Method for selecting cardiomyocytes with high proliferation potential

Abstract

A method for producing cardiomyocytes that comprises a step for collecting cardiomyocytes highly expressing CD105 from a population of cardiomyocytes and a step for extensively culturing the cardiomyocytes thus collected.

Description

[Technical Field]

[0001] The present invention relates to a method for selecting cardiomyocytes with high proliferation potential. More specifically, the present invention relates to a method for selecting cardiomyocytes with high proliferation potential from a cardiomyocyte population, a method for selecting cardiomyocytes with activated cell cycle from a cardiomyocyte population, a method for producing cardiomyocytes, and a method for producing cardiac tissue. This application claims priority to U.S. Patent Application No. 63 / 029,564, provisionally filed in the U.S. on May 25, 2020, the contents of which are incorporated herein by reference. [Background technology]

[0002] For patients with severely impaired cardiac function, heart transplantation is often the only treatment option. However, there is a chronic shortage of donor hearts. Therefore, alternative therapies to heart transplantation are needed. Cardiac cell therapy is expected to be one alternative treatment.

[0003] However, it is known that cardiomyocytes in adult cardiac tissue do not have the ability to proliferate, and that cardiomyocytes differentiated in vitro lose their proliferative ability with continued culture. Therefore, active efforts are being made to develop techniques to increase the proliferative ability of cardiomyocytes (see, for example, Non-Patent Document 1).

[0004] Although knockout mice for the genes encoding the transcription factors HAND1 and HAND2 exhibit left and right ventricular hypoplasia, respectively, the functions and expression of these transcription factors during human cardiac development remain unclear. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Mohamed TMA, et al. Regulation of Cell Cycle to Stimulate Adult Cardiomyocyte Proliferation and Cardiac Regeneration, Cell, 173 (1), 104-116, 2018. Summary of the Invention [Problem to be solved by the invention]

[0006] In the method of Non-Patent Document 1, in order to increase the proliferation capacity of cardiomyocytes, it is necessary to overexpress cyclin-dependent kinase 1 (CDK1), CDK4, cyclin B1, and cyclin D1 in cardiomyocytes by gene transfer. Therefore, an object of the present invention is to provide a technique for obtaining cardiomyocytes with high proliferation capacity without gene transfer. [Means for solving the problem]

[0007] The present invention includes the following aspects. [1] A method for producing cardiomyocytes, comprising the steps of recovering cardiomyocytes that highly express CD105 from a population of cardiomyocytes, and expanding the recovered cardiomyocytes. [2] A method for producing cardiac tissue, comprising the steps of: recovering cardiomyocytes that highly express CD105 from a population of cardiomyocytes; and culturing the recovered cardiomyocytes to produce cardiac tissue. [3] The method of production described in [1] or [2], wherein the cardiomyocyte population is derived from pluripotent stem cells. [4] The method of [3], wherein the pluripotent stem cells are human iPS cells. [5] A method for selecting cardiomyocytes with high proliferation potential from a cardiomyocyte population, the method comprising the step of selecting cardiomyocytes that highly express CD105 from the cardiomyocyte population. [6] A method for selecting cardiomyocytes with an activated cell cycle from a cardiomyocyte population, the method comprising the step of selecting cardiomyocytes that highly express CD105 from the cardiomyocyte population. [7] The method according to [5] or [6], wherein the cardiomyocyte population is derived from pluripotent stem cells. [8] The method according to [7], wherein the pluripotent stem cells are human iPS cells.

[0008] The present invention can also be said to include the following aspects. [P1] A method for selecting cardiomyocytes with high proliferation potential from a population of cardiomyocytes derived from pluripotent stem cells, the method comprising a step of selecting cardiomyocytes that highly express CD105 from the population of cardiomyocytes, wherein the selected cardiomyocytes are cardiomyocytes with high proliferation potential. [P2] A method for selecting cardiomyocytes with an activated cell cycle from a population of cardiomyocytes derived from pluripotent stem cells, the method comprising a step of selecting cardiomyocytes that highly express CD105 from the population of cardiomyocytes, wherein the selected cardiomyocytes are cardiomyocytes with an activated cell cycle. [P3] A method for determining the maturity of cardiomyocytes derived from pluripotent stem cells, comprising a step of measuring the expression level of CD105 in the cardiomyocytes, wherein a higher expression level compared to a control indicates that the cell cycle of the cardiomyocytes is activated and the cardiomyocytes are less mature, and a lower expression level compared to a control indicates that the cell cycle of the cardiomyocytes is not activated and the cardiomyocytes are more mature. [P4] A method for treating cardiac disease, comprising the steps of recovering cardiomyocytes that highly express CD105 or cardiomyocytes that lowly express CD105 from a population of cardiomyocytes derived from pluripotent stem cells, and transplanting the recovered cardiomyocytes into a patient's heart, wherein the cardiomyocytes that highly express CD105 are transplanted to patients who require the transplantation of cardiomyocytes with high proliferation ability or cardiomyocytes with an activated cell cycle and low maturity as cardiomyocytes, and the cardiomyocytes that lowly express CD105 are transplanted to patients who require the transplantation of cardiomyocytes with an inactive cell cycle and high maturity as cardiomyocytes. [P5] A method for producing large quantities of cardiomyocytes, comprising the steps of recovering cardiomyocytes that highly express CD105 from a population of cardiomyocytes derived from pluripotent stem cells, and expanding the recovered cardiomyocytes in vitro. [P6] A method for highly efficient production of cardiac tissue, comprising the steps of recovering cardiomyocytes with an activated cell cycle from a population of cardiomyocytes derived from pluripotent stem cells, and culturing the recovered cardiomyocytes to produce cardiac tissue, wherein the cardiomyocytes with an activated cell cycle are cardiomyocytes that highly express CD105. [Effects of the Invention]

