3. Put the T12.5 flasks into the biaxial drive rotator and place

them back into CO2 incubator for further 3 days of cell

cultivation.

4. Change the medium with a half-volume fresh hematopoietic

progenitor cells medium every other day.

5. After 3 days of culture in hematopoietic progenitor cells

medium, many grape-like clusters are forming (Fig. 4a) and

many round cells are floating in medium (Fig. 4b). The differ-

entiated hematopoietic progenitor cells are characterized by

surface marker CD34 and CD43 (Fig. 4c, d).

3.5

Characterization

of Differentiated Cells

Derived from hESCs in

Bioreactor

3.5.1

Immunofluorescence

Staining

1. At the culture of day 2, 5, and 8, the differentiated cells in flasks

under different culture conditions are collected and prepared

for immunostaining.

2. Remove the medium of the flasks and add PBS to the cells for

washing 1–2 times.

3. Aspirate the PBS and then fix with 4% paraformaldehyde solu-

tion at room temperature for 20–30 min.

4. Wash the cells one time with PBS and the bottom of the flasks is

divided into small pieces (1 cm  1 cm size) by sharp blades.

5. Put each small piece containing cells into the 24-well cell

culture dish and add PBS to the well.

6. Permeabilize the cells for 20 min with 0.2% Triton X-100 and

1% donkey serum at room temperature (see Note 7).

7. Wash the cells two times with PBS and add the 5% donkey

serum blocking solution into the well for 1 h blocking at 37
C.

8. Prepare the primary antibody in 5% donkey serum at dilutions

recommended by the manufacturer.

Fig. 3 Hemogenic endothelium induction from human embryonic stem cells (hESCs) in RPM. (a) Representa-

tive morphology of differentiated cells after further 3 days culture. Several clusters of grape-like appear at day

5 (Arrows). Scale bars, 50 μm. (b) Representative immunostaining images of day 5 cells for CD34 and CD31.

Scale bars, 50 μm. (c) Representative flow cytometry results of surface markers CD31and CD34 at day 5

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Xiaohua Lei et al.