hESC (Electronics)

Your Guide to
Maximize Your Plurip tential

Table of Contents
3 Introduction
4 mTeSR™1: Defined, Feeder-Free Maintenance medium for Human Pluripotent Stem Cells
6 TeSR™-E8™ and Vitronectin XF™
8 TeSR™2 and StemAdhere™ Defined Matrix for hPSC
10 mFreSR™ and CryoStor™CS10

11 Antibodies for Characterization of Human Pluripotent Stem Cells
12 EasySep™
14 AggreWell™
15 STEMdiff™ Definitive Endoderm Kit
16 STEMdiff™ Neural Induction Medium
17 STEMdiff™ APEL™ Medium
18 Support Reagents
19 References
Your Guide to
Reduce Variation With The Most Complete, Defined System For Pluripotent Stem Cell Culture
2
Products for Human Pluripotent Stem Cells
Human Pluripotent Stem Cells

Products for Research
Introduction
Human embryonic and induced pluripotent stem cells,
collectively known as human pluripotent stem cells (hPSCs),
are defined by the potential for unlimited expansion and the
ability to generate cells of all three germ layers – endoderm,
mesoderm and ectoderm – that can then further differentiate
into specific cell lineages.1-5 This ability has spurred their use
for a variety of clinical applications and for the study of human
cellular and developmental systems.
Embryonic stem cells (ES cells) are derived from the inner
cell mass of pre-implantation blastocysts, whereas induced
pluripotent stem cells (iPS cells) are somatic cells reprogrammed
by the transient overexpression of a small number of genes.2-4
The establishment of standardized methods for generating and
characterizing these cells is crucial to fully harnessing their
therapeutic potential in the future.
STEMCELL Technologies provides a full range of products
that support the standardization of ES and iPS cell research.
From isolation and characterization to maintenance and
differentiation, see how you can “Maximize Your Pluripotential”
and reduce variation with the most complete, defined system
for human embryonic and induced pluripotent stem cell culture.
3
mTeSR™1
Defined, Feeder-Free Maintenance Medium for Human Pluripotent Stem Cells
Developed at the: Advantages of mTeSR™1

Consistency. Defined, feeder-free ES and iPS cell
culture medium.
Reproducibilit y. Standardizes culture methods
for more reproducible data.
Convenience. Saves time and effort needed
for feeder and conditioned media preparation.
A Complete Formul ation. No supplements
or other growth factors required.
mTeSR™1 is a standardized medium for the feeder-free

maintenance of human ES cells and iPS cells. It is a complete,
serum-free, defined formulation based on Ludwig et al.6 and
developed under license from the WiCell® Research Institute.
With pre-screened raw materials that ensure batch-to-batch
consistency and robust feeder-free protocols for ES and iPS
cell culture, mTeSR™1 provides more consistent cultures with
mTeSR™1 is the most widely homogeneous, undifferentiated phenotypes.
published feeder-free pluripotent

stem cell medium in the world
Visit www.stemcell.com/en/promotions/mTeSR1 for a fully
searchable list of publications and interviews with leading
pluripotent researchers.
Table 1. mTeSR™1 Has Been Tested and Published Extensively for the Long-Term Maintenance and Expansion of Various Cell Lines,
Including Those Listed Below (*Unpublished Customer Reports)
hESC and hiPSC Lines Maintained in mTeSR™1
H1, H9, H7 H13*, H14, H15*, H16* BG01, BG02, BG03, BG04* Shef1*, Shef4*
HESM01*, HESM02*, HS237, HS239, HS360,

HUES1, HUES3, HUES6, HUES8*, HUES9 Regea 06/015*
HESM03*, HESM04* HS401
Banked at WISC bank at WiCell:

MA01*, CA1*, CA1T*, iPS-DF19-9-11T.H, iPS-DF19-9-7T,
NCL-3, HSF-6*, HES2, iPSC(IMR90)-3, MSC-
KCL1*, Man1*, MEL-1*, iPS-DF4-3-7T, iPS-DF6-9-9T.B,
HES3 (ES03), HES4* iPSC1
MEL-2* iPS(Foreskin)-1 (Clone 1), iPS IMR90-1,
iPS(IMR90)-4 (Clone 4)
4
A B
400 µm 100 µm
Figure 1. Morphology of Human ES Cells Cultured in mTeSR™1

H1 human ES cells grow as colonies with (A) defined edges and (B) high
nucleus to cytoplasm ratio.
A B
Figure 4. Human ES Cells Cultured in mTeSR™1 Retain Normal

Karyotype Following Long-Term Passaging
1000 μm 1000 μm
Chromosomal analysis of H1 ES cells cultured in mTeSR™1 for 48 passages
shows that normal karyotype is retained during long-term passaging.
Figure 2. Morphology of Human iPS Cells Cultured in mTeSR™1 Data from Cytogenetics Lab, WiCell® Research Institute.
Human iPS cell photographs courtesy of M. O'Connor and C. Eaves, The

Vancouver Human Embryonic Stem Cell Core Facility. The human iPS cell
lines (A) iPSC(IMR90)-3 and (B) MSC-iPSC1 maintained in mTeSR™1 show
similar morphological characteristics. product quantity catalog #
500 mL 05850
1L 05857
A B mTeSR™1
10 Kits 05870
25 Kits 05875
1000 µm 1000 µm
VIDEO
mTeSR™1: Standardized Medium for the Feeder-
Independent Maintenance of hESCs & hiPSCs
1000 µm
www.stemcell.com/mTeSR1Video
Figure 3. Morphology of Human ES Cells Cultured in mTeSR™1

