Methods for reprogramming cells

Abstract

To provide methods for reprogramming human somatic cells into cells exhibiting at least one characteristic of trophoblast stem cells (MSCs).SOLUTION: The method of the present invention comprises the following steps, in order: a) increasing protein expression of one or more factors in somatic cells, the factors being for reprogramming the somatic cells toward a pluripotent state; b) culturing the cells for a time and under conditions sufficient to allow the cells to be reprogrammed toward a pluripotent state; c) contacting the cells with a culture medium suitable for maintaining trophoblast stem cells (TSCs); and d) culturing the cells in a TSC medium for a time and under conditions sufficient to allow the cells to exhibit at least one characteristic of TSCs, thereby reprogramming the somatic cells into cells exhibiting at least one characteristic of TSCs.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to methods and compositions for generating induced trophoblast stem cells from cells.

[0002] Related Applications This application claims priority from Australian Provisional Patent Application No. 2019904283, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] Mammalian embryonic development begins with a totipotent zygote capable of developing into a blastocyst, separated by an inner cell mass (ICM) and extraembryonic trophectoderm (TE). The ICM develops into the epiblast that gives rise to the embryo proper, and the primitive endoderm that gives rise to the yolk sac. The TE ultimately gives rise to the placenta.

[0004] The placenta is a key organ in human fetal development because trophoblast cells in the placenta can mediate interactions between the fetus and the mother at the fetal-maternal interface.

[0005] There are three major types of trophoblast-derived cells that can be found in the human placenta: undifferentiated cytotrophoblasts (CTs), which can give rise to extravillous trophoblasts (EVTs) and syncytiotrophoblasts (STs). All trophoblast cells arise from TE cells in the blastocyst, and their well-regulated proliferation and differentiation are essential for fetal development. Disruptions in trophoblast development and function can lead to various complications during pregnancy, including miscarriage, preeclampsia, and intrauterine growth restriction.

[0006] Despite advances in the research and clinical application of human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), the development of reproducible methods for generating trophoblast stem cells remains lacking. Furthermore, although the derivation of TSCs has been achieved, the isolation of these cells from preimplantation embryos or placentas does not provide access to genetic diversity.

[0007] There is a need for new and / or improved methods for generating human trophoblast stem cells, or cells that exhibit characteristics of trophoblast stem cells, without the need for a preimplantation embryo or placenta.

[0008] The reference to prior art herein is not an admission or suggestion that this prior art forms part of the common general knowledge in any jurisdiction, or that this prior art could reasonably be expected to be understood, considered relevant, and / or combined with other prior art by a person skilled in the art. Summary of the Invention [Means for solving the problem]

[0009] In a first aspect, the present invention provides a method for reprogramming a somatic cell into a cell exhibiting at least one characteristic of a trophoblast stem cell (TSC), the method comprising the steps of, in order: a) increasing the protein expression or amount of one or more factors in somatic cells, the factors being for reprogramming the somatic cells towards a dedifferentiated or pluripotent state; b) culturing the cells for a time and under conditions sufficient to allow dedifferentiation or reprogramming of the cells towards a pluripotent state; c) contacting the cells with a trophoblast stem cell (TSC) culture medium suitable for maintaining TSCs; d) culturing the cells in TSC medium for a time and under conditions sufficient to allow the cells to exhibit at least one characteristic of a TSC; This reprograms the somatic cell into a cell that exhibits at least one characteristic of a TSC.

[0010] Preferably, the TSC medium comprises a growth factor, preferably EGF, and a Rho kinase (ROCK) inhibitor. Accordingly, the present invention also provides a method for reprogramming somatic cells into cells exhibiting at least one characteristic of trophoblast stem cells (TSCs), the method comprising the steps of, in order: a) increasing the protein expression or amount of one or more factors in somatic cells, the factors being for reprogramming the somatic cells towards a dedifferentiated or pluripotent state; b) culturing the cells for a time and under conditions sufficient to allow dedifferentiation or reprogramming of the cells towards a pluripotent state; c) contacting the cells with a TSC culture medium containing a growth factor, preferably EGF, and a ROCK inhibitor; d) culturing the cells in TSC culture medium for a time and under conditions sufficient to allow the cells to exhibit at least one characteristic of a TSC; This reprograms the somatic cell into a cell that exhibits at least one characteristic of a TSC.

[0011] The present invention also provides a method for generating cells exhibiting at least one characteristic of trophoblast stem cells (TSCs) from somatic cells, the method comprising the steps of, in order: a) increasing the protein expression or amount of one or more factors in somatic cells, the factors being for reprogramming the somatic cells towards a dedifferentiated or pluripotent state; b) culturing the cells for a time and under conditions sufficient to allow dedifferentiation or reprogramming of the cells towards a pluripotent state; c) contacting the cells with a TSC culture medium containing a growth factor, preferably EGF, and a ROCK inhibitor; d) culturing the cells in TSC culture medium for a time and under conditions sufficient to allow the cells to exhibit at least one characteristic of a TSC; This generates cells from somatic cells that exhibit at least one characteristic of TSCs.

[0012] In a further embodiment, the present invention provides a method for reprogramming fibroblasts, the method comprising the steps of, in order: a) increasing the protein expression or amount of one or more factors in fibroblasts, the factors being for reprogramming the fibroblasts towards a dedifferentiated or pluripotent state; b) culturing the cells for a time and under conditions sufficient to allow dedifferentiation or reprogramming of the cells towards a pluripotent state; c) contacting the cells with a TSC culture medium containing a growth factor, preferably EGF, and a ROCK inhibitor; d) culturing the cells in TSC culture medium for a time and under conditions sufficient to allow the cells to exhibit at least one characteristic of a TSC; Here, the fibroblasts are reprogrammed to exhibit at least one characteristic of a TSC.

[0013] It is understood that any method for dedifferentiating or reprogramming somatic cells toward a pluripotent state (i.e., a method for performing steps a) and b) of the methods described herein) can be used in accordance with the methods of the present invention. Thus, the present invention is not limited by the particular method or culture conditions for increasing protein expression or amount of associated factors that can be used in accordance with steps a) and b) to enable somatic cells to initiate reprogramming toward plasticity or pluripotency. Such methods are known in the art and are further described herein.

[0014] In a preferred embodiment, the factor for dedifferentiating or reprogramming somatic cells toward a pluripotent state is a transcription factor. The transcription factor may comprise, consist of, or consist essentially of one or more of the following factors: OCT4, SOX2, KLF4, and MYC. In a particularly preferred embodiment, the transcription factor comprises all four of the factors OCT4, SOX2, KLF4, and MYC (OSKM), or variants thereof.

[0015] Thus, in a preferred embodiment, the present invention provides:

[0016] In a further embodiment, the present invention provides a method for reprogramming fibroblasts, the method comprising the steps of, in order: a) increasing the protein expression or amount of one or more of the transcription factors OCT4, SOX2, KLF4, and MYC (OSKM) in fibroblasts; b) culturing the cells for a time and under conditions sufficient to allow dedifferentiation or reprogramming of the cells towards a pluripotent state; c) contacting the cells with a TSC culture medium containing EGF and a ROCK inhibitor; d) culturing the cells in TSC medium for a time and under conditions sufficient to allow the cells to exhibit at least one characteristic of a TSC; wherein the fibroblasts are reprogrammed to exhibit at least one characteristic of a TSC;

[0017] In further embodiments, transcription factors for reprogramming somatic cells, such as fibroblasts, toward a dedifferentiated or pluripotent state may also include the factors LIN28 and / or NANOG. In certain embodiments, the protein expression of each of OCT4, SOX2, KLF4, MYC, LIN28, and NANOG is increased in somatic cells.

[0018] Typically, the protein expression or amount of a transcription factor described herein is increased by contacting a cell with an agent that increases expression of the transcription factor. Preferably, the agent is selected from the group consisting of nucleotide sequences, proteins, aptamers and small molecules, ribosomes, RNAi agents, and peptide nucleic acids (PNAs), and analogs or variants thereof. In some embodiments, the agent is exogenous. The present invention also contemplates the use of transcription activation systems (e.g., gRNAs for use in gene activation systems such as CRISPR / Cas9 or TALEN) to increase expression of one or more transcription factors.

[0019] Typically, the protein expression or amount of a transcription factor described herein is increased by introducing into a cell at least one nucleic acid comprising a nucleotide sequence encoding the transcription factor or a nucleotide sequence encoding a functional fragment thereof.

[0020] In a preferred embodiment of the present invention, the nucleic acid sequence encoding the transcription factor protein is introduced into the cell by a plasmid. One or more nucleic acids encoding one or more transcription factors can be used. Therefore, it is clear that one or more plasmids can be used for the purpose of increasing the expression or amount of one or more required transcription factors. In other words, the nucleic acid sequence can be in or on a single plasmid, or can be provided to the somatic cell in two or more plasmids.

[0021] In any embodiment of the invention, a plasmid comprising nucleic acid encoding one or more transcription factors for use in accordance with the invention may be an episomal plasmid.

[0022] In any embodiment of the invention, a detectable marker may also be introduced into the somatic cells to identify when the somatic cells have been reprogrammed to exhibit at least one characteristic of a TSC. The detectable marker may be a fluorescent reporter operably linked to a promoter or enhancer sequence.

[0023] Preferably, the nucleic acid further comprises a heterologous promoter. Preferably, the nucleic acid is in a vector, such as a viral vector or a non-viral vector. Preferably, the vector is a viral vector that contains a genome that does not integrate into the host cell genome. The viral vector can be a retroviral vector, a lentiviral vector, an adenovirus, or a Sendai virus.

[0024] In any embodiment of the invention, the protein expression or amount of a factor is increased in a somatic cell by contacting the somatic cell with one or more agents to increase expression of the factor in the cell. In certain embodiments, the protein expression or amount of a factor is increased in a somatic cell by transducing or transfecting the somatic cell with one or more vectors encoding the transcription factor. The vector may be a viral vector, including an integrative or non-integrative viral vector. In a further embodiment, the vector may be an episomal vector.

[0025] It will also be understood that the term "programming towards a pluripotent state" does not require that the cells be pluripotent upon contact with TSC medium. In other words, the somatic cells need not have completed reprogramming to a pluripotent state prior to contacting the cells with TSC medium. Rather, the cells are preferably in an intermediate state upon contact with TSC medium, transitioning from a differentiated state to a more plastic or mature state (i.e., towards a non-differentiated or dedifferentiated state).

[0026] Accordingly, the present invention further provides a method for reprogramming a somatic cell into a cell exhibiting at least one characteristic of a trophoblast stem cell (TSC), the method comprising the steps of, in order: a) increasing the protein expression or amount of one or more factors in somatic cells, the factors being for reprogramming the cells towards a dedifferentiated or pluripotent state; b) culturing the cells for a time and under conditions sufficient to allow reprogramming of the cells to a reprogramming intermediate state between differentiation and pluripotency; c) contacting the cells with a trophoblast stem cell (TSC) culture medium suitable for maintaining TSCs; d) culturing the cells in TSC medium for a time and under conditions sufficient to allow the cells to exhibit at least one characteristic of a TSC; This reprograms the somatic cell into a cell that exhibits at least one characteristic of a TSC.

[0027] As used herein, reprogramming to an intermediate state between differentiation and pluripotency may also be understood to refer to reprogramming towards a less "mature" cell state.

[0028] In any embodiment, the period between increasing the protein expression or amount of the factor and contacting the cells with TSC culture medium can be any period, as long as it allows for a decrease in markers associated with somatic cells. In a further example, the period between increasing the protein expression or amount of the factor and contacting the cells with TSC culture medium can be any period, as long as it allows the cells to progress through a mesenchymal to epithelial transition state. In further or alternative embodiments, this period can be any period, as long as it allows for upregulation of expression of trophectoderm (TE)-associated transcription factors in the cells. Such transcription factors include TFAP2C and GATA2.

[0029] In certain embodiments, the culture period at which cells begin dedifferentiation or reprogramming toward a pluripotent state is at least one day after increasing protein expression or amount of one or more factors. The period can be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 25, 28, 30, 35, 42 days or more after increasing protein expression or amount of one or more factors. In any embodiment, the culture period at which cells begin dedifferentiation or reprogramming toward a pluripotent state can be any period that allows for a decrease in markers associated with somatic cells.

[0030] In a further embodiment of the invention, the above method comprises culturing the cells towards a pluripotent state in a medium selected from a medium suitable for culturing somatic cells (e.g., fibroblast or similar medium), a priming pluripotency medium, a naive pluripotency medium, or an expansion pluripotency medium (e.g., LCDM medium) prior to contacting and culturing the cells with TSC medium.

[0031] Still further, the above method may include culturing the cells toward a pluripotent state in primed medium, followed by transferring the cells to a medium selected from a naive medium or an expansion medium (e.g., LCDM medium), and then contacting and culturing the cells with TSC medium.

[0032] The present invention also provides methods for deriving TSCs from naive and expanded pluripotent stem cells.

[0033] Thus, in a further aspect, the present invention provides a method for generating cells exhibiting at least one characteristic of trophoblast stem cells (TSCs), the method comprising the steps of, in order: a) providing established naive pluripotent stem cells (iPSCs) or expanded induced pluripotent stem cells (EPSCs); b) contacting EPSCs or naive iPSCs with a TSC culture medium containing a growth factor, preferably EGF, and a ROCK inhibitor; c) culturing the cells in TSC medium for a time and under conditions sufficient to allow the cells to exhibit at least one characteristic of a TSC; EPSCs or naive iPSCs are thereby reprogrammed into cells that exhibit at least one characteristic of TSCs.

[0034] In any of the above aspects, the cells are iPSCs obtained by reprogramming somatic cells by any method known in the art. In a preferred embodiment, the iPSCs are obtained from reprogrammed fibroblasts. In a further embodiment, the iPSCs are obtained by reprogramming fibroblasts by increasing the expression of one or more of the transcription factors OCT4, SOX2, KLF4, and MYC (OSKM) in the fibroblasts.

[0035] In a further aspect, the present invention provides a method for generating cells exhibiting at least one characteristic of trophoblast stem cells (TSCs), the method comprising the steps of, in order: a) providing established primed expanded pluripotent stem cells (EPSCs) or naive induced pluripotent stem cells (iPSCs); b) contacting EPSCs or naive iPSCs with naive medium or LCDM medium; c) contacting EPSCs or iPSCs with a TSC culture medium containing a growth factor, preferably EGF, and a ROCK inhibitor; d) culturing the cells in TSC medium for a time and under conditions sufficient to allow the cells to exhibit at least one characteristic of a TSC; The EPSC or naive iPSC cell is thereby reprogrammed into a cell that exhibits at least one characteristic of a TSC.

[0036] In any of the above aspects, the cells are naive iPSCs / EPSCs obtained by reprogramming somatic cells by any method known in the art. In preferred embodiments, the naive iPSCs / EPSCs are obtained from reprogrammed fibroblasts or other somatic cell types. In further embodiments, the iPSCs are obtained by reprogramming fibroblasts by increasing the expression of one or more of the transcription factors OCT4, SOX2, KLF4, and MYC (OSKM) in the fibroblasts.

[0037] In any embodiment of the present invention, the TSC culture medium comprises the ROCK inhibitor trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632), or a salt thereof.

[0038] In further embodiments, the TSC culture medium further comprises one or more of the following: 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]benzamide (SB 431542) or its salts; - 6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]mycotinonitrile (CHIR 99021) or its salts - A83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide) or its salts;

[0039] In a further embodiment, the TSC medium also includes an agent for stimulating Wnt and one or more inhibitors of TGFβ.

[0040] In a preferred embodiment, the TSC medium comprises A83-01, SB431542, EGF, CHIR, a ROCK inhibitor, ascorbic acid, and valproic acid (the medium is sometimes referred to as ASECRiAV).

[0041] As used herein, characteristics of a TSC are understood to include: - undifferentiated and bipotent state with the ability to differentiate into cells that exhibit one or more characteristics of extravillous trophoblast (EVT) or syncytiotrophoblast (ST); - Cobblestone colony appearance; - Methylation patterns similar to blastocyst-derived TSCs as determined by bisulfite assay or genome-wide DNA methylation profiling techniques; - expression of one or more biochemical markers of TSC, determined by immunohistochemistry and / or PCR assays, preferably where the markers are selected from the group consisting of CD249 (aminopeptidase A), CD49f (ITGA6); nuclear GATA2 / 3, TFAP2C, P63 and NR2F2; - levels of chromatin accessibility similar to blastocyst-derived TSCs as determined using ATAC-seq; - Histone modification profile similar to blastocyst-derived TSCs (e.g., H3K4me3, H3K27ac gene modifications); - Proteome or metabolomic profile similar to blastocyst-derived TSCs.

[0042] In further embodiments, cells exhibiting at least one characteristic of TSC are characterized by the absence of markers characterizing somatic cells, hi certain embodiments, cells exhibiting at least one characteristic of TSC do not express one or more of the following markers: OCT4 (also known as POU5F1), NANOG, SOX2, SALL2, OTX2, BANCR, KLF17, DPPA3, ARGFX, and DNMT3L.

[0043] In any embodiment, the TSC cells produced according to the present invention are characterized by at least one, at least two, at least three, at least four, or at least five characteristics of TSCs described herein.

[0044] In a preferred embodiment, the method defined herein comprises the steps of: - have the ability to differentiate into cells that exhibit one or more characteristics of extravillous trophoblast (EVT) or syncytiotrophoblast (ST); - expresses one or more biochemical markers of TSC selected from the group consisting of nuclear GATA2 / 3, TFAP2C, P63, and NR2F2 It is for reprogramming cells.

[0045] As used herein, characteristics of ST include one or more of: SDC1+ multinucleated cells and increased expression of one or more of the markers CGA, CGB, PSG1, CSH1, HSD3B1, CYP19A1, SDC1, and INHA compared to TSC.

[0046] As used herein, characteristics of EVT include one or more of increased expression of one or more of the markers HLA-G, PRG2, PAPPA2, MMP2, ITGA5, and ATGA1 compared to TSC.

[0047] In a further embodiment, cells exhibiting at least one characteristic of a TSC retain their undifferentiated state when maintained in subculture.

[0048] Preferably, cells having at least one characteristic of a TSC retain at least one characteristic of a TSC for at least 5, at least 10, at least 15, at least 20, at least 40 or more cell culture passages.

[0049] Somatic cells can be any cell type described herein, including disease cells. Somatic cells can be adult cells or cells derived from adults that exhibit one or more detectable characteristics of adult or non-embryonic cells. Disease cells can be cells that exhibit one or more detectable characteristics of a disease or condition.

[0050] In a preferred embodiment, the somatic cells are fibroblasts.

[0051] Typically, conditions suitable for reprogramming somatic cells include culturing the cells in an appropriate medium for a sufficient period of time, which may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.

[0052] In any of the methods described herein, the method may further comprise expanding the cells that exhibit at least one characteristic of a TSC to increase the proportion of cells in the population that exhibit at least one characteristic of a TSC. Expanding the cells may comprise culturing the cells for a time and under conditions sufficient to generate the population of cells, as described below.

[0053] In any of the methods described herein, the method may further include differentiating the cells exhibiting at least one characteristic of a TSC to produce cells exhibiting at least one characteristic of an EVT or a ST. Differentiating the cells may include culturing the cells for a time and under conditions sufficient to produce cells having at least one characteristic of an EVT or a ST, as described herein.

[0054] Still further, the method may further comprise differentiating cells exhibiting at least one characteristic of a TSC into an extraplacental cell type for use in regenerative medicine.

[0055] The present invention also provides a mammalian undifferentiated progenitor cell having at least one characteristic of a TSC, wherein the cell is obtained by any of the methods defined herein.

[0056] The present invention also provides cells exhibiting at least one characteristic of a TSC produced by the methods described herein.

[0057] In any of the methods described herein, the method may further comprise isolating cells that exhibit at least one characteristic of a TSC.

[0058] Thus, in a further embodiment, there is provided an isolated TSC obtainable or obtained by any of the methods described herein.

[0059] The present invention also provides an isolated EVT or ST derived from or differentiated from a cell exhibiting at least one characteristic of a TSC, obtained according to the method of the present invention.

[0060] The present invention also provides a population of cells in which at least 5% of the cells exhibit at least one characteristic of a TSC, and the cells are generated by the methods described herein. Preferably, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the population exhibit at least one characteristic of a TSC.

[0061] The present invention also provides a population of cells in which at least 5% of the cells are STs or EVTs differentiated from cells exhibiting at least one characteristic of a TSC generated by the methods described herein. Preferably, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells in the population are STs or EVTs obtained by differentiating cells exhibiting at least one characteristic of a TSC obtained according to the present invention.

[0062] The present invention also provides a pharmaceutical composition comprising: - a cell exhibiting at least one characteristic of a TSC, the cell being obtained or obtainable according to the method of the invention, and - pharmaceutically acceptable excipients.

[0063] The present invention also provides a pharmaceutical composition comprising: - cells exhibiting at least one characteristic of ST or EVT, which are obtained or obtainable by differentiating cells having at least one characteristic of TSC obtained or obtainable according to the method of the invention, and - pharmaceutically acceptable excipients.

[0064] The present invention also provides a homeopathic or dietary supplement comprising composition comprising: - a cell or cell population that exhibits at least one characteristic of a TSC, said cell or cell population being obtained or obtainable according to the method of the invention, and / or - a cell or cell population exhibiting at least one characteristic of ST or EVT, which cell or cell population is obtained or obtainable by differentiating a cell having at least one characteristic of TSC obtained or obtainable according to the method of the invention, and / or - organoids derived from cells or cell populations that exhibit at least one characteristic of ST or EVT, said cells being obtained or obtainable by differentiating cells with at least one characteristic of TSCs obtained or obtainable according to the method of the invention; and - pharmaceutically acceptable excipients.

[0065] The present invention further provides compositions comprising a homeopathic or dietary supplement derived from: - a cell or cell population that exhibits at least one characteristic of a TSC, said cell or cell population being obtained or obtainable according to the method of the invention, and - a cell or cell population that exhibits at least one characteristic of a ST or an EVT, said cell or cell population being obtained or obtainable by differentiating a cell having at least one characteristic of a TSC obtained or obtainable according to the method of the invention, and - an organoid derived from a cell or cell population that exhibits at least one characteristic of a TSC or an EVT, wherein the cell or cell population is obtained or is obtainable by differentiating a cell that has at least one characteristic of a TSC that is obtained or obtainable according to the method of the invention.

