Chapter 12
Manipulating the Avian Epiblast and Epiblast-Derived
Stem Cells
Cantas Alev, Mikiharu Nakano, Yuping Wu, Hiroyuki Horiuchi,
and Guojun Sheng
Abstract
Compared to eutherian mammals, birds retain a primitive form of epiblast development. Molecular studiesof the avian epiblast can provide valuable insight for mammalian epiblast research. Here, we introduce
several basic techniques in handling epiblast-stage embryos of the chick, the major model organism foravian developmental biology studies. We describe how to collect embryos for RNA extraction and gene
expression analysis, to set up ex ovo New culture for overexpression, bead graft and small molecule-based
inhibitor studies, and to carry out whole-mount RNA in situ hybridization analysis. We introduce a noveland simple method for molecular perturbation of the epiblast differentiation in ovo. We also describe how
to perform primary chicken epiblast cell culture, to establish stable epiblast stem cell (Epi-SC) lines, andto assay for pluripotency in primary epiblast cells and Epi-SCs.
Key words Avian, Chicken, Epiblast, Stem cells, Pluripotency, New culture, Bead graft, In situ
hybridization, Chicken ES cells, Epiblast stem cells

1

Introduction
Epiblast is a term used to describe the primitive ectoderm inamniotes (mammals, birds, and reptiles). In birds, it includes cells
in both the area pellucida (equivalent to the mammalian epiblast)
and the area opaca (similar to mammalian mural trophectoderm).
Mammalian polar trophectoderm does not have an avian counterpart. Here, we will use the chick as the avian model and only concern ourselves with the area pellucida epiblast. We will refer to thispart as the epiblast. It should be kept in mind that in addition to
the three germ layers in the embryo proper, the epiblast in boththe mammals and birds contributes to the extraembryonic mesoderm in its entirety and to the extraembryonic ectoderm partially.
The first day of chicken development takes place intrauterally.
A fertilized and freshly laid hen’s egg contains approximately

Ramiro Alberio (ed.), Epiblast Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1074,
DOI 10.1007/978-1-62703-628-3_12, © Springer Science+Business Media New York 2013

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40,000 cells and has reached the Eyal-Giladi and Kochav (EGK)stage X [1]. Intrauteral development (EGK I-X) and post-laying
pre-streak development (EGK XI-XIV) are traditionally staged
according to the EGK criteria [1] and correspond collectively toStage 1 of the Hamburger and Hamilton (HH) system [2]. Afterthe appearance of the primitive streak at HH2 (about 6–8 h ofincubation post-laying), chicken development is staged according
to the HH criteria [2]. Although the later half of the intrauteraldevelopment is relevant to issues concerning epiblast formationand pluripotency, technical limitations in obtaining these embryos
preclude their routine use in labs without access to a poultry farm.
In this chapter, we mainly discuss about how to handle chicken
embryos during the first 18 h of its post-laying development (fromstage EGK-X up to stage HH5). Most pluripotency genes aremarkedly down-regulated in the epiblast by HH5 [3, 4]. This
period of chicken development corresponds roughly to E5.5–E7.5
of mouse development.

2

Materials

2.1 Manipulation
and Analysis of
Epiblast-Stage
Chicken Embryos

1. Pannett-Compton Solution: H2O, 40 ml of solution-A, 60 mlof solution-B in 1 l final volume. Pannett-Compton Solution-A:
H2O, 121 g NaCl, 15.5 g KCl, 10.42 g CaCl2–2H2O, and12.7 g MgCl2–6H2O in 1 l final volume. Autoclave. PannettCompton Solution-B: H2O, 1.88 g Na2HPO4, and 0.188 g
NaH2PO4–2H2O in 1 l final volume. Autoclave.
2. 20× PBS: H2O, 175.3 g NaCl, 22.7 g Na2HPO4, and 4.7 g
NaH2PO4–H2O in 1 l final volume. Autoclave. For 1× PBT: 1×
PBS with 0.1 % Tween-20.
3. 4 % PFA: Add 4 % w/v of paraformaldehyde powder with
continuous stirring to preheated (65 °C) 1× PBS. Adjust pH
to 7.5 with 1 N NaOH. Cool to RT and add EGTA to final
concentration of 2 mM. Alternatively, premade 4 %Paraformaldehyde Phosphate Buffer Solution can be purchased from commercial sources.
4. Postfix: 4 % formaldehyde and 0.1 % glutaraldehyde in 1× PBT.
5. Pre-hybridization solution: H2O, 50 % formamide, 1.3× SSC,
5 mM EDTA, 50 μg/ml yeast RNA, 0.002 % Tween-20,
0.005 % CHAPS, and 100 μg/ml Heparin. All glassware andtools in making this solution should be baked. Aliquot into
50 ml tubes. Store at −20 °C.
6. 20× SSC: H2O, 175.3 g NaCl, 88.2 g Na3Citrate–2H2O in 1 l
final volume. Adjust pH to 5.3 with citric acid.
7. 10× TBST: H2O, 8 g NaCl, 0.2 g KCl, 25 ml of 1 M Tris–HCl
pH 7.5, and 11 g Tween-20 in 100 ml final volume.