[0009] According to the present invention, a technique for obtaining cardiomyocytes with high proliferation potential can be provided without gene transfer. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram illustrating an outline of the production of HAND1-mCherry reporter hiPSCs in Experimental Example 1. [Figure 2] FIG. 2 is a schematic diagram illustrating an outline of the production of HAND2-EGFP reporter hiPSCs in Experimental Example 1. [Figure 3] FIG. 3 is a schematic diagram showing the structure of the MYH6-iRFP670 transposon vector used in Experimental Example 2. [Figure 4] FIG. 4 is a heat map showing the results of Experimental Example 4. [Figure 5] 5 is a graph showing the results of flow cytometer analysis (FACS analysis) in Experimental Example 5. The horizontal axis represents EGFP fluorescence intensity, and the vertical axis represents APC fluorescence intensity. In the figure, "High" indicates MYH6-EGFP-positive cells and cardiomyocytes with high CD105-APC expression, and "Low" indicates MYH6-EGFP-positive cells and cardiomyocytes with low CD105-APC expression. [Figure 6] 6 is a graph showing the results of the EdU assay in Experimental Example 5. In the figure, "High" indicates the results for cardiomyocytes with high CD105 expression, and "Low" indicates the results for cardiomyocytes with low CD105 expression. The vertical axis indicates the percentage of EdU-positive cells. [Figure 7]7 is a graph showing the results of the EdU assay in Experimental Example 6. In the figure, "High" indicates the results for cardiomyocytes with high CD105 expression, and "Low" indicates the results for cardiomyocytes with low CD105 expression. The vertical axis indicates the percentage of EdU-positive cells. [Figure 8] 8 is a graph showing the results of the EdU assay in Experimental Example 6. In the figure, "High" indicates the results for cardiomyocytes with high CD105 expression, and "Low" indicates the results for cardiomyocytes with low CD105 expression. The vertical axis indicates the percentage of EdU-positive cells. [Figure 9] 9 is a graph showing the results of quantitative real-time PCR in Experimental Example 7. In the figure, "High" indicates the result for cardiomyocytes with high CD105 expression, and "Low" indicates the result for cardiomyocytes with low CD105 expression. In the figure, the vertical axis shows the relative value, where the expression level of the GAPDH gene was used as an internal control, and the expression level of the HAND1 gene in cardiomyocytes with low CD105 expression was determined by the ddCt method, and set to 1. [Figure 10] 10 is a graph showing the results of quantitative real-time PCR in Experimental Example 8. In the figure, "High" indicates the result for cardiomyocytes with high CD105 expression, and "Low" indicates the result for cardiomyocytes with low CD105 expression. In the figure, the vertical axis shows the relative value, where the expression level of the GAPDH gene was used as an internal control, and the expression level of the HAND1 gene in cardiomyocytes with low CD105 expression was determined by the ddCt method, and set to 1. [Figure 11] 11 is a graph showing the results of quantitative real-time PCR in Experimental Example 8. In the figure, "High" indicates the result for cardiomyocytes with high CD105 expression, and "Low" indicates the result for cardiomyocytes with low CD105 expression. In the figure, the vertical axis shows the relative value, where the expression level of the GAPDH gene was used as an internal control, and the expression level of the HAND1 gene in cardiomyocytes with low CD105 expression was determined by the ddCt method, and set to 1. DETAILED DESCRIPTION OF THE INVENTION

[0011] In this specification, a positive result may be represented by "+" and a negative result by "-". A positive result means that the expression level of a target gene or target protein in a cell is high, or that the cell is stained to a high degree under specified conditions. A negative result means that the expression level of a target gene or target protein in a cell is low, or that the cell is stained to a low degree under specified conditions.

[0012] [Method for selecting cardiomyocytes with high proliferation potential] In one embodiment, the present invention provides a method for selecting cardiomyocytes with high proliferation potential from a cardiomyocyte population, the method comprising a step of selecting cardiomyocytes that highly express CD105 from the cardiomyocyte population. The selected cardiomyocytes are cardiomyocytes with high proliferation potential.

[0013] According to the method of this embodiment, cardiomyocytes with high proliferation potential can be selected without gene transfer into the cardiomyocytes. Because gene transfer is not performed, safe cardiomyocytes that can be transplanted into humans can be easily obtained. Cardiomyocytes selected and collected by the method of this embodiment can be used to elucidate biological mechanisms and for cell transplantation therapy in the field of regenerative medicine.

[0014] The method of this embodiment can be described as a method for selecting cardiomyocytes having proliferation capacity from a cardiomyocyte population, a method for selecting cardiomyocytes whose cell cycle is activated from a cardiomyocyte population, or the like.

