KR101852304B1 - Method for enhancement of maturation and differentiation efficiency of induced pluripotent stem cell derived cardiomyocytes - Google Patents

Abstract

The present inventors have conducted intensive studies to improve the efficiency of differentiation of stem cell-derived myocardial cells and improve maturity. As a result, not only did the efficiency of differentiation into stem cell-based myocardial cells increased in the NecroX treated group, Can be obtained in a short time, and the present invention has been completed. According to the present invention, the stem cell-derived myocardial cell according to the present invention has a larger and thicker cell size than that of the control group, and is robust and has a dense actin structure. NecroX-induced stem cell- And thus, NecroX treatment can induce high efficiency and high purity differentiation of myocardial cells derived from stem cells. Accordingly, NecroX of the present invention is expected to be useful for establishing cardiac disease model by establishing differentiation of cardiomyocytes in patient-customized pluripotent stem cells as a useful substance that can increase the differentiation efficiency and maturity of differentiated cells.

Description

TECHNICAL FIELD The present invention relates to a stem cell-derived cardiomyocyte differentiation efficiency and an improved pluripotent stem cell derived cardiomyocyte,

The present invention relates to a method for differentiating a high proportion of myocardial cells selectively by a method of inducing differentiation of stem cell-derived cardiomyocytes by treating NecroX, a scavenger of ROS (Reactive Oxygen Species) derived from mitochondria, It is about how to mature.

In Korea, the mortality rate due to cardiovascular disease has been increasing rapidly. Among them, the prognosis of patients with heart failure is worse than that of patients with gastric cancer, so that the development and research of medicines capable of improving the treatment and prognosis of cardiovascular diseases are actively carried out. Myocardial cell damage through myocardial infarction will ultimately lead to heart failure because it reduces the structural and functional capacity of the heart to send the blood to the periphery. Research to replace damaged myocardial cells with healthy myocardial cells to restore damaged myocardium has begun to be studied as a potential method of cardiac regeneration. As a substitute for myocardial cells, adult stem cells such as skeletal myoblasts, various bone marrow-derived stem cells, and cardiac stem cells existing in the heart have been studied. However, these cells have the ability to restore cardiac function It was difficult to acquire a sufficient amount of pure myocardial cells for restoration of normal function of the heart. Since then, the method of differentiating myocardial cells using embryonic stem cells has been rapidly developed, and myocardial cells through embryonic stem cells have been shown to be most functional and structural in terms of their contractility and action potential, like the original myocardial cells, There is an ethical and immune problem for other people to use acquired cardiomyocytes.

However, the development of inducible pluripotent stem cell (iPSC) production method using four genes developed by Professor Yamanaka of Japan in 2006 showed the possibility of new development in the patient-customized cell therapy market and disease development mechanism research The use of inducible pluripotent stem cells as a resource to produce a large number of myocardial cells has also been actively studied. Various studies have been conducted to improve the efficiency of induction of pluripotent stem cells into myocardial cells. In recent years, studies have been actively conducted to obtain a large amount of myocardial cells economically using microcompounds that are not recombinant cytokines for myocardial cell differentiation . In addition, a method of increasing the purity of differentiated myocardial cells by removing other cells outside of myocardial cells when differentiating into pluripotent myocardial cells from induced pluripotent stem cells has been studied. It is mainly a cell sorting method using surface markers of myocardial cells or a selection method using metabolic difference of adult myocardial cells.

Since differentiated myocardial cells exhibit similar properties to neonatal myocardial cells, they do not reflect accurate adult myocardial cells. In the case of a disease, the molecular, structural, and metabolic characteristics of various adult myocardial cells It has been found that it has various reactions. Therefore, if the maturity of myocardial cells for analysis of toxicity and drug effects requiring precise analysis is low, there is a high possibility that errors are involved in obtaining biased results or in deriving accurate results due to different baseline. In 2011, Hom JR developed mitochondrial permeability transition pore (mitochondrial permeability transition pore) of mitochondria in neonatal myocardial cells in the Developmental Cell, resulting in depolarization of the mitochondrial membrane potential to increase the level of ROS (Reactive Oxygen Species) Mature myocardial cells, mPTP is closed, mitochondrial membrane potential is stabilized, and ROS production is reduced.

