] According to certain embodiments, the iTSCs do not express exogenous GATA3, OCT4, KLF4, KLF5, and c-MYC transcription factors as determined by PCR, Western blot, and / or flow cytometry.

[0132] According to certain embodiments, the iTSCs express exogenous transcription factors that are not in the natural location (i.e., locus) and / or expression level (e.g., copy number and / or cellular localization) of the transcription factor's native gene.

[0133] According to certain embodiments, the iTSCs contain ectopic DNA for an exogenous transcription factor that is integrated into the genome of the cell but not in its natural location (ie, locus) and / or copy number.

[0134] According to certain embodiments, the transcription factor is selected from the group consisting of GATA3, OCT4, KLF, and c-MYC.

[0135] According to certain embodiments, the transcription factor is selected from the group consisting of GATA3, OCT4, KLF4, and c-MYC.

[0136] According to certain embodiments, the transcription factor is selected from the group consisting of GATA3, OCT4, KLF4, KLF5, and c-MYC.

[0137] As described, the present inventors have developed a novel method for generating human iTSCs.

[0138] Thus, according to a further or alternative aspect of the present invention, there is provided a method for generating induced trophoblast stem cells (iTSCs) from human cells, the method comprising expressing exogenous GATA3 and OCT4 transcription factors in the cells under conditions that allow for the generation of iTSCs from the cells, thereby generating iTSCs from the cells.

[0139] According to certain embodiments, the method further comprises expressing an exogenous c-MYC transcription factor in said cell.

[0140] According to certain embodiments, the method further comprises expressing an exogenous KLF transcription factor in said cell.

[0141] According to certain embodiments, the method further comprises expressing an exogenous KLF4 transcription factor in said cell.

[0142] According to certain embodiments, the method further comprises expressing an exogenous KLF5 transcription factor in said cell.

[0143] According to certain embodiments, the method further comprises expressing at least one of exogenous KLF4 and KLF5 transcription factors in said cell.

[0144] According to certain embodiments, the method further comprises expressing exogenous KLF4 and KLF5 transcription factors in said cells.

[0145] According to an aspect of some embodiments of the present invention there is provided an isolated human induced trophoblast stem cell (iTSC) obtainable by the method disclosed herein.

[0146] Furthermore, since expression of key regulators of TSC disclosed herein induced reprogramming of somatic cells into pluripotent cells, certain embodiments suggest using GATA3, OCT4, and optionally KLFs (e.g., KLF4, KLF5, KLF6, KLF15) and / or c-MYC for dedifferentiation of cells.

[0147] Furthermore, epigenetic changes such as DNA methylation have been proposed as a major cause of age-related diseases such as cognitive decline and cardiovascular disorders. Since expression of the master regulators of TSC disclosed herein induced reprogramming of somatic cells into pluripotent cells, while influencing DNA methylation, thereby reversing the epigenetic clock, certain embodiments suggest using GATA3, OCT4, and optionally KLFs (e.g., KLF4, KLF5, KLF6, KLF15) and / or c-MYC to rejuvenate aged cells.

[0148] Thus, according to a further or alternative aspect of the present invention there is provided a method for rejuvenating and / or de-differentiating a human cell, the method comprising expressing exogenous GATA3 and OCT4 transcription factors in the cell under conditions allowing the rejuvenation and / or de-differentiation of the cell, thereby generating a rejuvenated and / or de-differentiated cell.

[0149] According to certain embodiments, the method further comprises expressing an exogenous c-MYC transcription factor in said cell.

[0150] According to certain embodiments, the method further comprises expressing an exogenous KLF transcription factor in said cell.

[0151] According to certain embodiments, the method further comprises expressing an exogenous KLF4 transcription factor in said cell.

[0152] According to certain embodiments, the method further comprises expressing an exogenous KLF5 transcription factor in said cell.

[0153] According to certain embodiments, the method further comprises expressing at least one of exogenous KLF4 and KLF5 transcription factors in said cell.

[0154] According to certain embodiments, the method further comprises expressing exogenous KLF4 and KLF5 transcription factors in said cells.

[0155] According to an aspect of some embodiments of the present invention there is provided an isolated human rejuvenated and / or dedifferentiated cell obtainable by the methods disclosed herein.

[0156] As used herein, the term "dedifferentiated cells" refers to cells obtained by dedifferentiation or reprogramming of cells to a less specialized and earlier developmental stage within the same lineage. Methods for determining dedifferentiation are known in the art and are further described below. These methods include, but are not limited to, morphological assessment, expression of markers, in vitro and / or in vivo differentiation compared to the cell from which it was derived (i.e., a cell of the same type that has not been subjected to the method).

[0157] As used herein, the term "aged cells" refers to cells derived from an adult organism, for example a human subject that is at least 20 years of age.

[0158] As used herein, the term "rejuvenated cell" refers to a cell that is at the same lineage and differentiation / developmental state as the cell from which it was derived, but has characteristics of a younger age, which can be determined, for example, by an epigenetic signature.

[0159] According to certain embodiments, the rejuvenated cell has improved functionality compared to the cell from which it was derived.

[0160] According to certain embodiments, the rejuvenated cells are (i) The morphology of cells of the same type and developmental stage that have not been subjected to the method; (ii) markers for cells of the same type and developmental stage that were not subjected to the method, as determined by immunostaining, Western blot, and / or PCR assays; (iii) a transcriptome, with the exception of age-associated genes, similar to cells of the same type and developmental stage that were not subjected to the method, with the exception of genes determined by RNA sequencing assays; (iv) a methylation pattern, as determined by a bisulfate assay, that is distinct from cells of the same type and developmental stage that have not been subjected to the method. It is characterized by at least one of the following:

[0161] According to certain embodiments, the rejuvenated or de-differentiated cells do not express exogenous transcription factors as determined by PCR, Western blot, and / or flow cytometry.

[0162] According to certain embodiments, the rejuvenated or de-differentiated cells do not express exogenous GATA3, OCT4, KLF, and c-MYC transcription factors as determined by PCR, Western blot, and / or flow cytometry.

[0163] According to certain embodiments, the rejuvenated or de-differentiated cells do not express exogenous GATA3, OCT4, KLF4, and c-MYC transcription factors as determined by PCR, Western blot, and / or flow cytometry.

[0164] According to certain embodiments, the rejuvenated or de-differentiated cells do not express exogenous GATA3, OCT4, KLF4, KLF5, and c-MYC transcription factors as determined by PCR, Western blot, and / or flow cytometry.

[0165] According to an aspect of some embodiments of the present invention there is provided an isolated human rejuvenated cell obtainable by the method disclosed herein.

[0166] According to an aspect of some embodiments of the present invention there is provided an isolated human de-differentiated cell obtainable by the method disclosed herein.

[0167] As used herein, the term "cell" refers to any cell derived from an organism, including adult cells, fetal cells, somatic cells, and stem cells.

[0168] According to certain embodiments, the cells are aged cells.

[0169] According to certain embodiments, the cells are stem cells.

[0170] As used herein, the phrase "stem cell" refers to a cell that is not terminally differentiated, i.e., capable of differentiating into other cell types (e.g., fully differentiated cells) that have a more specific, specialized function. The term encompasses embryonic stem cells, fetal stem cells, adult stem cells, or committed / progenitor cells.

[0171] According to certain embodiments, the cell is a somatic cell.

[0172] As used herein, the phrase "somatic cell" refers to a terminally differentiated cell. Non-limiting examples of somatic cells include fibroblasts, blood cells, endothelial cells, hepatocytes, pancreatic cells, chondrocytes, muscle cells, cardiac muscle cells, smooth muscle cells, keratinocytes, neural cells, retinal cells, epidermal cells, epithelial cells (e.g., isolated from the oral cavity), or cells isolated from the placenta.

[0173] According to certain embodiments, the somatic cells are selected from the group consisting of fibroblasts, blood cells, keratinocytes, epithelial cells, such as cells isolated from the oral cavity, or cells isolated from the placenta.

[0174] According to a particular embodiment, the somatic cells are fibroblasts.

[0175] According to certain embodiments, the cells are selected from the group consisting of keratinocytes, hematopoietic cells, retinal cells (e.g., PE, photoreceptors), fibroblasts, hepatocytes, cardiac cells, renal cells, pancreatic cells (e.g., alpha, beta), and neurons.

[0176] According to certain embodiments, the cells are hematopoietic cells or mesenchymal stem cells. According to certain embodiments, the cells are human cells.

[0177] According to certain embodiments, the cells are comprised in a homogenous population of cells, for example, at least about 80% of the cells in the population are iTSCs, rejuvenated cells, or de-differentiated cells.

[0178] Thus, according to one aspect of the present invention, an isolated population of cells is provided, wherein at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% of the cells are iTSCs as disclosed herein.

[0179] According to other specific embodiments, the cells are comprised in a heterogeneous population of cells, i.e., a population comprising two or more cell types, of which at least 5%, at least 10%, at least 15%, at least 20%, at least 30% are iTSCs.

[0180] According to further or alternative aspects of the present invention, there is provided an isolated population of cells, wherein at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% of the cells are rejuvenated cells as disclosed herein.

[0181] According to other specific embodiments, the cells are comprised in a heterogeneous population of cells, i.e., a population comprising two or more cell types, of which at least 5%, at least 10%, at least 15%, at least 20%, at least 30% are rejuvenated cells.

[0182] According to further or alternative aspects of the present invention, there is provided an isolated population of cells, wherein at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% of the cells are dedifferentiated cells as disclosed herein.

[0183] According to other specific embodiments, the cells are comprised in a heterogeneous population of cells, i.e., a population comprising two or more cell types, of which at least 5%, at least 10%, at least 15%, at least 20%, at least 30% are dedifferentiated cells.

[0184] As stated, the exogenous transcription factor is expressed within the cell.

[0185] As used herein, the term "transcription factor" refers to a cellular factor that controls the transcription of genes. According to certain embodiments, a transcription factor is a polypeptide that has the ability to bind to a particular nucleic acid sequence (i.e., a binding site) that is specific for a particular transcription factor. Non-limiting examples of transcription factors include GATA3, OCT4, KLF (e.g., KLF4, KLF5, KLF6, KLF15), and c-MYC.

[0186] As used herein, the term "GATA3", also known as GATA binding protein 3 and HDRS, refers to the polynucleotides and expression products, e.g., polypeptides, of the GATA3 gene. According to certain embodiments, GATA3 refers to human GATA3, e.g., as provided in the following GeneBank Numbers NP_001002295 and NM_001002295 (SEQ ID NOs: 1-2). A functional expression product of GATA3, optionally together with other factors described herein, can support the generation of iTSCs.

[0187] As used herein, the term "OCT4 (Octamer-binding transcription factor 4)", also known as POU5F1, refers to the polynucleotides and expression products, e.g., polypeptides, of the POU5F1 gene. According to certain embodiments, OCT4 refers to human OCT4 as provided in the following GeneBank Numbers NP_001167002, NP_001272915, NP_001272916, NP_002692, NP_976034, NM_203289, NM_001173531, NM_001285986, NM_001285987, and NM_002701 (SEQ ID NOs: 3-12). Functional expression products of OCT4, optionally with other factors described herein, can support the generation of iTSCs.

[0188] As used herein, the term "KLF" refers to the polynucleotides and expression products, e.g., polypeptides, of any one of the Kruppel-like family of transcription factors, which are a set of C2H2 zinc finger DNA binding proteins that control gene expression. According to certain embodiments, KLF refers to human KLF. A functional expression product of KLF, optionally with other factors described herein, can support the generation of iTSCs. Non-limiting examples of KLF transcription factors include KLF1, KLF2, KLF3, KLF4, KLF5, KLF6, KLF7, KLF8, KLF9, KLF10, KLF11, KLF12, KLF13, KLT14, KLF15, KLF16, KLF17.

[0189] According to certain embodiments, the KLF transcription factor comprises at least one KLF transcription factor.

[0190] According to certain embodiments, the KLF transcription factor is selected from the group consisting of KLF4, KLF5, KLF6, and KLF15.

[0191] According to certain embodiments, the KLF transcription factor is selected from the group consisting of KLF4 and KLF5.

[0192] According to certain embodiments, at least one of the KLF transcription factors comprises at least two distinct KLF transcription factors.

[0193] According to certain embodiments, the KLF transcription factors include both KLF4 and KLF5.

[0194] According to certain embodiments, the KLF transcription factor comprises KLF4.

[0195] As used herein, the term "KLF4 (Kruppel-like factor 4)", also known as GKLF and EZF, refers to the polynucleotides and expression products, e.g., polypeptides, of the KLF4 gene. According to certain embodiments, KLF4 refers to human KLF4, e.g., as provided in the following GeneBank Numbers NP_004226 and NM_004235 (SEQ ID NOs: 13-14). Functional expression products of KLF4, optionally together with other factors described herein, can support the generation of iTSCs.

[0196] According to certain embodiments, the KLF transcription factor comprises KLF5.

[0197] As used herein, the term "KLF5 (Kruppel-like factor 5)", also known as BTEB2, CKLF, and IKLF, refers to the polynucleotides and expression products, e.g., polypeptides, of the KLF5 gene. According to certain embodiments, KLF5 refers to human KLF5, e.g., as provided in the following GeneBank Numbers NP_001273747, NP_001721, NM_001730, and NM_001286818 (SEQ ID NOs: 81-84). A functional expression product of KLF5, optionally with other factors described herein, can support the generation of iTSCs.

[0198] According to certain embodiments, the KLF transcription factor comprises KLF6.

[0199] As used herein, the term "KLF6 (Kruppel-like factor 6)", also known as BCD1, CBA1, COPEB, PAC1, ST12, and ZF9, refers to the polynucleotides and expression products, e.g., polypeptides, of the KLF6 gene. According to certain embodiments, KLF6 refers to human KLF6, e.g., as provided in the following GeneBank Numbers NP_001153596, NP_001153597, NP_001291, NM_001008490, NM_001160124, NM_001160125, and NM_001300 (SEQ ID NOs: 85-91). A functional expression product of KLF6, optionally with other factors described herein, can support the generation of iTSCs.

[0200] According to certain embodiments, the KLF transcription factor comprises KLF15.

[0201] As used herein, the term "KLF15 (Kruppel-like factor 615)" refers to polynucleotides and expression products, e.g., polypeptides, of the KLF15 gene. According to certain embodiments, KLF15 refers to human KLF15, e.g., as provided in the following GeneBank Numbers NP_054798, NM_014079 (SEQ ID NOs: 92-93). A functional expression product of KLF15, optionally together with other factors described herein, can support the generation of iTSCs.

[0202] As used herein, the term "c-MYC", also known as V-Myc avian myelocytomatosis viral oncogene homolog, class E basic helix-loop-helix protein 39, transcription factor P64, BHLHe39, MRTL, and MYCC, refers to the polynucleotides and expression products, e.g., polypeptides, of the MYC gene. According to certain embodiments, c-MYC refers to human c-MYC, e.g., as provided in the following GeneBank Numbers NP_002458 and NM_002467 (SEQ ID NOs: 15-16). A functional expression product of c-MYC, optionally with other factors described herein, can support the generation of iTSCs.

