Culture Conditions Research Articles

Assessing the mass-culture system and proximate content of Arthrospira maxima local for biogas and biohydrogen productions

Arthrospira maxima is a type of local cyanobacteria that has shown success in being grown massively in Indonesia. This research is aimed at looking at the resilience and ability to grow in outdoor conditions using cheap media and following environmental patterns in terms of temperature, sunlight and aeration. In specific purpose is utilization of the obtained biomass for biogas or biohydrogen production. Also, media optimization research from chemical pour grade analyzes into commercial types has been carried out at laboratory scale and field scale. The results show that at the scaling up process from laboratorial to outdoor condition the amount of biomass ratio has decreased by 2–10%, whereas proximate of protein has decreased from 46% to 38%, in contrast starch tends to increase from 10% to 23% and lipid was increase from around 9%–12%. Freshwater and marine water basic media also showing difference in ratio of biomass production and main proximate. The total calory and protein seems higher if A. maxima cultivate using marine water. Cultivation time outdoors turns out to be faster growth when compared to controlled conditions indoors. Biomass analysis results show that the largest content is protein, followed by carbohydrates and lipid. Considering the composition of the proximate, the cyanobacteria biomass tends to be used for biogas conversion rather than alcohol, due to the high protein with less carbohydrate content. Moreover, Arthrospira is cyanobacteria which is capable for producing heterocyst cells that can be excite gas. It was found the cyanobacteria release hydrogen gas around 54 μL/g wet biomasses/day mature culture. However, it should be remembered that this strain is still original or wild type, and can still be developed using both environmental and molecular manipulation approaches. Survival rate from laboratory scale to field scale shows that the microalgae is strong and worthy of consideration for industrial applications. Apart from that, high pH culture conditions are very good for removing contaminants. Also changing the media from pour analyses grade chemicals to commercial chemicals grade only reduces the biomass composition by 2–5% so it is worth considering for commercial applications. Testing various reactors to make biomass as needed with high productivity will be a challenge in the future.

Enhancement of Mycelial Biomass Production and Bioactive Properties Related to Cosmeceuticals of Calocybe indica (Milky Mushroom) by Optimization of Submerged Culture Conditions

Calocybe indica or milky mushroom grown in submerged culture was investigated as a new source of antioxidants and anti-collagenase substances, and for its ability to inhibit three bacteria that trigger inflammatory acne, Cutibacterium acnes, Staphylococcus epidermidis, and S. aureus. To facilitate C. indica submerged cultivation and achieve high productivity, a three-level four-factor Box-Behnken design was utilized to find the optimal culture conditions for its mycelial biomass production. The model estimated the optimal conditions as 30 °C, a medium pH of 5.9, and incubation for 14 days under static culture, with results verifi ed by validation experiments. The optimal conditions gave mycelial biomass of 7.12 g/L, which is 5-fold higher than the original condition. This statistical method facilitated rapid identification and integration of key factors for C. indica high mycelial biomass production with significantly improved cosmeceutical properties in terms of antioxidant and antibacterial activities against acne-causing bacteria, especially C. acnes at 19-fold and 18-fold, respectively, compared to the original condition. An ethanolic extract derived from C. indica mycelia was nontoxic to human skin keratinocytes (HaCaT). These diverse functionalities indicate that the mycelial extract of C. indica may represent a promising source of bioactive substances for cosmeceutical applications.

A dynamically loaded ex vivo model to study neocartilage and integration in human cartilage repair.

