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Platelet Biology & its Disorders
Anti-apoptotic BCL2L2 increases megakaryocyte proplatelet formation in cultures of human cord blood
Ferrata Storti Foundation
Haematologica 2019 Volume 104(10):2075-2083
Seema Bhatlekar,1 Indranil Basak,1 Leonard C. Edelstein,2 Robert A. Campbell,1 Cory R. Lindsey,2 Joseph E. Italiano Jr.,3 Andrew S. Weyrich,1 Jesse W. Rowley,1 Matthew T. Rondina,1,4 Martha Sola-Visner5 and Paul F. Bray1,6
1Program in Molecular Medicine and Department of Internal Medicine, University of Utah, Salt Lake City, UT; 2Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, PA; 3Brigham and Women’s Hospital, Harvard University, Boston, MA; 4George E. Wahlen VAMC GRECC, Salt Lake City, UT; 5Boston Children’s Hospital, Harvard University, Boston, MA and 6Division of Hematology and Hematologic Malignancies, Department of Internal Medicine, University of Utah, Salt Lake City, UT, USA
ABSTRACT
Apoptosis is a recognized limitation to generating large numbers of megakaryocytes in culture. The genes responsible have been rigor- ously studied in vivo in mice, but are poorly characterized in human culture systems. As CD34-positive (+) cells isolated from human umbilical vein cord blood were differentiated into megakaryocytes in culture, two distinct cell populations were identified by flow cytometric forward and side scatter: larger size, lower granularity (LLG), and smaller size, higher granularity (SHG). The LLG cells were CD41aHigh CD42aHigh phosphatidylserineLow, had an electron microscopic morphology similar to mature bone marrow megakaryocytes, developed proplatelets, and dis- played a signaling response to platelet agonists. The SHG cells were CD41aLowCD42aLowphosphatidylserineHigh, had a distinctly apoptotic mor- phology, were unable to develop proplatelets, and showed no signaling response. Screens of differentiating megakaryocytes for expression of 24 apoptosis genes identified BCL2L2 as a novel candidate megakaryocyte apoptosis regulator. Lentiviral BCL2L2 overexpression decreased megakary- ocyte apoptosis, increased CD41a+ LLG cells, and increased proplatelet for- mation by 58%. An association study in 154 healthy donors identified a significant positive correlation between platelet number and platelet BCL2L2 mRNA levels. This finding was consistent with the observed increase in platelet-like particles derived from cultured megakaryocytes over-expressing BCL2L2. BCL2L2 also induced small, but significant increases in thrombin-induced platelet-like particle αIIbβ3 activation and P- selectin expression. Thus, BCL2L2 restrains apoptosis in cultured megakaryocytes, promotes proplatelet formation, and is associated with platelet number. BCL2L2 is a novel target for improving megakaryocyte and platelet yields in in vitro culture systems.
Introduction
Hematopoietic stem cells (HSC) are essential for reconstituting hematopoiesis in the setting of bone marrow (BM) transplantation and are of great value for studies of the basic biology of hematopoiesis. Differentiating HSC into megakaryocytes (MK) in culture has become a standard approach to study megakaryocytopoiesis (MKpoiesis). MK culture systems are also powerful tools for the functional assess- ment of novel platelet genes, gene variants, protein-coding transcripts, and microRNA associated with platelet reactivity and clinical hemorrhagic or throm- botic disorders. Recent advancements in MKpoiesis research have opened up a new, promising field of ex vivo manufacturing of platelets,1-4 with the ultimate goal of infusing these products into patients with thrombocytopenia or qualitative
Correspondence:
PAUL F. BRAY
paul.bray@hsc.utah.edu
Received: August 16, 2018. Accepted: January 30, 2019. Pre-published: February 7, 2019.
doi:10.3324/haematol.2018.204685
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/104/10/2075
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haematologica | 2019; 104(10)
2075
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