Transdifferentiation of erythroblasts to megakaryocytes using FLI1 and ERG transcription factors

Darin Siripin; Pakpoom Kheolamai; Yaowalak U-Pratya; Aungkura Supokawej; Methichit Wattanapanitch; Nuttha Klincumhom; Chuti Laowtammathron; Surapol Issaragrisil

doi:10.1160/TH14-12-1090

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2015; 114(03): 593-602
DOI: 10.1160/TH14-12-1090

Cellular Haemostasis and Platelets

Schattauer GmbH

Transdifferentiation of erythroblasts to megakaryocytes using FLI1 and ERG transcription factors

Darin Siripin

¹Faculty of Medical Technology, Mahidol University, Bangkok, Thailand

⁵Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

,

Pakpoom Kheolamai

²Division of Cell Biology, Faculty of Medicine, Thammasat University, Pathumthani, Thailand

³Center of Excellence in Stem Cell Research, Faculty of Medicine, Thammasat University, Pathumthani, Thailand

⁵Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

,

Yaowalak U-Pratya

⁴Division of Hematology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

⁵Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

,

Aungkura Supokawej

¹Faculty of Medical Technology, Mahidol University, Bangkok, Thailand

,

Methichit Wattanapanitch

⁵Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

,

Nuttha Klincumhom

⁵Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

,

Chuti Laowtammathron

⁵Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

,

Surapol Issaragrisil

⁴Division of Hematology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

⁵Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

› Author Affiliations

Abstract

Summary

Platelet transfusion has been widely used to prevent and treat life-threatening thrombocytopenia; however, preparation of a unit of concentrated platelet for transfusion requires at least 4–6 units of whole blood. At present, a platelet unit from a single donor can be prepared using apheresis, but lack of donors is still a major problem. Several approaches to produce platelets from other sources, such as haematopoietic stem cells and pluripotent stem cells, have been attempted but the system is extremely complicated, time-consuming and expensive. We now report a novel and simpler technology to obtain platelets using transdifferentiation of human bone marrow erythroblasts to megakaryocytes with overexpression of the FLI1 and ERG genes. The obtained transdifferentiated erythroblasts (both from CD71⁺ and GPA⁺ erythroblast subpopulations) exhibit typical features of megakaryocytes including morphology, expression of specific genes (cMPL and TUBB1) and a marker protein (CD41). They also have the ability to generate megakaryocytic CFU in culture and produce functional platelets, which aggregate with normal human platelets to form a normal-looking clot. Overexpression of FLI1 and ERG genes is sufficient to transdifferentiate erythroblasts to megakaryocytes that can produce functional platelets.

Keywords

Transdifferentiation - erythroblast - megakaryocyte - platelet - transcription factors

