br Experimental Procedures br Author Contributions N N G
Author Contributions N.N.G. performed most of the experimental work, with the help of M.G., J.L., and P.G.P. C.S.J. helped perform in vivo cell transplantation experiments. S.A.M. and F.R. were responsible for inducible Pax7 lineage-tracing experiments. A.A. performed qRT-PCR analyses. J.J.C. generated B195AP-Cre-expressing mice and together with M.L.M. characterized this transgenic line. P.G.B. and J.M.G.V. performed ultrastructural analyses. L.T., V.A.L., and M.J.A.B. did the transcriptomics and their corresponding analyses, respectively. D.H. and A.B. performed bone marrow transplantation experiments. A.M. and A.L.M. provided helpful guidance, reagents, and suggestions. N.N.G. and A.I. wrote the manuscript, which was approved by all authors prior to submission. P.G.P. and A.I. directed all experimental work.
Acknowledgments We thank investigators for monoclonal EDC.HCl A4.1025 (H.M. Blau) and F5D (W.E. Wright), which were obtained from the Developmental Studies Hybridoma Bank (developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA). Special thanks to G. Cossu for critical reading of the manuscript. We are also grateful to F. Costantini, C.-M. Fan, C. Lepper, M.J. Sánchez-Sanz, H. Sakai, and S. Tajbakhsh for kindly providing study materials; S. Lamarre of the GeT-Biochip facility for help in the microarray data; D. Ortiz de Urbina, J.C. Mazabuel, and A. Guisasola for help with irradiation protocol; and A. Aduriz, A. Pavón, and M. P. López-Mato for help with FACS analyses. This work was supported by grants from Instituto de Salud Carlos III (ISCIII; PS09/00660, PI13/02172, and PI14/7436), Gobierno Vasco (SAIO12-PE12BN008) from Spain and the European Union (POCTEFA-INTERREG IV A program; REFBIO13/BIOD/006 and REFBIO13/BIOD/009). N.N.G. received a studentship from the Department of Education, University and Research of the Basque Government (PRE2013-1-1168). P.G.P. received fellowships from the Department of Health of the Basque government (2013011016), EMBO (Short-Term; ASTF 542–2013), and Boehringer Ingelheim Fonds. M.L.M. and J.J.C. were supported by a Marie Curie Career Integration Grant from the European Commission (PEOPLE-CIG/1590). A.I. was supported by the Programa I3SNS (CES09/015) from ISCIII and by Osakidetza-Servicio Vasco de Salud (Spain). M.G. and S.A.M. contributed equally to this work.
Introduction Immunodeficient mice are widely used as hosts for xenogeneic transplantation to study human hematopoiesis in vivo (Doulatov et al., 2012; Goyama et al., 2015; Ishikawa, 2013; Rongvaux et al., 2013; Shultz et al., 2012). Elimination of the mouse lymphoid system, including T, B, and natural killer (NK) cells, is necessary to prevent human graft rejection. Thus, mice with the scid mutation (Greiner et al., 1998; McCune et al., 1988; Shultz et al., 1995) or those lacking recombination activating gene 1 or 2 (Rag1 or Rag2) (Goldman et al., 1998; Shultz et al., 2000, 2003) have been used. In addition, these mice also had mutations in the interleukin-2 (IL-2) receptor common γ-chain subunit (Il2rg) gene (Ishikawa et al., 2005; Ito et al., 2002; Shultz et al., 2005). Previous studies reported that a non-obese diabetic (NOD) or BALB/c genetic background facilitates human hematopoietic stem cell (HSC) engraftment (Brehm et al., 2010). Therefore, these strains with complete lymphoid depletion, such as NOD-scid Il2rgnull (NSG/NOG) (Ito et al., 2002; Shultz et al., 2005), NOD.Rag1nullIl2rgnull (NOD-RG) (Pearson et al., 2008), and BALB/c.Rag1/2nullIl2rgnull (BALB-RG) (Brehm et al., 2010; Traggiai et al., 2004) mice, have been commonly used for recent xenotransplantation experiments. We found that the strain-specific genetic determinant of human HSC engraftment was a polymorphism in the signal-regulatory protein α (Sirpa) gene (Iwamoto et al., 2014; Takenaka et al., 2007; Yamauchi et al., 2013). SIRPA is expressed on the surface of macrophages and binds to its ligand, CD47, which is ubiquitously expressed (Matozaki et al., 2009). This binding activates inhibitory signals for phagocytosis of CD47-expressing cells, termed the “don\'t eat me signal” (Jaiswal et al., 2009; Kuriyama et al., 2012; Majeti et al., 2009; Oldenborg et al., 2000). In the setting of human-to-mouse xenotransplantation, this inhibitory signal is necessary to prevent engulfment of human graft cells by host macrophages. Although the binding of SIRPA and CD47 is species specific (Subramanian et al., 2006), NOD and BALB/c mouse strains contain unique SIRPA immunoglobulin (Ig) domains that cross-react with human CD47 (Iwamoto et al., 2014; Takenaka et al., 2007). With regard to human CD47, NOD-SIRPA has the strongest affinity while BALB/c-SIRPA has an intermediate affinity (Iwamoto et al., 2014), and it is not recognized by C57BL/6-SIRPA. Based on these data, we developed a C57BL/6.Rag2nullIl2rgnull mouse line harboring NOD-Sirpa (BRGS) (Yamauchi et al., 2013). The efficiency of human cell engraftment in BRGS mice is significantly greater than in NOD-RG mice. Because BRGS mice do not possess a number of other NOD-specific abnormalities, including complement 5 deficiency (Yamauchi et al., 2013), they are also useful for testing the complement-dependent cytotoxic activity of antibodies in vivo (Kikushige and Miyamoto, 2013). Thus, the BRGS mouse line is one of the most efficient and convenient immunodeficient mouse strains for xenotransplantation.