Cellular Fate Research Articles

In Situ Cellular Localization of Nonfluorescent [60]Fullerene Nanomaterial in MCF-7 Breast Cancer Cells.

Cellular localization of carbon nanomaterials in cancer cells is essential information for better understanding their interaction with biological targets and a crucial factor for further evaluating their biological properties as nanovehicles or nanotherapeutics. Recently, increasing efforts to develop promising fullerene nanotherapeutics for cancer nanotechnology have been made. However, the main challenge regarding studying their cellular effects is the lack of effective methods for their visualization and determining their cellular fate due to the limited fluorescence of buckyball scaffolds. Herein, we developed a method for cellular localization of nonfluorescent and water-soluble fullerene nanomaterials using the in vitro click chemistry approach. First, we synthesized a triple-bonded fullerene probe (TBC60ser), which was further used as a starting material for 1,3-dipolar cycloaddition using 3-azido-7-hydroxycoumarin and sulfo-cyanine5 azide fluorophores to create fluorescent fullerene triazoles. In this work, we characterized the structurally triple-bonded [60]fullerene derivative and confirmed its high symmetry (Th) and the successful formation of fullerene triazoles by spectroscopic techniques (i.e., ultraviolet–visible, fluorescence, and Fourier transform infrared spectroscopies) and mass spectrometry. The created fluorescent fullerene triazoles were successfully localized in the MCF-7 breast cancer cell line using fluorescent microscopy. Overall, our findings demonstrate that TBC60ser localizes in the lysosomes of MCF-7 cells, with only a small affinity to mitochondria.

Engineered models for placental toxicology: Emerging approaches based on tissue decellularization

Recent increases in prescriptions and illegal drug use as well as exposure to environmental contaminants during pregnancy have highlighted the critical importance of placental toxicology in understanding and identifying risks to both mother and fetus. Although advantageous for basic science, current in vitro models often fail to capture the complexity of placental response, likely due to their inability to recreate and monitor aspects of the microenvironment including physical properties, mechanical forces and stiffness, protein composition, cell-cell interactions, soluble and physicochemical factors, and other exogenous cues. Tissue engineering holds great promise in addressing these challenges and provides an avenue to better understand basic biology, effects of toxic compounds and potential therapeutics. The key to success lies in effectively recreating the microenvironment. One strategy to do this would be to recreate individual components and then combine them. However, this becomes challenging due to variables present according to conditions such as tissue location, age, health status and lifestyle. The extracellular matrix (ECM) is known to influence cellular fate by working as a storage of factors. Decellularized ECM (dECM) is a recent tool that allows usage of the original ECM in a refurbished form, providing a relatively reliable representation of the microenvironment. This review focuses on using dECM in modified forms such as whole organs, scaffold sheets, electrospun nanofibers, hydrogels, 3D printing, and combinations as building blocks to recreate aspects of the microenvironment to address general tissue engineering and toxicology challenges, thus illustrating their potential as tools for future placental toxicology studies.

Changing Fate: Reprogramming Cells via Engineered Nanoscale Delivery Materials.

The incorporation of nanotechnology in regenerative medicine is at the nexus of fundamental innovations and early-stage breakthroughs, enabling exciting biomedical advances. One of the most exciting recent developments is the use of nanoscale constructs to influence the fate of cells, which are the basic building blocks of healthy function. Appropriate cell types can be effectively manipulated by direct cell reprogramming; a robust technique to manipulate cellular function and fate, underpinning burgeoning advances in drug delivery systems, regenerative medicine, and disease remodeling. Individual transcription factors, or combinations thereof, can be introduced into cells using both viral and nonviral delivery systems. Existing approaches have inherent limitations. Viral-based tools include issues of viral integration into the genome of the cells, the propensity for uncontrollable silencing, reduced copy potential and cell specificity, and neutralization via the immune response. Current nonviral cell reprogramming tools generally suffer from inferior expression efficiency. Nanomaterials are increasingly being explored to address these challenges and improve the efficacy of both viral and nonviral delivery because of their unique properties such as small size and high surface area. This review presents the state-of-the-art research in cell reprogramming, focused on recent breakthroughs in the deployment of nanomaterials as cell reprogramming delivery tools.

Mitochondria as Cellular and Organismal Signaling Hubs.

