Culture Period Research Articles

Nutritional Values of Foxtail Millet (Setaria italica) and other Millets Used for Common Diseases and Management

Abstract: The ever-increasing population and malnutrition in tropical and sub-tropical countries upsurge the demand and security for poor people’s food and nutrition. In erratic climatic conditions, millets can survive as they require less water, short cultivation period and are somewhat tolerant to biotic (bacteria and fungi) and biotic stresses (drought, salinity, nutrient deficiency, etc). Millets possess several nutrient rich components and several health benefits. Despite having superior nutritional, health and agricultural advantages, millets have not received their due attention. Among the millets, foxtail millet is one of the major millets in terms of its second in global production and millets’ yield, quality, and quantity are affected by various diseases. This review provides an overview of the origin and distribution of eight millets, their morphological characteristics with chemical composition, potential health benefits and the fungal, bacterial and viral diseases affecting the growth and yield of millets and their effective management in the framework of ensuring food and nutritional securities in the tropical and sub-tropical countries.

Astragaloside IV Protects Against IL-1β-Induced Chondrocyte Damage via Activating Autophagy.

Osteoarthritis (OA) is a chronic inflammatory condition that affects the articular cartilage. Astragaloside IV (AS-IV) constitutes the primary active component of the Chinese herbal medicine Huangqi (Radix Astragali Mongolici). AS-IV demonstrates anti-inflammatory and anti-apoptotic attributes, exhibiting therapeutic potential across various inflammatory and apoptosis-related disorders. Nevertheless, its pharmaceutical effects in OA are yet to be fully defined. This study aimed to investigate the protective impact of AS-IV on rat chondrocytes treated with IL-1β and ascertain whether autophagy plays a role in this effect. Chondrocytes were isolated and cultivated from the knee joints of neonatal SD mice. The study included the blank control group, the model group, and the AS-IV concentration gradient group (50, 100, 200 μmol/L) to intervene with chondrocytes. The MTT assay was employed to assess cell viability at varying culture periods, enabling the determination of suitable concentration and duration. Subsequently, chondrocytes were treated with the optimal AS-IV concentration and divided into three groups: the model group replicated IL-1β-induced inflammatory chondrocyte injury, the AS-IV group received a co-culture of AS-IV and IL-1β, and a blank control group was established. Changes in cell morphology and structure were observed using ghost pen cyclic peptide staining. ELISA was used to measure TNF-α and GAG levels in cell supernatants. RT-qPCR assessed p62 and LC3 mRNA expression, while Western Blot evaluated p62 and LC3Ⅱ/Ⅰ protein expression. AS-IV promoted chondrocyte proliferation and concurrently inhibited cell apoptosis. An optimal AS-IV dose of 200 μmol/L and a suitable reaction time of 48 h were identified. Ghost pen cyclic peptide staining indicated that the model group's cytoskeleton exhibited fusiform changes with reduced immunofluorescence intensity, as opposed to the blank control group. The AS-IV group displayed more polygonal cytoskeletal morphology with increased immunofluorescence intensity. AS-IV reduced TNF-α levels and elevated GAG levels in the culture supernatant. Additionally, AS-IV lowered p62 mRNA and protein expression while increasing LC3 mRNA expression in cultured chondrocytes. Our findings suggest that AS-IV mitigates inflammatory chondrocyte injury, safeguarding chondrocytes through a potential autophagy suppression mechanism. These results imply that AS-IV could offer preventive advantages for OA.

Microfluidic device-assisted 3D cell spheroids isolation, staining and embedding

3D cell spheroids have emerged as indispensable tools in cell biology research. In this study, the efficacy of an open-close switchable microfluidic device for on-chip staining and agar-embedding of 3D spheroids was thoroughly validated, especially in preparation for immunohistochemistry (IHC) analysis. The on-chip staining procedure, employing Trypan blue and Calcein-AM/PI staining, was demonstrated to evaluate the cell viability of spheroids. During overextended culture periods, a discernible decline in cell viability was observed at the central region of the spheroids, offering valuable information on the temporal aspects of spheroid development. On-chip agar embedding stands out for its precise control over the location and orientation of three-dimensional spheroids, a key factor in exploring cellular architecture, especially in fused spheroids using IHC analysis. In comparison to tube-based staining, this method significantly cuts down on reagent use and analysis time. This not only boosts efficiency but also lowers the risk of spheroid damage or loss during staining, highlighting its potential for handling miniaturized tissue samples and biopsies in cell assays. The on-chip staining and agar-embedding techniques presented here not only provide valuable insights into spheroids’ behavior but also pave the way for further developments in miniaturized tissue analysis and cell-based assays.

