Patent Description:
A continuum of pluripotent configurations represents changes occurring during in vivo transition of naive pre-implantation pluripotency toward that of primed post-implantation pluripotent state, can be captured in vitro to various extents. Many naive and primed pluripotency properties can be individually characterized and attributed to pluripotent stem cells expanded in distinct conditions. In mice, defined serum free 2i/LIF conditions have been extensively characterized where many naive molecular and functional properties are endowed by these conditions. The latter include global DNA hypomethylation, loss of bivalency over developmental genes, exclusive nuclear localization of TFE3 transcription factor, tolerance of lack of exogenous L-glutamine, tolerance for loss of repressors like DNMT1, METTL3 and DGCR8 (or DICER). Mouse ESCs expanded Fetal Bovine Serum (FBS)/Lif are also considered naive and possess features such as retention of pre-X inactivation state, ability to tolerate lack of repressors like Mettl3, Dnmt1 and Dgcr8. However, they do not retain a global hypermethylated epigenome, and acquire H3K27me3 over developmental genes consistent with retaining a relatively less naive state. Rodent EpiSCs expanded in Fgf2/Activin A show further consolidation and acquisition of their milieu of primed pluripotency characteristics, thus exemplifying how mouse naive and primed PSCs can have different mix of naive and primed pluripotent states. EpiSC lines are heterogeneous in their epigenetic and transcriptional patterns, and while they are pluripotent and give rise to differentiated cells from all three germ layers, they are epigenetically restricted as evident for example in their reduced ability, after long term/permanent maintenance in FGF2/ACTIVIN A conditions, to differentiate into primordial germ cells (PGCs) or contribute to chimera formation when injected in the pre-implantation ICM.

While conventional human embryonic stem cells (hESCs) and iPSCs (hiPSCs) growth conditions entailed FGF/TGFB as typical for murine EpiSC, these two cell types are not identical, and hESC share several molecular features with naive mESCs including expression of E-CADHERIN (rather than N-CADHERIN). Further, conventional human ESCs express high levels of PRDM14 and NANOG as murine naive ESCs, and they are functionally dependent on their expression. Still however, hESCs retain a variety of epigenetic properties that are consistent with possessing a primed pluripotent state. This includes inability to tolerate MEK/ERK signaling inhibition, predominant (yet non-exclusive) utilization of the proximal enhancer element to maintain OCT4 expression, tendency for initiation of X chromosome inactivation in most female ESC lines, pronounced increase in DNA methylation, prominent deposition of H3K27me3 and bivalency acquisition on lineage commitment regulators.

The ability of human zygotes to develop into blastocysts in the presence of MEK/ERKi and the proof of concept for the metastability between naive and primed state in rodents, have raised the possibility that the human genetic background is more "stringent" in regards to requirement for exogenous factors provided in allowing preservation of ground state-naive pluripotency in comparison to rodents.

Condition to derive naive MEK/ERK signaling-independent, genetically unmodified human pluripotent cells via iPSC generation, from established conventional ESC lines or directly from human blastocysts are described in <CIT>. Specifically, NHSM conditions do not require the use of exogenous transgenes or feeder cells, maintain teratoma formation competence and entail the following components: LIF, 2i, P38i/JNKi, PKCi, ROCKi, TGFB1/ACTIVIN A and FGF2. NHSM conditions endow human PSCs with variety with naïve features including maintain pluripotency while MEK/ERK signaling is inhibited, predominant TFE3 nuclear localization, resolution of bivalent domains over developmental regulators, in vitro reconstitution of human PGCLC and a mild reduction of demethylation. The latter effect was profoundly weaker than that seen in mouse pluripotent cells, suggesting sub-optimal human naïve pluripotency growth conditions.

<NPL>, describe alternative conditions that generate MEK independent human naïve cells and retain a more compelling milieu of transcriptional markers expressed in the human ICM. Several components found in NHSM conditions (2i, ROCK inhibitor, FGF/ACTIVIN) were supplemented with BRAF inhibitors, to generate MEF obligatory dependent naive cell lines (different conditions termed: 5iLA-, 5iLAF-, 6i/LA- and 4i/LA-MEF conditions). Globally these conditions generated more pronounced downregulation in DNA methylation and upregulation of naive pluripotent cell markers. However, the hypomethylation in these conditions is however accompanied by immediate and global deterministic loss of imprinting (<NPL>) and obligatory confounding chromosomal abnormalities in nearly <NUM>% of the line generated by <NUM> passages only (<NPL>).

Derivation of human naive ESC in t2iL-Go conditions has been reported, however these results have not yet been reproduced without exogenous transgenes. In both cases, the reported cell line do not form teratomas in vivo and can only differentiate in vitro after an extended <NUM>-week transfer to primed conditions, thus questioning their pluripotent functionality and stability (<NPL>; <NPL>; <NPL>). The latter is in striking difference from rodent ground state naïve PSCs, which are fully pluripotent and can initiate differentiation in vivo following autologous induction of the needed priming signals toward differentiation.

Additional background art includes <CIT>.

Some aspects of the disclosure are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of aspects of the disclosure. In this regard, the description taken with the drawings makes apparent to those skilled in the art how aspects of the disclosure may be practiced.

The present disclosure, in some aspects thereof, relates to culture media for culturing pluripotent stem cells more particularly, but not exclusively, to naive pluripotent stem cells.

Before explaining at least one aspect of the disclosure in detail, it is to be understood that the disclosure is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The disclosure is capable of other aspects or of being practiced or carried out in various ways.

The present inventors have uncovered novel conditions, which are required for isolating and generating primate (e.g., human) pluripotent stem cells (PSCs), and maintaining them in their pluripotent state.

As shown in the Examples section which follows, the present inventors have uncovered through laborious experimentation, particular combinations of factors that are required for maintaining PSCs in a pluripotent state in general and more specifically, in a "naive state". Unlike combinations of factors previously disclosed, (see for example <CIT>), the present combinations were shown to maintain the pluripotent stem cell in a hypomethylated state.

Thus, according to a first aspect of the present disclosure, there is provided a culture medium comprising a Wingless/Integrated (WNT) inhibitor, a SRC Proto-Oncogene, Non-Receptor Tyrosine Kinase (SRC) inhibitor and a protein kinase C (PKC) inhibitor, the medium being devoid of an amount of GSK3β inhibitor that increases β-catenin translocation to the nucleus of a pluripotent stem cell being cultured in the culture medium.

According to another aspect of the present disclosure, there is provided a culture medium comprising a WNT inhibitor, a Notch inhibitor and a protein kinase C (PKC) inhibitor, the medium being devoid of an amount of GSK3β inhibitor that increases β-catenin translocation to the nucleus of a pluripotent stem cell being cultured in said culture medium.

As used herein the phrase "culture medium" refers to a solid or a liquid substance used to support the growth of stem cells and maintain them in an undifferentiated state. Preferably, the phrase "culture medium" as used herein refers to a liquid substance capable of maintaining the stem cells in an undifferentiated state. The culture medium used by the present disclosure can be a water-based medium which includes a combination of substances such as salts, nutrients, minerals, vitamins, amino acids, nucleic acids, proteins such as cytokines, growth factors and hormones, all of which are needed for cell proliferation and are capable of maintaining the stem cells in an undifferentiated state. For example, a culture medium can be a synthetic tissue culture medium such as KO-DMEM (Gibco-Invitrogen Corporation products, Grand Island, NY, USA), DMEM/F12 (Gibco-Invitrogen Corporation products, Grand Island, NY, USA), Neurobasal medium (Invitrogen Corporation products, Grand Island, NY, USA <NUM>-<NUM>) or DMEM/F12 (without HEPES; Biological Industries, Biet Haemek, Israel), supplemented with the necessary additives as is further described hereinunder.

According to a particular aspect, the medium is a <NUM>:<NUM> mix of Neurobasal medium and DMEM F/<NUM>.

Preferably, all ingredients included in the culture medium of the present disclosure are substantially pure, with a tissue culture grade.

According to some aspects of the disclosure, the culture medium is devoid of serum, e.g., devoid of any animal serum.

According to some aspects of the disclosure, the culture medium is devoid of any animal contaminants, i.e., animal cells, fluid or pathogens (e.g., viruses infecting animal cells), e.g., being xeno-free.

According to some aspects of the disclosure, the culture medium is devoid of human derived serum.

According to some aspects of the disclosure, the culture medium further comprises a serum replacement (i.e., a substitute of serum) such as KNOCKOUT™ Serum Replacement (Gibco-Invitrogen Corporation, Grand Island, NY USA), ALBUMAX®II (Gibco®; Life Technologies - Invitrogen, Catalogue No. <NUM>-<NUM>;
Lipid-rich bovine serum albumin for cell culture) or a chemically defined lipid concentrate (Gibco®; Invitrogen, Life Technologies - Invitrogen, Catalogue No. <NUM>-<NUM>).

According to some aspects of the disclosure, the culture medium further comprises N2 supplement (Gibco®; Life Technologies - Invitrogen, Catalogue No. <NUM>-<NUM>) a chemically defined, serum-free supplement. For a <NUM> of culture medium <NUM> of the N2 mix (Invitrogen) can be added.

Alternatively, the following materials (substitute the N2 supplement) can be added to a <NUM> culture medium: Recombinant Insulin (Sigma I-<NUM>) at a <NUM> microg/ml (µg/ml) final concentration; Apo-Transferrin (Sigma T-<NUM>) at a <NUM>µg/ml final concentration; Progesterone (Sigma- P8783) at a <NUM>µg/ml final concentration; Putrescine (Sigma- P5780) at a <NUM>µg/ml final concentration; and <NUM> microL (µl) of <NUM> stock of Sodium Selenite (Sigma - S5261) are added per <NUM> culture medium (e.g., the WIS-NHSM).

According to some aspects of the disclosure, the KNOCKOUT™ Serum Replacement is provided at a concentration of at least <NUM>%, e.g., in the range of about <NUM>%-<NUM>%, e.g., about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>% or about <NUM>%.

According to some aspects of the disclosure, the ALBUMAX™ is provided at a concentration of at least <NUM>%, e.g., in the range of about <NUM>%-<NUM>%, e.g., about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>% or about <NUM>%, e.g., <NUM>%.

According to some aspects of the disclosure, the defined lipid concentrate is provided at a concentration of at least about <NUM>%, e.g., in the range of <NUM>-<NUM>%, e.g., about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, e.g., <NUM>%.

According to some aspects of the disclosure, the culture medium comprises the N2 supplement (e.g., <NUM> N2 per <NUM> of culture medium) and the defined lipid concentrate (<NUM> defined lipid concentrate per <NUM> medium).

According to some aspects of the disclosure, the culture medium can further include antibiotics (e.g., PEN-STREP), sodium pyruvate, B27, NEAA (non-essential amino acids).

The culture medium may comprise glutamine or be devoid of glutamine (e.g. only comprise trace amounts (e.g. less than <NUM>/<NUM>th of the amount that is typically present in base media such that it does not bring about a biological effect). In one aspect, the medium is completely devoid of exogenously added glutamine.

The present inventors contemplate addition of a combination of specific inhibitors to the medium disclosed. Such inhibitors are preferably specific towards their target. In one aspect, they are capable of binding the named target with a higher affinity (at least <NUM> %, <NUM> %, <NUM> %, <NUM> % <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> % or even <NUM> % higher affinity) than another protein which is expressed in the cell.

