Contribution of peripheral blood mononuclear cells isolated by advanced filtration system to myogenesis of human bone marrow mesenchymal stem cells co-cultured with myoblasts
Pasqualina Scala,
Paola Manzo,
Raffaele Longo,
Valentina Giudice,
Maria Camilla Ciardulli,
Bianca Serio,
Carmine Selleri,
Liberata Guadagno,
Laura Rehak,
Nicola Maffulli,
Giovanna Della Porta
Affiliations
Pasqualina Scala
Department of Medicine, Surgery and Dentistry, University of Salerno, Via S. Allende, 43, 84081 Baronissi SA, Italy
Paola Manzo
Hematology and Transplant Center, University Hospital “San Giovanni di Dio e Ruggi D'Aragona”, Largo Città d'Ippocrate, 1, 84131 Salerno SA, Italy
Raffaele Longo
Department of Industrial Engineering, University of Salerno, Via Giovanni Paolo II, 132, 84084 Fisciano SA, Italy
Valentina Giudice
Department of Medicine, Surgery and Dentistry, University of Salerno, Via S. Allende, 43, 84081 Baronissi SA, Italy; Hematology and Transplant Center, University Hospital “San Giovanni di Dio e Ruggi D'Aragona”, Largo Città d'Ippocrate, 1, 84131 Salerno SA, Italy
Maria Camilla Ciardulli
Department of Medicine, Surgery and Dentistry, University of Salerno, Via S. Allende, 43, 84081 Baronissi SA, Italy
Bianca Serio
Hematology and Transplant Center, University Hospital “San Giovanni di Dio e Ruggi D'Aragona”, Largo Città d'Ippocrate, 1, 84131 Salerno SA, Italy
Carmine Selleri
Department of Medicine, Surgery and Dentistry, University of Salerno, Via S. Allende, 43, 84081 Baronissi SA, Italy; Hematology and Transplant Center, University Hospital “San Giovanni di Dio e Ruggi D'Aragona”, Largo Città d'Ippocrate, 1, 84131 Salerno SA, Italy
Liberata Guadagno
Department of Industrial Engineering, University of Salerno, Via Giovanni Paolo II, 132, 84084 Fisciano SA, Italy
Laura Rehak
Athena Biomedical Innovations, Viale Europa 139, Florence, 50126, Italy
Nicola Maffulli
Department of Medicine, Surgery and Dentistry, University of Salerno, Via S. Allende, 43, 84081 Baronissi SA, Italy; Centre for Sports and Exercise Medicine, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, 275 Bancroft Road, London E1 4DG, UK
Giovanna Della Porta
Department of Medicine, Surgery and Dentistry, University of Salerno, Via S. Allende, 43, 84081 Baronissi SA, Italy; Interdepartment Centre BIONAM, Università di Salerno, via Giovanni Paolo II, 132, 84084 Fisciano SA, Italy; Corresponding author. Department of Medicine, Surgery and Dentistry, University of Salerno, Via S. Allende, Baronissi SA 84084, Italy. ;
Background: Contribution of peripheral blood mononuclear cells (PBMCs) in myogenesis is still under debate, even though blood filtration systems are commonly used in clinical practice for successfully management of critic limb ischemia. Objectives: A commercial blood filter used for autologous human PBMC transplantation procedures is characterized and used to collect PBMCs, that are then added to well-established 2D in vitro myogenic models assembled with a co-culture of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and skeletal myoblasts (hSkMs) whit the aim of investigating their potential contribution to stem cell myogenic commitment. Methods: A commercial blood filter was physically and chemically studied to understand its morphological characteristics and composition. PBMCs were concentrated using this system, further isolated by Ficoll-Paque density gradient centrifugation, and then added in an upper transwell chamber to a 2D co-culture of hBM-MSCs and hSkMs. Myogenic commitment was investigated by RT-PCR, immunofluorescence, and flow cytometry immunophenotyping. Cytokine levels were monitored by ELISA assay in culture media. Results: The blood filtration system was disassembled and appeared to be formed by twelve membranes of poly-butylene terephthalate fibers (diameters, 0.9–4.0 μm) with pore size distribution of 1–20 μm. Filter functional characterization was achieved by characterizing collected cells by flow cytometry. Subsequently, collected PBMCs fraction was added to an in-vitro model of hBM-MSC myogenic commitment. In the presence of PBMCs, stem cells significantly upregulated myogenic genes, such as Desmin and MYH2, as confirmed by qRT-PCR and expressed related proteins by immunofluorescence (IF) assay, while downregulated pro-inflammatory cytokines (IL12A at day 14) along the 21 days of culture. Novelty: Our work highlights chemical-physical properties of commercial blood filter and suggests that blood filtrated fraction of PBMC might modulate cytokine expression in response to muscle injury and promote myogenic events, supporting their clinical use in autologous transplantation.
