Kultivisanje Hes

Human Embryonic Stem Cell Culture Protocols

Manual for Essential Human Embryonic Stem Cell Culture Methods Course

UNIVERSITY OF MINNESOTA 2001 6th Street SE, MC 2873, Minneapolis, MN 55455 p: 612-625-0602 f: 612-624-2436 e: [email protected] w: www.stemcell.umn.edu

Human Embryonic Stem Cell Protocols April 2007 University of Minnesota

TABLE OF CONTENTS

Introduction 1

Human embryonic stem cells (hESC) Setting up a hESC lab Characteristics of hESC cultures

Feeder Cultures

Introduction to feeder cells 3

Fibroblast feeder preparation 4

Passaging fibroblasts 6

Freezing, thawing and irradiation of stock and feeder fibroblast cells 7

Plating fibroblast cells as feeders 11

Human embryonic stem cells

Introduction to hESC culture methods 12

Passaging hESC 13

Freezing and thawing hESC 15 APPENDIX 1: Required materials list A1.1 APPENDIX 2: Reagents A2.1 APPENDIX 3: Tips on growing hESC A3.1 APPENDIX 4: Photographs of hESC colonies A4.1 APPENDIX 5: Creating hESC banks A5.1 APPENDIX 6: Reading list A6.1 APPENDIX 7: Additional Protocols A7.1

Human Embryonic Stem Cell Protocols April 2007

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Human Embryonic Stem Cell Culture Protocols April 2007

INTRODUCTION These protocols were compiled to assist with the initiation and maintenance of human embryonic stem (hESC) cultures. The protocols are collected from those our laboratories uses to grow the HSF-6 and HSF-1 lines derived by the Firpo lab, as well as hESC lines from other labs. These protocols are designed for those with extensive tissue culture experience. While protocols from most labs distributing hESC are similar, you should carefully follow the protocols provided with the cells you receive. Human embryonic stem cells Human ESC are self-renewing, pluripotent cells derived from the inner cell mass of blastocyst stage human embryos. Human ESC are similar in potential and self renewal capacity to mouse ESC (mESC), although in vivo tests of chimera and germline contribution are not possible in humans. In the absence of feeder fibroblasts, hESC, like mESC, spontaneously differentiate into numerous cell types derived from all three embryonic germ layers (Thomson, 1998; Rubinoff, 2000). In fact the differentiation potential of hESC may be greater than that of mESC, because trophectoderm has been found in vitro in addition to tissues of the embryo proper (Xu, 2002). If grown in suspension culture, these differentiating cells aggregate into structures known as embryoid bodies (EBs). Interestingly, the temporal expression profile of molecular markers in EBs is similar to that observed during embryonic development. If hESC are injected into immunodeficient mice, teratomas, benign tumors containing multiple tissue types form. The study of hESC holds much promise for understanding human development and the generation of novel or improved cellular therapies. However, hESC have specific requirements for growth conditions in order to maintain their unique qualities. Setting up a hESC lab Establishing a hESC lab does not require special equipment other than that found in most cell culture labs. It is not necessary to devote specific equipment to use with hESC as long as settings are appropriate and care is taken to prevent cross-contamination. Care should be taken, however, to identify the source of funding used to purchase equipment if culturing hESC lines not eligible for federal funding. A CO2 incubator that has not been treated with detergents or agents to control contamination is required. For best results to control contamination, excellent sterile technique should be used. Incubators may be cleaned with 70% ethanol. A biocontainment hood is required to control the introduction of microorganisms. In order to clearly see the hESC and assess cell and colony morphology, an inverted microscope with phase-contrast optics should be used. A refrigerated centrifuge for washing cells in 15 and 50ml tubes, as well as refrigerator, and –20C and –80C freezers are required, and a liquid nitrogen freezer is required to store stocks of hESC and feeders. A small 37C waterbath is needed for thawing stocks of frozen cells. A list of equipment can be found in appendix A.

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Characteristics of hESC cultures The success of maintaining hESC begins with the propagation of healthy feeder cultures. We recommend the use of mouse embryonic feeder fibroblasts (MEFs). Mouse fibroblast cells grown and passaged before being plated for hESC cultures are referred to as stock fibroblasts, whereas cells plated to support the maintenance of hESC, and have been rendered mitotically inactive via irradiation or chemical treatment are referred to as feeder fibroblasts. If the feeder cells are too sparse, they may not maintain the hESC without differentiation, and the hESC may not attach well. At too high density, the feeder layer may detach from the plate, and the culture will be lost. Maintaining hESC morphology and density promotes healthy, pluripotent, cell growth. We maintain our cultures at approximately 300-500 cells per colony and densities of several hundred colonies per 10cm plate. If there are too many colonies, the cells are likely to differentiate. If most of the colonies are touching, the colonies are either too big or too dense. If there are too few colonies, the cells will grow very slowly. It is important that hESC remain in an undifferentiated state. The individual cells should have a high nuclear/cytoplasmic ratio, and prominent nucleoli. The cells should be tightly packed within the colony, and maintain a defined border at the periphery of the colony. We have observed colonies both appearing to grow on top of the feeders, as well as growing among feeders, pushing them outward as they proliferate. Frequent passage of hESC, within these parameters, onto fresh feeders promotes healthy undifferentiated growth. Finally, we recommend that you make banks of characterized cells before you begin performing experiments with them. When cultures differentiate, it is too late to save them. Throw them away and begin again with frozen stocks. For banking strategies, refer to appendix 5.

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FEEDER CULTURES

The function of feeder cultures is to support the undifferentiated growth of hESC.

