Title: Congenital disease-specific human embryonic stem cells reveal developmental defects in the hematopoietic lineage
Description: Human embryonic stem cells (hESCs) enable the study of uniquely human aspects of development. The process of in vitro hematopoietic differentiation from hESCs recapitulates many key aspects of the cell fate and lineage specification decisions that occur during fetal hematopoiesis. Therefore, hESCs could serve as a unique platform for the study of hematopoietic development and its dysfunction in hematological disease. In this dissertation, we present hESC-based models of Fanconi anemia (FA) and Shwachman-Diamond syndrome (SDS), two inherited pediatric bone marrow failure disorders whose pathogeneses are poorly understood due to the lack of appropriate animal models. FA is a genetically heterogeneous, autosomal recessive disorder characterized by progressive marrow aplasia. Mouse models deficient in FA genes do not develop bone marrow failure and thus fail to recreate the central feature of the human disease. We have created a human-specific system to study the developmental aspects of FA using a lentiviral RNAi strategy in hESCs. We show that knockdown of FANCA and FANCD2 in hESCs leads to a reduction in hematopoietic fates reflective of the dysfunction seen in FA patients. Our data indicates that hematopoiesis is impaired in FA from the very earliest stages of development, a novel contribution to our understanding of the disease pathogenesis.

SDS is characterized by prominent neutropenia, exocrine pancreatic insufficiency, and an increased risk of acute myeloid leukemia. It is caused by mutations in the Shwachman-Bodian-Diamond syndrome ( SBDS ) gene. Knockout of the SBDS gene in mice resulted in early embryonic lethality and thus a model system for studying the pathogenesis of neutropenia and bone marrow failure in SDS is lacking. Knockdown of SBDS in hESCs revealed a specific reduction in granulocytic fates, without a concomitant decrease in overall hematopoietic development. Therefore, our results establish a platform for further mechanistic dissection of the development of neutropenia in SDS. This dissertation presents hESCs as a powerful new model system for the study of hematopoietic dysfunction in human congenital disease. Our results provide insight into the disease pathogenesis of FA and SDS and more generally, demonstrate the utility of hESCs in furthering our understanding of complex hereditary illness.