WO2018034519A1 - Method for enhancing differentiation efficiency and maturation level of stem cell-derived cardiac muscle cells by using necrox - Google Patents

Abstract

The present inventors completed the present invention by performing formal research in order to promote differentiation efficiency of stem cell-derived cardiac muscle cells and enhance the maturation level of the same and, as a result, and confirming that, in a group treated with NecroX, the efficiency of differentiation into stem cell-derived cardiac muscle cells is increased and more mature cardiac muscle cells can be obtained in a short period of time. According to the present invention, the stem cell-derived cardiac muscle cells treated with NecroX have a larger and thicker cell size compared to those of control groups, are strong, and display a dense actin structure, thereby indicating that stem cell-derived cardiac muscle cells induced through NecroX treatment can be prepared into cardiac muscle cells with increased maturation level, and thus when NecroX is treated, the differentiation of stem cell-derived cardiac muscle cells can be induced with high efficiency and high purity. Therefore, it is expected that NecroX of the present invention is helpful as a useful material, which can also increase the differentiation efficiency and maturation level of other differentiated cells, for the preparation of a heart disease model by establishing cardiac muscle cell differentiation from patient-specific induced pluripotent stem cells.

Description

Method for Enhancing Differentiation Efficiency and Maturity of Stem Cell-derived Cardiomyocytes Using NECROX

The present invention selectively differentiates a high proportion of cardiomyocytes by a method of inducing differentiation of stem cell-derived cardiomyocytes by treating NecroX, a scavenger of mitochondrial-derived ROS (Reactive Oxygen Species). It is about how to mature.

In Korea, mortality from cardiovascular disease has been increasing rapidly. Among them, the prognosis of patients suffering from heart failure is worse than that of patients with gastric cancer, and the development and research of medicines that can improve the treatment and prognosis of cardiovascular diseases have been actively conducted.

Damage to cardiomyocytes, such as myocardial infarction, eventually leads to heart failure because it reduces the structural and functional capacity of the heart to send blood peripherally. The replacement of damaged cardiomyocytes with healthy cardiomyocytes for the restoration of damaged myocardium has begun to be investigated as a potential method of heart regeneration. As myocardial cell replacement cells, skeletal myoblasts, adult bone marrow-derived stem cells, and cardiac stem cells present in the heart have been studied. However, these cells have the ability to restore the function of the heart compared to the actual cardiomyocytes. It is difficult to obtain a sufficient amount of pure cardiomyocytes for the normal functioning of the heart due to the large number of cell regeneration ability or the limitation of cell regeneration. Since the differentiation of cardiomyocytes using embryonic stem cells has been rapidly developed, cardiomyocytes through embryonic stem cells have received the most attention in terms of functional and structural aspects because they have contractile capacity or activity potential like the original cardiomyocytes. Therefore, there are ethical and immune problems for others to use the acquired cardiomyocytes.

However, the development of a method for producing induced pluripotent stem cells (iPSCs) using four genes developed by Professor Yamanaka of Japan in 2006 suggests new development potential in the market for patient-specific cell therapy and research on disease development mechanism. The use of induced pluripotent stem cells as a resource for producing a large number of cardiomyocytes has also been actively studied. Through various studies, the efficiency of differentiation of induced pluripotent stem cells into cardiomyocytes has been improved. Recently, studies have been actively conducted to economically acquire large amounts of cardiomyocytes by using micro-compounds other than recombinant cytokines for cardiomyocyte differentiation. . In addition, a method of increasing the purity of differentiated cardiomyocytes by removing other cells generated outside the cardiomyocytes during differentiation from induced pluripotent stem cells to cardiomyocytes has been studied. It is mainly a cell salting method using surface markers of cardiomyocytes or a screening method using metabolic differences of adult cardiomyocytes.