Articular cartilage injuries in the knee can lead to post-traumatic osteoarthritis if untreated, causing debilitating problems later in life. Standard surgical treatments fail to ensure long lasting repair of damaged cartilage, often resulting in fibrotic tissue. While there is a vast amount of research into cartilage regeneration, integrating engineered implants with cartilage remains a challenge. As cartilage is a load bearing tissue, it is imperative to evaluate tissue repair strategies and their ability to integrate under mechanical loading. This work established a dynamically loaded ex vivo model of cartilage repair using human cartilage explants. The model was used to assess the efficacy of a stem cell therapy delivered in a bioadhesive hydrogel comprised of photocrosslinkable gelatin methacryloyl (GelMA) and microbial transglutaminase to repair the model defect. Extensive neocartilage production and integration were observed via histology and immunohistochemistry after 28days chondrogenic culture. Analysis of culture media allowed monitoring of glycosaminoglycan and type II collagen production over time. A mechanical assessment of integration via a push out test showed a 15-fold increase in push out strength over the culture duration. The model was successful in exhibiting robust chondrogenesis with transglutaminase or without, and under both culture conditions. The work also highlights several limitations of ex vivo models and challenges of working with bioreactors that must be overcome to increase their utility. This ex vivo model has the potential to delay the need for costly pre-clinical studies and provide a more nuanced assessment of cartilage repair strategies than is possible in vivo.

Harnessing Emotional Intelligence: Enhancing Employee Performance in Kerala’s Retail Textile Industry

This research explores the effects of EI (Emotional Intelligence) on employee performance in the retail textile industry in Kerala with specific reference to the moderating roles of organizational culture and leadership style. With the ever-growing demands of the retail sector, it is imperative to demonstrate the efficiency of EI in improving employee performance to support a sustained competitive advantage. This research adopts a descriptive research design, whereby both quantitative and qualitative data will be collected to capture the goals and objectives of the study. Structural equation Modelling (SEM) was used to determine the relationship between EI and the level of performance of employees, as well as the moderating role of organizational culture and leadership style. This study focuses on textile manufacturing employees of the selected retail outlets of Kerala, with a sample size of 250. The results are expected to help explain how EI can enhance performance, especially under conditions of leadership support and organizational culture. The implications of these findings should offer explicit counsel to managers in the retail textile industry to facilitate a work climate that boosts the emotional capabilities of workers, resulting in increased work efficiency and contentment. Apart from extending the body of knowledge on emotional intelligence and employee performance, the findings of this study provide an important guideline for the retail sector of Kerala with regard to emotional intelligence that must be developed to enhance performance and sustain business success in the future.

Cytotoxic Effect of Bajong LN Rice Methanol Extract on Human Squamous Cell Carcinoma, ORL-48

Oral squamous cell carcinoma (OSCC), a major oral cancer, significantly challenges treatments and impacts patient quality of life; current therapies often cause severe side effects, highlighting the urgent need for gentler alternatives. Rice stands as one of the primary cereal grains providing the daily caloric intake for more than half of the global population. Extensive research has demonstrated the significant health benefits derived from rice, attributed to its abundance of bioactive compounds. This study endeavours to explore the potential cytotoxic effects of Bajong LN rice, a pigmented purple rice indigenous to Sarawak, on human squamous cell carcinoma, ORL-48 cells. Cells were cultured in complete DMEM/F-12 media and incubated under standard culture conditions. Upon reaching 80% confluency, the cells were treated to varied concentrations (ranging from 0 μg/ml to 2000 μg/ml) of Bajong LN rice methanol extract (BLN-ME) and cisplatin. Subsequently, the cells were incubated for 48 and 72 hours, and their cytotoxicity was assessed using the MTS assay. Results demonstrated that cisplatin inhibited ORL-48 cells with an IC50 of 7.483 μg/ml and 3.877 μg/ml; and an IC80 of 40.649 μg/ml; and 17.543 μg/ml for 48 and 72 hours, respectively. Correspondingly, BLN-ME exhibited a notable cytotoxic effect against ORL-48 cells at 48- and 72-hour intervals, with an IC50 of 354.4 μg/ml and 342.0 μg/ml; and an IC80 of 450.3 μg/ml and 423.63 μg/ml, respectively. The cytotoxic activity of BLN-ME against ORL-48 cells was observed in both a time and dose-dependent manner. Morphological analysis and the Trypan blue exclusion assay corroborated the MTS assay's findings. Our preliminary findings provide the first scientific evidence of the cytotoxic effect of BLN-ME specifically against human squamous cell carcinoma, ORL-48 cells. This study suggests the potential of BLN-ME as a promising anti-cancer agent, presenting opportunities for further investigation into its underlying cytotoxic mechanisms.

Effects of Ethylene Inhibitors on the Long-Term Maintenance of the Embryogenic Callus of Vitis vinifera L.

