THROUGH THE EFFECTS OF RETINOIC ACID ANALOGUE ΠΤΝΡΒ RHO-ASSOCIATED KINASE INHIBITOR fY-27632 AND PPARvAGONIST

fPIOGLITAZONEl

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claim priority to U.S. patent application number US 62/572,888, filed October 16, 2017, the entire contents of which is hereby incorporated by reference. FIELD

[0002] The present disclosure relates generally to enhanced transfection efficiency of human pluripotent stem cells through the effects of retinoic acid analogue (TTNPB), Rho- associated kinase inhibitor (Y-27632), and PPARy agonist pioglitazone.

BACKGROUND

[0003] When combined with genome engineering, pluripotent stem cells (PSCs) offer significant potential for enabling personalized/precision medicine. Genome engineering can allow the correction of disease genes in patient derived PSCs, which in turn can be used for regenerative medicine/gene therapy purposes. Similarly, PSC genome engineering can be used address mechanistic questions in disease modelling/drug development. While genome engineering mouse PSCs has been straightforward, it has been very challenging in human (h) PSCs. Unlike mouse, hPSCs are prone to anoikis, a form of cell death that occurs when cells are enzymatically dissociated.

SUMMARY

[0004] In one aspect there is described a method for an in vitro transfection of eukaryotic cells in cell culture, comprising:

[0005] - providing said eukaryotic cells in a culture medium;

[0006] - adding TTNPB and Y-27632 to said culture medium;

[0007] - adding a polynucleotide molecule, or a plurality of polynucleotide molecules, to be transfected into said cells, to said culture medium; and

[0008] - incubating said cells, TTNPB, Y-27632, and nucleotide for an incubation period. [0009] In one example, the TTNPB, Y-27632 are added before and/or after transfection.

[0010] In one aspect there is described a method for an in vitro transfection of eukaryotic cells in cell culture, comprising:

[0011] - providing said eukaryotic cells in a culture medium;

[0012] - adding a polynucleotide molecule, or a plurality of polynucleotide molecules, to be transfected into said cells, to said culture medium;

[0013] - incubating said culture medium containing said polynucleotide molecule or said plurality of polynucleotide molecules with TTNPB, Y-27632 and Pioglitazone, for an incubation period.

[0014] In one aspect there is described a method for an in vitro transfection of eukaryotic cells in cell culture, comprising:

[0015] - providing said eukaryotic cells in a culture medium;

[0016] - adding a polynucleotide molecule, or a plurality of polynucleotide molecules, to be transfected into said cells, to said culture medium;

[0017] - incubating said cells and polynucleotide with TTNPB and Pioglitazone, for an incubation period.

[0018] In one aspect there is described a method for an in vitro transfection of eukaryotic cells in cell culture, comprising:

[0019] - providing said eukaryotic cells in a culture medium;

[0020] - adding a polynucleotide molecule, or a plurality of polynucleotide molecules, to be transfected into said cells, to said culture medium;

[0021] - incubating said cells and polynucleotide with TTNPB and Y-27632, for an incubation period.

[0022] In one aspect there is described a method for an in vitro transfection of eukaryotic cells in cell culture, comprising:

[0023] - providing said eukaryotic cells in a culture medium;

[0024] - adding a polynucleotide molecule, or a plurality of polynucleotide molecules, to be transfected into said cells, to said culture medium;

[0025] - incubating said cells and polynucleotide with Pioglitazone and Y-27632, for an incubation period.

[0026] In one aspect there is described a method for an in vitro transfection of eukaryotic cells in cell culture, comprising: [0027] - providing said eukaryotic cells in a culture medium;

[0028] - adding a polynucleotide molecule, or a plurality of polynucleotide molecules, to be transfected into said cells, to said culture medium;

[0029] - incubating said cells and polynucleotide with Pioglitazone, for an incubation period.

[0030] In one aspect there is described a method for an in vitro transfection of eukaryotic cells in cell culture, comprising:

[0031] - providing said eukaryotic cells in a culture medium;

[0032] - adding a polynucleotide molecule, or a plurality of polynucleotide molecules, to be transfected into said cells, to said culture medium;

[0033] - incubating said cells and polynucleotide with Y-27632, for an incubation period.

[0034] In one aspect there is described a method for an in vitro transfection of eukaryotic cells in cell culture, comprising:

[0035] - providing said eukaryotic cells in a culture medium;

[0036] - adding a polynucleotide molecule, or a plurality of polynucleotide molecules, to be transfected into said cells, to said culture medium;

[0037] - incubating said cells and polynucleotide with TTNPB, for an incubation period.

[0038] In one example, said incubation is about 24 hours.

[0039] In one example, said incubation is at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours.

[0040] In one example, the concentration of TTNPB in said culture medium is about

0.125 to about 1 μΜ.

[0041] In one example, wherein the concentration of Y-27632 is about 5μΜ to about

20μΜ, or about 2μΜ to about 16μΜ.

[0042] In one example, the concentration of Y-27632 is about 10 μΜ.

[0043] In one example, the concentration of pioglitazone is about 2 μΜ to about 16 μΜ

[0044] In one example, the concentration of pioglitazone is about 8 μΜ.

[0045] In one example, said eukaryotic cell is a human cell.

