Methods for selective inhibition of pluripotent stem cells
Inventors
Gundry, Rebekah L. • Boheler, Kenneth R. • Kropp, Erin M.
Assignees
Medical College of Wisconsin • US Department of Health and Human Services
Publication Number
US-10316287-B2
Publication Date
2019-06-11
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 of reducing or eliminating undifferentiated pluripotent stem cells by contacting a heterogeneous cell population comprising both differentiated cell types and undifferentiated pluripotent stem cells with an effective amount of a compound. The compound selectively reduces or eliminates the undifferentiated pluripotent stem cells without damaging the differentiated cells. Known compounds for this method include STF-31, FK866, and other inhibitors of nicotinamide phosphoribosyltransferase (NAMPT). The effective amounts of such compounds range between about 0.1 μM and about 100 μM.
The problem being solved is the presence of undifferentiated pluripotent stem cells in stem cell-derived populations which can cause tumorigenicity, including teratoma formation after transplantation. Current differentiation protocols yield heterogeneous populations containing these potentially tumorigenic cells, posing a significant obstacle for clinical applications of pluripotent stem cell-derived therapies. Existing methods to test pluripotent stem cell potency are either impractical for high-throughput applications or insufficient to ensure elimination of tumorigenic cells.
The invention overcomes limitations of prior chemical and physical methods by providing compounds that selectively target undifferentiated pluripotent stem cells based on their metabolic reliance on an NAD+ salvage pathway mediated by NAMPT. This method allows selective toxicity against pluripotent cells while sparing differentiated progeny. Additionally, the invention provides methods for obtaining cell populations substantially free of undifferentiated pluripotent stem cells, suitable for research and clinical applications, with the ability to expand these populations as single cell clones.
Claims Coverage
The patent includes one independent claim with several dependent claims, covering methods of reducing or eliminating undifferentiated pluripotent stem cells using NAMPT inhibitors at defined effective amounts.
Selective reduction or elimination of undifferentiated pluripotent stem cells using NAMPT inhibitors
A method comprising contacting a heterogeneous cell population containing differentiated and undifferentiated pluripotent stem cells with an effective amount of a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor compound to selectively reduce or eliminate the undifferentiated pluripotent stem cells.
Use of specific NAMPT inhibitors
The NAMPT inhibitor used in the method is selected from the group consisting of STF-31, FK866, GMX-1778, GNE-617, and GNE-618.
Defined effective compound concentrations for selective inhibition
Effective amounts of the compound for inducing selective elimination range between about 1 nanomolar (nM) and about 100 micromolar (μM), with STF-31 effective at about 5 μM, and FK866 effective at about 25 nM.
The claims cover a method of selectively eliminating undifferentiated pluripotent stem cells from a mixture by treatment with defined amounts of specific NAMPT inhibitors, highlighting STF-31 and FK866 as exemplary compounds with effective concentration ranges.
Stated Advantages
STF-31 exhibits robust selective toxicity against undifferentiated pluripotent stem cells across varying culture conditions and cell densities, including confluent monolayers where other compounds are ineffective.
The method spares differentiated progeny and terminally differentiated cells such as cardiomyocytes, neural progenitors, fibroblasts, and retinal pigmented epithelial cells, preserving their function and viability.
STF-31’s mode of action targets NAMPT in the NAD+ salvage pathway, providing rapid, scalable, and efficient elimination of pluripotent stem cells, thus mitigating tumorigenicity risks.
A short pulse treatment (24-48 hours) with STF-31 is sufficient to eliminate undifferentiated cells, simplifying treatment protocols and improving safety for stem cell-derived therapies.
Documented Applications
Obtaining populations of stem cell-derived cell types substantially free of undifferentiated pluripotent stem cells for research and clinical transplantation applications.
Formulating therapeutic compositions comprising NAMPT inhibitors for in vitro or in vivo reduction of tumorigenic pluripotent stem cells in cell populations to be administered to subjects.
Suppressing tumorigenicity of transplanted pluripotent stem cell-derived cell compositions by pre-treating with compounds that selectively eliminate tumorigenic undifferentiated cells.
Use in regenerative medicine and tissue engineering to produce safer pluripotent stem cell-derived tissues and cell products with reduced risk of teratoma formation.
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