Quantum Sensing of Free Radical Generation in Mitochondria of Single Heart Muscle Cells during Hypoxia and Reoxygenation.

Cells are damaged during hypoxia (blood supply deprivation) and reoxygenation (oxygen return). This damage occurs in conditions such as cardiovascular diseases, cancer, and organ transplantation, potentially harming the tissue and organs. The role of free radicals in cellular metabolic reprogramming under hypoxia is under debate, but their measurement is challenging due to their short lifespan and limited diffusion range. In this study, we employed a quantum sensing technique to measure the real-time production of free radicals at the subcellular level. We utilize fluorescent nanodiamonds (FNDs) that exhibit changes in their optical properties based on the surrounding magnetic noise. This way, we were able to detect the presence of free radicals. To specifically monitor radical generation near mitochondria, we coated the FNDs with an antibody targeting voltage-dependent anion channel 2 (anti-VDAC2), which is located in the outer membrane of mitochondria. We observed a significant increase in the radical load on the mitochondrial membrane when cells were exposed to hypoxia. Subsequently, during reoxygenation, the levels of radicals gradually decreased back to the normoxia state. Overall, by applying a quantum sensing technique, the connections among hypoxia, free radicals, and the cellular redox status has been revealed.

Development of curcumin-loaded zein nanoparticles for transport across the blood-brain barrier and inhibition of glioblastoma cell growth.

Glioblastoma (GBM) is a devastating primary brain tumor resistant to conventional therapies. A major obstacle to GBM treatment is the blood-brain barrier (BBB), or blood-glioma barrier, which prevents the transport of systemically administered (chemotherapeutic) drugs into the tumor. This study reports the design of dodecamer peptide (G23)-functionalized polydopamine (pD)-coated curcumin-loaded zein nanoparticles (CUR-ZpD-G23 NPs) that efficiently traversed the BBB, and delivered curcumin to glioblastoma cells. The NPs enhanced the cellular uptake of curcumin by C6 glioma cells compared to free curcumin, and showed high penetration into 3D tumor spheroids. Functionalization of the NPs with G23 stimulated BBB crossing and tumor spheroid penetration. Moreover, the NPs markedly inhibited proliferation and migration and induced cell death in liquid and soft agar models of C6 glioma cell growth. Fluorescence microscopy and flow cytometry studies showed that the CUR-ZpD-G23 NPs increased cellular ROS production and induced apoptosis of C6 glioma cells. Following in vivo intravenous injection in zebrafish, ZpD-G23 NPs demonstrated the ability to circulate, which is a first prerequisite for their use in targeted drug delivery. In conclusion, zein-polydopamine-G23 NPs show potential as a drug delivery platform for therapy of GBM, which requires further validation in in vivo glioblastoma models.

Topography induced stiffness alteration of stem cells influences osteogenic differentiation.

Topography-driven alterations in cell morphology tremendously influence cell biological processes, particularly stem cell differentiation. Aligned topography is known to alter the cell shape, which we anticipated to also induce altered physical properties of the cell. Here, we show that topography has a significant influence on single cell stiffness of human bone marrow derived-Mesenchymal Stem Cells (hBM-MSCs) and the osteogenic differentiation of these. Aligned topographies were used to control the cell elongation, depicted as the cell aspect ratio (CAR). Intriguingly, an equal CAR elicited from different topographies, resulted in highly altered differentiation behavior and the underlying single cell mechanics was found to be critical. The cell behavior was found to be focal adhesion-mediated and induced stiffness alterations rather than just influencing the cell elongation. The effect was further corroborated by investigations of the transcriptional regulators YAP. Our study provides insight into how mechanical properties of the cell, which are stimulated by topography, modulate the osteogenesis of hBM-MSCs, which is beneficial for improving the understanding of interactions between stem cells and topography for developing applications of tissue engineering and regenerative medicine.

Long-Term In Vitro Expansion of Salivary Gland Stem Cells Driven by Wnt Signals.

