iPS CELL, iPS CELL BANK, AND METHOD FOR PROVIDING iPS CELL INFORMATION

Abstract

The present invention addresses the problem of providing iPS cells in which HLA-ABC expression is negative.　The problem can be solved by providing iPS cells in which HLA-ABC expression is negative (except for iPS cells in which HLA-ABC expression is made negative by modifying a gene expressing HLA-ABC).

Description

iPS cells, iPS cell bank and iPS cell information providing method

[0001] The disclosure of the present application relates to iPS cells, an iPS cell bank comprising said iPS cells, and a method for providing iPS cell information from said iPS cell bank.

[0002] Examples of clinical trials for regenerative medicine include age-related macular degeneration, Parkinson's disease, severe heart failure / cardiomyopathy, thrombocytopenia, and spinal cord injury, and clinical trials for various other conditions are also under consideration. For iPS cells used for transplantation, cases of using autologous cells (autologous transplantation) are known due to immune rejection after transplantation. However, in the case of autologous transplantation, iPS cells are first prepared using autologous cells, then differentiated into cells for transplantation before being used for treatment, which has the problem of requiring a large amount of time and high cost.

[0003] In order to solve the above problem, cases using allogeneic cells (allogeneic transplantation) are also known (see Patent Document 1). Patent Document 1 describes that an iPS cell bank is prepared by preparing a plurality of iPS cells having a predetermined typing of Human Leukocyte Antigen (HLA), and iPS cells suitable for a patient are selected.

[0004] Japanese Unexamined Patent Application Publication No. 2019-4702

[0005] As described above, it is already known to prepare an iPS cell bank using iPS cells having predetermined typing. Incidentally, among HLAs, HLA-ABC is a transmembrane protein expressed on most nucleated cells and platelets, and is known to be an important immune mechanism involved in the discrimination between self and non-self. That is, strong expression of HLA-ABC increases the risk of rejection during iPS cell transplantation. Therefore, it is preferable that iPS cells used for transplantation have negative HLA-ABC expression (no expression, or low expression even if expressed).

[0006] On the other hand, HLA-G binds to KIR2DL4, an NK cell inhibitory receptor, and helps to evade attack by NK cells. In other words, it is known that positive HLA-G expression (high expression) promotes immune tolerance.

[0007] Therefore, when using iPS cells for regenerative medicine, it is preferable that they are negative for HLA-ABC expression, which is involved in the recognition of self and non-self, and optionally positive for HLA-G expression.

[0008] However, currently, no iPS cells that are negative for HLA-ABC expression (excluding those created by modifying the gene that expresses HLA-ABC) are known.

[0009] The disclosures in this application are made to solve the above-mentioned problems. The inventors have conducted diligent research and have newly created iPS cells that are negative for HLA-ABC expression and optionally positive for HLA-G expression, without modifying the genes that express HLA-ABC.

[0010] In other words, the purpose of the disclosure in this application is to provide iPS cells that are negative for HLA-ABC expression without modifying the genes that express HLA-ABC, and optionally positive for HLA-G expression, an iPS cell bank containing said iPS cells, and a method for providing iPS cell information from said iPS cell bank.

[0011] The disclosures in this application relate to iPS cells, iPS cell banks, and methods for providing iPS cell information, as described below.

[0012] (1) iPS cells that are negative for HLA-ABC expression (excluding cases where HLA-ABC expression is made negative by modifying the gene that expresses HLA-ABC). (2) iPS cells as described in (1) above that are positive for HLA-G expression. (3) iPS cells as described in (1) or (2) above, wherein the iPS cells are produced from cells collected from placental tissue excluding the amniotic membrane. (4) iPS cells as described in (3) above, wherein the collected cells are at least one selected from the group consisting of trophoblast stem cells, trophoblast stem cells, syncytiotrophoblast cells, and extrachorionic trophoblast cells. (5) iPS cells as described in any one of (1) to (4) above, wherein the iPS cells are of the naive type. (6) iPS cells as described in any one of (1) to (4) above, wherein the iPS cells are of the prime type. (7) iPS cells according to any one of (1) to (4) above, wherein the iPS cells are capable of dedifferentiating from primed to naive. (8) iPS cells according to any one of (1) to (7) above, wherein the iPS cells are cultured using serum-free medium. (9) iPS cell bank containing iPS cells according to any one of (1) to (8) above, wherein the iPS cell bank contains two or more types of iPS cells in which at least one of the HLA typings of HLA-A locus, HLA-B locus, and HLA-DR locus is different. (10) The iPS cell bank contains at least one selected from the group consisting of HLA-A locus typings A*24:02, A*02:01, A*11:01, A*26:01, A*33:03 and A*31:01, at least one selected from the group consisting of HLA-B locus typings B*52:01, B*54:01, B*51:01, B*40:01, B*40:02, B*46:01, B*44:03, B*15:01 and B*07:02, and The iPS cell bank described in (9) above, comprising iPS cells having at least one typing selected from the group consisting of DRB1*04:05, DRB1*09:01, DRB1*15:02, DRB1*08:03, DRB1*13:02, DRB1*14:03 and DRB1*01:01, which are HLA-DR locus typings.(11) The iPS cell bank according to (9) above, wherein the iPS cell bank includes iPS cells having at least one halotype selected from the group consisting of A*24:02-B*52:01-DRB1*15:02, A*33:03-B*44:03-DRB1*13:02, A*24:02-B*07:02-DRB1*01:01, and A*24:02-B*54:01-DRB1*04:05. (12) A method for providing iPS cell information, which provides information on iPS cells stored in an iPS cell bank described in any one of (9) to (11) above, wherein each iPS cell constituting the iPS cell bank is stored in association with typing information of at least HLA-A, HLA-B, and HLA-DR loci among the HLA typings, and the iPS cell information provision method comprises: an HLA typing information input step in which typing information of at least the subject's HLA-A, HLA-B, and HLA-DR loci is input; and, based on the input subject's HLA typing information, iPS cell information in which two of the HLA-A, HLA-B, and HLA-DR loci typings match among the stored iPS cells, and A method for providing iPS cell information, wherein at least one piece of information is provided selected from the group consisting of iPS cell information in which one of the typing information entries for HLA-A, HLA-B, and HLA-DR loci matches.

