Ruttachuk Rungsiwiwut

Assisted reproductive technologies (ART) provide opportunities for conservation of endangered wildlife, but their use is still limited by species-specific reproductive physiology, variable gamete competence, cryoinjury, and lack of validation of new approaches. This narrative review covers ART, biobanking, somatic cell nuclear transfer (SCNT), induced pluripotent stem cells (iPSCs), in vitro gametogenesis (IVG) and conservation genomics in vertebrates. Key molecular barriers include oxidative and mitochondrial damage, aberrant embryonic genome activation, incomplete epigenetic reprogramming, nuclear-mitochondrial incompatibility and defective germ-cell differentiation. Technological readiness is used to evaluate the evidence and separate demonstrated reproductive outcomes from experimental proof of concept, and indirect findings from domestic or laboratory models. Semen cryopreservation and artificial insemination are established in selected species, while embryo-based interventions remain species-dependent and stem-cell-derived reproductive approaches require functional validation. We propose an integrated decision framework linking reproductive diagnosis, biobanking, genomic donor selection, technological readiness, animal welfare, and resource allocation with reproductive and population-level outcomes. Importantly, laboratory milestones alone cannot demonstrate conservation success without sustained genetic contribution across multiple subsequent generations. Successful translation depends on species-specific protocols, standardized endpoints, and evidence of genetically valuable offspring survival, reproduction, and contribution to long-term population viability.

Induced pluripotent stem cell (iPSC)-based in vitro gametogenesis (IVG) provides a possible route from cryobanked or collected somatic cells to functional gametes and, ultimately, offspring. However, full offspring-level proof of concept has so far been largely demonstrated in mice. In livestock, non-human primates and threatened species, iPSC-based IVG remains prospective, with most studies restricted to derivation of pluripotent cells, specification of primordial germ cell-like cells or partial germline development. If successfully developed, this technology could increase the representation of founders in threatened populations, recover genetic contributions from non-reproductive or deceased animals, and potentially support shorter generation intervals and targeted trait introgression in livestock. Here, we critically evaluate the biological and translational requirements for iPSC-based IVG. These include reprogramming quality, germline specification, epigenetic reprogramming, imprint erasure, meiosis, sex-specific gonadal niches, gamete maturation, embryo development and long-term offspring assessment. iPSC-based IVG should be integrated with cryobanking, population-genetic management, assisted reproduction, animal welfare and habitat recovery for conservation. In livestock, applications include management of genetic diversity, recovery of rare breeds, shorter generation intervals, and targeted trait introgression. The current evidence shows that it is research and protocol development, not commercial deployment. A stage-gated validation framework and a cross-species roadmap are proposed. Reproductive application should require evidence from genomics, epigenomics, cytology, function, development, and multiple generations.

Background/Aim: Andrographolide, a compound derived from Andrographis paniculata, has exhibited promising antineoplastic effects; however, its effects on pluripotency and cancer stemness remain unclear. Since pluripotency and cancer stemness are key factors in treatment resistance and relapse, it is important to investigate how andrographolide affects these properties. Materials and Methods: MCF7 cells were treated with andrographolide (7.5-120 μM) for 24 or 48 h. Cell viability was evaluated using the MTT assay. Mitochondrial membrane potential was measured using TMRE. Gene expression of TOMM20, caspase-3, BAX, BCL2 and OCT4 was analyzed using RT-qPCR. Mammosphere and colony formation assays were performed to assess self-renewal and proliferation. Results: MTT assays revealed a dose- and time-dependent reduction in the cell viability following andrographolide treatment. TMRE staining revealed a decrease in the mitochondrial membrane potential of andrographolide-treated MCF7 cells, indicating mitochondrial dysfunction. RT-qPCR analysis showed down-regulation of TOMM20 and caspase-3 expression, although MCF7 cells are typically caspase-3 deficient. Notably, BAX expression was up-regulated following treatment with 60 μM andrographolide, suggesting the induction of apoptosis. A dose-dependent increase in OCT4 expression was observed, with a 13.52-fold rise at 60 μM, indicating a potential shift toward a pluripotent-like state. Gene correlation analysis revealed a negative correlation between TOMM20 and OCT4 expression, suggesting that TOMM20 down-regulation enhances pluripotency. The positive correlation between OCT4 and BAX expression suggests a complex link between apoptosis and stemness. Immunocytochemistry confirmed TOMM20 localization in the cytoplasm. Reduced mammosphere and colony formation indicated decreased self-renewal and proliferation. Conclusion: Andrographolide exerts anti-cancer effects through mitochondrial dysfunction and apoptosis, while paradoxically increases OCT4 expression. These findings may indicate an adaptive cellular survival mechanism, in which OCT4 up-regulation and TOMM20 down-regulation are correlated with cancer-like characteristics.

