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.

Anticancer Activity of Novel Difluorinated Curcumin Analog and Its Inclusion Complex with 2-Hydroxypropyl-ß-Cyclodextrin against Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is the primary reason for cancer-related deaths in the US. Genetic mutations, drug resistance, the involvement of multiple signaling pathways, cancer stem cells (CSCs), and desmoplastic stroma, which hinders drug penetrance, contribute to poor chemotherapeutic efficacy. Hence, there is a need to identify novel drugs with improved delivery to improve treatment outcomes. Curcumin is one such compound that can inhibit multiple signaling pathways and CSCs. However, curcumin's clinical applicability for treating PDAC is limited because of its poor solubility in water and metabolic instability. Hence, we developed a difluorinated curcumin (CDF) analog that accumulates selectively in the pancreas and inhibits PDAC growth in vitro and in vivo. In the present work, we developed its 2-hydroxy-propyl-ß-cyclodextrin (HCD) inclusion complex to increase its water solubility and hydrolytic stability. The CDFHCD inclusion complex was characterized by spectroscopic, thermal, and microscopic techniques. The inclusion complex exhibited increased aqueous solubility, hydrolytic stability, and antiproliferative activity compared to parent CDF. Moreover, CDF and CDFHCD inhibited colony and spheroid formation, and induced cell cycle and apoptosis in PDAC cell lines. Hence, CDFHCD self-assembly is an efficient approach to increase water solubility and anticancer therapeutic efficacy, which now warrants advancement towards a clinical proof of concept in PDAC patients.

Biomarkers and molecular endotypes of sarcoidosis: lessons from omics and non-omics studies.

Sarcoidosis is a chronic granulomatous disorder characterized by unknown etiology, undetermined mechanisms, and non-specific therapies except TNF blockade. To improve our understanding of the pathogenicity and to predict the outcomes of the disease, the identification of new biomarkers and molecular endotypes is sorely needed. In this study, we systematically evaluate the biomarkers identified through Omics and non-Omics approaches in sarcoidosis. Most of the currently documented biomarkers for sarcoidosis are mainly identified through conventional "one-for-all" non-Omics targeted studies. Although the application of machine learning algorithms to identify biomarkers and endotypes from unbiased comprehensive Omics studies is still in its infancy, a series of biomarkers, overwhelmingly for diagnosis to differentiate sarcoidosis from healthy controls have been reported. In view of the fact that current biomarker profiles in sarcoidosis are scarce, fragmented and mostly not validated, there is an urgent need to identify novel sarcoidosis biomarkers and molecular endotypes using more advanced Omics approaches to facilitate disease diagnosis and prognosis, resolve disease heterogeneity, and facilitate personalized medicine.

Wnt5a/ß-catenin axis is involved in the downregulation of AT2 lineage by PAI-1.

Failure to regenerate injured alveoli functionally and promptly causes a high incidence of fatality in coronavirus disease 2019 (COVID-19). How elevated plasminogen activator inhibitor-1 (PAI-1) regulates the lineage of alveolar type 2 (AT2) cells for re-alveolarization has not been studied. This study aimed to examine the role of PAI-1-Wnt5a-ß catenin cascades in AT2 fate. Dramatic reduction in AT2 yield was observed in Serpine1Tg mice. Elevated PAI-1 level suppressed organoid number, development efficiency, and total surface area in vitro. Anti-PAI-1 neutralizing antibody restored organoid number, proliferation and differentiation of AT2 cells, and ß-catenin level in organoids. Both Wnt family member 5A (Wnt5a) and Wnt5a-derived N-butyloxycarbonyl hexapeptide (Box5) altered the lineage of AT2 cells. This study demonstrates that elevated PAI-1 regulates AT2 proliferation and differentiation via the Wnt5a/ß catenin cascades. PAI-1 could serve as autocrine signaling for lung injury repair.

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.

Tissue-specific mesenchymal stem cell-dependent osteogenesis in highly porous chitosan-based bone analogs.

