Bone's capacity for self-repair is limited when large defects arise from trauma or infection. Traditional grafting methods like autografts and allografts often face challenges like immune rejection and limited availability. Traditional scaffold manufacturing techniques for bone tissue engineering frequently lack precise control over the constructs' material composition and pore architecture. Recently, 3D printing technology, particularly with interpenetrating polymer networks (IPNs), has successfully addressed these limitations, improving biocompatibility, strength, and degradation. Our study investigated gelatin methacryloyl (GelMA)/Alginate IPNs laden with beta tri-calcium phosphate (β-TCP) particles in a 3D-printed format to optimize cell proliferation and tissue regeneration conditions. Rheology studies showed shear-thinning viscosity and fast recovery (~90%) to primary viscosity after stress removal, confirming the inks' suitability for extrusion-based printing. Both inks demonstrated high resolution and acceptable printability (0.9-1). Incorporating β-TCP increased the compressive modulus (0.09±0.01 MPa for the control group vs. 0.15±0.01 MPa for 15% (w/v) β-TCP, ***p < 0.001) and swelling ratio, decreasing biodegradation over 35 days. Cell assays showed enhanced cell proliferation over 7 days, with no significant differences between groups. Compared to basal and osteogenic media controls, higher mineralization and osteogenic gene expression were observed in 15% β-TCP-laden 3D-printed constructs on days 14 and 21. Histological analysis in vivo showed no signs of inflammation after three weeks, suggesting favorable tissue compatibility. Furthermore, calcium carbonate deposits were identified, evidencing the successful differentiation of mesenchymal stem cells into cells capable of producing a mineralized matrix. This study demonstrated that the (GelMA)/Alginate IPN containing β-TCP could be a successful biomaterial ink with promising bioactive properties for bone tissue engineering.

Rheumatic disease is a chronic inflammatory disease that imposes significant societal and economic burdens. Accumulating evidence has demonstrated that cellular senescence plays an important role in inflammation-induced rheumatic diseases. Due to the lack of effective therapies, there is an urgent need for a deeper understanding of the etiopathogenesis of rheumatic diseases. In this review, we systematically summarized the role of cellular senescence in rheumatic diseases. We first focused on the mechanisms and hallmarks of cellular senescence, and then summarized evidence that can induce or aggravate cellular senescence, as well as related signaling pathways. Next, we discussed the mechanisms of interaction between cellular senescence and rheumatic diseases. Additionally, we focused on and elucidated the mechanisms and impacts of chondrocyte senescence and mesenchymal stem cell (MSC) senescence in osteoarthritis (OA) and systemic lupus erythematosus (SLE), respectively. Finally, we highlighted the potential of therapies targeting senescent cells in rheumatic diseases as a strategy, especially the multi-target effect of traditional Chinese medicine.

Since its advent about ten years ago, the CRISPR-Cas9 system has been frequently used in biomedical applications. It has advanced various fields, and CRISPR-Cas9-based therapeutics have shown promising results in the treatment of specific hematological diseases. Furthermore, CRISPR gene editing technologies have revolutionized cancer research by enabling a broad range of genetic perturbations, including genetic knockouts and precise single nucleotide changes. This perspective focuses on the state-of-the-art methodology of CRISPR knock-ins to engineer immune cells. Since this technique relies on homology-directed repair (HDR) of double-strand breaks (DSBs) induced by the Cas9 enzyme, it can be used to introduce specific mutations into the target genome. Therefore, this methodology offers a valuable opportunity to functionally study specific mutations and to uncover their impacts not only on overall cell functions but also on the mechanisms behind cancer-related alterations in common signaling pathways. This article highlights CRISPR knock-in strategies, protocols, and applications in cancer and immune research, with a focus on diffuse large B cell lymphoma.

Circulating tumor cells (CTCs) are cancer cells that are detached from the primary and metastatic tumor site and invade the bloodstream. Most importantly, CTCs are the key players in the development of metastasis. As one of the main components of liquid biopsy, they may significantly contribute to improvements in early cancer diagnosis, monitoring response to therapy, and predicting recurrence of the disease. Although identifying and analyzing CTCs offers the potential for a real-time liquid biopsy, their detection is associated with a number of challenges, which mainly stem from three sources: complexity of the CTCs, complexity of the media (blood), and performance of the detection assays. Particularly, low concentration of the CTCs and the presence of a vast population of hematopoietic cells in the blood make their detection technically complex. The heterogeneity of the target cells and not enough sensitivity of the measuring platforms are also among major technical challenges in CTC detection. Therefore, this review aims to give an update on various methods developed for CTC isolation, including chip-based assays and biosensors. The work will elucidate various challenges associated with the isolation and detection of CTCs and showcase the studies that aimed to tackle them. A number of available commercial platforms for CTC detection and hurdles associated with their widespread applications in clinical settings will also be discussed.

