Method for producing mesenchymal stem cells from human pluripotent stem cells and mesenchymal stem cells produced thereby

Abstract

The present invention relates to a method for producing mesenchymal stem cells from human pluripotent stem cells, and more particularly, to a method for producing mesenchymal stem cells by differentiating from embryoid bodies of a certain size in a xeno-free and serum-free environment, thereby improving safety and maintaining the characteristics of mesenchymal stem cells for a long period of time. The method for producing mesenchymal stem cells from human pluripotent stem cells according to the present invention eliminates the problem of contamination with exogenous animal-derived substances through a feeder cell-free, xeno-free, and serum-free culture environment, producing safe mesenchymal stem cells. At the same time, it forms mature embryoid bodies of a uniform shape and size using spherical embryoid bodies, which has the revolutionary effect of improving the efficiency of differentiation into mesenchymal stem cells and stably maintaining the characteristics of mesenchymal stem cells even after long-term subculture of 20 or more passages. This enables mass production of mesenchymal stem cells derived from human pluripotent stem cells, which is useful for commercializing safe and efficient cell therapy agents.

Description

[Technical Field]

[0001] The present invention relates to a method for producing mesenchymal stem cells from human pluripotent stem cells, and more particularly to a method for producing mesenchymal stem cells that are safer and maintain the characteristics of mesenchymal stem cells for a long period of time even after multiple subcultures, through the formation of mature embryoid bodies of uniform morphology and size using a xeno-free, serum-free mesenchymal stem cell production environment and spherical embryoid bodies. [Background technology]

[0002] Stem cells are cells that can differentiate into various cells that make up biological tissues. They are a collective term for undifferentiated cells in the pre-differentiation stage that can be obtained from various tissues of embryos, fetuses, and adults. Stem cells differentiate into specific cells in response to differentiation stimuli (environment). Unlike cells that have completed differentiation and stopped cell division, stem cells have the ability to self-renew and proliferate through cell division. They are also characterized by their plasticity in differentiation, being able to differentiate into different cells in response to different environments or different differentiation stimuli.

[0003] Stem cells are classified into pluripotent, multipotent, and unipotent stem cells based on their differentiation potential. Pluripotent stem cells are pluripotent cells that have the potential to differentiate into all cell types, and include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Multipotent and / or unipotent stem cells include adult stem cells.

[0004] Embryonic stem cells are formed from the inner cell mass during the blastocyst stage, an early stage of embryonic development. They have the potential to differentiate into all types of cells and can differentiate into any tissue cell. They can also be cultured in an immortal, undifferentiated state. Unlike adult stem cells, they can also produce germ cells, making them inheritable to the next generation (Thomson et al., Science, 282:1145-1147, 1998; Reubinoff et al., Nat Biotechnol, 18:399-404, 2000).

[0005] Human embryonic stem cells are produced by isolating and culturing only the inner cell mass during the formation of a human embryo, but the human embryonic stem cells currently produced worldwide are derived from frozen embryos left over after sterilization. Various attempts have been made to use human embryonic stem cells, which have the pluripotency to differentiate into all cell types, as cell therapy agents, but the current situation is that the risk of cancer development and the high barrier of immune rejection have not yet been fully overcome.

[0006] Meanwhile, induced pluripotent stem cells (iPSCs), which are included in the concept of pluripotent stem cells, are cells that have been reverted to a pluripotent stem cell state, an early stage of differentiation, by dedifferentiating adult cells through various methods. To date, it has been reported that dedifferentiated cells exhibit almost the same characteristics as embryonic stem cells, which are pluripotent stem cells, in terms of gene expression and differentiation potential. However, even in the case of iPSCs, although the risk of immune rejection can be eliminated by using autologous cells, the risk of cancer development remains a challenge that must be addressed.

[0007] As an alternative to overcome these issues, mesenchymal stem cells (MSCs), which have immune-regulating properties and no risk of cancer development, have been proposed. Mesenchymal stem cells are multipotent cells capable of differentiating into adipocytes, osteocytes, chondrocytes, muscle cells, neurons, and cardiomyocytes. They have also been reported to regulate immune responses. While mesenchymal stem cells can be isolated and cultured from a variety of tissues, their abilities and cell surface markers vary slightly depending on their origin, making it difficult to clearly define them. However, cells that can differentiate into osteocytes, chondrocytes, and muscle cells, have a spindle-shaped morphology, and express the basic cell surface markers CD73(+), CD105(+), CD34(-), and CD45(-) are generally defined as mesenchymal stem cells.

[0008] Furthermore, for mesenchymal stem cells to be used as cell therapy agents, the minimum number of cells required in the fields of regenerative medicine and / or cell therapy (approximately 1 × 10 9 However, the number of cells required increases even further when considering experiments to establish appropriate conditions and standards. Therefore, to obtain this amount of mesenchymal stem cells from various sources, at least 10 passages are required in in vitro experiments. In this case, the cells may senesce and deform, making them unsuitable for achieving the intended purpose of cell therapy. This results in a difference between the cells used in clinical trials and those undergoing quality evaluation, and there are drawbacks to this method, such as the inability to eliminate risks during post-mortem testing. Therefore, for use as cell therapy, it is important to maintain the characteristics of mesenchymal stem cells without deformation even after repeated passages over a long period of time.

[0009] As an alternative to adult-derived mesenchymal stem cells, methods for inducing differentiation of human pluripotent stem cells into mesenchymal stem cells have been proposed. However, existing methods have drawbacks, such as the need for expensive induction processes using specific cytokines (e.g., BMPs) that require concentration control, the need for xenofeeders that pose a risk of xenopathogens, and the use of fetal bovine serum (FBS). The conventional method for inducing differentiation of human pluripotent stem cells into mesenchymal stem cells (WO2011052818) requires culturing human pluripotent stem cells on xenofeeders, which can pose a risk of xenopathogens, thereby increasing the risk of the introduction of foreign cells through the use of trophoblasts. Furthermore, due to concerns about zoonotic diseases caused by the use of animal-derived sera, such as bovine serum or fetal calf serum, in the culture of pluripotent stem cells, improved xeno-free and serum-free culture methods that do not use foreign animal sera are required for the development of future cell therapy agents.

[0010] Therefore, the present inventors have made extensive efforts to resolve the issues of stem cell safety and mass production in order to commercialize mesenchymal stem cells, which are differentiated from human pluripotent stem cells, as cell therapeutic agents. As a result, they have maximized cell safety through a xeno-free and serum-free culture environment, formed mature embryoid bodies of uniform shape and size using spherical embryoid bodies, and confirmed that mesenchymal stem cells induced to differentiate from these embryoid bodies maintain the characteristics of mesenchymal stem cells for a long period of time even after repeated subculture, thereby completing the present invention. Summary of the Invention [Problem to be solved by the invention]

[0011] The object of the present invention is to provide a method for producing mesenchymal stem cells from human pluripotent stem cells that can be used as a cell therapy agent, by producing mesenchymal stem cells that have improved safety against contamination with foreign cells and foreign animal-derived substances and that maintain the characteristics of mesenchymal stem cells for a long period of time even after repeated subculture.

[0012] Another object of the present invention is to provide mesenchymal stem cells prepared by the above method and a cell therapy agent using the same. [Means for solving the problem]

[0013] In order to achieve the above object, the present invention provides (a) culturing human pluripotent stem cells in a serum-free pluripotent stem cell culture medium without xeno-feeder cells to obtain colonies of human pluripotent stem cells, and isolating human pluripotent stem cells from the colonies; (b) suspending the isolated pluripotent stem cells in an embryoid body formation medium and culturing them so that the pluripotent stem cells aggregate to form a single spherical embryoid body; (c) culturing the embryoid bodies in suspension in an embryoid body maturation medium to form mature embryoid bodies; (d) culturing the embryoid bodies in a xeno-free, serum-free mesenchymal stem cell culture medium to induce their differentiation into mesenchymal stem cells; and (e) A method for producing mesenchymal stem cells that maintain long-term subculture stability from human pluripotent stem cells is provided, which method includes a step of proliferating and culturing the differentiated mesenchymal stem cells in a xeno-free, serum-free mesenchymal stem cell culture medium while maintaining the identity of the mesenchymal stem cells.

