Method for inducing aneuploidy and / or chromosomal instability in human or non-human primate pluripotent stem cells and diagnostic / therapeutic uses thereof

Abstract

The invention provides a novel screening platform and aneuploidal pluripotent stem cells and compositions containing for use in the identification of genes / pathways affected by different ploidy states or chromosomal stability and therapeutic agents that selectively act on cells of different ploidy states or chromosomal stability. In particular the present invention provides novel methods for inducing aneuploidy and / or chromosomal instability in human pluripotent stem cells (hPSCs) or non-human pluripotent stem cells (nhpPSCs). The present application also relates to the use of such methods and aneuploid hPSCs or nhpPSCs obtained thereby for identifying genes which are affected by different ploidy states and / or chromosomal stability and for identifying therapeutic agents that selectively or preferentially act on cells of different ploidy states and / or which affect chromosomal stability.

Description

Method for Inducing Aneuploidy and / or Chromosomal Instability in Human or NonHuman Primate Pluripotent Stem Cells and Diagnostic / Therapeutic Uses ThereofRELATED APPLICATIONS[1] The present application claims benefit of priority to US Provisional Application Nos.: 63 / 597,834, filed on November 10, 2023, and 63 / 642,042 filed May 3, 2024, the contents of each and ail of which are incorporated by reference in its entirety.GRANTS[2] [1] The instant application was made with government support under grant no.R01HD101436 awarded by the National Institute Child Health and Development ("NICHD"). The government has certain rights in the invention.FIELD[3] The invention provides a method for producing aneuploidal pluripotent stem ceils and compositions and cell cultures containing which may be used in the identification of genes / pathways affected by different pioidy states or chromosomal stability and therapeutic agents that selectively act on cells of different pioidy states or chromosomal stability. In particular the present invention provides novel methods for inducing aneuploidy and / or chromosomal instability in human pluripotent stem cells (hPSCs) or non-human pluripotent stem cells (nhpPSCs). The present application also relates to the use of such methods and aneuploid hPSCs or nhpPSCs obtained thereby for identifying genes which are affected by different pioidy states and / or chromosomal stability. The present application further relates to use of such methods and aneuploid hPSCs obtained thereby for identifying therapeutic agents that selectively or preferentially act on cells of different pioidy states and / or which affect chromosomal stability.BACKGROUND[4] Euploidy refers to the normal chromosome complement of cells and is an exact multiple of the haploid chromosome number (n), which is defined as a single set ofchromosomes. In human cells, the euploid number of chromosomes is 46 which consists of 23 chromosome pairs. Hence for human cells, the normal euploid chromosome complement is a diploid state (2n = 46) or double the haploid number (n = 23). In contrast, aneuploidy is defined as a state of abnormal chromosome numbers and is subdivided into numerical whole chromosome or structural aneuploidies. Numerical whole chromosome aneuploidies are due to the gain or loss of whole chromosomes resulting from chromosome missegregation during ceil division (meiosis for gamete formation or mitosis for non- reproductive cells). In addition, cells with more than two complete sets of whole chromosomes are referred to as polyploids. Polyploidy can be the natural state of euploidy for some human cells (e.g. hepatocytes) whereas for most human cell types polyploidy is an abnormal state that is caused by errors in ceil division (e.g. mitotic slippage) or non-ceil division events (e.g. cell fusion). While structural aneuploidies are segmental chromosome copy number alterations caused by DNA damage (Orr B et al., "Aneuploidy", Current Biology. 2015;25(13):R538-~R542. PMID: 26126276).[5] Overall, aneuploidy is associated with a variety of human conditions and diseases including, but not limited to, cancer, congenital birth defects, infertility, aging, and rare diseases (e.g. mosaic variegated aneuploidy). In cancers, 90% of solid tumors and 75% of hematological malignancies are estimated to be aneuploid (Weaver BA, Cleveland DW, "Does aneuploidy cause cancer?", Current Opinion in Cell Biology. 2006;18(6):658~667. PMID: 17046232; Zasadil LM et al., "2n or not 2n: Aneuploidy, polyploidy and chromosomal instability in primary and tumor cells", Semin Cell Dev Biol. 2013;24(4):370~379. PMCID: PMC3736819), making aneuploidy a near universal feature of cancers. Furthermore, cancer ceils frequently display the related phenomenon of chromosomal instability (CIN) which is a persistent and high rate of chromosome missegregation that when coupled with the continued survival and propagation of aneuploid cancer ceils leads to intra- and inter- genetic heterogeneity among cancer ceils (Godek KM et al., "Chromosomal Instability Affects the Tumorigenicity of Glioblastoma Tumor-Initiating Ceils", Cancer Discovery. 2016;6(5):532~545. PMID: 27001151; Burrell RA et al., "The causes and consequences of genetic heterogeneity in cancer evolution", Nature, 2013; 501(7467): naturel2625, PMID: 24048066). The genetic heterogeneity caused by aneuploidy and CIN also contributes to cancer metastasis (Vasudevan A et al., "Aneuploidy as a promoter and suppressor ofmalignant growth", Nat Rev Cancer, 2021;21(2):89-103, PMID: 33432169), resistance to cancer treatment methods and therapeutics (Ippolito MR et al., "Gene copy-number changes and chromosomal instability induced by aneuploidy confer resistance to chemotherapy”, Dev Cell, 2021; PMID: 34352223; Lukow DA et al., "Chromosomal instability accelerates the evolution of resistance to anti-cancer therapies”, Dev Ceil, 2021; PMID: 34352222), and immune evasion (Vasudevan A et al, 2021 (Id.)), all of which promote disease progression and poor patient prognosis.[6] Moreover, in the context of human fertility and development, aneuploidy is the primary cause of miscarriages, occurring in ~50%, and congenital birth defects ((Orr B et al., 2006, (Id.)); Roper RJ, Reeves RH., "Understanding the Basis for Down Syndrome Phenotypes", PLoS Genetics, 2006;2(3):e50. PMID: 16596169). The most common congenital birth defect in humans, Down Syndrome, is caused by having three copies of chromosome 21 (trisomy 21) (Roper RJ, Reeves RH., 2006 (Id.). Moreover, the prevalence of aneuploidy also increases during natural human aging where it is associated with adverse characteristic such as cellular senescence (Macedo JC et al., "FoxMl repression during human aging leads to mitotic decline and aneuploidy-d riven full senescence", Mat Commun. 2018;9(l):2834. PMCID: PMC605342511).[7] Also, aneuploidy is a cause of some rare human diseases such as mosaic variegated aneuploidy (MVA) which is estimated to affect less than 1,000 people in the United States (Orr B et al., 2006, (Id.)). Furthermore, aneuploidy poses a major challenge to regenerative medicine approaches that use human pluripotent stem cells (hPSCs), including embryonic and induced PSCs, because during prolonged in vitro propagation spontaneously arising aneupioid hPSCs often overtake a population due to culture adaptation that selects for clonal and stable aneupioid karyotypes with a fitness advantage over diploid hPSCs, raising concerns for their therapeutic use (Baker D.E.C. et al., (2007), "Adaptation to culture of human embryonic stem cells and oncogenesis in vivo", Nature Biotechnology 25, nbtl285. 10.1038 / nbtl285; Mayshar Y et al, (2010). Identification and Classification of Chromosomal Aberrations in Human Induced Pluripotent Stem Cells. Cell Stem Cell i, 521- 531. 10.1016 / j. stem.2010.07.017; Price C.J. et al, (2021), "Genetically variant human pluripotent stem cells selectively eliminate wild-type counterparts through YAP-mediated cell competition", Dev Cell 56, 2455-2470.el0. 10.1016 / j.devcel.2021.07.019; Taapken, S.M.et al, (2011), "Karyotypic abnormalities in human induced pluripotent stem cells and embryonic stem cells”. Nature Biotechnology 29, 313. 10.1038 / nbt.l835). Currently, there is a substantial unmet clinical need to identify genes and pathways that cause aneuploidy and / or CIN and which contribute to their pathological consequences as well as developing therapeutic agents that selectively act on cells of different ploidy states and / or chromosomal stability.[8] Based on the foregoing, new and improved methods and compositions for use in identifying genes and pathways that cause aneuploidy and / or CIN and which contribute to their pathological consequences as well as developing therapeutic agents that selectively act on ceils of different ploidy states and / or chromosomal stability are sorely needed. This invention meets these objectives as the invention provides novel screening methods and compositions for use therein that potentially may be used to identify genes and pathways that cause aneuploidy and / or CIN which contribute to their pathological consequences as well as the identification of therapeutic agents that selectively act on cells of different ploidy states and / or chromosomal stability.SUMMARY[9] The present application generally relates to methods for inducing aneuploidy and / or chromosomal instability in human pluripotent stem cells (hPSCs) or non-human primate (nhpPSCs). The present application also relates to use of such methods for identifying genes which are affected by different ploidy states and / or chromosomal stability. The present application further relates to use of such methods for identifying therapeutic agents that selectively or preferentially act on cells of different ploidy states and / or which affect chromosomal stability. Therapeutic discoveries which may be realized using the inventive screening methods may include developing agents that selectively eliminate cells with a particular ploidy state and / or chromosomal stability, suppressing or enhancing chromosomal instability, or alleviating the pathological consequences of having a specific ploidy state and / or chromosomal stability.

[0010] Specifically, as is disclosed in detail infra, the inventors have developed a novel method for creating mixed ceil cultures comprising aneupioid and chromosomaily unstablehuman pluripotent stem cells that is advantageous over existing methods used to create aneuploid and / or chromosomaily unstable hPSCs and other aneuploid / chromosomally unstable human cell types.OBJECTS OF THE INVENTION

[0011] Accordingly, it is an object of the invention to provide novel methods for inducing aneuploidy and / or chromosomal instability in human or non-human primate pluripotent stem cells (hPSCs or nhpPSCs).

[0012] It is another object of the invention to use these methods for identifying genes which are affected by different ploidy states and / or chromosomal stability.

[0013] It is another object of the invention to use these methods for identifying therapeutic agents that selectively or preferentially act on cells of different ploidy states and / or chromosomal stability and / or which suppress or enhance the rate of chromosomal instability.

[0014] It is a specific object of the invention to provide a method for creating a mixed ceil population comprising human pluripotent stem cells (hPSCs) or non-human primate pluripotent stem cells (nhpPSCs) which are aneuploidal and / or exhibit chromosomal instability (CIN) by contacting a cell sample comprising eupioid (diploid) hPSCs or nhpPSCs, optionally wherein all or substantially all of the hPSCs or nhpPSCs in the cell sample are eupioid (diploid) prior to contacting the cell sample comprising eupioid (diploid) hPSCs or nhpPSCs with at least one compound that induces chromosome missegregation, which method comprises contacting said cell sample comprising eupioid (diploid) hPSCs or nhpPSCs with at least one compound that induces chromosome missegregation for a time sufficient to produce a mixed cell population wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more of the hPSCs or nhpPSCs in the cell sample are aneuploidal and / or exhibit chromosomal instability (CIN).

[0015] It is a specific object of the invention to provide a method as in any of the above, wherein all or substantially all (at least 90, 95, 99% or more) of the human pluripotent stem cells (hPSCs) or non-human primate pluripotent stem cells (nhpPSCs) in the cell sample are eupioid (diploid) prior to contacting with said at least one compound.

[0016] It is a specific object of the invention to provide a method as in any of the above, wherein the cell sample prior to contacting with said at least one compound comprises at least 103, 103, 105, 10s, 107, 108, 109, or more euploid (diploid) hPSCs or nhpPSCs.

[0017] It is a specific object of the invention to provide a method as in any of the above, wherein the dosage of said at least one compound results in at least 10%, 20%, 30%, 40%, 50%, or the majority (over 50%) of the hPSCs or nhpPSCs after treatment becoming aneuploidal and / or exhibiting chromosomal instability (CIN).

[0018] It is a specific object of the invention to provide a method as in any of the above, wherein the dosage of said at least one compound results in at least 60%, 70%, 80% or 90% of the human pluripotent stem cells (hPSCs) or non-human primate pluripotent stem cells (nhpPSCs) becoming aneuploidal and / or exhibiting chromosomal instability (CIN).

[0019] It is a specific object of the invention to provide a method as in any of the above, wherein the human and non-human primate embryonic stem ceils (hESCs) are derived from:(i) human or non-human primate blastocysts,(ii) embryos unused after a fertility treatment,(iii) human or non-human primate nuclear transfer embryos,(iv) non-human primate or human induced pluripotent stem cells (hiPSCs) reprogrammed from a somatic state to a pluripotent state,(v) an existing human or non-human primate ESC line, optionally Hl; or(vi) any combination of the foregoing.

[0020] It is a specific object of the invention to provide a method as in any of the above, wherein the at least one compound that causes chromosome missegregation comprises a microtubule inhibitor, optionally an inhibitor of microtubule dynamics or an inhibitor of microtubule ~ kinetochore attachment.

[0021] It is a specific object of the invention to provide a method as in any of the above, wherein the at least one compound that causes chromosome missegregation includes one or more of taxanes, vinca alkaloids, optionally one or more of eribulin, paclitaxel, docetaxel, Ixabepiione, Cabazitaxei, antibody drug conjugates comprising vedotion, e.g., Enfortumabvedotin,, Tisotumab vedotin, or Brentuximab vedotin; Trastuzumab emtansine, Tirbanibulin, Paclitaxel ceribate, Vinblastine, vincristine, Vinorelbine, vinflunine, Cryptophycin 52, Halichondrin B, Dolastatins, e.g, Dolastatins 10 and 15; Hemiasterlins, e.g., Hemiasterlin A and Hemiasterlin B; Colchicine, Combretastatins, E7010, a sulfonamide antimitotic agent, 2- Methoxyestradiol, Docetaxel, Paclitaxel, Epothilones, e.g., Epothilone A and epothilone B; and Discodermolide.

[0022] It is a specific object of the invention to provide a method as in any of the above, wherein the at least one compound that causes chromosome missegregation comprises reversine or another inhibitor of a mitotic kinase optionally an inhibitor of monopolar spindle 1 (Mpsl) kinase.

[0023] It is a specific object of the invention to provide a method as in any of the above, wherein the at least one compound that causes chromosome missegregation comprises a compound which inhibits one or more kinases or microtubule motor proteins, optionally a compound which inhibits one or more of AurkA, AurkB, Pan-AurK, Eg5, PLK1, Mpsl, CENPE and CDK1.

