Corneal Epithelial Development and the Role of Induced Pluripotent Stem Cells for Regeneration.

Severe corneal disorders due to infective aetiologies, trauma, chemical injuries, and chronic cicatricial inflammations, are among vision-threatening pathologies leading to permanent corneal scarring. The whole cornea or lamellar corneal transplantation is often used as a last resort to restore vision. However, limited autologous tissue sources and potential adverse post-allotransplantation sequalae urge the need for more robust and strategic alternatives. Contemporary management using cultivated corneal epithelial transplantation has paved the way for utilizing stem cells as a regenerative potential. Humaninduced pluripotent stem cells (hiPSCs) can generate ectodermal progenitors and potentially be used for ocular surface regeneration. This review summarizes the process of corneal morphogenesis and the signaling pathways underlying the development of corneal epithelium, which is key to translating the maturation and differentiation process of hiPSCs in vitro. The current state of knowledge and methodology for driving efficient corneal epithelial cell differentiation from pluripotent stem cells are highlighted.

Limonoids and alkaloids from Tetradium austrosinense (Hand.-Mazz.) T.G.Hartley.

A pair of new enantiomeric indolopyridoquinazoline-type alkaloids, (+)-1,7S,8R- and (-)-1,7R,8S-trihydroxyrutaecarpine (3a and 3b), and a new limonoid-tyrosamine hybrid, austrosinin (8), along with six known alkaloids and limonoids, were isolated from the stems with leaves of Tetradium austrosinense. Their structures were elucidated on the basis of analysis of MS, NMR, ECD and time-dependent density functional theory-based electronic circular dichroism (TDDFT-ECD) calculations, as well as proposed biosynthetic pathway. An anti-inflammatory bioassay in vitro showed 8 had significant immunosuppressive effect against the production of pro-inflammatory cytokine TNF-α in lipopolysaccharide (LPS)-stimulated RAW264.7 cells.

Overexpression of the Chemosensory Protein CSP7 Gene Contributed to Lambda-Cyhalothrin Resistance in the Bird Cherry-Oat Aphid Rhopalosiphum padi.

Lambda-cyhalothrin is one of the most important pyrethroids used for controlling wheat aphids. Extensive spraying of lambda-cyhalothrin has led to the development of high resistance to this pyrethroid inRhopalosiphum padi. The mechanisms of resistance are complex and not fully understood. In this study, we found that a laboratory-selected strain of R. padi showed extremely high resistance to lambda-cyhalothrin and cross-resistance to bifenthrin and deltamethrin. The expression level of RpCSP7 was significantly elevated in the resistant strain compared to that in the susceptible strain. Knockdown of RpCSP7 increased the susceptibility of R. padi to lambda-cyhalothrin, whereas the susceptibility to bifenthrin and deltamethrin was not significantly changed. The recombinant RpCSP7 displayed a high affinity for lambda-cyhalothrin but no affinities to bifenthrin and deltamethrin. These findings suggest that the overexpression of RpCSP7 contributes to the resistance of R. padi to lambda-cyhalothrin. This study provides valuable insights into CSP-mediated insecticide resistance in insects.

B-seco Limonoids with anti-inflammatory activity from Tetradium fraxinifolium (Hook.) T.G.Hartley.

Fraxinifolines A-F (1-6), six new B-seco limonoids, together with four known A,D-di-seco ones, were isolated from the twigs with leaves of Tetradium fraxinifolium. Their structures with absolute configurations were elucidated on the basis of analysis of MS, NMR, single-crystal X-ray diffraction and biogenetic pathway. An anti-inflammatory bioassay in vitro showed limonoids 1-3 had significant immunosuppressive effect against the production of pro-inflammatory cytokines (IL-1ß and/or TNF-α) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells.

Network Pharmacology and Molecular Docking Analysis of Active Compounds in Tualang Honey against Atherosclerosis.

