Mass Spectrometry Imaging for the Characterization of C═C Localization in Unsaturated Lipid Isomers at the Single-Cell Level.

Unsaturated lipids with carbon-carbon double bonds (C═C) have been implicated in the pathogenesis of various diseases. While mass spectrometry imaging (MSI) has been employed to map the distribution of lipid isomers in tissue sections, the identification of lipid C═C positional isomers at the single-cell level using MSI poses a significant challenge. In this study, we developed a novel approach utilizing ToF-SIMS in conjunction with the Paternò-Büchi (P-B) photochemical reaction to characterize the C═C localization in unsaturated lipid isomers at the single-cell level. The P-B reaction was employed to produce adduct products, which were subsequently subjected to collision-induced dissociation by the primary ion beam of ToF-SIMS to generate characteristic ion pairs indicative of the presence of C═C bonds. Utilizing this approach, lipid isomers in brain and skeletal tissues from mice, as well as different cell lines, were visualized at single-cell resolution. Furthermore, distinct variations in the composition of FA 18:1 isomers across different microregions and cell types were revealed. Our P-B ToF-SIMS approach enables the accurate identification and characterization of complex lipid structures with remarkable spatial resolution and can be helpful in understanding the physiological role of these C═C positional isomers.

Allogeneic CAR-T cells with of HLA-A/B and TRAC disruption exhibit promising antitumor capacity against B cell malignancies.

BACKGROUND: Although chimeric antigen receptor T (CAR-T) cells have been proven to be an effective way of treating B cell malignancies, a lot of patients could not benefit from it because of failure in CAR-T cell manufacturing, disease progression, and unaffordable price. The study aimed to explore universal CAR-T cell products to extend the clinical accessibility. METHODS: The antitumor activity of CRISPR/Cas9-edited allogeneic anti-CD19 CAR-T (CAR-T19) cells was assessed in vitro, in animal models, and in patients with relapsed/refractory (R/R) acute B cell lymphoblastic leukemia (B-ALL) or diffuse large B cell lymphoma. RESULTS: B2M-/TRAC- universal CAR-T19 (U-CAR-T19) cells exhibited powerful anti-leukemia abilities both in vitro and in animal models, as did primary CD19+ leukemia cells from leukemia patients. However, expansion, antitumor efficacy, or graft-versus-host-disease (GvHD) was not observed in six patients with R/R B cell malignancies after U-CAR-T19 cell infusion. Accordingly, significant activation of natural killer (NK) cells by U-CAR-T19 cells was proven both clinically and in vitro. HLA-A-/B-/TRAC- novel CAR-T19 (nU-CAR-T19) cells were constructed with similar tumoricidal capacity but resistance to NK cells in vitro. Surprisingly, robust expansion of nU-CAR-T19 cells, along with rapid eradication of CD19+ abnormal B cells, was observed in the peripheral blood and bone marrow of another three patients with R/R B-ALL. The patients achieved complete remission with no detectable minimal residual disease 14 days after the infusion of nU-CAR-T19 cells. Two of the three patients had grade 2 cytokine release syndrome, which were managed using an IL-6 receptor blocker. Most importantly, GvHD was not observed in any patient, suggesting the safety of TRAC-disrupted CAR-T cells generated using the CRISPR/Cas9 method for clinical application. CONCLUSIONS: The nU-CAR-T19 cells showed a strong response in R/R B-ALL. nU-CAR-T19 cells have the potential to be a promising new approach for treating R/R B cell malignancies.

Irradiated microparticles suppress prostate cancer by tumor microenvironment reprogramming and ferroptosis.