[0024] It is a specific object of the invention to provide a method as in any of the above, wherein the at least one compound includes one or more of Alisertib (MLN8237), ENMD- 981693 / ENMD-2076, Danusertib (PHA- 739358), MLN8054, MK-5108 (VX-689), MK-8745, Barasertib (AZD1152-HQPA, AZD2811), BI811283, GSK1070916, PF-03814735, ZM-447439, XL-228, TozasertibfVX- 680 / MK-0457), CYC-116, SNS-314, AMG 900, JN.i-7706621, Cenisertib (AS703569, MSC1992371A), AT9283, Rigosertib (ON 01910), Volasertib (BI6727), Onvansertib (NMS- P937), BI2.536, GSK461364, Tak9960, Poloxin-2, S81694 (NMS-P153), BOS172722, Empesertib (BAY1161909), BAY1217389, Mpsl-IN-1, Mpsl-IN-2, Mpsl-IN-3, TC- Mpsl- 12, AZ3146, NMS-P715, MPI-0479605, CCT251455lspinesib (SB-715992), Filanesib (ARRY-520) SB-743921, Litronesib (LY252335), ARQ-621, MK-0731, EMD-534085. 4SC- 205, acin, PF-2771, GSK-923295, APC / C, 4SC-205, 4SC-205, Danusertib (PHA-739358), MLN8054, MK-5108 (VX-689). MK-8745, Alisertib (MLN8237), ENMD-981693 / ENMD- 2076, Danusertib (PHA-739358), MLN8054, MK-5108 (VX-689), MK-8745, Barasertib (AZD1152-HQPA, AZD2811), BI811283, GSK1070916, PF-03814735, ZM-447439, XL-228, Tozasertib(VX- 680 / MK-0457), CYC-116, SNS-314, AMG 900, JNJ-7706621, Cenisertib (AS703569, MSC1992371A), AT9283, Rigosertib (ON 01910), Volasertib (BI6727), Onvansertib (NMS-P937), BI2536, GSK461364, Tak9960, Poloxin-2, S81694 (NMS-P153), BOS172722, Empesertib (BAY1161909), BAY1217389, Mpsl-IN-1, Mpsl-IN-2, Mpsl-IN-3, TC-Mpsl-12, AZ3146, NMS-P715, M PI-0479605, CCT251455, Ispinesib (SB-715992), Fiianesib (ARRY-520), SB-743921, AZD4877, Litronesib (LY2523355), ARQ-621, MK-0731, EMD-534085, 4SC-205, PF-2771, GSK-923295, Compound-A, APC / C and Apcin.

[0025] It is a specific object of the invention to provide a method as in any of the above, wherein the dosage amount of the at least one compound that causes chromosome missegregation, optionally reversine, ranges from about 1 nM to 10,0000 nM, 10-1000 nM, 100-400 nM, 100-300 nM or 100-200 nM.

[0026] It is a specific object of the invention to provide a method as in any of the above, wherein the at least one compound that causes chromosome missegregation comprises or consists of reversine, and the hPSCs or nhpPSCs are contacted therewith, optionally at a dosage of 100-400 nM, further optionally 100-200 nM, ~150 nM or~100nm for at least 4 hours, at least 8 hours, least 12 hours, least 16 hours, least 24 hours, least 32 hours, least 40 hours, least 48 hours, or longer.

[0027] It is a specific object of the invention to provide a method as in any of the above, wherein the at least one compound that causes chromosome missegregation is removed or inactivated after at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more of the hPSCs or nhpPSCs in the cell sample are aneuploidal and / or exhibit chromosomal instability (CIN) and / or after the majority (over 50%) of the hPSCs or nhpPSCs in the cell sample are aneuploidal and / or exhibit chromosomal instability (CIN).

[0028] It is a specific object of the invention to provide a method as in any of the above, wherein a substantial number, optionally at least 10-40% or 30-50% of the resultant aneuploidal hPSCs or nhpPSCs retain aneuploidy for a prolonged time after the at least one compound that induces chromosome missegregation, optionally reversine, is removed or inactivated, optionally by washing.

[0029] It is a specific object of the invention to provide a method as in any of the above, wherein a substantial number, optionally 10-40% or at least 30-50% of the resultant aneuploidal hPSCs or nhpPSCs retain aneuploidy for at least a week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or more after the at least one compound that induceschromosome missegregation, optionally reversine, is removed or inactivated, optionally by washing.

[0030] It is a specific object of the invention to provide a method as in any of the above, wherein a substantial number, optionally at least 10-40% or at least 30-50% of the resultant aneuploidal hPSCs or nhpPSCs in the cell sample remain viable (continue to proliferate) when cultured in the presence of reservine or another compound that induces chromosome missegregation for a prolonged duration, optionally for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or more after creation.

[0031] It is a specific object of the invention to provide a method as in any of the above, wherein the aneuploidal hPSCs or nhpPSCs express pluripotency transcription factors, optionally one or all of MYC, SOX2, NANOG, or OCT4 and / or cell cycle arrest proteins optionally p53 and p21, comparably to diploid hPSCs or nhpPSCs for prolonged duration, optionally for at least 1 week, 2 weeks, 3 weeks, 4 weeks or more after creation.

[0032] It is a specific object of the invention to provide a method as in any of the above, which generates a population of hPSCs or nhpPSCs comprising random, variable aneuploid karyotypes.

[0033] It is a specific object of the invention to provide a method as in any of the above, which generates a population of hPSCs or nhpPSCs comprising different ploidy states and / or chromosomal stabilities.

[0034] It is a specific object of the invention to provide a method as in any of the above, which generates a population of hPSCs or nhpPSCs that are tolerant of chromosomal instability for a prolonged duration, optionally for at least 1 week, 2 weeks, 3 weeks, 4 weeks or more after creation.

[0035] It is a specific object of the invention to provide a method as in any of the above, wherein the resultant aneuploidal hPSCs or nhpPSCs and / or hPSCs or nhpPSCs exhibiting chromosomal instability (Cl N) when cultured produce confluent cultures which may be passaged one or more times.

[0036] It is a specific object of the invention to provide a method as in any of the above, which further includes culturing the resultant aneuploidal hPSCs or nhpPSCs and / or hPSCsor nhpPSCs exhibiting chromosomal instability (CIN) on a solid surface or plate, optionally comprising a matrix of Matrigei or Laminin-521.

[0037] It is a specific object of the invention to provide a method as in any of the above, which further includes culturing the resultant aneuploidal hPSCs or nhpPSCs and / or hPSCs or nhpPSCs exhibiting chromosomal instability (CIN) on a solid surface or plate, optionally comprising a matrix of Matrigei or Laminin-521, wherein after the cultured cells result in a confluent culture, optionally at around 3-5 days, they are passaged one or more times.

[0038] It is a specific object of the invention to provide a method as in any of the above, wherein the aneuploidal hPSCs or nhpPSCs and / or hPSCs or nhpPSCs exhibiting chromosomal instability (CIN) are cultured in the presence of the compound that resulted in chromosome missegregation, optionally reversine, and continue to express pluripotency transcription factors, optionally MYC, SOX2, NANOG, or OCT4 and cell cycle arrest proteins, optionally p53 and p21, characteristic of normal (diploid) hPSCs or nhpPSCs.

[0039] It is a specific object of the invention to provide an isolated mixed cell population exhibiting aneuploidal hPSCs or nhpPSCs and / or hPSCs or nhpPSCs comprising chromosomal instability (CIN) produced by the method of any one of the above.

[0040] It is a specific object of the invention to provide an isolated mixed cell population exhibiting aneuploidal hPSCs or nhpPSCs and / or hPSCs or nhpPSCs comprising chromosomal instability (CIN) produced by the method of any one of the above, which is derived using a specific hPSC line, optionally Hl, H9, or WTC-11 hPSCs.

[0041] It is a specific object of the invention to provide an isolated mixed cell population as above, wherein at least 30%, 40%, 50%, 60%, 70%, 80% or 90% or more of the hPSCs or nhpPSCs are aneuploidal and / or exhibit chromosomal instability (CIN).

[0042] It is a specific object of the invention to provide an isolated mixed cell population as in any of the above, wherein the aneuploidal hPSCs or nhpPSCs are not genetically modified by any other means.

[0043] It is a specific object of the invention to provide an isolated mixed cell population as in any of the above, wherein the aneuploidal hPSCs or nhpPSCs are engineered to comprise a marker, optionally H2B-GFP, that permits monitoring of mitotic errors and / or proliferation which does not itself induce aneuploidy / CIN.

[0044] It is a specific object of the invention to provide an isolated mixed cell population as in any of the above, which is modified after production to include diploid PSCs, optionally of the same HLA or genotype (matched cells), further optionally wherein the ratio of aneuploid to normal diploid cells mimics that of a particular disease condition, optionally mosaic variegated aneuploidy (MVA).

[0045] It is also an object of the invention to provide a confluent cell culture comprising the mixed cell population of any one of the above.

[0046] It is also an object of the invention to provide the mixed cell population or confluent cell culture of any one of the above which comprises at least one compound that induces chromosome missegregation, optionally reversine.

[0047] It is also an object of the invention to provide a method of using human pluripotent stem cells (hPSCs) or non-human primate pluripotent stem cells (nhpPSCs) which are aneuploidal and / or comprise chromosomal instability (CIN ) produced according to any one of the above or the mixed cell population or cell culture of any of the above in drug studies.

[0048] It is also an object of the invention to provide a method of using human pluripotent stem cells (hPSCs) or non-human primate pluripotent stem cells (nhpPSCs) which are aneuploidal and / or comprise chromosomal instability (CIN) produced according to any one of the above or the mixed ceil population or cell culture of any of the above for modeling mixed environments of diploid and aneuploid cells using matched cells, optionally for study of diseases such as MVA and / or for identifying drugs for treatment thereof.

[0049] It is also an object of the invention to provide a method of using human pluripotent stem ceils (hPSCs) or non-human primate pluripotent stem cells (nhpPSCs) which are aneuploidal and / or comprise chromosomal instability (CIN) produced according to any one of the above or the mixed cell population or cell culture of any of the above in screening assays for identifying drugs which selectively kill aneuploid cells and / or cells exhibiting chromosomal instability (CIN).

[0050] It is also an object of the invention to provide a method of using human pluripotent stem cells (hPSCs) or non-human primate pluripotent stem cells (nhpPSCs) which are aneuploidal and / or comprise chromosomal instability (CIN) produced according toany one of the above or the mixed cell population or cell culture of any of the above in screening assays for study of different ploidy states and / or chromosomal instabilities and / or genes involved in aneuploidy or chromosomal instability.

[0051] It is also an object of the invention to provide a method of using human pluripotent stem cells (hPSCs) or non-human primate pluripotent stem cells (nhpPSCs) which are aneuploidal and / or comprise chromosomal instability (ON) as above, which includes comparing the effect of at least one screened moiety, optionally a drug, on human pluripotent stem ceils (hPSCs) or non-human primate pluripotent stem cells (nhpPSCs) which are aneuploidal and / or exhibit chromosomal instability (ON) to the effect of the same screened moiety on normal (diploidaI) hPSCs or nhpPSCs, optionally normal (diploidal) hPSCs or nhpPSCs of the same HLA or genotype (matched cells).

[0052] It is another object of the invention to provide a method of using human pluripotent stem cells (hPSCs) or non-human primate pluripotent stem cells (nhpPSCs) which are aneuploidal and / or exhibit chromosomal instability (CIN) produced according to any of the previous methods in screening methods for identifying agents that selectively eliminate ceils with a particular ploidy state and / or chromosomal stability, that suppress or enhance chromosomal instability, and / or which alleviate the pathological consequences of having a specific ploidy state and / or chromosomal stability.BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIGURE 1A-H contains the results of experiments demonstrating that inhibiting MPS1 kinase increases mitotic errors and aneuploidy in hPSCs in experiments designed to test if inhibiting MPS1 kinase increases mitotic errors and aneuploidy in hPSCs. FIGURE 1A shows the percent of anaphase errors quantified from time-lapse live-cell microscopy of Hl H2B-GFP hPSCs treated with DMSO, 75 nM, or 100 nM reversine (Rev). In these experiments Hl H2B-GFP hPSCs were imaged for 6-7 hours in the presence of DMSO or reversine and mitotic errors were quantified during this time period. Lagging and unaligned chromosomes cause numerical, whole chromosome aneuploidies while chromosome bridges cause structural aneuploidies. n > 45 cells. FIGURES 1B-H show copy numbers of chromosomes 2,8, 10, 12, or 17 in Hl H2B-GFP hPSCs treated with DMSO or 100 nM reversine for 24h, 48h, or 96h. Copy numbers were determined using fluorescent in situ hybridization (FISH) and interphase cells were scored. FIGURE IB contains representative images which show aneuploid trisomic (white arrow) or monosomic (yellow arrowhead) cells. Scale bars: 10 pm. FIGURE 1C shows the aneusomic percent, including gains and losses, of chromosomes 2, 8, 10, 12, and 17. For each chromosome, the aneusomic percent for reversine treated cells was compared to their respective DMSO controls, with 48h reversine compared to 24h DMSO. The copy number distribution of chromosomes 2 (FIGURE ID), 8 (FIGURE IE), 10 (FIGURE IF), 12 (FIGURE 1G), or 17 (FIGURE 1H). n = 1500 cells pooled per FISH probe; mean ± SD. Three independent experiments; ns p > 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 using a two-tailed Student's t-test.

[0054] FIGURE 2A-E shows pluripotency transcription factor expression, karyotype analysis, anaphase error rates, and mitotic durations in Hl hPSCs, Hl H2B-GFP hPSCs, or RPE-1 H2B-GFP cells relating to the experiments in FIGURE 1A-H and contain experimental results showing pluripotency transcription factor expression, karyotype analysis, anaphase error rates, and mitotic durations in Hl hPSCs, Hl H2B-GFP hPSCs, or somatic RPE-1 H2B- GFP cells. FIGURE 2A shows representative immunofluorescence (IF) images of somatic RPE- 1 H2B-GFP cells, Hl H2B-GFP hPSCs, and Hl hPSCs that show DNA (cyan or yellow), OCT4 (magenta), NANOG (green), SOX2 (magenta), or MYC (magenta). Scale bars: 100 pm. FIGURE 2B shows the quantification of OCT4, NANOG, SOX2, or MYC protein levels by IF in somatic RPE-1 H2B-GFP cells, Hl H2B-GFP hPSCs, and Hl hPSCs. n = 300 cells; mean ± SD. FIGURE 2C is a G-banding karyotype analysis of twenty Hl H2B-GFP hPSCs showing no clonal abnormalities. FIGURE 2D contains representative IF images of mitotic errors that cause numerical or structural aneuploidies from time-lapse live-cell microscopy of Hl H2B-GFP hPSCs treated with 100 nM reversine. Scale bar: 10 pm. FIGURE 2E shows mitotic duration (left), prometaphase duration (middle) and metaphase duration (right) of Hl H2B-GFP hPSCs treated with DMSO, 75 nM, or 100 nM reversine. NEB, nuclear envelope breakdown; AO, anaphase onset; n > 45 cells; mean ± SD. Three independent experiments; ns p > 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 using a one-way ANOVA and Tukey's multiple comparisons test (FIGURE 2B) or a one-way ANOVA and Dunnett's multiple comparisons test (FIGURE 2E).