[0015] In the method of this embodiment, the cardiomyocyte population may be a cardiomyocyte population derived from a living organism, or may be obtained by inducing differentiation from pluripotent stem cells. Cardiomyocyte populations include cardiomyocytes with high proliferation potential, cardiomyocytes with low proliferation potential, and cardiomyocytes that have lost proliferation potential. The method for inducing differentiation of pluripotent stem cells into cardiomyocytes is not particularly limited and may be a method commonly used in the art. Pluripotent stem cells are preferably human-derived cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). In the case of a cell population induced to differentiate from pluripotent stem cells, cells obtained on days 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 after the start of differentiation induction are used.

[0016] High proliferation ability of cardiomyocytes, cardiomyocytes having proliferation ability, or cardiomyocyte cell cycle activation means, for example, that the amount of new DNA synthesis in cardiomyocytes is 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, 10.0-fold, 11.0-fold, 12.0-fold, 13.0-fold, 14.0-fold, 15.0-fold, 16.0-fold, 17.0-fold, 18.0-fold, 19.0-fold, 20.0-fold, 21.0-fold, 22.0-fold, 23.0-fold, 24.0-fold, 25.0-fold, 26.0-fold, 27.0-fold, 28.0-fold, 29.0-fold, 30.0-fold, 31.0-fold, 32.0-fold, 33.0-fold, 34.0-fold, 35.0-fold, 36.0-fold, 37.0-fold, 38.0-fold, 39.0-fold, 40.0-fold, 41.0-fold, 42.0-fold, 43.0-fold, 44.0-fold, 45.0-fold, 46.0-fold, The control cell may be 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, or 10.0-fold higher than that of the control cell. Here, the control cell may be a cell that has been previously shown to have low cell proliferation ability, such as cardiomyocytes derived from adult cardiac tissue or cardiomyocytes with low CD105 expression.

[0017] The amount of newly synthesized DNA can be measured, for example, by an EdU assay, which measures the incorporation of the nucleoside analogue EdU (5-ethynyl-2'-deoxyuridine) into DNA.

[0018] CD105 is a cell membrane surface protein encoded by the ENG gene. Hereinafter, the ENG gene may be referred to as the CD105 gene. NCBI accession numbers for human CD105 protein include NP_000109.1, NP_001108225.1, and NP_001265067.1. NCBI accession numbers for human CD105 mRNA include NM_000118.3, NM_001114753.3, and NM_001278138.2.

[0019] A low expression level of CD105 or low expression of CD105 may mean, for example, that the degree of staining with an anti-CD105 antibody is the same as that of cardiomyocytes not stained with an anti-CD105 antibody.

[0020] Furthermore, "high CD105 expression" or "high CD105 expression" may mean that the expression level of CD105 at the mRNA level or protein level is significantly higher than that of control cells, or may mean that the expression level is 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, or 10.0-fold higher than that of control cells. Here, cells previously shown to have low cell proliferation ability can be used as the control cells, as described above.

[0021] The expression level of CD105 at the gene level can be measured, for example, by quantitative real-time PCR, etc. The expression level of CD105 at the protein level can be measured, for example, by cell staining with a fluorescently labeled anti-CD105 antibody and flow cytometer analysis (FACS analysis), etc.

[0022] In the method of this embodiment, the step of selecting cardiomyocytes highly expressing CD105 can be performed, for example, by staining a population of cardiomyocytes with an anti-CD105 antibody and analyzing the result using a flow cytometer, etc. Alternatively, cardiomyocytes with high proliferation potential may be collected using the sorting function of a flow cytometer.

[0023] As described in the Examples section below, we established human induced pluripotent stem cells (hiPSCs) expressing three reporter genes, HAND1, HAND2, and MYH6, and analyzed the expression of these genes during cardiomyocyte differentiation. RNA sequencing (RNA-seq) and EdU analysis revealed that HAND1 expression correlated with cell proliferation. Furthermore, we focused on a cell surface protein specifically expressed in HAND1-positive cells and identified CD105. Therefore, CD105 can be considered a novel marker for proliferative cardiomyocytes.

[0024] To date, no technology has been known for isolating cardiomyocytes exhibiting proliferation potential from hiPSC-derived cardiomyocytes. The method of this embodiment makes it possible to easily obtain cardiomyocytes with high proliferation potential without gene transfer.

[0025] [Method of producing cardiomyocytes] In one embodiment, the present invention provides a method for producing cardiomyocytes, comprising the steps of recovering cardiomyocytes that highly express CD105 from a population of cardiomyocytes and expanding the recovered cardiomyocytes in vitro.

[0026] In the method of this embodiment, the cardiomyocyte population is the same as that described above. Furthermore, the step of recovering cardiomyocytes highly expressing CD105 from the cardiomyocyte population can be performed in the same manner as described above. More specifically, the cardiomyocyte population can be stained with an anti-CD105 antibody, and cardiomyocytes with high proliferation potential can be recovered, for example, using the sorting function of a flow cytometer. Alternatively, cardiomyocytes highly expressing CD105 can be recovered by contacting the cardiomyocyte population with magnetic beads bound to anti-CD105 antibodies, and then recovering the cells bound to the magnetic beads using a magnetic stand.

