Generation of Pancreatic Hormone-Expressing Islet-Like Cell Aggregates from Murine Adipose Tissue-Derived Stem Cells

Abstract

The success of cell replacement therapy for diabetes depends on the availability and generation of an adequate number of islets, preferably from an autologous origin. Stem cells are now being probed for the generation of physiologically competent, insulin-producing cells. In this investigation, we explored the potential of adipose tissue-derived stem cells (ASCs) to differentiate into pancreatic hormone-expressing islet-like cell aggregates (ICAs). We initiated ASC culture from epididymal fat pads of Swiss albino mice to obtain mesenchymal cells, murine epididymal (mE)-ASCs. Subsequent single-cell cloning resulted in a homogeneous cell population with a CD29+CD44+Sca-1+ surface antigen expression profile. We formulated a 10-day differentiation protocol to generate insulin-expressing ICAs from mE-ASCs by progressively changing the differentiation cocktail on day 1, day 3, and day 5. Our stage-specific approach successfully differentiated mesodermic mE-ASCs into definitive endoderm (cells expressing Sox17, Foxa2, GATA-4, and cytokeratin [CK]-19), then into pancreatic endoderm (cells expressing pancreatic and duodenal homeobox [PDX]-1, Ngn3, NeuroD, Pax4, and glucose transporter 2), and finally into cells expressing pancreatic hormones (insulin, glucagon, somatostatin). Fluorescence-activated cell sorting analysis showed that day 5 ICAs contained 64.84% ± 7.03% PDX-1+ cells, and in day 10 mature ICAs, 48.17% ± 3% of cells expressed C-peptide. Day 10 ICAs released C-peptide in a glucose-dependent manner, exhibiting in vitro functionality. Electron microscopy of day 10 ICAs revealed the presence of numerous secretory granules within the cell cytoplasm. Calcium alginate-encapsulated day 10 ICAs (1,000–1,200), when transplanted i.p. into streptozotocin-induced diabetic mice, restored normoglycemia within 2 weeks. The data presented here demonstrate the feasibility of using ASCs as a source of autologous stem cells to differentiate into the pancreatic lineage. Disclosure of potential conflicts of interest is found at the end of this article.

Betacellulin Overexpression in Mesenchymal Stem Cells Induces Insulin Secretion In Vitro and Ameliorates Streptozotocin-Induced Hyperglycemia in Rats

“…5A, B) similar to the structures described by other authors during specific differentiation protocols [29][30][31].…”

Production of islet-like insulin-producing cell clusters in vitro from adiposederived stem cells

“…Moreover, they can differentiate into specific cells such as fat cells, osteocytes, chondrocytes and myocytes. Importantly, these cells of mesodermal origin can be driven to differentiate into endodermal cells such as insulin-producing cells via trans-differentiation (Chandra et al, 2009;Dave et al, 2013Dave et al, , 2014Fedyunina et al, 2011;Hwang et al, 2011;Marappagounder et al, 2013;Mohamad Buang et al, 2012;Moshtagh et al, 2013;Timper et al, 2006). This study aimed to develop and test an efficient protocol to produce functional islets that can be used in research and application.…”

Differentiation of Stem Cells into Insulin-Producing Cells: Current Status and Challenges

“…Moreover, matured ICAs exhibited expression of Pdx-1, C-peptide, insulin, glucagon, somatostatin, pancreatic polypeptide and Glut-2. ICAs transplantation in streptozotocine induced diabetic mice restored normoglycemia within 2 weeks (Chandra 2009). Similar three-step differentiation protocol was able to differentiate the human adipose tissue-derived stem cells into IPCs (Chandra et al 2011).…”

“…Upregulation of pancreatic transcription factors IPF-1, ISL-1, NGN3 and islet hormones was observed during the differentiation period (Timper et al 2006). Chandra (2009) demonstrated three-step differentiation protocol of IPCs from murine epididymal adipose stem cells (mE-ASCs). The mE-ASCs were first converted into definitive endoderm (activin-A, sodium butyrate, b-mercaptoethanol), then into pancreatic endoderm (taurine) and finally into pancreatic hormone-expressing islet-like cell aggregates (ICAs) (taurine, GLP-1, nicotinamide, NEAA).…”