A General and Convenient Peptide Self-Assembling Mechanism for Developing Supramolecular Versatile Nanomaterials Based on The Biosynthetic Hybrid Amyloid-Resilin Protein.

Self-assembling peptides are valuable building blocks to fabricate supramolecular biomaterials, which have broad applications from biomedicine to biotechnology. However, limited choices to induce different globular proteins into hydrogels hinder these designs. Here, an easy-to-implement and tunable self-assembling strategy, which employs Ure2 amyloidogenic peptide, are described to induce any target proteins to assemble into supramolecular hydrogels alone or in combination with notable compositional control. Furthermore, the collective effect of nanoscale interactions among amyloid nanofibrils and partially disordered elastomeric polypeptides are investigated. This led to many useful macroscopic material properties simultaneously emerging from one pure protein material, i.e. strong adhesion to any substrates under wet conditions, rapidly self--assembling into robust and porous hydrogels, adaptation to remodeling processes, strongly promoting cell adhesion, proliferation and differentiation. Moreover, he demonstrated this supramolecular material's robust performance in vitro and vivo for tissue engineering, cosmetic and hemostasis applications and exhibited superior performance compared to corresponding commercial counterparts. To the best of his knowledge, few pure protein-based materials could meet such seemingly mutually exclusive properties simultaneously. Such versatility renders this novel supramolecular nanomaterial as next-generation functional protein-based materials, and he demonstrated the sequence level modulation of structural order and disorder as an untapped principle to design new proteins.

Morphology-Based Prediction of Proliferation and Differentiation Potencies of Porcine Muscle Stem Cells for Cultured Meat Production.

Inconsistent efficiency of cell production caused by cellular quality variations has become a significant problem in the cultured meat industry. In our study, morphological information on passages 5-9 of porcine muscle stem cells (pMuSCs) from three lots was analyzed and used as input data in prediction models. Cell proliferation and differentiation potencies were measured by cell growth rate and average stained area of the myosin heavy chain. Analysis of PCA and heatmap showed that the morphological parameters could be used to discriminate the differences of passages and lots. Various morphological parameters were analyzed, which revealed that accumulating time-course information regarding morphological heterogeneity in cell populations is crucial to predicting the potencies. Based on the 36 and 60 h morphological profiles, the best proliferation potency prediction model (R2 = 0.95, RMSE = 1.1) and differentiation potency prediction model (R2 = 0.74, RMSE = 1.2) were explored. Correlation analysis demonstrated that morphological parameters selected in models are related to the quality of porcine muscle stem cells.

Tea polyphenols coated sodium alginate-gelatin 3D edible scaffold for cultured meat.

This study aimed to use edible scaffolds as a platform for animal stem cell expansion, thus constructing block-shaped cell culture meat. The tea polyphenols (TP)-coated 3D scaffolds were constructed of sodium alginate (SA) and gelatin (Gel) with good biocompatibility and mechanical support. Initially, the physicochemical properties and mechanical properties of SA-Gel-TP scaffolds were measured, and the biocompatibility of the scaffolds was evaluated by C2C12 cells. SEM results showed that the scaffold had a porous laminar structure with TP particles attached to the surface, while FT-IR results also demonstrated the encapsulation of TP coating on the scaffold. In addition, the porosity of all scaffolds was higher than 40% and the degradation rate during the incubation cycle was less than 40% and the S2-G1-TP0.1-3 h scaffold has excellent cell adhesion and extension. Subsequently, we inoculated rabbit skeletal muscle myoblasts (RbSkMC) on the scaffold and induced differentiation. The results showed good adhesion and extension behavior of RbSkMC on S2-G1-TP0.1-3 h scaffolds with high expression of myogenic differentiation proteins and genes, and SEM results confirmed the formation of myotubes. Additionally, the adhesion rate of cells on scaffolds with TP coating was 1.5 times higher than that on scaffolds without coating, which significantly improved the cell proliferation rate and the morphology of cells with extension on the scaffolds. Furthermore, rabbit-derived cultured meat had similar appearance and textural characteristics to fresh meat. These conclusions indicate the high potential of the scaffolds with TP coating as a platform for the production of cultured meat products.

The Effect of Long-Term Passage on Porcine SMCs' Function and the Improvement of TGF-ß1 on Porcine SMCs' Secretory Function in Late Passage.

