A Report from a Workshop of the International Stem Cell Banking Initiative, Held in Collaboration of Global Alliance for iPSC Therapies and the Harvard Stem Cell Institute, Boston, 2017.

This report summarizes the recent activity of the International Stem Cell Banking Initiative held at Harvard Stem Cell Institute, Boston, MA, USA, on June 18, 2017. In this meeting, we aimed to find consensus on ongoing issues of quality control (QC), safety, and efficacy of human pluripotent stem cell banks and their derivative cell therapy products for the global harmonization. In particular, assays for the QC testing such as pluripotency assays test and general QC testing criteria were intensively discussed. Moreover, the recent activities of global stem cell banking centers and the regulatory bodies were briefly summarized to provide an overview on global developments and issues. Stem Cells 2019;37:1130-1135.

Direct Visualization and Quantification of Maternal Transfer of Silver Nanoparticles in Zooplankton.

The immense application of silver nanoparticles (AgNPs) in biomedical fields is likely to increase the exposure of humans. However, little is known about whether these nanoparticles can be maternally transferred, especially regarding their biodistribution in the younger generation, maternal transfer efficiency, and toxic effects. In the present study, maternal transfer of AgNPs in model zooplankton (Daphnia magna) was for the first time visualized and quantified. We found that AgNPs were transferred from mother to offspring and mainly accumulated in the lipids due to the strong colocalization with lipid droplets, which were the major energy sources of Daphnia embryos. In contrast, Ag+ was irregularly distributed in different sites, probably due to the mobility and reactivity of Ag+. The maternal transfer efficiency quantified by the radiolabeling methodology was 2.37 ± 0.25 and 6.05 ± 0.89% for 110mAgNPs and 110mAg, respectively. Furthermore, AgNPs and Ag+ significantly inhibited the reproduction capability of F0 and F1 generations, but such maternal toxic effect inhibition was only found within the first two broods of F0 and F1 generations. Our bioimaging findings demonstrated that AgNPs could be maternally transferred to the next generation; thus, it is critical to produce AgNPs with lower toxic effects, higher delivery efficacy, and more precise targeting.

Changes in cell morphology guide identification of tubulin as the off-target for protein kinase inhibitors.

In the field of kinase inhibitors for applications in cancer research, tubulin is emerging as a targeted cellular protein that can significantly contribute to their activities. However, investigation of kinase inhibitors beyond the kinome is an area often neglected. Herein, we describe the results of pharmacological studies using drugs targeting kinases, tubulin or both. A key finding is that if cells are treated with a kinase inhibitor unintentionally targeting tubulin, their characteristic shape will diminish within a short timeframe. These changes in cell morphology are not seen when cells are treated with bona fide kinase inhibitors that do not directly target tubulin. Thus, early changes in cell morphology upon treatments are a strong indication that the inhibitor is directly targeting tubulin. Recognizing tubulin as a target of kinase inhibitors will build confidence in the future mechanistic studies using kinase inhibitors.

Effect of mid-follicular phase recombinant LH versus urinary HCG supplementation in poor ovarian responders undergoing IVF - a prospective double-blinded randomized study.

Luteinizing hormone (LH) is crucial for the development of follicular growth and oocyte maturation, especially in the management of poor ovarian responders (PORs). This study presents the results of a prospective double-blinded randomized study to compare the effect of mid-follicular phase recombinant LH (rLH) supplementation with urinary human chorionic gonadotrophin (uHCG) supplementation when using a fixed gonadotrophin-releasing hormone (GnRH) antagonist protocol in IVF cycles. A total of 49 women with poor ovarian response (POR) according to the Bologna criteria were recruited. This study showed no statistically significant difference in cycle cancellation rates, numbers of oocytes retrieved per cycle initiated, fertilization rates, the numbers of embryos obtained per cycle initiated, implantation, clinical pregnancy and live birth rates, although the live birth rate per cycle initiated in the uHCG group (29.2%) was 3.6 times that of the rLH group (8.0%). Further studies are required to verify if uHCG supplementation produces better clinical outcomes compared with rLH in women with POR.

Coronary Flow Velocity Reserve Declines After Anthracycline Therapy in Breast Cancer Patients.

Anthracycline therapy (ANT) is associated with cancer therapy-related cardiac dysfunction. Coronary flow velocity reserve (CFVR) has shown prognostic utility in non-cancer cohorts, but no data have been obtained in a cardio-oncology setting. We investigated the acute effect of ANT on CFVR in breast cancer patients. A total of 12 female breast cancer patients undergoing ANT had pre- and post-ANT CFVR assessment. A significant decline in CFVR occurred (baseline: 2.66 ± 0.41 vs post-ANT: 2.47 ± 0.37, P = 0.016). This prospective study is the first to identify ANT-related coronary physiology changes in humans. Further studies are required to determine their clinical significance.

