UNIT 2.22 Murine Embryonic Stem Cell Derivation, In Vitro Pluripotency Characterization, and In Vivo Teratoma Formation

 

Introduction

This unit describes the methods to properly characterize newly derived embryonic stem (ES) cell lines before extensively using them for any intended research projects. Since frequent subculturing, media change, and preparation of feeder layers, etc. are time consuming and require considerable attention, the key to successfully obtaining and consistently maintaining healthy ES cell cultures is careful and meticulous attention to detail during all stages. The most important part of the procedure is to ensure that ES cells retain their pluripotent state. However, it is also imperative that the cell lines have a normal karyotype and differentiate appropriately as determined by the teratoma assay and embryoid body formation (Fuegemann et al., 2010). Assays to evaluate pluripotency markers and teratoma formation are described in the protocols below, while the methods for embryoid formation may be found in Cerdan et al. (2007); Figure 2.22.1 offers an example of embryoid bodies formed from cells derived by the author's own work.

Figure 2.22.1 Differentiation of GFP^+ mouse ES cells: Embryoid bodies (EBs) culture. EB culture is used to differentiate ES cells in vitro into different cell lineages. There are two steps in the process. The first step is the hanging drop culture to form EBs followed by growing EBs in suspension culture (low attachment dish) on an orbital shaker. EBs are then dissociated and a single cell suspension is sorted by the FACSAria for the desired cell populations. (Image taken with 10× objective.) For additional details, see Cerdan et al. (2007).

In this unit, murine ES cells are derived from embryonic day 3.5 (E3.5) embryos (also called blastocyst stage embryos; Basic Protocol 1). To sustain pluripotency, ES cell lines are maintained on mouse embryonic fibroblasts (MEF) feeder layers with the addition of leukemia inhibitory factor (LIF) in the culture medium. Once stable cell lines are established, they are then subject to molecular characterization by evaluating Oct4, Sox2, Nanog, and SSEA1 expression (see Basic Protocol 2). Finally, the teratoma assay confirms the differentiation potential of the new ES cell lines to form three germ layers in vivo (see Basic Protocol 3). Emphasis is placed on validating ES cells, as well-characterized ES cell lines with low passage numbers are essential starting materials for scientists interested in generation of transgenic mice, studying specific genes during embryonic development, or using differentiated ES cells in pharmacological and toxicological screens.

NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

 

Basic Protocol 1: Murine Embryonic Stem (ES) Cell Derivation

The techniques to derive and culture murine ES cells from inner cell mass (ICM) of blastocyst embryos were first reported almost 30 years ago (Evans and Kaufman, 1981; Martin, 1981). The standard procedures used in laboratories worldwide (Turksen, 2006a; Yabuuchi et al., 2008) are outlined in Basic Protocol 1. Generating cultures of murine ES cells that retain the correct karyotype in a proliferating and undifferentiated state is a multistep process. The following methods provide step-by-step instruction on how to derive quality murine ES cell lines.

 Materials

• 129SvEv female and male mice (3- to 4-week–old females and 8-week-old males; Taconic)
• Pregnant mare serum gonadotropin (PMSG; see recipe)
• Human chorionic gonadotropin (hCG; see recipe)
• Mouse embryonic stem (mES) cell culture medium (see recipe)
• Mouse embryonic fibroblast (MEF) culture medium (see recipe)
• 0.2% (w/v) gelatin solution in Milli-Q water (see recipe)
• Mouse embryonic fibroblasts (MEFs) treated with mytomycin C from CF1 strain (Chemicon/Millipore)
• 1× EmbryoMax M2 medium with phenol red (Millipore)
• Iodine or combination of 70% ethanol/surfactant
• Acid tyrode solution (Millipore)
• 1× FHM HEPES–buffered medium without phenol red (Millipore)
• 0.25% (1×) trypsin/EDTA (Invitrogen)
• Phosphate buffered saline without Ca²⁺ and Mg²⁺ (PBS^–; Invitrogen)
• C57BL/6-TgN(ACTbEGFP) male mice (Jackson Laboratories); only for generation of GFP-positive ES cell lines (ES cell lines are F1 cross between C57BL/6 and 129SvEv; B6/129 F1 hybrid; also called V6.5), optional

