Title:
Identification and isolation of pure mesenchymal stem cells by flowcytometry

Yumi Matsuzaki
Associate Professor
Center for integrated Medical Research, Keio University , School of Medicine

Abstract:
Mesenchymal stem/stromal cells (MSCs) are self-renewing cells with the ability to differentiate into osteocytes, chondrocytes and adipocytes, but also neuron-like cells . These multipotent cells are found in various adult and fetal tissues including bone marrow, umbilical cord blood, liver, dental pulp, and term placenta. Because of their multipotency and low immunogenicity, MSCs are considered a potential candidate for clinical applications including cartilage reconstitution , and the therapy of inherited diseases such as osteogenesis imperfecta.

MSC can be identified by their ability to form colony forming units-fibroblast (CFU-F) in vitro . Traditionally, the isolation of MSCs from unfractionated bone marrow relies on their adherence to plastic. This technique gives riseto heterogeneous MSC populations, frequently contaminated with other cell populations includingosteoblasts and/or osteoprogenitor cells, fat cells, reticularcells, macrophages, endothelial cells and haematopoietic cells. Prolonged culture is often required to remove contaminants and obtain a reasonably pure population of self-renewing and multipotent MSCs. Unfortunately,during this process, the plasticity and proliferative ability of CFU-Fs gradually diminishes, as these cells differentiate to a more mature phenotype.

In an effort to over come these problems, there has been intense effort to identify reliable MSC surface markers that will facilitate the prospective isolation of a pure population of cells. We perform a comprehensive screen of MSC markers in order to select those that are most predictive of a pure multipotent MSCs population in bone marrow. We detail for the first time an innovative method that allows prospective isolation of MSCs from human BM based on the expression of LNGFR and Thy-1.The MSCs from this isolation were significantly more potent with respect to growth and differentiation compared to traditionally isolated MSCs.