Atherosclerosis, a pathological condition marked by the accumulation of lipids and fibrous substances in the arterial walls, is a leading cause of heart failure and death. The present study aimed to utilize network pharmacology to assess the potential pharmacological effects of bioactive compounds in Tualang honey on atherosclerosis. This is significant as previous studies have indicated the cardioprotective effects of Tualang honey, yet a comprehensive evaluation using network pharmacology has yet to be conducted. The bioactive compounds in Tualang honey were screened and the potential gene targets for these compounds were predicted through Swiss Target Prediction and SuperPred databases. Atherosclerosis genes were retrieved from the OMIM, DisGeNet, and GeneCards databases. The interaction between these compounds and atherosclerosis genes was established through protein-protein interaction, gene ontology, and KEGG pathway analysis. The results of these analyses were then further confirmed through molecular docking studies using the AutoDock Tools software. The results revealed that 6 out of 103 compounds in Tualang honey met the screening criteria, with a total of 336 potential gene targets, 238 of which were shared with atherosclerosis. Further analysis showed that these active compounds had a good affinity with key targets and were associated with biological processes related to protein phosphorylation and inflammation as well as pathways related to lipid and atherosclerosis and other signaling pathways. In conclusion, the study provides insight into the potential pharmacological effects of Tualang honey bioactive compounds on atherosclerosis, supporting its use as a promising treatment for the disease.

Large animal models for cardiac remuscularization studies: A methodological review.

Myocardial infarction is the most common cause of heart failure, one of the most fatal non-communicable diseases worldwide. The disease could potentially be treated if the dead, ischemic heart tissues are regenerated and replaced with viable and functional cardiomyocytes. Pluripotent stem cells have proven the ability to derive specific and functional cardiomyocytes in large quantities for therapy. To test the remuscularization hypothesis, the strategy to model the disease in animals must resemble the pathophysiological conditions of myocardial infarction as in humans, to enable thorough testing of the safety and efficacy of the cardiomyocyte therapy before embarking on human trials. Rigorous experiments and in vivo findings using large mammals are increasingly important to simulate clinical reality and increase translatability into clinical practice. Hence, this review focus on large animal models which have been used in cardiac remuscularization studies using cardiomyocytes derived from human pluripotent stem cells. The commonly used methodologies in developing the myocardial infarction model, the choice of animal species, the pre-operative antiarrhythmics prophylaxis, the choice of perioperative sedative, anaesthesia and analgesia, the immunosuppressive strategies in allowing xenotransplantation, the source of cells, number and delivery method are discussed.

Vaginal Infections during Pregnancy Increase Breast Milk Microbiome Alpha Diversity and Alter Taxonomic Composition.

We previously reported that breast milk from women with (W) or without (WO) vaginal yeast infection during pregnancy differs in its immunological and antimicrobial properties, especially against pathogenic vaginal Candida sp.. Here, we investigated the differences in microbiota profiles of breast milk from these groups. Seventy-two breast milk samples were collected from lactating mothers (W, n=37; WO, n=35). The DNA of bacteria was extracted from each breast milk sample for microbiota profiling by 16S rRNA gene sequencing. Breast milk from the W-group exhibited higher alpha diversity than that from the WO-group across different taxonomic levels of class (P=0.015), order (P=0.011), family (P=0.020), and genus (P=0.030). Compositional differences between groups as determined via beta diversity showed marginal differences at taxonomic levels of phylum (P=0.087), family (P=0.064), and genus (P=0.067). The W-group showed higher abundances of families Moraxellaceae (P=0.010) and Xanthomonadaceae (P=0.008), and their genera Acinetobacter (P=0.015), Enhydrobacter (P=0.015), and Stenotrophomonas (P=0.007). Meanwhile, the WO-group showed higher abundances of genus Staphylococcus (P=0.046) and species Streptococcus infantis (P=0.025). This study shows that, although breast milk composition is affected by vaginal infection during pregnancy, this may not pose a threat to infant growth and development.

In-depth profiling of di(2-ethylhexyl) phthalate metabolic footprints in rats using click chemistry-mass spectrometry probes.

