Composition for inducing formation of aggregates of stem cells

Abstract

The present invention relates to a composition for promoting the formation of embryoid bodies of pluripotent stem cells, and a method for promoting the formation of embryoid bodies using same. The present invention, through a simple process of adding an aminopyridine (aminopyrimidine) derivative compound to a culture medium at a specific time point before pluripotent stem cells form embryoid bodies, enables promoting the formation of the embryoid bodies which are important mediators for lineage-specific differentiation, and enables maintaining structural stability so as to enable more efficient differentiation into target cells.

Description

Composition for inducing stem cell aggregate formation

[0001] The present invention relates to a composition for inducing aggregate formation in stem cells comprising an aminopyrimidine derivative compound as an active ingredient, and a method for forming embryonic bodies of pluripotent stem cells using the same.

[0002]

[0003] Pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), can proliferate indefinitely while maintaining the pluripotency to differentiate into all types of somatic cells, making them highly valuable as an unlimited source of cells in cell therapy and regenerative medicine. Accordingly, research is actively being conducted on various differentiation induction methods, methods for isolating differentiated cells, and methods for removing undifferentiated cells to specifically differentiate them into therapeutic immune cells or cells suitable for tissues to be regenerated.

[0004] Meanwhile, immunotherapy utilizing the induction of a patient's immune function is being actively researched to treat various tumors and infectious diseases. In particular, regarding therapies using immune cells, natural killer (NK) cells, a lymphoid cell type that plays a crucial role in the immune response, are receiving significant attention as a therapeutic cell resource. Unlike T cells, NK cells do not require antigen presentation for target cell recognition, and their importance as an immunotherapy agent is being emphasized due to the advantage of being free from graft-versus-host disease (GVHD). While securing a large number of NK cells is necessary to utilize them as effective therapeutic cells, NK cells account for only about 10% of blood cells, and their number, differentiation ability, and function are further reduced, especially in patients, making it practically difficult to secure a therapeutically effective amount.

[0005] NK cells originate from hematopoietic stem cells (HSCs) in bone marrow. Although methods have been reported to induce differentiation into NK cells by isolating hematopoietic stem cells for in vitro culture and treating them with appropriate cytokines, the increase in proliferation rates using currently developed in vitro proliferation methods remains minimal. Consequently, methods to differentiate NK cells from pluripotent stem cells capable of infinite proliferation are being actively researched. However, most methods exhibit limited differentiation efficiency, yield, and purity that are difficult to apply to industrial-scale mass production, making it challenging to secure therapeutic cell resources on an industrial scale. In particular, the culture techniques required to maintain and proliferate undifferentiated human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are not only very demanding but also have the disadvantage of requiring a considerably long time to increase the number of cells, which is the biggest obstacle to obtaining NK cells in large quantities by inducing differentiation from pluripotent stem cells. In most universal differentiation induction techniques using hESCs and hiPSCs, differentiation induction methods involving the production of embryoid bodies (EBs) are utilized. That is, the embryoid body formation stage, in which cells spontaneously aggregate into spherical shapes through in vitro hESC and hiPSC suspension culture, must be completed first, and these embryoid bodies serve as important mediators for lineage-specific differentiation induction. Embryoid bodies produced by conventional methods are very difficult to produce in a homogeneous form with uniform diameter, shape, and cell count, and above all, have been limited by their dependence on the number of starting cells (pluripotent stem cells), which are subject to limited mass supply.

[0006]

[0007] Throughout this specification, numerous papers and patent documents are referenced and cited. The disclosures of the cited papers and patent documents are incorporated by reference into this specification in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention.

[0008]

[0009] The inventors have made diligent research efforts to develop a method for efficiently and stably forming an embryoid body, which serves as a key mediator in the process of inducing lineage-specific differentiation of stem cells, specifically pluripotent stem cells, into specific cells. As a result, the present invention was completed by confirming that when a compound represented by Chemical Formula 1 described below is added to a culture medium of stem cells, the formation of an embryoid body is promoted and structural stability is maintained, thereby enabling efficient differentiation into a target cell without the need for stabilization and adaptation processes after thawing frozen stem cells.

[0010] Therefore, the objective of the present invention is to provide a composition for promoting the formation of aggregates of stem cells, specifically pluripotent stem cells, and a method for promoting the formation of stem cell aggregates using the same.