As mentioned, the media of the present disclosure comprise a Wnt inhibitor.

The term "Wnt inhibitor" as used refers to any agent, including any compound and/or protein that inhibits Wnt signaling, including but not limited to Wnt antagonists that bind either to the Wnt ligand itself, or to Wnt receptors, such as Dickkopf (Dkk) proteins, Wnt Inhibitory Factor-<NUM> (WIF-<NUM>), and secreted Frizzled-Related Proteins (sFRPs), as well as Wnt inverse agonists (e.g. an agent that binds to the same receptor as an agonist but induces a pharmacological response opposite to that of an agonist).

According to a particular aspect, the Wnt inhibitor is a small molecule.

In one aspect, the Wnt inhibitor brings about its effect by stabilizing the AXIN/APC complex which in turn degrades β-catenin, thereby inhibiting Wnt signaling.

Exemplary Wnt inhibitors include, but are not limited to ICG-<NUM>, IWR-<NUM>, IWP2, XAV939, Wnt-C59 (C59), IWP-L6, iCRT3, LF3, PNU-<NUM>, KYA1797K, PRI-<NUM> and WIKI <NUM>, all of which are commercially available from Selleckchem and/or Tocris.

According to a particular aspect, the Wnt inhibitor is a Tankyrase inhibitor (e.g. IWR-<NUM> - Sigma Aldrich <NUM>; and XAV939 - TOCRIS Cat. No. <NUM>). In one aspect, the Tankyrase inhibitor is one which blocks the PARP domain of Tankyrase (which ultimately leads to an increase in the stability of AXIN1 and AXIN2 and therefore inhibition of canonical Wnt signaling).

Another exemplary WNT inhibitor is a small molecule inhibitor for Porcupine enzyme which is responsible for processing and secretion of all Wnt signaling ligands (e.g. IWP2).

The Wnt inhibitor is typically present in the medium in an amount such that the overall net effect thereof is a reduction in the amount of β-catenin in the nucleus of a pluripotent stem cell which is cultured within. It will be appreciated that the medium is typically devoid of agents which promote β-catenin translocation to the nucleus. Thus, according to this aspect of the present disclosure, the medium is devoid of an amount of GSK3β inhibitor that increases β-catenin translocation to the nucleus of a pluripotent stem cell being cultured in the culture medium. For example, the medium of the present disclosure should not contain more than <NUM> of a GSK3β inhibitor and preferably not more than <NUM> of a GSK3β inhibitor. It will be appreciated that the phrase "being devoid of a GSK3β inhibitor" refers to a medium in which no GSK3β inhibitor has been positively added to a medium and does not mean to exclude that a trace amount of GSK3 inhibitor is contained in the base medium.

Exemplary amounts of Wnt inhibitor (e.g. XAV939) are between <NUM> - <NUM>, more preferably between <NUM> - <NUM>, <NUM> - <NUM>, and more preferably between <NUM> - <NUM> - about <NUM>.

As mentioned, a NOTCH signaling inhibitor is contemplated to be included in the media of the present disclosure. Preferably, the NOTCH signaling inhibitor is added when the medium comprises less than <NUM>, for example about <NUM>, <NUM>, <NUM> or <NUM> ERK1/<NUM> inhibitor. In a particular aspect, the NOTCH signaling inhibitor is added to a medium which is devoid of an ERK1/<NUM> inhibitor.

NOTCH signaling inhibitors include, but are not limited to the following gamma secretase inhibitors: DAPT (Axon Medchem <NUM> - <NUM>-<NUM> final concentration), LY2886721 hydrochloride (Axon Medchem <NUM> - <NUM>-<NUM> final concentration)], DBZ (Axon Medchem - Axon <NUM>- <NUM>-<NUM> final concentration).

According to a particular aspect, the NOTCH signaling inhibitor is one which inhibits the transcription factor RBPJ - Recombination Signal Binding Protein For Immunoglobulin Kappa J Region. An example of such an inhibitor is RIN1 (see for example <NPL>). An exemplary concentration of RIN1 is <NUM>-<NUM> and more preferably between <NUM> and <NUM>.

The medium of this aspect of the present disclosure may further comprise a SRC inhibitor, also referred to herein as a src family kinase inhibitor.

The phrase "src family kinase inhibitor" refers to any agent which impedes or inhibits the function of a member of the src kinase family. Such agents include, without limitation, small molecules, chemical compounds and nucleic acid molecules which function to down regulate expression of target genes and inhibit the function of direct and indirect c-Src substrates, such as the focal adhesion kinase, signal transducer and activator of transcription <NUM> (STAT3), vascular endothelial growth factor (VEGF), paxillin, Cas, p190RhoGAP, RRas, E-cadherin, c-Jun amino-terminal kinase, NEDD9, and others. Exemplary agents include dasatinib, SU6656, and AZD05530. Src inhibitors are also available from Wyeth and include for example, <NUM>-[(<NUM>,<NUM>-Dichloro-<NUM>-methoxyphenyl)amino]-<NUM>-[<NUM>-(<NUM>-ethyl-<NUM>-piperazinyl)propo- xy]-<NUM>-methoxy-<NUM>-quinolinecarbonitrile; <NUM>-[(<NUM>,<NUM>-Dichloro-<NUM>-methoxyphenyl)amino]-<NUM>-methoxy-<NUM>-[<NUM>-(<NUM>-methyl-<NUM>-pipera- zinyl)ethoxy]-<NUM>-quinolinecarbonitrile; <NUM>-[(<NUM>,<NUM>-Dichloro-<NUM>-methoxyphenyl)amino]-<NUM>-[<NUM>-(<NUM>-ethyl-<NUM>-piperazinyl)ethox- y]-<NUM>-methoxy-<NUM>-quinolinecarbonitrile; <NUM>-[(<NUM>,<NUM>-Dichloro-<NUM>-methoxyphenyl)amino]-<NUM>-methoxy-<NUM>-[(<NUM>-methylpiperidin-<NUM>- -yl)methoxy]-<NUM>-quinolinecarbonitrile; <NUM>-[(<NUM>,<NUM>-Dichloro-<NUM>-methoxyphenyl)amino]-<NUM>-methoxy-<NUM>-[<NUM>-(<NUM>-methylpiperidin- -<NUM>-yl)ethoxy]-<NUM>-quinolinecarbonitrile; <NUM>-[(<NUM>,<NUM>-Dichloro-<NUM>-methoxyphenyl)amino]-<NUM>-methoxy-<NUM>-[<NUM>-(<NUM>-methylpiperidin- -<NUM>-yl)propoxy]quinoline-<NUM>-carbonitrile; <NUM>-[(<NUM>,<NUM>-Dichloro-<NUM>-methoxyphenyl)amino]-<NUM>-[(<NUM>-ethylpiperidin-<NUM>-yl)methoxy-]-<NUM>-methoxyquinoline-<NUM>-carbonitrile; <NUM>-[(<NUM>,<NUM>-Dichloro-<NUM>-methoxyphenyl)amino]-<NUM>-ethoxy-<NUM>-[<NUM>-(<NUM>-methylpiperazin-- <NUM>-yl)propoxy]quinoline-<NUM>-carbonitrile; <NUM>-[(<NUM>,<NUM>-Dichloro-<NUM>-methoxyphenyl)amino]-<NUM>-ethoxy-<NUM>-[(<NUM>-methylpiperidin-<NUM>-- yl)methoxy]quinoline-<NUM>-carbonitrile; <NUM>-[(<NUM>,<NUM>-Dichloro-<NUM>-methoxyphenyl)amino]-<NUM>-ethoxy-<NUM>-[<NUM>-(<NUM>-ethylpiperazin-<NUM>- -yl)propoxy]quinoline-<NUM>-carbonitrile; <NUM>-[(<NUM>,<NUM>-Dichloro-<NUM>-methoxyphenyl)amino]-<NUM>-ethoxy-<NUM>-[<NUM>-(<NUM>-methylpiperidin-- <NUM>-yl)propoxy]quinoline-<NUM>-carbonitrile; <NUM>-[(<NUM>,<NUM>-Dichloro-<NUM>-methoxyphenyl)amino]-<NUM>-ethoxy-<NUM>-[<NUM>-(<NUM>-methyl-<NUM>-piperaz- inyl)ethoxy]quinoline-<NUM>-carbonitrile; <NUM>-[(<NUM>,<NUM>-Dichloro-<NUM>-methoxyphenyl)amino]-<NUM>-ethoxy-<NUM>-[<NUM>-(<NUM>-methylpiperidin-- <NUM>-yl)ethoxy]quinoline-<NUM>-carbonitrile; or <NUM>-[(<NUM>,<NUM>-Dichloro-<NUM>-methoxyphenyl)amino]-<NUM>-methoxy-<NUM>-[<NUM>-(<NUM>-propyl-<NUM>-pipera- zinyl)propoxy]-<NUM>-quinolinecarbonitrile; and pharmaceutically acceptable salts thereof.

According to a particular aspect, the agent which possesses inhibitory activity against the Src family kinase is a small molecule agent.

According to a particular aspect, the agent which possesses inhibitory activity against the Src family kinase is a chemical agent.

Suitable compounds possessing inhibitory activity against the Src family of non-receptor tyrosine kinases include the quinazoline derivatives disclosed in <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT> (arising from <CIT>), <CIT> (arising from <CIT>) and <CIT> (arising from <CIT>), the quinoline derivatives described in <CIT> (arising from <CIT>), <CIT> and <CIT> and the quinazoline derivatives described in <CIT>) and <CIT>).

It is disclosed in <NPL> that certain <NUM>-anilino-<NUM>-cyanoquinoline derivatives are useful for the inhibition of Src-dependent cell proliferation. The <NUM>-anilino-<NUM>-cyanoquinoline Src inhibitor known as SKI <NUM> is described in <NPL>.

Other compounds which possess Src kinase inhibitory properties are described in, for example, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT> and <CIT>.

Other compounds which possess Src kinase inhibitory properties are described in, for example, <NPL>, <NPL>, <NPL>, <NPL> and <NPL>.

Particular Src kinase inhibitors include the following:.

Other compounds which may possess Src kinase inhibitory properties are described in, for example, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT> and <CIT>.

Particular Src inhibitors include those provided in <CIT>.

Further particular Src inhibitors include the following compounds from <CIT>, <CIT>, <CIT> and <CIT>.

Exemplary agents include, without limitation, dasatinib, and AZD0530.

Other exemplary agents include CGP77675 (AXON MEDCHEM <NUM>), SU <NUM>, AZD0530, Dasatinib, Bosutinib and WH-<NUM>-<NUM>.

According to some aspects of the disclosure, the Src family kinase inhibitor (e.g. CGP77675) is provided at a concentration range of between about <NUM>-<NUM>, e.g., from about <NUM> to about <NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., about <NUM>-<NUM>, e.g., about <NUM>.

Since SRC inhibition leads to NPKβ signaling inhibition, the present inventors contemplate use of NFKβ pathway inhibitors instead of the SRC inhibitors.

Examples of small molecule NFKβ inhibitors include, but are not limited to Rolipram, JSH-<NUM> and LY <NUM>. Exemplary concentrations the NFKβ inhibitors may be used is between <NUM>-<NUM>.

