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Human induced pluripotent stem cell (iPSC)-derived macrophages (IDMs) present a valuable substitute for monocyte-derived macrophages (MDMs) in order to study inflammation pathways in vitro. Through optimization of an IDM differentiation protocol, a six-fold increase in the production yield of myeloid progenitors was achieved. The derived IDMs were further characterized with respect to nucleotide-binding oligomerization domain (NOD) and receptor-interacting serine/threonine-protein kinase 2 (RIPK2) signaling, a key regulatory pathway for autoimmune diseases. The IDM cells recapitulated MDM biology with respect to the proinflammatory chemokine and inflammatory cytokine fingerprint more closely than THP-1 cells. When assessing RIPK2 modulation effect on tumor necrosis factor α (TNF-α), a cardinal mediator of inflammation, a similar pharmacological effect of RIPK2 inhibitors was observed in IDMs and MDMs. Additionally, IDMs and MDMs displayed a similar transcription and pathway profile in response to NOD1/2 stimulation and pharmacological inhibition of RIPK2. In summary, the enhanced myeloid production yield in the improved IDM differentiation protocol offers new opportunities for utilizing physiologically relevant macrophage models in the context of inflammatory diseases., Competing Interests: Declaration of competing interest Authors were either employees of Boehringer Ingelheim Pharma GmbH & Co KG or of Boehringer Ingelheim Pharmaceuticals, Inc., at the time this study was performed. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Macrophages play a pivotal role in drug discovery due to their key regulatory functions in health and disease. Overcoming the limited availability and donor variability of human monocyte-derived macrophages (MDMs), human induced pluripotent stem cell (iPSC)-derived macrophages (IDMs) could provide a promising tool for both disease modeling and drug discovery. To access large numbers of model cells for medium- to high-throughput application purposes, an upscaled protocol was established for differentiation of iPSCs into progenitor cells and subsequent maturation into functional macrophages. These IDM cells resembled MDMs both with respect to surface marker expression and phago- as well as efferocytotic function. A statistically robust high-content-imaging assay was developed to quantify the efferocytosis rate of IDMs and MDMs allowing for measurements both in the 384- and 1536-well microplate format. Validating the applicability of the assay, inhibitors of spleen tyrosine kinase (Syk) were shown to modulate efferocytosis in IDMs and MDMs with comparable pharmacology. The miniaturized cellular assay with the upscaled provision of macrophages opens new routes to pharmaceutical drug discovery in the context of efferocytosis-modulating substances., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Tumor-associated neutrophils (TANs) regulate many processes associated with tumor progression, and depending on the microenvironment, they can exhibit pro- or antitumor functions. However, the molecular mechanisms regulating their tumorigenicity are not clear. Using transplantable tumor models, we showed here that nicotinamide phosphoribosyltransferase (NAMPT), a molecule involved in CSF3R downstream signaling, is essential for tumorigenic conversion of TANs and their pro-angiogenic switch. As a result tumor vascularization and growth are strongly supported by these cells. Inhibition of NAMPT in TANs leads to their antitumor conversion. Adoptive transfer of such TANs into B16F10-tumor bearing mice attenuates tumor angiogenesis and growth. Of note, we observe that the regulation of NAMPT signaling in TANs, and its effect on the neutrophil tumorigenicity, are analogous in mice and human. NAMPT is up-regulated in TANs from melanoma and head-and-neck tumor patients, and its expression positively correlates with tumor stage. Mechanistically, we found that targeting of NAMPT suppresses neutrophil tumorigenicity by inhibiting SIRT1 signaling, thereby blocking transcription of pro-angiogenic genes. Based on these results, we propose that NAMPT regulatory axis is important for neutrophils to activate angiogenic switch during early stages of tumorigenesis. Thus, identification of NAMPT as the critical molecule priming protumor functions of neutrophils provides not only mechanistic insight into the regulation of neutrophil tumorigenicity, but also identifies a potential pathway that may be targeted therapeutically in neutrophils. This, in turn, may be utilized as a novel mode of cancer immunotherapy., (© 2018 UICC.)

