American Society of Hematology

Figure 3.

$Figure 3. HSC culture with V+ P− stromal cells supports the expansion of CD41+ CD42+ polyploid MKs that efficiently produce proplatelets. (A) Fetal liver stromal cells were sorted confluent adherent layers developed over 2 weeks. HSCs (50 SLAM cells) were cocultured with sorted stromal cells for 7 to 13 days and assayed for MK production. At days 9 to 13, a fraction of the cells were subcultured in proplatelet medium. (B) Total number of cells produced from 50 SLAM cells in the absence of stroma or with the different stroma. Values represent mean ± SD from 5 independent experiments. *P < .01 vs the nonstroma group for the same day; # P < .05 vs stroma V+P− for the same day. Other comparisons were not statistically significant. (C) Total number of MKs, identified as cells expressing CD41 and CD42c, produced from 50 SLAM cells. Values represent mean ± SD from 5 independent experiments *P < .01 vs the nonstroma group for the same day; #P < .001 vs stroma V+P− for the same day. Other comparisons were not statistically significant. (D) Representative histogram of the DNA content analysis of MKs (CD41+CD42c+) top) and all cells (bottom) produced during a 13-day coculture on V+P− cells. (E) Representative phase contrast image of cells first cultured on V+P− cells and replated for 3 to 4 days in proplatelet medium. White arrows indicate MKs, and black arrows indicate proplatelet extension. Scale bar represents 100 μm. (F) Immunostaining confirmed the presence of tubulin β1 in the proplatelet extension. Scale bar represents 25 μm. (G) Cells from day 11 coculture were harvested and plated in proplatelet medium using a limiting dilution scheme. The frequency of cells produced from day 11 cocultures capable of producing proplatelet-bearing MKs is presented. Values represent mean ± SD from 4 independent experiments *P < .01 vs the nonstroma group for the same day; #P < .01 vs stroma V+P− for the same day. Other comparisons were not statistically significant. CK, cytokine; D, day.$

HSC culture with V⁺P⁻ stromal cells supports the expansion of CD41⁺CD42⁺polyploid MKs that efficiently produce proplatelets. (A) Fetal liver stromal cells were sorted confluent adherent layers developed over 2 weeks. HSCs (50 SLAM cells) were cocultured with sorted stromal cells for 7 to 13 days and assayed for MK production. At days 9 to 13, a fraction of the cells were subcultured in proplatelet medium. (B) Total number of cells produced from 50 SLAM cells in the absence of stroma or with the different stroma. Values represent mean ± SD from 5 independent experiments. *P < .01 vs the nonstroma group for the same day; # P < .05 vs stroma V⁺P⁻ for the same day. Other comparisons were not statistically significant. (C) Total number of MKs, identified as cells expressing CD41 and CD42c, produced from 50 SLAM cells. Values represent mean ± SD from 5 independent experiments *P < .01 vs the nonstroma group for the same day; #P < .001 vs stroma V⁺P⁻ for the same day. Other comparisons were not statistically significant. (D) Representative histogram of the DNA content analysis of MKs (CD41⁺CD42c⁺) top) and all cells (bottom) produced during a 13-day coculture on V⁺P⁻ cells. (E) Representative phase contrast image of cells first cultured on V⁺P⁻ cells and replated for 3 to 4 days in proplatelet medium. White arrows indicate MKs, and black arrows indicate proplatelet extension. Scale bar represents 100 μm. (F) Immunostaining confirmed the presence of tubulin β1 in the proplatelet extension. Scale bar represents 25 μm. (G) Cells from day 11 coculture were harvested and plated in proplatelet medium using a limiting dilution scheme. The frequency of cells produced from day 11 cocultures capable of producing proplatelet-bearing MKs is presented. Values represent mean ± SD from 4 independent experiments *P < .01 vs the nonstroma group for the same day; #P < .01 vs stroma V⁺P⁻ for the same day. Other comparisons were not statistically significant. CK, cytokine; D, day.

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