[0055] FIGURES 3A-E show the results of experiments designed to test the aneuploidy tolerance of hPSCs which demonstrate that HPSCs tolerate aneuploidy. FIGURE 3A shows the experimental schemes to measure the proliferation, p53 and p21 protein levels, pluripotency transcription factor protein levels, or proportion of aneuploid ceils for somatic RPE-1 cells grown in reversine (top panel) or hPSCs grown in the presence and upon the removal of reversine (bottom panel). FIGURE 38 compares the proliferation of diploid (DMSO controls) vs. aneuploid (reversine) cells. RPE-1 H2B-GFP cells, Hl and Hl H2B-GFP hPSCs, H9 and H9 H2B-GFP hPSCs, and WTC-11 and WTC-11 H2B-GFP hPSCs were treated with DMSO or 100 nM reversine for 96h to induce aneuploidy. During the course of treatment, total cell numbers were measured daily. Also, for hPSCs, DMSO or reversine was washed out and total cell numbers were measured daily for an additional 96h. Each time point is normalized to Oh. Mean ± SD, FIGURE 3C shows the quantification of MYC, OCT4, NANOG, SOX2, p53, or p21 protein levels by IF in diploid (DMSO controls) Hl and Hl H2B- GFP hPSCs vs. aneuploid (reversine) Hl H2B-GFP hPSCs. n = 300 cells; mean ± SD, FIGURE 3D shows the quantification of MYC, OCT4, NANOG, SOX2, p53, or p21 protein levels by IF in diploid (DMSO controls) H9 and H9 H2B-GFP or WTC-11 and WTC-11 H2B-GFP hPSCs vs. aneuploid (reversine) H9 H2B-GFP or WTC-11 H2B-GFP hPSCs. n - 300 cells; mean ± SD. FIGURE 3E shows aneusomic percent, including gains and losses, determined using FISH for chromosomes 2, 8, 10, 12, or 17 in Hl and Hl H2B-GFP hPSCs, H9 and H9 H2B-GFP hPSCs, or WTC-11 and WTC-11 H2B-GFP hPSCs upon removal of DMSO or reversine for 96h. For each chromosome, the aneusomic percent of their respective DMSO and reversine treated cells were compared, n - 1500 cells per FISH probe; meant SD. Three independent experiments; ns p > 0.05, ***p < 0.001, and ****p < 0.0001 a one-way ANOVA with Tukey's correction (FIGURES 3C and D) or a two-tailed Student's t-test (FIGURE 3E).

[0056] FIGURE 4A-J show mitotic duration, anaphase error rates, aneusomic rates, and p53 and p21 levels in somatic RPE-1 H2B-GFP cells. Hl hPSCs, or Hl H2B-GFP hPSCs relating to the experiments of FIGURE 3A-E. FIGURE 4A contains representative IF images of RPE-1 H2B-GFP or Hl H2B-GFP hPSCs treated with DMSO or reversine for 96h. Shown is DNA (cyan), p53 (yellow), and p21 (magenta). Scale bars: 10 pm. FIGURE 4B shows quantification of p53 and p21 protein levels by IF in diploid (DMSO control) vs. aneuploid (reversine) RPE-1 H2B-GFP cells, n = 300 cells; mean ± SD. FIGURE 4C shows mean mitotic duration,prometaphase duration, or metaphase duration, and mitotic error rates of RPE-1 H2B-GFP ceils treated with DMSO or 100 nM reversine. FIGURE 4D shows copy numbers of chromosomes 2, 8, 10, or 12 in RPE-1 H2B-GFP ceiis treated with DMSO or 100 nM reversine for 24h determined using FISH. interphase ceiis were scored for copy numbers.Representative FISH images show aneupioid trisomic (white arrows) or monosomic (yeiiow arrowheads) ceiis. Scale bars: 10 pm. FIGURE 4E shows aneusomic percent, including gains and losses, and copy number distribution of chromosomes 2, 8, 10, or 12 for RPE-1 H2B GFP cells. For each chromosome, the aneusomic percent of their respective DMSO and reversine treated ceils were compared (left panel). Copy number distribution of chromosomes 2, 8, 10, and 12 (right panels), n = 1500 cells pooled per FISH probe; meant SD. FIGURE 4F contains representative IF images of p53 protein levels in somatic RPE-1 H2B-GFP cells, Hl H2B-GFP hPSCs, and Hl hPSCs treated with DMSO or 1 pM doxorubicin for 4h. Shown is DNA (cyan) and p53 (green). FIGURE 4G shows the quantification of p53 protein levels in somatic RPE-1 H2B-GFP cells, Hl H2B-GFP hPSCs, and Hl hPSCs treated with DMSO or 1 pM doxorubicin for 4h. n = 300 cells; mean ± SD. FIGURE 4H contains representative IF images of p21 protein levels in somatic RPE-1 H2B-GFP ceils, Hl H2B-GFP hPSCs, and Hl hPSCs treated with DMSO or 1 pM doxorubicin for 4h. Shown is DNA (cyan) and p21 (magenta). FIGURE 41 shows the quantification of p21 protein levels in somatic RPE-1 H2B-GFP ceils, Hl H2B-GFP hPSCs, and Hl hPSCs treated with DMSO or 1 pM doxorubicin for 4h. n = 300 cells; mean ± SD. FIGURE 4J shows the percent of live Hl or Hl H2B-GFP hPSCs after DMSO or 1 pM doxorubicin for 24h. Mean ± SD. Three independent experiments (FIGURES 48, E, G, and I) and two independent experiments (FIGURE 4C and J); *p < 0.05, ** p < 0.01, and0.0001 using a two-tailed Student’s t-test.

[0057] FIGURE 5A-D contains experimental results demonstrating that aneupioid hPSCs are chromosomally unstable and that hPSCs proliferate upon long-term MPS1 inhibition obtained in experiments designed to assess whether induction of aneuploidy in hPSCs fuels further chromosomal instability, aneupioid hPSCs maintain an undifferentiated state, and hPSCs tolerate persistent chromosome missegregation upon long-term reversine (Rev) treatment with growth comparable to normal hPSCs. FIGURE 5A contains the experimental scheme to measure mitotic error rates and pluripotency transcription factor protein levels for hPSCs grown in the presence and upon the removal of reversine. FIGURE 58 containsexperimental resuits wherein Hl hPSCs were treated with DMSO or 100 nM reversine for 96h to induce aneuploidy, subsequently, the DMSO or reversine was washed out and Hl hPSCs were grown for an additional 96h. Mitotic errors, including lagging chromosomes, chromosome bridges, acentric DNA fragments, unaligned chromosomes, multipolar anaphases, and a combination of errors, were quantified using IF. n > 300 anaphases scored. FIGURE 5C shows the quantification of OCT4 or NANOG protein levels by IF in diploid (DMSO controls) vs. aneuploid (reversine) Hl hPSCs 96h after removal of DMSO or reversine, n = 300 cells; mean ± SD. FIGURE 5D contains the results of experiments wherein Hl H2B-GFP hPSCs were grown continuously in 100 nM reversine for 13 days to persistently drive chromosome missegregation, and the growth of the culture was monitored daily, reaching confluency every 4 days followed by splitting and passaging. Three independent experiments (FIGURES 58 and C) or one replicate (FIGURE 5D); ns p > 0.05, **p < 0.01, and ****p < 0.0001 using a two-tailed Fisher's exact test (FIGURE 58) or two-taiied Student's t- test (FIGURE 5C).

[0058] FIGURE 6A-C contain experimental results showing the creation of defined compositions of diploid and aneuploid hPSC populations which demonstrate the feasibility of the inventive methods to create mixed populations of diploid and aneuploid hPSCs at defined ratios and to follow the fate of diploid and aneuploid hPSCs over time, FIGURE 6A contains the experimental scheme wherein prior to mixing, WTC-11 hPSCs were treated for 96 h with DMSO and WTC-11 H2B-GFP hPSCs were treated with DMSO or reversine for 96 h. Following this, WTC-11 diploid (DMSO) hPSCs were mixed with either WTC-11 H2B-GFP diploid (DMSO) or aneuploid (reversine) hPSCs at a defined ratio of 2:1, The proportion of - GFP or +GFP diploid and +GFP aneuploid hPSCs was measured over time. FIGURE 68 contains representative IF image of a mixed population showing -GFP diploid and +GFP aneuploid WTC-11 hPSCs. Shown is DAPI (purple) and GFP (green). FIGURE 6C shows the percent of -GFP diploid or +GFP diploid and +GFP aneuploid hPSCs were measured 96 h and ~2 weeks following mixing. Two biological replicates were performed at 96 h time point post-mixing and one replicate was performed ~2 weeks post-mixing.DEFINITIONS

[0059] As used in the description herein and throughout the ciaims that follow, the meaning of "a," "an," and "the" includes plural reference unless the context clearly dictates otherwise.

[0060] "Aneuploid," as used herein, refers to cells having an abnormal number of chromosomes.

[0061] "Diploid," as used herein, refers to cells having a normal number of chromosomes, i.e., 2 of each chromosome, and in the case of diploid human ceils 46 chromosomes.

[0062] "Chromosome instability" or "CIN" refers to a persistent rate of errors in chromosome segregation during mitosis leading to structural and numerical chromosomal abnormalities. Chromosome instability is a hallmark of human cancer and is often associated with poor prognosis, metastasis, and therapeutic resistance.

[0063] "Embryonic stem cells"' or "ESCs" or "pluripotent stem cells" or "PSCs" refer to pluripotent ceils which may be derived from the inner cell mass of a blastocyst, or an early- stage pre- implantation embryo, optionally human or non-human primate blastocysts, embryos unused after fertility treatment, nuclear transfer embryos, non-human primate or human induced pluripotent stem cells (optionally hiPSCs) reprogrammed from a somatic state to a pluripotent state or from an existing ESC line, optionally Hl. These ceils characteristically express pluripotency transcription factors (MYC, SOX2, NANOG, and OCT4) and express minimal levels of the cell cycle arrest proteins (p53 and p21) under normal conditions.

[0064] "Human Embryonic stem ceils" or "hESCs" refer to embryonic stem cells as defined above which are human, "Human induced pluripotent stem cells" or "hiPSCs" refer to cells as defined above which are human. "Human pluripotent stem cells" or "hPSCs" refers to both hESCs and hiPSCs.

[0065] "Non-human Primate Embryonic stem cells" or "nhpESCs" or "non-human primate pluripotent stem cells" or "nhpPSCs" refer to ESCs as defined above which are derived from a non-human primate, e.g., a chimpanzee, cyno or macaque, baboon. Rhesus monkey, ape, et seq.

[0066] "Cancer / ' as used herein, refers broadly to any neoplastic disease (whether invasive or metastatic) characterized by abnormal and uncontrolled cell division causing malignant growth or tumor.

[0067] "Control amount," as used herein, refers broadly to a marker can be any amount or a range of amounts to be compared against a test amount of a marker. For example, a control amount of a marker may be the amount of a marker in a patient with a particular disease or condition or a person without such a disease or condition. A control amount can be either in absolute amount (e.g., microgram / ml) or a relative amount (e.g., relative intensity of signals).

[0068] "Differentially present," as used herein, refers broadly to differences in the quantity or quality of a marker present in a sample taken from patients having a disease or condition as compared to a comparable sample taken from patients who do not have one of the diseases or conditions. For example, a nucleic acid fragment may optionally be differentially present between the two samples if the amount of the nucleic acid fragment in one sample is significantly different from the amount of the nucleic acid fragment in the other sample, for example as measured by hybridization and / or NAT-based assays. A polypeptide is differentially present between the two samples if the amount of the polypeptide in one sample is significantly different from the amount of the polypeptide in the other sample. It should be noted that if the marker is detectable in one sample and not detectable in the other, then such a marker may be considered to be differentially present. Optionally, a relatively low amount of up-regulation may serve as the marker.

[0069] "Diagnostic," as used herein, refers broadly to identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and who test negative in the assay are termed "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.

[0070] "Diagnosing / ' as used herein, refers broadly to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and / or prospects of recovery. The term "detecting” may also optionally encompass any of the foregoing.

[0071] An "effective amount" of a composition such as a polypeptide, drug, siRNA or analog thereof, antibody, pharmaceutical, small molecule, or other compound, or an agonist or antagonist thereof, is an amount sufficient to carry out a specifically stated purpose. An "effective amount" may be determined empirically and in a routine manner, in relation to the stated purpose.

[0072] "Immunoassay," as used herein, refers broadly to an assay that uses an antibody to specifically bind an antigen. The immunoassay may be characterized by the use of specific binding properties of a particular antibody to isolate, target, and / or quantify the antigen.

[0073] "Isolated," as used herein, refers broadly to material removed from its original environment in which it naturally occurs, and thus is altered by the hand of man from its natural environment. Isolated material may be, for example, exogenous nucleic acid included in a vector system, exogenous nucleic acid contained within a host ceil, or any material which has been removed from its original environment and thus altered by the hand of man (e.g., "isolated ESCs").

[0074] "Label" or a "detectable moiety" as used herein, refers broadly to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.

[0075] "Patient,” as used herein, refers broadly to any animal, typically human or nonhuman primate, who is in need of treatment either to alleviate a disease state or to prevent the occurrence or reoccurrence of a disease state. The term "subject" may be used interchangeably with the term "patient". Generally, herein this term refers to a human subject.

