HUMAN IPSC CULTURE PROTOCOLS

HUMAN IPSC CULTURE PROTOCOLS FOR TRAINING COURSE IXCELLS BIOTECHNOLOGIES USA, LLC 10340 CAMINO SANTA FE, SUITE C, SAN DIEGO, CA 92121

TABLE OF CONTENTS CHAPTER 1 BEFORE STARTING 2-7 SECTION 1.1 COATING PLATES WITH DIFFERENT MATRICES 2-3 SUBSECTION 1.1.A COATING PLATES WITH 0.1% GELATIN 2 SUBSECTION 1.1.B COATING PLATES WITH MATRIGEL 3 SECTION 1.2 SEEDING MEF FEEDERS 4-8 SECTION 1.3 PREPARING HUMAN IPSC CULTURE MEDIUM 6-7 CHAPTER 2 IPSC MAINTENANCE 8-15 SECTION 2.1 RECOVERING HUMAN IPSC 8 SECTION 2.2 MAINTAINING HUMAN IPSC 9-14 SUBSECTION 2.2.A PURIFYING HUMAN IPSC 9-10 SUBSECTION 2.2.B SUBCLONING HUMAN IPSC 11 SUBSECTION 2.2.C PASSAGING HUMAN IPSC AS CLUMPS 12-13 SUBSECTION 2.2 D PASSAGING HUMAN IPSC AS SINGLE CELLS 14 SECTION 2.3 FREEZING DOWN HUMAN IPSC 15 CHAPTER 3 IPSC CHARACTERIZATION 16-17 SECTION 3.1 ANTIBODY STAINING 16 SECTION 3.2 FLOW CYTOMETRY 17 Page 1 of 17

CHAPTER 1. BEFORE STARTING SECTION 1.1. COATING PLATES WITH DIFFERENT MATRICES SECTION 1.1.A. COATING PLATES WITH 0.1% GELATIN 1.1. 0.1% Gelatin: Stem Cell Technologies, Cat # 07903. 0.1% Gelatin 2.1. Place one 6-well plate into the hood. 2.2. Add 1ml 0.1% gelatin into each well of a 6-well plate. 2.3. Incubate at 37 C CO2 incubator for at least 1hr (up to 24 hours). 2.4. Before use, place the 6-well plate into the hood. 2.5. Aspirate the solution before seeding cells. Note: It is not necessary to air dry or wash the plate. Page 2 of 17

SECTION 1.1.B. COATING PLATES WITH MATRIGEL 1.1. Matrigel Matrices (Growth Factor Reduced): BD Biosciences, Cat# 356230. 1.2. DMEM/F12: Thermo Fisher Scientific, Cat# 10565042. Matrigel Matrices 2.1. Thaw the whole bottle at 4 C overnight. 2.2. Find the concentration of Matrigel for each lot, from CoA provided by the vendor. 2.3. Place the Matrigel bottle on ice. 2.4. Transfer the Matrigel to a pre-chilled 250 ml bottle. 2.5. Dilute the Matrigel into 1 mg/ml using cold DMEM/F12 media. 2.6. Aliquot the Matrigel into pre-chilled 15ml cortical tubes at 1ml/tube. 2.7. Store the aliquots at -20 C for up to 6 months. 2.8. Before use, thaw one tube of aliquoted Matrigel (1 mg/ml, 1 ml aliquot) on ice (or 4 C) for at least 2 hours. 2.9. Add 11 ml cold DMEM/F12 to the tube. 2.10. Mix well by inverting the tube several times. 2.11. Add 1 ml of diluted Matrigel into each well of a 6-well plate. 2.12. Wrap the plates with Parafilm. 2.13. Incubate at RT for at least two hours before use. 2.14. The plates can be stored at 4 C for up to 2 weeks. 2.15. Aspirate the solution before seeding cells. Note: It is not necessary to air dry or wash the plate. Page 3 of 17

