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

E XP E RI M ENT A L C E L L R E SE A RC H 3 1 8 (2 0 1 2) 2 5 –3 2

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Research Article

Hirudin and heparin enable efficient megakaryocyte differentiation of mouse bone marrow progenitors Catherine Strassel, Anita Eckly, Catherine Léon, Sylvie Moog, Jean-Pierre Cazenave, Christian Gachet, François Lanza⁎ UMR_S949 INSERM-Université de Strasbourg, EFS-Alsace, Strasbourg, France

A R T I C L E I N F O R M A T I O N

A B S T R A C T

Article Chronology:

Hematopoietic progenitors from murine fetal liver efficiently differentiate in culture into proplatelet-

Received 18 May 2011

producing megakaryocytes and have proved valuable to study platelet biogenesis. In contrast, megakar-

Revised version received

yocyte maturation is far less efficient in cultured bone marrow progenitors, which hampers studies in

12 September 2011

adult animals. It is shown here that addition of hirudin to media containing thrombopoietin and

Accepted 1 October 2011

serum yielded a proportion of proplatelet-forming megakaryocytes similar to that in fetal liver cultures

Available online 8 October 2011

(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

Keywords:

added in the early stages of differentiation. Hirugen, which targets the thrombin anion binding exosite

Bone marrow

I, and argatroban, a selective active site blocker, also promoted proplatelet formation albeit less efficient-

Proplatelets

ly than hirudin. Heparin, an indirect thrombin blocker, and OTR1500, a stable heparin-like synthetic

Culture

glycosaminoglycan generated proplatelets at levels comparable to hirudin. Heparin with low affinity

Megakaryocytes

for antithrombin was equally as effective as standard heparin, which indicates antithrombin indepen-

Hirudin

dent effects. Use of hirudin and heparin compounds should lead to improved culture conditions and

Heparin

facilitate studies of platelet biogenesis in adult mice. © 2011 Elsevier Inc. All rights reserved.

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 [2–5]. 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 [6–9]. 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

⁎ Corresponding author at: INSERM U.949, Etablissement Français du Sang-Alsace (EFS-Alsace), 10, rue Spielmann, B.P. N°36, 67065 Strasbourg Cedex, France. Fax: +33 388 21 25 21. E-mail address: [email protected] (F. Lanza). 0014-4827/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2011.10.003

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yields of proplatelet-producing megakaryocytes to levels obtained in fetal-derived cells.

Materials and methods 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 (RAM.2-488) and glycoprotein (GP)Ibβ (RAM.1-488) were produced and labeled in our laboratory [13]. RNAse A, propidium iodide, BSA, DAPI and tubulin 1β (clone SAP-4G5), chondroitin sulfate and hyaluronic acid were from Sigma-Aldrich (Rueil-Malmaison, France). Recombinant hirudin rHV2-Lys47 (r-hirudin) was kindly provided by Transgène (Strasbourg, France). Standard heparin (150 USP/mg) was a gift from Sanofi Choay (Paris, France). Hirugen ([Tyr(SO3H)63]-hirudin fragment 54–65) was purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France) and argatroban from Glaxo Smith Kline (Research Triangle Park, NC). Heparin with low affinity for antithrombin (<5% of standard heparin) was kindly provided by Maurice Petitou (Endotis Pharma, Romainville, France). The defined glycosaminoglycan mimetic OTR1500 was kindly provided by OTR3 SARL (Paris, France) [14]. Bovine plasma fibronectin was purchased from Calbiochem (Nottingham, UK).

In some experiments, Lin− cells were cultured with 2.6% serumsupplemented StemPro medium with complements, murine thrombopoietin (TPO) (50 ng/mL) in the presence or absence of hirudin or heparin at 37 °C for 4 days. To evaluate the effect of plating with an extracellular matrix, bone marrow cells were cultured in DMEM containing 2 mM L- glutamine, penicillin/streptomycin, 10% FBS and 50 ng/ml TPO with addition of hirudin for 2 days at 37 °C. Purified megakaryocytes were obtained using a 2–4% BSA gradient for 40 min at room temperature and were then plated on a fibronectin coated surface for 3 h at 37 °C.