[0076] "Solid support," "support," "substrate," or "solid phase" as used herein, refer to a non-aqueous matrix to which pluripotent cells of the present invention can adhere or attach. Examples of solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Pat. No. 4,275,149. The definition broadly includes any material that provides a solid or semi-solid structure with which another material can be attached including but not limited to smooth supports (e.g., metal, glass, plastic, silicon, and ceramic surfaces) as well as textured and porous materials.

[0077] "Subjects” as used herein, refers broadly to anyone suitable to be treated according to the present invention include, but are not limited to, avian and mammalian subjects, and are preferably mammalian. Mammals of the present invention include, but are not limited to, canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g., rats and mice), lagomorphs, primates, and humans. Generally, it will refer to a human or non-human primate subject. "Subjects" is used interchangeably with "patients."

[0078] "Symptoms" of disease as used herein, refers broadly to any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by the patient and indicative of disease., e, g., cancer or another condition involving aneuploidal cells or cells comprising CIN.

[0079] "Treating" or "treatment" or "alleviation" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject or mammal is successfully "treated" for a cancer if, after receiving a therapeutic amount of a drug shows observable and / or measurable reduction in or absence of one or more of the following: reduction in the number of cancer cells or absence of the cancer cells; reduction in the tumor size; inhibition (i.e., slow to some extent and preferably stop) of cancer cell infiltration into peripheral organs including the spread of cancer into soft tissue and bone; inhibition (i.e., slow to some extent and preferably stop) of tumor metastasis; inhibition, to some extent, of tumor growth; and / or relief to some extent, one or more of the symptoms associated with the specific cancer; reduced morbidity and mortality, and improvement in quality of life issues.To the extent the antibody may prevent growth and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. Reduction of these signs or symptoms may also be feit by the patient.

[0080] The term "therapeutically effective amount" refers to an amount of a drug effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve to some extent one or more of the symptoms associated with the cancer.

[0081] The term "synergistic effect" refers to the result achieved using a combination being greater than the sum of the results that would be achieved using the individual components of the combination. For example, in the context of treatment of cancer a synergistic effect indicates that the effect of two or more treatments in combination elicit more than just the additive effect of the individual agents, optionally as evidenced by reduced tumor burden, increased mean survival, or another endpoint known in the art.

[0082] DETAILED DESCRIPTION OF THE INVENTION

[0083] In order that the invention described herein may be fully understood, the following detailed description is set forth.

[0084] As is disclosed in detail in the examples infra, and the FIGURES referenced therein, the inventors have developed a novel method for obtaining primate aneuploidal cells, particularly human aneuploidal cells, and / or primate or human cells exhibiting chromosomal instability (CIN), using primate pluripotent stem cells. In the exemplified methods human PSCs are treated with reversine, a mitotic kinase (MPS1) inhibitor which has been used to study chromosome segregation. Unexpectedly the inventive methods have been demonstrated to result in a diverse population of aneuploidal human PSCs which proliferate for prolonged duration, comprise a diverse array of different aneuploidies, exhibit chromosomal instability, and which moreover appear to be otherwise equivalent to normal primate pluripotent stem cells, e.g., they express similar levels of pluripotencytranscription markers, similar low levels of cell cycle arrest proteins, produce confluent cultures which may be passaged, and like normal primate pluripotent stem ceils, e.g., human pluripotent stem cells may be cultured for prolonged periods.

[0085] As has been previously mentioned, aneuploidy is associated with a variety of human conditions and diseases most particularly cancer, since aneuploidy is nearly a universal feature of cancers. Also, as further previously mentioned, cancer cells also frequently display the related phenomenon of chromosomal instability (CIN) which is a persistent and high rate of chromosome missegregation that when coupled with the continued survival and propagation of aneuploid cancer cells leads to intra- and inter- genetic heterogeneity among cancer ceils and which aneuploidy and CIN contribute to cancer metastasis, treatment resistance, and immune evasion, all of which promote disease progression and poor patient prognosis.

[0086] Moreover, as previously mentioned, aneuploidy poses a major challenge to the use of hPSCs in regenerative medicine therapies because during prolonged in vitro propagation spontaneously arising aneuploid hPSCs often overtake a population due to culture adaptation that selects for clonal and stable aneuploid karyotypes with a fitness advantage over diploid hPSCs, raising concerns for their therapeutic use.

[0087] Accordingly, because of the commonality of aneuploidy and CIN across a spectrum of human conditions and diseases aneuploidal and / or chromosomally unstable human or non-human primate, produced using the inventive methods should be well suited for identifying genes and pathways that cause aneuploidy and / or CIN and / or which contribute to their pathological consequences. Also, aneuploid and / or chromosomally unstable human or non-human primate cells, produced using the inventive methods should be useful for identifying therapeutic agents that selectively act on cells of different ploidy states and / or chromosomal stability.

[0088] For example, aneuploid and / or chromosomally unstable human or non-human primate ceils, produced using the inventive methods cells may be used to identify agents that selectively eliminate cells with a particular ploidy state and / or chromosomal stability, agents that selectively suppress or enhance chromosomal instability, or agents that selectively alleviate the pathological consequences of having a specific ploidy state and / or chromosomal stability.

[0089] These methods are feasible using aneuploidal human or non-human primate cells, and / or primate or human cells exhibiting chromosomal instability (ON), produced using the inventive methods because the inventive methods unexpectedly yield a diverse population of pluripotent cells comprising different states of aneuploidy wherein the majority of the cells are aneuploid and exhibit chromosomal instability (CIN). Based thereon these diverse cell populations and particular cells derived therefrom should be well suited for identifying different genes / pathways which are affected by changes in ploidy and / or chromosomal stability and for identifying potential therapeutic agents that selectively affect ceils of various ploidy states or chromosomal stabilities.

[0090] As is shown infra, the inventive methods for creating aneuploid and chromosomally unstable human or non-human primate pluripotent stem ceils offer significant advantages over conventional methodologies for creating aneuploidal and / or chromosomally unstable cells which are currently used in screening methods for identifying genes and pathways that cause aneuploidy and / or CIN and in drug studies.

[0091] In the present invention human or non-human pluripotent stem cells (nhpPSCs or hPSCs) which may be used to obtain aneuploidal human or non-human primate cells, and / or primate or human cells exhibiting chromosomal instability (CIN), includes both human and non-human primate embryonic stem cells (hESCs) derived from human or non- human primate blastocysts as well as human or non-human primate induced pluripotent stem cells (hiPSCs) reprogrammed from a somatic state to a pluripotent state or obtained via nuclear transfer or from existing hESC lines which are commercially available. HPSCs are normal, naturally immortal cells that are capable of differentiating to the three embryonic germ lineages (Angeles ADL et al., "Hallmarks of pluripotency", Nature, 2015;525(7570):469. PMID: 26399828).

[0092] In contrast to normal human somatic cells, hPSCs inherently display a higher rate of mitotic errors with the most frequent mitotic error being lagging chromosomes in anaphase (Deng C, Ya A, Compton DA, Godek KM. "A pluripotent developmental state confers a low fidelity of chromosome segregation", Stem Cell Rep. 2023;18(2):475--488, PMID: 36638786). Lagging chromosomes in anaphase are one of the most common sources of chromosome missegregation in human cells and hence causes of aneuploidy and CIN (Thompson SL, Compton DA., "Examining the link between chromosomal instability andaneuploidy in human cells", The Journal of Cell Biology, 2008;180(4):665~672. PMCID: PMC2265570). The inventors' prior discovery that hPSCs exhibit a higher rate of mitotic errors than somatic cells led us to further investigate aneuploidy in hPSCs.

[0093] Normal diploid human somatic ceils do not tolerate aneuploidy and CIN and fail to proliferate because chromosome imbalances induce proteotoxic and metabolic stress, genomic instability, and cell cycle arrest (Hinchcliffe EH et al., "Chromosome missegregation during anaphase triggers p53 cell cycle arrest through histone H3.3 Ser31 phosphorylation", Nature Cell Biology, 2016;18(6):668-675. PMID: 27136267; Santaguida S, Amon A., "Short- and long-term effects of chromosome mis-segregation and aneuploidy", Nature Reviews Molecular Cell Biology, 2015;16(8):nrm4025. PMID: 26204159; Ben-David U, Amon A., "Context is everything: aneuploidy in cancer", Nature Reviews Genetics, 2019; PMID: 31548659; Thompson SL, Compton DA. "Proliferation of aneuploid human cells is limited by a p53-dependent mechanism", The Journal of Cell Biology, 2010;188(3):369~381. PMCID: PMC2819684).

[0094] By contrast, the inventive method for the induction of aneuploidy / CIN in hPSCs results in a durable aneuploid / CIN phenotype. To the best of the inventors' knowledge this is the first example of a durable tolerance for aneuploidy and CIN in an otherwise normal, non-transformed, and naturally diploid human cell type.

[0095] Furthermore, the inventive method creates aneuploid / chromosomally unstable hPSCs that continue to propagate and otherwise maintain normal characteristics including expression of markers of an undifferentiated state. This method therefore provides a novel source of aneuploid / chromosomally unstable human cells for identifying genes / pathways affected by ploidy changes or chromosomal stability and therapeutic agents that selectively act on cells of different ploidy states and / or chromosomal stability with minimal other confounding changes.

[0096] In the inventive method, we have shown that aneuploidy may be induced in hPSCs, e.g., by treatment with a MPS1 kinase inhibitor, reversine, which increases chromosome missegregation. Particularly in exemplary embodiments (see experimental examples and FIGURES referenced therein) we determined a minimum concentration of reversine that resulted in a significant increase in the mitotic error rate, measuring both sources of whole chromosome numerical and structural aneuploidies (FIGURE 1A).However, it is expected that aneupioidy may be induced in hPSCs or nhpPSCs using other agents, e.g, other MPS1 kinase inhibitors or compounds that induce chromosome missegregation so as to create a durabie aneuploidy / chromosomally unstabie phenotype in hPSCs. For exampie, other compounds that interfere with microtubule dynamics or microtubule - kinetochore attachment and induce chromosome mis-segregation may be used,

[0097] In the exemplary embodiments, we showed that treating hPSCs with 100 nM reversine for prolonged duration (e.g, 96 hours) resulted in a significant increase in the whole chromosome missegregation rate. Further when we examined the missegregation of multiple different chromosomes we found no bias with respect to which chromosomes were being missegregated indicating that our method generates a population of hPSCs comprising random, variable aneuploid karyotypes (FIGURES

[0098] We also determined that after prolonged (e.g., 96-hour reversine treatment) that the vast majority of the population was aneuploid (FIGURE 3.C) which is essential for producing a sufficient proportion of aneuploid hPSCs for any strategy which seeks to compare different ploidy states and / or chromosomal stabilities.

[0099] In contrast to typical conventional methodologies, our means for the induction of aneupioidy and / or CIN in human ceils does not rely on permanent genetic alterations, the selection of a rare population of cells the overcome the fitness barriers of aneupioidy to proliferate (Ippolito MR et al, "'Gene copy-number changes and chromosomal instability induced by aneupioidy confer resistance to chemotherapy", Dev Cell, 2021; PMID: 34352223; Lukow DA et al, "Chromosomal instability accelerates the evolution of resistance to anti-cancer therapies", Dev Cell, 2021; PMID: 34352222; Stingele S et al, "Global analysis of genome, transcriptome and proteome reveals the response to aneupioidy in human ceils", Molecular Systems Biology, 2012;8(l):608, PMCID: PMC3472693; Taylor AM et al, "Genomic and Functional Approaches to Understanding Cancer Aneupioidy" Cancer Cell, 2018; PMID: 29622463; Truong MA et al, "A kinesin-based approach for inducing chromosome-specific mis-segregation in human cells", EMBOJ., 2023;42(10):elll559. PMCID: PMC10183822; Bosco N, et al, "A CRISPR-based technology to study chromosomespecific aneupioidy by targeting human centromeres", Cell, 2023;186(9):1985-2001,el9. PMID: 37075754; Girish V et al, "Oncogene-like addiction to aneupioidy in human cancers",Science, 2023;381(6660):eadg4521. PMID: 37410869; Price CJ. et al, (2021), "Genetically variant human pluripotent stem cells selectively eliminate wild-type counterparts through YAP-mediated ceil competition", Dev Ceil 56, 2455-2470.el0.10.1016 / j.devcel.2021.07,019), or the use of transformed cancer ceils that have additional mutations (Ippolito MR et al, "Gene copy-number changes and chromosomal instability induced by aneuploidy confer resistance to chemotherapy", Dev Cell, 2021; PMID: 34352223; Lukow DA, et al., "Chromosomal instability accelerates the evolution of resistance to anti-cancer therapies", Dev Cell, 2021; PMID: 34352222).

[0100] The inventive method circumvents these limitations as we surprisingly discovered that hPSCs, including embryonic and induced, treated for a prolonged duration (e.g., 96 h or longer) with reversine, optionally 100 nM reversine, followed by reversine removal continue to proliferate unlike somatic RPE-1 ceils (FIGURES 3A and B). Moreover, hPSCs maintained minimal amounts of cell cycle arrest proteins upon induction of aneuploidy using reversine (FIGURES 3C and D). Also, the inventors discovered that the induction of aneuploidy using reversine did not result in a loss of markers of an undifferentiated state or in the amounts of such markers of an undifferentiated state (FIGURES 3C and D). Thus, aneuploid hPSCs otherwise retain the characteristics of normal, diploid PSCs defined by the expression of the pluripotency transcription factors OCT4, NANOG, MYC, and SOX2 comparably to untreated hPSCs (FIGURES 3C and D). Furthermore, hPSCs remain aneuploid for prolonged duration, i.e., at least up to 96h post-reversine removal (FIGURE 3E) demonstrating that survival of aneuploid hPSCs is independent on MSP1 inhibition and that hPSCs are inherently tolerate an aneuploid genome. Thus, the inventive method for inducing aneuploidy unexpectedly results in a durable population of aneuploid hPSCs that, although their ploidy status is altered, maintain other characteristics of normal diploid hPSCs.

[0101] Also, the inventors further surprisingly discovered that aneuploidal hPSCs exhibit an increase in the frequency of mitotic errors, particularly lagging chromosomes, upon removal of reversine (FIGURE 5A and B) demonstrating that induction of aneuploidy increased chromosomal instability. Moreover, aneuploidy and an increased mitotic error rate did not result in a loss of markers of an undifferentiated state defined by amounts of the pluripotency transcription factors OCT4 and NANOG that was comparable to untreatedhPSCs (FIGURE SC). Furthermore, longer-term induction of aneuploidy and ongoing chromosome missegregation in the continual presence of reversine resulted in aneuploidal hPSCs which continue to proliferate with the expected growth rate [of normal diploid hPSCs], for prolonged duration, e.g., when cultured in the continual presence of reversine for at least 13 days (FIGURE 5D). Collectively, these data suggest that hPSCs treated according to the inventive methods can tolerate both an aneuploid state and chromosomal instability (FIGURES 5A-D).