[0066] use In any of the methods described herein, the method may further comprise administering to a subject in need thereof: - a cell exhibiting at least one characteristic of a TSC produced according to the methods described herein, or - a cell population comprising cells exhibiting at least one characteristic of a TSC produced according to the methods described herein, or - A differentiated cell or population of differentiated cells obtained from cells exhibiting at least one characteristic of a TSC produced according to the invention.

[0067] In some embodiments, methods of enhancing a placenta or blastocyst are provided, comprising introducing into the placenta or blastocyst: - cells exhibiting at least one characteristic of a TSC produced according to the methods described herein; - a cell population comprising cells exhibiting at least one characteristic of a TSC, produced according to the methods described herein. - A differentiated cell or population of differentiated cells obtained from cells exhibiting at least one characteristic of a TSC produced according to the invention.

[0068] The present invention also provides organoids obtained from cells exhibiting at least one characteristic of a TSC produced according to the methods described herein, and / or differentiated cells (e.g., STs or EVTs) obtained from cells exhibiting at least one characteristic of a TSC produced according to the methods described herein.

[0069] In a further aspect, there is provided a method for treating and / or preventing a disorder associated with trophoblast development and / or activity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a cell or cell population exhibiting at least one characteristic of a TSC produced according to the methods described herein, a differentiated ST or EVT obtained from a cell exhibiting at least one characteristic of a TSC produced according to the methods described herein, or a pharmaceutical product according to the invention, thereby treating and / or preventing a disorder associated with trophoblast development and / or activity in the subject.

[0070] The present invention also provides the use of cells produced according to the present invention, or cell populations comprising cells, that exhibit at least one characteristic of TSC, in the manufacture of a medicament for treating a placental disease / disorder. For example, the reprogrammed cells of the present invention can be introduced (transplanted) into an individual to ameliorate a placental disease or condition. Alternatively, cells that exhibit at least one characteristic of TSC can be differentiated before use in the manufacture of a medicament described herein.

[0071] In embodiments of any of the above aspects, the disease is selected from the group consisting of recurrent miscarriage, pre-eclampsia, fetal growth restriction (FGR), hydratiform mole, and choriocarcinoma. The present invention also provides a method for identifying an agent capable of modulating trophoblast development and / or activity, the method comprising: - contacting isolated TSCs or placenta produced according to the present invention with a candidate agent; - comparing the development and / or activity of the isolated TSCs or placenta after contact with the agent with the development and / or activity of the TSCs or placenta without the agent; An effect of the agent on TSC or placental development and / or activity that is greater than a predetermined level compared to TSC or placental development and / or activity without the agent indicates that the agent modulates trophoblast development and / or activity.

[0072] Furthermore, the present invention provides a method for obtaining a compound produced by trophoblasts, which method comprises culturing a cell or cell population comprising at least one characteristic of a TSC according to the present invention, or a culture comprising the same, and isolating a compound secreted by the cells from the culture medium, thereby obtaining the compound produced by the trophoblasts.

[0073] The present invention also provides a method for obtaining a compound or particle secreted by an EVT or ST, the method comprising culturing an ST or EVT, or a population of STs or EVTs, obtained by differentiating a cell comprising at least one characteristic of a TSC according to the present invention, and isolating the compound or particle secreted by the cell from the culture medium, thereby obtaining the compound or particle produced by the EVT or ST.

[0074] The compound may be a hormone or a growth factor. The particle secreted by the cell may be an extracellular vesicle, such as an exosome.

[0075] The present invention also relates to kits for generating cells exhibiting at least one characteristic of TSCs as disclosed herein. In some embodiments, the kits include somatic cells, reprogramming factors, and TSC culture medium as disclosed herein. Preferably, the kits can be used to generate cells exhibiting at least one characteristic of TSCs. Preferably, the kits can be used with somatic cells that are fibroblasts. In some embodiments, the kits further include instructions for reprogramming somatic cells into cells exhibiting at least one characteristic of TSCs according to the methods disclosed herein. Preferably, the present invention provides kits for use in the methods of the present invention described herein.

[0076] The kit may also include one or more agents suitable for differentiating TSCs toward an EVT or ST fate, or other non-placental differentiated cell type.

[0077] Any of the methods described herein may have one or more or all steps performed in vitro, ex vivo or in vivo.

[0078] As used herein, unless the context otherwise requires, the term "comprise" and variations of that term, such as "comprising," "comprises," and "comprised," are not intended to exclude additional additives, components, integers, or steps.

