products for human hescs and hipscs Standardized Tools and Reagents

Transcription

1 products for human PLURIPOTENT stem cells hescs and hipscs Standardized Tools and Reagents

2 Table of Contents 3 Introduction 4 Human Pluripotent Stem Cell Research: Product Overview 5 STEMcircles : Non-Viral, Non-Integrating Somatic Cell Reprogramming to hipscs 6 mtesr 1: Defined, Feeder-Independent Maintenance Medium for Human Pluripotent Stem Cells 9 TeSR 2: Animal Protein-Free, Defined, Feeder-Independent Maintenance Medium for Human Pluripotent Stem Cells 11 mfresr and CryoStor CS10: Defined Cryopreservation Media for Human Pluripotent Stem Cells 12 Antibodies for Characterization of Human Pluripotent Stem Cells 14 Fast and Easy Cell Separation of Human Pluripotent Stem Cells 15 AggreWell : Reproducible Production of Uniformly-Sized Embryoid Bodies 17 Primary Human Cells: Products for Culture and Expansion 18 Support Reagents 19 References Standardizing human pluripotent stem cell culture. STEMCELL Technologies provides standardized tools and reagents to streamline your work with human embryonic and induced pluripotent stem cells. 22

3 PRODUCTS FOR HUMAN PLURIPOTENT STEM CELLS Human Pluripotent Stem Cells Products for Research Introduction Human pluripotent stem cells are defined by the potential for unlimited expansion with the retention of normal karyotype 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. Human pluripotent stem cells can be divided into two broad categories: human embryonic stem cells (hescs), which are derived from the inner cell mass of pre-implantation blastocysts, and induced human pluripotent stem cells (hipscs), which are reprogrammed from somatic cells 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. 3

4 Human Pluripotent Stem Cell Research Product Overview STEMCELL Technologies provides a full range of products that support every step of your research on undifferentiated and differentiated cells from isolation and characterization to maintenance and proliferation. Reprogram STEMcircles (pg. 5) Culture and Freeze mtesr 1 and TeSR 2 (pg. 7-10) mfresr and CryoStor CS10 (pg. 11) Isolate and Characterize Antibodies for Characterization (pg ) EasySep SSEA-4 Positive Selection Kit (pg. 14) Form Embryoid Bodies AggreWell (pg ) Differentiate Products for Primary Cells Isolate, culture and characterize hematopoietic, neural and mesenchymal progenitor cells (pg. 17). 44

5 PRODUCTS FOR HUMAN PLURIPOTENT STEM CELLS STEMcircles Non-Viral, Non-Integrating Somatic Cell Reprogramming to hipscs Figure 1. The STEMcircles TM -LGNSO Vector SOX2 OCT3/4 NANOG CMV Expression of the four reprogramming factors and GFP are driven by the CMV promoter. 8 GFP LIN28 STEMcircles -LGNSO minicircle DNA contains the minimal DNA sequences required for reprogramming human somatic cells: the four factors LIN28, NANOG, SOX2 and OCT3/4, and a gfp reporter gene. Unlike conventional DNA plasmids, the STEMcircles minicircle vector contains no bacterial DNA, and can therefore evade silencing mechanisms that cells naturally use against foreign DNA. 6,7 The result is more robust and prolonged gene expression, and superior reprogramming over that of regular DNA plasmids. This non-viral and non-integrating method thus enables the effective generation of human induced pluripotent stem cells (hipscs). STEMcircles -LGNSO has been used to derive hipscs from adipose-derived stem cells and neonatal fibroblasts. 8 However, STEMcircles -LGNSO has not yet been successful in generating hipscs from adult dermal fibroblasts, or other low efficiency cell types. 19 Figure 2. Robust Expression of Reprogramming Factors Using STEMcircles A 293 transfected 293 transfected no RT 293 untransfected B H9 hes (+ control) 293 untransfected 293 transfected Why Use STEMcircles : SAFETY. Non-viral and non-integrating. LIN28 LIN28 CONVENIENCE. No antibiotic selection required. NANOG NANOG CAPACITY FOR SELECTION. Uses gfp reporter gene to monitor transfection efficiency. EASE OF USE. Single vector contains all reprogramming factors. SOX2 SOX2 OCT3/4 OCT3/4 GAPDH GAPDH PRODUCT Description Catalog # 293 cells were transfected with 5 μg of STEMcircles -LGNSO using Lipofectamine. FACS analysis after 48 h revealed 20-30% GFP + cells (not shown), and RNA and protein analyses were performed on unsorted cells. RT-PCR (A) and Western blot (B) data both indicate strong RNA and protein expression of the crucial reprogramming factors in 293 cells post-transfection. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was measured as a loading control for the amount of RNA or protein. STEMcircles -LGNSO 100 µg vial of minicircle DNA Lipofectamine is a trademark of Invitrogen