SECTION 1.2. SEEDING MEF FEEDERS 1.1. Gelatin-coated plates (See Section 1.1.A.). 1.2. Matrigel-coated plates (optional) (See Section 1.1.B.). 1.3. DMEM: VWR/Corning, Cat# 10-013-C. 1.4. Fetal Bovine Serum: VWR/Seradigm, Cat# 1300-500. 1.5. Antibiotic-antimycotic (Anti-Anti): Thermo Fisher Scientific, Cat# 15240-062 (optional). 1.6. CF1 MEF Feeder (5x10 6 cells/vial) (ixcells Biotechnologies, Cat#10MU-001). 1.7. Fibroblast Growth Medium (ixcells Biotechnologies, Cat# MDFGM). Fibroblast Growth Medium 2.1. Coating plates with 0.1% gelatin (See Section 1.1.A.) 2.2. Prepare Fibroblast Growth Medium: ixcells provides ready-to-use optimized Fibroblast Growth Medium (Cat# MDFGM), or you can make your own medium using the following formula (500 ml): DMEM ------------------------------------------------------------------------------------ 450 ml Fetal Bovine Serum -------------------------------------------------------------------- 50 ml Anti-Anti (optional) ---------------------------------------------------------------------- 5 ml 2.3. Seeding CF1 MEF feeders: 2.3.1. Thaw the frozen vial of CF1 MEF feeder in 37 C water bath for 30-45 seconds. 2.3.2. Use 70% ethanol to spray the vial. 2.3.3. Use paper towel to wipe out the ethanol. 2.3.4. Put the cryogenic vial inside the hood. 2.3.5. Add 10ml pre-warmed Fibroblast Growth Medium to a 50 ml conical tube. 2.3.6. Transfer the cells from the cryogenic vial into the 50 ml conical tube. 2.3.7. Use 1 ml Fibroblast Growth Medium to rinse the vial, and transfer the remaining cells to the same tube. 2.3.8. Centrifuge at 200 g (~1,000 rpm) for 5 minutes at RT. Page 4 of 17

2.3.9. Aspirate the supernatant. 2.3.10. Re-suspend the cells with 5 ml Fibroblast Growth Medium. 2.3.11. Mix well with 5 ml serological pipet. 2.3.12. Bring the volume up to desired volume. Normally a vial containing 5x10 6 cells goes to 36 ml Fibroblast Growth Medium, then goes to 18 wells at 2 ml/well. The density in each well should be 250,000 cells/well. 2.3.13. Aspirate gelatin from the coated plates. 2.3.14. Seed 2 ml of suspended cells onto each well. 2.3.15. Mix well by rotating the dishes several times. 2.3.16. Put the dishes in 37 C CO2 incubator. 2.3.17. Incubate overnight. 2.3.18. The feeders should be used in 1-3 days after seeding. ixcells imef Feeder (CF1), irradiated Page 5 of 17

