Development of a Three-Dimensional Multicellular Model of Human Neuroblastoma Using Matrigel as an Extracellular Matrix Analogue.

Neuroblastoma, the most prevalent extracranial solid tumor in children, poses therapeutic challenges due to its variable clinical course and propensity for metastasis. Despite advances in treatment strategies like chemotherapy, drug resistance remains a significant concern, highlighting the need for improved models to study tumor behavior and drug responses. This chapter proposes the development of a three-dimensional multicellular model of human neuroblastoma using Matrigel as an ECM analogue. Such models aim to replicate the complexity of the tumor microenvironment, providing valuable insights into tumor progression and drug resistance mechanisms. By recapitulating key features of neuroblastoma within a physiologically relevant context, these models offer a platform for preclinical drug screening and therapeutic development.

Eternal Youth: A Comprehensive Exploration of Gene, Cellular, and Pharmacological Anti-Aging Strategies.

The improvement of human living conditions has led to an increase in average life expectancy, creating a new social and medical problem-aging, which diminishes the overall quality of human life. The aging process of the body begins with the activation of effector signaling pathways of aging in cells, resulting in the loss of their normal functions and deleterious effects on the microenvironment. This, in turn, leads to chronic inflammation and similar transformations in neighboring cells. The cumulative retention of these senescent cells over a prolonged period results in the deterioration of tissues and organs, ultimately leading to a reduced quality of life and an elevated risk of mortality. Among the most promising methods for addressing aging and age-related illnesses are pharmacological, genetic, and cellular therapies. Elevating the activity of aging-suppressing genes, employing specific groups of native and genetically modified cells, and utilizing senolytic medications may offer the potential to delay aging and age-related ailments over the long term. This review explores strategies and advancements in the field of anti-aging therapies currently under investigation, with a particular emphasis on gene therapy involving adeno-associated vectors and cell-based therapeutic approaches.

The Potential of Dendritic Cell Subsets in the Development of Personalized Immunotherapy for Cancer Treatment.

Since the discovery of dendritic cells (DCs) in 1973 by Ralph Steinman, a tremendous amount of knowledge regarding these innate immunity cells has been accumulating. Their role in regulating both innate and adaptive immune processes is gradually being uncovered. DCs are proficient antigen-presenting cells capable of activating naive T-lymphocytes to initiate and generate effective anti-tumor responses. Although DC-based immunotherapy has not yielded significant results, the substantial number of ongoing clinical trials underscores the relevance of DC vaccines, particularly as adjunctive therapy or in combination with other treatment options. This review presents an overview of current knowledge regarding human DCs, their classification, and the functions of distinct DC populations. The stepwise process of developing therapeutic DC vaccines to treat oncological diseases is discussed, along with speculation on the potential of combined therapy approaches and the role of DC vaccines in modern immunotherapy.

Emerging Gene Therapy Approaches in the Management of Spinal Muscular Atrophy (SMA): An Overview of Clinical Trials and Patent Landscape.

Spinal muscular atrophy (SMA) is a rare autosomal recessive neuromuscular disease that is characterized by progressive muscle atrophy (degeneration), including skeletal muscles in charge of the ability to move. SMA is caused by defects in the SMN1 gene (Survival of Motor Neuron 1) which encodes a protein crucial for the survival and functionality of neuron cells called motor neurons. Decreased level of functioning SMN protein leads to progressive degeneration of alpha-motor neurons performing muscular motility. Over the past decade, many strategies directed for SMN-level-restoration emerged, such as gene replacement therapy (GRT), CRISPR/Cas9-based gene editing, usage of antisense oligonucleotides and small-molecule modulators, and all have been showing their perspectives in SMA therapy. In this review, modern SMA therapy strategies are described, making it a valuable resource for researchers, clinicians and everyone interested in the progress of therapy of this serious disorder.

Safety and Efficacy of Intravenous and Intrathecal Delivery of AAV9-Mediated ARSA in Minipigs.

Metachromatic leukodystrophy (MLD) is a hereditary neurodegenerative disease characterized by demyelination and motor and cognitive impairments due to deficiencies of the lysosomal enzyme arylsulfatase A (ARSA) or the saposin B activator protein (SapB). Current treatments are limited; however, gene therapy using adeno-associated virus (AAV) vectors for ARSA delivery has shown promising results. The main challenges for MLD gene therapy include optimizing the AAV dosage, selecting the most effective serotype, and determining the best route of administration for ARSA delivery into the central nervous system. This study aims to evaluate the safety and efficacy of AAV serotype 9 encoding ARSA (AAV9-ARSA) gene therapy when administered intravenously or intrathecally in minipigs, a large animal model with anatomical and physiological similarities to humans. By comparing these two administration methods, this study contributes to the understanding of how to improve the effectiveness of MLD gene therapy and offers valuable insights for future clinical applications.

