CD41 /CD45 Cells Without Acetylcholinesterase Activity Are Immature and a Major - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:56 |只看该作者 |倒序浏览 |打印

作者：Kuniko Matsumura-Takedaa,d, Shinji Sogoa, Yoshimasa Isakaria,d, Yasuo Haradaa, Kinue Nishiokaa, Takuma Kawakamib, Toshihide Onoc, Takao Takia
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      【摘要】
   Murine megakaryocytes (MKs) are defined by CD41/CD61 expression and acetylcholinesterase (AChE) activity; however, their stages of differentiation in bone marrow (BM) have not been fully elucidated. In murine lineage-negative (Lin¨C)/CD45  BM cells, we found CD41  MKs without AChE activity (AChE¨C) except for CD41 MKs with AChE activity (AChE ), in which CD61 expression was similar to their CD41 level. Lin¨C/CD41 /CD45 /AChE¨C MKs could differentiate into AChE , with an accompanying increase in CD41/CD61 during in vitro culture. Both proplatelet formation (PPF) and platelet (PLT) production for Lin¨C/CD41 /CD45 /AChE¨C MKs were observed later than for Lin¨C/CD41  /CD45 /AChE  MKs, whereas MK progenitors were scarcely detected in both subpopulations. GeneChip and semiquantitative polymerase chain reaction analyses revealed that the Lin¨C/CD41 /CD45 /AChE¨C MKs are assigned at the stage between the progenitor and PPF preparation phases in respect to the many MK/PLT-specific gene expressions, including &szlig;1-tubulin. In normal mice, the number of Lin¨C/CD41 /CD45 /AChE¨C MKs was 100 times higher than that of AChE  MKs in BM. When MK destruction and consequent thrombocytopenia were caused by an antitumor agent, mitomycin-C, Lin¨C/CD41 /CD45 /AChE¨C MKs led to an increase in AChE  MKs and subsequent PLT recovery with interleukin-11 administration. It was concluded that MKs in murine BM at least in part consist of immature Lin¨C/CD41 /CD45 /AChE¨C MKs and more differentiated Lin¨C/CD41  /CD45 /AChE  MKs. Immature Lin¨C/CD41 /CD45 /AChE¨C MKs are a major MK population compared with AChE  MKs in BM and play an important role in rapid PLT recovery in vivo.7 [5 z3 S" [8 E( I+ f

Disclosure of potential conflicts of interest is found at the end of this article.
      【关键词】 Bone marrow Thrombopoiesis Mouse Microarray Megakaryocytes* G  j1 r! W6 M7 F3 e% \2 m
   INTRODUCTION
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Megakaryocytes (MKs) are specialized differentiated cells that produce platelets (PLTs). In megakaryopoiesis, hematopoietic stem cells (HSCs) differentiate into MKs through their progenitors, including colony-forming unit (CFU)-MK . Therefore, the in vivo mechanisms of their actions are poorly understood, because of the difficulty in precisely estimating the individual MK differentiation stages within BM.$ x5 N9 a6 G$ O0 ^1 J; F8 z

In hematopoietic differentiation, specific surface markers define various stages. In particular, pluripotent hematopoietic stem cells are defined by CD34 or c-kit molecules , but the analysis failed to fully disclose how many stages and which state(s) are involved in BM differentiation, especially the differentiation of MK progenitor to AChE  cells./ _; W8 \% L% a* b& e

Another issue to be addressed for MK maturation is the state from which MKs start to prepare for PPF, which is a filamentous structure with the filament composed of ß1-tubulin , suggesting that ß1-tubulin is essential for PPF. However, at which stage in MK differentiation ß1-tubulin synthesis begins remains unknown.
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In this report, we describe the isolation of MKs in murine BM, focusing on surface markers, AChE activity, and PLT production, using fine immunomagnetic depletion of other lineage cells. Isolated MKs were clearly divided into two subpopulations, Lin¨C/CD41 /CD45 /AChE¨C and Lin¨C/CD41  /CD45 /AChE , which were quite different from each other, in terms of both their MK ability and expression of MK-specific markers, including ß1-tubulin. In particular, Lin¨C/CD41 /CD45 /AChE¨C was a more immature MK population than Lin¨C/CD41  /CD45 /AChE . The in vivo characterization of this immature MK population was examined in IL-11-induced megakaryopoiesis.2 u0 C9 ?0 a' Z# \& c: [
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MATERIALS AND METHODS7 d# j; X2 E( S6 Q7 K! G