SECTION 1.3. PREPARING HUMAN IPSC CULTURE MEDIA 1.1. DMEM/F12: Thermo Fisher Scientific, Cat#10565042. 1.2. Knockout TM Serum Replacement: Thermo Fisher Scientific, Cat# 10828-028. 1.3. Non-Essential Amino Acids: Thermo Fisher Scientific, Cat# 11140-050. 1.4. -Mercaptoethanol (55mM): Thermo Fisher Scientific, Cat# 21985-023. 1.5. Antibiotic-antimycotic (Anti-Anti): Thermo Fisher Scientific, Cat# 15240-062 (optional). 1.6. Fibroblast Growth Factor-basic (hbfgf): ixcells Biotechnologies Cat# MDFGF. 1.7. D-PBS: VWR/Corning, Cat# 21-031-CV. 1.8. BSA fraction V (7.5%): Thermo Fisher Scientific, Cat# 15260-037. 1.9. ixcells optimized ready-to-use Human ipsc Culture Medium (select one of the following media): 1.9.1. Human ipsc Growth Medium: ixcells Biotechnologies, Cat# MDPSGM. 1.9.2. MEF Conditioned Medium: ixcells Biotechnologies, Cat# MDMEFC. 1.9.3. Human ipsc Feeder-Free Growth Medium: ixcells Biotechnologies, Cat# MDPFGM. 1.10. Medium Selection Guideline: Medium Name ixcells Cat # Coating Matrices Feeder layers Selection Guide KOSR Medium Recipe See Section 1.3. 0.1% Gelatin (See Section 1.1.A.) Required (See Section 1.2.) $ Need to seed feeders the day before Need to prepare medium freshly Human ipsc Growth Medium MDPSGM 0.1% Gelatin (See Section 1.1.A.) Required (See Section 1.2.) $ Need to seed feeders the day before Ready-to-use complete medium MEF Conditioned Medium MDMEFC Matrigel (See Section 1.1.B.) Not necessary $$ No Need to prepare feeders Ready-to-use complete medium Human ipsc Feeder-Free Growth Medium MDPFGM Matrigel (See Section 1.1.B.) Not necessary $$$ No Need to prepare feeders Ready-to-use complete medium Defined medium Highest consistency Page 6 of 17

Human ipsc Growth Medium MEF Conditioned Medium Human ipsc Feeder-Free Growth Medium 2.1. Preparation of hbfgf 2.1.1. Prepare 0.2% BSA solution in PBS and filter through a 0.22 m filter. 2.1.2. Add 10 ml 0.2% BSA solution to re-constitute 1mg hbfgf, the stock concentration is: 100 g/ml. 2.1.3. Pre-wet a 0.22 m filter by filtering 5 ml 10% BSA solution through the filter. Discard the 10 ml BSA wash. 2.1.4. Filter the hbfgf through the pre-washed filter. 2.1.5. Aliquot the hbfgf in sterile Eppendorf tubes and store at 20 C. 2.2. Preparation of KOSR Medium: (500 ml): DMEM/F12 -------------------------------------------------------------------------------------- 400ml Knockout TM Serum Replacement -------------------------------------------------------- 100ml Non-Essential Amino Acids -------------------------------------------------------------------- 5ml -Mercaptoethanol ----------------------------------------------------------------------------- 0.9ml Anti-Anti (optional) ------------------------------------------------------------------------------- 5ml hbfgf (100 g/ml) ------------------------------------------------------------------------------- 50 l Note: ixcells provides three ready-to-use optimized Human ipsc Culture Medium (Cat# MDPSGM, MDMEFC, MDPFGM). See above table for guideline of ipsc culture medium selection for your applications. Page 7 of 17

CHAPTER 2. IPSC MAINTENANCE SECTION 2.1. RECOVERING HUMAN IPSC 1.1. MEF feeder plates (See Section 1.2.). 1.2. Matrigel-coated plates (See Section 1.1.B.). 1.3. ipsc Culture Medium (select one of the following media): 1.3.1. Human ipsc Growth Medium: ixcells Biotechnologies, Cat# MDPSGM. 1.3.2. MEF Conditioned Medium: ixcells Biotechnologies, Cat# MDMEFC. 1.3.3. Human ipsc Feeder-Free Growth Medium: ixcells Biotechnologies, Cat# MDPFGM. Note: Human ipsc Growth Medium is for on-feeder culture, MEF Conditioned Medium and Human ipsc Feeder-Free Growth Medium are for feeder-free culture. 1.4. Y27632 (10 mm in DMSO): ixcells Biotechnologies Cat# MD-0020. 2.1. Thaw the frozen vial of human ipscs in 37 C water bath for 30-45 seconds. 2.2. Use 70% ethanol to spray the vial. 2.3. Use paper towel to wipe out the ethanol. 2.4. Put the cryogenic vial inside the hood. 2.5. Add 10 ml pre-warmed Human ipsc Culture Medium to a 15 ml conical tube. 2.6. Transfer the cells from the cryogenic vial into the 15 ml conical tube. 2.7. Use 1 ml Human ipsc Culture Medium to rinse the vial, and transfer the remaining cells to the same tube. 2.8. Centrifuge at 50 g (~250 rpm) for 5 minutes at RT. 2.9. Aspirate the supernatant. 2.10. Re-suspend the cells with 2ml Human ipsc Culture Medium supplemented with 10 M Y27632. 2.11. Mix well with 5ml serological pipet. 2.12. Remove the Fibroblast Growth Medium from the feeder plate or Matrigel from the Matrigel coated plate. 2.13. Gently transfer the cell suspension into the wells, normally the cells recovered from one frozen vial go to one well of a 6-well plate 2.14. Mix well by rotating the plates several times and incubate in 37 C CO2 incubator overnight 2.15. The next day, change to Human ipsc Culture Medium without Y27632. 2.16. Change media daily until the colonies are big Figure 1. ipsc colony recovered for 2 days enough to be passaged. Page 8 of 17