[0079] Further aspects of the invention, and further embodiments of the aspects described in the preceding paragraphs, will become apparent from the following description, given by way of example, and with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0080] [Figure 1-1]Trophectoderm (TE) signatures are upregulated during human reprogramming. a) Heatmap of the intensity of the TE gene signature during fibroblast reprogramming to naive iPSCs, as defined by Petropoulos et al. (2016), Cell, 165:1012-26. (b) In vivo TE signatures on FDL projections overlaid with single-cell trajectories constructed using Monocle3 (black line). Blue arrows indicate TE-enriched cell populations. (c) Gene set enrichment analysis (GSEA) of EPI, PE, and TE gene signatures in reprogramming intermediates and iPSCs reprogrammed under primed and several naive culture conditions (Methods). (d) Normalized gene expression (average z-scaling) of genes associated with ATAC-seq cluster peaks. (e) Transcription factor motif enrichment analysis of ATAC-seq peak clusters. Motif enrichment (-log(P value)) heatmap by color and size, which is the percentage of sequences in the cluster that feature the motif. Red arrows point to OCT4, SOX2, NANOG, and KLF4 motifs in the transient ATAC-seq cluster (C3). Blue arrows indicate enrichment of TE-associated transcription factors TFAP2C and GATA2 (C7 and C8). [Figure 1-2]Trophectoderm (TE) signatures are upregulated during human reprogramming. a) Heatmap of the intensity of the TE gene signature during fibroblast reprogramming to naive iPSCs, as defined by Petropoulos et al. (2016), Cell, 165:1012-26. (b) In vivo TE signatures on FDL projections overlaid with single-cell trajectories constructed using Monocle3 (black line). Blue arrows indicate TE-enriched cell populations. (c) Gene set enrichment analysis (GSEA) of EPI, PE, and TE gene signatures in reprogramming intermediates and iPSCs reprogrammed under primed and several naive culture conditions (Methods). (d) Normalized gene expression (average z-scaling) of genes associated with ATAC-seq cluster peaks. (e) Transcription factor motif enrichment analysis of ATAC-seq peak clusters. Motif enrichment (-log(P value)) heatmap by color and size, which is the percentage of sequences in the cluster that feature the motif. Red arrows point to OCT4, SOX2, NANOG, and KLF4 motifs in the transient ATAC-seq cluster (C3). Blue arrows indicate enrichment of TE-associated transcription factors TFAP2C and GATA2 (C7 and C8). [Figure 1-3]Trophectoderm (TE) signatures are upregulated during human reprogramming. a) Heatmap of the intensity of the TE gene signature during fibroblast reprogramming to naive iPSCs, as defined by Petropoulos et al. (2016), Cell, 165:1012-26. (b) In vivo TE signatures on FDL projections overlaid with single-cell trajectories constructed using Monocle3 (black line). Blue arrows indicate TE-enriched cell populations. (c) Gene set enrichment analysis (GSEA) of EPI, PE, and TE gene signatures in reprogramming intermediates and iPSCs reprogrammed under primed and several naive culture conditions (Methods). (d) Normalized gene expression (average z-scaling) of genes associated with ATAC-seq cluster peaks. (e) Transcription factor motif enrichment analysis of ATAC-seq peak clusters. Motif enrichment (-log(P value)) heatmap by color and size, which is the percentage of sequences in the cluster that feature the motif. Red arrows point to OCT4, SOX2, NANOG, and KLF4 motifs in the transient ATAC-seq cluster (C3). Blue arrows indicate enrichment of TE-associated transcription factors TFAP2C and GATA2 (C7 and C8). [Figure 2] Characterization of iTSCs. A) Immunofluorescence (IF) labeling confirms that iTSCs express TE markers CK7, GATA3, nuclear VGLL1, and TFAP2C, but not NANOG (PSC marker). B) iTSC-derived EVT express HLA-G, and iTSC-ST express SDC1 and show multinucleation (arrows). DAPI nuclear staining is in blue. [Figure 3-1]Stabilization of TE signatures during naive programming for the derivation of human iTSCs. (a) Schematic of the experimental design for capturing and stabilizing naive intermediates in TSC medium. (b) Phase-contrast images of generated iTSCs. (c) Immunostaining of fibroblasts, primed, and naive t2iL GoY iPS cells with P63, TFAP2C, GATA2, and KRT7, n=2. Scale bar, 100 μm. (d) Immunostaining of iTSCd21n with P63, TFAP2C, GATA2, and KRT7. Scale bar, 100 μm. Representative image from n=4. (e) Gene expression of trophoblast genes in fibroblasts, primed, and naive t2iL GoY iPS cells, iTSCd21n, and TS cells (TSCs) derived from human blastocysts (TSCblasts) and first-trimester placental trophoblasts (TSCCTs), average of replicates, n=2. (f) Phase-contrast images of ST and EVT cells differentiated from iTSCd21n, n = 4. Scale bar, 100 μm. (g) SDC1 and HLA-G immunostaining of syncytiotrophoblast (ST) (left) and extravillous trophoblast (EVT) (right) cells differentiated from iTSCd21n, respectively. Scale bar, 100 μm. Representative images from n = 4. (h) Fusion index of iTSCd21n ST and iTSCd21n, n = 5. Data are expressed as mean ± sem. P values ​​by two-tailed unpaired Student's t-test. (i) Representative results of a commercially available hCG pregnancy test for the culture medium of ST cells differentiated from iTSCd21n and control culture medium, n = 6. (j) hCG levels in iTSCd21n- and iTSCd21n-ST conditioned medium detected by ELISA, n = 4. (k) Representation of iTSCd21n engraftment assay by injection into NOD-SCID mice. Urine, serum, and lesions were examined 9 days post-injection (dpi). Representative positive results for hCG pregnancy test from urine samples collected from iTSCd21n-injected mice compared to vehicle control. n=3. (l) hCG levels in mouse serum detected by ELISA, n=4. (m) Lesions collected from iTSCd21n subcutaneously engrafted into NOD-SCID mice, n=4. (n) H&E staining, immunohistochemical staining for KRT7, SDC1, and HLA-G in lesions collected from iTSCd21n engraftments in NOD-SCID mice.No obvious lesions were observed in the vehicle control. Arrows indicate HLA-G-positive trophoblast cells. Scale bar, 200 μm. Representative images from n=4. [Figure 3-2]Stabilization of TE signatures during naive programming for the derivation of human iTSCs. (a) Schematic of the experimental design for capturing and stabilizing naive intermediates in TSC medium. (b) Phase-contrast images of generated iTSCs. (c) Immunostaining of fibroblasts, primed, and naive t2iL GoY iPS cells with P63, TFAP2C, GATA2, and KRT7, n=2. Scale bar, 100 μm. (d) Immunostaining of iTSCd21n with P63, TFAP2C, GATA2, and KRT7. Scale bar, 100 μm. Representative image from n=4. (e) Gene expression of trophoblast genes in fibroblasts, primed, and naive t2iL GoY iPS cells, iTSCd21n, and TS cells (TSCs) derived from human blastocysts (TSCblasts) and first-trimester placental trophoblasts (TSCCTs), average of replicates, n=2. (f) Phase-contrast images of ST and EVT cells differentiated from iTSCd21n, n = 4. Scale bar, 100 μm. (g) SDC1 and HLA-G immunostaining of syncytiotrophoblast (ST) (left) and extravillous trophoblast (EVT) (right) cells differentiated from iTSCd21n, respectively. Scale bar, 100 μm. Representative images from n = 4. (h) Fusion index of iTSCd21n ST and iTSCd21n, n = 5. Data are expressed as mean ± sem. P values ​​by two-tailed unpaired Student's t-test. (i) Representative results of a commercially available hCG pregnancy test for the culture medium of ST cells differentiated from iTSCd21n and control culture medium, n = 6. (j) hCG levels in iTSCd21n- and iTSCd21n-ST conditioned medium detected by ELISA, n = 4. (k) Representation of iTSCd21n engraftment assay by injection into NOD-SCID mice. Urine, serum, and lesions were examined 9 days post-injection (dpi). Representative positive results for hCG pregnancy test from urine samples collected from iTSCd21n-injected mice compared to vehicle control. n=3. (l) hCG levels in mouse serum detected by ELISA, n=4. (m) Lesions collected from iTSCd21n subcutaneously engrafted into NOD-SCID mice, n=4. (n) H&E staining, immunohistochemical staining for KRT7, SDC1, and HLA-G in lesions collected from iTSCd21n engraftments in NOD-SCID mice.No obvious lesions were observed in the vehicle control. Arrows indicate HLA-G-positive trophoblast cells. Scale bar, 200 μm. Representative images from n=4. [Figure 3-3]Stabilization of TE signatures during naive programming for the derivation of human iTSCs. (a) Schematic of the experimental design for capturing and stabilizing naive intermediates in TSC medium. (b) Phase-contrast images of generated iTSCs. (c) Immunostaining of fibroblasts, primed, and naive t2iL GoY iPS cells with P63, TFAP2C, GATA2, and KRT7, n=2. Scale bar, 100 μm. (d) Immunostaining of iTSCd21n with P63, TFAP2C, GATA2, and KRT7. Scale bar, 100 μm. Representative image from n=4. (e) Gene expression of trophoblast genes in fibroblasts, primed, and naive t2iL GoY iPS cells, iTSCd21n, and TS cells (TSCs) derived from human blastocysts (TSCblasts) and first-trimester placental trophoblasts (TSCCTs), average of replicates, n=2. (f) Phase-contrast images of ST and EVT cells differentiated from iTSCd21n, n = 4. Scale bar, 100 μm. (g) SDC1 and HLA-G immunostaining of syncytiotrophoblast (ST) (left) and extravillous trophoblast (EVT) (right) cells differentiated from iTSCd21n, respectively. Scale bar, 100 μm. Representative images from n = 4. (h) Fusion index of iTSCd21n ST and iTSCd21n, n = 5. Data are expressed as mean ± sem. P values ​​by two-tailed unpaired Student's t-test. (i) Representative results of a commercially available hCG pregnancy test for the culture medium of ST cells differentiated from iTSCd21n and control culture medium, n = 6. (j) hCG levels in iTSCd21n- and iTSCd21n-ST conditioned medium detected by ELISA, n = 4. (k) Representation of iTSCd21n engraftment assay by injection into NOD-SCID mice. Urine, serum, and lesions were examined 9 days post-injection (dpi). Representative positive results for hCG pregnancy test from urine samples collected from iTSCd21n-injected mice compared to vehicle control. n=3. (l) hCG levels in mouse serum detected by ELISA, n=4. (m) Lesions collected from iTSCd21n subcutaneously engrafted into NOD-SCID mice, n=4. (n) H&E staining, immunohistochemical staining for KRT7, SDC1, and HLA-G in lesions collected from iTSCd21n engraftments in NOD-SCID mice.No obvious lesions were observed in the vehicle control. Arrows indicate HLA-G-positive trophoblast cells. Scale bar, 200 μm. Representative images from n=4. [Figure 4-1] Cellular heterogeneity of fibroblasts and iTSCd8 reprogramming intermediates revealed by scRNA-seq. (a) Experimental design and scRNA-seq library preparation for day 21 fibroblasts, naive, and TSCd8 reprogramming intermediates. (b) FDL representation of the scRNA-seq library for day 21 reprogramming intermediates (10,518 cells). (c) Intensity of the EPI signature on the FDL (10,518 cells). Cell populations not enriched for the EPI signature but enriched for the TE signature are indicated by purple arrows. (d) TE signature on the FDL projection. TE-like populations are highlighted and colored by the library. (e) Representation of 13 cell clusters from unsupervised clustering projected onto FDL: fibroblast medium cell cluster: D21fm1-D21fm7; naive programming cell cluster: D21nr1-D21nr3; trophoblast reprogramming cell cluster: D21tr1-D21tr3. (f) Contribution (%) of each scRNA-seq library to the composition of cell clusters. The D21tr1 cluster is indicated by a purple arrow. [Figure 4-2]Cellular heterogeneity of fibroblasts and iTSCd8 reprogramming intermediates revealed by scRNA-seq. (a) Experimental design and scRNA-seq library preparation for day 21 fibroblasts, naive, and TSCd8 reprogramming intermediates. (b) FDL representation of the scRNA-seq library for day 21 reprogramming intermediates (10,518 cells). (c) Intensity of the EPI signature on the FDL (10,518 cells). Cell populations not enriched for the EPI signature but enriched for the TE signature are indicated by purple arrows. (d) TE signature on the FDL projection. TE-like populations are highlighted and colored by the library. (e) Representation of 13 cell clusters from unsupervised clustering projected onto FDL: fibroblast medium cell cluster: D21fm1-D21fm7; naive programming cell cluster: D21nr1-D21nr3; trophoblast reprogramming cell cluster: D21tr1-D21tr3. (f) Contribution (%) of each scRNA-seq library to the composition of cell clusters. The D21tr1 cluster is indicated by a purple arrow. [Figure 5-1]Direct induction of iTSCs from human fibroblasts. (a) Experimental design for direct induction of iTSCd8 from fibroblasts. (b) Phase-contrast image of iTSCd8. Scale bar, 100 μm. (c) Immunostaining of iTSCd8 for several TS cell markers. Scale bar, 100 μm. (d) Sendai virus transgene in iTS cell lines with positive and negative controls, n = 6. (e) Gene expression of trophoblast genes in fibroblasts, primed iPSCs, naive t2iL GoY iPCs, iTSCd8, and iTSCd21n was compared with TSCs derived from human blastocysts (TSCblasts) and first-trimester placental trophoblasts (TSCCTs). Data are presented as averages (n = 2). (f) Spearman correlation between transcriptomes from this study and published datasets. Biological replicates (n ≥ 2) were averaged before correlation. Blast, blastocyst; blast / CT, blastocyst / cytotrophoblast; CT, cytotrophoblast. (g, h) Spearman correlation of transcriptomes of fibroblasts, primed and naive t2iL GoY iPS cells, iTSCd8 and iTSCd21n, iTSCd8 ST and iTSCd21n ST, iTSCd8 EVT, and iTSCd21n EVT, generated in this study using trophoblast organoid samples. (i) Expression of miRNAs from the chromosome 19 miRNA cluster normalized to 1,000× miR-103a expression in the indicated cell lines. Mean ± semND, not detected. The red dotted line indicates the level in primed iPS cells. n=2. (j) ATAC-seq signal at the ELF5 region in the indicated cell types. Mean of replicates (n=2). Peaks in TS cells are marked in gray. (k,l) Phase contrast and immunostaining of ST (k) and EVT (l) cells differentiated from iTSCd8. Scale bar, 100 μm. n=4. (m) Cell fusion index of iTSCd8 ST and iTSCd8. n=5. Data are mean ± sem. P values ​​by two-tailed unpaired Student's t-test. (n) Representative results of hCG pregnancy test obtained from the culture medium of ST cells differentiated from iTSCd8, n=6. (o) hCG levels in iTSCd8- and iTSCd8-ST-conditioned medium detected by ELISA, n=4.(p) Representative flow cytometry analysis of pan-HLA-A, B, C class I markers (W6 / 32), HLA-Bw4, and HLA-G in fibroblasts and EVT, n=4. (q) Representative flow cytometry analysis of pan-HLA class I markers (W6 / 32) and HLA-G in iTSCd8 EVT and iTSCd21n EVT. (r, s) Expression of ST genes in iTSCd8- and iTSCd21n-derived ST cells (r) and EVT gene expression in iTSCd8- and iTSCd21n-derived EVT cells (s). (t) Representative hCG testing of urine samples collected from iTSCd8-injected mice, n=3. (u) Lesions collected from iTSCd8 subcutaneously engrafted in NOD-SCID mice, n=4. (v) hCG protein levels detected by hCG ELISA using mouse serum samples, n=4. (w) Hematoxylin and eosin (H&E) and immunohistochemical staining for KRT7, SDC1, and HLA-G in lesions harvested from iTSCd8 grafts in NOD-SCID mice, n = 4. No obvious lesions were observed in vehicle controls. Scale bar, 200 μm. [Figure 5-2]Direct induction of iTSCs from human fibroblasts. (a) Experimental design for direct induction of iTSCd8 from fibroblasts. (b) Phase-contrast image of iTSCd8. Scale bar, 100 μm. (c) Immunostaining of iTSCd8 for several TS cell markers. Scale bar, 100 μm. (d) Sendai virus transgene in iTS cell lines with positive and negative controls, n = 6. (e) Gene expression of trophoblast genes in fibroblasts, primed iPSCs, naive t2iL GoY iPCs, iTSCd8, and iTSCd21n was compared with TSCs derived from human blastocysts (TSCblasts) and first-trimester placental trophoblasts (TSCCTs). Data are presented as averages (n = 2). (f) Spearman correlation between transcriptomes from this study and published datasets. Biological replicates (n ≥ 2) were averaged before correlation. Blast, blastocyst; blast / CT, blastocyst / cytotrophoblast; CT, cytotrophoblast. (g, h) Spearman correlation of transcriptomes of fibroblasts, primed and naive t2iL GoY iPS cells, iTSCd8 and iTSCd21n, iTSCd8 ST and iTSCd21n ST, iTSCd8 EVT, and iTSCd21n EVT, generated in this study using trophoblast organoid samples. (i) Expression of miRNAs from the chromosome 19 miRNA cluster normalized to 1,000× miR-103a expression in the indicated cell lines. Mean ± semND, not detected. The red dotted line indicates the level in primed iPS cells. n=2. (j) ATAC-seq signal at the ELF5 region in the indicated cell types. Mean of replicates (n=2). Peaks in TS cells are marked in gray. (k,l) Phase contrast and immunostaining of ST (k) and EVT (l) cells differentiated from iTSCd8. Scale bar, 100 μm. n=4. (m) Cell fusion index of iTSCd8 ST and iTSCd8. n=5. Data are mean ± sem. P values ​​by two-tailed unpaired Student's t-test. (n) Representative results of hCG pregnancy test obtained from the culture medium of ST cells differentiated from iTSCd8, n=6. (o) hCG levels in iTSCd8- and iTSCd8-ST-conditioned medium detected by ELISA, n=4.(p) Representative flow cytometry analysis of pan-HLA-A, B, C class I markers (W6 / 32), HLA-Bw4, and HLA-G in fibroblasts and EVT, n=4. (q) Representative flow cytometry analysis of pan-HLA class I markers (W6 / 32) and HLA-G in iTSCd8 EVT and iTSCd21n EVT. (r, s) Expression of ST genes in iTSCd8- and iTSCd21n-derived ST cells (r) and EVT gene expression in iTSCd8- and iTSCd21n-derived EVT cells (s). (t) Representative hCG testing of urine samples collected from iTSCd8-injected mice, n=3. (u) Lesions collected from iTSCd8 subcutaneously engrafted in NOD-SCID mice, n=4. (v) hCG protein levels detected by hCG ELISA using mouse serum samples, n=4. (w) Hematoxylin and eosin (H&E) and immunohistochemical staining for KRT7, SDC1, and HLA-G in lesions harvested from iTSCd8 grafts in NOD-SCID mice, n = 4. No obvious lesions were observed in vehicle controls. Scale bar, 200 μm. [Figure 5-3]Direct induction of iTSCs from human fibroblasts. (a) Experimental design for direct induction of iTSCd8 from fibroblasts. (b) Phase-contrast image of iTSCd8. Scale bar, 100 μm. (c) Immunostaining of iTSCd8 for several TS cell markers. Scale bar, 100 μm. (d) Sendai virus transgene in iTS cell lines with positive and negative controls, n = 6. (e) Gene expression of trophoblast genes in fibroblasts, primed iPSCs, naive t2iL GoY iPCs, iTSCd8, and iTSCd21n was compared with TSCs derived from human blastocysts (TSCblasts) and first-trimester placental trophoblasts (TSCCTs). Data are presented as averages (n = 2). (f) Spearman correlation between transcriptomes from this study and published datasets. Biological replicates (n ≥ 2) were averaged before correlation. Blast, blastocyst; blast / CT, blastocyst / cytotrophoblast; CT, cytotrophoblast. (g, h) Spearman correlation of transcriptomes of fibroblasts, primed and naive t2iL GoY iPS cells, iTSCd8 and iTSCd21n, iTSCd8 ST and iTSCd21n ST, iTSCd8 EVT, and iTSCd21n EVT, generated in this study using trophoblast organoid samples. (i) Expression of miRNAs from the chromosome 19 miRNA cluster normalized to 1,000× miR-103a expression in the indicated cell lines. Mean ± semND, not detected. The red dotted line indicates the level in primed iPS cells. n=2. (j) ATAC-seq signal at the ELF5 region in the indicated cell types. Mean of replicates (n=2). Peaks in TS cells are marked in gray. (k,l) Phase contrast and immunostaining of ST (k) and EVT (l) cells differentiated from iTSCd8. Scale bar, 100 μm. n=4. (m) Cell fusion index of iTSCd8 ST and iTSCd8. n=5. Data are mean ± sem. P values ​​by two-tailed unpaired Student's t-test. (n) Representative results of hCG pregnancy test obtained from the culture medium of ST cells differentiated from iTSCd8, n=6. (o) hCG levels in iTSCd8- and iTSCd8-ST-conditioned medium detected by ELISA, n=4.(p) Representative flow cytometry analysis of pan-HLA-A, B, C class I markers (W6 / 32), HLA-Bw4, and HLA-G in fibroblasts and EVT, n=4. (q) Representative flow cytometry analysis of pan-HLA class I markers (W6 / 32) and HLA-G in iTSCd8 EVT and iTSCd21n EVT. (r, s) Expression of ST genes in iTSCd8- and iTSCd21n-derived ST cells (r) and EVT gene expression in iTSCd8- and iTSCd21n-derived EVT cells (s). (t) Representative hCG testing of urine samples collected from iTSCd8-injected mice, n=3. (u) Lesions collected from iTSCd8 subcutaneously engrafted in NOD-SCID mice, n=4. (v) hCG protein levels detected by hCG ELISA using mouse serum samples, n=4. (w) Hematoxylin and eosin (H&E) and immunohistochemical staining for KRT7, SDC1, and HLA-G in lesions harvested from iTSCd8 grafts in NOD-SCID mice, n = 4. No obvious lesions were observed in vehicle controls. Scale bar, 200 μm. [Figure 5-4]Direct induction of iTSCs from human fibroblasts. (a) Experimental design for direct induction of iTSCd8 from fibroblasts. (b) Phase-contrast image of iTSCd8. Scale bar, 100 μm. (c) Immunostaining of iTSCd8 for several TS cell markers. Scale bar, 100 μm. (d) Sendai virus transgene in iTS cell lines with positive and negative controls, n = 6. (e) Gene expression of trophoblast genes in fibroblasts, primed iPSCs, naive t2iL GoY iPCs, iTSCd8, and iTSCd21n was compared with TSCs derived from human blastocysts (TSCblasts) and first-trimester placental trophoblasts (TSCCTs). Data are presented as averages (n = 2). (f) Spearman correlation between transcriptomes from this study and published datasets. Biological replicates (n ≥ 2) were averaged before correlation. Blast, blastocyst; blast / CT, blastocyst / cytotrophoblast; CT, cytotrophoblast. (g, h) Spearman correlation of transcriptomes of fibroblasts, primed and naive t2iL GoY iPS cells, iTSCd8 and iTSCd21n, iTSCd8 ST and iTSCd21n ST, iTSCd8 EVT, and iTSCd21n EVT, generated in this study using trophoblast organoid samples. (i) Expression of miRNAs from the chromosome 19 miRNA cluster normalized to 1,000× miR-103a expression in the indicated cell lines. Mean ± semND, not detected. The red dotted line indicates the level in primed iPS cells. n=2. (j) ATAC-seq signal at the ELF5 region in the indicated cell types. Mean of replicates (n=2). Peaks in TS cells are marked in gray. (k,l) Phase contrast and immunostaining of ST (k) and EVT (l) cells differentiated from iTSCd8. Scale bar, 100 μm. n=4. (m) Cell fusion index of iTSCd8 ST and iTSCd8. n=5. Data are mean ± sem. P values ​​by two-tailed unpaired Student's t-test. (n) Representative results of hCG pregnancy test obtained from the culture medium of ST cells differentiated from iTSCd8, n=6. (o) hCG levels in iTSCd8- and iTSCd8-ST-conditioned medium detected by ELISA, n=4.(p) Representative flow cytometry analysis of pan-HLA-A, B, C class I markers (W6 / 32), HLA-Bw4, and HLA-G in fibroblasts and EVT, n=4. (q) Representative flow cytometry analysis of pan-HLA class I markers (W6 / 32) and HLA-G in iTSCd8 EVT and iTSCd21n EVT. (r, s) Expression of ST genes in iTSCd8- and iTSCd21n-derived ST cells (r) and EVT gene expression in iTSCd8- and iTSCd21n-derived EVT cells (s). (t) Representative hCG testing of urine samples collected from iTSCd8-injected mice, n=3. (u) Lesions collected from iTSCd8 subcutaneously engrafted in NOD-SCID mice, n=4. (v) hCG protein levels detected by hCG ELISA using mouse serum samples, n=4. (w) Hematoxylin and eosin (H&E) and immunohistochemical staining for KRT7, SDC1, and HLA-G in lesions harvested from iTSCd8 grafts in NOD-SCID mice, n = 4. No obvious lesions were observed in vehicle controls. Scale bar, 200 μm. [Figure 5-5]Direct induction of iTSCs from human fibroblasts. (a) Experimental design for direct induction of iTSCd8 from fibroblasts. (b) Phase-contrast image of iTSCd8. Scale bar, 100 μm. (c) Immunostaining of iTSCd8 for several TS cell markers. Scale bar, 100 μm. (d) Sendai virus transgene in iTS cell lines with positive and negative controls, n = 6. (e) Gene expression of trophoblast genes in fibroblasts, primed iPSCs, naive t2iL GoY iPCs, iTSCd8, and iTSCd21n was compared with TSCs derived from human blastocysts (TSCblasts) and first-trimester placental trophoblasts (TSCCTs). Data are presented as averages (n = 2). (f) Spearman correlation between transcriptomes from this study and published datasets. Biological replicates (n ≥ 2) were averaged before correlation. Blast, blastocyst; blast / CT, blastocyst / cytotrophoblast; CT, cytotrophoblast. (g, h) Spearman correlation of transcriptomes of fibroblasts, primed and naive t2iL GoY iPS cells, iTSCd8 and iTSCd21n, iTSCd8 ST and iTSCd21n ST, iTSCd8 EVT, and iTSCd21n EVT, generated in this study using trophoblast organoid samples. (i) Expression of miRNAs from the chromosome 19 miRNA cluster normalized to 1,000× miR-103a expression in the indicated cell lines. Mean ± semND, not detected. The red dotted line indicates the level in primed iPS cells. n=2. (j) ATAC-seq signal at the ELF5 region in the indicated cell types. Mean of replicates (n=2). Peaks in TS cells are marked in gray. (k,l) Phase contrast and immunostaining of ST (k) and EVT (l) cells differentiated from iTSCd8. Scale bar, 100 μm. n=4. (m) Cell fusion index of iTSCd8 ST and iTSCd8. n=5. Data are mean ± sem. P values ​​by two-tailed unpaired Student's t-test. (n) Representative results of hCG pregnancy test obtained from the culture medium of ST cells differentiated from iTSCd8, n=6. (o) hCG levels in iTSCd8- and iTSCd8-ST-conditioned medium detected by ELISA, n=4.(p) Representative flow cytometry analysis of pan-HLA-A, B, C class I markers (W6 / 32), HLA-Bw4, and HLA-G in fibroblasts and EVT, n=4. (q) Representative flow cytometry analysis of pan-HLA class I markers (W6 / 32) and HLA-G in iTSCd8 EVT and iTSCd21n EVT. (r, s) Expression of ST genes in iTSCd8- and iTSCd21n-derived ST cells (r) and EVT gene expression in iTSCd8- and iTSCd21n-derived EVT cells (s). (t) Representative hCG testing of urine samples collected from iTSCd8-injected mice, n=3. (u) Lesions collected from iTSCd8 subcutaneously engrafted in NOD-SCID mice, n=4. (v) hCG protein levels detected by hCG ELISA using mouse serum samples, n=4. (w) Hematoxylin and eosin (H&E) and immunohistochemical staining for KRT7, SDC1, and HLA-G in lesions harvested from iTSCd8 grafts in NOD-SCID mice, n = 4. No obvious lesions were observed in vehicle controls. Scale bar, 200 μm. [Figure 5-6]Direct induction of iTSCs from human fibroblasts. (a) Experimental design for direct induction of iTSCd8 from fibroblasts. (b) Phase-contrast image of iTSCd8. Scale bar, 100 μm. (c) Immunostaining of iTSCd8 for several TS cell markers. Scale bar, 100 μm. (d) Sendai virus transgene in iTS cell lines with positive and negative controls, n = 6. (e) Gene expression of trophoblast genes in fibroblasts, primed iPSCs, naive t2iL GoY iPCs, iTSCd8, and iTSCd21n was compared with TSCs derived from human blastocysts (TSCblasts) and first-trimester placental trophoblasts (TSCCTs). Data are presented as averages (n = 2). (f) Spearman correlation between transcriptomes from this study and published datasets. Biological replicates (n ≥ 2) were averaged before correlation. Blast, blastocyst; blast / CT, blastocyst / cytotrophoblast; CT, cytotrophoblast. (g, h) Spearman correlation of transcriptomes of fibroblasts, primed and naive t2iL GoY iPS cells, iTSCd8 and iTSCd21n, iTSCd8 ST and iTSCd21n ST, iTSCd8 EVT, and iTSCd21n EVT, generated in this study using trophoblast organoid samples. (i) Expression of miRNAs from the chromosome 19 miRNA cluster normalized to 1,000× miR-103a expression in the indicated cell lines. Mean ± semND, not detected. The red dotted line indicates the level in primed iPS cells. n=2. (j) ATAC-seq signal at the ELF5 region in the indicated cell types. Mean of replicates (n=2). Peaks in TS cells are marked in gray. (k,l) Phase contrast and immunostaining of ST (k) and EVT (l) cells differentiated from iTSCd8. Scale bar, 100 μm. n=4. (m) Cell fusion index of iTSCd8 ST and iTSCd8. n=5. Data are mean ± sem. P values ​​by two-tailed unpaired Student's t-test. (n) Representative results of hCG pregnancy test obtained from the culture medium of ST cells differentiated from iTSCd8, n=6. (o) hCG levels in iTSCd8- and iTSCd8-ST-conditioned medium detected by ELISA, n=4.(p) Representative flow cytometry analysis of pan-HLA-A, B, C class I markers (W6 / 32), HLA-Bw4, and HLA-G in fibroblasts and EVT, n=4. (q) Representative flow cytometry analysis of pan-HLA class I markers (W6 / 32) and HLA-G in iTSCd8 EVT and iTSCd21n EVT. (r, s) Expression of ST genes in iTSCd8- and iTSCd21n-derived ST cells (r) and EVT gene expression in iTSCd8- and iTSCd21n-derived EVT cells (s). (t) Representative hCG testing of urine samples collected from iTSCd8-injected mice, n=3. (u) Lesions collected from iTSCd8 subcutaneously engrafted in NOD-SCID mice, n=4. (v) hCG protein levels detected by hCG ELISA using mouse serum samples, n=4. (w) Hematoxylin and eosin (H&E) and immunohistochemical staining for KRT7, SDC1, and HLA-G in lesions harvested from iTSCd8 grafts in NOD-SCID mice, n = 4. No obvious lesions were observed in vehicle controls. Scale bar, 200 μm. [Figure 5-7]Direct induction of iTSCs from human fibroblasts. (a) Experimental design for direct induction of iTSCd8 from fibroblasts. (b) Phase-contrast image of iTSCd8. Scale bar, 100 μm. (c) Immunostaining of iTSCd8 for several TS cell markers. Scale bar, 100 μm. (d) Sendai virus transgene in iTS cell lines with positive and negative controls, n = 6. (e) Gene expression of trophoblast genes in fibroblasts, primed iPSCs, naive t2iL GoY iPCs, iTSCd8, and iTSCd21n was compared with TSCs derived from human blastocysts (TSCblasts) and first-trimester placental trophoblasts (TSCCTs). Data are presented as averages (n = 2). (f) Spearman correlation between transcriptomes from this study and published datasets. Biological replicates (n ≥ 2) were averaged before correlation. Blast, blastocyst; blast / CT, blastocyst / cytotrophoblast; CT, cytotrophoblast. (g, h) Spearman correlation of transcriptomes of fibroblasts, primed and naive t2iL GoY iPS cells, iTSCd8 and iTSCd21n, iTSCd8 ST and iTSCd21n ST, iTSCd8 EVT, and iTSCd21n EVT, generated in this study using trophoblast organoid samples. (i) Expression of miRNAs from the chromosome 19 miRNA cluster normalized to 1,000× miR-103a expression in the indicated cell lines. Mean ± semND, not detected. The red dotted line indicates the level in primed iPS cells. n=2. (j) ATAC-seq signal at the ELF5 region in the indicated cell types. Mean of replicates (n=2). Peaks in TS cells are marked in gray. (k,l) Phase contrast and immunostaining of ST (k) and EVT (l) cells differentiated from iTSCd8. Scale bar, 100 μm. n=4. (m) Cell fusion index of iTSCd8 ST and iTSCd8. n=5. Data are mean ± sem. P values ​​by two-tailed unpaired Student's t-test. (n) Representative results of hCG pregnancy test obtained from the culture medium of ST cells differentiated from iTSCd8, n=6. (o) hCG levels in iTSCd8- and iTSCd8-ST-conditioned medium detected by ELISA, n=4.(p) Representative flow cytometry analysis of pan-HLA-A, B, C class I markers (W6 / 32), HLA-Bw4, and HLA-G in fibroblasts and EVT, n=4. (q) Representative flow cytometry analysis of pan-HLA class I markers (W6 / 32) and HLA-G in iTSCd8 EVT and iTSCd21n EVT. (r, s) Expression of ST genes in iTSCd8- and iTSCd21n-derived ST cells (r) and EVT gene expression in iTSCd8- and iTSCd21n-derived EVT cells (s). (t) Representative hCG testing of urine samples collected from iTSCd8-injected mice, n=3. (u) Lesions collected from iTSCd8 subcutaneously engrafted in NOD-SCID mice, n=4. (v) hCG protein levels detected by hCG ELISA using mouse serum samples, n=4. (w) Hematoxylin and eosin (H&E) and immunohistochemical staining for KRT7, SDC1, and HLA-G in lesions harvested from iTSCd8 grafts in NOD-SCID mice, n = 4. No obvious lesions were observed in vehicle controls. Scale bar, 200 μm. [Figure 5-8]Direct induction of iTSCs from human fibroblasts. (a) Experimental design for direct induction of iTSCd8 from fibroblasts. (b) Phase-contrast image of iTSCd8. Scale bar, 100 μm. (c) Immunostaining of iTSCd8 for several TS cell markers. Scale bar, 100 μm. (d) Sendai virus transgene in iTS cell lines with positive and negative controls, n = 6. (e) Gene expression of trophoblast genes in fibroblasts, primed iPSCs, naive t2iL GoY iPCs, iTSCd8, and iTSCd21n was compared with TSCs derived from human blastocysts (TSCblasts) and first-trimester placental trophoblasts (TSCCTs). Data are presented as averages (n = 2). (f) Spearman correlation between transcriptomes from this study and published datasets. Biological replicates (n ≥ 2) were averaged before correlation. Blast, blastocyst; blast / CT, blastocyst / cytotrophoblast; CT, cytotrophoblast. (g, h) Spearman correlation of transcriptomes of fibroblasts, primed and naive t2iL GoY iPS cells, iTSCd8 and iTSCd21n, iTSCd8 ST and iTSCd21n ST, iTSCd8 EVT, and iTSCd21n EVT, generated in this study using trophoblast organoid samples. (i) Expression of miRNAs from the chromosome 19 miRNA cluster normalized to 1,000× miR-103a expression in the indicated cell lines. Mean ± semND, not detected. The red dotted line indicates the level in primed iPS cells. n=2. (j) ATAC-seq signal at the ELF5 region in the indicated cell types. Mean of replicates (n=2). Peaks in TS cells are marked in gray. (k,l) Phase contrast and immunostaining of ST (k) and EVT (l) cells differentiated from iTSCd8. Scale bar, 100 μm. n=4. (m) Cell fusion index of iTSCd8 ST and iTSCd8. n=5. Data are mean ± sem. P values ​​by two-tailed unpaired Student's t-test. (n) Representative results of hCG pregnancy test obtained from the culture medium of ST cells differentiated from iTSCd8, n=6. (o) hCG levels in iTSCd8- and iTSCd8-ST-conditioned medium detected by ELISA, n=4.(p) Representative flow cytometry analysis of pan-HLA-A, B, C class I markers (W6 / 32), HLA-Bw4, and HLA-G in fibroblasts and EVT, n=4. (q) Representative flow cytometry analysis of pan-HLA class I markers (W6 / 32) and HLA-G in iTSCd8 EVT and iTSCd21n EVT. (r, s) Expression of ST genes in iTSCd8- and iTSCd21n-derived ST cells (r) and EVT gene expression in iTSCd8- and iTSCd21n-derived EVT cells (s). (t) Representative hCG testing of urine samples collected from iTSCd8-injected mice, n=3. (u) Lesions collected from iTSCd8 subcutaneously engrafted in NOD-SCID mice, n=4. (v) hCG protein levels detected by hCG ELISA using mouse serum samples, n=4. (w) Hematoxylin and eosin (H&E) and immunohistochemical staining for KRT7, SDC1, and HLA-G in lesions harvested from iTSCd8 grafts in NOD-SCID mice, n = 4. No obvious lesions were observed in vehicle controls. Scale bar, 200 μm. [Figure 6] Derivation of iTSCs from day 21 human fibroblast reprogramming intermediates. (a) Experimental design to verify the potential of day 21 fibroblast reprogramming intermediates for the derivation of primed and naive iPSCs and iTSCs. (b) Phase-contrast images of primed and naive iPSCs and iTSCs generated from day 21 fibroblast reprogramming intermediates, n = 2. Scale bar, 50 μm. Immunostaining of primed and naive iPSCs and iTSCs using NANOG, KLF17, NR2F2, KRT7, and DAPI for nuclear staining, n = 2. Scale bar, 200 μm. (c) Reverse transcription-qPCR analysis of NANOG, ZIC2, KLF17, DPPA3, GATA2, and KRT7 expression in primed and naive iPSCs and iTSCs generated from day 21 fibroblast reprogramming intermediates, n = 3. Data are mean ± sem. [Figure 7-1]Derivation of iTSCs from partially reprogrammed intermediates, conversion of naive and expanded iPSCs to TSCs. (a) Schematic of the reprogramming protocol. Phase-contrast photographs show changes in cell morphology. Placenta-derived hTSCs are shown as a control. (b) Schematic of the conversion protocol. Phase-contrast photographs show changes in cell morphology. (c) Heatmap of Pearson correlation coefficients for hPSCs, hEPS, hNPSCs, hiTSCs, hcTSCs, and hTSC lines, as well as STs and EVTs differentiated from hTSCs. Correlations are determined from 2770 majority comparisons across the distributed genes of the dataset (see Methods). Samples are clustered from the Euclidean distance of correlations by hierarchical clustering using Ward's method. (d) Gene expression levels of the indicated lineage markers are shown for hPSCs, hEPS, hNPSCs, hiTSCs, hcTSCs, and previously established embryo- and placenta-derived hTSC lines. Differentiated STs and EVTs are included as controls. Expression levels are given as transcripts per million mRNA molecules. A Wilcoxon-Mann-Whitney statistical test was performed for each type of hPSC and hTSC, with embryo- and placenta-derived hTSCs as the reference group. Asterisks indicate statistical significance of differences: *p<0.05; **p<0.01; ***p<0.001. (e) Immunofluorescence images of hTSCs, hiTSCs, hcTSCs, hNPSCs, and hEPS stained for the trophoblast-associated transcription factors GATA2 and NR2F2 and the pluripotency-associated transcription factor SOX2. Nuclei were stained with DAPI. Scale bar: 100 μm. (f) Schematic of the EVT differentiation protocol (left). Brightfield photograph of EVT progeny of h(i / c)TSCs (right). (g) Schematic of the 3D-ST differentiation protocol (left). Brightfield image of a 3D-ST structure derived from h(i / c)TSCs (right). (h) RT-qPCR quantification of markers associated with ST (CGA, CGB, SDC1), EVT (HLA-G, MMP2, ASCL2), and hTSC (LRP2, CDKN3). Wilcoxon-Mann-Whitney statistical tests were performed for each type of hTSC and differentiated cell progeny. Asterisks indicate statistical significance of differences: * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001.(i) Immunofluorescence images of 3D-ST structures derived from hiTSCs, hcTSCs, and hTSC lines stained for GATA3 and the membrane-associated protein desmoplakin (DSP), as well as the syncytiotrophoblast-associated marker CGB, highlighting syncytia. Nuclei were stained with DAPI. (j) Immunofluorescence images of EVT differentiated from hiTSCs, hcTSCs, and placenta-derived hTSC lines stained for the trophoblast-associated transcription factor GATA3 and the extravillous trophoblast-specific surface marker HLA-G. Nuclei were stained with DAPI. Scale bar (fg): 100 μm; (ij): 30 μm. [Figure 7-2]Derivation of iTSCs from partially reprogrammed intermediates, conversion of naive and expanded iPSCs to TSCs. (a) Schematic of the reprogramming protocol. Phase-contrast photographs show changes in cell morphology. Placenta-derived hTSCs are shown as a control. (b) Schematic of the conversion protocol. Phase-contrast photographs show changes in cell morphology. (c) Heatmap of Pearson correlation coefficients for hPSCs, hEPS, hNPSCs, hiTSCs, hcTSCs, and hTSC lines, as well as STs and EVTs differentiated from hTSCs. Correlations are determined from 2770 majority comparisons across the distributed genes of the dataset (see Methods). Samples are clustered from the Euclidean distance of correlations by hierarchical clustering using Ward's method. (d) Gene expression levels of the indicated lineage markers are shown for hPSCs, hEPS, hNPSCs, hiTSCs, hcTSCs, and previously established embryo- and placenta-derived hTSC lines. Differentiated STs and EVTs are included as controls. Expression levels are given as transcripts per million mRNA molecules. A Wilcoxon-Mann-Whitney statistical test was performed for each type of hPSC and hTSC, with embryo- and placenta-derived hTSCs as the reference group. Asterisks indicate statistical significance of differences: *p<0.05; **p<0.01; ***p<0.001. (e) Immunofluorescence images of hTSCs, hiTSCs, hcTSCs, hNPSCs, and hEPS stained for the trophoblast-associated transcription factors GATA2 and NR2F2 and the pluripotency-associated transcription factor SOX2. Nuclei were stained with DAPI. Scale bar: 100 μm. (f) Schematic of the EVT differentiation protocol (left). Brightfield photograph of EVT progeny of h(i / c)TSCs (right). (g) Schematic of the 3D-ST differentiation protocol (left). Brightfield image of a 3D-ST structure derived from h(i / c)TSCs (right). (h) RT-qPCR quantification of markers associated with ST (CGA, CGB, SDC1), EVT (HLA-G, MMP2, ASCL2), and hTSC (LRP2, CDKN3). Wilcoxon-Mann-Whitney statistical tests were performed for each type of hTSC and differentiated cell progeny. Asterisks indicate statistical significance of differences: * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001.(i) Immunofluorescence images of 3D-ST structures derived from hiTSCs, hcTSCs, and hTSC lines stained for GATA3 and the membrane-associated protein desmoplakin (DSP), as well as the syncytiotrophoblast-associated marker CGB, highlighting syncytia. Nuclei were stained with DAPI. (j) Immunofluorescence images of EVT differentiated from hiTSCs, hcTSCs, and placenta-derived hTSC lines stained for the trophoblast-associated transcription factor GATA3 and the extravillous trophoblast-specific surface marker HLA-G. Nuclei were stained with DAPI. Scale bar (fg): 100 μm; (ij): 30 μm. [Figure 7-3]Derivation of iTSCs from partially reprogrammed intermediates, conversion of naive and expanded iPSCs to TSCs. (a) Schematic of the reprogramming protocol. Phase-contrast photographs show changes in cell morphology. Placenta-derived hTSCs are shown as a control. (b) Schematic of the conversion protocol. Phase-contrast photographs show changes in cell morphology. (c) Heatmap of Pearson correlation coefficients for hPSCs, hEPS, hNPSCs, hiTSCs, hcTSCs, and hTSC lines, as well as STs and EVTs differentiated from hTSCs. Correlations are determined from 2770 majority comparisons across the distributed genes of the dataset (see Methods). Samples are clustered from the Euclidean distance of correlations by hierarchical clustering using Ward's method. (d) Gene expression levels of the indicated lineage markers are shown for hPSCs, hEPS, hNPSCs, hiTSCs, hcTSCs, and previously established embryo- and placenta-derived hTSC lines. Differentiated STs and EVTs are included as controls. Expression levels are given as transcripts per million mRNA molecules. A Wilcoxon-Mann-Whitney statistical test was performed for each type of hPSC and hTSC, with embryo- and placenta-derived hTSCs as the reference group. Asterisks indicate statistical significance of differences: *p<0.05; **p<0.01; ***p<0.001. (e) Immunofluorescence images of hTSCs, hiTSCs, hcTSCs, hNPSCs, and hEPS stained for the trophoblast-associated transcription factors GATA2 and NR2F2 and the pluripotency-associated transcription factor SOX2. Nuclei were stained with DAPI. Scale bar: 100 μm. (f) Schematic of the EVT differentiation protocol (left). Brightfield photograph of EVT progeny of h(i / c)TSCs (right). (g) Schematic of the 3D-ST differentiation protocol (left). Brightfield image of a 3D-ST structure derived from h(i / c)TSCs (right). (h) RT-qPCR quantification of markers associated with ST (CGA, CGB, SDC1), EVT (HLA-G, MMP2, ASCL2), and hTSC (LRP2, CDKN3). Wilcoxon-Mann-Whitney statistical tests were performed for each type of hTSC and differentiated cell progeny. Asterisks indicate statistical significance of differences: * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001.(i) Immunofluorescence images of 3D-ST structures derived from hiTSCs, hcTSCs, and hTSC lines stained for GATA3 and the membrane-associated protein desmoplakin (DSP), as well as the syncytiotrophoblast-associated marker CGB, highlighting syncytia. Nuclei were stained with DAPI. (j) Immunofluorescence images of EVT differentiated from hiTSCs, hcTSCs, and placenta-derived hTSC lines stained for the trophoblast-associated transcription factor GATA3 and the extravillous trophoblast-specific surface marker HLA-G. Nuclei were stained with DAPI. Scale bar (fg): 100 μm; (ij): 30 μm. [Figure 7-4]Derivation of iTSCs from partially reprogrammed intermediates, conversion of naive and expanded iPSCs to TSCs. (a) Schematic of the reprogramming protocol. Phase-contrast photographs show changes in cell morphology. Placenta-derived hTSCs are shown as a control. (b) Schematic of the conversion protocol. Phase-contrast photographs show changes in cell morphology. (c) Heatmap of Pearson correlation coefficients for hPSCs, hEPS, hNPSCs, hiTSCs, hcTSCs, and hTSC lines, as well as STs and EVTs differentiated from hTSCs. Correlations are determined from 2770 majority comparisons across the distributed genes of the dataset (see Methods). Samples are clustered from the Euclidean distance of correlations by hierarchical clustering using Ward's method. (d) Gene expression levels of the indicated lineage markers are shown for hPSCs, hEPS, hNPSCs, hiTSCs, hcTSCs, and previously established embryo- and placenta-derived hTSC lines. Differentiated STs and EVTs are included as controls. Expression levels are given as transcripts per million mRNA molecules. A Wilcoxon-Mann-Whitney statistical test was performed for each type of hPSC and hTSC, with embryo- and placenta-derived hTSCs as the reference group. Asterisks indicate statistical significance of differences: *p<0.05; **p<0.01; ***p<0.001. (e) Immunofluorescence images of hTSCs, hiTSCs, hcTSCs, hNPSCs, and hEPS stained for the trophoblast-associated transcription factors GATA2 and NR2F2 and the pluripotency-associated transcription factor SOX2. Nuclei were stained with DAPI. Scale bar: 100 μm. (f) Schematic of the EVT differentiation protocol (left). Brightfield photograph of EVT progeny of h(i / c)TSCs (right). (g) Schematic of the 3D-ST differentiation protocol (left). Brightfield image of a 3D-ST structure derived from h(i / c)TSCs (right). (h) RT-qPCR quantification of markers associated with ST (CGA, CGB, SDC1), EVT (HLA-G, MMP2, ASCL2), and hTSC (LRP2, CDKN3). Wilcoxon-Mann-Whitney statistical tests were performed for each type of hTSC and differentiated cell progeny. Asterisks indicate statistical significance of differences: * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001.(i) Immunofluorescence images of 3D-ST structures derived from hiTSCs, hcTSCs, and hTSC lines stained for GATA3 and the membrane-associated protein desmoplakin (DSP), as well as the syncytiotrophoblast-associated marker CGB, highlighting syncytia. Nuclei were stained with DAPI. (j) Immunofluorescence images of EVT differentiated from hiTSCs, hcTSCs, and placenta-derived hTSC lines stained for the trophoblast-associated transcription factor GATA3 and the extravillous trophoblast-specific surface marker HLA-G. Nuclei were stained with DAPI. Scale bar (fg): 100 μm; (ij): 30 μm. [Figure 7-5]Derivation of iTSCs from partially reprogrammed intermediates, conversion of naive and expanded iPSCs to TSCs. (a) Schematic of the reprogramming protocol. Phase-contrast photographs show changes in cell morphology. Placenta-derived hTSCs are shown as a control. (b) Schematic of the conversion protocol. Phase-contrast photographs show changes in cell morphology. (c) Heatmap of Pearson correlation coefficients for hPSCs, hEPS, hNPSCs, hiTSCs, hcTSCs, and hTSC lines, as well as STs and EVTs differentiated from hTSCs. Correlations are determined from 2770 majority comparisons across the distributed genes of the dataset (see Methods). Samples are clustered from the Euclidean distance of correlations by hierarchical clustering using Ward's method. (d) Gene expression levels of the indicated lineage markers are shown for hPSCs, hEPS, hNPSCs, hiTSCs, hcTSCs, and previously established embryo- and placenta-derived hTSC lines. Differentiated STs and EVTs are included as controls. Expression levels are given as transcripts per million mRNA molecules. A Wilcoxon-Mann-Whitney statistical test was performed for each type of hPSC and hTSC, with embryo- and placenta-derived hTSCs as the reference group. Asterisks indicate statistical significance of differences: *p<0.05; **p<0.01; ***p<0.001. (e) Immunofluorescence images of hTSCs, hiTSCs, hcTSCs, hNPSCs, and hEPS stained for the trophoblast-associated transcription factors GATA2 and NR2F2 and the pluripotency-associated transcription factor SOX2. Nuclei were stained with DAPI. Scale bar: 100 μm. (f) Schematic of the EVT differentiation protocol (left). Brightfield photograph of EVT progeny of h(i / c)TSCs (right). (g) Schematic of the 3D-ST differentiation protocol (left). Brightfield image of a 3D-ST structure derived from h(i / c)TSCs (right). (h) RT-qPCR quantification of markers associated with ST (CGA, CGB, SDC1), EVT (HLA-G, MMP2, ASCL2), and hTSC (LRP2, CDKN3). Wilcoxon-Mann-Whitney statistical tests were performed for each type of hTSC and differentiated cell progeny. Asterisks indicate statistical significance of differences: * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001.(i) Immunofluorescence images of 3D-ST structures derived from hiTSCs, hcTSCs, and hTSC lines stained for GATA3 and the membrane-associated protein desmoplakin (DSP), as well as the syncytiotrophoblast-associated marker CGB, highlighting syncytia. Nuclei were stained with DAPI. (j) Immunofluorescence images of EVT differentiated from hiTSCs, hcTSCs, and placenta-derived hTSC lines stained for the trophoblast-associated transcription factor GATA3 and the extravillous trophoblast-specific surface marker HLA-G. Nuclei were stained with DAPI. Scale bar (fg): 100 μm; (ij): 30 μm. DETAILED DESCRIPTION OF THE INVENTION

