Biotechnological Applications of Stem Cells

 

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.

 

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.

 

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.

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.

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

*In collaboration with Prof. Oded Shoseyov

 

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.

 

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