Differentiated cardiomyocytes have similar properties to neonatal cardiomyocytes, so they do not reflect accurate adult cardiomyocytes.In case of disease, the molecular, structural, and metabolic characteristics of various adult cardiomyocytes are determined according to the conditions of the disease. It has been found to have various reactions. Therefore, the low maturity of cardiomyocytes for the analysis of toxicity and drug effects that require precise analysis may lead to biased results or different baselines, leading to errors in obtaining accurate results. In 2011, Hom JR et al. Reported that neonatal cardiomyocytes in developmental cells open mitochondrial permeability transition pores (mPTPs) in mitochondria, which depolarizes the mitochondrial membrane potential and increases intracellular reactive oxygen species (ROS) levels. In mature cardiomyocytes, mPTP was closed to stabilize the mitochondrial membrane potential and reduce ROS production.

NecroX plays a role similar to that of normal fibroblast-induced pluripotent stem cells by enhancing the differentiation of normal fibroblast-induced pluripotent stem cell-derived differentiated cardiomyocytes as well as the efficiency of other mesoderm-derived induced pluripotent stem cell cardiomyocyte differentiation. NecroX is used not only for cardiomyocyte differentiation but also for differentiation efficiency and maturation of smooth muscle cells and muscle cells according to further studies. It is expected to be wider as it is also applied to differentiation.

In addition, various studies are underway to induce differentiation of stem cells (see Korean Patent Registration No. 10-1465354), but studies on how to efficiently induce cardiomyocytes from stem cells are still insufficient.

In order to solve the above problems, the present inventors earnestly studied to improve the differentiation efficiency of stem cell-derived cardiomyocytes and to improve maturity, and NecroX mPTP in the previous experiments using NecroX. It has been shown that NecroX contributes to increased expression of calcium channels while inhibiting the opening of cells and maintaining the mitochondrial membrane potential, and inhibiting necrosis as a scavenger of mitochondrial ROS (Reactive Oxygen Species). As a result of examining how these various functions of NecroX affect the differentiation efficiency and maturity of stem cell-derived cardiomyocytes, NecroX-7 (NecX), a scavenger of mitochondrial ROS (Reactive Oxygen Species) When treated during cardiomyocyte differentiation, NecroX-treated group increased the differentiation efficiency of stem cell-based cardiomyocytes. In addition to confirming that more mature cardiomyocytes can be obtained in a short time, the present invention was completed.

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

In addition, another object of the present invention is to provide a composition for inducing differentiation from stem cells containing necrox to cardiomyocytes.

In addition, the present invention is differentiated by the above method to a) express cardiac Troponin T (cTnT) and α-sarcomeric actin; And b) to provide a cardiomyocyte and a cell therapy for the treatment of heart disease containing the cardiomyocytes as an active ingredient, characterized in that the characteristics of the beating cells.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention provides a method for differentiating stem cells into cardiomyocytes comprising culturing stem cells in a medium containing NecroX.

The present invention also provides a composition for inducing differentiation of stem cells from the necrox to cardiomyocytes.

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

 In addition, the present invention is differentiated by the above method to a) express cardiac Troponin T (cTnT) and α-sarcomeric actin; And b) provides a cardiomyocyte and a cell therapy for the treatment of heart disease containing the cardiomyocytes as an active ingredient, characterized in that the characteristics of the beating cells.

The inventors have confirmed that the treatment of NecroX can induce differentiation of high-purity cardiomyocytes more efficiently from stem cells, and completed the present invention. According to the present invention, it is possible to establish the differentiation of cardiomyocytes in patient-specific induced pluripotent stem cells using NecroX, and further, to manufacture a cardiac disease model using the same, which may contribute to diagnosis technology and new drug development. In addition, it is expected that if the induced pluripotent stem cells that can be applied to the human body are developed in the future, the conditions for conducting clinical trials can be created by using them as cell therapeutics.

Figure 1 is a simplified diagram of the process of differentiating cardiomyocytes in stem cells with a medium and a reagent that requires treatment in time order.