• 1-ml syringes (BD Biosciences)
• 27-G needles (BD Biosciences)
• Fine forceps and fine scissors
• IVF 4-well plates (Nunc/Fisher)
• 37°C, 5% CO₂ incubator with water tray
• 37°C water bath
• 35- and 60-mm tissue culture dishes (Corning/Fisher)
• Inverted light microscope
• 0.22-µm bottle top filter (Corning/Fisher)

 Prepare animals

1.	House 3- to 4-week-old female mice in the animal facility 2 to 3 days before hormone induction for superovulation. p type = annotation The most convenient lighting cycle for the mouse facility is darkness from 1900 to 0500 and light from 0500 to 1900.
2.	Day 1: At 1400, administer 100 µl PMSG via intraperitoneal (i.p.) injection into each female mouse using a 1-ml syringe 27-G needle.
3.	Day 3: At 1400, 48 hr after PMSG administration, administer 100 µl hCG via i.p. injection into each female mouse using a 1-ml syringe and 27-G needle.
4.	Place one female in a cage with one male. p type = annotation It is the best to use stud males with a good plugging performance.
5.	Day 4: Before 0800, check all females for copulation plugs with forceps or pipet tips (this is considered 0.5-day post-coitum, dpc). Group the plugged females in new cages and label the cage cards.

 Prepare for ES cell isolation

6.	Day 6: Prepare mES culture medium and MEF medium according to recipes.
7.	Coat 4-well plates with 0.2% gelatin solution (500 µl/well). Place plates under a tissue culture hood for at least 15 min at room temperature, then aspirate gelatin solution.
8.	Prepare MEF feeder plates by plating MEFs on gelatin-coated plates according to manufacturer's instructions (Chemicon or other MEF manufacturer) to ensure correct cell density. Culture overnight in a 37°C, 5% CO2 incubator so that the plated MEFs attach and develop a spindle-shaped morphology. p type = annotation The correct cell density is that which will ensure confluent monolayers by the time ES cells are ready to be plated on the MEF feeder plates. p type = annotation MEFs can also be prepared in the laboratory from 13.5 dpc embryos. However, the consistency of the MEF quality would need to be ensured. To check the quality of MEFs, see Hogan et al. (1994) and Schmidt (2001).
9.	Autoclave surgical tools in preparation for animal surgery. p type = annotation At minimum, several pairs of forceps and scissors will be required.

 Perform surgery

10.	Day 7: Warm up mES medium for 15 min in a 37°C water bath. Remove the 4-well plates seeded with MEFs on day 6 from incubator. Aspirate the old medium and add fresh, warm mES medium (500 µl/well). Place MEFs back into the 37°C, 5% CO2 incubator. Additionally, warm 5 ml of 1× EmbryoMax M2 medium for a minimum of 1 hr in the 37°C, 5% CO2 incubator.
11.	Sacrifice pregnant female mice according to institutional guidelines and in accordance with the animal regulatory board. Immediately following sacrifice, place animals onto their backs on top of a hard surface covered with absorbent padding. Clean fur with iodine or a combination of 70% ethanol and a surfactant.
12.	Open the abdominal cavity. Use different pairs of forceps and scissors to cut the skin and the body wall (peritoneum) to minimize any contamination. Remove the uterus by carefully trimming away the mesometrium and place into a 35-mm tissue culture dish containing M2 medium.

 Collect ES cells and start initial culture of ES derivation plates

 13.

Using a 25-G or 27-G needle attached to a 1-ml syringe, flush blastocysts out of uterine horns with ~200 µl of 1× EmbryoMax M2 medium on each side into an empty 35-mm tissue culture dish.

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The flushing step can be repeated with a lower volume of medium (e.g., 100 µl).

 14.

Collect the blastocysts from all the pregnant mice, either in the same 35-mm tissue culture dish or separate ones (if different genetic backgrounds of mice are used). Place the 35-mm tissue culture dish(es) into a 37°C incubator. Work as quickly and aseptically as possible during blastocyst embryo harvest.

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The blastocysts can be placed up to 2 hr in the 37°C incubator. However, it is recommended to proceed to next step without delay.

 15.

Remove the 35-mm tissue culture dish and assess the blastocysts' quality and quantity under microscope (Fig. 2.22.2A,B).

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The quality of blastocysts may be evaluated by the morphological characteristics of significant expansion and an enlarged cavity almost filling the embryo.

