Differential Efficiency Research Articles

Integrated transcriptomic and metabolomic analyses elucidate the mechanism by which grafting impacts potassium utilization efficiency in tobacco

BackgroundPotassium plays a crucial role in determining the quality of flue-cured tobacco leaves. Our prior investigations have demonstrated that using potassium-efficient rootstocks through grafting offers a viable solution to the prevalent issue of low potassium levels in Chinese flue-cured tobacco leaves. Nevertheless, the specific molecular mechanisms responsible for the increase in potassium content following grafting in tobacco leaves have yet to be elucidated. This study revealing for the first time how grafting improves potassium utilization efficiency through combined transcriptome and metabolome analysis.ResultsThis study selected Wufeng NO. 2, a potassium-efficient variety, and Yunyan 87, a main cultivar, as the research subjects to investigate the underlying reasons for differential potassium utilization efficiency among different tobacco rootstocks through transcriptome and metabolic data analysis of grafted tobacco. The results showed a considerable increment of 90.1% in the potassium content of the grafted tobacco leaves. Overall, 2044 differentially expressed genes were identified through transcriptome analysis, with the majority being enriched in plant hormone signal transduction and the MAPK pathway. Metabolome analysis revealed 175 metabolites with significant differences, primarily involving primary metabolites such as amino acids and carbohydrates. Among these, there was an increase in the metabolites levels related to glycolysis, amino acid metabolism, and the TCA cycle pathway in grafted tobacco leaves. The key metabolites and genes in the above pathways were selected for Mantel-Pearson correlation analysis, leading to the identification of 2 genes and 3 metabolites, including IAA, CIP1, D-fructose, Fumaric acid and Oxoglutaric acid, that were significantly associated with the increased potassium content in grafted tobacco.ConclusionsThis study uncovers the intricate molecular mechanism behind grafting tobacco to enhance potassium utilization efficiency, thereby offering theoretical support for enhancing crop nutrient utilization efficiency through grafting technology.

Imeglimin, unlike metformin, does not perturb differentiation of human induced pluripotent stem cells towards pancreatic β-like cells and rather enhances gain in β cell identity gene sets.

Metformin treatment for hyperglycemia in pregnancy (HIP) beneficially improves maternal glucose metabolism and reduces perinatal complications. However, metformin could impede pancreatic β cell development via impaired mitochondrial function. A new anti-diabetes drug imeglimin, developed based on metformin, improves mitochondrial function. Here we examine the effect of imeglimin on β cell differentiation using human induced pluripotent stem cell (iPSC)-derived pancreatic islet-like spheroid (SC-islet) models. Human iPSCs are differentiated into SC-islets by three-dimensional culture with and without imeglimin or metformin. Differentiation efficiencies of SC-islets were analyzed by flow cytometry, immunostaining, quantitative PCR, and insulin secretion assay. RNA sequencing and oxygen consumption rate were obtained for further characterization of SC-islets. SC-islets were cultured with proinflammatory cytokines, in part mimicking the uterus environment in HIP. Metformin perturbed SC-islet differentiation while imeglimin did not alter it. Furthermore, imeglimin enhanced the gene expressions of β cell lineage markers. Maintenance of mitochondrial function and optimization of TGF-β and Wnt signaling were considered potential mechanisms for augmented β cell maturation by imeglimin. In the presence of proinflammatory cytokines, imeglimin ameliorated β cell differentiation impaired by cytokines and metformin. Imeglimin does not perturb differentiation of SC-islet cells and rather enhances gain in β cell identity gene sets in contrast to metformin. This may lead to the improvement of in vitro β cell differentiation protocols.

Efficient differentiation of human iPSCs into Leydig-like cells capable of long-term stable secretion of testosterone.

