Biomedical Research and Therapy

Human In Vitro and Ex Vivo Models in Angiogenesis Research: Advances and Challenges

Maisarah Md Razmi — 2025-04-30

Angiogenesis, the process of new blood vessel formation, is a complex phenomenon that plays a crucial role in various physiological and pathological processes, including embryonic development, tissue repair, vascular homeostasis, and tumor microenvironments. The utilization of human in vitro and ex vivo models to study angiogenesis is an actively investigated area that holds great promise for offering novel insights and prospects for developing methods to treat angiogenesis-related diseases, such as cancer and cardiovascular disorders. Combining in vitro and ex vivo models using human samples can enhance the understanding of the complex process of angiogenesis in the human body. This integrative strategy facilitates a holistic exploration of angiogenesis, bridging the gap between simplified in vitro systems and the complexities inherent in in vivo settings, thereby augmenting the translational prospects of research outcomes for clinical applications. However, ethical constraints, inherent individual variability in human samples, challenges in obtaining tissue samples, technical issues in tissue handling, and the high cost involved are key limitations to consider.

Murine Models of Allergic Asthma: Methodological Insights into Allergen Sensitization and Challenge Protocols

Solehah Mohd Rosdan Bushra — 2025-04-30

Asthma represents a chronic inflammatory airway disease with a steadily increasing global prevalence in recent decades. Animal models have proven invaluable in elucidating the underlying disease mechanisms and identifying innovative therapeutic approaches. The murine model is extensively used to investigate key characteristics of allergic asthma, including airway inflammation, airway hyperresponsiveness (AHR), and airway remodeling. Classic protocols involving sensitizing and challenging animals with different types of allergens and modes of administration are major factors in inducing asthmatic features in a mouse model. The present review critically analyzes the commonly used sensitization and allergen challenge protocols for inducing acute and chronic inflammation in the airways of mouse models of asthma, emphasizing their potential in advancing therapeutic development for allergic asthma studies.

Efficient Enrichment of Muse Cells from Human Umbilical Cord Mesenchymal Stem Cells Using Collagenase and Hypoxic Stress

Thuan Minh Le — 2025-04-30

Background: Muse (multilineage-differentiating stress-enduring) cells are a pluripotent subpopulation of mesenchymal stem cells (MSCs), characterized by their stress tolerance and significant potential in regenerative medicine. However, their low abundance poses challenges for isolation. This study aims to evaluate the efficacy of severe stress conditions—including low temperature, severe hypoxia, and collagenase treatment (LHC)—in isolating Muse cells.

Methods: Human umbilical cord-derived MSCs (hUCMSCs) were treated with 0.1% collagenase D in DMEM at 37 °C for 30 minutes, then incubated at 4 °C for 16 hours in sealed containers completely filled with the collagenase-containing medium. Muse cell enrichment following this treatment was quantified by flow cytometry. Morphological characteristics of the Muse-enriched-cell (MEC) populations were examined under adherent and suspension culture conditions. Their trilineage differentiation potential into adipocytes (mesoderm), hepatocyte-like cells (endoderm), and neuron-like cells (ectoderm) was evaluated. Additionally, the expression of pluripotency-associated genes (Sox2, Nanog, and Oct4) was assessed via RT-qPCR, and chromosomal stability was confirmed through G-banding karyotype analysis.

Results: The percentage of SSEA-3⁺ cells in MEC populations (53.47 ± 17.16%) was significantly higher than in native hUCMSCs (3.43 ± 1.50%). MECs formed clusters resembling embryonic stem cells in suspension culture and differentiated into adipocytes (lipid droplet⁺), hepatocyte-like cells (cytokeratin-7⁺), and neuron-like cells (MAP-2⁺). MEC-SC populations exhibited significantly higher mRNA expression of Nanog, Sox2, and Oct4 compared to MEC-AC populations and native hUCMSCs.

Conclusion: The LHC method provides a promising and efficient approach for isolating Muse cells, which could significantly advance their applications in regenerative medicine.

