Differentiation of bone marrow stroma-derived mesenchymal cells.

Stem cell biology is a promising, fast-growing field that aims to develop cell-based tools for the treatment of a wide range of diseases. Special attention is paid to bone marrow cells that play a role in the musculoskeletal system. Bone marrow contains hematopoietic stem cells (HSCs) and stromal cells that form the reticular network that supports HSCs formation. Stromal cells also contain mesenchymal stem cells (MSCs) that differentiate to various lineages. The major obstacles in utilizing stem cells are identifying these cells and following the signaling pathways that control the cells' fate. The interactions of stem cells with an extracellular matrix and other cells create a tissue-specific niche that is responsible for regulating cell differentiation. It is important to identify biomarkers that will enable the isolation, selection and expansion of stem cells in vitro to allow their use for cell therapy. The study of stem cells' differentiation is based on various techniques, including the generation of antibodies to specific cellular sub-populations and high throughput analysis at transcriptome and proteome levels. Advances in transcriptome and proteome profiling will establish the molecular signature and will allow the discovery of the cell type specific differentiation programming. Such analysis identified several proteins related to chromatin remodeling factors, cell adhesion molecules and extracellular matrix ligands that localize cells at the specific niche. Detailed interpretation of the transcriptional and translational expression patterns will provide understanding of key properties of MSCs and allow their future use in regenerative medicine.

Characterization and functional analysis of CReMM, a novel chromodomain helicase DNA-binding protein.

The present study describes a newly identified protein named CReMM (chromatin-related mesenchymal modulator). The protein was studied by bioinformatic means and classified as a member of the third subfamily of chromodomain helicase DNA-binding proteins (CHD). In silico translation defined CReMM as a multiple domains protein including two chromodomains, SNF2/ATPase, helicase C domain and an A/T-DNA-binding domain (DBD). Predicted extensive post-translation phosphorylation on serine and tyrosine residues was demonstrated by Western blot in the presence and in the absence of phosphatase inhibitors using specific antibodies. Immunoprecipitated CReMM disclosed a DNA-dependent ATPase activity quantified by colorimetric assay. Electrophoresis mobility-shift assay (EMSA) validated that CReMM binds to A/T-rich DNA. CReMM is expressed in mesenchymal progenitors, as shown in vitro and in vivo. CReMM protein structural motifs and proven biochemical activities highlight its role in chromatin remodeling. Further delineation of the function of this protein will provide information about its dynamics in transcriptional regulation of mesenchymal cells.

Molecular and cellular characterization of mesenchymal progenitors for skeletal biomedical devices.

Mesenchymal cells are successfully used to create cell-loaded devices in tissue engineering. Molecular properties of the cells and interaction with polymer scaffolds regulate the development of desired tissues. The present study compared the molecular markers in mesenchymal pleuripotent C3H10T1/2 and osteogenic MBA-15 cells. The cells express transcription factors (TF) of chondro-ostegenic pathway (cbfa-1 and c-fos) and MyoD - TF of muscle differentiation pathway, but not myogenin. Analyzed cells expressed receptors for glucocorticoids, growth hormone, prolactin, and PTH, which indicates their potential responsiveness to systemic signals. Analysis of mRNA encoding for receptors of TGFbeta, TNF, and various interleukins revealed differential expression of IL-2r and TGFbeta-1r receptors, which were expressed by MBA-15 but not by C3H10T1/2 cells. Expression of functional genes indicates differences in the stages of cell differentiation: ALK was present in MBA-15 only, while both cell types expressed collagen-I. Furthermore, we evaluated the expression of adhesion proteins that mediate cell-polymer interactions by flow cytometry analysis. Cell adhesion molecules (CAMs) analyzed were integrinalpha-M (CD11b), selectin-E (CD62E), and PECAM-1 (CD31), which have shown differential expression on cells cultured on plastic, poly(L-lactic acid) (PLLA) or poly(DL-lactide-glycolide acid) (PDLGA) polymer films. Detailed molecular characterization of mesenchymal cells will enable optimization of culture conditions for successful creation of implantable cell-loaded constructs.

Adhesion molecule expression by osteogenic cells cultured on various biodegradable scaffolds.