Late-onset hypogonadism (LOH) syndrome is characterized by age-related testosterone deficiency and negatively affects the quality of life of older men. A promising therapeutic approach for LOH syndrome is transplantation of testosterone-producing Leydig-like cells (LLCs) derived from human induced pluripotent stem cells (hiPSCs). However, previous studies have encountered obstacles, such as limited cell longevity, insufficient testosterone production, and inefficiency of differentiation. To address these issues, we developed a novel protocol that includes forced NR5A1 expression, a cytokine cocktail promoting mesoderm differentiation, and a transitional shift from 3D to 2D cultures. The resultant cells survived on culture dishes for over 16weeks, produced 22-fold more testosterone than the conventional method, and constituted a homogeneous population of LLCs with a differentiation efficiency exceeding 99% without purification. Furthermore, these LLCs were successfully engrafted subcutaneously into mice, resulting in increased serum testosterone levels. Our study will facilitate innovative therapeutic strategies for LOH syndrome.

Dual-stimuli-responsive nanoparticles for the co-delivery of small molecules to promote neural differentiation of human iPSCs.

The differentiation of human induced pluripotent stem cells (hiPSCs) into neural progenitor cells (NPCs) is a promising approach for the treatment of neurodegenerative diseases and regenerative medicine. Dual-SMAD inhibition using small molecules has been identified as a key strategy for directing the differentiation of hiPSCs into NPCs by regulating specific cell signaling pathways. However, conventional culture methods are time-consuming and exhibit low differentiation efficiency in neural differentiation. Nanocarriers can address these obstacles as an efficient platform for the controlled release and accurate delivery of small molecules. In this paper, we developed calcium phosphate-coated mesoporous silica nanoparticles capable of delivering multiple small molecules, including LDN193189 as a bone morphogenetic protein (BMP) inhibitor and SB431542 as a transforming growth factor (TGF)-beta inhibitor, for direct differentiation of hiPSC-mediated NPCs. Our results demonstrated that this nanocarrier-mediated small molecule release system not only enhanced the in vitro formation of neural rosettes but also modulated the expression levels of key markers. In particular, it downregulated OCT4, a marker of pluripotency, while upregulating PAX6, a critical marker for the neuroectoderm. These findings suggest that this controlled small molecule release system holds significant potential for therapeutic applications in neural development and neurodegenerative diseases.

Comparative diagnostic accuracy of ChatGPT-4 and machine learning in differentiating spinal tuberculosis and spinal tumors.

In clinical practice, distinguishing between spinal tuberculosis (STB) and spinal tumors (ST) poses a significant diagnostic challenge. The application of AI-driven large language models (LLMs) shows great potential for improving the accuracy of this differential diagnosis. To evaluate the performance of various machine learning models and ChatGPT-4 in distinguishing between STB and ST. A retrospective cohort study. 143 STB cases and 153 ST cases admitted to Xiangya Hospital Central South University, from January 2016 to June 2023 were collected. This study incorporates basic patient information, standard laboratory results, serum tumor markers, and comprehensive imaging records, including Magnetic Resonance Imaging (MRI) and Computed Tomography (CT), for individuals diagnosed with STB and ST. Machine learning techniques and ChatGPT-4 were utilized to distinguish between STB and ST separately. Six distinct machine learning models, along with ChatGPT-4, were employed to evaluate their differential diagnostic effectiveness. Among the 6 machine learning models, the Gradient Boosting Machine (GBM) algorithm model demonstrated the highest differential diagnostic efficiency. In the training cohort, the GBM model achieved a sensitivity of 98.84% and a specificity of 100.00% in distinguishing STB from ST. In the testing cohort, its sensitivity was 98.25%, and specificity was 91.80%. ChatGPT-4 exhibited a sensitivity of 70.37% and a specificity of 90.65% for differential diagnosis. In single-question cases, ChatGPT-4's sensitivity and specificity were 71.67% and 92.55%, respectively, while in re-questioning cases, they were 44.44% and 76.92%. The GBM model demonstrates significant value in the differential diagnosis of STB and ST, whereas the diagnostic performance of ChatGPT-4 remains suboptimal.