Figure 2 is a photograph of the specific morphology of differentiated cells shown at day 12, the end point of differentiation of stem cells to cardiomyocytes by NecroX (NecX) treatment concentration.

Figure 3 is a flow cytometric result showing the ratio of cTNT-positive cells, which are markers of cardiomyocytes, in differentiated cells by necroX (NeX) treatment concentrations and their quantification on day 12, when stem cells differentiated from cardiomyocytes. It is a graph.

 Figure 4 shows a-sarcomeric actin and mitochondria, which stain the myocardium actin in the control group (Vehicle) and experimental group (NecroX; NecX) at day 22 of differentiation of stem cells to cardiomyocytes, and staining the nuclei of cells. Immunostaining results of DAPI.

5 shows the gene expression of calcium channels (RYR2, CAV2.1, SERCA2) in the control group (Vehicle) and the experimental group (NecX) on day 22 of differentiation of stem cells to cardiomyocytes.

FIG. 6 shows regular expression in the differentiated myocardium using FLUO-4, a staining solution that binds to intracellular calcium in experiments using the control group (Vehicle) and the experimental group (NecroX; NecX) on day 22 of differentiation of stem cells to cardiomyocytes. The results show the concentration of calcium released (ΔF / F).

7 is a diagram illustrating the effect of NecroX (NecX) treatment on the differentiation of cardiomyocytes in stem cells.

The present inventors have studied diligently to enhance the differentiation efficiency and improve the maturity of stem cell-derived cardiomyocytes, not only increased the differentiation efficiency from the NecroX treatment group to stem cell-based cardiomyocytes but also more mature cardiomyocytes. Confirmed that can be obtained in a short time, the present invention was completed.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for differentiating stem cells into cardiomyocytes comprising culturing stem cells in a medium containing NecroX.

In the present invention, "NecroX" is a low-molecular compound having a strong cell protection effect against necrosis and a function of inhibiting inflammation, and a mechanism of removing excessively accumulated free radicals and calcium has been confirmed, and mPTP of mitochondria has been confirmed. It inhibits opening and maintains the mitochondrial membrane voltage of the cell and suppresses oxidative stress as a reactive oxygen species (ROS) scavenger. In addition, the action of inhibiting JNK pathyway is known.

In addition, in the present invention, "ROS" is an abbreviation of reactive oxygen species, means active oxygen, and reactive oxygen species refers to all kinds of modified oxygen that damages cells. Free radicals are not harmful to our bodies unconditionally, for example, the hydroxyl radicals generated as a result of the decomposition of hydrogen peroxide in the body indiscriminately attack the pathogens and act as a disinfectant.

The present invention also relates to a composition for inducing differentiation of stem cells from cardiac myocytes containing NecroX.

In addition, the present invention is differentiated by the above method to a) express cardiac Troponin T (cTnT) and α-sarcomeric actin; And b) relates to a cardiomyocyte and a cell therapy for the treatment of heart disease containing the cardiomyocytes as an active ingredient, characterized in that the characteristics of the beating cells.

 In one embodiment of the present invention, in order to enhance the differentiation efficiency of stem cell-derived cardiomyocytes and improve maturity, starting with a medium containing no insulin, after treatment with Wnt inhibitor (CHIR99021) for 2 days, 10ng / ml concentration Activin A and its double amount of bFGF (20ng / ml) were treated for one day, the medium was changed after one day, the next day the GSK inhibitor IWR1 was treated for two days, then two days later insulin The medium was changed once every two days with the medium containing this, and the cardiomyocyte differentiation medium to which NecroX was added was observed (see Example 1).

In another embodiment of the present invention, flow cytometry was performed to confirm the increase of cardiomyocyte marker expressing cells (see Example 2), and immunofluorescent staining was performed on α-sarcomeric actin and mitochondria (Tom20) in cardiomyocytes. (See Example 3), and Realtime PCR was performed to investigate the gene expression pattern in the cardiomyocytes (see Example 4).

