1.1] The H3.3 coding genes have distinct roles in retroviral silencing in ESC.

Histone 3 variant, H3.3, obtains different modifications according to its position in the chromatin. It was shown to occupy gene body and active transcription zones, as well as centromeres and highly conserved repeat elements, which are mostly repressed. Although H3.3 was shown to occupy endogenous retroviruses sequences, the contribution of this variant to retroviral silencing in embryonic stem cells (ESC) is not yet clear.

Here we show that H3.3 depletion disrupts retroviral sequences' silencing in mouse ESC. Interestingly, our results show a differential impact on the depletion of the two genes coding for H3.3, H3f3a, and H3f3b, on retroviral expression. By infecting H3.3A-depleted cells with a retroviral vector, we demonstrate a transient upregulation of incoming retroviral expression, as well as that of ERVs. Conversely, H3.3B- knock-out did not show a similar effect, and retroviral repression was maintained. Notably, the depletion of both genes activated retroviral expression in a stable manner.

The upregulation of retroviral expression was associated with the depletion of the silencing mediator Trim28 and the H3K9me3 chromatin mark from the retroviral sequences. Deletion of DAXX, a specific H3.3 chaperon, did not affect either the expression or H3.3 loading on retroviral sequences, whereas depletion of Trim28 did affect both. Without Trim28, retroviral expression is upregulated and the H3.3 accumulation on the retroviral promoter is abolished.  Thus, our results show for the first time a distinct function for the two H3.3 genes in retroviral regulation in ESC and suggest a functional interplay between Trim28 recruitment and H3.3 loading.

1.2] Smarcad1 is essential for H3.3 deposition and Trim28 dependent silencing on retroviral sequences in mouse embryonic stem cells.

Moloney murine leukemia virus (MLV) replication is suppressed in mouse embryonic stem cells by the Trim28-ESET complex. The chromatin remodeler Smarcad1 interacts with Trim28 and is located at similar genomic sites. Moreover, Smarcad1 evicts nucleosomes from endogenous retroviruses and enables the loading of the histone variant H3.3 onto those sequences. Therefore, we wished to examine the role of Smarcad1 in MLV repression and elucidate its mechanism. To address the functional role of Smarcad1 in MLV repression, we use shRNA to deplete Smarcad1 before or after MLV infection and test the change in expression of the reporter GFP. A marked increase in expression is observed following Smarcad1 depletion, attesting to its involvement in MLV silencing. Next, we map the effect of Smarcad1 depletion on H3.3 deposition, Trim28 recruitment, and H3K9me3 heterochromatin marking on the retroviral sequences. The observed reductions suggest that the epigenetic silencing of retroviral sequences is dependent on Smarcad1. Last, we examine the interactions among H3.3, its chaperons, and the proteins that comprise retroviral silencing complex, in Smarcad1 depleted cells. This study provides evidence for the role of Smarcad1 in H3.3 and Trim28 dependent retroviral repression in embryonic stem cells.

 

1.3] Bovine-induced pluripotent stem cells

​Pluripotent stem cells are cells that, by definition, are capable of differentiating to all three germ layers and self-renew indefinitely. There are two methods to deriving pluripotent stem cells: 1) Harvesting from the inner cell mass of a blastocyst or 2) reprogramming differentiated cells using external factors to induce pluripotency or in other words induced pluripotent stem cells. In mice and humans,​​ the protocol to reprogram differentiated cells is already well established. However, there is no established and agreed-upon method for reprogramming cattle cells. In our research, we aim to define and establish a protocol for reprogramming bovine cells.

Derivation of bovine pluripotent cells can be applied in drug selection, human disease modeling and agriculture‐related applications. For example, the main issue in agriculture is the decreased fertility of cows due to exposure to environmental stress. Currently, the study of early developmental problems requires in vitro fertilization and maturation of blastocysts. This is both costly, time-consuming, and requires extensive training. Using pluripotent stem cells will allow us to study the effects of stress on reproduction in a more simple and controlled system. Another example is the cultured meat industry. Reprogrammed cells can self-renew indefinitely and can differentiate into any cell in the body, including muscle. Companies could then keep one line of cells that could turn into any tissue without the need to renew and re-check new cells harvested from a living cow. This would lower costs and make lab-grown meat more available, vegan and environmentally friendly. Moreover, pluripotent cells can serve as a new source for regenerative medicine in the medical and veterinary fields. With induced pluripotent cells, new avenues of stem cell therapy can be examined and tried, using the patient's own cells and the new technology of Crisper/CAS9 gene editing.

 

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.

 

 

3.1] Mesenchymal stromal cells modulate infection and inflammation in the uterus and mammary gland

The use of mesenchymal stromal cells (MSCs) is emerging as an efficacious and safe treatment for many infectious and non-infectious inflammatory diseases in human and veterinary medicine. Such use could be done to treat mastitis and metritis, which are the most common disease conditions affecting dairy cows leading to considerable economic losses and reduced animal welfare. Currently, both disease conditions are commonly treated using local and systemic administration of antibiotics. However, this strategy has many disadvantages including low cure rates and the public health hazards. Looking for alternative approaches, we investigated the properties of MSCs using in-vitro mammary and endometrial cell systems and in-vivo mastitis and metritis murine model systems. In-vitro, co-culture of mammary and uterus epithelial cells constructed with NF-kB reporter system, the master regulator of inflammation, demonstrated their anti-inflammatory effects in response to.LPS. In vivo, we challenge animals with field strains of mammary and utero pathogenic Escherichia coli and evaluated the effects of local and systemic application of MSC in the animal models. Disease outcome was evaluated using histological analysis, bacterial counts and gene expression of inflammatory markers. We show that MSC treatment reduced bacterial load in metritis and significantly modulated the inflammatory response of the uterus and mammary gland to bacterial infection. Most notably are the immune modulatory effects of remotely engrafted intravenous MSCs, which open new avenues to the development of MSC-based cell-free therapies.   

