Exploring the Therapeutic Potential of Extracellular Vesicles Derived from Human Immature Dental Pulp Cells on Papillary Thyroid Cancer.

Mesenchymal stem-cell-derived extracellular vesicles (MSC-EVs) have been increasingly investigated for cancer therapy and drug delivery, and they offer an advanced cell-free therapeutic option. However, their overall effects and efficacy depend on various factors, including the MSC source and cargo content. In this study, we isolated EVs from the conditioned medium of human immature dental pulp stem cells (hIDPSC-EVs) and investigated their effects on two papillary thyroid cancer (PTC) cell lines (BCPAP and TPC1). We observed efficient uptake of hIDPSC-EVs by both PTC cell lines, with a notable impact on gene regulation, particularly in the Wnt signaling pathway in BCPAP cells. However, no significant effects on cell proliferation were observed. Conversely, hIDPSC-EVs significantly reduced the invasive capacity of both PTC cell lines after 120 h of treatment. These in vitro findings suggest the therapeutic potential of hIDPSC-EVs in cancer management and emphasize the need for further research to develop novel and effective treatment strategies. Furthermore, the successful internalization of hIDPSC-EVs by PTC cell lines underscores their potential use as nanocarriers for anti-cancer agents.

How does the age of control individuals hinder the identification of target genes for Huntington's disease?

Several studies have compared the transcriptome across various brain regions in Huntington's disease (HD) gene-positive and neurologically normal individuals to identify potential differentially expressed genes (DEGs) that could be pharmaceutical or prognostic targets for HD. Despite adhering to technical recommendations for optimal RNA-Seq analysis, none of the genes identified as upregulated in these studies have yet demonstrated success as prognostic or therapeutic targets for HD. Earlier studies included samples from neurologically normal individuals older than the HD gene-positive group. Considering the gradual transcriptional changes induced by aging in the brain, we posited that utilizing samples from older controls could result in the misidentification of DEGs. To validate our hypothesis, we reanalyzed 146 samples from this study, accessible on the SRA database, and employed Propensity Score Matching (PSM) to create a "virtual" control group with a statistically comparable age distribution to the HD gene-positive group. Our study underscores the adverse impact of using neurologically normal individuals over 75 as controls in gene differential expression analysis, resulting in false positives and negatives. We conclusively demonstrate that using such old controls leads to the misidentification of DEGs, detrimentally affecting the discovery of potential pharmaceutical and prognostic markers. This underscores the pivotal role of considering the age of control samples in RNA-Seq analysis and emphasizes its inclusion in evaluating best practices for such investigations. Although our primary focus is HD, our findings suggest that judiciously selecting age-appropriate control samples can significantly improve best practices in differential expression analysis.

The impact of interleukin-6 (IL-6) and mesenchymal stem cell-derived IL-6 on neurological conditions.

Interleukin-6 (IL-6) is a versatile cytokine crucial for immune response modulation, inflammation regulation, and various physiological processes in the body. Its wide-ranging functions underscore its importance in maintaining health. Dysregulated IL-6 is closely associated with many diseases, making it a key research and therapeutic target. Elevated IL-6 levels in the central nervous system worsen neuroinflammation in neurodegenerative diseases by activating microglia and astrocytes and releasing pro-inflammatory cytokines and neurotoxic molecules. Moreover, dysregulated IL-6 weakens the blood-brain barrier, exacerbating neuroinflammation and neuronal damage by allowing peripheral immune cells and inflammatory mediators to enter the brain. Mesenchymal stem cells (MSCs) show promise in modulating neuroinflammation by regulating IL-6 levels. They effectively suppress pro-inflammatory cytokines, including IL-6, while promoting anti-inflammatory factors. This therapeutic approach highlights the importance of targeting IL-6 and other inflammatory mediators to alleviate neuroinflammation and its adverse effects on neurological disorders. This review provides a comprehensive overview of IL-6's involvement in neurological disorders, examining endogenous IL-6 and IL-6 derived from MSCs. We explore IL-6's mechanisms affecting neuronal function, survival, and immune modulation in the central nervous system. Additionally, we discuss the potential of MSC-derived IL-6 in neuroregeneration and neuroprotection. By elucidating IL-6's interplay with neurological pathologies, this review offers insights into novel therapeutic strategies targeting IL-6 signaling pathways for neurological disorders.

