Biomedical Engineering Reference
In-Depth Information
Blastocyst
Totipotent
Morula
Oocyte
Human fetus
Sperm
Pluripotent
Inner mass cells
Examples:
Immune system
Circulatory system
Nervous system
Unipotent
FIGURE 7.17
Pluripotency.of.ES.cells..(Figure.contributed.by.Michael.D..Jones,.MSc.)
Without optimal culture conditions or genetic manipulation, ES stem cells diferentiate rap-
idly. Hence, there have been great research eforts (many microluidic-based) into optimizing the
culture and growth conditions of ES stem cell lines. It is also possible to form
embryoid bodies
, or
aggregates of cells from ES cell lines, that although they are largely disorganized compared with
a real embryo, they recapitulate some of the normal events of embryonic development (beating
heart muscle cells and neurons appear commonly in embryoid bodies). We have seen how Shuichi
Takayama's group was able to immobilize ES cells and form embryoid bodies in particular locations
of microchannels using a hydrodynamic trapping technique (see
Figure 2.42
in Section 2.6.2.6).
Conventional techniques for forming embryoid bodies do not allow for a precise control over
the size of the initial ES cell aggregates. Robert Langer's group at MIT cocultured hESCs on
mouse embryonic ibroblast feeder layers inside 200-μm-diameter PDMS microwells to produce
hESC aggregates of controlled size (
Figure 7.18
). Within the microwells, the hESCs maintained
their undiferentiated state (as conirmed by Oct-4 and ALP immunostaining) and displayed
a similar viability but superior homogeneity in aggregate size compared with lat substrates
(±8300 mm
2
within microwells versus ±46,000 mm
2
on lat surfaces).
A newer version of this process using PEG microwells instead of PDMS microwells has fur-
ther improved the homogeneity in size and shape of the embryoid bodies achieved, without the
need for a ibroblast feeder layer (
Figure 7.19
).
A team led by Maish Yarmush at the Massachusetts General Hospital in Boston performed
an in-depth study of mouse ES cell aggregate formation in PDMS microwells. he microwells
were produced by applying a PDMS stencil that contained holes ranging from 100 to 500 μm in
diameter (
Figure 7.20a
). hey found that germ layer diferentiation (ater 20 days of induction of
diferentiation), assessed by gene and protein expression assays as well as biochemical functions,
depends on the initial size of the ES cell aggregate. he smallest (100 μm) aggregates showed an
increased expression of ectodermal markers compared with the largest (500 μm) aggregates.
On the other hand, the 500-μm aggregates showed an increased expression of mesodermal and
endodermal markers compared with 100 μm aggregates. Hence, the initial conditions on which
the embryoid bodies are formed can be critical for the extent of diferentiation.
Several groups have already started developing microluidic cell culture laboratories (see
Section 5.4), and it can hardly be a coincidence that most of the devices are being applied to