Among conventional fabrication techniques, freeze-drying process has widely been investigated for polymeric implants. However, the understanding of the stem cell progenitor-dependent cell functionality modulation and quantitative analysis of early osseointegration of highly porous scaffolds have not been explored. Here, we developed a novel, highly porous, multimaterial composite, chitosan/hydroxyapatite/polycaprolactone (CHT/HA/PCL). The in vitro studies have been performed using mesenchymal stem cells (MSCs) from three tissue sources: human bone marrow-derived MSCs (BM-MSCs), adipose-derived MSCs (AD-MSCs), and Wharton's jelly-derived MSCs (WJ-MSCs). Although cell attachment and metabolic activity [3-4,5-dimethylthiazol-2yl-(2,5 diphenyl-2H-tetrazoliumbromide) assay] were ore enhanced in WJ-MSC-laden CHT/HA/PCL composites, scanning electron microscopy, real-time gene expression (alkaline phosphatase [ALP], collagen type I [Col I], osteocalcin [OCN], and bone morphogenetic protein 4 [BMP-4]), and immunostaining (COL I, ß-CATENIN, OCN, and SCLEROSTIN [SOST]) demonstrated pronounced osteogenesis with terminal differentiation on BM-MSC-laden CHT/HA/PCL composites only. The enhanced cell functionality on CHT/HA/PCL composites was explained in terms of interplay among the surface properties and the optimal source of MSCs. In addition, osteogenesis in rat tibial model over 6 weeks confirmed a better ratio of bone volume to the total volume for BM-MSC-laden composites over scaffold-only and defect-only groups. The clinically conformant combination of 3D porous architecture with pore sizes varying in the range of 20 to 200 µm together with controlled in vitro degradation and early osseointegration establish the potential of CHT/HA/PCL composite as a potential cancellous bone analog.

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.

Proliferative regulation of alveolar epithelial type 2 progenitor cells by human Scnn1d gene.

Lung epithelial sodium channel (ENaC) encoded by Scnn1 genes is essential for maintaining transepithelial salt and fluid homeostasis in the airway and the lung. Compared to α, ß, and γ subunits, the role of respiratory δ-ENaC has not been studied in vivo due to the lack of animal models. Methods: We characterized full-length human δ802-ENaC expressed in both Xenopus oocytes and humanized transgenic mice. AT2 proliferation and differentiation in 3D organoids were analysed with FACS and a confocal microscope. Both two-electrode voltage clamp and Ussing chamber systems were applied to digitize δ802-ENaC channel activity. Immunoblotting was utilized to analyse δ802-ENaC protein. Transcripts of individual ENaC subunits in human lung tissues were quantitated with qPCR. Results: The results indicate that δ802-ENaC functions as an amiloride-inhibitable Na+ channel. Inhibitory peptide α-13 distinguishes δ802- from α-type ENaC channels. Modified proteolysis of γ-ENaC by plasmin and aprotinin did not alter the inhibition of amiloride and α-13 peptide. Expression of δ802-ENaC at the apical membrane of respiratory epithelium was detected with biophysical features similar to those of heterologously expressed channels in oocytes. δ802-ENaC regulated alveologenesis through facilitating the proliferation of alveolar type 2 epithelial cells. Conclusion: The humanized mouse line conditionally expressing human δ802-ENaC is a novel model for studying the expression and function of this protein in vivo .

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.

Repositioning of Misplaced Central Venous Catheter with Saline Injection Under C-Arm Imaging.

Malposition of central venous catheter is a well known technical complication. Misplaced catheter often requires reinsertion for proper placement of the catheter in the superior vena cava (SVC) to support safe delivery of care and minimize complications. But reinsertion exposes the patient once again to risks of complications related to the procedure including potential of misplacement. Literature describes only a few techniques for repositioning a misplaced central venous catheter (CVC). We tried old simple method of saline injection with force under image intensifier using hydrostatic force of intravenous fluid to straighten the CVC. We could successfully reposition two misplaced CVC's using this method.

Culture & differentiation of mesenchymal stem cell into osteoblast on degradable biomedical composite scaffold: In vitro study.

