Embryonic and Induced Pluripotent Stem Cell Workflow Solutions Isolate and Culture

Pluripotent stem cells can be efficiently expanded in culture using specialized media and growth factors. It is important to begin experimentation with a verified, homogeneous, and pluripotent ES/iPS starter population to maintain confidence in cell differentiation and subsequent data interpretation. With this necessity in mind, R&D Systems offers high quality options for the expansion and characterization of ES/iPS cells, including culture media, feeder-cells, culture matrix, and cryopreservation media.

Product Catalog # StemXVivo® Mouse Pluripotent Stem Cell Media CCM025 Irradiated Mouse Embryonic Fibroblasts PSC001 Mouse Embryonic Fibroblast Conditioned Media AR005 CryoDefend®-Stem Cells Media CCM018

A. B. 104 Embryonic Stem Cells Grown in Mouse Pluripotent Stem Cell Media Express Pluripotency Stem Cell Markers, SSEA-1 and Oct-3/4, and Lack SSEA-4. v6.5 mouse embryonic stem cells (Novus Biologicals, Catalog # NBP1-41162) were 3 10 cultured in StemXVivo® Mouse Pluripotent Stem Cell Media (R&D Systems, Catalog # CCM025, A) Expression of SSEA-1 and SSEA-4 were detected using PE-conjugated Mouse Anti- SSEA-1 Monoclonal Antibody (R&D Systems, Catalog # 102 FAB2155P) and APC-conjugated Mouse Anti-SSEA-4 Monoclonal Antibody (R&D Systems, Catalog # FAB1435A,

SSEA-4-APC B) Expression of Oct-3/4 was detected using Rat Anti- Human/Mouse Oct-3/4 Monoclonal Antibody (R&D 101 Systems, Catalog # MAB1759) followed by NorthernLights™ (NL)557-conjugated Goat Anti-Rat Secondary Antibody (R&D Systems, Catalog # NL013). The nuclei were counterstained with DAPI (blue). 100 100 101 102 103 104 SSEA-1-PE

Serum and Media for the Culture of Stem Cells

Fetal bovine serum (FBS) is the ideal growth supplement for cell culture media and is generally preferred over other types of sera due to its high levels of nutrients and optimal combination of growth factors. Our FBS is produced utilizing strict manufacturing processes and quality assurance procedures, which guarantees unsur- passed quality and consistency from lot-to-lot. It is processed according to validated and approved cGMP and ISO 9001 guidelines and is globally sourced and collected in USDA-approved countries and documented to be Fetal Bovine exotic disease-free. Serum Embryonic Stem Cell Qualified

Triple 0.1 µm filtered

0.2 µm filtered • Embryonic Stem Cell Qualified FBS is available Cat. No.: S10250 Lot. No.: FBS123 Volume: 500 mL • Our Serum Testing Program enables the free screening of serum lots in your specific application Store at -5C to -20C For In Vitro Research Use Only • Lot Matching Program assists in selecting serum based on the researcher’s serum specifications and performance criteria Learn more | rndsystems.com/selectFBS

Cultrex® BME for Stem Cell and Organoid Cultures

Cultrex basement membranes play an essential role in tissue organization by influencing cell adhesion, migration, proliferation, and differentiation.

Name Size Catalog # Application Cultrex BME 5 mL 3432-005-01 General use including 2-D cultures, 3-D cultures, cell attachment and cell invasion Cultrex Stem Cell Qualified RGF BME 5 mL 3434-005-02 Stem Cells Cultrex Ready BME, Stem Cell Qualified RGF 50 mL 3434-050-RTU Ready-to-use pre-diluted Cultrex Stem Cell Qualified RGF BME Cultrex Organoid Qualified RGF BME, Type 2 5 mL 3533-005-02 Robust organoid cultures Cultrex Organoid Qualified RGF BME, Type R1 5 mL 3433-005-R1 Difficult to culture organoids Cultrex 3-D Culture Matrix™, RGF BME 5 mL 3445-005-01 3-D Tissue Spheroids

Learn more | rndsystems.com/cultrex Small Molecules for Reprogramming and Expansion

Small molecules can provide an additional level of control over iPS cell reprogramming as well as ES/iPS cell maintenance and expansion.