6 mtesr 1 Defined, Feeder-Independent Maintenance Medium for Human Pluripotent Stem Cells Developed at the: mtesr 1 is a standardized medium for the feeder-independent maintenance of hescs and hipscs. 3 It is a complete, serumfree, defined formulation based on Ludwig et al. 9 and developed under license from the WiCell Research Institute. To date, mtesr 1 has been used successfully to maintain over 50 independently derived hesc and hipsc lines. mtesr 1 is designed to be used with BD Matrigel hescqualified Matrix (BD Catalog #354277) as a substrate. STEMCELL Technologies pre-qualifies each batch of BD Matrigel to ensure consistency, reproducibility and reliability in performance. mtesr 1 provides: Consistency: Defined, feeder-free hesc/hipsc culture medium. Reproducibility: Standardizes culture methods for more reproducible data. mtesr 1 is the most widely published feeder-free pluripotent cell medium in the world. Visit for a list of references. Convenience: Saves time and effort needed for feeder and conditioned media preparation. A Complete Formulation: No supplements or other growth factors required. product quantity catalog # 500 ml mtesr 1 10 x 500 ml x 500 ml L NEW! 66 7

7 PRODUCTS FOR HUMAN PLURIPOTENT STEM CELLS mtesr 1 Expansion and Morphology figure 3. mtesr 1 Cultures Show Consistent Expansion H1 cell line H9 cell line Average Fold Expansion H1 and H9 hescs were expanded in mtesr 1 for 19 and 18 passages respectively. Cultures show 7- to 10-fold expansion consistently across passages. Figure 4. Morphology of hescs and hipscs Cultured in mtesr 1 A B C D 400 µm 100 µm 1000 μm 1000 µm E F G 1000 µm 1000 µm 1000 µm DAY 2. Colonies are small and less densely packed, making them appear translucent under phase contrast. DAY 4. Colonies rapidly increase in size and start to develop phase-bright centers when viewed under phase contrast. DAY 6. Colonies begin to merge and have phase-bright centers that are densely packed with cells. These colonies are ready to passage. H1 hescs grow as colonies with (A) defined edges and (B) high nucleus to cytoplasm ratio. The hipsc lines (C) ipsc(imr90)-3 and (D) MSC-iPSC1 maintained in mtesr 1 show similar morphological characteristics. (E-G) H9 hescs are routinely passaged every 5-7 days. The morphology of hescs in culture varies slightly compared to feeder-containing or conditioned medium cultures. hipsc photographs courtesy of M. O'Connor and C. Eaves, The Vancouver Human Embryonic Stem Cell Core Facility. 7

8 mtesr 1 Karyotype & Pluripotency FIGURE 5. hescs Cultured in mtesr 1 Retain Normal Karyotype Following Long-Term Passage FIGURE 6. hescs Cultured in mtesr 1 are Pluripotent Cartilage Chromosomal analysis of H1 hescs cultured in mtesr 1 for 48 passages shows that normal karyotype is retained during long-term passaging. Data from Cytogenetics Lab, WiCell Research Institute. Muscle Neural H9 hescs were cultured for 6 passages in mtesr 1 then injected subcutaneously into immunocompromised mice. The resulting teratoma contained cell types from all 3 germ layers. Representative tissue types are shown. 88