[0046] In one example, said eukaryotic cell is a stem cell.

[0047] In one example, said stem sell is a pluripotent stem cell or embryonic stem cell.

[0048] In one example, said pluripotent stem cell is a human pluripotent stem cell

(hESC). [0049] In one example, said pluripotent stem cell is an hESC or hiPSC.

[0050] In one aspect there is described a use of a composition comprising TTNPB, Y-

27632 and Pioglitazone, for the transfection of a polynucleotide in a eukaryotic cell.

[0051] In one aspect there is described a use of a composition comprising TTNPB and

Pioglitazone for the transfection of a polynucleotide in a eukaryotic cell.

[0052] In one aspect there is described a use of a composition comprising Y-27632 and Pioglitazone, for the transfection of a polynucleotide in a eukaryotic cell.

[0053] In one aspect there is described a use of a composition comprising

Pioglitazone, for the transfection of a polynucleotide in a eukaryotic cell.

[0054] In one aspect there is described a use of a composition comprising TTNPB and

Y-27632, for the transfection of a polynucleotide in a eukaryotic cell.

[0055] In one aspect there is described a use of a composition comprising Y-27632, for the transfection of a polynucleotide in a eukaryotic cell.

[0056] In one aspect there is described a use of a composition comprising TTNPB, for the transfection of a polynucleotide in a eukaryotic cell.

[0057] In one example, said eukaryotic cell is a human cell.

[0058] In one example, said eukaryotic cell is a stem cell.

[0059] In one example, said stem sell is a pluripotent stem cell.

[0060] In one example, said pluripotent stem cell is a human pluripotent stem cell

(hESC).

[0061] In one example, said pluripotent stem cell is an hESC or hiPSC.

[0062] In one aspect there is described a kit, comprising:

[0063] - TTNPB, and

[0064] - Y-27632; and optionally a container.

[0065] In one example, further comprising PPARy agonists such as

Pioglitazone/rosiglitazone.

[0066] In one example, further comprising subjecting said cells to cell sorting to isolate transfected cells and plating transfected cells on hPSC feeder cells with hPSC conditioned media.

[0067] In one example, wherein said cell sorting is carried out with flow sorting.

[0068] In one example, wherein said polynucleotide comprises a polynucleotide encoding green fluorescent protein (GFP). BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.

[0070] Fig. 1 shows hPSC transfection efficiency. The efficiency is increased in hPSCs treated with TTNPB plus Y-27632 (C), vs. Y-27836 alone (B) or untreated (A).

[0071] Fig. 2 shows improved transfection efficiency in hPSCs treated TTNPB plus Y-

27632 (C, D) compared with Y-27632 alone (A, B). A and C are fluorescence images. B and

D are brightfield plus fluorescence (overlay of brightfield and fluorescence images).

[0072] Fig. 3 depicts hPSCs transfection efficiency. The graphs show the dot plot and histogram of flowcytometry analysis for human pluripotent stem cells (hPSCs) transfected with

GFP-DNA (pmaxGFP). The histogram and dot plot graphs demonstrate the percentage of GFP positive cells and mean fluorescent intensity (both are indications of transfection efficiency) in hPSCs following transfection and treatment with TTNPB, Y-27632, and Pioglitazone through various combinations. A) The mock transfected cells (cells transfected with transfection reagent only and not with DNA, treated with TTNPB, Y-27632, and Pioglitazone) did not show any GFP positive cells as expected. B) The transfection efficiency is increased in hPSCs treated with all three small molecules; TTNPB, Y-27632, and Pioglitazone (76.7%) in comparison to the other combinations, and in comparison, to the untreated cells (Mock transfected). Accordingly, the mean fluorescent intensity (expression level of GFP protein) is the highest in hPSCs treated with all three small molecules; TTNPB, Y-27632, and Pioglitazone

(26,051). The percentage of GFP positive cells in other combinations of small molecules are as followings: C) TY (49.3%), D) TP (70.2%), E) YP (48.2%), F) T (64.6%), G) Y (40.5%), H)

P (56.8%). The mean fluorescent intensity in the cells transfected with other combinations are as followings: TY (7,671), TP (13,670), YP (7,338), T (10,844), Y (6,804), P (8,009). T =

TTNPB, Y = Y-27632, P = Pioglitazone, TYP = TTNPB + Y-27632 + Pioglitazone, TY = TTNPB

+ Y-27632, TP = TTNPB + Pioglitazone, YP = Y-27632 + Pioglitazone.

[0073] Fig. 4 depicts the expression of GFP in hPSCs 24 h after transfection. The upper panel shows the brightfield microscopic images, and the lower panel shows the fluorescence images of hPSCs transfected with GFP 24 h after transfection. The cells were treated with various combinations of small molecules for 24 h following transfection. The expression of GFP has shown in the fluorescent images. The increased expression of GFP was observed in the cells treated with the three small molecules, TTNPB, Y-27632, and

Pioglitazone (fluorescent images, lower panel). T = TTNPB, Y = Y-27632, P = Pioglitazone, TYP = TTNPB + Y-27632 + Pioglitazone, TY = TTNPB + Y-27632, TP = TTNPB + Pioglitazone, YP = Y-27632 + Pioglitazone. The percentage of GFP positive cells was: TYP = 76.7%, TY = 49.3%, TP = 70.2%, YP = 48.2%, T = 64.6%, Y = 40.5%, and P = 56.8%. The mean fluorescent intensity was: TYP = 26,051 ; TY = 7,671 ; TP = 13,670; YP = 7,338; T = 10,844; Y = 6,804; and P = 8,009. The highest % of GFP positive cells in order: TPY (76.7%); TP (70.2%); T (64.6%).

