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and store insulin.^33 However, the cells produced from this protocol
lacked definitive beta cell function as they coexpressed other hor-
mones such as glucagon. They also exhibited other functional differ-
ences from adult beta cells including lowered processing efficiency of
proinsulin and minimal C-peptide secretion response to changes in
glucose.^33 This method was later refined from a five-stage differentia-
tion to a four-stage process resulting in pancreatic endoderm and
endocrine precursors.^34
In 2007, Shim et al. demonstrated a 3D embryoid body culture
technique to derive pancreatic endodermal cells from human pluripo-
tent stem cells (hPSCs) using the sequential additions of serum,
Activin A, and retinoic acid to the culture.^35 They established that the
cells resulting from grafts transplanted into mice produced insulin,
proinsulin, C-peptide, and glucagon and rescued a hyperglycemic
state suggesting the ability to further differentiate into beta-like cells
in vivo.^35 However, the uncontrolled nature of the final differentiation
stage made it difficult to develop a definitive understanding of the
complete process directing beta cell differentiation. Jiang et al. uti-
lized a three-stage protocol and suspension culture system to derive
insulin-producing cells from hESCs.^36 Their methods employed
Activin A to produce the definitive endoderm and retinoic acid to
induce pancreatic commitment. Supplements added during the matu-
ration stage of this differentiation included bFGF and nicotinamide.^36
The resulting insulin-producing cells exhibited expression of several
beta cell markers, including Pdx1, insulin, C-peptide, glut2, gluca-
gon, and amylase, and the cells secreted insulin in response to changes
in glucose level.^36 The efficiency of this differentiation method was
reported at 15%, leaving room for optimization.^36
Two years later, Jiang et al. demonstrated that an improved ver-
sion of their differentiation protocol could be used to derive insulin
producing cells in vitro from both hESCs and iPSCs.^37 This protocol
implemented a pancreatic progenitor expansion stage by adding epi-
dermal growth factor (EGF) prior to the maturation stage, resulting
in a 25% yield of insulin-producing cells.^37 The chemically defined
protocol represented an advantage to this differentiation method;
however, improvements in efficiency would be beneficial in
