WO2021241524A1 - Method for selecting cardiomyocytes having high proliferation ability - Google Patents

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

How to select cardiomyocytes with high proliferative capacity

The present invention relates to a method for selecting cardiomyocytes having high proliferative ability. More specifically, the present invention relates to a method for selecting cardiomyocytes having high proliferative ability from a cardiomyocyte population, a method for selecting cardiomyocytes having an activated cell cycle from a cardiomyocyte population, a method for producing cardiomyocytes, and a heart. Regarding the method of manufacturing the tissue. This application claims priority based on US Patent Application No. 63 / 029,564, which was provisionally filed in the United States on May 25, 2020, the contents of which are incorporated herein by reference.

Heart transplantation is often the only treatment option for patients with markedly impaired cardiac function. However, the donor's heart is chronically deficient. Therefore, there is a demand for a treatment method that can replace heart transplantation. Cardiac cell therapy is expected as an alternative to heart transplantation.

However, it is known that cardiomyocytes in adult heart tissue do not have proliferative ability, and cardiomyocytes induced to differentiate in vitro also lose proliferative ability by continuing culturing. Therefore, the development of a technique for increasing the proliferative capacity of cardiomyocytes is being actively carried out (see, for example, Non-Patent Document 1).

By the way, it is known that knockout mice of genes encoding the transcription factors HAND1 and HAND2 show hypoplasia of the left ventricle and the right ventricle, respectively. However, the function and expression of these transcription factors in human cardiac development has not been fully elucidated.

In the method of Non-Patent Document 1, in order to increase the proliferative ability 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 having high proliferative ability without introducing a gene.

The present invention includes the following aspects.
[1] A method for producing cardiomyocytes, which comprises a step of recovering cardiomyocytes highly expressing CD105 from a cardiomyocyte population and a step of expanding and culturing the recovered cardiomyocytes.
[2] A method for producing a heart tissue, comprising a step of recovering cardiomyocytes highly expressing CD105 from a cardiomyocyte population and a step of culturing the recovered cardiomyocytes to prepare a heart tissue.
[3] The production method according to [1] or [2], wherein the cardiomyocyte population is derived from pluripotent stem cells.
[4] The production method according to [3], wherein the pluripotent stem cell is a human iPS cell.
[5] A method for selecting cardiomyocytes having high proliferative ability from a cardiomyocyte population, which comprises a step of selecting cardiomyocytes highly expressing CD105 from the cardiomyocyte population.
[6] A method for selecting cardiomyocytes having an activated cell cycle from a cardiomyocyte population, which comprises a 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 cell is a human iPS cell.

It can also be said that the present invention includes the following aspects.
[P1] A method for selecting cardiomyocytes having high proliferative ability from a cardiomyocyte population derived from pluripotent stem cells, which comprises a step of selecting cardiomyocytes highly expressing CD105 from the cardiomyocyte population. A method in which cardiomyocytes are highly proliferative cardiomyocytes.
[P2] A method for selecting cardiomyocytes having an activated cell cycle from a cardiomyocyte population derived from pluripotent stem cells, which comprises a step of selecting cardiomyocytes highly expressing CD105 from the cardiomyocyte population. A method, wherein the selected cardiomyocytes are cardiomyocytes in which the cell cycle is activated.
[P3] A method for determining the maturity of cardiomyocytes derived from pluripotent stem cells, which comprises a step of measuring the expression level of CD105 in the cardiomyocytes, and the expression level is higher than that of a control. It indicates that the cell cycle of the cardiomyocytes is activated and the maturity as a cardiomyocyte is low, and that the expression level is lower than that of the control means that the activation of the cell cycle of the cardiomyocytes is not observed and the cardiomyocyte is myocardial. A method that indicates a high degree of maturity as a cell.
[P4] A step of recovering cardiomyocytes that highly express CD105 or cardiomyocytes that express CD105 from a population of cardiomyocytes derived from pluripotent stem cells, which is a method for treating heart disease, and the collected cardiomyocytes. CD105 is highly expressed in patients who need to transplant cardiomyocytes with high proliferative capacity or cardiomyocytes with activated cell cycle and low maturity as cardiomyocytes in the process of transplanting to the patient's heart. For patients who need to transplant the cardiomyocytes and transplant the cardiomyocytes with high maturity as cardiomyocytes without activation of the cell cycle, the step of transplanting the cardiomyocytes expressing low CD105. And, including treatment methods.
[P5] Manufacture of cardiomyocytes in large quantities, including a step of recovering cardiomyocytes highly expressing CD105 from a pluripotent stem cell-derived cardiomyocyte population and a step of expanding and culturing the recovered cardiomyocytes in vitro. how to.
[P6] The cells include a step of recovering cardiomyocytes whose cell cycle has been activated from a cardiomyocyte population derived from pluripotent stem cells, and a step of culturing the recovered cardiomyocytes to prepare a heart tissue. Cardiomyocytes whose cycle is activated are cardiomyocytes that highly express CD105, which is a method for producing heart tissue with high efficiency.