Vitis vinifera is an important fruit crop which is mainly consumed fresh or used for the production of wine. Genetic improvement programs through New Genomic Techniques (NGTs) aim to develop grapevine varieties resistant to biotic and abiotic stresses or enhancing nutraceutical properties. In order to apply NGTs, maintaining embryogenic calluses from flower tissues is critical. Optimizing culture conditions—pH, gelling agents, temperature, light, growth regulators, and gas composition—is essential for inducing efficient embryogenic responses tailored to each genotype/explant. Ethylene, a pivotal gaseous plant hormone, significantly influences tissue culture by affecting organogenesis and embryogenesis processes in several plants. Modulating ethylene levels shows promise for improving tissue culture vitality. This study evaluates in Vitis vinifera the effects of silver thiosulfate (STS) and salicylic acid (SA) on embryogenic callus growth, specifically investigating their roles in maintaining and inducing embryogenic competence. STS, particularly at 40 µM and 60 µM concentrations, effectively preserved embryogenic competence in Italia and Red Globe calluses, while high SA concentrations showed varied and occasionally adverse effects. At the same time, STS markedly suppressed the non-embryogenic callus growth in recalcitrant variety Italia, potentially increasing the ratio between embryogenic to non-embryogenic calluses development.

Banked Primary Progenitor Cells for Allogeneic Intervertebral Disc (IVD) Therapy: Preclinical Qualification and Functional Optimization within a Cell Spheroid Formulation Process.

Background/Objectives: Biological products are emerging as therapeutic management options for intervertebral disc (IVD) degenerative affections and lower back pain. Autologous and allogeneic cell therapy protocols have been clinically implemented for IVD repair. Therein, several manufacturing process design considerations were shown to significantly influence clinical outcomes. The primary objective of this study was to preclinically qualify (chondrogenic potential, safety, resistance to hypoxic and inflammatory stimuli) cryopreserved primary progenitor cells (clinical grade FE002-Disc cells) as a potential cell source in IVD repair/regeneration. The secondary objective of this study was to assess the cell source's delivery potential as cell spheroids (optimization of culture conditions, potential storage solutions). Methods/Results: Safety (soft agar transformation, β-galactosidase, telomerase activity) and functionality-related assays (hypoxic and inflammatory challenge) confirmed that the investigated cellular active substance was highly sustainable in defined cell banking workflows, despite possessing a finite in vitro lifespan. Functionality-related assays confirmed that the retained manufacturing process yielded strong collagen II and glycosaminoglycan (GAG) synthesis in the spheroids in 3-week chondrogenic induction. Then, the impacts of various process parameters (induction medium composition, hypoxic incubation, terminal spheroid lyophilization) were studied to gain insights on their criticality. Finally, an optimal set of technical specifications (use of 10 nM dexamethasone for chondrogenic induction, 2% O2 incubation of spheroids) was set forth, based on specific fine tuning of finished product critical functional attributes. Conclusions: Generally, this study qualified the considered FE002-Disc progenitor cell source for further preclinical investigation based on safety, quality, and functionality datasets. The novelty and significance of this study resided in the establishment of defined processes for preparing fresh, off-the-freezer, or off-the-shelf IVD spheroids using a preclinically qualified allogeneic human cell source. Overall, this study underscored the importance of using robust product components and optimal manufacturing process variants for maximization of finished cell-based formulation quality attributes.

Microbial production of levulinic acid from glucose by engineered Pseudomonas putida KT2440

