Patent Application
20210363495
The ASCII file, entitled 88060SequenceListing.txt, created on Jul. 22, 2021, comprising 126,913 bytes, submitted concurrently with the filing of this application is incorporated herein by reference. The sequence listing submitted herewith is identical to the sequence listing forming part of the international application.
The present invention, in some embodiments thereof, relates to culture media for culturing pluripotent stem cells more particularly, but not exclusively, to naïve pluripotent stem cells.
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 naïve 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 naïve 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 naïve and possess features such as retention of pre-X inactivation state, ability to tolerate lack of repressors like Mett13, Dnmt1 and Dgcr8. However, they do not retain a global hypermethylated epigenome, and acquire H3K27me3 over developmental genes consistent with retaining a relatively less naïve state. Rodent EpiSCs expanded in Fgf2/Activin A show further consolidation and acquisition of their milieu of primed pluripotency characteristics, thus exemplifying how mouse naïve and primed PSCs can have different mix of naïve 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 naïve mESCs including expression of E-CADHERIN (rather than N-CADHERIN). Further, conventional human ESCs express high levels of PRDM14 and NANOG as murine naïve 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 naïve 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-naïve pluripotency in comparison to rodents.
Condition to derive naïve 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 WO2016/016894. 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.
Theunissen et al., 2014; Cell Stem Cell 1-17, 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 naïve 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 naïve pluripotent cell markers. However, the hypomethylation in these conditions is however accompanied by immediate and global deterministic loss of imprinting (Theunissen, 2016; Cell Stem Cell 1-49) and obligatory confounding chromosomal abnormalities in nearly 100% of the line generated by 10 passages only (Liu et al., 2017, Nat. Methods 14, 1-14).
Derivation of human naïve 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 2-week transfer to primed conditions, thus questioning their pluripotent functionality and stability (Guo et al., 2016, Stem Cell Reports 1-19; Liu et al., 2017, Nat. Methods 14, 1-14; Takashima et al., 2014, Cell 158, 1254-1269). 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 WO2014/174470.
According to an aspect of the present invention there is provided a culture medium comprising a WNT inhibitor, a 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 an aspect of the present invention there is provided a culture medium comprising a WNT inhibitor, a Notch inhibitor and a protein kinase C (PKC) inhibitor, said 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.
According to an aspect of the present invention there is provided a cell culture comprising cells and the culture medium disclosed herein.
According to an aspect of the present invention there is provided a method of expanding pluripotent stem cells (PSCs), comprising culturing the pluripotent stem cell in the culture medium disclosed herein, thereby culturing the pluripotent stem cells.
According to an aspect of the present invention there is provided a method of generating an induced pluripotent stem cell (iPSC) from a somatic cell, comprising:
(a) expressing within the somatic cell a first factor selected from the group consisting of Nanog, ESRRB, KLF17, TFAP2C, TBX3, ERAS and a second factor selected from the group consisting of Nanog, ESRRB, KLF17, TBX3, ERAS, Oct4, Sox2, Klf4, c-Myc, wherein the first and second factor are non-identical; and
(b) culturing the somatic cell in the culture medium of any one of claims 1-12 under conditions that promote the generation of an iPSC, thereby generating the iPSC from a somatic cell.
According to an aspect of the present invention there is provided a method of generating a naive pluripotent stem cell (PSC), comprising culturing a non-naive PSC cell in the culture medium disclosed herein, under conditions which allow generation of the naive PSC from the non-naive PSC, thereby generating the naive PSC.
According to embodiments of the present invention, the culture medium further comprises a STAT3 activator.
According to embodiments of the present invention, the culture medium further comprises a SRC inhibitor.
According to embodiments of the present invention, the culture medium further comprises an ERK inhibitor.
According to embodiments of the present invention, the culture medium further comprises at least one agent selected from the group consisting of a STAT3 activator, a SRC inhibitor and an ERK inhibitor.
According to embodiments of the present invention, the culture medium further comprises at least one agent selected from the group consisting of a STAT3 activator, an ERK inhibitor, a p38 inhibitor, a JNK inhibitor and a ROCK inhibitor.
According to embodiments of the present invention, the culture medium further comprises a STAT3 activator, an ERK inhibitor, a p38 inhibitor, a JNK inhibitor and a ROCK inhibitor.
According to embodiments of the present invention, the culture medium further comprises a Notch inhibitor.
According to embodiments of the present invention, the Notch inhibitor comprises a gamma secretase inhibitor and/or an RBPj inhibitor.
According to embodiments of the present invention, the culture medium further comprises a STAT3 activator, a p38 inhibitor and a ROCK inhibitor.
According to embodiments of the present invention, the medium is devoid of an amount of basic fibroblast growth factor (bFGF) that has a mitogenic activity on a pluripotent stem cell being cultured in the medium.
According to embodiments of the present invention, the medium is devoid of L-glutamine.
According to embodiments of the present invention, the culture medium further comprises Activin A.
According to embodiments of the present invention, the STAT3 activator is selected from the group consisting of leukemia inhibitory factor (LIF) and interleukin 6 (IL6).
According to embodiments of the present invention, the culture medium is devoid of animal serum.
According to embodiments of the present invention, the culture medium comprises serum replacement.
According to embodiments of the present invention, the cells are non-genetically modified.
According to embodiments of the present invention, the medium is capable of maintaining pluripotent stem cells in an undifferentiated state for at least 2 passages.
According to embodiments of the present invention, the cells comprise pluripotent stem cells.
According to embodiments of the present invention, the pluripotent stem cells comprise naïve pluripotent stem cells.
According to embodiments of the present invention, the pluripotent stem cells comprise primate or swine pluripotent stem cells.
According to embodiments of the present invention, the primate pluripotent stem cells comprise human pluripotent stem cells.
According to embodiments of the present invention, the pluripotent stem cells are not rodent pluripotent stem cells.
According to embodiments of the present invention, the culturing is effected on an adherent surface.
According to embodiments of the present invention, the culturing is effected in the absence of MEFs.
According to embodiments of the present invention, the adherent surface is selected from the group consisting of Matrigel™, Geltrex™, Biolaminin™, fibronectin and gelatin.
According to embodiments of the present invention, the pluripotent stem cell is non-genetically modified.
According to embodiments of the present invention, the pluripotent stem cell is a primate or swine pluripotent stem cell.
According to embodiments of the present invention, the primate pluripotent stem cell is a human pluripotent stem cell.
According to embodiments of the present invention, the pluripotent stem cell is not a rodent pluripotent stem cell.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Some embodiments of the invention 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 embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
In the drawings:
I. FACS analysis showing preservation of ΔPE-OCT4-GFP naïve marker expression in both WT and DGCR8 KO human ESCS expanded in ENHSM conditions.
E. Western blot analysis for validation of TFAP2C KO generation in primed human WIBR3-ΔPE-O4G hESCs.
Tile scale bar 500 μm. Zoomed-in scale bar 100 μm. Lung-E17.5-Frozen section-injected WT hiPSCs; 100 μm scale bars. Tile: scale bar 500 μm.
Tile scale bar 200 μm. Inset scale bar 50 μm.
Tile scale bar 1000 μm. Zoomed-in region scale bar 100 μm.
Tile scale bar 1000 μm. Zoomed-in region scale bar 100 μm.
Tile scale bar 1000 μm. Zoomed-in region scale bar 100 μm.
Wi=WNTi=TNKi (XAV939 3 μM)
Si=SRCi=CGP77675 1.2 μM
Pi=PKCi=Go6983 2 μM
Ni=NOTCHi=DBZ 0.3 μM
The present invention, in some embodiments thereof, relates to culture media for culturing pluripotent stem cells more particularly, but not exclusively, to naïve pluripotent stem cells.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments 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 WO2014/174470), the present combinations were shown to maintain the pluripotent stem cell in a hypomethylated state.