[0011]

[0012] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings.

[0013]

[0014] According to one aspect of the present invention, the present invention provides a composition for promoting the formation of stem cell aggregates comprising, as an active ingredient, a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof:

[0015] Chemical formula 1

[0016]

[0017] In the above chemical formula, R1 is a C1-C3 alkyl, R2 to R4 are each independently a C1-C3 alkyl, hydrogen, or halogen, and n is an integer from 0 to 2.

[0018] The inventors have made diligent research efforts to develop a method for efficiently and stably forming embryoid bodies, which serve as key mediators in the process of inducing lineage-specific differentiation of stem cells, specifically pluripotent stem cells, into specific cells. As a result, they have discovered for the first time that when an aminopyrimidine derivative compound of Formula 1 is added to the stem cell culture medium at a specific time before embryoid bodies are formed, the spontaneous formation of aggregates of stem cells in suspension culture is significantly promoted, and the structural stability of each cell constituting the aggregate is improved, thereby ultimately maximizing the efficiency of differentiation into the target cell.

[0019] In this specification, the term “stem cell” refers collectively to undifferentiated cells in a stage prior to differentiation into the individual cells constituting a tissue, which possess the ability to differentiate into specific cells under specific differentiation stimuli (environments). Unlike differentiated cells whose cell division has ceased, stem cells are characterized by their ability to self-renewal (produce identical cells through cell division) and possess differentiation plasticity, which allows them to differentiate into various cells depending on the nature of the stimulus when a differentiation stimulus is applied.

[0020] According to a specific embodiment of the present invention, the stem cell is a pluripotent stem cell.

[0021] In this specification, the term “pluripotent stem cell” refers to a stem cell that is in a state of development more advanced than a fertilized egg and possesses pluripotency capable of differentiating into all cells constituting the endoderm, mesenchyme, and ectoderm.

[0022] According to a specific embodiment of the present invention, the pluripotent stem cells used in the present invention are one or more selected from the group consisting of embryonic stem cells (ESC), embryonic germ cells, embryonic carcinoma cells, and induced pluripotent stem cells (iPSC), more specifically are embryonic stem cells or induced pluripotent stem cells, and most specifically are induced pluripotent stem cells.

[0023] In this specification, the term “induced pluripotent stem cell” refers to a pluripotent stem cell artificially induced by inserting a specific gene associated with an undifferentiated or pluripotent phenotype into a non-pluripotent cell (e.g., somatic cell). Induced pluripotent stem cells are considered to have the same phenotype, physiological characteristics, and developmental characteristics as natural pluripotent stem cells, such as embryonic stem cells, in that they possess stem cell gene expression, chromosome methylation, doubling time, embryoid formation, teratoma formation, viability chimera formation, hybridization, and differentiation. Induced pluripotent stem cells can be obtained, for example, by introducing one or more pluripotent inducing transcription factors selected from the group consisting of Oct3 / 4 (octamer-binding transcription factor 3 / 4), Sox2 (sex determining region Y-box 2), c-Myc (cellular myelocytomatosis oncogene), and Klf4 (Kruppel-like factor 4) into somatic cells, but are not limited thereto, and all methods known in the art that can induce an undifferentiated state by causing dedifferentiation of somatic cells can be used.

[0024] In this specification, the term “differentiation of stem cells” includes not only cases where complete differentiation into specific cells is induced from undifferentiated stem cells, but also the formation of precursor cells formed at an intermediate stage prior to complete differentiation from stem cells into specific cells. Accordingly, “target cells differentiated from pluripotent stem cells” encompasses both cells that have completed differentiation into target cells and precursor cells prior to differentiation by culturing the aforementioned pluripotent stem cells, specifically induced pluripotent stem cells, in a microenvironment containing an effective amount of appropriate target cell-specific differentiation-inducing factors.

[0025] For example, when the target cell is a natural killer cell, the differentiation induction medium for this may be a medium containing the components of Table 1 and / or Table 2 described below, for example, but is not limited thereto, and various culture mixtures known in the art to induce differentiation into natural killer cells may be used.