Immunogenic cell death (ICD) plays a crucial role in triggering the antitumor immune response in the tumor microenvironment (TME). Recently, considerable attention has been dedicated to ferroptosis, a type of ICD that is induced by intracellular iron and has been demonstrated to change the immune desert status of the TME. However, among cancers that are characterized by an immune desert, such as prostate cancer, strategies for inducing high levels of ferroptosis remain limited. Radiated tumor cell-derived microparticles (RMPs) are radiotherapy mimetics that have been shown to activate the cGAS-STING pathway, induce tumor cell ferroptosis, and inhibit M2 macrophage polarization. RMPs can also act as carriers of agents with biocompatibility. In the present study, we designed a therapeutic system wherein the ferroptosis inducer RSL-3 was loaded into RMPs, which were tested in in vitro and in vivo prostate carcinoma models established using RM-1 cells. The apoptosis inducer CT20 peptide (CT20p) was also added to the RMPs to aggravate ferroptosis. Our results showed that RSL-3- and CT20p-loaded RMPs (RC@RMPs) led to ferroptosis and apoptosis of RM-1 cells. Moreover, CT20p had a synergistic effect on ferroptosis by promoting reactive oxygen species (ROS) production, lipid hydroperoxide production, and mitochondrial instability. RC@RMPs elevated dendritic cell (DC) expression of MHCII, CD80, and CD86 and facilitated M1 macrophage polarization. In a subcutaneously transplanted RM-1 tumor model in mice, RC@RMPs inhibited tumor growth and prolonged survival time via DC activation, macrophage reprogramming, enhancement of CD8+ T cell infiltration, and proinflammatory cytokine production in the tumor. Moreover, combination treatment with anti-PD-1 improved RM-1 tumor inhibition. This study provides a strategy for the synergistic enhancement of ferroptosis for prostate cancer immunotherapies.

Acute myocardial infarction due to coronary embolism caused by a metastatic mass from lung cancer.

BACKGROUND: Acute arterial embolism due to tumor embolus is a rare complication in cancer patients, even rarer is lung tumor embolization leading to acute myocardial infarction. We report a patient who had a diagnosis of acute myocardial infarction(AMI)which was brought on by a coronary artery embolism by a metastatic lung cancer tumor. Clinicians need to be aware that tumor embolism can result in AMI. CASE PRESENTATION: An 80-yeal-old male patient presented with persistent chest pain for 2 h and his electrocardiogram(ECG)showed anterior ST-segment elevation myocardial infarction. Instead of implanting a stent, thrombus aspiration was performed. Pathological examination of coronary artery thrombosis showed that a few sporadic atypical epithelial cells were scattered in the thrombus-like tissue. Combined with immune phenotype and clinical history, metastatic squamous cell carcinoma is more likely. CONCLUSIONS: We report a rare case of a patient who was diagnosed of AMI due to a coronary artery embolism by a metastatic mass from lung cancer. Since there is no evidence-based protocol available for the treatment of isolated coronary thrombosis, we used thrombus aspiration to treat thrombosis rather than implanting a stent.

Scutellarein protects against cardiac hypertrophy via suppressing TRAF2/NF-κB signaling pathway.

BACKGROUND: Scutellarein, a widely studied ingredient of scutellaria herbs, has higher bioavailability and solubility than that of scutellarin. Although the scutellarein had been reported to modulate numerous biological functions, its ability in suppressing cardiac hypertrophy remains unclear. Hence, the present study attempted to investigate whether scutellarein played critical roles in preventing phenylephrine (PE)-induced cardiac hypertrophy. METHODS AND RESULTS: Immunocytochemistry (ICC) was employed for evaluating the morphology of the treated cardiomyocytes. Real-time PCR and western blot were respectively applied to assess the mRNA levels and protein expression of the relevant molecules. Bioinformatics analyses were carried out to investigate the potential mechanisms by which scutellarein modulated the PE-induced cardiac hypertrophy. The results showed that Scutellarein treatment significantly inhibited PE-induced increase in H9c2 and AC16 cardiomyocyte size. Besides, scutellarein treatment also dramatically suppressed the expression of the cardiac hypertrophic markers: ANP, BNP and ß-MHC. Furthermore, the effects of scutellarein on attenuating the cardiac hypertrophy might be mediated by suppressing the activity of TRAF2/NF-κB signaling pathway. CONCLUSIONS: Collectively, our data indicated that scutellarein could protect against PE-induced cardiac hypertrophy via regulating TRAF2/NF-κB signaling pathway using in vitro experiments.

