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Colloids and Surfaces. B, Biointerfaces Nov 2021Induced pluripotent stem cells (iPSCs) can be used to generate desired types of cells that belong to the three germ layers (i.e., ectoderm, endoderm and mesoderm). These... (Review)
Review
Induced pluripotent stem cells (iPSCs) can be used to generate desired types of cells that belong to the three germ layers (i.e., ectoderm, endoderm and mesoderm). These cells possess great potential in regenerative medicine. Before iPSCs are used in various biomedical applications, the existing xenogeneic culture methods must be improved to meet the technical standards of safety, cost effectiveness, and ease of handling. In addition to commonly used 2D substrates, a culture system that mimics the native cellular environment in tissues will be a good choice when culturing iPS cells and differentiating them into different lineages. Hydrogels are potential candidates that recapitulate the native complex three-dimensional microenvironment. They possess mechanical properties similar to those of many soft tissues. Moreover, hydrogels support iPSC adhesion, proliferation and differentiation to various cell types. They are xeno-free and cost-effective. In addition to other substrates, such as mouse embryonic fibroblast (MEF), Matrigel, and vitronectin, the use of hydrogel-based substrates for iPSC culture and differentiation may help generate large numbers of clinical-grade cells that can be used in potential clinical applications. This review mainly focuses on the use of hydrogels for the culture and differentiation of iPSCs into various cell types and their potential applications in regenerative medicine.
Topics: Animals; Cell Differentiation; Fibroblasts; Hydrogels; Induced Pluripotent Stem Cells; Mice
PubMed: 34333302
DOI: 10.1016/j.colsurfb.2021.111991 -
Molecular Biology of the Cell Jan 2021We performed a high-throughput whole-genome RNAi screen to identify novel inhibitors of ciliogenesis in normal and basal breast cancer cells. Our screen uncovered a...
We performed a high-throughput whole-genome RNAi screen to identify novel inhibitors of ciliogenesis in normal and basal breast cancer cells. Our screen uncovered a previously undisclosed, extensive network of genes linking integrin signaling and cellular adhesion to the extracellular matrix (ECM) with inhibition of ciliation in both normal and cancer cells. Surprisingly, a cohort of genes encoding ECM proteins was also identified. We characterized several ciliation inhibitory genes and showed that their silencing was accompanied by altered cytoskeletal organization and induction of ciliation, which restricts cell growth and migration in normal and breast cancer cells. Conversely, supplying an integrin ligand, vitronectin, to the ECM rescued the enhanced ciliation observed on silencing this gene. Aberrant ciliation could also be suppressed through hyperactivation of the YAP/TAZ pathway, indicating a potential mechanistic basis for our findings. Our findings suggest an unanticipated reciprocal relationship between ciliation and cellular adhesion to the ECM and provide a resource that could vastly expand our understanding of controls involving "outside-in" and "inside-out" signaling that restrain cilium assembly.
Topics: Actins; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cilia; Extracellular Matrix; Female; Focal Adhesions; Gene Silencing; Genetic Association Studies; Genetic Testing; Genome, Human; Humans; Integrins; Ligands; Organogenesis; RNA, Small Interfering; Signal Transduction; Suppression, Genetic
PubMed: 33206585
DOI: 10.1091/mbc.E20-02-0111 -
Journal of Materials Chemistry. B Oct 2021Human pluripotent stem cells (hPSCs) are typically cultivated on extracellular matrix (ECM) protein-coated dishes in xeno-free culture conditions. We supplemented mixed...
Laminin-511 and recombinant vitronectin supplementation enables human pluripotent stem cell culture and differentiation on conventional tissue culture polystyrene surfaces in xeno-free conditions.
Human pluripotent stem cells (hPSCs) are typically cultivated on extracellular matrix (ECM) protein-coated dishes in xeno-free culture conditions. We supplemented mixed ECM proteins (laminin-511 and recombinant vitronectin, rVT) in culture medium for hPSC culture on conventional polystyrene dishes. Three hPSC cell lines were successfully cultivated on uncoated polystyrene dishes in medium supplemented with optimal conditions of laminin-511 and rVT. Excellent colony shape and colony size as well as high expansion fold of hPSCs were found under these conditions, whereas the colony size was small and poor expansion fold was found solely on L-511-coated dishes. A small portion of L-511 in the culture medium supported hPSC adhesion and prevented the adhesion from being too strong on the uncoated dishes, and rVT in the culture medium further supported adhesion of hPSCs on the dishes by maintaining their pluripotency. Having the optimal composition of L-511 and rVT in the culture medium was important for generating good hPSC colony shapes and sizes as well as a high expansion fold. After long-term culture of hPSCs on uncoated dishes supplemented with the mixed proteins, the hPSCs successfully showed pluripotent markers and could differentiate into a specific lineage of cells, cardiomyocytes, with high efficiency.
