title: Reprogramming of human peripheral blood monocytes to erythroid lineage by blocking of the PU-1 gene expression.
abstract: In hematopoietic system development, PU.1 and GATA-1 as lineage-specific transcription factors (TF) are expressed in common myeloid progenitors. The cross antagonism between them ascertains gene expression programs of monocytic and erythroid cells, respectively. This concept in transdifferentiation approaches has not been well considered yet, especially in intralineage conversion systems. To demonstrate whether PU.1 suppression induces monocyte lineage conversion into red blood cells, a combination of three PU.1-specific siRNAs was implemented to knock down PU.1 gene expression and generate the balance in favor of GATA-1 expression to induce erythroid differentiation. For this purpose, monocytes were isolated from human peripheral blood and transfected by PU.1 siRNAs. In transfected monocytes, the rate of PU.1 expression in mRNA level was significantly decreased until 0.38 ± 0.118 when compared to untreated monocytes at 72 h (p value ≤0.05) which resulted in significant overexpression of GATA1 of 16.1 ± 0.343-fold compared to the untreated group (p value ≤0.01). Subsequently, overexpression of hemoglobin (α 13.26 ± 1.34-fold; p value≤0.0001) and β-globin (37.55 ± 16.56-fold; p value≤0.0001) was observed when compared to control groups. The results of western immunoblotting confirm those findings too. While, reduced expression of monocyte, CD14 gene, was observed in qRT-PCR and flow cytometry results. Our results suggest that manipulating the ratio of the two TFs in bifurcation differentiation pathways via applying siRNA technology can possibly change the cells' fate as a safe way for therapeutics application.
authors.affiliation: Department of Molecular Medicine and Biochemistry, National Institute of Genetic Engineering and Biotechnology Km. 17, Karaj-Tehran freeway Pajouhesh Blvd., P.O.Box 14155-6343, Tehran, Iran.
authors.forename: Masoumeh
authors.initials: M
authors.lastname: Nouri
authors.affiliation: Department of Molecular Medicine and Biochemistry, National Institute of Genetic Engineering and Biotechnology Km. 17, Karaj-Tehran freeway Pajouhesh Blvd., P.O.Box 14155-6343, Tehran, Iran. deezagi@nigeb.ac.ir.
authors.forename: Abdolkhalegh
authors.initials: A
authors.lastname: Deezagi
authors.affiliation: Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
authors.forename: Marzieh
authors.initials: M
authors.lastname: Ebrahimi
mesh_terms: D019070:Cell Lineage / Q000502:physiology*; D000067470:Cellular Reprogramming Techniques / Q000379:methods*; D015672:Erythroid Precursor Cells / Q000378:metabolism*; D050982:GATA1 Transcription Factor / Q000096:biosynthesis*; D005786:Gene Expression Regulation; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D009000:Monocytes / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis*; D015534:Trans-Activators / Q000096:biosynthesis*; D000099232:Proto-Oncogene Protein Spi-1
chemical_list: D050982:GATA1 Transcription Factor; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1; C494688:GATA1 protein, human
keywords: Cell reprogramming; Erythrocytes; GATA1; Monocytes; PU-1 siRNA