Activated monocytes resist elimination by retinal pigment epithelium and downregulate their OTX2 expression via TNF‐α

Orthodenticle homeobox 2 (OTX2) controls essential, homeostatic retinal pigment epithelial (RPE) genes in the adult. Using cocultures of human CD14⁺ blood monocytes (Mos) and primary porcine RPE cells and a fully humanized system using human‐induced pluripotent stem cell‐derived RPE cells, we show that activated Mos markedly inhibit RPEOTX2 expression and resist elimination in contact with the immunosuppressive RPE. Mechanistically, we demonstrate that TNF‐α, secreted from activated Mos, mediates the downregulation of OTX2 and essential RPE genes of the visual cycle among others. Our data show how subretinal, chronic inflammation and in particular TNF‐α can affect RPE function, which might contribute to the visual dysfunctions in diseases such as age‐related macular degeneration (AMD) where subretinal macrophages are observed. Our findings provide important mechanistic insights into the regulation of OTX2 under inflammatory conditions. Therapeutic restoration of OTX2 expression might help revive RPE and visual function in retinal diseases such as AMD.     

New Type of Sendai Virus Vector Provides Transgene-Free iPS Cells Derived from Chimpanzee Blood

Induced pluripotent stem cells (iPSCs) are potentially valuable cell sources for disease models and future therapeutic applications; however, inefficient generation and the presence of integrated transgenes remain as problems limiting their current use. Here, we developed a new Sendai virus vector, TS12KOS, which has improved efficiency, does not integrate into the cellular DNA, and can be easily eliminated. TS12KOS carries KLF4, OCT3/4, and SOX2 in a single vector and can easily generate iPSCs from human blood cells. Using TS12KOS, we established iPSC lines from chimpanzee blood, and used DNA array analysis to show that the global gene-expression pattern of chimpanzee iPSCs is similar to those of human embryonic stem cell and iPSC lines. These results demonstrated that our new vector is useful for generating iPSCs from the blood cells of both human and chimpanzee. In addition, the chimpanzee iPSCs are expected to facilitate unique studies into human physiology and disease.     

Modeling of hemophilia A using patient-specific induced pluripotent stem cells derived from urine cells

Aims

Hemophilia A (HA) is a severe, congenital bleeding disorder caused by the deficiency of clotting factor VIII (FVIII). For years, traditional laboratory animals have been used to study HA and its therapies, although animal models may not entirely mirror the human pathophysiology. Human induced pluripotent stem cells (iPSCs) can undergo unlimited self-renewal and differentiate into all cell types. This study aims to generate hemophilia A (HA) patient-specific iPSCs that differentiate into disease-affected hepatocyte cells. These hepatocytes are potentially useful for in vitro disease modeling and provide an applicable cell source for autologous cell therapy after genetic correction.

Main methods

In this study, we mainly generated iPSCs from urine collected from HA patients with integration-free episomal vectors PEP4-EO2S-ET2K containing human genes OCT4, SOX2, SV40LT and KLF4, and differentiated these iPSCs into hepatocyte-like cells. We further identified the genetic phenotype of the FVIII genes and the FVIII activity in the patient-specific iPSC derived hepatic cells.

Key findings

HA patient-specific iPSCs (HA-iPSCs) exhibited typical pluripotent properties evident by immunostaining, in vitro assays and in vivo assays. Importantly, we showed that HA-iPSCs could differentiate into functional hepatocyte-like cells and the HA-iPSC-derived hepatocytes failed to produce FVIII, but otherwise functioned normally, recapitulating the phenotype of HA disease in vitro.

Significance

HA-iPSCs, particular those generated from the urine using a non-viral approach, provide an efficient way for modeling HA in vitro. Furthermore, HA-iPSCs and their derivatives serve as an invaluable cell source that can be used for gene and cell therapy in regenerative medicine.     

PIWI Proteins Are Dispensable for Mouse Somatic Development and Reprogramming of Fibroblasts into Pluripotent Stem Cells

PIWI proteins play essential and conserved roles in germline development, including germline stem cell maintenance and meiosis. Because germline regulators such as OCT4, NANOG, and SOX2 are known to be potent factors that reprogram differentiated somatic cells into induced pluripotent stem cells (iPSCs), we investigated whether the PIWI protein family is involved in iPSC production. We find that all three mouse Piwi genes, Miwi, Mili, and Miwi2, are expressed in embryonic stem cells (ESCs) at higher levels than in fibroblasts, with Mili being the highest. However, mice lacking all three Piwi genes are viable and female fertile, and are only male sterile. Furthermore, embryonic fibroblasts derived from Miwi/Mili/Miwi2 triple knockout embryos can be efficiently reprogrammed into iPS cells. These iPS cells expressed pluripotency markers and were capable of differentiating into all three germ layers in teratoma assays. Genome-wide expression profiling reveals that the triple knockout iPS cells are very similar to littermate control iPS cells. These results indicate that PIWI proteins are dispensable for direct reprogramming of mouse fibroblasts.     

Up-regulation of the embryonic self-renewal network through reversible polyploidy in irradiated p53-mutant tumour cells

We have previously documented that transient polyploidy is a potential cell survival strategy underlying the clonogenic re-growth of tumour cells after genotoxic treatment. In an attempt to better define this mechanism, we recently documented the key role of meiotic genes in regulating the DNA repair and return of the endopolyploid tumour cells (ETC) to diploidy through reduction divisions after irradiation. Here, we studied the role of the pluripotency and self-renewal stem cell genes NANOG, OCT4 and SOX2 in this polyploidy-dependent survival mechanism. In irradiation-resistant p53-mutated lymphoma cell-lines (Namalwa and WI-L2-NS) but not sensitive p53 wild-type counterparts (TK6), low background expression of OCT4 and NANOG was up-regulated by ionising radiation with protein accumulation evident in ETC as detected by OCT4/DNA flow cytometry and immunofluorescence (IF). IF analysis also showed that the ETC generate PML bodies that appear to concentrate OCT4, NANOG and SOX2 proteins, which extend into complex nuclear networks. These polyploid tumour cells resist apoptosis, overcome cellular senescence and undergo bi- and multi-polar divisions transmitting the up-regulated OCT4, NANOG and SOX2 self-renewal cassette to their descendents. Altogether, our observations indicate that irradiation-induced ETC up-regulate key components of germ-line cells, which potentially facilitate survival and propagation of the tumour cell population.