DETAILED DESCRIPTION

[0074] In one aspect, there is described herein compounds, compositions, and method of transfecting cells.

[0075] In one aspect, there is described a method of transfecting a stem cell.

[0076] In one aspect, a cell is transfected with a polynucleotide.

[0077] The polynucleotide described herein may be used in a vector.

[0078] The term "vector" as used herein refers to any nucleic acid molecule for the cloning of and/or transfer of a nucleic acid into a cell. A vector may be a replicon to which another nucleotide sequence may be attached to allow for replication of the attached nucleotide sequence. A "replicon" can be any genetic element (for example a plasmid, phage, cosmid, chromosome, viral genome) that functions as an autonomous unit of nucleic acid replication in vivo, and for example, is capable of replication under its own control. The term "vector" includes both viral and nonviral (e.g., plasmid) nucleic acid molecules for introducing a nucleic acid into a cell in vitro, ex vivo, and/or in vivo. A large number of vectors known in the art may be used to manipulate nucleic acids, incorporate response elements and promoters into genes, and the like. For example, the insertion of the nucleic acid fragments corresponding to response elements and promoters into a suitable vector can be accomplished by ligating the appropriate nucleic acid fragments into a chosen vector that has complementary cohesive termini. Alternatively, the ends of the nucleic acid molecules may be enzymatically modified or any site may be produced by ligating nucleotide sequences (linkers) to the nucleic acid termini. Such vectors may be engineered to contain sequences encoding selectable markers that provide for the selection of cells that contain the vector and/or have incorporated the nucleic acid of the vector into the cellular genome. Such markers allow identification and/or selection of host cells that incorporate and express the proteins encoded by the marker. A "recombinant" vector refers to a viral or non- viral vector that comprises one or more heterologous nucleotide sequences. [0079] As used herein, the term "isolated" refers to material, for example a

polynucleotide, a polypeptide, or a cell, that is substantially or essentially free from

components that normally accompany it in its native state.

[0080] The term "introducing" as used herein in the context of a cell, or organism, refers to presenting the polynucleotide to the cell, and/or organism, in such a manner that the nucleic acid molecule gains access to the interior of a cell. Where more than one nucleic acid molecule is to be introduced these nucleic acid molecules can be assembled as part of a single polynucleotide or nucleic acid construct, or as separate polynucleotide or nucleic acid constructs, and can be located on the same or different nucleic acid constructs. Accordingly, these polynucleotides can be introduced into cells in a single transformation event or in separate transformation events.

[0081] Thus, the term "transformation" as used herein refers to the introduction of a heterologous nucleic acid into a cell and/or homologous nucleic acid into a cell. Transformation of a cell may be stable or transient.

[0082] The term "transient transformation" as used herein in the context of a polynucleotide refers to a polynucleotide that may be introduced into the cell and does not integrate into the genome of the cell.

[0083] The term "stably introducing" or "stably introduced" as used herein in the context of a polynucleotide introduced into a cell refers to a polynucleotide that may be stably incorporated into the genome of the cell, and thus the cell is stably transformed with the polynucleotide.

[0084] As used herein, the terms "contacting" refers to a process by which, for example, a compound may be delivered to a cell. The compound may be administered in a number of ways, including, but not limited to, direct introduction into a cell (i.e., intracellularly) and/or extracellular introduction into a cavity, interstitial space, or into the circulation of the organism.

[0085] A "cell" or "host cell" refers to an individual cell or cell culture that can be or has been a recipient of any recombinant vector(s), or isolated polynucleotide. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or a polynucleotide of the invention. A host cell which comprises a recombinant vector of the invention is a recombinant host cell. [0086] In one example, the host cell is a cell obtained or derived from a subject.

[0087] The term "subject", as used herein, refers to an animal, and can include, for example, domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. The subject may be an infant, a child, an adult, or elderly. In a specific example, the subject is a human.

[0088] In one example the cell host is a human cell.

[0089] In one example, the cell host is a stem cell.

[0090] In one example, the cell host is a pluripotent stem cell.

[0091] The term "stem cell" refers to cells that have the capacity to self-renew and to generate differentiated progeny.

[0092] The term "pluripotent stem cells" refers to stem cells that can give rise to cells of all three germ layers (endoderm, mesoderm and ectoderm), but do not have the capacity to give rise to a complete organism.

[0093] Examples of pluripotent stem cells (PSCs) include, but are not limited to, human embryonic stem cells (hESCs), human induced pluripotent stem cells (hiPSCs), non-human primate embryonic stem cells (nhpESCs), non-human primate induced pluripotent stem cells (nhpiPSCs).

[0094] As used herein, "embryonic stem cells" or "ESCs" mean a pluripotent cell or population of pluripotent cells derived from an inner cell mass of a blastocyst. In some examples, ESCs are commercially available from sources.