Le traitement par l'anthracycline est associé à une dysfonction cardiaque liée au traitement anticancéreux. La réserve de débit coronaire a démontré son utilité pronostique dans les cohortes sans cancer, mais aucune donnée n'a été obtenue dans un contexte de cardio-oncologie. Nous avons étudié l'effet aigu de l'anthracycline sur la réserve de débit coronaire chez des patientes atteintes d'un cancer du sein. La réserve de débit coronaire a été évaluée avant et après le traitement par l'anthracycline chez un total de 12 femmes atteintes d'un cancer du sein. Un déclin important de la réserve de débit coronaire est survenu (valeur initiale de 2,66 ± 0,41 par rapport à 2,47 ± 0,37 après le traitement par l'anthracycline, p = 0,016). Cette étude prospective est la première à déceler des changements dans la physiologie coronarienne liés à l'anthracycline chez les humains. D'autres études sont nécessaires pour en déterminer la portée clinique.

Differential gene expression during floral transition in pineapple.

Pineapple (Ananas comosus var. comosus) and ornamental bromeliads are commercially induced to flower by treatment with ethylene or its analogs. The apex is transformed from a vegetative to a floral meristem and shows morphological changes in 8 to 10 days, with flowers developing 8 to 10 weeks later. During eight sampling stages ranging from 6 h to 8 days after treatment, 7961 genes were found to exhibit differential expression (DE) after the application of ethylene. In the first 3 days after treatment, there was little change in ethylene synthesis or in the early stages of the ethylene response. Subsequently, three ethylene response transcription factors (ERTF) were up-regulated and the potential gene targets were predicted to be the positive flowering regulator CONSTANS-like 3 (CO), a WUSCHEL gene, two APETALA1/FRUITFULL (AP1/FUL) genes, an epidermal patterning gene, and a jasmonic acid synthesis gene. We confirm that pineapple has lost the flowering repressor FLOWERING LOCUS C. At the initial stages, the SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) was not significantly involved in this transition. Another WUSCHEL gene and a PHD homeobox transcription factor, though not apparent direct targets of ERTF, were up-regulated within a day of treatment, their predicted targets being the up-regulated CO, auxin response factors, SQUAMOSA, and histone H3 genes with suppression of abscisic acid response genes. The FLOWERING LOCUS T (FT), TERMINAL FLOWER (TFL), AGAMOUS-like APETELAR (AP2), and SEPETALA (SEP) increased rapidly within 2 to 3 days after ethylene treatment. Two FT genes were up-regulated at the apex and not at the leaf bases after treatment, suggesting that transport did not occur. These results indicated that the ethylene response in pineapple and possibly most bromeliads act directly to promote the vegetative to flower transition via APETALA1/FRUITFULL (AP1/FUL) and its interaction with SPL, FT, TFL, SEP, and AP2. A model based on AP2/ERTF DE and predicted DE target genes was developed to give focus to future research. The identified candidate genes are potential targets for genetic manipulation to determine their molecular role in flower transition.

Low intensity ultrasound stimulates osteoblast migration at different frequencies.

This study investigated the effects of different frequencies of low intensity ultrasound on osteoblast migration using an in vitro scratch-wound healing assay. Mouse calvarial-derived MC3T3-E1 osteoblasts in culture were exposed to continuous 45 kHz ultrasound (25 mW/cm(2)) or pulsed 1 MHz ultrasound (250 mW/cm(2)) for 30 min followed by 2 days' culture. Ultrasound treatment with either kHz or MHz output similarly and significantly increased cell numbers after 2 days in culture compared with untreated control cultures. In the scratch-wound healing assay the presence of the cell proliferation inhibitor mitomycin C (MMC) did not influence scratch-wound closure in control cultures indicating that cell migration was responsible for the in vitro wound healing. Application of ultrasound significantly stimulated wound closure. MMC did not affect kHz-stimulated in vitro wound healing; however, MMC reduced in part the scratch-wound closure rate in MHz-treated cultures suggesting that enhanced cell proliferation as well as migration was involved in the healing promoted by MHz ultrasound. In conclusion, both continuous kHz and pulsed MHz ultrasound promoted osteoblastic migration; however, subtle differences were apparent in the manner the different ultrasound regimens enhanced in vitro scratch-wound healing.

Iron Polymaltose Infusion Effects on Bone Scintigraphy and Remodeling.