Phenotypic modulation of vascular smooth muscle cells in the corpus spongiosum surrounding the urethral plate in hypospadias.

Hypospadias is an abnormal ventral development of the penis caused by incomplete virilization of the male genital tubercle. This study investigated the phenotypic modulation of vascular smooth muscle cells (VSMCs) in the corpus spongiosum surrounding the urethral plate in hypospadias. The urethral corpus spongiosum tissue was collected for HE, Masson and α-SMA immunohistochemical staining. Spongiosum VSMCs were cultured and identified by α-SMA fluorescence. qRT-PCR and Western blotting and fluorescence were performed. The results showed that the vascular lumen of the corpus spongiosum around the urethral plate was larger and that the vascular smooth muscle layer was thicker in hypospadias. The expression of the contractile markers α-SMA and Calponin 1 in VSMCs was decreased, the expression of the synthetic marker OPN was increased, and the transcription of the phenotypic switching factors SRF and MYOCD was decreased. The expression of Ki67, PCNA and BAX was increased, and the expression of Bcl-2 was decreased. The phenotype of corpus spongiosum VSMCs in hypospadias changed from the contractional type to the synthetic type. This phenotypic modulation was associated with increased proliferation and apoptosis rates. SRF and MYOCD may be the main factors mediating the phenotypic modulation of urethral corpus spongiosum VSMCs.

Hierarchical Supramolecular Self-Assembly: Fabrication and Visualization of Multiblock Microstructures.

Due to the fast dynamics and re-equilibration of supramolecular self-assembly, bottom-up molecular strategies to fabricate well-defined and controllable multiblock structures are rare. Herein, we propose a new concept for fabrication of fluorescent multiblock microcolumns containing 1 to 7 blocks via hierarchical supramolecular self-assembly based on cucurbit[8]uril (CB[8]), NaBr and an AIEgen guest. Through the complexation between CB[8] and different numbers of AIEgen guests (2, 1, 0), the competitive displacement caused by the binding of the sodium cation to the CB[8] portal, and the reversible assembly of positively charged guests in salt solutions, one-pot hierarchical supramolecular self-assembly is realized. The molecular structure of each block is analyzed by single-crystal X-ray diffraction. The AIEgen enables the self-assembly of multiblocks to be visualized, understood, and regulated.

Identification of miRNA-Target Gene Pairs in the Parietal and Frontal Lobes of the Brain in Patients with Alzheimer's Disease Using Bioinformatic Analyses.

Alzheimer's disease (AD) is a growing health concern worldwide. MicroRNAs (miRNAs) have been extensively studied in many diseases, including AD. To identify differentially expressed miRNAs (DEmiRNAs) and genes specific to AD, we used bioinformatic analyses to investigate candidate miRNA-mRNA pairs involved in the pathogenesis of AD. We focused on differentially expressed genes (DEGs) that are targets of DEmiRNAs. The GEO2R tool and the HISAT2-DESeq2 software were used to identify DEmiRNAs and DEGs. Bioinformatic tools available online, such as TAM and the Database for Annotation, Visualization and Integrated Discovery (DAVID), were used to perform functional annotation and enrichment analysis. Targets of miRNAs were predicted using the miRTarBase. The Search Tool for the Retrieval of Interacting Genes (STRING) and Cytoscape, which are available online, were utilized to construct protein-protein interaction (PPI) networks and identify hub genes. Furthermore, transcription factors (TFs) encoded by the DEGs were predicted using the TransmiR database and TF-miRNA-mRNA networks were constructed. Finally, the expression profile of a hub gene in peripheral blood mononuclear cells was compared between healthy individuals and AD patients. We identified 26 correlated miRNA-mRNA pairs. In the parietal lobe, miRNA-mRNA pairs involved in protein folding were enriched, and in the frontal lobe, miRNA-mRNA pairs involved in synaptic transmission, abnormal protein degradation, and apoptosis were enriched. In addition, HSP90AB1 in peripheral blood mononuclear cells was found to be significantly downregulated in AD patients, and this was consistent with its expression profile in the parietal lobe of AD patients. Our results provide brain region-specific changes in miRNA-mRNA associations in AD patients, further our understanding of potential underlying molecular mechanisms of AD, and reveal promising diagnostic and therapeutic targets for AD.

