RAISE THE BARWith the New Standard for
hPSC Maintenance

4

The Evolution of mTeSR™

6

Versatility Through Stabilization

8

Enhanced Growth and Morphology

10

Validation of Pluripotency

12

Genetic Analysis

14

Guide to Genome Editing in mTeSR™ Plus

16

Ectoderm Differentiation

18

Mesoderm Differentiation

20

Endoderm Differentiation

22

Frequently Asked Questions

23

References

Why Use mTeSR™ Plus?
STABILIZED.
Enhanced buffering and stabilized components, including FGF2, support cell quality
while allowing for alternate feeding schedules.
ENHANCED GROWTH.
Supports superior culture morphology and cell growth characteristics.
EFFICIENT CLONING.
Enables heightened single-cell survival when supplemented with CloneR™.
STREAMLINED.
Fully compatible with established genome editing and differentiation protocols.

Catalog #05825 | www.stemcell.com/mTeSRPlus

THE EVOLUTION OF mTeSR™

“TeSR1” was created in the laboratory of Prof. James Thomson at the University of Wisconsin-Madison in 2006 by Dr. Tenneille Ludwig
and colleagues. This was the first feeder-independent cell culture medium for human embryonic stem (ES) cells containing protein
components derived solely from recombinant sources or purified from human material. First manufactured for commercial sale by
STEMCELL Technologies in 2007, “modified” TeSR1 (mTeSR™1) went on to become the global standard for feeder-free human pluripotent
stem cell (hPSC) maintenance, supporting thousands of scientific publications and discoveries in the stem cell research field.

1998
The first human ES cell lines are derived in Prof.
James Thomson’s laboratory at the Universityof Wisconsin-Madison. Cells are maintained in
undefined serum-containing medium on a bed ofmouse embryonic fibroblast (MEF) “feeder” cells.¹

2000
Michal Amit and colleagues discoverthat, in the absence of serum, FGF2 isrequired to maintain human ES cells in theundifferentiated state.²

2001
Chunhui Xu establishes the first feeder-freemethod for human ES cell maintenance using
MEF-conditioned medium.³

2006
2007

Researchers in the James Thomson laboratory
report the first feeder-independent human ES
cell culture medium, “TeSR1”, containing protein
components derived solely from recombinantsources or purified from human material.4

Shinya Yamanaka demonstrates the generationof induced pluripotent stem (iPS) cells fromadult human dermal fibroblasts with four
factors: Oct3/4, Sox2, Klf4, and c-Myc.5

2011

2013

Researchers in the James Thomson laboratory
develop E8™ medium, a minimal formulation
containing only the critical components requiredfor hPSC maintenance.6 STEMCELL Technologies
went on to release “TeSR™-E8™” in 2013.

Feng Zhang describes Cas9-mediatedgenome editing in human ES cells
maintained in mTeSR™1.7

2019
mTeSR™ Plus is developed with enhanced stability
to ensure cell quality while allowing for truly
versatile hPSC maintenance.

4

STEMCELL Technologies Inc.

Research & Development, Pluripotent Stem Cell Biology
As scientists, we know that there’s nothing more important thanhaving results that speak the truth. Supporting the work of hPSC
researchers over the past decade has inspired us to continue
innovating the highest quality of tools available — so that you
always have confidence while pursuing your next breakthrough.

VERSATILITY
THROUGHSTABILIZATION

Medium color indicates the pH
difference after hPSCs were
cultured for 72 hours without
feeding in either mTeSR™1 (left)
or mTeSR™ Plus (right). For eachmedium, cells were at a densityof approximately 2 x 106 cells perwell of a 6-well plate at the endof the culture period.

6

STEMCELL Technologies Inc.

Work Smarter, Not Harder.
Free up your days with just two golden rules, and nothing more.

Maintain hPSCs On Your Own Schedule Skip 2 days = Double feed
 Skip 1 day = Regular feed
The possibilities are endless. Use your regular schedule, or
try something new to free up your days.

PASSAGINGFREQUENCY

MON

TUE

WED

THU

FRI

SAT

SUN

7d

P

F

F

F

F

F

F

repeat

7d

P

F

F

F

2F

x

x

repeat

6d

P

x

2F

x

x

F

5d

P

F

2F

x

x

3d/4d

P

F

x

P

2F

repeatrepeatxx

repeatFill OutYour Own

P = Passage; F = Single Feed; 2F = Double Feed

Sustained pH and Stabilized Components, Including FGF2
mTeSR™1% FGF remaining relative to T=0

mTeSR™ Plus
Competitor S

7.5

pH

pH relative to T=0% FGF remaining

mTeSR™1

100

50

7.0

6.5
0

00

20

1× 10 6 40

2× 10
60

6

3× 10 680

NumberCells After 72 hours
Timeof(hours)

100

mTeSR™ Plus
Competitor S

50

0

0

20

40

Time (hours)

