FIELD OF THE INVENTION

The present invention relates to the use of one or more type 7 long terminal repeat (LTR7) nucleic acid sequences of type H human endogenous retroviruses (HERVH) (“LTR7/HERVH nucleic acid sequences”) for identifying primate naive pluripotent stem cells. The invention is directed to the use of LTR7/HERVH nucleic acid sequences as a marker, wherein LTR7/HERVH-associated transcription is used as a marker for primate naive pluripotent stem cells. The invention also relates to a reporter construct comprising LTR7/HERVH nucleic acid sequences in addition to the use of said reporter, in particular for optimizing culture conditions for naïve primate pluripotent stem cells. The invention also relates to a cell growth medium for cultivation of primate naive pluripotent stem cells that preferably exhibit elevated levels of LTR7/HERVH-associated transcription in comparison to control cells.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing submitted as an ASCII text file via EFS-Web is hereby incorporated by reference in accordance with 35 U.S.C. § 1.52(e). The name of the ASCII text file for the Sequence Listing is 31273065_1.TXT, the date of creation of the ASCII text file is Sep. 6, 2019, and the size of the ASCII text file is 5.9 KB.

BACKGROUND OF THE INVENTION

Naïve embryonic stem cells (ESCs) hold great promise for research and therapeutics as they have broad and robust developmental potential. While such cells are readily derived from mouse blastocysts it has been impossible to easily isolate human equivalents1,2, although human naïve-like cells have been artificially generated (rather than extracted) by coercion of human primed ES cells by modifying culture conditions2-4or through transgenic modifications5.

Despite the progress made in establishing culture conditions for selecting and maintaining naïve pluripotent stem cells (PSCs), improvements are required that enable a more reliable identification and subsequent prolonged culturing of said cells from stem cell populations.

Transcription of LTR sequences has been observed in stem cell populations, but has not been proposed as an effective marker for identifying and/or selecting naïve stem cells. Ohnuki et al (PNAS, 2014, v. 111, no. 34.) discloses transient hyperactivation of LTR7 sequences during iPSC generation. Induction of LTR7 expression is mediated by OCT3/4, SOX2, and KLF4. Ohnuki et al state that when reprogramming is complete and cells acquire full pluripotency, LTR7 activity decreases to levels comparable with those in ESCs. According to Ohnuki et al, failure to reduce the LTR7 activity is postulated to lead to a differentiation defective phenotype, thereby teaching that LTR7 transcription is not suitable as a marker for naïve PSCs.

WO 2013/014929 discloses a method and means for screening iPSC for differentiation resistance using large intergenic non-coding RNAs or specific mRNA sequences. According to WO 2013/014929, iPSCs without differentiation resistance are characterised by the absence of expression of particular LTR7 sequences that exhibit increased levels of DNA methylation and reduced expression in iPSCs without differentiation resistance. In summary, both Ohnuki et al and WO 2013/014929 fail to identify the relevance of LTR transcription with respect to the identification and maintenance of the naïve state of naive PSCs.

Alternative approaches described in the prior art have employed a reporter system for naive human pluripotency based on OCT4 distal enhancer activity combined with an optimized culture medium for cultivation of naïve PSCs27(Theunissen et al., Cell Stem Cell. 2014, 15(4): 471). Although some success has been achieved using such approaches, the cells obtained by these methods show reduced genome stability that is disadvantageous for later use (such as therapeutic use) of the cells or cells derived therefrom.

SUMMARY OF THE INVENTION

The present invention demonstrates that a sub-population of cells within cultures of human ESCs (hESCs) and induced pluripotent stem cells (hiPSCs) manifest key properties of naïve state cells. These “naïve-like” cells (or naïve pluripotent stem cells) can be identified by elevated transcription of HERVH, a primate-specific endogenous retrovirus (ERV). HERVH elements provide functional binding sites for a combination of naïve pluripotency transcription factors, including LBP9, OCT4, NANOG and KLF4. LBP9 was recently recognized as relevant to naivety in mice6. LBP9/HERVH drives hESC-specific alternative and chimeric transcripts, including pluripotency modulating long non-coding RNAs (IncRNAs). Disruption of LBP9, HERVH and HERVH-derived transcripts compromises self-renewal. These observations define HERVH expression as a feature of naïve hESCs, establish novel primate-specific transcriptional activity regulating pluripotency and enable the use of HERVH elements in the identification and/or separation of naïve-like hESCs from a cell mixture, such as embryonic cells or extracts thereof, or from hiPS cells.

In light of the prior art the technical problem underlying the present invention is to provide improved or alternative means for identifying and/or maintaining primate naïve pluripotent stem cells in culture.

This problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

The invention therefore relates to the in vitro use of one or more type 7 long terminal repeat (LTR7) nucleic acid sequences of type H human endogenous retroviruses (HERVH) (“LTR7/HERVH nucleic acid sequences”) for identifying and/or isolating primate naive pluripotent stem cells.

Preferred embodiments of the LTR7/HERVH nucleic acid sequences are disclosed herein as the sequences according to SEQ ID NO 1, 2 and/or 3.

It was unexpected at the time of developing the present invention that LTR7 sequence transcription may be utilized as a marker for naïve PSCs. The prior art in this field had suggested that maintained LTR sequence transcription in SCs lead to a differentiation defective phenotype (a loss of pluripotency; Ohnuki et al) or that cells without LTR7 expression showed maintained pluripotency (differentiation resistance; WO 2013/014929).

The invention therefore relates to a method for identifying and/or isolating primate naive pluripotent stem cells comprising an analysis (measurement, detection, identification and/or determination) of LTR7/HERVH-associated transcription.

In one embodiment the method of the invention and use of LTR7/HERVH sequences as described herein is characterised in that the method comprises analysis of LTR7/HERVH-associated transcription, wherein LTR7/HERVH-associated transcription is used as a positive marker for primate naive pluripotent stem cells.

Analysis of LTR7/HERVH-associated transcription may employ any appropriate technical means, such as a quantitative or semi-quantitative RNA method, in particular measuring the RNA produced from said transcription. This RNA may be assessed by PCR amplification of reverse transcribed DNA molecules corresponding to LTR7 transcripts. Appropriate primers may be selected by a skilled person using means known in the art. For example, RT-PCR may be employed, or sequencing-based methods may be applied that are capable of sequencing and/or quantifying reverse transcribed DNA corresponding to LTR7 transcripts.

In one embodiment the use of LTR7/HERVH sequences as described herein is characterised in that the LTR7/HERVH nucleic acid sequence comprises a LBP9 binding motif, preferably wherein the LTR7/HERVH nucleic acid sequence comprises a binding motif for one or more (preferably all) of the following transcription factors: LBP9, OCT4, NANOG and/or KLF4.

In another aspect the invention relates to an in vitro method for isolating primate naive pluripotent stem cells comprising an analysis of LTR7/HERVH-associated transcription and isolation of cells in which LTR7/HERVH-associated transcription is elevated in comparison to control cells, wherein control cells are preferably primed pluripotent stem cells or differentiated cells.

