Embryonic Stem Cell Transplant (ESCT)-Derived Mixed Hematopoietic Chimerism Prevents Diabetes in NOD Mice.

Abstract

NOD mice are a widely used animal model for studying type I diabetes mellitus characterized by lymphocytic infiltration of pancreatic islets following by development of diabetes by age 3 to 4 months. It has been shown that allogeneic bone marrow transplantation can prevent insulitis and overt diabetes. We have recently demonstrated that embryonic stem cell-derived hematopoietic stem cells (ESC-derived HSC) can reconstitute bone marrow in lethally irradiated mice across MHC barriers without graft versus host disease (GVHD). Herein we report a new ESC-derived HSCT approach for the prevention of autoimmune diabetes in NOD mice. 6-weeks old female mice were sublethally irradiated (8.0 Gy) and transplanted with ESC-derived HSC. To induce differentiation toward HSC in vitro, ESC (R1) were cultured in methylcellulose-based medium supplemented with SCF, IL-3 and IL-6. An enriched c-kit+CD45+ ESC-derived suspension was delivered intra bone marrow (IBM) (mean 0.5 x 106/mouse) (N=8) or intravenously (mean 1 x 106/mouse) (N=6). Six NOD mice were held as control. Mice were followed by blood glucose measurements and chimerism analysis until onset of diabetes or until 32 weeks after transplantation. 7 out of 8 mice from IBM group and 4 out of 6 from IV group did not become hyperglycemic in contrast to control group where 5 out of 6 mice were euthanized because of diabetes. The level of chimerism achieved after transplantation was 8.2%±5.76 in IBM group and 3.2%±2.75 in IV group. Histological examination showed that most of islets were replaced by lymphocytic infiltration or fibrous tissue in controls (even in case of a mouse without clinical evidence of diabetes). In 78% (11/14) of animals from ESC-derived hematopoiesis, remission was confirmed by histology revealing absence of insulinitis and normal immunohistochemical staining of islet cells for insulin. Prevention of diabetes/insulinitis was predicted by the percentage ESC-derived hematopoietic chimerism. All mice with > 5% ESC-derived chimerism remained free of diabetes and insulinitis. These results suggest that allogeneic chimerism achieved with embryonic stem cells can prevent diabetes in NOD mice without evidence of GVHD or use of post transplant immune suppression.