Methods for selective inhibition of pluripotent stem cells
Inventors
Gundry, Rebekah L. • Boheler, Kenneth R. • Kropp, Erin M.
Assignees
Government Of United States, Represented By Secretary Of Department Of Health And Human Services Nih Office Of Technology Transfer AS • Medical College of Wisconsin • National Institutes of Health NIH
Publication Number
US-11959096-B2
Publication Date
2024-04-16
Expiration Date
2035-01-21
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Abstract
Provided herein are methods of reducing or eliminating undifferentiated pluripotent stem cells, where the methods comprise contacting an effective amount of a compound to a heterogeneous cell population or sample comprising or suspected of comprising differentiated cell types and undifferentiated pluripotent stem cells, whereby the contacting selectively reduces or eliminates undifferentiated pluripotent stem cells from the cell population or sample. Also provided are methods for obtaining a population of stem cell-derived cell types substantially free of undifferentiated pluripotent stem cells as well as isolated populations of such of stem cell-derived cell types.
Core Innovation
The invention provides methods for reducing or eliminating undifferentiated pluripotent stem cells from heterogeneous cell populations containing both differentiated and undifferentiated cells. This is achieved by contacting the cell population with an effective amount of a compound, specifically a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor such as STF-31 or FK866, which selectively targets and depletes the undifferentiated pluripotent stem cells while sparing differentiated progeny.
The key problem being addressed is the risk associated with the presence of undifferentiated pluripotent stem cells in stem cell-derived therapy products, which includes the potential for tumorigenicity such as teratoma formation after transplantation. Current tests and elimination strategies for undifferentiated pluripotent stem cells are impractical for large-scale or high-throughput applications and can be inconsistent across different culture conditions, cell lines, and differentiation states.
Accordingly, the claimed methods enable the selective removal of undifferentiated pluripotent stem cells by exploiting their metabolic dependence on the NAD+ salvage pathway mediated by NAMPT. An effective concentration of a NAMPT inhibitor is applied to the mixed population, and after sufficient exposure, the remaining cells are substantially free of the undifferentiated population. This approach is applicable across varying conditions, media, cell lines, and densities, and also allows for the isolation and expansion of safe, differentiated stem cell-derived cell types for use in research or clinical settings.
Claims Coverage
The patent has one independent claim that describes a method involving three core inventive features.
Inducing differentiation or partial differentiation of pluripotent stem cells
The method starts by inducing undifferentiated pluripotent stem cells to differentiate or partially differentiate into one or more stem cell-derived cell types. This step prepares the cell population for selective depletion of undifferentiated cells in subsequent steps.
Selective elimination using a NAMPT inhibitor
An effective amount of a compound that is a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor is contacted with the induced cell population. Examples of such inhibitors include STF-31, FK866, GMX-1778, GNE-617, and GNE-618. This step selectively reduces or eliminates undifferentiated pluripotent stem cells from the population, relying on the specific vulnerability of these cells to NAMPT inhibition.
Isolation of differentiated, substantially pluripotent stem cell-free populations
After contacting the cells with the NAMPT inhibitor, the treated cells are isolated to obtain a population of one or more stem cell-derived cell types. The resulting population is substantially free of undifferentiated pluripotent stem cells, improving the safety and suitability of the population for downstream applications.
In summary, the independent claim defines a comprehensive method for producing stem cell-derived populations substantially free of undifferentiated pluripotent stem cells using NAMPT inhibitors, with steps covering differentiation, selective elimination, and isolation.
Stated Advantages
Provides selective elimination of undifferentiated pluripotent stem cells without affecting differentiated progeny.
Reduces the risk of tumorigenicity, such as teratoma formation, from stem cell-derived products.
Applicable across a wide range of culture conditions, cell lines, densities, and media compositions.
Provides a rapid, scalable, and inexpensive method for eliminating pluripotent stem cells.
Enhances the safety of stem cell-derived cell populations for research and clinical applications.
Works independently of cell proliferation rates and is effective in confluent cultures.
Documented Applications
Production of stem cell-derived cell populations for research and clinical applications, including tissue engineering.
Depletion of undifferentiated pluripotent stem cells in vitro to obtain cell populations suitable for transplantation.
Suppression of tumorigenicity in cell compositions administered to a subject, for the prevention of teratoma or other tumor formation.
Elimination or reduction of tumorigenic cells from pluripotent stem cell-derived progeny prior to in vitro or in vivo downstream applications, including transplantation of derivatives such as cardiomyocytes, neurons, hepatocytes, or retinal pigmented epithelial cells.
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