Neurotrophic extracellular matrix proteins promote neuronal and iPSC astrocyte progenitor cell- and nano-scale process extension for neural repair applications.

The extracellular matrix plays a critical role in modulating cell behaviour in the developing and adult central nervous system influencing neural cell morphology, function and growth. Neurons and astrocytes, play vital roles in neural signalling and support respectively and respond to cues from the surrounding matrix environment. However, a better understanding of the impact of specific individual extracellular matrix proteins on both neurons and astrocytes is critical for advancing the development of matrix-based scaffolds for neural repair applications. This study aimed to provide an in-depth analysis of how different commonly used extracellular matrix proteins- laminin-1, Fn, collagen IV, and collagen I-affect the morphology and growth of trophic induced pluripotent stem cell (iPSC)-derived astrocyte progenitors and mouse motor neuron-like cells. Following a 7-day culture period, morphological assessments revealed that laminin-1, fibronectin, and collagen-IV, but not collagen I, promoted increased process extension and a stellate morphology in astrocytes, with collagen-IV yielding the greatest increases. Subsequent analysis of neurons grown on the different extracellular matrix proteins revealed a similar pattern with laminin-1, fibronectin, and collagen-IV supporting robust neurite outgrowth. fibronectin promoted the greatest increase in neurite extension, while collagen-I did not enhance neurite growth compared to poly-L-lysine controls. Super-resolution microscopy highlighted extracellular matrix-specific nanoscale changes in cytoskeletal organization, with distinct patterns of actin filament distribution where the three basement membrane-associated proteins (laminin-1, fibronectin, and collagen-IV) promoted the extension of fine cellular processes. Overall, this study demonstrates the potent effect of laminin-1, fibronectin and collagen-IV to promote both iPSC-derived astrocyte progenitor and neuronal growth, yielding detailed insights into the effect of extracellular matrix proteins on neural cell morphology at both the whole cell and nanoscale levels. The ability of laminin-1, collagen-IV and fibronectin to elicit strong growth-promoting effects highlight their suitability as optimal extracellular matrix proteins to incorporate into neurotrophic biomaterial scaffolds for the delivery of cell cargoes for neural repair.

A life course study of the Beixin culture residents from the Neolithic site of Xiaheqiadong, Shandong Province, China

AbstractThe Xiaheqiadong site located in Zhangqiu district, Jinan city, Shandong province, China, was excavated in 2016 by the Jinan Institute of Archaeology, which revealed six single burials dating to the Beixin culture period (ca. 5,300–4,500 BC to ca. 4,100–3,600 BC). This paper used bioarchaeological methods to study the human skeletal remains to reconstruct the life course of the residents of the Beixin culture at the Xiaheqiadong site, focusing on stable isotope analysis, evaluation of stress, tooth ablation, and intentional skull modification. Based on stable isotope analysis of carbon, it is evident that the dietary structure of the ancient residents of Xiaheqiadong was mainly dependent on C4 plants or animals that ate mainly C4 plants. The stable isotope analysis of nitrogen indicated that the ancient residents of the Xiaheqiadong site exhibited a relatively sufficient consumption of animal protein. Furthermore, this result also reveals that a subsistence economic model was established consisting of farming, gathering, and hunting. Observations of the stress indicators (including cribra orbitalia, porotic hyperostosis, and linear enamel hypoplasia) revealed that they experienced poor health conditions and were under high levels of stress during infancy and early childhood. However, this situation improved with increasing age, suggesting that residents from the Xiaheqiadong site had the capacity to withstand severe living conditions and adapt after experiencing stress events. The tooth ablation and occipital modification cases from the Xiaheqiadong site are the earliest cases among the individuals with accurate dating results found in China.

