title: Generating Rho-0 Cells Using Mesenchymal Stem Cell Lines.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.affiliation: Servicio de Reumatología, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, Universidade da Coruña (UDC), A Coruña, Spain.
authors.forename: Mercedes
authors.initials: M
authors.lastname: Fernández-Moreno
authors.affiliation: Servicio de Reumatología, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, Universidade da Coruña (UDC), A Coruña, Spain.
authors.forename: Tamara
authors.initials: T
authors.lastname: Hermida-Gómez
authors.affiliation: Departamento de Bioquímica, Instituto de Investigaciones Biomédicas "Alberto Sols" UAM-CSIC, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain.
authors.forename: M Esther
authors.initials: ME
authors.lastname: Gallardo
authors.affiliation: Servicio de Reumatología, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, Universidade da Coruña (UDC), A Coruña, Spain.
authors.forename: Andrea
authors.initials: A
authors.lastname: Dalmao-Fernández
authors.affiliation: Servicio de Reumatología, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, Universidade da Coruña (UDC), A Coruña, Spain.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Rego-Pérez
authors.affiliation: Departamento de Bioquímica, Instituto de Investigaciones Biomédicas "Alberto Sols" UAM-CSIC, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain.
authors.forename: Rafael
authors.initials: R
authors.lastname: Garesse
authors.affiliation: Servicio de Reumatología, Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas, Universidade da Coruña (UDC), A Coruña, Spain.
authors.forename: Francisco J
authors.identifier: http://orcid.org/0000-0001-9821-7635
authors.initials: FJ
authors.lastname: Blanco
mesh_terms: D017209:Apoptosis; D002454:Cell Differentiation; D002460:Cell Line; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D053078:Membrane Potential, Mitochondrial; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D010641:Phenotype; D017382:Reactive Oxygen Species / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D004272:DNA, Mitochondrial; D017382:Reactive Oxygen Species; D004247:DNA