Di(2-ethylhexyl) phthalate (DEHP), the most widely used plasticizers in the world, has been regarded as an endocrine disrupting chemical with serious adverse health outcomes. Accumulating evidence strongly suggests that the undesirable biological effects of DEHP are meditated by its metabolites rather than itself. However, the metabolic footprints of DEHP in vivo are still unclear. Here we developed a click chemistry-assisted mass spectrometry (CC-MS) strategy for in-depth profiling DEHP metabolites in rats. An alkyne-modified DEHP analogue (alkyne-DEHP) was synthesized as a tracer for in vivo tracing, and a pair of MS probes (4-azido-nphenylbenzamide, 4-ANPA, and its deuterated reagent d5-4-ANPA) were prepared to specifically label the alkyne-DEHP metabolites, and prominently improve their detection sensitivity and selectivity. Using the CC-MS strategy, we successfully screened 247 alkyne-DEHP metabolites from rat urine, feces, and serum, including many unrevealed metabolites, such as oxidized phthalate diester metabolites and glucuronides of phthalate monoester metabolites. The discovery of new DEHP metabolites provides additional insights for understanding the metabolism of DEHP, which may be beneficial in exploring the mechanism underlying DEHP induced-toxicity in the future.

Breast milk from healthy women has higher anti-Candida properties than women with vaginal infections during pregnancy.

The aim of this study was to investigate the different immunological and antimicrobial properties of breast milk from women with (W) or without (WO) vaginal yeast infections during pregnancy in 85 lactating women (W, n = 43; WO, n = 42). Concentrations of IL-10, IgA, IgM, IgG, EGF, and TGF-α were similar in both groups. However, breast milk of women aged below 31 years old from the W-group showed higher concentration of EGF than the WO-group (p = 0.031). Breast milk from WO-group exhibited higher anti-Candida properties than W-group, both via growth inhibition and aggregation of yeast cells (p < 0.001). Correlation analysis showed that breast milk concentration of TGF-α positively correlated with concentrations of IL-10 (p = 0.001) and IgA (p = 0.021) in the W-group. Data from our present study shows that although breast milk from women with vaginal infections during pregnancy may not sufficiently hinder Candida growth, other immuno-modulatory bioactives may substitute for such a protective effect.

Mesenchymal Stromal Cells-Derived Exosome and the Roles in the Treatment of Traumatic Brain Injury.

Traumatic brain injury (TBI) could result in life-long disabilities and death. Though the mechanical insult causes primary injury, the secondary injury due to dysregulated responses following neuronal apoptosis and inflammation is often the cause for more detrimental consequences. Mesenchymal stromal cell (MSC) has been extensively investigated as the emerging therapeutic for TBI, and the functional properties are chiefly attributed to their secretome, especially the exosomes. Delivering these nanosize exosomes have shown to ameliorate post-traumatic injury and restore brain functions. Recent technology advances also allow engineering MSC-derived exosomes to carry specific biomolecules of interest to augment their therapeutic outcome. In this review, we discuss the pathophysiology of TBI and summarize the recent progress in the applications of MSCs-derived exosomes, the roles and the signalling mechanisms underlying the protective effects in the treatment of the TBI.

Adverse Effects of Bisphenol A on the Liver and Its Underlying Mechanisms: Evidence from In Vivo and In Vitro Studies.

BPA is a known endocrine-disrupting agent that is capable of binding to the estrogen receptor and has exhibited adverse effects in many laboratory animal and in vitro studies. Moreover, it also been shown to have estrogenic, antiandrogenic, inflammatory, and oxidative properties. The widespread presence of BPA in the environment presents a considerable threat to humans. BPA has been shown to be leached into the human ecosystem, where it is commonly found in food products consumed by humans. Although the concentration is relatively low, its prolonged consumption may cause a variety of deleterious health effects. The liver is an important organ for metabolizing and detoxifying toxic metabolites to protect organisms from potentially toxic chemical insults. BPA that is ingested will be eliminated by the liver. However, it has also induced hepatoxicity and injury via various mechanisms. To find research demonstrating the effects of BPA on kidney, a number of databases, including Google Scholar, MEDLINE, PubMed, and the Directory of Open Access Journals, were searched. Thus, this review summarizes the research on the relationship between BPA and its effects on the liver-derived from animals and cellular studies. The underlying mechanism of liver injury caused by BPA is also elucidated.