SECTION 2.2. MAINTAIN HUMAN IPSC SECTION 2.2.A. PURIFYING HUMAN IPSC 1. Material: 1.1. Human ipsc Culture Medium (select one of the following media): 1.1.1. Human ipsc Growth Medium: ixcells Biotechnologies, Cat# MDPSGM. 1.1.2. MEF Conditioned Medium: ixcells Biotechnologies, Cat# MDMEFC. 1.1.3. Human ipsc Feeder-Free Growth Medium: ixcells Biotechnologies, Cat# MDPFGM. Note: Human ipsc Growth Medium is for on-feeder culture, MEF Conditioned Medium and Human ipsc Feeder-Free Growth Medium are for feeder-free culture. 1.2. D-PBS: VWR/Corning, Cat# 21-031-CV. 1.3. ReLeSR TM Enzyme-Free Human ES and ips Cell Selection and Passaging Reagent: Stem Cell Technologies, Cat# 05873. 2.1. Manually purify human ipsc culture. 2.1.1. Connect a 1ml pipette tip with a 200 l pipette tip. 2.1.2. Under a dissection microscope, manually detach the differentiating colonies using the connected tip. 5x 20x Figure 2. ipsc colonies with different morphologies. Left: undifferentiated colonies; Middle: partially differentiatiated colonies; Right: Completely differentiated colonies. 2.1.3. Remove the floating colonies by changing medium. 2.2. Purify ipsc culture using ReLeSR TM. 2.2.1. Remove the culture media from the plates. 2.2.2. Wash the cells once with D-PBS. 2.2.3. Add 1ml ReLeSR TM to each well of a 6-well plate. Page 9 of 17

2.2.4. Rotate the plates for 30-60 seconds. 2.2.5. Remove the ReLeSR TM from the plates. 2.2.6. Leave the plates in 37 C CO2 incubator for 2-5 minutes. 2.2.7. Check the cells under microscope occasionally. 2.2.8. Once the cells are dissociated, add cell culture media to each well. 2.2.9. Gently shake the plates several times. 2.2.10. Check under microscope to make sure most of the undifferentiated cells are detached from the plates. 2.2.11. Gently transfer the media to a 15 ml conical tube. 2.2.12. Centrifuge at 50 g (~250 rpm) for 5 minutes at RT. 2.2.13. Aspirate the supernatant. 2.2.14. Re-suspend the cells with 2ml Human ipsc Culture Medium supplemented with 10 M Y27632. 2.2.15. Mix well with 5 ml serological pipet. 2.2.16. Remove the Fibroblast Growth Medium from the feeder plate or Matrigel from the Matrigel coated plate. 2.2.17. Gently transfer the cell suspension into the wells. 2.2.18. Mix well by rotating the plates several times and incubate in 37 C CO2 incubator overnight. 2.2.19. The next day, change to Human ipsc Culture Medium without Y27632. 2.2.20. Change media daily until the colonies are big enough to be passaged. Page 10 of 17

