Artículos de revistas: "4D cell culture"

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Hilderbrand, Amber M., Elisa M. Ovadia, Matthew S. Rehmann, Prathamesh M. Kharkar, Chen Guo y April M. Kloxin. "Biomaterials for 4D stem cell culture". Current Opinion in Solid State and Materials Science 20, n.º 4 (agosto de 2016): 212–24. http://dx.doi.org/10.1016/j.cossms.2016.03.002.

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Nies, Cordula, Tobias Rubner, Hanna Lorig, Vera Colditz, Helen Seelmann, Andreas Müller y Eric Gottwald. "A Microcavity Array-Based 4D Cell Culture Platform". Bioengineering 6, n.º 2 (31 de mayo de 2019): 50. http://dx.doi.org/10.3390/bioengineering6020050.

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(1) Background: We describe a 4D cell culture platform with which we tried to detect and to characterize migration dynamics of single hematopoietic stem cells in polymer film microcavity arrays integrated into a microtiter plate. (2) Methods: The system was set up with CD34-expressing KG-1a cells as a surrogate for hematopoietic stem cells. We then evaluated the system as an artificial hematopoietic stem cell niche model comprised of a co-culture of human hematopoietic stem cells from cord blood (cord blood CD34+ cells, hHSCs) and human mesenchymal stromal cells (hMSCs) from bone marrow over a period of 21 days. We used a software-based cell detection method to count single hematopoietic stem cells (HSCs) in microcavities. (3) Results: It was possible to detect single HSCs and their migration behavior within single microcavities. The HSCs displayed a pronounced migration behavior with one population of CD34-expressing cells located at the bottom of the microcavities and one population located in the middle of the microcavities at day 14. However, at day 21 the two populations seemed to unite again so that no clear distinction between the two was possible anymore. (4) Conclusions: Single cell migration detection was possible but microscopy and flow cytometry delivered non-uniform data sets. Further optimization is currently being developed.

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Miao, Shida, Haitao Cui, Timothy Esworthy, Bhushan Mahadik, Se‐jun Lee, Xuan Zhou, Sung Yun Hann, John P. Fisher y Lijie Grace Zhang. "4D Self‐Morphing Culture Substrate for Modulating Cell Differentiation". Advanced Science 7, n.º 6 (marzo de 2020): 1902403. http://dx.doi.org/10.1002/advs.201902403.

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Zheng, Yijun, Mitchell Kim Liong Han, Qiyang Jiang, Bin Li, Jun Feng y Aránzazu del Campo. "4D hydrogel for dynamic cell culture with orthogonal, wavelength-dependent mechanical and biochemical cues". Materials Horizons 7, n.º 1 (2020): 111–16. http://dx.doi.org/10.1039/c9mh00665f.

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Miao, Shida, Haitao Cui, Timothy Esworthy, Bhushan Mahadik, Se‐jun Lee, Xuan Zhou, Sung Yun Hann, John P. Fisher y Lijie Grace Zhang. "Programmable Culture Substrates: 4D Self‐Morphing Culture Substrate for Modulating Cell Differentiation (Adv. Sci. 5/2020)". Advanced Science 7, n.º 6 (marzo de 2020): 2070034. http://dx.doi.org/10.1002/advs.202070034.

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Yang, Chen, Jeffrey Luo, Marianne Polunas, Nikola Bosnjak, Sy‐Tsong Dean Chueng, Michelle Chadwick, Hatem E. Sabaawy, Shawn A. Chester, Ki‐Bum Lee y Howon Lee. "4D‐Printed Transformable Tube Array for High‐Throughput 3D Cell Culture and Histology". Advanced Materials 32, n.º 40 (31 de agosto de 2020): 2004285. http://dx.doi.org/10.1002/adma.202004285.

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Suvannasankha, Attaya, Colin D. Crean, Douglas R. Tompkins, Jesus Delgado-Calle, Teresita M. Bellido, G. David Roodman y John M. Chirgwin. "Regulation of Osteoblast Function in Myeloma Bone Disease By Semaphorin 4D". Blood 128, n.º 22 (2 de diciembre de 2016): 4439. http://dx.doi.org/10.1182/blood.v128.22.4439.4439.

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Abstract Multiple myeloma (MM) bone disease (MMBD) is characterized by activation of osteoclasts and suppression of osteoblastic differentiation, with these changes in the bone microenvironment supporting MM cell growth and drug resistance. These complex interactions between MM cells and bone cells are incompletely understood. Current bone targeted therapy with bisphosphonates or Denosumab only blocks bone resorption but has no effect on osteoblast activity and only modest effects on MM growth. Therefore, new MMBD treatments are needed. Semaphorin-4D (Sema4D; CD100), is made by osteoclasts and inhibits osteoblasts by binding to the Plexin B receptor. Breast cancers also express Sema4d, and silencing sema4D in MDA-MB-231 breast cancer cells suppresses bone metastasis (Yang Y et al, PLoS One 2016). Since breast cancers and MM both cause osteolytic bone destruction and soluble Sema4D and Plexin B levels are increased in sera of MM patients (Terpos et al, 2012), we tested if sema4D contributed to MMBD. qPCR analysis of human MM cell lines and primary CD138+ cells showed MM cells express high levels of sema4D mRNA, comparing to the MDA-MB-231 breast cancer cells. Analysis of previously reported gene expression array data confirmed that MM cells express sema4D at a higher level compared to bone marrow plasma cells of MGUS and healthy donors (GenomicScape.com; Zhan F et al, Blood 2007; Mattiolo M et al, Oncogene, 2005). These results plus those of Terpos et al suggest that MM cells commonly express Sema4D. We next asked if the bone microenvironment increases MM expression of Sema4D. We co-cultured human MM cell lines RPMI8226 and JJN3 with mouse bones. Species -specific changes in tumor and bone were evaluated by quantitative RT-PCR. MM cells engrafted onto mouse bones, increasing markers of osteolysis similar to those seen in MM bone disease. After a week of co-culture, Sema4D expression was increased in MM cells (mean ±SD; 4.2±0.4; p=0.023), compared to MM cells grown alone. In addition, bones co-cultured with MM cells expressed higher Sema4D mRNA than bones alone (mean ±SD; 3.6±0.21; p=0.03). While co-culture increased both MM and bone Sema4D, markers of osteoblast activity, Col1a1, alkaline phosphatase and osteocalcin were suppressed. Preliminary experiments suggest that osteocytes are a major source of Sema4D expression in bone, in addition to active osteoclasts, which are much rarer cells than osteocytes. The induction of Sema4D in bone was only partially inhibited by 100nM zoledronic acid to inhibit osteoclast activity. Since osteocytes can physically interact with MM cells in vivo (Delgado Calle, Cancer Res 2016), we then tested the effect of MM cells on osteocyte sema4D expression in co-cultures of RPMI 8226 and JJN3 MM cells with MOL-Y4 osteocytic cells, separated by transwells. Both MM cell lines increased the Sema4D mRNA content of MLO-Y4 cells (mean ±SD; 3.1±0.4; p=0.036), suggesting that myeloma-secreted factors regulate osteocyte Sema4D expression. Since Sema4D is a potent osteoblast inhibitor, our data suggest that osteocyte -derived Sema4D may be a major contributor to MMBD, and that neutralization of Sema4D activity should improve the suppressed bone formation in MM. Disclosures Roodman: Amgen: Consultancy.

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Burgstaller, Gerald, Sarah Vierkotten, Michael Lindner, Melanie Königshoff y Oliver Eickelberg. "Multidimensional immunolabeling and 4D time-lapse imaging of vital ex vivo lung tissue". American Journal of Physiology-Lung Cellular and Molecular Physiology 309, n.º 4 (15 de agosto de 2015): L323—L332. http://dx.doi.org/10.1152/ajplung.00061.2015.

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During the last decades, the study of cell behavior was largely accomplished in uncoated or extracellular matrix (ECM)-coated plastic dishes. To date, considerable cell biological efforts have tried to model in vitro the natural microenvironment found in vivo. For the lung, explants cultured ex vivo as lung tissue cultures (LTCs) provide a three-dimensional (3D) tissue model containing all cells in their natural microenvironment. Techniques for assessing the dynamic live interaction between ECM and cellular tissue components, however, are still missing. Here, we describe specific multidimensional immunolabeling of living 3D-LTCs, derived from healthy and fibrotic mouse lungs, as well as patient-derived 3D-LTCs, and concomitant real-time four-dimensional multichannel imaging thereof. This approach allowed the evaluation of dynamic interactions between mesenchymal cells and macrophages with their ECM. Furthermore, fibroblasts transiently expressing focal adhesions markers incorporated into the 3D-LTCs, paving new ways for studying the dynamic interaction between cellular adhesions and their natural-derived ECM. A novel protein transfer technology (FuseIt/Ibidi) shuttled fluorescently labeled α-smooth muscle actin antibodies into the native cells of living 3D-LTCs, enabling live monitoring of α-smooth muscle actin-positive stress fibers in native tissue myofibroblasts residing in fibrotic lesions of 3D-LTCs. Finally, this technique can be applied to healthy and diseased human lung tissue, as well as to adherent cells in conventional two-dimensional cell culture. This novel method will provide valuable new insights into the dynamics of ECM (patho)biology, studying in detail the interaction between ECM and cellular tissue components in their natural microenvironment.

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Angelats Lobo y Ginestra. "Cell Bioprinting: The 3D-Bioplotter™ Case". Materials 12, n.º 23 (2 de diciembre de 2019): 4005. http://dx.doi.org/10.3390/ma12234005.

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The classic cell culture involves the use of support in two dimensions, such as a well plate or a Petri dish, that allows the culture of different types of cells. However, this technique does not mimic the natural microenvironment where the cells are exposed to. To solve that, three-dimensional bioprinting techniques were implemented, which involves the use of biopolymers and/or synthetic materials and cells. Because of a lack of information between data sources, the objective of this review paper is, to sum up, all the available information on the topic of bioprinting and to help researchers with the problematics with 3D bioprinters, such as the 3D-Bioplotter™. The 3D-Bioplotter™ has been used in the pre-clinical field since 2000 and could allow the printing of more than one material at the same time, and therefore to increase the complexity of the 3D structure manufactured. It is also very precise with maximum flexibility and a user-friendly and stable software that allows the optimization of the bioprinting process on the technological point of view. Different applications have resulted from the research on this field, mainly focused on regenerative medicine, but the lack of information and/or the possible misunderstandings between papers makes the reproducibility of the tests difficult. Nowadays, the 3D Bioprinting is evolving into another technology called 4D Bioprinting, which promises to be the next step in the bioprinting field and might promote great applications in the future.

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Gerner, E. W., P. S. Mamont, A. Bernhardt y M. Siat. "Post-translational modification of the protein-synthesis initiation factor eIF-4D by spermidine in rat hepatoma cells". Biochemical Journal 239, n.º 2 (15 de octubre de 1986): 379–86. http://dx.doi.org/10.1042/bj2390379.

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The rates of synthesis and turnover of the rare amino acid hypusine [N6-(4-amino-2-hydroxybutyl)-2,6-diaminohexanoic acid] in protein were studied in relationship to polyamine metabolism and growth rates in rat hepatoma tissue-culture (HTC) cells. Hypusine is selectively formed in the eukaryotic translation initiation factor eIF-4D, by a post-translational mechanism involving spermidine [Cooper, Park, Folk, Safer & Braverman (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1854-1857]. The half-life of the hypusine-containing protein was longer than 24 h. In cells whose intracellular spermidine pools had been initially depleted, by using DL-alpha-difluoromethylornithine (DFMO), maximum synthesis rates of hypusine in protein were 5-10 times higher, on restoration of endogenous spermidine contents by exogenous addition, than those observed in untreated exponential-phase cultures. In cells pretreated with DFMO, the rate of hypusine synthesis was constant for up to 1 h after the addition of 5 microM-spermidine, whereas endogenous spermidine contents varied from less than 1 to more than 10 nmol/mg of protein. However, the overall amount of hypusine formed, during the first 1 h after the addition of various concentrations of spermidine (0.05-10 microM) to the culture medium, was markedly dependent on the final endogenous spermidine content achieved at the end of the 1 h measurement interval. Early in exponential-phase growth, protein-bound hypusine was synthesized at a rate of 1-2 pmol/h per mg of protein. This rate decreased to less than 0.5 pmol/h per mg of protein when cell growth rates decreased as cultures reached high cell densities. Analysis of the polyamine substrate specificity for hypusine formation showed that N1-acetylspermidine did not compete with spermidine in the reaction, nor did N1-(buta-2,3-dienyl)-N2-methylbutane-1,4-diamine, and irreversible inhibitor of polyamine oxidase, block the reaction. On the basis of comparative radiolabelling experiments, spermine was either a poor substrate, or not a substrate, for hypusine formation. These results confirm that spermidine is the likely precursor of the aminohydroxybutyl moiety of hypusine, and show that overall hypusine formation, but not necessarily the synthesis rate, is dependent on the endogenous spermidine concentration, especially under conditions where spermidine concentrations are initially low, as is the case after DFMO treatment, and then increase.

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Dejani, Naiara, Felipe Fortino, Victoria Nino y Alexandra Ivo Medeiros. "‘Efferocytosis of infected apoptotic cells inhibits Th17 differentiation via PGE2-EP4 signaling’". Journal of Immunology 196, n.º 1_Supplement (1 de mayo de 2016): 133.34. http://dx.doi.org/10.4049/jimmunol.196.supp.133.34.