Meanwhile, NecroX has a role similar to that of normal fibroblast-derived pluripotent stem cells, such as promoting the differentiation of differentiated myocardial cells derived from normal fibroblast-derived pluripotent stem cells as well as the efficiency of differentiated pluripotent stem cells derived from pluripotent stem cells . In addition, NecroX has been used to increase the differentiation efficiency and maturation of smooth muscle cells and muscle cells as well as myocardial cell differentiation, and its utility has been studied not only in myocardial cells but also in other types of cells It is also expected to be wider as it is applied to differentiation.

In addition, although various studies are under way to induce the differentiation of stem cells (see Korean Patent No. 10-1465354), studies on a method for efficiently inducing myocardial cells from stem cells are still insufficient.

The inventors of the present invention conducted extensive studies to improve the efficiency of differentiation of stem cell-derived cardiomyocytes and improve maturity in order to solve the conventional problems as described above. As a result of previous experiments using NecroX, NecroX showed mPTP (NACROX) as a scavenger of ROS (Reactive Oxygen Species) while inhibiting the opening of the mitochondrial membrane and maintaining the mitochondrial membrane potential. The effect of various NecroX functions on stem cell-derived cardiomyocyte differentiation efficiency and maturation was examined. As a result, NecroX-7 (NecX-7, an inhibitor of cytotoxicity of mitochondrial ROS (Reactive Oxygen Species) ) Was treated with myocardial cell differentiation, the efficiency of differentiation into stem cell-based myocardial cells was increased in the NecroX treated group Not only was a, confirmed that it is possible to obtain a more mature cardiomyocytes in a short period of time, and have accomplished the present invention.

Accordingly, it is an object of the present invention to provide a method for differentiating stem cells into myocardial cells including culturing stem cells in a medium containing NecroX.

It is another object of the present invention to provide a composition for inducing differentiation of stem cells into myocardial cells including Necro X.

The present invention also relates to a method of differentiating by a) expressing cardiac Troponin T (cTnT) and a-sarcomeric actin; And b) a pulsatile cell. The present invention also provides a cell therapy agent for treating a heart disease, which contains the myocardial cell as an active ingredient.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to accomplish the above object, the present invention provides a method for differentiating stem cells into myocardial cells including culturing stem cells in a medium containing NecroX.

In addition, the present invention provides a composition for inducing differentiation of stem cells into cardiomyocytes comprising necrox.

In one embodiment of the present invention, the Necro X may be included at a concentration of preferably 5 nM to 500 nM, but is not limited thereto.

 The present invention also relates to a method of differentiating by a) expressing cardiac Troponin T (cTnT) and a-sarcomeric actin; And b) a pulsatile cell. The present invention also provides a cell therapy agent for treating a heart disease comprising the myocardial cell as an active ingredient.

The inventors of the present invention confirmed that treatment of NecroX can induce differentiation of cardiomyocytes with high purity from stem cells more efficiently and completed the present invention. According to the present invention, it is possible to establish the differentiation of myocardial cells in patient-customized induced pluripotent stem cells using NecroX, and furthermore, it is possible to produce a cardiac disease model using the same, thereby contributing to the development of diagnostic techniques and new drugs. In addition, when induction pluripotent stem cells capable of being applied to the human body are developed in the future, it is expected that conditions for conducting clinical trials can be established immediately by using them as a cell therapy agent.

FIG. 1 is a schematic representation of the process of differentiating myocardial cells from stem cells into media and reagents that require treatment in time sequence.
FIG. 2 is a photograph showing a specific type of differentiated cells observed at day 12, which is the end of differentiation from stem cells to myocardial cells, by NecroX (NecX) treatment concentration.
FIG. 3 is a graph showing the results of flow cytometry analysis of the percentage of cTNT-positive cells, which are markers of cardiac myocytes in all differentiated cells, by NecroX (NecX) treatment concentration on the 12th day after the end of differentiation from stem cells to myocardial cells Graph.
Fig. 4 shows a-sarcomeric actin that stains myocardial actin in the control group (Vehicle) and experimental group (NecroX; NecX) at day 22 differentiation from stem cells to myocardial cells, Tom20 which indicates mitochondria, It is the result of immunological staining of DAPI.
FIG. 5 shows the gene expression of calcium channels (RYR2, CAV2.1, SERCA2) in the vehicle (Vehicle) and the experimental group (NecX) on the 22nd day from the stem cell to the myocardial cell differentiation.
FIG. 6 shows the results of the experiment using the vehicle (Vehicle) and the experimental group (NecroX; NecX) on the 22nd day from the stem cell to the myocardial cell using FLUO-4, And the concentration of calcium released (? F / F).
Figure 7 is an illustration of the effect of NecroX (NecX) treatment on the differentiation process of cardiomyocytes in stem cells.

