243. Transplantation of Neonatal NOD/SCID/MPSVII Mice with Human Mesenchymal Stem Cells

Abstract

In this study we assessed the transfer and expression of the gene for beta-glucuronidase (GUSB) into multiple tissues by infusing normal human mesenchymal stem cells (MSC) into unconditioned neonatal NOD/SCID/MPSVII (GUSB null) mice. In this study, pups were transplanted before 3 days of age using the facial vein injection strategy. We hypothesized that neonatal pups would provide a rapidly growing environment for engraftment and expansion of transplanted normal human MSC into different tissues. The Sands laboratory has used the MPSVII mouse for many years to study disease progression, including neural defects, and to study correction of the disease by gene replacement therapies. In order to study cellular therapies using normal human cells, the murine MPSVII mutation was backcrossed for ten generations onto the NOD/SCID strain. Human umbilical cord blood CD34+ cells engraft in the resultant NOD/SCID/GUSB null strain to levels of 86% marrow replacement, similar to the NOD/SCID parent (Hofling 2004). The tissues of the mice can be homogenized and subjected to a GUSB enzyme assay, and plotted against a standard curve composed of normal human and GUSB null mouse cells, to quantitate the percentage of human cells that had been residing in the tissue of interest at the time of harvest. There is also a FACS|[ndash]|based assay to detect levels of the GUSB protein, and to allow sorting of donor-derived cells from any tissue after transplantation. The aspect of the NOD/SCID/GUSB null strain that makes it so particularly well suited for the study of human stem cell therapy is the clear-cut enzymatic stain for normal, GUSB positive human cells transplanted into the mice. On tissue slides prepared from mice that had been transplanted at birth with human mesenchymal stem cells, the histochemical stain for GUSB revealed normal cells in multiple tissues. Almost all tissues surveyed (N=19) demonstrated the presence of human MSC by staining for GUSB. The stain is quite specific, and although the released enzyme can be taken up by neighboring cells, it appears to be in very low abundance or, more likely, in a processed form where it is no longer detectable by the histochemical analysis. Thus, the individual transplanted human cells stand out vividly against the background murine tissues. In summary, the use of the NOD/SCID/MPSVII neonatal injection model has allowed accurate tracking and quantitation of human MSC into multiple tissues of mice injected by facial vein within three days of birth. This study may set the stage for neonatal gene/cell therapy for newborns with lysosomal storage diseases.