[0095] As used herein, "induced pluripotent stem cells" or "iPS cells" refers to a pluripotent cell or population of pluripotent cells that may vary with respect to their differentiated somatic cell of origin, that may vary with respect to a specific set of potency-determining factors and that may vary with respect to culture conditions used to isolate them, or may vary with respect to the method (reprogramming method) used to generate them, but nonetheless are substantially genetically identical to their respective differentiated somatic cell of origin and display characteristics similar to higher potency cells, and display characteristics similar to ESCs. Induced pluripotent stem cells exhibit morphological properties (e.g., round shape, large nucleoli and scant cytoplasm) and growth properties. In addition, iPS cells may express pluripotent cell-specific markers. Induced pluripotent stem cells, however, are not immediately derived from embryos. [0096] As used herein, "not immediately derived from embryos" means that the starting cell type for producing iPS cells is a non-embryonic, non-pluripotent cell, such as a multipotent cell or terminally differentiated cell, such as somatic cells obtained from a postnatal individual.

[0097] In some examples, iPS cells may be obtained by reprogramming a somatic cell of a particular human subject according to methods known in the art.

[0098] Subject-specific somatic cells for reprogramming into iPS cells may be obtained or isolated from a target tissue of interest by biopsy or other tissue sampling methods. In some cases, subject-specific cells are manipulated in vitro prior to use. For example, subject-specific cells can be expanded, differentiated, genetically modified, contacted to polypeptides, nucleic acids, or other factors, cryo- preserved, or otherwise modified.

[0099] The culturing of cells and suitable culture media are known.

[00100] Chemically defined culture medium and substrate conditions for culturing stem cells, such as pluripotent stem cells, are known in the art.

[00101] In some example, a serum-free, chemically defined culture medium is used. As used herein, the terms "chemically-defined culture conditions," "fully defined, growth factor free culture conditions," and "fully-defined conditions" indicate that the identity and quantity of each medium ingredient is known and the identity and quantity of supportive surface is known. As used herein, "serum-free" means that a medium does not contain serum or serum replacement, or that it contains essentially no serum or serum replacement. For example, an essentially serum-free medium can contain less than about 0.5%, 0.4%, 0.3%, 0.2% or 0.1 % serum.

[00102] In some examples, the culture medium is capable of maintaining the stem cells in an undifferentiated and pluripotent state for at least 10 passages. The cell culture may be maintained in vitro, under culturing conditions, in which the cells are being passaged for extended periods of time (e.g., for at least 20 passages, e.g., at least about 30, 40, 50, 60, 70, 80, 90, 100 passages or more), while maintaining the cells in their undifferentiated state.

[00103] 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.

[00104] In some examples, the culture medium used by may 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. In some examples, a culture medium may be a synthetic tissue culture medium, supplemented with the necessary additives. In some examples, all ingredients included in the culture medium of the present invention are substantially pure, with a tissue culture grade.

[00105] The terms "defined culture medium," "defined medium," and the like, as used herein, indicate that the identity and quantity of each medium ingredient is known. The term "defined," when used in relation to a culture medium or a culture condition, refers to a culture medium or a culture condition in which the nature and amounts of approximately all the components are known.

[00106] In one example, transfections of cells carried in culture media comprising TTNPB and Y-27632.

[00107] TTNPB (Arotinoid acid) is a retinoic acid analogue, which has previously been shown to enhance chemical reprogramming of mouse iPSCs [5].

[00108] TTNPB is a ligand of the retinoid X receptor gamma (RXRy), which heterodimerizes with the Peroxisome Proliferator Activator Receptor gamma (PPARy) to influence gene expression [6]. Previously PPARy ligands including rosiglitazone have been found to cause the upregulation of Bcl-2, which in turn stabilizes mitochondrial membranes and suppresses apoptosis [7, 8]. While not wishing to be bound by theory, it may be that that TTNPB enhances transfection by suppressing apoptosis via its interaction with PPARy pathway. We have accumulated circumstantial evidence that supports this hypothesis. Recently we have observed that TTNPB enhances the cloning efficiency of hPSCs [9]. A separate study suggests similarly that the PPARy ligand pioglitazone also enhances hPSC cloning efficiency [10].

[00109] In one example, cells may be exposed to TTNPB following nucleofection.

[00110] In one example, cells may be exposed to TTNPB cells before enzymatic dissociation.

[00111] In one example, it is described herein that the retinoic acid analogue, TTNPB, when supplemented at concentrations between about 0.125 to about 1 μηι, or about 0.25 - about 0.5 μΜ, for 4 days, in combination with Y-27632, can synergistically reduce

dissociation-induced apoptosis, increase clonal colony forming efficiency and transfection efficiency by two times (17.6% to 38.9%). [00112] In one example, the number of the number of seeded cells for human pluripotent stem cells (e.g., ESCs and iPSCs) used in transfection is from about 1 x 10 ⁶ - 3 x 10 ⁶ .

[00113] In one example, the Electroporation / transfection system may be the

Amaxa™ biosystems Nucleofector™ II (Lonza) or Neon Transfection System (Thermo Fisher), with different settings (transfection program) to have high transfection efficiency in human pluripotent stem cells (ESCs & iPSCs). In one example, the effective range is greater than 40% efficiency. In some examples, for maximum transfection efficiency, the settings / program needed to be optimized for each cell line.

