Department of Pathology, The Ohio State University, Columbus, OH 43210, USA.
Biochem Biophys Res Commun. 2010 Jul 30;398(3):377-82. doi: 10.1016/j.bbrc.2010.06.075. Epub 2010 Jun 22.
In order to produce clinically useful quantities of platelets ex vivo we may need to firstly enhance early self-renewal of hematopoietic stem cells (HSCs) and/or megakaryocyte (Mk) progenitors. The homeodomain transcription factor HoxB4 has been shown to be an important regulator of stem cell renewal and hematopoiesis; however, its effect on megakaryopoiesis is unclear. In this study, we investigated the effect of HoxB4 overexpression or RNA silencing on megakaryocytic development in the human TF1 progenitor cell line; we then used recombinant tPTD-HoxB4 fusion protein to study the effect of exogenous HoxB4 on megakaryocytic development of human CD34 positively-selected cord blood cells. We found that ectopic HoxB4 in TF1 cells increased the antigen expression of CD61and CD41a, increased the gene expression of thrombopoietin receptor (TpoR), Scl-1, Cyclin D1, Fog-1 and Fli-1 while it decreased c-Myb expression. HoxB4 RNA silencing in TF1 cells decreased the expression of CD61 and CD41a and decreased Fli-1 expression while it increased the expression of c-Myb. Recombinant tPTD-HoxB4 fusion protein increased the percentages and absolute numbers of CD41a and CD61 positive cells during megakaryocytic differentiation of CD34 positively-selected cord blood cells and increased the numbers of colony-forming unit-megakaryocyte (CFU-Mk). Adding tPTD-HoxB4 fusion protein increased the gene expression of TpoR, Cyclin D1, Fog-1 and Fli-1 while it inhibited c-Myb expression. Our data suggest that increased HoxB4 enhanced early megakaryocytic development in human TF1 cells and CD34 positively-selected cord blood cells primarily by upregulating TpoR and Fli-1 expression and downregulating c-Myb expression. Increasing HoxB4 expression or adding recombinant HoxB4 protein might be a way to expand Mks for the production of platelets for use in transfusion medicine.
为了在体外产生临床上有用数量的血小板，我们可能需要首先增强造血干细胞（HSCs）和/或巨核细胞（Mk）祖细胞的早期自我更新。同源盒转录因子 HoxB4 已被证明是干细胞更新和造血的重要调节因子；然而，其对巨核细胞生成的影响尚不清楚。在这项研究中，我们研究了 HoxB4 过表达或 RNA 沉默对人类 TF1 祖细胞系巨核细胞发育的影响；然后，我们使用重组 tPTD-HoxB4 融合蛋白研究外源性 HoxB4 对人 CD34 阳性选择脐血细胞巨核细胞发育的影响。我们发现，TF1 细胞中的异位 HoxB4 增加了 CD61 和 CD41a 的抗原表达，增加了血小板生成素受体（TpoR）、Scl-1、细胞周期蛋白 D1、Fog-1 和 Fli-1 的基因表达，同时降低了 c-Myb 的表达。TF1 细胞中的 HoxB4 RNA 沉默降低了 CD61 和 CD41a 的表达，并降低了 Fli-1 的表达，同时增加了 c-Myb 的表达。重组 tPTD-HoxB4 融合蛋白增加了 CD34 阳性选择脐血细胞巨核细胞分化过程中 CD41a 和 CD61 阳性细胞的百分比和绝对数量，并增加了集落形成单位-巨核细胞（CFU-Mk）的数量。添加 tPTD-HoxB4 融合蛋白增加了 TpoR、细胞周期蛋白 D1、Fog-1 和 Fli-1 的基因表达，同时抑制了 c-Myb 的表达。我们的数据表明，增加 HoxB4 主要通过上调 TpoR 和 Fli-1 的表达和下调 c-Myb 的表达，增强了人类 TF1 细胞和 CD34 阳性选择脐血细胞的早期巨核细胞发育。增加 HoxB4 表达或添加重组 HoxB4 蛋白可能是一种扩大巨核细胞以用于输血医学中血小板生产的方法。
