Clonal hematopoiesis (CH) is characterized by the presence of hematopoietic stem cells (HSCs) with the potential of clonally expanding and giving rise to hematological malignancies. Clonal hematopoiesis of indeterminate potential (CHIP) is the outgrowth of a single HSC clone with an acquired somatic mutation in the absence of hematological abnormalities. CHIP variants occur with a variant allele frequency (VAF) of at least 2% in peripheral blood. This definition does not account for less frequent mutations that give rise to hematopoietic clones. Previous studies indicate an association between CH and secondary hematologic malignancies in cancer patients who receive chemotherapy.

Rationale: Induced pluripotent stem cells (iPSCs) share transcriptomic similarities with cancer cells and express tumor-specific and tumor-associated antigens, highlighting their potential as cancer vaccines. Our previous study demonstrated that an iPSC-based vaccine effectively prevented tumor growth in various mouse models, including melanoma, breast, lung, and pancreatic cancers. However, the underlying mechanisms and the therapeutic efficacy of the iPSC-based vaccine remain unclear. Colorectal cancer (CRC), the third most common cancer with a rising incidence worldwide, presents an urgent need for novel strategies to prevent and treat CRC. Methods: Allograft mouse models were established to evaluate the antitumor effects of the iPSC-based vaccine. CpG oligonucleotide (ODN) 1826 served as a vaccine adjuvant. Bulk RNA-Sequencing (RNA-Seq) and the Microenvironment Cell Population counter (MCP-Counter) algorithm were performed to analyze transcriptomic changes. Liquid chromatography-mass spectrometry (LC-MS) combined with in silico strategies was employed to identify potential antigen proteins. Chinese Hamster Ovary (CHO-K1) models were utilized to express candidate neoantigen proteins. Mouse bone marrow-derived dendritic cells (BMDCs) were used to investigate T cell priming in response to iPSC-associated proteins. Immune cell profiles were characterized by flow cytometry. Results: The combination of CpG and iPSC vaccination demonstrated both prophylactic and therapeutic efficacy in reducing tumor growth in CRC mouse models. Vaccination significantly increased CD8+ T cell infiltration within tumor regions, while T cell depletion abrogated the antitumor effects, underscoring the critical role of T cells in mediating these responses. Proteomic analysis identified two iPSC-associated proteins, heterogeneous nuclear ribonucleoprotein U (HNRNPU) and nucleolin (NCL), as key drivers of the observed immune responses. Vaccination with HNRNPU or NCL, in combination with CpG, enhanced dendritic cell activation, induced antigen-specific CD8+ T cell cytotoxicity, and promoted the formation of central memory CD8+ T cells, collectively leading to significant CRC tumor shrinkage. Conclusions: Our findings reveal potential mechanisms underlying the efficacy of iPSC-based vaccines in cancer immunotherapy. Additionally, HNRNPU and NCL were identified as key antigen proteins in iPSC, demonstrating promise for the development of peptide-based vaccines for both the prevention and treatment of CRC.

Background: The regeneration of functional hair cells (HCs) remains a critical challenge in addressing sensorineural hearing loss. This study aimed to investigate the molecular and functional mechanisms driving stereocilia maturation within inner ear organoids (IEO) derived from homogenic Lgr5-positive progenitor cells (LPCs) and to compare outcomes with traditional heterotypic cultures. Methods: Mouse cochlear LPCs were isolated via magnetic-activated cell sorting (MACS) to establish homotypic cultures, ensuring purity and eliminating the heterotypic influences present in traditional manual isolation (MI) methods. Differentiation into HCs was induced through Wnt and Notch signaling modulation. Transcriptomic profiling using bulk and single-cell RNA sequencing (scRNA-seq) identified gene expression changes linked to stereocilia development. A Sonic Hedgehog (Shh) agonist was applied to enhance structural maturation of HCs. Functional assessment included electron microscopy, FM1-43 uptake assays, and microelectrode array recordings in assembloids of IEO with primary spiral ganglion neurons (SGN) co-cultures. Results: While homotypic LPC-derived IEOs successfully differentiated into HC-like cells, initial morphological assessment revealed immature stereocilia structures. Bulk RNA-seq analysis highlighted a downregulation of morphogenesis-related genes in these organoids. The application of a Shh agonist, acting as a key morphogen, promoted stereocilia development, as evidenced by enhanced ultrastructural features and increased expression of cuticular plate-associated genes (Pls1, Lmo7 and Lrba). Single-cell RNA sequencing (scRNA-seq) further identified distinct cell clusters, which exhibited robust expression of stereocilia-related genes (Espn, Lhfpl5, Loxhd1 and Tmc1), indicative of advanced HC maturation. Electrophysiological assessments of IEO-SGN assembloids using microelectrode arrays confirmed functional mechanoelectrical transduction between cells. Conclusion: This integrated approach elucidates critical pathways and cellular dynamics underpinning stereocilia maturation and functional HC development in EIOs. These findings provide new insights into the molecular regulation of HC maturation and support the utility of Shh-modulated IEOs as a promising platform for inner ear regeneration and therapeutic development for inner ear regenerative therapies.