[0026] In this specification, "Natural Killer Cell" refers to an immune cell that differentiates in the bone marrow, lymph nodes, spleen, tonsils, and thymus, and is an innate immune cell that performs the body's primary defense function of eliminating pathogen infections such as viruses and bacteria, as well as abnormal self cells (such as cancer cells). Since the number of natural killer cells naturally present in human blood falls far short of the number of cells required to achieve an effective therapeutic effect, there is an increasing demand for the development of an efficient process to secure a therapeutically effective amount of natural killer cells for use as an effective cell therapy.

[0027] In this specification, "culture medium" or "culture medium" refers collectively to a mixture capable of inducing cell growth and proliferation in vitro by containing sugars, amino acids, minerals, and various nutrients essential for cell growth and proliferation. Cell culture mediums can be optimized according to the characteristics of the cells intended for culture; examples include basic culture media prepared to support cell growth, recombinant protein production media prepared to promote protein production from cells, media for inducing differentiation of undifferentiated cells into specific target cells, and concentrated media made by concentrating nutrients to high concentrations. In the present invention, the medium to which the aforementioned cytokines are added as a cell culture medium for proliferating pluripotent stem cell-derived natural killer cells in vitro may be artificially manufactured or commercially available. Commercially available cell culture media include, for example, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), cellgro SCGM, X-VIVO, and AIM-V, but are not limited thereto.

[0028] In this specification, the term “suspension culture” refers to culturing cells to be cultured in a state where they are floating in a culture medium without being fixed to a substrate or the like. Accordingly, the term “suspension culture” is used with the same meaning as “three-dimensional culture.” Stem cells, which are adhesion-dependent, undergo cell aggregation during suspension culture; cells that are not included in this aggregation and float alone undergo apoptosis and die, so an environment suitable for the cell's adhesion characteristics must be established.

[0029] In this specification, the term “cell aggregate” refers to a cluster of cell aggregates with a three-dimensional structure formed by self-aggregation of cells cultured in an environment, such as suspension culture, where three-dimensional growth is permitted rather than in a monolayer. Cell aggregates produced as a result of three-dimensional culture provide an environment similar to in vivo tissues from which stem cells are derived, and may be spherical or non-spherical in shape depending on their size and the number of self-assembled cells. Spherical cell aggregates are called spheroids, but spheroids do not need to be geometrically perfect spheres.

[0030] According to a specific embodiment of the present invention, the aggregate of the stem cells is an embryoid body. In this specification, the term "embryoid body" refers to a spherical mass of cells derived from pluripotent stem cells produced under suspension culture conditions, and is used as a precursor in the differentiation induction process for lineage-specific differentiation by possessing the potential ability to differentiate into endoderm, mesoderm, and ectoderm. If the efficient formation or proliferation of the embryoid body itself is promoted, the yield of differentiated cells derived from pluripotent stem cells can be dramatically improved.

[0031]

[0032] In this specification, the term “alkyl” means a straight-chain or branched saturated hydrocarbon group, including, for example, methyl, ethyl, propyl, isopropyl, etc. C1-C3 alkyl means an alkyl group having alkyl units having 1 to 3 carbon atoms, and when C1-C3 alkyl is substituted, the number of carbon atoms of the substituent is not included.

[0033] In this specification, the term “halogen” refers to a halogen group element, including, for example, fluoro, chloro, bromo, and iodo.

[0034] According to a specific embodiment of the present invention, R1 is a C1 alkyl, R2 and R4 are halogens, R3 is hydrogen, and n is 1. Most specifically, R2 and R4 are chlorine.

[0035] The compound of Formula 1, in which R1 is C1 alkyl (methyl), R2 and R4 are chlorine, R3 is hydrogen, and n is 1, is an aminopyridine compound having the IUPAC name “6-((2-((4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazole-2-yl)pyrimidine-yl)amino)ethyl)amino)nicotinonitrile” and is also referred to throughout this specification by the abbreviation “CHIR99021”. CHIR99021 is a selective inhibitor of GSK (glycogen synthase kinase) 3 and a Wnt activator, and is a compound that has never been proposed or reported to promote the stable formation of embryos when added to the culture process of pluripotent stem cells, and is proposed for the first time through the present invention for the purpose of promoting embryonic body formation.

[0036] According to a specific embodiment of the present invention, the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof is included in the composition at a concentration of 2 μM to 12 μM. More specifically, at a concentration of 3 μM to 11 μM, even more specifically at a concentration of 4 μM to 11 μM, and most specifically at a concentration of 5 μM to 10 μM.

