Publications
2018
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(2018) Cell Death and Disease. 9, 695. Abstract
Fas-L is a TNF family member known to trigger cell death. It has recently become evident that Fas-L can transduce also non-apoptotic signals. Mesenchymal stem cells (MSCs) are multipotent cells that are derived from various adult tissues. Although MSCs from different tissues display common properties they also display tissue-specific characteristics. Previous works have demonstrated massive apoptosis following Fas-L treatment of bone marrow-derived MSCs both in vitro and following their administration in vivo. We therefore set to examine Fas-L-induced responses in adipose-derived stem cells (ASCs). Human ASCs were isolated from lipoaspirates and their reactivity to Fas-L treatment was examined. ASCs responded to Fas-L by simultaneous apoptosis and proliferation, which yielded a net doubling of cell quantities and a phenotypic shift, including reduced expression of CD105 and increased expression of CD73, in association with increased bone differentiation potential. Treatment of freshly isolated ASCs led to an increase in large colony forming unit fibroblasts, likely produced by early stem cell progenitor cells. Fas-L-induced apoptosis and proliferation signaling were found to be independent as caspase inhibition attenuated Fas-L-induced apoptosis without impacting proliferation, whereas inhibition of PI3K and MEK, but not of JNK, attenuated Fas-L-dependent proliferation, but not apoptosis. Thus, Fas-L signaling in ASCs leads to their expansion and phenotypic shift toward a more potent stem cell state. We speculate that these reactions ensure the survival of ASC progenitor cells encountering Fas-L-enriched environments during tissue damage and inflammation and may also enhance ASC survival following their administration in vivo.
2016
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(2016) Oncotarget. 7, 41, p. 67061-67070 Abstract
Introduction: Multiple myeloma is still incurable in most cases. Polyclonal anti T lymphocyte globulins (ATG) have been reported to kill human myeloma cells in vitro and in mouse models. Methods: Anti-human-myeloma globulins (AMG) were produced by immunizing rabbits with human myeloma cell lines RPMI-8226 (AMG-8226) or KMS-12-BM (AMG- 12-BM). Cytotoxicity of the polyclonal antibodies was analyzed in vitro and in a xenograft NOD-SCID mouse model. Results: Both AMG had stronger cytotoxicity against myeloma cells compared to ATG. In primary T cells, AMG-8226 showed greater complement-dependent cytotoxicity (CDC) than ATG, whereas complement-independent cytotoxicity did not differ. Effects on non-hematopoietic cell lines were also similar. Competitive blocking assays revealed fourfold more antibodies against CD38 in AMG-8226 compared to ATG. Low concentrations of AMG-8226 and ATG increased ADCC. At higher concentrations, ATG inhibited ADCC more potently than AMG-8226. Combinations of ATG and AMG- 8226 with melphalan or bortezomib showed additive to synergistic cytotoxicity on myeloma cells. The cytotoxic effects of AMG and ATG were confirmed in the xenograft NOD-SCID mouse model. Conclusion: Our data show more potent antimyeloma effects of AMG compared to ATG. These results lay the ground for the development of polyclonal antibodies for the treatment of multiple myeloma.
2015
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(2015) RNA & disease. 2, Supplement 1, e780. Abstract
Mesenchymal stem/stromal cells (MSCs) are multipotent progenitors that are derived from most adult tissue as well as cord blood and placenta. MSCs are defined by their adherent nature, ability to propagate in culture and capacity to differentiate into bone fat and cartilage. However, many studies have shown that MSCs, derived from different tissues, differ both in their in situ and in vitro phenotypes. Despite abundance of MSCs studies, little is known about the molecular events that control their tissue specific nature. Two recent studies comparing MSCs derived from different tissues have now found clues to the molecular mechanisms that control the tissue specific nature ofthese cells. In the first, the superior genomic stability of adipose derived MSCs (ASCs), compared to bone marrow (BM) MSCs, was explained by reduced H19, a long non-coding RNA expression and increased p53 activity of ASCs. In the second, a compression of abdominal and subcutaneous ASCs reveals poor propagation, differentiation and migration capacities of abdominal ASCs that is explained by their increased tendency to over-accumulate reactive oxygen species (ROS) in culture. ROS over production in abdominal ASCs was shown to be controlled by the NADPH oxidase NOX1. The unique features of MSCs derived from different tissues suggest a tissue specific molecular signature arising from the tissue of origin that is retained during culture. The implications of this phenomenon on our understanding of the role and function of MSCs in situ as well as on their clinical utilization, is discussed.
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(2015) Stem Cell Reviews and Reports. 11, 6, p. 826-840 Abstract
Mesenchymal stem cells (MSCs) serve as supporting and regulatory cells, by providing tissues with multiple factors and are also known for their immunosuppressive capabilities. Our laboratory had previously shown that MSCs expressed toll-like receptor (TLR) 2 and are activated by its ligand Pam3Cys. TLR2 is an important component of the innate immune system, as it recognizes bacterial lipopeptides, thus priming a pro-inflammatory immune response. This study showed that Pam3Cys attached extensively to cells of both wild-type and TLR2 deficient cultured MSCs, thus, independently of TLR2. The TLR2 independent binding occurred through the adsorption of the palmitoyl moieties of Pam3Cys. It was further showed that Pam3Cys was transferred from cultured MSCs to immune cells. Moreover, Pam3Cys provided to the immune cells induced a pro-inflammatory response in vitro and in vivo. Overall, it is demonstrated herein that a TLR2 ligand bound to MSCs also through a TLR2 independent mechanism. Furthermore, the ligand incorporated by MSCs is subsequently released to stimulate an immune response both in vitro and in vivo. It is thus suggested that during bacterial infection, stromal cells may retain a reservoir of the TLR2 ligands, in a long-term manner, and release them slowly to maintain an immune response.
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(2015) Biochimica et Biophysica Acta - Gene Regulatory Mechanisms. 1849, 4, p. 371-377 Abstract
Over a decade of intensive investigation of the possible plasticity of mammalian cells has eventually substantiated that mammalian species are endowed with a remarkable capacity to change mature cell fates. We review below the evidence for the occurrence of processes such as dedifferentiation and transdifferentiation within mammalian tissues in vivo, and in cells removed from their protective microenvironment and seeded in culture under conditions poorly resembling their physiological state in situ. Overall, these studies point to one major conclusion: stressful conditions, whether due to in vivo tissue damage or otherwise to isolation of cells from their in vivo restrictive niches, lead to extreme fate changes. Some examples of dedifferentiation are discussed in detail showing that rare cells within the population tend to turn back into less mature ones due to severe cell damage. It is proposed that cell stress, mechanistically sensed by isolation from neighboring cells, leads to dedifferentiation, in an attempt to build a new stem cell reservoir for subsequent regeneration of the damaged tissue. This article is part of a Special Issue entitled: Stress as a fundamental theme in cell plasticity.
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2014
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(2014) Stem Cells. 32, 8, p. 2008-2020 Abstract
Mesenchymal stromal cell populations include a fraction, termed mesenchymal stem cells, exhibiting multipotency. Other cells within this population possess a lesser differentiation range. This was assumed to be due to a mesenchymal cellular cascade topped by a multipotent cell, which gives rise to progeny with diminishing differentiation potentials. Here, we show that mesenchymal cells, a priori exhibiting a limited differentiation potential, may gain new capacities and become multipotent following single-cell isolation. These fate changes were accompanied by upregulation of differentiation promoting genes, many of which also became H4K20me1 methylated. Early events in the process included TGFβ and Wnt modulation, and downregulation of hypoxia signaling. Indeed, hypoxic conditions inhibited the observed cell changes. Overall, cell isolation from neighboring partners caused major molecular changes and particularly, a newly established epigenetic state, ultimately leading to the acquisition of new differentiation potentials and an altered cell fate.
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(2014) Stem Cell Reviews and Reports. 10, 3, p. 376-388 Abstract
The outstanding heterogeneity of stem cell populations is a major obstacle on the way to their clinical application. It is therefore paramount to identify the molecular mechanisms that underlay this heterogeneity. Individually derived bone marrow mesenchymal stromal cells (MSCs) preparations, studied here, diverged markedly in various properties, despite of being all tripotent in their differentiation potential. Microarray analysis showed that MSC diversity is evident also in highly variable gene expression patterns. Differentially expressed genes were significantly enriched in toll-like receptors (TLRs) and differentiation pathways. Marked differences were observed in LPS binding protein (LBP) and transforming growth factor (TGF)β1 expression. These differences correlated with MSC functionality. Therefore, the possible contribution of these molecules to MSC diversity was examined. In the TLR signaling pathway, LBP levels predicted the ability of specific MSCs to secrete interleukin (IL)-6 in response to LPS. A relatively higher expression of TGFβ1 endowed MSCs with a capacity to respond to IL-1β by reduced osteogenic differentiation. This study thus demonstrates major diversity within MSC isolates, which appears early on following derivation and persists following long-term culture. MSC heterogeneity results from highly variable transcriptome. Differential expression of LBP and TGFβ1, along with other genes, in different MSC preparations, produces the variable responses to external stimuli.