Over Time
H9 ES cells are routinely passaged every 5 - 7 days. The morphology of ES
cells in culture varies slightly compared to feeder-containing or conditioned
medium cultures. On day 2 (A), colonies are small and less densely packed.
By day 4 (B) colonies rapidly increase in size and start to become multilayered.
By day 6 (C), colonies are multilayered, densely packed and beginning to
merge. These colonies are ready to passage.
5
New TeSR™ -E8™ and Vitronectin XF™
Low Protein, Highly Defined, Feeder-Free Maintenance
of Human Pluripotent Stem Cells
New TeSR™-E8™ is a highly defined feeder-free culture

medium for human pluripotent stem cells. It is based on the E8
formulation published by the laboratory of Dr. James Thomson Advantages of TeSR™ -E8™ and
(University of Wisconsin- Madison), the lead research group Vitronectin XF™:
behind the design of mTeSR™1.
• Completely defined xeno-free culture system
Like mTeSR™1, TeSR™-E8™ is made with the highest level
• Contains only the essential components for
of quality and care. TeSR™-E8™ contains only the essential
feeder-free culture
components required for maintenance of ES cells and iPS cells,
providing a simpler medium for the culture of pluripotent stem • Enables flexible passaging schedule
cells. It can be used with a surface coating of Matrigel™ hESC-
Qualified Matrix or with Vitronectin XF™.
A Component mTeSR™1 E8*
DMEM-F12 (DF12) • •
NaHCO3 • •
500 µm 25 µm L-Ascorbic Acid • •
Selenium • •
B
Transferrin • •
Insulin • •
FGF2 • •
500 µm 25 µm
TGF-β • •
Figure 5. Normal human ES and iPS cell morphology is observed Bovine Serum Albumin (BSA) •
in TeSR™-E8™ cultures.
Undifferentiated (A) human ES (H9) and (B) human iPS (WLS-1C) cells Glutathione •
cultured on Matrigel™ in TeSR™-E8™ retain the prominent nucleoli and high
nuclear to cytoplasm ratio characteristic of this cell type. Densely packed
Trace Elements •
cells and multilayering are prominent when cells are ready to passage.
β-mercaptoethanol (BME) •
Pipecolic Acid •
GABA •
Lithium Chloride •
Defined Lipids •
*As published in Chen et al. 2011, the base design for TeSR™-E8™.
6
Customize Your Passaging 45
Schedule: Passage Anytime 40
Average Fold Expansion/Passage

Between Days 4 & 7 35
30
The amount of time until your next passage depends largely on
25
the size and density of initially plated cell aggregates, as well as
20
on the colony density at time of passaging. The following table
indicates how these three variables can be manipulated to allow 15
human ES and iPS cells cultured in TeSR™-E8™ medium to 10
be passaged between 4 to 7 days after plating. Regardless of
5
the plating density or cell aggregate size used, the majority of
0
colonies should be densely packed and multilayered in the center
when ready for passaging. See Figure 5 for a representative
0
1
1)
1)
70
D
H
-1
™
-4
LS
13
SR
SR
LS
W
A
Te
Te
W
example indicating the optimal window for passaging.
m
1(
9(
H
H
hiPSCs hESCs
Adjustable Parameters That Affect Time
to Next Passage Figure 6. High expansion rates are observed in TeSR™-E8™
cultures.
AT TIME OF PLATING AT TIME OF PASSAGING
Graph shows the average fold expansion per passage +/- SEM obtained
for human ES and iPS cells cultured in TeSR™-E8™ with BD Matrigel™ over
CELL
PLATING COLONY DAYS IN 10 passages (brown). Representative data is also shown for human ES
AGGREGATE
DENSITY DENSITY CULTURE cells cultured in mTeSR™1 using a similar protocol for comparison (gray).
SIZE
Expansion was determined by enumerating the cell aggregates obtained at
harvest and dividing by the number of cell aggregates seeded. Note that this
Large High High 4-5
data is representative of cultures passaged after 6-7 days in culture; lower
expansion should be expected if using shorter culture times.
Large Low Low
Medium Medium Medium 5-6
Small High High A Day 1 (AggreWell™ 400 plate) B Day 2 (ULA plate)
Small Low Low 6-7
For a detailed guide to customizing your passaging schedule,

100X 100X
please view section 7.0 of the TeSR™-E8™ manual (Document
#29267) at www.stemcell.com. The manual contains examples
of cell aggregate sizes, colony densities, and cultures that are Figure 7. Uniformly-sized embryoid bodies differentiated from cells
ready for passaging at several timepoints. cultured in TeSR™-E8™.
H1 cells cultured using TeSR™-E8™ on BD Matrigel™ were dissociated to
single cells using standard techniques, then placed in an AggreWell™ 400
product SIZE catalog # plate containing AggreWell™ medium and 10 µM Y-27632 for 24 hours (A),
after which they were transferred to an ultra low-adherence (ULA) plate
TeSR-E8 1 Kit 05840 for inspection (B). This protocol can be found in the AggreWell™ manual
(Document #29146) at www.stemcell.com.
Vitronectin XF™ 1 Kit 07190
Vitronectin XF™ is a trademark of Primorigen Biosciences®. Vitronectin XF™ is developed and manufactured by
Primorigen Biosciences®. All other trademarks and registered trademarks are the property of their respective holders.
the stem cell company™
7
TeSR™2 and StemAdhere™ Defined Matrix for hPSC
Defined Culture Systems
Advantages of TeSR™2 and

StemAdhere™ Defined Matrix
for hPSC
• Completely defined culture system minimizes
experimental variability
• Combination of animal protein-free medium
and human recombinant protein matrix takes
A a significant step towards a fully humanized
system
• No preparation of feeders or conditioned media
required
• StemAdhere™ is growth factor-free
100 µm • Maintains phenotypically homogeneous and
karyotypically normal cells after long-term
passaging
B
Given current interest in using ES and iPS cells for applications