[0037] According to another specific embodiment of the present invention, the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof is included in the composition at a concentration of 2 μM to 10 μM. More specifically, at a concentration of 3 μM to 8 μM, even more specifically at a concentration of 4 μM to 6 μM, and most specifically at a concentration of about 5 μM.

[0038] According to a specific embodiment of the present invention, the composition of the present invention is added to the culture medium of the stem cells 1 to 3 days before the formation of the stem cell aggregates begins. More specifically, it is added 30 to 66 hours before, more specifically 36 to 60 hours before, even more specifically 42 to 54 hours before, and most specifically about 48 hours before.

[0039] In this specification, the point in time when “stem cell aggregate formation is initiated” means the point in time when appropriate stimulation capable of inducing stem cell aggregate formation begins to be applied, including suspension culture and / or culture in human pluripotent cell expansion (or differentiation) medium. The human pluripotent cell expansion (or differentiation) media that can be used in the present invention are, for example, mTeSR1, mTeSR™ Plus, mTeSR™3D, TeSR™-E8™, TeSR™-E8™3D, TeSR™-AOF, RSeT™ Feeder-Free Medium, StemFit™ Basic03, StemFit™ Basic04, Essential 8 (E8) Medium, Essential 8 Flex, Essential 6 (E6) Medium, NutriStem hPSC XF Medium, PluriSTEM™, StemMACS iPS-Brew XF, Primorigen PluriQ™ hPSC Medium, StemPro hESC SFM, iPS-Brew GMP Medium, ReproFF2, EZStem Medium, STEMdiff™ APEL Medium, STEMdiff™ APEL™2 Medium, StemPro-34 SFM, DMEM / F12 + Supplements, RPMI 1640 + B27 and N2, EGM-2, Neurobasal media, NEuroCult Neural stem cell medium, M15 medium, Keratinocyte Medium, AdipoDuc media, StemSpan™ SFEM II, McCoy's 5A Medium, Alpha MEM + supplements, GyneCult™ Fallopian Tube Organoid Medium and BrainPhys™ Neuronal Medium are available, but are not limited to.

[0040] In this specification, for example, “added to the culture medium of stem cells two days prior to the initiation of aggregate formation” means that the composition of the present invention (or the compound represented by Formula 1 in the composition or a pharmaceutically acceptable salt thereof) is added at a predetermined concentration one to three days prior to the time when stimulation for the formation of aggregates in the stem cells is initiated, and this encompasses a process of maintaining said concentration until the time when stimulation for the formation of aggregates in the stem cells is initiated (for example, in the case of exchanging the medium, a process of including the composition of the present invention or the compound represented by Formula 1 in the composition or a pharmaceutically acceptable salt thereof at the same concentration in the exchanged medium). Accordingly, “added two days prior to the initiation of aggregate formation” is used with the same meaning as “treatment for two days prior to the initiation of aggregate formation.”

[0041]

[0042] According to another aspect of the present invention, the present invention provides a method for promoting the formation of stem cell aggregates comprising the following steps:

[0043] (a) a step of culturing stem cells isolated from an individual or stem cells dedifferentiated from cells isolated from an individual; and

[0044] (b) a step of adding a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof to the culture medium of the stem cells:

[0045] Chemical formula 1

[0046]

[0047] In the above chemical formula, R1 is a C1-C3 alkyl, R2 to R4 are each independently a C1-C3 alkyl, hydrogen, or halogen, and n is an integer from 0 to 2.

[0048] As the stem cells used in the present invention, the aggregates formed therefrom, the compound of Formula 1, and the concentration of the compound used to promote the formation of stem cell aggregates have already been described above, the description thereof is omitted to avoid excessive duplication.

[0049] According to a specific embodiment of the present invention, the method further comprises, prior to step (a), a step of freezing stem cells separated from the individual or stem cells dedifferentiated from cells separated from the individual, and a step of thawing the frozen cells.