Mitochondria are traditionally known as the powerhouse of the cell, but their functions extend far beyond energy production. They are vital in cellular and organismal pathways that direct metabolism, stress responses, immunity, and cellular fate. To accomplish these tasks, mitochondria have established networks of both intra- and extracellular communication. Intracellularly, these communication routes comprise direct contacts between mitochondria and other subcellular components as well as indirect vesicle transport of ions, metabolites, and other intracellular messengers. Extracellularly, mitochondria can induce stress responses or other cellular changes that secrete mitochondrial cytokine (mitokine) factors that can travel between tissues as well as respond to immune challenges from extracellular sources. Here we provide a current perspective on the major routes of communication for mitochondrial signaling, including their mechanisms and physiological impact. We also review the major diseases and age-related disorders associated with defects in these signaling pathways. An understanding of how mitochondrial signaling controls cellular homeostasis will bring greater insight into how dysfunctional mitochondria affect health in disease and aging.

Differentiation of Pluripotent Stem Cells Into Thymic Epithelial Cells and Generation of Thymic Organoids: Applications for Therapeutic Strategies Against APECED

The thymus is a primary lymphoid organ essential for the induction of central immune tolerance. Maturing T cells undergo several steps of expansion and selection mediated by thymic epithelial cells (TECs). In APECED and other congenital pathologies, a deficiency in genes that regulate TEC development or their ability to select non auto-reactive thymocytes results in a defective immune balance, and consequently in a general autoimmune syndrome. Restoration of thymic function is thus crucial for the emergence of curative treatments. The last decade has seen remarkable progress in both gene editing and pluripotent stem cell differentiation, with the emergence of CRISPR-based gene correction, the trivialization of reprogramming of somatic cells to induced pluripotent stem cells (iPSc) and their subsequent differentiation into multiple cellular fates. The combination of these two approaches has paved the way to the generation of genetically corrected thymic organoids and their use to control thymic genetic pathologies affecting self-tolerance. Here we review the recent advances in differentiation of iPSc into TECs and the ability of the latter to support a proper and efficient maturation of thymocytes into functional and non-autoreactive T cells. A special focus is given on thymus organogenesis and pathway modulation during iPSc differentiation, on the impact of the 2/3D structure on the generated TECs, and on perspectives for therapeutic strategies in APECED based on patient-derived iPSc corrected for AIRE gene mutations.

Gpx3 and Egr1 Are Involved in Regulating the Differentiation Fate of Cardiac Fibroblasts under Pressure Overload.

Objectives Although myocardial fibrosis is a common pathophysiological process associated with many heart diseases, the molecular mechanisms regulating the development of fibrosis have not been fully determined. Recently, single cell RNA sequencing (scRNA-seq) analysis has been used to examine cellular fate and function during cellular differentiation and has contributed to elucidating the mechanisms of various diseases. The main purpose of this study was to characterize the fate of cardiac fibroblasts (CFs) and the dynamic gene expression patterns in a model of cardiac pressure overload using scRNA-seq analysis. Methods The public scRNA-seq dataset of the transverse aortic coarctation (TAC) model in mice was downloaded from the GEO database, GSE155882. First, we performed quality control, dimensionality reduction, clustering, and annotation of the data through the Seurat R package (v4.0.5). Then, we constructed the pseudotime trajectory of cell development and identified key regulatory genes using the Monocle R package (v2.22.0). Different cell fates and groups were fully characterized by Gene Set Enrichment Analysis (GSEA) analysis and Transcription factor (TF) activity analysis. Finally, we used Cytoscape (3.9.1) to extensively examine the gene regulatory network related to cell fate. Results Pseudotime analysis showed that CFs differentiated into two distinct cell fates, one of which produced activated myofibroblasts, and the other which produced protective cells that were associated with reduced fibrosis levels, increased antioxidative stress responses, and the ability to promote angiogenesis. In the TAC model, activated CFs were significantly upregulated, while protective cells were downregulated. Treatment with the bromodomain inhibitor JQ1 reversed this change and improved fibrosis. Analysis of dynamic gene expression revealed that Gpx3 was significantly upregulated during cell differentiation into protective cells. Gpx3 expression was affected by JQ1 treatment. Furthermore, Gpx3 expression levels were negatively correlated with the different levels of fibrosis observed in the various treatment groups. Finally, we found that transcription factors Jun, Fos, Atf3, and Egr1 were upregulated in protective cells, especially Egr1 was predicted to be involved in the regulation of genes related to antioxidant stress and angiogenesis, suggesting a role in promoting differentiation into this cell phenotype. Conclusions The scRNA-seq analysis was used to characterize the dynamic changes associated with fibroblast differentiation and identified Gpx3 as a factor that might be involved in the regulation of myocardial fibrosis under cardiac pressure overload. These findings will help to further understanding of the mechanism of fibrosis and provide potential intervention targets.