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If there is no colony picking hood set up in the laboratory, as an alternative, transfer the inverted microscope to the tissue culture hood and wipe everything with 70% ethanol.

Figure 2.22.2 Collection of mouse blastocyst embryos. (A) Low magnification, bright-field image (4× objective). Blastocysts have been flushed out of uterine horns with M2 media. (B) Higher magnification (30× objective) image of preimplantation mouse blastocysts. The ICM and trophectoderm can be easily identified. The trophectoderm serves as the interface between the blastocyst and the oviduct/uterine environment. The ICM can give rise to ES cells. The quality of blastocysts may be evaluated by the morphological characteristics of significant expansion and an enlarged cavity that almost fills the embryo.

 16.

Treat blastocyst embryos with 0.5 ml acid tyrode solution (1:8 acid tyrode solution/total volume of medium) in a 35-mm tissue culture dish until zona pellucida (Figure 2.22.2B) dissolves (<1 min). Perform this step under the dissecting microscope to determine when to stop the reaction by adding 2 ml FHM HEPES–buffered medium to the dish. Collect all the embryos and rinse them again in a 35-mm tissue culture dish filled with 2 ml FHM HEPES–buffered medium.

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If some of the zona pellucida remain intact, these embryos may not satisfactorily give rise to ES cells after transferring them onto MEF feeder plates.

 17.

Transfer each blastocyst individually, using a 10-µl pipet, into a single well of an MEF-seeded 4-well plate. Repeat the process until all the blastocysts are transferred to individual wells of the MEF-seeded plates.

 18.

Culture in a 37°C, 5% CO₂ incubator. Monitor daily under microscope.

 19.

Days 8 and 9: After 24 to 48 hr of cell culture, the inner cell mass (ICM) should be enlarged and the embryo should hatch on MEFs. Observe ICM outgrowth under the microscope. Do not change medium and continue culturing for an additional 24 hr.

 20.

Day 10: Add an additional 200 µl of mES medium to each well. Do not change medium and continue culturing. Monitor cells daily to determine when they will be ready for subculturing.

 Subculture ES cells

 21.

Days 12 to 14 (5 to 7 days after blastocyst harvest): 1 day before subculturing, prepare fresh MEF feeder plates as described in steps 7 and 8.

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In most cases, the ICM will have grown to a distinct cellular mass/clump after 5 days of blastocyst culture and will be ready to be dissociated. In some cases however the ICM proliferation will require more time due to the incomplete removal of the zona pellucida.

 22.

Days 12 to 14 (morning of subculturing): In a 37°C water bath, warm the 0.25% trypsin/EDTA and PBS in the water bath. Carefully aspirate the old medium from ES-derivation plates using a 1000-µl pipet and rinse cells one time with warm PBS.

 23.

Add 75 µl warm trypsin/EDTA per well and allow plates to stand 5 to 10 min in the 37°C incubator.

 24.

Vigorously pipet up and down to dissociate the cell clumps to obtain a single cell suspension. Add 100 µl mES culture medium to neutralize the trypsin.

 25.

Pipet the contents of each individual well to a corresponding well in the MEF feeder plate.

 26.

Days 13 to 15: Change medium with fresh mES medium (200 µl/well) on the next day of subculturing.

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Initially, it may be difficult to see obvious ES colonies, but they will begin to appear in the next day or two.

 27.

Days 14 to 16: ES colonies will begin to appear 2 days after subculturing. Change medium daily and expand cells as previously described (Evans and Kaufman, 1981). Monitor under microscope and carefully determine if subculturing is necessary before subculturing.

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An established ES cell line (Fig. 2.22.3) requires careful subculture at 2-day intervals at a 1:10 ratio, although this depends on the growth rate.

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Figure 2.22.4 represents ES cells derived from a B6/129 F1 hybrid blastocyst embryo expressing green fluorescent protein (GFP). More detailed steps of ES culturing, subculturing, and freezing down cell aliquots can be found in Schmidt (2001).

Figure 2.22.3 Morphology of mouse ES cell colonies. Undifferentiated and healthy mouse ES colonies grow on a mitotically inactivated MEF feeder monolayer. Mouse ES cells grow as tightly clustered colonies with smooth phase bright borders. Mouse ES cells proliferate very quickly and require daily monitoring. The image in A represents classical morphology of the mES cell line (image taken with 20× objective). The image in B illustrates an example of an established ES cell line ready for subculture (image taken with 10× objective).

