title: Monitoring live human mesenchymal stromal cell differentiation and subsequent selection using fluorescent RNA-based probes.
abstract: Investigating mesenchymal stromal cell differentiation requires time and multiple samples due to destructive endpoint assays. Osteogenesis of human bone marrow derived mesenchymal stromal cells (hBMSCs) has been widely studied for bone tissue engineering. Recent studies show that the osteogenic differentiation of hBMSCs can be assessed by quantifying the ratio of two important transcription factors (Runx2/Sox9). We demonstrate a method to observe mRNA expression of two genes in individual live cells using fluorescent probes specific for Runx2 and Sox9 mRNA. The changes of mRNA expression in cells can be observed in a non-destructive manner. In addition, the osteogenic hBMSCs can be prospectively identified and obtained based on the relative intracellular fluorescence of Sox9 in relation to Runx2 using fluorescence activated cell sorting. Relatively homogeneous cell populations with high osteogenic potential can be isolated from the original heterogeneous osteogenically induced hBMSCs within the first week of induction. This offers a more detailed analysis of the effectiveness of new therapeutics both at the individual cell level and the response of the population as a whole. By identifying and isolating differentiating cells at early time points, prospective analysis of differentiation is also possible, which will lead to a greater understanding of MSC differentiation.
authors.affiliation: AO Research Institute Davos, 7270, Davos Platz, Switzerland.
authors.forename: Bojun
authors.initials: B
authors.lastname: Li
authors.affiliation: AO Research Institute Davos, 7270, Davos Platz, Switzerland.
authors.forename: Ursula
authors.initials: U
authors.lastname: Menzel
authors.affiliation: AO Research Institute Davos, 7270, Davos Platz, Switzerland.
authors.forename: Claudia
authors.initials: C
authors.lastname: Loebel
authors.affiliation: Department of Orthopedics and Traumatology, University Medical Center, Albert-Ludwigs University Freiburg, Germany.|Department of Orthopaedics and Traumatology, Odense University Hospital and Department of Clinical Research, University of Southern Denmark, Denmark.
authors.forename: Hagen
authors.initials: H
authors.lastname: Schmal
authors.affiliation: AO Research Institute Davos, 7270, Davos Platz, Switzerland.
authors.forename: Mauro
authors.initials: M
authors.lastname: Alini
authors.affiliation: AO Research Institute Davos, 7270, Davos Platz, Switzerland.|Department of Orthopedics and Traumatology, University Medical Center, Albert-Ludwigs University Freiburg, Germany.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Stoddart
mesh_terms: D001854:Bone Marrow Cells / Q000502:physiology; D001861:Bone Regeneration; D002454:Cell Differentiation; D002469:Cell Separation; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics*; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D015675:Osteocalcin / Q000378:metabolism; D010012:Osteogenesis; D015347:RNA Probes / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D055758:SOX9 Transcription Factor / Q000235:genetics*; D059010:Single-Cell Analysis / Q000379:methods*; D023822:Tissue Engineering
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D005456:Fluorescent Dyes; D015347:RNA Probes; D012333:RNA, Messenger; C493709:RUNX2 protein, human; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; D015675:Osteocalcin