Standard Operating Procedures (SOPs) for the production and testing of embryonic stem cell lines in Taiwan Stem Cell Bank

Standard Operating Procedures (SOPs) for the production and testing of embryonic stem cell lines in Taiwan Stem Cell Bank Bioresource Collection and Research Centre (BCRC) at Food Industry Research and Development Institute (FIRDI) in Taiwan is committed to stem cell banking. Our goal is to provide the researchers and clinicians with the human embryonic stem (hes) cells that meet standards of the scientific communities, therefore we established Standard Operating Procedures (SOPs) for the production and testing of our banked stem cell lines. All the reference materials are listed on last page. Version 1 4/17/2009 By S.Y.C. 1

Table of Contents Title of SOP PAGE 1. Preparation of human embryonic stem cell culture medium 3 2. Preparation of Collagenase Solution 5 3. Preparation of mouse embryonic fibroblast (MEF) Medium 6 4. Derivation of new mouse embryonic fibroblast (MEF) cells 8 5. Plating mouse embryonic fibroblast (MEF) cells onto gelatin coated plates 10 6. Propagation and Distribution of mouse embryonic fibroblast (MEF) cells 11 7. Thawing mouse embryonic fibroblast (MEF) vials 12 8. Thawing human embryonic stem cells 13 9. Cryopreservation of hesc BG01 V 14 10. Cryopreservation of inactivated mouse embryonic fibroblast (MEF) cells 15 11. Propagation and distribution of hesc BG01 V 17 12. Thawing, propagation, and cryopreservation of hesc TW1 19 13. Embryonic stem cell assessment by flow cytometry 22 14. Immunofluorescence staining of Oct-4 and Nanog in undifferentiated human embryonic stem cells. 23 15. Passage of 2102Ep human EC cell line 25 References 26 2

1. Preparation of hes Cell Culture Medium NOTE This SOP details how to prepare 250ml of completed medium for hes cell culture on MEFs. This SOP was adapted from the protocols developed by the U.S. National Stem Cell Bank. 1. MATERIALS DMEM-F12 media Invitrogen 11330-032 Knockout Serum Replacer (KOSR) Invitrogen 10828-028 L-glutamine, non-animal, cell culture tested Sigma G-8540 MEM Non-Essential Amino acid solution Invitrogen 11140-050 Basic Fibroblast Growth Factor (β-fgf) Invitrogen PHG0021 PBS without CaCl 2 or MgCl 2 Invitrogen 14190-250 PBS with CaCl 2 and MgCl 2 Invitrogen 14040-141 Bovine Serum Albumin Sigma A2153 2-Mercaptoethanol Sigma M7522 2. METHODS 2.1. 0.1% BSA in PBS 2.1.1. Weigh out 250mg of Bovine Serum Albumin (BSA) and add the BSA to 250ml PBS with CaCl 2 and MgCl 2. 2.1.2. Sterilize it by Stericup filter and transfer into sterile 50ml centrifuge tubes. Keep the working aliquot at 2-8 C, and freeze the other aliquots for up to 12 months at -20 C. Record the date prepared, expiration date, and your name on each tube. 2.2. β-fgf solution 2.2.1. Briefly centrifuge the β-fgf vial (should contain 100 ug of β-fgf). 2.2.2. Add 50ml 0.1% BSA in PBS +Ca /+Mg to a 50ml conical tube. 2.2.3. Remove 500µl of the PBS with a 1000µl pipetman. Gently re-suspend the β-fgf, and add it into the 50ml conical tube. 2.2.4. Remove 500µl of the reconstituted β-fgf solution and return it to the vial to collect any residual proteins, if any. Return the solution back to the 50ml conical. 2.2.5. Aliquot the reconstituted β-fgf into 1.0ml volumes in sterile 1.5ml microcentrifuge tubes. 2.2.6. Record the lot number of the β-fgf, the date, the concentration, and the initials on each vial. 2.2.7. Store aliquots in the -80 C freezer. They expire after 6 months. 2.3. 100mM L-Glutamine +BME solution 2.3.1. Weigh out 0.073g of L-Glutamine and put it in a 15ml conical tube. Wrap the tube in foil. 2.3.2. Add 5ml of PBS without CaCl 2 and MgCl 2. 3