Figure 2.22.4 Mouse ES cells with green fluorescence marker. This particular line of ES cells express green fluorescent protein under control of a chicken beta-actin promoter, which allows differentiation between the fluorescent ES colonies and the nonfluorescent MEF cells. GFP-positive mouse ES cells are useful for tracking or imaging the fate and engraftment of the transplanted ES cell derivatives in vivo. (Image taken with 20× objective.)

 

Basic Protocol 2: In Vitro Pluripotency Characterization

A critical step in ES cell culture and maintenance is characterization of cells to ensure maintenance of pluripotency. The methods outlined in Basic Protocol 2 are well-established in vitro screens that may be used to verify pluripotency. These methods are less time-consuming than in vivo functional assays (i.e., teratoma formation; Basic Protocol 3), and are therefore ideal screening assays; however, the importance of validating ES cells by assessing their competence in in vivo teratoma formation must be stressed. The in vitro assays detailed below (alkaline phosphatase staining, immunofluorescent staining, and FACS analysis) are presented together, although each assay may be performed independently of the others. For thoroughness, it is strongly recommended that ES cells be tested against as many established parameters as is practical.

 Materials

• ES cells
• Phosphate buffered saline (PBS; Invitrogen)
• 4% paraformaldehyde (PFA; Sigma)
• 0.05% (w/w) Tween-20 (Sigma or Bio-Rad) in PBS
• Alkaline phosphatase (AP) staining kit (Chemicon)
• 3% BSA (Sigma) and 0.1% Triton X-100 (Sigma) in PBS
• Primary antibodies:
  • Oct4 antibody (Santa Cruz)
  • Sox2 antibody (Chemicon)
  • Nanog antibody (Abcam)
• Secondary antibodies conjugated with desired fluorochromes (Molecular Probe)
• Vectashield mounting medium with DAPI (Vector Laboratories)
• RPMI medium (Invitrogen) containing 0.5% FBS, ice cold
• Anti-SSEA1 antibody (Hybridoma bank at the University of Iowa)
• PE-conjugated rat anti-mouse IgM (BD Biosciences)

• 35-mm tissue culture dishes or 6-well plates (Corning/Fisher)
• Fluorescent microscope with digital camera
• Coverslips
• FACSCalibur analyzer or FACSAria cell sorter (BD Biosciences)

 Stain with alkaline phosphatase

 1a.

Culture ES cells on 35-mm tissue culture plates or 6-well plates until ~70% confluent.

 2a.

Aspirate the medium and rinse once with PBS. Add 2 ml of 4% PFA in PBS to each well to fix ES cells for 1 to 2 min at room temperature.

 3a.

Remove PFA and wash cells three times with 2 ml of 0.05% Tween-20 in PBS at 1-min intervals.

 4a.

Prepare staining solution as follows: mix fast red violet with naphthol AS-BI phosphate solution from AP staining kit and water in a 2:1:1 ratio.

 5a.

Add 2 ml staining solution to a 35-mm culture plate or one well of 6-well plate and incubate in the dark (cover with aluminum foil) 15 to 20 min at room temperature or until the red color has developed.

 6a.

Remove staining solution. Wash plates with 2 ml of 0.05% Tween-20 in PBS two times at 1-min intervals and fill the plate with 2 ml PBS. Observe under light microscope and take representative images.

 7a.

For more detail about the alkaline phosphatase staining protocol, see the AP kit manufacturer's data sheet.

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An image of positive AP staining can be found in Figure 2.22.5.

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True ES cells are AP positive (Pease et al., 1990).

Figure 2.22.5 Mouse ES cells with alkaline phosphatase staining. Pluripotent mouse ES cells express alkaline phosphatase enzyme in the cell membrane and the colonies are stained positively in red.

