A serum-free media formulation for cultured meat production supports bovine satellite cell differentiation in the absence of serum starvation [SF_Diff]
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ABSTRACT: Production of cultured meat requires the robust differentiation of satellite cells into mature muscle fibers without the use of animal-derived components. Current protocols induce myogenic differentiation in vitro through serum starvation, an abrupt reduction in serum concentration. Here, we used RNA sequencing to investigate the transcriptomic remodelling of bovine satellite cells during myogenic differentiation induced by serum starvation. We characterized canonical myogenic gene expression, and identified surface receptors upregulated during the early phase of differentiation. Supplementation of ligands to these receptors enabled the formulation of a chemically defined media that induced differentiation in the absence of serum starvation and/or transgene expression. Serum-free myogenic differentiation was of similar extent to that induced by serum starvation, as evaluated by transcriptome analysis, protein expression and the presence of a functional contractile apparatus. Moreover, the serum-free differentiation media supported the fabrication of mature three-dimensional bioartificial muscle constructs, demonstrating its suitability for cultured beef production.
Project description:Production of cultured meat requires the robust differentiation of satellite cells into mature muscle fibers without the use of animal-derived components. Current protocols induce myogenic differentiation in vitro through serum starvation, an abrupt reduction in serum concentration. Here, we used RNA sequencing to investigate the transcriptomic remodelling of bovine satellite cells during myogenic differentiation induced by serum starvation. We characterized canonical myogenic gene expression, and identified surface receptors upregulated during the early phase of differentiation. Supplementation of ligands to these receptors enabled the formulation of a chemically defined media that induced differentiation in the absence of serum starvation and/or transgene expression. Serum-free myogenic differentiation was of similar extent to that induced by serum starvation, as evaluated by transcriptome analysis, protein expression and the presence of a functional contractile apparatus. Moreover, the serum-free differentiation media supported the fabrication of mature three-dimensional bioartificial muscle constructs, demonstrating its suitability for cultured beef production.
Project description:A serum-free media formulation for cultured meat production supports bovine satellite cell differentiation in the absence of serum starvation
Project description:A serum-free media formulation for cultured meat production supports bovine satellite cell differentiation in the absence of serum starvation [SF_Diff]
Project description:A serum-free media formulation for cultured meat production supports bovine satellite cell differentiation in the absence of serum starvation [Timecourse]
Project description:Traditional differentiation of myoblasts is usually induced by serum starvation method, in which 2%HS is used to induce myoblasts to differentiate into myotubes. But, traditional serum starvation method is difficult to differentiate large yellow croaker muscle satellite cells efficiently. Although myotubes were induced within 2-3 days with medium containing 2% horse serum (HS), they detached shortly and died soon after. To improve differentiation efficiency and survival rate, different combinations of basal medium, serum ratios and myogenic factors were evaluated. Finally, the F12 medium containing 8% HS, 10 ng/ml IGF-1, 50 nM necrosulfonamide and 200 μM ascorbic acid was identified as an effective recipe for myogenic differentiation. On day 3 of culture in this differentiation medium, elongated myotubes began to appear, and on day 6, striations similar to skeletal muscle were observed in some of myotubes. But it is still inefficient, the fusion index (the proportion of nuclei in multinucleated myotubes) was 6.39% on day 3 and 1.30% on day 6 (1.30%) .Therefore, we need to find a more efficient method to induce myogenic differentiation. We then performed gene expression profiling analysis using data obtained from RNA-seq of Large yellow croaker muscle satellite cells during differentiation at three time points(day0,day3,day6).
Project description:Production of cultured meat requires defined medium formulations for the robust differentiation of myogenic cells into mature skeletal muscle fibres in vitro. Whilst such formulations can drive myogenic differentiation to an extent similar to serum-starvation based protocols, these cultures are visually heterogeneous in nature, with a significant proportion of cells not participating in myofusion, limiting the maturation of the muscle. Here, we use RNA sequencing to characterise this heterogeneity at single-nucleus resolution, identifying distinct cellular subpopulations, including proliferative cells that fail to exit the cell cycle, and 'reserve cells' that do not commit to myogenic differentiation. By targeting the ERK, RXR and NOTCH pathways, we show that cell cycle exit can be promoted whilst simultaneously abrogating reserve cell formation. Under these improved culture conditions, fusion indices close to 100% can be robustly obtained in 2D culture. We further show that this translates to higher levels of myotube formation and muscle protein accumulation in animal-free bioartificial muscle (BAM) constructs, providing proof of principle for the generation of highly differentiated cultured muscle with excellent mimicry to traditional meat.
Project description:We sought to identify microRNAs that were differentially regulated in cultured primary human aortic smooth muscle cells (SMCs) when exposed to growth arrest via serum starvation over two time points - 48 hours and 72 hours, when compared with serum-fed cells. This treatment leads to increased expression of SMC differentiation markers such as ACTA2, MYH11 and TAGLN. We identified 31 significantly regulated miRNA candidates during this process, 28 rising and 3 falling. Human aortic SMCs (AoSMCs) were propagated in growth media. Control plates were harvested, and the remaining plates were serum starved for 48 (SS48) or 72 hours (SS72) in serum-free basal media. Several arrays were excluded from final analysis after QC, resulting in a final set of 10.
Project description:This is an Affy experiment that was conducted on human derived pancreatic islet cells that were expanded in serum-containing medium to produce hIPC cells (human Islet-derived Precursor Cells). These cells, when subjected to 1 day of serum starvation, transition into hormone-expressing ICAs (Islet-like Cell Aggregates). In this experiment, proliferative hIPCs were trypsin-harvested and transferred in either serum-containing media (C) or serum-free media (F). After 24 hours, cells from both conditions were harvested and RNA was extracted. This experiment was meant to identify gene expression changes between hIPC early differentiation (condition F) and hIPC proliferation (condition C) with cells being in culture for the same amount of time. There were 3 replicates each for each of the two conditions, of which the 3rd was a pool of the first 2 replicates.
Project description:Introduction: The changing composition of non-cell autonomous circulating factors in blood as humans age is believed to play a role in muscle mass and strength loss. The mechanisms through which these circulating factors act in age-related skeletal muscle changes is not fully understood. In this study, we used human myogenic progenitors derived from human pluripotent stem cells to study non-cell autonomous roles of circulating factors during the process of myogenic differentiation. Methods: Myogenic progenitors from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) were supplemented with serum samples from aged or young Fischer 344 × Brown Norway F1-hybrid rats. The effect of aged or young serum supplementation on myogenic progenitor proliferation, myotube formation capacity, differentiation, and early transcriptomic profiles were analyzed. Results: We found that aged rat serum supplementation significantly reduced cell proliferation and increased cell death in both ESC- and iPSC-derived myogenic progenitors. Next, we found that the supplementation of aged rat serum inhibited myotube formation and maturation during terminal differentiation from progenitors to skeletal myocytes when compared to the cells treated with young adult rat serum. Lastly, we identified that gene expression profiles were affected following serum supplementation in culture. Discussion: Together, aged serum supplementation caused cellular and transcriptomic changes in human myogenic progenitors. The current data from our in vitro model possibly simulate non-cell autonomous contributions of blood composition to age-related processes in human skeletal muscle.
Project description:The study aimed to identify the differentially regulated microRNAs during serum starvation. We identified miRNAs exhibiting altered expression in asynchronous, serum starved and serum re-supplemented human cells. We identified several miRNAs that were upregulated following serum starvation and downregulated upon serum re-supplementation.