[00114] In one example, the amount of DNA plasmid (e.g. GFP-DNA) used for transfection is about 1 μg - 5 μg. In some example, for maximum transfection efficient, the amount of transfected DNA/nucleotide needed to be optimized for each cell line.

[00115] In one example, treatment of the cells with TTNPB and Y-27632 may be made before transfection.

[00116] In some examples, in the case of exposure of the cells to TTNPB before transfection, TTNPB may added about 1 hour to about 24 hours before transfection.

[00117] In some examples, in the case of exposure of the cells to TTNPB and Y- 27632 before transfection, TTNPB and Y-27632 may be added about 1 hour to about 24 hours before transfection.

[00118] In some examples, in the case of exposure of the cells to Y-27632 before transfection, Y-27632 may added about 1 hour to about 24 hours before transfection.

[00119] In some examples, treatment of the cells with TTNPB and Y-27632 may be made after transfection.

[00120] In some examples, in the case of exposure of the cells to TTNPB after transfection, TTNPB may be added about 1 hour to about 24 to 72 hours after transfection.

[00121] In some examples, in the case of exposure of the cells to TTNPB and Y- 27632 after transfection, TTNPB and Y-27632 may be added about 1 hour to about 24 to 72 hours after transfection.

[00122] In some examples, in the case of exposure of the cells to Y-27632 after transfection, Y-27632 may be added about 1 hour to about 24 to 72 hours after transfection.

[00123] In one example the concentration of TTNPB is about 0.25 μηι to about 0.5 μηι.

[00124] In one example the concentration of Y-27632 is about 2 μηι to about 16 μηι.

[00125] In one example the concentration of Y-27632 is about 10 μηι. [00126] In one example, the concentration of pioglitazone is about 2 μΜ to about 16 μΜ, or about 5μΜ to about 20μΜ.

[00127] In on example, the concentration of pioglitazone is about 8 μΜ.

[00128] In one example there is described a method for an in vitro transfection of eukaryotic cells in cell culture, comprising:

[00129] - providing said eukaryotic cells in a culture medium;

[00130] - adding a polynucleotide molecule, or a plurality of polynucleotide molecules, to be transfected into said cells, to said culture medium;

[00131] - incubating said culture medium containing said polynucleotide molecule or said plurality of polynucleotide molecules with TTNPB, Y-27632 and Pioglitazone, for an incubation period.

[00132] In one example there is described a method for an in vitro transfection of eukaryotic cells in cell culture, comprising:

[00133] - providing said eukaryotic cells in a culture medium;

[00134] - adding a polynucleotide molecule, or a plurality of polynucleotide molecules, to be transfected into said cells, to said culture medium;

[00135] - incubating said cells and polynucleotide with TTNPB and Pioglitazone, for an incubation period.

[00136] In one example there is described a method for an in vitro transfection of eukaryotic cells in cell culture, comprising:

[00137] - providing said eukaryotic cells in a culture medium;

[00138] - adding a polynucleotide molecule, or a plurality of polynucleotide molecules, to be transfected into said cells, to said culture medium;

[00139] - incubating said cells and polynucleotide with TTNPB and Y-27632, for an incubation period.

[00140] In one example there is described a method for an in vitro transfection of eukaryotic cells in cell culture, comprising:

[00141] - providing said eukaryotic cells in a culture medium;

[00142] - adding a polynucleotide molecule, or a plurality of polynucleotide molecules, to be transfected into said cells, to said culture medium;

[00143] - incubating said cells and polynucleotide with Pioglitazone and Y-27632, for an incubation period. [00144] In one example there is described a method for an in vitro transfection of eukaryotic cells in cell culture, comprising:

[00145] - providing said eukaryotic cells in a culture medium;

[00146] - adding a polynucleotide molecule, or a plurality of polynucleotide molecules, to be transfected into said cells, to said culture medium;

[00147] - incubating said cells and polynucleotide with Pioglitazone, for an incubation period.

[00148] In one example there is described a method for an in vitro transfection of eukaryotic cells in cell culture, comprising:

[00149] - providing said eukaryotic cells in a culture medium;

[00150] - adding a polynucleotide molecule, or a plurality of polynucleotide molecules, to be transfected into said cells, to said culture medium;

[00151] - incubating said cells and polynucleotide with Y-27632, for an incubation period.

[00152] In one example there is described a method for an in vitro transfection of eukaryotic cells in cell culture, comprising:

[00153] - providing said eukaryotic cells in a culture medium;

[00154] - adding a polynucleotide molecule, or a plurality of polynucleotide molecules, to be transfected into said cells, to said culture medium;

[00155] - incubating said cells and polynucleotide with TTNPB, for an incubation period.

[00156] In one example, said incubation is about 24 hours.

[00157] In one example, said incubation is at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours.

[00158] In one example, the number of seed cells after sorting is about 100-150 cells/60 mm tissue culture plate (for example to pick up a positive pure single clone).

[00159] In another example, the combination of TTNPB and Y-27632 may allow us to isolate individual clones after FACS sorting. Although a single cell needs other cells in its microenvironment to survive, this could be resolved using hPSC conditioned media or by creating a feeder layer of mitostatic hPSCs (by treating them with mitomycin C).