Optimizing Growth of Melon (Cucumis melo L. cv. Madesta) in Nutrient Film Technique and Drip Irrigation Hydroponics with Varied Substrates

Hydroponic systems offer a promising solution for urban farming and the utilization of unproductive land. Successful implementation, however, requires careful optimization to select the most effective hydroponic system tailored to specific plants and environmental conditions. This study aims to compare the growth and physiological responses of Madesta melons (Cucumis melo L. cv. Madesta) cultivated using the nutrient film technique (NFT) and drip irrigation system (DIS) with variations in growth media. The Madesta melon seeds underwent a two-week germination phase in coco peat media, followed by transplanting into NFT and DIS setups utilizing diverse growth media, including rice husk, rice husk mixed with compost, and compost only. Over four weeks post-cultivation, assessments were conducted on key growth metrics such as leaf count, leaf diameter, plant height, and stem diameter. Plant physiological responses were also analyzed, encompassing chlorophyll and nitrogen levels, along with the mineral composition within leaves and fruits. Results revealed that the DIS cultivation outperformed the NFT in terms of growth outcomes. Among the varied media combinations, the rice husk and compost blend supported growth most effectively. Notably, no significant differences were observed in leaf and fruit nitrogen content between the DIS and NFT systems, and the overall mineral content of the media remained relatively stable before and after the cultivation period. Mineral content analysis revealed calcium as the predominant element in the leaves, while potassium emerged as the most abundant mineral in the fruits. This research sheds light on the potential of hydroponic systems, specifically the DIS method, for enhancing melon cultivation, emphasizing the importance of selecting appropriate growth media to maximize plant growth.

Integrated RNA Sequencing Analysis Revealed Early Gene Expression Shifts Associated with Cancer Progression in MCF-7 Breast Cancer Cells Cocultured with Adipose-Derived Stem Cells

Breast cancer remains a prevalent global health challenge, with tumor-removal surgeries being among the most common treatments but often leading to aesthetic defects. Adipose-derived stem cell (ADSC)-enriched fat grafting in breast reconstruction offers promising therapeutic benefits. However, concerns about its oncological safety persist, particularly regarding the potential risks of promoting cancer recurrence. This study investigated the effects of ADSCs on breast cancer progression by coculturing ADSCs with the MCF-7 breast cancer cell line for a short cell cultivation period of 3 days. We performed an RNA-seq analysis to identify significant transcriptomic changes in cocultured MCF-7 cells and carried out functional enrichment analyses to uncover key biological pathways influenced by ADSCs. Our findings revealed that transcriptomic alterations in MCF-7 cells are linked to aggressive cancer traits, including the upregulation of epithelial–mesenchymal transition (EMT) and the HIF-1 signaling pathway, which indicate a shift toward aerobic glycolysis. Some of the observed gene expression changes also correlated with relapse risk and mortality. These findings underscore the need for further research to explore the implications of these genes and pathways in driving aggressive cancer phenotypes and assess the safety of ADSCs in clinical settings.

In vitro growth of secondary follicles from cryopreserved-thawed ovarian cortex.

Can secondary follicles be obtained from cultured cryopreserved-thawed human ovarian cortical tissue? We obtained high-quality secondary follicles from cultured cryopreserved-thawed human ovarian cortical tissue from cis female donors (cOVA), but not from trans masculine donors (tOVA) in the same culture conditions. The in vitro growth of oocytes present in unilaminar follicles into metaphase II stage (MII) oocytes has been previously achieved starting from freshly obtained ovarian cortical tissue from adult cis female donors. This involved a multi-step culture protocol and the first step included the transition from unilaminar follicles to multilayered secondary follicles. Given that the ovarian cortex (from both cis female and trans masculine donors) used for fertility preservation is cryopreserved, it is crucial to investigate the potential of unilaminar follicles from cryopreserved-thawed ovarian cortex to grow in culture. Cryopreserved-thawed ovarian cortical tissue from adult trans masculine donors (n = 3) and adult cis female donors (n = 3) was used for in vitro culture following the first culture step described in two published culture protocols (7-8 days and 21 days) and compared to freshly isolated ovarian cortex from trans masculine donors (n = 3) and to ovarian cortex prior to culture. Ovarian cortical tissue was obtained from adult trans masculine donors undergoing gender-affirming surgery while using testosterone, and from adult cis female donors undergoing oophorectomy for fertility preservation purposes before chemotherapy. The ovarian cortex was fixed either prior (day 0) or after the culture period. Follicular survival, growth, and morphology were assessed through histology and immunofluorescence. We quantified the different stages of follicular development (primordial, primary, secondary, and atretic) after culture and observed an increase in the percentage of secondary follicles as well as an increase in COLIV deposition in the stromal compartment regardless of the culture media used. The quality of the secondary follicles obtained from cOVA was comparable to those prior to culture. However, in the same culture conditions, the secondary follicles from tOVA (fresh and cryo) showed low-quality secondary follicles, containing oocytes with small diameter, granulosa cells that expressed abnormal levels of KRT19 and steroidogenic-marker STAR and lacked ACTA2+ theca cells, when compared to tOVA secondary follicles prior to culture. The number of different donors used was limited. Our study revealed that cryopreserved-thawed cOVA can be used to generate high-quality secondary follicles after culture and those can now be further tested to evaluate their potential to generate functional MII oocytes that could be used in the clinic. However, using the same culture protocol on tOVA (fresh and cryo) did not yield high-quality secondary follicles, suggesting that either the testosterone treatment affects follicular quality or adapted culture protocols are necessary to obtain high-quality secondary follicles from tOVA. Importantly, caution must be taken when using tOVA to optimize folliculogenesis in vitro. This research was funded by the European Research Council Consolidator Grant OVOGROWTH (ERC-CoG-2016-725722 to J.S.D.V. and S.M.C.D.S.L.), the Novo Nordisk Foundation (reNEW NNF21CC0073729 to H.C., F.W., J.S.D.V., S.M.C.D.S.L.), and China Scholarship Council (CSC 202008320362 and CSC 202008450034 to H.C. and F.W.), respectively. The authors have no conflicts of interest to declare. N/A.

A Non-Destructive Measurement Approach for the Internal Temperature of Shiitake Mushroom Sticks Based on a Data–Physics Hybrid-Driven Model

This study aimed to develop a non-destructive measurement method utilizing acoustic sensors for the efficient determination of the internal temperature of shiitake mushroom sticks during the cultivation period. In this research, the sound speed, air temperature, and moisture content of the mushroom sticks were employed as model inputs, while the temperature of the mushroom sticks served as the model output. A data–physics hybrid-driven model for temperature measurement based on XGBoost was constructed by integrating monotonicity constraints between the temperature of the mushroom sticks and sound speed, along with the condition that limited the difference between air temperature and stick temperature to less than 2 °C. The experimental results indicated that the optimal eigenfrequency for applying this model was 850 Hz, the optimal distance between the sound source and the shiitake mushroom sticks was 8.7 cm, and the temperature measurement accuracy was highest when the moisture content of the shiitake mushroom sticks was in the range of 56~66%. Compared to purely data-driven models, our proposed model demonstrated significant improvements in performance; specifically, RMSE, MAE, and MAPE decreased by 74.86%, 77.22%, and 69.30%, respectively, while R2 increased by 1.86%. The introduction of physical knowledge constraints has notably enhanced key performance metrics in machine learning-based acoustic thermometry, facilitating efficient, accurate, rapid, and non-destructive measurements of internal temperatures in shiitake mushroom sticks.

From Organotypic Mouse Brain Slices to Human Alzheimer’s Plasma Biomarkers: A Focus on Nerve Fiber Outgrowth

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by memory loss and progressive deterioration of cognitive functions. Being able to identify reliable biomarkers in easily available body fluids such as blood plasma is vital for the disease. To achieve this, we used a technique that applied human plasma to organotypic brain slice culture via microcontact printing. After a 2-week culture period, we performed immunolabeling for neurofilament and myelin oligodendrocyte glycoprotein (MOG) to visualize newly formed nerve fibers and oligodendrocytes. There was no significant change in the number of new nerve fibers in the AD plasma group compared to the healthy control group, while the length of the produced fibers significantly decreased. A significant increase in the number of MOG+ dots around these new fibers was detected in the patient group. According to our hypothesis, there are factors in the plasma of AD patients that affect the growth of new nerve fibers, which also affect the oligodendrocytes. Based on these findings, we selected the most promising plasma samples and conducted mass spectrometry using a differential approach and we identified three putative biomarkers: aldehyde-dehydrogenase 1A1, alpha-synuclein and protein S100-A4. Our method represents a novel and innovative approach for translating research findings from mouse models to human applications.

