Ex Vivo Large-Scale Generation of Human Platelets from Cord Blood CD34 Cells - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-4 23:57 |只看该作者 |倒序浏览 |打印

作者：Takuya Matsunagaa, Ikuta Tanakaa, Masayoshi Kobunea, Yutaka Kawanoa, Maki Tanakaa, Kageaki Kuribayashia, Satoshi Iyamaa, Tsutomu Satoa, Yasushi Satoa, Rishu Takimotoa, Tetsuji Takayamaa, Junji Katoa, Takafumi Ninomiyab, Hirofumi Hamadac, Yoshiro Niitsua

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      【摘要】
   In the present investigation, we generated platelets (PLTs) from cord blood (CB) CD34  cells using a three-phase culture system. We first cultured 500 CB CD34  cells on telomerase gene-transduced human stromal cells (hTERT stroma) in serum-free medium supplemented with stem cell factor (SCF), Flt-3/Flk-2 ligand (FL), and thrombopoietin (TPO) for 14 days. We then transferred the cells to hTERT stroma and cultured for another 14 days with fresh medium containing interleukin-11 (IL-11) in addition to the original cytokine cocktail. Subsequently, we cultured the cells in a liquid culture medium containing SCF, FL, TPO, and IL-11 for another 5 days to recover PLT fractions from the supernatant, which were then gel-filtered to purify the PLTs. The calculated yield of PLTs from 1.0 unit of CB (5 x 106 CD34  cells) was 1.26 x 1011¨C1.68 x 1011 PLTs. These numbers of PLTs are equivalent to 2.5¨C3.4 units of random donor-derived PLTs or 2/5¨C6/10 of single-apheresis PLTs. The CB-derived PLTs exhibited features quite similar to those from peripheral blood in morphology, as revealed by electron micrographs, and in function, as revealed by fibrinogen/ADP aggregation, with the appearance of P-selectin and activated glycoprotein IIb-IIIa antigens. Thus, this culture system may be applicable for large-scale generation of PLTs for future clinical use. 5 B/ ?. W, {5 h8 Q
      【关键词】 Platelet Megakaryocyte Cord blood CD  cell Stromal cell
   INTRODUCTION

Platelet (PLT) transfusion has been increasingly needed for severe thrombocytopenic patients after chemotherapy or hematopoietic stem cell (HSC) transplantation. PLTs may also be used for wound healing and regeneration of tissue, including those in the periodontal area . However, most medical facilities often suffer from a shortage of PLTs products since PLT concentration (PC) for transfusion supplied by blood centers is derived from volunteer donors. In addition, there is inevitable risk of pathogenic contamination in the products from donors. Thus, a new source of PLTs is urgently required.
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Cord blood (CB) represents a large and readily available source of HSC. Several investigators  to routine clinical use.
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Zauli et al.  were able to generate megakaryocytes from peripheral blood CD34  cells using a serum-free liquid culture system in the presence of TPO and stromal-derived factor-1; this technique is considered to be feasible for clinical use. However, it may be not applicable to clinical PLT transfusion since the number of generated megakaryocytes was only 12-fold that of the input CD34  cells.; d8 T- g9 S5 C; L) K8 S
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On the other hand, Eto et al.  developed methods to generate a large amount of PLTs in vitro from murine ESCs. Their techniques may be useful for PLT transfusion in the future. At present, however, some serious obstacles need to be overcome before their methods can be used for clinical PLT transfusion since, in addition to the fact that they did not present the data for PLTs produced from human ESCs, ethical issues remain unresolved as to the clinical application of human ESCs.# n8 u3 a" \: m7 [3 t2 p- ^
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We have previously established a long-term serum-free coculture system  of CB CD34  cells with human telomerase catalytic subunit gene-transduced stromal cells (hTERT stroma) using a cytokine cocktail of stem cell factor (SCF), Flt-3/Flk-2 ligand (FL) and TPO. Using this method in the present study, we expanded hematopoietic progenitor/stem cells (first phase), which were further cultured in the presence of SCF, FL, TPO, and interleukin-11 (IL-11) on hTERT stroma to give rise to megakaryocytic lineage differentiation and expansion (second phase) and then finally cultured in a liquid culture system containing SCF, FL, TPO, and IL-11 to generate PLTs from megakaryocytes (third phase). With this three-phase culture system, we succeeded in producing an estimated 1.68 x 1011 PLTs (equivalent to 3.4 units of random donor-derived PLTs or 6/10 of single-apheresis PLTs) from 1.0 CB unit (5 x 106 CD34  cells). PLTs thus obtained exhibited features quite similar to those of PLTs from peripheral blood in both morphology and function.. H1 K- M7 F, L+ g. v  |7 {5 ]