[0027] The recovered cardiomyocytes are then expanded in vitro. As described in the Examples below, cardiomyocytes highly expressing CD105 have the ability to proliferate. Therefore, they can be cultured to proliferate and increase the cell number. As a result, cardiomyocytes can be prepared in large quantities. Here, "preparing in large quantities" may mean expanding the number of cardiomyocytes at the start of culture to, for example, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0 times the number of cardiomyocytes at the start of culture.

[0028] The method of this embodiment can be described as a method for growing cardiomyocytes, a method for mass-culturing cardiomyocytes, or the like.

[0029] [Method of manufacturing cardiac tissue] In one embodiment, the present invention provides a method for producing cardiac tissue, comprising the steps of recovering cardiomyocytes that highly express CD105 from a population of cardiomyocytes, and culturing the recovered cardiomyocytes to produce cardiac tissue.

[0030] In the method of this embodiment, the cardiomyocyte population is the same as that described above, and the step of recovering cardiomyocytes highly expressing CD105 from the cardiomyocyte population is also the same as that described above.

[0031] The collected cardiomyocytes are then cultured to produce cardiac tissue. Cardiac tissue can be produced, for example, by densely seeding cardiomyocytes on a culture dish to form a monolayer, and then stacking the monolayer cultures to form a multilayer myocardial sheet, or by arranging cardiomyocytes using a 3D printer.

[0032] [Other embodiments] In one embodiment, the present invention provides a method for treating cardiac disease, comprising the step of transplanting an effective amount of cardiomyocytes highly expressing CD105 into the heart of a subject in need thereof. The cardiomyocytes highly expressing CD105 are preferably selected and collected from a population of cardiomyocytes derived from human pluripotent stem cells without gene transfer.

[0033] In one embodiment, the present invention provides cardiomyocytes that highly express CD105 for use in the treatment of cardiac disease. The cardiomyocytes of this embodiment are preferably formulated as a cell medicine. Furthermore, the cardiomyocytes are preferably cells selected and recovered from a population of cardiomyocytes derived from human pluripotent stem cells without gene transfer.

[0034] In one embodiment, the present invention provides use of cardiomyocytes highly expressing CD105 in the manufacture of a cellular medicine for treating cardiac disease. Preferably, the cardiomyocytes highly expressing CD105 are cells selected and collected from a population of cardiomyocytes derived from human pluripotent stem cells without gene transfer. [Example]

[0035] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0036] Materials and Methods (hiPSC culture and differentiation into cardiomyocytes) hiPSCs were cultured on SNL feeder cells in primate ES cell medium (catalog number “#RCHEMD001”, ReproCell) supplemented with 4 ng / mL bFGF (catalog number “#060-04543”, Fujifilm Wako Pure Chemical Industries).

[0037] All differentiation media were based on StemPro34 medium (catalog number #10640-019, Thermo Fisher Scientific) and supplements (catalog number #10641-025, Thermo Fisher Scientific), containing 50 μg / mL ascorbic acid (catalog number #A4544-25G, Sigma), 2 mM L-glutamine (catalog number #25030164, Thermo Fisher Scientific), 150 μg / mL transferrin (catalog number #10652202001, Sigma), 4 × 10 -4 The solution contained M monothioglycerol (catalog number "#M6145-25ML", Sigma) and 0.5% penicillin / streptomycin (catalog number "#15140-122", Thermo Fisher Scientific).

[0038] On day 0 of differentiation induction, hiPSCs were dissociated into single cells by treatment with Accumax (catalog number "#AM-105", Innovative Cell Technologies) at 37°C for 5 minutes.

[0039] The cells were then pipetted into a 15 mL tube with 6 mL of IMDM (1x) (catalog number #12440-053, Thermo Fisher Scientific). The tube was then centrifuged at 800 rpm for 5 minutes, and the pellet was suspended in a medium containing 10 μM Y-27632 (catalog number #036-24023, Fujifilm Wako Pure Chemical Industries), 2 ng / mL BMP4 (catalog number #314-BP, R&D Systems), and Matrigel (day 0 medium). 2 x 10 cells were then plated onto a Hema (catalog number #P3932-25G, Sigma)-coated 96-well round-bottom plate. 4 The cells were seeded at a density of 10 cells / well and allowed to aggregate.

[0040] On day 1 of differentiation induction, 12 ng / mL human recombinant activin A (catalog number "#338-AC-500", R&D Systems), 18 ng / mL BMP4, and 10 ng / mL bFGF (catalog number "#233-FB", R&D Systems) were added to the day 0 medium.

[0041] On day 3 of differentiation, embryoid bodies were collected into 15 mL tubes, dissociated with Accumax, washed with 6 mL of IMDM, and centrifuged. They were then cultured for 4 days in differentiation medium containing 10 ng / mL VEGF (Cat. No. #293-VE, R&D Systems) and 1 μM IWP-3 (Cat. No. #04-0035, Stemgent) to regenerate embryoid bodies.

[0042] On day 7 of differentiation induction, embryoid bodies were collected onto Hema-coated 6-well plates in differentiation medium containing 5 ng / mL VEGF. The medium was then changed every 2–3 days until day 20. During differentiation induction, cells were cultured under hypoxic conditions (5% O2) from days 0–12. Then, on day 12, plates were transferred to normoxic conditions.