60

80

Figure 1. mTeSR™ Plus Maintains Optimal pH Levels Throughout
a Weekend-Free Protocol

Figure 2. mTeSR™ Plus Maintains Consistent Levels of FGF2
Throughout a Weekend-Free Protocol

The pH of spent medium from hPSCs cultured in mTeSR™ Plus is higher than thatof hPSCs cultured in mTeSR™1 and other flexible-feeding medium at similar celldensities. pH and cell numbers were measured after a 72-hour period without
feeding. Range of cell numbers shown represent different densities that would
be observed throughout a typical passage. This demonstrates that feeds can beskipped for two days at any time during routine maintenance using mTeSR™ Plus
while maintaining a pH above 7.0. Note: Cultures were fed double the standard
medium volume prior to the 72-hour period without feeds in all media and cellnumbers are from one well of a 6-well plate.

FGF2 levels remain high in mTeSR™ Plus when kept at 37°C over a 72-hour time
period. Measured by ELISA.

Tested to WorkTo test the limits of mTeSR™ Plus and to ensure that the medium upholds cell quality under rigorous standards, all mTeSR™ Plus performance testing
was completed using a highly reduced feeding schedule, as shown below.

Restricted

MON

TUE

WED

THU

FRI

SAT

SUN

P

-

F

-

2F

-

-


WWW.STEMCELL.COM

7

ENHANCEDGROWTH AND
MORPHOLOGY

Human ES cells (H9)cultured for 15 passages
in mTeSR™ Plus. DAPI
(cyan), tubulin (orange),phalloidin (magenta), andspindle pole (CDK5RAP2;
yellow).

8

STEMCELL Technologies Inc.

Superior Morphology of Undifferentiated hPSC Colonies
Day 5

A

B

Day 6

Day 7

mTeSR™1

Uniform, densely packed
colonies with defined borders
are indicators of high cell
quality.

mTeSR™ Plus

Dense centers that arephase-bright indicate healthycolonies that are ready forpassaging.

Figure 3. Human ES Cells in mTeSR™ Plus Display Improved Morphology and Larger Colony Size When Feeding is Restricted
Human ES cells (H9) cultured in (A) mTeSR™1 or (B) mTeSR™ Plus with restricted feeding on Corning® Matrigel®. Images were taken on days 5, 6, and 7 after seeding. PSC
colonies grown in mTeSR™ Plus are larger in size and exhibit tighter cellular packing at the edges, resulting in greater border definition compared to mTeSR™1 when feedingis restricted.

80
70
60
50

H1

90
80
70
60
50

H9H1

M001
H9

WLS-1C
M001

B

90
80
70
60
50

WLS-1C100% TRA 1-60 Expression

90

100

% TRA 1-60 Expression% OCT4 Expression

100% OCT4 Expression

100

A

H1

mTeSR™1mTeSR™1mTeSR™ Plus mTeSR™ Plus

90
80
70
60
50

M001
H9

H9H1

WLS-1C
M001

WLS-1C

Figure 4. Cells Cultured in mTeSR™ Plus Medium with Restricted Feeding Express Undifferentiated Cell Markers
Human ES (H1, H9) and iPS (WLS-1C, STiPS-M001) cells were characterized using flow cytometry for undifferentiated cell markers, (A) OCT3/4 and (B) TRA-1-60. Graphs show
average expression (± SEM) results from analyses of duplicate wells every 5 passages for up to 10-15 passages in mTeSR™1 (daily feeds), or mTeSR™ Plus (restricted feeds).

Larger Colony Size and More Rapid Expansion
4× 10 6mTeSR™1

Colony Size (um)mTeSR™ Plus
3000

2000

Daily Feeding
3× 10 6

Restricted Feeding

2× 10 6mTeSR™ Plus
Daily Feeding
Restricted Feeding

Number of Cells

mTeSR™1

4000

1× 10 6

1000

0

0

H1

H9

M001

WLS-1C

0

2

4

6

8

Days in Culture

Figure 5. Larger Colonies Are Observed in mTeSR™ Plus

Figure 6. mTeSR™ Plus Supports Higher Cell Numbers

The average colony size per passage (± SEM) was obtained for human ES (H1,
H9) and iPS (STiPS-M001, WLS-1C) cells cultured in mTeSR™1 (daily feeds) or
mTeSR™ Plus (restricted feeds) on Corning® Matrigel® over 10 passages. Size
was determined by measuring representative colony diameters at harvest. Notethat this data is representative of cultures passaged at a 7-day passaging interval;
smaller colony size should be expected if using shorter passaging intervals.

Growth curves were obtained for human ES (H9) cells cultured in mTeSR™1 or
mTeSR™ Plus on Corning® Matrigel® matrix over 7 days with either daily feeds
or restricted feeds. Growth curves were determined by seeding 20,000 cells perwell of a 6-well plate as aggregates and counting the cell numbers each day induplicate wells.

