Using alternating electric fields in stem cell-based therapy to prevent and treat teratoma formation

Abstract

In the case of a stem cell-based therapy in which differentiated cells are derived from pluripotent stem cells, the present application discloses a method for eliminating residual pluripotent stem cells that may remain in a batch of differentiated offspring cells. When the differentiated cells are ultimately used in the therapy, this advantageously prevents the formation of teratomas. This can be achieved by exposing this batch of differentiated offspring cells and the residual pluripotent stem cells to an alternating electric field for a period of time. The frequency and field strength of the alternating electric field cause the pluripotent stem cells to die in succession due to exposure to the alternating electric field, while the differentiated cells remain substantially undamaged. This produces purified batches of differentiated offspring cells so that they can be safely used in the stem cell-based therapy.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 749,305, filed October 23, 2018, which is incorporated herein by reference in its entirety.

[0003] background

[0004] Regenerative medicine is a game-changing field of medicine that involves the process of creating living, functional tissue to repair or replace tissue or organ function lost due to age, disease, injury or congenital defects. The field holds the promise of repairing or replacing damaged tissues and organs in the body by introducing foreign cells, tissues or even entire organs to integrate and become part of the tissue or replace the entire organ. Importantly, regenerative medicine has the potential to address the shortage of donor organs for patients in need of life-saving organ transplants.

[0005] A key to the success of regenerative medicine strategies is the ability to separate and produce stem cells including pluripotent stem cells. Pluripotent stem cells, including embryonic stem cells (ESC or ES cells) and induced pluripotent stem cells (iPSC or iPS cells), are leading candidates for cell-based therapies due to their unlimited self-renewal ability and their ability to differentiate into any cell type in the body (including any cell type required to replace tissues damaged by disease or injury). For example, this type of treatment can be used to replace neurons damaged by stroke, spinal cord injury, Alzheimer's disease, Parkinson's disease or other nervous system disorders. Pancreatic cells that grow to produce insulin can treat people with diabetes, and heart cells can repair damage after a heart attack. It is conceivable that this list may include any tissue that is damaged or sick. Stem cell therapy can also be used to help the body's own cells fight cancer by engineering the immune system to fight cancer through stem cells.

[0006] Despite its therapeutic prospects, an important safety issue for the clinical implementation of human pluripotent stem cells is the possibility of forming teratomas in vivo. Teratomas are complex tumors caused by residual pluripotent stem cells that escape the differentiation process contaminating cells derived from therapeutic stem cells. Even injecting a small amount of undifferentiated pluripotent stem cells into tissues (e.g., injecting 10,000 ES cells into skeletal muscle) may also lead to teratomas. See, for example, Lee et al. (2009) CellCycle 8: 2608-2612. Therefore, this obstacle must be overcome before stem cell therapy becomes accepted for human use.

[0007] Several attempts have been made to selectively remove residual undifferentiated pluripotent stem cells from cells prior to transplantation while retaining their differentiated progeny. These approaches include the use of cytotoxic antibodies (Tan et al., 2009; Choo et al., 2008), specific antibody cell sorting (Tang et al., 2011; Fong et al., 2009), genetic manipulation (including introduction of suicide genes) (Blum et al., 2009; Schuldiner et al., 2003), pharmacological approaches (Lee et al., 2013; Ben-David et al., 2013; Lin et al., 2017) and radiotherapy (Lee et al., 2017). However, each of these methods has significant disadvantages, such as high cost (cytotoxic antibodies and specific antibody cell sorting), batch-to-batch variability (cytotoxic antibodies and specific antibody cell sorting), nonspecific binding (cytotoxic antibodies), the need for genetic manipulation and stable integration of toxic genes (genetic manipulation), time-consuming procedures (genetic manipulation, specific antibody cell sorting, and cytotoxic antibodies), and the use of ionizing radiation (radiotherapy). Although many studies have attempted to prevent teratoma formation from residual pluripotent stem cells, no clinically feasible strategy to eliminate teratoma formation has yet been developed. SUMMARY OF THE INVENTION

[0009] One aspect of the present invention relates to a first method for preventing iatrogenic teratomas in stem cell-based cancer therapy by eliminating residual pluripotent stem cells from a batch of differentiated progeny cells. The first method includes exposing the batch of differentiated progeny cells and the residual pluripotent stem cells to an alternating electric field for a period of time. The alternating electric field has a frequency and field strength such that (a) the pluripotent stem cells die one after another due to exposure to the alternating electric field for the period of time, thereby producing a purified batch of differentiated progeny cells that are safe for subsequent use in stem cell-based therapy, and (b) the differentiated cells exposed to the alternating electric field for the period of time remain substantially undamaged. The first method also includes, after exposure, using the purified batch of differentiated progeny cells to treat cancer.

[0010] In certain instances of the first method, the stem cell-based cancer therapy is a stem cell-based therapy for leukemia. In certain instances of the first method, the stem cell-based cancer therapy is a stem cell-based therapy for lymphoma.

[0011] Another aspect of the present invention relates to a second method of eliminating residual pluripotent stem cells from a batch of differentiated progeny cells to prevent the formation of teratomas in stem cell-based therapies. The second method includes exposing the batch of differentiated progeny cells and residual pluripotent stem cells to an alternating electric field for a period of time. The alternating electric field has a frequency and field strength such that (a) due to exposure to the alternating electric field for the period of time, the pluripotent stem cells die one after another, thereby producing a purified batch of differentiated progeny cells, making it safe for subsequent use in stem cell-based therapies, and (b) the differentiated cells exposed to the alternating electric field for the period of time remain substantially undamaged.

[0012] In some cases of the second method, the purified batch contains less than 100,000 pluripotent stem cells. In some cases of the second method, the purified batch contains less than 10,000 pluripotent stem cells. In some cases of the second method, the purified batch contains less than 1,000 pluripotent stem cells. In some cases of the second method, the purified batch contains less than 100 pluripotent stem cells. In some cases of the second method, the purified batch contains less than 100 pluripotent stem cells. In some cases of the second method, the purified batch contains less than 100 pluripotent stem cells. In some cases of the second method, the time period is at least 12 hours. In some cases of the second method, the time period is at least 2 days. In some cases of the second method, the alternating electric field has an orientation that switches from time to time between at least two different directions within the time period.

[0013] In some cases of the second method, the frequency of the alternating electric field is between 50 kHz and 500 kHz. In some of these cases, the field strength of the alternating electric field is at least 1 V / cm.

[0014] In some cases of the second method, the frequency of the alternating electric field is between 250 kHz and 350 kHz and the field strength is at least 1 V / cm. In some cases of the second method, the frequency of the alternating electric field is between 50 kHz and 500 kHz and the field strength is at least 1 V / cm, and the time period is at least 12 hours. In some cases of the second method, the frequency of the alternating electric field is between 250 kHz and 350 kHz and the field strength is at least 1 V / cm, and the time period is at least 3 days.

[0015] Certain aspects of the second method further include, after the exposing, using the batch of differentiated cells for therapeutic or diagnostic purposes.

[0016] In certain cases of the second method, prior to exposing, the population of differentiated progeny cells is obtained by expanding pluripotent stem cells and differentiating the expanded pluripotent stem cells into the population of differentiated progeny cells.

[0017] In some cases of the second method, the exposing step is performed in vitro. In some cases of the second method, the exposing step is performed in vivo. In some cases of the second method, the pluripotent stem cells include one or more of induced pluripotent stem cells, embryonic stem cells, cancer stem cells, and embryonic germ cells. In some cases of the second method, the differentiated progeny cells include cardiomyocytes or cardiomyocyte progenitor cells. In some cases of the second method, the pluripotent stem cells and the differentiated progeny cells are human cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A flow chart depicting the steps of an exemplary method for stem cell-based therapy to significantly reduce the risk of teratoma formation.

[0020] Figure 2A H7 human embryonic stem cells (H7-ESCs) exposed to alternating electric fields of varying frequencies are depicted over time and compared to non-exposed controls as assessed by trypan blue cell staining.

[0021] Figure 2B Figure 2. H7-ESCs exposed to alternating electric fields of different frequencies over time. Figure 2A A magnified version of the data shown in .

[0022] Figure 3 Plotted over time the cell viability of human ESCs (H7 line) when exposed to alternating electric fields of varying frequencies and compared to non-exposed controls. The luminescence output is positively correlated with the number of cells (R 2 =0.942). RLU stands for relative luminescence unit.

