Effect of increased HoxB4 on human megakaryocytic development

Abstract

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.

Fig. 1

HoxB4 overexpression in TF1 cells increased the expression of CD61 and CD41a by primarily upregulating TpoR expression and downregulating c-Myb expression. (A) Percentages of CD61 or CD41a positive cells in untreated TF1 cells. (B) Percentages of CD61 or CD41a positive cells in TF1 cells treated by PDB and TPO. (C) Cell growth of untreated TF1 cells was inhibited by HoxB4 overexpression. (D) Absolute numbers of CD61 or CD41a positive cells in untreated TF1 cells. (E) Absolute numbers of CD61 or CD41a positive cells in TF1 cells treated by PDB and TPO. (F) HoxB4 expression in TF1 cells. (G) Gene expression in TF1 control and TF1-HoxB4 cells. (H) Gene expression in TF1 control and TF1-HoxB4 shRNA cells. Data is the averages of three independent experiments.

Fig. 2

Recombinant tPTD-HoxB4 fusion protein increased the expression of CD61 and CD41a during Mk development from CD34 positively-selected cord blood cells by primarily increasing TpoR expression and decreasing c-Myb expression. (A) Percentages of CD61 or CD41a positive cells on Day 5. (B) Percentages of CD61 or CD41a positive cells on Day 8. (C) Absolute numbers of CD61 or CD41a positive cells on Day 5. (D) Absolute numbers of CD61 or CD41a positive cells on Day 8. (E) Cell growth was inhibited by HoxB4 fusion protein. (F) Gene expression on Day 8. Data is from two independent experiments.

Fig. 3

CFU-Mk assays. (A) Numbers of CFU-Mk per 5,000 cells on Day 5 and Day 8. (B) Absolute numbers of CFU-Mk on Day 5 and Day 8. Data is from two independent experiments.