According to the present invention, it is possible to provide a technique for obtaining cardiomyocytes having high proliferative ability without performing gene transfer.

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

In this specification, positive may be referred to as "+" and negative may be referred to as "-". Further, being positive means that the expression level of the target gene or the target protein in the cell is high, or the degree to which the cell is stained under a predetermined condition is high. Further, negative means that the expression level of the target gene or the target protein in the cell is low, or the degree to which the cell is stained under a predetermined condition is low.

[Method of selecting cardiomyocytes with high proliferative capacity]
In one embodiment, the present invention provides a method for selecting cardiomyocytes having high proliferative ability from a cardiomyocyte population, which comprises a step of selecting cardiomyocytes highly expressing CD105 from the cardiomyocyte population. .. The selected cardiomyocytes are cardiomyocytes having high proliferative ability.

According to the method of this embodiment, cardiomyocytes having high proliferative ability can be selected without introducing a gene into cardiomyocytes. Since no gene transfer is performed, safe cardiomyocytes that can be transplanted into humans can be easily obtained. The cardiomyocytes selected and collected by the method of the present embodiment can be used for elucidation of the biological mechanism, cell transplantation therapy in the field of regenerative medicine, and the like.

The method of the present embodiment can be rephrased as a method of selecting cardiomyocytes having proliferative ability from a cardiomyocyte population, a method of selecting cardiomyocytes having an activated cell cycle from a cardiomyocyte population, and the like.

In the method of the present embodiment, the cardiomyocyte population may be a living body-derived cardiomyocyte population or may be obtained by inducing differentiation from pluripotent stem cells. The cardiomyocyte population includes cardiomyocytes having high proliferative ability, cardiomyocytes having low proliferative ability, cardiomyocytes having lost proliferative ability, and the like. The method for inducing differentiation of pluripotent stem cells into cardiomyocytes is not particularly limited, and may be a method usually used in the art. The pluripotent stem cells are preferably cells derived from humans, and examples thereof include embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC). In the case of a cell population induced to differentiate from pluripotent stem cells, for example, 8th, 9th, 10th, 11th, 12th, 13th, 14th, and 15th days from the start of differentiation induction. , 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th The day, 29th, 30th, 40th, and 50th days are used.

The proliferative capacity of myocardial cells is high, the myocardial cells have proliferative capacity, or the cell cycle of myocardial cells is activated. For example, the amount of nascent DNA synthesis of myocardial cells is higher than that of control cells. 3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.2 times, 2. 3. times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3.0 times, 4.0 times, 5.0 times, 6. It may be 0 times, 7.0 times, 8.0 times, 9.0 times, 10.0 times higher, and the cell proliferation amount of myocardial cells is 1.3 times, 1 times that of the control cell. 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2 4.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3.0 times, 4.0 times, 5.0 times, 6.0 times, 7 It may be 0.0 times, 8.0 times, 9.0 times, or 10.0 times higher. Here, as the control cell, cells having a low cell proliferation ability can be used, and examples thereof include cardiomyocytes derived from adult heart tissue and cardiomyocytes having a low expression level of CD105. Be done.

The amount of nascent DNA synthesis can be measured, for example, by an EdU assay or the like, which measures the uptake of EdU (5-ethynyl-2'-deoxyuridine), which is a nucleoside analog, into DNA.

CD105 is one of the cell membrane surface proteins encoded by the ENG gene. Hereinafter, the ENG gene may be referred to as a CD105 gene. The NCBI accession numbers for the human CD105 protein are NP_1000109.1, NP_001108225.1, NP_001265067.1, and the like. The NCBI accession numbers of the mRNA of the human CD105 gene are NM_000118.3., NM_00111475.3, NM_001278138.2 and the like.

The low expression level of CD105 or the low expression of CD105 may mean, for example, that the degree of staining with the anti-CD105 antibody is similar to that of cardiomyocytes not stained with the anti-CD105 antibody. ..

