Successful containment of unwanted cell cycle progression in tumors such as glioblastoma (GBM) requires targeted therapeutic approaches, which rely on understanding cell cycle dynamics in response to microenvironmental stimuli. Glioma Stem Cells (GSCs) can drive tumor initiation, recurrence, therapy resistance, and are often attributed to the heterogeneity and plasticity of GBM. In vitro models, using patient-derived GSCs, provide a life relevant tool for exploration of complex molecular mechanisms underlying the aggressive characteristics of GBM. Introduction of 3D tissue culture systems permits the study of spatial complexity of the tumor mass and enables control over diverse conditions within the surrounding microenvironment. This chapter demonstrates detailed methods to study spatiotemporal changes to the cell cycle dynamics using available fluorescent cell cycle reporter systems in combination with bioinformatics-based signal intensity and localization analysis. We present a successful approach that investigates the 3D cell cycle dynamics of GSC populations. This approach utilizes GBM neurosphere and organoid cultures, which are assessed over time and under therapeutic pressure. These models can be further explored, manipulated, and customized to serve specific experimental designs.

Brain tumors are among the most heterogeneous tumors characterized. This heterogeneity drives resistance to single-drug therapies, making combination treatment a more promising approach to improve treatment outcomes. To optimize drug combinations and guide treatment development in this new era of combinatorial therapy, methods for quantifying synergistic, additive or antagonistic interactions between treatments are needed. In this chapter, we describe an in vitro assay to identify synergistic drug combinations in brain-tumor-derived cell cultures. As the gold-standard assay for the in vitro evaluation of drug combination efficacy, this protocol is a fast, inexpensive, and highly reproducible method for identifying and validating synergistic drug combinations.

A major hurdle in brain tumor therapy development is a lack of adequate controls to ensure therapeutic safety in human patients. Neural stem cells (NSCs) are healthy brain cells that are commonly used by neurobiologists and neuroscientists as controls for therapy development. Consequently, it is important that these cells are properly isolated and cultured to maintain cell integrity to efficiently uncover potential toxicities associated with developing therapies for downstream applications, including omics studies. In this chapter, we describe the optimized techniques for the isolation, acquisition, and maintenance of NSCs from fetal brain samples. For further details on the use of this protocol, please refer to Suk et al. (STAR Protoc 3:101628. https://doi.org/10.1016/J.XPRO.2022.101628 , 2022).

Utilization of human embryonic stem cells (hESCs) as a model system to study highly malignant pediatric cancers has led to significant insight into the molecular mechanisms governing tumor progression and has revealed novel therapeutic targets for these devastating diseases. Here, we describe our method for previously generating heterogeneous populations of neural precursors from both normal and neoplastic hESCs and the subsequent injection of neoplastic human embryonic neural cells (hENs) into intracerebellar xenograft models. Histopathologically, neural tumors derived from neoplastic hENs exhibit features similar to more aggressive medulloblastoma, the most common malignant primary pediatric brain tumor. In this chapter, we will outline the detailed methods for culturing normal and neoplastic neural precursor cells in both adherent and tumorsphere formats and the full characterization of the brain tumors generated from these cells in nonobese diabetic severe combined immunodeficiency (NOD SCID) mice.

Shortly after the discovery of cancer stem cells in acute myeloid leukemia, cancer stem cells in several cancer types were discovered, including brain cancer. Brain tumor stem cells (BTSCs), initially characterized by the expression of CD133, are a population of cells that can proliferate and form spheres in vitro and form tumors in vivo. Due to the belief that they are treatment-resistant and seed-recurrent diseases, they are an ideal preclinical model that can be used to study brain cancer. In this chapter, we describe how to process, maintain, and propagate a BTSC culture from brain tumor tissue samples.