Typically, primary fibroblasts are used for this purpose. We prepare our fibroblast feeder cells from CF-1 mice and are certain that this strain is effective. We recommend using CF-1 mice for feeder fibroblast cells for routine expansion of hESC to ensure optimal growth of hESC without differentiation. CF-1 mice can be purchased from Charles River Laboratories. CF-1 mouse embryonic fibroblasts (MEFs) are recommended for several reasons. First, MEFs from CF-1 mice function well for making feeders, and are readily available and are inexpensive. In addition, mouse cells allow detection, and exclusion of contaminating feeder transcripts in PCR experiments. Similar benefits may be obtained for protein and immunostaining studies. Other methods of culturing hESC on human feeders and on extracellular matrices are listed in the reference section. Stock fibroblasts are isolated from E13-E14 mouse embryos and are used as feeders between passages three and seven. Prior to passage 3, fibroblast cultures may be able to support hESC growth, but also may still contain other cell types from the mouse fetus. After passage seven, fibroblasts lose their ability to maintain hESC in an undifferentiated state, and the fibroblasts themselves may begin to senesce. The MEFs are typically irradiated using a cesium source gamma irradiator. We have also included instructions to irradiate feeders using an x-ray machine, and chemical inactivation using mitomycin C if a gamma source irradiator is not available to you. We recommend irradiation over mitomycin for inactivation of fibroblasts for several reasons: irradiation is more reliable at inactivating cells, and is less likely to result in feeders expanding during hESC culture. Mitomycin is extremely toxic to both cells and humans. Medium containing mitomycin as well as the first wash of cells that have been exposed to mitomycin-containing medium must be disposed of as hazardous waste. Embryo cells are very sensitive to even small amounts of mitomycin (as well as many chemicals, such as antibiotics). For best results, trypsinize fibroblasts, and wash several times by centrifugation after mitomycin treatment. Feeder density is crucial for ESC culture success. Characteristics of CF-1 MEFs: Fibroblasts are used as feeders between passages 3 and 5. Irradiated feeder cells should be kept frozen no longer than 4 months before use to

avoid reduced quality. Once fibroblast cells have been plated as feeders, the plates should be ready for

hESC within 24 hours. Plated irradiated feeder cells should be used within one week, before the quality of

the cells begins to degenerate.

Note: In the following protocols, recipes for reagents found in green can be found in Appendix 2. Other reagents are listed in the Required Materials List (Appendix 1). Procedures in found in blue refer to other protocols found in the same section.

FIBROBLAST FEEDER PREPARATION

Required Materials Ca++/Mg++ free PBS 10 cm Culture Dishes 70% Ethanol Trypsin Scalpel 50ml Centrifuge Tubes DNAseI (optional) T175 Flasks Feeder Medium 0.1% Gelatin sterile forceps (2) Sterile scissors (2) The first steps of this procedure can be done on the bench or in a biocontainment hood. Dissection can be done under a dissecting microscope if desired.

1. Place ~ 10ml PBS in 6 10cm culture dishes. 2. Sacrifice pregnant mice by cervical dislocation. 3. Sterilize mouse abdominal area with 70% ethanol. 4. Open abdominal cavity using sterile instruments (one forceps and one scissors). 5. With second set of sterile instruments, remove uterine horns and place in petri

dish with PBS (fetuses may come free of the uterus, if they do not you may dissect off the uterine muscle).

6. Transfer fetuses to fresh PBS in another dish, repeating until dish is clear of all blood, approximately 3 times.

7. Under the dissecting microscope, cut off head of fetuses and remove visceral organs, saving the remaining carcass.

Note: It is recommended that the remaining steps be completed in a biocontainment hood.

8. Transfer carcasses into fresh petri dish containing PBS and repeat until free of all blood, approximately twice.

9. Transfer to a clean petri dish containing 5ml Trypsin per 10 fetuses. 10. Cut tissues into small pieces using dissecting scissors or scalpel, or shear through

10ml syringe (this may cause gel formation due to DNA from broken cells. Pieces should be no larger than 1-2mm2.

11. Transfer minced tissue slurry to a 50ml centrifuge tube. 12. Incubate 15-20 minutes in 5% CO2 at 37C. 13. Neutralize Trypsin solution with Feeder Medium equal to 2 times the volume of

the Trypsin solution.

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14. Pass the solution repeatedly through a 10ml pipet with moderate force (you may expect some additional gelling at this point as more DNA is released. You may use a small amount of DNAseI equaling 1g/ml and incubate for a few minutes at 37C, if desired).

15. Centrifuge 5 minutes at 1000 RPM. 16. Aspirate supernatant and resuspend in 10ml fresh medium. 17. Repeat steps 15 and 16 until supernatant is clear of all blood, typically twice. 18. Plate cells in large, gelatin-coated flasks in 30ml Feeder Medium (one T175 flask

per fetus). 19. One day after plating, collect and replace medium with fresh feeder medium. 20. Passage cells every 2-6 days (when confluent) and freeze early passages (up to

seven passages) for later use as stock fibroblasts.

PASSAGING FIBROBLASTS When feeder cells cover more than 90% of the surface of the flask, the cells are confluent and should be passaged. Each passage should be at a 1:3 ratio.

Required Materials Ca++/Mg++ free PBS Trypsin Feeder Medium 50ml Centrifuge Tubes 15ml Centrifuge Tubes T175 Flasks 0.1% Gelatin

1. Aspirate medium from flask. 2. Rinse cells on flask with 10ml PBS and aspirate. 3. Add 5ml Trypsin to flask and gently swirl to ensure cells are covered with the

solution. 4. Incubate for 5 minutes at 37C. 5. Tap side of flask to dislodge cells and check under microscope to ensure that

cells are in single-cell suspension. If not, incubate for an additional 5 minutes.

Note: To be sure the cells are in a single-cell suspension, check under inverted microscope before the next step.

6. Neutralize Trypsin by adding 10ml Feeder Medium to flask, and transfer the flask contents to a 50ml centrifuge tube (1 tube per flask).

7. Centrifuge for 5 minutes at 1000 RPM. 8. Aspirate supernatant and resuspend cells in 20ml fresh Feeder Medium. 9. Prepare flasks with 10ml Feeder Medium. 10. Plate suspension in 3 T175 gelatin-coated flasks containing Feeder Medium to

final volume 30ml per flask. 11. Incubate until growth is confluent.