[0013] The iPS cells disclosed in this application are negative for HLA-ABC expression, which is involved in the recognition of self and non-self, without modification of the gene expressing HLA-ABC, and are optionally positive for HLA-G expression, which is known to work in the direction of immune tolerance. Therefore, when the iPS cells disclosed in this application are used in regenerative medicine, it is expected that immune rejection will be less likely to occur.

[0014] Figure 1 shows a schematic diagram of the placenta. Figure 2 is a substitute photograph of cells cultured in the first culture step of Example 1. Figure 3 is a substitute photograph of iPS cells produced in Example 1 after subculturing, and a photograph of iPS cells in the third subculturing stage stained with alkaline phosphatase (ALP). Figure 4 is a substitute photograph of iPS cells produced in Example 1 after immunostaining using various antibodies. Figure 5 shows the results of flow cytometry analysis of SSEA-4, a membrane protein of iPS cells produced in Example 1. Figure 6 shows the results of qPCR analysis of iPS cell markers contained in iPS cells produced in Example 1 and cells before iPS treatment. Figure 7 shows the results of flow cytometry analysis of HLA-ABC and HLA-G of iPS cells produced in Example 1. Figure 8 is a substitute photograph showing dedifferentiation from primed iPS cells to naive iPS cells produced in Example 1. Figure 9 is a table summarizing the HLA-A and HLA-B typing of the nine types of iPS cells produced in Example 3.

[0015] The iPS cells, iPS cell bank, and iPS cell information provision method disclosed in this application will be described in detail below.

[0016] Furthermore, in this specification, (1) a numerical range expressed using "~" means a range that includes the numerical values ​​written before and after "~" as the lower and upper limits, (2) numerical values, numerical ranges, and qualitative expressions (for example, expressions such as "identical" and "same") indicate numerical values, numerical ranges, and properties that include errors that are generally acceptable in the relevant technical field, and (3) when it is written as "approximately ○○ shape", it is interpreted to include not only the exact ○○ shape but also a shape that is understood to be approximately ○○ shape.

[0017] (Embodiment of iPS Cells) The iPS cells according to this embodiment are negative for HLA-ABC expression without modifying the gene that expresses HLA-ABC (hereinafter, this may simply be referred to as "negative for HLA-ABC expression"). In other words, the HLA-ABC gene remains in the wild type, but HLA-ABC expression is negative. Furthermore, in addition to being negative for HLA-ABC expression, the iPS cells according to this embodiment may also be positive for HLA-G expression. Regarding positive HLA-G expression, the HLA-G expression is positive without modifying the gene that expresses HLA-G; in other words, the HLA-G gene remains in the wild type, but HLA-G expression is positive. Furthermore, in this specification, "negative HLA-ABC expression" means that the genes for expressing HLA-A, HLA-B, and HLA-C antigens are present, but no antigens are expressed from those genes, or the antigens are expressed from the genes, but at a low level that does not affect the distinction between self and non-self. As mentioned above, a threshold for low expression levels can be appropriately determined within a range that does not affect the distinction between self and non-self. Although not limited to this, when measuring the amount of antigen expression as fluorescence intensity using a flow cytometer, the autofluorescence (negative control) of the iPS cells themselves may be used as the reference value, and cells may be considered "negative" if the autofluorescence is 10 times or less, 9 times or less, 8 times or less, 7 times or less, 6 times or less, 5 times or less, 4 times or less, 3 times or less, 2 times or less, or 1 time or less of the reference value. The autofluorescence (reference value) may be the median value of the fluorescence intensity measured using a flow cytometer with an Isotype control antibody of HLA-ABC antibody on iPS cells. Then, the fluorescence intensity of the target iPS cells measured using a flow cytometer with HLA-ABC antibody is compared to the reference value, and if it is 10 times or less, it should be considered "negative".

[0018] "Positive HLA-G expression" means that the HLA-G antigen is expressed. Since immune tolerance is achieved through HLA-G expression, there are no particular restrictions as long as HLA-G is expressed, but a higher expression level is preferable. The median fluorescence intensity measured using an Isotype control antibody of HLA-G is used as the reference value, and when comparing the fluorescence intensity of the target iPS cells measured using an HLA-G antibody with the reference value, it is preferable, though not limited to, that the fluorescence intensity is 1.1 times or more, 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, or 10 times or more.

[0019] The above description is merely an example of a method for setting thresholds based on measurements taken with a flow cytometer, and is not limited to this method. Alternatively, other methods, such as Western blotting, may be used, as long as the measurement value obtained using a reagent that does not react with HLA-ABC antigen and HLA-G antigen can be compared to the reference value. Alternatively, negative control cells that have been knocked down to not express the antigen may be prepared, and the measurement value obtained when the negative control cells are reacted with an antibody may be used as the reference value. In any method, if the measurement value obtained using a reagent that reacts with HLA-ABC antigen is 10 times or less of the reference value, it should be considered "negative for HLA-ABC expression." For HLA-G antigen, if the measurement value obtained using a reagent that reacts with HLA-G antigen is greater than the reference value, it should be considered "positive for HLA-G expression," with a value of 1.1 times or more being considered more favorably positive.