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8. Color reaction solution: NTMT (1 ml) + NBT (2.3 μl) + BCIP
(2.3 μl). Scale up accordingly. NBT: 4-nitro-blue tetrazolium
chloride solution (Roche). BCIP: 5-bromo-4-chloro-3indolyl-phosphate-toluidine salt solution (Roche). NTMT:
H2O, 1 ml of 5 M NaCl, 2.5 ml of 2 M Tris–HCl (pH 9.5),
1.25 ml of 2 M MgCl2, 5 ml of 10 % Tween-20 in 50 ml final
volume. Make fresh each time.
9. Affigel beads: Affi-Gel Blue beads (BioRad). AG1 ion exchange
beads: AG1-X2 ion exchange beads (BioRad). Heparin-coatedbeads: Heparin immobilized on acrylic beads (Sigma).
10. Antibody blocking solution: 5 % heat-inactivated goat serum(30 min at 55 °C) and 1 mg/ml BSA in TBST.
11. pGEM-T Vector: pGEM-T Easy Vector System I (Promega).
12. Glass ring: Outer diameter 26 mm and inner diameter 23 mm.
Custom made by local company (URIN, Kyoto, Japan).
2.2 Manipulation
and Analysisof Epiblast Cells
in Culture

1. Epiblast cell culture medium (ECC medium): KnockOutDMEM (Invitrogen) supplemented with 20 % knockout serum
replacement (KSR, Invitrogen), 2 % chicken serum, 1 mM
sodium pyruvate, 1× nonessential amino acids, 2 mM Lglutamine, 1× nucleoside solution, 0.1 mM β-mercaptoethanol,
20 ng/ml recombinant chicken leukemia inhibitory factor.
2. 0.1 % gelatin solution: H2O, 1 g gelatin in 1,000 ml final volume, followed by sterile filtration.
3. Ham-F12: F12 Nutrient Mixture.
4. Lipofectamine 2000 reagent: Lipofectamine™
Transfection Reagent (Invitrogen).

2000

5. Mitomycin C solution: 1× PBS with 2 mg/ml mitomycin C.
6. 100× nucleoside solution: H2O, 80 mg adenosine, 73 mg cytidine, 85 mg guanosine, 24 mg thymidine, and 73 mg uridine
in 100 ml final volume, sterilized by passing through a 0.22 μm
disposable filter.
7. Stem cell passaging tool: StemPro EZPassage (Invitrogen).
8. SuperScript™ III: SuperScript™ III First-strand Synthesis
System (Invitrogen).
9. TOP10F E. coli strain: One Shot® TOP10 ChemicallyCompetent E. coli (Invitrogen).
10. Zeocin: Zeocin™ Selection Reagent (Invitrogen).
11. ProBond Resin: ProBond™ Purification System with Antibody(Invitrogen).
12. pSecTag2A Vector: pSecTag2 Expression and Purification kit(Invitrogen).
13. Anti-chicken Nanog polyclonal rabbit antibody: supplied by
Hiroshima University [5].

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Methods

3.1 Obtaining
Epiblast-Stage
Chicken Embryos

Basic embryological precautions should be taken in staging andhanding early-stage chicken embryos (see Note 1).
1. Prepare spoon, scissors, and coarse and fine forceps (Fig. 1a).
Regular wash (soap, tap water, distilled water, and 70 % ethanol) of these tools is sufficient for most experiments.
2. Eggs are flipped upside down from their storage/incubation
position. This way the yolk is positioned lower inside the egg.
An incision is made with the tip of coarse forceps (Fig. 1b, c).
3. Top part of the egg shell is opened and removed (Fig. 1c, d)and most of the albumen removed (or collected in a small beaker if needed) (Fig. 1d, e).
4. Position the embryo at the center by gently moving the yolk
with a spoon or the back of forceps. Do not touch the embryo
directly. Use scissors to make 3–4 quick cuts around theembryo (Fig. 1e, f).
5. Scoop up the embryo, together with the vitelline membrane
above and some yolk below, with a spoon. Dip the embryo
into PBS or Pannett-Compton solution (Fig. 1g).
6. Remove the vitelline membrane (with embryo) from the yolk
with fine forceps (Fig. 1h). Move slowly over the embryo area.
7. In most cases after more than a few hours of incubation, theembryo stays attached to the vitelline membrane (Fig. 1h). In
cases when the embryo stays attached to the yolk, use the bluntend of a Pasteur pipette to suck up and transfer the embryo(Fig. 1i).
8. Use coarse forceps to hold down the vitelline membrane andfine forceps (with tips closed) to gently dislodge the embryo
from the vitelline membrane by scraping the surface of thevitelline membrane towards to the embryo (Fig. 1j). This way
the edges of the embryo are dislodged from the vitelline membrane, while most regions of the embryo are not touched by
the forceps (Fig. 1k).
9. Once freed from the vitelline membrane (Fig. 1l), the embryo
can be transported using the blunt end of a Pasteur pipettefrom dish to dish with minimal damage, so long as it does notcome into the liquid/air interface. Excessive yolk can beremoved with forceps or gentle blows using a Pasteur or Gilson
pipette.
10. If needed (e.g., for RT-PCR or large-scale transcriptomic analysis), the epiblast region can be cut out from the embryo(Fig. 1m). In most cases, embryos will be fixed for later use.
Fix them flat. This is very important. The epiblast is slightly

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Fig. 1 Obtaining epiblast stage chick embryos. Unincubated eggs are opened (a–d) and embryos (red arrow )cut out and separated from the yolk as shown (e-h). Embryos are carefully detached from the vitelline membrane (i–l). The epiblast region can be cut out from freshly isolated embryos (m) or embryos can be fixed andused for subsequent analysis (n, o)156Cantas Alev et al.

stretched in ovo, so it shrinks a bit in solution and can also foldup very easily. Flatten each embryo (it does not matter whichside is facing up) with fine forceps, and further stretch it flat byremoving all Pannett-Compton (or PBS) liquid around theembryo. Using a Pasteur pipette, gently add 1–2 drops of fixative (4 % PFA) on top (Fig. 1n). After 1–2 min, add a few
drops. After 5–10 min of fixation, embryos can be handledquite roughly. With the blunt end of a Pasteur pipette, transfer
embryos to a glass vial, fix for a couple of hours at RT or overnight at 4 °C (Fig. 1o).
3.2 Setting Up the
New Culture

