1. Different ploidy levels of megakaryocytes generated from peripheral or cord blood CD34+ cells are correlated with different levels of platelet release
by Gianfranco Mattia, Francesca Vulcano, Luisa Milazzo, Alessandra Barca, Giampiero Macioce, Adele Giampaolo, and H. Jane Hassan
Blood
Volume 99(3):
February 1, 2002
©2002 by American Society of Hematology

2. Morphologic differences between CB- and PB-derived megakaryocytic cells.Morphology of MKs derived from CB- (left panels) and PB-CD34+ cells (right panels) induced to differentiate in serum-free medium in the presence of TPO (100 ng/mL) is presented.
Morphologic differences between CB- and PB-derived megakaryocytic cells.Morphology of MKs derived from CB- (left panels) and PB-CD34+ cells (right panels) induced to differentiate in serum-free medium in the presence of TPO (100 ng/mL) is presented. Cytospins were observed at different days by light microscopy after May-Grünwald staining. (Bar = 10 μm.)‏
Gianfranco Mattia et al. Blood 2002;99:
©2002 by American Society of Hematology

3. Delay in the appearance of CD61 and CD41a expression in MKs generated from CB with respect to PB.Comparative analysis of the megakaryocytic markers CD61, CD41a, and CD42b during MK differentiation of the CD34+ cells cultured in the presence of TPO (100 ng/m...
Delay in the appearance of CD61 and CD41a expression in MKs generated from CB with respect to PB.Comparative analysis of the megakaryocytic markers CD61, CD41a, and CD42b during MK differentiation of the CD34+ cells cultured in the presence of TPO (100 ng/mL). Cells were labeled with either PE- or FITC-conjugated MoAbs and analyzed by flow cytometry. ●, CB; ○, PB. (Mean ± SEM values from 5 experiments.)‏
Gianfranco Mattia et al. Blood 2002;99:
©2002 by American Society of Hematology

4. Similar expression of NF-E2, GATA-1, and GpIbα but delay in the GpIIIa expression in MKs derived from CB compared with PB.(A) RT-PCR analysis of GpIIIa, GpIbα, GATA-1, and NF-E2 mRNA expression during in vitro megakaryocytic differentiation of CD34+ cells g...
Similar expression of NF-E2, GATA-1, and GpIbα but delay in the GpIIIa expression in MKs derived from CB compared with PB.(A) RT-PCR analysis of GpIIIa, GpIbα, GATA-1, and NF-E2 mRNA expression during in vitro megakaryocytic differentiation of CD34+ cells generated from CB (left panels) and PB (right panels). S26 controls are presented. Representative results from 3 independent experiments are shown. (B) Densitometric analysis (Instant Imager, Packard, Canberra) of GATA-1 and NF-E2 expression normalized to the S26 controls. ●, 1:1, ▪, 1:2; ▴, 1:4 sample dilution. (See also “Materials and methods.”)‏
Gianfranco Mattia et al. Blood 2002;99:
©2002 by American Society of Hematology

5. Increased proliferation of MKs derived from CB with respect to PB CD34+ cells.Cells were grown in serum-free liquid suspension culture in presence of TPO (100 ng/mL) at 4 × 104cells/mL and enumerated at different days of culture. ●, CB; ○, PB. Mean ± SEM va...
Increased proliferation of MKs derived from CB with respect to PB CD34+ cells.Cells were grown in serum-free liquid suspension culture in presence of TPO (100 ng/mL) at 4 × 104cells/mL and enumerated at different days of culture. ●, CB; ○, PB. Mean ± SEM values from 5 experiments are reported.
Gianfranco Mattia et al. Blood 2002;99:
©2002 by American Society of Hematology

6. Morphologic aspects of CB and PB megakaryocytic colonies
Morphologic aspects of CB and PB megakaryocytic colonies.CD34+ cells purified from CB or PB were assessed for their capacity to give rise to MK colonies when cultured in 0.9% methylcellulose using the same serum-free medium of the liquid suspension culture ...
Morphologic aspects of CB and PB megakaryocytic colonies.CD34+ cells purified from CB or PB were assessed for their capacity to give rise to MK colonies when cultured in 0.9% methylcellulose using the same serum-free medium of the liquid suspension culture containing 50 ng/mL TPO. (A) CB-derived multifocal BFU-MK colony after 16 days of culture, showing > 100 small cells. (B) PB-derived CFU-MK colony at day 12 of culture, with few large cells. Colonies were evaluated by inverted light microscopy. (Original magnification, × 32.)‏
Gianfranco Mattia et al. Blood 2002;99:
©2002 by American Society of Hematology

7. Low DNA content of MKs differentiated from CB in comparison with PB-derived CD34+ cells.CD34+ cells were cultured for 12 days in serum-free media with TPO (100 ng/mL).
Low DNA content of MKs differentiated from CB in comparison with PB-derived CD34+ cells.CD34+ cells were cultured for 12 days in serum-free media with TPO (100 ng/mL). MK ploidy was measured by a double-staining technique and flow cytometry (left panels). Histograms of DNA content (right panels) were obtained gating the cells double-labeled with anti-CD61 MoAb and propidium iodide (see also “Materials and methods”). Representative experiment of 5 independent ones is presented.
Gianfranco Mattia et al. Blood 2002;99:
©2002 by American Society of Hematology