The present inventors have conducted intensive studies to improve the efficiency of differentiation of stem cell-derived myocardial cells and improve maturity. As a result, not only did the efficiency of differentiation into stem cell-based myocardial cells increased in the NecroX treated group, Can be obtained in a short time, and the present invention has been completed.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method of differentiating stem cells into myocardial cells, comprising culturing the stem cells in a medium containing NecroX.

In the present invention, "NecroX" is a low-molecular compound having a strong cytoprotective effect against necrosis and an inflammation-suppressing function, and it has been confirmed that the excessive accumulation of active oxygen and calcium is eliminated, and mitochondrial mPTP It inhibits the opening and maintains the mitochondrial membrane potential of the cells and inhibits oxidative stress as a reactive oxygen species (ROS) scavenger. In addition, actions that inhibit the JNK pathway are known.

Also, in the present invention, "ROS" is an abbreviation of reactive oxygen species, which means active oxygen, and reactive oxygen species refers to all kinds of modified oxygen that damage cells. Active oxygen is not harmful to our body unconditionally. For example, hydroxyl radicals generated as a result of decomposition of hydrogen peroxide in the body act as a disinfectant by indiscriminately attacking pathogens and the like.

The present invention also relates to a composition for inducing differentiation of stem cells into cardiomyocytes comprising NecroX.

The present invention also relates to a method of differentiating by a) expressing cardiac Troponin T (cTnT) and a-sarcomeric actin; And b) a cell that is pulsatile. The present invention also relates to a cell therapy agent for treating a heart disease comprising the myocardial cell as an active ingredient.

 In one embodiment of the present invention, the Wnt inhibitor (CHIR99021) was treated for 2 days, starting with medium lacking insulin, to enhance the differentiation efficiency of stem cell-derived myocardial cells and to improve maturation, (20 ng / ml) was treated for one day, and the day after that, the medium was shaved. The next day, the GSK inhibitor, IWR1, was treated for two days, and two days later, insulin (NecroX) -mediated cardiomyocyte differentiation medium was observed (see Example 1) while changing the medium every two days.

In another embodiment of the present invention, flow cytometry analysis was performed to ascertain myocardial cell marker expressing cells (see Example 2). Immunofluorescent staining was performed to determine the presence of α-sarcomeric actin and mitochondria (Tom 20) (See Example 3). Real-time PCR was performed to examine the expression pattern of the gene in myocardial cells (see Example 4).

In another embodiment of the present invention, in order to measure changes in differentiated myocardial calcium, functional differences of differentiated myocardial cells were compared using FLUO-4, a calcium-specific staining reagent, and NecroX of the present invention It was confirmed that the cardiac muscle cells were induced to increase the calcium channel, so that the heart rate was faster and stronger, and myocardial cells were able to be obtained earlier than the control group and provided more purity myocardial cells (see Example 5).

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1 Establishment of high-efficiency high-purity differentiation method of human dermal fibroblast-induced pluripotent stem cell myocardial cells

In order to improve the differentiation efficiency of stem cell - derived myocardial cells and to improve the maturity, faulted differentiation conditions were established in normal pluripotent stem cells derived from dermal fibroblasts. As shown in Fig. 1, 10 mM of ROCK inhibitor was added to a 35 mm culture dish coated with Matrigel, and induction pluripotent stem cells isolated with single cells were seeded at 2 x ¹⁰ < ⁶ > The cells were changed to mTeSR ^TM 1 (STEMCELL Technologies) until the 35 mm petri dish was filled, and when the inducible pluripotent stem cells were filled in 35 mm culture dishes, they were inoculated into RPMI 1640 medium supplemented with B27 minus insulin (Thermo Fisher Scientific) ), CHIR99021 (Cayman Chemical), which is a GSK-3 inhibitor, was added to a concentration of 6 μM, and then cardiac cell differentiation medium supplemented with 5 nM, 100 nM, and 500 nM of NecroX was prepared. After lightly washing once, 2 ml of the myocardial cell differentiation medium was added and cultured for 24 hours.

Thereafter, the same day, the cells were rinsed with RPMI 1640 medium one time Were replaced with the same myocardial cell differentiation medium supplemented with 5 nM, 100 nM, and 500 nM NecroX, respectively, and cultured for 24 hours. The following day, 10 ng / ml of Activin A (R & D) and 20 ng / ml of bFGF were added to RPMI 1640 medium supplemented with B27 minus insulin, and 5 nM, 100 nM and 500 nM of NecroX were added to each well. After washing once with 1640 medium, the medium was changed and cultured for 24 hours.

