Method and probe for determining the possibility of the presence of a teratoma derived from pluripotent stem cells

Abstract

This invention provides a method and probe for effectively determining the possibility of the presence of a teratoma derived from pluripotent stem cells. [Solution] A method for determining the possibility of the presence of a teratoma derived from pluripotent stem cells includes: a first step of preparing one or more images showing the distribution of the labeled amino acid in the host body, which is obtained in advance using a device for detecting the labeled amino acid from a host to which a population of cells differentiated in vitro from pluripotent stem cells has been transplanted, and to which a probe containing a labeled amino acid has been administered after the transplantation, with the amount of the labeled amino acid uptake in the teratoma being greater than the amount uptake in normal parts of the body; a second step of selecting from the one or more images a region of interest showing a part of the host body in which a teratoma derived from undifferentiated cells that may be included in the cell population may exist, and a control region showing a normal part of the host body; a third step of comparing the amount of the labeled amino acid in the region of interest with the amount of the labeled amino acid in the control region based on an index that reflects the amount of the labeled amino acid in the images; and a fourth step of determining that if the amount of the labeled amino acid in the region of interest is greater than the amount of the labeled amino acid in the control region, there is a possibility that the teratoma actually exists in the part of the host body shown in the region of interest.

Description

Technical Field

[0001] The present invention relates to a method and a probe for determining the possibility of the presence of teratomas derived from pluripotent stem cells.

Background Art

[0002] Development of a regenerative medicine technique for transplanting a cell population induced to differentiate in vitro from pluripotent stem cells has been underway. On the other hand, it is also known that when undifferentiated cells remain in the transplanted cell population, teratomas derived from the undifferentiated cells can be formed in the host into which the cell population has been transplanted.

[0003] In this regard, in Non-Patent Document 1, as a result of an animal experiment using rats, it has been suggested that teratomas larger than 8 mm derived from human pluripotent stem cells can be detected by MRI (magnetic resonance imaging). , , ,

[0007] , , , 18 , , , ,

[0004] , ,<0​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Riegler, J., Ebert, A., Qin, X., Shen, Q., Wang, M., Ameen, M., Kodo, K., Ong, S.G., Lee, W.H., Lee, G., et al. (2016). Comparison of magnetic resonance imaging and serum biomarkers for detection of human pluripotent stem cell-derived teratomas. Stem Cell Reports 6, 176-187. 10.1016 / j.stemcr.2015.12.008.

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

[0008] However, the experimental results using MRI described in Non-Patent Document 1 are from experiments with small animals using a magnetic field stronger than that used in human clinical settings. On the other hand, human clinical MRI requires the use of a weaker magnetic field, which may mean that only teratomas that have grown to a considerably large size can be detected by MRI.

[0009] [ 18 Regarding PET using [F]FDG as a probe, detection of teratomas derived from human pluripotent stem cells is not easy because they are basically benign tumors.

[0010] For PET / SPECT using probes that bind to integrins, the manufacturing of these probes is not easy due to the complex molecular structure of the compounds used in their production.

[0011] Regarding methods using genetically modified pluripotent stem cells, there are concerns about the safety of transplanting these genetically modified pluripotent stem cells into humans.

[0012] This invention has been made in view of the above problems, and one of its objectives is to provide a method and probe for effectively determining the possibility of the presence of a teratoma derived from pluripotent stem cells. [Means for solving the problem]

[0013] [1] A method for determining the possibility of the presence of a teratoma derived from pluripotent stem cells according to one embodiment of the present invention for solving the above problems includes: a first step of preparing one or more images showing the distribution of the labeled amino acid in the body of a host to which a population of cells differentiated in vitro from pluripotent stem cells has been transplanted, and to which a probe containing a labeled amino acid has been administered after the transplantation, with the amount of uptake in the teratoma being greater than the amount uptake in a normal part of the body, using a device for detecting the labeled amino acid; a second step of selecting from the one or more images a region of interest showing a part of the body of the host in which a teratoma derived from undifferentiated cells that may be included in the population of cells may exist, and a control region showing a normal part of the body of the host; a third step of comparing the amount of the labeled amino acid in the region of interest with the amount of the labeled amino acid in the control region based on an index that reflects the amount of the labeled amino acid in the images; and a fourth step of determining that there is a possibility that the teratoma actually exists in the part of the body of the host shown in the region of interest if the amount of the labeled amino acid in the region of interest is greater than the amount of the labeled amino acid in the control region. The present invention provides a method for effectively determining the possibility of the presence of a teratoma derived from pluripotent stem cells.

[0014] [2] In the method of [1], the labeled amino acid may be one or more selected from the group consisting of labeled phenylalanine, labeled tyrosine, labeled tryptophan, labeled methionine, and labeled glycine. [3] In the method of [1] or [2], the labeled amino acid may be an amino acid labeled with a radioisotope or a fluorescent dye. [3] In any of the methods of [1] to [3], the labeled amino acid is an amino acid labeled with a radioisotope, the device for detecting the labeled amino acid is a positron emission tomography (PET) device or a single-photon emission tomography (SPECT) device, and the image may be a PET image or a SPECT image.

[0015] [5] A probe for determining the possibility of the presence of a teratoma derived from pluripotent stem cells, according to one embodiment of the present invention for solving the above problems, contains a labeled amino acid that is uptaken in a larger amount in the teratoma compared to the amount uptaken in a normal part of the body. According to the present invention, a probe for effectively determining the possibility of the presence of a teratoma derived from pluripotent stem cells is provided.

[0016] [6] In the probe of [5], the labeled amino acid may be one or more selected from the group consisting of labeled phenylalanine, labeled tyrosine, labeled tryptophan, labeled methionine, and labeled glycine. [7] In the probe of [5] or [6], the labeled amino acid may be an amino acid labeled with a radioisotope or a fluorescent dye. [8] Any of the probes of [5] to [7] is a positron emission tomography (PET) probe or a single-photon emission tomography (SPECT) probe, and the labeled amino acid may be an amino acid labeled with a radioisotope. [Effects of the Invention]

[0017] The present invention provides a method and probe for effectively determining the possibility of the presence of a teratoma derived from pluripotent stem cells. [Brief explanation of the drawing]

[0018] [Figure 1] This diagram illustrates the results of evaluating the gene expression level of amino acid transporters by RNA sequencing in an embodiment of this theory. [Figure 2A] This diagram illustrates an example of the results obtained by imaging mass spectrometry in an embodiment of this theory, showing the amount of amino acid accumulation. [Figure 2B] This diagram illustrates another example of the results obtained by imaging mass spectrometry in the embodiment of this theory, showing the amount of amino acid accumulation. [Figure 3A]This is an explanatory diagram showing an example of the results obtained by performing PET imaging in an embodiment according to this embodiment. [Figure 3B] This is an explanatory diagram showing another example of the results obtained by performing PET imaging in the embodiment of this present invention. [Figure 4] This diagram illustrates the results of ex vivo autoradiography and HE staining performed in the embodiment of this model. [Modes for carrying out the invention]

[0019] One embodiment of the present invention is described below. However, the present invention is not limited to this embodiment.

[0020] As one aspect of this embodiment, in The method for determining the possibility of the presence of teratomas derived from pluripotent stem cells (hereinafter referred to as "this method") includes a first step of preparing one or more images showing the distribution of the labeled amino acid in the host body, which has been previously obtained using a device for detecting the labeled amino acid from a host to which a population of cells differentiated in vitro has been transplanted, and to which a probe containing a labeled amino acid has been administered after the transplantation, with the amount of the labeled amino acid uptake in the teratoma being greater than the amount uptake in normal parts of the body; a second step of selecting from the one or more images a region of interest showing a part of the host body in which a teratoma derived from undifferentiated cells that may be included in the cell population may exist, and a control region showing a normal part of the host body; a third step of comparing the amount of the labeled amino acid in the region of interest with the amount of the labeled amino acid in the control region based on an index that reflects the amount of the labeled amino acid in the image; and a fourth step of determining that if the amount of the labeled amino acid in the region of interest is greater than the amount of the labeled amino acid in the control region, there is a possibility that the teratoma actually exists in the part of the host body shown in the region of interest.

[0021] In other words, as described above, cell populations differentiated in vitro from pluripotent stem cells may contain undifferentiated cells that can form teratomas in the host body after transplantation.

[0022] However, conventionally, there has been no non-invasive technical means to safely and early detect teratomas originating from undifferentiated cells that may be present in a host to which a cell population differentiated in vitro from pluripotent stem cells has been transplanted.