Brain cancer remains to be one of the most formidable challenges in oncology, due to its complexity, heterogeneity, and aggressive nature. Despite advances in treatment, many patients continue to face poor prognoses, underscoring the dire need for ongoing research and innovation to address this clinical enigma. Brain tumor stem cells (BTSCs) have shown to play a significant factor in the transformation and progression of brain tumors. This distinct subpopulation of cancer cells exhibit key characteristics remnant of neural stem cells (NSCs) including self-renewal and asymmetric differentiation. They are also characterized by abhorrent signaling pathways, metabolic processes, and immune surveillance. Current methods utilize fluorescent-activated cell sorting (FAC) methods to isolate and enrich for BTSCs based on cell surface markers. In this chapter, we review the current challenges in BTSC research, highlighting the anomalies of BTSCs and their surrounding niche that lead to therapeutic resistance and relapse. Current and prospective research are explored to address these challenges, including innovative technologies such as single cell RNA sequencing captures genetic and transcriptomic information on the single-cell level. Screening methods utilizing CRISPR technology are discussed to identify targetable vulnerabilities. Finally, with the advent of artificial intelligence (AI), current research utilizing AI and machine learning are explored to introduce novel therapeutic targets and improved diagnostic tools.

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss among the elderly in developed countries, manifesting in two primary forms: neovascular (wet) AMD and non-neovascular (dry) AMD. Current treatments, such as anti-VEGF therapy, offer limited efficacy, particularly for dry AMD, highlighting the urgent need for alternative strategies. Advancements in contemporary treatment strategies for these eye conditions are a pressing medical concern. Stem cell-based therapies have emerged as a promising approach for retinal regeneration due to their capacity for self-renewal and differentiation into retinal cell types, including retinal pigment epithelial (RPE) cells and photoreceptors, two key cell populations damaged in AMD. Among the various sources, pluripotent stem cells such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) show significant potential in generating functional RPE cells and restoring retinal architecture and function. Preclinical and early clinical studies have demonstrated promising outcomes, including improved visual acuity and anatomical integration. However, challenges such as immune rejection, tumorigenicity, limited long-term integration, and ethical concerns continue to impede clinical translation. This review critically evaluates current stem cell-based therapeutic strategies for AMD, including advances in mesenchymal stem cells, retinal organoids, and combinatorial approaches with gene and nanotherapy. Furthermore, this review outlines the translational bottlenecks and future directions required to advance these therapies toward clinical application.

Stem cell therapy has emerged as a promising approach for treating severe chronic liver diseases, including decompensated liver cirrhosis (DLC) and liver failure(LF), with mesenchymal stem cells (MSCs) being the most extensively investigated cell type in both preclinical and clinical trials.

Efficient lymph flow is ensured by lymphatic valves (LVs). The mechanisms that regulate LV development are incompletely understood. Here, we show that the deletion of the GPCR sphingosine 1-phosphate receptor-1 (S1PR1) from lymphatic endothelial cells (LECs) results in fewer LVs. Interestingly, LVs that remained in the terminal ileum-draining lymphatic vessels were specifically dysfunctional. Furthermore, tertiary lymphoid organs (TLOs) formed in the terminal ileum of the mutant mice. TLOs in this location are associated with ileitis in humans and mice. However, mice lacking S1PR1 did not develop obvious characteristics of ileitis. Mechanistically, S1PR1 regulates shear stress signaling and the expression of the valve-regulatory molecules FOXC2 and connexin-37. Importantly, Foxc2+/- mice, a model for lymphedema-distichiasis syndrome, also develop TLOs in the terminal ileum. Thus, we have discovered S1PR1 as a previously unknown regulator of LV and TLO development. We also suggest that TLOs are a sign of subclinical inflammation that can form due to lymphatic disorders in the absence of ileitis.

Hair follicles regenerate spontaneously through a cycle of anagen, catagen, and telogen, and this cycle is driven by hair follicle stem cells (HFSCs). Long non-coding RNAs (lncRNAs) have previously been implicated in hair follicle cycling processes. According to the previous lncRNA sequencing results of cashmere goats, an annotated lncRNA XR_310056.1, referred to as lnc056, was found to be differentially expressed during the hair follicle cycle. Here, the purpose of this study was to determine whether lnc056 affects the proliferation of HFSCs by regulating thyroid hormone receptor interactor 6 (TRIP6) expression in combination with the transcription factor HNRNPUL1. The expression of lnc056 in HFSCs was detected by RT-qPCR. HFSCs were then treated with lnc056 and TRIP6 overexpressing adenovirus, si-HNRNPUL1, and si-TRIP6 to detect cell viability and proliferation. In addition, we investigated the binding between lnc056 and HNRNPUL1 or HNRNPUL1 and TRIP6. Finally, the biological function of lnc056 through the HNRNPUL1/TRIP6 axis was verified by target gene recovery experiments. Lnc056 was expressed in the nuclei of HFSCs, and its overexpression promoted the proliferation of cells. Moreover, lnc056 was found to bind to the transcription factor HNRNPUL1 and promoted TRIP6 expression. Furthermore, recovery assays demonstrated that lnc056 promoted the proliferation of HFSCs via the HNRNPUL1/TRIP6 axis. In summary, the results of this study suggested that lnc056 up-regulated the expression of TRIP6 by binding to the transcription factor HNRNPUL1, thereby accelerating the proliferation of HFSCs. This study enriches the molecular mechanism of lncRNA in the hair follicle cycle and provides a potential therapeutic target for hair loss.

