3.2

Mesoderm

Induction from hPSCs

1. Before mesoderm induction, coat each T12.5 flasks with

1.5 mL diluted corning Matrigel Growth Factor Reduced

Basement Membrane Matrix (see Subheading 2.2) at room

temperature for 30 min and then incubated the flasks in

37
C for another 30 min.

2. At the time of cell passaging in Subheading 3.1, digest the

hPSCs colonies into small cell aggregates by ReLeSRTM

(STEMCELL Technologies) at 37
C for about 3 min. Accord-

ing to the manual described (see Note 2).

3. Stop digestion with TeSR™-E8™, pat the cell dish gently to

make sure the small cell aggregates fall off from the bottom of

the dish.

4. Harvest the cell aggregates to 15 mL centrifuge tube and the

cell aggregate suspension is pipetted up and down gently about

3–5 times to adjust the size of aggregates to 100–200 cells/

aggregate (see Note 3).

5. Centrifuge the harvested aggregates at 500 rpm for 5 min at

room temperature, remove the supernatant of 15 mL centri-

fuge tube and resuspend the small cell aggregates in 2 mL of

TeSR™-E8™.

6. Remove the liquid of the Corning coated 12.5 flasks and add

2 mL TeSR™-E8™into the flask.

7. Transfer the cell aggregates onto Corning Matrigel-coated

T12.5 flask at a density of about 100 aggregates/cm² in

TeSR™-E8™(see Note 4).

8. Place the flasks in CO2 incubator (37
C, 5% CO2 and 95%

humidity) and shake the flasks in several quick sides to side,

forward

to

back

motions

to

uniformly

distribute

the

aggregates.

9. Check the growth status of the cell colonies on the second day,

if the size of colonies exceeds 300 μm that should be ready to

be replaced into differentiation medium.

10. Aspirate medium from the T12.5 flask and add 5 mL of meso-

derm induction medium (see Subheading 2.2), place the flasks

in an incubator for culturing 2 days.

11. After 2 days of culture in mesoderm induction medium, we

have found that almost all of the colonies exhibit loose colony

appearance and elongated cell morphology (Fig. 2a) and give

rise to differentiated Brachyury+ cells (Fig. 2b).

3.3

Generation of

Hemogenic

Endothelium

Progenitor

After 2 days of mesoderm induction, the cells are further cultured

by changing with hemogenic endothelium progenitor cell differen-

tiation medium (see Subheading 2.2). Some of the T12.5 flasks with

mesoderm-induced cells are put into the biaxial drive rotator. The

60

Xiaohua Lei et al.