[0014] The present invention also provides mesenchymal stem cells differentiated from the human pluripotent stem cells produced by the above method, and a cell therapy agent comprising the mesenchymal stem cells. [Brief explanation of the drawings]

[0015] [Figure 1]FIG. 1 is a diagram comparing a conventional method for producing mesenchymal stem cells derived from pluripotent stem cells ( WO2011052818 ) with the improved method for producing mesenchymal stem cells derived from pluripotent stem cells of the present invention. [Figure 2] Figure 2 shows the morphology of human pluripotent stem cell colonies obtained by culturing human pluripotent stem cells in serum-free pluripotent stem cell culture medium without xeno-feeder cells. (A) These colonies were uniformly sectioned into 200 μm × 200 μm pieces, and (B) the cells were isolated. [Figure 3] Figure 3 shows the results of immunofluorescence staining of pluripotency markers, OCT-4 and SSEA-4, to analyze the characteristics of pluripotent stem cells cultured in a nutrient-free, xeno-free, and serum-free environment. [Figure 4] FIG. 4 is a diagram comparing the size and morphology of embryoid bodies produced by a conventional production method (WO2011052818) and embryoid bodies produced by the improved method of the present invention. [Figure 5] FIG. 5 is a graph comparing the differentiation efficiency into mesenchymal stem cells and cell morphology of embryoid bodies produced by a conventional production method (WO2011052818) and embryoid bodies produced by the improved method of the present invention. [Figure 6] FIG. 6 shows the results of cell surface marker expression analysis of pluripotent stem cell-derived mesenchymal stem cells cultured in a nutrient-free, xeno-free, and serum-free environment. [Figure 7] FIG. 7 shows the results of an analysis of the differentiation potential of pluripotent stem cell-derived mesenchymal stem cells cultured in a nutrient-free, xeno-free, and serum-free environment. [Figure 8] FIG. 8 shows the results of G-band karyotype analysis of pluripotent stem cell-derived mesenchymal stem cells cultured in a nutrient-free, xeno-free, and serum-free environment. [Figure 9] Figure 9 is a diagram comparing the cell morphology of pluripotent stem cell-derived mesenchymal stem cells at passages 7 and 12 produced by a conventional method for producing pluripotent stem cell-derived mesenchymal stem cells (WO2011052818) and pluripotent stem cell-derived mesenchymal stem cells produced by the improved method for producing pluripotent stem cell-derived mesenchymal stem cells of the present invention. [Figure 10] Figure 10 shows the differentiation ability into bone cells, chondrocytes, and adipocytes of mesenchymal stem cells derived from pluripotent stem cells produced by a conventional method (WO2011052818) and the improved mesenchymal stem cells derived from pluripotent stem cells of the present invention. [Figure 11] Figure 11 is a schematic diagram showing the method for producing mesenchymal stem cells derived from (A) Western embryonic stem cells and (B) Oriental induced pluripotent stem cells cultured through the mature embryoid body formation stage in a nutrient-free, xeno-free, and serum-free environment. [Figure 12] FIG. 12 shows an analysis of the pluripotency of (A) Western embryonic stem cells and (B) Oriental induced pluripotent stem cells cultured in a nutrient-free, xeno-free, and serum-free environment. DETAILED DESCRIPTION OF THE INVENTION

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the nomenclature used herein is that which is well known and commonly used in the art.

[0017] As used herein, the term "stem cell" refers to a master cell that can reproduce without restriction to form specialized cells of tissues and organs. Stem cells are pluripotent or multipotent cells capable of development. Stem cells can divide into two daughter stem cells, or one daughter stem cell and one origin ("transit") cell, and then proliferate into mature, fully formed cells of tissues. These stem cells can be classified in various ways. One of the most frequently used methods is based on their differentiation potential, which divides them into pluripotent stem cells, which can differentiate into three germ layers; multipotent stem cells, which are limited to differentiation into more than a specific germ layer; and unipotent stem cells, which can differentiate only into a specific germ layer.

[0018] The term "pluripotent stem cells" as used herein refers to stem cells that have the ability to differentiate into all three germ layers that constitute the body, and generally includes embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Adult stem cells are classified as multipotent or unipotent stem cells.

[0019] The term "mesenchymal stem cells" as used herein refers to stem cells that have the multipotency to differentiate into cells such as adipocytes, osteocytes, chondrocytes, muscle cells, nerve cells, and cardiac muscle cells.

[0020] As used herein, the term "differentiation" refers to the phenomenon in which cell structure and function become specialized as cells divide and grow. Pluripotent mesenchymal stem cells differentiate into lineage-restricted progenitor cells (e.g., mesodermal cells), which can then differentiate into other types of progenitor cells (e.g., osteoblasts), and then into terminally differentiated cells (e.g., adipocytes, osteocytes, chondrocytes, etc.) that play characteristic roles in specific tissues (e.g., bone).

[0021] As used herein, the term "embryoid body (EB)" refers to an aggregate of pluripotent stem cells generated to induce differentiation of pluripotent stem cells. As used herein, "mature embryoid body" refers to an aggregate of pluripotent stem cells, i.e., an embryoid body that has grown in size through repeated division in suspension culture. In the present invention, mature embryoid bodies are used as materials for inducing differentiation into mesenchymal stem cells.

[0022] The term "cell therapy" as used herein refers to pharmaceutical products (as defined by the U.S. FDA) that are made from cells and tissues isolated from humans, cultured, and specially processed for therapeutic, diagnostic, and preventive purposes. These products are used for therapeutic, diagnostic, and preventive purposes through a series of actions, such as the ex vivo proliferation and selection of living autologous, allogeneic, or xenogeneic cells, or the alteration of the biological properties of cells by other methods, in order to restore the function of cells or tissues. Cell therapy products are broadly classified into somatic cell therapy products and stem cell therapy products depending on the degree of cell differentiation, and the present invention particularly relates to stem cell therapy products.

[0023] In this invention, to commercialize mesenchymal stem cells differentiated from human pluripotent stem cells as cell therapy agents, we solved the issues of cell safety and mass production by inducing differentiation of human pluripotent stem cells into mesenchymal stem cells in a feeder-free, xeno-free, and serum-free environment. Furthermore, we induced the formation of single spherical embryoid bodies through cell aggregation of human pluripotent stem cells, and then cultured these embryoid bodies in suspension to form mature embryoid bodies of uniform morphology and size, thereby minimizing quality differences between mature embryoid bodies and thereby improving the efficiency and consistency of differentiation into mesenchymal stem cells. The mesenchymal stem cells thus produced showed the groundbreaking effect of stably maintaining the characteristics of mesenchymal stem cells even after repeated subculture for more than 20 passages. That is, the present invention provides human pluripotent stem cell-derived mesenchymal stem cells that are uniform in quality, have excellent long-term subculture stability, and are suitable for mass production by forming mature embryoid bodies of uniform morphology and size using safe, spherical embryoid bodies without the contamination of foreign cells or foreign animal-derived substances. Therefore, the mesenchymal stem cells produced by the method of the present invention, with improved productivity and safety, enable the continuous mass supply of mesenchymal stem cells needed in the fields of regenerative medicine and cell therapy.

[0024] Thus, the present invention in one aspect provides a method for producing a composition comprising: (a) culturing human pluripotent stem cells in a serum-free pluripotent stem cell culture medium without xeno-feeder cells to obtain colonies of human pluripotent stem cells, and isolating human pluripotent stem cells from the colonies; (b) suspending the isolated pluripotent stem cells in an embryoid body formation medium and culturing them so that the pluripotent stem cells aggregate to form a single spherical embryoid body; (c) culturing the embryoid bodies in suspension in an embryoid body maturation medium to form mature embryoid bodies; (d) culturing the embryoid bodies in a xeno-free, serum-free mesenchymal stem cell culture medium to induce their differentiation into mesenchymal stem cells; and (e) A method for producing mesenchymal stem cells from human pluripotent stem cells, comprising the step of proliferating and culturing the differentiated mesenchymal stem cells in a xeno-free, serum-free mesenchymal stem cell culture medium while maintaining the identity of the mesenchymal stem cells.