[0203] The terms "GATA3", "OCT4", "KLF4", "KLF", and "c-MYC" also refer to functional GATA3, OCT4, KLF (e.g., KLF4, KLF5, KLF6, KLF15) and c-MYC homologs that exhibit a desired activity (i.e., dedifferentiate or reprogram a cell to an iTSC). Such homologs may be at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the polypeptides of, for example, SEQ ID NOs: 1, 3-7, 13, 81-93, and 15, respectively. 00% identical or homologous, or may be 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polynucleotide encoding it (as described further hereinafter).

[0204] A homologue can refer to an ortholog, a substitution variant including deletions, insertions, or amino acid substitutions, so long as it retains activity.

[0205] Sequence identity or homology can be determined using any protein or nucleic acid sequence alignment algorithm, such as Blast, ClustalW, and MUSCLE.

[0206] Certain embodiments of the present invention contemplate expressing at least GATA3 and OCT4 and optionally c-MYC and / or KLF transcription factors.

[0207] Certain embodiments of the present invention contemplate expressing at least GATA3 and OCT4 and optionally c-MYC, KLF4, and / or KLF5 transcription factors.

[0208] Certain embodiments of the present invention contemplate expressing at least GATA3 and OCT4 and optionally c-MYC and / or KLF4 transcription factors.

[0209] According to certain embodiments, two, three, or all of the transcription factors are exogenously expressed in the cell, such as GATA3+OCT4, GATA3+OCT4+c-MYC, GATA3+OCT4+KLF (e.g., GATA3+OCT4+KLF4, GATA3+OCT4+KLF5, GATA3+OCT4+KLF4+KLF5), GATA3+OCT4+c-MYC+KLF (e.g., GATA3+OCT4+c-MYC+KLF4, GATA3+OCT4+c-MYC+KLF5, GATA3+OCT4+c-MYC+KLF4+KLF5).

[0210] According to certain embodiments, all of the transcription factors are exogenously expressed in the cell, i.e., GATA3+OCT4+c-MYC+KLF (e.g., GATA3+OCT4+c-MYC+KLF4+KLF5).

[0211] As used herein, the term "expressing" or "expression" refers to gene expression at the RNA and / or protein level. This term also refers to upregulating gene expression by expressing DNA or RNA, or upregulating protein levels by direct administration of the protein to a cell.

[0212] As used herein, the term "exogenous" refers to a heterologous polynucleotide or polypeptide that is not naturally expressed in a cell or whose overexpression in a cell is desired. Exogenous polynucleotides and / or polypeptides may be introduced into a cell in a stable or transient manner. In the case of polynucleotides, the introduction results in the production of ribonucleic acid (RNA) molecules and / or polypeptide molecules. According to certain embodiments, expressing includes transiently expressing. It is noted that exogenous polynucleotides and / or polypeptides may comprise nucleic acid sequences and / or amino acid sequences that are identical or partially homologous to endogenous nucleic acid sequences and / or endogenous amino acid sequences, respectively, of the cell. Methods for expressing exogenous nucleic acid and / or amino acid sequences are known in the art and include, for example, those described in the Materials and Methods section below, as well as in Mansour et al., 2012; Warren et al., 2010, and Hongyan Zhou et al., Cell Stem Cell (2009) 4(6): 581; Rabinovich and Weissman (2013) Methods Mol Biol. 969:3-28; International Application Publication No. WO 2013049389, and U.S. Patent No. 8,557,972, which are incorporated herein by reference in their entireties.

[0213] Further description of the preparation of expression vectors and their modes of administration to cells is provided below.

[0214] According to certain embodiments, the expressing step is not at the natural location (i.e., locus) and / or expression level (e.g., copy number and / or cellular localization) of the native gene of the transcription factor.

[0215] According to other particular embodiments, the expressing is not at the native location and / or copy number of the native gene of the transcription factor within the genome.

[0216] Alternatively, or in addition, exogenous expression of transcription factors is driven by activation of the endogenous locus of these genes, whereby the transcription factors are overexpressed in the cells. Methods for activating and overexpressing endogenous genes are well known in the art [see, e.g., Menke D. Genesis (2013) 51: - 618; Capecchi, Science (1989) 244: 1288-1292; Santiago et al., Proc Natl Acad Sci USA (2008) 105: 5809-5814; International Patent Application Nos. WO 2014085593, WO 2009071334, and WO 2011146121; U.S. Patent Nos. 8,771,945, 8,586,526, 6,774,279, and U.S. Patent Application Publication Nos. 20030232410, 20050026157, and US20060014264. The contents of which are incorporated by reference in their entirety], including but not limited to targeted homologous recombination (e.g., "hit and run", "double replacement"), site-specific recombinases (e.g., Cre recombinase and Flp recombinase), PB transposases (e.g., Sleeping Beauty, piggyBac, Tol2, or Frog Prince), genome editing with engineered nucleases (e.g., meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR / Cas systems), and genome editing using recombinant adeno-associated virus (rAAV) platforms (rAAV), and small molecules. Agents for introducing nucleic acid modifications into a gene of interest can be designed using publicly available sources or commercially available from Transposagen, Addgene, and Sangamo Biosciences.

[0217] The term "endogenous" as used herein means a polynucleotide or polypeptide that is present and / or naturally expressed within a cell.

[0218] Distinguishing cells that express an exogenous polynucleotide and / or polypeptide (e.g., a transcription factor) from cells that do not express the exogenous polynucleotide and / or polypeptide can be accomplished, for example, by determining the level and / or distribution of RNA and / or protein molecules in the cell, the location of DNA integration in the genome of the cell, and / or the number of gene copy numbers. Methods for determining the presence of an exogenous polynucleotide and / or polypeptide in a cell are well known in the art and include, for example, PCR, DNA and RNA sequencing, Southern blot, Western blot, immunoprecipitation, immunocytochemistry, flow cytometry, and imaging.

[0219] As used herein, the term "polynucleotide" refers to a single- or double-stranded nucleic acid sequence in the form of an RNA sequence (e.g., mRNA), a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence (e.g., a sequence isolated from a chromosome), a composite polynucleotide sequence (e.g., a combination of the above), or a mimetic or analog thereof. The term includes polynucleotides and / or oligonucleotides derived from naturally occurring nucleic acid molecules (e.g., RNA or DNA), synthetic polynucleotide and / or oligonucleotide molecules composed of naturally occurring bases, sugars and covalent internucleoside linkages (e.g., backbones), and synthetic polynucleotides and / or oligonucleotides having non-naturally occurring portions that function in a similar manner to the respective naturally occurring portions.

[0220] According to certain embodiments, the polynucleotide is a modified polynucleotide, such as modified RNA.

[0221] Such modified polynucleotides may contain modifications in either the backbone, the internucleoside linkage, or the bases. Modified polynucleotides may contain naturally modified nucleotides or synthetic nucleoside analogs. Modified polynucleotides are preferred over natural forms in certain embodiments due to desirable properties such as enhanced cellular uptake, enhanced affinity for nucleic acid targets, increased stability in the presence of nucleases, and reduced immunogenicity.

[0222] Such modifications include, but are not limited to, 5-methoxyuridine, pseudouridine, 5-methylcytidine, N6-methyladenosine, 2'-O-methyl, 2'-O-methyl 3' phosphorothioate, 2'-O-methyl 3' thiophosphonoacetate, locked nucleic acid (LNA).

[0223] Methods for stabilizing mRNA are known in the art and include adjusting the length of the polyadenine tail found at the 3' end of the mRNA transcript. Alternatively or additionally, the RNA cap found at the 5' end of the molecule can be modified. Naturally occurring cap structures typical of mammalian cells tend to be improperly incorporated into in vitro synthesized RNA, making them less effective. Synthetic "anti-reverse cap analogs" (available commercially, for example from Thermo Fisher Scientific) can prevent this mis-incorporation, resulting in more stable RNA with improved translation efficiency. To reduce immunogenicity, certain nucleotide substitutions can be replaced with chemically modified alternatives such as 5-methylcytosine or pseudouridine. Such substitutions can also silence immune responses while enhancing mRNA stability and translation efficiency. Other exemplary chemically modified nucleotides are described herein above.

[0224] Alternatively, or in addition, the mRNA may be encapsulated in lipid-based particles to enhance fusion with lipid cell membranes.

[0225] According to certain embodiments, the polynucleotide is an isolated polynucleotide.

[0226] Polynucleotides designed according to the teachings of some embodiments of the present invention can be produced according to any method of polynucleotide synthesis known in the art, such as enzymatic synthesis or solid phase synthesis. Equipment and reagents for carrying out solid phase synthesis are commercially available, for example from Applied Biosystems. Any other means for such synthesis may also be employed. The actual synthesis of polynucleotides is well within the capabilities of one of ordinary skill in the art and can be accomplished using solid phase chemistry, e.g., cyanoethyl phosphoramidites followed by deprotection, desalting, and purification, e.g., by automated trityl-ion methods or HPLC, according to established methodologies, as detailed, e.g., in "Molecular Cloning: A laboratory Manual" by Sambrook et al. (1989); "Current Protocols in Molecular Biology" Volumes I-III edited by Ausubel, RM (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988), and "Oligonucleotide Synthesis" edited by Gait, MJ (1984).

[0227] The term "polypeptide" or "protein" as used herein includes natural peptides (either degradation products, synthetically synthesized peptides, or recombinant peptides) and peptidomimetics (typically synthetically synthesized peptides), as well as peptoids and semipeptoids, which are peptide analogs that may have modifications, for example, that make the peptide more stable in the body or more permeable to cells. Such modifications include, but are not limited to, modifications of the N-terminus, modifications of the C-terminus, modifications of peptide bonds, modifications of the backbone, and modifications of residues. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, CA Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein.

[0228] The peptide bond (-CO-NH-) in a peptide may be replaced by, for example, an N-methylated amide bond (-N(CH3)-CO-), an ester bond (-C(=O)-O-), a ketomethylene bond (-CO-CH2-), a sulfinylmethylene bond (-S(=O)-CH2-), an α-aza bond (-NH-N(R)-CO-) (where R is any alkyl (e.g., methyl)), an amine bond (-CH2-NH-), a sulfide bond (-CH2-S-), an ethylene bond (-CH2-CH2-), a hydroxyethylene bond (-CH(OH)-CH2-), a thioamide bond (-CS-NH-), an olefinic double bond (-CH=CH-), a fluorinated olefinic double bond (-CF=CH-), a retroamide bond (-NH-CO-), a peptide derivative (-N(R)-CH2-CO-) (where R is a naturally occurring "normal" side chain on a carbon atom).

[0229] These modifications may occur at any bond along the polypeptide chain or at several (2-3) bonds simultaneously.

[0230] The natural aromatic amino acids Trp, Tyr, and Phe may be substituted with unnatural aromatic amino acids such as 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), naphthylalanine, ring-methylated derivatives of Phe, halogenated derivatives of Phe, or O-methyl-Tyr.

[0231] Polypeptides of some embodiments of the invention may contain one or more modified amino acids or one or more non-amino acid monomers (eg, fatty acids, complex carbohydrates, etc.).

[0232] The term "amino acid" is understood to include the 20 naturally occurring amino acids, those that are frequently post-translationally modified in vivo, including, for example, hydroxyproline, phosphoserine, and phosphothreonine, as well as other unconventional amino acids, including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, norvaline, norleucine, and ornithine. Additionally, the term "amino acid" includes both D- and L-amino acids.

[0233] Polypeptides of some embodiments of the present invention may be synthesized by any technique known to those skilled in the art of peptide synthesis, including, for example, but not limited to, recombinant DNA techniques or solid phase peptide synthesis.

[0234] The following is a non-limiting description of expression vectors that can be used to express a polypeptide of interest in a cell (e.g., any of the proteins described above and below, such as GATA3, OCT4, KLF (e.g., KLF4, KLF5, KLF6, FKL15), and c-MYC) and the manner in which they can be administered into a cell.

[0235] According to certain embodiments, the expressing step comprises introducing into the cell a polynucleotide encoding the polypeptide of interest (eg, a transcription factor).

[0236] According to a particular embodiment, the polynucleotide is DNA.

[0237] In certain embodiments, the polynucleotide is RNA.Typically, the mRNA introduced into cells is present only in the cytoplasm, does not cause genome perturbation, and is essentially transient.Unless the expression of mRNA epigenetically alters cells, transient transfection is limited to the time that the mRNA and associated proteins persist in the cell, and does not continue after the degradation of associated proteins.

[0238] To express an exogenous protein in a mammalian cell, a polynucleotide sequence encoding a polypeptide of interest is preferably ligated into a nucleic acid construct suitable for expression in a mammalian cell.

[0239] The present teachings further contemplate that the polynucleotide may be part of a nucleic acid construct system in which a polypeptide of interest is expressed from multiple constructs.

[0240] It will be appreciated that overexpression or elimination of genes can be achieved using knock-in and / or knock-out constructs [see, e.g., Fukushige, S. and Ikeda, JE: Trapping of mammalian promoters by Cre-lox site-specific recombination. DNA Res 3 (1996) 73-50; Bedell, MA, Jerkins, NA and Copeland, NG: Mouse models of human disease. Part I: Techniques and resources for genetic analysis in mice. Genes and Development 11 (1997) 1-11; Bermingham, JJ, Scherer, SS, O'Connell, S., Arroyo, E., Kalla, KA, Powell, FL and Rosenfeld, MG: Tst-1 / Oct-6 / SCIP regulates a unique step in peripheral myelination and is required for normal respiration. Genes Dev 10 (1996) 1751-62].

[0241] Thus, according to one aspect of the present invention, there is provided a nucleic acid construct or system comprising at least one polynucleotide comprising a nucleic acid sequence encoding the GATA3 and OCT4 transcription factors.

[0242] According to certain embodiments, the at least one polynucleotide further comprises a nucleic acid sequence encoding a c-MYC transcription factor.

[0243] According to certain embodiments, the at least one polynucleotide further comprises a nucleic acid sequence encoding a KLF transcription factor.

[0244] According to certain embodiments, the at least one polynucleotide further comprises a nucleic acid sequence encoding a KLF4 transcription factor.

[0245] According to certain embodiments, the at least one polynucleotide further comprises a nucleic acid sequence encoding a KLF5 transcription factor.

[0246] According to certain embodiments, the at least one polynucleotide further comprises a nucleic acid sequence encoding at least one of the KLF4 and KLF5 transcription factors.

[0247] According to certain embodiments, the at least one polynucleotide further comprises a nucleic acid sequence encoding the KLF4 and KLF5 transcription factors.

[0248] According to certain embodiments, two, three, or all of the transcription factors are encoded by a polynucleotide, e.g., GATA3+OCT4; GATA3+OCT4+c-MYC, GATA3+OCT4+KLF (e.g., GATA3+OCT4+KLF4, GATA3+OCT4+KLF5, GATA3+OCT4+KLF4+KLF5), or GATA3+OCT4+c-MYC+KLF (e.g., GATA3+OCT4+c-MYC+KLF4, GATA3+OCT4+c-MYC+KLF5 GATA3+OCT4+c-MYC+KLF4+KLF5).