BACKGROUND & OBJECTIVES: There is a significant bone tissue loss in patients from diseases and traumatic injury. The current autograft transplantation gold standard treatment has drawbacks, namely donor site morbidity and limited supply. The field of tissue engineering has emerged with a goal to provide alternative sources for transplantations to bridge this gap between the need and lack of bone graft. The aim of this study was to prepare biocomposite scaffolds based on chitosan (CHT), polycaprolactone (PCL) and hydroxyapatite (HAP) by freeze drying method and to assess the role of scaffolds in spatial organization, proliferation, and osteogenic differentiation of human mesenchymal stem cells (hMSCs) in vitro, in order to achieve bone graft substitutes with improved physical-chemical and biological properties. METHODS: Pure chitosan (100CHT) and composites (40CHT/HAP, 30CHT/HAP/PCL and 25CHT/HAP/PCL scaffolds containing 40, 30, 25 parts per hundred resin (phr) filler, respectively) in acetic acid were freeze dried and the porous foams were studied for physicochemical and in vitro biological properties. RESULTS: Scanning electron microscope (SEM) images of the scaffolds showed porous microstructure (20-300 µm) with uniform pore distribution in all compositions. Materials were tested under compressive load in wet condition (using phosphate buffered saline at pH 7.4). The in vitro studies showed that all the scaffold compositions supported mesenchymal stem cell attachment, proliferation and differentiation as visible from SEM images, [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay, alkaline phosphatase (ALP) assay and quantitative reverse transcription (qRT)-PCR. INTERPRETATION & CONCLUSIONS: Scaffold composition 25CHT/HAP/PCL showed better biomechanical and osteoinductive properties as evident by mechanical test and alkaline phosphatase activity and osteoblast specific gene expression studies. This study suggests that this novel degradable 3D composite may have great potential to be used as scaffold in bone tissue engineering.

Endovascular treatment in spinal perimedullary arteriovenous fistula.

This study includes 20 patients with 21 spinal perimedullary fistulae. There were nine Type IVa (42.8%) lesions, ten Type IVb (47.6%) and two Type IVc (9.5%) lesions. The dominant arterial supply was from the anterior spinal artery (47.6%), posterior spinal artery (19%) and directly from the radiculomedullary artery (28.5%). Sixteen lesions in 15 patients were treated by endovascular route using n-butyl-2-cyanoacrylate. Endovascular treatment was not feasible in five patients. Of the ten patients with microfistulae, catheterization failed/was not attempted in 40%, complete obliteration of the lesion was seen in 60% but clinical improvement was seen in 40% of patients. Catheterization was feasible in all ten patients with macrofistulae (nine type IVb and two type IVc lesions). Complete obliteration of the lesions was seen in 60% and residue in 30%. Clinical improvement was seen in 80% and clinical deterioration in 10%. In conclusion, endovascular glue embolization is safe and efficacious in type IVb and IVc spinal perimedullary fistulae and should be considered the first option of treatment. It is also feasible in many of the type IVa lesions.

Prevalence of MR imaging abnormalities in vitamin B12 deficiency patients presenting with clinical features of subacute combined degeneration of the spinal cord.

BACKGROUND AND AIMS: Subacute combined degeneration (SACD) of the spinal cord, characterized by degeneration of lateral and posterior columns, is often found in vitamin B12 deficiency. Our aim was to look for sensitivity of imaging in depicting the spinal cord abnormality in vitamin B12 deficient patients and to find any correlation of vitamin B12 levels with clinical scores/severity at time of presentation. MATERIAL AND METHODS: A total 54 patients with biochemically proven vitamin B12 deficiency were included in the study. In all these patients MR study of cervico-dorsal spine was done. All the patients after initiation of appropriate treatment were followed up for a minimum of two months. RESULTS: MRI showed cord signal abnormality in only 8 patients out of 54 patients with low sensitivity of 14.8%. After appropriate therapy, complete resolution of cord signal abnormalities was observed in all these 8 patients, on follow-up MR imaging. Significant negative correlation (r=-0.503, p<0.000) was seen between the clinical severity scores and initial vitamin B12 levels. CONCLUSION: Conventional MRI may not be a useful tool for the diagnosis of SACD as it has very low sensitivity. Inverse correlation of Vitamin B12 levels with clinical scoring suggests that initial serum vitamin B12 levels may help in predicting the clinical severity.