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Abstract Clearance of infected apoptotic cells (IAC) by dendritic cells (DC) triggers the production of Th17-related cytokines. Moreover, we have found that prostaglandin E2 (PGE2) is also produced during efferocytosis containing microbial products, but the effect of this prostanoid on Th17-differentiation is still unknown. The purpose of this study was to evaluate the role of PGE2, produced during recognition of IAC by DC, on Th17 differentiation. As source of IAC, Raw 264-7 cells were cultured with Escherichia coli during 2h for phagocytosis and apoptosis was induced by UV irradiation. BMDC were co-culture with IAC during 18h, and the supernatant from this co-culture was used to differentiate naïve CD4+ T cells for 4d, plus anti-CD3/anti-CD28. Phagocytosis of IAC by DC promotes the production of high levels of PGE2 (15±0.7 ng/ml), IL-6 (26±0.4 ng/ml), TGF-β (3.5±0.09 ng/ml) and IL-1β (5.4±0.1 ng/ml), when compared to phagocytosis of uninfected-AC. The treatment with COX inhibitor significantly decreased PGE2 level and promotes Th17 cell differentiation at least 4 times higher (16%) than untreated co-cultures (4%). Moreover, adding back exogenous PGE2 decreased drastically the percentage of Th17 cells (2%). Moreover, the treatment of naive T cells with antagonists of PGE2 receptor (EP1/EP2/EP4) suggests that the suppressor effect of this prostanoid on Th17 cell differentiation is mainly mediated by EP4/cAMP/PKA activation pathway. Taken all together, our results show that PGE2 is able to suppress Th17 differentiation in vitro through EP4 signaling. Further studies are needed to clarify how PGE2 can modulate the Th17 differentiation using in vivo model. Financial Support: FAPEP 14/17374-3, 12/23580-0, 11/17611-7.

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Batalov, Ivan, Kelly R. Stevens y Cole A. DeForest. "Photopatterned biomolecule immobilization to guide three-dimensional cell fate in natural protein-based hydrogels". Proceedings of the National Academy of Sciences 118, n.º 4 (19 de enero de 2021): e2014194118. http://dx.doi.org/10.1073/pnas.2014194118.

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Hydrogel biomaterials derived from natural biopolymers (e.g., fibrin, collagen, decellularized extracellular matrix) are regularly utilized in three-dimensional (3D) cell culture and tissue engineering. In contrast to those based on synthetic polymers, natural materials permit enhanced cytocompatibility, matrix remodeling, and biological integration. Despite these advantages, natural protein-based gels have lagged behind synthetic alternatives in their tunability; methods to selectively modulate the biochemical properties of these networks in a user-defined and heterogeneous fashion that can drive encapsulated cell function have not yet been established. Here, we report a generalizable strategy utilizing a photomediated oxime ligation to covalently decorate naturally derived hydrogels with bioactive proteins including growth factors. This bioorthogonal photofunctionalization is readily amenable to mask-based and laser-scanning lithographic patterning, enabling full four-dimensional (4D) control over protein immobilization within virtually any natural protein-based biomaterial. Such versatility affords exciting opportunities to probe and direct advanced cell fates inaccessible using purely synthetic approaches in response to anisotropic environmental signaling.

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Strach, Madeleine Cornelia, Nicole Yeung, Hui-Ming Lin, Nabila Ansari, Cherry Koh, Joo-Shik Shin, James Kench et al. "Patient-derived explant model of appendiceal cancer." Journal of Clinical Oncology 40, n.º 16_suppl (1 de junio de 2022): 4160. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.4160.

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4160 Background: Appendiceal cancers (AC) are rare with 5-year survival of 15% for high grade (HG) adenocarcinomas and 75% for low grade (LG) mucinous neoplasms. There is limited literature on the AC tumour microenvironment (TME) in disease progression and drug resistance. Ex vivo cultures, such as patient derived explants (PDEs), have been used for other solid tumours to test anticancer agents, explore the TME, and are being developed as personalised models. The aim of our study was to develop a PDE model of AC, preserve the TME, study the biological profile of AC and test novel therapies. Methods: Fresh tissue was collected during cytoreductive surgery (CRS) from consenting patients with AC with peritoneal disease, Jul 2020-Mar 2021. Tissues from 10 patients were dissected and cultured as PDEs under varying conditions of tissue size, media, matrix support and duration (Table). Uncultured Day 0 tissues and PDEs were fixed in formalin prior to paraffin embedding (FFPE). Immunohistochemical staining was performed on sections of FFPE tissue to assess viability with antibodies against the tumour marker, Cytokeratin-20 (CK20), and cell death marker, cleaved caspase 3 (CC3). Tissue architecture was rated on a 4-point scale (MS, J-SS). Cancer cells were counted in 6 Day 0 samples and PDEs from 3 patients using QuPath v0.3.2. Apoptotic index (AI) was calculated as the proportion of cells positive for CC3 divided by the number of total CK20 positive cells. Results: The mean proportion of tumour and mucin in the tissues was 3% (0-60%) and 39% (0-95%) respectively. Day 0 samples were viable with mean AI of cancer cells 7% (0-4%). Tissue architecture was maintained, as compared to Day 0 control, for varying sizes of PDE and culture durations up to 4d. More small or medium-sized PDEs had improved architecture compared to large sized PDEs (Table). PDEs at 4d had poorer architecture compared to at 1-3d. There was improved architecture in PDEs using enriched media with support factors compared to base media, and in specimens with matrix support compared to none. The mean AI of PDE cancer cells was 21% (0-92%) and 51% of PDEs had no cancer cell death. Conclusions: This is the first study demonstrating that AC tissue is amenable to ex vivo culture as PDEs. The optimal PDE model was <10mm tissue placed on a gelatine sponge in enriched media for 1-3d. PDEs had preserved tissue architecture and viability compared to uncultured tissue. Protein expression was in keeping with the original tumour. We plan to use this model to test anticancer agents and explore the TME of AC.[Table: see text]

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Sukparangsi, W., R. Bootsri, W. Sikeao, S. Karoon y A. Thongphakdee. "181 Establishment of Induced Pluripotent Stem Cells from Fishing Cat and Clouded Leopard Using Integration-Free Method for Wildlife Conservation". Reproduction, Fertility and Development 30, n.º 1 (2018): 230. http://dx.doi.org/10.1071/rdv30n1ab181.

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Fishing cat (Prionailurus viverrinus) and clouded leopard (Neofelis nebulosa) are wild felids, currently in vulnerable status according to the International Union for Conservation of Nature red list (2017). Several measures in assisted reproductive technology (e.g. AI, embryo transfer) have been used by the Zoological Park Organization of Thailand (ZPO) to increase their offspring in captivity. Recently, the generation of induced pluripotent stem cell (iPS cells) becomes popular and provides alternative way to preserve good genetics in the form of cell with diverse capacities. This great potential of iPS cells is unlimited self-renewal and pluripotency, similar to embryonic stem cells (ESC). Under the right cell culture conditions, pluripotent stem cells can differentiate into all cell types of the body. Here, we aimed to find the optimal condition to generate integration-free iPS cells from fishing cat and clouded leopard. At first, to obtain somatic cells for cellular reprogramming, adult dermal fibroblast cell lines from both species were established from belly skin tissues. Subsequently, several nucleofection programs of AmaxaTM 4D-nucleofectorTM (Lonza, Basel, Switzerland) were examined to introduce integration-free DNA vectors carrying reprogramming factors into the felid fibroblasts. The transfected cells were cultured under numerous conditions: (1) matrix/defined surface including irradiated mouse embryonic fibroblast, gelatin, vitronectin, and Geltrex® (Thermo Fisher Scientific, Waltham, MA, USA); (2) ESC/iPS cell medium including Essential 8TM (Thermo Fisher Scientific) DMEM containing KnockOutTM Serum Replacement (KOSR; Thermo Fisher Scientific) and/or fetal bovine serum (FBS); and (3) supplement including basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), l-ascorbic acid, nicotinamide, ALK5 inhibitor (A83-01) and RevitaCellTM (Thermo Fisher Scientific). We found that optimal nucleofection programs for human dermal fibroblast including FF-135 and EN-150 were able to transfer episomal vectors and excisable piggyBAC transposon carrying reprogramming factors into fishing cat and clouded leopard fibroblasts, respectively. The iPS-like colonies appeared around 26 to 30 days post-nucleofection. The culture of transfected cells on either Geltrex® or Vitronectin-coated surface supports the formation of iPS-like colonies with different derivation efficiency (0.01 and 0.005%, respectively). In addition, all colonies were formed under medium containing FBS, together with both bFGF and LIF supplements. Taken together, we have developed a platform to generate iPS cells from tissue collection to the establishment of iPS cell culture. This will further enable us to apply the technique to obtain iPS cells from other endangered and vulnerable felid species.

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Schöneberg, Johannes, Daphné Dambournet, Tsung-Li Liu, Ryan Forster, Dirk Hockemeyer, Eric Betzig y David G. Drubin. "4D cell biology: big data image analytics and lattice light-sheet imaging reveal dynamics of clathrin-mediated endocytosis in stem cell–derived intestinal organoids". Molecular Biology of the Cell 29, n.º 24 (26 de noviembre de 2018): 2959–68. http://dx.doi.org/10.1091/mbc.e18-06-0375.

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New methods in stem cell 3D organoid tissue culture, advanced imaging, and big data image analytics now allow tissue-scale 4D cell biology, but currently available analytical pipelines are inadequate for handing and analyzing the resulting gigabytes and terabytes of high-content imaging data. We expressed fluorescent protein fusions of clathrin and dynamin2 at endogenous levels in genome-edited human embryonic stem cells, which were differentiated into hESC-derived intestinal epithelial organoids. Lattice light-sheet imaging with adaptive optics (AO-LLSM) allowed us to image large volumes of these organoids (70 × 60 × 40 µm xyz) at 5.7 s/frame. We developed an open-source data analysis package termed pyLattice to process the resulting large (∼60 Gb) movie data sets and to track clathrin-mediated endocytosis (CME) events. CME tracks could be recorded from ∼35 cells at a time, resulting in ∼4000 processed tracks per movie. On the basis of their localization in the organoid, we classified CME tracks into apical, lateral, and basal events and found that CME dynamics is similar for all three classes, despite reported differences in membrane tension. pyLattice coupled with AO-LLSM makes possible quantitative high temporal and spatial resolution analysis of subcellular events within tissues.

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MUNASINGHE, VARUNI S., D. STARK y J. T. ELLIS. "New advances in thein-vitroculture ofDientamoeba fragilis". Parasitology 139, n.º 7 (16 de febrero de 2012): 864–69. http://dx.doi.org/10.1017/s0031182012000145.

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SUMMARYDientamoeba fragilisis an intestinal protozoan in humans that is commonly associated with diarrhoea and other gastrointestinal complaints. Studies conducted to investigate the biology of this parasite are limited by methods forin vitrocultivation. The objective of this study was to improve a biphasic culture medium, based on the Loeffler's slope, by further supplementation in order to increase the yield of trophozoites in culture. The currentin vitroculture ofD. fragilisis a xenic culture with a mix of bacteria. Three different liquid overlays were evaluated including Earle's balanced salt solution (EBSS), PBS and Dulbecco's modified PBS (DPBS), for their ability to support thein vitrogrowth ofD. fragilistrophozoites. Out of these 3 overlays EBSS gave the highest increase in the trophozoite numbers. The effect of supplementation was analysed by supplementing EBSS with ascorbic acid, ferric ammonium citrate, L-cysteine, cholesterol and alpha-lipoic acid and quantification ofin vitrogrowth by cell counts. A new liquid overlay is here described based upon EBSS supplemented with cholesterol and ferric ammonium citrate that, in conjunction with the Loeffler's slope, supports the growth ofD. fragilistrophozoitesin vitro. This modified overlay supported a 2-fold increase in the numbers of trophozoite in culture from all 4D. fragilisisolates tested, when compared to a PBS overlay. These advances enable the harvest of a larger number of trophozoites needed for further studies on this parasite.

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Górnicki, Tomasz, Jakub Lambrinow, Afsaneh Golkar-Narenji, Krzysztof Data, Dominika Domagała, Julia Niebora, Maryam Farzaneh et al. "Biomimetic Scaffolds—A Novel Approach to Three Dimensional Cell Culture Techniques for Potential Implementation in Tissue Engineering". Nanomaterials 14, n.º 6 (16 de marzo de 2024): 531. http://dx.doi.org/10.3390/nano14060531.

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Biomimetic scaffolds imitate native tissue and can take a multidimensional form. They are biocompatible and can influence cellular metabolism, making them attractive bioengineering platforms. The use of biomimetic scaffolds adds complexity to traditional cell cultivation methods. The most commonly used technique involves cultivating cells on a flat surface in a two-dimensional format due to its simplicity. A three-dimensional (3D) format can provide a microenvironment for surrounding cells. There are two main techniques for obtaining 3D structures based on the presence of scaffolding. Scaffold-free techniques consist of spheroid technologies. Meanwhile, scaffold techniques contain organoids and all constructs that use various types of scaffolds, ranging from decellularized extracellular matrix (dECM) through hydrogels that are one of the most extensively studied forms of potential scaffolds for 3D culture up to 4D bioprinted biomaterials. 3D bioprinting is one of the most important techniques used to create biomimetic scaffolds. The versatility of this technique allows the use of many different types of inks, mainly hydrogels, as well as cells and inorganic substances. Increasing amounts of data provide evidence of vast potential of biomimetic scaffolds usage in tissue engineering and personalized medicine, with the main area of potential application being the regeneration of skin and musculoskeletal systems. Recent papers also indicate increasing amounts of in vivo tests of products based on biomimetic scaffolds, which further strengthen the importance of this branch of tissue engineering and emphasize the need for extensive research to provide safe for humansbiomimetic tissues and organs. In this review article, we provide a review of the recent advancements in the field of biomimetic scaffolds preceded by an overview of cell culture technologies that led to the development of biomimetic scaffold techniques as the most complex type of cell culture.

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Liu, Haiyan, Bo Hu y Yuan Song. "The role of donor memory-like NK cell infusion in allogeneic hematopoietic stem cell transplantation". Journal of Immunology 196, n.º 1_Supplement (1 de mayo de 2016): 140.1. http://dx.doi.org/10.4049/jimmunol.196.supp.140.1.

