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					        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: kauf020@umn.edu
  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
                               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 –20C and –80C freezers are required, and a liquid nitrogen freezer is
required to store stocks of hESC and feeders. A small 37C waterbath is needed for
thawing stocks of frozen cells. A list of equipment can be found in appendix A.



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Human Embryonic Stem Cell Culture Protocols                               April 2007
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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Human Embryonic Stem Cell Culture Protocols                                 April 2007
                             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.


                                             3

Human Embryonic Stem Cell Culture Protocols                                 April 2007
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 37C.
   13. Neutralize Trypsin solution with Feeder Medium equal to 2 times the volume of
       the Trypsin solution.

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Human Embryonic Stem Cell Culture Protocols                                April 2007
   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 1g/ml and incubate for a few minutes
       at 37C, 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.




                                           5

Human Embryonic Stem Cell Culture Protocols                              April 2007
                      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 37C.
   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.




                                            6

Human Embryonic Stem Cell Culture Protocols                                April 2007
 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 -80C freezer overnight, or up
      to 24 hours.
   5. Transfer to liquid nitrogen freezer for up to four months.




                                           7

Human Embryonic Stem Cell Culture Protocols                              April 2007
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                         37C waterbath

   1. Prepare a 50ml centrifuge tube with 10ml Feeder Medium.
   2. Remove vial from nitrogen freezer.
   3. Place vial in 37C 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.




                                            8

Human Embryonic Stem Cell Culture Protocols                                April 2007
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 -80C 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.


                                             9

Human Embryonic Stem Cell Culture Protocols                                  April 2007
Thawing and plating frozen irradiated feeder cells
Required Materials
Feeder Medium        15ml Centrifuge Tubes                6-well or 10cm Plates
0.1% Gelatin         37C 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.




                                          10

Human Embryonic Stem Cell Culture Protocols                              April 2007
         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.




                                           11

Human Embryonic Stem Cell Culture Protocols                               April 2007
                   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.




                                        12

Human Embryonic Stem Cell Culture Protocols                  April 2007
                                 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.



                                          13

Human Embryonic Stem Cell Culture Protocols                              April 2007
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.




                                        14

Human Embryonic Stem Cell Culture Protocols                          April 2007
                 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 4C).
   4. Transfer cell suspension to a precooled (4C) cryogenic vial.
   5. Place vial in room temperature isopropanol freezing containers, and place
      containers in a -80C 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 37C waterbath until ice has nearly thawed. Move immediately to
      the biocontainment hood.
   4. Resuspend cells in vial by carefully pipetting once.



                                         15

Human Embryonic Stem Cell Culture Protocols                             April 2007
   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.




                                          16

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 3                             A1.1

        Human Embryonic Stem Cell Culture Protocols                  April 2007
                                       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             3.5l         14.3M           0.1mM
FGF-2                         0.2ml         10g/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                             A2.1

      Human Embryonic Stem Cell Culture Protocols
           April 2007
                                  Volume      Stock     Final Conc.

Gelatin (500ml)
De-ionized Water                  500ml       -------   -------
Gelatin                           0.5g        -------   0.1%

Note: Dissolve gelatin in water, warm in 37C water bath until completely
dissolved, filter sterilize or autoclave and store at 4C 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




Appendix 2                             A2.2

Human Embryonic Stem Cell Culture Protocols                               April 2007
   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
Appendix 3                                A3.1

Human Embryonic Stem Cell Culture Protocols                                         April 2007
                                 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.




Appendix 4                             A4.1

Human Embryonic Stem Cell Culture Protocols                             April 2007
                                   QuickTime™ and a
                             TIFF (PackBits) decompressor
                             are needed to see this picture.




Appendix 4                                A4.2

Human Embryonic Stem Cell Culture Protocols                    April 2007
                                   QuickTime™ and a
                             TIFF (PackBits) decompressor
                             are needed to see this picture.




Appendix 4                                A4.3

Human Embryonic Stem Cell Culture Protocols                    April 2007
                        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

                                                                   3-5
Thaw vial 1                                                       Times
into 2 wells
                                                Split 1:2




                                                             …
                     Expand to 20 100mm plates




                                                   Repeat expansion
          Characterize and Freeze                and characterization
               as cell bank                       for working stocks
  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
                     ADDITIONAL PROTOCOLS

Estimating Cell Suspension Concentration Using a
Hemocytometer

      -   Suspend the cells in a known volume of medium.
      -   Pipet 10L of the cell suspension in to 40L 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 @ 4C for 5 days, or @ -20C 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                               A7.1

Human Embryonic Stem Cell Protocols                                          April 2007
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 37C 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 37C.
   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.




Appendix 7                                A7.2

Human Embryonic Stem Cell Protocols                                           April 2007
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 37C 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 37C 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 4C
      -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 65C on slide warmer over night.




Appendix 7                               A7.3

Human Embryonic Stem Cell Protocols                                            April 2007
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), 100M
MEM Non-Essential Amino Acids (Invitrogen), 2mM glutamine (Invitrogen), and 100M
-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 37C 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 50L of PBS. With a 1cc syringe and 25g needle, 50L
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).




Appendix 7                               A7.4

Human Embryonic Stem Cell Protocols                                          April 2007
Total RNA Isolation (RNeasy protocol)

       ****Use Qiagen Rneasy mini kit****

   Before Beginning: Prepare the Buffer RLT by adding 10l -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 350L Buffer RLT (600L 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 350L 70% EtOH (or 600L) to the cell lysate, and mix.
   6) Load up to 700L of the cell lysate onto an Rneasy mini column, and
       centrifuge for ~15 seconds at maximum speed. Discard the flow through.
   7) Add 700L 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 37C, mix, and place at RT.
       - Heat Buffer OEB to 70C.
       - If Buffer OBB has precipitated, re-dissolve at 37C and place @RT.
Appendix 7                              A7.5

Human Embryonic Stem Cell Protocols                                         April 2007
   1) Determine the starting volume of total RNA. Add RNase-free water to the total
       RNA, such that the final volume is 250L.
   2) Add 250 L Buffer OBB, and 15 L Oligotex Suspension to the tube and mix.
   3) Incubate the tube for 3 min at 70C.
   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.




Appendix 7                             A7.6

Human Embryonic Stem Cell Protocols                                       April 2007
cDNA Synthesis

   1) Start with up to 22L of mRNA. If less than 22L mRNA, add RNase free water
      to bring the volume to 22L.
   2) In a PCR reaction tube, add: 22 L mRNA
                                     2L 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 70C for 10 minutes. Remove the
      tubes from the PCR machine and chill on ice for 2 minutes.
   4) To each tube, add:             8L 5x First Strand Buffer
                                     4L 0.1M DTT
                                     2 L 10mM dNTP mix
   5) Mix tube and place back in the PCR machine and continue at 25C for 5 minutes.
   6) Add 2 L Superscript III Reverse Transcriptase to the tube, and continue at
                              25C for 10 minutes
                              42C for 50 minutes
                              70C 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 :    94C x 3:00 min

38 cycles:   93C x 1:00 min
             55C x 1:00 min
             72C x 1:30 min

1 cycle:     72C x 10:00 min



Appendix 7                             A7.7

Human Embryonic Stem Cell Protocols                                       April 2007
PCR Primer Optimization

Ingredients (per 100L 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.
Appendix 7                                 A7.8

Human Embryonic Stem Cell Protocols                                              April 2007
   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 4C 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.




Appendix 7                              A7.9

Human Embryonic Stem Cell Protocols                                        April 2007

				
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