Cultured meat is one of the meat substitutes produced through tissue engineering and other technologies. Large-scale cell culture is the key for cultured meat products to enter the market. Therefore, this study is aimed to explore the effect of long-term passage in vitro on smooth muscle cells (SMCs) and the effect of transforming growth factor-ß1 (TGF-ß1) on SMCs in the late passage. Multiple passages lead to the decline of the proliferation rate of SMCs in the proliferation stage and the differentiation ability in the differentiation stage. Transcriptome results showed that the ECM pathway and aging-related signaling pathways were significantly up-regulated in the late passage period. TGF-ß1 did not promote SMCs of late passage proliferation at the proliferation stage but promoted the gene and protein expression of collagen as the main protein of the extracellular matrix proteins at the differentiation stage. In addition, proteomic analysis revealed that TGF-ß1 promoted the expression of cell adhesion molecules which activate the Hippo signaling pathway and the HIF-1 signaling pathway and further promoted the production of collagen-containing extracellular matrix proteins. This could provide ideas for large-scale production of cultured meat products using SMCs.

Gellan gum-gelatin scaffolds with Ca2+ crosslinking for constructing a structured cell cultured meat model.

As an emerging technology to obtain protein by culturing animal-derived cells in vitro, it is crucial to construct 3D edible scaffolds to prepare structured cell cultured meat products. In this study, a scaffold based on gellan gum (GG)-gelatin (Gel) was prepared and further cross-linked with Ca2+. FTIR confirmed the electrostatic interaction between GG and Gel and the ionic cross-linking of Ca2+ and carboxyl groups, and SEM images showed the porous structure of the scaffolds. The staining results showed that scaffolds with high concentrations of Ca2+ had higher biocompatibility than scaffolds with low concentrations of Ca2+ and non-crosslinked scaffolds, and scaffolds Ca2+-GG2-Gel3-0.5 adhered to more cells and were more conducive to cell spreading. The immunofluorescence staining, SEM images, Western blot, and RT-qPCR showed that the scaffolds supported the proliferation and myogenic differentiation of chicken skeletal muscle satellite cells (CSMSCs) and myotubes were formed on the scaffolds. Finally, the scaffolds were stained and fried after culturing. The results of the textural and chromatic analysis showed that the texture and color of the scaffolds were similar to fresh meat and meat products. These results showed that ionically crosslinked GG-Gel scaffolds are biocompatible and stable for structured cell cultured meat models.

Preparation and tribological properties of multi-layer graphene/silicon dioxide composites-based solid lubricant coatings at elevated temperatures.

The solid lubricating coatings have an important role in hot metal forming. However, traditional lubricants cannot be applied to the harsh working conditions. In this investigation, the novel solid lubricant coatings including multi-layer graphene (MLG)/silicon dioxide (SiO2) composites and sodium metaphosphate phosphate were prepared. The high-temperature tribological properties of the solid lubricant coatings were investigated by friction and wear tester. The experimental results showed that SiO2 nanoparticles were evenly grafted by sol-gel method on the surface of MLG, forming MLG/SiO2 composites. MLG/SiO2 composites presented excellent thermal stability at 800°C. In the range of 400-800°C, the average coefficients of friction (COFs) were decreased from 0.3936 to 0.3663, and then increased from 0.3663 to 0.4226. Based on the analysis of wear scar, the lubrication mechanisms of the solid lubricating coatings were proposed. The low interlayer shear of MLG and the ball bearing of SiO2 nanoparticles are the main reason for the reduction of COFs. In addition, the tribo-chemical reaction film formed on the frictional interface could protect the contact surfaces from severe damage. The findings would be beneficial for developing novel lubricants for hot metal forming process.

Preparation and Quality Evaluation of Cultured Fat.

Cultured meat is rapidly developing as an emerging meat production technology. Adipose tissue plays an essential role in the flavor of meat products. In this study, cultured fat was produced by cultured adipose-derived stem cells (ADSCs) based on collagen in vitro, with a 3D model. The research showed that ADSCs could attach to collagen hydrogels and differentiate into mature adipocytes. Texture analysis demonstrated that the springiness, cohesiveness, and resilience of cultured fat were consistent with porcine subcutaneous fat. Moreover, 28 volatile organic compounds (VOCs) were detected by headspace gas chromatography-ion mobility spectrometry. The relative contents of 17 VOCs in cultured fat were significantly higher than porcine subcutaneous fat and empty collagen hydrogels, and the relative contents of 5 VOCs in cultured fat were not significantly different from porcine subcutaneous fat. These findings assert the promising application of cultured fat in cultured meat production.