[0023] In this regard, the inventors of the present invention have diligently studied technical means for determining the possibility of the presence of teratomas originating from undifferentiated cells in a host to which a cell population differentiated in vitro from pluripotent stem cells has been transplanted.

[0024] As a result, the inventors of the present invention discovered that there are amino acids that are taken up in greater amounts in teratomas compared to normal parts of the body. Furthermore, they confirmed that the possibility of the presence of teratomas in the host body can be safely, early, and non-invasively determined by using a probe containing a labeled amino acid, thus completing the present invention.

[0025] Therefore, this embodiment also includes, in other aspects, a probe (hereinafter sometimes referred to as "this probe") for determining the possibility of the presence of a teratoma derived from pluripotent stem cells, which contains a labeled amino acid that is uptaken in greater amounts in teratomas compared to normal parts of the body.

[0026] More specifically, this probe is used to determine the possibility of the presence of teratomas originating from undifferentiated cells (i.e., undifferentiated cells derived from the pluripotent stem cells) in a host to which a cell population differentiated in vitro from pluripotent stem cells has been transplanted. This probe is preferably used in this method as a probe containing labeled amino acids.

[0027] The cell population transplanted into the host includes differentiated cells induced in vitro from pluripotent stem cells. That is, this cell population is transplanted into the host body to compensate for structural and / or functional defects in a part of the host's body (organ or tissue) using the differentiated cells contained within the population.

[0028] The differentiated cells for transplantation included in the cell population are not particularly limited as long as they can compensate for structural and / or functional defects in a part of the host body, but may include, for example, cardiomyocytes (e.g., one or more cardiomyocytes selected from the group consisting of ventricular myocytes, atrial myocytes, and pacemaker cells), retinal cells, corneal cells, nerve cells, skin cells (e.g., epidermal cells and / or dermal cells), hepatocytes (e.g., hepatocytes), pancreatic cells (e.g., pancreatic β-cells), chondrocytes, lung cells (e.g., alveolar epithelial cells), or renal cells (e.g., tubular cells).

[0029] The ratio of differentiated cells in a cell population to the total number of cells in the transplanted cell population is not particularly limited as long as the effects of the present invention are obtained, but is preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more.

[0030] Differentiated cells included in the transplanted cell population may form cell aggregates (e.g., spheroids, cell sheets, or other organoids). In other words, transplantation of a cell population into a host may be the transplantation of cell aggregates of differentiated cells included in that cell population.

[0031] Cell aggregates are formed by culturing differentiated cells induced in vitro from pluripotent stem cells under conditions suitable for the formation of such cell aggregates. The method for forming the cell aggregates is not particularly limited as long as the effects of the present invention are obtained, but known methods are preferably used, for example.

[0032] The pluripotent stem cells used in the production of the cell population are not particularly limited as long as they differentiate into differentiated cells to be transplanted into a host through differentiation induction in vitro, but are preferably induced pluripotent stem (iPS) cells or embryonic stem (ES) cells. The method for inducing differentiated cells from pluripotent stem cells in vitro is not particularly limited as long as the effects of the present invention are obtained, but are preferably known methods.

[0033] The cell population is transplanted into a site in the host body where the differentiated cells contained within that cell population can play a role in compensating for structural and / or functional defects. In other words, the cell population is transplanted into a site in the host body where the differentiated cells contained within that cell population naturally reside.

[0034] Specifically, for example, a group of cells containing cardiomyocytes is transplanted into the heart, a group of cells containing retinal cells into the retina, a group of cells containing corneal cells into the cornea, a group of cells containing nerve cells into nerve tissue (e.g., the spinal cord), a group of cells containing skin cells into the skin, a group of cells containing hepatocytes into the liver, a group of cells containing pancreatic cells into the pancreas, a group of cells containing chondrocytes into the cartilage, lung cells into the lungs, or kidney cells into the kidneys.

[0035] Furthermore, the cell population may be ectopically transplanted to a site in the host body other than the site where the differentiated cells included in the cell population originally reside. Specifically, for example, pancreatic β-cells are expected to engraft and function even when transplanted to a site other than the pancreas in the host body. Therefore, a cell population containing pancreatic β-cells differentiated in vitro from pluripotent stem cells may be transplanted to a site other than the pancreas, such as subcutaneous tissue or muscle tissue (e.g., skeletal muscle tissue).

[0036] As described above, the cell population may include undifferentiated cells derived from pluripotent stem cells used to create the cell population. In other words, the transplanted cell population may include differentiated cells induced in vitro from pluripotent stem cells, and may also include undifferentiated cells derived from said pluripotent stem cells.

[0037] Specifically, the transplanted cell population may include, as undifferentiated cells, for example, pluripotent stem cells used to create the cell population, and / or undifferentiated cells derived from the pluripotent stem cells that have not yet fully differentiated into differentiated cells included in the cell population.

[0038] Furthermore, undifferentiated cells within a cell population may form teratomas in the host's body when the cell population is transplanted into the host. Teratomas formed in the host's body are tumors that originate from undifferentiated cells that were part of the cell population transplanted into the host, and contain tissues derived from one or more selected from the group consisting of endoderm, mesoderm, and ectoderm.

[0039] More specifically, a teratoma derived from undifferentiated cells that were part of a cell population transplanted into a host includes, for example, one or more differentiated cells derived from one or more germ layers that are different from the differentiated cells intended for transplantation that were part of the cell population.

[0040] The cells constituting the cell population transplanted into the host, that is, the differentiated and undifferentiated cells included in the cell population, may be cells from animals other than humans, but it is preferable that they be human cells. In other words, it is preferable that the cell population transplanted into the host be a cell population (human cell population) differentiated in vitro from human pluripotent stem cells.

[0041] Furthermore, the host to which the cell population is transplanted may be an animal other than a human, but it is preferable that it be human (for example, a human patient). If the host is human, it is preferable that the cells constituting the cell population transplanted into the host are human cells. In other words, it is preferable that the host is a human patient to whom a cell population differentiated in vitro from human pluripotent stem cells has been transplanted.

[0042] Furthermore, animals other than humans are preferably mammals, and more specifically, they are preferably primates (e.g., monkeys), rodents (e.g., mice, rats, hamsters, guinea pigs, or rabbits), carnivores (e.g., dogs, cats), or ungulates (e.g., pigs, cows, horses, goats, or sheep).

[0043] The method for transplanting a cell population into a host is not particularly limited as long as the effects of the present invention are obtained. For example, if the cell population includes dispersed cells or dispersible cell aggregates (e.g., spheroids), a preferred method is to inject a composition (e.g., suspension) containing the dispersed cells or cell aggregates into the organs or tissues of the host. Alternatively, if the cell population includes cell aggregates having a predetermined shape, such as cell sheets, a preferred method is to attach the cell aggregates to the organs or tissues of the host.

[0044] The probe used in this method (this probe) contains a labeled amino acid that is uptaken in greater amounts in teratomas compared to normal parts of the body. Amino acids that are uptaken in greater amounts in teratomas compared to normal parts of the body can be identified experimentally.

[0045] In other words, in a host (human or non-human animal) to which a cell population differentiated in vitro from pluripotent stem cells has been transplanted, and a teratoma has formed derived from the undifferentiated cells contained in that cell population, it is possible to identify amino acids that are uptaken in greater amounts in the teratoma than in the normal parts of the host's body by comparing the uptake levels of multiple types of amino acids in the normal parts of the host's body with the uptake levels of the same multiple types of amino acids in the teratoma. Then, by labeling the amino acids thus identified, labeled amino acids can be obtained as probe components.

[0046] The normal body parts used for comparison with the teratoma in terms of the uptake of labeled amino acids are not particularly limited as long as the uptake of the labeled amino acids is smaller than that in the teratoma. For example, these may be normal parts of the organ or tissue to which the cell population is transplanted, or normal parts of the body where the uptake of the labeled amino acids is smaller than in other parts of the body (for example, subcutaneous tissue, muscle tissue (for example, skeletal muscle tissue), or normal parts of the lungs).

[0047] Specifically, the amount of labeled amino acid taken up in teratomas may be more than 1.0 times the amount taken up in normal parts of the body, preferably 1.1 times or more, more preferably 1.2 times or more, and particularly preferably 1.3 times or more.

