Cell lineage analysis of the mammalian female germline.

Fundamental aspects of embryonic and post-natal development, including maintenance of the mammalian female germline, are largely unknown. Here we employ a retrospective, phylogenetic-based method for reconstructing cell lineage trees utilizing somatic mutations accumulated in microsatellites, to study female germline dynamics in mice. Reconstructed cell lineage trees can be used to estimate lineage relationships between different cell types, as well as cell depth (number of cell divisions since the zygote). We show that, in the reconstructed mouse cell lineage trees, oocytes form clusters that are separate from hematopoietic and mesenchymal stem cells, both in young and old mice, indicating that these populations belong to distinct lineages. Furthermore, while cumulus cells sampled from different ovarian follicles are distinctly clustered on the reconstructed trees, oocytes from the left and right ovaries are not, suggesting a mixing of their progenitor pools. We also observed an increase in oocyte depth with mouse age, which can be explained either by depth-guided selection of oocytes for ovulation or by post-natal renewal. Overall, our study sheds light on substantial novel aspects of female germline preservation and development.

Estimating cell depth from somatic mutations.

The depth of a cell of a multicellular organism is the number of cell divisions it underwent since the zygote, and knowing this basic cell property would help address fundamental problems in several areas of biology. At present, the depths of the vast majority of human and mouse cell types are unknown. Here, we show a method for estimating the depth of a cell by analyzing somatic mutations in its microsatellites, and provide to our knowledge for the first time reliable depth estimates for several cells types in mice. According to our estimates, the average depth of oocytes is 29, consistent with previous estimates. The average depth of B cells ranges from 34 to 79, linearly related to the mouse age, suggesting a rate of one cell division per day. In contrast, various types of adult stem cells underwent on average fewer cell divisions, supporting the notion that adult stem cells are relatively quiescent. Our method for depth estimation opens a window for revealing tissue turnover rates in animals, including humans, which has important implications for our knowledge of the body under physiological and pathological conditions.

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.

Application of the laser capture microdissection technique for molecular definition of skeletal cell differentiation in vivo.

Laser capture microdissection (LCM) method allows selection of individual or clustered cells from intact tissues. This technology enables one to pick cells from tissues that are difficult to study individually, sort the anatomical complexity of these tissues, and make the cells available for molecular analyses. Following the cells' extraction, the nucleic acids and proteins can be isolated and used for multiple applications that provide an opportunity to uncover the molecular control of cellular fate in the natural microenvironment. Utilization of LCM for the molecular analysis of cells from skeletal tissues will enable one to study differential patterns of gene expression in the native intact skeletal tissue with reliable interpretation of function for known genes as well as to discover novel genes. Variability between samples may be caused either by differences in the tissue samples (different areas isolated from the same section) or some variances in sample handling. LCM is a multi-task technology that combines histology, microscopy work, and dedicated molecular biology. The LCM application will provide results that will pave the way toward high throughput profiling of tissue-specific gene expression using Gene Chip arrays. Detailed description of in vivo molecular pathways will make it possible to elaborate on control systems to apply for the repair of genetic or metabolic diseases of skeletal tissues.

Insights on the functional role of chromatin remodelers in osteogenic cells.

Control of eukaryotic gene expression requires interaction between sequence-specific transcription factors and their regulatory elements on the particular promoter. The dynamic alteration of chromatin structure regulates the accessibility of these elements in the genome and therefore contributes to the control of transcriptional activity. Here we discuss chromatin remodelling in the context of osteoblast lineage regulation. This review specifically highlights the role of the protein chromatin-related mesenchymal modulator (CReMM/CHD9), a recently identified chromatin remodeler, in osteogenic cell differentiation.

Transcriptional profiling of mesenchymal stromal cells from young and old rats in response to Dexamethasone.

BACKGROUND: Marrow-derived stromal cells (MSCs) maintain the capability of self-renewal and differentiation into multiple lineages in adult life. Age-related changes are recognized by a decline in the stemness potential that result in reduced regeneration potential of the skeleton. To explore the molecular events that underline skeletal physiology during aging we catalogued the profile of gene expression in ex vivo cultured MSCs derived from 3 and 15 month old rats. The ex vivo cultured cells were analyzed following challenge with or without Dexamethasone (Dex). RNA retrieved from these cells was analyzed using Affymetrix Gene Chips to compare the effect of Dex on gene expression in both age groups. RESULTS: The molecular mechanisms that underline skeletal senescence were studied by gene expression analysis of RNA harvested from MSCs. The analysis resulted in complex profiles of gene expression of various differentiation pathways. We revealed changes of lineage-specific gene expression; in general the pattern of expression included repression of proliferation and induction of differentiation. The functional analysis of genes clustered were related to major pathways; an increase in bone remodeling, osteogenesis and muscle formation, coupled with a decrease in adipogenesis. We demonstrated a Dex-related decrease in immune response and in genes that regulate bone resorption and an increase in osteoblastic differentiation. Myogenic-related genes and genes that regulate cell cycle were induced by Dex. While Dex repressed genes related to adipogenesis and catabolism, this decrease was complementary to an increase in expression of genes related to osteogenesis. CONCLUSION: This study summarizes the genes expressed in the ex vivo cultured mesenchymal cells and their response to Dex. Functional clustering highlights the complexity of gene expression in MSCs and will advance the understanding of major pathways that trigger the natural changes underlining physiological aging. The high throughput analysis shed light on the anabolic effect of Dex and the relationship between osteogenesis, myogenesis and adipogenesis in the bone marrow cells.

