Contribution of peripheral blood mononuclear cells isolated by advanced filtration system to myogenesis of human bone marrow mesenchymal stem cells co-cultured with myoblasts.

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 PBMC transplantation procedures is characterized and used to collect PBMCs, that are then added to well-established 2D myogenic models assembled with a co-culture of bone marrow-derived mesenchymal stem cells (BM-MSCs) and skeletal myoblasts (SkMs) 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 BM-MSCs and SkMs. 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 model of BM-MSC myogenic commitment. In the presence of PBMCs, stem cells significantly upregulated myogenic genes, such as and , as confirmed by qRT-PCR and expressed related proteins by immunofluorescence (IF) assay, while downregulated pro-inflammatory cytokines ( 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.