Further, a high expression level of CD105 or a high expression level of CD105 may mean that the expression level of CD105 at the mRNA level or the protein level is significantly higher than that of the control cell, or 1.3. Double, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.2 times, 2.3 times Double, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3.0 times, 4.0 times, 5.0 times, 6.0 times It may be times, 7.0 times, 8.0 times, 9.0 times, and 10.0 times higher. Here, as the control cell, a cell whose cell proliferation ability is known to be low can be used, and is the same as that described above.

The expression level of CD105 at the gene level can be measured, for example, by quantitative real-time PCR or the like. 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).

In the method of the present embodiment, the step of selecting cardiomyocytes that highly express CD105 can be performed, for example, by staining the cardiomyocyte population with an anti-CD105 antibody and analyzing it with a flow cytometer or the like. In addition, cardiomyocytes having high proliferative ability may be collected by using the sorting function of the flow cytometer.

As will be described later in the Examples, the inventors established human induced pluripotent stem cells (hiPSC) expressing three reporter genes, HAND1, HAND2 and MYH6, and expressed these genes in the differentiation of myocardial cells. Analyzed. As a result of RNA sequence (RNA-seq) and EdU analysis, it was revealed that the expression of HAND1 correlates with cell proliferation. Furthermore, the inventors focused on cell surface proteins specifically expressed in HAND1-positive cells and identified CD105. Therefore, it can be said that CD105 is a novel marker of proliferative cardiomyocytes.

To date, no technique has been known for separating cardiomyocytes exhibiting proliferative ability from hiPSC-derived cardiomyocytes. By the method of this embodiment, cardiomyocytes having high proliferative ability can be easily obtained without performing gene transfer.

[Method for manufacturing cardiomyocytes]
In one embodiment, the present invention provides a method for producing cardiomyocytes, which comprises a step of recovering cardiomyocytes highly expressing CD105 from a cardiomyocyte population and a step of expanding and culturing the recovered cardiomyocytes in vitro. do.

In the method of this embodiment, the cardiomyocyte population is the same as described above. In addition, 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 the anti-CD105 antibody, and for example, the sorting function of the flow cytometer can be used to recover the cardiomyocytes having high proliferative ability. Alternatively, cardiomyocytes that highly express CD105 may be recovered by contacting the magnetic beads bound with the anti-CD105 antibody with the cardiomyocyte population and then recovering the cells bound to the magnetic beads at a magnetic stand.

Subsequently, the collected cardiomyocytes are expanded and cultured in vitro. As will be described later in the examples, cardiomyocytes that highly express CD105 have proliferative ability. Therefore, it is possible to proliferate and increase the number of cells by culturing. As a result, a large amount of cardiomyocytes can be prepared. Here, to prepare a large amount means to increase the number of myocardial cells at the start of culture, for example, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times. 1.9 times, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times By multiplying by 2.9 times, 3.0 times, 4.0 times, 5.0 times, 6.0 times, 7.0 times, 8.0 times, 9.0 times, 10.0 times It may be there.

The method of this embodiment can be rephrased as a method of proliferating cardiomyocytes, a method of culturing a large amount of cardiomyocytes, and the like.

[Manufacturing method of heart tissue]
In one embodiment, the present invention comprises a step of recovering cardiomyocytes highly expressing CD105 from a cardiomyocyte population and a step of culturing the recovered cardiomyocytes to prepare a heart tissue. Provide a manufacturing method.

In the method of this embodiment, the cardiomyocyte population is the same as described above. Further, the step of recovering cardiomyocytes highly expressing CD105 from the cardiomyocyte population is the same as described above.

Subsequently, the collected cardiomyocytes are cultured to prepare heart tissue. To prepare the heart tissue, for example, a method in which cardiomyocytes are densely seeded on a culture dish and cultured in a single layer, and then the cultures cultured in the single layer are laminated to form a multi-layered myocardial sheet, using a 3D printer. This can be done by a method of arranging cardiomyocytes or the like.

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

In one embodiment, the invention provides cardiomyocytes that highly express CD105 for use in the treatment of heart disease. The cardiomyocytes of the present embodiment are preferably formulated as a cell medicine. Further, it is preferable that the cells are selected and recovered from the cardiomyocyte population derived from human pluripotent stem cells without gene transfer.

