Abstract

Adult mammalian bone marrow (BM) contains at least two distinct stem cell populations; the stem cells of the hemopoietic lineage and a second population termed mesenchymal stem cells (MSC) whose function is to maintain the non-hemopoietic BM elements as well as skeletal homeostasis. MSC have been implicated as potential targets in a range of cellular therapies for treatment of defects of both the hemopoietic and skeletal systems, and as vehicles for gene therapy. In order to evaluate the potential of these cells in various therapies, a pre-clinical animal model in which both the biology and potential therapeutic applications of these cells can be assessed is of fundamental importance. The goal of the current study was to develop a robust and reproducible method for the isolation of MSC from murine hemopoietic tissues. Tibiae and femurs harvested from C57BL6/J mice were gently crushed with a pestle to release the marrow. The bone fragments were subsequently cut into small pieces with a scalpel and digested in a solution containing 3mg/ml Type I collagenase to yield a population of compact bone (CB) derived cells. Mesenchymal progenitor cells (MPC) were detected using an in vitro assay for fibroblast-colony forming cells (CFU-F). CB cells were plated at 1000 or 5000 cells per cm2 in complete MesenCult™ medium for 12 days, after which CFU-F-derived colonies were enumerated. We show that CFU-F are present at a significantly higher frequency in mouse CB than in the BM (433±225 vs 11.7±3.5 colonies/106 cells respectively, n=3). Based on these data we developed a simple and robust immunomagnetic selection method to highly enrich MPC from mouse CB by depleting essentially all nucleated hemopoietic cells (CD45+) and red blood cells (Ter119+) using magnetic particles and antibodies to CD45 and Ter119, respectively. Target CD45Ter119 cells initially comprised 1.1±0.5% (n=9) of the total CB fraction as assayed by FACS. Following depletion, CD45Ter119 cells comprised 74.5±16% (n=6) of the cells and were enriched 205 fold for CFU-F compared to the starting population, with a CFU-F frequency of 1 per 11 cells plated, and a total CFU-F recovery of 57.9 ± 18.5%. Analysis of CD45Ter119Sca-1+ cells, a phenotype previously shown to enrich for MPC, revealed that these cells were enriched 50 fold following depletion, from 0.53±0.5 to 26.5±8.23% (n=3). The enriched MPCs cultured at low O2 tension were devoid of hemopoietic contaminants at passage 1 and 2 as shown by lack of CD45, Ter119 and CD11b expression. The cultured CB-derived MPCs were capable of extensive in vitro proliferation and maintained the ability to differentiate into cells of the osteogenic, adipogenic and chondrogenic lineages. Furthermore, irradiated cultured mesenchymal cells supported long-term culture-initiating cells (LTC-IC) in 4-week cultures of Sca-1+ BM cells under limiting dilution conditions, at frequencies similar to those detected using irradiated primary BM feeders (i.e. 1 per 1600). These data provide a rapid, reproducible method by which multipotent mesenchymal cells devoid of contaminating hemopoietic cells can be readily obtained from limited numbers of mice to study the biology of MSC as well as the use of these cells as therapeutic agents in a preclinical animal model.

Author notes

Disclosure:Employment: Allen C. Eaves is president and CEO of StemCell Technologies Inc.