[0020] Generally, primed iPS cells are considered more stable. Therefore, the iPS cells according to the embodiment may be provided in the primed form. On the other hand, there are biological tissues, such as placental tissue, that can differentiate from naive iPS cells but not from primed iPS cells. In other words, naive iPS cells have greater diversity in the biological tissues they can differentiate into compared to primed iPS cells. Therefore, the iPS cells according to the embodiment may be provided in the naive form. Furthermore, as shown in the examples described later, the iPS cells disclosed in this application can be dedifferentiated from primed iPS cells to naive iPS cells. Therefore, depending on the therapeutic purpose using the iPS cells, they may be provided in either primed or naive form. Note that cells can be provided in an isolated state, but they are generally provided in a cultured state (a state in which multiple cells exist). In this specification, when "iPS cells" is mentioned, it means that the term includes both isolated single iPS cells and a state in which multiple iPS cells are mixed.

[0021] Whether iPS cells are naive or primed can be determined by examining known markers. Known markers for naive cells include CDX2, KLF2 (Kruppel-like factor 2), KLF4 (Kruppel-like factor 4), Nanog, Tbx3, and Lin28. Known markers for primed cells include GATA3, TFAP2C, ITGA6, KRT7, Oct4 (POU5F1), Tcf3 (TCF3), Sox17, and Fgf2. By examining the markers expressed in iPS cells using known methods such as immunohistochemistry, quantitative PCR (qPCR), Western blotting, and flow cytometry, it is possible to determine whether iPS cells are naive or primed.

[0022] The iPS cells according to this embodiment are not particularly limited in their production method, as long as they are negative for HLA-ABC expression and optionally positive for HLA-G expression. For example, they can be produced by a biological tissue collection step, an enzyme treatment step, a first culture step, a cell isolation step in which cells negative for HLA-ABC expression and optionally positive for HLA-G expression (hereinafter sometimes referred to as "pre-iPS processed cells") are isolated, an iPS cell production step, and a second culture step.

[0023] While there are no particular restrictions on the type of biological tissue collected during the tissue collection process, placental tissue, which exists as a site of immune tolerance between mother and child, is preferred. Figure 1 shows a schematic representation of the placenta. The placenta is composed of the amnion, chorionic villi (chorionic layer), and basal decidua (decidua) from the fetal side. The decidua is tissue that has been transformed from the endometrium, and therefore contains many maternal cells. Pre-iPS cell preparations are thought to be predominantly found on the chorionic side of the decidua and in the chorionic layer, but are also thought to be present throughout the chorionic layer. Therefore, pre-iPS cell preparations are preferably isolated from placental tissue excluding the amnion. Among the placental tissue excluding the amnion, the chorionic layer and decidua on the decidua side, or the chorionic layer directly beneath the amnion, are more preferred. Furthermore, the placenta is said to be fully formed around 15-16 weeks of gestation. Therefore, placental tissue from 16 weeks of gestation onwards is suitable for collecting cells before iPS cell processing, and from the perspective of ease of acquisition, placenta from 28 weeks of gestation onwards to full term is suitable.

[0024] The enzyme treatment process involves cutting placental tissue, collected from the placenta, into strips, for example, 5 mm or less, and then treating them with enzymes. The enzyme used in the enzyme treatment process is not particularly limited, as long as it is an enzyme commonly used in the field of cell culture. Examples of such enzymes, though not limited, include collagenase, trypsin, papain, and dispase. As mentioned above, the placental tissue should be the placental tissue excluding the amniotic membrane, with the chorionic layer and decidua on the decidual side, or the chorionic layer directly beneath the amniotic membrane, being preferred. Furthermore, the placenta should be from 16 weeks of gestation to full term.

[0025] The first culture step involves culturing cells derived from placental tissue obtained by enzyme treatment. It is preferable to culture the cells in the first culture step using a serum-free medium. While serum-free medium is preferable, any medium commonly used for cell culture can be used; however, it is not limited to such media. Examples of suitable media include DMEM liquid medium, DMEM / F12 liquid medium, and Neurobasal medium.

[0026] The cell isolation step before iPS cell treatment involves separating cells that are negative for HLA-ABC expression (and optionally positive for HLA-G expression) from the cultured cells. Separation can be performed based on cell morphology or using antibodies. While not limited to these methods, cells negative for HLA-ABC expression can be obtained by removing HLA-ABC-positive cells using an HLA-ABC antibody. As shown in the examples described later, cells obtained from placental tissue are positive for HLA-G expression. Therefore, separation using antibodies is not required for HLA-G, but cells expressing HLA-G may be isolated using an HLA-G antibody.

[0027] The iPS cell isolation step is intended to reduce the probability of producing iPS cells that do not possess the characteristics disclosed in this application when the cells are converted into iPS cells in the iPS cell production step described later. Since it is also possible to isolate iPS cells possessing the characteristics disclosed in this application after carrying out the iPS cell production step described later, the iPS cell isolation step is an optional additional step.

[0028] The iPS cell generation process can be carried out by converting cultured cells into iPS cells using a known method. Alternatively, the iPS cell generation process can be carried out using commercially available kits or the like.