Dielectric screening of early differentiation patterns in mesenchymal stem cells induced by steroid hormones.

In the framework of this study, target identification and localization of differentiation patterns by means of dielectric spectroscopy is presented. Here, a primary pre-osteoblastic bone marrow-derived MBA-15 cellular system was used to study the variations in the dielectric properties of mesenchymal stem cells while exposed to differentiation regulators. Using the fundamentals of mixed dielectric theories combined with finite numerical tools, the permittivity spectra of MBA-15 cell suspensions have been uniquely analyzed after being activated by steroid hormones to express osteogenic phenotypes. Following the spectral analysis, significant variations were revealed in the dielectric properties of the activated cells in comparison to the untreated populations. Based on the differentiation patterns of MBA-15, the electrical modifications were found to be highly correlated with the activation of specific cellular mechanisms which directly react to the hormonal inductions. In addition, by describing the dielectric dispersion in terms of transfer functions, it is shown that the spectral perturbations are well adapted to variations in the electrical characteristics of the cells. The reported findings vastly emphasize the tight correlation between the cellular and electrical state of the differentiated cells. It therefore emphasizes the vast abilities of impedance-based techniques as potential screening tools for stem cell analysis.

Insights into the transcriptional and chromatin regulation of mesenchymal stem cells in musculo-skeletal tissues.

Utilizing adult stem cells for regenerative medicine of skeletal tissues requires the development of molecular and biochemical tools that will allow isolation of these cells and direction of their differentiation towards a desired lineage and tissue formation. Stem cell commitment and fate decision into specialized functional cells involve coordinated activation and silencing of lineage-specific genes. Transcription factors and chromatin-remodeling proteins are key players in the control process of lineage commitment and differentiation during embryogenesis and adulthood. Transcription factors act in cooperation with co-regulator proteins to generate tissue-specific responses that elicits the tissue specific gene expression. Consequently, one of the main challenges of today's research is to characterize molecular pathways that coordinate the lineage-specific differentiation. Epigenetic regulation includes chromatin remodeling that control structural changes of DNA required for the binding of transcription factors to promoter regions. Revealing the mechanisms of action of such factors will provide understanding of how transcription and chromatin regulatory factors function together to regulate stem cell lineage fate decision.

Insights into chromatin remodelers in mesenchymal stem cells and differentiation.

Acquisition of lineage specific fate depends on the well orchestrated performance of master transcription factors and on dynamic changes in chromatin structure that account for epigenetic regulation. Epigenetic mechanisms regulate transcription at the promoter level and involve the recruitment of numerous chromatin modifiers in order to permit tissue-selective gene transcription. The dynamics of chromatin structural changes are achieved by the actions of two classes of enzymes: ATP-dependent chromatin remodelers, and histone modifying enzymes. The enzymes are partners in multi-protein complexes that activate or repress transcription depending on the composition of the protein complex. It is fully appreciated now that mechanisms triggering changes in chromatin structure are an integral in determining the stem cell fate. Elucidating the nature of cross talk between chromatin remodelers and master genes is important for identifying pathways that govern stem cell fate and lineage decision.

The effect of surface treatment on the surface texture and contact angle of electrochemically deposited hydroxyapatite coating and on its interaction with bone-forming cells.

This work demonstrates the effects of both surface preparation and surface post-treatment by exposure to electron beam on the surface texture, contact angle and the interaction with bone-forming cells of electrochemically deposited hydroxyapatite (HAp) coating. Both the surface texture and the contact angle of the ground titanium substrate changed as a result of either heat treatment following soaking in NaOH solution or soaking in H(2)O(2) solution. Consequently, the shape of the current transients during potentiostatic deposition of HAp changed, and the resulting coatings exhibited different surface textures and contact angles. The developed interfacial area ratio Sdr and the core fluid retention index Sci were found more reliable than the mean roughness R(a) and the root-mean-square roughness Z(rms) in correlating the adhesion of the coating to the metal substrate and the cellular response with surface texture. The NaOH pretreatment provided the highest surface area and induced the highest cell attachment, even though the H(2)O(2) treatment provided the highest hydrophilicity to the metal substrate. Electrodeposition at pH 6 was found preferable compared to electrodeposition at pH 4.2. The ability to modify the cellular response by exposure to unique electron-beam surface treatment was demonstrated. The very high hydrophilicity of the as-deposited HAp coating enhanced its bioactivity.

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.

The effect of irregularity on the dielectric dispersion characteristics of spherical cellular suspension.