[0023] Figure 4 Depicted are cell counts as assessed by trypan blue cell staining of ESC-derived cardiomyocytes after exposure to alternating electric fields of varying frequencies and compared to non-exposed control cardiomyocytes. For any of the five frequencies tested, there were no significant differences in cardiomyocyte counts before and after application of the alternating electric field.

[0024] Figure 5A Depicted are the results of a contractility assay measuring the beating rate of ESC-derived cardiomyocytes following exposure to alternating electric fields and compared to non-exposed (untreated) controls. There were no significant differences in beating rate between ESC-CMs exposed to alternating electric fields and those that were not exposed.

[0025] Figure 5BDepicted are the results of a contractility assay measuring the contraction velocity of ESC-derived cardiomyocytes following exposure to alternating electric fields and compared to non-exposed (untreated) controls. There were no significant differences in contraction velocity between ESC-CMs exposed to alternating electric fields and those that were not exposed.

[0026] Figure 5C Depicted are the results of a contractility assay measuring the acceleration of ESC-derived cardiomyocytes following exposure to alternating electric fields and compared to non-exposed (untreated) controls. There were no significant differences in acceleration between ESC-CMs exposed to alternating electric fields and those that were not exposed.

[0027] Figures 6A-6C Depict the BLI signal at 3 different times after cell injection of H7-ESC in living mice. Note that there is a huge difference in BLI signal between the ESC injection side treated with alternating electric field and the non-treated ESC injection side. No BLI signal was detected at all in the left flank of mice injected with ESC treated with alternating electric field.

[0028] 7A-7B depict control ESCs that were not exposed to an alternating electric field ( Fig. 7A ) and ESCs subjected to 300 kHz alternating electric field treatment for 3 days ( Figure 7B ) of the cell cycle analysis results.

[0029] Various embodiments are described in detail below with reference to the drawings, wherein like reference numerals represent like elements.

[0030] DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Eliminating the risk of residual pluripotent stem cells (which could give rise to teratomas) in previous stem cell-based therapies has previously been extremely difficult. The embodiments described herein use a novel approach to overcome this difficulty.

[0032] "Pluripotency" and pluripotent stem cells mean that such cells have the ability to differentiate into all cell types in an organism. The term "induced pluripotent stem cells" encompasses pluripotent cells, just like embryonic stem (ES) cells, which can be cultured for a long time while maintaining the ability to differentiate into all cell types in an organism, but unlike ES cells (derived from the inner cell mass of the blastocyst), which are derived from differentiated somatic cells, i.e., cells with narrower, more defined potentials and that cannot produce all cell types in an organism without experimental manipulation. iPS cells have an ESC-like morphology and grow into flat colonies with a large nuclear-cytoplasmic ratio, defined boundaries, and prominent nuclei. In addition, iPS cells express one or more key pluripotency markers known to those of ordinary skill in the art, including but not limited to alkaline phosphatase, SSEA3, SSEA4, Sox2, Oct3 / 4, Nanog, TRA160, TRA181, TDGF 1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and Zfp42. In addition, iPS cells are capable of forming teratomas. In addition, they are capable of forming or contributing to ectodermal, mesodermal or endodermal tissues in living organisms.

[0033] Figure 1 A flowchart depicting the steps of an exemplary method for significantly reducing the risk of teratoma formation for stem cell-based therapy. (Abbreviations: ESC - embryonic stem cells, iPSC - induced pluripotent stem cells, PPSC - pluripotent stem cells) The first 4 steps (i.e., steps 20-26) are similar to the corresponding steps in the prior art methods of stem cell-based therapy. The remaining steps (i.e., steps 30-35) provide improvements over the prior art methods.

[0034] Figure 1 The entry point of the flow chart depends on whether the process relies on induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC). If the process relies on iPSC, the process starts at step 20, where cells are isolated from the subject to obtain an initial cell population, which will be reprogrammed to generate pluripotent stem cells, from which therapeutic cells are derived for stem cell-based therapies. Alternatively, cells can be extracted / isolated from different donors. Cells can initially be isolated from a subject (or other donor), for example, by a skin sample or by drawing blood from the person.

[0035] "Starting cell population" or "initial cell population" refers to somatic cells, typically primary or non-transformed somatic cells, that have undergone nuclear reprogramming for pluripotency. The starting cell population may belong to any mammalian species, but particularly includes human cells. The source of the starting cell population includes individuals who wish to undergo cell therapy, individuals with target genetic defects for research, etc.

[0036] In some embodiments, human cells obtained from a subject for regenerative purposes can be selected from any human cell type, including fibroblasts, adipose tissue cells, mesenchymal cells, bone marrow cells, gastric cells, hepatocytes, epithelial cells, nasal epithelial cells, mucosal epithelial cells, follicular cells, connective tissue cells, muscle cells, bone cells, chondrocytes, gastrointestinal cells, spleen cells, kidney cells, lung cells, testicular cells, neural tissue cells, etc. In some embodiments, the human cell type is a fibroblast, which can be conveniently obtained from a subject by punch biopsy.

[0037] Next, in step 22, the initial cell population is reprogrammed to generate induced pluripotent stem cells (e.g., iPSC). This can be implemented using any of a variety of methods apparent to those skilled in the relevant art. "Reprogramming factors" as used herein refer to one or more (i.e., mixtures) of bioactive factors, which act on cells to change transcription, thereby reprogramming cells to pluripotency or multipotency. Reprogramming factors can be provided to cells alone or as a single composition of reprogramming factors (i.e., as a premixed composition), for example, from cells of individuals with a family history of heart disease or target genetic composition, such as fibroblasts, adipocytes, etc. The factors can be provided in the same molar ratio or in different molar ratios. Factors can be provided once or multiple times during the process of culturing cells. In some embodiments, the reprogramming factors are transcription factors, including, but not limited to, Oct3 / 4, Sox2, Klf4, c-Myc, Nanog, and Lin-28.

[0038] Somatic cells are contacted with the reprogramming factors as defined above in a combination and quantity sufficient to reprogram cells into pluripotency. Reprogramming factors can be provided to somatic cells individually or as a single composition of reprogramming factors (i.e. as a premixed composition). In some embodiments, reprogramming factors are provided as multiple encoding sequences on a carrier.

[0039] Optionally, genes can be introduced into somatic cells or iPS cells derived therefrom for a variety of purposes, such as to replace genes with functional inactivation mutations, provide marker genes, etc. Alternatively, vectors expressing antisense mRNA or ribozymes are introduced to block the expression of undesirable genes. Other methods of gene therapy are to introduce drug-resistant genes so that normal progenitor cells can have advantages and withstand selection pressure, such as multidrug resistance genes (MDR) or anti-apoptotic genes, such as bcl-2. As described above, nucleic acids can be introduced into target cells using various techniques known in the art, such as electroporation, calcium-precipitated DNA, fusion, transfection, lipofection, infection, etc. The specific method of introducing DNA is not critical.

[0040] Note that instead of starting with somatic cells that have been isolated from a subject or other donor and reprogrammed to form induced pluripotent stem cells (as described above in conjunction with steps 20-22), alternative methods can be used to obtain pluripotent stem cells for use in subsequent steps. Examples include using any conventional method to obtain embryonic stem cells to obtain such cells. In this case, Figure 1 The entry point of the flow chart is step 20' where the ESC is isolated.

[0041] After obtaining pluripotent stem cells (PPSCs) as described above (i.e., using steps 20-22 in the case of induced pluripotent stem cells, or using step 20' in the case of embryonic stem cells), step 24 is performed. In this step 24, the initial number of pluripotent stem cells is expanded using any of a variety of methods that will be appreciated by those skilled in the relevant art. For example, the initial number of pluripotent stem cells can be cultured and fed with an appropriate nutrient medium until a batch of pluripotent stem cells sufficient for the intended application is obtained.

[0042] Next, in step 26, the batch of pluripotent stem cells is differentiated to form a batch of specialized cells of the desired differentiation type, which will depend on the nature of the treatment ultimately desired. For example, if the ultimate goal is to introduce cardiomyocytes into a patient suffering from heart disease, the differentiation process in step 26 should differentiate the pluripotent stem cells into cardiomyocytes.