Animals7 f& Z( }2 ~% g  m/ t- e7 Q

Male Balb/c mice (6¨C10 weeks old) were purchased from Charles River Japan (Shiga, Japan, http://www.criver.com). Animals were housed and experiments were conducted according to the Otsuka Pharmaceutical Co., Ltd. (Tokyo, http://www.otsuka.co.jp), Guidelines for Animal Care and Use.
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Antibodies and Cytokines
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All antibodies were purchased from BD Pharmingen (San Diego, http://www.bdbiosciences.com/pharmingen), Serotec (Raleigh, NC, http://www.serotec.com), Cedar Lane (Hornby, ON, Canada, http://www.cedarlanelabs.com), Emfret Analytics (Wurzburg, Germany, http://www.emfret.com), or eBioscience (San Diego, http://www.ebioscience.com). Purified or biotin-conjugated monoclonal antibodies (mAbs) were used specifically for the following murine lineage markers: CD2 (clone, RM2-5), CD4 (RM4-5), CD8 (53-6.7), Thy1.2 (53-2.1), B220 (RA3-6B2), CD11b (M1/70), Gr-1(RB6-8C5), CD71 (C2), TER119, F4/80, and 7/4. For flow cytometry, fluorescein isothiocyanate (FITC)-conjugated anti-CD41 mAb (MWReg30), FITC-conjugated anti-CD42b mAb (Xia.G5), FITC-conjugated anti-CD42a mAb (Xia.B4), FITC-conjugated anti-FcRI mAb (MAR-1), phycoerythrin (PE)-conjugated anti-CD45 mAb (30-F11), biotin-conjugated anti-CD61 mAb (2C9.G2), PE-conjugated anti-Thy1.2 mAb (30H-12), biotin-conjugated anti-c-kit mAb (2B8), and Streptavidin (SA)-QuantumRed (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) were used throughout the experiments.5 O$ @: K) a  y0 Q$ [. v9 J
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For liquid and semisolid culture, the following recombinant cytokines were purchased: human TPO (hTPO) (Peprotech, Rocky Hill, NJ, http://www.peprotech.com), mouse IL-3 (mIL-3) (Genzyme, Cambridge, MA, http://www.genzyme.com), human IL (hIL)-6 (R&D Systems Inc., Minneapolis, http://www.rndsystems.com), and hIL-11 (Peprotech).4 U/ n, I- X7 v9 O( P
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Isolation of Megakaryocytic Cells, J4 q) H2 Q7 E, j
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BM cells were harvested by flushing femurs of mice with Hanks' balanced salt solution (Sigma-Aldrich). The cell suspension was passed three times through a 26-gauge needle and finally through a 100-µm cell strainer (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) to obtain a single-cell suspension.& q# n1 W6 z: Q) @' D- O4 v% m2 q

BM cells were stained with antibodies specific for the following lineage markers (Lin): CD4, CD8, Thy1.2, B220, TER119, CD71, F4/80, CD11b, Gr-1, and 7/4. To obtain Lin¨C cells, Lin  cells were depleted using sheep anti-rat IgG polyclonal antibody-conjugated magnetic beads (Dynal Biotech, ASA, Oslo, Norway, http://www.dynalbiotech.com). This depletion procedure was repeated twice. We then confirmed that the contaminating Lin  cells were
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Lin¨C cells were stained with various FITC-conjugated mAbs (anti-CD41 mAb, anti-CD42b mAb, anti-CD42a mAb, or anti-FcRI mAb), PE-conjugated anti-CD45 mAb, and biotin-conjugated anti-CD61mAb, and CD61 was visualized by SA-QuantumRed. Stained cells were analyzed or sorted using a single laser (488-nm argon laser), Epics ELITE using Elite 4.02 software (Beckman Coulter, Miami, FL, http://www.beckmancoulter.com).
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Cell Culture2 }2 q- ^! N: s$ X1 L5 _: }

Lin¨C cells were seeded at 2 x 105 cells per 500 µl in 48-well plates. Sorted cells were seeded at 3 x 103 cells per 500 µl in 48-well plates, at 1 x 103 cells per 100 µl in 96-well plates, and at 200 cells per 10 µl in Terasaki plates. These cells were cultured in Iscove's modified Dulbecco's medium (Invitrogen, Grand Island, NY, http://www.invitrogen.com) containing 10% fetal bovine serum (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) with 10 ng/ml hTPO. All cultures were performed at 37¡ãC in a fully humidified atmosphere of 5% CO2. After the culture, the cells' smear preparations were made using Cytospin2 (Thermo Shandon Inc., Pittsburgh, http://www.thermo.com) and stained by the Wright-Giemsa method or the AChE staining method .- b- l) P) r( `# C7 F+ c

Counting of Culture-Derived PLTs

The number of PLTs derived from megakaryocytic cell culture was counted using an Epics XL-MCL flow cytometer (Beckman Coulter). After 500 mM EDTA/phosphate-buffered saline (PBS) was added to each culture (final concentration, 10 mM), the cells were collected, centrifuged (1,710g for 10 minutes at 4¡ãC), and stained with FITC-conjugated anti-CD41 mAb. Twenty thousand units of Flow Count (Beckman Coulter) were added to each sample as an internal control. The culture-derived PLTs per 1,000 units of Flow Count were enumerated as CD41  events with the same forward/side scatter properties as murine blood PLTs.
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Quantitative Measurement of AChE Activity
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AChE activity of sorted cells was measured by the microplate method .

