Cellular expansion of MSCs: Shifting the regenerative potential

Mesenchymal-derived stromal or progenitor cells, commonly called “MSCs,” have attracted significant clinical interest for their remarkable abilities to promote tissue regeneration and reduce inflammation. Recent studies have shown that MSCs' therapeutic effects, originally attributed to the cells' direct differentiation capacity into the tissue of interest, are largely driven by the biomolecules the cells secrete, including cytokines, chemokines, growth factors, and extracellular vesicles containing miRNA. This secretome coordinates upregulation of endogenous repair and immunomodulation in the local microenvironment through crosstalk of MSCs with host tissue cells. Therapeutic applications for MSCs and their secretome-derived products often involve in vitro monolayer expansion. However, consecutive passaging of MSCs significantly alters their therapeutic potential, inducing a broad shift from a pro-regenerative to a pro-inflammatory phenotype. A consistent by-product of in vitro expansion of MSCs is the onset of replicative senescence, a state of cell arrest characterized by an increased release of proinflammatory cytokines and growth factors. However, little is known about changes in the secretome profile at different stages of in vitro expansion. Some culture conditions and bioprocessing techniques have shown promise in more effectively retaining the pro-regenerative and anti-inflammatory MSC phenotype throughout expansion. Understanding how in vitro expansion conditions influence the nature and function of MSCs, and their associated secretome, may provide key insights into the underlying mechanisms driving these alterations. Elucidating the dynamic and diverse changes in the MSC secretome at each stage of in vitro expansion is a critical next step in the development of standardized, safe, and effective MSC-based therapies.     

Cytoskeleton stiffness regulates cellular senescence and innate immune response in Hutchinson–Gilford Progeria Syndrome

Hutchinson–Gilford progeria syndrome (HGPS) is caused by the accumulation of mutant prelamin A (progerin) in the nuclear lamina, resulting in increased nuclear stiffness and abnormal nuclear architecture. Nuclear mechanics are tightly coupled to cytoskeletal mechanics via lamin A/C. However, the role of cytoskeletal/nuclear mechanical properties in mediating cellular senescence and the relationship between cytoskeletal stiffness, nuclear abnormalities, and senescent phenotypes remain largely unknown. Here, using muscle‐derived mesenchymal stromal/stem cells (MSCs) from the Zmpste24^?/? (Z24^?/?) mouse (a model for HGPS) and human HGPS fibroblasts, we investigated the mechanical mechanism of progerin‐induced cellular senescence, involving the role and interaction of mechanical sensors RhoA and Sun1/2 in regulating F‐actin cytoskeleton stiffness, nuclear blebbing, micronuclei formation, and the innate immune response. We observed that increased cytoskeletal stiffness and RhoA activation in progeria cells were directly coupled with increased nuclear blebbing, Sun2 expression, and micronuclei‐induced cGAS‐Sting activation, part of the innate immune response. Expression of constitutively active RhoA promoted, while the inhibition of RhoA/ROCK reduced cytoskeletal stiffness, Sun2 expression, the innate immune response, and cellular senescence. Silencing of Sun2 expression by siRNA also repressed RhoA activation, cytoskeletal stiffness and cellular senescence. Treatment of Zmpste24^?/? mice with a RhoA inhibitor repressed cellular senescence and improved muscle regeneration. These results reveal novel mechanical roles and correlation of cytoskeletal/nuclear stiffness, RhoA, Sun2, and the innate immune response in promoting aging and cellular senescence in HGPS progeria.     

Global co-occurrence of methanogenic archaea and methanotrophic bacteria in Microcystis aggregates

Global warming and eutrophication contribute to the worldwide increase in cyanobacterial blooms, and the level of cyanobacterial biomass is strongly associated with rises in methane emissions from surface lake waters. Hence, methane-metabolizing microorganisms may be important for modulating carbon flow in cyanobacterial blooms. Here, we surveyed methanogenic and methanotrophic communities associated with floating Microcystis aggregates in 10 lakes spanning four continents, through sequencing of 16S rRNA and functional marker genes. Methanogenic archaea (mainly Methanoregula and Methanosaeta) were detectable in 5 of the 10 lakes and constituted the majority (~50%–90%) of the archaeal community in these lakes. Three of the 10 lakes contained relatively more abundant methanotrophs than the other seven lakes, with the methanotrophic genera Methyloparacoccus, Crenothrix, and an uncultured species related to Methylobacter dominating and nearly exclusively found in each of those three lakes. These three are among the five lakes in which methanogens were observed. Operational taxonomic unit (OTU) richness and abundance of methanotrophs were strongly positively correlated with those of methanogens, suggesting that their activities may be coupled. These Microcystis-aggregate-associated methanotrophs may be responsible for a hitherto overlooked sink for methane in surface freshwaters, and their co-occurrence with methanogens sheds light on the methane cycle in cyanobacterial aggregates.     