Design of tissue-engineered cell-loaded device involves cells seeding onto scaffolds in vitro, allowing them to settle and grow before in vivo transplantation. Interaction between scaffold and cells is important in the development of desired tissues. The present study aimed to investigate the effect of cell-polymer interactions on cell morphology and expression of surface markers of osteogenic MBA-15 cells cultured on various bioresorbable polymers. In this study, we used various polymers: poly(L-lactic acid) (PLLA), poly(DL-lactic acid) (PDLLA), poly(L-lactic-glycolic acid) (PLGA), and poly(DL-lactide-glycolide acid) PDLGA1 and PDLGA2. Expression of integrinalpha-M (CD11b), selectin-E (CD62E), and PECAM-1 (CD31), important in cell-cell and cell-matrix interactions, were quantified by flow-cytometry analysis. Cells grown on PDLGA1 films demonstrated fivefold increase in CD62E expression and two-folds increase in CD11b expression. None of the polymers affected the levels of CD31. Identified differential effect of polymers on the expression of cell-adhesion molecules by osteoprogenitors in vitro might help to choose optimal parameters for successful engraftment of cell-loaded constructs.

Sensitivity of hematopoietic progenitors of acute myeloblastic leukemia to new compounds derived from marine organisms.

Results of chemotherapy in acute myeloid leukemia (AML) have improved slowly or not at all in the last decade. We evaluated the effect of Eilatin and Norsegoline, two new aromatic alkaloids derived from the Red Sea purple tunicate Eudistoma sp., on in vitro proliferation and differentiation of leukemic cell lines and blast cells of three AML patients. These biological properties were studied in two complementary culture methods. The first is a clonogenic assay that supports colony formation in agar and reflects terminal divisions. The second is a suspension assay where clonogenic cells increase exponentially and reflects self-renewal. Eilatin and Norsegoline, at micromolar concentrations, suppressed, in a dose-dependent manner, both primary colony formation in agar and the recovery of clonogenic cells from suspension culture in the investigated cell lines and in fresh blasts. Furthermore, both alkaloids were more effective in inhibiting clonogenic cells grown in suspension than primary colonies grown in agar. In addition, these agents were able to induce immunophenotypic maturation of leukemic cell lines (upregulation of CD14 and CD11 and down-regulation of CD34 antigens). Our results indicate that Eilatin and Norsegoline significantly inhibit self-renewal capacity of leukemic progenitors and may provide a useful new tool for the treatment of AML patients.

Growth modulation and differentiation of acute myeloid leukemia cells by jaspamide.

We have examined the effect of Jaspamide, a peptide isolated from the marine sponge Hemiastrella minor, on in vitro proliferation and differentiation of leukemic cell lines and blast cells of three AML patients and compared it to that of cytosine arabinoside (ARA-C). The biological properties were studied in two complementary culture methods. The first is a clonogenic assay that supports colony formation in agar and reflects terminal divisions. The second is a suspension assay in which clonogenic cells increase exponentially and which reflects self-renewal. Jaspamide, at micromolar concentrations and in a dose-dependent manner, suppressed both primary colony formation in agar and the recovery of clonogenic cells from suspension culture in the investigated cell lines and in fresh blasts. Furthermore, Jaspamide was more effective in inhibiting clonogenic cells grown in suspension than primary colonies grown in agar. In addition, Jaspamide, similarly to ARA-C, was able to induce immunophenotypic maturation of leukemic cell lines (upregulation of CD14 and CD11 and downregulation of CD34 antigens). Our results indicate that Jaspamide significantly inhibits the self-renewal capacity of leukemic progenitors and may provide a new useful tool for the treatment of acute myeloid leukemia (AML) patients.

Alternatively spliced isoforms of a novel stromal RNA regulating factor.

Bone marrow stromal cells (MSC) are pluripotent cells that possess a unique capacity to differentiate under appropriate conditions into various lineages. The MSC differentiation is dependent on factors that can switch on and maintain a relevant genetic program to make a particular cell type. The present study describes the cloning and molecular analysis of a novel gene, SRRF (Stromal RNA Regulating Factor), suggested to be involved in RNA processing in MSC. We cloned two alternatively spliced isoforms of this gene, transcripts A and B, from the marrow stromal cells expression library. Differential expression analysis demonstrated a restricted expression of the transcripts to MSC, while other spliced forms of this gene were detected in other tissues. The bioinformatic analysis of the two isoforms revealed RNA binding motifs (RRM), protein-protein and protein-DNA interaction motifs. Participation of SRRF isoforms in post-transcriptional events in MSC is believed to govern the tissue specificity of RNA transcription and to have an important role in regulation of the RNA expression that directs the MSC differentiation pathway.