Colony-Forming Assay

CFU-MK assay was performed in semisolid culture medium (MegaCult; Stem Cell Technologies) supplemented with cytokines (10 ng/ml mIL-3, 50 ng/ml hIL-11, 20 ng/ml hIL-6, and hTPO). Cells were cultured in the medium for 7 days at 37¡ãC in 5% CO2. Aggregates that consisted of more than three AChE  cells were counted as colonies by observation with an inverted microscope. CFU-granulocytes/macrophage (GM) and burst forming unit erythroid (BFU-E) assays were performed in methylcellulose medium (MethoCult M3231; Stem Cell Technologies) supplemented with cytokines (50 ng/ml murine stem cell factor, 10 ng/ml mIL-3, 10 ng/ml hIL-6, and 3 U/ml human erythropoietin). Cells were cultured in the medium for 13 days at 37¡ãC, 5% CO2. Colonies were scored at day 13 for CFU-GM and BFU-E with an inverted microscope. These assays were performed according to the manufacturer's instructions.5 v3 A2 B- {* [8 G6 o2 m( z
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Isolation of HSC/Progenitor Cells and PLTs for Gene Expression Profiling
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Although Lin¨C/c-kit /Sca-1  was often used as a marker for HSCs . No contamination of this PLT fraction with CD3  cells was detected by reverse transcription-polymerase chain reaction (PCR).1 w4 [0 h7 T1 r2 m5 P( F

Gene Expression Profiling

Total RNA was isolated from HSC/progenitor cells (S), Lin¨C/CD41 /CD45 /AChE¨C cells (IM), and Lin¨C/CD41  /CD45 /AChE  cells (M) using an RNeasy Mini Kit (Qiagen, Valencia, CA, http://www1.qiagen.com) and from PLTs using the TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Biotin-labeled target cRNA was prepared from 10 µg of total RNA from each sample and hybridized to Murine Genome U74A, B, C v2 GeneChip (Affymetrix, Santa Clara, CA, http://www.affymetrix.com). The hybridized chips were washed, stained with SA-PE solution, and then scanned at a wavelength of 570 nm using an Affymetrix HP GeneArray Scanner. Data were analyzed with GeneSpring 6.0 (Silicongenetics, Redwood, CA, http://www.silicongenetics.com). At first, values below 0.01 were set to 0.01. Data were normalized in two ways: per chip normalization and per gene normalization. For per chip normalization, all expression data on a chip were normalized to the 50th percentile of all values on that chip. For per gene normalization, all samples were normalized against the measurement of the control sample, HSC/progenitor (measurements for each gene in those samples was divided by the measurement of the control sample.).7 x5 A7 Q! m9 i* A
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Semiquantitative PCR
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Total RNA extracted from each cell pellet was reverse transcribed into cDNA using Superscript III RTase (Invitrogen) and random primer (Takara, Shiga, Japan, http://www.takara.co.jp) in a 20-µl mixture. cDNAs from the same amount of total RNA were serially diluted. Subsequent PCR amplification (50 µL) was performed with 5 µl of cDNA using TaKaRa Ex Taq in the presence of specific primer pairs. Each cycle of PCR consisted of 30 seconds at 95¡ãC, 30 seconds at 58¡ãC, and 1 minute at 72¡ãC. The following oligonucleotides were used as primers: ß1-tubulin, sense primer, 5'-GCATCCGATCCAGCAGATTGGGAGTCC-3', antisense primer, 5'-CTCTATCAGCTGGTGGATGGATAGCACG-3'; CD62P, sense primer, 5'-GATCTACATCAAGAGTAACTCGGCCCCTGG-3', antisense primer, 5'-CACATTTTCAGATCCCAGTAGCTCAAAGTC-3'; CD42b (gpIb), sense primer, 5'-AAGCTGAAGAAACTCAATCTGG-3', antisense primer, 5'-ATGGTCTGGGTTCAGGTTGGAG-3'; CD41 (gpIIb), sense primer, 5'-AGTGCAACCCGTTGCTCTTCGACC-3', antisense primer, 5'-AGCTTGCGGTCTGCCCTGCTCTC-3'; CD42a (gpIX), sense primer, 5'-TGCGACCACAGATACTCAGGCT-3', antisense primer, 5'-TAGACCAGCCAGCAGAATCAGG-3'; and glyceraldehyde-3-phosphate dehydrogenase, sense primer, 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3', antisense primer, 5'-CATGTAGGCCATGAGGTCCACCAC-3'.
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Thrombocytopenia Models