MATERIALS AND METHODS

Cytokines and Monoclonal Antibodies
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Recombinant human SCF, TPO, and IL-3 were gifts from Kirin Brewery (Takasaki, Japan). Recombinant human FL, IL-11, platelet-derived growth factor (PDGF), stromal cell-derived factor-1 (SDF-1), IL-6, fibroblast growth factor-4 (FGF-4), and IL-1ß were purchased from R&D Systems (Minneapolis, http://www.rndsystems.com). Unless otherwise specified, the concentrations of cytokines used were as follows: SCF, 10 ng/ml; TPO, 50 ng/ml; IL-3, 20 ng/ml; FL, 50 ng/ml; IL-11, 20 ng/ml; PDGF, 50 ng/ml; SDF-1, 1 µg/ml; IL-6, 20 ng/ml; FGF-4, 50 ng/ml; IL-1ß, 10 ng/ml. Anti-human transforming growth factor-ß1 antibody (TGF-ß ab) was purchased from Genzyme-Techne (Minneapolis, http://www.genzyme.com). Fluorescein isothiocyanate (FITC)-conjugated anti-CD41 antibody, phycoerythrin (PE)-conjugated anti-CD41 antibody, PE-conjugated anti-P-selectin (CD62P) antibody, neutralizing anti-glycoprotein (GP)IIb-IIIa antibody, FITC-conjugated anti-human leukocyte antigen (HLA)-class I (ABC) antibody, FITC-conjugated anti-HLA-class II (DR) antibody, and FITC-conjugated anti-CD106 antibody were purchased from Immunotech (Marseille, France, http://www.immunotech.com). FITC-conjugated anti-activated GPIIb-IIIa (PAC-1) antibody was purchased from Becton, Dickinson and Company (San Jose, CA, http://www.bd.com). FITC-conjugated anti-CD73 antibody was purchased from Alexis Biochemicals (San Diego, CA, http://www.alexis-corp.com). FITC-conjugated anti-CD105 antibody was purchased from Ancell (Bayport, MN, http://www.ancell.com).
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Purification of Human CB CD34  Cells: N& d  F8 [9 T
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We obtained CB from normal full-term deliveries after obtaining written informed consent that was accepted by the Sapporo Medical University institutional review board according to the Declaration of Helsinki. We first separated low-density mononuclear cells by Histopaque-1077 (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) centrifugation and then purified CB CD34  cells from the mononuclear cells by positive selection using a MACS Direct CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com) according to the manufacturer's instructions. More than 90% of the enriched cells were CD34-positive as confirmed by fluorescence-activated cell sorting (FACS).
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Cultivation of CB CD34  Cells on hTERT Stroma for Expansion of Hematopoietic Progenitor/Stem Cells ("First Phase Culture")
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We expanded hematopoietic progenitor/stem cells from CB CD34  cells in first phase culture as described previously . In brief, we seeded 5 x 102 CB CD34  cells on a monolayer of hTERT stroma that had been plated in a 75-cm2 flask (Greiner Bio-One, Frickenhausen, Germany, http://www.gbo.com/en) in 10 ml of serum-free medium, X-VIVO10 (BioWhittaker, Walkersville, MD, http://www.cambrex.com), supplemented with SCF, TPO, and FL at 37¡ãC in 5% CO2. After 7 days, we added 10 ml of fresh complete medium containing the same concentration of cytokines and continued cultivation for another 7 days (Fig. 1, first phase of protocol C).
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Figure 1. Culture protocols of this study. Three types of culture protocols were performed in this study. Protocol A: Cord blood (CB) CD34  cells were cocultured with bone marrow stromal cells transduced with the human telomerase catalytic subunit gene (hTERT stroma) for 14 days in the presence of stem cell factor (SCF), thrombopoietin (TPO), Flt-3/Flk-2 ligand (FL), and interleukin (IL)-11 for megakaryocytic lineage expansion (second phase). Protocol B: CB CD34  cells were cocultured with hTERT stroma for 14 days in the presence of SCF, TPO, FL, and IL-11 for megakaryocytic lineage expansion (second phase). Unseparated cells, including megakaryocytic lineage cells obtained from the second phase, were cultured without hTERT stroma for 7 days in the presence of SCF, TPO, FL, and IL-11 for platelet production (third phase). Protocol C: CB CD34  cells were cocultured with hTERT stroma for 14 days in the presence of SCF, TPO, and FL for hematopoietic progenitor/stem cell expansion (first phase). Unseparated cells, including hematopoietic progenitor/stem cells obtained from the first phase, were cultured with hTERT stroma for 14 days in the presence of SCF, TPO, FL, and IL-11 for megakaryocytic lineage expansion (second phase). Unseparated cells, including megakaryocytic lineage cells obtained from the second phase, were cultured without hTERT stroma for 5 days in the presence of SCF, TPO, FL, and IL-11 for platelet production (third phase). Abbreviation: CB, cord blood.! r2 \, i  ^1 G3 U2 N% {, E3 U