Full Text

References

References
1 Lu SJ, Li F, Yin H. et al. Platelets generated from human embryonic stem cells are functional in vitro and in the microcirculation of living mice. Cell Res 2011; 21: 530-545.
2 Reems J-A, Pineault N, Sun S. In Vitro Megakaryocyte Production and Platelet Biogenesis: State of the Art. Transfusion Med Rev 2010; 24: 33-43.
3 Greening DW, Sparrow RL, Simpson RJ. Preparation of Platelet Concentrates #. T Serum/Plasma Proteomics. In Methods in Molecular Biology 7282011. Springer; New York: 2011. pp. 267-278.
4 Kaufman RM. Platelets: testing, dosing and the storage lesion--recent advances. Haematology Am Soc Haematol Educ Program 2006; 492-496.
5 Chen TW, Yao CL, Chu IM. et al. Large generation of megakaryocytes from serum-free expanded human CD34+ cells. Biochem Biophys Res Commun 2009; 378: 112-117.
6 Guerriero R, Mattia G, Testa U. et al. Stromal cell-derived factor 1alpha increases polyploidization of megakaryocytes generated by human haematopoietic progenitor cells. Blood 2001; 97: 2587-2595.
7 Matsunaga T, Tanaka I, Kobune M. et al. Ex vivo large-scale generation of human platelets from cord blood CD34+ cells. Stem Cells 2006; 24: 2877-2887.
8 Gaur M, Kamata T, Wang S. et al. Megakaryocytes derived from human embryonic stem cells: a genetically tractable system to study megakaryocytopoiesis and integrin function. J Thromb Haemost 2006; 04: 436-442.
9 Takayama N, Nishikii H, Usui J. et al. Generation of functional platelets from human embryonic stem cells in vitro via ES-sacs, VEGF-promoted structures that concentrate haematopoietic progenitors. Blood 2008; 111: 5298-5306.
10 Nakamura S, Takayama N, Hirata S. et al. Expandable megakaryocyte cell lines enable clinically applicable generation of platelets from human induced pluripo-tent stem cells. Cell Stem Cell 2014; 14: 535-548.
11 Takayama N, Eto K. In Vitro Generation of Megakaryocytes and Platelets from Human Embryonic Stem Cells and Induced Pluripotent Stem Cells. In: Methods in Molecular Biology 788. Springer; New York: 2012. pp. 205-217.
12 Deutsch VR, Tomer A. Megakaryocyte development and platelet production. Br J Haematol 2006; 134: 453-466.
13 Pang L, Weiss MJ, Poncz M. Megakaryocyte biology and related disorders. J Clin Invest 2005; 115: 3332-3338.
14 Charbord P. [The haematopoietic stem cell and the stromal microenvironment]. Therapie 2001; 56: 383-384.
15 Tsai FY, Keller G, Kuo FC. et al. An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature 1994; 371: 221-226.
16 Warren AJ, Colledge WH, Carlton MB. et al. The oncogenic cysteine-rich LIM domain protein rbtn2 is essential for erythroid development. Cell 1994; 78: 45-57.
17 Porcher C, Swat W, Rockwell K. et al. The T cell leukemia oncoprotein SCL/tal-1 is essential for development of all haematopoietic lineages. Cell 1996; 86: 47-57.
18 Athanasiou M, Clausen PA, Mavrothalassitis GJ. et al. Increased expression of the ETS-related transcription factor FLI-1/ERGB correlates with and can induce the megakaryocytic phenotype. Cell Growth Differ 1996; 07: 1525-1534.
19 Stankiewicz MJ, Crispino JD. ETS2 and ERG promote megakaryopoiesis and synergize with alterations in GATA-1 to immortalize haematopoietic progenitor cells. Blood 2009; 113: 3337-3347.
20 Xie H, Ye M, Feng R. et al. Stepwise Reprogramming of B Cells into Macrophages. Cell 2004; 117: 663-676.
21 Sharrocks AD, Brown AL, Ling Y. et al. The ETS-domain transcription factor family. Intern J Biochem Cell Biol 1997; 29: 1371-1387.
22 Wei G-H, Badis G, Berger MF. et al. Genome-wide analysis of ETS-family DNA-binding in vitro and in vivo. EMBO J 2010; 29: 2147-2160.
23 Hart A, Melet F, Grossfeld P. et al. Fli-1 is required for murine vascular and megakaryocytic development and is hemizygously deleted in patients with thrombocytopenia. Immunity 2000; 13: 167-177.
24 Raslova H, Komura E, Le Couedic JP. et al. FLI1 monoallelic expression combined with its hemizygous loss underlies Paris-Trousseau/Jacobsen thrombopenia. J Clin Invest 2004; 114: 77-84.
25 Spyropoulos DD, Pharr PN, Lavenburg KR. et al. Haemorrhage, impaired haematopoiesis, and lethality in mouse embryos carrying a targeted disruption of the Fli1 transcription factor. Mol Cell Biol 2000; 20: 5643-5652.
26 Loughran SJ, Kruse EA, Hacking DF. et al. The transcription factor Erg is essential for definitive haematopoiesis and the function of adult haematopoietic stem cells. Nature Immunol 2008; 09: 810-819.
27 Shimizu K, Ichikawa H, Tojo A. et al. An ets-related gene, ERG, is rearranged in human myeloid leukemia with t(16;21) chromosomal translocation. Proc Natl Acad Sci USA 1993; 90: 10280-10284.
28 Sorensen PH, Lessnick SL, Lopez-Terrada D. et al. A second Ewing’s sarcoma translocation, t(21;22), fuses the EWS gene to another ETS-family transcription factor, ERG. Nature Genet 1994; 06: 146-151.
29 Tomlins SA, Rhodes DR, Perner S. et al. Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science 2005; 310: 644-648.
30 Kruse EA, Loughran SJ, Baldwin TM. et al. Dual requirement for the ETS transcription factors Fli-1 and Erg in haematopoietic stem cells and the megakaryocyte lineage. Proc Natl Acad Sci USA 2009; 106: 13814-13819.
31 Ono Y, Wang Y, Suzuki H. et al. Induction of functional platelets from mouse and human fibroblasts by p45NF-E2/Maf. Blood 2012; 120: 3812-3821.
32 Long MW. Megakaryocyte differentiation events. Semin Haematol 1998; 35: 192-199.