title: Lentiviral-mediated HoxB4 expression in human embryonic stem cells initiates early hematopoiesis in a dose-dependent manner but does not promote myeloid differentiation.
abstract: The variation of HoxB4 expression levels might be a key regulatory mechanism in the differentiation of human embryonic stem cell (hESC)-derived hematopoietic stem cells (HSCs). In this study, hESCs ectopically expressing high and low levels of HoxB4 were obtained using lentiviral gene transfer. Quantification throughout differentiation revealed a steady increase in transcription levels from our constructs. The effects of the two expression levels of HoxB4 were compared regarding the differentiation potential into HSCs. High levels of HoxB4 expression correlated to an improved yield of cells expressing CD34, CD38, the stem cell leukemia gene, and vascular epithelium-cadherin. However, no improvement in myeloid cell maturation was observed, as determined by colony formation assays. In contrast, hESCs with low HoxB4 levels did not show any elevated hematopoietic development. In addition, we found that the total population of HoxB4-expressing cells, on both levels, decreased in developing embryoid bodies. Notably, a high HoxB4 expression in hESCs also seemed to interfere with the formation of germ layers after xenografting into immunodeficient mice. These data suggest that HoxB4-induced effects on hESC-derived HSCs are concentration-dependent during in vitro development and reduce proliferation of other cell types in vitro and in vivo. The application of the transcription factor HoxB4 during early hematopoiesis from hESCs might provide new means for regenerative medicine, allowing efficient differentiation and engraftment of genetically modified hESC clones. Our study highlights the importance of HoxB4 dosage and points to the need for experimental systems allowing controlled gene expression. Disclosure of potential conflicts of interest is found at the end of this article.
authors.affiliation: Department of Medicine, M54, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden.
authors.forename: Christian
authors.initials: C
authors.lastname: Unger
authors.forename: Elerin
authors.initials: E
authors.lastname: Kärner
authors.forename: Alexandra
authors.initials: A
authors.lastname: Treschow
authors.forename: Birgitta
authors.initials: B
authors.lastname: Stellan
authors.forename: Ulrika
authors.initials: U
authors.lastname: Felldin
authors.forename: Hernan
authors.initials: H
authors.lastname: Concha
authors.forename: Mikael
authors.initials: M
authors.lastname: Wendel
authors.forename: Outi
authors.initials: O
authors.lastname: Hovatta
authors.forename: Alar
authors.initials: A
authors.lastname: Aints
authors.forename: Lars
authors.initials: L
authors.lastname: Ahrlund-Richter
authors.forename: M Sirac
authors.initials: MS
authors.lastname: Dilber
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D003114:Colony-Forming Units Assay; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006410:Hematopoiesis / Q000235:genetics*; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008297:Male; D051379:Mice; D016513:Mice, SCID; D022423:Myeloid Cells / Q000166:cytology*; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D013724:Teratoma / Q000473:pathology; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014161:Transduction, Genetic
chemical_list: D015415:Biomarkers; C090460:HOXB4 protein, human; D018398:Homeodomain Proteins; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D014157:Transcription Factors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins