The bone marrow (BM) niche is a highly specialized and dynamic microenvironment that tightly regulates hematopoiesis in both health and disease. In this chapter, we present a protocol for generating patient-specific 3D BM-mimicking assembloids, which offer precise control over cellular composition and genetic background. This in vitro platform enables the dissection of mechanisms underlying hematopoietic regulation and BM niche remodeling. We describe, in detail, the stepwise differentiation of induced pluripotent stem cells (iPSCs) into hematopoietic and endothelial lineages, the isolation of human primary mesenchymal stromal cells (MSCs) from femoral heads, and the assembly of BM-mimicking 3D assembloids. Single-cell RNA sequencing of these assembloids identified key myeloid populations and non-hematopoietic lineages such as endothelial cells and various MSC clusters, all crucial for stem cell fate determination and niche maintenance. Furthermore, assembloids harboring the JAK2V617F driver mutation successfully recapitulated key features of myeloproliferative neoplasms, demonstrating the platform's potential for mechanistic studies in human hematopoiesis. This approach provides a powerful tool to model both physiological and neoplastic BM niches, facilitating preclinical research and drug development while potentially reducing reliance on animal models.

Organoids have emerged as a powerful in vitro model system for biomedical research, providing a physiologically relevant alternative to traditional cell lines. Intestinal organoids recapitulate the complex cellular composition and function of the intestinal epithelium, making them valuable for studying intestinal biology and disease. These three-dimensional (3D) cultures can be differentiated to contain all the major intestinal cell types-including enterocytes, Paneth cells, goblet cells, stem cells, enteroendocrine cells, and tuft cells-allowing for more accurate modeling of intestinal function. However, their 3D structure presents challenges for high-resolution imaging and histological analysis. Common methods for embedding intestinal organoids, such as frozen sectioning or pre-embedding in semi-solid gels, can compromise morphology and sectioning integrity. To address these limitations, we present an optimized paraffin-embedding protocol that provides robust immunofluorescent staining and imaging of intestinal organoids while preserving cellular architecture. This approach provides researchers with an improved tool for analyzing organoid-based models of intestinal function and disease.

The treatment of advanced osteoarthritis (OA) remains clinically intractable due to the inability to regenerate multifocal cartilage defects, stemming from poor targeted recruitment of bone marrow-derived mesenchymal stem cells (BMSCs) and the absence of a sustained chondrogenic microenvironment at the injury sites. In this work, an antibody-mediated gelatin methacrylate-based hydrogel microsphere modified by TGFβ-affinity peptides (TRG microsphere) is developed, to precisely target and repair scattered cartilage injuries by only intraarticular injection without any surgical assistance. By leveraging the specific expression of type I collagen in OA cartilage lesions, the type I collagen antibodies anchoring on TRG's surface enable the specific and accurate targeting of the multiple injury areas that need regeneration. In the meantime, the TGFβ-affinity peptides incorporated in the TRG microsphere can capture the endogenous TGFβ, a growth factor that can recruit BMSCs and promote their differentiation, to precisely induce the hyaline-like cartilage regeneration locally. In a rat model of advanced OA, a single intra-articular injection of TRG microspheres can repair scattered cartilage defects, restore glycosaminoglycan deposition, and alleviate joint dysfunction. This study proposes an injection-based strategy that enables continuous recruitment of endogenous BMSCs for precise cartilage regeneration, eliminating complex invasive procedures and patient discomfort.

The subventricular zone (SVZ), the brain's largest neural stem cells reservoir, plays a critical role in glioblastoma development and progression. This study aims to investigate the association between MRI features and SVZ contact in IDH-wild-type glioblastoma, as well as their prognostic significance to guide personalized diagnosis and treatment.