[0050] In this specification, the terms “freezing” or “cryopreservation” refer to maintaining cells stably for a short or long period in a low-temperature state, specifically in an ultra-low temperature state of -70°C to -200°C. Since cells generally undergo mutations at a rate of about one in ten thousand during culture, and if cell passages are continued over a long period, the cells may degenerate into a population different from the original cell population, lose their inherent biological activity and function, and the risk of infection by Mycoplasma, etc., increases proportionally with the duration, the method of freezing cells is widely used to preserve the inherent characteristics of the cells. Cell freezing can be performed by treating the cells with a cryoprotectant that minimizes cell damage caused by the formation of ice crystals and imbalances in ions and osmotic pressure, which inevitably accompany the freezing and thawing processes. The freezing medium may contain, for example, dextran, albumin, and / or DMSO to enhance the safety and stability of the cells during freezing.

[0051] In this specification, the term “thawing” refers to the process of raising the temperature of frozen or cryopreserved cells until they become living cells in a soft state so that they can resume life activities. Thawing can typically be carried out rapidly by removing ultra-low temperature frozen cells from a liquid nitrogen tank, etc., and placing them in a constant temperature water bath at 37°C.

[0052] According to a more specific embodiment of the present invention, the method further comprises a step of stabilizing the thawed cells for 15 to 25 days after thawing. More specifically, it further comprises a step of stabilizing for 17 to 23 days, even more specifically for 20 to 22 days, and most specifically for about 21 days.

[0053] According to a specific embodiment of the present invention, the method further includes a step of inducing aggregate formation by culturing the stem cells in three dimensions after step (b).

[0054] According to a more specific embodiment of the present invention, step (b) is performed 1 to 3 days before the step of inducing the formation of aggregations of the stem cells. More specifically, it is performed 30 to 66 hours before, more specifically 36 to 60 hours before, more specifically 42 to 54 hours before, and most specifically about 48 hours before.

[0055] According to a more specific embodiment of the present invention, the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof is added to the culture medium of the stem cells at a concentration of 2 μM to 12 μM. More specifically, it is added at a concentration of 3 μM to 11 μM, even more specifically at a concentration of 4 μM to 11 μM, and most specifically at a concentration of 5 μM to 10 μM.

[0056] According to another specific embodiment of the present invention, the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof is added to the culture medium of the stem cells at a concentration of 2 μM to 10 μM. More specifically, it is added at a concentration of 3 μM to 8 μM, even more specifically at a concentration of 4 μM to 6 μM, and most specifically at a concentration of about 5 μM.

[0057]

[0058] The features and advantages of the present invention are summarized as follows:

[0059] (a) The present invention provides a composition for promoting embryoid body formation of pluripotent stem cells and a method for promoting embryoid body formation using the same.

[0060] (b) The present invention promotes the formation of embryos that serve as important mediators for lineage-specific differentiation and sustains structural stability through a simple process of adding an aminopyrimidine derivative compound to the culture medium at a specific time before pluripotent stem cells form embryos, thereby enabling more efficient differentiation into target cells.

[0061]

[0062] Figure 1 is a schematic diagram summarizing the step-by-step process of forming an embryoid body (EB) from an iPSC and differentiating into an NK cell.

[0063] Figure 2 shows representative images (Figure 2a) showing whether iPSCs form EB and differentiate into target cells (NK cells) among the control group, the CHIR99021 pretreatment group (Day 2), and the CHIR99021 6-day treatment group at various differentiation stages (Day 1, Day 6, Day 13, Day 28); EB images when CHIR99021 was pretreated at various concentrations (0, 5, 10, 20 μM) 1, 2, and 3 days before EB formation (Figure 2b); and the results of measuring NK cell marker expression in each group by flow cytometry (FACS) (Figure 2c).

[0064] Figure 3a shows representative images from Day 1, Day 6, and Day 13 showing the results of inducing differentiation into NK cells in the control group and the CHIR99021 pretreatment group without the mTeSR adaptation process. Figure 3b shows representative images showing the results of EB formation and differentiation into NK cells in two iPSC cell lines (Ff-I14s04 and Ff-I01s04) pretreated with CHIR99021 without the mTeSR adaptation process. Figure 3c shows the results of FACS analysis for NK cell markers after the completion of differentiation in the two iPSC cell lines.

[0065] Figure 4a is an image comparing the degree of EB formation directly formed from iPSCs on Day 6 between the CHIR99021 pretreatment group and the control group not treated with CHIR99021. Figure 4b is a figure showing the FACS analysis results of NK cells differentiated from EB directly formed from iPSCs. Figure 4c shows the results comparing the reproducibility of NK differentiation between each experiment.