The fate of fertilizer-derived phosphorus under different long-term fertilization regimes: A phosphate oxygen isotope study.

Understanding the fate of exogenous fertilizer-derived inorganic phosphorus (Pi) is essential for effective P management. Hence, this study carried out a 180-day incubation experiment with or without KH2P18O4 in soils with four different fertilization regimes [without fertilizer (CK), mineral P and K fertilizer (PK), mineral N, P, and K fertilizer (NPK), compost (OM)]. We analyzed the atom % excess in phosphate oxygen isotope of sequentially extracted Pi pools (H2O-Pi, NaHCO3-Pi, NaOH-Pi, and HCl-Pi), soil respiration, potential phosphatase activities, and microbial biomass. Our results showed that exogenous phosphate fertilizer was immediately transformed into the H2O-Pi and NaHCO3-Pi pools and gradually partially immobilized in the HCl-Pi pool. Additionally, biotransformation plays an important role in the turnover of fertilizer-derived Pi. After the 180-day incubation, the biologically transformed H2O-Pi content was significantly (P<0.05) reduced by 63.2 % on average, with the largest reduction in PK. The NaHCO3-Pi gradually increased in both CK and OM through biotic processes. However, it continuously decreased in PK and NPK, likely due to the strong adsorption and microbial fixation. The NaOH-Pi fluctuated slightly in CK, NPK, and OM while gradually decreasing in PK. At the end of the incubation, 28.6 %, 37.0 %, 61.2 %, and 75.2 % of the Pi increment in CK, OM, NPK, and PK were stored in the HCl-Pi pool, respectively. Overall, these findings provide important information on the dynamics of fertilizer-derived Pi, delivering new insights into rational phosphate fertilizer management and sustainable agricultural development.

Identification of porcine adipose progenitor cells by fluorescence-activated cell sorting for the preparation of cultured fat by 3D bioprinting.

Cultured meat is an emerging technology that is friendly for the environment and animal welfare. As a novel food ingredient, cultured fat is essential for the flavor and nutrition of cultured meat. In this study, we purified adipose progenitor cell (APC) from freshly isolated porcine stromal vessel fraction (SVF) by fluorescence-activated cell sorting (FACS) and identified the transcriptome characteristics of APC by RNA sequencing (RNA-seq). The results showed that APC had characteristics of high-efficiency proliferation and adipogenic differentiation and was distinct from SVF cell in transcriptome profiles. Subsequently, APC was used to prepare cultured fat by 3D bioprinting and to evaluate the differences in fatty acid composition between cultured fat and porcine subcutaneous adipose tissue (pSAT). The results indicated that the fatty acid composition and content of cultured fat had a certain similarity with pSAT; specifically, the content of key monounsaturated fatty acid (MUFA) that create pork flavor in cultured fat, such as C18:1(n-12), C18:1(n-9) and C19:1(n-9)T, were close to that of pSAT. Therefore, this research indicated that APC is a promising candidate cell type for the production of cultured fat.

Large-Scale Expansion of Porcine Adipose-Derived Stem Cells Based on Microcarriers System for Cultured Meat Production.

Cultured meat is an innovative meat-production technology that does not rely on animal husbandry. As a new food component, cultured fat is of great significance to cultured meat. In this study, we isolated adipose-derived stem cells (ADSCs) and identified the purity by immunofluorescence staining of ADSC-specific surface marker proteins CD44 and CD29 and showed that most of the cells were positive for CD29 and CD44. In addition, we detected the expression of FABP4 and Plin1 to confirm that ADSCs differentiated into mature adipocytes at 10 days post-induction. Subsequently, the culture conditions of ADSCs on microcarriers (MCs) were optimized and showed that cell density of living cells reached their highest after 5 days when continuously stirring at 50 rpm. Finally, the expression of FABP4 and PPARγ was detected to confirm the adipogenic differentiation ability of ADSCs on 2D and 3D culture systems and showed that ADSCs maintained their adipogenic differentiation ability after expansion on MCs. In conclusion, this research demonstrated that reliance on MCs to expand ADSCs was a promising approach for production of cultured fat.

