Normal Hemopoiesis

Caspase activation is involved in early megakaryocyte differentiation but not in platelet production from megakaryocytes

Abstract

To elucidate whether caspase activation is involved in megakaryopoiesis, we characterized megakaryocytes (MKs) in vav-bcl-2 transgenic (Tg) mice, in which Bcl-2 is overexpressed in hematopoietic cells. To exclude the effect of splenomegaly in Tg mice on megakaryopoiesis, splenectomy was performed. After splenectomy, basal platelet counts in peripheral blood were not significantly different between Tg and wild-type (WT) mice. However, when experimental thrombocytopenia was induced by injecting 5-fluorouracil into splenectomized mice, overshoot of platelet counts during the recovery phase was hardly observed in Tg mice. Analyses of MK ploidy during the recovery phase showed that MKs less than 16 N ploidy were significantly decreased in Tg mice, suggesting that MK supply from progenitors is impaired. Supporting this, differentiation of CD34−/c-kit+/Sca-1+/Lineage− stem cells into MKs was significantly hampered in Tg mice, whereas megakaryocyte-erythroid progenitors (MEPs) normally differentiated into MKs. It suggests that differentiation into MKs is impaired in Tg mice before the stage of MEP. Furthermore, MK colony formation in WT cells was dose-dependently inhibited in the presence of a caspase inhibitor. Contrary, Bcl-2-overexpressing MKs showed normal ability for in vitro platelet production. We thus believe that caspase activation is involved in the differentiation of progenitors into megakaryocytic lineage but not in platelet production.

Introduction

Megakaryocytes (MKs) differentiate from hematopoietic stem cells under the control of a lineage-specific cytokine, thrombopoietin (TPO), in concert with MK-specific transcription factors, including GATA-1, FOG-1 and p45 NF-E2.1, 2 Maturation of MKs is a complex process that is morphologically characterized by polyploidization and the expansion of cytoplasmic mass, which is mediated by a unique type of cell division, endomitosis. Final stage of the maturation is characterized by platelet release from the ends of long thin cytoplasmic processes called proplatelets.3 Indeed, mature MKs contain tubularly invaginated cytoplasmic membranes called a demarcation membrane system in the abundant cytoplasm, which is assumed to be a reservoir for proplatelets. In addition, recent observation of murine megakaryopoiesis using multiphoton intravital microscopy demonstrated that, even in vivo, platelets are released from proplatelets protruding into vascular lumen of bone marrow (BM) sinus under the force of shear stress.4 These findings have collectively established that proplatelet formation is a physiological and relevant process that is required for platelet production, although other hypotheses, such as cytoplasmic fragmentation, have not been completely rejected.5

Several lines of evidence have suggested that cellular apoptosis is involved in the process of platelet production. The first observation supporting for the apoptotic process in MKs was reported by Zauli et al.,6 who argued that cultured mature MKs in comparison with immature cells are prone to apoptosis even in the presence of TPO. Subsequently, Sanz et al.7 reported that antiapoptotic protein Bcl-xL becomes absent in senescent MKs, although it is abundantly expressed in cultured MKs derived from CD34+ progenitors. Corresponding with this, the activation of caspase-3 and -9, and permeabilization of the mitochondrial membrane were reported in mature MKs and a megakaryocytic cell line forming proplatelet-like processes.8, 9 Furthermore, bcl-2- or bcl-xL-transfected MKs, which should be resistant to caspase-3 activation, were reported to be significantly less capable of forming proplatelets.9, 10 Contrary to these findings, our recent in vitro and in vivo observations suggested that Bcl-xL protein is highly expressed throughout megakaryopoiesis until the platelet-producing late stage of maturation,11 which raised the question about the apoptosis theory of platelet production. On the other hand, recent studies have established that caspase activation is involved in nonapoptotic cellular functions, such as the proliferation and differentiation of various types of cells.12 Although involvement of apoptotic process was demonstrated in the differentiation of erythroblasts,13, 14 it has never been reported in MKs except for platelet production from MKs. We are thus urged to elucidate whether an apoptotic process is involved in megakaryopoiesis. To approach this issue, we used Bcl-2-overexpressing transgenic (Tg) mice under the control of the vav promoter.15 As Bcl-2-overexpessing cells were expected to be resistant to the activation of caspases, we analyzed the in vitro and in vivo behavior of MKs and their progenitors obtained from the Tg mice.

