Comparative Study of Human Hematopoietic Cell Engraftment into Balb/c and C57BL/6 Strain of Rag-2/Jak3 Double-Deficient Mice

Immunodeficient mice are becoming invaluable tools in human stem cell and tumor research. In this study, we generated Rag-2/Jak3 double-deficient (Rag-2−/−Jak3−/−) mice with a C57/BL6 and Balb/c genetic background and compared the human lymphohematopoietic cell engraftment rate. Human cord blood-derived CD34+ hematopoietic stem cells were successfully engrafted into Balb/c Rag-2−/−Jak3−/− mice; however, the engraftment rate was far lower in C57/BL6 Rag-2−/−Jak3−/− mice. Transplantation of human peripheral blood mononuclear cells resulted in the same tendency. Thus, a Balb/c background offers superior engraftment capacity than a C57/BL6 background and provides an attractive model for human hematopoietic cell engraftment.


Introduction
Animal models with a human hematopoietic and immune system are now important tools for studying the human immune system as well as human-specific infections, such as HIV-1 [1,2]. Recent approaches have involved the use of genetically immunodeficient mice that have been reconstituted using human-derived hematopoietic stem cells (HSCs) (known as humanized mice). Several immunodeficient mice have been developed and NOD/Scid mice with NK defective genetically modified mice are mainly used for this approach [3][4][5], because they exhibit deficiencies in macrophage and dendritic cell function, and complement as well as T and B lymphocyte deficiencies [6]. Rag-2 −/− γc −/− mice are also commonly used for establishing humanized mice [7,8], and it is known that the genetic background of Rag-2 −/− γc −/− mice is influenced by the efficiency of human cell engraftment.
JAK3 is a non-receptor-type tyrosine kinase crucial for mediating signaling from the common γ-chain of cytokine receptors, and Jak3-deficient mice and common γ-chain-deficient mice show the same phenotype [9]. Based on these findings, we generated Rag-2 −/− Jak3 −/− mice with C57/BL6 and Balb/c genetic backgrounds. These mice showed a lack of mature T and B lymphocytes and NK cells. Balb/c Rag-2 −/− Jak3 −/− mice showed high efficiency of both human CD34 + hematopoietic stem cell (HSC) and peripheral blood mononuclear cell (PBMC) transplantation compared with C57/BL6 Rag-2 −/− Jak3 −/− mice. Our data show that the genetic background of the mice influences human cell engraftment and Balb/c Rag-2 −/− Jak3 −/− mice are becoming an alternative source of humanized mice.

Cell Preparation and Transplantation
. Peripheral blood and umbilical cord blood samples were collected after obtaining written informed consent and approval from the Ethics Committee of Kumamoto University Faculty of Medical and Pharmaceutical Sciences. CD34 + cells were isolated using the CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec, Sunnyvale, CA) and transplanted into the liver of irradiated (4.0 Gy) newborn mice. At 12 weeks after transplantation, peripheral blood was obtained and analyzed by flow cytometry.

Statistical Analysis.
The statistical significance of differences was determined using Student's t-test. P values less than .05 were defined as significant.
Humanized mice with reconstituted human hematopoietic and immune cells are becoming a powerful tool for the investigation of human biological systems and for  translational research [2]. NOD/Scid mice have been used for these purposes; however, human hematopoietic stem cell engraftment is not sufficient and functionally mature lymphocytes are not developed in these mice, mostly due to residual NK activity in this mouse. To overcome these limitations, NOD/Scid mice were intercrossed with NK defective genetically modified mice, and several strains of mice suitable for humanized mice were established [3][4][5]. Among them, NOD/Scid/γc −/− (NOG and NSG) mice are becoming the gold standard for generating humanized mice. Recently, Spira gene polymorphism has been identified as a determinant influencing the efficacy of human hematopoietic stem cell engraftment into the NOD/Scid strain [12]. Meanwhile, Stroma cells of the Balb/c strain have been shown to support human hematopoiesis to a lesser extent but not other strains (C57/BL6, C3H, ICR), indicating that other mechanisms support human cell engraftment in immunodeficient mice than Spira gene polymorphism. In fact, Manz and colleagues described the reconstitution of human acquired and innate immunity in Balb/c Rag-2 −/− γc −/− mice [7]. Although no study has compared reconstitution efficiency directly between NOD/Scid/γc −/− mice and Balb/c Rag-2 −/− γc −/− mice, the efficiency of human hematopoietic stem cell engraftment seems to be higher in NOD background from the previous studies [1,2]. However, the efficiency of reconstitution in Balb/c Rag-2 −/− γc −/− mice is high enough for human immune and hematopoietic cell research (Figures 2 and 3) and Balb/c background has several advantages compared with NOD/Scid background.
The NOD/Scid strain has several disadvantages [1]. Mice with the scid mutation lack functional T and B lymphocytes resulting from the absence of gene recombination events in T-cell receptors (TCRs) and immunoglobulins; however, they are known to show a leaky phenomenon in which functional T and B lymphocytes are produced with aging or ionized irradiation [13,14]. In addition, the Prkdc gene disrupted by the scid mutation is expressed broadly and is involved in DNA repair, while expressions of Rag-1 and Rag-2 are limited to hematopoietic cells and are involved only in DNA recombination of T-and B-cell receptor genes, indicating that scid mice are more sensitive to radiation-induced DNA damage than their Rag-1 −/− or Rag-2 −/− counterparts. Recently, T. Pearson et al. established radioresistant NOD Rag-1 −/− γc −/− mice and showed that they support a similar level of human lymphohematopoietic cell engraftment [15], which could overcome the disadvantages of the scid mutation. The NOD/Scid strain has a predisposition to thymic lymphoma due to an endogenous ectopic provirus (Emv-30) [16]. NOD/Scid and NOD/Scid/β2m −/− mice have a short mean life span for this reason [6,17], while NOD/Scid/γc −/− (NOG and NSG) mice do not develop thymic lymphoma for an unknown reason [4]. Balb/c strain could overcome these disadvantages of NOD/Scid strain, although efficiency of human hematopoietic stem cell engraftment is not better than NOD/Scid strain [1].
As the strain background is known to affect human-cell engraftment and to function markedly in scid mice [1,2,18], we established and directly compared C57/BL6 and Balb/c strains of Rag-
Journal of Biomedicine and Biotechnology