FactorStem Reprogramming Kit. Part Number FBRK010

Transcription

1 FactorStem Reprogramming Kit Part Number May 18, 2015

2 Table of Contents Sec on Page About This Manual 2 Kit Contents 3 Materials Required 3 Introduc on 4 Notes Before Star ng 4 Reprogramming Protocol Summary 6 Detailed Reprogramming Protocol 7 Example Results 9 Troubleshoo ng 13 Legal Informa on 15 Contact Informa on 16 About This Manual This manual assumes that the user has experience with mammalian cell culture, including the culture of human pluripotent stem cells. We strongly recommend reading through the en re manual before using the FactorStem Reprogramming Kit for the first me. Technical support can be obtained by at techsupport@factorbio.com. Explana on of Keywords and Symbols Used in This Manual: NOTE! CAUTION! Important informa on specific to this kit. A step that may be technically challenging and/or a step that, if performed incorrectly, can cause the experiment to fail. 2

3 Kit Contents FactorStem Reprogramming RNA 4 x 5µg (4 x 25µL at 200ng/µL), enough for 2 wells FactorStem Reprogramming Medium 25mL FactorPlex Complexa on Buffer 2 x 500µL FactorCoat Coa ng Matrix 20µL, enough for 10 wells of a 24 well plate NOTE! Store all components at 4C. Components are stable for at least 3 months at 4C. NOTE! FactorStem Reprogramming Medium may arrive par ally frozen. Thaw completely and mix well before use. NOTE! Before each use, mix the contents of each tube by flicking gently, and spin briefly to collect the material at the bo om of the tube. NOTE! Use of the included FactorCoat Coa ng Matrix is op onal. If desired, the FactorCoat Coa ng Matrix may be used to coat wells in prepara on for transferring reprogrammed cells (i.e., AFTER reprogramming). Cells to be reprogrammed should be plated on uncoated wells (see detailed protocol on Pages 7 8). NOTE! For best results, warm the FactorStem Reprogramming Medium by placing the bo le with the cap loosened in the incubator in which the cells will be cultured. Materials Required Human fibroblasts An established culture of ac vely dividing cells Transfec on reagent (we recommend Lipofectamine 3000, Life Technologies Part# L ) Cell culture incubator (37 C, 5% CO 2 required, 5% O 2 strongly recommended) Cell culture plas cware including 6 well plates Cell dissocia on solu on for ini al pla ng of fibroblasts (e.g., trypsin, etc.) Fibroblast pla ng medium (e.g., DMEM + 10% FBS) Human pluripotent stem cell culture medium: FactorStem Reprogramming Medium can be used as a pluripotent stem cell maintenance medium. When used as a maintenance medium, FactorStem Reprogramming Medium does not require condi oning. We do not recommend the use of low protein media (such as E8 medium), which o en require lengthy acclima on to achieve op mal growth. 3

