Differentiation of Human Embryonic Stem Cells into Insulin‐Producing Clusters

Abstract

Type I diabetes mellitus is caused by an autoimmune destruction of the insulin-producing β β cells. The major obstacle in using transplantation for curing the disease is the limited source of insulin-producing cells. The isolation of human embryonic stem (hES) cells introduced a new prospect for obtaining a sufficient number of β β cells for transplantation.We present here a method for forming immature islet-like clusters of insulin-producing cells derived from hES cells.The protocol consisted of several steps. Embryoid bodies were first cultured and plated in insulin-transferrin-selenium-fibronectin medium, followed by medium supplemented with N2, B27, and basic fibroblast growth factor (bFGF). Next, the glucose concentration in the medium was lowered, bFGF was withdrawn, and nicotinamide was added. Dissociating the cells and growing them in suspension resulted in the formation of clusters which exhibited higher insulin secretion and had longer durability than cells grown as monolayers.Reverse transcription-polymerase chain reaction detected an enhanced expression of pancreatic genes in the differentiated cells. Immunofluorescence and in situ hybridization analyses revealed a high percentage of insulin-expressing cells in the clusters. In addition to insulin, most cells also coexpressed glucagon or somatostatin, indicating a similarity to immature pancreatic cells.Further improvement of this insulin-producing cell protocol may lead to the formation of an unlimited source of cells suitable for transplantation.

Directed differentiation of human embryonic stem cells towards a pancreatic cell fate

“…To date, only a few studies have suggested the potential of hESCs to differentiate into insulin-producing cells [5][6][7][8]. It was previously reported that insulin-producing cells were found in EBs after spontaneous in vitro differentiation of hESCs [5].…”

“…Based on these findings, recent studies have also suggested the potential of hESCs to differentiate into insulin-producing cells through spontaneous in vitro differentiation [5] or the use of a multi-stage protocol [6]. However, several studies have claimed that insulin staining of ESC-derived cells is likely due to insulin uptake by apoptotic cells from the culture medium [19][20][21], suggesting that more reliable analyses should be considered in terms of beta cell physiology and from an embryological standpoint.…”

“…To date, while many studies have reported that hESCs could be efficiently induced to differentiate into mesodermal and ectodermal lineages, such as cardiomyocytes [1], endothelial cells [2], blood cells [3] and nerve cells [4], few reports have focused on the conversion of hESCs into endodermal lineages, particularly into pancreatic cell types [5][6][7][8], which may provide a limitless stock of insulin-producing cells to treat type 1 diabetes. Moreover, despite increasing knowledge on pancreatic development, there is still debate regarding the origin of beta cells and the markers that can be used to identify their progenitors [9].…”

Gene Therapy Progress and Prospects: Gene therapy for diabetes mellitus

“…ES cells have been touted to be able to differentiate into multiple cell lineages including the pancreatic endocrine cell lineages. 54,70,71 Notwithstanding the ethical and current legal controversies regarding the use of ES cells, there are significant potential advantages and some disadvantages of using stem cells for cell therapy for diabetes. ES cells by nature are totipotent and in theory could reconstitute the entire islet and thus be the ideal replacement therapy for type I diabetes.…”

Detecting de novo insulin synthesis in embryonic stem cell-derived populations

“…These unique characteristics make ES cells a potential source of insulin-producing cells for transplantation therapy [1][2][3]. Since 2000, several important and exciting reports have been published describing the derivation of insulin-positive cells from both mouse [4][5][6][7][8][9] and, to a lesser extent, human ES cell lines [10,11]. Several of these studies have been based on the derivation of insulinproducing cells using modified protocols that were originally designed to generate neurons, on the premise that the pancreas and central nervous system share developmental pathways [12][13][14].…”