9 PRODUCTS FOR HUMAN PLURIPOTENT STEM CELLS TeSR 2 Animal Protein-Free, Defined, Feeder-Independent Maintenance Medium For Human Pluripotent Stem Cells Given current interest in using hescs and hipscs for applications in regenerative medicine, the development of fully humanized and defined systems for the maintenance of hescs and hipscs is critical. TeSR 2 takes a significant step in this direction by allowing hescs and hipscs to be cultured feeder-free in animal protein-free, defined medium. 10 Advantages of TeSR 2: Animal protein-free, defined Reduced variability in hesc and hipsc culture No need to grow feeders product quantity catalog # TeSR ml x 500 ml Expansion & Morphology FIGURE 7. Expansion of hescs Cultured in TeSR 2 FIGURE 8. Morphology of hipscs Cultured in TeSR 2 12 H1 cell line A B Average Fold Expansion H9 cell line 1000 µm hipscs cultured in TeSR 2 grow as colonies with defined edges 200 and µm high nucleus to cytoplasm ratio. ips(imr90)-1 cell line cultured (A) for 8 passages in TeSR 2 and (B) for 10 passages in TeSR 2. Photographs courtesy of Dr. T. Ludwig, WiCell Research Institute. H1 and H9 hescs were expanded in TeSR 2 for 26 and 27 passages respectively. hescs cultured in TeSR 2 demonstrate 6- to 8-fold expansion consistently across passages. 9

10 TeSR 2 Karyotype & Pluripotency FIGURE 9. hescs Cultured Long-Term in TeSR 2 Retain Normal Karyotype H1 hescs after 19 passages in TeSR 2 H9 hescs after 22 passages in TeSR 2 FIGURE 10. hescs Cultured in TeSR 2 are Pluripotent Muscle Neural Cartilage Gut Epithelia Muscle H9 hescs were cultured for 11 passages in TeSR 2 then injected subcutaneously into NOD-SCID mice. The resulting teratomas contained cell types from all 3 germ layers. Representative tissue types are shown. 10

11 PRODUCTS FOR HUMAN PLURIPOTENT STEM CELLS mfresr and CryoStor CS10 Defined Cryopreservation Media for Human Pluripotent Stem Cells 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 hescs and hipscs, that has been shown to improve thawing efficiencies 5- to 10-fold over other reported methods CryoStor CS10 is a cgmp-manufactured, animal-protein-free cryopreservation medium for human pluripotent cells. Combine these serum-free cryopreservation media with our feeder-free maintenance media mtesr 1 and TeSR 2 to minimize variability in your human pluripotent cell lines. mfresr Serum-free Research use only CryoStor CS10 Serum-free and xeno-free cgmp-compliant product quantity catalog # mfresr 10 x 5 ml ml Ideally used after culture in mtesr 1 or TeSR 2 CryoStor CS ml FIGURE 11. Clump Survival Data for mfresr FIGURE 12. Clump Survival Data for CryoStor CS10 10% 10% % clumps giving rise to undifferentiated colonies 5% % clumps giving rise to undifferentiated colonies 5% 0% p64 p74 p74 p40 p40 p38 p38 p42 p42 0% p53 p53 p65 p65 p61 p61 H9 hescs were cryopreserved in mfresr at the indicated passage after culture with mtesr 1. Thawing efficiencies were determined by counting the number of clumps after thawing, and the number of resultant undifferentiated colonies. H9 hescs were cryopreserved with CryoStor CS10 at the indicated passage after culture with TeSR 2. Thawing efficiencies were determined by counting the number of clumps after thawing, and the number of resultant undifferentiated colonies. 11