In another aspect the invention relates to an isolated population of primate naive pluripotent stem cells in which LTR7/HERVH-associated transcription is elevated in comparison to control cells, wherein control cells are preferably primed pluripotent stem cells or differentiated cells.

A description of primed cells is provided in25in addition to2-4and the cells used in the examples disclosed herein. Primed PSCs may be identified without difficulties by a person skilled in the art.

To the knowledge of the inventors, an isolated population of naïve stem cells has been neither described nor suggested in the art. The naïve PSCs that exhibit elevated LTR7 expression are naïve in the sense that they reflect very closely the expression profile of cells from the ICM and show no pre-disposition to differentiate in any particular differentiation fate.

According to the present invention the expression profile of cells may be used to identify “naivety” in a PSC. For example cells that resemble closely the inner cell mass (ICM) may be considered as a naïve or naïve-like PSC. To this end, the cells described herein, enriched using the HERVH reporter, are good representatives of naïve cells as they cluster nearer to cells of the ICM when compared with the ‘novel naïve’ cells obtained in reference 4 (FIG. 4e, refer alsoFIG. 20). The HERVH-driven transcriptional profiles in the current naïve-like hPSC lines (including the GFP(high) cells; also referred to interchangeably as GFPhigh) are only slightly different from human ICM. The reporter construct described herein therefore represents a powerful tool for isolating naïve PSCs, culturing naïve PSCs and for enabling optimization of naïve-like hPSC culture conditions.

As used herein, the term naïve pluripotent stem cell relates preferably to the LTR7-expressing naïve pluripotent stem cell as described in detail herein. These cells may be referred as “naïve pluripotent stem cell” or “naïve-like PCSs” due to the closeness of their expression profiles to cells of the ICM, thereby indicating a “true” state or naivety.

The cells of the invention show unexpectedly good properties with respect to long culture times without priming towards a differentiation fate or the occurrence of differentiation. The cells may be maintained in culture and later differentiated to particular precursors, as is desired according to the intended therapy. The cells are also particularly suitable and robust when stored under cryopreservation or whilst being maintained in culture. The frequency of transposition in the isolated cell population of the invention is low compared to previously described “naïve SC populations” (compared for example to reference 27).

In another aspect the invention relates to a nucleic acid reporter construct comprising a nucleic acid sequence region encoding one or more marker or reporter molecules operably linked to a sequence comprising one or more LTR7/HERVH nucleic acid sequences.

A marker molecule relates preferably to a fluorescent protein, preferably green fluorescent protein or other proteins capable of being used as a reporter, and/or other selectable proteins, such as antibiotic resistance genes.

According to the present invention the term reporter construct relates to a nucleic acid molecule capable of selective identification of a particular cellular or sub-cellular state, preferably a reporter construct is capable of expression of a marker protein upon entering a particular state. For example, the reporter construct described herein is preferably defined by induced expression of a reporter (or marker protein, such as GFP) after LTR7 sequence expression as a marker for the naïve pluripotent stem cell state.

Fluorescent proteins are, without limitation, preferably selected from the group consisting of GFP (wt), Green Fluorescent Proteins, EGFP, Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, T-Sapphire, Blue Fluorescent Proteins, EBFP, EBFP2, Azurite, mTagBFP, Cyan Fluorescent Proteins, ECFP, mECFP, Cerulean, mTurquoise, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, mTFP1 (Teal), Yellow Fluorescent Proteins, EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, mBanana, Orange Fluorescent Proteins, Kusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express (T1), DsRed-Monomer, mTangerine, Red Fluorescent Proteins, mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum and AQ143.

Suitable antibiotic resistance genes are, without limitation, preferably selected from the group consisting of Blasticidin, Zeocin, Puromycin, G418, Hygromycin B Gold and Phleomycin.

The reporter may therefore be introduced into stem cells or populations of stem cells in vitro, and subsequently used to identify and/or separate naïve stem cells based on activation of the reporter. Activation of the reporter may be detected via fluorescence microscopy, FACS, survival of cells after antibiotic treatment, or other suitable means.

In one embodiment the invention relates to an isolated cell comprising a reporter construct as described herein, wherein the reporter construct is preferably comprised by a nucleic acid vector, wherein the vector preferably comprises transposon sequences.

The invention also relates to the in vitro use of the reporter construct, nucleic acid vector and/or cell as described herein in a method for optimizing a cell growth medium for primate naive pluripotent stem cells.

As described in the examples in more detail, the invention enables the optimization of cell growth medium by monitoring the expression of the reporter of the invention whilst modifying cell culture conditions or medium components, in order to maintain a “read-out” on the naïve status of the cultured pluripotent stem cells.

In one embodiment the method for optimizing a cell growth medium as described herein comprises:In vitro cultivation of primate naive pluripotent stem cells in a cell growth medium for primate naive pluripotent stem cells, wherein said cells comprise a reporter construct as described herein;Modification of the presence and/or concentration of one or more components of said cell growth medium or other cell culture conditions; andanalysis of expression of the reporter molecule encoded by said construct, preferably comprising a comparison in reporter molecule expression between the modified (according to step b.) and unmodified cell growth medium.

In another aspect the invention relates to a cell growth medium for cultivation of primate naive pluripotent stem cells produced by the optimization method described herein.

In another aspect the invention relates to a cell growth medium for cultivation of primate naive pluripotent stem cells. The medium may be optimized for enabling cultivation of said cells. The optimization involves the addition or modification of the concentration of various medium components.

The initial examples of the present invention employ human 2i/LIF medium, which is based on mouse 2i/LIF medium. The human medium contains, in one embodiment, by way of example, knockout DMEM, 20% knockout serum supplement, 1 mM L-Glutamine, 1% nonessential amino acids, 0.1 mM 2-mercaptoethanol, 10 ng/ml LIF, 1 μM CHIR99021, 1 μM PD0325901 and primocin, and the medium was supplemented with 10 ng/ml bFGF.

The contents of this medium may therefore be modified with respect to the presence and/or concentration of any one or more of its components as described above, or with respect to the following chemical inhibitors, or other medium components, such as cytokines, or other common components known to a skilled person, in order to assess whether any given change leads to an effect on expression of the LTR7/HERVH nucleic acid sequences as described herein.

In a preferred embodiment of the method for optimizing cell culture conditions, an improved cell culture medium or culture conditions has been identified when the expression of the reporter construct described herein is greater in the modified culture medium/condition in comparison to an unmodified cell growth medium/condition.

In a further embodiment of the invention the medium comprises a combination of basal medium, cytokines and small molecules, such as the inhibitors described herein, for example in the form of a cocktail of inhibitors.

In another aspect the invention relates to a kit for producing a cell growth medium for cultivation of primate naive pluripotent stem cells. The medium may be provided prior to its preparation a kit comprising components suitable for producing the medium upon their combination. For example, the kit of the invention may comprise the various components of the medium as described herein, either as single components or in pre-prepared mixtures. Pre-pared mixtures preferably relate to the basal medium, cytokines and a cocktail of multiple small molecules.