[0081] Reference will now be made in detail to specific embodiments of the invention. While the invention will be described in connection with the embodiments, it will be understood that the invention is not limited to these embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents which may be included within the scope of the present invention as defined by the claims.

[0082] Those skilled in the art will recognize many methods and materials that are similar or equivalent to those described herein. The present invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

[0083] For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0084] The present invention relates to a novel method for generating human trophoblast stem cells (hTSCs) directly from somatic cells by nuclear reprogramming. Previously, published methods for generating TSCs have only involved isolating TSCs from primary tissues (e.g., first-trimester placenta or blastocysts) or differentiating "extended potential stem cells" (EPSCs) into TSCs. The present invention enables the generation of human "induced" trophoblast stem cells (iTSCs) from easily accessible patient-specific sources (e.g., fibroblasts), thus eliminating the reliance on obtaining tissue sources of such cells (e.g., placenta) or difficult-to-generate EPSCs.

[0085] While studying the reprogramming of fibroblasts to different pluripotent states, we identified an intermediate cell population that transiently upregulates the trophectoderm (TE) transcriptional network. By stabilizing the intermediate TE state and transferring the reprogrammed cells from pluripotency medium to a medium capable of supporting TSCs, we devised a method for generating "induced trophoblast stem cells" (iTSCs) from somatic cells.

[0086] Characterization of iTSCs generated according to our methods demonstrates that these cells express key factors that define human TEs and TSCs. For example, the cells express nuclear NR2F2, a marker of PCNA+ proliferating cells in the human placenta. TSCs generated according to the present invention are proliferative and maintain TSC characteristics through multiple passages without a decline in growth rate. Furthermore, these cells closely resemble TSCs derived from human blastocysts or first-trimester placentas, both molecularly and functionally. Functional characterization of TSCs generated according to the present methods demonstrates that these cells can differentiate into either syncytiotrophoblasts (ST) or extravillous trophoblasts (EVT). Importantly, these assays demonstrate that iTSCs generated according to the present methods are indeed bipotential and can differentiate into ST and EVT cells.

[0087] Human trophoblast stem cells (TSCs) derived from primary placental tissue or human blastocysts are difficult to access and highly regulated. Therefore, having stable, self-renewing iTSC lines that can be derived from adult cells offers a unique opportunity to study not only human trophoblast development, but also its relationship to pluripotent cells and its role in regulating events associated with early human embryonic development in an in vitro setting where modern biochemical and molecular techniques can be applied at scale. iTSC lines can also be used for disease modeling, drug screening, and regenerative medicine. Furthermore, iTSCs may be useful for generating organoids that resemble the placenta.

[0088] Cells generated according to the present invention may also find utility in a variety of other clinical applications, including the generation of trophoblast organoids to study maternal-fetal transmission of xenobiotics, drugs and pathogens, proteins, and hormones. Furthermore, human TSCs / iTSCs and iPSCs derived from the somatic cells of the same healthy individual or patient can be used to assemble human blastocyst-like structures. This provides an unlimited source of synthetic human blastocyst-like organoids for large-scale screening studies, including those to treat infertility and improve IVF success rates. Furthermore, iTSCs generated according to the present invention can be used in regenerative medicine. For example, placental cells have recently been shown to be useful for regenerating cardiac tissue. The iTSCs of the present invention can be used to generate such placental cells without the need to obtain cells directly from the placenta.

[0089] Compared with current methods of deriving human TSCs from human placenta or embryos, our reprogramming approach to generate human iTSCs is more accessible, labor- and cost-effective, without ethical constraints, and allows for an unlimited supply of isogenic iTSCs for large-scale screening studies using disease-specific iTSCs generated from patients.

[0090] cell Cells for use in accordance with the methods of the present invention can be any cell type described herein, including somatic cells or diseased cells. Somatic cells can be adult cells or cells derived from adults that exhibit one or more detectable characteristics of adult or non-embryonic cells. Diseased cells can be cells that exhibit one or more detectable characteristics of a disease or condition.

[0091] Somatic cells for use in accordance with the present invention may be derived from iPSCs or other embryonic or adult stem cells, or may be derived from tissue explants derived from a subject.

[0092] In a preferred embodiment, the somatic cell is a fibroblast (preferably a dermal fibroblast or a cardiac fibroblast), a keratinocyte (preferably an epidermal keratinocyte), a monocyte, or an endothelial cell.

[0093] As used herein, the term "stem cell" refers to a cell that is not terminally differentiated, i.e., capable of differentiating into other cell types with more specific, specialized functions. The term encompasses embryonic stem cells, fetal stem cells, adult stem cells, or committed / progenitor cells.

[0094] As used herein, "somatic cell" refers to a cell that has finally differentiated. As used herein, the term "somatic cell" refers to any cell that forms the body of an organism, as opposed to a germline cell. In mammals, germline cells (also known as "gametes") are sperm and eggs, which fuse at fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. All other cell types in a mammal's body (except sperm and eggs, the cells from which sperm and eggs are made (gametocytes), and undifferentiated stem cells) are somatic cells. The organs, skin, bone, blood, and connective tissues in the body are all made up of somatic cells. In some embodiments, a somatic cell is a "non-embryonic somatic cell," which means a somatic cell that is not present in or obtained from an embryo and does not result from the propagation of such a cell in vitro. In some embodiments, a somatic cell is an "adult somatic cell," which means a cell that is present in or obtained from an organism other than an embryo or fetus, or a cell that results from the propagation of such a cell in vitro. Somatic cells can be immortalized to provide an unlimited supply of cells, for example, by increasing the levels of telomerase reverse transcriptase (TERT). For example, the levels of TERT can be increased by increasing the transcription of TERT from an endogenous gene or by introducing a transgene via any gene delivery method or system.

[0095] Differentiated somatic cells, including cells derived from humans, including fetal, neonatal, juvenile, or adult primates, are suitable somatic cells for the methods of the present invention. Suitable somatic cells include, but are not limited to, bone marrow cells, epithelial cells, endothelial cells, fibroblasts, hematopoietic cells, keratinocytes, hepatocytes, intestinal cells, mesenchymal cells, bone marrow progenitor cells, and spleen cells. Alternatively, somatic cells can be cells that can proliferate and differentiate into other types of cells, including blood stem cells, muscle / bone stem cells, brain stem cells, and liver stem cells. Suitable somatic cells are either competent or can be made competent to incorporate transcription factors, including genetic material encoding the transcription factors, using methods commonly known in the scientific literature. The uptake enhancement method may vary depending on the cell type and expression system. Exemplary conditions used to prepare competent cells with suitable transduction efficiency are well known to those skilled in the art. Starting somatic cells can have a doubling time of approximately 24 hours.

[0096] As used herein, the term "isolated cell" refers to a cell that has been removed from an organism in which it was originally found, or the progeny of such a cell. Optionally, the cell has been cultured in vitro, e.g., in the presence of other cells. Optionally, the cell is later introduced into a second organism, or reintroduced into the organism from which it was isolated (or the cell from which it was derived).

[0097] As used herein, the term "isolated population" in reference to an isolated cell population refers to a population of cells that has been removed and separated from a mixed or heterogeneous population of cells. In some embodiments, an isolated population is a substantially pure population of cells as compared to the heterogeneous population from which the cells are isolated or enriched.

[0098] The term "substantially pure," with respect to a particular cell population, refers to a population of cells that is at least about 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure, with respect to the cells that make up the entire cell population. Rephrased, the terms "substantially pure" or "essentially purified," with respect to a population of target cells, i.e., cells that exhibit at least one characteristic of a TSC, refer to a population of cells that contains less than about 20%, more preferably about 15%, 10%, 8%, 7%, and most preferably about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not target cells or their progeny as defined by the terms herein.

[0099] Those skilled in the art will also be familiar with means for distinguishing somatic cell characteristics from TSC characteristics (i.e., examining the disappearance of somatic cell phenotypes). For example, as provided in the Examples herein, suitable somatic cells for generating TSCs include fibroblasts. Those skilled in the art can easily distinguish the characteristics of fibroblasts from TSCs; for example, fibroblasts are typically positive for CD44. Those skilled in the art can determine the conversion of fibroblasts to TSCs by observing decreased expression of this marker.

[0100] Other markers whose expression changed during the conversion of somatic cells to a pluripotent state include TWIST1, TWIST2, SNAI1, SNAI2, ZEB1 and ZEB2.

[0101] One skilled in the art will also be able to determine when a somatic cell has begun reprogramming toward a pluripotent state when it loses morphological characteristics typical of somatic cells. Again, one skilled in the art is familiar with the morphological characteristics of various somatic cells, including those of fibroblasts.

[0102] A somatic cell can also be determined to be reprogrammed to a pluripotent state if it exhibits at least one characteristic of a pluripotent cell (e.g., iPSC or ESC). The one or more characteristics of a pluripotent cell (e.g., iPSC or ESC) include upregulation of any one or more ESC markers and / or changes in cell morphology. Typically, a cell converted into an iPSC / EPSC-like cell exhibits one, two, three, four, five, six, seven, eight, or more characteristics of an iPSC / EPSC.

[0103] A somatic cell is determined to have been converted to a TSC if it exhibits at least one characteristic of a TSC. For example, a fibroblast is identified as having been converted to a TSC if the fibroblast treated according to the methods of the present invention exhibits at least one characteristic of a TSC.

[0104] The one or more characteristics of a TSC include upregulation of any one or more trophoblast markers and / or changes in cell morphology. Typically, cells that have converted to a TSC exhibit one, two, three, four, five, six, seven, eight or more characteristics of a TSC.

[0105] As used herein, cells produced according to the present invention that exhibit at least one characteristic of TSCs may also be referred to as "induced trophoblast stem cells" or iTSCs.

[0106] In any embodiment of the invention, protein markers characteristic of TSC nuclear CD49f (iTGA6), CD249 (aminopeptidase A), and nuclear NR2F2, TFAP2C, GATA2 / 3 and p63.

[0107] In further embodiments, cells exhibiting at least one characteristic of a TSC do not express one or more of the following markers: OCT4 (POU5F1), NANOG, SOX2, SALL2, OTX2, BANCR, KLF17, DPPA3, and DNMT3L.

[0108] Furthermore, cells that have at least one characteristic of a TSC have the potential to differentiate into cells that exhibit one or more characteristics of an EVT or ST.

