29063_hesc(electronics)

Maximize Your Plurip tentialYour Guide to

2Table of Contents 3 Introduction

4 mTeSR1: Defined, Feeder-Free Maintenance medium for Human Pluripotent Stem Cells

6 TeSR-E8 and Vitronectin XF

8 TeSR2 and StemAdhere Defined Matrix for hPSC

10 mFreSR and CryoStorCS10

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 toReduce Variation With The Most Complete, Defined System For Pluripotent Stem Cell Culture

3Products for Human Pluripotent Stem Cells

IntroductionHuman 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.

Human Pluripotent Stem CellsProducts for Research

4Developed at the:

mTeSR1Defined, Feeder-Free Maintenance Medium for Human Pluripotent Stem Cells

hESC and hiPSC LinES MaintainEd in mteSR1

H1, H9, H7 H13*, H14, H15*, H16* BG01, BG02, BG03, BG04* Shef1*, Shef4*

HESM01*, HESM02*, HESM03*, HESM04*

HS237, HS239, HS360, HS401

HUES1, HUES3, HUES6, HUES8*, HUES9 Regea 06/015*

MA01*, CA1*, CA1T*, KCL1*, Man1*, MEL-1*, MEL-2*

NCL-3, HSF-6*, HES2, HES3 (ES03), HES4*

Banked at WISC bank at WiCell: iPS-DF19-9-11T.H, iPS-DF19-9-7T, iPS-DF4-3-7T, iPS-DF6-9-9T.B, iPS(Foreskin)-1 (Clone 1), iPS IMR90-1, iPS(IMR90)-4 (Clone 4)

iPSC(IMR90)-3, MSC-iPSC1

Table 1. mTeSR1 Has Been Tested and Published Extensively for the Long-Term Maintenance and Expansion of Various Cell Lines, Including Those Listed Below (*Unpublished Customer Reports)

mTeSR1 is the most widely 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.

Advantages of mTeSR1

ConSiSTenCy. Defined, feeder-free ES and iPS cell culture medium.

RePRoduCibiliTy. Standardizes culture methods for more reproducible data.

ConvenienCe. Saves time and effort needed for feeder and conditioned media preparation.

A ComPleTe FoRmulATion. No supplements or other growth factors required.

mTeSR1 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, mTeSR1 provides more consistent cultures with homogeneous, undifferentiated phenotypes.


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Figure 3. Morphology of Human ES Cells Cultured in mTeSR1 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.

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500 mL 05850

1 L 05857

10 Kits 05870

25 Kits 05875

Figure 4. Human ES Cells Cultured in mTeSR1 Retain Normal Karyotype Following Long-Term Passaging

Chromosomal analysis of H1 ES cells cultured in mTeSR1 for 48 passages shows that normal karyotype is retained during long-term passaging.Data from Cytogenetics Lab, WiCell Research Institute.

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Figure 1. Morphology of Human ES Cells Cultured in mTeSR1

H1 human ES cells grow as colonies with (A) defined edges and (B) high nucleus to cytoplasm ratio.

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Figure 2. Morphology of Human iPS Cells Cultured in mTeSR1

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 mTeSR1 show similar morphological characteristics.

videomTeSR1: Standardized Medium for the Feeder-Independent Maintenance of hESCs & hiPSCs www.stemcell.com/mteSR1Video

6New TeSR-e8 and vitronectin XFLow Protein, Highly Defined, Feeder-Free Maintenance of Human Pluripotent Stem Cells

Advantages of TeSR-E8 and Vitronectin XF:

Completely defined xeno-free culture system

Contains only the essential components for feeder-free culture

Enables flexible passaging schedule

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 (University of Wisconsin- Madison), the lead research group behind the design of mTeSR1.

Like mTeSR1, TeSR-E8 is made with the highest level of quality and care. TeSR-E8 contains only the essential components required for maintenance of ES cells and iPS cells, providing a simpler medium for the culture of pluripotent stem cells. It can be used with a surface coating of Matrigel hESC-Qualified Matrix or with Vitronectin XF.

Figure 5. Normal human ES and iPS cell morphology is observed in TeSR-E8 cultures.

Undifferentiated (A) human ES (H9) and (B) human iPS (WLS-1C) cells cultured on Matrigel in TeSR-E8 retain the prominent nucleoli and high nuclear to cytoplasm ratio characteristic of this cell type. Densely packed cells and multilayering are prominent when cells are ready to passage.