Note: There are some variations from lab to lab for this procedure. The Kaufman lab eliminates the use of gelatin for non-inactivated feeders, and passages the cells at 1:3 ratio or less in T75 flasks. What is important is to culture the feeders carefully so that they never overgrow and the medium is not depleted of nutrients or too acidic.

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FREEZING, THAWING AND IRRADIATION OF STOCK AND FEEDER FIBROBLAST CELLS

Note: Fibroblasts that have not yet been irradiated are considered “stock” fibroblasts. Fibroblasts may be frozen for stock within low passage numbers (1-3) without irradiation for later use in plating for further passaging. At passages higher than passage 3, fibroblasts may be irradiated and used as “feeder” cells for supporting hESC. Freezing of stock fibroblasts Required Materials Ca++/Mg++ free PBS Trypsin Feeder Medium 50ml Centrifuge Tubes T175 Flasks Cryogenic Vials 15ml Centrifuge Tubes Isopropanol freezing Freezing medium

containers

1. Follow steps 1 through 7 of protocol for Passaging of Fibroblasts. 2. Aspirate medium from tube and resuspend cells in 1ml freshly made Freezing

Medium (previously cooled on ice). 3. Transfer cell suspension to cryogenic vials in 1ml per vial. One T175 flask should

yield 1 cryogenic vial. 4. Working quickly, place closed cryotubes in room-temperature isopropanol

freezing containers. Place freezing containers in a -80C freezer overnight, or up to 24 hours.

5. Transfer to liquid nitrogen freezer for up to four months.

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Thawing stock fibroblasts Note: It is essential that cells are handled very carefully after thawing, or viability will be drastically reduced: Once thawed, cells must be handled gently, as they are quite fragile. In addition, freezing medium must be diluted immediately after it is thawed. Required Materials 50ml Centrifuge Tubes Feeder Medium T175 Flasks 0.1% Gelatin 37C waterbath

1. Prepare a 50ml centrifuge tube with 10ml Feeder Medium. 2. Remove vial from nitrogen freezer. 3. Place vial in 37C waterbath until ice is nearly thawed. Move quickly to the

biocontainment hood so that when the last ice crystal is thawed, you are ready to dilute the cells.

4. Resuspend cells in vial by gently pipetting once. 5. Transfer cell suspension from one vial to previously prepared centrifuge tube by

adding a few drops at a time, and swirling centrifuge tube gently until entire cell suspension has been transferred to tube.

6. Centrifuge 5 minutes at 800 RPM. 7. Aspirate supernatant and resuspend pellet with 20ml Feeder Medium. 8. Transfer cell suspension to a gelatin-coated T175cm flask containing 10ml

Feeder Medium. 9. After 24 hours, replace medium. 10. Incubate until cells are confluent.

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Irradiating stock fibroblasts Required Materials Ca++/Mg++ free PBS Trypsin Feeder Medium 50ml Centrifuge Tubes T175 Flasks 0.1% Gelatin

1. Follow steps 1 through 7 from protocol for Passaging of Fibroblasts. 2. Aspirate supernatant and resuspend cells in 10ml Feeder Medium and transfer to

50ml centrifugation tube (multiple flasks may be irradiated in one centrifuge tube by pooling flask contents at this step).

3. Irradiate cell suspension with 3000 rad (30Gy) X irradiation or gamma irradiation using a Cesium-source irradiator.

4. Centrifuge for 5 minutes at 1000 RPM. 5. Follow protocol for Freezing Irradiated Feeder Cells (page 11) or Plating

Fibroblast Cells as Feeders (page 12).

Note: If you are not sure about the radiation dose for your irradiator, or are not sure about the accuracy of calibration, a radiation titration (klll curve) should be done to determine the timing for inactivation of feeders by an individual irradiator. Freezing irradiated feeder cells Required Materials Ca++/Mg++ free PBS Trypsin Feeder Medium 50ml Centrifuge Tubes 15ml Centrifuge Tubes T175 Flasks Freezing Medium Cryogenic Vials 0.1% Gelatin Isopropanol freezing containers

1. Complete protocol for Irradiation of Stock Fibroblasts. 2. Aspirate medium and resuspend cells with 1ml per flask freshly made Freezing

Medium (previously cooled on ice) slowly and with a swirling motion. 3. Transfer cell suspension to a cryogenic vial (one flask should yield one vial). 4. Place vial in room temperature isopropanol freezing containers and place

containers in -80C overnight, or up to 24 hours. 5. Transfer vials to liquid nitrogen for up to 4 months.

Note: Your feeders may last more or less than 4 months, depending on the consistency of temperature they encounter in storage.

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Thawing and plating frozen irradiated feeder cells Required Materials Feeder Medium 15ml Centrifuge Tubes 6-well or 10cm Plates 0.1% Gelatin 37C waterbath

1. Prepare 15ml Centrifuge tube with 10ml Feeder Medium. 2. Remove vial (from 1 confluent T175 flask) from nitrogen freezer. 3. Place vial in waterbath until ice is nearly thawed. 4. Resuspend cells in vial by carefully pipetting. 5. Transfer cell suspension to previously prepared centrifuge tube by adding a few

drops and swirling gently until entire cell suspension has been transferred to tube.

6. Centrifuge for 5 minutes at 800 RPM. 7. Aspirate the supernatant and gently resuspend the cells in 10ml Feeder Medium. 8. Plate 6-10 drops per well into 3 gelatin-coated 6-well plates containing 3ml

Feeder Medium OR

Plate 2.5 ml per plate into 4 gelatin-coated 10cm plates containing 7.5ml Feeder Medium.

9. After 24 hours, replace medium with fresh feeder medium. 10. This method should yield approximately 7.5x104 cells per mL. 11. Thawed, irradiated feeders can be used for up to 7 days.