[0043] Examples of differentiated cells include any differentiated cells from ectodermal (e.g., neurons and fibroblasts), mesoderm (e.g., cardiomyocytes), or endodermal (e.g., pancreatic cells) lineages. The differentiated cells may be one or more of cholinergic, serotonergic, GABAergic, glutamatergic neuronal cells, pancreatic β cells, neural stem cells, neurons (e.g., dopaminergic neurons), retinal ganglion cells, photoreceptor cells (rods and cones), retinal pigment epithelial cells, oligodendrocytes, oligodendrocyte progenitor cells, hepatocytes, hepatic stem cells, chondrocytes, osteocytes, connective tissue cells, glial cells, astrocytes, myocytes, hematopoietic cells, egg or sperm cells, pacemaker cells, or cardiomyocytes.

[0044] Although the methods described herein are not limited to producing cardiomyocytes, the following example of differentiating a batch of pluripotent stem cells into a batch of cardiomyocytes is provided for illustrative purposes. Pluripotent cells can differentiate into somatic cells, including, but not limited to, cardiomyocytes. Inhibition of bone morphogenetic protein (BMP) signaling can lead to the production of cardiac muscle cells (or cardiomyocytes), see, for example, Yuasa et al., (2005), Nat. Biotechnol., 23(5):607-11. Therefore, in one embodiment, pluripotent cells are cultured for about 2 to about 6 days, for example, about 2, about 3, about 4, about 5 or about 6 days, in the presence of noggin before allowing embryoid bodies to form, and the embryoid bodies are cultured for about 1 week to about 4 weeks, for example, about 1, about 2, about 3 or about 4 weeks.

[0045] Cardiomyocyte differentiation can be promoted by including cardiotropic agents in the culture, such as activin A and / or bone morphogenetic protein 4 (see examples herein, Xu et al. Regen Med. 2011 Jan; 6(1):53-66; Mignone et al. Circ J. 2010 74(12):2517-26; Takei et al. Am J Physiol Heart Circ Physiol. 2009 296(6):H1793-803, each specifically incorporated herein by reference). Examples of such protocols also include, for example, optionally adding a Wnt agonist such as Wnt 3A in the presence of cytokines such as BMP4, VEGF, and activin A; and then culturing in the presence of a Wnt antagonist such as soluble frizzled protein. However, any suitable method for inducing cardiomyocyte differentiation may be used, such as cyclosporine A as described by Fujiwara et al. PLoS One. 2011 6(2):e16734; Dambrot et al. Biochem J. 2011 434(1):25-35; isometric cyclic stretch, angiotensin II, and phenylephrine (PE) as described by Foldes et al. J Mol Cell Cardiol. 2011 50(2):367-76; ascorbic acid, dimethyl sulfoxide, and 5-aza-2'-deoxycytidine as described by Wang et al. Sci China Life Sci. 2010 53(5):581-9; endothelial cells as described by Chen et al. J Cell Biochem. 2010 111(1):29-39, etc., which are specifically incorporated herein by reference.

[0046] Cells are harvested at an appropriate developmental stage, which can be determined based on the expression of markers and phenotypic characteristics of the desired cell type (e.g., at about 1-4 weeks). Cultures can be tested empirically by staining for the presence of markers of interest, as determined by morphology, etc. Cells are optionally enriched before or after a positive selection step, by drug selection, panning, density gradient centrifugation, etc.

[0047] "Cardiomyocyte precursors" are defined as cells that are capable of producing progeny including cardiomyocytes. Cardiomyocyte phenotypes that appear during mammalian heart development can be distinguished: primary cardiomyocytes, nodal cardiomyocytes, conducting cardiomyocytes, and working cardiomyocytes. All cardiomyocytes have sarcomeres and sarcoplasmic reticulum (SR), are coupled by gap junctions, and display automaticity. The cells of the primitive heart tube are characterized by high automaticity, low conduction velocity, low contractility, and low SR activity. This phenotype persists mainly in the ganglion cells. In contrast, atrial and ventricular working cardiomyocytes show little automaticity, are well coupled between cells, have well-developed sarcomeres, and have high SR activity. Conducting cells from the atrioventricular bundle, bundle branches, and the surrounding ventricular conduction system have poorly developed sarcomeres, low SR activity, but are well coupled and display high automaticity.

[0048] Developmental transitions have been observed in differentiated ES cell cultures for α-Mhc, β-Mhc, and cardiac troponin I and slow skeletal troponin I. Expression of Mlc2v and Anf is often used to distinguish ventricular-like cells from atrial-like cells, respectively, in ES cell cultures, although Anf expression in ES-derived cells (ESDCs) does not specifically identify atrial cardiomyocytes and may be a general marker for working cardiomyocytes.

[0049] At this point in the process (i.e., the end of step 26), we have a batch of differentiated progeny cells of the desired type (e.g., cardiomyocytes). However, since the differentiated progeny cells are produced using a process involving pluripotent stem cells, it is likely that not all pluripotent stem cells have differentiated into the desired cell type. This means that the residual pluripotent stem cells are likely to still be mixed with the batch of differentiated cells. Therefore, before using this batch of differentiated progeny cells for therapeutic purposes, the batch of differentiated progeny cells should be purified by eliminating the residual pluripotent stem cells that are still mixed with the batch of differentiated cells (preferably to the greatest extent possible). When this batch of differentiated progeny cells is ultimately used in stem cell-based therapies, this will prevent the formation of teratomas.

[0050] Step 30 is where this purification is performed. More specifically, in step 30, the batch of differentiated progeny cells and any residual pluripotent stem cells mixed therein are exposed to an alternating electric field for a period of time. The alternating electric field has a frequency and field strength characteristic. The frequency and field strength of the alternating electric field are such that the pluripotent stem cells die one after another due to exposure to the alternating electric field for the period of time, thereby producing a purified batch of differentiated progeny cells that are safe for subsequent use in stem cell-based therapies. At the same time, the frequency and field strength of the alternating electric field also cause the differentiated cells exposed to the alternating electric field for the period of time to remain substantially undamaged.

[0051] Importantly, it is not necessary to kill all pluripotent stem cells to make the batch of purification safe. On the contrary, a small amount of pluripotent stem cells can be retained, as long as the number is small enough, so that the offspring cells using the differentiation of the purified batch in the therapy based on stem cells will not cause the inappropriate risk of teratoma formation. In the case of mice, an example of this aspect represented by numbers is less than 10,000 pluripotent stem cells are injected into skeletal muscle and less than 100,000 pluripotent stem cells are injected into myocardium. In some preferred embodiments, the number of pluripotent stem cells is reduced as much as possible (for example, less than 1000 or less than 100 pluripotent stem cells).

[0052] A set of experiments was performed to determine suitable exemplary frequencies and field strengths for the electric field. Those experiments showed that frequencies between 50 kHz-500 kHz and field strengths of 1-4 V / cm can be effectively used to purify differentiated progeny cells by eliminating rapidly dividing pluripotent stem cells from the initial pre-purification batch.

[0053] Tumor Treatment Fields (TTFields) is an FDA-approved therapy for solid tumors that operates by delivering low-intensity (e.g., 1-4 V / cm) alternating electric fields in the mid-frequency range (e.g., 50-500 kHz) to the tumor. Furthermore, any known method of applying TTFields to a culture in vitro or to a subject in vivo can be used to apply an alternating electric field to the batch of differentiated progeny cells mixed in residual pluripotent stem cells to purify the batch of differentiated cells, thereby preventing the formation of teratomas. Since TTFields are alternating electric fields, an example of a method that can be used to eliminate pluripotent stem cells from a batch of differentiated progeny cells containing residual pluripotent stem cells is using the Novocure INOVITRO™ system. The INOVITRO™ system includes a software-controlled TTFields generator and a substrate with an ultra-high dielectric constant ceramic culture dish. The INOVITRO™ system allows researchers to set target TTFields intensity and frequency in each ceramic dish and is used to conduct the in vitro experiments described herein.

[0054] The core unit of the TTFields in vitro application system is a high dielectric constant ceramic (i.e., lead magnesium niobate-lead titanate [PMN-PT]) culture dish. Two pairs of electrodes are printed perpendicularly on a circuit board glued to the bottom of the TTFields ceramic dish to allow the electric field to be applied from two perpendicular directions parallel to the plane of the dish. This configuration optimizes the proportion of cells that will be exposed to the alternating electric field in metaphase spread. Because cell division can occur at any time, long exposure to the electric field is required to achieve maximum effect.