Temperature, concentration, and surface modification influence the cellular uptake and the protein corona of polystyrene nanoparticles

The composition of the protein corona varies depending on several parameters and influences the cellular fate of the nanocarriers. Here, we investigated the influence of three key parameters (surface charge, temperature, and plasma concentration) on the formation and composition of the protein corona of polystyrene nanoparticles and ultimately on the cellular uptake of pre-coated nanoparticles. At a fixed temperature and concentration, the surface charge, and surfactant influence its composition. We observed that the composition of the corona formed at low temperatures (4°C) is different from that formed at physiological temperatures (37°C). At low plasma concentrations (up to 25%), the corona consists of more diverse proteins than at higher concentrations. Finally, we concluded that regardless of the nanoparticle formulation, the degree of uptake by model cancer and endothelial cells of the nanoparticles decreased when pre-coated at increasing temperature or plasma concentration. Statement of significanceDrug delivery through nanocarriers is an increasingly important concept in research and medicine. One problem in the application of nanocarriers in medicine is the protein corona that forms around the nanocarriers when they get in contact with protein-containing fluids. So far, several factors have been identified that influence the composition of the protein corona and thus the biological identity of the particles. However, lacking comparability remains between the studies because different concentrations or temperatures of the protein solutions are used. In this study we demonstrate how the incubation temperature or the concentration of plasma influences the protein corona and thus the cellular uptake of polystyrene nanoparticles.

Beyond microplastics - investigation on health impacts of submicron and nanoplastic particles after oral uptake in vitro

The continuously increasing use of plastics is supposed to result in a rising exposure of MNPs to humans. Available data on human health risks of microplastics after oral uptake increased immensely in the past years and indicates very likely only low risks after oral consumption. Concerning nanoplastics, uptake, transport and potential adverse effects after oral uptake are less well understood. This study aims to investigate differences between microplastic particles and particles in the submicron- and nanoscaled size derived from food-relevant polymers with a particle size range consistent with higher potential for cellular uptake, fate, and effects when applied to human intestinal and liver cells. This work includes the development of cellular and subcellular detection methods for synthetic polymeric particles in the micro- and nanometer-range, using Scanning Electron Microscopy, Small-Angle X-ray and Dynamic Light Scattering methods, Asymmetric Flow Field Flow Fractionation, octanol-water fractionation, fluorescence microscopy and flow cytometry. Polylactic acid (250 nm and 2 μm (polydisperse)), melamine formaldehyde (366 nm) and polymethylmethacrylate (25 nm) were thoroughly characterized. The submicro- and nanoplastic test particles showed an increased uptake and transport quantity through intestinal cells. Both types of particles resulted in observed differences of uptake behavior, most likely influenced by different lipophilicity, which varied between the polymeric test materials. Toxic effects were detected after 24 h only in overload situations for the particles in the submicrometer range. This study provides further evidence for gastrointestinal uptake of submicro- and nanoplastics and points towards differences regarding bioavailability between microplastics and smaller plastic particles that may result following the ingestion of contaminated food and beverages. Furthermore, the results reinforce the importance for studying nanoplastics of different materials of varying size, surface properties, polymer composition and hydrophobicity.

