Stemgent mRNA Reprogramming System - Miltenyi Biotec

mRNA Reprogramming System
Non-integrating human iPS cell generation
Miltenyi Biotec is the authorized distributor
of Stemgent products

Non-integrating reprogramming technology
The discovery that induced pluripotent stem (iPS) cells can
be generated from somatic cells through the over
expression of transcription factors holds the promise of
treating diseases, such as diabetes and Parkinson’s disease.
However, conventional DNA-based reprogramming
technologies carry the risk of disrupting the cell’s genome
and leading it to become cancerous and are therefore not
suitable for clinical applications, such as disease modeling,
drug discovery, and regenerative medicine.
In November 2010, Warren et al. described the ability to
reprogram human cells using modified mRNA with
conversion efficiencies and kinetics superior to DNA-based
methods. This technology is a significant leap forward in the
drive to safely and effectively reprogram mature human
cells. As part of our continuing effort to provide cutting
edge reprogramming technology, Stemgent® is the first to
offer the mRNA Reprogramming System with a validated
protocol that has been proven on normal and diseasespecific
cells to be highly efficient and reproducible.
For ordering information and the latest news on
all our stem cell products refer to
macs-stemcells.com
These Stemgent products are not distributed by
Miltenyi Biotec in the US or Israel.

Stemgent mRNA Reprogramming System
The Stemgent mRNA Reprogramming System enables
non-viral, non-integrating, clinically-relevant reprogramming
of human cells. Messenger RNA-based reprogramming
completely eliminates the risk of genomic integration and
mutagenesis inherent to DNA and viral-based technologies
while offering a highly robust and efficient reprogramming
system. Stemgent’s mRNA Reprogramming System and
validated protocol combines everything you need for
successful reprogramming of your cells.
• Non-integrative technology eliminates risk
of insertional mutagenesis
• Transient protein expression levels are easily regulated
• Faster reprogramming, colonies appear within 16 days
• Proven reprogramming protocol using normal and
diseased cells
• Suitable for clinical applications; disease modeling,
drug discovery, and regenerative medicine
Antibody, +DAPI
Oct4
Klf4
Sox2 c-Myc
Lin-28 nGFP
Stemgent mRNA Reprogramming
Factors Set: hOKSML
The hOKSML Set consists of in vitro transcribed mRNA
synthetically modified to minimize innate antiviral
responses to single stranded RNA. mRNA does not integrate
into the host cell DNA and therefore allows for transient
re-programming factor expression required to reprogram
cells.
Stemgent Pluriton
mRNA Reprogramming Medium
The Pluriton Medium was designed specifically to support
mRNA based reprogramming. Pluriton Medium is an
essential component of the mRNA Reprogramming System.
Each lot is qualified for mRNA-based reprogramming of
human fibroblasts.
Stemgent mRNA Reprogramming –
Qualified Kit
The mRNA Reprogramming-Qualified Kit includes the
Stemgent mRNA Reprogramming Factor Set: hOKSML,
Pluriton mRNA Reprogramming Medium, and B18R, a
critical component required to inhibit interferon alpha
(IFN-α). All components in the kit have been tested together
to successfully reprogram BJ fibroblasts.
Figure 1: Expression of mRNA reprogramming factors in BJ
fibroblasts. BJ fibroblasts were transfected with Stemgent mRNA
encoding Oct4, Klf4, Sox2, c-Myc, Lin-28, and nGFP as a control. Protein
expression and localization were analyzed by immunocytochemistry
of Oct4, Klf4, Sox2, c-Myc (all shown in red) and Lin-28, nGFP (shown in
green). All images show nuclear counterstain using DAPI (blue).

