Alveolar Type 2 Epithelial Cell Organoids: Focus on Culture Methods.

Lung diseases rank third in terms of mortality and represent a significant economic burden globally. Scientists have been conducting research to better understand respiratory diseases and find treatments for them. An ideal in vitro model must mimic the in vivo organ structure, physiology, and pathology. Organoids are self-organizing, three-dimensional (3D) structures originating from adult stem cells, embryonic lung bud progenitors, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs). These 3D organoid cultures may provide a platform for exploring tissue development, the regulatory mechanisms related to the repair of lung epithelia, pathophysiological and immunomodulatory responses to different respiratory conditions, and screening compounds for new drugs. To create 3D lung organoids in vitro, both co-culture and feeder-free methods have been used. However, there exists substantial heterogeneity in the organoid culture methods, including the sources of AT2 cells, media composition, and feeder cell origins. This article highlights the currently available methods for growing AT2 organoids and prospective improvements to improve the available culture techniques/conditions. Further, we discuss various applications, particularly those aimed at modeling human distal lung diseases and cell therapy.

Phage Display-Derived Peptide for the Specific Binding of SARS-CoV-2.

Beginning from the end of 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic swept all over the world and is still afflicting the whole global population. Given that the vaccine-manufacturing ability is limited and the virus can evolve quickly, vaccination alone may not be able to end the pandemic, thus developing fast and accurate diagnoses and effective therapeutics will always be unmet needs. Phage display peptide library has been used in screening antigen-specific peptides for the invention of novel mimic receptors/ligands. Here, we report that a 12-mer phage display peptide library has been screened against the SARS-CoV-2 receptor-binding domain (RBD), and five of the screened peptides show binding ability with the RBD protein by the enzyme-linked immune sorbent assay. The surface plasmon resonance assay further demonstrates that peptide no. 1 can specifically bind to SARS-CoV-2 RBD with a binding affinity constant (K d) of 5.8 µM. Transmission electron microscopy coupled with a magnetic bead assay further confirms that the screened peptide can specifically bind the inactivated SARS-CoV-2 virus. This SARS-CoV-2-specific peptide holds great promise as a new bioreceptor/ligand for the rapid and accurate detection of SARS-CoV-2.

Graphene nanofiber composites for enhanced neuronal differentiation of human mesenchymal stem cells.

Aim: To differentiate mesenchymal stem cells into functional dopaminergic neurons using an electrospun polycaprolactone (PCL) and graphene (G) nanocomposite. Methods: A one-step approach was used to electrospin the PCL nanocomposite, with varying G concentrations, followed by evaluating their biocompatibility and neuronal differentiation. Results: PCL with exiguous graphene demonstrated an ideal nanotopography with an unprecedented combination of guidance stimuli and substrate cues, aiding the enhanced differentiation of mesenchymal stem cells into dopaminergic neurons. These newly differentiated neurons were seen to exhibit unique neuronal arborization, enhanced intracellular Ca2+ influx and dopamine secretion. Conclusion: Having cost-effective fabrication and room-temperature storage, the PCL-G nanocomposites could pave the way for enhanced neuronal differentiation, thereby opening a new horizon for an array of applications in neural regenerative medicine.

In vitro degradation behaviour, cytocompatibility and hemocompatibility of topologically ordered porous iron scaffold prepared using 3D printing and pressureless microwave sintering.

Biodegradable porous iron having topologically ordered porosity and tailorable properties as per the required application has been the major requirement in the field of biodegradable biomaterials. Hence, in the present study, iron scaffolds with the topologically ordered porous structure were developed and for the first time, the effect of the variation in the topology on the in vitro degradation behaviour, cytocompatibility and hemocompatibility were investigated. Iron scaffold samples were fabricated using a novel process based on the combination of 3D printing and pressureless microwave sintering. To investigate the effect of topology, two different types of topological structures namely Truncated Octahedron (TO) (with variable strut size) and Cubic (C) were used. From the morphological characterization, it was found that fabricated iron scaffold possessed interconnected porosity varying from 50.70%-80.97% which included the random microporosities in the strut and designed macroporosity. Furthermore, it was inferred that the topology of the iron scaffold significantly affected its degradation properties and cytocompatibility. Increase in the weight loss, corrosion rate and reduction in cell viability with the reduction in porosity were obtained. The maximum corrosion rate and weight loss achieved was 1.64 mmpy and 6.4% respectively. Direct cytotoxicity test results revealed cytotoxicity, while prepared iron scaffold samples exhibited excellent hemocompatibility and anti-platelet adhesion property. A comparative study with relevant literature was performed and it was established that the developed iron scaffold exhibited favorable degradation and biological properties which could be tailored to suit appropriate bone tissue engineering applications.

