Method of making conditioned medium from immortalized dental pulp stem cells
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.
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 β1 (TGF-β1) 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.