Flow cytometry Platelet-size elements generated in the culture suspension were analyzed in a Gallios flow cytometer (Beckman Coulter, Villepinte, France). Cells were centrifuged, washed, and stained for 20 min with RAM.1-488 at 5 μg/ml. Then, events were accumulated for 180 s and positive cells were enumerated in a region predefined using the forward/side scatter region of murine blood platelets.

Immunofluorescence microscopy Cultured megakaryocytes were fixed in 4% paraformaldehyde (PFA) for 15 min and cytospun onto poly L-lysine-coated slides. The cells were then permeabilized with 0.05% saponin in PBS containing 0.2% BSA for 15 min and incubated sequentially for 30 min with 10 μg/ml of Mab against β-tubulin, Cy3-GAM, and RAM.1-488 in the same buffer and the nuclei were counterstained with DAPI. The cells were washed thoroughly at each step. The slides were mounted in Mowiol for examination and proplatelet counting.

Differential interference contrast microscopy Culture of megakaryocytes from mouse fetal liver progenitor cells Megakaryocytes were cultured essentially as described previously [7]. Briefly, livers were recovered from mouse fetuses between embryonic days 13 and 15. After lineage immunodepletion as recommended by the manufacturer (Stem Cell Technologies), Lin− cell suspensions were cultured for 4 days in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin and 50 ng/ml rhTPO.

Culture of megakaryocytes derived from mouse bone marrow progenitor cells Bone marrow cells were flushed from the femurs and tibias of 2 to 4 month-old male C57Bl/6 mice and successively passed through 21-, 23- and 25-gauge needles. The cells were spun down and nucleated cells were counted manually after diluting the bone marrow cells 50 fold in 3% acetic acid. The cells (typically 1.5 to 3 × 107 per mouse) were then pelleted at 1200 rpm for 7 min and resuspended at 1 × 108 cells/ml in PBS supplemented with 5% (v/v) rat serum and 2 mM EDTA to perform a Lin selection (Stem Cell Technologies). The Lin− population was adjusted to 5 × 106 cells/ml in DMEM containing 2 mM L-glutamine, penicillin/streptomycin, 10% FBS and 50 ng/ ml TPO with or without addition of hirudin or heparin. Cultures were usually performed in 12-well tissue culture plates with 700 μl of cell suspension per well and incubated at 37 °C under a 5% CO2 atmosphere for up to 5 days.

Cultured megakaryocytes were fixed in 4% PFA for 15 min and cytospun onto poly L-lysine-coated slides. Differential interference contrast (DIC) images were acquired using a Leica DM4000B microscope (Leica Microsystems) with a 20×/0.6 NA lens coupled to a CoolSNAP photometrics HQ camera (Roper Scientific, Ottobrunn, Germany). The surface covered by megakaryocytes bearing proplatelets was determined in 8 random fields by image analysis with Metamorph software (Molecular Devices, Downingtown, PA).

Statistical analysis All statistical analyses were performed using GraphPad Prism Version 5 (GraphPad Software Inc, La Jolla, CA). Results were expressed as the mean (±SEM). Unpaired Student t tests were used for Figs. 1 and 2A. One-way ANOVA was used for Figs. 2C,D and Figs. 3–4. P values of less than 0.05 were considered to be statistically significant.

Results 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

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A

fetal liver

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bone marrow

B % CD41+ MKs with proplatelets

75

50

25

0

fetal liver

bone marrow

Fig. 1 – Proplatelet formation in cultures of mouse fetal liver and adult bone marrow progenitor cells. (A) Representative images on day 4 of culture of megakaryocytes (MKs) differentiated from fetal liver or bone marrow Lin− progenitors, stained for β-tubulin, showing a lower proportion of proplatelet-bearing cells in the bone marrow sample (Bar = 25 μm). (B) Quantification of the percentage of CD41+ megakaryocytes extending proplatelets (PP) on day 4 of culture (mean ± SEM in 3 separate experiments, ***p< 0.001).

