In regenerative medicine, bone marrow is a promising source of mesenchymal stem cells (MSCs) for a broad range of cellular therapies. This research addresses a basic prerequisite to realize the therapeutic potential of MSCs by developing a novel high-capacity assay to quantify the clonal heterogeneity in potency that is inherent to MSC preparations. The assay utilizes a 96-well format to (1) classify MSCs according to colony-forming efficiency as a measure of proliferation capacity and trilineage potential to exhibit adipo-, chondro-, and osteogenesis as a measure of multipotency and (2) preserve a frozen template of MSC clones of known potency for future use. The heterogeneity in trilineage potential of normal bone marrow MSCs is more complex than previously reported: all eight possible categories of trilineage potential were detected. In this study, the average colony-forming efficiency of MSC preparations was 55-62%, and tripotent MSCs accounted for nearly 50% of the colony-forming cells. The multiple phenotypes detected in this study infer a more convoluted hierarchy of lineage commitment than described in the literature. Greater cell amplification, colony-forming efficiency, and colony diameter for tri- versus unipotent clones suggest that MSC proliferation may be a function of potency. CD146 may be a marker of multipotency, with approximately 2-fold difference in mean fluorescence intensity between tri- and unipotent clones. The significance of these findings is discussed in the context of the efficacy of MSC therapies. The in vitro assay described herein will likely have numerous applications given the importance of heterogeneity to the therapeutic potential of MSCs.

Directed cell adhesion remains an important goal of implant and tissue engineering technology. In this study, surface energy and surface roughness were investigated to ascertain which of these properties show more overall influence on biomaterial-cell adhesion and colonization. Jet impingement was used to quantify cellular adhesion strength. Cellular proliferation and extracellular matrix secretion were used to characterize colonization of 3T3MC fibroblasts on: HS25 (a cobalt based implant alloy, ASTM F75), 316L stainless steel, Ti-6Al4V (a titanium implant alloy), commercially pure tantalum (Ta), polytetrafluoroethylene (PTFE), silicone rubber (SR), and high-density polyethylene (HDPE). The metals exhibited a nearly five-fold greater adhesion strength than the polymeric materials tested. Generally, surface energy was proportional to cellular adhesion strength. Only polymeric materials demonstrated significant increased adhesion strength associated with increased surface roughness. Cellular adhesion on metals demonstrated a linear correlation with surface energy. Less than half as much cellular proliferation was detected on polymeric materials compared to the metals. However the polymers tested demonstrated greater than twice the amount of secreted extracellular matrix (ECM) proteins on a per cell basis than the metallic materials. Thus, surface energy may be a more important determinant of cell adhesion and proliferation, and may be more useful than surface roughness for directing cell adhesion and cell colonization onto engineered tissue scaffoldings.

Adult stem cells have the potential to revolutionize regenerative medicine with their unique abilities to self-renew and differentiate into various phenotypes. This review examines progress and challenges in ex vivo tissue engineering with adult stem cells. These rare cells are harvested from a variety of tissues, including bone marrow, adipose, skeletal muscle, and placenta, and differentiate into cells of their own lineage and in some cases atypical lineages. Insight into the stem cell niche leads to the identification of matrix components, soluble factors, and physiological conditions that enhance the ex vivo amplification and differentiation of stem cells. Scaffolds composed of metals, naturally occurring materials, and synthetic polymers organize stem cells into complex spatial groupings that mimic native tissue. Cell signals from covalently bound ligands and slowly released regulatory factors in scaffolds direct stem cell fate. Future advances in stem cell biology and scaffold design will ultimately improve the efficacy of tissue substitutes as implants, in research, and as extracorporeal devices.

We detected cell‐to‐cell communication via intercellular bridges in DU 145 human prostate cancer cells by fluorescence microscopy. Since DU 145 cells have deficient gap junctions, intercellular bridges may have a prominent role in the transfer of chemical signals between these cells. In culture, DU 145 cells are contiguous over several cell diameters through filopodial extensions, and directly communicate with adjacent cells across intercellular bridges. These structures range from 100 nm to 5 μm in diameter, and from a few microns to at least 50–100 μm in length. Time‐lapse imagery revealed that (1) filopodia rapidly move at a rate of microns per minute to contact neighboring cells and (2) intercellular bridges are conduits for transport of membrane vesicles (1–3 μm in diameter) between adjacent cells. Immunofluorescence detected alpha‐tubulin in intercellular bridges and filopodia, indicative of microtubule bundles, greater than a micron in diameter. The functional meaning, interrelationship of these membrane extensions are discussed, along with the significance of these findings for other culture systems such as stem cells. Potential applications of this work include the development of anticancer therapies that target intercellular communication and controlling formation of cancer spheroids for drug testing.

Human mesenchymal stem cells (MSCs) from bone marrow are a heterogeneous ensemble of progenitors and lineage-committed cells, with a broad range of regenerative properties. Ex vivo expansion to produce sufficient quantities of MSCs is essential for most therapeutic applications. The present study resolves the relationship between proliferation potential of MSCs and their potency. Clonal analysis generated single-cell derived colonies of MSCs that were classified according to their trilineage potential to exhibit adipo- (A), chondro-(C) and osteogenesis (O) as a measure of potency. Multipotent OAC clones were highly proliferative with colony-forming efficiencies that ranged from 35% to 90%; whereas, O clones formed colonies with an efficiency of 5% or less (P<0.01). Similar trends were evident during ex vivo expansion: for example, the median specific growth rate was 0.85 day−1 (20 h doubling time) for cultures inoculated with OAC clones and was 5-fold less for inocula of O clones (P<0.01). OA and OC clones had similar proliferation potentials. More than 75% of cells in subconfluent cultures inoculated with O clones stained positive for senescence-associated β-galactosidase activity vs. less than 10% for OAC clones (P<0.001). Apoptotic cells were in the minority for all potency groups. Preliminary data generated during clonal analysis suggest that osteogenic potential of MSCs to produce mineralized matrix is a function of potency, as well. These results are discussed in the context of the preparation of efficacious MSC therapies by ex vivo expansion.