[0048] The labeled amino acid, which is uptaken in greater amounts in teratomas compared to normal parts of the body, is preferably one or more selected from the group consisting of labeled phenylalanine, labeled tyrosine, labeled tryptophan, labeled methionine, and labeled glycine, more preferably one or more selected from the group consisting of labeled phenylalanine, labeled tyrosine, labeled tryptophan, and labeled glycine, and particularly preferably labeled phenylalanine.

[0049] Specifically, labeled phenylalanine may be, for example, a phenylalanine molecule without a functional group, or a phenylalanine molecule with a functional group. The phenylalanine molecule with a functional group may be, for example, a phenylalanine molecule with a vonono group (i.e., a vonophenylalanine molecule), or a phenylalanine molecule with an alkyl group (i.e., an alkylphenylalanine molecule). The phenylalanine molecule with an alkyl group may be, for example, a phenylalanine molecule with a methyl group (i.e., a methylphenylalanine molecule), a phenylalanine molecule with an ethyl group (i.e., an ethylphenylalanine molecule), a phenylalanine molecule with a propyl group (i.e., a propylphenylalanine molecule), or a phenylalanine molecule with a butyl group (i.e., a butylphenylalanine molecule).

[0050] Preferably, the labeled amino acid used is one that has high selectivity for amino acid transporters (e.g., LAT1, ASCT2, xCT, or TAT1) whose expression is upregulated in teratomas compared to normal parts of the body.

[0051] Specifically, for example, labeled phenylalanine, such as labeled boronophenylalanine, labeled methylphenylalanine, and labeled ethylphenylalanine, are labeled amino acids that exhibit high selectivity for the amino acid transporter LAT1, which was confirmed to be upregulated in teratomas in the examples described later.

[0052] The labeling of the labeled amino acid is not particularly limited as long as it enables non-invasive detection of the labeled amino acid administered to the host, but it is preferable that the labeled amino acid is labeled with, for example, a radioactive isotope or a fluorescent dye, and it is particularly preferable that it is labeled with a radioactive isotope.

[0053] As the radioisotope element, for example, a positron-emitting nuclide used for a positron emission tomography (PET) probe and / or a single photon-emitting nuclide used for a single photon emission computed tomography (SPECT) probe are preferably used.

[0054] The positron-emitting nuclide used for labeling an amino acid is, for example, fluorine-18 ( 18 F), carbon-11 ( 11 C), nitrogen-13 ( 13 N), oxygen-15 ( 15 O), gallium-68 ( 68 Ga), rubidium-82 ( 82 Rb), copper-62 ( 62 Cu), or iodine-124 ( 124 I), and preferably 18 F, 11 C, 13 N or 15 O, more preferably 18 F.

[0055] Specifically, 18 phenylalanine labeled with 18 F may be, for example, labeled phenylalanine in which a hydrogen atom (for example, a hydrogen molecule of an aromatic ring) of a phenylalanine molecule is substituted with

[0056] In this case, 18 F-labeled phenylalanine may be, for example, labeled phenylalanine in which a hydrogen atom (for example, a hydrogen molecule of an aromatic ring) of a phenylalanine molecule without an added functional group is substituted with 18 F (for example, 18 F] fluorophenylalanine), or labeled phenylalanine in which a hydrogen atom (for example, a hydrogen molecule of an aromatic ring) of a phenylalanine molecule with an added functional group is substituted with 18 F. The labeled phenylalanine in which a hydrogen atom of a phenylalanine molecule with an added functional group is substituted with 18 F is, for example, a labeled phenylalanine in which a hydrogen atom (for example, a hydrogen molecule of an aromatic ring) of a phenylalanine molecule with a borono group added (that is, a boronophenylalanine molecule) is18 Labeled phenylalanine substituted with F (e.g., [ 18 A hydrogen atom (e.g., a hydrogen atom of an aromatic ring) of a phenylalanine molecule with an alkyl group attached (i.e., an alkylphenylalanine molecule) 18 Labeled phenylalanine substituted with F (e.g., [ 18 [F]alkylfluorophenylalanine) may also be used. 18 [F]alkylfluorophenylalanine is, for example, a phenylalanine molecule to which a methyl group has been added (i.e., a methylphenylalanine molecule) has hydrogen atoms (e.g., hydrogen atoms of the aromatic ring) 18 Labeled phenylalanine substituted with F (e.g., [ 18 [F]methylfluorophenylalanine), the hydrogen atoms (for example, the hydrogen atoms of the aromatic ring) of a phenylalanine molecule to which an ethyl group has been added (i.e., an ethylphenylalanine molecule) 18 Labeled phenylalanine substituted with F (e.g., [ 18 [F]ethylfluorophenylalanine), the hydrogen atoms (for example, the hydrogen atoms of the aromatic ring) of a phenylalanine molecule to which a propyl group has been added (i.e., a propylphenylalanine molecule) 18 Labeled phenylalanine substituted with F (e.g., [ 18 The hydrogen atoms (e.g., hydrogen atoms of the aromatic ring) of a phenylalanine molecule with a butyl group attached (i.e., a butylphenylalanine molecule) 18 Labeled phenylalanine substituted with F (e.g., [ 18 It may also be [F]butylfluorophenylalanine).

[0057] Also, 11 In phenylalanine labeled with C, for example, the carbon atom bonded to the aromatic ring of the phenylalanine molecule (the C of the CH2 group bonded to the aromatic ring) 11 It may also be labeled phenylalanine substituted with C.

[0058] Single-photon emitting nuclides used for labeling amino acids include, for example, iodine-123.123 I) Iodine-131 ( 131 I) Technetium-99m( 99m Tc), Indium-111( 111 In), Thallium-201 ( 201 Tl), or Gallium-67 ( 67 It is preferable that it be Ga. 123 I or 99m It is especially preferable that it be Tc.

[0059] The method for labeling amino acids with radioactive isotopes is not particularly limited as long as the effects of the present invention are obtained, but known methods used in the manufacture of PET probes or SPECT probes are preferably used.

[0060] As fluorescent dyes used for labeling amino acids, for example, fluorescent dyes used in in vivo imaging probes are preferably used. As fluorescent dyes used in in vivo imaging probes, for example, near-infrared fluorescent dyes or short-wave infrared fluorescent dyes are preferably used. That is, the phenylalanine labeled with a fluorescent dye is preferably phenylalanine labeled with a near-infrared fluorescent dye or a short-wave infrared fluorescent dye.

[0061] The method for labeling amino acids with a fluorescent dye is not particularly limited as long as the effects of the present invention are obtained, but known methods used for the manufacture of in vivo imaging probes are preferably used.

[0062] Because this probe contains labeled amino acids, which are synthesized by labeling amino acids with small molecular weights and simple molecular structures, as its probe component, it is easier to manufacture and safer than probes that contain labeled compounds with larger molecular weights or more complex molecular structures.

[0063] This probe is a composition administered to a host. Specifically, this probe comprises a labeled amino acid and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for administering the labeled amino acid to the host, but it is preferably a water-soluble carrier that dissolves the labeled amino acid (e.g., physiological saline).

[0064] If the probe contains an amino acid labeled with a radioactive isotope, it is preferable that the probe be used for PET or SPECT. For example, a probe containing an amino acid labeled with a positron-emitting nuclide is preferably used as a PET probe. Furthermore, a probe containing an amino acid labeled with a single-photon-emitting nuclide is preferably used as a SPECT probe.

[0065] Furthermore, if the probe contains an amino acid labeled with a fluorescent dye as the labeled amino acid, it is preferable that the probe be used for in vivo imaging. That is, for example, a probe containing an amino acid labeled with a near-infrared fluorescent dye or a short-wave infrared fluorescent dye is preferably used as an in vivo imaging probe.

[0066] The method of administering this probe to the host is not particularly limited as long as the effects of the present invention are obtained, but intravascular administration is preferred, and intravenous administration is particularly preferred.

[0067] The one or more images (hereinafter referred to as "distribution images") showing the distribution of labeled amino acids in the host body, which are prepared in the first step of this method, are obtained from the host to which the probe has been administered, using a device that detects the labeled amino acids contained in the probe.

[0068] The apparatus for detecting labeled amino acids is not particularly limited as long as it is capable of detecting such labeled amino acids. However, for example, when detecting amino acids labeled with radioactive isotopes, a PET or SPECT apparatus is preferably used.

[0069] In other words, when detecting amino acids labeled with positron-emitting nuclides, a PET scanner is preferably used. Furthermore, when detecting amino acids labeled with single-photon-emitting nuclides, a SPECT scanner is preferably used.

