Our laboratory is using human induced pluripotent stem cells (iPSC) for disease modeling and drug discovery. By viral-mediated introduction of the 4 Yamanaka factors (Oct4, Sox2, Klf4 and c-Myc) we have reprogrammed patient fibroblasts into iPSC representative of all 3 clinical subtypes of Gaucher disease. This is an autosomal recessive disorder caused by mutations in the acid beta-glucocerebrosidase (GCase) gene (GBA1). GCase deficiency results in the accumulation of glucosylsphingolipids, leading to phenotypic abnormalities in a number of cell types, including macrophages, neuronal cells, osteoblasts, and hematopoietic cells. The enzyme deficiency in Gaucher patients results in hepatosplenomegaly, hematologic abnormalities, bone disease, and there is a 50-fold increased propensity for development of B cell lymphomas and multiple myeloma. The more severe mutations in types 2 and 3 Gaucher disease also cause neurological manifestations that are fatal. The importance of this enzyme extends well beyond Gaucher disease. 10% of all Parkinson’s disease cases also have mutations in GBA1, making them the highest known risk factor for Parkinson’s disease. By directed differentiation of patient-derived iPSC to the affected cell types, we have been able to recapitulate characteristic hallmarks of the disease, and obtained new mechanistic insights with important clinical implications.
We found that the abnormal phenotype of Gaucher cells is not merely due to an accumulation of lipid, but there is also a defect in intracellular membrane transport, which blocks the flow of cargo (phagocytosed material, protein aggregates, organelles for recycling) to the lysosome. The mutant iPSC-derived macrophages exhibit a striking delay in clearing of phagocytosed red blood cells, which recapitulates a characteristic hallmark of the disease. In mutant iPSC-derived neurons there is an autophagy block that prevents fusion of autophagosomes with lysosomes, leading to neuronal cell death. Analysis of the mechanisms involved showed that mutant GBA1 interferes with lysosomal biogenesis by downregulation and destabilization of TFEB, the master regulator of lysosomal and autophagy genes (Awad et al., submitted).
Further analysis using this "disease-in-a-dish" iPSC model showed that in addition to deregulation of essential lysosomal functions, mutant GBA1 causes developmental defects, recapitulating the cytopenias and other clinical manifestations observed in patients. We are now using the tools of molecular genetics to elucidate the mechanisms by which GBA1 mutations interfere with critical developmental pathways in different cell types affected by the disease. The mutant GBA1 iPSC lines and directed differentiation protocols we developed, will enable us to use functional, cell-based assays to identify new drugs for dual use in preventing or reversing the clinical manifestations of Gaucher and Parkinson’s disease.
Previous work in the laboratory involved the use transgenic mice (SCL-TVA), which allowed targeted delivery of genes to hematopoietic and vascular endothelial stem cells in the intact animal. One application of this gene delivery system is in vivo. 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. This flexible experimental system circumvents the problems associated with conventional in vitro manipulation of bone marrow stem cells and transplantation into lethally irradiated animals. A goal of our laboratory is to combine our expertise in reprogramming technology and targeted delivery of genes to specific tissues, for in vivo reprogramming, gene therapy, and regenerative medicine.
A postdoctoral position is available in the Department of Microbiology and Immunology at the University of Maryland School of Medicine, to model inherited lipid storage diseases using iPS cell technology. Patient-derived iPS cells will be differentiated into different cell types, including hematopoietic and neuronal lineages, and used to study the molecular mechanisms leading to abnormal cell function, and to develop gene- and stem cell-based therapy. The candidate should have a PhD or equivalent degree, experience in Molecular and Cell Biology, excellent oral and written communication skills, and be able to work independently. Experience in hES/iPS cell technology and hematopoietic or neural development is desirable. Our laboratory is part of the newly established Center for Stem Cell Biology & Regenerative Medicine at UMB. This is an excellent opportunity for broad-based training in reprogramming biology and regenerative medicine. Salary is commensurate with experience. To apply, please send resume and contact information for three references to Dr. Ricardo A. Feldman