The New culture technique takes some practice to master. But it is
the best ex ovo culture technique available for epiblast-stage
chicken embryos and its versatility (see Note 2) justifies the initialsteep learning curve.
1. Prepare glass tray, glass rings, watch glasses, forceps, and scissors (Fig. 2a). Regular wash of these tools (as in Subheading 3.1)
is sufficient, but avoid using too much detergent and rinse withtap water thoroughly.
2. Pour 1–1.5 l of Pannett-Compton solution into a glass tray.
Open eggs, collect thin albumen, remove as much thick albumen as possible without damaging the yolk. Pour intact yolk
gently into the solution. Prepare a few yolks this way (up to 20
if you are experienced) and put several watch glasses inside thetray (Fig. 2b).
3. Use the back end of forceps to rotate the yolk so that theembryo is positioned on the top. Starting from one side of theyolk equator (Fig. 2c, e), cut all around the yolk with scissors(Fig. 2d, f).
4. Peel off the vitelline membrane with forceps gently and steadily(Fig. 2g). If the egg has been incubated for several hours, theembryo should stay attached to the vitelline membrane(Fig. 2f). If unincubated, the embryo tends to be detachedfrom the vitelline membrane. In such cases, collect the embryo
from the yolk using the blunt end of a Pasteur pipette, wash
excessive yolk off the embryo and put the embryo back on to
the vitelline membrane after the latter has been assembled. In
the meantime, continue with the assembly of the vitelline
membrane.
5. Move the vitelline membrane over a watch glass (yolky side up)(Fig. 2h) and position a glass ring on the membrane (Fig. 2i).
6. Use forceps to grab the membrane gently around the ring sothat there is membrane margin all around the ring (Fig. 2j).
Move the watch glass (with the embryo and the ring) out of
the tray and on to the bench.

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Fig. 2 Setting up New Culture. Egg yolks are collected in a glass tray filled with Pannett-Compton solution(a–c). Embryos attached to the vitelline membrane are obtained by generous circumferential cutting and careful dislodgement of the membrane plus embryos from the underlying yolk (d–h). The vitelline membrane (yolkside up) is carefully wrapped around and attached to a glass ring as shown (i–m). The assembled embryo isplaced onto a 35 mm dish containing albumen (o–r). Albumen can be mixed with, e.g., small molecule-based
inhibitors following successive dilution steps (n). Representative images of control and TGF-beta inhibitor(25 μM SB431542)-treated embryos reveal clear reduction in the expression of the pluripotency marker Nanog
in the central epiblast upon New culture treatment in the presence of TGF-beta inhibition (s, t)

7. Reposition the ring as it may have shifted its position during
the transfer. Use a Pasteur pipette to remove a bit of solution
so that the top of the ring is exposed to the air. Use coarse
forceps first, and then Pasteur pipettes (flame-polished toremove sharp edges) to grab the outer edges of the vitelline
membrane over the ring (Fig. 2k). Because the top of the ringis exposed to the air, the membrane will (after 10–30 min) be
attached to the ring after drying up. During this time window,

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the membrane can be adjusted. An ideal outcome is that thevitelline membrane is taut under the ring and wraps around the
top surface of the ring (Fig. 2l).
8. Cut off excessive vitelline membrane inside the ring (most lateral bits of the membrane) with scissors. Large pieces of yolkon the vitelline membrane inside the ring are removed with a
Pasteur pipette (Fig. 2l, m). Always leave some liquid to cover
the embryo. The assembly process may be stopped here for up
to a couple of hours before moving on to the next step. Also at
this step, the embryo can be removed for electroporation,
detached embryos can be put back, and for bead graft, a few
beads can be put onto the embryo.
9. Aliquot 2–3 ml of thin albumen in each 35 mm dish. For whole
embryo treatment with small molecules, the chemical stock
solution (or control solution), often dissolved in DMSO, isdiluted in PBS first, followed by further dilution in the thinalbumen (Fig. 2n). The volume of albumen in each dish can be
reduced to 0.5–1 ml if cost of the chemical is a concern (e.g.,
SU5402).
10. Remove all Pannett-Compton solution from both inside thering and on the watch glass outside the ring (Fig. 2o).
11. Move the ring (with the embryo) using coarse forceps fromthe watch glass to the 35 mm dish with albumen (Fig. 2p, q).
12. The vitelline membrane under the ring should bulge slightly
upward. The embryo should be in the middle, at the highest
point (Fig. 2r). The ring should naturally touch the bottom(remove some albumen if not) of the dish instead of floatingon the albumen. Remove Pannett-Compton solution that has
drained to the lowest point inside the ring. No albumen should
leak inside the ring. Discard if so.
13. Put the lid on and put the petri dish inside a box moisturizedwith a wet towel. Incubate in a normal incubator (no need tohave CO2 or humidity control).
14. After a desired period of incubation, embryos are fixed andprocessed for further analysis. For example (Fig. 2s, t), embryos
treated with a TGF-beta inhibitor from early-streak stage
showed a reduction of Nanog expression in the central epiblast(Fig. 2t), whereas control-treated embryos exhibited normal
expression (Fig. 2s).
3.3 Bead Graft andSubgerminal Cavity
Injection

Epiblast-stage embryos can be experimentally manipulated using a
number of techniques (see Note 3). Here we describe two of them,
the bead graft technique and the subgerminal cavity injection
technique.