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Abstract Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative therapy for patients with chemotherapy-resistant hematologic malignancies and some solid tumors. However, relapse after allo-HSCT is common in those with high-risk disease and typically portends a poor prognosis. Previous studies showed that donor NK cell infusion could prevent disease recurrence possibly through killing residual leukemia cells. However, NK cell-based immunotherapy is hampered by their transient effector function. Recently, IL-12/15/18-preactivated memory-like NK cells were shown to persist with sustained effector function, we investigated whether memory-like donor NK cells could improve donor NK cell’s GVL effect in murine allo-HSCT. NK cells preactivated with IL-12/18 or IL-12/15/18 produce high levels of IFN-γ upon restimulation by IL-12/15 after 4d of in vitro culture. The infusion of IL-12/15/18 or IL-12/18-preactivated NK cells significantly promoted the GVL effects compared to donor HSCs infusion alone. After adoptive transfer, IL-12/18 and IL-12/15/18 preactivated NK cells were found in higher numbers in spleen, liver and lung compared with the control NK cells. The proliferation of the transferred NK cells was greatly promoted by IL-12/18 and IL-12/15/18 preactivation. Despite of the strong GVL effect of the IL-12/15/18-preactivated NK cells, they aggravated acute GVHD whereas IL-12/18-preactivated NK cell infusion significantly prolonged the GVHD-related survival compared with the HSCs alone or control NK cells. Therefore, IL-12/18-preactivated memory-like donor NK cell infusion may be a more effective and safer strategy for preventing or treating relapse after allo-HSCT.

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Yang, Rui, Boyang Zhang, Yueqi Wang, Yan Zhang, Yansen Zhao, Daozhen Jiang, Lanxin Chen, Bo Tang y Xueming Zhang. "H3K9me3 Levels Affect the Proliferation of Bovine Spermatogonial Stem Cells". International Journal of Molecular Sciences 25, n.º 17 (25 de agosto de 2024): 9215. http://dx.doi.org/10.3390/ijms25179215.

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Spermatogonial stem cells (SSCs) possess the characteristics of self-renewal and differentiation, as well as the ability to generate functional sperm. Their unique stemness has broad applications in male infertility treatment and species preservation. In rodents, research on SSCs has been widely reported, but progress is slow in large livestock such as cattle and pigs due to long growth cycles, difficult proliferation in vitro, and significant species differences. Previously, we showed that histone 3 (H3) lysine 9 (K9) trimethylation (H3K9me3) is associated with the proliferation of bovine SSCs. Here, we isolated and purified SSCs from calf testicular tissues and investigated the impact of different H3K9me3 levels on the in vitro proliferation of bovine SSCs. The enriched SSCs eventually formed classical stem cell clones in vitro in our feeder-free culture system. These clones expressed glial cell-derived neurotrophic factor family receptor alpha-1 (GFRα1, specific marker for SSCs), NANOG (pluripotency protein), C-KIT (germ cell marker), and strong alkaline phosphatase (AKP) positivity. qRT-PCR analysis further showed that these clones expressed the pluripotency genes NANOG and SOX2, and the SSC-specific marker gene GFRα1. To investigate the dynamic relationship between H3K9me3 levels and SSC proliferation, H3K9me3 levels in bovine SSCs were first downregulated using the methyltransferase inhibitor, chaetocin, or transfection with the siRNA of H3K9 methyltransferase suppressor of variegation 3-9 homologue 1 (SUV39H1). The EDU (5-Ethynyl-2′-deoxyuridine) assay revealed that SSC proliferation was inhibited. Conversely, when H3K9me3 levels in bovine SSCs were upregulated by transfecting lysine demethylase 4D (KDM4D) siRNA, the EDU assay showed a promotion of cell proliferation. In summary, this study established a feeder-free culture system to obtain bovine SSCs and explored its effects on the proliferation of bovine SSCs by regulating H3K9me3 levels, laying the foundation for elucidating the regulatory mechanism underlying histone methylation modification in the proliferation of bovine SSCs.

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Zhang, Zhenhe, Miles Freeman, Yiqiang Zhang, Danny El-Nachef, George Davenport, Allison Williams y W. Robb MacLellan. "Hippo signaling and histone methylation control cardiomyocyte cell cycle re-entry through distinct transcriptional pathways". PLOS ONE 18, n.º 2 (13 de febrero de 2023): e0281610. http://dx.doi.org/10.1371/journal.pone.0281610.

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Aims Accumulating data demonstrates that new adult cardiomyocytes (CMs) are generated throughout life from pre-existing CMs, although the absolute magnitude of CM self-renewal is very low. Modifying epigenetic histone modifications or activating the Hippo-Yap pathway have been shown to promote adult CM cycling and proliferation. Whether these interventions work through common pathways or act independently is unknown. For the first time we have determined whether lysine demethylase 4D (KDM4D)-mediated CM-specific H3K9 demethylation and Hippo pathways inhibition have additive or redundant roles in promoting CM cell cycle re-entry. Methods and results We found that activating Yap1 in cultured neonatal rat ventricular myocytes (NRVM) through overexpressing Hippo pathway inhibitor, miR-199, preferentially increased S-phase CMs, while H3K9me3 demethylase KDM4D preferentially increased G2/M markers in CMs. Together KDM4D and miR-199 further increased total cell number of NRVMs in culture. Inhibition of Hippo signaling via knock-down of Salvador Family WW Domain Containing Protein 1 (Sav1) also led to S-phase reactivation and additional cell cycle re-entry was seen when combined with KDM4D overexpression. Inducible activating KDM4D (iKDM4D) in adult transgenic mice together with shRNA mediated knock-down of Sav1 (iKDM4D+Sav1-sh) resulted in a significant increase in cycling CMs compared to either intervention alone. KDM4D preferentially induced expression of genes regulating late (G2/M) phases of the cell cycle, while miR-199 and si-Sav1 preferentially up-regulated genes involved in G1/S phase. KDM4D upregulated E2F1 and FoxM1 expression, whereas miR-199 and si-Sav1 induced Myc. Using transgenic mice over-expressing KDM4D together with Myc, we demonstrated that KDM4D/Myc significantly increased CM cell cycling but did not affect cardiac function. Conclusions KDM4D effects on CM cell cycle activity are additive with the Hippo-Yap1 pathway and appear to preferentially regulate different cell cycle regulators. This may have important implications for strategies that target cardiac regeneration in treating heart disease

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Drakul, Marija, Sergej Tomić, Marina Bekić, Dušan Mihajlović, Miloš Vasiljević, Sara Rakočević, Jelena Đokić, Nikola Popović, Dejan Bokonjić y Miodrag Čolić. "Sitagliptin Induces Tolerogenic Human Dendritic Cells". International Journal of Molecular Sciences 24, n.º 23 (27 de noviembre de 2023): 16829. http://dx.doi.org/10.3390/ijms242316829.

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Sitagliptin, an anti-diabetic drug, is a dipeptidyl peptidase (DPP)-4/CD26 inhibitor with additional anti-inflammatory and immunomodulatory properties. In this study, we investigated for the first time the effect of sitagliptin on the differentiation and functions of human dendritic cells generated from monocytes (MoDCs) for 4 days using the standard GM-CSF/IL-4 procedure. LPS/IFN-γ treatment for an additional 24 h was used for maturation induction of MoDCs. Sitagliptin was added at the highest non-cytotoxic concentration (500 µg/mL) either at the beginning (sita 0d protocol) or after MoDC differentiation (sita 4d protocol). Sitagliptin impaired differentiation and maturation of MoDCs as judged with the lower expression of CD40, CD83, CD86, NLRP3, and HLA-DR, retention of CD14 expression, and inhibited production of IL-β, IL-12p70, IL-23, and IL-27. In contrast, the expression of CD26, tolerogenic DC markers (ILT4 and IDO1), and production of immunoregulatory cytokines (IL-10 and TGF-β) were increased. Generally, the sita 0d protocol was more efficient. Sitagliptin-treated MoDCs were poorer allostimulators of T-cells in MoDC/T-cell co-culture and inhibited Th1 and Th17 but augmented Th2 and Treg responses. Tolerogenic properties of sitagliptin-treated MoDCs were additionally confirmed by an increased frequency of CD4+CD25+CD127- FoxP3+ Tregs and Tr1 cells (CD4+IL-10+FoxP3-) in MoDC/T-cell co-culture. The differentiation of IL-10+ and TGF-β+ Tregs depended on the sitagliptin protocol used. A Western blot analysis showed that sitagliptin inhibited p65 expression of NF-kB and p38MAPK during the maturation of MoDCs. In conclusion, sitagliptin induces differentiation of tolerogenic DCs, and the effect is important when considering sitagliptin for treating autoimmune diseases and allotransplant rejection.

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Munawaroh, Zahrotul, Putri Kemala Sari, Bagas Setiyo Pambudi y Rahmi Faradisya Ekapti. "DEVELOPMENT OF THE ETINTEC STUDENT BOOK (ETHNOSCIENCE-BASED INTERACTIVE TEACHING STUDENT BOOK) AS AN INTERACTIVE LEARNING MEDIA BASED ON THE TYPICAL PONOROGO CULTURE ON ECOLOGY AND BIODIVERSITY MATERIALS". INSECTA: Integrative Science Education and Teaching Activity Journal 3, n.º 2 (30 de noviembre de 2022): 158–67. http://dx.doi.org/10.21154/insecta.v3i2.5143.

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Science learning is still often considered difficult. But basically, it will feel easy if it is based on interest. Ecology and biodiversity material, which mostly contains theory, requires a strategy to get students interested, namely by associating something around them with that material. Based on this, research was carried out with the aim of developing an interactive student book called the Etintec Student Book on ecology and biodiversity based on the ethnoscience of culture typical of Ponorogo Regency. This research is development research with a modified model from 4D to 3D with stages including defining, designing, and developing. In the define stage, things are found that become a reference for designing innovative products, starting from learning problems to development goals. At the design stage, the Etintec Student Book product was produced including layout design, material preparation (narration, comics, Ponorogo cultural reviews), evaluation preparation consisting of crossword puzzles (TTS), digital evaluation with quizizz (QR Code), and project evaluation. And at the development stage validation was carried out on several learning media experts from science teachers and lecturers with a score of 3.58, meaning that this product can be used with minor improvements. This research can be continued with the field practice test phase and the effectiveness test so that later the Etintec Student Book can be widely used and the dissemination stage can be carried out.

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23

Guerra, Antonio J., Hernan Lara-Padilla, Matthew L. Becker, Ciro A. Rodriguez y David Dean. "Photopolymerizable Resins for 3D-Printing Solid-Cured Tissue Engineered Implants". Current Drug Targets 20, n.º 8 (10 de mayo de 2019): 823–38. http://dx.doi.org/10.2174/1389450120666190114122815.

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With the advent of inexpensive and highly accurate 3D printing devices, a tremendous flurry of research activity has been unleashed into new resorbable, polymeric materials that can be printed using three approaches: hydrogels for bioprinting and bioplotting, sintered polymer powders, and solid cured (photocrosslinked) resins. Additionally, there is a race to understand the role of extracellular matrix components and cell signalling molecules and to fashion ways to incorporate these materials into resorbable implants. These chimeric materials along with microfluidic devices to study organs or create labs on chips, are all receiving intense attention despite the limited number of polymer systems that can accommodate the biofabrication processes necessary to render these constructs. Perhaps most telling is the limited number of photo-crosslinkable, resorbable polymers and fabrication additives (e.g., photoinitiators, solvents, dyes, dispersants, emulsifiers, or bioactive molecules such as micro-RNAs, peptides, proteins, exosomes, micelles, or ceramic crystals) available to create resins that have been validated as biocompatible. Advances are needed to manipulate 4D properties of 3D printed scaffolds such as pre-implantation cell culture, mechanical properties, resorption kinetics, drug delivery, scaffold surface functionalization, cell attachment, cell proliferation, cell maturation, or tissue remodelling; all of which are necessary for regenerative medicine applications along with expanding the small set of materials in clinical use. This manuscript presents a review of the foundation of the most common photopolymerizable resins for solidcured scaffolds and medical devices, namely, polyethylene glycol (PEG), poly(D, L-lactide) (PDLLA), poly-ε-caprolactone (PCL), and poly(propylene fumarate) (PPF), along with methodological advances for 3D Printing tissue engineered implants (e.g., via stereolithography [SLA], continuous Digital Light Processing [cDLP], and Liquid Crystal Display [LCD]).

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Perisse, I. V., G. Almeida-Porada, C. D. Porada, K. L. White y I. A. Polejaeva. "93 A sheep model of sickle cell disease using CRISPR/Cas9 and somatic cell nuclear transfer". Reproduction, Fertility and Development 33, n.º 2 (2021): 154. http://dx.doi.org/10.1071/rdv33n2ab93.

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Sickle cell disease (SCD) is the most common inherited hemoglobinopathy, with more than 2 million people in the United States alone carrying the sickle gene. Approximately 100 000 of these people are homozygous and suffer from SCD. Worldwide, there are ∼4.4 million people with SCD. SCD is caused by a single A to T nucleotide replacement at the sixth codon of the β-globin gene, which results in the substitution of a valine for glutamate in the β-globin protein. This causes the resultant tetrameric haemoglobin molecule to be unstable and the red cells carrying this aberrant protein to “sickle,” decreasing the ability of these cells to carry oxygen. Sheep and humans exhibit a high degree of homology at the level of the genome. In addition, their anatomy, organ physiology, and immune system development closely parallel that of humans during fetal life. The ovine β-globin (HBB) gene shares 87.5% similarity with human HBB. Therefore, we hypothesised that the introduction of the “sickle” mutation in the sheep genome would lead to the SCD phenotype in sheep that could provide a valuable platform for evaluating prenatal and postnatal drug and gene therapies for this disease. In this study, we used a CRISPR/Cas9 gene-editing approach to introduce the SCD mutation into the sheep β-globin/HBB gene. We designed a single guide (sg)RNA targeting exon 1 of the sheep β-globin/HBB gene using the Benchling software (https://benchling.com/academic). The sgRNA was synthesised by Synthego and Cas9 purchased from IDT. Using the Lonza-4D-Nucleofector system, the Cas9/sgRNA ribonucleoprotein complex was transfected into sheep fetal fibroblasts (SFFs) along with 101-bp single-stranded oligodeoxynucleotides, flanking the sickle cell mutation to enable homology-directed repair. The transfected SFFs were then cultured in Dulbecco’s modified Eagle medium, supplemented with 15% fetal bovine serum and 1% penicillin, and incubated at 38.5°C. After 2 days, DNA was extracted from one-third of the SFFs and the remainder were seeded individually into five 96-well plates by limited dilution. After 7 days of culture, individual colonies were expanded into 24-well plates and cultured for an additional 3 days. PCR-restriction fragment length polymorphism (RFLP) analysis using Image J software demonstrated a high rate of mutations (∼70%) by either indels or SCD mutation that led to the loss of the restriction enzyme site, which was further supported by the analysis of cell colonies. We isolated 59 single cell-derived SFF colonies and, based on PCR/RLFP assay, 31/59 (52%) of them contained biallelic mutations (either indels or point mutations) and were subsequently submitted for Sanger sequencing. The sequencing demonstrated that 3 colonies (9.6%) contained biallelic SCD mutations in the β-globin/HBB gene. These data demonstrate that we successfully introduced the SCD mutation into SFFs. These cells will be used in the production of the first large animal (sheep) SCD model by somatic cell nuclear transfer in fall of 2020. This research was supported by UAES project 1343 and by USDA/NIFA multistate research project W-4171.