8. Reduced level of platelet production in CB-derived MK culture
Reduced level of platelet production in CB-derived MK culture.(A) Platelets released from CB- (▪) and PB-derived (■) cultures.
Reduced level of platelet production in CB-derived MK culture.(A) Platelets released from CB- (▪) and PB-derived (■) cultures. Platelets were enumerated using anti-CD61 FITC-conjugated MoAb; see also panel B. Histograms were the mean ± SD of 5 independent experiments. Statistical significance was established by using the Student t test, P < .002. Corresponding CB (●) and PB (▵) megakaryocytic growth is indicated (right axis); see also Figure 4. (B) Platelets obtained from CB-MKs (upper panels) and PB-MKs (bottom panels) were analyzed at days 12 and 14 by flow cytometry. In vitro–produced platelets were evidenced by using anti-CD61 FITC-conjugated MoAb (solid line). Expression of P-selectin on the surface of platelets before (dotted line) and after (dashed line) stimulation with 2 U/mL thrombin at 37°C for 15 minutes was analyzed by using anti-CD62 PE-conjugated MoAb. Isotype control is also presented (thin line). Representative experiment of 5 independent ones is presented.
Gianfranco Mattia et al. Blood 2002;99:
©2002 by American Society of Hematology

9. Ploidy MKs release more platelets than diploid MKs
Ploidy MKs release more platelets than diploid MKs.(A) MKs from PB culture were sorted on the basis of the cell size at day 8.
Ploidy MKs release more platelets than diploid MKs.(A) MKs from PB culture were sorted on the basis of the cell size at day 8. The R1 obtained population (∼10%) was recultured in serum-free medium in the presence of TPO (100 ng/mL). (B) Morphology of the megakaryocytic-sorted fraction after Wright-Giemsa staining (Bar = 30 μm). (C) ▪: percentage of proplatelets forming MKs on PB-R1 fraction and day 12 CB-derived MKs. ■: platelets produced by the same populations were enumerated at days 12 and 14, respectively. (See also “Materials and methods” and “Results.”)‏
Gianfranco Mattia et al. Blood 2002;99:
©2002 by American Society of Hematology

10. Late release of PF4 and βTG in the supernatant of megakaryocytic cell cultured from CB-CD34+ cells.The concentration of PF4 and βTG was measured by immunoenzymatic assay in the supernatants of MK cultures obtained from CB (▪) and PB (■) CD34+ cells in the p...
Late release of PF4 and βTG in the supernatant of megakaryocytic cell cultured from CB-CD34+ cells.The concentration of PF4 and βTG was measured by immunoenzymatic assay in the supernatants of MK cultures obtained from CB (▪) and PB (■) CD34+ cells in the presence of TPO (100 ng/mL). The supernatant volume corresponding to 10 000 cells was used. Protein levels were expressed as international units per milliliter at the indicated days of culture. Histograms were the mean ± SD of 3 different experiments. Asterisk indicates a significant difference established, using the Student t test (P < .05).
Gianfranco Mattia et al. Blood 2002;99:
©2002 by American Society of Hematology

11. The expression patterns of cyclin D3 and cyclin B1 differ in PB- and CB-derived MKs.Immunofluorescence analysis by confocal laser microscopy revealed up-regulation of cyclin D3 in PB-derived MKs (green, right panels) from day 5 to the end of the culture.
The expression patterns of cyclin D3 and cyclin B1 differ in PB- and CB-derived MKs.Immunofluorescence analysis by confocal laser microscopy revealed up-regulation of cyclin D3 in PB-derived MKs (green, right panels) from day 5 to the end of the culture. In CB-derived culture (green, left panels) cyclin D3 was evident at day 9 in only 5% of the cells and reaching not more than 50% at day 12. Cyclin B1 immunofluorescence expression at nuclear level decreased at late endomitotic stage (from day 9) in PB culture (red, right panels), whereas its expression was constant in whole CB cultures (red, left panels). A total of 300 cells per slide were enumerated. These expression patterns were confirmed in 2 other experiments. (Bar = 10 μm.)‏
Gianfranco Mattia et al. Blood 2002;99:
©2002 by American Society of Hematology

12. Cyclin B1 expression is decreased in high polyploid MKs in metaphase stage.(A) Basic pattern of cell-cycle stages observed in megakaryocytic cells stained with anti–phospho-histone H1 + H3 antibodies; the different cell-cycle stages (G1, S, G2, metaphase, a...
Cyclin B1 expression is decreased in high polyploid MKs in metaphase stage.(A) Basic pattern of cell-cycle stages observed in megakaryocytic cells stained with anti–phospho-histone H1 + H3 antibodies; the different cell-cycle stages (G1, S, G2, metaphase, and anaphase) are indicated. (B) Double-immunofluorescence analysis by confocal laser microscopy of MKs at the metaphase stage, displaying different ploidy level, was performed by anti-H1 + H3 antibodies (in green, left panels) and anti–cyclin B1 MoAb (in red, middle panels). The yellow area (merge, right panels) indicates colocalization of histones and cyclin B1. A total of 100 MKs in the metaphase stage at different ploidy levels were analyzed. (Bar = 10 μm.)‏
Gianfranco Mattia et al. Blood 2002;99:
©2002 by American Society of Hematology