[0025] Each step will be explained in detail below.

[0026] (a) culturing human pluripotent stem cells in a serum-free pluripotent stem cell culture medium without xeno-feeder cells to obtain colonies of human pluripotent stem cells, and isolating human pluripotent stem cells from the colonies; In the present invention, the human pluripotent stem cells may be embryonic stem cells or induced pluripotent stem cells. The human pluripotent stem cells are in an undifferentiated state.

[0027] Generally, in the past, a scaffold was required for culturing human pluripotent stem cells to maintain their undifferentiated state. Mouse embryonic fibroblasts have been primarily used as a scaffold for human pluripotent stem cells. However, the introduction of various pathogens between different species has been recognized as a problem when attempting to use pluripotent stem cells clinically. As a solution, the possibility of using scaffolds for various human-derived cells has been reported. However, even this approach has limitations, such as the inability to completely eliminate allogeneic pathogens, the essential need for exogenous factors (e.g., bFGF, IGF, activin, etc.) to maintain the undifferentiated state, and the inability to continuously supply cells for long-term culture.

[0028] However, the method of the present invention allows human pluripotent stem cells to be maintained in an undifferentiated state even when cultured in a serum-free medium using a vitronectin-coated culture vessel without xeno-feeder cells.

[0029] In the present invention, the pluripotent stem cells in step (a) are preferably cells cultured in a culture vessel coated with a human-derived extracellular matrix, an extracellular matrix containing no animal-derived components, or a synthetic material that can replace the extracellular matrix, but are not limited thereto.

[0030] The human-derived extracellular matrix is ​​preferably vitronectin, collagen, or laminin, and the extracellular matrix that does not contain animal-derived components other than human is preferably animal component-free matrigel. Furthermore, the synthetic substance that can replace the extracellular matrix is ​​preferably, but is not limited to, heparan sulfate proteoglycan.

[0031] That is, the method of the present invention is characterized in that all steps are carried out in a xeno-feeder cell-, cytokine-, and xenogeneic substance-free medium, i.e., in a feeder cell-free, xeno-free, and serum-free environment.

[0032] In particular, while the conventional method (WO2011052818) cultured pluripotent stem cells using xeno-feeder cells cultured in a medium containing FBS, the present invention is characterized by using human pluripotent stem cells cultured in a serum-free medium without xeno-feeder cells.

[0033] In addition, while conventional pluripotent stem cell culture media have used DMEM / F12 containing KSR, NEAA, β-mercaptoethanol, and bFGF, the present invention uses a serum-free pluripotent stem cell culture medium.

[0034] In the present invention, the serum-free medium for culturing human pluripotent stem cells in step (a) may be, but is not limited to, TeSR-E8 (essential 8 media), TeSR-2, StemMACS iPS-Brew XF, or Human medium.

[0035] (b) suspending the isolated pluripotent stem cells in an embryoid body formation medium and culturing them to aggregate, thereby forming a single spherical embryoid body; The present invention is characterized by the use of mature embryoid bodies with uniform morphology and size for inducing differentiation of human pluripotent stem cells into mesenchymal stem cells. Conventional methods have been unable to produce embryoid bodies with uniform morphology and size, making it difficult to expect consistent differentiation efficiency into mesenchymal stem cells. However, the present invention overcomes this technical limitation by forming single spherical embryoid bodies through cell-to-cell aggregation of human pluripotent stem cells, and then culturing these embryoid bodies in suspension to form mature embryoid bodies with uniform morphology and size. This not only improved the differentiation efficiency of mesenchymal stem cells derived from human pluripotent stem cells, but also produced revolutionary mesenchymal stem cells that maintain stem cell characteristics for long periods of time, even after repeated subculture.

[0036] In the conventional method (WO2011052818), pluripotent stem cells were cultured in suspension during embryoid body formation, but in the present invention, pluripotent stem cells were seeded onto the lid of a culture vessel and then cultured in hanging drops for 24 hours by turning the vessel upside down to allow the cells to aggregate by gravity, so that the pluripotent stem cells would aggregate together to form single spherical embryoid bodies. The cell aggregates thus formed, i.e., embryoid bodies, were further cultured in suspension to mature into mature embryoid bodies. As a result, mature embryoid bodies with uniform morphology and size were formed, as shown in Figure 1, and these mature embryoid bodies exhibited consistent differentiation efficiency into mesenchymal stem cells and stability during subculture.

[0037] Figure 4 shows that, while the size and morphology of mature embryoid bodies produced by conventional methods are not uniform, the mature embryoid bodies produced by the method of the present invention are uniform in size (300-500 μm) and have a consistent spherical morphology. Furthermore, it can be seen that mesenchymal stem cells induced to differentiate from the mature embryoid bodies produced in this manner have consistent differentiation efficiency and mesenchymal stem cell size. However, mesenchymal stem cells induced to differentiate from embryoid bodies produced by conventional methods are not consistent in differentiation efficiency or cell morphology (Figure 5).

[0038] Furthermore, mesenchymal stem cells induced to differentiate from mature embryoid bodies with uniform morphology and size surprisingly showed expression of mesenchymal stem cell surface markers CD29, CD44, CD73, and CD105 at over 90% up to passage 20. Furthermore, expression of cell surface markers CD45, a hematopoietic stem cell-specific surface marker, HLA-DR, an MHC class II marker, and SSEA-3, TRA-1-60, and TRA-1-81, a pluripotent stem cell-specific surface marker, was shown to be less than 2%. Thus, mesenchymal stem cells prepared by the method of the present invention can be subcultured for long periods and maintain high purity, enabling the mass production of stem cells suitable for use as cell therapy agents. Mesenchymal stem cells prepared by conventional methods undergo senescence and transformation once they reach the standard for use as cell therapy agents, making them no longer suitable for achieving their intended purpose as cell therapy agents. Ultimately, there is a drawback in that cells used in clinical practice differ from cells undergoing quality evaluation. However, the mesenchymal stem cells of the present invention do not deform and maintain the quality characteristics of mesenchymal stem cells even when repeatedly subcultured for a long period of time for use as a cell therapy agent, making them extremely useful for commercializing cell therapy agents.

[0039] Although methods for controlling embryoid body size (KR10-2013-0013537) and forming uniformly sized embryoid bodies through hanging drop culture (KR10-2007-0075006) have been reported, the formation of uniformly sized embryoid bodies is only known to affect cell differentiation efficiency, and its effect on aging or suppression of cell transformation in mesenchymal stem cells differentiated from uniformly sized embryoid bodies is unknown. However, in the present invention, we formed mature embryoid bodies with uniform morphology and size, and demonstrated that the mesenchymal stem cell characteristics of mesenchymal stem cells induced to differentiate from these embryoid bodies were maintained for up to 20 passages. In particular, we confirmed that mesenchymal stem cells differentiated from embryoid bodies prepared by conventional methods showed a decrease in CD105 expression to 43.6% after 6 passages and 26.6% after 12 passages (Table 2). That is, while stem cells produced by conventional methods do not maintain the characteristics of mesenchymal stem cells for up to 20 passages, the stem cells of the present invention can maintain the characteristics of mesenchymal stem cells for a long period of time, up to 20 passages, through the formation of embryoid bodies of uniform size.

[0040] Therefore, the present invention produces safe mesenchymal stem cells by eliminating the problem of contamination with exogenous animal-derived substances through a feeder cell-free, xeno-free, and serum-free culture environment. At the same time, the present invention produces human pluripotent stem cell-derived mesenchymal stem cells with the groundbreaking effect of improving the efficiency of differentiation into mesenchymal stem cells and stably maintaining the characteristics of mesenchymal stem cells even after long-term subculture of more than 20 passages by forming mature embryoid bodies of uniform shape and size using spherical embryoid bodies.