4 Introduc on Soma c cells can be reprogrammed to a pluripotent stem cell state by expressing certain proteins ( reprogramming factors ) in the cells. Many reprogramming factors are proteins that are highly expressed in pluripotent stem cells, and can cause downregula on of soma c cell associated genes and upregula on of pluripotent stem cell associated genes. When reprogramming factors are expressed in ac vely dividing fibroblasts for several days, the fibroblasts undergo morphological changes resembling mesenchymal to epithelial transi on (MET), and eventually resemble embryonic stem cells in morphology, gene expression, and the ability to differen ate into cell types of all three germ layers both in vitro and in vivo. The FactorStem Reprogramming Kit uses patented RNA Reprogramming technology to reprogram human fibroblasts to a pluripotent stem cell state (U.S. Pat. No. 8,497,124). The kit contains two main components: 1. FactorStem Reprogramming RNA, a powerful mixture of synthe c RNA encoding reprogramming factors, and 2. FactorStem Reprogramming Medium, a proprietary, animal component free medium required for RNA based reprogramming. Together, these components enable reprogramming of human fibroblasts, including primary, adult human fibroblasts, to pluripotency with unprecedented speed, efficiency, and ease, and without the need for feeders, condi oning, immunosuppressants or mid protocol passaging. When following the protocol provided in this manual, fibroblasts are plated on Day 0, six daily transfec ons are performed on Days 1 8, and colonies can be picked as early as Day 10. Reprogramming ac vely dividing, low passage, primary adult human fibroblasts using the FactorStem Reprogramming Kit typically yields between 20 and 100 pickable colonies per well of a 6 well plate from 1000 star ng cells, corresponding to a reprogramming efficiency of 2 10%. Notes Before Star ng Achieving the best possible results with the FactorStem Reprogramming Kit requires special care to ensure that the large numbers of rapidly dividing cells undergoing reprogramming remain healthy throughout the reprogramming process. For this reason, it is important to adhere closely to the protocol, and to use cell culture best prac ces throughout. Following is a list of prac ces that should be used for best results: 1. Warm medium in incubator with cap loosened. The correct color of the FactorStem Reprogramming Medium is orange (Fig. 1). The orange color indicates that the medium has the correct ph. The correct ph and orange color will be maintained if the medium is kept equilibrated at 5% CO 2. This is most easily achieved by warming the medium before each use by placing the bo le with the cap loosened in the incubator (i.e., instead of using a waterbath). If the medium is not kept equilibrated at 5% CO 2, it will begin to turn pink, and performance will decrease. The medium can be divided into aliquots, if desired, to speed warming. 4

5 Figure 1. The correct color of the FactorStem Reprogramming Medium is orange. 2. Place plates directly on incubator shelf (i.e., do not stack). Stacking plates in the incubator can increase the me required for the top plate to reach 37C. 3. Minimize the me that the cells are out of the incubator. Cells can be observed just before each transfec on, and should be returned to the incubator as quickly as possible. If allowed to cool significantly, the cells will begin to detach from the plate. 4. Avoid bumping or jostling the plate between transfec ons. Handling the plate between transfec ons can increase transfec on associated toxicity, and decrease reprogramming efficiency. The plate should only be removed from the incubator to perform the transfec ons. 5. Add medium dropwise. Adding medium too quickly can cause the cells to detatch from the plate. Add the medium as slowly as possible, but do not let the cells dry out. Adding the medium to the same spot of the well each me can help to localize any damage to the cell layer that may occur. 6. Use healthy fibroblasts. Fibroblasts that are not passaged regularly (every 2 4 days) or that have been allowed to grow to confluency will become senescent and difficult to reprogram. Plate fibroblasts 2 3 days a er thawing or passaging. 5

6 Reprogramming Protocol Summary This protocol is designed to reprogram human fibroblasts in 1 well of a 6 well plate. Volumes can be scaled up to reprogram cells in more than 1 well. Reprogramming cells in other well sizes/formats (24 well, 96 well plates, etc.) is NOT recommended. CAUTION! CAUTION! CAUTION! Perform the transfec ons at the same me each day. Do not replace the medium or disturb the plate on Day 2 or Day 3. Do not replace the medium for 2 days a er the last transfec on. Table 1. Transfec on Schedule: FactorPlex for Transfec on Reagent Total Volume of Complexed RNA Day µg of RNA FactorPlex for RNA RNA Solu on Transfec on Reagent µg 5µL 1.25µL 6µL 0.25µL 12.5µL µg 20µL 5µL 24µL 1µL 50µL 5 1µg 20µL 5µL 24µL 1µL 50µL 6 2µg 40µL 10µL 48µL 2µL 100µL 7 2µg 40µL 10µL 48µL 2µL 100µL 8 2µg 40µL 10µL 48µL 2µL 100µL

