Methods for reprogramming cells
The present invention provides a method for reprogramming a human somatic cell to a cell exhibiting at least one characteristic of a trophoblast stem cell (TSC), the method comprising the following steps in order: a) increasing the protein expression of one or more factors in the somatic cell, wherein the factors are for reprogramming the somatic cell towards a pluripotent state; b) culturing the cell for a sufficient time and under conditions to allow the reprogramming of the cell towards a pluripotent state; c) contacting the cell with a culture medium suitable for sustaining trophoblast stem cells (TSC); and d) culturing the cell in the TSC medium for a sufficient time and under conditions to allow the cell to exhibit at least one characteristic of a TSC, thereby reprogramming the somatic cell to a cell exhibiting at least one characteristic of a TSC.
1 . A method for reprogramming a human somatic cell to a cell exhibiting at least one characteristic of a human trophoblast stem cell (TSC), the method comprising the following steps in order:
a) increasing the protein expression or amount of one or more factors in the somatic cell, wherein the factors are for reprogramming the cell towards a de-differentiated or pluripotent state;
b) culturing the cell for a sufficient time and under conditions to allow the reprogramming of the cell towards a de-differentiated or pluripotent state;
c) contacting the cell with a TSC culture medium suitable for sustaining trophoblast stem cells (TSC), wherein the cell is contacted with the TSC medium during the reprogramming process, and prior to completion of reprogramming of the cell to a pluripotent state; and
d) culturing the cell in the TSC medium for a sufficient time and under conditions to allow the cell to exhibit at least one characteristic of a TSC,
thereby reprogramming the somatic cell to a cell exhibiting at least one characteristic of a TSC, wherein the protein expression, or amount, of the factor(s) for reprogramming a somatic cell towards a de-differentiated or pluripotent state, is/are increased by:
contacting the cell with an agent which increases the expression of the factor, wherein the agent is selected from the group consisting of: a nucleotide sequence, a protein, an aptamer and small molecule, ribosome, RNAi agent and peptide-nucleic acid (PNA) and analogues or variants thereof; or
introducing at least one nucleic acid comprising a nucleotide sequence encoding the factor, or encoding a functional fragment thereof, in the cell;
and
wherein the at least one characteristic of a TSC comprises one or more of:
an undifferentiated, bipotential state;
cobblestone-shaped colony appearance;
the ability to differentiate into a cell exhibiting one or more characteristics of an extravillous trophoblast (EVT) or syncytiotrophoblast (ST);
a methylation pattern similar to a blastocyst-derived TSC, as determined by a bisulfite assay; and
the expression of one or more biochemical markers of a TSC, as determined by an immunohistochemistry and/or PCR assay.
2 . The method according to claim 1 , wherein the TSC culture medium comprises a growth factor, and a Rho-kinase (ROCK) inhibitor.
3 . The method according to claim 1 , wherein the TSC medium comprises a growth factor, and a Rho-kinase (ROCK) inhibitor and wherein the growth factor is selected from: Epidermal Growth Factor (EGF), insulin, transforming growth factor (TGF) and/or wherein the ROCK inhibitor is trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632), or a salt thereof.
4 . The method according to claim 1 , wherein the TSC medium comprises a growth factor, and a Rho-kinase (ROCK) inhibitor and wherein the TSC culture medium additionally comprises one or more of:
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.
5 . The method according to claim 1 , wherein the TSC culture is ASECRiAV and comprises: A83-01, SB431542, EGF, CHIR, a ROCK inhibitor, ascorbic acid and valproic acid.
6 . The method according to claim 1 , wherein the somatic cell is selected from: a fibroblast, a dermal fibroblast, an epidermal cell, a keratinocyte, or a monocyte.
7 . The method according to claim 1 , wherein the factors for reprogramming the somatic cell towards a de-differentiated or pluripotent state, are one or more of: OCT4, SOX2, KLF4 and MYC, or wherein the factors are OCT4, SOX2, KLF4 and MYC.
8 . The method according to claim 1 , wherein the factors for reprogramming the somatic cell towards a de-differentiated or pluripotent state, are OCT4, SOX2, KLF4, MYC and LIN28 or, OCT4, SOX2, KLF4, MYC and NANOG, or OCT4, SOX2, KLF4, MYC, LIN28 and NANOG.
9 . The method according to claim 1 , wherein the somatic cell is contacted with the TSC medium at least 1 days, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42 or more days after increasing the protein expression or amount of one or more factors for reprogramming the somatic towards a de-differentiated or pluripotent state.