Metabolic disorders could cause dysregulated glucose and lipid at the systemic level, but how inter-tissue/organ communications contribute to glucolipotoxicity is difficult to dissect in animal models. To solve this problem, myocardium and nerve tissues were modelled by 3D engineered heart tissues (EHTs) and neural organoids (NOs), which were co-cultured in a generalised medium with normal or elevated glucose/fatty acid contents. Morphology, gene expression, cell death and functional assessments detected no apparent alterations of EHTs and NOs in co-culture under normal conditions. By contrast, NOs significantly ameliorated glucolipotoxicity in EHTs. Transcriptomic and protein secretion assays identified the extracellular matrix protein versican as a key molecule that was transferred from NOs into EHTs in the high-glucose/fatty acid condition. Recombinant versican protein treatment was sufficient to reduce glucolipotoxicity in EHTs. Adeno-associated virus-delivered versican overexpression was sufficient to ameliorate cardiac dysfunction in a murine model of diabetic cardiomyopathy. These data provide the proof-of-concept evidence that inter-tissue/organ communications exist in the co-culture of engineered tissues and organoids, which could be systemically studied to explore potential pathological mechanisms and therapeutic strategies for multi-organ diseases in vitro.

Adult hippocampal neurogenesis (AHN) is the process by which new neurons are continuously generated from neural stem and progenitor cells (NSPCs) in the adult dentate gyrus. AHN plays a pivotal role in cognitive functions, including learning, memory, and mood regulation. Transcription factors regulate AHN by maintaining the NSPC pool and facilitating lineage progression. The nuclear factor I (NFI) transcription factor family member NFIA is critical for neurogenesis and gliogenesis during early brain development, but its role in adult neurogenesis remains poorly understood. Here, we generated an inducible Nfia loss-of-function mouse model to investigate the role of NFIA in Ascl1-lineage adult-born neurons. By tracking lineage progression from NSPCs to mature neurons, we found that NFIA deletion significantly reduced neurogenesis. Populations of NSPCs, neuroblasts, and mature granule neurons were all similarly diminished, indicating a primary defect in NSPC maintenance. Behaviorally, NFIA loss impaired hippocampal-dependent contextual fear memory without affecting locomotor activity, anxiety levels, spatial memory, or cued fear memory. Our findings demonstrate that NFIA is crucial for AHN and hippocampus-dependent contextual memory, thereby providing insights into its role in adult neurogenesis.

Schizothorax prenanti is an important economic Cyprinidae fish endemic to the upper reaches of the Yangtze River in China. The wild population of S. prenanti continues to decline and has been listed as an endangered fish because of environmental pollution and overfishing. Herein, the skin cell line (SPSK) of S. prenanti was established using the tissue block method to aid in protecting S. prenanti at the cellular level and provide a skin cell line that can be applied in functional genomics and disease aetiology of the spring viraemia of carp virus (SVCV), which is highly infectious in Cyprinidae fish. The SPSK cell line was sub-cultured to more than 30 generations at 24°C in L-15 medium supplemented with 15% fetal bovine serum (FBS). Karyotype analysis further revealed that the chromosome number of SPSK ranged between 140 and 149, with 146 accounting for the highest proportion. Significant fluorescent signals were observed after transfection of SPSK with pEGFP-N1 and Cy3-siRNA, with a 30% and 90% transfection efficiency, respectively. Severe cytopathic effects (CPE) were also observed when SPSK was infected with SVCV, and the SVCV glycoprotein gene was detected by RT-PCR, indicating that SPSK was susceptible to SVCV. To further explore the mechanism of bacterial infection, transcriptome analysis was conducted for LPS treated SPSK cells and 9099 differentially expressed genes were identified. These genes significantly enriched into pathways including the Haematopoietic Cell Lineage and Primary immunodeficiency. Furthermore, seven predominantly expressed epidermal maker genes were identified by transcriptomic data, suggesting that SPSK cells were mainly derived from skin epidermis, composed of epidermal stem cell, Merkel cell, and immune cell. The establishment and characterisation of SPSK revealed its application in functional genomics and aetiology studies, making it a favourable tool for exploring disease control in S. prenanti and recovering fish resources.

