Genetic aberrations in iPSCs are introduced by a transient G1/S cell cycle checkpoint deficiency.

A number of point mutations have been identified in reprogrammed pluripotent stem cells such as iPSCs and ntESCs. The molecular basis for these mutations has remained elusive however, which is a considerable impediment to their potential medical application. Here we report a specific stage at which iPSC generation is not reduced in response to ionizing radiation, i.e. radio-resistance. Quite intriguingly, a G1/S cell cycle checkpoint deficiency occurs in a transient fashion at the initial stage of the genome reprogramming process. These cancer-like phenomena, i.e. a cell cycle checkpoint deficiency resulting in the accumulation of point mutations, suggest a common developmental pathway between iPSC generation and tumorigenesis. This notion is supported by the identification of specific cancer mutational signatures in these cells. We describe efficient generation of human integration-free iPSCs using erythroblast cells, which have only a small number of point mutations and INDELs, none of which are in coding regions.

Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites.

The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism.

Differentiation and Molecular Properties of Mesenchymal Stem Cells Derived from Murine Induced Pluripotent Stem Cells Derived on Gelatin or Collagen.

The generation of induced-pluripotential stem cells- (iPSCs-) derived mesenchymal stem cells (iMSCs) is an attractive and promising approach for preparing large, uniform batches of applicable MSCs that can serve as an alternative cell source of primary MSCs. Appropriate culture surfaces may influence their growth and differentiation potentials during iMSC derivation. The present study compared molecular properties and differentiation potential of derived mouse iPS-MSCs by deriving on gelatin or collagen-coated surfaces. The cells were derived by a one-step method and expressed CD73 and CD90, but CD105 was downregulated in iMSCs cultured only on gelatin-coated plates with increasing numbers of passages. A pairwise scatter analysis revealed similar expression of MSC-specific genes in iMSCs derived on gelatin and on collagen surfaces as well as in primary mouse bone marrow MSCs. Deriving iMSCs on gelatin and collagen dictated their osteogenic and adipose differentiation potentials, respectively. Derived iMSCs on gelatin upregulated Bmp2 and Lif prior to induction of osteogenic or adipose differentiation, while PPARγ was upregulated by deriving on collagen. Our results suggest that extracellular matrix components such as gelatin biases generated iMSC differentiation potential towards adipose or bone tissue in their derivation process via up- or downregulation of these master genes.

Intra- and extracellular plasminogen activator inhibitor-1 regulate effect of vitronectin against radiation-induced endothelial cell death.

Plasminogen activator inhibitor-1 (PAI-1) is induced by radiation resulting in endothelial cell impairment, potentially leading to multiple organ failure. Vitronectin (VN) is a 75-kDa glycoprotein (VN75) cleaved into two forms (VN75 or VN65/10) by furin, which is regulated by intracellular PAI-1. VN protects against radiation-induced endothelial cell death, but the mechanisms involved in VN processing and its interactions with intra- and extracellular PAI-1 remain unclear. We examined these processes in cells in vitro using recombinant proteins or overexpression of VN and PAI-1 genes, including furin-susceptible (T381) and furin-resistant VN (A381). VN processing was analyzed using a mutant PAI-1 with relatively weaker binding to VN. VN function was evaluated by survival of radiation-damaged endothelial cells. Wild-type, but not mutant PAI-1 inhibited furin-dependent VN processing. Gene transfer revealed that furin-susceptible VN was processed more than the furin-resistant form, but processing of both was inhibited by PAI-1 overexpression. Intracellular PAI-1 formed a complex with VN75 (T381) in cells and media, and the VN75 form was secreted preferentially. Only VN75 protected against radiation-induced endothelial cell death, in which its effect was abolished by wild-type but not mutant PAI-1. These findings indicate that intracellular PAI-1 inhibits VN processing and protects against radiation-induced endothelial cell death.

Characteristics of three-dimensional prospectively isolated mouse bone marrow mesenchymal stem/stromal cell aggregates on nanoculture plates.