ERO1L promotes NSCLC development by modulating cell cycle-related molecules.

Lung cancer is the leading cause of cancer-related death worldwide. Previous studies revealed that endoplasmic reticulum oxidoreductase 1 alpha (ERO1L) played critical roles in the malignant behaviors of several cancer types, but its role in non-small cell lung cancer (NSCLC) remained unclear. In this study, we identified 26 upregulated and 102 downregulated genes in NSCLC using bioinformatics analyses, and these genes were enriched in the biological processes of the cell cycle. ERO1L was remarkably upregulated in NSCLC and overexpression of ERO1L was associated with poor prognosis of NSCLC. ERO1L deficiency markedly suppressed NSCLC cell proliferation, colony formation, migration, and invasion. ERO1L depletion caused a dramatically decreased expression of cell cycle-related factors in NSCLC cells. Collectively, our data validated that ERO1L could function as a tumor promoter in NSCLC, indicating the potential of targeting ERO1L for the treatment of NSCLC.

Functionalized Acrylonitriles with Aggregation-Induced Emission: Structure Tuning by Simple Reaction-Condition Variation, Efficient Red Emission, and Two-Photon Bioimaging.

Acrylonitriles with aggregation-induced emission (AIE) characteristics have been found to show promising applications in two-photon biomedical imaging. Generally, elaborate synthetic efforts are required to achieve different acrylonitriles with distinct functionalities. In this work, we first reported the synthesis of two different group-functionalized AIE-active acrylonitriles (TPAT-AN-XF and 2TPAT-AN) obtained simply by mixing the same reactants at different temperatures using a facile and transition metal-free synthetic method. These two AIE luminogens (AIEgens) exhibit unique properties such as bright red emission in the solid state, large Stokes shift, and large two-photon absorption cross section. Water-soluble nanoparticles (NPs) of 2TPAT-AN were prepared by a nanoprecipitation method. In vitro imaging data show that 2TPAT-AN NPs can selectively stain lysosome in live cells. Besides one-photon imaging, remarkable two-photon imaging of live tumor tissues can be achieved with high resolution and deep tissue penetration. 2TPAT-AN NPs show high biocompatibility and are successfully utilized in in vivo long-term imaging of mouse tumors with a high signal-to-noise ratio. Thus, the present work is anticipated to shed light on the preparation of a library of AIE-active functionalized acrylonitriles with intriguing properties for biomedical applications.

Effects of alprostadil combined with calcium dobesilate in patients with diabetic peripheral neuropathy.

OBJECTIVE: To observe the clinical curative effects of alprostadil combined with calcium dobesilate in type 2 diabetes patients with peripheral neuropathy. METHODS: We randomly divided 120 type 2 diabetes patients with diabetic peripheral neuropathy into two groups. The treatment group was prescribed alprostadil (10 µg, once daily) and oral calcium dobesilate (0.5 g, 3 times daily), and the control group was prescribed alprostadil (10 µg, once daily) for a total treatment duration of 2 weeks. The Michigan Diabetic Neuropathy Score (MDNS) and the Michigan Neuropathy Screening Instrument (MNSI) were used to evaluate differences between the two groups before and after treatment. RESULTS: Following 2 weeks of treatment, the total effective rate in the treatment group was significantly better than that of the control group (p<0.05) and the MDNS and MNSI scores in the treatment group were significantly lower than those in the control group (p<0.05 or p<0.01). CONCLUSION: Combined alprostadil and calcium dobesilate treatment for type 2 diabetic peripheral neuropathy showed good clinical efficacy and an improved curative effect than single alprostadil treatment.