Integrase deficient lentiviral vector: prospects for safe clinical applications.

HIV-1 derived lentiviral vector is an efficient transporter for delivering desired genetic materials into the targeted cells among many viral vectors. Genetic material transduced by lentiviral vector is integrated into the cell genome to introduce new functions, repair defective cell metabolism, and stimulate certain cell functions. Various measures have been administered in different generations of lentiviral vector systems to reduce the vector's replicating capabilities. Despite numerous demonstrations of an excellent safety profile of integrative lentiviral vectors, the precautionary approach has prompted the development of integrase-deficient versions of these vectors. The generation of integrase-deficient lentiviral vectors by abrogating integrase activity in lentiviral vector systems reduces the rate of transgenes integration into host genomes. With this feature, the integrase-deficient lentiviral vector is advantageous for therapeutic implementation and widens its clinical applications. This short review delineates the biology of HIV-1-erived lentiviral vector, generation of integrase-deficient lentiviral vector, recent studies involving integrase-deficient lentiviral vectors, limitations, and prospects for neoteric clinical use.

Tribulus terrestris L. extract ameliorates atherosclerosis by inhibition of vascular smooth muscle cell proliferation in ApoE-/- mice and A7r5 cells via suppression of Akt/MEK/ERK signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Atherosclerosis (AS) is one of major threatens of death worldwide, and vascular smooth muscle cell (VSMC) proliferation is an important characteristic in the progression of AS. Tribulus terrestris L. is a well-known Chinese Materia Medica for treating skin pruritus, vertigo and cardiovascular diseases in traditional Chinese medicine. However, its anti-AS activity and inhibition effect on VSMC proliferation are not fully elucidated. AIMS: We hypothesize that the furostanol saponins enriched extract (FSEE) of T. terrestris L. presents anti-AS effect by inhibition of VSMC proliferation. The molecular action mechanism underlying the anti-VSMC proliferation effect of FSEE is also investigated. MATERIALS AND METHODS: Apolipoprotein-E deficient (ApoE-/-) mice and rat thoracic smooth muscle cell A7r5 were employed as the in vivo and in vitro models respectively to evaluate the anti- AS and VSMC proliferation effects of FSEE. In ApoE-/- mice, the amounts of total cholesterol, triglyceride, low density lipoprotein and high density lipoprotein in serum were measured by commercially available kits. The size of atherosclerotic plaque was observed by hematoxylin & eosin staining. The protein expressions of α-smooth muscle actin (α-SMA) and osteopontin (OPN) in the plaque were examined by immunohistochemistry. In A7r5 cells, the cell viability and proliferation were tested by MTT and Real Time Cell Analysis assays. The cell migration was evaluated by wound healing assay. Propidium iodide staining followed by flow cytometry was used to analyze the cell cycle progression. The expression of intracellular total and phosphorylated proteins including protein kinase B (Akt) and mitogen-activated protein kinases (MAPKs), such as mitogen-activated extracellular signal-regulated kinase (MEK), extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), were detected by western blotting analysis. RESULTS: FSEE significantly reduced the area of atherosclerotic plaque in high-fat diet-fed ApoE-/- mice. And FSEE increased the protein expression level of α-SMA and decreased the level of OPN in atherosclerotic plaque, which revealed the inhibition of VSMC phenotype switching and proliferation. In A7r5 cells, FSEE suppressed fetal bovine serum (FBS) or oxidized low density lipoprotein (oxLDL)-triggered VSMC proliferation and migration in a concentration dependent manner. FSEE protected against the elevation of cell numbers in S phase induced by FBS or oxLDL and the reduction of cell numbers in G0/G1 phase induced by oxLDL. Moreover, the phosphorylation of Akt and MAPKs including MEK, ERK and JNK could be facilitated by FBS or oxLDL, while co-treatment of FSEE attenuated the phosphorylation of Akt induced by oxLDL as well as the phosphorylation of MEK and ERK induced by FBS. In addition, (25R)-terrestrinin B (JL-6), which was the main ingredient of FSEE, and its potential active pharmaceutical ingredients tigogenin (Tigo) and hecogenin (Heco) also significantly attenuated FBS or oxLDL-induced VSMC proliferation in A7r5 cells. CONCLUSION: FSEE presents potent anti- AS and VSMC proliferation activities and the underlying mechanism is likely to the suppression of Akt/MEK/ERK signaling. The active components of FSEE are JL-6 and its potential active pharmaceutical ingredients Tigo and Heco. So, FSEE and its active compounds may be potential therapeutic drug candidates for AS.

