title: Knockdown of Pu.1 by small interfering RNA in CD34+ embryoid body cells derived from mouse ES cells turns cell fate determination to pro-B cells.
abstract: The factors that regulate murine ES cell-derived hematopoietic progenitor cell (HPC) commitment to the B lymphocyte lineage remain unclear. Pu.1 plays an essential role in the development of all lymphoid lineages; however, it also regulates commitment to other blood cell lineages. In this study, we found evidence for early B cell lineage commitment as determined by coexpression of CD19 and CD45R (B220) when Pu.1 expression was knocked down in HPC by specific small interfering RNA (siRNA); moreover, the expression of early B cell factor (Ebf) and paired box protein 5 (Pax-5) transcription factors was induced when cells were treated by Pu.1 siRNA, but not by control siRNA. We also found that siRNA-mediated knockdown of Pu.1 expression was more efficient in generating progenitor B cells (pro-B cells) compared with the more common in vitro method of B lymphoid development by means of coculture of CD34+ embryoid body (EB) cells with OP9 stromal cells. To investigate whether this phenomenon also exists in HPC from other sources, we then knocked down Pu.1 gene expression in CD34+ murine bone marrow cells and found a similar effect of increased production of CD19+CD43+CD45R+ progenitor B cells upon the siRNA-mediated decrease in Pu.1 expression. We conclude that, in early B cell development from ES cell-derived HPC, constitutive Pu.1 expression inhibits the earliest B cell development through repressing early B cell factor and paired box protein 5 expression, although lower levels of Pu.1 expression in HPC play a key role in promoting B cell fate determination.
authors.affiliation: Section of Hematology/Oncology, Department of Medicine, University of Chicago Medical Center, 5841 South Maryland Avenue, MC2115, Chicago, IL 60637, USA.
authors.forename: Gang-Ming
authors.initials: GM
authors.lastname: Zou
authors.forename: Jian-Jun
authors.initials: JJ
authors.lastname: Chen
authors.forename: Mervin C
authors.initials: MC
authors.lastname: Yoder
authors.forename: Wei
authors.initials: W
authors.lastname: Wu
authors.forename: Janet D
authors.initials: JD
authors.lastname: Rowley
mesh_terms: D000818:Animals; D018952:Antigens, CD34; D001402:B-Lymphocytes / Q000166:cytology*; D019070:Cell Lineage; D018920:Coculture Techniques; D004268:DNA-Binding Proteins / Q000235:genetics; D015536:Down-Regulation; D004622:Embryo, Mammalian / Q000166:cytology; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000502:physiology*; D051379:Mice; D051757:PAX5 Transcription Factor; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D034741:RNA, Small Interfering / Q000494:pharmacology*; D017154:Stromal Cells / Q000166:cytology; D015534:Trans-Activators / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics; D000099232:Proto-Oncogene Protein Spi-1
chemical_list: D018952:Antigens, CD34; D004268:DNA-Binding Proteins; D051757:PAX5 Transcription Factor; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D015534:Trans-Activators; D014157:Transcription Factors; D000099232:Proto-Oncogene Protein Spi-1; C489921:Ebf1 protein, mouse; C497282:Pax5 protein, mouse