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25

Perisse, I. V., G. Almeida-Porada, C. D. Porada, K. L. White y I. A. Polejaeva. "93 A sheep model of sickle cell disease using CRISPR/Cas9 and somatic cell nuclear transfer". Reproduction, Fertility and Development 33, n.º 2 (2021): 154. http://dx.doi.org/10.1071/rdv33n2ab93.

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Sickle cell disease (SCD) is the most common inherited hemoglobinopathy, with more than 2 million people in the United States alone carrying the sickle gene. Approximately 100 000 of these people are homozygous and suffer from SCD. Worldwide, there are ∼4.4 million people with SCD. SCD is caused by a single A to T nucleotide replacement at the sixth codon of the β-globin gene, which results in the substitution of a valine for glutamate in the β-globin protein. This causes the resultant tetrameric haemoglobin molecule to be unstable and the red cells carrying this aberrant protein to “sickle,” decreasing the ability of these cells to carry oxygen. Sheep and humans exhibit a high degree of homology at the level of the genome. In addition, their anatomy, organ physiology, and immune system development closely parallel that of humans during fetal life. The ovine β-globin (HBB) gene shares 87.5% similarity with human HBB. Therefore, we hypothesised that the introduction of the “sickle” mutation in the sheep genome would lead to the SCD phenotype in sheep that could provide a valuable platform for evaluating prenatal and postnatal drug and gene therapies for this disease. In this study, we used a CRISPR/Cas9 gene-editing approach to introduce the SCD mutation into the sheep β-globin/HBB gene. We designed a single guide (sg)RNA targeting exon 1 of the sheep β-globin/HBB gene using the Benchling software (https://benchling.com/academic). The sgRNA was synthesised by Synthego and Cas9 purchased from IDT. Using the Lonza-4D-Nucleofector system, the Cas9/sgRNA ribonucleoprotein complex was transfected into sheep fetal fibroblasts (SFFs) along with 101-bp single-stranded oligodeoxynucleotides, flanking the sickle cell mutation to enable homology-directed repair. The transfected SFFs were then cultured in Dulbecco’s modified Eagle medium, supplemented with 15% fetal bovine serum and 1% penicillin, and incubated at 38.5°C. After 2 days, DNA was extracted from one-third of the SFFs and the remainder were seeded individually into five 96-well plates by limited dilution. After 7 days of culture, individual colonies were expanded into 24-well plates and cultured for an additional 3 days. PCR-restriction fragment length polymorphism (RFLP) analysis using Image J software demonstrated a high rate of mutations (∼70%) by either indels or SCD mutation that led to the loss of the restriction enzyme site, which was further supported by the analysis of cell colonies. We isolated 59 single cell-derived SFF colonies and, based on PCR/RLFP assay, 31/59 (52%) of them contained biallelic mutations (either indels or point mutations) and were subsequently submitted for Sanger sequencing. The sequencing demonstrated that 3 colonies (9.6%) contained biallelic SCD mutations in the β-globin/HBB gene. These data demonstrate that we successfully introduced the SCD mutation into SFFs. These cells will be used in the production of the first large animal (sheep) SCD model by somatic cell nuclear transfer in fall of 2020. This research was supported by UAES project 1343 and by USDA/NIFA multistate research project W-4171.

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26

Rajzer, Izabella, Anna Kurowska, Jarosław Janusz, Maksymilian Maślanka, Adam Jabłoński, Piotr Szczygieł, Janusz Fabia et al. "Four-Dimensional Printing of β-Tricalcium Phosphate-Modified Shape Memory Polymers for Bone Scaffolds in Osteochondral Regeneration". Materials 18, n.º 2 (11 de enero de 2025): 306. https://doi.org/10.3390/ma18020306.

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The use of scaffolds for osteochondral tissue regeneration requires an appropriate selection of materials and manufacturing techniques that provide the basis for supporting both cartilage and bone tissue formation. As scaffolds are designed to replicate a part of the replaced tissue and ensure cell growth and differentiation, implantable materials have to meet various biological requirements, e.g., biocompatibility, biodegradability, and mechanical properties. Osteoconductive materials such as tricalcium phosphate ceramics and some biodegradable polymers appear to be a perfect choice. The present work evaluates the structural, mechanical, thermal, and functional properties of a shape memory terpolymer modified with β-tricalcium phosphate (β-TCP). A new approach is using the developed materials for 4D printing, with a particular focus on its applicability in manufacturing medical implants. In this study, the manufacturing parameters of the scaffold components were developed. The scaffolds were examined via scanning electron microscopy with energy dispersive spectroscopy (SEM-EDS), Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and mechanical testing. The cytotoxicity result was obtained with an MTT assay, and the alkaline phosphatase (ALP) activity was measured. The structural and microstructural investigations confirmed the integration of β-TCP into the filament matrix and scaffolds. Thermal stability was enhanced as β-TCP delayed depolymerization of the polymer matrix. The shape memory studies demonstrated effective recovery. The in vitro cell culture studies revealed the significantly increased cell viability and alkaline phosphatase (ALP) activity of the β-TCP-modified terpolymer after 3 weeks. The developed terpolymer can be tailored for applications in which partial shape recovery is acceptable, such as bone scaffolds.

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27

Yamaki, T., T. Uede, N. Shijubo y K. Kikuchi. "Functional analysis of mononuclear cells infiltrating into tumors. III. Soluble factors involved in the regulation of T lymphocyte infiltration into tumors." Journal of Immunology 140, n.º 12 (15 de junio de 1988): 4388–96. http://dx.doi.org/10.4049/jimmunol.140.12.4388.

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Abstract We have analyzed the mechanisms controlling the accumulation of T lymphocytes in tumor tissues. Spleen cells, left or right popliteal lymph node cells, and tumor-infiltrating cells were obtained from tumor-inoculated rats and were cultured for 24 h. Culture supernatants were obtained and assessed for lymphocyte migration factor (LMF) activity with the use of a modified Boyden chamber. We found that tumor-infiltrating cells derived from T-9-sensitized rats produced LMF. Two waves of LMF production were observed. The first wave of LMF production was detected between 6 and 12 h (LMF-a) and the second wave of LMF production was detected between 4 and 6 days (LMF-4d and -6d) after tumor inoculation. The tumor-infiltrating cells consisted of heterogenous cell populations. We found that only tumor-infiltrating neutrophils of T-9-sensitized rats produced LMF-a. Five peaks of LMF (A through E) were detected upon fractionation of LMF-a using Mono Q anion exchange column chromatography. Peak D exhibited the strongest activity. The action of peak D was chemotactic, but not chemokinetic. The m.w. of peak D was 33,000 and 70,000. Only W3/25 (+) (helper/inducer) T cells were found to be sensitive to peak D. The production of LMF-a by purified tumor-infiltrating neutrophils in vitro is in agreement with the histologic observation that the infiltration of neutrophils precedes the appearance of W3/25 (+) T cells in tumor tissues of T-9-sensitized rats. It is thus likely that peak D of LMF-a is responsible for the infiltration of T lymphocytes into tumor tissues.

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Soper, Brian W., Mark D. Lessard, Travis L. Alley, Jennifer L. Proctor, Anthony J. Mourino y Jane E. Barker. "Treatment of Neurological Dysfunction in MPS VII and Batten Disease by Transplantation of Lentivirally Transduced Neuronal Stem Cells Cultured from Hematopoietic Tissue." Blood 106, n.º 11 (16 de noviembre de 2005): 1284. http://dx.doi.org/10.1182/blood.v106.11.1284.1284.

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Abstract The subventricular zone (SVZ) of lateral ventricles in brain contains neuronal stem cells (NSC) that form neurospheres when cultured with EGF and/or bFGF. Progeny from transplanted NSC migrate throughout the brain and replace multiple differentiated cells. Our goal is to develop this phenomenon into a cellular/gene therapeutic approach for treatment of neurological disease. Progeny of normal or gene transduced NSC can replace defective host cells or act as enzyme delivery vehicles. One model for testing this approach is the Mucopolysaccharidosis Type VII (MPS VII) mouse that is deficient in β-glucuronidase (GUS) expression, causing lysosomal storage disease (Sly Syndrome). Undegraded substrates accumulate in brain, causing cognitive dysfunction. A second model is deficient in palmitoyl-protein thioesterase 1 (PPT1), causing neurodegeneration in humans and mice known as infantile neuronal ceroid lipofuscinosis, or Battens Disease. By 24–30wks of age, mice develop motor abnormalities and seizures. Obstacles to NSC therapy include suitable source of donor cells and immunological rejection. Since mesenchymal stem cells (MSC) derived from bone marrow and cord blood have neuronal differentiation capacity, we believe these tissues can generate NSC. If so, self bone marrow or cord blood derived NSC could be transduced with a lentivirus that restores enzyme expression, solving both obstacles. We cultured 15d fetal liver (the murine equivalent of cord blood) from eGFP transgenic mice in neurosphere medium containing EGF and bFGF +/− Noggin/Fc. Noggin is an antagonist for bone morphogenic protein, is expressed in ependymal cells of the SVZ, upregulates neurogenesis, and inhibits bone formation. Growth factors were added days 1–5, and alternating days thereafter. Half the media was replaced every 4d. At 14d, the +Noggin culture had 2–3x the confluency of the -Noggin culture and contained spindle-shaped cells resembling MSC. After another 14d with EGF and bFGF alone, the +Noggin cells formed neurospheres. Neurospheres reformed after trituration, indicating self-renewal capacity. The neurospheres were Nestin+, a marker for NSC, and upon differentiation with serum, β-NGF, BDNF, and NT-3, stained positive for neurons (MAP2) and astrocytes (GFAP). Next, we transplanted 250,000 normal eGFP fetal liver derived NSC (FL NSC) into the lateral ventricles of PPT1−/ − neonatal recipients. Brains 18–54 days post transplant revealed cells in the SVZ of the lateral, dorsolateral, and third ventricles. Donor cells migrated away from the ventricles, into the hippocampal fimbria, under the corpus callosum, and into the rostral migratory stream. Later time points revealed increased migration away from ventricles. We also tested transduction of NSC from MPS VII fetal brain using a lentiviral SIN vector driving human GUS from a PGK promoter. Transduction efficiency was 88–90%. Transplantation of 250,000 transduced NSC into neonatal MPS VII recipients revealed a similar pattern of transplantation as described above for FL NSC. Donor GUS+ cells were detected 4mo post transplant, indicating long-term gene expression and donor cell survival. In conclusion, NSC are efficiently transduced with lentivirus, can be cultured from hematopoietic tissue, and engraft long-term following neonatal injection. These results demonstrate a method to circumvent both donor cell availability and immune barriers to transplantation, providing hope for patients with devastating neurological disorders.

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Yoshimi, Mayumi, Susumu Goyama, Masahiro Nakagawa, Takashi Asai, Motoshi Ichikawa y Mineo Kurokawa. "Phosphorylation of AML1/Runx1 Is Essential for T Cell Differentiation and Early Hematopoietic Development." Blood 110, n.º 11 (16 de noviembre de 2007): 2189. http://dx.doi.org/10.1182/blood.v110.11.2189.2189.

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Abstract AML1/Runx1 is one of the most frequent targets of gene aberrations associated with human acute myelogenous leukemia and myelodysplastic syndrome. Furthermore, gene-targeting studies in mice demonstrated that AML1 is a critical regulator for T-cell differentiation and early development of definitive hematopoiesis. It has been shown that phosphorylation of AML1 at specific serine/threonine residues (276, 293, 300, 303) controls both transcriptional activity and rate of degradation. However, the biological consequences of phosphorylation with respect to AML1 function are unclear. In this study, we evaluated hematopoietic activities of AML1 mutants which harbor serine/threonine-to-alanine (A) or serine/threonine-to-asparatic acid (D) mutations at these phosphorylation sites using primary culture systems. We first evaluated the biologic effects of AML1 phosphorylation on T-cell differentiation using the fetal liver (FL)/OP9-DL1 coculture system. In this system, AML1-excised FL cells failed to undergo normal T-cell differentiation, which is successfully restored by the reintroduction of wild-type AML1. AML1-4A, which cannot be phosphorylated at any of the four serine/threonine residues, showed a severely impaired capacity to rescue the defective T-cell differentiation of AML1-deficient cells. We also demonstrate that blocking the ERK-mediated phosphorylation of AML1 by the MEK inhibitor significantly suppressed the AML1-induced T-cell differentiation. Next, we assessed the effect of AML1 phosphorylation on early hematopoietic development using para-aortic splanchnopleural (P-Sp) region/OP9 coculture system. In this system, AML1-deficient P-Sp cells failed to produce any hematopoietic cells and this hematopoietic defect can be rescued by retrovirally transferred AML1. AML1–4A could rescue the hematopoietic defect of AML1-deficient P-Sp cells, but the emergence of hematopoietic cells in cultures infected with AML1–4A was reproducibly delayed two or more days compared with those infected with wild-type AML1. Because it was shown that serine 462 is phosphorylated upon phorbol ester treatment together with the four phosphorylation sites, we then generated AML1 mutants in which serine 462 is substituted with alanine (A) or aspartic acid (D) in AML1–4A or AML1–4D respectively (AML1–5A or AML1-5D). Interestingly, introduction of AML1–5A into AML1-deficient P-Sp cells did not generate any hematopoietic cells. Moreover, AML1–5A completely lost its T-cell differentiation activity. In contrast, the phospho-mimic protein, AML1–5D rescued the hematopoietic defects of AML1-deficient cells as efficiently as wild-type AML1. Taken together, these results suggest that phosphorylation of AML1 at the five serine/threonine residues (276, 293, 300, 303 and 462) is essential for T cell differentiation and early hematopoietic development.