WWW.STEMCELL.COM

9

VALIDATION OF
PLURIPOTENCY
Efficient Differentiation Into the Embryonic Germ Layers

hPSC

NANOG
POU5F1
DNMT3B
CDH1
SOX2
PAX6

Ectoderm

OTX2
NES
TFAP2A
FABP7
DLK1
LHX2
RAX
HES5

T
HAND1
PDGFRA
TBX6
CDX2
APLNR
MIXL1
CXCR4
KIT
GSC

0

16

100

Mesoderm(T+NCAM+)

80

mTeSR™1mTeSR™ Plus

60
40
20
0

Figure 8. Cells Maintained in mTeSR™ Plus with Restricted
Feeding Have Comparable Differentiation Efficiencies to Cells
Maintained in mTeSR™1Human ES (H1, H9) and iPS (WLS-1C, STiPS-M001) cells were maintainedin mTeSR™1 (daily feeds) or mTeSR™ Plus (restricted feeds). Cells weredifferentiated using directed differentiation protocols and subjected toflow cytometry analysis. Graphs show average expression (± SEM) resultsfrom the 4 cell lines. The markers used for flow cytometry for each germlayer are listed in the bar titles.

CER1
EOMES
FOXA2
GATA4
GATA6
HHEX
LEFTY1AFP
SOX7

Figure 7. Molecular Analysis of hPSCs Cultured in mTeSR™ Pluswith Restricted Feeding Show Efficient Differentiation to All
Three Germ Layers
Human iPS (WLS-1C) cells were maintained in mTeSR™ Plus on a restricted
feeding schedule and directed to each of the three germ layers using directeddifferentiation protocols. Undifferentiated cells, differentiated ectoderm,
mesoderm, and endoderm cells were then analyzed using the hPSC Trilineage
Differentiation qPCR Array to evaluate expression levels of key germ layer
markers. Cells cultured with mTeSR™ Plus and differentiated showed clear
upregulation of appropriate germ layer-specific markers.

How is Pluripotency Assessed?
The potency of hPSCs, and their ability to differentiate
into the three primary germ layers, can be evaluated using
a number of established strategies. While the teratoma
assay is known as the “gold standard” technique, it can belengthy, cost prohibitive, and variable. Another common
method, embryoid body differentiation, is also inherently
variable. Fortunately, recent advances in monolayer
trilineage differentiation protocols have enabled the rapid
validation of pluripotency with more ease-of-use andspecificity than before.

STEMdiff™ Trilineage Differentiation Kit • Format: 1 Kit • Catalog # 05230
hPSC Trilineage Differentiation qPCR Array • Format: 384-Well Plate • Catalog # 07515

10

Endoderm(CXCR4+SOX17+)

SOX17

Endoderm

Normalized Gene
Expression (rlog 2)-8

Mesoderm

NCAM1% Positive Cells

Ectoderm(PAX6+Nestin+)

STEMCELL Technologies Inc.

ECTODERM

MESODERMENDODERM

Human iPS (WLS-1C) cells cultured with mTeSR™ Plus were differentiated following directed differentiation
protocols. Immunohistological analysis of each germ layer displays lineage-specific markers PAX6 (purple),
Brachyury (cyan), and SOX17 (red). Actin is counterstained with Phalloidin (green).

GENETICANALYSIS

Karyotype of human iPS (WLS-1C) cells at passage 12, cultured in mTeSR™ Plus.

12

STEMCELL Technologies Inc.

Maintenance of Genomic Integrity
H1

WLS-1C

Chromosome Analysis Report: 073921

Copy Number

Date Reported: Monday, November 19, 2018
Cell Line: H1
Passage#: 37(P12)
Date of Sample: 11/14/2018
Specimen:

Cell Line Sex:

Copy NumberA

Human ES

Results: 46,XY
Nonclonal Findings: 47,XY,+22

Male

Figure 9. hPSCs Cultured in mTeSR™
Plus with Restricted Feeding Maintain
Normal Karyotype
Chromosomea Analysis
Report: 073920

Human ES (H1) and iPS (WLS-1C) cells were
cultured in mTeSR™ Plus for 15 passages. No
Date Reported: Tuesday, November 20, 2018
Cellcommon
Line Sex:
Male were detected by theabnormalities
Investigator:
Matthew Wong, StemCell Technologies
Cell Line:
1C
Reasonfor Testing:Routine(A) hPSC
Genetic Analysis
KitKaryotyping
at p5, 10, or 15(passage 15 shown) and cultures displayed a
Passage#: 32(P12)
normal karyotype
by (B)Wong,
G-bandingat p12.Technologies
Date of Sample: 11/14/2018
Investigator:
Matthew
StemCellReason for Testing: Routine Karyotyping

Specimen:

Human IPS

Results: 46,XY

B
Cell: 38

Cell: 6

Slide: G02
Slide Type: Karyotype
Total Counted:

20

Slide: G03
Slide Type: Karyotype
Total Counted:

20

Total Analyzed: 8

Total Analyzed: 8

Total Karyogrammed: 4

Total Karyogrammed: 4Band Resolution: 450 - 500

Band Resolution: 425 - 525Interpretation:

Interpretation:This
is a normal
karyotype;no clonal
were detected at the stated band level of resolution.
Consistent
hPSC
Gene
Expression
ProfileThis is a normal
karyotype;no clonal
abnormalities
weredetectedat the statedbandabnormalities
level of resolution.

