Our laboratory uses the tools of molecular genetics to study the self-renewal, mobilization, differentiation and tissue regeneration properties of adult and embryonic stem cells. In one of our projects we use transgenic mice (SCL-TVA) that allow targeted delivery of genes to hematopoietic and vascular endothelial stem cells in the intact animal. This is done by injection of SCL-TVA mice with tissue-specific retroviral vectors that home to stem cells in their bone marrow niche. One application of this gene delivery system is in vivo labeling of long-term self-renewing stem cells with markers such as luciferase and EGFP. This allowed us to visualize the location of hemangioblasts and hematopoietic stem cells in their natural locations by bioluminescence imaging of live animals, and to follow the fate and mobilization of these stem cells in response to injury or biotherapeutic agents, in real time. In another application we delivered an oncogene to bone marrow stem cells in vivo, and recapitulated the first steps in the conversion of hemangioblasts to hemangioma stem cells that occur in pediatric and adult hemangiomas. We found that this oncogenic event involved the activation of the PI 3-kinase and the Rapamycin-sensitive mTor pathways. The ability to deliver regulatory genes and shRNAs to stem cells will be used to identify the critical signaling molecules that regulate self-renewal, mobilization and differentiation of stem cells in an in vivo model, and in real time. This flexible experimental system circumvents the problems associated with conventional in vitro manipulation of bone marrow stem cells and transplantation into lethally irradiated animals.

Another major interest of our laboratory is the generation of disease-specific human embryonic stem cells, for modeling and treating Gaucher’s disease. This is the most frequent inherited lipid-storage disorder, and is caused by mutations in the acid beta-glucocerebrosidase gene. This enzyme deficiency results in the accumulation of glucosylceramide in the lysosomes of macrophages and other cells of the reticuloendothelial system. The accumulation of lipid in lysosomes leads to hepatomegaly, splenomegaly, hematologic abnormalities, bone disease, and in some cases neurological involvement. Disease modeling is done by two different approaches. One involves knockdown of the glucocerebrosidase gene using shRNA-encoding lentiviruses. The other is based on the reprogramming of fibroblasts from Gaucher patients harboring mutations in the glucocerebrosidase gene, into induced pluripotent stem (iPS) cells. As iPS cells can give rise to any cell type, their controlled differentiation into the affected cell types provides an unlimited supply of patient-specific cells for disease modeling and drug discovery. Our ultimate goal is to repair the genetic defect of the Gaucher-specific iPS cells, differentiate them into long-term self-renewing hemangioblasts, and engraft repaired autologous hemangioblasts into patients for cure of the disease.