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Sousa, Ingrid Grazielle, Priscila Keiko Matsumoto Martin, Dulcinéia Martins de Albuquerque, Carolina Lanaro, Ryo Kurita, Yukio Nakamura y Fernando Ferreira Costa. "Knockdown of HNF4A Gene Increases Fetal Hemoglobin Synthesis in Hudep-2". Blood 134, Supplement_1 (13 de noviembre de 2019): 968. http://dx.doi.org/10.1182/blood-2019-130920.

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Sickle cell anemia is a recessive inherited disease caused by a single nucleotide polymorphism in the β-globin gene the resulting substitution of glutamic acid by valine causes red blood cell sickling when deoxygenated. Some hypomethylating agents are able to induce the expression of γ-globin by inhibiting DNA methylation at the gene promoter. However, it is not completely understood how this regulation occurs and which genes are involved in this process. To understand fetal hemoglobin (HbF) regulation, CD34+ cells were treated with 1µM of decitabine on the 9th day of culture to induce HbF production. The expression levels of transcription factors and chromatin modifiers were evaluated through the PCR Array platform (Qiagen™ Germany). The transcription factor, HNF4A (Hepatocyte Nuclear Factor 4 Alpha), was highly upregulated in cells treated with decitabine, compared to the control cells, and was chosen as a candidate for CRISPR/Cas9 knockout in HUDEP-2 cells (immortalized human erythroid progenitor cells). The HNF4A gene has been reported as a transcription factor, which regulates the expression of several hepatic genes, and is able to play a role in the development of the liver, kidney, and intestines. Moreover, HNF4A is expressed in the hematopoietic tissue. To the best of our knowledge, the association between HNF4A and gamma globin gene synthesis has not been previously described. HUDEP-2 cells were cultured and then treated with 50nM decitabine. After 72 hours, HbF levels were measured with anti-HbF antibody by flow cytometry in three biological replicates. The percentage of cells positive for HbF in decitabine-treated HUDEP-2 were 12.27 ± 0.7%, N=3, while in control cells the percentage was 1.0 ± 0.06%, N=3 (p<0.0001). These results corroborate the increased expression measured in CD34+ cells. To knock out HNF4A in HUDEP-2 cells, we used the CRISPR/Cas9 system. We generated INDELs in heterozygosity for HNF4A. Briefly, HUDEP-2 cells were nucleofected with Cas9 high fidelity ribonucleoprotein (104 pmol), crRNA:tracrRNA (120 pmol) complex and 1µM of gRNA HNF4A using a CD34+ human cell kit and the E-001 program in an AMAXA Nucleofector 4D-device (Lonza). Two days after nucleofection, edited HUDEP-2 cells were submitted to clonal selection and expanded for approximately 28 days. Genomic DNA from clones was analyzed by a Sanger Sequencer, and four edited HNF4A-HUDEP-2 clones were selected with INDELs in the fourth exon of HNF4A gene. These clones were expanded in culture with controls, and the HbF levels were quantified by flow cytometry. HbF levels in these four clones were 8.2 ± 2.4%, while in the wild type HUDEP-2, HbF was 0.9 ± 0.02% (p< 0.05). The edited clones expressed significantly more HbF than the controls, although this expression was not homogeneous. Western blotting of edited HNF4A-HUDEP-2 clones demonstrated decreased HNF4A at the protein level (0.35 ± 0.9 AU N=8), compared to controls (2.02± 1.1 AU N=3, p< 0.05). Results suggest that HNF4A may play a role in the gamma globin gene transcription in HUPED-2 cells. The mechanism of how HNF4A may regulate gamma globin gene expression remains to be clarified. Disclosures No relevant conflicts of interest to declare.

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Lai, Qiao-Ling, Wei-Dong Zheng y Yan Huang. "Regulation role of miR-204 on SIRT1/VEGF in metabolic memory induced by high glucose in human retinal pigment epithelial cells". International Journal of Ophthalmology 17, n.º 7 (18 de julio de 2024): 1232–37. http://dx.doi.org/10.18240/ijo.2024.07.06.

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AIM: To examine the regulatory role of microRNA-204 (miR-204) on silent information regulator 1 (SIRT1) and vascular endothelial growth factor (VEGF) under high-glucose-induced metabolic memory in human retinal pigment epithelial (hRPE) cells. METHODS: Cells were cultured with either normal (5 mmol/L) or high D-glucose (25 mmol/L) concentrations for 8d to establish control and high-glucose groups, respectively. To induce metabolic memory, cells were cultured with 25 mmol/L D-glucose for 4d followed by culture with 5 mmol/L D-glucose for 4d. In addition, exposed in 25 mmol/L D-glucose for 4d and then transfected with 100 nmol/L miR-204 control, miR-204 inhibitor or miR-204 mimic in 5 mmol/L D-glucose for 4d. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect miR-204 mRNA levels. SIRT1 and VEGF protein levels were assessed by immunohistochemical and Western blot. Flow cytometry was used to investigate apoptosis rate. RESULTS: It was found that high glucose promoted miR-204 and VEGF expression, and inhibited SIRT1 activity, even after the return to normal glucose culture conditions. Upregulation of miR-204 promoted apoptosis inhibiting SIRT1 and increasing VEGF expression. However, downregulation of miR-204 produced the opposite effects. CONCLUSION: The study identifies that miR-204 is the upstream target of SIRT1and VEGF, and that miR-204 can protect hRPE cells from the damage caused by metabolic memory through increasing SIRT1 and inhibiting VEGF expression.

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Naeimi Kararoudi, Meisam, Ezgi Elmas, Margaret Lamb, Nitin Chakravarti, Prashant Trikha y Dean Anthony Lee. "Disruption of SOCS3 Promotes the Anti-Cancer Efficacy of Primary NK Cells". Blood 132, Supplement 1 (29 de noviembre de 2018): 5687. http://dx.doi.org/10.1182/blood-2018-99-116621.

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Abstract Genetic modification of NK cells to enhance cancer immunotherapy has potential application to treat a wide range of cancers. Recently, we developed a new strategy in which CRISPR/Cas9 elements are introduced into NK cells as ribonucleoproteins (RNPs) via electroporation, followed by expansion on feeder cells expressing 4-1BBL and membrane-bound IL-21 to generate large numbers of genetically modified NK cells. This method was used to genetically modify several genes in primary and expanded NK cells including suppressor of cytokine signaling 3 (SOCS3). SOCS3 negatively regulates cytokine signaling through the JAK/STAT pathway. We hypothesized that disruption of SOCS3 in primary NK cells using Cas9/RNPs could potentially maintain STAT3 signaling levels and subsequently increase their proliferation and cytotoxic function. We designed gRNAs to target exon 2 of the SOCS3 gene and electroporated them along with Cas9 protein as Cas9/RNPs into primary NK cells using the Lonza 4D electroporator. We tested 6 different conditions of gRNAs alone or in combination. The NK cells in the control group were electroporated with no Cas9/RNPs. After electroporation, the cells were rested in culture media supplemented with 100 IU of human IL-2 for 48 hours and then expanded using irradiated feeder cells. At day 7, an equal number of cells were restimulated with irradiated feeder cells to test the effect of SOCS3 KO on proliferation. Western blot was used to assay the knock out efficacy at the protein level. Calcein assay and IncuCyte Zoom (Essen) were performed to measure cytotoxicity against two cancer cell lines, K562 and Daoy. Our results showed a significant reduction in SOCS3 protein levels in 3 conditions (gRNA1, gRNA3 and gRNA1+ gRNA3) in comparison to the control group. The calcein assay and IncuCyte zoom showed the modified SOCS3 KO NK cells could kill the tumors more efficiently in comparison to the control. Proliferation data showed the SOCS3 KO cells can grow faster than the control group. In conclusion, our data demonstrate the role of SOCS3 and JAK/STAT pathway in NK cell function and suggests that SOCS3 may be a good target for genetic modification to improve cancer immunotherapy using NK cells. Disclosures Lee: CytoSen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck, Sharp, and Dohme: Consultancy; Courier Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Smith, Eric L., Sham Mailankody, Arnab Ghosh, Reed Masakayan, Mette Staehr, Terence J. Purdon, Elizabeth Halton et al. "Development and Evaluation of a Human Single Chain Variable Fragment (scFv) Derived Bcma Targeted CAR T Cell Vector Leads to a High Objective Response Rate in Patients with Advanced MM". Blood 130, Suppl_1 (7 de diciembre de 2017): 742. http://dx.doi.org/10.1182/blood.v130.suppl_1.742.742.

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Abstract Patients with relapsed/refractory MM (RRMM) rarely obtain durable remissions with available therapies. Clinical use of BCMA targeted CAR T cell therapy was first reported in 12/2015 for RRMM, and based on small numbers, preliminary results appear promising. Given that host immune anti-murine CAR responses have limited the efficacy of repeat dosing (Turtle C. Sci Trans Med 2016), our goal was to develop a human BCMA targeted CAR T cell vector for clinical translation. We screened a human B cell derived scFv phage display library containing 6x1010 scFvs with BCMA expressing NIH 3T3 cells, and validated results on human MM cell lines. 57 unique and diverse BCMA specific scFvs were identified containing light and heavy chain CDR's each covering 6 subfamilies, with HCDR3 length ranges from 5-18 amino acids. 17 scFvs met stringent specificity criteria, and a diverse set was cloned into CAR vectors with either a CD28 or a 4-1BB co-stimulatory domain. Donor T cells transduced with BCMA targeted CAR vectors that conveyed particularly desirable properties over multiple in vitro assays, including: cytotoxicity on human MM cell lines at low E:T ratios (>90% lysis, 1:1, 16h), robust proliferation after repeat antigen stimulation (up to 700 fold, stimulation q3-4d for 14d), and active cytokine profiling, were selected for in vivo studies using a marrow predominant human MM cell line model in NSG mice. A single IV injection of CAR T cells, either early (4d) or late (21d) after MM engraftment was evaluated. In both cases survival was increased when treated with BCMA targeted CAR T cells vs CD19 targeted CAR T cells (median OS at 60d NR vs 35d p<0.05). Tumor and CAR T cells were imaged in vivo by taking advantage of luciferase constructs with different substrates. Results show rapid tumor clearance, peak (>10,000 fold) CAR T expansion at day 6, followed by contraction of CAR T cells after MM clearance, confirming the efficacy of the anti-BCMA scFv/4-1BB containing construct. Co-culture with primary cells from a range of normal tissues did not activate CAR T cells as noted by a lack of IFN release. Co-culture of 293 cells expressing this scFv with those expressing a library of other TNFRSF or Ig receptor members demonstrated specific binding to BCMA. GLP toxicity studies in mice showed no unexpected adverse events. We generated a retroviral construct for clinical use including a truncated epithelial growth factor receptor (EGFRt) elimination gene: EGFRt/hBCMA-41BBz. Clinical investigation of this construct is underway in a dose escalation, single institution trial. Enrollment is completed on 2/4 planned dose levels (DL). On DL1 pts received cyclophosphamide conditioning (3g/m2 x1) and 72x106 mean CAR+ T cells. On DL2 pts received lower dose cyclophosphamide/fludarabine (300/30 mg/m2 x3) and 137x106 mean CAR+ T cells. All pts screened for BCMA expression by IHC were eligible. High risk cytogenetics were present in 4/6 pts. Median prior lines of therapy was 7; all pts had IMiD, PI, high dose melphalan, and CD38 directed therapies. With a data cut off of 7/20/17, 6 pts are evaluable for safety. There were no DLT's. At DL1, grade 1 CRS, not requiring intervention, occurred in 1/3 pts. At DL2, grade 1/2 CRS occurred in 2/3 pts; both received IL6R directed Tocilizumab (Toci) with near immediate resolution. In these 2 pts time to onset of fever was a mean 2d, Tmax was 39.4-41.1 C, peak CRP was 25-27mg/dl, peak IL6 level pre and post Toci were 558-632 and 3375-9071 pg/ml, respectively. Additional serum cytokines increased >10 fold from baseline in both pts include: IFNg, GM CSF, Fractalkine, IL5, IL8, and IP10. Increases in ferritin were limited, and there were no cases of hypofibrinogenemia. There were no grade 3-5 CRS and no neurotoxicities or cerebral edema. No pts received steroids or Cetuximab. Median time to count recovery after neutropenia was 10d (range 6-15d). Objective responses by IMWG criteria after a single dose of CAR T cells were observed across both DLs. At DL1, of 3 pts, responses were 1 VGPR, 1 SD, and 1 pt treated with baseline Mspike 0.46, thus not evaluable by IMWG criteria, had >50% reduction in Mspike, and normalization of K/L ratio. At DL2, 2/2 pts had objective responses with 1 PR and 1 VGPR (baseline 95% marrow involvement); 1 pt is too early to evaluate. As we are employing a human CAR, the study was designed to allow for an optional second dose in pts that do not reach CR. We have treated 2 pts with a second dose, and longer follow up data is pending. Figure 1 Figure 1. Disclosures Smith: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: BCMA targeted CAR T cells, Research Funding. Almo: Cue Biopharma: Other: Founder, head of SABequity holder; Institute for Protein Innovation: Consultancy; AKIN GUMP STRAUSS HAUER & FELD LLP: Consultancy. Wang: Eureka Therapeutics Inc.: Employment, Equity Ownership. Xu: Eureka Therapeutics, Inc: Employment, Equity Ownership. Park: Amgen: Consultancy. Curran: Juno Therapeutics: Research Funding; Novartis: Consultancy. Dogan: Celgene: Consultancy; Peer Review Institute: Consultancy; Roche Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Liu: Eureka Therpeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

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Nasri, Masoud, Perihan Mir, Benjamin Dannenmann, Diana Amend, Yun Xu, Anna Solovyeva, Sylwia Stefanczyk et al. "A Method to Fluorescently Label the CRISPR/Cas9-gRNA RNP Complexes Enables Enrichment of Clinical-Grade Gene-Edited Primary Hematopoietic Stem Cells and iPSCs". Blood 132, Supplement 1 (29 de noviembre de 2018): 1108. http://dx.doi.org/10.1182/blood-2018-99-114844.

