# US 12,000,825 B2 · Method of selecting stem cells having ability to produce extracellular vesicles with high efficiency using activation of protease-activated receptor-mediated signaling pathways

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- Number: US 12,000,825 B2
- Document ID: US12000825B2
- Status: Granted
- Kind: B2
- Application number: 16/961,851
- Filed: 2020-04-28
- Published: 2021-08-19
- Granted: 2024-06-04
- Earliest claimed: 2019-04-29
- Assignees: Samsung Life Public Welfare Foundation
- Applicants: Samsung Life Public Welfare Foundation
- Inventors: Yun Sil Chang; Won Soon Park; Dong Kyung Sung
- Web page: https://patentgrep.com/patent/US12000825B2

## CPC

- G01N 33/5023: {on expression patterns}
- C12N 5/0665: {Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood}
- G01N 33/5073: {Stem cells}
- G01N 33/573: for enzymes or isoenzymes
- C12N 2501/734: Proteases (EC 3.4.)
- G01N 2333/91215: with a definite EC number (2.7.1.-)
- G01N 2333/914: Hydrolases (3)
- G01N 2333/974: Thrombin
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present disclosure relates to a method of selecting stem cells having the ability to produce extracellular vesicles with high efficiency, the method including the step of measuring the activity of protease-activated receptor (PAR)-mediated signaling pathways, stem cells selected by the method, and a method of screening an inducer for the production of extracellular vesicles. According to the present disclosure, upon treatment of stem cells with thrombin, the production of extracellular vesicles in the stem cells and the levels of proteins in the extracellular vesicles are significantly increased via PAR-mediated signaling pathways, and thus stem cells having the ability to produce extracellular vesicles with high efficiency can be efficiently selected by treating stem cells with thrombin and measuring an activation level of a PAR-mediated signaling pathway, and stem cells selected by this method can be effectively used in related research and clinical fields.

## Claims

### Claim 1

A method of selecting a stem cell having an ability to produce extracellular vesicles with high efficiency, the method comprising measuring an activation level of a protease-activated receptor (PAR)-mediated signaling pathway,
wherein the PAR is PAR1 or PAR3,
wherein the ability to produce extracellular vesicles with high efficiency is promotion of the production of extracellular vesicles or promotion of a level of a protein in extracellular vesicles, and
wherein the measurement of the activation level of the PAR-mediated signaling pathway comprises the following processes:
(a) culturing stem cells and then treating the stem cells with thrombin;
(b) measuring an expression level of one or more selected from the group consisting of Ras-related protein Rab-5 (Rab-5), early endosome antigen-1 (EEA-1), phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2), and phospho-protein kinase B (p-AKT) in the thrombin-treated stem cells; and
(c) selecting a stem cell in which the expression level has been increased, as a stem cell having an ability to produce extracellular vesicles with high efficiency.

### Claim 2 (depends on 1)

The method of claim 1, wherein the protein comprises one or more selected from the group consisting of a vascular endothelial growth factor (VEGF), a hepatocyte growth factor (HGF), angiogenin, and angiopoietin-1.

### Claim 3 (depends on 1)

The method of claim 1, wherein the stem cell is an embryonic stem cell or an adult stem cell.

### Claim 4 (depends on 3)

The method of claim 3, wherein the adult stem cell comprises one or more selected from the group consisting of a mesenchymal stem cell, a human tissue-derived mesenchymal stromal cell, a human tissue-derived mesenchymal stem cell, and a multipotent stem cell.

### Claim 5 (depends on 4)

The method of claim 4, wherein the mesenchymal stem cell is derived from one or more tissues selected from the group consisting of an umbilical cord, umbilical cord blood, bone marrow, fat, muscle, skin, an amniotic membrane, and a placenta.

### Claim 6 (depends on 1)

The method of claim 1, wherein the thrombin is treated at 1 U/mL to 10 U/mL.