On the following day, only 5 nM, 100 nM, and 500 nM of NecroX were added to the RPMI1640 medium supplemented with B27 minus insulin supplement, washed lightly with RPMI 1640 medium, and the medium was ground and cultured for 24 hours. On the next day, each of 5 nM, 100 nM, and 500 nM of NecroX was added to RPMI 1640 medium supplemented with B27 minus insulin. IWR1 (Sigma Aldrich), a 5 μM Wnt inhibitor, was rinsed once with RPMI 1640 lightly, Lt; / RTI > Then, only 5 nM, 100 nM, and 500 nM of NecroX were added to the RPMI1640 medium supplemented with B27 minus insulin supplement. The cells were washed once with RPMI 1640 medium and cultured for 24 hours. The same procedure was repeated the next day. Then, 5nM, 100nM, and 500nM NecroX were added to the medium supplemented with B27 (Thermo Fisher Scientific) supplements and washed lightly once with RPMI 1640, and the medium was ground and cultured for 48 hours. When the medium was supplemented with B27 supplement, the myocardial cells appeared on the day. The number of myocardial cells and the ratio of NecroX concentration were increased.

As a result, as shown in FIG. 2, in the group treated with NecroX, it was possible to more uniformly observe myocardial cells distant from the bottom of the culture dish, and it was confirmed that the differentiation efficiency of myocardial cells was increased.

Example 2. Confirmation of Increase of Myocardial Cell Marker (cTnT) Expressing Cell by Flow Cytometry

In order to confirm the increase of myocardial cell marker expressing cells, flow cytometry analysis was carried out. The dish containing the induced pluripotent stem cells was rinsed twice with PBS, treated with 0.25% Trypsin-EDTA solution, Gathered. The cells collected in the test tube were rinsed with FACS buffer supplemented with PBS + 2.5% FBS (Fetal Bovine Serum) using a centrifuge at 1000 rpm for 3 minutes, the cell pellet was loosened, and the cells were incubated with 1% paraformaldehyde for 10 minutes at room temperature To fix the cells. After the fixation, the cells were rinsed by centrifugation at 1000 rpm for 3 minutes, followed by vortexing, and the cells were permeabilized for 10 minutes at 4 ° C with methanol so that the final concentration was 90%. After perfection, the upper part of the FACS tube was filled with FACS buffer, centrifuged at 1000 rpm for 3 minutes, rinsed with excess methanol, and washed once more with FACS buffer. After the cells were dispensed, cardiac troponin T primary antibody was added at a concentration of 1: 100 and incubated on ice for 30 minutes. FACS buffer was added and centrifuged at 1800 rpm for 3 minutes to remove excess antibody. Each antibody was incubated on ice for 30 minutes in the same manner as that of the second antibody with the corresponding fluorescence at 1: 300. FACS buffer was further added thereto, and the cells were rinsed by centrifugation at 1800 rpm for 3 minutes. The cells were then pelleted and analyzed using a FACS instrument.

As a result, as shown in Fig. 3, in the NecroX treated group, pulsatile myocardial cells were observed as early as 12 days after the differentiation, and when cardiac Troponin T (cTnT) expression was examined through flow cytometry Compared with the control group, the cTnT-positive cells were more increased in the NecroX-treated group, and the increase rate of the positive cells in the 100 nM NecroX-treated group was 5-fold or more.

Example  3. Day 22 of differentiation In myocardial cells  α- sarcomeric  actin and mitochondria ( Tom20 )of Immunofluorescent staining