Adult stem cells are the ultimate source for replenishment of salivary gland (SG) tissue. Self-renewal ability of stem cells is dependent on extrinsic niche signals that have not been unraveled for the SG. The ductal compartment in SG has been identified as the location harboring stem cells. Here, we report that rare SG ductal EpCAM(+) cells express nuclear ß-catenin, indicating active Wnt signaling. In cell culture experiments, EpCAM(high) cells respond potently to Wnt signals stimulating self-renewal and long-term expansion of SG organoids, containing all differentiated SG cell types. Conversely, Wnt inhibition ablated long-term organoid cultures. Finally, transplantation of cells pre-treated with Wnt agonists into submandibular glands of irradiated mice successfully and robustly restored saliva secretion and increased the number of functional acini in vivo. Collectively, these results identify Wnt signaling as a key driver of adult SG stem cells, allowing extensive in vitro expansion and enabling restoration of SG function upon transplantation.

Similar ex vivo expansion and post-irradiation regenerative potential of juvenile and aged salivary gland stem cells.

BACKGROUND AND PURPOSE: Salivary gland dysfunction is a major side effect of radiotherapy for head and neck cancer patients, which in the future might be salvaged by autologous adult salivary gland stem cell (SGSC) therapy. Since frail elderly patients may have decreased activity of SGSCs, we aimed to characterize the potential of aged SGSC-population in a murine model. MATERIALS AND METHODS: Salivary glands and salisphere-derived cells from young and old mice were tested for CD24 and CD29 stem cell marker expression using FACS. Moreover, in vitro expansion capability and in vivo regeneration potential upon post-irradiation transplantation of young and aged SGSCs were measured. RESULTS: An increase in CD24(hi)/CD29(hi) putative stem cells was detected in aged salivary glands albeit with a decrease in functional ability to form salispheres. However, the salispheres formed from aged mice salivary glands expressed CD24(hi)/CD29(hi) to the same extent as the ones from young mice. Moreover, following exposure to adequate growth conditions old and young SGSCs exhibited similar in vitro expansion- and in vivo regeneration potential. CONCLUSIONS: Aged SGSCs although reduced in number are in vitro indistinguishable from young SGSCs and could potentially be used to ameliorate age- or treatment related salivary gland dysfunction.

High and low LET radiation differentially induce normal tissue damage signals.

PURPOSE: Radiotherapy using high linear energy transfer (LET) radiation is aimed at efficiently killing tumor cells while minimizing dose (biological effective) to normal tissues to prevent toxicity. It is well established that high LET radiation results in lower cell survival per absorbed dose than low LET radiation. However, whether various mechanisms involved in the development of normal tissue damage may be regulated differentially is not known. Therefore the aim of this study was to investigate whether two actions related to normal tissue toxicity, p53-induced apoptosis and expression of the profibrotic gene PAI-1 (plasminogen activator inhibitor 1), are differentially induced by high and low LET radiation. METHODS AND MATERIALS: Cells were irradiated with high LET carbon ions or low LET photons. Cell survival assays were performed, profibrotic PAI-1 expression was monitored by quantitative polymerase chain reaction, and apoptosis was assayed by annexin V staining. Activation of p53 by phosphorylation at serine 315 and serine 37 was monitored by Western blotting. Transfections of plasmids expressing p53 mutated at serines 315 and 37 were used to test the requirement of these residues for apoptosis and expression of PAI-1. RESULTS: As expected, cell survival was lower and induction of apoptosis was higher in high -LET irradiated cells. Interestingly, induction of the profibrotic PAI-1 gene was similar with high and low LET radiation. In agreement with this finding, phosphorylation of p53 at serine 315 involved in PAI-1 expression was similar with high and low LET radiation, whereas phosphorylation of p53 at serine 37, involved in apoptosis induction, was much higher after high LET irradiation. CONCLUSIONS: Our results indicate that diverse mechanisms involved in the development of normal tissue damage may be differentially affected by high and low LET radiation. This may have consequences for the development and manifestation of normal tissue damage.