In one embodiment, the invention provides the use of cardiomyocytes that highly express CD105 in the manufacture of cell medicines for the treatment of heart disease. The cardiomyocytes that highly express CD105 are preferably cells selected and recovered from the cardiomyocyte population derived from human pluripotent stem cells without gene transfer.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[Materials and methods]
(Culture of hiPSC and induction of 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, Ltd.). ..

All differentiation media are based on StemPro34 medium (catalog number "# 10640-019", Thermo Fisher Scientific) and supplements (catalog number "# 10641-025", Thermo Fisher Scientific), 50 μg / mL ascorbic acid. (Catalog number "# A4544-25G", Sigma), 2 mM L-glutamine (Catalog number "# 25030164", Thermo Fisher Scientific), 150 μg / mL transferase (Catalog number "# 106522012001", Sigma), 4 × 10 ^-4 M monothioglycerol (catalog number "# M6145-25ML", Sigma) and 0.5% penicillin / streptomycin (catalog number "# 1510-1122", Thermo Fisher Scientific) were included.

On the 0th day of differentiation induction, hiPSC was dissociated into single cells by treatment with Accumax (catalog number "# AM-105", Innovative Cell Technologies) at 37 ° C. for 5 minutes.

The cells were then pipetted and collected in a 15 mL tube with 6 mL IMDM (1x) (catalog number "# 12440-053", Thermo Fisher Scientific). Subsequently, the tube was centrifuged at 800 rpm for 5 minutes, and the pellet was subjected to 10 μM Y-27632 (catalog number “# 036-24023”, Fujifilm Wako Pure Chemical Industries, Ltd.), 2 ng / mL BMP4 (catalog number “# 314-BP” R & D). Suspended in medium containing Systems) and Matrigel (day 0 medium) and seeded on a Hema (catalog number "# P3932-25G", Sigma) -coated 96-well round bottom plate at a cell density of ^{2 x 10 4 cells / well.} And aggregated.

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

On the 3rd day of differentiation induction, germ layers were collected in a 15 mL tube, dissociated with Accumax, washed with 6 mL IMDM and centrifuged. Subsequently, the cells were cultured in a differentiation medium containing 10 ng / mL VEGF (catalog number “# 293-VE”, R & D Systems) and 1 μM IWP-3 (catalog number “# 04-0035”, Stemgent) for 4 days, and germ layers were again cultured. Formed a body.

On day 7 of induction of differentiation, germ layers were collected in Hema-coated 6-well plates in differentiation medium containing 5 ng / mL VEGF. Subsequently, the medium was changed every 2 to 3 days until the 20th day. During the induction of differentiation, cells were _{cultured under hypoxic conditions (5% O 2} ) on days 0-12. Subsequently, on day 12, the plates were transferred to normal oxygen conditions.

(RNA extraction and quantitative real-time PCR)
RNA was purified using the QIAZOL reagent and miRNeasy Micro Kit (catalog number "# 217084], Qiagen), followed by RiverTra Ace (R) (catalog number"# TRT-101 ", Toyobo) and poly-T primers, or , RiverTra Ace (R) qPCR RT Master Mix with gDNA Reagent (catalog number "# FSQ-301", Toyobo) was used to synthesize cDNA.

The HAND1 gene uses the TaqMan probe (catalog number "# 4331182", Hs02330376_m1, Thermo Fisher Scientific) and the TaqMan ^TM Universal Master Mix II, with UNG (catalog number "# 4440044, Thermo Fisher Scientific). , StepOne (catalog number "# 4376374, Thermo Fisher Scientific)" was used to perform real-time quantitative PCR analysis.

The gene expression level is determined by the ddCt method using the GAPDH gene (catalog number "# 4331182", Hs99999905_m1, Thermo Fisher Scientific) or the ACTB gene (catalog number "# 4331182", Hs0035733_g1, thermo Fisher scientific) as an internal control. Estimated. Statistical analysis of qPCR results was performed by Welch's test using Prism7 (GraphPad).

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

Subsequently, using 200 μg of RNA, TruSeq Stranded total RNA with Rivo-Zero Gold LT Single Prep Kit (catalog number “# 20020598”, Illumina) and TruSeq RNA Single Index 92 And "# 20020493", Illumina) to make a library.

The sequence was performed using NextSeq 500/550 High Output Kit v2 (75 Cycles) (catalog number "# 200024906", Illumina).

Subsequently, the adapter sequence was removed from the reads using cutapt-1.15, and the reads less than 20 bp were discarded. Subsequently, using bowtie2 (ver. 2.2.2.5), the reads from which the adapter sequence was removed were mapped to human ribosomal RNA and transfer RNA. Subsequently, using samtools and bam2fastq, unmapped bum files were extracted and translated into fastq files.