Materials and methods

Animal experiments

vav-bcl-2 Tg mice, expressing a human bcl-2 cDNA under control of vav promoter on an inbred C57BL/6J background, were provided by Professor J Adams (The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia). To induce thrombocytopenia, 5-fluorouracil (5-FU, 150 mg/kg) was intraperitoneally injected. Blood samples obtained from the tail vein were diluted to 1:200 in 1.25% ammonium oxalate and platelets were counted under a phase contrast microscope. Animal experiments were performed under the approval of the Institutional Animal Experiment Committee.

Preparation and/or analysis of the progenitors, megakaryocytes and platelets

c-kit+/Lineage (Lin)− cells were prepared as described previously.11 CD34−/c-kit+/Sca-1+/Lin− (KSL) cells were stained by fluorescein isothiocyanate (FITC)-conjugated anti-CD34 (Becton and Dickinson, San Jose, CA, USA), FITC-conjugated anti-CD117 (eBioscience, San Diego, CA, USA), phycoerythrin (PE)-conjugated anti-Sca-1 (Becton and Dickinson) and biotinylated anti-Lin monoclonal antibodies (mAbs) followed by streptavidin-PE/Cy5 (BioLegend, San Diego, CA, USA). Megakaryocyte-erythroid progenitors (MEPs) were stained by FITC-conjugated anti-CD34 (Becton and Dickinson), PE-conjugated anti-Sca-1, APC-conjugated anti-CD117, PE/Cy7-conjugated anti-CD16/CD32 (eBioscience) and biotinylated anti-Lin mAbs followed by streptavidin-PE/Cy5 (BioLegend) as described previously.16 Analyses and sorting of KSL cells, CD34−/KSL cells or MEPs were performed by using a CyAn flow cytometry (Dako, Glostrup, Denmark) and a MoFlo sorter (Dako), respectively. To obtain primary MKs, BM mononuclear cells (MNCs) were loaded onto 50% Percoll, prepared by mixing an equal volume of CATCH medium (Hank's balanced salt solution containing 1 mM adenosine, 2 mM theophylline and 0.38% sodium citrate, pH 7.2) and Percoll (Pharmacia, Uppsala, Sweden), and centrifuged at 200 g for 20 min at 20 °C. Cells harvested from the interface were washed with 0.1% bovine serum albumin (BSA)/CATCH (at 90 g for 10 min) and loaded onto discontinuous BSA (2, 3, 4 and 16%)/CATCH gravity sedimentation. MK-rich samples harvested from 4 and 16% fractions were washed and resuspended in Iscove's modified Dulbecco's medium. Washed platelets were prepared from murine platelet-rich plasma as described previously.17

Liquid culture

Stem cells or MK progenitors were cultured in STEM-PRO 34 SFM medium (Invitrogen, Carlsbad, CA, USA) with human recombinant TPO (Kirin Brewery, Tokyo, Japan) alone or in combination with mouse recombinant stem cell factor (SCF, PeproTech, London, UK).

Colony assay

Megakaryocyte colony was developed using a MegaCult-C culture kit (StemCell Technologies, Vancouver, Canada). MK progenitors were cultured with 50 ng/ml TPO, 10 ng/ml mouse IL-3 (PeproTech) and 20 ng/ml mouse IL-6 (PeproTech) for 6 days in chamber slides. After acetylcholine esterase (AchE) staining, MK colonies, defined as the clusters of more than two AchE-positive cells, were counted under a microscope. For the assay of erythroid colony, c-kit+/Lin− cells (5 × 104 cells) were cultured in MethoCult M3231 medium (StemCell Technologies) with 1 U/ml human recombinant erythropoietin (Chugai Pharmaceutical, Tokyo, Japan) for 4 days, and benzidine-positive colonies were counted. For the assay of granulocyte/macrophage (GM) colony, c-kit+/Lin− cells (1 × 104 cells) were cultured in MethoCult M3231 medium with 50 ng/ml SCF, 10 ng/ml rIL-3 and 10 ng/ml rIL-6 for 6 days. AchE and benzidine stainings were performed by conventional methods.

