A Simple Method for Isolation, Propagation, Characterization, and Differentiation of Adult Mouse Bone Marrow-Derived Multipotent Mesenchymal Stem Cells

Abstract

Mesenchymal stem cells (MSCs) are spindle-shaped, adherent, clonogenic, non-phagocytic, fibroblastic and multipotent in nature with the intrinsic ability of self-renewal and proliferation. Bone marrow is the richest source of MSCs for studying the underlying processes of proliferation, self-renewal, and multiple-lineage differentiation. There have been several studies to improve the method of isolation, propagation, characterization and differentiation of mesenchymal stem cells from the mouse bone marrow, but none are widely acceptable. Owing to unavailability of universally acceptable method of MSCs culture continuous efforts are being made in this direction. Here, we report a simple method with some subtle modifications aiming to improve the overall method of isolation, culture, propagation and differentiation of MSCs in vitro. Following this protocol, we have isolated MSCs with typical spindle-shaped morphology as shown by scanning electron microscopy. These cells also showed expression of MSC-specific markers, CD29 (98.94% ± 0.67%), CD44 (84.27% ± 7.77%), Sca-1 (92.70% ± 3.81%) with negligible expression of HSC-specific markers such as CD45 (0.40% ± 0.10%), CD34 (0.15% ± 0.05%) and CD11b (0.45% ± 0.15%). MSCs were also found to differentiate into mesodermal lineages such as adipocytes, osteocytes and chondrocytes as well as ectodermal neuron-like cells. Moreover, MSCs showed differential basal expression of pluripotency-associated transcription factors such as Oct4, Nanog, Sox2 and Myc. Based on the above findings, we propose a simple protocol that can be used to isolate, culture, propagate and characterize multipotent MSCs from mouse bone marrow for experimental and application purposes.