At present, the development of nanoplatforms that integrate infection control, inflammation resolution, and tissue repair for the treatment of periodontitis remains an enormous challenge. Based on the intricate pathogenesis of periodontitis and the advantages of mild photothermal therapy (PTT) and nanotherapy, a multifunctional photothermal nanoplatform (designated BR@C3G-Cu) was rationally developed by coating bilirubin nanoparticles (BR NPs) with copper ion coordinated cyanidin-3-O-glucoside metal-polyphenol networks (MPNs) in this study. The synergistic consequences of the photothermal conversion properties and metal-dependent functionality of this MPNs contributed to the effective and persistent elimination of periodontal pathogens under safe and mild conditions, meeting the heterogeneous antibacterial requirements of periodontitis. Further, photothermal BR@C3G-Cu NPs restored the macrophage polarization balance and mitigated periodontal inflammation by countering oxidative stress damage and blocking the cGAS-STING pathway, thereby normalizing the periodontal local osteoimmune microenvironment and promoting the osteogenic differentiation of in situ stem cells. BR@C3G-Cu NPs exposed to near-infrared irradiation also offered beneficial therapeutic outcomes in a mouse periodontitis model, including inhibiting alveolar bone resorption and promoting periodontal tissue remodeling. Overall, this work highlights the potential of MPN-coated engineered NPs (i.e., BR@C3G-Cu) for the treatment of periodontitis through the integration of mild PTT and immunomodulatory therapy to eliminate pathogenic bacteria, alleviate inflammation, and promote regeneration.

Osteoarthritis is a common cause of disability worldwide. Exosomes are extracellular vesicles and can exert paracrine and endocrine actions. DPSCs exosomes offer a new avenue of research that may elucidate various functions related to cell proliferation, differentiation, and immunomodulation. We hypothesized that DPSC exosomes produced under hypoxia-induced culture conditions may have an anti-inflammatory effect on osteoarthritic chondrocytes and may re-regulate the inflammatory response that is increased in osteoarthritis. We also hypothesized that the decreased glycosaminoglycan production in osteoarthritis may be re-induced by DPSC exosomes produced under hypoxia.

The methodologies and applications of ex vivo expansion of umbilical cord blood cells represent a significant research domain, primarily owing to the distinctive characteristics of these cells and their prospective uses in regenerative medicine and hematopoietic stem cell transplantation. We searched PubMed and Web of Science using the terms "umbilical cord blood cells," "cytokine," "haematopoietic cells," "umbilical cord mesenchymal stem cells," "in vitro expansion," and "microenvironment," and the selected literature was organized and analyzed. We present a narrative review of the mechanisms by which umbilical cord mesenchymal stem cells enhance the in vitro expansion of umbilical cord blood-derived hematopoietic stem cells, focusing on three primary aspects: secretion of diverse cytokines to replicate the natural hematopoietic microenvironment, transmission of critical signals via direct cell-to-cell contact, and exertion of immunomodulatory effects to alleviate environmental stress. Although these processes can significantly promote the proliferation and survival of hematopoietic stem cells, the challenge of concurrently preserving the long-term stemness of these cells in an in vitro environment remains a critical issue for future research.