Various AAV Serotypes and Their Applications in Gene Therapy: An Overview.

Despite scientific discoveries in the field of gene and cell therapy, some diseases still have no effective treatment. Advances in genetic engineering methods have enabled the development of effective gene therapy methods for various diseases based on adeno-associated viruses (AAVs). Today, many AAV-based gene therapy medications are being investigated in preclinical and clinical trials, and new ones are appearing on the market. In this article, we present a review of AAV discovery, properties, different serotypes, and tropism, and a following detailed explanation of their uses in gene therapy for disease of different organs and systems.

Cell Immunotherapy against Melanoma: Clinical Trials Review.

Melanoma is one of the most aggressive and therapy-resistant types of cancer, the incidence rate of which grows every year. However, conventional methods of chemo- and radiotherapy do not allow for completely removing neoplasm, resulting in local, regional, and distant relapses. In this case, adjuvant therapy can be used to reduce the risk of recurrence. One of the types of maintenance cancer therapy is cell-based immunotherapy, in which immune cells, such as T-cells, NKT-cells, B cells, NK cells, macrophages, and dendritic cells are used to recognize and mobilize the immune system to kill cancer cells. These cells can be isolated from the patient's peripheral blood or biopsy material and genetically modified, cultured ex vivo, following infusion back into the patient for powerful induction of an anti-tumor immune response. In this review, the advantages and problems of the most relevant methods of cell-based therapy and ongoing clinical trials of adjuvant therapy of melanoma are discussed.

Gene Therapy of Sphingolipid Metabolic Disorders.

Sphingolipidoses are defined as a group of rare hereditary diseases resulting from mutations in the genes encoding lysosomal enzymes. This group of lysosomal storage diseases includes more than 10 genetic disorders, including GM1-gangliosidosis, Tay-Sachs disease, Sandhoff disease, the AB variant of GM2-gangliosidosis, Fabry disease, Gaucher disease, metachromatic leukodystrophy, Krabbe disease, Niemann-Pick disease, Farber disease, etc. Enzyme deficiency results in accumulation of sphingolipids in various cell types, and the nervous system is also usually affected. There are currently no known effective methods for the treatment of sphingolipidoses; however, gene therapy seems to be a promising therapeutic variant for this group of diseases. In this review, we discuss gene therapy approaches for sphingolipidoses that are currently being investigated in clinical trials, among which adeno-associated viral vector-based approaches and transplantation of hematopoietic stem cells genetically modified with lentiviral vectors seem to be the most effective.

Cytochalasin B-Induced Membrane Vesicles from TRAIL-Overexpressing Mesenchymal Stem Cells Induce Extrinsic Pathway of Apoptosis in Breast Cancer Mouse Model.

Tumor-necrosis-factor-associated apoptosis-inducing ligand (TRAIL) is one of the most promising therapeutic cytokines that selectively induce apoptosis in tumor cells. It is known that membrane vesicles (MVs) can carry the surface markers of parental cells. Therefore, MVs are of interest as a tool for cell-free cancer therapy. In this study, membrane vesicles were isolated from TRAIL-overexpressing mesenchymal stem cells using cytochalasin B treatment (CIMVs). To evaluate the antitumor effect of CIMVs-TRAIL in vivo, a breast cancer mouse model was produced. The animals were intratumorally injected with 50 µg of native CIMVs or CIMVs-TRAIL for 12 days with an interval of two days. Then, tumor growth rate, tumor necrotic area, the expression of the apoptosis-related genes CASP8, BCL-2, and BAX and the level of CASP8 protein were analyzed. A 1.8-fold increase in the CAS8 gene mRNA and a 1.7-fold increase in the CASP8 protein level were observed in the tumors injected with CIMVs-TRAIL. The expression of the anti-apoptotic BCL-2 gene in the CIMV-TRAIL group remained unchanged, while the mRNA level of the pro-apoptotic BAX gene was increased by 1.4 times, which indicated apoptosis activation in the tumor tissue. Thus, CIMVs-TRAIL were able to activate the extrinsic apoptosis pathway and induce tumor cell death in the breast cancer mouse model.

Characteristics and Resistance to Cisplatin of Human Neuroblastoma Cells Co-Cultivated with Immune and Stromal Cells.