[0109] Further markers that can be used to determine whether somatic cells have been reprogrammed or converted into TSCs are known to those skilled in the art. Examples of suitable markers are disclosed, for example, in Okae et al., (2018) Cell Stem Cell 22:50-63, Deglincerti et al., (2016) Nature, 533:751-4, Shahbazi et al., (2016 Nature Cell Biology 18:700-708 and Niakan & Eggan (2013) Dev Biol 375:54-64), the entire contents of which are incorporated herein by reference.

[0110] Those skilled in the art are familiar with methods for determining the conversion of fibroblasts to TSCs by observing changes in cell morphology, for example, adult fibroblasts are characterized by having an elongated, branched cytoplasm surrounding an oval, speckled nucleus.

[0111] In contrast, TSCs typically form large, cobblestone-like colonies.

[0112] In any embodiment of the present invention, TSC characteristics may be determined by analysis of cell morphology, gene expression profile, activity assay, protein expression profile, surface marker profile, differentiation capacity, or a combination thereof. Exemplary characteristics or markers include those described herein and known to those of skill in the art.

[0113] Reprogramming Various methods are known in the art for reprogramming somatic cells toward a pluripotent state. Reprogramming of somatic cells typically involves the expression of reprogramming factors (including transcription factors), followed by culture in specific conditions to promote the loss of somatic markers (i.e., loss of differentiation) and the potential to become cell types of early embryonic origin.

[0114] Examples of suitable methods for reprogramming somatic cells are abundant in the art and are exemplified in WO 2009 / 101407, WO 2014 / 200030, WO 2015 / 056804, WO 2014 / 200114, WO 2014 / 065435, WO 2013 / 176233, WO 2012 / 060473, WO 2012 / 036299, WO 2011 / 158967, WO 2011 / 055851, WO 2011 / 037270, WO 2011 / 090221, the contents of which are incorporated herein by reference.

[0115] Particularly preferred transcription factors and their nucleic acid sequences that can be used to reprogram somatic cells (e.g., fibroblasts) according to the methods of the present invention are set forth below in Table 1. However, it will be understood that the present invention is not limited to the use of the transcription factors listed in Table 1 to reprogram somatic cells.

[0116] Transcription factors and other protein factors referred to herein are referred to by HUGO Gene Nomenclature Committee (HGNC) symbols. Table 1 provides exemplary Ensembl Gene IDs and Uniprot IDs for the transcription factors listed herein. Nucleotide sequences are derived from the Ensembl database (Flicek et al. (2014). Nucleic Acids Research Volume 42, Issue D1. Pp. D749-D755) version 83. Any homologs, orthologs, or paralogs of the transcription factors referred to herein are also contemplated for use in the present invention.

[0117] Those skilled in the art will also be familiar with the ability to reprogram somatic cells toward a naive pluripotent state (as compared to a primed pluripotent state). It will be understood that the methods of the present invention apply to cells that are treated to promote reprogramming toward a naive or primed pluripotent state. In a preferred embodiment of the present invention, the methods comprise increasing protein expression of one or more factors in somatic cells to reprogram the somatic cells toward a naive pluripotent state.

[0118] Those skilled in the art will understand that this information can be used in practicing the methods of the invention, for example, to provide increased amounts of a transcription factor in a somatic cell, or to provide nucleic acids for recombinantly expressing a transcription factor in a somatic cell, etc.

[0119] [Table 1]

[0120] The term "variant" refers to a polypeptide that is at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the full-length polypeptide. The present invention contemplates the use of variants of the transcription factors described herein. A variant may be a fragment of a full-length polypeptide or a naturally occurring splice variant. A variant may be a polypeptide that is at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to a fragment of a polypeptide, where the fragment is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99%, so long as the full-length wild-type polypeptide or a domain thereof retains the desired functional activity, such as the ability to promote conversion of a somatic cell type to a target cell type. In some embodiments, the domain is at least 100, 200, 300, or 400 amino acids in length, beginning at any amino acid position in the sequence and extending toward the C-terminus. Variations known in the art to eliminate or substantially reduce the activity of a protein are preferably avoided. In some embodiments, variants lack N- and / or C-terminal portions of the full-length polypeptide, e.g., up to 10, 20, or 50 amino acids from either end. In some embodiments, the polypeptide has the sequence of a mature (full-length) polypeptide, meaning a polypeptide from which one or more portions, such as a signal peptide, have been removed during normal intracellular proteolytic processing (e.g., during co-translational or post-translational processing). In some embodiments, where the protein is produced other than by purifying it from cells that naturally express it, the protein is a chimeric polypeptide, meaning that it contains portions from two or more different species. In some embodiments, where the protein is produced other than by purifying it from cells that naturally express it, the protein is a derivative, meaning that the protein contains additional sequences not associated with the protein, so long as they do not substantially reduce the biological activity of the protein. Those of skill in the art will recognize or be able to readily confirm whether a particular polypeptide variant, fragment, or derivative is functional using assays known in the art.For example, the ability of a transcription factor variant to convert a somatic cell into a target cell type can be assessed using the assays disclosed in the Examples herein. Another convenient assay involves measuring the ability to activate transcription of a reporter construct containing a transcription factor binding site operably linked to a nucleic acid sequence encoding a detectable marker, such as luciferase. In certain embodiments of the invention, functional variants or fragments have at least 50%, 60%, 70%, 80%, 90%, 95%, or more of the activity of the full-length wild-type polypeptide.

[0121] The term "increasing the amount" in relation to increasing the amount of a transcription factor refers to increasing the amount of the transcription factor in a cell of interest (e.g., a somatic cell such as a fibroblast). In some embodiments, the amount of a transcription factor is "increased" in a cell of interest (e.g., a cell into which an expression cassette directing the expression of one or more transcription factor-encoding polynucleotides has been introduced) if the amount of the transcription factor is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to a control (e.g., a fibroblast into which none of the expression cassettes have been introduced). However, any method of increasing the amount of a transcription factor is contemplated, including any method that increases the amount, rate, or efficiency of transcription, translation, stability, or activity of a transcription factor (or pre-mRNA or mRNA encoding it). Additionally, downregulation or interference with negative regulators of transcription expression, increasing the efficiency of existing translation (e.g., SINEUPs), are also contemplated.

[0122] As used herein, the term "agent" refers to any compound or substance, including, but not limited to, a small molecule, nucleic acid, polypeptide, peptide, drug, ion, etc. An "agent" can be any chemical entity, entity, or moiety, including, but not limited to, synthetic and naturally occurring proteinaceous and non-proteinaceous entities. In some embodiments, an agent is a nucleic acid, nucleic acid analog, protein, antibody, peptide, aptamer, oligomer of nucleic acid, amino acid, or carbohydrate, including, but not limited to, a protein, oligonucleotide, ribozyme, DNAzyme, glycoprotein, siRNA, lipoprotein, aptamer, and modifications and combinations thereof. In certain embodiments, an agent is a small molecule having a chemical moiety. For example, the chemical moiety includes an unsubstituted or substituted alkyl, aromatic, or heterocyclic moiety (including macrolides, leptomycin, and related natural products or analogs thereof). The compound can be known to have a desired activity and / or property, or can be selected from a diverse library of compounds.

[0123] The term "exogenous," when used with reference to a protein, gene, nucleic acid, or polynucleotide in a cell or organism, refers to a protein, gene, nucleic acid, or polynucleotide that has been introduced into the cell or organism by artificial or natural means; or, when used with reference to a cell, refers to a cell that has been isolated and then introduced into another cell or organism by artificial or natural means. An exogenous nucleic acid can be derived from a different organism or cell, or can be one or more additional copies of a nucleic acid that naturally occurs in the organism or cell. An exogenous cell can be derived from a different organism or from the same organism. As a non-limiting example, an exogenous nucleic acid is a nucleic acid that is at a chromosomal location that is different from that of a native cell, or a nucleic acid that is adjacent to a nucleic acid sequence that is different from that found in nature. An exogenous nucleic acid can also be extrachromosomal, such as an episomal vector.

[0124] Screening one or more candidate factors for their ability to increase the amount of one or more transcription factors required to reprogram a somatic cell type to a pluripotent state can include contacting a system that allows for the production or expression of the transcription factor with the candidate factor and determining whether the amount of the transcription factor is increased. The system can be in vivo, e.g., a tissue or cell in an organism, or in vitro, e.g., a cell isolated from an organism, or an in vitro transcription assay, or ex vivo in a cell or tissue. The amount of the transcription factor is measured directly or indirectly by measuring the amount of protein or RNA (e.g., mRNA or pre-mRNA). A candidate agent can function to increase the amount of a transcription factor by increasing any step in the transcription of the gene encoding the transcription factor or by increasing translation of the corresponding mRNA. Alternatively, a candidate agent may reduce the inhibitory activity of a repressor of transcription of the gene encoding the transcription factor, or the activity of a molecule that causes degradation of the mRNA encoding the transcription factor or the transcription factor protein itself.

[0125] Suitable detection means include the use of labels such as radionucleotides, enzymes, coenzymes, fluorescers, chemiluminescers, chromogens, enzyme substrates or cofactors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes, and the like. Such labeled reagents can be used in a variety of well-known assays, such as radioimmunoassays, enzyme immunoassays (e.g., ELISAs), fluorescence immunoassays, and the like. See, e.g., U.S. Patent Nos. 3,766,162, 3,791,932, 3,817,837, and 4,233,402.

[0126] The methods of the present invention include high-throughput screening applications. For example, high-throughput screening assays can be used, including any of the assays according to the present invention, in which aliquots of a system allowing for the production or expression of a transcription factor are exposed to multiple candidate agents in different wells of a multi-well plate. Furthermore, high-throughput screening assays according to the present disclosure include aliquots of a system allowing for the production or expression of a transcription factor exposed to multiple candidate agents in any kind of miniaturized assay system.

[0127] The methods of the present disclosure can be "miniaturized" in assay systems by any acceptable miniaturization method, including, but not limited to, multiwell plates, such as 24, 48, 96, or 384 wells per plate, microchips, or slides. Assays can be reduced in size to be performed on microchip supports, advantageously involving smaller amounts of reagents and other materials. Any miniaturization of the process that facilitates high-throughput screening is within the scope of the present invention.

[0128] In any of the methods of the present invention, cells having at least one characteristic of TSCs can be transferred into the same mammal from which the somatic cells were obtained. In other words, the somatic cells used in the methods of the present invention can be autologous, i.e., can be obtained from the same individual to which the target cells are administered. Alternatively, the target cells can be allogeneically transferred to another individual. Preferably, the cells are autologous to the subject in methods for treating or preventing a medical condition in an individual.

[0129] Culture medium The term "cell culture medium" (also referred to herein as "culture medium" or "culture medium"), as referred to herein, is a medium for culturing cells, containing nutrients that maintain cell viability and support growth. Cell culture media may contain any of the following in appropriate combinations: salts, buffers, amino acids, glucose or other sugars, antibiotics, serum or serum substitutes, and other components such as peptide growth factors. Cell culture media commonly used for particular cell types are known to those of skill in the art. Exemplary cell culture media for use in the methods of the present invention are shown in Table 2.

[0130] [Table 2]

[0131] [Table 3]

[0132] A key discovery of the present invention is that once the reprogramming process has begun, somatic cells undergoing (but not completing) reprogramming can be transferred to a medium that supports the growth and proliferation of TSCs. The inventors have found that contacting cells with such a medium allows the generation of stable TSC-like cells (which may be interchangeably referred to as induced trophoblast stem cells or iTSCs).

[0133] Therefore, the present inventors developed a novel method for generating human TSCs directly from somatic cells by exposing the cells to TSC medium during the reprogramming process.

[0134] It is understood that, according to the methods of the present invention, once the reprogramming process has begun, the somatic cells can be contacted with TSC medium for any time, for example, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 10, or more days (including 14, 21, 28, 35, or more days) after the initiation of reprogramming toward a pluripotent fate.

[0135] The initial medium used during the early stages of reprogramming also need not be a pluripotency medium. For example, in a preferred method of the invention, cells are exposed to a reprogramming agent in fibroblast medium (or other medium that supports the growth of somatic cells) before being transferred to TSC medium at a later stage, but before completion of reprogramming.

[0136] Alternatively, once reprogramming has begun, somatic cells may be cultured in naive or priming medium, or expansion medium, as described herein, for a period of time (which may or may not include completion of the reprogramming process) to generate induced pluripotent stem cells (iPSCs). After this time, the cells may be transferred to TSC medium.

[0137] Suitable culture media for supporting TSCs and their proliferation are preferably those described in Okae (2018) Cell Stem Cell and WO 2016 / 143866, the contents of which are incorporated herein by reference.

[0138] The basal medium composition for TSCs can be any commonly used basal medium, including DMEM, MEM, RPMI 1640, etc. The medium can contain serum, growth factors, pyruvate, amino acids, antibiotics, etc. as appropriate.

[0139] The TSC culture preferably contains at least one growth factor and at least one ROCK inhibitor.

[0140] As used herein, the growth factor may be any growth factor, preferably one selected from epidermal growth factor (EGF), insulin, and transforming growth factor (TGF). The amount of the growth factor may be any amount, for example, 0.1 to 1000 ng / ml, preferably 10 to 100 ng / ml.

[0141] As used herein, a ROCK inhibitor refers to an inhibitor of Rho-associated kinase. Examples of such inhibitors include ((1R,4r)-4-(((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide, Abcam) (also known as trans-N-(4-(1-aminoethyl)-cyclohexanecarboxamide), 1-(5-isoquinolinyl)sulfonyl)homopiperazine (1-(5-isoquinolinylsulfonyl)homopiperazine). Typically, the amount of the ROCK inhibitor is about 0.1 to 50 μM, preferably about 1 to 10 μM.

[0142] In certain preferred embodiments, TSC cultures are in ASECRiAV medium as described by Okae et al., which contains A83-01, SB431542, EGF, CHIR, a ROCK inhibitor, ascorbic acid, and valproic acid.

[0143] Most preferably, the TSC medium comprises: - DMEM / F-12, - GlutaMAX™ (ThermoFisher) supplemented with 0.3% BSA (Sigma) - 0.2% FBS (ThermoFisher), - 1% ITS-X supplement (ThermoFisher), - 0.1 mM 2-mercaptoethanol (ThermoFisher), - 0.5% Pen-Strep (ThermoFisher), 1.5 μg / ml L-ascorbic acid (Sigma), 5 μM Y27632 ((1R,4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide, Abcam), 2 μM CHIR99021 (6-((2-(4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-yl)amino)ethyl)amino)nicotinonitrile Miltenyi Biotec), 0.5 μM A83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide, Sigma), - 1 μM SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide) - 50ng / ml EGF (Peprotech) and - 0.8 mM valproic acid (VPA, Sigma).

[0144] differentiation The invention also includes methods of generating differentiated cells, comprising subjecting cells exhibiting at least one characteristic of TSCs produced according to the invention to differentiation conditions. According to certain embodiments, TSC cells produced according to the invention can be used to isolate lineage-specific cells, such as STs and EVTs.

[0145] Those skilled in the art will be familiar with the standard methods for differentiating the TSCs generated according to the present invention. Briefly, TSCs can be differentiated into syncytiotrophoblasts (STs) by subjecting TSCs to MEFBAP treatment (MEF-conditioned medium, BMP4, TGFβi, FGFRi, as described in Amita et al., 2013 PNAS, 110:E1212-1221). Alternatively, TSCs can be exposed to forskolin treatment to differentiate TSCs into ST fate.

[0146] Further methods for differentiation are disclosed in Kidder (2014) Methods Mol Biol, 1150-201-12; Lei et al., (2017) Placenta, 28:14-12; and Chen et al., (2013) Biochemical and Biophysical Research Communications, 431; 179-202. The method may include culturing cells in a medium lacking GFG4 and heparin. The method may also include genetically modifying the cells in a medium containing differentiation factors.

[0147] Successful differentiation into STs can be determined by measuring basal β-hCG (human chorionic gonadotropin) secretion and expression of the human placental lactogen gene, or by the presence of SDC1+ multinucleated cells.

[0148] Successful differentiation towards an EVT fate can be confirmed by determining the protein expression of one or more markers selected from HLA-G, PRG2 and PAPP2.

[0149] Nucleic acids and vectors The nucleic acids described herein or vectors containing the nucleic acids may include one or more of the sequences referred to in Table 1 above.

[0150] The term "expression", as applicable, refers to the cellular processes involved in the production of RNA and proteins, including, but not limited to, transcription, translation, folding, modification and processing, and optionally, protein secretion.

[0151] As used herein, the terms "isolated" or "partially purified" refer to a nucleic acid or polypeptide that has been separated from at least one other component (e.g., nucleic acid or polypeptide) that is present with the nucleic acid or polypeptide when it is found in its natural source and / or when it is expressed or, in the case of a secreted polypeptide, secreted by a cell. A nucleic acid or polypeptide that is chemically synthesized or synthesized using in vitro transcription / translation is considered "isolated."

[0152] The term "vector" refers to a carrier DNA molecule into which a DNA sequence can be inserted for introduction into a host or somatic cell. Preferred vectors are those capable of autonomous replication and / or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operably linked are referred to herein as "expression vectors." Thus, an "expression vector" is a specialized vector containing the necessary regulatory regions required for the expression of a gene of interest in a host cell. In some embodiments, the gene of interest is operably linked to another sequence in the vector. The vector can be a viral vector or a non-viral vector. When a viral vector is used, the viral vector is preferably replication-deficient, which can be achieved, for example, by removing all viral nucleic acid encoding replication. Replication-deficient viral vectors still retain their infectivity and enter cells in a manner similar to replicating adenoviral vectors, but once inside the cell, they do not replicate or propagate. Vectors also encompass liposomes and nanoparticles, as well as other means for delivering DNA molecules to cells.

[0153] The term "operably linked" means that regulatory sequences required for expression of a coding sequence are positioned in a DNA molecule in the appropriate position relative to the coding sequence so as to effect expression of the coding sequence. This same definition can also be applied to the arrangement of coding sequences and transcription control elements (e.g., promoters, enhancers, and termination elements) in an expression vector. The term "operably linked" includes having an appropriate initiation signal (e.g., ATG) in front of the polynucleotide sequence to be expressed, and maintaining the correct reading frame to allow expression of the polynucleotide sequence under the control of the expression control sequences and production of the desired polypeptide encoded by the polynucleotide sequence.

[0154] The term "viral vector" refers to the use of a virus or virus-associated vector as a carrier of a nucleic acid construct into a cell. The construct can be incorporated into and packaged in a non-replicating defective viral genome, such as adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV), or others, including retroviral and lentiviral vectors, for infection or transduction of the cell. The vector may or may not be integrated into the genome of the cell. The construct can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into a vector capable of episomal replication, such as EPV and EBV vectors.

[0155] As used herein, the term "adenovirus" refers to viruses of the Adenoviridae family. Adenoviruses are medium-sized (90-100 nm), non-enveloped (naked), icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA genome.

[0156] As used herein, the term "non-integrating viral vector" refers to a viral vector that does not integrate into the host genome, and the expression of genes delivered by the viral vector is transient. Because there is little or no integration into the host genome, non-integrating viral vectors have the advantage of not generating DNA mutations by inserting at random points in the genome. For example, non-integrating viral vectors remain extrachromosomal and do not insert their genes into the host genome, potentially disrupting the expression of endogenous genes. Non-integrating viral vectors may include, but are not limited to, adenoviruses, alphaviruses, picornaviruses, and vaccinia viruses. These viral vectors are "non-integrating" viral vectors, as the term is used herein, even though any of them may, in some rare circumstances, integrate viral nucleic acid into the genome of a host cell. Importantly, the viral vectors used in the methods described herein do not integrate their nucleic acid into the genome of a host cell, either as a rule or as a major part of their life cycle under the conditions used.

[0157] The vectors described herein can be constructed and engineered using methods generally known in the scientific literature to increase their safety for therapeutic use, to include selection and enrichment markers, if desired, and to optimize expression of the nucleotide sequences contained therein. The vectors should contain components that allow the vector to self-replicate in somatic cell types. For example, the known Epstein-Barr oriP / Nuclear Antigen-1 (EBNA-I) combination (see, e.g., Lindner, S.E. and B. Sugden, "The plasmid replicon of Epstein-Barr virus: mechanistic insights into efficient, licensed, extrachromosomal replication in human cells," Plasmid 58:1 (2007)) is sufficient to support vector self-replication, and other combinations known to function in mammalian, particularly primate, cells can also be used. Standard techniques for constructing expression vectors suitable for use in the present invention are well known to those of skill in the art and can be found in publications such as Sambrook J, et al., "Molecular cloning: a laboratory manual", (3rd ed. Cold Spring Harbor Press, Cold Spring Harbor, NY 2001), which is incorporated herein by reference in its entirety as if set forth herein.

[0158] In the methods of the present invention, genetic material encoding the relevant transcription factors required for conversion is delivered to somatic cells via one or more reprogramming vectors. Each transcription factor can be introduced into the somatic cell as a polynucleotide transgene encoding the transcription factor operably linked to a heterologous promoter capable of driving expression of the polynucleotide in the somatic cell.

[0159] Suitable reprogramming vectors include any of those described herein, including episomal vectors (e.g., plasmids) that do not encode all or part of a viral genome sufficient to produce infectious or replication-competent virus, although the vector may contain structural elements from more than one virus. One or more reprogramming vectors can be introduced into a single somatic cell. One or more transgenes can be provided on a single reprogramming vector. A single strong constitutive transcription promoter can provide transcriptional control for multiple transgenes, which can be provided as expression cassettes. Separate expression cassettes on a vector can be under the transcriptional control of separate strong constitutive promoters, which can be copies of the same promoter or separate promoters. A variety of heterologous promoters are known in the art and can be used depending on factors such as the desired expression level of transcription factors. As exemplified below, it can be advantageous to use different promoters with different strengths to control the transcription of separate expression cassettes in v-cells. Another consideration when selecting a transcription promoter is the rate at which the promoter is silenced. Those skilled in the art will understand that it may be advantageous to reduce the expression of one or more transgenes or transgene expression cassettes after the gene product has completed or substantially completed its role in the reprogramming method. Exemplary promoters include the human EF1α elongation factor promoter, the CMV cytomegalovirus immediate-early promoter, and the CAG chicken albumin promoter, as well as corresponding homologous promoters from other species. In human somatic cells, both EF1α and CMV are strong promoters, but the CMV promoter is silenced more efficiently than the EF1α promoter, such that expression of transgenes under the former control is turned off earlier than expression of transgenes under the latter control. Transcription factors can be expressed in somatic cells in relative ratios that can be varied to adjust reprogramming efficiency. Preferably, when multiple transgenes are encoded on a single transcript, an internal ribosome entry site is provided upstream of the transgene, distal from the transcription promoter.The relative ratios of the factors may vary depending on the factors being delivered, but the optimal ratios of factors can be determined by one of skill in the art in possession of this disclosure.