in regenerative medicine, the development of humanized and
defined systems for the maintenance of ES and iPS cells is
critical. TeSR™2 takes a significant step in this direction by
500 µm
allowing hES and iPS cells to be cultured feeder-free in an
animal protein-free, defined medium.7
C TeSR™2 can be used with StemAdhere™ Defined Matrix
for hPSC, developed and manufactured by Primorigen
Biosciences®, for a completely defined culture system that
allows for complete control over the culture environment.8
StemAdhere™ is an alternative to the commonly used Matrigel™
and has the advantage of being a defined, recombinant human
100 µm protein, which allows for more consistent cell populations and
more reproducible results in downstream applications.
Figure 8. Undifferentiated hPSCs Grown in TeSR™2
and mTeSR™1 on StemAdhere™ Defined Matrix for hPSC
hPSCs grown in TeSR™2 and mTeSR™1 on StemAdhere™ Defined Matrix for
hPSC have a slightly different morphology compared to hPSCs grown on other
matrices. This difference is not indicative of culture quality or pluripotency. (A)
H9 ES cells at Day 5 of culture in TeSR™2 on StemAdhere™ Defined Matrix
for hPSC tend to form colonies that are more loosely packed, with noticeable
cellular spreading at the edges of the colonies and (B) are ready to passage
when the colonies are large, beginning to merge and have centers that are
dense and phase-bright compared to their edges. (C) WLS-4D1 iPS cells at
Day 6 of culture in TeSR™2 on StemAdhere™ Defined Matrix for hPSC.
8
A A
1000 µm 25 µm
B B
200 µm 25 µm
FIGURE 9. Morphology of iPS Cells Cultured in TeSR™2 FIGURE 10. Morphology of hPSCs Cultured in mTeSR™1
Human iPS cells cultured in TeSR™2 grow as colonies with defined edges or TeSR™2 on StemAdhere™ Defined Matrix for hPSC
and high nucleus to cytoplasm ratio. iPS(IMR90)-1 cell line cultured (A) for Undifferentiated hPSCs cultured in mTeSR™1 or TeSR™2 on StemAdhere™
8 passages in TeSR™2 and (B) for 10 passages in TeSR™2. Defined Matrix for hPSC exhibit high nuclear to cytoplasmic ratio and
Photographs courtesy of Dr. T. Ludwig, WiCell Research Institute. prominent nucleoli. (A) H9 ES cells at Day 5 of culture in TeSR™2 on
StemAdhere™ Defined Matrix for hPSC. (B) H9 ES cells at Day 6 of culture
in mTeSR™1 on StemAdhere™ Defined Matrix for hPSC.
PRODUCT SIZE Catalog #
TeSR™2 500 mL Kit 05860
StemAdhere™ Defined
1 Kit 07170
Matrix for hPSC
StemAdhere™ is a trademark of Primorigen Biosciences®. StemAdhere™ is developed and manufactured by

Primorigen Biosciences®. All other trademarks and registered trademarks are the property of their respective holders.
the stem cell company™
9
mFreSR™ and CryoStor™ CS10
Defined Cryopreservation Media for Human Pluripotent Stem Cells
mFreSR™ CryoStor™ CS10

• Serum-free • Serum-free and xeno-free
• Research use only • cGMP-compliant
Ideally used after culture in mTeSR™1 or TeSR™2
Conventional cryopreservation methods for human pluripotent

stem cells use fetal bovine serum, introducing an undefined
component into the culture media. mFreSR™ is a defined
cryopreservation medium designed specifically for use with
hPSCs, that has been shown to improve thawing efficiencies
5- to 10-fold over other reported methods.10-14 CryoStor™ CS10
is a cGMP-manufactured, animal-protein-free cryopreservation
medium for human pluripotent cells. Combine these serum-
free cryopreservation media with our feeder-free maintenance
media mTeSR™1 and TeSR™2 to minimize variability in your
human pluripotent cell lines.
10% 10%
to undifferentiated colonies
to undifferentiated colonies
% clumps giving rise
% clumps giving rise
5% 5%
0% 0%
p64 p74 p74 p40 p40 p38 p38 p42 p42 p53
p53 p53
p53 p65
p65 p65
p65 p61
p61 p61
p61
Figure 11. Clump Survival Data for mFreSR™ Figure 12. Clump Survival Data for CryoStor™ CS10
H9 ES cells were cryopreserved in mFreSR™ at the indicated passage after H9 ES cells were cryopreserved with CryoStor™ CS10 at the indicated
culture with mTeSR™1. Thawing efficiencies were determined by counting the passage after culture with TeSR™2. Thawing efficiencies were determined
number of clumps after thawing, and the number of resultant undifferentiated by counting the number of clumps after thawing, and the number of resultant
colonies. undifferentiated colonies.
10
Antibodies for Characterization

of Human Pluripotent Stem Cells
STEMCELL Technologies offers a wide range of primary and secondary antibodies suitable for the characterization of human ES
and iPS cells during culture maintenance and expansion. Undifferentiated human ES and iPS cells express high levels of certain
pluripotency markers, such as Oct-3/4 and SSEA-3, whose detection can assist in determining the undifferentiated state of a particular
ES or iPS cell population.
A B C
FIGURE 13. Immunocytochemistry of H1 ES Cells cultured in mTeSR™1 reveals expression of (A) Oct-3/4 and (B) TRA-1-60.
(A) Oct3/4 (Catalog #01550/01551) with FITC-conjugated secondary antibody (Catalog #10210). (B) TRA-1-60R, PE conjugated antibody (Catalog #60064PE). (C)
Flow cytometry analysis of human H1 ES cells (filled histogram) or HT1080 cells (negative control; dashed line) labeled with TRA-1-60R, PE. Labeling with Mouse
IgM, kappa Isotype Control Antibody, Clone MM-30, PE is shown (Catalog #60069PE; solid line histogram).
Alexa
Primary Antibody Unconjugated PE Conjugated Fluor® 488 Species Reactivity Isotype
Conjugated
Catalog #
Oct-3/4 Antibody 01550 Human, Mouse IgG1 κ (Mouse)
Anti-Mouse SSEA-1
60060 60060PE 60060AD Human, Mouse Rat IgM κ (Mouse)
Antibody, Clone MC-480
Anti-Mouse SSEA-3 Human, Mouse, Rat,
60061 60061PE 60060AD IgM κ (Rat)
Antibody, Clone MC-631 Rhesus
Human, Mouse, Rat,