Exosomes as a Nano-Carrier for Chemotherapeutics: A New Era of Oncology.

Despite the considerable advancements in oncology, cancer remains one of the leading causes of death worldwide. Drug resistance mechanisms acquired by cancer cells and inefficient drug delivery limit the therapeutic efficacy of available chemotherapeutics drugs. However, studies have demonstrated that nano-drug carriers (NDCs) can overcome these limitations. In this sense, exosomes emerge as potential candidates for NDCs. This is because exosomes have better organotropism, homing capacity, cellular uptake, and cargo release ability than synthetic NDCs. In addition, exosomes can serve as NDCs for both hydrophilic and hydrophobic chemotherapeutic drugs. Thus, this review aimed to summarize the latest advances in cell-free therapy, describing how the exosomes can contribute to each step of the carcinogenesis process and discussing how these nanosized vesicles could be explored as nano-drug carriers for chemotherapeutics.

Advanced cell therapy with low tissue factor loaded product NestaCell® does not confer thrombogenic risk for critically ill COVID-19 heparin-treated patients.

Since the COVID-19 pandemic started, mesenchymal stromal cells (MSC) appeared as a therapeutic option to reduce the over-activated inflammatory response and promote recovery of lung damage. Most clinical studies use intravenous injection for MSC delivery, raising several concerns of thrombogenic risk due to MSC procoagulant activity (PCA) linked to the expression of tissue factor (TF/CD142). This is the first study that demonstrated procoagulant activity of TF+ human immature dental pulp stromal cells (hIDPSC, NestaCell® product) with the percentage of TF+ cells varied from 0.2% to 63.9% in plasma of healthy donors and COVID-19 heparin-treated patients. Thrombogenic risk of TF+ hIDPSCs was evaluated by rotational thromboelastometry (in vitro) and in critically ill COVID-19 patients (clinical trial). We showed that the thromboelastography is not enough to predict the risk of TF+ MSC therapies. Using TF-negative HUVEC cells, we demonstrated that TF is not a unique factor responsible for the cell's procoagulant activity. However, heparin treatment minimizes MSC procoagulant (in vitro). We also showed that the intravenous infusion of hIDPSCs with prophylactic enoxaparin administration in moderate to critically ill COVID-19 patients did not change the values of D-dimer, neither in the PT and PTT times. Our COVID-19 clinical study measured and selected the therapeutic cells with low TF (less than 25% of TF+ hIDPSCs). Our data indicate that the concomitant administration of enoxaparin and low TF-loaded is safe even for critically ill COVID-19 patients.

Therapeutic Potential of Human Immature Dental Pulp Stem Cells Observed in Mouse Model for Acquired Aplastic Anemia.

Aplastic anemia (AA) is a rare and serious disorder of hematopoietic stem cells (HSCs) that results in the loss of blood cells due to the failure of the bone marrow (BM). Although BM transplantation is used to treat AA, its use is limited by donor availability. In this sense, mesenchymal stem cells (MSCs) can offer a novel therapeutic approach for AA. This is because the MSCs contribute to the hematopoietic niche organization through their repopulating. In our study, we used the human immature dental pulp stem cell (hIDPSC), an MSC-like cell, to explore an alternative therapeutic approach for AA. For this, isogenic C57BL/6 mice were exposed to total body irradiation (TBI) to induce the AA. After 48 h of TBI, the mice were intraperitoneally treated with hIDPSC. The immunohistochemistry analyses confirmed that the hIDPSCs migrated and grafted in the mouse bone marrow (BM) and spleen, providing rapid support to hematopoiesis recovery compared to the group exposed to radiation, but not to those treated with the cells as well as the hematological parameters. Six months after the last hIDPSC transplantation, the BM showed long-term stable hematopoiesis. Our data highlight the therapeutic plasticity and hematoprotective role of hIDPSC for AA and potentially for other hematopoietic failures.