A Non-Canonical Role for IRE1α Links ER and Mitochondria as Key Regulators of Astrocyte Dysfunction: Implications in Methamphetamine use and HIV-Associated Neurocognitive Disorders

Astrocytes are one of the most numerous glial cells in the central nervous system (CNS) and provide essential support to neurons to ensure CNS health and function. During a neuropathological challenge, such as during human immunodeficiency virus (HIV)-1 infection or (METH)amphetamine exposure, astrocytes shift their neuroprotective functions and can become neurotoxic. Identifying cellular and molecular mechanisms underlying astrocyte dysfunction are of heightened importance to optimize the coupling between astrocytes and neurons and ensure neuronal fitness against CNS pathology, including HIV-1-associated neurocognitive disorders (HAND) and METH use disorder. Mitochondria are essential organelles for regulating metabolic, antioxidant, and inflammatory profiles. Moreover, endoplasmic reticulum (ER)-associated signaling pathways, such as calcium and the unfolded protein response (UPR), are important messengers for cellular fate and function, including inflammation and mitochondrial homeostasis. Increasing evidence supports that the three arms of the UPR are involved in the direct contact and communication between ER and mitochondria through mitochondria-associated ER membranes (MAMs). The current study investigated the effects of HIV-1 infection and chronic METH exposure on astrocyte ER and mitochondrial homeostasis and then examined the three UPR messengers as potential regulators of astrocyte mitochondrial dysfunction. Using primary human astrocytes infected with pseudotyped HIV-1 or exposed to low doses of METH for 7 days, astrocytes had increased mitochondrial oxygen consumption rate (OCR), cytosolic calcium flux and protein expression of UPR mediators. Notably, inositol-requiring protein 1α (IRE1α) was most prominently upregulated following both HIV-1 infection and chronic METH exposure. Moreover, pharmacological inhibition of the three UPR arms highlighted IRE1α as a key regulator of astrocyte metabolic function. To further explore the regulatory role of astrocyte IRE1α, astrocytes were transfected with an IRE1α overexpression vector followed by activation with the proinflammatory cytokine interleukin 1β. Overall, our findings confirm IRE1α modulates astrocyte mitochondrial respiration, glycolytic function, morphological activation, inflammation, and glutamate uptake, highlighting a novel potential target for regulating astrocyte dysfunction. Finally, these findings suggest both canonical and non-canonical UPR mechanisms of astrocyte IRE1α. Thus, additional studies are needed to determine how to best balance astrocyte IRE1α functions to both promote astrocyte neuroprotective properties while preventing neurotoxic properties during CNS pathologies.

Abstract 582: Pre-clinical development of CAR Monocytes (CAR Mono) for solid tumor immunotherapy

Abstract Introduction: Engineered cell therapies have demonstrated significant clinical activity against hematologic malignancies, but responses have been rare in solid tumors. Our previously developed human chimeric antigen receptor macrophage (CAR-M) platform has shown potent anti-tumor activity in pre-clinical solid tumor models1, and the anti-HER2 CAR-M CT-0508 is currently being evaluated in a Phase I trial. The use of myeloid cells as a platform for cell therapy provides the tools to overcome critical solid tumor challenges such as infiltration, immunosuppression within the tumor microenvironment, lymphocyte exclusion, and target antigen heterogeneity. Currently, CAR-M are generated in a week-long ex-vivo process in which peripheral blood monocytes are differentiated into macrophages prior to genetic manipulation. Here, we demonstrate the production feasibility, phenotype, pharmacokinetics, cellular fate, specificity, and anti-tumor activity of human CD14+ CAR monocytes. Experimental: Using the chimeric adenoviral vector Ad5f35, we engineered primary human CD14+ monocytes to express a CAR (CAR-mono) targeted against HER2. We established a process that allowed for same day manufacturing (from Leukopak to cryopreserved CAR-mono cell product). Results: CAR-mono showed high CAR expression and viability (>90%), and efficiently differentiated into CAR-expressing macrophages. Adenoviral transduction led to pre-conditioning of CAR-mono, resulting in a strong M1 phenotype upon differentiation into CAR-M. CAR-mono derived macrophages demonstrated potent anti-tumor activity regardless of exposure to GM-CSF or M-CSF, and were protected against M2 switching by immunosuppressive factors. Treating CAR-mono with GM-CSF and IL-4 resulted in their differentiation to monocyte-derived CAR-DCs with an activated phenotype, indicating that these cells retained their myeloid differentiation potential. In vivo, CAR-mono induced anti-tumor activity in various HER2+ solid tumor xenograft models. Following IV administration, CAR-mono demonstrated the ability to traffic to both GM-CSFhigh and GM-CSFlow expressing tumors. Notably, CAR-mono showed long-term CAR expression and persistence (>180 days) in both NSG and NSG-S mouse models, demonstrating lasting persistence irrespective of human cytokine support. Conclusions: The CAR-mono platform enables an automated, same-day manufacturing process while maintaining the key characteristics of CAR-M therapy. The use of Ad5f35 for human monocyte transduction primes the cells toward M1 macrophage differentiation and produces a cell population phenotypically and functionally similar to our established CAR-M platform. These data provide strong pre-clinical support to advance the CAR-mono platform into clinical testing.1Klichinsky M, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nature Biotechnology. March 2020. Citation Format: Daniel Blumenthal, Linara Gabitova, Brett Menchel, Patricia Reyes-Uribe, Sabrina Ceeraz DeLong, Sascha Abramson, Michael Klichinsky. Pre-clinical development of CAR Monocytes (CAR Mono) for solid tumor immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 582.