Anti-Human SSEA-4
60062 60062PE 60060AD Rhesus, Cat, Chicken, IgG3 κ (Mouse)
Antibody, Clone MC-813-70
Dog, Rabbit
Anti-Human TRA-1-60
60064 60064PE 60064AD Human, Rhesus, Rabbit IgM κ (Mouse)
Antibody, Clone TRA-1-60R
Anti-Human TRA-1-81
60065 60065PE Human, Rat, Rhesus IgM κ (Mouse)
Antibody, Clone TRA-1-81
Human, Non-Human
Anti-Human TRA-2-49
60066 Primate, Monkey, Cat, Pig, IgG1 κ (Mouse)
Antibody, Clone TRA-2-49/6E
Rabbit, Tiger
Anti-Human TRA-2-54 Human, Chimpanzee,

60067 IgG1 κ (Mouse)
Antibody, Clone TRA-2-54/2J Monkey, Cat, Pig, Rabbit
11
EasySep™
Fast and Easy Cell Isolation of Human Pluripotent Stem Cells
Advantages of EasySep™
FAST AND EASY. No columns or washes.
High Purit y. Reliably obtain purities of

up to 99%.
FUNCTIONAL CELLS. Gentle procedure enables the
isolation of functional and viable cells.
FLOW CY TOMETRY-COMPATIBLE. Isolated cells are
immediately available for use as EasySep™ magnetic
particles do not interfere with flow cytometry.
Pluripotent stem cells can be enriched from a mixed population

containing differentiated and undifferentiated cells or
Start: 12% SSEA-4+ H9 Cells Selected: 99% SSEA-4+ H9 Cells
reprogrammed and non-reprogrammed cells, based on their
100 100 100 100
surface expression of SSEA-4 or TRA-1-60. EasySep™ is a
80 80 80 80
powerful cell isolation platform that combines the specificity
Counts
Counts
60 60 60 60
40 40 40 40
of monoclonal antibodies with the simplicity and speed of a
20 20 20 20 column-free, immunomagnetic system. Use the EasySep™
0
0
0
0 1 1 2 2 3 3
0
0
0
0 1 1 2 2 3 3
hESC/hiPSC SSEA-4 Positive Selection Kit or the EasySep™
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Human ES/iPS Cell TRA-1-60 Positive Selection Kit to isolate
SSEA-4 PE SSEA-4 PE
highly purified human pluripotent cells quickly and easily.
Figure 14. Typical Flow Cytometry Histogram Results Using

EasySep™ hESC/hiPSC SSEA-4 Antibody Positive Selection Kit product Capacity catalog #
Starting with a mixture of H9 ES cells and HT1080 fibroblast cells, the EasySep™ hESC/hiPSC for 1 x 109
SSEA-4+ cell content of the enriched fraction typically ranges from 95 - 99%. 18165
SSEA-4 Positive Selection Kit* cells
EasySep™ Human ES/

abeling up to
iPS Cell TRA-1-60 Positive 18166
1 x 109cells
Selection Kit*
*Required equipment: EasySep™ Magnet (Catalog #18000)
FREE SAMPLE KIT

EasySep™ hESC/hiPSC SSEA-4
Positive Selection Kit
www.stemcell.com/SSEA4SampleKit
12
EasySep™
Cell Separation for Cells Differentiated from Human Pluripotent Stem Cells
EasySep™ can be used to quickly and easily perform positive
or negative selection of virtually any cell type from any source.
Add EasySep™ selection
The EasySep™ hESC-derived CD34 Positive Selection Kit
cocktail to cells
(Catalog #18167) is designed to purify CD34+ cells during
hematopoietic differentiation protocols. Alternatively, customiza-
tions for your specific experimental needs are also available.
Incubate 15 minutes
EasySep™ Custom Selection Kits
Use any conjugated antibody with EasySep™ PE (Catalog
#18551), FITC (Catalog #18552), Biotin (Catalog #18553), or
APC (Catalog #18451) Selection Kits to select or deplete your
cells of interest.
Fully Automated Cell Separation Add EasySep™

with RoboSep™ magnetic particles
RoboSep™ (Catalog #20000), the fully automated cell separa-

tor, uses EasySep™ technology for high throughput sample
processing with minimal handling. RoboSep™ is often the
instrument of choice for large research facilities focused on Incubate 10 minutes
disease samples.
Start: 8.3% CD34+ Cells Selected: 97.2% CD34+ Cells
100 200
Place tube in magnet for 10
minutes
80
150
Counts
Counts
60
100
Pour off supernatant.

40
50
20
Positively selected cells
0
0 1 2 3 4
0
0 1 2 3 4
remain in tube.
10 10 10 10 10 10 10 10 10 10
CD34 CD34
Figure 15. Typical Flow Cytometry Histogram Results Figure 16. EasySep™ Positive Selection Protocol Diagram
with EasySep™ hESC-Derived CD34 Positive Selection Kit
Starting with a differentiated population containing at least 5% CD34+ cells,
the CD34+ cell content of the enriched fraction typically ranges from 84% -
99%.
product Capacity catalog #
EasySep™ hESC-Derived CD34 for 1 x 109

18167
Positive Selection Kit* cells
*Required equipment: EasySep™ Magnet (Catalog #18000)
13
AggreWell™
Reproducible Production of Uniformly-Sized Embryoid Bodies
Advantages of AggreWell™
• Forms EBs of uniform size and shape
• Enables the user to control size from 10 cells to
20,000 cells per EB
• Reduces variability in differentiation protocols
that utilize EBs
• High-throughput
Many pluripotent stem cell differentiation protocols begin with

the formation of 3-dimensional aggregates of cells called
embryoid bodies (EBs). Conventional EB formation methods15,16
result in EBs that are heterogeneous in size and shape, leading
to inefficient and uncontrolled differentiation.17 AggreWell™
plates solve this issue by aggregating pluripotent stem cells
into EBs of defined size using microwells, making differentiation
experiments more reproducible.
EB formation is accomplished by adding a single cell

suspension to the plate, centrifuging to distribute the cells
evenly among the microwells, and then culturing for a minimum
of 24 hours to allow aggregation of the cells within each
microwell. The resulting EBs are highly uniform in size and
shape (Figure 17) and can be efficiently differentiated into a
Figure 17. Uniformly Sized Embryoid Bodies Generated variety of cell types.18
with AggreWell™
EBs formed using AggreWell™ plates are uniform in size and consistently 500 cells/EB 2,000 cells/EB scraped EBs
spherical in shape. Shown are EBs generated with 2,000 human ES cells
each, using AggreWell™400.
PRODUCT SIZE CATALOG #
AggreWell™400 Plate 1/pack 27845