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25 m500 m

25 m500 m

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CoMPonEnt mteSR1 E8*

DMEM-F12 (DF12)

NaHCO3

L-Ascorbic Acid

Selenium

Transferrin

Insulin

FGF2

TGF-

Bovine Serum Albumin (BSA)

Glutathione

Trace Elements

-mercaptoethanol (BME)

Pipecolic Acid

GABA

Lithium Chloride

Defined Lipids

*As published in Chen et al. 2011, the base design for TeSR-E8.


the stem cell company

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.

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Figure 6. High expansion rates are observed in TeSR-E8 cultures.

Graph shows the average fold expansion per passage +/- SEM obtained for human ES and iPS cells cultured in TeSR-E8 with BD Matrigel over 10 passages (brown). Representative data is also shown for human ES cells cultured in mTeSR1 using a similar protocol for comparison (gray). Expansion was determined by enumerating the cell aggregates obtained at harvest and dividing by the number of cell aggregates seeded. Note that this data is representative of cultures passaged after 6-7 days in culture; lower expansion should be expected if using shorter culture times.

Customize Your Passaging Schedule: Passage Anytime Between Days 4 & 7The amount of time until your next passage depends largely on the size and density of initially plated cell aggregates, as well as on the colony density at time of passaging. The following table indicates how these three variables can be manipulated to allow human ES and iPS cells cultured in TeSR-E8 medium to be passaged between 4 to 7 days after plating. Regardless of the plating density or cell aggregate size used, the majority of colonies should be densely packed and multilayered in the center when ready for passaging. See Figure 5 for a representative example indicating the optimal window for passaging.

Adjustable Parameters That Affect Time to Next Passage

at tiME oF PLating at tiME oF PaSSaging

CELL AGGREGATE SIZE

PLATING DENSITY

COLONY DENSITY

DAYS IN CULTURE

Large High High 4 - 5

Large Low Low

5 - 6Medium Medium Medium

Small High High

Small Low Low 6 - 7

For a detailed guide to customizing your passaging schedule, 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 ready for passaging at several timepoints.

Figure 7. Uniformly-sized embryoid bodies differentiated from cells 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 plate containing AggreWell medium and 10 M Y-27632 for 24 hours (A), after which they were transferred to an ultra low-adherence (ULA) plate for inspection (B). This protocol can be found in the AggreWell manual (Document #29146) at www.stemcell.com.

100X100X

A bDay 1 (AggreWell 400 plate) Day 2 (ULA plate)

PRoduCt SiZE CataLog #

TeSR-E8 1 Kit 05840

Vitronectin XF 1 Kit 07190

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Advantages of TeSR2 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 significant step towards a fully humanized system

No preparation of feeders or conditioned media required

StemAdhere is growth factor-free

Maintains phenotypically homogeneous and karyotypically normal cells after long-term passaging

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. TeSR2 takes a significant step in this direction by allowing hES and iPS cells to be cultured feeder-free in an animal protein-free, defined medium.7

TeSR2 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 protein, which allows for more consistent cell populations and more reproducible results in downstream applications.

TeSR2 and StemAdhere defined matrix for hPSCDefined Culture Systems

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Figure 8. Undifferentiated hPSCs Grown in TeSR2 and mTeSR1 on StemAdhere Defined Matrix for hPSC

hPSCs grown in TeSR2 and mTeSR1 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 TeSR2 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 TeSR2 on StemAdhere Defined Matrix for hPSC.


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FiGuRe 9. Morphology of iPS Cells Cultured in TeSR2

Human iPS cells cultured in TeSR2 grow as colonies with defined edges and high nucleus to cytoplasm ratio. iPS(IMR90)-1 cell line cultured (A) for 8 passages in TeSR2 and (B) for 10 passages in TeSR2.Photographs courtesy of Dr. T. Ludwig, WiCell Research Institute.

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FiGuRe 10. Morphology of hPSCs Cultured in mTeSR1 or TeSR2 on StemAdhere Defined Matrix for hPSC

Undifferentiated hPSCs cultured in mTeSR1 or TeSR2 on StemAdhere Defined Matrix for hPSC exhibit high nuclear to cytoplasmic ratio and prominent nucleoli. (A) H9 ES cells at Day 5 of culture in TeSR2 on StemAdhere Defined Matrix for hPSC. (B) H9 ES cells at Day 6 of culture in mTeSR1 on StemAdhere Defined Matrix for hPSC.

25 m

25 m


TeSR2 500 mL Kit 05860

StemAdhere Defined Matrix for hPSC

1 Kit 07170

the stem cell company

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.