Human Immature Dental Pulp Stem Cells Did Not Graft into a Preexisting Human Lung Adenocarcinoma.

Mesenchymal stem cell (MSC)-based therapies have been considered an attractive approach for treating Huntington's disease (HD). However, due to the pulmonary first-passage effect associated with intravenous infusion (the most commonly used route of MSC administration), there is a rising concern that the cells could be entrapped in the lungs and grafted (homing) into preexisting lung cancer. Herein, we report the case of a patient with HD enrolled in a cell therapy phase I clinical trial for HD treatment having a preexisting pulmonary nodule. The nodule was found at the trial screening. The patient was referred to a pulmonologist who considered the nodule non-cancer and authorized enrollment. The patient received four intravenous administrations of human immature dental pulp stem cells (hIDPSCs) at the dose of 1 × 106 cells/kg of body weight within 2 years. One month after the last dose, a computerized tomography scan showed nodule growth. A bronchoscopy biopsy showed primary lung adenocarcinoma. The neoplasm was surgically excised (lung superior right lobectomy). The patient is cured of the neoplasm. The tumor was sectioned into six fragments, which were subjected to RNA-seq. The transcriptome of each tumor section was compared with the transcriptome of infused hIDPSCs using two statistical approaches: principal component analysis and NOIseq. Both results demonstrated a linear distance between the hIDPSCs and the lung adenocarcinoma. These results suggest that the infused hIDPSCs neither home nor graft within the pulmonary nodule.

Restoration of BDNF, DARPP32, and D2R Expression Following Intravenous Infusion of Human Immature Dental Pulp Stem Cells in Huntington's Disease 3-NP Rat Model.

Huntington's disease (HD) is a neurodegenerative inherited genetic disorder, which leads to the onset of motor, neuropsychiatric and cognitive disturbances. HD is characterized by the loss of gamma-aminobutyric acid (GABA)ergic medium spiny neurons (MSNs). To date, there is no treatment for HD. Mesenchymal stem cells (MSCs) provide a substantial therapeutic opportunity for the HD treatment. Herein, we investigated the therapeutic potential of human immature dental pulp stem cells (hIDPSC), a special type of MSC originated from the neural crest, for HD treatment. Two different doses of hIDPSC were intravenously administrated in a subacute 3-nitropropionic acid (3NP)-induced rat model. We demonstrated hIDPSC homing in the striatum, cortex and subventricular zone using specific markers for human cells. Thirty days after hIDPSC administration, the cells found in the brain are still express hallmarks of undifferentiated MSC. Immunohistochemistry quantities analysis revealed a significant increase in the number of BDNF, DARPP32 and D2R positive stained cells in the striatum and cortex in the groups that received hIDPSC. The differences were more expressive in animals that received only one administration of hIDPSC. Altogether, these data suggest that the intravenous administration of hIDPSCs can restore the BDNF, DARPP32 and D2R expression, promoting neuroprotection and neurogenesis.

Exosomes in the Tumor Microenvironment: From Biology to Clinical Applications.

Cancer is one of the most important health problems and the second leading cause of death worldwide. Despite the advances in oncology, cancer heterogeneity remains challenging to therapeutics. This is because the exosome-mediated crosstalk between cancer and non-cancer cells within the tumor microenvironment (TME) contributes to the acquisition of all hallmarks of cancer and leads to the formation of cancer stem cells (CSCs), which exhibit resistance to a range of anticancer drugs. Thus, this review aims to summarize the role of TME-derived exosomes in cancer biology and explore the clinical potential of mesenchymal stem-cell-derived exosomes as a cancer treatment, discussing future prospects of cell-free therapy for cancer treatment and challenges to be overcome.

Stem Cell-Derived Exosomes as Therapeutic Approach for Neurodegenerative Disorders: From Biology to Biotechnology.