Count
(8 inserts in 24 wells) 5/pack 27945
AggreWell™400Ex Plate 1/pack 27840

(6 wells) 5/pack 27940
AggreWell™ 800 Plate 1/pack 27865

(8 inserts in 24 wells) 5/pack 27965 2 3
Area (micron ) x 10
AggreWell™ Medium 100 mL 05893 Figure 18. The Size of EBs Can Easily be Adjusted Using
AggreWell™ Rinsing Solution 100 mL 07010 AggreWell™ Plates
(C) H9 ES cells were centrifuged into AggreWell™400 plates and cultured
37 µm Reversible Strainers, Small 20/box 27215
for 24 hours prior to EB harvest. EB size is tightly controlled with AggreWell™
37 µm Reversible Strainers, Large 12/box 27250 (light and dark grey), unlike with scraping protocols (brown) that give a wider
distribution.22
14
STEMdiff™ Definitive Endoderm Kit

Defined, Animal Component-Free System for hPSC Differentiation
to Definitive Endoderm
Advantages of STEMdiff™
Definitive Endoderm Kit
• Fully defined, serum-free and animal component-free
• Optimized for use with cells maintained in mTeSR™1

or TeSR™2
• Efficient and reproducible differentiation of multiple

ES cell and iPS cell lines
• Differentiated cells co-express SOX17, CXCR4,

FOXA2 and c-KIT
• Convenient, user-friendly format and protocol
100
The STEMdiff™ Definitive Endoderm Kit, optimized for use with
% CXCR4+/SOX17+ Cells
80
mTeSR™1 and TeSR™2, allows for the differentiation of hPSCs
60
to multipotent definitive endoderm. It is a defined, animal
40
component-free system. SOX17 FOXA2
20
0 hPSCs differentiated using this kit are highly enriched for
16 6 11 4 1 definitive endoderm, as indicated by co-expression of SOX17,
H9
h9 h1 WLS-4D1 A13700 WLS-1C
CXCR4, FOXA2 andSOX17
c-KIT, and can be used to generate multipleMerged
FOXA2
downstream endodermal cell types, including hepatocytes and
SOX17
pancreatic precursors. FOXA2 Merged
100
% CXCR4+/SOX17+ Cells
80 H9 H9 WLS-4D1
SOX17 FOXA2 Merged DAPI
60
H9 4D1
40 SOX17
SOX17 FOXA2 Merged DAPI
20
H9
0 SOX17 FOXA2 Merged DAPI
8 4 4 1 Representative images of SOX17 and FOXA2 immu
H9 4D1 following 4 days of differentiation to definitive endoderm
SOX17 FOXA2
FOXA2 Merged DAPI
images show extensive co-localization of these two ma
h9 h1 WLS-4D1 A13700 4D1
H9
Figure 19. Definitive Endoderm Differentiation is Efficient Across Representative images of SOX17 and FOXA2 immunoreactivity in hES (H9
H9 4D1
Multiple ES and iPS Cell Lines
SOX17 FOXA2 Merged following 4 days
Merged of differentiation to definitive endoderm using STEMdiff™ Defin
DAPI
Representative images of SOX17 and FOXA2 immunoreactivity in hES (H9
images show extensive co-localization of these two markers. Scale bar, 100 µm
Quantitative analysis of definitive endoderm formation in multiple hPSC 4D1 lines
following 4 days of differentiation to definitive endoderm using STEMdiff™ Defin
as measured by co-expression of CXCR4 and SOX17. Prior to differentiation
images show extensive co-localization of these two markers. Scale bar, 100 µm
using STEMdiff™ Definitive Endoderm Kit, cells were maintainedRepresentative
in their images of SOX17 and FOXA2 immunoreactivity in hES (H9) and hiPS (4D1) cells
4D1
pluripotent stateH9
by culturing in mTeSR™1 on Matrigel™ (A) and following TeSR™2 4 days of differentiation to definitive endoderm using STEMdiff™ Definitive Endoderm. Merged
DAPI
Representative
on Matrigel (B). Data are expressed as the mean percent of cells expressing
4D1 images images co-localization
show extensive of SOX17 and of FOXA2
these twoimmunoreactivity in hES
markers. Scale bar, 100 (H9)
µm. and hiPS (4D1) cells
following 4 days of differentiation to definitive endoderm using STEMdiff™ Definitive Endoderm. Merged
both markers. Error bars indicate SEM; n values for each cell line are indicated
by the white number within each bar. images show extensive co-localization of these two markers. Scale bar, 100 µm.
Representative images of SOX17 and FOXA2 immunoreactivity in hES (H9) and hiPS (4D1) cells
Figureendoderm
following 4 days of differentiation to definitive 20. Expression of Key Definitive
using STEMdiff ™
DefinitiveEndoderm Markers
Endoderm. Merged
product 4D1 Representative
images images
SIZE show extensive
catalog of SOX17 andofinFOXA2
co-localization
# Human
these two ES and iPS
markers. Cells
immunoreactivity
Scale in ishES
bar, Widespread
100 (H9)
µm. and hiPS (4D1) cells
following 4 days of differentiation to definitive endoderm
Representative using of
images STEMdiff ™
SOX17 and Definitive
FOXA2 Endoderm. Merged
immunoreactivity in hES (H9)
STEMdiff™ Definitive images show extensive co-localization of these two markers. Scale bar, 100 µm.
1 Kit 05110 and hiPS (WLS-4D1) cells following 4 days of differentiation to definitive
Endoderm Kit
endoderm using STEMdiff™ Definitive Endoderm Kit. Merged images show
Representative images of SOX17 and FOXA2 immunoreactivity extensive
in hESco-localization
(H9) and hiPS of these twocells
(4D1) markers. Scale bar: 100 µm.
following 4 days of differentiation to definitive endoderm using STEMdiff™ Definitive Endoderm. Merged
images show extensive co-localization of these two markers. Scale bar, 100 µm.
15
STEMdiff™ Neural Induction Medium
Defined, Serum-Free Medium for hPSC Differentiation to Neural Progenitor Cells
Advantages of STEMdiff™ Neural

