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Characterization of Transcription Factor Networks Involved in Umbilical Cord Blood CD34+ Stem Cells-Derived Erythropoiesis

ABSTRACT: The characteristics of global gene expression patterns during umbilical cord blood (UCB)-CD34+ stem cell-derived erythropoiesis are not clearly elucidated. In this study, UCB-stem cells were grown in liquid culture as a model for this process. We observed a high proliferative capacity of UCB-stem cells producing over 10 billion viable cells in culture. Normal erythroid maturation was confirmed by increased expression of CD71 and CD235a biomarkers. Furthermore, the gamma- to beta-globin gene switch was observed around day 45 indicating extended gamma-globin gene expression in fetal erythroid cells. To study global gene expression patterns, microarray analysis was performed on days 21, 42, 49 and 56. Forty-five transcription factors were silenced during the culture period (Profile-1) including CUX1 and HES5 among others. Conversely, 30 transcription factors were activated by day 56 (Profile-2) such as KLF1, GATA1, and MAFB. Both datasets were analyzed further using the MIMI Cytoscape platform which defined transcription factor networks around KLF4 and GATA2 from Profile-1 genes and KLF1 and GATA1 for Profile-2 genes. The characteristics of UCB-CD34+ stem cells including high proliferative capacity and prolonged gamma-globin expression combined with novel transcription factor networks suggest novel mechanisms of fetal UCB-stem cell-derived erythropoiesis.

ORGANISM(S): Homo sapiens

PROVIDER: GSE49438 | GEO | 2013/08/02

SECONDARY ACCESSION(S): PRJNA213913

REPOSITORIES: GEO

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Project description:The Krüppel-like factors, KLF1 and KLF2, positively regulate embryonic β-globin expression, and have additional overlapping roles in embryonic (primitive) erythropoiesis. KLF1-/-KLF2-/- double knockout mice are anemic at embryonic day 10.5 (E10.5) and die by E11.5, in contrast to single knockouts. To investigate the combined roles of KLF1 and KLF2 in primitive erythropoiesis, expression profiling of E9.5 erythroid cells was performed. A limited number of genes had a significantly decreasing trend of expression in wild-type, KLF1-/- and KLF1-/-KLF2-/-. Among these, c-myc emerged as a central node in the most significant gene network. c-myc expression is synergistically regulated by KLF1 and KLF2, and both factors bind the c-myc promoters. To characterize the role of c-myc in primitive erythropoiesis, ablation was performed specifically in mouse embryonic proerythroblast cells. After E9.5, these embryos exhibit an arrest in the normal expansion of circulating red cells and develop anemia analogous to KLF1-/-KLF2-/-. In the absence of c-myc, circulating erythroid cells do not show the normal increase in α- and β-like globin expression, but interestingly, have accelerated erythroid maturation, between E9.5 and E11.5. This study reveals a novel regulatory network by which KLF1 and KLF2 regulate c-myc, to control the primitive erythropoietic program. Timed-pregnant KLF1+/-, KLF1+/- KLF2+/- females were anesthetized and sacrificed. E9.5 yolk sacs were dissected from the embryo, cryoprotected in 20% sucrose in PBS and frozen in OCT media. A small portion of the embryo tail was used for PCR genotyping. Eight micron frozen yolk sac sections were obtained and laser capture microdissection (LCM) was used to isolate primitive erythroid precursors. For each biological replicate, 2 to 4 yolk sacs from 2 different litters were used. Total RNA was isolated from 8 different wild-type, 3 KLF1-/-, 3 KLF1-/- KLF2-/- erythroid samples and hybridized to Affymetrix 430 A 2.0 microarrays.

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Characterization of Transcription Factor Networks Involved in Umbilical Cord Blood CD34+ Stem Cells-Derived Erythropoiesis

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