[0041] In the present invention, "embryoid bodies in the form of spheroids" refers to cell aggregates in the form of round spheres, and the spheroid form is often also referred to as a spheroid form.

[0042] In the present invention, the embryoid bodies of step (b) can be formed by any culture method capable of inducing intercellular aggregation of pluripotent stem cells. Any culture method capable of producing pluripotent stem cell aggregates, i.e., single spherical embryoid bodies, can be used without limitation. For example, the embryoid bodies of step (b) can be formed by hanging drop culture, or culture using a V-shaped tube, a round 96-well plate, or a conical tube.

[0043] In the present invention, the embryoid body formation medium in step (b) can be any medium that can induce cell aggregation between pluripotent stem cells. For example, the embryoid body formation medium in step (b) may be Aggrewell EB formation medium, Gibco Essential 6 Medium, CTS Essential 6 Medium, or TeSR-E6. The culture in step (b) can be for 18 to 30 hours, for example, 20 to 28 hours, 22 to 26 hours, or for example, 24 hours.

[0044] (c) culturing the embryoid bodies in suspension in an embryoid body maturation medium to form mature embryoid bodies; In step (c), the embryoid bodies are grown in suspension culture, and the mature embryoid bodies obtained at this stage are characterized by having uniform morphology and size.

[0045] The expression "uniform morphology and size" for mature embryoid bodies means that mature cultures obtained by suspension culture of spherical embryoid bodies are uniform in morphology and size. The morphology of the mature cultures is spherical, just like the embryoid bodies, and the size of the mature embryoid bodies is uniform, being within ±15% of the average size of the entire mature embryoid bodies. That is, when the average size of the mature embryoid bodies is 100%, the minimum size of the mature embryoid bodies is within 90% and the maximum size of the mature embryoid bodies is within 120%, meaning that the sizes are very uniform. Preferably, the size of the mature embryoid bodies is uniform, being within ±10% of the average size of the entire mature embryoid bodies. In this case, when the average size of the mature embryoid bodies is 100%, the minimum size of the mature embryoid bodies is within 90% and the maximum size of the mature embryoid bodies is within 110%. The average size of mature embryoid bodies may vary depending on the maturation culture conditions and period of the embryoid bodies, but the average size of mature embryoid bodies suitable for inducing differentiation into stem cells may be 350 to 450 μm, for example, 380 to 420 μm. Preferably, mature embryoid bodies have a uniform size of 300 to 500 μm.

[0046] In the present invention, the suspension culture in step (c) is performed using an embryoid body maturation medium for embryoid body maturation. A commonly known medium can be used as the embryoid body maturation medium, but it is not particularly limited thereto. In a specific example of the present invention, the embryoid body maturation medium in step (c) may be a basal medium containing knockout serum replacement (KSR), non-essential amino acid (NEAA), and β-mercaptoethanol. The basal medium may be, but is not limited to, DMEM / F12, alpha MEM, Ham's F12 media, or DMEM.

[0047] The suspension culture in step (c) may be cultured for 10 to 18 days, for example, 12 to 16 days, for example, 14 days, but is not limited thereto.

[0048] (d) culturing the embryoid body in a xeno-free, serum-free mesenchymal stem cell culture medium to induce differentiation into mesenchymal stem cells. When inducing differentiation of human pluripotent stem cells into mesenchymal stem cells, differentiation induction is generally initiated by external addition of cytokines such as bone morphogenetic protein (BMP). However, in the present invention, it was confirmed that differentiation into mesenchymal stem cells was naturally induced without the addition of BMP. In a conventional method (WO2011052818), FBS-containing DMEM medium was used for differentiation of mesenchymal stem cells, and FBS-containing EGM2-MV medium was used for proliferation and culture. However, in the present invention, mesenchymal stem cells were propagated and cultured in a xeno-free and serum-free environment.

[0049] In the present invention, the mesenchymal stem cell culture medium used in step (d) may be a xeno-free serum-free medium containing L-glutamine. The total concentration of L-glutamine in the medium is preferably 2 to 4 mM, but is not limited thereto.

[0050] Examples of the xeno-free, serum-free mesenchymal stem cell culture medium used in step (d) include, but are not limited to, Stempro SFM Xeno-free medium, PRIME-XV MSC Expansion XSFM medium, Human Mesenchymal-XF Expansion medium, MSC Nutristem XF medium, StemMACS MSC expansion media kit XF, human medium, or a medium containing 5 to 20% human platelet lysate instead of FBS.

[0051] Furthermore, in the present invention, the differentiation induction into mesenchymal stem cells in step (d) may be for 12 to 20 days, for example, 14 to 18 days, for example, 16 days, but is not limited thereto.

[0052] In one example of the present invention, differentiation of mesenchymal stem cells was induced in Stempro MSC SFM Xeno-free medium, which does not contain differentiation inducers or serum.

[0053] (e) Proliferating and culturing the differentiated mesenchymal stem cells in a xeno-free, serum-free mesenchymal stem cell culture medium while maintaining the identity of the mesenchymal stem cells. Step (e) is the step of expanding and culturing the differentiation-induced mesenchymal stem cells. To commercialize cell therapy containing human mesenchymal stem cells, it is extremely important to secure a sufficient amount of mesenchymal stem cells obtained at this step while at the same time maintaining the identity of the mesenchymal stem cells, i.e., their characteristics as mesenchymal stem cells.

[0054] In the present invention, when the differentiation-induced mesenchymal stem cells are cultured for proliferation, they are cultured in a xeno-free medium to which no additional differentiation-inducing factors or FBS (fetal bovine serum) are added.

[0055] As in step (d), the mesenchymal stem cell culture medium used in step (e) of the present invention may be a xeno-free serum-free medium containing L-glutamine. The total concentration of L-glutamine in the medium is preferably, but not limited to, 2 to 4 mM.

[0056] Examples of the xeno-free, serum-free mesenchymal stem cell culture medium used in step (e) include, but are not limited to, Stempro SFM Xeno-free medium, PRIME-XV MSC Expansion XSFM medium, Human Mesenchymal-XF Expansion medium, MSC Nutristem XF medium, StemMACS MSC expansion media kit XF, human medium, or a medium containing 5 to 20% human platelet lysate instead of FBS.

[0057] In one embodiment of the present invention, Stempro MSC SFM medium containing no FBS was used as the mesenchymal stem cell proliferation medium, but the present invention is not limited to this, and a xeno-free medium not containing xenogeneic substances such as xenogeneic proteins may also be used.

[0058] As explained above, in order to use mesenchymal stem cells as cell therapy agents, a priority must be given to providing a sufficient number of cells, which requires subculturing mesenchymal stem cells. However, continued subculturing can lead to senescence, loss of division ability, and loss of activity (differentiation potential). In this regard, the present invention has demonstrated that the characteristics and activity of mesenchymal stem cells can be maintained for more than 20 passages during in vitro culture in a xeno-free and serum-free medium (Table 1). Thus, mesenchymal stem cells produced by the method of the present invention can maintain their characteristics for a long period of time, enabling their use as cell therapy agents through mass production. These characteristics can be achieved through a xeno-free, serum-free mesenchymal stem cell production environment and the production of uniformly sized embryoid bodies.

[0059] Mesenchymal stem cells are defined by a uniform spindle-shaped fingerprint pattern and the expression of basic cell surface markers such as CD73(+), CD105(+), CD34(-), and CD45(-), and can differentiate into bone cells, chondrocytes, adipocytes, etc.

[0060] In the present invention, the mesenchymal stem cells in step (e) are characterized by being mesenchymal stem cells that have multipotency and can differentiate into cells selected from the group consisting of adipocytes, osteocytes, chondrocytes, muscle cells, nerve cells, and cardiomyocytes.