Subsequently, using STAR (ver. 2.5.4a), the fastq file was aligned with the GRCh38 human reference genome downloaded from the UCSC Genome Browser.

Read counting and normalization were performed using the iGenome gtf files of HTSeq (ver. 0.9.1), DESeq2 (R package) and R-3.6.1. It is also listed in "Outside the Protoplasmic Membrane" (GO: 0009897) by Heatmap (R Package) and is differentially expressed in 4 subpopulations by likelihood ratio test, adjusted P value <0.05. We created a heatmap of the expression level of the genes that were shown to be.

(Sorting of proliferative / non-proliferative cardiomyocytes using anti-CD105 antibody)
Germ layers obtained from hiPSCs with cardiomyocyte reporters (eg, MYH6-EGFP) were first treated with collagenase type I (catalog number "# C-0130", Sigma) for 6-12 hours into 15 mL tubes. collected.

Subsequently, collagenase was removed and 1 to 2 mL of Accumax was added. After incubating at 37 ° C. for 15 minutes, the germ layer was gently pipetted and dissected. Subsequently, 6 mL of IMDM was added to a 15 mL tube and centrifuged at 800 rpm for 5 minutes. After removing the supernatant, the cells were suspended in anti-CD105 antibody (catalog number "# 130-099-125", Miltenyi Biotec) diluted 1:50 with FACS buffer (5% FBS-PBS) and at room temperature. It was incubated for 30 minutes and stained.

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

The suspended cells were then filtered and placed in a 5 mL FACS tube and the cells were sorted using FACS Maria II (Becton Dickinson). First, DAPI-positive cells were removed, and then EGFP-positive CD105 ^high or CD105 ^low cardiomyocytes were isolated as proliferative and non-proliferative cardiomyocytes, respectively.

(EdU Assay)
EdU assays were performed using Click-iT (R) EdU Flow Cytometry Assay Kits (catalog number "# C10418", Thermo Fisher Scientific).

Sorting cardiomyocytes fibronectin (catalog number "# F4759-5MG", Sigma) in 6-well plates or 12-well plates coated were seeded at each 6 × 10 ⁵ cells / well or 2 × 10 ⁵ / well. Subsequently, 2 days later, 1 μM EdU was added and incubated for 18 hours, and the assay was performed according to the instructions.

[Experimental Example 1]
(Establishment of HAND1-mCherry / HAND2-EGFP double reporter hiPSC)
A double reporter hiPSC was established using the CRISPR / Cas9 system. First, guide RNAs (gRNAs) were designed near the stop codons of the HAND1 gene and the HAND2 gene, respectively. Subsequently, each gRNA was cloned into a pHL-H1-cdB-mEF1a-RiH vector (catalog number "# 60601", Addgene), respectively.

In addition, the flag tag, 2A peptide, and mCherry gene were designed to be knocked in to the stop codon of the HAND1 gene, and a targeting vector was prepared. In addition, the HA tag, 2A peptide, and EGFP gene were designed to knock in to the stop codon of the HAND2 gene, and a targeting vector was prepared. These targeting vectors had an antibiotic selection cassette with a PGK promoter and a puromycin or neomycin resistance gene between the LoxP sequences. These sequences were placed between the homologous arms in the regions 1,000 bases upstream and 1,000 bases downstream from the stop codons of the HAND1 and HAND2 genes for homologous recombination.

FIG. 1 is a schematic diagram illustrating an outline of the production of the HAND1-mCherry reporter hiPSC. Further, FIG. 2 is a schematic diagram illustrating an outline of the production of the HAND2-EGFP reporter hiPSC. In FIGS. 1 and 2, "5arm" indicates a 5'homologous arm, "3arm" indicates a 3'homologous arm, "2A" indicates a 2A peptide, "flag" indicates a flag tag, and "HA" indicates a flag tag. "PGK" indicates the promoter sequence of phosphoglycerate kinase 1, "PuroR" indicates the puromycin resistance gene, "NeoR" indicates the neomycin resistance gene, and "H" was used for Southern blotting. Indicates the HindIII cleavage site, where "Probe" indicates the probe used for Southern blotting. The NCBI accession number of the mRNA of the human HAND1 gene is NM_004821.3. The NCBI accession number of the mRNA of the human HAND2 gene is NM_021973.3.