[0160] Those skilled in the art will appreciate the advantageous efficiency of introducing all factors via a single vector rather than via multiple vectors, but will also appreciate that as the total vector size increases, it becomes increasingly difficult to introduce the vector. Those skilled in the art will also appreciate that the location of transcription factors within a polycistronic cassette on a vector can affect their temporal expression and the resulting reprogramming efficiency. Thus, various combinations of factors for vector combinations can be used. Some such combinations are shown herein to support reprogramming.

[0161] After introduction of the reprogramming vector, and while the somatic cell is being reprogrammed, the vector can persist in the target cell while the introduced transcriptional gene is transcribed and translated. Transgene expression can be advantageously downregulated or turned off in cells reprogrammed to the target cell type. The reprogramming vector can remain extrachromosomal. With very low efficiency, the vector can integrate into the cell's genome. The following examples are intended to illustrate, but in no way limit, the present invention.

[0162] Suitable methods for nucleic acid delivery for transformation of cells, tissues, or organisms for use in the present invention are intended to include virtually any method by which nucleic acid (e.g., DNA) can be introduced into a cell, tissue, or organism, as described herein or known to those of skill in the art (e.g., Stadtfeld and Hochedlinger, Nature Methods 6(5):329-330 (2009); Yusa et al., Nat. Methods 6:363-369 (2009); Woltjen, et al., Nature 458,766-770 (9 Apr. 2009)). Such methods include, for example, microinjection (Harland and Weintraub, J. Cell 2001), optionally with ex vivo transfection using lipid-based transfection reagents such as Fugene 6 (Roche) or Lipofectamine (Invitrogen) (Wilson et al., Science 244:1344-1346, 1989; Nabel and Baltimore, Nature 326:711-713, 1987). Biol., 101:1094-1099, 1985; by injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859, each of which is incorporated by reference herein), including U.S. Pat. No. 5,789,215, incorporated by reference herein; by electroporation (U.S. Pat. No. 5,384,253, incorporated by reference herein; Tur-Kaspa et al., Mol. Cell Biol., 6:716-718, 1986; Potter et al., al., Proc. Nat'l Acad. Sci. USA, 81:7161-7165, 1984); calcium phosphate precipitation (Graham and Van Der Eb, Virology, 52:456-467, 1973; Chen and Okayama, Mol. Cell Biol.,7(8):2745-2752, 1987; Rippe et al., Mol. Cell Biol.,10:689-695, 1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, Mol. Cell Biol.,5:1188-1190, 1985); by direct sonication (Fechheimer et al., Proc. Nat'l Acad. Sci. USA,84:8463-8467, 1987); liposome-mediated transfection (Nicolau and Sene, Biochim. Biophys. Acta,721:185-190, 1982; Fraley et al., Proc. Nat'l Acad. Sci. USA,76:3348-3352, 1979; Nicolau et al., Methods Enzymol., 149:157-176, 1987; Wong et al., Gene, 10:87-94, 1980; Kaneda et al., Science, 243:375-378, 1989; Kato et al., J. Biol. Chem., 266:3361-3364, 1991) and direct delivery of DNA by receptor-mediated transfection (Wu and Wu, Biochemistry, 27:887-892, 1988; Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987); and any combination of these methods (each of which is incorporated herein by reference).

[0163] A number of polypeptides capable of mediating the introduction of relevant molecules into cells have been previously described and can be adapted for the present invention, see, e.g., Langel (2002) Cell Penetrating Peptides: Processes and Applications, CRC Press, Pharmacology and Toxicology Series. Examples of polypeptide sequences that enhance transport across membranes include the Drosophila homeoprotein Antennapedia transcription protein (AntHD) (Joliot et al., New Biol. 3:1121-34, 1991; Joliot et al., Proc. Natl. Acad. Sci. USA, 88:1864-8, 1991; Le Roux et al., Proc. Natl. Acad. Sci. USA, 90:9120-4, 1993), herpes simplex virus structural protein VP22 (Elliott and O'Hare, Cell 88:223-33, 1997); the HIV-1 transcriptional activator TAT protein (Green and Loewenstein, Cell 55:1179-1188, 1988; Frankel and Pabo, Cell 55:1 289-1193, 1988); Kaposi's FGF signal sequence (kFGF); protein transduction domain-4 (PTD4); Penetratin, M918, Transportan-10; nuclear localization sequence, PEP-I peptide; amphipathic peptides (e.g., MPG peptide); delivery-enhancing transporters such as those described in U.S. Pat. No. 6,730,293 (including, but not limited to, peptide sequences containing at least 5-25 or more consecutive arginines or 5-25 or more arginines in a contiguous set of 30, 40, or 50 amino acids; including, but not limited to, peptides with sufficient, e.g., at least 5, guanidino or amidino moieties); and the commercially available Penetratin™ 1 peptide; and the Diatos Peptide Vector ("DPV") of the Vectocell® platform available from Daitos SA, Paris, France.See also WO 2005 / 084158 and WO 2007 / 123667 and the additional transporters described therein. Not only are these proteins able to cross the plasma membrane, but the attachment of other proteins, such as the transcription factors described herein, is sufficient to stimulate cellular uptake of these complexes.

[0164] Pharmaceutical Compositions and Other Uses According to certain embodiments, the present invention contemplates the use of any cell, tissue, and / or organ / organoid generated using cells (i.e., iTSCs) having at least one characteristic of a TSC produced according to the present invention. The present invention further contemplates the use of any particle or compound secreted by a TSC produced according to the present invention, or by an EVT or ST differentiated from a TSC produced according to the present invention.

[0165] The isolated cells of the present invention can further be used for disease modeling, drug screening and patient-specific cell-based therapy.

[0166] Thus, according to certain aspects of the present invention, there are provided isolated placenta-like organoids or blastocyst-like cells comprising TSCs produced according to the methods of the present invention.

[0167] According to a further aspect, there is provided a method for enhancing a placenta or blastocyst, comprising introducing into the placenta or blastocyst a cell or cell population produced according to the present invention. The cell or cell population may be a cell exhibiting at least one characteristic of a TSC produced according to the method of the present invention. Alternatively, the cell or cell population may be a differentiated cell or differentiated cell population produced from a cell exhibiting at least one characteristic of a TSC of the present invention.

[0168] Introduction of the cells can be accomplished in vitro or ex vivo via direct injection or aggregation within the developing host placenta or embryo.

[0169] Cells having at least one characteristic of TSCs produced according to the present invention may also be used to prepare model systems for disorders associated with trophoblast development and / or activity, which find utility in screening for genes expressed in or essential for trophoblast differentiation and / or activity, screening for agents and conditions (such as culture conditions and manipulations) that achieve trophoblast development and / or activity, production of specific growth factors and hormones, or as cellular therapies for disorders associated with trophoblast development and / or activity.

[0170] Thus, according to one aspect of the present invention there is provided a method for identifying an agent capable of modulating trophoblast development and / or activity, the method comprising: (i) contacting a TSC produced according to the present invention, or an organoid derived from said TSC (such as a blastocyst-like organoid or a placenta-like organoid), with a candidate agent; (ii) comparing the development and / or activity of the TSCs or organoids after contact with the agent with the development and / or activity of the TSCs or organoids without the agent; An effect of the agent on the development and / or activity of the TSCs or organoids that exceeds a predetermined level compared to the development and / or activity of the TSCs or organoids without the agent indicates that the agent regulates trophoblast development and / or activity.

[0171] As used herein, the term "modulate" refers to altering trophoblast development and / or activity by inhibiting or promoting.

[0172] According to certain embodiments, the modulation is inhibiting development and / or activity.

[0173] According to certain embodiments, the modulation is promoting development and / or activity.

[0174] Under the same culture conditions, the effect of a candidate agent on trophoblast development and / or activity 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 control.

[0175] As used herein, the phrase "an effect exceeding a predetermined level" refers to a change in trophoblast development and / or activity or organoid development after contact with a candidate agent that is greater than a predetermined level, such as greater than about 10%, for example greater than about 20%, for example greater than about 30%, for example greater than about 40%, for example greater than about 50%, for example greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 2-fold, greater than about 3-fold, greater than about 4-fold, greater than about 5-fold, greater than about 6-fold, greater than about 7-fold, greater than about 8-fold, greater than about 9-fold, greater than about 20-fold, greater than about 50-fold, greater than about 100-fold, greater than about 200-fold, greater than about 350-fold, greater than about 500-fold, greater than about 1000-fold, or greater than a predetermined level, compared to the expression level before contact with the candidate agent.

[0176] According to certain embodiments, the candidate agent may be any compound, including but not limited to, chemical compounds, small molecules, polypeptides, and polynucleotides.

[0177] According to certain embodiments, selected agents may further be used to treat a variety of conditions requiring modulation of trophoblast development or activity, such as those conditions described below.

[0178] Recurrent miscarriage and fetal growth restriction (FGR) are associated with placental insufficiency and contribute to handicap and, in severe cases, death. Cell transplantation of intact and healthy TSCs holds great promise in the clinic, as the transplanted cells may be able to rescue some of these fetuses by supporting the underdeveloped / damaged placenta.

[0179] According to another aspect of the present invention, there is provided a method for treating and / or preventing disorders associated with trophoblast development and / or activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of TSCs produced according to the present invention, thereby treating and / or preventing disorders associated with trophoblast development and / or activity in the subject.

[0180] The term "treating" or "treatment" refers to inhibiting, preventing, or halting the occurrence of a pathology (e.g., recurrent miscarriage) and / or causing a reduction, remission, or regression of the pathology. Those skilled in the art will understand that a variety of methodologies and assays can be used to assess the development of a pathology, and similarly, a variety of methodologies and assays can be used to assess the reduction, remission, or regression of a pathology.

[0181] As used herein, the phrase "subject in need thereof" refers to a mammalian subject (e.g., a human) diagnosed with a pathology. In certain embodiments, the term encompasses individuals at risk of developing the pathology. Veterinary uses are also intended. The subject can be of any gender or age, including newborn, infant, juvenile, adolescent, adult, and elderly adult. According to certain embodiments, the subject is female.

[0182] This aspect of the invention is intended to treat disorders associated with trophoblast development and / or activity. According to certain embodiments, the disease is selected from the group consisting of recurrent miscarriage, pre-eclampsia, fetal growth restriction (FGR), hydatiform mole, and choriocarcinoma.

[0183] Because trophoblasts produce several secreted growth factors and hormones, as well as particles such as exosomes according to another aspect of the present invention, there is provided a method for obtaining compounds or particles produced by trophoblasts, comprising culturing a TSC or TCS cell culture of the present invention and isolating compounds or particles secreted by the cells from the culture medium, thereby obtaining compounds or particles produced by trophoblasts.

[0184] According to certain embodiments, the compound is a growth factor or hormone, such as, but not limited to, human chorionic gonadotropin (hCG). The particle may be an exosome.

[0185] The cells of the present invention may be implanted into a subject either by themselves or in a pharmaceutical composition in which they are mixed with a suitable carrier or excipient. Similarly, the constructs of the present invention may be administered to a subject either by themselves or in a pharmaceutical composition.

[0186] As used herein, a "pharmaceutical composition" refers to a preparation of one or more 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.

[0187] As used herein, the term "active ingredient" refers to the cells or organoids of the present invention that are responsible for the biological effect.

[0188] Hereinafter, the expressions "physiologically acceptable carrier" and "pharmaceutically acceptable carrier", which may be used interchangeably, refer to a carrier or diluent that does not cause significant irritation to an organism and does not abolish the biological activity and properties of the administered compound.

[0189] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0190] The pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0191] Thus, pharmaceutical compositions for use in accordance with 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 ingredient into pharmaceutically usable preparations. The appropriate formulation will depend on the route of administration selected.

[0192] 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.

[0193] Typically, pharmaceutical compositions are administered in a local rather than systemic manner, for example, by injecting the pharmaceutical composition directly into a tissue region of the patient.

[0194] The pharmaceutical compositions described herein can be formulated for parenteral administration, for example, by bolus injection or continuous infusion. Injectable preparations can be provided in unit dosage form, for example, in ampoules or multi-dose containers, optionally with the addition of preservatives. The compositions can be suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending agents, stabilizing agents, and / or dispersing agents.

[0195] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Furthermore, suspensions of the active ingredient can be prepared as appropriate oily or aqueous 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 can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the active ingredient, allowing for the preparation of highly concentrated solutions.

[0196] Pharmaceutical compositions suitable for use in the context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve its intended purpose, 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.

[0197] The compositions of the present invention may, if desired, be presented in a pack or dispenser device (e.g., an FDA-approved kit), which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise 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 also contain a notice associated with the container in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects approval by the agency of the composition or form for human or veterinary administration. Such notice may, for example, be that of labeling approved by the U.S. Food and Drug Administration for prescription drugs or an approved product insert. Compositions comprising the preparations of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as further detailed above.

[0198] The present invention includes the following non-limiting examples. [Example]

[0199] Example 1: Human iTSCs generated from somatic cell reprogramming Primary adult human dermal fibroblasts (HDFa) were obtained from Life Technologies. For nuclear reprogramming experiments, HDFa were grown in Medium 106 (Life Technologies) supplemented with LSGS (Gibco). Subsequently, early passage (<P6) fibroblasts were seeded at 50,000 - 70,000 cells per well in a 6-well plate and then transduced in fibroblast medium containing DMEM (Gibco), 10% FBS (Hyclone), 1% non-essential amino acids (Gibco), 1 mM GlutaMAX (Gibco), 1% Pen-strep (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), and 1 mM sodium pyruvate (Gibco).

[0200] After 48 hours, the cells in one well were trypsinized for counting to determine the volume of virus (MOI) required for transduction. Transduction was performed using the CytoTune 2.0 iPSC Sendai Reprogramming Kit (Invitrogen) consisting of four transcription factors, OCT4, SOX2, cMYC, KLF4 (OKSM). After 24 hours, the virus was removed and the medium was changed every other day.

[0201] Seven days after transduction, the cells were harvested using TrypLE Express (Life tech) and re-seeded onto a layer of irradiated MEF feeders in fibroblast medium. The next day, the medium was changed to 1) iTSC medium, 2) naive medium, 3) priming medium, or 4) LCDM medium as further described below.

[0202] <​​​​For cells cultured in medium 2-4 (i.e., naive, primed, or LCDM medium), cells were cultured in this medium for 5-13 days before being transferred to TSC medium. Cells cultured in naive medium on days 8-21 can be referred to as "partially reprogrammed intermediates," "naive reprogramming intermediates," or D21 naive intermediates. Cells cultured in Essential 7 (E7) medium on days 8-21 can also be referred to as "partially reprogrammed intermediates" (or "primed reprogramming intermediates" or D21 primed intermediates).

[0204] When iTSCs were cultured in TSC medium, they were passaged every 3–4 days at a ratio of 1:2–1:4 once they became confluent and palpable. For the first four passages, iTSCs were passaged onto iMEF feeders using TrpLE Express (Lifetech) and cultured at 37°C in a 5% O2, 5% CO2 incubator. Starting at passage 5, iTSCs were passaged onto tissue culture flasks pre-coated with 5 μg / ml Col IV (Sigma) for at least 1 hour at 37°C in a 20% O2, 5% CO2 incubator.

[0205] In yet a further example, cells were maintained in fibroblast medium for up to 21 days before being transferred to TSC medium. These cells are also referred to herein as iTSCd28 cells (see Figure 6).

[0206] It will be appreciated that, generally, regardless of the medium used, the methods of the present invention can be used to obtain iTSCs from partially reprogrammed intermediates.

[0207] Media used in this example: 1. iTSC medium is as described in Okae et al., Cell Stem Cell, 2018, and contains: - DMEM / F-12, GlutaMAX™ (ThermoFisher) supplemented with 0.3% BSA (Sigma), - 0.2% FBS (ThermoFisher), - 1% ITS-X supplement (ThermoFisher), - 0.1 mM 2-mercaptoethanol (ThermoFisher), - 0.5% Pen-Strep (ThermoFisher), 1.5 μg / ml L-ascorbic acid (Sigma), 5 μM Y27632 ((1R,4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide, Abcam), 2 μM CHIR99021 (6-((2-((4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-yl)amino)ethyl)amino)nicotinonitrile Miltenyi Biotec), 0.5 μM A83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide, Sigma), - 1 μM SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide) - 50ng / ml EGF (Peprotech) and - 0.8 mM valproic acid (VPA, Sigma).

[0208] 2. Naive medium. Naive t2iLGoY medium as reported in Guo et al., (2016) Stem Cell Reports, 6(4):P437-446, containing: - A 50:50 mixture of DMEM / F-12 (Gibco) and Neurobasal Medium (Gibco) supplemented with 2 mM L-glutamine (Gibco). 0.1 mM 2-mercaptoethanol (Gibco), - 0.5% N2 supplement (Gibco), - 1% B27 supplement (Gibco), - 1% Pen-strep (Gibco), - 10ng / ml human LIF (homemade), 250 μM L-ascorbic acid (Sigma), 10 μg / ml recombinant human insulin (Sigma), - 1μM PD0325901 (Miltenyi Biotec), - 1μM CHIR99021 (Miltenyi Biotec), - 2.5 μM Goe6983 (Tocris), - 10 μM Y-27632 (Abcam). Other examples of naive media include 5iLAF, RSeT, and PXGL.

[0209] 3. Priming medium. An exemplary priming medium includes: - DMEM / F12 (ThermoFisher), - 20% knockout serum replacement (KSR, ThermoFisher). - 1mM GlutaMAX (ThermoFisher), - 55 μM 2-mercaptoethanol (ThermoFisher), - 1% non-essential amino acids (ThermoFisher), - 50ng / mL recombinant human FGF2 (Miltenyi Biotec), - 1% Pen-strep (ThermoFisher). The media KSR / FGF2, E8, mTeSR, AKIT, B8 are further examples of priming media.

[0210] 4. LCDM medium: - a 50:50 mixture of DMEM / F-12 (ThermoFisher) and Neurobasal medium (ThermoFisher) supplemented with 0.5% N2 supplement (ThermoFisher); - 1% B27 supplement (ThermoFisher), - 1% non-essential amino acids (ThermoFisher), - 1mM GlutaMAX (ThermoFisher), - 1% Pen-strep(ThermoFisher), - 0.1 mM 2-mercaptoethanol (ThermoFisher), - 0.5% KSR (ThermoFisher), - 10ng / ml human LIF (homemade), - 1μM CHIR99021 (Miltenyi Biotec), 2 μM (S)-(+)-dimethindene maleate (Tocris), - 2 μM minocycline hydrochloride (Tocris), - 1 μM IWR-endo-1 (Selleckchem) and - 2 μM Y-27632 (Abcam).

[0211] Example 2: Conversion of established human pluripotent stem cells into TSCs: Established naive EPSCs / iPSCs were cultured under the t2itLGoY conditions described above. Naive hEPSCs / hiPSCs were seeded onto a layer of MEF feeders or collagen IV-coated plates. When TSCs became confluent and distinct, they were passaged at a ratio of 1:2 to 1:4 every 3–4 days. TSCs were passaged onto iMEF feeders or Collagen IV (Sigma) using TrpLE Express (Life Technologies) and cultured at 37°C in a 20% O2 and 5% CO2 incubator.

[0212] The primed human EPSCs / iPSCs are preferably contacted with naive medium before being contacted with TSC medium.

[0213] Established human LCDM EPSCs or iPSCs (also referred to as "expanded" EPSCs or iPSCs) were cultured in LCDM medium as described above. Cells were seeded onto a layer of MEF feeders or collagen IV-coated plates. When TSCs became confluent and distinct, they were passaged at a ratio of 1:2 to 1:4 every 3 to 4 days. TSCs were passaged onto iMEF feeders or Col IV (Sigma) using TrpLE Express (Lifetech) and cultured at 37°C in a 20% O2 and 5% CO2 incubator.

[0214] The primed human EPSCs / iPSCs are preferably contacted with LCDM medium before being contacted with TSC medium.

[0215] Example 3: Direct conversion of somatic cells into iTSCs Figure 3 shows the results of characterization of TSCs generated by stabilization of TE signatures during naive programming for the derivation of human iTSCs.

[0216] Figure 3a provides a schematic of the experimental design for capturing and stabilizing naive intermediates in TSC medium.

[0217] Briefly, the results show that it is possible to capture and stabilize intermediates in TSC medium during reprogramming of fibroblasts to a dedifferentiated state, allowing for the generation of iTSCs.

[0218] Phase contrast images of cells generated through this process show that the cells have a morphology similar to TSCs (cobblestone-like appearance, Figure 3b).

[0219] Immunostaining of fibroblast, primed, and naive t2iLiGoY iPSCs with p63, TFAP2C, GATA2, KRT7, and DAPI for nuclear staining demonstrates markers characteristic of TSCs as shown in Figure 3c. Figure 3d shows immunostaining of iTSCs generated from d21 naive programming intermediates.

[0220] Cells generated from D21-primed and naive intermediates were subjected to flow cytometry. APA+ITGA6+ double-positive cells were gated from the TRA-1-60-negative cell population and sorted to purify iTSCs from bulk reprogramming cells (not shown). Figure 3D also shows the percentage of TRA-1-60-negative, APA-positive, and ITGA6-positive iTSCs generated from D21-primed and naive reprogramming intermediates.

[0221] The expression levels of TSC markers were determined in human fibroblasts, primed iPSCs, naive iPSCs, and iTSCs generated according to the present invention and compared with TSCTs and TSblasts obtained from blastocysts (Okae et al., 2018). The results are shown in Figure 3e.

[0222] ST cells were obtained by differentiating TSCs generated according to the present invention using standard methods. Phase contrast images and SDC1 immunostaining of differentiated cells show that the cells exhibit ST characteristics (Figures 3f and 3g).

[0223] Figure 3i shows a positive result for ST cells differentiated from iTSCs using an hCG pregnancy test stick. ELISA was also used to detect the levels of hCG secreted by TSCs generated according to the present invention and by STs obtained by differentiating these TSCs (Figure 3j).

[0224] Figure 3f shows a phase contrast image of EVT cells differentiated from iTSCs generated according to the present invention. HLA-G immunostaining was also performed (Figure 3g).