[0061] In one embodiment of the present invention, to confirm the consistency of stem cell characteristics through subculture of pluripotent stem cell-derived mesenchymal stem cells, changes in cell surface markers were comparatively analyzed up to passage 20. Expression of mesenchymal stem cell surface markers CD29, CD44, CD73, and CD105, hematopoietic stem cell-specific surface marker CD45, MHC class type II marker HLA-DR, and pluripotent stem cell-specific surface markers SSEA-3, TRA-1-60, and TRA-1-81 was comparatively analyzed from passages 12 to 20. As a result, it was confirmed that the expression of mesenchymal stem cell surface markers CD29, CD44, CD73, and CD105 was maintained at 90% or more up to passage 20 (Table 1). Furthermore, mesenchymal stem cells derived from pluripotent stem cells prepared using conventional methods (WO2011052818) showed CD105 expression of less than 50% at passage 6, and in particular, it was confirmed to decrease to 26.6% after 12 passages (Table 2). That is, stem cells prepared using conventional methods do not maintain the characteristics of mesenchymal stem cells up to passage 20, but the stem cells of the present invention can maintain the characteristics of mesenchymal stem cells for a long period of time up to passage 20 through the formation of mature embryoid bodies with uniform morphology and size.

[0062] CD105 is known to be a specific surface marker of mesenchymal stem cells. In 2003, Duff SE et al. reported that CD105 plays an important role in vascular regeneration by mesenchymal stem cells (The FASEB Journal 2003;17(9):984-992). Furthermore, CD105 expression is known to be downregulated during mesenchymal stem cell differentiation into osteocytes (Levi B et al., The Journal of Biological Chemistry. 2011;286(45):39497-39509), and also plays an important role in maintaining the stemness of mesenchymal stem cells by decreasing expression after differentiation into osteocytes, chondrocytes, and adipocytes (Jin HJ et al., BBRC 2009;381(4):676-681).

[0063] In the present invention, the mesenchymal stem cells in the step (e) are characterized by expressing the cell surface markers CD29(+), CD44(+), CD73(+) and CD105(+).

[0064] In the present invention, it is preferable that the expression of the cell surface marker is maintained at 90% or more in mesenchymal stem cells for 20 or more passages, and more preferably, the expression of the CD105(+) cell surface marker is maintained at 90% or more in mesenchymal stem cells for 20 or more passages, but is not limited thereto.

[0065] In the present invention, the mesenchymal stem cells in step (e) are characterized as being CD34(-), CD45(-), HLA-DR(-), TRA-1-60(-), and TRA-1-81(-) mesenchymal stem cells.

[0066] In another aspect, the present invention relates to mesenchymal stem cells induced to differentiate from human pluripotent stem cells produced by the above-mentioned method.

[0067] Mesenchymal stem cells induced to differentiate from human pluripotent stem cells produced by the method of the present invention yielded the same results, regardless of the differences in origin (Eastern or Western) and type (embryonic stem cells or induced pluripotent stem cells). The present invention provides a standardized differentiation induction and proliferation culture method that can be generally used to produce mesenchymal stem cells from human pluripotent stem cells of diverse genetic origins. That is, the standardized method of the present invention is a method that can be generally used to induce differentiation into mesodermal stem cells from pluripotent stem cells with diverse genetic backgrounds and / or culture environments.

[0068] In one embodiment of the present invention, mesenchymal stem cells were produced using SNUhES35 / hES12011003 embryonic stem cells registered in the Stem Cell Bank of the National Stem Cell Regeneration Center as human pluripotent stem cells, but are not limited thereto.

[0069] In another embodiment of the present invention, mesenchymal stem cells were produced using Western embryonic stem cells ESI-017 / hES22014005 and ESI-035 / hES22014006, which are registered in the Stem Cell Bank of the National Stem Cell Regeneration Center. However, this is not limited to these, and embryonic stem cells such as ESI-049 / hES22014007, ESI-051 / hES22014008, and ESI-053 / hES22014009 can also be used.

[0070] In yet another embodiment of the present invention, mesenchymal stem cells were produced using induced pluripotent stem cells from Oriental individuals, but the present invention is not limited thereto.

[0071] In yet another aspect, the present invention relates to a cell therapy agent containing, as an active ingredient, mesenchymal stem cells induced to differentiate from human pluripotent stem cells produced by the above-mentioned method. The cell therapy agent may contain water for injection in addition to mesenchymal stem cells. The cell therapy agent may also contain a freezing excipient used for freezing. The freezing excipient is preferably CryoStor 10 (CS10) or STEM-CELLBANKER DMSO Free GMP grade, which does not contain animal-derived components, but is not limited thereto. Typically, a cell therapy agent containing mesenchymal stem cells contains 1×10 6 ~1×10 8 The cell therapy agent containing mesenchymal stem cells according to the present invention is administered to a subject at a dose of 1000 cells / kg. The cell therapy agent containing mesenchymal stem cells according to the present invention can be used without limitation for the treatment of various diseases generally known to be effective in mesenchymal stem cell transplantation therapy. In particular, the cell therapy agent containing mesenchymal stem cells according to the present invention can be used to treat patients infected with the COVID-19 virus, severe acute pancreatitis (SAP), etc. [Example]

[0072] The present invention will be described in more detail below through examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the present invention.

[0073] Example 1: Method for producing mesenchymal stem cells from human pluripotent stem cells 1-1: Cultivation of pluripotent stem cells in a feeder cell-free, xeno-free, and serum-free environment and confirmation of pluripotency To confirm whether pluripotent stem cells can be cultured in vitro while maintaining pluripotency in a nutrient-free, xeno-free, and serum-free environment, tissue culture vessels were coated with human vitronectin, a component of the human extracellular matrix, at a final concentration of 10 μg / mL. Undifferentiated human pluripotent stem cells (Korean embryonic stem cells: SNUhES35 / hES12011003) were cultured in TeSR-2 or TeSR-E8 (essential 8 media), a xeno-free, serum-free medium, to obtain colonies of pluripotent stem cells (Figure 1, left photo of ESC culture). At 60% confluency (more than 20 colonies), pluripotent stem cell colonies were uniformly sectioned into approximately 200 μm x 200 μm sections using the EZPassage Passaging Tool (Figure 2A). The sections were then detached from the culture vessel using a pipette, transferred to a conical tube, and allowed to settle. The supernatant was then removed. The isolated pluripotent stem cells were confirmed to be spherical cells with a diameter of approximately 10-15 μm (Figure 2B).

[0074] To confirm the pluripotency of pluripotent stem cells cultured in a nutrient-free, xeno-free, and serum-free environment, we examined the expression of OCT-4 and SSEA-4, which are pluripotency markers for stem cells, through immunofluorescence staining.

[0075] As a result, pluripotent stem cells cultured in a nutrient-free, xeno-free, and serum-free environment expressed OCT-4 and SSEA-4, markers of pluripotency, confirming that the cultured pluripotent stem cells maintained their pluripotency in a nutrient-free, xeno-free, and serum-free environment (Figure 3).

[0076] 1-2: Formation of mature embryoid bodies from pluripotent stem cells A fixed volume of Aggrewell EB Formation Medium containing pluripotent stem cells was inoculated onto the lid of a petri dish and cultured in hanging drops to form cell aggregates, or embryoid bodies. The embryoid bodies were then inoculated into embryoid maturation medium and cultured in suspension to form mature embryoid bodies of a certain size.

[0077] Specifically, embryoid body formation medium (Aggrewell EB Formation Medium) (400 μl per 20 human pluripotent stem cell colonies) was added to the conical tube containing the isolated pluripotent stem cells, and the isolated pluripotent stem cells were suspended in the embryoid body formation medium by pipetting to form single cells. 20 μl of the embryoid body formation medium containing the suspended pluripotent stem cells was then inoculated into the lid of a tissue culture vessel, which was then inverted and cultured in hanging drops at 37°C and in a 5% CO2 incubator for 24 hours to allow the cells to aggregate by gravity. After 24 hours of culture, single spherical cell aggregates, i.e., embryoid bodies, were formed.

