Journal: The Journal of Experimental Medicine

Article Title: Metalloproteinase regulation improves in vitro generation of efficacious platelets from mouse embryonic stem cells

doi: 10.1084/jem.20071482

Figure Lengend Snippet: c-Kit + αIIb + cell selection from day 6 EB determines the megakaryocytic lineage. (A) Protocol used in this study. (B) EB formation or OP9 co-culture was used for ESC differentiation from day 0 through day 6 in vitro. On the indicated days of culture, expression of Flk-1, integrin αIIb subunit (CD41), or CD31 was examined in both groups of cultured cells. The graph shows mean ± SEM from three independent experiments. (C) GPIbα and GFP expression in GPIbα-ESCs is regulated by the human GPIbα promoter. GFP is detectable in cultured cells that exhibit megakaryopoiesis (day 9) but not in progenitors (day 6). In the bottom box, cells with surface-expressed GFP also express human GPIbα. The cells on day 9 were fixed and stained to mark human GPIbα to confirm the colocalization with GFP expression at the membrane of the cells. Bars, 10 μm. (D) Representative dot plots for the expression of individual antigens on 10,000 living cells from day 6 EB. c-Kit + αIIb + cells corresponded with Flk-1 + cells (not depicted) but not with VE-cadherin + cells. And >96% of c-Kit + αIIb + cells are found in the CD9 + Sca-1 − fraction. (E) EB cells were sorted as shown in D, and 20,000 cells per well of a 6-well plate were seeded onto OP9 stroma cells in the presence of TPO. The left graph shows the number of generated GFP + cells (mean ± SEM) per well of a 6-well plate on day 12 of culture. Representative images from day 12 culture dishes (phase or fluorescence microscopy [insets: bar, 50 μm] of four different fraction-derived cells at day 6; i, c-Kit + /αIIb + ; ii, c-Kit − /αIIb + ; iii, c-Kit + /αIIb − ; iv, c-Kit − /αIIb − [phase: bar, 50 μm]) and of Wright-Giemsa–stained cytospin preparation on day 12 derived from c-Kit + /αIIb + fraction of day 6 were also shown. Bar, 50 μm. Results indicate that c-Kit + /αIIb + fraction of day 6 (i) selectively yields megakaryocytes on day 12. Similar results have been obtained from three independent experiments. (F) Expression of genes related to the megakaryocytic lineage as determined by RT-PCR of cells from day 0, 6, or 8 cultures as indicated. (G) Wright-Giemsa–stained cytospin preparation of sorted c-Kit + αIIb + cells from EB at day 6 is shown. Bar, 10 μm.

Article Snippet: Purified human fibrinogen was from American Diagnostica Inc. FITC- and allophycocyanin (APC)-conjugated, PE-conjugated, and unconjugated anti–mouse integrin αIIb subunit and APC-conjugated anti–c-Kit, PE-conjugated anti-CD31, PE-conjugated anti–Sca-1, biotin-conjugated anti-CD9, FITC-conjugated anti–P-selectin, unconjugated human anti-GPIbα, and streptoavidin-APC–cyanine 7 (APC-Cy7) antibodies were from BD Biosciences.

Techniques: Selection, Co-Culture Assay, In Vitro, Expressing, Cell Culture, Staining, Generated, Fluorescence, Microscopy, Derivative Assay, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Experimental Medicine

Article Title: Metalloproteinase regulation improves in vitro generation of efficacious platelets from mouse embryonic stem cells

doi: 10.1084/jem.20071482

Figure Lengend Snippet: GPIbα expression is reduced on cultured ESPs but not megakaryocytes. (A) On day 12 of the culture, ESC-derived megakaryocytes bearing proplatelets are represented in panel (i) (phase contrast image in culture dish). Bar, 100 μm. Panels (ii) and (iii) show TEM images of ESPs. The subcellular structure of ESPs showed an open canalicular system, dense granules (arrowhead), and α granules (arrow), which were similar to those of peripheral blood platelets. Bars, 1 μm. (B) Mouse platelets (12 wk old) or ESPs (day 12) were subjected to flow cytometry. Graphs show representative forward scatter (FSC) or side scatter (SSC) dot plots. ESPs vary in size compared with mouse platelets. The remaining six graphs show surface expression of αIIb integrin subunit, GPIbα, GPIbβ, GPV, GPVI, and GPIX on mouse platelets (red lines) or ESPs (blue lines) with control IgG (black lines). (C) (i) On the indicated days of culture (x axis), expression of GPIbα and GPIbβ in αIIb + megakaryocytes derived from ESCs and ESPs is shown. Panel (ii) shows the number of αIIb + ESPs on days 8 and 12 that are either GPIbα + (black bar) or GPIbα − (white bar). All results are mean ± SEM from four independent experiments.

Article Snippet: Purified human fibrinogen was from American Diagnostica Inc. FITC- and allophycocyanin (APC)-conjugated, PE-conjugated, and unconjugated anti–mouse integrin αIIb subunit and APC-conjugated anti–c-Kit, PE-conjugated anti-CD31, PE-conjugated anti–Sca-1, biotin-conjugated anti-CD9, FITC-conjugated anti–P-selectin, unconjugated human anti-GPIbα, and streptoavidin-APC–cyanine 7 (APC-Cy7) antibodies were from BD Biosciences.

Techniques: Expressing, Cell Culture, Derivative Assay, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Metalloproteinase regulation improves in vitro generation of efficacious platelets from mouse embryonic stem cells

doi: 10.1084/jem.20071482

Figure Lengend Snippet: Inhibition of metalloproteinases is required for platelet function mediated through integrin αIIbβ3 in ESPs. (A) (i) Representative flow cytometry dot plots showing three mM MnCl 2 -stimulated or 0.1 U/ml thrombin-stimulated fibrinogen binding to integrin αIIbβ3 in ESPs after pretreatment with 1% DMSO as a vehicle or 100 μM GM6001. (ii) The graphs show specific fibrinogen binding to αIIbβ3 stimulated with 3 mM MnCl 2 (reference ), 0.1 U/ml thrombin, or 500 μM adenosine diphosphate. The value of control (vehicle) is defined as 100%. Results are the mean ± SEM from three independent experiments. (B) On day 14 of culture, washed ESPs pretreated with 1% DMSO or 100 μM GM6001 were plated on fibrinogen-coated coverslips for 45 min. An aliquot of each preparation was assayed in the presence of 1 U/ml thrombin. Cells were fixed, permeabilized, and stained with Alexa 488–phalloidin to stain F-actin (green) and with an anti-αIIb antibody followed by Alexa 567 (red). Bar, 10 μm. The value of control (vehicle) is defined as 100%. The right graph summarizes three independent experiments (mean ± SEM).

Article Snippet: Purified human fibrinogen was from American Diagnostica Inc. FITC- and allophycocyanin (APC)-conjugated, PE-conjugated, and unconjugated anti–mouse integrin αIIb subunit and APC-conjugated anti–c-Kit, PE-conjugated anti-CD31, PE-conjugated anti–Sca-1, biotin-conjugated anti-CD9, FITC-conjugated anti–P-selectin, unconjugated human anti-GPIbα, and streptoavidin-APC–cyanine 7 (APC-Cy7) antibodies were from BD Biosciences.

Techniques: Inhibition, Flow Cytometry, Binding Assay, Staining