To perform immunofluorescence staining, the dish containing the induced pluripotent stem cell-derived differentiated myocardial cells was rinsed twice with PBS, and the cold 100% methanol stored at -20 ° C was added to 1 ml of dish, And the cells were allowed to stand for 10 minutes. After 10 min, the cells were removed and the methanol was discarded. The cells were washed three times with 0.05% TBS-T (1 × TBS: Tris-Buffered Saline, 0.05% tween 20) for 5 min each time and the area to be stained was marked with a Dako pen. Then, the blocking solution prepared by dissolving 1% BSA (Bovine Serum Albumin) and 0.05% Triton X-100 in PBS and filtering with a 0.22 μm filter was applied to the area where the cells were not dripped, Blocking process. Next, the primary antibody against α-sarcomeric actin was diluted 1: 100 in the diluent to prepare the primary antibody solution, the blocking solution was removed, the primary antibody solution was treated, and the reaction was carried out at 4 ° C. overnight . The next day, the cells were rinsed with 0.05% TBS-T 3 times for 10 minutes. Then, the secondary antibody solution (1: 100) corresponding to each antibody was prepared so that the cells did not dry, treated with each cell, Lt; / RTI > Then, for the second antibody staining, the first antibody against Tom20 was treated again after the blocking process, and then the staining was carried out through the same procedure. Finally, rinse with 0.05% TBS-T three times for 10 minutes at room temperature. For nuclear staining, 1 mg / ml DAPI (4 ', 6-diamidino-2-phenylindole) After incubation, cells were shaded and reacted at room temperature for 10 minutes. Fluorescence microscopy confirmed that DAPI was stained with nuclei, rinsed 3 times with 0.05% TBS-T, and mounted with a fluorescence mounting medium (DAKO) using a cover slip. After mounting, fluorescence was taken using a laser scanning confocal microscope 710 (Zeiss, Germany) and analyzed with Zeiss Zen software.

As a result of observation with an optical microscope, as shown in Fig. 4, when α-sarcomeric actin staining was performed through immunofluorescence staining at 22 days after differentiation, NecroX-treated differentiated myocardial cells And the pattern of staining of α-sarcomeric actin was also observed to be thicker and more pronounced. Mitochondria stained with Tom20 were observed to be slightly darker in the NecroX treated group than in the NecroX treated group.

Example 4. Investigation of Gene Expression in Myocardial Cells on Day 22 of Differentiation

In order to investigate the expression pattern of the gene in myocardial cells, in order to perform Realtime PCR, the induced pluripotent stem cell differentiated myocardial cells were dissolved in Trizol reagent to obtain RNA. RNA of myocardial cells obtained was quantified and cDNA was synthesized by reverse transcription using 1 μg of RNA. 1 μl of this cDNA product was mixed with SYBR® Green PCR Master Mix and real-time PCR was performed using RYR2, CaV2.1, and SERCA2 primers.

As a result, as shown in FIG. 5, when the expression pattern of the gene was examined at the time of 22 days after the differentiation, the NecroX-treated group showed a higher level of RYR2, CaV2.1, We could observe that the SERCA2 (RYR2; calcium glass ion channel, CaV2.1; L-type calcium channel, SERCA2, a pump that pumps calcium into the sarcomeric reticulum (SR)

Example 5. Measurement of calcium change in differentiated myocardial cells using FLUO-4, a calcium-dyeing reagent

Induced pluripotent stem cell differentiated myocardial cells were rinsed twice with D-PBS and probenecid, an organic anion-transport inhibitor, was added to 1 μM FBS-treated PBS containing 1 mM of FLUO-4, a calcium indicator The resulting solution was treated with the cells. After FLUO-4 treatment, the cells were incubated at 37 ° C for 30 minutes, and then rinsed twice with PBS containing 1% FBS. Then, the cells were changed with an extracellular solution (Nacl, Kcl, HEPES, MgCl ₂ ) containing 1.8 mM CaCl ₂ and 5 mM glucose, incubated at room temperature for 30 minutes, and then the intensity of Ca 2+ was measured with a live imaging device.

As a result, as shown in FIG. 6, when comparing the functional differences of differentiated myocardial cells using FLUO-4, a calcium-specific staining reagent, the NecroX-treated group showed a more regular and higher ΔF / F value Respectively. In other words, it was observed that the amount of calcium reaching the maximum value and the rate of decrease from the maximum value to the base level increased in the NecroX treated group. On the other hand, the time to reach 90% of the maximum calcium emission appeared to decrease in the NecroX treated group.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (6)

A method of differentiating stem cells into myocardial cells, comprising culturing the stem cells in a medium containing NecroX,
Wherein the myocardial cell obtained by the above method is characterized in that the beating is promoted as the intracellular calcium is increased.
The method according to claim 1,
Wherein the necro X is contained at a concentration of 5 nM to 500 nM.
A composition for inducing differentiation of stem cells into myocardial cells, comprising NecroX,
Wherein the myocardial cells are stimulated with an increase in intracellular calcium.
The method of claim 3,
Wherein the necrox is contained at a concentration of 5 nM to 500 nM.
The method according to claim 1,
Wherein the Necro X promotes an increase in calcium in myocardial cells to promote beating.
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