title: Enforced Expression of HOXB4 in Human Embryonic Stem Cells Enhances the Production of Hematopoietic Progenitors but Has No Effect on the Maturation of Red Blood Cells.
abstract: : We have developed a robust, Good Manufacturing Practice-compatible differentiation protocol capable of producing scalable quantities of red blood cells (RBCs) from human pluripotent stem cells (hPSCs). However, translation of this protocol to the clinic has been compromised because the RBCs produced are not fully mature; thus, they express embryonic and fetal, rather than adult globins, and they do not enucleate efficiently. Based on previous studies, we predicted that activation of exogenous HOXB4 would increase the production of hematopoietic progenitor cells (HPCs) from hPSCs and hypothesized that it might also promote the production of more mature, definitive RBCs. Using a tamoxifen-inducible HOXB4-ER(T2) expression system, we first demonstrated that activation of HOXB4 does increase the production of HPCs from hPSCs as determined by colony-forming unit culture activity and the presence of CD43(+)CD34(+) progenitors. Activation of HOXB4 caused a modest, but significant, increase in the proportion of immature CD235a(+)/CD71(+) erythroid cells. However, this did not result in a significant increase in more mature CD235a(+)/CD71(-) cells. RBCs produced in the presence of enhanced HOXB4 activity expressed embryonic (ε) and fetal (γ) but not adult (β) globins, and the proportion of enucleated cells was comparable to that of the control cultures. We conclude that programming with the transcription factor HOXB4 increases the production of hematopoietic progenitors and immature erythroid cells but does not resolve the inherent challenges associated with the production of mature adult-like enucleated RBCs.
  
  
  
  As worldwide blood donations decrease and transfusable transmitted infections increase, intense interest has ensued in deriving red blood cells (RBCs) in vitro from alternative sources such as pluripotent stem cells. A translatable protocol was developed to generate RBCs; however, these RBCs have an immature phenotype. It was hypothesized that the transcription factor HOXB4 could enhance their production and maturation. Although HOXB4 increased the production of erythroid progenitors, it did not promote their maturation. Despite the remaining challenges, a robust system has been established to test other candidates and add to the knowledge base in this field.
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: Rui
authors.initials: R
authors.lastname: Ma
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: Richard A
authors.initials: RA
authors.lastname: Axton
authors.affiliation: Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Easterbrook
authors.affiliation: Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Maria
authors.initials: M
authors.lastname: Kydonaki
authors.affiliation: Institute of Cardiovascular and Medical Sciences, British Heart Foundation Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, United Kingdom Scottish National Blood Transfusion Service, Edinburgh, United Kingdom.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Olivier
authors.affiliation: Institute of Cardiovascular and Medical Sciences, British Heart Foundation Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, United Kingdom Scottish National Blood Transfusion Service, Edinburgh, United Kingdom.
authors.forename: Lamin
authors.initials: L
authors.lastname: Marenah
authors.affiliation: Murdoch Childrens Research Institute, The Royal Children's Hospital, Parkville, Victoria, Australia Department of Anatomy and Developmental Biology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, Victoria, Australia Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Edouard G
authors.initials: EG
authors.lastname: Stanley
authors.affiliation: Murdoch Childrens Research Institute, The Royal Children's Hospital, Parkville, Victoria, Australia Department of Anatomy and Developmental Biology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, Victoria, Australia Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Elefanty
authors.affiliation: Institute of Cardiovascular and Medical Sciences, British Heart Foundation Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, United Kingdom Scottish National Blood Transfusion Service, Edinburgh, United Kingdom.
authors.forename: Joanne C
authors.initials: JC
authors.lastname: Mountford
authors.affiliation: Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom L.Forrester@ed.ac.uk.
authors.forename: Lesley M
authors.initials: LM
authors.lastname: Forrester
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D019070:Cell Lineage; D049109:Cell Proliferation; D002478:Cells, Cultured; D065150:Cellular Reprogramming; D000067470:Cellular Reprogramming Techniques; D053595:Embryonic Stem Cells / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010641:Phenotype; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D015854:Up-Regulation
chemical_list: D015415:Biomarkers; C090460:HOXB4 protein, human; D018398:Homeodomain Proteins; D014157:Transcription Factors
keywords: CD34+; Cell surface markers; Colony formation; Differentiation; Electroporation; Embryonic stem cells; Erythroid differentiation; Transcription factor