Cultivation of Hematopoietic Progenitor/Stem Cells Obtained from First Phase Culture on hTERT Stroma for Megakaryocytic Lineage Expansion ("Second Phase Culture")
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We cultured 5 x 103 unseparated cells including hematopoietic progenitor/stem cells obtained from first phase culture (Fig. 1, protocol C) on a monolayer of hTERT stroma in a 75-cm2 flask in 10 ml of serum-free medium, X-VIVO10 supplemented with SCF, TPO, FL, and IL-11 for 7 days at 37¡ãC in 5% CO2, after which we added 10 ml of complete medium containing the same concentration of cytokines and continued cultivation for another 7 days (Fig. 1, second phase of protocol C). In an experiment to determine the optimal cytokine combination for megakaryocytic lineage expansion, we used, in addition to the above cytokine cocktail, SDF-1, PDGF, IL-3, IL-6, and TGF-ß ab (5 mg/ml) in various combinations and 5 x 103 CB CD34  cells instead of the cells expanded by first phase culture under cultivation conditions that were otherwise the same as described above (Fig. 1, protocol A). At the end of this cultivation period, we assessed the numbers of total cells, CD41  cells, megakaryocytes, and colony-forming unit megakaryocytes (CFU-Megs) by the method described below.

Cultivation of Megakaryocytic Lineage Cells Obtained from Second Phase Culture with or Without Human Umbilical Endothelial Cells for PLT Production ("Third Phase Culture")
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We cultured 1 x 106 unseparated cells including megakaryocytic lineage cells obtained from second phase culture (Fig. 1, protocol B) in a six-well tissue culture plate (Nunc, Roskilde, Denmark, http://www.nuncbrand.com) in 4 ml of X-VIVO10 containing SCF, TPO, FL, and IL-11 or the same cytokines plus SDF-1 and FGF-4 with or without human umbilical endothelial cell (HUVEC) (American Type Culture Collection, Manassas, VA, http://www.atcc.org) layers that had been plated at 37¡ãC in 5% CO2 (Fig. 1, third phase of protocol B). On day 4, we added 4 ml of fresh complete medium containing the same concentration of cytokines to the medium and continued cultivation another 3 days. During this cultivation period, we assessed the numbers of total cells, CD41  cells, CFU-Megs, and megakaryocytes on days 0, 3, 5, and 7 by the methods described below.

Assessment of Cell Viability" ]% z" G% k8 v5 w( N, H

Viable cell numbers were measured by the trypan blue dye exclusion method.

CFU-Meg Assay

We assayed the clonogenic potentials of megakaryocytic progenitors using the serum-free collagen-based system MegaCult-C (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) according to the manufacturer's instructions. The cytokines used were 50 ng/ml TPO, 10 ng/ml IL-6, and 10 ng/ml IL-3. After incubation for 12 days on double-chamber culture slides, megakaryocytic colonies were differentiated by immunostaining for CD41 and counted.
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Identification of Megakaryocytic Lineage Cells by FACS Using the Anti-CD41 Antibody

We collected cultured cells by centrifugation at 350g (1,300 rpm) for 5 minutes and resuspended them in phosphate-buffered saline (PBS). We then identified and quantified megakaryocytic lineage cells with a FACSCalibur flow cytometer (Becton Dickinson) using the FITC-conjugated anti-CD41 antibody according to the procedure described in Zauli et al. .