2.3.3. Add 3.5µl of 2-Mercaptoethanol using the 10µl pipetman in the chemical fume hood. Combine the ingredients and mix well. 2.3.4. Use the L-glutamine solution immediately, and aspirate any excess solution into the liquid waste container. 2.4. Preparation of Medium Components 2.4.1. Prepare the 100mM L-Glutamine solution as 2.3.4 shown above. 2.4.2. Please note that in the case that the KOSR will not be used in a two week period, you have to freeze back 40ml aliquots. Also be sure to wrap the bottle in foil to prevent light degradation. 2.4.3. To make 250ml of hes Cell Culture Medium, combine the following in a 250ml Stericup. 2.4.4. Filter the medium with the Stericup. 2.4.5. Record all lot numbers of medium components and from the Stericup filter unit used on the data capture sheet in the Medium Preparation Log Book. 2.4.6. Assign a lot number to the completed medium 2.4.7. The label of each bottle should include assigned lot number, hes cell media, Expiration date ( two weeks after preparation), your name. 2.4.8. Store at 2-8 C and use for up to two weeks. 4

2. Preparation of Collagenase NOTE Collagenase can remove hes cells from the MEF feeder. This SOP details how to prepare 150ml of a 1mg/ml Collagenase Solution. 1. MATERIALS DMEM-F12 Invitrogen 11330-057 Collagenase Type IV Invitrogen 17104-019 2. METHODS 2.1. Preparation of Collagenase Solution 2.1.1. Weigh 150mg of Collagenase Type IV powder. 2.1.2. Add 150ml of room temperature DMEM-F12 medium into the upper part of a 150ml 0.22µm Stericup filter unit. 2.1.3. Add the Collagenase to the medium, it should dissolve instantly. 2.1.4. Filter the solution and keep it sterile. 2.1.5. Cut open the uterine horns with a sharp Iris scissors. 2.1.6. Label the flask with Collagenase Solution, your name, Expiration date (14 days after media preparation). 2.1.7. Store at 2-8 C 5

3. Preparation of MEF Medium NOTE This SOP, adapted from the protocols developed by the U.S. National Stem Cell Bank, details how to prepare 500ml of completed medium for the use in MEF culture and plating. 1. MATERIALS DMEM (liquid) Invitrogen 11965-118 MEM Non-Essential Amino acid solution Invitrogen 11140-050 Fetal Bovine Serum, certified Invitrogen 16000-069 2. METHODS 2.1. Preparation of Media components 2.1.1. Ensure the FBS is heat inactivated prior to medium preparation. 2.1.2. In a biosafety cabinet, combine the following in a 500ml Stericup filter unit: 2.1.3. Filter the media with the Stericup filter. 2.1.4. Record lot numbers of all media components used in the media. 2.1.5. Assign a lot number to the completed media. 2.1.6. The label of each bottle should include assigned lot number, MEF media, Expiration date (two weeks after preparation), and your name. 2.1.7. Store at 2-8 C and use for up to two weeks. 6

4. Derivation of Mouse Embryonic Fibroblasts (MEF) NOTE Human Embryonic Stem (hes) cells require MEF cells as a feeder layer for culture. The MEF cells are mortal cell line derived from pregnant mice. They may only be cultured for up to 5 passages, because their ability to support hes growth declines from passage 7 onward. Hence National Stem Cell Bank suggests that new MEF derivation needs to be done one to three times a year. This SOP outlines a derivation procedure that is a fusion methodology of protocols developed by the U.S. National Stem Cell Bank and Industrial Technology Research Institute, R.O.C. 1. MATERIALS Pregnant ICR mice: 11- to 14-day (13-day of conception is recommended.) 70% Alcohol Watchmaker s forceps: 2 Sharp Iris scissors: 1 big, 1 small, 1 curved Bacteriological Petri dish: 100 mm Glass Petri dish : 60 mm Trypsin-EDTA PBS Conical tubes: 15ml, 50ml Culture flask: T75 MEF culture medium: 90% DMEM with 10% FBS and 1x Penicillin-Streptomycin. 37 water bath CO2 incubator 2. METHODS 2.1. Harvest the embryo 2.1.1.Wipe abdomen with 70% ethanol. 2.1.2. Cut the skin to expose the peritoneum and further cut the peritoneal wall to expose the uterine horns. 2.1.3. Remove the uterine horns into a PBS-containing100 mm Petri dish and bring them into a sterile biosafety cabinet. 2.1.4. Wash the uterine horns three times with 10ml PBS and transfer the horns into a sterile Petri dish. 2.1.5. Cut open the uterine horns with a sharp Iris scissors. 2.1.6. Release the embryos by a watchmaker s forceps. 2.1.7. Wash the embryos three times with 10ml of PBS. 2.1.8. Use tips of sharp forceps to separate the visceral tissue (darker red color) from the embryos. 7

