Insulin-like growth factor 1 (IGF-1), the most abundant growth factor in the bone matrix, regulates bone mass in adulthood. We report that IGF-1 released from bone matrix stimulates osteoblastic differentiation of mesenchymal stem cell (MSCs) by activation of mTOR during bone remodeling. Mice knockout of IGF-1 receptor (Igf1r) in the preosteoblastic cells exhibited low bone mass and reduced mineral deposition rates. The MSCs recruited to the bone surface were unable to differentiate into osteoblasts. In age-related osteoporosis in humans, we found that marrow IGF-1 levels were 40% lower than controls. Similarly, the levels of IGF-1 in the bone matrix and marrow of aged rats were also decreased and directly correlated with the age-related decrease in bone mass. Notably, injection of IGF-1 with IGF binding protein 3 (IGFBP3), not IGF-1 alone, increased the level of IGF-1 in the bone matrix and stimulated new bone formation in old rats. Thus, IGF-1 released during bone resorption from bone matrix activates mTOR to induce osteoblast differentiation of MSCs in maintaining bone micro-architecture and mass.

Bone marrow adipogenesis is a normal physiologic process in all mammals. However, its function is unknown. The mesenchymal stem cell is the marrow precursor for adipocytes as well as osteoblasts, and PPARγ is an essential differentiation factor for entrance into the fat lineage. Mouse models have provided significant insight into the molecular cues that define stromal cell fate. In humans, accelerated marrow adipogenesis has been associated with aging and several chronic conditions including diabetes mellitus and osteoporosis. Newer imaging techniques have been used to determine the developmental time course of fat generation in bone marrow. However, more studies are needed to understand the interrelationship among hematopoietic, osteoblastic, and adipogenic cells within the marrow niche.

Human MSCs have been studied to define the mechanisms involved in normal bone remodeling and the regulation of osteogenesis. During osteogenic differentiation, MSCs change from their characteristic fibroblast-like phenotype to near spherical shape. In this study, we analyzed the correlation between the organization of cytoskeleton of MSCs, changes in cell morphology, and the expression of specific markers (alkaline phosphatase activity and calcium deposition) of osteogenic differentiation. For osteoblastic differentiation, cells were cultured in a culture medium supplemented with 100 nM dexamethasone, 10 mM beta- glycerophosphate, and 50 microg/ml ascorbic acid. The organization of microfilaments and microtubules was examined by inmunofluorescence using Alexa fluor 594 phalloidin and anti alpha-tubulin monoclonal antibody. Cytochalasin D and nocodazole were used to alter reversibly the cytoskeleton dynamic. A remarkable change in cytoskeleton organization was observed in human MSCs during osteogenic differentiation. Actin cytoskeleton changed from a large number of thin, parallel microfilament bundles extending across the entire cytoplasm in undifferentiated MSCs to a few thick actin filament bundles located at the outermost periphery in differentiated cells. Under osteogenic culture conditions, a reversible reorganization of microfilaments induced by an initial treatment with cytochalasin D but not with nocodazole reduced the expression of differentiation markers, without affecting the final morphology of the cells. The results indicate that changes in the assembly and disassembly kinetics of microfilaments dynamic of actin network formation may be critical in supporting the osteogenic differentiation of human MSCs; also indicated that the organization of microtubules appears to have a regulatory role on the kinetic of this process.

The formation, maintenance, and repair of bone tissue involve close interlinks between two stem cell types housed in the bone marrow: the hematologic stem cell originating osteoclasts and mesenchymal stromal cells (MSCs) generating osteoblasts. In this review, we consider malfunctioning of MSCs as essential for osteoporosis. In osteoporosis, increased bone fragility and susceptibility to fractures result from increased osteoclastogenesis and insuffi cient osteoblastogenesis. MSCs are the common precursors for both osteoblasts and adipocytes, among other cell types. MSCs´ commitment towards either the osteoblast or adipocyte lineages depends on suitable regulatory factors activating lineage-specifi c transcriptional regulators. In osteoporosis, the reciprocal balance between the two diff erentiation pathways is altered, facilitating adipose accretion in bone marrow at the expense of osteoblast formation; suggesting that under this condition MSCs activity and their microenvironment may be disturbed. We summarize research on the properties of MSCs isolated from the bone marrow of control and osteoporotic post-menopausal women. Our observations indicate that intrinsic properties of MSCs are disturbed in osteoporosis. Moreover, we found that the regulatory conditions in the bone marrow fl uid of control and osteoporotic patients are signifi cantly diff erent. These conclusions should be relevant for the use of MSCs in therapeutic applications.

Mesenchymal stem cells (MSCs), precursor cells resident in the bone marrow, have the capacity to differentiate into bone, cartilage, fat, and connective tissue. We have recently reported that MSCs from "healthy" donors differ from cells obtained from osteoporotic postmenopausal women in their proliferation rate, mitogenic response to osteogenic growth factors, and potential to mineralize. The purpose of this study was to examine the factors that explain the differential capacity of MSCs derived from "healthy" control and osteoporotic postmenopausal women to support mineralization. In addition, we examined the factors that regulate the differentiation of osteoporotic cells into adipocytes. For this purpose, we isolated MSCs from bone marrow of donors and analyzed the synthesis and deposition of type I collagen, the main component of bone extracellular matrix, the time course of gelatinolytic activity expression, the deposition of transforming growth factor beta (TGF-beta), and the ability of cells to differentiate into adipocytes. Our results indicate that cells derived from osteoporotic donors synthesized 50% less type I collagen than normal cells and maintained higher levels of gelatinolytic activity under differentiation conditions (70% versus 15% after 14 days in culture). MSCs derived from osteoporotic women produced 60-65% less TGF-beta and expressed higher adipogenic capacity. We conclude that the capacity of MSCs derived from osteoporotic postmenopausal women to generate and maintain type I collagen-rich extracellular matrix is decreased, favoring their adipogenic differentiation. These observations may explain the decreased mineralization previously observed in these types of cells.