BALB/c mice were randomly separated into groups (n = 5) for each treatment protocol. Mitomycin C (MMC) (6 mg/kg; Kyowa Hakko, Tokyo, Japan, http://www.kyowa.co.jp/eng) or rabbit anti-murine PLT (x100 dilution, 0.1 ml/10 g; Inter Cell Technologies Inc., Jupiter, FL) were injected via the tail vein at a dose of 6 mg/kg at day 0. From day 1 to day 5, recombinant hIL-11 (Neumega, Madison, NJ, http://www.wyeth.com; 10 µg/mouse/day) or saline was administered subcutaneously. At each time point, peripheral blood of each mouse was collected into a Microtainer EDTA (Becton, Dickinson and Company) for measurement of hematological parameters, and a femur was obtained from each mouse. BM cells were flushed from each femur into 2 ml of PBS containing 5 mM EDTA and suspended. PLT count and the number of BM cells were enumerated using an automatic microcell counter K-4500 (Toa Medical Electronics, Kobe, Japan, http://www.sysmex.co.jp). The cytospin of each BM sample (2 x 105 cells per slide) was prepared and stained by the AChE staining protocol. The ratio of AChE-positive MKs was estimated using a microscope. Each BM sample (1 x 106 cells) was preincubated with 2 µl of Fc blocking antibody (2.4G2, BD Pharmingen) for 15 minutes and then stained with FITC-conjugated anti-CD41 mAb, PE-conjugated anti-CD45 mAb, and biotin-conjugated mAbs against lineage markers (CD4, CD8, Thy1.2, B220, TER119, CD71, F4/80, CD11b, Gr-1, and 7/4), followed by staining with SA-QuantumRed to visualize the lineage markers. The ratio of immature MKs (Lin¨C/CD41 /CD45  cells) was estimated in 100,000 BM cells of each mouse using an Epics XL-MCL flow cytometer. Then, the number of AChE  MKs or immature MKs per femur was calculated.
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RESULTS4 j" l/ A& p# _  m7 L9 c
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Isolation of Megakaryocytic Subpopulations in Lin¨C BM Cells# B1 A1 \" J9 m3 ~2 l

The isolation of murine MKs was attempted by focusing on the surface markers and PLT production ability. First, we depleted other lineage cells beside MKs using immunomagnetic beads. The Lin¨C BM cells thus obtained were cultured with or without 10 ng/ml hTPO. In the presence of hTPO, enlarged megakaryocytic cells were observed on day 3 (Fig. 1A), and culture-derived PLTs were increased time dependently (Fig. 1B), but not in the culture without hTPO.
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Figure 1. Primary megakaryocytes (MKs) enriched in Lin¨C murine bone marrow (BM) cells. Lin¨C murine BM cells were cultured with or without human TPO (hTPO) (10 ng/ml) for 3¨C6 days. After 3 days, enlarged cells were observed in hTPO culture (A). Culture-derived PLTs were counted by Epics XL-MCL (closed circles, hTPO 10 ng/ml; open circles, control) (B). Experiments were done in triplicate, and data are presented as mean ¡À SE. Abbreviations: PLT, platelet; TPO, thrombopoietin.+ d$ U; j+ q: o
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To identify a megakaryocytic population in the Lin¨C BM cells, we analyzed the expression pattern of CD41, CD61, and CD45. The CD45 /CD61  cells were found in the Lin¨C BM cells (Fig. 2A, R1), being subdivided into two subpopulations based on their CD41 and CD45 expression (Fig. 2A, middle panel, R2 and R3). The expressions of CD41 and CD61 were similar in each subpopulation (Fig. 2A, right upper and lower panels). In the various purification steps, cell frequencies were as follows: whole bone marrow cells, 2.8 x 107 cells per femur; Lin¨C bone marrow cells (BMC), 2.8 x 105 cells per femur; Lin¨C/CD41 /CD45  cells, 1.8 x 104 cells per femur; Lin¨C/CD41  /CD45  cells, 0.4 x 104 cells per femur (Table 1). The sorted Lin¨C/CD41  /CD45  cells varied in size (10¨C50 µm in diameter), containing large and AChE  cells (24.3% ¡À 4.3%) (Fig. 2B, 2C; Table 1). Lin¨C/CD41 /CD45  cells were a homogenous population (10¨C20 µm diameter) with no detectable signs of either cytoplasmic maturation or AChE activity (Fig. 2B, 2C; Table 1). In both subpopulations, granulocytes were detected by Giemsa staining at a level of 1.3% ¡À 0.3% for the former and 2% ¡À 0.6% for the latter (Table 1). To examine whether these subpopulations contained MK progenitors, semisolid culture (CFU-MK) assay was performed. In serum-free conditions, CFU-MK were scarcely detected in either Lin¨C/CD41  /CD45  (0.3 ¡À 0.3 colonies per 1 x 103 cells plated, 25.5 ¡À 3.4 colonies per 3 x 103 cells plated) or Lin¨C/CD41 /CD45  (1.0 ¡À 0.6 colonies per 1 x 103 cells plated, 13.8 ¡À 4.2 colonies per 3 x 103 cells plated), whereas MK colonies were detected in whole BMC (5.9 ¡À 2.0 colonies per 1 x 105 cells plated) (Table 1). To further estimate the content of MK progenitors in Lin¨C/CD41 /CD45  cells, sorted Lin¨C/CD41 /CD45  cells were cultured in methylcellulose medium with TPO and serum (supplemental online Table 1). Emerged colonies (TPO-dependent colonies) were then estimated. The number of TPO-dependent colonies increased and reached a peak on days 6¨C7 (6.8%¨C7.4%). CD41 /Fc receptor-I (FcRI)¨C cells were 59.5% of the cultured cells. In addition, Lin¨C/CD41 /CD45  included erythroid progenitor cells (BFU-E, 0.1% ¡À 0.0%) and myeloid progenitor cells (CFU-GM, 7.3% ¡À 0.6%). On the other hand, CD42b (gpIb) was expressed in Lin¨C/CD41  /CD45  (71.3%) or Lin¨C/CD41 /CD45  (44.4%) and CD42a (gpIX) was expressed in Lin¨C/CD41  /CD45  (73.5%) or Lin¨C/CD41 /CD45  (64.4%), whereas none of the FcRI expressed cells were detected in either population (data not shown). These megakaryocytic subpopulations were noted as Lin¨C/CD41 /CD45 /AChE¨C and Lin¨C/CD41  /CD45 /AChE .