The invention therefore relates to a kit for the provision of a cell growth medium comprising the following components in separated compartments in proximity to one another: a basal medium, comprising neurobasal medium and DMEM, optionally comprising at least one or more cytokines of the IL-6 family, and a cocktail of inhibitors, comprising at least one or more MEK/ERK inhibitors, one or more a GSK3 inhibitors, one or more Axin stabilizers and one or more PKC inhibitors.

The invention further relates to the in vitro use of the cell growth medium as described herein for culturing, maintaining and/or enriching LTR7-expressing primate naive pluripotent stem cells, in which LTR7/HERVH-associated transcription is elevated in comparison to control cells, wherein control cells are preferably primed pluripotent stem cells or differentiated cells, from a stem cell population.

The invention further relates to a method for enriching LTR7-expressing primate naive pluripotent stem cells, in which LTR7/HERVH-associated transcription is elevated in comparison to control cells, wherein control cells are preferably primed pluripotent stem cells or differentiated cells, from a stem cell population by culturing a stem cell population in the cell growth medium as described herein.

Any disclosure provided herein directed to the kit, reporter, medium or any other aspect are to be understood in their context with each other. The features provided for one aspect of the invention may be used to define other aspects of the invention as required. In particular, the particular embodiments of inhibitors described herein are considered to be disclosed in any given combination of components and concentrations, as understood by a skilled person. The kit was developed specifically for the provision of the medium as described herein and the features disclosed in the context of the medium are correspondingly disclosed for the kit. The features as described in the context of the medium are also relevant for the methods and uses as described herein.

In one embodiment the basal medium comprises neurobasal medium. Neurobasal medium is known in the art and relates preferably to products that are commercially available, such as Neurobasal®-A Medium (Gibco/ThermoFisher), which is a basal medium formulated to meet the special cell culture requirements of post-natal and adult brain neuronal cells when used with GIBCO® B-27® Supplement. Neurobasal medium typically allows for both long and short term maintenance of homogeneous populations of neuronal cells without the need of an astrocyte feeder layer.

In one embodiment the basal medium comprises Dulbecco's Modified Eagle Medium (DMEM), which is a standard mammalian cell culture medium, or in DMEM/F12, which comprises DMEM with Nutrient Mixture F-12, as available from Gibco/ThermoFisher.

In further embodiments of the invention the basal medium of the cell culture medium of the invention comprises L-glutamine, Non-essential amino acids (NEAA), N2 supplement, B27 supplement without Vitamin A, and/or Vitamin C. The basal medium may optionally comprise insulin, 2-Mercaptoethanol and/or antibiotics.

In one embodiment the basal medium comprises a combination of commercially available components: Neurobasal medium, DMEM/F12, L-glutamine, NEAA, N2 supplement, B27 supplement (w/o Vitamin A), Vitamin C and 2-Mercaptoethanol. By way of example, the medium may be made to a 500 mL volume. In one embodiment the basal medium comprises 200-300 mL of neurobasal medium, preferably 220-270 mL. In one embodiment the basal medium comprises 200-300 mL of DMEM or DMEM/F12, preferably 220-270 mL. In one embodiment the basal medium comprises between 1 mL and 10 mL of a 100× stock of L-glutamine. In one embodiment the basal medium comprises between 1 mL and 10 mL of a 100× stock of NEAA. In one embodiment the basal medium comprises between 1 mL and 10 mL of a 100× stock of N2 supplement. In one embodiment the basal medium comprises between 2 mL and 20 mL of a 50× stock of B27 supplement, preferably without Vitamin A. In one embodiment the basal medium comprises between 10 and 500 mg/mL of Vitamin C, preferably 50-100 mg/mL.

In one embodiment the basal medium comprises between 0 and 100 ug/mL of insulin, preferably 20-50 mg/mL. In one embodiment the basal medium comprises between 0 and 1 mM, preferably 0.01 to 0.5 mM of 2-Mercaptoethanol. Other agents that reduce disulfide bonds may be used at an appropriate concentration.

In one embodiment the cytokines of the medium comprise one or more cytokines of the IL-6 family. Cytokines of the IL-6 family are known as IL-6, IL-11, oncostatin M (OSM) and LIF. Cytokines of the IL-6 family may be provided at a concentration of 1 to 1000 ng/mL, preferably 10 100 ng/mL.

In one embodiment the cytokines of the medium comprise human IL6 at 1 to 100 ng/mL, preferably 10 to 50 ng/mL.

In one embodiment the cytokines of the medium comprise human sIL-6R (soluble IL-6 receptor), at 1 to 100 ng/mL, preferably 10 to 50 ng/mL.

In one embodiment the cytokines of the medium comprise human LIF at 1 to 100 ng/mL, preferably 10 to 50 ng/mL.

In one embodiment the cytokines of the medium comprise optionally human Activin A at 0 or 1 to 100 ng/mL, preferably 10 to 50 ng/mL.

In one embodiment the cytokines of the medium comprise optionally human IL-11 at 0 or 1 to 100 ng/mL, preferably 10 to 50 ng/mL.

In one embodiment the cytokines of the medium comprise optionally human bFGF at 0 or 1 to 100 ng/mL, preferably 5 to 50 ng/mL.

In one embodiment the medium (or cocktail of inhibitors of the kit) comprises small molecules that comprise a MEK/ERK inhibitor, a B-raf inhibitor, a JNK inhibitor, a GSK3 inhibitor, a Axin stabilizer, a PKC inhibitor, a Notch inhibitor, a Sonic Hedgehog inhibitor, a BMP inhibitor, a TGFbeta inhibitor, a mitochondrial pyruvate dehydrogenase kinase inhibitor, a histone methyltransferase inhibitor, and/or a histone deacetylase inhibitor.

In one embodiment the medium (or corresponding cocktail of inhibitors of the kit) comprises a MEK/ERK inhibitor PD0325901, preferably 0.01-10 μM, more preferably 0.2-1 μM.

In one embodiment the medium (or cocktail of inhibitors) comprises a B-raf inhibitor SB590885: preferably 0.01-5 μM, more preferably 0.1-0.5 μM.

In one embodiment the medium (or cocktail of inhibitors) comprises a JNK inhibitor TCS-JNK-6o: preferably 0.05-50 μM, more preferably 0.2-10, or 0.5-5 μM.

In one embodiment the medium (or cocktail of inhibitors) comprises a GSK3 inhibitor BIO: preferably 0.01-5 μM, more preferably 0.05-0.5 μM; or CHIR99021: preferably 0.01-10 μM, more preferably 0.1-1 uM.

In one embodiment the medium (or cocktail of inhibitors) comprises an Axin stabilizer XAV939: preferably 0.1-50 μM, more preferably 1-10, or 2-5 μM; or endo-IWR1: preferably 0.1-50 μM, more preferably 1-5 μM.

In one embodiment the medium (or cocktail of inhibitors) comprises a PKC inhibitor Go6983: preferably 0.01-50 μM, more preferably 1-5, or 2-4 μM.

