“9.61x6.69” b2815 Tissue Engineering and Nanotheranostics
Directed Differentiation of Human Pluripotent Stem Cells 83
system. As opposed to ectoderm, endoderm induction does not rely
as heavily on inhibiting the signals for other germ layers. Instead, it
relies on a complex network of signaling unique to its own tissue. The
directed differentiation techniques used to promote stem cell differ-
entiation to beta cells, lung progenitors, and hepatocytes follow.
3.2.1. Beta cells
Beta cells, characterized by their specialized ability to secrete and
store insulin for the regulation of blood glucose levels as well as other
substances like C-peptide, compose the pancreatic islets of the pan-
creas. The loss of functional beta cells typifies type I diabetes. As a
result, the potential for an inexhaustible source of functional beta cells
for type 1 diabetes therapy is the primary reason researchers are inter-
ested in the directed differentiation of beta cells from hESCs. Several
groups have developed and directed differentiation protocols to
derive beta cells or functionally similar cells.
The first step in the differentiation from a hESC to a beta cell is
dermal layer specification, in this case, endodermal specification. This
requires cellular signaling to move first from pluripotency to mesen-
doderm and then to a definitive endodermal cell type characterized by
high levels of Sox17 and FOXA2 expression.^30 In practice, this first
transition is commonly accomplished by the addition of Activin A to
a low serum content culture medium.^30 Activin A, a TGF-β related
growth factor, mimics the function of the nodal ligand to trigger the
same cell signaling cascade that would be endogenously activated.^31
From the definitive endoderm, the next step of differentiation
requires pancreatic progenitor specification and further differentia-
tion to a beta cell.
Several protocols have been published using a variety of different
factors to induce pancreatic progenitor and later insulin producing
cell specification. One of the common induction factors, retinoic acid,
has been reported to be involved in endoderm differentiation and
pancreatic bud formation.^32 In 2006, D’Amour et al. published a five-
stage protocol for differentiating a 2D monolayer culture of pluripo-
tent stem cells into endocrine hormone-expressing cells that secrete