[0102] Thus, the invention provides a novel method using hPSCs or nhpPSCs that allows for the creation of a population of aneuploid / chromosomally unstable human or nonhuman primate cells derived from hPSCs or nhpPSCs that does not rely on cancerous transformation, other genetic alterations and / or genetic manipulations, or the selection of a rare population of cells. Based on the foregoing advantages, the invention provides a novel platform for the direct comparison of isogenic diploid vs. aneuploid / chromosomally unstable human cells that can be used to identify genes / pathways affected by changes in ploidy or chromosomal stability and therapeutic agents that selectively act on different ploidy states and / or chromosomal stability.

[0103] Particularly, the inventive method described herein, and the aneuploid / chromosomally unstable human cells derived using these methods, offer significant advantages to current strategies employed for constructing aneuploid and chromosomally unstable cells. For example, previous approaches for studying the effects of aneuploidy have used genetically engineered aneuploid yeast (Torres EM et al, "Identification of Aneuploidy-Tolerating Mutations", Cell, 2010;143(l):71-83. PMID: 2085017624), or mouse cells (Tang YC, Williams BR, Siegel JJ, Amon A., "Identification of Aneuploidy-Selective Antiproliferation Compounds", Ce / / , 2010;144(4):499-512. PMID: 21315436), and not human cells, to identify genes affected by ploidy changes and aneuploidy tolerating drugs. Unfortunately, there are serious questions of how well findings in yeast and mice correlate to human diseases and conditions.

[0104] Moreover, although the induction of aneuploidy and / or chromosomal instability is feasible in normal human somatic cells, current approaches rely on genetic engineering strategies that permanently alter cells and / or the selection of a rare population of somatic cells that overcome the fitness barriers of aneuploidy to continue proliferating (Ippolito MRet al, "Gene copy-number changes and chromosomal instability induced by aneuploidy confer resistance to chemotherapy", Dev Cell, 2021; PMID: 34352223; Lukow DA et al., "Chromosomal instability accelerates the evolution of resistance to anti-cancer therapies", Dev Cell, 2021; PMID: 34352222; Stingele S et al, "Global analysis of genome, transcriptome and proteome reveals the response to aneuploidy in human cells", Molecular Systems Biology, 2012;8(l):608, PMCID: PMC3472693; Taylor AM et al, "Genomic and Functional Approaches to Understanding Cancer Aneuploidy", Cancer Cell, 2018; PMID: 29622463; Truong MA et al., "A kinesin-based approach for inducing chromosome-specific missegregation in human cells", EMBOJ., 2023;42(10):elll559. PMCID: PMC10183822; Bosco N, et al., "A CRISPR-based technology to study chromosome-specific aneuploidy by targeting human centromeres", Cell, 2023;186(9):1985-2001.el9. PMID: 37075754; Girish V et al,, "Oncogene-like addiction to aneuploidy in human cancers", Science, 2023;381(6660):eadg4521. PMID: 37410869), or the use of transformed cancer cells that have additional mutations (Ippolito MR et al, "Gene copy-number changes and chromosomal instability induced by aneuploidy confer resistance to chemotherapy", Dev Cell, 2021; PMID: 34352223; Lukow DA, et al., "Chromosomal instability accelerates the evolution of resistance to anti-cancer therapies", Dev Cell, 2021; PMID: 34352.2.2.2). Further, while transformed cancer cells frequently exhibit aneuploidy and chromosomal instability, their use is confounded by the presence of additional mutations. By contrast, the present invention uses hPSCs or nhpPSCs that are otherwise normal and non-transformed, and that are naturally immortal (unlike cancer ceils that become immortalized by genetic alterations contributing to the transformation steps or artificially immortalized somatic ceils via exogenous expression of telomerase). Importantly, we show that hPSCs treated by the inventive methods are inherently tolerant of an aneuploid state and chromosomal instability. Critically, and unlike for normal somatic cells or spontaneously arising aneuploid hPSCs, the present invention does not require us to isolate a select population of aneuploid / chromosomally unstable ceils and therefore avoids potential introduction of rare genetic variants. This is a powerful discovery that permits the identification of genes / pathways affected by ploidy changes and / or chromosomal stability and therapeutic agents that selectively act on different ploidy states and / or chromosomal stability without other confounding alterations due to selection pressures or transformation.

[0105] Yet additionally, the present invention advantageously produces random, variable karyotypes modeling the genetic diversity found in tumors (Godek KM, et al., "Chromosomal Instability Affects the Tumorigenicity of Glioblastoma Tumor-Initiating Cells'', Cancer Discovery, 2016;6(5):532-545. PMID: 27001151), human preimplantation embryos (Vanneste E, et al., "Chromosome instability is common in human cleavage-stage embryos", Nature Medicine, 2009;15(5):nm.l924, PMID: 19396175), and which are present in ceils of individuals with MVA1.

[0106] Accordingly, the present invention provides a unique and improved approach compared to gene editing methods that genetically engineer ceils with specific aneuploid karyotypes (Taylor AM et al, "Genomic and Functional Approaches to Understanding Cancer Aneuploidy", Cancer Ceil, 2018; PMID: 29622463; Truong MA et al., "A kinesin-based approach for inducing chromosome-specific mis-segregation in human ceils", EMBOJ., 2023;42(10):elll559. PMCID: PMC10183822; Bosco N, et al., "A CRISPR-based technology to study chromosome-specific aneuploidy by targeting human centromeres", Ceil, 2023;186(9):1985-2001.el9. PMID: 37075754; Girish V et al., "Oncogene-like addiction to aneuploidy in human cancers", Science, 2023;381(6660):eadg4521. PMID: 37410869),

[0107] Yet another advantage of the invention is that it allows for modeling mixed environments of diploid and aneuploid ceils using matched ceils with differences attributed to their pioidy status and not confounding alterations due to additional mutations or selection. This is a unique application of our invention. For example, in diseases such as MVA approximately 25% of cells in individuals are aneuploid and 75% are diploid and the present invention can be used to model this mixed environment. We provide supporting data demonstrating the feasibility of our invention to model mixed environments (FIGURES 6A-C).

[0108] As is disclosed in the examples the inventors have produced our aneuploidai hPSCs using Hl, H9, and WTC-11 hPSCs. We anticipate that equivalent results, may be obtained using other hPSC or nhpPSCs lines.

[0109] Furthermore, in the experiments disclosed in the examples the inventors modified these Hl, H9, or WTC-11 hPSCs to express H2B-GFP so that we could readily monitor mitotic errors and / or proliferation and not induce aneuploidy / CIN in these cells unlike other methods. The inventors have no reason to believe that this specificmodification impacts the rigor and reproducibility of our results. In support, we showed that Hl H2B-GFP, H9 H2B-GFP, WTC-11 H2B-GFP hPSCs were comparable to their non-modified counterparts in terms of pluripotency transcription factor expression (FIGURES 2A-B and 3C- D), genomic stability (FIGURE 2Cj, and growth rates (FIGURE 38). This can be corroborated using hPSC lines that do not carry this specific modification or which comprise another modification that facilitates the monitoring of mitotic errors and which modification does not itself induce aneupioidy and / or CIN.

[0110] Yet additionally, we have only tested a single method of inducing chromosome missegregation (reversine) in hPSCs. However, as is noted supra, the present invention is not limited to the specific method for causing chromosome missegregation in hPSCs or nhpPSCs and embraces the use of other compounds that induce chromosome missegregation so as to create a durable aneuploidy / chromosomaliy unstable phenotype in hPSCs. For example, other compounds that interfere with microtubule dynamics or microtubule - kinetochore attachment and induce chromosome mis-segregation may be used.

[0111] Also, other microtubule inhibitors may be used such as taxanes, vinca alkaloids, such as eribuiin, paclitaxel, docetaxel, Ixabepiione, Cabazitaxei, Enfortumab vedotin, Trastuzumab emtansine, Ti rbanibulin, Paclitaxel ceribate, Vinblastine, vincristine, Vinorelbine, vinflunine, Cryptophycin 52, Halichondrin B, Dolastatins, e.g., Dolastatins 10 and 15, Hemiasterlins, Hemiasterlin A and hemiasterlin B, Colchicine, Combretastatins, E7010, a sulfonamide antimitotic agent, 2-Methoxyestradiol, Docetaxel, Paclitaxel, Epothilones, Epothilone A and epothilone B, Discodermolide, among others.

[0112] Yet further, other compounds which like reversine inhibit mitotic kinases such as MPS1 may be used to elicit chromosome missegregation such as CDKN2A. Additionally, other mitotic inhibitors may be used such as those which inhibit one or more kinases or microtubule motor proteins selected from AurkA, AurkB, Pan-AurK, Eg5, PLK1, MPS1, CENPE, and CDK1. Such inhibitor compounds are known in the art and include by way of example: Alisertib (MLN8237), ENMD-981693 / ENMD-2076, Danusertib (PHA-739358), MLN8054, MK-5108 (VX-689), MK-8745, Barasertib (AZD1152-HQPA, AZD2811), 81811283, GSK1070916, PF-03814735, ZM-447439, XL-228, Tozasertib (VX-680 / MK-0457), CYC-116, SNS-314, AMG 900, JNJ-7706621, Cenisertib (AS703569, MSC1992371A), AT9283, Rigosertib(ON 01910), Volasertib (BI6727), Onvansertib (NMS-P937), BI2536, GSK461364, Tak9960, Poioxin-2, 581694 (NMS-P153), BOS172722, Empesertib (BAY1161909), BAY1217389, Mpsl- IN-1, Mpsl-IN-2, Mpsl-IN-3, TC-Mpsl-12, AZ3146, NMS-P715, MPI-0479605, CCT251455lspinesib (SB-715992). Filanesib (ARRY-520) SB-743921, Litronesib (LY252335), ARQ-621, MK-0731. EMD-534085. 4SC-205, acin, PF-2771, GSK-923295, APC / C, 4SC-205, 4SC-205, Danusertib (PHA-739358), MLN8054, MK-5108 (VX-689), MK-8745, Alisertib (MLN8237), ENMD-981693 / ENMD-2076, Danusertib (PHA-739358), MLN8054, MK-5108 (VX-689), MK-8745, Barasertib (AZD1152-HQPA, AZD2811), BI811283, GSK1070916, PF- 03814735, ZM-447439, XL-228, Tozasertib (VX-680 / MK-0457), CYC-116, SNS-314, AMG 900, JNJ-7706621, Cenisertib (AS703569, MSC1992371A), AT9283, Rigosertib (ON 01910), Volasertib (BI6727), Onvansertib (NMS-P937), BI2536, GSK461364, Tak9960, Poloxin-2, S81694 (NMS-P153), BOS172722, Empesertib (BAY1161909), BAY1217389, Mpsl-IN-1, Mpsl-IN-2, Mpsl-IN-3, TC-Mpsl-12, AZ3146, NMS-P715, MPS-0479605, CCT251455, Ispi nesib (SB-715992), Fi lanesib (ARRY-520), SB-743921, AZD4877, Litronesib (LY2523355), ARQ-621, MK-0731, EMD-534085, 4SC-205, PF-2771, GSK-923295, Compound-A, APC / C and Apcin (see, Alfonso Serrano-del Valle et al., "Future prospects for mitosis-targeted antitumor therapies". Biochemical Pharmacology, Vol. 190, August 2021, 114655).

[0113] In order that the invention described herein may be fully understood, various embodiments of the invention are described in detail and further illustrated by the provided examples. The examples provided are intended to be illustrative, rather than limiting, on the scope of the invention, which is limited only by the scope of the claims provided after the examples.EXAMPLES

[0114] EXAMPLE 1: Creation of aneuploid hPSCs

[0115] To induce aneuploidy in hPSCs, we treated Hl hPSCs (purchased from WiCell, NIH registration # NIHhESC-10-0043) with a commercially available MPS1 kinase inhibitor, reversine, to increase chromosome missegregation. As is shown in FIGURE 1A we determined the lowest possible concentration of reversine that resulted in a significantincrease in the mitotic error rate, measuring both sources of whole chromosome numerical and structural aneuploidies (FIGURES 1A and 2D),

[0116] In the experiments in FIGURE 1A, time-lapse live-cell imaging of the Hl H2B-GFP hPSCs treated with DMSO or 100 nM reversine was performed. Hl H2B-GFP hPSCs were imaged for 6-7 hours in the presence of DMSO or reversine and mitotic errors were quantified during this time period. Lagging chromosomes and unaligned chromosomes are sources of numerical whole chromosome aneuploidies while chromosome bridges are a source of structural aneuploidies (Orr B et al., "Aneuploidy", Current Biology, 2015;25(13):R538~R542. PMID: 26126276). As can be seen treatment with 100 nM reversine significantly increased the mitotic error rate (**p < 0.01; Fisher's Exact Test). Furthermore, overall mitotic timing was unchanged (FIGURE 2E) with lOOnM reversine treatment demonstrating that MPS1 activity is partially compromised at this dose since further inhibition with higher concentrations of reversine accelerate mitosis (Santaguida S et al., (2010), "Dissecting the role of MPS1 in chromosome biorientation and the spindle checkpoint through the small molecule inhibitor reversine", J. Cell Biol. 190, 73-87.10.1083 / jcb.201001036).

[0117] EX AM PLE 2 : Generation of hPSCs with random and variable aneuploid karyotypes

[0118] We next conducted experiments to assess whether treating hPSCs with 100 nM reversine ranging between 24-96 hours resulted in a significant increase in the whole chromosome missegregation rate and whether there was any bias with respect to which chromosomes were being missegregated. We determined individual copy numbers of chromosomes 2, 8, 10, 12, and 17 in single Hl H2B-GFP hPSCs using fluorescence in situ hybridization (FISH) and representative images are shown in FIGURE IB. The bar graphs in the FIGURES 1C and D report the percentage of Hl H2B-GFP hPSCs deviating from the expected diploid copy number of 2 for each chromosome and show that the percent of aneuploid Hl H2B-GFP hPSCs significantly increased with the duration of reversine treatment due to both chromosome gains and losses compared to DMSO controls. Furthermore, we observed no significant difference in the aneusomic rate (one-way ANOVA p > 0.05) among chromosomes 2, 8, 10, 12, or 17 (FIGURE 1C) demonstrating that chromosome missegregation was random in Hl H2B-GFP hPSCs. Moreover, given that theaneusomic frequency was ~3-4% per chromosome after 96 hr reversine treatment, this indicates that nearly all ceils in the population become aneuploid when accounting for all 46 chromosomes. Thus, our inventive method generates a uniformly aneuploid population of hPSCs with diverse karyotypes.