Efficacy and Mechanism of Highly Active Umbilical Cord Mesenchymal Stem Cells in the Treatment of Osteoporosis in Rats.

A rat model of osteoporosis was induced with dexamethasone sodium phosphate. Highly active umbilical cord mesenchymal stem cells (HA-UCMSCs) and UCMSCs were isolated, cultured, identified, and infused intravenously once at a dose of 2.29 × 106 cells/kg. In the 4th week of treatment, bone mineral density (BMD) was evaluated via cross-micro-CT, tibial structure was observed via HE staining, osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) was examined via alizarin red staining, and carboxy-terminal cross-linked telopeptide (CTX), nuclear factor-κβ ligand (RANKL), procollagen type 1 N-terminal propeptide (PINP) and osteoprotegerin (OPG) levels were investigated via enzyme-linked immunosorbent assays (ELISAs). BMMSCs were treated with 10-6 mol/L dexamethasone and cocultured with HA-UCMSCs and UCMSCs in transwells. The osteogenic and adipogenic differentiation of BMMSCs was subsequently examined through directional induction culture. The protein expression levels of WNT, β-catenin, RUNX2, IFN-γ and IL-17 in the bone tissue were measured via Western blotting. The BMD in the healthy group was higher than that in the model group. Both UCMSCs and HA-UCMSCs exhibited a fusiform morphology; swirling growth; high expression of CD73, CD90 and CD105; and low expression of CD34 and CD45 and could differentiate into adipocytes, osteoblasts and chondrocytes, while HA-UCMSCs were smaller in size; had a higher nuclear percentage; and higher differentiation efficiency. Compared with those in the model group, the BMD increased, the bone structure improved, the trabecular area, number, and perimeter increased, the osteogenic differentiation of BMMSCs increased, RANKL expression decreased, and PINP expression increased after UCMSC and HA-UCMSC treatment for 4 weeks. Furthermore, the BMD, trabecular area, number and perimeter, calcareous nodule counts, and OPG/RANKL ratio were higher in the HA-UCMSC treatment group than in the UCMSC treatment group. The osteogenic and adipogenic differentiation of dexamethasone-treated BMMSCs was enhanced after the coculture of UCMSCs and HA-UCMSCs, and the HA-UCMSC group exhibited better effects than the UCMSC coculture group. The protein expression of WNT, β-catenin, and runx2 was upregulated, and IFN-γ and IL-17 expression was downregulated after UCMSC and HA-UCMSC treatment. HA-UCMSCs have a stronger therapeutic effect on osteoporosis compared with that of UCMSCs. These effects include an improved bone structure, increased BMD, an increased number and perimeter of trabeculae, and enhanced osteogenic differentiation of BMMSCs via activation of the WNT/β-catenin pathway and inhibition of inflammation.</p>.

Synthesis and characterization of turmeric extract-β-cyclodextrin inclusion complexes: Metabolite profiling and antioxidant activity.

Turmeric (Curcuma longa L.) has gained significant attention for its medicinal properties, yet its therapeutic applications are often limited by low aqueous solubility and susceptibility to environmental factors. This study investigates the formulation of a curcumin-rich turmeric extract-β-cyclodextrin inclusion complex (TUE-β-CD) to enhance its bioactivity and stability. Structural characterization and metabolite profiling of the inclusion complex were conducted using field-emission scanning electron microscopy, thermogravimetric analysis, X-ray diffraction, Fourier transform infrared spectroscopy, and ultrahigh-performance liquid chromatography coupled with LTQ-Orbitrap-mass spectrometry (UHPLC-LTQ-Orbitrap-MS). Results revealed that the inclusion complexes exhibit distinct morphological, spectroscopic, crystalline, and thermal properties compared to both curcumin-rich TUE and β-CD, confirming successful encapsulation of turmeric metabolites within β-cyclodextrin cavities. Results of UHPLC-LTQ-Orbitrap-MS confirmed that β-CD had differential encapsulation efficiencies for bioactive compounds of turmeric. The results suggest that the inclusion complex significantly improves the thermal stability and bioactivity of turmeric extract, thereby enhancing its potential applications as a functional component in both food and non-food industries.