Thus, according to a first aspect of the present invention, 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 invention, 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 invention 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, N.Y., USA), DMEM/F12 (Gibco-Invitrogen Corporation products, Grand Island, N.Y., USA), Neurobasal medium (Invitrogen Corporation products, Grand Island, N.Y., USA 21103-049) or DMEM/F12 (without HEPES; Biological Industries, Biet Haemek, Israel), supplemented with the necessary additives as is further described hereinunder.
According to a particular embodiment, the medium is a 1:1 mix of Neurobasal medium and DMEM F/12.
Preferably, all ingredients included in the culture medium of the present invention are substantially pure, with a tissue culture grade.
According to some embodiments of the invention, the culture medium is devoid of serum, e.g., devoid of any animal serum.
According to some embodiments of the invention, 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 embodiments of the invention, the culture medium is devoid of human derived serum.
According to some embodiments of the invention, the culture medium further comprises a serum replacement (i.e., a substitute of serum) such as KNOCKOUT™ Serum Replacement (Gibco-Invitrogen Corporation, Grand Island, N.Y. USA), ALBUMAX®II (Gibco®; Life Technologies—Invitrogen, Catalogue No. 11021-029;
Lipid-rich bovine serum albumin for cell culture) or a chemically defined lipid concentrate (Gibco®; Invitrogen, Life Technologies—Invitrogen, Catalogue No. 11905-031).
According to some embodiments of the invention, the culture medium further comprises N2 supplement (Gibco®; Life Technologies—Invitrogen, Catalogue No. 17502-048) a chemically defined, serum-free supplement. For a 500 ml of culture medium 5 ml of the N2 mix (Invitrogen) can be added.
Alternatively, the following materials (substitute the N2 supplement) can be added to a 500 ml culture medium: Recombinant Insulin (Sigma 1-1882) at a 12.5 microg/ml (μg/ml) final concentration; Apo-Transferrin (Sigma T-1147) at a 500 μg/ml final concentration; Progesterone (Sigma-P8783) at a 0.02 μg/ml final concentration; Putrescine (Sigma-P5780) at a 16 μg/ml final concentration; and 5 microL (μ1) of 3 mM stock of Sodium Selenite (Sigma—S5261) are added per 500 ml culture medium (e.g., the WIS-NHSM).
According to some embodiments of the invention, the KNOCKOUT™ Serum Replacement is provided at a concentration of at least 0.5%, e.g., in the range of about 0.5%-25%, e.g., about 5%, about 10%, about 15%, about 20% or about 25%.
According to some embodiments of the invention, the ALBUMAX™ is provided at a concentration of at least 0.01%, e.g., in the range of about 0.01%-10%, e.g., about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9% or about 10%, e.g., 1%.
According to some embodiments of the invention, the defined lipid concentrate is provided at a concentration of at least about 0.1%, e.g., in the range of 0.1-5%, e.g., about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, e.g., 1%.
According to some embodiments of the invention, the culture medium comprises the N2 supplement (e.g., 5 ml N2 per 500 ml of culture medium) and the defined lipid concentrate (5 ml defined lipid concentrate per 500 ml medium).
According to some embodiments of the invention, 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 1/10th of the amount that is typically present in base media such that it does not bring about a biological effect). In one embodiment, 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 embodiment, they are capable of binding the named target with a higher affinity (at least 10%, 20%, 30%, 40% 50%, 60%, 70%, 80%, 90% or even 100% higher affinity) than another protein which is expressed in the cell.
As mentioned, the media of the present invention 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-1 (WIF-1), 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 embodiment, the Wnt inhibitor is a small molecule.
In one embodiment, 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-001, IWR-1, IWP2, XAV939, Wnt-059 (C59), IWP-L6, iCRT3, LF3, PNU-74654, KYA1797K, PRI-724 and WIKI 4, all of which are commercially available from Selleckchem and/or Tocris.
According to a particular embodiment, the Wnt inhibitor is a Tankyrase inhibitor (e.g. IWR-1—Sigma Aldrich 10161; and XAV939—TOCRIS Cat. No. 3748). In one embodiment, 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 invention, 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 invention should not contain more than 0.5 μM of a GSK3β inhibitor and preferably not more than 0.1 μM 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 0.1 μM-100 μM, more preferably between 1 μM-100 μM, 0.1 μM-10 μM, and more preferably between 1 μM-10 μM-about 3 μM.
As mentioned, a NOTCH signaling inhibitor is contemplated to be included in the media of the present invention. Preferably, the NOTCH signaling inhibitor is added when the medium comprises less than 0.5 μM, for example about 0.4 μM, 0.3 μM, 0.2 μM or 0.1 μM ERK1/2 inhibitor. In a particular embodiment, the NOTCH signaling inhibitor is added to a medium which is devoid of an ERK1/2 inhibitor.
NOTCH signaling inhibitors include, but are not limited to the following gamma secretase inhibitors: DAPT (Axon Medchem 1484—0.05-50 μM final concentration), LY2886721 hydrochloride (Axon Medchem 1964—0.05-50 μM final concentration)], DBZ (Axon Medchem—Axon 1488-0.05-50 μM final concentration).
According to a particular embodiment, 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 Hurtado et al., Scientific Reports, Volume 9, Article number: 10811 (2019). An exemplary concentration of RIN1 is 0.1-10 μM and more preferably between 0.1 and 1 μM.
The medium of this aspect of the present invention 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 3 (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, 4-[(2,4-Dichloro-5-methoxyphenyl)amino]-7-[3-(4-ethyl-1-piperazinyl)propo-xy]-6-methoxy-3-quinolinecarbonitrile; 4-[(2,4-Dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[2-(4-methyl-1-pipera-zinyl)ethoxy]-3-quinolinecarbonitrile; 4-[(2,4-Dichloro-5-methoxyphenyl)amino]-7-[2-(4-ethyl-1-piperazinyl)ethox-y]-6-methoxy-3-quinolinecarbonitrile; 4-[(2,4-Dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]-3-quinolinecarbonitrile; 4-[(2,4-Dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[2-(1-methylpiperidin-4-yl)ethoxy]-3-quinolinecarbonitrile; 4-[(2,4-Dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(1-methylpiperidin-4-yl)propoxy]quinoline-3-carbonitrile; 4-[(2,4-Dichloro-5-methoxyphenyl)amino]-7-[(1-ethylpiperidin-4-yl)methoxy-]-6-methoxyquinoline-3-carbonitrile; 4-[(2,4-Dichloro-5-methoxyphenyl)amino]-6-ethoxy-7-[3-(4-methylpiperazin-1-yl)propoxy]quinoline-3-carbonitrile; 4-[(2,4-Dichloro-5-methoxyphenyl)amino]-6-ethoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinoline-3-carbonitrile; 4-[(2,4-Dichloro-5-methoxyphenyl)amino]-6-ethoxy-7-[3-(4-ethylpiperazin-1-yl)propoxy]quinoline-3-carbonitrile; 4-[(2,4-Dichloro-5-methoxyphenyl)amino]-6-ethoxy-7-[3-(1-methylpiperidin-4-yl)propoxy]quinoline-3-carbonitrile; 4-[(2,4-Dichloro-5-methoxyphenyl)amino]-6-ethoxy-7-[2-(4-methyl-1-piperaz-inyl)ethoxy]quinoline-3-carbonitrile; 4-[(2,4-Dichloro-5-methoxyphenyl)amino]-6-ethoxy-7-[2-(1-methylpiperidin-4-yl)ethoxy]quinoline-3-carbonitrile; or 4-[(2,4-Dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-propyl-1-pipera-zinyl)propoxy]-3-quinolinecarbonitrile; and pharmaceutically acceptable salts thereof.