[0043] (RNA extraction and quantitative real-time PCR) RNA was purified using QIAZOL reagent and the miRNeasy Micro Kit (Cat. No. #217084, Qiagen). cDNA was then synthesized using ReverTra Ace® (Cat. No. #TRT-101, Toyobo) and poly-T primers or ReverTra Ace® qPCR RT Master Mix with gDNA Remover (Cat. No. #FSQ-301, Toyobo).

[0044] The HAND1 gene was analyzed using a TaqMan probe (catalog number #4331182, Hs02330376_m1, Thermo Fisher Scientific) and TaqMan TMAmplification was performed using Universal Master Mix II with UNG (catalog number #4440044, Thermo Fisher Scientific), and real-time quantitative PCR analysis was performed using StepOne (catalog number #4376374, Thermo Fisher Scientific).

[0045] Gene expression levels were estimated by the ddCt method using the GAPDH gene (catalog number "#4331182", Hs99999905_m1, Thermo Fisher Scientific) or ACTB gene (catalog number "#4331182", Hs0035733_g1, Thermo Fisher Scientific) as an internal control. Statistical analysis of qPCR results was performed using the Welch test using Prism7 (GraphPad).

[0046] (RNA extraction, RNA sequencing and analysis) Cardiomyocytes were separated from the cells on day 20 after the start of differentiation induction by FACS, and total RNA was extracted and purified using the miRNeasy Micro Kit with RNase-Free DNase Set (catalog number "#79254", Qiagen).

[0047] Next, 200 μg of RNA was used to generate libraries using the TruSeq Stranded total RNA with Ribo-Zero Gold LT Sample Prep Kit (Cat. No. 20020598, Illumina) and TruSeq RNA Single Indexes Set A and B (Cat. Nos. 20020492 and 20020493, Illumina).

[0048] Sequencing was performed using the NextSeq 500 / 550 High Output Kit v2 (75 cycles) (catalog number #20024906, Illumina).

[0049] Next, we used cutadapt-1.15 to remove adapter sequences from the reads, and discarded reads shorter than 20 bp. We then used bowtie2 (ver. 2.2.2.5) to map the adapter-stripped reads to human ribosomal RNA and transfer RNA. We then used samtools and bam2fastq to extract unmapped bam files and translate them into fastq files.

[0050] Subsequently, fastq files were aligned to the GRCh38 human reference genome downloaded from the UCSC Genome Browser using STAR (ver. 2.5.4a).

[0051] Read counting and normalization were performed using HTSeq (ver. 0.9.1), DESeq2 (R package), and the iGenome gtf file in R-3.6.1. Additionally, Pheatmap (R package) was used to generate a heatmap of the expression levels of genes listed in "outside the plasma membrane" (GO:0009897) that were shown to be differentially expressed in the four subpopulations by likelihood ratio tests with adjusted P values ​​of <0.05.

[0052] (Sorting of proliferating / non-proliferating cardiomyocytes using anti-CD105 antibody) Embryoid bodies obtained from hiPSCs carrying a cardiomyocyte reporter (e.g., MYH6-EGFP) were first treated with collagenase type I (catalog number "#C-0130", Sigma) for 6–12 h and collected in a 15 mL tube.

[0053] The collagenase was then removed and 1–2 mL of Accumax was added. After incubation at 37°C for 15 minutes, the embryoid bodies were dissociated by gentle pipetting. Then, 6 mL of IMDM was added to the 15 mL tube and the tube was centrifuged at 800 rpm for 5 minutes. After removing the supernatant, the cells were suspended in anti-CD105 antibody (Cat. No. 130-099-125, Miltenyi Biotec) diluted 1:50 in FACS buffer (5% FBS-PBS) and incubated at room temperature for 30 minutes for staining.

[0054] Subsequently, after washing twice with FACS buffer, the cells were suspended in FACS buffer containing DNase (catalog number "#260913", Calbiochem) and DAPI (catalog number "#D1306", Thermo Fisher Scientific).

[0055] The suspended cells were then passed through a filter and placed in a 5 mL FACS tube. The cells were then sorted using a FACS Aria II (Becton Dickinson). First, DAPI-positive cells were removed, followed by EGFP-positive CD105 cells. high or CD105 low Cardiomyocytes were isolated as proliferating and non-proliferating cardiomyocytes, respectively.

[0056] (EdU assay) The EdU assay was performed using Click-iT® EdU Flow Cytometry Assay Kits (catalog number #C10418, Thermo Fisher Scientific).

[0057] The sorted cardiomyocytes were plated onto fibronectin (catalog number "F4759-5MG", Sigma)-coated 6-well or 12-well plates at 6 × 10 5 pcs / well or 2 x 10 5 Two days later, 1 μM EdU was added and the mixture was incubated for 18 hours, and the assay was performed according to the manufacturer's instructions.

