Research Article
Hirudin and heparin enable efficient megakaryocyte differentiation of mouse bone marrow progenitors

https://doi.org/10.1016/j.yexcr.2011.10.003Get rights and content

Abstract

Hematopoietic progenitors from murine fetal liver efficiently differentiate in culture into proplatelet-producing megakaryocytes and have proved valuable to study platelet biogenesis. In contrast, megakaryocyte maturation is far less efficient in cultured bone marrow progenitors, which hampers studies in adult animals. It is shown here that addition of hirudin to media containing thrombopoietin and serum yielded a proportion of proplatelet-forming megakaryocytes similar to that in fetal liver cultures (approximately 50%) with well developed extensions and increased the release of platelet particles in the media. The effect of hirudin was maximal at 100 U/ml, and was more pronounced when it was added in the early stages of differentiation. Hirugen, which targets the thrombin anion binding exosite I, and argatroban, a selective active site blocker, also promoted proplatelet formation albeit less efficiently than hirudin. Heparin, an indirect thrombin blocker, and OTR1500, a stable heparin-like synthetic glycosaminoglycan generated proplatelets at levels comparable to hirudin. Heparin with low affinity for antithrombin was equally as effective as standard heparin, which indicates antithrombin independent effects. Use of hirudin and heparin compounds should lead to improved culture conditions and facilitate studies of platelet biogenesis in adult mice.

Introduction

Megakaryopoiesis is an elaborate process leading to platelet production following expansion and differentiation of hematopoietic stem cells [1]. In the final steps, the megakaryocytes develop cytoplasmic projections, the proplatelets, which give rise to the platelets delivered to the circulation [2], [3], [4], [5]. In vitro studies of these final events have been facilitated by improved culture procedures and imaging techniques and their use in genetically manipulated mice. This has resulted in the identification of key regulators of proplatelet formation, including tubulin, GPIb, myosin and filamin [6], [7], [8], [9]. Most of the results have been obtained from fetal liver progenitors cultured in media containing thrombopoietin (TPO) and serum [10]. Under these conditions, a large percentage of megakaryocytes reach the proplatelet stage after 4 days of culture.

Use of progenitors from adult animals is desirable to alleviate difficulties related to embryo collection and low fertility problems in some mouse strains. In addition, a comparison of embryonic and adult megakaryopoiesis would also be advisable to avoid potential misinterpretation [11]. Unfortunately, mouse bone marrow progenitors poorly differentiate to the proplatelet stage in suspension using the current one-step culture conditions [10], [12], with reported yields of typically only 5 to 10%. A procedure is described here where addition of hirudin or heparin compounds increased the yields of proplatelet-producing megakaryocytes to levels obtained in fetal-derived cells.

Section snippets

Materials

DMEM medium, StemPro medium, penicillin, streptomycin and glutamine were from Invitrogen (Cergy-Pontoise, France). Recombinant human TPO (rhTPO), fetal bovine serum (FBS) and a mouse hematopoietic progenitor cell enrichment kit were purchased from Stem Cell Technologies (Vancouver, BC, Canada). Cy3-conjugated goat anti-mouse immunoglobulin G (IgG) was from Jackson ImmunoResearch (West Grove, PA) and purified rat IgG1 from Pharmingen (Le Pont de Claix, France). Mabs against mouse integrin αIIbβ3

Inefficient proplatelet formation of megakaryocytes differentiated from mouse bone marrow progenitors

The capacity of mouse bone marrow progenitor cells (Lin) to differentiate into proplatelet-producing megakaryocytes was evaluated using culture conditions previously employed for fetal liver progenitors, in a medium containing 50 ng/mL TPO and 10% FBS [7], [15]. On day 4, bone marrow derived megakaryocytes predominantly displayed a round morphology with only 18.8 ± 1% of the cells extending proplatelets (Fig. 1). In contrast, a large percentage of fetal-derived CD41+ megakaryocytes (50.3 ± 4%)

Discussion

Proplatelet-forming megakaryocytes are efficiently cultured from murine fetal liver progenitors. In contrast, 3-time lower yields were obtained when the same culture conditions were applied to adult bone marrow derived progenitors (Fig. 1). This is consistent with reports of lower megakaryocyte yields and slower differentiation in mouse bone marrow cultures [10], [11], [12]. To provide material for studies of proplatelet formation, a two-step procedure has been described where megakaryocytes

Conflict of interest disclosure

The authors declare no potential conflict of interest.

Acknowledgments

We thank A. Bull and J. Y. Rinckel for expert technical assistance, M. Freund for taking care of the animals, J.M. Mulvihil for reviewing the English of the manuscript, and P. Albanese and D. Papy-Garcia for providing the OTR compound and for useful discussions.

This study was supported by ARMESA (Association de Recherche et Développement en Médecine et Santé Publique, Paris, France) and ANR (Agence National pour la Recherche, Grant no. ANR-07-MRAR-016-01).

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