PLATING FIBROBLAST CELLS AS FEEDERS

Required Materials Ca++/Mg++ free PBS Trypsin Feeder Medium 50ml Centrifuge Tubes 6-well or 10cm Plates 0.1% Gelatin

1. Irradiated fibroblasts from fresh or frozen stocks can be used. 2. If using frozen stocks, see instructions for Thawing and Plating of Frozen

Irradiated Fibroblast cells (page 11). 3. If using fresh stocks, follow instructions 1-7 of Irradiation of Stock Fibroblasts. 4. Resuspend cells from one T175 flask in 10ml Feeder Medium. 5. Plate 6-10 drops per well into 3 gelatin-coated 6-well plates containing Feeder

Medium to final volume 3ml per well OR

Plate 2.5 ml per plate into 4 gelatin-coated 10cm plates containing 7.5 ml Feeder Medium.

Note: the final density of feeders should be approximately 2x105 cells per well of a 6-well plate. A protocol for counting cells can be found in Appendix 6.

6. After 24 hours, replace medium with fresh Feeder Medium. 7. Irradiated feeders can be used for up to 7 days.

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HUMAN EMBRYONIC STEM CELLS Achieving optimal growth of hESC requires maintaining several

parameters. The feeders must be healthy, the colony size should be kept relatively small to reduce differentiation, and the colonies should be kept relatively high in density (without touching) to improve growth rate.

Introduction to ESC culture methods:

Human embryonic stem (hESC) should be passaged when colonies reach an estimated average size of 300-500 cells regardless of the number of colonies on the plate (approximately every 4-6 days). Passage times should be determined by colony size and density, as well as the quality of the feeders in the dish. Allowing colonies to get too large will result in differentiation.

Plates should be passaged for expansion cultures at a ratio of 1:2 or

1:3 (if colonies are very few then plate at ratio of 1:1 to reduce colony size and increase density of colonies on the plate).

Human embryonic stem cells should always be passaged to a plate

of feeder cells less than seven days old. Check the feeders before use to be sure they are healthy (more than 90% viable, and with normal fibroblast morphology). The Feeder Medium should be aspirated from the plate and replaced with ESC medium prior to use.

At no time should colonies be allowed to reach a size or density high

enough to touch each other, as this will lead to increased differentiation.

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PASSAGING hESC

Note: These protocols are for established hESC cultures. For natal thawing and establishment of hESC cultures, follow the instructions provided by the hESC supplier. These instructions are for a 1:2 passage. Adjust volumes for other ratios. Scraping the plate to harvest cells maximizes hESC yield, but may cause some feeder carryover into the new culture. For expansion of hESC, this is not normally a problem. If cells are being passaged into different growth conditions (for example, differentiation cultures), hESC can be collected by washing the plate by pipetting medium over the surface with medium after incubation with collagenase, rather than by scraping the plate. This will allow feeder cells to remain in the dish, but may reduce the efficiency of hESC collection.

Required Materials ESC Medium Collagenase type IV 10cmPlates 15ml Centrifuge Tubes Cell Scraper (optional) 0.1% Gelatin Ca++/Mg++ free PBS

1. Aspirate medium from plate. 2. Wash once with 3 ml PBS. 3. Replace with 1ml ESC Medium containing Collagenase (less than 1 week old) per

well. 4. Incubate 10-15 minutes. 5. Scrape plate gently with cell scraper to dislodge cells from plate. Alternatively,

ESC can be dislodged by vigorous pipetting. 6. Using a 5ml or 10ml pipet, remove cell suspension from plate making sure to

break up cell clumps by pipetting (colonies should be reduced to approximately 50-100 cells).

Note: To be sure the colonies are small enough, check them by closing the lid tightly and looking at colonies under an inverted microscope before the next step. Colonies that are not broken up are likely to differentiate.

7. Place cell suspension in 15ml centrifuge tube and centrifuge for 5 minutes at 1000 RPM.

8. Aspirate supernatant and resuspend cell pellet in 10ml of fresh ESC Medium. 9. Centrifuge for 5 minutes at 1000 RPM.

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Note: it is important to wash cells twice to remove collagenase from the culture. If collagenase is not removed, the hESC colonies will not adhere well to the feeders, resulting in reduced plating efficiency and differentiation.

10. Aspirate supernatant and resuspend cells in 10ml ESC Medium. 11. Plate into two 10cm feeder plates (Feeder Medium removed) containing ESC

Medium to final volume of 10ml. 12. After 24 hours, replace medium with fresh hESC medium.

Note: after 24 hours, check colonies in the plate. If any are too large to completely adhere to feeders, remove by aspirating medium, then aspirating the large colony using a sterile Pasteur pipet with a sterile p20 pipet tip on the end while holding the plate at an angle to help see the large colony. The same method can be used to remove differentiated colonies prior to passaging. If differentiation is extensive (more than a few differentiated colonies in each plate), it is better to discard the plate and begin again with banked vials.

FREEZING AND THAWING hESC

Human ESC are extremely fragile following thawing. Handle them gently. Prepare all needed reagents ahead of time, so that as soon as the last ice crystal is thawed, you are diluting the freezing medium. Freezing hESC Note: These instructions are for a single well of a 6 well plate, or 10cm plate. Fewer cells can be frozen per vial, but this will reduce the yield after thawing. Required Materials hESC Medium Collagenase Freezing Medium Cryogenic Vials Isopropanol freezing containers

1. Collect cells by following steps 1-10 of Passaging hESC. 2. Aspirate supernatant. 3. Resuspend cells in 1-2ml Freezing Medium (pre-cooled to 4C). 4. Transfer cell suspension to a precooled (4C) cryogenic vial. 5. Place vial in room temperature isopropanol freezing containers, and place

containers in a -80C freezer overnight, or up to 24 hours. 6. Remove vial from freezer and transfer immediately to liquid nitrogen.

Thawing hESC

Required Materials hESC Medium 15ml Centrifuge Tubes 6-well Plates Irradiated Feeder Cells in 6 well plate

1. Prepare 15 ml centrifuge tube with 10ml ESC Medium. 2. Remove vial (containing 1/2 of a 10cm plate or fewer cells) from nitrogen

freezer. 3. Place vial in 37C waterbath until ice has nearly thawed. Move immediately to

the biocontainment hood. 4. Resuspend cells in vial by carefully pipetting once.

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5. Transfer cell suspension to previously prepared centrifuge tube by adding a few drops and swirling centrifuge tube gently until entire cell suspension has been transferred to tube.