This study investigated the potential of scale-up mango leaf extract (MLE) as a treatment for diabetes, a global public health concern. MLE was prepared by boiling in water, yielding 12.07% (w/w), with a bioactive mangiferin content of 165.67 ± 10.88 μg/g in the crude powder. Mechanistically, MLE demonstrated a hypoglycemic effect by stimulating glucagon-like peptide-1 (GLP-1) secretion in NCI-H716 L-cells. This occurred through activation of the MAPK signaling pathway, evidenced by increased p-ERK1/2, p-p38, and p-c-Jun expression, and the Wnt signaling pathway, shown by increased β-catenin and decreased GSK-3β and Axin1 expression, consistent with molecular docking. In a type 2 diabetic rat model, MLE administration (40 mg/kg) significantly reduced metabolic parameters, including fasting blood glucose (FBG), body weight, cholesterol (CHOL), triglycerides (TGs), and HbA1c. Notably, MLE lowered serum insulin and the HOMA-IR index, and reduced serum dipeptidyl peptidase-IV (DPP-IV) levels, resulting in increased serum GLP-1, comparable to the drug sitagliptin. These findings suggest that MLE has great potential to lower blood glucose by inducing GLP-1 secretion via MAPKs and Wnt signaling pathways, positioning it as a promising candidate for alternative diabetes treatment or development as a dietary supplement.

Culturing stem cells in species-specific serum ensures physiological relevance, reduces variability, and supports safer clinical use, highlighting the urgent need to develop reliable, species-matched systems for research and therapy. To address the concept of species specificity in cell culture, we investigated the effects of goat serum (GS) on the derivation of goat muscle stem cells (MuSCs). First, MuSCs were isolated from goat muscle tissue and cultured in media supplemented with either 10% goat GS or fetal bovine serum (FBS). Next, the isolated cells underwent characterization and differentiation. Finally, the effects of varying concentrations of GS and FBS on cell proliferation were evaluated. The results demonstrated that goat MuSCs grew in a GS-containing medium and were positively immunostained for CD29 and Pax7. Gene expression analysis revealed no significant differences in the expression of Pax7, MyoD, and MyoG genes between goat MuSCs grown in GS- or FBS-containing medium. Cells grown in GS-containing medium showed more efficient differentiation toward myogenic and adipogenic lineages than those grown in FBS. Supplementing the culture medium with 10% GS resulted in the greatest enhancement of goat MuSC proliferation, as evidenced by the MTT assay, increased Ki67 expression, and a higher number of colony-forming units. This study demonstrated that GS supplementation is notably beneficial for the proliferation of goat MuSCs.

Background Aromatherapy with a blend of essential oils is an alternative treatment for stress and sleep disturbances, providing stress relief and sleep enhancement, while increasing the efficacy and scent appeal. This study aimed to formulate an essential oil blend with the potential to reduce stress and enhance sleep quality, and to identify the most preferred blend based on participant satisfaction. Methods A cross-sectional study was conducted involving 63 female volunteers aged 25 - 50 years. Three formulations (A, B, and C) of essential oil blends were developed, each with a distinct scent profile—floral, unisex, and aromatic. A preference test was conducted using a sensory evaluation method, assessing participants’ satisfaction of the blends through inhalation. Preferences were quantified using a structured scoring system. Formula B was the most popular and was further refined into two recipes, B1 and B2. B2 emerged as the preferred option, and its primary component was identified using gas chromatography-mass spectrometry (GC–MS). Results Formulation B, rich in bergamot oil, received significantly higher hedonic ratings than Formulation A (p<0.05) and was the most preferred in interviews. Participants recommended a milder, more balanced scent for relaxation and sleep. While B1 and B2 showed no significant differences, B2 was chosen for its higher bergamot content, fresher unisex profile, and reduced floral notes. GC–MS analysis identified limonene, menthol, and linalool as its main components. Conclusion This study demonstrated that an essential oil blend predominantly composed of bergamot oil was most favored by the volunteers, indicating its potential benefits for promoting relaxation.

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Graphical Mitochondrial DNA (mtDNA) from embryonic cells is released into the spent culture medium (SCM) during cellular processes, providing a potential biomarker of embryo health. Analysing mtDNA levels in SCM enables a non-invasive evaluation of embryo quality and potential developmental abnormalities. In this retrospective study, we aimed to investigate the relationship between relative mtDNA copy number in embryo SCM and key factors, including embryo fragmentation, morphological quality and chromosomal abnormalities. Fertilised embryos produced through intracytoplasmic sperm injection were cultured to the blastocyst stage in an incubator. Embryo fragmentation was assessed on day 3 using the Istanbul criteria, while morphological grading was evaluated on day 5 using the Gardner criteria. On day 5, trophectoderm (TE) biopsies were performed for preimplantation genetic testing for aneuploidy, followed by embryo cryopreservation and collection of embryo SCM. The mtDNA was quantified using quantitative PCR. Statistical analyses using the Mann–Whitney U and Kruskal–Wallis tests (significance at P < 0.05) showed that relative mtDNA copy number did not significantly differ among embryos with fragmentation levels 25% (P > 0.05). For blastocyst grading, which evaluates the inner cell mass (ICM) and TE, no significant difference was observed in relative mtDNA copy number between grades B and C for ICM (P = 0.190) and TE (P = 0.289). Furthermore, a trend towards higher relative mtDNA levels was observed in aneuploid than in euploid embryos, although the difference was not statistically significant. Thus, relative mtDNA copy number in SCM may not accurately reflect embryo characteristics, such as fragmentation, morphological grading or chromosomal abnormalities. Lay summary: This study examined whether the amount of mitochondrial DNA (mtDNA) in the fluid used to culture embryos in the laboratory could indicate embryo quality. We assessed various factors, including the appearance of the embryos, the presence of fragmented cells and the occurrence of chromosomal abnormalities. Fertilized eggs were cultured until they developed into blastocysts, and the amount of mtDNA in the culture fluid was measured using a machine that detects genetic material. The results revealed no clear association between mtDNA levels and embryo appearance or fragmentation. Although embryos with chromosomal abnormalities had slightly more mtDNA, the difference was not statistically significant. These findings suggest that mtDNA in the culture fluid may not be a reliable marker for assessing embryo quality or chromosomal status.

