Biomedical Engineering Reference
In-Depth Information
and successfully expand undifferentiated hESCs as aggregates by
a stirred suspension culture system without microcarriers [21].
During the expansion culture, hESC aggregates were dissociated
every six days in the presence of a ROCK inhibitor to keep the
aggregate size small, which was also indispensable to prevent
the differentiation and keep pluripotency of hESCs in aggregates.
Steiner et al
demonstrated that the use of a Neurobasal® Medium
(Invitrogen™) supplemented with a KnockOut™ Serum Replacement
(Invitrogen), fibroblast growth factor 2 (FGF2), and activin A
prevented the differentiation of hESCs in aggregates and enabled
the expansion of undifferentiated hESCs as aggregates in a stirred
suspension culture system, although they did not completely identify
the essential requirements for this result [22]. They also found that
the supplementation of Nutridoma-CS (Roche) and extracellular
matrix such as fibronectin and laminin significantly promoted the
expansion rate of undifferentiated hESCs. They applied this culture
method to three hESC lines (HES1, HES2, and H7) and successfully
expanded the hESCs of all these three lines for 10 weeks. Expansion
culture of undifferentiated ESCs/iPSCs as aggregates does not
require the procedure of separating microcarriers from cells during
passaging and thus a further simplified method. Additionally, the
culture system can be subsequently applied for the differentiation
culture of ESCs/iPSCs as it is after the expansion culture, because
the differentiaion of ESCs/iPSCs is generally induced by aggregate
formation. On the other hand, this culture method requires some
specific medium components such as rapamycin to prevent ESC/iPSC
differentiation in aggregates. The effect of such medium components
on the properties of ESCs/iPSCs should be examined carefully.
.
2c.3
Induction of Cardiac Differentiation
As previously described, cardiac differentiation of hESCs/iPSCs is
generally induced by the formation of hESC/iPSC aggregates called
embryoid bodies (EBs). There are also methods to induce cardiac
differentiation of hESCs/iPSCs by monolayer culture with specific
feeder cells and/or the use of some differentiation-inductive
biochemical agents [23, 24]. However, the method of forming
EBs is still a gold standard for inducing cardiac differentiation
and is adaptable to a stirred suspension culture system, which