[0055] The current through the electrodes is obtained by mounting the ceramic dish on a base plate, which in turn is hooked up to a power supply box connected to a sine waveform generator and amplifier. This setup enables TTFields to be applied at frequencies ranging from 50-500 kHz and electric field strengths of 1-4 V / cm. To dissipate excess heat, the TTFields dish is maintained in a temperature-controlled incubator pre-set at 20-27°C. The temperature of the culture medium in the wells of the ceramic dish is controlled by constantly monitoring the temperature of the dish via a temperature probe printed on a circuit board on the bottom of the dish. The temperature and electric field strength are kept constant by computer-controlled adjustments of the voltage and current applied to the bottom of the circuit board by the INOVITRO™ system.

[0056] A typical workflow for cardiac stem cell therapy experiments using the INOVITRO™ system will now be described. Depending on the nature of the experiment, (a) ESC-derived cardiomyocytes or (b) ESCs are seeded onto Matrigel-coated coverslips. The coverslips are then transferred to ceramic dishes pre-filled with the appropriate culture medium (~2 mL). During the TTFields experiment, a lid is placed on the ceramic dish to mitigate evaporation. The ceramic dish is then mounted on the TTFields baseplate. The experimental dish, placed on the baseplate, is transferred to a special incubator for alternating electric field conditions. The baseplate is then connected to the TTFields generator using a flat cable connector. After setting the treatment settings using the Inovitro™ software, the experiment is started. TTFields set to any value in the range of 1-4 V / cm are applied by the Inovitro™ power generator. The selected TTFields frequencies include 50 kHz, 100 kHz, 200 kHz, 300 kHz, 400 kHz, and 500 kHz. When TTFields were applied, the incubation temperature of the ceramic dishes ranged from 20-27°C, with a target temperature of 37°C. The culture medium was manually replaced every 24 hours throughout the experiment. The treatment time lasted for any number of days from 3-4 days, after which the coverslips were removed and cell counts or cell viability assays were performed. Control dishes were placed in a separate tissue culture incubator set to 37°C, 95% air and 5% CO2. Unless otherwise stated, all experiments were performed with triplicate samples per condition.

[0057] For each experiment, the same number of cells was seeded onto each coverslip. Depending on the individual experiment, a seeding density of 50,000 - 500,000 cells per coverslip was used.

[0058] Importantly, although the in vitro experiments described herein were performed using the INOVITRO™ system, the methods described herein are not limited to the use of the INOVITRO™ system to apply the alternating electric field. Rather, the alternating electric field may be applied using any of a variety of alternative methods, including, but not limited to, systems that are similar to the INOVITRO™ system but scaled up to increase the number of cells to which the alternating electric field is applied.

[0059] The genus pluripotent stem cells includes both embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC). Also, although the experiments described herein were performed on embryonic stem cells, the results should also apply to other types of pluripotent stem cells.

[0060] In certain experiments, H7-ESCs were transfected with a lentiviral vector expressing luciferase and tomato red under the EF1a promoter. Transfection of embryonic stem cells with this lentiviral vector is advantageous because tomato red causes ESCs to become visible when illuminated with light of the appropriate wavelength, while luciferase causes ESCs to become visible when luciferin is available to the cells.

[0061] Figure 2A and 2B Depicts the results of a first experiment measuring cell number counts over time of H7 human embryonic stem cells when exposed to alternating electric fields of 50 kHz, 100 kHz, 200 kHz, 300 kHz, 400 kHz, and 500 kHz, and a control in which no alternating electric field was applied. ( Figure 2A and 2B The same data are plotted with different Y-axis scales. ) After 3 days in the incubator, the cell number of non-treated control ESCs increased by > 3600% (i.e., jumped from ~45,000 ESCs at day 0 [baseline] to ~1.7 million cells at day 3). ( Figure 2B ) Among the frequencies tested, the frequency that showed the highest ESC killing was 300 kHz. It is believed that long-term exposure to alternating electric fields will eradicate almost all ESCs.

[0062] Figure 3Depicts the cell viability of ESCs over time when exposed to alternating electric fields of different frequencies. At the baseline before the start of alternating electric field therapy and 3 days after the start of alternating electric field therapy, viability was determined based on the luminescent signal obtained from the Cell Titer Glo assay. After 3 days in the incubator, the luminescent output of non-treated control ESCs increased by> 450%. However, ESCs to which alternating electric fields were applied for 3 days at all 5 frequencies shown were almost completely eradicated. (Note that Figure 3 The data points for 100 kHz, 200 kHz, 300 kHz, 400 kHz, and 500 kHz all overlap at values ​​very close to zero. ) showed that the frequency of highest ESC killing was 300 kHz (>99.9% cell killing compared to their baseline starting value). Cell number correlated with luminescence output (R 2 = 0.942). RLU stands for relative luminescence unit.

[0063] In another experiment, bright field images are also used to observe the influence of alternating electric fields on embryonic stem cells. In this experiment, bright field images of ESC dishes are obtained at the baseline before the experiment begins. Some dishes are exposed to alternating electric fields for 3 days at a frequency of 300 kHz, while other dishes (i.e., control dishes) are not exposed to alternating electric fields in the same time period. At the end of the experiment, visual observation of the bright field images of the dishes shows that the number of embryonic stem cells in the dishes that are not exposed to the alternating electric field increases rapidly, but in the dishes that are exposed to the alternating electric field, it seems to be completely eradicated.

[0064] These experiments showed that exposing pluripotent stem cells to an alternating electric field at the above frequency resulted in the death of the pluripotent stem cells.

[0065] Additional experiments were performed to determine what happens when differentiated cells derived from pluripotent stem cells are exposed to alternating electric fields of the same frequency. More specifically, in these experiments, dishes of ESC-derived cardiomyocytes (ESC-CMs) were visually observed before and after alternating electric fields were applied to those cells for 3 days at 5 different frequencies (50, 100, 200, 300, and 400 kHz). In addition, control dishes were visually observed before and after a 3-day period during which alternating electric fields were not applied. Visual observation of the dish did not show significant differences in cardiomyocyte pores before and after the application of alternating electric fields.

[0066] In addition to visual observation, cell counts were performed and Figure 4Depicts the results of cell counting of ESC-derived cardiomyocytes (ESC-CMs) after exposure to alternating electric fields of different frequencies. Cell counting was performed using a trypan blue cell counting assay at the end of a 3-day period. Cell counting showed that there was no statistically significant difference in cardiomyocyte counts before and after application of an alternating electric field for any of the 5 frequencies tested.

[0067] ESC-derived cardiomyocytes were also observed to beat both before and after application of the alternating electric field, indicating a fully functional phenotype (ie, no deleterious effects caused by the alternating electric field).

[0068] It was also observed that ESC-derived cardiomyocytes had reduced beat-to-beat variability following exposure to alternating electric fields, suggesting that the improved electromechanical properties observed may be indicative of more mature cardiomyocyte characteristics.

[0069] Figures 5A-5C Depict the results of a contractility assay measuring the beating rate, contraction velocity, and acceleration of ESC-derived cardiomyocytes after exposure to an alternating electric field at 300 kHz and control ESC-CMs that were not exposed to an alternating electric field. No statistically significant differences were found in the beating rate, contraction velocity, and acceleration of the cardiomyocytes between ESC-CMs exposed to an alternating electric field and those that were not exposed. This provides additional evidence that cardiomyocytes are not harmed by alternating electric fields.

[0070] In summary, these experiments show that exposing a population of stem cell-derived differentiated cells (e.g., cardiomyocytes) to alternating electric fields of the above frequencies leaves those cells essentially unharmed. Of note, for the differentiated progeny cells (which are not damaged by the alternating electric fields, see e.g. Figure 4 ) are similar to the results observed for stem cells (which die when exposed to alternating electric fields, see e.g. Figure 3 ) are in stark contrast to the results observed.

