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RNA-seq of human iPS derived macrophages with or without KLF1- transcription factor Activation

ABSTRACT: Red blood cells (RBCs) mature within a specialized niche (the erythroblastic island (EI)), which consists of a central macrophage surrounded by differentiating erythroblasts. Human Induced Pluripotent Stem Cell derived macrophages (iPSC-DMs) enhance proliferation and terminal maturation of Umbilical Cord Blood (UCB) CD34+ derived erythroid cells and iPSC derived erythroid cells. These effects are further increased when an inducible KLF1-ERT2 fusion protein is activated in iPSC-DMs. To assess the mechanism of action, we sought to compare the transcriptome of iPSC-DMs with and without KLF1 activation. For this, we used an inducible IPSC line (iKLF1.2) in which upon tamoxifen addition, the KLF1 transcription factor is translocated to nucleus and consequently KLF1 downstream targets are expressed. The identification and characterisation of could identify factors involved in erythroid maturation and thus helpful to improve current protocols to manufacture RBCs in vitro.

ORGANISM(S): Homo sapiens

PROVIDER: GSE125150 | GEO | 2019/01/16

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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RNA-seq of human iPS derived macrophages with or without KLF1- transcription factor Activation

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