Abstract 1740: Determining the intracellular fate of ligand-conjugated therapeutics using nanogold labeling

Abstract Recent developments in ligand-conjugated RNAi therapeutics have led to improved stability of RNAi molecules, reducing effective therapeutic dose, and avoiding delivery vehicle-associated toxicity. However, as our group has shown in previous studies with FolamiR (Folate-conjugated miR-34a), endosomal entrapment of the therapeutic molecule is a rate-limiting factor in RNAi activity. To improve endosomal release of ligand-conjugates, several approaches are being explored such as incorporation of endosomal escape agents (nigericin), chemical modification to enhance stability of miRNA/siRNA within the acidic compartments, and modification of ligand-receptor interaction to enable adequate release from the receptor upon internalization. Current fluorescent microscopy techniques are only able to approximately estimate the cellular fate of FolamiR without providing enough resolution to determine the endosomal stage, receptor disengagement and other dynamics involved in the endosomal entrapment/release of FolamiR. To overcome this, we developed Folate-conjugated nanogold construct (FolaGold) that mimics FolamiR molecule. The nanogold in FolaGold is used as a proxy for miR-34a and electron microscopy can be performed to pinpoint the cellular fate of nanogold including receptor disengagement process, the endosomal stage as well as cytosolic enrichment at the nanometer resolution. In this study, FolaGold is validated to bind to folate receptor in a competition assay and is confirmed to be present in specific endosomal compartments in MB231 cells, unlike unconjugated nanogold. We further plan to use FolaGold to determine changes in intracellular fate of nanogold upon incorporation of endosomal escape agents (nigericin) and modification of ligand-receptor interaction by using a lower affinity binding ligand that facilitates ligand-receptor disengagement following internalization. While we are using nanogold labeling to determine the intracellular fate of ligand-conjugated RNAi therapeutics, the labeling can also help improve our understanding of endosomal release dynamics and activity of other ligand-conjugate based therapeutics. Citation Format: Ikjot S. Sohal, Ahmed M. Abdelaal, Andrea L. Kasinski. Determining the intracellular fate of ligand-conjugated therapeutics using nanogold labeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1740.

Abstract 144: Survivin, a novel mediator of the UPR identified by a functional genome wide CRISPR/Cas9 based knock out screen