ABSTRACT: A 73-year-old woman with metastatic breast cancer and known widespread skeletal metastases was referred for bone scintigraphy. Delayed images acquired at 2 and 5 hours postinjection of 30 mCi (1089 MBq) 99mTc-HDP demonstrated markedly reduced bony uptake, markedly increased renal activity, and significantly increased soft tissue accumulation. By contrast, appropriate skeletal uptake of 99mTc-HDP was seen in prior bone scans. The patient had been treated for iron deficiency anemia with an infusion of 1 g of iron polymaltose approximately 22 hours before injection of 99mTc-HDP. This phenomenon may be due to transient reduced bone resorption with increased FGF23 release secondary to IV iron supplementation.

Taro raphide-associated proteins: Allergens and crystal growth.

Calcium oxalate raphide crystals are found in bundles in intravacuolar membrane chambers of specialized idioblasts cells of most plant families. Aroid raphides are proposed to cause acridity in crops such as taro (Colocasia esculenta (L.) Schott). Acridity is irritation that causes itchiness and pain when raw/insufficiently cooked tissues are eaten. Since raphides do not always cause acridity and since acridity can be inactivated by cooking and/or protease treatment, it is possible that a toxin or allergen-like compound is associated with the crystals. Using two-dimensional (2D) gel electrophoresis and mass spectrometry (MS) peptide sequencing of selected peptides from purified raphides and taro apex transcriptome sequencing, we showed the presence on the raphides of peptides normally associated with mitochrondria (ATP synthase), chloroplasts (chaperonin ~60 kDa), cytoplasm (actin, profilin), and vacuole (V-type ATPase) that indicates a multistage biocrystallation process ending with possible invagination of the tonoplast and addition of mucilage that may be derived from the Golgi. Actin might play a crucial role in the generation of the needle-like raphides. One of the five raphide profilins genes was highly expressed in the apex and had a 17-amino acid insert that significantly increased that profilin's antigenic epitope peak. A second profilin had a 2-amino acid insert and also had a greater B-cell epitope prediction. Taro profilins showed 83% to 92% similarity to known characterized profilins. Further, commercial allergen test strips for hazelnuts, where profilin is a secondary allergen, have potential for screening in a taro germplasm to reduce acridity and during food processing to avoid overcooking.

Fats and Mets, KRAS-Driven Lipid Dysregulation Affects Metastatic Potential in Pancreatic Cancer.

In this issue of Cancer Research, Rozeveld and colleagues present intriguing evidence of the importance of lipid droplets and hormone-sensitive lipase (HSL) in regulating the aggressive nature of pancreatic cancer. Initially demonstrating a dependency of preloaded lipids on an invasive phenotype, the authors then establish that oncogenic KRAS mutation downregulates HSL, thereby facilitating lipid storage during steady state. Thereafter, a phenotypic switch to oxidative metabolism with lipid utilization to fuel invasion and metastasis occurs. Experimentally, blocking the KRAS-HSL axis results in fewer lipid droplets, as well as metabolic reprogramming of the invasive cell phenotype, effectively reducing invasive capacity of KRAS-mutant pancreatic cancer. Of note, HSL overexpression in tumor cells also inhibited invasion, due to depletion of lipid droplets and the stored lipids, which are essential during invasion. Collectively, these novel findings highlight the importance of energy metabolism and its dynamic regulation in the evolution of the metastatic capacity of pancreatic cancer.See related article by Rozeveld et al., p. 4932.

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.

Targeting the complexity of Src signalling in the tumour microenvironment of pancreatic cancer: from mechanism to therapy.

Pancreatic cancer, a disease with extremely poor prognosis, has been notoriously resistant to virtually all forms of treatment. The dynamic crosstalk that occurs between tumour cells and the surrounding stroma, frequently mediated by intricate Src/FAK signalling, is increasingly recognised as a key player in pancreatic tumourigenesis, disease progression and therapeutic resistance. These important cues are fundamental for defining the invasive potential of pancreatic tumours, and several components of the Src and downstream effector signalling have been proposed as potent anticancer therapeutic targets. Consequently, numerous agents that block this complex network are being extensively investigated as potential antiinvasive and antimetastatic therapeutic agents for this disease. In this review, we will discuss the latest evidence of Src signalling in PDAC progression, fibrotic response and resistance to therapy. We will examine future opportunities for the development and implementation of more effective combination regimens, targeting key components of the oncogenic Src signalling axis, and in the context of a precision medicine-guided approach.

PAK inhibition by PF-3758309 enhanced the sensitivity of multiple chemotherapeutic reagents in patient-derived pancreatic cancer cell lines.

