T12.5 flasks and the biaxial drive rotator are placed into CO2
incubator for cell cultivation (Fig. 1c).
1. Aspirate the mesoderm induction medium and add 30 mL
hemogenic
endothelium
progenitor
cell
differentiation
medium into the T12.5 flasks of RPM group and add 5 mL
medium into the T12.5 flasks of control group (see Note 5).
2. Put the T12.5 flasks of the RPM group into the biaxial drive
rotator.
3. Place the biaxial drive rotator with T12.5 flasks and the T12.5
flasks of the control group into the CO2 incubator at 37 C
with 5% CO2 and 95% humidity for cell inoculation and
cultivation.
4. Turn on the power of the electronic control system and set it in
random models of speed (0.1–10 rpm).
5. Change the medium with a half-volume fresh hemogenic endo-
thelium progenitor cell differentiation medium every other day
(see Note 6).
6. After 3 days of culture in hemogenic endothelium progenitor
cell differentiation medium, the cell with endothelial morphol-
ogy and tube-like structures emerged in the RPM group
(Fig. 3a). Some differentiated cells contained round cells with
a “grape-like” clusters (Fig. 3a, arrow). The differentiated cells
are characterized by surface marker CD31 and CD34.
3.4
Generation of
Hematopoietic
Progenitor Cells
For the further induction of hematopoietic progenitor cells, the
cells are growth in hematopoietic progenitor cells medium for
another 3–5 days.
1. Turn off the power the electronic control system and carefully
remove the T12.5 flasks from the biaxial drive rotator.
2. Aspirate the hemogenic endothelium progenitor cell differen-
tiation medium and add about 30 mL hematopoietic progeni-
tor cells medium into the T12.5 flasks of RPM group.
Fig. 2 Mesoderm induction from human embryonic stem cells (hESCs). (a) Representative morphology of
differentiated cells after 2 days culture. Scale bars, 100 μm. (b) Representative immunostaining images of day
2 cells for brachyury. Scale bars, 50 μm
Hematopoietic Stem/Progenitor Cell Differentiation in Random Positioning. . .
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