10 . The method according to claim 1 , wherein the somatic cell is cultured in non-pluripotent medium during the reprogramming process, and prior to being contacted with the TSC medium.
11 . The method according to claim 1 , wherein the step of culturing the cell for a sufficient time and under conditions to allow the reprogramming of the cell towards a de-differentiated or pluripotent state, comprises culturing the cell in media for supporting growth of the somatic cell a period of at least 7 days and no more than 22 days, wherein the somatic cell has not achieved pluripotency prior to being contacted with a TSC culture medium suitable for sustaining trophoblast stem cells (TSC).
12 . The method according to claim 1 , wherein the method comprises:
a) contacting somatic cells with an agent for increasing the amount or expression of OCT4, SOX2, KLF4 and MYC in the cell;
b) culturing the cells in culture medium for supporting the growth of the somatic cell, for a sufficient time and under conditions to allow the generation of reprogramming intermediate cells;
c) contacting the reprogramming intermediate cells with a TSC culture medium suitable for sustaining trophoblast stem cells (TSCs), thereby producing a cell exhibiting at least one characteristic of a TSC from a somatic cell.
13 . The method according to claim 12 , wherein the medium comprises A83-01, SB431542, EGF, CHIR, a ROCK inhibitor, ascorbic acid and valproic acid.
14 . The method according to claim 1 , wherein the somatic cell is further cultured in naïve media or extended media prior to contacting and culturing the cells with TSC medium.
15 . The method of claim 1 , wherein the at least one characteristic of a TSC further comprises one or more of:
the expression of one or more biochemical markers of a TSC, as determined by an immunohistochemistry and/or PCR assay, wherein the markers are selected from the group consisting of: CD49f (iTGA6), CD249, nuclear GATA2/3, TFAP2C, P63, and NR2F2;
an absence of the markers characterising the somatic cell, optionally wherein the cell exhibiting at least one characteristic of a TSC does not express one or more of the following markers: OCT4 (also called POU5F1), NANOG, SOX2, SALL2, OTX2, BANCR, KLF17, DPPA3, ARGFX and DNMT3L;
retains its undifferentiated state when maintained in subculture;
the ability to differentiate into a cell exhibiting one or more characteristics of an extravillous trophoblast (EVT) or syncytiotrophoblast (ST); and
expression of one or more biochemical markers of a TSC selected from the group consisting of: nuclear GATA2/3, TFAP2C, P63, and NR2F2;
and wherein the cell retains 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.
16 . The method of claim 1 wherein the method further comprises the step of differentiating the cells exhibiting at least one characteristic of a TSC to generate a cell exhibiting at least one characteristic of an EVT or an ST or to a non-placental cell type, for use in regenerative medicine.
17 . A pharmaceutical composition or supplement comprising:
a cell obtained according to claim 1 ;
a population of cells obtained according to claim 1 ;
a differentiated cell or population of differentiated cells obtained according to claim 1 , followed by a step of differentiation; or
an organoid obtained according to the method of claim 1 , or part thereof;
and a pharmaceutically acceptable excipient.
18 . A method of treating and/or preventing a disorder associated with the development and/or activity of trophoblasts in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of:
a cell obtained according to the method of claim 1 ;
a population of cells obtained according to the method of claim 1 ;
a differentiated cell or population of differentiated cells obtained according to the method of claim 1 , followed by a step of differentiation;
an organoid obtained according to the method of claim 1 ; or
a pharmaceutical composition comprising a cell obtained according to claim 1 ,
to a subject in need thereof, thereby treating and/or preventing the disorder associated with the development and/or activity of trophoblasts in the subject.
19 . The method according to claim 1 , wherein the cell exhibiting at least one characteristic of a human TSC is further introduced into a placenta or blastocyst thereby augmenting the placenta or the blastocyst.
20 . The method according to claim 1 , wherein the cell exhibiting at least one characteristic of a human TSC is further contacted with an agent,
wherein the development and/or activity of the cell, following the contacting with the agent is compared to the development and/or activity of the cell, without the agent to identify the agent capable of modulating trophoblast development and/or activity, and
wherein the effect of the agent on the development and/or activity of the cell above a predetermined level relative to the development of the cell without the agent is indicative that the agent modulates trophoblast development and/or activity.
21 . The method according to claim 1 , wherein the cell exhibiting at least one characteristic of a human TSC is further cultured and a compound secreted by the cells is isolated from the culture medium.