SECTION 2.2.B. SUBCLONING HUMAN IPSC 1.1. Human ipsc Culture Medium (select one of the following media): 1.1.1. Human ipsc Growth Medium: ixcells Biotechnologies, Cat# MDPSGM 1.1.2. MEF Conditioned Medium: ixcells Biotechnologies, Cat# MDMEFC 1.1.3. Human ipsc Feeder-Free Growth Medium: ixcells Biotechnologies, Cat# MDPFGM Note: Human ipsc Growth Medium is for on-feeder culture, MEF Conditioned Medium and Human ipsc Feeder-Free Growth Medium are for feeder-free culture. 1.2. Y27632 (10 mm in DMSO): ixcells Biotechnologies, Cat# MD-0020. 2.1. The day before colony pickup, prepare a feeder plate or Matrigel-coated plate (See Section 1.1.A. and 1.1.B.). 2.2. Before colony pickup, remove Fibroblast Growth Medium from the feeder plate, or remove Matrigel from Matrigel-coated plate. 2.3. Add Human ipsc Culture Medium supplemented with 10 M Y27632 to the feeder plate or Matrigel plate. 2.4. Under a dissection microscope, manually aspirate the single colony using a p200 pipette tip. 2.5. Transfer the cells into a 1.5 ml Eppendorf tube. 2.6. Dissociate the cells by pipetting up and down several times. 2.7. Transfer the pieces of the colonies from the Eppendorf tubes to the wells, one clone goes to one well. 2.8. Incubate overnight at 37 C CO2 incubator. 2.9. The next day, change to Human ipsc Culture Medium without Y27632. 2.10. Change media daily until the colonies are big enough to be passaged. Page 11 of 17

SECTION 2.2.C. PASSAGING HUMAN IPSC AS CLUMPS 1.1. DMEM/F12: Thermo Fisher Scientific, Cat#10565042. 1.2. Human ipsc Culture Medium (select one of the following media): 1.2.1. Human ipsc Growth Medium: ixcells Biotechnologies, Cat# MDPSGM. 1.2.2. MEF Conditioned Medium: ixcells Biotechnologies, Cat# MDMEFC. 1.2.3. Human ipsc Feeder-Free Growth Medium: ixcells Biotechnologies, Cat# MDPFGM. Note: Human ipsc Growth Medium is for on-feeder culture, MEF Conditioned Medium and Human ipsc Feeder-Free Growth Medium are for feeder-free culture. 1.3. Collagenase IV: Thermo Fisher Scientific, Cat# 17104-019. 1.4. Dispase II: Thermo Fisher Scientific, Cat# 17105-041. 1.5. D-PBS: VWR/Corning, Cat# 21-031-CV. 1.6. ReLeSR TM Enzyme-Free Human ES and ips Cell Selection and Passaging Reagent: Stem Cell Technologies Cat# 05873. 2.1. Passage the cells using Collagenase IV or Dispase II. 2.1.1. Preparation of Collagenase IV or Dispase II solution. 2.1.1.1. Dissolve 10 mg Collagenase IV or Dispase II in 10mL DMEM/F12 medium. 2.1.1.2. Filter the enzyme through a 0.22 m filter. 2.1.1.3. Aliquot the enzyme and store at 20 C. 2.1.2. Thaw one aliquot of enzyme in 37 C water bath. 2.1.3. Remove the medium from the plates. 2.1.4. Add 1 mg/ml Collagenase IV or Dispase II to the wells. Note: It s not necessary to wash the plates with D-PBS 2.1.5. Incubate at 37 C for 5-10 minutes. 2.1.6. Check the cells under microscope. The edges of the colonies should start to peel off from the plates. 2.1.7. Remove the enzyme from the plates. 2.1.8. Add 2 ml of Human ipsc Culture Medium to each well and detach the colonies by scratching the wells using 5 ml pipette tip. 2.1.9. Transfer the cells to a 15 ml conical tube. 2.1.10. Centrifuge at 50 g (~250 rpm) for 5 minutes at RT. 2.1.11. Aspirate the supernatant from the tube. 2.1.12. Add Human ipsc Culture Medium supplemented with 10 M Y27632 to each tube and re-suspend the cells by gently pipetting up and down several times. Figure 3. Image of the Colony Treated with Dispase for 5 minutes Page 12 of 17