Assay of MK ploidy

Cells were stained with FITC-conjugated anti-CD41 mAb (Becton and Dickinson), permeabilized in 70% ethanol for 30 min on ice and resuspended in 2% fetal bovine serum (FBS)/CATCH containing 0.1% sodium citrate. DNA was stained with 6 μg/ml propidium iodide for 30 min on ice, followed by treatment with 25 μg/ml RNase for 30 min at room temperature (RT). Ploidy of CD41+ cells was analyzed by flow cytometry.

Cell-cycle analyses

Cell cycle was analyzed using an APC BrdU Flow kit (Becton and Dickinson). For in vivo assay, BMMNCs were harvested after various hours of intraperitoneal injection of 5-bromo-2-deoxyuridine (BrdU, 1 mg per body). For in vitro assay, c-kit+/Lin− cells were cultured in STEM-PRO 34 SFM medium with TPO, incubated with 10 μM BrdU for the last 45 min. After staining with APC-conjugated anti-BrdU and FITC-conjugated anti-CD117mAbs, BrdU incorporation into CD117-positive cells was analyzed by flow cytometry.

Immunocytochemistry

Immunofluorescence staining was performed as described previously.11 Briefly, MKs cultured in eight-well chamber slides (Nunc; Lab-Tek, Naperville, IL, USA) were fixed in 3% paraformaldehyde for 10 min, washed with Tris-buffered saline (TBS, 20 mM Tris-HCl, 150 mM NaCl, pH 7.4), permeabilized in 0.1% Triton X in TBS for 5 min and washed again. After blocking, cells were stained with the first Ab (anti-active caspase-3 Ab (Promega, Madison, WI, USA) or anti-Bcl-2 Ab (Santa Cruz Biotechnology, Santa Cruz, CA, USA)) for 4 h at RT, washed and stained with Alexa Fluor 546-conjugated goat anti-rabbit Ig Ab (Invitrogen) for 1 h at RT. MKs were detected by staining with FITC-conjugated anti-CD41 mAb. After fixation with Vectashield mounting medium containing DAPI (Vector, Burlingame, CA, USA), cells were observed under a fluorescence microscope.

Immunohistochemistry

Bone marrow samples were fixed in 3% paraformaldehyde for 4 h at 4 °C, decalcified and embedded in paraffin. Paraffin-embedded sections were deparaffinized and antigen-retrieved in 1 mM EDTA solution (pH 8.0) at 95 °C for 30 min. After washing with 0.1% Tween 20/TBS, MKs were stained with rabbit anti-von Willebrand factor (vWF) polyclonal Ab (Chemicon, Temecula, CA, USA) for 2 h at RT. Positive staining was detected by Vectastain ABC kit (Vector). For nuclear staining, hematoxylin staining was added.

Detection of caspase activation by flow cytometry

Cells were fixed in 3% paraformaldehyde for 10 min at 37 °C, permeabilized in 90% methanol for 30 min on ice, resuspended in 2% FBS/phosphate-buffered saline (PBS), stained with anti-cleaved caspase-3 Ab (Cell Signaling, Beverly, MA, USA) for 1 h at RT and analyzed by flow cytometry.

Assay of proplatelet formation

Megakaryocytes cultured in eight-well chamber slides (Lab-Tek) were stained with FITC-conjugated anti-mouse CD41 mAb. CD41+ cells with cytoplasmic processes longer than the diameter of the cytoplasm were defined as proplatelet-forming MKs.

Platelet counts in culture supernatants

Cells were recovered from the supernatants of MKs cultured in 48-well microtiter plates, washed with 1% FBS/PBS (136.9 mm NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4), stained with FITC-conjugated anti-CD41 mAb and resuspended in 1 ml PBS. After adding 100 μl of Flow-Count Fluorospheres (Beckman Coulter, Fullerton, CA, USA), platelet numbers were determined by flow cytometry following the manufacturer’s instructions.