[0071] Figures 6A-6C Depicted are BLI signals at three different time points after cell injection of -500,000 human H7-ESCs in the right flank of mice ( Fig. 6A : Day 1, Figure 6B : Day 3, and Figure 6C: Day 7). BLI signals at 3 different time points were also shown for ESCs treated with alternating electric fields injected in the left flank of mice (treated with alternating electric fields at 300 kHz for 3 days) and matrigel injections alone in the left scapula of mice (used as our control). Of note, there was a huge difference in BLI signal between the ESC injection side treated with alternating electric fields and the non-treated ESC injection side. No BLI signal was detected at all in the left flank of mice injected with ESCs treated with alternating electric fields. The luminescent signal is measured in photons per second per square centimeter per steradian (photons / sec / cm 2 This indicates that exposing pluripotent stem cells to an alternating electric field reduces the number of pluripotent stem cells to the point where they do not form teratomas in vivo.

[0072] Figure 7A-7B Depicts the results of cell cycle analysis, showing the difference between ESCs and untreated control ESCs ( Fig. 7A ) compared to the ESCs treated with an alternating electric field at 300kHz for 3 days ( Figure 7B ) are arrested in the G2 / M checkpoint of the cell cycle. This provides important insights into the mechanism of action of alternating electric fields on rapidly dividing cells, such as stem cells and tumor cells.

[0073] "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.

[0074] "Mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport or pet animals, such as dogs, horses, cats, cows, etc. In some preferred embodiments, the mammal is a human.

[0075] The application describes methods for eliminating residual pluripotent stem cells from a population of differentiated progeny cells to prevent the formation of teratomas in stem cell-based therapies. In some embodiments, the cell population purified by the methods described herein is purified to a concentration of 10 7 In some embodiments, the cell population purified by the methods described herein is purified to the extent that there are less than 100,000 pluripotent stem cells therein. In some embodiments, the cell population purified by the methods described herein is purified to the extent that there are less than 10,000 pluripotent stem cells therein. In some embodiments, the cell population purified by the methods described herein is purified to the extent that there are less than 1,000 pluripotent stem cells therein. In some embodiments, the cell population purified by the methods described herein is purified to the extent that there are less than 100 pluripotent stem cells therein.

[0076] For in vitro embodiments, the cell population may include any population suspected of containing a mixture of differentiated and pluripotent cells. Of particular interest are cultures of differentiated cells prepared as therapeutics, e.g., cells differentiated into a pathway of interest, where the differentiated cells are derived from stem cells, and where residual stem cells may be present in the population of differentiated cells.

[0077] In order to eliminate pluripotent cells from a batch of differentiated progeny cells, an appropriate solution can be used for dispersion or suspension. Such a solution is usually a balanced salt solution, such as physiological saline, PBS, Hank's balanced salt solution, etc., which can be conveniently supplemented with fetal bovine serum or other naturally occurring factors, and an acceptable low concentration buffer (usually 5-25 mM). Convenient buffers include HEPES, phosphate buffer, lactate buffer, etc.

[0078] Pluripotent cells can be eliminated from a complex mixture of cells by suspending the cells in a culture medium and optionally allowing the cells to adhere to a surface. The cells are then subjected to a low intensity alternating electric field in the medium frequency range (50-500 kHz) for a period of time.

[0079] In some embodiments, the frequency of the alternating electric field is about 200 kHz to about 400 kHz, about 250 kHz to about 350 kHz, and can be about 300 kHz. In some embodiments, the electric field is at least 1 V / cm. In some embodiments, the electric field is between 1-4 V / m. In other embodiments, a combination of field strengths is applied, such as combining two or more frequencies simultaneously, and / or applying two or more frequencies at different times.

[0080] The exposure may last for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours or longer.

[0081] In some embodiments, the cell culture comprises an artificial organ cultured in vitro, for example, wherein one or more different tissue types or tissue layers are co-cultured, wherein the artificial organ is treated to eliminate pluripotent cells by exposure to an alternating electric field prior to implantation of the organ in vivo.

[0082] In some embodiments, the alternating electric field is combined with an effective dose of a substance that targets dividing cells, including, but not limited to, an alkylating agent, etc. It is known that alkylating agents act by alkylation of macromolecules such as the DNA of cancer cells, and are generally strong electrophilic agents. This activity can disrupt DNA synthesis and cell division. Examples of alkylating agents suitable for use herein include nitrogen mustards and their analogs and derivatives, including cyclophosphamide, ifosfamide, chlorambucil, estramustine, mechlorethamine hydrochloride, melphalan, and uracil mustard. Other examples of alkylating agents include alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, and streptozocin), triazenes (e.g., dacarbazine and temozolomide), ethylenimines / methylmelamines (e.g., altretamine and thiotepa), and methylhydrazine derivatives (e.g., procarbazine). Included in the group of alkylating agents are alkylating-like platinum-containing drugs, including carboplatin, cisplatin, and oxaliplatin.

[0083] Additional substances may include, but are not limited to, analogs and derivatives of folic acid, pyrimidines, purines, and cytidine. Members of the folic acid group of substances suitable for use herein include, but are not limited to, methotrexate (amethopterin), pemetrexed, and analogs and derivatives thereof. Pyrimidine substances suitable for use herein include, but are not limited to, cytarabine, floxuridine, fluorouracil (5-fluorouracil), capecitabine, gemcitabine, and analogs and derivatives thereof. Purine substances suitable for use herein include, but are not limited to, mercaptopurine (6-mercaptopurine), pentostatin, thioguanine, cladribine, and analogs and derivatives thereof. Suitable cytidines for use herein include, but are not limited to, cytarabine (cytosine arabinodside), azacitidine (5-azacytidine), and analogs and derivatives thereof.

[0084] Suitable anti-mitotic agents for use herein include, but are not limited to, vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine, and analogs and derivatives thereof) and podophyllotoxins (including, but not limited to, etoposide, teniposide, and analogs and derivatives thereof). Suitable anti-tumor agents for use herein include, but are not limited to, belomycin, dactinomycin, doxorubicin, idarubicin, epirubicin, mitomycin, mitoxantrone, pentostatin, plicamycin, and analogs and derivatives thereof. Camptothecin analogs and derivatives are suitable for use herein and include camptothecin, topotecan, and irinotecan.

[0085] The number of pluripotent cells remaining in the population can be assessed, for example, by culturing an aliquot of the population for the presence of teratoma-forming cells, or by analyzing markers of pluripotency, for example, by staining for the presence of markers such as Oct-4, Sox2, Nanog, KLF4, TRA-1-60 / TRA-1-81 / TRA-2-54, SSEA1, SSEA4, etc. For example, an aliquot of the cells can be stained with an antibody that binds to one or more such markers indicative of pluripotency. The antibody is added to the cell suspension and incubated for a period of time sufficient to bind to available cell surface antigens, and cells bound to such an antibody are quantified.

[0086] As defined above, the offspring cells exposed to the differentiation of the alternating electric field remain substantially unharmed. However, it is noted that this will be analyzed from an overall perspective, and a small portion of the offspring cells of differentiation may die. Preferably, the offspring cells less than 5% differentiation will die. In an alternative embodiment, the offspring cells less than 10%, 15% or 25% differentiation will die. In other words, there may be some killing of the offspring cells of differentiation, but it is expected to have at least 75%, at least 85%, at least 90%, at least 95% or higher differentiation offspring cell viability after the purification process.

[0087] The purified batch of differentiated progeny cells lacking residual pluripotent stem cells can be resuspended in any appropriate culture medium that maintains cell viability. Various culture media are commercially available and can be used according to the properties of the cells, including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., usually supplemented with fetal bovine serum.

[0088] The purified batches of differentiated progeny cells can be used immediately. Alternatively, the cells can be frozen at liquid nitrogen temperatures and stored for a long time, thawed and can be reused. In this case, the cells are usually frozen in 10% DMSO, 50% serum, 40% buffered medium or some other such solutions as commonly used in the art to preserve the cells at such freezing temperatures and thawed in a manner as commonly known in the art for thawing frozen cells.

[0089] return Figure 1 The purified batch of differentiated progeny cells can be used therapeutically in step 35 to treat the subject or for diagnostic purposes. The therapy can be directed to treating the cause of the disease; alternatively, the therapy can be to treat the effects of the disease or condition. The induced cells can be transferred to or near the site of injury in the subject; or the cells can be introduced into the subject in a manner that allows the cells to migrate or home to the site of injury. The transferred cells can advantageously replace damaged or injured cells and allow for improvement of the overall condition of the subject. In some cases, the transferred cells can stimulate tissue regeneration or repair.

