Method of making conditioned medium from immortalized dental pulp stem cells

• US 10,639,355 B2
• Filed: 01/23/2015
• Issued: 05/05/2020
• Est. Priority Date: 01/24/2014
• Status: Active Grant

• Current Assignee: Cysay Incorporated
• Original Assignee: Quarrymen & Co. Inc.
• Inventors: Ueda, Minoru
• Primary Examiner(s): Wilson, Michael C
• Application Number:
  US15/113,646
• Publication Number:
  US 20170007677A1
• Time in Patent Office: 1,929 Days
• Field of Search: 435325, 435455
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61K 35/32 Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
• CPC Class Codes: A61K 38/1841 Transforming growth factor [TGF]
• CPC Class Codes: A61K 38/1866 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: A61K 38/195 Chemokines, e.g. RANTES
• CPC Class Codes: A61K 38/30 Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2 insulin-like growth factor binding protein A61K38/1754
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C12N 2500/02 Atmosphere, e.g. low oxygen conditions
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2500/99 Serum-free medium
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/165 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: C12N 2501/21 Chemokines, e.g. MIP-1, MIP-2, RANTES, MCP, PF-4
• CPC Class Codes: C12N 2501/60 Transcription factors
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2506/1361 from dental pulp or dental follicle stem cells
• CPC Class Codes: C12N 2510/04 Immortalised cells
• CPC Class Codes: C12N 5/0664 Dental pulp stem cells, Dental follicle stem cells
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

The invention provides a novel anticancer therapeutic having a high effect against solid cancers, lacking the side effects of chemotherapeutics, and being unlikely to cause resistance. Provided is a pharmaceutical composition for cancer treatment prepared by a method having: a stem cell production step for making immortalized stem cells by introducing four types of genes into deciduous tooth dental pulp stem cells obtained from the dental pulp of mammals; and a condition medium preparation step for culturing the immortalized stem cells for a predetermined length of time in serum-free medium at 23-27° C. under conditions of low oxygen concentration at an oxygen concentration of from 0.5% to less than 20%, the pharmaceutical composition containing 1.5 times or more of insulin-like growth factor (IGF-1) and vascular endothelial growth factor (VEGF) than is contained in condition medium prepared when culturing at an oxygen concentration of 20% and otherwise identical conditions.

Claims

1. A method of making conditioned medium, the method comprising:

(a) transfecting dens deciduous dental pulp stem cells with nucleic acid sequences encoding hTERT, bmi-1, E6 and E7;

(b) selecting an immortalized dens deciduous dental pulp stem cell or cells that has at least one of;

(1) a 1.5 to 3 times higher expression ratio of STRO-1 positive cells as compared to non-transfected dens deciduous dental pulp stem cells at population doubling times 20 or 40, (2) a neonatal bone quantity production ability five times greater than that of a primary dens deciduous dental pulp stem cell at population doubling times of 20, or (3) combinations thereof;

c) culturing immortalized dens deciduous dental pulp stem cells selected in step b) in serum free medium in 0.5% to 1% oxygen at 23-27°

C.; and

d) isolating conditioned medium from the immortalized dens deciduous dental pulp stem cells cultured in step c),

wherein the conditioned medium comprises more than 1.5 times higher concentration of both insulin like growth factor 1 (IGF-1) and vascular endothelial cell growth factor (VEGF) as compared to conditioned medium prepared by culturing immortalized dens deciduous dental pulp stem cells selected in step b) in serum free medium in 20% oxygen at 23-27°

C.

2. The method according to claim 1, wherein said culturing in step c) is for 40 to 56 hours.

3. The method according to claim 1, wherein the conditioned medium comprises at least 5 times higher concentration of transforming growth factor β

1 (TGF-β

1) as compared to conditioned medium prepared by culturing immortalized dens deciduous dental pulp stem cells selected in step b) in serum free medium in 20% oxygen at 23-27°

C.

4. The method according to claim 1, wherein the conditioned medium comprises at least 3 times higher concentration of stromal cell-derived factor 1 (SDF-1) as compared to conditioned medium prepared by culturing immortalized dens deciduous dental pulp stem cells selected in step b) in serum free medium in 20% oxygen at 23-27°

C.