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 experimenta- tion 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 manu- facturing 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 t/SX1/DPI racyury/H/DPI SX17/GATA-4/DPI 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).