As mentioned, the media described herein also comprise a protein kinase C inhibitor.

As used herein the term "protein kinase C inhibitor" refers to any molecule capable of inhibiting the activity of protein kinase C as determined by reducing the levels of phosphorylated versus non phosphorylated PKC isoforms. According to a particular aspect, the PKC inhibitor is a small molecule inhibitor.

A non-limiting example of a protein kinase C inhibitor is Go6983 (<NPL>), a potent, cell-permeable, reversible, and ATP-competitive inhibitor of protein kinase C (PKC) with a broad spectrum protein kinase C (PKC) inhibitor (IC50 values are <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> for PKCα, PKCβ, PKCγ, PKCδ, PKCζ and PKCµ respectively). Go6983 is available from various suppliers such as Calbiochem (Catalogue number <NUM>-500UG), and TOCRIS (Catalogue number <NUM>).

According to some aspects of the disclosure, Go6983 is provided at a concentration range of between about <NUM>-<NUM>, e.g., from about <NUM> to about <NUM>, e.g., between about <NUM>-<NUM>, <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., about <NUM>.

Additional agents that may be added to the medium include a STAT3 activator, an ERK inhibitor, a p38 inhibitor and a ROCK inhibitor each of which will be described herein below.

As used herein the term "STATS" refers to the signal transducer and activator of transcription <NUM> gene product (acute-phase response factor) (Gene ID <NUM>). In response to cytokines and growth factors, STAT family members are phosphorylated by the receptor associated kinases, and then form homo - or heterodimers that translocate to the cell nucleus where they act as transcription activators. Known STAT3 activators include, but are not limited to, interferon (IFN), epidermal growth factor (EGF), interleukin <NUM> (IL5), interleukin <NUM> (II,<NUM>), hepatocyte growth factor (HGF), leukemia inhibitory factor (LIF) and bone morphogenetic protein <NUM> (BMP2).

According to some aspects of the disclosure, the STAT3 activator, which is used in the medium of some aspects of the disclosure is selected from the group consisting of LIF, IL6 and EGF.

According to some aspects of the disclosure, the STAT3 activator, which is used in the medium of some aspects of the disclosure is selected from the group consisting of LIF and IL6.

According to some aspects of the disclosure, the STAT3 activator, which is used in the medium of some aspects of the disclosure is LIF.

As used herein the term "leukemia inhibitor factor (LIF)" refers to a polypeptide which comprises the amino acid sequence as set forth by GenBank Accession No. NP_001244064. <NUM> (SEQ ID NO: <NUM>), encoded by the nucleotide sequence set forth in GenBank Accession No. NM_001257135 (SEQ ID NO: <NUM>). Preferably, the LIF used by the method according to some aspects of the disclosure is capable of supporting, along with other factors which are described herein, the undifferentiated growth of naive primate (e.g., human) PSCs, while maintaining their pluripotent capacity. LIF can be obtained from various manufacturers such as Millipore, Peprotech, and R&D systems.

According to some aspects of the disclosure, LIF is provided at a concentration range from about <NUM> nanogram per milliliter (ng/ml) to about <NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM> ng/ml.

As used herein the term "interleukin <NUM> (II,<NUM>)" refers to a polypeptide which comprises the amino acid sequence set forth by GenBank Accession No. NP_000591. <NUM> (SEQ ID NO: <NUM>), which is encoded by the nucleic acid set forth by GenBank Accession No. NM_000600. <NUM> (SEQ ID NO: <NUM>). Preferably, the IL6 used by the method according to some aspects of the disclosure is capable of supporting, along with other factors which are described herein, the undifferentiated growth of naive primate (e.g., human) PSCs, while maintaining their pluripotent capacity. II,<NUM> can be obtained from various manufacturers such as Speed BioSystems, Millipore, Peprotech, and R&D systems.

According to some aspects of the disclosure, IL6 is provided at a concentration range from about <NUM> ng/ml to about <NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM>-<NUM> ng/ml, e.g., about <NUM> ng/ml.

As used herein the term "p38" refers to the "p38α (alpha)" mitogen-activated protein kinase <NUM> (MAPK14), which includes MAPK14 isoform <NUM> set forth by GenBank Accession No. NP_001306. <NUM> (SEQ ID NO: <NUM>), MAPK14 isoform <NUM> set forth by GenBank Accession No. NP_620581. <NUM> (SEQ ID NO: <NUM>), MAPK14 isoform <NUM> set forth by GenBank Accession No. NP_620582. <NUM> (SEQ ID NO: <NUM>) and MAPK14 isoform <NUM> set forth by GenBank Accession No. NP_620583. <NUM> (SEQ ID NO: <NUM>); "ρ38β (beta)" (MAPK11), which is set forth by GenBank Accession No. NP_002742. <NUM> (SEQ ID NO: <NUM>); "ρ38γ (gamma)" (MAPK12) which is set forth by GenBank Accession No. NP_002960. <NUM> (SEQ ID NO: <NUM>); and/or "p38δ (delta)" (MAPK13) which is set forth in GenBank Accession No. NP_002745. <NUM> (SEQ ID NO: <NUM>), all of them having kinase activity and involved in signal transduction.

As used herein the term "p38 inhibitor" refers to any molecule (e.g., small molecules or proteins) capable of inhibiting the activity of p38 family members as determined by Western blot quantification of phosphorylated p38 levels.

Non-limiting examples of p38 inhibitors include SB203580 (AXONMEDCHEM - Axon <NUM>), and SB <NUM> (AXONMEDCHEM - Axon <NUM>), LY <NUM> (AXONMEDCHEM-Axon <NUM>), BIRB0796 (Axon Medchem <NUM>) and PD169316 (AXONMEDCHEM - Axon <NUM>).

As BMP signaling is an activator for p38 signaling, examples of p38 inhibitors also include BMP inhibitors like Dorsomorphin (AXONMEDCHEM - Axon <NUM>) and LDN193189 (AXON MEDCHEM AXON <NUM>) or other inhibitors of the BMP pathway such as recombinant NOGGIN protein [GenBank Accession No. NP_005441. <NUM> (SEQ ID NO: <NUM>] can be used to replace small molecule inhibitors of BMP signaling.

According to some aspects of the disclosure, SB203580 is provided at a concentration range of between about <NUM>-<NUM>, e.g., from about <NUM> to about <NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., about <NUM>, e.g., about <NUM>.

According to some aspects of the disclosure, SB <NUM> is provided at a concentration range of between about <NUM> to about <NUM>, e.g., from about <NUM> to about <NUM>, e.g., from about <NUM> to about <NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., about <NUM>.

According to some aspects of the disclosure, BIRB0796 is provided at a concentration range of between about <NUM> to about <NUM>, e.g., from about <NUM> to about <NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., about <NUM>.

As used herein the term "ROCK" refers to the protein set forth by GenBank Accession No. NP_005397. <NUM> (P160ROCK; SEQ ID NO: <NUM>); and NP_004841. <NUM> (ROCK2; SEQ ID NO: <NUM>) having the serine/threonine kinase activity, and regulates cytokinesis, smooth muscle contraction, the formation of actin stress fibers and focal adhesions, and the activation of the c-fos serum response element.

As used herein the term "ROCK inhibitor" refers to any molecule capable of inhibiting the activity of ROCK as determined by inhibition of ROCK phosphorylation levels (detected by western blot analysis).

According to a particular aspect, the ROCK inhibitor is a small molecule agent.

Non-limiting examples of ROCK inhibitors include Y27632 (TOCRIS, Catalogue number <NUM>).

According to some aspects of the disclosure, Y27632 is provided at a concentration range of between about <NUM>-<NUM>, e.g., from about <NUM> to about <NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g. between about <NUM>-<NUM>, e.g., about <NUM>.

It will be appreciated that instead of a ROCK inhibitor, the present inventors contemplate using an inhibitor of JNK.

As used herein the term "JNK" refers to the mitogen-activated protein kinase <NUM> (MAPK8) protein set forth by GenBank Accession Nos. <NUM> (isoform alpha2) (SEQ ID NO: <NUM>), NP_620635. <NUM> (isoform beta2) (SEQ ID NO: <NUM>), NP_620634. <NUM> (isoform beta1) (SEQ ID NO: <NUM>), NP_002741. <NUM> (isoform alpha1) (SEQ ID NO: <NUM>) which are involved in a wide variety of cellular processes such as proliferation, differentiation, transcription regulation and development.

As used herein the term "JNK inhibitor" refers to any molecule (e.g. small molecule) capable of inhibiting the activity of JNK as determined by phosphorylation of JNK family member protein by western blot analysis.

Non-limiting examples of JNK inhibitors include SP600125 (TOCRIS - Cat no. <NUM>), AEG3482 (AXONMEDCHEM- AXON <NUM>), BIX02189, BRAFi (SB590885) and BIRB796 (AXONMEDCHEM - Axon <NUM>).

According to some aspects of the disclosure, SP600125 is provided at a concentration range of between about <NUM>-<NUM>, e.g., from about <NUM> to about <NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., about <NUM>.

According to some aspects of the disclosure, BIX02189 is provided at a concentration range of between about <NUM>-<NUM>, e.g., from about <NUM> to about <NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., about <NUM>.

According to some aspects of the disclosure, BRAFi (SB590885) is provided at a concentration range of between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., about <NUM>.

As used herein the term "ERK1" refers to the mitogen-activated protein kinase <NUM> (MAPK3) isoform <NUM> set forth by GenBank Accession No. NP_002737. <NUM> (SEQ ID NO: <NUM>), the MAPK3 isoform <NUM> set forth by GenBank Accession No. NP_001035145. <NUM> (SEQ ID NO: <NUM>), the MAPK3 isoform <NUM> set forth by GenBank Accession No. NP_001103361. <NUM> (SEQ ID NO: <NUM>) and/or ERK1 set forth in GenBank Accession No. M84490 (SEQ ID NO: <NUM>) having the MAPK signaling activity.

As used herein the term "ERK2" refers to the mitogen-activated protein kinase <NUM> (MAPK1) set forth by GenBank Accession No. NP_002736. <NUM> (SEQ ID NO: <NUM>) and/or GenBank Accession No. NP_620407. <NUM> (SEQ ID NO: <NUM>) having the MAPK signaling activity.

As used herein the term "ERK1/<NUM> inhibitor" refers to any molecule capable of inhibiting the activity of ERK1/<NUM> as determined by Western blot protein detection of phosphorylated ERK1/<NUM> proteins. According to a particular aspect, the ERK1/<NUM> inhibitor is a small molecule agent.

Non-limiting examples of ERK1/<NUM> inhibitors (also known as MEK1/<NUM> inhibitors) include PD0325901 (AXONMEDCHEM - AXON <NUM>), PD98059 (AXONMEDCHEM - Axon <NUM>), and PD184352 (AXONMEDCHEM - AXON <NUM>); and/or even inhibitors of RAF (which is upstream of MEK/ERK pathway) such as Sorafenib tosylate (also known as BAY <NUM>-<NUM> AXONMEDCHEM -AXON <NUM>) or SB <NUM> (TOCRIS #<NUM>).

According to some aspects of the disclosure, PD0325901 is provided at a concentration range from about <NUM> microM (µM) to about <NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., about <NUM>.

According to some aspects of the disclosure, PD98059 is provided at a concentration range from about <NUM> microM (µM) to about <NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., about <NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>.