Granulocyte-macrophage colony-stimulating factor dependent monocyte-mediated cytotoxicity post-autologous bone marrow transplantation.

We investigated the in vitro antitumor activity of monocytes derived from autologous bone marrow transplanted (ABMT) patients treated in vivo with granulocyte-macrophage colony-stimulating factor (GM-CSF). Thirty-four patients (17 female, 17 male), median age 42 (range 3-57) years, were enrolled in the study. Fourteen patients were diagnosed with non-Hodgkin's lymphoma (NHL), eight with Hodgkin's disease (HD), nine with breast cancer and three with neuroblastoma. Six patients who did not receive GM-CSF post-ABMT served as controls. We assessed cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC), expression of the activation antigen CD16, and cytokine production by an enriched population of monocytes (> 90% CD+14) pre-, during and post-GM-CSF administration. Within the group of patients receiving treatment, ADCC was significantly higher during in vivo GM-CSF administration than post-therapy (P < 0.05) and in 50% of these patients, ADCC increased during in vivo GM-CSF administration over pretreatment values. In addition, in vivo GM-CSF administration caused the monocytes to secrete elevated levels of tumor necrosis factor-alpha (TNF-alpha) and GM-CSF (P < 0.05). We conclude that GM-CSF augments monocyte-mediated cytotoxicity post-ABMT, and therefore may have a role in controlling minimal residual disease post-transplant.

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 17beta-Estradiol (17betaE2). 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 17betaE2 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 17betaE2 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.

In vivo association of CReMM/CHD9 with promoters in osteogenic cells.

Molecular mechanisms that control cell differentiation involve with chromatin remodeling activities. We recently identified Chromatin Related Mesenchymal Modulator (CReMM), a CHD protein expressed by mesenchymal cells. In this study, we analyzed CReMM expression on RNA and protein levels during embryonic development in mouse skeletal tissues. CReMM appears transiently during mesenchymal cell differentiation, being detected first in osteoprogenitors and declining in mature cells. A novel aspect of the study elaborates on in vivo association of CReMM with promoters in cells obtained by laser capture micro-dissection (LCM) technique from periosteum and endochondreal ossification regions. Using chromatin immunoprecipitation (ChIP), we proved that CReMM binds to skeletal tissue-specific promoters: CBFA1, biglycan, osteocalcin (OC), collagen-II, and myosin in a differential manner. The results imply that CReMM selectively interacts with analyzed promoters activated in the tissue at the appropriate time of development. The identification of CReMM and its tissue distribution and function provides an attractive clue for the study of transcriptional regulation of osteogenic cells' maturation.

Expression and regulation of CReMM, a chromodomain helicase-DNA-binding (CHD), in marrow stroma derived osteoprogenitors.

This study follows the expression of CReMM, a new CHD family member, in osteoprogenitors. CReMM expression was analyzed in primary cultured mesnchymal cells from rat and human. Analysis in ex vivo cultured marrow stromal cells (MSC) from rats revealed higher level of CReMM in cells from young (3 months), when compared to cells from old (15 months) rats. CReMM level was higher in human MSC then in mature trabecular bone cells (TBC). Within the MSC population, osteogenic clones showed higher levels of CReMM then non-osteogenic ones. We used bone marrow derived osteogenic cell line (MBA-15) to elaborate on the regulation of CReMM expression in correlation with cell proliferation and co-expression with alkaline phosphatase (ALK). CReMM is highly expressed in proliferating cells and is inversely related to expression of ALK. MBA-15 cells were challenged with dexamethasone (Dex) or 17beta-estradiol and quantification of CReMM at the protein (ELISA) and mRNA (RT-PCR) levels had shown that Dex upregulated CReMM levels. Since CReMM is regulated by Dex, we analyzed the interaction of CReMM with the glucocorticoid receptor (GR), which mediates Dex action. Co-immunopercipitation (Co-IP) demonstrated an association between CReMM and GR. In summary, CReMM is a CHD protein expressed by osteoprogenitors, and we suggest it plays a role in mediating transcriptional response to hormones that coordinate osteoblast function.