Iron oxide labeling does not affect differentiation potential of human bone marrow mesenchymal stem cells exhibited by their differentiation into cardiac and neuronal cells.

Mesenchymal stem cells (MSCs) have shown promising outcomes in cardiac and neuronal diseases. Efficient and noninvasive tracking of MSCs is essential to harness their therapeutic potential. Iron oxide nanoparticles (IONPs) have emerged as effective means to label stem cells and visualize them using magnetic resonance imaging (MRI). It is known that IONPs do not affect viability and cell proliferation of stem cells. However, very few studies have demonstrated differentiation potential of iron oxide-labeled MSCs and their differentiation into specific lineages that can contribute to cellular therapies. The differentiation of IONP-labeled human bone marrow mesenchymal stem cells (hBM-MSCs) into cardiac and neuronal lineages has never been studied. In this study, we have shown that IONP-labeled hBM-MSCs retain their differentiation potential to cardiac and neuronal cell lineages. We also confirmed that labeling hBM-MSCs with IONP does not affect their characteristic properties such as viability, cellular proliferation rate, surface marker profiling, and trilineage differentiation capacity. This study shows that IONP can be efficiently tracked, and its labeling does not alter stemness and differentiation potential of hBM-MSCs. Thus, the labeled hBM-MSCs can be used in clinical therapies and regenerative medicine.

TGFß1 contributes to cardiomyogenic-like differentiation of human bone marrow mesenchymal stem cells.

BACKGROUND: The majority of the protocols for cardiomyocyte differentiation of MSC use 5-azacytidine as an inducer. As transforming growth factor ß1 and 5-azacytidine share similar target signaling pathways, we examined whether transforming growth factor ß1 can play a role in cardiac differentiation process in human mesenchymal stem cell of bone marrow origin. METHODS: The differentiation protocol involving transforming growth factor ß1 was compared with that of 5-azacytidine in these cells. The two differentiation regimes were compared using reverse transcriptase PCR, flow cytometry, and quantitative PCR. RESULTS: We observed that in both cases, acquired morphological features were similar. Protein and gene expression assays also indicated similar cardiac marker expression profile in both the differentiation conditions. Furthermore, transforming growth factor ß1 and 5-azacytidine allowed the acquisition of comparable levels of cardiac cell like molecular characteristic as attested by evaluation of myosin light chain-2v expression. CONCLUSION: In conclusion, we demonstrate that transforming growth factor ß1 can play a similar role in cardiac differentiation process of human bone marrow mesenchymal stem cells.

Cell adhesion and proliferation studies on semi-interpenetrating polymeric networks (semi-IPNs) of polyacrylamide and gelatin.

In this study, the effect of feed composition, degree of hydrophilicity, and internal morphology has been investigated for cell proliferation potential of the polyacrylamide/gelatin (PAm/G) semi-interpenetrating polymeric network (semi-IPNs). Polycaprolactone diacrylate was used to cross-link polyacrylamide chains. Scanning electron microscopy (SEM) micrographs demonstrate uniformly distributed porous structure with internal diameter in the range of 75-175 µm, dependent on matrix compositions. Water-air contact angle was found in the range of 49° ± 0.22 to 89° ± 0.14 (p < 0.02) suggesting varying degree of hydrophilicity of the hydrogel surface. In addition, protein adsorption study showed 45 ± 0.14 µg to 64 ± 0.12 µg (p < 0.01) of protein adsorbed per cm² of hydrogel. Quantitative estimation of cell adhesion and proliferation was carried out by DNA quantification using fluorimetric assay method (p < 0.02). Microscopic images of proliferative cells on semi-IPNs by fluorescent and inverted phase contrast supported the findings of DNA quantification. Contact angle in the range of 63-69° in association with 52-59 µg/cm² protein absorption and 115-150 µm pore size was found optimum for fibroblast proliferation on PAm/G semi-IPN scaffolds. The newly developed semi-interpenetrating network may serve as a potential scaffold for soft tissue-engineering applications.