12 Antibodies for Characterization of Human Pluripotent Stem Cells STEMCELL offers a wide range of primary and secondary antibodies suitable for the characterization of hescs and hipscs during culture maintenance and expansion. Undifferentiated hescs and hipscs 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 hesc or hipsc population. FIGURE 13. Immunohistochemistry of H1 hescs cultured in mtesr 1 reveal uniform expression of pluripotency markers throughout the colony. Oct-3/4(Catalog #01550/01551) with FITC-conjugated secondary antibody (Catalog #10210). Data courtesy of Dr. T Ludwig, WiCell Research Institute. SSEA-4 (Catalog #01554) with FITC-conjugated secondary antibody (Catalog #10210); DAPI staining shows nuclei. Primary Antibodies Target Antigen Clone Isotype Catalog # Quantity Oct-3/4 40 Mouse IgG µg 150 µg SSEA-1 MC-480 Mouse IgM tests SSEA-3 MC-631 Rat IgM tests SSEA Mouse IgG tests TRA-1-60 TRA-1-60 Mouse IgM tests TRA-1-81 TRA-1-81 Mouse IgM tests TRA-2-49 TRA-2-49/6E Mouse IgG tests TRA-2-54 TRA-2-54/2J Mouse IgG tests Secondary Antibodies Target Antigen Host Species Format Catalog # Quantity For use with Mouse IgG Goat FITC mg Mouse IgM Goat FITC mg Anti-TRA-2-49 Anti-TRA-2-54 Anti-SSEA-4 Anti-Oct-3/4 Anti-SSEA-1 Anti-TRA-1-60 Anti-TRA-1-81 Rat IgM Goat APC mg Anti-SSEA-3 12

13 PRODUCTS FOR HUMAN PLURIPOTENT STEM CELLS FIGURE 14. hescs Cultured in either mtesr 1 or TeSR 2 Express High Levels of Pluripotent Markers and Low Levels of Differentiation Markers Oct-3/4 SSEA-3 SSEA-4 % of Maximum % of Maximum 95.6% 97.7% 96.1% % of Maximum TRA-1-60 TRA-1-81 SSEA-1 % of Maximum % of Maximum 92.9% 95.5% 0.2% % of Maximum Flow cytometric analysis of H9 hescs maintained in mtesr 1 for 17 passages Oct-3/4 SSEA-3 % of Maximum 99.67% % of Maximum 92.71% SSEA-4 TRA-1-81 % of Maximum 99.96% % of Maximum 94.95% Flow cytometric analysis of H1 hescs maintained in TeSR 2 for 26 passages 13

14 Fast and Easy Cell Separation of Human Pluripotent Stem Cells 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. 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 to isolate highly purified human pluripotent cells quickly and easily. 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. FIGURE 15. Highly Purified SSEA-4 + Cells Isolated with EasySep Typical FACS Histogram Results Using EasySep hesc/hipsc SSEA-4 Antibody Positive Selection Kit Start: 6% SSEA-4 + H9 Cells Selected: 99% SSEA-4 + H9 Cells Counts Counts SSEA-4 PE SSEA-4 PE Starting with a mixture of H9 hescs and HT1080 fibroblast cells, the SSEA-4 + cell content of the enriched fraction typically ranges from 95-99%. product DESCRIPTION catalog # EasySep hesc/hipsc SSEA-4 Positive Selection Kit* Optimized kit for the isolation of highly purified hescs and hipscs using SSEA-4 antibody *Required equipment: EasySep Magnet (Catalog #18000) Additional Cell Separation Products for Differentiated Human Cells EasySep Cell Separation Kits EasySep can be used for the positive or negative selection of almost any cell type from any source, in as little as 25 minutes. Customizations for your specific experimental needs are available. RoboSep (Catalog #20000) The fully automated cell separator 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. 14