Three-dimensional (3-D) aggregate culturing is useful for investigating the functional properties of mesenchymal stem/stromal cells (MSCs). For 3-D MSC analysis, however, pre-expansion of MSCs with two-dimensional (2-D) monolayer culturing must first be performed, which might abolish their endogenous properties. To avoid the need for 2-D expansion, we used prospectively isolated mouse bone marrow (BM)-MSCs and examined the differences in the biological properties of 2-D and 3-D MSC cultures. The BM-MSCs self-assembled into aggregates on nanoculture plates (NCP) that have nanoimprinted patterns with a low-cellular binding texture. The 3-D MSCs proliferated at the same rate as 2-D-cultured cells by only diffusion culture and secreted higher levels of pro-angiogenic factors such as vascular endothelial growth factor and hepatocyte growth factor (HGF). Conditioned medium from 3-D MSC cultures promoted more capillary formation than that of 2-D MSCs in an in vitro tube formation assay. Matrigel-implanted 3-D MSC aggregates tended to induce angiogenesis in host mice. The 3-D culturing on NCP induced alpha-fetoprotein (AFP) expression in MSCs without the application of AFP- or endodermal-inducible factors, possibly via an HGF-autocrine mechanism, and maintained their differentiation ability for adipocytes, osteocytes, and chondrocytes. Prospectively isolated mouse BM-MSCs expressed low/negative stemness-related genes including Oct3/4, Nanog, and Sox2, which were not enhanced by NCP-based 3-D culturing, suggesting that some of these cells differentiate into meso-endodermal layer cells. Culturing of prospectively isolated MSCs on NCP in 3-D allows the analysis of the biological properties of more closely endogenous BM-MSCs and might contribute to tissue engineering and repair.

Estimation of secondary measles transmission from a healthcare worker in a hospital setting.

Measles among healthcare workers (HCWs) is associated with a significant risk of nosocomial transmission to susceptible patients. When a measles case occurs in the healthcare setting, most guidelines recommend exhaustive measures. To evaluate the effects of measures against measles transmission in the healthcare setting precisely, it is essential to determine whether secondary transmission generally occurs. This study describes, for the first time, the actual secondary transmission rate for a measles-infected HCW in a ward with no special air ventilation capacity. The routine treatment of a number of immunocompromised patients occurs in this ward, and thus patients as well as HCWs have a thorough understanding and practice of standard and extended precautions. Our paired serum sample study revealed that none of the people in the ward exposed to the HCW at the catarrhal stage over a period of 4 days exhibited elevated levels of antibodies against measles. We suggest that strict adherence to standard and expanded precautions among patients and HCWs may be effective for preventing the transmission of a highly airborne disease, such as measles.

Radiation increases the cellular uptake of exosomes through CD29/CD81 complex formation.

Exosomes mediate intercellular communication, and mesenchymal stem cells (MSC) or their secreted exosomes affect a number of pathophysiologic states. Clinical applications of MSC and exosomes are increasingly anticipated. Radiation therapy is the main therapeutic tool for a number of various conditions. The cellular uptake mechanisms of exosomes and the effects of radiation on exosome-cell interactions are crucial, but they are not well understood. Here we examined the basic mechanisms and effects of radiation on exosome uptake processes in MSC. Radiation increased the cellular uptake of exosomes. Radiation markedly enhanced the initial cellular attachment to exosomes and induced the colocalization of integrin CD29 and tetraspanin CD81 on the cell surface without affecting their expression levels. Exosomes dominantly bound to the CD29/CD81 complex. Knockdown of CD29 completely inhibited the radiation-induced uptake, and additional or single knockdown of CD81 inhibited basal uptake as well as the increase in radiation-induced uptake. We also examined possible exosome uptake processes affected by radiation. Radiation-induced changes did not involve dynamin2, reactive oxygen species, or their evoked p38 mitogen-activated protein kinase-dependent endocytic or pinocytic pathways. Radiation increased the cellular uptake of exosomes through CD29/CD81 complex formation. These findings provide essential basic insights for potential therapeutic applications of exosomes or MSC in combination with radiation.