Why Monitor
Genetic Stability?

Completedby: may result fromLeah
George,CG(ASCP)
There is a nonclonal finding, listed above. Nonclonalfindings
technical
artifact,but may be
H9M001
due to a developing clonal abnormality
or to low-levelmosaicism.
Reviewed
and Interpreted by: Jennifer Laffin, PhD, FACMG
mTeSR™1

Completed by:

mTeSR™ PlusmTeSR™1

Kate Bird, CG(ASCP)mTeSR™ Plus

hPSCs have the capacity to
QC Review By: ____
acquire karyotypic abnormalities
Limitations: This assay allows for microscopic visualization of numerical and structural chromosome
abnormalities.
The
size
of
structuralabnormality that can be detected
in a non-random and sporadicis >3-10Mb, dependent upon the G-band resolution obtained from this specimen. For the purposes of this report, band level is defined as the number of G-bands per
haploid genome. It is documented here as “band level”, i.e., the range of bands determined from the four karyograms in this assay. Detection of heterogeneity of clonalmanner during routine culture.
Date:_______________________ Sent By:____ cellSent
To:_____________________
Review
By: ____
populationsin this specimen (i.e.,mosaicism) is limited byQC
the numberof metaphasecells examined, documented here as “# of cells counted”.
Themechanisms
behindtheseThisstructural
assay wasconducted solely
for listed investigator/institution.
results of that
this assay
for researchuse only. Unlessotherwise
mutually agreed in writing, theLimitations: This assay allows for microscopic visualization of numerical andchromosome
abnormalities.
The size of structuralTheabnormality
can beare
detected
services For
provided
to you hereunderby WiCell
Institute,
aregovernedsolely by WiCell’s Terms and Conditions of Service, found atis >3-10Mb, dependent upon the G-band resolution obtained from this specimen.
the purposesof this report,
bandResearchlevel is definedasInc.
the(“WiCell”)
number of
G-bands
percytogenetic
changesremainwww.wicell.org/privacyandterms. Any terms you may attach to a purchase order or other document that are inconsistent, add to, or conflict with WiCell’s Terms andhaploid genome. It is documented here as “band level”, i.e., the range of bands determined from the four karyograms in this assay. Detection of heterogeneity of clonal
Conditions of Service are null and void and of no legal force or effect.
cell populations in this specimen (i.e.,mosaicism) is limited by the number of metaphase cells examined, documented here as “# of cells counted”.
unknown. A number of theseThis assay was conducted solely for listed investigator/institution. The results of this assay are for research use only. Unless otherwise mutually agreed in writing, recurrent
theabnormalities areservices provided to you hereunder by WiCell Research Institute, Inc. (“WiCell”) are governed solely by WiCell’s Terms and Conditions of Service, found atwww.wicell.org/privacyandterms. Any terms you may attach to a purchase order or other document that are inconsistent, add to, or conflict with WiCell’s Terms andalso observed in humanConditions of Service are null and void and of no legal force or effect.
cancers, raising safety concerns
over using these cells fortherapeutic applications. The
most commonly affected
chromosomes in hPSCs include
1, 8, 10, 12, 17, 18, 20, and
X. Recent studies suggest that
Page 1 of 1
WiCell Research Institute - WiCell Characterization- POResearchBox 7365Institute
Madison- Wisconsin- 53707-7365
acidosisof culture
mediummay
Page 1 of 1 Lab
WiCell- WiCell
Characterization
Lab - PO Box
7365 Madison- Wisconsin- 53707-7365
be associated with increased
levels of DNA damage, genomic
Figure 10. Cells Maintained in mTeSR™ Plus with Restricted Feeding Are Equivalent to Cells
instability, and growth arrest of
Maintained in mTeSR™1 with Daily Feeding
hPSCs. Due to the stabilization
Human ES (H9) and iPS (STiPS-M001) cells were cultured for at least 10 passages with either mTeSR™1 (daily feeds)
enhancements of mTeSR™
or mTeSR™ Plus (restricted feeds). Transcriptome analysis of hPSCs maintained in mTeSR™ Plus using RNAseq shows
Plus, medium pH is sustained,a gene expression profile indistinguishable from cultures maintained in mTeSR™1. Heat map displays all 19,665
reducing the rapid onset ofgenes measured for each condition.
acidosis.
18

Normalized Gene
Expression (rlog 2)