Abstract Tumor growth and metastasis involve a complex relationship with the tissue microenvironment. A proliferating tumor encounters several stresses from the microenvironment like hypoxia, lack of nutrients and acidosis. To cope with these conditions, cancer cells can co-opt existing cytoprotective mechanisms, such as the Unfolded Protein Response (UPR). The UPR involves transcriptional and translational activation of signaling pathways designed to relieve cellular stress and attenuate cell death; although, in the case of unresolved or chronic ER stress UPR can promote cell death. However, the mechanisms by which the UPR influences cell fate in the presence of ER stress are poorly understood. To address this, we have used a functional CRISPR-based genetic knockout screen, to determine novel regulators of UPR and the mechanisms involved to control cellular fate following chronic ER stress. We delivered a lentiviral genome-wide CRISPR-Cas9 knockout library to two cell lines and identified sgRNAs which are over-represented (pro-apoptotic genes), or underrepresented (pro-survival genes) following ER stress induced by thapsigargin and tunicamycin. One of the highest-ranking gene candidates was Survivin/BIRC5, a protein which is overexpressed in tumor cells compared to normal tissues. Survivin was first identified as an inhibitor of apoptosis but also participates in the regulation of the cell cycle. Survivin held a high position in the negative ranking scale of all screens suggesting a possible prosurvival role in response to ER stress. In agreement, our results show that genetic knock down or chemical inhibition of Survivin led to sensitization of cells to ER stress. Moreover, both in vitro an in vivo data support that PERK (a key sensor of the third arm of the UPR cascade) is responsible for this sensitization. Interestingly, we observed that ER stress in turn leads to downregulation of Survivin in a dose and time dependent fashion, both in the level of protein but also RNA, employing a variety of mechanisms like protein translation, degradation and RNA synthesis. Foreseeably, PERK knock down reverses the aforementioned effect supporting its role as a key participant in the interplay between Survivin and ER stress. Though the balance between those two is still to be understood our results demonstrate that Survivin holds a novel function as an ER stress regulator but also targeting of PERK and Survivin in a clinical setting could reveal new windows in therapeutic intervention in malignancy. Citation Format: Nektaria Maria Leli, Souvik Dey, Lauren Brady, Carlo Salas Salinas, Jerome Lin, Giorgos Skoufos, Ioannis Verginadis, Artemis Chatzigeorgiou, Constantinos Koumenis. Survivin, a novel mediator of the UPR identified by a functional genome wide CRISPR/Cas9 based knock out screen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 144.

Abstract 211: Hypofractionated radiation therapy promotes distinct resistant cellular fates as a survival mechanism in two colorectal cell lines: senescence or transient dormancy followed by heterogeneous repopulation

Abstract Radiotherapy (RT) is an effective non-surgical treatment used in colorectal cancer (CRC), which is one of the major causes of cancer mortality. In patients with advanced rectal cancer, this approach is used as neoadjuvant therapy to reduce local recurrence risk. However, RT has a small impact on metastasis formation, which remains an obstacle in clinical practice, being responsible for the high mortality rates. Also, local tumor recurrence after RT tends to be more aggressive, with shorter survival expectancy. This suggests that RT surviving cells develop resistance mechanisms that allow their survival and increase metastatic potential. Despite efforts in this field, the knowledge concerning the post-irradiation behavior and the radioresistant cancer cells phenotype is still limited. Therefore, it is important to elucidate the mechanisms that contribute to the acquisition of this resistant phenotype to improve therapeutic success. In this study, we investigated the characteristics of surviving cells that were submitted to a clinically relevant treatment protocol of hypofractionated irradiation. The cell lines were chosen due to the different levels of genomic instability and genetic mutations and were exposed to 25 Gy delivery in 5 daily fractions. Irradiation surviving cells (ISC) from both cell lines, SW-480 and HCT-116, gain resistance to re-irradiation, increasing their autophagic process and assuming a polyploid giant cancer cell phenotype. In addition, HCT-116 ISC became senescent, while SW-480 ISC show an increase in mesenchymal markers and a decrease in epithelial markers. Long-term evaluation of the ISCs confirmed senescence in HCT-116, while SW-480 exhibited a transient dormancy state, resuming its growth after 14 days. SW-480 ISC also generates a heterogeneous progeny with three distinct phenotypes: rounded and highly proliferative cells, elongated mesenchymal cells, and polyploid giant cancer cells. Furthermore, both ISC enhance the secretion of extracellular vesicles and cytokines (like IL-8, MCP-1, VEGF, and GM-CSF) in a cell-specific manner. Based on these results and the identification of proteins and pathways differentially regulated in ISCs we can contribute to the identification of therapeutic targets for neoadjuvant therapies, in order to improve the prognosis of CRC patients. Citation Format: Josiane Weber Tessmann, Murilo Ramos Rocha, Renata Ivo Vasconcelos, Danielle Lazarin-Bidoia, Raphael Rodrigues Corrêa, Rafael Soares Lindoso, Paulo Emílio Corrêa Leite, Celso Vataru Nakamura, Jose Andres Morgado-Diaz. Hypofractionated radiation therapy promotes distinct resistant cellular fates as a survival mechanism in two colorectal cell lines: senescence or transient dormancy followed by heterogeneous repopulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 211.