In one embodiment the medium (or cocktail of inhibitors) comprises a Notch inhibitor DAPT: preferably 0.1-100, more preferably 1-50, or 2-10 μM.

In one embodiment the medium (or cocktail of inhibitors) comprises a Sonic Hedgehog inhibitor HPI1: preferably 0.1-50, more preferably 1-5 μM.

In one embodiment the medium (or cocktail of inhibitors) comprises a BMP inhibitor K02288: preferably 0.1-50, more preferably 1-5 μM.

In one embodiment the medium (or cocktail of inhibitors) comprises a TGFbeta inhibitor A83-01: preferably 0.01-10, more preferably 0.1-1.0, or 0.2-0.5 μM.

In one embodiment the medium (or cocktail of inhibitors) comprises a mitochondrial pyruvate dehydrogenase kinase inhibitor DCA: preferably 0.1-100, more preferably 2-10 μM.

In one embodiment the medium (or cocktail of inhibitors) comprises a histone methyltransferase inhibitor DZNep: preferably 0.001-10, more preferably 0.005-1, or 0.01-0.1 μM.

In one embodiment the medium (or cocktail of inhibitors) comprises a histone deacetylase inhibitor Sodium butyrate: preferably 0.01-10, more preferably 0.1-0.5 mM; or SAHA: preferably 0.001-0.5, more preferably 0.01-0.05 μM.

In one embodiment the medium (or cocktail of inhibitors) comprises at least one or more MEK/ERK inhibitors, one or more a GSK3 inhibitors, one or more Axin stabilizers and one or more PKC inhibitors, preferably of those mentioned above in the concentrations mentioned above. The use of the basal medium, cytokines and small molecules of these classes (MEK/ERK inhibitors, GSK3 inhibitors, Axin stabilizers and PKC inhibitors) leads to a “4i” medium. The 4i medium is characterized by the ability to maintain naïve PSCs for long periods in culture without the need for re-sorting (such as using FACS) and also provides cells with increased genome stability, for example the Line1, SVA and other transposable elements show reduced mobility (reduced retrotransposition) in the genomes of naïve PSCs cultures after culturing in this medium.

Surprisingly, the invention provides an “exno-free” and feeder-free medium suitable for long-term culturing, maintenance and/or enrichment of naïve PSCs. Until the present time feeder cells were required when culturing naïve PSCs, leading to enhanced complication, cost and risk of contamination during culturing, which represents a significant disadvantage especially with respect to culturing the cells for later therapeutic use.

The medium as described herein is free of, or substantially free of, animal-derived components, thereby also reducing the disadvantages of most commonly used systems. Stem cell culture systems that rely on undefined animal-derived components introduce variability to the cultures and complicate their therapeutic use.

One aspect of the invention relates to a method for enriching LTR7-expressing primate naive pluripotent stem cells from a stem cell population by culturing a stem cell population in the cell growth medium as described herein. In particular, the medium described herein is capable of providing enrichment of LTR7-expressing naïve PSCs in culture. The medium is therefore defined by a set of features, namely LTR7-expression in naïve PSCs, that represent a common and unexpected concept linking all aspects of the present invention.

The invention therefore relates to the use of the medium described herein for the culturing of LTR7-expressing naïve PSCs. In one embodiment the oxygen content during cell culture can be reduced to approx. 5% oxygen (+/−3%), in order to additionally maintain the naïve state of the LTR-7-expressing naïve PSCs. Oxygen conditions during culture are therefore at approx. 20% (+/−5%), below 20%, below 15%, below 10%, such as between 2 and 8%, such as 5% oxygen.

The naive cells described herein are particularly useful for the provision of therapeutic material in the future by initiating differentiation programs as desired, in order to create cell therapy products, without have to use cells primed towards certain fates.

In one embodiment the medium (or cocktail of inhibitors) comprises, in addition to the one or more MEK/ERK inhibitors, one or more a GSK3 inhibitors, one or more Axin1 stabilizers and one or more PKC inhibitors, additionally one or more B-raf inhibitors, Notch inhibitors, Sonic Hedgehog inhibitors, JNK inhibitors, and one or more BMP inhibitors, preferably at the concentrations and specific examples provided above.

In one embodiment the medium (or cocktail of inhibitors) comprises at least one or more GSK3 inhibitors and one or more Axin stabilizers. The combination of these two classes of molecules provides unexpected results that are advantageous for the culturing of the LTR7-expressin naïve cells of the present invention.

The GSK3 inhibitor, such as BIO, leads to an activation of Wnt-signalling, whereas the Axin-1-stabilisor, such as XAV939, leads to an inhibition of Wnt-Signalling. Wnt-signalling is well-known to a skilled person and requires no detailed explanation in this context. The Wnt signaling pathway encompasses a group of signal transduction pathways that pass signals from outside of a cell through cell surface receptors inside the cell. Wnt-signalling is highly evolutionarily conserved in animals.

Through the combination of one or more GSK3 inhibitors and one or more Axin-1-stabilisors Wnt-signalling is repressed, but a low level of Wnt-singalling is maintained. This balanced activity leads to beneficial and surprising results. In particular, the combination of these two components, preferably with one or more MEK/ERK inhibitors and PKC inhibitors, provides long term maintenance of naïve PSCs in culture, up to for example 60 passages, without the need for re-sorting the cells according to expression of the LTR7 expression. This combination of factors leads to an enrichment during in vitro cell culture of naïve PSCs without any other sorting (such as FACS) steps. This combination of factors, i. e. the maintenance of a low level of Wnt-signalling, leads to maintenance and/or re-programming of PSCs into the naïve PSCs as defined by increased LTR7 expression compared to primed PSCs. Naïve PSCs may therefore be cultured in the medium described herein without the presence of the LTR7-reporter construct described herein. Independent of the use of the LTR7 reporter, the LTR7 transcription will be enhanced in the cell population cultured in the medium of the invention.

Information on the involved signals is provided inFIGS. 17-19.

One of the proposed mechanisms for the importance of Wnt-signaling is the proportion of Beta-Catenin that is free to act as a transcriptional regulator and the amount that functions in the cytosol but at the membrane in E-Cadherin complexes, which are important in cell-cell contact. This mechanism suggest that Wnt-signalling may be reduced, but not removed entirely, partially perhaps due to the requirement of the E-Cadherin function in forming cell colonies in culture. Some cytosolic fraction of B-catenin should be maintained in order to keep these functions in order.

In a preferred embodiment the Wnt-Signalling is modulated to correspond to an activity defined by administration of a GSK3 inhibitor, such as BIO, and an Axin-1-stabilisor, such as XAV939, at a ratio of 1:1000 to 1:1, preferably 1:200 to 1:10, more preferably 1:150 to 1:50.

