title: Activation of KLF1 Enhances the Differentiation and Maturation of Red Blood Cells from Human Pluripotent Stem Cells.
abstract: Blood transfusion is widely used in the clinic but the source of red blood cells (RBCs) is dependent on donors, procedures are susceptible to transfusion-transmitted infections and complications can arise from immunological incompatibility. Clinically-compatible and scalable protocols that allow the production of RBCs from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been described but progress to translation has been hampered by poor maturation and fragility of the resultant cells. Genetic programming using transcription factors has been used to drive lineage determination and differentiation so we used this approach to assess whether exogenous expression of the Erythroid Krüppel-like factor 1 (EKLF/KLF1) could augment the differentiation and stability of iPSC-derived RBCs. To activate KLF1 at defined time points during later stages of the differentiation process and to avoid transgene silencing that is commonly observed in differentiating pluripotent stem cells, we targeted a tamoxifen-inducible KLF1-ERT2expression cassette into the AAVS1 locus. Activation of KLF1 at day 10 of the differentiation process when hematopoietic progenitor cells were present, enhanced erythroid commitment and differentiation. Continued culture resulted the appearance of more enucleated cells when KLF1 was activated which is possibly due to their more robust morphology. Globin profiling indicated that these conditions produced embryonic-like erythroid cells. This study demonstrates the successful use of an inducible genetic programing strategy that could be applied to the production of many other cell lineages from human induced pluripotent stem cells with the integration of programming factors into the AAVS1 locus providing a safer and more reproducible route to the clinic. Stem Cells 2017;35:886-897.
authors.affiliation: Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Cheng-Tao
authors.initials: CT
authors.lastname: Yang
authors.affiliation: Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Rui
authors.initials: R
authors.lastname: Ma
authors.affiliation: Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Axton
authors.affiliation: Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Melany
authors.initials: M
authors.lastname: Jackson
authors.affiliation: Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: A Helen
authors.initials: AH
authors.lastname: Taylor
authors.affiliation: Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Antonella
authors.initials: A
authors.lastname: Fidanza
authors.affiliation: Institute of Cardiovascular & Medical Sciences, University of Glasgow, Glasgow, United Kingdom.|Scottish National Blood Transfusion Service, Scotland, United Kingdom.
authors.forename: Lamin
authors.initials: L
authors.lastname: Marenah
authors.affiliation: Department of Biochemistry, University of Bristol, United Kingdom.
authors.forename: Jan
authors.initials: J
authors.lastname: Frayne
authors.affiliation: Institute of Cardiovascular & Medical Sciences, University of Glasgow, Glasgow, United Kingdom.|Scottish National Blood Transfusion Service, Scotland, United Kingdom.
authors.forename: Joanne C
authors.initials: JC
authors.lastname: Mountford
authors.affiliation: Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Lesley M
authors.initials: LM
authors.lastname: Forrester
mesh_terms: D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D004912:Erythrocytes / Q000166:cytology* / Q000378:metabolism*; D004920:Erythropoiesis / Q000235:genetics; D005786:Gene Expression Regulation; D005914:Globins / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D020014:K562 Cells; D051741:Kruppel-Like Transcription Factors / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D051741:Kruppel-Like Transcription Factors; D011993:Recombinant Fusion Proteins; C080774:erythroid Kruppel-like factor; D005914:Globins
keywords: Erythroid differentiation; Gene delivery systems in vivo or in vitro; Induced pluripotent stem cells; Transcription factors