Method for producing retinal pigment epithelial cells
View Patent ↗The present invention provides a production method of a retinal pigment epithelial cell containing the following steps: (1) a first step for culturing a pluripotent stem cell in a medium containing at least one kind selected from the group consisting of an FGF receptor inhibitor and an MEK inhibitor for a period of not more than 30 days, and (2) a second step for culturing the cell obtained in the first step in a medium containing at least one kind selected from the group consisting of a Rho signal transduction pathway inhibitor and an apoptosis inhibitor to form a retinal pigment epithelial cell. The method can produce retinal pigment epithelial cells from pluripotent stem cells more efficiently and conveniently.
1. A method of producing a retinal pigment epithelial cell comprising the following steps:
(1) a first step for culturing a pluripotent stem cell in a medium comprising at least one kind selected from the group consisting of a fibroblast growth factor (FGF) receptor inhibitor and a mitogen-activated protein kinase kinase (MEK) inhibitor for a period of not more than 30 days, wherein the FGF receptor inhibitor is at least one kind selected from the group consisting of PD173074, SU5402, and PD161570, wherein the MEK inhibitor is at least one kind selected from the group consisting of PD0325901, PD184352, PD98059, U0126, MEK162, SL327, TAK-733, and AZD-8330, and wherein the culture period in the first step is sufficient for inducing gene expression of at least one of eye field transcription factors, and
(2) a second step for culturing the cell obtained in the first step in a medium containing at least one kind selected from the group consisting of a Rho signal transduction pathway inhibitor and an apoptosis inhibitor to form a retinal pigment epithelial cell, wherein the Rho signal transduction pathway inhibitor is at least one kind selected from the group consisting of Y-27632, Fasudil, H-1152, and Blebbistatin, and wherein the apoptosis inhibitor at least one kind selected from the group consisting of Z-VAD-FMK, Emricasan, Z-DEVD-FMK, procaspase activating compound 1 (PAC-1), Z-IETD-FMK, and Z-LEHD-FMK.
2. The method according to claim 1 , wherein the first step is performed in serum-free conditions.
3. The method according to claim 1 , wherein the first step is performed in the absence of feeder cells.
4. The method according to claim 1 , wherein
the medium in the first step further comprises a factor for maintaining undifferentiated state, wherein the factor for maintaining undifferentiated state is at least one kind selected from the group consisting of bFGF, FGF4, FGF8, TGFβ1, TGFβ2, Nodal, ActivinA, and ActivinB.
5. The method according to claim 4 , wherein the factor for maintaining undifferentiated state is a FGF signal transduction pathway agonist, wherein the FGF signal transduction pathway agonist is at least one kind selected from the group consisting of bFGF, FGF4, and FGF8.
6. The method according to claim 5 , wherein the FGF signal transduction pathway agonist is bFGF.
7. The method according to claim 1 , wherein the medium in the second step does not contain either an exogenous Nodal signal transduction pathway inhibitor or an exogenous Wnt signal transduction pathway inhibitor.
8. The method according to claim 1 , wherein the medium in the second step does not contain an exogenous substance that influences differentiation induction of pluripotent stem cells, which is other than a Rho signal transduction pathway inhibitor and an apoptosis inhibitor.
9. The method according to claim 1 , wherein the medium in the second step does not contain an exogenous substance that influences differentiation induction of pluripotent stem cells into ectodermal cells, which is other than a Rho signal transduction pathway inhibitor and an apoptosis inhibitor.
10. The method according to claim 1 , wherein the apoptosis inhibitor is Z-VAD-FMK.
11. The method according to claim 1 , wherein the pluripotent stem cell is a primate pluripotent stem cell.
12. The method according to claim 1 , wherein the pluripotent stem cell is a human pluripotent stem cell.
13. The method according to claim 1 , wherein the culture period in the first step is for 2 days-13 days.
14. The method according to claim 1 , wherein the culture period in the first step is for 4 days-6 days.
15. The method according to claim 1 , wherein the culture period in the first step is a period sufficient for inducing gene expression of at least one of Paired box 6 (PAX6), LIM Homeobox 2 (LHX2), and SIX Homeobox 3 (SIX3).
16. The method according to claim 1 , wherein the medium in the second step does not contain either FGF receptor inhibitor or MEK inhibitor.
17. The method according to claim 1 , wherein the medium in the second step does not contain either bone morphogenetic protein (BMP) signal transduction pathway agonist or Wnt signal transduction pathway agonist.