Nanoelectrode-Mediated Extracellular Electrical Stimulation Directing Dopaminergic Neuronal Differentiation of Stem Cells for Improved Parkinson's Disease Therapy.

Parkinson's disease (PD) is a neurodegenerative disease caused by the dysfunction and death of dopaminergic neurons. Neural-stem-cell (NSC)-based therapy is a promising approach for the treatment of PD but its therapeutic performance is limited by low efficiency of differentiation of NSCs to dopaminergic neurons. Although electrical stimulation can promote neuronal differentiation, it is not verified whether it can induce the NSCs to specifically differentiate into dopaminergic neurons. Meanwhile, it is a great challenge to precisely apply electrical stimulation to dynamically migrating NSCs after transplantation. Here, electrochemically exfoliated graphene nanosheets are designed to anchor to the membrane of NSCs to serve as wireless nanoelectrodes. After anchoring to the cell membrane, these nanoelectrodes are able to migrate together with the cells and precisely apply extracellular electrical stimulation to the receptors or ion transport channels on the membrane of transplanted cells under alternating magnetic field. The nanoelectrode-mediated electrical stimulation induces 38.46% of the NSCs to specifically differentiate into dopaminergic neurons, while the percentage is only 5.82% for NSCs without the nanoelectrode stimulation. Transplantation of NSCs anchored with the nanoelectrodes effectively improves the recovery of the motor and memory ability of PD mice under alternating magnetic field within 2 weeks.

Development of differential diagnostic models for distinguishing between limb-girdle muscular dystrophy and idiopathic inflammatory myopathy

ObjectiveLimb-girdle muscular dystrophy (LGMD) is usually confused with idiopathic inflammatory myopathy (IIM) in clinical practice. Our study aimed to establish convenient and reliable diagnostic models for distinguishing between LGMD and IIM.MethodsA total of 71 IIM patients, 24 LGMDR2 patients and 22 LGMDR1 patients diagnosed at our neuromuscular center were enrolled. Differences in clinical, laboratory and histopathological characteristics were comprehensively compared. A nomogram and a decision tree were developed to distinguish between LGMD and IIM patients.ResultsCompared to patients with LGMD, IIM patients exhibited a significantly older age of onset, a higher prevalence of cervical flexor weakness and a more commonly diffuse MHC-I expression on muscle pathology. The ratio of synchronous serum myoglobin (Mb, ng/ml) to creatine kinase (CK, U/L) before immunotherapy was significantly higher in IIM patients than in LGMD patients. Receiver operating characteristic analysis indicated a high differential diagnostic efficiency of synchronous Mb/CK with a cutoff value of 0.18. A nomogram prediction model and a decision tree were developed based on four independent indicators (age of onset, cervical flexor weakness, synchronous Mb/CK and diffuse MHC-I expression). Five-fold cross-validation and bootstrapping techniques substantiated the discriminate efficacy of the nomograph and decision tree.ConclusionWe developed two practical differential diagnosis models for LGMD and IIM based on the analysis of four accessible indicators, including the age of onset, cervical flexor weakness, the ratio of synchronous Mb/CK values and diffuse MHC-I expression. Further studies with larger samples are needed to refine the predictive efficiency of the differential diagnostic models.