[0070] Furthermore, a PET / CT system or SPECT / CT system equipped with X-ray computed tomography (CT) functionality is particularly preferred as the PET or SPECT system. Alternatively, a PET / MRI system or SPECT / MRI system equipped with magnetic resonance imaging (MRI) functionality may also be used as the PET or SPECT system.

[0071] Furthermore, when detecting amino acids labeled with fluorescent dyes, an in vivo imaging device is preferably used. That is, when detecting amino acids labeled with near-infrared fluorescent dyes or short-wave infrared fluorescent dyes, an in vivo imaging device is preferably used.

[0072] The time from transplanting a cell population into a host to acquiring a distribution image is not particularly limited as long as the effects of the present invention are obtained. However, the distribution image may be acquired, for example, at a time when 7 days or more, preferably 14 days or more, more preferably 21 days or more, even more preferably 28 days or more, even more preferably 35 days or more, even more preferably 42 days or more, even more preferably 49 days or more, even more preferably 56 days or more, and particularly preferably 63 days or more have elapsed since the transplantation.

[0073] The timing for starting the acquisition of distribution images after the administration of the probe is not particularly limited as long as the effects of the present invention are obtained. For example, the acquisition of distribution images may be started after the start of probe administration to the host, or it may be started after the completion of probe administration to the host.

[0074] In other words, for example, the probe may first be administered to the host (e.g., bolus administration), and then, after the completion of said administration, the acquisition of a distribution image showing the distribution of labeled amino acids derived from the probe in the host's body may begin. The time required for bolus administration of the probe to the host is usually a few seconds.

[0075] The distribution image prepared in the first step of this method is not particularly limited as long as it is a distribution image obtained from a host to which the probe was administered. For example, it may include a distribution image obtained at a time of 30 seconds or more, preferably 30 minutes or more, more preferably 40 minutes or more, and especially preferably 60 minutes or more, from the time when the administration of the probe was started or finished.

[0076] The preparation of the distribution image in the first step of this method is the preparation of the distribution image to be used in the downstream steps described later. That is, in the first step, for example, the distribution image acquired in advance by a device for detecting labeled amino acids is prepared by storing it in a storage device connected to a computer used for determination by this method, and / or displaying it on a display device (for example, a liquid crystal display) connected to the computer.

[0077] In the first step, one distribution image may be prepared, or multiple distribution images may be prepared, to be used in the downstream steps described later. The distribution image may be, for example, a PET image or SPECT image taken using a PET or SPECT device, showing the distribution of amino acids labeled with radioisotopes in the host body, or an in vivo imaging image taken using an in vivo imaging device, showing the distribution of amino acids labeled with fluorescent dyes in the host body.

[0078] The distribution image prepared in the first step displays the distribution of labeled amino acids in the host body in a manner corresponding to the amount of each labeled amino acid present. In other words, the distribution image is preferably an image that displays the distribution of labeled amino acids using differences in color and / or color intensity corresponding to the amount of each labeled amino acid present (for example, a color heatmap image or a monochrome heatmap image such as grayscale).

[0079] In the second step of this method, a region of interest showing a part of the host body in which a teratoma may be present, and a control region showing a normal part of the host body are selected from one or more distribution images prepared in the first step.

[0080] The region of interest selected in a distribution image is an image region that constitutes part or all of the distribution image and shows the part of the host body where a teratoma may exist. In other words, the region of interest is, for example, the region that shows part or all of the site where the cell population of the host body has been transplanted.

[0081] Furthermore, undifferentiated cells included in the transplanted cell population may, for example, travel through the bloodstream to a different site in the host body from the site of transplantation, potentially forming an ectopic teratoma. For this reason, the region of interest may be, for example, the area in the host body where an ectopic teratoma may form.

[0082] Ectopic teratomas can form in any organ or tissue of the host body, but specifically, one or more sites in the host body where teratomas can form ectopically include, for example, subcutaneous tissue, muscle tissue (e.g., skeletal muscle tissue), and the lungs.

[0083] Furthermore, in areas where the uptake of labeled amino acids is lower compared to other areas, if a teratoma forms there, the uptake of the labeled amino acids in the surrounding normal areas is low, making it easier to detect the teratoma, which has a relatively high uptake of the labeled amino acids. For this reason, regardless of the site to which the cell population is transplanted, it may be advisable to select a region of interest in the host body where the uptake of labeled amino acids is relatively low.

[0084] Specifically, sites with relatively low uptake of labeled amino acids include, for example, one or more selected from the group consisting of subcutaneous tissue, muscle tissue (e.g., skeletal muscle tissue), and the lungs.

[0085] Furthermore, when selecting a region of interest in a distribution image, which is a PET or SPECT image showing the distribution of labeled amino acids in the host body, the region in the PET or SPECT image corresponding to the region in the CT image acquired from the host using a CT device (for example, a CT image acquired by the PET / CT or SPECT / CT device used to acquire the PET or SPECT image) that shows a mass that may be a teratoma may be selected as the region of interest. In addition, MRI images may be used in combination with PET or SPECT images, either in place of or in addition to CT images.

[0086] The control region selected in the distribution image is a region that constitutes part or all of the distribution image and contains a normal part of the host body, which should be compared with the region of interest.

[0087] The control region is, for example, a region in the vicinity of a normal area of ​​the host body that is visible in the region of interest (for example, a region in the vicinity of the region of interest that does not include the region of interest itself (for example, part or all of the region surrounding the region of interest)). In other words, for example, a region in which a normal area of ​​the same organ or tissue as the organ or tissue visible in the region of interest of the host body is selected as the control region.

[0088] Specifically, for example, if a region of interest is selected that shows the site where a population of host body cells has been transplanted, a region showing the normal area surrounding the transplanted site may be selected as a control region.

[0089] Furthermore, the control region may be selected as a control region if it is a normal region showing a smaller uptake of labeled amino acids compared to other regions, regardless of whether it is the site where the cell population was transplanted and / or the selected region of interest.

[0090] Specifically, normal sites in which the uptake of labeled amino acids is relatively small include, for example, one or more normal sites selected from the group consisting of subcutaneous tissue, muscle tissue (e.g., skeletal muscle tissue), and the lungs.

[0091] The selection of the region of interest and / or the control region in the second step may be performed by a computer using a trained model that, upon inputting a distribution image, outputs results identifying the regions in which teratomas may exist and / or regions in which normal areas are depicted.

[0092] Such pre-trained models are generated, for example, by machine learning using distribution images as training data. The computer processor used in this method then inputs the distribution image to be judged into the pre-trained model read from storage, for example, and selects the region of interest and / or the control region in the distribution image based on the specific result output from the pre-trained model.

[0093] The region of interest and the control region may be different regions in a single distribution image, or, among multiple distribution images, the region of interest may be selected in a first distribution image, and the control region may be selected in a second distribution image different from the first distribution image.

[0094] In other words, for example, if a single distribution image shows both a region where a teratoma may exist and a normal region suitable for comparison, the region showing the teratoma may be selected as the region of interest, and the region showing the normal region may be selected as the control region.

[0095] Furthermore, for example, if, among multiple distribution images acquired sequentially after a single probe administration (for example, multiple PET or SPECT images acquired sequentially by a PET or SPECT device after a single bolus administration of the probe), the first image shows a region in which a teratoma may exist, and a second image, different from the first image, shows a normal region suitable for comparison, then the region in the first image showing the teratoma may be selected as the region of interest, and the region in the second image showing the normal region may be selected as the control region.

[0096] Furthermore, in the second step, it is preferable to select a region of interest and / or a control region from one or more distribution images acquired at a time of 30 seconds or more, preferably 30 minutes or more, more preferably 40 minutes or more, and particularly preferably 60 minutes or more, from the time when the administration of the probe to the host is started, preferably from the time when the administration of the probe is finished.

[0097] In the third step of this method, the amount of the labeled amino acid in the region of interest is compared with the amount of the labeled amino acid in the control region, based on an index that reflects the amount of the labeled amino acid in the distribution image.

[0098] The index used for comparison in the third step, which reflects the amount of labeled amino acid present, is not particularly limited as long as the effects of the present invention are obtained. However, it is preferable that it be one or more selected from the group consisting of the color displayed according to the amount of labeled amino acid present (e.g., the type of color displayed in each pixel constituting a color heat map), the intensity of the color (e.g., the intensity of the color displayed in each pixel constituting a monochrome color map such as grayscale), and the corresponding numerical values ​​(e.g., numerical values ​​that reflect the amount of labeled amino acid present, associated with each pixel constituting the distribution image (e.g., numerical values ​​that indicate the intensity of the detected signal)).

