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

Cell-cell interaction between acinar-like cells and islet endocrine cells encapsulated by common BMs.

<p><b>A:</b> A desmosomal junction (arrowhead) is observed between acinar-like cells (AC) and alpha cells (A), which are encapsulated by common BMs and ECM. <b>B</b>: Coated-pit-like structure is observed between an acinar-like cell (AC) and an alpha cell (A) touching directly and covered by common BMs (arrows). Inset shows a magnified view of the coated-pit-like structure. <b>C</b>: Excretion of vesicles from acinar-like cells (AC) to a beta cell (B). Note the vesicular membrane of the AC is dissolved (arrow), and the vesicular content is released to the beta cell (B) touching it. <b>D1</b>: Exocytotic features of vesicles in acinar-like cells (AC) to beta cells (B), which are in contact with each other. Arrowhead indicates BMs/ECM encapsulating acinar-like cells and beta cells, V: vasculature. <b>D2:</b> Higher magnified view of D1. The vesicle is internalized to the beta cell. AC: acinar-like cell, B: beta cell. <b>E:</b> Vesicles of acinar-like cells (AC) are internalized to touching alpha cells (A) and beta cell (B) shown by arrows. Arrowheads indicate BMs/ECM surrounding beta cell (B), alpha cell (A), and acinar-like cell (AC). Inset shows magnified view of (E). LB: lipofuscin body, ER: endoplasmic reticulum.</p