In another embodiment of the present invention, in order to measure the change in calcium in differentiated cardiomyocytes, the functional differences of differentiated cardiomyocytes were compared using calcium-specific staining reagent FLUO-4, and NecroX of the present invention was differentiated. It was confirmed that the cardiomyocytes obtained by inducing an increase in the calcium channel of the cardiomyocytes and having a faster and stronger pulsation rate can be obtained earlier than the control group and provide higher purity cardiomyocytes (see Example 5).

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

EXAMPLE

Example 1 Establishment of a Highly Efficient, High-Purity Differentiation Method of Human Skin Fibroblast-Induced Pluripotent Stem Cell Cardiomyocytes

In order to enhance the differentiation efficiency of stem cell-derived cardiomyocytes and to improve maturity, monolayer differentiation conditions were established in normal dermal fibroblast-derived induced pluripotent stem cells. As shown in FIG. 1, ROCK inhibitor was added to a Matrigel-coated 35 mm culture dish at a concentration of 10 μM, and seeded induced pluripotent stem cells, which were separated into single cells using accutase, were seeded into 2 × 10 ⁶ cells. Change to mTeSR ^TM 1 (STEMCELL Technologies) until the cells fill the 35 mm petri dish, and when the induced pluripotent stem cells fill the 35 mm petri dish, RPMI 1640 medium with B27 minus insulin (Thermo Fisher Scientific) supplement (Thermo Fisher Scientific) ) Was added to the GSK-3 inhibitor CHIR99021 (Cayman Chemical) to a concentration of 6μM, and then the cardiomyocyte differentiation medium with 5nM, 100nM, and 500nM of NecroX was also prepared, and RPMI 1640 medium was prepared. After washing lightly once, 2 ml of the above-mentioned cardiomyocyte differentiation medium was incubated for 24 hours.

After that, the next day, wash lightly once with RPMI 1640 medium. The same cardiomyocyte differentiation medium to which 5 nM, 100 nM, and 500 nM of NecroX were added was changed and incubated for 24 hours. The next day, 10 ng / ml Activin A (R & D) and 20 ng / ml bFGF were added to RPMI1640 medium with B27 minus insulin supplement and 5 nM, 100 nM, and 500 nM NecroX were added as in the previous experiment. After washing lightly with RPMI 1640 medium, the medium was changed and incubated for 24 hours.

The next day, only 5nM, 100nM, and 500nM of NecroX were added to RPMI1640 medium containing B27 minus insulin supplement, washed once with RPMI 1640 medium, and the medium was incubated for 24 hours. Next day, 5 nM, 100 nM, and 500 nM of NecroX were added to RPMI1640 medium containing B27 minus insulin supplement, and 5 μM of Wnt inhibitor IWR1 (SigmaAldrich) was added. Incubated for Thereafter, only 5 nM, 100 nM, and 500 nM of NecroX were added to RPMI1640 medium containing B27 minus insulin supplement, washed lightly once with RPMI 1640 medium, and the medium was ground for 24 hours, and the same process was repeated the next day. Thereafter, 5 nM, 100 nM, and 500 nM of NecroX were added to a medium containing B27 (Thermo Fisher Scientific) supplement, washed briefly with RPMI 1640, and the medium was ground and incubated for 48 hours. In the medium containing B27 supplementation, cardiomyocytes appeared on the day, and the heart rate and the rate of cardiomyocytes were increased depending on NecroX concentration.

As a result, as shown in Figure 2, in the NecroX-treated group was able to observe the cardiomyocytes more uniformly from the bottom of the culture dish, it was confirmed that the differentiation efficiency of the cardiomyocytes increased.

