Compositions and methods for enrichment of neural stem cells using ceramide analogs
View Patent ↗The present invention provides compositions and methods for human neural cell production. More particularly, the present invention provides cellular differentiation methods employing amphiphilic lipid compounds, preferably ceramide analogs of the β-hydroxyalkylamine type and optionally employing an essentially serum free MEDII conditioned medium for the generation of human neural cells from pluripotent human cells. The methods alternatively comprise modulating apoptosis by modifying the levels of PAR-4, with or without the presence of amphiphilic lipid compounds and optionally employing MEDII conditioned medium. The methods alternatively encompass modulating apoptosis by modulating the intracellular concentration of endogenous lipid second messengers, such as ceramide.
1. A method of producing a human neural cell comprising,
a) providing a pluripotent human cell; and
b) culturing the pluripotent human cell with a composition comprising a ceramide compound selected from the group consisting of N-(2-hydroxy-1-(hydroxymethyl)ethyl)-palmitoylamide (“S16”), N-(2-hydroxy-1-(hydroxymethyl)ethyl)-oleoylamide (“S18”), N,N-bis(2-hydroxyethyl)palmitoylamide (“B16”), N,N-bis(2-hydroxyethyl)oleoylamide (“B18”) N-tris(hydroxymethyl)methyl-palmitoylamide (“T16”), N-tris(hydroxymethyl)methyl-oleoylamide (“T18”), N-acetyl sphingosine (“C2-ceramide”), and N-hexanoylsphingosine (“C6-ceramide”) to produce the human neural cell.
2. The method of claim 1 , wherein the pluripotent human cell is a differentiating pluripotent human cell.
3. The method of claim 1 , comprising the intermediate step of forming an embryoid body comprising the pluripotent human cell prior to culturing a cell from the embryoid body with the ceramide compound.
4. The method of claim 3 , wherein the embryoid body is formed by culturing the pluripotent human cell with an essentially serum free medium.
5. The method of claim 4 , wherein the essentially serum free medium is a MEDII conditioned medium.
6. The method of claim 5 , comprising the additional steps of,
a) dispersing the embryoid body to an essentially single cell suspension;
b) culturing the essentially single cell suspension comprising the pluripotent human cell in an adherent culture with a composition comprising the ceramide compound.
7. The method of claim 6 , wherein the composition comprising the ceramide compound further comprises a MEDII conditioned medium.
8. The method of claim 5 wherein the MEDII conditioned medium is a Hep G2 conditioned medium.
9. The method of claim 7 , wherein the composition comprising the ceramide compound is essentially serum free.
10. A method of producing a human neural cell comprising,
a) providing a pluripotent human cell; and
b) culturing the pluripotent human cell with a composition comprising a ceramide compound of the structure
11. A method of producing a human neural cell comprising,
a) providing a pluripotent human cell; and
b) culturing the pluripotent human cell with a composition comprising a ceramide compound of the structure
12. The method of claim 1 , wherein the concentration of the ceramide compound is from approximately 0.1 μM to approximately 1000 μM.
13. The method of claim 1 , wherein the concentration of the ceramide compound is from approximately 1 μM to approximately 100 μM.
14. The method of claim 1 , wherein the concentration of the ceramide compound is from approximately 5 μM to approximately 50 μM.
15. The method of claim 1 , wherein the concentration of the ceramide compound is approximately 10 μM.
16. The method of claim 1 , wherein the duration of culturing the human pluripotent cell with the ceramide compound is from approximately 6 hours to 10 days.
17. The method of claim 1 , wherein the pluripotent human cell is selected from the group consisting of a human embryonic stem cell, a human inner cell mass (ICM)/epiblast cell, a human primitive ectoderm cell, and a human primordial germ cell.
18. The method of claim 1 , wherein the pluripotent human cell is a human embryonic stem cell.
19. The method of claim 1 , wherein the human pluripotent cell is a multipotent cell.
20. The method of claim 19 , wherein the multipotent cell is a neural precursor cell.