Human ESC-derived Neural Progenitor Expansion Starter Panel Catalog Number SC016 Reagents for the expansion of human neural progenitors. This package insert must be read in its entirety before using this product. FOR LABORATORY RESEARCH USE ONLY. NOT FOR DIAGNOSTIC USE. THE SAFETY AND EFFICACY OF THIS PRODUCT IN DIAGNOSTIC OR OTHER CLINICAL USES HAS NOT BEEN ESTABLISHED.
Contents TABLE OF CONTENTS INTRODUCTION 2 DESIGN OF THE KIT...................................... 2 LIMITATIONS OF THE PROCEDURE 3 PRECAUTIONS......................................... 3 MATERIALS PROVIDED 3 STORAGE............................................ 3 OTHER SUPPLIES REQUIRED 4 REAGENT AND MATERIAL PREPARATION.......................... 4 PROCEDURE 5 Human Neural Progenitor Expansion........................... 5 Human Neural Progenitor Passage 6 Human Neural Progenitor Characterization........................ 6 SAMPLE DATA 6 REFERENCES.......................................... 7 Page MANUFACTURED AND DISTRIBUTED BY: R&D Systems, Inc. TELEPHONE: (800) 343-7475 614 McKinley Place NE (612) 379-2956 Minneapolis, MN 55413 FAX: (612) 656-4400 United States of America E-MAIL: info@rndsystems.com DISTRIBUTED BY: R&D Systems Europe, Ltd. 19 Barton Lane TELEPHONE: +44 (0)1235 529449 Abingdon Science Park FAX: +44 (0)1235 533420 Abingdon, OX14 3NB E-MAIL: info@rndsystems.co.uk United Kingdom R&D Systems GmbH TELEPHONE: +49 (0)6122 90980 Borsigstrasse 7 FAX: +49 (0)6122 909819 65205 Wiesbaden-Nordenstadt E-MAIL: infogmbh@rndsystems.co.uk Germany R&D Systems Europe 77 boulevard Vauban FREEPHONE: +0800 90 72 49 59041 LILLE CEDEX FAX: +0800 77 16 68 France E-MAIL: info@rndsystems.co.uk
INTRODUCTION Neural progenitors (NPs), with the potential to self-renew and differentiate into neurons and glial cells, have been identified from different regions of the developing and adult brain (1-6). NPs can also be derived from human embryonic stem cells (ESCs) and grown in vitro in the presence of mitogen. These in vitro propagated NPs can proliferate in culture while retaining the potency to differentiate into neurons, oligodendrocytes, and glia. In vitro human neural progenitor expansion makes it possible to grow sufficient numbers of NPs for researchers to study basic developmental biology, drug discovery, and preclinical neural transplantation. Figure 1: Expanded human NPs in the monolayer system on day 3 after passage 10. DESIGN OF THE KIT The Human Embryonic Stem Cell-Derived Neural Progenitor (NP) Expansion Starter Panel is a monolayer system designed for in vitro NP expansion in a serum-free environment. The kit contains a specially formulated N-2 Plus Supplement that has been optimized for NP expansion (7). Human Fibroblast Growth Factor basic (FGF basic) and Neuregulin1-1 (NRG1-1) are included to promote the in vitro proliferation of NPs. Bovine Fibronectin, used to coat plates, is also included. The quantity of each component provided in the kit is sufficient to make 500 ml of growth media for cell expansion. This kit also contains mouse anti-human Nestin antibody. Cells propagated using this kit are Nestin-positive (8) and retain their potency to differentiate into neurons, oligodendrocytes, and glia as identified after differentiation induction by staining with antibodies specific to Tuj1, O4, and GFAP, respectively. This product is sold under license from NovoCell, Inc. 2
LIMITATIONS OF THE PROCEDURE FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. The safety and efficacy of this product in diagnostic or other clinical uses has not been established. This kit should not be used beyond the expiration date on the kit label. The quality of the neural stem cells and any variations in the procedure can cause variation in the efficiency of cell expansion and/or differentiation. PRECAUTIONS The acute and chronic effects of over-exposure to reagents of this kit are unknown. Safe laboratory procedures should be followed, and protective clothing should be worn when handling kit reagents. The N-2 Plus Supplement contains human transferrin. This transferrin was tested at the donor level using an FDA licensed method and found to be non-reactive for anti-hiv-1/2 and Hepatitis B