Identification and Quantification of CB CD34  Cell-Derived PLTs

We identified and quantified CB CD34  cell-derived PLTs (CB-PLTs) in culture supernatant by FACS according to the procedures described by Choi et al. . In brief, we labeled the plasma-derived PLTs with the FITC-conjugated anti-CD41 antibody and applied them to FACS to create a single "PLT gate." Next, we collected CB-PLTs as pellets from the medium of third phase culture on days 0, 3, 5, and 7 (Fig. 1, protocol B) or on days 0, 3, and 5 (Fig. 1, protocol C). The culture medium was centrifuged at 100g (700 rpm) for 10 minutes to remove nucleated large cells as pellets. The supernatant was then subjected to centrifugation at 1,880g (3,000 rpm) for 5 minutes to obtain CB-PLT pellets. After washing the pellets with PBS, we suspended them in 500 µl of PBS. We then labeled PLT-like fragments in the suspension with FITC-conjugated anti-CD41 antibodies and applied them to FACS to create a single PLT gate for quantification.$ X; m/ R+ y: Q' r- W7 k
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Morphological Analyses of Megakaryocytes and PLTs5 Q) B( g" d% `
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We identified and quantified megakaryocytes expanded in culture with a conventional microscope after May-Gr¨¹nwald-Giemsa staining. For visualizing in an electron microscope, we first stabilized CB CD34  cell-derived megakaryocytes (CB-Megs), CB-PLTs, and plasma-derived PLTs from healthy volunteers with 0.38% formaldehyde and 0.6% acid citrate dextrose (Sigma-Aldrich) and fixed them with 2.5% glutaraldehyde solution in PBS for 60 minutes. After rinsing with PBS, we postfixed the megakaryocytes and PLTs with 1% osmium tetraoxide and 1.5% potassium ferrocyanide in PBS for 60 minutes, dehydrated them with ethanol, and embedded them in Epons (Taab Laboratories Equipment, Reading, U.K., http://www.taab.co.uk). Then, we cut the Epons with a diamond knife into ultrathin sections, stained them with uranyl acetate and lead citrate, and viewed them under a JEOL 1200EX electron microscope (Japan Electron Optics Laboratory Cooperation, Tokyo, http://www.jeol.co.jp) at 80 kV .
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Detection of P-Selectin and Activated GPIIb-IIIa on PLTs by FACS

We isolated CB-PLTs or plasma-derived PLTs as described above, which we then fixed immediately with an equal volume of cold 1% paraformaldehyde, pH 7.4, for at least 2 hours (resting PLTs). We stimulated another set of PLTs with 40 µmol/l ADP for 10 minutes and then fixed it as above (activated PLTs). We washed the resting PLTs and the activated PLTs with PBS and incubated them for 30 minutes at room temperature with PE-conjugated anti-P-selectin (CD62P) monoclonal antibodies or FITC-conjugated anti-activated GPIIb-IIIa (PAC-1) antibodies according to the manufacture's instructions. We washed the PLTs with PBS and incubated them with FITC-conjugated anti-CD41 antibodies or PE-conjugated anti-CD41 antibodies for 30 minutes, after which they were washed again and analyzed by FACS.
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Preparation of PLTs for Aggregation Analyses
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For aggregation analyses, we prepared CB-PLTs and plasma-derived PLTs by gel filtration according to the methods of Tangen et al.  on Sepharose-2B (Amersham Biosciences, Princeton, NJ, http://www.amersham.biosciences.com) in gel filtration buffer (137 mmol/l NaCl, 2.7 mmol/l KCl, 1 mmol/l MgCl2, 5.6 mmol/l glucose, 1 mg/ml bovine serum albumin, and 20 mmol/l HEPES, pH 7.4).2 J4 b1 X5 f% s
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Analyses of PLT Aggregation  C% H) a5 l3 I- K4 C- h

We incubated gel-filtered PLTs suspended in 50 µl of PBS with 600 µg/ml human fibrinogen (Sigma-Aldrich) and 2 mmol/l ADP (Sigma-Aldrich) for 5 minutes. Then, we pipetted 10 µl of PLTs suspension (1 x 105 PLTs) into wells of a 72-well Terasaki plate (Greiner Bio-One) and viewed the PLT aggregation under a TS-100 phase contrast microscope (Nikon, Tokyo, http://www.nikon.com). In an experiment to examine the inhibitory effect of neutralizing the anti-GPIIb-IIIa antibody on the PLT aggregation , we incubated PLTs with 50 µg/ml of the antibody or control antibody (anti-CD34) for 5 minutes at room temperature and then conducted the aggregation procedure described above.7 w7 `3 G0 x# a; Y
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Analyses of DNA Ploidy

We analyzed the ploidy of cultured cells by FACS after DNA staining with 40 µg/ml propidium iodide (PI) (Sigma-Aldrich) according to the procedure described by Guerriero et al. .
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FACS for HLA Expression on PLTs: o$ R" F8 K! r- U2 m0 N/ x

We isolated CB-PLTs or plasma-derived PLTs by centrifugation as described above and labeled them with FITC-conjugated anti-HLA-class I (ABC) or HLA-class II (DR) antibodies and analyzed them by FACS.
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Analyses of hTERT Stroma Contamination in Cultured Cells

We stained cultured cells with FITC-conjugated anti-CD73, anti-CD105, or anti-CD106 antibodies, which recognize the specific surface antigens of stromal cells, and analyzed them for hTERT stroma contamination by FACS.