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%) exhibited numerous extensions characteristic of the proplatelet stage. These yields were obtained under continuous culture, without cell sub-sampling. Prolongation of the bone marrow culture for 1–2 days did not improve the proplatelet yields (data not shown) but rather resulted in important cell death, indicating that the low yields were not due to a slower process but rather to impaired maturation. These results support previous observations that the culture conditions developed for fetal liver cells are not suitable for adult progenitors [10,12].

marrow cultures (data not shown), hirudin was added to prevent activation by traces of thrombin. Unexpectedly, this treatment increased the proportion of bone marrow megakaryocytes reaching the proplatelet stage, from 11.9 ± 4% to 46.5 ± 4% for the highest hirudin concentration (Figs. 2A–C). This resulted in enhanced coverage of cytospin slides by the proplatelet membranous network, clearly visible by DIC microscopy (Figs. 2B,D). Increased surface coverage was not due to increased complexity of the proplatelets since the ratio of proplatelet surface/cell body surface remained unchanged (Supplementary information, Fig. S1). The effect of hirudin was maximal when it was added early after progenitor seeding and was weaker when the treatment was started on day 2 (Fig. 2E).

Effects of other antithrombin agents Increased proplatelet formation in media supplemented with hirudin Following the observation at day 4 of abnormally shaped proplatelets with spherical hairy platelet buds in fetal as well as bone

Deshirudin (Revasc), another hirudin used in orthopedic surgery, was equally as potent in promoting proplatelet formation (data not shown). Hirugen, a negatively charged C-terminal dodecapeptide of

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hirudin, partially reproduced the effect of the entire molecule (38.3± 6% proplatelet-forming/CD41+ cells) (Fig. 3). Argatroban, a selective active site blocker, also had a partial effect (30±8% proplateletforming/CD41+ cells). We also evaluated heparin, an indirect inhibitor, which has previously been shown to potentiate differentiation of human CD34+ progenitors. Very similarly to hirudin, heparin increased the yield of proplatelet-forming megakaryocytes (Figs. 3B–C), with a maximal effect observed at 70 U/mL. To determine whether heparin acted through its thrombin blocking activity, we evaluated a fraction with low antithrombin binding capacity. Low affinity heparin had an equivalent ability to potentiate proplatelet production at a mass concentration corresponding to 14 U/ml of heparin (Figs. 4A–B), indicating that it can

A % CD41+ MKs with proplatelets

75

enhance proplatelet formation independently of its thrombin inhibitory property. Heparin has a heterogeneous chemical formulation, requires purification from animal tissues and can be degraded by heparanases. To circumvent this, several synthetic glycosaminoglycans have been developed in recent years with well defined chemical composition and increased stability [14]. We evaluated the effect of one such compound, OTR1500, and observed that it also potentiated proplatelet formation at 1 μM concentration (Figs. 4A–B), To evaluate if more efficient proplatelet formation translated into increased platelet release in the media, these were enumerated on day 4 using flow cytometry. Platelet sized elements were more abundant following addition of hirudin, heparin or OTR1500 in comparison to control culture media (Fig. 5).