Water retention characteristics and mechanical properties of vegetated biopolymer and biochar-reinforced sandy loam

Vegetation is a sustainable strategy for erosion control and slope stabilization, though its initial cultivation can be lengthy and potentially weaken soil structures. This study compared two bio-mediated ground improvement techniques, biopolymer and biochar, known for their supportive effects on vegetation growth. Additionally, a novel treatment combining biopolymer and biochar was examined for its potential in vegetated-engineering practices. Engineering performance was assessed through soil water characteristic curve, vegetation growth, direct shear testing, and rainfall simulation. The results revealed that biopolymer and biochar treatments enhanced soil water capacity but negatively impacted vegetation germination rates and shear strength of the reinforced soil, attributed to hydrogel formation and increased soil water content from irrigation. In comparison, soil reinforced with the combined method showed a promotion in the vegetation while maintaining the soil’s mechanical performance throughout the cultivation period and exhibited only minor reductions in the shear strength compared to other reinforced soils. Moreover, the new treatment showed improved soil erodibility under a majority of rainfall occasions, regardless of the vegetation coverage. This enhanced engineering performance by the new treatment is believed to be the polymerisation between the biopolymer hydrogel, biochar and soil particles.

External nutrients fattening promotes hepatopancreases development, metabolism, nutrient deposition and flavor yield during the maturation stage in female Chinese mitten crab (Eriocheir sinensis)

The nutritive value and maturity status of hepatopancreases dominates the quality of Chinese mitten crab, Eriocheir sinensis. However, the underlying function and regulatory mechanism, as well as the external nutrient factors on the development of hepatopancreases in different growth stages of E. sinensis remains unclear. In this study, we first revealed the function of hepatopancreases feeding with iced trash fish (ITF) during the whole culture period; and further investigated the underlying mechanism on nutrient deposition and flavor yield feeding with formulated feed (FEED) in replacement of ITF in female E. sinensis. From the results, growth performance and metabolism indicate E. sinensis was robustly developed, and ITF promotes growth and hepatopancreases development during the maturation period. Meanwhile, activation of fatty acid and amino acid metabolism in hepatopancreases resulted in depletion of monounsaturated fatty acids (MUFAs) but promoted protein deposition during the maturation stage. Integrated analysis of transcriptome and metabolome reveal metabolism, biosynthesis, and flavor presentation were intensively active during maturation, evidenced by the expression of key genes of ALDH, TBXAs, HADHA, UPB1, CYP2, GST, and γ-GT-X1; as well as the key metabolites of GMP and IMP. Additionally, substitution of ITF with FEED further confirm full price artificial feed could meet the nutrient requirements during the maturation period, evidenced by the growth performance, fatty acid and amino acid derived nutrient and flavor yield in hepatopancreases. In conclusion, nutrient and flavor deposition was associated with the fast development of hepatopancreases, and fatten with full price compound feed contributes to the nutrient requirement and flavor yield during the maturation stage.

A human ex vivo skin model breaking boundaries

Ex vivo human skin models are valuable tools in skin research due to their physiological relevance. Traditionally, standard cultivation is performed in a cell culture incubator with a defined temperature of 37 °C and a specific atmosphere enriched with CO2 to ensure media stability. Maintaining the model under these specific conditions limits its flexibility in assessing exposures to which the skin is exposed to in daily life, for example changes in atmospheric compositions. In this study we demonstrated that the foreskin-derived skin model can be successfully cultured at room temperature outside a CO2 incubator using a CO2-independent, serum-free media. Over a cultivation period of three days, the integrity of the tissue and the preservation of immune cells is well maintained, indicating the model’s stability and resilience under the given conditions. Exposing our Medical University of Graz – human Organotypic Skin Explant Culture (MUG-hOSEC) model to cytotoxic and inflammatory stimuli results in responses analyzable within the supernatant. Besides the common analysis of released proteins upon treatment, such as cytokines and enzymes, we have included extracellular vesicle to obtain a more comprehensive picture of cell communication.

