2.1] Short heat shock has a long-term effect on mesenchymal stem cells’ transcriptome

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^oC 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 cluster of genes that are transiently upregulated following 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.

2.2] Transposable elements regulation in response to viral infection

Transposable elements (TEs) are induced in response to viral infections. TEs induction triggers a robust and durable interferon (IFN) response, providing a host defense mechanism. Still, the connection between SARS-CoV-2 IFN response and TEs remained unexplored. Here, we analyzed TE expression changes in response to SARS-CoV-2 infection in different human cellular models. We find that compared to other viruses, which cause global upregulation of TEs, SARS-CoV-2 infection results in a significantly milder TE response in both primary lung epithelial cells and in iPSC-derived lung alveolar type 2 cells. In general, we observe that TE activation correlates with, and precedes, the induction of IFN-related genes, suggesting that the failure to activate TEs may be the reason for the weak IFN response. Moreover, we identify two variables that explain most of the observed diverseness in immune responses: basal expression levels of TEs in the pre-infected cell, and the viral load. Since basal TE levels increase with age, we propose that ‘TE desensitization’ leads to age-related death from COVID19. This work provides a mechanistic explanation for SARS-CoV-2’s success in its fight against the host immune system, and suggests that TEs could be used as sensors or serve as potential drug targets for COVID-19.

2.3] Transmitted epigenetic changes associated with heat stress in cattle

It has been shown that heat stress may have a long-lasting impact on the bovine fetus and its progeny. Heat stress related in-utero programming may result in reduced birth weight of calves, altered mammary development and impaired innate and cellular immunity; effects may be transmitted to the second generation. The mechanisms responsible for this programming are unknown.

Transcriptional and epigenetic patterns form primarily during embryonic development and cell differentiation, and environmental factors have been shown to influence those patterns. Often, these changes are detrimental. If environmental factors induce epigenetic alterations in the gametes, they may affect not only the phenotype but also the epigenetic patterns of the offspring. A dult stem cells are the longest living cell population in the body, and are therefore exposed to many stressful environmental conditions that might affect their function. The main goal of this research is to delineate the effect of heat stress on stem cell function and to identify the mechanisms underlying the epigenetic aberrations. I will focus primarily on mesenchymal stem cells (MSC), which can differentiate into various lineages and may suppress inflammation, because they are essential for the homeostasis and regeneration of many tissues in the body, and therefore may be most susceptible to the cellular and epigenetic alterations and their phenotypic consequences. We study for the first time the functional and epigenomic changes in the fetus and three subsequent generations caused by in utero heat stress, using cultured bovine MSC from various tissues.We plan to investigate the altered gene expression programs, the aberrant CpG methylation patterns and chromatin accessibilities of MSCs by whole-transcriptome RNA-seq analysis, reduced representation bisulfite sequencing (RRBS), and an assay for transposase-accessible chromatin using sequencing (ATAC-seq). MSCs will be isolated from four relevant tissues (uterus, placenta, mammary gland and umbilical cord), cultured and their molecular signatures analyzed. The general hypothesis is that a major reason that heat stress has such a profound detrimental effect on cattle is because it results in aberrant epigenetic patterns in MSC and thereby compromises their function. Results from this project will, for the first time, establish the foundation for transgenerational epigenetic studies in large animals, and ultimately may provide a basis for important practical agricultural ramifications. At the basic level, they will expand our understanding of the underlying molecular mechanisms and epigenetic alterations that are involved in the cellular response to heat stress and its long term effects.