Faster iPS cell generation
Stemgent’s mRNA Reprogramming protocol has been
validated on normal and disease-specific human fibroblasts.
Viable iPS cell colonies appear within 16 days after the
initiation of reprogramming. This is more than a week
earlier than many viral-based systems, which typically
yield colonies between 30–40 days after the initiation
of reprogramming. For successful mRNA reprogramming
of human fibroblasts, cells are transfected daily (figure 2A).
An example of iPS colonies can be seen in figure 2B.
Generating diseased-patient
iPS cell lines
The Stemgent mRNA Reprogramming System has been
used to generate diseased-patient specific iPS cells. In
collaboration with the laboratory of Dr. Rudolf Jaenisch,
iPS cell lines were generated from dermal fibroblasts
derived from a patient with Parkinson’s disease (figure 2B).
A
Plate Feeders
Plate Cells
Transfect with Oct4, Klf4, Sox2, c-Myc,
Lin-28 and nGFP mRNA cocktail
Pick
Colonies
-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 +
Day 14 – 10x Day 20 – 10x (Tra-1-81)
Figure 2: Reprogramming timeline for mRNA reprogramming.
(A) Cells are transfected daily using a 1.2 ug hOKSML Set mRNA cocktail.
(B) iPS colony generated using the Stemgent mRNA Reprogramming
Factor Set on Parkinson’s disease patient dermal fibroblasts. Colonies
are shown at day 14 (left) and day 20 (right). Day 20 primary iPS colony
pluripotency evaluated using Tra-1-81 StainAlive antibody.
Validated protocol for high
efficiency reprogramming
The Stemgent mRNA Reprogramming System includes
a validated, easy-to-use protocol that allows for the
reproducible and efficient generation of iPS cells. The
Pluriton mRNA Reprogramming Medium is an essential
component of the reprogramming protocol. This medium
allows for reproducible colony formation resulting in
up to 588 iPS cell colonies per 10K target cells plated,
as compared to other media that failed to support mRNA
reprogramming (figure 3). Induced pluripotent stem cell
colonies generated from BJ fibroblasts using this protocol
express pluripotency markers (figure 4) and maintain a
normal karyotype.
B Figure 3: mRNA Reprogramming in Stemgent Pluriton mRNA
Reprogramming Medium. mRNA iPS derivation media comparison
of Stemgent Pluriton mRNA Reprogramming Medium and other common
human ES culture media. Different target cell densities (50K, 25K,
and 10K for Pluriton; 50K and 10K for all others) of BJ fibroblasts were
plated on human fibroblast feeders (250K/well) in a single well of a
6-well plate. Each condition was transfected for 16 days with 1.2 ug of
reprogramming cocktail generated using the mRNA Reprogramming
Factor Set. Tra-1-81 positive colonies were counted on day 19, three
days after the last mRNA transfection. Each bar in the graph is
individually labeled with the number of iPS cell colonies generated.
For a detailed protocol and more information
on mRNA reprogramming please visit
macs-stemcells.com/reprogramming

Proven portfolio for mRNA Reprogramming
Nanog/Tra-1-60 Oct4/Tra-1-81
Rex1/SSEA4
Figure 4: Human iPS cell colonies generated with the Stemgent
mRNA Reprogramming System. Pluripotency immunocytochemistry
images (10X) from an mRNA iPS cell line derived from BJ fibroblasts.
Nuclear pluripotency markers (Nanog, Oct4, Rex 1) shown in red. Cell
surface pluri-potency markers (Tra-1-60, Tra-1-81, SSEA4) shown in
green.
Product List
Description Cat. No.
Stemgent mRNA Reprogramming Factors Set: hOKSML
Contains: 2 vials of human Oct4 mRNA, 1 vial of human Klf4, Sox2, c-Myc,
Lin-28 mRNA, and 1 vial nGFP mRNA
Stemgent mRNA Reprogramming-Qualified Kit
Includes B18R Recombinant Protein Carrier-Free*
*B18R is manufactured by eBioscience®.
References
130–096–528
coming soon
Pluriton mRNA Reprogramming Medium 130–096–820
Stemgent Oct4 mRNA, Human 130–096–524
Stemgent Klf4 mRNA, Human 130–096–526
Stemgent Sox2 mRNA, Human 130–096–527
Stemgent Lin-28 mRNA, Human 130–096–525
Stemgent c-Myc mRNA, Human 130–096–523
Stemgent nGFP mRNA 130–096–522
Stemgent eGFP mRNA Transfection Control 130–096–812
1. Warren, L. et al. (2010) Highly efficient reprogramming to pluri-potency
and directed differentiation of human cells with synthetic modified
mRNA. Cell Stem Cell 7: 618–30.
2. Angel, M. and Yanik, M.F. (2010) Innate immune Suppression Enables
Frequent Transfection with RNA Encoding Reprogramming Proteins.
PloS One 5: e11756.
3. Yakubov, E. et al. (2010): reprogramming of Human Fibroblasts to
Pluripotent Stem Cells using mRNA of Four Transcription factors.
Biochem. Biophys. Res. Commun.: 394–189.