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(2014) Stem Cells and Development. 23, 6, p. 676-686 Abstract
Umbilical cord blood (UCB) is a good source of hematopoietic progenitors with increasing implementation in the clinical transplant setting. This study evaluates the molecular mechanisms of progenitor resistance to apoptosis triggered by Fas cross-linking. CD34(+) and lineage-negative progenitors survive short-term ex vivo incubation and are not induced into apoptosis by Fas cross-linking. Furthermore, brief exposure of UCB cells to Fas-ligand for 24-48 h does not impair quantitative severe combine immune deficiency (SCID) reconstitution activity and appears to foster myelomonocyte reconstitution. The transcriptome of Fas receptor-positive CD34(+) cells that survived an apoptotic challenge showed significant transcriptional upregulation of caspase-8, mucosa-associated lymphoid tissue lymphoma translocation gene-1 (MALT1), HtrA2, and GSK3 beta in addition to higher levels of c-FLICE inhibitory protein (FLIP), Bcl-2, and cytosolic inhibitor of apoptosis protein (cIAP) in all Fas-positive cells. Most prominent is the transcriptional upregulation of several key components the NF kappa B1 pathway including the membrane receptors TGF-beta, interleukin-1 (IL-1), and TCR, the associated factor TNF receptor-associated factor-6 (TRAF6), and the converting enzymes TGF-beta-activated kinase-1 (TAK1), double-stranded RNA-activated protein kinase (PKR), and alpha-catalytic subunit of I kappa B kinase (IKK alpha), that promote activation and nuclear translocation of this transcription factor. These data indicate that hematopoietic progenitors are not insensitive to apoptosis but are actively shielded from the extrinsic and intrinsic apoptotic pathways. This may occur through inherent transcriptional upregulation of the entire NF kappa B pathway in the presence of competent apoptotic signaling.
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(2014) Stem Cell Research & Therapy. 5, 6, 139. Abstract
Introduction: Mesenchymal stem cells (MSCs) are multipotent and have been derived from various tissues. Although MSCs share many basic features, they often display subtle tissue specific differences. We previously demonstrated that bone marrow (BM) MSCs frequently become polyploid in culture. This tendency was mediated by a reduction in the expression of H19 long non-coding RNA during the transition from a diploid to a polyploid state. Methods: MSCs were derived from both BM and adipose tissue of mice and expanded under normoxic and hypoxic culture conditions. Cells were stained by propidium iodide and their ploidy was evaluated by FACS. Gene expression of independent MSC preparations was compared by quantitative real time PCR and protein expression levels by Western blot analysis. p53 silencing in MSCs was performed by a specific small hairpin RNA (shRNA). Results: We set to examine whether genomic instability is common to MSCs originating from different tissues. It is demonstrated that adipose derived MSCs (ASCs) tend to remain diploid during culture while a vast majority of BM MSCs become polyploid. The diploid phenotype of ASCs is correlated with reduced H19 expression compared to BM MSCs. Under hypoxic conditions (3% oxygen) both ASCs and BM MSCs demonstrate increased RNA expression of H19 and Vascular endothelial growth factor A. Importantly, ASC gene expression is significantly less variable than BM MSCs under both oxygen conditions, indicating to their superior homogeneity. Gene expression analysis revealed that p53 target genes, often induced by DNA damage, are up-regulated in ASCs under basal conditions. However, p53 activation following treatment with DNA damaging agents was strongly elevated in BM MSCs compared to ASCs. We found that p53 is involved in maintaining the stable diploid state of ASCs as p53 shRNA induced ploidy changes in ASCs but not in BM MSCs. Conclusions: The increased genomic stability of murine ASCs together with their lower H19 expression and relative homogeneity suggest a tissue specific higher stability of ASCs compared to BM MSCs, possibly due to higher activity of p53. The tissue specific differences between MSCs from a different tissue source may have important consequences on the use of various MSCs both in vitro and in vivo.
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Self-Renewal, Induced Proliferation, and Autonomous Cell Growth Represent Distinct Modes of Cell Multiplication: Relevance to the Cancer Stem Cell Theory(2014) CANCER STEM CELLS. p. 39-47 Abstract
2013
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(2013) Journal of the American Heart Association. 2, 5, e000253. Abstract
Background--Human mesenchymal stromal cells (hMSCs) from adipose cardiac tissue have attracted considerable interest in regard to cell-based therapies. We aimed to test the hypothesis that hMSCs from the heart and epicardial fat would be better cells for infarct repair. Methods and Results--We isolated and grew hMSCs from patients with ischemic heart disease from 4 locations: epicardial fat, pericardial fat, subcutaneous fat, and the right atrium. Significantly, hMSCs from the right atrium and epicardial fat secreted the highest amounts of trophic and inflammatory cytokines, while hMSCs from pericardial and subcutaneous fat secreted the lowest. Relative expression of inflammation- and fibrosis-related genes was considerably higher in hMSCs from the right atrium and epicardial fat than in subcutaneous fat hMSCs. To determine the functional effects of hMSCs, we allocated rats to hMSC transplantation 7 days after myocardial infarction. Atrial hMSCs induced greatest infarct vascularization as well as highest inflammation score 27 days after transplantation. Surprisingly, cardiac dysfunction was worst after transplantation of hMSCs from atrium and epicardial fat and minimal after transplantation of hMSCs from subcutaneous fat. These findings were confirmed by using hMSC transplantation in immunocompromised mice after myocardial infarction. Notably, there was a correlation between tumor necrosis factor- α secretion from hMSCs and posttransplantation left ventricular remodeling and dysfunction. Conclusions--Because of their proinflammatory properties, hMSCs from the right atrium and epicardial fat of cardiac patients could impair heart function after myocardial infarction. Our findings might be relevant to autologous mesenchymal stromal cell therapy and development and progression of ischemic heart disease.
2012
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(2012) Cancer Research. 72, 24, p. 6403-6413 Abstract
Mesenchymal stromal cells (MSC) are used extensively in clinical trials; however, the possibility that MSCs have a potential for malignant transformation was raised. We examined the genomic stability versus the tumor-forming capacity of multiple mouse MSCs. Murine MSCs have been shown to be less stable and more prone to malignant transformation than their human counterparts. A large series of independently isolated MSC populations exhibited low tumorigenic potential under syngeneic conditions, which increased in immunocompromised animals. Unexpectedly, higher ploidy correlated with reduced tumor-forming capacity. Furthermore, in both cultured MSCs and primary hepatocytes, polyploidization was associated with a dramatic decrease in the expression of the long noncoding RNA H19. Direct knockdown of H19 expression in diploid cells resulted in acquisition of polyploid cell traits. Moreover, artificial tetraploidization of diploid cancer cells led to a reduction of H19 levels, as well as to an attenuation of the tumorigenic potential. Polyploidy might therefore serve as a protective mechanism aimed at reducing malignant transformation through the involvement of the H19 regulatory long noncoding RNA.
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(2012) Stem Cell Reviews and Reports. 8, 2, p. 343-354 Abstract
In human multiple myeloma (MM), the tumor cells exhibit strict dependence on bone marrow (BM) stromal elements. It has been suggested that, in turn, MM cells modify multipotent stromal cells (MSCs), diverting them to support the myeloma. We investigated MM-derived MSCs by comparing their toll-like receptor (TLR) responses to those of MSCs derived from healthy controls. We now report that MM-derived MSCs manifested intact proliferation responses and IL-6 secretion and their adipose and osteogenic differentiation responses to TLR ligands were also similar to those of healthy controls, ranging from augmentation to inhibition. However, MM-derived MSCs were found to be defective in IL-8 secretion and ERK1/2 phosphorylation following TLR-2 activation. Moreover, MM-derived MSCs failed to respond to EGF by elevation of ERK1/2 phosphorylation. The persistence of these changes in extensively cultured MM-derived MSCs, suggests that these cells are stably, if not irreversibly modified.