Time-efficient strategies in human iPS cell-derived pancreatic progenitor differentiation and cryopreservation: advancing towards practical applications

BackgroundDifferentiation of patient-specific induced pluripotent stem cells (iPS) helps researchers to study the individual sensibility to drugs. However, differentiation protocols are time-consuming, and not all tissues have been studied. Few works are available regarding pancreatic exocrine differentiation of iPS cells, and little is known on culturing and cryopreserving these cells.MethodsWe differentiated the iPS cells of two pediatric Crohn’s disease patients into pancreatic progenitors and exocrine cells, adapting and shortening a protocol for differentiating embryonic stem cells. We analyzed the expression of key genes and proteins of the differentiation process by qPCR and immunofluorescence, respectively. We explored the possibility of keeping differentiated cells in culture and freezing and thawing them to shorten the time needed for the differentiation. We analyzed the cell cycle of undifferentiated and differentiated cells by flow cytometry.ResultsThe analysis of mRNA levels of key pancreatic differentiation genes PDX1 and pancreatic amylase indicate that iPS cells were successfully differentiated into pancreatic exocrine cells with expression of PDX1 (one way ANOVA p < 0.0001), and the two isoforms of amylase (one way ANOVA p < 0.05) significantly higher in exocrine cells in comparison to iPS cells. Differentiation efficiency was also confirmed by immunofluorescence analysis of PDX1 and amylase. We confirmed the possibility of shortening the time necessary for obtaining pancreatic cells without losing differentiation efficiency. Pancreatic progenitors and exocrine cells were maintained in culture and cryopreserved. Interestingly, the stemness marker OCT4 resulted significantly lower after subculturing (OCT4 p < 0.001; one-way ANOVA) and after freezing and thawing procedures (p < 0.05, one-way ANOVA) suggesting a reduction of undifferentiated stem cells leading to a purer population of pancreatic progenitor cells. Also, the stemness marker NANOG resulted lower after passaging, corroborating this result.ConclusionsIn this work, we optimized the generation of patient-specific pancreatic differentiated cells and laid the foundation for creating a bank of patient-specific pancreatic lines exploitable for tailored pharmacological assays.Trial registrationThe study was approved by the Ethical Committee of the Institute of Maternal and Child Health IRCCS Burlo Garofolo, with approval number 1556 (internal ID RC 44/22).

Transcriptome and epigenome dynamics of the clonal heterogeneity of human induced pluripotent stem cells for cardiac differentiation

Human induced pluripotent stem cells (hiPSCs) generate multiple clones with inherent heterogeneity, leading to variations in their differentiation capacity. Previous studies have primarily addressed line-to-line variations in differentiation capacity, leaving a gap in the comprehensive understanding of clonal heterogeneity. Here, we aimed to profile the heterogeneity of hiPSC clones and identify predictive biomarkers for cardiomyocyte (CM) differentiation capacity by integrating transcriptomic, epigenomic, endogenous retroelement, and protein kinase phosphorylation profiles. We generated multiple clones from a single donor and validated that these clones exhibited comparable levels of pluripotency markers. The clones were classified into two groups based on their differentiation efficiency to CMs—productive clone (PC) and non-productive clone (NPC). We performed RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin with sequencing (ATAC-seq). NPC was enriched in vasculogenesis and cell adhesion, accompanied by elevated levels of phosphorylated ERK1/2. Conversely, PC exhibited enrichment in embryonic organ development and transcription factor activation, accompanied by increased chromatin accessibility near transcription start site (TSS) regions. Integrative analysis of RNA-seq and ATAC-seq revealed 14 candidate genes correlated with cardiac differentiation potential. Notably, TEK and SDR42E1 were upregulated in NPC. Our integrative profiles enhance the understanding of clonal heterogeneity and highlight two novel biomarkers associated with CM differentiation. This insight may facilitate the identification of suboptimal hiPSC clones, thereby mitigating adverse outcomes in clinical applications.Graphical

Использование комплексной дифференциации и кластеризации геолого-промысловой информации при прогнозировании продуктивности залежей Южно-Татарского свода