Quality evaluation of cultured meat with plant protein scaffold.

Cultured meat technology is a promising new technology to solve the negative problems brought by traditional animal husbandry. Cultured meat should be further developed to appear on consumers' tables as alternative protein product. Therefore, this study used food grade peanut wire-drawing protein as scaffold to culture smooth muscle cells (SMCs) in vitro to obtain cultured meat productions containing both animal protein and plant protein. Multiple passages lead to the decline of the proliferation rate of SMCs in the proliferation stage and the differentiation ability in the differentiation stage, which means that the plasticity of cells decreased in the later stage of passage. SMCs can well adhere to the peanut wire-drawing protein scaffold and produce extracellular matrix protein and muscle protein, so as to form a cultured meat product with rich protein composition. This study provides a theoretical basis for the production of nutrient-rich cultured meat products.

Effects of selected flavonoids oncellproliferation and differentiation of porcine muscle stem cells for cultured meat production.

Stemness decline of muscle stem cells (MuSCs) is a significant problem in cultured meat processing. In the present study, three flavonoids (quercetin, icariin, and 3,2'-dihydroxyflavone) with multi concentrations were evaluated to promote the proliferation and differentiation of porcine muscle stem cells. In the proliferation phase, 3,2'-dihydroxyflavone (10 µM) significantly amplified the cells by 34% and up-regulated the expression of paired box transcription factor 7 (PAX7) by 60%, which was higher than quercetin (75 nM) and icariin (7.5 nM). In the differentiation phase, quercetin (50 nM) showed the best pro-differentiation effect and up-regulated the expression of myosin heavy chain (MYHC) by 4.73-fold compared with the control group. These results indicated that flavonoids had a significant impact on promoting the proliferation and differentiation of porcine MuSCs, and 3,2'-dihydroxyflavone (10 µM) for proliferation and quercetin (50 nM) for differentiation were the optimal combinations.

Production of cultured fat with peanut wire-drawing protein scaffold and quality evaluation based on texture and volatile compounds analysis.

Cultured meat is an emergent technology that cultivates cells in three-dimensional scaffolds to generate tissue for consumption. Fat makes an important contribution to the flavor and texture of traditional meat, but there are few reports on cultured fat. Here, we demonstrated the construction of cultured fat by inoculating porcine adipose-derived mesenchymal stem cell (ADSC) on peanut wire-drawing protein (PWP) scaffolds. First, we demonstrated that basic fibroblast growth factor (bFGF) promoted cell proliferation and maintained adipogenic differentiation ability. Then, we generated cultured fat and found that cultured fat decreased the texture of PWP scaffolds. Moreover, 43 volatile compounds were detected by headspace gas chromatography-ion mobility spectrometry (GC-IMS), of which 17 volatile compounds showed no significant differences between cultured fat and porcine subcutaneous adipose tissue (pSAT), which indicated that cultured fat and pSAT had certain similarities. Collectively, this research has great promise for improving the quality of cultured meat.

Production of cultured meat by culturing porcine smooth muscle cells in vitro with food grade peanut wire-drawing protein scaffold.

Cultivating meat is a promising solution to the negative problems brought by traditional animal husbandry. To make cultured meat have the sensory and nutritional characteristics of conventional meat as much as possible, many studies have been conducted on various cell types and scaffold characteristics. Therefore, this study aims to produce a low-cost cultured meat with a quality closer to that of conventional meat. Tissue generation requires three-dimensional (3D) scaffolds to support cells and simulate extracellular matrix (ECM). Here, we used peanut wire-drawing protein (a biomaterial based on edible porous protein) as a new culture meat scaffold to culture cells. The scaffold can support cell attachment and proliferation to create 3D engineered porcine muscle tissue. The differentiation of smooth muscle cells (SMCs) was induced by a low serum medium to produce more extracellular matrix proteins. After differentiation, it was found that peanut wire-drawing protein scaffolds could be used for porcine smooth muscle cell adhesion and growth. The ECM protein and muscle protein produced by SMCs can endow cultured meat with better quality. This technology provides an innovative pathway for the industrialized production of cultured meat.

Production of cultured meat from pig muscle stem cells.