Levulinic acid(LA) is produced through acid-catalyzed hydrolysis and dehydration of lignocellulosic biomass. It is a key platform chemical used as an intermediate in various industries including biofuels, cosmetics, pharmaceuticals, and polymers. Traditional LA production uses chemical conversion, which requires high temperatures and pressures, strong acids, and produces undesirable side reactions, repolymerization products, and waste problems Therefore, we designed an integrated process to produce LA from glucose through metabolic engineering of Pseudomonas putida KT2440. As a metabolic engineering strategy, codon optimized phospho-2-dehydro-3-deoxyheptonate aldolase (AroG), 3-dehydroshikimate dehydratase (AsbF), and acetoacetate decarboxylase (Adc) were introduced to express genes of the shikimate and β-ketoadipic acid pathways, and the 3-oxoadipate CoA-transferase (pcaIJ) gene was deleted to prevent loss of biosynthetic intermediates. To increase the accumulation of the produced LA, the lva operon encoding levulinyl-CoA synthetase (LvaE) was deleted resulting in the high LA-producing strain P. putida HP203. Culture conditions such as medium, temperature, glucose concentration, and nitrogen source were optimized, and under optimal conditions, P. putida HP203 strain biosynthesized 36.3 mM (4.2 g/L) LA from glucose in a fed-batch fermentation system. When lignocellulosic biomass hydrolysate was used as the substrate, this strain produced 7.31 mM of LA. This is the first report of microbial production of LA from glucose by P. putida. This study suggests the possibility of manipulating biosynthetic pathway to produce biological products from glucose for various applications.

Tumor Cell Spatial Organization Directs EGFR/RAS/RAF Pathway Primary Therapy Resistance through YAP Signaling.

Non-small cell lung cancers (NSCLC) harboring common mutations in EGFR and KRAS characteristically respond transiently to targeted therapies against those mutations, but invariably, tumors recur and progress. Resistance often emerges through mutations in the therapeutic target or activation of alternative signaling pathways. Mechanisms of acute tumor cell resistance to initial EGFR (EGFRi) or KRASG12C (G12Ci) pathway inhibition remain poorly understood. Our study reveals that acute response to EGFR/RAS/RAF-pathway inhibition is spatial and culture context specific. In vivo, EGFR mutant tumor xenografts shrink by > 90% following acute EGFRi therapy, and residual tumor cells are associated with dense stroma and have increased nuclear YAP. Interestingly, in vitro EGFRi induced cell cycle arrest in NSCLC cells grown in monolayer, while 3D spheroids preferentially die upon inhibitor treatment. We find differential YAP nuclear localization and activity, driven by the distinct culture conditions, as a common resistance mechanism for selective EGFR/KRAS/BRAF pathway therapies. Forced expression of the YAPS127A mutant partially protects cells from EGFR-mediated cell death in spheroid culture. These studies identify YAP activation in monolayer culture as a non-genetic mechanism of acute EGFR/KRAS/BRAF therapy resistance, highlighting that monolayer vs spheroid cell culture systems can model distinct stages of patient cancer progression.

Statistical Optimisation of Streptomyces sp. DZ 06 Keratinase Production by Submerged Fermentation of Chicken Feather Meal

This study focused on the isolation of actinobacteria capable of producing extracellular keratinase from keratin-rich residues, which led to the selection of an actinobacterial strain referenced as Streptomyces strain DZ 06 (ES41). The Plackett–Burman screening plan was used for the statistical optimization of the enzymatic production medium, leading to the identification of five key parameters that achieved a maximum activity of 180.1 U/mL. Further refinement using response surface methodology (RSM) with a Box–Behnken design enhanced enzyme production to approximately 458 U/mL. Model validation, based on the statistical predictions, demonstrated that optimal keratinase activity of 489.24 U/mL could be attained with 6.13 g/L of chicken feather meal, a pH of 6.25, incubation at 40.65 °C for 4.11 days, and an inoculum size of 3.98 × 107 spores/mL. The optimized culture conditions yielded a 21.67-fold increase in keratinase compared with the initial non-optimized standard conditions. The results show that this bacterium is an excellent candidate for industrial applications when optimal conditions are used to minimize the overall costs of the enzyme production process.