Each year, 1 in every 700 babies is born with an orofacial cleft in the USA. Despite a well-established protocol for early cleft repair, the alveolar cleft persists during craniofacial growth. Current surgical treatments with bone grafts for alveolar cleft often provide inadequate nasal base support and insufficient alveolar bone volume for permanent tooth eruption. Here, we developed 3-dimensionally printed polycaprolactone scaffolds with controlled delivery of icariin (ICA) to facilitate bone reconstruction. After establishing a reliable fabrication process, we determined the optimal loading dose and release kinetics of ICA for induced osteogenic differentiation of bone marrow mesenchymal stem/progenitor cells and mineralized tissue formation in vitro. Then, the ICA-releasing polycaprolactone scaffolds with the preoptimized dose were implanted into rats with full-thickness maxillary defects. Up to 8 weeks, micro-computed tomography analyses demonstrated significantly accelerated bone healing and defect closure with an ICA-releasing scaffold compared to scaffold alone and defect controls. Histology consistently confirmed the formation of dense woven bone with ICA-releasing scaffolds in contrast to unclosed gaps and soft tissue infiltration in controls. Our findings suggest the significant potential of ICA-releasing 3-dimensionally printed scaffolds to serve as a patient-focused and custom-built bone graft to improve the clinical outcome of alveolar cleft reconstruction.

Gastric stem cells (GSCs) and chief cells are vital for maintaining gastric epithelial homeostasis. However, under pathological conditions, these cells undergo significant functional changes, contributing to the progression of precancerous lesions of gastric cancer (PLGC). Dysregulation of key signaling pathways such as WNT, NF-κB, and YAP leads to aberrant cellular behaviors, which are implicated in the early stages of gastric carcinogenesis. This study aimed to elucidate the roles of GSCs and chief cells in maintaining gastric epithelial integrity, their contributions to the development of precancerous lesions, and the molecular mechanisms that regulate their behavior during disease progression.

Cancer stem cells (CSCs), harboring stem cell-like properties involving self-renewal and aberrant differentiation potential, have been known to be one of the determining factors that contribute to therapeutic resistance and tumor recurrence. However, much remains to be understood about the reprogramming network leading to the generation of CSCs driven by chemotherapy. In this study, guided by bioinformatics study, we uncover and provide deeper insight into the CSC enrichment mechanism driven by cisplatin (CDDP) treatment. We discover that CDDP can repopulate the level of CSC by activating AKT1 oncogenic pathway that is further enhanced by COX-2 inflammatory signaling. Simultaneously blocking these two pathways can synergistically restrain the number of CSCs. Under the guidance of a series of advanced hierarchical computational modeling, including molecular docking, molecular dynamics (MD) simulation and binding free energy analysis, MK-2206 is selected as the AKT1 inhibitor to achieve optimal codelivery of CDDP, MK-2206 and 5-ASA (COX-2 inhibitor) through the use of 5-ASA-derivatized dual functional immunostimulatory nanocarrier (PASA). This triple combination (PASA/CDDP/MK-2206) significantly reduces tumor burden in both orthotopic and metastatic lung cancer models. Mechanistic studies show that this improved therapeutic activity is due to elimination of CSCs and reversal of the immunosuppressive tumor microenvironment. Our study suggests that PASA/CDDP/MK-2206 may represent a simple and effective lung cancer therapy via reversing CSCs-associated chemoresistance.

Pathological scars, including hypertrophic, keloid, and atrophic scars, remain challenging to treat, with current therapies offering limited success. Adipose-derived stem cell (ADSC) products, classified into cell-based therapies (stromal vascular fraction [SVF], ADSCs) and cell-free therapies (adipose tissue extract [ATE], secretomes, exosomes, extracellular vesicles), have emerged as potential treatments.

Estimating the prognosis of spontaneous intracerebral hemorrhage (ICH) is of great importance. It has not been conclusively clarified whether sarcopenia is predictive for the functional outcome in ICH. Determining the temporalis muscle thickness (TMT) may be helpful for estimating sarcopenia. An association of TMT with outcome (mRS) has been shown in cerebellar ischemia and traumatic brain injury.