[0066]

[0067] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0068]

[0069] Examples

[0070] Experimental method

[0071] iPSC thawing, stabilization, and embryoid body (EB) formation

[0072] In this invention, three iPSC cell lines (Ff-WJs531, Ff-I14s04, Ff-I01s04) provided by the CiRA Foundation were used. The iPSCs were StemFit Basic 03 (Ajinomoto) and iMatrix (Nippi, concentration 0.5 μg / cm³). 2 After thawing, it was stabilized for 3 weeks. 80 ng / mL of bFGF (Basic fibroblast growth factor) was added to the culture medium. The inoculation density for each cell line was 10 cm² for Ff-WJs531. 2 14,000 cells per cell, 10 cm² for Ff-I14s04 2 8,000 cells per 10 cm², and 10,000 cells per 10 cm² for Ff-I01s04.

[0073] To induce embryoid formation, iPSCs were acclimated to mTeSR (STEMCELL Technologies) and Matrigel (Corning), as embryoids cannot be formed directly in StemFit medium. Three weeks after thawing and stabilization, cells were isolated according to standard passage procedures, switched to mTeSR, and maintained for an additional three weeks. Once acclimation was complete, the cells were washed with PBS, separated by TrypLE for 5 minutes, centrifuged, washed with PBS, and resuspended in APEL2 medium supplemented with SCF 40 ng / mL, VEGF 20 ng / mL, BMP4 20 ng / mL, and Y-27632 10 μM. Subsequently, 100 μL of medium was used to inoculate 96-well U-bottom plates at a density of 8,000 cells per well.

[0074]

[0075] EB stabilization through CHIR99021 processing

[0076] To evaluate the effect of CHIR99021 on the structural stability of EB, 5 μM of CHIR99021 was added at various stages of the EB formation process:

[0077] (1) General procedure: While iPSCs were being maintained in mTeSR, CHIR99021 (5 μM) was added two days before EB formation using APEL2 medium.

[0078] (2) Omission of mTeSR adaptation process: After the stabilization period following thawing, CHIR99021 (5μM) was added two days before EB formation using APEL2 medium, and the cells were immediately separated and EB formation was carried out.

[0079] (3) Stabilization after thawing omitted: After thawing, iPSCs were stabilized for one day, then treated with CHIR99021 (5μM) two days before EB formation using APEL2 medium, and then the cells were isolated to induce EB formation.

[0080]

[0081] Experiment comparing the effects of CHIR99021 according to the timing of treatment

[0082] After thawing, iPSCs were stabilized for one day, then treated with CHIR99021 at various concentrations (0, 5, 10, 20 μM) in APEL2 medium 1, 2, and 3 days prior to EB formation, and then the cells were isolated to induce EB formation.

[0083]

[0084] Differentiation into NK cells

[0085] To differentiate EB into NK cells, EB formation was carried out for 6 days and then collected in tubes. After natural sedimentation, the medium was removed, and NK differentiation induction medium containing the basic components summarized in Table 1 below was added. After mixing all reagents, the medium was filtered through a 0.22 μm filter. The additional components summarized in Table 2 were added.

[0086] NK Differentiation Medium (Basic Components) Component Concentration DMEM (High Glucose) 2 parts Ham's F121 part ABSR (Alpha-Bovine Serum Replacement) 20% P / S (Penicillin / Streptomycin) 1% L-Glutamine (200 mM) 2 mM Sodium Acetate (5 μg / mL) 5 ng / mL Ethanolamine (16.6 M) 50 μM Ascorbic Acid (20 mg / mL) 20 mg / L

[0087] Newly Added Components to NK Differentiation Medium Component Concentration β-Mercaptoethanol (55 mM) 25 μMIL-3 (100 μg / mL) 5 ng / mL (first week only) IL-7 (100 μg / mL) 20 ng / mL IL-15 (100 μg / mL) 10 ng / mL LSCF (100 μg / mL) 20 ng / mL LFLT 3L (100 μg / mL) 10 ng / mL

[0088] EB is 10 cm 2 New culture vessels were inoculated with approximately 16 EB cells per cell and cultured until attached to the surface. After one week, the medium was replaced with a medium excluding IL-3. From the third week onwards, the medium was replaced twice a week. After approximately four weeks of NK cell differentiation, suspension cells were collected, and the expression of NK cell markers such as CD3 (negative), CD45, CD56, CD16, NKG2A, NKG2D, and NKp30 was analyzed.

