Assessment of the Impact of Post-Thaw Stress Pathway Modulation on Cell Recovery following Cryopreservation in a Hematopoietic Progenitor Cell Model

The development and use of complex cell-based products in clinical and discovery science continues to grow at an unprecedented pace. To this end, cryopreservation plays a critical role, serving as an enabling process, providing on-demand access to biological material, facilitating large scale production, storage, and distribution of living materials. Despite serving a critical role and substantial improvements over the last several decades, cryopreservation often remains a bottleneck impacting numerous areas including cell therapy, tissue engineering, and tissue banking. Studies have illustrated the impact and benefit of controlling cryopreservation-induced delayed-onset cell death (CIDOCD) through various “front end” strategies, such as specialized media, new cryoprotective agents, and molecular control during cryopreservation. While proving highly successful, a substantial level of cell death and loss of cell function remains associated with cryopreservation. Recently, we focused on developing technologies (RevitalICE™) designed to reduce the impact of CIDOCD through buffering the cell stress response during the post-thaw recovery phase in an effort to improve the recovery of previously cryopreserved samples. In this study, we investigated the impact of modulating apoptotic caspase activation, oxidative stress, unfolded protein response, and free radical damage in the initial 24 h post-thaw on overall cell survival. Human hematopoietic progenitor cells in vitro cryopreserved in both traditional extracellular-type and intracellular-type cryopreservation freeze media were utilized as a model cell system to assess impact on survival. Our findings demonstrated that through the modulation of several of these pathways, improvements in cell recovery were obtained, regardless of the freeze media and dimethyl sulfoxide concentration utilized. Specifically, through the use of oxidative stress inhibitors, an average increase of 20% in overall viability was observed. Furthermore, the results demonstrated that by using the post-thaw recovery reagent on samples cryopreserved in intracellular-type media (Unisol™), improvements in overall cell survival approaching 80% of non-frozen controls were attained. While improvements in overall survival were obtained, an assessment on the impact of specific cell subpopulations and functionality remains to be completed. While work remains, these results represent an important step forward in the development of improved cryopreservation processes for use in discovery science, and commercial and clinical settings.

On the Role of the Carboxyl Group to the Protective Effect of o-Dihydroxybenzoic Acids to Saccharomyces cerevisiae Cells upon Induced Oxidative Stress

In the present work, the role of the carboxyl group of o-dihydroxybenzoic acids (pyrocatechuic, 2,3-diOH-BA and protocatechuic, 3,4-diOH-BA) on the protection against induced oxidative stress in Saccharomyces cerevisiae was examined. Catechol (3,4-diOH-B) was included for comparison. Cell survival, antioxidant enzyme activities, and TBARS level were used to evaluate the efficiency upon the stress induced by H2O2 or cumene hydroperoxide. Theoretical calculation of atomic charge values, dipole moment, and a set of indices relevant to the redox properties of the compounds was also carried out in the liquid phase (water). Irrespective of the oxidant used, 2,3-diOH-BA required by far the lowest concentration (3–5 μM) to facilitate cell survival. The two acids did not activate catalase but reduced superoxide dismutase activity (3,4-diOH-BA>2,3-diOH-BA). TBARS assay showed an antioxidant effect only when H2O2 was used; equal activity for the two acids and inferior to that of 3,4-diOH B. Overall, theoretical and experimental findings suggest that the 2,3-diOH-BA high activity should be governed by metal chelation. In the case of 3,4-diOH BA, radical scavenging increases, and chelation capacity decreases. The lack of carboxyl moiety (3,4-diOH B) adds to radical scavenging, interaction with lipophilic free radicals, and antioxidant enzymes. The present study adds to our knowledge of the antioxidant mechanism of dietary phenols in biological systems.

The complex interplay between autophagy and cell death pathways

Autophagy is a universal cellular homeostatic process, required for the clearance of dysfunctional macromolecules or organelles. This self-digestion mechanism modulates cell survival, either directly by targeting cell death players, or indirectly by maintaining cellular balance and bioenergetics. Nevertheless, under acute or accumulated stress, autophagy can also contribute to promote different modes of cell death, either through highly regulated signalling events, or in a more uncontrolled inflammatory manner. Conversely, apoptotic or necroptotic factors have also been implicated in the regulation of autophagy, while specific factors regulate both processes. Here, we survey both earlier and recent findings, highlighting the intricate interaction of autophagic and cell death pathways. We, Furthermore, discuss paradigms, where this cross-talk is disrupted, in the context of disease.