The dielectric dispersion characteristics of cellular suspensions are fundamentally determined based on the analogy to composite dielectric materials when periodically and discrete arrangement of cells is assumed. However, under native physiological conditions, when flocculation and clamping events usually occur, those assumptions are usually not valid. In the framework of this study, an examination of irregularity effect on the dispersion characteristics of spherical cellular suspensions is presented. Here, the permittivity spectra of the suspensions have been determined by both measurements of living K562 cell suspensions and finite numerical simulations. Based on the measured and simulated spectra, the dispersion characteristics of the suspensions, for several destinies and arrangements of cells, have been quantitatively analyzed using the Havriliak-Negami empirical formula. Generally, a strong correlation between the low dispersion characteristics was observed as the concentration and density of the cells was increased. In addition, all characteristics found to be significantly deviated in comparison to the characteristics of a periodically arrayed suspension. However, when low-dense arrangement was assumed, the correlation found to be much lower when all characteristics found to be less perturbated. Based on a simple model of interacting cells, it is suggested that those deviations are related to intercellular interactions between adjacent cells.

Reconstruction of cell lineage trees in mice.

The cell lineage tree of a multicellular organism represents its history of cell divisions from the very first cell, the zygote. A new method for high-resolution reconstruction of parts of such cell lineage trees was recently developed based on phylogenetic analysis of somatic mutations accumulated during normal development of an organism. In this study we apply this method in mice to reconstruct the lineage trees of distinct cell types. We address for the first time basic questions in developmental biology of higher organisms, namely what is the correlation between the lineage relation among cells and their (1) function, (2) physical proximity and (3) anatomical proximity. We analyzed B-cells, kidney-, mesenchymal- and hematopoietic-stem cells, as well as satellite cells, which are adult skeletal muscle stem cells isolated from their niche on the muscle fibers (myofibers) from various skeletal muscles. Our results demonstrate that all analyzed cell types are intermingled in the lineage tree, indicating that none of these cell types are single exclusive clones. We also show a significant correlation between the physical proximity of satellite cells within muscles and their lineage. Furthermore, we show that satellite cells obtained from a single myofiber are significantly clustered in the lineage tree, reflecting their common developmental origin. Lineage analysis based on somatic mutations enables performing high resolution reconstruction of lineage trees in mice and humans, which can provide fundamental insights to many aspects of their development and tissue maintenance.

Dynamic interactions of chromatin-related mesenchymal modulator, a chromodomain helicase-DNA-binding protein, with promoters in osteoprogenitors.

The newly identified protein chromatin-related mesenchymal modulator (CReMM) is expressed by marrow stromal progenitors in vivo and ex vivo. CReMM belongs to a recently identified subgroup of chromodomain helicase-DNA-binding proteins composed of multiple domains including chromodomains, SNF2/ATPase, helicase-C domain, SANT, and A/T-hook-DNA binding domain. Chromatin immunoprecipitation assay was applied to follow the dynamics of CReMM binding to A/T-rich regions on promoters of genes that play a role in osteoblast maturation. CReMM interaction with BMP4 and biglycan promoters in the marrow stromal cells was challenged with transforming growth factor-beta. Treatment with 17beta-estradiol enhanced the binding to estrogen receptor and abolished binding to the prolactin receptor promoters; CReMM interaction with osteocalcin promoter was identified constantly. CReMM binding to the analyzed endogenous promoters suggests its direct role in the transcriptional program activated during osteogenic cell differentiation, which may be a useful tool for following the molecular mechanism of the "stemness" of mesenchymal cells.

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.

Analysis of mesenchymal cells derived from an chondrodysplasia punctuate patient and donors.

Conradi-Hunermann syndrome (CDPX2) is X-linked dominant disorder appeared with aberrant punctuate calcification. The skeletal cells derived from the marrow stroma are active in maintaining the skeletal formation. We obtained mesenchymal stem cells from a patient with CDPX2 and studied the formation of colony forming unit-fibroblast (CFU-F) in vitro in comparison cells obtained from normal donors. Cultured cells were studied morphologically and subjected to gene expression analysis. Marrow stromal cells (MSC)-chondrodysplasia punctuate (CDP) cells from CDPX2 were identified by their mosaic morphology formed three phenotypically distinct types of CFU-F colonies. One type consisted of normal fibroblasts with developed cell body and cellular processes; the second type contained pathological small cells without processes; and the third type comprised of mixed cells. We compared gene expression by the MSC-CDP to cells from normal donors. Transcription factors analyzed proliferation potential were similar in both normal and mixed colonies of MSC-CDP and similar to normal MSCs. The message expression for cytokines and extra cellular matrix (ECM) proteins revealed similar expression for biglycan, osteocalcin, and osteonectin, while IL-6, IL-11, and M-CSF mRNA levels were significantly higher in normal cells than in MSC-CDP. Mixed cells had elevated levels for IL-6 and M-CSF mRNA, but expressed IL-11 at the normal range. The studied genes were expressed at lower levels by the pathological (MSC-CDP) cells compared to normal ones. Hence, MSC-CDP was demonstrated to display abnormal morphology and transcription of several investigated genes. This study further illuminates the basis of the mosaic pattern of mesenchymal cells derived from a patient affected with CDPX2, and their gene expression involvement.