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Recombinant protein therapeutics have become important components of the modern medicine. Majority of them are produced with mammalian cells that are cultured either through adherent culturing, in which cells are cultured on substrates, or suspension culturing, in which cells are suspended and cultured in agitated cell culture medium in a culture vessel. The adherent cell culturing method is limited by its low yield. In suspension culturing, cells need extensive genetic manipulation to grow as single cells at high density, which is time- and labor-consuming. Here, we report a new method, which utilizes a thermoreversible hydrogel as the scaffold for culturing protein-expressing cells. The hydrogel scaffolds not only provide 3D spaces for the cells, but also act as physical barriers to prevent excessive cellular agglomeration and protect cells from the hydrodynamic stresses. As a result, cells can grow at high viability, high growth rate, and extremely high yield even without genetic manipulations. The cell yield in the hydrogels is around 20 times of the suspension culturing. In addition, the protein productivity per cell per day in the hydrogel is higher than the adherent culturing method. This new method is simple, scalable and defined. It will be of great value for both the research laboratories and pharmaceutical industry for producing proteins.

Long QT syndromes (LQTS) are heritable diseases characterized by prolongation of the QT interval on an electrocardiogram, which often leads to syncope and sudden cardiac death. Here we report the generation of induced pluripotent stems (iPS) cells from two patients with LQTS type 3 carrying a different point mutation in a sodium channel Nav1.5 (p.V240M and p.R535Q) and functional characterization of cardiomyocytes (CM) derived from them. The iPS cells exhibited all characteristic properties of pluripotent stem cells, maintained the disease-specific mutation and readily differentiated to CM. The duration of action potentials at 50% and 90% repolarization was longer in LQTS-3 CM as compared to control CM but this difference did not reach statistical significance due to high variations among cells. Sodium current recordings demonstrated longer time to peak and longer time to 90% of inactivation of the Na+ channel in the LQTS-3 CM. This hints at a defective Na+ channel caused by deficiency in open-state inactivation of the Na+ channel that is characteristic of LQTS-3. These analyses suggest that the effect of channel mutation in the diseased CM is demonstrated in vitro and that the iPS cell-derived CM can serve as a model system for studying the pathophysiology of LQTS-3, toxicity testing and design of novel therapeutics. However, further improvements in the model are still required to reduce cell-to-cell and cell line-to-cell line variability. [ABSTRACT FROM AUTHOR]

Pluripotent stem cells evade replicative senescence, whereas other primary cells lose their proliferation and differentiation potential after a limited number of cell divisions, and this is accompanied by specific senescence- associated DNA methylation (SA-DNAm) changes. Here, we investigate SA-DNAm changes in mesenchymal stromal cells (MSC) upon long-term culture, irradiation-induced senescence, immortalization, and reprogramming into induced pluripotent stem cells (iPSC) using high-density HumanMethylation450 BeadChips. SA-DNAm changes are highly reproducible and they are enriched in intergenic and nonpromoter regions of developmental genes. Furthermore, SA-hypomethylation in particular appears to be associated with H3K9me3, H3K27me3, and Polycomb-group 2 target genes. We demonstrate that ionizing irradiation, although associated with a senescence phenotype, does not affect SA-DNAm. Furthermore, overexpression of the catalytic subunit of the human telomerase (TERT) or conditional immortalization with a doxycycline-inducible system (TERT and SV40-TAg) result in telomere extension, but do not prevent SA-DNAm. In contrast, we demonstrate that reprogramming into iPSC prevents almost the entire set of SA-DNAm changes. Our results indicate that long-term culture is associated with an epigenetically controlled process that stalls cells in a particular functional state, whereas irradiation-induced senescence and immortalization are not causally related to this process. Absence of SA-DNAm in pluripotent cells may play a central role for their escape from cellular senescence. [ABSTRACT FROM AUTHOR]

Induced pluripotent stem cell technology, also termed iPS, is an emerging approach to reprogram cells into an embryonic stem cell-like state by viral transduction with defined combinations of factors. iPS cells share most characteristics of embryonic stem cells, counting pluripotency and self-renewal, and have so far been obtained from mouse and humans, including patients with genetic diseases. Remarkably, autologous transplantation of cell lineages derived from iPS cells will eliminate the possibility of immunological rejection, as well as current ethical issues surrounding human embryonic stem cell research. However, before iPS can be used for clinical purposes, technical problems must be overcome. Among other considerations, full and homogeneous iPS reprogramming is an important prerequisite. However, despite the fact that cells from several mouse tissues can be successfully induced to iPS, the overall efficiency of chimera formation of these clones remains low even if selection for Oct4 or Nanog expression is applied. In this report, we demonstrate that cells from the mouse meningeal membranes express elevated levels of the embryonic master regulator Sox2 and are highly amenable to iPS. Meningeal iPS clones, generated without selection, are fully and homogeneously reprogrammed based on DNA methylation analysis and 100% chimera competent. Our results define a population of somatic cells that are ready to undergo iPS, thus highlighting a very attractive cell type for iPS research and application. [ABSTRACT FROM AUTHOR]

Metallothionein (MT) is the protein that has been shown to bind heavy metals, scavenge free radicals, protect DNA from radiation damage, and alleviate disease symptoms. However, only very limited success has been achieved in expression and production of active recombinant metallothionein. In this study, human metallothionein 1A (hMT1A) was transformed into yeast Pichia pastoris for expression with secretion of the protein into the medium. The expression system was optimized to obtain the targeted protein in active form at 335 mg per litre culture. hMT1A showed the character of extreme instability in the experiment. High concentration, aeration and heavy metal ions are the main factors affecting hMT1A stability.