[0089]

[0090] Experimental results

[0091] Improvement of EB stability through CHIR99021 pretreatment

[0092] It was confirmed that pretreatment with CHIR99021 two days prior to EB formation improved EB stability and enhanced NK cell marker expression, thereby promoting efficient differentiation into target cells. Specifically, the effect of CHIR99021 on EB stability during the NK cell differentiation process was evaluated by comparing the control group, the CHIR99021 2-day pretreatment group, and the CHIR99021 6-day treatment group. As a result, the pretreatment group and the 6-day treatment group formed larger EBs than the control group during the EB formation stage on Day 1 and Day 6 (Fig. 2a). On Day 13 of differentiation, both the control group and the pretreatment group showed EBs attached to the culture surface and floating cells, indicating the presence of potential NK cells. In contrast, in the 6-day treatment group, the EBs detached from the culture surface, losing structural integrity, and only floating cells appeared. On Day 28, the experiment was terminated as further differentiation was impossible for the 6-day treatment group. However, the control group and the pretreatment group were able to successfully complete the differentiation process. These results showed that while long-term treatment with CHIR99021 excessively interfered with EB stability and had a negative effect on NK cell differentiation, 2-day pretreatment improved EB stability while maintaining NK cell differentiation ability.

[0093]

[0094] CHIR99021 Searching for the optimal time for preprocessing

[0095] To determine the optimal timing for CHIR99021 treatment to maximize EB formation efficiency, CHIR99021 was applied at concentrations of 0, 5, 10, and 20 μM 1, 2, and 3 days prior to the initiation of EB formation using APEL2 medium, and EB formation was induced while maintaining these concentrations. As a result, as shown in Figure 2b, when CHIR99021 was applied 2 days prior to EB formation (2-day treatment), not only were embryonic bodies with the largest and clearest structures formed at all concentrations, but numerous cystic structures, which serve as markers of active differentiation, were also formed (arrow in Figure 2b), confirming that more efficient differentiation into target cells via EB formation was possible.

[0096]

[0097] Changes in NK cell differentiation activity induced by CHIR99021 pretreatment

[0098] Meanwhile, flow cytometry analysis (FACS) further confirmed that pretreatment with CHIR99021 did not interfere with NK cell differentiation; on the contrary, the expression levels of some NK cell markers in the pretreatment group were higher than those in the control group (Fig. 2c). This increase in marker expression is believed to have resulted from the promotion of the NK cell differentiation process through improved EB stability. These results indicate that while long-term exposure to CHIR99021 (6-day treatment) has a somewhat negative effect on EB stability and NK cell differentiation, 2-day pretreatment improves EB stability and more efficiently induces differentiation into target cells.

[0099]

[0100] Omission of mTeSR adaptation process via CHIR99021 preprocessing

[0101] As a result of verifying whether pretreatment with CHIR99021 could skip the mTeSR adaptation stage during NK cell differentiation, larger and clearer EBs were formed in the CHIR99021 pretreatment group on both Day 1 and Day 6, whereas the control group exhibited incomplete EBs that were smaller and lacked proper morphology, indicating that CHIR99021 pretreatment promoted the formation and stabilization of EBs (Fig. 3a). By Day 13, the EBs of the pretreatment group had properly attached to the culture surface, and floating cells appeared, indicating that the differentiation process had proceeded successfully. In contrast, the control group failed to pass this stage, and the experiment was terminated (Fig. 3a).

[0102] To further verify the reproducibility of the culture method of the present invention using CHIR99021, the same experimental process was repeated on two iPSC cell lines (Ff-I14s04, Ff-I01s04), and as a result, unlike the control group, EB was efficiently formed with CHIR99021 pretreatment in both cell lines and successfully differentiated into NK cells (Fig. 3b).

[0103] In addition, flow cytometry (FACS) results confirmed successful NK cell differentiation, with over 90% of the cells highly expressing various NK markers, including CD56, a major NK cell marker (Fig. 3c). These results indicate that CHIR99021 pretreatment significantly increases EB stability and efficiently induces appropriate EB formation, particularly when the mTeSR adaptation step is omitted, thereby greatly improving the yield of cells targeted for differentiation.