The problem of reliable calculation and reasonable interpretation of various productivity coefficient forecasting models is complicated by the presence of hidden patterns between geological and field parameters. The object of the study was the carbonate reservoirs of the deposits of the South Tatar arch, the data on which were obtained as a result of generalization and integration of the results of operating more than 700 wells for various purposes. Twenty geological and physical parameters were identified for the study, using which a discriminant analysis was successfully carried out. Multidimensional regression modeling was carried out for three selected groups of homogeneous objects. The simulation results have shown the efficiency of complex differentiation and clustering of geological and field information, which is based on the hierarchical structuring of parameters, which makes it possible, even with a minimum amount of data at a relevant level, to successfully identify the main trends in the influence of model parameters on the productivity coefficient, expanding the scope of their application to solve a range of basic and indirect problems of rational development of oil fields.

A deep learning approach to predict differentiation outcomes in hypothalamic-pituitary organoids

We use three-dimensional culture systems of human pluripotent stem cells for differentiation into pituitary organoids. Three-dimensional culture is inherently characterized by its ability to induce heterogeneous cell populations, making it difficult to maintain constant differentiation efficiency. That is why the culture process involves empirical aspects. In this study, we use deep-learning technology to create a model that can predict from images of organoids whether differentiation is progressing appropriately. Our models using EfficientNetV2-S or Vision Transformer, employing VENUS-coupled RAX expression, predictively class bright-field images of organoids into three categories with 70% accuracy, superior to expert-observer predictions. Furthermore, the model obtained by ensemble learning with the two algorithms can predict RAX expression in cells without RAX::VENUS, suggesting that our model can be deployed in clinical applications such as transplantation.

Dynamic three dimensional environment for efficient and large scale generation of smooth muscle cells from hiPSCs

BackgroundChronic ischemic limb disease often leads to amputation, which remains a significant clinical problem. Smooth-muscle cells (SMCs) are crucially involved in the development and progression of many cardiovascular diseases, but studies with primary human SMCs have been limited by a lack of availability. Here, we evaluated the efficiency of two novel protocols for differentiating human induced-pluripotent stem cells (hiPSCs) into SMCs and assessed their potency for the treatment of ischemic limb disease.MethodshiPSCs were differentiated into SMCs via a conventional two-dimensional (2D) protocol that was conducted entirely with cell monolayers, or via two protocols that consisted of an initial five-day three-dimensional (3D) spheroid phase followed by a six-day 2D monolayer phase (3D + 2D differentiation). The 3D phases were conducted in shaker flasks on an orbital shaker (the 3D + 2D shaker protocol) or in a PBS bioreactor (the 3D + 2D bioreactor protocol). Differentiation efficiency was evaluated via the expression of SMC markers (smooth-muscle actin [SMA], smooth muscle protein 22 [SM22], and Calponin-1), and the biological activity of the differentiated hiPSC-SMCs was evaluated via in-vitro assessments of migration (scratch assay), contraction in response to the treatment with a prostaglandin H2 analog (U46619), and tube formation on Geltrex, as well as in-vivo measurements of perfusion (fluorescence angiography) and vessel density in the limbs of mice that were treated with hiPSC-SMCs after experimentally induced hind-limb ischemia (HLI).ResultsBoth 3D + 2D protocols yielded > 5.6 × 107 hiPSC-SMCs/differentiation, which was ~ nine-fold more than that produced via 2D differentiation, and flow cytometry analyses confirmed that > 98% of the 3D + 2D-differentiated hiPSC-SMCs expressed SMA, > 81% expressed SM22, and > 89% expressed Calponin-1. hiPSC-SMCs obtained via the 3D + 2D shaker protocol also displayed typical SMC-like migratory, contraction, and tube-formation activity in-vitro and significantly improved measurements of perfusion, vessel density, and SMA-positive arterial density in the ischemic limb of mouse HLI model.ConclusionsOur dynamic 3D + 2D protocols produced an exceptionally high yield of hiPSC-SMCs. Transplantation of these hiPSC-SMCs results in significantly improved recovery of ischemic limb after ischemic injury in mice.