[0078] The formed embryoid bodies were cultured in suspension in a petri dish using embryoid maturation medium (DMEM / F12, 20% knockout serum replacement (KSR), 0.1 mM non-essential amino acid (NEAA), 0.1 mM β-mercaptoethanol) for 14 days, with medium changes every 2–3 days (see Figure 1).

[0079] As a result, it was confirmed that mature embryoid bodies with a uniform spherical morphology and a size of 300 to 500 μm were formed (Figure 4, photograph of the improved method on the right).

[0080] 1-3: Induction of differentiation of mature embryoid bodies into mesenchymal stem cells and proliferation Mature embryoid bodies cultured for 14 days were grown on a xeno-free substrate, CellStart TM (Thermo Fisher Scientific) (78 μL / cm 2 The embryoid bodies were then seeded into each well of a 6-well plate (at a concentration of 1000 x 1000 x 1000 mm). After seeding, 4-5 embryoid bodies were seeded into each well, and the cells were cultured in StemPro MSC SFM XenoFree, a xenogeneic and serum-free mesenchymal stem cell culture medium, to induce differentiation into mesenchymal stem cells. TM (Thermo Fisher Scientific) was used to induce differentiation.

[0081] For the induction of differentiation into mesenchymal stem cells and for the initial culture, the culture was continued for 16 days without subculture, with fresh medium being replaced every 2 to 3 days.

[0082] After confirming sufficient proliferation of mesenchymal stem cells differentiated from the attached mature embryoid bodies under a microscope, subculture was carried out.

[0083] Furthermore, the pluripotent stem cell-derived mesenchymal stem cells produced in this manner were subcultured and cultured in a 5% CO2 incubator at 37°C using StemPro MSC SFM XenoFree medium for differentiation and proliferation in a xeno-free and serum-free environment.

[0084] Figure 5 compares the differentiation efficiency into mesenchymal stem cells and cell morphology between embryoid bodies produced by a conventional production method (WO2011052818) and embryoid bodies produced by the improved method of the present invention. According to the method of the present invention, the differentiation efficiency into mesenchymal stem cells and cell morphology were consistent due to the uniform morphology and size of the embryoid bodies (bottom panel of Figure 5). However, mesenchymal stem cells induced to differentiate from embryoid bodies produced by the conventional method were not consistent in differentiation efficiency or cell morphology (top panel of Figure 5).

[0085] Example 2: Analysis of the characteristics of mesenchymal stem cells derived from pluripotent stem cells 2-1: Analysis of cell surface marker expression in mesenchymal stem cells To confirm whether the pluripotent stem cell-derived mesenchymal stem cells prepared by the method of Example 1 have the characteristics of mesenchymal stem cells, cell surface markers were analyzed using flow cytometry, and whether they could differentiate into osteocytes, chondrocytes, and adipocytes was analyzed.

[0086] FIG. 6 shows the results of cell surface marker expression analysis of pluripotent stem cell-derived mesenchymal stem cells cultured in a nutrient-free, xeno-free, and serum-free environment.

[0087] As a result, it was confirmed that the pluripotent stem cell-derived mesenchymal stem cells positively expressed the mesenchymal stem cell-specific surface markers CD73 and CD105, but negatively expressed HLA-DR, an MHC class type II cell surface marker associated with immune responses, and CD34 and CD45, hematopoietic stem cell-specific cell surface markers.

[0088] Furthermore, to check for contamination with pluripotent stem cells, the expression of TRA-1-60, a cell surface marker specific to pluripotent stem cells, was checked. TRA-1-60 expression was found to be negative, confirming that the pluripotent stem cell-derived mesenchymal stem cells produced express the specific cell surface marker of general mesenchymal stem cells (Figure 6).

[0089] 2-2: Analysis of the differentiation potential of mesenchymal stem cells To analyze the differentiation potential of the pluripotent stem cell-derived mesenchymal stem cells prepared by the method of Example 1, they were induced to differentiate into adipocytes, osteocytes, and chondrocytes for 14 days, and then their differentiation potential was examined using specific chemical staining methods.

[0090] After 14 days of induction of adipocyte differentiation, Oil-Red-O staining confirmed that the pluripotent stem cell-derived mesenchymal stem cells had differentiated into adipocytes. After 14 days of induction of osteocyte differentiation, Alizarin-Red-S and Alkaline Phosphatase staining confirmed that the pluripotent stem cell-derived mesenchymal stem cells had differentiated into adipocytes. After 14 days of induction of chondrocyte differentiation, Alcian Blue staining confirmed that the pluripotent stem cell-derived mesenchymal stem cells had differentiated into chondrocytes (Figure 7).

[0091] This indicates that the mesenchymal stem cells derived from pluripotent stem cells prepared in Example 1 have differentiation potential similar to that of general mesenchymal stem cells.

[0092] 2-3: Analysis of chromosomal abnormalities in mesenchymal stem cells To confirm whether chromosomal abnormalities occur during the differentiation of the pluripotent stem cell-derived mesenchymal stem cells prepared by the method of Example 1, G-banding karyotyping (Saccone et al., Proc Natl Acad Sci USA, 89:4913-4917, 1992) was performed.

[0093] As a result, the normal karyotype of 46XY was confirmed in the prepared pluripotent stem cell-derived mesenchymal stem cells, indicating that no chromosomal abnormalities were induced during the differentiation process of the pluripotent stem cell-derived mesenchymal stem cells prepared in Example 1 (Figure 8).

[0094] 2-4: Analysis of the persistence of stem cell properties of mesenchymal stem cells To confirm the consistency of stem cell characteristics through subculture of the pluripotent stem cell-derived mesenchymal stem cells prepared by the method of Example 1, continuous subculture was carried out in a culture vessel, and changes in cell surface markers were comparatively analyzed up to 20 passages.

[0095] The expression of cell surface markers for mesenchymal stem cells (CD29, CD44, CD73, CD105), hematopoietic stem cell-specific surface marker CD45, MHC class type II marker HLA-DR, and pluripotent stem cell-specific surface markers SSEA-3, TRA-1-60, and TRA-1-81 was comparatively analyzed from passages 12 to 20 (Table 1).

[0096] As a result, it was confirmed that the pluripotent stem cell-derived mesenchymal stem cells prepared by the method of Example 1 maintained 90% or more expression of mesenchymal stem cell surface markers CD29, CD44, CD73, and CD105 from passage 12 to passage 20. It was also confirmed that the expression of hematopoietic stem cell-specific cell surface marker CD45, MHC class type II marker HLA-DR, and pluripotent stem cell-specific cell surface markers SSEA-3, TRA-1-60, and TRA-1-81 remained negative.

[0097] These results suggest that the mesenchymal stem cells derived from pluripotent stem cells produced by the method of Example 1 maintain the properties of mesenchymal stem cells up to 20 passages, and they are highly valuable as an important cell resource not only for the future mass culture of therapeutic agents using mesenchymal stem cells, but also for the development of stem cell therapeutic agents that enhance cell function through gene transfer.

[0098] [Table 1]

[0099] Example 3: Comparison of the characteristics of mesenchymal stem cells derived from pluripotent stem cells depending on the production method 3-1: Comparison of cell morphology The cell morphology of two types of pluripotent stem cell-derived mesenchymal stem cells produced from the same pluripotent stem cells using different mesenchymal stem cell production methods was compared using a microscope. Morphological comparisons were conducted on the 7th and 12th passages of the pluripotent stem cell-derived mesenchymal stem cells produced.

[0100] Both the mesenchymal stem cells derived from pluripotent stem cells prepared by the conventional method (WO2011052818) and the mesenchymal stem cells derived from pluripotent stem cells prepared in the above examples had a spindle-shaped morphology, and it was confirmed that the cell morphology was maintained similarly up to passage 7. However, in the case of the mesenchymal stem cells derived from pluripotent stem cells prepared by the conventional method, cell clumping occurred at passage 12.

