Kimio Kobayashi

To establish a more appropriate animal recipient for xenotransplantation, NOD/SCID/gamma(c)(null) mice double homozygous for the severe combined immunodeficiency (SCID) mutation and interleukin-2Rgamma (IL-2Rgamma) allelic mutation (gamma(c)(null)) were generated by 8 backcross matings of C57BL/6J-gamma(c)(null) mice and NOD/Shi-scid mice. When human CD34+ cells from umbilical cord blood were transplanted into this strain, the engraftment rate in the peripheral circulation, spleen, and bone marrow were significantly higher than that in NOD/Shi-scid mice treated with anti-asialo GM1 antibody or in the beta2-microglobulin-deficient NOD/LtSz-scid (NOD/SCID/beta2m(null)) mice, which were as completely defective in NK cell activity as NOD/SCID/gamma(c)(null) mice. The same high engraftment rate of human mature cells was observed in ascites when peripheral blood mononuclear cells were intraperitoneally transferred. In addition to the high engraftment rate, multilineage cell differentiation was also observed. Further, even 1 x 10(2) CD34+ cells could grow and differentiate in this strain. These results suggest that NOD/SCID/gamma(c)(null) mice were superior animal recipients for xenotransplantation and were especially valuable for human stem cell assay. To elucidate the mechanisms involved in the superior engraftment rate in NOD/SCID/gamma(c)(null) mice, cytokine production of spleen cells stimulated with Listeria monocytogenes antigens was compared among these 3 strains of mice. The interferon-gamma production from dendritic cells from the NOD/SCID/gamma(c)(null) mouse spleen was significantly suppressed in comparison with findings in 2 other strains of mice. It is suggested that multiple immunological dysfunctions, including cytokine production capability, in addition to functional incompetence of T, B, and NK cells, may lead to the high engraftment levels of xenograft in NOD/SCID/gamma(c)(null) mice.

Here, we demonstrate a significant ex vivo expansion of human hematopoietic stem cells capable of repopulating in NOD/SCID mice. Using a combination of stem cell factor (SCF), Flk2/Flt3 ligand (FL), thrombopoietin (TPO), and a complex of IL-6 and soluble IL-6 receptor (IL-6/sIL-6R), we cultured cord blood CD34(+) cells for 7 days and transplanted these cells into NOD/SCID mice. Bone marrow engraftment was judged successful when recipient animals contained measurable numbers of human CD45(+) cells 10-12 weeks after transplantation. When cells were cultured with SCF+FL+TPO+IL-6/sIL-6R, 13 of 16 recipients were successfully engrafted, and CD45(+) cells represented 11.5% of bone marrow cells in engrafted recipients. Cells cultured with a subset of these factors were less efficiently engrafted, both as measured by frequency of successful transplantations and prevalence of CD45(+) cells. In animals receiving cells cultured with all 4 factors, human CD45(+) cells represented various lineages, including a large number of CD34(+) cells. The proportion of CD45(+) cells in recipient marrow was 10 times higher in animals receiving these cultured cells than in those receiving comparable numbers of fresh CD34(+) cells, and the expansion rate was estimated at 4.2-fold by a limiting dilution method. Addition of IL-3 to the cytokine combination abrogated the repopulating ability of the expanded cells. The present study may provide a novel culture method for the expansion of human transplantable hematopoietic stem cells suitable for clinical applications.

The role of sex in biomedical studies has often been overlooked, despite evidence of sexually dimorphic effects in some biological studies. Here, we used high-throughput phenotype data from 14,250 wildtype and 40,192 mutant mice (representing 2,186 knockout lines), analysed for up to 234 traits, and found a large proportion of mammalian traits both in wildtype and mutants are influenced by sex. This result has implications for interpreting disease phenotypes in animal models and humans.

Establishment of an assay capable of generating all classes of human lymphocytes from hematopoietic stem cells (HSCs) will provide new insight into the mechanism of human lymphopoiesis. We report ontogenic, functional, and histologic examination results of reconstituted human lymphocytes in NOD/SCID/ gammacnull mice after the transplantation of human cord blood (CB) CD34+ cells. After transplantation, human B, natural killer (NK), and T cells were invariably identified in these mice, even though no human tissues were cotransplanted. Immature B cells resided mainly in bone marrow (BM), whereas mature B cells with surface immunoglobulins were preferentially found in spleen. NK cells were identified in BM and spleen. T cells were observed in various lymphoid organs, but serial examinations after transplantation confirmed human T lymphopoiesis occurring in the thymus. These human lymphocytes were also functionally competent. Human immunoglobulin M (IgM), IgA, and IgG were detected in the sera of these mice. T cells showed a diverse repertoire of T-cell-receptor Vbeta (TCR Vbeta) chains, proliferated in response to phytohemagglutinin, and were cytotoxic against cell lines. NK activity was demonstrated using the K562 cell line. Immunohistochemical analysis revealed that human lymphocytes formed organized structures in spleen and thymus that were analogous to those seen in humans. In the thymus, CD4 and CD8 double-positive T cells were predominant and coexpressed CD1a and Ki-67, thereby supporting the notion that T lymphopoiesis was taking place. NOD/SCID/ gammacnull mice provide a unique model to investigate human lymphopoiesis without the cotransplantation of human tissues.