Comparability of automated human induced pluripotent stem cell culture: a pilot study.

Consistent and robust manufacturing is essential for the translation of cell therapies, and the utilisation automation throughout the manufacturing process may allow for improvements in quality control, scalability, reproducibility and economics of the process. The aim of this study was to measure and establish the comparability between alternative process steps for the culture of hiPSCs. Consequently, the effects of manual centrifugation and automated non-centrifugation process steps, performed using TAP Biosystems' CompacT SelecT automated cell culture platform, upon the culture of a human induced pluripotent stem cell (hiPSC) line (VAX001024c07) were compared. This study, has demonstrated that comparable morphologies and cell diameters were observed in hiPSCs cultured using either manual or automated process steps. However, non-centrifugation hiPSC populations exhibited greater cell yields, greater aggregate rates, increased pluripotency marker expression, and decreased differentiation marker expression compared to centrifugation hiPSCs. A trend for decreased variability in cell yield was also observed after the utilisation of the automated process step. This study also highlights the detrimental effect of the cryopreservation and thawing processes upon the growth and characteristics of hiPSC cultures, and demonstrates that automated hiPSC manufacturing protocols can be successfully transferred between independent laboratories.

The Future of Regenerative Medicine: Cell Therapy Using Pluripotent Stem Cells and Acellular Therapies Based on Extracellular Vesicles.

The rapid progress in the field of stem cell research has laid strong foundations for their use in regenerative medicine applications of injured or diseased tissues. Growing evidences indicate that some observed therapeutic outcomes of stem cell-based therapy are due to paracrine effects rather than long-term engraftment and survival of transplanted cells. Given their ability to cross biological barriers and mediate intercellular information transfer of bioactive molecules, extracellular vesicles are being explored as potential cell-free therapeutic agents. In this review, we first discuss the state of the art of regenerative medicine and its current limitations and challenges, with particular attention on pluripotent stem cell-derived products to repair organs like the eye, heart, skeletal muscle and skin. We then focus on emerging beneficial roles of extracellular vesicles to alleviate these pathological conditions and address hurdles and operational issues of this acellular strategy. Finally, we discuss future directions and examine how careful integration of different approaches presented in this review could help to potentiate therapeutic results in preclinical models and their good manufacturing practice (GMP) implementation for future clinical trials.

Distributed automated manufacturing of pluripotent stem cell products.

Establishing how to effectively manufacture cell therapies is an industry-level problem. Decentralised manufacturing is of increasing importance, and its challenges are recognised by healthcare regulators with deviations and comparability issues receiving specific attention from them. This paper is the first to report the deviations and other risks encountered when implementing the expansion of human pluripotent stem cells (hPSCs) in an automated three international site-decentralised manufacturing setting. An experimental demonstrator project expanded a human embryonal carcinoma cell line (2102Ep) at three development sites in France, Germany and the UK using the CompacT SelecT (Sartorius Stedim, Royston, UK) automated cell culture platform. Anticipated variations between sites spanned material input, features of the process itself and production system details including different quality management systems and personnel. Where possible, these were pre-addressed by implementing strategies including standardisation, cell bank mycoplasma testing and specific engineering and process improvements. However, despite such measures, unexpected deviations occurred between sites including software incompatibility and machine/process errors together with uncharacteristic contaminations. Many only became apparent during process proving or during the process run. Further, parameters including growth rate and viability discrepancies could only be determined post-run, preventing 'live' corrective measures. The work confirms the critical nature of approaches usually taken in Good Manufacturing Practice (GMP) manufacturing settings and especially emphasises the requirement for monitoring steps to be included within the production system. Real-time process monitoring coupled with carefully structured quality systems is essential for multiple site working including clarity of decision-making roles. Additionally, an over-reliance upon post-process visual microscopic comparisons has major limitations; it is difficult for non-experts to detect deleterious culture changes and such detection is slow.

Cell death in protists without mitochondria.