We investigated the features of the morphology and cytokine profiles of neuroblastoma SH-SY5Y cells, bone marrow-derived mesenchymal stromal/stem cells (BM-MSCs), and peripheral blood mononuclear cells (PBMCs) in double (BM-MSCs + SH-SY5Y cells) and triple (BM-MSCs + SH-SY5Y cells + PBMCs) co-cultures incubated on plastic and Matrigel. Cells in the co-cultures communicated by vesicular transport and by exchanging membrane and cytoplasmic components. The cytokine profile of double and triple co-cultures incubated on Matrigel and plastic had differences and showed the highest concentration of a number of chemokines/cytokines, such as CXCL8/IL-8, I-TAC/CXCL11, IP10/CXCL10, MDC/CCL22, MIP-1α/CCL3, IL-1ß, ENA-78/CXCL5, Gro-α/CXCL1, MCP-1/CCL2, TERC/CCL25, CXCL8/IL-8, and IL-6. High concentrations of inflammatory chemokines/cytokines in the conditioned medium of triple co-culture form a chronic inflammation, which brings the presented co-cultivation system closer to a natural tumor. Triple co-cultures were more resistant to cisplatin (CDDP) than the double- and monoculture of SH-SY5Y. The mRNA levels of BCL2, BCL2L1, RAC1, CAV1, CASP3, and BAX genes were changed in cells after co-culturing and CDDP treatment in double and triple co-cultures. The expression of the BCL2, BAX, CAV1, and CASP3 proteins in SH-SY5Y cells after the triple co-culture and CAV1 and BAX protein expression in SH-SY5Y cells after the double co-culture were determined. This study demonstrated the nature of the cellular interactions between components of tumor niche and the intercellular influence on chemoresistance observed in our tumor model, which should enable the development of novel test systems for anti-tumor agents.

Comparative Analysis of Natural and Cytochalasin B-Induced Membrane Vesicles from Tumor Cells and Mesenchymal Stem Cells.

To date, there are numerous protocols for the isolation of extracellular vesicles (EVs). Depending on the isolation method, it is possible to obtain vesicles with different characteristics, enriched with specific groups of proteins, DNA and RNA, which affect similar types of cells in the opposite way. Therefore, it is important to study and compare methods of vesicle isolation. Moreover, the differences between the EVs derived from tumor and mesenchymal stem cells are still poorly understood. This article compares EVs from human glioblastoma cells and mesenchymal stem cells (MSCs) obtained by two different methods, ultracentrifugation and cytochalasin B-mediated induction. The size of the vesicles, the presence of the main EV markers, the presence of nuclear and mitochondrial components, and the molecular composition of the vesicles were determined. It has been shown that EVs obtained by both ultracentrifugation and cytochalasin B treatment have similar features, contain particles of endogenous and membrane origin and can interact with monolayer cultures of tumor cells.

Contribution of Tumor-Derived Extracellular Vesicles to Malignant Transformation of Normal Cells.

Tumor-cell-derived extracellular vesicles (EVs) are known to carry biologically active molecules of parental cells, which can actively modulate the tumor microenvironment. EVs produced by tumor cells play significant roles in the development and maintenance of tumor growth, metastasis, immune escape, and other important processes. However, the ability of EVs to induce the transformation of normal cells has hardly been investigated. This review discusses studies that describe the ability of tumor-cell-derived EVs to alter the metabolism and morphology of normal cells, causing changes associated with malignant transformation. Additionally, the horizontal transfer of oncogenes through EVs of tumor cells and the induction of epigenetic changes in normal cells, which leads to genomic instability and subsequent oncogenic transformation of normal cells, are also discussed.

The Dual Role of Mesenchymal Stromal Cells and Their Extracellular Vesicles in Carcinogenesis.

Mesenchymal stem cells (MSCs) are a major component of the tumor microenvironment (TME) and play an important role in tumor progression. MSCs remodel the extracellular matrix, participate in the epithelial-mesenchymal transition, promote the spread of metastases, and inhibit antitumor immune responses in the TME; however, there are also data pertaining to the antitumor effects of MSCs. MSCs activate the cell death mechanism by modulating the expression of proteins involved in the regulation of the cell cycle, angiogenesis receptors, and proapoptotic proteins. One of the main ways in which MSCs and TME interact is through the production of extracellular vesicles (EVs) by cells. Currently, data on the effects of both MSCs and their EVs on tumor cells are rather contradictory. Various studies have reported that EVs from MSCs can have either antitumor or pro-tumor effects, depending on both the tumor type and developmental stage. In this review, we discuss published data on EV MSCs and their effect on tumor cells. The molecular composition of vesicles obtained from MSCs is also presented in the review. In addition, the use of EV MSCs for the development of new methods for treating oncological diseases is described.