[0090] The differentiated progeny cells can be transferred to subjects with a wide range of diseases or disorders. Degenerative heart diseases such as ischemic cardiomyopathy, conduction diseases and congenital defects may benefit from regenerative cell therapy, see, e.g., Janssens et al., (2006), Lancet, 367: 113-121.

[0091] In some embodiments, the purified batch of differentiated progeny cells includes cardiomyocytes differentiated in vitro from a population of pluripotent stem cells. It was found that after treatment with an alternating electric field, the beat-to-beat variations of ESC-derived cardiomyocytes were reduced, indicating improved electromechanical properties, indicating more mature cardiomyocyte characteristics. In some embodiments, the cardiomyocytes show enhanced structural and functional maturation relative to similarly differentiated cardiomyocytes that were not exposed to an alternating electric field.

[0092] In those embodiments involving cardiomyocytes, a purified batch of differentiated cardiomyocytes or cardiomyocyte progenitor cell colonies can be administered to the heart of a subject in need thereof by injection. In one embodiment, the injection is intramyocardial. Those skilled in the art will be fully aware of the advantages of delivering cardiac stem cells by intramyocardial injection, because the heart is a working muscle. Intramyocardial injection minimizes the loss of injected cells due to the contractile motion of the heart. Alternatively, the treated cells can be administered by transendocardial or transepicardial injection. In one embodiment, a catheter-based method is used to deliver transendocardial injection. The use of catheters excludes more invasive delivery methods in which the chest cavity will need to be opened. As those skilled in the art will appreciate, optimal recovery time will be allowed by more minimally invasive surgery. The catheter method may involve the use of technology such as the NOGA catheter or similar systems. The NOGA catheter system promotes guided administration by providing an electromechanical map of the target area and a retractable needle that can be used to deliver targeted injections or bathe the target area with a therapeutic agent. The method described herein can be performed by using such a system to deliver injections. Those skilled in the art will recognize an alternative system that also provides the ability to provide targeted therapy by integrating imaging and catheter delivery systems that can be used with the methods described herein. Information about the use of NOGA and similar systems can be found, for example, in Sherman (2003) Basic Appl. Myol. 13: 11-14; Patel et al. (2005) The Journal of Thoracic and Cardiovascular Surgery 130: 1631-38; and Perrin et al. (2003) Circulation 107: 2294-2302. In another embodiment, stem cell-derived cardiac cells can be administered via an intracoronary administration route. Those skilled in the art will recognize other useful delivery or implantation methods that can be used with the methods described herein, including those described in Dawn et al. (2005) Proc. Natl. Acad. Sci. USA 102, 3766-3771.

[0093] Subjects suffering from nervous system diseases or disorders benefit from regenerative cell therapy. In some methods, purified batches of differentiated progeny cells are transplanted into the site of injury to treat neural stem cells or neural cells for nervous system disorders, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, cerebral infarction, spinal cord injury or other central nervous system disorders, see, for example, Morizane et al., (2008), Cell Tissue Res., 331(1):323-326; Coutts and Keirstead (2008), Exp. Neurol., 209(2):368-377; Goswami and Rao (2007), Drugs, 10(10):713-719.

[0094] Endothelial cells can be used, for example, to improve vascular structure and function in peripheral arterial disease, enhance angiogenesis and improve perfusion. Islet cells (or primary cells of the islets of Langerhans) can be transplanted into subjects with diabetes (e.g., type 1 diabetes), see, for example, Burns et al., (2006) Curr. Stem Cell Res. Ther., 2: 255-266. In some embodiments, pancreatic beta cells are transplanted into subjects with diabetes (e.g., type 1 diabetes). In other examples, hepatocytes or progenitor cells are transplanted into subjects with liver diseases such as hepatitis, cirrhosis, or liver failure.

[0095] Hematopoietic cells or hematopoietic stem cells (HSC) can be transplanted into subjects with blood cancer or other blood or immune disorders. Examples of blood cancers that hematopoietic cells or HSC may treat include: acute lymphoblastic leukemia, acute myeloblastic leukemia, chronic myeloid leukemia (CML), Hodgkin's disease, multiple myeloma and non-Hodgkin's lymphoma. Generally, subjects with this disease must undergo radiation and / or chemotherapeutic treatment to kill rapidly dividing blood cells. HSC derived from the methods described herein is introduced into these subjects to help regenerate exhausted cell reservoirs. In some cases, hematopoietic cells or HSC derived by transdifferentiation can also be used to directly fight cancer. For example, the transplantation of allogeneic HSC has shown promise in the treatment of renal cancer, see, for example, Childs et al., (2000), N. Engl. J. Med., 343:750-758. In some embodiments, allogeneic or even autologous HSCs derived from inductive cells can be introduced into a subject to treat renal or other cancers. Hematopoietic cells or HSCs derived from inductive cells can also be introduced into a subject to generate or repair cells or tissues other than blood cells, such as muscle, blood vessels, or bone. Such treatments may be useful for a variety of disorders.

[0096] The number of times a treatment is administered to a subject may vary. Introducing a purified batch of differentiated progeny cells into a subject may be a one-time event; however, in some cases, such treatment may elicit improvement for a limited period of time and require a series of repeated treatments to be continued. In other cases, multiple administrations of cells may be required to observe an effect. The exact regimen depends on the disease or condition, the stage of the disease, and the parameters of the individual subject being treated.

[0097] The cells can be introduced into the subject by any of the following routes: parenterally, intravenously, intra-arterially, intramuscularly, subcutaneously, transdermally, intratracheally, intraperitoneally, or into the cerebrospinal fluid.

[0098] The purified batches of the offspring cells of differentiation can be given in any physiologically acceptable culture medium. They can be provided alone or together with a suitable substrate or matrix, for example to support their growth and / or organization in the tissue to be transplanted. Cells can be introduced by injection, catheter, etc. Cells can be frozen and stored for a long time at liquid nitrogen temperature and can be used when thawed. If frozen, cells will be stored in 10% DMSO, 50% FCS, 40% RPMI 1640 culture medium usually. Once thawed, cells can be expanded by using growth factors and / or stromal cells related to progenitor cell proliferation and differentiation.

[0099] Although the above in vitro experiments were conducted using the above frequencies, field strengths, and durations, those parameters may vary. For example, the frequency may be between 50-500 kHz, the electric field strength may be between 0.5-5 V / cm; and the duration may be any time longer than 12 hours.

[0100] In in vitro experiments using the Inovitro™ system described herein, the direction of the alternating electric field was switched between two perpendicular directions at one second intervals. However, in alternative embodiments, the direction of the alternating electric field can be switched at a faster rate (e.g., at intervals between 1-1000 ms) or at a slower rate (e.g., at intervals between 1-100 seconds).

[0101] In an in vitro experiment using the Inovitro™ system described herein, the direction of the alternating electric field is switched between two perpendicular directions by applying an AC voltage in an alternating order to two pairs of electrodes arranged 90° apart from each other in 2D space. However, in an alternative embodiment, the direction of the alternating electric field can be switched between two non-perpendicular directions or between three or more directions (assuming that additional electrode pairs are provided) by relocating the electrode pairs. For example, the direction of the alternating electric field can be switched between three directions, each of which is determined by the placement of its own electrode pairs. Optionally, the three pairs of electrodes can be arranged so that the generated electric field is arranged 90° apart from each other in 3D space. In other alternative embodiments, the direction of the electric field remains constant.

[0102] In in vitro experiments using the Inovitro™ system described herein, the electric field is capacitively coupled into the culture because the Inovitro™ system uses conductive electrodes disposed on the outer surface of the dish sidewalls, and the ceramic material of the sidewalls acts as a dielectric. However, in alternative embodiments, the electric field can be applied directly to the cells without capacitive coupling (e.g., by modifying the Inovitro™ system configuration so that the conductive electrodes are disposed on the inner surface of the sidewalls rather than the outer surface of the sidewalls).