[0119] EXAMPLE 3: Generation of a durable population of aneuploid hPSCs

[0120] In these experiments, somatic RPE-1 H2B-GFP, Hl or Hl H2B-GFP, H9 (purchased from WiCell, NIH registration # NIHhESC-10-0062) or H9 H2B-GFP, and WTC-11 (purchased from the Coriel I Institute for Medical Research) or WTC-11 H2B-GFP hPSCs were treated with DMSO or 100 nM reversine for 96 hours (FIGURE 3A). The total cell numbers were measured daily. As expected, and in agreement with prior studies (Janssen A et al., (2011), "Chromosome Segregation Errors as a Cause of DNA Damage and Structural Chromosome Aberrations", Science 333, 1895-1898. 10.1126 / science.1210214; Santaguida S et al., (2017), "Chromosome Mis-segregation Generates Cell-Cycle-Arrested Celis with Complex Karyotypes that Are Eliminated by the Immune System”, Developmental Ceil 41, 638-651. e5. 10.1016 / j.devcel.2017.05.022; Garribba L et al. (2023), “Short-term molecular consequences of chromosome mis-segregation for genome stability", Nat Commun 14, 1353. 10.1038 / s41467-023-37095-7; Passerini V et al., (2016), "The presence of extra chromosomes leads to genomic instability”, Nature Communications 7, 10754.10.1038 / ncommsl0754; Hinchcliffe E.H et al., (2016), "Chromosome missegregation during anaphase triggers p53 cell cycle arrest through histone H3.3 Ser31 phosphorylation", Nature Cell Biology 18, 668-675. 10.1038 / ncb3348; Thompson, S.L., and Compton, D.A. (2010), “Proliferation of aneuploid human cells is limited by a p53-dependent mechanism", The Journal of Cell Biology 188, 369-381. 10.1083 / jcb.200905057; Soto M et al,, (2017), "p53 Prohibits Propagation of Chromosome Segregation Errors that Produce Structural Aneuploidies", Cell Reports 19, 2423-2431. 10.1016 / j.celrep.2017.05.055), somatic RPE-1 H2B-GFP cells failed to proliferate upon induction of chromosome missegregation and aneupioidy using lOOnM reversine (FIGURES 3B, 4C-E). In contrast, Hl H2B-GFP, H9 H2B- GFP, and WTC-11 H2B-GFP continued to proliferate upon induction of chromosome missegregation and aneupioidy using lOOnM reversine comparable to that of DMSO control treated cells (FIGURE 3B). We reasoned that inhibition of MPS1 using reversine may allow for the survival and growth of aneuploid hPSCs, so we removed the drug and measured theproliferation of hPSCs daiiy for an additionai 96h (FIGURE 3A). As we had hoped, removal of reversine did not alter the proliferation of aneuploid hPSCs (FIGURE 3B). Moreover, we confirmed that aneuploid hPSCs persisted after reversing removal by determining individual copy numbers of chromosomes 2, 8, 10, 12, and 17 using FISH to measure the percent of the population that deviated from the expected diploid copy number of 2 for each chromosome. The bar graph is FIGURE 3E shows that at 96h after reversine removal the percent of aneuploid hPSCs remained significantly increased due to both chromosome gains and losses compared to DMSO controls. Collectively, these results indicate that, unlike somatic ceils, the inventive method generates a population of hPSCs where after 96-hour reversine treatment the vast majority of the population was aneuploid (FIGURE 1C) and that the population of aneuploid hPSCs is durable at least 96 h after reversine removal (FIGURE 3E). This is a key requirement for producing a sufficient proportion of aneuploid hPSCs for any strategy comparing different ploidy states and / or chromosomal stabilities. A limitation of many current methodologies for producing aneuploidal human cells is that the induction of aneuploidy and / or CIN in human ceils relies on permanent genetic alterations, the selection of a rare population of cells the overcome the fitness barriers of aneuploidy to proliferate, or the use of transformed cancer cells that have additional mutations. By contrast, the results in FIGURES 1C and 3E demonstrate that the inventive method circumvents these limitations as a substantial percentage of hPSCs treated for 96 h with 100 nM reversine followed by reversine removal continue to proliferate and remain aneuploid.

[0121] EXAMPLE 4: DNA damage, but not aneuploidy, inhibits the growth of hPSCs

[0122] In somatic RPE-1 cells, induction of aneuploidy with 100 nM reversine ceased proliferation (FIGURE 3B) correlating with a significant increase in p53 and p21 protein levels (FIGURES 4A and B) that are well-known to mediate cell cycle arrest (Thompson, S.L, and Compton, D.A. (2010), "Proliferation of aneuploid human cells is limited by a p53- dependent mechanism", The Journal of Cell Biology 188, 369-381. 10.1083 / jcb.200905057). In contrast, induction of aneuploidy in hPSCs did not cause a proliferation defect (FIGURE 3B) or a significant increase in p53 or p21 protein levels (FIGURES 3C and D). Hence, we considered the possibility that aneuploid hPSCs proliferated due to the clonal acquisition of p53 mutations for two reasons. First, in somatic and cancer cells, p53 deletion or mutation permits the survival of aneuploid cells (Thompson, S.L., and Compton, D.A. (2010),“Proliferation of aneuploid human cells is limited by a p53-dependent mechanism", The Journal of Cell Biology 188, 369-381, 10.1083 / jcb.200905057; Taylor A.M et al,, (2018), "Genomic and Functional Approaches to Understanding Cancer Aneuploidy", Cancer Ceil. 10.1016 / j.ccell.2018.03.007). Second, hPSCs frequently acquire p53 mutations during in vitro propagation (Merkle, F.T et al., (2017), "Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations". Nature 545, 229.10.1038 / nature22312; Lezmi, E, Jung, J, and Benvenisty, N. (2024), "High prevalence of acquired cancer-related mutations in 146 human pluripotent stem cell lines and their differentiated derivatives", Nat. Biotechnol., 1-5. 10.1038 / s41587-023-02090-2). We tested for wild-type p53 functionality in RPE-1 somatic and Hl hPSCs by treating RPE-1 H2B-GFP and Hl or Hl H2B-GFP hPSCs with the DNA damaging agent doxorubicin. DNA damage significantly increased p53 and p21 protein levels in somatic RPE-1 H2B-GFP cells as expected (Janssen A et al., (2011), "Chromosome Segregation Errors as a Cause of DNA Damage and Structural Chromosome Aberrations", Science 333, 1895-1898, 10.1126 / science.1210214; Santaguida S et al., (2017), "Chromosome Mis-segregation Generates Cell-Cycle-Arrested Cells with Complex Karyotypes that Are Eliminated by the Immune System", Developmental Ceil 41, 638-651.e5. 10.1016 / j.devcel.2017,05.022;Garribba L et al., (2023), "Short-term molecular consequences of chromosome mis- segregation for genome stability", Nat Commun 14, 1353. 10.1038 / s41467-023-37095-7; Passerini V et al., (2016), "The presence of extra chromosomes leads to genomic instability" Nature Communications 7, 10754, 10.1038 / ncommsl0754) (FIGURES 4F-I). Moreover, DNA damage induced similar p53 levels but attenuated p21 levels in Hl H2B-GFP hPSCs compared to RPE-1 somatic ceils in agreement with previous reports (Itahana Y et al,, (2016), "Histone modifications and p53 binding poise the p21 promoter for activation in human embryonic stem cells", Scientific Reports 6, srep28112. 10.1038 / srep28112; Liu, J.C., Lerou, P.H., and Lahav, G. (2014), "Stem cells: balancing resistance and sensitivity to DNA damage" Trends in Cell Biology 24, 268-274. 10.1016 / j.tcb.2014.03.002; Barta T et al, (2010), "Human Embryonic Stem Ceils Are Capable of Executing Gl / S Checkpoint Activation", STEM CELLS 28, 1143-1152. 10.1002 / stem.451; Dolezalova D et al, (2012),"MicroRNAs Regulate p21Wafl / Cipl Protein Expression and the DNA Damage Response in Human Embryonic Stem Cells", STEM CELLS 30, 1362-1372.10.1002 / stem,1108) (FIGURES 4F-4 ) suggesting that p53 is functional. Also, we measured thesurvival of Hl H2B-GFP hPSCs after 24 hr doxorubicin treatment because p53 mutations confer resistance to apoptosis upon DNA damage induced by doxorubicin (Vries A et al., (2002), "Targeted point mutations of p53 lead to dominant-negative inhibition of wild-type p53 function", Proc. Natl. Acad. Sci. 99, 2948-2953. 10.107.3 / pnas.052713099). Hl H2.B-GFP hPSCs all died by 24 hrs in the presence of doxorubicin (FIGURE 4J). Collectively, these results suggest that p53 exhibits wild-type function in Hl H2B-GFP hPSCs and a clonal p53 mutation is unlikely to account for the proliferation and survival of aneuploid hPSCs. Thus, our inventive method overcomes a limitation of many current methodologies for producing aneuploidal human cells in that the induction of aneupioidy and / or CIN in human cells relies on permanent genetic alterations, particularly knock-out or depletion of p53 (Lukow DA et al., "Chromosomal instability accelerates the evolution of resistance to anti-cancer therapies", Dev Cell, 2021; PMID: 34352222, Crasta K et al., (2012), "DNA breaks and chromosome pulverization from errors in mitosis", Nature 482,53. https: / / doi.org / 10.1038 / naturel0802). Also, our method is advantageous because it provides a platform that separates aneupioidy and / or chromosomal instability from a DNA damage response.

[0123] EXAMPLE 4: Aneuploid hPSCs also exhibit chromosomal instability

[0124] In these experiments, we assessed whether aneuploid Hl hPSCs also exhibited chromosomal instability (CIN), which is a persistent and high rate of chromosome missegregation. We compared the frequency of mitotic errors for diploid (DMSO control) vs. aneuploid (reversine) Hl hPSCs 96 h post-DMSO or reversine removal (FIGURE 5A). Induction of aneupioidy resulted in a significant increase in the frequency of lagging chromosomes in anaphase (FIGURE 5B). Previous studies demonstrated that lagging chromosomes cause whole chromosome missegregation and that the rate of lagging chromosomes is proportional to the chromosome missegregation rate (Thompson SL, Compton DA., "Examining the link between chromosomal instability and aneupioidy in human cells". The Journal of Cell Biology. 2008;180(4):665~672. PMCID: PMC2265570). Thus, induction of aneupioidy in Hl hPSCs increases chromosomal instability. Furthermore, we showed that Hl hPSCs tolerate longer-term induction of aneupioidy and ongoing CIN. Hl hPSCs were grown in the continual presence of reversine for 13 days and continued to proliferate with the expected growth rate of normal diploid hPSCs (FIGURE 5D). Collectively,these data indicate that hPSCs treated according to the inventive methods can tolerate both an aneupioid state and exhibit chromosomal instability (FIGURES 5A-D).

[0125] EXAMPLE 5: Aneupioid and chromosomaliy unstable hPSCs maintain an undifferentiated state

[0126] As is disclosed in the above examples, we shown that the inventive method results in a stable, diverse population of aneupioid hPSCs that are chromosomaliy unstable. We also assessed in the experiments shown in FIGURES 3C and D and FIGURE SC whether the induction of aneuploidy and CIN resulted in a loss of any markers of an undifferentiated state. In the experiments in FIGURES 3C and D Hl or Hl H2B-GFP hPSCs, H9 or H9 H2B-GFP, and WTC-11 or WTC-11 H2B-GFP hPSCs were treated with DMSO or 100 nM reversine for 96 h and quantitative IF was performed to measure the abundance of the pluripotency transcription factors MYC, SOX2, NANOG, or OCT4. As shown there was no discernable difference in the levels of any of these pluripotency transcription factors (FIGURES 3C and D). Also, we performed quantitative IF to measure the abundance of the pluripotency transcription factors NANOG and OCT4 in Hl hPSCs treated with DMSO or 100 nM reversine for 96 h followed by wash out of DMSO or reversine for an additional 96 h. As shown in FIGURE 5C there was no discernable difference in the levels of NANOG between DMSO and reversine treated Hl hPSCs. There was a slight, but significant, decrease in OCT4 abundance for reversine treated Hl hPSCs. However, the slight decrease in OCT4 abundance is not comparable to the complete loss of OCT4 in differentiated somatic cells (FIGURE 2B). Collectively, aneupioid and chromosomaliy unstable hPSCs produced by the inventive methods express comparable levels of pluripotency transcription factors to normal (diploid) hPSCs.

[0127] EXAMPLE 6: Creating defined compositions of mixed diploid and aneupioid hPSC populations

[0128] We conducted experiments to assess the feasibility of creating defined compositions of mixed diploid and aneupioid populations of hPSCs. Separate populations of WTC-11 hPSCs were treated for 96 h with DMSO and WTC-11 H2B-GFP hPSCs were treated with DMSO or reversine for 96 h and then -GFP diploid and +GFP diploid or -GFP diploid and +GFP aneupioid hPSCs were mixed at a defined ratio of 2:1 (FIGURE 6A). The proportion of - GFP or +GFP diploid and +GFP aneupioid hPSCs was measured at 96 h and ~2 weeksfoliowing mixing (FIGURES 6B and C). The relative proportion of +GFP aneuploid hPSCs to GFP diploid hPSCs remained constant during the tested time period. Thus, our inventive method allows for modeling mixed environments of diploid and aneuploid ceils using matched cells with differences attributed to their pioidy status and not confounding alterations due to additional mutations or selection.CONCLUSIONS

[0129] The present invention provides a unique and improved approach compared to conventional methods for producing aneuploidal cells that may be used in drug and disease studies and other methods where aneuploidal ceils find known usage.

[0130] Unlike conventional methods for producing aneuploidal cells, the inventive method is relatively straightforward, reproducible and avoids the need for complicated genetic engineering and / or transformation methods.