[0099] In other words, for example, if the distribution of a labeled amino acid in a distribution image is displayed in a color corresponding to the amount of that labeled amino acid present, the amount of that labeled amino acid in the area shown in the region of interest (the area where a teratoma may exist) is compared with the amount of that labeled amino acid in the area shown in the control area (a normal area) based on the difference between the color shown in the region of interest and the color shown in the control area.

[0100] Furthermore, for example, in a distribution image, if the distribution of labeled amino acids is displayed with a color intensity corresponding to the amount of the labeled amino acid present, the amount of the labeled amino acid in the region of interest is compared with the amount of the labeled amino acid in the control region based on the difference between the color intensity displayed in the region of interest and the color intensity displayed in the control region.

[0101] Furthermore, for example, if each pixel constituting the distribution image is associated with a numerical value corresponding to the amount of labeled amino acid present (for example, if numerical data reflecting the amount of the labeled amino acid present corresponding to each pixel constituting the distribution image is stored in a computer), then the amount of the labeled amino acid present in the region of interest is compared with the amount of the labeled amino acid present in the region of the control, based on the difference between the representative value (e.g., mean or maximum) of the numerical value associated with the pixels constituting the region of interest and the representative value (e.g., mean or maximum) of the numerical value associated with the pixels constituting the control region.

[0102] In the fourth step of this method, based on the comparison results from the third step, if the amount of labeled amino acid in the region of interest is greater than the amount of the same labeled amino acid in the control region, it is determined that there is a possibility that a teratoma is actually present in the part of the host body reflected in the region of interest.

[0103] In other words, for example, if the distribution of a labeled amino acid in a distribution image is displayed in a color corresponding to the amount of that labeled amino acid present, and the amount of that labeled amino acid corresponding to the color displayed in the region of interest is greater than the amount of that labeled amino acid corresponding to the color displayed in the control region, then it is determined that there is a possibility that a teratoma actually exists in the area shown in the region of interest.

[0104] Furthermore, for example, in a distribution image, if the distribution of labeled amino acids is displayed with a color intensity corresponding to the amount of those labeled amino acids present, and the amount of those labeled amino acids corresponding to the color intensity displayed in the region of interest is greater than the amount of those labeled amino acids corresponding to the color intensity displayed in the control region, then it is determined that there is a possibility that a teratoma actually exists in the area shown in the region of interest.

[0105] Furthermore, for example, if each pixel constituting the distribution image is associated with a numerical value corresponding to the amount of labeled amino acid present, then if the representative value (e.g., mean or maximum) of the numerical value associated with the pixels constituting the region of interest is greater than the representative value (e.g., mean or maximum) of the numerical value associated with the pixels constituting the control region, it is determined that there is a possibility that a teratoma actually exists in the area shown in the region of interest.

[0106] Furthermore, if each pixel constituting the distribution image is associated with a numerical value corresponding to the amount of labeled amino acid present, it may be determined that there is a possibility that a teratoma actually exists in the area shown in the region of interest if the representative value (e.g., mean or maximum value) of the numerical value associated with the pixels constituting the region of interest is greater than 1.0 times, preferably 1.1 times or more, more preferably 1.2 times or more, and particularly preferably 1.3 times or more, than the representative value (e.g., mean or maximum value) of the numerical value associated with the pixels constituting the control region. In this case, the greater the representative value of the numerical value associated with the pixels constituting the region of interest is compared to the representative value of the numerical value associated with the pixels constituting the control region, the higher the probability that a teratoma actually exists in the area shown in the region of interest.

[0107] Furthermore, if the amount of labeled amino acid in the region of interest is not greater than the amount of the same labeled amino acid in the control region, it may be determined, for example, that there is no possibility, or a low probability, of a teratoma actually existing in the area shown in the region of interest.

[0108] Thus, according to the present invention, by comparing an objective indicator reflecting the abundance of labeled amino acids in a distribution image obtained from a host potentially containing teratomas derived from pluripotent stem cells between a region of interest and a control region, the possibility of the presence of such teratomas in the host's body can be safely, early, and non-invasively determined.

[0109] Furthermore, if the possibility of a teratoma being present in the host's body is confirmed, treatment for the teratoma can be carried out at an early stage. In this case, for example, surgical removal of the teratoma and / or chemotherapy (e.g., anticancer drug treatment) can be effectively implemented.

[0110] Next, a specific example of this embodiment will be described. [Examples]

[0111] [Maintenance and proliferation of human iPS cells] The 253G4 human iPS cell line was provided by the Center for iPS Cell Research and Application, Kyoto University. The cells were cultured at 37°C in a humidified 5% CO2 incubator and regularly tested for mycoplasma infection. The cells were maintained in AS103C (Ajinomoto Co., Inc., Tokyo, Japan), a modified StemFit® medium.

[0112] Cells were passaged every 4 to 7 days. During passage, cells were washed with Dulbecco phosphate-buffered saline (D-PBS) (Fujifilm Wako Pure Chemical Industries, Osaka, Japan, 045-29795) and incubated at 37°C for 3 minutes using TrypLE Select® (Thermo Fisher Scientific, Waltham, MA, USA, 12563-011).

[0113] Subsequently, the cells were collected in AS103C containing 10 μM Y-27632 (Fujifilm Wako Pure Chemical Industries, Osaka, Japan, 034-24024) and centrifuged at 300 rcf (relative centrifugal force) for 3 minutes. Next, the cells were resuspended and the cell count was performed using Vi-CELL® (Beckman Coulter, Brea, CA, USA). The cells were then seeded onto culture plates coated with Matrigel® (CORNING, Corning, NY, USA, 354230).

[0114] [Induction of differentiation of human iPS cells into cardiomyocytes] On day 0, human iPS cells were washed with D-PBS and cultured for 1 day in RPMI-1640 medium (FUJIFILM Wako Pure Chemical, Osaka, Japan, 189-02025) supplemented with 2% B27 supplement (without insulin) (Thermo Fisher Scientific, Waltham, MA, USA, A1895601), 6 μM CHIR99021 (FUJIFILM Wako Pure Chemical, Osaka, Japan, 034-23103), and 1 ng / mL BMP4 (R&D Systems, Minneapolis, MN, USA, 314-BP).

[0115] On day 1, cells were washed with D-PBS and cultured in RPMI-1640 medium supplemented with B27 supplement (without insulin). On day 3, cells were washed with D-PBS and cultured in RPMI-1640 medium supplemented with B27 supplement (without insulin) and 5 μM IWR-1 (Sigma-Aldrich, St. Louis, MO, USA, I0161-25MG). On day 6, cells were washed with D-PBS and cultured in RPMI-1640 medium supplemented with B27 supplement (without insulin). On day 7, cells were cultured in a medium prepared by supplementing MEM-α (Thermo Fisher Scientific, Waltham, MA, USA, 12571-048) with 5% fetal bovine serum (FBS) (Biowest, France, S1560-500) and 2 mM sodium pyruvate (Sigma-Aldrich, St. Louis, MO, USA, S8636-100ML).

[0116] On day 10, the cells were washed with D-PBS and then detached from the culture substrate surface by incubation in a 0.25% trypsin / ethylenediaminetetraacetic acid (EDTA) (NACALAI TESQUE, Kyoto, Japan, 3554-64) solution for 5 minutes. The cells were then collected in MEM-α supplemented with 5% FBS and 2 mM sodium pyruvate, and centrifuged at 300 rcf for 3 minutes to precipitate. Next, the cells were resuspended in MEM-α supplemented with 5% FBS, 2 mM sodium pyruvate, and 6 μM orlistat (Sigma-Aldrich, St. Louis, MO, USA, O4139-25MG), and the cell count was calculated using Vi-CELL. The cells were seeded onto dishes coated with collagen type I (AGC TECHNO GLASS, Shizuoka, Japan, 4030-010).

[0117] On day 13, glucose and glutamine were removed and the medium was replaced with AS501 medium (Ajinomoto, Tokyo, Japan) with 4 mM lactate added. From day 14 to day 16, the medium was changed daily with fresh AS501. On day 17, the cells were incubated in D-PBS for 3 minutes, then incubated in 0.25% trypsin / EDTA solution for 5 minutes, and the cells were detached from the bottom of the dish. The cells were collected in MEM-α supplemented with 5% FBS and 2 mM sodium pyruvate, and centrifuged at 300 rcf for 3 minutes to precipitate the cells. The cells were dispensed into microcentrifuge tubes for cryopreservation using CryoStor® CS10 (STEMCELL Technologies, Vancouver, Canada, ST-07931) and cryopreserved.

