adjusted by altering the number of times the cell aggregate

mixture is pipetted up and down. Take care to maintain the

cells as aggregates.

4. Seeding the cell aggregates at an optimal density to the flask

(about 100 aggregates/cm²). Do not seed too dense or too

sparse.

5. The flask should be filled with a cell culture medium and make

sure the bubble is pulled out in the RPM group.

6. The power of the electronic control system should be turn off

when prepare to change the cell medium.

7. This step can be omitted when we stain the cells with CD31,

CD34 or CD43 antibodies.

Acknowledgments

This work has been funded by the Chinese Manned Space Station

Application Project (YYWT-0901-EXP-15, to XL), CAS Key Tech-

nology Talent Program (to XL), and the NSFC Grant (31471287,

to XL).

References

1. Kaufman

DS,

Hanson

ET,

Lewis

RL,

Auerbach R, Thomson JA (2001) Hemato-

poietic

colony-forming

cells

derived

from

human embryonic stem cells. Proc Natl Acad

Sci U S A 98(19):10716–10721

2. Chadwick K, Wang L, Li L, Menendez P,

Murdoch B, Rouleau A, Bhatia M (2003)

Cytokines and BMP-4 promote hematopoietic

differentiation of human embryonic stem cells.

Blood 102(3):906–915

3. Vodyanik MA, Bork JA, Thomson JA, Slukvin

II (2005) Human embryonic stem cell-derived

CD34+ cells: efficient production in the cocul-

ture with OP9 stromal cells and analysis of

lymphohematopoietic potential. Blood 105

(2):617–626

4. Vo LT, Daley GQ (2015) De novo generation

of HSCs from somatic and pluripotent stem

cell sources. Blood 125(17):2641–2648

5. Choi KD, Vodyanik M, Slukvin II (2011)

Hematopoietic differentiation and production

of mature myeloid cells from human pluripo-

tent stem cells. Nat Protoc 6(3):296–313

6. Wang H, Liu C, Liu X, Wang M, Wu D, Gao J,

Su P, Nakahata T, Zhou W, Xu Y et al (2018)

MEIS1 regulates Hemogenic endothelial gen-

eration, Megakaryopoiesis, and Thrombopoi-

esis

in

human

pluripotent

stem

cells

by

targeting TAL1 and FLI1. Stem Cell Rep 10

(2):447–460

7. Huang X,

Gschweng

E,

Van

Handel

B,

Cheng D, Mikkola HK, Witte ON (2011)

Regulated

expression

of

microRNAs-126/

126*

inhibits

erythropoiesis

from

human

embryonic

stem

cells.

Blood

117

(7):2157–2165

8. Wang CY, Tang XM, Sun XM, Miao ZC, Lv

YX, Yang YL, Zhang HD, Zhang PB, Liu Y,

Du LY et al (2012) TGF beta inhibition

enhances the generation of hematopoietic pro-

genitors from human ES cell-derived hemo-

genic

endothelial

cells

using

a

stepwise

strategy. Cell Res 22(1):194–207

9. Kennedy

M,

Awong

G,

Sturgeon

CM,

Ditadi A, LaMotte-Mohs R, Zuniga-Pflucker

JC, Keller G (2012) T lymphocyte potential

marks the emergence of definitive hematopoie-

tic progenitors in human pluripotent stem cell

differentiation

cultures.

Cell

Rep

2

(6):1722–1735

10. Woll PS, Morris JK, Painschab MS, Marcus RK,

Kohn AD, Biechele TL, Moon RT, Kaufman

DS (2008) Wnt signaling promotes hematoen-

dothelial cell development from human embry-

onic stem cells. Blood 111(1):122–131

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

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