Optimizing Continuous Wave Semiconductor Laser toward Achieving (100’s) mW Power for Use in Medical Application: Theoretical Work

Diode laser is very efficient in medical applications, especially in photodynamic therapy (PDT), a type of laser phototherapy. Conventional laser diodes are classified as low-power lasers that emit laser radiation with a power of few 10s-mW. Extending the laser diode to further applications requires enhancing the emitted power. In this theoretical research, we aim to optimize laser diode's continuous-wave (CW) parameters that control the light-current (L-I) characteristics toward achieving higher slope efficiency and output power. The study is based on numerical solution of the rate equations and evaluating the steady state value of the emitted power, which is then used in the relationships among the total slope efficiency, differential quantum efficiency, threshold current, and emitted power. The numerical calculations indicated that power in the range of 100s-mW is predicted by designing the laser to have an anti-reflecting front facet, high-reflecting back facet, and short cavity with low material loss and high confinement of the lasing field. Therefore, the outcome of this study is to offer a guide for designing a high-power laser diode for use in PDT.

Unraveling the Differential Efficiency of Dorsal and Ventral Pathways in Visual Semantic Decoding.

Visual semantic decoding aims to extract perceived semantic information from the visual responses of the human brain and convert it into interpretable semantic labels. Although significant progress has been made in semantic decoding across individual visual cortices, studies on the semantic decoding of the ventral and dorsal cortical visual pathways remain limited. This study proposed a graph neural network (GNN)-based semantic decoding model on a natural scene dataset (NSD) to investigate the decoding differences between the dorsal and ventral pathways in process various parts of speech, including verbs, nouns, and adjectives. Our results indicate that the decoding accuracies for verbs and nouns with motion attributes were significantly higher for the dorsal pathway as compared to those for the ventral pathway. Comparative analyses reveal that the dorsal pathway significantly outperformed the ventral pathway in terms of decoding performance for verbs and nouns with motion attributes, with evidence showing that this superiority largely stemmed from higher-level visual cortices rather than lower-level ones. Furthermore, these two pathways appear to converge in their heightened sensitivity toward semantic content related to actions. These findings reveal unique visual neural mechanisms through which the dorsal and ventral cortical pathways segregate and converge when processing stimuli with different semantic categories.

A small molecule K-3 promotes PDX1 expression and potentiates the differentiation of pluripotent stem cells into insulin-producing pancreatic β cells

Abstract Insulin-producing pancreatic β-like cells derived from human pluripotent stem cells (PSCs) are anticipated as a novel cell source for cell replacement therapy for diabetes patients. Here, we describe the identification of small molecule compounds that promote the differentiation of the PSCs into insulin-producing cells by high throughput screening with a chemical library composed of 55,000 compounds. The initial hit compound K-1 and one derivative K-3 increased the proportion of PSC-derived insulin-positive endocrine cells and their glucose-stimulated insulin secretory (GSIS) functions. K-3 preferentially acts on stage 3 pancreatic progenitor cells and increases the population expressing high levels of PDX1. As a result, the ratios of the PSC-derived PDX1 / NKX6.1 double-positive endocrine progenitor and INS / NKX6.1 double-positive mono-hormonal endocrine cells were increased. K-3 enhances the expression of functional pancreatic β cell markers and affects biological processes concerning organ development. K-3 also increased the yield of endocrine cells at the end of stage 5. The novel compound is a beneficial new tool for efficiently generating PSC-derived insulin-producing cells with high functionality and differentiation efficiency.

Controlling spheroid attachment improves pancreatic beta cell differentiation from human iPS cells.

