Description

Immune system humanized mice are a powerful tool to study human hematopoietic and immune system in vivo. Human lymphoid cells including T cells, B cells and NK cells can be well reconstituted by transplanting human CD34+ HSCs into B-NDG mice or B-NDG hIL15 mice. This reconstituted model can be used to study the mechanism of various human lymphoid cells and evaluate drug efficacy. However, human myeloid cells, including monocytes/macrophages, dendritic cells, granulocytes, and red blood cells, were under-reconstituted in these humanized mouse models, mainly due to the insufficient cross-reactivity of cytokines that promote the reconstituted myeloid cells between human and mice. Existing data have shown that the expression of human cytokine IL3, human granulocyte-macrophage colony-stimulating factor (GM-CSF) and human macrophage colony-stimulating factor (M-CSF) in mice can promote the development and differentiation of dendritic cells and mononuclear/macrophage cells. Human thrombopoietin (THPO) can promote functional human hematopoietic stem cells to maintain their multilineage differentiation potential and promote their long-term engraftment in mouse bone marrow.

Biocytogen developed B-NDG MGMT3 mice in which the full-length sequences of mouse Il3, GM-CSF, M-CSF, and Thpo genes were replaced with the full-length coding sequences of the corresponding human genes in B-NDG mice. The severely immunodeficient mice humanized with four cytokines were obtained by secondary gene editing and mutual mating. After engraftment of human CD34+ HSCs, reconstitution of various types of human myeloid cells, including monocytes/macrophages, dendritic cells, and reconstitution of human lymphoid cells, including CD4+ T cells, Tregs and NK cells, was significantly enhanced compared with B-NDG mice. B-NDG MGMT3 mice can be used to study the mechanism of hematopoietic development and differentiation, and to study the pathogenic mechanism and efficacy evaluation of immune-related diseases such as tumors, autoimmunity, infection and metabolism.

Protein expression analysis

Strain specific GM-CSF, CSF1 and THPO expression analysis in wild-type B-NDG mice and homozygous B-NDG MGMT3 mice by ELISA. Serum was collected from the two mice stimulated with LPS in vivo and analyzed by ELISA (n=3). Mouse GM-CSF, CSF1 and THPO were only detectable in B-NDG mice (+/+) but not in B-NDG MGMT3 mice (H/H). Human GM-CSF, CSF1 and THPO were only detectable in B-NDG MGMT3 mice. As IL3 is mainly expressed in activated T cells and there is no mature T cells in B-NDG background mice, mouse or human IL3 was not detectable in both of the two mice.

Human CD34+ HSCs engraftment for human immune system reconstitution  

Human CD34+ HSCs (3E4) were intravenous (temporal vein) engrafted into wild-type B-NDG mice and homozygous B-NDG MGMT3 mice (both sex, 24-72 hr after birth, n=15). B-NDG mice were treated with 1.0 Gy-irradiation. B-NDG MGMT3 mice were not irradiated. A. Survival rates of the mice were analyzed with Kaplan Meier survival curves. B. Body weight. Results showed that the survival rate of B-NDG MGMT3 mice was similar to that of B-NDG mice until 18 weeks after human CD34+ HSCs engraftment and then decreased to 42.85% at 24 weeks post engraftment. But the body weight of B-NDG MGMT3 mice was significantly higher than that of B-NDG mice and increased steadily during the whole reconstitution. Values are expressed as mean ± SEM. HSCs: hematopoietic stem cells.

Human CD34+ HSCs (3E4) were intravenous (temporal vein) engrafted into wild-type B-NDG mice and homozygous B-NDG MGMT3 mice (both sex, 24-72 hr after birth, n=15). B-NDG mice were treated with 1.0 Gy-irradiation. B-NDG MGMT3 mice were not irradiated. Peripheral blood lymphocytes from the two mice after engraftment with human CD34+ HSCs were analyzed with flow cytometry. Results showed that the cell numbers of all the cells analyzed from 12 weeks after engraftment in B-NDG MGMT3 mice were higher than that in B-NDG mice. Values are expressed as mean ± SEM. Values are expressed as mean ± SEM.