2.1.13. Transfer the cells to the wells pre-seeded with feeders or pre-coated with Matrigel. Split ration depends on the confluency of the plates and the culture medium used. 1:3-6 split ratio is recommended. 2.1.14. Incubate overnight at 37 C CO2 incubator. 2.1.15. The next day, change to Human ipsc Culture Medium without Y27632. 2.1.16. Change media daily until the colonies are big enough to be passaged. 2.2. Passage the cells using ReLeSR TM. 2.2.1. Remove the culture media from the plates. 2.2.2. Wash the cells once with D-PBS. 2.2.3. Add 1ml ReLeSR TM to each well of a 6-well plate. 2.2.4. Rotate the plates for 30-60 seconds. 2.2.5. Remove the ReLeSR TM from the plates. 2.2.6. Leave the plates in 37 C CO2 incubator for 2-5 minutes. 2.2.7. Check the cells under microscope occasionally. 2.2.8. Once the cells are dissociated, add cell culture media to each well. 2.2.9. Gently shake the plates several times. 2.2.10. Check under microscope to make sure most of the undifferentiated cells are detached from the plates. 2.2.11. Gently transfer the media to a 15 ml conical tube. 2.2.12. Centrifuge at 50 g (~250 rpm) for 5 minutes at RT. 2.2.13. Aspirate the supernatant. 2.2.14. Re-suspend the cells with 2 ml Human ipsc Culture Medium supplemented with 10 M Y27632. 2.2.15. Mix well with 5 ml serological pipet. 2.2.16. Remove the Fibroblast Growth Medium from the feeder plate or Matrigel from the Matrigel coated plate. 2.2.17. Gently transfer the cell suspension into the wells. 2.2.18. Mix well by rotating the plates several times and incubate in 37 C CO2 incubator overnight. 2.2.19. The next day, change to Human ipsc Culture Medium without Y27632. 2.2.20. Change media daily until the colonies are big enough to be passaged. Page 13 of 17

SECTION 2.2.D. PASSAGING HUMAN IPSC AS SINGLE CELLS 1.1. Human ipsc Culture Medium (select one of the following media): 1.1.1. Human ipsc Growth Medium: ixcells Biotechnologies, Cat# MDPSGM. 1.1.2. MEF Conditioned Medium: ixcells Biotechnologies, Cat# MDMEFC. 1.1.3. Human ipsc Feeder-Free Growth Medium: ixcells Biotechnologies, Cat# MDPFGM. Note: Human ipsc Growth Medium is for on-feeder culture, MEF Conditioned Medium and Human ipsc Feeder-Free Growth Medium are for feeder-free culture. 1.2. D-PBS: VWR/Corning, Cat# 21-031-CV. 1.3. StemPro Accutase Cell Dissociation Reagent: Themo Fisher Scientific Cat# 11105-01. 1.4. TrypLE TM Express Enzyme (1x): Themo Fisher Scientific Cat# 12605-010. 1.5. Falcon cell strainer (40 m): VWR Cat# 21008-949. 2.1. 2 hours before splitting the cells, add 10 M Y27632 into the cell culture medium. 2.2. After incubating the cells with Y27632 for 2 hours, remove the medium from the plates. 2.3. Wash the cells once with D-PBS. 2.4. Add 1ml Accutase or TrypLE to each of the wells. 2.5. Incubate at 37 C for 5-10 minutes. 2.6. Check the cells under microscope. Most of the cells should start to detach from the plates. 2.7. Add 2 ml of Human ipsc Culture Medium to each well and detach the colonies by pipetting up and down several times using a 5 ml pipet tip. 2.8. Transfer the cells to a 15 ml conical tube. 2.9. Centrifuge at 200 g (~1,000 rpm) for 5 minutes at RT. 2.10. Aspirate the supernatant. 2.11. Add 2 ml of Human ipsc Culture Medium supplemented with 10 M Y27632 to each tube and re-suspend the cells by gently pipetting up and down several times. 2.12. Filter the cells through a 40 m cell strainer. 2.13. Count the cell number. 2.14. Dilute the cells to the desired concentration using ipsc culture media supplemented with 10 M Y27632. 2.15. Transfer the cells the wells of 6-well plates with feeders or Matrigel coated. 2.16. Incubate the cells overnight at 37 C CO2 incubator. 2.17. The next day, change to Human ipsc Culture Medium without Y27632. 2.18. Change media daily until the colonies are big enough to be passaged. Page 14 of 17