Figure 2. Two megakaryocytic subpopulations in Lin¨C murine BM cells. Expression profiles of CD41, CD45 and CD61 on Lin¨C BM cells. The gated CD61  cells (R1) were subdivided into CD41 /CD45  (R2) and CD41  /CD45  (R3). CD41/CD61 expressions of the R2 and R3 gated cells are represented by blue and red colored dots, respectively (, upper and lower right panels). Lin¨C/CD41 /CD45  and Lin¨C/CD41  /CD45  cells were sorted by the R1 R2 and R1 R3 gates, respectively (A). Sorted cells were stained by the Wright-Giemsa or AChE method (B). Specific AChE activity of Lin¨C/CD41 /CD45  (1,000 cells), Lin¨C/CD41  /CD45  (1,000 cells), or peripheral PLTs (1 x 105) was estimated by a quantitative method of AChE activity (C). Experiments were done in triplicate, and data are presented as mean ¡À SE. Abbreviations: AChE, acetylcholinesterase; BM, bone marrow; ND, not detected; OD, optical density; PLT, platelet.
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Table 1. Frequency and profiles of each population
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In Vitro Differentiation of Lin¨C/CD41 /CD45 /AChE¨C and Lin¨C/CD41  /CD45 /AChE

To assess the megakaryocytic ability of each subpopulation, we estimated PPF, AChE activity, and PLT production in the presence of hTPO. As shown in Figure 3A, the number of AChE  cells and PPF were increased in the Lin¨C/CD41  /CD45 /AChE  cells. For the Lin¨C/CD41 /CD45 /AChE¨C cells, enlarged AChE  cells emerged with subsequent PPF. As cell culture progressed, CD41 expression increased significantly, as did CD61. The appearance of these phenomena began later in Lin¨C/CD41 /CD45 /AChE¨C cells. Kinetic studies showed that PPF increased 38¨C72 hours after the start of culture of Lin¨C/CD41  /CD45 /AChE  and at 48¨C96 hours for Lin¨C/CD41 /CD45 /AChE¨C cells (Fig. 3B). A similar tendency was observed for PLT production: 38¨C91.5 hours for the former and 60.5¨C115.5 hours for the latter, whereas the increment rates between subpopulations were similar (Fig. 3C). On the other hand, no PLT production occurred with the CD41¨C/CD45 /CD61¨C subpopulation of Lin¨C BM cells (data not shown).