Induction Medium
• Fully defined and serum-free
• Optimized for use with cells maintained in
mTeSR™1 or TeSR™2
• Efficient and reproducible differentiation of
multiple ES cell and iPS cell lines
• Rapid neural induction: neural rosettes within
6 days
• Convenient, user-friendly format and protocol
STEMdiff™ Neural Induction Medium is a defined, serum-free

medium that enables the differentiation of hPSCs to neural
progenitor cells (NPCs). These NPCs can then differentiate to
more mature cell types of the central nervous system (CNS).
Neural aggregates in STEMdiff™ Neural Induction Medium

rapidly and efficiently form neural rosettes (Figure 21) – which
can then be isolated for further experiments using enzyme-free
Figure 21. Neural Induction is Efficient Using STEMdiff™ Neural STEMdiff™ Neural Rosette Selection Reagent (Figure 22). This
Induction Medium is a complete and highly efficient system to obtain enriched
Neural aggregates of 10,000 cells each were formed and cultured in an NPCs within 12 days without manual rosette isolation.
AggreWell™800 plate in STEMdiff™ Neural Induction Medium for 5 days
with daily medium changes. Neural aggregates were then harvested and
plated onto poly-L-ornithine/laminin (PLO/L)-coated plates. Attached neural
aggregates are shown 2 days after attachment with neural rosette structures
clearly visible. In this example, 100% of attached neural aggregates are filled
with neural rosettes. (Obj. 2x)
Figure 23. Neural Progenitor Cells Derived in STEMdiff™ Neural

Induction Medium After Single Cell Passage
Neural progenitor cells derived in STEMdiff™ Neural Induction Medium
immunostained for the neural progenitor markers SOX1 (green) and ZO-1
(red). (Obj. 10x)
Figure 22. Neural Rosettes are Isolated from the Surrounding
Flat Cells After Treatment with STEMdiff™ Neural Rosette PRODUCT SIZE Catalog #
Selection Reagent
The STEMdiff™ Neural Rosette Selection Reagent selectively lifts neural STEMdiff™ Neural
100 mL 05831
rosette structures from attached neural aggregates , leaving the surrounding Induction Medium
flat cells behind. (Obj. 2x)
STEMdiff™ Neural Rosette
100 mL 05832
Selection Reagent
16
STEMdiff™ APEL™ Medium

Defined, Animal Component-Free Basal Medium for hPSC Differentiation
Advantages of STEMdiff™ APEL™

Medium
• Defined and animal component-free
• Optimized for use with cells maintained in mTeSR™1
• Versatile, growth factor-free formulation
• Published protocols for differentiation to ectoderm,
endoderm and mesoderm
STEMdiff™ APEL™ Medium is a defined, serum-free and animal

component-free medium specifically developed to support
hPSC differentiation along ectoderm, mesoderm and endoderm
lineages. Developed by STEMCELL Technologies and based
100
on a published formulation,22 STEMdiff™ APEL™ Medium
90
is a versatile, growth factor-free, basal medium that is to be
80
Percent Beating EBs
70 supplemented with specific lineage-inducing factors. Optimized

60 to be used following maintenance in either mTeSR™1 or TeSR™2
50
media, this medium can be used to differentiate cells in EB-
40
based (AggreWell™) or adherent cell-based systems.
30
20
10
WLS-4D1 hiPSC line H9 hESC line
0
1 2 3 4 5 6 7 8 9 10 4 10 4
2.45% 5.63% 6.10% 5.30%
Experiment Number
10 3 10 3
Figure 24. Differentiation of Human ES Cells into Cardiomyocytes

CD45 APC
CD45 APC
102 102
Using STEMdiff™ APEL™ Medium
H9 cells were differentiated into cardiomyocytes based on Yang et al.20 with 101 101
the following changes: (1) STEMdiff™ APEL™ Medium was substituted for
80.4% 11.5% 69.5% 19.1%
StemPro®-34 SFM as a basal medium; (2) cells were maintained prior to 10 0 10 0
0 101 102 10 3 10 4 0 101 102 10 3 10 4
differentiation for at least 10 passages in mTeSR™1 Medium on Matrigel™; CD34 PE CD34 PE
(3) EBs were formed in AggreWell™400 plates, and (4) the entire procedure
was carried out within the AggreWell™400 plate. Beating EBs were counted
on Day 16 of culture, and varied between 5% and 95% of total EBs (n=9). Figure 26. Differentiation of hPSCs into Hematopoietic Cells Using
STEMdiff™ APEL™ Medium
WLS-4D1 cells (left) and H9 cells (right) were differentiated based on Ng
10 4 H9 cells were differentiated based et al.22 and Chadwick et al.,23 with the following changes: (1) STEMdiff™
on Rezania et al.21 with the following APEL™ Medium was substituted for DMEM with 20% FBS as a basal medium;
changes: (1) STEMdiff™ APEL™
10 3 (2) cells were maintained prior to differentiation for at least 10 passages in
Medium was substituted for RPMI
CXCR4-PE
with 2% BSA as a basal medium; mTeSR™1 Maintenance Medium on Matrigel™; (3) the entire differentiation
102 procedure was performed in adherent cell culture, on a Matrigel™ coated
and (2) cells were maintained prior
to the differentiation for at least surface instead of an EB-based method. Cells were analyzed by flow
101 10 passages in mTeSR™1 Medium cytometry on Day 13 of differentiation culture for expression of hematopoietic
on Matrigel™. On Day 4, cells were markers CD34 and CD45.
10 0 highly positive for CXCR4 and
10 0 101 102 10 3 10 4 SOX17 markers, which together
SOX17-APC characterize definitive endoderm. PRODUCT SIZE Catalog #
STEMdiff™ APEL™
Figure 25. Differentiation of hPSCs into Definitive Endoderm Using 100 mL 05210
Medium
STEMdiff™ APEL™ Medium and Published Cytokines
17
Support Reagents
A variety of support products are available to accompany StemCell Technologies’ array of specialized products for human ES and
iPS cell research. Please visit www.stemcell.com for more details and a full list of tissue culture reagents and supplies.
Tissue Culture Media Miscellaneous Tissue Culture Reagents & Supplies