The aging population has contributed to the rapid rise in the global incidence of neurodegenerative diseases. Despite the medical advances, there are no effective treatments for these disorders. Therefore, there is an urgent need for new treatments for these diseases. In this sense, cell therapy has been recognized as the best candidate for treating incurable diseases, such as neurodegenerative disorders. However, the therapeutic use of these cells can be limited by several factors. Thus, there has been a rediscovery that extracellular vesicles, including exosomes, can be alternatively explored in the treatment of these diseases, overcoming the limits of cell-based therapy. In this sense, this review aims to revisit all areas from biology, including biogenesis and the content of exosomes, to biotechnology, proposing the minimal information required to isolate, characterize, and study the content of these vesicles for scientific and/or clinical purposes.

LIMD2 Regulates Key Steps of Metastasis Cascade in Papillary Thyroid Cancer Cells via MAPK Crosstalk.

Although papillary thyroid carcinoma (PTC) has a good prognosis, 20-90% of patients show metastasis to regional lymph nodes and 10-15% of patients show metastasis to distant sites. Metastatic disease represents the main clinical challenge that impacts survival rate. We previously showed that LIMD2 was a novel metastasis-associated gene. In this study, to interrogate the role of LIMD2 in cancer invasion and metastasis, we used CRISPR-mediated knockout (KO) of LIMD2 in PTC cells (BCPAP and TPC1). Western blot and high-content screening (HCS) analysis confirmed functional KO of LIMD2. LIMD2 KO reduced in vitro invasion and migration. Ultrastructural analyses showed that cell polarity and mitochondria function and morphology were restored in LIMD2 KO cells. To unveil the signals supervising these phenotypic changes, we employed phospho-protein array. Several members of the MAPK superfamily showed robust reduction in phosphorylation. A Venn diagram displayed the overlap of kinases with reduced phosphorylation in both cell lines and showed that they were able to initiate or sustain the epithelial-mesenchymal transition (EMT) and DNA damage checkpoint. Flow cytometry and HCS validation analyses further corroborated the phospho-protein array data. Collectively, our findings show that LIMD2 enhances phosphorylation of kinases associated with EMT and invasion. Through cooperation with different kinases, it contributes to the increased genomic instability that ultimately promotes PTC progression.

Identification of very small cancer stem cells expressing hallmarks of pluripotency in B16F10 melanoma cells and their reoccurrence in B16F10-derived clones.

Melanoma is characterized by high heterogeneity and plasticity, most likely due to the presence of mutated melanocyte stem cells or immature progenitor cells in the skin that serves as precursors to melanoma. In the present study, for the first time, we identified rare cells in the murine melanoma B16F10, and human A2058 and SK-MEL-28 cell lines that express pluripotency markers, including Oct4, Nanog, Sox2 and a marker of melanoma cancer cells (ALDH1/2). These cells are very small with round morphology and they grow onto melanoma cells, thereby demonstrating feeder layer dependence similar to that of other pluripotent cells. These cells underwent self-renewal, symmetric and asymmetric division. We called these cells murine very small cancer stem cells (VSCSC). VSCSC were also found in B16F10-derived clones after 3-5 consecutive passages, where they occur as single cells or as small colonies, nevertheless, always using melanoma cells as feeders. These cells formed melanospheres enriched with Oct4-and ALDH1/2-positive cells. We also evaluated the possible effect of VSCSC that presented in the parental cell line (B16F10) and in clones based on their functional characteristics. We found that VCSCS present in the B16F10 cell line reappearing in their clones were required for continuous tumor growth and were responsible for melanoma cell heterogeneity and plasticity rather than directly affecting functional characteristics of melanoma cells. Our data, together with those of previous reports suggested the existence of melanoma-competent melanocyte stem cells, which corroborate the hypothesis of the existence of tumor-initiating cells and cancer stem cell hierarchies, at least in melanoma.

Medical applications of clustered regularly interspaced short palindromic repeats (CRISPR/Cas) tool: A comprehensive overview.