Drug-inducible synergistic gene silencing with multiple small hairpin RNA molecules for gene function study in animal model.

Gene targeting is a critical tool for construction of disease models. However, the application of traditional homologous recombination-mediated gene knockout technology is limited by the absence of rapid frequency-guaranteed targeting methods. Although conventional small hairpin RNA (shRNA)-mediated gene silencing offers an alternative for gene targeting, its application is frequently compromised by lower expression efficiency via RNA interference compared to gene knockout. Here we provide an efficient gene targeting strategy involving drug-inducible synergistic silencing with multiple shRNA molecules. On induction, the levels of the target proteins decreased to undetectable levels in all the tested stable transgenic mammalian cell lines, including HEK293 and embryonic stem cell-derived progenies carrying shRNA silencing cassettes. In a transgenic mouse model carrying a silencing cassette targeting the rhodopsin gene, short-time inducer treatment was sufficient to ablate the rhodopsin protein in the retina, resulting in similar retinal phenotypic changes as those observed in rhodopsin mutant mice. Therefore, on a broad basis, this inducible shRNA gene targeting strategy offers a true gene knockout alternative comparable to conventional RNA interference approaches.

Regulation of Protein Binding Capability of Surfaces via Host-Guest Interactions: Effects of Localized and Average Ligand Density.

The protein binding capability of biomaterial surfaces can significantly affect subsequent biological responses, and appropriate ligand presentation is often required to guarantee the best functions. Herein, a new facile method for regulating this capability by varying the localized and average ligand density is presented. Binding between lysine and plasminogen relevant to a fibrinolysis system was chosen as a model. We integrated different lysine-modified ß-cyclodextrin (ß-CD) derivatives onto bioinert copolymer brushes via host-guest interactions. The localized and average lysine density can be conveniently modulated by changing the lysine valency on ß-CD scaffolds and by diluting lysine-persubstituted ß-CD with pure ß-CD, respectively. Both the plasminogen adsorption and the plasminogen binding affinity were enhanced by lysine-persubstituted ß-CD compared with those of lysine-monosubstituted ß-CD, which is possibly due to the higher localized lysine density and the multivalent binding of plasminogen on lysine-persubstituted ß-CD surfaces. With a change in the ratio of lysine-persubstituted ß-CD to ß-CD, the average lysine density can be tuned, leading to the linear regulation of the adsorption of plasminogen on surfaces.

Emodin-mediated cross-linking enhancement for extracellular matrix homeostasis.

The extracellular matrix (ECM) is an essential element of mammalian organisms, and its cross-linking formation plays a vital role in ECM development and postnatal homeostasis. Defects in cross-link formation caused by aging, genetic, or environmental factors are known to cause numerous diseases in mammals. To augment the cross-linking formation of ECM, the present study established a ZsGreen reporter system controlled by the promoter of lysyl oxidase-like 1 gene (LOXL1), which serves as both a scaffold element and a cross-linking enzyme in the ECM. By using this system in a drug screen, we identified emodin as a strong enhancer of LOXL1 expression that promoted cross-linking formation of ECM in all the tested systems, including human fibroblast cells, cultured human skin tissues, and animals that received long-term emodin treatment. Collectively, the results suggest that emodin may serve as an effective drug or supplement for ECM homeostasis.

Development of molecularly imprinted electrochemical sensors based on Fe3O4@MWNT-COOH/CS nanocomposite layers for detecting traces of acephate and trichlorfon.