6. Centrifuge 5 minutes at 800 RPM. 7. Aspirate supernatant and gently resuspend pellet with 3ml fresh ESC Medium. 8. Place suspension into one well of a gelatin-coated 6-well plate containing

confluent irradiated fibroblast feeders. 9. After 24 hours, replace medium with fresh hESC medium. 10. Incubate until growth is confluent.

Appendix 3 A1.1 Human Embryonic Stem Cell Culture Protocols April 2007

REQUIRED MATERIALS LIST

REAGENT MANUFACTURER CATALOG NUMBER -Mercaptoethanol 14.3M (1000X) GIBCO/BRL M-6250

10ml Serological Pipettes Fisher Scientific 13-678-12E 25ml Serological Pipettes Fisher Scientific 13-678-14B 2ml Serological Pipettes Fisher Scientific 13-678-12C 5ml Serological Pipettes Fisher Scientific 13-678-12D Acrodisc - 25mm/0.2mm Fisher Scientific 09-730-218

Amino acids, non-essential 10mM (100X) GIBCO/BRL 11140050 Cell Scraper – 3/4 inch blade Fisher Scientific 08-773-2

Collagenase, Type IV GIBCO/BRL 17104019 Corning 100ml Glass Bottle Fisher Scientific 06-414-1A Corning 2ml Cryogenic Vials Fisher Scientific 09-761-72 Corning 500 ml Glass Bottle Fisher Scientific 06-414-1C

Corning Centrifuge Tubes – 15ml Fisher Scientific 05-538-51F Corning Centrifuge Tubes – 50ml Fisher Scientific 05-538-55A

De-ionized Water Sigma W 1503 Dimethylsulfoxide Sigma D 2650

Dispase GIBCO/BRL 17105-041 DMEM/F12 GIBCO/BRL 11960-044

Falcon Tissue Culture Flasks – 750ml Fisher Scientific 10-126 Falcon Tissue Culture Plates - 100x20mm Fisher Scientific 08-772E

Falcon Tissue Culture Plates - 6 Well Fisher Scientific 08-772-1B Fetal Calf Serum, Characterized Hyclone SH30071.03

FGF-2, Human R&D Systems 233-FB-025 Filter Units - 150ml/0.2mm Fisher Scientific 09-740-1A Filter Units - 500ml/0.2mm Fisher Scientific 09-740-25A

Gelatin, Porcine Sigma G 1890 L-Glutamine 200mM (100X) GIBCO/BRL 25030081

Knockout DMEM GIBCO/BRL 10829018 Knockout Serum Replacer GIBCO/BRL 10828028 Pasteur Pipettes – 9 inch Fisher Scientific 13-678-20D PBS (Ca++ Mg++ Free) GIBCO/BRL 14190-144

Penicillin/Streptomycin (100X) GIBCO/BRL 15140-122 Scalpel – blade #10 Fisher Scientific 08-927-5A

Trypsin - 0.25% Trypsin/0.02% EDTA GIBCO/BRL 25300054

Appendix 2 A2.1


REAGENTS

Volume Stock Final Conc. hESC Medium (500ml) DMEM/F12 medium 413ml ------- ------- Knockout Serum Replacer 75ml ------- 15% Glutamine 5ml 200mM 2mM Nonessential Amino Acids 5ml 10mM 0.1mM -Mercaptoethanol 3.5l 14.3M 0.1mM FGF-2 0.2ml 10g/ml 4ng/ml Note: FGF-2 should be added immediately prior to use Collagenase (100ml) HESC 100ml ------- ------- Collagenase type IV 100mg ------- 1mg/ml Note: Dissolve powder in medium and sterile filter prior to use Freezing Medium (10ml) Fetal Calf Serum (FCS) 9ml ------- 90% DMSO 1ml ------- 10% Note: Make up fresh, and cool on ice prior to use Fibroblast Feeder Medium (500ml) DMEM High Glucose 450ml ------- ------- FCS 50ml ------- 10% Penicillin/Streptomycin 5ml ------- 1% L-Glutamine 5ml 200mM 2mM

Appendix 2 A2.2


Volume Stock Final Conc. Gelatin (500ml) De-ionized Water 500ml ------- ------- Gelatin 0.5g ------- 0.1% Note: Dissolve gelatin in water, warm in 37C water bath until completely dissolved, filter sterilize or autoclave and store at 4C until ready for use Note: All flasks and plates referred to in this protocol manual should be coated with gelatin prior to plating with fibroblast or hESC.

1. Using a 10ml or 25ml pipet, add enough gelatin to the flask or plate to coat the bottom

2. Let stand flat at room temperature for 20 minutes 3. Remove remaining liquid gelatin by aspiration prior to use


TIPS ON GROWING HUMAN EMBRYONIC STEM CELLS Variables to consider:

Reagent quality (age, pH, concentration). Feeder density. Feeder quality. ES Cell line. (HSF6, HSF1, H9 etc.) The condition/quality of the cells you are passaging. Length of time the cells are incubated with collagenase. Method of dislodging colonies from the plate. (pipetting vs. scraping) Amount of pressure applied when pipetting cells. The density of the colonies on the plate. (split 1:1 or 1:2) ???????????

Before you start:

How do your Feeders look? What condition are the ES cells in? What is your desired outcome? (expansion, selection, maintenance, etc.)

Tips:

Take good notes. While you get used to working with the ES cells, take notes on the specific methods you use each time you passage, your observations about colony morphology before and after passaging, and the condition of the feeders you’re using. Over time, your observations may help you find the methods that work best for you, and help you to be consistent.

Freeze, freeze, freeze. When you get to a point where you have really good

looking colonies, freeze some of them for later use! Even if the cells just look mediocre, freeze some of them. In a process that can be so unpredictable at times, it is extremely important to have “back-ups” frozen for later use. That is, unless you want to shell out another $5000……

Be Attentive. Especially in the beginning, be sure to check your cells every day.

(Unfortunately, the word “weekend” means nothing to these colonies.) Eventually, you may be able to plan ahead based on the patterns of growth you observe, but check them every day anyway.

