Prospective isolation of mesenchymal stem cells from mouse compact bone.

Bone marrow from numerous species, including rodents and man, has been shown to contain a rare population of cells known as marrow stromal cells or mesenchymal stem cells (MSC). Given the innate ability of these cells to give rise to multiple tissue types including bone, fat and cartilage, there is considerable interest in utilizing MSC in a broad repertoire of cell-based therapies for the treatment of human disease. In order for such therapies to be realized, a preclinical animal model in which to refine strategies utilizing MSC is required.We have described methodology allowing for the prospective isolation by fluorescence activated cell sorting (FACS) of a highly purified population of MSC from murine compact bone (CB). These cells are multipotent and capable of extensive proliferation in vitro and thus represent an ideal source of cells with which to explore both the fundamental biology of MSC and their efficacy in a variety of cellular therapies.

Mesenchymal stem cells.

It has become clear that adult mammalian bone marrow contains not one but two ostensibly discrete populations of adult stem cells. The first and by far the most fully characterized are the hematopoietic stem cells responsible for maintaining lifelong production of blood cells. The biological characteristics and properties of the second marrow resident population of stem cells, variously termed bone marrow stromal cells or mesenchymal stem cells, are in contrast much less well understood. In vitro, cultures established from single-cell suspensions of bone marrow from a wide range of mammalian species generate colonies of adherent marrow stromal cells, each derived from a single precursor cell termed a colony-forming unit-fibroblast (CFU-F). Culture conditions have been developed to expand marrow stromal cells in vitro while maintaining the capacity of these cells to differentiate into bone, fat, and cartilage. A significant portion of our current knowledge of this population of cells is based on analysis of the properties of these culture expanded cells, not on the primary colony-initiating cells. In this article, we will focus on methodologies to prospectively isolate stromal progenitors from mouse and human bone marrow and will review current data that suggest stromal progenitors in the bone marrow in situ are associated with the outer surfaces of blood vessels and may share identity with vascular pericytes.

Isolation, enumeration, and expansion of human mesenchymal stem cells in culture.

Human bone marrow (BM) contains a population of non-hematopoietic stem cells also termed stromal cells, mesenchymal cells or multipotent mesenchymal stromal cells (MSCs). These cells have unique stem cell-like properties including their ability to self-renew, differentiate into multiple tissue types, and modulate immune cell responses through paracrine effects. These properties have positioned mesenchymal cells as biological agents in clinical trials for various diseases since the 1990s. Mesenchymal cells have been isolated from various tissues and cultured using various media and methods resulting in a lack of standardization in culture methods for these cells. Consequently, cells cultured in different laboratories exhibit different characteristics of MSC-like cells. This chapter outlines protocols for optimal isolation, enumeration, and expansion of human MSCs from BM in fetal bovine serum (FBS)-containing medium, as well as in xeno-free medium.

Isolation and culture of mesenchymal stem cells from mouse compact bone.

The bone marrow (BM) of numerous species, including rodents and man, contains a rare population of cells termed marrow stromal cells or mesenchymal stem cells (MSC). Given the ability of these cells to differentiate into cells of the osteogenic, chondrogenic and adipogenic lineages, there is considerable interest in utilizing MSCs in a broad repertoire of cell-based therapies for the treatment of human disease. Before such potential therapies can be realized, a preclinical animal model in which to test and refine strategies utilizing MSC is required. Here we describe methods for the isolation of a highly enriched population of MSC from mouse cortical/compact bone (CB), quantitation using the colony forming unit-fibroblast assay (CFU-F) and in vitro expansion. These cells are both multipotent and capable of extensive in vitro expansion and thus represent an ideal cellular source to explore both the biological properties of MSC as well as their potential efficacy in a variety of cellular therapies.

G-CSF increases mesenchymal precursor cell numbers in the bone marrow via an indirect mechanism involving osteoclast-mediated bone resorption.

During the course of studies to investigate whether MPC circulate in response to G-CSF, the agent most frequently used to induce mobilization of hematopoietic progenitors, we observed that while G-CSF failed to increase the number of MPC in circulation (assayed in vitro as fibroblast colony-forming cells, CFU-F), G-CSF administration nevertheless resulted in a time-dependent increase in the absolute number of CFU-F within the BM, peaking at Day 7. Treatment of BM cells from G-CSF-treated mice with hydroxyurea did not alter CFU-F numbers, suggesting that the increase in their numbers in response to G-CSF administration is not due to proliferation of existing CFU-F. Given previous studies demonstrating that G-CSF potently induces bone turnover in mice, we hypothesized that the increase in CFU-F may be triggered by the bone resorption that occurs following G-CSF administration. In accord with this hypothesis, administration of an inhibitor of osteoclast differentiation, osteoprotegerin (OPG), prevented the increase of CFU-F numbers induced by G-CSF. In conclusion, these data indicate that the cytokine treatment routinely used to mobilize hematopoietic stem cells could provide a readily applicable method to induce in vivo expansion of MPC for clinical applications.

G-CSF potently inhibits osteoblast activity and CXCL12 mRNA expression in the bone marrow.

Accumulating evidence indicates that interaction of stromal cell-derived factor 1 (SDF-1/CXCL12 [CXC motif, ligand 12]) with its cognate receptor, CXCR4 (CXC motif, receptor 4), generates signals that regulate hematopoietic progenitor cell (HPC) trafficking in the bone marrow. During granulocyte colony-stimulating factor (G-CSF)-induced HPC mobilization, CXCL12 protein expression in the bone marrow decreases. Herein, we show that in a series of transgenic mice carrying targeted mutations of their G-CSF receptor and displaying markedly different G-CSF-induced HPC mobilization responses, the decrease in bone marrow CXCL12 protein expression closely correlates with the degree of HPC mobilization. G-CSF treatment induced a decrease in bone marrow CXCL12 mRNA that closely mirrored the fall in CXCL12 protein. Cell sorting experiments showed that osteoblasts and to a lesser degree endothelial cells are the major sources of CXCL12 production in the bone marrow. Interestingly, osteoblast activity, as measured by histomorphometry and osteocalcin expression, is strongly down-regulated during G-CSF treatment. However, the G-CSF receptor is not expressed on osteoblasts; accordingly, G-CSF had no direct effect on osteoblast function. Collectively, these data suggest a model in which G-CSF, through an indirect mechanism, potently inhibits osteoblast activity resulting in decreased CXCL12 expression in the bone marrow. The consequent attenuation of CXCR4 signaling ultimately leads to HPC mobilization.