BACKGROUND/OBJECTIVE: Pancreatic ductal adenocarcinoma (PDA) remains the most lethal malignancy due to lack of an effective treatment. P21-activated kinases (PAKs) play a key role not only in cell proliferation and migration, but also in mediating chemo-resistance in PDA. The aim of this study was to investigate the combined effect of a PAK inhibitor PF-3758309 with multiple chemotherapeutic reagents on a panel of patient-derived PDA cell lines, and potential mechanisms involved. METHODS: Cells were treated with PF-3758309 plus or minus gemcitabine, 5-fluorouracil (5-FU) or abraxane, and cell growth was determined using a cell proliferation assay kit. Protein expression profiles were measured by Western blot. PDA cells were subcutaneously injected into the flanks of SCID mice which were then treated with saline, gemcitabine, PF-3758309, gemcitabine plus PF-3758309 or abraxane. Tumour growth was measured by volume and weight. RESULTS: PAK1 was correlated with CK19 expression, and PAK4 with α-SMA and palladin expression. Combination of PF-3758309 with 5-FU, gemcitabine or abraxane further suppressed cell growth of patient-derived PDA cell lines in vitro. The combination of PF-3758309 with gemcitabine maximally inhibited tumour growth in vivo by suppressing cell proliferation. PF-3758309 inhibited the expression of HIF-1α, palladin and α-SMA both in vitro and in vivo. CONCLUSIONS: PAK inhibitor PF-3758309 can enhance anti-tumour effects of multiple chemotherapeutic reagents on a panel of patient-derived PDA cell lines. Combination of PF-3758309 with gemcitabine achieves comparable efficacy to combination of gemcitabine with abraxane, and thus provides a potential targeted therapy in the management of PDA.

The Evolving Understanding of the Molecular and Therapeutic Landscape of Pancreatic Ductal Adenocarcinoma.

Pancreatic cancer is the third leading cause of cancer-related deaths, characterised by poor survival, marked molecular heterogeneity and high intrinsic and acquired chemoresistance. Only 10⁻20% of pancreatic cancer patients present with surgically resectable disease and even then, 80% die within 5 years. Our increasing understanding of the genomic heterogeneity of cancer suggests that the failure of definitive clinical trials to demonstrate efficacy in the majority of cases is likely due to the low proportion of responsive molecular subtypes. As a consequence, novel treatment strategies to approach this disease are urgently needed. Significant developments in the field of precision oncology have led to increasing molecular stratification of cancers into subtypes, where individual cancers are selected for optimal therapy depending on their molecular or genomic fingerprint. This review provides an overview of the current status of clinically used and emerging treatment strategies, and discusses the advances in and the potential for the implementation of precision medicine in this highly lethal malignancy, for which there are currently no curative systemic therapies.

Preservation and stability of cell therapy products: recommendations from an expert workshop.

If the field of regenerative medicine is to deliver therapies, rapid expansion and delivery over considerable distances to large numbers of patients is needed. This will demand efficient stabilization and shipment of cell products. However, cryopreservation science is poorly understood by life-scientists in general and in recent decades only limited progress has been made in the technology of preservation and storage of cells. Rapid translation of new developments to a broader range of cell types will be vital, as will assuring a deeper knowledge of the fundamental cell biology relating to successful preservation and recovery of cell cultures. This report presents expert consensus on these and other issues which need to be addressed for more efficient delivery of cell therapies.

Low-intensity low-frequency ultrasound promotes proliferation and differentiation of odontoblast-like cells.

INTRODUCTION: Ultrasound is a potential therapeutic tool for dental tissue repair, but its biological effects on odontoblasts have not been well characterized. In this study, the effects of low-intensity low-frequency ultrasound on the viability, proliferation, and differentiation of odontoblast-like cells were investigated. METHODS: Cell viability and proliferation were assessed after the treatment of adherent clonal MDPC-23 odontoblast-like cells with a 25-mW/cm(2) 45-kHz ultrasound. An in vitro scratch wound healing assay was used to investigate the ultrasound effects on cell migration. Long-term cultures were used to study odontogenic differentiation and extracellular mineralization. RESULTS: Ultrasound exposure for up to 30 minutes did not significantly affect odontoblast-like cell viability but significantly increased cell numbers after 2 days in culture. Ultrasound did not influence the scratch wound closure rate in the absence or presence of the mitogen inhibitor mitomycin C, indicating that ultrasound did not influence cellular migration. Single and consecutive exposures to ultrasound resulted in the enhancement of in vitro mineralization after 14 days in culture with an osteogenic differentiation medium. This coincided with the up-regulation of gene expression of collagen type I, osteoadherin, dentine matrix protein 1, and osteocalcin as well as the expression of cell markers alkaline phosphatase and nestin. CONCLUSIONS: These findings indicate that low-frequency ultrasound is able to influence proliferation and differentiation of odontoblast-like cells and may potentially be considered as a therapeutic tool for dental pulp and dentine repair.