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Journal of Stem Cells & Regenerative... 2022Coronary heart disease (CHD) is a leading cause of death globally, while its current management is limited to reducing the myocardial infarction area without actually...
Coronary heart disease (CHD) is a leading cause of death globally, while its current management is limited to reducing the myocardial infarction area without actually replacing dead cardiomyocytes. Direct cell reprogramming is a method of cellular cardiomyoplasty which aims for myocardial tissue regeneration, and CD34 cells are one of the potential sources due to their shared embryonic origin with cardiomyocytes. However, the isolation and culture of non-adherent CD34 cells is crucial to obtain adequate cells for high-efficiency genetic modification. This study aimed to investigate the optimal method for isolation and culture of CD34 peripheral blood cells using certain culture media. A peripheral blood sample was obtained from a healthy subject and underwent pre-enrichment, isolation, and expansion. The culture was subsequently observed for their viability, adherence, and confluence. Day 0 observation of the culture showed a healthy CD34 cell with a round cell shape, without any adherent cells present yet. Day 4 of observation showed that CD34 cells within the blood plasma medium became adherent, indicated by their transformations into spindle or oval morphologies. Meanwhile, CD34 cells in vitronectin and fibronectin media showed no adherent cells and many of them died. Day 7 observation revealed more adherent CD34 cells in blood plasma medium, and which had 75% of confluence. In conclusion, the CD34 cells that were isolated using a combination of density and magnetic methods may be viable and adequately adhere in culture using blood plasma medium, but not in cultures using fibronectin and vitronectin.
PubMed: 36003658
DOI: 10.46582/jsrm.1801004 -
Bioinformatics and Biology Insights 2022Some studies in the literature show that viruses can affect bacteria directly or indirectly, and viruses use their own specific ways to do these interactions....
Some studies in the literature show that viruses can affect bacteria directly or indirectly, and viruses use their own specific ways to do these interactions. Furthermore, it is said that bacteria are prone to attachment mammalian cells during a viral illness using their surface proteins that bind to host extracellular matrix proteins such as fibronectin, fibrinogen, vitronectin, and elastin. A recent study identified the cooperation between bacteria and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in silico, in vitro, and in vivo. Like this study, we hypothesized that more bacteria protein might help SARS-CoV-2 transport and attach to angiotensin-converting enzyme 2 (ACE2). The bacteria's outer membrane proteins (OMPs) we chose were not random; they had to be on the outer surface of the bacteria because these proteins on the outer surface should have a high probability of interacting with both the spike protein and ACE2. We obtained by using bioinformatics tools that there may be binding between both ACE2 and spike protein of these bacteria's OMPs. Protein-protein interaction results also supported our hypothesis. Therefore, based on our predicted results, these bacteria OMPs may help SARS-CoV-2 move in our body, and both find and attach to ACE2. It is expected that these inferences obtained from the bioinformatics results may play a role in the SARS-CoV-2 virus reaching host cells. Thus, it may bring a different perspective to studies on how the virus can infect host cells.
PubMed: 35966808
DOI: 10.1177/11779322221116320 -
ELife Aug 2022The membrane attack complex (MAC or C5b-9) is an important effector of the immune system to kill invading microbes. MAC formation is initiated when complement enzymes on...
The membrane attack complex (MAC or C5b-9) is an important effector of the immune system to kill invading microbes. MAC formation is initiated when complement enzymes on the bacterial surface convert complement component C5 into C5b. Although the MAC is a membrane-inserted complex, soluble forms of MAC (sMAC), or terminal complement complex (TCC), are often detected in sera of patients suffering from infections. Consequently, sMAC has been proposed as a biomarker, but it remains unclear when and how it is formed during infections. Here, we studied mechanisms of MAC formation on different Gram-negative and Gram-positive bacteria and found that sMAC is primarily formed in human serum by bacteria resistant to MAC-dependent killing. Surprisingly, C5 was converted into C5b more potently by MAC-resistant compared to MAC-sensitive strains. In addition, we found that MAC precursors are released from the surface of MAC-resistant bacteria during MAC assembly. Although release of MAC precursors from bacteria induced lysis of bystander human erythrocytes, serum regulators vitronectin (Vn) and clusterin (Clu) can prevent this. Combining size exclusion chromatography with mass spectrometry profiling, we show that sMAC released from bacteria in serum is a heterogeneous mixture of complexes composed of C5b-8, up to three copies of C9 and multiple copies of Vn and Clu. Altogether, our data provide molecular insight into how sMAC is generated during bacterial infections. This fundamental knowledge could form the basis for exploring the use of sMAC as biomarker.