Garlic-Induced Enhancement of Bifidobacterium: Enterotype-Specific Modulation of Gut Microbiota and Probiotic Populations

The gut microbiome is a dynamic ecosystem crucial for maintaining its host’s health by regulating various immune and metabolic functions. Since diet plays a fundamental role in shaping the gut microbiome, understanding the relationship between food consumption and microbiome structure is essential. Although medicinal plants are widely recognized for their broad health benefits, their specific impact on the gut microbiome remains unclear. In this study, we investigated the effects of garlic (Allium sativum) on the gut microbiome using an in vitro human fecal incubation model. Our findings revealed that the impact of garlic on gut microbial structure varied depending on the dominant gut microbiome components (enterotypes). The Bacteroides-dominant enterotype exhibited significant changes in overall microbial diversity in response to garlic, while the Prevotella-dominant enterotype remained unaffected. Additionally, the garlic treatment led to specific alterations in microbiota composition, such as an increase in beneficial probiotics like Bifidobacterium. We validated garlic’s prebiotic potential by promoting the growth of Bifidobacterium adolescentis under in vitro culture conditions. Our study highlights the importance of understanding enterotype-specific responses to diet and suggests that garlic may serve as a dietary supplement for modulating gut microbiota and promoting the growth of beneficial probiotics.

Forward–reverse mutation cycles in cancer cell lines under chemical treatments

Spontaneous forward–reverse mutations were reported by us earlier in clinical samples from various types of cancers and in HeLa cells under normal culture conditions. To investigate the effects of chemical stimulations on such mutation cycles, the present study examined single nucleotide variations (SNVs) and copy number variations (CNVs) in HeLa and A549 cells exposed to wogonin-containing or acidic medium. In wogonin, both cell lines showed a mutation cycle during days 16–18. In acidic medium, both cell lines displayed multiple mutation cycles of different magnitudes. Genomic feature colocalization analysis suggests that CNVs tend to occur in expanded and unstable regions, and near promoters, histones, and non-coding transcription sites. Moreover, phenotypic variations in cell morphology occurred during the forward–reverse mutation cycles under both types of chemical treatments. In conclusion, chemical stresses imposed by wogonin or acidity promoted cyclic forward–reverse mutations in both HeLa and A549 cells to different extents.

Design and validation of cell-based potency assays for frataxin supplementation treatments

Friedreich's ataxia (FRDA) is a multisystem, autosomal recessive disorder caused by mutations in the frataxin (FXN) gene. As FRDA is considered a FXN deficiency disorder, numerous therapeutic approaches in development or clinical trials aim to supplement FXN or restore endogenous FXN expression. These include gene therapy, protein supplementation, genome editing or upregulation of FXN transcription. To evaluate efficacy of these therapies, potency assays capable of quantitative determination of FXN biological activity are needed. Herein, we evaluate the suitability of mouse embryonic fibroblasts derived from Fxn G127V knock-in mice (MUT MEFs) as a candidate for cell-based potency assays. We demonstrate that these cells, when immortalized, continue to express minute amounts of Fxn and exhibit a broad range of phenotypes that result from severe Fxn deficiency. Exogenous FXN supplementation reverses these phenotypes. Thus, immortalized MUT MEFs are an excellent tool for developing potency assays to validate novel FRDA therapies. Care needs to be exercised while utilizing these cell lines, as extended passaging results in molecular changes that spontaneously reverse FRDA-like phenotypes without increasing Fxn expression. Based on transcriptome analyses, we identified the Warburg effect as the mechanism allowing cells expressing a minimal level of Fxn to thrive under standard cell culture conditions.

Vitamin B6 Pathway Maintains Glioblastoma Cell Survival in 3D Spheroid Cultures.

Glioblastoma (GBM) is a deadly brain cancer. The prognosis of GBM patients has marginally improved over the last three decades. The response of GBMs to initial treatment is inevitably followed by relapse. Thus, there is an urgent need to identify and develop new therapeutics to target this cancer and improve both patient outcomes and long-term survival. Metabolic reprogramming is considered one of the hallmarks of cancers. However, cell-based studies fail to accurately recapitulate the in vivo tumour microenvironment that influences metabolic signalling and rewiring. Against this backdrop, we conducted global, untargeted metabolomics analysis of the G7 and R24 GBM 2D monolayers and 3D spheroid cultures under identical cell culture conditions. Our studies revealed that the levels of multiple metabolites associated with the vitamin B6 pathway were significantly altered in 3D spheroids compared to the 2D monolayer cultures. Importantly, we show that pharmacological intervention with hydralazine, a small molecule that reduces vitamin B6 levels, resulted in the cell death of 3D GBM spheroid cultures. Thus, our study shows that inhibition of the vitamin B6 pathway is a novel therapeutic strategy for the development of targeted therapies in GBMs.