[0029] The second culture step involves culturing the cells processed in the iPS cell production step. If the pre-iPS cell isolation step has not been performed, iPS cells having the characteristics disclosed in this application may be selected and isolated from the cells cultured in the second culture step, using cell morphology, iPS markers, HLA-ABC negativity, and optionally HLA-G positivity as indicators, as needed. The culture medium used in the second culture step may be the same as or different from that used in the first culture step, but it is preferable that it be serum-free. When providing iPS cells, it is not a problem if a small number of cells lacking the desired characteristics are included, as long as the majority of the iPS cells are negative for HLA-ABC expression (optionally positive for HLA-G expression). However, from the viewpoint of reducing unexpected risks, it is preferable to remove as many cells other than the target iPS cells as possible. Optionally, iPS cells having the desired characteristics can also be isolated by repeating the second culture step and selection / isolation.

[0030] The iPS cells isolated from placental tissue excluding the amniotic membrane include trophoblast stem cells, trophoblast stem cells, syncytiotrophoblast cells conjugated with trophoblast stem cells, and extravillous trophoblast cells differentiated from trophoblast stem cells. Therefore, the iPS cells according to this embodiment can be defined as iPS cells such as trophoblast stem cells, trophoblast stem cells, syncytiotrophoblast cells, and extravillous trophoblast cells.

[0031] When iPS cells are transplanted for therapeutic purposes, the diseases that can be expected to be treated are not limited to age-related macular degeneration, Parkinson's disease, severe heart failure / cardiomyopathy, thrombocytopenia, spinal cord injury, and corneal diseases requiring corneal transplantation. Furthermore, the transplantation of iPS cells for therapeutic purposes is not limited to humans. In this specification, "iPS cells" refers not only to human iPS cells but also to iPS cells obtained from mammals such as pets (dogs, cats, mice, etc.), livestock (cattle, horses, goats, sheep, etc.).

[0032] The iPS cells according to this embodiment exhibit the following effects: (1) They are negative for HLA-ABC expression, which is involved in the recognition of self and non-self, and positive for HLA-G expression, which is optionally known to work in the direction of immune tolerance. Therefore, even when used as cells for allogeneic transplantation, immune rejection is suppressed, and they are expected to be used in regenerative medicine. (2) Some conventional iPS cells cannot dedifferentiate from primed to naive type. On the other hand, the iPS cells disclosed in this application can dedifferentiate from primed to naive type. Therefore, the types of biological tissues that can be differentiated from iPS cells increase, thus increasing the range of diseases that can be treated. (3) Since the placenta is naturally shed and expelled from the mother's uterus during childbirth, collecting the placenta is not considered an intervention. Furthermore, the placenta is a type of medical waste and is usually treated as infectious waste because it is in contact with blood and bodily fluids. Therefore, it is easy to obtain consent from donors, and it is possible to produce iPS cells that are negative for various HLA-ABC expression.

[0033] (Embodiment of the iPS Cell Bank) Next, an iPS cell bank according to the embodiment (hereinafter sometimes simply referred to as the "bank") will be described. The bank according to the embodiment includes two or more types of iPS cells according to the embodiment described above, in which at least one of the HLA typings (sometimes referred to as "HLA type") of the HLA-A locus, HLA-B locus, and HLA-DR locus is different; in other words, two or more types of iPS cells with different halotypes, which are combinations of HLA-A locus, HLA-B locus, and HLA-DR locus.

[0034] As described above, the iPS cells according to the embodiment are negative for HLA-ABC expression, which is involved in the recognition of self and non-self, making immune rejection less likely. However, although the iPS cells according to the embodiment are negative for HLA-ABC expression, they do possess the genes that express HLA-A antigen, HLA-B antigen, and HLA-C antigen. Therefore, from the viewpoint of suppressing the risk of immune rejection as much as possible, it is preferable to provide patients with iPS cells with the same HLA typing.

[0035] The HLA type of mammalian cells is determined by the combination of two inherited HLA types, one from the father and one from the mother. A combination of identical HLA types is called HLA homozygosity, while a combination of different HLA types is called HLA heterozygosity. When iPS cells (or induced biological tissue) are transplanted into a patient, immune rejection is less likely to occur if one of the HLA types in the transplanted iPS cells matches one of the patient's HLA types. Therefore, HLA homozygosity is preferable for iPS cells stocked in a bank because it increases the range of patients that can be covered by a single HLA type. However, even HLA heterozygous iPS cells are less likely to cause immune rejection if they perfectly match the patient's HLA type (combination of two types). Therefore, iPS cells included in a bank may also be HLA heterozygous.

[0036] Human HLAs are classified into six types based on differences in function and structure: HLA-A, HLA-B, and HLA-C (class I molecules), and HLA-DR, HLA-DQ, and HLA-DP (class II molecules). To minimize immune rejection, it is desirable to match three of the six types: HLA-A, HLA-B, and HLA-DR.

[0037] Incidentally, it is known that there are numerous genetic variations in the HLA-A, HLA-B, and HLA-DR loci. Furthermore, it is known that the tendencies of genetic variations differ depending on race. For example, in the allele group of Japanese people, it is known that (1) the HLA-A locus has a relatively large number of genotypes such as HLA-A*02, HLA-A*24, and HLA-A*01, (2) the HLA-B locus has a relatively large number of genotypes such as HLA-B*40, HLA-B*46, HLA-B*15, and HLA-B*51, and (3) the HLA-DR locus has a relatively large number of genotypes such as DRB1*04, DRB1*01, and DRB1*15.

[0038] It is desirable that iPS cells included in the bank be stored in association with at least the genotype information of the HLA-A locus, the HLA-B locus, the HLA-DR locus, and the halotype, which is a combination of the HLA-A, HLA-B, and HLA-DR loci. If at least one of the genotypes (typings) of the HLA-A, HLA-B, and HLA-DR loci is different, then the iPS cells can be considered to be of different HLA types (different halotypes).