Fish-derived biomaterials, such as collagen, polyunsaturated fatty acids, and antimicrobial peptides, have emerged as promising candidates for scaffold development in stem cell therapies and tissue engineering due to their excellent biocompatibility and low immunogenicity. Although good bioactivity is a prerequisite for biomedical substitutes, scaffold design is necessary for the successful development of bioconstructs used in tissue regeneration. However, the limited processability of fish biomaterials poses a substantial challenge to the development of diverse scaffold structures. In this review, unlike previous reviews that primarily focused on the bioactivities of fish-derived components, we placed greater emphasis on scaffold fabrication and its applications in tissue regeneration. Specifically, we examined various cross-linking strategies to enhance the structural integrity of fish biomaterials and address challenges, such as poor processability, low mechanical strength, and rapid degradation. Furthermore, we demonstrated the potential of fish scaffolds in stem cell therapies, particularly their capacity to support stem cell growth and modulate the cellular microenvironment. Finally, this review provides future directions for the application of these scaffolds in cancer therapy.

Rationale: Following the structural destruction of annulus fibrosus (AF), the early-stage damage manifests as symptoms such as an inflammatory phenotype and loss of mechanical support. The microenvironmental deterioration at the injury site, the limited population, and the inadequate differentiation of intrinsic stem/progenitor cells impede the efficient repair of AF. To address the aforementioned challenges, we developed a dual-drug-loaded hydrogel system to achieve systematic and functional annulus fibrosus tissue repair. Methods: A tannic acid-crosslinked gelatin-based hydrogel scaffold with the addition of Mn2+ was designed to work as a platform to provide mechanical support, antioxidant capacity, and immune-modulating function. The kartogenin-loaded nanofiber and SDF-1α mimic peptide were also incorporated into the hydrogel system to facilitate the recruitment of endogenous stem cells and direct AF tissue regeneration. Results: The resulting hydrogel scaffolds exhibit excellent biogenic properties while achieving mechanical properties similar to those of AF. The composite scaffold also enhances ROS clearance and promotes M2 polarization of macrophages to improve the inflammatory microenvironment during early-stage injury. Furthermore, the sustained release of kartogenin-loaded nanofiber and SDF-1α mimic peptide effectively enhances endogenous stem cell recruitment, promotes cartilage differentiation, and facilitates specific extracellular matrix deposition, thus meeting requirements for late-stage AF repair. Conclusion: The findings demonstrate the potential of a multifunctional, high-strength supramolecular hydrogel loaded with dual drugs for the functional regeneration of AF tissue.

Cadherin-based biomaterials play a pivotal role in influencing the fate of mesenchymal stem cells (MSC). Enhancing the adhesion of adipose tissue-derived MSCs has been shown to augment their paracrine effects while N-cadherin biomaterials have been suggested to regulate the paracrine effects of MSCs via specific growth factors although the precise mechanisms underlying this regulation remain insufficiently understood. This study aims to compare the effects of a 3D N-cadherin mimetic environment on cell viability, apoptosis, extracellular matrix regulation, and growth factor expression with those observed in traditional 2D and 3D spheroid cultures. Additionally, the study seeks to evaluate the effects of conditioned media derived from the N-cadherin mimetic environment on the viability and migration of endothelial cells.

Retinal dehydrogenase 12 (RDH12) is a photoreceptor NADPH-dependent retinal reductase enzyme, converting all-trans-retinal to all-trans-retinol. Heterozygous variants in RDH12 cause a rare autosomal dominant (AD) retinitis pigmentosa.

