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Unstimulated diagnostic marrow tap – a minimally invasive and reliable source for mesenchymal stem cells

Authors

  • Reddy Purushotham Koppula,

    1. CTRACER, Hyderabad Eye Research Foundation, L.V. Prasad Eye Institute, Hyderabad500034, India
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  • Naresh Polisetti,

    1. CTRACER, Hyderabad Eye Research Foundation, L.V. Prasad Eye Institute, Hyderabad500034, India
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  • Geeta K. Vemuganti

    Corresponding author
    1. CTRACER, Hyderabad Eye Research Foundation, L.V. Prasad Eye Institute, Hyderabad500034, India
    2. Ophthalmic Pathology Service, Sudhakar and Sreekanth Ravi Stem Cell Biology Laboratory, Champalimaud Translational Centre, Professor Brien Holden Eye Research Centre, Hyderabad Eye Research Foundation, L.V. Prasad Eye Institute, L.V. Prasad Marg, Banjara Hills, Hyderabad500034, India
      email geeta@lvpei.org
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email geeta@lvpei.org

Abstract

BMMNCs (bone marrow mononuclear cells) were isolated by density gradient centrifugation from unstimulated diagnostic marrow tap to propagate and characterize hBMSCs (human bone marrow stromal cells) and to explore their plasticity towards neuronal and other lineages. hBMSCs were characterized by flow cytometry for established markers, serially passaged and differentiated into adipo, osteo, chondro and neuronal lineages. Neural differentiation was analysed by RT-PCR (reverse transcriptase-PCR), ICC (immunocytochemistry) and Western blotting. The hBMSCs (n=39) were spindle-shaped and immunoreactive for mesenchymal markers such as CD71, CD106, CD105, CD90 and Vimentin and negative for haematopoietic markers such as CD11c, CD34 and CD45. These cells showed differentiation into adipocytes, osteocytes and chondrocytes. Upon neuronal differentiation, hBMSCs expressed neuronal markers, i.e. β-III tubulin, GAP43 (growth-associated proteins), neurofilament by ICC, RT-PCR and Western blotting. Our study demonstrates that minimal volumes of unstimulated diagnostic marrow tap forms a minimally invasive and reliable source for isolation of BMMNCs to establish cultures of mesenchymal stem cells and expand them. The plasticity observed in these cells towards mesenchymal (adipogenic, osteogenic and chondrocytic) and non-mesenchymal lineage (neural) substantiates the nature of mesenchymal stem cells and warrants further studies to evaluate their functional role

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