2.1.9. Place the embryos into a new plate; wash the embryos with 10ml PBS until visceral tissue is no longer present. Carefully remove the PBS using a serological pipet each time. 2.2. Mince the embryo 2.2.1. Mince tissue with Iris dissecting scissors to break the embryos into tiny pieces. 2.2.2. Add 5ml Trypsin-EDTA to the dish. 2.2.3. Place the dish into the incubator for 10 minutes. 2.2.4. Add 5 ml of Trypsin, pipet up and down several times and return the plate to the incubator. 2.2.5. Incubate cells for 10 minutes. 2.2.6. Vigorously pipet the mixture up and down until the majority of the cells is in a fine suspension. 2.2.7. Add 20ml of MEF Medium to the minced tissue and transfer the contents to a sterile 50ml plastic conical tube. 2.2.8. Centrifuge at 1,000 rpm for 5 minutes. 2.2.9. Resuspend in 40 ml fresh medium, and incubate overnight at 37 C. 2.3. Observe Fibroblast Cultures and Refresh Medium 2.3.1. Inspect the cell layer covering the flask surface. NOTE: If at least 90% of the flask surface is covered with a cell layer, harvest the cells. If not, allow the cells to grow for an additional day. Discard them if you observe obvious bone formation or beating cells. 8

5. Plating Mouse Embryonic Fibroblast (MEF) cells onto gelatin coated plates NOTE Irradiated or mitomycin C-treated MEF cells must be grown on gelatin coated plates. This SOP outlines the preparation of gelatin and cell plating methods. 1. MATERIALS Gelatin (Type A from porcine skin): Sigma G-1890 Double distilled or Milli-Q grade pure water 500 ml sterile glass bottle 2. METHODS 2.1 Prepare 1% Gelatin Stock Solution 2.1.1. Add 0.4 g of gelatin power into a glass bottle containing 40 ml sterile water. 2.1.2. Mix well. 2.1.3. Cover the lid with foil and have it loosen. 2.1.4. Autoclave at 121 C for 20 minutes. 2.1.5. Store 20 ml aliquots at -20 C. 2.2. Prepare 0.1% Gelatin working solution 2.2.1. Combine 20 ml of the 1% gelatin stock solution with 180 ml sterile water. 2.3. Coat the plates 2.3.1. Cover plating dish by 0.1% gelatin solution as the following table suggests: Plate or dish Volume of gelatin (ml) 4 well 0.5 6 well 2 35 mm dish 2 25 cm2 5 100 mm dish 10 2.3.2. Leave at room temperature or in 37 C incubator for at least 2 hours. 2.3.3. Aspirate the gelatin solution before use. 9

2.4. Plate MEF cells 2.4.1. Add cell suspensions (mitomycin C treated) on gelatin-coated culture dishes. No. of Mitomycin C treated Flask/Plate Growth area (cm2) MEF cells T225 225 18.0 X 10 6 T75 75 7.0 X 10 6 T25 25 3.0X10 6 6 well 9.5 5.0 X 10 5 2.4.2. After plating theses MEF cells, please allow one day to plate hes cells. 2.4.3. Label the passage number, the date plated, and your name. 10