In further embodiments, one or more of the following inhibitors may be added to the medium or the inhibitor cocktail as described herein, and the concentration thereof preferably modified in order to assess whether expression of the LTR7/HERVH nucleic acid sequences, as a marker for the primate naive pluripotent stem cells, is affected:

Mitogen-activated protein kinase kinase (MAP2K, MEK, MAPKK) inhibitor, WNT signalling activator, mitogen-activated protein (MAP) Kinase Inhibitor, c-Jun N-terminal kinases (JNK) inhibitor, Protein kinase C (PKC) inhibitor, Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, Glycogen synthase kinase 3 (GSK-3) inhibitor, Bone morphogenetic protein (BMP) signalling inhibitor, histone deacetylase (HDAC) inhibitor, B-Raf kinase inhibitor, Lck/Src inhibitor, RasGAP inhibitor, ERK1 or ERK2 (extracellular-signal-regulated kinases (ERK) 1/2) inhibitor, histone-lysine methyltransferase (HMTase) inhibitor and/or DNA methyltransferase inhibitor.

MEK/ERK inhibitors include but are not limited to PD98059 (Pfizer), U0126 (DuPont), PD184352 [CI-1040] (Pfizer), PD0325901 (Pfizer), Selumetinib (a.k.a., ARRY-142886, AZD6244) (Astra-Zeneca), GDC-0994 and RDEA119 (Ardea Biosciences) and PD0325901.

GSK3 inhibitors include but are not limited to Valproic acid sodium salt, Staurosporine, KT 5720, GSK-3 Inhibitor IX, Ro 31-8220, SB-216763, CID 755673, Kenpaullone, Lithium Chloride, GSK-3β Inhibitor XII, TWS119, GSK-3 Inhibitor XVI, 10Z-Hymenialdisine, Indirubin, CHIR-98014, GSK-3β Inhibitor VI, Manzamine A, Indirubin-3′-monoxime, GSK-3 Inhibitor X, GSK-3 Inhibitor XV, SB-415286, 1-Azakenpaullone, TWS 119 ditrifluoroacetate, 5-Iodo-indirubin-3′-monoxime, GSK-3β Inhibitor I, 9-Cyanopaullone, 5-Iodo-Indirubin-3′-monoxime, Indirubin-5-sulfonic acid sodium salt, GSK-3β Inhibitor VII, Cdk1/5 Inhibitor, Bisindolylmaleimide X hydrochloride, Isogranulatimide, Raf Kinase Inhibitor IV, L-779,450, Indirubin-3′-monoxime-5-sulphonic Acid, GSK-3 Inhibitor II, GSK-3β Inhibitor VIII, Aloisine A, GSK-3β Inhibitor XI, GSK-3 Inhibitor IX, Control, MeBIO, Alsterpaullone, 2-Cyanoethyl, TCS 2002, TCS 21311, Enzastaurin, MeBIO, Cdk2/9 Inhibitor, Cdk1/2 Inhibitor III, PHA 767491 hydrochloride, AR-AO 14418-d3, Hymenialdisine Analogue 1 and BIO.

Axin stabilizers include but are not limited to IWR-1-endo, IWR-1-exo and XAV939.

PKC inhibitors include but are not limited to Calphostin C, CGP 53353, Chelerythrine chloride, Dihydrosphingosine, GF 109203X, Go 6976, Go 6983, K-252c, LY 333531 hydrochloride, [Ala107]-MBP (104-118), [Ala113]-MBP (104-118), Melittin, (±)-Palmitoylcarnitine chloride, PKC (19-36), [Glu27]-PKC (19-36), Inactive control peptide for PKC (19-36), PKC 412, PKC β pseudosubstrate, PKC ζ pseudosubstrate, Ro 32-0432 hydrochloride, Rottlerin, D-erythro-Sphingosine (synthetic), Go6983 and TCS 21311.

B-raf inhibitors include but are not limited to Vemurafenib (PLX4032, RG7204), Sorafenib Tosylate, PLX-4720, Dabrafenib (GSK2118436), GDC-0879, LY3009120, RAF265 (CHIR-265), AZ 628, NVP-BHG712 and SB590885.

Notch inhibitors include but are not limited to FLI-06, RO4929097, Semagacestat (LY450139), LY411575, YO-01027 (Dibenzazepine), DAPT and Avagacestat (BMS-708163).

Sonic Hedgehog inhibitors include but are not limited to GANT61, Vismodegib (GDC-0449), Taladegib (LY2940680), TAI-1, HPI1 and Pimasertib (AS-703026).

JNK inhibitors include but are not limited to AEG 3482, Anisomycin, BI 78D3, CEP 1347, c-JUN peptide, IQ 3, JIP-1 (153-163), SR 3576, SU 3327, TCS-JNK-6o and TCS JNK 5a.

BMP inhibitors include but are not limited to Dorsomorphin dihydrochloride, K 02288, ML 347, NBMPR and UK 383367.

The medium of the present invention may therefore comprise one or more of the above mentioned inhibitors. All possible combinations of each of the various inhibitors or classes of inhibitors disclosed herein are considered for use in the medium of the present invention.

As examples of such inhibitors, one or more of the following components, which are not limiting to the inhibitor classes mentioned above, may be utilized during optimization (presence and/or concentration varied during testing), and/or may be present in the medium of the present invention:

PD0325901, at preferred concentration of 0.01 to 100 μM, more preferred 0.1 to 10 μM, such as 1 or 0.5 μM. PD0325901 is an orally bioavailable, synthetic organic molecule targeting mitogen-activated protein kinase kinase (MAPK/ERK kinase or MEK) with potential antineoplastic activity. MEK inhibitor PD325901 is a derivative of MEK inhibitor CI-1040, selectively binds to and inhibits MEK, which may result in the inhibition of the phosphorylation and activation of MAPK/ERK and the inhibition of tumor cell proliferation. The dual specific threonine/tyrosine kinase MEK is a key component of the RAS/RAF/MEK/ERK signaling pathway that is frequently activated in human tumors.

CHIR99021, at preferred concentration of 0.01 to 300 μM, more preferred 0.1 to 30 μM, most preferred 1-3 μM. CHIR99021 is an aminopyrimidine derivative that is an extremely potent inhibitor of GSK3, inhibiting GSK3β (IC50=6.7 nM) and GSK3α (IC50=10 nM) and functions as a WNT activator. It is the most selective inhibitor of GSK3 reported so far. Used in cardiomyocyte differentiation from human embryonic stem (ES) and induced pluripotent stem (iPS) cells. CHIR99021 maintains undifferentiated mouse ES cells in combination with PD0325901, in the absence of LIF. CHIR99021 maintains human and mouse hematopoietic stem cells in cytokine-free conditions, in combination with rapamycin. CHIR99021 enables chemical reprogramming (without genetic factors) of mouse embryonic fibroblasts to iPS cells, in combination with Forskolin, Tranylcypromine, Valproic Acid, 3-Deazaneplanocin A, and E-616452. Generates mouse-like or “ground state” iPS cells from human and rat somatic cells, in combination with PD0325901 and A83-01.