Western blotting

Western blotting was performed as described previously11 by using anti-Bcl-2 mAb (Becton and Dickinson).

Statistic analysis

Comparisons of two means were performed using Student’s t-test.

Results and discussion

Bcl-2-overexpresing cells are resistant to caspase activation

To confirm the overexpression of Bcl-2 protein throughout megakaryopoiesis, KSL stem cells were obtained from wild-type (WT) and vav-bcl-2 Tg mice and MKs were differentiated by culturing with TPO. As shown in Figure 1a, overexpression of Bcl-2 protein in both stem cells and MKs obtained from Tg mice was demonstrated by immunocytochemistry. Similarly, Bcl-2 overexpression in platelets was confirmed by western blotting.

Figure 1
figure1

Bcl-2-overexpressing cells are resistant to caspase activation. (a) Purified KSL stem cells or megakaryocytes (MKs) derived from KSL cells by culturing with thrombopoietin (TPO, 200 ng/ml) were stained for Bcl-2, and expression of Bcl-2 was observed under a fluorescence microscope (left and middle panels). MKs were identified by positive staining with CD41. Expression of Bcl-2 in platelets was observed by western blotting (right panel). (b) CD34−/KSL stem cells maintained in the presence of 50 ng/ml stem cell factor (SCF) and 50 ng/ml TPO for 2 days or MKs derived from c-kit+/Lin− progenitor cells by culturing with 200 ng/ml TPO for 3 days were depleted of cytokines for the indicated duration. After immunostaining for cleaved (active) caspase-3, positive cells were observed under a fluorescence microscope. Data indicating the mean±s.d. (n=3) are representative of three similar experiments.

To ascertain whether Bcl-2-overexpressing CD34−/KSL stem cells and MKs are resistant to apoptosis, we observed caspase-3 activation induced by cytokine depletion. For this purpose, CD34−/KSL stem cells maintained with SCF and TPO or MKs derived from c-kit+/Lin− progenitor cells by culturing with TPO were stained for cleaved caspase-3 after cytokine depletion. As was expected, both Bcl-2-overexpresing CD34−/KSL stem cells and MKs were highly resistant to caspase-3 activation in comparison with WT cells (Figure 1b).

Early MK maturation is impaired in vav-bcl-2 Tg mice

We assayed platelet counts in vav-bcl-2 Tg and WT mice. Platelet counts in peripheral blood were significantly decreased in vav-bcl-2 Tg mice (mean±s.d.; Tg: 60.7±12.7 per μl vs WT: 96.3±21.2 per μl), however, as vav-bcl-2 Tg mice manifested with giant splenomegaly, splenectomy was performed. Several weeks after splenectomy, platelet counts became stable, and there was no significant difference in platelet counts between Tg and WT mice (Figure 2a).

Figure 2
figure2

Megakaryopoiesis following experimental thrombocytopenia is impaired in vav-bcl-2 transgenic (Tg) mice. (a, b) Platelet counts in peripheral blood of wild-type (WT) and vav-bcl-2 Tg mice were sequentially assayed after splenectomy (a) or after 5-fluorouracil (5-FU) injection into splenectomized mice (b). (c) Bone marrow (BM) specimens obtained from 5-FU-injected splenectomized WT and Tg mice in the preinjection (day 0) and the recovery phase (day 8) were stained with anti-vWF Ab (left panel: original magnification, × 200). Numbers of megakaryocytes (MKs) per HPF were analyzed under a microscope (right panel). Data indicating the mean±s.d. (n=3) are representative of three similar experiments (ac). (d) MK ploidy in the preinjection and the recovery phase BM obtained from WT and Tg splenectomized mice was analyzed by flow cytometry. Data shown are representative of three similar experiments.