According to a particular embodiment, the agent which possesses inhibitory activity against the Src family kinase is a small molecule agent.
According to a particular embodiment, 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 International Patent Applications WO 01/94341, WO 02/16352, WO 02/30924, WO 02/30926, WO 02/34744, WO 02/085895, WO 02/092577 (arising from PCT/GB 02/02117), WO 02/092578 (arising from PCT/GB 02/02124) and WO 02/092579 (arising from PCT/GB 02/02128), the quinoline derivatives described in WO 03/008409 (arising from PCT/GB 02/03177), WO 03/047584 and WO 03/048159 and the quinazoline derivatives described in European Patent Applications 02292736.2 (filed 4 Nov. 2002) and 03290900.4 (filed 10 Apr. 2003).
It is disclosed in Journal Medicinal Chemistry, 2001, 44, 822-833 and 3965-3977 that certain 4-anilino-3-cyanoquinoline derivatives are useful for the inhibition of Src-dependent cell proliferation. The 4-anilino-3-cyanoquinoline Src inhibitor known as SKI 606 is described in Cancer Research, 2003, 63, 375.
Other compounds which possess Src kinase inhibitory properties are described in, for example, International Patent Applications WO 96/10028, WO 97/07131, WO 97/08193, WO 97/16452, WO 97/28161, WO 97/32879 and WO 97/49706.
Other compounds which possess Src kinase inhibitory properties are described in, for example, J Bone Mineral Research, 1999, 14 (Suppl. 1), 5487, Molecular Cell, 1999, 3, 639-647, Journal Medicinal Chemistry, 1997, 40, 2296-2303, Journal Medicinal Chemistry, 1998, 41, 3276-3292 and Bioorganic & Medicinal Chemistry Letters, 2002, 12, 1361 and 3153.
Particular Src kinase inhibitors include the following:
(i) 4-amino-5-(3-methoxyphenyl)-7-{(4-[2-(2-methoxyethylamino)ethox-y]phenyl)-}-pyrrolo[2,3-d]pyrimidine and 4-amino-5-(3-methoxyphenyl)-7-(4-{(2-[di-(2-methoxyethyl)amino]ethoxy}phe-nyl)pyrrolo[2,3-d]pyrimidine which are obtainable by methods described in International Patent Application WO 96/10028:
(ii) 4-amino-7-tert-butyl-5-(4-tolyl)pyrazolo[3,4-d]pyrimidine which is also known as PP1 and is described in Molecular Cell, 1999, 3, 639-648;
(iii) 2-(2,6-dichloroanilino)-6,7-dimethyl-1,8-dihydroimidazo[4,5-h]isoquinolin-9-one and 2-(2,6-dichloroanilino)-7-[(E)-3-diethylaminoprop-1-enyl]-6-met-hyl-1,8-dihydroimidazo[4,5-h]isoquinolin-9-one which are obtainable by methods described in Journal Medicinal Chemistry, 2002, 45, 3394;
(iv) 1-[6-(2,6-dichlorophenyl)-2-(4-diethylaminobutyl)pyrido[2,3-d]pyrimidin-7-yl]-3-ethylurea which is obtainable by methods described in Journal Medicinal Chemistry, 1997, 40, 2296-2303 and Journal Medicinal Chemistry, 2001, 44, 1915;
(v) 6-(2,6-dichlorophenyl)-2-[4-(2-diethylaminoethoxy)anilino]-8-me-thyl-8H-pyrido[2,3-d]pyrimidin-7-one which is also known as PD166285 and is described in J. Pharmacol. Exp. Ther., 1997, 283, 1433-1444;
(vi) the compound known as PD 162531 which is described in Mol. Biol. Cell, 2000, 11, 51-64;
(vii) the compound known as PD166326 which is described in Biochem Pharmacol., 2000, 60, 885-898; and
(viii) the compound known as PD173955 which is described in Cancer Research, 1999, 59, 6145-6152.
Other compounds which may possess Src kinase inhibitory properties are described in, for example, International Patent Applications WO 02/079192, WO 03/000188, WO 03/000266, WO 03/000705, WO 02/083668, WO 02/092573, WO 03/004492, WO 00/49018, WO 03/013541, WO 01/00207, WO 01/00213 and WO 01/00214.
Particular Src inhibitors include those provided in International Patent Application WO 01/94341.
Further particular Src inhibitors include the following compounds from International Patent Application WO 02/16352, WO 02/30924, WO 02/30926 and WO 02/34744.
Exemplary agents include, without limitation, dasatinib, and AZD0530.
Other exemplary agents include CGP77675 (AXON MEDCHEM 2097), SU 6656, AZD0530, Dasatinib, Bosutinib and WH-4-023.
According to some embodiments of the invention, the Src family kinase inhibitor (e.g. CGP77675) is provided at a concentration range of between about 0.1-70 μM, e.g., from about 0.2 μM to about 70 μM, e.g., between about 0.2-60 μM, e.g., between about 0.2-55 μM, e.g., between about 0.2-50 μM, e.g., between about 0.2-45 μM, e.g., between about 0.2-40 μM, e.g., between about 0.2-35 μM, e.g., between about 0.2-30 μM, e.g., between about 0.2-25 μM, e.g., between about 0.2-20 μM, e.g., between about 0.2-15 μM, e.g., between about 0.2-10 μM, e.g., between about 0.3-10 μM, e.g., between about 0.4-10 μM, e.g., between about 0.5-10 μM, e.g., between about 0.6-10 μM, e.g., between about 0.7-10 μM, e.g., between 0.8-10 μM, e.g., between 0.9-10 μM, e.g., between 0.9-9 μM, e.g., between 1-8 μM, e.g., between 1-7 μM, e.g., between 1-6 μM, e.g., between 1-5 μM, e.g., about 1-3 μM, e.g., about 1.5 μM.
Since SRC inhibition leads to NFKβ 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-23 and LY 294002. Exemplary concentrations the NFKβ inhibitors may be used is between 0.1-10 μM.
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 embodiment, the PKC inhibitor is a small molecule inhibitor.
A non-limiting example of a protein kinase C inhibitor is Go6983 (CAS 133053-19-7), 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 7, 7, 6, 10, 60 and 20000 nM for PKCα, PKCβ, PKCγ, PKCγ, PKCζ and PKCμ respectively). Go6983 is available from various suppliers such as Calbiochem (Catalogue number 365251-500UG), and TOCRIS (Catalogue number 2285).
According to some embodiments of the invention, Go6983 is provided at a concentration range of between about 0.1-100 μM, e.g., from about 0.5 μM to about 100 μM, e.g., between about 0.5-50 μM, 0.5-25 μM, e.g., between about 1-20 μM, e.g., between about 1-10 μM, e.g., between about 1-5 μM, e.g., about 2 μM.
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 “STAT3” refers to the signal transducer and activator of transcription 3 gene product (acute-phase response factor) (Gene ID 6774). 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 5 (IL5), interleukin 6 (IL6), hepatocyte growth factor (HGF), leukemia inhibitory factor (LIF) and bone morphogenetic protein 2 (BMP2).
According to some embodiments of the invention, the STAT3 activator, which is used in the medium of some embodiments of the invention is selected from the group consisting of LIF, IL6 and EGF.
