title: Priming 3D cultures of human mesenchymal stromal cells toward cartilage formation via developmental pathways.
abstract: The field of regenerative medicine has increasingly recognized the importance to be inspired by developmental processes to identify signaling pathways crucial for 3D organogenesis and tissue regeneration. Here, we aimed at recapitulating the first events occurring during limb development (ie, cell condensation and expansion of an undifferentiated mesenchymal cell population) to prime 3D cultures of human bone marrow-derived mesenchymal stromal/stem cells (hBM-MSC) toward the chondrogenic route. Based on embryonic development studies, we hypothesized that Wnt3a and fibroblast growth factor 2 (FGF2) induce hBM-MSC to proliferate in 3D culture as an undifferentiated pool of progenitors (defined by clonogenic capacity and expression of typical markers), retaining chondrogenic potential upon induction by suitable morphogens. hBM-MSC were responsive to Wnt signaling in 3D pellet culture, as assessed by significant upregulation of main target genes and increase of unphosphorylated β-catenin levels. Wnt3a was able to induce a five-fold increase in the number of proliferating hBM-MSC (6.4% vs. 1.3% in the vehicle condition), although total DNA content of the 3D construct was decreasing over time. Preconditioning with Wnt3a improved transforming growth factor-β1 mediated chondrogenesis (30% more glycosaminoglycans/cell in average). In contrast to developmental and 2D MSC culture models, FGF2 antagonized the Wnt-mediated effects. Interestingly, the CD146⁺ subpopulation was found to be more responsive to Wnt3a. The presented data indicate a possible strategy to prime 3D cultures of hBM-MSC by invoking a "developmental engineering" approach. The study also identifies some opportunities and challenges to cross-fertilize skeletal development models and 3D hBM-MSC culture systems.
authors.affiliation: Departments of Surgery and Biomedicine, University Hospital Basel, University of Basel , Basel, Switzerland .
authors.forename: Matteo
authors.initials: M
authors.lastname: Centola
authors.forename: Beatrice
authors.initials: B
authors.lastname: Tonnarelli
authors.forename: Stefan
authors.initials: S
authors.lastname: Schären
authors.forename: Nicolas
authors.initials: N
authors.lastname: Glaser
authors.forename: Andrea
authors.initials: A
authors.lastname: Barbero
authors.forename: Ivan
authors.initials: I
authors.lastname: Martin
mesh_terms: D000328:Adult; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D002356:Cartilage / Q000166:cytology / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism; D020219:Chondrogenesis / Q000187:drug effects / Q000235:genetics; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D060449:Wnt Signaling Pathway / Q000187:drug effects / Q000235:genetics; D060509:Wnt3A Protein / Q000494:pharmacology
chemical_list: D060509:Wnt3A Protein; D016222:Fibroblast Growth Factor 2