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Karyotype analysis of the cell line. Representative G-band karyotype (A) and corresponding metaphase spread (B) from passage 50 cells of the hBSM cell line are shown. Chromosomes were
Source publication
We report a cell line (hBSM) established from human urinary bladder wall smooth muscle that maintains most of the phenotypic characteristics of smooth muscle cells. Cells were dissociated from the muscular layer with collagenase (1 mg/ml) and collected and grown in M199 supplemented with 10% fetal calf serum and 1% antibiotic-antimycotic. Primary c...
Citations
... Confocal microscopy images of the immunostained urothelial tubes after 14 d showed the formation of mature monolayer of spreading HUCs, with the expression of both ZO-1 and E-cadherin, confirming the presence of critical tight junction protein-1 and cellcell adhesion molecules necessary for proper urothelial function. In addition, expression of α-SMA by HBdSMCs [36] was also observed ( Figure 4E). ...
Despite advances in the bioprinting technology, biofabrication of circumferentially multilayered tubular tissues or organs with cellular heterogeneity, such as blood vessels, trachea, intestine, colon, ureter, and urethra, remains a challenge. Herein, a promising multichannel coaxial extrusion system (MCCES) for microfluidic bioprinting of circumferentially multilayered tubular tissues in a single step, using customized bioinks constituting gelatin methacryloyl, alginate, and eight‐arm poly(ethylene glycol) acrylate with a tripentaerythritol core, is presented. These perfusable cannular constructs can be continuously tuned up from monolayer to triple layers at regular intervals across the length of a bioprinted tube. Using customized bioink and MCCES, bioprinting of several tubular tissue constructs using relevant cell types with adequate biofunctionality including cell viability, proliferation, and differentiation is demonstrated. Specifically, cannular urothelial tissue constructs are bioprinted, using human urothelial cells and human bladder smooth muscle cells, as well as vascular tissue constructs, using human umbilical vein endothelial cells and human smooth muscle cells. These bioprinted cannular tissues can be actively perfused with fluids and nutrients to promote growth and proliferation of the embedded cell types. The fabrication of such tunable and perfusable circumferentially multilayered tissues represents a fundamental step toward creating human cannular tissues.
... Human bSMCs purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) were cultured in smooth muscle growth medium supplemented with 10% fetal bovine serum, 1× smooth muscle growth factors, 1× 1000 U/ml penicillin, and 1000 mg/ml streptomycin from ScienCell Research Laboratories (CA). The phenotype of the cells was confirmed by culture characteristics coupled with the expression of bladder smooth muscle markers; high molecular weight caldesmon, desmin, α smooth muscle actin (αSMA) as well as the absence of light molecule weight caldesmon as previously established [9,15,16]. ...
Purpose:
Partial bladder outlet obstruction is a multifactorial urological condition in which hypoxia plays a significant role. We recently investigated hypoxia's role as a single stressor and found that hypoxia induced an intense inflammatory and profibrotic switch in bladder smooth muscle cells (bSMCs). With the immunomodulatory capacity of mesenchymal stem cells (MSCs), we aimed to investigate if the hypoxia-signaling pathways can be mitigated using MSCs.
Methods:
Bladder smooth muscle cells were cultured in 3% oxygen tension for 72 h with either the direct or indirect co-culture with bone marrow derived MSCs. High pore density transwells were used for indirect co-cultures. Total RNA was extracted for gene expression analysis and the Mesoscale multiplex assay was used for secreted cytokines and growth factor measurements. Total collagen contents were determined using the Sirius Red collagen assay.
Results:
Hypoxia induced increase of HIF3α, VEGF, TGFβ1, TNFα, IL-1β, IL-6, αSMA, and total collagen expression and decreased IL-10 levels in bSMCs. Both direct and indirect MSCs co-cultures inhibited > 50% of hypoxia-induced TGFβ1 and IL-6 expression (p < 0.005) in a HIF-independent manner. Also, both MSCs co-culture techniques induced > 200% increase in IL-10 protein (p < 0.005) and inhibited hypoxia-induced αSMA, collagen I and III transcripts as well as total collagen proteins (p < 0.0001). Contrastingly, the hypoxia-induced IL-1β and TNFα were inhibited by only the direct co-cultures (p < 0.05).
Conclusions:
MSCs co-culture with bSMCs potently mitigates hypoxia-induced inflammatory and profibrotic pathways. This work has elucidated the role of cell-cell contact and paracrine immunomodulatory mechanisms of MSCs action and opened avenues for therapeutic intervention.
... To develop reproducible in vitro cellular models, cell lines have been established from human [9,10] and animal SMCs [11,12]; however, these cells lines do not show bidirectional phenotypic modulation. The establishment of in vitro human SMC systems is required for the causal analysis of symptoms and therapeutic approaches of human smooth muscle diseases, including bladder dysfunction and atherosclerosis. ...
... These cell lines may have undergone multiple alterations outside of the p16 INK4a -Rb pathway. The previously established human SMC lines exhibit an abortive karyotype and/or extraordinarily prolonged doubling time [10,16]. ...
Previous studies have shown that phenotypic modulation of smooth muscle cells (SMCs) plays a pivotal role in human diseases. However, the molecular mechanisms underlying the reversible differentiation of SMCs remain elusive particularly because cultured SMCs that reproducibly exhibit bidirectional phenotypic modulation have not been established. Here we established an immortalized human bladder SMC line designated as hBS11. Under differentiation-inducing conditions, hBS11 cells underwent smooth muscle differentiation accompanied by the robust expression of smooth muscle differentiation markers and isoform-dependent reorganization of actin bundles. The cholinergic receptor agonist carbachol increased intracellular calcium in differentiated hBS11 cells in an acetylcholine muscarinic receptor-dependent manner. Differentiated hBS11 cells displayed contractile properties depending on the elevation in the levels of intracellular calcium. Depolarization of membrane potential triggered inward sodium current in differentiated hBS11 cells. However, differentiated hBS11 cells lost the differentiated phenotype and resumed mitosis when re-fed with growth medium. Our study provides direct evidence pertaining to the human bladder SMCs being able to retain the capacity of reversible differentiation and that the reorganization of actin bundles is involved in the reinstatement of contractility. Moreover, we have established a human SMC line retaining high proliferating potential without compromising differentiation potential.