SECTION 2.3. FREEZING DOWN HUMAN IPSC 1.1. Human ipsc Culture Medium(select one of the following media): 1.1.1. Human ipsc Growth Medium: ixcells Biotechnologies, Cat# MDPSGM. 1.1.2. MEF Conditioned Medium: ixcells Biotechnologies, Cat# MDMEFC. 1.1.3. Human ipsc Feeder-Free Growth Medium: ixcells Biotechnologies, Cat# MDPFGM. Note: Human ipsc Growth Medium is for on-feeder culture, MEF Conditioned Medium and Human ipsc Feeder-Free Growth Medium are for feeder-free culture. 1.2. Collagenase IV: Thermo Fisher Scientific, Cat# 17104-019. 1.3. Dispase II: Thermo Fisher Scientific, Cat# 17105-041. 1.4. D-PBS: VWR/Corning, Cat# 21-031-CV. 1.5. ReLeSR TM Enzyme-Free Human ES and ips Cell Selection and Passaging Reagent: Stem Cell Technologies Cat# 05873. 1.6. StemPro Accutase Cell Dissociation Reagent: Themo Fisher Scientific Cat# 11105-01. 1.7. TrypLE TM Express Enzyme (1x): Themo Fisher Scientific, Cat# 12605-010. 1.8. Falcon cell strainer (40 m): VWR, Cat# 21008-949. 1.9. Dimethyl Sulfoxide (DMSO): Sigma-Aldrich, Cat # D2650. 2.1. Prepare 2x cryopreservation medium by adding 20% DMSO into Human ipsc Culture Medium and leave the medium on ice. 2.2. Manually remove the differentiated colonies before cryopreservation (See Section 2.2.A.). 2.3. Detach the cells into small clumps (See Section 2.2.C.) or into single cells (See Section 2.2.D.). 2.4. Centrifuge at 50 g (250 rpm, for small clumps) or 200 g (1,000 rpm, for single cells) for 5 minutes at RT. 2.5. Aspirate the supernatant. 2.6. Add ipsc culture media to the cell pellet and re-suspend the cell pellets by pipetting up and down several times. 2.7. Dropwise add equal volume of 2x cryopreservation medium to the re-suspended cells 2.8. Mix well by gently pipetting up and down several times. 2.9. Aliquot to the cryogenic vials at 1 ml/vial. 2.10. Put the cells in Freezing Containers to achieve a rate of cooling at -1 C /minute. 2.11. Leave the containers in -80 C freezer overnight. 2.12. On the following day, transfer the cells to liquid N2 tank for long-term storage. Page 15 of 17