[0101] However, it was confirmed that the pluripotent stem cell-derived mesenchymal stem cells prepared in the above example maintained a good spindle-shaped cell morphology even after 12 passages (FIG. 9).

[0102] 3-2: Comparison of cell surface marker expression As in Example 3-1, the expression of cell surface markers was compared between mesenchymal stem cells derived from pluripotent stem cells prepared by a conventional method (WO2011052818) and mesenchymal stem cells derived from pluripotent stem cells prepared according to the present invention.

[0103] The mesenchymal stem cells derived from pluripotent stem cells prepared by the method of the present invention were confirmed to be positive for the expression of mesenchymal stem cell-specific cell surface markers CD29, CD44, CD73, and CD105 in over 90% of cases up to passages 6, 8, and 12. Furthermore, the expression of hematopoietic stem cell-specific cell surface markers CD34 and CD45, the MHC class type II marker HLA-DR, and the pluripotent stem cell-specific cell surface marker TRA-1-60 was confirmed to be negative (Table 2).

[0104] In contrast, the expression of cell surface markers, except for CD105, of mesenchymal stem cells derived from pluripotent stem cells prepared by conventional methods was maintained at a similar level to that of mesenchymal stem cells derived from pluripotent stem cells prepared by the method of the present invention up to passages 6, 8, and 12. However, CD105 expression was shown to be less than 50% at passage 6, and in particular, it was confirmed to decrease to 26.6% after 12 passages (Table 2).

[0105] [Table 2]

[0106] 3-3: Comparison of osteocyte, chondrocyte, and adipocyte differentiation potential As in Example 3-1, the differentiation ability of pluripotent stem cell-derived mesenchymal stem cells prepared by a conventional method (WO2011052818) and pluripotent stem cell-derived mesenchymal stem cells prepared according to the present invention into osteocytes, chondrocytes, and adipocytes was compared.

[0107] It was confirmed that the mesenchymal stem cells derived from pluripotent stem cells prepared by the method of the present invention have a higher differentiation potential into osteocytes, chondrocytes, and adipocytes than the mesenchymal stem cells derived from pluripotent stem cells prepared by conventional methods (Figure 10).

[0108] Example 4: Comparison of mesenchymal stem cells based on different types of pluripotent stem cells 4-1: Mesenchymal stem cells derived from Western human embryonic stem cells To confirm whether mesenchymal stem cells produced by the method of the present invention differ depending on the race of origin of the pluripotent stem cells, Western human embryonic stem cells (ESI-017 / hES22014005, ESI-035 / hES22014006) were cultured in a nutrient-free, xeno-free, and serum-free environment.

[0109] In the same manner as in Example 1-1, human vitronectin, a component of human extracellular matrix, was coated onto a tissue culture vessel to a final concentration of 10 μg / mL, and undifferentiated Western human pluripotent stem cells were cultured in TeSR-2 medium, a xeno-free, serum-free medium (FIG. 11A).

[0110] To confirm the pluripotency of pluripotent stem cells cultured in a nutrient-free, xeno-free, and serum-free environment, we performed immunofluorescence staining to examine the expression of OCT-4 and SSEA-4, which are stem cell pluripotency markers. We found that OCT-4 and SSEA-4, which indicate pluripotency, were expressed in Western human pluripotent stem cells. Furthermore, we confirmed the expression of alkaline phosphatase, demonstrating that the cultured Western human pluripotent stem cells maintained their pluripotency (Figure 12A).

[0111] To induce differentiation of mesenchymal stem cells from cultured Western human pluripotent stem cells, mature embryoid bodies were formed using the same method and medium as in Examples 1-2. These mature embryoid bodies, cultured for 14 days, were then differentiated into mesenchymal stem cells using the same method and medium as in Examples 1-3. After confirming sufficient proliferation of mesenchymal stem cells differentiated from the attached mature embryoid bodies under a microscope, subculture was performed, and the cells were cultured in a 5% CO2 incubator at 37°C using StemPro MSC SFM Xeno-free medium in a xeno-free and serum-free environment for differentiation and proliferation (Figure 11A).

[0112] Therefore, it was confirmed that the mesenchymal stem cells produced by the method of the present invention do not differ depending on the race of origin of the pluripotent stem cells.

[0113] 4-2: Mesenchymal stem cells derived from induced pluripotent stem cells To confirm whether there are differences between the mesenchymal stem cells produced by the method of the present invention depending on the type of pluripotent stem cells, induced pluripotent stem cells (iPSCs) were cultured in a nutrient-free, xeno-free, and serum-free environment.

[0114] In the same manner as in Example 1-1, a tissue culture vessel was coated with human vitronectin, a component of human extracellular matrix, to a final concentration of 10 μg / mL, and undifferentiated induced pluripotent stem cells were cultured in TeSR-2 medium, a xeno-free, serum-free medium (FIG. 11B).

[0115] To confirm the pluripotency of pluripotent stem cells cultured in a nutrient-free, xeno-free, and serum-free environment, the expression of OCT-4 and SSEA-4, which are pluripotency markers of stem cells, was confirmed through immunofluorescence staining. As a result, OCT-4 and SSEA-4, which indicate pluripotency, were expressed in induced pluripotent stem cells. In other words, it was found that the cultured induced pluripotent stem cells maintained pluripotency (Figure 12B).

[0116] To induce differentiation of mesenchymal stem cells from the cultured induced pluripotent stem cells, mature embryoid bodies were formed in the same manner as in Examples 1-2, and these mature embryoid bodies cultured for 14 days were differentiated into mesenchymal stem cells in the same manner as in Examples 1-3. After confirming sufficient proliferation of mesenchymal stem cells differentiated from the attached embryoid bodies under a microscope, subculture was performed, and the cells were cultured in a 5% CO2 incubator at 37°C using Stempro MSC SFM Xeno-free medium for differentiation and proliferation in a xeno-free and serum-free environment (Figure 11B).

[0117] Therefore, it was confirmed that there is no difference between the types of pluripotent stem cells produced by the method of the present invention.

[0118] While the specific details of the present invention have been described above, it will be apparent to those skilled in the art that these specific details are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents. [Industrial Applicability]