According to some embodiments of the invention, the STAT3 activator, which is used in the medium of some embodiments of the invention is selected from the group consisting of LIF and IL6.
According to some embodiments of the invention, the STAT3 activator, which is used in the medium of some embodiments of the invention 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_001244064.1 (SEQ ID NO: 11), encoded by the nucleotide sequence set forth in GenBank Accession No. NM_001257135 (SEQ ID NO: 12). Preferably, the LIF used by the method according to some embodiments of the invention 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 embodiments of the invention, LIF is provided at a concentration range from about 0.5 nanogram per milliliter (ng/ml) to about 1000 ng/ml, e.g., about 1-1000 ng/ml, e.g., about 1-900 ng/ml, e.g., about 1-800 ng/ml, e.g., about 1-700 ng/ml, e.g., about 1-600 ng/ml, e.g., about 1-500 ng/ml, e.g., about 1-400 ng/ml, e.g., about 1-300 ng/ml, e.g., about 1-200 ng/ml, e.g., about 1-100 ng/ml, e.g., about 1-50 ng/ml, e.g., about 2-50 ng/ml, e.g., about 4-50 ng/ml, e.g., about 5-50 ng/ml, e.g., about 10-50 ng/ml, e.g., about 10-40 ng/ml, e.g., about 10-30 ng/ml, e.g., about 20 ng/ml.
As used herein the term “interleukin 6 (IL6)” refers to a polypeptide which comprises the amino acid sequence set forth by GenBank Accession No. NP_000591.1 (SEQ ID NO: 13), which is encoded by the nucleic acid set forth by GenBank Accession No. NM_000600.3 (SEQ ID NO: 14). Preferably, the IL6 used by the method according to some embodiments of the invention 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. IL6 can be obtained from various manufacturers such as Speed BioSystems, Millipore, Peprotech, and R&D systems.
According to some embodiments of the invention, IL6 is provided at a concentration range from about 0.1 ng/ml to about 100 ng/ml, e.g., about 0.1-90 ng/ml, e.g., about 0.1-80 ng/ml, e.g., about 0.1-70 ng/ml, e.g., about 0.1-50 ng/ml, e.g., about 0.1-40 ng/ml, e.g., about 0.1-30 ng/ml, e.g., about 0.1-20 ng/ml, e.g., about 0.1-10 ng/ml, e.g., about 0.1-8 ng/ml, e.g., about 0.1-7 ng/ml, e.g., about 0.1-6 ng/ml, e.g., about 0.1-5 ng/ml, e.g., about 0.1-4 ng/ml, e.g., about 0.1-3 ng/ml, e.g., about 0.1-4 ng/ml, e.g., about 0.5-4 ng/ml, e.g., about 0.5-4 ng/ml, e.g., about 3 ng/ml.
As used herein the term “p38” refers to the “p38α (alpha)” mitogen-activated protein kinase 14 (MAPK14), which includes MAPK14 isoform 1 set forth by GenBank Accession No. NP_001306.1 (SEQ ID NO: 15), MAPK14 isoform 2 set forth by GenBank Accession No. NP_620581.1 (SEQ ID NO: 16), MAPK14 isoform 3 set forth by GenBank Accession No. NP_620582.1 (SEQ ID NO: 17) and MAPK14 isoform 4 set forth by GenBank Accession No. NP_620583.1 (SEQ ID NO: 18); “p38β (beta)” (MAPK11), which is set forth by GenBank Accession No. NP_002742.3 (SEQ ID NO:19); “p38γ (gamma)” (MAPK12) which is set forth by GenBank Accession No. NP_002960.2 (SEQ ID NO: 20); and/or “p38δ (delta)” (MAPK13) which is set forth in GenBank Accession No. NP_002745.1 (SEQ ID NO: 21), 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 1363), and SB 202190 (AXONMEDCHEM—Axon 1364), LY 2228820 (AXONMEDCHEM—Axon 1895), BIRB0796 (Axon Medchem 1358) and PD169316 (AXONMEDCHEM—Axon 1365).
As BMP signaling is an activator for p38 signaling, examples of p38 inhibitors also include BMP inhibitors like Dorsomorphin (AXONMEDCHEM—Axon 2150) and LDN193189 (AXON MEDCHEM AXON 1509) or other inhibitors of the BMP pathway such as recombinant NOGGIN protein [GenBank Accession No. NP_005441.1 (SEQ ID NO: 22] can be used to replace small molecule inhibitors of BMP signaling.
According to some embodiments of the invention, SB203580 is provided at a concentration range of between about 0.5-70 μM, e.g., from about 1 μM to about 70 μM, e.g., between about 1-60 μM, e.g., between about 1-55 μM, e.g., between about 1-50 μM, e.g., between about 1-45 μM, e.g., between about 1-40 μM, e.g., between about 1-35 μM, e.g., between about 1-30 μM, e.g., between about 1-25 μM, e.g., between about 1-20 μM, e.g., between about 1-15 μM, e.g., between about 1-10 μM, e.g., between about 2-10 μM, e.g., between about 3-10 μM, e.g., between about 4-10 μM, e.g., between about 4-6 μM, e.g., about 5 μM, e.g., about 10 μM.
According to some embodiments of the invention, SB 202190 is provided at a concentration range of between about 0.1 μM to about 50 μM, e.g., from about 0.5 μM to about 50 μM, e.g., from about 1 μM to about 50 μM, e.g., between about 1-45 μM, e.g., between about 1-40 μM, e.g., between about 1-35 μM, e.g., between about 1-30 μM, e.g., between about 1-25 μM, e.g., between about 1-20 μM, e.g., between about 1-15 μM, e.g., between about 1-10 μM, e.g., between about 1-9 μM, e.g., between about 1-8 μM, e.g., between about 1-7 μM, e.g., between about 2-7 μM, e.g., between about 3-7 μM, e.g., between about 4-7 μM, e.g., between about 4-6 μM, e.g., about 5 μM.
According to some embodiments of the invention, BIRB0796 is provided at a concentration range of between about 0.05 to about 30 μM, e.g., from about 0.1 to about 30 μM, e.g., between about 0.2-30 μM, e.g., between about 0.2-25 μM, e.g., between about 0.2-20 μM, e.g., between about 0.2-15 μM, e.g., between about 0.2-10 μM, e.g., between about 0.2-8 μM, e.g., between about 0.2-6 μM, e.g., between about 0.5-6 μM, e.g., between about 0.5-5 μM, e.g., between about 0.5-4 μM, e.g., between about 0.5-3 μM, e.g., between about 0.5-2 μM, e.g., between about 1-3 μM, e.g., between about 1-2.5 μM, e.g., about 2 μM.
As used herein the term “ROCK” refers to the protein set forth by GenBank Accession No. NP_005397.1 (P160ROCK; SEQ ID NO: 23); and NP_004841.2 (ROCK2; SEQ ID NO: 24) 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 embodiment, the ROCK inhibitor is a small molecule agent.
Non-limiting examples of ROCK inhibitors include Y27632 (TOCRIS, Catalogue number 1254).
According to some embodiments of the invention, Y27632 is provided at a concentration range of between about 0.1-100 μM, e.g., from about 0.1 μM to about 90 μM, e.g., between about 0.1-85 μM, e.g., between about 0.1-80 μM, e.g., between about 0.1-70 μM, e.g., between about 0.1-60 μM, e.g., between about 0.1-55 μM, e.g., between about 0.1-50 μM, e.g., between about 0.1-45 μM, e.g., between about 0.1-40 μM, e.g., between about 0.1-35 μM, e.g., between about 0.1-30 μM, e.g., between about 0.1-25 μM, e.g., between about 0.1-10 μM, e.g., between about 0.1-5 μM, e.g., between about 0.5-5 μM, e.g., between about 0.5-2 μM, e.g. between about 1-5 μM, e.g., about 1 μM.