RESULTS1 \% W7 \/ [0 s4 T+ E- M: ~
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Optimal Cytokine Combination in Second Phase

Since we have previously established the cultivation conditions for expansion of hematopoietic progenitor/stem cells from CD34  cells (first phase) (Fig. 1, protocol C)  as an enhancer of megakaryocyte ploidy and examined their effectiveness in combination with the first phase cytokine cocktail, SCF, TPO, and FL (SCF/TPO/FL). The total cell count with the first phase cocktail alone (SCF/TPO/FL) (Fig. 1, protocol C) was significantly lower than with the other cytokine cocktails (Table 1). The combinations SCF/TPO/FL/IL-11/IL-3/IL-6 and SCF/TPO/FL/IL-11/TGF-ß ab/SDF-1 showed similarly high expansion rates, followed by SCF/TPO/FL/IL-11/TGF-ß ab, SCF/TPO/FL/IL-11/SDF-1, and SCF/TPO/FL/IL-11. Significant increases in CD41  cells were observed in cultures with SCF/TPO/FL/IL-11/SDF-1 and SCF/TPO/FL/IL-11 as compared with that with SCF/TPO/FL. The expansion rate of CD41  cells by SCF/TPO/FL/IL-11 was higher than that of SCF/TPO/FL/IL-11/SDF-1, followed by SCF/TPO/FL/IL-11/IL-3/IL-6 and SCF/TPO/FL/IL-11/TGF-ß ab. SCF/TPO/FL/IL-11/PDGF and SCF/TPO/FL/IL-11/TGF-ß ab/SDF-1 showed lower expansion of CD41  cells than SCF/TPO/FL. The expansion rate of CFU-Megs by SCF/TPO/FL/IL-11 was higher than that of SCF/TPO/FL, followed by SCF/TPO/FL/IL-11/SDF-1, SCF/TPO/FL/IL-11/IL-3/IL-6, and SCF/TPO/FL/IL-11/TGF-ß ab. The combinations SCF/TPO/FL/IL-11/PDGF and SCF/TPO/FL/IL-11/TGF-ß ab/SDF-1 gave rise to lower expansion of CFU-Megs than did SCF/TPO/FL alone. The megakaryocyte count as determined by May-Gr¨¹nwald-Giemsa staining was significantly lower in culture treated with a cocktail of SCF/TPO/FL than with other cytokine cocktails. Combinations of SCF/TPO/FL/IL-11 brought about the highest expansion rate of megakaryocyte count, followed by SCF/TPO/FL/IL-11/IL-3/IL-6, SCF/TPO/FL/IL-11/SDF-1, and SCF/TPO/FL/IL-11/TGF-ß ab. These results revealed that SCF/TPO/FL/IL-11 was the optimal cytokine cocktail for megakaryocyte expansion in second phase (Fig. 1, protocol C).  E- g) U5 S7 c
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Table 1. Generation of megakaryocytic progenitors and megakaryocytes from CB CD34  cells on hTERT stroma with various cytokine combinations

Effect of Input Number of CD34  Cells on Recovery of Total Cells, CD41  Cells, CFU-Megs, and Megakaryocytes in Second Phase

Next, we examined the effect of various concentrations of CD34  cells on the expansion of megakaryocytic lineage cells in second phase using protocol A as shown in Figure 1. The concentrations of cell numbers used were 50, 500, and 5,000 cells per 10-ml/75-cm2 flask. The most efficient expansion rate of total cells, CD41  cells, CFU-Megs, and megakaryocytes was observed with 500 CD34  cells per 10-ml/75-cm2 flask (Table 2).8 O  P9 H- w1 t$ v7 O0 Q8 Q, |
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Table 2. Effect of input CB CD34  cell number on megakaryocytic progenitors and megakaryocytes combinations
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Differentiation of Megakaryocytic Lineage Cells and Generation of PLTs in Third Phase* y7 i  n8 r7 Z* l