7 Detailed Reprogramming Protocol Day 0 Day 1 Plate 1,000 fibroblasts in one uncoated well of a 6 well plate in DMEM + 10% FBS. Allow cells to a ach and spread in the incubator overnight. 1. Replace the culture medium with 2mL of warm FactorStem Reprogramming Medium and return the plate to the incubator. NOTE! Ensure that the FactorStem Medium is orange as shown on Page Transfect cells with 0.25µg FactorStem Reprogramming RNA: A. Prepare 2 sterile, RNase free 1.5mL tubes. Label one tube RNA, and the other tube Transfec on Reagent. B. Add 5µL of FactorPlex to the tube labeled RNA, and 6µL of FactorPlex to the tube labeled Transfec on Reagent. C. Add 1.25µL of FactorStem Reprogramming RNA to the tube labeled RNA and mix by pipe ng. D. Add 0.25µL of Transfec on Reagent to the tube labeled Transfec on Reagent, mix by pipe ng, and incubate at room temperature for 30 seconds. E. Transfer the en re contents of the tube labeled Transfec on Reagent to the tube labeled RNA and mix by rapidly pipe ng up and down 10 mes. Incubate at room temperature for 10 minutes. F. A er the 10 minute incuba on, remove the plate from the incubator, and apply the complexed RNA to the cells dropwise, keeping the pipet p above the surface of the medium. Distribute the complexed RNA by alternately shaking the plate back and forth, then side to side. Repeat the shaking mo ons 10 mes. Return the plate to the incubator. Do not disturb the plate un l Day 4. Days 2 3 Do not disturb the plate on Day 2 or Day 3. 7

8 Days Each day, replace the medium and transfect the cells with FactorStem Reprogramming RNA as on Day 1 (Day 1, Steps 1, 2A 2F), using the volumes shown in Table 1 for the corresponding day. CAUTION! Do not replace the medium or disturb the cells on Day 9 or Day 10. Day Beginning on Day 11, the medium can be replaced with FactorStem Reprogramming Medium or with standard human pluripotent stem cell culture medium. 2. Colonies can be picked or passaged onto plates coated with FactorCoat Coa ng Matrix (included) as soon as they are large enough to isolate. To coat 1 well of a 6 well plate, dilute 10µL of FactorCoat in 1mL of PBS, mix, add to the well, and incubate for 1 hour at room temperature. A er the 1 hour incuba on, aspirate the FactorCoat solu on, and replace with warm FactorStem Reprogramming Medium or standard human pluripotent stem cell culture medium, being careful not to let the well dry out. CAUTION! A er picking colonies or passaging, cells should be handled using standard human pluripotent stem cell culture techniques. Cells must be cultured in FactorStem Reprogramming Medium or standard human pluripotent stem cell culture medium, on plates coated with an appropriate matrix, for example, FactorCoat Coa ng Matrix (included). 8

9 Example Results (Using Posi ve Control Cells, Part# MA001SK) Day 1 Day 4 Day 5 9

10 Day 6 Day 7 Day 8 10

11 factor Reprogramming efficiencies greater than 2% are typical (1000 cells/well were plated on Day 0; colonies were stained for SSEA4 on Day 15): 11

12 Colonies can be picked as soon as they are large enough to isolate. Established lines stain posi ve for Oct4 and SSEA4 (colonies were picked on Day 10): Merge Oct4 SSEA4 Nucleus Visit for addi onal data. 12

13 Troubleshoo ng Problem Possible Cause Solu on Excessive toxicity on Days 1 4 RNA transfec on reagent complex forma on protocol steps not performed in order and/or with the prescribed ming. Closely follow the complex forma on protocol, performing all pipe ng steps in the order and with the ming described. No morphological changes Complexes not evenly distributed. Unhealthy fibroblast culture. RNA transfec on reagent complex forma on protocol steps not performed in order and/or with the prescribed ming. Complexes not evenly distributed. Unhealthy fibroblast culture. Miscalibrated CO 2 incubator. Add complexes to cells dropwise. Shake plate as described in the protocol immediately a er adding RNA complexes to cells. Ensure that fibroblasts are ac vely dividing with a doubling me of 18 24h. If unhealthy fibroblasts must be used, increase the number of cells plated on Day 0. Closely follow the complex forma on protocol, performing all pipe ng steps in the order and with the ming described. Add complexes to cells dropwise. Shake plate as described in the protocol immediately a er adding RNA complexes to cells. Ensure that fibroblasts are ac vely dividing with a doubling me of 18 24h. Calibrate the incubator. Note that the incubator must be set at 5% CO 2. 13