Signal Transduction and Gene Regulation in the Endothelium.

Extracellular signals act on G-protein-coupled receptors (GPCRs) to regulate homeostasis and adapt to stress. This involves rapid intracellular post-translational responses and long-lasting gene-expression changes that ultimately determine cellular phenotype and fate changes. The lipid mediator sphingosine 1-phosphate (S1P) and its receptors (S1PRs) are examples of well-studied GPCR signaling axis essential for vascular development, homeostasis, and diseases. The biochemical cascades involved in rapid S1P signaling are well understood. However, gene-expression regulation by S1PRs are less understood. In this review, we focus our attention to how S1PRs regulate nuclear chromatin changes and gene transcription to modulate vascular and lymphatic endothelial phenotypic changes during embryonic development and adult homeostasis. Because S1PR-targeted drugs approved for use in the treatment of autoimmune diseases cause substantial vascular-related adverse events, these findings are critical not only for general understanding of stimulus-evoked gene regulation in the vascular endothelium, but also for therapeutic development of drugs for autoimmune and perhaps vascular diseases.

Adaptive liquid interfaces induce neuronal differentiation of mesenchymal stem cells through lipid raft assembly

Stem cells and their microenvironment interact cooperatively to dictate their fates. Biomaterials are dynamically remodeled by stem cells, and stem cells sense and translate the changes into cell fate decisions. We have previously reported that adaptive biomaterials composed of fibronectin inserted into protein nanosheets at a liquid interface enhance neuronal differentiation of human mesenchymal stem cells (hMSCs). However, we could not decouple clearly the effect of ligand density from that of fibrillary structure on cellular function and fate. Here we present an adaptive biomaterial based on two-dimensional networks of protein nanofibrils at a liquid–liquid interface. Compared with flat protein nanosheets, this biomaterial enhances neuronal differentiation of hMSCs through a signaling mechanism involving focal adhesion kinase. Lipid raft microdomains in plasma membrane are found to play a central role in which hMSCs rapidly adapt to the dynamic microenvironment at the fluid interface. Our finding has substantial implications for regenerative medicine and tissue engineering.

Anti-Müllerian Hormone Signal Transduction involved in Müllerian Duct Regression.

Over seventy years ago it was proposed that the fetal testis produces a hormone distinct from testosterone that is required for complete male sexual development. At the time the hormone had not yet been identified but was invoked by Alfred Jost to explain why the Müllerian duct, which develops into the female reproductive tract, regresses in the male fetus. That hormone, anti-Müllerian hormone (AMH), and its specific receptor, AMHR2, have now been extensively characterized and belong to the transforming growth factor-β families of protein ligands and receptors involved in growth and differentiation. Much is now known about the downstream events set in motion after AMH engages AMHR2 at the surface of specific Müllerian duct cells and initiates a cascade of molecular interactions that ultimately terminate in the nucleus as activated transcription factors. The signals generated by the AMH signaling pathway are then integrated with signals coming from other pathways and culminate in a complex gene regulatory program that redirects cellular functions and fates and leads to Müllerian duct regression.

Decellularized Human Umbilical Tissue-Derived Hydrogels Promote Proliferation and Chondrogenic Differentiation of Mesenchymal Stem Cells

Tissue engineering is a promising approach for the repair and regeneration of cartilaginous tissue. Appropriate three-dimensional scaffolding materials that mimic cartilage are ideal for the repair of chondral defects. The emerging decellularized tissue-based scaffolds have the potential to provide essential biochemical signals and structural integrity, which mimics the natural tissue environment and directs cellular fate. Umbilical cord-derived hydrogels function as 3D scaffolding material, which support adherence, proliferation, migration, and differentiation of cells due to their similar biochemical composition to cartilage. Therefore, the present study aimed to establish a protocol for the formulation of a hydrogel from decellularized human umbilical cord (DUC) tissue, and assess its application in the proliferation and differentiation of UC-MSCs along chondrogenic lineage. The results showed that the umbilical cord was efficiently decellularized. Subsequently, DUC hydrogel was prepared, and in vitro chondral differentiation of MSCs seeded on the scaffold was determined. The developed protocol efficiently removed the cellular and nuclear content while retaining the extracellular matrix (ECM). DUC tissue, pre-gel, and hydrogels were evaluated by FTIR spectroscopy, which confirmed the gelation from pre-gel to hydrogel. SEM analysis revealed the fibril morphology and porosity of the DUC hydrogel. Calcein AM and Alamar blue assays confirmed the MSC survival, attachment, and proliferation in the DUC hydrogels. Following seeding of UC-MSCs in the hydrogels, they were cultured in stromal or chondrogenic media for 28 days, and the expression of chondrogenic marker genes including TGF-β1, BMP2, SOX-9, SIX-1, GDF-5, and AGGRECAN was significantly increased (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Moreover, the hydrogel concentration was found to significantly affect the expression of chondrogenic marker genes. The overall results indicate that the DUC-hydrogel is compatible with MSCs and supports their chondrogenic differentiation in vitro.

