Medium composition for culturing porcine pluripotent stem cells
A medium composition for porcine pluripotent stem cells, containing bFGF, activin A, CHIR99021 and IWR-1 is disclosed. The composition is prepared from specific ingredients, excluding non-specific ingredients, thereby facilitating quality control, and thus is suitable for the study of porcine pluripotent stem cells. Also disclosed is a method for culturing, maintaining and preserving porcine pluripotent stem cells having the ability to form teratomas, by using the composition.
1 . A culture medium composition for culturing porcine pluripotent stem cells, comprising:
a. the culture medium composition comprising
3.03×10 −4 to 6.06×10 −3 μM basic fibroblast growth factor (bFGF);
1.50×10 −4 to 5.40×10 −4 μM Activin A;
0.5 to 2.50 μM CHIR99021 (6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile); and
1.20 to 3.00 μM IWR-1 (4-(1,3,3a,4,7,7a-Hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-Benzamide), and
b. porcine pluripotent stem cells,
wherein the culture medium composition is free of fetal bovine serum and the culture medium composition is capable of maintaining porcine pluripotent stem cells in an undifferentiated state and allowing for their long-term cryopreservation, and
wherein the culture medium composition has the ability to maintain the pluripotent stem cells in an undifferentiated state with in vivo differentiation capacity, and the porcine pluripotent stem cells cultured in the culture medium composition form teratomas and/or chimeras.
2 . The culture medium composition of claim 1 , wherein the culture medium further comprises Dulbecco's Modified Eagle's Medium (DMEM) comprising a serum replacement, lipid concentrate (LC), L-arginine and/or L-glutamine, Modified Eagle's Medium (MEM) nonessential amino acids, antibacterial agents, antifungal agents, and beta-mercaptoethanol.
3 . The culture medium composition of claim 1 , comprising:
about 6.06×10 −4 μM bFGF;
about 3.85×10 −4 μM Activin A;
about 0.5 μM CHIR99021; and
about 1.5 μM IWR-1, and
wherein the culture medium further comprises Dulbecco's Modified Eagle's Medium (DMEM) comprising about 20% (v/v) of bovine serum replacement; about 0.1% (v/v) of lipid concentrate (LC); about 1% (v/v) of L-arginine and/or L-glutamine; about 1% (v/v) of Modified Eagle's Medium (MEM) nonessential amino acids; about 1% (v/v) of antibiotics-antimycotics; and about 0.1 mM β-mercaptoethanol.
4 . The culture medium composition of claim 1 , wherein the culture medium is Dulbecco's Modified Eagle's Medium (DMEM) comprising about 15% (v/v) of the serum replacement; about 0.1% (v/v) of the lipid concentrate (LC); about 1% (v/v) of L-arginine and/or L-glutamine; about 1% (v/v) of Modified Eagle's Medium (MEM) nonessential amino acids; about 1% (v/v) of antibiotics-antimycotics; about 0.1 mM β-mercaptoethanol; about 1.21×10 −3 μM bFGF; about 3.85×10 −4 μM activin A; about 1.5 μM CHIR99021; and about 2.5 μM IWR-1.
5 . A cryopreservation composition for cryopreserving porcine pluripotent stem cells, the cryopreservation composition comprising about 10% (v/v) of dimethyl sulfoxide (DMSO), about 10% (v/v) of ethylene glycol, and about 80% (v/v) of the culture medium composition according to claim 4 .
6 . A cryopreservation composition for cryopreserving porcine pluripotent stem cells, the cryopreservation composition comprising about 20% (v/v) of dimethyl sulfoxide (DMSO), about 20% (v/v) of ethylene glycol, about 0.5M sugar, and about 60% (v/v) of the culture medium composition according to claim 4 .
7 . A method for culturing porcine pluripotent stem cells comprising culturing the porcine pluripotent stem cells in the culture medium composition according to claim 1 .
8 . A method for maintaining porcine pluripotent stem cells comprising maintaining the porcine pluripotent stem cells in the culture medium composition of claim 1 .
9 . A method for preserving porcine pluripotent stem cells comprising preserving the porcine pluripotent stem cells in the culture medium composition of claim 1 .
10 . The culture medium composition of claim 2 , wherein the culture medium is DMEM comprising about 20% (v/v) of the serum replacement; about 0.1% (v/v) of the LC; about 1% (v/v) of L-arginine-L-glutamine; about 1% (v/v) of MEM nonessential amino acids; about 1% (v/v) of antibiotics-antimycotics; about 0.1 mM β-mercaptoethanol; about 6.06×10 −4 μM bFGF; about 3.85×10 −4 μM Activin A; about 0.5 μM CHIR99021; and about 1.5 μM IWR-1.
11 . The culture medium composition of claim 1 , wherein the culture medium is DMEM comprising about 15% (v/v) of the serum replacement; about 0.1% (v/v) of the LC; about 1% (v/v) of L-arginine-L-glutamine; about 1% (v/v) of MEM nonessential amino acids; about 1% (v/v) of antibiotics-antimycotics; about 0.1 mM β-mercaptoethanol; about 1.21×10 −3 μM bFGF; about 3.85×10 −4 μM activin A; about 1.5 μM CHIR99021; and about 2.5 μM IWR-1.
12 . A cryopreservation composition for cryopreserving porcine pluripotent stem cells, the cryopreservation composition comprising about 10% (v/v) of dimethyl sulfoxide (DMSO), about 10% (v/v) of ethylene glycol, and about 80% (v/v) of the culture medium composition according to claim 11 .
13 . A cryopreservation composition for cryopreserving porcine pluripotent stem cells, the cryopreservation composition comprising about 20% (v/v) of dimethyl sulfoxide (DMSO), about 20% (v/v) of ethylene glycol, about 0.5M sugar, and about 60% (v/v) of the culture medium composition according to claim 11 .
14 . A method for culturing porcine pluripotent stem cells comprising culturing the porcine pluripotent stem cells in the culture medium composition according to claim 2 .
15 . A method for maintaining porcine pluripotent stem cells comprising maintaining the porcine pluripotent stem cells in the culture medium composition of claim 2 .
16 . A method for preserving porcine pluripotent stem cells comprising preserving the porcine pluripotent stem cells in the culture medium composition of claim 2 .
17 . The culture medium composition according to claim 1 , wherein the culture medium composition has an ability to maintain pluripotency of the porcine pluripotent stem cells, and the porcine pluripotent stem cells cultured in the culture medium composition express SSEA4, TRA-1-60 and TRA-1-81.