Figure 3. Differentiation of the two megakaryocyte subpopulations in vitro. Sorted Lin¨C/CD41 /CD45 /AChE¨C and Lin¨C/CD41  /CD45 /AChE  cells were cultured with human thrombopoietin (10 ng/ml). At each time point (Pre and days 1, 2, and 3), both PPF and AChE  cells were observed, and the expressions of CD41/CD61 were measured by flow cytometry (arrowhead, PPF) (A). The numbers of PPF (B) and culture-derived PLTs (C) were measured in the Lin¨C/CD41 /CD45 /AChE¨C (closed circles) and Lin¨C/CD41  /CD45 /AChE  cultures (open circles). Experiments were done in quadruplicate, and data are presented as mean ¡À SE. Abbreviations: AChE, acetylcholinesterase; PLT, platelet; PPF, proplatelet formation; Pre, preculture.2 `' T; n) l% L  X2 P1 A  A. G
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Gene Expression Profiling of Megakaryocytic Lineage Cells# i* M" q& R8 R& O8 A
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To investigate the maturation stage of the two subpopulations in terms of mRNA level, we performed gene expression profiling of megakaryocytic lineage cells using GeneChip. To profile in detail, the following fractions were analyzed: (a) HSC/progenitor cells (S); (b) Lin¨C/CD41 /CD45 /AChE¨C cells (IM); (c) Lin¨C/CD41  /CD45 /AChE  cells (M); and (d) blood platelets (P). The expression of individual genes in these samples was calculated against the rate in the control sample (HSC/progenitor cells). Expressions of selected genes are shown in Figure 4.
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Figure 4. Gene expression profiling of megakaryocytic lineage cells. GeneChip analyses were performed on four fractions: S, IM, M, and P. The expression of each gene was calculated as the ratio against that in S. Each graph shows the expression of the selected genes as follows: megakaryocyte (MK)/platelet (PLT)-related cell surface markers (A, B), PLT functional receptors (C), cytokine/chemokine receptors (D), other lineage markers (E), and MK-related transcription factors (F). Abbreviations: AChE, acetylcholinesterase; EPO-R, erythropoietin receptor; GMCSFR-, granulocyte macrophage-colony stimulating factor receptor; IL, interleukin; IM, Lin¨C/CD41 /CD45 /AChE¨C; M, Lin¨C/CD41  /CD45 /AChE ; P, platelet; PGHS-1, prostaglandin H synthase 1; S, hematopoietic stem cell/progenitor; TXS, thromboxane synthase; vWF, von Willebrand factor.
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Gene expression for MK/PLT-specific cell surface markers CD41 (gpIIb), CD42a (gpIX), CD42b (gpIb), CD61 (gpIIIa), GPV, and CD9 increased clearly from HSC/progenitor to PLT, in the following order: S, IM, M, P (Fig. 4A). CD62P and AChE expression increased from HSC/progenitor to Lin¨C/CD41  /CD45 /AChE  cells, in the following order: S, IM, M (Fig. 4A, 4B). Moreover, in both cases, expression of the following genes for MK/PLT-related molecules also increased: vWF, prostaglandin H synthase 1, thromboxane synthase, ADP receptors (P2Y1 and P2Y12), thrombin receptors (PAR-1, PAR-3, and PAR-4), NAP-2, and ENA-78, which all function in PLTs (Fig. 4B, 4C). In particular, in the megakaryopoietic cytokine/chemokine receptors, gene expressions of c-mpl, a TPO receptor, and c-kit, a stem cell factor (SCF) receptor, increased and gradually decreased, respectively, from HSC/progenitor to PLT (Fig. 4D). Unexpectedly, IL-6R and CXCR-4, a SDF-1 receptor, gene expressions were unchanged, and neither IL-3R nor IL-11R was detected. Other lineage surface markers, such as CD7, CD14, CD16, CD19, CD56, FcR, IL-7R, granulocyte macrophage-colony stimulating factor receptor-, and erythropoietin receptor, did not show any changes among all of the samples (Fig. 4D, 4E). CD3, CD13, and granulocyte-colony stimulating factor receptor were not detected. In addition, MK-related transcription factors, such as GATA-1, GATA-2, MafG, MafK, and p45 NF-E2, were expressed but not changed in all samples (Fig. 4F).

Differences in expression for CD41, CD42a, CD42b, CD62P, and ß1-tubulin between Lin¨C/CD41  /CD45 /AChE  and Lin¨C/CD41 /CD45 /AChE¨C cells were confirmed by semiquantitative PCR. As shown in Figure 5, these genes were expressed at higher levels in Lin¨C/CD41  /CD45 /AChE  than in Lin¨C/CD41 /CD45 /AChE¨C cells. It is notable that the expression of ß1-tubulin was induced 10 times more highly in Lin¨C/CD41  /CD45 /AChE  than in Lin¨C/CD41 /CD45 /AChE¨C cells, as was expression of CD42a, CD42b, and CD62P. Taken together, these data reveal that Lin¨C/CD41 /CD45 /AChE¨C cells were more immature MKs than Lin¨C/CD41  /CD45 /AChE  ones.; P: K; i3 b; G3 R

Figure 5. Semiquantitative polymerase chain reaction (PCR) analyses of megakaryocyte/platelet-related genes in two subpopulations. The presence of G3PDH (internal control), CD41 (gpIIb), CD42a (gpIX), CD42b (gpIb), CD62P, and ß1-tubulin transcripts was assessed by semiquantitative PCR analysis. cDNAs prepared from the same amount of total RNA from two subpopulations (IM and M) were serially diluted as indicated and amplified. The products were analyzed on 1% agarose gels. Abbreviations: G3PDH, glyceraldehyde-3-phosphate dehydrogenase; IM, Lin¨C/CD41 /CD45 /acetylcholinesterase¨C; M, Lin¨C/CD41  /CD45 /acetylcholinesterase .9 k; `$ d: q& Q7 x( T& x

In Vivo Dynamics of Lin¨C/CD41 /CD45 /AChE¨C MKs in Thrombocytopenia Models# w) T4 n! _1 K( @