bone marrow

50

25

0

control

+ hirudin

B control

10 U/ml

+ hirudin 1 U/ml

100 U/ml

Fig. 2 – Effect of hirudin on proplatelet formation in cultures of bone marrow progenitors. (A) Effect of hirudin (100 U/ml) on the percentage of proplatelet-forming MKs derived from bone marrow Lin− progenitors (mean ± SEM in 3 separate experiments, **p < 0.01). (B–D) Effects of a range of hirudin concentrations. (B) Representative DIC microscopy images of the cells on day 4 (Bar = 50 μm). (C) Quantification of the percentage of CD41+ MKs extending proplatelets on day 4 (18.5 ± 2% in control, 29.1 ± 8% with hirudin 1 U/ml, 38.3 ± 4% with hirudin 10 U/ml and 44.3 ± 3% with hirudin 100 U/ml; mean ± SEM in 5 separate experiments, ns p > 0.05). (D) Quantification of the surface covered by MKs extending proplatelets as a percentage of the area analyzed (5.8 ± 2% in control, 9.0 ± 1% with hirudin 1 U/ml, 13.1 ± 1% with hirudin 10 U/ml and 19.2 ± 1% with hirudin 100 U/ml; mean ± SEM in 3 separate experiments, ns p > 0.05, *p < 0.05, ***p < 0.001. (E) Quantification of the surface covered by MKs extending proplatelets after addition of hirudin at the start of culture (D0) or after 1 or 2 days (D1, D2) of culture (mean ± SEM in 3 separate experiments).

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% CD41+ MKs with proplatelets

C

% surface covered with MKs

D

% surface covered with MKs

ns ns

25

0

control

1 U/ml

10 U/ml

100 U/ml

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***

20

* ns 10

0

E

ns

50

control

1 U/ml

10 U/ml

100 U/ml

10

5

0 control

D0

D1

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time of hirudin addition Fig. 2 (continued).

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 [10–12]. To provide material for studies of proplatelet formation, a two-step procedure has been described where megakaryocytes are concentrated on a BSA gradient and then re-cultured, but this method is still hampered by low proplatelet yields [11]. We report here a one-step procedure where addition of a single component, i.e. hirudin or heparin, greatly enhances proplatelet formation in adult bone marrow derived cells. This method avoids cell manipulation, is more representative of the entire cell population, and more importantly, this readily applicable technique will greatly facilitate platelet biogenesis studies in adult mice. We also evaluated the effects of hirudin and heparin using culture conditions reported

by others, using StemPro media or after plating cells on fibronectin coated wells [16–18]. A potentiating effect was again noted upon adding hirudin, but lower proplatelet yields were obtained in comparison with the procedure reported here (Figs. S2, S3). Bone marrow-derived cells grown with hirudin or heparin showed differentiation profiles that were very similar to fetal liver-derived cells with regard to timing, yields and morphologies of proplateletforming megakaryocytes and production of platelet-sized particles. These results indicate that adult cells have a preserved thrombopoiesis potential that can be revealed upon optimized culture conditions. This also suggests that conclusions made in fetal-derived cultures could also apply to adult cells [19]. Indeed, anomalies caused by GPIb-IX deficiency in fetal liver-derived cells, i.e. enlarged proplatelet buds and over developed microtubular coil [7], were entirely recapitulated in bone marrow cultures (data not shown). A possible explanation for the effect of hirudin is the observation that serum derived thrombin inhibited proplatelet formation in guinea pig bone marrow cultures whereas proplatelets formed efficiently in serum-free media [20]. In human CD34+ cells, addition of thrombin led to contradictory results, with no effect on the

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number of megakaryocyte colonies or ploidy [21] or an inhibition of megakaryocyte growth [22]. We observed here in the mouse, that hirudin was effective when added early upon cell seeding, suggesting a role of thrombin already at the progenitor stage. Thrombin could originate from the serum added to the culture media or during bone marrow collection. The former appears more likely since proplatelet formation was increased in media containing heatinactivated serum but not when hirudin was added during bone marrow collection. Heat-inactivated serum was nevertheless less