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 8 (MAPK8) protein set forth by GenBank Accession Nos. NP_620637.1 (isoform alpha2) (SEQ ID NO: 25), NP_620635.1 (isoform beta2) (SEQ ID NO: 26), NP_620634.1 (isoform beta1) (SEQ ID NO: 27), NP_002741.1 (isoform alpha1) (SEQ ID NO: 28) 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. 1496), AEG3482 (AXONMEDCHEM—AXON 1291), BIX02189, BRAFi (SB590885) and BIRB796 (AXONMEDCHEM—Axon 1358).
According to some embodiments of the invention, SP600125 is provided at a concentration range of between about 0.5-100 μM, e.g., from about 1 μM to about 100 μM, e.g., between about 1-90 μM, e.g., between about 1-80 μM, e.g., between about 1-70 μM, e.g., between about 1-60 μM, e.g., between about 1-55 μM, e.g., between about 1-50 μM, e.g., between about 1-45 μM, e.g., between about 1-40 μM, e.g., between about 1-35 μM, e.g., between about 1-30 μM, e.g., between about 1-25 μM, e.g., between about 1-20 μM, e.g., between about 1-15 μM, e.g., between about 1-10 μM, e.g., between about 2-10 μM, e.g., between about 3-10 μM, e.g., between about 4-10 μM, e.g., between about 4-6 μM, e.g., about 5 μM.
According to some embodiments of the invention, BIX02189 is provided at a concentration range of between about 0.5-100 μM, e.g., from about 1 μM to about 100 μM, e.g., between about 1-90 μM, e.g., between about 1-80 μM, e.g., between about 1-70 μM, e.g., between about 1-60 μM, e.g., between about 1-55 μM, e.g., between about 1-50 μM, e.g., between about 1-45 μM, e.g., between about 1-40 μM, e.g., between about 1-35 μM, e.g., between about 1-30 μM, e.g., between about 1-25 μM, e.g., between about 1-20 μM, e.g., between about 1-15 μM, e.g., between about 1-10 μM, e.g., between about 2-10 μM, e.g., between about 3-10 μM, e.g., between about 4-10 μM, e.g., between about 4-6 μM, e.g., about 5 μm.
According to some embodiments of the invention, BRAFi (SB590885) is provided at a concentration range of between about 0.1-100 μM, e.g., between about 0.1-90 μM, e.g., between about 0.1-80 μM, e.g., between about 0.1-70 μM, e.g., between about 0.1-60 μM, e.g., between about 0.1-50 μM, e.g., between about 0.1-40 μM, e.g., between about 0.1-30 μM, e.g., between about 0.1-20 μM, e.g., between about 0.1-10 μM, e.g., between about 0.1-5 μM, e.g., between about 0.1-2 μM, e.g., between about 0.1-1 μM, e.g., about 0.5 μM.
As used herein the term “ERK1” refers to the mitogen-activated protein kinase 3 (MAPK3) isoform 1 set forth by GenBank Accession No. NP_002737.2 (SEQ ID NO: 29), the MAPK3 isoform 2 set forth by GenBank Accession No. NP_001035145.1 (SEQ ID NO: 30), the MAPK3 isoform 3 set forth by GenBank Accession No. NP_001103361.1 (SEQ ID NO: 31) and/or ERK1 set forth in GenBank Accession No. M84490 (SEQ ID NO: 32) having the MAPK signaling activity.
As used herein the term “ERK2” refers to the mitogen-activated protein kinase 1 (MAPK1) set forth by GenBank Accession No. NP_002736.3 (SEQ ID NO: 33) and/or GenBank Accession No. NP_620407.1 (SEQ ID NO: 34) having the MAPK signaling activity.
As used herein the term “ERK1/2 inhibitor” refers to any molecule capable of inhibiting the activity of ERK1/2 as determined by Western blot protein detection of phosphorylated ERK1/2 proteins.
According to a particular embodiment, the ERK1/2 inhibitor is a small molecule agent.
Non-limiting examples of ERK1/2 inhibitors (also known as MEK1/2 inhibitors) include PD0325901 (AXONMEDCHEM—AXON 1408), PD98059 (AXONMEDCHEM—Axon 1223), and PD184352 (AXONMEDCHEM—AXON 1368); and/or even inhibitors of RAF (which is upstream of MEK/ERK pathway) such as Sorafenib tosylate (also known as BAY 43-9006 AXONMEDCHEM—AXON 1397) or SB 590885 (TOCRIS #2650).
According to some embodiments of the invention, PD0325901 is provided at a concentration range from about 0.01 microM (μM) to about 50 μM, e.g., between about 0.05-45 μM, e.g., between about 0.1-50 μM, e.g., between about 0.1-45 μM, e.g., between about 0.1-40 μM, e.g., between about 0.1-35 μM, e.g., between about 0.1-30 μM, e.g., between about 0.1-25 μM, e.g., between about 0.1-20 μM, e.g., between about 0.1-15 μM, e.g., between about 0.1-10 μM, e.g., between about 0.2-10 μM, e.g., between about 0.3-10 μM, e.g., between about 0.4-10 μM, e.g., between about 0.5-10 μM, e.g., between about 0.6-10 μM, e.g., between about 0.7-10 μM, e.g., between 0.8-10 μM, e.g., between 0.9-10 μM, e.g., between 0.9-9 μM, e.g., between 0.9-8 μM, e.g., between 0.9-7 μM, e.g., between 0.9-6 μM, e.g., between 0.8-5 μM, e.g., between 0.8-4 μM, e.g., between 0.8-3 μM, e.g., between 0.8-2 μM, e.g., between 0.8-1.5 μM, e.g., between 0.9-1.2 μM, e.g., about 1 μM.
According to some embodiments of the invention, PD98059 is provided at a concentration range from about 0.1 microM (μM) to about 70 μM, e.g., between about 0.1-65 μM, e.g., between about 0.1-55 μM, e.g., between about 0.1-50 μM, e.g., between about 0.1-45 μM, e.g., between about 0.1-40 μM, e.g., between about 0.1-35 μM, e.g., between about 0.1-30 μM, e.g., between about 0.1-25 μM, e.g., between about 0.1-20 μM, e.g., between about 0.1-15 μM, e.g., between about 2-20 μM, e.g., between about 5-15 μM, e.g., about 10 μM, e.g., between about 0.1-10 μM, e.g., between about 0.2-10 μM, e.g., between about 0.3-10 μM, e.g., between about 0.4-10 μM, e.g., between about 0.5-10 μM, e.g., between about 0.6-10 μM, e.g., between about 0.7-10 μM, e.g., between 0.8-10 μM, e.g., between 0.9-10 μM, e.g., between 0.9-9 μM, e.g., between 0.9-8 μM, e.g., between 0.9-7 μM, e.g., between 0.9-6 μM, e.g., between 0.8-5 μM, e.g., between 0.8-4 μM, e.g., between 0.8-3 μM, e.g., between 0.8-2 μM, e.g., between 0.8-1.5 μM, e.g., between 0.9-1.2 μM.