Cancer stem cells (CSCs) drive tumor initiation, relapse, and metastasis. Our research team developed polycaprolactone electrospun (PCL-ES) scaffolds for enriching lung CSCs (LCSCs) since monolayer culture do not allow the study of this malignant population. The upregulation of fatty acid synthase (FASN) correlates with resistance to tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR), and its inhibition induces cytotoxicity in EGFR-mutated (EGFRm) non-small cell lung cancer (NSCLC) cells. Therefore, this study aims to elucidate the role of FASN and related signaling pathways in LCSCs cultured in PCL-ES scaffolds and to evaluate the effectiveness of FASN inhibitor G28, a synthetic derivative of (-)-epigallocatechin-3-gallate (EGCG), against this population.

Myelodysplastic syndrome is a clonal disorder of hematopoietic stem cells leading to dysplasia and ineffective hematopoiesis. The typical presentation is asymptomatic with incidental lab findings consistent with varying degrees of pancytopenia. This syndrome is commonly diagnosed in older individuals and tends to result from chemical and/or radiation exposures; however, this patient presents as a unique manifestation of myelodysplastic syndrome in a six-month-old male. For curative management, the patient had an initial haploidentical hematopoietic stem cell transplant, followed by another, due to early graft rejection. In the following years leading to the present time, the patient has had complications due to a persistent immunodeficient state. He has been evaluated and managed for seizures, anemia, chemotherapy-induced cardiomyopathy, hypogammaglobinemia, failure to thrive, recurrent infections, graft versus host disease, malnutrition, and hypertransaminasemia with hepatosplenomegaly. He has had resolution of his cardiomyopathy and seizures but continues to manage his persistent immunodeficiencies, hepatomegaly, and slow weight gain. It is hard to discern if the multitude of dysfunctions arises from pharmacologically induced immunodeficiency, from parental and environmental neglect, if the manifestations compounded one another, or if the above factors combined to create the unique constellation of clinical manifestations. This patient provides insight into the complexities associated with understanding unique manifestations in dysplastic and oncological conditions, as well as managing complications arising from treatment. This case raises awareness of medical complications in the context of parental neglect and the importance of managing them both. The authors received consent from the patient's guardian to use their data for this report.

ASCL1 is a potent proneural factor with paradoxical functions during development, promoting both progenitor pool expansion and neuronal differentiation. How a single factor executes and switches between these potentially opposing functions remain to be understood. Using neuroblastoma cells as a model system, we show that ASCL1 exhibits cell cycle phase-dependent chromatin binding patterns. In cycling cells, S/G2/M phase-enriched binding occurs at promoters of transcribed pro-mitotic genes, while G1 phase-enriched binding of ASCL1 is associated with the priming of pro-neuronal enhancer loci. Prolonged G1 arrest is further required to activate these ASCL1-bound and primed neuronal enhancers to drive neuronal differentiation. Thus, we reveal that the same transcription factor can control distinct transcriptional programmes at different cell cycle stages, and demonstrate how lengthening of G1 allows engagement of a differentiation programme by turning unproductive factor binding into productive interactions.

In the early zebrafish neural plate, proneural cluster domains are defined by surrounding neural progenitor pools (NPPs), generating primary neurogenesis patterns. In each NPP, several Notch-independent Hes/her-type genes are expressed in distinct manners. Previous knockdown (KD) experiments induced ectopic neurogenesis in NPPs where only the targeted her genes were expressed, with other her genes absent, suggesting cooperative functions of Notch-independent her genes. In this study, to overcome the inherent limitations in KD approaches, we knocked out (KO) three her genes, her3, her5, and her11, using genome editing techniques. The resulting mutants exhibited ectopic neurogenesis patterns at the end of gastrulation, similar to those observed in KD experiments. KOs of her5 and her11 induced ectopic neurogenesis around the midbrain-hindbrain boundary, whereas her3 KO led to ectopic neurogenesis in rhombomere 1/2 and r4. In these cases, the expression of other Notch-independent her genes was not affected, except for her11, whose expression depended on her5. Analyses of compound mutants revealed that their phenotypes were essentially the sum of those of individual her mutants, indicating independent suppression of neurogenesis by Notch-independent her genes. In conclusion, different Notch-independent her genes collectively define the characteristic pattern of primary neurogenesis in the neural plate.