14 Morphological changes are visible, but cells revert to a fibroblast morphology a er the last transfec on Cell sheet li s off of the well surface Slowly responding cells. Miscalibrated CO 2 incubator. Excessive me in culture. Some cells may require more than 6 transfec ons to generate colonies. Addi onal daily 2µg transfec ons may be performed beyond Day 8. Note, however, that even in an experiment that yields hundreds of colonies, most of the cells surrounding the colonies will normally revert to a fibroblast morphology. Calibrate the incubator. Note that the incubator must be set at 5% CO 2. Pick colonies as soon as they are large enough to pick. If cells do li off, cells can be recovered by passaging into FactorStem Reprogramming Medium. Note that cells must be passaged onto plates coated with an appropriate matrix, for example, FactorCoat Coa ng Matrix (included, see Page 3). 14

15 Legal Informa on Limited Use License The FactorStem Reprogramming Kit, FactorStem Reprogramming RNA, FactorStem Reprogramming Medium, FactorPlex Complexa on Buffer, FactorCoat Coa ng Matrix, and Posi ve Control Cells, and their components (herea er "the products") are for research use only, and are not intended for any human or animal diagnos c or therapeu c use. By purchasing the products, the purchaser agrees to use the products to reprogram cells for in vitro research use only, and to not use the products or cells treated with or produced using the products for any other use, including any human in vivo or diagnos c use, for any screening use or for any commercial use, including any manufacturing use. The user also agrees not to subject the products to chemical or other analysis (i.e. "reverse engineering") or to sequence, amplify or a empt to sequence or amplify the products. The user agrees not to sell, transfer or otherwise make available the products or cells treated with or produced using the products (including any and all cells derived from cells treated with or produced using the products) to any third party without the express prior wri en permission of Factor Bioscience Inc. Use of the products or any part thereof for any purpose whatsoever cons tutes acceptance of these terms. If the terms of this limited use license are not accepted, the original purchaser may return the products unopened within 30 days of receipt for a full refund. Limita on of Liability No warranty, either express or implied, is provided with the products, and Factor Bioscience Inc. assumes no liability for any loss, damage or injury resul ng from their use. The products and cells treated with and/or produced using the products should be handled in accordance with all applicable safety prac ces, included those that may be required by law. Factor Bioscience Inc. makes no warranty of reprogramming speed, efficiency, effec veness or that the products will successfully reprogram the user's cells. Factor Bioscience Inc. agrees to provide the original purchaser of the FactorStem Reprogramming Kit with technical support, free of charge, for a period of one (1) year from the original date of purchase, which shall include feedback on data provided to Factor Bioscience Inc. by the original purchaser, such as images of the user's cells treated with the products. Intellectual Property The products and their use are protected by U.S. Pat. No. 8,497,124, which is owned by Factor Bioscience Inc., and by mul ple pending U.S. and foreign patents including PCT/US2012/ Purchase of the products includes a limited, non transferrable license for the original purchaser to use the products to prac ce the inven ons disclosed in these issued and pending patents for a period of one (1) year from the original date of purchase only in the manner and for the purpose described on this page under the heading "Limited Use License". Any other use of the products may infringe these and/or other issued or pending patents. Factor Bioscience Inc. assumes no liability for customer's infringement of intellectual property, including intellectual property owned by third par es. Contact Factor Bioscience Inc. at or licensing@factorbio.com for addi onal licensing informa on. FactorStem is a trademark of Factor Bioscience Inc. Lipofectamine is a registered trademark of Thermo Fisher Scien fic, Inc. 15

16 Contact Informa on Sales: Customer Support: Technical Support: Licensing: All Other Inquiries: Phone: (781) Address: Factor Bioscience Inc Cambridge Street, Suite 10B Cambridge, MA