Topics: Complement Activation; Complement C5; Complement Membrane Attack Complex; Escherichia coli; Escherichia coli Infections; Gram-Positive Bacteria; Humans; Vitronectin
PubMed: 35947526
DOI: 10.7554/eLife.77503 -
Materials Today. Bio Dec 2022Mesenchymal stem cell (MSC)-based tissue engineering strategies are of interest in the field of bone tissue regenerative medicine. MSCs are commonly investigated in...
Mesenchymal stem cell (MSC)-based tissue engineering strategies are of interest in the field of bone tissue regenerative medicine. MSCs are commonly investigated in combination with growth factors (GFs) and biomaterials to provide a regenerative environment for the cells. However, optimizing how biomaterials interact with MSCs and efficiently deliver GFs, remains a challenge. Here, via plasma polymerization, tissue culture plates are coated with a layer of poly (ethyl acrylate) (PEA), which is able to spontaneously permit fibronectin (FN) to form fibrillar nanonetworks. However, vitronectin (VN), another important extracellular matrix (ECM) protein forms multimeric globules on the polymer, thus not displaying functional groups to cells. Interestingly, when FN and VN are co-absorbed onto PEA surfaces, VN can be entrapped within the FN fibrillar nanonetwork in the monomeric form providing a heterogeneous, open ECM network. The combination of FN and VN promote MSC adhesion and leads to enhanced GF binding; here we demonstrate this with bone morphogenetic protein-2 (BMP2). Moreover, MSC differentiation into osteoblasts is enhanced, with elevated expression of osteopontin (OPN) and osteocalcin (OCN) quantified by immunostaining, and increased mineralization observed by von Kossa staining. Osteogenic intracellular signalling is also induced, with increased activity in the SMAD pathway. The study emphasizes the need of recapitulating the complexity of native ECM to achieve optimal cell-material interactions.
PubMed: 35937570
DOI: 10.1016/j.mtbio.2022.100367 -
Journal of Extracellular Vesicles Aug 2022Breast cancer cells release a large quantity of biocargo-bearing extracellular vesicles (EVs), which mediate intercellular communication within the tumour...
Breast cancer cells release a large quantity of biocargo-bearing extracellular vesicles (EVs), which mediate intercellular communication within the tumour microenvironment and promote metastasis. To identify EV-bound proteins related to metastasis, we used mass spectrometry to profile EVs from highly and poorly metastatic breast cancer lines of human and mouse origins. Comparative mass spectrometry indicated that integrins, including αv and β1 subunits, are preferentially enriched in EVs of highly metastatic origin over those of poorly metastatic origin. These results are consistent with our histopathological findings, which show that integrin αv is associated with disease progression in breast cancer patients. Integrin αv colocalizes with the multivesicular-body marker CD63 at a higher frequency in the tumour and is enriched in circulating EVs of breast cancer patients at late stages when compared with circulating EVs from early-stage patients. With a magnetic bead-based flow cytometry assay, we confirmed that integrins αv and β1 are enriched in the CD63 subsets of EVs from both human and mouse highly metastatic cells. By analysing the level of integrin αv on circulating EVs, this assay could predict the metastatic potential of a xenografted mouse model. To explore the export mechanism of integrins into EVs, we performed immunoprecipitation mass spectrometry and identified members of the galectin family as potential shuttlers of integrin αvβ1 into EVs. In particular, knockdown of galectin-3, but not galectin-1, causes a reduction in the levels of cell surface integrins β1 and αv, and decreases the colocalization of these integrins with CD63. Importantly, knockdown of galectin-3 leads to a decrease of integrin αvβ1 export into the EVs concomitant with a decrease in the metastatic potential of breast cancer cells. Moreover, inhibition of the integrin αvβ1 complex leads to a reduction in the binding of EVs to fibronectin, suggesting that integrin αvβ1 is important for EV retention in the extracellular matrix. EVs retained in the extracellular matrix are taken up by fibroblasts, which differentiate into cancer associated fibroblasts. In summary, our data indicate an important link between EV-bound integrin αvβ1 with breast cancer metastasis and provide additional insights into the export of integrin αvβ1 into EVs in the context of metastasis.
Topics: Animals; Breast Neoplasms; Extracellular Vesicles; Female; Galectin 3; Humans; Integrin alphaV; Melanoma; Mice; Receptors, Vitronectin; Skin Neoplasms; Tumor Microenvironment; Melanoma, Cutaneous Malignant
PubMed: 35923105
DOI: 10.1002/jev2.12234 -
RSC Advances Jul 2022Surface treatment is known as a very efficient measure by which to modulate the surface properties of biomaterials in terms of grain structure, topography, roughness and...
Using a two-step method of surface mechanical attrition treatment and calcium ion implantation to promote the osteogenic activity of mesenchymal stem cells as well as biomineralization on a β-titanium surface.