According to some aspects of the disclosure, PD184352 is provided at a concentration range from about <NUM> microM (µM) to about <NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., about <NUM>. e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>.

According to some aspects of the disclosure, Sorafenib is provided at a concentration range from about <NUM> microM (µM) to about <NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between about <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., about <NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>, e.g., between <NUM>-<NUM>.

A particular contemplated media is one which comprises each of the following components: LIF, WNT inhibitor, ERK inhibitor, P38 inhibitor, PKC inhibitor SRC inhibitor and Rock inhibitor.

In some cases the amount of ERK1/<NUM> inhibitor present in the medium is less than <NUM>, for example about <NUM>, <NUM>, <NUM> or <NUM>. In some cases, the medium is devoid of ERK1/<NUM> inhibitor. It will be appreciated that the phrase "being devoid of ERK1/<NUM> inhibitors" refers to a medium in which no ERK1/<NUM> inhibitors have been positively added to a medium and does not mean to exclude trace amounts of ERK1/<NUM> inhibitors contained in the base medium.

The present inventors contemplate addition of an activator of the TGF-Activin pathway to the medium when the ERK1/<NUM> in the medium is less than <NUM>.

According to some aspects of the disclosure, activators of TGF/ACTIVIN pathway including ACTIVIN A (also known as Inhibin beta A, INHBA, Gene ID: <NUM>; GenBank Accession No. NM_002192. <NUM> (SEQ ID NO: <NUM>), which encodes GenBank Accession No. NP_002183. <NUM>; SEQ ID NO: <NUM>).

Preferably the amount of ACTIVIN A added is between <NUM>-<NUM> ng/ml and more preferably between <NUM>-<NUM> ng/ml (for example about <NUM> ng/ml).

NOTCH signaling inhibitors may also be included in the media of the present disclosure. Preferably, the NOTCH signaling inhibitor is added when the medium comprises less than <NUM>, for example about <NUM>, <NUM>, <NUM> or <NUM> ERK1/<NUM> inhibitor. NOTCH signaling inhibitors include, but are not limited to the following gamma secretase inhibitors: DAPT (Axon Medchem <NUM> - <NUM>-<NUM> final concentration), LY2886721 hydrochloride (Axon Medchem <NUM> - <NUM>-<NUM> final concentration)], DBZ (Axon Medchem - Axon <NUM>-<NUM>-<NUM> final concentration).

A particular contemplated media is one which comprises each of the following components: LIF, WNT inhibitor, Notch inhibitor, P38 inhibitor, PKC inhibitor SRC inhibitor, Activin A and Rock inhibitor.

In one aspect, the media of the present disclosure are devoid of exogenously added TGF (e.g. TGFβ1, TGFβ2) and FGF (e.g. bFGF). A medium devoid of TGF or FGF refers to a medium which does not comprise TGF or FGF in an amount that has an effect on the mitogenic activity of pluripotent cells cultured within. In one aspect, "being devoid of TGF or FGF" refers to a medium in which no TGF or FGF has been positively added to a medium and does not mean to exclude trace amounts TGF or FGF contained in the base medium.

Additional agents that may be added to the media of the present disclosure include at least one, at least two, at least three, at least four, at least five, at least six or more of the following agents: a ROCK inhibitor, Ascorbic acid, NFKb inhibitor, a YAP/TAZ inhibitor, an SHH inhibitor, a TGFβR inhibitor, a BMP inhibitor, an FGFR inhibitor, a JNK inhibitor, an ERK5 inhibitor, a BRAF inhibitor, an ARAFi, a CRAFi, a p38 inhibitor, an LSD1 inhibitor, a PI3K activator, a SMAD activator and a DOT1L inhibitor, Forskolin, Kenpaullone, BayK8644, an inhibitor of G9a, an inhibitor of Glp, stem cell factor (SCF), insulin-like growth factor <NUM> (IGF1), insulin-like growth factor II (IGFII), Mbd3/Gatad2a/NuRD complex inhibitor, HDAC inhibitor, Recombinant human Vitronectin, Recombinant human Laminin and Recombinant human Biolaminin.

Additional components that may be added to the media of this aspect of the present are disclosed in <CIT>.

The media described herein can be used to culture cells. Thus, according to an aspect of some aspects of the disclosure, there is provided a cell culture comprising cells and the culture medium of some aspects of the disclosure.

The cells may be any cells, e.g., prokaryotic or eukaryotic cells, e.g., primate cells, e.g., mammalian cells, e.g., human cells.

According to some aspects of the disclosure, the cells are somatic cells, pluripotent stem cells (PSCs), primed pluripotent stem cells, non-naive pluripotent stem cell and/or naive pluripotent stem cells.

According to some aspects of the disclosure, the culture medium is capable of maintaining pluripotent stem cells in an undifferentiated state for at least <NUM> passages, e.g., for at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> passages. The pluripotent stem cells cultured in the presently disclosed media retain their hypomethylated state for the number of passages.

According to some aspects of the disclosure, the pluripotent stem cells are primate pluripotent stem cell (homo sapiens (human), monkey, chimpanzee, Gorillas, Rhesus and/or Baboon). Other pluripotent stem cells contemplated by the present disclosure are swine (porcine) pluripotent stem cells.

Preferably, the pluripotent stem cells are not rodent pluripotent stem cells.

In one aspect, the pluripotent stem cell is a naive pluripotent stem cell.

The phrase "naive pluripotent stem cell (PSC) " refers to a cell capable of forming a PSC, and that exhibits a pre-X-inactivation state, and therefore is considered to be the origin of the PSC.

The pre-X-inactivation state according to some aspects of the disclosure is characterized by presence of two unmethylated alleles of an X-inactive specific transcript (XIST) gene in the female cell, and presence an unmethylated allele of the XIST gene in a male cell.

The XIST gene is located on human Xq13. <NUM> chromosome and has the sequence depicted in clone NC_000023. <NUM> (<NUM>, complement, based on GenBank version GRCh37. The XIST gene has a non-coding RNA which is provided in GenBank Accession NO. <NUM> (SEQ ID NO: <NUM>).

According to some aspects of the disclosure, presence of two unmethylated alleles of XIST gene in a female cell refers to having below about <NUM>% of CpG methylated reads sequenced in the XIST promoter, e.g., below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, e.g., <NUM>% (e.g., complete absence) of CpG methylated reads sequenced in the XIST promoter.

According to some aspects of the disclosure, presence of one unmethylated allele of XIST gene in a male cell refers to having below about <NUM>% of CpG methylated reads sequenced in the XIST promoter, e.g., below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, below about <NUM>%, e.g., <NUM>% of CpG methylated reads sequenced in the XIST promoter.

A non-limited example of the XIST promoter which includes CpG islands which can be either methylated or unmethylated is provided in the XIST promoter amplicon set forth by SEQ ID NO: <NUM>.

According to some aspects of the disclosure, the human naive PSC is characterized by a reduced methylation of CpG islands as compared to a level of methylation of the CpG islands in a human primed PSC.

Some human naive ESCs are characterized by significantly low levels of total methylated cytosine out of the total guanine nucleotides in each cell (e.g., <NUM>-<NUM>%,) as determined by Liquid Chromatography - Mass Spectrometry (LC-MS) quantitative analysis.

According to some aspects of the disclosure, the human naive PSC is characterized by <NUM>-<NUM>% of total methylated cytosine out of the total Guanine nucleotides in the naive PSC cell. For comparison, the primed PSC or a somatic cell has between <NUM>%-<NUM>% of total methylated cytosine out of the total Guanine nucleotides in the primed PSC cell.

Thus, the naive pluripotent stem cell of some aspects of the disclosure is in a naive, hypomethylated state (relating to global levels of DNA methylation). For example in one aspect, less than <NUM> % of the cytosines of a CG sequence of the DNA of the naive pluripotent stem cell are methylated, less than <NUM> % of the cytosines of a CG sequence of the DNA of the naive pluripotent stem cell are methylated, less than <NUM> % of the cytosines of a CG sequence of the DNA of the naive pluripotent stem cell are methylated.

As used herein the phrase "naive state" refers to being in an undifferentiated state wherein both alleles of the X-inactive specific transcript (XIST) gene of the female cell are unmethylated, or wherein the XIST allele of the male cell is unmethylated.

It should be noted that the naive PSCs of some aspects of the disclosure (which are in a pre-X inactivation and a naive state) can upon differentiation inactivate one of the X chromosome alleles and methylate one of the XIST genes.

As used herein the term "isolated" refers to at least partially separated from the natural environment e.g., from the primate (e.g., mammalian) embryo or the primate (e.g., mammalian) body.

According to some aspects of the disclosure, the non-naive PSC is selected from the group consisting of a primed PSC, an embryonic stem cell, a non-human blastocyst, an induced pluripotent stem cell (a primed iPSC) and a somatic cell.

The phrase "embryonic stem cells" refers to embryonic cells which are capable of differentiating into cells of all three embryonic germ layers (i.e., endoderm, ectoderm and mesoderm), or remaining in an undifferentiated state. The phrase "embryonic stem cells" may comprise cells which are obtained from the non-human embryonic tissue formed after gestation (e.g., blastocyst) before implantation of the non-human embryo (i.e., a pre-implantation blastocyst), extended non-human blastocyst cells (EBCs) which are obtained from a post-implantation/pre-gastrulation stage blastocyst (see <CIT>) and embryonic germ (EG) cells which are obtained from the genital tissue of a fetus any time during gestation, preferably before <NUM> weeks of gestation.

Induced pluripotent stem cells (iPS; embryonic-like stem cells), are cells obtained by de-differentiation of adult somatic cells which are endowed with pluripotency (i.e., being capable of differentiating into the three embryonic germ cell layers, i.e., endoderm, ectoderm and mesoderm). According to some aspects of the disclosure, such cells are obtained from a differentiated tissue (e.g., a somatic tissue such as skin) and undergo de-differentiation by genetic manipulation which re-program the cell to acquire embryonic stem cells characteristics. According to some aspects of the disclosure, the induced pluripotent stem cells are formed by inducing the expression of Oct-<NUM>, Sox2, Kfl4 and c-Myc in a somatic stem cell.

The embryonic stem cells of some aspects of the disclosure can be obtained using well-known cell-culture methods. For reference only, human embryonic stem cells can be isolated from human blastocysts. Human blastocysts are typically obtained from human in vivo preimplantation embryos or from in vitro fertilized (IVF) embryos. Alternatively, a single cell human embryo can be expanded to the blastocyst stage. For the isolation of human ES cells the zona pellucida is removed from the blastocyst and the inner cell mass (ICM) is isolated by immunosurgery, in which the trophectoderm cells are lysed and removed from the intact ICM by gentle pipetting. The ICM is then plated in a tissue culture flask containing the appropriate medium which enables its outgrowth. Following <NUM> to <NUM> days, the ICM derived outgrowth is dissociated into clumps either by a mechanical dissociation or by an enzymatic degradation and the cells are then re-plated on a fresh tissue culture medium. Colonies demonstrating undifferentiated morphology are individually selected by micropipette, mechanically dissociated into clumps, and re-plated. Resulting ES cells are then routinely split every <NUM>-<NUM> days. For further details on methods of preparation human ES cells see <CIT>; <NPL>; <NPL>; <NPL>]; <NPL>]; and <NPL>].

