Embryoid body (EB) formation and
detection of three lineage markers
While iPSCs must function as
pluripotent cells, they must also
demonstrate the ability to differentiate
into any cell type in the body. To
demonstrate the iPSCs can differentiate
into cell types of all three germ layers
in vitro, the expanded clones maintained
on MEF dishes were harvested
using collagenase IV and cultured in
suspension in EB medium (DMEM/F-12
supplemented with KSR without bFGF,
as bFGF is used to keep the cells
undifferentiated). EB medium contains
the same components as iPSC medium
but it does not contain bFGF, as bFGF is
used to keep the cells undifferentiated.
When the cells are in EB medium in
a suspended environment they begin
to form EBs, spheroid structures that
expand into cell types of the three germ
layer lineages: endoderm, medoderm,
and ectoderm. After 14 or 20 days,
EBs are then seeded on Geltrex®coated chamber slides for 7 days prior
to staining for markers of each of the
three lineages. The expression of the
markers α-fetoprotein (endoderm),
β-III tubulin (ectoderm), and smooth
muscle actin (mesoderm) confirm the
tri-lineage capabilities of the iPSCs. All
clones tested were able to differentiate
into cell types of three germ layers.
Representative images are shown in
Figure 6. We observed that varying
CULTURE
ENGINEERING
DIFFERENTIATION
CHARACTERIZATION
culture times can vary the degree of
differentiation into a specific lineage
between cell lines. When working
with different disease lines, we highly
recommend optimizing the duration of
EB suspension culture, the duration of
post-suspension adherent culture, or
both, to achieve differentiation into all
three germ layers.
A BC
D EF
19 Life Technologies | Parkinson's cell model
Figure 6. iPSCs can form embryoid bodies that can differentiate and
express markers from the 3 germ layers. iPSCs from PD-2 donor
(A, B, C) or Ctrl-2 (D, E, F) were cultured in EB medium in suspension
for 14 to 20 days. Cells were then cultured in Geltrex®-coated chamber
slides for 7 days. Cells were fixed, permeabilized, and stained for
α-fetoprotein (A and D, green), β-III tubulin (B and E, green), and smooth
muscle actin (C and F, green). Hoechst 33342 DNA staining is in blue.
Images were taken using the FLoid® Cell Imaging Station.