SP600125, at preferred concentration of 0.01 to 1000 μM, more preferred 0.1 to 100 μM, most preferred 10 μM. SP600125 is a potent, cell-permeable, selective and reversible inhibitor of c-Jun N-terminal kinase (JNK). It inhibits in a dose-dependent manner the phosphorylation of JNK. JNK is a member of the mitogen-activated protein kinase (MAPK) family and plays an essential role in TLR mediated inflammatory responses. Inhibition of JNK activity by SP600125 is usually associated with downregulation of Beclin-1 and reduced autophagy.

SB 202190, at preferred concentration of 0.01 to 1000 μM, more preferred 0.1 to 50 μM, most preferred 5 μM. SB 202190 is a potent, reversible, competitive, and cell-permeable inhibitor of p38 MAP kinase.

Go6983, at preferred concentration of 0.01 to 1000 μM, more preferred 0.1 to 50 μM, most preferred 1 to 10 μM, or 5 μM. Go6983 is a PKC inhibitor and has been shown to selectively inhibit several PKC isoenzymes (IC50=7 nM for PKCα and PKCβ; 6 nM for PKCγ; 10 nM for PKCδ; 60 nM for PKCζ). The compound does not effectively inhibit PKCμ (IC50=20 μM) and therefore can be used to differentiate PKCμ from other isoforms.

ROCK inhibitor Y-27632, at preferred concentration of 0.01 to 1000 μM, more preferred 0.1 to 100 μM, most preferred 10 μM. Y-27632 is a cell-permeable, highly potent and selective inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK). Y-27632 inhibits both ROCKI (Ki=220 nM) and ROCKII (Ki=300 nM) by competing with ATP for binding to the catalytic site. It enhances survival of human embryonic stem (ES) cells when they are dissociated to single cells by preventing dissociation-induced apoptosis (anoikis), thus increasing their cloning efficiency. Improves embryoid body formation using forced-aggregation protocols. Increases the survival of cryopreserved single human ES cells after thawing.

BIO, at preferred concentration of 0.001 to 1000 μM, more preferred 0.05 to 0.1 μM, most preferred 2 μM. 6-bromoindirubin-3-oxime (BIO) is a potent, reversible and ATP-competitive GSK-3α/β inhibitor and the first pharmacological agent shown to maintain self-renewal in human and mouse embryonic stem cells. Human embryonic stem cells (hESCs) are maintained in the undifferentiated state through treatment with a GSK-3 inhibitor, BIO, under a feeder-free condition.

Dorsomorphin, at preferred concentration of 0.01 to 1000 μM, more preferred 0.1 to 100 μM, most preferred 2 μM. Dorsomorphin is a selective inhibitor of Bone morphogenetic protein (BMP) signaling. It has been found to inhibit BMP signals required for embryogenesis and promoted significant neural differentiation from human pluripotent stem cell (hPSC) lines. Dorsomorphin also acts as a potent, selective, reversible, and ATP-competitive inhibitor of AMPK (AMP-activated protein kinase); Ki=109 nM in the presence of 5 μM ATP and the absence of AMP).

Sodium butyrate, at preferred concentration of 0.01 to 100 mM, more preferred 0.1 to 10 mM, most preferred 0.1 mM. Sodium butyrate is a compound with formula Na(C3H7COO). It is the sodium salt of butyric acid. It has various effects on cultured mammalian cells including inhibition of proliferation, induction of differentiation and induction or repression of gene expression. As such, it can be used in lab to bring about any of these effects. Specifically, butyrate treatment of cells results in histone hyperacetylation, and butyrate itself inhibits HDAC activity. Butyrate has been an essential vehicle for determining the role of histone acetylation in chromatin structure and function. Inhibition of HDAC activity is estimated to affect the expression of only 2% of mammalian genes.

SAHA, at preferred concentration of 0.01 to 1000 nM, more preferred 0.1 to 100 nM, most preferred 50 nM. SAHA or Vorinostat facilitates the transcription of genes that result in apoptosis, differentiation and growth arrest. It has been observed to give beneficial results in lymphoma but not in solid tumors. Vorinostat or suberoylanilide hydroxamic acid (SAHA) is a potent, reversible pan-histone deacetylase (HDAC) inhibitor. It inhibits both class I and class II HDACs, altering gene transcription and inducing cell cycle arrest and/or apoptosis in a wide variety of transformed cells.

SB590885, at preferred concentration of 0.01 to 100 μM, more preferred 0.1 to 10 μM, most preferred 0.5 μM. SB-590885 is a potent and selective ATP competitive inhibitor of B-Raf kinase with Kd=300 pM for B-Raf, and >1000-fold selectivity over a panel of 22 commonly studied cellular kinases

WH-4-023, at preferred concentration of 0.01 to 1000 μM, more preferred 0.1 to 100 μM, most preferred 1 μM. WH-4-023 is a potent and selective dual Lck/Src inhibitor with IC50 of 2 nM/6 nM for Lck and Src kinase respectively; shows little inhibition on p38α and KDR.

IM-12, at preferred concentration of 0.01 to 1000 μM, more preferred 0.1 to 100 μM, most preferred 1 μM. IM-12 is a selective GSK-3β inhibitor with IC50 of 53 nM, and also enhances canonical Wnt signalling.

Pluripotin, at preferred concentration of 0.01 to 1000 μM, more preferred 0.1 to 100 μM, most preferred 2 μM. Pluripotin is an activator of murine embryonic stem (ES) cell self-renewal. It appears that pluripotin mediates the activity by dual RasGAP and ERK1 inhibition.

FR 180204, at preferred concentration of 0.01 to 1000 μM, more preferred 0.1 to 100 μM, most preferred 1 to 10 μM. FR180204 is a potent, cell-permeable, ATP-competitive inhibitor of ERK1 and ERK2 (mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinases (ERK) 1/2).

BIX 01294, at preferred concentration of 0.01 to 1000 μM, more preferred 0.1 to 100 μM, most preferred 1 to 10 μM. BIX-01294, a diazepin-quinazolinamine derivative, is a histone-lysine methyltransferase (HMTase) inhibitor that modulates the epigenetic status of chromatin. BIX-01294 inhibits the G9aHMTase dependent levels of histone-3 lysine (9) methylation (H3K9me). Bix-01294 and valproic acid, a histone deacetylase (HDAC) inhibitor, may replace the requirement for ectopic OCT4 (POU5F1) and cMyc respectively in pluripotent stem cell induction (iPS) recipes. BIX 01294 is a selective histone methyl transferase inhibitor. In its inhibition of the histone lysine methyltransferases, BIX 01294 does not compete with cofactor S-adenosylmethionine. The target enzyme is G9a, and it selectively impairs G9a HMTase and the generation of H3K9me2 in vitro.

Decitabine, at preferred concentration of 0.01 to 1000 μM, more preferred 0.1 to 100 μM, most preferred 0.5 to 10 μM. Decitabine (trade name Dacogen), or 5-aza-2′-deoxycytidine, is a drug for the treatment of myelodysplastic syndromes, a class of conditions where certain blood cells are dysfunctional, and for acute myeloid leukemia (AML). Chemically, it is a cytidine analog. Decitabine is a hypomethylating agent. It hypomethylates DNA by inhibiting DNA methyltransferase. It functions in a similar manner to azacitidine, although decitabine can only be incorporated into DNA strands while azacitidine can be incorporated into both DNA and RNA chains.