Cultured meat is meat for consumption produced in a more sustainable way. It involves cell harvesting and expansion, differentiation into myotubes, construction into muscle fibres and meat structuring. We isolated 5.3 × 104 porcine muscle stem cells from 1 g of neonatal pig muscle tissue. According to calculations, we need to expand muscle stem cells 106-107 times to produce 100 g or 1 kg of cultured meat. However, the cells gradually lost the ability to express stemness and mature muscle cell markers (PAX7, MyHC). To tackle this critical issue and maintain cell function during cell expansion, we found that long-term culture with (100 µM) l-Ascorbic acid 2-phosphate (Asc-2P) accelerated cell proliferation while preserving the muscle cell differentiation. We further optimized a scalable PDMS mold. Porcine muscle stem cells formed structurally-organized myotubes similar to muscle fibres in the mold. Asc-2P enhanced porcine muscle cells grown as 3D tissue networks that can produce a relatively large 3D tissue networks as cultured meat building blocks, which showed improved texture and amino acid content. These results established a realistic workflow for the production of cultured meat that mimics the pork meat structurally and is potentially scalable for industry.

Differential diagnosis of acute and chronic colitis in mice by optical coherence tomography.

Background: The differential diagnosis of acute and chronic colitis remains a common clinical problem. Optical coherence tomography (OCT) is a non-invasive, high-resolution imaging technique that can be used to measure morphological changes in the intestinal wall and estimate intestinal inflammation. We aimed to conduct an ex vivo experiment on a mouse model investigate the value of OCT as a tool for the differential diagnosis of acute and chronic colitis. Methods: Mice were administered dextran sulfate sodium salt (DSS) to construct acute and chronic colitis models. Acutely- and chronically-affected intestinal walls were scanned by OCT, and then the scanned colonic tissue samples were stained with hematoxylin and eosin (HE). Structural and morphological changes indicating inflammation in the intestinal wall were evaluated in the HE sections and OCT images using different parameters. The parameters were used in one-way analysis of variance (ANOVA) to screen for a differential diagnosis of acute or chronic colitis. Results: For the HE sections, the angle of the mucosal folds, length of the basilar part, and submucosal height and area were statistically significant parameters in the comparisons between the mice with acute colitis and the control-group mice (P<0.05). In the comparisons between chronic colitis mice and control-group mice, the angle of the mucosal folds, length of the basilar part, submucosal height and area, muscularis thickness, submucosal height + muscularis thickness, and mucosal thickness were statistically significant parameters (P<0.05). Finally, in the comparisons between acute colitis mice and those with chronic colitis, the angle of the mucosal folds, submucosal height and area, muscularis thickness, submucosal height + muscularis thickness, and mucosal thickness were statistically significant parameters (P<0.05). For the OCT images, only the length of the basilar part and submucosal height + muscularis thickness were statistically significant parameters between the acute colitis mice and control-group mice (P<0.05). The length of the basilar part and submucosal height + muscularis thickness were statistically significant between chronic colitis mice and control-group mice (P<0.05). In the comparisons between acute colitis mice and those with chronic colitis, only submucosal height + muscularis thickness was a statistically significant parameter (P<0.05). Conclusions: Certain intestinal wall parameters in OCT can be used to make a differential diagnosis between acute and chronic colitis possible. This study contributes to constructing a potential diagnostic system for evaluating colorectal inflammation using OCT.

Engineered meatballs via scalable skeletal muscle cell expansion and modular micro-tissue assembly using porous gelatin micro-carriers.

The emerging field of cultured meat faces several technical hurdles, including the scale-up production of quality muscle and adipose progenitor cells, and the differentiation and bioengineering of these cellular materials into large, meat-like tissue. Here, we present edible, 3D porous gelatin micro-carriers (PoGelat-MCs), as efficient cell expansion scaffolds, as well as modular tissue-engineering building blocks for lab-grown meat. PoGelat-MC culture in spinner flasks, not only facilitated the scalable expansion of porcine skeletal muscle satellite cells and murine myoblasts, but also triggered their spontaneous myogenesis, in the absence of myogenic reagents. Using 3D-printed mold and transglutaminase, we bio-assembled pork muscle micro-tissues into centimeter-scale meatballs, which exhibited similar mechanical property and higher protein content compared to conventional ground pork meatballs. PoGelat-MCs also supported the expansion and differentiation of 3T3L1 murine pre-adipocytes into mature adipose micro-tissues, which could be used as modular assembly unit for engineered fat-containing meat products. Together, our results highlight PoGelat-MCs, in combination with dynamic bioreactors, as a scalable culture system to produce large quantity of highly-viable muscle and fat micro-tissues, which could be further bio-assembled into ground meat analogues.