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3.3.1

Bead Graft

159

1. Embryos are prepared using the New culture method (Fig. 3a).
2. Prepare beads to be grafted. Beads come in different sizes. Pickthe right bead size and pore size (for AG1 beads) suitable foryour experiments. Bead preparation takes time, so carry out
this step before or during New culture preparation. Aliquot
10–100 μl of beads to an Eppendorf tube. AG1 resins come as
dry powdery beads, so aliquot an equivalent volume. These
beads are relatively inexpensive and one bottle can last for

Fig. 3 Bead graft and subgerminal cavity injection. Embryos are prepared using the New culture setting.
Growth factor-soaked beads are carefully placed on top of the embryo (b, b′) and positioned to the desired
area using a sharp needle or metal tips (c, d, d′). Two types of beads can be combined (e, e′) with an example
for such an experiment shown; induction of Brachyury by FGF can be inhibited when SU5402-soaked beads
are placed in close proximity to FGF-loaded beads (f, f′). Unincubated eggs (embryo on top) are placed into
prepared foster egg shells (g, h). Growth factor and/or small molecule solutions are injected into the subgerminal cavity using a fine pulled capillary and mouth pipetting (i, j). The injected egg is filled with albumen andsealed using plastic wrap, custom made rings, and rubber bands (k, l). Injected embryos are incubated horizontally and under rotation (90° every 90′). Examples of injected embryos are shown, with normal distribution
of VASA positive PGCs in control-injected embryos (m), and abnormal number and location of PGCs after BMP
injection (n)160Cantas Alev et al.

many years. If the protein/small molecule to be loaded is dissolved in water-based solution as stock, wash the beads with
PBS several times (each time a few minutes followed by shortspins). If the stock is dissolved in DMSO, use DMSO to wash.
3. The stock solutions are normally kept as 5–10 μl aliquots, so
only use equal or less volume of beads for loading. Mix beads
and stock solution. Leave either on ice (for proteins) or RT(for small molecules) for at least a couple of hours.
4. Before use, wash beads 3× in PBS or Pannett-Compton solution. We wash by taking a couple of microliters out from the
Eppendorf tube, and transferring successively into fresh wash
solution three times.
5. Several (1–5) beads or bead combinations can be grafted on toeach embryo. Beads can be added to the New culture after
final assembly (Fig. 3b, b′) or at the step shown in Fig. 2m.
Use fine forceps, a P20 pipette or any fine tips to add beads.
Some beads may be sticky, add these beads as close to targeted
locations as possible.
6. Use a 1 ml syringe needle or other fine metal tips to movebeads to targeted areas (Fig. 3c, d, d′).
7. If a combination of beads is required, add a second type ofbeads (Fig. 3e, e′).
8. When all beads are in their targeted locations, remove excessive liquid from over the embryo and inside the ring using acapillary and mouth pipetting. Press beads down gently tomake sure all beads are in contact with the embryo, and not
floating on top of it.
9. Incubate. After intended period of incubation, make surebeads are still in place. If not, discard.
10. Fix the embryo and analyze the effect of the bead graft. An
example is shown in Fig. 3f, in which three FGF-loaded beads
induce Brachyury gene expression, and this induction is inhibited when both FGF- and SU5402-loaded beads are grafted.
3.3.2 Subgerminal Cavity
Injection

1. Prepare foster egg shells (top part evenly cut with a circularsaw) and large plastic rings as shown in Fig. 3g.
2. Transfer egg content from an unincubated egg to the foster
egg shell (Fig. 3h).
3. Reposition the yolk with a spoon so that the embryo is on the
top.
4. Use a fine pulled capillary and mouth pipetting, take up 5–10 μlof solution to be injected (growth factors or small molecules
diluted in PBS or Pannett-Compton Solution, with a bit of fastgreen dye for visualization).

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5. Inject 1–1.5 μl of solution per embryo into the subgerminal
cavity (Fig. 3i, j). Do not inject above the epiblast or into theyolk. This is easy to achieve with only a little practice. The capillary opening should be large enough so that 1–1.5 μl can beinjected in 2–3 s, but small enough so that no injected content
leaks out afterwards.
6. Fill the injected egg with albumen (use a 10 ml syringe) to thevery top. Cover the egg with a small piece of plastic wrap.
Leave no trapped air bubbles. Cover the wrap with anotherplastic ring (Fig. 3k).
7. Tie together the top and bottom rings with rubber bands(Fig. 3k, l).
8. Incubate horizontally with 45–90° rotation every 90′.
9. After incubation, pour out the content, obtain the embryo as
shown in Fig. 1 and analyze the effect of injected materials.
10. An example is shown in Fig. 3m, n. Embryo with control injection shows normal PGC specification (Vasa staining), whereas
abnormal location and number of PGCs are seen in BMPinjected embryo.
3.4 RNA In Situ
Hybridization

Whole-mount RNA in situ hybridization is the most basic technique in gene expression analysis. To achieve good results, follow
basic precautions for RNA in situ analysis (see Note 4).
1. Make DIG or Fluorescein-labeled probes. This is an important
step, but is the same for any type of RNA in situ analysis. So it
is only briefly explained here. First, clone the region you intend
to make the antisense probe of. We routinely use PCR method
and pGEM-T-Easy as the host vector. Second, confirm thesequence and check the orientation by sequencing. Keep eachconstruct as a stock. Third, either linearize the construct bydigestion for antisense-directed transcription, or PCR theinsert out using M13F/R, purify the DNA. Fourth, using the
DIG or Fluorescein labeling kit, transcribe the antisense probe.
Fifth, purify the probe. Keep it as a concentrated stock in a few
milliliters of hybridization solution. Sixth, test each probe
before experimental use.
2. Collect embryos as shown in Subheading 3.1. Fix them flat.
3. Remove the fixative completely and add 100 % methanol (withone rinse). Embryos can be stored this way for several weeks at−20 °C. Strong and clean probes may work with embryos kept
this way for a couple of years.
4. Remove methanol, rehydrate in PBT by rinsing a couple oftimes and washing (nutating) for 30 min (Fig. 4a). Do notworry about yolk materials attached to the embryo. They will