Example 2 Confirmation of Increase of Cardiomyocyte Marker (cTnT) Expressing Cells by Flow Cytometry

Flow cytometry was performed to confirm the increase of cardiomyocyte marker expressing cells. The dishes with differentiated cardiomyocytes derived from induced pluripotent stem cells were rinsed twice with PBS and treated with 0.25% Trypsin-EDTA solution to transfer the cells into FACS tubes. Collected. Rinse the cells collected in the test tube using a centrifuge at 1000 rpm for 3 minutes in FACS buffer containing PBS + 2.5% FBS (Fetal bovine serum), release the cell pellet well, and then wait for 10 minutes at room temperature with 1% paraformaldehyde in final concentration. Fixed cells. After fixation, FACS buffer was added, the cells were rinsed by centrifugation at 1000 rpm for 3 minutes, and the cells were permeabilized at 4 ° C. for 10 minutes with Methanol added slowly to a final concentration of 90% while vortexing. After permeabilization, the FACS buffer was filled up to the top of the FACS tube, centrifuged at 1000 rpm for 3 minutes, the excess methanol was rinsed off, and the cells were washed once more with FACS buffer. After dispensing the cells, the cells were incubated for 30 minutes on ice with a cardiac troponin T primary antibody at a concentration of 1: 100, and then the extra antibodies were removed by centrifugation at 1800 rpm for 3 minutes with FACS buffer. Each antibody was incubated for 30 minutes on ice in the same manner, and the secondary antibody with the corresponding fluorescent substance 1: 300 was again filled with FACS buffer, and the cells were rinsed by centrifugation at 1800 rpm for 3 minutes. The cell pellet was then well released and analyzed using a FACS instrument.

As a result, as shown in Figure 3, NecroX-treated group was observed beating cardiomyocytes as early as 12 days after the differentiation, when the expression of cardiac Troponin T (cTnT), a cardiomyocyte marker through flow cytometry Compared with the control group, the NecroX-treated group showed more cTnT-positive cells. In the 100-NM NecroX-treated group, the percentage of positive cells increased more than five times.

Example  3. Day 22 of Eruption In cardiomyocytes  α- sarcomeric  actin and mitochondria ( Tom20 )of Immunofluorescence Staining

To carry out immunofluorescence staining, the dish containing induced pluripotent stem cell-derived differentiated cardiomyocytes was rinsed twice with PBS and cold 100% methanol stored at −20 ° C. was added to 1 ml dish and immediately the cells were treated at −20 ° C. Transfer was allowed to stand for 10 minutes to fix the cells. After 10 minutes, the cells were removed, discarded methanol, washed three times with 0.05% TBS-T (1XTBS: Tris-Buffered Saline, 0.05% tween 20) three times for 5 minutes, and then circled with Dako pen on the area to be stained. Then, a blocking solution prepared by dissolving 1% BSA (Bovine Serum Albumin) and 0.05% Triton X-100 in PBS and filtered through a 0.22 μm filter was covered with the cells in a dry place, and then allowed to stand at room temperature for 30 minutes. The blocking procedure was carried out. Next, the primary antibody against α-sarcomeric actin was diluted 1: 100 in a diluent to prepare a primary antibody solution, the blocking solution was removed, the primary antibody solution was treated, and then reacted at 4 ° C. overnight. . The next day, after rinsing three times with 0.05% TBS-T for 10 minutes three times, a secondary antibody solution (1: 100) corresponding to each antibody was prepared so as not to dry, and then treated to each cell, and then shaded at room temperature for 2 hours. Reacted for a while. Then, the second antibody staining was performed again from the blocking process to treat the primary antibody against Tom20, and then staining was performed through the same process. Finally, rinse three times for 10 minutes at room temperature with 0.05% TBS-T, and then mix 1 mg / mL DAPI (4 ', 6-diamidino-2-phenylindole) solution with dilutions to 1: 1000 for nuclear staining. The cells were placed in a cell, shielded, and reacted at room temperature for 10 minutes. After confirming that DAPI was stained in the nucleus by fluorescence microscope, rinsed three times with 0.05% TBS-T, and then mounted with a fluorescence mounting medium (DAKO) using a cover slip. After mounting, fluorescence was performed using a Laser scanning confocal microscopy 710 (Zeiss, Germany) and analyzed by Zeiss Zen software.