surface antigen. As no testing can offer complete assurance of freedom from infectious agents, these reagents should be handled as if capable of transmitting infection. MATERIALS PROVIDED N-2 Plus Supplement (Part 390155) - 5 ml of a 100X concentrated solution containing bovine insulin, human transferrin, sodium selenite, putrescine, and progesterone. Human FGF basic (Part 390314) - 10 g of lyophilized recombinant human FGF basic in Tris buffer. Human NRG1-1 (Part 390492) - 10 g of lyophilized recombinant human NRG1-1 in Tris buffer. Bovine Fibronectin (Part 390438) - 250 L of a 100X solution (100 g/ml) of purified bovine fibronectin in Tris buffer. Mouse Anti-human Nestin Antibody (Part 964671) - 25 g of lyophilized mouse anti-human Nestin monoclonal antibody; enough to prepare 5 ml of 0.5 g/100 L staining solution. STORAGE Unopened Kit Opened or Reconstituted Reagents Store at -20 C in a manual defrost freezer. Do not use beyond the kit expiration date. N-2 Plus Supplement Human FGF basic Human NRG1-1 Bovine Fibronectin (100X Stock) Mouse Anti-human Nestin Antibody *Provided this is within the expiration date of the kit. Store at 2-8 C for up to 1 month, or aliquot and store at -20 C in a manual defrost freezer for up to 3 months.* Avoid repeated freeze-thaw cycles. 3
OTHER SUPPLIES REQUIRED Materials Human ESC-derived Neural Progenitor cells (R&D Systems, Catalog # PSC002 or equivalent) 0.2 m, 500 ml filter unit (Nalgene, VWR Catalog # 28198-505 or equivalent) 0.2 m, 1000 ml filter unit (Nalgene, VWR Catalog # 28198-496 or equivalent) 0.2 m syringe filter (PALL Gelman Laboratories, Catalog # 4187 or equivalent) 10 ml syringes (Becton Dickinson, Catalog # 309604 or equivalent) 60 mm tissue culture dishes (Falcon, Catalog # 353002 or equivalent) 15 ml centrifuge tubes (Corning, Catalog # 430052 or equivalent) Pipettes and pipette tips Reagents DMEM/F-12, no HEPES (Invitrogen, Catalog # 12500-062 or equivalent) Phosphate Buffered Saline (PBS) (Invitrogen, Catalog # 10010-023 or equivalent) Glucose (Sigma, Catalog # G6152 or equivalent) L-Glutamine (Sigma, Catalog # G5763 or equivalent) Sodium Bicarbonate (NaHCO 3 ) (Sigma, Catalog # S5761 or equivalent) Penicillin-Streptomycin, 100X (Invitrogen, Catalog # 15140-148 or equivalent) Poly-L-Ornithine (Sigma, Catalog # P3655 or equivalent) BSA (Millipore, Catalog # 81-068-3 or equivalent) Probumin BSA (Millipore, Catalog # 82-047-3 or equivalent) Accutase (Innovative Cell Technologies, Catalog # AT104 or equivalent) Trypan blue (Invitrogen, Catalog # 15250-061) Sterile deionized water Equipment 37 C CO 2 incubator Centrifuge Hemocytometer Microscope REAGENT AND MATERIAL PREPARATION Probumin BSA Stock (20% w/v) - Dissolve 20 grams of Probumin BSA powder in 100 ml of DMEM/F-12. Aliquot and store at -80 C for long-term storage. N-2 Plus Medium - Mix the following ingredients with deionized water to make 500 ml of medium. Adjust ph to 7.2 0.2. Sterile filter the solution using a 500 ml 0.2 m filter unit, and add 5 ml of 100X penicillin-streptomycin. Store in the dark at 2-8 C for up to 2 weeks. DMEM/F-12 Glucose L-Glutamine NaHCO 3 6 mg 775 mg 36.5 mg 845 mg N-2 Plus Supplement (100X) 5 ml Probumin BSA Stock 5 ml Probumin is a trademark of Millipore, Inc. 4
0.1% BSA - Dissolve 10 mg of BSA in 10 ml of PBS. Sterile filter the solution using a 0.2 m syringe filter and store at 2-8 C for up to 3 months. FGF basic Stock (1000X) - Add 0.5 ml of 0.1% BSA to the human FGF basic vial. Store at 2-8 C for up to 1 month, or aliquot and store at -20 C in a manual defrost freezer for up to 3 months. Avoid repeated freeze-thaw cycles. NRG1-1 Stock (1000X) - Add 0.5 ml of 0.1% BSA to the human NRG1-1 vial. Store at 2-8 C for up to 1 month, or aliquot and store at -20 C in a manual defrost freezer for up to 3 months. Avoid repeated freeze-thaw cycles. Human NP Completed Medium - Dilute FGF basic Stock and NRG1-1 Stock 1000-fold in N-2 Plus Medium. Prepare fresh as needed. Poly-L-Ornithine Stock (100X) - Dissolve Poly-L-Ornithine in sterile deionized H 2 O to make a 10 mg/ml stock. Aliquot and store at -20 C in a manual defrost freezer for up to 6 months. Avoid repeated freeze-thaw cycles. Poly-L-Ornithine