In vitro cultured meat is one of the future foods that might revolutionize meat production by allowing food manufacturers to sidestep traditional animal farming. Despite being in early development, in vitro cultured meat faces challenges such as technological limitations and consumer acceptance, hindering its market integration. Beyond being an ethical alternative to conventional meat, it resembles conventionally farmed meat, making it a focus on healthcare and food safety. From a health perspective, it theoretically presents intriguing possibilities in the fields of functional foods, tailored dietary therapy, and public health. In functional foods, its unique composition allows precise nutritional manipulation for specific dietary needs, aligning with personalized nutrition. In dietary therapy, in vitro cultured meat might offer customization for individuals with dietary restrictions or health conditions, providing a novel therapeutic avenue. From a public health standpoint, it holds promise in mitigating environmental and health challenges linked to conventional meat production, aligning with sustainability and disease prevention initiatives. However, the potential health risks of in vitro cultured meat include concerns about the use of genetically modified starting cells, fetal bovine serum, growth factors, scaffolding materials, and antibiotics during the production process. These additives may have unforeseen long-term health effects if not properly regulated. Additionally, in vitro cultured meat may lack some of the natural nutrients found in conventional meat, which could lead to nutritional imbalances. There are also uncertainties about how the texture and bioavailability of nutrients in in vitro cultured meat might affect digestion and absorption in the human body. Nevertheless, specific aspects of research in the field of in vitro cultured meat demand increased focus from researchers to guarantee the optimal safety standards. An in-depth study on safety-oriented research of in vitro cultured meat should be emphasized to ensure benefits for individual and public health. HIGHLIGHTS In theory, in vitro cultured meat offers potential as a novel solution for developing functional foods by allowing precise manipulation of nutritional content, enabling the creation of health-optimized products. In vitro cultured meat could help mitigate the risk of antimicrobial resistance by minimizing the need for antimicrobial agents, reducing overall exposure and the potential for the development and spread of antimicrobial-resistant microorganisms. Starting cells derived from genetically modified cells in the in vitro cultured meat may pose risks of allergenicity and contamination, as well as potential immune responses to novel proteins. Strict safety protocols and thorough testing are crucial to address these risks and ensure the final product is safe for consumption. GRAPHICAL ABSTRACT

Spinal cord injury (SCI) causes inflammation and neuronal degeneration, resulting in functional movement loss. Since the availability of SCI treatments is still limited, stem cell therapy is an alternative clinical treatment for SCI and neurodegenerative disorders. Human umbilical cord Wharton’s jelly-derived mesenchymal stem cells (hWJ-MSCs) are an excellent option for cell therapy. This study aimed to induce hWJ-MSCs into neural stem/progenitor cells in sphere formation (neurospheres) by using neurogenesis-enhancing small molecules (P7C3 and Isx9) and transplant to recover an SCI in a rat model. Inducted neurospheres were characterized by immunocytochemistry (ICC) and gene expression analysis. The best condition group was selected for transplantation. The results showed that the neurospheres induced by 10 µM Isx9 for 7 days produced neural stem/progenitor cell markers such as Nestin and β-tubulin 3 through the Wnt3A signaling pathway regulation markers (β-catenin and NeuroD1 gene expression). The neurospheres from the 7-day Isx9 group were selected to be transplanted into 9-day-old SCI rats. Eight weeks after transplantation, rats transplanted with the neurospheres could move normally, as shown by behavioral tests. MSCs and neurosphere cells were detected in the injured spinal cord tissue and produced neurotransmitter activity. Neurosphere-transplanted rats showed the lowest cavity size of the SCI tissue resulting from the injury recovery mechanism. In conclusion, hWJ-MSCs could differentiate into neurospheres using 10 µM Isx9 media through the Wnt3A signaling pathway. The locomotion and tissue recovery of the SCI rats with neurosphere transplantation were better than those without transplantation.