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  • This has important implications for competitive transplantat

    2018-11-08

    This has important implications for competitive transplantation experiments. Because they have the same reconstitution potential, modified HSPCs from any C57BL/6N or C57BL/6J strain can be directed compared with our CD45.1STEM strain without an additional control group. This contrasts with the situation when using the current B6.SJL competitor strain. Given the inherent disadvantage of WT B6.SJL HSPCs, one requires both a test competitive transplant (CD45.2 test versus B6.SJL) as well as a control competitive transplant (CD45.2 WT versus B6.SJL) in order to correctly determine HSPC fitness. As such, the C57BL/6N-CD45.1STEM strain provides a better competitor and offers the possibility of reducing the number of mice and cost associated with competitive HSPC transplantation assays. We would like to highlight a few important methodological variables in performing competitive HSPC transplantation assays. (1) We stress the importance of pooling bone marrow Cy3 hydrazide from a minimum of three donor mice. Despite the fact that these mice may be genetically identical WT littermates, there exist subtle variations in reconstitution ability between individuals that can be minimized by pooling multiple donors. (2) Slight differences in reconstitution ability are exacerbated by transplanting low numbers of HSPCs (Lacombe et al., 2010). As we transplanted only 500,000 bone marrow cells from each strain, this led to impressive mouse to mouse variability that was overcome by performing multiple biological replicates (Figure S3A). (3) The high fractionated dose of radiation (2 × 600 cGy) given as pre-transplant conditioning ensures that there are few residual endogenous recipient cells, although in all cases we would advocate the use of F1 hybrid CD45.1∗CD45.2 recipients to permit distinguishing the doubly labeled residual host cells (Figure S3B). In summary, the C57BL/6N-CD45.1STEM mouse represents a technical advance for investigators who wish to accurately quantify the fitness of genetically, or otherwise modified, HSPCs derived in the common background of the C57BL/6N or C57BL/6J mouse strains. Unlike the previous competitor strain, HSPCs from the CD45.1STEM mouse have comparable reconstitution potential, eliminating the need for a separate control group. For these reasons, we propose that the CD57Bl/6N-CD45.1STEM mouse should become the standard competitor in competitive bone marrow transplantation assays.
    Experimental Procedures
    Author Contributions
    Acknowledgments We would like to thank Youmna Kfoury, Jonathan Hoggatt, Jacqueline Bachand, Cam Dung Le, Katrina Maxcy, and Alexa Carver. We would like to thank the staff of ingenious Targeting Laboratories, especially AnnMarie Degruccio and Anne Schirmer. David Tan provided the mouse graphic in Figure 1. Brian D. Sykes created the ribbon structure in Figure 2A. Jiantao Shi helped with the statistical analysis. We would also like to thank the Harvard Stem Cell Institute (HSCI) for the Seed Grant that provided funds for the generation of this mouse strain as well as members of the CRM/HSCI Flow Cytometry Core. F.E.M. was supported by a Clinician-Scientist training award from the Canadian Institutes of Health Research. D.B.S. was supported by the American Society of Hematology, the Leukemia and Lymphoma Foundation, and Alex\'s Lemonade Stand Foundation. D.T.S. was supported by the Gerald and Darlene Jordan Chair in Medicine at Harvard University.
    Introduction With the seminal discovery of human pluripotent stem cells (hPSCs) (Thomson et al., 1998; Takahashi et al., 2007), human cells that would be difficult or impossible to obtain can be produced using in vitro cell-culture techniques. This in turn has raised hopes that hPSCs can be used to study and treat different forms of disease, including neurological and neuropsychiatric disorders (Dolmetsch and Geschwind, 2011; Fox et al., 2014; Han et al., 2011; Imaizumi and Okano, 2014; Kanning et al., 2010; Liu and Zhang, 2010; Mariani et al., 2015). However, a key step in the utilization of hPSCs for these purposes is the ability to obtain cell types of interest. This has often proved to be challenging for several reasons including neural diversity, culture-to-culture and line-to-line variability, and limitations on large-scale cell production.