To describe principles of ultrasound-guided injection techniques for intralesional delivery of regenerative orthobiologics into equine tendon and ligament core lesions.

Prolonged cytopenias are a common complication after chimeric antigen receptor (CAR) T-cell therapy for multiple myeloma, increasing the risk of severe infection. Infusion of previously collected autologous stem cells may mitigate this risk, but the clinical and economic implications of proactive collection remain uncertain.

Aging is associated with impaired B lymphopoiesis and T lymphopoiesis, contributing to immunosenescence and poor immune recovery. Although this decline can be attributed to intrinsic hematopoietic stem cell aging, growing evidence indicates that lymphoid failure reflects constraints operating across multiple levels of the hematopoietic system. This review frames age-associated lymphopoiesis decline as a systems-level problem and outlines conceptual avenues for therapeutic intervention.

Experimental approaches for measuring single-cell gene expression can observe each cell at only one time point, requiring computational approaches for reconstructing the dynamics of gene expression during cell fate transitions. RNA velocity is a promising computational approach for this problem, but existing inference methods fail to capture key aspects of real data, limiting their utility. To address these limitations, we developed VeloVAE, a Bayesian model for RNA velocity inference. VeloVAE uses variational Bayesian inference to estimate the posterior distribution of latent time, latent cell state, and kinetic rate parameters for each cell. Our approach can incorporate prior distributions on rate parameters and time points; model lineage bifurcations using branching differential equations; and directly model discrete count data. We show that VeloVAE significantly outperforms previous approaches in terms of data fit, accuracy of inferred differentiation directions, and transcription rate estimation. These improvements allow VeloVAE to accurately model gene expression dynamics in complex biological systems, including hematopoiesis, induced pluripotent stem cell reprogramming, the developing mouse brain, and the entire mouse embryo. We find that the latent time automatically inferred using all cells can even outperform pseudotime inferred using manually chosen cell subsets and root cells. Our work provides important new tools for modeling sequential changes in gene expression from single-cell expression data.

Small cell carcinoma is a highly lethal cancer variant often found with neuroendocrine (NE) features, as exemplified by small cell lung cancer and small cell NE prostate cancer (SCPC). A genome-wide CRISPR dependency screen using SCPC models generated through human prostate cell transformation identifies a requirement for the transcription factor E2F3. E2F3 dependency is linked to RB inactivation, a near universal occurrence across small cell cancers. The requirement for E2F3 is shared by RB-deficient cells originating from the prostate, lung, and adnexa. In RB-deficient cancer cells, E2F3 inhibition restrains cell cycle progression, proliferation, and tumor growth in vivo. Inhibition of de novo pyrimidine synthesis limits E2F3 expression and suppresses small cell carcinoma proliferation in culture. Directly or indirectly targeting E2F3 to leverage a pan-cancer synthetic lethality resulting from RB inactivation represents a potential treatment strategy.

Single-cell RNA sequencing (scRNA-seq) provides an important means to reveal the heterogeneity and dynamic processes of tissues, organisms, and complex diseases, but technical capture loss (dropout) often obscures true biological expression, and existing imputation methods have difficulty distinguishing biological zeros (silent expression) from technical noise. To address this, we propose the imputation framework scZN. scZN assumes that the observed scRNA-seq data arise from a combination of RNA's two-state transcription process and dropout, and formulates imputation as nonnegative factorization: decomposing the raw count matrix into two interpretable nonnegative factors, performing learning and optimization under constraints from prior knowledge and multiple regularizations, thereby reconstructing the cellular expression landscape. Experiments show that scZN can capture the true distributional characteristics at both the gene and cell levels and significantly suppress spurious activation of genes that should not be expressed. Across multiple real datasets, it outperforms dozens of state-of-the-art methods. Especially in complex experimental design scenarios, scZN markedly improves trajectory inference for embryonic stem cells and mouse dentate gyrus data. In Alzheimer's disease data, scZN can also effectively recover pathways related to neuroinflammation, improving downstream scRNA-seq analysis. Overall, scZN provides a unified framework for missing-value imputation and expression reconstruction that combines accuracy and interpretability.