PRODUCT Size Catalog # PRODUCT Size Catalog #
DMEM with 4500 mg/L D-glucose 500 mL 36250 3% Acetic Acid with
100 mL 07060
Methylene Blue
DMEM with 1000 mg/L D-glucose 500 mL 36253
Collagen Solution (3 mg/mL) 35 mL 04902
DMEM/F-12 500 mL 36254
Fibronectin (1 mg/mL) 1 mL 07159
Iscove’s MDM (IMDM) 500 mL 36150
Gelatin (0.1% in water) 500 mL 07903
Hypoxia Chamber 1 chamber 27310
Balanced Salt Solutions Rat Serum
2 mL 13551
5 x 2 mL 13561
PRODUCT Size Catalog #
Sodium Pyruvate (100 mM) 100 mL 07000
D-PBS 500 mL 37350 Trypan Blue 100 mL 07050
D-PBS, 10X 500 mL 37354 1 mg 07171
Y-27632 (ROCK inhibitor)
HBSS, Ca ++
& Mg
++
free 500 mL 37250 5 mg 07172
HBSS, without Phenol Red 500 mL 37150

Tissue Culture Dishes
PRODUCT Size Catalog #
Enzymes
10/pack 27115
PRODUCT Size Catalog # 35 mm Diameter
500/case 27116
AccutaseTM 100 mL 07920 10/pack 27120
60 mm Diameter
400/case 27121
Collagenase 5 mL 07902
10/pack 27125
Collagenase Type IV 100 mL 07909 100 mm Diameter
240/case 27127
Dispase (1 mg/mL) 100 mL 07923 4/pack 27140
245 mm x 245 mm
16/case 27141
Dispase (5 mg/mL) 100 mL 07913
1/pack 27135
DNase I (1 mg/mL) 1 mL 07900 96-Well Plates
50/case 27136
Trypsin-EDTA (0.05%) 500 mL 07910
Trypsin-EDTA (0.25%) 500 mL 07901 Recombinant Cytokines

Trypsin in Citrate Saline 100 mL 07400 PRODUCT Size Catalog #
Activin A 10 µg 02528
BAFF 20 µg 02517
Antibiotics
Bone Morphogenetic Protein-2 10 µg 02523
PRODUCT Size Catalog # Bone Morphogenetic Protein-4 10 µg 02524
Neomycin (G418) 250 mg 03812 Noggin 20 µg 02525
Hygromycin B 100 mg 03813 Basic Fibroblast