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Fig. 4 RNA in situ hybridization. PFA and methanol fixed embryos (a–c) are pre-hybridized at 68 °C for several
hours following Proteinase K treatment, post-fixation, and multiple PBT washes. Removal and addition of liquids
should always be done from the side of the vial (d–f ). Following pre-hybridization diluted DIG and/or Fluoresceinlabeled probes are added to vials and incubated over night using a water bath at 68 °C (g). After multiple washing steps prior and post-incubation with an anti-DIG and/or anti-Fluorescein antibody solution, color development
is initiated using a NTMT + BCIP + NBT solution (h, i). Following several hours up to several days of color development at RT or 4 °C, embryos are washed with TBST and used for further analysis. Representative in situ
hybridization results of the pluripotency associated genes Nanog ( j), Dnmt3B (k), and Lin28 (l)come off during washes in subsequent steps (Fig. 4b) andembryos will eventually be free of yolk (Fig. 4c). When changing solutions in a vial, remove liquid from the side (do nottouch or aspirate the embryos) and add fresh liquid to the side
(do not drop directly onto the embryos) (Fig. 4d, e, f).

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5. Digest the embryos with Proteinase K (Sigma) (10 μg/ml) for
15–30 min at RT. Leave the vials standing up, do not nutate.
During and after this treatment, embryos are very fragile, butshould not disintegrate (if so, adjust Proteinase K concentration or digestion time).
6. Remove Proteinase K very carefully, replace with postfix solution. Fix for 30 min to 1 h.
7. Remove postfix and wash with PBT (rinse 2× and wash 2×
10 min). Change into pre-hybridization solution.
8. Pre-hybridize in a water bath set at 68 °C (Fig. 4g) for at least
2 h. This step can be extended by several hours, or stopped bystoring embryos in pre-hybridization solution at −20 °C for up
to a couple of years.
9. Dilute labeled probes with pre-hybridization solution todesired dilutions (this should be tested out beforehand foreach probe and diluted probes can be reused for many months).
Diluted probe in pre-hybridization solution is called hybridization solution. Remove the pre-hybridization solution and add
warmed-up hybridization solution. Hybridize overnight.
10. Remove the hybridization solution (and reuse) and wash withwarmed-up pre-hybridization solution (3× rinses and 2×
30 min washes). Avoid contamination when working withmultiple probes.
11. Change into 1:1 mix of pre-hybridization solution and TBST
solution. Keep for another 30 min at 68 °C.
12. Wash in TBST (2× rinses and 3× 30 min washes) at RT.
13. Block in the blocking solution for 2 h at RT.
14. Incubate in the antibody solution (blocking solution + antiDIG or anti-Fluorescein antibody (1:5,000 dilution)) overnight at 4 °C.
15. Wash in TBST (3× rinses and 3× 60 min washes) at RT.
16. Wash in NTMT for 10 min. Proceed to color development(NTMT + NBT + BCIP solution, protect from light) (Fig. 4h, i).
17. Monitor the color development in the first hour at RT. Decideon how long to continue at RT or move to 4 °C. If there is no
signal in the first hour, the reaction can be left for several more
hours at RT. If there are weak signals in the first hour, monitor
closely every hour or so at RT or leave at 4 °C overnight.
A good probe for a highly expressed gene will reach ideal signal/background ratio in 1–2 h. But for most moderately andspecifically expressed genes, an ideal outcome is to have thebest signal/background ratio within from several hours toovernight. Sometimes color reaction may take a few days. It is
still good if there is no background or trapping signals.

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18. To stop the color reaction, wash with TBST a few times (overnight if staining is strong). Change into fix solution and store
at 4 °C for further analysis (photography, secondary staining,or sectioning).
19. Examples of in situ hybridization of pluripotency genes areshown in Fig. 4j (Nanog), Fig. 4k (Dnmt3B), and Fig. 4l(Lin28).
3.5 Primary Cultureof Dissociated Epiblast
Cells and Subcultureof Epi-SCs

Refer to Note 5 for a general description of avian epiblast-derived
ES cells. Here we show how to make recombinant chicken LIF,
prepare feeder layer, generate primary epiblast cell culture, andmaintain Epi-SCs.

3.5.1 Production of
Recombinant Chicken LIF

1. Total RNA is extracted from epiblast cells (see Subheading 3.1)
with Trizol reagent and is used for reverse transcription PCR(RT-PCR) with the SuperScript™ III. Design and synthesize
the appropriate oligonucleotide primers for amplification ofchicken LIF. For cloning into pSecTag2A vector, modify the
5′-end sequence of the primers: forward primer
5′-GCGCTAGCCATGAGGCTCATCCCC-3′ (italics, NheI
restriction site; underline, start codon); reverse primer
5′-GCGTCGACCGCGGGGCTGAGGTGAGG-3′(italics,
SalI restriction site).
2. Prepare the PCR mixture (50 μl final volume) in an amplification tube: 1.0 μl template DNA (approx. 100 ng), 5.0 μl
2.5 mM dNTPs, 5.0 μl 10× Ex Taq buffer, 1.0 μl forward
primer (10 pmol/μl), 1.0 μl reverse primer (10 pmol/μl),
0.5 μl Takara Ex Taq DNA polymerase (5 U/μl), and 36.5 μl
H2O. Using a thermal cycler, heat the samples to 94 °C for
5 min and then run 30 amplification cycles in the linear rangeof 30 s at 94 °C (denaturation), 30 s at 60 °C (annealing), and1 min at 72 °C (polymerization). Finally, hold for 10 min at
72 °C as an extension step and then store at 4 °C.
3. Digest both pSecTag2A and PCR products with NheI and
SalI, and purify the DNAs using a commercially available kit.
The myc-epitope in pSecTag2A is excised from the vector
using restriction enzymes. Ligate the PCR products into thepSecTag2A vector using a commercially available kit. Transformthe recombinant vector to TOP10F E. coli strain and purify therecombinant vector using EndoFree Plasmid Maxi Kit.
4. Grow up CHO cells in Ham-F12 supplemented 10 % FBS or
CHCC-OU2 cells in DMEM supplemented with 10 % FBS in
a 6-well culture plate. The seeded cells are 90–95 % confluent
at the time of transfection. Transfect 2.0 μg/ml of the recombinant vector using Lipofectamine 2000 reagent.
5. After incubation for 24 h, remove the medium including therecombinant vector and add fresh medium supplemented with