10

mFreSR and CryoStorCS10Defined Cryopreservation Media for Human Pluripotent Stem Cells

Figure 11. Clump Survival Data for mFreSR

H9 ES cells were cryopreserved in mFreSR at the indicated passage after culture with mTeSR1. Thawing efficiencies were determined by counting the number of clumps after thawing, and the number of resultant undifferentiated colonies.

Figure 12. Clump Survival Data for CryoStor CS10

H9 ES cells were cryopreserved with CryoStor CS10 at the indicated passage after culture with TeSR2. Thawing efficiencies were determined by counting the number of clumps after thawing, and the number of resultant undifferentiated colonies.

mFreSR CryoStorCS10 Serum-free Serum-free and xeno-free Research use only cGMP-compliant

Ideally used after culture in mTeSR1 or TeSR2

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 mTeSR1 and TeSR2 to minimize variability in your human pluripotent cell lines.

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Products for 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.

Antibodies for Characterizationof Human Pluripotent Stem Cells

PRiMaRy antibody unConjugatEd PE ConjugatEdaLExa FLuoR 488 ConjugatEd

SPECiES REaCtiVity iSotyPE

CataLog #

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

Anti-Mouse SSEA-1 Antibody, Clone MC-480

60060 60060PE 60060AD Human, Mouse Rat IgM (Mouse)

Anti-Mouse SSEA-3 Antibody, Clone MC-631

60061 60061PE 60060ADHuman, Mouse, Rat, Rhesus

IgM (Rat)

Anti-Human SSEA-4 Antibody, Clone MC-813-70

60062 60062PE 60060ADHuman, Mouse, Rat, Rhesus, Cat, Chicken, Dog, Rabbit

IgG3 (Mouse)

Anti-Human TRA-1-60 Antibody, Clone TRA-1-60R

60064 60064PE 60064AD Human, Rhesus, Rabbit IgM (Mouse)

Anti-Human TRA-1-81 Antibody, Clone TRA-1-81

60065 60065PE Human, Rat, Rhesus IgM (Mouse)

Anti-Human TRA-2-49 Antibody, Clone TRA-2-49/6E

60066Human, Non-Human Primate, Monkey, Cat, Pig, Rabbit, Tiger

IgG1 (Mouse)

Anti-Human TRA-2-54 Antibody, Clone TRA-2-54/2J

60067Human, Chimpanzee, Monkey, Cat, Pig, Rabbit

IgG1 (Mouse)

FiGuRe 13. Immunocytochemistry of H1 ES Cells cultured in mTeSR1 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).

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Pluripotent stem cells can be enriched from a mixed population containing differentiated and undifferentiated cells or reprogrammed and non-reprogrammed cells, based on their surface expression of SSEA-4 or TRA-1-60. EasySep is a powerful cell isolation platform that combines the specificity of monoclonal antibodies with the simplicity and speed of a column-free, immunomagnetic system. Use the EasySep hESC/hiPSC SSEA-4 Positive Selection Kit or the EasySep Human ES/iPS Cell TRA-1-60 Positive Selection Kit to isolate highly purified human pluripotent cells quickly and easily.

easySep Fast and Easy Cell Isolation of Human Pluripotent Stem Cells

Figure 14. Typical Flow Cytometry Histogram Results Using EasySep hESC/hiPSC SSEA-4 Antibody Positive Selection Kit

Starting with a mixture of H9 ES cells and HT1080 fibroblast cells, the SSEA-4+ cell content of the enriched fraction typically ranges from 95 - 99%.

FRee SAmPle KiTEasySep hESC/hiPSC SSEA-4 Positive Selection Kitwww.stemcell.com/SSEa4SampleKit

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EasySep hESC/hiPSC SSEA-4 Positive Selection Kit*

for 1 x 109 cells

18165

EasySep Human ES/iPS Cell TRA-1-60 Positive Selection Kit*

abeling up to 1 x 109cells

18166

*Required equipment: EasySep Magnet (Catalog #18000)

Advantages of EasySep

FAST And eASy. No columns or washes.

HiGH PuRiTy. Reliably obtain purities of up to 99%.

FunCTionAl CellS. Gentle procedure enables the isolation of functional and viable cells.

FloW CyTomeTRy-ComPATible. Isolated cells are immediately available for use as EasySep magnetic particles do not interfere with flow cytometry.

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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. The EasySep hESC-derived CD34 Positive Selection Kit (Catalog #18167) is designed to purify CD34+ cells during hematopoietic differentiation protocols. Alternatively, customiza-tions for your specific experimental needs are also available.

