Figure 2.
Figure 2. Hematopoietic differentiation from recultured hEB cells initiates with expansion of adherent MHE colonies. Single-cell suspensions of hEB cells differentiated for 3 to 25 days were plated in modified serum-free methylcellulose medium containing hematopoietic growth factors as described in “Materials and methods.” (A) Within 1 to 3 weeks following day 7 to 12 hEB cell suspension CFC assay plating, a population of semiadherent MHE colonies expanded either from plastic-adherent hEB clonogenic progenitors, or rarely from attached secondary hEBs. Plastic-adherent MHE colonies (i; magnification × 200) differentiate by lateral endothelioid expansion accompanied by budding, hemoglobinizing hematopoietic blasts (arrows) that are intimately intermixed with adherent cells (ii; magnification × 40). MHE clusters can become quite prolific after 3 to 5 weeks (iii; magnification × 200) if refreshed with fresh medium with growth factors every 1 to 2 weeks. Wright stains (iv; magnification × 600, oil) of budding, nonadherent cells from prolific MHE clusters revealed abundant hematopoietic blasts (Bl), primitive nucleated erythrocytes (EryP), foamy primitive macrophages (Macro-P), and rare definitive cells including granulocytes and definitive erythroid cells (not shown). (B) Kinetics of MHE colony appearance from differentiating hEBs (mean and standard deviations of 3 independent experiments). Loosely adherent cells from prolific HE clusters were picked and analyzed by FACS (C) and found to express abundant levels of early erythroid (CD71), myeloid (CD13), and panhematopoietic (CD45) markers. Mature (3- to 5-week-old) MHE colonies were extensively washed of budding, nonadherent hematopoietic cells, and remaining plastic-adherent cells were incubated with 10 μg/mL acetylated Dil-LDL overnight and then fixed, permeabilized, and further evaluated for expression of endothelial-specific and mesodermal markers. Approximately 15% to 20% of elongated, plastic-adherent cells that formed the base of MHE colonies were shown by in situ immunofluorescence (D) or FACS analysis (as single cells) (E) to be capable of simultaneously taking up acetylated Dil-LDL and costaining with either Ulex europaeus agglutinin-1–FITC (D; UEA1, magnification × 100) (E) or VE-cadherin plus FITC-conjugated secondary antibody (D; magnification × 200). The remainder of the adherent cells that surrounded intermixed, robust Dil-LDL–positive endothelial clusters stained brightly for intracellular vimentin, thus identifying them as mesodermal-mesenchymal (nonendothelial) in lineage (D, right 4 panels; magnification × 100, × 200, respectively). Shown are merged images of phase and red and/or green immunofluorescent filters.

Hematopoietic differentiation from recultured hEB cells initiates with expansion of adherent MHE colonies. Single-cell suspensions of hEB cells differentiated for 3 to 25 days were plated in modified serum-free methylcellulose medium containing hematopoietic growth factors as described in “Materials and methods.” (A) Within 1 to 3 weeks following day 7 to 12 hEB cell suspension CFC assay plating, a population of semiadherent MHE colonies expanded either from plastic-adherent hEB clonogenic progenitors, or rarely from attached secondary hEBs. Plastic-adherent MHE colonies (i; magnification × 200) differentiate by lateral endothelioid expansion accompanied by budding, hemoglobinizing hematopoietic blasts (arrows) that are intimately intermixed with adherent cells (ii; magnification × 40). MHE clusters can become quite prolific after 3 to 5 weeks (iii; magnification × 200) if refreshed with fresh medium with growth factors every 1 to 2 weeks. Wright stains (iv; magnification × 600, oil) of budding, nonadherent cells from prolific MHE clusters revealed abundant hematopoietic blasts (Bl), primitive nucleated erythrocytes (EryP), foamy primitive macrophages (Macro-P), and rare definitive cells including granulocytes and definitive erythroid cells (not shown). (B) Kinetics of MHE colony appearance from differentiating hEBs (mean and standard deviations of 3 independent experiments). Loosely adherent cells from prolific HE clusters were picked and analyzed by FACS (C) and found to express abundant levels of early erythroid (CD71), myeloid (CD13), and panhematopoietic (CD45) markers. Mature (3- to 5-week-old) MHE colonies were extensively washed of budding, nonadherent hematopoietic cells, and remaining plastic-adherent cells were incubated with 10 μg/mL acetylated Dil-LDL overnight and then fixed, permeabilized, and further evaluated for expression of endothelial-specific and mesodermal markers. Approximately 15% to 20% of elongated, plastic-adherent cells that formed the base of MHE colonies were shown by in situ immunofluorescence (D) or FACS analysis (as single cells) (E) to be capable of simultaneously taking up acetylated Dil-LDL and costaining with either Ulex europaeus agglutinin-1–FITC (D; UEA1, magnification × 100) (E) or VE-cadherin plus FITC-conjugated secondary antibody (D; magnification × 200). The remainder of the adherent cells that surrounded intermixed, robust Dil-LDL–positive endothelial clusters stained brightly for intracellular vimentin, thus identifying them as mesodermal-mesenchymal (nonendothelial) in lineage (D, right 4 panels; magnification × 100, × 200, respectively). Shown are merged images of phase and red and/or green immunofluorescent filters.

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