LNGFR+THY-1+VCAM-1hi+ Cells Reveal Functionally Distinct Subpopulations in Mesenchymal Stem Cells

摘要

class="head no_bottom_margin" id="sec1title">IntroductionMesenchymal stem/stromal cells (MSCs) are defined as nonhematopoietic, plastic-adherent, self-renewing cells that are capable of in vitro trilineage differentiation into fat, bone, and cartilage (). Additional plasticity of MSCs has been suggested by experiments demonstrating their in vitro differentiation into myocytes, neuron-like cells, and hepatocytes (). Despite these data, the term “MSCs” has been controversial, as a definitive demonstration of their “stemness” by single-cell isolation and in vivo serial transplantation experiments has been lacking (). These multipotent cells are found in various fetal and adult human tissues, including bone marrow (BM), umbilical cord blood (UCB), liver, and term placenta (). MSCs are multipotent and have low immunogenicity, and therefore are considered as potential candidates for a variety of clinical applications (), including cartilage reconstitution and the treatment of rheumatoid arthritis, acute osteochondral fractures, spinal disk injuries, and inherited diseases such as osteogenesis imperfecta (). However, to date, these cells have been poorly characterized, which raises significant concerns because human trials using MSCs are currently under way.MSCs can be retrospectively identified based on their ability to form colony-forming unit fibroblasts (CFU-Fs) in vitro (). Traditionally, the isolation of MSCs from unfractionated whole BM (WBM) has relied on their adherence to plastic dishes. This technique gives rise to heterogeneous cell populations that frequently are contaminated with osteoblasts and/or osteoprogenitor cells, fat cells, reticular cells, macrophages, endothelial cells, and hematopoietic cells (). Prolonged culture is often required to remove these contaminants and obtain a reasonably pure population of MSCs. However, during this process, the differentiation, proliferation, and migration potency of the MSCs gradually diminishes as the cells acquire a more mature phenotype (). In an effort to overcome these problems, investigators have made an intense effort to identify reliable MSC surface markers that could facilitate the prospective isolation of colony-initiating cells.Various surface markers, including CD49a, CD73, CD105, CD106 (VCAM-1), CD140b, CD146, CD271 (LNGFR), MSCA-1, and STRO-1, have been used alone or in combination to isolate human MSCs (hMSCs) (). CD49a, CD73, CD140b, and CD146 are widely expressed in stromal cells (e.g., pericytes and reticular cells) and thus are not unique to MSCs. STRO-1 is a popular MSC marker and is often used in combination with VCAM-1 for MSC isolation. However, these markers are also found on some hematopoietic cells, and additional markers, including CD45 and Glycophorin A (GPA), are required to exclude contaminating cells (). Therefore, the identification of a combination of cell surface markers specific to hMSCs has remained an important prerequisite for the repeated isolation of purified multipotent MSC fractions.In the present study, we performed a comprehensive screening of putative surface markers to select the most useful ones for prospectively identifying a pure MSC population in human BM. We describe a significantly improved method that enables the simple and reliable prospective isolation of MSCs based on their expression of LNGFR, THY-1, and VCAM-1.