Célula x

glucagón, somatostatina y PP se expresaron sólo en

pequeños subconjuntos de células que se mezclaron con secretores de insulina

células. Esto es consistente con el patrón de expresión hormonal.

de islotes maduros determinación de la secreción de insulina del islote

organoides en prueba de glucosa

etermination of insulin secretion from islet

organoids on glucose challenge

Finally, we detected glucose-responsive insulin secretion

from islet organoids developed within C-M scaffolds. Cell

clusters collected at Stage IV were exposed to 5.5, 15, and

25 mM of glucose, respectively. We observed a sharp increase

in insulin secretion from the cell clusters when exposed to a

high concentration of glucose (Fig. 6C). We detected the se-

cretion of 44 – 4.9 mIU (international unit) insulin/mg cellular

proteins from 3D-induced cell clusters when exposed to 25 mM

glucose. This represents a seven-fold increase in insulin se-

cretion, as compared with what was detected in cells exposed to

a low concentration of glucose (5.5 mM). In contrast, 2D-

formed insulin-secreting cells are less sensitive to glucose

challenge. Only 10.8 – 3.2 mIU insulin/mg cellular protein

secretion was detected when 2D-induced cells were ex-

posed to 25 mM glucose, representing a 3.7-fold increase in

insulin secretion, as compared with that detected in cells

exposed to a low concentration of glucose (5.5 mM). These

results demonstrated that 3D-induced insulin-secreting

cells are more sensitive to a glucose challenge due to their

elevated maturity.

Discussion

The study of hESC pancreatic differentiation has been fo-

cused mainly on mimicking the sequence of signaling events

that underlie cell commitment and tissue formation during

pancreatogenesis. Apart from soluble growth factors that are

the keys to cell lineage specification, the importance of ECM

in hESC pancreatic differentiation and islet development has

not been fully elucidated. In this study, we demonstrated the

feasibility of generating islet organoids from hESCs by using

a biomimetic ECM scaffold. We investigated the combina-

tory effects of physiological cues and ILV offered by collagen

and Matrigel scaffolds on the generation of islet organoids

from hESCs. As previously shown [13,28], collagen I scaf-

folds offer better niches for promoting in vitro differentiation

and maturation of mESC-derived pancreatic endocrine b

cells. Nevertheless, a collagen scaffold is not strong enough to

support hESC pancreatic differentiation. These scaffolds

collapse during differentiation. The addition of Matrigel to

collagen scaffolds seems to improve the mechanical strength

of the scaffolds. Although we did not measure the Young’s

modulus of these scaffolds, it has been discovered that the

Young’s moduli of collagen-Matrigel scaffolds is propor-

tional to the concentration of Matrigel incorporated [29].

Thus, we speculated that the addition of Matrigel to a collagen

scaffold improves its mechanical strength, which better sup-

ports the development of islet organoids from hESCs. The

addition of Matrigel not only improves the mechanic strength

of the scaffolds but also affects the fibrillary structure of the

scaffolds. The SEM analysis suggested that