Reprogramming of Somatic Cells to iPSCs* Maintenance of ESCs and iPSCs Name Description Catalog # Name Description Catalog # CHIR 98014 Highly potent and selective GSK-3 inhibitor 6695 A 83-01 Selective inhibitor of TGF-βRI, ALK4 and ALK7 2939 CHIR 99021 Highly selective GSK-3 inhibitor 4423 BIO Potent GSK-3 inhibitor; also inhibits cdks 3194 ROCK inhibitor ; improves the efficiency of fibroblast 16, Thiazovivin reprogramming and induction of iPSCs 3845 16-Dimethyl Synthetic prostaglandin E2 derivative; regulates HSC Prostaglandin development 4027 PD 0325901 Potent inhibitor of MEK1/2 4192 E2 RepSox Potent and selective inhibitor of TGF-βRI 3742 Go 6983 Broad spectrum PKC inhibitor 2285 DBZ γ-secretase inhibitor; inhibits Notch pathway 4489 PD 98059 MEK inhibitor 1213 Trichostatin A Potent histone deacetylase inhibitor 1406 SB 202190 Potent, selective inhibitor of p38 MAPK 1264 Potent, selective inhibitor of TGF-βRI, ALK4 and Y-27632 Selective p160ROCK inhibitor 1254 SB 431542 ALK7 1614 * Use of all 7 together found to reprogram somatic cells to iPS cells. Hou, P. et al. (2013) Science 341:651.

ProDot® Proteins for Stem Cell Culture

Simplify your cell culture preparation. ProDots® Proteins are highly bioactive proteins packaged into easy-to-use lyophilized “dots” that take the place of frozen aliquots. Lyophilized dots easily roll out of the vial into a cell culture media bottle and dissolve instantly.

Proteins for Pluripotent Stem Cell Expansion ProDots® Proteins, Recombinant Catalog # Catalog # Proteins Human Mouse Rat Human • High Bioactivity: R&D Systems® proteins are know for their industry-leading bioactivity Activin A 338-AC 338-AC 338-AC PRD338 BMP-4 314-BP 5020-BP PRD314 • Consistent: tested for lot-to-lot consistency FGF basic 233-FB 3139-FB 3339-FB PRD233 LIF 7734-LF 8878-LF PRD7734 TGF-β1 7754-BH 7666-MB PRD240 Nodal 3218-ND 1315-ND Wnt-3a 5036-WN 1324-WN

GMP Reagents for Stem Cell Therapy Reserach

Progressing your cancer discovery into cell or immune therapy requires transitioning into a GMP- compliant manufacturing process. Bio-Techne provides industry-leading quality and regulatory support for GMP ancillary and raw materials, including recombinant cytokines, growth factors, and small molecules used in ex vivo cell therapy manufacturing. Our experienced quality and manufacturing teams are focused on quality, innovation, supply chain continuity, and traceability, Bio Techne GMP reagents are the reliable solution for your cell culture expansion and differentiation protocols.

GMP Proteins GMP Small Molecules GMP Media and Supplements Activin A Y-27632 GMP N-2 Media Supplement NEW! EGF SB 431542 Custom Media Options FGF basic CHIR 99021 IL-6 DAPT Wnt-3a and more!

Learn more | bio-techne.com/GMPCellCulture Verify

Confidence in ES/iPS cell pluripotency prior to expansion and differentiation is essential for downstream experimentation and data interpretation. Beginning an experiment with suboptimal, unverified populations will put the investigator at risk for inconsistent results, thus wast- ing time and reagents. R&D Systems offers a selection of kits to verify ES/iPS cell pluripotency through either functional differentiation or cell-specific marker expression.