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Abstract Although proven to be an excellent method for gene editing, CRISPR/Cas9-mediated technology still has some limitations for the applications in primary hematopoietic stem cells and progenitor cells (HSPCs) as well as in human induced pluripotent stem cells (hiPSCs). Delivery of Cas9 protein in a form of ribonucleoprotein (RNP) in a complex with guide RNA (gRNA) provides a DNA free methodology, but a big hinderance of this application is that it is not possible to sort and enrich gene edited cells for further applications. Here we report the establishment of a new protocol of fluorescent labeling of the Cas9/gRNA ribonucleoprotein complex (CRISPR/Cas9-gRNA RNP). We designed crRNA for exon 1 of GADD45b gene, annealed this crRNA with transactivating crRNA (tracrRNA) to form gRNA and covalently introduced one fluorchrome agent (CX-rhodamine or fluorescein) per approximately every 20 nucleotides. HEK293FT cells, Jurkat T-ALL cell line, bone marrow CD34+ HSPCs, and iPSCs were transfected with fluorescently-labeled GADD45b CRISPR/Cas9-gRNA RNP by means of cathionic polymer based transfection reagent for HEK293FT cells and Lonza 4D nucleofection for Jurkat T-ALL cell line, CD34+ HSPCs, and iPSCs. We detected CX-rhodamine- or fluorescein intracellular signals 12 hours after transfection that disappeared approximately 48 hours post transfection. Transfection efficiency varied between 40 % and 80 %, depending on the cell type. Labeling did not affect integrity of crRNA/tracRNA duplex formation, gene editing efficiency and off-target activities of CRISPR/Cas9-gRNA RNP, as assessed by Sanger sequencing and TIDE assay of transfected HEK293FT cells, Jurkat cells, CD34+ HSPCs and human iPSCs. Using fluorescein- or CX-rhodamine signal of labeled CRISPR/Cas9-gRNA RNP, we sorted and enriched gene-edited cells. Gene modification efficiency in sorted cells was between 40 and 70 %, based on the cell type. Of note, we detected much lower transfection and editing efficiency of the fused Cas9-EGFP protein assembled with GADD45b targeting gRNA, as compared to CRISPR/Cas9-gRNA RNP. Most probably, conjugation of EGFP tag is affecting functions of CRISPR/Cas9- gRNA RNP. GADD45b (Growth Arrest And DNA Damage Inducible Beta), also termed myeloid differentiation primary response 118 gene (MyD118), belongs to a family of evolutionarily conserved GADD45 proteins (GADD45a, GADD45b and GADD45g) that function as stress sensors regulating cell cycle, survival and apoptosis in response to stress stimulus as ultraviolet (UV)-induced DNA damage and genotoxic stress. We further performed functional studies of the effect of GADD45b knockout on cell growth and sensitivity to UV-induced DNA damage. Remarkably, we detected severe diminished viability of GADD45b-deficient HEK293FT, Jurkat cells, iPSCs and CD34+ HSPCs as compared to control transfected cells. We also found markedly elevated susceptibility of GADD45b-deficient Jurkat cells, CD34+ HSPCs and iPSCs to UV induced DNA damage, as documented by elevated levels of γH2AX (pSer139). Based on these observations, we conclude that GADD45b knockout using transfection of cells with labeled GADD45b-targeting CRISPR/Cas9-gRNA RNP led to increased susceptibility to DNA damage. Moreover, GADD45b deficient iPSCs retained pluripotency, but they failed to differentiate to mature neutrophils in embryoid body (EB)-based culture. Taken together, this is the first report describing transfection and sorting of primary hematopoietic cells and iPSCs using fluorescently-labeled CRISPR/Cas9-RNP, which is simple, safe and efficient method, and therefore may strongly expand the therapeutic avenues for gene-edited cells. Disclosures No relevant conflicts of interest to declare.

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Agyekum, Davies G., Zhong Chen, Haiyan Xiao, Megan Yu, Niren Patel, Betty S. Pace y Steffen E. Meiler. "Resveratrol Regulates Fetal Hemoglobin Production by Inhibiting KLF1 and BCL11A in KU812 Cells". Blood 120, n.º 21 (16 de noviembre de 2012): 1000. http://dx.doi.org/10.1182/blood.v120.21.1000.1000.

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Abstract Abstract 1000 Objective: The use of small molecules to reactive fetal hemoglobin (HbF) expression in patients with sickle cell disease (SCD) is a promising clinical strategy. Erythroid Kruppel-like factor (KLF1) and B cell lymphoma 11A (BCL11A) are key regulatory components of the developmental switch from fetal to adult hemoglobin. Current models suggest that KLF1 positively regulates the expression of the γ-globin gene repressor BCL11A by interacting with the BCL11A promoter. Knockdown studies of KLF1 in adult erythroid progenitors are consistent with this model and result in low BCL11A levels and derepression of γ-globin gene expression. Based on the critical role of KLF1 in regulating HbF production, we performed a limited screen of small molecules for their ability to suppress KLF1 expression. We identified resveratrol, a small polyphenolic compound, as the most potent suppressor of KLF1. The following studies were performed to confirm RSV's mechanism of action. Methods: Cell Culture. KU812 leukemia cells express both fetal and adult hemglobin and erythroid markers similar to days 7–14 primary erythroid cells, which makes them a useful in vitro model for testing the activity of HbF-inducing compounds. KU812 cells (ATTC) were cultured at a density of 5×105 cells/mL for 72h in the presence of vehicle or RSV (50 μM). Flow cytometry. Fixed KU812 cells were labeled with monoclonal anti-human γ-globin (Invitrogen) and β-globin (Santa Cruz) antibodies to determine the γ- to β-globin mean fluorescent ratio. Western Blot. Whole cell lysates were prepared in RIPA buffer containing protease inhibitors and quantified by BCA protein assay. Protein samples (40 μg per lane) were resolved on a 4–15% SDS-PAGE gradient gel and transferred onto a PVDF membrane. Blots were incubated on at 4°C in primary antibody (KLF1, 1:200, Santa Cruz; BCL11A, 1:1000, Novus Biologicals) followed by 1h incubation with an HRP-conjugated secondary antibody at RT. Protein bands were visualized by chemiluminescence and analyzed by densitometry using Image J software. Transient transfections. KU812 cells were transfected with a constitutively active KLF1 expression construct (pCMVSPORT6-KLF1) or an empty vector control (pCMV-EV) using the Amaxa 4D Nucleofector kit (Lonza). 12 h post transfection, KU812 cells (5×105 cells/mL) were incubated with vehicle or RSV (50 μM) for 72 h. Statistical analysis. One-Way ANOVA followed by Student-Newman-Kuels (Sigma Stat). Data are reported as the mean ± SEM. A p-value of <0.05 was considered significant. Results: RSV (50μM) induced a significant increase in HbF expression compared to vehicle (γ-/β-globin ratio: Veh: 7.09±0.18; RSV: 11.02±0.84; p<0.001). Parallel measurements revealed a 65% decrease of KLF1 protein expression in response to RSV (KLF1/actin ratio: Veh: 1.04±0.06; RSV: 0.39±0.007; p<0.001). Concurrently, RSV reduced BCL11A protein levels by 77% (BCL11A/actin: Veh: 1.00±0.05; RSV: 0.23±0.02; p<0.001). Overexpression of KLF1 completely abrogated the induction of HbF by RSV (γ-/β-globin ratio: pCMV-EV+vehicle 7.97±0.67; pCMV-EV+RSV (50 μM) 14.06±0.61; pCMV-KLF1+RSV (50 μM) 8.83±0.89; p<0.001). Summary & Conclusions: Our results demonstrate that RSV, a natural polyphenol found in grapes and red wine, is a potent inducer of HbF in KU812 cells. The mechanism of HbF induction is mediated in part through suppression of KLF1 and BCL11A expression. This view is strengthened by the fact that overexpression of a constitutively active form of KLF1 completely abrogated RSV's HbF activity. These findings support further investigations of RSV's bioactivity in primary CD34+ erythroid progenitor cells and preclinical models of SCD. Disclosures: No relevant conflicts of interest to declare.

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Chae, Hee-Don y Kathleen Sakamoto. "Replication Factor C3 Is a Direct Target Of CREB, Promotes G1/S Transition Of Acute Myeloid Leukemia Cells, and Increases Hematopoietic Stem/Progenitor Cell Self-Renewal". Blood 122, n.º 21 (15 de noviembre de 2013): 3754. http://dx.doi.org/10.1182/blood.v122.21.3754.3754.

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Abstract CREB (cAMP Response Element Binding protein) promotes cellular transformation of hematopoietic cells and proliferation of myeloid leukemia cells. However, the underlying mechanisms of CREB function in leukemic transformation and hematopoiesis are not fully understood. To address this, we have investigated the downstream pathways of CREB activation in proliferation using a human acute myeloid leukemia (AML) cell line KG1 cells knocked-down for CREB with specific shRNAs. The CREB-knockdown KG1 cells were significantly defective in proliferative capability compared to control cells [cell number after 4d (X105), seeding (1X105), control vs. CREB-knockdown: 34.18 +/– 1.27 vs. 14.52 +/– 0.46, n=3, p< 0.01, mean +/– SEM]. In order to characterize the specific role of CREB in cell proliferation, we analyzed cell cycle progression patterns of CREB-knockdown and control KG1 cells after release from mitotic arrest. Our results indicated that G1 to S phase transition as assessed by % S phase was impeded by CREB-knockdown [S phase (%), control vs. CREB-knockdown cells, 8h after release: 53.29 +/– 0.54 vs. 23.57 +/– 1.69; 12h: 66.92 +/– 0.63 vs. 45.16 +/– 0.50, n=3, p< 0.01, mean +/– SEM]. To identify potential CREB target genes, we chose several cell cycle related genes such as CCNE1, CCNA1, CCNB1 and PCNA and compared their RNA expression levels in the CREB-knockdown with those in control KG1 cells after release from mitotic arrest. To our surprise, we failed to detect any noticeable differences in the mRNA expression levels of those genes between CREB-knockdown and control KG1 cells. In an effort to search for CREB responsive target genes, we analyzed additional CREB targets previously identified from microarray data (Pellegrini et al BMC Cancer 2008). We found that expression of replication factor C3 (RFC3), a 38kDa subunit of the RFC complex involved in DNA replication and repair processes, was significantly reduced in CREB-knockdown cells compared to control cells [38 +/– 1% of control, n=3, p<0.01]. CREB-knockdown also inhibited RFC3 mRNA expression in U937 and HL60 AML cell lines. Consistent with these results, mRNA expression levels of RFC3 appeared to be closely correlated with those of CREB when we examined bone marrow samples obtained from AML patients [n = 16, Pearson coefficient = 0.6366, p = 0.0008]. Moreover, we found that CREB directly interacted with the CRE site in the RFC3 promoter region in vivo, as assessed by chromatin immunoprecipitation assays. Exogenous overexpression of RFC3 in CREB-knockdown KG1 cells restored the defective G1/S progression [S phase (%), CREB-knockdown vs. CREB-knockdown with RFC3 overexpression, 9h after release: 38.97 +/– 0.45 vs. 62.24 +/– 1.06; 12h: 48.12 +/– 0.60 vs. 67.70 +/– 1.15, n=3, p< 0.01, mean +/– SEM]. Taken together, these results suggest that RFC3 may act as a novel downstream oncogenic target of activated CREB in AML cells. We previously reported that CREB is a critical regulator of normal myelopoiesis (Cheng et al Blood 2008). To determine whether RFC3 could exert similar effects on normal hematopoiesis, we compared human umbilical cord blood derived CD34-positive cells with and without RFC3 overexpression for the capacity to form hematopoietic colonies. Overexpression of RFC3 in the CD34-positive cells resulted in significant increases of multi-potential CFU-GEMM colony numbers [without vs. with overexpression of RFC3 (per 1000 cells): 3.2 +/– 1.3 vs. 22.3 +/– 3.3, n=3, p< 0.01, mean +/– SEM]. The RFC3 effect on stimulating colony formation was magnified in secondary colony forming assays [without vs. with overexpression of RFC3 (per 100,000 cells): 10.7 +/– 3.5 vs. 180.2 +/– 44.4, n=3, p< 0.05, mean +/– SEM]. Since the formation of secondary colonies was derived mainly from residual stem/progenitor cell populations after long-term culture, RFC3 overexpression may enhance self-renewal of stem/progenitor cells. In conclusion, our results suggest that RFC3 is able to promote G1/S transition in a human AML cell line downstream of CREB activation. In addition, we provide evidence that RFC3 is involved in normal hematopoiesis and contributes to increased self-renewal potential of hematopoietic stem/progenitor cells. Our data demonstrate that RFC3 plays multiple roles in promoting AML cells proliferation as well as normal myelopoiesis through increasing the self-renewal potential of hematopoietic stem/progenitor cells in response to CREB activation. Disclosures: No relevant conflicts of interest to declare.

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Martin, Priscila Keiko Matsumoto, Dulcinéia Martins de Albuquerque, Carolina Lanaro, Ryo Kurita, Yukio Nakamura y Fernando F. Costa. "A Single -195 C < G HBG1 Promoter Mediated By CRISPR/Cas9 Genome Editing Induces Fetal Hemoglobin Synthesis in Hudep-2". Blood 132, Supplement 1 (29 de noviembre de 2018): 3481. http://dx.doi.org/10.1182/blood-2018-99-118534.