Germany/Austria/
Switzerland
Miltenyi Biotec GmbH
Friedrich-Ebert-Straße 68
51429 Bergisch Gladbach
Germany
Phone +49 2204 8306-0
Fax +49 2204 85197
macs@miltenyibiotec.de
USA/Canada
Miltenyi Biotec Inc.
2303 Lindbergh Street
Auburn, CA 95602, USA
Phone 800 FOR MACS
Phone +1 530 888 8871
Fax +1 530 888 8925
macs@miltenyibiotec.com
www.miltenyibiotec.com
Australia
Miltenyi Biotec
Australia Pty. Ltd.
Unit 16A, 2 Eden Park Drive
North Ryde, NSW 2113
Australia
Phone +61 2 8877 7400
Fax +61 2 9889 5044
macs@miltenyibiotec.com.au
Benelux
Miltenyi Biotec B.V.
Schipholweg 68 H, 2316 Leiden
The Netherlands
macs@miltenyibiotec.nl
Customer service Netherlands
Phone 0800 4020120
Fax 0800 4020100
Customer service Belgium
Phone 0800 94016
Fax 0800 99626
Customer service Luxembourg
Phone 800 24971
Fax 800 24984
China
Miltenyi Biotec GmbH
Shanghai Office
Rm. 2309–2310,
No. 319 Xianxia Rd.
Shanghai 200051, P.R. China
Phone +86 21 62351005
Fax +86 21 62350953
macs@miltenyibiotec.com.cn
France
Miltenyi Biotec SAS
10 rue Mercoeur
75011 Paris, France
Phone +33 1 56 98 16 16
Fax +33 1 56 98 16 17
macs@miltenyibiotec.fr
Italy
Miltenyi Biotec S.r.l.
Via Persicetana, 2/D
40012 Calderara di Reno (BO)
Italy
Phone +39 051 6 460 411
Fax +39 051 6 460 499
macs@miltenyibiotec.it
Japan
Miltenyi Biotec K.K.
Nittsu-Eitai Building 5F
16-10 Fuyuki, Koto-ku, Tokyo
135-0041, Japan
Phone +81 3 5646 8910
Fax +81 3 5646 8911
macs@miltenyibiotec.jp
Singapore
Miltenyi Biotec
Asia Pacific Pte Ltd.
100 Beach Road
#28-06 to 28-08 Shaw Tower
Singapore 189702
Phone +65 6238 8183
Fax +65 6238 0302
macs@miltenyibiotec.com.sg
Miltenyi Biotec provides products and services worldwide. Visit www.miltenyibiotec.com/local to find your nearest Miltenyi Biotec contact.
Spain
Miltenyi Biotec S.L.
C/Luis Buñuel 2
Ciudad de la Imagen
28223 Pozuelo de Alarcón
(Madrid), Spain
Phone +34 91 512 12 90
Fax +34 91 512 12 91
macs@miltenyibiotec.es
United Kingdom
Miltenyi Biotec Ltd.
Almac House, Church Lane
Bisley
Surrey GU24 9DR, UK
Phone +44 1483 799 800
Fax +44 1483 799 811
macs@miltenyibiotec.co.uk
MACS and the MACS logo are registered trademarks of of Miltenyi Biotec GmbH. Unless otherwise specifically indicated, Miltenyi Biotec products and services are
for research use only and not for therapeutic or diagnostic use. Stemgent, StainAlive, and Pluriton are either registered trademarks or trademarks of Stemgent Inc.
eBioscience is a registered trademark of eBioscience CORPORATION CALIFORNIA. Copyright © 2011 Miltenyi Biotec GmbH. All rights reserved.
130-096-791