Construction of Aspergillus oryzae strains with organelles labeled with fluorescence and observation of organelle morphology

The organelles in the multi-nucleated filamentous fungus Aspergillus oryzae present polymorphism. To observe the organelle morphology in A. oryzae and provide references for the localization prediction of unknown proteins and the disclosure of biological reaction pathways in A. oryzae, we fused different subcellular localization signals with green fluorescent protein (GFP) to obtain different subcellular localization vectors, which were then transferred into A. oryzae by Agrobacterium tumefaciens-mediated transformation. The A. oryzae reporter strains with fluorescence-labeled nuclei, mitochondria, endoplasmic reticulum, vacuole, lipid droplets, peroxisome, and Golgi apparatus were successfully constructed. Furthermore, staining with small-molecule specific dyes was carried out to validate the co-localization of fluorescence-labeled mitochondria, nuclei, and lipid droplets in the reporter strains, which further confirmed that the reporter strains were successfully constructed. The distribution and morphology of fluorescence-labeled organelles were observed at different growth stages and under different culture conditions. The constructed reporter strains provide basic tools for studying the organelle morphology, localization of unknown target proteins, and subcellular localization in A. oryzae.

Training entrepreneurs in culturally diverse countries. Influence of Social Norms on Entrepreneurial Intention

One of the most influential factors in people's lives is the place of birth. At the educational and employment level, our future is often determined by social and cultural factors associated with the country or region where we are born. This study explores the extent to which National Culture conditions the influence of Social Norms on the Entrepreneurial Intention of business students. We focus on the components of the Theory of Planned Behaviour proposed by Ajzen and on Hofstede's dimensions of National Culture. To test our hypotheses we surveyed 352 business students at universities or business schools in seven countries with large cultural differences. The results of PLS analyses show that the effect of Social Norms on Entrepreneurial Intention is larger among individuals from national cultures with high Power Distance, low Individualism and Short-Term Orientation. These effects of national culture should be considered when designing entrepreneurship education programmes.

Chromogranin B Protects Human Umbilical Endothelial Cells against Oxidative Stress

Chromogranin B (CgB) is involved in the control of the cardiovascular system through the regulation of catecholamine release. Whether CgB can exert direct actions on the endothelium has not yet been clarified. Here, we aimed to investigate the effects of CgB on cell viability, mitochondrial membrane potential, reactive oxygen species (ROS), glutathione (GSH), nitric oxide (NO) release, and the cytosolic calcium concentration ([Ca2+]c) in human vascular endothelial cells (HUVECs) cultured under both physiological and peroxidative conditions. In HUVECs, experiments were conducted to establish the proper concentration and timing of CgB stimulation. Thereafter, specific assays were used to evaluate the response of HUVECs cultured in physiologic or oxidative stress conditions to CgB in the presence or absence of β-adrenergic receptor agonists and antagonists and intracellular pathways blockers. Analysis of cell viability, mitochondrial membrane potential, and NO release revealed that CgB was able to cause increased effects in HUVECs cultured in physiological conditions. Additionally, the same analyses performed in HUVECs cultured with H2O2, showed protective effects exerted by CgB, which was also able to counteract ROS release and maintain GSH levels. Furthermore, CgB played a dual role on the [Ca2+]c depending on the physiological or peroxidative cell culturing conditions. In conclusion, our data provide new information about the direct role of CgB in the physiological regulation of endothelial function and highlight its potential as a protective agent against peroxidative conditions, such as those found in cardiovascular diseases.