Product Catalog # Human Pluripotent Stem Cell Functional Identification Kit SC027B Human Three Germ Layer 3-Color Immunocytochemistry Kit SC022 GloLIVE™ Human Pluripotent Stem Cell Live Cell Imaging Kit SC023B Human Pluripotent Stem Cell Marker Antibody Panel SC008 Human Pluripotent Stem Cell Marker Antibody Panel Plus SC009

Human Pluripotent Stem Cell Functional Identification Kit

• Efficient: differentiation and identification in 5 days • Reliable: induces ES/iPS tri-lineage differentiation with kit-provided supplements • Complete: contains germ layer-specific antibodies to confirm successful differentiation

Human Three Germ Layer 3-Color Immunocytochemistry Kit

• Efficient: includes fluorochrome-conjugated antibodies to detect ectoderm, mesoderm, and endoderm • Thorough: contains 2 antibodies to confirm each tri-lineage cell-type • Fewer variables: detects heterogeneity of starting cell populations

Ectoderm Mesoderm Endoderm

tSPI racyuryHPI S-PI

Functional Pluripotency of Human iPS Cells Confirmed with the Human Three Germ Layer conjugated Otx2 (red) and NL493-conjugated SOX1 (green). Mesoderm differentiated cells were 3-Color Immunocytochemistry Kit. Human iPS2 cells were differentiated into each of the three simultaneously stained with NL557-conjugated Brachyury (red) and NL637-conjugated HAND1 germ layers using the Human Pluripotent Stem Cell Functional Identification Kit (Catalog # (green). Endoderm differentiated cells were simultaneously stained with NL637-conjugated SC027B). Germ layer differentiation was verified using the six fluorochrome-conjugated SOX17 (red) and NL493-conjugated GATA-4 (green). All nuclei were counterstained with DAPI antibodies provided in the Human Three Germ Layer 3-Color Immunocytochemistry Kit. (blue). Nuclear expression of each marker was detected in their respective cell lineage. Ectoderm differentiated cells were simultaneously stained with NorthernLights™ (NL)557-

Learn more | rndsystems.com/esips GloLIVE™ Human Pluripotent Stem Cell Live Cell Imaging Kit

• Quick: verify pluripotency of live human stem cells in 30 minutes • Flexible: contains 4 fluorochrome-conjugated pluripotent stem cell antibodies for versatile staining combinations • Convenient: no adverse effects on proliferation or differentiation following staining • Cost-effective: great value for all stem cell researchers

Verification of iPS Pluripotency using the ™GloLIVE Pluripotent Stem Cell Live Cell Imaging Kit. Human iPS2 cells were grown on Irradiated Mouse Embryonic Fibroblasts (Catalog # PSC001). NorthernLights™ (NL)493-conjugated SSEA-4 (green) and NL557-conjugated TRA-1-60(R) (red) antibodies from the GloLIVE™ Pluripotent Stem Cell Live Cell Imaging Kit (Catalog # SC023B) were added to live cultures to label pluripotent stem cells. Cells were fixed and stained with Hoechst 33342 (blue) prior to imaging. Positive staining for SSEA-4 and TRA-1-60(R) combined with negative staining for SSEA-1 (not shown), indicates that cultures primarily contain undifferentiated human iPS cells.

GloLIVE™ Antibodies

Azide-free, stem cell marker antibodies conjugated to NorthernLights™ (NL) fluorochromes that can be used for single-step, direct immunocytochemical staining of live, unfixed pluripotent stem cells.

Marker Human Stem Cells Mouse Stem Cells Conjugated to NL493 (green) Conjugated to NL557 (red) SSEA-1 Negative Marker Positive Marker NLLC2155G NLLC2155R SSEA-4 Positive Marker Negative Marker NLLC1435G NLLC1435R

TRA-1-60(R) Positive Marker Not applicable NLLC4770G NLLC4770R Detection of SSEA-4 in Human ES cells. The pluripotent TRA-1-81 Positive Marker Not applicable NLLC16581G NLLC16581R stem cell marker SSEA-4 was visualized in live BG01V human embryonic stem cells using the GloLIVE™ NL557- conjugated Mouse Anti-Human/Mouse SSEA-4 Monoclonal Antibody (Catalog # NLLC1435R; red). The cells were counterstained with Hoechst 33342 (blue).

