# US 2021/0340498 A1 · Medium for direct differentiation of pluripotent stem cell-derived mesenchymal stem cell, method for preparing mesenchymal stem cell by using same, and mesenchymal stem cell prepared thereby

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- Number: US 2021/0340498 A1
- Document ID: US20210340498A1
- Status: Published application
- Kind: A1
- Application number: 17/274,029
- Filed: 2019-09-06
- Published: 2021-11-04
- Earliest claimed: 2018-09-07
- Assignees: Cha Biotech Co., Ltd.
- Applicants: Cha Biotech Co., Ltd.
- Inventors: Dong Ryul LEE; Jeong Eun LEE; Soo Kyung JUNG; Kyung Soon PARK; Ji Hoon PARK
- Web page: https://patentgrep.com/patent/US20210340498A1

## CPC

- C12N 5/0668: {Mesenchymal stem cells from other natural sources}
- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- C12N 2500/30: Organic components
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/15: Transforming growth factor beta (TGF-β)
- C12N 2501/727: Kinases (EC 2.7.)
- C12N 2501/73: Hydrolases (EC 3.)
- C12N 2501/734: Proteases (EC 3.4.)
- C12N 2502/1323: Adult fibroblasts
- C12N 2506/02: from embryonic cells
- C12N 2513/00: 3D culture
- C12N 2527/00: Culture process characterised by the use of mechanical forces, e.g. strain, vibration
- C12N 2533/00: Supports or coatings for cell culture, characterised by material
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

The present invention relates to a medium for direct differentiation of embryonic stem cell-derived mesenchymal stem cells, a method of preparing mesenchymal stem cells by using same, mesenchymal stem cells prepared thereby, and a cell therapy product comprising the same mesenchymal stem cells. In a medium composition and a method according to an embodiment, mesenchymal stem cells may be prepared at high yield within a short period of time. In addition, the method is simple in preparation procedure because of the absence of an embryoid body formation step and allows homogeneous cells to be prepared, thus advantageously providing a cell therapy product within a reduce period of time, compared to conventional methods.

## Claims

### Claim 1

A medium composition for inducing differentiation of pluripotent stem cells into mesenchymal stem cells comprising a DNA repair agent and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor.

### Claim 2 (depends on 1)

The medium composition of claim 1, wherein the DNA repair agent is 3-[(benzylamino)sulfonyl]-4-bromo-N-(4-bromophenyl)benzamide or 4-bromo-N-(4-bromophenyl)-3-[[phenylmethyl)amino]sulfonyl]-benzamide.

### Claim 3 (depends on 1)

The method of claim 1, wherein the ROCK inhibitor is one selected from Fasudil, Ripasudil, 4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide, 4-(1-aminoethyl)-N-(1H-pyrrolo(2,3-b)pyridin-4-yl)cyclohexanecarboxamide dihydrochloride, N-(6-fluoro-1H-indazol-5-yl)-1,4,5,6-tetrahydro-2-methyl-6-oxo-4-[4-(trifluoromethyl)phenyl]-3-pyridinecarboxamide, 1-(3-hydroxybenzyl)-3-[4-(pyridin-4-yl)thiazol-2-yl]urea, 2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride, N-[2-[2-(dimethylamino)ethoxy]-4-(1H-pyrazol-4-yl)phenyl-2,3-dihydro-1,4-benzodioxin-2-carboxamide dihydrochloride], 2-fluoro-N-[[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)phenyl]methyl]benzenemethanamine dihydrochloride, N-[3[[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-6-yl]oxy]phenyl]-4-[2-(4-morpholinyl)ethoxy]benzamide, (3S)-1-[[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-7-yl]carbonyl]-3-pyrrolidinamine dihydrochloride, N-[(1 S)-2-hydroxy-1-phenylethyl]-N′4-[4-(4-pyridinyl)phenyl]-urea, Azaindole-1, and Narciclasine.

### Claim 4 (depends on 1)

The medium composition of claim 1, further comprising a TGF-β inhibitor.