Reviewed and Interpreted by: Jennifer Laffin, PhD,
FACMG
Date:_______________________
Sent By:____ Sent To:_____________________
7-2

hPSC Genetic Analysis Kit • Format: 1 Kit • Catalog # 07550

WWW.STEMCELL.COM

13

GUIDE TO
GENOME EDITING
IN mTeSR Plus™

ArciTect™ Cas9 Nuclease • Catalog #76002
ArciTect™ crRNA • Catalog #76010
ArciTect™ tracrRNA Kit • Catalog #76016

Step 1: Culture Cells and Prepare
Single-Cell Suspension

Step 2: Form Ribonucleoprotein(RNP) Complex

Step 3: Deliver RNP Complex
into hPSCs

gRNA

Cas9

For optimal results, collect cells during
exponential growth phase

Allow 10-20 minutes for RNP formation

Electroporate single cell suspensionof hPSCs

B

A

Knockout Efficiency(% GFP- Cells)

50
40
30
20
10
0

mTeSR™1 mTeSR™ Plus

Figure 11. Representative Cell Morphology 24 Hours After RNP
Electroporation in mTeSR™1 and mTeSR™ Plus
H1-eGFP ES cells were plated in (A) mTeSR™1 and (B) mTeSR™ Plus andsupplemented with CloneR™ immediately following RNP electroporation. Images
were taken 24 hours after electroporation.

14

STEMCELL Technologies Inc.

Figure 12. CRISPR-Cas9 Genome Editing Efficiency is Comparable
for Cells Cultured in mTeSR™1 with Daily Feeding and mTeSR™
Plus with Restricted Feeding
H1-eGFP ES cells were electroporated with RNP complexes targeting the eGFP
transgene (15:30 pmol Cas9:gRNA) and knockout efficiency measured 72 hours
following electroporation. Knockout efficiency(± SEM) was measured as % GFP negative (GFP-) cells in test condition subtracted
by % GFP- cells in non-electroporated controls.
n = 3 biological replicates.

Efficient Genome Editing of hPSCs
The ease-of-use and versatility of CRISPR-Cas9 has been pivotal to furthering ourunderstanding of how individual genes and genetic variants impact biology and disease
pathogenesis. Used in combination with stem cells, it has revolutionized the field. For
complex cell types like hPSCs, pre‑ and post-editing culture are critical steps that must
be optimized for experimental success during genome editing. mTeSR™ Plus works
seamlessly with our ArciTect™ CRISPR-Cas9 system for high-efficiency editing, and,
when supplemented with CloneR™, provides a robust culture system to support highlevels of post-editing survival for effective clonal derivation and expansion.

CloneR™ • Size: 10 mL • Catalog # 05888

Step 4: Recovery/Editing

Step 5: Analysis of Editing
Efficiency

Step 6: Derivation & Characterizationof Clonal hPSC Lines

Plate cells into mTeSR™ Plus media
supplemented with CloneR™ andallow cells to recover for 48 - 72 hours
following RNP delivery.

Depending on experimental design,
editing efficiency can be assessed using
the ArciTect™ T7 Endonuclease I Kit, throughflow cytometry-based approaches, or bygenomic DNA sequencing.

Plate cells at clonal density and use CloneR™
to enhance cloning efficiency. Use editing
efficiency calculated in Step 5 to estimate theapproximate number of clones to select for further
characterization.

50

Cloning Efficiency (%)

mTeSR™140

mTeSR™ Plus

30

A

B

20
10

ROCK Inhibitor (Y-27632)

W
LS
-1
C

H
9

H
1

W
LS
-1
C

H
9

H
1

0

CloneR™

Figure 13. High Cloning Efficiency of hPSCs in mTeSR™ Plus
Supplemented with CloneR™
hPSCs (H1, H9, and WLS-1C) plated in mTeSR™ Plus with CloneR™ demonstratecloning efficiencies equal to or greater than hPSCs in mTeSR™1 with CloneR™.
Cells were seeded at clonal density (25 cells/cm²) in mTeSR™1 or mTeSR™ Plus on
CellAdhere™ Vitronectin™ XF™-coated plates.

Figure 14. Clones Derived in mTeSR™ Plus are Larger and Readyto Be Picked at an Earlier Timepoint
Representative images of human ES (H9) colonies taken 8 days following singlecell plating at clonal density (25 cells/cm²) in either (A) mTeSR™1 or (B) mTeSR™
Plus supplemented with CloneR™ on CellAdhere™ Vitronectin™ XF™-coatedplates.

WWW.STEMCELL.COM

15

ECTODERM
DIFFERENTIATION

16STEMCELL Technologies Inc.