efficient than normal serum supplemented with hirudin (data not shown). In addition, a serum-free medium did not recapitulate the potentiating effects and resulted instead in a poor differentiation capacity of bone marrow progenitors (data not shown). It is therefore possible that hirudin could also promote cell differentiation through mechanisms independent of its thrombin blocking activity. The high hirudin concentration for maximal effect (100 U/ml) and the lower efficacy of argatroban also suggest thrombin independent effects. Antithrombin dependent and independent mechanisms can also be envisaged for heparin. Amelioration of megakaryocyte maturation by standard as well as low antithrombin affinity heparin favors a mechanism unrelated to thrombin blockade. Heparin is a known multi functional compound, affecting responses to growth factors, cytokines, matrix proteins and adhesion molecules. In their studies on human CD34+ cells, Vainchenker's group reported decreased numbers of megakaryocytes, proplatelets and platelets in the presence of plasma, an effect which was partly reversed by heparin [23]. Heparin is known to neutralize PF4 and to abolish its inhibitory effect on megakaryocyte colony formation [24]. In vivo, heparan sulfates are present in the bone marrow stroma and could modulate hematopoiesis by retaining and compartmentalizing growth factors and cytokines potentiating megakaryocyte differentiation such as TPO, SCF and Flt-3 [25]. In CD34+ cells grown in a serum-free medium in the presence of TPO, the effects of heparin on megakaryocyte maturation were reproduced by chondroitin sulfate, dermatan sulfate, or hyaluronic acid [26]. Here, we observed that a synthetic heparin-like GAG likewise increased proplatelet formation in mouse bone marrow progenitors, however, these effects were not reproduced following addition of hyaluronic acid or chondroitin sulfate (Fig. S4). These results indicate that the hypothesis of an effect mediated by the charge of the compounds might not on its own explain the effects of heparin. The difference might be due to the degree of sulfatation, since hyaluronic acid is devoid of sulfates and chondroitin sulfate has a lower sulfate to carbohydrate ratio as compared to heparin. In conclusion, and despite remaining questions on the mechanisms by which hirudin and heparin facilitate the production of proplatelet-bearing megakaryocytes, their generalized use is expected to greatly facilitate future studies of megakaryopoiesis in the adult mouse.

Fig. 3 – Effects of other antithrombin agents on proplatelet formation in bone marrow derived mouse megakaryocytes. (A) Effects of hirudin (100 U/ml), hirugen (20 μM) and argatroban (3 μg/ml). Quantification of the percentage of CD41+ megakaryocytes extending proplatelets on day 4 of culture (mean±SEM in 4 separate experiments, 17.7±4% in control, 59.7±3% with hirudin, 38.3±6% with hirugen and 22.6±8% with argatroban; ns p>0.05, ***p<0.001). (B–C) Effects of heparin (7–70 U/ml). (B) Representative DIC microscopy images of the cells on day 4 (Bar=50 μm). (C) Quantification of the percentage of CD41+ megakaryocytes extending proplatelets on day 4 (mean±SEM in 4 separate experiments, 11.6±4% in control, 33.3±3% with heparin 7 U/ml, 42.7±4% with heparin 14 U/ml and 49.0±7% with heparin 70 U/ml; ns p>0.05, **p<0.01, ***p<0.001).

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Fig. 5 – Effects of hirudin, heparin and OTR1500 on platelet release. Platelet release on day 4 was expressed as a ratio of control culture media. CD42c+ platelet-sized particles were quantified by flow cytometry as described in the Methods (values ± SEM in 4 separate experiments, 1 in control, 2.7 ± 0.4 in hirudin (100 U/ml), 3.2 ± 0.3 in heparin (14 U/ml) and 1.7 ± 0.3 in OTR1500 (1 μmol/L) supplemented media, respectively; ns p > 0.05, **p < 0.01).

Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10. 1016/j.yexcr.2011.10.003.

Fig. 4 – Effects of low antithrombin affinity heparin and a synthetic glycosaminoglycan on proplatelet formation. (A–B) Effects of heparin (14 U/ml), low antithrombin affinity heparin (14 U/ml equivalent) and OTR1500 (1 μM). (A) Representative DIC microscopy images of the cells on day 4 (Bar = 50 μm). (B) Quantification of the percentage of CD41+ megakaryocytes extending proplatelets on day 4 (mean ± SEM in 3 separate experiments).

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-MRAR016-01).

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