2.4

Immunostaining

and Flow Cytometry

1. Blocking solution: 5% bovine serum albumin (BSA, Cat#

A1933, Sigma) in PBS or 5% donkey serum in PBS. Store at

20
C.

2. Primary antibody: PE Mouse Anti-Human CD31 (Cat#

560983, BD), FITC Mouse Anti-Human CD34 (Cat#

560942,

BD),

APC

Mouse

Anti-Human

CD43

(Cat#

560198, BD), and Goat Anti-Human Brachyury Polyclonal

Antibody (Cat# AF2085, R&D systems).

3. Secondary antibodies: Alexa Fluor^® 568 Donkey Anti-Goat

secondary antibody (Cat# A-11057, ThermoFisher Scientific).

2.5

Other Material

T12.5 cell culture Flasks (TCF-012-025, JET BIOFIL), 4% PFA

(Cat# P-6148, Sigma) in 100 mL PBS, 5% Normal Donkey Serum

(Cat#ab7475, abcam) in PBS, 0.1–1 μg/mL of Hoechst 33342

(Cat# B2261, Sigma) in PBS, Nikon Ti microscope (Nikon, Japan).

3

Methods

3.1

Feeder-Free

Expansion of Human

PSCs

Before differentiation of human ESCs, undifferentiated feeder-

dependent human ESCs are cultured in feeder-free and chemically

defined conditions with the use of TeSR™-E8™medium (STEM-

CELL Technologies) on Matrigel Matrix HESC-qualified (Bio-

Coat) coated plates as the protocols described in the manual. We

can skip this step if human ESCs are maintained in TeSR™-E8™

medium. The following instructions are used in our experiments.

1. Before human ESC thawing or passing, coat a new 6-well plate

with Matrigel Matrix solution (see Subheading 2.2), and pre-

incubate at room temperature (25
C) for at least 1 h before use

(see Note 1).

2. Remove the Matrigel Matrix solution using a pipette or by

aspiration.

3. Add 2 mL TeSR™-E8™medium to 6-well plate.

4. Put the small cell aggregate mixture (500–1000 aggregate/

wells) onto the 6-well plate containing TeSR™-E8™and

observe the state of cell aggregate under the microscope to

ensure the desired cell density.

5. Place the 6-well plate in a 37
C CO2 incubator. Move the plate

in a back-and-forth and side-to-side motions to evenly distrib-

ute the small cell aggregates.

6. Change medium daily with TeSR™-E8™and monitor cell

growth daily until the cell next passaging or cell differentiation.

Hematopoietic Stem/Progenitor Cell Differentiation in Random Positioning. . .

59