In this study, we developed a novel biomimetic electrochemical sensor sensitized with a Fe3O4@carboxyl-functionalized multiwalled carbon nanotube/chitosan nanocomposite layer using a molecularly imprinted film as a recognition element for the rapid detection of acephate and trichlorfon. The performance of the imprinted sensor was investigated using cyclic voltammetry and differential pulse voltammetry, and the results indicated that the sensor exhibited fast responses to both acephate and trichlorfon. The imprinted sensor had good linear current responses to acephate and trichlorfon concentrations in the ranges from 1.0 × 10(-4) to 1.0 × 10(-10) M and 1.0 × 10(-5) to 1.0 × 10(-11) M, respectively. Under optimal conditions, the imprinted sensor had low limits of detection (signal to noise ratio, S/N = 3) of 6.81 × 10(-11) M for acephate and 8.94 × 10(-12) M for trichlorfon. The developed method was successfully applied to detect acephate and trichlorfon spiked in fortified kidney bean and cucumber samples with good recoveries ranging from 85.7% to 94.9% and relative standard deviations of 3.46-5.18%.

The effect of gold nanoparticles on the proliferation and differentiation of murine osteoblast: a study of MC3T3-E1 cells in vitro.

The current study involves in identification and molecular levels characterization of optimal size and concentration of gold nanoparticles (AuNPs). Stable, gold nanoparticles were synthesized and characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS). The concentration and size dependent effects of the gold nanoparticles on proliferation of pre-osteoblast cells MC3T3-E1 was evaluated employing MTT cell proliferation assay. The results revealed that 30 nm diameter gold nanoparticles at a concentration of 10(-11) ppm were the most effective in promoting cell proliferation. Assay for alkaline phosphatase (ALP) activity and ALP staining were also used to confirm the effect of gold nanoparticles on osteoblast proliferation and differentiation. Moreover, reverse transcriptase polymerase chain reaction (RT-PCR) was used to measure the expression of the osteogenic genes Runx2, ALP, OCN and OPN as response gold nanoparticles. The data demonstrated that 30 nm gold nanoparticles at a concentration of 10(-11) ppm was the best combination of size and concentration to promote the proliferation and differentiation of osteoblasts, as indicated by an increase in the ALP activity and expression of the osteogenic genes Runx2, ALP, OCN and OPN. Collectively the results of this study suggest that gold nanoparticles can promote the proliferation and differentiation of osteoblasts and could be used effectively in treatments promoting bone regeneration.

Shen'ge Formula Protects Cardiac Function in Rats with Pressure Overload-Induced Heart Failure.

Background: In China, Shen'ge formula (SGF), a Traditional Chinese Medicine blend crafted from ginseng and gecko, holds a revered place in the treatment of cardiovascular diseases. However, despite its prevalent use, the precise cardioprotective mechanisms of SGF remain largely uncharted. This study aims to fill this gap by delving deeper into SGF's therapeutic potential and underlying action mechanism, thus giving its traditional use a solid scientific grounding. Methods: In this study, rats were subjected to abdominal aortic constriction (AAC) to generate pressure overload. Following AAC, we administered SGF and bisoprolol intragastrically at specified doses for two distinct durations: 8 and 24 weeks. The cardiac function post-treatment was thoroughly analyzed using echocardiography and histological examinations, offering insights into SGF's influence on vital cardiovascular metrics, and signaling pathways central to cardiac health. Results: SGF exhibited promising results, significantly enhanced cardiac functions over both 8 and 24-week periods, evidenced by improved ejection fraction and fractional shortening while moderating left ventricular parameters. Noteworthy was SGF's role in the significant mitigation of myocardial hypertrophy and in fostering the expression of vital proteins essential for heart health by the 24-week mark. This intervention markedly altered the dynamics of the Akt/HIF-1α/p53 pathway, inhibiting detrimental processes while promoting protective mechanisms. Conclusion: Our research casts SGF in a promising light as a cardioprotective agent in heart failure conditions induced by pressure overload in rats. Central to this protective shield is the modulation of the Akt/HIF-1α/p53 pathway, pointing to a therapeutic trajectory that leverages HIF-1α promotion and p53 nuclear transport inhibition.