[00160] In one example, one GFP positive cell (single cell) will be plated/seeded on each well of a 96-well tissue culture plate (one well / 96-well plate) during sorting procure.

[00161] The effective and variable parameters may be as follows. [00162] One single cell will be plated on one well (one well of 96-well tissue culture plate) pre-coated with hPSC conditioned media, and combination of TTNPB and Y-27632

[00163] The hPSCs conditioned media will be a media which will be exposed to hPSC for 24h, collected after 24h, and added to one well of 96-well plate before sorting of single cell

[00164] One single cell will be plated on one well (one well of 96-well tissue culture plate) pre-coated with feeder layer of mitotically inactivated hPSC, and combination of TTNPB and Y-27632

[00165] The feeder layer of mitotically inactivated hPSC will be a layer of hPSCs inactivated with Mitomycine C or gamma-irradiation and added to one well of 96-well plate before sorting of single cell

[00166] One single cell will be plated on one well (one well of 96-well tissue culture plate) pre-coated with feeder layer of mitotically inactivated hPSC-derived fibroblasts, and combination of TTNPB and Y-27632

[00167] The feeder layer of mitotically inactivated hPSC-derived fibroblasts will be a layer of fibroblast cells derived from hPSCs inactivated with Mitomycine C or gamma- irradiation and added to one well of 96-well plate before sorting of single cell

[00168] One single cell will be plated on one well (one well of 96-well tissue culture plate) pre-coated with matrigel, hPSCs media (mTeSR™1) and combination of TTNPB and Y-27632 (this can be control group)

[00169] In one example, transferred cells are cloned using cell sorting and plating on hPSC feeders with hPSC conditioned media. In one example the cells are transfected with a cector comprising GFP. In one example, cell soring is carried out using FACS. In one example, cell sorting is carried our using FACS on cells transfected with a vector comprising GFP.

[00170] In one example, following cell sorting of cells transfected with a vector comprising GFP, for Imaging, an epi-fluorescent microscope from Olympus model: IX70 was used.

[00171] Method of the invention are conveniently practiced by providing the compounds and/or compositions used in such method in the form of a kit. Such kit preferably contains the composition. Such a kit preferably contains instructions for the use thereof.

[00172] Kits may include a polynucleotide, a vector, and/or a cell. [00173] Kits may also include one or more reagents, buffers, culture medium, or neuronal or other type of cell.

[00174] To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in any way.

[00175] EXAMPLES

[00176] Example 1

[00177] When combined with genome engineering, pluripotent stem cells (PSCs) offer significant potential for enabling personalized/precision medicine. Genome engineering can allow the correction of disease genes in patient derived PSCs, which in turn can be used for regenerative medicine/gene therapy purposes. Similarly, PSC genome engineering can be used address mechanistic questions in disease modelling/drug development. While genome engineering mouse PSCs has been straightforward, it has been very challenging in human (h) PSCs. Unlike mouse, hPSCs are prone to anoikis, a form of cell death that occurs when cells are enzymatically dissociated. To be efficiently transfected for genome engineering, cells need to be dissociated, which possess a significant problem for genome engineering of hPSCs.

[00178] While genome engineering mPSCs has been straightforward, it has been very challenging in hPSCs. Unlike mouse, hPSCs are prone to anoikis, a form of cell death that occurs when cells are enzymatically dissociated [1]. To be efficiently transfected for genome engineering, cells need to be dissociated, which possess a significant problem for genome engineering of hPSCs. Although the rho kinase inhibitor Y-27632 suppresses anoikis [2] and in turn allows some transfection to occur, it is rather inefficient making it difficult to generate enough clones to screen for mutations [3]. One solution, which has recently been suggested, is to reprogram hPSCs to the naive pluripotent state since naive hiPSCs are not sensitive to enzymatic dissociation [4]. While we have successfully used this approach, we believe it is tedious and time-consuming.

[00179] Although the rho kinase inhibitor Y-27632 suppresses anoikis and in turn allows some transfection to occur, it is rather inefficient making it difficult to generate enough clones to screen for mutations.

[00180] We have identified a small molecule (TTNPB), which substantially improves hPSC transfection efficiency when combined with Y-27632, following nucleofection of GFP plasmid. [00181] Compared to untreated cells, which result in almost no GFP ⁺ cells and Y- 27632-treated cells, which only give rise to 17.6% transfection efficiency, TTNPB results in an almost 40% transfection efficiency, when combined with Y-27632 (Fig. 1).

[00182] Moreover, we observe that more GFP is expressed in cells, suggesting that either more plasmid is entering the cell or it is more stable following transfection (Fig. 2).

[00183] Example 2

[00184] In this example, enhanced transfection efficiency of human pluripotent stem cells through the combinatorial effects of retinoic acid analogue (TTNPB), Rho-associated kinase inhibitor (Y-27632), and PPARy agonist (Pioglitazone).

[00185] The concentration of TTNPB may be from about 0.125 to about 1 μΜ. In the specific example here, the working concentration of 0.5 μΜ was used.

[00186] The working concentration of Y-27632 used has 10 μΜ.