[0249] According to certain embodiments, the nucleic acid construct or system comprises at least one polynucleotide comprising a nucleic acid sequence encoding GATA3, OCT4, c-MYC, and KLF.

[0250] According to certain embodiments, the nucleic acid construct or system comprises at least one polynucleotide comprising a nucleic acid sequence encoding GATA3, OCT4, c-MYC, and KLF4.

[0251] According to certain embodiments, the nucleic acid construct or system comprises at least one polynucleotide comprising a nucleic acid sequence encoding GATA3, OCT4, c-MYC, KLF4, and KLF5.

[0252] Thus, according to certain embodiments, the nucleic acid construct system comprises a separate nucleic acid construct for each transcription factor.

[0253] According to other particular embodiments, a single construct comprises several transcription factors.

[0254] Such a nucleic acid construct or system comprises at least one cis-acting control element for directing the expression of a nucleic acid sequence. Cis-acting control sequences include sequences that direct the constitutive expression of a nucleotide sequence, as well as sequences that direct the inducible expression of a nucleotide sequence only under certain conditions. That is, a promoter sequence is included in the nucleic acid construct to direct the transcription of a polynucleotide sequence in a cell, for example, in a constitutive or inducible manner. In the case of mRNA, since gene expression from an RNA source does not require transcription, it is not necessary to include a promoter sequence or additional sequences in the transcription described below.

[0255] The nucleic acid constructs or systems (also referred to herein as "expression vectors") of some embodiments of the present invention contain additional sequences that make the vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). Additionally, a typical cloning vector may contain transcription and / or translation initiation sequences, transcription and / or translation terminators, and polyadenylation signals. By way of example, such constructs typically contain a 5' LTR, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis, and a 3' LTR, or portions thereof.

[0256] Eukaryotic promoters typically contain two types of recognition sequences: the TATA box and upstream promoter elements. The TATA box is located 25-30 base pairs upstream of the transcription start site and is thought to be involved in directing RNA polymerase to begin RNA synthesis. Other upstream promoter elements determine the rate at which transcription is initiated.

[0257] Enhancer elements can stimulate transcription up to 1,000-fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription start site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer / promoter combinations suitable for some embodiments of the invention include those derived from long terminal repeats from various retroviruses, such as polyoma virus, human or mouse cytomegalovirus (CMV), murine leukemia virus, mouse or Rous sarcoma virus, and HIV. See Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, NY 1983, which is incorporated herein by reference.

[0258] In constructing an expression vector, a promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting, although, as is known in the art, some variation in this distance can be accommodated without loss of promoter function.

[0259] Polyadenylation sequences can also be added to expression vectors to increase the efficiency of mRNA translation. Two distinct sequence elements are required for accurate and efficient polyadenylation: a GU- or U-rich sequence located downstream from the polyadenylation site, and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream. Termination and polyadenylation signals suitable for some embodiments of the invention include those derived from SV40.

[0260] In addition to the elements already mentioned, the expression vectors of some embodiments of the invention may contain other specialized elements, typically intended to increase the level of expression of the cloned nucleic acid or to facilitate the identification of cells carrying the recombinant DNA. For example, some animal viruses contain DNA sequences that promote the extrachromosomal replication of the viral genome in permissive cell types. Plasmids carrying these viral replicons will replicate episomally as long as the appropriate factors are provided by genes carried on the plasmid or with the genome of the host cell.

[0261] The vector may or may not contain a eukaryotic replicon. If a eukaryotic replicon is present, the vector can be amplified in eukaryotic cells using an appropriate selectable marker. If the vector does not contain a eukaryotic replicon, episomal amplification is not possible. Instead, the recombinant DNA is integrated into the genome of the engineered cell, where the promoter directs the expression of the desired nucleic acid.

[0262] The expression vectors of some embodiments of the invention may further comprise additional polynucleotide sequences allowing the translation of several proteins from a single mRNA, such as, for example, an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.

[0263] It will be appreciated that individual elements contained in expression vectors can be arranged in a variety of configurations. For example, enhancer elements, promoters, etc., and even polynucleotide sequences encoding proteins of interest can be arranged in a "head-to-tail" configuration, can exist in a complementary configuration as reverse complements, or antiparallel strands. Although such various configurations are likely to occur with non-coding elements of expression vectors, alternative configurations of coding sequences in expression vectors are also envisioned.

[0264] In addition to containing the necessary elements for the transcription and translation of the inserted coding sequence, the expression constructs of some embodiments of the invention may contain sequences engineered to increase the stability, productivity, purification, yield, or toxicity of the expressed peptide.

[0265] According to certain embodiments, the expression construct comprises a label for imaging in the cell, such as a fluorescent label.

[0266] Examples of mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+ / -), pGL3, pZeoSV2(+ / -), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81 available from Invitrogen, pCI available from Promega, pMbac, pPbac, pBK-RSV, and pBK-CMV available from Strategene, pTRES available from Clontech, and derivatives thereof.

[0267] Expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses can also be used. SV40 vectors include pSVT7 and pMT2. Vectors derived from bovine papilloma virus include pBV-1MTHA, and vectors derived from Epstein-Barr virus include pHEBO and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector that allows expression of a protein under the direction of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown to be effective for expression in eukaryotic cells.

[0268] As mentioned above, viruses are highly specialized infectious agents that have evolved to often evade the host's defense mechanisms. Typically, viruses infect and grow in specific cell types. The targeting specificity of viral vectors exploits their natural specificity to specifically target a given cell type, thereby introducing recombinant genes into the infected cells. That is, the type of vector used in some embodiments of the present invention will depend on the cell type to be transformed. It is well within the capabilities of those skilled in the art to select a suitable vector depending on the cell type to be transformed, and therefore a general description of the considerations for selection is not provided here.

[0269] A variety of methods can be used to introduce polynucleotides or polypeptides of some embodiments of the invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989); Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995); Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995); Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988), and Gilboa et al., Biotechniques 4(6): 504-512, 1986, and include, for example, stable or transient transfection, lipofection, electroporation, nucleofection, microinjection, and infection with recombinant viral vectors. See also U.S. Patent Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.Currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, herpes simplex I virus, or adeno-associated virus (AAV), and lipid-based systems.

[0270] Naked DNA or RNA, cell penetrating peptides, or viral or non-viral vectors (such as, but not limited to, liposomes, nanoparticles, mammalian vectors, etc.) may be utilized as delivery vehicles in the delivery of polynucleotides or polypeptides, as is known in the art. According to certain embodiments of the invention, the delivery systems used are biocompatible and non-toxic.

[0271] The following are exemplary embodiments suitable for increasing the penetration of exogenous polynucleotides or polypeptides into cells.

[0272] According to one exemplary embodiment, naked DNA or RNA [e.g., naked plasmid DNA (pDNA)] is a non-viral vector that can be produced in bacteria and manipulated using standard recombinant DNA techniques. It does not induce an antibody response against itself (i.e., anti-DNA or anti-RNA antibodies are not generated) and allows long-term gene expression without chromosomal integration. Naked DNA or RNA can be introduced by many means, including but not limited to intravascular or electroporation techniques [Wolff JA, Budker V, 2005, Adv. Genet. 54: 3-20] or jet injection [Walther W, et al., 2004, Mol. Biotechnol. 28: 121-8].

[0273] According to another exemplary embodiment, a mammalian vector is used, as further described above.

[0274] According to certain embodiments, the polynucleotide is contained in a viral vector. The introduction of nucleic acid by viral infection offers several advantages over other methods such as lipofection and electroporation, since the infectivity of the virus allows for high transfection efficiency. The viral vector may be a virus with a DNA-based genome or a virus with an RNA-based genome (i.e., positive and negative single-stranded RNA viruses). Examples of viral vectors include, but are not limited to, lentiviruses, adenoviruses, and retroviruses.

[0275] Viral constructs, such as retroviral constructs, contain at least one transcription promoter / enhancer or locus-defining element, or other elements that control gene expression by other means, such as alternative splicing, nuclear RNA export, or post-translational modification of messengers. Such vector constructs also contain packaging signals, long terminal repeats (LTRs), or portions thereof, as well as positive and negative strand primer binding sites appropriate for the virus used, unless they are already present in the viral construct. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found, for example, in Current Protocols in Molecular Biology, edited by Ausubel et al., Greene Publishing Associates, (1989). Other suitable expression vectors may be adenoviruses, lentiviruses, herpes simplex I viruses, or adeno-associated viruses (AAV).

[0276] Control elements can also be included that limit expression to particular cell types. Such features include, for example, promoters and control elements that are specific for the desired cell type.

[0277] According to certain embodiments, the expressing step comprises introducing the polypeptide of interest (eg, a transcription factor) into the cell.

[0278] That is, according to one aspect of the present invention, there is provided a protein preparation comprising GATA3 and OCT4 transcription factor polypeptides.

[0279] According to certain embodiments, the protein formulation further comprises a c-MYC transcription factor polypeptide.

[0280] According to certain embodiments, the protein formulation further comprises a KLF transcription factor polypeptide.

[0281] According to certain embodiments, the protein formulation further comprises a KLF4 transcription factor polypeptide.

[0282] According to certain embodiments, the protein formulation further comprises a KLF5 transcription factor polypeptide.

[0283] According to certain embodiments, the protein formulation further comprises at least one of a KLF4 and a KLF5 transcription factor polypeptide.

[0284] According to certain embodiments, the protein formulation further comprises KLF4 and KLF5 transcription factor polypeptides.

[0285] According to certain embodiments, two, three or all of the transcription factors are comprised in a protein formulation, such as GATA3+OCT4; GATA3+OCT4+c-MYC, GATA3+OCT4+KLF (e.g., GATA3+OCT4+KLF4, GATA3+OCT4+KLF5, GATA3+OCT4+KLF4+KLF5) or GATA3+OCT4+c-MYC+KLF (e.g., GATA3+OCT4+c-MYC+KLF4, GATA3+OCT4+c-MYC+KLF5, GATA3+OCT4+c-MYC+KLF4+KLF5).

[0286] According to certain embodiments, the protein formulation comprises GATA3, OCT4, c-MYC, and KLF4 polypeptides.

[0287] According to certain embodiments, the protein formulation comprises GATA3, OCT4, c-MYC, KLF4, and KLF5 polypeptides.

[0288] According to certain embodiments, the protein formulation comprises each of the transcription factors at levels above residual levels (eg, above 0.1%).

[0289] According to certain embodiments, the protein preparation contains each of the transcription factors at a purity level of at least 10%.

[0290] According to certain embodiments, the protein preparation contains GATA3 and OCT4 at a purity level of at least 20%.

[0291] According to certain embodiments, the protein formulation comprises all of the transcription factors contained in the formulation at a purity level of at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%.

[0292] According to certain embodiments, a protein preparation contains all of the transcription factor polypeptides contained in the preparation at a purity level of at least 90%.

[0293] That is, according to certain embodiments, each of the polypeptides in the protein formulation is provided in an individual formulation.

[0294] According to other specific embodiments, the polypeptides in the protein formulation are provided in a coformulation.

[0295] According to certain embodiments, the polypeptide is provided in a formulation suitable for cell penetration to enhance intracellular delivery of the polypeptide, as further described below.

[0296] Cell-penetrating peptides (CPPs) are short peptides (less than 40 amino acids) that have the ability to access the interior of almost any cell. They are highly cationic and usually rich in arginine and lysine amino acids. They have excellent properties to carry a wide variety of covalently and non-covalently conjugated cargoes into cells, such as proteins, oligonucleotides, and even 200 nm liposomes. Thus, according to further exemplary embodiments, CPPs can be used to transport polynucleotides or polypeptides into the interior of cells.

[0297] TAT (transcriptional activator from HIV-1), pAntp (homeodomain transcription factor from Drosophila antennapedia, also named penetratin), and VP22 (from herpes simplex virus) are examples of CPPs that can enter cells non-toxically and efficiently and may be suitable for use in some embodiments of the present invention. Protocols for producing CPP-cargo conjugates and infecting cells with the conjugates can be found, for example, in L Theodore et al. [The Journal of Neuroscience, (1995) 15(11): 7158-7167], Fawell S, et al. [Proc Natl Acad Sci USA, (1994) 91: 664-668], and Jing Bian et al. [Circulation Research. (2007) 100: 1626-1633].

[0298] The expression levels and / or activity levels of exogenous polynucleotides and / or polypeptides expressed in cells of some embodiments of the present invention can be determined using methods known in the art, such as, but not limited to, Northern blot analysis, PCR analysis, Western blot analysis, immunohistochemistry, and fluorescence activated cell sorting (FACS).

[0299] "Conditions that allow for the generation of iTSCs from said cells" refers to culture conditions that affect cell dedifferentiation / reprogramming and maintenance of the TSC phenotype for at least 20 passages. Non-limiting examples of such conditions include culture time, medium composition, oxygen concentration, expression of small molecules, cytokines, and exogenous transcription factors.

[0300] "Conditions that allow cell rejuvenation" refers to culture conditions that affect cell rejuvenation without affecting the lineage and differentiation state of the cells. Non-limiting examples of such conditions include culture time, medium composition, oxygen concentration, small molecules, cytokines, and expression of exogenous transcription factors. "Conditions that allow cell dedifferentiation" refers to culture conditions that affect cell dedifferentiation without affecting the lineage of the cells. These conditions include culture time, medium composition, oxygen concentration, small molecules, cytokines, and expression of exogenous transcription factors.

[0301] According to a particular embodiment, the conditions are such that expression is transient.

[0302] That is, according to certain embodiments, the iTSCs, rejuvenated cells, or de-differentiated cells do not express exogenous transcription factors as determined by PCR, Western blot, and / or flow cytometry.

[0303] According to certain embodiments, the iTSCs, rejuvenated cells, or de-differentiated cells are free of exogenous transcription factors as determined by PCR, Western blot, and / or flow cytometry.

[0304] According to certain embodiments, the conditions are such that expression extends for at least 14 days, at least 15 days, at least 20 days, at least 25 days following introduction of the exogenous transcription factor into the cell.

[0305] According to certain embodiments, the conditions are such that expression extends for at least 14 days following introduction of the exogenous transcription factor into the cell.

[0306] According to certain embodiments, the conditions are such that expression occurs for no more than 28 days, no more than 30 days, or no more than 40 days following introduction of the exogenous transcription factor into the cell.

[0307] According to certain embodiments, the conditions are such that expression does not exceed 28 days following introduction of the exogenous transcription factor into the cell.

[0308] According to certain embodiments, the conditions are such that expression does not exceed 30 days following introduction of the exogenous transcription factor into the cell.

[0309] According to certain embodiments, the conditions are such that expression extends for 14 to 28 days following introduction of the exogenous transcription factor into the cells.

[0310] According to certain embodiments, the conditions are such that expression extends for at least 1 day, at least 3 days, at least 6 days, at least 9 days, at least 12 days, or at least 18 days following introduction of the exogenous transcription factor into the cell.

[0311] According to certain embodiments, the conditions are such that expression occurs for no more than 30 days, no more than 25 days, no more than 20 days, no more than 15 days following introduction of the exogenous transcription factor into the cell.

[0312] According to certain embodiments, the conditions are such that expression extends for less than 14 days following introduction of the exogenous transcription factor into the cell.