[0225] An iTSC engraftment assay was performed by injecting iTSCs generated according to the present invention into NOD-SCID mice. Nine days after injection, urine, lesions, and serum were examined. Figure 3k shows that urine samples collected from iTSC-injected mice showed positive hCG results on a pregnancy test stick compared with vehicle controls. hCG levels were also detected by hCG ELISA using serum samples (Figure 3l).

[0226] FIG. 3n shows hematoxylin and eosin and immunohistochemical staining for KRT7, SDC1, and HLA-G in lesions harvested from iTSC-engrafted NOD-SCID mice; no obvious lesions were observed in vehicle controls.

[0227] Figure 3m shows the results observed after engraftment of TSCs generated according to the present invention in NOD-SCID mice. Briefly, iTSCs with 80% confluency were dissociated with TrypLE express (ThermoFisher) and counted. 7 iTSCs were resuspended in 200 μl of a 1:2 mixture of Matrigel (Corning) and DMEM / F-12, GlutaMAX™ (ThermoFisher) supplemented with 0.3% BSA (Sigma) and 1% ITS-X supplement (ThermoFisher). The cell mixture was then subcutaneously injected into the dorsal flank of NOD-SCID mice (100 μl of the mixture in each flank). Nine days after injection, urine, lesions, and serum were collected from the mice for analysis. Mouse urine and serum were used to detect and measure hCG secretion, as detailed in the following section. The collected lesions were fixed overnight in 4% paraformaldehyde (PFA, Sigma) and subsequently embedded in paraffin. Paraffin-embedded tissues were sectioned at 5 μm and stained with hematoxylin-eosin (H&E) or proceeded to immunostaining as described above.

[0228] Figure 4 shows the cellular heterogeneity of fibroblasts and reprogramming intermediates (iTSCd8) revealed by scRNA-seq.

[0229] To test whether iTSCs could be derived directly from human fibroblasts, we initiated a reprogramming experiment. Day 8 iTSCs were transferred to TSC medium, naive medium, or maintained in fibroblast medium. We then performed scRNA-seq on day 21 to assess cellular heterogeneity (Figure 4a). A population of TE-like cells was observed, and closer examination revealed that this TE-like population contained cells from all three reprogramming conditions (Figure 4b-d).

[0230] Example 4: Direct derivation of iTSCs from human fibroblasts Reprogramming of fibroblasts was initiated according to the method described in Example 1. Briefly, 8 days after transfection with Sendai virus expressing the transcription factor OKSM, cells were transferred to TSC medium as described herein (FIG. 5a).

[0231] Phase contrast images of cells generated through this process show that the cells have a morphology similar to TSCs (cobblestone-like appearance, Figure 5b).

[0232] Figure 5c shows immunostaining of iTSCs generated from d8 fibroblast intermediates. These iTSCs are capable of self-renewal in transgene-free conditions (Figure 5d).

[0233] The expression levels of TSC markers were determined in human fibroblasts, primed iPSCs, naive iPSCs, and iTSCs generated according to the present invention and compared with TSCTs and TSblasts obtained from blastocysts (Okae et al., 2018). The results are shown in Figure 5e. Furthermore, iTSCs and iTSC-derived syncytiotrophoblasts (STs) and extravillous trophoblasts (EVTs) share common transcriptome profiles with corresponding primary cell types from other published datasets (Figures 5f-h).

[0234] iTSCs also exhibit higher expression levels of microRNAs (miRNAs) from the chromosome 19 miRNA cluster compared with fibroblasts and iPS cells—a unique feature of primary trophoblasts ( Figure 5 i).

[0235] iTSCs exhibit specific open chromatin accessibility in the promoter and putative enhancer regions of the ELF5 locus, as seen in TSCBT5 (derived from human blastocysts) (Fig. 5j).

[0236] ST cells were obtained by differentiating iTSCs generated according to the present invention using standard methods. Phase contrast images and SDC1 immunostaining of differentiated cells show that the cells exhibit ST characteristics (Figure 5k).

[0237] Figure 5n shows a positive result for ST cells differentiated from iTSCs using an hCG pregnancy test stick. ELISA was also used to detect the levels of hCG secreted by TSCs generated according to the present invention and by STs obtained by differentiating those iTSCs (Figure 5o).

[0238] Figure 5l shows a phase-contrast image of EVT cells differentiated from iTSCs generated according to the present invention. HLA-G immunostaining was also performed (Figure 5I). Furthermore, expression of HLA-A, -B, and -C pan-markers (W632) was detected in iTS cells, similar to that previously reported in blastocyst-derived TS cells (Okae et al., 2018) (Figures 5p and 5q).

[0239] Figures 5r and 5s show that iTS cell-derived syncytiotrophoblast and extravillous trophoblast show expression of relevant marker genes.

[0240] Figure 5t shows a schematic diagram of an iTSC engraftment assay performed by injecting iTSCs generated according to the present invention into NOD-SCID mice. Nine days after injection, urine, lesions, and serum were examined. Figure 5t shows that urine samples collected from iTSC-injected mice showed positive hCG results on a pregnancy test stick compared to vehicle controls. hCG levels were also detected by hCG ELISA using serum samples (Figure 5v).

[0241] FIG. 5w shows hematoxylin and eosin and immunohistochemical staining for KRT7, SDC1, and HLA-G in lesions harvested from iTSC-engrafted NOD-SCID mice; no obvious lesions were observed in vehicle controls.

[0242] Figure 5u shows the results observed after engraftment of TSCs generated according to the present invention in NOD-SCID mice. Briefly, iTSCs with 80% confluency were dissociated with TrypLE express (ThermoFisher) and counted. 7 iTSCs were resuspended in 200 μl of a 1:2 mixture of Matrigel (Corning) and DMEM / F-12, GlutaMAX™ (ThermoFisher) supplemented with 0.3% BSA (Sigma) and 1% ITS-X supplement (ThermoFisher). The cell mixture was then subcutaneously injected into the dorsal flank of NOD-SCID mice (100 μl of the mixture in each flank). Nine days after injection, urine, lesions, and serum were collected from the mice for analysis. Mouse urine and serum were used to detect and measure hCG secretion, as detailed in the following section. The collected lesions were fixed overnight in 4% paraformaldehyde (PFA, Sigma) and subsequently embedded in paraffin. Paraffin-embedded tissues were sectioned at 5 μm and stained with hematoxylin-eosin (H&E) or proceeded to immunostaining as described above.

[0243] Example 5: Derivation of iTSCs from day 21 human fibroblast reprogramming intermediates Reprogramming of fibroblasts was initiated according to the method described in Example 1. Briefly, 21 days after transfection with Sendai virus expressing the transcription factor OKSM, cells were transferred to TSC medium as described herein.

[0244] The intermediates of the fibroblast cultures at day 21 also consisted of cells with strong epiblast, primed, and naive signatures, and thus, these intermediates were capable of giving rise to pluripotent and TS cell lines (Figure 6a-c).

[0245] Example 6: Derivation of iTSCs from Partially Reprogrammed Intermediates and Derivation of TSCs from Established Naive and Expanded iPSCs Reprogramming of fibroblasts to obtain reprogramming intermediates was initiated according to the method described in Example 1. Briefly, 7 days after transfection with Sendai virus expressing the transcription factor OKSM, cells were transferred to E7 medium as described herein. Figure 7a shows a schematic diagram of the reprogramming protocol and phase-contrast images of cells at each stage.

[0246] TSCs derived from established naive and expanded iPSCs were obtained according to the method described in Example 2. An outline of the conversion protocol and phase-contrast images of cells at each stage are shown in Figure 7b.

[0247] Figure 7c shows hierarchical clustering, which defined three cell groups: (1) naive hPSCs, (2) expanded and primed hPSCs, and (3) trophoblasts. Both induced and converted hTSCs clustered with previously established embryo- and placenta-derived hTSCs to form the trophoblast group. This group was subdivided among established, induced, and converted hTSCs, including ST and EVT hTSCs. Pearson correlation analysis further confirmed the proximity of induced and converted embryo- and placenta-derived hTSCs. Expanded and primed hPSCs exhibited a high degree of transcriptional similarity despite their relative differences in their potential to form hTSCs.

[0248] Further analysis confirmed that the induced and transformed hTSCs expressed key trophoblast-lineage markers. In particular, the expression levels of GATA3, KRT7, and VGL1 were similar to those observed in previously established embryo- and placenta-derived hTSCs (absolute gene expression ranging from 10 to 300 UPM). We also identified genes associated with hTSC stemness, including PEG10, NR2F2, and LRP2. These genes were expressed at similar levels in hTSCs, hiTSCs, and hcTSCs, but not in the differentiated ST and EVT (absolute gene expression ranging from 20 to 200 UPM in hTSCs, and less than 10 UPM in hTSC-ST and hTSC-EVT) (Figure 7d).

[0249] Figure 7e shows immunostaining of hiTSCs and hcTSCs for the trophoblast markers NR2F2 and GATA2, which are expressed in the trophectoderm of human blastocysts. NR2F2 and GATA2 were highly expressed and localized to the nuclei of all cells. Conversely, SOX2 was highly expressed in hPSCs but absent in hTSCs (Figure 1e). These expression patterns were comparable among hiTSCs, hcTSCs, and placenta-derived hTSCs. These results confirm that hi / cTSCs share a similar expression profile to previously established hTSCs.

[0250] Figures 7f-g show the optimized ST / EVT assay protocol. During optimized ST differentiation, cells upregulated the expression of CGA, CGB, and SDC1, which are not expressed in hTSCs (relative gene expression ranging from 10- to 1,300-fold change). In contrast, HLA-G, MMP2, and ASCL2 were generally increased under EVT differentiation conditions (relative gene expression ranging from 10- to 700-fold change). Finally, LRP2 and CDKN3, which were primarily expressed in hTSCs, were downregulated during differentiation (relative gene expression ranging from 2- to 70-fold change). Importantly, the gene expression pattern was comparable to that of placental cells, which was confirmed by statistical analysis (Figure 7h).

[0251] Figure 7i shows the structure of a multinucleated syncytium expressing DESMOPLAKIN (DSP) and CGB, typically containing 6 to 10 nuclei.

[0252] Immunostaining for GATA3 and HLA-G confirmed the identity of EVT differentiated from induced and converted placenta-derived hTSCs (Fig. 7j).

[0253] Example 7: General Methods Cell culture conditions Primary human adult dermal fibroblasts from three female donors were obtained from ThermoFisher (catalog numbers C-013-5C and lot number 1029000 for 38F, lot number 1528526 for 55F, and lot number 1569390 for 32F); cells were harvested and plated in Medium 106 (ThermoFisher) supplemented with low serum growth supplement (LSGS) (ThermoFisher) for expansion. Culture conditions used for human somatic cell reprogramming were prepared as previously described. Fibroblast medium: DMEM (ThermoFisher), 10% fetal bovine serum (FBS) (Hyclone), 1% non-essential amino acids (ThermoFisher), 1 mM GlutaMAX (ThermoFisher), 1% penicillin-streptomycin (ThermoFisher), 55 μM 2-mercaptoethanol (ThermoFisher), and 1 mM sodium pyruvate (ThermoFisher). Priming medium: DMEM / F12 (ThermoFisher), 20% knockout serum replacement (KSR) (ThermoFisher), 1 mM GlutaMAX (ThermoFisher), 0.1 mM 2-mercaptoethanol (ThermoFisher), 1% non-essential amino acids (ThermoFisher), 50 ng / ml recombinant human FGF2 (Miltenyi Biotec), 1% penicillin-streptomycin (ThermoFisher).Naive medium (t2iLGoY): 2 mM l-glutamine (ThermoFisher), 0.1 mM 2-mercaptoethanol (ThermoFisher), 0.5% N2 supplement (ThermoFisher), 1% B27 supplement (ThermoFisher), 1% penicillin-streptomycin (ThermoFisher), 10 ng / ml human leukemia inhibitory factor (LIF) (homemade), 250 μM l-ascorbic acid (Sigma), 10 μg / ml recombinant human insulin (Sigma), 1 μM PD0325901 (Miltenyi Biotec), 1 μM CHIR99021 (Miltenyi Biotec), 2.5 μM Goe6983 (Tocris), 10 μM A 50:50 mixture of DMEM / F-12 (ThermoFisher) and Neurobasal Medium (ThermoFisher) supplemented with Y-27632 (Abcam). Naive Human Stem Cell Medium (NHSM): Culture conditions adapted from Gafni, O. et al. "Derivation of novel human ground state naive pluripotent stem cells." Nature 504, 282-286, with suggested modifications from the J. Hanna Laboratory webpage, 2014.10 mg / ml AlbuMAX I (ThermoFisher), 1% penicillin-streptomycin (ThermoFisher), 1 mM GlutaMAX (ThermoFisher), 1% non-essential amino acids (ThermoFisher), 10% KSR (ThermoFisher), 1% N2 supplement (ThermoFisher), 12.5 μg / ml recombinant human insulin (Sigma), 50 μg / ml l-ascorbic acid (Sigma), 20 ng / ml recombinant human LIF (homemade), 8 ng / ml FGF2 (Peprotech), 2 ng / ml recombinant TGFβ1 (Peprotech), 20 ng / ml human LR3-IGF1 (Prospec), and small molecule inhibitors: 1 μM PD0325901 (Miltenyi Biotec), 3 μM CHIR99021 (Miltenyi Biotec), 5 μM SP600125 (Tocris), 2 μM BIRB796 (Axon), 0.4 μM LDN193189 (Axon), 10 μM Y-27632 (medium replenished daily from a freshly thawed stock aliquot) (Abcam), and 1 μM Goe6983 (medium replenished daily from a freshly thawed stock aliquot) (Tocris). 1% N2 supplement (ThermoFisher), 2% B27 supplement (ThermoFisher), 1% non-essential amino acids (ThermoFisher), 1 mM GlutaMAX (ThermoFisher), 1% penicillin-streptomycin (ThermoFisher), 0.1 mM 2-mercaptoethanol (ThermoFisher), 50 μg / ml bovine serum albumin (ThermoFisher), 1 μM PD0325901 (Miltenyi Biotec), 1 μM IM-12 (Millipore), 0.5 μM SB590885 (Tocris), 1 μM WH-4-023 (A Chemtek), 10 μM Y-27632 (Abcam), 20 ng / ml activin A (Peprotech).A 50:50 mixture of DMEM / F-12 (ThermoFisher) and neurobasal medium (ThermoFisher) supplemented with 8 ng / ml FGF2 (Miltenyi Biotec), 20 ng / ml human LIF (in-house), and 0.5% KSR (ThermoFisher). Naive RSeT medium: 100 ml of RSeT 5x supplement, 1 ml of RSeT 500x supplement, and 0.5 ml of RSeT 1,000x supplement were placed in 398.5 ml of RSeT basal medium (Stem Cell Technologies) supplemented with 1% penicillin-streptomycin (ThermoFisher). Human TS cell medium 7: GlutaMAX (ThermoFisher) supplemented with DMEM / F-12, 0.3% BSA (Sigma), 0.2% FBS (ThermoFisher), 1% ITS-X supplement (ThermoFisher), 0.1 mM 2-mercaptoethanol (ThermoFisher), 0.5% penicillin-streptomycin (ThermoFisher), 1.5 μg / ml l-ascorbic acid (Sigma), 5 μM Y27632 (Abcam), 2 μM CHIR99021 (Miltenyi Biotec), 0.5 μM A83-01 (Sigma), 1 μM SB431542, 50 ng / ml EGF (Peprotech), and 0.8 mM valproic acid (VPA) (Sigma).

[0254] Reprogramming experiments t2itLGoY medium was used for naive reprogramming because it has previously been shown to be capable of reprogramming fibroblasts into naive iPS cells, which possess all the characteristics of naive pluripotency and maintain a more stable karyotype compared to other conditions. Human somatic cell reprogramming experiments to primed and naive pluripotent states, as well as subsequent culture of primed and naive iPS cells, were performed as previously described. Briefly, human fibroblast reprogramming was performed using the CytoTune-iPS 2.0 Sendai Reprogramming Kit according to the manufacturer's instructions (ThermoFisher).

[0255] Primary human adult dermal fibroblasts were cultured in fibroblast medium at approximately 5–10 × 10 4 Cells were seeded at a density of 1000 x g. Cells were transduced with Sendai virus in fibroblast medium at the following multiplicities of infection (MOI): KLF4, OCT4, and SOX2, MOI = 5 or 10; MYC, MOI = 5 or 10; and KLF4, MOI = 6 or 12. Cells were replated on a layer of iMEF feeders on day 7 and transferred to different culture media (primed, t2itLGoY, NHMS, RSeT, or 5iLAF) the following day. After 18–21 days, iPSCs were passaged and expanded as previously described. For induction of iTSCd21n during primed or naive reprogramming, day 21 primed or naive t2iLGoY reprogramming intermediates were transferred to TS cell medium. After 4–5 days, cells were passaged every 3–4 days at a ratio of 1:2–1:4 using TrypLE express (ThermoFisher). For the first four passages, iTSCs were passaged onto iMEF feeders and cultured in a 37°C, 5% O2, and 5% CO2 incubator.

[0256] Starting at passage 5, iTSCd21n were passaged (for at least 1 hour at 37°C) into tissue culture flasks pre-coated with 5 μg / ml collagen IV (Sigma) and cultured in a 37°C, 20% O2, and 5% CO2 incubator. For direct induction of iTSCd8 from human fibroblasts, day 8 fibroblast reprogramming intermediates were transferred to TSC medium. After 10–13 days, iTSCd8 were passaged onto iMEF feeders and cultured in a 37°C, 5% O2, and 5% CO2 incubator, as described for iTSCd21n. Sendai detection in established iTSC lines was performed as described in the Sendai reprogramming protocol (ThermoFisher). For the derivation of primed, naive iPSCs and iTSCs from day 21 fibroblast reprogramming intermediates, day 21 fibroblast reprogramming intermediates were transferred to primed, naive, or TSC medium and then cultured and expanded as described.

[0257] For the data shown in Figure 7, human adult fibroblasts were reprogrammed using the CytoTune-iPS 2.0 Sendai Reprogramming Kit (Life Technologies™). Two days before infection, 3.0–4.0 × 10 cells were added per well. 4fibroblasts were seeded onto Matrigel-coated 12-well plates. On day 0, cells were infected with three vectors: polycistronic Klf4-Oct4-Sox2, Myc, and Klf4, at a multiplicity of infection (MOI) of 5:5:3 or 3:3:3, respectively. On day 9 postinfection, cells were dissociated with TrypLE (5 min, 37°C, Life Technologies™) and plated onto MEFs in 35 mm dishes. The next day, cells were transferred to E7 reprogramming medium (STEMCELL Technologies™). From day 21 onwards, cells were transferred to hTSC medium. Induced hTSC lines (hiTSCs) were routinely cultured at 37°C under hypoxic conditions (5% O2, 5% CO2). Somatic cell reprogramming into hiNPSCs, hiEPS, and hiPSCs was performed as described previously (Kilens et al., 2018 Nature Communications 9:360; Yang et al., 2017, Cell, 169:243-257).

[0258] Conversion of hNPSCs and hEPS to hcTS hNPSCs and hEPS were dissociated with TrypLE (5 min, 37°C, Life Technologies™) at 0.6–1.7 × 10 cells per dish. 5 Cells were seeded onto MEFs in 35 mm dishes at a density of 0.5–1.25 × 10 cells per dish. Cells were maintained in their original medium supplemented with 10 μM Y27632 for 1 day. From day 2 onwards, cells were transferred to hTSC medium. Transformed hTSC lines (hcTSCs) were routinely cultured at 37°C under hypoxic conditions (5% O2, 5% CO2). Primed hPSCs included in the transformation experiments were initially cultured in KSR+FGF2 or iPS-BREW. Ten colonies were picked (KSR+FGF2) or cells were passaged in TrypLE (iPS-BREW) and plated at 0.5–1.25 × 10 cells per dish into MEF-coated 35 mm dishes for the transformation assay. 5 The cells were seeded at a density of 1000×.

[0259] In vitro differentiation of iTSCd21n and iTSCd8 into ST and EVT Differentiation of iTSCs into ST and EVT was performed as previously described (Okae, H. et al. (2018) Derivation of human trophoblast stem cells. Cell Stem Cell 22, 50-63). For differentiation of iTSCs into ST, iTSCs were cultured at 1 × 10 per well. 5 Cells were seeded onto 6-well plates pre-coated with 2.5 μg / ml collagen IV (Sigma) at a density of 1000 μg / ml and incubated with 0.3% BSA (Sigma), 4% KSR (ThermoFisher), 1% ITS-X supplement (ThermoFisher), 0.1 mM 2-mercaptoethanol (ThermoFisher), 0.5% penicillin-streptomycin (ThermoFisher), 2.5 μM iTSCs were cultured in 2 ml of ST differentiation medium (DMEM / F-12, GlutaMAX (ThermoFisher) supplemented with Y27632 (Abcam) and 2 μM forskolin (Selleckchem). The medium was changed daily for the first 4 days, and cells were analyzed on day 6. The fusion index was used to quantify the efficiency of cell fusion; it was calculated by subtracting the number of syncytia from the number of nuclei counted in the syncytia, then dividing by the total number of nuclei counted. To differentiate iTSCs into EVTs, 0.75 × 10 iTSCs were cultured per well. 5Cells were seeded onto 6-well plates pre-coated with 1 μg / ml collagen IV (Sigma) at a density of 1000 μg / ml and cultured in 2 ml of EVT differentiation medium (DMEM / F-12, GlutaMAX (ThermoFisher)) supplemented with 0.3% BSA (Sigma), 4% KSR (ThermoFisher), 1% ITS-X supplement (ThermoFisher), 0.1 mM 2-mercaptoethanol (ThermoFisher), 0.5% penicillin-streptomycin (ThermoFisher), 2.5 μM Y27632 (Abcam), 100 ng / ml NRG1 (Cell Signaling), and 7.5 μM A83-01 (Sigma). Immediately after suspending the cells in EVT differentiation medium, Matrigel (Corning) was overlaid to a final concentration of 2%. On day 3 of differentiation, human NRG1 (Cell Signaling) was added to the 2 ml EVT differentiation medium. EVT differentiation medium without NRG1 (Cell Signaling) or KSR (ThermoFisher) was added to a final concentration of 0.5%. On day 6 of differentiation, the EVT differentiation medium was replaced without NRG1 (Cell Signaling) or KSR (ThermoFisher), and Matrigel (Corning) was added to a final concentration of 0.5%. Cells were cultured for an additional 2 days before analysis.