Potential Protection of Mesenchymal Stem Cells Against Oxidative Stress With Hypoxia Preconditioning and Lipopolysaccharide Exposure

Fadhilla Maulany El Fajri — 2025-04-30

Background: Mesenchymal stem cells (MSCs) are among the most promising therapeutic options for degenerative diseases. However, the low viability and prolonged doubling time of MSCs during ex vivo expansion remain obstacles in their application in MSC-based therapies. MSC preconditioning has been considered a potential strategy to overcome challenges in MSC culture and to increase the therapeutic effects of MSCs. To mimic an inflammatory microenvironment and enhance stress resilience, lipopolysaccharide (LPS) was combined with hypoxia preconditioning. The aim of this study was to examine the effects of hypoxia and LPS preconditioning on viability, population doubling time, and expression levels of antioxidant genes in MSCs.

Methods: MSCs were isolated from Wharton’s jelly of the human umbilical cord tissue. Hypoxia was simulated using 100 μM CoCl₂, a chemical hypoxia mimetic, and cells were supplemented with 10 ng/mL LPS. To test oxidative stress resilience, preconditioned MSCs were exposed to 100 μM H₂O₂ for 24 hours. Cell viability was assessed using the CCK-8 assay, and population doubling time was calculated from cell counts obtained via trypan blue exclusion assays at 24-hour intervals. The mRNA expression levels of superoxide dismutase 1 (SOD1), catalase (CAT), and hypoxia-inducible factor 1α (HIF1α) were quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). MSCs were divided into four groups: (1) normoxia (control), (2) hypoxia (CoCl₂), (3) LPS (10 ng/mL), and (4) hypoxia + LPS. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test.

Results: The results showed that the hypoxia + LPS preconditioning group significantly increased MSC viability compared with the hypoxia-alone group (p < 0.05). Similarly, MSCs in the hypoxia + LPS group exhibited a shorter population doubling time than untreated controls (p < 0.05). Preconditioned MSCs also demonstrated increased resistance to H₂O₂-induced oxidative stress, with higher viability compared to non-preconditioned cells (p < 0.05).

Conclusion: MSC preconditioning with hypoxia and LPS enhanced cell viability, proliferation capacity, and resistance to oxidative stress induced by H₂O₂. These findings suggest that combining hypoxia mimetics with inflammatory stimuli could optimize MSC culture conditions and improve therapeutic outcomes in degenerative diseases.

Surface Display of Alpha-Toxin HlaH35LH48L on Bacillus subtilis Cells for Oral Vaccine Delivery in Mice

Nhi NY Nguyen — 2025-04-30

Introduction: Surface display of proteins on Bacillus subtilis has emerged as a promising strategy in vaccinology, leveraging its safety, gastrointestinal resilience, and capacity for efficient antigen presentation. Targeting Staphylococcus aureus, a pathogen reliant on alpha-toxin (Hla) for virulence, this study focuses on a detoxified variant, Hla_H35LH48L, to potentially neutralize toxicity while preserving immunogenicity. We investigated B. subtilis as an oral vaccine vector to display Hla_H35LH48L and elicit mucosal and systemic immune responses in mice.

Methods: The hla_H35LH48L gene was fused to the yhcR anchoring motif and integrated into the amyE locus of B. subtilis HT800F via double-crossover recombination, generating strain BsHT2315. Successful chromosomal integration was confirmed by PCR. Surface display of Hla_H35LH48L was verified through Western blot and bacterial-enzyme-linked immunosorbent assay (bactELISA). Swiss mice were orally administered BsHT2315, wild-type B. subtilis, or PBS (control). Serum IgG and intestinal IgA levels were quantified by ELISA.

Results: Western blot and bactELISA confirmed robust surface expression of Hla_H35LH48L on BsHT2315. Oral immunization with BsHT2315 induced a significant two-fold increase in intestinal IgA compared to controls (p < 0.05), indicative of mucosal immunity. Serum IgG levels also showed a modest but significant elevation (1.5-fold, p < 0.01), suggesting systemic response activation.