A

NeuralProgenitor
Cells

mTeSR™1B

mTeSR™ Plus

STiPS-M001

H9

Figure 15. Generation of Neural Progenitor Cells from hPSCs Maintained in mTeSR™ Plus
Human ES (H9) and iPS (STiPS-M001) cells were maintained in (A) mTeSR™1 with daily feeds or (B) mTeSR™ Plus with restricted feeds and differentiated using an embryoid
body (EB)-based protocol with STEMdiff™ SMADi Neural Induction Kit. Neural progenitor cells derived from hPSCs maintained in either mTeSR™1 or mTeSR™ Plus clearly
display neural rosettes (arrowheads) after replating EBs.

STEMdiff™ SMADi Neural Induction Kit • Format: 1 Kit • Catalog # 08581

A

mTeSR™1B

mTeSR™ Plus

Astrocytes
STiPS-M001

H9

Figure 16. Downstream Astrocyte Differentiation of Neural Progenitor Cells Derived from hPSCs Maintained in mTeSR™ Plus
Neural progenitor cells generated from human ES (H9) and iPS (STiPS-M001) cells maintained in (A) mTeSR™1 with daily feeds or (B) mTeSR™ Plus with restricted feeds
were differentiated and matured to astrocytes using the STEMdiff™ Astrocyte Differentiation Kit for 25 - 26 days and STEMdiff™ Astrocyte Maturation Kit for 18 days. The
astrocytes express GFAP (green), S100B (red), and DCX (blue).

STEMdiff™ Astrocyte Differentiation Kit • Format: 1 Kit • Catalog # 08540
STEMdiff™ Astrocyte Maturation Kit • Format: 1 Kit • Catalog # 08550

Cerebral Organoids
Human ES (H9) cells were cultured with mTeSR™ Plus and directed to cerebral organoids using the STEMdiff™ Cerebral Organoid Kit.
Image shows apical progenitor marker SOX2 (purple) and neuronal marker TBR1 (green).

STEMdiff™ Cerebral Organoid Kit • Format: 1 Kit • Catalog # 08570

WWW.STEMCELL.COM

17

MESODERM
DIFFERENTIATION18STEMCELL Technologies Inc.

Hematopoietic Progenitor Cells
A

B

Hematopoietic Progenitors

C

Total Cell Yield
500000# Viable Cells/cm2% CD34+CD45+ Cells

60

40

20

0

mTeSR™1

4000003000002000001000000mTeSR™ Plus
n=5

Figure 17. Generation of Hematopoietic Progenitor Cells from hPSCs Maintained in mTeSR™ PlusmTeSR™1mTeSR™ Plusn=4

Human ES (H1, H9) and iPS (STiPS-M001, WLS-1C) cell lines maintained in mTeSR™1 (daily feeds) or mTeSR™ Plus (restricted feeds) were differentiated to hematopoietic
progenitor cells using the STEMdiff™ Hematopoietic Kit. At the end of the differentiation period, cells were harvested from the supernatant and analyzed by flow cytometry
for co-expression of CD34 and CD45. (A) Representative density plots showing CD34+ and CD45+ expression, (B) percentage of cells co-expressing CD34+ and CD45+, and(C) total number of viable cells harvested are shown. Data are expressed as the mean (± SEM); n=4.

STEMdiff™ Hematopoietic Kit • Format: 1 Kit • Catalog # 05310

Cardiomyocytes
Human iPS (STiPS-M001) cells maintained with mTeSR™ Plus were directed to cardiomyocytes using the STEMdiff™ Cardiomyocyte Differentiation Kit.
Image shows markers of cardiomyocyte markers cardiac troponin T (cTNT; red) and alpha-actinin (green). Nuclei are counterstained with DAPI (blue).

A

B

1.5

C

Voltage (mV)

1

0.5

0

-0.5

0

5

10

15

Time (s)

20

25

30

Figure 18. Generation of Cardiomyocytes from hPSCs Maintained in mTeSR™ Plus
Human ES (H9) and iPS (WLS-1C) cells were maintained in mTeSR™1 (daily feeds) or mTeSR™ Plus (restricted feeds) and differentiated to cardiomyocytes using the
STEMdiff™ Cardiomyocyte Differentiation Kit. At the end of the differentiation period, cells were harvested and analyzed by microelectrode array (MEA) and flow cytometry.
(A) Representative MEA voltage recordings of cardiomyocytes (day 20) demonstrate a characteristic electrical profile and stable beat rate. (B) Percentages of cells expressingcTNT and (C) total number of viable cells harvested are shown. Data are expressed as the mean (± SEM); n=2.

