HuStem media are highly qualified feeder-free and xeno pathogen-free media which can be used to culture

human pluripotent stem cells [human embryonic stem cells (hESC) and human induced pluripotent stem cells

(hiPSC)] and to generate human induced pluripotent stem cell lines.

Dong-Youn Hwang (Professor, CHA University)

Education & Job experience

● Seoul National University, B.S. & M.S.

● University of Pittsburgh, PhD

● Harvard University, Post. Doc.

● Harvard University, an instructor

● Harvard University, an Assistant Professor (tenure track)

Inventor’s information

Patent information

Patent Number

Patent title

Patent Number

Patent title

Patent Number

Patent title

KR10-2011-0033789

Method for generating induced pluripotent stem cells using

reprogramming-enhancing agents

KR10-2011-0038838

Culture media for maintaining stemness of human embryonic stem cells or

induced pluripotent stem cells and culture method using the same

US13/051,660

Stem cell culture media for maintaining stemness or inducing differentiation

of stem cells

New xeno-free & feeder-free media

for the derivation and maintenance of human

pluripotent stem cells

Abstract

1. Xeno-free & feeder-free

- For clinical use of stem cells

- Less variance in performance

- Easy standardization of tissue culture protocols

- Significant reduction in time, effort, and expense

2. Long-term maintenance of hESCs or hiPSCs; H9-hESCs were maintained undifferentiated for more than

50 passages

3. Efficient derivation of xeno-free hiPSCs

This technology can be used for long-term culture and derivation of xeno-free human pluripotent stem cells.

Advantages of this technology

1

2

3

● Hwang D.-Y. “Chapter 1. Feeder-Free Growth of Undifferentiated Human Embryonic Stem Cells” in the book

of Human Embryonic and Induced Pluripotent Stem Cells, Ye, Kaiming, & Jin, Sha (Eds), 2011, 1st Edition, Humana

Press, ISBN 978-1-61779-266-3

● Yoon T.-M., Chang B., Jee J.-H., Kim H.-T., Kim D.-W., and Hwang D.-Y., Human embryonic stem cells (hESCs)

cultured under distinctive feeder-free culture conditions display global gene expression patterns similar to

hESCs from feeder-dependent culture conditions. Stem Cell Review and Reports, 2010 Sep.; 6:425-437

● Hwang K.-C., Kim J. Y., Chang W., Ha H.-Y., Lim S., Kim D.-S., Song B.-W., Kang S.-M., Choi I.-G., Hwang D.-Y., Song

H., Jang Y., Chung N., Kim S.-H., Kim D.-W. Chemicals that modulate stem cell differentiation. P.N.A.S., 2008 May;

105:7467-7471

● Korea Patent Application No.: 10-2008-0072099, Gene constructs comprising two or more reprogramming

-inducing genes and method for preparing induced pluripotent stem cells using the same

● Korea Patent Application No.: 10-2008-0072101, Method for producing induced pluripotent stem cells using

herpes simplex amplicon viruses containing reprogramming-inducing genes

● Culture media for human pluripotent stem cells

Key Technology Highlights

1

Related Publications%

Co

lon

y U

nd

iffer

enti

ated

(Un

diff

eren

tiat

ed c

olo

ny

no.

/To

tal c

olo

ny

no.

x 1

00)

120%

100%

80%

60%

40%

20%

0%P1 P2 P3 P4 P5 P6 P7 P8 P9 P10

mTeSR1 (Not xeno-free; Stemcell Technologies)

HuStem1 (Xeno-free)

HuStem2 (Xeno-free)

HuStem3 (Xeno-free)

HuStem4 (Xeno-free)

H9-hESC line

% C

olo

ny

Un

diff

eren

tiat

ed(U

nd

iffer

enti

ated

co

lon

y n

o./

Tota

l co

lon

y n

o. x

100

)

120%

100%

80%

60%

40%

20%

0%

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14

NutriStem (Xeno-free; Stemgent)

mTeSR1 (Not xeno-free; Stemcell Technologies)

Invitrogen XF-medium (with cocktail)

TeSR2 (Xeno-free; Stemcell Technologies)

HuStem1 (Xeno-free)

H9-hESC line

Fig 1. The HuStem1-4 (xeno-free & feeder-free) media supported undifferentiated growth of hESCs as efficiently as

mTeSR1 (feeder-free, but not xeno-free; Stemcell Technologies).

Fig 2. The HuStem1 medium was superior in culturing H9-hESC compared to other commercially available

xeno-free & feeder-free products.

hiPSC line, HuStem1, p27

DIC

X100 X200

DAPI Oct4

DAPI Sox2

DAPI SSEA4

DAPI Tra1-60

DAPI Tra1-81

H9-hESC line, HuStem1, p20 hiPSC line, HuStem4, p22

Fig 4. A normal karyotype was observed for H9-hESC after 20 passages in HuStem1 as well as for a hiPSC line after

22 passages in HuStem4.

Fig 3. After the 27th passage, a hiPSC line maintained in the HuStem1 medium showed no sign of differentiation in

morphology and expressed pluripotent cell-specific markers such as Oct4, Sox2, SSEA4, Tra1-60, and Tra1-81.

1 2 3 4 5

6 7 8 9 10 11 12

13 14 15 16 17 18

19 20 21 22 Y X

1 2 3 4 5

6 7 8 9 10 11 12

13 14 15 16 17 18

19 20 21 22 Y X

(a)

(b)

Tra1-60 staining

Fig 5. (a) The number of hiPSC colonies formed in HuStem1 medium was higher than that generated by the

conventional MEF-mediated method. The colonies were immunostained with antibody against Tra1-60. (b) The

number of hiPSC colonies generated by each method was expressed in a graph. (n=5, p=0.017).

● Xeno-free & feeder-free hiPSC derivation using HuStem1

MEF(Conventional method)

HuStem1 (Xeno-free & feeder-free)

No.

of h

ESC

-lik

e co

lon

y / 1

X 1

04

cel

ls

25

20

15

10

5

0

MEF(Conventional method)

HuStem1 (Xeno-free & feeder-free)

* p=0.017

● A H9-hESC line maintained in the HuStem1 and mTeSR1 medium showed no differentiation for 14 passages.

On the other hand, a H9-hESC line maintained in other commercially available xeno-free & feeder-free media

(NutriStem, Invitrogen XF-medium and TeSR2) showed instability and differentiation.

● HuStem1 medium can be used to culture pluripotent stem cells for at least 20 passages while retaining

pluripotency marker expression, and robust proliferation with a normal karyotype.

Comparative analysis