Suggested Method Reprogramming MEF Cells into pips Cells using the Stemgent Recombinant Human Protein Set: OSKM-11R

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1 Page 1 Reprogramming MEF Cells into pips Cells using the Cat. No OVERVIEW The procedure outlined below describes the reprogramming of mouse embryonic fibroblasts (MEF) by the addition of four recombinant transcription factor proteins. Hongyan Zhou and colleagues in the Sheng Ding laboratory at the Scripps Research Institute demonstrated the use of recombinant transcription factor proteins to generate protein-induced pluripotent stem (pips) cells. 1 Reprogramming with recombinant proteins is an undeveloped technology and the subject of ongoing research investigations. Scientists in Sheng Ding s laboratory and at have developed the following optimized suggested method based on the original publication. This recombinant protein set comes with four vials of each protein. If following the suggested method, one vial of each protein is needed to complete the procedure. Due to the low reprogramming efficiency, however, further optimization is required in the experimental design. See the discussion section at the end of this protocol for additional comments. Materials Name Cat. No. Size Storage Recombinant Human Oct4-11R x 50 µl -20 C Recombinant Human Sox2-11R x 50 µl -20 C Recombinant Human Klf4-11R x 50 µl -20 C Recombinant Human c-myc-11r x 50 µl -20 C ADDITIONAL MATERIALS REQUIRED DMEM medium FBS 100X non-essential amino acids 200 mm L-glutamine Gelatin Knockout DMEM Medium (Invitrogen Cat. No ) Knockout Serum Replacement (Invitrogen Cat. No ) ESGRO (LIF) (Millipore Cat. No. ESG1107) 55 mm β-mercaptoethanol Irradiated CF-1 MEFs OG2 MEFs, logo, and all other trademarks are property of Inc Inc.

2 Page 2 Reprogramming MEF Cells into pips Cells using the 0.05% Trypsin/EDTA 0.22 µm pore size filter units 6-well tissue culture plates MATERIAL PREPARATION Preparation of MEF Medium: Supplement 450 ml of DMEM with 10% FBS, 1X of 100X non-essential amino acids, and 2 mm of 200 mm L-glutamine. Store MEF Medium at 4 C. MEF Medium must be warmed to room temperature before use. Preparation of Gelatin Coated Plates: Dilute gelatin to a final concentration of 0.1% in warmed dh 2 O. Let gelatin solution cool to room temperature then filter sterilize with a 0.22 µm pore size filter unit for long term storage at 4 C. Add 2 ml of 0.1% gelatin solution to each well of a 6-well plate and incubate at 37 C for a minimum of 1 hour. Gelatin Coated Plates can be stored at 37 C for up to 1 week. Preparation of ES Cell Medium: Supplement 400 ml of Knockout DMEM with 20% Knockout Serum Replacement, 1X of 100X non-essential amino acids, 2 mm of 200 mm L-glutamine, 0.1 µm of 55 mm β-mercaptoethanol, and 10 3 units of ESGRO (LIF). ES Cell Medium can be stored at 4 C for up to 2 weeks. ES Cell Medium must be warmed to room temperature before use. Preparation of MEF Feeder Plates: Plate irradiated CF-1 MEFs onto 6-well plates at a concentration of 1 x 10 6 to 1.5 x 10 6 cells per well in ES Cell Medium. Incubate overnight at 37 C and 5% CO 2. After incubation, MEF Feeder Plates are ready to use. REPROGRAMMING OG2 MEFs INTO pips CELLS Seeding Cells for Reprogramming (Day 0) The MEF cells used in this suggested method are isolated from the embryos of OG2 transgenic mice carrying GFP under the control of the Oct4 promoter. The mouse B6;CBA_Tg ( Pou5f1-EGFP ) 2Mnn / J strain can be obtained from The Jackson Laboratory (stock number ), and MEF cells should be prepared according to the protocol reported on the WiCell Research Institute website: Introduction to human embryonic stem cell culture methods. 1. Seed OG2 MEFs at a density of 5 x 10 4 cells per well in MEF Medium in a 6-well Gelatin Coated Plate. 2. Incubate overnight at 37 C and 5% CO 2. Note: It is critical to use MEFs at a low passage number. If MEFs are cultured for too many passages they may accumulate DNA damage, proliferation begins to slow, and they could develop medium-induced epigenetic changes. Using primary cells or cells with a low passage number will ensure the best chance of achieving a reprogramming efficiency similar to what is seen in publication., logo, and all other trademarks are property of Inc Inc.

3 Page 3 Reprogramming MEF Cells into pips Cells using the Recombinant Protein Treatment (Day 1 to Day 10) 1. Thaw one vial of each of the 4 recombinant proteins: Oct4-11R, Sox2-11R, Klf4-11R, and c-myc-11r. 2. Aspirate the MEF Medium and add 1 ml per well of ES Cell Medium to the cells. 3. Add 8 µl of each protein directly to the well for a final concentration of 8 µg/ml each. Note: 1 mm of Valproic Acid (VPA) compound ( Cat. No ) can be added to the ES Cell Medium during the overnight incubation with recombinant proteins to increase reprogramming efficiency. Thawed proteins may be stored at 4 C for up to 7 days. Multiple freeze/thaw cycles are not recommended. If necessary, thaw additional vials of each protein. 4. Incubate overnight at 37 C and 5% CO The next day, aspirate the medium and add 1 ml per well of ES Cell Medium without reprogramming proteins. 6. Incubate for 36 hrs at 37 C and 5% CO Repeat steps 2 through 6 three times for a total of 4 cycles. Cell Culture and pips Cell Colony Identification (Day 10 to Day 40) 1. Split the cells onto a 6-well MEF Feeder Plate in ES Cell Medium using 0.05% Trypsin/EDTA. Cells should be split at a 1:2 to 1:3 ratio, however, they may require a different split ratio at each passage. Determine the appropriate ratio by observing the culture density and growth rate after each passage. Note: Any cells not re-plated can be frozen using standard cryopreservation techniques. 2. After splitting, change the medium every 48 hrs and split the cells when they become confluent, cutting the cell clumps into small pieces if they are sticky after trypsination. Note: Recombinant proteins may be added every 10 days by incubating overnight with 1 ml of ES Cell Medium containing 8 µg/ml of each recombinant protein. 3. pips colonies should start to form around day 30. Once a pips colony begins to form, change the medium every 24 hrs. Note: 1 µm of PD ( Cat. No ) can be added to the ES Cell Medium starting on day 21 to help keep the pips colonies stable. 4. Culture the colonies, splitting when necessary, until they become GFP positive. Pick GFP positive colonies and plate onto a 6-well MEF Feeder Plate. 5. Continue culturing the colonies using standard ES cell culture techniques., logo, and all other trademarks are property of Inc Inc.

4 Page 4 Reprogramming MEF Cells into pips Cells using the DISCUSSION Optimizing the efficiency and timescales of crucial events occurring during in vitro reprogramming to pluripotency in both viral mediated and protein derived methods has been problematic due to the cellular and genetic heterogeneity of somatic cells and the stochastic process of the reprogramming event. 2 Although the suggested method described above was tested in mouse OG2 MEFs, it can be modified in several ways depending on the goal of the experiment. The amounts of proteins added to the cells may need to be optimized for different experimental conditions, especially if using cells and media other than what is described above. Both published research and in house testing showed that the proteins readily enter cells at concentrations of 0.5 to 8 µg/ml, however, the optimal amounts and ratios to use for the most efficient reprogramming are unknown at this time. The amount of time the cells are exposed to the proteins may also be adjusted to attempt to increase reprogramming efficiency. While mouse cells are used for reprogramming in this method, the recombinant proteins are derived from human sources and may also work to reprogram human cells. The recombinant proteins can also be used in single protein replacement experiments. In these studies, three transcription factors are introduced to the cells using a viral delivery system, while the fourth transcription factor is introduced as a recombinant protein. Both Sox2-11R and Klf4-11R replacement studies have given rise to similar reprogramming efficiencies when compared to their control viral vectors. For these experiments, 8 µg/ml of protein was used to successfully replace the corresponding viral vector in the Reprogramming Ecotropic Retrovirus Set: OSKM (Cat. No ). Currently, the optimal protein concentration conditions for the single protein replacement of Oct4-11R and cmyc-11r are still being determined for OG2 MEFs. REFERENCES 1. Zhou, H., Wu, S., Joo, J.Y., Zhu, S., Han, D.W., Lin, T., Trauger, S., Bien, G., Yao, S., Zhu, Y., Siuzdak, G., Scholer, H.R., Duan, L., Ding, S. (2009) Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell 4: Hanna, J., Saha, K., Pando, B., van Zon, J., Lengner C.J., Creyghton, M.P., van Oudenaarden, A., Jaenisch, R. Direct cell reprogramming is a stochastic process amenable to acceleration. (2009) Nature 462: Knockout DMEM and Knockout Serum Replacement are Trademarks of Gibco. GlutaMAX is a trademark of Gibco. EmbryoMAX is a trademark of the Millipore Corp. ESGRO is a trademark of the Millipore Corp., logo, and all other trademarks are property of Inc Inc.

5 Page 5 Reprogramming MEF Cells into pips Cells using the Recombinant Human Protein Set: OSKM-11R t for use in diagnostic procedures. and all other trademarks are property of Inc Inc.