Biology Reference
In-Depth Information
3.8. Analysis of Differentiation Abilities
of Homozygous ES Cells in Embryo Bodies and Teratomas
In addition to the study of chimeric tissue formation, the differentiation abili-
ties of ES cells can also be tested by in vitro differentiation assays and by the
induction of ES-derived teratomas. The extraordinary potential of ES cells to
differentiate in vitro can be utilized to study particular aspects of mutant phe-
notypes that cannot be easily approached in in vivo models. The case of
embryonic lethal phenotypes provide the typical situation in which the study of
the developmental abnormalities can be greatly extended with biochemical and
cellular studies on differentiated homozygous cells. Analysis of the differen-
tiation state at different time-points and comparison of mutant and wild-type
cultures may give essential clues on the effects of the mutation
(36)
.
Several protocols have been developed to differentiate cultured ES cells and
most of them are specialized to generate a particular cell type. Nonetheless, ES
cells can be aggregated in vitro to form so-called embryo bodies in which vari-
ous tissues with distinct embryonic origin start to form. These cell aggregates
can then be grown for several days and the formed tissues may be analyzed
with the classical tools of biochemistry and cell biology.
An alternative method to generate ES cells-derived differentiated tissues,
consists of injecting ES cells ectopically in syngeneic male mice (for example,
under the skin). In these conditions, ES cells can form a teratoma, a noninfil-
trating, benign tumors containing large numbers of highly differentiated cells
often organized in epithelia, glands, vessels, and even nerves.
3.8.1. Generation of ES Cells-Derived Embryoid Bodies
Grow ES cells with standard methods to obtain at least 10
6
cells.
1.
2.
Wash cells two times with PBS and incubate with trypsin for 5 min at 37
C. Add
ES medium and disperse aggregates by gently pipeting cells up and down. Add
ES medium if needed and let feeder cells attach to the culture dish for 30 min in
the incubator.
°
3.
Gently wash the plate to collect nonadherent ES cells and save the supernatant in
a sterile tube. Count the cells with a Burcker's chamber. Dilute cells so that
500-1000 are contained in 20
µ
L.
4.
Pipet on the inside the lid of a sterile 9-cm Petri dish several 20-
L drops of cells.
Fill the Petri dish with 5-10 mL of PBS. Gently turn the lid upside-down and
close the Petri dish so that cells are held in suspension in the hanging drops.
µ
5.
Wait for 2 d until aggregates of ES form. Collect aggregates (embryoid bodies)
by washing the lids with EF medium. Plate aggregates on bacterial culture Petri
dishes in EF medium (supplement of serum to 20% may be required).
6.
Embryoid bodies can be grown in suspension up to 30 d. Alternatively, they can
be trypsinized and cells can be plated in tissue-culture dishes.
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