The generation of Rho-0 cells requires the use of an immortalization process, or tumor cell selection, followed by culture in the presence of ethidium bromide (EtBr), incurring the drawbacks its use entails. The purpose of this work was to generate Rho-0 cells using human mesenchymal stem cells (hMSCs) with reagents having the ability to remove mitochondrial DNA (mtDNA) more safely than by using EtBr. Two immortalized hMSC lines (3a6 and KP) were used; 143B.TK-Rho-0 cells were used as reference control. For generation of Rho-0 hMSCs, cells were cultured in medium supplemented with each tested reagent. Total DNA was isolated and mtDNA content was measured by real-time polymerase chain reaction (PCR). Phenotypic characterization and gene expression assays were performed to determine whether 3a6 Rho-0 hMSCs maintain the same stem properties as untreated 3a6 hMSCs. To evaluate whether 3a6 Rho-0 hMSCs had a phenotype similar to that of 143B.TK-Rho-0 cells, in terms of reactive oxygen species (ROS) production, apoptotic levels and mitochondrial membrane potential (Δψm) were measured by flow cytometry and mitochondrial respiration was evaluated using a SeaHorse XFp Extracellular Flux Analyzer. The differentiation capacity of 3a6 and 3a6 Rho-0 hMSCs was evaluated using real-time PCR, comparing the relative expression of genes involved in osteogenesis, adipogenesis and chondrogenesis. The results showed the capacity of the 3a6 cell line to deplete its mtDNA and to survive in culture with uridine. Of all tested drugs, Stavudine (dt4) was the most effective in producing 3a6-Rho cells. The data indicate that hMSC Rho-0 cells continue to express the characteristic MSC cell surface receptor pattern. Phenotypic characterization showed that 3a6 Rho-0 cells resembled 143B.TK-Rho-0 cells, indicating that hMSC Rho-0 cells are Rho-0 cells. While the adipogenic capability was higher in 3a6 Rho-0 cells than in 3a6 cells, the osteogenic and chondrogenic capacities were lower. Among the drugs and conditions tested, the use of d4t was the best option for producing Rho-0 cells from hMSCs. Rho-0 cells are useful for studying the role of mitochondria in hMSC differentiation.

Authors have demonstrated that apoptosis activation is a pathway related to cartilage degradation characteristics of the OA process. Autophagy is an adaptive response to protect cells from various environmental changes, and defects in autophagy are linked to cell death. In this sense, decreased autophagy of chondrocytes has been observed in OA articular cartilage. The aim of this work was to study the role of OA mitochondria in apoptosis, autophagy, and senescence, using OA and Normal (N) transmitochondrial cybrids. Results: OA cybrids incubated with menadione showed a higher percentage of late apoptosis and necrosis than N cybrids. Stimulation of cybrids with staurosporine and IL-1β showed that OA cybrids were more susceptible to undergoing apoptosis than N cybrids. An analysis of the antioxidant response using menadione on gene expression revealed a lower expression of nuclear factor erythroid 2-like 2 and superoxide dismutase 2 in OA than N cybrids. Activation of microtubule-associated protein 1A/1B-light chain 3 was reduced in OA compared to N cybrids. However, the percentage of senescent cells was higher in OA than N cybrids. Conclusion: This work suggests that mitochondria from OA patients could be involved in the apoptosis, autophagy, and senescence of chondrocytes described in OA cartilage.

Osteoarthritis (OA) is the most frequent joint disease; however, the etiopathogenesis is still unclear. Chondrocytes rely primarily on glycolysis to meet cellular energy demand, but studies implicate impaired mitochondrial function in OA pathogenesis. The relationship between mitochondrial dysfunction and OA has been established. The aim of the study was to examine the differences in glucose and Fatty Acids (FA) metabolism, especially with regards to metabolic flexibility, in cybrids from healthy (N) or OA donors. Glucose and FA metabolism were studied using D-[¹⁴C(U)]glucose and [1-¹⁴C]oleic acid, respectively. There were no differences in glucose metabolism among the cybrids. Osteoarthritis cybrids had lower acid-soluble metabolites, reflecting incomplete FA β-oxidation but higher incorporation of oleic acid into triacylglycerol. Co-incubation with glucose and oleic acid showed that N but not OA cybrids increased their glucose metabolism. When treating with the mitochondrial inhibitor etomoxir, N cybrids still maintained higher glucose oxidation. Furthermore, OA cybrids had higher oxidative stress response. Combined, this indicated that N cybrids had higher metabolic flexibility than OA cybrids. Healthy donors maintained the glycolytic phenotype, whereas OA donors showed a preference towards oleic acid metabolism. Interestingly, the results indicated that cybrids from OA patients had mitochondrial impairments and reduced metabolic flexibility compared to N cybrids.

Introduction: Skeletal muscle is a major contributor to whole-body energy homeostasis and the utilization of fatty acids and glucose. At present, 2D cell models have been the most used cellular models to study skeletal muscle energy metabolism. However, the transferability of the results to in vivo might be limited. This project aimed to develop and characterize a skeletal muscle 3D cell model (myospheres) as an easy and low-cost tool to study molecular mechanisms of energy metabolism.Methods and results: We demonstrated that human primary myoblasts form myospheres without external matrix support and carry structural and molecular characteristics of mature skeletal muscle after 10 days of differentiation. We found significant metabolic differences between the 2D myotubes model and myospheres. In particular, myospheres showed increased lipid oxidative metabolism than the 2D myotubes model, which oxidized relatively more glucose and accumulated more oleic acid.Discussion and conclusion: These analyses demonstrate model differences that can have an impact and should be taken into consideration for studying energy metabolism and metabolic disorders in skeletal muscle.