CHAPTER 3. IPSC CHARACTERIZATION SECTION 3.1. ANTIBODY STAINING 1.1. Primary Antibodies (e.g. Oct4, Sox2, Nanog, SSEA3, SSEA4, Tra-1-60, Tra-1-81). 1.2. Secondary Antibodies (e.g. Anti-mouse IgG Alexa 488). 1.3. D-PBS: VWR/Corning, Cat# 21-031-CV. 1.4. BSA. 1.5. Triton-X100. 2.1. Prepare blocking buffer: 2.1.1. Prepare D-PBS containing 5% BSA for surface markers. 2.1.2. Prepare D-PBS containing 5% BSA and 0.1% Triton-X 100 for nuclear markers. 2.2. Antibody staining of hipscs in a 24-well plate: 2.2.1. Aspirate the medium from the wells. 2.2.2. Wash the cells twice with D-PBS. 2.2.3. Add 1 ml 4% PFA solution to each of the wells. 2.2.4. Incubate for >15 minutes at RT. 2.2.5. Aspirate the PFA solution. 2.2.6. Wash the cells twice with D-PBS. 2.2.7. Add 1 ml Blocking Buffer to each well. 2.2.8. Incubate for >1 hour at RT. 2.2.9. Aspirate the Blocking Buffer. 2.2.10. Add 0.2 ml of diluted primary antibody (diluted with blocking buffer) to the wells. 2.2.11. Incubate at least 1 hour at RT or overnight at 4 C. 2.2.12. Aspirate the primary antibody from the wells and wash the cells three times with D- PBS. 2.2.13. Add 0.2 ml diluted secondary antibody (diluted with blocking buffer) to the wells. 2.2.14. Protect from the light with aluminum foil and incubate >1 hour at RT. 2.2.15. Aspirate the secondary antibody and wash cells three times with D-PBS. 2.2.16. Add 0.2 ml/well diluted DAPI (diluted with blocking buffer) to the wells. 2.2.17. Cover with aluminum foil and incubate 10 minutes at RT 2.2.18. Aspirate DAPI and wash cells twice with D- PBS 2.2.19. Aspirate and add 1 ml/well D-PBS. 2.3. ICC analysis by microscopy: Figure 4. Antibody staining images of ipscs. 2.3.1. Take representative images of each well with the digital camera. Page 16 of 17

SECTION 3.2. FLOW CYTOMETRY 1.1. Fluoresence conjugate antibodies (e.g. Alexa 488 conjugate SSEA-4 antibody, etc.). 1.2. D-PBS: VWR/Corning, Cat# 21-031-CV. 1.3. StemPro Accutase Cell Dissociation Reagent: Themo Fisher Scientific, Cat# 11105-01. 1.4. Human ipsc Culture Medium: 1.4.1. Human ipsc Growth Medium: ixcells Biotechnologies, Cat# MDPSGM. 1.4.2. MEF Conditioned Medium: ixcells Biotechnologies, Cat# MDMEFC. 1.4.3. Human ipsc Feeder-Free Growth Medium: ixcells Biotechnologies, Cat# MDPFGM. Note: Human ipsc Growth Medium is for on-feeder culture, MEF Conditioned Medium and Human ipsc Feeder-Free Growth Medium are for feeder-free culture. 1.5. D-PBS. 2.1. Prepare cells: 2.1.1. Use Accutase to dissociate the cells into single cells (See Section 2.2.D.). 2.1.2. Count the cell number. 2.1.3. Re-suspend the cells in cell culture media at 1x10 6 cells/ml. 2.2. Flow cytometry of hipscs: 2.2.1. Add Fluoresence conjugate cell surface antibodies to the cells. 2.2.2. Incubate at least 1 hour on ice. 2.2.3. Wash the cells three times with D-PBS. 2.2.4. Re-suspend the cells in D-PBS. 2.3. IF analysis by Flow Cytometer: 2.3.1. Count the positive cells vs total cell number using Flow Cytometer. Figure 5. Representative image of Flow Cytometry Analysis Using SSEA4 Antibody Page 17 of 17