### Claim 5 (depends on 4)

The medium composition of claim 4, wherein the TGF-β inhibitor is one selected from 4-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]-pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide hydrate, 4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline, 2-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]-1,5-naphthyridine, 4-(5-benzol[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide hydrate, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide hydrate, 44-[4-(3,4-methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide hydrate, 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide, 2-(3-(6-methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine, 4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline, 2-[4-(1,3-benzodioxol-5-yl)-2-(1,1-dimethylethyl)-1H-imidazol-5-yl]-6-methyl-pyridine, 2-(5-chloro-2-fluorophenyl)-4-[(4-pyridyl)amino]pteridine, 6-[2-tert-butyl-5-(6-methyl-pyridin-2-yl)-1H-imidazol-4-yl]-quinoxaline, 4-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]-pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide hydrate, 4-[2-fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazole-1-ethanol, and 3-[[5-(6-methyl-2-pyridinyl)-4-(6-quinoxalinyl)-1H-imidazol-2-yl]methyl]benzamide.

### Claim 6 (depends on 1)

The medium composition of claim 1, wherein the DNA repair agent is contained in a concentration of 5 μM to 20 μM.

### Claim 7 (depends on 1)

The medium composition of claim 1, wherein the ROCK inhibitor is contained in a concentration of 5 μM to 20 μM.

### Claim 8 (depends on 4)

The medium composition of claim 4, wherein the TGF-β inhibitor is contained in a low concentration of 0.2 μM to 2.0 μM.

### Claim 9

A method of preparing pluripotent stem cell-derived mesenchymal stem cells, the method comprising:
culturing isolated pluripotent stem cells; and
inducing differentiation of the cultured pluripotent stem cells into mesenchymal stem cells by culturing the pluripotent stem cells in a medium comprising a DNA repair agent and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor.

### Claim 10 (depends on 9)

The method of claim 9, further comprising differentiating the cultured embryonic stem cells into mesodermal cells in the presence of a TGF-β inhibitor.

### Claim 11 (depends on 9)

The method of claim 9, further comprising: culturing the differentiation-induced mesenchymal stem cells in a mesenchymal stem cell maturation medium; or subculturing the mesenchymal stem cells cultured in the maturation medium.

### Claim 12 (depends on 9)

The method of claim 9, wherein the culturing of isolated pluripotent stem cells is performed in a culture dish coated with a cell adhesion enhancer in the absence of feeder cells.

### Claim 13 (depends on 9)

The method of claim 9, wherein the method does not comprise a process of forming an embryoid body.

### Claim 14 (depends on 9)

The method of claim 9, wherein the DNA repair agent is 3-[(benzylamino)sulfonyl]-4-bromo-N-(4-bromophenyl)benzamide or 4-bromo-N-(4-bromophenyl)-3-[[phenylmethyl)amino]sulfonyl]-benzamide.

### Claim 15 (depends on 9)

The method of claim 9, wherein the ROCK inhibitor is one selected from Fasudil, Ripasudil, 4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide, 4-(1-aminoethyl)-N-(1H-pyrrolo(2,3-b)pyridin-4-yl)cyclohexanecarboxamide dihydrochloride, N-(6-fluoro-1H-indazol-5-yl)-1,4,5,6-tetrahydro-2-methyl-6-oxo-4-[4-(trifluoromethyl)phenyl]-3-pyridinecarboxamide, 1-(3-hydroxybenzyl)-3-[4-(pyridin-4-yl)thiazol-2-yl]urea, 2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride, N-[2-[2-(d imethylamino)ethoxy]-4-(1H-pyrazol-4-yl)phenyl-2,3-dihydro-1,4-benzodioxin-2-carboxamide dihydrochloride], 2-fluoro-N-[[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)phenyl]methyl]benzenemethanamine dihydrochloride, N-[3-[[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-6-yl]oxy]phenyl]-4-[2-(4-morpholinyl)ethoxy]benzamide, (3S)-1-[[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-7-yl]carbonyl]-3-pyrrolidinamine dihydrochloride, N-[(1S)-2-hydroxy-1-phenylethyl]-N′4-[4-(4-pyridinyl)phenyl]-urea, Azaindole-1, and Narciclasine.