[0058] [Experimental Example 1] (Establishment of HAND1-mCherry / HAND2-EGFP double reporter hiPSCs) We established double-reporter hiPSCs using the CRISPR / Cas9 system. First, guide RNAs (gRNAs) were designed near the stop codons of the HAND1 and HAND2 genes, respectively. Each gRNA was then cloned into the pHL-H1-ccdB-mEF1a-RiH vector (catalog number #60601, Addgene).

[0059] In addition, targeting vectors were constructed by knocking in a flag tag, 2A peptide, and mCherry gene at the stop codon of the HAND1 gene. In addition, targeting vectors were constructed by knocking in an HA tag, 2A peptide, and EGFP gene at the stop codon of the HAND2 gene. These targeting vectors contained an antibiotic selection cassette carrying a PGK promoter and a puromycin or neomycin resistance gene between LoxP sequences. These sequences were placed between the homologous arms 1,000 bases upstream and 1,000 bases downstream of the stop codons of the HAND1 and HAND2 genes for homologous recombination.

[0060] Figure 1 is a schematic diagram outlining the construction of HAND1-mCherry reporter hiPSCs. Figure 2 is a schematic diagram outlining the construction of HAND2-EGFP reporter hiPSCs. In Figures 1 and 2, "5arm" indicates the 5' homology arm, "3arm" indicates the 3' homology arm, "2A" indicates the 2A peptide, "flag" indicates the flag tag, "HA" indicates the HA peptide, "PGK" indicates the promoter sequence of phosphoglycerate kinase 1, "PuroR" indicates the puromycin resistance gene, "NeoR" indicates the neomycin resistance gene, "H" indicates the HindIII cleavage site used for Southern blotting, and "Probe" indicates the probe used for Southern blotting. The NCBI accession numbers for the human HAND1 gene mRNA are NM_004821.3, etc., and the NCBI accession numbers for the human HAND2 gene mRNA are NM_021973.3, etc.

[0061] 409B2 hiPSCs were established by episomal culture. 409B2 hiPSCs were cultured on puromycin- and neomycin-resistant STO cells. 50 × 10 hiPSCs were cultured by electroporation (product name "NEPA21" by NEPAGENE). 4 Each cell was simultaneously transfected with 2.5 μg of Cas9 expression vector (pHL-EF1a-SphcCas9-iP-A, catalog number #60599, Addgene), 3 μg of each targeting vector, and 2.5 μg of each of the HAND1 gRNA vector and HAND2 gRNA vector.

[0062] After 48 hours, 0.5 μg / mL puromycin was added and the cells were treated for 5 days. Subsequently, 50 μg / mL neomycin (product name: Geneticin TM Selective Antibiotic (G418 Sulfate) (50 mg / mL), catalog number #10131027, Thermo Fisher Scientific) was added.

[0063] Surviving cells were subsequently cloned, and the knock-in sequences were identified by PCR, and sequences within and outside the homologous arms were sequenced by Southern blotting using HindIII (NEB) and four probes corresponding to the internal and external regions of the knock-in sequences.

[0064] The selection cassette was then removed. Specifically, Matrigel (Corning, Catalog No. #354230) was diluted to 10 μg / mL with DMEM / F-12 (Thermo Fisher Scientific, Catalog No. #11320033) and coated onto a dish. The dish was incubated for 1 hour. Next, hiPSCs were transfected with 1 μg / mL of the Cre expression vector (pCAG-Cre-Blast, provided by Dr. Keisuke Okita) using FuGENE HD Transfection Reagent (Promega, Catalog No. #E2311) and seeded onto the above-mentioned dish.

[0065] Next, hiPSCs were cultured in MEF-conditioned medium and incubated with 10 μg / mL blasticidin (Funakoshi, Catalog No. KK-400) for 2 days. The hiPSCs were then recloned and identified by PCR and Southern blotting.

[0066] [Experimental Example 2] (Establishment of MYH6-iRFP670 / HAND1-mCherry / HAND2-EGFP triple reporter hiPSCs) The MYH6 reporter gene was introduced into the genome of HAND1-mCherry / HAND2-EGFP double reporter hiPSCs.

[0067] Specifically, we constructed a PiggyBac transposon vector (PB-hMYH6-iRFP670-IPPNL) containing the human MYH6 promoter (-4391 to +1051), an iRFP670-IRES-puromycin resistance gene cassette, and a neomycin resistance cassette flanked by LoxP sequences and controlled by the PGK promoter. Figure 3 shows the structure of this transposon vector.

[0068] Next, the transposon vector (PB-hMYH6-iRFP670-IPPNL) and PiggyBac transposase expression vector were transfected using FuGENE HD Transfection Reagent (catalog number "E2311", Promega). Transfected cells were then selected with neomycin (50 μg / mL) to obtain MYH6-iRFP670 / HAND1-mCherry / HAND2-EGFP triple reporter hiPSCs. The NCBI accession number for the human MYH6 mRNA is NM_002471.4.

[0069] [Experimental Example 3] (Establishment of MYH6-EGFP reporter hiPSCs) A transposon vector having the EGFP gene in place of the iRFP670 gene was constructed using the transposon vector whose structure is shown in FIG.

[0070] Next, the transposon vector and PiggyBac transposase expression vector were transfected into 201B7 hiPSCs using FuGENE HD Transfection Reagent (Cat. No. E2311, Promega). Transfected cells were then selected with neomycin (50 μg / mL) to obtain MYH6-EGFP reporter hiPSCs. These MYH6-EGFP reporter hiPSCs express EGFP upon differentiation into cardiomyocytes (MYH6-positive cells).