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10 % FBS and 250 μg/ml of Zeocin. Feed the cells with theselective medium every 3 days until foci can be identified. Pickand expand 20–40 foci to obtain recombinant chicken LIF
producing cells. Check the producing cells by immunoblotting
analysis using the culture supernatant and anti-His HRP antibody (supplied with the kit) (Fig. 5a). Grow up the cells in
DMEM supplemented with 10 % FBS and collect the culture
supernatant of 500 ml.
6. Recombinant chicken LIF is expressed as a fusion protein
linked to a 6× Histidine tag and purified by affinity chromatography method using ProBond Resin. Add 10 ml of the washedresin to 500 ml of the culture supernatant, and aliquot thesupernatant in 50 ml tubes. Use gentle agitation such as endover-end rotation for 2 h at 4 °C. Pour the resin in a
2.5 cm × 10 cm column. Wash the column with 100 ml native
wash buffer (supplied with the kit). Repeat washing three moretimes.
7. Elute the protein with native elution buffer (supplied with thekit). Collect 10 ml fractions and analyze with SDS-PAGE(Fig. 5b). Pool the major fractions containing the recombinant
chicken LIF in a clean tube. Dialyze against cold PBS at 4 °C
overnight. If necessary, concentrate to about 100 μg/ml using
Amicon Ultra-15.
8. Aliquot the recombinant chicken LIF into 1.5 ml tubes, store
at −80 °C until use. Using this method, 0.5–2 mg of purified
recombinant chicken LIF can be obtained.
3.5.2 Preparationof Feeder Layer

1. Grow STO cells in DMEM supplemented with 10 % FBS until
confluent.
2. Remove medium and add fresh medium supplemented with
10 % FBS and 10 μg/ml of mitomycin C.
3. After incubation for 2 h, remove the medium, and wash theculture dishes in PBS (4× rinses) at RT.
4. Add PBS with 0.025 % trypsin and 1 mM EGTA, and incubate
for 5 min.
5. Resuspend the cells in a volume of DMEM supplemented with
10 % FBS and spin the cells in the DMEM for 5 min at 500 × g(3× rinses) at RT.
6. Plate the cells to gelatin-coated culture dishes at a concentration of 10,000–15,000 cells/cm2 and culture at 37 °C with
5 % CO2 for 24 h. The culture dishes are gelatinized with 0.1 %gelatin solution for a minimum of 30 min before use.
7. STO feeder layers should be prepared and used within 4 days.

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Fig. 5 Primary culture of dissociated epiblast cells and subculture of Epi-SCs. Recombinant chicken LIF in
the supernatant was analyzed using 12.5 % SDS-PAGE, blotted to blotting membrane, and reacted with antiHis HRP antibody. Lanes 1 and 2 were incubated with negative control antibody and anti-His HRP antibody,respectively (a). The fractions were analyzed using 15 % SDS-PAGE and the silver-staining method. MolecularChicken Epiblast and EpiSCs
3.5.3 Primary Cultureof Epiblast Cells

167

1. Warm ECC medium and KO-DMEM at 37 °C and keep themwarm until ready for use.
2. Pick up dissected epiblast tissues (see Subheading 3.1 andFig. 1m), and transfer them to a 1.5 ml siliconized tube.
3. Centrifuge at 120 × g for 3 min, and then remove thesupernatant.
4. Add 0.5 ml of ECC medium and gently suspend with amicropipette.
5. Remove the culture medium from STO feeder layer plated on
a 35 mm culture dish, wash the dish with KO-DMEM, andadd 2 ml of ECC medium.
6. Add 0.5 ml of the cell suspension to the culture dish and culture at 37 °C with 5 % CO2 and 3 % O2.
7. After 2 days add 0.5 ml of ECC medium. The cultured epiblast
cells form small and round colonies (Fig. 5c).
8. Change one-half of the total medium every 2 days. After 5–6
days, the colonies grow to the size of approximately 1 mm indiameter (Fig. 5d).

3.5.4 Subculture andMaintenance of Epi-SCs

1. After 5–6 days, the colonies of cultured epiblast cells are readyfor mechanical passaging by using a razor or a stem cell passaging tool. Do not cut up the colonies too finely at this step.
2. Warm ECC medium and KO-DMEM at 37 °C and keep themwarm until ready for use.
3. Aspirate the medium from the epiblast cell cultured dish. Wash
once with 2 ml of ECC medium. Replace with 1 ml of ECC
medium.
4. Cut the colonies using a razor or a stem cell passaging tool
under the stereomicroscope (Fig. 5e). Collect the segmented
colonies with a micropipette (Fig. 5f) and break up cell clumps
by gentle pipetting.
5. Seed these cells to new 35 mm dish(es) plated with feeder layercells (typically at a 1:1 or 1:2 passaging ratio).
6. After 1 day, small colonies start to form (Fig. 5g). After
3 days from passaging, both healthy (arrow) and unhealthy

Fig. 5 (continued) size markers: lane M. The molecular mass of recombinant chicken LIF is 20–35 kDa (b).
When dissociated epiblast cells are cultured with ECC medium, they form small and round colonies after 2days (c). After 6 days, the colonies grow to the size of over 1 mm in diameter (d). For subculture, colonies are
cut with a stem cell passaging tool (e). The segmented colonies are collected with a micropipette (f). After 1
day from passaging, small colonies start to form (g). After 3 days from passaging, both healthy (arrow) andunhealthy (arrowhead) large colonies start to form (h)168Cantas Alev et al.