Solution (1X) - Dissolve Poly-L-Ornithine Stock 100-fold in sterile deionized H 2 O to make a 1X solution (100 g/ml). Prepare fresh as needed. Fibronectin Solution (1X) - Gently dilute Fibronectin Stock 100-fold in sterile deionized H 2 O to make a 1X solution (1 g/ml). Prepare fresh as needed. Poly-L-Ornithine/Fibronectin Coated Plates 1. Add 2 ml of Poly-L-Ornithine Solution (1X) to each 60 mm tissue culture dish. Incubate overnight at 2-8 C. 2. Discard the Poly-L-Ornithine Solution. Wash each dish 3 times with 2 ml of sterile deionized H 2 O. 3. Add 2 ml of Fibronectin Solution (1X) to each dish. Incubate at 37 C for a minimum of 3 hours and up to 24 hours. 4. Discard the Fibronectin Solution. Wash each dish once with 2 ml of sterile deionized H 2 O and once with 2 ml of DMEM/F-12 media before use.* *Coated plates can also be stored with PBS at 2-8 C for up to 2 weeks. Do not let the plates dry out. PROCEDURE Human Neural Progenitor Expansion 1. Seed 1.0-1.5 x 10 6 NPs in 2 ml of human NP Completed Medium on a Poly-L-Ornithine/Fibronectin Coated Plate. 2. Incubate the cells at 37 C and 5% CO 2. 3. Supplement the medium with FGF basic (1X) and NRG1-1 (1X) each day. Every second day, replace the medium with fresh human NP Completed Medium. 4. Pass the cells at 90% confluence (approximately 4 days after initial plating) according to the procedure described on page 6. 5
Human Neural Progenitor Passage 1. Pre-warm the Accutase and 15 ml of human NP Completed Medium at 37 C. 2. Remove the medium from the cells. Wash once in 2 ml PBS. 3. Add 1 ml of Accutase to the 60 mm tissue culture plate. Incubate cells at 37 C for 5-15 minutes until cells round up (check frequently). 4. Gently tap the sides of the plate to dislodge cells. 5. Gently triturate using a 2 ml pipette and transfer the cells to a 15 ml centrifuge tube containing 10 ml of pre-warmed human NP Completed Medium. 6. Centrifuge for 5 minutes at 200 x g. 7. Remove the supernatant and resuspend the cells with 5 ml of human NP Completed Medium by slowly pipetting up and down approximately 5 times with a 5 ml pipette. 8. Mix 10 L of the cell suspension with 10 L of Trypan blue and count the live cells on a hemocytometer. Human Neural Progenitor Characterization Human NPs can be characterized after expansion for Nestin expression using mouse anti-human Nestin antibody by following standard immunocytochemistry and intracellular flow cytometry protocols (www.rndsystems.com/pdf/mab1259.pdf). SAMPLE DATA For full color images, refer to www.rndsystems.com/pdf/sc016.pdf Figure 2: ESC-derived human NPs stained with mouse anti-human Nestin monoclonal antibody (R&D Systems, Catalog # MAB1259; green) followed by NorthernLights anti-mouse IgG-NL637 (R&D Systems, Catalog # NL008) and goat anti-human SOX2 (R&D Systems, Catalog # AF2018; red) followed by NorthernLights anti-goat IgG-NL557 (R&D Systems, Catalog # NL001). Cells were counter stained with Dapi (blue). 6
Figure 3: ESC-derived human NPs stained with (A) PE-conjugated mouse anti-human Nestin monoclonal antibody (R&D Systems, Catalog # IC1259P; green histogram) with isotype control (R&D Systems, Catalog # IC002P; black histogram) and (B) APC-conjugated mouse anti-human SOX2 monoclonal antibody (R&D Systems, Catalog # IC2018A; green histogram) with isotype control (R&D Systems, Catalog # IC003A; black histogram). Figure 4: Differentiated ESC-derived human NPs stained with goat anti-human GFAP polyclonal antibody (R&D Systems, Catalog # AF2594; red) followed by NorthernLights anti-goat IgG-NL557 (R&D Systems, Catalog # NL001) and mouse anti-tuj1 (R&D Systems, Catalog # MAB1195; green) followed by NorthernLights anti-mouse IgG-NL493 (R&D Systems, Catalog # NL003). Cells were counter-stained with Dapi (blue). REFERENCES 1. Alvarez-Buylla, A. et al. (1998) J. Neurobiol. 36:105. 2. McKay, R.D. (1997) Science 276:66. 3. Reynolds, B.A. et al. (1992) Science 255:1707. 4. Johansson, C.B. et al. (1999) Cell 96:25. 5. Kilpatrick, T.J. et al. (1993) Neuron 10:255. 6. Davis, A.A. et al. (1994) Nature 372:263. 7. Johe, K.K. et al. (1996) Genes & Development 10:3129. 8. Lendahl, U. et al. (1990) Cell 60:585. 2008 R&D Systems, Inc. 02.08 725819.0 2/08 7