Since the discovery of the double helix and the introduction of genetic engineering, the possibility to develop new strategies to manipulate the genome has fascinated scientists around the world. Currently scientists have the knowledge andabilitytoedit the genomes. Several methodologies of gene editing have been established, all of them working like "scissor", creating double strand breaks at specific spots. The introduction of a new technology, which was adapted from the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas bacterial immune system, has revolutionized the genetic therapy field, as it allows a much more precise editing of gene than the previously described tools and, therefore, to prevent and treat disease in humans. This review aims to revisit the genome editing history that led to the rediscovery of the CRISPR/Cas technology and to explore the technical aspects, applications and perspectives of this fascinating, powerful, precise, simpler and cheaper technology in different fields.

FAM129A regulates autophagy in thyroid carcinomas in an oncogene-dependent manner.

We previously proposed that high expression of FAM129A can be used as a thyroid carcinoma biomarker in preoperative diagnostic exams of thyroid nodules. Here, we identify that FAM129A expression is increased under nutrient and growth factor depletion in a normal thyroid cell line (PCCL3), overlapping with increased expression of autophagy-related protein and inhibition of AKT/mTOR/p70S6K. Supplementation of insulin, TSH and serum to the medium was able to reduce the expression of both FAM129A and autophagy-related protein and reestablish the AKT/mTOR/p70S6K axis. To determine the direct role of FAM129A on autophagy, FAM129A was transfected into PCCL3 cells. Its overexpression induced autophagic vesicles formation, evidenced by transmission electron microscopy. Co-expression of FAM129A and mCherry-EGFP-LC3B in PCCL3 showed an increased yellow puncta formation, suggesting that FAM129Ainduces autophagy. To further confirm its role on autophagy, we knockdown FAM129A in two thyroid carcinoma cell lines (TPC1 and FTC-236). Unexpectedly, FAM129A silencing increased autophagic flux, suggesting that FAM129A inhibits autophagy in these models. We next co-transfected PCCL3 cells with FAM129A and RET/PTC1 and tested autophagy in this context. Co-expression of FAM129A and RET/PTC1 oncogene in PCCL3 cells, inhibited RET/PTC1-induced autophagy. Together, our data suggest that, in normal cells FAM129A induces autophagy in order to maintain cell homeostasis and provide substrates under starvation conditions. Instead, in cancer cells, decreased autophagy may help the cells to overcome cell death. FAM129A regulates autophagy in a cell- and/or context-dependent manner. Our data reinforce the concept that autophagy can be used as a strategy for cancer treatment.

The human papillomavirus (HPV)-related cancer biology: An overview.

Despite the novel diagnostic methods and therapies implemented in oncology, the number of patients that succumb by the cancer remains high globally. Currently studies point out that 20-25% of all human malignancies are related to micro-organism infections. Among these cancer-related pathogens, the human papillomavirus (HPV) has a prominent position, since the virus is responsible for about 30% of all infectious agent-related cancers. Thus, an amount of cancers could be avoided by means prophylactic and/or therapeutic measures. However, these measures required a holistic comprehension about HPV-related cancer biology. Based on this, this review aims to summarize the last evidences of HPV on cancer biology (from initiation to metastasis), focus on molecular and biochemical deregulations associated with viral infection, and discuss the viral etiology in different malignancies.

Analysis of antioxidant, cytotoxic and mutagenic potential of Agave sisalana Perrine extracts using Vero cells, human lymphocytes and mice polychromatic erythrocytes.

Brazilian Northeast is the world's largest producer of Agave sisalana Perrine for the supply of the sisal fiber. About 95% of plant biomass, which comprise the mucilage and sisal juice, is considered a waste residual is discarded in the soil. However, the sisal juice is rich in steroidal saponins, which exhibits different pharmacological properties. Despite this, natural products are not necessarily safe. Based on this, this study analyzed the antioxidant, cytotoxic and mutagenic potential of three extracts derived from acid hydrolysis (AHAS), dried precipitate (DPAS) and hexanic of A. sisalana (HAS). These analyses were performed by in vitro and in vivo methods, using Vero cells, human lymphocytes and mice. Results showed that AHAS 50 and 100 can be considered a useful antineoplastic candidate due to their antioxidant and cytotoxic activity, with no genotoxic/clastogenic potential in Vero cells and mice. Although the comet assay in human lymphocytes has showed that the AHAS 25, AHAS 50 and AHAS 100 can lead to DNA breaks, these extracts did not promote DNA damages in mice bone marrow. Considering the different mutagenic responses obtained with the different methods employed, this study suggest that the metabolizing pathways can produce by-products harmful to health. For this reason, it is mandatory to analyze the mutagenic potential by both in vitro and in vivo techniques, using cells derived from different species and origins.