25 µg 02634
Growth Factor (bFGF)
2 µg 02647
Transforming Growth Factor-β1
10 µg 02847
18
References
1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, 13. Ware CB, Nelson AM, Blau CA. Controlled-rate freezing of
Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell human ES cells. Biotechniques 38:879-80, 882-3, 2005
lines derived from human blastocysts. Science 282:1145-7, 14. Richards M, Fong CY, Tan S, Chan WK, Bongso A. An
1998 efficient and safe xeno-free cryopreservation method for the
2. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, storage of human embryonic stem cells. Stem Cell 22:779-89,
Tomoda K, Yamanaka S. Induction of pluripotent stem cells 2004
from adult human 15. Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka
fibroblasts by defined factors. Cell 131:861-72, 2007 O, Amit M, Soreq H, Benvenisty N. Differentiation of human
3. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, embryonic stem cells into embryoid bodies compromising the
Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, three embryonic germ layers. Mol Med:6:88-95, 2000
Slukvin II, Thomson JA. Induced pluripotent stem cell lines 16. Kurosawa H. Methods for inducing embryoid body formation:
derived from human somatic cells. Science 318:1917-20, in vitro differentiation system of embryonic stem cells. J Biosci
2007 Bioeng 103:389-398, 2007
4. Park IH, Zhao R, West JA, Yabuuchi A, Hu H, Ince TA, Lerou 17. Bauwens C L et al. Control of human embryonic stem
PH, Lensch MW, Daley GQ. Reprogramming of human cell colony and aggregate size heterogeneity influences
somatic cells to pluripotency with defined factors. Nature differentiation trajectories. Stem Cells 26:2300-2310, 2008
451:141-6, 2008
18. Ungrin MD, Joshi C, Nica A, Bauwens C, Zandstra PW.
5. Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A. Reproducible, ultra-high throughput formation of multicellular
Embryonic stem cell lines from human blastocysts: somatic organization from single cell suspension-derived human
differentiation in vitro. Nat Biotechnol 18:399-404, 2000 embryonic stem cell aggregates. PLoS One 3(2):e1565, 2008
6. Ludwig TE, Bergendahl V, Levenstein ME, Yu J, Probasco 19. Markway BD et al. Enhanced chondrogenic differentiation
MD, Thomson JA. Feeder-independent culture of human of human bone marrow-derived. Mesenchymal stem
embryonic stem cells. Nat Methods 3:637-46, 2006 cells in low oxygen environment micropellet cultures. Cell
7. Ludwig TE, Levenstein ME, Jones JM, Berggren WT, Mitchen Transplantation. 19:29–42, 2010
ER, Frane JL, Crandall LJ, Daigh CA, Conard KR, Piekarczyk 20. Yang L, Soonpaa MH, Adler ED, Roepke TK, Kattman SJ,
MS, Llanas RA, Thomson JA. Derivation of human embryonic Kennedy M, Henckaerts E, Bonham K, Abbott GW, Linden
stem cells in defined conditions. Nat Biotechnol 24:185-7, RM, Field LJ, Keller GM. Human cardiovascular progenitor
2006 cells develop from a KDR+ embryonic-stem-cell-derived
8. Nagaoka M, Si-Tayeb K, Akaike T, Duncan SA. Culture of population. Nature. 453:524-528, 2008
human pluripotent stem cells using completely defined 21. Rezania A, Riedel MJ, Wideman RD, Karanu F, Ao Z,
conditions on a recombinant E-cadherin substratum. BMC Warnock GL, Kieffer TJ. Production of functional glucagon-
Dev Biol 10:60, 2010 secreting cells from human embryonic stem cells. Diabetes.
9. Chen G, Gulbranson DR, Hou Z, Bolin JM, Ruotti V, Probasco 60:239-247, 2011
MD, Smuga-Otto K, Howden SE, Diol NR, Propson NE, 22. Ng ES, Davis R, Stanley EG, Elefanty AG. A protocol
Wagner R, Lee GO, Antosiewicz-Bourget J, Teng JM, describing the use of a recombinant protein-based, animal
Thomson JA. Chemically defined conditions for human iPSC product-free medium (APEL) for human embryonic stem cell
derivation and culture. Nat Methods.8(5):424-429, 2011 differentiation as spin embryoid bodies. Nat Protoc. 3:768-
10. Fujioka T, Yasuchika K, Nakamura Y, Nakatsuji N, Suemori H. 776, 2008
A simple and efficient cryopreservation method for primate 23. Chadwick K, Wang L, Li L, Menendez P, Murdoch B, Rouleau
embryonic stem cells. Int J Dev Biol 48:1149-54, 2004 A, Bhatia M. Cytokines and BMP-4 promote hematopoietic
11. Ha SY, Jee BC, Suh CS, Kim HS, Oh SK, Kim SH, Moon SY. differentiation of human embryonic stem cells. Blood.
Cryopreservation of human embryonic stem cells without the 102:906-915, 2003
use of
a programmable freezer. Hum Reprod 20:1779-85, 2005
12. Ji L, de Pablo JJ, Palecek SP. Cryopreservation of adherent
human embryonic stem cells. Biotechnol Bioeng 88:299-312,
2004
19
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INFO@STEMCELL.COM • TECHSUPPORT@STEMCELL.COM
FOR GLOBAL CONTACT DETAILS VISIT OUR WEBSITE
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DOCUMENT #29063 VERSION 5.1.0 apRil 2013
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Your Guide to

Maximize Your Plurip tential

6 TeSR™-E8™ and Vitronectin XF™

8 TeSR™2 and StemAdhere™ Defined Matrix for hPSC

10 mFreSR™ and CryoStor™CS10

Human Pluripotent Stem Cells

Developed at the: Advantages of mTeSR™1

mTeSR™1 is a standardized medium for the feeder-free

published feeder-free pluripotent

hESC and hiPSC Lines Maintained in mTeSR™1

HESM01*, HESM02*, HS237, HS239, HS360,

Banked at WISC bank at WiCell:

Figure 1. Morphology of Human ES Cells Cultured in mTeSR™1

Figure 4. Human ES Cells Cultured in mTeSR™1 Retain Normal

Human iPS cell photographs courtesy of M. O'Connor and C. Eaves, The

Figure 3. Morphology of Human ES Cells Cultured in mTeSR™1

New TeSR™-E8™ is a highly defined feeder-free culture

A Component mTeSR™1 E8*

500 µm 25 µm L-Ascorbic Acid • •

Customize Your Passaging 45

Schedule: Passage Anytime 40

Average Fold Expansion/Passage

Medium Medium Medium 5-6

Small Low Low 6-7

For a detailed guide to customizing your passaging schedule,

Advantages of TeSR™2 and

Given current interest in using ES and iPS cells for applications

PRODUCT SIZE Catalog #

TeSR™2 500 mL Kit 05860

StemAdhere™ is a trademark of Primorigen Biosciences®. StemAdhere™ is developed and manufactured by

mFreSR™ CryoStor™ CS10

Ideally used after culture in mTeSR™1 or TeSR™2

Conventional cryopreservation methods for human pluripotent

% clumps giving rise

Antibodies for Characterization

Oct-3/4 Antibody 01550 Human, Mouse IgG1 κ (Mouse)

Human, Mouse, Rat,

Anti-Human TRA-2-54 Human, Chimpanzee,

High Purit y. Reliably obtain purities of

Pluripotent stem cells can be enriched from a mixed population

Figure 14. Typical Flow Cytometry Histogram Results Using

EasySep™ Human ES/

*Required equipment: EasySep™ Magnet (Catalog #18000)

FREE SAMPLE KIT

Fully Automated Cell Separation Add EasySep™

RoboSep™ (Catalog #20000), the fully automated cell separa-

Start: 8.3% CD34+ Cells Selected: 97.2% CD34+ Cells

Pour off supernatant.

product Capacity catalog #

EasySep™ hESC-Derived CD34 for 1 x 109

*Required equipment: EasySep™ Magnet (Catalog #18000)

Many pluripotent stem cell differentiation protocols begin with

EB formation is accomplished by adding a single cell

PRODUCT SIZE CATALOG #

AggreWell™400 Plate 1/pack 27845

(8 inserts in 24 wells) 5/pack 27945

AggreWell™400Ex Plate 1/pack 27840

AggreWell™ 800 Plate 1/pack 27865

STEMdiff™ Definitive Endoderm Kit

• Optimized for use with cells maintained in mTeSR™1

• Efficient and reproducible differentiation of multiple

• Differentiated cells co-express SOX17, CXCR4,

• Convenient, user-friendly format and protocol

HESM01, HESM02, HS237, HS239, HS360,