Changes in Water Quality and Soil Property in the Rice–Freshwater Animal Co-Culturing System

This study investigated the effects of integrating various fish species in a rice field co-culture system on water quality and soil properties. The species included common carp (Cyprinus carpio), Nile tilapia (Oreochromis niloticus), silver barb (Barbonymus gonionotus), snakeskin gourami (Trichopodus pectoralis), and giant freshwater prawn (Macrobrachium rosenbergii). The key water quality parameters measured included water temperature, dissolved oxygen, pH, transparency, ammonia, and nitrite. Soil properties were evaluated through pH, electrical conductivity, organic matter, nitrogen, phosphorus, and potassium. All the selected aquatic animals showed high adaptability in co-culture systems, with survival rates exceeding 80%. Additionally, rice yields increased by approximately 16%, with the highest yield observed in plots stocked with prawns. The results indicated that the presence of aquatic animals enhanced nutrient cycling, leading to significant improvements in both water quality and soil fertility. Differences in water quality and soil properties throughout the culture period were specific to the species present. These findings suggest that rice-fish co-culture systems serve as an effective nature-based solution for enhancing productivity, sustainability, and food security.

Crop Coefficients and Irrigation Demand in Response to Climate-Change-Induced Alterations in Phenology and Growing Season of Vegetable Crops

This study investigates the effects of climate change on the irrigation demand of vegetable crops caused by alteration of climate parameters affecting evapotranspiration (ET), plant development, and growing periods in Central Europe. Utilizing a model framework comprising two varying climate scenarios (RCP 2.6 and RCP 8.5) and two regional climate models (COSMO C-CLM and WETTREG 2013), we calculate the daily crop water balance (CWBc) as a measure for irrigation demand based on reference ET and the temperature-driven duration of crop coefficients until 2100. Our findings for onion show that rising temperatures may shorten cultivation periods by 5 to 17 days; however, the irrigation demand may increase by 5 to 71 mm due to higher ET. By reaching the base temperatures for onion growth earlier in the year, cultivation start can be advanced by up to 30 days. Greater utilization of winter soil moisture reduces the irrigation demand by up to 21 mm, though earlier cultivation is restricted by frost risks. The cultivation of thermophilic crops, however, cannot be advanced to the same extent, as shown for bush beans, and plants will transpire more strongly due to longer dry periods simulated for summer. The results underscore the need for adaptive crop and water management strategies to counteract the simulated changes in phenology and irrigation demand of vegetable crops. Therefore, special consideration must be given to the regional-specific and model- and scenario-dependent simulation results.

Computation of evapotranspiration using crop simulation models and comparison with leaf area index from multiple sources

The study demonstrated the computation of evapotranspiration (ET) in cultivation of groundnut (Arachis hypogaea) through the application of crop simulation models, alongside a comparative analysis with leaf area index (LAI) from various sources. The cultivation period for groundnut was conducted during the calendar year 2023, during which 2 distinct growth patterns were noted, attributed to variations in environmental conditions. The study analyses the estimated evapotranspiration from AquaCrop model, utilizing crop coefficient (Kc) values in comparison with evapotranspiration data derived from satellite observations provided by the MOD16A2v061 product. Furthermore, LAI was measured through 3 methodologies: an empirical equation based on field data, the Li-Cor 2200 plant canopy analyzer and LAI calculations derived from cloud-free normalized difference vegetation index (NDVI). LAI obtained from Li-Cor 2200 instrument exhibited a higher degree of consistency in correlation with empirical LAI derived from ground observations. Conversely, LAI values calculated using the normalized difference vegetation index (NDVI) demonstrated a greater degree of variability, especially during times of cloud cover. The study emphasizes the relationship between LAI and ET and magnitude of LAI in amount of total evapotranspiration.

Magnetically Driven Hydrogel Surfaces for Modulating Macrophage Behavior.