2011
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(2011) Molecular Immunology. 49, 2-Jan, p. 239-252 Abstract
The existence of incomplete T cell receptor (TCR) mRNA forms, including germline transcripts and products of unfruitful TCR rearrangements, has long been known. However, it is unclear whether these molecules are functional. We have previously shown that T cells also contain truncated TCR beta peptides that lack the N-terminal part and contain C-terminus sequences. These partial forms of TCR beta, target the mitochondrion and induce apoptosis, exhibiting a novel mode of TCR mediated cell death. Here we aimed at analyzing the minimal TCR sequences that direct the peptide to the mitochondrion. It is shown that truncated TCR beta, targets mitochondria and induces mitochondrial perinuclear clustering, in both monkey COS-7 and human 293 cells. These phenomena are mediated by the C-terminus of the molecule. Whereas the positively charged amino acids flanking the transmembrane domain (TMD) of TCR beta are beneficial for this process, they are not essential. Indeed, the isolated TMD of TCR beta serves as a sufficient mitochondrial targeting sequence. These results indicate that any given partial form of TCR beta, that contains the TMD, is bound to be sequestered by the mitochondrion. This may assure that incomplete TCR forms would not interfere with correct TCR complex formation. (C) 2011 Elsevier Ltd. All rights reserved.
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(2011) Stem Cell Reviews. 7, 3, p. 560-568 Abstract
Cultured mesenchymal stromal cell (MSC) populations are best characterized by the capacity of some cells within this population to differentiate into mesodermal derivatives such as osteoblasts, chondrocytes and adipocytes. However, this progenitor property is not shared by all cells within the MSC population. Furthermore, MSCs exhibit variability in their phenotypes, including proliferation capacity, expression of cell surface markers and ability to secrete cytokines. These facts raise three major questions: (1) Does the in vitro observed variability reflect the existence of MSC subsets in vivo? (2) What is the molecular basis of the in vitro observed heterogeneity? and (3) What is the biological significance of this variability? This review considers the possibility that the variable nature of MSC populations contributes to the capacity of adult mammalian tissues to adapt to varying microenvironmental demands.
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Towards a molecular rather than a descriptive definition of stemness(2011) M S-Medecine Sciences. 27, 3, p. 303-307 Abstract
Towards a molecular rather than a descriptive definition of sternness Stem cells are a non-coherent group of cells that have little in common Despite the fact that these diverse cell types are regarded as belonging to the some category, they do not share molecular markers. The definition of sternness is therefore descriptive, relating to potentials of the cells rather than to the actual properties that they harbor. This situation is confusing and causes unnecessary debates in this field of research. It is therefore of paramount importance to find a new, molecular definition of sternness, that would consist of the cellular machineries which constitute the stem cell state
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(2011) Stem cell reviews and reports.. 7, 3, p. 488-493 Abstract
Differentiation cascades are arranged hierarchically; stem cells positioned at the top of the hierarchy generate committed progenitors that, in turn, proliferate and further differentiate stepwise into mature progeny. This rigid, irreversible structure ensures the phenotypic stability of adult tissues. However, such rigidity may be problematic under conditions of tissue damage when reconstitution is required. Although it may seem unlikely that the restrictions on changes in cell phenotypes would be lifted to enable tissue reconstitution, it is nevertheless possible that mammalian tissues are endowed with sufficient flexibility to enable their adaptation to extreme conditions.
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(2011) Cell Stem Cell. 8, 1, p. 10-11 Abstract
Mesenchymal stromal cells (MSCs) are capable of differentiating into bone-forming osteoblasts. A recent Nature Medicine study (Medici et al., 2010) shows that the mislocalized bone in the human disease fibrodisplasia ossificans progressiva (FOP) originates from vascular endothelium that gives rise to MSCs.
2010
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(2010) From Molecular To Modular Tumor Therapy: Tumors Are Reconstructible Communicatively Evolving Systems. p. 75-96 Abstract
Mesenchymal stromal cells were first isolated from the bone marrow, where they serve as a component of the tissue microenvironment. These cells provide a physical support for the other cells of the tissue; i.e., the hemopoietic cell lineage, and further participate in the formation of bone structures. Most importantly, stromal cells regulate the growth and differentiation of hemopoietic stem cells. The mesenchyme is not specific to the bone marrow: such cells are found body-wide, and serve similar regulatory functions. By the same token, the mesenchymal stroma contributes to tumor formation by providing regulatory signals. In addition, the stromal cells themselves may undergo transformation, and subsequently form tumors. This chapter discusses these two major aspects of stromal cell involvement in the tumorigenic process.
2009
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(2009) Leukemia. 23, 8, p. 1378-1388 Abstract
Cytopenia represents a significant complication after chemotherapy, irradiation before bone marrow (BM) transplantation or as a therapy for cancer. The mechanisms that determine the pace of BM recovery are not fully understood. During the recovery phase after chemotherapy or irradiation, the signals for retention of white blood cells within the BM increase significantly. This leads to a delay in the release of WBC, which can be overcome by targeting the CXCR4 axis with the antagonist 4F-benzoyl-TN14003 (T140). The delay in the release of WBC is also accompanied by suppression in the production of progenitor cells and mature cells by the BM stroma. Administration of T140 to mice transplanted with BM cells stimulates the production of all types of progenitors and mature cells, and increases the exit of mature cells to the periphery. Moreover, addition of T140, but not AMD3100, to BM stromal cultures stimulates the production of mature cells and progenitors from all lineages. The unique ability of the CXCR4 antagonist, T140 to stimulate the production and exit of WBC cells may be used as a novel therapeutic approach to overcome cytopenia associated with treatments for cancer and BM transplantation. Leukemia (2009) 23, 1378-1388; doi:10.1038/leu.2009.56; published online 26 March 2009
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(2009) Experimental Cell Research. 315, 11, p. 1904-1913 Abstract
Multiple myeloma is characterized by the malignant growth of immunoglobulin producing plasma cells, predominantly in the bone marrow. The effects of primary human mesenchymal stromal cells on the differentiation phenotype of multiple myeloma cells were studied by co-culture experiments. The incubation of multiple myeloma cells with bone marrow-derived mesenchymal stromal cells resulted in significant reduction of the expression of the predominant plasma cell differentiation markers CD38 and CD138, and cell surface immunoglobulin light chain. While the down-regulation of CD138 by stromal cells was completely dependent on their adhesive interactions with the multiple myeloma cells, interleukin-6 induced specific down-regulation of CD38. Mesenchymal stromal cells or their conditioned media inhibited the growth of multiple myeloma cell line, thereby reducing the overall amounts of secreted light chains. Analysis of primary multiple myeloma bone marrow samples reveled that the expression of CD38 on multiple myeloma cells was not affected by adhesive interactions. The ex vivo propagation of primary multiple myeloma cells resulted in significant increase in their differentiation markers. Overall, the data indicate that the bone marrow-derived mesenchymal stromal cells revert multiple myeloma cells to less differentiated phenotype by the combined activities of adhesive interactions and interleukin-6.
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(2009) Blood. 113, 15, p. 3530-3541 Abstract
The default pathway of cell-surface T-cell receptor (TCR) complex formation, and the subsequent transport to the membrane, is thought to entail endoplasmic reticulum (ER) localization followed by proteasome degradation of the unassembled chains. We show herein an alternative pathway: short, incomplete peptide versions of TCR beta naturally occur in the thymus. Such peptides, which have minimally lost the leader sequence or have been massively truncated, leaving only the very C terminus intact, are sorted preferentially to the mitochondrion. As a consequence of the mitochondrial localization, apoptotic cell death is induced. Structure function analysis showed that both the specific localization and induction of apoptosis depend on the transmembrane domain (TMD) and associated residues at the COOH-terminus of TCR. Truncated forms of TCR, such as the short peptides that we detected in the thymus, may be products of protein degradation within thymocytes. Alternatively, they may occur through the translation of truncated mRNAs resulting from unfruitful rearrangement or from germline transcription. It is proposed that mitochondria serve as a subcellular sequestration site for incomplete TCR molecules. (Blood. 2009; 113: 3530-3541)
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(2009) Heidelberg: . (trueStem Cell Biology and Regenerative Medicine). Abstract
Biology of Stem Cells and the Molecular Basis of the Stem State concentrates upon adult stem cells, particularly on mesenchymal cell populations, which are the authors area of expertise. The text offers the reader a detailed description of the emergence of stem cell research and the dogmas that were created during the first decades of analysis of stem cell properties, particularly those of hemopoietic stem cells. Biology of Stem Cells and the Molecular Basis of the Stem State also introduces the reader to the commonly accepted notions regarding stem cell biology, with an emphasis on an alternative view of stemness, i.e. the stem state. In keeping with the popularity of this topic, Biology of Stem Cells and the Molecular Basis of the Stem State addresses the major controversies and points of dispute, among researchers in the stem cell field. Overall, Biology of Stem Cells and the Molecular Basis of the Stem State presents a well-rounded dialogue about stem cells as it not only concentrates upon the biological elements of stem cell, but also addresses the controversy and hype currently enveloping this popular subject.