[0104]

[0105] CHIR99021 pretreatment enables EB formation and NK differentiation immediately after iPSC thawing.

[0106] The inventors sought to determine whether normal EB formation was possible without an additional stabilization step after thawing frozen iPSCs when pretreated with CHIR99021. As a result, as shown in Fig. 4a, normal EB formation failed when EB formation was attempted with thawed iPSCs without CHIR99021 pretreatment, whereas intact and stable EBs were successfully formed when CHIR99021 was applied 2 days prior to EB formation. After EB formation in the CHIR99021 pretreatment group, differentiation into NK cells was induced, and flow cytometry confirmed that major NK cell markers were significantly expressed (Fig. 4b).

[0107] To confirm the reproducibility of the above results, the same experiment was repeated one month later using the same iPSC cells, and identical and consistent results were obtained (Fig. 4c). This showed that CHIR99021 pretreatment can efficiently induce EB formation and NK cell differentiation even without stabilization after thawing.

[0108] In conclusion, CHIR99021 pretreatment plays a crucial role in promoting EB formation from iPSCs and significantly improves the structural stability of the cells. Two days of CHIR99021 pretreatment leads to the formation of larger and clearer EBs, which allows for the omission of both mTeSR adaptation and post-thaw stabilization steps. The improved stability provided by CHIR99021 enables more efficient and reliable EB formation, simplifying the process of differentiation into various target cells using pluripotent cells and significantly improving efficiency.

[0109]

[0110] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A composition for promoting the formation of stem cell aggregates comprising, as an active ingredient, a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: Chemical formula 1 In the above chemical formula, R1 is a C1-C3 alkyl, R2 to R4 are each independently a C1-C3 alkyl, hydrogen, or halogen, and n is an integer from 0 to 2.

2. A composition according to claim 1, characterized in that the stem cells are pluripotent stem cells.

3. A composition according to claim 2, characterized in that the pluripotent stem cells are one or more selected from the group consisting of embryonic stem cells (ESC), embryonic germ cells, embryonic carcinoma cells, and induced pluripotent stem cells (iPSC).

4. A composition according to claim 1, characterized in that the aggregate of the stem cells is an embryoid body.

5. A composition according to claim 1, characterized in that R1 is a C1 alkyl, R2 and R4 are halogens, R3 is hydrogen, and n is 1.

6. A composition according to claim 1, characterized in that the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof is included in the composition at a concentration of 2 μM to 12 μM.

7. The composition according to claim 1, characterized in that the composition is added to the culture medium of the stem cells 1 to 3 days before the formation of the stem cell aggregates begins.

8. A method for promoting stem cell aggregate formation comprising the following steps: (a) a step of culturing stem cells isolated from an individual or stem cells dedifferentiated from cells isolated from an individual; and (b) a step of adding a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof to the culture medium of the stem cells: Chemical formula 1 In the above chemical formula, R1 is a C1-C3 alkyl, R2 to R4 are each independently a C1-C3 alkyl, hydrogen, or halogen, and n is an integer from 0 to 2.

9. The method of claim 8, wherein the method further comprises, prior to step (a), a step of freezing stem cells separated from the individual or stem cells dedifferentiated from cells separated from the individual, and a step of thawing the frozen cells.

10. The method of claim 9, wherein the method further comprises the step of stabilizing the thawed cells for 15 to 25 days after thawing.

11. The method of claim 8, wherein the method further comprises the step of inducing aggregate formation by culturing the stem cells in three dimensions after step (b).

12. A method according to claim 11, wherein step (b) is performed 1 to 3 days prior to the step of inducing the formation of aggregations of the stem cells.

13. A method according to claim 8, characterized in that the stem cells are pluripotent stem cells.

14. The method according to claim 11, wherein the pluripotent stem cells are one or more selected from the group consisting of embryonic stem cells (ESC), embryonic germ cells, embryonic carcinoma cells, and induced pluripotent stem cells (iPSC).

15. A method according to claim 8, characterized in that the aggregate of the stem cells is an embryoid body.

16. A method according to claim 8, characterized in that R1 is a C1 alkyl, R2 and R4 are halogens, R3 is hydrogen, and n is 1.

17. The method according to claim 8, characterized in that the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof is added to the culture medium of the stem cells at a concentration of 2 μM to 12 μM.

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