Autism spectrum disorder (ASD) and congenital heart disease (CHD) frequently co-occur, yet the underlying molecular mechanisms of this comorbidity remain unknown. Given that children with CHD are identified as newborns, understanding which CHD variants are associated with autism could help select individuals for early intervention. Autism gene perturbations commonly dysregulate neural progenitor cell (NPC) biology, so we hypothesized that CHD genes disrupting neurogenesis are more likely to increase ASD risk. Therefore, we performed an in vitro pooled CRISPR interference screen to identify CHD genes disrupting NPC biology and identified 45 CHD genes. A cluster of ASD and CHD genes are enriched for ciliary biology, and perturbing any one of seven such genes (CEP290, CHD4, KMT2E, NSD1, OFD1, RFX3 and TAOK1) impairs primary cilia formation in vitro. In vivo investigation of TAOK1 in Xenopus tropicalis reveals a role in motile cilia formation and heart development, supporting its prediction as a CHD gene. Together, our findings highlight a set of CHD genes that may carry risk for ASD and underscore the role of cilia in shared ASD and CHD biology.

Growth factors enhance survival and integration of transplanted Mesenchymal Stromal Cells (MSC), but successful supplementation often requires supraphysiological growth factor doses, risking off-target effects. Short peptide mimics like the knuckle epitope (KE) of Bone Morphogenetic Protein 2 (BMP-2) can be covalently immobilized to biomaterials, localizing bioactivity at the delivery site. However, these short peptides often lack the potency of full-length growth factors. We sought to improve the potency of alginate-grafted KE to encourage MSC osteogenic differentiation. When alginate gels co-presented KE and integrin-binding cyclo-RGD (cRGD) peptides, MSC expressed early markers of osteogenesis (Runt-related Transcription Factor2, RUNX2, Alkaline Phosphatase, ALP, and osteocalcin, OCN) in a KE-dose dependent manner. When co-presented with cRGD, high concentrations of KE partially mimicked the osteogenic potential (ALP induction) of full-length BMP-2. Proximity between KE and cRGD may be the mechanism through which high dose KE induces osteogenesis in the presence of cRGD. To investigate this possibility, we used orthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) and maleimide-thiol chemistries to graft KE and cRGD in a bivalent (same alginate chain) and a monovalent (different alginate chain) manner, at constant bulk peptide concentration. Bivalent presentation of peptides (separation distance of 5.5 ± 0.5 nm verified by FRET) ultimately increased RUNX2 and ALP expression compared to monovalent presentation. This platform technology can be used in future studies to control peptide nanopatterning to enhance potency, in the context of MSC-based therapies and beyond.

Currently, autologous nerve (AN) transplantation remains the gold standard for treating peripheral nerve injuries (PNIs). However, its inherent limitations, including donor site morbidity and immune rejection risks associated with allografts, have prompted the exploration of alternative therapeutic strategies. Among these, tissue engineering approaches have gained significant attention, with nerve conduit design emerging as a particularly promising research direction. Electrospinning technology has been widely adopted for its ability to fabricate nanofibrous scaffolds that closely mimic the native extracellular matrix. In this study, we engineered an aligned nanofiber conduit utilizing polylactic acid and gelatin through electrospinning, and integrated a sodium alginate hydrogel enriched with Schwann cells (SCs) and neural stem cells (NSCs) via coaxial bioprinting. The three-dimensional (3D) hydrogel microenvironment facilitated synergistic interactions between SCs and NSCs, augmenting the secretion of neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). A dynamic perfusion culture system was further employed to optimize cell viability and functionality. In vivo studies revealed that the implantation of this conduit in a sciatic nerve defect model markedly enhanced motor function recovery, nerve regeneration, and muscle morphology. These improvements were substantiated by an increased sciatic functional index (SFI), heightened expression of S-100 and NF-200, and greater myelin thickness and axon diameter. Although the efficacy of the 3D-aligned nanofiber conduit cocultured with SCs and NSCs approximated that of AN transplantation, further research is imperative to identify more efficient seed cells and biocompatible 3D carriers to achieve optimal nerve regeneration. This study highlights the potential of tissue-engineered nerve conduits as a viable alternative for PNI repair, paving the way for future advancements in the field.

Attention-deficit/hyperactivity disorder (ADHD) is one of the most common neurodevelopmental disorders, with symptoms including hyperactivity, inattention and impulsivity. Moreover, ADHD persists into adulthood in ∼50% cases, significantly affecting quality of life. Currently, the complex aetiology of ADHD remains unclear. Single nucleotide polymorphisms (SNPs) in the adhesion G protein-coupled receptor isoform L3 gene (ADGRL3) have been associated with ADHD development, with the rs1397547 SNP found associated with altered ADGRL3 transcription in fibroblast cells. However, ADGRL3 function has not been investigated in human neurodevelopment.