Another method for preparing ES cells is described in <NPL>. This method comprises removing a single cell from an embryo during an in vitro fertilization process. The embryo is not destroyed in this process.

It will be appreciated that commercially available stem cells can also be used according to some aspects of the disclosure. Human ES cells can be purchased from the NIH human embryonic stem cells registry [Hypertext Transfer Protocol://grants (dot) nih (dot) gov/stem_cells/registry/current (dot) htm]. For reference, non-limiting examples of commercially available embryonic stem cell lines are BG01, BG02, BG03, BG04, CY12, CY30, CY92, CY10, TE03, TE32, CHB-<NUM>, CHB-<NUM>, CHB-<NUM>, CHB-<NUM>, CHB-<NUM>, CHB-<NUM>, CHB-<NUM>, CHB-<NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, HUES <NUM>, CyT49, RUES3, WA01, UCSF4, NYUES1, NYUES2, NYUES3, NYUES4, NYUES5, NYUES6, NYUES7, UCLA <NUM>, UCLA <NUM>, UCLA <NUM>, WA077 (H7), WA09 (H9), WA13 (H13), WA14 (H14), HUES <NUM>, HUES <NUM>, HUES <NUM>, CT1, CT2, CT3, CT4, MA135, Eneavour-<NUM>, WIBR1, WIBR2, WIBR3, WIBR4, WIBR5, WIBR6, HUES <NUM>, Shef <NUM>, Shef <NUM>, BJNhem <NUM>, BJNhem20, SA001, SA001.

In addition, ES cells can be obtained from other species as well, including mouse (Mills and Bradley, <NUM>), golden hamster [<NPL>], rat [<NPL>] rabbit [<NPL>; <NPL>], several domestic animal species [<NPL>; <NPL>; <NPL>] and non-human primate species (Rhesus monkey and marmoset) [<NPL>; <NPL>].

Extended non-human blastocyst cells (EBCs) can be obtained from a non-human blastocyst of at least nine days post fertilization at a stage prior to gastrulation. Prior to culturing the blastocyst, the zona pellucida is digested [for example by Tyrode's acidic solution (Sigma Aldrich, St Louis, MO, USA)] so as to expose the inner cell mass. The blastocysts are then cultured as whole embryos for at least nine and no more than fourteen days post fertilization (i.e., prior to the gastrulation event) in vitro using standard embryonic stem cell culturing methods.

EG cells are prepared from the primordial germ cells obtained from fetuses of about <NUM>-<NUM> weeks of gestation (in the case of a human fetus) using laboratory techniques known to anyone skilled in the arts. The genital ridges are dissociated and cut into small chunks which are thereafter disaggregated into cells by mechanical dissociation. The EG cells are then grown in tissue culture flasks with the appropriate medium. The cells are cultured with daily replacement of medium until a cell morphology consistent with EG cells is observed, typically after <NUM>-<NUM> days or <NUM>-<NUM> passages. For additional details on methods of preparation human EG cells see<NPL>] and <CIT>.

Induced pluripotent stem cells (iPS) (embryonic-like stem cells) can be generated from somatic cells by genetic manipulation of somatic cells, e.g., by retroviral transduction of somatic cells such as fibroblasts, hepatocytes, gastric epithelial cells with transcription factors such as Oct-<NUM>/<NUM>, Sox2, c-Myc, and KLF4 [<NPL>;<NPL>. (Epub ahead of print); <NPL>;<NPL>]. Other embryonic-like stem cells can be generated by non-human nuclear transfer to oocytes, fusion with embryonic stem cells or non-human nuclear transfer into zygotes if the recipient cells are arrested in mitosis.

Culturing the cells in the media described herein may be effected in any vesicle, e.g. plate, chamber, bioreactor etc..

The number of cells that may be selected and/or cultured according to the present disclosure may be any number including small batches - e.g. <NUM> ×<NUM><NUM> cells to larger batches - e.g. <NUM> ×<NUM><NUM> or <NUM> × <NUM><NUM> cells.

The cells may be cultured in a bioreactor (or in multi-level industrial flasks), the size of which is selected according to the number of cells being cultured.

As used herein, the term "bioreactor" refers to any device in which biological and/or biochemical processes develop under monitored and controlled environmental and operating conditions, for example, pH, temperature, pressure, nutrient supply and waste removal. According to one aspect of the disclosure, the basic classes of bioreactors suitable for use with the present disclosure include static bioreactors, stirred flask bioreactors, rotating wall bioreactors, hollow fiber bioreactors and direct perfusion bioreactors.

According to a particular aspect, the cells are cultured (i.e. expanded) on an adherent surface.

Examples of such surfaces are provided herein under.

The culture media described in the present application may be used for a myriad of purposes.

According to a particular aspect, the culture media are used for expanding (i.e. increasing the number of) cells - e,g, expanding PSCs. The present inventors have noted that expansion of pluripotent stem cells in the presently disclosed media maintains the pluripotent state of the cells and further ensures that less than <NUM> % of the Cs of a CG sequence in the DNA are not methylated. In some aspects less than <NUM> % of the Cs of a CG sequence in the DNA are not methylated.

It should be noted that culturing PSC involves replacing the culture medium with a "fresh" medium (of identical composition) every <NUM>-<NUM> hours, and passaging each culture dish (e.g., a plate) to <NUM> or <NUM> culture dishes (e.g., plates) every <NUM>-<NUM> days. Thus, when cells in the culture reach about <NUM>-<NUM>% confluence the supernatant is discarded, the culture dishes are washed [e.g., with phosphate buffered saline (PBS)] and the cells are subjected to enzymatic dissociation from the culture dish, e.g., using trypsinization (<NUM>% or <NUM>% Try sin + EDTA), e.g., until single cells or cell clumps are separated from each other.

The culture media described herein can be used in the generation of iPSCs from somatic cells. Methods of generating iPSCs are known in the art and include for example genetically modifying the somatic cells to express at least one dedifferentiating factor selected from the group consisting of KLF4, c-MYC, OCT4, SOX2, Nanog, and LIN28. Alternatively, the somatic cells can be provided directly with the RNA that encodes the transcription factors.

According to a particular aspect, the generation of iPSCs comprises expressing in the somatic cells at least two dedifferentiating factors - the first factor selected from the group consisting of Nanog, ESRRB, KLF2, KLF17, TBX3, TFAP2C, ERAS and the second factor selected from the group consisting of Nanog, ESRRB, KLF2, KLF17, TFAP2C, TBX3, ERAS, Oct4, Sox2, Klf4c-Myc.

Methods of DNA transfections into mammalian cells are known in the art and include those described in Reference (Mansour et al. Further description of preparation of expression vectors and modes of administering them into cells are provided hereinunder.

According to some aspects of the disclosure, expressing the factors is performed using RNA transfection of the growth factors.

Methods of RNA transfections into mammalian cells are known in the art and include those described for example in (Warren et al.

Examples of somatic cell types retinal pigment epithelial cells, cardiomyocytes, epithelial cells such as keratin-containing cells, hepatocytes, pancreatic cells (e.g. pancreatic beta cells), muscle cells, blood cells, fat cells, bone cells, chondrocytes, neurons, astrocytes and oligodendrocytes.

The culture media described herein can be used in the generation of naive pluripotent stem cells from non-naïve pluripotent stem cells. Preferably the media used for generation or maintenance of naive pluripotent stem cells comprises: LIF, WNT inhibitor, Notch inhibitor, P38 inhibitor, PKC inhibitor SRC inhibitor, Activin A and Rock inhibitor.

Thus, according to another aspect, the culture media described herein are used to generate naive pluripotent stem cells from non-naïve cells.

More specifically, according to another aspect of the present disclosure there is provided a method of generating a naive pluripotent stem cell (PSC), comprising:.

It is expected that during the life of a patent maturing from this application many relevant WNT inhibitors, SRC inhibitors and protein kinase C (PKC) inhibitors will be developed and the scope of these terms is intended to include all such new technologies a priori.

As used herein the term "about" refers to ± <NUM> %
The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

Throughout this application, various aspects of this disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure.

It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, may also be provided in combination in a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, may also be provided separately or in any suitable subcombination or as suitable in any other described aspect of the disclosure. Certain features described in the context of various aspects are not to be considered essential features of those aspects, unless the aspect is inoperative without those elements.

Various aspects and aspects of the present disclosure as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

Reference is now made to the following examples, which together with the above descriptions illustrate some aspects of the disclosure in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present disclosure include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "<NPL>); "<NPL>); <NPL>); <NPL>); <NPL>; <NPL>); methodologies as set forth in <CIT>; <CIT>; <CIT>; <CIT> and <CIT>; "<NPL>); "<NPL>on; "<NPL>); <NPL>); <NPL>); available immunoassays are extensively described in the patent and scientific literature, see, for example, <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT> and <CIT>; "<NPL>); "<NPL>); "<NPL>); "<NPL>); "<NPL>); "<NPL>) and "<NPL>; "<NPL>); <NPL>). Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader.

PolyA-RNA-seq library preparation: Total RNA was isolated from indicated cell lines and extracted from Trizol pellets by chloroform-phenol extraction protocol, then utilized for RNA-Seq by ScriptSeq Preparation Kit v2 (Illumina) according to manufacturer's instruction.

ATAC-seq library preparation: Cells were trypsinized and counted, <NUM>,<NUM> cells were centrifuged at <NUM> for <NUM>, followed by a wash using <NUM>µl of cold PBS and centrifugation at <NUM> for <NUM>. Cells were lysed using cold lysis buffer (<NUM> Tris-HCl, pH <NUM>, <NUM> NaCl, <NUM> MgCl<NUM> and <NUM>% IGEPAL CA-<NUM>). Immediately after lysis, nuclei were spun at <NUM> for <NUM> using a refrigerated centrifuge. Next, the pellet was resuspended in the transposase reaction mix (<NUM>µl <NUM>× TD buffer, <NUM>µl transposase (Illumina) and <NUM>µl nuclease-free water). The transposition reaction was carried out for <NUM> at <NUM> and immediately put on ice. Directly afterwards, the sample was purified using a Qiagen MinElute kit. Following purification, the library fragments were amplified using custom Nextera PCR primers <NUM> and <NUM> for a total of <NUM> cycles. Following PCR amplification, the libraries were purified using a QiagenMinElute Kit and sequenced.

Whole-Genome Bisulfite Sequencing (WGBS) Library preparation: DNA was isolated from cells using the Quick-gDNA miniprep kit (Zymo). DNA (50ng) was then converted by bisulfite using the EZ DNA Methylation-Gold kit (Zymo). Libraries were prepared using the TruSeq kit (Illumina) and length distribution of each library was measured using the Bioanalyzer and product concentration was measured using Qubit Fluorometric Quantitation. For sequencing, the libraries, NextSeq <NUM>/<NUM> High Output v2 kit (<NUM> cycles) was used.

ChIP-seq library preparation: Cells were crosslinked in formaldehyde (<NUM>% final concentration, <NUM> at room temperature), and then quenched with glycine (<NUM> at room temperature). Antibodies detailed in Table <NUM> were then lysed in <NUM> HEPES KOH pH <NUM>, <NUM> NaCl, <NUM> EDTA, <NUM>% glycerol, <NUM>% NP-<NUM> alternative, <NUM>% Triton supplemented with protease inhibitor at <NUM> (Roche, <NUM>) for <NUM>, and later centrifuged at <NUM> for <NUM>.