Current non-surgical managements of osteoarthritis (OA) do not change the clinical course or arrest the progression of the disease, while joint replacement is indicated for end-stage disease. Given these limitations, there is an unmet clinical demand for new treatment modalities that can improve the pain and quality of life of patients suffering from OA without surgery. The recent surge of interest in regenerative medicine (RM) for OA is based on these circumstances. Unlike traditional medicine, RM products may be accompanied by many uncertainties and long-term consequences. Considering that OA directly affects quality of life rather than life and death, the 'first do no harm' principle is more important when applying RM technology to the disease. Presently, culture-expanded mesenchymal stromal cells (MSCs) and orthobiologics, including bone marrow aspirate concentrate, stromal vascular fraction from adipose tissue and platelet-rich plasma have been applied to patients in clinical trials. Results of randomized clinical trials using MSCs have demonstrated that structural improvement and reversal of the pathologic process in OA are not definitely shown, while symptomatic relief is apparent. Orthobiologics seem to have efficiency comparable to those of culture-expanded MSCs. With the advantage of avoiding the approval process from regulation agencies, orthobiologics might provide a less expensive and handier option to culture-expanded MSCs. High-quality data from a large number of patients and head-to-head comparisons of several RM products will be necessary to define the place of culture-expanded MSCs or orthobiologics for OA treatment and resolve the reimbursement issue.

Measurable residual disease (MRD) is a major prognostic factor in newly diagnosed multiple myeloma. An assessment of an MRD-guided consolidation strategy in patients who are eligible for autologous stem-cell transplantation (ASCT) may be useful.

The control of gluconeogenesis is critical for glucose homeostasis and the pathology of type 2 diabetes (T2D). Here, we uncover a novel function of TET2 in the regulation of gluconeogenesis. In mice, both fasting and a high-fat diet (HFD) stimulate the expression of TET2, and TET2 knockout impairs glucose production. Mechanistically, FBP1, a rate-limiting enzyme in gluconeogenesis, is positively regulated by TET2 in liver cells. TET2 is recruited by HNF4α, contributing to the demethylation of the FBP1 promoter and activating its expression in response to glucagon stimulation. Moreover, metformin treatment increases the phosphorylation of HNF4α on Ser313, which prevents its interaction with TET2, thereby decreasing the expression level of FBP1 and ameliorating the pathology of T2D. Collectively, we identify an HNF4α-TET2-FBP1 axis in the control of gluconeogenesis, which contributes to the therapeutic effect of metformin on T2D and provides a potential target for the clinical treatment of T2D.

Recent evidence indicates the potential of gamma-irradiated (γi) Staphylococcus aureus to be used as an osteo-immunomodulator for bone regeneration. This study aims at characterizing the inflammatory milieu caused by the stimulation of γi S. aureus in immune cells and investigates its effects on MSC osteogenic differentiation. Furthermore, we aimed to recreate the immune-modulatory response exhibited by γi S. aureus by using a mixture of various synthetic pathogen recognition receptor (PRR) ligands consisting of TLR2, TLR8, TLR9, and NOD2 agonists. Human peripheral blood mononuclear cells (hPBMCs), isolated from healthy human donors, were exposed to γi S. aureus or seven different ligand mixtures. After 24 h, the conditioned medium (CM) from the hPBMCs was collected and its effects on hMSC osteogenic differentiation were investigated by assessing alkaline phosphatase (ALP) activity and matrix mineralization. The hPBMCs and their CM were also analyzed by bulk RNA sequencing and for cytokine secretion. CM from the γi S. aureus and the mixture consisting of Pam3CSK4, C-class CpG oligodeoxynucleotide (CpG ODN C), and murabutide targeting TLR2, TLR9, and NOD2 showed a fivefold increase in ALP and matrix mineralization in a donor-dependent manner. These effects were due to the upregulation of inflammatory signaling pathways, which led to an increase in cytokines and chemokines TNF, interleukin (IL)-6, IFN-γ, IL-1α, CXCL10, CCL18, CCL17, CXCL1, and CCL5. Upregulation of genes like BMP2R, BMP6R, BGLAP, and others contributed to the upregulation of osteogenic pathways in the hPBMCs stimulated with γi S. aureus and the aforementioned mix. Thus, formulations with mixtures of PRR ligands may serve as immune-modulatory osteogenesis-enhancing agents.