Mitochondrial translation release factor in rescue (MTRFR) catalyzes a termination step in protein synthesis, facilitating release of the nascent chain from mitoribosomes. Pathogenic variants in MTRFR cause MTRFR deficiency and are loss-of-function variants. Here, we tested gene replacement as a possible therapeutic strategy. A truncating mutation (K155*) was generated in mice; however, homozygotes die embryonically whereas mice heterozygous for this K155* allele are normal. We also generated transgenic strains expressing either wild-type human MTRFR or a partially functional MTRFR. Despite dose-dependent phenotypes from overexpression in vitro, neither transgene caused adverse effects in vivo. In K155* homozygous mice, the wild-type MTRFR transgene completely rescued the phenotype with only one copy present, whereas the mutant transgene rescued less efficiently. Detailed evaluation of mice rescued with the wild-type MTRFR transgene revealed no abnormalities. In human induced pluripotent stem cell (hiPSC)-derived knockdown neurons, mitochondrial phenotypes were corrected by AAV9-mediated delivery of MTRFR. Thus, we find no toxicity from truncated gene products or overexpression of MTRFR in vivo, and expression of MTRFR corrects phenotypes in both mouse and hiPSC models.

Mesenchymal stem cell-derived exosome (MSC-exo) has garnered increasing attention because of its therapeutic potential for acute kidney injury (AKI). The aim of this study is to investigate the regulatory mechanism of MSC-exo in ischaemic AKI.

One of the most prevalent cancers in the world is colorectal carcinoma (CRC). Aggressive cancer forms and a poor prognosis are linked to cancer stem cell (CSC) markers. The study aimed to determine whether the co-expression of the CSC markers CD133 and CD44 could predict an increased risk of metastasis in colorectal cancer.

Non-clinical pharmacological safety studies are conducted using cells and animals to ensure the safety of pharmaceuticals in humans. Following these studies, drug candidates are administered to humans during clinical trials. Safety must be sufficiently confirmed in non-clinical studies to ensure that test participants suffer no adverse health effects. However, due to species differences, low ability to extrapolate from in vitro to in vivo evaluation methods, and other problems, health hazards may unfortunately still occur. Therefore, sophisticated in vitro evaluation systems using human cells are actively being pursued. The main challenge remains the lack of a reliable methodology for extrapolating in vitro results to in vivo settings. We have attempted to extract parameters that can be predictably translated from in vitro [contractile evaluation in three-dimensional (3D) heart tissue] to in vivo (guinea pig echocardiography) conditions, using cardiac contractile dysfunction induced by anticancer drugs as an example. In this review, we introduce the in vitro methods developed to date to evaluate this cardiac contractile dysfunction, analyze the factors enabling highly accurate prediction of torsades de pointes in humans based on past proarrhythmic risk prediction methods using human induced pluripotent stem cell-derived cardiomyocytes, and apply them to evaluate cardiac contractile dysfunction caused by anticancer drugs using three-dimensional heart tissue. We also introduce the proposed strategy for this evaluation method in this section.

To increase success rates of clinical studies, preclinical evaluation systems have been expected to improve human predictability. In addition, future preclinical studies need to become more sophisticated and efficient on the back ground of the adoption of FDA Modernization Act 2.0 and the 3R principle promotion of animal tests. In this review, we will discuss about the efficiency of in vivo imaging in preclinical studies taking 'an attempt to establishment of in vitro in vivo extraporation (IVIVE) model for seizure risk assessment using microphysiological system (MPS) and magnetic resonance imaging (MRI)', and 'an attempt to predict drug delivery to the alveoli' as examples. In the seizure risk assessment of new drugs so far, primary cultures of rodent neurons and in vivo behavioral observation have been mainly used, however, since the human induced pluripotent stem cell (iPSC) technology was reported, the need for IVIVE model is more and more increasing to improve human predictability. As an MPS, we here introduce microelectrode array (MEA) system recording of primary culture of rodent neurons, while as in vivo experiments, we here introduce the measurement of cerebrospinal fluid (CSF) concentrations and MRI imaging of forebrains of the rats i.p. injected with seizurogenic compounds. In case of inhalation drugs, it has been difficult to confirm whether or not the drugs surely reach alveoli. We visualized two-dimensional spatial localization of inhaled ciclesonide (CIC) in rat lungs after administration of a single dose of a CIC aerosol using by desorption electrospray ionization-time of flight mass spectrometry imaging (DESI-MSI).

To investigate the efficacy of oral fenofibrate in the amelioration of ocular surface inflammation in diabetes mellitus (DM).