Topics: Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Humans; Laminin; Particle Size; Pluripotent Stem Cells; Polystyrenes; Recombinant Proteins; Surface Properties; Vitronectin
PubMed: 34605523
DOI: 10.1039/d1tb01878g -
Scientific Reports May 2021Bovine embryonic stem cells (bESCs) extend the lifespan of the transient pluripotent bovine inner cell mass in vitro. After years of research, derivation of stable bESCs...
Bovine embryonic stem cells (bESCs) extend the lifespan of the transient pluripotent bovine inner cell mass in vitro. After years of research, derivation of stable bESCs was only recently reported. Although successful, bESC culture relies on complex culture conditions that require a custom-made base medium and mouse embryonic fibroblasts (MEF) feeders, limiting the widespread use of bESCs. We report here simplified bESC culture conditions based on replacing custom base medium with a commercially available alternative and eliminating the need for MEF feeders by using a chemically-defined substrate. bESC lines were cultured and derived using a base medium consisting of N2B27 supplements and 1% BSA (NBFR-bESCs). Newly derived bESC lines were easy to establish, simple to propagate and stable after long-term culture. These cells expressed pluripotency markers and actively proliferated for more than 35 passages while maintaining normal karyotype and the ability to differentiate into derivatives of all three germ lineages in embryoid bodies and teratomas. In addition, NBFR-bESCs grew for multiple passages in a feeder-free culture system based on vitronectin and Activin A medium supplementation while maintaining pluripotency. Simplified conditions will facilitate the use of bESCs for gene editing applications and pluripotency and lineage commitment studies.
Topics: Animals; Cattle; Cell Culture Techniques; Cell Differentiation; Cell Lineage; Culture Media; Embryonic Stem Cells; Fibroblasts
PubMed: 34040070
DOI: 10.1038/s41598-021-90422-0 -
Experimental and Therapeutic Medicine Jan 2022Neurogenic bladder (NGB) is an important complication of urinary tract dysfunction after spinal cord injury (SCI). However, using urodynamics and urography to guide...
Neurogenic bladder (NGB) is an important complication of urinary tract dysfunction after spinal cord injury (SCI). However, using urodynamics and urography to guide therapy remains invasive and complicated. Therefore, the present study aimed to identify potential noninvasive biomarkers from urinary exosomes that can facilitate diagnosis and guide prognosis of patients with NGB subsequent to SCI. Urinary exosomes were isolated, and their proteome profile was analyzed by mass spectrometry. Transmission electron microscopy and Nanoparticle Tracking Analysis confirmed the size and morphological characteristics of urinary exosomes. In addition, bioinformatics analysis and parallel reaction monitoring (PRM) were used to screen candidate biomarkers. The selected biomarkers were validated using western blotting and ELISA. Mass spectrometry identified 134 upregulated proteins and 99 downregulated proteins between the vesicoureteral reflux (VUR) and non-VUR groups. A total of 18 candidate proteins were selected for PRM validation, but only vitronectin (VTN) and α-1 type I collagen (COL1A1) demonstrated significant differences. In the validation experiments using western blotting and ELISA, VTN was exclusively highly expressed in VUR patients compared with non-VUR patients. However, the ELISA results of COL1A1 revealed no significant difference when a larger sample size was used. Furthermore, a receiver operating characteristic curve of ELISA-based VTN demonstrated an area under the curve of 0.795 and 80% sensitivity at a threshold set to give 82.9% specificity. Collectively, these results suggested that VTN in urinary exosomes may be used as a biomarker to predict the progression and guide the prognosis of NGB.
PubMed: 34934436
DOI: 10.3892/etm.2021.10988 -
Journal of Cerebral Blood Flow and... Oct 2022We found that blood vitronectin (VTN) leaks into the brain and exacerbates tissue loss after stroke by increasing pro-inflammatory IL-6 expression in female, but not...