[0118] Thus, human iPS cell-derived cardiomyocytes were obtained by differentiating human iPS cells in vitro. The differentiation efficiency and purity of the obtained human iPS cell-derived cardiomyocytes were evaluated by flow cytometry using an anti-troponin T antibody (clone: ​​REA400; Miltenyi Biotec, Auburn, CA, USA, 130-129-225). The results showed that the proportion of these human iPS cell-derived cardiomyocytes to the total number of cells differentiated in vitro from human iPS cells as described above was over 95%.

[0119] [Creating Spheroids] Human iPS cell-derived cardiomyocytes (hiPSC-CMs) obtained as described above were cultured in microwells (CORNING, Corning, NY, USA, No 4440 or Kuraray, Tokyo, Japan, Elplasia 400 560 CL6) for one week to produce hiPSC-CM spheroids (hiPSC-CM spheroids). Each hiPSC-CM spheroid was formed from approximately 1000 hiPSC-CMs and had a diameter of approximately 200 μm. Similarly, human iPS cells were cultured in microwells for one day to produce human iPS cell spheroids (hiPSC-spheroids).

[0120] [Spheroid transplantation] The protocol for this experiment was approved by the Animal Experiment Committee and Ethics Review Committee of Keio University and the National Institutes for Quantum Science and Technology, respectively, based on the Basic Guidelines for the Proper Conduct of Animal Experiments (Ministry of Education, Culture, Sports, Science and Technology of Japan). All animals were properly cared for in accordance with the "Guidelines for the Rearing and Use of Laboratory Animals." In this experiment, adult male NOG mice (InVivoScience Co., Ltd., Tokyo) were used as immunodeficient mice.

[0121] For the transplantation of hiPSC-CM-spheroids or hiPSC-spheroids into the heart, mice were anesthetized with a low dose of isoflurane. The anesthetized mice were connected to a rodent ventilator (Shinano, Tokyo, Japan, SN-480-7). An approach was made from the left fourth intercostal space, and the left chest was incised. 1 × 10 6 pieces~3×10 6 hiPSC-CM-spheroids or hiPSC-spheroids, equivalent to individual cells, were mixed with 60 μL of PBS using a 27G syringe (Terumo, Tokyo, Japan, SS-10M2713) and transplanted into the hearts of mice. Subsequently, the intercostal spaces and skin were sutured with nylon (Natsume, Tokyo, Japan, AR15-40N3).

[0122] For the transplantation of hiPSC-spheroids into subcutaneous tissue, mice were anesthetized with a low dose of isoflurane. 3 × 10 7 pieces~4×10 7 hiPSC-spheroids, equivalent to individual cells, were mixed with either 300 μL of a solution prepared by mixing PBS and Matrigel® (CORNING, Corning, NY, USA, 354230) in a 1:1 ratio, or 300 μL of a solution prepared by mixing PBS and Vitronectin (Thermo Fisher Scientific, Waltham, MA, USA, A14700) in a 1:1 ratio, and then transplanted into the subcutaneous tissue of the back of mice.

[0123] [Immunohistochemistry] Frozen sections fixed with 4% paraformaldehyde, or formalin-fixed, paraffin-embedded sections, were prepared. The tissue sections were immunostained using the following antibodies. The primary antibodies used for immunostaining of human iPS cell-derived cardiomyocyte tissue (hiPSC-cardiac tissue) and human iPS cell-derived teratomas (hiPSC-teratomas) obtained from mice transplanted with hiPSC-CM-spheroids were: cardiac troponin I (Abcam, Cambridge, MA, USA, ab52862), troponin T cardiac isoform Ab-1 (Clone 13-11) (Thermo Fisher Scientific, Waltham, MA, USA, MS-295-P), α-actinin (Abcam, Cambridge, MA, USA, ab137346), myosin light chain 2a (MLC2a) (Synaptic Systems, Germany, SYSY 311-011), myosin light chain 2v (MLC2v) (Abcam, Cambridge, USA, ab 79935), and human cell nucleus (Merck). Antibodies were used against the following proteins found in proliferating cells: Millipore, Burlington, MA, USA, MAB1281; CD31 (Abcam, Cambridge, USA, ab28364); or Ki-67 (Sigma-Aldrich, St. Louis, MO, USA, P6834). Secondary antibodies used were Alexa Fluor® 594 donkey-derived anti-mouse IgG (Invitrogen, Waltham, MA, USA, A21207), Alexa Fluor® 488 donkey-derived anti-mouse IgG (Invitrogen, Waltham, MA, USA, A21202), or Alexa Fluor® 488 donkey-derived anti-goat IgM (Invitrogen, Waltham, MA, USA, A21042). Cell nuclei were stained with Hoechst® 33342 (Thermo Fisher Scientific, Waltham, MA, USA, H3570).All images were acquired using a fluorescence microscope (BIOREVO, BZ-X710, Keyence, Osaka, Japan) and analyzed with ImageJ software.

[0124] [Laser Microdissection] The hearts of mice transplanted with spheroids were embedded in OCT compound (Tissue-Tek, SAKURA, Tokyo, Japan, 4583) or Super Cryoembedding Medium (SCEM) and frozen on dry ice. From the frozen heart samples, 7 μm thick tissue sections were prepared using a cryostat (Leica, Wetzlar, Germany) and mounted on membrane-coated slides. These tissue sections were fixed in 95% ethanol for 2 minutes. Subsequently, they were stained with 0.1% toluidine blue to identify hiPSC-myocardial tissue and hiPSC-teratomas. Furthermore, these hiPSC-myocardial tissue and hiPSC-teratomas were excised using laser microdissection (PALM MB-IV, Zeiss, Oberkochen, Germany) and collected in tubes. 350 μL of Trizole (trademark) (Life Technologies, Carlsbad, CA, USA, 15596026) was added and vortexed for 30 minutes.

[0125] [RNA sequencing] Total RNA from hiPSC-cardiocardial tissue and hiPSC-teratomas, both transplanted from mice with hiPSC-CM spheroids, was extracted using the miRNeasy™ Micro Kit (QIAGEN, Hilden, Germany). Complementary DNA was synthesized using the SMART-Seq™ Stranded Kit (Clontech, Mountain View, CA, USA). Libraries were prepared using NextSeq500™ (Illumina, San Diego, CA, USA). Human (hg38) and mouse (mm10) genome data were integrated in silico. Reads were mapped using STAR 2.7.5c (GitHub, San Francisco, CA, USA). The proportion of reads assigned to hg38 or mm10 was counted. Reads assigned to hg38 were classified as human reads, and alignment files (BAMs) were created. Aligned reads were analyzed using StrandNGS® 3.4 software (Agilent Technologies, Inc., CA, USA). The number of reads assigned to each gene and transcript (Ensembl Database 2016.12.01) was quantified using the Transcripts Per Million (TPM) method. Data analysis was performed using Python 3 [version 3.8] and R [version 4.2.2]. Heatmaps were created using pandas [version 1.2.4], matplotlib [version 3.2.1], and gplots [version 3.1.3]. Principal component analysis was performed using pandas [version 1.2.4], numpy [version 1.18.5], and sklearn [version 0.23.1]. For GSEA analysis, we used org.Hs.eg.db [version 3.16.0], DOSE [version 3.24.2], and ClusterProfiler [version 4.2.2]. In gene ontology analysis, GO terms were extracted from a list of genes whose expression differed by more than twofold.