As a result of observing with light microscopy, as shown in FIG. 4, the differentiation of α-sarcomeric actin through immunofluorescence staining at 22 days after differentiation resulted in differentiation cardiomyocytes of NecroX-treated group compared to vehicle group. It was observed that the larger size and the pattern of α-sarcomeric actin staining tend to be thicker and more pronounced. Mitochondria stained with Tom20 showed little difference between the two groups, but the mycochondria of the NecroX-treated group were observed to be thicker and increased in volume.

Example 4. Investigation of gene expression in cardiomyocytes on day 22 of differentiation

To investigate the gene expression patterns in cardiomyocytes, RNA was obtained by melting differentiated cardiomyocytes derived from induced pluripotent stem cells with Trizol reagent. RNA of the obtained cardiomyocytes 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 realtime PCR was performed using primers of RYR2, CaV2.1, and SERCA2.

As a result, as shown in Figure 5, when the gene expression pattern was examined 22 days after differentiation, NecroX-treated group RYR2, CaV2.1, and genes related to intracellular calcium handling compared to the control group SERCA2 (RYR2; calcium free ion channel in SR membrane, CaV2.1; L-type calcium channel, SERCA2; pump that injects calcium into sarcomeric reticulum (SR)) was observed.

Example 5 Measurement Results of Calcium Changes in Differentiated Cardiomyocytes Using Calcium Staining Reagent FLUO-4

After rinsing the induced pluripotent stem cell-derived differentiated cardiomyocytes twice with D-PBS, the calcium indicator FLUO-4 was added to the final concentration of 1uM in PBS containing 1% FBS treated with 1mM of organic anion-transport inhibitors. The solution entered was treated with cells. After incubation at 37 ° C. for 30 minutes with FLUO-4 treatment, the cells were rinsed twice with PBS containing 1% FBS. Afterwards, the cells were changed to extracellular solutions (Nacl, Kcl, HEPES, MgCl ₂ ) containing 1.8 mM CaCl ₂ and 5 mM glucose, incubated for 30 minutes at room temperature, and the intensity change of Ca2 + was measured by a live imaging device.

As a result, as shown in Figure 6, even when comparing the functional differences of differentiated cardiomyocytes using calcium-specific staining reagent FLUO-4, NecroX treated group was more regular and higher ΔF / F value than the control group Showed. In other words, it was observed that the amount of calcium reaching the maximum and the rate of decrease from the maximum to the basal level were increased in the NecroX-treated group. On the other hand, the time to reach 90% of the maximum calcium emissions appeared to be reduced in the NecroX treated group.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Treatment of NecroX according to the present invention can induce differentiation of highly purified cardiomyocytes from stem cells more efficiently. According to the present invention, differentiation of cardiomyocytes from patient-specific induced pluripotent stem cells using NecroX according to the present invention. Once established, it is expected to be able to produce heart disease models using it, which will contribute to diagnostic technology and new drug development.

Claims (6)

1. A method for differentiating stem cells into cardiomyocytes comprising culturing stem cells in a medium containing NecroX.
2. The method of claim 1,

   The necrox is characterized in that it is included at a concentration of 5nM to 500nM.
3. A composition for inducing differentiation from stem cells to cardiomyocytes, including necrox.
4. The method of claim 3,

   The necrox, characterized in that contained in a concentration of 5nM to 500nM, the composition.
5. Cardiomyocytes, characterized in that differentiated by the method of claim 1 or claim 2 having the following characteristics:

   a) expressing cardiac Troponin T (cTnT) and α-sarcomeric actin; And

   b) beating cells.
6. Cell therapy for heart disease containing the cardiomyocyte of claim 5 as an active ingredient.

Images