[00187] The working concentration of Pioglitazone was 8 μΜ.

[00188] The incubation of the cells with the three small molecules (TTNPB, Y-27632, and Pioglitazone) was 24 h. The small molecules were added to the cells right after transfection (using nucleofection), and were incubated with the cells for 24 h.

[00189] Detailed method: The following method was used.

[00190] Human embryonic stem cells (H9) were harvested using Accutase (StemPro®

Accutase® Cell Dissociation Reagent, Thermo Fisher Scientific, Cat.No. A11 10501)

[00191] The cells were made single using accuatse (4-5 min in 37°C).

[00192] Centrifuge at 200 g, 5 min, at room temperature (RT).

[00193] Count the cells and take an aliquot contain 3 million cells (3 x 10 ⁶ )

[00194] Wash the cells (3 million cells) with PBS- (DPBS, no calcium, no magnesium,

Thermo Fisher Scientific, Cat.No. 14190144), centrifuge at 200 g, 5 min, RT

[00195] Prepare mixed buffer using Nucleofector solution kit (Mouse ES Cell

Nucleofector® Kit (25 RCT), Lonza, Cat. No. VPH-1001) as following:

[00196] Add 91 μΙ supplement to 409 μΙ Nucleofector solution, keep at room temperature

[00197] Add 100 μΙ of mixed buffer to 3 μg GFP-DNA (pmaxGFP, Lonza) in a

RNAase/DNAase-free tube, and mix gently.

[00198] Remove PBS- and add mixed buffer/DNA to the cells.

[00199] Gently pipet up and down (no bubble). [00200] Transfer the cells/DNA mixture into the Cuvette placed in Cuvette holder

[00201] Use Amaxa™ biosystems Nucleofector™ II for electroporation/transfection

[00202] Following program was used for transfection: Mouse ES cells A-023

[00203] After transfection, the cells were seeded on Matrigel (Corning® Matrigel® hESC-Qualified Matrix, BD354277) - coated plates contain pre-warmed mTeSRI media with the following small molecule combinations:

[00204] One plate containing TTNPB + Y-27632 + Pioglitazone

[00205] One plate containing TTNPB + Y-27632

[00206] One plate containing TTNPB + Pioglitazone

[00207] One plate containing Y-27632 + Pioglitazone

[00208] One plate containing TTNPB

[00209] One plate containing Y-27632

[00210] One plate containing Pioglitazone

[00211] Harvest the cells 24 h after transfection using Accutase (the same procedure)

[00212] Asses the percentage of GFP positive cells 24 h after transfection

[00213] Figure 3 depicts hPSCs transfection efficiency. The graphs show the dot plot and histogram of flowcytometry analysis for human pluripotent stem cells (hPSCs) transfected with GFP-DNA (pmaxGFP). The histogram and dot plot graphs demonstrate the percentage of GFP positive cells and mean fluorescent intensity (both are indications of transfection efficiency) in hPSCs following transfection and treatment with TTNPB, Y-27632, and Pioglitazone through various combinations. A) The mock transfected cells (cells transfected with transfection reagent only and not with DNA, treated with TTNPB, Y-27632, and Pioglitazone) did not show any GFP positive cells as expected. B) The transfection efficiency is increased in hPSCs treated with all three small molecules; TTNPB, Y-27632, and Pioglitazone (76.7%) in comparison to the other combinations, and in comparison, to the untreated cells (Mock transfected). Accordingly, the mean fluorescent intensity (expression level of GFP protein) is the highest in hPSCs treated with all three small molecules; TTNPB, Y-27632, and Pioglitazone (26,051). The percentage of GFP positive cells in other combinations of small molecules are as followings: C) TY (49.3%), D) TP (70.2%), E) YP (48.2%), F) T (64.6%), G) Y (40.5%), H) P (56.8%). The mean fluorescent intensity in the cells transfected with other combinations are as followings: TY (7,671), TP (13,670), YP (7,338), T (10,844), Y (6,804), P (8,009). T = TTNPB, Y = Y-27632, P = Pioglitazone, TYP = TTNPB + Y- 27632 + Pioglitazone, TY = TTNPB + Y-27632, TP = TTNPB + Pioglitazone, YP = Y-27632 + Pioglitazone. As shown in Figure 3, the highest % of GFP positive cells in order was: TPY (76.7%); TP (70.2%); T (64.6%)

[00214] Fig. 4 depicts the expression of GFP in hPSCs 24 h after transfection. As shown in Figure 4, the upper panel shows the brightfield microscopic images, and the lower panel shows the fluorescence images of hPSCs transfected with GFP 24 h after transfection. The cells were treated with various combinations of small molecules for 24 h following transfection. The expression of GFP has shown in the fluorescent images. The increased expression of GFP was observed in the cells treated with the three small molecules, TTNPB, Y-27632, and Pioglitazone (fluorescent images, lower panel). T = TTNPB, Y = Y-27632, P = Pioglitazone, TYP = TTNPB + Y-27632 + Pioglitazone, TY = TTNPB + Y-27632, TP = TTNPB + Pioglitazone, YP = Y-27632 + Pioglitazone.

[00215] As shown herein, TTNPB helps to increase the transfection efficiency (% of GFP positive cells) (either alone or in combination), since the groups with TTNPB have the highest percentage of GFP. TTNPB helps for more fluorescent intensity: the groups contain TTNPB show the highest fluorescent intensity.

