Preparation method of human-derived cardiac stem cell spheroid and use thereof

Patent metadata

Field	Value
Publication number	US-11692171-B2
Application number	US-201816627258-A
Country	US
Kind code	B2
Filing date	Jun 29, 2018
Priority date	Jun 30, 2017
Publication date	Jul 4, 2023
Grant date	Jul 4, 2023

Abstract

The present invention relates to a preparation method of a spheroid using human-derived cardiac stem cells and a therapeutic use for ischemic heart disease using the myocardial regeneration effect thereof. The spheroid using the cardiac stem cells provided in the present invention has excellent myocardial differentiation ability and regenerative therapeutic ability as compared to existing cardiac stem cells, and thus may be used for the treatment of ischemic heart disease such as myocardial infarction.

First claim

The invention claimed is:

1. A method for preparing a cardiac stem cell spheroid, comprising,
culturing the cardiac stem cells (CSCs) within 3 days in a culture vessel that inhibits cell adhesion,
wherein the culture vessel comprises a medium comprising serum at a concentration of 0.1% to 1%, and
wherein the CSCs are seeded at a density of between 1×10 2 cells/cm 2 to 2×10 5 cells/cm 2 in the medium.

2. The method of claim 1 , wherein the spheroid prepared above has a size of 50 μm to 200 μm.

3. The method of claim 1 , wherein the spheroid prepared above has a cell number of 100 to 2,000.

4. The method of claim 1 , wherein the prepared cardiac stem cell spheroid has a low level of caspase 3/7 activity compared to a cardiac stem cell spheroid prepared by seeding at a seeding density of 2.5×10 5 cells/cm 2 or higher.

5. The method of claim 1 , wherein the prepared cardiac stem cell spheroid has less than ½ folds of caspase 3/7 activity compared to a cardiac stem cell spheroid prepared by seeding at a seeding density of 5×10 5 cells/cm 2 or higher.

6. The method of claim 1 , wherein the prepared cardiac stem cell spheroid is characterized in that the expression of cardiac muscle differentiation factors BMP2, BMP6, SOX4 and SOX9 and angiogenesis factors GPNMB, ESM1, FMNL3, HEY1, PTGS2 and MGP is increased; the expression of an apoptotic enzyme CASP1 mRNA is decreased; and the expression of anti-inflammatory and immuno-suppressive cytokine IL-24 is increased, compared to cardiac stem cells cultured by monolayer culture.

7. The method of claim 1 , wherein the prepared cardiac stem cell spheroid is characterized in that the expression of proangiogenic factors amphiregulin, DPPIV, FGF-7, HGF, IGFBP-1, prolactin and VEGF-C is increased; and the expression of anti-angiogenenic factors platelet factors 4(PF4) and thrombospondin-1 (TSP-1) is decreased, compared to cardiac stem cells cultured by monolayer culture.

8. The method of claim 1 , wherein the prepared cardiac stem cell spheroid is characterized in that the expression levels of IGF, VEGF, HGF, and Ang-1 are increased compared to human cardiac stem cells cultured by monolayer culture.

9. The method of claim 1 , wherein the prepared cardiac stem cell spheroid is characterized in that the expression of myosin heavy chain (MHC), desmin, troponin (TNI) and alpha-sarcomeric actinin (α-SA) is increased by Wnt signaling compared to cardiac stem cells cultured by monolayer culture.

Assignees

• Univ Inje Ind Acad Coop Found
• Hierabio Inc

Inventors

• Lee Seung Jin
• Yang Young-Il

Classifications

• C12N2513/00

  3D culture · CPC title

• A61K35/34 Primary

  Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes (vascular smooth muscle A61K35/44) · CPC title

• C12N5/0657 Primary

  Cardiomyocytes; Heart cells · CPC title

• C12N5/0662 Primary

  Stem cells · CPC title

• C12N2501/415

  Wnt; Frizzeled · CPC title