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(2009) Regulatory Networks in Stem Cells. Vemuri M. C. & Rajasekhar V. K.(eds.). Totowa, NJ: . p. 175-184 Abstract
Mesenchymal progenitor cells are widespread in the organism and are implicated in a variety of physiological and pathological processes. As such, these cells should be able to respond to microenvironmental signals. Here we review some of the conditions that modulate the biological functions of mesenchymal progenitors, particularly during inflammation and stress.
2008
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(2008) PLoS ONE. 3, 11, p. 1-15 e3707. Abstract
Background: The tumor suppressor p53 is an important regulator that control various cellular networks, including cell differentiation. Interestingly, some studies suggest that p53 facilitates cell differentiation, whereas others claim that it suppresses differentiation. Therefore, it is critical to evaluate whether this inconsistency represents an authentic differential p53 activity manifested in the various differentiation programs. Methodology/Principal Findings: To clarify this important issue, we conducted a comparative study of several mesenchymal differentiation programs. The effects of p53 knockdown or enhanced activity were analyzed in mouse and human mesenchymal cells, representing various stages of several differentiation programs. We found that p53 down-regulated the expression of master differentiation-inducing transcription factors, thereby inhibiting osteogenic, adipogenic and smooth muscle differentiation of multiple mesenchymall cell types. In contrast, p53 is essential for skeletal muscle differentiation and osteogenic re-programming of skeletal muscle committed cells. Conclusions: These comparative studies suggest that, depending on the specific cell type and the specific differentiation program p53 may exert a positive or a negative effect, and thus can be referred as a "guardian of differentiation" at large.
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Increased regulatory versus effector T cell development is associated with thymus atrophy in mouse models of multiple myeloma(2008) Journal of Immunology. 181, 5, p. 3714-3724 Abstract
CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) play a central role in cancer tolerance. However, mechanisms leading to their accumulation in cancer remain unknown. Although the thymus is the main site of Treg development, thymic contribution to Treg expansion in cancer has not been directly examined. Herein, we used two murine models of multiple myeloma (MM), 5T2 MM and 5T33 MM, to examine Treg accumulation in peripheral lymphoid organs, including spleen, lymph nodes, bone marrow, and blood, and to explore thymic Treg development during malignancy. We found that peripheral ratios of suppressive-functional Tregs increased in both models of MM-inflicted mice. We found that thymic ratios of Treg development in MM increased, in strong association with thymus atrophy and altered developmental processes in the thymus. The CD4(+)CD8(+) double-positive population, normally the largest thymocyte subset, is significantly decreased, whereas the CD4(-)CD8(-) double-negative population is increased. Administration of thymocytes from MM-inflicted mice compared with control thymocytes resulted in increased progression of the disease, and this effect was shown to be mediated by Tregs in the thymus of MM-inflicted mice. Our data suggest that increased ratios of Treg development in the thymus may contribute to disease progression in MM-inflicted mice.
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(2008) Stem Cells. 26, 9, p. 2275-2286 Abstract
Cultured bone marrow stromal cells create an in vitro milieu supportive of long-term hemopoiesis and serve as a source for multipotent mesenchymal progenitor cells defined by their ability to differentiate into a variety of mesodermal derivatives. This study aims to examine whether the capacity to support myelopoiesis is coupled with the multipotency. Our results show that the myelopoietic supportive ability of stromal cells, whether from the bone marrow or from embryo origin, is not linked with multipotency; cell populations that possess multipotent capacity may or may not support myelopoiesis, whereas others, lacking multipotency, may possess full myelopoietic supportive ability. However, upon differentiation, the ability of multipotent mesenchymal progenitors to support myelopoiesis is varied. Osteogenic differentiation did not affect myelopoietic supportive capacity, whereas adipogenesis resulted in reduced ability to support the maintenance of myeloid progenitor cells. These differences were accompanied by a divergence in glycosylation patterns, as measured by binding to lectin microarrays; osteogenic differentiation was associated with an increased level of antennarity of N-linked glycans, whereas adipogenic differentiation caused a decrease in antennarity. Inhibition of glycosylation prior to seeding the stroma with bone marrow cells resulted in reduced capacity of the stromal cells to support the formation of cobblestone areas. Our data show that myelopoietic support is unrelated to the multipotent phenotype of cultured mesenchymal progenitors but is dependent on the choice of differentiation pathway and upon correct glycosylation of the stromal cells.
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(2008) Stem Cells and Development. 17, 1, p. 93-105 Abstract
In vitro and in vivo studies implicate a series of cytokines in regulation of lymphohematopoiesis. However, direct indications for a local role of most of these cytokines within the bone marrow is lacking. In the present study, we aimed to test the contribution of a specific cytokine, activin A, a member of the transforming growth factor-beta (TGF-beta) family, to lymphohematopoiesis in mouse bone marrow. We show that mouse embryonic fibroblasts (MEFs) are indistinguishable from multipotent stromal cells (MSCs). Such MEFs overexpressing activin A, supported in vitro myelopoiesis in long-term bone marrow cultures as effectively as control MEFs. In contrast, activin A-overexpressing MEFs interfered with the in vitro generation of B lineage cells in such cultures. Thus, excessive expression in vitro of activin A, by supportive stromal cells, causes preferential maturation of myeloid rather than lymphoid cells. Moreover, the activin A-overexpressing MEFs caused an increased incidence in vivo of relatively immature B lineage cells; upon transplantation through the spleen route, MEFs engrafted the bone marrow specifically. Activin A-overexpressing MEFs accumulated in the bone marrow compartment and slowed down the progression of B cell precursors along the differentiation pathway, while sparing the myeloid population. The assay system described in this paper provides a means to assess the contribution of a wide range of molecules to hematopoiesis without perturbing the constitution of other organs.
2007
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(2007) Journal of Clinical Anesthesia. 19, 3, p. 168-174 Abstract
Study Objective: To investigate the immunohistochemical localization of beta A subunit of activin A in human term placenta, as a marker for placental infection/inflammation and elevated temperature, in parturients laboring during two analgesic regimens. Design: Prospective, randomized controlled study. Setting: Delivery room. Patients: 56 healthy, ASA physical status I and II primiparous women in labor. Interventions: Parturients were assigned to receive patient-controlled epidural analgesia (PCEA) with 0.2% ropivacaine or patient-controlled intravenous analgesia (PCA) with meperidine. Measurements: Histologic and immunohistochemical placental evaluation for white blood cell infiltration and activin beta A staining were made. Maternal temperature elevation above 37.6 degrees C and leukocytosis above 15 000/mu L were recorded. Main Results: Temperature was not significantly increased in parturients receiving PCEA over those who received PCA with meperidine (31% vs 11%, respectively; P = 0.1). There was also no association between temperature elevation during epidural analgesia and increased white blood cell count (> 15000/mu L) or presence of polymorphonuclear and/or lymphocyte aggregation in the placenta. Immunohistochemical staining with antisera against the beta A subunit of activin was present mainly in the placental cytotrophoblast, syncytiotrophoblast, and vascular endothelium, and was not associated with an increase in maternal temperature. No significant difference was noted between the two analgesic techniques with regard to maternal temperature elevation. Intrapartum temperature elevation was not associated with histologic signs of placental inflammation or with expression of activin beta A in the placenta. Conclusion: Other mechanisms may be involved in the etiology of temperature elevation during labor. (c) 2007 Elsevier Inc. All rights reserved.
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(2007) Stem Cells. 25, 3, p. 761-770 Abstract
Stem cells exhibit a promiscuous gene expression pattern. We show herein that the early embryo and adult MSCs express B-cell receptor component mRNAs. To examine possible bearings of these genes on the expressing cells, we studied immunoglobulin mu chain-deficient mice. Pregnant mu chain-deficient females were found to produce a higher percentage of defective morulae compared with control females. Structure analysis indicated that the mu mRNA species found in embryos and in mesenchyme consist of the constant region of the mu heavy chain that encodes a recombinant 50-kDa protein. In situ hybridization localized the constant mu gene expression to loose mesenchymal tissues within the day-12.5 embryo proper and the yolk sac. In early embryo and in adult mesenchyme from mu-deficient mice, delta replaced mu chain, implying a possible requirement of these alternative molecules for embryo development and mesenchymal functions. Indeed, overexpression of the mesenchymal-truncated mu heavy chain in 293T cells resulted in specific subcellular localization and in G(1) growth arrest. The lack of such occurrence following overexpression of a complete, rearranged form of mu chain suggests that the mesenchymal version of this mRNA may possess unique functions.