6. Propagation and Distribution of MEF cells NOTE Before you start this procedure, please observe the cells under microscope. The flasks should be approximately 90% confluent for passaging. You may expect to get 3 T-75 flasks from the flask being passaged if your purpose of doing this procedure is to routinely subculture/maintain MEF cells. The MEF cells should be passaged approximately every 3-4 days. The MEF cells do not require daily medium replacement. 1. MATERIALS Pre-warmed PBS without CaCl 2 and MgCl 2 Invitrogen 14190-250 Pre-warmed 0.05% Trypsin-EDTA Invitrogen 25300-054 Pre-warmed MEF Culture Medium (see SOP: Preparation of MEF medium) 2. METHODS 2.1. Passage MEF cells 2.1.1. Aspirate MEF medium. 2.1.2. Add enough room temperature PBS to cover the flask and then aspirate. 2.1.3. Add 2ml of Trypsin-EDTA to each of the T75 flasks, and incubate for 3-5 minutes at 37 C. 2.1.4. Tighten the cap and then tap the side of the flask 3 times to dislodge the MEF cells from the flasks. 2.1.5. Add at least 2ml MEF Culture Medium to each T75 flask to inhibit the action of the Trypsin-EDTA. Pipet the liquid along the back of the flask to make sure you dislodge all the MEF cells. 2.1.6. Collect the cells from the flasks in a sterile tube. 2.1.7. Centrifuge the cell suspension at 200 g for 3 minutes. 2.1.8. Remove the supernatant and re-suspend the pellet in 6 ml of MEF culture medium and mix well. 2.1.9. Count cells and re-suspend them in desired medium volume to meet your experimental need. If these are used as a feeder layer for hes cells, the density should be 0.75x10 5 MEF cells/ml. If these cells are used to condition medium for hes cells grown on Matrigel, the density should be 2.12x10 5 MEF cells/ml. Cap each new flask and label with the date, MEF, your name, and new passage number. 11

7. Thawing MEF cells NOTE The MEF vials are frozen at passage 1, and are only viable to passage 4-6; therefore a fresh vial of cells can support hes cells for about 2-3 weeks. 1. Determine location of the desired cell vial. 2. Wear eye protection and ultra low temperature cryo gloves. Remove MEF cell vial(s) from the liquid nitrogen tank. 3. Immerse the vial in a 37 C water bath, but keep the cap above the water until you see only an ice crystal remains. 4. Use 95% ethanol to sterilize the outside of the tube. 5. Pipet cells gently into a sterile 15ml conical tube using a 1ml glass pipet. 6. Slowly add 5ml of MEF Cell Culture Medium drop-wise to cells in the 15ml conical tube. While adding the medium, gently move the tube back and forth to mix the MEF cells. 7. Centrifuge the cells at 200 x g for 5 minutes. 8. Discard the supernatant. 9. Re-suspend the pellet in 10 ml MEF Cell Culture Medium and transfer to one T75 flask. 10. Label the flask with the following: MEF, P1, the date, and your name. 12

8. Thawing hes cells 1. Determine location of the desired cell vial. 2. Wear eye protection and ultra low temperature cryo gloves. Remove hes cell vial(s) from the liquid nitrogen tank. 3. Immerse the vial in a 37 C water bath, but keep the cap above the water until you see only an ice crystal remains. 4. Use 95% ethanol to sterilize the outside of the tube. 5. Pipet cells gently into a sterile 15ml conical tube using a 1ml glass pipet. 6. Slowly add 4ml of hes Cell Culture Medium drop-wise to cells in the 15ml conical tube. While adding the medium, gently move the tube back and forth to mix the hes cells. 7. Centrifuge the cells at 200 x g for 5 minutes. 8. Discard the supernatant. 9. Re-suspend the pellet in 2.5ml hes Cell Culture Medium. 10. Label a 6-well plate containing inactivated MEF cells with the following: The passage number from the vial, the date, and your name. 11. Aspirate the MEF medium from one well, and slowly add the hes cell suspension drop-wise into the well. 13

9. Cryopreservation of hesc BG01V NOTE We obtained the cells from ATCC, and thus use the cryopreservation procedure indicated on the product information sheet. 1. MATERIALS Cell suspension prepared by Collagenase method. 1: 1 solution of 50% complete growth medium and 50% FBS. DMSO Sigma Aldrich D-2650 2. METHODS 2.1. Centrifuge the cell suspension for 5 minutes at 200 g. 2.2. Aspirate the supernatant. 2.3. Re-suspend the pellet in a 1: 1 solution of 50% complete growth medium and 50% FBS. 2.4. Use P1000 tips to pipet up and down to break the colonies. 2.5. Slowly add an equal volume of complete growth medium with 20% DMSO. Mix gently. 2.6. Distribute 1 ml of the cell suspension into each cryovial. 14

