Optimizing the production of erythroid cells from human embryonic stem cells
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Red blood cell (RBC) transfusion is the major treatment for patients suffering from trauma or severe anaemias, and life-long transfusion may be needed to alleviate symptoms and maintain body functioning. However, with a relatively low portion of people are donating, shortage in blood supply is becoming a life-threatening issue in the aging society. Among attempts to identify novel sources for transfusion medicine, human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are currently the most promising candidate, which is capable of producing donor-independent, pathogen-free and immunologically compatible RBCs. Currently, hESC-derived erythropoiesis in vitro is considered to mimic the very primitive yolk sac haematopoiesis, indicated by a low or absent level of β globin production and incomplete enucleation. Thus these cells are not mature enough to be used in transfusion medicine. The aim of this PhD project was to overexpress a key erythroid transcription factor, Erythroid Krüppel-like factor (EKLF or KLF1) in an inducible manner to improve the maturation of hESC-derived erythroid cells. EKLF is a member of the Krüppel-like factor family, which is characterized by three C2H2 type zinc finger motifs. EKLF expression in vivo is highly restricted to erythroid cells in yolk sac, fetal liver, spleen and the bone marrow, although recently a low-level of expression was found in haematopoietic precursors. Published reports demonstrate that EKLF can 1) activate β globin expression by binding to the CACCC box in its promoter or by altering β-like globin locus chromatin structure; 2) exert a role in MEP (common progenitor for erythrocytes and megakaryocytes) stage by favouring erythroid differentiation against megakaryocyte differentiation; 3) promote enucleation by affecting the DNase II-alpha expression in the central macrophage of a fetal liver erythroblastic island; 4) act as an instructive factor for lineage commitment towards erythroid fate in HSCs. In this project, 1) We tested and evaluated a feeder-free, serum-free differentiation system for deriving erythroid cells from hESCs; 2) We constructed constitutive and inducible EKLF expression vectors and validated them in K562 cells; 3) We generated hESC lines carrying these EKLF expression vectors and assessed their effects on erythrocyte production and maturation. We found that our differentiation system was capable of generating haematopoietic progenitors (HPCs) and erythroid cells at high efficiency. Using this differentiation system, we concluded that enhanced expression of EKLF upregulated adult β globin expression selectively, without altering expressions of other globins. This finding provides hints for the development of novel approaches to “reprogramme” hESCs towards a certain fate and overexpression of EKLF in this differentiation system may be beneficial for resolving issues in future transfusion medicine.