The purpose of a biobank is to process, organize, and maintain various types of biospecimens that are to be utilized for both clinical and research-based services. There are different types of biobanks, so the goals of the biobank should be delineated at the outset of forming a biobank. The startup of a biobank benefits from accreditation and stringent adherence to standards of practice. Fundamental to these practices is the protection of privacy and informed consent. A budget must be developed, and sources of funding should be obtained to properly equip the designated space and personnel. The appropriate space for freezers and for biospecimen processing should be identified. Information technology is also a critical part of the biobank and effort should be expended to ensure that this aspect is effective and secure. Given the ethical concerns surrounding biospecimens, engagement with the public is also highly valuable.

Akt1 is known to promote non-homologous end-joining (NHEJ)-mediated DNA double-strand break (DSB) repair by stimulation of DNA-PKcs. In the present study, we investigated the effect of Akt1 on homologous recombination (HR)-dependent repair of radiation-induced DSBs in non-small cell lung cancer (NSCLC) cells A549 and H460. Akt1-knockdown (Akt1-KD) significantly reduced Rad51 protein level, Rad51 foci formation and its colocalization with γH2AX foci after irradiation. Moreover, Akt1-KD decreased clonogenicity after treatment with Mitomycin C and HR repair, as tested by an HR-reporter assay. Double knockdown of Akt1 and Rad51 did not lead to a further decrease in HR compared to the single knockdown of Rad51. Consequently, Akt1-KD significantly increased the number of residual DSBs after irradiation partially independent of the kinase activity of DNA-PKcs. Likewise, the number of residual BRCA1 foci, indicating unsuccessful HR events, also significantly increased in the irradiated cells after Akt1-KD. Together, the results of the study indicate that Akt1 seems to be a regulatory component in the HR repair of DSBs in a Rad51-dependent manner. Thus, based on this novel role of Akt1 in HR and the previously described role of Akt1 in NHEJ, we propose that targeting Akt1 could be an effective approach to selectively improve the killing of tumor cells by DSB-inducing cytotoxic agents, such as ionizing radiation., Competing Interests: The authors declare no conflict of interest.

Cross-talk among oncogenic signaling and metabolic pathways may create opportunities for new therapeutic strategies in cancer. Here we show that although acute inhibition of EGFR-driven glucose metabolism induces only minimal cell death, it lowers the apoptotic threshold in a subset of patient-derived glioblastoma (GBM) cells. Mechanistic studies revealed that after attenuated glucose consumption, Bcl-xL blocks cytoplasmic p53 from triggering intrinsic apoptosis. Consequently, targeting of EGFR-driven glucose metabolism in combination with pharmacological stabilization of p53 with the brain-penetrant small molecule idasanutlin resulted in synthetic lethality in orthotopic glioblastoma xenograft models. Notably, neither the degree of EGFR-signaling inhibition nor genetic analysis of EGFR was sufficient to predict sensitivity to this therapeutic combination. However, detection of rapid inhibitory effects on [ 18 F]fluorodeoxyglucose uptake, assessed through noninvasive positron emission tomography, was an effective predictive biomarker of response in vivo. Together, these studies identify a crucial link among oncogene signaling, glucose metabolism, and cytoplasmic p53, which may potentially be exploited for combination therapy in GBM and possibly other malignancies.

Objectives: Recently cell therapy is a promising therapeutic modality for many types of disease including acute kidney injury (AKI). Due to the unique biological properties, mesenchymal stem cells (MSCs) are attractive cells in this regard. This study aims to transplant MSCs equipped with nuclear factor E2-related factor 2 (Nrf2) in rat experimental models of acute kidney and evaluate regeneration potential of injured kidney especially expression of injury and repaired biomarkers., Materials and Methods: Nrf2 was overexpressed in bone marrow-derived MSCs by pcDNA.3.1 plasmid. AKI was induced using glycerol in rat models. The regenerative potential of Nrf2-overexpressed MSCs was evaluated in AKI-Induced animal models using biochemical and histological methods after transplantation. Expression of repaired genes, AQP1 and CK-18, as well as injury markers, Kim-1 and Cystatin C, was also assayed in engrafted kidney sections., Results: Our results revealed that transplantation of Nrf2-overexpressed MSCs into AKI-induced rats decreased blood urea nitrogen and creatinine and ameliorated kidney regeneration throughout 14 days. Upregulation of repaired markers and downregulation of injury markers were considerable 14 days after transplantation., Conclusions: Overexpression of Nrf2 in MSCs suggests a new strategy to increase efficiency of MSC-based cell therapy in AKI.