According to some embodiments of the invention, PD184352 is provided at a concentration range from about 0.1 microM (μM) to about 70 μM, e.g., between about 0.1-60 μM, e.g., between about 0.1-50 μM, e.g., between about 0.5-50 μM, e.g., between about 0.5-45 μM, e.g., between about 0.5-40 μM, e.g., between about 0.1-35 μM, e.g., between about 0.5-30 μM, e.g., between about 0.5-25 μM, e.g., between about 0.5-20 μM, e.g., between about 0.5-15 μM, e.g., between about 0.5-10 μM, e.g., between 0.5-9 μM, e.g., between 0.5-8 μM, e.g., between 0.5-7 μM, e.g., between 0.9-6 μM, e.g., between 0.8-5 μM, e.g., between 0.8-4 μM, e.g., between 0.8-3 μM, e.g., about 3 μM. e.g., between 0.8-2 μM, e.g., between 0.8-1.5 μM, e.g., between 0.9-1.2 μM.
According to some embodiments of the invention, Sorafenib is provided at a concentration range from about 0.1 microM (μM) to about 70 μM, e.g., between about 0.1-60 μM, e.g., between about 0.1-50 μM, e.g., between about 0.5-50 μM, e.g., between about 0.5-45 μM, e.g., between about 0.5-40 μM, e.g., between about 0.1-35 μM, e.g., between about 0.5-30 μM, e.g., between about 0.5-25 μM, e.g., between about 0.5-20 μM, e.g., between about 0.5-15 μM, e.g., between about 0.5-10 μM, e.g., between 0.5-9 μM, e.g., between 0.5-8 μM, e.g., between 0.5-7 μM, e.g., between 0.9-6 μM, e.g., between 0.8-5 μM, e.g., about 5 μM, e.g., between 0.8-4 μM, e.g., between 0.8-3 μM, e.g., between 0.8-2 μM, e.g., between 0.8-1.5 μM, e.g., between 0.9-1.2 μM.
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 ERK1/2 inhibitor present in the medium is less than 0.5 μM, for example about 0.4 μM, 0.3 μM, 0.2 μM or 0.1 μM. In some cases, the medium is devoid of ERK1/2 inhibitor. It will be appreciated that the phrase “being devoid of ERK1/2 inhibitors” refers to a medium in which no ERK1/2 inhibitors have been positively added to a medium and does not mean to exclude trace amounts of ERK1/2 inhibitors contained in the base medium.
The present inventors contemplate addition of an activator of the TGF-Activin pathway to the medium when the ERK1/2 in the medium is less than 0.5 μM.
According to some embodiments of the invention, activators of TGF/ACTIVIN pathway including ACTIVIN A (also known as Inhibin beta A, INHBA, Gene ID: 3624; GenBank Accession No. NM_002192.2 (SEQ ID NO: 35), which encodes GenBank Accession No. NP_002183.1; SEQ ID NO: 36).
Preferably the amount of ACTIVIN A added is between 1-100 ng/ml and more preferably between 1-10 ng/ml (for example about 4 ng/ml).
NOTCH signaling inhibitors may also be included in the media of the present invention. Preferably, the NOTCH signaling inhibitor is added when the medium comprises less than 0.5 μM, for example about 0.4 μM, 0.3 μM, 0.2 μM or 0.1 μM ERK1/2 inhibitor. NOTCH signaling inhibitors include, but are not limited to the following gamma secretase inhibitors: DAPT (Axon Medchem 1484—0.05-50 μM final concentration), LY2886721 hydrochloride (Axon Medchem 1964—0.05-50 μM final concentration)], DBZ (Axon Medchem—Axon 1488-0.05-50 μM 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 embodiment, the media of the present invention 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 embodiment, “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 invention 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 TGFI3R 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 1 (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 WO2014/174470, the contents of which can be incorporated herein by reference.
The media described herein can be used to culture cells. Thus, according to an aspect of some embodiments of the invention, there is provided a cell culture comprising cells and the culture medium of some embodiments of the invention.
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 embodiments of the invention, the cells are somatic cells, pluripotent stem cells (PSCs), primed pluripotent stem cells, non-naïve pluripotent stem cell and/or naive pluripotent stem cells.
According to some embodiments of the invention, the culture medium is capable of maintaining pluripotent stem cells in an undifferentiated state for at least 2 passages, e.g., for at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 passages. The pluripotent stem cells cultured in the presently disclosed media retain their hypomethylated state for the number of passages.
According to some embodiments of the invention, 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 invention are swine (porcine) pluripotent stem cells.
Preferably, the pluripotent stem cells are not rodent pluripotent stem cells.
In one embodiment, the pluripotent stem cell is a naïve 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 embodiments of the invention 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 Xq13.2 chromosome and has the sequence depicted in clone NC_000023.10 (73040486.73072588, complement, based on GenBank version GRCh37.p10. The XIST gene has a non-coding RNA which is provided in GenBank Accession NO. NR_001564.2 (SEQ ID NO: 37).
According to some embodiments of the invention, presence of two unmethylated alleles of XIST gene in a female cell refers to having below about 20% of CpG methylated reads sequenced in the XIST promoter, e.g., below about 19%, below about 18%, below about 17%, below about 16%, below about 15%, below about 14%, below about 13%, below about 12%, below about 11%, below about 10%, below about 9%, below about 8%, below about 7%, below about 6%, below about 5%, below about 4%, below about 3%, below about 2%, below about 1%, e.g., 0% (e.g., complete absence) of CpG methylated reads sequenced in the XIST promoter.
According to some embodiments of the invention, presence of one unmethylated allele of XIST gene in a male cell refers to having below about 20% of CpG methylated reads sequenced in the XIST promoter, e.g., below about 19%, below about 18%, below about 17%, below about 16%, below about 15%, below about 14%, below about 13%, below about 12%, below about 11%, below about 10%, below about 9%, below about 8%, below about 7%, below about 6%, below about 5%, below about 4%, below about 3%, below about 2%, below about 1%, e.g., 0% 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: 38.
According to some embodiments of the invention, 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., 1-2%) as determined by Liquid Chromatography—Mass Spectrometry (LC-MS) quantitative analysis.
According to some embodiments of the invention, the human naive PSC is characterized by 0-3% 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 3.5%-5% of total methylated cytosine out of the total Guanine nucleotides in the primed PSC cell.
Thus, the naive pluripotent stem cell of some embodiments of the invention is in a naïve, hypomethylated state (relating to global levels of DNA methylation). For example in one embodiment, less than 70% of the cytosines of a CG sequence of the DNA of the naïve pluripotent stem cell are methylated, less than 60% of the cytosines of a CG sequence of the DNA of the naïve pluripotent stem cell are methylated, less than 50% of the cytosines of a CG sequence of the DNA of the naïve 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 embodiments of the invention (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 embodiments of the invention, the non-naive PSC is selected from the group consisting of a primed PSC, an embryonic stem cell, a 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 embryonic tissue formed after gestation (e.g., blastocyst) before implantation of the embryo (i.e., a pre-implantation blastocyst), extended blastocyst cells (EBCs) which are obtained from a post-implantation/pre-gastrulation stage blastocyst (see WO2006/040763) and embryonic germ (EG) cells which are obtained from the genital tissue of a fetus any time during gestation, preferably before 10 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 embodiments of the invention, 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 embodiments of the invention, the induced pluripotent stem cells are formed by inducing the expression of Oct-4, Sox2, Kfl4 and c-Myc in a somatic stem cell.
The embryonic stem cells of some embodiments of the invention can be obtained using well-known cell-culture methods. For example, 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 9 to 15 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 4-7 days. For further details on methods of preparation human ES cells see Thomson et al., [U.S. Pat. No. 5,843,780; Science 282: 1145, 1998; Curr. Top. Dev. Biol. 38: 133, 1998; Proc. Natl. Acad. Sci. USA 92: 7844, 1995]; Bongso et al., [Hum Reprod 4: 706, 1989]; and Gardner et al., [Fertil. Steril. 69: 84, 1998].