[0313] According to certain embodiments, the conditions are such that the reprogramming is performed in the absence of eggs, embryos, embryonic stem cells (ESCs), or iPSCs, i.e., none of these components are present in the culture system.

[0314] According to certain embodiments, the conditions include a low oxygen concentration, for example between 2-10% oxygen, for example about 5% oxygen.

[0315] According to certain embodiments, the conditions include culture medium containing EGF, CHIR99021, A83-01, SB431542, Y27632, and / or VPA or TSA.

[0316] According to certain embodiments, the conditions include DMEM / F12 culture medium containing 2-mercaptoethanol, FBS, penicillin-streptomycin, BSA, ITS supplements, L-ascorbic acid, EGF, CHIR99021, A83-01, SB431542, VPA or TSA, and Y27632, as further described below.

[0317] According to certain embodiments, the method includes a step of isolating iTSCs, rejuvenated cells, or de-differentiated cells.

[0318] Methods for isolating cells are well known in the art and include mechanical and marker-based techniques. Non-limiting examples of isolation techniques include cell sorting by fluorescence activated cell sorting (FACS), magnetic separation using magnetically labeled antibodies and magnetic separation columns (e.g., MACS, Miltenyi), and manual collection under a microscope.

[0319] According to certain embodiments, cell isolation is achieved by picking iTSC colonies under a binocular / microscope, followed by trypsinization and culturing on plates containing feeder cells.

[0320] According to certain embodiments, the isolation process results in a population comprising at least about 10%, at least about 12%, at least about 14%, at least about 16%, at least about 18%, at least about 20%, at least about 22%, at least about 24%, at least about 26%, at least about 28%, at least about 30%, at least about 32%, at least about 34%, at least about 36%, at least about 38%, at least about 40%, at least about 42%, at least about 44%, at least about 46%, at least about 48%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, e.g., 100%, iTSCs, rejuvenated cells, or de-differentiated cells of some embodiments of the invention.

[0321] According to certain embodiments, the method is accomplished ex vivo or in vitro.

[0322] Since the cells disclosed herein (iTSCs, rejuvenated cells, de-differentiated cells) are generated by expressing the transcription factors disclosed herein in cells, another aspect of the invention provides an isolated human cell expressing exogenous GATA3 and OCT4 transcription factors.

[0323] According to certain embodiments, the isolated cells further express an exogenous c-MYC transcription factor.

[0324] According to certain embodiments, the isolated cells further express an exogenous KLF transcription factor.

[0325] According to certain embodiments, the isolated cells further express an exogenous KLF4 transcription factor.

[0326] According to certain embodiments, the isolated cells further express an exogenous KLF5 transcription factor.

[0327] According to certain embodiments, the isolated cells further express at least one of exogenous KLF4 and KLF5 transcription factors.

[0328] According to certain embodiments, the isolated cells further express exogenous KLF4 and KLF5 transcription factors.

[0329] According to certain embodiments, the cells are comprised in a homogenous population of cells, and thus according to one aspect of the invention, an isolated population of cells is provided, wherein at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% of the cells are cells disclosed herein.

[0330] According to other specific embodiments, the cells are comprised in a heterogeneous population of cells, i.e., in a population comprising two or more cell types, of which at least 5%, at least 10%, at least 15%, at least 20%, at least 30% are cells disclosed herein.

[0331] According to certain embodiments, the isolated cells express two, three, or all of the transcription factors disclosed herein, such as GATA3+OCT4; GATA3+OCT4+c-MYC, GATA3+OCT4+KLF4 (e.g., GATA3+OCT4+KLF4, GATA3+OCT4+KLF5, GATA3+OCT4+KLF4+KLF5), GATA3+OCT4+c-MYC+KLF (e.g., GATA3+OCT4+c-MYC+KLF4, GATA3+OCT4+c-MYC+KLF5, GATA3+OCT4+c-MYC+KLF4+KLF5).

[0332] According to certain embodiments, the isolated cells express GATA3, OCT4, c-MYC, and KLF4.

[0333] According to certain embodiments, the isolated cells express GATA3, OCT4, c-MYC, KLF4, and KLF5.

[0334] According to certain embodiments, the isolated cells express a DNA molecule encoding a transcription factor as disclosed herein.Methods for assessing the presence of an exogenous DNA molecule are known in the art and include, but are not limited to, DNA sequencing, Southern blot, FISH, and PCR.

[0335] According to certain embodiments, the isolated cells contain an RNA molecule encoding a transcription factor as disclosed herein. Methods for assessing the presence of exogenous RNA are known in the art and include, but are not limited to, RNA sequencing, Northern blot, and PCR.

[0336] According to certain embodiments, the isolated cells contain protein molecules of the transcription factors disclosed herein. Methods for assessing the presence of exogenous protein molecules are known in the art and include, but are not limited to, Western blot, immunoprecipitation, immunocytochemistry, and flow cytometry.

[0337] According to certain embodiments, the isolated cells are de-differentiated from somatic cells, and such cells may sometimes still contain markers of origin, i.e., markers of the somatic cell of origin.

[0338] According to certain embodiments, once obtained, the cells are cultured in culture medium and serially passaged.

[0339] Thus, according to one aspect of the invention, there is provided a cell culture comprising the isolated cells of some embodiments of the invention and a culture medium.

[0340] According to one aspect of the invention, a cell culture is provided comprising isolated iTSCs and a culture medium.

[0341] According to one aspect of the invention, a cell culture is provided comprising isolated rejuvenated cells and a culture medium.

[0342] According to one aspect of the invention, a cell culture is provided comprising isolated dedifferentiated cells and a culture medium.

[0343] According to certain embodiments, the culture comprises a feeder cell layer, such as, but not limited to, mouse embryonic feeder (MEF) cells, human embryonic fibroblasts, or adult fallopian tube epithelial cells, and a human foreskin feeder layer. Typically, the feeder cell layer secretes factors necessary for the proliferation of stem cells while at the same time inhibiting the differentiation of stem cells.

[0344] In some embodiments, the cell cultures can be maintained in vitro under culture conditions that allow the cells to be passaged for extended periods of time (e.g., at least 20 passages, e.g., at least 30, 40, 50, 60, 70, 80, 90, 100 passages, or more), while maintaining the level of differentiation of the cells (i.e., the undifferentiated TSC state of the cells).

[0345] It should be noted that culturing cells (e.g., iTSCs) includes replacing the culture medium with "fresh" medium (of the same composition) every 24-72 hours and passaging each culture dish (e.g., plate) 1-3 times a week. That is, when the cells in culture reach about 60-90% confluence, the supernatant is discarded, the culture dish is washed [e.g., with phosphate buffered saline (PBS)], and the cells are subjected to enzymatic dissociation from the culture dish, e.g., using trypsinization (0.25% or 0.05% Trysin + EDTA or TrypLE® Select Enzyme Gibco), e.g., until single cells or cell clumps are separated from each other.

[0346] It should be noted that the culture conditions of some embodiments allow for the maintenance of iTSCs in their undifferentiated state without the need for further exogenous expression of transcription factors.

[0347] According to certain embodiments, the method includes assaying the generation of iTSCs, rejuvenation, or dedifferentiation.

[0348] Non-limiting examples of assays that can be used to evaluate iTSCs are described in detail above and below, as well as in the Examples section below.

[0349] According to certain embodiments, during the culturing process, the cells are further monitored for their differentiation state. Differentiation or dedifferentiation of cells can be determined by evaluating cell morphology or by examining cell or tissue specific markers, which are known to be indicative of differentiation. For example, undifferentiated human iTSCs may express TSC specific markers KRT7, GATA2, GATA3, TFAP2A, TFAP2C, TP63. In contrast, differentiated cells express other specific markers, such as fibroblast specific markers including THY1, ZEB1, VIM, ACTA2, and cardiomyocyte specific markers including Troponin 2.

[0350] Tissue / cell specific markers can be detected using immunological techniques well known in the art [Thomson JA et al. (1998). Science 282: 1145-7]. Examples include, but are not limited to, flow cytometry for membrane-bound and also intracellular markers, immunohistochemistry for extracellular and intracellular markers, and enzyme immunoassays for secreted molecular markers.

[0351] Useful methods for monitoring the expression levels of particular genes are well known in the art and include RT-PCR, semi-quantitative RT-PCR, Northern blot, RNA in situ hybridization, Western blot analysis, and immunohistochemistry.

[0352] Determining the undifferentiated or dedifferentiated state can also be accomplished by assessing the differentiation potential of cells both in vitro and in vivo.

[0353] For example, determination of the undifferentiated state of iTSCs can be accomplished by growing the cells in a specified differentiation culture medium and assessing their differentiation potential both in vitro and in vivo by methods well known in the art, such as, but not limited to, formation of trophoblast hemorrhagic lesions, localization to the extraembryonic region of the blastocyst, or localization to the placenta of the developing embryo.

[0354] In addition to monitoring the differentiation state, cells are often monitored for genomic stability, transcriptome, and / or methylation patterns by methods well known in the art and compared to corresponding species.

[0355] Non-limiting examples of assays that can be used to assess rejuvenation are described in detail above and below.

[0356] For example, cell identity is assessed by morphology, immunohistochemistry, transcriptome analysis (RNA-seq), etc., while rejuvenation is assessed by DNA methylation clock, telomere length, histone marks, mitochondrial activity, gene expression, and functional assays, e.g., using bisulfite sequencing.

[0357] The functionality of the rejuvenated cells may also be evaluated. Non-limiting examples of functional assays that can be accomplished in connection with the rejuvenation aspects disclosed herein include mitochondrial activity using MitoSOX reagents and / or Seahorse XF analyzer; DNA damage response using quantification of γH2A.X foci radix and staining for DNA damage biomarkers ATM, 53BP1, RAD51; and / or senescence by β-gal staining. Rejuvenated mesenchymal cells may be further evaluated by wound healing assays using IncuCyte® S3 and transwell migration. Rejuvenated MSCs may be further evaluated for improved immunosuppression by co-culturing the cells with peripheral blood mononuclear cells (PBMCs) and examining their proliferation rate. With regard to CD34+ stem cells, umbilical cord blood contains approximately 50% B cells and 20% myeloid cells, while adult blood contains more than 50% myeloid cells and approximately 10-15% B cells. Therefore, one way to demonstrate cell rejuvenation of CD34+ cells is to obtain more than 20% B cells after rejuvenation, as opposed to control cells that should show about 10% B cells. Additionally or alternatively, the presence of CD5+ cells is evaluated to explore the possibility of generating B1 cells, which are most restricted to the youngest (fetal liver) HSCs, as opposed to "adult" B2 cells, which are CD5- cells. The functionality of rejuvenated CD34+ cells may be further evaluated in vivo by transplantation into NSG mice. Furthermore, the tumorigenic potential of rejuvenated cells may be evaluated by subcutaneous transplantation into NOD / SCID mice.

[0358] As used herein, the phrase "culture medium" refers to a solid or liquid substance used to support the growth of cells. According to certain embodiments, the culture medium is a liquid medium.

[0359] According to certain embodiments, the culture medium comprises a composition of components that have been shown to support the culture of human TSCs, as further described herein.

[0360] According to certain embodiments, the culture medium is capable of maintaining the iTSCs in their differentiated state (ie, undifferentiated state).

[0361] According to certain embodiments, the culture medium is capable of maintaining iTSCs at their level of differentiation for at least 20 passages, such as at least about 30, 40, 50, 60, 70, 80, 90, 100 passages or more.

[0362] According to certain embodiments, the culture medium is capable of maintaining iTSCs at their differentiation level for at least 20 passages.

[0363] The culture medium used by some embodiments of the present invention may be an aqueous medium containing a combination of substances such as salts, nutrients, minerals, vitamins, amino acids, nucleic acids, proteins, such as cytokines, growth factors, and hormones, all of which are necessary for cell proliferation and capable of maintaining stem cells in an undifferentiated state. For example, the culture medium may be a synthetic tissue culture medium such as RPMI (a product of Gibco-Invitrogen Corporation, Grand Island, NY, USA), Ko-DMEM (a product of Gibco-Invitrogen Corporation, Grand Island, NY, USA), DMEM / F12 (a product of Gibco-Invitrogen Corporation, Grand Island, NY, USA), or DMEM / F12 (Biological Industries, Biet Haemek, Israel), supplemented with necessary additives as further described hereinafter. Preferably, all components contained in the culture medium are substantially pure and tissue culture grade.

[0364] According to a particular embodiment, the culture medium is DMEM / F12.

[0365] It will be appreciated that any of the proteinaceous factors used in the culture media of some embodiments of the present invention can be recombinantly expressed or biochemically synthesized. Furthermore, naturally occurring proteinaceous factors can be purified from biological samples (e.g., from human serum, cell cultures) using methods well known in the art.

[0366] According to certain embodiments, the culture medium comprises conditioned medium, which is the growth medium of a monolayer of cell cultures (i.e., feeder cells) that exists after a certain culture period. The conditioned medium comprises growth factors and cytokines secreted by the monolayer cells in culture.

[0367] According to certain embodiments, the culture medium is devoid of conditioned medium.

[0368] According to some embodiments of the invention, the culture medium lacks serum, for example lacks any animal serum.

[0369] According to some embodiments of the invention, the culture medium is devoid of any animal contaminants, i.e. animal cells, body fluids, or pathogens (e.g. viruses that infect animal cells), e.g. xenobiotic-free.

[0370] According to some embodiments of the invention, the culture medium is devoid of human-derived serum.

[0371] According to some embodiments of the invention, the culture medium further comprises a serum replacer such as, but not limited to, KNOCKOUT™ Serum Replacement (Gibco-Invitrogen Corporation, Grand Island, NY USA), ALBUMAX® II (Gibco®; Life Technologies - Invitrogen, Cat. No. 11021-029; lipid-enriched bovine serum albumin for cell culture), or a chemically defined lipid concentrate (Gibco®; Invitrogen, Life Technologies - Invitrogen, Cat. No. 11905-031).

[0372] According to certain embodiments, the culture medium lacks a serum substitute.

[0373] According to some embodiments of the present invention, the culture medium may further comprise an antibiotic (e.g., PEN-STREP), L-glutamine, and NEAA (non-essential amino acids).

[0374] According to certain embodiments, the medium comprises 2-mercaptoethanol, FBS, penicillin-streptomycin, BSA, ITS supplement, L-ascorbic acid, EGF, CHIR99021, A83-01, SB431542, and / or VPA or TSA and Y27632.

[0375] According to certain embodiments, the medium comprises 0.1 mM 2-mercaptoethanol, 0.2% FBS, 0.5% penicillin-streptomycin, 0.3% BSA, 1% ITS supplement, 1.5 μg / ml L-aspartic acid, 50 ng / ml EGF, 2 μM CHIR99021, 0.5 μM A83-01, 1 μM SB431542, 0.8 mM VPA or 10 nM TSA, and 5 μM Y27632 as described in Okae et al., Cell Stem Cell. (2018) Jan 4;22(1):50-63.

[0376] In addition to primary cultures, the isolated cells, iTSCs, rejuvenated cells, and / or dedifferentiated cells disclosed herein can be used to generate cell lines, iTSC lines, rejuvenated cell lines, or dedifferentiated cell lines, which can be expanded indefinitely in culture.