One of the major obstacles in cancer therapy is the lack of anticancer agent specificity to tumor tissues. The strategy of cell-based therapy is a promising therapeutic option for cancer treatment. The specific tumor-oriented migration of mesenchymal stem cells (MSCs) makes them a useful vehicle to deliver anticancer agents. In this study, we genetically manipulated bone marrow-derived mesenchymal stem cells with their lipocalin 2 (Lcn2) in order to inhibit liver metastasis of colon cancer in nude mice. Lcn2 was successfully overexpressed in transfected MSCs. The PCR results of SRY gene confirmed the presence of MSCs in cancer liver tissue. This study showed that Lcn2-engineered MSCs (MSC-Lcn2) not only inhibited liver metastasis of colon cancer but also downregulated the expression of vascular endothelial growth factor (VEGF) in the liver. Overall, MSCs by innate tropism toward cancer cells can deliver the therapeutic agent, Lcn2, and inhibit cancer metastasis. Hence, it could be a new modality for efficient targeted delivery of anticancer agent to liver metastasis.

Background: Mesenchymal stem cells (MSCs) are valuable for cell-based therapy. However, their application is limited owing to their low survival rate when exposed to stressful conditions. Autophagy, the process by which cells recycle the cytoplasm and dispose of defective organelles, is activated by stress stimuli to adapt, tolerate adverse conditions, or trigger the apoptotic machinery. This study aimed to determine whether regulation of autophagy would affect the survival of MSCs under stress conditions., Methods: Autophagy was induced in bone marrow-derived MSCs (BM-MSCs) by rapamycin, and was inhibited via shRNA-mediated knockdown of the autophagy specific gene, ATG7. ATG7 expression in BM-MSCs was evaluated by reverse transcription polymerase chain reaction (RT-PCR), western blot, and quantitative PCR (qPCR). Cells were then exposed to harsh microenvironments, and a water-soluble tetrazolium salt (WST)-1 assay was performed to determine the cytotoxic effects of the stressful conditions on cells., Results: Of 4 specific ATG7-inhibitor clones analyzed, only shRNA clone 3 decreased ATG7 expression. Under normal conditions, the induction of autophagy slightly increased the viability of MSCs while autophagy inhibition decreased their viability. However, under stressful conditions such as hypoxia, serum deprivation, and oxidative stress, the induction of autophagy resulted in cell death, while its inhibition potentiated MSCs to withstand the stress conditions. The viability of autophagy-suppressed MSCs was significantly higher than that of relevant controls (P<0.05, P<0.01 and P<0.001)., Conclusion: Autophagy modulation in MSCs can be proposed as a new strategy to improve their survival rate in stressful microenvironments.

Objectives: Lipocalin2 (Lcn2) gene is highly expressed in response to various types of cellular stresses. The precise role of Lcn2 has not been fully understood yet. However, it plays a key role in controlling vital cellular processes such as proliferation, apoptosis and metabolism. Recently it was shown that Lcn2 decreases senescence and increases proliferation of mesenchymal stem cells (MSC) with finite life span under either normal or oxidative stress conditions. However, Lcn2 effects on immortal cell line with infinite proliferation are not defined completely. Materials and., Materials and Methods: HEK-293 cells were transfected with recombinant pcDNA3.1 containing Lcn2 fragment (pcDNA3.1-Lcn2). Expression of lipocalin2 in transfected cells was evaluated by RT-PCR, real time RT-PCR, and ELISA. Different cell groups were treated with H2O2 and WST-1 assay was performed to determine their proliferation rate. Senescence was studied by β-galactosidase and gimsa staining methods as well as evaluation of the expression of senescence-related genes by real time RT-PCR., Results: Lcn2 increased cell proliferation under normal culture condition, while the proliferation slightly decreased under oxidative stress. This decrease was further found to be attributed to senescence., Conclusion: Our findings indicated that under harmful conditions, Lcn2 gene is responsible for the regulation of cell survival through senescence.