Methods and compositions to maintain stem cell quiescence
View Patent ↗Compositions and methods are provided for induction and maintenance of quiescence of stem cells.
1. A serum-free liquid cell culture medium that provides for quiescence of stem cells during in vitro culture, the medium comprising each of components:
elcatonin; MGCD-265, JNJ-7706621, Forskolin, Somatostatin, SB203580, SU5402, TGFbeta and serum replacement; and
a hydrogel substrate.
2. The medium of claim 1 , wherein the hydrogel substrate comprises artificial muscle fibers of 10-100 μm in diameter and 50-5000 μm in length.
3. A method of culturing quiescent stem cells, comprising culturing the cells in the medium according to claim 1 for a period of at least 6 hours.
4. The method of claim 3 , wherein quiescent cells are seeded into the medium.
5. The method of claim 3 , wherein proliferating blast cells are seeded into the medium and induced to quiescence.
6. The method of claim 5 , wherein the blast cells are myoblasts.
7. The method of claim 5 , wherein the blast cells have been expanded in vitro.
8. The method of claim 3 , further comprising contacting the cells with a polynucleotide to genetically modify the cells.
9. The method of claim 3 , further comprising transplanting the cells into a mammalian recipient.
10. The liquid culture medium of claim 1 , wherein the hydrogel substrate comprises a polymer selected from a group comprising polyethylene glycol, poly(vinyl acetate), poly(ethylene acrylate), polyaliphatic polyurethane, polyether polyurethane, polyester polyurethane, polyethylene copolymer, polyamide, polyvinyl alcohol, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate), poly(hydroxyethyl methacrylate), poly (glycolic acid), poly (DL-lactic-co-glycolic acid), polyhydroxyalkanoate, poly(4-hydroxybutirate), sulfonated polymers, polygluconic acid, poly(acrylic acid), polyphosphazenes, polysaccharides, proteins, collagen, elastin, alginate, fibrin, fibronectin, laminin, hyaluronic acid, or another biologically-occurring polymer, polypeptide sequences cleavable by proteases including matrix metalloproteases, or copolymers formed from monomers of one or more of these.
11. The liquid culture medium of claim 1 , wherein the hydrogel stiffness has an elasticity between 1 and 2 kPa.
12. A serum-free liquid culture medium that, when combined with a hydrogel substrate, provides for quiescence of human muscle stem cells during in vitro culture, the medium comprising each of components: elcatonin; Forskolin, SB203580, SU5402, TGFβ, insulin-transferrin-selenium and serum replacement.
13. The liquid culture medium of claim 12 , wherein elcatonin is present at a concentration of from 0.1 to 10 μM, SB203580 is present at a concentration of from 1 to 100 μM, SU5402 is present at a concentration of from 0.1 to 10 μM, Forskolin is present at a concentration of from 0.1 to 10 μM, TGFβ is present at a concentration of from 0.5 to 25 nM, serum-replacement is present at a concentration of from 0.1 to about 10%, and insulin-transferrin-selenium is present at a concentration of from 0.1 to about 10%.
14. A serum-free liquid cell culture medium that provides for quiescence of human muscle stem cells during in vitro culture, the medium comprising each of components:
elcatonin; Forskolin, SB203580, SU5402, TGFβ, insulin-transferrin-selenium and serum replacement; and
a hydrogel substrate at least 5 μm thick.
15. The medium of claim 14 , wherein the hydrogel substrate comprises artificial muscle fibers of 10-100 μm in diameter and 50-5000 μm in length.
16. The liquid culture medium of claim 15 , wherein the hydrogel substrate comprises a polymer selected from a group comprising polyethylene glycol, poly(vinyl acetate), poly(ethylene acrylate), polyaliphatic polyurethane, polyether polyurethane, polyester polyurethane, polyethylene copolymer, polyamide, polyvinyl alcohol, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate), poly(hydroxyethyl methacrylate), poly (glycolic acid), poly (DL-lactic-co-glycolic acid), polyhydroxyalkanoate, poly(4-hydroxybutirate), sulfonated polymers, polygluconic acid, poly(acrylic acid), polyphosphazenes, polysaccharides, proteins, collagen, elastin, alginate, fibrin, fibronectin, laminin, hyaluronic acid, or another biologically-occurring polymer, polypeptide sequences cleavable by proteases including matrix metalloproteases, or copolymers formed from monomers of one or more of these.
17. The liquid culture medium of claim 15 , wherein the hydrogel stiffness has an elasticity between 1 and 2 kPa.
18. The liquid culture medium of claim 14 , wherein elcatonin is present at a concentration of from 0.1 to 10 μM, SB203580 is present at a concentration of from 1 to 100 μM, SU5402 is present at a concentration of from 0.1 to 10 μM, Forskolin is present at a concentration of from 0.1 to 10 μM, TGFβ is present at a concentration of from 0.5 to 25 nM, serum-replacement is present at a concentration of from 0.1 to about 10%, and insulin-transferrin-selenium is present at a concentration of from 0.1 to about 10%.
19. A serum-free liquid culture medium that, when combined with a hydrogel substrate, provides for quiescence of mouse muscle stem cells during in vitro culture, the medium comprising each of components: elcatonin; Forskolin, SB203580, SU5402, TGFβ, insulin-transferrin-selenium and serum replacement.
20. A serum-free liquid cell culture medium that provides for quiescence of mouse muscle stem cells during in vitro culture, the medium comprising each of components:
elcatonin; MGCD-265, JNJ-7706621, Forskolin, somatostatin, SB203580, SU5402, TGFbeta and serum replacement; and
a hydrogel substrate at least 5 μm thick.
21. The medium of claim 20 , wherein the hydrogel substrate comprises artificial muscle fibers of 10-100 μm in diameter and 50-5000 μm in length.
22. The liquid culture medium of claim 21 , wherein the hydrogel substrate comprises a polymer selected from a group comprising polyethylene glycol, poly(vinyl acetate), poly(ethylene acrylate), polyaliphatic polyurethane, polyether polyurethane, polyester polyurethane, polyethylene copolymer, polyamide, polyvinyl alcohol, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate), poly(hydroxyethyl methacrylate), poly (glycolic acid), poly (DL-lactic-co-glycolic acid), polyhydroxyalkanoate, poly(4-hydroxybutirate), sulfonated polymers, polygluconic acid, poly(acrylic acid), polyphosphazenes, polysaccharides, proteins, collagen, elastin, alginate, fibrin, fibronectin, laminin, hyaluronic acid, or another biologically-occurring polymer, polypeptide sequences cleavable by proteases including matrix metalloproteases, or copolymers formed from monomers of one or more of these.
23. The liquid culture medium of claim 22 , wherein the hydrogel stiffness has an elasticity between 1 and 2 kPa.