EasySep Custom Selection KitsUse 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 with RoboSepRoboSep (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 disease samples.

Figure 15. Typical Flow Cytometry Histogram Results 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%.

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Figure 16. EasySep Positive Selection Protocol Diagram

Add EasySep selection cocktail to cells

Incubate 15 minutes

Add EasySep magnetic particles

Incubate 10 minutes

Place tube in magnet for 10 minutes

Pour off supernatant. Positively selected cells remain in tube.

PRoduCt CaPaCity CataLog #

EasySep hESC-Derived CD34 Positive Selection Kit*

for 1 x 109 cells

18167

*Required equipment: EasySep Magnet (Catalog #18000)

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AggreWellReproducible Production of Uniformly-Sized Embryoid Bodies

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 variety of cell types.18

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

Figure 17. Uniformly Sized Embryoid Bodies Generated with AggreWell

EBs formed using AggreWell plates are uniform in size and consistently spherical in shape. Shown are EBs generated with 2,000 human ES cells each, using AggreWell400.

Figure 18. The Size of EBs Can Easily be Adjusted Using AggreWell Plates

(C) H9 ES cells were centrifuged into AggreWell400 plates and cultured for 24 hours prior to EB harvest. EB size is tightly controlled with AggreWell (light and dark grey), unlike with scraping protocols (brown) that give a wider distribution.22

500 cells/EB 2,000 cells/EB scraped EBs


AggreWell400 Plate (8 inserts in 24 wells)

1/pack 27845

5/pack 27945

AggreWell400Ex Plate (6 wells)

1/pack 27840

5/pack 27940

AggreWell 800 Plate (8 inserts in 24 wells)

1/pack 27865

5/pack 27965

AggreWell Medium 100 mL 05893

AggreWell Rinsing Solution 100 mL 07010

37 m Reversible Strainers, Small 20/box 27215

37 m Reversible Strainers, Large 12/box 27250

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The STEMdiff Definitive Endoderm Kit, optimized for use with mTeSR1 and TeSR2, allows for the differentiation of hPSCs to multipotent definitive endoderm. It is a defined, animal component-free system.

hPSCs differentiated using this kit are highly enriched for definitive endoderm, as indicated by co-expression of SOX17, CXCR4, FOXA2 and c-KIT, and can be used to generate multiple downstream endodermal cell types, including hepatocytes and pancreatic precursors.

STemdiff definitive endoderm KitDefined, 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 mTeSR1 or TeSR2

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

Figure 19. Definitive Endoderm Differentiation is Efficient Across Multiple ES and iPS Cell Lines

Quantitative analysis of definitive endoderm formation in multiple hPSC lines as measured by co-expression of CXCR4 and SOX17. Prior to differentiation using STEMdiff Definitive Endoderm Kit, cells were maintained in their pluripotent state by culturing in mTeSR1 on Matrigel (A) and TeSR2 on Matrigel (B). Data are expressed as the mean percent of cells expressing both markers. Error bars indicate SEM; n values for each cell line are indicated by the white number within each bar.

Figure 20. Expression of Key Definitive Endoderm Markers in Human ES and iPS Cells is Widespread

Representative images of SOX17 and FOXA2 immunoreactivity in hES (H9) and hiPS (WLS-4D1) cells following 4 days of differentiation to definitive endoderm using STEMdiff Definitive Endoderm Kit. Merged images show extensive co-localization of these two markers. Scale bar: 100 m.

H9 WLS-4D1

H9

Representative images of SOX17 and FOXA2 immunoreactivity in hES (H9) and hiPS (4D1) cells 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 m.

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1 Kit 05110

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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 STEMdiff Neural Rosette Selection Reagent (Figure 22). This is a complete and highly efficient system to obtain enriched NPCs within 12 days without manual rosette isolation.

