Adult Donor-Derived Human CD34⁺ Cell Engraftment and Hemato-Lymphoid System Development in 3rd Generation Humanized Mice

Transplantation of human CD34⁺ hematopoietic stem and progenitor cells into severe immunocompromised newborn mice allows the development of a human hemato-lymphoid system (HHLS) in vivo (Rongvaux et al. Ann. Rev. Immunol. 2013). While fetal liver- or cord blood- derived CD34⁺ cells lead to high levels of engraftment, adult donor-derived CD34⁺ cell transplantation usually led to low levels of engraftment in existing humanized mice models. We recently generated novel mouse strains called 3^rd generation humanized mice (3^rd gen. huMice) in which human versions of cytokines (M-CSF and TPO with or without IL-3/GM-CSF) are knocked into Balb/c Rag2^-/-γ_C^-/- strains (MISTRG or MSTRG, respectively). In addition, human Sirpα, which is a critical factor to prevent donor cell to be eliminated by host macrophages, is expressed as transgene in both strains (Rongvaux et al., Nat. Biotechnol. 2014).

To evaluate human adult CD34⁺ cell engraftment in 3^rd gen. huMice, CD34⁺ cells obtained from peripheral blood after G-CSF administration (3.0 – 5.5 x10⁵ cells) were i.h. injected into sub-lethally irradiated newborn MISTRG or MSTRG and NOD/scid/γ_C^-/- (NSG) mice or Rag2^-/-γ_C^-/-hSirpα^Tg (RGS) mice as controls. Seventeen of 18 (94%) MISTRG/MSTRG mice showed human CD45⁺ cell engraftment (>1% of total CD45⁺ cells in BM) 10-16 weeks after injection, whereas 4 of 11 (36%) NSG/RGS mice supported human cell engraftment. Percentages of human cells in the BM of the engrafted MISTRG/MSTRG were 7- to 8 fold higher than in the BM of engrafted NSG/RGS mice (30.2% ± 6.9 vs 4.1% ± 0.9, respectively). MISTRG/MSTRG mice supported significantly increased numbers of non-classical monocytes and NKp46⁺ cells in BM compared with NSG/RGS mice. Moreover, we observed significantly increased numbers of CD34⁺ and CD34⁺CD38^- cells, a population enriched for human early progenitor cells and HSCs, in the BM of MISTRG/MSTRG mice. In addition, MISTRG/MSTRG mice supported higher level of human thymocyte development compared to NSG/RGS mice. Besides lymphoid organs, we further observed increased human CD45⁺ cells, mostly myeloid lineage cells, in the liver and lung of MISTRG/MSTRG mice compared to NSG/RGS mice.

Taken together, this study demonstrates that our 3^rd gen. huMice models support adult donor-derived HSC engraftment and development of myeloid as well as lymphoid lineage cells at high levels in primary lymphoid and non-lymphoid organs. These models thus have the potential for personalized studies of healthy hematopoiesis as well as hemato-immune system diseases from adult individuals.