To analyze megakaryopoiesis during and after experimental thrombocytopenia, 5-FU was injected into splenectomized mice. Interestingly, in vav-bcl-2 Tg mice, overshoot of platelet counts in the recovery phase was hardly observed (Figure 2b). To gain insights about the pathogenesis underlying impaired thrombopoiesis in vav-bcl-2 Tg mice, preinjection (day 0) and the recovery phase (day 8) BM specimens were prepared and analyzed by staining with anti-vWF Ab (Figure 2c). Before 5-FU injection, there was no difference in MK numbers between WT and vav-bcl-2 Tg BM specimens. In the recovery phase, abundant numbers of MKs were observed in WT mice, whereas the numbers were significantly lower in Tg mice. We also analyzed MK ploidy, as it directly reflects endomitosis and thus can be a reasonable indicator of MK maturation (Figure 2d). In the preinjection steady state, the percentages of immature MKs possessing 2–4 N ploidy were slightly increased in vav-bcl-2 Tg mice in comparison with WT mice. In the recovery phase, mature MKs with more than or equal to 16 N ploidy increased in both vav-bcl-2 Tg and WT BM. However, importantly, the percentages of MKs with less than 16 N ploidy were remarkably decreased in vav-bcl-2 Tg mice. We thus considered that the supply of MKs from the progenitors might be impaired in vav-bcl-2 Tg mice, especially in excessively platelet-producing phase.

To further characterize this abnormal phenotype, TPO-induced in vitro differentiation of MKs from purified stem cells was evaluated (Figure 3a). CD34−/KSL stem cells were cultured in the presence of TPO and the resultant production of AchE+ cells was analyzed. We observed significantly decreased numbers of AchE+ cells and MK colonies in vav-bcl-2 Tg mice. In a sharp contrast, when MEPs were cultured in the presence of TPO, we did not observe the difference. The data suggest that TPO-induced differentiation of stem cells into MKs was impaired in the early phase of the differentiation, presumably before the stage of the MEP. Corresponding with this, the percentages of CD34−/KSL stem cells in the BM were significantly increased in vav-bcl-2 Tg mice. Although the percentages of MEPs were also increased in Tg mice compared to WT mice, the difference was not so profound as was seen in CD34−/KSL cells (Figure 3b). We consider that the increased percentage of MEPs in the Tg BM is reflected into the increased percentage of MKs with 2–4 N ploidy, which is presented in Figure 2d. To clarify whether Bcl-2-overexpressing MKs mature normally, CD34−/KSL cells were cultured with TPO and polyploidization of MKs was analyzed. There was no difference in the distribution of ploidy between WT and Bcl-2-overexpressing cells (data not shown). These data collectively suggest that differentiation of stem cells into MKs is impaired in vav-bcl-2 Tg mice, but once differentiated into MKs, these cells mature normally.

Figure 3
figure3

Differentiation of megakaryocytes (MKs) from stem cells is impaired in Bcl-2-overexpressing cells. (a) CD34−/KSL stem cells (left panel: 100 cells) and the megakaryocyte-erythroid progenitors (MEPs, right panel: 150 cells) purified from wild-type (WT) and vav-bcl-2 transgenic (Tg) bone marrow (BM) were cultured under the culture condition for developing MK colonies for 5 days. After acetylcholine esterase (AchE) staining, numbers of AchE+ cells and MK colonies were counted under a microscope. (b) Percentages of CD34−/KSL stem cells (left panel) and the MEPs (right panel) among BMMNCs were assayed by flow cytometry. Data indicating the mean±s.d. (n=3) are representative of three similar experiments.