STemdiff neural induction mediumDefined, Serum-Free Medium for hPSC Differentiation to Neural Progenitor Cells

Figure 21. Neural Induction is Efficient Using STEMdiff Neural Induction Medium

Neural aggregates of 10,000 cells each were formed and cultured in an AggreWell800 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)

Advantages of STEMdiff Neural Induction Medium

Fully defined and serum-free

Optimized for use with cells maintained in mTeSR1 or TeSR2

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

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 Selection Reagent

The STEMdiff Neural Rosette Selection Reagent selectively lifts neural rosette structures from attached neural aggregates , leaving the surrounding flat cells behind. (Obj. 2x)


STEMdiff Neural Induction Medium

100 mL 05831

STEMdiff Neural Rosette Selection Reagent

100 mL 05832

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2.45% 5.63%

11.5%80.4%

6.10% 5.30%

19.1%69.5%

STemdiff APel mediumDefined, Animal Component-Free Basal Medium for hPSC Differentiation

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 on a published formulation,22 STEMdiff APEL Medium is a versatile, growth factor-free, basal medium that is to be supplemented with specific lineage-inducing factors. Optimized to be used following maintenance in either mTeSR1 or TeSR2 media, this medium can be used to differentiate cells in EB-based (AggreWell) or adherent cell-based systems.

Advantages of STEMdiff APEL Medium

Defined and animal component-free

Optimized for use with cells maintained in mTeSR1

Versatile, growth factor-free formulation

Published protocols for differentiation to ectoderm, endoderm and mesoderm

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 et al.22 and Chadwick et al.,23 with the following changes: (1) STEMdiff APEL Medium was substituted for DMEM with 20% FBS as a basal medium; (2) cells were maintained prior to differentiation for at least 10 passages in mTeSR1 Maintenance Medium on Matrigel; (3) the entire differentiation procedure was performed in adherent cell culture, on a Matrigel coated surface instead of an EB-based method. Cells were analyzed by flow cytometry on Day 13 of differentiation culture for expression of hematopoietic markers CD34 and CD45.

CD

45

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H9 hESC lineWLS-4D1 hiPSC line

Figure 24. Differentiation of Human ES Cells into Cardiomyocytes Using STEMdiff APEL Medium

H9 cells were differentiated into cardiomyocytes based on Yang et al.20 with the following changes: (1) STEMdiff APEL Medium was substituted for StemPro-34 SFM as a basal medium; (2) cells were maintained prior to differentiation for at least 10 passages in mTeSR1 Medium on Matrigel; (3) EBs were formed in AggreWell400 plates, and (4) the entire procedure was carried out within the AggreWell400 plate. Beating EBs were counted on Day 16 of culture, and varied between 5% and 95% of total EBs (n=9).

H9 cells were differentiated based on Rezania et al.21 with the following changes: (1) STEMdiff APEL Medium was substituted for RPMI with 2% BSA as a basal medium; and (2) cells were maintained prior to the differentiation for at least 10 passages in mTeSR1 Medium on Matrigel. On Day 4, cells were highly positive for CXCR4 and SOX17 markers, which together characterize definitive endoderm.

Figure 25. Differentiation of hPSCs into Definitive Endoderm Using STEMdiff APEL Medium and Published Cytokines

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STEMdiff APEL Medium

100 mL 05210

18

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.


DMEM with 4500 mg/L D-glucose 500 mL 36250

DMEM with 1000 mg/L D-glucose 500 mL 36253

DMEM/F-12 500 mL 36254

Iscoves MDM (IMDM) 500 mL 36150

Tissue Culture Media


Activin A 10 g 02528

BAFF 20 g 02517

Bone Morphogenetic Protein-2 10 g 02523

Bone Morphogenetic Protein-4 10 g 02524

Noggin 20 g 02525

Basic Fibroblast Growth Factor (bFGF)