[0377] Cell lines of some embodiments of the invention can be generated by immortalizing isolated cells, iTSCs, rejuvenated cells, and / or dedifferentiated cells by methods known in the art, including, for example, expressing the telomerase gene in the cells (Wei, W. et al., 2003. Mol Cell Biol. 23: 2859-2870) or co-culturing the cells with NIH 3T3 hph-HOX11 retroviral producer cells (Hawley, RG et al., 1994. Oncogene 9: 1-12).

[0378] According to an aspect of some embodiments of the present invention, there is provided a method of generating differentiated cells, comprising subjecting iTSCs or de-differentiated cells of some embodiments of the present invention to differentiation conditions, thereby generating differentiated cells. Methods of differentiating iTSCs into specific cell types are known in the art, and the present invention contemplates all such methods, for example as disclosed in Okae et al., Cell Stem Cell. 2018 Jan 4;22(1):50-63 and Haider et al., Stem Cell Reports. 2018 Aug 14;11(2):537-551, the contents of which are fully incorporated herein by reference, including culturing the cells in a medium lacking factors that support an undifferentiated state, such as when culturing in DMEM medium containing 10% FBS or in a medium that promotes directed differentiation. The method may include genetic modification of the cells and / or culturing the cells in a medium containing differentiation factors. It will be appreciated that the re-differentiating step may result in the generation of fully differentiated cells or partially differentiated cells along a particular lineage.

[0379] According to certain embodiments of the invention, the iTSCs of some embodiments of the invention can be used to isolate lineage-specific cells.

[0380] As used herein, the phrase "isolated lineage-specific cells" refers to enrichment of a mixed population of cells in culture with cells that exhibit at least one characteristic primarily associated with a particular lineage phenotype. That is, for example, iTSCs can differentiate into any of the trophoblast cell lineages. Lineage-specific cells can be obtained by directly inducing expanded undifferentiated iTSCs into culture conditions suitable for differentiation of a particular cell lineage, by methods well known in the art. It will be understood that culture conditions suitable for differentiation and proliferation of isolated lineage-specific cells include a variety of tissue culture media, growth factors, antibiotics, amino acids, etc., and that it is within the capabilities of one of ordinary skill in the art to determine which conditions should be applied to proliferate and differentiate a particular cell type and / or cell lineage.

[0381] The present invention, according to some embodiments, contemplates using cells, tissues, and organs generated from the iTSCs disclosed herein using any differentiation protocol known in the art.

[0382] The isolated cells and constructs disclosed herein may be further used, for example, for disease modeling, drug screening, and patient-specific cell-based therapies.

[0383] Thus, according to one aspect of the present invention, there are provided isolated aggregates, organoids, placentas, developing embryos, and synthetic embryos comprising the iTSCs, constructs, or protein preparations disclosed herein.

[0384] According to another aspect of the invention, there is provided a method of enhancing a placenta, developing embryo, or synthetic embryo, comprising the step of introducing an iTSC, construct, or protein preparation disclosed herein into the placenta, developing embryo, or synthetic embryo.

[0385] As used herein, the term "developing embryo" refers to an embryo at any stage of development, including an embryo at the 4-cell stage, 8-cell stage, 16-cell stage, early morula, late morula, early blastocyst, and / or late blastocyst.

[0386] Methods for administering cells to the placenta of a developing animal embryo in vitro or in vivo are well known in the art, e.g., Gafni O et al., Nature. 2013 Dec 12;504(7479):282-6; and Manipulating the Mouse Embryo: A Laboratory Manual, 4th Edition, Richard Behringer; Marina Gertsenstein; Kristina Vintersten Nagy; Andras Nagy, each of which is fully incorporated herein by reference, and are also disclosed in the Materials and Methods section of the Examples below.

[0387] According to some embodiments of the invention, introduction into cells is performed in vitro or ex vivo by direct injection or association with the placenta or embryo of a developing host.

[0388] According to another aspect of the invention, there is provided a method for generating aggregates or organoids comprising trophoblast cells, the method comprising introducing iTSCs, constructs, or protein preparations disclosed herein into a scaffold or matrix.

[0389] The iTSCs and iTSC-derived cell preparations and chimeric placentas can be used to prepare model systems for disorders associated with trophoblast cell development and / or activity, to screen for genes expressed in or essential for trophoblast cell differentiation and / or activity, to screen for agents or conditions (e.g., culture conditions or manipulations) that effect trophoblast cell differentiation and / or activity, to produce trophoblast cell-specific growth factors and hormones, and as cell therapy for disorders associated with trophoblast cell development and / or activity.

[0390] Thus, the cell preparations and chimeric placentas may be used to screen for agents that may modulate trophoblast cell development or activity, such as invasion or proliferation.

[0391] Thus, according to one aspect of the present invention there is provided a method for identifying an agent capable of modulating trophoblast cell development and / or activity, the method comprising: (i) contacting an isolated iTSC, a population of cells comprising iTSC, an aggregate, an organoid, or a placenta as disclosed herein with a candidate agent; and (ii) comparing the development and / or activity of the isolated iTSCs, cell populations, aggregates, organoids, or placenta following said contact with the agent to the development and / or activity of the isolated iTSCs, cell populations, aggregates, organoids, or placenta in the absence of the agent. Including, An effect of the agent on the development and / or activity of the isolated iTSCs, cell population, aggregate, organoid, or placenta that is greater than a predetermined level compared to the development and / or activity of the isolated iTSCs, cell population, aggregate, organoid, or placenta in the absence of the agent indicates that the drug modulates the development and / or activity of trophoblast cells.

[0392] As used herein, the term "modulating" means to alter the development and / or activity of trophoblast cells by inhibiting or promoting.

[0393] According to certain embodiments, modulating is inhibiting development and / or activity.

[0394] According to certain embodiments, modulating is promoting development and / or activity.

[0395] For the same culture conditions, the effect of a candidate agent on the development and / or activity of trophoblast cells is generally expressed relative to the development and / or activity in cells of the same species but not contacted with the candidate agent or with a vehicle control, also referred to as the control.

[0396] As used herein, the phrase "effect exceeding a predetermined threshold" refers to a change in trophoblast cell development and / or activity following contact with a compound that is, for example, about 10% higher, for example, about 20% higher, for example, about 30% higher, for example, about 40% higher, for example, about 50% higher, for example, about 60% higher, about 70% higher, about 80% higher, about 90% higher, about 2-fold higher, about 3-fold higher, about 4-fold higher, about 5-fold higher, about 6-fold higher, about 7-fold higher, about 8-fold higher, about 9-fold higher, about 20-fold higher, about 50-fold higher, about 100-fold higher, about 200-fold higher, about 350-fold higher, about 500-fold higher, about 1000-fold higher, or more than a predetermined threshold, relative to the level of expression prior to contact with the compound.

[0397] According to certain embodiments, the candidate agent may be any compound, including but not limited to, chemical compounds, small molecules, polypeptides, and polynucleotides.

[0398] Cell preparations, aggregates, organoids, and placentas can also be used to identify genes and substances important for trophoblast cell development and / or activity.Isolated iTSCs can also be modified by introducing mutations into genes in the cells or by introducing transgenes into the cells.

[0399] According to certain embodiments, selected agents may further be used to treat a variety of conditions requiring regulation of trophoblast cell development or activity, such as those conditions described below.

[0400] Recurrent miscarriage and fetal growth restriction (FGR) are associated with placental insufficiency, causing disability and in severe cases death. Cell transplantation of intact and healthy TSCs holds great promise for clinical applications, as the transplanted cells may be able to rescue some of these fetuses by supporting the non-developing / damaged placenta.

[0401] Thus, according to another aspect of the present invention, there is provided a method for treating and / or preventing a disorder associated with trophoblast cell development and / or activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an iTSC, construct, or protein preparation disclosed herein, thereby treating and / or preventing a disorder associated with trophoblast cell development and / or activity in the subject.

[0402] According to further or alternative aspects of the present invention, there is provided an iTSC, construct or protein preparation as disclosed herein for use in the treatment and / or prevention of a disorder associated with trophoblast cell development and / or activity in a subject in need thereof.

[0403] This aspect of the invention is intended to treat disorders related to the development and / or activity of trophoblast cells. Malfunctioning trophoblast cells can affect the mother on the one hand and the fetus on the other hand. Thus, both conditions are contemplated. Non-limiting examples of such disorders include recurrent miscarriage, pre-eclampsia, fetal growth restriction (FGR), hydatidiform mole, and choriomas.

[0404] The term "treating" or "treatment" refers to inhibiting or preventing the progression of a lesion (e.g., recurrent miscarriage) and / or causing the reduction, remission, or regression of a lesion. Those skilled in the art will appreciate that a variety of methodologies and assays can be used to assess the progression of a lesion, and similarly a variety of methodologies and assays can be used to assess the reduction, remission, or regression of a lesion.

[0405] As used herein, the term "preventing" means preventing a disease (or pathology) from occurring in a subject who may be at risk for the disease but has not yet been diagnosed as having the disease.

[0406] As used herein, the phrase "subject in need thereof" refers to a mammalian subject (e.g., a human) who has been diagnosed with a pathology. In certain embodiments, the term encompasses individuals who are at risk for developing a pathology. Veterinary applications are also contemplated. The subject may be of any gender or age, including neonates, infants, juveniles, adolescents, adults, and older adults. According to certain embodiments, the subject is female.

[0407] According to certain embodiments, the subject is at least 20 years of age.

[0408] According to certain embodiments, the subject is at least 40 years of age.

[0409] According to certain embodiments, the subject is at least 50 years of age.

[0410] According to certain embodiments, the subject is at least 60 years of age.

[0411] According to certain embodiments, the subject is at least 70 years of age.

[0412] Since trophoblast cells produce several secreted growth factors and hormones, according to another aspect of the present invention, there is provided a method for obtaining a compound produced by trophoblast cells, the method comprising culturing an isolated iTSC, a population of cells comprising iTSC, or an iTSC cell culture as disclosed herein, and isolating a compound secreted by the cells from the culture medium, thereby obtaining the compound produced by the trophoblast cells.

[0413] In certain embodiments, the compound is a growth factor or hormone, including but not limited to, human chorionic gonadotropin (hCG).

[0414] According to a further or alternative aspect of the present invention, there is provided a method of treating and / or preventing an aging-associated disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a rejuvenated cell, dedifferentiated cell, construct or protein preparation disclosed herein, thereby treating and / or preventing the disease in the subject.

[0415] According to further or alternative aspects of the present invention, there is provided a rejuvenated cell, dedifferentiated cell, construct or protein preparation as disclosed herein for use in the treatment and / or prevention of an age-related disease in a subject in need thereof.

[0416] This aspect of the invention is intended to treat diseases associated with aging, non-limiting examples of which include glaucoma, cataracts, high myopia, retinitis pigmentosa, cone dystrophy, cone-rod dystrophy, Usher syndrome, Stargardt's disease, Bardet-Biedl syndrome, Best disease, hereditary macular degeneration, myelodysplastic syndromes (MDS), cancer, graft rejection, graft versus host disease (GVHD), infectious diseases, cytokine storm, radiation injury, neurodegenerative diseases, and wounds.

[0417] According to certain embodiments, the aging-related disease results from increased aging.

[0418] According to certain embodiments, the disease is a vision-related disease.

[0419] According to certain embodiments, the disease is selected from the group consisting of glaucoma, cataracts, high myopia, retinitis pigmentosa, cone dystrophy, cone-rod dystrophy, Usher syndrome, Stargardt's disease, Bardet-Biedl syndrome, Best disease, and hereditary macular degeneration.

[0420] According to certain embodiments, the disease is selected from the group consisting of myelodysplastic syndromes (MDS), cancer, graft rejection, graft versus host disease (GVHD), infectious diseases, cytokine storm, radiation injury, neurodegenerative diseases, and wounds.

[0421] Since the constructs and protein formulations disclosed herein induce cellular rejuvenation and dedifferentiation, the inventors contemplate another use thereof as an anti-aging agent, e.g., in cosmetic compositions for skin rejuvenation.

[0422] Thus, according to a further or alternative aspect of the present invention, there is provided a method of performing cosmetic care in a subject in need thereof, the method comprising the step of applying a therapeutically effective amount of a construct or protein formulation disclosed herein to the skin of the subject, thereby performing cosmetic care.

[0423] The cells, constructs, and protein preparations disclosed herein may be implanted into a subject by themselves, or may be formulated into compositions intended for a particular use. Similarly, the constructs and protein preparations disclosed herein may be administered to a subject by themselves, or may be formulated into compositions intended for a particular use.

[0424] For the treatment of disease, the cell, construct or protein preparations disclosed herein may be formulated into pharmaceutical compositions in which they are mixed with suitable carriers or excipients.

[0425] As used herein, a "pharmaceutical composition" refers to a formulation of one or more of the active ingredients described herein with other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0426] As used herein, the term "active ingredient" refers to a cell (e.g., iTSC, rejuvenated cell, dedifferentiated cell), construct, or protein preparation disclosed herein that is recognized to be responsible for a biological effect.

[0427] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutical acceptable carrier" are used interchangeably to mean a carrier or diluent that does not cause significant irritation to a living organism and does not abolish the biological activity and properties of the administered compound. As used herein, the term "excipient" means an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples of excipients include, without limitation, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0428] Techniques for formulating and administering drugs can be found in the latest edition of "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., which is incorporated herein by reference.

[0429] Pharmaceutical compositions of some embodiments of the present invention can be manufactured by processes well known in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, elutriating, emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0430] Thus, pharmaceutical compositions for use according to some embodiments of the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and adjuvants that facilitate processing of the active ingredients into a formulation and that are pharma- ceutically acceptable. The proper formulation will depend on the chosen route of administration.

[0431] For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.

[0432] Suitable routes of administration include, for example, oral, rectal, mucosal, particularly nasal, enteral, or intramuscular, subcutaneous, and intramedullary injection, as well as parenteral delivery, including intrathecal, directly intraventricular, e.g., into the right or left ventricle, into the common coronary arteries, intracardiac, intravenous, intraperitoneal, intranasal, or intraocular injection.

[0433] According to certain embodiments, the pharmaceutical compositions are administered locally rather than systemically, for example, by injection of the pharmaceutical composition directly into a tissue region of the patient.

[0434] The pharmaceutical compositions described herein may be formulated for parenteral administration, for example, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, for example in ampoules or multi-dose containers, with optional added preservatives. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents.

[0435] Pharmaceutical compositions for parenteral administration include aqueous solutions of active formulations in water-soluble form. Furthermore, suspensions of active ingredients may be prepared as suitable oil-based or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may contain suitable stabilizers or agents that increase the solubility of the active ingredients, allowing for the preparation of highly concentrated solutions.

[0436] Suitable pharmaceutical compositions for use in connection with the present invention include compositions in which the active ingredient is contained in an amount effective to achieve its intended purpose. More specifically, a therapeutically effective amount refers to an amount of active ingredient (e.g., iTSC) effective to prevent, reduce, or ameliorate symptoms of a disorder (e.g., recurrent miscarriage) or prolong the survival of the subject being treated.