[0039] To provide iPS cells that match the HLA type of many patients, the bank should contain at least two different types of iPS cells with different HLA types. The more types of iPS cells with different HLA types included in the bank, the wider the range of patients to whom cells can be provided. Therefore, the number of types of iPS cells included in the bank may be 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1000 or more.

[0040] Furthermore, it is desirable for the bank to include iPS cells with HLA types that match the HLA types that are prevalent in humans, as this broadens the range of suitable patients. For example, the website of the Japanese Society for Histocompatibility (https: / / drive.google.com / file / d / 1tn5Sgd2fkFf1bO0lnSBjmY4wiipZMgWD / view) lists the HLA types that frequently occur in Japanese people at the subtype level. According to the website, the most frequent subtypes are as follows: HLA-A: A*24:02, A*02:01, A*11:01, A*26:01, A*33:03, A*31:01. Among these, A*24:02 is the most frequent. - HLA-B: B*52:01, B*54:01, B*51:01, B*40:01, B*40:02, B*46:01, B*44:03, B*15:01, B*07:02. Among these, B*52:01 is the most frequent. - HLA-DR: DRB1*04:05, DRB1*09:01, DRB1*15:02, DRB1*08:03, DRB1*13:02, DRB1*14:03, DRB1*01:01. Among these, DRB1*04:05 is the most frequent.

[0041] Therefore, it is preferable that the bank contains iPS cells having the following typings: • For HLA-A locus typing, it is preferable that at least one selected from the group consisting of A*24:02, A*02:01, A*11:01, A*26:01, A*33:03, and A*31:01 is included, and it is more preferable that A*24:02 is included. • For HLA-B locus typing, it is preferable that at least one selected from the group consisting of B*52:01, B*54:01, B*51:01, B*40:01, B*40:02, B*46:01, B*44:03, B*15:01, and B*07:02 is included, and it is more preferable that B*52:01 is included. - For the typing of the HLA-DR seats, it is preferable that at least one selected from the group consisting of DRB1*04:05, DRB1*09:01, DRB1*15:02, DRB1*08:03, DRB1*13:02, DRB1*14:03, and DRB1*01:01 is included, and it is more preferable that DRB1*04:05 is included.

[0042] Furthermore, the Kyoto University iPS Cell Research Foundation website (https: / / www.cira-foundation.or.jp / j / provision-of-ips-cells / homozygous / ) states that the most common HLA-A, B, and DR halotypes are, in order: A*24:02-B*52:01-DRB1*15:02, A*33:03-B*44:03-DRB1*13:02, A*24:02-B*07:02-DRB1*01:01, and A*24:02-B*54:01-DRB1*04:05. Therefore, when identifying iPS cells contained in the bank by HLA halotype, it is preferable that at least one selected from the group consisting of A*24:02-B*52:01-DRB1*15:02, A*33:03-B*44:03-DRB1*13:02, A*24:02-B*07:02-DRB1*01:01, and A*24:02-B*54:01-DRB1*04:05 is included, and it is more preferable that A*24:02-B*52:01-DRB1*15:02 is included.

[0043] When the iPS cells are heterozygous, at least one of the HLA types may include the above-described HLA-A locus typing, HLA-B locus typing, HLA-DRB1 typing, and HLA haplotypes. The above-described typing is for an example of Japanese people. When producing a bank for people of races other than Japanese, the typing frequency in the race may be investigated, such that the bank contains iPS cells having high-frequency typing.

[0044] (Embodiment of iPS cell information providing method) Next, an iPS cell information providing method according to an embodiment will be described. The iPS cell information providing method according to the embodiment is a method for providing iPS cell information contained in the iPS cell bank according to the above-described embodiment. As described above, each iPS cell constituting the iPS cell bank is stored in association with at least typing information of HLA-A locus, HLA-B locus and HLA-DR locus among HLA typings, and optionally additionally with a haplotype that is a combination of HLA-A locus, B locus and DR locus. In addition to the above-described HLA-A locus, B locus, DR locus and haplotype information, the iPS cells may optionally further be stored in association with information such as typing information of HLA-C locus, DQ locus and DP locus, cell type, race, number of passages, and the like. Hereinafter, information associated with iPS cells may be collectively referred to as "iPS cell information".

[0045] The iPS cell information providing method includes at least an HLA typing information input step of inputting HLA typing information of a subject, and an iPS cell information providing step.

[0046] The HLA typing information input step only requires inputting typing information for at least the subject's HLA-A, HLA-B, and HLA-DR loci. If the subject is heterozygous, typing information for both alleles' HLA-A, HLA-B, and HLA-DR loci may also be input. The iPS cell information provision step, based on the input subject's HLA typing information, provides information on iPS cells with a matching halotype if any of the stored iPS cells contain matching halotypes. If the halotypes do not match, information on iPS cells with matching HLA-A, HLA-B, and HLA-DR locus typing information, or iPS cell information with matching HLA-A, HLA-B, and HLA-DR locus typing information, may be provided. Furthermore, if the typing of the subject's HLA-A, HLA-B, and HLA-DR loci does not match any of the stored iPS cells, it is sufficient to provide information indicating that no iPS cells matching at least one of the subject's typings are stored.

[0047] Furthermore, as described above, the iPS cells according to this embodiment are cells that are less likely to cause immune rejection. After clinical trials and other studies have gathered evidence that even if the halotype does not match, if any of the HLA-A, HLA-B, and HLA-DR locus typing information matches, immune rejection is less likely to occur, a predetermined standard value may be set, and a selection process may be carried out to identify iPS cells that can be provided to the subject from among the iPS cells stored in the bank, based on the subject's HLA typing information. The selection process may identify the iPS cells most suitable for the subject, or it may identify multiple types of cells that can be provided to the subject. After carrying out the selection process, a provision process for the identified cells may be carried out. Of course, if the halotype is a perfect match, iPS cells that match the subject's halotype may be provided without carrying out the selection process.