### Claim 16 (depends on 9)

The method of claim 9, wherein the TGF-β inhibitor is one selected from 4-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]-pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide hydrate, 4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline, 2-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]-1,5-naphthyridine, 4-(5-benzol[1,3]dioxo1-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide hydrate, 4-[4-(1,3-benzodioxo1-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide hydrate, 4-[4-(3,4-methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide hydrate, 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide, 2-(3-(6-methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine, 4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline, 2-[4-(1,3-benzodioxo1-5-yl)-2-(1,1-dimethylethyl)-1H-imidazol-5-yl]-6-methyl-pyridine, 2-(5-chloro-2-fluorophenyl)-4-[(4-pyridyl)amino]pteridine, 6-[2-tert-butyl-5-(6-methyl-pyridin-2-yl)-1H-imidazol-4-yl]-quinoxaline, 4-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]-pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide hydrate, 4-[2-fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazole-1-ethanol, and 3-[[5-(6-methyl-2-pyridinyl)-4-(6-quinoxalinyl)-1H-imidazol-2-yl]methyl]benzamide.

### Claim 17 (depends on 10)

The method of claim 10, wherein the differentiating the cultured embryonic stem cells into the mesodermal cells in the presence of the TGF-β inhibitor comprises culturing the cells in a medium containing the TGF-β inhibitor for 2 days to 7 days.

### Claim 18 (depends on 9)

The method of claim 9, wherein the inducing of differentiation in the medium comprises inducing differentiation in a medium comprising a DNA repair agent, a ROCK inhibitor, and a TGF-β inhibitor for 1 day to 2 days.

### Claim 19 (depends on 11)

The method of claim 11, wherein the culturing of the differentiated mesenchymal stem cells in the maturation medium comprises: treating the cells with trypsin; culturing the cells in a Dulbecco's Modified Eagle Medium:Nutrient Mixture F-12 (DMEM/F12) maturation medium; and centrifuging the cells cultured in the maturation medium.

### Claim 20 (depends on 11)

The method of claim 11, wherein the subculturing is performed by subculturing the cells in a culture dish coated with a cell adhesion enhancer in the presence of trypsin.

### Claim 21 (depends on 20)

The method of claim 20, wherein the subculturing is performed from 1 passage to 10 passages.

### Claim 22 (depends on 11)

The method of claim 11, wherein the subculturing comprises removing exfoliated non-mesodermal stem cells and isolating mesodermal stem cells therefrom.

### Claim 23 (depends on 9)

The method of claim 9, wherein the pluripotent stem cells are nuclear transfer pluripotent stem cells (NT-hPSC), parthenote-derived human pluripotent stem cells (pn-hPSC), induced pluripotent stem cells (iPSC), or embryonic stem cells (ESC).

### Claim 24 (depends on 9)

The method of claim 9, wherein, in the inducing of differentiation into the mesenchymal stem cells, the mesenchymal stem cells are immature mesenchymal stem cells and prepared via the immature mesenchymal stem cells.

### Claim 25 (depends on 24)

The method of claim 24, wherein 10% or less of the immature mesenchymal stem cells in the population express a hemangioblast marker VEGFR2 and the differentiation occurs not via hemangioblasts.

### Claim 26 (depends on 9)

The method of claim 9, wherein 70% or more of the prepared mesenchymal stem cells in the population are positive for CD29, CD44, CD90, or CD105 marker and 10% or less of the mesenchymal stem cells in the population are negative for TRA-160, CD34, or VEFGR2 marker.

### Claim 27 (depends on 9)

The method of claim 9, wherein the mesenchymal stem cells have the ability to differentiate into at least one selected from hematopoietic stem cells, myocytes, cardiomyocytes, hepatocytes, chondrocytes, epithelial cells, urinary cells, renal cells, vascular cells, retinal cells, and neuronal cells.

### Claim 28

Pluripotent stem cell-derived mesenchymal stem cells differentiation-induced in the presence of a DNA repair agent and a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor.

### Claim 29 (depends on 28)

The mesenchymal stem cells of claim 28, wherein 70% or more of the mesenchymal stem cells in the population are positive for CD29, CD44, CD90, or CD105 marker and 10% or less of the mesenchymal stem cells in the population are negative for TRA-160, CD34, or VEFGR2 marker.

### Claim 30 (depends on 9)

A method of preparing a cell therapy product comprising mesenchymal stem cells, the method comprising subculturing the mesenchymal stem cells prepared according to the method of claim 9 for at least 3 passages, wherein a period during which differentiation of pluripotent stem cells is induced and culturing is performed for application to the cell therapy product is from 20 days to 40 days.