Neural stem and progenitor cells (NSPCs) are crucial for nervous system development and self-renewal. However, their properties are sensitive to environmental and chemical factors, including chemotherapy agents like cisplatin, an FDA-approved drug used to treat cancer. Cisplatin inhibits DNA replication but can cause side effects such as nephrotoxicity, ototoxicity, and neurotoxicity. While its cytotoxic effects are well understood, the impact of non-cytotoxic cisplatin concentrations on NSPC differentiation remains unclear.

Prime editing offers remarkable versatility in genome editing, but its efficiency remains a major bottleneck. While continuous optimization of the prime editing enzymes and guide RNAs (pegRNAs) has improved editing outcomes, the method of delivery also plays a crucial role in overall performance. To maximize prime editing efficiency, we implemented a series of systematic optimizations, achieving up to 80% editing efficiency across multiple loci and cell lines. Beyond integrating the latest advancements in prime editing, our approach combined stable genomic integration of prime editors via the piggyBac transposon system, selection of integrated single clones, the use of an enhanced promoter, and lentiviral delivery of pegRNAs, ensuring robust, ubiquitous, and sustained expression of both prime editors and pegRNAs. To further assess its efficacy in challenging cell types, we validated our optimized system in human pluripotent stem cells (hPSCs) in both primed and naïve states, achieving substantial editing efficiencies of up to 50%. Collectively, our optimized prime editing strategy provides a highly efficient and versatile framework for genome engineering in vitro, serving as a roadmap for refining prime editing technologies and expanding their applications in genetic research and therapeutic development.

The development of human organotypic models of cartilage provides essential insights into chondrogenesis and chondrocyte hypertrophy while enabling advanced applications in drug discovery, gene editing, and tissue regeneration. Here, we present a robust and efficient protocol for differentiating human expanded pluripotent stem cells (hEPSCs) into hypertrophic chondrocytes through a sclerotome intermediate. The protocol involves initial sclerotome induction, followed by 3D chondrogenic culture and subsequent hypertrophic maturation induced by bone morphogenetic protein-4 (BMP4), thyroid hormone (T3), and β-glycerophosphate. This protocol also allows for sensitive testing of the effects of various compounds on hypertrophic differentiation during the maturation process. Furthermore, we identify an α-adrenergic receptor antagonist, phentolamine, as an inhibitor of hypertrophic differentiation. This organoid system provides a practical platform for exploring cartilage hypertrophy mechanisms and testing therapeutic strategies for cartilage regeneration. Key features • This differentiation protocol generates hypertrophic chondrocytes from hEPSCs through a sclerotome intermediate. • This protocol facilitates sensitive testing of compounds during the hypertrophic maturation stage, enabling the study of molecular mechanisms and therapeutic interventions for cartilage hypertrophy. • This protocol identifies the α-adrenergic receptor antagonist phentolamine as a modulator of hypertrophic differentiation.

Neurons and oligodendrocytes are the building blocks of the brain. Neurons form synaptic connections and transmit signals, while oligodendrocytes, including oligodendrocyte precursor cells (OPCs) and their derivatives, are vital for central nervous system maintenance and myelination. The demand for human-specific neuron-oligodendrocyte model systems to study these interactions has grown, yet co-culture protocols remain limited. Recent advancements in the field provide methods for deriving co-cultures of neurons and OPCs from human induced pluripotent stem cells (hiPSC), each with distinct benefits and challenges. This study presents a time-efficient, reproducible method to derive neurons and O4-expressing oligodendrocytes, followed by a straightforward co-culture system that minimizes astrocyte differentiation and ensures robust neuron and oligodendrocyte populations. Key features • Reliable, stable generation of neurons and O4-expressing oligodendrocytes within a practical timeframe. • Co-culture system utilizing hIPSC-derived neurons and O4-expressing oligodendrocytes. • Maturation of neurons and oligodendrocytes achieved within 10 days of co-culturing. Graphical overview Graphical overview. The diagram outlines the sequential steps involved in the preparation, differentiation, and analysis phases. Key stages include the differentiation of neural progenitor cells (NPCs) into O4-expressing oligodendrocytes and neurons separately and then combining them into a co-culture, which can then be used for further experiments.

