Pluripotent stem cells are central tools to many regenerative medicine strategies due to their ability to differentiate toward the three embryonic germ layers. One challenge remains in providing ...control over their differentiation into specific lineages, such as cardiac commitment. Here, the possibility of directing cardiomyogenesis of embryonic stem cells using a microfabricated magnetic pattern is demonstrated. The stem cells are labeled with magnetic nanoparticles, aggregated into embryoid bodies (EBs) onto the pattern, and stimulated with a local magnetic force applied via the pattern. The EBs formed on this magnetic device experience the same differentiation profile as the ones created by the common hanging drop approach, while it allows high‐throughput production of hundreds of EBs. Further on/off cyclic magnetic force stimulation mediated by the same device is sufficient to enhance cardiomyogenesis in a way that almost all EBs develop spontaneous beating, confirmed by the overexpression of α‐actin and troponin proteins, and by the upregulation (twofold to fivefold) of genes involved in mesoderm differentiation (Nkx2.5, Gata4, and Gata6), and more specifically cardiac lineage (Tnnt2, Myh6, and Myl‐2). Beyond holding high application‐level potential, this work confirms that physical forces, and specifically on/off dynamic ones can be sufficient to govern cell function.
Using a pattern of magnetic microtips and magnetically labeled embryonic stem cells, high‐throughput cell aggregation into embryoid bodies can be achieved. The magnetic pattern allows an on/off magneto‐mechanical stimulation of the stem cells, which impressively enhances their differentiation into cardiomyocytes when compared to a conventional hanging drop method, with over 90% of embryoid bodies developing spontaneous beating.
The ability to create a 3D tissue structure from individual cells and then to stimulate it at will is a major goal for both the biophysics and regenerative medicine communities. Here we show an ...integrated set of magnetic techniques that meet this challenge using embryonic stem cells (ESCs). We assessed the impact of magnetic nanoparticles internalization on ESCs viability, proliferation, pluripotency and differentiation profiles. We developed magnetic attractors capable of aggregating the cells remotely into a 3D embryoid body. This magnetic approach to embryoid body formation has no discernible impact on ESC differentiation pathways, as compared to the hanging drop method. It is also the base of the final magnetic device, composed of opposing magnetic attractors in order to form embryoid bodies in situ, then stretch them, and mechanically stimulate them at will. These stretched and cyclic purely mechanical stimulations were sufficient to drive ESCs differentiation towards the mesodermal cardiac pathway.The development of embryoid bodies that are responsive to external stimuli is of great interest in tissue engineering. Here, the authors culture embryonic stem cells with magnetic nanoparticles and show that the presence of magnetic fields could affect their aggregation and differentiation.
Photodynamic therapy is an emerging cancer treatment that is particularly adapted for localized malignant tumor. The phototherapeutic agent is generally injected in the bloodstream and circulates in ...the whole organism as a chemotherapeutic agent, but needs light triggering to induce localized therapeutic effects. We found that one of the responses of in vitro and in vivo cancer cells to photodynamic therapy was a massive production and emission of extracellular vesicles (EVs): only 1 hour after the photo-activation, thousands of vesicles per cell were emitted in the extracellular medium. A similar effect has been found after treatment with Doxorubicin (chemotherapy), but far less EVs were produced, even 24 hours after the treatment. Furthermore, we found that the released EVs could transfer extracellular membrane components, drugs and even large intracellular objects to naive target cells. In vivo, photodynamic treatment and chemotherapy increased the levels of circulating EVs several fold, confirming the vast induction of cancer cell vesiculation triggered by anti-cancer therapies.