During the host response toward implanted biomaterials, macrophages can shift phenotypes rapidly upon changes in their microenvironment within the host tissue. Exploration of this phenomenon can benefit significantly from the development of adequate tools. Creating cell microenvironment alterations on classical hydrogel substrates presents challenges, particularly when integrating them with cell cultivation and monitoring processes. However, having the capability to dynamically manipulate the cell microenvironment on biomaterial surfaces holds significant potential. We introduce magnetically actuated hydrogels (MadSurface) tailored to induce reversible stiffness changes on polyacrylamide hydrogel substrates with embedded magnetic microparticles in a time-controllable manner. Our investigation focused on exploring the potential of magnetic fields and MadSurfaces in dynamically modulating macrophage behavior in a programmable manner. We achieved a consistent modulation by subjecting the MadSurface to a pulsed magnetic field with a frequency of 0.1 Hz and a magnetic field flux density of 50 mT and analyzed exposed cells using flow cytometry and ELISA. At the single-cell level, we identified a subpopulation for which the dynamic stiffness conditions in conjunction with the pulsed magnetic field increased the expression of CD206 in M1-activated THP-1 cells, indicating a consistent shift toward the M2 anti-inflammatory phenotype on MadSurface. At the population level, this effect was mostly hindered in the culture period utilized in this work. The MadSurface approach advances our understanding of the interplay between magnetic field, cell microenvironment alterations, and macrophage behavior.

9194 Generation of a Human-Induced Pluripotent Stem Cell Line From a Patient With Hypopituitarism and a Novel Nonsense Variant in FOXA2

Abstract Disclosure: M.A. Camilletti: None. G.T. Chirino Felker: None. G. Amin: None. S. Castañeda: None. F. Zabalegui: None. C. Romano Florit: None. A. Waisman: None. M. Ciaccio: None. M.I. Di Palma: None. E. Vaiani: None. A. Belgorosky: None. S.G. Miriuka: None. L.N. Moro: None. M.I. Perez-Millan: None. Congenital Hypopituitarism (CH) is a genetically complex disease characterized by one or more pituitary hormone deficiencies. Forkhead Box A2 (FOXA2, 600288) is a pioneer transcription factor associated with development of multiple tissues in humans and mice. Heterozygous loss of function mutations in FOXA2 were recently shown to cause CH with hyperinsulinemic hypoglycemia, but little is known about the molecular mechanism of FOXA2 action during pituitary development. We identified a novel, heterozygous nonsense variant in FOXA2 (c.686C<A; p.S229X) in a patient diagnosed with GH deficiency at 1.7 years of age, with anterior lobe hypoplasia and absent pituitary stalk, asymptomatic hypoglycemia, and craniofacial malformations. To discover the mechanism of FOXA2 regulation of pituitary development we generated an induced pluripotent stem cell line (FOXA2mut-iPSC) from the patient’s polymorphonuclear blood cells (PBMCs) using an EF1a-hSTEMCCA-loxP vector containing the pluripotency genes OCT-4, SOX-2, c-MYC and KLF4. FOXA2mut-iPSC clone R1 had a normal karyotype and embryonic stem cell-like morphology. These cells express endogenous pluripotency-associated markers NANOG, SOX2, and OCT4, have alkaline phosphatase activity, and can differentiate into all three developmental layers, indicating that these cells are pluripotent. Short tandem repeat (STR) analysis confirmed that FOXA2mut-iPSC R1 clone profiles matched those of the donor PBMCs. Next, we compared pituitary differentiation of FOXA2mut-iPSC vs. control-iPSCs in vitro. Cells were cultured in a differentiation medium containing BMP4, the smoothened agonist SAG, and fibroblast growth factor (FGF), as described in Zimmer et al., 2016 (1). Gene expression analysis (by qRT-PCR) of samples collected on days 0, 4, 7, and 15 of the differentiation protocol showed an increased expression of genes associated with anterior pituitary development including OTX2, PITX1/2, and SIX1 in both cell lines (n=2). These preliminary data suggest that pituitary cell fate can be induced in the heterozygous FOXA2mut-iPSC clone. Ongoing studies will assess pituitary hormone-producing cell differentiation after longer periods of FOXA2mut-iPSC cell culture. In sum, patient iPSC differentiation is a promising tool for assessment of normal and variant FOXA2 function in human pituitary development, and for enlightening the mechanisms of FOXA2 action in human pituitary development. (1) Zimmer et al., Stem Cell Reports. 2016 Jun 14;6(6):858-872. Presentation: 6/1/2024

7151 Generation of a Human-Induced Pluripotent Stem Cell Line From a Patient With Hypopituitarism and a Novel Nonsense Variant in FOXA2