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(2007) Blood. 109, 4, p. 1422-1432 Abstract
Mesenchymal stem cells (MSCs) are widespread in adult organisms and may be involved in tissue maintenance and repair as well as in the regulation of hematopoiesis and immunologic responses. Thus, it is important to discover the factors controlling MSC renewal and differentiation. Here we report that adult MSCs express functional Toll-like receptors (TLRs), con-firmed by the responses of MSCs to TLR ligands. Pam3Cys, a prototypic TLR-2 ligand, augmented interleukin-6 secretion by MSC, induced nuclear factor κ B (NF-κB) translocation, reduced MSC basal motility, and increased MSC proliferation. The hallmark of MSC function is the capacity to differentiate into several mesodermal lineages. We show herein that Pam3Cys inhibited MSC differentiation into osteogenic, adipogenic, and chondrogenic cells while sparing their immunosuppressive effect. Our study therefore shows that a TLR ligand can antagonize MSC differentiation triggered by exogenous mediators and consequently maintains the cells in an undifferentiated and proliferating state in vitro. Moreover, MSCs derived from myeloid factor 88 (MyD88)-deficient mice lacked the capacity to differentiate effectively into osteogenic and chondrogenic cells. It appears that TLRs and their ligands can serve as regulators of MSC proliferation and differentiation and might affect the maintenance of MSC multipotency
2006
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(2006) Cancer and Metastasis Reviews. 25, 3, p. 459-467 Abstract
Tissues and organs harbor a component of supportive mesenchymal stroma. The organ stroma is vital for normal functioning since it expresses factors instructing growth and differentiation along with molecules that restrain these processes. Similarly, the growth of tumors is strictly dependent on the tumor stroma. This review first discusses the possibility of developing tools to block the propagation of the tumor-associated stroma, that may halt tumor progression. It further describes how the tropism of mesenchymal stroma to tumor sites may be utilized to cause regression of the cancerous tissue. Mesenchyme can be genetically modified to overexpress specific regulatory molecules with known effects on specific tumors, such as interferon beta, studied in the context of melanoma and glioma and activin A, a transforming growth factor beta cytokine, examined in multiple myeloma. These studies point to the possibility that genetically modified mesenchymal cells may be used as a therapeutic modality for incurable human diseases. It is proposed that further development of methods of tumor stroma targeting, or alternatively the use of stromal mesenchyme as a cell or cell/gene therapy modalities, may yield novel clinical tools for the treatment of human cancers.
2005
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(2005) Stem Cells. 23, 6, p. 719-726 Abstract
The prevailing stein cell concept is derived from the large body of evidence available on the structure of the blood-generating system. Hemopoiesis is organized such that a multipotent stein cell, endowed with self-renewal capacity, is viewed as being positioned at the origin of a hierarchical tree of branching specificities, increasing maturity and decreasing self-renewal ability. Data accumulated in recent years on various stem cell systems often contradict this traditional view of stem cells and are reviewed herein. It is suggested that other options should be considered and put to experimental scrutiny; it is argued that the organization of the hemopoietic system may not represent a general structure of stem cell systems. The basic trait of the stem state is proposed to be plasticity. Self-renewal is not a specific stem cell trait; rather, it is exhibited by some mature cell types, whereas other particular stein cells are endowed with relatively poor renewal ability. Hierarchical structuring is also proposed to be an optional stem cell trait and may exist only in specific tissues where it serves the need for rapid expansion. The stem state is therefore defined by the highest degree of plasticity of a cell, within the repertoire of cell types present in the organism.
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Dynamic sorting of nuclear components into distinct nucleolar caps during transcriptional inhibition(2005) Molecular Biology of the Cell. 16, 5, p. 2395-2413 Abstract
Nucleolar segregation is observed under some physiological conditions of transcriptional arrest. This process can be mimicked by transcriptional arrest after actinomycin D treatment leading to the segregation of nucleolar components and the formation of unique structures termed nucleolar caps surrounding a central body. These nucleolar caps have been proposed to arise from the segregation of nucleolar components. We show that contrary to prevailing notion, a group of nucleoplasmic proteins, mostly RNA binding proteins, relocalized from the nucleoplasm to a specific nucleolar cap during transcriptional inhibition. For instance, an exclusively nucleoplasmic protein, the splicing factor PSF, localized to nucleolar caps under these conditions. This structure also contained pre-rRNA transcripts, but other caps contained either nucleolar proteins, PML, or Cajal body proteins and in addition nucleolar or Cajal body RNAs. In contrast to the capping of the nucleoplasmic components, nucleolar granular component proteins dispersed into the nucleoplasm, although at least two (p14/ARF and MRP RNA) were retained in the central body. The nucleolar caps are dynamic structures as determined using photobleaching and require energy for their formation. These findings demonstrate that the process of nucleolar segregation and capping involves energy-dependent repositioning of nuclear proteins and RNAs and emphasize the dynamic characteristics of nuclear domain formation in response to cellular stress.
2004
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(2004) Blood Cells Molecules And Diseases. 33, 3, p. 211-215 Abstract
Plastic behavior of cells is a hallmark of embryonic development. The emergence of primary mesenchyme from within the inner cell mass entails the first epithelial-mesenchymal transition step that is then followed by sequential transitions; the formation of new tissues and organs requires transitions from mesenchyme into epithelium and vice versa. Although it is currently believed that in the adult such transitions do not persist, the frequent occurrence of mesenchymal stem cells (MSCs) in various tissues of the adult organisms, and the reported plasticity of such adult mesenchymal cells, raises the question as to whether the frequency of mesenchymal epithelial transitions in the adult have been underestimated. Indeed, adult mesenchymal stem cells have been reported to differentiate in culture into a multitude of mature cell types including epithelial cells. This opens the way to the use of these stem cells for the construction of new tissues and organs for therapeutic purposes, but the question is still open as to whether mesenchymal stem cells transdifferentiate also in vivo. The molecular mechanism that underlies the plasticity of mesenchymal stem cells and their capacity to transdifferentiate is unresolved. We found that these cells have a promiscuous gene expression pattern; mesenchymal cells, whether primary or cloned cell lines, express T cell receptor (TCR) beta and a genes, along with other components of the TCR complex. These cells may therefore be in a standby state, in which many gene families are expressed at a low level thereby making the cell readily capable of shifting fates. (C) 2004 Elsevier Inc. All rights reserved.
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(2004) Nature Reviews Genetics. 5, 11, p. 873-878 Abstract
Stem cells are endowed with self-renewal and multipotential differentiation capacities. Contrary to the expectation that stem cells would selectively express specific genes, these cells have a highly promiscuous gene-expression pattern. Here, I suggest that the transient stem cell state, termed the 'stem state', may be assumed by any cell and that the search for specific genes expressed by all stem cells, which would characterize the stem cell as a cell type, might be futile.
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(2004) Blood. 103, 8, p. 2892-2899 Abstract
Epithelial mesenchymal transitions are a remarkable example of cellular plasticity. These transitions are the hallmark of embryo development, are pivotal in cancer progression, and seem to occur infrequently in adult organisms. The reduced incidence of transitions in the adult could result from restrictive functions of the microenvironment that stabilizes adult cell phenotypes and prevents plastic behavior. Multipotential progenitor cells exhibiting a mesenchymal phenotype have been derived from various adult tissues. The ability of these cells to differentiate into all germ layer cell types, raises the question as to whether mesenchymal epithelial transitions occur in the adult organism more frequently than presently appreciated. A series of cytokines are known to promote the transitions between epithelium and mesenchyme. Moreover, several transcription factors and other intracellular regulator molecules have been conclusively shown to mediate these transitions. However, the exact molecular basis of these transitions is yet to be resolved. The identification of the restrictive mechanisms that prevent cellular transitions in adult organisms, which seem to be unleashed in cancerous tissues, may lead to the development of tools for therapeutic tissue repair and effective tumor suppression. (C) 2004 by The American Society of Hematology.
2003
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(2003) Circulation. 108, 7, p. 863-868 Abstract
Background-Systemic delivery of bone marrow-derived mesenchymal stem cells (BM-MSCs) is an attractive approach for myocardial repair. We aimed to test this strategy in a rat model after myocardial infarction (MI). Methods and Results-BM-MSCs were obtained from rat bone marrow, expanded in vitro to a purity of >50%, and labeled with Tc-99m exametazime, fluorescent dye, LacZ marker gene, or bromodeoxyuridine. Rats were subjected to MI by transient coronary artery occlusion or to sham MI. Tc-99m-labeled cells (4x10(6)) were transfused into the left ventricular cavity of MI rats either at 2 or 10 to 14 days after MI and were compared with sham-MI rats or MI rats treated with intravenous infusion. Gamma camera imaging and isolated organ counting 4 hours after intravenous infusion revealed uptake of the Tc-99m-labeled cells mainly in the lungs, with significantly smaller amounts in the liver, heart, and spleen. Delivery by left ventricular cavity infusion resulted in drastically lower lung uptake, better uptake in the heart, and specifically higher uptake in infarcted compared with sham-MI hearts. Histological examination at 1 week after infusion identified labeled cells either in the infarcted or border zone but not in remote viable myocardium or sham-MI hearts. Labeled cells were also identified in the lung, liver, spleen, and bone marrow. Conclusions-Systemic intravenous delivery of BM-MSCs to rats after MI, although feasible, is limited by entrapment of the donor cells in the lungs. Direct left ventricular cavity infusion enhances migration and colonization of the cells preferentially to the ischemic myocardium.