We report the construct of a nanosystem based on multi-walled carbon nanotubes (MWCNT) and the photosensitizer m-tetrahydroxyphenylchlorin (mTHPC) for cancer treatment by combination of photodynamic ...(PDT) and photothermal (PTT) therapy. The photoactivity of the complex mTHPC/MWCNT was studied revealing quenching of the photosensitizer associated to CNT and almost total release of the photosensitizer into the cell cytoplasm after uptake by SKOV3 ovarian cancer cells. The photothermal and photodynamic cytotoxicity of these mTHPC/MWCNT on/off complexes was assessed at the cell level by viability test, imaging flow cytometry, confocal microscopy and transmission electron microscopy and at the molecular level by a proteomic analysis of apoptosis-related proteins and genomic analysis of 84 genes involved in oxidative stress. Cytotoxicity correlated at the cell level to the uptake of mTHPC/MWCNT, while PDT and PTT treatment induced different signaling pathways leading to cell apoptosis. For the first time, the mechanisms of PDT/PTT synergy in eradication of cancer cells were studied revealing that distinct cell responses to PDT and PTT defeat the cell defense to oxidative stress.
Abstract Inspired by microvesicle-mediated intercellular communication, we propose a hybrid vector for magnetic drug delivery. It consists of macrophage-derived microvesicles engineered to enclose ...different therapeutic agents together with iron oxide nanoparticles. Here, we investigated in vitro how magnetic nanoparticles may influence the vector effectiveness in terms of drug uptake and targeting. Human macrophages were loaded with iron oxide nanoparticles and different therapeutic agents: a chemotherapeutic agent (doxorubicin), tissue-plasminogen activator (t-PA) and two photosensitizers (disulfonated tetraphenyl chlorin-TPCS2a and 5,10,15,20-tetra(m-hydroxyphenyl)chlorin-mTHPC). The hybrid cell microvesicles were magnetically responsive, readily manipulated by magnetic forces and MRI-detectable. Using photosensitizer-loaded vesicles, we showed that the uptake of microvesicles by cancer cells could be kinetically modulated and spatially controlled under magnetic field and that cancer cell death was enhanced by the magnetic targeting. From the Clinical Editor In this article, the authors devised a biogenic method using macrophages to produce microvesicles containing both iron oxide and chemotherapeutic agents. They showed that the microvesicles could be manipulated by magnetic force for targeting and subsequent delivery of the drug payload against cancer cells. This smart method could provide a novel way for future fight against cancer.
Recent advances in cell therapy and tissue engineering opened new windows for regenerative medicine, but still necessitate innovative noninvasive imaging technologies. We demonstrate that ...high-resolution magnetic resonance imaging (MRI) allows combining cellular-scale resolution with the ability to detect two cell types simultaneously at any tissue depth. Two contrast agents, based on iron oxide and gadolinium oxide rigid nanoplatforms, were used to “tattoo” endothelial cells and stem cells, respectively, with no impact on cell functions, including their capacity for differentiation. The labeled cells’ contrast properties were optimized for simultaneous MRI detection: endothelial cells and stem cells seeded together in a polysaccharide-based scaffold material for tissue engineering appeared respectively in black and white and could be tracked, at the cellular level, both in vitro and in vivo. In addition, endothelial cells labeled with iron oxide nanoparticles could be remotely manipulated by applying a magnetic field, allowing the creation of vessel substitutes with in-depth detection of individual cellular components.
The labeling of stem cells with iron oxide nanoparticles is increasingly used to enable MRI cell tracking and magnetic cell manipulation, stimulating the fields of tissue engineering and cell ...therapy. However, the impact of magnetic labeling on stem-cell differentiation is still controversial. One compromising factor for successful differentiation may arise from early interactions of nanoparticles with cells during the labeling procedure. It is hypothesized that the lack of control over nanoparticle colloidal stability in biological media may lead to undesirable nanoparticle localization, overestimation of cellular uptake, misleading MRI cell tracking, and further impairment of differentiation. Herein a method is described for labeling mesenchymal stem cells (MSC), in which the physical state of citrate-coated nanoparticles (dispersed versus aggregated) can be kinetically tuned through electrostatic and magnetic triggers, as monitored by diffusion light scattering in the extracellular medium and by optical and electronic microscopy in cells. A set of statistical cell-by-cell measurements (flow cytometry, single-cell magnetophoresis, and high-resolution MRI cellular detection) is used to independently quantify the nanoparticle cell uptake and the effects of nanoparticle aggregation. Such aggregation confounds MRI cell detection as well as global iron quantification and has adverse effects on chondrogenetic differentiation. Magnetic labeling conditions with perfectly stable nanoparticles-suitable for obtaining differentiation-capable magnetic stem cells for use in cell therapy-are subsequently identified.