[0437] Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0438] For any formulation used in the methods of the invention, the therapeutically effective amount or dose can be extrapolated from animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0439] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in experimental animals. Data obtained from these animal studies can be used in formulating a range of dosages for use in humans. Dosages can vary depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage can be selected by the individual clinician in view of the patient's condition (see, for example, Fingl et al., 1975, "The Pharmacological Basis of Therapeutics", Chapter 1, page 1).

[0440] Dosage amount and interval may be adjusted individually to provide a level of active ingredient sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC varies for each formulation but can be estimated from in vitro data. The dosage required to achieve the MEC depends on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations of C-peptide and / or insulin.

[0441] The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing clinician, etc.

[0442] The compositions of the invention may be presented in a pack or dispenser device, such as an FDA approved kit, containing one or more unit dosage forms containing the active ingredient, if desired. The pack may comprise, for example, metal or plastic foil, such as a blister pack. The pack or dispenser device may be a syringe. The syringe may be pre-filled with cells. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may contain notices associated with the container in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects the approval by the government agency of the form of the composition or its administration to humans or veterinarians. Such notices may be, for example, labels approved by the U.S. Food and Drug Administration for prescription drugs or approved product inserts. Compositions containing the formulations of the invention formulated in a compatible pharmaceutical carrier may be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as described in more detail above.

[0443] For cosmetic purposes, the constructs or protein preparations disclosed herein may be formulated into cosmetic compositions, which are mixed with suitable, e.g., dermatologically acceptable, carriers or excipients suitable for external topical application.

[0444] According to certain embodiments, the cosmetic composition is formulated as a cream, a facial mask, a scrub, a soap, a wash, or a gel.

[0445] Cosmetic compositions according to some embodiments of the present invention may further comprise at least one pharmaceutical adjuvant known to those skilled in the art, selected from thickeners, preservatives, fragrances, colorants, chemical or mineral filters, moisturizers, spring waters, and the like.

[0446] The composition may comprise at least one agent selected from sebum regulating agents, antibacterial agents, antifungal agents, keratolytic agents, keratoregulating agents, astringents, anti-inflammatory / anti-irritant agents, antioxidants / free radical scavengers, scarring agents, anti-aging agents, and / or moisturizing agents.

[0447] The term "sebum regulator" refers, for example, to 5-α-reductase inhibitors, notably the active agent 5-α-Avocuta® marketed by Laboratoires Expanscience. Zinc and its gluconate salts, salicylates and pyroglutamic acid also have sebum suppressing activity. Mention is also made of spironolactone, an antiandrogen and aldosterone antagonist, which significantly reduces the rate of sebum secretion after 12 weeks of application. Other molecules extracted, for example, from Cucurbita pepo seeds, pumpkin seed oil and from palm cabbage, also limit the production of sebum, by inhibiting the transcription and activity of 5-α-reductase. Other sebum regulators of lipid origin, acting on the quality of sebum, such as linoleic acid, are also of interest.

[0448] The terms "antibacterial" and "antifungal" refer to molecules that limit the growth of or kill pathogenic microorganisms, such as certain bacteria, such as P. acnes, or certain fungi, such as Malassezia furfur. The most traditional are preservatives commonly used in cosmetics or dietary supplements, molecules with antibacterial activity (pseudopreservatives), such as caprylic acid derivatives (capryloyl glycine, glyceryl caprylate, etc.), such as hexanediol and sodium levulinate, zinc and copper derivatives (gluconate and PCA), phytosphingosine and its derivatives, benzoyl peroxide, piroctone olamine, zinc pyrithione, selenium sulfide, econazole, ketoconazole, or topical antibiotics such as erythromycin and clindamycin.

[0449] The terms "keratoregulator" and "keratolytic agent" refer to agents that regulate or assist in the removal of dead cells of the stratum corneum of the epidermis. The most commonly used keratoregulators / keratolytic agents include fruit alpha hydroxy acids (AHA) (citric acid, glycolic acid, malic acid, lactic acid, etc.), AHA esters, combinations of AHA with other molecules such as malic acid with almond protein (Keratolite®), glycolic or lactic acid with arginine, or combinations of hydroxy acids with lipid molecules such as LHA® (lipo-hydroxy acid), amphoteric hydroxy acid complexes (AHCare), willow bark (Salix alba bark extract), azelaic acid and its salts and esters, salicylic acid and its derivatives such as capryloyl salicylic acid, or combinations with other molecules such as salicylic acid with polysaccharides (beta hydroxy acids or BHA), tazarotene, adapalene, and molecules of the retinoid family such as tretinoin, retinaldehyde, isotretinoin, and retinol.

[0450] The term "astringent" refers to agents that help tighten pores, the most commonly used being polyphenols, zinc derivatives, and witch hazel.

[0451] The term "anti-inflammatory / anti-irritant" refers to an agent that suppresses the inflammatory response caused by cytokines or arachidonic acid metabolic mediators and has soothing and anti-irritant properties. The most traditional are: glycyrrhetinic acid (licorice derivative) and its salts and esters, alpha-bisabolol, ginkgo biloba, calendula, lipoic acid, beta-carotene, vitamin B3 (niacinamide, nicotinamide), vitamin E, vitamin C, vitamin B12, flavonoids (green tea, quercetin, etc.), lycopene or lutein, avocado sugar, avocado oil distillate, arabinogalactan, lupin peptides, total lupin extract, quinoa peptide extract, Cycloceramide® (oxazoline derivative), anti-glycation agents such as carnosine, N-acetylcysteine, isoflavones such as genistein / genistin, daidzein / daidzin, spring or thermal waters (Eau d'Abène, Eau de la Roche-Possé, Eau de Saint-Gervais, Eau du Liage, Eau de Gamalde), wolfberry extract (Lycium barbarum), plant amino acid peptides or complexes, topical dapsone, or anti-inflammatory drugs.

[0452] The term "antioxidant" refers to a molecule that reduces or prevents the oxidation of other chemicals. Antioxidants / free radical scavengers to be used in combination are advantageously thiols and phenols, licorice derivatives such as glycyrrhetinic acid and its salts and esters, alpha-bisabolol, Ginkgo biloba extract, calendula extract, Cycloceramide® (oxazoline derivative), avocado peptides, trace elements such as copper, zinc and selenium, lipoic acid, vitamin B12, vitamin B3 (niacinamide, nicotinamide), vitamin C, vitamin E, coenzyme Q10, krill, glutathione, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), lycopene or lutein, beta-carotene, the family of polyphenols such as tannins, phenolic acids, anthocyanins, flavonoids such as those from green tea, red berries, cocoa, grapes, Passiflora incarnata, or citrus extracts, or isoflavones, such as genistein / genistin, and daidzein / daidzin. The group of antioxidants further includes anti-glycation agents such as carnosine or certain peptides, N-acetylcysteine, and antioxidants or free radical scavenging enzymes such as superoxide dismutase (SOD), catalase, glutathione peroxidase, thioredoxin reductase, and their agonists.

[0453] The agents which may be used in combination to form scars and restore the barrier function are advantageously vitamin A, panthenol (vitamin B5), Avocado Furan®, avocado sugar, lupeol, maca peptide extract, quinoa peptide extract, arabinogalactan, zinc oxide, magnesium, silicon, madecassic or asiatic acid, dextran sulfate, coenzyme Q10, glucosamine and its derivatives, chondroitin sulfate, and glycosaminoglycans (GAGs) as a whole, dextran sulfate, ceramides, cholesterol, squalane, phospholipids, marine, vegetable or biotechnological polysaccharides such as fermented or unfermented soy peptides, plant peptides, algae extracts or fern extracts, trace elements, extracts of plants rich in tannins such as tannins derived from gallic acid, called gallic, first discovered in oak galls, or hydrolysable tannins, as well as the extracts of plants whose model is the catechu (Acacia The catechin tannins result from the polymerization of flavan units provided by the catechu. The trace elements used are advantageously selected from the group consisting of copper, magnesium, manganese, chromium, selenium, silicon, zinc, and mixtures thereof.

[0454] Anti-aging agents that may act in combination with the constructs and protein formulations disclosed herein include antioxidants, particularly vitamin C, vitamin A, retinol, retinal, hyaluronic acid of any molecular weight, Avocado Furan®, lupin peptides, and maca peptide extracts.

[0455] The most commonly used moisturizers / emollients are glycerin or its derivatives, urea, pyrrolidone carboxylic acid and its derivatives, hyaluronic acid of any molecular weight, glycosaminoglycans and any other polysaccharides of marine, vegetable or biotechnological origin, such as xantham gum, Fucogel®, certain fatty acids, such as lauric acid, myristic acid, mono- and poly-saturated omega-3, -6, -7 and -9 fatty acids (linoleic acid, palmitoleic acid, etc.), sunflower oil distillates, avocado peptides, and cupuaçu butter.

[0456] As used herein, the term "about" means ±10%.

[0457] The terms "comprises," "comprising," "includes," "including," "having" and their conjugations mean "including but not limited to."

[0458] The term "consisting of" means "including and limited to."

[0459] The term "consisting essentially of" means that the composition, method, or structure may include additional components, steps, and / or moieties, but only if the additional components, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0460] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include multiple compounds, including mixtures thereof.

[0461] Throughout this application, various embodiments of the present invention are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as inflexibly limiting the scope of the present invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0462] Whenever a numerical range is given herein, it is meant that any recited numerical values ​​(fractional or integer) are included within the indicated range. The phrases "ranging 'between' a first indicated number and a second indicated number" and "ranging 'from' a first indicated number to a second indicated number" are used interchangeably herein and are meant to include the first and second indicated numbers and all fractions and integers therebetween.

[0463] As used herein, the term "method" means manners, means, techniques, and procedures for accomplishing a given task, including but not limited to manners, means, techniques, and procedures known or readily developed from known manners, means, techniques, and procedures to practitioners of the chemical, pharmacological, biological, biochemical, and medical arts.

[0464] When referring to a particular sequence listing, it should be understood that such reference also encompasses sequences that substantially correspond to their complementary sequences, including minor sequence variations due to, for example, sequencing errors, cloning errors, or other changes resulting in base substitutions, deletions, or additions, provided that the frequency of variation is less than 1 in 50 nucleotides, alternatively less than 1 in 100 nucleotides, alternatively less than 1 in 200 nucleotides, alternatively less than 1 in 500 nucleotides, alternatively less than 1 in 1000 nucleotides, alternatively less than 1 in 5,000 nucleotides, alternatively less than 1 in 10,000 nucleotides.

[0465] It will be appreciated that certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination, or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be regarded as essential elements of those embodiments, unless the embodiment is inoperable without those elements.

[0466] Various embodiments and aspects of the present invention as described hereinabove and as claimed in the claims section below are found to be experimentally supported in the following examples. EXAMPLES

[0467] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting manner.

[0468] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques, which are fully explained in the literature.See, e.g., Molecular Cloning: A laboratory Manual, Sambrook et al. (1989); Current Protocols in Molecular Biology, vols. I-III, Ausubel, RM ed. (1994); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989); Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988); Watson et al., Recombinant DNA, Scientific American Books, New York; Birren et al. (eds.), Genome Analysis: A Laboratory Manual Series, vols. 1-4, Cold Spring Harbor Laboratory Press, New York. New York (1998); the methodologies described in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659, and 5,272,057; Cell Biology: A Laboratory Handbook, volumes I-III, ed. Cellis, JE (1994); Culture of Animal Cells - A Manual of Basic Technique, 3rd Edition, by Freshney, Wiley-Liss, NY (1994); Current Protocols in Immunology, volumes I-III, ed. Coligan JE (1994); Stites et al. (eds.), Basic and Clinical Immunology, 8th Edition, Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds.), Selected Methods in Cellular Immunology, WH Freeman and Co., New York (1980). Available immunoassays have been described extensively in the patent and scientific literature.For example, U.S. Patent Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; No. 5,074; No. 3,984,533; No. 3,996,345; No. 4,034,074; No. 4,098,876; No. 4,879,219; No. 5,011,771 and No. 5,281,521; "Synthesis" Gait, MJ (1984); "Nucleic Acid Hybridization" Hames, BD, and Higgins SJ (1985); "Transcription and See, "Animal Cell Culture," edited by Hames, BD, and Higgins SJ (1984); "Animal Cell Culture," edited by Freshney, RI (1986); "Immobilized Cells and Enzymes," IRL Press, (1986); "A Practical Guide to Molecular Cloning," Perbal, B., (1984) and "Methods in Enzymology," volumes 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications," Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual," CSHL Press (1996), all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All information contained therein is incorporated by reference herein.

[0469] Materials and Methods Derivation of human trophoblast stem cells from human blastocysts - To generate human blastocyst-derived TSC (hbdTSC) control lines, human blastocysts were seeded onto mitomycin C-treated MEF feeders and cultured in human TSC medium as described by Okae et al. [Cell stem cell (2018) 22, pp. 50-63 e56]. After blastocyst expansion, cells were trypsinized and transferred to new mitomycin C-treated mouse embryonic fibroblast (MEF) feeder plates. Cells were passaged several times until stable proliferative hbdTSCs emerged.

[0470] Molecular cloning and reprogramming of hiTSCs and hiPSCs - dox-inducible factors were generated by cloning the open reading frames of the respective factors obtained by reverse transcription with specific primers (see primer list in Table 1 below) into pMINI vector (NEB), then restricted with EcoRI or MfeI and inserted into FUW-TetO expression vector. For transient expression of transcription factors, the dox-dependent system of lentiviral vectors was utilized. KLF5 coding sequence was synthesized by TWIST and subcloned into FUW-TetO by EcoRI. For infection, different reprogramming factors and ratios (GOKM 2:3:3:2 or 1:1:1:0.3 for reprogramming of hiTSCs, GOK 4 K 5Replication-incompetent lentiviruses containing psPAX2 and pDGM.M (1:1:1:1:0.3) for reprogramming of hiPSCs and OKSM STEMCCA cassette) were packaged with lentiviral packaging mix (7.5 μg of psPAX2 and 2.5 μg of pDGM.2) into 293T cells and harvested 48, 60, 72, and 84 hours after transfection. The supernatant was filtered through a 0.45 μm filter, supplemented with 8 μg / ml of polybrene, and then used to infect human foreskin fibroblasts (HFFs). 12 hours after the fourth infection, the medium was replaced with fresh DMEM containing 10% FBS. For reprogramming of hiTSCs, 2 μg / ml of doxycycline was added to the medium after 6 hours. For reprogramming of hiTSCs, basic reprogramming medium (BRM) consisting of DMEM supplemented with 10% FBS was replaced every other day for 14 days, followed by 7 days in medium consisting of 50% BRM and 50% hTSC medium as described in Okae et al., 2018, followed by 7 days in hTSC medium as described in Okae et al., 2018, after which dox was removed. 7–10 days after dox removal, plates were screened for primary hiTSC colonies. Individual colonies were isolated, trypsinized by TrypLE (Gibco), and seeded on feeder cells in separate wells of a 6-well plate. Cells were passaged several times until stable, proliferative hiTSC colonies emerged.

