Epigenetic-scale comparison of human iPSCs generated by retrovirus, Sendai virus or episomal vectors

Human induced pluripotent stem cells (iPSCs) are established by introducing several reprogramming factors, such as OCT3/4, SOX2, KLF4, c-MYC. Because of their pluripotency and immortality, iPSCs are considered to be a powerful tool for regenerative medicine. To date, iPSCs have been established all over the world by various gene delivery methods. All methods induced high-quality iPSCs, but epigenetic analysis of abnormalities derived from differences in the gene delivery methods has not yet been performed. Here, we generated genetically matched human iPSCs from menstrual blood cells by using three kinds of vectors, i.e., retrovirus, Sendai virus, and episomal vectors, and compared genome-wide DNA methylation profiles among them. Although comparison of aberrant methylation revealed that iPSCs generated by Sendai virus vector have lowest number of aberrant methylation sites among the three vectors, the iPSCs generated by non-integrating methods did not show vector-specific aberrant methylation. However, the differences between the iPSC lines were determined to be the number of random aberrant hypermethylated regions compared with embryonic stem cells. These random aberrant hypermethylations might be a cause of the differences in the properties of each of the iPSC lines

Distinct cell clusters touching islet cells induce islet cell replication in association with over-expression of Regenerating Gene (REG) protein in fulminant type 1 diabetes.

BACKGROUND: Pancreatic islet endocrine cell-supporting architectures, including islet encapsulating basement membranes (BMs), extracellular matrix (ECM), and possible cell clusters, are unclear. PROCEDURES: The architectures around islet cell clusters, including BMs, ECM, and pancreatic acinar-like cell clusters, were studied in the non-diabetic state and in the inflamed milieu of fulminant type 1 diabetes in humans. RESULT: Immunohistochemical and electron microscopy analyses demonstrated that human islet cell clusters and acinar-like cell clusters adhere directly to each other with desmosomal structures and coated-pit-like structures between the two cell clusters. The two cell-clusters are encapsulated by a continuous capsule composed of common BMs/ECM. The acinar-like cell clusters have vesicles containing regenerating (REG) Iα protein. The vesicles containing REG Iα protein are directly secreted to islet cells. In the inflamed milieu of fulminant type 1 diabetes, the acinar-like cell clusters over-expressed REG Iα protein. Islet endocrine cells, including beta-cells and non-beta cells, which were packed with the acinar-like cell clusters, show self-replication with a markedly increased number of Ki67-positive cells. CONCLUSION: The acinar-like cell clusters touching islet endocrine cells are distinct, because the cell clusters are packed with pancreatic islet clusters and surrounded by common BMs/ECM. Furthermore, the acinar-like cell clusters express REG Iα protein and secrete directly to neighboring islet endocrine cells in the non-diabetic state, and the cell clusters over-express REG Iα in the inflamed milieu of fulminant type 1 diabetes with marked self-replication of islet cells

Pathological features of the pancreas affected by fulminant type 1 diabetes (FT1DM).

<p><b>A:</b> CD8 + T cells (red) infiltrate from outside the islet, disrupting vascular BMs (green, arrows) through the interstitial space between the vasculature and islets. Arrowheads indicate BMs (green) of exocrine pancreatic cells. <b>B:</b> BMs and ECM surrounding islets (green) are markedly disrupted and punctuated (arrows) in FT1DM. The vasculatures of the islets show marked dilation, and the vascular BMs have lost the human-specific double membrane profile <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095110#pone.0095110-Virtanen1" target="_blank">[15]</a> (arrowheads). <b>C:</b> Acinar-like cell cluster touching Langerhans islets with thin interstitial surrounding (ATLANTIS) shows marked expression of REG Iα (green) in FT1DM. BMs (red, arrows) encapsulating the islet beta cells (blue) and ATLANTIS (green) are disrupted and discontinuous in some parts. <b>D:</b> Double immunostaining for amylase (red) and REG Iα (green) shows that amylase expression in the ATLANTIS (in circle) in inflamed FT1DM becomes faint in inverse relation to REG Iα over-expression. I: Islet, PAC: pancreatic acinar cells. <b>E:</b> Triple immunostaining for REG Iα (green), glucagon + somatostatin (SS) + pancreatic polypeptide (PP) (red), and insulin (blue) demonstrates that REG Iα-positive cells are not beta, glucagon, SS, or PP cells. <b>F:</b> Serum levels of REG Iα are increased in the patients with FT1DM of duration less than 2 weeks. **p<0.01 vs. controls.</p

REG Iα-positive cell cluster contacting with beta cell cluster and surrounded by common BMs and ECM.

<p><b>A–D</b>: Immunostaining for REG Iα (green) (<b>A</b>), acinar-like cell marker, amylase (blue). (<b>B</b>), and double immunostaining with insulin (brown) and fibronectin (red) (<b>C</b>). Auto-fluorescence of collagen fiber surrounding islet is observed (<b>A</b>, arrowheads). The merged image (<b>D</b>) shows that amylase-positive acinar-like cells that are in contact with beta cells express REG Iα protein (light blue: arrowheads), and the acinar-like cell cluster is surrounded by BMs/ECM (red, fibronectin). <b>E</b>: Electron-immunostaining with immuno-gold for REG Iα (20 nm: arrowheads) in acinar-like cell touching a beta cell containing insulin (5 nm: arrows). REG Iα is mainly localized in the center of the vesicle that is near the beta cell wall. <b>F</b>: Immuno-electron microscopy with immunogold for REG Iα (25 nm: arrowheads) and insulin (5 nm: arrows). Densely stained REG Iα vesicle (*) is just beside the cell wall touching a beta cell. Dissolved vesicles positive for REG Iα (arrowheads) are observed in insulin-containing beta cells.</p