Methods for detection of rare subpopulations of cells and highly purified compositions of cells
View Patent ↗Methods are provided for detection of a target cell type within a cell population, and compositions are provided comprising cells and an indicator that indicates the number of cells of the target cell type in the cell population. Examples are provided in which these methods are used to detect human embryonic stem cells within a differentiated cell population with exquisite sensitivity. Differentiated cells produced from embryonic stem cells can be characterized by these methods before transplantation into a recipient, thereby providing further assurance of safety.
1. A method of confirming absence of contaminating pluripotent stem cells in a preparation of differentiated cells generated by differentiation of said pluripotent stem cells, comprising:
(a) providing a preparation of differentiated cells produced by in vitro differentiation of pluripotent stem cells in a differentiation medium, and culturing said preparation of differentiated cells in a stem cell medium, wherein the stem cell medium maintains pluripotent stem cells in their pluripotent state;
(b) applying a first stain and a second stain to said preparation of differentiated cells, wherein
said first stain detects a first embryonic stem cell marker, expression of which is indicative of presence of said contaminating pluripotent stem cells, and
said second stain detects a second embryonic stem cell marker, expression of which is indicative of presence of said contaminating pluripotent stem cells,
wherein said first embryonic stem cell marker is different from said second embryonic stem cell marker,
wherein said first embryonic stem cell maker is alkaline phosphatase; and
(c) confirming absence of contaminating pluripotent stem cells, if no cells positive for both said first stain and said second stain are identified by microscopic observation.
2. The method of claim 1 , wherein said first stain is observable under visible light and said second stain is observable under ultraviolet light.
3. The method of claim 1 , wherein said first stain comprises an antibody that specifically binds to said first embryonic stem cell marker.
4. The method of claim 1 , wherein said first stain comprises an alkaline phosphatase substrate.
5. The method of claim 1 , wherein said second stain comprises a primary antibody that specifically binds to said second embryonic stem cell marker, wherein said primary antibody is directly or indirectly coupled to a fluorescent label.
6. The method of claim 1 , wherein said second embryonic stem cell marker is selected from the group consisting of: Oct-4, Nanog, Stage-specific embryonic antigen-3 (SSEA-3), Stage-specific embryonic antigen-4 (SSEA-4), TRA-1-60, TRA-1-81, TRA-2-49/6E, Sox2, and Lin28.
7. The method of claim 1 , wherein said preparation of differentiated cells contains at least 10 5 cells, at least 10 6 cells, at least 10 7 cells, at least 10 8 cells, at least 10 9 cells, at least 10 10 cells, or between 10 5 and 10 10 cells.
8. The method of claim 1 , wherein said contaminating pluripotent stem cells are embryonic stem cells or induced pluripotent (iPS) cells.
9. The method of claim 1 , wherein said preparation of differentiated cells comprises retinal pigment epithelium (RPE) cells differentiated from pluripotent stem cells.
10. The method of claim 9 , wherein said RPE cells are human RPE cells.
11. The method of claim 9 , wherein said RPE cells are made by a method comprising:
(a) providing pluripotent stem cells;
(b) culturing the pluripotent stem cells to form a multilayer population of pluripotent stem cells or embryoid bodies comprising pluripotent stem cells;
(c) culturing the multilayer population or embryoid bodies in the differentiation medium for a sufficient duration for RPE cells to appear in the culture of cells; and
(d) isolating the RPE cells from the culture.
12. The method of claim 1 , wherein the second embryonic stem cell marker is Nanog.
13. The method of claim 1 , wherein the second embryonic stem cell marker is Oct-4.
14. The method of claim 1 , wherein said preparation of differentiated cells is cultured on feeder cells.
15. The method of claim 14 , wherein said feeder cells are murine embryonic fibroblasts (MEFs) or human adult skin fibroblasts.
16. The method of claim 1 , wherein the pluripotent stem cells are human embryonic stem cells or human induced pluripotent stem cells, and the preparation of differentiated cells comprises human RPE cells differentiated in vitro from said pluripotent stem cells.
17. The method of claim 16 , wherein the second embryonic cell marker is Oct-4.
18. The method of claim 1 , wherein the method detects contaminating pluripotent stem cells that are present at as little as 0.0008% of the preparation of differentiated cells.
19. The method of claim 17 , wherein the method detects contaminating pluripotent stem cells that are present at as little as 0.0008% of the preparation of differentiated cells.
20. The method of claim 1 , wherein said preparation of differentiated cells is cultured in conditions that comprise leukemia inhibitory factor (LIF).
21. The method of claim 1 , wherein the preparation of differentiated cells comprises at least about 95% differentiated RPE cells.
22. The method of claim 1 , wherein said first stain and said second stain are visually distinguishable.