[0071] [Experimental Example 4] (RNA sequencing analysis) The MYH6-iRFP670 / HAND1-mCherry / HAND2-EGFP triple reporter hiPSCs prepared in Experimental Example 2 were induced to differentiate into cardiomyocytes. - EGFP - , mCherry + EGFP - , mCherry + EGFP + , mCherry - EGFP + iRF670-positive cells (cardiomyocytes) were sorted by FACS and subjected to RNA sequencing. Experiments were performed in triplicate.

[0072] Based on the results of RNA sequencing, we performed gene ontology (GO) analysis. As a result, HAND1-positive cells (mCherry + Furthermore, EdU assay confirmed that the percentage of EdU-positive cells, which indicates that the cell cycle is activated, is higher in HAND1-positive cells than in HAND1-negative cells.

[0073] Next, using RNA sequencing data, we extracted cell surface-exposed genes from the AmiGO database (GO:0009897) and created a heat map for these genes. Figure 4 shows the heat map. The color shading in the heat map corresponds to the Z-score of gene expression. In Figure 4, "ex1," "ex2," and "ex3" represent the results of the first, second, and third experiments, respectively.

[0074] Based on the heatmap shown in Figure 4, cardiomyocytes expressing HAND1 but not HAND2, i.e., mCherry + EGFP - We focused on CD105 as a gene whose expression level in these cells is higher than that in other cells.

[0075] [Experimental Example 5] (EdU assay of CD105-high expressing cardiomyocytes 1) The 201B7 hiPSC-derived MYH6-EGFP reporter hiPSCs prepared in Experimental Example 3 were induced to differentiate into cardiomyocytes. The MYH6-EGFP reporter hiPSCs were cultured on SNL feeder cells.

[0076] Twenty days after the start of differentiation induction, cells were sorted by FACS sorting using anti-CD105 antibody (CD105-APC) to separate high-CD105 expressing cardiomyocytes (top 30%) and low-CD105 expressing cardiomyocytes (bottom 30%). Figure 5 is a graph showing the results of FACS analysis. In Figure 5, the horizontal axis represents EGFP fluorescence intensity, and the vertical axis represents APC fluorescence intensity. The area enclosed by the box marked "High" represents the region of recovered CD105-high expressing cardiomyocytes, and the area enclosed by the box marked "Low" represents the region of recovered CD105-low expressing cardiomyocytes. The "Negative control" shows the results for cells not stained with anti-CD105 antibody.

[0077] Next, an EdU assay was performed on each of the sorted cells. Figure 6 is a graph showing the percentage of EdU-positive cells on day 23 after the start of differentiation induction (n = 3). In Figure 6, "High" indicates the result for cardiomyocytes with high CD105 expression, and "Low" indicates the result for cardiomyocytes with low CD105 expression. In addition, "***" indicates a significant difference (p < 0.001) in the t-test for unpaired samples.

[0078] The results revealed that a significantly higher proportion of EdU-positive cells were present among the CD105-high expressing cells, indicating that cardiomyocytes with high CD105 expression are in an activated cell cycle.

[0079] [Experimental Example 6] (EdU assay of CD105-high expressing cardiomyocytes 2) Different hiPSC lines from those used in Experimental Example 5 were induced to differentiate into cardiomyocytes, and EdU assays were performed on cardiomyocytes with high CD105 expression. The hiPSC lines used were 692D2 and 1390D4. The 692D2 line was cultured on SNL feeder cells. The 1390D4 line was cultured feeder-free.

[0080] From each cell line on day 20 after the start of differentiation induction, lineage-negative, SIRPA-positive, high-CD105-expressing cardiomyocytes (top 30%) and low-expressing cardiomyocytes (bottom 30%) were isolated by FACS sorting using an anti-CD105 antibody (CD105-APC), an antibody against a lineage marker, and an antibody against the cardiomyocyte marker SIRPA (CD172a) (anti-CD172a / b antibody, CD172a / b-PE / Cyanine7, catalog number "323808", BioLegend). Antibodies against lineage markers were used in combination with anti-CD140b antibody (CD104b-PE, catalog number 558821, BD Biosciences), anti-CD49a antibody (CD49a-PE, catalog number 559596, BD Biosciences), anti-CD31 antibody (CD31-PE, catalog number 555446, BD Biosciences), and anti-CD90 antibody (CD90-PE, catalog number 555596, BD Biosciences).

[0081] Next, EdU assays were performed on each of the sorted cells. Figures 7 and 8 are graphs showing the percentage of EdU-positive cells on day 23 after the start of differentiation induction (n = 3). Figure 7 shows the results for 692D2 hiPSCs, and Figure 8 shows the results for 1390D4 hiPSCs. In Figures 7 and 8, "High" indicates the results for cardiomyocytes with high CD105 expression, and "Low" indicates the results for cardiomyocytes with low CD105 expression. Additionally, "*" and "**" indicate significant differences at p<0.05 and p<0.01, respectively, based on the results of a t-test for unpaired samples.

