1.1] The dynamic control of retroviral suppression during the transition into pluripotency

Pluripotency undergoes dynamic transitions starting from the inner cell mass (ICM) of the blastocyst through the epiblast stage, encompassing both naïve and primed states in mouse embryonic stem cells (ESCs). These states exhibit distinct gene expressions, DNA methylation status and chromatin structures. Recently, our research demonstrated the essential role of H3.3 deposition in silencing repressed retroviral sequences in naïve mouse ESCs. However, understanding of how H3.3 dynamics and epigenetic differences regulate retroviral silencing during the transition from naïve to primed pluripotency remains limited.

Sharon Schlesinger

Here, we focus on evaluating how H3.3 impacts the transcription of retroviral sequences during this pluripotency transition in mouse ESCs. We compared the transcriptional statuses of newly integrated Moloney leukemia virus (MLV) provirus between naïve and formative pluripotent states using wild-type (wt) and H3.3 null ESCs. Our findings reveal that MLV provirus repression is compromised in naïve (2i/LIF) ESCs upon H3.3 depletion, whereas wt ESCs effectively suppress viral expression. In contrast, epiblast-like cells (EpiLC- FGF2/Activin) infected with MLV show reduced silencing in wt and H3.3 null cells.

1.2] 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.

1.3] Bovine pluripotent stem cells

Mammalian development follows fundamental principles, yet species-specific variations critically influence early embryogenesis. By utilizing blastoids—lab-derived structures mimicking early, pluripotent embryos—we aim to investigate interspecies differences in pluripotency, early cell lineage commitment, and molecular mechanisms underlying embryogenesis. A key focus of our research is the successful generation of bovine blastoids to (1) Examine critical developmental events, such as early lineage specification, and better understand the molecular control of lineage specification in bovine embryos compared to other mammals. (2) Study epigenetic modifications during early development. For instance, bovine blastoids provide a controlled model to explore how metabolic states influence epigenetic reprogramming, offering novel insights into these dynamic processes. (3) Analyze the impact of environmental conditions on early developmental stages, which were previously inaccessible for detailed investigation.
Bovine blastoids offer significant ethical and practical advantages by reducing dependency on natural embryos, addressing ethical concerns, and enabling high-throughput research. They facilitate systematic cross-species comparisons under controlled conditions, revealing species-specific adaptations and evolutionary conservation of gene regulatory networks and implantation mechanisms.
Potential applications in livestock breeding include overcoming the bottleneck of limited embryo availability for artificial reproductive technologies (ART). Bovine blastoids could accelerate genetic improvement programs by enabling scalable embryo production without reliance on IVF or somatic cell nuclear transfer (SCNT), transforming animal breeding and genetics.
Future directions involve further optimization and validation of bovine blastoid models through comparisons with in vivo embryos to ensure fidelity and applicability. These advancements promise to enhance both fundamental biological understanding and practical applications in agriculture and biomedicine.

Sharon Schlesinger

2.1] Heat Shock-Induced Epigenetic Modifications Enhance Adipogenic Differentiation in Bovine Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) present remarkable potential for applications in cell therapy and cultured meat (CM) production, thanks to their accessibility, multilineage differentiation capabilities, and relatively simple, cost-effective cultivation techniques. However, their limited self-renewal and slow differentiation rates pose significant hurdles for scalable production. This highlights the need for advancements in culture and differentiation methodologies. Building on our prior research indicating that brief heat shock (HS) induces epigenetic changes influencing cell fate, our current findings demonstrate that a short, well-timed thermal shock significantly accelerates adipogenic differentiation in bovine MSCs. Not only do HS-preconditioned cells differentiate more rapidly, but they also produce fat cells with an improved fatty acid composition. These results suggest that pre-treating MSCs with heat shock can substantially alter cellular identity and developmental trajectories. By exploring the underlying mechanisms of this effect, we seek to reduce dependency on chemical additives and growth factors in culture media. Specifically, optimizing MSC adipogenesis through cost-effective HS preconditioning could revolutionize their application in the CM industry. This advancement holds promise for streamlining differentiation processes, reducing production timelines, increasing yield, and enhancing both meat quality and differentiation efficiency in CM production. On a broader scale, our results offer valuable insights into evolutionary development and regenerative processes in cattle.