 Perform immunofluorescent staining

1b.	Culture ES cells on 35-mm tissue culture plates or 6-well plates.
2b.	Aspirate the medium and rinse once with PBS. Fix ES cells with 4% PFA in PBS for 10 min.
3b.	Wash cells three times with PBS (5 min/each wash). Block and permeabilize the cells in 3% BSA, 0.1% Triton X-100 in PBS (blocking solution) for 30 min.
4b.	Dilute primary antibodies (Oct4, Sox2, or Nanog) to working concentration in 3% BSA according to manufacturer's recommendations. Aspirate the blocking solution and add the diluted antibodies to each culture well (three different antibodies to three different wells). Incubate 1 hr at room temperature.
5b.	Remove the solution containing primary antibodies. Wash cells with PBS three times (5 min/each wash to wash away residual primary antibodies). Prepare each secondary antibody against Oct4, Sox2, or Nanog separately. Incubate cells with secondary antibodies conjugated with desired fluorochromes (e.g., FITC, Texas Red, Alexa 488 and 594, etc.) for 1 hr in the dark (cover with aluminum foil) at room temperature. Include a control sample, which is incubated with secondary antibody alone. p type = annotation The selection of secondary antibodies is based on the species in which the primary antibodies are derived. For example, if the primary antibody is raised in goat, the secondary antibody should be anti-goat.
6b.	Wash cells with PBS three times (5 min/each wash).
7b.	Aspirate PBS solution. Pipet 100 µl Vectashield mounting medium with DAPI solution over the center of the plate and place a coverslip over the mounting medium.
8b.	Keep in dark. Wait 5 min and observe the plates under a fluorescent microscope. Take representative images at different magnifications. p type = annotation True ES cells are positive for Oct4, Sox2, and Nanog expressions.

 Perform FACS analysis

1c.	Collect ES cells by trypsinization and resuspend in ice-cold RPMI containing 0.5% FBS. p type = annotation Although fixed cells can be used for FACS analysis, this protocol does not require the fixation step.
2c.	Collect the trypsinized single cell suspension in a 15-ml tube and aspirate the supernatant after centrifuging cells. Resuspend cells with 500 µl anti-SSEA1 antibody in hybridoma (no dilution) and incubate 30 min on ice.
3c.	Wash cells two times with RPMI containing 0.5% FBS and then incubate with PE-conjugated rat anti-mouse IgM for 30 min on ice. p type = annotation Since the anti-SSEA1 hybridoma is isolated from mice, the secondary antibodies must be anti-mouse.
4c.	Wash cells two times with RPMI containing 0.5% FBS, resuspend in RPMI containing 0.5% FBS, and analyze on a FACSCalibur analyzer or FACSAria cell sorter. p type = annotation True ES cells are SSEA-1 positive. A distinct population of PE-positive cells should be observed on the FACS plot.

 

Basic Protocol 3: In Vivo Teratoma Formation

The ability of ES cells to generate teratomas is considered to be a functional hallmark of pluripotency (Thomson et al., 1998). Teratomas contain all three germ layers, and their growth may be induced in immunodeficient mice (such as the NOD.CB17-Prkdc^scid/J line) by injection of ES cells into a variety of locations (Gertow et al., 2007). Growth of a teratoma followed by careful histological analysis will allow for confirmation of the differentiation potential, and thus, pluripotency of derived ES cells.

 Materials

• ES cells
• Bovine collagen (StemCell Technologies)
• Matrigel (Sigma)
• MEF medium (see recipe)
• NOD.CB17-Prkdc^scid/J male mice (3 to 5 weeks old; Jackson Laboratories)
• Isoflurane (Fisher)
• 4% paraformaldehyde (PFA; Sigma)
• 70% ethanol

• 100-mm tissue culture dish (Corning/Fisher)
• Hemacytometer
• 50-ml tubes (Falcon)
• 1-ml syringe (BD Biosciences)
• 23-G needle (BD Biosciences)

 Prepare ES cells for injection

1.	Trypsinize a semi-confluent to confluent 100-mm plate of ES cells cultured on MEFs. Count cell number using a hemacytometer.
2.	Resuspend 1 × 10^6 ES cells in 100 µl of a 1:1:2 mixture of bovine collagen, matrigel, and MEF medium and keep mixture on ice. Inject the cells within 1 hr.

 Inject ES cells

3.	Anesthetize SCID mice with isoflurane (nose-cone using a 50-ml tube). p type = annotation There are alternative ways to anesthetize mice—consult the animal facility staff for other methods.
4.	In a sterilized area, e.g., hood, inject ES cell suspension subcutaneously by the scruff near the base of the mouse head (the location as mouse is restrained by hand) with a 1-ml syringe and 23-G needle. Provide sterile food and water and sterile bedding for mice. p type = annotation Teratoma will appear as a firm mass in 4 to 6 weeks.

