Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: Bioprocess intensification and scaling-up approaches.

Human induced pluripotent stem cells (hiPSC) are attractive tools for drug screening and disease modeling and promising candidates for cell therapy applications. However, to achieve the high numbers of cells required for these purposes, scalable and clinical-grade technologies must be established. In this study, we use environmentally controlled stirred-tank bioreactors operating in perfusion as a powerful tool for bioprocess intensification of hiPSC production. We demonstrate the importance of controlling the dissolved oxygen concentration at low levels (4%) and perfusion at 1.3day-1 dilution rate to improve hiPSC growth as aggregates in a xeno-free medium. This strategy allowed for increased cell specific growth rate, maximum volumetric concentrations (4.7×106cell/mL) and expansion factors (approximately 19 in total cells), resulting in a 2.6-fold overall improvement in cell yields. Extensive cell characterization, including whole proteomic analysis, was performed to confirm that cells' pluripotent phenotype was maintained during culture. A scalable protocol for continuous expansion of hiPSC aggregates in bioreactors was implemented using mechanical dissociation for aggregate disruption and cell passaging. A total expansion factor of 1100 in viable cells was obtained in 11days of culture, while cells maintained their proliferation capacity, pluripotent phenotype and potential as well as genomic stability after 3 sequential passages in bioreactors.

[Community-based screening: a motivating experience for drug users]. / Dépister hors les murs d’un centre de dépistage?: une expérience mobilisatrice pour des usagers de drogues.

OBJECTIVES: Hepatitis C virus infection (HCV) is a major public health problem among drug users. Screening for hepatitis C virus in this population is complicated. The aim of the study was to describe a community-based screening experience conducted by the Tours university hospital addiction medicine team. METHODS: Between 2008 and 2010, a free 14-day HCV, hepatitis B virus (HBV) and HIV community-based screening programme was conducted by the addiction medicine and prevention team. A questionnaire collected the main risk factors for transmission of these viruses and the subject's viral serology status. RESULTS: 76% of the 219 screened subjects reported being drug users. HCV prevalence was 20%. Risk factors for HCV infection were exclusive intravenous use and the use of several routes of administration. Among the 30 HCV patients with positive RNA, 83% were followed up. CONCLUSIONS: The prevalence of HCV infection was similar to that reported in the literature for drug users, whereas the number of patients treated and followed up was higher than in the literature. A community-based screening experience facilitated initiation and follow-up of medical care.

Phototoxic action spectrum on a retinal pigment epithelium model of age-related macular degeneration exposed to sunlight normalized conditions.

Among the identified risk factors of age-related macular degeneration, sunlight is known to induce cumulative damage to the retina. A photosensitive derivative of the visual pigment, N-retinylidene-N-retinylethanolamine (A2E), may be involved in this phototoxicity. The high energy visible light between 380 nm and 500 nm (blue light) is incriminated. Our aim was to define the most toxic wavelengths in the blue-green range on an in vitro model of the disease. Primary cultures of porcine retinal pigment epithelium cells were incubated for 6 hours with different A2E concentrations and exposed for 18 hours to 10 nm illumination bands centered from 380 to 520 nm in 10 nm increments. Light irradiances were normalized with respect to the natural sunlight reaching the retina. Six hours after light exposure, cell viability, necrosis and apoptosis were assessed using the Apotox-Glo Triplex™ assay. Retinal pigment epithelium cells incubated with A2E displayed fluorescent bodies within the cytoplasm. Their absorption and emission spectra were similar to those of A2E. Exposure to 10 nm illumination bands induced a loss in cell viability with a dose dependence upon A2E concentrations. Irrespective of A2E concentration, the loss of cell viability was maximal for wavelengths from 415 to 455 nm. Cell viability decrease was correlated to an increase in cell apoptosis indicated by caspase-3/7 activities in the same spectral range. No light-elicited necrosis was measured as compared to control cells maintained in darkness. Our results defined the precise spectrum of light retinal toxicity in physiological irradiance conditions on an in vitro model of age-related macular degeneration. Surprisingly, a narrow bandwidth in blue light generated the greatest phototoxic risk to retinal pigment epithelium cells. This phototoxic spectrum may be advantageously valued in designing selective photoprotection ophthalmic filters, without disrupting essential visual and non-visual functions of the eye.