[0131] Also, unlike most conventional methods for producing aneuploidal ceils, the inventive method results in human or non-human primate aneuploidal cells which should be superior to non-human aneuploidal ceils for drug and other studies.

[0132] Another advantage of the invention is that it allows for modeling mixed environments of diploid and aneuploid cells using matched cells with differences attributed to their pioidy status and not confounding alterations due to additional mutations or selection. Such mixed environments of diploid and aneuploid cells should provide a superior disease model for study of diseases such as MVA and therapeutics for potential usage therein wherein some of the subject's cells are normal and others are aneuploid (e.g., in MVA approximately 25% of cells in individuals are aneuploid and 75% are diploid). Essentially, the present invention will enable this mixed environment to be appropriately modeled.

[0133] Another advantage of the invention is that it yields a mixed population of aneuploidal hPSCs wherein the vast majority of the cells are aneuploid thereby providing many cells available for drug and other studies.

[0134] Yet another advantage of the invention is that aneuploidal hPSCs remain viable (continue to proliferate) for prolonged duration after creation, and after reservine washout, further facilitating drug and other studies.

[0135] Still another advantage of the invention is that the inventive method generates a population of hPSCs comprising random, variable aneuploid karyotypes further facilitating the use of these cells for study of different ploidy states and / or chromosomal instabilities.

[0136] Another advantage of the invention is that the inventive method generates a population of hPSCs that appear to be tolerant of chromosomal instability as they proliferate comparably to normal hPSCs for prolonged duration (at least 13 days) after creation.EXEMPLARY EMBODIMENTSEMBODIMENT 1: A method for creating human pluripotent stem cells (hPSCs) or nonhuman primate pluripotent stem cells (nhpPSCs) which are aneuploidal and / or exhibit chromosomal instability (C!N) by contacting the hPSCs or nhpPSCs with at least one compound that wherein at least 30%, 40%, 50%, 60%, 70%, 80% or 90% or more are aneuploidal and / or exhibit chromosomal instability (ON).EMBODIMENT 2: The method of EMBODIMENT 1, which uses a dose of the compound such that the majority (over 50%) of the hPSCs or nhpPSCs after treatment are aneuploidal and / or exhibit chromosomal instability (CIN).EMBODIMENT 3: The method of EMBODIMENT 1, which uses a dose of the compound resulting in at least 80% or 90% of the human pluripotent stem cells (hPSCs) or nonhuman primate pluripotent stem cells (nhpPSCs) being aneuploidal and / or exhibit chromosomal instability (CIN).EMBODIMENT 4: The method of EMBODIMENT 1, 2 or 3, wherein the human and non-human primate embryonic stem cells (hESCs) are derived from:(i) human or non-human primate blastocycsts,(ii) embryos unused after fertility treatment,(iii) human or non-human primate nuclear transfer embryos,(iv) non-human primate or human induced pluripotent stem cells (hiPSCs) reprogrammed from a somatic state to a pluripotent state, or(v) an existing human or non-human primate ESC line, optionally Hl.EMBODIMENT 5: The method of any of the previous EMBODIMENTS, wherein the compound that causes chromosome missegregation is a microtubule inhibitor, optionally an inhibitor of microtubule dynamics or an inhibitor of microtubule ~ kinetochore attachment.EMBODIMENT 6: The method of any of the previous EMBODIMENTS, wherein the compound that causes chromosome missegregation is selected from taxanes, vinca alkaloids, such as eribulin, paclitaxel, docetaxel, Ixabepilone, Cabazitaxel, Enfortumab vedotin, Trastuzumab emtansine, Tirbanibulin, Paclitaxel ceribate, Vinblastine, vincristine, Vinorelbine, vinflunine, Cryptophycin 52, Halichondrin B, Doiastatins, e.g., Dolastatins 10 and 15, Hemiasterlins, Hemiasterlin A and hemiasterlin B, Colchicine, Combretastatins, E7010, a sulfonamide antimitotic agent, 2-Methoxyestradiol, Docetaxel, Paclitaxel, Epothilones, Epothilone A and epothilone B, and Discodermolide.EMBODIMENT 7: The method of any of the previous EMBODIMENTS, wherein the compound that causes chromosome missegregation is reversine or another inhibitor of a mitotic kinases such as MPS1.EMBODIMENT 8: The method of any of the previous EMBODIMENTS, wherein the compound that causes chromosome missegregation is a compound which inhibits one or more kinases or microtubule motor proteins, optionally one or more of AurkA, AurkB, Pan-AurK, Eg5, PLKl, Mpsl, CENPE and CDK1.EMBODIMENT 9: The method of EMBODIMENT 8, wherein the compound that inhibits one or more kinases or microtubule motor proteins, optionally one or more of AurkA, AurkB, Pan-AurK, Eg5, PLKl, Mpsl, CENPE, and CDK1, is selected from Alisertib (MLN8237), ENMD-981693 / ENMD-2076, Danusertib (PHA- 739358), MLN8054, MK- 5108 (VX-689), MK-8745, Barasertib (AZD1152-HQPA, AZD2811), BI811283, GSK1070916, PF-03814735, ZM-447439, XL-228, Tozasertib(VX- 680 / MK-0457), CYC-116, SNS-314, AMG 900, JNJ-7706621, Cenisertib (AS703569, MSC1992371A), AT9283, Rigosertib (ON 01910), Volasertib (BI6727), Onvansertib (NMS- P937), BI2536, GSK461364, Tak9960, Poloxin-2, S81694 (NMS-P153), BOS172722, Empesertib (BAY1161909), BAY1217389, Mpsl-IN-1, Mpsl-IN-2, Mpsl-IN-3, TC-Mpsl- 12, AZ3146, NMS-P715, MPI-0479605, CCT251455lspinesib (SB-715992), Filanesib (ARRY-520) SB-743921. Litronesib (LY252335), ARQ-621, MK-0731, EMD-534085. 4SC- 205, acin, PF- 2771, GSK-923295, APC / C, 4SC-205, 4SC-205, Danusertib (PHA-739358), MLN8054, MK- 5108 (VX-689). MK-8745, Alisertib (MLN8237), ENMD-981693 / ENMD- 2076, Danusertib (PHA-739358), MLN8054, MK-5108 (VX-689), MK-8745, Barasertib (AZD1152-HQPA, AZD2811), 81811283, GSK1070916, PF-03814735, ZM-447439, XL-228, Tozasertib(VX- 680 / MK-0457), CYC-116, SNS-314, AMG 900, JNJ-7706621, Cenisertib (AS703569, MSC1992371A), AT9283, Rigosertib (ON 01910), Volasertib (BI6727), Onvansertib (NMS- P937), BI2536, GSK461364, Tak9960, Poloxin-2, S81694 (NMS-P153), BOS172722, Empesertib (BAY1161909), BAY1217389, Mpsl-IN-1, Mpsl-IN-2, Mpsl-IN-3, TC-Mpsl- 12, AZ3146, NMS-P715, MPi-0479605, CCT251455, Ispinesib (SB-715992), Fiianesib (ARRY-520), SB-743921, AZD4877, Litronesi b (LY2523355), ARQ-621, MK-0731, EMD- 534085, 4SC-205, PF-2771, GSK-923295, Compound-A, APC / C and Apcin.EMBODIMENT 10: The method of any of the previous EMBODIMENTS, wherein the amount if the compound that causes chromosome missegregation, optionally reversine ranges from about 1 nM to 10,0000 nM, preferably from 10-1000 nM, more preferably ranges from 100-400 nM or 100-300 nM or 100-200 nM,EMBODIMENT ll:The method of EMBODIMENT 10, wherein the compound that causes chromosome missegregation is reversine, and the hPSCs or nhpPSCs are contacted therewith, optionally at a dosage of 100-400 nM, further optionally 100-200 nM, ~150 nM or~100nm for at least 4 hours, at least 8 hours, least 12 hours, least 16 hours, least 24 hours, least 32 hours, least 40 hours, least 48 hours, or longer.EMBODIMENT 1.2: The method of any of the previous EMBODIMENTS, wherein the compound that causes chromosome missegregation is removed after the majority (over 50%) of the hPSCs or nhpPSCs are aneuploidal and / or exhibit chromosomal instability (CIN).EMBODIMENT 1.3: The method of any of the previous EMBODIMENTS, wherein a substantial number, optionally at least 30-50% of the resultant aneuploidal hPSCs or nhpPSCs retain aneuploidy after the compound that induces chromosome missegregation, optionally reversine, is removed, optionally by washing, further optionally for at least a week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or more.EMBODIMENT 14: The method of any of the previous EMBODIMENTS, wherein a substantial number, optionally at least 30-50% of the resultant aneuploidal hPSCs or nhpPSCs remain viable (continue to proliferate) when cultured in the presence of reserving or another compound that induces chromosome missegregation for prolonged duration, optionally for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or more after creation.EMBODIMENT 15: The method of any of the previous EMBODIMENTS, wherein the aneuploidal hPSCs or nhpPSCs express pluripotency transcription factors, optionally one or all of MYC, SOX2, NANOG, or OCT4 and / or cell cycle arrest proteins optionally p53 and p21, comparable to diploid hPSCs or nhpPSCs for prolonged duration, optionally for at least 1 week, or 2 weeks after creation.EMBODIMENT 16: The method of any of the previous EMBODIMENTS, which generates a population of hPSCs or nhpPSCs comprising random, variable aneuploid karyotypes.EMBODIMENT 17: The method of any of the previous EMBODIMENTS , which generates a population of hPSCs or nhpPSCs comprising different ploidy states and / or chromosomal stabilities.EMBODIMENT 18: The method of any of the previous EMBODIMENTS, which generates a population of hPSCs or nhpPSCs that are tolerant of chromosomal instability for prolonged duration, optionally for at least 1 week, or 2 weeks after creation.EMBODIMENT 19: The method of any of the previous EMBODIMENTS, wherein the resultant aneuploidal hPSCs or nhpPSCs and / or hPSCs or nhpPSCs exhibiting chromosomal instability (CIN) when cultured produce confluent cultures which may be passaged one or more times.EMBODIMENT 20: The method of any of the previous EMBODIMENTS, which includes culturing the resultant aneuploidal hPSCs or nhpPSCs and / or hPSCs or nhpPSCs exhibiting chromosomal instability (CIN) on a solid surface or plate, optionally comprising a matrix of Matrigel or Laminin-521.EMBODIMENT 21: The method of EMBODIMENT 20, wherein after the cultured cells result in a confluent culture, optionally at around 3-5 days, they are passaged one or more times.EMBODIMENT 22: The method of EMBODIMENT 20 or 21, wherein the aneuploidal hPSCs or nhpPSCs and / or hPSCs or nhpPSCs exhibiting chromosomal instability (CIN) are cultured in the presence of the compound that resulted in chromosome missegregation, optionally reversine, and continue to express pluripotency transcription factors, optionally MYC, SOX2, NANOG, or OCT4 and cell cycle arrest proteins, optionally p53 and p21, characteristic of normal (diploid) hPSCs or nhpPSCs.EMBODIMENT 23: An isolated mixed cell population exhibiting aneuploidal hPSCs or nhpPSCs and / or hPSCs or nhpPSCs comprising chromosomal instability (CIN) produced by the method of any of the any of the previous EMBODIMENTS.EMBODIMENT 24:The isolated mixed cell population of EMBODIMENT 23, which is derived using a specific hPSC line, optionally an Hl, H9, or WTC-11 hPSC ceil line.EMBODIMENT 25: The mixed cell population of EMBODIMENT 23 or 24, wherein at least 30%, 40%, 50%, 60%, 70%, 80% or 90% or more of the hPSCs or nhpPSCs are aneuploidal and / or exhibit chromosomal instability (CIN).EMBODIMENT 26: The mixed cell population of any of EMBODIMENTS 23-25, wherein the aneuploidal hPSCs or nhpPSCs are not genetically modified by any other means.EMBODIMENT 27: The mixed cell population of EMBODIMENT 23, 24 or 25, wherein the aneuploidal hPSCs or nhpPSCs are engineered to comprise a marker, optionally H2B- GFP, that permits monitoring of mitotic errors and / or proliferation which does not itself induce aneuploidy / CIN.EMBODIMENT 28: The mixed cell population of any one of EMBODIMENTS 23-27, which is modified to include diploid PSCs, optionally of the same HLA or genotype (matched cells), further optionally wherein the ratio of aneuploid to normal diploid cells mimics that of a particular disease condition, optionally mosaic variegated aneuploidy (MVA).EMBODIMENT 29: A confluent cell culture comprising the mixed cell population of any one of EMBODIMENTS: 23-28.EMBODIMENT 30:The mixed cell population or confluent ceil culture of any of EMBODIMENTS 23-29 which comprises a compound that induces chromosome missegregation, optionally reversine.EMBODIMENT 31: A method of using human pluripotent stem cells (hPSCs) or nonhuman primate pluripotent stem cells (nhpPSCs) which are aneuploidal and / or comprise chromosomal instability (CIN) produced according to any of the previous EMBODIMENTS in drug studies.EMBODIMENT 32: A method of using human pluripotent stem cells (hPSCs) or nonhuman primate pluripotent stem cells (nhpPSCs) which are aneuploidal and / or comprise chromosomal instability (CIN) produced according to any of the previous EMBODIMENTS for modeling mixed environments of diploid and aneuploid ceils using matched ceils, optionally for study of diseases such as MVA and / or for identifying drugs for treatment thereof.EMBODIMENT 33: A method of using human pluripotent stem cells (hPSCs) or nonhuman primate pluripotent stem cells (nhpPSCs) which are aneuploidal and / or exhibit chromosomal instability (CIN) produced according to any of the previous methods in screening assays for identifying drugs which selectively kill aneuploid ceils and / or cells exhibiting chromosomal instability (CIN).EMBODIMENT 34: A method of using human pluripotent stem cells (hPSCs) or non-human primate pluripotent stem cells (nhpPSCs) which are aneuploidal and / or exhibit chromosomal instability (CIN) produced according to any of the previous EMBODIMENTS in screening assays for study of different ploidy states and / or chromosomal instabilities.EMBODIMENT 35: The method of any one of EMBODIMENTS 31-34, which compares the effect of the screened moiety on human pluripotent stem cells (hPSCs) or non-human primate pluripotent stem cells (nhpPSCs) which are aneuploidal and / or exhibit chromosomal instability (CIN) to normal (dipioidal) hPSCs or nhpPSCs, optionally normal (diploidal) hPSCs or nhpPSCs of the same HLA or genotype (matched ceils).References Cited in Application:

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[0163] Each document cited herein, including all patent and non-patent literature, published or unpublished patent applications, abstracts, and any other document cited, is hereby incorporated by reference in its entirety.