The interplay between skeletal muscle and bone is primarily mechanical; however, biochemical crosstalk by secreted mediators has recently gained increased attention. The aim of this study was to investigate metabolic effects of conditioned medium from osteoblasts (OB-CM) on myotubes and vice versa. Human skeletal muscle cells incubated with OB-CM showed increased glucose uptake and oxidation, and mRNA expression of the glucose transporter (GLUT) 1, while fatty acid uptake and oxidation, and mRNA expression of the fatty acid transporter CD36 were decreased. This was supported by proteomic analysis, where expression of proteins involved in glucose uptake, glycolytic pathways, and the TCA cycle were enhanced, and expression of several proteins involved in fatty acid metabolism were reduced. Similar effects on energy metabolism were observed in human bone marrow stromal cells differentiated to osteoblastic cells incubated with conditioned medium from myotubes (SKM-CM), with increased glucose uptake and reduced oleic acid uptake. Proteomic analyses of the two conditioned media revealed many common proteins. Thus, our data may indicate a shift in fuel preference from fatty acid to glucose metabolism in both cell types, induced by conditioned media from the opposite cell type, possibly indicating a more general pattern in communication between these tissues.

Frontiers' correction template for authors A correction refers to a change to their article that the author wishes to publish after publication. The publication of this article is subject to Frontiers' editorial approval. Instructions: ● Please read through all the templates before choosing ● Pick the most relevant text template(s) from the following page and delete all others. ● Edit the text as necessary, ensuring that the original incorrect text is included for the record, please see the below. ● Please do not use any extra formatting when editing the templates, and only modify the red text unless absolutely necessary ● Submit to Frontiers following the instructions on this page. When the original text contained incorrect information, to preserve the scientific record, please include that text when editing the below templates. For example: There was a mistake in the Funding statement, an incorrect number was used. The correct number is "2015C03Bd051.". The publisher apologizes for this mistake. The original version of this article has been updated. In the published article, there was a mistake in the Funding statement. The funding statement for the Key Development Project of the Department of Science and Technology was displayed as "2015CBd051". The correct statement is "Key Development Project of Department of Science and Technology (2015C03Bd051).'' Correction: Development of three-dimensional primary human myospheres as culture model of skeletal muscle cells for metabolic studies Correction on: Dalmao-Fernandez A, Aizenshtadt A, Bakke HG, Krauss S, Rustan AC, Thoresen GH, Kase ET. Development of three-dimensional primary human myospheres as culture model of skeletal muscle cells for metabolic studies. Front Bioeng Biotechnol. 2023 Mar 23;11:1130693. doi: 10.3389/fbioe.2023.1130693. PMID: 37034250; PMCID: PMC10076718. Error in figure/table Wrong content There was a mistake in figure [1] as published. One of the gene names is misspelled: solute carrier family 26 member 4 (SLC26A4), the correct form is: solute carrier family 2 member 4 (SLC2A4). The corrected figure [1] appears below. "Evaluation of 3D morphological parameters and comparison of muscle cell differentiation markers in 2D and 3D models. Myospheres were formed in the ultra-low attachment treatment (ULA) 96-well plate system and differentiation was carried out between 0 and 21 days. After 10 days of differentiation, mRNA was isolated, and gene expression was analyzed by qPCR. (A) Phase-contrast photos of myospheres during 3, 5, 10 and 12 of differentiation. Area (B) and diameter (C) were analyzed by AnaSP for up to 21 days of differentiation. (D) mRNA expression of the muscle differentiation markers MYOD, MYOG, MYH2, MYH7, and SLC2A4 before (day 0) and after differentiation (day 10), normalized to housekeeping gene (RPLP0). Scale bar = 200 µm. Results are presented as mean ± SEM. *p < 0.05 **p < 0.01 ****p < 0.0001 by ordinary one-way ANOVA test." "The process of muscle differentiation is regulated through different phases which stimulate myoblasts into fusion and maturation to become myotubes (Schmidt et al., 2019; Isesele and Mazurak, 2021). The differentiation process is initiated by an increased expression of myogenic differentiation 1 (MYOD) which induces gene expression of myogenin (MYOG) and subsequent expression of differentiation markers such as the myosin-heavy chains 2 (MYH2) and 7 (MYH7), and maturation factors related to metabolic muscle function like the insulin-regulated facilitative glucose transporter, solute carrier family 2 member 4 (SLC2A4). Comparison of expression of MYOD, MYH2, MYH7, and SLC2A4 revealed higher mRNA expression levels in 3D than 2D myotube models (Figure 1C). This relevant finding demonstrated a higher efficiency of cell maturation during differentiation in 3D than in 2D myotube models. In both cell models, mRNA expression levels of MYOD, MYOG, MYH2, MYH7, and SLC2A4 tended to increase after 10 days of differentiation (Figure 1C)".