Another method for preparing ES cells is described in Chung et al., Cell Stem Cell, Volume 2, Issue 2, 113-117, 7 Feb. 2008. 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 embodiments of the invention. 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]. Non-limiting examples of commercially available embryonic stem cell lines are BG01, BG02, BG03, BG04, CY12, CY30, CY92, CY10, TE03, TE32, CHB-4, CHB-5, CHB-6, CHB-8, CHB-9, CHB-10, CHB-11, CHB-12, HUES 1, HUES 2, HUES 3, HUES 4, HUES 5, HUES 6, HUES 7, HUES 8, HUES 9, HUES 10, HUES 11, HUES 12, HUES 13, HUES 14, HUES 15, HUES 16, HUES 17, HUES 18, HUES 19, HUES 20, HUES 21, HUES 22, HUES 23, HUES 24, HUES 25, HUES 26, HUES 27, HUES 28, CyT49, RUES3, WA01, UCSF4, NYUES1, NYUES2, NYUES3, NYUES4, NYUES5, NYUES6, NYUES7, UCLA 1, UCLA 2, UCLA 3, WA077 (H7), WA09 (H9), WA13 (H13), WA14 (H14), HUES 62, HUES 63, HUES 64, CT1, CT2, CT3, CT4, MA135, Eneavour-2, WIBR1, WIBR2, WIBR3, WIBR4, WIBR5, WIBR6, HUES 45, Shef 3, Shef 6, BJNhem19, BJNhem20, SA001, SA001.
In addition, ES cells can be obtained from other species as well, including mouse (Mills and Bradley, 2001), golden hamster [Doetschman et al., 1988, Dev Biol. 127: 224-7], rat [Iannaccone et al., 1994, Dev Biol. 163: 288-92] rabbit [Giles et al. 1993, Mol Reprod Dev. 36: 130-8; Graves & Moreadith, 1993, Mol Reprod Dev. 1993, 36: 424-33], several domestic animal species [Notarianni et al., 1991, J Reprod Fertil Suppl. 43: 255-60; Wheeler 1994, Reprod Fertil Dev. 6: 563-8; Mitalipova et al., 2001, Cloning. 3: 59-67] and non-human primate species (Rhesus monkey and marmoset) [Thomson et al., 1995, Proc Natl Acad Sci USA. 92: 7844-8; Thomson et al., 1996, Biol Reprod. 55: 254-9].
Extended blastocyst cells (EBCs) can be obtained from a 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 8-11 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 7-30 days or 1-4 passages. For additional details on methods of preparation human EG cells see Shamblott et al., [Proc. Natl. Acad. Sci. USA 95: 13726, 1998] and U.S. Pat. No. 6,090,622.
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-3/4, Sox2, c-Myc, and KLF4 [Yamanaka S, Cell Stem Cell. 2007, 1(1):39-49; Aoi T, et al., Generation of Pluripotent Stem Cells from Adult Mouse Liver and Stomach Cells. Science. 2008 Feb. 14. (Epub ahead of print); IH Park, Zhao R, West J A, et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 2008; 451:141-146; K Takahashi, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007; 131:861-872]. Other embryonic-like stem cells can be generated by nuclear transfer to oocytes, fusion with embryonic stem cells or 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 method of the present invention may be any number including small batches—e.g. 100×104 cells to larger batches—e.g. 100×106 or 100×107 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 embodiment of the invention, the basic classes of bioreactors suitable for use with the present invention include static bioreactors, stirred flask bioreactors, rotating wall bioreactors, hollow fiber bioreactors and direct perfusion bioreactors.
According to a particular embodiment, the cells are cultured (i.e. expanded) on an adherent surface.
Examples of such surfaces are provided herein under.
1. Laminin/Fibronectin coated plates. Sources for Fibronectin: (Sigma Aldrich Bovine Fibronectin F1141, or human Fibronectin Millipore FC010). Sources for Laminin (Sigma Aldrich Ewing Sarcoma derived Laminin L2020).
2. Cells can be expanded on gelatin and vitronectin coated plates (e.g. 0.2% gelatin and 1 μg/ml Vitronectin coated plates).
3. Cells can be expanded on plates coated with 0.2% gelatin/irradiated mouse or human fibroblast feeder cells.
4. Human naïve cells can be expanded on plates coated with only 0.2% gelatin coated plates.
5. Human naïve cells can be expanded on plates coated with only Matrigel or Geltrex (BD Biosciences).
6. Human naïve and primed cells can be expanded in suspension in plates, flasks or plastic bags with rocking or rotation movements.
The culture media described in the present application may be used for a myriad of purposes.
According to a particular embodiment, 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 80% of the Cs of a CG sequence in the DNA are not methylated. In some embodiments less than 70% 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 24-48 hours, and passaging each culture dish (e.g., a plate) to 2 or 3 culture dishes (e.g., plates) every 3-5 days. Thus, when cells in the culture reach about 60-90% 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 (0.25% or 0.05% 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 embodiment, 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. 2012), which is fully incorporated herein by reference in its entirety. Further description of preparation of expression vectors and modes of administering them into cells are provided hereinunder.
According to some embodiments of the invention, 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. 2010) which is fully incorporated herein by reference in its entirety.
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 naïve pluripotent stem cells from non-naïve pluripotent stem cells. Preferably the media used for generation or maintenance of naïve 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 naïve pluripotent stem cells from non-naïve cells.
More specifically, according to another aspect of the present invention there is provided a method of generating a naive pluripotent stem cell (PSC), comprising:
incubating a non-naive PSC cell in the culture medium described herein, the culture medium allowing generation of the naive PSC from the non-naive PSC, wherein:
(i) when the naive PSC is a female PSC, then the naive female PSC has two unmethylated alleles of an X-inactive specific transcript (XIST) gene; and
(ii) when the naive PSC is a male PSC, then the naive male PSC has an unmethylated allele of the XIST gene; and/or
an expression level of transcription factor E3 (TFE3) in the naive PSC is characterized by a nucleus to cytoplasm expression ratio which is equal to or higher than 1 as determined by an immunostaining assay, thereby generating the naive PSC.
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 ±10%.
The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.
The term “consisting of” means “including and limited to”.
The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.
Throughout this application, various embodiments of this invention 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 invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention 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 embodiments of the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique” by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
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, 50,000 cells were centrifuged at 500 g for 3 min, followed by a wash using 50 μl of cold PBS and centrifugation at 500 g for 3 min. Cells were lysed using cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630). Immediately after lysis, nuclei were spun at 500 g for 10 min using a refrigerated centrifuge. Next, the pellet was resuspended in the transposase reaction mix (25 μl 2×TD buffer, 2.5 μl transposase (Illumina) and 22.5 μl nuclease-free water). The transposition reaction was carried out for 30 min at 37° C. 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 1 and 2 for a total of 12 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 (50 ng) 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 500/550 High Output v2 kit (150 cycles) was used.
ChIP-seq library preparation: Cells were crosslinked in formaldehyde (1% final concentration, 10 min at room temperature), and then quenched with glycine (5 min at room temperature). Antibodies detailed in Table 1 were then lysed in 50 mM HEPES KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40 alternative, 0.25% Triton supplemented with protease inhibitor at 4° C. (Roche, 04693159001) for 10 min, and later centrifuged at 950 g for 10 min.