Caspase activation is involved in early MK maturation

As several lines of evidence have suggested that Bcl-2 overexpression causes delayed cell-cycle progression from G1 to S phase,18 we analyzed this possibility in Bcl-2-overexpressing progenitors. BrdU was injected into mice and its incorporation into stem cells was analyzed by flow cytometry. Unexpectedly, there was no difference in BrdU incorporation into KSL stem cells between WT and vav-bcl-2 Tg mice (data not shown). Furthermore, levels of in vitro BrdU incorporation into c-kit+/Lin− cells cultured in the presence of TPO were not different between WT and Bcl-2-overexpressing progenitors (data not shown). These data suggest that Bcl-2 overexpression in progenitors does not affect TPO-driven cell-cycle progression. We thus pursued another possibility, namely that caspase activation, which is at least partially inhibited by Bcl-2 overexpression, is involved in early MK maturation. To prove this possibility, we assayed the production of MK colonies from c-kit+/Lin− progenitors in the presence of a broad caspase inhibitor. When z-VAD-fmk was added, numbers of MK colonies derived from c-kit+/Lin− cells were dose-dependently decreased in WT mice (Figure 4a). Similar inhibitory effect was also observed in vav-bcl-2 Tg mice. It may be because Bcl-2 overexpression, which only inhibits the pathway of mitochondria-mediated caspase activation, does not abrogate activation of caspase-3 totally. To test the involvement of caspase activation in the differentiation of other lineages of hematopoietic cells, erythroid and GM colony formation from c-kit+/Lin− progenitor cells was assayed. The addition of z-VAD-fmk significantly inhibited erythroid colony formation (Figure 4b), corresponding with the previous results,13 whereas GM colony formation was not affected (data not shown). Collectively, we believe that caspase-3 activation is involved in the commitment of stem cells into megakaryocytic lineage as well as erythroid lineage.

Figure 4
figure4

Caspase activation is involved in early megakaryocyte (MK) differentiation. (a) Numbers of MK colonies derived from wild-type (WT) or vav-bcl-2 transgenic (Tg) progenitors (1 × 104 c-kit+/Lin− cells) were assayed in the presence of various concentrations of a broad caspase inhibitor, z-VAD-fmk. (b) Numbers of erythroid colonies derived from WT progenitors (c-kit+/Lin− cells) were assayed in the presence of various concentrations of z-VAD-fmk. (c) c-kit+/Lin− cells obtained from WT and vav-bcl-2 Tg mice were cultured with 200 ng/ml thrombopoietin (TPO) for the indicated durations. Cells were stained with anti-cleaved caspase-3 Ab and analyzed by flow cytometry. Data indicating the mean±s.d. (n=3) are representative of three similar experiments.

In an attempt to assess directly the activation of caspase-3 during early megakaryopoiesis, c-kit+/Lin− progenitor cells were cultured in the presence of TPO and cleaved caspase-3 was detected by flow cytometry. As shown in Figure 4c, activation of caspase-3 after 6 h of the addition of TPO was significantly suppressed in Bcl-2-overexpressing cells. However, in a parallel experiment, as the decreased numbers of Annexin-V-positive cells were observed in vav-bcl-2 Tg mice (data not shown), we could not exclude the possibility that the decreased caspase-3 activation observed in Tg mice simply reflects the decreased cellular apoptosis.

Although it is well established that caspase-3 is one of the central players for the process of cellular apoptosis, nonapoptotic function of caspases in the setting of cellular proliferation, differentiation and cell-cycle progression has recently attracted many researchers’ interest.12 As regards hematopoietic cells, caspase activation was reported to be involved in the differentiation of monocytes into macrophages.19 Transient activation of caspase is also required for early13, 20 as well as terminal14 erythroid differentiation. In line with these previous results focusing on the importance of caspases in hematopoiesis, we have here demonstrated for the first time that caspase activation is involved in early MK differentiation. Remaining questions to be solved are what is the substrate for activated caspases and what is the downstream signaling pathway during early MK differentiation. Thus far, diverse proteins, including cytokines, kinases and transcription factors, have been identified as substrates for the nonapoptotic function of caspases in various cell types.21 In this context, it is interesting that mammalian sterile 20-like kinase (MST1) is activated through proteolysis by caspase-3, and differentiation defect of caspase-3-deficient myoblasts was rescued by introducing the active form of MST1.22 These findings suggest that MST1 activated by caspase-3 can be a regulator of cellular differentiation. Interestingly, a previous report showed that the expression of MST1 is induced by Mpl stimulation in MKs, and that forced expression of MST1 facilitates MK maturation, indicating the possible relevant role of this kinase in MK differentiation.23 The downstream signaling of caspase activation in megakaryopoiesis should be clarified in future experiments.