Role of whole-body magnetic resonance imaging in the evaluation of extrapulmonary tuberculosis in immunocompetent patients.

OBJECTIVE: The aim of this study was to evaluate the role of whole-body magnetic resonance imaging (WB-MRI) in imaging of extrapulmonary tuberculosis. METHODS: Eighteen patients with single-site extrapulmonary tuberculosis were evaluated with contrast-enhanced dedicated MRI of the clinically symptomatic site followed by WB-MRI using contrast-enhanced 3-dimensional (3D) modified DIXON technique (m-DIXON) and diffusion-weighted WB imaging with background body signal suppression (DWIBS) sequences. Studies were read by 2 experienced radiologists, and additional lesions seen on WB-MRI were separately charted. RESULTS: Of 18 patients, 14 were found to have asymptomatic involvement of other organs on WB-MRI. In 5 patients, the information was helpful in choosing an easily accessible site for biopsy/aspiration. Postcontrast 3D m-DIXON was better in picking up brain and lymph nodal lesions, whereas DWIBS was better in detecting vertebral lesions. CONCLUSIONS: Whole-body MRI may be used for assessing the asymptomatic involvement of other body organs in tuberculosis. The combination of postcontrast 3D m-DIXON and DWIBS is complementary and may provide a road map for biopsy of accessible lesions.

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.

Imaging of meningitis and ventriculitis.

Central nervous system infections account for 1% of primary hospital admissions and 2% of nosocomial infections and when encountered require prompt diagnosis and initiation of specific treatment. Imaging findings are mostly nonspecific with respect to the causative pathogen. This article describes the anatomy of cranial meninges and extra-axial spaces of the brain. Characteristic findings and recent advances in neuroimaging of meningitis and its complications and ventriculitis are summarized, and certain noninfectious causes of meningitis and meningitis mimics are described.

Dose-dependent facilitation of peripheral nerve regeneration by bone marrow-derived mononuclear cells: a randomized controlled study: laboratory investigation.

OBJECT: Bone marrow-derived stem cells enhance the rate of regeneration of neuronal cells leading to clinical improvement in nerve injury, spinal cord injury, and brain infarction. Recent experiments in the local application of bone marrow-derived mononuclear cells (BM-MNCs) in models of sciatic nerve transection in rats have suggested their beneficial role in nerve regeneration, although the effects of variable doses of stem cells on peripheral nerve regeneration have never been specifically evaluated in the literature. In this paper, the authors evaluated the dose-dependent role of BM-MNCs in peripheral nerve regeneration in a model of sciatic nerve transection in rats. METHODS: The right sciatic nerve of 60 adult female Wistar rats (randomized into 2 test groups and 1 control group, 20 rats in each group) underwent transection under an operating microscope. The cut ends of the nerve were approximated using 2 epineural microsutures. The gap was filled with low-dose (5 million BM-MNCs/100 µl phosphate-buffered saline [PBS]) rat BM-MNCs in one group, high-dose (10 million BM-MNCs/100 µl PBS) rat BM-MNCs in another group, and only PBS in the control group, and the approximated nerve ends were sealed using fibrin glue. Histological assessment was performed after 30 days by using semiquantitative and morphometric analyses and was done to assess axonal regeneration, percentage of myelinated fibers, axonal diameter, fiber diameter, and myelin thickness at distal-most sites (10 mm from site of repair), intermediate distal sites (5 mm distal to the repair site), and site of repair. RESULTS: The recovery of nerve cell architecture after nerve anastomosis was far better in the high-dose BM-MNC group than in the low-dose BM-MNC and control groups, and it was most evident (p < 0.02 in the majority of the parameters [3 of 4]) at the distal-most site. Overall, the improvement in myelin thickness was most significant with incremental dosage of BM-MNCs, and was evident at the repair, intermediate distal, and distal-most sites (p = 0.001). CONCLUSIONS: This study emphasizes the role of BM-MNCs, which can be isolated easily from bone marrow aspirates, in peripheral nerve injury and highlights their dose-dependent facilitation of nerve regeneration.