The 409B2 hiPSC established by the episomal method was used. 409B2 hiPSCs were cultured on puromycin-resistant and neomycin-resistant STO cells. By electroporation (product name "NEPA21", NEPAGENE), 2.5 μg Cas9 expression vector (pHL-EF1a-SphcCas9-iP-A, catalog number "# 60599", Addgene) was ^{added to 50 × 10 4 cells.} , 3 μg of each targeting vector, 2.5 μg of HAND1 gRNA vector and HAND2 gRNA vector, respectively, were simultaneously introduced.

After 48 hours, 0.5 μg / mL puromycin was added and 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.

Subsequently, the surviving cells were cloned, the sequence knocked in by PCR was identified, and the nucleotide sequences inside and outside the homologous arm were sequenced. Southern blotting was also performed using HindIII (NEB) and four probes corresponding to the inner and outer regions of the knocked-in sequence.

Subsequently, the selected cassette was removed. Specifically, first, dilute Matrigel (catalog number "# 354230", Corning) with DMEM / F-12 (catalog number "# 1132333", Thermo Fisher Scientific) to 10 μg / mL to make a dish. Put in, incubated for 1 hour and coated. Subsequently, a 1 μg / mL Cre expression vector (pCAG-Cre-Blast, provided by Dr. Keisuke Okita) was introduced into hiPSC using FuGENE HD Transfection Reagent (catalog number “# E2311”, Promega). , Sown in the above-mentioned dish.

Subsequently, hiPSCs were cultured in MEF-conditioned medium, 10 μg / mL blastsaidin (catalog number “# KK-400”, Funakoshi) was added, and the mixture was incubated for 2 days. Subsequently, hiPSCs were cloned again and identified by PCR and Southern blotting.

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

Specifically, the PiggyBac transposon vector (PB-hMYH6-) having a human MYH6 promoter (-4391 to +1051), an iRFP670-IRES-puromycin resistance gene cassette, and a neomycin resistance cassette sandwiched between LuxP sequences and controlled by the PGK promoter. iRFP670-IPPNL) was prepared. FIG. 3 is a schematic diagram showing the structure of the transposon vector.

Subsequently, a transposon vector (PB-hMYH6-iRFP670-IPPNL) and a PiggyBac transposase expression vector were introduced using FuGENE HD Transfection Reagent (catalog number “# E2311”, Promega). Subsequently, cells into which the transposon vector was introduced were selected with neomycin (50 μg / mL) to obtain MYH6-iRFP670 / HAND1-mCherry / HAND2-EGFP triple reporter hiPSC. The NCBI accession number of the mRNA of the human MYH6 gene is NM_002471.4 or the like.

[Experimental Example 3]
(Establishment of MYH6-EGFP reporter hiPSC)
In the transposon vector whose structure is shown in FIG. 3, a transposon vector having an EGFP gene instead of the iRFP670 gene was prepared.

Subsequently, a transposon vector and a PiggyBac transposase expression vector were introduced into 201B7 hiPSC using FuGENE HD Transfection Reagent (catalog number “# E2311”, Promega). Subsequently, the cells into which the transposon vector was introduced were selected with neomycin (50 μg / mL) to obtain a MYH6-EGFP reporter hiPSC. The MYH6-EGFP reporter hiPSC expresses EGFP when differentiated into cardiomyocytes (MYH6-positive cells).

[Experimental Example 4]
(RNA sequence analysis)
The MYH6-iRFP670 / HAND1-mCherry / HAND2-EGFP triple reporter hiPSC prepared in Experimental Example 2 was induced to differentiate into cardiomyocytes. Then, from day 20 cells from the start of differentiation ^{induction, mCherry - EGFP -, mCherry +} EGFP -, mCherry + EGFP +, mCherry - is EGFP ^+, the iRF670 positive cells (cardiomyocytes), Shi fractionated by FACS , Each RNA sequence was performed. The experiment was performed 3 times each.

Gene ontology (GO) analysis was performed based on the results of RNA sequencing. As a result, cell cycle-related terms were enriched in HAND1-positive cells (mCherry ^{+ cells).} Furthermore, when the EdU assay was performed, it was confirmed that the proportion of EdU-positive cells showing that the cell cycle was activated was higher in the HAND1-positive cells than in the HAND1-negative cells.

Subsequently, using the RNA sequence data, genes exposed on the cell surface were extracted from the AmiGO database (GO: 0009897), and heat maps were created for these genes. FIG. 4 is a created heat map. The shades of color in the heatmap correspond to the Z-score of gene expression. In FIG. 4, "ex1", "ex2", and "ex3" indicate that they are the results of the first, second, and third experiments, respectively.