Taurine deficiency damages retinal neurones: cone photoreceptors and retinal ganglion cells.

In 1970s, taurine deficiency was reported to induce photoreceptor degeneration in cats and rats. Recently, we found that taurine deficiency contributes to the retinal toxicity of vigabatrin, an antiepileptic drug. However, in this toxicity, retinal ganglion cells were degenerating in parallel to cone photoreceptors. The aim of this study was to re-assess a classic mouse model of taurine deficiency following a treatment with guanidoethane sulfonate (GES), a taurine transporter inhibitor to determine whether retinal ganglion cells are also affected. GES treatment induced a significant reduction in the taurine plasma levels and a lower weight increase. At the functional level, photopic electroretinograms were reduced indicating a dysfunction in the cone pathway. A change in the autofluorescence appearance of the eye fundus was explained on histological sections by an increased autofluorescence of the retinal pigment epithelium. Although the general morphology of the retina was not affected, cell damages were indicated by the general increase in glial fibrillary acidic protein expression. When cell quantification was achieved on retinal sections, the number of outer/inner segments of cone photoreceptors was reduced (20 %) as the number of retinal ganglion cells (19 %). An abnormal synaptic plasticity of rod bipolar cell dendrites was also observed in GES-treated mice. These results indicate that taurine deficiency can not only lead to photoreceptor degeneration but also to retinal ganglion cell loss. Cone photoreceptors and retinal ganglion cells appear as the most sensitive cells to taurine deficiency. These results may explain the recent therapeutic interest of taurine in retinal degenerative pathologies.

Review: Lamin A/C, caspase-6, and chromatin configuration during meiosis resumption in the mouse oocyte.

After in vitro maturation (IVM), isolation of the healthiest oocytes is essential for successful in vitro fertilization. As germinal vesicle (GV) oocytes resume meiosis through healthy or apoptotic pathways without discernable morphological criteria, we checked for an apoptotic element acting at the nucleus level. We hypothesized that caspase-6 with its corresponding substrate, lamin A/C, could be a potential target candidate, because caspase-6 is the only functional caspase for lamin A/C. We used immunohistochemistry methods, Western blots, and a specific caspase-6 inhibitor to determine the presence of lamin A/C and caspase-6 during oogenesis and in isolated oocytes. Our results demonstrated that these proteins were always present and that their distributions were related to oocyte maturity, determined by chromatin configuration and oocyte diameter. Caspase-6 inhibition slowed meiosis resumption suggesting the involvement of caspase-6 in the oocyte apoptotic pathway. Lamin A/C and caspase-6 could be valuable tools in the knowledge of oocyte in vitro destiny.

Caspase-2(L), caspase-9, and caspase-3 during in vitro maturation and fragmentation of the mouse oocyte.

Several studies have shown that apoptotic pathways control fragmentation of unfertilized ovulated oocyte, induced by doxorubicin. But very few have investigated the basis of this process, from prophase I to later stages. Our results revealed the presence of caspase-2(L), caspase-9, and caspase-3 in their zymogen and cleaved forms in the oocyte before meiosis resumption. Caspase-2(L) and caspase-9 were detected in the nucleus of GV-oocytes in a distribution related to chromatin configuration. The inhibition of caspase activity by Z-VAD-fmk accelerated the transition from metaphase I to metaphase II, and caspase-9 and caspase-3 were detected along the meiotic spindle. Surprisingly, Western blot analysis revealed that the three cleaved caspases were present in similar amounts in healthy and fragmented oocytes and caspase inhibition did not prevent doxorubicin-induced apoptosis. Our results suggest that, if cleaved, caspases may be dispensable for final oocyte death and they could be involved in regulating the maturation process.

Natural uranium disturbs mouse folliculogenesis in vivo and oocyte meiosis in vitro.