Gene and Cell Therapy for Epilepsy: A Mini Review.

Epilepsy is a chronic non-infectious disease of the brain, characterized primarily by recurrent unprovoked seizures, defined as an episode of disturbance of motor, sensory, autonomic, or mental functions resulting from excessive neuronal discharge. Despite the advances in the treatment achieved with the use of antiepileptic drugs and other non-pharmacological therapies, about 30% of patients suffer from uncontrolled seizures. This review summarizes the currently available methods of gene and cell therapy for epilepsy and discusses the development of these approaches. Currently, gene therapy for epilepsy is predominantly adeno-associated virus (AAV)-mediated delivery of genes encoding neuro-modulatory peptides, neurotrophic factors, enzymes, and potassium channels. Cell therapy for epilepsy is represented by the transplantation of several types of cells such as mesenchymal stem cells (MSCs), bone marrow mononuclear cells, neural stem cells, and MSC-derived exosomes. Another approach is encapsulated cell biodelivery, which is the transplantation of genetically modified cells placed in capsules and secreting various therapeutic agents. The use of gene and cell therapy approaches can significantly improve the condition of patient with epilepsy. Therefore, preclinical, and clinical studies have been actively conducted in recent years to prove the benefits and safety of these strategies.

Mesenchymal Stem Cell-Based Therapy for Lysosomal Storage Diseases and Other Neurodegenerative Disorders.

Lysosomal storage diseases (LSDs) are a group of approximately 50 genetic disorders caused by mutations in genes coding enzymes that are involved in cell degradation and transferring lipids and other macromolecules. Accumulation of lipids and other macromolecules in lysosomes leads to the destruction of affected cells. Although the clinical manifestations of different LSDs vary greatly, more than half of LSDs have symptoms of central nervous system neurodegeneration, and within each disorder there is a considerable variation, ranging from severe, infantile-onset forms to attenuated adult-onset disease, sometimes with distinct clinical features. To date, treatment options for this group of diseases remain limited, which highlights the need for further development of innovative therapeutic approaches, that can not only improve the patients' quality of life, but also provide full recovery for them. In many LSDs stem cell-based therapy showed promising results in preclinical researches. This review discusses using mesenchymal stem cells for different LSDs therapy and other neurodegenerative diseases and their possible limitations.

Analysis of the Interaction of Human Neuroblastoma Cell-Derived Cytochalasin B Induced Membrane Vesicles with Mesenchymal Stem Cells Using Imaging Flow Cytometry.

At present, there is an increasing interest in the potential role of extracellular vesicles (EVs), acting as multi-signal messengers of the tumor stroma, in the development and progression of tumor. Tumor cell-derived EVs are considered a potential vector for the targeted delivery of antitumor agents due to the ability to fuse with parental cells through endocytosis and release their contents into the cytoplasm of the recipient cell. Tumor cell-derived EVs could be also used for priming immune cells and therapeutic vaccine development. It is also known that mesenchymal stem cells (MSCs) have a tropism toward tumor niches. It is believed that MSC migration to the tumor is due to its inflammatory signaling. Presumably, with the accumulation of MSCs at tumor sites, these cells differentiate into pericytes or tumor-associated fibroblasts, thereby forming a supporting tumor growth microenvironment. However, besides the ability to promote tumor progression, MSCs can also suppress its growth by inhibiting proliferation and cell cycle progression, and angiogenesis. Thus, the further studies of the MSC role in TME and MSC interaction with other cells of the tumor stroma, including through EVs, are of particular interest. To increase the yield of vesicles the isolation method based on pharmacological disorganization of the actin cytoskeleton induced by treating with cytochalasin B was used in this study. In this investigation the interaction of SH-SY5Y neuroblastoma cell-derived membrane vesicles, obtained using cytochalasin B (CIMVs), with human bone marrow-derived MSCs was analyzed using imaging flow cytometry. Using transmission electron microscopy, it was shown that CIMVs have a size similar to that of natural microvesicles, which is 100-1000 nm. Using imaging flow cytometry, it was shown that after 24 h of co-cultivation 6% of the MSCs contained a large number of CIMVs, and 42% of the MSCs contained a small amount of CIMVs. Cultivation of MSCs with SH-SY5Y cell-derived CIMVs also induced dose-dependent decrease in the expression of CD markers typical for MSCs. Thus, the internalization of SH-SY5Y cell-derived CIMVs within MSCs and the ability of the CIMVs to modulate immunophenotype of the recipient cells were shown. However, further studies are required to determine the effect of CIMVs on pro- or antioncogenic phenotype and function of MSCs.