Sharon Schlesinger

2.2] Stress Can Rewire Mesenchymal Stem Cell Memory

Mesenchymal stem cells (MSCs) are central to tissue repair and homeostasis, adapting to environmental stressors through a poorly understood mechanism of cellular memory. Our study reveals how heat stress (HS) triggers long-lasting transcriptional and epigenetic changes in MSCs, effectively “rewiring” their response to future challenges.
We found that bovine umbilical cord-derived MSCs exposed to HS exhibit slowed cell cycles, heightened oxidative stress, and persistent changes in gene expression, driven by dynamic alterations in chromatin accessibility and DNA methylation. Sequential HS treatments demonstrated that these changes create a form of epigenetic memory, enabling MSCs to respond more efficiently—or, in some cases, more sensitively—to repeated stress. Through transcriptomic and epigenomic profiling, we aim to identify key memory-associated pathways, including oxidative stress response networks and chromatin remodeling factors, that mediate adaptive resilience.
Mechanistically, transcription factors and chromatin remodelers, such as histone deacetylases and histone methyltransferases, were shown to be crucial for embedding and maintaining stress memory. Functional disruption of these regulators using CRISPR/Cas9 and RNAi will confirm their role in modulating MSC resilience and plasticity during subsequent stress events.
Our findings will provide critical insights into how environmental stress imprints long-term epigenetic memory in MSCs, with significant implications for regenerative medicine and the development of stress-resilient therapies.

Sharon Schlesinger

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. Adult 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.

Sharon Schlesinger

Sharon Schlesinger

3.1] Genetically Engineered Stem Cells for Improved Growth and Muscle Differentiation

The growing global population and increasing demand for protein drive the need for sustainable alternatives to conventional livestock farming. Cellular agriculture leverages stem cells to develop scalable food production systems, yet the limited proliferative and differentiation capacity of these cells poses a major challenge to the cultured meat (CM) industry.
In this study, we utilize bovine mesenchymal stem cells (MSCs) for their multipotency and ease of culture, exploring the potential of hTERT-immortalized MSCs (iMSCs) for myogenic differentiation. iMSCs were genetically engineered via lentiviral transduction to express MyoD, a master regulator of muscle differentiation. The system incorporates a tetracycline-inducible (Tet-On) mechanism, allowing precise temporal control over MyoD activation. While MyoD induction alone was insufficient to drive myotube fusion, myogenesis was significantly enhanced using a combination of chemical compounds and growth factors. Notably, this approach increased differentiation efficiency from 20% to over 60% and reduced differentiation time from 21 days to just 10 days.

Sharon Schlesinger

These findings provide key insights into optimizing muscle differentiation strategies for CM production, offering a scalable, efficient approach to engineering sustainable protein sources.

3.2] Reprogramming Muscle Cells for Proliferation and Differentiation: A Promising Approach for Cultured Meat Production*

*In Collaboration with Prof. Benjamin Dekel and Prof. Eran Meshorer

Cultured meat is gaining increased attention in recent years, providing exciting opportunities to reduce and ultimately replace animal farm pollution and animal suffering. Specifically, cow farming, which generates excess methane, is a main contributor to ozone layer disruption, and growing public awareness is calling to reduce farm pollution. 
In recent years, several companies have begun to offer meat replacement solutions. The ones that can be found in supermarkets today are currently still plant-based, but several cultured meat-based products are beginning to emerge, seeking FDA approval. Despite these advances, several major caveats remain unsolved. On the one hand, the muscle cells themselves have a very limited proliferation capacity. Therefore, other cell types, most notably fibroblasts, are usually cultured and expanded from bovine muscle tissue. While these cell types offer bovine-based material, they are extremely remote from the desired end product, and significant manipulation is required to make them edible. On the other hand, when satellite muscle stem cells are used, their large-scale expansion is very difficult to achieve, and it remains challenging to reach the required cell numbers for industrial-level production. Here we offer a solution to address these issues, and produce proliferating bovine muscle cells using partial reprogramming. In a recent study (Omer D et al., Mol Ther Methods Clin Dev. 2023; PMID: 37214315) we showed that by introducing OCT4 into primary renal cells we are able to induce partial dedifferentiation and obtain self-renewing kidney progenitors. We showed that while these cells can be expanded at large numbers, they retain their renal identity both in vitro and in vivo and readily differentiate into kidney cells and spheroids. To test this idea in muscle cells, we will introduce OCT4 and other established reprogramming factors into muscle satellite cells to evaluate their potential for enhancing cell proliferation, specifically by assessing the population doubling time and the maximum time these cells can be cultured. Additionally, we will investigate the effectiveness and rate of differentiation into muscle myotubes, which resemble those found in edible skeletal muscle. We will utilize fluorescence microscopy and RT-qPCR to analyze markers such as PAX7 for satellite cell identity and Myosin heavy chain for myotube identity, respectively. 
Once we have determined the factors capable of reprogramming satellite cells, we will acquire modified mRNA molecules and utilize them as a gene delivery system to temporarily increase the expression of the selected genes. Modified mRNA is a desirable method of gene delivery as it enables safe, transient, and high-level gene expression without genetically altering the cells. We will evaluate the reprogramming factors identified in the previous step both individually and in various combinations. One outcome of this investigation will be the development of a reprogramming protocol that is rapid, efficient and user-friendly. The second outcome will be several non-genetically modified proliferating bovine satellite cell lines.
Our final objective is to assess the validated protocol for application in other species. Initially, we will investigate the impact on satellite cells derived from other mammals, specifically ovine and swine muscles satellite cells. In the subsequent phase, we will adapt the protocol and examine its effectiveness on cells from different vertebrate and invertebrate species, such as fish and mollusks.