Backgrounds & Aims:
Hepcidin is an acute-phase response peptide. We have investigated the possible involvement of hepcidin in massive obesity, a state of chronic low-grade inflammation. Three groups ...of severely obese patients with or without diabetes or nonalcoholic steatohepatitis were investigated.
Methods:
Hepcidin expression was studied in liver and adipose tissue of these patients. Hepcidin regulation was investigated in vitro by adipose tissue explant stimulation studies.
Results:
Hepcidin was expressed not only in the liver but also at the messenger RNA (mRNA) and the protein levels in adipose tissue. Moreover, mRNA expression was increased in adipose tissue of obese patients. The presence of diabetes or NASH did not modify the hepcidin expression levels in liver and adipose tissue. In adipose tissue, mRNA expression correlated with indexes of inflammation, interleukin-6, and C-reactive protein. Interleukin-6 also promoted in vitro hepcidin expression. A low transferrin saturation ratio was observed in 68% of the obese patients; moreover, 24% of these patients presented with anemia. The observed changes in iron status could be due to the role of hepcidin as a negative regulator of intestinal iron absorption and macrophage iron efflux. Interestingly, a feedback control mechanism on hepcidin expression related to low transferrin saturation occurred in the liver but not in the adipose tissue.
Conclusions:
Hepcidin is a proinflammatory adipokine and may play an important role in hypoferremia of inflammation in obese condition.
There are very few methods to investigate how nanoparticles (NPs) are taken up and processed by cells in the organism in the short and long terms. We propose a nanomagnetism approach, in combination ...with electron microscopy, to document the magnetic outcome of iron oxide-based P904 NPs injected intravenously into mice. The NP superparamagnetic properties are shown to be modified by cell internalization, due to magnetic interactions between NPs sequestered within intracellular organelles. These modifications of magnetic behaviour are observed in vivo after NP uptake by resident macrophages in spleen and liver or by inflammatory macrophages in adipose tissue as well as in vitro in monocyte-derived macrophages. The dynamical magnetic response of cell-internalized NPs is theoretically and experimentally evidenced as a global signature of their local organization in the intracellular compartments. The clustering of NPs and their magnetism become dependent on the targeted organ, on the dose administrated and on the time elapsed since their injection. Nanomagnetism probes the intracellular clustering of iron-oxide NPs and sheds light on the impact of cellular metabolism on their magnetic responsivity.
A growing tumor is submitted to ever-evolving mechanical stress. Endoscopic procedures add additional constraints. However, the impact of mechanical forces on cancer progression is still debated. ...Herein, a set of magnetic methods is proposed to form tumor spheroids and to subject them to remote deformation, mimicking stent-imposed compression. Upon application of a permanent magnet, the magnetic tumor spheroids (formed from colon cancer cells or from glioblastoma cells) are compressed by 50% of their initial diameters. Such significant deformation triggers an increase in the spheroid proliferation for both cell lines, correlated with an increase in the number of proliferating cells toward its center and associated with an overexpression of the matrix metalloproteinase-9 (MMP-9). In vivo peritoneal injection of the spheroids made from colon cancer cells confirmed the increased aggressiveness of the compressed spheroids, with almost a doubling of the peritoneal cancer index (PCI), as compared with non-stimulated spheroids. Moreover, liver metastasis of labeled cells was observed only in animals grafted with stimulated spheroids. Altogether, these results demonstrate that a large compression of tumor spheroids enhances cancer proliferation and metastatic process and could have implications in clinical procedures where tumor compression plays a role.
PDF
