Shaikh, Seema2011-05-022011-05-022010-12https://hdl.handle.net/11299/103451University of Minnesota M.S. thesis. December 2010. Major: Stem cell biology. Advisor: Dr. Michael Kyba. 1 computer file (PDF); vii, 67 pages. Ill. (some col.)A single fertilized cell has the ability to develop into any cell depending on the various cues it responds to. This ability to differentiate into desired cell types can be made use of in the field of developmental biology for studying early embryonic development and for regenerative medicine. Previous work in the lab showed that mouse ES cells engineered with an inducible construct co-expressing the hematopoietic regulatory factors SCL, LMO2 and GATA2 give very efficient hematopoiesis. In monolayer differentiation where hematopoiesis does not occur because the majority of cells differentiate towards ectoderm, expression of these 3 factors diverted cells towards hematopoietic lineage. My work in the lab addressed two questions: Can we optimize the system to obtain hematopoietic progenitors instead of differentiated blood cells? and How does the SCL complex reprogram cells at the molecular level? I added a cocktail of cytokines to the serum free, growth factor free medium to obtain undifferentiated hematopoietic progenitors. A short pulse of induction was sufficient to obtain large number of CD41+, hemoglobin expressing, round semi adherent cells. This treatment gave rise to progenitors of myeloid, erythroid and megakaryocytic lineages proving the multipotent nature of the blood cells that differentiated to both hematopoietic progenitors and committed erythroid cells. To understand the reprogramming potential of the SCL complex at the transcriptional level I performed two RNA sequencing experiments. The first experiment evaluated the early changes and showed that the SCL complex up-regulated many important hematopoietic genes including SCL, LMO2, GATA2, Lyl1 and Gfi1 within 6 hours, but other genes (globins) required a longer period of induction. The second experiment evaluated cells 3 days after a reprogramming pulse. The data showed that the non-reprogrammed (CD41-) cells expressed hematopoietic genes, but at lower levels compared to the fully reprogrammed cells (CD41+) indicating that the cells were not completely reprogrammed. Surprisingly the CD41- cells had higher expression of endodermal genes indicating that the cell could have reverted endoderm, a nearby lineage during the chase period. The data also showed a decrease in the ectodermal genes in both CD41+ and CD41- cells suggesting that the triple construct may be stably erasing the ectodermal program even in non-reprogrammed cells. These results improved the efficiency of the system and shed light on the mechanism of the SCL-LMO2-GATA2 action in lineage specific reprogramming.en-USFertilized cellEmbryonic developmentHematopoietic lineageRNAStem cell biologyThesis or Dissertation