[0471] Quantitative PCR (qPCR) for mRNA expression and analysis of genomic integration of transgenes - For analysis of mRNA expression using qPCR, total RNA was isolated using the Macherey-Nagel kit (Ornat). 500-2000 ng of total RNA was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad). Quantitative PCR analysis was performed in duplicate with SYBR green Fast qPCR Mix (Applied Biosystems) using 1 / 100 of the reverse transcription reaction in StepOnePlus (Applied Biosystems). Specific primers were designed for the different genes (see Table 1 below). All quantitative real-time PCR experiments were normalized to the expression of GAPDH and expressed as the mean ± standard deviation of two duplicates.

[0472] For analysis of transgene integration into genomic DNA using qPCR, genomic DNA was isolated by incubating trypsinized cell pellets overnight with 400 μg / ml proteinase K (Axxora) in lysis buffer containing 100 mM Tris (pH 8.0), 5 mM EDTA, 0.2% SDS, and 200 mM NaCl at 37° C. for 1 hour, followed by incubation at 55° C. for 1 hour. Genomic DNA was then precipitated with isopropanol, washed with 70% ethanol, and resuspended in ultrapure water (BI). A forward primer for the end of the last exon of the cloned gene was used in conjunction with a reverse primer for the FUW vector in the region immediately downstream of the cloned gene (see Table 1 below). Results were normalized to the intronic region of the GAPDH gene and expressed as the mean ± standard deviation of two duplicate determinations.

[0473] Immunostaining and flow cytometry of PFA-fixed cells - Cells were fixed with 4% paraformaldehyde (in PBS) for 20 min, rinsed three times with PBS, and blocked for 1 h with PBS containing 0.1% Triton X-100 and 5% FBS. Cells were incubated with primary antibodies (1:200) overnight at 4°C. The antibodies used were anti-KRT7 (Abcam, ab215855), anti-GATA3 (Abcam, ab106625), anti-GATA2 (Abcam, ab173817), anti-TFAP2C (Santa Cruz Biotechnologies, sc-8977), anti-KRT18 (Santa Cruz Biotechnologies, sc-51582), anti-E-cadherin (Santa Cruz Biotechnologies, sc-7870), and anti-vimentin (Cell Signaling, Inc., San Jose, CA) diluted in PBS containing 0.1% Triton X-100 and 1% FBS. The antibodies were anti-EpCAM (Abcam, ab71916), anti-SDC1 (Abcam, ab128936), anti-CSH1 (Abcam, ab15554), and anti-HLA-G (Abcam, ab52455). The following day, cells were washed three times and incubated for 1 hour with the relevant (Alexa) secondary antibody (1:500 dilution) in PBS containing 0.1% Triton X-100 and 1% FBS. DAPI was added 10 minutes before the end of incubation. Negative controls included incubation with secondary antibody without the primary antibody.

[0474] For flow cytometric analysis of HLA class I expression, cells were trypsinized and blocked for 10 min in incubation buffer containing 0.5% bovine serum albumin (BSA) (Sigma Aldrich) in PBS. Cells were then centrifuged and resuspended in incubation buffer with anti-HLA class I (Abcam, ab22432) (1:100) for 1 h. Cells were then washed with incubation buffer and incubated with the relevant (Alexa) secondary antibody for 30 min, after which cells were washed, resuspended in incubation buffer and analyzed by FACS (Beckman Coulter). Results were analyzed using Kaluza software. Each sample was also incubated with secondary antibody alone as a negative control.

[0475] Detection of hCG using a commercial pregnancy test - A commercial rapid pregnancy test ("Uni Test", Core Technologies) was used to detect the presence of hCG in the cell culture medium. Each cell line was seeded in a well of a 6-well plate with 2 ml of the appropriate medium until it reached 60-80% confluence (40-50% for iPSCs), and 0.5 ml was taken after 24 hours. HFFs not infected with GOKM were seeded in a 15 cm plate with 25 ml of medium. From this, 0.5 ml was taken at 70% confluence after 72 hours.

[0476] RNA and RRBS Libraries and Sequencing- For RNAseq, total RNA was isolated using the Qiagen RNeasy kit. mRNA libraries were prepared using the SENSE mRNA-seq Library Prep Kit V2 (Lexogen), and pooled libraries were sequenced on an Illumina NextSeq 500 platform to generate 75-bp single-end reads. For RRBS, DNA was isolated from samples and incubated in lysis buffer (25 mM Tris-HCl, pH 8, 2 mM EDTA, 0.2% SDS, 200 mM NaCl) supplemented with 300 μg / mL proteinase K (Roche), followed by phenol:chloroform extraction and ethanol precipitation. To eliminate the presence of MEF feeder cells, hiTSC and hbdTSC colonies were passaged twice on Matrigel. RRBS libraries were prepared as described in Boyle et al., Genome Biol. 2012 Oct 3;13(10):R92. Samples were processed on a HiSeq 2500 (Illumina).

[0477] RNA-seq and RRBS analysis - For analysis of RNA-seq results, raw reads (fastq files) were quality trimmed using an in-house Perl script and adapters were removed using cutadapt (version 1.12). Processed fastq files were mapped to the human transcriptome and genome using TopHat (v2.1.1). The genome version was GRCh38 and annotation from Ensembl was release 89. Quantification was performed using htseq-count (version 0.6.1). Genes with a sum of counts less than 10 across all samples were excluded, leaving 25596 genes. Normalization was performed with the DESeq2 package (version 1.16.1).

[0478] For analysis of RRBS results, paired-end reads were aligned to the human genome (hg19) using BSMAP v 2.9, and adapters and low-quality sequences were trimmed using Trim Galore. Methylation ratios of CpGs with a sequencing depth of at least 10 reads were calculated based on 100 bp tiles.

[0479] Differentiation of hiTSCs and staining with PI - Two hbdTSCs and two hiTSC lines were seeded in hTSC medium on Matrigel-coated 6-well plates and allowed to reach 70% confluency. The medium was then replaced with basal differentiation medium of DMEM supplemented with 10% FBS, supplemented with 1% L-glutamine solution (BI) and antibiotics (BDM). Cells were harvested from six identical wells on day 0 and every day for 5 days for gene expression analysis.

[0480] For directed differentiation into ST, approximately 4 × 10 5 Cells were seeded onto Matrigel-coated 6-well plates at a concentration of 1:30 under ambient oxygen conditions in a medium consisting of DMEM / F12 supplemented with 0.1 mM 2-mercaptoethanol, 0.5% penicillin-streptomycin, 0.3% BSA, 1% ITS additive, 2.5 μM Y27632, 2 μM forskolin, and 4% KSR. Cells were harvested on days 2 and 6 for analysis of mRNA expression using qPCR as described above. Cells were also collected at approximately 10 per plate. 5 The cells were seeded at a density of 100x in 12-well plates, cultured in the same manner, and fixed with 4% PFA for immunostaining as described above.

[0481] For directed differentiation into EVT, approximately 4 × 10 5Cells were seeded onto Matrigel-coated 6-well plates at a concentration of 1:100 in DMEM / F12 medium supplemented with 0.1 mM 2-mercaptoethanol, 0.5% penicillin-streptomycin, 0.3% BSA, 1% ITS additive, 100 ng / ml NRG1, 7.5 μM A83-01, 2.5 μM Y27632, and 4% knockout serum replacement, 5% oxygen, and Matrigel was added to a final concentration of 2%. On day 3, the medium was replaced with EVT medium without NRG1, and Matrigel was added to a final concentration of 0.5%. On day 6, cells were harvested for analysis of mRNA expression using qPCR or suspended in EVT medium without NRG1 and KSR and Matrigel was added to a final concentration of 0.5% as described in Okae et al. Cell Stem Cell. 2018 Jan 4;22(1):50-63 until cells were harvested on day 14. Cells were also plated at approximately 10 5 The cells were seeded at a density of 100x in 12-well plates, cultured in the same manner, and fixed with 4% PFA for immunostaining as described above.

[0482] Directed differentiation was repeated three times with similar results.

[0483] For staining with PI, 1 × 10 cells from two hbdTSC and two hiTSC lines were 6 Cells were seeded on Matrigel-coated 10 cm plates in hTSC medium. After 2–4 days, the medium was replaced with BDM. For PI staining, cells were fixed with ethanol on days 0, 4, and 8 and stored at -20°C. On the day of staining, all samples were washed with PBS and resuspended in staining mixture containing 50 μg / ml RNAse A (Sigma-Aldrich) and 50 μg / ml PI (BD). After 30 min of incubation, cells were analyzed by FACS (Beckman Coulter). Results were analyzed using Kaluza software.

[0484] Formation of trophoblast cell organoids with bdTSCs and hiTSCs - bdTSCs and hiTSCs were suspended in trophoblast cell organoid medium (TOM) consisting of DMEM / F12 supplemented with 10 mM HEPES, 1x B27, 1x N2, 10 mM L-glutamine, 100 ng / mL R-spondin, 1 μM A83-01, 100 ng / mL recombinant human epidermal growth factor (rhEGF), 50 ng / mL recombinant mouse hepatocyte growth factor (rmHGF), 2.5 μM prostaglandin E2, 3 μM CHIR99021, and 100 ng / mL Noggin, as described in Haider et al., Stem Cell Reports. 2018 Aug 14;11(2):537-551. Growth factor reduced matrigel (GFR-M) was added to reach a final concentration of 60%. 10 4 ~10 5 A solution (40 μL) containing bdTSCs / hiTSCs was added to the center of a 24-well plate. After 2 min at 37 °C, the plate was inverted upside down to ensure even spreading of the cells in the solidifying GFR-M-forming dome. After 15 min, the plate was inverted again and 500 μL of pre-warmed TOM was carefully overlaid onto the dome. The cells were cultured in 5% oxygen for 10–19 days and then subjected to immunostaining.

[0485] Immunostaining of bdTSC and hiTSC trophoblast cell organoids - Matrigel domes containing organoids were fixed overnight in 4% PFA. The domes were then washed twice with PBS for 15 min. The domes were immersed overnight at 4°C in blocking solution containing 3% bovine serum albumin (BSA), 5% fetal bovine serum (FBS), 0.1% Triton X-100 in PBS. The tissues were then incubated with primary antibodies containing anti-Ki67 (1:200, Abcam, ab15580) and anti-KRT7 (1:200, Abcam, ab215855) diluted in PBS containing 1% BSA and 0.1% Triton X-100 on a rocking plate at 4°C for 2 nights. The plates were then transferred to room temperature, rocking continued for at least 2 more hours, and washed overnight in PBS containing 0.1% Triton X-100 with at least 5 changes of buffer. The next day, the domes were incubated overnight at 4°C on a rocking plate in secondary antibody solution containing the relevant (Alexa) secondary antibody (1:200) diluted in 1% BSA and 0.1% Triton X-100. The domes were washed again overnight with PBS containing 0.1% Triton X-100 with at least 5 changes of buffer. Finally, the domes were incubated with DAPI for 1 hour and stored in PBS at 4°C until imaging. Imaging was performed using a spinning disk confocal microscope equipped with a Nikon Eclipse Ti2 CSU-W1, a Yokogawa confocal scanning unit, an Andor Zyla sCMOS camera, and a Nikon Plan Apo VC 20X NA 0.75 lens. Maximum intensity projection images were generated using NIS-Elements microscope imaging software.

[0486] Transplantation of hiTSCs into NOD-SCID mice and immunohistochemistry (IHC) - Approximately 4 × 10 6were trypsinized with TrypLE, washed twice with PBS, resuspended in 150 μl of a 1:2 mixture of Matrigel and PBS, and injected subcutaneously into NOD-SCID mice. Nine days after injection, lesions were harvested, dissected, fixed overnight in 4% paraformaldehyde, paraffin-embedded, sectioned, and mounted on slides. Some slides were stained with H&E, while others were subjected to IHC staining. For IHC, slides were deparaffinized in xylene and rehydrated in a decreasing ethanol gradient. Antigen retrieval was performed in sodium citrate buffer, and slides were heated at 110–120 °C for 3 min. After a brief incubation in 3% hydrogen peroxide, sections were incubated overnight in CAS-block (Invitrogen) with the primary antibody anti-KRT7 (1:1000) (Abcam, ab215855). Sections were then incubated with the appropriate HRP-conjugated secondary antibody (Vector Laboratories) for 30 min, and immunohistochemistry was performed using a DAB peroxidase substrate kit (Vector Laboratories). Slides were lightly counterstained with hematoxylin.

[0487] [Table 1A]

[0488] [Table 1B]

[0489] Example 1 Generation of human induced trophoblast stem cell-like cells (hiTSCs) from fibroblasts by ectopic expression of GATA3, OCT4, KLF4, and c-MYC To reprogram fibroblasts into human induced trophoblast stem cells (hiTSCs), seven genes, namely GATA3, TFAP2C, ESRRB, OCT4, KLF4, SOX2, and MYC, were cloned into a doxycycline (dox)-inducible lentiviral vector and used to infect human foreskin fibroblasts (HHFs). Cells were kept in hypoxic conditions and treated with dox in basic reprogramming medium (DMEM + 10% FBS) for 2 weeks, and the medium was gradually replaced with hTSC medium (Okae et al., 2018, Figure 1A). After 4 weeks of reprogramming, dox was removed from the induced cells and allowed to stabilize for 7–10 days, after which individual epithelial-like colonies were manually transferred to separate plates for expansion and analysis. Transgene integration analysis revealed that the only integrated transgenes among all colonies tested were GATA3, OCT4, KLF4, and MYC (herein referred to as "GOKM") (Figure 7A). Indeed, infection of two primary HFF lines, KEN and PCS201, with GOKM factors (Figure 7B) produced stable, dox-independent, epithelial-like colonies that exhibited morphology remarkably similar to that of mTSCs and human blastocyst-derived TSCs (hbdTSCs) after passaging (Figure 1B). Reprogramming efficiency ranged from 0.000002 to 0.00005% depending on infection efficiency, with only 2 × 10 6 Approximately 5 to 100 colonies were obtained from each HHF.

[0490] To assess the identity of the resulting colonies, expression of hTSC markers was evaluated. Quantitative PCR (qPCR) revealed active transcription of known trophoblast cell markers such as GATA2, TFAP2A, TFAP2C, KRT7, and TP63, as well as endogenous expression of GATA3, in a manner similar to hbdTSCs (Figures 1C and 7C). Furthermore, expression of the HLA class I gene HLA-A was absent from all hiTSC and hbdTSC lines (Figure 7D). As expected, the resulting hiTSC colonies showed a marked downregulation of mesenchymal markers and upregulation of epithelial markers, indicating successful mesenchymal-to-epithelial transition (MET) (Figures 1D and 7E). Notably, similar to mouse (Benchetrit et al., 2015), the epithelial marker KRT18 distinguished human epithelial cells of pluripotent origin (i.e., ESCs and iPSCs) from epithelial cells of trophectodermal origin (i.e., hbdTSCs and hiTSCs). The expression of hTSC markers, i.e., GATA3, GATA2, TFAP2C, and KRT7, epithelial markers, i.e., CDH1 and KRT18, and the absence of mesenchymal markers VIM and classical HLA class I proteins (HLA-ABC), were also confirmed at the protein level (Figure 1E and Figure 7F-G).

