Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Anayah I. Ferris - Univeristy of the Virgin Islands
Co-Author(s): Anne D. Koniski, University of Rochester Medical School, Rochester, NY; Kathleen E. McGrath, University of Rochester Medical School, Rochester, NY; James Palis, University of Rochester Medical School, Rochester, NY
Embryonic stem cells (ESC) are maintained as undifferentiated stem cells, and when placed into specific culture conditions, can be induced to become erythroid cells. Scientists would like to use these cells as a source of blood cells for transfusions as well as to study erythropoiesis. However, during maturation in the mouse embryo overlapping waves of primitive (embryonic) and definitive (adult-like) erythroid cells are observed which express different globin genes. Our lab has evidence that both primitive and definitive erythroid cells are formed in overlapping waves in murine ESC, but globin analysis of one of the most well characterized systems of human ESC differentiation only looked at very late stages of maturation. Based on literature and our findings, we hypothesize that human ESC differentiation will produce both primitive and definitive erythroid cells. We aimed to answer the following questions in this study: 1) can we observe globin gene expression changes during ESC maturation similar to what is observed in embryos and 2) can we identify specific progenitors (CFC) that have a primitive or definitive globin expression patterns and do they change in prevalence during ESC development? Human ESC were differentiated and globin gene expression was analyzed at early and late time points. Changes in globin expression were found that support an overlap in the primitive and definitive erythroid waves, similar to that in the mammalian embryo. Additionally cells were sorted based on immunophenotype, and assayed for erythroid progenitors by being plated in methylcellulose at Day 7 & Day 14, where they will grow and form colonies. Progenitor colonies were plucked according to morphology and assayed to determine globin expression using qPCR. No correlation between colony morphology or tested immunophenotype and progenitor colony globin expression was found. However, there is a significant difference between the expression of embryonic globins on day 7 versus day 14 colonies (P values= 0.0001 and 0.0003, respectively). Thus, globin expression of single progenitor colonies also supports overlapping primitive and definitive erythroid waves during ESC differentiation.Not Submitted
Funder Acknowledgement(s): R01 HL120670 NYSTEM IDEAS U01 HL1346996 Michael Napoleone Memorial Foundation Grant
Faculty Advisor: Kathleen E. McGrath, firstname.lastname@example.org
Role: I took part in all aspects of the research. My responsibilities included differentiating the human embryonic stem cells to erythroid cells, analyzing the globin gene expression, sorting cells based on morphology and immunophenotype, and assaying for erythroid progenitors.