Chitosan­sodium alginate-collagen/gelatin three-dimensional edible scaffolds for building a structured model for cell cultured meat.

Cell cultured meat (CCM) production is an innovative technology that does not depend on livestock farming practices to produce meat. The construction of structured CCM requires a three-dimensional (3D) scaffold to mimic the extracellular matrix to provide mechanical support for the cells. Furthermore, the 3D scaffolds should be edible and have good biocompatibility and tissue-like texture. Here, we demonstrated a 3D edible chitosan­sodium alginate-collagen/gelatin (CS-SA-Col/Gel) scaffold that can support the adhesion and proliferation of porcine skeletal muscle satellite cells, culminating in the construction of a structured CCM model. The 3D edible scaffolds were prepared by freeze-drying using electrostatic interactions between chitosan and sodium alginate. Initially, the physicochemical properties and structural characteristics of different scaffolds were explored, and the biocompatibility of the scaffolds was evaluated using the C2C12 cell model. The results showed that the 2-CS-SA-Col1-Gel scaffold provided stable mechanical support and abundant adhesion sites for the cells. Subsequently, we inoculated porcine skeletal muscle satellite cells on the 2-CS-SA-Col1-Gel scaffold and induced differentiation for a total of 14 days. Immunofluorescence staining results showed cytoskeleton formation, and Western blotting (WB) and qPCR results showed upregulation of skeletal proteins and myogenic genes. Ultimately, the structured CCM model has similar textural properties (chewiness, springiness and resilience) and appearance to those of fresh pork. In conclusion, the method of constructing 3D edible scaffolds to prepare structured CCM models exhibits the potential to produce cell cultured meat.

Evaluation of the effect of smooth muscle cells on the quality of cultured meat in a model for cultured meat.

While the research on improving the meat quality of cultured meat is in full swing, few studies have focused on the effect of smooth muscle cells (SMCs) on the meat quality of cultured meat. Therefore, this study aimed at building a cultured meat model containing smooth muscle cells, and further evaluating the effect of smooth muscle cells on the quality of cultured meat, so as to reveal the contribution of smooth muscle cells in the production of cultured meat. In this study, we isolated high purity of smooth muscle cells from vascular tissues. The addition of basic fibroblast growth factor (bFGF) to the medium significantly increased the growth rate of smooth muscle cells and the expression of extracellular matrix related genes, especially collagen and elastin. Smooth muscle cells were seeded in a collagen gel to construct a culture meat model. It was found that the pressure loss of the model meat significantly decreased from 98.5 % in control group to 54 % with the extension of culture time for 9 days, while the total collagen content of model meat increased significantly (P < 0.05). In addition, the hydrogel tissue with smooth muscle cells compacted more dramatically and were more tightly, accompanied by significantly increased hardness, springiness and chewiness compared to the control one (P < 0.05). These results indicate that smooth muscle cells can secrete extracellular matrix proteins such as collagen, which can significantly enhance the texture of cultured meat models prepared by hydrogel.

YAP Promotes Cell Proliferation and Stemness Maintenance of Porcine Muscle Stem Cells under High-Density Condition.

Muscle stem cells (MuSCs) isolated ex vivo are essential original cells to produce cultured meat. Currently, one of the main obstacles for cultured meat production derives from the limited capacity of large-scale amplification of MuSCs, especially under high-density culture condition. Here, we show that at higher cell densities, proliferation and differentiation capacities of porcine MuSCs are impaired. We investigate the roles of Hippo-YAP signaling, which is important regulators in response to cell contact inhibition. Interestingly, abundant but not functional YAP proteins are accumulated in MuSCs seeded at high density. When treated with lysophosphatidic acid (LPA), the activator of YAP, porcine MuSCs exhibit increased proliferation and elevated differentiation potential compared with control cells. Moreover, constitutively active YAP with deactivated phosphorylation sites, but not intact YAP, promotes cell proliferation and stemness maintenance of MuSCs. Together, we reveal a potential molecular target that enables massive MuSCs expansion for large-scale cultured meat production under high-density condition.