Human mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells were isolated from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro. These adult stem cells could be induced to differentiate exclusively into the adipocytic, chondrocytic, or osteocytic lineages. Individual stem cells were identified that, when expanded to colonies, retained their multilineage potential.

Mesenchymal stem cells are multipotent cells that can be isolated from adult bone marrow and can be induced in vitro and in vivo to differentiate into a variety of mesenchymal tissues, including bone, cartilage, tendon, fat, bone marrow stroma, and muscle. Despite their potential clinical utility for cellular and gene therapy, the fate of mesenchymal stem cells after systemic administration is mostly unknown. To address this, we transplanted a well-characterized human mesenchymal stem cell population into fetal sheep early in gestation, before and after the expected development of immunologic competence. In this xenogeneic system, human mesenchymal stem cells engrafted and persisted in multiple tissues for as long as 13 months after transplantation. Transplanted human cells underwent site-specific differentiation into chondrocytes, adipocytes, myocytes and cardiomyocytes, bone marrow stromal cells and thymic stroma. Unexpectedly, there was long-term engraftment even when cells were transplanted after the expected development of immunocompetence. Thus, mesenchymal stem cells maintain their multipotential capacity after transplantation, and seem to have unique immunologic characteristics that allow persistence in a xenogeneic environment. Our data support the possibility of the transplantability of mesenchymal stem cells and their potential utility in tissue engineering, and cellular and gene therapy applications.

Adult human mesenchymal stem cells are primary, multipotent cells capable of differentiating to osteocytic, chondrocytic, and adipocytic lineages when stimulated under appropriate conditions. To characterize the molecular mechanisms that regulate osteogenic differentiation, we examined the contribution of mitogenactivated protein kinase family members, ERK, JNK, and p38. Treatment of these stem cells with osteogenic supplements resulted in a sustained phase of ERK activation from day 7 to day 11 that coincided with differentiation, before decreasing to basal levels. Activation of JNK occurred much later (day 13 to day 17) in the osteogenic differentiation process. This JNK activation was associated with extracellular matrix synthesis and increased calcium deposition, the two hallmarks of bone formation. Inhibition of ERK activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked the osteogenic differentiation in a dose-dependent manner, as did transfection with a dominant negative form of MAP kinase kinase (MEK-1). Significantly, the blockage of osteogenic differentiation resulted in the adipogenic differentiation of the stem cells and the expression of adipose-specific mRNAs peroxisome proliferator-activated receptor ␥2, aP2, and lipoprotein lipase. These observations provide a potential mechanism involving MAP kinase activation in osteogenic differentiation of adult stem cells and suggest that commitment of hMSCs into osteogenic or adipogenic lineages is governed by activation or inhibition of ERK, respectively.Human bone marrow-derived mesenchymal stem cells (hMSCs) 1 are multipotent, capable of differentiating into at least three lineages (osteogenic, chondrogenic, and adipogenic) when cultured under defined in vitro conditions (1-3). The hMSCs do not differentiate spontaneously, and their in vitro and in vivo osteogenic potential has been very well characterized by us and others (4 -6). When cultured in the presence of the synthetic glucocorticoid dexamethasone, ascorbic acid, and ␤-glycerophosphate (osteogenic supplements, OS), hMSCs differentiate to the osteogenic lineage, producing bone-like nodules with a mineralized extracellular matrix containing hydroxyapatite (4). The similar developmental phenomenon has also been described by others (7, 8) using bone marrow-derived cells. Other than the osteoinductive effect that OS has on MSCs, OS also acts as a mitogen (9). Presumably, the osteoinductive and mitogenic effects are due to dexamethasone present in OS because glucocorticoids are potent regulators of cellular growth and differentiation (10). However, the underlying molecular mechanisms of OS-induced mitogenic and osteogenic differentiation are presently unknown.In order to acquire a new cell phenotype, uncommitted hMSCs must undergo proliferative and differentiative changes, the two most fundamental biological processes in the life cycle of cells. One of the potential signal transduction pathways that might regulate the proliferation and differentiation of hMSCs is the MAP kin...