[0103] The methods described herein can also be applied to in vivo situations by applying an alternating electric field to a target area of ​​a living subject's body to prevent teratoma formation or to treat a teratoma that has begun to form. For example, this can be achieved by applying an alternating electric field to the body of a living subject using the Novocure Optune system (or a variant thereof). See, for example, U.S. Patent 7,565,205, which is incorporated herein by reference. In this case, electrodes are placed on the subject's body or implanted in the subject's body (e.g., directly below the subject's skin or near the target area), and an AC voltage is applied between those electrodes to apply an alternating electric field in the target area of ​​the subject's body. Optionally, the orientation of the alternating electric field can be periodically switched between two or more different directions.

[0104] In this in vivo situation, pluripotent stem cells are eliminated in vivo to prevent the formation of teratomas or to treat existing teratomas. This is achieved by exposing the pluripotent stem cells to an alternating electric field for a period of time, the alternating electric field having a frequency and a field strength. The frequency and field strength of the alternating electric field are such that the pluripotent stem cells die off one after another due to exposure to the alternating electric field for the period of time until the number of pluripotent stem cells is sufficiently small to (a) prevent the formation of teratomas or (b) treat teratomas that have already formed. The frequency and field strength of the alternating electric field are such that differentiated cells exposed to the alternating electric field in the subject remain substantially undamaged.

[0105] experiment

[0106] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to implement the methods described herein, and they are neither intended to limit the scope of what the inventors regard as their invention nor to represent that the following experiments are all or the only experiments performed. Every effort has been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is degrees Celsius, and pressure is at or near atmospheric pressure.

[0107] Example 1

[0108] Seeding cells onto coverslips. Human pluripotent stem cells (including human embryonic stem cells [ESCs] and human induced pluripotent stem cells [iPSCs]) are maintained in Essential 8 (E8) medium supplemented with 10 μM ROCK inhibitor Y-27632. ESCs (WA07 [H7] strain) or iPSCs are seeded onto the center of a 22 mm diameter glass or plastic coverslip in a 6-well plate. The coverslips have been previously coated with Matrigel diluted 1:200 in DMEM / F12 for at least 1 hour at 37°C in a conventional tissue culture incubator (37°C, 95% air, 5% CO2). Once the cells have adhered to the coverslips, a total of 2 mL of E8 medium supplemented with 10 μM ROCK inhibitor Y-27632 is added to each well. As cells recovered, the medium was changed daily for 1-2 days, and the coverslips were then transferred to the ceramic dish of an Inovitro™ TTFields apparatus (Novocure Inc., Haifa, Israel).

[0109] The procedure for seeding human ESC- or iPSC-derived cardiomyocytes (ESC-CMs or iPSC-CMs) was similar to that for pluripotent stem cells, except that the recovery period after seeding lasted longer (~5 days) until obvious spontaneous beating was observed. ESC-CMs and iPSC-CMs were also maintained in RPMI 1640 medium containing B27 supplement plus insulin. A monolayer of beating cardiac cells was visualized on the coverslip before starting the tumor treatment electric field experiment. Cell imaging was performed using a LeicaDM IL LED inverted fluorescence microscope (Leica Microsystems, Buffalo Grove, IL) or a Revolve microscope (Echo laboratories, San Diego, CA).

[0110] Trypan blue cell counting is measured using an automatic cell counter.Except for two 2 mL PBS washings, the coverslip in the ceramic dish is prepared for cell counting by removing the culture medium.Then the coverslip is transferred to a 6-well plate, followed by the addition of 0.5 mL trypsin-LE (TrypLE).The detached cells are suspended in 15 mL Falcon tubes, containing 4.5 mL of E8 culture medium (for ESC and iPSC) supplemented with 10 μM ROCK inhibitor Y-27632 or RPMI 1640 culture medium (for ESC-CM and iPSC-CM) containing B27 supplement plus insulin.Cells are centrifuged at room temperature for 5 minutes at 300 g.Once precipitation is obtained, supernatant is sucked out, and cells are resuspended in 10 mL Essential 8 or RPMI 1640 culture medium to perform cell counting.

[0111] After mixing the cell culture suspension, a 10 μL aliquot was taken and placed in a 1.5 mL Eppendorf tube. 10 μL of 0.4% trypan blue solution was then added to the cell culture aliquot, mixed by pipetting up and down, and loaded into one of the wells of a cell counting slide. Cells were counted using a LUNA-FL dual fluorescence automated cell counter (Logos Biosystems, Annandale, VA, USA). For each experimental condition (e.g., control [no TTF], 50 kHz, 100 kHz, 200 kHz, 300 kHz, 400 kHz, and 500 kHz), the total live and dead cell counts were calculated from 3 technical replicates and averaged. Total cell counts were extrapolated based on a 1:10 dilution factor of the initial cell suspension.

[0112] Quantitative measurement of cell viability using the CELLTITER-GLO 2.0 assay. Cell viability of human pluripotent stem cells and stem cell-derived cardiomyocytes was quantitatively measured using the CellTiter-Glo 2.0 assay (Promega, Madison, WI, USA). The CellTiter-Glo 2.0 assay provides a homogeneous method to determine the number of viable cells in culture by quantifying the amount of ATP present, which indicates the presence of metabolically active cells. 2+, ATP (contributed by living cells) and molecular oxygen, luciferase catalyzes the single oxidation of luciferin.Add the CellTiter-Glo 2.0 reagent volume equal to the cell culture medium volume present in each well (for example, 0.5 mL CellTiter-Glo 2.0 reagent is added to 0.5 mL of the culture medium containing cells).The contents are mixed on an orbital oscillator for 2 minutes to induce cell lysis.6-well plates are incubated at room temperature for 10 minutes to stabilize the luminescent signal.Afterwards, 50 μl from each well are transferred to white 96-well plates in triplicate.Luminescence is recorded using 1 second integration time on Synergy HTX multi-mode plate reader (BioTek, Winooski, VT, USA).Luminescent signals are analyzed using BioTek Gen5 3.03 software.

[0113] Contractility Assays. Contractility measurements of stem cell-derived cardiomyocytes were evaluated by a Sony SI8000 live cell imaging system (Sony Biotechnology, San Jose, CA) before and after application of tumor therapeutic electric fields. ESC-CMs or iPSC-CMs were treated at various alternating electric field frequencies (e.g., 50 kHz, 100 kHz, 200 kHz, 300 kHz, 400 kHz, and 500 kHz) for 72 hours. Control dishes did not have any alternating electric fields applied to them. Data were acquired using a high-performance camera that captured the motion of the cells with high spatiotemporal fidelity using unique motion vector software. After image acquisition, motion detection algorithms developed by Sony Biotechnology were used to calculate the displacement and amplitude of cell motion. Regions of interest (ROIs) were positioned on individual cells or clusters of cardiac cells, and various contractile and diastolic parameters were calculated.

[0114] Fluorescence-activated cell sorting (FACS) and cell cycle analysis. For FACS and cell cycle analysis studies, we harvested H7 ESCs after the indicated alternating electric field experimental conditions and washed them with PBS. We then fixed the cells in ice-cold 70% ethanol at −20°C for 2 h. After fixation, we collected the cells by centrifugation and stained them with 1.0 mL of PBS containing 10 μg / mL propidium iodide (PI), 100 μg / mL RNase A, and 0.05% Triton X-100. The cells were incubated in PI solution for 15 min at room temperature in the dark and washed once with PBS, resuspended in 0.5 ml PBS, and evaluated for live and dead cells using InCyte software in a Guava FACS analyzer (EMD Millipore, Burlington, MA), and cell cycle status using Guava cell cycle analysis software. We analyzed the results using FlowJo software (TreeStar, Ashland, OR) to measure live / dead cells and cell cycle status.

[0115] Culture and maintenance of human pluripotent stem cells (hESC and hiPSC)

[0116] Generation of iPSCs. Dermal fibroblasts from healthy human donors were reprogrammed into iPSCs using non-integrating Sendai virus vectors carrying the following transcription factors: Oct4, Sox2, Nanog, and cMyc.

[0117] ESC culture and maintenance. The human ESC line used in these experiments was the WA07 (H7) line transfected with a lentiviral vector expressing luciferase and tomato red under the EF1a promoter. ESCs were grown to 90% confluence on Matrigel-coated plates (ES Qualified, BD Biosciences, San Diego, CA) using chemically defined E8 medium as previously described. The medium was changed daily, and cells were passaged every 3-4 days with EDTA (Thermo Fisher Scientific, CA). Prior to plating, dissociated cells were supplemented with 10 μM ROCK inhibitor Y-27632 to prevent apoptosis.

