Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro

Abstract

Human bone marrow contains a population of cells capable of differentiating along multiple mesenchymal cell lineages. Recently, techniques for the purification and culture-expansion of these human marrow-derived Mesenchymal Stem Cells (MSCs) have been developed. The goals of the current study were to establish a reproducible system for the in vitro osteogenic differentiation of human MSCs, and to characterize the effect of changes in the microenvironment upon the process. MSCs derived from 2nd or 3rd passage were cultured for 16 days in various base media containing 1 to 1000 nM dexamethasone (Dex), 0.01 to 4 mM L-ascorbic acid-2-phosphate (AsAP) or 0.25 mM ascorbic acid, and 1 to 10 mM beta-glycerophosphate (beta GP). Optimal osteogenic differentiation, as determined by osteoblastic morphology, expression of alkaline phosphatase (APase), reactivity with anti-osteogenic cell surface monoclonal antibodies, modulation of osteocalcin mRNA production, and the formation of a mineralized extracellular matrix containing hydroxyapatite was achieved with DMEM base medium plus 100 nM Dex, 0.05 mM AsAP, and 10 mM beta GP. The formation of a continuously interconnected network of APase-positive cells and mineralized matrix supports the characterization of this progenitor population as homogeneous. While higher initial seeding densities did not affect cell number of APase activity, significantly more mineral was deposited in these cultures, suggesting that events which occur early in the differentiation process are linked to end-stage phenotypic expression. Furthermore, cultures allowed to concentrate their soluble products in the media produced more mineralized matrix, thereby implying a role for autocrine or paracrine factors synthesized by human MSCs undergoing osteoblastic lineage progression. This culture system is responsive to subtle manipulations including the basal nutrient medium, dose of physiologic supplements, cell seeding density, and volume of tissue culture medium. Cultured human MSCs provide a useful model for evaluating the multiple factors responsible for the step-wise progression of cells from undifferentiated precursors to secretory osteoblasts, and eventually terminally differentiated osteocytes.

Regulation of Breast Cancer Stem Cells by Mesenchymal Stem Cells in the Metastatic Niche

“…The classic osteogenic differentiation of MSCs in different tissues requires distinct signals (Jaiswal et al, 1997;Pittenger et al, 1999). It has been demonstrated that only one-third of initial adherent bone marrow-derived MSC colonies are pluripotent and capable of differentiating into osteoblastic, chondrocytic and adipocytic cell lineages, while the majority of MSCs exhibits a bilineage (osteo/chondro) or unilineage (osteo) potential.…”

Surface Engineered Carboxymethylchitosan/Poly(amidoamine) Dendrimer Nanoparticles for Intracellular Targeting

“…This range of concentrations of Dex has been shown to be effective for the osteogenic differentiation of MSCs. [41,50] It is known that ALP, a cell surface glycoprotein, is an early marker of osteogenic differentiation. [47,51] To confirm the osteogenic differentiation of RBMSCs exposed to Dex-loaded CMCht/PAMAM dendrimer nanoparticles, we carried out an ALP staining (Fig.…”

Retroviral marking of human bone marrow fibroblasts: In vitro expansion and localization in calvarial sites after subcutaneous transplantation in vivo

“…We and others have shown previously the ability to modulate the phenotype of human bone marrow stromal ®broblasts into a variety of cell and tissue types (Caplan, 1991;Jaiswal et al, 1997;Horwitz et al, 1999;Oreffo et al, 1997;Pittenger et al, 1999). Magni®cation  100. c: Osteocalcin expression in human bone marrow cells as detected by immunocytochemistry using monoclonal antibody, OS 35, which shows speci®c reactivity with osteocalcin in cultured cells.…”

“…However, the isolation and characterization of such stem cells has been hampered by the lack of speci®cally reactive antibodies and the very low incidence of these cells. Current approaches have centered on the isolation and expansion of these early progenitors from marrow ®broblast populations Jaiswal et al, 1997;Kadiyala et al, 1997). Recent animal and human studies indicate that mesenchymal stem cell therapy may be a realistic therapeutic target for a variety of skeletal, muscular, and haemopoietic diseases (Prockop, 1997;Baltzer et al, 1999;Marx et al, 1999;Oyama et al, 1999;Horwitz et al, 1999).…”