PDGF-D−PDGFRβ signaling enhances IL-15–mediated human natural killer cell survival

The axis of platelet-derived growth factor (PDGF) and PDGF receptor-beta (PDGFRβ) plays prominent roles in cell growth and motility. In addition, PDGF-D enhances human natural killer (NK) cell effector functions when binding to the NKp44 receptor. Here, we report an additional but previously unknown role of PDGF-D, whereby it mediates interleukin-15 (IL-15)–induced human NK cell survival but not effector functions via its binding to PDGFRβ but independent of its binding to NKp44. Resting NK cells express no PDGFRβ and only a low level of PDGF-D, but both are significantly up-regulated by IL-15, via the nuclear factor κB signaling pathway, to promote cell survival in an autocrine manner. Both ectopic and IL-15–induced expression of PDGFRβ improves NK cell survival in response to treatment with PDGF-D. Our results suggest that the PDGF-D−PDGFRβ signaling pathway is a mechanism by which IL-15 selectively regulates the survival of human NK cells without modulating their effector functions.

PVT1, a YAP1 dependent stress responsive lncRNA drives ovarian cancer metastasis and chemoresistance

Metastatic growth of ovarian cancer cells into the peritoneal cavity requires adaptation to various cellular stress factors to facilitate cell survival and growth. Here we demonstrate the role of PVT1, one such stress induced long non-coding RNA, in ovarian cancer growth and metastasis. PVT1 is an amplified and overexpressed lncRNA in ovarian cancer with strong predictive value for survival and response to targeted therapeutics. We find that expression of PVT1 is regulated by ovarian tumor cells in response to cellular stress, particularly loss of cell-cell contacts and changes in matrix rigidity occurring in a YAP1 dependent manner. Induction of PVT1 promotes tumor cell survival, growth, and migration. Conversely, reducing PVT1 levels robustly abrogates metastatic behavior and tumor cell dissemination in cell lines and syngeneic transplantation models in vivo. We find that reducing PVT1 causes widespread transcriptome changes leading to alterations in cellular stress response and metabolic pathways including doxorubicin metabolism, which directly impacts chemosensitivity. Together, these findings implicate PVT1 as a promising therapeutic target to suppress metastasis and avoid chemoresistance in ovarian cancer.

Reciprocal epigenetic Sox2 regulation by SMAD1-SMAD3 is critical for anoikis resistance and metastasis in cancer

Growth factors in the tumor environment are key regulators of cell survival and anoikis resistance during metastasis. Here we reveal significant dichotomy between TGF-β superfamily growth factors BMP and TGF-β/activin and their downstream SMAD effectors in regulation of anchorage-independent tumor cell survival in ovarian cancer. Gene expression profiling uncovered the transcription factor Sox2 as a key signaling node regulated in an opposing manner by anoikis-promoting BMP2 4 and 9 and anoikis-suppressing TGF-β and activin A. Mechanistically, repression of Sox2 by BMPs is mediated by type I receptors ALK2 and ALK3 induced SMAD1 activation, leading to SMAD1-dependent histone H3K27me3 recruitment and DNA methylation at SOX2s promoter. Conversely, TGF-β and activin A promote Sox2 expression directly by ALK5-mediated SMAD3 activation and histone H3K4me3 recruitment. Increased Sox2 expression promotes anoikis resistance, while decreasing Sox2 levels conversely reduces anoikis resistance and activates cell death pathways. Additionally, administrating BMP9 as a strategy to reduce Sox2 robustly inhibits intraperitoneal tumor burden and increases survival in multiple ovarian cancer xenograft models. Importantly, BMP-driven SMAD1 signaling can override the effects of TGF-β and activin on Sox2 regulation, which has potential clinical significance as we find high TGF-β levels in patient ascites. Our findings highlight the contrasting regulation of anoikis by distinct SMAD signaling pathways that are dependent on a novel dichotomous regulation of Sox2 and implicate the use of a subset of BMPs as a therapeutic strategy in cancer

Effect of miR-384-targeting LINC00491 on proliferation, migration and invasion of tongue squamous cell carcinoma cells

