Equine amniotic fluid-derived multipotent stem cells and a method for producing the same

1. A method for preparing equine amniotic fluid-derived multipotent stem cells, comprising

Step 1 of isolating mononuclear cells from equine amniotic

Step 2 of culturing the mononuclear cells in basal culture media for a period time, wherein the cultured mononuclear cells are adherent; and

wherein the basal culture media comprises an endothelial cell growth media containing vascular endothelial growth factor (VEGF), long R 3 -insulin-like growth factor; (R 3 -IGF), and human epidermal growth factor(h-EGF); and

Step 3 of culturing the adherent cells in Dulbecco's Modified Eagle's Medium (DMEM) after culturing in the basal media; and

Step 4 of recovering the stem cells from the culture, wherein the recovered multipotent stem cells:

(a) are negative for the human markers CD19, CD20, CD28, CD31, CD34, CD38, CD41a, CD62L, CD62P, and CD200, and

(b) are positive the human markers CD44, CD90, and CD105;

(c) have the ability to differentiate into, mesoderm derived cells;

(d) have the ability to maintain an undifferentiated state for 14 passages or more; and

(e) are more homogeneous than those before culture.

2. The method according to claim 1 , isolation of the mononuclear cells is performed with a density-gradient solution and the mononuclear cells is performed with a density-gradient solution and the mononuclear cells are isolated from a middle layer.

3. The method according to claim 1 , wherein the cells are adherent and cultured in monolayer.

4. The method according to claim 1 , wherein the basal culture media contains fetal bovine serum (FBS).

5. The method according to claim 1 , wherein the DMEM is low glucose-DMEM containing FBS.

6. A method for differentiating multipotent stem cells into chondrocytes, comprising:

preparing equine amniotic fluid-derived multipotent stem cells by the method of claim 1 ; and

culturing the multipotent stem cells in a chondrogenic induction medium.

7. A method for differentiating multipotent stem cells into osteocytes, comprising:

preparing equine amniotic fluid-derived multipotent stem cells by the method of claim 1 ; and

culturing the multipotent stem cells in a culture medium comprising dexamethasone, beta-glycerophosphate and ascorbic acid 2-phosphate.

8. A method for differentiating multipotent stem cells into adipocytes, comprising:

preparing equine amniotic fluid-derived multipotent stem cells by the method of claim 1 ; and

culturing the multipotent stem cells in a culture medium comprising dexamethasone, indomethacin, 3-isobutyl methylxanthine and insulin to make adipocytes.