Caspase activation is not involved in platelet production from MKs

As previous reports have suggested that caspase activation is involved in platelet production from MKs,8, 9, 10 we assayed platelet production from Bcl-2-overexpressssing MKs. As shown in Figure 5a, Bcl-2-overexpressing MKs showed comparable ability of proplatelet formation with WT MKs. A similar tendency was observed when MKs were derived from c-kit+/Lin− progenitor cells by culturing with TPO and the resultant proplatelet formation was analyzed after 6 days (data not shown). Supporting these results, the addition of z-VAD-fmk (100 μM) did not affect proplatelet formation in WT primary MKs (Figure 5b). We also evaluated the ability of platelet release into culture supernatants of MKs differentiated from c-kit+/Lin− progenitor cells. As shown in Figure 5c, platelet production per MKs assayed by flow cytometry on day 6 was rather increased in Bcl-2-overexpressing MKs. These data collectively suggest that caspase activation is not involved in platelet production from MKs, which is strikingly different from the previous report by De Botton et al.9 in which MKs differentiated from retrovirally bcl-2-transfected human CD34+ cells were analyzed for their ability of proplatelet formation. One possible explanation for the discrepancy is that bcl-2 transfection into CD34+ cells may have significantly inhibited the megakaryocytic differentiation of these cells, as was observed in our present experiments (Figure 3a). Nevertheless, they assayed the percentages of proplatelet-forming cells among GFP-positive cells, namely successfully bcl-2-transfected cells. This assay may have caused spuriously reduced percentages of proplatelet formation because of the increased GFP+ non-megakaryocytic cells by bcl-2 transfection. Similarly, Kaluzhny et al.10 used cultured MKs obtained from the BM of bcl-xl Tg mice, observed MKs under phase contrast microscope, and concluded that the percentages of proplatelet-forming MKs were reduced by Bcl-xL overexpression. We speculate that Bcl-xL overexpression may have hampered differentiation of progenitors into MKs, which again resulted in spuriously reduced proplatelet formation.

Figure 5
figure5

Bcl-2-overexpressing megakaryocytes (MKs) show normal ability of in vitro platelet production. (a, b) Primary MKs obtained from wild-type (WT) and vav-bcl-2 transgenic (Tg) mice were cultured in chamber slides for 6 or 24 h (a). A typical proplatelet-forming MK is shown in the left panel. Primary WT MKs were cultured with various concentrations of z-VAD-fmk for 24 h (b). After the culture, proplatelet formation among CD41+ cells was analyzed under a fluorescence microscope. Data indicating the mean±s.d. (n=3) are representative of three similar experiments. (c) c-kit+/Lin− cells obtained from WT and vav-bcl-2 Tg mice were cultured with 200 ng/ml thrombopoietin (TPO) in 48-well microtiter plates, and the numbers of platelets released into the culture supernatants were assayed by flow cytometry as described in Materials and methods. Panels (i) and (ii) show typical dot plots of WT cells on days 2 and 6 of the culture, respectively. Note that numerous dots are observed within the gated area in panel (ii), whereas those are hardly observed on day 2. Gated dots in panel (ii) were replotted in panel (iii) by FSC–SSC axis presentation, which corresponds well with the dot plot of peripheral blood platelets shown in panel (iv). Panel (v) shows numbers of platelets per 3000 MKs obtained from day 6 culture supernatant of WT and vav-bcl-2 Tg c-kit+/Lin− cells. Numbers of MKs were determined by counting CD41+ cells in a parallel experiment under fluorescence microscope. Data indicating the mean±s.d. (n=3) are representative of three similar experiments.

Taken together, we believe that caspase activation is involved in early MK differentiation but not in platelet production.

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Acknowledgements

This work was supported in part by a grant from the Japanese Ministry of Education, Culture, Sports, Science and Technology (HK), and Mitsubishi Pharma Research Foundation (HK).

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Correspondence to H Kojima.

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Kozuma, Y., Yuki, S., Ninomiya, H. et al. Caspase activation is involved in early megakaryocyte differentiation but not in platelet production from megakaryocytes. Leukemia 23, 1080–1086 (2009). https://doi.org/10.1038/leu.2009.7

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Keywords

  • megakaryocyte
  • platelet
  • caspase
  • Bcl-2

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