Molecular and cellular characterization of SEL-OB/SVEP1 in osteogenic cells in vivo and in vitro.

We describe a novel human gene, named SEL-OB/SVEP1, expressed by skeletal tissues in vivo and by cultured osteogenic cells. The mRNA expression was analyzed on frozen tissues retrieved by laser-capture microscope dissection (LCM) and was detected in osteogenic tissues (periosteum and bone) but not in cartilage or skeletal muscle. The SEL-OB/SVEP1 cDNA of 11,139 bp was in silico translated into a 3574AA protein with expected molecular weight of 370 kDa. The protein is composed of multiple domains including complement control protein (CCP) modules with selectin superfamily signature; sushi and other domains, such as vWA, EGF, PTX, and HYR. Stromal osteogenic cells were analyzed for the protein expression using anti-SEL-OB/SVEP1 for immuno-precipitation and Western blot application confirm the presence of high molecular weight protein. Immuno-histochemistry and fluorescence-activated cell sorting (FACS) were applied to detect SEL-OB/SVEP1 on the surface of stromal cells. ELISA quantified the dependence of protein expression on cell density. Bioinformatic analysis of SEL-OB/SVEP1 revealed domains compositions recognized in cell surface molecules and suggested its role in cell adhesion. Analysis of mesechymal osteogenic cells' adhesion in presence of anti-SEL-OB/SVEP1 antibody demonstrated its interference with initial adhesion stages. In summary, present study describes novel SEL-OB/SVEP1 protein with a unique composition of functional domains, restricted pattern of expression in skeletal cells and demonstrated involvement in attachment of mesenchymal cells. The unusual composition of functional domains puts SEL-OB/SVEP1 in the discrete new group of membrane proteins involved in cell adhesion processes. All together makes SEL-OB/SVEP1 an attractive marker for studying the role of stromal osteogenic cells and their interactions within the bone marrow microenvironment creating a network that regulates the skeletal homeostasis.

Characterization of adhesion and differentiation markers of osteogenic marrow stromal cells.

Marrow stroma cells (MSC) play a major role in osteogenesis. The potential of the MSC to differentiate to bone-forming cells relies upon molecular regulation. This study analyzed MBA-15 cells for the expression of genes and proteins that are key regulators of osteoblast differentiation. These cells express Cbfa1 and c-fos transcription factors (TF) of osteoprogenitor proliferating cells. RT-PCR and immunohistochemistry were used to demonstrate the message and protein expression of extracellular matrix proteins that are a prerequisite for matrix formation and mineralization, including alkaline phosphatase (ALP), osteocalcin, osteopontin, biglycan, and bone sialoprotein (BSP). The activity of ALP was correlated at various cell densities with co-expression of osteocalcin or osteopontin. Adhering cells must attach to the appropriate matrix to enable survival and differentiation. Using attachment assays, we demonstrated that MBA-15 cells adhered to collagenous matrix and the effect on survival measured by changes in intracellular calcium (Ca) levels. The cells' adhesion to matrix is mediated via cell surface molecules. We quantified the expression of cells surface molecules that are important players in mediating cell-matrix interaction. Flow cytometry analysis (FACS) was used to determine the expression of CD-31 (36%), and lower levels were identified for CD-62E and CD11b. In summary, the present study demonstrates the expression of molecular markers that are distinctive for the osteoblastic phenotype in MBA-15 marrow stroma cells and have crucial role in cell-matrix interaction, in establishing the cellular osteogenic phenotype and their survival.

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.

Hormonal changes affect the bone and bone marrow cells in a rat model.