Background: Recently, intra-articular injection of mesenchymal stem cells (MSCs) had been proposed as a conservative treatment for hip osteoarthritis (HOA). Adipose tissue was demonstrated as a viable source of MSCs because of the high concentration of cells and the easy access to the donor site. The purpose of this study was to evaluate the time-related results of a single intra-articular injection of autologous adipose-derived stem cells (aASCs) in a series of patients with HOA. Methods: A retrospective study was conducted on 30 patients with HOA, who underwent an intra-articular injection of aASCs between September 2018 and January 2021. Inclusion criteria for the procedure were as follows: onset of symptoms of the affected hip in the prior six or more months ago, failure of the conservative treatment (NSAIDs and/or physiotherapy) and age > 18 years. Exclusion criteria were trauma in the affected hip occurred in the previous 3 months, recent arthroscopic treatment, infectious joint disease, chondromatosis of the hip or any other secondary HOA, malignancy, hyaluronic acid or other injections in the previous 6 months and incomplete follow-up. Because a low BMI makes extremely difficult to harvest enough adipose tissue, patients with a BMI < 18 were also excluded. The Oxford Hip Score, the 12-item Short Form Survey and Visual Analogue Scale were used to evaluate the results of the proposed treatment at regular intervals. Results: In 27/30 patients, a constant improvement in pain relief, hip function and quality of life was observed during the entire follow-up period of 12 months. Two patients underwent a subsequent total hip arthroplasty. Conclusion: The single injection of aAMSCs seems to be a valuable treatment for HOA. A constant amelioration of pain and function could be observed in most patients at 12 months of follow-up.

Chronic fatigue syndrome (CFS) is a disorder characterized by severe unexplained fatigue and is associated with various factors including infections, immune responses, genetics, and environmental influences. However, the underlying mechanisms and possible interventions for CFS remain unclear. We used a two-mediated MR method to investigate causal relationships between diet, lipid levels, immune cells, and CFS. Our findings suggest that certain lipids, specifically low-density cholesterol, apolipoprotein E, and apolipoprotein B, contribute to CFS development. Conversely, high-density lipoprotein cholesterol and apolipoprotein A1 may delay the onset of the syndrome. Additionally, we explored how lipids affect fatigue through immune cell mediation. Factors, such as hematopoietic stem cell absolute count, the percentage of CD3-natural killer lymphocytes, and IgD presence on IgD+ CD38+ B cells may mediate the causal pathway linking lipids to CFS. Furthermore, we examined the relationship between diet, lipids, and CFS. This indicated that specific dietary selections, like alcohol intake, a preference for chili peppers, and an affinity for breakfast, contributed to CFS. Conversely, cheese and pork consumption were protective factors against CFS. The protective effect of cheese consumption on CFS was mediated by apolipoprotein A1 and high-density lipoprotein cholesterol. In conclusion, the study established an ecological chain: cheese consumption leads to increased high-density lipoprotein cholesterol and alters immune cell phenotypes-specifically, increasing the percentage of CD3-lymphocytes and IgD on IgD+ CD38+ B cells-ultimately influencing the development of CFS. These findings enhance our understanding of how lipid levels and immune factors are related to CFS and how dietary choices can potentially mitigate the syndrome.

Regulation of vesicle biology and trafficking plays a critical role in cell viability. Vesicular trafficking is a process that entails vesicle biogenesis, transport, and sorting of materials such as proteins, enzymes, hormones, and neurotransmitters to different cellular compartments. This phenomenon is especially important in cells of the central nervous system, including neural progenitors, neurons, and glial cell populations, because of their highly polarized architecture. In line with that, disruption in vesicular trafficking during cortical development affects progenitor proliferation and differentiation and leads to brain malformations. On the other hand, neuronal cells require long-range vesicular trafficking to reach distant locations, such as the distal part of the axons, and synaptic vesicles are essential for cell-cell communication. Neurons have high energy demands. Therefore, any malfunction in vesicular trafficking is a trigger to spiraling into neurodegeneration. Here, we give a comprehensive review of the role of intracellular and extracellular vesicles in cortical development and neurodegeneration, and we discuss how trafficking between organelles in specific cell types contributes to brain pathologies. Finally, we highlight the emerging evidence linking disruption in vesicular trafficking to neurological disorders such as Alzheimer's disease, Parkinson's disease, and autism.