[0119] The method for producing mesenchymal stem cells from human pluripotent stem cells according to the present invention eliminates the problem of contamination with exogenous animal-derived substances through a feeder cell-free, xeno-free, and serum-free culture environment, and enables the mass production of mesenchymal stem cells whose cell characteristics do not change even after repeated subculture over a long period of time through the formation of mature embryoid bodies with uniform morphology and size using spherical embryoid bodies, making it useful for commercializing safe and efficient cell therapy agents. Further aspects of the present invention are described below: [Section 1] A method for producing mesenchymal stem cells from human pluripotent stem cells, comprising the steps of: (a) culturing human pluripotent stem cells in a serum-free pluripotent stem cell culture medium without xeno-feeder cells to obtain colonies of human pluripotent stem cells, and isolating human pluripotent stem cells from the colonies; (b) suspending the isolated pluripotent stem cells in an embryoid body formation medium and culturing them so that the pluripotent stem cells aggregate to form a single spherical embryoid body; (c) culturing the embryoid bodies in suspension in an embryoid body maturation medium to form mature embryoid bodies; (d) culturing the embryoid bodies in a xeno-free, serum-free mesenchymal stem cell culture medium to induce their differentiation into mesenchymal stem cells; and (e) Proliferating and culturing the differentiated mesenchymal stem cells in a xeno-free, serum-free mesenchymal stem cell culture medium while maintaining the identity of the mesenchymal stem cells. [Section 2] Item 1. The method according to Item 1, wherein the human pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells. [Section 3] Item 1. The method according to Item 1, wherein the human pluripotent stem cells are in an undifferentiated state. [Section 4] Item 1. The method according to Item 1, wherein the human pluripotent stem cells in step (a) are cultured in a culture vessel coated with a substance selected from the group consisting of vitronectin, collagen, laminin, matrigel, and heparan sulfate proteoglycan. [Section 5] Item 2. The method according to Item 1, wherein the serum-free medium used to culture the human pluripotent stem cells in step (a) is TeSR-E8 (essential 8 media), TeSR-2, StemMACS iPS-Brew XF, or Human medium. [Section 6] The method according to item 1, wherein the embryoid bodies at stage (b) are formed by hanging drop culture, or culture using a V-shaped tube, a round 96-well plate, or a conical tube. [Section 7] Item 2. The method according to Item 1, wherein the embryoid body formation medium in step (b) is Aggrewell EB formation medium, Gibco Essential 6 Medium, CTS Essential 6 Medium, or TeSR-E6. [Section 8] Item 1. The method according to Item 1, wherein the culture in step (b) is carried out for 18 to 30 hours. [Section 9] Item 2. The method according to Item 1, wherein the embryoid body maturation medium in step (c) is a basal medium containing KSR (knock out serum replacement), NEAA (non-essential amino acid), and β-mercaptoethanol. [Section 10] Item 10. The method according to Item 9, wherein the basal medium in step (c) is DMEM / F12, alpha MEM, Ham's F12 media, or DMEM. [Section 11] Item 1. The method according to Item 1, wherein the suspension culture in step (c) is carried out for 10 to 18 days. [Section 12] Item 2. The method according to Item 1, wherein the average size of the mature embryoid bodies obtained in step (c) is 350 to 450 μm. [Section 13] Item 1. The method according to Item 1, wherein the mesenchymal stem cell culture medium in steps (d) and (e) is a xeno-free, serum-free medium containing L-glutamine. [Section 14] Item 14. The method according to Item 13, wherein the xeno-free, serum-free medium is Stempro SFM Xeno-free medium, PRIME-XV MSC Expansion XSFM medium, Human Mesenchymal-XF Expansion medium, MSC Nutristem XF medium, StemMACS MSC expansion media kit XF, or human medium. [Section 15] Item 1. The method according to Item 1, wherein the differentiation induction into mesenchymal stem cells in step (d) is carried out for 12 to 20 days. [Section 16] Item 1. The method according to Item 1, wherein the mesenchymal stem cells obtained in step (e) are mesenchymal stem cells that have multipotency and can differentiate into cells selected from the group consisting of adipocytes, bone cells, chondrocytes, muscle cells, nerve cells, and cardiomyocytes. [Section 17] Item 1. The method according to Item 1, wherein the mesenchymal stem cells obtained in step (e) express the cell surface markers CD29(+), CD44(+), CD73(+), and CD105(+). [Section 18] Item 18. The method according to Item 17, wherein the expression of the cell surface marker is maintained at 90% or more in mesenchymal stem cells for 20 or more passages. [Section 19] Item 1. The method according to Item 1, wherein the mesenchymal stem cells obtained in step (e) are CD34(-), CD45(-), HLA-DR(-), TRA-1-60(-), and TRA-1-81(-) mesenchymal stem cells. [Section 20] Item 1. A mesenchymal stem cell induced to differentiate from a human pluripotent stem cell produced by the method described in Item 1. [Section 21] Item 21. The mesenchymal stem cells according to Item 20, which have the multipotency to differentiate into cells selected from the group consisting of adipocytes, bone cells, chondrocytes, muscle cells, nerve cells and cardiac muscle cells. [Section 22] Item 21. The mesenchymal stem cell according to Item 20, which expresses the cell surface markers CD29(+), CD44(+), CD73(+), and CD105(+). [Section 23] Item 21. The mesenchymal stem cells according to Item 20, wherein the expression of the cell surface marker is maintained at 90% or more in mesenchymal stem cells for 20 or more passages. [Section 24] 21. The mesenchymal stem cells according to item 20, which are CD34(-), CD45(-), HLA-DR(-), TRA-1-60(-), and TRA-1-81(-). [Section 25] Item 21. A cell therapy agent comprising the mesenchymal stem cells according to Item 20 as an active ingredient. [Section 26] Item 26. The cell therapeutic agent according to Item 25, wherein the cell therapeutic agent is for the prevention or treatment of COVID-19 infection or severe acute pancreatitis (SAP).

Claims

1. A method for producing mesenchymal stem cells from human pluripotent stem cells, comprising the steps of: (a) culturing human pluripotent stem cells in a serum-free pluripotent stem cell culture medium without xenofeeder cells to obtain colonies of human pluripotent stem cells, and isolating human pluripotent stem cells from the colonies; (b) suspending the isolated pluripotent stem cells in an embryoid body formation medium and culturing them in a hanging drop culture, or in a V-shaped tube, a round 96-well plate, or a conical tube to aggregate the pluripotent stem cells and form a single spherical embryoid body; (c) culturing the embryoid bodies in suspension in an embryoid body maturation medium to form mature embryoid bodies; (d) culturing the embryoid body in a xeno-free, serum-free mesenchymal stem cell culture medium to induce differentiation into mesenchymal stem cells; and (e) Proliferating and culturing the differentiated mesenchymal stem cells in a xeno-free, serum-free mesenchymal stem cell culture medium while maintaining the identity of the mesenchymal stem cells.

2. The method of claim 1 , wherein the human pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

3. The method of claim 1 , wherein the human pluripotent stem cells are in an undifferentiated state.

4. 2. The method of claim 1, wherein the human pluripotent stem cells in step (a) are cultured in a culture vessel coated with a material selected from the group consisting of vitronectin, collagen, laminin, matrigel, and heparan sulfate proteoglycan.

5. 2. The method of claim 1, wherein the serum-free medium used to culture the human pluripotent stem cells in step (a) is TeSR™-E8™ (essential 8 media), TeSR™-2, or StemMACS™ iPS-Brew XF, Human Medium.

6. 2. The method of claim 1, wherein the embryoid body formation medium in step (b) is Aggrewell™ EB formation medium, Gibco™ Essential 6™ Medium, CTS™ Essential 6™ Medium, or TeSR™-E6.

7. The method according to claim 1, wherein the culturing in step (b) is carried out for 18 to 30 hours.

8. 2. The method of claim 1, wherein the embryoid body maturation medium in step (c) is a basal medium containing Knock Out™ serum replacement (KSR), non-essential amino acid (NEAA), and β-mercaptoethanol.

9. The method according to claim 8, wherein the basal medium in step (c) is DMEM / F12, alpha MEM, Ham's F12 media, or DMEM.

10. The method according to claim 1, wherein the suspension culture in step (c) is carried out for 10 to 18 days.

11. The method of claim 1, wherein the average size of the mature embryoid bodies obtained in step (c) is 350 to 450 μm.

12. 2. The method of claim 1, wherein the mesenchymal stem cell culture medium in steps (d) and (e) is a xeno-free, serum-free medium containing L-glutamine.

13. 13. The method of claim 12, wherein the xeno-free, serum-free medium is Stenpro® MSC SFM Xeno-free medium, PRIME-XV® MSC Expansion XSFM medium, Human Mesenchymal-XF™ Expansion medium, MSC Nutristem® XF medium, or StemMACS™ MSC expansion media kit XF, human medium.

14. The method of claim 1, wherein the differentiation into mesenchymal stem cells in step (d) is carried out for 12 to 20 days.

15. 2. The method of claim 1, wherein the mesenchymal stem cells obtained in step (e) are mesenchymal stem cells that have multipotency and can differentiate into cells selected from the group consisting of adipocytes, osteocytes, chondrocytes, muscle cells, nerve cells, and cardiomyocytes.

16. The method of claim 1, wherein the mesenchymal stem cells obtained in step (e) express the cell surface markers CD29(+), CD44(+), CD73(+), and CD105(+).

17. The method according to claim 16, wherein the cell surface marker expression is maintained at 90% or more in mesenchymal stem cells for 20 or more passages.

18. 2. The method of claim 1, wherein the mesenchymal stem cells obtained in step (e) are CD34(-), CD45(-), HLA-DR(-), TRA-1-60(-), and TRA-1-81(-) mesenchymal stem cells.

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