Accurate CD34+ cell enumeration is essential in stem cell transplantation. Flow cytometry (FC) is the standard method to determine optimal collection time of CD34+ stem cells harvesting for peripheral blood stem cell (PBSC) transplantation but is costly and requires skilled personnel. The ADAMII-CD34 cell counter may be a feasible alternative for PBSC apheresis samples and for cryopreserved stem cell products which are assessed by manual cell viability counting using the trypan blue exclusion method.

Mesenchymal stem cells derived from Wharton's Jelly (WJ-MSCs) are an attractive cell source for regenerative medicine due to high proliferative capacity, non-invasive accessibility, and minimal ethical constraints. However, their therapeutic efficacy may vary with isolation technique and culture conditions.

This study evaluated the antitumor efficacy of atorvastatin (ATO) and luteolin (LUT), administered individually or in combination with or without doxorubicin (DOX), in mice bearing solid Ehrlich carcinoma (SEC). Additionally, we examined the effects of these treatments on ATP-binding cassette (ABC) transporters, telomerase reverse transcriptase (TERT), and cancer stem cell-related markers as potential mechanisms underlying chemoresistance. SEC tumors were induced in 70 Swiss albino female mice, which were randomly assigned into seven groups (n = 10/group): SEC control, DOX (4 mg/kg, i.p.), ATO (20 mg/kg, i.p.), LUT (40 mg/kg, i.p.), ATO+ DOX, LUT+DOX, and ATO + LUT. At the end of the study, tumors were excised, weighed, and processed for histopathological evaluation, immunohistochemical analysis of CD44, quantitative assessment of ABCB1 and ABCG2 gene expression, and protein analysis of both the non-phosphorylated (TERT) and phosphorylated form (P-TERT). Co-treated groups exhibited a greater reduction in tumor growth compared to the control and single-agent groups. These effects were accompanied by marked downregulation of ABCB1 and ABCG2 gene expression and suppression of TERT and P-TERT protein levels. Histopathological findings revealed increased apoptotic features, including karyorrhexis, and reduced mitotic activity in the co-treated groups. CD44 immunostaining was strong in the SEC and DOX groups, moderate in the ATO- or LUT-treated groups, and weak in the co-treated groups. Combining ATO or LUT with DOX enhanced antitumor efficacy and attenuated molecular determinants associated with chemoresistance in SEC. Notably, the ATO+ LUT combination without DOX demonstrated the greatest antitumor efficacy compared to DOX-containing regimens, highlighting its potential as a multi-target, non-cytotoxic therapeutic strategy.

Cell senescence has been widely demonstrated to limit the osteogenic and odontogenic potential of human dental pulp stem cells (DPSCs). This study aimed to elucidate the regulatory mechanism by which E2F transcription factor 2 (E2F2) influences senescence and osteogenic differentiation in young DPSCs (yDPSCs). yDPSCs and old DPSCs (oDPSCs) were isolated and characterized by flow cytometry. The endogenous expression levels of E2F2, methyltransferase-like 3 (METTL3), and senescence- or osteogenesis-related markers were determined by qRT-PCR and Western blot. Senescence-associated β-galactosidase (SA-β-gal) staining was performed to assess cell senescence, while Alizarin Red S (ARS) and alkaline phosphatase (ALP) staining were used to evaluate the osteogenic differentiation capacity of yDPSCs. Dual-luciferase reporter and chromatin immunoprecipitation assays were conducted to confirm molecular interactions. Both yDPSCs and oDPSCs were positive for CD73 and CD105 and negative for HLA-DR. Compared with oDPSCs, yDPSCs exhibited stronger osteogenic differentiation potential and higher E2F2 expression. Knockdown of E2F2 increased the proportion of SA-β-gal-positive cells and upregulated the senescence markers p21 and p16, while it suppressed mineralization, ALP activity, and the expression of osteogenesis-associated markers (BMP2, OCN, OPN, Runx2, Osterix) in yDPSCs. Mechanistically, METTL3 was identified as a transcriptional target of E2F2. Overexpression of METTL3 reversed the inhibitory effects of E2F2 knockdown on odontogenic/osteogenic differentiation and the promoting effects on cell senescence in yDPSCs. E2F2 plays a critical role in suppressing DPSC senescence and promoting their osteogenic differentiation.