Surface treatment is known as a very efficient measure by which to modulate the surface properties of biomaterials in terms of grain structure, topography, roughness and chemistry to determine the osseointegration of implants. In this work, a two-step method of surface modification was employed to impart high osteogenic activity and biomineralization capacity on a Ti-25Nb-3Mo-2Sn-3Zr alloy (a type of β-titanium named TLM). The preliminary surface mechanical attrition treatment (SMAT) refined the average grain size from 170 ± 19 μm to 74 ± 8 nm in the TLM surface layer and promoted the surface to be much rougher and more hydrophilic. The subsequent Ca-ion implantation did not change the surface roughness and topography obviously, but enhanced the surface wettability of the SMAT-treated TLM alloy. The evaluations of the adhesion, proliferation, osteogenic genes (RUNX2, ALP, BMP-2, OPN, OCN and COL-I) and protein (ALP, OPN, OCN and COL-I) expressions, as well as extracellular matrix (ECM) mineralization of mesenchymal stem cells (MSCs) revealed that the initial SMAT-treated sample significantly enhanced the adhesion and osteogenic functions of MSCs compared to an untreated TLM sample, and the subsequent introduction of Ca ions onto the SMAT-derived nanograined sample further promotes the MSC adhesion, proliferation, osteo-differentiation and ECM mineralization due to the adsorption of more proteins such as laminin (Ln), fibronectin (Fn) and vitronectin (Vn) on the surface, as well as the increase in extracellular Ca concentrations. In addition, the biomineralization capacity of the samples was also evaluated by soaking them in simulated bodily fluid (SBF) at 37 °C for 28 days, and the results showed that the Ca-ion implanted sample significantly boosted the deposition of Ca and P containing minerals on its surface, which was associated with the generation of more Ti-OH groups on the surface after ion implantation. The combination of the SMAT technique and Ca-ion implantation thus endowed the TLM alloy with outstanding osteogenic and biomineralization properties, providing a potential means for its future use in the orthopedic field.
PubMed: 35919615
DOI: 10.1039/d2ra00032f -
Annals of Thoracic and Cardiovascular... Oct 2022Circular RNAs are associated with non-small cell lung cancer (NSCLC) development and radiosensitivity. Nevertheless, the function and regulation mechanism of...
BACKGROUND
Circular RNAs are associated with non-small cell lung cancer (NSCLC) development and radiosensitivity. Nevertheless, the function and regulation mechanism of hsa_circ_0079530 (circ_0079530) in NSCLC development and radiosensitivity are largely unknown.
METHODS
The abundances of circ_0079530, microRNA (miR-409-3p), aquaporin 4 (AQP4), E-cadherin, intercellular adhesion molecule-1, vitronectin, proliferating cell nuclear antigen, and matrix metalloproteinase 9 were determined via quantitative reverse transcription polymerase chain reaction or western blotting. Cell proliferation, survival fraction, cycle process, migration, invasion, and in vivo growth were examined by cell counting kit-8, colony formation, flow cytometry, transwell, and xenograft analyses. The binding relationship was assessed via dual-luciferase reporter assay and RNA immunoprecipitation assay.
RESULTS
Circ_0079530 expression was increased in NSCLC tissues and radioresistant samples. Circ_0079530 knockdown restrained cell proliferation, migration, and invasion, and facilitated radiosensitivity. Circ_0079530 silence decreased tumor growth with or without radiation treatment. Circ_0079530 was verified as a miR-409-3p sponge, and miR-409-3p downregulation mitigated the effects of circ_0079530 interference on NSCLC cell malignancy and radiosensitivity. AQP4 was directly targeted by miR-409-3p. MiR-409-3p restrained cell proliferation, migration, and invasion, and enhanced radiosensitivity by decreasing AQP4 expression. Notably, circ_0079530 silence decreased AQP4 expression by regulating miR-409-3p expression.
CONCLUSION
Circ_0079530 silence repressed cell proliferation, migration, and invasion, and facilitated radiosensitivity in NSCLC cells by mediating miR-409-3p/AQP4 axis.
Topics: Humans; Aquaporin 4; Cadherins; Carcinoma, Non-Small-Cell Lung; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Intercellular Adhesion Molecule-1; Lung Neoplasms; Matrix Metalloproteinase 9; MicroRNAs; Proliferating Cell Nuclear Antigen; Radiation Tolerance; RNA, Circular; Treatment Outcome; Vitronectin
PubMed: 35896371
DOI: 10.5761/atcs.oa.21-00237 -
Bioengineering (Basel, Switzerland) Jul 2022Dry age-related macular degeneration (AMD) is estimated to impact nearly 300 million individuals globally by 2040. While no treatment options are currently available,...