Conclusion: This study demonstrated the successful development of B. subtilis BsHT2315 as an oral vaccine vehicle for Hla_H35LH48L delivery. The strain triggered potent mucosal and systemic antibody responses, underscoring B. subtilis’s potential for cost-effective, needle-free vaccine platforms. Future work will explore protective efficacy against Staphylococcus aureus infection and scalability for clinical translation.

Investigating the Immunomodulatory Effects of Tieu U Hoan Herbal Preparation in a Murine Hepatocellular Carcinoma Model

Hung Truong Thanh Luu — 2025-04-30

Introduction: Traditional medicine treatments (TMT) are widely recognized as effective complementary therapies in cancer care, including hepatocellular carcinoma (HCC). Tieu u hoan (TUH), an herbal formulation derived from TMT principles and clinical expertise, has shown promise in preclinical and clinical studies as a supportive therapy for HCC. This study investigates the immunomodulatory effects of TUH in immunodeficient nude mice engrafted with human Hep3B liver cancer cells.

Methods: Immune responses were evaluated in Hep3B tumor-bearing nude mice treated with TUH. Parameters included relative spleen weight, splenic histopathology (lymphoid follicle proliferation in white pulp), proportions of NK cells, dendritic cells (DCs), and macrophages in the spleen, plasma concentrations of IL-2 and TNF-α, and leukocyte/lymphocyte counts in peripheral blood.

Results: Administration of TUH (3.53 g/kg) significantly increased relative spleen weight (p < 0.05), enhanced lymphoid follicle proliferation in splenic white pulp, and elevated proportions of NK cells, DCs, and macrophages (p < 0.05). TUH also raised plasma TNF-α and IL-2 levels (p < 0.05) and increased peripheral leukocyte and lymphocyte counts (p < 0.05), suggesting potentiation of both innate and adaptive immunity.

Conclusion: TUH demonstrates significant immunoenhancing effects in a murine HCC model, providing a scientific basis for its use as a complementary therapy in HCC management. These findings underscore its potential to augment immune responses during conventional cancer treatment.

Plerixafor for Stem Cell Mobilization in Autologous Haematopoietic Stem Cell Transplantation: A Case Series of Lymphoma Patients from a Northeastern Malaysian Teaching Hospital

Razan Hayati Zulkeflee — 2025-04-30

Background: In recent years, plerixafor, a CXCR4 chemokine receptor inhibitor, has emerged as a promising agent for the mobilization of hematopoietic stem cells (HSCs) when combined with other mobilizers such as granulocyte colony-stimulating factor (G-CSF) and chemotherapy in patients with multiple myeloma and lymphoma undergoing autologous peripheral blood stem cell transplantation (APBSCT). Our facility has recently implemented plerixafor as a specialized rescue treatment in lymphoma patients who are at risk or have experienced mobilization failure with G-CSF.

Case Series: We present five cases of lymphoma in young adult patients (26 to 49 years old), comprising two cases of Hodgkin lymphoma and three cases of diffuse large B-cell lymphoma. All five patients presented with advanced stage IV disease. Three patients received plerixafor following initial mobilization failure with G-CSF-based protocols, one patient received plerixafor preemptively, and one patient received it as an upfront treatment strategy.

Outcomes: All five cases achieved a collection of CD34⁺ cells exceeding 2 × 10⁶ cells/kg (ranging from 2.67 to 3.95 × 10⁶ cells/kg) after a single mobilization involving plerixafor, and no adverse reactions were reported.

Conclusion: Our findings highlight the significant enhancement of HSCs mobilization achieved with plerixafor compared to traditional methods. Plerixafor is not only highly effective but also safe for use in lymphoma patients. These case series findings underscore its value as a key tool in optimizing HSCs collection for successful APBSCT.