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Abstract Sickle cell disease (SCD) is a serious condition, chronic and undoubtedly represents a public health problem worldwide. SCD is caused by a point mutation in codon 6 of the β globin gene resulting in the production of a structurally abnormal hemoglobin, hemoglobin S. Although the cause of the disease has been known for more than fifty years, therapeutic options are still quite limited. High levels of fetal hemoglobin (HbF) in the blood are associated with a better clinical outcome in SCD patients. In some individuals, the expression of γ-globin gene persists into adulthood in elevated levels, which is called hereditary persistence of fetal hemoglobin (HPFH). A single nucleotide mutation from C to G at position -195 of the HBG1 gene promoter, called non deletional HPFH Brazilian type (nd-HPFH-B), augments the levels of HbF in patients in 7%- 30%. Nd-HPFH-B has been described by our group, but the mechanism and how this single mutation rises HbF levels differently in red blood cells is still unknown. Genome editing using CRISPR/Cas9 in HUDEP-2 cell, an erythroid precursor line, has been developed through homologous direct repair from a small single DNA strand containing the guanine in -195 position at HBG1 gene promotor. All the other genes, including the second HBG1 allele were unaltered. This point mutation has been carried out by CRISPR/Cas9 high fidelity system, capable of performing a specific break in the DNA target sequence, that improves homologous recombination rate of the donor sequence containing the -195 C<G mutation (ssODN -195). For the first time, we generated a HUDEP-2 cell line with the -195 C>T mutation in HBG1 promoter using CRISPR/Cas9 genome editing. The HUDEP-2 cells were nucleofected with Cas9 high fidelity ribonucleoprotein (104 pmol), crRNA:tracrRNA (120 pmol) complex and 1uM ssODN -195, using CD34+ human cell kit and program E-001 in AMAXA Nucleofector 4D- device (Lonza). Seven days after nucleofection, the transformed cells were submitted to clonal selection for 25 days. The genomic DNA from 48 clones were submitted by Sanger Sequencing. The sequencing analysis showed highest Crispr/Cas9 efficiency in genomic DNA cut (77.08%; 37/48) and satisfactory ssODN -195 homologous recombination (10.4%; 5/48). Five nd-HPFH-B HUDEP-2 clones and three other clones without the mutation, but with indels after Cas9 DNA cut (controls), were expanded in culture and the HbF levels were measure with anti-HbF antibody by flow cytometry in two biological replicates. HbF levels in nd-HPFH-B HUDEP-2 clones were 6.02%±1.4, 8.25% ± 0.28, 10.18% ± 3.71, 11.95% ± 0.49, 26,3% ± 4,6 while in controls were 1.69% ± 0.26, 1.66% ± 0.26, 0.59% ± 0.06. Two nd-HPFH-B clones were differentiated into erythrocyte in vitro, and fetal hemoglobin levels persisted at high levels seen previously. In addition, α-globin, β-globin and γ-globin mRNA levels were evaluated in three nd-HPFH-B HUDEP-2 clones and two control clones. The mRNA HBG1/HBG1+HBB percentage in nd-HPFH-B were 96.16% ± 4.10 against 22.63% ± 9.64 in controls. The monoallelic single nucleotide mutation -195 C>G is capable to increase the fetal hemoglobin levels up to 30% in nd-HPFH-B HUDEP-2, and our results shows that this is a potential experimental in vitro model to be used in future studies. Disclosures No relevant conflicts of interest to declare.

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Lontos, Konstantinos, Juraj Adamik, Peng Zhang, Quanhong Sun, David Roodman, Attaya Suvannasankha y Deborah Lynn Galson. "Semaphorin 4D to suppress bone formation in multiple myeloma." Journal of Clinical Oncology 35, n.º 15_suppl (20 de mayo de 2017): 8039. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.8039.

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8039 Background: Myeloma bone disease is characterized by osteoclast activation and long-term osteoblast suppression. We investigated if Semaphorin 4D (Sema4D; CD100) plays a role in these processes. Sema4D has been shown to be a potent osteoblast inhibitor (Negishi-Koga T et al, Nat Med. 2011). A study recently identified that the breast cancer cell line MDA-MB-231 utilizes Sema4D to create osteolysis (Yang Y et al, PLOS One 2016). There have been previous data that Sema4D is increased in the serum of myeloma patients (Terpos et al, Blood 2012) and that co-culturing myeloma cell lines with osteocytes increases the expression of Sema4D mRNA in both (Suvannasankha et al, Blood 2016). We sought to investigate if myeloma cells are using Sema4D to suppress bone formation and if they affect the levels of Sema4D produced by osteoclasts. Methods: We used lentivirus carrying shRNA for Sema4D or control (Scr) to knock down the level of the protein in the 5TGM1 murine myeloma cell line. Knockdown was verified by qPCR and Western Blot. We subsequently co-cultured the 5TGM1 cells with the MC3T3-subclone M4 (MC4) murine stromal cell line for 2 days, removed the myeloma cells and then differentiated the MC4 cells using ascorbic acid and β-glycerolphosphate. At day 5, we analyzed the cells for Runx2 (a critical gene for the differentiation of stromal cells into osteoblasts) expression utilizing qPCR. Also, we performed qPCR in primary osteoclast (OCL) mouse cells differentiating into OCL with RANKL with or without pre-treatment with myeloma-conditioned media for 3 days before the addition of RANKL. Results: When 5TGM1-Scr were co-cultured with MC4 cells the expression of Runx2 on day 5 was decreased (p=0.02). Strikingly, the 5TGM1-shSema4D cells when co-cultured with MC4s did not have the same effect and allowed the upregulation of Runx2 expression on day 5 (p=0.01). Myeloma-conditioned media increased Sema4D expression by OCL throughout the 5 days of differentiation 2 to 3-fold (p=0.01 for day 5). Conclusions: The myeloma cells seem to be utilizing Sema4D both directly and indirectly to inhibit bone formation. Targeted therapy against Sema4D may improve outcomes and fracture-free survival for multiple myeloma patients.

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Aung-Htut, May T., Craig S. McIntosh, Kristin A. West, Sue Fletcher y Steve D. Wilton. "In Vitro Validation of Phosphorodiamidate Morpholino Oligomers". Molecules 24, n.º 16 (12 de agosto de 2019): 2922. http://dx.doi.org/10.3390/molecules24162922.

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One of the crucial aspects of screening antisense oligonucleotides destined for therapeutic application is confidence that the antisense oligomer is delivered efficiently into cultured cells. Efficient delivery is particularly vital for antisense phosphorodiamidate morpholino oligomers, which have a neutral backbone, and are known to show poor gymnotic uptake. Here, we report several methods to deliver these oligomers into cultured cells. Although 4D-Nucleofector™ or Neon™ electroporation systems provide efficient delivery and use lower amounts of phosphorodiamidate morpholino oligomer, both systems are costly. We show that some readily available transfection reagents can be used to deliver phosphorodiamidate morpholino oligomers as efficiently as the electroporation systems. Among the transfection reagents tested, we recommend Lipofectamine 3000™ for delivering phosphorodiamidate morpholino oligomers into fibroblasts and Lipofectamine 3000™ or Lipofectamine 2000™ for myoblasts/myotubes. We also provide optimal programs for nucleofection into various cell lines using the P3 Primary Cell 4D-Nucleofector™ X Kit (Lonza), as well as antisense oligomers that redirect expression of ubiquitously expressed genes that may be used as positive treatments for human and murine cell transfections.

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40

Neeson, Paul J., Alexander James Davenport, Joseph A. Trapani, Michael Kershaw, Ryan Cross, H. Miles Prince, Ricky W. Johnstone, David Ritchie, Phil Darcy y Misty R. Jenkins. "Bigger, Stronger, Faster: Chimeric Antigen Receptor T Cells Are Olympic Killers". Blood 128, n.º 22 (2 de diciembre de 2016): 814. http://dx.doi.org/10.1182/blood.v128.22.814.814.

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Abstract Chimeric antigen receptor T cells (CAR T) re-directed to CD19, have induced remarkable responses in clinical trials for patients with B-cell malignancies. Patients have responded to therapy with a CAR T dose which is a fraction of the pre-existing tumor burden. Explanations for this observation include studies which show the proliferative potential of the CAR T cells (Kalos M et al Sci Transl Med 2011), as well as our recent study which showed that individual CAR T cells can serial kill tumor cells (Davenport AJ et al CIR 2015, Figure 6). Using our dual antigen receptor model (OT-I T cell receptor and 2nd generation HER-2 CAR in the same T cell, termed CAR.OTI cells), we also observed a reproducible and significantly shorter time from CAR- vs TCR-mediated activation to detachment from dying tumor cells (Davenport AJ et al CIR 2015, Figure 4D)suggesting that CAR-mediated individual killing events are actually faster. To explore how this may occur, we examined the immune synapse structure at 20 minutes of CAR.OTI CTL co-culture with tumor cells expressing the cognate antigen for either the CAR or TCR. At this timepoint, CAR.OTI CTL, activated via the TCR, formed a conventional bull's eye immune synapse with accumulation of LCK and actin clearance (Figure 1). Surprisingly, CAR.OTI CTL activated via their CAR had an immune synapse with no or diffuse LCK and small actin rings (Figure 1). At the same timepoint, CAR-activated CAR.OTI CTL conjugates with tumor cells were characterized by a microtubule organizing center (MTOC) distant from the immune synapse LCK accumulation. In contrast TCR activated CAR.OTI CTL conjugates consistently had the MTOC proximal to the LCK accumulation (Figure 2A). Despite this, CAR-mediated CAR.OTI CTL killing of tumor targets was inhibited by a protein kinase C zeta inhibitor and is, therefore, MTOC dependent (Figure 2B). The MTOC circumnavigates the activated CTL nucleus and moves to the immune synapse, bringing cytotoxic granules with it. Using time lapse live video (TLLV) microscopy, we compared CAR.OTI CTL cytotoxic granule movement when the CAR.OTI were activated via the TCR vs CAR. We showed that following CAR vs TCR activation, CAR.OTI cytotoxic granules moved with a significantly higher velocity, and had a shorter time lapse to reach the immune synapse following activation (Ca2+ flux), and a significantly shorter time to detachment from the dying tumor cell (Figure 2C). We then re-examined immune synapse formation at an earlier timepoint, to explore whether the data from Figures 1-2 could be explained by a more rapid CTL response following CAR-mediated signaling. In contrast to our observations at 20 minutes, at five minutes we showed CAR-activated CAR.OTI CTL formed conjugates with tumor targets and the immune synapse showed distinct LCK accumulation and actin clearance (Figure 3A). Finally, we explored CAR.OTI CTL signaling and showed that CAR-mediated activation induced a significantly lower number of Ca2+ fluxes, however each Ca2+ flux amplitude was not different (Figure 3B). We also examined changes in proximal (phospho-LCK, pLCK) and distal (phospho-ERK, pERK) signals in CAR- versus TCR- activated CAR.OTI cells, and showed that CAR-mediated activation induced more rapid proximal and distal activation signals (Figure 3C). In conclusion, this study showed that compared to activation by TCR ligation, T cells respond to CAR ligation with faster phospho-protein signaling, Ca2+ flux, formation of an immune synapse and a more rapid movement of the MTOC and delivery of the cytotoxic granules to kill the tumor cells. Furthermore, LFA-1 did not accumulate at the immune synapse following CAR activation, therefore, reduced adhesion may facilitate the observed rapid detachment from the dying tumor cell, and enable the CAR T to rapidly move onto the next tumor target for 'bigger, stronger, faster' killing. Disclosures No relevant conflicts of interest to declare.

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Oseghale, Aluya Richard, Alice Bertaina y Matthew Porteus. "Towards Automated Engineering of Donor-Derived T Lymphocytes into CRISPR/Cas9-Mediated CAR T Cells in a Closed-System". Blood 142, Supplement 1 (28 de noviembre de 2023): 4826. http://dx.doi.org/10.1182/blood-2023-189883.

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Chimeric antigen receptor (CAR)-expressing T cells have revolutionized immunotherapy of cancer. However, some patients derive no benefit from approved CARTcell therapies due to limitations such as toxicity and use of autologous cells which are sometimes unfit and risk manufacturing failures. Additional or more effective CARTcell therapies are needed. Harnessing the endogenous homology-directed DNA repair pathway via precision genome editing based on Cas9RNP/rAAV6, we and others have demonstrated the feasibility of targeting transgene(s) integration into specific gene site(s) including their incorporation under endogenous regulatory elements. Using this strategy, we aimed to engineer healthy donor-derived CAR T cells from αβ+ T lymphocytes. For enhanced safety, we metabolically reprogrammed the CAR T cells by inactivating the UMPS gene needed for uridine biosynthesis. The UMPS-/-CAR T cells become dependent on exogenous uridine thereby serving as a cell control mechanism. We hypothesized that donor-derived αβ+ T lymphocytes can be engineered using the Cas9RNP-rAAV6 strategy in an automated closed-system into controllable CAR T cells. We inactivated TRAC and UMPS gene loci in αβ+T lymphocytes using Lonza4D nucleofector. Electroporated cells were simultaneously transduced with CD19CAR-containing rAAV6 DNA vector. We obtained TRAC gene knockout up to 97% with knock-in up to 89%. Disruption of the UMPS gene would transform the cells to being auxotrophic and depend on uridine supply for viability and growth. To evaluate the CART cells' antitumor potency, we cocultured the CD19CART cells with GFP-expressing Nalm6 leukemia cell lines. The CD19CART cells eradicated the leukemia cells within 72 hours. To mimic relapse, we re-challenged cocultured CART with Nalm6 cells and the tumor cells were again depleted within 72 hours. Expectedly, IL-2 production was elevated in CD19CART cells challenged with Nalm6. Next, we tested the ability to control the cytotoxicity of the CD19CART cells by coculturing them with Nalm6 cells in separate groups with/without exogenous uridine. Interestingly, the CD19CART cells cocultured without uridine eradicated the target cells within 72 hours but lost their potency upon subsequent challenge (Figure 1). In contrast, CD19CAR T cells supplied with uridine remained potent and repeatedly eradicated target cells indicating the ability to turn off the potency of CART cells with uridine supply/withdrawal. To establish an automated closed-system for CART cells, we adopted the Miltenyi CliniMACS Prodigy with in-line electroporator (Elpo) for process engineering. First, we determined optimized parameters for electroporating αβ+ T lymphocytes on Elpo(Table1). Cells electroporated with these settings were (>80% viability) comparable to untreated cells. We first engineered CART cells with Elpo/Miltenyi reagents in an open system(i.e.cultivated Elpo-electroporated cells in culture vessels/incubators). We obtained 95% of TRAC and UMPS gene knockouts and up to 90% CD19CAR transgene knock-in to the TRAC locus - an efficiency slightly higher than using Lonza 4D nucleofector. To scale up the Cas9-based T cell engineering into a single automated process on the CliniMACS Prodigy, the Cas9 and guide RNA reagents would be scaled up 1000fold. To make the process cost-effective, we tested AZD7468, a small molecule DNAPKcs inhibitor. We optimized/reduced the amounts of reagents upto 90% less of Cas9RNP used without any significant reduction in gene editing outcomes. AZD7648 reduced the MOI needed for viral transduction by 50% while maintaining 80% or higher levels of transgene knock-in efficiency. Next, we commenced pilot studies to automate the CAR T cell engineering process in closed-system. Currently, we have achieved an automated TRAC gene knockout of over 90% and CAR transgene knock-in of slightly >10%. Further optimization studies are underway to improve automated gene knock-in efficiency. We have engineered donor-derived αβ+ T lymphocytes into potent/controllable CD19CART cells. The UMPS-deficient CD19CART cells lose antileukemia activity without uridine. We show that metabolic reprogramming can be combined with precision genome editing to generate controllable CART cells. Finally, our data demonstrate the feasibility of automating CRISPR/Cas9-mediated engineering of CAR T cells within closed-system.