Current Strategies for the Gene Therapy of Autosomal Recessive Congenital Ichthyosis and Other Types of Inherited Ichthyosis.

Mutations in genes such as transglutaminase-1 (TGM1), which are responsible for the formation and normal functioning of a lipid barrier, lead to the development of autosomal recessive congenital ichthyosis (ARCI). ARCIs are characterized by varying degrees of hyperkeratosis and the presence of scales on the body surface since birth. The quality of life of patients is often significantly affected, and in order to alleviate the manifestations of the disease, symptomatic therapy with moisturizers, keratolytics, retinoids and other cosmetic substances is often used to improve the condition of the patients' skin. Graft transplantation is commonly used to correct defects of the eye. However, these approaches offer symptomatic treatment that does not restore the lost protein function or provide a long-term skin barrier. Gene and cell therapies are evolving as promising therapy for ARCIs that can correct the functional activity of altered proteins. However, these approaches are still at an early stage of development. This review discusses current studies of gene and cell therapy approaches for various types of ichthyosis and their further prospects for patient treatment.

Intrinsic and Extrinsic Factors Impacting Cancer Stemness and Tumor Progression.

Tumor heterogeneity represents an important limitation to the development of effective cancer therapies. The presence of cancer stem cells (CSCs) and their differentiation hierarchies contribute to cancer complexity and confer tumors the ability to grow, resist treatment, survive unfavorable conditions, and invade neighboring and distant tissues. A large body of research is currently focusing on understanding the properties of CSCs, including their cellular and molecular origin, as well as their biological behavior in different tumor types. In turn, this knowledge informs strategies for targeting these tumor initiating cells and related cancer stemness. Cancer stemness is modulated by the tumor microenvironment, which influences CSC function and survival. Several advanced in vitro models are currently being developed to study cancer stemness in order to advance new knowledge of the key molecular pathways involved in CSC self-renewal and dormancy, as well as to mimic the complexity of patients' tumors in pre-clinical drug testing. In this review, we discuss CSCs and the modulation of cancer stemness by the tumor microenvironment, stemness factors and signaling pathways. In addition, we introduce current models that allow the study of CSCs for the development of new targeted therapies.

From 3D printing to 3D bioprinting: the material properties of polymeric material and its derived bioink for achieving tissue specific architectures.

The application of 3D printing technologies fields for biological tissues, organs, and cells in the context of medical and biotechnology applications requires a significant amount of innovation in a narrow printability range. 3D bioprinting is one such way of addressing critical design challenges in tissue engineering. In a more general sense, 3D printing has become essential in customized implant designing, faithful reproduction of microenvironmental niches, sustainable development of implants, in the capacity to address issues of effective cellular integration, and long-term stability of the cellular constructs in tissue engineering. This review covers various aspects of 3D bioprinting, describes the current state-of-the-art solutions for all aforementioned critical issues, and includes various illustrative representations of technologies supporting the development of phases of 3D bioprinting. It also demonstrates several bio-inks and their properties crucial for being used for 3D printing applications. The review focus on bringing together different examples and current trends in tissue engineering applications, including bone, cartilage, muscles, neuron, skin, esophagus, trachea, tympanic membrane, cornea, blood vessel, immune system, and tumor models utilizing 3D printing technology and to provide an outlook of the future potentials and barriers.

The Role of Cancer Stem Cells and Their Extracellular Vesicles in the Modulation of the Antitumor Immunity.

Cancer stem cells (CSCs) are a population of tumor cells that share similar properties to normal stem cells. CSCs are able to promote tumor progression and recurrence due to their resistance to chemotherapy and ability to stimulate angiogenesis and differentiate into non-CSCs. Cancer stem cells can also create a significant immunosuppressive environment around themselves by suppressing the activity of effector immune cells and recruiting cells that support tumor escape from immune response. The immunosuppressive effect of CSCs can be mediated by receptors located on their surface, as well as by secreted molecules, which transfer immunosuppressive signals to the cells of tumor microenvironment. In this article, the ability of CSCs to regulate the antitumor immune response and a contribution of CSC-derived EVs into the avoidance of the immune response are discussed.