Based on the heat map shown in FIG. 4, the myocardial cells that do not express the hand2 HAND1 is not expressed, i.e. mCherry ⁺ EGFP ^- gene expression in cells, as high gene as compared to other cells, focusing on CD105 bottom.

[Experimental Example 5]
(EdU Assay 1 of CD105 Highly Expressed Cardiomyocytes)
The MYH6-EGFP reporter hiPSC derived from 201B7 hiPSC prepared in Experimental Example 3 was induced to differentiate into cardiomyocytes. The MYH6-EGFP reporter hiPSC was cultured on SNL feeder cells.

CD105 high-expressing cardiomyocytes (top 30%) and low-expressing cardiomyocytes (bottom 30%) were separated from cells 20 days after the start of differentiation induction by FACS sorting using an anti-CD105 antibody (CD105-APC). bottom. FIG. 5 is a graph showing the results of FACS analysis. In FIG. 5, the horizontal axis shows the fluorescence intensity of EGFP, and the vertical axis shows the fluorescence intensity of APC. Further, the region surrounded by the square indicated by "High" indicates the region of the recovered CD105 high-expressing cardiomyocytes, and the region surrounded by the square indicated by "Low" indicates the region of the recovered CD105 low-expressing cardiomyocytes. In addition, "Negative control" shows the results of cells not stained with the anti-CD105 antibody.

Subsequently, an EdU assay was performed on each of the separated cells. FIG. 6 is a graph showing the proportion of EdU-positive cells on the 23rd day from the start of differentiation induction (n = 3). In FIG. 6, "High" indicates the result of CD105 high expression cardiomyocytes, and "Low" indicates the result of CD105 low expression cardiomyocytes. Further, "***" indicates that there is a significant difference at p <0.001 as a result of the t-test of the unpaired sample.

As a result, it was clarified that EdU-positive cells were present in a significantly high proportion among the CD105 high-expressing cells. This result indicates that cardiomyocytes highly expressing CD105 have an activated cell cycle.

[Experimental Example 6]
(EdU Assay 2 of CD105 Highly Expressed Cardiomyocytes)
A hiPSC strain different from Experimental Example 5 was induced to differentiate into cardiomyocytes, and an EdU assay for CD105 high-expressing cardiomyocytes was performed. As the hiPSC strain, 692D2 strain and 1390D4 strain were used. The 692D2 strain was cultured on SNL feeder cells. The 1390D4 strain was cultured in a feeder-free manner.

Anti-CD105 antibody (CD105-APC), antibody against Lineage marker, antibody against myocardial cell marker SIRPA (CD172a) (anti-CD172a / b antibody, CD172a / b-PE /) from each cell on the 20th day from the start of differentiation induction. By FACS sorting using Antibody7, Catalog No. "323808", BioLegend), Lineage-negative SIRPA-positive CD105 high-expressing myocardial cells (upper 30%) and low-expressing myocardial cells (lower 30%) were separated, respectively. Antibodies to the Lineage marker include anti-CD140b antibody (CD104b-PE, catalog number "558821", BD Biosciences), anti-CD49a antibody (CD49a-PE, catalog number "559596", BD Biosciences), and anti-CD31 antibody (CD31-PE). , Catalog number "555446", BD Biosciences), anti-CD90 antibody (CD90-PE, catalog number "555596", BD Biosciences) were mixed and used.

Subsequently, an EdU assay was performed on each of the separated cells. 7 and 8 are graphs showing the proportion of EdU-positive cells on the 23rd day from the start of differentiation induction (n = 3). FIG. 7 is the result of 692D2 hiPSC, and FIG. 8 is the result of 1390D4 hiPSC. In FIGS. 7 and 8, "High" indicates the result of CD105 high expression cardiomyocytes, and "Low" indicates the result of CD105 low expression cardiomyocytes. Further, "*" and "**" indicate that there is a significant difference in p <0.05 and p <0.01 as a result of the t-test of the unpaired sample.

As a result, it was clarified that EdU-positive cells were present in a significantly high rate among the CD105 high-expressing cells even in the hiPSC strains other than 201B7. This result further supports that the cardiomyocytes highly expressing CD105 have an activated cell cycle.

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

EGFP-positive CD105 high-expressing cardiomyocytes (top 30%) and low-expressing cardiomyocytes (bottom 30%) were obtained from cells 20 days after the start of differentiation induction by FACS sorting using an anti-CD105 antibody (CD105-APC). Each was separated.