Characterization of oral and gut microbiome and plasma metabolomics in COVID-19 patients after 1-year follow-up.

BACKGROUND: Due to the outbreak and rapid spread of coronavirus disease 2019 (COVID-19), more than 160 million patients have become convalescents worldwide to date. Significant alterations have occurred in the gut and oral microbiome and metabonomics of patients with COVID-19. However, it is unknown whether their characteristics return to normal after the 1-year recovery. METHODS: We recruited 35 confirmed patients to provide specimens at discharge and one year later, as well as 160 healthy controls. A total of 497 samples were prospectively collected, including 219 tongue-coating, 129 stool and 149 plasma samples. Tongue-coating and stool samples were subjected to 16S rRNA sequencing, and plasma samples were subjected to untargeted metabolomics testing. RESULTS: The oral and gut microbiome and metabolomics characteristics of the 1-year convalescents were restored to a large extent but did not completely return to normal. In the recovery process, the microbial diversity gradually increased. Butyric acid-producing microbes and Bifidobacterium gradually increased, whereas lipopolysaccharide-producing microbes gradually decreased. In addition, sphingosine-1-phosphate, which is closely related to the inflammatory factor storm of COVID-19, increased significantly during the recovery process. Moreover, the predictive models established based on the microbiome and metabolites of patients at the time of discharge reached high efficacy in predicting their neutralizing antibody levels one year later. CONCLUSIONS: This study is the first to characterize the oral and gut microbiome and metabonomics in 1-year convalescents of COVID-19. The key microbiome and metabolites in the process of recovery were identified, and provided new treatment ideas for accelerating recovery. And the predictive models based on the microbiome and metabolomics afford new insights for predicting the recovery situation which benefited affected individuals and healthcare.

Vascular Protective Effect and Its Possible Mechanism of Action on Selected Active Phytocompounds: A Review.

Vascular endothelial dysfunction is characterized by an imbalance of vasodilation and vasoconstriction, deficiency of nitric oxide (NO) bioavailability and elevated reactive oxygen species (ROS), and proinflammatory factors. This dysfunction is a key to the early pathological development of major cardiovascular diseases including hypertension, atherosclerosis, and diabetes. Therefore, modulation of the vascular endothelium is considered an important therapeutic strategy to maintain the health of the cardiovascular system. Epidemiological studies have shown that regular consumption of medicinal plants, fruits, and vegetables promotes vascular health, lowering the risk of cardiovascular diseases. This is mainly attributed to the phytochemical compounds contained in these resources. Various databases, including Google Scholar, MEDLINE, PubMed, and the Directory of Open Access Journals, were searched to identify studies demonstrating the vascular protective effects of phytochemical compounds. The literature had revealed abundant data on phytochemical compounds protecting and improving the vascular system. Of the numerous compounds reported, curcumin, resveratrol, cyanidin-3-glucoside, berberine, epigallocatechin-3-gallate, and quercetin are discussed in this review to provide recent information on their vascular protective mechanisms in vivo and in vitro. Phytochemical compounds are promising therapeutic agents for vascular dysfunction due to their antioxidative mechanisms. However, future human studies will be necessary to confirm the clinical effects of these vascular protective mechanisms.

