A. Alex Hofling

The potential for human adipose-derived mesenchymal stem cells (AMSC) to traffic into various tissue compartments was examined using three murine xenotransplantation models: nonobese diabetic/severe combined immunodeficient (NOD/SCID), nude/NOD/SCID, and NOD/SCID/MPSVII mice. Enhanced green fluorescent protein was introduced into purified AMSC via retroviral vectors to assist in identification of cells after transplantation. Transduced cells were administered to sublethally irradiated immune-deficient mice through i.v., intraperitoneal, or subcutaneous injection. Up to 75 days after transplantation, tissues were harvested and DNA polymerase chain reaction (PCR) was performed for specific vector sequences as well as for human Alu repeat sequences. Duplex quantitative PCR using human beta-globin and murine rapsyn primers assessed the contribution of human cells to each tissue. The use of the novel NOD/SCID/MPSVII mouse as a recipient allowed rapid identification of human cells in the murine tissues, using an enzyme reaction that was independent of surface protein expression or transduction with an exogenous transgene. For up to 75 days after transplantation, donor-derived cells were observed in multiple tissues, consistently across the various administration routes and independent of transduction parameters. Tissue localization studies showed that the primary MSC did not proliferate extensively at the sites of lodgement. We conclude that human AMSC represent a population of stem cells with a ubiquitous pattern of tissue distribution after administration. AMSC are easily obtained and highly amenable to current transduction protocols for retroviral transduction, making them an excellent avenue for cell-based therapies that involve a wide range of end tissue targets.

Recent evidence suggests that bone marrow-derived angioblasts or endothelial progenitor cells circulate in peripheral blood and can incorporate at sites of pathologic neovascularization or during the ovarian cycle. However, the incorporation of endothelial progenitor cells into vessels of nonischemic tissues in adult animals has not been observed. We hypothesized that the vascular microenvironment differs between newborn and adult animals, and that donor endothelial cell progenitors would engraft in rapidly growing normal tissues during the neonatal period. After nonablative administration of bone marrow cells either at birth or at 4 weeks of age, donor-derived endothelial cells were found only in the neovasculature of the newborn recipients. Both the incorporation of donor endothelial cells into the newborn neovasculature as well as tissue vascularity were significantly increased by coadministering vascular endothelial growth factor with bone marrow cells. These findings suggest that bone marrow-derived endothelial progenitor cells can contribute to neovascularization during the newborn period and are responsive to vascular endothelial growth factor.

A novel murine system was developed to study the in vivo localization of xenotransplanted human cells and assess their therapeutic effect in an authentic model of disease. The beta-glucuronidase (GUSB) mutation of the mucopolysaccharidosis type VII (MPSVII) mouse was backcrossed onto the nonobese diabetic/severe combined immunodeficient (NOD/SCID) xenotransplantation strain. The resulting NOD/SCID/MPSVII mice displayed the characteristic features of lysosomal storage disease because of GUSB deficiency and were also capable of engrafting human cells. Human CD34+ hematopoietic progenitor cells from healthy, GUSB+ donors engrafted NOD/SCID/MPSVII mice in a manner similar to that of standard NOD/SCID mice. Six to 12 weeks following transplantation, 1% to 86% of the host bone marrow was positive for human CD45. By using a GUSB-specific histochemical assay, human engraftment was detected with single-cell sensitivity not only in well-characterized hematopoietic tissues like bone marrow, spleen, lymph node, and thymus, but also in other nonhematopoietic organs like liver, kidney, lung, heart, brain, and eye. Quantitative measurements of GUSB activity confirmed this expansive tissue distribution. The GUSB-specific assays were validated for their accuracy in identifying human cells through colocalization of human CD45 expression with GUSB activity in tissues of mice receiving transplants. An analysis of the therapeutic effects of engrafted human cells revealed a reduction of pathologic storage material in host organs, including the bone, spleen, and liver. Such xenotransplantation experiments in the NOD/SCID/MPSVII mouse represent a powerful approach to both study the in vivo biology of human cells and gather preclinical data regarding treatment approaches for a human disease.

Bone marrow transplantation (BMT) is relatively effective for the treatment of lysosomal storage diseases. To better understand the contribution of specific hematopoietic lineages to the efficacy of BMT, we transplanted beta-glucuronidase-positive mononuclear phagocytes derived from either the peritoneum or from bone marrow in vitro into syngeneic recipients with mucopolysaccharidosis type VII (MPS VII). Cell surface marking studies indicate that the bone marrow-derived cells are less mature than the peritoneal macrophages. However, both cell types retain the ability to home to tissues rich in cells of the reticuloendothelial system after intravenous injection into MPS VII mice. The half-life of both types of donor macrophages is approximately 7 days, and some cells persist for at least 30 days. In several tissues, therapeutic levels of beta-glucuronidase are present, and histopathologic analysis demonstrates that lysosomal storage is dramatically reduced in the liver and spleen. Macrophages intravenously injected into newborn MPS VII mice localize to the same tissues as adult mice but are also observed in the meninges and parenchyma of the brain. These data suggest that macrophages play a significant role in the therapeutic efficacy of BMT for lysosomal storage diseases and may have implications for treatments such as gene therapy.