Supernatant was discarded and pellet was resuspended in RIPA-<NUM> (<NUM>% SDS, <NUM> EDTA, <NUM>% DOC, <NUM> NaCl and <NUM> Tris-HCl) with protease inhibitor. Cells were then fragmented with a Branson Sonifier (model S-450D) at - <NUM> to size ranges between <NUM> and <NUM> bp and centrifugation at max speed for <NUM>. Supp lysate was extracted and diluted with RIPA <NUM>-<NUM>-fold (<NUM>% SDS, <NUM> EDTA, <NUM>% DOC, Triton <NUM>%, <NUM> NaCl and <NUM> Tris-HCl). Small amount of lysate were saved for whole cell extract at this point. Antibody was pre-bound by incubating with Protein-G Dynabeads (Invitrogen 10004D) in blocking buffer (PBS supplemented with <NUM>% TWEEN and <NUM>% BSA) for <NUM> at room temperature. Washed beads were added to the lysate for incubation. Samples were washed five times with RIPA buffer, twice with RIPA buffer supplemented with <NUM> NaCl, twice with LiCl buffer (<NUM> TE, <NUM> LiCl, <NUM>% NP-<NUM>, <NUM>% DOC), once with TE (<NUM> Tris-HCl pH <NUM>, <NUM> EDTA), and then eluted in <NUM>% SDS, <NUM> NaCl, <NUM> EDTA, <NUM> Tris HCl pH <NUM>. Eluate was incubated treated sequentially with RNaseA (Roche, <NUM>) for <NUM> in <NUM> and proteinase K (NEB, P8102S) for <NUM> in <NUM> and de-crosslinked in <NUM> for <NUM>. DNA was purified with The Agencourt AMPure XP system (Beckman Coulter Genomics, A63881). Libraries of cross-reversed ChIP DNA samples were prepared according to a modified version of the Illumina Genomic DNA protocol, as described previously (Rais et al.

PolyA-RNA analysis: hESCs grown in naive and primed conditions, from different cell lines (LIS41, LIS49, WIBR2) were used for RNA-seq analysis. STAR software version <NUM>. 2b was used to align reads to human GRCh38 reference genome (<NUM>), using the following flags: - -outFilterMultimapNmax <NUM> --outReadsUnmapped Fastx --twopassMode Basic-outSAMstrandField intronMotif. FPKM values were estimated with HTSeq software over all genes in GRCh38 assembly using the following flags: -a <NUM> -s no -t exon -i gene_id. Genes with accumulated expression of FPKM><NUM> over all samples, were selected for analysis. The filtering was done independently in each analysis, therefore the number of genes included may change, as it is dependent on the samples that were included for that analysis.

FPKM values were further normalized using R DESeq software, and corrected for batch effects using R limma package. Hierarchical clustering was carried out using R pheatmap command. PCA analysis was carried out using R prcomp command.

Differentially expressed genes between naive and primed samples were selected from HTSeq output in the following parameters: FC><NUM> of FC<<NUM>, and adjusted p-value<<NUM>.

Whole-Genome Bisulfite Sequencing (WGBS) analysis: The sequencing reads were aligned to the human hg19 reference genome (UCSC, <NUM>), using a proprietary script based on Bowtie2. In cases where the two reads were not aligned in a concordant manner, the reads were discarded. Methylation levels of CpGs calculated by WGBS were unified. Mean methylation was calculated for each CpG that was covered by at least <NUM> distinct reads (X5). Average methylation level was calculating by taking the average over all covered X5 covered CpG sites in that genome.

ChIP-seq analysis: Chip-seq data of the following DNA-binding proteins was analyzed: NANOG, SOX2, OCT4, KLF4, KLF17, TFAP2C, H3K27AC. For alignment and peak detection, bowtie2 software was used to align reads to human hg19 reference genome (UCSC, <NUM>), with default parameters. Enriched intervals of all measured proteins were analyzed using MACS version <NUM>. <NUM>-<NUM>. Sequencing of whole-cell extract was used as control to define a background model. Duplicate reads aligned to the exact same location were excluded by MACS default configuration. Peaks were assigned to genes using Homer software.

ATAC-seq analysis: Reads were aligned to hg19 human genome using Bowtie2 with the parameter -X2000 (allowing fragments up to <NUM> kb to align). Duplicated aligned reads were removed using Picard MarkDuplicates tool with the command REMOVE_DUPLICATES=true. To identify chromatin accessibility signal we considered only short reads (≤ 120bp) that correspond to nucleosome free region. To detect and separate accessible loci in each sample, we used MACS version <NUM>. <NUM>-<NUM> with --call-subpeaks flag (PeakSplitter version <NUM>).

Enhancer Identification: H3K27ac peaks were detected using MACS version <NUM>. <NUM>-<NUM> and merged for each condition (naive and primed) using bedtools merge command. All ATAC peaks were filtered to include only peaks which co-localized with the merged H3K27ac peaks in at least one condition. Finally, peaks that co-localized with promoter or exon regions based on hg19 assembly (UCSC, <NUM>) were filtered out. Finally, the data was confined to defined genomic intervals which was annotated as enhancers.

Motif analysis: Enriched binding motifs were searched in chromatin accessible loci using findMotifsGenome function from homer software package version <NUM>, using the software default parameters.

In order to identify culture conditions which capture human naive PSC, the present inventors looked for agents which are capable of maintaining stem cells in a pluripotent state in the absence of defined epigenetic repressors. Human knock-in WIBR3 hESC lines with conditional inducible ablation expression of METTL3 were engineered (<FIG>). This was carried out by introducing an exogenous METTL3 transgene under the regulation of Tet-OFF promoter (Liao et al. , <NUM>), followed by CRISPR/Cas9 mediated ablation of both endogenous human METTL3 alleles. Two resultant clones were validated for METTL3 expression only from the exogenous allele, which can be shut off by addition of DOX to the media (called Tet-OFF-METTL3 lines) (<FIG>).

Primed Tet-OFF-METTL3 hESCs expanded in TeSR or KSR/FGF2 primed conditions could not be sustained in the presence of DOX for more than four passages (both on MEF or on Geltrex coated dishes) and resulted in massive cell death and differentiation (<FIG>). In the presence of MEFs, Tet-OFF-METTL3 could be stably maintained in NHSM conditions, but not in 4iLA-MEF, 5iLAF-MEF, 5iLA-MEF, 6iLA-MEF, TESR/3iL-MEF conditions (<FIG>). The latter further support rewiring toward naïve pluripotency in NHSM conditions. However, in the absence of MEFs, NHSM conditions could not support maintenance of pluripotency when METTL3 was ablated, suggesting that NHSM conditions can be enhanced to endow the cells with such ability (<FIG>). Candidate molecules were tested for the ability to enrich NHSM conditions that allow Tet-OFF-METTL3 to be maintained on Geltrex coated plates in the presence of DOX.

NHSM conditions were supplemented with individual small molecules (<FIG>). Remarkably, supplementing NHSM conditions with the Tankyrase inhibitor named IWR1, but not any of the other <NUM> compounds tested, enabled the expansion of Tet-OFF-METTL3 on DOX with great homogeneity (<FIG>). IWR1 is a WNT inhibitor (WNTi) small molecule that stabilizes AXIN protein in the cytoplasm by inhibiting Tankyrase enzyme (abbreviated herein as TNK inhibitor - TNKi). An additional TNKi, XAV939, yielded a similar effect, while using exo-IWR1 an inactive modified version of IWR1 failed to do so, supporting specific inhibition of Tankyrase as the target yielding stability of these pluripotent cells (<FIG>).

Two additional cell lines based on WIBR3 hESC line carrying knock in ΔPE-OCT4-GFP reporter (Theunissen et al. , 2014a) were used in parallel to optimize and enhance NHSM conditions (<FIG>). Supplementation of TNKi to NHSM conditions yielded a dramatic increase in GFP signal when compared to primed, NHSM or 4i-LA conditions (<FIG>, <FIG>). Consistent with studies conducted in mice (Kim et al. , <NUM>), including TNKi rendered exogenous supplementation of FGF2 dispensable even in feeder free conditions (<FIG>). Further, as XAV939 inhibits WNT signaling, the present inventors validated that including GSK3 inhibitor is dispensable and, in fact, compromises the intensity of ΔPE-OCT4-GFP signal (<FIG>). Pluripotent cells could be maintained in the absence of JNK and P38 inhibitors, they boosted naive pluripotency marker expression and therefore were maintained in the media used herein (<FIG>). Importantly, after optimizing NHSM conditions, the present inventors attempted to substitute TNKi with other components included in the screen, to exclude the possibility that the latter optimizations may facilitate a different screening result. However, none of them allowed expanding METTL3 depleted cells in vitro as seen with supplementing TNKi (including VPA, BRAFi, Forskolin, Kenpaullone, SHHi, DOT1Li, LSD1i, TGFRi, ERK5i) (<FIG>).

Under the above described conditions, human ESCs maintained uniformly high ΔPE-OCT4-GFP levels only in the presence of exogenous ACTIVIN A, and consistently differentiated when TGFR inhibitor was provided (<FIG>, <FIG>). Thus, the present inventors for the identification of a small molecule whose supplementation will render human PSCs that are independent of exogenous ACTIVIN/TGF supplementation. It should be noted that none of the previously described human naive conditions have been able to maintain teratoma competent pluripotent cells that can be maintained long term and validated for their naive identity after prolonged specific inhibition of ACTIVIN/NODAL signaling. To do this, the latter TNKi supplemented and modified conditions were used in the absence of ACTIVIN A, and candidate molecules were added to allow expanding OCT4+ PSCS independent of METTL3 expression (on DOX) (<FIG>). While under most conditions, Oct4+ cell fraction rapidly deteriorated, it was noted that a validated SRCi (CGP77675) dramatically maintained the stability of dome like cells that were uniformly OCT4+ (<FIG>). This led to the assembly of a defined FGF/TGF/ACTIVINV/MEF free and independent growth conditions which is referred to herein as Enhanced NHSM - "ENHSM" (<FIG>). Of note, supplementation of SRCi in ACTIVIN A containing conditions, although not essential to maintain ΔPE-OCT4-GFP+ when ACTIVIN was provided, it did support consistency and domed like morphology among naive cells (<FIG>) (conditions referred to as ENHSM-ACT).

Following METTL3 depletion in ENHSM conditions, WIBR3 cells maintained their typical domed lie morphology and uniformly expressed pluripotency markers including KLF17 that is specific to the naive state both with and without METTL3 depletion (<FIG>). Measurement of m<NUM>A on mRNA showed over <NUM>% depletion of total levels after DOX addition (<FIG>), comparable to those seen upon knockout of the Mettl3/<NUM> complex in mouse naive ESCs. ESCs maintained in the absence of METTL3 for over <NUM> passages remained pluripotent and were capable of generating mature teratomas in vivo without the need to passage them first for a period of time under primed conditions (<FIG>). The latter validates maintenance of naive pluripotency in human PSCs expanded in ENHSM without METTL3 protein and ablated m<NUM>A levels deposited on mRNA in the cells.