Chaetocin, at preferred concentration of 0.01 to 1000 μM, more preferred 0.1 to 100 μM, most preferred 1 to 10 μM. Chaetocin is a fungal metabolite with antimicrobial and cytostatic activity. It belongs to the 3,6-epidithio-diketopiperazines class of which gliotoxin, sporidesmin, aranotin, oryzachloride, verticillin A and the melinacidins are members.1,3 Chaetocin is a molecular dimer of two five-membered rings cis fused. Interestingly, the chirality of the 3,6-epidithio-diketopiperazine moiety in chaetocin is opposite to the chirality in gliotoxin, sporidesmin, aranotin and oryzachloride and while the later compounds show antiviral activity, chaetocin does not. This fungal toxin showed strong cytotoxicity against HeLa cells (IC50=0.05 μg/ml). Chaetocin was found to be a specific inhibitor of the lysine-specific histone methyltransferase SU(VAR)3-9 (IC50=0.6 μM) ofDrosophila melanogasterand of its human ortholog (IC50=0.8 μM), and acts as a competitive inhibitor for S-adenosylmethionine.

XAV939 at a preferred concentration of 0.1 μM to 100 μM, preferably 1 to 10 μM. XAV939 antagonizes Wnt signaling via stimulation of β-catenin degradation and stabilization of axin. Inhibits proliferation of the β-catenin-dependent colon carcinoma cell line DLD-1. It promotes cardiomyogenic development in mesoderm progenitor cells.

DAPT at a preferred concentration of 0.11 to 100 μM, preferably 1 to 50, more preferably 2 to 10 μM. DAPT is a γ-secretase inhibitor and indirectly an inhibitor of Notch, a γ-secretase substrate. DAPT has been shown to inhibit Notch signaling in studies of autoimmune and lymphoproliferative diseases, such as ALPS and lupus erythematosus (SLE), as well as in cancer cell growth.

HPI1 at a preferred concentration of 0.01 to 500 μM, preferably 0.1 to 50 μM, more preferably 1 to 5 μM. HPI1 relates to Hedgehog (Hh) signaling inhibitor. Inhibits Sonic hedgehog (Shh)-, SAG- and Gli-induced Hh pathway activation in Shh-LIGHT2 cells. It does not inhibit Wnt signaling.

TCS-JNK-60 at a preferred concentration of 0.01 to 500 μM, preferably 0.1 to 50 μM, more preferably 0.5 to 5 μM. TCS-JNK-60 is an ATP-competitive c-Jun N-terminal kinase (JNK) inhibitor. It inhibits c-Jun phosphorylation and prevents collagen-induced platelet aggregation in vitro.

K02288 at a preferred concentration of 0.01 to 500 μM, preferably 0.1 to 50 μM, more preferably 0.5 to 5 μM. K02288 is a potent and selective inhibitor of type I bone morphogenic protein (BMP) receptors.

TABLE 1Summary of potential inhibitors for the medium of the invention:PreferredAmountVolumeStockFinal.InhibitorSourceCat. No.W.M.(mg)(μl)SolventCon.Con.PD0325901AxonAxon 1408482.1951036.9DMSO10mM1μMMedchemCHIR99021Axon1386465.3521432.6DMSO3mM1-3μMMedchemSP600125Tocris1496220.2310908.1DMSO50mM10μMSB 202190AxonAxon 1364331.35101207.2DMSO25mM5μMMedchemGo6983Tocris2285447.01101656.7DMSO10mM5μMROCKIMillipore688000338.3102956H2O10mM10μMBIOSigmaB1686-5MG356.1751403.8DMSO10mM2μMDorsomorphinSigmaP5499-5MG399.4951251.6DMSO10mM2μMK02288BMPTocris4986352.3810 mgDMSO10mM2μMSodiumSigmaB5887110.12502270.7H2O1000mM0.1mMbutyrateSAHACayman10009929264.3100015000DMSO250mM50nMSB590885Tocris2650453.54104410DMSO5mM0.5μMWH-4-023AH620061568.67101758DMSO10mM1μMChemtek,IM-12EnzoBML-WN102-377.451324DMSO10mM1μM0005PluripotinToris4433550.545908DMSO10mM2μMFR 180204Toris3706327.3451527DMSO10mM1-10μMGDC-0994SelleckechemS7554439.855 mg1137 μlDMSO10mM2μMSCH772984SelleckechemS7101587.675 mg851 μlDMSO10mM0.5-1μMBIX 01294Toris3364600.025833DMSO10mM1-10μMDecitabineToris2624228.2152191DMSO10mM0.5-10μMChaetocinToris4504696.845718DMSO10mM1-10μM

Furthermore, the medium of the invention may comprise one or more cytokines. The cytokines may be adjusted or optimized according to expression of the LTR7/HERVH nucleic acid sequences as described herein.

TABLE 2Summary of potential cytokines for the medium of the invention:CytokinesBrand/companyCat. No.Final. Con.LIFMilliporeLIF101010 ng/μlbFGFPeproTechAF-100-18B10 ng/μlIL11PeproTech200-1110 ng/μlsIL6RPeproTech20 ng/μlIL6PeproTechAF-200-0620 ng/μl

The preferred LTR7/HERVH sequences used in the present invention are the following:

Reporter sequences:1) LTR7_long version (human; corresponds toLTR7#2; SEQ ID NO 1):ATGCTGCGAGATGGGAAACACATACAAAATCTTCAACCTTCAGTAAGTAAAAACCTTCTCTATTAAAATCTGCAAAGTGTATTCATTTGTTCTAAAATTATTTGCTAAGTGCCCACACAGCACTAGGAATGAAACATAAAAAAATCTCTTCCCTCACTTAGCTTCGTATTCTCTTTGGGAATGTCAGGCCTCTGAGCCCAAGCCAAGCCATCGCATCCCCTATGACATGCACGTACACGCCCAGATGGCCTGAAGTAACTGAAGAATCACAAAAGAAGTGAATATGCCCTGCCCCACCTTAACTGATGACATTCCACCACAAAAGAAGTGTAAATGGCCAGTCCTTGCCTTAACTGATGACATTACCTTGTGAAAGTCCTTTTCCTGGCTCATCCTGGCTCAAAAAGCACCCCCACTGAGCACCTTGCGACCCCCCGCTCCTACCCGCCAGAGAACAAACCCCCTTTGACTGTAATTTTCCTTTACCTAACCAAATCCTATAAAACGGCCCCACCCTTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAGCCCGCCTGCACCCAGGTGAAATAAACAGCCTCGTTGCTCACACAAAGCCTGTTTGGTGGTCTCTTCACACGGACGCGCATGAAATTTGGTGCCGTGACTCGGATCGGGGGACCTCCCTTGGGAGATCAATCCCCTGTCCTCCTGCTCTTTGCTCCGTGAGAAAGATCCACCTACGACCTCAGGTCCTCAGACCAACCAGCCCAAGAAACATCTCACCAATTTCAAATCCGGTAAGCGGCCTCTTTTTACTCTGTTCTCCAACCTCCCTCACTATCCCTCAACCTCTTTCTCCTTTCAATCTTGGCGCCACACTTCAATCTCTCCCTTCTCTTAATTTCAATTCCTTTCATTCTCTGGTAGAGACAAAAGAGACATGTTTTATCCGTGAACCCAAAACTCCGGCGCCGGTCACGGACTGGGAAGGCAGTCTTCCCTTGGTGTTTAATCATTGCAGGGACGCCTCTCTGATTTCACGTTTCAGACCACGCAGGGATGCCTGCCTTGGTCCTTCACCCTTAGCGGCAAGTCCCGCTTTCCTGGGGCAGGGGCAAGTACCCCTCAACCCCTTCTCCTTCACCCTTAGCGGCAAGTCCCGCTTTTCTGGGGCAGGGGCAAGTACCCCTCAACCCCTTCTCCTTCACCC2) LTR7_short version (human; corresponds toLTR7#1; SEQ ID NO 2):TGCTAAGTGCCCACACAGCACTAGGAATGAAACATAAAAAAATCTCTTCCCTCACTTAGCTTCGTATTCTCTTTGGGAATGTCAGGCCTCTGAGCCCAAGCCAAGCCATCGCATCCCCTATGACATGCACGTACACGCCCAGATGGCCTGAAGTAACTGAAGAATCACAAAAGAAGTGAATATGCCCTGCCCCACCTTAACTGATGACATTCCACCACAAAAGAAGTGTAAATGGCCAGTCCTTGCCTTAACTGATGACATTACCTTGTGAAAGTCCTTTTCCTGGCTCATCCTGGCTCAAAAAGCACCCCCACTGAGCACCTTGCGACCCCCCGCTCCTACCCGCCAGAGAACAAACCCCCTTTGACTGTAATTTTCCTTTACCTAACCAAATCCTATAAAACGGCCCCACCCTTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAGCCCGCCTGCACCCAGGTGAAATAAACAGCCTCGTTGCTCACACAAAGCCTGTTTGGTGGTCTCTTCACACGGACGCGCATGAAATTTGGTGCCGTGACTCGGATCGGGGGACCTCCC3) LTR7Y: (human; corresponds to a variant ofLTR7. Preliminary data suggest that in addition toconstructs #1 and #2 it would be useful tooptimize culture conditions; SEQ ID NO 3):TGTCAGGCCTCTGAGCCCAGGCCAGGCCATCGCATCCCCTGTGACTTGCACGTATACATCCAGATGGCCTGAAGTAACTGAAGATCCACAAAAGAAGTAAAAACAGCCTTAACTGATGACATTCCACCATTGTGATTTGTTCCTGCCCCACCCTAACTGATCAATGTACTTTGCAATCTCCCCCACCCTTAAGAAGGTTCTTTGTAATTCTCCCCACCCTTGAGAATGTACTTTGTGAGATCCACCCCTGCCCACCAGAGAACAACCCCCTTTGACTGTAATTTTCCATTACCTTCCCAAATCCTATAAAACGGCCCCACCCCTATCTCCCTTCGCTGACTCTCTTTTCGGACTCAGCCCGCCTGCACCCAGGTGAAATAAACAGCCATGTTGCTCACACAAAGCCTGTTTGGTGGTCTCTTCACACGGACGCGCATGAAA

The invention as described herein is not limited to the specific LTR7 sequences as disclosed above, but to functionally analogous sequences that exhibit essentially the same desired properties as shown for these particular examples. Sequence variants with a sequence identity of at least 70%, 75%, 80%, 85%, 90% or 95% to the specific sequences listed, in addition to complementary sequences, corresponding RNA or other nucleic acid sequences, or other derivatives, are also encompassed within the scope of the present invention. The determination of sequence identity can be carried out by a skilled person without undue effort, for example using sequence comparison tools such as BLAST or Clustal.

The sequences provided above relate to human LTR7 sequences. Analogous sequences, for example those derived from other primate species, are encompassed by the present invention.

Primates refer to placental mammals of the order Primates, typically having hands and feet with opposable digits, and a highly developed brain. Primates include, without limitation, humans, lemurs, lorises, monkeys and other apes, in particular humans (genusHomo), chimpanzees (genusPan), gorillas (genusGorilla), orangutans (subfamily Ponginae), gibbons (family Hylobatidae), Old World monkeys (superfamily Cercopithecoidea), New World monkeys (parvorder Platyrrhini), tarsiers (superfamily Tarsioidea), lemurs (superfamily Lemuroidea), lorises (superfamily Lorisoidea).

Definition of Naïve Pluripotent Stem Cells:

Murine naive ESCs have a series of unusual properties: both X chromosomes are active, they form 3D rounded clusters, resembling a E4.5 epiblast of preimplantation blastocyst1, and they don't expresses genes typical of differentiated cells. Human characteristics may however differ.

The GFPhighcell line that the inventors have established shows the above features. In the 2i/LIF condition the GFPhighcells stably maintain naive-like morphology for a good time (followed for Passage 20, over 100 days, and ongoing) (for passage 9 seeFIGS. 4a, 12a-d). In an analysis of 5 culture media, for longer-term culture 3iL4medium proved beneficial (FIGS. 11e-h), but with room for optimization. Additional optimization of the medium has provided the 4i-medium, which is capable of maintaining the naïve state, as evidenced by LTR7 transcription, for long periods of time without the need for re-sorting.

This invention establishes that much of the circuitry regulating pluripotency in hPSCs is primate/human specific. This observation could explain why some currently identified human naïve-like cells2-6 are not identical to the murine state. Thus, we cannot expect the human naïve cells to have the same defining features as murine naïve cells7. More particularly, recent studies reveal that certain murine naïve phenotypes, including the absence of X inactivation8or 3D morphology (FIG. 11j) appear to be imperfect to characterize cultured human naïve cells.

Alternatively, naïvety may be defined by functionality. Behavior within a chimera is thought to be one of the most stringent functional assays. Consistent with this view, in contrast to EpiSCs, naïve mESCs can efficiently integrate into the ICM of blastocyst and generate normal chimeras, indicating their full developmental potential.

According to the present invention the expression profile of cells may be used to identify “naivety”, for example those cells that closely resemble cells of the inner cell mass (ICM) may be considered as a naïve or naïve-like PSC. To this end, the cells described herein, enriched using the HERVH reporter, are good representatives of naïve cells as they cluster nearest to ICM when compared with the ‘novel naïve’ cells obtained in reference 4 (FIG. 4e). As used herein, the term naïve pluripotent stem cell relates preferably to the LTR7-expressing naïve pluripotent stem cell as described in detail herein. These cells may be referred as “naïve pluripotent stem cell” due to the closeness of their expression profiles to cells of the ICM.