Background: CXCR4 enhances the homing of mesenchymal stem cells (MSCs), thereby potentially improving outcomes in myocardial infarction (MI). However, the molecular mechanisms underlying MSC homing remain poorly understood. Methods: The identity of MSCs was confirmed through flow cytometry, utilizing their cluster of differentiation (CD) marker profile. Migration and invasion were assessed using wound healing and transwell assays. In a rat MI model, myocardial function, hemodynamic parameters, and the degree of myocardial fiber damage were evaluated post-MSC treatment, along with the observation of MSC homing. Luciferase assays identified binding sites between SOX10 and the CXCR4 promoter, and the effects of SOX10 on MSC migration, invasion, and homing were explored both in vitro and in vivo. Results: Overexpression of CXCR4 significantly enhanced MSC migration, invasion, and homing. MSCs overexpressing CXCR4 improved cardiac function and reduced infarct size in the rat MI model. A direct interaction between SOX10 and CXCR4 was confirmed, with SOX10 acting as a transcription factor to upregulate CXCR4 expression, thereby enhancing MSC homing and ameliorating MI in rats. Knockdown of SOX10 reversed the beneficial effects of CXCR4-overexpressing MSCs on MI therapy, as well as the functional impact of CXCR4 on MSCs. Conclusion: In conclusion, SOX10 facilitates MSC homing by upregulating CXCR4 expression, offering a potential therapeutic approach for MI treatment.

Type 1 diabetes (T1D) is not only a disorder of insulin production from beta cell destruction, but also a progressive condition that brings about life-threatening complications such as diabetic nephropathy, impaired wound recovery, and cardiovascular disease. Mesenchymal stromal cell (MSC) use has recently become an encouraging new way to treat these complications and can result in better health outcomes for T1D patients. Some research has shown that MSC injections into mice and rat models have resulted in reduced mesangial cell thickening, inflammatory mediator recruitment, proteinuria, and fibrosis normally seen in diabetic nephropathy. Other studies have demonstrated that MSCs aid wound healing by increasing anti-inflammatory M2 macrophage differentiation, stimulating angiogenesis and collagen synthesis, and signaling the proliferation and migration of dermal fibroblasts toward injury sites. Additionally, there is evidence that MSCs are capable of activating the PI3K pathway and exhibiting antioxidant effects in murine models experiencing diabetic-related heart disease. However, given these efforts, further research is needed to establish the prolonged safety and efficacy of MSC use in humans to treat T1D.

Deepening the genetic mechanisms underlying Normal Hearing Function (NHF) has proven challenging, despite extensive efforts through Genome-Wide Association Studies (GWAS).

Quiescence is a conserved, reversible state of proliferative arrest, characterized by changes in cell physiology and metabolism. Many cells spend a considerable part of their lifetime in quiescence, including adult stem cells or microorganisms facing unfavorable environmental conditions. Cells can remain quiescent for long periods of time while retaining their viability and reproductive capacity, indicating a need to maintain protein homeostasis. Given the changes in intracellular organization, it has been unclear how protein quality control (PQC) functions in quiescent cells. In our recent study, we examined model misfolded proteins expressed in glucose-depleted quiescent yeast cells and found that quiescent cells maintain an active PQC that relies primarily on selective protein degradation, requiring the ubiquitin-proteasome system, intact nucleus-vacuole junctions and autophagy. Our results highlight the relevance of mitigating misfolded proteins in quiescence.

