Marrow stromal cells are adult stem cells from bone marrow that can differentiate into multiple nonhematopoietic cell lineages. Previous reports demonstrated that single-cell-derived colonies of marrow stromal cells contained two morphologically distinct cell types: spindle-shaped cells and large flat cells. Here we found that early colonies also contain a third kind of cell: very small round cells that rapidly self-renew. Samples enriched for the small cells had a greater potential for multipotential differentiation than samples enriched for the large cells. Also, the small cells expressed a series of surface epitopes and other proteins that potentially can be used to distinguish the small cells from the large cells. The results suggested it will be important to distinguish the major subpopulations of marrow stromal cells in defining their biology and their potential for cell and gene therapy.B one marrow contains at least two kinds of stem cells, hematopoietic stem cells and stem cells for nonhematopoietic tissues (1-26), variously referred to as mesenchymal stem cells or marrow stromal cells (MSCs). MSCs are of interest because they are easily isolated from a small aspirate of bone marrow and readily generate single-cell-derived colonies (1-5, 25, 27); the single-cell-derived colonies can be expanded through as many as 50 population doublings in about 10 weeks (25), and they can differentiate into osteoblasts, adipocytes, chondrocytes (1-13), myocytes (9), astrocytes, oligodendrocytes, and neurons (17,23,26,27). For these reasons, the cells are currently being tested for their potential use in cell and gene therapy for a number of human diseases (22,24). Previous reports (3,11,12) demonstrated that single-cell-derived colonies of human MSCs are heterogeneous in that they contain at least two morphologically distinct kinds of cells: spindle-shaped cells and large cuboidal or flattened cells. Here we have extended our previous observations (25) to demonstrate that the colonies also contain extremely small cells that are rapidly self-renewing (RS cells). The RS cells appear to be the earliest progenitors in the cultures and have the greatest potential for multilineage differentiation. They differ from more mature cells in the same cultures by a series of surface epitopes and expressed proteins. MethodsIsolation and Growth of MSCs. To isolate human MSCs, bone marrow aspirates of 10-20 ml were taken from the iliac crest of normal donors ranging in age from 19 to 49 years old under an Institutional Review Board approved protocol. Nucleated cells were isolated with a density gradient [Ficoll͞Paque (Pharmacia)] and resuspended in complete culture medium [␣ MEM, GIBCO͞BRL; 20% FBS, lot-selected for rapid growth of MSCs (Atlanta Biologicals); 100 units/ml of penicillin͞100 g/ml of streptomycin͞2 mM L-glutamine (GIBCO͞BRL)]. All of the cells were plated in 25 ml of medium in a 175-cm 2 culture dish (Falcon) and incubated at 37°C with 5% humidified CO 2 . After 24 h, nonadherent cells were discarded, and adherent cells ...

Summary. Marrow stromal cells (MSCs) were isolated from bone marrow obtained by aspirates of the iliac crest of normal volunteers. The cells were isolated by their adherence to plastic and then passed in culture. Some of the samples expanded through over 15 cell doublings from the time frozen stocks were prepared. Others ceased replicating after about four cell doublings. The replicative potential of the cells in culture was best predicted by a simple colonyforming assay in which samples from early passages were plated at low densities of about 10 cells per cm 2 . Samples with high colony-forming ef®ciency exhibited the greatest replicative potential. The colonies obtained by plating early passage cells at low density varied in size and morphology. The large colonies readily differentiated into osteoblasts and adipocytes when incubated in the appropriate medium. As samples were expanded in culture and approached senescence, they retained their ability to differentiate into osteoblasts. However, the cells failed to differentiate into adipocytes. The loss of multipotentiality following serial passage in culture may have important implications for the use of expanded MSCs for cell and gene therapy.