[0048] There are no particular restrictions on the method for providing iPS cell information, as long as each of the above steps is carried out. For example, the iPS cell information can be stored in a computer, and the HLA typing information input step, the iPS cell information provision step, and optionally an additional discrimination step can be performed.

[0049] The iPS cell bank and the iPS cell information providing method according to the embodiment produce the following effects. (1) iPS cells that are less likely to cause immune rejection can be provided to patients. (2) As described above, the iPS cells according to the embodiment are cells that are less likely to cause immune rejection. The iPS cells can also be used for drug discovery research and the like while taking into account the aforementioned characteristics and HLA typing. (3) Since the iPS cells according to the embodiment are produced from placenta-derived cells, at least one of the HLA types is the same for at least the mother or the child of the placenta donor. Therefore, by associating donor information with the iPS cells, the iPS cells can be suitably used when treatment using iPS cells is required for the donor's mother or child in the future.

[0050] Examples are given below to specifically describe the embodiments disclosed in the present application, but these examples are merely for the purpose of describing the embodiments. They are not intended to limit or restrict the technical scope disclosed in the present application.

[0051] [Preparation of iPS Cells] <Example 1> iPS cells were prepared from placental tissue by the following procedure.

[0052] <Cell Culture from Placental Tissue (Biological Tissue Collection Process, Enzyme Treatment Process, First Culture Process)> (1) Placental tissue (decidua including part of the chorionic layer) was excised from a placenta at 38 weeks of gestation, cut into strips of 5 mm or less, and transferred to a tube. (2) Enzyme treatment was performed using collagenase (Sigma-Aldrich). (3) CELLOTION (registered trademark, Takara Bio Inc.) and PBS were added and the mixture was diluted to the required volume and centrifuged at 1,000 rpm for 5 minutes at 4°C. (4) Tissue fibers that were not digested by the enzyme treatment were removed using sterile gauze and a cell strainer with a mesh size of 100 μm. (5) Ficol-Paque (registered trademark, Merck) was added to the cell suspension and centrifuged by specific gravity to recover the nucleated cell layer. (6) CELLOTION and PBS were added and the mixture was centrifuged at 1,000 rpm for 5 minutes at 4°C. (7) Remove the supernatant and add 10 times the volume of erythrocyte hemolysis reagent (RBC Lysis Buffer, pluriSelect Life Science) to the cell suspension, then hemolyze at 4°C for 15 minutes. (8) Add CELLOTION and PBS and centrifuge at 1,000 rpm for 5 minutes at 4°C. (9) Remove the supernatant and suspend the precipitated cells by pipetting, then culture them in a cell dish using serum-free medium. The medium used was an equal mixture of serum-free DMEM / F12 medium (Cat No. 11320033) and Neurobasal medium (Cat No. 21103049). The medium was changed every 2-3 days. Figure 2 shows a photograph of the cultured cells. The cell population indicated by the arrow in Figure 2 exhibits a cobblestone-like epithelial cell morphology and is characterized by dense adhesion between cells forming colonies, indicating that it is a placental trophoblast cell.

[0053] <Cell Isolation Process Before iPS Cell Treatment> (1) Cells cultured using enzymes were detached from the cell dish, centrifuged and washed with PBS, and then labeled with HLA-ABC-FITC antibody (1:10 dilution, Beckman) by reacting at 4°C for 30 minutes. After centrifuging and washing with PBS, the cells were labeled with Anti-FITC MicroBeads (1:10 dilution, Milltenyi BioTec) by reacting at 4°C for 30 minutes. (2) After centrifuging and washing with PBS, positive selection was performed using a magnetic column (MS Column, Milltenyi BioTec) to remove cells expressing HLA-ABC (HLA-ABC positive cells). Furthermore, the HLA-ABC-FITC antibody is an antibody that reacts with any of HLA-A, HLA-B, and HLA-C. Therefore, if cells that react with the HLA-ABC-FITC antibody are removed, the remaining cells will be negative for HLA-ABC expression. Also, as shown in the data described later, the cells obtained from placental tissue are positive for HLA-G. Therefore, HLA-G was not labeled or isolated. (3) The HLA-ABC negative cells isolated by magnetic column were cultured again in the serum-free medium described above.

[0054] <iPS Cell Production Process> (1) The cells cultured in the cell isolation process before iPS treatment described above were introduced with four Yamanaka factors using a Sendai virus vector-based cell reprogramming vector (CytoTune-iPS2.0L). The gene transfer reagent kit used was CytoTune-iPS2.0L (Nacalai Tesque Co., Ltd.), and the experiment was conducted according to the instructions (https: / / www.nacalai.co.jp / products / 306 / ). (2) The gene-transferred cells were cloned to produce iPS cells.

[0055] <Verification of iPS cells> (1) Alkaline phosphatase (ALP) staining The prepared iPS cells were cultured for three passages using the serum-free medium described above. The third passage iPS cells were fixed with 4% paraformaldehyde, and in order to confirm the expression of alkaline phosphatase (ALP), a common marker that is highly expressed in all pluripotent stem cells, including ES cells and iPS cells, they were stained using the Red-Color® AP Staining Kit (System Biosciences, Palo Alto, CA, USA) according to the manufacturer's protocol, and observed under a microscope (Power IX-71 and DP-71; OLYMPUS).