In this study, we used a rat model to investigate the effects of gonad hormones and replacement therapy on bone structure and the immune system. In the first phase of the study, 3- and 11-month-old F344 rats underwent ovariectomy (OVX) or were sham operated. Three months later, severe osteopenia was histologically observed in OVX rats of both age groups. The changes in the bone marrow structure of OVX rats included deterioration of cancellous bone that was associated with a remarkable increase of adipocyte cells. Furthermore, differential analyses for the expression of cell surface antigens by lymph-myeloid cells was studied using flow cytometry (FACS). The number of myeloid cells expressing ED-9(+) or CD-44(+) was similar in both age groups, and unaffected by OVX. However, an augmentation of T-lymphoid cells expressing CD4(+), CD5(+), or both, were observed with age, as well as after OVX. In the second phase of the study, 11-month-old rats were divided into five experimental groups: sham-operated, OVX, and OVX treated with sustained-release pellets of 17beta-estradiol (OVX-E), progesterone (OVX-P), or both (OVX-E/P). Hormone replacement therapy maintained low physiological levels, and rats were tested 12 weeks after treatment initiation. Administration of 17beta-E, with or without the addition of progesterone, prevented the rise of T lymphoid cells observed in OVX rats, whereas progesterone alone had no effect. In agreement with findings from the first phase, neither OVX nor replacement therapy affected the myeloid cells expression of ED-9 or CD-44. In summary, the cellular changes in the bone marrow of OVX rats were associated with an increase in adipocytes that was correlated with bone atrophy. An augmentation of T-lymphopoiesis was noted with increase in age or after OVX. This increase was reversed to baseline levels by 17beta-E treatment.

Differential gene expression of cultured human osteoblasts.

Human cells with osteogenic capacity were studied for differential gene expression. In the first part of the study we compared gene expression of marrow stroma cells (MSC) in comparison to matured osteoblasts cultured from trabecular bone (TBC) that were analyzed by RT-PCR for series of messages. High expression was detected for PTH-r, TGFb1 and biglycan in TBC compared to MSC's. The messages for c-MYC, IL-6, IL-11, M-CSF, osteonectin, and osteocalcin were expressed at the same level in the two populations of cells. In the second part of the study, we analyzed gene expression within the MSC derived from 25 donors (2.5-49 years old) with respect to donors' age and gender. Increased message levels for M-CSF and biglycan were measured in correlation with age of the donors. Gender differences did not affect the expression of cytokines studied (IL-6, IL-11, MCSF, TGFb1). We investigated the effect of Dexamethasone treatment on MSC and monitored an increased expression of IL-11, M-CSF, biglycan, and osteocalcin messages. This study employs primary cell systems (MSC and TBC) to illustrate differential gene expression by osteoblastic cells. The expression was correlated with maturation status of the cells with respect to differences between donors.

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.

Cellular and molecular properties associated with osteosarcoma cells.

Osteosarcoma cells are recognized by abnormal function that causes a primary bone tumor. Osteosarcoma cells U(2)OS and SAOS-2 were analyzed for the expression of cell surface markers. High expression was quantified for hyaloronidase receptor (CD-44) > moderate for integrins (CD-51 and -61), > and lower for selectins (CD-62). High mitotic capacity were demonstrated by gene expression (measured by RT-PCR) and the protein level (measured by FACS) for cFOS, cMYC, and cJUN. The basic definition of osteosarcoma is excessive production of pathological osteoid. Expression of mRNA for matrix genes osteocalcin, osteonectin, and biglycan was studied. Osteocalcin and osteonectin were detected in RNA from primary cultured marrow stromal, trabecular bone cells, and osteosarcoma cell lines (U(2)OS, SAOS-2). mRNA for biglycan was detected only in primary cells and MG-63 cell line and was undetectable in RNA from U(2)OS, SAOS-2 osteosarcoma cell lines and by RNA extracted from bone biopsies of osteosarcoma patients. The absence of biglycan message observed in osteosarcoma samples provides evidence for the alterations in the extra cellular matrix which result with non-mineralized osteoid produced by the osteosarcoma cells.

[Detection of staphylococcal protein A in broth cultures using counter electrophoresis]. / Vyiavlenie proteina A stafilokokkov v bul'onnykh kul'turakh metodom vstrechnogo élektroforeza.

The diagnostic value of counter-current electrophoresis for the detection of Staphylococcus aureus protein A in the material from patients, carriers, and environmental objects is demonstrated: this method identifies 99.8% of S. aureus cultures. Microorganisms of other species and physiologic groups, not incorporating protein A, are undetectable by counter-current electrophoresis.