More Like this

1. Differentiation-dependent chromosomal organization changes in normal myogenic cells are absent in rhabdomyosarcoma cells

   https://doi.org/10.3389/fcell.2023.1293891  

   Ibarra, Joe; Hershenhouse, Tyler; Almassalha, Luay; Walterhouse, David; Backman, Vadim; MacQuarrie, Kyle L (November 2023, Frontiers in Cell and Developmental Biology)

   Myogenesis, the progression of proliferating skeletal myoblasts to terminally differentiated myotubes, regulates thousands of target genes. Uninterrupted linear arrays of such genes are differentially associated with specific chromosomes, suggesting chromosome specific regulatory roles in myogenesis. Rhabdomyosarcoma (RMS), a tumor of skeletal muscle, shares common features with normal muscle cells. We hypothesized that RMS and myogenic cells possess differences in chromosomal organization related to myogenic gene arrangement. We compared the organizational characteristics of chromosomes 2 and 18, chosen for their difference in myogenic gene arrangement, in cultured RMS cell lines and normal myoblasts and myotubes. We found chromosome-specific differences in organization during normal myogenesis, with increased area occupied and a shift in peripheral localization specifically for chromosome 2. Most strikingly, we found a differentiation-dependent difference in positioning of chromosome 2 relative to the nuclear axis, with preferential positioning along the major nuclear axis present only in myotubes. RMS cells demonstrated no preference for such axial positioning, but induced differentiation through transfection of the pro-myogenic miRNA miR-206 resulted in an increase of major axial positioning of chromosome 2. Our findings identify both a differentiation-dependent, chromosome-specific change in organization in normal myogenesis, and highlight the role of chromosomal spatial organization in myogenic differentiation. 

   more » « less
2. Edible mycelium as proliferation and differentiation support for anchorage-dependent animal cells in cultivated meat production

   https://doi.org/10.1038/s41538-024-00263-0  

   Ogawa, Minami; Kermani, Alex S; Huynh, Mayrene J; Baar, Keith; Leach, J Kent; Block, David E (December 2024, npj Science of Food)

   Abstract Cultivated meat production requires bioprocess optimization to achieve cell densities that are multiple orders of magnitude higher compared to conventional cell culture techniques. These processes must maximize resource efficiency and cost-effectiveness by attaining high cell growth productivity per unit of medium. Microcarriers, or carriers, are compatible with large-scale bioreactor use, and offer a large surface-area-to-volume ratio for the adhesion and proliferation of anchorage-dependent animal cells. An ongoing challenge persists in the efficient retrieval of cells from the carriers, with conflicting reports on the effectiveness of trypsinization and the need for additional optimization measures such as carrier sieving. To surmount this issue, edible carriers have been proposed, offering the advantage of integration into the final food product while providing opportunities for texture, flavor, and nutritional incorporation. Recently, a proof of concept (POC) utilizing inactivated mycelium biomass derived from edible filamentous fungus demonstrated its potential as a support structure for myoblasts. However, this POC relied on a model mammalian cell line combination with a single mycelium species, limiting realistic applicability to cultivated meat production. This study aims to advance the POC. We found that the species of fungi composing the carriers impacts C2C12 myoblast cell attachment—with carriers derived fromAspergillus oryzaepromoting the best proliferation. C2C12 myoblasts effectively differentiated on mycelium carriers when induced in myogenic differentiation media. Mycelium carriers also supported proliferation and differentiation of bovine satellite cells. These findings demonstrate the potential of edible mycelium carrier technology to be readily adapted in product development within the cultivated meat industry. 

   more » « less
3. Cysteine Rich Intestinal Protein 2 is a copper-responsive regulator of skeletal muscle differentiation and metal homeostasis

   https://doi.org/10.1371/journal.pgen.1011495  

   Verdejo-Torres, Odette; Klein, David C; Novoa-Aponte, Lorena; Carrazco-Carrillo, Jaime; Bonilla-Pinto, Denzel; Rivera, Antonio; Bakhshian, Arpie; Fitisemanu, Fa’alataitaua M; Jiménez-González, Martha L; Flinn, Lyra; et al (December 2024, PLOS Genetics)

   Bhagwat, Ashok (Ed.)

