Method of deriving progenitor cell line
View Patent ↗We disclose a method comprising: (a) providing an embryonic stem (ES) cell; and (b) establishing a progenitor cell line from the embryonic stem cell; in which the progenitor cell line is selected based on its ability to self-renew. Preferably, the method selects against somatic cells based on their inability to self-renew. Preferably, the progenitor cell line is derived or established in the absence of co-culture, preferably in the absence of feeder cells, which preferably selects against embryonic stem cells. Optionally, the method comprises (d) deriving a differentiated cell from the progenitor cell line.
1. A method of screening a candidate molecule for its effect on a human mesenchymal progenitor cell, the method comprising:
(a) providing a parental embryonic stem cell or descendants of a parental embryonic stem cell obtained by dispersing an embryonic stem cell colony with trypsin, wherein the parental embryonic stem cell is a human embryonic stem cell; and
(b) culturing the parental embryonic stem cell in the absence of feeder cells in rich media comprising (i) essential nutrients required for cell growth and (ii) serum or serum replacement, wherein the rich media does not comprise additional grown regulators or hormones that promote growth of embryonic stem cells, and wherein the culturing produces mesenchymal progenitor cells which self renew;
thereby establishing in the absence of transformation a mesenchymal progenitor cell line from the mesenchymal progenitor cells which self-renew; wherein the mesenchymal progenitor cell line is maintainable in cell culture for more than 20 generations, and wherein the mesenchymal progenitor cell line is lineage restricted compared to the parental embryonic stem cell;
(c) culturing cells from the mesenchymal progenitor cell line in the presence of a candidate molecule; and
(d) determining the effect of a candidate molecule on cells from the mesenchymal progenitor cell line treated with the candidate molecule compared with untreated cells from the mesenchymal progenitor cell line or cells from the mesenchymal progenitor cell line treated with an inert compound.
2. The method according to claim 1 , wherein the mesenchymal progenitor cell line is maintainable in cell culture for more than 25 generations.
3. The method according to claim 1 , wherein the mesenchymal progenitor cell line is maintainable in cell culture for more than 30 generations.
4. The method according to claim 1 , wherein the mesenchymal progenitor cell line is maintainable in cell culture for more than 35 generations.
5. The method according to claim 1 , wherein the mesenchymal progenitor cell line is maintainable in cell culture for more than 40 generations.
6. A method of screening a candidate molecule for its effect on a cell differentiated from a human mesenchymal progenitor cell line, the method comprising:
(a) providing a parental embryonic stem cell or descendants of a parental embryonic stem cell obtained by dispersing an embryonic stem cell colony with trypsin, wherein the parental embryonic stem cell is a human embryonic stem cell; and
(b) culturing the parental embryonic stem cell in the absence of feeder cells in rich media comprising (i) essential nutrients required for cell growth and (ii) serum or serum replacement, wherein the rich media does not comprise additional grown regulators or hormones that promote growth of embryonic stem cells, and wherein the culturing produces mesenchymal progenitor cells which self-renew;
thereby establishing in the absence of transformation a mesenchymal progenitor cell line from the mesenchymal progenitor cells which self-renew; wherein the mesenchymal progenitor cell line is maintainable in cell culture for more than 20 generations, and wherein the mesenchymal progenitor cell line is lineage restricted compared to the parental embryonic stem cell;
(c) deriving a differentiated cell from the mesenchymal progenitor cell line;
(d) treating the differentiated cell with a candidate molecule;
and
(e) determining the effect of a candidate molecule on the differentiated cell treated with the candidate molecule compared with untreated differentiated cells or differentiated cells treated with an inert compound.
7. The method according to claim 6 , wherein the mesenchymal progenitor cell line is maintainable in cell culture for more than 25 generations.
8. The method according to claim 6 , wherein the mesenchymal progenitor cell line is maintainable in cell culture for more than 30 generations.
9. The method according to claim 6 , wherein the mesenchymal progenitor cell line is maintainable in cell culture for more than 35 generations.
10. The method according to claim 6 , wherein the mesenchymal progenitor cell line is maintainable in cell culture for more than 40 generations.
11. The method according to claim 1 , wherein the rich media is DMEM with D-glucose, fetal calf serum, non-essential amino acids, L-glutamine, and β-mercaptoethanol.
12. The method according to claim 6 , wherein the rich media is DMEM with D-glucose, fetal calf serum, non-essential amino acids, L-glutamine, and β-mercaptoethanol.
13. The method according to claim 1 , wherein the mesenchymal progenitor cells which self-renew are CD29 + , CD44 + , CD105 + , CD166 + , CD34 − , and CD45 − .
14. The method according to claim 6 , wherein the mesenchymal progenitor cells which self-renew are CD29 + , CD44 + , CD105 + , CD166 + , CD34 − , and CD45 − .