To characterize immature Lin¨C/CD41 /CD45 /AChE¨C MKs in vivo, cell dynamics were compared with those of AChE , mature MKs, and PLTs in two different thrombocytopenia mouse models using an antitumor agent, MMC, or anti-PLT antibody (Ab). First, MMC treatment caused the destruction of megakaryocytic cells and resulted in thrombocytopenia. The administration of IL-11 from 1 day after MMC injection stimulated megakaryopoiesis, followed by an increase in AChE  MKs and PLTs . MMC was injected into mice on day 0, and then IL-11 (10 µg/mouse/day) or saline was administered from day 1 to day 5. As shown in Figure 6A and 6B, the number of Lin¨C/CD41 /CD45 /AChE¨C MKs was 100 times higher (0.5¨C2.0 x 105 cells per femur) than that of AChE  MKs (0.6¨C3.2 x 103 cells per femur) in BM. In a control group, PLT number was decreased to a nadir at day 9, but both Lin¨C/CD41 /CD45 /AChE¨C MK and AChE  MK numbers decreased more quickly to a nadir at day 7, and all three parameters were increased in a similar fashion (Fig. 6, open circles). In contrast, IL-11 accelerated the increase of both AChE  MKs and PLTs (Fig. 6B, 6C, closed circles) without changing the total number of BM cells (data not shown). However, the dynamics of Lin¨C/CD41 /CD45 /AChE¨C MKs in IL-11-treated mice were different from those of other parameters. The larger number of Lin¨C/CD41 /CD45 /AChE¨C MKs was detected at day 7, and the number then gradually decreased (Fig. 6A, closed circle). From day 7 to day 14, the number of AChE  MKs increased markedly following the increase in PLTs (Fig. 6B, 6C, closed circles). The decrease in Lin¨C/CD41 /CD45 /AChE¨C MKs and the increase in AChE  MKs were inversely correlated.7 X6 Q! D* ^+ w$ u! Y6 V

Figure 6. Dynamics of immature MKs in thrombocytopenic mice. Mitomycin C (6 mg/kg) (A¨CC) or rabbit anti-murine PLT antiserum (x100 dilution) (D¨CF) was injected into BALB/c mice on day 0. Recombinant hIL-11 (10 µg/mouse/day; closed circles) or solvent (open circles) was administered subcutaneously from day 1 to day 5. The ratio of immature MKs was measured by flow cytometry and calculated as the number of Lin¨C/CD41 /CD45  cells per femur (A, D). The ratio of mature MKs was measured by AChE staining of bone marrow cells and was calculated as the number of AChE-positive cells per femur (B, E). The number of PLTs was measured by Sysmex (C, F). Each data point is presented as mean ¡À SE; n = 5 mice per time point. Abbreviations: AChE, acetylcholinesterase; MK, megakaryocyte; PLT, platelet.

On the other hand, anti-PLT Ab injection on day 0 caused PLTs to decrease without the destruction of megakaryocytic cells. Then, the number of PLTs increased from day 2 and reached a peak on day 5 at a level two times higher than the normal one (Fig. 6F). The number of AChE  MKs increased from day 2 and reached a peak on day 4 prior to the PLT peak (Fig. 6E). However, the number of immature MKs did not increase but slightly decreased on days 1 and 3. In this model, the administration of IL-11 did not accelerate the recovery of PLTs (data not shown). Our data revealed that the increase of AChE  MKs was well correlated with PLT increase in both thrombocytopenia models. However, immature Lin¨C/CD41 /CD45 /AChE¨C MKs, which are a larger MK population in BM, did not increase but rather decreased just prior to a significant increase of AChE  MKs in rapid PLT recovery in vivo.

DISCUSSION8 J) d/ Z8 O3 ]* p" g; R
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Rapid recovery of peripheral PLTs, without thrombocytosis, is needed for the cure of patients with thrombocytopenia. For this purpose, it is essential to understand the mechanism(s) for the acceleration of megakaryopoiesis during differentiation of MKs in BM. However, details of this process have not been elucidated because of experimental difficulties, such as the fragility of primary MKs, their varying size, and their rarity in BM. Cell surface phenotype is an effective index representing individual stages of differentiation . We therefore undertook to characterize the phenotype of BM MKs, based on CD41/CD45/CD61 expression, the potency of PLT production, and AChE activity.
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In the present investigation, we succeeded for the first time in isolating two subpopulations of murine BM MKs, Lin¨C/CD41 /CD45 /AChE¨C and Lin¨C/CD41  /CD45 /AChE , by sequential isolation following immunomagnetic depletion and flow cytometry sorting, coming to the conclusion that CD45  BM MKs consist of at least two subpopulations that are closely related but differ in cell surface phenotypes and AChE activities. Both subpopulations had the MK-specific maker CD42a/b, but they had no, or very few, contaminating various progenitors, granulocytes, or mast cells. Moreover, the expression of CD42a/b increased concomitantly with that of CD41. Further evidence of the high purity and their differentiation stages was obtained by in vitro kinetics of PPF and PLT production, gene expression profiling against four fractions (in the following order: S, IM, M, P), and semiquantitative PCR. More importantly, these analyses provided crucial evidence that both subpopulations were pure and committed MKs and that Lin¨C/CD41 /CD45 /AChE¨C was a more immature subpopulation than Lin¨C/CD41  /CD45 /AChE .

The assignment of these two subpopulations to their respective differentiation stages was made after detecting a remarkable increase in the mRNA level of both CD62P and ß1-tubulin upon changing from Lin¨C/CD41 /CD45 /AChE¨C to Lin¨C/CD41  /CD45 /AChE  MKs. In addition, CD62P was expressed on the surface of Lin¨C/CD41  /CD45 /AChE  MKs, distinct from other cells (data not shown). CD62P is expressed at the late stage of megakaryocytic maturation . NF-E2 is a heterodimer composed of a p45 NF-E2 and a small Maf protein (MafK or MafG), and it is a major regulator of thrombopoiesis. The expression of NF-E2 remained unchanged among the four fractions, indicating that ß1-tubulin is not quantitatively regulated by NF-E2 but is somehow controlled by post-translational modification in MKs.