STEMdiff™ Cardiomyocyte Kit • Format: 1 Kit • Catalog # 05120

WWW.STEMCELL.COM

19

ENDODERM
DIFFERENTIATION

20STEMCELL Technologies Inc.

mTeSR™1
2.76%

5

10

94.16%4

CXCR4 PECXCR4 PE

10

2

10

1

3

10

2

10

1

10

0

92.65%

10

3

10

2.92%

4

10

Definitive
Endoderm

Definitive Endoderm

mTeSR™ Plus
% CXCR4+SOX17+ Cells

5

10

10

1.99%
0

10

1.10%
1

10

2

10

3

10

SOX17 APC

4

10

0

10

5

0

10

1.42%

3.01%

10

1

10

2

10

3

10

SOX17 APC

4

10

5

10

100
80
60
40
20
0

mTeSR™1mTeSR™ Plus

Figure 19. Generation of Definitive Endoderm from hPSCs Maintained in mTeSR™ Plus(A) Representative density plots showing CXCR4 and SOX17 expression in cells cultured in mTeSR™1 (daily feeds) or mTeSR™ Plus (restricted feeds), following 5 days ofdifferentiation using the STEMdiff™ Definitive Endoderm Kit. (B) Quantitative analysis of definitive endoderm formation in multiple hPSC lines (H9, STiPS-M001, WLS-1C)
maintained with mTeSR™1 or mTeSR™ Plus as measured by co-expression of CXCR4 and SOX17. Data are expressed as the mean percentage of cells (± SEM) expressing bothmarkers; n=3.

STEMdiff™ Definitive Endoderm Kit • Format: 1 Kit • Catalog # 05110

7.48%

5

10

80.56%

4

PDX1 PE

10

2

10

1

72.34%

3

10

2

10

1

10
10

6.05%

10

3

0

Pancreatic Progenitors

mTeSR™ Plus

4

10

PDX1 PE

Pancreatic
Progenitor
Cells

mTeSR™1% PDX1+NKX6.1+ Cells

5

10

10

10.59%
0

10

1

10

1.37%
2

10

3

10

NKX6.1 APC

4

10

0

5

10

10

19.33%
0

10

1

10

2.28%
2

10

3

10

NKX6.1 APC

4

10

5

10

100
80
60
40
20
0

mTeSR™1mTeSR™ Plus

Figure 20. Generation of Pancreatic Progenitors from hPSCs Maintained in mTeSR™ Plus(A) Representative density plots showing PDX-1 and NKX6.1 expression in cells cultured in mTeSR™1 (daily feeds) or mTeSR™ Plus (restricted feeds), following differentiation
using the STEMdiff™ Pancreatic Progenitor Kit. (B) Quantitative analysis of pancreatic progenitor formation in multiple hPS (H9, STiPS-M001, WLS-1C) cell lines maintainedwith mTeSR™1 or mTeSR™ Plus as measured by co-expression of PDX-1 and NKX6.1. Data are expressed as the mean percentage of cells (± SEM) expressing both markers;
n=3.

STEMdiff™ Pancreatic Progenitor Kit • Format: 1 Kit • Catalog # 05120

Intestinal Organoids
Human ES (H9) cells were cultured with mTeSR™ Plus and directed to intestinal organoids using the STEMdiff™ Intestinal Organoid Kit. Image shows
markers of the intestinal epithelium EpCAM (green) and CDX2 (red), and intestinal mesenchyme marker vimentin (white). Nuclei are counterstainedwith DAPI (blue).