(arrowhead) large colonies start to form (Fig. 5h). Remove theunhealthy colonies with a micropipette.
7. After 4–5 days from passaging, remove the unhealthy colonies
and subculture only the healthy colonies. Change half of thetotal medium every 2 days.
8. Repeat steps 3–7. After about 3 weeks of primary culture,these cells can grow stably and can be considered to be chicken
Epi-SCs.
3.6 Evaluationof Pluripotency
in Epi-SCs

Currently, tools for evaluating the pluripotency of cultured chicken
Epi-SCs are limited (see Note 6). This is due primarily to the lackof good antibodies for pluripotency markers and of suitable in vivo
differentiation assays. Here we describe two basic evaluation protocols: RT-PCR analysis for Oct4 and Nanog, and immunostaining analysis for Nanog.

3.6.1 mRNA Expression
Analysis of Oct4 and
Nanog

1. Using a micropipette, collect the Epi-SC colonies from a
60 mm culture dish into a 1.5 ml Eppendorf tube.
2. Wash the cells in RNase-free PBS by mild centrifugation.
Remove the supernatant carefully.
3. Total RNA is extracted from the Epi-SCs (Trizol method) andis used for reverse transcription PCR (RT-PCR) with
SuperScript™ III. The primers for expression analysis ofchicken Oct4, Nanog, and GAPDH (internal control) are
shown in Fig. 6a.
4. Prepare the PCR mixture (50 μl final volume): 1.0 μl template
DNA (approx. 100 ng), 5.0 μl 2.5 mM dNTPs, 5.0 μl 10× Ex
Taq buffer, 1.0 μl forward primer (10 pmol/μl), 1.0 μl reverse
primer (10 pmol/μl), 0.5 μl Takara Ex Taq DNA polymerase(5 U/μl), and 36.5 μl H2O.
5. Using a thermal cycler, heat the samples to 94 °C for 5 min
and then run optimal amplification cycles in the linear range of
30 s at 94 °C (denaturation), 30 s at optimal temperature(annealing, see Fig. 6a), and 1 min at 72 °C (polymerization).
Finally, hold for 10 min at 72 °C as an extension step and thenstore at 4 °C.
6. Analyze the amplification products on a 1.5–2 % agarose gel(Fig. 6b).

3.6.2 Immunostainingof Nanog

1. Gelatinize chamber slides with 0.1 % gelatin solution for 1 day.
2. Plate mitomycin C-treated STO cells (see Subheading 3.5.2) tothe slides at a concentration of 10,000–15,000 cells/cm2 andculture at 37 °C with 5 % CO2 for 24 h.
3. Aspirate the medium from the slides. Wash once with ECC
medium and replace with ECC medium.

Fig. 6 Evaluation of pluripotency in Epi-SCs. Primer information for expression analysis of Nanog and Oct4 is shown in (a). RNAs are isolated from Epi-SCs successively
subcultured for 30 days (#1 in b) and for 50 days (#2 in b). RNAs isolated from uncultured epiblast cells (positive control) and STO cells (negative control) are also
examined with the same primers. Chicken GAPDH gene is used as an internal control. In immuno-analysis, Nanog is seen to be expressed in the nucleus of most cellsof an Epi-SC colony (c–e)

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4. Seed the segmented colonies of Epi-SCs to the slides andculture at 37 °C with 5 % CO2 and 3 % O2. After 2 or 3 days,
aspirate the medium from the slides and wash with cold PBS(3× rinses).
5. Add 4 % PFA and incubate for 30 min at RT. Wash in 10 mM
glycine–PBS (3× rinses) and rewash in cold PBS (2× rinses).
6. Add 0.1 % Triton-X-100–PBS and incubate for 5 min at RT.
Wash in cold PBS (3× rinses).
7. Block in 3 % BSA–PBS for 15 min at RT. Aspirate the blocking
solution and add ×100 of anti-chicken Nanog polyclonal rabbit antibody diluted in 1 % BSA–PBS for 1 h at RT. Wash in
0.1 % BSA–PBS (6× rinses).
8. Add ×100 of Alexa Fluor 594-conjugated anti-rabbit IgG antibody and incubate for 1 h at RT. Wash in 0.1 % BSA–PBS (6×rinses).
9. Mount the slide with Vectashield mounting media. The sample
is examined using fluorescence microscopy (Fig. 6c–e).
3.7

General Remarks

4

Notes

The chick model is an essential complement to the mouse/humanmodels in epiblast biology. The mammals and reptiles (including
birds) are two main branches of the amniotic vertebrates, anddevelopment of primitive mammals has a lot in common with thatof the reptiles. Eutherian mammals (humans, mice, etc.) have
many derived features in their early development which are difficult
to understand without the knowledge of comparative amniote
embryology. Studies using the chick, the main nonmammalianmodel organism, can therefore offer essential insights in understanding the formation, differentiation, and pluripotency maintenance of the epiblast and epiblast stem cells.

1. Fertilized and freshly laid eggs purchased from poultry farms
can be stored at 16 °C for up to 1 week. Eggs are either warmed
up briefly (30′) for the unincubated stage or incubated at
38.5 °C for up to 18 h to reach desired stages. Depending on
the weather and flock conditions, developmental stage ofembryos from unincubated eggs varies, and this needs to betaken into consideration when designing an experiment.
Epiblast-stage embryos are fragile and easily damaged by metal
tools or at the liquid/air interface. Keep embryos submergedin liquid and do not grab them directly with forceps. A basic
understanding of how to stage and operate on early-streak
stage embryos is necessary [6].