The regenerative capacity of hepatocyte organoids following long-term cryopreservation in Republic of Korea

Background: Hepatocyte organoids (HOs) are more functionally versatile than bile duct epithelial cell organoids, but have a shorter lifespan. This limitation has been improved by optimizing culture methods, but could remain a barrier to their wider application in research and therapy, especially for use at the appropriate time.Methods: This study aimed to prolong the lifespan of pig HOs and assess their viability and functionality after a year of cryopreservation. Genes involved in apoptosis (TP53, P21, CASP8) and liver function (ALB, CYP3A29, EPCAM) were analyzed to evaluate the impact of adipose-derived mesenchymal stem cell (A-MSC) co-culture before and after freezing on cryoresistance. HOs were cut into fragments, cryopreserved for a year, and cultured alone or co-cultured with A-MSCs in Matrigel post-thawing.Results: After thawing, co-cultured HOs exhibited a higher development rate than those cultured alone. P21 expression increased irrespective of pre-freezing culture conditions. The ALB and CYP3A29 expression patterns resembled those of non-frozen HOs, with similar effects from co-culture. EPCAM expression surged post-freezing.Conclusion: This study demonstrates that HOs maintain liver function post-preservation, showing increased EPCAM expression and enhanced regenerative capacity against cryoinjury. These findings suggest that HOs may be a valuable cell source for drug development in animals and research on medications for human diseases.

Marmoset and human trophoblast stem cells differ in signaling requirements and recapitulate divergent modes of trophoblast invasion

Early human trophoblast development has remained elusive due to the inaccessibility of the early conceptus. Non-human primate models recapitulate many features of human development and allow access to early postimplantation stages. Here, we tracked the pre- to postimplantation transition of the trophoblast lineage in superficially implanting marmoset embryos invivo. We differentiated marmoset naive pluripotent stem cells into trophoblast stem cells (TSCs), which exhibited trophoblast-specific transcriptome, methylome, differentiation potential, and long-term self-renewal. Notably, human TSC culture conditions failed to support marmoset TSC derivation, instead inducing an extraembryonic mesoderm-like fate in marmoset cells. We show that combined MEK, TGF-β/NODAL, and histone deacetylase inhibition stabilizes a periimplantation trophoblast-like identity in marmoset TSCs. By contrast, these conditions differentiated human TSCs toward extravillous trophoblasts. Our work presents a paradigm to harness the evolutionary divergence in implantation strategies to elucidate human trophoblast development and invasion.

Expression patterns of folate metabolism-related enzymes in the bovine oviduct: estrous cycle-dependent modulation and responsiveness to folic acid

Folate metabolism is required for important biochemical processes that regulate cell functioning, but its role in female reproductive physiology in cattle during peri- and post-conceptional periods has not been thoroughly explored. Previous studies have shown the presence of folate in bovine oviductal fluid, as well as finely regulated gene expression of folate receptors and transporters in bovine oviduct epithelial cells (BOECs). Additionally, extracellular folic acid (FA) affects the transcriptional levels of genes important for the functioning of BOECs. However, it remains unknown whether the anatomical and cyclic features inherent to the oviduct affect regulation of folate metabolism. The present study aimed to characterize the gene expression pattern of folate cycle enzymes in BOECs from different anatomical regions during the estrous cycle and to determine the transcriptional response of these genes to increasing concentrations of exogenous FA. A first PCR screening showed the presence of transcripts encoding dihydrofolate reductase (DHFR), methylenetetrahydrofolate reductase (MTHFR), and methionine synthase (MTR) in bovine reproductive tissues (ovary, oviduct and uterus), with expression levels in oviductal tissues comparable to, or even higher than, those detected in ovarian and uterine tissues. Moreover, expression analysis through RT-qPCR in BOECs from the ampulla and isthmus during different stages of the estrous cycle demonstrated that folate metabolism-related enzymes exhibited cycle-dependent variations. In both anatomical regions, DHFR was upregulated during the preovulatory stage, while MTHFR and MTR exhibited increased expression levels during the postovulatory stage. Under in vitro culture conditions, ampullary and isthmic cells were cultured in the presence of 10, 50, and 100 μM FA for 24 h. Under these conditions, isthmus epithelial cells exhibited a unique transcriptional response to exogenous FA, showing a pronounced increase in MTR expression levels. Our results suggest that the expression of folate metabolism-related genes in BOECs is differentially regulated during the estrous cycle and may respond to exogenous levels of folate. This offers a new perspective on the transcriptional regulation of genes associated with the folate cycle in oviductal cells and provides groundwork for future studies on their functional and epigenetic implications within the oviductal microenvironment.