It has been reported that mouse bone marrow stromal cells inhibited differentiation of megakaryocytic lineage cells to release PLTs when they were cocultured  reported that nitric oxide (NO) could efficiently release PLTs from megakaryocytes (Meg-01 cell line), we added NO to the culture medium to facilitate PLT release from megakaryocytic lineage cells. However, PLT recovery was not affected by NO in our culture system (data not shown). Thus, we decided on the liquid culture system with the cytokine combination SCF/TPO/FL/IL-11 for third phase culture (Fig. 1, protocol C). From day 0 to day 3 of the third phase of protocol B, the number of CFU-Meg and the total cell number were stationary, whereas the number of CD41-positive cells and megakaryocytes rapidly increased with the appearance of PLTs, indicating differentiation (maturation) of megakaryocytic lineage cells during this period (Fig. 2B). This observation was compatible with the change of DNA ploidy, that is, a higher ploidy at day 3 than at day 0 (Fig. 2C). At day 5, the numbers of all cellular components (CD41-positive cells, CFU-Megs, and megakaryocytes) decreased (Fig. 2B), although the DNA ploidy pattern was almost the same as that on day 3, whereas the number of PLTs peaked; at day 7, PLTs decreased. We found that a significant amount of PLTs was trapped in the pellet when the culture supernatant was centrifuged to remove nucleated large cells.& X. _' t5 Q7 y

Figure 2. Serial changes in amplification of megakaryocytic lineage cells and platelets and ploidy distribution of cultured cells in third phase. (A): Detection of CD34  cell-derived platelets by fluorescence-activated cell sorting (FACS). a, FACS analysis data of plasma-derived platelets. b, FACS analysis data of CD34  cell-derived platelets. (B): Serial changes in the number of cultured cells. Data are shown as mean ¡À SD in triplicate culture and are representative of three independent experiments. (C): Ploidy distribution of cultured cells. Open bars, 2N; checkered bars, 4N; horizontal lined bars, 8N; dotted bars, 16N; diagonal lined bars, 32N; grid lined bars, 64N. Abbreviations: FSC, forward scatter; SSC, side scatter.$ i' g- C6 d  I" |  _

Table 3. Generation of platelets from megakaryocytes with two different cytokine combinations in the presence or absence of HUVECs
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Electron Micrograph of CB-Meg and CB-PLTs
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We examined CB-Meg obtained after 3 days of liquid culturing and PLTs after 5 days of culturing (Fig. 1, third phase of protocol B) by electron microscopy (Fig. 3). Both  granule and demarcation membrane systems, which are characteristics of mature human megakaryocytes, were evident in CB-Meg (Fig. 3A, 3B). CB-PLTs (Fig. 3C) showed quite similar characteristics to those from plasma-derived platelets (Fig. 3D) in that it contained  and dense granules.! e3 e5 L$ M( F  m4 ~

Figure 3. Electron micrographs of CD34  cell-derived megakaryocytes and platelets. (A): Electron micrograph of cultured megakaryocytes (magnification, x4,000). (B): Magnified image of a rectangle in (A) (magnification, x12,000). Arrow,  granule; arrowhead, demarcation membrane. (C): Electron micrograph of plasma-derived platelets (magnification, x30,000). (D): Electron micrograph of CD34  cell-derived platelets (magnification, x30,000). Arrowhead, dense granule; arrow,  granule.! g; {/ ?" Z$ ~( R+ ]

Functional Assessment of CB-PLTs
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To validate the function of CB-PLTs produced using protocol B as shown in Figure 1, we first treated them with ADP. As revealed by FACS, the expression of P-selectin and activated GPIIb-IIIa on CB-PLTs was clearly evoked by this treatment (Fig. 4A, a, b, I) in a fashion similar to those from the healthy volunteer (Fig. 4A, a, b, II). To separate pure PLTs from the debris of nucleated cells, CB-PLTs and plasma-derived PLTs from healthy volunteers were gel-filtered as described by Tangen et al. . We then treated gel-filtered CB-PLTs (Fig. 4B, a, I, 4C) and plasma-derived PLTs from healthy volunteers (Fig. 4B, b, I, 4C) with ADP and fibrinogen. When these PLTs were observed under a phase contrast microscope, aggregation was clearly observed (Fig. 4B, a, b, II, 4C). This aggregation was blocked by preincubation with neutralizing anti-GPIIb-IIIa antibodies (Fig. 4B, a, b, III, 4C).* c- U1 k/ n3 @$ U* [5 E

