R. Socher

Identification of cultured progenitor cells from human marrow stroma

The marrow stromal cells (MSC) are essential for regulation of bone remodeling and hematopoiesis. It is of prime importance to isolate MSC and to expand the proliferating cells ex vivo. In this study, we analyzed cultured MSC for various cellular parameters, including cell morphology, cell cycle, and expression of cell surface antigens by flow cytometry. MSC were divided based on cell size to small (S‐cells) and large (L‐cells) and were visualized by light and electron microscope. The S‐cells were proliferating cells correlated with G0/G1 phase of cell cycle, and expressed cFOS. The expression of surface markers CD‐34, ‐44, ‐51, ‐61, ‐62E, ‐62P, ‐62L was quantified using flow cytometry. CD‐44 was ubiquitously expressed by S and L cells, CD‐51 and ‐61 were expressed by 30%–38% of S‐cells. CD‐34 and ‐62 expressed 20% positive of the analyzed cells that were of the proliferating progenitors (S‐cells). This study enables the identification of subpopulations from MSC with special attention paid to the proliferating cells from ex vivo cultures of marrow stroma. J. Cell. Biochem. 87: 51–57, 2002.

Cell-based screening for membranal and cytoplasmatic markers using dielectric spectroscopy

Dielectric spectroscopy (DS) of living biological cells is based on the analysis of the complex dielectric permittivity of cells suspended in a physiological medium. It provides knowledge on the polarization-relaxation response of cells to external electric field as function of the excitation frequency. This response is strongly affected by both structural and molecular properties of cells and therefore, can reveal rare insights on cell physiology and behaviour. This study demonstrates the mapping potential of DS after cytoplasmatic and membranal markers for cell-based screening analysis. The effect of membrane permittivity and cytoplasm conductivity was examined using tagged MBA and MDCK cell lines respectively. Comparing the permittivity spectra of tagged and native cell lines reveals clear differences between the analyzed suspensions. In addition, differences on the matching dielectric properties of cells were obtained. Those findings support the high distinction resolution and sensitivity of DS after fine molecular and cellular changes, and hence, highlight the high potential of DS as non invasive screening tool in cell biology research.

SVEP1 expression is regulated in estrogen-dependent manner

The SVEP1 protein comprises modules related to the selectin super family and other motifs found in cell surface molecules. Earlier, we demonstrated that SVEP1 is expressed in osteogenic cells both in vivo and in vitro; in the current study we elaborate on the regulation of SVEP1 by 17β‐Estradiol (17βE2). SVEP1 message is expressed in vivo by bone marrow cells of sham‐operated rats, but not in estrogen‐depleted ovariectomized (OVX) rats. We demonstrated that 17βE2 treatment increases the level of the SVEP1 expression in cultured osteoblasts. SVEP1 was identified also in breast carcinoma (BC) cells known to reside in bone when metastasized from the primary tumor. SVEP1 expression was demonstrated by immunohistochemistry and fluorescence‐activated cell sorting (FACS) on various BC cell lines. The chromatin immunoprecipitation (ChIP) assay was applied to analyze the estrogen receptor (ER) binding to the putative SVEP1 promoter. We demonstrated that treatment with 17βE2 or ICI 182,780 affects this binding and regulates the mRNA and protein levels of SVEP1 in BC cells. We propose that SVEP1 may serve as a useful biomarker for studying the mechanism of cells interactions within the local microenvironment affected by estrogen. J. Cell. Physiol. 210: 732–739, 2007.

Dexamethasone regulation of cFos mRNA in osteoprogenitors.

The glucocorticoid, dexamethasone (Dex), has a beneficial effect on osteogenesis by increasing the activation and differentiation of osteoblastic cells. We investigated the effect of Dex on osteoblasts and detected changes in pattern of expression of cFos mRNA. cFos is an early response gene that is expressed as unspliced and spliced mRNAs. We analyzed the regulation of cFos mRNA in correlation with the cell proliferation status and following the induction by Dex. Treatment of osteoblastic cells with Dex for 30 min resulted in elevated levels of spliced form of cFos mRNA, which was maintained with extended treatment of cultured cells for 24 h. In addition, we demonstrated interaction between glucocorticoid receptor (GR) and cFos mRNA. This study provides evidence for the action of Dex on osteoblasts through dynamic cell responses involving with cFos.

