Platelet Biology & its Disorders ARTICLE Anti-apoptotic BCL2L2 increases megakaryocyte proplatelet formation Ferrata Storti Foundation in cultures of human cord blood 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-Visner 5 and Paul F. Bray 1,6 1Program in Molecular Medicine and Department of Internal Medicine, University of 2 Utah, Salt Lake City, UT; Cardeza Foundation for Hematologic Research, Thomas Haematologica 2019 Jefferson University, Philadelphia, PA; 3Brigham and Women’s Hospital, Harvard University, Boston, MA; 4George E. Wahlen VAMC GRECC, Salt Lake City, UT; 5Boston Volume 104(10):2075-2083 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 poptosis 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 A + 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 CD41a High CD42a High phosphatidylserine Low , 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 CD41a Low CD42a Low phosphatidylserine High , had a distinctly apoptotic mor - Correspondence: phology, were unable to develop proplatelets, and showed no signaling response. Screens of differentiating megakaryocytes for expression of 24 PAUL F. BRAY apoptosis genes identified BCL2L2 as a novel candidate megakaryocyte [email protected] apoptosis regulator. Lentiviral BCL2L2 overexpression decreased megakary - ocyte apoptosis, increased CD41a + LLG cells, and increased proplatelet for - Received: August 16, 2018. mation by 58%. An association study in 154 healthy donors identified a Accepted: January 30, 2019. significant positive correlation between platelet number and platelet Pre-published: February 7, 2019. 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 doi:10.3324/ haematol.2018.204685 increases in thrombin-induced platelet-like particle αIIb β3 activation and P- selectin expression. Thus, BCL2L2 restrains apoptosis in cultured Check the online version for the most updated megakaryocytes, promotes proplatelet formation, and is associated with information on this article, online supplements, and information on authorship & disclosures: platelet number. BCL2L2 is a novel target for improving megakaryocyte and www.haematologica.org/content/104/10/2075 platelet yields in in vitro culture systems. ©2019 Ferrata Storti Foundation Introduction Material published in Haematologica is covered by copyright. All rights are reserved to the Ferrata Storti Foundation. Use of published material is allowed under the following terms and Hematopoietic stem cells (HSC) are essential for reconstituting hematopoiesis in conditions: the setting of bone marrow (BM) transplantation and are of great value for studies https://creativecommons.org/licenses/by-nc/4.0/legalcode. of the basic biology of hematopoiesis. Differentiating HSC into megakaryocytes Copies of published material are allowed for personal or inter - (MK) in culture has become a standard approach to study megakaryocytopoiesis nal use. Sharing published material for non-commercial pur - poses is subject to the following conditions: (MKpoiesis). MK culture systems are also powerful tools for the functional assess - https://creativecommons.org/licenses/by-nc/4.0/legalcode, ment of novel platelet genes, gene variants, protein-coding transcripts, and sect. 3. Reproducing and sharing published material for com - microRNA associated with platelet reactivity and clinical hemorrhagic or throm - mercial purposes is not allowed without permission in writing botic disorders. Recent advancements in MKpoiesis research have opened up a from the publisher. 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 haematologica | 2019; 104(10) 2075 S. Bhatlekar et al. platelet disorders. Improvements in this technology may IN, USA) or 10 μg/mL CRP (synthesized at Baylor College of also allow 'designer' platelets to be engineered that over - Medicine and cross-linked with glutaraldehyde) for 20 minutes come immunological incompatibility and infection issues. 5 (min) at 37°C, followed by 4% paraformaldehyde fixation at A major limitation of all MK culture systems is the rela - room temperature. Cells were analyzed on a Cytoflex or BD tively short time period for which the differentiating MK Accuri C6 flow cytometer. can be kept viable before they die through apoptosis. 