Figure 4. Functional assessment and HLA expression of CD34  cell-derived platelets. (A): P-selectin (CD62P) (a) and activated GPIIb-IIIa (PAC1) (b) expression on ADP-stimulated platelets (PLTs). CD34  cell-derived (I) or plasma-derived (II) PLTs were incubated with anti-CD62P or anti-PAC1 antibody as either resting populations (pre-ADP) or as activated populations (post-ADP). (B, C): CD34  cell-derived PLTs (CB-PLTs) aggregated in response to ADP/fibrinogen. Phase micrographs of CB-PLTs (B, a, I) and plasma-derived PLTs (B, b, I) before ADP/fibrinogen addition, and CB-PLTs (B, a, II) and plasma-derived PLTs (B, b, II) after ADP/fibrinogen addition. Inhibition of PLT aggregation by neutralizing anti-GPIIb-IIIa antibody of CB-PLTs (B, a, III) and plasma-derived PLTs (B, b, III). (C): The number of PLT aggregates counted under an inverted microscope. Data are shown as mean ¡À SD in three visual fields. Before, before ADP/fibrinogen addition; after, after ADP/fibrinogen addition; GPIIb/IIIa Ab, pre-incubation with neutralizing anti-GPIIb-IIIa Ab. (D): Expressions of HLA on CB-PLTs (a) and plasma-derived PLTs (b). Green line, isotypic control antibody; blue line, anti-HLA-class I antibody; red line, anti-HLA-class II antibody. Abbreviations: Ab, antibody; GP, glycoprotein; ND, not detected; PLT, platelet.# m: q5 t; G- T

HLA Expression of CB-PLTs

To further characterize the properties of CB-PLTs produced using protocol B as shown in Figure 1, we analyzed their HLA expression by FACS. The percentages of HLA-class I  and HLA-class II  CB-PLTs were more than 99% and less than 0.1%, respectively, and were similar to those of plasma-derived PLTs (Fig. 4D).
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Generation of PLTs by Combination of Three-Phase Culture System8 B: x) w5 e. w# @. Z
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We finally combined first, second, and third phase culture systems and examined the recovery of PLTs (Fig. 1, protocol C). For first phase culturing, 500 CD34  cells were incubated in 10 ml of medium for days 0¨C7 and 20 ml for days 8¨C14 on hTERT stroma with SCF/TPO/FL, from which we obtained 5.2 x 106 total cells that contained 5.2 x 105 CD34  cells (Table 4). Since the data in Table 2 showed that 500 CD34  cells per 10 ml was the optimal concentration for second phase, 5,000 unseparated cells obtained from first phase containing 500 CD34  cells were cocultured with hTERT stroma in 10 ml of medium for days 0¨C7 and 20 ml for days 8¨C14 with SCF/TPO/FL/IL-11. Finally, the third phase consisted of 1 x 106 unseparated cells from second phase, which included megakaryocytic lineage cells, being cultured in 4 ml of medium with SCF/TPO/FL/IL-11 but without hTERT stroma for 5 days (third phase), since the data in Table 4 indicated that the recovery of PLTs peaked on day 5. The total cell number peaked at day 28 and decreased on day 33 (Table 4). The number of CFU-Megs and megakaryocytes on day 28 was also higher than those on day 33, whereas the number of PLTs on day 33 increased to 2.1 x 107 ¡À 0.1 x 107 cells per 4 ml from 2.3 x 106 ¡À 0.4 x 106 cells per 4 ml on day 28 (Table 4). The expansion rate of PLTs with this three-phase culture system (Fig. 1, protocol C) was 5.8 times greater than with the above-mentioned two-phase culture system (Fig. 1, protocol B). Since scaling up is not straight forward in our culture system, actual and reliable yields of PLTs were not obtainable. Therefore, we estimated the yield by assuming that the recovery of cells or PLTs at each phase was 100%. Thus the calculated yield of PLTs obtained from 1.0 unit of CB (5 x 106 CD34  cells) by three-phase culturing was 2.1 x 1011. Finally, we gel-filtered the PLTs from CB CD34  cells and found that the recovery rates from gel filtration were between 60% and 80%, in agreement with the report by Tangen et al. . Therefore, the final yield of PLTs after gel filtration was calculated to be 1.26 x 1011¨C1.68 x 1011.+ W5 h0 O3 K6 @5 q* r

Table 4. Recovery of megakaryocytic lineage cells and platelets in three-phase culture system, A5 a" q7 J( f# x' {# F7 @4 V( k

Proportion of Megakaryocytes and Remaining Cells During Three-Phase Culture System

In the three-phase culture system (Fig. 1, protocol C), we analyzed the composition of the cell population. At the end of first phase (day 14), a majority of the cells were blast-like cells (92%), and the remaining cells were monocytes/macrophages (8%); there were no megakaryocytes. By the end of second phase (day 28), the number of blast-like cells had decreased (70%), whereas that of monocytes/macrophages had increased (25%), with the appearance of myeloid cells (promyelocytes, 3.25%; myelocytes, 1.25%; and metamyelocytes 0.25%) and megakaryocytes (0.25%). By the end of third phase (day 33), the number of blast-like cells had decreased remarkably (2.5%), whereas that of monocytes/macrophages had increased remarkably (85%); also observed was an increase of myeloid cells (myelocytes, 8.5%; metamyelocytes, 3.5%), whereas megakaryocytes increased to 0.5% of total cells.
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CB-PLTs Were Free of hTERT Stroma Contamination- f' d! q. o0 ~# J- J6 N
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To confirm that CB-PLTs were free of hTERT stroma contamination, we examined the stromal-specific antigens CD73, CD105, and CD106 of cultured cells on day 33 (Fig. 1, protocol C) by FACS and confirmed that all these antigens were negative (data not shown).