To extend the previous findings to another repressor machinery, OCT4-GFP-WIBR3 reporter ESCs were targeted by TALENs to generate DGCR8 null cells (<FIG>). While conducting such targeting on primed cells did not yield any null cells (<FIG>), DGCR8 null cells could be obtained when the targeted cells were expanded in ENHSM and ENHSM-ACT conditions (<FIG>, <FIG>). To test which of the naive conditions enable expanding human naive PSCs in the absence of DNMT1 cells, a similar approach to that applied for making Tet-OFF-METTL3 herein, has been recently used to generate TET-OFF DNMT1 in HUES64 ESC line (<FIG>). Cells expanded in previously described naïve conditions including NHSM-MEF, 4i/LA-MEF, 5i/LA-MEF, 6i/LA-MEF, 5i/LAF-MEF, TESR/3iL-MEF and NHSM could not be maintained in the presence of DOX for more than <NUM> passages (<FIG>). ENHSM and ENHSM-ACT conditions allowed stable and unlimited expansion of DNMT1 depleted human naive ESCs both in feeder and feeder free conditions (<FIG>). Whole Genome Bisulfite Sequencing (WGBS) confirmed global loss of methylation in naive DNMT1 depleted cells expanded in ENHSM conditions and analyzed at passage <NUM> (<FIG>) and maintained expression of pluripotency markers (<FIG>). Collectively these results demonstrate that ENHSM conditions mimic mouse naive ESC and for the first time enable generation of human PSCs ablated for epigenetic repressors (both in feeder and feeder free conditions) and that are independent from ACTIVIN/TGF signaling.

Murine naive ESCs retain bivalent metabolic capability utilizing both Oxidative phosphorylation (OXPHOS) and Glycolytic metabolism, while upon priming then become dependent only on glycolytic metabolism. As shown previously, NHSM, 5i-LA and transgene containing reset cells increase OXPHOS activity leading to retaining bivalent metabolic profile. ENHSM condition were similarly tested herein and by measuring basal oxygen consumption rate (OCR) it was substantially higher in ENHSM conditions than in conventional PSC (<FIG>). Higher electron transport chain activity in ENHSM was evidenced by a greater OCR increase in response to the mitochondrial uncoupler FCCP (<FIG>). Cells expanded in ENHSM condition displayed more intense staining with tetramethylrhodamine methyl ester (TMRE) (<FIG>), indicative of mitochondrial membrane depolarization. The latter supports the conclusion that this is a naive feature is relatively less stringent and can be obtained in a variety of human naive protocols devised so far.

However, a newly identified stringent metabolic feature recently identified in naive ESCs in 2i or 2i/LIF is that they can endogenously synthesize glutamine at sufficient levels to maintain adequate alpha-ketoglutarate (αKG) levels. While they benefit form exogenous L-Glutamine supplementation, it is not essential for their stability or pluripotency as they can metabolically synthesize it internally as part of their altered metabolic configuration. FBS/LIF naive murine ESCs or primed EpiSCs cannot be maintained in the absence of exogenous L-Glutamine. To compare the latter observation and apply them on distinct human pluripotent states, WIBR3-OCT4-GFP knock-in ESC line, ΔPE-WIBR3-OCT4-GFP knock-in ESC line, H9-NANOG-GFP ESC lines were then tested for their ability to maintain pluripotency in the presence and absence of L-Glutamine (<FIG>). Importantly, the present inventors failed to maintain primed PSCs or other naive PSCs in the absence of L-Glutamine (in NHSM, 4i/LA-MEF, Si/LAF-MEF, 6iLA-MEF, TESR/3iL-MEF) even when MEFs were used (<FIG>). However, ΔPE-WIBR3-OCT4-GFP expanded in ENHSM was not compromised when L-glutamine was not included in ENHSM conditions (<FIG>) and GFP signal was positive for H9-NANOG-GFP ESC both in the presence and absence of L-Glutamine (both on feeder and feeder free conditions) (<FIG>). Cells expressed general and naive specific pluripotency markers in ENHSM with and without exogenous L-Glutamine and generated differentiated teratomas without the need for passaging in vitro in primed conditions (<FIG>). Collectively, these results validate that ENHSM conditions can maintain naive pluripotency characteristics and endow the cells with ability to be expanded in the absence of exogenous L-Glutamine, as with murine 2i/LIF naïve PSCs.

The present inventors next aimed to convert previously established primed PSCs lines and to derive new lines directly in ENHSM-ACT and ENHSM conditions from the ICM of human blastocysts. Human blastocysts were plated on mouse embryonic fibroblast (MEF) coated plates and medium successfully generated domed cell outgrowths following <NUM>-<NUM> days of plating. ICM derived outgrowths were then trypsinized and passaged. Subsequently, <NUM> new stem cell lines termed LIS36, LIS42 and LIS46 were derived in ENHSM-ACT; LIS41 and LIS49 ESCs in ENHSM conditions (<FIG>). Multiple conventional (hereafter will be named "primed") hESC lines (WIBR1, WIBR2, WIBR3, HUES64, H9) were plated on Geltrex coated dishes in ENHSM or ENHSM-ACT medium (<FIG>). Within <NUM>-<NUM> days of applying this protocol, dome-shaped colonies with packed round cell morphology, typical of mESCs, could be readily isolated and further expanded (<FIG>). Adult human dermal fibroblast cells or peripheral blood cells were reprogrammed to iPSCs in ENHSM conditions following either lentiviral transduction with DOX inducible OKSM factors (BF1 hiPSC) or by non-integrating sendai viruses (JH1, BC1 and MECP5 hiPSC) (<FIG>). All polyclonal and subcloned hESC and iPSC lines expanded in ENHSM conditions were uniformly positive for pluripotent markers AP, OCT4, NANOG, SSEA4, TRA1-<NUM> and TRA1-<NUM> (representative images in <FIG>) and robustly formed mature teratomas in vivo without the need for short- or long-term exposure to primed growth conditions, and as typically observed with rodent ground state naive PSCs (<FIG>). Naive lines were passaged with TryplE every <NUM>-<NUM> days and had single cell cloning efficient up to <NUM>-<NUM>%, while primed cell single cell cloning increased only up to <NUM>-<NUM>% when Y27632 was used. Human naive pluripotent lines maintained normal karyotype after extended passaging in ENHSM-ACT or ENHSM in most lines of tested (<FIG>). In some cultures, minor aneuploidy cells were observed; however no recurrent abnormality was observed between any of these lines as determined by G-banding of metaphase chromosomes (<FIG>). The results indicate that epigenetic resetting in ENHSM does not cause obligatory chromosomal abnormalities nor select for pre-existing variants, as had been observed for other naive conditions like 5iLA-MEF conditions (<NUM>-<NUM>% chromosomal abnormality by passage <NUM> only) (Liu et al.

Global gene expression patterns were compared between naive and primed hESCs and hiPSCs, many of which were genetically matched. Unbiased clustering of genome-wide expression profiles demonstrated that naive hESC and hiPSCs possess a distinct gene expression pattern and clustered separately from conventional/primed hESCs and hiPSCs (<FIG>). Transcripts associated with naive pluripotency were significantly upregulated in naive cells. The later included NANOG, TFCP2L1, KLF17, KLF4, STELLA (DPPA3), DPPA5 (<FIG>). RT-PCR analysis validated the dramatic upregulation in naive PSCs expanded both in ENHSM and ENHSM-ACT conditions (<FIG>). When including naive datasets generated in 5iLA-MEF, 4i-LAF and t2i-LIF-GO-NK2, it was noted that cells generated in ENHSM and ENHSM-ACT conditions clustered with all the latter naïve conditions and not with primed samples (<FIG>, <FIG>). FACS analysis confirmed upregulation of previously identified human naïve pluripotency markers CD77 and CD130 in ENHSM conditions, and primed pluripotency marker CD24 was downregulated in ENHSM conditions (<FIG>) (Collier et al. , <NUM>; Shakiba et al. Importantly, naive pluripotent cells had profoundly down regulated transcripts associated with lineage commitment genes including T, ZIC2 and VIM1 that are expressed at low, but appreciable, levels in primed hESCs (<FIG>, <FIG>). STELLA-CFP knock-in allele was introduced via CRISPR/Cas9 (<FIG>), to monitor pluripotency maintenance in the different tested conditions, and STELLA-CFP was induced in both ENHSM and previously described 5iLA conditions (<FIG>). Similar to its upregulation and importance in maintaining human naive pluripotency in 5iLA conditions (Pastor et al. , <NUM>), TFAP2C KO lines showed that it is essential for deriving and maintain human naïve PSCs in both ENHSM and ENHSM ACT conditions (<FIG>, <FIG>). The latter results confirm that ENHSM conditions attain consensus transcriptional feature observed in other previously published naive hPSCs studies or in vivo human embryo data.

Transposable Element (TE)-derived transcripts were profiled and compared in conventional and naive human PSCS expanded in ENHSM conditions (Theunissen et al. The top <NUM>,<NUM> TEs with largest SD separated naive and primed samples both in hierarchical clustering (<FIG>) and in PCA based analysis (<FIG>). Members of the SINE-VTR-Alu (SVA) family of TEs and HERVK- associated LTR were transcribed almost exclusively in ENHSM conditions similar to previously obtained in 5i/LA and transgene dependent t2iLGo conditions (<FIG>) (Theunissen et al. TE profiling was used to measure the degree to which ENHSM and primed conditions resemble pluripotent cells in early human embryos in vivo. Naive, but not primed cells, demonstrated the most significant overlap with the human morula and epiblast stages when looking TEs (<FIG>), as was similarly shows for coding genes. These results support the endowment of late pre-implantation like transposon expression profile in PSCs expanded in ENHSM conditions in vitro.

ENHSM conditions were tested to see whether they endow human naive PSCs with a pre-X chromosome configuration. Primed human WIBR2 hESC carrying knock-in MECP2-dTomato and MECP2-mCherry alleles were used (Theunissen et al. Correctly targeted clone #<NUM> expresses only the red allele, however upon transferring the cells into ENHSM conditions ><NUM>% of cells expressed bother fluorescent markers consistent with reactivation of both x chromosome alleles. Transferring the cells into primed media allowed inactivation of X chromosome in a non-random manner as evident by obtaining GFP-/tdTomato+ pattern ><NUM>% of the reprised cells (<FIG>). FISH analysis was carried out for ATRX in Female cells as this locus is expressed from one copy even in human primed WIBR3 hESCs that have undergone erosion of X chromosome (Xe). Indeed, two ATRX foci could be uniformly found in naive, but not primed, human female PSCs supporting reactivation of X chromosome (<FIG>). Primed human iPSCs were obtained from donor fibroblast carrying a null mutation in only one of the MECP2 allele, and this primed clone was validated to inactivate the wild-type MECP2 allele and thus lacks MECP2 protein expression (Sahakyan et al. Expanding the cells in ENHSM or ENHSM-ACT for <NUM> passages was sufficient to yield ><NUM>% of iPSC cultures as positive for MECP2+. Subsequently naive cells were re-primed for <NUM> passages and stained negative for Mecp2 upon repriming, thus indicating ability to inactive X chromosome upon reprogramming. SNP based analysis of X chromosome allele expression as detected in RNA-seq datasets showed biallelic expression of X chromosome encoded genes in ENHSM but not primed conditions, consistent with functional reactivation of X chromosome in female naive PSCS induced in ENHSM conditions. Collectively, the above findings indicate that ENHSM and ENHSM-ACT conditions consolidate human naive pluripotency identity and endows them with nearly all known naive pluripotency features that have been attributed to human ICM in vivo, previously derived human naive cells and murine ground state naive cells.