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2. New culture was invented by Denis New [7] with several
modifications made afterwards [8]. It has been the most powerful ex ovo culture method for pre-streak and early-streak
stage chicken embryos. Using this culture method, young
embryos can be experimented upon using a variety of techniques (tissue graft, bead graft, DNA electroporation, timelapse imaging, and whole embryo treatment with small
molecules). Although it is tedious to set up and takes practice
to master, the New culture and its versatile applications areindispensible for avian epiblast-related studies.
3. To study a specific gene or pathway during early avian development, localized treatment is often preferred. The New culture method (Subheading 3.2) can be modified for such
purpose. Localized effects can be achieved through bead graft
or targeted DNA electroporation. For protein absorption,
Heparin-coated agarose/acrylic beads or Affigel blue beads
are commonly used. We have also successfully used hydrogel
to embed and release proteins locally. For absorption of small
molecules, AG1 ion exchange beads are used. A great varietyof small molecules (e.g., agonists or antagonists of kinases,
receptors, or membrane channels) are currently available. But
proteins are limited to secreted growth factors. They are often
expensive to acquire from commercial sources and in general
their efficacy in such experimental settings has not beentested. This limitation is circumvented by DNA electroporation. DNA constructs expressing any gene of interest can beelectroporated and their effect analyzed locally in cells expressing the gene of interest, either through a protein tag,
2A-peptide-mediated co-expression of a marker gene, or coelectroporation of a marker gene-encoding construct. The
drawback of electroporation, especially for early-stageembryos, is that there is a minimum of 2 h (for GFP) needed
to transcribe and translate introduced genes. Any effect froman electroporated gene may not be evident until at least 4–6 hculture after electroporation. In addition to the New culturebased methods, we have also tested a method for in ovo analysis of the epiblast-stage embryos. We call it the subgerminal
cavity method. The subgerminal cavity is the space between
the yolk cell membrane and the epiblast/hypoblast. Its lateral
margin is the germ wall where the yolk cell and deep layercells of the germ wall adhere to each other. In unincubatedeggs, epiblast cells are directly exposed to the subgerminal
cavity. Growth factors and/or small molecules can be injected
into this space, and their influence on epiblast cell differentiation can be analyzed after a short period of incubation. The
advantage of this method is that it is in ovo, easy to learn andquick to perform (compared to the New culture) and its effect172

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on epiblast or embryonic development is rapid (compared tothe electroporation method).
4. There are a few protocols for RNA in situ hybridization ofchicken embryos. The main differences are in the ingredientsfor the hybridization solution and the hybridization temperature. We follow the protocol of the Stern lab [9]. This protocol
has worked well for us. We have also used the same protocol
successfully for reptilian and other avian embryos. Pregastrulation and early gastrulation-stage embryos are fragile,
so throughout the entire in situ process (solution changes,
washing, etc.), care should be taken to avoid damage to theembryos. The “tissues” at these stages are only a few cell-layers
thick, so there is minimal worry of tissue trapping or trappingrelated background staining. Key to success is the quality of
DIG- or Fluorescein-labeled probes and the washes after
hybridization and antibody incubation. Routine precautions
for RNA-related work (baking glassware and metal tools andusing DEPC-treated water) should be taken when preparing
stock solutions.
5. Chicken epiblast cells derived from a stage X (EGK) embryo
have the capacity to contribute to all three germ layers andgerm cells when transplanted into a recipient embryo of thesame developmental stage [10, 11]. Thus, attempts have been
made to establish chicken embryonic stem (ES) cells by in vitro
culture of early epiblast cells [5, 12–14]. All of the established
ES cells are of the epiblast origin, and we describe them as
epiblast stem cells (Epi-SC). The pluripotency and growth ofcultured epiblast cells can be maintained by the addition ofchicken leukemia inhibitory factor (LIF) [15] or of conditioned medium from buffalo rat liver cells [13] to the culture
medium. We follow the protocol of epiblast culture using
chicken LIF. Recombinant chicken LIF protein [5, 16] isobtained from recombinant CHO-K1 cells (ATCC #CCL-61)
or CHCC-OU2 cells (supplied by the United States
Department of Agriculture [17]) transfected with the chicken
LIF gene. The epiblast cells or Epi-SCs are plated onto a layerof mitotically inactivated Sandoz inbred mouse-derived thioguanine-resistant and ouabain-resistant (STO) cell (ATCC#CRL-1503) feeders. Chicken epiblast stem cells in culture aresimilar in morphology to primate ES cells and consist of circular colonies with clear contour and each individual cell visible.
6. Mouse and human ES cells express a number of pluripotency
markers (Oct4, Nanog, Sox2, Rex1, Utf, etc.). In chickens,
however, only Oct4 and Nanog have been reported [3, 5].
These factors are required for the maintenance of pluripotency
and self-renewal of chicken ES cells [16]. Our unpublishedresults indicate that several other pluripotency markers, such as

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Dnmt3B and Lin28, can also serve as good markers for avian
Epi-SCs. The list will undoubtedly increase as more wholegenome analyses are being performed for early-stage chicken
embryos. Pluripotency of cultured chicken Epi-SCs can beevaluated by assaying the expression level of these genes using
RT-PCR or immunostaining. For RT-PCR analysis, the
GenBank accession numbers for chicken Oct4 and Nanog are
DQ867024 and DQ867025, respectively. For immunostaining, unfortunately, specific antibodies against these proteins
are not commercially available, and the commercially supplied
antibodies against their mammalian orthologues do not show
satisfactory cross-reactivity.
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