Hematopoietic stem cells (HSCs) self-renew and differentiate into all blood cells maintaining the hematopoietic system. Age-related HSC dysfunction impacts all of hematopoiesis, with DNA methylation alterations in aged HSCs contributing to altered function. Growth Arrest and DNA Damage-inducible proteins (Gadd45a, Gadd45b, and Gadd45g) are expressed in HSC activation, and Gadd45b has been reported to induce DNA demethylation. Thus, we explored the relationship between Gadd45b, DNA methylation and age-related HSC changes. WGBS on HSCs from GADD45B knockout mice demonstrated young knockout HSCs have increased DNA methylation, with both unique and overlapping methylation changes compared to aged wild-type HSCs without reflecting aging transcriptional changes. Peripheral blood and bone marrow analysis, competitive transplants, and single-cell culture analyses showed no significant loss of functional potential in the aberrantly methylated GADD45B knockout HSCs. We concluded these altered methylation sites don't alter HSC potential. We generated a searchable HSC DNA methylation database incorporating available datasets and present a truncated list of methylation sites associated with changes in HSC function for prioritization to target for resetting the age-associated loss of HSC potential.

In vitro reconstruction of gametogenesis is an important research direction in germ cell biology and assisted reproduction. While substantial progress has been made in the in vitro oogenesis of mammalian germ cells, analogous approaches in avian species remain unexplored. In this study, we investigated the capacity of chicken primordial germ cells (PGCs) and oogonial stem cells (OSCs) to differentiate into oocyte-like cells in vitro. Under meiotic induction with retinoic acid and vitamin C, both PGCs and OSCs initiated meiosis but arrested at the prophase I stage. Subsequent exposure to follicle-stimulating hormone, human chorionic gonadotropin, and progesterone facilitated the resumption of meiosis, leading to the formation of secondary oocytes. Next, parthenogenetic activation experiments showed that these germ cell-derived secondary oocytes were capable of undergoing cleavage and developing into blastomeres. Furthermore, in the aged and stopped-laying ovaries, OSCs retained the proliferation ability but lost their differentiation potential to increase the risk of germ cell tumor. The current in vitro oogenesis system enabled evaluation of healthy and compromised oocytes in avian species. This study not only provides direct evidence for the in vitro recapitulation of oogenesis in birds but also offers new avenues for preserving female germ cells and mass production of chicken oocytes.

Recent advances in stem cell biology and tissue engineering have profoundly renewed the field of liver organoids. More mature and complex, they now incorporate functional canalicular networks, metabolic gradients, and co-cultures including cholangiocytes, stellate cells, and macrophages. Advances in vascularization, bioprinting, and microfluidics are improving their stability and functionality, thereby enhancing their usefulness for disease modeling, pharmacology, personalized medicine, and future therapeutic applications.

High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) remains the standard of care for fit patients with newly diagnosed multiple myeloma (MM). The increasing use of CD38 antibody-based quadruplet induction regimens such as daratumumab-VTd (Dara-VTd) has raised concerns regarding impaired stem cell mobilization.