Supernatant was discarded and pellet was resuspended in RIPA-1 (0.2% SDS, 1 mM EDTA, 0.1% DOC, 140 mM NaCl and 10 mM Tris-HCl) with protease inhibitor. Cells were then fragmented with a Branson Sonifier (model S-450D) at −4° C. to size ranges between 200 and 800 bp and centrifugation at max speed for 10 min. Supp lysate was extracted and diluted with RIPA 2-3-fold (0.1% SDS, 1 mM EDTA, 0.1% DOC, Triton 1%, 140 mM NaCl and 10 mM 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 0.5% TWEEN and 0.5% BSA) for 1 h 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 500 mM NaCl, twice with LiCl buffer (10 mM TE, 250 mM LiCl, 0.5% NP-40, 0.5% DOC), once with TE (10Mm Tris-HCl pH 8.0, 1 mM EDTA), and then eluted in 0.5% SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris HCl pH 8.0. Eluate was incubated treated sequentially with RNaseA (Roche, 11119915001) for 30 min in 37° C. and proteinase K (NEB, P8102S) for 2 h in 37° C. and de-crosslinked in 65° C. for 8 h. 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., 2013).
PolyA-RNA analysis: hESCs grown in naïve and primed conditions, from different cell lines (LIS41, LIS49, WIBR2) were used for RNA-seq analysis. STAR software version 2.5.2b was used to align reads to human GRCh38 reference genome (2013), using the following flags: —outFilterMultimapNmax 1—outReadsUnmapped Fastx—twopassMode Basic—outSAMstrandField intronMotif. FPKM values were estimated with HTSeq software over all genes in GRCh38 assembly using the following flags: -a 10-s no -t exon -i gene_id. Genes with accumulated expression of FPKM>10 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 naïve and primed samples were selected from HTSeq output in the following parameters: FC>2 of FC<0.5, and adjusted p-value<0.1.
Whole-Genome Bisulfite Sequencing (WGBS) analysis: The sequencing reads were aligned to the human hg19 reference genome (UCSC, 2009), 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 5 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, 2009), with default parameters. Enriched intervals of all measured proteins were analyzed using MACS version 1.4.2-1. 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 2 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 (≤120 bp) that correspond to nucleosome free region. To detect and separate accessible loci in each sample, we used MACS version 1.4.2-1 with—call-subpeaks flag (PeakSplitter version 1.0).
Enhancer Identification: H3K27ac peaks were detected using MACS version 1.4.2-1 and merged for each condition (naïve 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, 2009) 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 4.7, using the software default parameters.
Culture Medium:
Enhanced NHSM Composition
WIS-NHSM media (B27, vitamin C and N2 based) (No Activin/TGF/FGF)
Primary Cytokines+Inhibitors:
Primary Cytokines+Inhibitors:
Enhanced NHSM Composition with Low or No ERKi (tENHSM or 0ENHSM)
WIS-NHSM media (B27, vitamin C and N2 based) (No Activin/TGF/FGF)
Primary Cytokines+Inhibitors:
Primary Cytokines+Inhibitors:
Human PSCs (H9 female 46XX human ESC line) were expanded for passages in N2B27 defined base media supplemented with:
Results
In order to identify culture conditions which capture human naïve 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 (
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 (
NHSM conditions were supplemented with individual small molecules (
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 (
Defining Human Naïve Pluripotency Conditions Independent of TGF/ACTIVIN/NODAL Signaling
Under the above described conditions, human ESCs maintained uniformly high APE-OCT4-GFP levels only in the presence of exogenous ACTIVIN A, and consistently differentiated when TGFR inhibitor was provided (
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 naïve state both with and without METTL3 depletion (
To extend the previous findings to another repressor machinery, OCT4-GFP-WIBR3 reporter ESCs were targeted by TALENs to generate DGCR8 null cells (
Tolerance for Absence of Exogenous L-Glutamine in ENHSM Conditions
Murine naïve 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 (
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 naïve 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, APE-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 (
Transcriptional Characterization of Human PSCs in ENHSM Conditions
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 6-8 days of plating. ICM derived outgrowths were then trypsinized and passaged. Subsequently, 3 new stem cell lines termed LIS36, LIS42 and LIS46 were derived in ENHSM-ACT; LIS41 and LIS49 ESCs in ENHSM conditions (
Global gene expression patterns were compared between naïve and primed hESCs and hiPSCs, many of which were genetically matched. Unbiased clustering of genome-wide expression profiles demonstrated that naïve hESC and hiPSCs possess a distinct gene expression pattern and clustered separately from conventional/primed hESCs and hiPSCs (
Transposable Element (TE)-derived transcripts were profiled and compared in conventional and naïve human PSCS expanded in ENHSM conditions (Theunissen et al., 2016). The top 5,000 TEs with largest SD separated naïve and primed samples both in hierarchical clustering (
Epigenetic Characterization of Human PSCs in ENHSM Conditions
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., 2016). Correctly targeted clone #9 expresses only the red allele, however upon transferring the cells into ENHSM conditions >99% 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 >95% of the reprised cells (
Human naïve 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 82% in primed hPSCs to 65% in ENHSM expanded human hPSCs and down to 53% when Activin was supplemented (ENHSM-ACT conditions) (
WNT/ß-CATENIN and SRC/NFkB Signaling are Major Priming Pathways Compromising Human Naïve Pluripotency
The results above indicate that functional naïve 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 4 components compromised naïve pluripotency hallmarks like X chromosome inactivation in female cell lines (
The present inventors next aimed to define the signaling pathway downstream of Tankyrase inhibition facilitating human naïve 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 naïve 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 naïve 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, naïve 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 naïve 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 naïve pluripotency.
In mouse ground state naïve conditions, LIF/Stat3 has been shown to be a booster for naïve marker expression however they can be omitted without entire collapse of the naïve PSC circuit (Ying et al., 2008). By omitting LIF from ENHSM conditions and by generating STAT3 KO human naïve 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 naïve identity even in the absence of LIF/STAT3 signaling (
Inhibition of NOTCH Pathway Facilitates Maintenance of Human Naïve Pluripotency without Use of MEK/ERK Inhibition
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., 2017). In mice, using alternative naïve 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., 2017). The latter murine cells are fully naïve and are capable of generate all-iPS mice with contribution to the germline, and thus provide a safer route for exploiting defined mouse naïve PSCs (Choi et al., 2017).
Although ENHSM conditions had modest levels of hypomethylation, and erosion of imprinting was slow and sporadic on few loci and only after extended passaging (
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 (
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 (
ENHSM-Derived hiPSCs Give Rise to Interspecies Chimaeras
Naïve hiPSCs can contribute to interspecies chimaerism with highly variable and limited efficiency—Gafni et al. Nature 2013. 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 3 passages in ENHSM before being micro-injected into E2.5 mouse morulas. Following transplantation into pseudo-pregnant foster mothers the day after, their survival and integration was assayed throughout 14 days using various imaging techniques. ENHSM-derived hiPSCs are able to colonize mouse embryos up to E17.5 at various anatomic regions of different embryonic germ layers as shown in (
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 (
Additional culture conditions were analyzed to determine if additional media are capable of capturing human naïve PSCs.
The culture conditions are described in the Materials and methods section and labeled conditions 1-8.
As illustrated in
WIBR3 female human ESCs were expanded for 10 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 2, herein below.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
It is the intent of the applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is/are hereby incorporated herein by reference in its/their entirety.
This application is a Continuation of PCT Patent Application No. PCT/IL2020/050095, having international filing date of Jan. 23, 2020 which claims the benefit of priority under 35 USC § 119(e) of U.S. Provisional Patent Application No. 62/795,626 filed on Jan. 23, 2019. The contents of the above applications are all incorporated by reference as if fully set forth herein in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62795626 | Jan 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/IL2020/050095 | Jan 2020 | US |
| Child | 17382500 | US |