6 The role of apoptosis during MKpoiesis is somewhat con - Statistical analysis troversial, 7 with some data supporting a role for the intrin - All statistical analyses were performed using GraphPad Prism 6 sic pathway of apoptosis in platelet production, 8-10 while software version 10.1 (La Jolla, CA, USA) and reported as other studies show that MK must restrain apoptosis to Mean±Standard Error of Mean (SEM). Fold changes for BCL2L2 survive and progress safely through proplatelet formation levels over time in cultures and for lentiviral overexpression were 11-13 and platelet generation. However, the apoptosis regula - presented as log 2 (fold change) compared to their respective con - tors of human cord blood-derived (CB)-MK cultures trols (day 6 for changes in BCL2L2 levels over time and empty vec - remain poorly understood. During the course of our stud - tor control for overexpression) and analyzed by a one-sample t- ies with cultured CB MK, we observed two distinct popu - test. Log 2 transformation was adopted to have a normally distrib - lations of cells by forward and side scatter flow cytometry. uted fold-change data, determined by Kolmogorov-Smirnov nor - The aims of the current study were to determine: (i) mality test. The relationship between platelet BCL2L2 mRNA whether there were viability and apoptosis differences expression levels and platelet counts in the 154 healthy human between these two populations; and (ii) molecular mech - donors in the Platelet RNA Expression Study 1 (PRAX-1) was anism(s) of apoptosis regulation during Mkpoiesis. We assessed by Pearson’s correlation with 95% Confidence Interval also wanted to begin to identify approaches that would (95%CI). For all other analyses, statistical significance was reduce MK apoptosis in order to produce greater yields of assessed using paired Student t-test. P<0.05 was considered statis - MK and platelet in cultures. We demonstrate that the tically significant. anti-apoptosis Bcl2 family member BCL2L2 (encoding Bcl- See Online Supplementary Appendix for further details of the w) regulates cultured MK apoptosis, promotes proplatelet experiments. formation, and is associated with human platelet number. Results Methods Subsets of cultured megakaryocytes show differing Primary megakaryocyte cultures viability Human umbilical cord CB was obtained from the New York CD34 + cells were isolated from human umbilical vein Blood Center (New York, NY, USA) under institutional review CB and cultured to generate MK as described previously. 14 board (IRB) approval (00108527). CD34 + hematopoietic stem and By day 13, CD34 + cells were differentiated into MK, as progenitor cells (HSPC) were isolated from human umbilical vein demonstrated by the expression of mature MK markers CB and cultured in serum free expansion media supplemented (Online Supplementary Figure S1A-D ). Day 13 MK were with 25 ng/mL of stem cell factor (SCF) and 20 ng/mL of throm - larger than day 0 HSC ( Online Supplementary Figure S1E ) bopoietin (TPO) (Peprotech, Rocky Hill, NJ, USA) for six days. and demonstrated polyploid MK ( Online Supplementary Cells were cultured with 50 ng/mL TPO only from days 6-13. 14 Figure S1F and G ). Thus, these culture conditions promot - Adult granulocyte-macrophage colony-stimulating factor (GM- ed generation of mature, polyploid MK. CSF) mobilized CD34 + HSPC were purchased from the Utah Cell Flow cytometric analysis of day 13 cultures revealed dis - Therapy and Regenerative Medicine Center (Salt Lake City, UT, tinct cell populations differing by forward scatter (FSC) USA) under the University of Utah IRB approval (00108527). and side scatter (SSC) characteristics (Figure 1A): larger MK with lower granularity [larger, lower granular (LLG)] Megakaryocyte proplatelet formation assay and smaller MK with higher granularity [smaller, higher Day 9 transduced cells were plated at 2x10 4 cells/mL in granular (SHG)]. Peripheral blood (PB) mobilized adult + 60 μ-Dish Grid-500 plates (Ibidi, Fitchburg, WI, USA) using fresh CD34 cells cultured under the same conditions also gen - medium supplemented with 50 ng/mL TPO. On day 13, the pro - erated similar subpopulations of MK ( Online Supplementary platelet forming