The Roles of ROS in Cancer Heterogeneity and Therapy.

Cancer comprises a group of heterogeneous diseases encompassing high rates of morbidity and mortality. Heterogeneity, which is a hallmark of cancer, is one of the main factors related to resistance to chemotherapeutic agents leading to poor prognosis. Heterogeneity is profoundly affected by increasing levels of ROS. Under low concentrations, ROS may function as signaling molecules favoring tumorigenesis and heterogeneity, while under high ROS concentrations, these species may work as cancer modulators due to their deleterious, genotoxic or even proapoptotic effect on cancer cells. This double-edged sword effect represented by ROS relies on their ability to cause genetic and epigenetic modifications in DNA structure. Antitumor therapeutic approaches may use molecules that prevent the ROS formation precluding carcinogenesis or use chemical agents that promote a sudden increase of ROS causing considerable oxidative stress inside tumor mass. Therefore, herein, we review what ROS are and how they are produced in normal and in cancer cells while providing an argumentative discussion about their role in cancer pathophysiology. We also describe the various sources of ROS in cancer and their role in tumor heterogeneity. Further, we also discuss some therapeutic strategies from the current landscape of cancer heterogeneity, ROS modulation, or ROS production.

Papillomaviruses: a systematic review

Abstract In the last decades, a group of viruses has received great attention due to its relationship with cancer development and its wide distribution throughout the vertebrates: the papillomaviruses. In this article, we aim to review some of the most relevant reports concerning the use of bovines as an experimental model for studies related to papillomaviruses. Moreover, the obtained data contributes to the development of strategies against the clinical consequences of bovine papillomaviruses (BPV) that have led to drastic hazards to the herds. To overcome the problem, the vaccines that we have been developing involve recombinant DNA technology, aiming at prophylactic and therapeutic procedures. It is important to point out that these strategies can be used as models for innovative procedures against HPV, as this virus is the main causal agent of cervical cancer, the second most fatal cancer in women.

Integrated analysis of recombinant BPV-1 L1 protein for the production of a bovine papillomavirus VLP vaccine.

Bovine papillomatosis is an infectious disease that is caused by bovine papillomavirus (BPV), which results in important economic losses. However, no BPV vaccines or effective treatment methods are commercially available to date. Moreover, the absence of papillomavirus replication in vitro makes the use of recombinant protein a promising candidate for vaccine formulations. Hence, we developed an integrated study on the L1 capsid protein of BPV-1, obtained from a bacterial expression system, regarding its purification, biosafety, thermostability and immunogenicity. The results indicated an absence of genotoxicity of the purified recombinant L1 protein, ß-sheet prevalence of secondary structure folding, protein stability under high temperatures as well as the presence of capsomeres and VLPs. In addition, preliminary experimental vaccination of calves showed the production of specific antibodies against BPV-1 L1.

Papillomaviruses: a systematic review.

In the last decades, a group of viruses has received great attention due to its relationship with cancer development and its wide distribution throughout the vertebrates: the papillomaviruses. In this article, we aim to review some of the most relevant reports concerning the use of bovines as an experimental model for studies related to papillomaviruses. Moreover, the obtained data contributes to the development of strategies against the clinical consequences of bovine papillomaviruses (BPV) that have led to drastic hazards to the herds. To overcome the problem, the vaccines that we have been developing involve recombinant DNA technology, aiming at prophylactic and therapeutic procedures. It is important to point out that these strategies can be used as models for innovative procedures against HPV, as this virus is the main causal agent of cervical cancer, the second most fatal cancer in women.