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The mesenchymal stroma negatively regulates B cell lymphopoiesis through the expression of activin A(2003) Hematopoietic Stem Cells 2002: Genetics And Function. 996, p. 245-260 Abstract
The negative control of B cell generation is only partially resolved. We assessed the role of activin A in regulation of B lymphopoiesis in view of its specific inhibitory effects on tumor B lineage cells. Activin A is constitutively expressed in mouse hemopoietic organs and in cultured mesenchymal cell lines. We observed an inverse relationship between activin A titer and B lineage cell production. In the spleen, the red pulp exhibited a relatively higher abundance of the protein as compared with the lymphoid follicles, wherein B cell accumulation occurs. Furthermore, a specific shut off in activin A expression was observed in bone marrow and spleen following in vivo induction of B lymphocyte polyclonal activation. We further substantiated these in vivo observations by in vitro studies of primary bone marrow cultures, in which the expression of functional activin A was found to be diminished prior to the onset of B lymphopoiesis. The reduction in functional activin A is shown to concomitantly occur with spontaneous induction of the expression of activin A specific inhibitors. We therefore propose that the mesenchymal organ stroma expresses activin A that negatively controls B cell lymphopoiesis.
2002
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(2002) FEBS Letters. 531, 2, p. 109-114 Abstract
Proteins are often referred to in accordance with the activity with which they were first associated or the organelle in which they were initially identified. However, a variety of nuclear factors act in multiple molecular reactions occurring simultaneously within the nucleus. This review describes the functions of the nuclear factors PSF (polypyrimidine tract-binding protein-associated splicing factor) and p54(nrb)/NonO. PSF was initially termed a splicing factor due to its association with the second step of pre-mRNA splicing while p54(nrb)/NonO was thought to participate in transcriptional regulation. Recent evidence shows that the simplistic categorization of PSIT and its homolog p54(nrb)/NonO to any single nuclear activity is not possible and in fact these proteins exhibit multi-functional characteristics in a variety of nuclear processes. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
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(2002) Experimental Hematology. 30, 7, p. 628-633 Abstract
Institute for Health and Consumer Protection, Joint Research Centre, Ispra, Italy, November 21-23, 2001The workshop on stem cells in therapy and toxicology is one of a series organized by the European Center for the Validation of Alternative Methods (ECVAM).
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(2002) Oncogene. 21, 13, p. 2029-2036 Abstract
The mesenchyme plays a crucial regulatory role in organ formation and maintenance. However, comprehensive molecular characterization of these cells is lacking. We found unexpectedly that primary mesenchyme, as well as mesenchymal cell clones, express T cell receptor (TCR)chibeta mRNAs, lacking the variable region. Immunological and genetic evidence support the expression of a corresponding TCRbeta protein. Additionally, mRNAs encoding TCR complex components including CD3 and chain are present. A relatively higher expression of the mesenchymal TCRbeta mRNA by cultured mesenchymal cell clones correlates with fast growth, whereas poorly expressing cells are slow growers and are contact inhibited. The clones that express relatively higher amount of the TCR mRNA exhibit an increased capacity to form tumors in nude mice. However, the expression of this mRNA in the mesenchyme is not per se leading to tumorigenesis, as demonstrated by primary mesenchyme that does not form tumors in mice while expressing moderate amounts of the TCR transcripts. The expression of mesencymal TCRbeta was confined to the G2/M phases of the cell cycle in the MBA-13 mesenchymal cell line. This cell cycle dependent expression, considered together with the correlation between growth properties and the level of TCR expression by cell clones, implies association of mesenchymal TCR with cell growth control.
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(2002) Stem Cells. 20, 6, p. 493-500 Abstract
Activin A, a cytokine member of the transforming growth factor-beta superfamily, is expressed locally by the mesenchymal component of the hemopoietic microenvironment. Its expression is regulated on the mRNA level by different cytokines, and the biological activity of the protein is tightly controlled by several inhibitory molecules. Activin A affects hemopoietic cells of various lineages, as evidenced by in vitro studies of leukemia and lymphoma cell lines, which were used to elucidate the mechanism of its action. In the B-cell lineage, activin A is a cell cycle inhibitor, a mediator of apoptosis, and a cytokine antagonist. Limited information is available on the effects of activin A on normal hemopoietic cells. Recent studies suggest that it might be a negative regulator of normal B lymphopoiesis. Whereas the functions of activin A in vitro are well established, further research tools are needed to elucidate its role within specific hemopoietic microenvironments in vivo.
2001
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Reduced expression of activin A in focal lymphoid agglomerates within nasal polyps(2001) Journal Of Histochemistry & Cytochemistry. 49, 10, p. 1245-1252 Abstract
It has been previously reported that activin A, a homodimer of the PA inhibin subunit, is secreted by stromal cells from mouse bone marrow and causes apoptotic death of mouse plasmacytoma tumor cells. Recent in vitro studies have also implicated this cytokine in the suppression of normal B-cell lymphopoiesis. In this study we examined the occurrence of activin A in nasal polyp tissues that present a combination of epithelium, mesenchyme, and vascular endothelium, with frequent massive hemopoietic infiltration. Anti-PA-chain antibodies strongly stained epithelial mucous glands and some endothelial cells, and diffusely stained the polyp stroma. Normal adult conchae were similarly stained, whereas activin A was not detected prenatally by immunostaining of nasal tissues. Staining specificity was substantiated by ligand competition assays. Detailed examination of the inflammatory polyp infiltrate showed that activin A staining was reduced in sites of focal infiltration of B-lymphoid cells. It is therefore implied that local accumulation of a large number of B-cells is associated with relatively low activin A expression.
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Nuclear relocalization of the pre-mRNA splicing factor PSF during apoptosis involves hyperphosphorylation, masking of antigenic epitopes, and changes in protein interactions(2001) Molecular Biology of the Cell. 12, 8, p. 2328-2340 Abstract
The spatial nuclear organization of regulatory proteins often reflects their functional state. PSF, a factor essential for pre-mRNA splicing, is visualized by the B92 mAb as discrete nuclear foci, which disappeared during apoptosis. Because this mode of cell death entails protein degradation, it was considered that PSF, which like other splicing factors is sensitive to proteolysis, might be degraded. Nonetheless, during the apoptotic process, PSF remained intact and was N-terminally hyperphosphorylated on serine and threonine residues. Retarded gel migration profiles suggested differential phosphorylation of the molecule in mitosis vs. apoptosis and under-phosphorylation during blockage of cells at G1/S. Experiments with the use of recombinant GFP-tagged PSF provided evidence that in the course of apoptosis the antigenic epitopes of PSF are masked and that PSF reorganizes into globular nuclear structures. In apoptotic cells, PSF dissociated from PTB and bound new partners, including the U1-70K and SR proteins and therefore may acquire new functions.
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(2001) Journal of Biological Chemistry. 276, 27, p. 24719-24725 Abstract
Activin A, a member of the transforming growth factor beta (TGF beta) superfamily, blocks interleukin (IL)-6 biological functions. The molecular basis of the influence of this TGF beta signaling on the IL-6 receptor triggered cascade is unknown. We studied IL-g-induced secretion of the acute phase protein haptoglobin by hepatoma cells. Overexpression of the C/EBP beta gene, a downstream effector in the IL-6 pathway, activated transcription from the haptoglobin promoter. This was abolished by either a constitutively active form of activin A type IB receptor (CAactRIB) or by a combination of Smad3 and Smad4, Similarly, Smads abolished transcriptional activation by co-stimulation with IL-6 and STAT3. The transcription co-activator p300 partially overcame the suppressive effect of Smads. Electrophoretic mobility shift assays indicated that C/EBP beta binding to haptoglobin promoter DNA was reduced by over-expression of CAactRIB and Smad4. We thus show that Smad proteins operate as transcription inhibitors on target genes of the IL-6 induced pathway. The effect of Smads is exerted on components of the transcription activation complex and may also involve interference with DNA binding. This study thus depicts molecular sites of interaction between the TGF beta superfamily and the IL-6 signaling cascades.