3.2] Cultured meat and alternative proteins

Climate change is threatening the future food availability of the world population, posing a major challenge faced by societies and governments. The awareness of emerging climate change increases the global demand for alternative protein sources, therefore a pressing need to develop more sustainable sources of proteins is arising. Cellular agriculture is an evolving branch of biotechnology that aims to develop new solutions and improve the old ones, including plant-based substitutes and cell-based meats and dairy products. One of the proposed solutions is cultured meat (CM), an alternative protein source that has flourished in recent years as the demand for CM products has risen. Cultured meat (CM) is an emerging technology based on the proliferation and differentiation of animal stem cells in vitro to produce edible tissues for human consumption. However, a significant improvement is needed for the process to become cost-effective and reliable enough to bring it to production on a scale suitable for the food supply. This session aims to address these challenges by bringing together scientists, policymakers and entrepreneurs to discuss aims to revolutionize the way we produce meat, showcase recent success by Israeli scientists and startups, and discuss future challenges. This session also addresses the ways governments and investors can focus on funding plans worldwide toward a sustainable way to feed humanity's future meat demands. The move from traditional farm-animal-agriculture-based nutrition to cultured meat will directly contribute to the efficient utilization of environmental resources and reduction of pollution, improved food safety, and a healthier diet. (Session and presentation at the UNFCCC- COP27 in Sharm el-Sheikh, Egypt)

 

3.3] Designing edible scaffolds and growing bovine 'whole cut' for the cultured meat

We aim to develop an innovative, cost-effective, and scalable approach for the production of bovine 'whole cut' cultured meat products. In addition, this multicellular 'whole cut' will have desired nutritional properties and support cellular proliferation and/or differentiation. Within the scope of the current research project, our primary goal is to produce a working prototype of one or more edible scaffolds and characterize their ability to support bovine mesenchymal stem cells proliferation and differentiation into muscle and fat tissues.

Cellular agriculture is an evolving branch of biotechnology that aims to improve traditional agriculture on issues related to environmental impacts, animal welfare and sustainability. One of the proposed solutions is cultured meat (CM) cultured meat - which does not yet exist in commercial production.

Cultured meat has flourished in recent years as the demand for CM products has risen. This is mainly due to the moral reluctance of a considerable percentage of the population from the breeding practices in the coops and farms as well as from the mass slaughter of livestock for meat. The awareness of emerging climate change increases the global demand for alternative protein sources.

Cultured meat (CM) is an emerging technology based on the proliferation and differentiation of animal stem cells in vitro to produce edible tissues for human consumption¹. First, a significant improvement is needed for the process to become cost-effective and reliable enough to bring it to production on a scale suitable for the food supply². Secondly, cultured meat is animal tissue grown outside the body, in vitro. In general, tissue growth requires three things:  (1) Source cells: stem cells of adult animals (removed by biopsy or extracted from excess tissue, e.g. placenta, umbilical cord, etc.) or embryonic stem cells. The cells must have the ability to differentiate to form mature muscle tissue. (2) Support: The cells should grow on a surface and adhere to it in order to create three-dimensional muscle tissue with a noticeable thickness. (3) Nutrition: Cells are grown in a medium containing essential amino acids and growth factors. Moreover, to produce tissue comparable to meat, muscle cells should be combined with other cells, such as fat cells, and the scaffold has to allow transportation of nutrients through a system similar to capillaries².

 

3.4] Cost-effective “Smart Scaffold” production for the cultivated meat (CM) industry application

With this research, we aim to develop an innovative approach in the field of tissue engineering. When mature, this technology will enable the cost-effective production of complex cultivated meat (CM) products that require multiple cell types to be organized in a preordered shape tissue (Figure 1.). Within the scope of the current research project, our main goal is to produce a working prototype of such “Smart Scaffold”. The Schlesinger lab has been growing and differentiating bovine umbilical cord MSC (BUC-MSC) and bovine adipose MSC (BAD-MSC) for almost three years. We have several lines of MSC that can be grown and differentiated into chondrocytes, osteocytes and adipocyte fates in the lab by standard protocols. We have shown that BUC-MSC are highly expandable since from 2cm of UC we get more than one million of cells, which double every 22-26 hours for more than 40 days and remain multipotent and ready to differentiate (Shimoni et al., 2020).  In addition to the high scalability of the cells, their maintenance in culture is relatively lowcost and simple, and cellular senescence (aging) can be delayed by adding antioxidants to their growth media.  Recently, we have examined and optimized two muscle differentiation protocols and were able to show efficient differentiation of BUC-MSC into muscle fibers. Additionally, we induce faster adipogenesis by pre-treatment with heat shock, therefore directing the cells toward fate commitment before seeding on Scaffold.