Abstract
Methods to identify and enumerate primitive, and typically rare, undifferentiated cells in normal tissue using functional endpoints are powerful tools for acquiring insights into the mechanisms that regulate normal tissue stem cell turnover and differentiation. In this paper, we describe a xenotransplantation-based protocol that allows mammary stem cells with in vivo tissue regenerative properties to be specifically detected and quantified among the heterogeneous cell populations obtained from dissociated normal human mammary tissue. This methodology involves implanting a collagen gel containing the test cells in combination with supportive fibroblasts under the kidney capsule of highly immune-deficient, hormone-supplemented mice and then, 4 weeks later, searching for regenerated human cells with in vitro clonogenic activity. Quantification of the input human mammary stem cells is achieved using standard limiting dilution transplant approaches. This approach circumvents the need to modify the mouse mammary fat pad, and is objective, rapid (∼5 weeks) and economical to perform.
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Acknowledgements
We acknowledge the excellent technical contributions of D. Wilkinson, G. Edin, the staff of the Flow Cytometry Facility of the Terry Fox Laboratory and the Centre for Translational and Applied Genomics. J. Emerman helped organize the accrual of the mammoplasty material, which was obtained with informed patient consent, with the assistance of J. Sproul, P. Lennox, N. Van Laeken and R. Warren. The project was funded by grants from Genome BC/Genome Canada, the Canadian Stem Cell Network, the Canadian Breast Cancer Foundation BC and Yukon Division and the Canadian Cancer Society. P.E. was a recipient of a US Department of Defense Breast Cancer Research Program Studentship, a Terry Fox Foundation Research Studentship from the National Cancer Institute of Canada, a Canadian Imperial Bank of Commerce interdisciplinary award and a Canadian Stem Cell Network Studentship. J.S. held a Canadian Breast Cancer Foundation BC and Yukon Division Fellowship and a National Science and Engineering Research Council Industrial Fellowship.
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A.K. and J.E.G. designed the research; all authors conducted experiments and contributed to the writing of the paper.
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Supplementary information
Supplementary Methods
Comparison of the statistical resolution of MRU frequency determinations by LDA versus “bulk CFC” endpoints. (DOC 30 kb)
Supplementary Figure 1
Simulation of the comparative ability of LDA and bulk CFC measurements to resolve differences in MRU frequencies that exist between 2 populations. The bars indicate the range of a measured MRU frequency for hypothetical sample #2 for which the null hypothesis of no statistical difference from measured MRU frequency = 100 for hypothetical sample #1 cannot be rejected with 95% confidence. These are obtained from Monte-Carlo simulations of experiments comprising 20 transplants (10 for each of the 2 samples). For the simulations using the LDA endpoint, transplants were assumed to be made at limiting dilution (i.e., an average of 1 MRU per transplant). Likelihood ratio tests for inequality in frequency between samples were carried out using the ELDA web tool2, which uses a generalized linear model assuming a single-hit Poisson process. For the simulations using the bulk CFC endpoint, the regenerated CFC output per transplant was assumed to follow a normal distribution with an average of 70 CFCs and a standard deviation of 30 CFCs, which is consistent with technical replicates series carried out by us previously (data not shown). Student t-tests were carried out to test for inequality. (PDF 21 kb)
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Eirew, P., Stingl, J. & Eaves, C. Quantitation of human mammary epithelial stem cells with in vivo regenerative properties using a subrenal capsule xenotransplantation assay. Nat Protoc 5, 1945–1956 (2010). https://doi.org/10.1038/nprot.2010.148
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DOI: https://doi.org/10.1038/nprot.2010.148
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