Purpose: To investigate the effect of long-chain non-coding RNA LINC00491 (LncRNA LINC00491) on the proliferation, migration and invasion of tongue squamous cell carcinoma (TSCC) cells, and the underlying mechanism. Methods: Real-time quantitative polymerase chain reaction (qRT-PCR) was applied to determine the expressions of LINC00491 and micro-RNA-384 (miR-384). Furthermore, LINC00491 and miR-384 were transfected into CAL-27 cells, while cell cycle was analyzed using flow cytometry. Cell proliferation was determined by methyl thiazolyl diphenyl-tetrazolium (MTT) assay. Cell migration and invasion were evaluated using Transwell experiments. The relationship between LINC00491 and miR-384 was confirmed using double luciferase reporting assay, while protein expression levels of P21, Ki67, E- cadherin, N-cadherin, and vimentin were assayed with Western blotting. Results: The expression of LINC00491 increased in TSCC tissues (p < 0.05). The proportion of cells in G1-phase increased, while the proportion of cells in S-phase decreased (p < 0.05). There was decrease in cell survival, cell migration and cell invasion (p < 0.05). The protein expression levels of Ki67, N- cadherin, and vimentin were lowered, while those of P21, E-cadherin protein were increased (p < 0.05). Transfection of LINC00491 and miR- 384 reduced the proportion of cells in G1 phase, but increased the proportion of cells in S-phase (p < 0.05). Moreover, cell survival, migration and invasion were increased. The protein expressions of Ki67, N-cadherin, and vimentin rose, while those of P21 and E-cadherin decreased (p < 0.05). Conclusion: LINC00491 promotes the proliferation, migration and invasion of TSCC cells by inhibiting miR-384. This finding provides a potential target for the treatment of TSCC.

Microglia-secreted TNF-α affects differentiation efficiency and viability of pluripotent stem cell-derived human dopaminergic precursors

Parkinson's Disease is a neurodegenerative disorder characterized by the progressive loss of dopaminergic cells of the substantia nigra pars compacta . Even though successful transplantation of dopamine-producing cells into the striatum exhibits favourable effects in animal models and clinical trials; transplanted cell survival is low. Since every transplant elicits an inflammatory response which can affect cell survival and differentiation, we aimed to study in vivo and in vitro the impact of the pro-inflammatory environment on human dopaminergic precursors. We first observed that transplanted human dopaminergic precursors into the striatum of immunosuppressed rats elicited an early and sustained activation of astroglial and microglial cells after 15 days post-transplant. This long-lasting response was associated with Tumor necrosis factor alpha expression in microglial cells. In vitro conditioned media from activated BV2 microglial cells increased cell death, decreased Tyrosine hydroxylase -positive cells and induced morphological alterations on human neural stem cells-derived dopaminergic precursors at two differentiation stages: 19 days and 28 days. Those effects were ameliorated by inhibition of Tumor necrosis factor alpha, a cytokine which was previously detected in vivo and in conditioned media from activated BV-2 cells. Our results suggest that a pro-inflammatory environment is sustained after transplantation under immunosuppression, providing a window of opportunity to modify this response to increase transplant survival and differentiation. In addition, our data show that the microglia-derived pro-inflammatory microenvironment has a negative impact on survival and differentiation of dopaminergic precursors. Finally, Tumor necrosis factor alpha plays a key role in these effects, suggesting that this cytokine could be an interesting target to increase the efficacy of human dopaminergic precursors transplantation in Parkinson's Disease.

Alteration of ribosome function upon 5-fluorouracil treatment favors cancer cell drug-tolerance

Mechanisms of drug-tolerance remain poorly understood and have been linked to genomic but also to non-genomic processes. 5-fluorouracil (5-FU), the most widely used chemotherapy in oncology is associated with resistance. While prescribed as an inhibitor of DNA replication, 5-FU alters all RNA pathways. Here, we show that 5-FU treatment leads to the production of fluorinated ribosomes exhibiting altered translational activities. 5-FU is incorporated into ribosomal RNAs of mature ribosomes in cancer cell lines, colorectal xenografts, and human tumors. Fluorinated ribosomes appear to be functional, yet, they display a selective translational activity towards mRNAs depending on the nature of their 5′-untranslated region. As a result, we find that sustained translation of IGF-1R mRNA, which encodes one of the most potent cell survival effectors, promotes the survival of 5-FU-treated colorectal cancer cells. Altogether, our results demonstrate that “man-made” fluorinated ribosomes favor the drug-tolerant cellular phenotype by promoting translation of survival genes.