Dry age-related macular degeneration (AMD) is estimated to impact nearly 300 million individuals globally by 2040. While no treatment options are currently available, multiple clinical trials investigating retinal pigmented epithelial cells derived from human pluripotent stem cells (hPSC-RPE) as a cellular replacement therapeutic are currently underway. It has been estimated that a production capacity of >109 RPE cells annually would be required to treat the afflicted population, but current manufacturing protocols are limited, being labor-intensive and time-consuming. Microcarrier technology has enabled high-density propagation of many adherent mammalian cell types via monolayer culture on surfaces of uM-diameter matrix spheres; however, few studies have explored microcarrier-based culture of RPE cells. Here, we provide an approach to the growth, maturation, and differentiation of hPSC-RPE cells on Cytodex 1 (C1) and Cytodex 3 (C3) microcarriers. We demonstrate that hPSC-RPE cells adhere to microcarriers coated with Matrigel, vitronectin or collagen, and mature in vitro to exhibit characteristic epithelial cell morphology and pigmentation. Microcarrier-grown hPSC-RPE cells (mcRPE) are viable; metabolically active; express RPE signature genes including BEST1, RPE65, TYRP1, and PMEL17; secrete the trophic factors PEDF and VEGF; and demonstrate phagocytosis of photoreceptor outer segments. Furthermore, we show that undifferentiated hESCs also adhere to Matrigel-coated microcarriers and are amenable to directed RPE differentiation. The capacity to support hPSC-RPE cell cultures using microcarriers enables efficient large-scale production of therapeutic RPE cells sufficient to meet the treatment demands of a large AMD patient population.
PubMed: 35877348
DOI: 10.3390/bioengineering9070297 -
PLoS Pathogens Jul 2022During hematogenously disseminated candidiasis, blood borne fungi must invade the endothelial cells that line the blood vessels to infect the deep tissues. Although...
During hematogenously disseminated candidiasis, blood borne fungi must invade the endothelial cells that line the blood vessels to infect the deep tissues. Although Candida albicans, which forms hyphae, readily invades endothelial cells, other medically important species of Candida are poorly invasive in standard in vitro assays and have low virulence in immunocompetent mouse models of disseminated infection. Here, we show that Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida krusei can bind to vitronectin and high molecular weight kininogen present in human serum. Acting as bridging molecules, vitronectin and kininogen bind to αv integrins and the globular C1q receptor (gC1qR), inducing human endothelial cells to endocytose the fungus. This mechanism of endothelial cell invasion is poorly supported by mouse endothelial cells but can be restored when mouse endothelial cells are engineered to express human gC1qR or αv integrin. Overall, these data indicate that bridging molecule-mediated endocytosis is a common pathogenic strategy used by many medically important Candida spp. to invade human vascular endothelial cells.
Topics: Animals; Candida; Candida albicans; Candidiasis; Endothelial Cells; Humans; Mice; Vitronectin
PubMed: 35797411
DOI: 10.1371/journal.ppat.1010681 -
ALTEX 2023Human induced pluripotent stem cells (hiPSCs) offer great opportunities within the 3R framework. In the field of toxicology, they may contribute greatly to the reduction... (Comparative Study)
Comparative Study
Human induced pluripotent stem cells (hiPSCs) offer great opportunities within the 3R framework. In the field of toxicology, they may contribute greatly to the reduction and eventually replacement of animal models. However, culturing hiPSCs as well as differentiation of hiPSCs into target cells that are used for toxicity testing depend on the presence of extracellular matrix (ECM) coating the growth surface. The most widely used ECM is MatrigelR, an animal product that is derived from mouse sarcoma. Drawbacks of Matrigel are widely recognized and include batch-to batch variations, use of animal rather than human material, and ethical concerns about its production. While alternative coatings exist, higher cost and limited characterizations may hinder their broader uptake by the scientific community. Here, we report an extensive comparison of three commercially available human ECM coatings, vitronectin, laminin-511, and laminin-521, to Matrigel in three different hiPSC lines in long-term culture (≥ 9 passages). Characterization included expression of pluripotent markers in a genome-wide transcriptomics study (TempO-Seq), capacity to differentiate into embryoid bodies, and karyotype stability assessed by analyzing copy number variations by shallow DNA sequencing. Furthermore, a low-cost, decellularized ECM produced by human neonatal dermal fibroblasts was tested. In addition, all alternative coatings were tested for hiPSC differentiation into renal podocyte-like cells in a genome-wide transcriptomics screen. Our results show that all tested coatings were highly comparable to animal-derived Matrigel for both hiPSC maintenance and differentiation into renal podocyte-like cells. Furthermore, decellularized fibroblast-ECM could be a novel, attractive low-cost coating material.
Topics: Animals; Humans; Infant, Newborn; Mice; Cell Differentiation; DNA Copy Number Variations; Extracellular Matrix; Fibroblasts; Induced Pluripotent Stem Cells; Laminin; Podocytes; Recombinant Proteins
PubMed: 35791294
DOI: 10.14573/altex.2112204