Heat stress can have a serious impact on the health of both humans and animals. A major question is how heat stress affects normal development and differentiation at both the cellular and the organism levels. Here we use an in vitro experimental system to address how heat shock treatment influences the properties of bovine mesenchymal stem cells (MSCs)-multipotent progenitor cells-which are found in most tissues. Because cattle are sensitive to harsh external temperatures, studying the effects of heat shock on MSCs provides a unique platform to address cellular stress in a physiologically relevant model organism. Following isolation and characterization of MSCs from the cow's umbilical cord, heat shock was induced either as a pulse (1 h) or continuously (3 days), and consequent effects on MSCs were characterized. Heat shock induced extensive phenotypic changes in MSCs and dramatically curtailed their capacity to proliferate and differentiate. These changes were associated with a partial arrest in the G1/S or G2/M checkpoints. Furthermore, MSCs lost their ability to resolve the inflammatory response of RAW macrophages in coculture. A possible explanation for this loss of function is the accumulation of reactive oxygen species and malfunction of the mitochondria in the treated cells. Heat shock treatments resulted in stressinduced premature senescence, affecting the MSCs' ability to proliferate properly for many cell passages to follow. Exposure to elevated external temperatures leads to mitochondrial damage and oxidative stress, which in turn conveys critical changes in the proliferation, differentiation, and immunomodulatory phenotype of heat-stressed MSCs. A better understanding of the effect of heat shock on humans and animals may result in important health and economic benefits.

Mesenchymal stem cells (MSC) have many roles that are important for the body’s proper functioning. When the MSC pool is damaged, it is often correlated with impaired development or health of the organism. MSC are known for their anti-inflammatory, immunomodulatory and trophic characteristics that play an important role in the physiological homeostasis of many tissues. Heat shock impairs MSC capacity by inducing the generation of reactive oxygen species and mitochondrial dysfunction, which, in turn, send the cells into a state of premature senescence. Here, we pre-exposed MSC to melatonin, resveratrol, or curcumin, which are natural antioxidative compounds, and tested the protective effects of these substances from oxidative stress and aging. Our data showed that pre-exposure of MSC to antioxidants decreased reactive oxygen species while mitochondrial damage remained high. Additionally, although the proliferation of the cells was slow, antioxidants protected the cells from premature senescence, and subsequent cytokine release was prevented. We conclude that while elevated temperatures directly cause mitochondrial damage, senescence is induced by elevated ROS levels. We suggest that heat shock alters cell and tissue homeostasis by several independent mechanisms; however, reducing tissue senescence will reduce damage and provide a pathway to overcome physiological challenges in animals.

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Background: Mesenchymal stem cells (MSCs) are multipotent stromal, non-hematopoietic cells with self-renewal and differentiation properties and are therefore a preferred source for cellular therapies. However, a better understanding of culture techniques is required to harness their full potential. Here we aim to compare the effects of short and long heat shock (HS) on the transcriptomic landscape of MSCs. Methods: MSCs were extracted from the umbilical cord of a bovine fetus, cultured, and validated as MSCs. Early passage cells were exposed to 40.5°C for six hours or three days. RNA sequencing and bioinformatics analysis were performed to systematically examine the transcriptional changes following each treatment and to identify specific biological features and processes. Results: The data indicates that while long heat stress influences many cell processes, such as immune response, cell cycle, and differentiation, the short HS mostly upregulates the cellular stress response. Once normothermia is resumed the long-term effects of the short HS can be revealed: although most genes revert to their original expression levels, a subgroup of epigenetically marked genes termed bivalent genes, maintains high expression levels. These genes are known to support cell lineage specification and are carefully regulated by a group of chromatin modifiers. One family of those chromatin modifiers, called MLL genes, is highly over-represented in the transiently upregulated cluster after six hours of HS. Therefore, our data provide a mechanistic explanation for the long-term phenotype of short HS on development-related genes and could be used to predict the long-term effect of HS on cell identity. Conclusions: Understanding the influence of culture conditions on morphology, phenotype, proliferative capacity, and fate decision of MSCs is needed to optimize culture conditions suitable for clinical or commercial use. Here, we suggest that simple and short stress can alter the cell's proliferation and differentiation capacities and, therefore, following future optimizations, be used to shift the cells toward a more desirable functionality.