10. Cryopreservation of Inactivated MEF Cells 1. MATERIALS MEF culture medium: 90% DMEM with 10% FBS and 1X Penicillin-Streptomycin. MEF freezing medium: 7% DMSO and 90% MEF medium. Freshly prepare. Trypsin-EDTA PBS DMSO Sigma Aldrich D-2650 2. METHODS 1. Prepare MEF freezing medium prior to beginning the procedure, keep it on ice. 2. Aspirate the medium and rinse once by PBS. 3. Add 2 ml of trypsin-edta. 4. Incubate for about 5 minutes. 5. Tap the side of the flask to help better detach the cells. Add 8 ml of MEF medium to neutralize the trypsin. 6. Collect cell suspension into new 15ml conical tube. 7. Centrifuge for 5 minutes at 1,000 rpm. 8. Aspirate the supernatant. 9. Re-suspend in desired volume of MEF freezing medium, pipette and mix gently. 10. Place 1 ml into two-ml cryogenic vials. (It is recommended to freeze 4 vials from 1 confluent T75 flask). 15

11. Propagation and Distribution of hesc BG01 V NOTE We obtained the hesc BG01V from ATCC. BG01V is characterized as a human embryonic stem cell line with an abnormal karyotype and requires medium renewal every day after the first 48 hours As U.S. National Stem Cell Bank suggested, split cells when the following occur: 1. MEF feeder is two weeks old. 2. hes Colonies are becoming too dense or too large. 3. Increased differentiation occurs. The split ratio is variable and needed to be adjusted by the experienced researcher. If the cells look healthy, split using the same ratio as how you passaged the cells last time. Cells will need to be split every 4-10 days based upon appearance. This SOP is a modified version of the protocols published on the product sheet of ATCC. 1. MATERIALS Pre-warmed complete growth medium: The cells were grown in DMEM/F12 (ATCC # 30-2006) Supplemented with: 2.0 mm L-Alanyl-L-Glutamine (ATCC #30-2115) 1x MEM Non-essential Amino Acid Solution (ATCC #30-2116) 0.1 mm 2-mercaptoethanol (Sigma #M-7522) 4 ng/ml bfgf (R& D Systems #233-FB) 5% knockout Serum Replacement (Invitrogen #10828) 15% fetal bovine serum (ATCC #SCRR-30-2020). This SCRR-30-2020 has been shown to provide optimal growth of ES cells. Use within 2 weeks after thawing. Pre-warmed 0.05% Trypsin-EDTA Invitrogen 25300-054 Pre-warmed MEF Culture Medium (see SOP Preparation of MEF medium) Collagenase IV solution (See SOP preparation of collagenase IV solution) 2. METHODS 2.1. Incubate cells with Collagenase IV Solution 2.1.1. Aspirate the medium from the wells. 2.1.2. Add Collagenase IV solution. Decide the amount by the table provided at the end of this document. 2.1.3. Incubate at 37 C for up to 2 hours. Check the cells after the first 30 minutes and then every 15 minutes. When the majority of the hesc colonies have completely detached or the edges of the colonies have folded back, add appropriate amount (see product sheet) of DMEM/F12 and wash gently using a pipette. 2.1.4. Collect cell suspensions into a 50 ml conical tube. 16

2.1.5. Centrifuge for 5 minutes at 200 x g at 25 C. 2.1.6. Remove the supernatant and re-suspend in complete growth medium. Pipette up and down to break the colonies to smaller clumps and evenly distribute cells to feeder-covered dishes/flasks. 2.1.7. Add 3 ml hescs culture medium to a well of 6 well-plate. 2.1.8. Incubate the cultures at 37 C CO 2 incubator. 2.1.9. Renew the culture medium every day after the first 48 hours of plating. Flask Growth area(cm2) Collagenase (ml) DMEM/F12 (ml) Growth Medium (ml) T225 225 10 10 30 T75 75 3.0 5 12 T25 25 2 5 6 6 well 9.5 0.5 1 3 17

