Pancreatic Islet-Like Three-Dimensional Aggregates Derived from Human Embryonic Stem Cells Ameliorate Hyperglycemia in Streptozotocin-Induced Diabetic Mice

Abstract

We previously reported the in vitro differentiation of human embryonic stem cells (hESCs) into pancreatic endoderm. Here we demonstrate that islet-like three-dimensional (3D) aggregates can be derived from the pancreatic endoderm by optimizing our previous protocol. Sequential treatment with Wnt3a, activin A, and noggin induced a transient upregulation of T and MixL1, followed by increased expression of endodermal genes, including FOXA2, SOX17, and CXCR4. Subsequent treatment with retinoic acid highly upregulated PDX1 expression. We also show that inhibition of sonic hedgehog signaling by bFGF/activin bB and cotreatment with VEGF and FGF7 produced many 3D cellular clusters that express both SOX17 and PDX1. We found for the first time that proteoglycans and vimentin + mesenchymal cells were mainly localized in hESC-derived PDX1 + clusters. Importantly, treatment with chlorate, an inhibitor of proteoglycan sulfation, together with inhibition of Notch signaling significantly increased the expression of Neurog3 and NeuroD1, promoting a transition from PDX1 + progenitor cells toward mature pancreatic endocrine cells. Purified dithizone + 3D aggregates generated by our refined protocol produced pancreatic hormones and released insulin in response to both glucose and pharmacological drugs in vitro. Furthermore, the islet-like 3D aggregates decreased blood glucose levels and continued to exhibit pancreatic features after transplantation into diabetic mice. Generation of islet-like 3D cell aggregates from human pluripotent stem cells may overcome the shortage of cadaveric donor islets for future cases of clinical islet transplantation.

Controlled induction of human pancreatic progenitors produces functional beta‐like cells in vitro

“…Consequently, numerous research efforts have focused on identifying abundant alternative sources of surrogate glucose‐responsive insulin‐producing cells (Zhou & Melton, ; Tudurí & Kieffer, ; Efrat & Russ, ; Hebrok, ; Nostro & Keller, ; Bouwens et al , ; Pagliuca & Melton, ). One of the most appealing approaches is the directed differentiation into insulin‐producing cells from pluripotent human embryonic stem cells (hESCs) (D'Amour et al , ; Chen et al , ; Mfopou et al , ; Nostro et al , ; Van Hoof et al , ; Xu et al , ; Guo et al , ; Shim et al , ) and, more recently, induced pluripotent stem cells (Maehr et al , ; Hua et al , ; Shang et al , ).…”

Pdx1 expression up-regulated by Tet-on system induces the mouse embryonic stem cells to pancreatic-like cells through Notch pathway

“…Our study indicates that Pdx1 can activate downstream target genes to induce differentiation into pancreatic cells. In recent years, ESCs have been induced to differentiate into insulin-secreting cells and islet-like cells [33,34] in vitro. Our results revealed that pancreatic precursor cells (Pdx1 + cells) can differentiate into several types of pancreatic-like cells: exocrine cells and ductal cells during the early stage and endocrine cells at a later stage.…”

Diabetes through a 3D lens: organoid models

“…Introduction of suspension cultures and an airliquid interface produced immature beta cells that did not secrete insulin [48,49]. In contrast, 3D islet-like organoids generated from either human embryonic stem cells [50] or from spontaneously formed endocrine cell clusters produced by stepwise differentiation of human PSCs [51] released insulin in response to glucose in vitro and in vivo. More recent efforts have introduced synthetic hydrogels [52] or co-cultures with HUVECs and mesenchymal stem cells (MSCs) combined with a self-condensation system [53].…”