[0260] Differentiation of hi / cTSCs into EVT and ST After at least 15 passages, cells were collected for differentiation assays. Prior to differentiation into ST and EVT, h(i / c)TSCs (initially cultured on MEFs) were transferred to fibronectin for at least three passages. EVT differentiation: 2–4 days before passage, h(i / c)TSCs were transferred to EVT pre-medium [DMEM / F12 supplemented with 0.1 mM 2-mercaptoethanol, 0.5% penicillin-streptomycin, 0.3% BSA, 1% ITS-X supplement, 4% KSR, 7.5 μM A83-01 (Tocris™), 2.5 μM Y27632, and 5 μM IWR-endo-1 (Miltenyi Biotec™)]. Cells were then cultured at 0.8–3.0 × 10 4 cells / cm 2Cells were passaged in TrypLE to a density of 100 μM. Before treatment, cells were placed in differentiation basal medium [DMEM / F12 containing 0.1 mM 2-mercaptoethanol, 0.5% penicillin-streptomycin, 0.3% BSA, and 1% ITS-X] supplemented with 10 μM ROCK inhibitor (Y27632). Within 6 hours, cells were transferred to EVT medium (Okae et al., 2018) [differentiation basal medium supplemented with 100 ng / ml NRG1, 7.5 mM A83-01, 2.5 mM Y27632, 4% knockout serum replacement, and 2% Matrigel] at a time appropriate for the strain. On day 3, the medium was replaced with EVT medium containing 0.5% Matrigel without NRG1. EVT formation was typically observed by days 4–5. On day 6, the medium was replaced with EVT medium containing 0.5% Matrigel without NRG1 and KSR. Cells were collected on day 8 for subsequent analysis. 2D-ST differentiation: h(i / c)TSCs were cultured at 2.0–6.0 × 10 4 cells / cm 2Cells were passaged in TrypLE to a density of 10 μM. Prior to treatment, cells were placed in differentiation basal medium [DMEM / F12 containing 0.1 mM mercaptoethanol, 0.5% penicillin-streptomycin, 0.3% BSA, and 1% ITS-X] supplemented with 10 μM ROCK inhibitor (Y27632). Within 3 hours, cells were transferred to ST medium (Okae et al., 2018) [differentiation basal medium supplemented with 2.5 mM Y27632, 2 mM forskolin, and 4% KSR] at a time appropriate for the strain. The medium was changed on day 3, and cells were analyzed on day 6. 3D-ST differentiation assay: Prior to the 3D differentiation assay, h(i / c)TSCs were transitioned into slightly modified trophoblast organoid medium (TOM) (Turco et al., 2018) [DMEM / F12 supplemented with 500 nM A83-01, 1.5 μM CHIR99021, 80 ng / ml human R-spondin 1, 50 ng / ml hEGF, 100 ng / ml hFGF2, 50 ng / ml hHGF, 2 μM Y-27632, 1X N2 supplement, 1X B27 supplement without vitamin A, 1.25 mM N-acetyl-L-cysteine, 1% GlutaMAX (Gibco™), 0.5% penicillin-streptomycin (TOM basal medium)]. 0.4–1.0 × 10 cells were passaged in TrypLE. 5 Cells were embedded in 150 μl droplets containing 50 μl Matrigel and 50 μl PBS+ / + along with 960 ng fibronectin, 50 ng laminin 521, and 50 μl TOM basal medium. The droplets were carefully deposited onto a sterile parafilm-covered dish and allowed to solidify at 37°C for 20 minutes. Complete TOM supplemented with 10 μM ROCK inhibitor (Y27632) was then added to cover the droplets. The medium was replaced with TOM every 3 days. 3D structures appeared within 1 week and were collected on day 14 for subsequent analysis.

[0261] In vivo engraftment assay of iTSCd21n and iTSCd8 The protocol and use of mice were approved by the Monash University Animal Welfare Committee and in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (2004) and Victoria's Animal Cruelty Prevention and Control Act. iTS cells at 80% confluency were dissociated with TrypLE express (ThermoFisher) and counted. Ten million iTS cells were resuspended in 200 μl of a 1:2 mixture of Matrigel (Corning) and DMEM / F-12, GlutaMAX (ThermoFisher) supplemented with 0.3% BSA (Sigma), and 1% ITS-X supplement (ThermoFisher). The cell mixture was then injected subcutaneously into the dorsal flank of male and female NOD / SCID IL-2R-γ knockout mice aged 5–20 weeks (100 μl per flank). Mice were randomly assigned between control and iTS cell treatments, but the investigators were not blinded. Nine days after injection, urine, blood, and lesions were collected from the mice for analysis. Mouse urine and serum were used to detect and measure hCG secretion, as detailed in "Pregnancy Test and hCG ELISA." The collected lesions were fixed overnight in 4% paraformaldehyde (PFA) (Sigma) and subsequently embedded in paraffin. The volume of the collected lesions was 1 cm. 3 Paraffin-embedded tissues were sectioned and stained with hematoxylin and eosin (H&E) or proceeded to immunohistochemical staining for KRT7, HLA-G, and SDC1.

[0262] Pregnancy test and hCG ELISA iTSCs were cultured at 0.5 x 10 per mL on 12-well plates for ST differentiation as detailed in "In Vitro Differentiation of iTSCd21n and iTSCd8 into ST and EVT". 5 The ST cell medium was replaced on day 4, and the conditioned medium was collected on day 6 and stored at -80°C. As a control, 0.5 × 10 cells were seeded per mL on a 12-well plate. 5iTSCs were seeded at a density of 1000 x 1000 cells and cultured in TSC medium. After two days, the conditioned medium was collected and stored at -80°C. The conditioned medium was then tested using a commercially available hCG pregnancy test stick (Freedom) according to the manufacturer's recommendations. Additionally, hCG levels in the medium were measured using an hCG ELISA kit (Abnova, ABNOKA4005) according to the manufacturer's instructions. After the iTSC engraftment assay, collected mouse urine was tested using a commercially available hCG pregnancy test stick, and serum hCG levels were measured using an hCG ELISA kit.

[0263] Flow cytometry analysis and fluorescence-activated cell sorting Cells were dissociated with TrypLE express (ThermoFisher), and DPBS (ThermoFisher) supplemented with 2% FBS (Hyclone) and 10 μM Y-27632 (Abcam) was used for antibody labeling and final resuspension of samples. For HLA experiments, cells were labeled with HLA-A, B, C (W6 / 32) or HLA-Bw4 (1:1, Purcell Labs) and then with AF647 goat anti-mouse IgG antibody (1:1,000, ThermoFisher). Alternatively, cells were labeled with (1) HLA-G MEG-G / 9 (1:500, Abcam); (2) AF488 goat anti-mouse IgG antibody (1:1,000, ThermoFisher); and (3) PE-Cy7 mouse anti-human HLA-A, B, WC, W6 / 32 (1:200, Biolegend).

[0264] scRNA-seq of human reprogramming intermediates For scRNA-seq experiments, Day 0, Day 3, Day 7, Day 13 primed, Day 13 naive, Day 21 primed, Day 21 naive, iPSC primed (passage 3), and iPSC naive (passage 3) samples were collected and cryopreserved. These collected samples were then subjected to FACS. Day 0, Day 3, Day 7, Day 13 primed, Day 13 naive, Day 21 primed, and Day 21 naive samples were sorted for PI-TRA-1-85 cells to remove photoreceptors and iMEF cells, and iPSC primed (passage 3) and iPSC naive (passage 3) samples were sorted for PI-TRA-1-85 CD13 F11R TRA-1-60 EPCAM cells to remove dead cells, iMEF cells, and differentiated cells. Three samples were prepared for subsequent library preparation (Extended Data Figure 1c): Sample 1 contained cells isolated from days 0, 3, and 7; Samples 2 and 3 contained cells from primed (days 13, 21, and iPSCs), and cells from naive reprogramming (days 13, 21, and iPSCs), respectively. A small number of cells from days 0, 3, and 7 were added to Samples 2 and 3 to capture the complete reprogramming trajectory and account for potential batch effects. Collected cells were isolated, encapsulated, and libraries were constructed using the Chromium controller (10X Genomics) according to the manufacturer's instructions (Chromium Single Cell 3' Reagent Kit V2 User Guide, 10X Genomics document number CG00052 Revision 33). A total of 12 cDNA amplification cycles were used. A total of 16 cycles of library amplification were used. Sequencing was performed using an Illumina NextSeq 500 using SBS V2 chemistry in high output mode according to the recommendations outlined by the 10x Genomics Chromium Single Cell 3' Reagent Kit V2 User Guide (10x Genomics document number CG00052 Revision 3), except that the second read was extended to 115 bp instead of 98 bp.Libraries were diluted according to the manufacturer's instructions (NextSeq 500 System User Guide, Illumina document number 15046563 v02) and loaded at 1.8 pM. Chromium barcodes were used for multiplexing, and FASTQ files were generated from the mkfastq pipeline using the Cellranger program (v.2.1.0). Alignment and UMI counting were performed against the hg19 genome according to snRNA-seq (except that mature mRNAs, rather than pre-mRNAs, were used, and UMIs were assigned to exons).

[0265] scRNA-seq of day 21 fibroblasts, naive and iTSCd8 reprogramming intermediates Day 21 fibroblasts, naive, and iTSCd8 reprogramming intermediates were collected and sorted for PI-TRA-1-85+ cells to remove dead and iMEF cells. Collected cells were isolated, encapsulated, and assembled using the Chromium controller (10x Genomics) according to the manufacturer's instructions (Chromium Next GEM Single Cell 3' Reagent Kit V3.3 User Guide). Sequencing was performed on an Illumina NovaSeq 6000 using a paired-end (R1 28 bp and R2 87 bp) sequencing strategy, aiming for 20,000 read pairs per cell. Chromium barcodes were used for multiplexing, and FASTQ files were generated from the mkfastq pipeline using the Cellranger program (v.3.1.0). Alignment and UMI counting were performed against the hg19 genome following the scRNA-seq experiment.

[0266] Example 8: Discussion These results demonstrate that the transient TE state observed during somatic cell reprogramming can be captured and stabilized through the transfer of reprogramming intermediates from pluripotent medium to a medium that supports TSC proliferation (e.g., the TSC medium described in Okae et al., Cell Stem Cell, 2018). Notably, human trophoblast stem cells (TSCs) were generated during reprogramming. Thus, this is the first report of direct reprogramming of human somatic cells to TSCs.

[0267] Further characterization indicates that TSCs generated according to this method express key markers that define human TE and TSC (Figure 3). These TSCs have undergone over 30 passages to date without a decline in growth rate. Furthermore, functional characterization of TSCs using in vitro and in vivo differentiation assays demonstrated that TSCs give rise to a) syncytiotrophoblasts (ST), defined SDC1+ multinucleated cells, and b) extravillous trophoblasts (EVT), defined by upregulated expression of HLA-G (an important histocompatibility molecule expressed in the placenta). Importantly, these assays demonstrate that TSCs are indeed bipotential and can differentiate into ST and EVT cells. Collectively, these results reveal previously uncharacterized extraembryonic potential for reprogramming intermediates and suggest that cell fate specification is highly dynamic and plastic during human somatic cell reprogramming to a naive state.

[0268] Having stable, self-renewing, fully isogenic human iPSC and iTSC lines that can be derived from adult cells will provide the entire field with a unique opportunity to study the human trophectoderm, its relationship to trophoblast development, pluripotent cells, and its role in regulating events related to cell fate decisions in early human embryonic development and developmental disorders in an in vitro setting where modern biochemical and molecular techniques can be applied on a large scale.

[0269] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of which different combinations constitute various alternative aspects of the invention.

Claims

1. 1. A method for reprogramming somatic cells into cells exhibiting at least one characteristic of trophoblast stem cells (TSCs), comprising the steps of: a) increasing the protein expression or amount of one or more factors in said somatic cells, said factors for reprogramming said cells towards a dedifferentiated or pluripotent state; b) culturing said cells for a time and under conditions sufficient to allow said reprogramming of said cells towards a dedifferentiated or pluripotent state; c) contacting the cells with a trophoblast stem cell (TSC) culture medium suitable for maintaining the TSCs; d) culturing said cells in said TSC medium for a time and under conditions sufficient to allow said cells to exhibit at least one characteristic of a TSC; in that order, thereby reprogramming said somatic cell into a cell exhibiting at least one characteristic of a TSC.

2. 1. A method for generating cells exhibiting at least one characteristic of trophoblast stem cells (TSCs) from somatic cells, comprising the steps of: a) increasing the protein expression or amount of one or more factors in said somatic cells, said factors for reprogramming said somatic cells towards a dedifferentiated or pluripotent state; b) culturing the cells for a time and under conditions sufficient to allow reprogramming of the cells towards a dedifferentiated or pluripotent state; c) contacting the cells with a trophoblast stem cell (TSC) culture medium suitable for maintaining the TSCs; d) culturing said cells in said TSC culture medium for a time and under conditions sufficient to allow said cells to exhibit at least one characteristic of a TSC; thereby generating from said somatic cells cells that exhibit at least one characteristic of a TSC.

3. The method of claim 1 or 2, wherein the TSC medium comprises a growth factor and a Rho kinase (ROCK) inhibitor.

4. 4. The method of claim 3, wherein the growth factor is selected from epidermal growth factor (EGF), insulin, and transforming growth factor (TGF).

5. The method of claim 4, wherein the growth factor is EGF.

6. The method according to any one of claims 3 to 5, wherein the ROCK inhibitor is trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) or a salt thereof.

7. The TSC culture medium comprises one or more of the following: 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]benzamide (SB 431542) or a salt thereof; 6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol_2-yl)-2-pyrimidinyl]amino]ethyl]amino]nicotinonitrile (CHIR 99021) or a salt thereof; and / or A83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide) or a salt thereof; The method of any one of claims 3 to 6, further comprising:

8. The method according to any one of claims 1 to 7, wherein the TSC culture is ASECRiAV and contains A83-01, SB431542, EGF, CHIR, a ROCK inhibitor, ascorbic acid, and valproic acid.

9. The method according to any one of claims 1 to 8, wherein the somatic cells are fibroblasts, epidermal cells, keratinocytes or monocytes, preferably dermal fibroblasts.

10. 10. The method of any one of claims 1 to 9, wherein the factors for reprogramming the somatic cells towards a dedifferentiated or pluripotent state are one or more of OCT4, SOX2, KLF4 and MYC.

11. The method of claim 10, wherein the factors are OCT4, SOX2, KLF4 and MYC.

12. The method of claim 11 , wherein the factors further comprise LIN28 and / or NANOG.

13. 13. The method of any one of claims 1 to 12, wherein the protein expression or amount of said factor for reprogramming somatic cells towards a dedifferentiated or pluripotent state is increased by contacting said cells with an agent that increases expression of said factor.

14. 14. The method of claim 13, wherein the agent is selected from the group consisting of a nucleotide sequence, a protein, an aptamer and a small molecule, a ribosome, an RNAi agent and a peptide nucleic acid (PNA), and analogs or variants thereof.

15. 15. The method of claim 14, wherein the protein expression or amount of the factor is increased by introducing into the cell at least one nucleic acid comprising a nucleotide sequence encoding the factor or encoding a functional fragment of the factor.

16. 16. The method of any one of claims 1 to 15, wherein the somatic cells are contacted with the TSC medium for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, or more after increasing the protein expression or amount of one or more factors for reprogramming the somatic cells towards a dedifferentiated or pluripotent state.

17. The method of any one of claims 1 to 16, wherein the somatic cells are contacted with the TSC medium during the reprogramming process and prior to completion of reprogramming of the cells to a pluripotent state.

18. The method of any one of claims 1 to 17, wherein during the reprogramming process and before contacting with the TSC medium, the somatic cells are cultured in a non-pluripotent medium.

19. 19. The method of any one of claims 1 to 18, wherein the step of culturing the cells for a time and under conditions sufficient to allow the reprogramming of the cells towards a dedifferentiated or pluripotent state comprises culturing the cells in a medium that supports the growth of the somatic cells for a period of at least 7 days and not more than 22 days, wherein the somatic cells have not achieved pluripotency prior to contacting them with trophoblast stem cell (TSC) culture medium suitable for maintaining TSCs.

20. The method comprising: a) contacting a somatic cell with an agent to increase the amount or expression of OCT4, SOX2, KLF4 and MYC in said cell; b) culturing said cells in a medium that supports the growth of said somatic cells for a time and under conditions sufficient to allow the generation of reprogramming intermediate somatic cells; c) contacting the reprogramming intermediate cells with a trophoblast stem cell (TSC) culture medium suitable for maintaining TSCs, preferably comprising A83-01, SB431542, EGF, CHIR, a ROCK inhibitor, ascorbic acid, and valproic acid; 20. The method of any one of claims 1 to 19, comprising:

21. The method of any one of claims 1 to 16, wherein the somatic cells are contacted with the TSC medium after being reprogrammed to a pluripotent state.

22. 22. The method of any one of claims 1 to 21, wherein the somatic cells are further cultured in naive medium or expansion medium before contacting and culturing the cells with TSC medium.

23. The at least one characteristic of the TSC is: - undifferentiated bipotential state; - Cobblestone colony appearance; - the ability to differentiate into cells that exhibit one or more characteristics of extravillous trophoblast (EVT) or syncytiotrophoblast (ST); - methylation pattern similar to blastocyst-derived TSCs as determined by bisulfite assay; - expression of one or more biochemical markers of TSC, as determined by immunohistochemistry and / or PCR assays, preferably said markers are selected from the group consisting of CD49f (iTGA6), CD249, nuclear GATA2 / 3, TFAP2C, P63, and NR2F2 The method of any one of claims 1 to 22, comprising one or more of:

24. 24. The method of any one of claims 1 to 23, wherein the cells exhibiting at least one characteristic of TSC are characterized by the absence of the markers characterizing the somatic cells, and optionally the cells exhibiting at least one characteristic of TSC do not express one or more of the following markers: OCT4 (also known as POU5F1), NANOG, SOX2, SALL2, OTX2, BANCR, KLF17, DPPA3, ARGFX, and DNMT3L.

25. The somatic cell: - have the ability to differentiate into cells that exhibit one or more characteristics of extravillous trophoblast (EVT) or syncytiotrophoblast (ST); - express one or more biochemical markers of TSC selected from the group consisting of nuclear GATA2 / 3, TFAP2C, P63, and NR2F2 The method according to any one of claims 1 to 24, wherein the cells are reprogrammed.

26. 26. The method of any one of claims 23 to 25, wherein the ST features include one or more of SDC1+ multinucleated cells.

27. The method of any one of claims 23 to 25, wherein the characteristics of EVT include one or more of increased expression of HLA-G, PRG2, and PAPPA2.

28. 28. The method of any one of claims 1 to 27, wherein the cells exhibiting at least one characteristic of a TSC retain their undifferentiated state when maintained in subculture.

29. 29. The method of claim 28, wherein the cells having at least one characteristic of a TSC retain the at least one characteristic of a TSC for at least 5, at least 10, at least 15, at least 20, at least 40 or more cell culture passages.

30. 30. The method of any one of claims 1 to 29, further comprising expanding the cells that exhibit at least one characteristic of TSC to increase the proportion of cells in the population that exhibit at least one characteristic of TSC.

31. An isolated cell exhibiting at least one characteristic of a TSC produced by the method of any one of claims 1 to 30.

32. 29. A cell population in which at least 5% of cells exhibit at least one characteristic of a TSC, the cells being produced by a method according to any one of claims 1 to 28, preferably wherein at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the population exhibit at least one characteristic of a TSC.

33. 33. The method of any one of claims 1 to 32, further comprising differentiating said cells exhibiting at least one characteristic of TSC to generate cells exhibiting at least one characteristic of EVT or ST.

34. 31. The method of any one of claims 1 to 30, further comprising differentiating cells exhibiting at least one characteristic of a TSC into a non-placental cell type for use in regenerative medicine.

35. 35. An isolated differentiated cell or population of differentiated cells produced by the method of claim 33 or 34.

36. Organoids derived from the cell population of claim 32 or the differentiated cell population of claim 35.

37. A pharmaceutical composition or supplement comprising: - a cell according to claim 31; - a cell population according to claim 32 - a differentiated cell or a differentiated cell population according to claim 35; - an organoid or part thereof according to claim 36; and pharmaceutically acceptable excipients.

38. 1. A method for treating and / or preventing disorders associated with trophoblast development and / or activity in a subject in need thereof, comprising administering to a subject a therapeutically effective amount of: - a cell according to claim 31; - a cell population according to claim 32; - a differentiated cell or a differentiated cell population according to claim 35; - an organoid according to claim 36; or - a pharmaceutical composition according to claim 37, to said subject, thereby treating and / or preventing said disorder associated with said development and / or activity of trophoblasts in said subject.

39. The placenta or blastocyst contains: - a cell according to claim 31; - a cell population according to claim 32; or - An isolated differentiated cell or differentiated cell population according to claim 35. A method for enhancing a placenta or blastocyst, comprising introducing

40. In the manufacture of a medicament for treating a disease or disorder associated with said development and / or activity of trophoblasts: - a cell according to claim 31; - a cell population according to claim 32; or - a differentiated cell or a differentiated cell population according to claim 35, - organoids according to claim 36; Use of.

41. 41. The method of claim 38; or the use of claim 40, wherein the disease or disorder is selected from the group consisting of recurrent miscarriage, pre-eclampsia, fetal growth restriction (FGR), hydratiform mole, and choriocarcinoma.

42. 1. A method for identifying an agent capable of modulating trophoblast development and / or activity, comprising: - contacting a cell or a cell population comprising at least one characteristic of a TSC obtained according to any one of claims 1 to 30 with a candidate drug; - comparing the development and / or activity of said cell or cell population after said contact with said agent with the development and / or activity of said cell or cell population without said agent; Including, A method in which the effect of the agent on the development and / or activity of the cell or cell population exceeds a predetermined level compared to the development of the cell or cell population without the agent indicates that the agent regulates trophoblast development and / or activity.

43. A method for obtaining a compound produced by trophoblasts, comprising culturing a cell or cell population comprising at least one characteristic of a TSC obtained by any one of claims 1 to 30, or a culture comprising said cell or cell population, and isolating a compound secreted by said cells from the culture medium, thereby obtaining said compound produced by said trophoblasts.

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