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(2001) Leukemia. 15, 7, p. 1102-1110 Abstract
The mesenchymal stroma has been shown to play a crucial role in the development of multiple myeloma, partly by secretion of interleukin (IL)-6, that serves as a growth factor for myeloma cells. However, it is still unclear which other stroma[ molecules are Involved in the pathogenesis of this disease. We chose, as a model system, a mouse plasmacytoma cell line, which does not respond to IL-6. We found that the formation of mouse plasmacytoma tumors, in an in vivo skin transplantation model, is facilitated by co-injection of these tumor cells along with a mesenchymal stromal cell. The tumor promoting effect of the stroma was reproduced in an in vitro model; stromal cells induced the proliferation of plasmacytoma cells under serum-free conditions. This growth promotion could not be mimicked by a series of cytokines including IL-6 and insulin-like growth factor (IGF)-I implying a role for yet unidentified stromal factors. The in vivo formation of plasmacytoma tumors was reduced following administration of activin A, a cytokine member of the transforming growth factor (TGF)beta superfamily. Furthermore, the in vitro growth promoting effect of the stroma was abrogated by basic fibroblast growth factor (bFGF) which induced a higher stromal expression of activin A. Our results thus show that mesenchymal stroma expresses plasmacytoma growth stimulating activities that overcome the low constitutive level of the plasmacytoma inhibitor, activin A. The expression of activin A is upregulated by bFGF rendering the stroma suppressive for plasmacytoma growth. The balance between the expression of these regulators may contribute to mesenchymal stroma activity and influence the progression of multiple myeloma.
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Role of activin A in negative regulation of normal and tumor B lymphocytes(2001) Journal of Leukocyte Biology. 69, 6, p. 867-873 Abstract
Activin A, a member of the transforming growth factor beta superfamily, has a wide spread expression pattern and pleiotropic functions. in this overview we summarize data that points to a role of activin A in negative regulation of B lineage lymphocytes. Experiments performed by us and by other groups revealed the capacity of activin A to cause apoptotic death of tumor myeloma cells, through mechanisms of cell cycle inhibition and antagonism with the survival signal of interleukin-6, in vitro studies on B lymphocyte generation from bone marrow stem cells and use of human nasal polyps as a model of inflamed tissue further demonstrate an inhibitory role of activin A in B cell spread and accumulation. These data are analyzed with respect to our model of tissue organization that we term the "restrictin model of cell growth regulation." This model assumes a morphogen-like role of activin A in the hematopoietic: system. Thus, the relative concentration of biologically functional activin Al in different parts of the tissue, may determine the local B cell content and functional state of these cells within a specific microenvironment.
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(2001) Journal of Cellular Biochemistry. 81, 3, p. 379-392 Abstract
Differentiation in several stem cell systems is associated with major morphological changes in global nuclear shape. We studied the fate of inner-nuclear structures, splicing factor-rich foci and Cajal (coiled) bodies in differentiating hemopoietic, testis and skin tissues. Using antibodies to the splicing factors PSF, U2AF(65) and snRNPs we find that these proteins localize in foci throughout the nuclei of immature bone marrow cells. Yet, when granulocytic cells differentiate and their nuclei condense and become segmented, the staining localizes in a unique compact and thread-like structure. The splicing factor-rich foci concentrate in the interior of these nuclei while the nuclear periphery and areas of highly compact chromatin remain devoid of these molecules. Differentiated myeloid cells do not stain for p80 coilin, the marker for Cajal bodies. Immature myeloid cells contain Cajal bodies although these usually do not coloclaize with PSF-rich foci. Following complete inhibition of transcription in myeloid cells, the threaded PSF pattern becomes localized in several foci in the different lobes of mature granulocytes while in human HL-60 immature myeloid leukemia cells PSF is found in the perinucleolar compartment. Studies of other differentiating stem cell systems show that PSF staining disappears completely in differentiated, transcriptionally inactive sperm cells, is scarce as cells migrate from the inner skin layers outward and is lost as cells of the hair follicle mature. We conclude that the formation and distribution of splicing factor-rich foci in the nucleus during differentiation of various cell lineages is dependent on the levels of chromatin condensation and the differentiation status of the cell. J. Cell. Biochem. 81:379-392, 2001. (C) 2001 Wiley-Liss, Inc.
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2000
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(2000) Experimental Hematology. 28, 9, p. 1029-1038 Abstract
Objective. Molecular identification and characterization of the bone marrow nuclear protein detected by the B92 monoclonal antibody. Materials and Methods. The protein was purified to homogeneity from acute myeloid leukemia cells and was subjected to peptide digestion and amino acid sequencing. Identified sequences were used to screen a bone marrow cDNA library in search of matching transcripts. The protein was further studied in different cells and tissues by examination of protease inhibitors and harsh lytic conditions and during apoptosis in HL-60 cells, Results. We found that the apparent bone marrow specific protein is a 47 kD proteolytic cleavage product of PSF, an essential pre-mRNA splicing factor, PSF is completely cleaved to p47 during lysis of immature myeloid cells due to potent proteolytic activity found in these cells but is rare in other cells and tissues. Furthermore, p47 is abundant in intact normal and tumor myeloid cells while in other cell types it is undetectable, The cleavage of PSF is accompanied by digestion of the PTB splicing regulator but not other proteins tested, In contrast, during apoptosis PTB is degraded while PSF remains intact, Conclusions, The bone marrow 47 kD protein is a fragment constituting the N-terminal, protease-resistant half of the splicing factor PSF, Proteolytic degradation of PSF specifically occurs in intact myeloid cells and this process is enhanced upon myeloid cell lysis, (C) 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc.
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(2000) Blood. 95, 11, p. 3289-3296 Abstract
Hematopoietic stem cell homing and engraftment require several adhesion interactions, which are not fully understood. Engraftment of nonobese/severe combined immunodeficiency (NOD/SCID) mice by human stem cells is dependent on the major integrins very late activation antigen-4 (VLA-4); VLA-5; and to a lesser degree, lymphocyte function associated antigen-1 (LFA-1). Treatment of human CD34+ cells with antibodies to either VLA-4 or VLA-5 prevented engraftment, and treatment with anti-LFA-1 antibodies significantly reduced the levels of engraftment. Activation of CD34+ cells, which bear the chemokine receptor CXCR4, with stromal derived factor 1 (SDF-1) led to firm adhesion and transendothelial migration, which was dependent on LFA-1/ICAM-1 (intracellular adhesion molecule-1) and VLA-4/VCAM-1 (vascular adhesion molecule-1). Furthermore, SDF-1-induced polarization and extravasation of CD34+/CXCR4+ cells through the extracellular matrix underlining the endothelium was dependent on both VLA-4 and VLA-5. Our results demonstrate that repopulating human stem cells functionally express LFA-1, VLA-4, and VLA-5. Furthermore, this study implies a novel approach to further advance clinical transplantation. (C) 2000 by The American Society of Hematology.
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Apoptosis induction of human myeloid leukemic cells by ultrasound exposure(2000) Cancer Research. 60, 4, p. 1014-1020 Abstract
Therapeutic ultrasound (ULS) and the resulting cavitation process has been shown to induce irreversible cell damage. In this study, we wanted to further investigate the mechanism of ULS-induced cell death and to determine whether apoptosis is involved. Nigh intensity focused pulsed ULS sonication at a frequency of 750 KHz was delivered to HL-60, K562, U937, and M1/2 leukemia cell line cultures. ULS exposure used with induction of transient cavitation in the focal area was delivered with an intensity level of 103.7 W/cm(2) and 54.6 W/cm(2) spatial-peak temporal-average intensity, As a control, ULS of lower intensity was delivered at 22.4 W/cm(2) spatial-peak temporal-average intensity, presumably without generation of cavitation. Our results indicated that DNA damage induced by ULS cavitation did not involve generation of free radicals in the culture media. Morphological alterations observed in cells after exposure to ULS included: cell shrinkage, membrane blebbing, chromatin condensation, nuclear fragmentation, and apoptotic body formation. Apoptotic cells were evaluated by fluorescence microscopy and detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, which identifies DNA breaks, and by the leakage of phosphatidylserine from the inner to the outer side of the membrane layer of treated cells. Some bioeffects induced on sonicated HL-60 cells, such as inhibition of cell proliferation, DNA repair, and cell-dependent apoptosis, were found to be similar to those produced by gamma-irradiation. Thus, much of the cell damage induced by therapeutic ULS in leukemia cells surviving ULS exposure appears to occur through an apoptotic mechanism.
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(2000) Journal of Clinical Investigation. 106, 11, p. 1331-1339 Abstract
The chemokine stromal-derived factor-1 (SDF-1) controls many aspects of stem cell function. Details of its regulation and sites of production are currently unknown. We report that in the bone marrow, SDF-1 is produced mainly by immature osteoblasts and endothelial cells. Conditioning with DNAdamaging agents (ionizing irradiation, cyclophosphamide, and 5-fluorouracil) caused an increase in SDF-1 expression and in CXCR4-dependent homing and repopulation by human stem cells transplanted into NOD/SCID mice. Our findings suggest that immature osteoblasts and endothelial cells control stem cell homing, retention, and repopulation by secreting SDF-1, which also participates in host defense responses to DNA damage.