Abstract Disclosure: M.A. Camilletti: None. G.T. Chirino Felker: None. G. Amin: None. S. Castañeda: None. F. Zabalegui: None. C. Romano Florit: None. A. Waisman: None. M. Ciaccio: None. M.I. Di Palma: None. E. Vaiani: None. A. Belgorosky: None. S.G. Miriuka: None. L.N. Moro: None. M.I. Perez-Millan: None. Congenital Hypopituitarism (CH) is a genetically complex disease characterized by one or more pituitary hormone deficiencies. Forkhead Box A2 (FOXA2, 600288) is a pioneer transcription factor associated with development of multiple tissues in humans and mice. Heterozygous loss of function mutations in FOXA2 were recently shown to cause CH with hyperinsulinemic hypoglycemia, but little is known about the molecular mechanism of FOXA2 action during pituitary development. We identified a novel, heterozygous nonsense variant in FOXA2 (c.686C<A; p.S229X) in a patient diagnosed with GH deficiency at 1.7 years of age, with anterior lobe hypoplasia and absent pituitary stalk, asymptomatic hypoglycemia, and craniofacial malformations. To discover the mechanism of FOXA2 regulation of pituitary development we generated an induced pluripotent stem cell line (FOXA2mut-iPSC) from the patient’s polymorphonuclear blood cells (PBMCs) using an EF1a-hSTEMCCA-loxP vector containing the pluripotency genes OCT-4, SOX-2, c-MYC and KLF4. FOXA2mut-iPSC clone R1 had a normal karyotype and embryonic stem cell-like morphology. These cells express endogenous pluripotency-associated markers NANOG, SOX2, and OCT4, have alkaline phosphatase activity, and can differentiate into all three developmental layers, indicating that these cells are pluripotent. Short tandem repeat (STR) analysis confirmed that FOXA2mut-iPSC R1 clone profiles matched those of the donor PBMCs. Next, we compared pituitary differentiation of FOXA2mut-iPSC vs. control-iPSCs in vitro. Cells were cultured in a differentiation medium containing BMP4, the smoothened agonist SAG, and fibroblast growth factor (FGF), as described in Zimmer et al., 2016 (1). Gene expression analysis (by qRT-PCR) of samples collected on days 0, 4, 7, and 15 of the differentiation protocol showed an increased expression of genes associated with anterior pituitary development including OTX2, PITX1/2, and SIX1 in both cell lines (n=2). These preliminary data suggest that pituitary cell fate can be induced in the heterozygous FOXA2mut-iPSC clone. Ongoing studies will assess pituitary hormone-producing cell differentiation after longer periods of FOXA2mut-iPSC cell culture. In sum, patient iPSC differentiation is a promising tool for assessment of normal and variant FOXA2 function in human pituitary development, and for enlightening the mechanisms of FOXA2 action in human pituitary development. (1) Zimmer et al., Stem Cell Reports. 2016 Jun 14;6(6):858-872. Presentation: 6/1/2024

Developing a workflow for the isolation of hybridoma cells producing fully human antigen-specific antibodies using a surface IgG detection method

The antigen-mediated B cell isolation method, based on the detection of surface IgG (sIgG), has increased the efficiency of therapeutic antibody (Ab) discovery. However, the reduction in sIgG expression on B cells during plasma cell differentiation presents challenges as it enables Ab production from only a small subset of B cells (e.g., memory B cells). The present study aimed to addressed this problem by developing a workflow to isolate human-IgG-secreting hybridoma cells produced by cell fusion, the majority of which express sIgG. We showed that our sIgG-based antigen-coated bead separation method efficiently enriched hybridoma cells expressing antigen-specific Abs with a yield of 83.5% (from the cell fusion pool) and a positive rate of 73.2%. Furthermore, because the separation could be performed after only a short (1–2-day) culture period following cell fusion, diverse hybridoma clones could be obtained, minimizing clonal selection and the incidence of duplicates. Given that the expression of membrane-bound IgG and sIgG are regulated by different splicing mechanisms, we speculate that the cell fusion step potentially attenuated the suppression of human sIgG expression. Overall, our proposed method is expected to markedly improve the efficiency of therapeutic Ab candidate production, which will have important clinical implications.