 Characterize teratoma

5.	Dissect teratoma by opening up mouse skin with scissors (the teratoma should appear as a well-defined tumor mass). Fix teratoma with 4% PFA overnight at 4°C. Following fixation, change the suspension solution to 70% ethanol and store at 4°C.
6.	Send out fixed teratoma for paraffin embedding, microtome sectioning, and histological staining to a service facility or others in the research center with prior experience. Alternatively, perform paraffin embedding, sectioning, and staining procedures according to Gertow et al. (2007).
7.	Consult with an experienced histologist or pathologist to correctly identify the structures within the teratoma. A list of examples of the histological markers to identify the three germ layers (ectoderm (karatinocyte or ganglion); mesoderm (cartilage); endoderm (gut)) may be found in Chou et al. (2008).

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Reagents and Solutions

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Use Milli-Q-purified water or equivalent in all recipes and protocol steps. For common stock solutions, see appendix 2A; for suppliers, see suppliers appendix.

 Gelatin solution in Milli-Q water, 0.2% (w/v)

To prepare a 100-ml solution in a suitable container, e.g., a volumetric flask or graduated cylinder, dispense 50 ml Milli-Q water, add 0.2 mg gelatin powder from porcine skin (Sigma), and mix. Bring up to a final volume of 100 ml with Milli-Q water.

Warm up the mixture in a 37°C water bath to ensure complete dissolution before filtering through a 0.22-µm sterile filter. Keep for up to 4 weeks at 4°C.

 Human chorionic gonadotropin (hCG)

Dissolve hCG (Sigma) in PBS and place on ice; prepare fresh and keep the unused portion at –20°C.

 Mouse embryonic fibroblast (MEF) culture medium

• To prepare 500 ml, add:
• 435 ml Dulbecco's modified Eagle's medium (DMEM; Invitrogen)
• 50 ml heat-inactivated fetal bovine serum (FBS; Invitrogen)
• 5 ml 200 mM glutamine, 100× concentrated stock (Gibco/Invitrogen)
• 5 ml 10 mM nonessential amino acids, 100× concentrated stock (Gibco/Invitrogen)
• 5 ml penicillin-streptomycin antibiotics, 100× concentrated stock (Invitrogen)

Filter sterilize using a 0.22-µm filter and store 2 to 3 weeks at 4°C

 Mouse embryonic stem (mES) cell culture medium

• To prepare 500 ml, add:
• 410 ml Dulbecco's modified Eagle's medium (DMEM; Invitrogen)
• 75 ml Heat inactivated fetal bovine serum (FBS; Invitrogen)
• 4 µl 14.3 M -mercaptoethanol (ME); tissue culture grade (Sigma)
• 5 ml 200 mM glutamine, 100× concentrated stock (Gibco/Invitrogen)
• 5 ml 10 mM nonessential amino acids, 100× concentrated stock (Gibco/Invitrogen)
• 5 ml penicillin/streptomycin antibiotics, 100× concentrated stock (Invitrogen)
• 50 µl ESGRO/LIF (10⁷ U) (Chemicon/Millipore)

Filter sterilize using a 0.22-µm filter and store for 2 weeks at 4°C

 Pregnant mare serum gonadotropin (PMSG)

Dissolve PMSG (Calbiochem) in PBS and place on ice; prepare fresh and keep the unused portion at –20°C.

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Commentary

 

Background Information

Following fertilization, the totipotent zygote undergoes rapid cleavage divisions to form a preimplantation blastocyst embryo, a hollow sphere consisting of two morphologically and functionally distinct cell types. An outer layer of trophectoderm cells encloses a small group of pluripotent cells known as the inner cell mass (ICM), from which the embryo proper will develop. At implantation, the ICM consists of pluripotent cells that give rise to all embryonic germ layers. It was the pioneering work of Martin and Evans that demonstrated that cells in the ICM can be propagated indefinitely in vitro in a stable pluripotent state as embryonic stem (ES) cells, while maintaining the ability to generate all tissues of the adult body (Martin and Evans, 1975; Evans and Kaufman, 1981; Martin, 1981).