12. Thawing, Propagation, and Cryopreservation of hesc TW1 NOTE This SOP is modified from the instruction of human embryonic stem cell-tw1. 1. MATERIALS 84% DMEM/F-12 or Knockout DMEM (ko-dmem, Invitrogen) 15% Serum replacement (SR, Invitrogen) 1% non-essential amino acid 1mM Glutamax or Glutamin (Invitrogen) 0.1mM b-mercaptoethanol 4 ng/ml bfgf Mitomycin C treated human forskin fibroblasts (HFF) or MEF (See SOP mitomycin C treatment of MEFs) 0.5 mg/ml collagenase IV solution (See SOP preparation of Collagenase) hescs freezing medium 1: 50% hescs culture medium and 50% FBS hescs freezing medium 2: hescs culture medium with 20% DMSO FBS DMSO 2. METHODS 2.1. Thawing TW1 2.1.1. Wear eye protection and ultra low temperature cryo gloves. Remove MEF cell vial(s) from the liquid nitrogen tank. 2.1.2. Immerse the vial in a 37oC water bath, but keep the cap above the water until you see only an ice crystal remains. 2.1.3. Use 95% ethanol to sterilize the outside of the tube. 2.1.4. Pipet cells gently into a sterile 15ml conical tube with 4 ml of culture medium using a 1ml glass pipet. 2.1.5. Use an additional 1 ml of medium to rinse the vial and transfer the liquid to the 15ml tube. 2.1.6. Add 4 ml medium to bring the total volume to 10 ml. 2.1.7. Centrifuge for 5 minutes at 800 rpm. 2.1.8. Remove supernatant and re-suspend cells in 3 ml medium. Then add the cell suspension to the culture vessels of mitomycin C treated feeder or Stematrix. 2.1.9. Label the flask with the following: TW1, Passage number, the date, and your name. Do not change the medium for the first 48 hours. After the first 48 hours, change the medium daily. 2.2. Propagation of TW1 cells by collagenase Plate mitomycin C treated feeder cells onto the culture vessels (6 well plate) at least 24 hours prior to each passage. 18

2.2.1. Remove medium from hescs culture vessels. Add appropriate volume of collagenase IV solution (see the table below), and incubate for at least 1 hour at 37 C. Flask/Plate Growth area (cm 2 ) Collagenase (ml) T225 225 10 T75 75 3 T25 25 2 6 well 9.5 0.5 2.2.2. Incubate the hess at 37 C for up to 2 hours. 2.2.3. Check the cells after the first 30 minutes and every 15 minutes later. 2.2.4. Add 1ml of DMEM/F12 medium per well when most colonies have completely detached or the edges of the colonies have rounded up. (Refer to the following table to determine the volume of DMEM/F12medium) Flask/Plate Growth area (cm 2 ) DMEM/F12 medium (ml) T225 225 10 T75 75 5 T25 25 5 6 well 9.5 1 2.2.5. Collect cell suspensions into a 15 ml conical tube, and centrifuge for 5 minutes at 800 rpm. 2.2.6. Remove the supernatant and re-suspend cells in culture medium. (Refer to the following table to determine the volume of culture medium) Flask/Plate Growth area (cm 2 ) HES culture medium (ml) T225 225 30 T75 75 12 T25 25 6 6 well 9.5 3 2.2.7. Pipet up and down to break the colonies into smaller clumps and plate directly plates covered with freeders or Stematrix. 19

2.2.8.Add hescs culture medium to achieve 3 ml per well of 6 well-plate. Label the flask with the following: TW1, Passage number, the date, and your name. 2.2.9. Incubate the cultures at 37 C, CO2 incubator. Renew the culture medium every day after the first 48 hours of plating. 2.3. Cryopreservation of hescs 2.3.1. Use collagenase to treat hess cell as described in2.2.1-2.2.5 2.3.2. Re-suspend the pellet in hescs freezing medium 1 (1:1 solution of 50% hescs culture medium and 50% FBS). The table below shows the examples of various culture vessels suggested for the number of frozen vials (1ml/per vial) when hescs have grown to 80-90% confluence therein. Flask / Plate Growth Area (cm2) Number of vials T225 225 16 T75 75 5 T25 25 2 6 well 9.5 1 2.3.3. Use P1000 tips to pipet up and down to break the colonies into small clumps. 2.3.4. Slowly add an equal volume of freezing medium 2 (hescs culture medium with 20% DMSO). Mix gently. 2.3.5. Put 1 ml of the cell suspension into each cryovial. 2.3.6. Freeze by Nalgene freezing box at 80 C overnight. 2.3.7.Transfer the cryovials to liquid nitrogen at next day for long term storage 20