[0126] [Imaging Mass Spectrometry] A matrix-assisted laser desorption / ionization quadrupole ion trap time-of-flight mass spectrometer (iMScope Trio®; Shimadzu Corp, Kyoto, Japan) was used to acquire the data. Tissue sections with a thickness of 10 μm were prepared from frozen heart samples using a cryostat (CM 3050S; Leica Microsystems, Wetzlar, Germany). The tissue sections were placed on indium tin oxide / MAS-coated slides (#SI0100M, Matsunami Glass, Osaka, Japan) and dried in a plastic tube containing silica gel. Next, 80% ethanol containing 5 mg / mL of 9-aminoacridine (Merck Millipore, Burlington, MA, USA) was sprayed for anion detection, and 50% acetonitrile containing 30 mg / mL of α-cyano-4-hydroxycinnamic acid (Sigma-Aldrich, St. Louis, MO, USA) was sprayed for cation detection. For the visualization of amino acids, tissue derivatization using an amino group derivatization reagent (pN,N,N,-trimethylammonioanilyl N′-hydroxysuccinimidyl carbamate iodide: TAHS) was applied to tissue sections. Mass spectrometry images were acquired using 80 lasers per spot in both cation mode (m / z 100-450, m / z 250-630 for amino acids) and anion mode (m / z 85-400). Mass spectra acquired from each measurement point at 25 μm intervals were reconstructed as a 2D heatmap using IMAGEREVEAL® MS software (Shimadzu Corp, Kyoto, Japan). After imaging mass spectrometry, tissue sections were washed with acetone and stained with HE according to the manufacturer's protocol (Muto Pure Chemicals, Tokyo, Japan). HE-stained slides were scanned using a virtual slide scanner (Nanozoomer-XR, Hamamatsu Photonics, Hamamatsu, Japan).In the cardiac tissue of host mice, normal myocardial tissue, hiPSC-myocardial tissue, and hiPSC-teratomas were identified in each image.

[0127] [PET Imaging] As a radiopharmaceutical for PET, deoxyglucose labeled with fluorine-18 is used. 18 F]Fluorodeoxyglucose([ 18 L-[methyl- FDG), a carbon-11 labeled methionine (L-[methyl- FDG]), is a carbon-11 labeled methionine. 11 C] Methionine ([ 11 L-4-[ phenylalanine labeled with fluorine-18), or C]Met) 18 F] Fluorophenylalanine ([ 18 F[fPhe) was used.

[0128] These probes were provided by the National Institutes for Quantum Science and Technology. For example, [ 18 F]fPhe was synthesized using a method based on known literature (e.g., Reference 1 (J. Am. Chem. Soc. 2021 May 12;143(18):6915-6921. DOI: 10.1021 / jacs.1c00523), Reference 2 (Org. Lett. 2015, 17, 5780-5783. DOI: 10.1021 / acs.orglett.5b02875)). Specifically, a commercially available QMA Light cartridge was first washed with distilled water and ethanol, and then dried by passing nitrogen gas through it. Next, the product was generated by a nuclear reaction. 18 [Fluoride ions were adsorbed by passing them through a dried QMA Light cartridge and recovered with a methanol solution (1.0 mg / mL) of tetraethylammonium bicarbonate (11268-25GF, manufactured by Sigma-Aldrich). The recovered [ 18A solution of [F]TEAF (Tetraethyl ammonium Fluoride) was concentrated under a nitrogen stream, and to the resulting residue, a solution (150 μL) of tert-butyl (S)-2-((tert-butoxycarbonyl)amino)-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propanoate (CAS RN 945259-94-9) (No. 273626, BioDuro) (B(pin) isomer, 2.2 mg) in a mixed solvent of DMAC (Dimethylacetamide) + nBuOH (1-Buthanol) (4 / 1) was added. Immediately after adding the solution, a 150 μL solution of tetrakis(pyridine)copper(II) triflate (Cu(OTf)2py4) (3.4 mg) in a DMAC + nBuOH (4 / 1) mixed solvent was added, and the mixture was reacted at 110°C for 15 minutes after passing dry air through it. After cooling to near room temperature, 0.4 mL of a 50 / 50 mixture of 5 N hydrochloric acid aqueous solution and methanol was added, and then the mixture was heated again and reacted at 110°C for 5 minutes to remove the protecting group. After further cooling to approximately room temperature, an appropriate amount of phosphate buffer was added, and the mixture was separated by HPLC (column: Capcell PAK AQ (10 mm × 250 mm, 5 μm) (Osaka Soda Co., Ltd.), mobile phase: 3% ethanol 20 mM phosphate buffer solution, flow rate: 4.0 mL / min, detection: UV-210 nm & RI (NaI), retention time: approximately 13 minutes). 11 C]Met is, for example, found on pages 87-94 of Reference 3 (Manufacturing and Quality Control of Radiopharmaceuticals for PET - A Guide to Synthesis and Clinical Applications, 6th Edition, edited by PET Chemistry Workshop, 2024) "7-1.[ 11 It is synthesized by the method described in "Methionine Synthesis Method" (C).

[0129] On the day of the experiment, prior to PET imaging, the mice were anesthetized with 5% isoflurane, and a homemade catheter with a needle was inserted into the tail vein. Anesthesia was then continued with 1.5% isoflurane, and the mice were placed in a homemade chamber and placed inside the PET scanner (Inveon, Siemens Medical Solutions, Knoxville, TN, USA). The mice's body temperature was maintained using a circulating water system (T / Pump TP401, Gaymar Industries, Orchard Park, NY, USA) with heated water (38°C~40°C).

[0130] [ 18 F]FDG or [ 11 In PET imaging using C]Met as a probe, the [ 18 F]FDG (dosage: 7-10 MBq / animal) or [ 11 C]Met (dosage: 35-40 MBq / mouse) was administered to mice via the tail vein, and a static radiographic scan was performed for 10 minutes 20 minutes after administration.

[0131] on the other hand,[ 18 In PET imaging using F]fPhe as a probe, the [ 18 F]fPhe (dosage: 13-15 MBq / mouse) was administered to mice by bolus injection via the tail vein. 30 seconds after the completion of the bolus administration, a dynamic radiographic scan was started in 3D list mode and performed for 60 minutes (1 minute x 4 frames, 2 minutes x 8 frames, 5 minutes x 8 frames).

[0132] Furthermore, to determine the anatomical position, after the PET imaging described above, the chamber containing the mouse was transferred to a small animal CT scanner (CosmoScan GX, Rigaku, Tokyo, Japan), and CT imaging was performed while the mouse was anesthetized with 2%-3% isoflurane. The CT images were acquired using an X-ray source set to 70kVp and 114μA, with a 16-second non-contrast scan.

[0133] and,[ 18In experiments where F]fPhe was administered, the [ 18 To obtain time-radioactivity curves for each tissue of F]fPhe, dynamic PET images and mean CT attenuation images were integrated using PMOD software (version 3.4, PMOD Technologies, Zurich, Switzerland), and regions of interest were set for the heart, lower extremity skeletal muscle tissue, and hiPSC-teratoma. The standardized uptake value (SUV) was calculated as follows: SUV = (radioactivity per 1 mL of tissue / administered radioactivity) × body weight (g).

[0134] [Ex vivo autoradiography] After performing the PET and CT imaging described above, the mice were anesthetized with 5% isoflurane, and saline solution was injected into their hearts to perfuse their entire blood system. Subsequently, the hiPSC teratomas formed in the mice's hearts and subcutaneous tissue were excised, embedded in OCT compound, and frozen on dry ice powder.

[0135] Using a microtome (NX70; Thermo Scientific, MA, USA), 20 μm thick tissue sections were prepared from frozen heart tissue or hiPSC-teratomas and placed on glass slides (Matsunami Glass, Tokyo, Japan). The tissue sections were then exposed to an imaging plate (BAS-MS2025; FUJIFILM, Tokyo, Japan), and radioactivity from the heart tissue or hiPSC-teratomas was detected by scanning the imaging plate.

[0136] [Statistical analysis] Statistical analysis was performed using GraphPad Prism 9 software. Data on the graft-to-host ratio in imaging mass spectrometry were obtained by analyzing Dunn's multiple comparisons test (n=5 at each time point). [hiPSC-teratoma and skeletal muscle tissue] 18 The average radioactivity of F]fPhe was obtained by analyzing it using the Wilcoxon Signed Rank Test (n=6).

[0137] [Result 1: Expression levels of amino acid transporter genes] Figure 1 shows a heatmap of the results of evaluating the gene expression levels of amino acid transporters by RNA sequencing in hiPSC-cardiac tissue derived from hiPSC-CM-spheroids transplanted into the hearts of NOG mice (labeled "Transplanted hiPSC-CMs" in the figure) and hiPSC-teratomas derived from hiPSC-spheroids transplanted into the hearts of NOG mice (labeled "Teratoma" in the figure).

[0138] As shown in Figure 1, the gene expression levels of amino acid transporters (LAT1, ASCT2, xCT, and TAT1) in hiPSC-teratomas were significantly higher than those in hiPSC-cardiac tissue.