[0056] Figure 3 shows photographs of the generated iPS cells (P0), first passage (P1), second passage (P2), third passage (P3), and ALP-stained third passage (P3 ALP). As shown in P3 ALP, it was confirmed that the generated iPS cells highly express alkaline phosphatase.

[0057] (2) A portion of the immunostained iPS cells were treated with 0.5% Triton® X-100 (Fujifilm Wako) for 5 minutes, then Protein Block Serum-Free Ready-To-Use (Agilent Technologies, Santa Clara, CA, USA) was added, and the cells were blocked at room temperature for 5 minutes. PBS containing 1% albumin was used with the following primary antibodies: anti-human SOX2 (nuclear iPS marker protein) monoclonal antibody (1:100; ThermoFisher Scientific, USA), anti-human OCT4 (nuclear iPS marker protein) polyclonal antibody (1:500; Medical & Biological Laboratories, Aichi, Japan), anti-human Keratan Sulfate (quality control marker indicating non-embryonic cancer cells) monoclonal antibody (1:100; Cosmo Bio LTD, USA), and anti-human SSEA-4 (iPS cell membrane protein marker) monoclonal antibody (1:100; Abcam, Cambridge, UK). Alexa Fluor® 594 or 488 labeled antibody (1:500; ThermoFisher Scientific) was added as a secondary antibody. Nuclear staining was performed using DAPI (Vectashield H-1200; Vector Laboratories). Immunostaining was evaluated using a fluorescence microscope (Power IX-71 and DP-71; Olympus). Figure 4 shows photographs observed with a fluorescence microscope. As shown in Figure 4, it was confirmed that the produced iPS cells expressed various iPS markers. Note that OCT4 in Figure 4 is a primed iPS cell marker. Also, the P2 iPS cells in Figure 3 show the morphological characteristics of primed cells, such as forming flat colonies. From these results, it was confirmed that the iPS cells produced in Example 1 were primed.

[0058] (3) Flow cytometry analysis of SSEA-4 Anti-human SSEA-4 monoclonal antibody was added to cells as the primary antibody, and Alexa Fluor® 488 (1:500; ThermoFisher Scientific) was added as the secondary antibody and incubated. A FACSCalibur flow cytometer (Becton Dickinson and Company, Franklin Lakes, NJ, USA) was used for cell analysis. Experiments using an isotype control antibody of the anti-human SSEA-4 monoclonal antibody were used as a comparative (negative) control. Figure 5 shows the analysis results. Since SSEA-4 is a membrane protein, flow cytometry confirmed that EESA-4 was highly expressed in the prepared iPS cell membrane.

[0059] (RNA Analysis) Gene expression analysis of iPS cells was performed using TaqMan® quantitative real-time polymerase chain reaction (qRT-PCR). TaqMan Gene Expression Cells-to-CT® Kit (ThermoFisher Scientific) was used for total RNA extraction and reverse transcription from cells. Subsequently, cDNA was used as a template for PCR amplification. For the TaqMan real-time PCR (qPCR) assay, qPCR was performed using the ABI PRISM 7900 HT Sequence Detection System (ThermoFisher Scientific). We used primers and probes for iPS cell marker genes, namely SOX2 (Hs00415716_m1), OCT3 / 4 (Hs00742896_s1), NANOG (Hs04260366_g1), and KLF4 (Hs00358836_m1), and used GAPDH (Hs999999905_m1) as an endogenous control.

[0060] Figure 6 shows the qPCR results for cells before iPS cell treatment (labeled "before introduction" in Figure 6) and iPS cells produced in the example (labeled "after introduction" in Figure 6). As is clear from Figure 6, the iPS cells produced in the example were confirmed to highly express iPS cell markers.

[0061] <Verification of negative HLA-ABC expression and positive HLA-G expression> (1) Flow cytometry analysis of HLA-ABC Anti-human HLA-ABC-FITC labeled antibody (Beckman) was added as the primary antibody to the prepared iPS cell solution and incubated. A flow cytometer, CytoFLEX (Beckman Coulter, Inc., Brea, CA, USA), was used for cell analysis. (2) Flow cytometry analysis of HLA-G The analysis was performed using the same procedure as described above for "Flow cytometry analysis of HLA-ABC", except that anti-human HLA-G-APC labeled antibody (1:200; Miltenyi BioTec) was used as the primary antibody.

[0062] Figure 7 shows the results of flow cytometry analysis of HLA-ABC and HLA-G. From the results in Figure 7, the negative rate for HLA-ABC was 90.13%, and the positive rate for HLA-G was 95.18%. From these results, it was confirmed that in Example 1, iPS cells that were negative for HLA-ABC expression and positive for HLA-G expression were obtained.

[0063] [Dedifferentiation from Prime Type to Naive Type] <Example 2> Prime type iPS cells prepared in Example 1 were cultured in a commercially available dedifferentiation medium (NaiveCult Induction Kit, VERITAS Corporation). The left side of Figure 8 shows a photograph of prime type iPS cells before dedifferentiation, and the right side shows a photograph of naive type iPS cells after dedifferentiation. Prime type iPS cells have the morphological characteristic of forming flat colonies. On the other hand, naive type iPS cells have the morphological characteristic of forming three-dimensional, monolayer colonies. As is clear from the photograph in Figure 8, morphological observation confirmed that the prime type iPS cells prepared in Example 1 were dedifferentiated to the naive type by culturing them in the dedifferentiation medium.