15 PRODUCTS FOR HUMAN PLURIPOTENT STEM CELLS AggreWell Reproducible Production of Uniformly-Sized Embryoid Bodies Many hesc and hipsc differentiation protocols begin with the formation of 3-dimensional aggregates of cells called embryoid bodies (EBs). 16 Conventionally, EBs are formed in suspension culture from uneven clumps of cells scraped off undifferentiated cell cultures. The EBs are heterogeneous in size and shape, leading to inefficient and uncontrolled differentiation. Using AggreWell plates, EBs form inside individual microwells that are inoculated with a single cell suspension. The resulting EBs are highly uniform in size (Figure 16) and can be efficiently differentiated into a variety of cell types. 17 AggreWell standardizes the production of EBs, making experiments more reproducible over time. Embryoid Bodies generated using AggreWell plates are: Uniform in size and shape Reproducible Size-controlled product DESCRIPTION quantity catalog # AggreWell 400 plate 8 wells, with approximately 1,200 microwells per well 1/pack /pack AggreWell 800 plate AggreWell Medium AggreWell Reversible Cell Strainer 8 wells, with approximately 300 microwells per well Defined, serum-free medium for generation and culture of embryoid bodies using AggreWell plates Sterile nylon mesh filter, fits standard 14 ml round bottom tubes or 15 ml conical tubes 1/pack /pack mL COMING SOON! Sterile nylon mesh filter, fits standard 50 ml conical tubes NEW! NEW! 15

16 FIGURE 16. EB Formation with AggreWell is Uniform and Easily Controlled 500 cells/eb 2000 cells/eb scraped EBs Count H9 hescs seeded onto AggreWell 400 plates at a density of 2000 cells per microwell. Area (micron 2 ) x 10 3 H9 hescs were centrifuged into AggreWell 400 plates and cultured for 24 hours prior to EB harvest. EB size is tightly controlled with AggreWell (grey), unlike with scraping protocols (brown) that give a wider distribution. 17 The resultant EBs are uniform in size and shape. Optimizing AggreWell Use Use AggreWell Medium and the AggreWell Reversible Cell Strainer to optimize the generation and isolation of EBs, respectively. AggreWell Medium is compatible with mtesr 1 & TeSR 2, and supports EB formation without serum or added growth factors. After formation in AggreWell, EBs of greater than 50 cells can be collected in and easily recovered from the AggreWell Reversible Cell Strainer, allowing for easy isolation of EBs from any residual non-incorporated cells. 16

17 PRODUCTS FOR HUMAN PLURIPOTENT STEM CELLS Primary Human Cells Products for Culture and Expansion STEMCELL Technologies offers a variety of products for the maintenance, expansion and characterization of multiple differentiated cell types. Our products are calibrated for optimal performance when used together. Hematopoietic Cells Isolate, culture and characterize human hematopoietic progenitors with: EasySep hesc-derived CD34 Positive Selection Kit - optimized kit for isolating hesc-derived CD34 + cells. MethoCult - methylcellulose-based media optimized for use in hematopoietic colony-forming cell (CFC) assays. Neural Cells Standardize the proliferation and differentiation of human neural stem and progenitor cells (NSCs) with NeuroCult : NeuroCult Proliferation Media - optimized media for prolonged and reproducible expansion of human neural stem cells. Available in serum-free and xeno-free formulations. NeuroCult SM1 Neuronal Supplement - serum-free neuronal supplement based on the B27 formulation 18 and optimized to give reproducibly high numbers of functional neurons with minimal glial cell contamination. Mesenchymal Cells Expand and differentiate human mesenchymal stem cells (MSCs) with: MesenCult -XF Medium - a xeno-free, serum-free culture medium for human MSCs that provides superior performance compared to traditional serumcontaining formulations. MesenCult Proliferation Kit (Human) - serum-containing medium optimized for cell expansion and CFU-F assays. Contains prescreened components that minimize lot-to-lot variability. 17