ES cells are pluripotent cells that can differentiate in vitro into neurons (Brüstle et al., 1999), hematopoietic cells (Choi et al., 1998), cardiomyocytes (Klug et al., 1996), and other cell types (Turksen, 2006b). The ability to induce and control differentiation of ES cells towards specific lineages provides powerful tools for investigation and development of treatments for diseases. In vitro cellular assays using ES cell derivatives have simplified discovery and toxicology studies by allowing for dismissal of drugs prior to more costly and time-consuming small animal studies (Gorba and Allsopp, 2003). ES cell tests (EST) can also serve as standards for the embryotoxicity test and represent a scientifically validated in vitro system for the classification of chemical compounds (Scholz et al., 1999).

 

Troubleshooting

To maximize the number of fertilized oocytes recovered from a super ovulated female, it is critical to use stud males. Stud males must be placed in individual cages to avoid fighting and injury. Male mice reach sexual maturity at ~8 weeks of age and should be replaced at 8 to 10 months of age.

Another important point is to prepare fresh hormones (on ice) right before the injections and avoid re-freezing of the unused portion. Poor quality hormones will result in low yields of oocytes and hence lower numbers of healthy blastocysts.

If ICM outgrowth is observed, but no ES cell colonies are isolated, this is likely attributed to the inadequate dissociation of the ICM outgrowth. The solution is to treat cells with trypsin until ICM is completely dispersed into single cells before replating.

If differentiation is observed in early passage ES cells, the reasons could be low MEF density or that the trypsin used to passage cells was not adequately neutralized with serum-containing medium.

The immunofluorescent staining protocol provided in this unit assumes that researchers will record the images immediately after staining under an inverted fluorescent microscope, and not store the slides/culture dishes. If samples are stored for too long of a period, secondary fluorescent antibody will be bleached. Even though it is not necessary to have a positive control, it is recommended to include an established ES cell line for in vitro characterization.

In some rare cases, ES cells will demonstrate a fibroblast-like differentiated morphology even though the density of MEFs is ideal and the cultured ES cells are not over-confluent. The possible reasons for this problem could be differences in serum lots or the quality of the growth factor LIF. Technically, the tissue culture reagents should pass standard quality controls before shipping to the research institutions, but mistakes may be made in any step of the manufacturing process. Thus, it is always safer to have some frozen vials of ES cells in stock when culturing them in the new lot of serum or LIF.

 

Anticipated Results

Newly derived ES cell morphology should exhibit very tight colonies as shown in Figures 2.22.3 and 2.22.4, and must be characterized properly before further use. In addition to the in vitro and in vivo assays to evaluate pluripotency described in this unit, it is recommended that ES cells are karyotyped every 10 to 15 passages. The normal karyotype is 2n = 40 in mice. Once stable ES cell lines are established, they can be frozen and stored in liquid nitrogen. The freezing medium and procedure can be found in Schmidt (2001).

Proper culture of ES cells is the key step before using them for any gene targeting projects. Daily medium change is the routine along with subculturing the cells every 2 to 3 days. Never allow the medium to turn orange or become yellowish in color. Color change of the medium usually means the cells are overgrown and this will soon lead to cell differentiation and loss of pluripotency. Therefore, it is necessary monitor the cell culture daily under a microscope.

It is also a good idea to freeze some vials of low passage and healthy ES cells. The derivation and characterization process of ES cells is tedious and demanding. But once the standard procedure is established, the skills and training will be highly beneficial for researchers interested in the fields of stem cells or reproductive medicine research.

 

Time Considerations

The derivation process to establish a novel mES cell line is ~2.5 weeks. To grow enough early passages of cells for frozen vials and in vitro characterization would likely take an additional 1.5 weeks. It is recommended that the in vivo characterization for teratoma formation be performed after the verification of in vitro results since the NOD-SCID mice are quite expensive. Particularly when multiple new mES lines are established, it would be more cost effective to pick the top rated (homogeneous) lines for the teratoma assay. The teratoma assay is estimated to take an additional 4 to 6 weeks.

It is critical to plan the experiments ahead. Order reagents and make reservations for equipment, especially the fluorescent microscope and FACSCalibur. It is recommended to have the mice delivered 2 to 3 days before the hormone induction and teratoma assay. This allows time for mice to adjust to the new environment (dark/light cycle) in the animal facility. The procedures of superovulation and blastocyst embryo retrieval require someone who is familiar with procedures to handle mice, as well as with the anatomy of female mice. Additional guidance can usually be acquired from institutional animal facility training workshops. Most importantly, all animal work should be covered under appropriate animal protocols approved by an oversight committee such as IACUC.