We investigated whether uranium intoxication affects female fertility by assessing its effects on ovarian function and on the oocyte. We treated two groups of female mice for 15 weeks with 5, 50 or 400 mg/L of uranyl nitrate in drinking water. In the first group, mice were euthanized immediately after intoxication. Mice of the second group were paired after intoxication with untreated males. Dams and their female pups were euthanized 3 months after the end of intoxication. We assayed the kidneys, femurs and one ovary per female for U content and collected the other ovary for histology. The number and size of all the ovarian follicles were analyzed. Mice from the first group and female pups had significantly fewer large antral follicles (Ø > 200 microm) than the untreated mice. By contrast, dams in the second group had more secondary and early preantral follicles (Ø 70-110 microm) than untreated mice. However, U had no effect on follicle atresia. We then analyzed the in vitro effects of U on oocyte maturation and fragmentation. GV-oocytes were cultured in the presence of 1mM uranyl acetate and observed for 72 h. Oocyte maturation was slowed down by U during resumption of meiosis and at metaphase II. However, the rhythm and rate of oocyte fragmentation were similar to those of control mice. Our findings demonstrate that U induces changes in folliculogenesis and oocyte maturation in mice and could consequently represent a risk for women who are chronically exposed.

The phosphoinositide-phospholipase C (PI-PLC) pathway in the mouse oocyte.

As highlighted in this review, the phosphoinositide-phospholipase C pathway is strongly implicated in the control of mouse oocyte meiosis. The pathway becomes progressively functional as oocyte growth advances, and it appears to play a role in the G2/M transition when meiosis resumes, at least in the in vitro spontaneous model. Even if the inositol 1,4,5-trisphosphate receptors are present from the beginning, they function and release Ca2+ when the follicular antrum appears. Phospholipase C beta1 (PLC beta 1) is first exclusively localized to the nucleus and then migrates to the cytoplasm when the oocyte is fully grown. During oocyte maturation PLC beta 1 is active in the cytoplasm before it migrates and becomes active in the nucleus just prior to germinal vesicle breakdown. Because a similar circuit is observed for protein kinase C alpha (PKC alpha), PKC beta 1, PKC beta 2, and active mitogen-activated protein kinase, it is tempting to envisage that a feedback loop occurs between these pathways as demonstrated in other cell types. The chronology of these molecular movements into the oocyte reveals the particular and important role of the nucleus phosphoinositide cycle during oocyte meiosis. It appears also that this chronology is crucial and that defects leading to an inappropriate intracellular localization can have dramatic consequences. Such anomalies can prevent the production of competent oocytes and lead to fertility problems.

Comparison of the expression patterns of five neural RNA binding proteins in the Xenopus retina.

An increasing body of evidence indicates that gene expression can be modulated by posttranscriptional mechanisms. RNA binding proteins, for instance, control gene expression at many regulatory levels including RNA splicing, transport, stability, and translation. Although numerous RNA binding proteins have been identified, very few have been studied extensively in the context of developmental processes. We focused our study on five neural RNA binding proteins: one Musashi homolog, Nrp-1, one member of the Bruno gene family, BruL-1 (also known as Etr-1), and three members of the ELAV/Hu family, ElrB, ElrC, and ElrD. As an initial step in addressing their function during Xenopus neurogenesis, we used in situ hybridization to determine their expression patterns during retinal development. We found that RNA binding proteins belonging to different families have distinct spatio-temporal expression. These combinatorial expression patterns are reminiscent of previously described cell type-specific expression patterns of transcription factors during retinal development. The distribution of RNA binding proteins within the retina suggests that these regulators of posttranscriptional events may play important roles in multiple steps of retinogenesis.

Retinal stem cells in vertebrates: parallels and divergences.

During the development of the nervous system, after a given number of divisions, progenitors exit the cell cycle and differentiate as neurons or glial cells. Some cells however do not obey this general rule and persist in a progenitor state. These cells, called stem cells, have the ability to self-renew and to generate different lineages. Understanding the mechanisms that allow stem cells to "resist" differentiating stimuli is currently one of the most fascinating research areas for biologists. The amphibian and fish retinas, known to contain stem cell populations, have been pioneering models for neural stem cell research. The Xenopus retina enabled the characterization of the genetic processes that occur in the path from a pluripotent stem cell to a committed progenitor to a differentiated neuron. More recently, the discovery that avian and mammalian retinas also contain stem cell populations, has contributed to the definitive view of the adult nervous system of upper vertebrates as a more dynamic and plastic structure than previously thought. This has attracted the attention of clinicians who are attempting to employ stem cells for transplantation into damaged tissue. Research in this area is promising and will represent a key instrument in the fight against blindness and retinal dystrophies. In this review, we will focus primarily on describing the main characteristics of various retinal stem cell populations, highlighting their divergences during evolution, and their potential for retinal cell transplantation. We will also give an overview of the signaling cascades that could modulate their potential and plasticity.