Vibrational Spectroscopy-Based Chemometrics Analysis of Clinacanthus nutans Extracts after Postharvest Processing and Extract Effects on Cardiac C-Kit Cells.

Chemical constituents in plants can be greatly affected by postharvest processing, and it is important to identify the factors that lead to significant changes in chemistry and bioactivity. In this study, attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy was used to analyze extracts of Clinacanthus nutan (C. nutans) leaves generated using different parameters (solvent polarities, solid-liquid ratios, ultrasonic durations, and cycles of extraction). In addition, the effects of these extracts on the viability of cardiac c-kit cells (CCs) were tested. The IR spectra were processed using SIMCA-P software. PCA results of all tested parameter sets were within acceptable values. Solvent polarity was identified as the most influential factor to observe the differences in chemical profile and activities of C. nutans extracts. Ideal extraction conditions were identified, for two sample groups (G1 and G2), as they showed optimal total phenolic content (TPC) yield of 44.66 ± 0.83 mg GAE/g dw and 45.99 ± 0.29 mg GAE/g dw and CC viability of 171.81 ± 4.06% and 147.53 ± 6.80%, respectively. Validation tools such as CV-ANOVA (p < 0.05) and permutation (R 2 and Q 2 plots were well intercepted to each other) have further affirmed the significance and reliability of the partial least square (PLS) model of solvent polarity employed in extraction. Hence, these approaches help optimize postharvest processes that encourage positive TPC and CCs results in C. nutans extracts.

The Zinc Finger Transcription Factor BbCmr1 Regulates Conidium Maturation in Beauveria bassiana.

The entomopathogenic fungus Beauveria bassiana is a typical filamentous fungus and has been used for pest biocontrol. Conidia are the main active agents of fungal pesticides; however, we know little about conidial developmental mechanisms and less about maturation mechanisms. We found that a Zn2Cys6 transcription factor of B. bassiana (named BbCmr1) was mainly expressed in late-stage conidia and was involved in conidium maturation regulation. Deletion of Bbcmr1 impaired the conidial cell wall and resulted in a lower conidial germination rate under UV (UV), heat shock, H2O2, Congo red (CR) and SDS stresses compared to the wild type. Transcription levels of the genes associated with conidial wall components and trehalose synthase were significantly reduced in the ΔBbcmr1 mutant. Further analysis found that BbCmr1 functions by upregulating BbWetA, a well-known transcription factor in the central development of BrlA-AbaA-WetA. The expression of Bbcmr1 was positively regulated by BbBrlA. These results indicated that BbCmr1 played important roles in conidium maturation by interacting with the central development pathway, which provided insight into the conidial development networks in B. bassiana. IMPORTANCE Conidium maturation is a pivotal event in conidial development and affects fungal survival ability under various biotic/abiotic stresses. Although many transcription factors have been reported to regulate conidial development, we know little about the molecular mechanism of conidium maturation. Here, we demonstrated that the transcription factor BbCmr1 of B. bassiana was involved in conidium maturation, regulating cell wall structure, the expression of cell wall-related proteins, and trehalose synthesis. BbCmr1 orchestrated conidium maturation by interplaying with the central development pathway BrlA-AbaA-WetA. BbBrlA positively regulated the expression of Bbcmr1, and the latter positively regulated BbwetA expression, which forms a regulatory network mediating conidial development. This finding was critical to understand the molecular regulatory networks of conidial development in B. bassiana and provided avenues to engineer insect fungal pathogens with high-quality conidia.

Monoterpene indole alkaloids from Melodinus cochinchinensis (Lour.) Merr.

Melodicochinines A - D (1-4), four new monoterpene indole alkaloids (MIAs), along with 21 known ones, were isolated from the stems and twigs of Melodinus cochinchinensis. Their structures were elucidated on the basis of extensive spectroscopic analysis. A ubiquitin-rhodamine 110 assay showed that 11-methyloxytabersonine had potential inhibitory effect against ubiquitin-specific protease 7 (USP7).