We found that blood vitronectin (VTN) leaks into the brain and exacerbates tissue loss after stroke by increasing pro-inflammatory IL-6 expression in female, but not male, mice. VTN signals through integrins and downstream focal adhesion kinase (FAK). Here, a two day systemic treatment with a small molecule FAK inhibitor starting 6 h after middle cerebral artery occlusion reduced ipsilateral brain injury size by ∼40-45% at 7 and 14 d, as well as inflammation and motor dysfunction in wild-type female, but not male, mice. FAK inhibition also reduced IL-6 expression in the injured female striatum at 24 h by 62%. Inducible selective gene deletion of FAK in astrocytes also reduced acute IL-6 expression by 72% only in females, and mitigated infarct size by ∼80% and inflammation at 14 d after stroke. Lastly, VTN-/- females had better outcomes, but FAK inhibitor treatment had no additional protective or anti-inflammatory effects. Altogether, this suggests that VTN is detrimental in females primarily through FAK and that FAK inhibition provides neuroprotection (cerebroprotection) by reducing VTN-induced IL-6 expression in astrocytes. Thus, VTN signaling can be targeted to mitigate harmful inflammation with relevance to treatments for women with ischemic stroke, who often have worse outcomes than men.
Topics: Animals; Anti-Inflammatory Agents; Brain Ischemia; Female; Focal Adhesion Protein-Tyrosine Kinases; Humans; Inflammation; Integrins; Interleukin-6; Ischemic Stroke; Mice; Mice, Inbred C57BL; Neuroprotection; Stroke; Vitronectin
PubMed: 35702047
DOI: 10.1177/0271678X221107871 -
Biomedicine & Pharmacotherapy =... Aug 2022Nephrotoxicity (NT) is a renal-specific situation caused by different toxins and drugs like non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs like diclofenac (DCF)...
Nephrotoxicity (NT) is a renal-specific situation caused by different toxins and drugs like non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs like diclofenac (DCF) lead to glomerular dysfunction. Pentoxifylline (PTX) and berberine (BER) have antioxidant and anti-inflammatory properties. Thus, the objective of the present study was to investigate the ameliorative effect of PTX, BER and their combination against DCF-mediated acute NT. Induction of acute NT was done via DCF injection (150 mg/kg I.P, for 6 days) in rats. PTX 200 mg/kg, BER 200 mg/kg and their combination were administrated for 6 days prior to DCF injection and concurrently with DCF for additional 6 days. Acute NT was evaluated biochemically and histopathologically by measuring blood urea (BU), serum creatinine (SCr), kidney injury molecule-1(KIM-1), integrin (ITG), and vitronectin (VTN), interleukin (IL)-18, Neutrophil gelatinase-associated lipocalin (NGAL), glomerular filtration rate (GFR), superoxide dismutase (SOD) and glutathione (GSH) and malondialdehyde (MDA) with the scoring of histopathological alterations. PTX, BER and their combination significantly (P < 0.05) attenuated biochemical and histopathological changes in DCF-mediated acute NT by amelioration of BU, SCr, KIM-1, ITG, VTN, IL-18, NGAL, GFR, SOD, GSH, MDA and scoring of histopathological alterations. The combined effects of PTX and BER produced more significant effects (P < 0.05) than either PTX or BER when used alone against DCF-induced acute NT. In conclusion, BER and BTX were found to have potential renoprotective effects against DCF-induced NT in rats by inhibiting inflammatory reactions and oxidative stress.
Topics: Animals; Anti-Inflammatory Agents; Anti-Inflammatory Agents, Non-Steroidal; Berberine; Diclofenac; Drug-Related Side Effects and Adverse Reactions; Glutathione; Inflammation; Kidney; Lipocalin-2; Male; Oxidative Stress; Pentoxifylline; Rats; Rats, Sprague-Dawley; Renal Insufficiency; Superoxide Dismutase
PubMed: 35671584
DOI: 10.1016/j.biopha.2022.113225 -
Molecular & Cellular Proteomics : MCP Sep 2023The asialoglycoprotein receptor (ASGPR) and the mannose receptor C-type 1 (MRC1) are well known for their selective recognition and clearance of circulating...