[0118] Cardiac Differentiation. Human pluripotent stem cells were grown to 90% confluence and subsequently differentiated into beating cardiomyocytes as previously described in Burridge et al. (2014); Burridge et al. (2015). Cardiac cell purity was 80-90% based on flow cytometry, where cells stained for cardiac markers such as troponin T and α-actin. Briefly, on day 0, cells were supplemented with basal medium (RPMI 1640 [Thermo Fisher Scientific] and 2% B27 insulin-free supplement [Thermo Fisher Scientific]) and 6 μM CHIR-99021 [Selleck Chemicals], a selective inhibitor of glycogen synthase kinase 3β, which activates the canonical Wnt signaling pathway. On day 2, the medium was replaced with basal medium without CHIR99021 supplementation. On day 3, cells were treated with 5 μM Wnt antagonist IWR-1 [Selleck Chemicals] for 2 days. On day 5 and every other day thereafter until harvest, the medium was replaced with fresh basal medium. 10X TrypLE was added to the cardiomyocytes at 37°C for 10 minutes to dissociate into single cells. After dissociation, ESC-CMs or iPSC-CMs were re-plated on fresh Matrigel-coated 6-well plates in RPMI 1640 medium supplemented with 10% knockout serum supplement, B27 supplement (containing insulin) and 1 μM Thiazovivin.

[0119] Cell injection in mice. Female immunodeficient nude (NU / NU) mice were anesthetized with 2% isoflurane and injected subcutaneously with approximately 500,000 H7 ESCs. The H7 ESCs were suspended in 100 μL Matrigel and then injected into the right flank of the mouse using a 28-gauge syringe needle. In addition, H7 ESCs treated with an alternating electric field (i.e., those ESCs treated at 300 kHz for 3-4 days) were injected subcutaneously into the left flank of the mouse. Matrigel-only injections in the left scapula were used as our controls. The syringe was kept on ice before injection to prevent Matrigel from solidifying at room temperature. All animal procedures were approved by the Stanford Administrative Panel on Laboratory Animal Care (APLAC).

[0120] Bioluminescence imaging (BLI) of transplanted cells to assess cell survival and teratoma formation. BLI was performed to track cell proliferation during the course of this study. In vivo BLI was performed on an IVIS Spectrum imaging system (Xenogen Corporation, Alameda, CA) for up to 5 weeks after cell injection. Cell survival and proliferation were monitored at 0, 1, 3, 7 days, and every 7 days until 35 days after cell transplantation. One gram of D-firefly luciferin potassium salt was diluted in 23 mL PBS, and 300 μl of the mixture was administered intraperitoneally using a 28-gauge insulin syringe needle. Ten minutes after the intraperitoneal injection, animals were imaged for 20 minutes using an acquisition window of 1 second to 5 minutes. Bioluminescence images at different time points were analyzed using Living Image software (Caliper LifeSciences, version 4.3.1). ROIs were drawn over the cell injection site and control area. The luminescence signal was measured in photons per second per square centimeter per steradian (photons / sec / cm 2 / sr) units of radiance.

[0121] Explantation and histology of teratomas. After teratomas grew to a size of approximately 15 mm in diameter, mice were euthanized and teratomas were excised, fixed with 4% paraformaldehyde, and sent to the pathology core laboratory for paraffin sectioning and H&E staining.

[0122] Statistical analysis. Statistical analyses were performed using GraphPad Prism (version 7.04) and SPSS (IBM, version 21). Tests with a significance alpha level set at P < 0.05 were considered significant. Unless otherwise indicated, data are reported as mean ± SD.

[0123] Materials List. The following materials were used in the experiments described in this article: Falcon 6-well clear flat-bottom plates (Corning, catalog number: 353046); Essential 8™ medium (ThermoFisher Scientific, catalog number: A1517001); RPMI 1640 medium (ThermoFisher Scientific, catalog number: 11875093); Phosphate-buffered saline, 10X solution, Fisher BioReagents™ (Thermo Fisher Scientific, catalog number: BP3994); CHIR-99021 (Selleck, catalog number: S2924); IWR-1 (Selleck, catalog number: S7086); B-27™ supplement (50X), serum-free (ThermoFisher Scientific, catalog number: 17504044); B-27™ supplement, insulin-free (ThermoFisher Scientific, catalog number: A1895601); Corning Matrigel Growth Factor Reduced (ThermoFisher Scientific, catalog number: 356231); TrypLE™ Select Enzyme (10X), Phenol Red Free (ThermoFisher Scientific, catalog number: A1217701); BD Lo-Dose U-100 Insulin Syringe 0.5 mL (ThermoFisher Scientific, catalog number: 329461); CellTiter-Glo 2.0 (Promega, catalog number: G9242); paraformaldehyde 20% solution EM grade (Electron Microscopy Sciences, catalog number: 15713-S); propidium iodide (Sigma-Aldrich, catalog number: P4170); RNase A (Thermo Fisher Scientific, catalog number: EN0531); Triton X-100 (Sigma-Aldrich, catalog number: X100); XenoLight D-luciferin potassium salt (PerkinElmer, catalog number: 122799); DM IL LED inverted fluorescence microscope (Leica Microsystems, Buffalo Grove, IL); Revolve microscope (Echo laboratories, San Diego, CA); Sony SI8000 live cell imaging system (Sony Biotechnology, San Jose, CA); Synergy HTX multi-mode plate reader (BioTek, Winooski, VT, USA); LUNA-FL dual fluorescence automated cell counter (Logos Biosystems, Annandale, VA, USA); Guava easyCyte HT flow cytometer (EMDMillipore, Burlington, MA); Inovitro™ in vitro TTField device (Novocure Inc., Haifa, Israel). .

[0124] References

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[0149] Although the present invention has been disclosed with reference to certain embodiments, various modifications, changes and variations may be made to the described embodiments without departing from the metes and bounds of the present invention as defined by the appended claims. Therefore, it is intended that the present invention is not limited to the described embodiments, but has the full scope defined by the language of the following claims and their equivalents.

Claims

1. A method for eliminating residual pluripotent stem cells from a population of differentiated progeny cells to prevent teratoma formation in stem cell-based therapy, the method comprising: exposing the differentiated progeny cells and the residual pluripotent stem cells to an alternating electric field for a period of time, the alternating electric field having a frequency and a field strength, wherein the frequency is between 50 kHz and 500 kHz and the field strength is at least 1 V / cm, wherein the frequency and field strength of the alternating electric field are such that the pluripotent stem cells die sequentially as a result of exposure to the alternating electric field for the period of time, thereby producing a purified batch of differentiated progeny cells that are safe for subsequent use in the stem cell-based therapy, wherein the frequency and field strength of the alternating electric field are such that the differentiated cells exposed to the alternating electric field for the period of time remain substantially undamaged, and The method described therein is not a therapeutic method.

2. The method of claim 1, wherein the purified batch contains less than 100,000 pluripotent stem cells.

3. The method of claim 1, wherein the purified batch contains less than 10,000 pluripotent stem cells.

4. The method of claim 1, wherein the time period is at least 12 hours.

5. The method of claim 1, wherein the time period is at least 2 days.

6. The method of claim 1, wherein the alternating electric field has an orientation that switches from time to time between at least two different directions during the time period.

7. The method of claim 1, wherein the frequency is between 250 kHz and 350 kHz and the field strength is at least 1 V / cm.

8. The method of claim 1, wherein the frequency is between 50 kHz and 500 kHz, the field strength is at least 1 V / cm, and the time period is at least 12 hours.

9. The method of claim 1, wherein the frequency is between 250 kHz and 350 kHz, the field strength is at least 1 V / cm, and the time period is at least 3 days.

10. The method of claim 1, wherein prior to said exposing, said population of differentiated progeny cells is obtained by expanding pluripotent stem cells and differentiating said expanded pluripotent stem cells into said population of differentiated progeny cells.

11. The method of claim 1, wherein the pluripotent stem cells comprise one or more of induced pluripotent stem cells, embryonic stem cells, cancer stem cells, and embryonic germ cells.

12. The method of claim 1, wherein the differentiated progeny cells comprise cardiomyocytes or cardiomyocyte progenitor cells.

13. The method of claim 1, wherein said pluripotent stem cells and said differentiated progeny cells are human cells.

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