Abstract
Abstract 908
The determinant factors leading from stem cells to megakaryocytes (MK) and the subsequent release of platelets have yet to be identified. Here we report that a combination of transcription factors p45NF-E2, MafG, and MafK converts mouse fibroblast cell line 3T3 and adult human dermal fibroblasts into MKs. We first performed screening for MK-inducing factors using the preadipocyte cell line 3T3-L1 known to be able to differentiate into MKs in a previously defined MK lineage induction media containing thrombopoietin (Zauli G et al, Blood 1997). Gene expression levels of candidate transcription factors were compared between 3T3 cells that do not differentiate into MKs and 3T3-L1 cells using quantitative real-time PCR. The expressions of p45NF-E2 and CEBPα were undetectable in 3T3 cells, whereas expression was seen in 3T3-L1 cells. Both cell lines had similar expression levels of GATA2, RUNX1, FOG1, and PPARγ and undetectable levels of GATA1. 3T3 cells transfected with p45NF-E2, its binding protein Maf, or CEBPα, and 3T3 cells transfected with any combination of these genes, were cultured in MK lineage induction media for 8 days. MK differentiation was assessed using CD41 expression in MK-sized cells by flow cytometry analysis. Frequency of CD41 expression in the 3T3 cells varied based on transgenic factors expressed: 19 ± 12% (p45NF-E2); 25 ± 13% (p45NF-E2/MafG/MafK); 18 ± 3% (p45NF-E2/MafG/MafK/CEBPα); 13 ± 2% (p45NF-E2/CEBPα); and 4 ± 1% (CEBPα). These results suggest that p45NF-E2 with or without Maf are defined factors for reprogramming of 3T3 fibroblast to MKs. We then tested whether other adult non-hematopoietic tissues could be forced into megakaryopoiesis by ectopic expression of p45NF-E2 and Maf. Adult human dermal fibroblasts (Cell Applications) were transfected with p45NF-E2/Maf genes and cultured in MK lineage induction media for 12 days. Large-sized cells were isolated using a 2-step density BSA gradient. By flow cytometry analysis, >99% of these cells expressed CD41. DNA ploidy of the induced MKs (iMKs) ranged from 2N to 16N with a mean of 4N. We then infused 5 × 105 large-sized iMKs into NOG mouse after induction of mild thrombocytopenia in them at day 7 after irradiation with 2.0 Gy. Tail vein samples were obtained from the tested female recipient NOG mice before and after 5 min, 30 min, 90 min, and 3h post iMK infusion. Flow cytometry analysis was performed on each sample stained with PE-conjugated anti-mouse CD41 and FITC-conjugated anti-human CD41 antibody. Human CD41-positive platelet-sized cells in the sample from before and after 5 min, 30 min, 90 min, and 3h represented 0.9%, 0.5%, 2.1%, 3.3%, and 4.0%, respectively, of the entire platelet pool. To examine whether the iMK-derived platelets were incorporated into ex vivo thrombus formation, FITC-anti human CD41 antibody-labeled blood sample from iMK-infused thrombocytopenic NOG mouse was perfused on type I collagen-coated chip under flow condition (Total Thrombus-formation Analyzing System). After perfusion for 10 min, we observed that cells expressing human CD41 were incorporated into thrombi on collagen surface. These findings demonstrate that p45NF-E2 is a key determinant factor for megakaryopoiesis and thrombopoiesis, and is sufficient to lead to platelet formation from a number of non-hematopoietic adult tissues. Generation of iMKs from fibroblasts could have important implications for studying the mechanisms of MK differentiation and platelet production.
Disclosures:
No relevant conflicts of interest to declare.
Induction of functional platelets from mouse and human fibroblasts by p45NF-E2/Maf