[00216] TTNPB and Pioglitazone have the same effect regarding the percentage of GFP positive cells (TPY, TP, T, P), however, TTNPB still shows higher mean fluorescent intensity.

[00217] TPY (76.7%) & TP (70.2%) show the highest percentage of GFP positive cells, however, TPY shows almost 2 times more fluorescent intensity compared to TP.

[00218] It seems that the best condition to increase transfection efficiency (both % and mean fluorescent intensity) is having all three molecules together.

[00219] While not wishing to be bound by theory, it may be that that TTNPB enhances transfection by suppressing apoptosis via its interaction with PPARy pathway. We have accumulated circumstantial evidence that supports this hypothesis. Recently we have observed that TTNPB enhances the cloning efficiency of hPSCs [9]. A separate study suggests similarly that the PPARy ligand pioglitazone also enhances hPSC cloning efficiency [10].

[00220] References

[00221] 1) Ohgushi M and Sasai Y (201 1) Lonely death dance of human pluripotent stem cells: ROCKing between metastable cell states. Trends Cell Biol 21 : 257- 320.

[00222] 2) Watanabe K, Ueno M, Kamiya D, Nishiyama A, Matsumura M, Wataya T, Takahashi JB, Nishikawa S, Nishikawa S, Muguruma K, and Sasai Y (2007) A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol 25: 681- 6.

[00223] 3) Chatterjee P, Cheung Y, and Liew C (2011) Transfecting and nucleofecting human induced pluripotent stem cells. J Vis Exp 56: 3110.

[00224] 4) Yang Y, Zhang X, Yi L, Hou Z, Chen J, Kou X, Zhao Y, Wang H, Sun XF, Jiang C, Wang Y, Gao S. (2016) Naive Induced Pluripotent Stem Cells Generated From β-Thalassemia Fibroblasts Allow Efficient Gene Correction Wth CRISPR/Cas9. Stem Cells Transl Med. 5: 8-19.

[00225] 5) Hou P, Li Y, Zhang X, Liu C, Guan J, Li H, Zhao T, Ye J, Yang W, Liu K, Ge J, Xu J, Zhang Q, Zhao Y, and Deng H (2013) Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science 341 : 651-4.

[00226] 6) Schulman IG, Shao G, and Heyman RA (1998) Transactivation by retinoid X receptor-peroxisome proliferator-activated receptor gamma (PPARgamma) heterodimers: intermolecular synergy requires only the PPARgamma hormone-dependent activation function. Mol Cell Biol 18: 3483-94.

[00227] 7) Wu JS, Lin TN, and Wu KK (2009) Rosiglitazone and PPAR-gamma overexpression protect mitochondrial membrane potential and prevent apoptosis by upregulating anti-apoptotic Bcl-2 family proteins. J Cell Physiol 220: 58-71.

[00228] 8) Fuenzalida K, Quintanilla R, Ramos P, Piderit D, Fuentealba RA, Martinez G, Inestrosa NC, and Bronfman M (2007) Peroxisome proliferator-activated receptor gamma up-regulates the Bcl-2 anti-apoptotic protein in neurons and induces mitochondrial stabilization and protection against oxidative stress and apoptosis. J Biol Chem. 282: 37006-15.

[00229] 9) Meng G, Hsu C and Rancourt DE (2017) Enhanced clonal colony formation of human pluripotent stem cells through the combinatorial effects of retinoic acid analogue and rho-associated kinase inhibitor. Till and McCulloch Meeting. October 24-26; Whistler, BC.

[00230] 10) Kajabadi NS, Ghoochani A, Peymani M, Ghaedi K, Kiani-Esfahani A, Hashemi MS, Nasr-Esfahani MH, and Baharvand H (2015) The Synergistic Enhancement of Cloning Efficiency in Individualized Human Pluripotent Stem Cells by Peroxisome

Proliferative-activated Receptor-γ (PPARv) Activation and Rho-associated Kinase (ROCK) Inhibition. J Biol Chem 290: 26303-13. [00231] 11) Butts BD, Tran NL, and Briehl MM (2004) Identification of a functional peroxisome proliferator activated receptor response element in the 3' untranslated region of the human bcl-2 gene. Int J Oncol. 24: 1305-10.

[00232] 12) Hsu CY and Rancourt DE (2015) En route to non-viral reprog ramming: A multiplexed approach to simultaneously track the kinetics of DNA uptake and transgene expression following non-viral gene delivery of multiple episomal plasmid DNAs. Till & McCulloch Meeting, Toronto, ON. Oct 26-28.

[00233] 13) Shen, Zhang W, Zhang J, Zhou J, Wang J, Chen L, Wang L, Hodgkins A, lyer V, Huang X, Skarnes WC. (2014) Efficient genome modification by CRISPR-Cas9 nickase with minimal off- target effects. Nat Methods. 11 : 399-402.

[00234] The embodiments described herein are intended to be examples only.

Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.

[00235] All publications, patents and patent applications mentioned in this

Specification are indicative of the level of skill those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication patent, or patent application was specifically and individually indicated to be incorporated by reference.

[00236] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modification as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.