13. Embryonic Stem Cell Assessment by Flow Cytometry NOTE The differentiation state of hes can be assessed for the expression of surface markers. This SOP details the method for staining the cell surface proteins in flow cytometry assay. We adapted the protocols from Stem Cells (2007) 25, pp. 437-446 2102Ep cells should be assayed with test samples to provide a standard and a positive control for the antibodies provided. Feeder cells should also be assayed with test samples as negative control. 1. MATERIALS 0.25% Trypsin/0.53 mm EDTA (ATCC 30-2101) 2% paraformaldehyde/1x PBS hes growth medium Wash buffer 1x PBS (Ca2+, Mg2+ free) /1% NGS (normal goat serum) and 0.1% sodium azide PBS (Ca2+, Mg2+ free) Primary antibodies SSEA-4, Clone: MC813-70 (1:50) (R&D Systems, Inc.) Mouse IgG TRA-1-60, Clone : TRA-1-60 (1:100) (Chemicon International) Mouse IgM TRA-1-81, Clone: TRA-1-81 (1:100) (Chemicon International) Mouse IgM All the primary antibodies are monoclonals and the same with the one used for the hes characterization studies conducted by international stem cell initiative. Secondary antibody Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) antibody (1:750) (Invitrogen Corporation). Healthy hes cell cultures Haemocytometer Centrifuge 15ml centrifuge tubes Round bottom 96 well plate Plate seals Flow cytometer Trypsin-EDTA Invitrogen 25300 Phosphate-buffered saline (Ca/Mg-free) 10% FBS/DMEM:F12 2. METHODS 2.1. Harvesting hes Cells for Flow Cytometry 2.1.1. Isolate cells and dissociate to single cell suspension by 0.25% Trypsin/0.53 mm EDTA. 2.1.2. Centrifuge for 5 minutes at 270g at room temperature. 21

2.1.3. Wash in 1x PBS, and then fix in 2% paraformaldehyde/1x PBS at room temperature for 20 minutes. 2.1.4. Wash in 1x PBS/1%. 2.1.5. Cells are incubated with monoclonal antibodies specific for SSEA-4 (1:50), TRA-1-60 (1:100), and TRA-1-81 (1:100) in PBS/3% NGS for 2 hours at room temperature. 2.1.6. Wash by PBS/1% NGS. 2.1.7. Cells are then incubated with an Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) antibody (1:750) (Invitrogen Corporation) for 1 hour at room temperature in dark. 2.1.8. Wash by 1x PBS/1% NGS and then re-suspend in 1x PBS. 2.1.9. Analyze the cells by a FACSCalibur flow cytometer (BD Biosciences). 2.2. Data analysis is performed by CellQuest software Please record the % cells positive and mean fluorescence of each cell line for each antibody, ensuring that data for 2102Ep cells, and for negative control antibodies for each cell line are reported. 2.3. The results below are the percentages you should get from 2102Ep cells. Phenotyping by flow cytometry of 2102Ep cells. Cells stained with primary antibodies and detected via indirect immunofluorescence are shown as filled histograms; negative controls (secondary antibody only) are shown as open histograms. The proportion of 2102Ep cells positive for SSEA-4 (A), TRA-1-60 (B), and TRA-1-81 (C) is shown. Modified from Stem Cells (2007) 25, pp. 437-446 22

14. Immunofluorescence staining of Oct-4 and Nanog in undifferentiated hes. NOTE Oct4 and Nanog are transcription factors expressed by undifferentiated embryonic stem cells. We adapted the protocols from the National Institutes of Health resource for stem cell research, U.S.A and Yu-Jen Chang s thesis, 2006. 1. MATERIALS 4% paraformaldehyde, Merck blocking buffer (10% goat serum, 0.3% Triton X-100) Mouse-anti-human Oct-4 (Santa Cruz biotechnology, 1:50 SC-5279) Fluorescein anti-mouse IgG, 1:200, Vector laboratory, FI-2000 PBS Goat-anti-human Nanog, 5 ug/ml, R&D system FIuorescein anti-goat IgG, 1:200, Vector laboratory Mounting media, Vector laboratory 4-6-Diamidino-2-phenylindole (DAPI), 500 ng/ml 2. METHODS 1. hes cells are washed with PBS once and then fixed with 4% paraformaldehyde for 20 minutes. 2. Wash 3x10min with PBS at room temperature. 3. The hes then are blocked in blocking buffer for 1 hour. 4. Wash 3x5min with PBS at room temperature. 5. Dilute the primary antibodies in a fresh solution of 5% NGS in PBS and incubate for 1hr at room temperature. 6. Wash 3x5min with PBS at room temperature. 7. Dilute secondary antibody in DAPI-containing PBS and incubate cells for 1-1.5 hr at room temperature. 8. Wash 3x5min at room temperature. 9. Mount in anti-fading mounting medium.. 23