On the other hand, Lin¨C/CD41 /CD45  MKs did not have AChE activity, in spite of having megakaryocytic surfaces (CD42a /CD42b /FcRI¨C). In vitro differentiation studies and gene expression profiling revealed that these Lin¨C/CD41 /CD45 /AChE¨C MKs were quite immature, thereby demonstrating that AChE mRNA was expressed without the activity or synthesis of its gene product (protein) and also had the capability to differentiate to AChE  MKs. Generally, murine MKs are defined as AChE  . In previous experiments, AChE¨C immature MKs were likely to have been wasted because the density isolation could not separate similar-sized cells from other progenitors. In the present study, we did not undertake comparative analysis between AChE¨C MKs and most large, mature AChE  MKs (collected by ordinary density isolation) in vitro, because the most mature MKs could not be isolated by flow cytometry sorting.
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Next, immature Lin¨C/CD41 /CD45 /AChE¨C MKs were directly detected from whole BM, unexpectedly demonstrating that immature Lin¨C/CD41 /CD45 /AChE¨C MKs are a major MK population in BM. The frequency was different from its in purification, because of the loss by immunomagnetic depletion (yield: 7.3%¨C30.5%). To clarify the role of immature Lin¨C/CD41 /CD45 /AChE¨C MKs, we analyzed their dynamics in two types of thrombocytopenia mouse models. At first, MMC injection caused the destruction of CFU-MK/AChE  MKs and consequent thrombocytopenia. In this model, the administration of IL-11 can stimulate CFU-MK and results in an increased PLT number; however, the increase of CFU-MK occurs after day 9 . Therefore, it is possible that the action of IL-11 until day 7 involves the rescue of immature Lin¨C/CD41 /CD45 /AChE¨C MKs rather than the acceleration of early megakaryopoiesis. In any case, large numbers of immature Lin¨C/CD41 /CD45 /AChE¨C MKs, induced by IL-11, lead to their differentiation into AChE  cells and an increase in rapid PLT recovery in vivo.

The differentiation stages of these megakaryocytic populations are summarized in Figure 7. In the present study, we clearly demonstrated that murine CD45  MKs were composed of immature MKs (phenotype shown in Fig. 7, box A) and maturing MKs (phenotype shown in Fig. 7, box B). To clarify the two MK populations, it is important to examine the regulatory mechanism(s) of starting PPF. As previously stated, the mechanism underlying the expression of ß1-tubulin might be post-translationally controlled, and the post-translational modification can be assessed by focusing on these two populations. In addition, our data revealed that a major MK population in BM was not ordinary AChE  MKs (phenotype shown in Fig. 7, box C, partially including maturing MKs) but immature MKs, which might behave not only as a precursor of AChE  MKs but as a PLT reservoir in vivo. For clinical applications, it is noted that the reservoir system will need to be defined to accomplish the goal of large-scale in vitro production of platelets for transfusion.

Figure 7. Relationships of each MK subpopulation among MK-lineage cells in murine bone marrow. Phenotypes characterizing each population are shown in boxes. Box C represents the phenotypes of terminal matured MKs. Each expression of the CD molecule represents the surface expression. The expression of ß1-tubulin represents the mRNA expression. AChE¨C means without AChE activity (mRNA-positive). Abbreviations: AChE, acetylcholinesterase; HSC, hematopoietic stem cell; MK, megakaryocyte; PLT, platelet.7 n9 o3 c- P7 n

Recently, gene expression profiling using GeneChip has become established as a useful tool for seeking candidate target molecules that are closely related to the mechanism of differentiation. As described earlier, we succeeded in gene expression profiling of MK lineages. Various efforts had been made to identify critical candidates responsible for differentiation toward PLTs . Several MK/PLT-related genes were detectable as highly upregulated, although accompanied by many others, making it difficult to specify a gene(s) exclusively or directly linked to MK/PLT development. In this regard, our profiling analyses were able to reflect gene alterations during megakaryocytic differentiation in detail because of our use of pure and multipoint samples, and they may in the future lead to the candidates, including novel regulators. To this end, it is essential that an appropriate validation system should be established, using the primary MK populations described here.+ |5 G: c  I* o3 E& c4 K' M

CONCLUSION. K! [5 X8 E2 A  R
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We demonstrated that MKs in murine CD45  BM consist of at least two subpopulations, immature Lin¨C/CD41 /CD45 /AChE¨C and maturing Lin¨C/CD41  /CD45 /AChE . The differentiation between the populations includes the start of preparing for PPF. Especially, immature AChE¨C MKs were a overwhelming majority in murine BM MK-lineage compared with AChE  MKs and play an important role in rapid PLT recovery in vivo.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS
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We thank Dr. H. Kiwada and Dr. T. Ishida (Institute of Health Biosciences, The University of Tokushima) for their generous support in the preparation of the manuscript.
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