18 Support Reagents A variety of support products are available to accompany StemCell Technologies array of specialized products for hesc and hipsc research. Please visit for more details and a full list of tissue culture reagents and supplies. Tissue Culture Media PRODUCT Catalog # Unit Size DMEM with 4500 mg/l D-glucose ml DMEM with 1000 mg/l D-glucose ml DMEM/F ml Iscove s MDM (IMDM) ml Balanced Salt Solutions PRODUCT Catalog # Unit Size D-PBS ml D-PBS, 10X ml HBSS, Ca ++ & Mg ++ free ml HBSS, without Phenol Red ml Enzymes PRODUCT Catalog # Unit Size Accutase ml Collagenase ml Collagenase Type IV ml Dispase (1 mg/ml) ml Dispase (5 mg/ml) ml DNase I (1 mg/ml) ml Trypsin-EDTA (0.05%) ml Trypsin-EDTA (0.25%) ml Trypsin in Citrate Saline ml Antibiotics PRODUCT Catalog # Unit Size Penicillin G and Streptomycin,100X ml Neomycin (G418) mg Hygromycin B mg ACCUTASE is a registered trademark of Innovative Cell Technologies. Miscellaneous Tissue Culture Reagents and Supplies PRODUCT Catalog # Unit Size 3% Acetic Acid with Methylene Blue Recombinant Cytokines PRODUCT Catalog # Unit Size Activin A µg BAFF µg Bone Morphogenetic Protein µg Bone Morphogenetic Protein µg Noggin µg Wnt-3a µg Basic Fibroblast Growth Factor (bfgf) Transforming Growth Factor-β ml Collagen Solution (3 mg/ml) ml Fibronectin (1 mg/ml) ml Gelatin (0.1% in water) ml Hypoxia Chamber chamber Rat Serum µg ml 5 x 2 ml Sodium Pyruvate (100 mm) ml Trypan Blue ml Y (ROCK inhibitor) Tissue Culture Dishes mg mg PRODUCT Catalog # Unit Size 35 mm Diameter 60 mm Diameter 100 mm Diameter 245 mm x 245 mm 96-Well Plates /pack 500/case 10/pack 400/case 10/pack 240/case 4/pack 16/case 1/pack 50/case 2 µg 10 µg 18

19 PRODUCTS FOR HUMAN PLURIPOTENT STEM CELLS References 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, 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, 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: , 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, Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18: , Chen ZY, He CY, Ehrhardt A, Kay MA. Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo. Mol. Ther. 8, ; Chen ZY, He CY, Kay MA. Improved production and purification of minicircle DNA vector free of plasmid bacterial sequences and capable of persistent transgene expression in vivo. Hum. Gene Ther. 16, ; Jia F, Wilson KD, Sun N, Gupta DM, Huang M, Li Z, Panetta NJ, Chen ZY, Robbins RC, Kay MA, Longaker MT, Wu JC. A nonviral minicircle vector for deriving human ips cells. Nature Methods 7(3): 197-9; 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, 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: , 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: , Ji L, de Pablo JJ, Palecek SP. Cryopreservation of adherent human embryonic stem cells. Biotechnol Bioeng 88: , Ware CB, Nelson AM, Blau CA. Controlled-rate freezing of human ES cells. Biotechniques 38:879-80, 882-3, 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, 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, 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, Brewer GJ, Torricelli JR, Evege EK, Price PJ. Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J Neurosci Res. 35: , Narsinh KH, Jia F, Robbins RC, Kay MA, Longaker MT, Wu JC. Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors. Nat Protoc. 6:78-88, 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 Copyright 2011 by STEMCELL Technologies Inc. All rights reserved including graphics and images. STEMCELL Technologies and Design, STEMCELL shield, THE CELL EXPERTS, STEMcircles, FreSR, AggreWell, EasySep, MethoCult, NeuroCult and MesenCult are trademarks of STEMCELL Technologies Inc. All other trademarks are the property of their respective holders. 19

20 THE CELL EXPERTS TOLL-FREE PHONE PHONE TOLL-FREE FAX FAX ORDERS@STEMCELL.COM INFO@STEMCELL.COM FOR FULL CONTACT DETAILS WORLDWIDE VISIT OUR WEBSITE FOR RESEARCH USE ONLY. NOT FOR THERAPEUTIC OR DIAGNOSTIC USE. CATALOG #29063 VERSION MARCH 2011