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42

Chapoval, Svetlana, EusebuisHenry Nkyimbeng-Takwi, Kathleen Shanks, Elizabeth Smith, Apoorva Iyer, Michael Lipsky, Louis DeTolla y Achsah Keegan. "Neuroimmune semaphorin 4D is necessary for optimal lung allergic inflammatory response (175.16)". Journal of Immunology 188, n.º 1_Supplement (1 de mayo de 2012): 175.16. http://dx.doi.org/10.4049/jimmunol.188.supp.175.16.

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Abstract Neuroimmune semaphorin 4D (Sema4D) was found to be expressed and function in the nervous and immune systems. In the immune system, Sema4D is constitutively expressed on T cells and regulates T cell priming. Currently available data suggest that Sema4D has crucial stimulatory functions in a broad range of cells including macrophages, DC, NK cells, and neutrophils. We hypothesized that Sema4D plays a critical non-redundant regulatory role in allergic airway response. To test our hypothesis, we employed Sema4D-/- and WT mice in the well-defined mouse model of OVA-induced experimental asthma. We observed a decrease in eosinophilic airway infiltration in allergen-treated Sema4D-/- mice relative to WT mice. This reduced allergic inflammatory response was associated with decreased BAL IL-5, IL-13, and TGFb1 levels. In addition, T cell proliferation in OVA323-339 - restimulated Sema4D-/- cell cultures were downregulated. However, airway hyperreactivity to methacholine challenges was not affected by Sema4D deficiency. These data provide a new insight into Sema4D biology and define Sema4D as an important regulator of Th2-driven lung pathophysiology.

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43

Wang, Yu-li. "Fluorescence microscopy of molecular organization and dynamics in cultured cells". Proceedings, annual meeting, Electron Microscopy Society of America 50, n.º 1 (agosto de 1992): 550–51. http://dx.doi.org/10.1017/s0424820100123155.

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Over the past ten years various technical advances have allowed the direct study of molecular activities in cultured cells under a fluorescence microscope. Fluorescent probes are well known for their high sensitivity, specificity and amenability to various spectroscopic analyses. When used in conjunction with low-light-level detectors and image processing computers, high resolution images of weak signals from single cells can be successfully acquired. In addition, the availability of digitized images has greatly facilitated the extraction of photometric and morphometric information.We use Zeiss inverted microscopes equipped with epi-illuminators and Dage-MTI ISIT video cameras or Photometrics cooled CCD cameras. Custom incubator systems built on the microscope stages allow the maintenance of live cells for up to several days. The signals are processed with image processing systems (Imaging Technologies) interfaced with graphics workstations (Silicon Graphics, Model 3130 or 4D/20) or personal computers (386/33). All images are acquired by frame averaging/signal integration, followed by subtraction of the dark noise, and storage as computer files. A variation of this simple processing strategy has allowed the detection of extremely weak signals that are essentially invisible on unprocessed ISIT images. Computer programs are then used to display sequences or images as motion pictures, to measure the linear dimension and angular orientation, and to integrate intensities over defined areas.

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44

Narożna, Maria, Violetta Krajka-Kuźniak, Robert Kleszcz y Wanda Baer-Dubowska. "Indomethacin and Diclofenac Hybrids with Oleanolic Acid Oximes Modulate Key Signaling Pathways in Pancreatic Cancer Cells". International Journal of Molecular Sciences 23, n.º 3 (22 de enero de 2022): 1230. http://dx.doi.org/10.3390/ijms23031230.

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Our earlier studies showed that coupling nonsteroidal anti-inflammatory drugs (NSAIDs) with oleanolic acid derivatives increased their anti-inflammatory activity in human hepatoma cells. The aim of this study was to evaluate their effect on the signaling pathways involved in inflammation processes in human pancreatic cancer (PC) cells. Cultured PSN-1 cells were exposed for 24 h (30 µM) to OA oxime (OAO) derivatives substituted with benzyl or morpholide groups and their conjugates with indomethacin (IND) or diclofenac (DCL). The activation of NF-κB and Nrf2 was assessed by the evaluation of the translocation of their active forms into the nucleus and their binding to specific DNA sequences via the ELISA assay. The expression of NF-κB and Nrf2 target genes was evaluated by R-T PCR and Western blot analysis. The conjugation of IND or DCL with OAO derivatives increased cytotoxicity and their effect on the tested signaling pathways. The most effective compound was the DCL hybrid with OAO morpholide (4d). This compound significantly reduced the activation and expression of NF-κB and enhanced the activation and expression of Nrf2. Increased expression of Nrf2 target genes led to reduced ROS production. Moreover, MAPKs and the related pathways were also affected. Therefore, conjugate 4d deserves more comprehensive studies as a potential PC therapeutic agent.

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Ji, Kyungmin, George J. Schwenkel, Raymond R. Mattingly, Harini G. Sundararaghavan, Zheng Gang Zhang y Michael Chopp. "A Fibroblast-Derived Secretome Stimulates the Growth and Invasiveness of 3D Plexiform Neurofibroma Spheroids". Cancers 16, n.º 14 (9 de julio de 2024): 2498. http://dx.doi.org/10.3390/cancers16142498.

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Plexiform neurofibromas (PNs) occur in about a half of neurofibromatosis type 1 (NF1) patients and have garnered significant research attention due to their capacity for growth and potential for malignant transformation. NF1 plexiform neurofibroma (pNF1) is a complex tumor composed of Schwann cell-derived tumor cells (Nf1−/−) and the tumor microenvironment (TME). Although it has been widely demonstrated that the TME is involved in the formation of neurofibromas, little is known about the effects of the TME on the subsequent progression of human pNF1. Elucidating the molecular interactions between tumor cells and the TME may provide new therapeutic targets to reduce the progression of pNF1. In the present study, we focused on the contributions of fibroblasts, the most abundant cell types in the TME, to the growth of pNF1. To simulate the TME, we used a three-dimensional (3D) coculture model of immortalized pNF1 tumor cells (Nf1−/−) and primary fibroblasts (Nf1+/−) derived from pNF1 patients. We performed live-cell imaging of 3D/4D (3D in real-time) cultures through confocal microscopy followed by 3D quantitative analyses using advanced imaging software. The growth of pNF1 spheroids in 3D cocultures with fibroblasts was significantly greater than that of pNF1 spheroids in 3D monocultures. An increase in the growth of pNF1 spheroids also occurred when they were cultured with conditioned media (CM) from fibroblasts. Moreover, fibroblast-derived CM increased the invasive outgrowth and further local invasion of pNF1 spheroids. Interestingly, when small extracellular vesicles (sEVs) were depleted from the fibroblast-derived CM, the stimulation of the growth of pNF1 spheroids was lost. Our results suggest that fibroblast-derived sEVs are a therapeutic target for reducing the growth of pNF1.

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Nishide, Masayuki, Satoshi Nojima, Daisuke Ito, Hyota Takamatsu, Shohei Koyama, Sujin Kang, Tetsuya Kimura et al. "Semaphorin 4D inhibits neutrophil activation and is involved in the pathogenesis of neutrophil-mediated autoimmune vasculitis". Annals of the Rheumatic Diseases 76, n.º 8 (17 de abril de 2017): 1440–48. http://dx.doi.org/10.1136/annrheumdis-2016-210706.

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ObjectivesInappropriate activation of neutrophils plays a pathological role in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). The aim of this study was to investigate the functions of semaphorin 4D (SEMA4D) in regulation of neutrophil activation, and its involvement in AAV pathogenesis.MethodsSerum levels of soluble SEMA4D were evaluated by ELISA. Blood cell-surface expression of membrane SEMA4D was evaluated by flow cytometry. To determine the functional interactions between neutrophil membrane SEMA4D and endothelial plexin B2, wild-type and SEMA4D−/− mice neutrophils were cultured with an endothelial cell line (MS1) stained with SYTOX green, and subjected to neutrophil extracellular trap (NET) formation assays. The efficacy of treating human neutrophils with recombinant plexin B2 was assessed by measuring the kinetic oxidative burst and NET formation assays.ResultsSerum levels of soluble SEMA4D were elevated in patients with AAV and correlated with disease activity scores. Cell-surface expression of SEMA4D was downregulated in neutrophils from patients with AAV, a consequence of proteolytic cleavage of membrane SEMA4D. Soluble SEMA4D exerted pro-inflammatory effects on endothelial cells. Membranous SEMA4D on neutrophils bound to plexin B2 on endothelial cells, and this interaction decreased NET formation. Recombinant plexin B2 suppressed neutrophil Rac1 activation through SEMA4D’s intracellular domain, and inhibited pathogen-induced or ANCA-induced oxidative burst and NET formation.ConclusionsNeutrophil surface SEMA4D functions as a negative regulator of neutrophil activation. Proteolytic cleavage of SEMA4D as observed in patients with AAV may amplify neutrophil-mediated inflammatory responses. SEMA4D is a promising biomarker and potential therapeutic target for AAV.

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Jain, Pramod T., J. Thomas Pento y Robert A. Magarian. "A comparison of the antitumor activity of two triarylcyclopropyl antiestrogens (compounds 4d and 5c) on human breast cancer cells in culture". Anti-Cancer Drugs 5, n.º 4 (agosto de 1994): 429–36. http://dx.doi.org/10.1097/00001813-199408000-00007.

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Hendrikson, Wilhelmus J., Jeroen Rouwkema, Federico Clementi, Clemens A. van Blitterswijk, Silvia Farè y Lorenzo Moroni. "Towards 4D printed scaffolds for tissue engineering: exploiting 3D shape memory polymers to deliver time-controlled stimulus on cultured cells". Biofabrication 9, n.º 3 (2 de agosto de 2017): 031001. http://dx.doi.org/10.1088/1758-5090/aa8114.

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Mierke, Claudia Tanja. "Bioprinting of Cells, Organoids and Organs-on-a-Chip Together with Hydrogels Improves Structural and Mechanical Cues". Cells 13, n.º 19 (1 de octubre de 2024): 1638. http://dx.doi.org/10.3390/cells13191638.

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The 3D bioprinting technique has made enormous progress in tissue engineering, regenerative medicine and research into diseases such as cancer. Apart from individual cells, a collection of cells, such as organoids, can be printed in combination with various hydrogels. It can be hypothesized that 3D bioprinting will even become a promising tool for mechanobiological analyses of cells, organoids and their matrix environments in highly defined and precisely structured 3D environments, in which the mechanical properties of the cell environment can be individually adjusted. Mechanical obstacles or bead markers can be integrated into bioprinted samples to analyze mechanical deformations and forces within these bioprinted constructs, such as 3D organoids, and to perform biophysical analysis in complex 3D systems, which are still not standard techniques. The review highlights the advances of 3D and 4D printing technologies in integrating mechanobiological cues so that the next step will be a detailed analysis of key future biophysical research directions in organoid generation for the development of disease model systems, tissue regeneration and drug testing from a biophysical perspective. Finally, the review highlights the combination of bioprinted hydrogels, such as pure natural or synthetic hydrogels and mixtures, with organoids, organoid–cell co-cultures, organ-on-a-chip systems and organoid-organ-on-a chip combinations and introduces the use of assembloids to determine the mutual interactions of different cell types and cell–matrix interferences in specific biological and mechanical environments.

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St-Denis, Nicole A., D. Richard Derksen y David W. Litchfield. "Evidence for Regulation of Mitotic Progression through Temporal Phosphorylation and Dephosphorylation of CK2α". Molecular and Cellular Biology 29, n.º 8 (2 de febrero de 2009): 2068–81. http://dx.doi.org/10.1128/mcb.01563-08.

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ABSTRACT Proper mitotic progression is crucial for maintenance of genomic integrity in proliferating cells and is regulated through an intricate series of events, including protein phosphorylation governed by a complex network of protein kinases. One kinase family implicated in the regulation of mitotic progression is protein kinase CK2, a small family of enzymes that is overexpressed in cancer and induces transformation in mice and cultured fibroblasts. CK2α, one isoform of the catalytic subunits of CK2, is maximally phosphorylated at four sites in nocodazole-treated cells. To investigate the effects of CK2α phosphorylation on mitotic progression, we generated phosphospecific antibodies against its mitotic phosphorylation sites. In U2OS cells released from S-phase arrest, these antibodies reveal that CK2α is most highly phosphorylated in prophase and metaphase. Phosphorylation gradually decreases during anaphase and becomes undetectable during telophase and cytokinesis. Stable expression of phosphomimetic CK2α (CK2α-4D, CK2α-4E) results in aberrant centrosome amplification and chromosomal segregation defects and loss of mitotic cells through mitotic catastrophe. Conversely, cells expressing nonphosphorylatable CK2α (CK2α-4A) show a decreased ability to arrest in mitosis following nocodazole treatment, suggesting involvement in the spindle assembly checkpoint. Collectively, these studies indicate that reversible phosphorylation of CK2α requires precise regulation to allow proper mitotic progression.

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