Recapitulating human cardio-pulmonary co-development using simultaneous multilineage differentiation of pluripotent stem cells.

The extensive crosstalk between the developing heart and lung is critical to their proper morphogenesis and maturation. However, there remains a lack of models that investigate the critical cardio-pulmonary mutual interaction during human embryogenesis. Here, we reported a novel stepwise strategy for directing the simultaneous induction of both mesoderm-derived cardiac and endoderm-derived lung epithelial lineages within a single differentiation of human-induced pluripotent stem cells (hiPSCs) via temporal specific tuning of WNT and nodal signaling in the absence of exogenous growth factors. Using 3D suspension culture, we established concentric cardio-pulmonary micro-Tissues (µTs), and expedited alveolar maturation in the presence of cardiac accompaniment. Upon withdrawal of WNT agonist, the cardiac and pulmonary components within each dual-lineage µT effectively segregated from each other with concurrent initiation of cardiac contraction. We expect that our multilineage differentiation model will offer an experimentally tractable system for investigating human cardio-pulmonary interaction and tissue boundary formation during embryogenesis.

Organs begin developing during the first few months of pregnancy, while the baby is still an embryo. These early stages of development are known as embryogenesis ­ a tightly organized process, during which the embryo forms different layers of stem cells. These cells can be activated to turn into a particular type of cell, such as a heart or a lung cell. The heart and lungs develop from different layers within the embryo, which must communicate with each other for the organs to form correctly. For example, chemical signals can be released from and travel between layers of the embryo, activating processes inside cells located in the different areas. In mouse models, chemical signals and cells travel between developing heart and lung, which helps both organs to form into the correct structure. But it is unclear how well the observations from mouse models translate to heart and lung development in humans. To find out more, Ng et al. developed a human model of heart and lung co-development during embryogenesis using human pluripotent stem cells. The laboratory-grown stem cells were treated with chemical signals, causing them to form different layers that developed into early forms of heart and lung cells. The cells were then transferred into a specific growing condition, where they arranged into three-dimensional structures termed microtissues. Ng et al. found that lung cells developed faster when grown in microtissues with accompanying developing heart cells compared to microtissues containing only developing lung cells. In addition, Ng et al. revealed that the co-developing heart and lung tissues automatically separate from each other during later stage, without the need for chemical signals. This human cell-based model of early forms of co-developing heart and lung cells may help provide researchers with new strategies to probe the underlying mechanisms of human heart and lung interaction during embryogenesis.

A guide in lentiviral vector production for hard-to-transfect cells, using cardiac-derived c-kit expressing cells as a model system.

Gene therapy revolves around modifying genetic makeup by inserting foreign nucleic acids into targeted cells via gene delivery methods to treat a particular disease. While the genes targeted play a key role in gene therapy, the gene delivery system used is also of utmost importance as it determines the success of gene therapy. As primary cells and stem cells are often the target cells for gene therapy in clinical trials, the delivery system would need to be robust, and viral-based entries such as lentiviral vectors work best at transporting the transgene into the cells. However, even within lentiviral vectors, several parameters can affect the functionality of the delivery system. Using cardiac-derived c-kit expressing cells (CCs) as a model system, this study aims to optimize lentiviral production by investigating various experimental factors such as the generation of the lentiviral system, concentration method, and type of selection marker. Our findings showed that the 2nd generation system with pCMV-dR8.2 dvpr as the packaging plasmid produced a 7.3-fold higher yield of lentiviral production compared to psPAX2. Concentrating the virus with ultracentrifuge produced a higher viral titer at greater than 5 × 105 infectious unit values/ml (IFU/ml). And lastly, the minimum inhibitory concentration (MIC) of puromycin selection marker was 10 µg/mL and 7 µg/mL for HEK293T and CCs, demonstrating the suitability of antibiotic selection for all cell types. This encouraging data can be extrapolated and applied to other difficult-to-transfect cells, such as different types of stem cells or primary cells.