Subsequently, the expression level of the HAND1 gene was measured by quantitative real-time PCR for each of the separated cells. FIG. 9 is a graph showing the results of quantitative real-time PCR. In FIG. 9, the vertical axis shows a relative value in which the expression level of the HAND1 gene in CD105 low-expressing cardiomyocytes is set to 1 by the ddCt method with the expression level of the GAPDH gene as an internal control. Further, "**" indicates that there is a significant difference at p <0.01 as a result of the t-test of the unpaired sample.

As a result, it was clarified that HAND1 was significantly highly expressed in cells with high expression of CD105. From the results of FIG. 4 and FIG. 9 of Experimental Example 4, it was shown that CD105 was also highly expressed in cardiomyocytes with high expression of HAND1 and that HAND1 was also highly expressed in cardiomyocytes with high expression of CD105.

[Experimental Example 8]
(Examination of HAND1 gene expression in CD105 high-expressing cardiomyocytes 2)
A hiPSC strain different from that of Experimental Example 7 was induced to differentiate into cardiomyocytes, and the expression level of the HAND1 gene in CD105 high-expressing cardiomyocytes was examined. As the hiPSC strain, 692D2 strain and 1390D4 strain were used. The 692D2 strain was cultured on SNL feeder cells. The 1390D4 strain was cultured in a feeder-free manner.

Anti-CD105 antibody (CD105-APC), antibody against Lineage marker, antibody against myocardial cell marker SIRPA (CD172a) (anti-CD172a / b antibody, CD172a / b-PE /) from each cell on the 20th day from the start of differentiation induction. By FACS sorting using Antibody7, Catalog No. "323808", BioLegend), Lineage-negative SIRPA-positive CD105 high-expressing myocardial cells (upper 30%) and low-expressing myocardial cells (lower 30%) were separated, respectively. Antibodies to the Lineage marker include anti-CD140b antibody (CD104b-PE, catalog number "558821", BD Biosciences), anti-CD49a antibody (CD49a-PE, catalog number "559596", BD Biosciences), and anti-CD31 antibody (CD31-PE). , Catalog number "555446", BD Biosciences), anti-CD90 antibody (CD90-PE, catalog number "555596", BD Biosciences) were mixed and used.

Subsequently, the expression level of the HAND1 gene was measured by quantitative real-time PCR for each of the separated cells. 10 and 11 are graphs showing the results of quantitative real-time PCR. In FIGS. 10 and 11, the vertical axis shows a relative value in which the expression level of the HAND1 gene in CD105 low-expressing cardiomyocytes is set to 1 by the ddCt method with the expression level of the GAPDH gene as an internal control. Further, "**" indicates that there is a significant difference at p <0.01 as a result of the t-test of the unpaired sample, and "ns" indicates that there is no significant difference.

As a result, it was clarified that HAND1 is highly expressed in cells with high expression of CD105 even in hiPSC strains other than 201B7. This result further supports the high expression of HAND1 in cardiomyocytes with high expression of CD105.

According to the present invention, it is possible to provide a technique for obtaining cardiomyocytes having high proliferative ability without performing gene transfer.

Claims (8)

1. A step of recovering cardiomyocytes highly expressing CD105 from a cardiomyocyte population, and
   The step of expanding and culturing the collected cardiomyocytes and
   A method for producing cardiomyocytes, including.
2. A step of recovering cardiomyocytes highly expressing CD105 from a cardiomyocyte population, and
   The step of culturing the collected cardiomyocytes to prepare heart tissue, and
   How to make heart tissue, including.
3. The production method according to claim 1 or 2, wherein the cardiomyocyte population is derived from pluripotent stem cells.
4. The production method according to claim 3, wherein the pluripotent stem cell is a human iPS cell.
5. It is a method of selecting cardiomyocytes with high proliferative ability from the cardiomyocyte population.
   A method comprising a step of selecting cardiomyocytes highly expressing CD105 from the cardiomyocyte population.
6. It is a method of selecting cardiomyocytes whose cell cycle is activated from the cardiomyocyte population.
   A method comprising a step of selecting cardiomyocytes highly expressing CD105 from the cardiomyocyte population.
7. The method according to claim 5 or 6, wherein the cardiomyocyte population is derived from pluripotent stem cells.
8. The method according to claim 7, wherein the pluripotent stem cell is a human iPS cell.

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