25 g 02634

Transforming Growth Factor-1 2 g10 g

0264702847

Recombinant Cytokines

Miscellaneous Tissue Culture Reagents & Supplies

Balanced Salt Solutions

Enzymes

Antibiotics


3% Acetic Acid with Methylene Blue

100 mL 07060

Collagen Solution (3 mg/mL) 35 mL 04902

Fibronectin (1 mg/mL) 1 mL 07159

Gelatin (0.1% in water) 500 mL 07903

Hypoxia Chamber 1 chamber 27310

Rat Serum2 mL 5 x 2 mL

13551 13561

Sodium Pyruvate (100 mM) 100 mL 07000

Trypan Blue 100 mL 07050

Y-27632 (ROCK inhibitor)1 mg 07171

5 mg 07172


D-PBS 500 mL 37350

D-PBS, 10X 500 mL 37354

HBSS, Ca++ & Mg++ free 500 mL 37250

HBSS, without Phenol Red 500 mL 37150


35 mm Diameter10/pack 500/case

2711527116

60 mm Diameter10/pack 400/case

2712027121

100 mm Diameter 10/pack 240/case

2712527127

245 mm x 245 mm4/pack 16/case

27140 27141

96-Well Plates1/pack 50/case

27135 27136


ACCUTASETM 100 mL 07920

Collagenase 5 mL 07902

Collagenase Type IV 100 mL 07909

Dispase (1 mg/mL) 100 mL 07923

Dispase (5 mg/mL) 100 mL 07913

DNase I (1 mg/mL) 1 mL 07900

Trypsin-EDTA (0.05%) 500 mL 07910

Trypsin-EDTA (0.25%) 500 mL 07901

Trypsin in Citrate Saline 100 mL 07400


Neomycin (G418) 250 mg 03812

Hygromycin B 100 mg 03813

Support Reagents

Tissue Culture Dishes

19


1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science 282:1145-7, 1998

2. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861-72, 2007

3. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA. Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917-20, 2007

4. Park IH, Zhao R, West JA, Yabuuchi A, Hu H, Ince TA, Lerou PH, Lensch MW, Daley GQ. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451:141-6, 2008

5. Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18:399-404, 2000

6. Ludwig TE, Bergendahl V, Levenstein ME, Yu J, Probasco MD, Thomson JA. Feeder-independent culture of human embryonic stem cells. Nat Methods 3:637-46, 2006

7. Ludwig TE, Levenstein ME, Jones JM, Berggren WT, Mitchen ER, Frane JL, Crandall LJ, Daigh CA, Conard KR, Piekarczyk MS, Llanas RA, Thomson JA. Derivation of human embryonic stem cells in defined conditions. Nat Biotechnol 24:185-7, 2006

8. Nagaoka M, Si-Tayeb K, Akaike T, Duncan SA. Culture of human pluripotent stem cells using completely defined conditions on a recombinant E-cadherin substratum. BMC Dev Biol 10:60, 2010

9. Chen G, Gulbranson DR, Hou Z, Bolin JM, Ruotti V, Probasco MD, Smuga-Otto K, Howden SE, Diol NR, Propson NE, Wagner R, Lee GO, Antosiewicz-Bourget J, Teng JM, Thomson JA. Chemically defined conditions for human iPSC derivation and culture. Nat Methods.8(5):424-429, 2011

10. Fujioka T, Yasuchika K, Nakamura Y, Nakatsuji N, Suemori H. A simple and efficient cryopreservation method for primate embryonic stem cells. Int J Dev Biol 48:1149-54, 2004

11. Ha SY, Jee BC, Suh CS, Kim HS, Oh SK, Kim SH, Moon SY. Cryopreservation of human embryonic stem cells without the 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

13. Ware CB, Nelson AM, Blau CA. Controlled-rate freezing of human ES cells. Biotechniques 38:879-80, 882-3, 2005

14. Richards M, Fong CY, Tan S, Chan WK, Bongso A. An efficient and safe xeno-free cryopreservation method for the storage of human embryonic stem cells. Stem Cell 22:779-89, 2004

15. Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, Amit M, Soreq H, Benvenisty N. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med:6:88-95, 2000

16. Kurosawa H. Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells. J Biosci Bioeng 103:389-398, 2007

17. Bauwens C L et al. Control of human embryonic stem cell colony and aggregate size heterogeneity influences differentiation trajectories. Stem Cells 26:2300-2310, 2008

18. Ungrin MD, Joshi C, Nica A, Bauwens C, Zandstra PW. Reproducible, ultra-high throughput formation of multicellular organization from single cell suspension-derived human embryonic stem cell aggregates. PLoS One 3(2):e1565, 2008

19. Markway BD et al. Enhanced chondrogenic differentiation of human bone marrow-derived. Mesenchymal stem cells in low oxygen environment micropellet cultures. Cell Transplantation. 19:2942, 2010

20. Yang L, Soonpaa MH, Adler ED, Roepke TK, Kattman SJ, Kennedy M, Henckaerts E, Bonham K, Abbott GW, Linden RM, Field LJ, Keller GM. Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature. 453:524-528, 2008

21. Rezania A, Riedel MJ, Wideman RD, Karanu F, Ao Z, Warnock GL, Kieffer TJ. Production of functional glucagon-secreting cells from human embryonic stem cells. Diabetes. 60:239-247, 2011

22. Ng ES, Davis R, Stanley EG, Elefanty AG. A protocol describing the use of a recombinant protein-based, animal product-free medium (APEL) for human embryonic stem cell differentiation as spin embryoid bodies. Nat Protoc. 3:768-776, 2008

23. Chadwick K, Wang L, Li L, Menendez P, Murdoch B, Rouleau A, Bhatia M. Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic stem cells. Blood. 102:906-915, 2003

References

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