[0491] Together, these data suggest that transient GOKM expression forces human fibroblasts to become stable, dox-independent epithelial colonies that resemble hbdTSCs in their morphology and expression of TSC markers.

[0492] Example 2 Characterization of generated hiTSCs The transcriptome of hiTSCs is highly similar to that of hbdTSCs. Extensive nuclear reprogramming during somatic cell conversion will result in the activation of newly established endogenous circuits in target cells (Buganim et al., 2013; Sebban and Buganim, 2016). Incomplete activation of endogenous circuits results in partially similar transcriptomes, as seen in some direct conversion models (Sebban and Buganim, 2016). To evaluate whether hiTSCs activate the endogenous circuits of TSCs, three hiTSC clones, herein referred to as hiTSC#1, hiTSC#4, and hiTSC#7, were subjected to RNA-sequencing (RNA-seq) analysis. Two hbdTSC lines, herein referred to as hbdTSC#2 and hbdTSC#9, parental HFF and hESC / hiPSC lines, were used as positive and negative controls, respectively. Notably, as shown by principal component analysis (Fig. 2A) and hierarchical correlation heatmap (Fig. 2B), various hiTSC clones clustered with hbdTSC lines and farther away from HFF and hESC / hiPSC controls. Notably, two hbdTSC lines clustered closer to hiTSC clones than to each other (i.e., hbdTSC#2 clustered with hiTSC#4, and hbdTSC#9 clustered with hiTSC#7) (Fig. 2B). Scatterplot analysis showed a significant R-squared between hbdTSCs and hiTSCs. 2 The results showed highly similar transcriptomes with a β-regression ratio (RRR) of >0.9, and key hTSC genes such as TP63 and GATA3 were highly expressed in all TSC samples but not in hESC or HFF negative controls (Figure 2C). Furthermore, gene expression differences between hTSCs (hbdTSCs or hiTSCs) and hESCs and HFFs revealed a significant enrichment of gene ontology terms related to placental and embryo-placental morphogenesis and development according to the Human Gene Atlas (Figures 8A-C).

[0493] Together, these data suggest that the transcriptome of induced hTSCs is highly similar to that of blastocyst-derived hTSCs.

[0494] The methylome and genome integrity of hiTSCs is comparable to that of hbdTSCs. Although the gene expression profile of hiTSCs is highly similar to that of hbdTSCs, we investigated whether the epigenetic landscape of hiTSCs is also similar to that of hbdTSCs. One of the epigenetic marks shown to be altered during the late stage of reprogramming to iPSCs by OSKM is DNA methylation (Apostolou and Hochedlinger, 2013). To test whether the DNA methylation landscape of hiTSCs is equivalent to that of hbdTSCs, four hiTSC clones, herein referred to as hiTSC#1, hiTSC#2, hiTSC#4, and hiTSC#11, were subjected to reduced representation bisulfite sequencing (RRBS) analysis. Two hbdTSC lines, namely hbdTSC#2 and hbdTSC#9, parental HFF and hESC lines, were used as positive and negative controls, respectively. Methylation analysis revealed 28,881 differentially methylated regions (DMRs), of which 4676 were hypomethylated in HFFs and hypermethylated in two hbdTSC lines, and 24205 DMRs were hypermethylated in HFFs and hypomethylated in two hbdTSC lines. Notably, analysis of the methylation status of the four hiTSC clones revealed that 4676 DMRs underwent robust de novo methylation in all four hiTSC clones (Figure 3A), while 24205 DMRs showed some variation among different colonies, with some regions remaining partially methylated (Figure 3B). Together, these results suggest that demethylation is less stringent in hiTSC reprogramming. Importantly, the overall methylation status of the hiTSC clones clustered tightly with hbdTSCs and was far removed from ESC and HFF controls in the case of both de novo methylated and demethylated DMRs.

[0495] During trans-differentiation of mouse ESCs to a TSC fate, several gatekeepers remain methylated, suggesting that they generate only TS-like cells (Cambuli et al., 2014). One of these gatekeepers is Elf5. Therefore, we investigated whether the ELF5 locus undergoes hypomethylation in hiTSCs. Analysis of RRBS data showed two DMRs at the ELF5 locus, with the proximal DMR (10 kb from the TSS, marked by a box) showing a similar pattern of hypomethylation in both hbdTSCs and all hiTSCs (Figure 3C). Similarly, the only DMR found at the pluripotency-specific locus NANOG (marked by a box) was fully hypomethylated in ESCs and to a lesser extent in HFFs, but was similarly methylated in both hbdTSCs and hiTSCs (Figure 3D). Interestingly, another DMR located at the neighboring locus, NANOGNB, showed a similar hypomethylation pattern in all hbdTSCs and hiTSCs, in contrast to the methylation patterns in HFFs and ESCs (Figure 3D). Together, these data suggest that DNA methylation is primarily recombined to the hTSC state in stable hiTSCs.

[0496] Next, we investigated whether the reprogramming process or long-term culture of hiTSCs is prone to genomic abnormalities. To this end, two hbdTSC lines (hbdTSC#2 and hbdTSC#9) and four hiTSC clones were subjected to sensitive karyotyping measurements using the Affymetrix CytoScan 750K array. Detailed analysis revealed that 50% of all colonies from both sources (i.e., hbdTSC or hiTSC) had intact karyotypes. The remaining 50% of colonies showed little abnormality in a small fraction of cells (Figure 9). These results indicate that hiTSC colonies with intact karyotypes can be isolated and expanded in culture, and that the reprogramming process does not promote genomic instability.

[0497] Example 3 The generated hiTSCs differentiate into all trophoblast cell types in vitro. Similar to natural placental stem cells, hbdTSCs can differentiate into multinucleated syncytial trophoblast cells (ST) and extravillous trophoblast cells (EVT) (Okae et al., 2018). Therefore, we investigated whether hiTSCs retain a similar potential to differentiate into ST and EVT. We first replaced the hTSC culture medium with DMEM + 10% FBS and allowed the cells to spontaneously differentiate. As in the mouse case, removing the stemness signal from the cells was sufficient to induce spontaneous differentiation into ST and EVT, as assessed by qPCR for markers specific for EVT (e.g., HLA-G, MMP2, and NOTCH1) and ST (e.g., ERVFRD-1, PSG1, SDC1, CGB, and CSH1), as well as PI staining for the presence of multinucleated cells followed by flow cytometry (Figure 4A and Figure 10A-B).

[0498] However, DMEM + 10% FBS medium is not optimal for growing trophoblast cells, and cells tend to die and detach from the plate after a few days in DMEM + 10% FBS medium. Therefore, direct differentiation into ST and EVT was performed using the protocol of Okae et al. (Okae et al., 2018) (Figure 4B and Figure 10C). hbdTSCs and hiTSCs were first differentiated into ST, while samples were taken after 2 and 6 days of differentiation. qPCR analysis for ST markers such as ERVFRD-1, CSH1, PSG1, and GCM1 showed a robust induction of ST markers equivalent to that in hbdTSCs (Figure 4C and Figure 10D). Notably, ERVFRD-1, which orchestrates fusion events early in the process, was upregulated after 2 days of differentiation, but returned to normal levels once cells completed fusion on day 6. Immunostaining for pan-trophoblast cells, KRT7, epithelial markers CDH1 and DAPI showed clear formation of large multinucleated cells after 6 days of differentiation in both hbdTSCs and hiTSCs. As expected, undifferentiated hiTSCs stained positive for CDH1, whereas multinucleated STs were negative for this. SDC1-positive three-dimensional ST structures were also observed in both cell types (Figures 4D-E and S10E-F). These data indicate that hiTSCs, like hbdTSCs, retain the ability to differentiate into STs.

[0499] We next directed the differentiation of hbdTSCs and hiTSCs into EVTs (Okae et al., 2018). Following seeding and cell attachment to the plate, cell aggregates were formed in the plate (Figure 5A). After 6 days of differentiation, spindle-like cells began to migrate out of the aggregates. qPCR analysis (Figure 5B) and immunostaining (Figure 5C) for key EVT genes (e.g., HLA-G and MMP2) confirmed the identity of the cells as EVTs. Together, these results suggest that hiTSCs retain the potential to differentiate into various placental cell types, similar to hbdTSCs.

[0500] Example 4 The generated hiTSCs proliferate and differentiate in vivo. When hbdTSCs were subcutaneously injected into nonobese diabetic (NOD)-severe combined immunodeficiency (SCID) mice, the cells formed KRT7-positive trophoblast cell lesions, but with little angiogenesis (Okae et al., 2018). To examine whether hiTSCs could form KRT7-positive trophoblast cell lesions, approximately 4 × 10 cells from two hiTSC clones (hiTSC#1 and hiTSC#3) and one hbdTSC control line (hbdTSC#2) were injected into the nucleus of the hiTSCs. 6 The cells were subcutaneously injected into NOD / SCID mice. After 9 days, lesions measuring approximately 5 mm in size had formed and were extracted (Figure 6A, Figure 11). Immunohistochemical staining showed areas of KRT7-positive cells with trophoblast cell morphology, similar to previously published findings (Okae et al., 2018). Secretion of hCG was confirmed in the culture medium using a commercially available pregnancy test (Figure 6B).

[0501] Example 5 The generated hiTSCs form trophoblast cell organoids. Recently, two trophoblast cell organoid systems have been developed and described (Haider et al., 2018; Turco et al., 2018). These studies demonstrated the ability of first trimester villous CTB cells to form three-dimensional structures containing both proliferating stem cells and differentiated cells. Close examination of the two systems revealed that many features of the early developmental program of the human placenta were also present in these organoids. Therefore, we investigated whether hiTSCs retained the same potential and would form trophoblast cell organoids. bdTSC#2 and hiTSC#4 were trypsinized, seeded inside droplets of Matrigel, and left to grow for 10 days. As shown for the villous CTB, both hbdTSCs and hiTSCs were able to form three-dimensional structures within a few days of culture. Immunostaining for key genes revealed that the organoids consisted of both proliferating stem cells and differentiated cells (Figure 6C). Together, these data demonstrate that hiTSCs can form functional organoids similar to their hbdTSC and villous CTB counterparts.

[0502] Example 6 Rejuvenation of human cells by ectopic expression of GATA3, OCT4, KLF4 / KLF5, and c-MYC This example provides a non-limiting example of a protocol for rejuvenation: GATA3, OCT4, at least one of KLF4 and KLF5, and optionally c-MYC, are introduced as mRNA molecules into CD34+ cells obtained from a human subject, for example a patient suffering from myelodysplastic syndrome (MDS). The cells are then cultured for 1-3 weeks to determine their genetic age and function. The rejuvenated cells are then transplanted back into the same patient.

[0503] Example 7 Generation of hiTSCs from fibroblasts by ectopic expression of GATA3, OCT4, KLF4, KLF5, and c-MYC The gene KLF5 was also identified as a potent booster for the reprogramming process into hiTSCs, for example when combined with GATA3, OCT4, KLF4, and c-MYC. Interestingly, KLF5 not only facilitated reprogramming into hTSCs, but also enabled the reprogramming of senescent fibroblasts (obtained from PromoCell, Cat no: C-12302) into hiTSCs (Figure 12).

[0504] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0505] It is the intention of the applicants that all publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety, as if each individual publication, patent, or patent application was specifically and individually noted to be incorporated herein by reference at the time of reference. Furthermore, citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. To the extent that they are used as section headings, they should not be construed as necessarily limiting. Additionally, any priority documents of this application are incorporated herein by reference in their entirety.

[0506] [References] TIFF2024534404000003.tif230165TIFF2024534404000004.tif109168

Claims

1. An in vitro method for generating induced trophoblast stem cells (iTSCs) from human cells, comprising the step of expressing exogenous GATA3 and OCT4 transcription factors intracellularly under conditions that enable the generation of iTSCs from said cells, thereby generating iTSCs from the cells.

2. An in vitro method for rejuvenating and / or dedifferentiating human cells, comprising the step of expressing exogenous GATA3 and OCT4 transcription factors intracellularly under conditions that enable the rejuvenation and / or dedifferentiation of said cells, thereby generating rejuvenated cells and / or dedifferentiated cells.

3. The method according to claim 1 or 2, wherein the step of expressing comprises a step of transient expression.

4. The method according to any one of claims 1 to 3, further comprising the step of expressing an exogenous c-MYC transcription factor and / or an exogenous KLF transcription factor intracellularly.

5. The above conditions are such that the step of expressing is at least 14 days after the introduction of the exogenous transcription factor into the cells, and / or The above conditions are such that the step of expressing does not exceed 30 days after the introduction of the exogenous transcription factor into the cells. The method according to any one of claims 1 to 4.

6. The above conditions are such that the step of expressing is at least 1 day after the introduction of the exogenous transcription factor into the cells, and / or The above conditions are such that the step of expressing is less than 25 days after the introduction of the exogenous transcription factor into the cells. The method according to any one of claims 2 to 4.

7. The method according to any one of claims 1 and 3 to 5, wherein when the iTSCs are determined by at least one of PCR, Western blot, and / or flow cytometry, the exogenous transcription factors are not expressed.

8. The method according to any one of claims 2 to 6, wherein when the rejuvenated cells and / or dedifferentiated cells are determined by at least one of PCR, Western blot, and / or flow cytometry, the exogenous transcription factors are not expressed.

9. A nucleic acid construct or system comprising at least one polynucleotide comprising a nucleic acid sequence encoding GATA3 and OCT4 transcription factors.

10. The nucleic acid construct or system according to claim 9, wherein the at least one polynucleotide further comprises a nucleic acid sequence encoding a c-MYC transcription factor and / or a KLF transcription factor.

11. The nucleic acid construct or system according to claim 9 or 10, wherein the at least one polynucleotide is RNA.

12. A protein preparation comprising GATA3 and OCT4 transcription factor polypeptides at a purity level of at least 20%.

13. The protein preparation according to claim 12, further comprising a c-MYC transcription factor polypeptide and / or a KLF transcription factor polypeptide.

14. An isolated human cell expressing exogenous GATA3 and OCT4 transcription factors.

15. The isolated cell according to claim 14, further expressing an exogenous c-MYC transcription factor and / or an exogenous KLF transcription factor.

16. An isolated induced trophoblast stem cell (iTSC) obtained according to the method of any one of claims 1 and 3 to 5.

17. An isolated rejuvenated and / or dedifferentiated cell obtained according to the method of any one of claims 2 to 6 and 8.

18. A method for generating an aggregate or organoid comprising trophoblast cells, the method comprising introducing the iTSC according to claim 16, the construct or system according to any one of claims 9 to 11, or the protein preparation according to claim 12 or 13 into a scaffold or matrix.

19. An agent for treating and / or preventing a disorder related to the generation and / or activity of trophoblast cells in a subject in need thereof, comprising the iTSC according to claim 16, the construct or system according to any one of claims 9 to 11, or the protein preparation according to claim 12 or 13.

20. An agent for treating and / or preventing an age-related disease in a subject in need thereof, comprising the cell according to claim 17, the construct or system according to any one of claims 9 to 11, or the protein preparation according to claim 12 or 13.

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