A self-assembled, genetically engineered, irradiated tumor cell debris vaccine.

Vaccine-based therapeutics for cancers face several challenges including lack of immunogenicity and tumor escape pathways for single antigen targets. It has been reported that radiotherapy has an in situ vaccine effect that provides tumor antigens following irradiation, helping to activate antigen-presenting cells (APCs). Herein, a new vaccine approach is developed by combining genetically engineered irradiated tumor cell debris (RTD) and hyaluronic acid (HA), termed HA@RTD. A cancer cell line is developed that overexpresses granulocyte-macrophage colony-stimulating factor (GM-CSF). A hydrogel was developed by covalent conjugation of HA with RTD proteins that acted as a potent vaccine system, the effects which were probed with T cell receptor sequencing. The engineered vaccine activated antitumor immunity responses and prevented tumor growth in mice even with a single immunization. HA@RTD vaccine efficacy was also assessed in therapeutic settings with established tumors and in combination with immune checkpoint blockade.

A broadly applicable protein-polymer adjuvant system for antiviral vaccines.

Although protein subunit vaccines generally have acceptable safety profiles with precise antigenic content, limited immunogenicity can lead to unsatisfactory humoral and cellular immunity and the need for vaccine adjuvants and delivery system. Herein, we assess a vaccine adjuvant system comprising Quillaja Saponaria-21(QS-21) and cobalt porphyrin polymeric micelles that enabling the display of His-tagged antigen on its surface. The nanoscale micelles promote antigen uptake and dendritic cell activation to induce robust cytotoxic T lymphocyte response and germinal center formation. Using the recombinant protein antigens from influenza A and rabies virus, the micelle adjuvant system elicited robust antiviral responses and protected mice from lethal challenge. In addition, this system could be combined with other antigens to induce high titers of neutralizing antibodies in models of three highly pathogenic viral pathogens: Ebola virus, Marburg virus, and Nipah virus. Collectively, our results demonstrate this polymeric micelle adjuvant system can be used as a potent nanoplatform for developing antiviral vaccine countermeasures that promote humoral and cellular immunity.

A comprehensive stem cell laboratory module with blended learning for medical students at Tongji University.

The laboratory practice "Primary culture and directional differentiation of rat bone marrow mesenchymal stem cells (BMSCs)" is part of a required course for sophomore medical students at Tongji university, which has been conducted since 2012. Blended learning has been widely applied in medical courses. Based on a student-centered teaching philosophy, we reconstructed a comprehensive stem cell laboratory module with blended learning in 2021, aiming to facilitate students in enhancing their understanding of the multi-lineage differentiation potential of stem cells and improve their experimental skills, self-directed learning ability, and innovative thinking. First, we constructed in-depth online study resources, including videos demonstrating laboratory procedures, a PowerPoint slide deck, and published literature on student self-learning before class. In class, students performed a primary culture of BMSCs, freely chose among adipogenic, osteogenic, or chondrogenic differentiation, and used cytochemical or immunofluorescence staining for identification. After class, the extracurricular part involved performing quantitative polymerase chain reaction to examine the expression of multi-lineage differentiation marker genes, which was designed as an elective. After 2 years of practice, positive feedback was obtained from both students and faculty members who achieved, the learning goal as expected. The reconstructed stem cell laboratory module provides comprehensive practice opportunities for students. Students have a better understanding of BMSC at the molecular, cellular, and functional levels and have improved their experimental skills, which forms a basis for scientific research for medical students. Introducing blended learning into other medical laboratory practices thus seems valuable.