[0164] Having fully described the invention, the following claims are now provided below

Claims

What is claimed is:

1. A method for creating a mixed ceil population comprising human pluripotent stem cells (hPSCs) or non-human primate pluripotent stem cells (nhpPSCs) which are aneuploidal and / or exhibit chromosomal instability (CIN) by contacting a ceil sample comprising euploid (diploid) hPSCs or nhpPSCs, optionally wherein all or substantially all of the hPSCs or nhpPSCs in the ceil sample are euploid (diploid) prior to contacting the cell sample comprising euploid (diploid) hPSCs or nhpPSCs with at least one compound that induces chromosome missegregation, which method comprises contacting said cell sample comprising euploid (diploid) hPSCs or nhpPSCs with at least one compound that induces chromosome missegregation for a time sufficient to produce a mixed cell population wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more of the hPSCs or nhpPSCs in the cell sample are aneuploidal and / or exhibit chromosomal instability (CI N).

2. The method of claim 1, wherein all or substantially all (at least 90, 95, 99% or more) of the human pluripotent stem cells (hPSCs) or non-human primate pluripotent stem cells (nhpPSCs) in the cell sample are euploid (diploid) prior to contacting with said at least one compound.

3. The method of claim 1 or 2, wherein the cell sample prior to contacting with said at least one compound comprises at least 103,103, 105, 10s, 107, 108, 109, 1010, 10u, or more euploid (diploid) hPSCs or nhpPSCs.

4. The method of claim 1, 2 or 3, which uses a dosage of said at least one compound that results in the majority (over 50%) of the hPSCs or nhpPSCs after treatment becoming aneuploidal and / or exhibiting chromosomal instability (CIN).

5. The method of claim 1, 2 or 3, which uses a dosage of said at least one compound that results in at least 80% or 90% of the human pluripotent stem ceils (hPSCs) or non-human primate pluripotent stem cells (nhpPSCs) becoming aneuploidal and / or exhibiting chromosomal instability (CIN).

6. The method of any one of the previous claims, wherein the human and non- human primate embryonic stem cells (hESCs) are derived from:(i) human or non-human primate blastocysts.(ii) embryos unused after a fertility treatment,(iii) human or non-human primate nuclear transfer embryos,(iv) non-human primate or human induced pluripotent stem cells (hiPSCs) reprogrammed from a somatic state to a pluripotent state,(v) an existing human or non-human primate ESC line, optionally Hl; or(vi) any combination of the foregoing.

7. The method of any one of the previous claims, wherein the at least one compound that causes chromosome missegregation comprises a microtubule inhibitor, optionally an inhibitor of microtubule dynamics or an inhibitor of microtubule ~ kinetochore attachment.

8. The method of any one of the previous claims, wherein the at least one compound that causes chromosome missegregation includes one or more of taxanes, vinca alkaloids, optionally one or more of eribulin, paclitaxel, docetaxel, Ixabepilone, Cabazitaxel, antibody drug conjugates comprising vedotion, e.g., Enfortumab vedotin, Tisotumab vedotin, or Brentuximab vedotin; Trastuzumab emtansine, Tirbanibu lin, Paclitaxel ceribate, Vinblastine, vincristine, Vinorelbine, vinflunine, Cryptophycin 52, Halichondrin B, Dolastatins, e.g., Doiastatins 10 and 15; Hemiasterlins, e.g., Hemiasterlin A and Hemiasterlin B; Colchicine, Combretastatins, E7010, a sulfonamide antimitotic agent, 2-Methoxyestradiol, Docetaxel, Paclitaxel, Epothilones, e.g., Epothiione A and epothilone B; and Discodermolide.

9. The method of any one of the previous claims, wherein the at least one compound that causes chromosome missegregation comprises reversine or another inhibitor of a mitotic kinase optionally an inhibitor of monopolar spindie 1 (Mpsl) kinase.

10. The method of any one of the previous claims, wherein the at least one compound that causes chromosome missegregation comprises a compound which inhibits one or more kinases or microtubule motor proteins, optionally a compound which inhibits one or more of AurkA, AurkB, Pan-AurK, Eg5, PLK1, Mpsl, CENPE and CDK1.

11. The method of claim 10, wherein the at least one compound includes one or more of Alisertib (MLN8237), ENMD-981693 / ENMD-2076, Danusertib (PHA- 739358), MLN8054, MK-5108 (VX-689), MK-8745, Barasertib (AZD1152-HQPA, AZD2811), 81811283,GSK1070916, PF-03814735, ZM-447439, XL-228, TozasertibfVX- 680 / MK-0457), CYC-116, SNS-314, AMG 900, JNJ-7706621, Cenisertib (AS703569, MSC1992371A), AT9283, Rigosertib (ON 01910), Voiasertib (BI6727), Onvansertib (NMS- P937), BI2536, GSK461364, Tak9960, Poloxin-2, S81694 (NMS-P153), BOS172722, Empesertib (BAY1161909), BAY1217389, Mpsl- IN-1, Mpsl-IN-2, Mpsl-IN-3, TC-Mpsl- 12, AZ3146, NMS-P715, MPI-0479605, CCT251455lspinesib (SB-715992), Fiianesib (ARRY-520) SB-743921, Litronesib (LY252335), ARQ-621, MK-0731, EMD-534085. 4SC- 205, acin, PF-2771, GSK-923295, APC / C, 4SC-205, 4SC-205, Danusertib (PHA-739358), MLN8054, MK-5108 (VX-689). MK-8745, Alisertib (MLN8237), ENMD-981693 / ENMD- 2076, Danusertib (PHA-739358), MLN8054, MK-5108 (VX-689), MK-8745, Barasertib (AZD1152-HQPA, AZD2811), BI811283, GSK1070916, PF- 03814735, ZM-447439, XL-228, Tozasertib(VX-680 / MK-0457), CYC-116, SNS-314, AMG 900, JNJ-7706621, Cenisertib (AS703569, MSC1992371A), AT9283, Rigosertib (ON 01910), Voiasertib (Bi6727), Onvansertib (NMS-P937), BI2536, GSK461364, Tak9960, Poioxin-2, S81694 (NMS-P153), BOS172722, Empesertib (BAY1161909), BAY1217389, Mpsl-IN-1, Mpsl-IN-2, Mpsl-IN-3, TC-Mpsl-12, AZ3146, NMS-P715, MPI-0479605, CCT251455, Ispinesib (SB-715992), Fiianesib (ARRY-520), SB-743921, AZD4877, Litronesib (LY2523355), ARQ-621, MK-0731, EMD-534085, 4SC-205, PF-2771, GSK-923295, Compound-A, APC / C and Apcin.

12. The method of any one of the previous claims, wherein the dosage amount of the at least one compound that causes chromosome missegregation, optionally reversine, ranges from about 1 nM to 10,0000 nM, 10-1000 nM, 100-400 nM, 100-300 nM or 100-200 nM.

13. The method of claim 12, wherein the at least one compound that causes chromosome missegregation comprises or consists of reversine, and the hPSCs or nhpPSCs are contacted therewith, optionally at a dosage of 100-400 nM, further optionally 100-200 nM, ~150 nM or~100nm for at least 4 hours, at least 8 hours, least 12 hours, least 16 hours, least 24 hours, least 32 hours, least 40 hours, least 48 hours, or longer.

14. The method of any one of the previous claims, wherein the at least one compound that causes chromosome missegregation is removed or inactivated after at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more of the hPSCs or nhpPSCs in the cell sample are aneuploidai and / or exhibit chromosomal instability (CIN) and / or after themajority (over 50%) of the hPSCs or nhpPSCs in the ceil sample are aneuploidal and / or exhi bit chromosomal instability (CIN),15. The method of any one of the previous claims, wherein a substantial number, optionally at least 30-50% of the resultant aneuploidal hPSCs or nhpPSCs retain aneupioidy for a prolonged time after the at least one compound that induces chromosome missegregation, optionally reversine, is removed or inactivated, optionally by washing.

16. The method of any one of the previous claims, wherein a substantial number, optionally at least 30-50% of the resultant aneuploidal hPSCs or nhpPSCs retain aneupioidy for at least a week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or more after the at least one compound that induces chromosome missegregation, optionally reversine, is removed or inactivated, optionally by washing.

17. The method of any one of the previous claims, wherein a substantial number, optionally at least 30-50% of the resultant aneuploidal hPSCs or nhpPSCs in the cell sample remain viable (continue to proliferate) when cultured in the presence of reservine or another compound that induces chromosome missegregation for a prolonged duration, optionally for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or more after creation.

18. The method of any one of the previous claims, wherein the aneuploidal hPSCs or nhpPSCs express pluripotency transcription factors, optionally one or all of MYC, SOX2, NANOG, or OCT4 and / or ceil cycle arrest proteins optionally p53 and p21, comparably to diploid hPSCs or nhpPSCs for prolonged duration, optionally for at least 1 week, 2 weeks, 3 weeks, 4 weeks or more after creation.

19. The method of any one of the previous claims, which generates a population of hPSCs or nhpPSCs comprising random, variable aneupioid karyotypes.

20. The method of any one of the previous claims, which generates a population of hPSCs or nhpPSCs comprising different ploidy states and / or chromosomal stabilities.

21. The method of any one of the previous claims, which generates a mixed population of hPSCs or nhpPSCs that are tolerant of chromosomal instability for a prolonged duration, optionally for at least 1 week, 2 weeks, 3 weeks, 4 weeks or more after creation.

22. The method of any one of the previous claims, wherein the resuitant aneuploidai hPSCs or nhpPSCs and / or hPSCs or nhpPSCs exhibiting chromosomal insta bility (CIN) when cultured produce confluent cultures which may be passaged one or more times.

23. The method of any one of the previous claims, which includes culturing the resultant aneuploidai hPSCs or nhpPSCs and / or hPSCs or nhpPSCs exhibiting chromosomal instability (CIN) on a solid surface or plate, optionally comprising a matrix of Matrigel or Laminin-521.

24. The method of claim 23, wherein after the cultured cells result in a confluent culture, optionally at around 3-5 days, they are passaged one or more times.

25. The method of claim 23 or 24, wherein the aneuploidai hPSCs or nhpPSCs and / or hPSCs or nhpPSCs exhibiting chromosomal instability (CIN) are cultured in the presence of the compound that resulted in chromosome missegregation, optionally reversine, and continue to express pluripotency transcription factors, optionally MYC, SOX2, NANOG, or OCT4 and cell cycle arrest proteins, optionally p53 and p21, characteristic of normal (diploid) hPSCs or nhpPSCs.

26. The method of any one of the previous claims, wherein one or more of the aneuploidai hPSCs or nhpPSCs and / or hPSCs or nhpPSCs exhibiting chromosomal instability are isolated from the culture and used to produce one or more other hPSCs or nhpPSC cultures.

27. An isolated cell population comprising aneuploidai hPSCs or nhpPSCs and / or hPSCs or nhpPSCs exhibiting chromosomal instability (CIN) produced by the method of any one of the any of the previous claims.

28. The isolated mixed cell population of claim 27, which is derived using a specific hPSC line, optionally Hl, H9, or WTC-11 hPSCs.

29. The mixed cell population of claim 27 or 28, wherein at least 30%, 40%, 50%, 60%, 70%, 80% or 90% or more of the hPSCs or nhpPSCs are aneuploidai and / or exhibit chromosomal instability (CIN).

30. The mixed cell population of any one of claims 27-29, wherein the aneuploidai hPSCs or nhpPSCs are not genetically modified by any other means.

31. The mixed cell population of any one of claims 27-29, wherein the aneuploidal hPSCs or nhpPSCs are engineered to comprise a marker, optionally H2B-GFP, that permits monitoring of mitotic errors and / or cell proliferation which does not itself induce aneuploidy / CIN.

32. The mixed cell population of any one of claims 27-31, which is modified after production to include diploid PSCs, optionally of the same HLA or genotype (matched cells), further optionally wherein the ratio of aneuploid to normal diploid cells mimics that of a particular disease condition, optionally mosaic variegated aneuploidy (MVA).

33. A confluent cell culture comprising the mixed ceil population of any one of claims 27-32.

34. The cell population or confluent cell culture of any one of claims 27-33, which comprises at least one compound that induces chromosome missegregation, optionally reversine.

35. A method of using human pluripotent stem cells (hPSCs) or non-human primate pluripotent stem cells (nhpPSCs) which are aneuploidal and / or comprise chromosomal instability (ON) produced according to any one of claims 1-26 or the cell population or cell culture of any one of claims 27-34 in drug studies.

36. A method of using human pluripotent stem cells (hPSCs) or non-human primate pluripotent stem cells (nhpPSCs) which are aneuploidal and / or comprise chromosomal instability (CIN) which are aneuploidal and / or comprise chromosomal instability (CIN) produced according to any one of claims 1-26 or the cell population or cell culture of any one of claims 27-34 for modeling mixed environments of diploid and aneuploid cells using matched cells, optionally for study of diseases such as MVA and / or for identifying drugs for treatment thereof.

37. A method of using human pluripotent stem cells (hPSCs) or non-human primate pluripotent stem ceils (nhpPSCs) which are aneuploidal and / or exhibit chromosomal instability (CIN) produced according to any one of claims 1-26 or the cell population or cell culture of any one of claims 27-34 in screening assays for identifying drugs which selectively kill aneuploid cells and / or cells exhibiting chromosomal instability (CIN).

38. A method of using human pluripotent stem ceils (hPSCs) or non-human primate pluripotent stem ceils (nhpPSCs) which are aneuploidal and / or exhibit chromosomal instability (CIN ) produced according to any one of daims 1-26 or the cel! population or cell culture of any one of claims 27-34 in screening assays for study of different ploidy states and / or chromosomal instabilities.

39. The method of any one of claims 35-38, which compares the effect of at least one screened moiety, optionally a drug, on said human pluripotent stem cells (hPSCs) or non-human primate pluripotent stem cells (nhpPSCs) which are aneuploidal and / or exhibit chromosomal instability (CIN) to the effect of the same screened moiety on normal (diploidal) hPSCs or nhpPSCs, optionally normal (diploidal) hPSCs or nhpPSCs of the same HLA or genotype (matched ceils).

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