Be gentle when freezing and thawing. Use less pressure when pipetting cells in

media that contains DMSO.

Plate extra irradiated feeders. Depending on the quality of the feeder cells, plating efficiency may vary. Try to plan ahead since feeders need to attach to the plate overnight.

Feed your cells after thawing or passaging. Lots of debris in the medium can

affect the growth of the ES cells. Give your cells fresh medium the day after thawing or passaging to remove the debris

PHOTOGRAPHS

Figure Legend Figure 1 hESC grown on CF-1 mouse embryonic fibroblast feeder cells. A) A healthy colony of HSF-1 cells, 40X. B, C, and D) Healthy colony of HSF-6 cells, 40X, 100X, and 200X respectively. Note that even at high magnification the cells remain tightly packed, cell/cell borders are difficult to discriminate, and the border around the edge of the colony remains tight and well defined. E and F) H9 cells grown under sub-optimal conditions starting to differentiate. E) Note that differentiating cells are larger, and cells have begun to separate from one another. F) A healthy colony is visible in the upper left corner, while on the right side of the photo the larger colony is differentiating. Note the differentiating colony has lost its defined border and the cells in the center of the colony have begun to grow on top of one another. Figure 2 hESC grown on CF-1 mouse embryonic fibroblast feeder cells. G) HSF-6 cells grown in sub-optimal conditions have differentiated. The feeder layer is almost invisible underneath the neural-like structures of the cells in the upper right. Cells can also be seen piling on top of one another in the lower left corner. H) A colony of H9 cells that is forming an embryoid body-like structure in the center. This is a common characteristic of cells that are differentiating. I) Differentiated HSF-6 cells. The feeder layer is visible in the lower left corner. The border between hESC and feeders is not visible, and the hESC are large and flat and have lost their characteristic shape. J) H9 cells, grown in sub-optimal conditions, have formed a crater-like structure in the center of the colony. This is characteristic of cells that have begun to differentiate. Note that the cells around the periphery of the colony have begun to elongate and is difficult to discern between the feeder layer and the hES colony. K and L) Differentiating HSF-6 cells. Note that it has become difficult to distinguish between the feeder layer and hESC.



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CREATING hESC BANKS

Before beginning to experiment with hESC, we recommend banking both archive and working banks of hESC. Cell banks can prevent the need for re-purchasing hESC from suppliers. Freezing the first few passages will give you an emergency backup in case of problems early on. Banks of cells reduces the need for frequent of characterization, which should be done every 10-15 passages. Characterization is time consuming and expensive, and necessary. Making working and archive banks, minimizes the impact of inevitable problems, since you can return to characterized stocks whenever contamination or differentiation reduces the quality of hESC cultures.

Freeze

Split 1:2

3-5 Times

Thaw vial 1

into 2 wells

…Expand to 20 100mm plates

Repeat expansion and characterization for working stocks

Characterize and Freeze as cell bank

Appendix 5 A5.1 Human Embryonic Stem Cell Protocols April 2007

READING LIST

Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science. 1998 282(5391):1145-7. Reubinoff BE, Itsykson P, Turetsky T, Pera MF, Reinhartz E, Itzik A, Ben-Hur T. Neural progenitors from human embryonic stem cells. Nat Biotechnol. 2001 12:1134-40. Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol. 2001 10:971-4. Pera MF, Trounson AO. Human embryonic stem cells: prospects for development. Development. 2004 131(22):5515-25. Keller G. Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev. 2005 19(10):1129-55. Xu RH, Peck RM, Li DS, Feng X, Ludwig T, Thomson JA. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Methods. 2005 2(3):185-90. Ludwig TE, Levenstein ME, Jones JM, Berggren WT, Mitchen ER, Frane JL, Crandall LJ, Daigh CA, Conard KR, Piekarczyk MS, Llanas RA, Thomson JA. Derivation of human embryonic stem cells in defined conditions. Nat Biotechnol. 2006 24(2):185-7

Appendix 6 A6.1 Human Embryonic Stem Cell Protocols April 2007

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=Abstract&list_uids=15509763&query_hl=2&itool=pubmed_docsum

ADDITIONAL PROTOCOLS Estimating Cell Suspension Concentration Using a Hemocytometer

- Suspend the cells in a known volume of medium. - Pipet 10L of the cell suspension in to 40L of Trypan Blue, and mix well.

(1:5 dilution) - Pipet 10 L of the trypan blue/cell mix onto one side of the hemocytometer. - Under the microscope, count the number of cells in 5 of the center squares

of the hemocytometer grid. Total the counts for all 5 squares. - Multiply the 5 square total by 104. This is the total cells per mL in your

original cell suspension. (For example: if you count 154 cells in 5 squares, your total cells/mL is 1.54x106.)

Mycoplasma detection assay (Cambrex MycoAlert kit)

Before beginning, reconstitute MycoAlert Reagent & Substrate in 600uL MycoAlert Buffer each. Allow to equilibrate @ RT for 15 minutes. (reconstituted reagents can be stored @ 4C for 5 days, or @ -20C for 2 months.)

1) Collect 2 mL of supernatant from the sample to be tested, and spin @ 1500 RPM for 5 minutes.

2) Transfer 100uL of the cleared supernatant to a round bottom luminometer tube. Also prepare 2 control tubes (for +and – controls`) with 100 uL media each.

3) Add 100 uL MycoAlert Reagent to each sample, and wait 5 minutes. 4) After 5 minutes, take “Reading A” on the luminometer: Record the reading for

each tube. 5) Add 100ul MycoAlert Substrate to each tube, and wait 10 minutes. 6) After 10 minutes, take “Reading B” on the luminometer. 7) Calculate the ratio of Reading B/ Reading A. Ratios of less than 1 indicate a

negative result (no mycoplasma). Ratios of 1-2 indicate a marginal result, and should be repeated for confirmation. Ratios larger than 2 indicate mycoplasma contamination.