STEMdiff™ Intestinal Organoid Kit • Format: 1 Kit • Catalog # 05140

WWW.STEMCELL.COM

21

FREQUENTLY ASKED QUESTIONS
Q: How do I transition my cell lines into mTeSR™ Plus?
A: Cells cultured in feeder-free media can be conveniently transitioned into mTeSR™ Plus without any adaptation step. We routinely transition cell lines
maintained in mTesR™1 on Matrigel® directly into mTeSR™ Plus on Matrigel® and cells do not demonstrate a lag period following the first passage.
Often, an enhanced growth rate is observed immediately, resulting in larger colonies and a higher confluency sooner after passaging.
Q: Do I still need to feed my cells every day?
A: You do not! To skip a single day (48 hours), we recommend performing a “single feed” as usual (for example, 2 mL per well of a 6-well plate). To skip
two consecutive days (72 hours), perform a “double feed” (for example, 4 mL per well of a 6-well plate). Of course, cultures can still be fed every day
with mTeSR™ Plus—it’s all up to your desired schedule. To see some examples of feeding schedules, please refer to page 7.
Q: Do cell lines behave differently in mTeSR™ Plus?
A: Population doubling time is reduced in mTeSR™ Plus compared with mTeSR™1. Colonies appear larger and more dense, with well-defined edges. As
a result, cell seeding densities or passaging intervals may need to be adjusted. To keep the same passaging interval, we recommend seeding ~20 - 25%
fewer clumps at passage than would normally be seeded into mTeSR™1. You may wish to test a number of split ratios to determine which best suits yourspecific method of hPSC maintenance. For more detailed guidance, feel free to contact us.
Q: Do I need to change how I use my passaging reagent?
A: In some cases, the passaging reagent may need to be exposed to cells for a longer period of time in order to successfully dissociate the larger colonies
grown in mTeSR™ Plus. We recommend observing your cells under the microscope to ensure that colonies have dissociated sufficiently prior to scrapingor spraying.
Q: Can I thaw cells directly into mTeSR™ Plus?
A: Cryopreserved hPSCs previously maintained in mTeSR™1 can be thawed directly into mTeSR™ Plus with no additional adaptation step. In fact, you arelikely to see enhanced recovery rates of cryopreserved hPSCs thawed directly into mTeSR™ Plus.
Q: How do I passage hPSCs as single cells using mTeSR™ Plus?
Extensive passaging of hPSCs as single cells is not recommended in any maintenance medium due to the increased risk of obtaining karyotypicabnormalities using this method. If single-cell passaging is required, we recommend using CellAdhere™ Laminin-521 cell culture matrix (Catalog#77003). If using a matrix other than CellAdhere™ Laminin-521, the addition of CloneR™ (Catalog #05888) or other survival agents such as Y-27632(Catalog #72302) is advised.
In order to obtain a highly viable population of single cells free of aggregates, incubate with enzymatic dissociation reagent (e.g. Accutase™ Cell
Detachment Solution [Catalog #07920]) at 37° for ~5 minutes. We recommend passaging cells when they reach 80 - 90% confluency.
Recommended cell seeding densities:
3 - 4 day passaging = 25,000 cells/cm2
4 - 5 day passaging = 12,500 cells/cm2
This protocol may need to be optimized for specific cell lines. Please contact us if you would like a more detailed protocol.
Q: How do I derive clonal hPSC lines in mTeSR™ Plus?
CloneR™ supplement (Catalog #05888) is formulated to significantly increase cloning efficiency of hPSCs. CloneR™ is recommended for use with
mTeSR™ Plus when seeding cells at a low density. If seeding multiple cells per well to derive clones, we recommend plating at a maximum density of
25 cells/cm2 to prevent generation of mosaic colonies. We advise withdrawing CloneR™ from mTeSR™ Plus after 4 days of exposure, as this duration is
sufficient for robust clone development. For a complete protocol detailing clonal hPSC line derivation, please refer to the CloneR™ Product Information
Sheet, available at www.stemcell.com/cloner.
Q: Will mTeSR™ Plus be available as cGMP?
mTeSR™ Plus has been prioritized in our GMP manufacturing pipeline and will be produced under a cGMP quality management system compliant to
CFR 820 in the near future. Please look out for a notification regarding this change towards the end of 2019. Until that point, mTeSR™ Plus will be
manufactured under a quality management system certified to ISO 13485, Medical Devices.

22

STEMCELL Technologies Inc.

References
1.

Thomson JA et al. (1998) Embryonic stem cell lines derived fromhuman blastocysts. Science 282(5391): 1145-7.

2.

Amit M et al. (2000) Clonally derived human embryonic stem
cell lines maintain pluripotency and proliferative potential for
prolonged periods of culture. Dev Biol 227(2): 271-8.

3.

Xu C et al. (2001) Feeder-free growth of undifferentiated humanembryonic stem cells. Nat Biotechnol 19(10): 971-4.

4.

Ludwig TE et al. (2006) Derivation of human embryonic stem cells
in defined conditions. Nat Biotechnol 24(2): 185-7.

5.

Takahashi K et al. (2007) Induction of pluripotent stem cells fromadult human fibroblasts by defined factors. Cell 131(5): 861-72.

6.

Chen G et al. (2011) Chemically defined conditions for human iPS
cell derivation and culture. Nat Methods 8(5): 424-9.

7.

Ran FA et al. (2013) Genome engineering using the CRISPR-Cas9
system. Nat Protoc 8(11): 2281-308.

Copyright © 2019 by STEMCELL Technologies Inc. All rights reserved including graphics
and images. STEMCELL Technologies & Design, STEMCELL Shield Design, Scientists Helping
Scientists, CellAdhere, CloneR, and STEMdiff are trademarks of STEMCELL Technologies Canada
Inc. ACCUTASE is a trademark of Innovative Cell Technologies, Inc. E8, TeSR, and mTeSR are
trademarks of WARF. Vitronectin XF is developed and manufactured by Primorigen Biosciences
Inc., and Vitronectin XF™ is a trademark of Primorigen Biosciences Inc. Corning and Matrigel areregistered trademarks of Corning Incorporated. All other trademarks are the property of theirrespective holders. While STEMCELL has made all reasonable efforts to ensure that the information provided by STEMCELL and its suppliers is correct, it makes no warranties or representations
as to the accuracy or completeness of such information.
STEMCELL TECHNOLOGIES INC.’S QUALITY MANAGEMENT SYSTEM IS CERTIFIED TO ISO 13485.
PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMALDIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED.

TOLL FREE PHONE 1 800 667 0322
PHONE +1 604 877 0713
INFO@STEMCELL.COM
TECHSUPPORT@STEMCELL.COM
FOR GLOBAL CONTACT DETAILS VISIT WWW.STEMCELL.COM

DOC#27158

VERSION 1.1.0

FEB 2019