Method for selecting highly efficient stem cell, using protein marker grp78

Patent metadata

Field	Value
Publication number	US-2021009946-A1
Application number	US-201816755370-A
Country	US
Kind code	A1
Filing date	Oct 15, 2018
Priority date	Oct 13, 2017
Publication date	Jan 14, 2021
Grant date	—

Abstract

The present invention relates to a method of screening highly efficiently stem cells using the protein marker GRP78, and more particularly, to a method of removing old stem cells with decreased expression of GRP78 and isolating only highly efficient stem cells. The use of the protein marker GRP78 according to the present invention makes it possible to isolate only highly efficient stem cells by removing old stem cells. Thus, the protein marker GRP78 may be used as an effective marker for controlling the quality of stem cell therapy products and evaluating the stability and effectiveness thereof, and is useful in the production of stem cell therapy products having excellent therapeutic efficacy.

First claim

1 . A method of isolating highly efficient stem cells using an antibody or aptamer specific for GRP78.

2 . The method of claim 1 , comprising steps of:
(a) binding a GRP78-specific primary antibody to stem cells; (b) binding a secondary antibody-immobilized magnetic bead to the primary antibody; and (c) isolating GRP78-expressing stem cells using MACS (magnetic-activated cell sorting).

3 . The method of claim 1 , wherein the highly efficient stem cells express GRP78 in the membrane.

4 . The method of claim 2 , wherein step (c) further comprises a step of removing stem cells expressing a low level of GRP78 in the membrane.

5 . The method of claim 4 , wherein the stem cells expressing a low level of GRP78 in the membrane are old stem cells.

6 . A composition for screening highly efficient stem cells, the composition comprising an agent capable of measuring the expression level of mRNA of GRP78 gene or a protein encoded by the gene.

7 . The composition of claim 6 , wherein the agent capable of measuring the expression level of the mRNA is a primer capable of amplifying the GRP78 gene or a probe that binds specifically to the GRP78 gene.

8 . The composition of claim 6 , wherein the agent capable of measuring the expression level of the protein is an antibody or aptamer specific for the protein encoded by the GRP78 gene.

9 . The composition of claim 6 , wherein the highly efficient stem cells are non-senescent young stem cells.

10 . A kit for screening highly efficient stem cells, the kit comprising the composition of claim 6 .

11 . The kit of claim 10 , wherein the kit is an RT-PCR kit, a DNA chip kit or a protein chip kit.

12 . A method for screening highly efficient stem cells, the method comprising steps of:
(a) extracting a DNA or protein from stem cells; (b) measuring the expression level of mRNA of GRP78 gene or a protein encoded by the gene; (c) comparing the measured expression level of mRNA of the gene or the protein encoded by the gene with a control; and (d) selecting the stem cells as highly efficient stem cells if the expression level increases compared to the control.

13 . The method of claim 12 , wherein the expression level of mRNA of the gene is measured by any one selected from the group consisting of polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, RNase protection assay (RPA), microarray, and Northern blotting.

14 . The method of claim 12 , wherein the expression level of the protein is measured by any one selected from the group consisting of Western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, flow cytometry, immunofluorescence assay, Ouchterlony assay, complement fixation assay, and protein chip assay.

15 . The method of claim 12 , wherein the control is old stem cells.

16 . The method of claim 12 , wherein the highly efficient stem cells are non-senescent young stem cells.

Assignees

• Nibec Co Ltd
• Nat Univ Chungbuk Ind Acad Coop Found
• Seoul Nat Univ R&Db Foundation

Inventors

• Park Yoon Shin
• Park Yoon Jeong
• Chung Chong-Pyoung
• Lee Jue-Yeon
• Choi Da Hyeon

Classifications

• G01N33/56966 Primary

  Animal cells · CPC title

• G01N33/5073

  Stem cells · CPC title

• C12Q2600/158

  Expression markers · CPC title

• C12N5/0081

  Purging biological preparations of unwanted cells · CPC title

• C07K16/28

  against receptors, cell surface antigens or cell surface determinants · CPC title