Cultures of plastic-adherent cells from bone marrow have attracted interest because of their ability to support growth of hematopoietic stem cells, their multipotentiality for differentiation, and their possible use for cell and gene therapy. Here we found that the cells grew most rapidly when they were initially plated at low densities (1.5 or 3.0 cells/cm 2 ) to generate single-cell derived colonies. The cultures displayed a lag phase of about 5 days, a log phase of rapid growth of about 5 days, and then a stationary phase. FACS analysis demonstrated that stationary cultures contained a major population of large and moderately granular cells and a minor population of small and agranular cells here referred to as recycling stem cells or RS-1 cells. During the lag phase, the RS-1 cells gave rise to a new population of small and densely granular cells (RS-2 cells). During the late log phase, the RS-2 cells decreased in number and regenerated the pool of RS-1 cells found in stationary cultures. In repeated passages in which the cells were plated at low density, they were amplified about 10 9 -fold in 6 wk. The cells retained their ability to generate single-cell derived colonies and therefore apparently retained their multipotentiality for differentiation.

ABSTRACT

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Three-dimensional (3D) cell culture is gaining acceptance in response to the need for cellular models that better mimic physiologic tissues. Spheroids are one such 3D model where clusters of cells will undergo self-assembly to form viable, 3D tumor-like structures. However, to date little is known about how spheroid biology compares to that of the more traditional and widely utilized 2D monolayer cultures. Therefore, the goal of this study was to characterize the phenotypic and functional differences between lung tumor cells grown as 2D monolayer cultures, versus cells grown as 3D spheroids. Eight lung tumor cell lines, displaying varying levels of epidermal growth factor receptor (EGFR) and cMET protein expression, were used to develop a 3D spheroid cell culture model using low attachment U-bottom plates. The 3D spheroids were compared with cells grown in monolayer for 1) EGFR and cMET receptor expression, as determined by flow cytometry, 2) EGFR and cMET phosphorylation by MSD assay, and 3) cell proliferation in response to epidermal growth factor (EGF) and hepatocyte growth factor (HGF). In addition, drug responsiveness to EGFR and cMET inhibitors (Erlotinib, Crizotinib, Cetuximab [Erbitux] and Onartuzumab [MetMab]) was evaluated by measuring the extent of cell proliferation and migration. Data showed that EGFR and cMET expression is reduced at day four of untreated spheroid culture compared to monolayer. Basal phosphorylation of EGFR and cMET was higher in spheroids compared to monolayer cultures. Spheroids showed reduced EGFR and cMET phosphorylation when stimulated with ligand compared to 2D cultures. Spheroids showed an altered cell proliferation response to HGF, as well as to EGFR and cMET inhibitors, compared to monolayer cultures. Finally, spheroid cultures showed exceptional utility in a cell migration assay. Overall, the 3D spheroid culture changed the cellular response to drugs and growth factors and may more accurately mimic the natural tumor microenvironment.

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Hypochlorous acid (HOCl) is produced by polymorphonuclear leukocytes that migrate and adhere to endothelial cells as part of the inflammatory response to tissue injury. HOCl is an extremely toxic oxidant that can react with a variety of cellular components, and concentrations reaching 200 microM have been reported in some tissues. In this study, we show that HOCl interacts with the skeletal sarcoplasmic reticulum Ca(2+)-adenosinetriphosphatase (ATPase), inhibiting transport function, HOCl inhibits sarcoplasmic reticulum Ca(2+)-ATPase activity in a concentration-dependent manner with a concentration required to inhibit ATPase activity by 50% of 170 microM and with complete inhibition of activity at 3 mM. A concomitant reduction in free sulfhydryl groups after HOCl treatment was observed, paralleling the inhibition of ATPase activity. It was also observed that HOCl inhibited the binding of the fluorescent probe fluorescein isothiocyanate to the ATPase protein, indicating some structural damage may have occurred. These findings suggest that the reactive oxygen species HOCl inhibits ATPase activity via a modification of sulfhydryl groups on the protein, supporting the contention that reactive oxygen species disrupt the normal Ca(2+)-handling kinetics in muscle cells.