The APOE gene, which encodes Apolipoprotein E (ApoE), is the strongest genetic risk locus for Alzheimer's disease (AD). A substantial fraction of AD risk genes converges on pathways controlling lipid metabolism and immune regulation, in which microglia serve as a central integrative hub in the brain. Although microglial phenotypes linked to different APOE genotypes have been extensively characterised, the fundamental question of how ApoE shapes the core functions of human microglia remains unresolved. Here, we generated APOE knockout (KO) microglia from AD patient-derived induced pluripotent stem cells (iPSCs) and characterised their cellular and molecular phenotypes. Ablation of APOE resulted in marked lipid droplet accumulation and increased NLRP3 inflammasome activation. Transcriptomic analysis further revealed downregulation of cell cycle-related pathways, accompanied by enrichment of an oxidative stress-associated pathway. Consistent with these transcriptomic signatures, APOE KO microglia exhibited elevated intracellular reactive oxygen species (ROS) levels and a marked reduction in proliferative capacity. Given the importance of microglial proliferation for maintaining immune homeostasis in the brain, our findings highlight ApoE as being an important regulator of this process, with potential consequences for the pathogenesis of neurodegenerative disorders.

Tetraploidy occurs infrequently in mammals but remains widespread in amphibians. Blastocyst complementation using xenogeneic transplantation of tetraploid embryonic stem cells (4 N-ESCs) represents a promising approach to mitigate organ shortages, yet robust generation of fully reconstituted organs in mammalian hosts remains elusive. In this study, CRISPR/Cas9, the Cre-LoxP system, and blastocyst complementation were combined to generate tetraploid mouse liver, heart, and pancreatic tissues. 4 N-ESCs (tdTomato-labeled) were established and shown to maintain stable pluripotency and tetraploidy, as confirmed by karyotyping and immunofluorescence analyses. Subsequently, these cells were microinjected into Hhex- and Pdx1-deficient blastocysts and Nkx2.5 lineage-ablated blastocysts, which were engineered to lack relevant organ-forming lineages. Tetraploid pups exhibited significantly reduced body mass and organ mass (liver and heart) relative to diploid controls ( P<0.05). Fluorescence-activated cell sorting demonstrated a significant 4 N-ESC (tdTomato-labeled) contribution within tetraploid organs (4N population) at E18.5, with tdTomato-positive fractions reaching 84.3% of hepatic cells, 67.8% of cardiac cells, and 73.4% of pancreatic cells. Single-cell transcriptome sequencing further revealed that tetraploidy markedly altered developmental trajectories and differentiation programs in liver and heart tissues, and 4 N-ESCs showed preferential integration into tetraploid liver and heart with a substantial contribution to pancreatic regeneration. Collectively, these findings support the feasibility of 4 N-ESC-based blastocyst complementation for human organ regeneration and establish a framework for developing strategies to alleviate organ shortages in clinical settings.

Neuroinflammation is involved in various neurodegenerative diseases, with glial cells playing crucial roles. It is known that neuroinflammation is initiated by microglia, which interact with astrocytes and neurons. However, the detailed molecular mechanisms underlying intercellular interactions during neuroinflammation are not fully understood. In this study, we developed a tri-culture system of neurons, astrocytes, and microglia derived from human induced pluripotent stem cells (iPSCs) to evaluate their relationships in neuroinflammation. Microglia cocultured with the astrocytes and neurons exhibited a morphology with branched processes compared to the monoculture system, suggesting a homeostatic state. By applying lipopolysaccharide (LPS) stimulation to induce inflammation, the microglial morphology shifted to an amoeboid shape, accompanied by an increase in the expression of pro-inflammatory cytokines. Additionally, nuclear translocation of NF-κB revealed that LPS specifically activates microglia through the TLR4 receptor, which subsequently releases TNF-α, leading to the activation of astrocytes. Furthermore, activated astrocytes were shown to enhance neuronal excitability. Using the tri-culture system, we elucidated a part of the cascade involving microglia, astrocytes, and neurons during neuroinflammation and demonstrated the amplification of inflammatory signals through cell communication. This culture system will be valuable for conducting detailed investigations into the interactions between glia and neurons, advancing research on neurodegenerative diseases associated with neuroinflammation.