One of the major factors contributing to aging and age-related diseases is the well-understood decline in the function of adult stem cells. Quantifying the degree of aging in adult stem cells is essential for advancing anti-aging mechanisms and developing anti-aging agents. However, no systematic approach to this exists. In this study, we developed a method to quantitatively assess the degree of aging in adult intestinal stem cells using a Drosophila midgut model and two aging markers. First, aging was induced in Drosophila with the desired genotype, and the anti-aging agent was administered 7 days before dissection. Then, the levels of two intestinal stem cell aging markers found in Drosophila (PH3 and γ-tubulin) were measured using immunohistochemistry. Finally, fluorescence microscopy was employed to count the number of aging markers and take images, which were analyzed using image analysis software. Using this approach, we quantitatively analyzed the effects of anti-aging agents on the aging of adult intestinal stem cells. This methodology is expected to significantly expedite the development of anti-aging agents and substantially reduce the research costs associated with aging-related studies. Key features • PH3 and γ-tubulin serve as reliable markers for quantitatively assessing aging in Drosophila intestinal stem cells. • This method for discovering anti-aging agents involves processes such as aging induction, treatment with anti-aging agents, dissection, fixation, antibody staining, and analysis of the results. • Vitamin D, similar to metformin and β-hydroxybutyrate, is an anti-aging agent. • Quantitative analysis of adult stem cell aging will enable the rapid and accurate identification of anti-aging agents and efficacy validation.

Immune thrombocytopenic purpura (ITP) is an autoimmune disorder characterized by the production of autoantibodies targeting platelet membrane antigens, leading to platelet destruction by the reticuloendothelial system. This results in a significant drop in platelet count to 100 × 10⁹/L or lower due to the formation of autoantibodies and immune complexes. Some studies suggest a potential link between Helicobacter pylori infection and ITP. This report presents a case of ITP in a patient with an H. pylori infection. To our knowledge, this is one of the unique and interesting reported cases of such a severe platelet deficiency, where the platelet count dropped to 1,000 cells/µL in the presence of an aggressive H. pylori infection. A 46-year-old male was admitted with mild gum bleeding and petechiae on his lower limbs. His medical history included chronic diabetes mellitus, dyslipidemia, and hypertension, though his clinical and vital signs were normal. Laboratory tests revealed a critically low platelet count of 3,000 cells/µL, leading to a provisional diagnosis of ITP. The patient was started on intravenous (IV) methylprednisolone (1 g for three days), IV immunoglobulin (0.4 g/kg for five days), proton pump inhibitors, calcium, and vitamin D supplements. However, there was no significant response to the treatment. Additional immunological and viral tests yielded negative results. Given this, an H. pylori test was conducted, which confirmed an infection. The patient was started on H. pylori eradication therapy. The platelet count improved to 48,000 cells/µL, but it dropped again to 1,000 cells/µL after a few days. Various treatment strategies were implemented to manage both ITP and H. pylori. After two months, the H. pylori urea breath test returned negative, and the patient's platelet count normalized. The patient was maintained on folic acid (5 mg daily) and eltrombopag (50 mg daily), with regular hematology follow-ups ensuring stable platelet levels. This case underscores a rare presentation of ITP associated with severe thrombocytopenia (1,000 cells/µL) and aggressive H. pylori infection. The findings emphasize the importance of considering H. pylori in the differential diagnosis of ITP and highlight the necessity of identifying underlying causes for effective treatment.

Lymph nodes are crucial for perioperative immunotherapy but have to be completely resected in surgery. Trials evaluating the safety and necessity of omitting systemic mediastinal lymph node (mLN) dissection in non-small cell lung cancer (NSCLC) are still absent.

Perianal fistulae are a common, recurrent and painful disease in dogs, particularly in German shepherd dogs, and significantly affect the quality-of-life for both the animal and its owner.

Herpes simplex virus (HSV) reactivation and acyclovir resistance are common complications in immunocompromised individuals. We report a pediatric hematopoietic stem cell transplantation (HSCT) recipient who received HSV-1-specific T cells from a partially human leukocyte antigen (HLA) matched donor for severe acyclovir-resistant HSV-1 mucositis resulting in clinical improvement, reduced plasma viral load, and emergence of antiviral T cells.

