American Society of Hematology

Figure 4

$Figure 4. Differential expression of CD41 and GPA permits subfractionation of a compartment of primitive embryonic erythro-megakaryocytic progenitors. (A) Sorting strategy used to fractionate the erythro-MK progenitors. H1 hES cells were analyzed by flow cytometry after 12 days of differentiation. Cells within the CD41+ cell population were examined for GPA coexpression and subdivided into CD41+GPA+ (R1), CD41−GPA+ (R2), CD41+GPA− (R3), and CD41−GPA− (R4) cell fractions. (B) Colony-forming potential of the different GPA/CD41 cell populations derived from H1 hES cells. Cells sorted at day 9, 12, and 14 were seeded into fibrin clot culture and tested for their colony-forming potential. Cultures were allowed to grow for 7 to 10 days, fixed, and stained with an anti-CD42 antibody. Colonies were scored under an inverted microscope for Ery-P (erythroid colony), MK (megakaryocytic colony), mixed E-MK (bipotent erythroid/MK), CFU-M (macrophage colony), and CFU-GM (granulo-macrophagic colony). Data represent the mean value ± SD from 3 experiments. Although mixed E-MK colonies were present in all tested subpopulations, multivariate analysis of variance showed that the highest frequency was found within the GPA+CD41+ fraction (P < .001). (C) Analysis of the cell composition of indivdual clones derived from H9 GPA+CD41+ cells. GPA+CD41+ cells were sorted after 12 days from hES /OP9 cocultures and seeded on MS5 feeder layer at 1 cell per well, in medium containing hES-tested FBS and a combination of SCF, EPO, TPO, and IL-3. The results presented are those obtained from 75 clones analyzed at day 7 and day 12 of culture after labeling with directly conjugated anti-GPA and anti-CD41 monoclonal antibodies. (D) Semisolid assays. (Top panel) Immunostaining of fibrin clot colonies grown in fibrin clots in the presence of a cocktail of cytokines. The primitive erythroid colony (Ery-P), mixed erythro-megakaryocytic colony (MEP), and pure megakaryocytic colony (CFU-MK) were stained with an anti-CD41 Ab (pink). (Bottom panel) Methylcellulose cultures showing typical primitive erythroid colonies (Ery-P) and mixed E-MK colonies containing erythroblasts and megakaryocytes (MEP). Colonies with typical morphologic features of BFU-E obtained from the double-negative population are clearly different from Ery-P–derived colonies. Colonies were examined under a Zeiss laser scanning microscope (LSM 510; Carl Zeiss) equipped with a 63×/1.4 numerical aperture (NA) oil objective (original magnifications 20× and 10×; MEP in fibrin clot and BFU-E).$

Differential expression of CD41 and GPA permits subfractionation of a compartment of primitive embryonic erythro-megakaryocytic progenitors. (A) Sorting strategy used to fractionate the erythro-MK progenitors. H1 hES cells were analyzed by flow cytometry after 12 days of differentiation. Cells within the CD41⁺ cell population were examined for GPA coexpression and subdivided into CD41⁺GPA⁺ (R1), CD41⁻GPA⁺ (R2), CD41⁺GPA⁻ (R3), and CD41⁻GPA⁻ (R4) cell fractions. (B) Colony-forming potential of the different GPA/CD41 cell populations derived from H1 hES cells. Cells sorted at day 9, 12, and 14 were seeded into fibrin clot culture and tested for their colony-forming potential. Cultures were allowed to grow for 7 to 10 days, fixed, and stained with an anti-CD42 antibody. Colonies were scored under an inverted microscope for Ery-P (erythroid colony), MK (megakaryocytic colony), mixed E-MK (bipotent erythroid/MK), CFU-M (macrophage colony), and CFU-GM (granulo-macrophagic colony). Data represent the mean value ± SD from 3 experiments. Although mixed E-MK colonies were present in all tested subpopulations, multivariate analysis of variance showed that the highest frequency was found within the GPA⁺CD41⁺ fraction (P < .001). (C) Analysis of the cell composition of indivdual clones derived from H9 GPA⁺CD41⁺ cells. GPA⁺CD41⁺ cells were sorted after 12 days from hES /OP9 cocultures and seeded on MS5 feeder layer at 1 cell per well, in medium containing hES-tested FBS and a combination of SCF, EPO, TPO, and IL-3. The results presented are those obtained from 75 clones analyzed at day 7 and day 12 of culture after labeling with directly conjugated anti-GPA and anti-CD41 monoclonal antibodies. (D) Semisolid assays. (Top panel) Immunostaining of fibrin clot colonies grown in fibrin clots in the presence of a cocktail of cytokines. The primitive erythroid colony (Ery-P), mixed erythro-megakaryocytic colony (MEP), and pure megakaryocytic colony (CFU-MK) were stained with an anti-CD41 Ab (pink). (Bottom panel) Methylcellulose cultures showing typical primitive erythroid colonies (Ery-P) and mixed E-MK colonies containing erythroblasts and megakaryocytes (MEP). Colonies with typical morphologic features of BFU-E obtained from the double-negative population are clearly different from Ery-P–derived colonies. Colonies were examined under a Zeiss laser scanning microscope (LSM 510; Carl Zeiss) equipped with a 63×/1.4 numerical aperture (NA) oil objective (original magnifications 20× and 10×; MEP in fibrin clot and BFU-E).

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