Dielectric dispersion of suspended cells using 3D reconstructed morphology model

In the framework of this study, novel method for dispersion analysis of cellular suspensions is presented. The method is fundamentally based on the ability to reconstruct the exact 3D morphology of a given cell with resolution accuracy of few nanometers using AFM imaging. By applying a reverse engineering approach, the morphology of the cell is constructed based on a set of measured spatial points that describes its geometry. The permittivity spectrum of the reconstructed cell is then directly derived based on computational solution of complex potential problem using 3D Boundary Element Method. The applicability of the method is demonstrated both theoretically and experimentally with tight comparison to the well known shell models. This comparison reveals significant deviations between the models, and hence, emphasises the vast effect of morphology in dispersion analysis of cellular suspensions.

Gene expression in skeletal tissues: application of laser capture microdissection.

Tissue differentiation is based on the expression of transcription factors, receptors for cytokines, and nuclear receptors that regulate a specific phenotype. The purpose of this study was to select cells from various skeletal tissues in order to analyse differential gene expression of cells in the native environment in vivo. It is a difficult task to obtain cells from skeletal tissues, such as cartilage, periost, bone and muscle, that are structured together and do not exist as individual organs. We used laser capture microdissection which permits the selection and isolation of individual cells from tissue sections. The RNA isolated from these tissues was used for reverse transcriptase‐polymerase chain reactions for molecular analysis. We analysed the expression of transcription factors (cFOS, cbfa1, MyoD), receptors for cytokines, nuclear receptors, alkaline phosphatase and the structural proteins osteocalcin and collagen II. The results obtained demonstrate differential patterns of gene expression according to the tissue arrangement in their native in vivo environment, with reliable interpretation of the functions of the analysed genes in the context of intact skeletal tissue physiology.

Comparative study using scanning electron techniques for imaging of micro-architecture and antigen appearance

We have used various scanning electron microscopy technologies for the analysis of structures and the appearance of antigen on eukaryotic cells. Various scanning electron microscopy including conventional scanning electron microscopy, high‐resolution scanning electron microscopy and environmental scanning electron microscopy applied for cell surface analysis allow obtaining sub‐micrometre surface features on cell membranes. We used cell systems for imaging along with molecular localization of a cell surface antigen, SVEP1 protein. SVEP1 was studied using the immuno‐gold technique for the identification of cell surface features and protein localization. We used an antibody to SVEP1 molecule that was labelled with secondary antibody conjugated with gold particles. The comparison between scanning electron methods enabled the visualization of surface structures and the molecular imaging for the distribution of SVEP1.

Tracking the molecular signature of developing skeletal tissues.

We isolated cells from their native in vivo microenvironment using the Laser Capture Micro dissection (LCM). Bone and cartilage tissues were studied from mouse embryonic (18dpc) processed by cry sections enabled the cell isolation from anatomical complexity of skeletal tissues using the LCM technique. RNA was purified from the isolated cells and followed with amplification stage to hybridize on gene array for high through (HT) put analysis to profile the tissues gene expression. Bioinformatics profiling of the differential expression performed according to the tissue origin highlighted the common and divergent genes in the regulation of these tissues. Specifically, we identified that genes related to cell replication and cell metabolism were more prominent in bone, while organic acid metabolism was more prominent in cartilage. This study has demonstrated the utility of applying HT microarray analysis using RNA from small number of cells isolated by LCM from skeletal tissues. The bioinformatics provides insight which has not yet been explored for the developing skeletal tissues. The power of LCM application provides a platform to make a broad molecular analysis using transcriptom analysis to reveal the molecular signature of tissues in their nature environment.

Localization of membrane-bond OPN using force spectroscopy analysis

At this study we present molecular recognition method which is based on force spectroscopy analysis for biological markers on the whole cell level. The presented method allows recognition of specific cell surface proteins and receptor sites by nanometer accuracy level. Here we demonstrate specific recognition of membrane-bond Osteopontin (OPN) sites over a whole Preosteogenic cell membrane. By merging specific force detection map of the proteins and topography image of the cell, we create a new image (recognition image), which demonstrate the exact locations of the proteins relative to the cell membrane. The recognition results indicate on the strong affinity between the modified tip and the target molecules, therefore, it enables the use of an AFM as a remarkable nanoscale tracking tool at the whole cell level.