A Proposed Framework for the Management of Water Quality in Arid-Region Lakes

For arid-region lakes, management conflicts are likely to occur between quantity and quality of water supplied: increasing quantity of water supply can lead to water quality deterioration. Such conflicts can best be resolved within an effective management program based on awareness and cooperation at all levels of water management from policy makers to experts. We propose a general framework for designing effective water resource management programs for lakes based on concrete definitions of management criteria such as water quality. The proposed system requires close interaction between policy makers, water resource managers, water suppliers and users, hydrological engineers and limnologists. The significance of mathematical modeling as a self-organizing tool of the management program is emphasized, especially with regards to designing limnological investigations directed toward lake management. We illustrate the application of this approach to water resource management in arid-region lakes (Lake Kinneret, Israel and Lake Sevan, Armenia), where artificial variability of lake morphometry due to water use is a forcing function affecting water quality.     

Lakes Hula and Agmon: destruction and creation of wetland ecosystems in northern Israel

A portion of the former Lake Hula wetland (northern Israel) was re-flooded in spring 1994 and the physical, chemical and biological developments within the resulting new lake and wetland complex (Agmon) was followed closely by a multi-disciplinary scientific team. The first three years of study relating to Lake Agmon are reported in this issue of Wetlands Ecology & Management. We provide in this paper a general background on the Lake Hula Draining Project in the 1950s and the recent re-flooding and creation of the Agmon wetland.     

Ablation of the Ferroptosis Inhibitor Glutathione Peroxidase 4 in Neurons Results in Rapid Motor Neuron Degeneration and Paralysis

Glutathione peroxidase 4 (GPX4), an antioxidant defense enzyme active in repairing oxidative damage to lipids, is a key inhibitor of ferroptosis, a non-apoptotic form of cell death involving lipid reactive oxygen species. Here we show that GPX4 is essential for motor neuron health and survival in vivo. Conditional ablation of Gpx4 in neurons of adult mice resulted in rapid onset and progression of paralysis and death. Pathological inspection revealed that the paralyzed mice had a dramatic degeneration of motor neurons in the spinal cord but had no overt neuron degeneration in the cerebral cortex. Consistent with the role of GPX4 as a ferroptosis inhibitor, spinal motor neuron degeneration induced by Gpx4 ablation exhibited features of ferroptosis, including no caspase-3 activation, no TUNEL staining, activation of ERKs, and elevated spinal inflammation. Supplementation with vitamin E, another inhibitor of ferroptosis, delayed the onset of paralysis and death induced by Gpx4 ablation. Also, lipid peroxidation and mitochondrial dysfunction appeared to be involved in ferroptosis of motor neurons induced by Gpx4 ablation. Taken together, the dramatic motor neuron degeneration and paralysis induced by Gpx4 ablation suggest that ferroptosis inhibition by GPX4 is essential for motor neuron health and survival in vivo.     

Water Quality Quantification: Basics and Implementation

Quantitative estimation of water quality and its relationships with management activities is a necessary step in efficient water resources management. However, water quality is typically defined in abstract terms and management activities are rarely quantified with respect to their impact on lake water quality. Here we show by demonstration of systems for Lake Kinneret, Israel and the Naroch Lakes of Belarus how water quality can be quantified in relation to lake management activities to be a part of sustainable management.     

Effects of zooplankton grazing and nutrients on the bloom-forming, N2-fixing cyanobacterium Aphanizomenon in Lake Kinneret

A bloom of the filamentous, N₂-fixing cyanobacterium Aphanizomenonovalisporum Forti occurred for the first time in Lake Kinneretduring late summer and fall 1994. During subsequent years (1995–1999),Aphanizomenon also appeared in late summer and fall, but didnot bloom. In outdoor microcosm experiments, we examined zooplanktongrazing on Lake Kinneret phytoplankton, with and without Aphanizomenonpresent, and the effects of N, P and N:P ratios on phytoplanktongrowth. In one-day feeding experiments, clearance and grazingrates of the ambient Lake Kinneret zooplankton assemblage feedingin lake water dominated by Aphanizomenon were 10-fold lowerthan in water without Aphanizomenon. We suspect that the lowgrazing rates were due to interference caused by the presenceof Aphanizomenon. In 9-day nutrient addition experiments, significantenhancement effects on phytoplankton were detected with additionsof either P or N; a high N:P was better for phytoplankton growththan a low N:P. After 7 days, bottles receiving low P and noN additions were dominated by Oscillatoria sp. and Closteriumacutum; few Aphanizomenon were present. In contrast, bottlesreceiving high P and N additions had large increases of Aphanizomenon,as well as Oscillatoria and Closterium. There was a tendencyfor more green algae and diatoms with increasing N additions.These results provide evidence that (i) non-grazeability ofAphanizomenon enabled it to gain a competitive advantage overgrazeable phytoplankton, and (ii) that nutrient limitation,but not grazing, was probably important in the eventual declineof the Aphanizomenon bloom.