DISCUSSION

In the present study, we have demonstrated that an estimated 1.26 x 1011¨C1.68 x 1011 PLTs are obtained from 1.0 CB unit (5 x 106 CD34  cells) using the "serum-free culture system." These numbers of PLTs are equivalent to 2.5¨C3.4 units of random donor-derived PLTs or 2/5¨C6/10 of single-apheresis PLTs. The recovery of PLTs by our method was approximately 210¨C350 times greater than the best yield of PLTs from CD34  cells by in vitro culture previously reported .
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Our method comprises three critical phases: (a) expansion of CD34  cells (Fig. 1, first phase of protocol C); (b) expansion of megakaryocytic lineage cells (Fig. 1, second phase of protocol C); and (c) maturation of megakaryocytes to produce PLTs (Fig. 1, third phase of protocol C). To expand CB CD34  cells with first phase culture, we used a method to make use of hTERT stroma that we established previously . Another procedure to improve the CD34  cell expansion rate may be to modify hTERT stroma to become more potent in supporting CD34  cell growth. We have shown that hematopoietic stem cells and progenitors could be expanded 3¨C5 times more than using hTERT stroma by using hTERT stroma transduced with the Indian hegdehog (Ihh) gene. Thus exploration to examine whether utilization of CAFCs and/or Ihh-transduced hTERT stroma indeed facilitates hematopoietic stem cells, which are capable of underlying megakaryocytic lineage differentiation and of producing PLTs is an important future undertaking.* \4 W" ?) G9 r2 n- X5 t8 g

Determining the optimal cytokine combination for second phase megakaryocytic lineage expansion and differentiation was the most crucial task in the present study. Among IL-11 . It therefore seemed reasonable to use IL-11 in combination with SCF/TPO/FL for second phase, and in fact, the addition of PDGF, SDF-1, IL-3, IL-6, or TGF-ß ab to the SCF/TPO/FL/IL-11 combination did not enhance the generation of megakaryocytic lineage cells. The cytokines and antibodies might have interfered with each other's ability to produce megakaryocytic lineage cells. It is also possible that PDGF, SDF-1, IL-3, IL-6, and TGF-ß ab were promoting maturation at the expense of proliferation of megakaryocytic lineage cells.

In the third phase, we chose a liquid culture system, eliminating hTERT stroma support, on the basis of our preliminary findings that maturation of megakaryocytes to produce PLTs did not occur in culture with hTERT stroma (data not shown). Our decision to eliminate hTERT stroma from third phase was also encouraged by the previous observation that bone marrow stromal cells functioned positively on megakaryocytopoiesis and negatively on PLT generation in mice . In the present study, we were not be able to reproduce this observation (Table 4), possibly because of the fact that considering the practical unfeasibility of using fresh BMECs, we instead used HUVECs. The establishment of immortalized BMECs to improve the efficiency of PLT release from CB-Meg may be an intriguing future task.2 ?1 p4 x9 F7 a
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With regard to the approach of using NO  and cultured megakaryocytes.
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Several investigators have previously reported that proplatelet formation (PPF) is observed in cultured megakaryocytes that are placed on glass or plastic and is triggered by contact with the solid substrate, initiating signal transduction from the surface of the megakaryocyte membrane. Some recent studies  reported that the ability to form proplatelets in megakaryocytes derived from cord blood is inferior to that of megakaryocytes derived from bone marrow. The stem cell source and cocultivation with bone marrow stromal cells in our culture system might have affected the PPF in CB-Meg.1 k" ^. V- {" {0 S

In conclusion, our technology to produce some appreciable quantity of human PLTs from CB in vitro under serum-free conditions may find applications in blood centers, dermatology, and plastic and reconstructive surgery in conjunction with good manufacturing practices and the solution of ethical issues for CB donation. Our culture system expanding CB to make PLTs might allow the ability to screen CB units and therefore PLTs for infection, which is a major drawback of CB transplantation.
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DISCLOSURES

The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS1 N+ ~1 G( ^5 o# K8 k

We thank Kevin Litton for editorial assistance. We also thank Dr. Koji Miyanishi for preparation of the manuscript.+ j( y9 K  O- x. Y. `) O
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