Mechanism of Blood Maturation Induced by Hedgehog Inhibition in Pluripotent Sources

Abstract

<p>The generation of hematopoietic progenitors from human pluripotent cell sources for use in personalized medicine is an attainable goal for the ease of clinical intervention using these cells. Furthermore, generated platelets and mature red blood cells are enucleated which allows for the use of induced pluripotent stem cells as a starting source or other sources of genetic manipulation. Generating these cells has proven difficult as the cells appear to be stuck in a primitive state of differentiation and do not mature into an adult phenotype. This thesis shows that inhibition of the hedgehog signaling pathway early in the differentiation of pluripotent stem cells induces a maturation towards definitive hematopoiesis. Generated erythroid cells were shown to express beta globin at the transcript as well as protein level. This maturation effect was confirmed to occur through central hedgehog repressor, Gli3R, through genetic manipulation. Further interrogation of this mechanism showed that globin regulation was not mediated by chromatin methylation by the polycomb repressive complex. Finally, Gli3R was also shown to not act as a transcription factor influencing globin expression directly and is therefore engaging separate regulatory mechanisms. This data provides great strides towards the generation of clinically relevant hematopoietic populations from pluripotent sources, however Gli3R’s direct mechanism of action remains to be determined.</p>