[0082] As a result, it was revealed that a significantly higher proportion of EdU-positive cells was present among the CD105-high expressing cells in hiPSC lines other than 201B7, further supporting the idea that cardiomyocytes with high CD105 expression are in an activated cell cycle.

[0083] [Experimental Example 7] (Examination of HAND1 gene expression in CD105-high expressing cardiomyocytes 1) The 201B7 hiPSC-derived MYH6-EGFP reporter hiPSCs prepared in Experimental Example 3 were induced to differentiate into cardiomyocytes.

[0084] From the cells on day 20 after the start of differentiation induction, EGFP-positive cardiomyocytes with high CD105 expression (top 30%) and low CD105 expression (bottom 30%) were separated by FACS sorting using anti-CD105 antibody (CD105-APC).

[0085] Next, the expression level of the HAND1 gene in each of the sorted cells was measured by quantitative real-time PCR. Figure 9 is a graph showing the results of quantitative real-time PCR. In Figure 9, the vertical axis shows the relative value, where the expression level of the GAPDH gene was used as an internal control and the expression level of the HAND1 gene in cardiomyocytes with low CD105 expression measured by the ddCt method was set to 1. In addition, "**" indicates that there was a significant difference at p<0.01 as a result of a t-test of unpaired samples.

[0086] As a result, it was revealed that HAND1 was significantly expressed at high levels in cells with high CD105 expression. The results in Figure 4 and Figure 9 of Experimental Example 4 showed that cardiomyocytes with high HAND1 expression also had high CD105 expression, and that cardiomyocytes with high CD105 expression also had high HAND1 expression.

[0087] [Experimental Example 8] (Examination of HAND1 gene expression in CD105-high expressing cardiomyocytes 2) Different hiPSC lines from those used in Experimental Example 7 were induced to differentiate into cardiomyocytes, and the expression level of the HAND1 gene in cardiomyocytes with high CD105 expression was examined. The hiPSC lines used were 692D2 and 1390D4. The 692D2 line was cultured on SNL feeder cells. The 1390D4 line was cultured feeder-free.

[0088] From each cell line on day 20 after the start of differentiation induction, lineage-negative, SIRPA-positive, high-CD105-expressing cardiomyocytes (top 30%) and low-expressing cardiomyocytes (bottom 30%) were isolated by FACS sorting using an anti-CD105 antibody (CD105-APC), an antibody against a lineage marker, and an antibody against the cardiomyocyte marker SIRPA (CD172a) (anti-CD172a / b antibody, CD172a / b-PE / Cyanine7, catalog number "323808", BioLegend). Antibodies against lineage markers were used in combination with anti-CD140b antibody (CD104b-PE, catalog number 558821, BD Biosciences), anti-CD49a antibody (CD49a-PE, catalog number 559596, BD Biosciences), anti-CD31 antibody (CD31-PE, catalog number 555446, BD Biosciences), and anti-CD90 antibody (CD90-PE, catalog number 555596, BD Biosciences).

[0089] Next, the expression level of the HAND1 gene for each of the sorted cells was measured by quantitative real-time PCR. Figures 10 and 11 are graphs showing the results of quantitative real-time PCR. In Figures 10 and 11, the vertical axis shows the relative value, where the expression level of the GAPDH gene was used as an internal control and the expression level of the HAND1 gene in cardiomyocytes with low CD105 expression measured by the ddCt method was set to 1. In addition, "**" indicates that there was a significant difference at p<0.01 as a result of a t-test of unpaired samples, and "ns" indicates that there was no significant difference.

[0090] As a result, it was revealed that HAND1 was highly expressed in cells with high CD105 expression in hiPSC lines other than 201B7, further supporting the idea that HAND1 is also highly expressed in cardiomyocytes with high CD105 expression. [Industrial Applicability]

[0091] According to the present invention, a technique for obtaining cardiomyocytes with high proliferation potential can be provided without gene transfer.

Claims

1. A method for producing a cardiac muscle cell comprising the steps of: recovering cardiac muscle cells highly expressing CD105 from a cardiac muscle cell population in vitro; a step of expanding the recovered cardiomyocytes; A method for producing cardiomyocytes, comprising:

2. A method for producing a cardiac muscle cell comprising: recovering cardiac muscle cells that highly express CD105 from a cardiac muscle cell population in vitro; Culturing the collected cardiomyocytes to prepare cardiac tissue; A method for producing cardiac tissue, comprising:

3. The method of claim 1 or 2, wherein the cardiomyocyte population is derived from pluripotent stem cells.

4. The method of claim 3 , wherein the pluripotent stem cells are human iPS cells.

5. A method for selecting cardiomyocytes with high proliferation potential from a population of cardiomyocytes in vitro, comprising: A method comprising a step of selecting cardiomyocytes that highly express CD105 from the cardiomyocyte population.

6. A method for selecting cardiomyocytes in which the cell cycle is activated from a population of cardiomyocytes in vitro, comprising: A method comprising a step of selecting cardiomyocytes that highly express CD105 from the cardiomyocyte population.

7. The method of claim 5 or 6, wherein the cardiomyocyte population is derived from pluripotent stem cells.

8. The method of claim 7, wherein the pluripotent stem cells are human iPS cells.

Images