chemical factors might further promote hematopoiesis [21]. In the

following section, we describe a protocol for using the random

positioning machine (RPM) bioreactor to culture hPSCs and the

large-scale production of HSPCs in a chemically defined system. We

identify the characteristics of the cells and analyze the efficiency of

hematopoietic differentiation in the RPM bioreactor by immuno-

histochemistry and flow cytometry.

2

Materials

2.1

Cell Lines

Human ESC line H1 is obtained from Wicell Research Institute,

Inc., Madison, WI.

2.2

Cell Culture

Medium and Reagents

1. Medium

for

undifferentiated

human

ESCs

expansion:

TeSR™-E8™medium (Cat# 05990, STEMCELL Technolo-

gies), basal medium store at 4
C, 25 supplement store at

20
C.

2. Matrigel for undifferentiated human ESCs expansion: Matrigel

Matrix HESC-qualified (BioCoat, 354277); Matrigel for

human ESCs differentiation: Corning Matrigel Growth Factor

Reduced

Basement

Membrane

Matrix

(Corning,

Cat#354230). The final dilution of Matrigel should be 1:50.

3. IF9S basal medium for human ESC differentiation: The for-

mulation of IF9S is list in Table 1 according to previously

described [22]. Store at 4
C.

4. Mesoderm induction medium: IF9S basal medium with

50 ng/mL BMP4 (PeproTech), 15 ng/mL ACTIVIN A

(PeproTech) and 1.5 μM CHIR99021 (STEMCELL™Tech-

nologies) . Stable for 1 week at 4
C.

5. Hemogenic

endothelium

progenitor

cell

differentiation

medium: IF9S basal medium with 10 μM SB431542 (R&D

Systems), 50 ng/mL VEGF (R&D Systems), 50 ng/mL bFGF

(PeproTech), and 50 ng/mL SCF (R&D Systems). Stable for

1 week at 4
C.

6. Hematopoietic progenitor cells medium: IF9S basal medium

with 50 ng/mL VEGF (R&D Systems), 50 ng/mL bFGF

(PeproTech), 50 ng/mL SCF (R&D Systems), 10 ng/mL

IL-3 (R&D Systems), 50 ng/mL IL-6 (R&D Systems), and

20 ng/mL TPO (Sino Biological). Stable for 1 week at 4
C.

2.3

Bioreactor

Equipment

The random positioning machine (SM-31J) for cell culture is

designed by the National Center of Space Science, Chinese Acad-

emy of Sciences, which is a microgravity simulated device. The

device included two parts: (1) biaxial drive rotator, which can

support 3D rotation of cell culture vessel (Fig. 1a), and (2) the

electronic control system, aims to supply power and to adjust the

rotation speed of rotator (Fig. 1b).

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

57