Sharon Schlesinger

In summary, we propose that leveraging our knowledge and comprehension of the reprogramming process in the field of cultured meat will enable us to establish an innovative protocol. This protocol will enable the conversion of numerous cell types, which are presently deemed unsuitable for the industry due to their limited proliferation capacity, into valuable cell sources for cultivated meat production.

3.3] Cultivation of bovine stem cells to create lipid chunks on Aloe vera scaffolds*

*In collaboration with Dr. Jonthat Giron and Prof. Oded Shoseyov

Cultivated meat, also known as lab-grown or cell-based meat, is a novel approach to produce animal-derived food products without animal slaughter. This technology has the potential to address some of the major environmental, ethical, and health issues associated with conventional animal agriculture. However, cultivated meat also faces significant technical, regulatory, and consumer acceptance challenges. One of the key bottlenecks is finding a suitable environment for cultured cells to organize, proliferate, and eventually form functional meat tissue. Aloe vera, known for its medicinal and food applications, offers a sustainable, scalable, and cost-effective alternative for cultured meat production. We developed a method to repurpose the Aloe vera parenchyma to produce sterile scaffolds with conducive porous structures that allow liquid retention. The scaffolds demonstrate good biocompatibility, supporting cell adhesion, proliferation, and extracellular matrix formation of bovine mesenchymal stem cells. Moreover, the addition of oleic acid resulted in lipid accumulation, suggesting the potential for the formation of 'lipid chunks’. This cultured bovine 3D tissue, enriched with lipids, can serve as a food additive in plant-based alternative meats, potentially contributing to their texture and flavor profile. Furthermore, to address the scalability challenge, a novel macrofluidic single-use bioreactor (MSUB) was used to culture the scaffolds, as an example of the potential of various scaling out or up possibilities, enabling sustainable and scalable production of cultured meat or to be used for regenerative medicine for tissue or cell-based therapies.

Sharon Schlesinger

3.4] Designing edible scaffolds and growing bovine 'whole cut' for the cultured meatRevolutionizing

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.

Sharon Schlesinger

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

*In collaboration with Prof. Oded Shoseyov

Sharon Schlesinger

Cultivated meat, which aims to replicate traditional meat using tissue engineering and stem cell biology, is a promising approach to supplementing traditional meat production to meet increasing global demand. The production of cultivated whole-cut meat is not trivial; it requires a complex structure that supports cell growth, enables nutrient and waste exchange, and mimics natural texture. Here, we develop a biocompatible, porose, and anisotropic scaffold, based on directional freezing of nano and microcrystalline cellulose, which supports the growth and differentiation of bovine mesenchymal stem cells toward fat and muscle lineages. Furthermore, we show that pre-loading the scaffolds with factors directing the cells for proliferation or differentiation (thus fabrication of ‘Smart Scaffolds’) is a promising alternative to conventional media delivery since these pretreated scaffolds yield similar proliferation and differentiation efficiencies using 10 to 100 times lower masses of prohibitively expensive factors, and thus significantly decrease production costs. Together, these findings propose a method for the production of cultivated whole-cut meat—a sustainable and ethically sound alternative to meet the growing demand for this highly sought-after product.

Sharon Schlesinger

Highlights:

• Engineering ‘whole cut’ marbled cultivated meat using bovine mesenchymal stem cells.
• Cellulose scaffolds are porous and customized for directional growth
• Myogenic and adipogenic differentiation on the scaffolds
• The 'Smart Scaffold' enhances cell growth and differentiation with only 1/10 of the growth factors