Recently, the use of hematopoietic stem cell transplantation (HSCT) for the treatment of hematological malignancies has increased because of its favorable therapeutic outcomes. The most common complication of HSCT is chronic graft-versus-host disease (GVHD). GVHD is an immune response caused by donor-derived cells that recognize the recipient as a foreign body (non-self) and attack the recipient's cells. GVHD manifests as lichen planus-like mucosal lesions in the oral cavity, and there have been reports of its malignant transformation to oral squamous cell carcinoma (OSCC). Chronic GVHD-associated OSCC may exhibit more aggressive behavior and a poorer prognosis than those of OSCC in patients who did not undergo HSCT. Here, we report three cases of GVHD after HSCT that transformed into OSCC during follow-up, in which early treatment achieved favorable outcomes. It is important to explain the risk of malignant transformation in patients with chronic GVHD and oral mucosal lesions and to monitor their progression.

Mesenchymal stem cells (MSCs) hold significant promise for bone tissue engineering, with adult bone marrow-derived MSCs (BMMSCs) widely used but limited by invasive harvesting and senescence-related osteogenic decline. Fetal-origin placental MSCs (PMSCs) offer a non-invasive and highly proliferative alternative, though their osteogenic capacity remains inconsistently reported. Because mitochondrial activity is closely linked to osteogenesis, and culture conditions strongly influence MSC function, we investigated how three-dimensional (3D) culture differentially modulates PMSCs and BMMSCs. Transcriptomic analyses revealed significant enrichment of cell adhesion and mitochondria-related pathways in PMSCs vs. BMMSCs; functional validation demonstrated larger viable spheroid formation and further augmentation of higher baseline mitochondrial activity in 3D-cultured PMSCs, along with upregulation of key osteogenic markers RUNX2 and osteoprotegerin. Surprisingly, even short-term 3D-priming led to elevated mitochondrial and osteogenic capacity in PMSCs but not BMMSCs. Single-cell RNA sequencing of PMSCs revealed that 3D culture promotes homogeneity, enriching for high mitochondrial- and osteogenic-expressing cell clusters. Functionally, inhibition of mitochondrial function suppressed osteogenic differentiation in 3D-primed PMSCs. Our findings reveal more robust baseline PMSC mitochondrial activity which can be further enhanced by even short-term, 1 day of 3D culture to significantly improve osteogenic commitment, demonstrating a practical strategy to improve MSC-based therapies for bone regeneration.

Intervertebral disc degeneration (IVDD) is characterized by the senescence and apoptosis of nucleus pulposus cells (NPCs), metabolic imbalance of the extracellular matrix (ECM), and local chronic inflammation, presenting a long-standing challenge in clinical treatment. Recent studies have confirmed that transplanted stem cells are prone to apoptosis in vivo, and the apoptotic extracellular vesicles (ApoEVs) they produce are key mediators of tissue repair. Given that both the physiological state of mesenchymal stem cells (MSCs) and the IVDD microenvironment exhibit hypoxic and inflammatory features, this study investigated the therapeutic effect and mechanism of MSCs-derived apoptotic bodies (I-ApoEVs) pretreated with a hypoxic-inflammatory composite microenvironment on IVDD. The results showed that compared with ApoEVs under conventional hypoxic conditions, I-ApoEVs more significantly inhibited NPCs senescence and promoted ECM synthesis. More importantly, they could target and regulate the STAT6 signaling pathway, induce macrophages to polarize towards the M2 anti-inflammatory phenotype, thereby remodeling the local inflammatory microenvironment of the intervertebral disc and alleviating inflammation-mediated degenerative damage. In conclusion, pretreatment with a hypoxic-inflammatory composite microenvironment enhances the therapeutic function of ApoEVs by regulating macrophage polarization, providing a novel and highly translatable therapeutic strategy for IVDD.