Human Pluripotent Stem Cell Marker Antibody Panel

• Cost-effective: includes 5 high-quality antibodies specific for established ES/iPS cell markers • Validated: manufactured and tested in-house for use with ES and iPS cells • Comprehensive: increases confidence in pluripotency status through the use of multiple markers

SSEA-1/DAPI Alkaline Phosphatase/Nanog Oct-3/4/SSEA-4

Expression of SSEA-1 in Mouse Embryonic Stem Cells. D3 Expression of Pluripotency Markers in Human Embryonic Stem Cells. BG01V human embryonic stem cells were stained with mouse embryonic stem cells were fixed and stained with the antibodies provided in this panel (Catalog # SC008). Pluripotency marker expression was analyzed by dual immunofluorescence SSEA-1 antibody supplied in this panel (Catalog # SC008). with the indicated primary antibodies. The cell were visualized using NorthernLights™ (NL)493- and NL557-conjugated The cells were visualized using NorthernLights™ (NL)493- Secondary Antibodies (green and red, respectively). Cells were counterstained with DAPI (blue). conjugated Secondary Antibody and were counterstained with DAPI.

Learn more | rndsystems.com/esips Differentiate

Efficient and consistent ES/iPS cell differentiation is essential for maximizing research productivity, increasing data reliability, and reducing the cost and labor associated with a sometimes lengthy differentiation process. These challenges are remedied by our StemXVivo® differentiation kits, which contain defined, premium quality factors to effectively drive differentiation of ES/iPS cells into ectoderm, mesoderm, endoderm, cardiomyocytes, hepatocytes, and neural progenitor cells.

Human Pluripotent Stem Cell Differentiation Kits and Media

• Robust: yields highly enriched, differentiated cell populations • Efficient: reproducibly induces differentiation using optimized growth factors and supplements • Complete: contains a marker antibody to verify differentiation status • Simple: involves validated and straightforward procedures

Human Pluripotent Stem Cell Differentiation Kits Species Catalog # StemXVivo® Neural Progenitor Differentiation Kit Human SC035 StemXVivo® Cardiomyocyte Differentiation Kit Human SC032B StemXVivo® Cardiomyocyte Maintenance Media Human AR011 StemXVivo® Hepatocyte Differentiation Kit Human SC033 iPSC-derived Neural Progenitor Cells Express SOX1. Human StemXVivo® Ectoderm Differentiation Kit Human SC031B iPSK3 induced pluripotent stem cells were differentiated StemXVivo® Mesoderm Differentiation Kit Human SC030B into neural progenitor cells (NPCs) using the StemXVivo® StemXVivo® Endoderm Differentiation Kit Human SC019B Neural Progenitor Cell Differentiation Kit (Catalog # SC035). In addition to the formation of neural rosettes, NPC Dopaminergic Neuron Differentiation Kit Human/Mouse SC001B differentiation was validated by labeling with Goat Anti- Oligodendrocyte Differentation Kit Mouse SC004 Human/Mouse/Rat SOX1 Antigen Affinity-purified Polyclonal Antibody (red; Catalog # AF3369) followed by NorthernLights™ 557-conjugated Donkey anti-Goat Secondary Antibody (R&D Systems, Catalog # NL001). The nuclei were Proteins for ES/iPS Cell Differentiation counterstained with DAPI (blue; Tocris, Catalog # 5748).