   Copper (Cu) is essential for respiration, neurotransmitter synthesis, oxidative stress response, and transcription regulation, with imbalances leading to neurological, cognitive, and muscular disorders. Here we show the role of a novel Cu-binding protein (Cu-BP) in mammalian transcriptional regulation, specifically on skeletal muscle differentiation using murine primary myoblasts. Utilizing synchrotron X-ray fluorescence-mass spectrometry, we identified murine cysteine-rich intestinal protein 2 (mCrip2) as a key Cu-BP abundant in both nuclear and cytosolic fractions. mCrip2 binds two to four Cu⁺ions with high affinity and presents limited redox potential. CRISPR/Cas9-mediated deletion ofmCrip2impaired myogenesis, likely due to Cu accumulation in cells. CUT&RUN and transcriptome analyses revealed its association with gene promoters, includingMyoD1andmetallothioneins, suggesting a novel Cu-responsive regulatory role for mCrip2. Our work describes the significance of mCrip2 in skeletal muscle differentiation and metal homeostasis, expanding understanding of the Cu-network in myoblasts. Copper (Cu) is essential for various cellular processes, including respiration and stress response, but imbalances can cause serious health issues. This study reveals a new Cu-binding protein (Cu-BP) involved in muscle development in primary myoblasts. Using unbiased metalloproteomic techniques and high throughput sequencing, we identified mCrip2 as a key Cu-BP found in cell nuclei and cytoplasm. mCrip2 binds up to four Cu⁺ions and has a limited redox potential. Deleting mCrip2 using CRISPR/Cas9 disrupted muscle formation due to Cu accumulation. Further analyses showed that mCrip2 regulates the expression of genes like MyoD1, essential for muscle differentiation, and metallothioneins in response to copper supplementation. This research highlights the importance of mCrip2 in muscle development and metal homeostasis, providing new insights into the Cu-network in cells. 

   more » « less
4. RBFOX2 regulated EYA3 isoforms partner with SIX4 or ZBTB1 to control transcription during myogenesis

   https://doi.org/10.1016/j.isci.2023.108258  

   Wiedner, Hannah J; Blue, R Eric; Sadovsky, Matheus; Mills, C Allie; Wehrens, Xander T; Herring, Laura E; Giudice, Jimena (November 2023, iScience)

   Alternative splicing is a prevalent gene-regulatory mechanism, with over 95% of multi-exon human genes estimated to be alternatively spliced. Here, we describe a tissue-specific, developmentally regulated, highly conserved, and disease-associated alternative splicing event in exon 7 of the eyes absent homolog 3 (Eya3) gene. We discovered that EYA3 expression is vital to the proliferation and differentiation of myoblasts. Genome-wide transcriptomic analysis and mass spectrometry-based proteomic studies identified SIX homeobox 4 (SIX4) and zinc finger and BTB-domain containing 1 (ZBTB1), as major transcription factors that interact with EYA3 to dictate gene expression. EYA3 isoforms differentially regulate transcription, indicating that splicing aids in temporal control of gene expression during muscle cell differentiation. Finally, we identified RNA-binding fox-1 homolog 2 (RBFOX2) as the main regulator of EYA3 splicing. Together, our findings illustrate the interplay between alternative splicing and transcription during myogenesis. 

   more » « less
5. 4D anisotropic skeletal muscle tissue constructs fabricated by staircase effect strategy

   https://doi.org/10.1088/1758-5090/ab1d07  

   Miao, Shida; Nowicki, Margaret; Cui, Haitao; Lee, Se-Jun; Zhou, Xuan; Mills, David K; Zhang, Lijie Grace (January 2019, Biofabrication)

   Like the morphology of native tissue fiber arrangement (such as skeletal muscle), unidirectional anisotropic scaffolds are highly desired as a means to guide cell behavior in anisotropic tissue engineering. In contrast, contour-like staircases exhibit directional topographical cues and are judged as an inevitable defect of fused deposition modeling (FDM). In this study, we will translate this staircase defect into an effective bioengineering strategy by integrating FDM with surface coating technique (FCT) to investigate the effect of topographical cues on regulating behaviors of human mesenchymal stem cells (hMSCs) toward skeletal muscle tissues. This integrated approach serves to fabricate shape-specific, multiple dimensional, anisotropic scaffolds using different biomaterials. 2D anisotropic scaffolds, first demonstrated with different polycaprolactone concentrations herein, efficiently direct hMSC alignment, especially when the scaffold is immobilized on a support ring. By surface coating the polymer solution inside FDM-printed sacrificial structures, 3D anisotropic scaffolds with thin wall features are developed and used to regulate seeded hMSCs through a self-established rotating bioreactor. Using layer-by-layer coating, along with a shape memory polymer, smart constructs exhibiting shape fix and recovery processes are prepared, bringing this study into the realm of 4D printing. Immunofluorescence staining and real-time quantitative polymerase chain reaction analysis confirm that the topographical cues created via FCT significantly enhance the expression of myogenic genes, including myoblast differentiation protein-1, desmin, and myosin heavy chain-2. We conclude that there are broad application potentials for this FCT strategy in tissue engineering as many tissues and organs, including skeletal muscle, possess highly organized and anisotropic extracellular matrix components. 

   more » « less