Appendix 7 Human Embryonic Stem Cell Protocols April 2007

A7.1

Mitomycin inactivation of feeders (alternative to irradiation) Per 10cm plate or T75 with 15 mL medium:

1) Remove 10 mL media. 2) Add 300ul of 500mg/mL mitomycin C (MMC) stock solution to 10mL media, mix

well. 3) Add this to cells in T75 (there will be 15mL of 10mg/mL mitomycin). 4) Incubate at 37C for 3 hrs. 5) Remove medium/MMC and wash 1x with 10mL fresh medium then 2x with 5 mL

Ca++/Mg++ free PBS. 6) Trypsinize with 2mL trypsin for 5 minutes at 37C. 7) Inactivate trypsin by adding 5mL medium. Wash and resuspend in 5mL medium. 8) Plate cells at desired concentration.

Plating feeder cells after MMC treatment

1) Count single cell suspension: ex. 1.2 x 106 cells per mL 2) Calculate number of cells needed:

For MEF plating:

Want 0.75 x 105 cells/mL x 15mL per 6-well plate = 1.125 x106 cells per plate. So for one 6-well plate use 0.940 mL cell solution (1.125/1.2) plus 14.06 mL media (15mL- 0.94mL) to plate.


A7.2

Metaphase spread preparation for FISH/Karyotype Colcemid- GIBCO Hypotonic solution: 1:1 0.4% KCl + 0.4% Sodium Citrate Fixative: Methanol : Acetic Acid (3:1) Protocol:

1. Harvest cells: -save supernatant in 15ml tube -wash cells with 1ml PBS (Ca++Mg++ free) -add 1ml Trypsin solution, incubate at 37C 5 minutes. -resuspend culture in collected media. -Spin at 1000 rpm for 5 minutes -aspirate media and leave small amount. -flick tube to resuspend cells.

2. Addition of hypotonic solution:

-add (drop wise against side of tube) 5 drops hypotonic solution, then bring volume up to 2ml. -incubate 7 minutes in 37C bath -spin at 1000 rpm for 5 minutes, aspirate solution, flick tube to resuspend cells.

3. Fixation:

-add dropwise as before, 5 drops of fixative, then bring final volume to 3ml -let stand at room temp 30 minutes. -spin at 1000 rpm for five minutes. Aspirate, fix and flick tube to resuspend. -add dropwise 5 drops fixative, bring volume to 2ml. -store at room temp for 20 minutes.

4. Slide Preparation:

-Prewash slides in Methanol at 4C -wash slides in ice water for 30 seconds…water should stick to slide in a sheet. -take slide out of ice bath, rinse with fixative. -drop cells onto angled slide. -Rinse slide with fixative, dropwise -aspirate loose fix with pipette wipe back of slide with paper towel -cure slides at 65C on slide warmer over night.


A7.3

In Vitro Differentiation to Embryoid Bodies (EBs)

Colonies of hES cells are dislodged from plates and centrifuged as described above in Passaging of hESC. Human ES cells are washed and resuspended in differentiation medium (DMEM High Glucose (Invitrogen), 20% FBS (Hyclone), 100M MEM Non-Essential Amino Acids (Invitrogen), 2mM glutamine (Invitrogen), and 100M -Mercaptoethanol (Invitrogen). Resuspended cells are plated in ultra-low attachment 100mm2 dishes or 6-well plates (Corning) with differentiation medium. Undifferentiated hES colonies spontaneously form EBs containing differentiating cells at 37C and 5% CO2 in suspension culture (see figure A). In Vivo Differentiation to Teratomas Human ES colonies are dislodged from plates as described above Passaging of hESC, washed three times in PBS, and resuspended in fresh PBS at an estimated concentration of 106 cells in each 50L of PBS. With a 1cc syringe and 25g needle, 50L of suspended hES colonies are injected into the quadriceps muscle of a immunodeficient mouse. Mice are observed for formation of teratomas for one to four months. The mice are sacrificed, and teratomas collected from the site of injection. Teratomas are then fixed overnight at 4°C in 4% paraformaldehyde, and subsequently embedded, sectioned and stained with hematoxylin/eosin for histological analysis (see figure B).


A7.4

Total RNA Isolation (RNeasy protocol) ****Use Qiagen Rneasy mini kit****

Before Beginning: Prepare the Buffer RLT by adding 10l -mercaptoetanol for every 1 mL of Buffer RLT. 1) Harvest cells and wash with PBS to make sure all traces of medium are gone. 2) Pellet cells in the centrifuge (1000 RPM x5min) and aspirate supernatant. Flick

the tube to loosen cell pellet. 3) In the biocontainment hood, add 350L Buffer RLT (600L for large amounts of

expected RNA – greater than 5x106) to each tube. Mix cells by pipetting. 4) Transfer cells to a QiaShredder column. Centrifuge at maximum speed x 2

minutes. After spinning, discard Qiashredder column and save flowthrough, which contains the homogenized cell lysate.

5) Add 350L 70% EtOH (or 600L) to the cell lysate, and mix. 6) Load up to 700L of the cell lysate onto an Rneasy mini column, and

centrifuge for ~15 seconds at maximum speed. Discard the flow through. 7) Add 700L Buffer RW1 to the column and spin for ~15 seconds at maximum

speed. Discard the flow through and transfer the column to a new 2ml collection tube.

8) Add 500 L Buffer RPE to the column and spin for ~15 seconds at maximum speed. Discard the flowthrough.

9) Add another 500 L Buffer RPE to the column and spin for 2 minutes at maximum speed to dry the column. Discard the flowthrough.

10) To elute the RNA, transfer the column to a new 1.5 mL collection tube and add 20-50 L RNase-free water to the silica gel in the column. LET COLUMN SIT WITH THE WATER IN IT FOR AT LEAST 3 MINUTES BEFORE CENTRIFUGING. Centrifuge for 1 minute at maximum speed. For maximum yield, add a second volume of RNase-free water to the column, and elute into the same collection tube. LET COLUMN SIT WITH THE WATER IN IT FOR AT LEAST 3 MINUTES BEFORE CENTRIFUGING. The resulting eluate should contain your total RNA.

mRNA Isolation (from total RNA, optional) ****Use Oligotex mRNA mini kit**** Before starting:

- Heat Oligotex Suspension to 37C, mix, and place at RT. - Heat Buffer OEB to 70C. - If Buffer OBB has precipitated, re-dissolve at 37C and place @RT.