The asialoglycoprotein receptor (ASGPR) and the mannose receptor C-type 1 (MRC1) are well known for their selective recognition and clearance of circulating glycoproteins. Terminal galactose and N-Acetylgalactosamine are recognized by ASGPR, while terminal mannose, fucose, and N-Acetylglucosamine are recognized by MRC1. The effects of ASGPR and MRC1 deficiency on the N-glycosylation of individual circulating proteins have been studied. However, the impact on the homeostasis of the major plasma glycoproteins is debated and their glycosylation has not been mapped with high molecular resolution in this context. Therefore, we evaluated the total plasma N-glycome and plasma proteome of ASGR1 and MRC1 deficient mice. ASGPR deficiency resulted in an increase in O-acetylation of sialic acids accompanied by higher levels of apolipoprotein D, haptoglobin, and vitronectin. MRC1 deficiency decreased fucosylation without affecting the abundance of the major circulating glycoproteins. Our findings confirm that concentrations and N-glycosylation of the major plasma proteins are tightly controlled and further suggest that glycan-binding receptors have redundancy, allowing compensation for the loss of one major clearance receptor.
Topics: Mice; Animals; Asialoglycoprotein Receptor; Mannose Receptor; Glycoproteins; Glycosylation; Protein Processing, Post-Translational; Carrier Proteins; Mannose
PubMed: 37414249
DOI: 10.1016/j.mcpro.2023.100615 -
Cells Aug 2021Male human fetal germ cells (hFGCs) give rise to spermatogonial stem cells (SSCs), which are the adult precursors of the male gametes. Human SSCs are a promising...
Male human fetal germ cells (hFGCs) give rise to spermatogonial stem cells (SSCs), which are the adult precursors of the male gametes. Human SSCs are a promising (autologous) source of cells for male fertility preservation; however, in contrast to mouse SSCs, we are still unable to culture them in the long term. Here, we investigated the effect of two different culture media and four substrates (laminin, gelatin, vitronectin and matrigel) in the culture of dissociated second trimester testes, enriched for hFGCs. After 6 days in culture, we quantified the presence of POU5F1 and DDX4 expressing hFGCs. We observed a pronounced difference in hFGC number in different substrates. The combination of gelatin-coated substrate and medium containing GDNF, LIF, FGF2 and EGF resulted in the highest percentage of hFGCs (10% of the total gonadal cells) after 6 days of culture. However, the vitronectin-coated substrate resulted in a comparable percentage of hFGCs regardless of the media used (3.3% of total cells in Zhou-medium and 4.8% of total cells in Shinohara-medium). We provide evidence that not only the choices of culture medium but also choices of the adequate substrate are crucial for optimizing culture protocols for male hFGCs. Optimizing culture conditions in order to improve the expansion of hFGCs will benefit the development of gametogenesis assays in vitro.
Topics: Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Collagen; Culture Media; DEAD-box RNA Helicases; Drug Combinations; Gelatin; Gene Expression Profiling; Germ Cells; Humans; Laminin; Male; Octamer Transcription Factor-3; Proteoglycans; RNA-Seq; Single-Cell Analysis; Stem Cells; Testis; Vitronectin
PubMed: 34440801
DOI: 10.3390/cells10082033 -
Diagnostics (Basel, Switzerland) Jan 2023Neurogenic lower urinary tract dysfunction requires lifelong surveillance and management for the perseveration of patients' quality of life and the prevention of... (Review)
Review
BACKGROUND
Neurogenic lower urinary tract dysfunction requires lifelong surveillance and management for the perseveration of patients' quality of life and the prevention of significant morbidity and mortality. Urine biomarkers are an attractive noninvasive method of surveillance for these patients. The aim of this systematic review is to search for and critically appraise studies that investigate the clinical usefulness of urine biomarkers in the management of neurogenic lower urinary tract dysfunction (NLUTD) in adults.
METHODS
This review was conducted according to PRISMA and MOOSE guidelines. Search strategy included PubMed, CENTRAL, and Scopus (until October 2022). Studies investigating potential urine biomarkers for the management of adults with NLUTD were included.
RESULTS
Fifteen studies fulfilled the criteria. To date, a variety of different urine molecules have been investigated for the diagnosis and management of neurogenic overactive bladder and detrusor overactivity (nerve growth factor, brain-derived neurotrophic factor, prostaglandin E2, prostaglandin F2α, transformation growth factor β-1, tissue inhibitor metalloproteinase-2, matrix metalloproteinase-2, substance P, microRNA), diagnosis of vesicoureteral reflux (exosomal vitronectin), urinary tract infection (neutrophil gelatinase-associated lipocalin, interleukin 6) and bladder cancer screening (cytology, BTA stat, survivin) in neurological patients.
CONCLUSION
Further studies are needed to specify the utility of each molecule in the management algorithm of adult NLUTD.
PubMed: 36766573
DOI: 10.3390/diagnostics13030468