The brain is the most complex human organ, and commonly used models, such as two-dimensionalcell cultures and animal brains, often lack the sophistication needed to accurately use in research. In this context, human cerebral organoids have emerged as valuable tools offering a more complex, versatile, and human-relevant system than traditional animal models, which are often unable to replicate the intricate architecture and functionality of the human brain. Since human cerebral organoids are a state-of-the-art model for the study of neurodevelopment and different pathologies affecting the brain, this field is currently under constant development, and work in this area is abundant. In this review, we give a complete overview of human cerebral organoids technology, starting from the different types of protocols that exist to generate different human cerebral organoids. We continue with the use of brain organoids for the study of brain pathologies, highlighting neurodevelopmental, psychiatric, neurodegenerative, brain tumor, and infectious diseases. Because of the potential value of human cerebral organoids, we describe their use in transplantation, drug screening, and toxicology assays. We also discuss the technologies available to study cell diversity and physiological characteristics of organoids. Finally, we summarize the limitations that currently exist in the field, such as the development of vasculature and microglia, and highlight some of the novel approaches being pursued through bioengineering.

The generation of human induced pluripotent stem cell-derived motor neurons overcomes limited access to human tissues and offers an unprecedented approach to modeling motor neuron diseases such as dystonia and amyotrophic lateral sclerosis. Motor neurons generated through different strategies may exhibit substantial differences in purity, maturation, characterization, and even neuronal identity, leading to variable outcomes in disease modeling and drug screening. However, very few comparative studies have been conducted to determine the similarities and differences among motor neurons prepared via different protocols. In this study, we prepared human induced pluripotent stem cell-motor neurons via lentiviral delivery of transcription factors and chemical induction and performed a systematic comparative analysis. We found that motor neurons generated by both approaches showed typical motor neuron morphology and robustly expressed motor neuron-specific markers, such as nuclear homeobox transcription factor 9 and choline acetyltransferase. The chemical induction protocol utilizes a combination of small molecules to induce motor neuron differentiation, offering a significantly faster maturation time of 35 days compared to 46 days with lentiviral delivery of transcription factors. Additionally, while lentiviral delivery of transcription factors are suitable for downstream biochemical analysis, chemical induction are more applicable for therapeutic approaches as they avoid the use of lentiviruses. Both approaches produce motor neurons with high purity (> 95%) and yield. No significant differences were found between chemical induction and lentiviral delivery of transcription factors in terms of motor neuron markers and maturation markers. These robust methodologies offer researchers powerful tools for investigating motor neuron diseases and potential therapeutic strategies.

Tobacco black shank, induced by Phytophthora nicotianae, ranks among the most destructive diseases threatening global tobacco production. Biological control constitutes a crucial method for the environmentally friendly management of this disease, with the discovery of biocontrol agents serving as the initial step in this endeavor. The present research seeks to uncover new biocontrol agents and plant growth promoters effective against P. nicotianae. A strain of endophytic actinomycete isolated from tobacco, designated W71, was identified as Streptomyces rochei. This strain exhibited strong IAA production capacity and inhibitory activity against P. nicotianae. In greenhouse trials, S. rochei W71 demonstrated significant plant growth promotion effects, markedly improving agronomic traits, root activity, root morphology indices, and antioxidant enzyme activities of tobacco plants. Field trials conducted at the rosette and prosperously growing stages revealed significant enhancements in several key crop parameters following the application of W71 treatment. These improvements encompassed increased maximum leaf length, maximum leaf width, stem girth, and plant height. Additionally, at harvest, W71 was found to facilitate a remarkable 98.91% boost in yield. In vitro inhibition tests demonstrated potent antagonism: live cells of S. rochei W71 suppressed P. nicotianae growth by 96.84%, outperforming the 70.89% inhibition rate of its cell-free culture filtrate. Greenhouse pot trials yielded an 87.53% disease control efficacy against tobacco black shank, and field trials resulted in a 72.68% control efficacy, indicating satisfactory performance. The study results demonstrate that S. rochei W71 possesses both plant growth-promoting properties and biocontrol capabilities against tobacco black shank, making it a promising candidate for use as a plant growth promoter and biological control agent.

Mesenchymal stromal cells (MSCs) are widely used to treat osteoarthritis (OA). Optimising dose, timing, and safety while comparing efficacy with standard therapies like hyaluronic acid (HA) is essential for their standardisation.