[0139] Specifically, the LAT1 gene expression level in hiPSC-teratomas was approximately 20 times higher than that in hiPSC-cardiocardial tissue. Furthermore, the ASCT2 gene expression level in hiPSC-teratomas was approximately 33 times higher than that in hiPSC-cardiocardial tissue. These results were calculated as the ratio of the average gene expression levels in hiPSC-teratomas (one individual at 2 weeks post-transplant, one individual at 4 weeks post-transplant, and one individual at 12 weeks post-transplant) to the average gene expression levels in hiPSC-cardiocardial tissue (one individual at 2 weeks post-transplant, two individuals at 4 weeks post-transplant, and three individuals at 12 weeks post-transplant).

[0140] [Result 2: Amount of amino acid accumulation] Figure 2A shows the results of evaluating the signal intensities of glycine ("Gly" in the figure), methionine ("Met" in the figure), tryptophan ("Trp" in the figure), phenylalanine ("Phe" in the figure), and tyrosine ("Tyr" in the figure) in hiPSC teratomas 2 weeks and 4 weeks after transplantation, using imaging mass spectrometry (IMS).

[0141] As shown in Figure 2A, four weeks after transplantation, the amino acid signal intensity of the hiPSC-teratoma (labeled "4wk" in the figure) was statistically significantly greater than that of the amino acid signal intensity of normal myocardial tissue from the host mouse heart (labeled "host" in the figure).

[0142] Specifically, with the amino acid signal intensity of normal myocardial tissue from a mouse heart set to "1," the relative amino acid signal intensities of hiPSC-teratomas were approximately 2.03 for glycine, 2.28 for methionine, 2.06 for tryptophan, 3.10 for phenylalanine, and 2.49 for tyrosine. In other words, the accumulation of these amino acids in hiPSC-teratomas was significantly greater than that in normal myocardial tissue from a mouse.

[0143] On the other hand, Figure 2B shows the results of evaluating the signal intensity of glycine, methionine, tryptophan, phenylalanine, and tyrosine in hiPSC-myocardial tissue at 2 weeks, 4 weeks, and 12 weeks after transplantation, using IMS.

[0144] As shown in Figure 2B, at 4 weeks after transplantation, no statistically significant difference was observed between the amino acid signal intensity of hiPSC-cardiac tissue (labeled "4wk" in the figure) and the amino acid signal intensity of normal cardiac cardiomyocytes from a mouse heart (labeled "host" in the figure).

[0145] In other words, the accumulation of the above amino acids in hiPSC-cardiac tissue was equivalent to that in normal mouse cardiac tissue.

[0146] As shown in Figures 2A and 2B, the accumulation of the above amino acids in hiPSC teratomas was reasonably found to be significantly larger than that in hiPSC myocardial tissue. Therefore, it was considered highly probable that the accumulation of the above amino acids in hiPSC teratomas was greater than that in normal human myocardial tissue.

[0147] [Result 3: PET Imaging] [ 18 PET imaging results using [F]FDG as a probe showed that in hiPSC teratomas formed by transplanting hiPSC spheroids into the heart of a mouse, [ 18 The amount of [F]FDG accumulated was not significantly different from that in normal myocardial tissue of the mice's heart (results not shown).

[0148] [ 11 PET imaging results using [C]Met as a probe revealed that in hiPSC teratomas formed by transplanting hiPSC spheroids into the heart of mice, and in hiPSC teratomas formed by transplanting hiPSC spheroids into the subcutaneous tissue of mice, [ 11 The accumulation of [C]Met was lower than that in normal myocardial tissue of the mice's hearts (results not shown).

[0149] Next, in the evaluation experiment using IMS described above, we focused on phenylalanine, which was the amino acid with the largest accumulation in hiPSC-teratomas, [ 18 PET imaging was performed using [F]fPhe as a probe.

[0150] Figure 3A shows the time elapsed (minutes) (horizontal axis) from the start of a PET scan of mice 9 weeks after transplantation of hiPSC-spheroids into the subcutaneous tissue, as well as the time elapsed (minutes) (horizontal axis) from the start of the PET scan of the normal skeletal muscle tissue ("Skeletal muscle" in the figure) and the hiPSC-teratoma ("Teratoma (intensive region)" in the subcutaneous tissue of the mice, respectively, and the time elapsed (left vertical axis), and the ratio of the radiation intensity of the hiPSC-teratoma to the radiation intensity of the skeletal muscle tissue (T / M ratio) ("Teratoma / muscle ratio" and "T / M ratio" in the figure) (right vertical axis) (n=6).

[0151] Figure 3B also shows the relative radiation intensity (i.e., T / M ratio) of hiPSC-teratomas ("Teratoma" in the figure) (vertical axis "Ratio" in the figure) at approximately 60 minutes after the start of the PET scan, with the radioactivity intensity of normal skeletal muscle tissue ("Skeletal muscle" in the figure) set to "1" (n=6).

[0152] As shown in Figure 3A, the T / M ratio increased over time. Then, as shown in Figures 3A and 3B, the T / M ratio reached approximately 1.35 about 60 minutes after the start of the PET scan.

[0153] Thus, in hiPSC teratomas formed in the subcutaneous tissue, [ 18 The uptake of [F]fPhe was significantly greater than that in normal skeletal muscle tissue. That is, [ 18 PET imaging using [F]fPhe allowed for the specific detection of hiPSC-teratomas.

[0154] [Result 4: ex vivo autoradiography] Figure 4 shows the above [ 18 The images show ex vivo autoradiography (ARG) and HE-stained images (HE) obtained from normal myocardial tissue ("Heart" in the figure) of a mouse heart, and from a hiPSC-teratoma ("Teratoma" in the figure) formed in the subcutaneous tissue of the mouse, respectively, after PET imaging using F]fPhe.

[0155] As shown in the autoradiography image in Figure 4, the radioactivity intensity in hiPSC-teratomas, i.e., [ 18 The uptake of [F]fPhe was greater than that in normal myocardial tissue. Furthermore, as shown in the HE image in Figure 4, various types of tissue were present in the hiPSC-teratoma.

Claims

1. A first step is to prepare one or more images showing the distribution of the labeled amino acid in the host body, which is obtained in advance using a device that detects the labeled amino acid, from a host to which a population of cells differentiated in vitro from pluripotent stem cells has been transplanted, and to which a probe containing a labeled amino acid has been administered after the transplantation, with the amount of the labeled amino acid uptake being greater in the teratoma compared to the amount uptake in normal parts of the body. A second step of selecting from the one or more images above a region of interest showing a part of the host body in which a teratoma derived from undifferentiated cells that may be included in the cell population may exist, and a control region showing a normal part of the host body. A third step involves comparing the amount of the labeled amino acid in the region of interest with the amount of the labeled amino acid in the control region, based on an index that reflects the amount of the labeled amino acid in the aforementioned image. A fourth step in which, if the amount of the labeled amino acid in the region of interest is greater than the amount of the labeled amino acid in the control region, it is determined that there is a possibility that the teratoma is actually present in a part of the host body visible in the region of interest, A method for determining the possibility of the presence of a teratoma derived from pluripotent stem cells, including [specific example].

2. The method according to claim 1, wherein the labeled amino acid is one or more selected from the group consisting of labeled phenylalanine, labeled tyrosine, labeled tryptophan, labeled methionine, and labeled glycine.

3. The method according to claim 1 or 2, wherein the labeled amino acid is an amino acid labeled with a radioactive isotope or a fluorescent dye.

4. The labeled amino acid is an amino acid labeled with a radioactive isotope element. The apparatus for detecting the labeled amino acid is a positron emission tomography (PET) system or a single-photon emission tomography (SPECT) system. The aforementioned image is a PET image or a SPECT image. The method according to claim 1 or 2.

5. A probe for determining the possibility of the presence of teratomas derived from pluripotent stem cells, containing a labeled amino acid that is uptaken in teratomas at a higher rate compared to normal body parts.

6. The probe according to claim 5, wherein the labeled amino acid is one or more selected from the group consisting of labeled phenylalanine, labeled tyrosine, labeled tryptophan, labeled methionine, and labeled glycine.

7. The probe according to claim 5 or 6, wherein the labeled amino acid is an amino acid labeled with a radioactive isotope or a fluorescent dye.

8. A probe for positron emission tomography (PET) or a probe for single-photon emission tomography (SPECT), The labeled amino acid is an amino acid labeled with a radioactive isotope. The probe according to claim 5 or 6.

Images