• High Bioactivity: R&D Systems® proteins are known for their industry-leading bioactivity • Consistent: tested for lot-to-lot consistency

ProDots® ProDots® Catalog # Proteins, Catalog # Proteins, Recombinant Proteins Catalog # Recombinant Proteins Catalog # Human Mouse Rat Human Human Mouse Rat Human Ectoderm Lineage Differentiation Endoderm Lineage Differentiation BMP-4 314-BP 5020-BP PRD314 FGF basic 233-FB 3139-FB 3339-FB PRD233 EGF 236-EG 2028-EG 3214-EG PRD236 FGF-4 235-F4 5846-F4 FGF basic 233-FB 3139-FB 3339-FB PRD233 FGF-10 345-FG 6224-FG 7804-FG PRD345 Mesoderm Lineage Differentiation FGF-18 8988-F18 Activin A 338-AC 338-AC 338-AC PRD338 HGF 294-HG 2207-HG BMP-4 314-BP 5020-BP PRD314 IGF-I 291-G1 791-MG 4326-RG PRD291 Dkk-1 5439-DK 5897-DK 4010-DK KGF/FGF-7 251-KG 5028-KG PRD251 FGF-3 1206-F3 PRD1206 Noggin 6057-NG 1967-NG PRD6057 FGF-10 345-FG 6224-FG 7804-FG PRD345 Sonic Hedgehog/Shh 1845-SH 464-SH PRD1845 FGF-13 319-FG 7400-FG Wnt-2b 3900-WN Flt-3 Ligand 308-FK 427-FL PRD308 Wnt-3a 5036-WN 1324-WNP IL-3 203-IL 403-ML 2524-RL PRD203 IL-6 7270-IL 406-ML 506-RL PRD206 IL-11 218-IL 418-ML PDGF-BB 220-BB 520-BB PRD220 SCF/c-kit Ligand 255-SC 455-MC 6998-SC PRD255 Thrombopoietin/Tpo 288-TPN 488-TO 7864-TP VEGF 165 293-VE PRD293 Wnt-5a 645-WN 645-WN

Learn more | rndsystems.com/esips Small Molecules for Differentiation

• Gain temporal control of differentiation pathways • Modulate cell fate by targeting specific signaling pathways • Minimize the use of animal-derived factors

ES and iPS Differentiation Name Description Catalog # Prostaglandin E2 Major endogenous prostanoid 2296 Dibutyryl-cAMP, Cell-permeable cAMP analog 1141 Small Molecules Promote iPS Cell Differentiation into Cells sodium salt of Pancreatic Lineage. Cells from the iPS cell line, iBJ7, were DAPT γ-secretase inhibitor; induces neuronal differentiation 2634 differentiated into pancreatic cells using a combination of growth factors as well as a series of small molecules, Dorsomorphin Potent AMPK inhibitor 3093 dihydrochloride including Retinoic acid (Catalog # 0695), Cyclopamine (Catalog # 1623), and Exendin-4 (Catalog # 1933). iBJ7 XAV 939 Potent tankyrase inhibitor 3748 cells were allowed to differentiate for 20 days and then SU 5402 Potent FGFR and VEGFR inhibitor 3300 stained with Mouse Anti-Human Insulin C-Peptide Monoclonal Antibody (red; Catalog # MAB14171) and Goat LDN 193189 promotes neural induction of human pluripotent stem cells 6053 Anti-Human SOX17 Polyclonal Antibody (green; Catalog # dihydrochloride AF1924). Primary antibodies were visualized using Donkey Forskolin Adenylyl cyclase activator 1099 Anti-Mouse NL557-conjugated (Catalog # NL007) and Donkey Anti-Goat NL493-conjugated (Catalog # NL003) SAG dihydrochloride Dihydrochloride salt of SAG; Smo receptor agonist 6390 secondary antibodies, respectively. The nuclei were labeled with DAPI (blue). Learn more | rndsystems.com/esips_smallmolecules

Investigate

After carefully validating ES/iPS pluripotency, expanding population size, and driving differentiation R&D Systems offers tools to investigate the function of terminally differentiated stem cells. Utilize our Proteome Profiler™ Array Kits to perform expedited protein analysis on your cells or ex- plore our vast selection of high quality antibodies validated for various scientific applications, including immunocytochemistry and flow cytometry.