Single-Cell Transcriptomics-Based Study of Transcriptional Regulatory Features in the Non-Obstructive Azoospermia Testis.

Non-obstructive azoospermia (NOA) is one of the most important causes of male infertility. Although many congenital factors have been identified, the aetiology in the majority of idiopathic NOA (iNOA) cases remains unknown. Herein, using single-cell RNA-Seq data sets (GSE149512) from the Gene Expression Omnibus (GEO) database, we constructed transcriptional regulatory networks (TRNs) to explain the mutual regulatory relationship and the causal relationship between transcription factors (TFs). We defined 10 testicular cell types by their marker genes and found that the proportion of Leydig cells (LCs) and macrophages (tMΦ) was significantly increased in iNOA testis. We identified specific TFs including LHX9, KLF8, KLF4, ARID5B and RXRG in iNOA LCs. In addition, we found specific TFs in iNOA tMΦ such as POU2F2, SPIB IRF5, CEBPA, ELK4 and KLF6. All these identified TFs are strongly engaged in cellular fate, function and homeostasis of the microenvironment. Changes in the activity of the above-mentioned TFs might affect the function of LCs and tMΦ and ultimately cause spermatogenesis failure. This study illustrate that these TFs play important regulatory roles in the occurrence and development of NOA.

A novel Cbx1, PurB, and Sp3 complex mediates long-term silencing of tissue- and lineage-specific genes

miRNA-based cellular fate reprogramming offers an opportunity to investigate the mechanisms of long-term gene silencing. To further understand how genes are silenced in a tissue-specific manner, we leveraged our miRNA-based method of reprogramming fibroblasts into cardiomyocytes. Through screening approaches, we identified three proteins that were downregulated during reprogramming of fibroblasts into cardiomyocytes: heterochromatin protein Cbx1, transcriptional activator protein PurB, and transcription factor Sp3. We show that knockdown of Cbx1, PurB, and Sp3 was sufficient to induce cardiomyocyte gene expression in fibroblasts. Similarly, gene editing to ablate Cbx1, PurB, and Sp3 expression induced fibroblasts to convert into cardiomyocytes in vivo. Furthermore, high-throughput DNA sequencing and coimmunoprecipitation experiments indicated that Cbx1, PurB, and Sp3 also bound together as a complex and were necessary to localize nucleosomes to cardiomyocyte genes on the chromosome. Finally, we found that the expression of these genes led to nucleosome modification via H3K27me3 (trimethylated histone-H3 lysine-27) deposition through an interaction with the polycomb repressive PRC2 complex. In summary, we conclude that Cbx1, PurB, and Sp3 control cell fate by actively repressing lineage-specific genes.

Midline represses Dpp signaling and target gene expression in Drosophila ventral leg development.

ABSTRACTVentral leg patterning in Drosophila is controlled by the expression of the redundant T-box Transcription factors midline (mid) and H15. Here, we show that mid represses the Dpp-activated gene Daughters against decapentaplegic (Dad) through a consensus T-box binding element (TBE) site in the minimal enhancer, Dad13. Mutating the Dad13 DNA sequence results in an increased and broadening of Dad expression. We also demonstrate that the engrailed-homology-1 domain of Mid is critical for regulating the levels of phospho-Mad, a transducer of Dpp-signaling. However, we find that mid does not affect all Dpp-target genes as we demonstrate that brinker (brk) expression is unresponsive to mid. This study further illuminates the interplay between mechanisms involved in determination of cellular fate and the varied roles of mid.