... The cells also exhibited a contractile response to agonist (bethanechol) stimulation. A human BSM cell line which expresses the smooth muscle phenotype, including the ability to contract in response to carbachol, has been established (71). However, karyotypic analysis of both rabbit and human bladder cell lines showed that these cells are tetraploid. ...
Smooth muscle cells, when subjected to culture, modulate from a contractile to a secretory phenotype. This has hampered the use of cell culture for molecular techniques to study the regulation of smooth muscle biology. The goal of this study was to develop a new organ culture model of bladder smooth muscle (BSM) that would maintain the contractile phenotype and aid in the study of BSM biology. Our results showed that strips of BSM subjected to up to nine days of organ culture maintained their contractile phenotype, including the ability to achieve near control levels of force with a temporal profile similar to that of non-cultured tissues. The technical aspects of our organ culture preparation that were responsible, in part, for the maintenance of the contractile phenotype were a slight longitudinal stretch during culture and subjecting the strips to a daily contraction/relaxation cycle. The tissues contained viable cells throughout the cross-section of the strips. There was an increase in extracellular collagenous matrix resulting in a leftward shift in the passive length-tension relationship. There were no significant changes in the content of smooth muscle specific α-actin, calponin, h-caldesmon, total myosin heavy chain, protein kinase G, ROCK-I, or the ratio of the SM1/SM2 myosin isoforms. Moreover the organ cultured tissues maintained functional voltage-gated calcium channels and BKCa potassium channels. Therefore, we propose that this novel BSM organ culture model maintains the contractile phenotype and will be a valuable tool for the use of cellular/molecular biology studies of bladder myocytes.
Vascular smooth muscle cells (VSMCs) play essential roles in regulating blood vessel form and function. Regeneration of functional vascular smooth muscle tissue to repair vascular diseases is an area of intense research in tissue engineering and regenerative medicine. For functional vascular smooth muscle tissue regeneration to become a practical therapy over the next decade, the field will need to have access to VSMC sources that are effective, robust and safe. While pluripotent stem cells hold good future promise to this end, more immediate translation is expected to come from approaches that generate functional VSMCs from adult sources of multipotent adipose-derived and bone marrow-derived mesenchymal stromal cells (ASCs and BMSCs). The research to this end is extensive and is dominated by studies relating to classical biochemical signalling molecules used to induce differentiation of ASCs and BMSCs. However, prolonged use of the biochemical induction factors is costly and can cause potential endotoxin contamination in the culture. Over recent years several non-traditional differentiation approaches have been devised to mimic defined aspects of the native micro-environment in which VSMCs reside to contribute to the differentiation of VSMC-like cells from ASCs and BMSCs. In this review, the promises and limitations of several non-traditional culture approaches (e.g., co-culture, biomechanical, and biomaterial stimuli) targeting VSMC differentiation are discussed. The extensive crosstalk between the underlying signalling cascades are delineated and put into a translational context. It is expected that this review will not only provide significant insight into VSMC differentiation strategies for vascular smooth muscle tissue engineering applications, but will also highlight the fundamental importance of engineering the cellular microenvironment on multiple scales (with consideration of different combinatorial pathways) in order to direct cell differentiation fate and obtain cells of a desired and stable phenotype. These strategies may ultimately be applied to different sources of stem cells in the future for a range of biomaterial and tissue engineering disciplines.
Partial bladder outlet obstruction (pBOO) is characterized by exaggerated stretch, hydrodynamic pressure, and inflammation which cause significant damage and fibrosis to the bladder wall. Several studies have implicated hypoxia in its pathophysiology. However, the isolated progressive effects of hypoxia on bladder cells are not yet defined. Sub-confluent normal human bladder smooth muscle cells (hbSMC) were cultured in 3% O2 tension for 2, 24, 48, and 72 h. RNA, cellular proteins, and secreted proteins were used for gene expression analysis, immunoblotting, and ELISA, respectively. Transcription of hypoxia-inducible factor (HIF)1α and HIF2α were transiently induced after 2 h of hypoxia (p < 0.05), whereas HIF3 was upregulated after 72 h (p < 0.005). HIF1 and HIF3α proteins were significantly induced after 2 and 72 h, respectively. VEGF mRNA increased significantly after 24 and 72 h (p < 0.005). The inflammatory cytokines, TGFB (protein and mRNA), IL 1β, 1L6, and TNFα (mRNA) demonstrated a time-dependent increased expression. Furthermore, the anti-inflammatory cytokine IL-10 was downregulated after 72 h (p < 0.05). Evidence of smooth muscle cell dedifferentiation included increased αSMA, vimentin, and desmin. Evidence of pro-fibrotic changes included increased CTGF, SMAD 2, and SMAD 3 as well as collagens 1, 2, 3, and 4, fibronectin, aggrecan, and TIMP 1 transcripts (p < 0.05). Total collagen proteins also increased time-dependently (p < 0.05). Together, these results show that exposure of hbSMC to low oxygen tension results in intense hypoxic cascade, including inflammation, de-differentiation, pro-fibrotic changes, and increased extracellular matrix expression. This elucidates mechanisms of hypoxia-driven bladder deterioration in bladder cells, which is important in tailoring in vivo experiments and may ultimately translate into improved clinical outcomes.