[0064] [HLA Typing] <Example 3> iPS cells were produced from the placentas of nine individuals using the same procedure as in Example 1 described above. The produced iPS cells were verified to be iPS cells using the same procedure as in Example 1, and it was confirmed that they were negative for HLA-ABC expression and positive for HLA-G expression. Next, the HLA-A typing of the nine types of produced iPS cells was examined using the SSOP typing method (see "SSOP Typing Method for HLA-A, B, and DR in a Japanese Population. MHC 8(3), 175-186, 2002. doi.org / 10.12667 / mhc.8.175").

[0065] The results are shown in Figure 9. The "Reference: Frequency in Japanese (%)" in Figure 9 is the value listed in the "List of Presumptive HLA Alleles (JSHI) 2024 Edition (pdf)" published on the website of the Japanese Society for Histocompatibility (https: / / drive.google.com / file / d / 1Ky173i-evE_CDBtr9hoQGysYkd_PwXRB / view). Note that, regarding the results for the HLA-A allele of iPS cell No. 2 in Figure 9, the above-mentioned SSOP typing method cannot distinguish between A*11:01 and A*11:02, so the two are listed together. The "Reference: Frequency in Japanese (%)" value for A*11:01 / 02 is the sum of the values ​​for A*11:01 and A*11:02 listed in the "List of Presumptive HLA Alleles (JSHI) 2024 Edition (pdf)". The same applies to iPS cell No. 8 A*26:01 / 02 in Figure 9. Of the nine types of iPS cells, iPS cells No. 1, 5, and 9 had the same HLA-A typing, accounting for approximately 33.3%. The typing of the other iPS cells differed, and the percentage of HLA-A typing confirmed in Example 3 was found to be close to the value described in the paper. Furthermore, all HLA-B typings were different. Note that, due to experimental constraints, only HLA-A and HLA-B typings were confirmed, but HLA-DR typing can be confirmed using a similar method.

[0066] As described above, the iPS cells disclosed in this application are negative for HLA-ABC expression and optionally positive for HLA-G expression, making them less likely to cause immune rejection during allogeneic transplantation. Furthermore, by creating a bank by associating the HLA typing information of at least the HLA-A, HLA-B, and HLA-DR loci of the produced iPS cells with the HLA typing information, it is possible to provide iPS cells suitable for the patient based on the patient's HLA typing information. The information identifying each iPS cell included in the bank and the HLA typing information of that iPS cell can be stored, for example, in a computer, and a program that can identify the iPS cells to be provided by inputting the patient's HLA typing information can be stored there.

[0067] The iPS cells disclosed in this application are negative for HLA-ABC expression, which is involved in the recognition of self and non-self, and optionally positive for HLA-G expression, which is known to promote immune tolerance. Therefore, even when used as cells for allogeneic transplantation, immune rejection can be expected to be suppressed, making them useful for the medical industry.

Claims

1. iPS cells that are negative for HLA-ABC expression (excluding cases where HLA-ABC expression is made negative by modifying the gene that expresses HLA-ABC).

2. The iPS cells according to claim 1, wherein HLA-G expression is positive.

3. The iPS cells according to claim 1 or 2, wherein the iPS cells are produced from cells collected from placental tissue excluding the amniotic membrane.

4. The iPS cells according to claim 3, wherein the collected cells are at least one selected from the group consisting of trophoblast stem cells, trophoblast stem cells, syncytiotrophoblast cells, and extravillous trophoblast cells.

5. The iPS cells according to claim 1 or 2, wherein the iPS cells are of the naive type.

6. The iPS cells according to claim 1 or 2, wherein the iPS cells are of the prime type.

7. The iPS cells according to claim 1 or 2, wherein the iPS cells are capable of dedifferentiating from primed to naive.

8. The iPS cells according to claim 1 or 2, wherein the iPS cells are cultured using a serum-free medium.

9. An iPS cell bank comprising iPS cells according to claim 1 or 2, wherein the iPS cell bank comprises two or more types of iPS cells in which at least one of the HLA typings of HLA-A locus, HLA-B locus, and HLA-DR locus is different.

10. The iPS cell bank contains at least one selected from the group consisting of HLA-A locus typings A*24:02, A*02:01, A*11:01, A*26:01, A*33:03 and A*31:01, at least one selected from the group consisting of HLA-B locus typings B*52:01, B*54:01, B*51:01, B*40:01, B*40:02, B*46:01, B*44:03, B*15:01 and B*07:02, and An iPS cell bank according to claim 9, comprising iPS cells having an iPS cell typing selected from the group consisting of DRB1*04:05, DRB1*09:01, DRB1*15:02, DRB1*08:03, DRB1*13:02, DRB1*14:03 and DRB1*01:01, which are HLA-DR locus typings.

11. The iPS cell bank according to claim 9, wherein the iPS cell bank includes iPS cells having at least one halotype selected from the group consisting of A*24:02-B*52:01-DRB1*15:02, A*33:03-B*44:03-DRB1*13:02, A*24:02-B*07:02-DRB1*01:01, and A*24:02-B*54:01-DRB1*04:

05.

12. A method for providing iPS cell information, which provides information on iPS cells stored in an iPS cell bank according to claim 9, wherein each iPS cell constituting the iPS cell bank is stored in association with typing information of at least HLA-A, HLA-B, and HLA-DR loci among the HLA typings, and the iPS cell information providing method comprises: an HLA typing information input step in which typing information of at least the subject's HLA-A, HLA-B, and HLA-DR loci is input; and, based on the input subject's HLA typing information, iPS cell information in which two of the HLA-A, HLA-B, and HLA-DR loci typings match among the stored iPS cells, and iPS cell information in which one of the HLA-A, HLA-B, and HLA-DR loci typings matches. A method for providing iPS cell information, wherein at least one piece of information selected from the group consisting of the following is provided.

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