Human naive and primed pluripotent cell's DNA methylation states were sampled by Whole genome Bisulfite Sequencing (WGBS). Lines tested displayed profound downregulation of global methylation levels from <NUM>% in primed hPSCs to <NUM>% in ENHSM expanded human hPSCs and down to <NUM>% when Activin was supplemented (ENHSM-ACT conditions) (<FIG>). DNMT1 methyltransferase is maintained in ENHSM, while UHRF1 protein is partially (%<NUM>-<NUM>%) depleted which may underlie the global downregulation in DNA methylation in ENHSM conditions (<FIG>). It is important to note that supplementing ENHSM conditions with BRAF inhibitor used in 5i/LAF conditions leads to dramatic downregulation in both DNMT1 and UHRF1 levels. This pattern is also observed in 5i/LAF condition which might explain the immediate and global loss of imprinting. As DNMT1 levels are maintained in ENHSM conditions and UHRF1 protein downregulation was partial, immediate global loss of imprints in ENHSM or ENHSM-ACT conditions was not observed (<FIG>), but they appeared sporadically and after at least <NUM> passages in ENHSM conditions.

The results above indicate that functional naive pluripotency in ENHSM composition not only relies on inhibition of ERKi and PKCi, but also on inhibition of TNK and SRC. Depletion of any of these <NUM> components compromised naïve pluripotency hallmarks like X chromosome inactivation in female cell lines (<FIG>). Combined depletion of TNKi and SRCi pushed human PSCs toward complete loss of pluripotency within a number of passaging, underlining the conclusions that ERKi together with tripartite inhibition of PKCi-SRCi-TNKi are essential for functional defined conditions for human naïve pluripotency.

The present inventors next aimed to define the signaling pathway downstream of Tankyrase inhibition facilitating human naive PSCs stabilization. Bcat-KO ESCs had higher levels of Oct4-GFP in ENHSM condition, and upon removal of XAV939 GFP level was not decreased in KO, but in WT ESCs. A similar trend was shown in RT-PCR analysis. Supplementing naive cells with WNT stimulator compromised delta-PE-OCT4-GFP levels, compromised their domed shape like morphology and their transcriptional profile. Using a tamoxifen induced Beta-Catenin-ERT transgene, the present inventors noted that delta-PE-OCT4-GFP signal and domed morphology were compromised upon tamoxifen stimulation. This is in striking contrast to mouse delta-PE-Oct4-GFP ESCs expanded in N2B27 LIF conditions that upon tamoxifen treatment induced deltaPe-Oct4-GFP reporter and nave characteristic domed like morphology. Similarly, while KO of TCF3 boosts mouse naive pluripotency and alleviates the need for WNT stimulation, TCF3 KO ESCs still required WNTi and/or SRCi to maintain their naïve identity in humans. Finally, Supplementing ENHSM conditions with CHIR compromised their ability to maintain pluripotency upon inhibition of TGFB inhibitor, depletion of DNA and RNA methylation or omitting L-Glutamine from the culture conditions. Collectively, these findings clearly establish WNT as a priming agent for human, but not mouse, naive pluripotency and establish that KO of beta-catenin can substitute for Tankyrase inhibition.

SRC inhibition has been shown previously to deplete activation of downstream effectors including ERK, PKC and NFKB signaling. Given that SRCi was needed in ENHSM conditions despite independent direct blocking of ERK and PKC pathways, this led the present inventors to focus on NFKB as a potential effector mediating the beneficial effect of the use of SRCi. Indeed, it was noted that the active subunit of NFKB, P65, was found predominantly in the nucleus of human and mouse primed PSCs, and was excluded to the cytoplasm upon transfer to naïve conditions. Transfection of NFKb signaling luciferase reporter showed high levels of activation in primed but not naive ENHSM conditions. Depletion of SRCi in ENHSM conditions induced nuclear P65 localization and a boost in luciferase reporter signal. Finally, the transfection of dominant negative NFKB subunit in Bcat-KO deltaPE-OCT4-GFP hESCs allowed maintenance of deltaPE-OCT4- GFP not only in ENHSM without TNKi but also without SRCi. These results establish that WNT/BCAT and SRC-NFKB pathways compromises human naive pluripotency.

In mouse ground state naive conditions, LIF/Stat3 has been shown to be a booster for naive marker expression however they can be omitted without entire collapse of the naive PSC circuit (Ying et al. By omitting LIF from ENHSM conditions and by generating STAT3 KO human naive PSCs, we show that LIF can slightly boost the purity of undifferentiated cells in culture and naïve marker expression by RT-PCR, however it is dispensable and human naive PSCs can maintain their naive identity even in the absence of LIF/STAT3 signaling (<FIG>) as has been previously shown for rodent ground state naïve PSCs.

As has been previously shown in mice, the use of ERK inhibition is the major mediator for inducing global hypomethylation which in turns leads to sporadic erosion of imprinting that gets more severe with extended passaging (Choi et al. In mice, using alternative naive conditions that do not employ ERK inhibitor or titrating ERKi allows isolating murine PSCs with all features of naivety except for global hypomethylation (Choi et al. The latter murine cells are fully naive and are capable of generate all-iPS mice with contribution to the germline, and thus provide a safer route for exploiting defined mouse naive PSCs (Choi et al.

Although ENHSM conditions had modest levels of hypomethylation, and erosion of imprinting was slow and sporadic on few loci and only after extended passaging (<FIG>), this may complicate the use of naive cells in future clinical applications if they are expanded in these conditions more than <NUM> passages. The present inventors thus aimed at defining conditions that allow human naive cell isolation but without global hypomethylation.

Withdrawal of ERK inhibitor form ENHSM conditions compromised the naivety of human ESC as evident be a decrease in deltaPE-Oct4-GFP levels and loss of x-reactivation state in most of the cells within the expanded population (<FIG>). Mere addition of ACTIVIN A upon omission of ERKi form ENHSM conditions did not block loss X reactivation in female cell lines upon depleting ERKi (<FIG>). Thus, the present inventors set out to screen for added compounds that would enable maintenance of pre-x inactivation upon omitting or depleting ERKi (<FIG>). Remarkably, it was noted that addition of gamma secretase inhibitor DBZ, which blocks NOTCH pathway allowed robust and feeder free maintenance of human naive cells when ERKi was omitted (0ENHSM conditions) or titrated down to <NUM>. 33microM termed (tENHSM conditions) (<FIG>). The use dominant negative Notch allowed maintenance of naive PSCs without adding DBZ, proving that Notch targeting is the effector mediator of maintaining robust naive pluripotency in human cells when ERK inhibition is depleted.

Human PSCs expanded in ENHSM conditions maintain deltaPE-OCT4-GFP signal equivalent to ENHSM conditions, and maintained pre-X inactivation state in female cell lines (<FIG>). RT-PCR analysis showed that the lines expressed naive pluripotency markers, although levels were less induced to concentration of ERKi used (<FIG>). Global gene expression analysis showed that the cells clustered with ENHSM naive PSCs rather than primed PSCs (<FIG>). At the functional levels, the cells were competent in making teratomas without any need for priming in vitro before injections (<FIG>). Cells could be maintained upon depletion of DNMT1, METTL3, DGCR8 or exogenous L-glutamine and these qualities depended on the presence of DBZ (<FIG>). WGBS analysis showed that these alternative 0ENHSM and tENHSM conditions did not show trends of global hypomethylation that were seen in ENHSM or ENHSM-ACT even after extended passaging and consistently do not show loss of imprinting (<FIG>). These results show that, similar to what was obtained in rodent in vitro PSCs with t2iL and a2iL conditions that endow naïve feature in murine PSCs without compromising global DNA methylation and imprinting regulation (Choi et al. , <NUM>), this can be obtained also in human naive PSCs as well.

The present inventors therefore examined whether the refined ENHSM conditions can endow hiPSCs to integrate and contribute to cross-species chimaerism more successfully and with higher propensity.

HiPSCs were labeled with GFP and maintained for at least <NUM> passages in ENHSM before being micro-injected into E2. <NUM> mouse morulas. Following transplantation into pseudo-pregnant foster mothers the day after, their survival and integration was assayed throughout <NUM> days using various imaging techniques. ENHSM-derived hiPSCs are able to colonize mouse embryos up to E17. <NUM> at various anatomic regions of different embryonic germ layers as shown in (<FIG>). Specific marker staining for human nuclei excluded any contamination (<FIG>). The present inventors next investigated whether this integration is based on random differentiation or following the respective tissue identity in-vivo. Immunofluorescence staining of frozen sections confirmed distinct expression of various lung antigens (CC10, Prosurfactant Protein C and Aquaporin <NUM>) substantiating proper lineage commitment and functionality of hiPSC-derived descendants (<FIG>).

In order to enhance and boost survival and integration of interspecies chimaera P53 was depleted. AAVS1-GFP labelled hiPSCs were CRISPR/Cas9 targeted for P53 and knock-out were generated extremely efficiently (<FIG>). These cells were injected analogously to WT cells and their integration behavior was examined in developing mouse embryos. Strikingly, not only was there an increased chimaeric yield per injection, but additionally a higher GFP+ contribution per embryo as illustrated in <FIG>. Notably high-degree chimaeras were generated with wide-spread GFP signal throughout the mouse embryo (<FIG>). Furthermore, live imaging of these embryos revealed a very large contribution especially in the heart and brain region (<FIG>). In order to obtain an estimate for overall contribution, whole-embryo FACS was performed, showing different degree of chimaeric percentages with up to <NUM>% GFP+ hiPSC-derivatives. Random spontaneous differentiation was ruled out by staining chimaeras for human-specific antigens (<FIG>) and most importantly for major developmental drivers covering all three embryonic germ layers overlapping extensively with human-derived GFP signal (<FIG>). Taken together, these results substantiate unequivocal generation of advanced human-mouse interspecies chimaera with various engraftment and functional integration in many defined lineages up to E17.

Additional culture conditions were analyzed to determine if additional media are capable of capturing human naive PSCs.

The culture conditions are described in the Materials and methods section and labeled conditions <NUM>-<NUM>.

As illustrated in <FIG>, all the media that were tested maintained the pluripotent stem cells in a hypomethylated state.

WIBR3 female human ESCs were expanded for <NUM> passages in the indicated conditions and immunostained for TFE3 protein expression. Nuclear/cytoplasmic ratio was calculated for each of the conditions. The results are provided in Table <NUM>, herein below.

Claim 1:
A culture medium comprising a WNT inhibitor selected from the group consisting of (i) Tankyrase inhibitor and (ii) a small molecule inhibitor for Porcupine enzyme, a SRC inhibitor, a protein kinase C (PKC) inhibitor and an ERK inhibitor, said medium does not contain more than <NUM> of a GSK3β inhibitor , and wherein said medium maintains human pluripotent stem cells in a naive, undifferentiated and pre-X-inactivation state for at least <NUM> passages, wherein said pre-X-inactivation is characterized by presence of two unmethylated alleles of an X-inactive specific transcript (XIST) gene in a female cell, and presence an unmethylated allele of the XIST gene in a male cell.