15. Passage of 2102Ep Human EC Cell Line NOTE The International Stem Cell Initiative (ISCI) had drawn the expertise of 17 laboratories from 11 countries to work on hes characterization. Many cell lines were analyzed for surface antigens by flow cytometry (http://www.stemcellforum.org/isci_project/browse_by_cell_line.cfm). To define the procedure for the growth and subculture of all cell line, ISCI chose 2102Ep cells, a human embryonic carcinoma cell line as the reference cell line. It keeps in an undifferentiated state and expresses all the key surface antigens of human ES cells when grown at high cell density. By contrast, at low cell densities, it undergoes limited differentiation and expresses SSEA1, which is not expressed by undifferentiated hes cells. Therefore 2102Ep cells are therefore a standard and positive control for the immunofluorescence-based assays. 1. MATERIALS Frozen ampoule of 2102Ep cells 2102Ep growth medium DMEM (high glucose) + 10% FCS + glutamine PBS (Ca2+, Mg2+ free) Trypsin-EDTA Pipettes, 5ml and 10ml Cell culture flasks, 25cm2 and 75cm2 Class II Microbiological Safety Cabinet (MSC) Cell culture microscope 15ml centrifuge tubes Centrifuge CO2 incubator 2. METHODS We will follow ISCI protocol and thus anticipate the following growth kinetics: The cultures should reach confluence and be ready for passage in 3-5 days with a yield of about 2 x 10 7 cells per 75cm 2. 2. 1. Establishing 2102Ep cultures from frozen stocks. 2.1.1 Rapidly thaw one ampoule of cells, and transfer to a centrifuge tube and slowly add 5 10ml growth medium. 2.1.2. Centrifuge at 200 x g for 5 min. Discard supernatant. 2.1.3. Re-suspend the cell pellet in 10ml growth medium, and transfer to 2x25cm2 flasks. 2.1.4. Incubate under a humidified atmosphere of 10% CO2 in air at 37ºC, until the cells are confluent. 2.2. To passage cells at high density (undifferentiated condition) 2.2.1. Aspirate medium from flask and wash cells with PBS. 2.2.2. Add 1ml Trypsin-EDTA per flask and incubate at 37 C for 5 minutes. 24

2.2.3. Tap flask to dislodge cells and break up any clumps. 2.2.4. Re-suspend the cells in 9ml growth medium. 2.2.5. Transfer to a 15ml centrifuge tube, and centrifuge at 200 x g for 5 minutes. 2.2.6. Re-suspend the pelleted cells in 10 ml medium, and count the number of cells. 2.2.7. Seed the cells into flasks at a density of 5 x10 6 cells per 75cm 2, with 15 ml medium. 2.2.8. Incubate under a humidified atmosphere of 10% CO2 in air at 37ºC. 2.3. Obtain partially differentiated cultures of 2102Ep cells. 2.3.1. Cells should be harvested from stock cultures, except they should be seeded at 10 5 cells per 75cm 2 flask. 2.3.2.Such cultures should be confluent by about 7 days. 25

References: 1. Nature Biotechnology 2007; 25:803-816 2. National Stem Cell Bank Protocols, http://www.wicell.org/index.php?option=com_content&task=category&sectionid=7&id=246&it emid=248 3. Protocols for Participating Laboratories, The International Stem Cell Initiative (ISCI) http://www.stemcellforum.org/isci_project/isci_data_set/isci_protocols.cfm 4. Culture Protocols, The National Institutes of Health resource for stem cell research http://stemcells.nih.gov/research/nihresearch/scunit/protocols.htm 5. Human Reproduction 2007; 22:567-577 6. Stem Cells 2007; 25, 437-446 7. 人 類 骨 髓 臍 帶 血 羊 水 與 羊 膜 複 能 性 間 葉 系 幹 細 胞 特 性 之 研 究, 張 育 甄 ; Yu-Jen Chang, 2006, 國 立 交 通 大 學 博 士 論 文 26