Hidradenitis Suppurativa (HS) is a chronic multifactorial inflammatory skin disease with a debilitating impact on quality of life. Here, we review the complex interplay of innate and adaptive immune dysregulation in HS pathogenesis, in the context of microbial dysbiosis, genetic predisposition, cellular dysfunction and epigenetic factors. Hyperactivation of the innate system triggered by follicular occlusion leads to a cascade of activated signaling pathways leading to persistent inflammation as the disease progresses. This immune hyperactivation is further complicated by microbiome dysbiosis, which is associated with dysregulation of inflammasomes and altered expression of host antimicrobial peptides. Keratinocytes, fibroblasts, macrophages, and neutrophils exhibit altered functions, and contribute to the inflammatory cascade and disease chronicity in HS. Epigenetic mechanisms including DNA methylation, histone modifications, and non-coding RNAs modulate immune responses and contribute to aberrant cytokine and chemokine expression that drive the persistent inflammatory state in HS pathogenesis. We highlight the need for future research to explore the concept of epigenetic memory in epidermal stem cells and inflammasome activation to gain a better understanding of these mechanisms and pave the way for development of future novel therapeutic targets and strategies to disrupt the persistent chronic inflammation cycle in this debilitating condition.

BCG vaccination can have heterologous or non-specific effects (NSE) that confer resistance against pathogens other than its target Mycobacterium tuberculosis, but the underlying mechanisms are not fully understood.

Recent advances in chimeric antigen receptors have provided an alternative approach for treating relapsed acute lymphocyte leukemia after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, relapsed patients who had undergone allogeneic umbilical cord blood transplantation (UCBT) have no chance of having CAR-T cells derived from donors due to lacking UCB. We present a case of a patient with Ph+ ALL who relapsed after UCBT and achieved complete morphological and molecular remission following treatment with CD19 CAR-T cells derived from the recipient post-UCBT. The patient had only grade I CRS. GVHD or neurotoxicity was not observed. More than 6 years after CAR-T cell infusion, the patient was still in hematologic and molecular complete remission with negative minimal residual disease (MRD). This case is the first to show a new strategy of practicality, efficacy, and safety of CD19 CAR-T cells derived from UCBT recipients for treating relapsed ALL after UCBT.

Autologous bone grafting, particularly using iliac crest bone grafts, historically been considered the gold standard for bone grafting. However, its limited availability and secondary surgical trauma associated with the procedure have restricted its widespread application.

Despite the regenerative and self-repair capabilities of bone tissues, significant bone loss can result in substantial bone defects. This study was aimed at investigating the role and underlying mechanisms of the mechanosensitive protein PDZ and LIM Domain 5 (PDLIM5) in the osteogenic differentiation of human adipose-derived stem cells (hASCs) under cyclic tensile stress conditions relevant to bone tissue repair. Utilising proteomics and single-cell RNA sequencing, we identified PDLIM5 and serpin E2 as key genes associated with the osteogenic differentiation of stem cells. To evaluate the expression levels of these genes and related proteins, we utilised western blotting, immunofluorescence and alkaline phosphatase (ALP) staining. Furthermore, lentiviral transfection, Cell Counting Kit-8 (CCK-8) assays, transwell migration assays, wound healing assays and protein-protein interaction analyses were conducted to evaluate changes in osteogenic differentiation under both chemical and physical stimuli, as well as to explore the relationship between serine protease inhibitor E2 (serpin E2) and its downstream effector, PDLIM5. The interactions among serpin E2, integrin β3 and PDLIM5 were confirmed through Haematoxylin and Eosin (H&E) staining, immunohistochemistry and immunofluorescence staining of bone tissues and primary adipose-derived stem cells isolated from integrin β3 knockout mice. Our findings indicate that PDLIM5 modulates the osteogenic differentiation of hASCs via a signalling pathway involving serpin E2, integrin β3 and lamin A.