A7.5

1) Determine the starting volume of total RNA. Add RNase-free water to the total

RNA, such that the final volume is 250L. 2) Add 250 L Buffer OBB, and 15 L Oligotex Suspension to the tube and mix. 3) Incubate the tube for 3 min at 70C. 4) Place tube at RT for 10 minutes. 5) Centrifuge at max speed for 2 minutes. 6) Remove the supernatant by pipetting, and resuspend the pellet in 400 L Buffer

OW2. 7) Pipet the mixture onto a small spin column in a fresh collection tube. 8) Centrifuge at max speed x 1 minute. 9) Transfer the spin column to a new collection tube, and add 400 L Buffer OW2

to the column. 10) Centrifuge at max speed x 1 minute. 11) Transfer the spin column to a fresh collection tube. Add 20-50 L HOT Buffer

OEB to the column, and pipet up and down to resuspend the resin. **If doing more than one sample, put the collection tubes on the heating block to ensure that the Buffer OEB stays hot.

12) Centrifuge at maximum speed for 1 minute. 13) To ensure maximum yield, add another 20-50 L Buffer OEB to the column, and

pipet up and down to resuspend the resin. 14) Centrifuge at maximum speed for 1 minute. The eluate should contain your

mRNA.


A7.6

cDNA Synthesis

1) Start with up to 22L of mRNA. If less than 22L mRNA, add RNase free water to bring the volume to 22L.

2) In a PCR reaction tube, add: 22 L mRNA 2L Random Primers

3) Place the tube in the PCR machine, and start the reverse transcription. In the first step of the program, heat the tube to 70C for 10 minutes. Remove the tubes from the PCR machine and chill on ice for 2 minutes.

4) To each tube, add: 8L 5x First Strand Buffer 4L 0.1M DTT 2 L 10mM dNTP mix

5) Mix tube and place back in the PCR machine and continue at 25C for 5 minutes. 6) Add 2 L Superscript III Reverse Transcriptase to the tube, and continue at

25C for 10 minutes 42C for 50 minutes 70C for 15 minutes.

PCR Reaction Mix (Semiquantitative) Ingredients (per 20 L rxn) 2.0 L 10x PCR Buffer (Invitrogen) 0.5 L 50 mM MgCl2 0.5 L 10mM dNTPs 11.9 L H2O 0.1 L Taq DNA Polymerase (5 units/uL) 4.0 L Primer (1mM) 1.0 L cDNA (50-1000 ng/ul) Temperature cycling (38 cycle PCR) 1 cycle : 94C x 3:00 min 38 cycles: 93C x 1:00 min 55C x 1:00 min 72C x 1:30 min 1 cycle: 72C x 10:00 min


A7.7

PCR Primer Optimization Ingredients (per 100L rxn) 10.0 L 10x PCR Buffer (Invitrogen) 2.5 L 50 mM MgCl2 2.5 L 10mM dNTPs 59.5 L H2O 0.5 L Taq DNA Polymerase (5 units/uL) 20.0 L Primer (1mM) 5.0 L cDNA (50-1000 ng/ul)

- Mix ingredients in one PCR tube, and run 38 cycle PCR program. - Remove 15 L sample from the tube at each of the following cycles: 18, 22,

26, 30, 34, 38. - Run samples on a 1.5% agarose gel with EtBr.

Immnohistochemistry Protocol for hES Cells

1) Plate feeders into 2-chamber slides, 4-well plates, 12-well plates, or 24-well plates at least one day before plating hES cells. Be sure to plate enough chambers/wells to allow for the negative controls. (i.e. at least one well that will not be treated with the primary antibody.)

2) Passage hES cells as usual onto the prepared plates. 3) Allow 2-4 days for the hES cells to grow. 4) After 2-4 days, fix the cells using 4% paraformaldehyde: 5) Aspirate the medium from the plates and wash 1x with PBS 6) Add enough 4% Paraformaldehyde to cover the surface of the plates, about

0.5mL, and leave at RT for 30min. 7) Aspirate the fixative, and wash the plates 3 times with PBS. 1st wash= 1 min, 2nd

wash=15 min, 3rd wash= 1 min. 8) Next, you need to add blocking solution. Use normal serum that is the same

isotype as the secondary antibody being used. For most purposes, the blocking solution needed is 5% normal goat serum in PBS:

9) Aspirate the last wash of PBS, and then replace with 5% normal goat serum. 10) Incubate at RT for an hour, or leave at 4 degrees overnight. 11) After the block, aspirate the blocking solution (except from the control chamber)

and wash the plates 3 times with PBS: 1st wash= 1 min, 2nd wash=15 min, 3rd wash= 1 min.

12) After blocking, add the primary antibodies. 13) Aspirate the last wash of PBS from the plates.


A7.8


A7.9

14) Add the diluted (for Chemicon antibodies SSEA-1, SSEA-3, SSEA-4, Tra-1-60, and Tra-1-81, 1:200 in PBS) primary antibody to each chamber, except for the control. ~0.5mL per chamber should be enough.

15) Store plates at 4C for at least 1 hour. (Overnight is OK.) 16) Treat with secondary antibody: 17) Aspirate primary antibodies and the blocking buffer from the control chamber. 18) wash 3x with PBS: 1st wash= 1 min, 2nd wash=15 min, 3rd wash= 1 min. 19) Add diluted (1:200 in PBS) FITC conjugated secondary antibodies to each

chamber. ~0.5mL per chamber, or enough to cover entire surface 20) Leave the plates COVERED with foil at RT for 1 hour. 21) After 1 hour, wash the plates 3 times with PBS: 1st wash= 1 min, 2nd wash=15

min, 3rd wash= 1 min. 22) Observe the plates under the fluorescent microscope using the GFP filter.

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