Proteome Profiler™ Human Pluripotent Stem Cell Array Kit

• Rapid: analyze the expression level of dozens of proteins simultaneously • Economical: contains 4 membranes—each protein is spotted in duplicate • Convenient: small sample size requirements

ndierentiated Ectoderm C Meoderm Endoderm

t Snail S

Proteome Profiler Uses Small Lysate Volume to Analyze Markers of Human Pluripotent buffer and analyzed using the Proteome Profiler Human Pluripotent Stem Cell Array Kit Stem Cell Differentiation. BG01V human embryonic stem cells were differentiated into (Catalog # ARY010). The differentiation supplements upregulated expression of markers for ectoderm, mesoderm, and endoderm using the differentiation supplements included in the ectoderm (B; Otx2), mesoderm (C; Snail), and endoderm (D; SOX17) compared to Human Pluripotent Stem Cell Functional Identification Kit (Catalog # SC027). Following undifferentiated cells (A). differentiation in a 24-well plate, a single well of confluent cells was lysed in 100 µL of lysis

Learn more | rndsystems.com/esips View Pathway

Blastocyst Adult cells

ES/iPS Cells

De-Differentiation Self-renewal factors

Activin A Activin A FGF basic BMP-4 Activin A LIF FGF basic FGF basic Noggin Wnt Nodal Ectoderm Wnt Wnt-3a

EGF Endoderm FGF basic Mesoderm

BMP-4 Activin FGF basic FGF-4 Neural IL-3 BMP-4 Progenitors Dkk-1 Insulin Wnt-3a IL-6 Collagen FGF-3, 10, PDGF-BB FGF-10 IL-11 FGF-4 11, 13, 15 TGF-beta 1 KGF/FGF-7 Intestinal HGF IL-3 SCF/c-kit Noggin Stem Cells Ligand Sonic IL-6 VEGF Epithelial SCF/c-kit Hepatocyte Hedgehog Progenitors Ligand Mesenchymal Progenitors (epidermal, corneal, Stem Cells VEGF mammary, follicular) Hematopoietic Wnt-3a Stem Cells BMP-4 Pancreatic FGF basic Progenitors BMP-4 FGF-10 Flt-3 Ligand FGF-18 Cardiac IL-3 SCF/c-kit Ligand KGF/FGF-7 BMP-2, 4 Progenitors IL-6 Thrombopoietin/Tpo Sonic Hedgehog Insulin SCF/c-kit VEGF Wnt-2a FGF-10 Ligand VEGF Wnt-2b Beta cells Cardiomyocytes Immature Hemangioblasts Lung Cells

Embryonic and Induced Pluripotent Stem Cells

Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are self-renewing progenitors that have the capacity to differentiate into cells of the ectoderm, mesoderm, and endoderm. Naturally existing ES cells can be isolated from the inner cell mass of a blastocyst while iPS cells are generated by inducing expression of specific transcription factors in terminally differentiated somatic cells. ES/iPS cells hold enormous potential in basic and clinical research through their ability to differentiate into a wide variety of cell types, including neurons, pancreatic beta cells, cardiomyocytes, and progenitor cells of the liver, lung, and skin. ES/iPS self-renewal and differentiation are regulated by a precise temporal sequence of growth factor presentation, intracellular signaling, and transcription factor expression. As a result, ES/iPS cell research can require weeks of culture before hypotheses can be tested. Bio-Techne present tools for the reliable isolation, differentiation, verification, and investigation of ES/ iPS cells. These products will reduce experimental variability, improve data consistency, and prevent wasted effort and reagents.

Global [email protected] bio-techne.com/find-us/distributors TEL +1 612 379 2956 North America TEL 800 343 7475 Europe | Middle East | Africa TEL +44 (0)1235 529449 China [email protected] TEL +86 (21) 52380373 bio-techne.com

For research use or manufacturing purposes only. Trademarks and registered trademarks are the property of their respective owners. BR_ESiPS_MKT20-10863 BG01V human embyronic stem cells are licensed from Viacyte, Inc.