Some protozoans, such as Trichomonad species, do not possess mitochondria. Most of the time, they harbor another type of membrane-bounded organelle, called hydrogenosome from its capacity to produce H(2). This is the case for the human parasite Trichomonas vaginalis. Some other parasites, such as the protist Giardia lamblia, do not harbor any of these organelles. From this observation arises naturally a naive question: How do cells die when the mitochondrion, the cornerstone of apoptotic process, is absent? Data strongly suggest that the mitochondrion and the hydrogenosome arose from a common ancestral endosymbiont. But hydrogenosomes do not appear to directly substitute for mitochondria in apoptotic functions. Thus, it appears judicious to examine more closely the genome of unicellular cells, which do not harbor mitochondria, and search for new molecules that could participate in the apoptotic process in these microorganisms.

A novel antiangiogenic and vascular normalization therapy targeted against human CD160 receptor.

Angiogenesis plays an essential role in several diseases of the eye and in the growth of solid tumors, but existing antiangiogenic therapies have limited benefits in several cases. We report the antiangiogenic effects of a monoclonal antibody, CL1-R2, in several animal models of neovascularization. CL1-R2 recognizes human CD160, a membrane receptor which is conserved in various mammal species. We show that CD160 is expressed on the endothelial cells of newly formed blood vessels in human colon carcinoma and mouse B16 melanoma but not in vessels of healthy tissues. CL1-R2 reduced fibroblast growth factor 2-induced neovascularization in the rabbit cornea, in a mouse model of oxygen-induced retinopathy, and in a mouse Matrigel plug assay. Treatment of B16 melanoma-bearing mice with CL1-R2 combined with cyclophosphamide chemotherapy caused regression of the tumor vasculature and normalization of the remaining vessels as shown by Doppler ultrasonography, intravital microscopy, and histology. These studies validate CD160 as a potential new target in cases of human pathological ocular and tumor neoangiogenesis that do not respond or become resistant to existing antiangiogenic drugs.

Distinct role of nox1, nox2, and p47phox in unstimulated versus angiotensin II-induced NADPH oxidase activity in human venous smooth muscle cells.

Human saphenous veins (SV) are used for coronary bypass surgery despite the higher rate of graft failure observed as compared to arteries. A higher production of reactive oxygen species (ROS) in SV than in internal mammary artery (IMA) has been incriminated as possibly implicated in graft failure. NADPH oxidase, involved in vascular ROS production, was therefore characterized in human smooth muscle cells from SV. ROS production was confirmed to be essentially NADPH oxidase dependent in cultured smooth muscle cells (SMC) from human SV and increased in comparison with IMA. To investigate the role of NADPH oxidase subunits, siRNA for nox1, nox2, or p47 mRNA were studied. In cultured venous SMC under unstimulated conditions, inhibition of nox1 or nox2 mRNA decreased ROS production, whereas p47 silencing increased it. During angiotensin II (AngII) activation, nox2 or p47 mRNA silencing decreased ROS production, while nox1 inhibition had no effect. Venous SMC express functional nox1 and nox2. Only nox2 is implicated in response to AngII whilst nox1 is involved in unstimulated ROS production. p47 negatively regulates ROS generation under basal conditions, whereas it enhances AngII increased ROS production. Thus, nox1, nox2, and p47 have distinct roles in NADPH oxidase activity in human veins.

A form of cell death with some features resembling apoptosis in the amitochondrial unicellular organism Trichomonas vaginalis.

One of hallmarks of apoptosis is the degradation and concomitant compaction of chromatin. It is assumed that caspases and caspase-independent pathways are rate limiting for the development of nuclear apoptosis. The caspase-independent pathway involves apoptosis-inducing factor (AIF) and leads to DNA fragmentation and peripheral chromatin condensation. Both pathways are the result of activation of death signals that the mitochondrion receives, integrates, and responds to with the release of various molecules (e.g., cytochrome c and AIF). In fact, both pathways have in common the final point of the DNA fragmentation and the mitochondrial origin of molecules that initiate the apoptotic events. Here, we examine the question of whether apoptosis or apoptotic-like processes exist in a unicellular organism that lacks mitochondria. We herein show that a form of cell death with some features resembling apoptosis is indeed present in Trichomonas vaginalis. Characterization of morphological aspects implicated in this event together with the preliminary biochemical data provided may lead to new insight about the evolutionary relationships between the different forms of programmed cell death identified so far.