Regenerative medicine using human induced pluripotent stem cells (hiPSCs) is available for treating type 1 diabetes; however, the efficiency and maturation of hiPSC differentiation into pancreatic beta cells requires improvement. Various protocols, including three-dimensional (3D) culture, have been developed to improve differentiation efficiency and maturation. Several methods for 3D culture have been reported; however, they require costly and complicated equipment, special materials, and complicated operations. To solve these problems, we developed a simple 3D culture method under static conditions using a cyclo-olefin polymer (COP) characterized by high moisture barrier properties, low surface energy, and hydrophobicity. Using this 3D method and our simple and low-cost protocol, we found that differentiation into the definitive endoderm (DE) was better when the spheroids were attached. Therefore, upon the addition of Y-27632, attached spheroids with unique shapes and cavities were formed, and the differentiation efficiency into DE increased. During DE differentiation, the attachment of spheroids to the substrate and their subsequent floating improved differentiation efficiency. We found that the amount of C-peptide in spheroids differentiated using COP dishes was greater than that in rotary culture. Furthermore, INSULIN was highly expressed in areas with low cell density, suggesting that the unique shape of the spheroids made from COP dishes improved differentiation efficiency. Our study suggests that a device-free, simple 3D culture method that controls spheroid attachment improves the efficiency of hiPSC differentiation into pancreatic beta cells.

Identification of apelin/APJ signaling dysregulation in a human iPSC-derived granulosa cell model of Turner syndrome

The interaction between germ cells and somatic cells in the ovaries plays a crucial role in establishing the follicle reserve in mammals. Turner syndrome (TS) predominantly affects females who have a partial or complete loss of one X chromosome. Our understanding of the role that granulosa cells (GCs) play in TS disease progression and pathogenesis remains limited. In this study, we achieved GC differentiation efficiency of up to 80% from iPSCs. When attempting to replicate the differentiation process of embryonic granulosa cells, we observed the downregulation of specific genes—GATA4, FOXL2, AMHR2, CYP19A1, and FSH—in Turner syndrome-derived granulosa cells (TS-GCs). Additionally, we identified dysregulation of the cell cycle in TS-GCs. To uncover the endogenous defects in TS-GCs, we compared global transcriptome patterns between iPSC-derived granulosa cells from healthy individuals and those with Turner syndrome. The apelin/APJ pathway exhibited differential signaling between the healthy and TS groups. Supplementation with apelin ligands and activation of apelin/APJ downstream signaling via Akt/PKB restored cell cycle progression and marker gene expression. We hypothesize that during early embryonic development, failures in apelin/APJ signaling in GCs of Turner syndrome patients lead to abnormalities in ovarian development, ultimately resulting in early oocyte loss and infertility.

Navigating stem cell culture: insights, techniques, challenges, and prospects.

Stem cell research holds huge promise for regenerative medicine and disease modeling, making the understanding and optimization of stem cell culture a critical aspect of advancing these therapeutic applications. This comprehensive review provides an in-depth overview of stem cell culture, including general information, contemporary techniques, encountered problems, and future perspectives. The article begins by explaining the fundamental characteristics of various stem cell types, elucidating the importance of proper culture conditions in maintaining pluripotency or lineage commitment. A detailed exploration of established culture techniques sheds light on the evolving landscape of stem cell culture methodologies. Common challenges such as genetic stability, heterogeneity, and differentiation efficiency are thoroughly discussed, with insights into cutting-edge strategies and technologies aimed at addressing these hurdles. Moreover, the article delves into the impact of substrate materials, culture media components, and biophysical cues on stem cell behavior, emphasizing the intricate interplay between the microenvironment and cell fate decisions. As stem cell research advances, ethical considerations and regulatory frameworks become increasingly important, prompting a critical examination of these aspects in the context of culture practices. Lastly, the article explores emerging perspectives, including the integration of artificial intelligence and machine learning in optimizing culture conditions, and the potential applications of stem cell-derived products in personalized medicine. This comprehensive overview aims to serve as a valuable resource for researchers and clinicians, fostering a deeper understanding of stem cell culture and its key role in advancing regenerative medicine and biomedical research.