1999
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(1999) Oncogene. 18, 26, p. 3886-3893 Abstract
The acute phase protein expression In HepG2 human hepatoma cells and promotes the growth of mouse B9 hybridoma. The signaling cascades leading to these biological functions are only partially known. We analysed the involvement of MAPK homologues in IL-6 transduction pathways and found that interleukin-6 triggered activation of p38 stress-activated protein kinase (p38) but not of jun kinase. p38 activity was required for biological functions including acute phase protein secretion from HepG2 hepatoma and proliferation of B9 hybridoma cells. Using a reporter gene construct containing a 190 bp promoter fragment of the acute phase protein haptoglobin we found that p38 is involved in transcriptional activation of the haptoglobin promoter by STAT3 but not by NF-IL6. Thus, we present evidence for a role of p38 in IL-6 induced functions and a possible cross-talk between this MAPK homologue and the STAT pathway.
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(1999) Experimental Hematology. 27, 5, p. 834-844 Abstract
We previously reported that among the various thymic lymphocyte subpopulations, the immature T cells preferentially adhere to mesenchymal bone marrow stroma, In the present study we examined the interactions between phenotypically defined populations of early T cells and stromal cell lines, The immature T cells segregated into two subpopulations according to their adhesive capacity. Whereas the majority of the adherent CD4(-)CD8(-) T cells were devoid of CD3/TCR alpha beta, most of the nonadherent CD4(-)CD8(-) T cells expressed this receptor complex. The adhesion of T cells to bone marrow stroma almost entirely was accounted for by CD49d and CD90, whereas that of adherent CD4(-)CD8 cells also was dependent on CD44, CD62L, and CD117 receptor. Blocking antibody combinations failed to reduce the adherence of these early T cells to less than 50% that of the control, On the other hand, the adhesion of unselected thymocytes to the stroma was reduced by 80%, using the same blocking antibodies. Therefore, the participation of additional molecules in the adhesion of early T cells to mesenchymal stroma is implicated. Comparison between the interaction of T cells with bone marrow mesenchymal or with thymus-derived epithelial stroma indicated that T cells utilize a selected set of adhesion molecules under each situation. Although CD49d and CD90 participated in both cases, CD11a, CD18, and CD2 receptors played a dominant role in the adhesion of T cells to thymic epithelium only, This study may point to a role of mesenchymal stroma in the regulation of early T-cell lymphopoiesis in the bone marrow. (C) 1999 International Society for Experimental Hematology. Published by Elsevier Science Inc.
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(1999) Science. 283, 5403, p. 845-848 Abstract
Stem cell homing and repopulation are not well understood. The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 were found to be critical for murine bone marrow engraftment by human severe combined immunodeficient (SCID) repopulating stem cells. Treatment of human cells with antibodies to CXCR4 prevented engraftment. In vitro CXCR4-dependent migration to SDF-1 of CD34+CD38-(/low) cells correlated with in vivo engraftment and stem cell function. Stem cell factor and interleukin-6 induced CXCR4 expression on CD34+ cells, which potentiated migration to SDF-1 and engraftment in primary and secondary transplanted mice. Thus, up-regulation of CXCR4 expression may be useful for improving engraftment of repopulating stem cells in clinical transplantation.
1998
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(1998) Gene. 206, 2, p. 283-285 Abstract
p. 155. Please replace the legend to Fig. 1 with the following new legend:Fig. 1. Nucleic acid sequence of full-length mouse SA-1 cDNA. Initiation codon, bp 297; termination codon, bp 4071. The nucleotide sequences have been submitted to EMBL Nucleotide Sequence Database with accession numbers: Z-75330 for HUMSA-1, Z-75331 for HUMSA-2 and Z-75332 for MUSSA-1.p. 156. The publisher regrets that, due to technical problems, many letters in Fig. 2 of this article did not reproduce satisfactorily. The complete Fig. 2 is reproduced on the following right-hand page.
1997
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(1997) Gene. 195, 2, p. 151-159 Abstract
We report the molecular cloning of a novel gene family. The first member of this family was cloned from a mouse lambda gt11 cDNA library using the B92 monoclonal antibody (mAb) raised against stromal cell extracts. This was followed by RACE-PCR using mRNA from the stromal cell line. A 4 kb cDNA was obtained encoding a unique protein sequence of 1258 aa, that we designate stromal antigen (SA)-1. The human SA-1 gene was cloned by homology from a thymus cDNA library and the sequence of the predicted protein was found to be highly homologous to the murine SA-1 (> 98.9%). Another cDNA was cloned and the deduced protein (SA-2) was 71% homologous to SA-1. Northern blot and PCR analysis indicated that on the mRNA level the SA-1 gene is expressed in all tissues analyzed and probably encodes a single transcript. The identification of SA-1 protein in tissues and cells required combined immunoprecipitation and Western blotting using a polyclonal antiserum raised against a predicted peptide of SA-1 and the B92 mAb. Using this assay we identified a protein of about 120 kDa in hemopoietic organs. Subcellular fractionation indicated that SA-1 is a nuclear protein. Thus, despite the ubiquitous expression on the mRNA level, the protein was predominantly detected in hemopoietic organs and may therefore be controlled on a post-transcriptional level. The SA-1 gene described in this study is highly conserved between mouse and man. This implies a crucial function for this protein. (C) 1997 Elsevier Science B.V.
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(1997) Hybridoma. 16, 4, p. 325-334 Abstract
We describe a novel mouse monoclonal antibody (PRA-72) that recognizes a nuclear antigen associated with cell proliferation. The monoclonal antibody stained the nuclei of logarithmically growing cultured stromal cells. The nuclear staining disappeared when these cells entered G(0) phase of the cell cycle. Western blot analysis revealed a nuclear protein which appeared as a doublet at 35-40 KD, which was undetectable in extracts from confluent cells. Immunocytological study of purified cell populations from various cell cycle phases revealed peripheral nuclear staining in all stages except mitosis, when the chromosomes were observed enveloped with the antigen. In co-cultures of quiescent stromal cells and proliferating hemopoietic precursors, only the latter showed nuclear staining by PRA-72 monoclonal antibody. Further indications for selective expression of the antigen by proliferating cells were found by an immunohistochemical study of various tissues including newborn mouse bone marrow and its surrounding connective tissue, mouse tongue epithelium, and human carcinoma of the colon. This antibody may, therefore, prove useful in the evaluation of human tumors.
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(1997) Oncogene. 15, 14, p. 1705-1711 Abstract
Cytokines are growth inhibitory in a target cell specific manner. The signaling pathways that characterize each cell type play a crucial role in determining the responsiveness to cytokine triggering. Activin A has been shown to suppress the growth of primary hepatocytes. Similarly, the human HepG2 hepatoma cell line was growth arrested by activin A as judged by lack of cell proliferation and suppression of DNA synthesis. In HepG2 cells activin A further induced accumulation of retinoblastoma protein in the hypophosphorylated form known to prevent entrance into S phase. This finding implies the involvement of cyclin dependent kinases and CDK inhibitors. Examination of HepG2 cells following addition of activin A revealed reduced expression of CDK4 and conversely, an increase in the CKI p21(WAF1/Cip1). This accumulation of p21(WAF1/Cip1) protein was partly due to increased transcriptional activity. Functional inactivation of p53, using a miniprotein that oligomerizes with p53 and abrogates DNA binding, abolished the ability of activin A to induce transcriptional activation from the p21(WAF1/Cip1) promoter. Thus, activin A, like transforming growth factor β, seems to suppress cell growth through the downstream target Rb. However, each of these cytokines seem to operate through a distinct pathway.
1996
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Cooperation between p53-dependent and p53-independent apoptotic pathways in myeloid cells(1996) Cancer Research. 56, 9, p. 2148-2156 Abstract
Apoptosis may involve p53-dependent and -independent pathways. Results presented here suggest a possible cooperation between these two types of pathways. M1/2 is a p53-nonproducer subclone that may undergo either a p53- independent apoptosis following growth factor deprivation, or a p53-dependent apoptosis following reconstitution of wild-type p53 expression. The p53- independent apoptosis in these cells is a slow process occurring after a G0- G1 arrest. In contrast, the p53-dependent apoptosis is much more rapid and is characterized by early and late apoptotic phases, taking place in cells arrested at G0-G1 and S phase. The transition from early to late apoptosis correlated with the levels of the p53 protein. Concomitant induction of both apoptotic pathways accelerated cell death and facilitated the transition from early to late apoptotic phase. The interaction between these pathways is further supported by the finding that mutant p53 interferes with p53- independent apoptosis. Thus, although apoptosis can occur via either p53- dependent or -independent pathways, under certain conditions the two pathways may interact with each other.
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Interactions between leukemia cells and bone marrow stromal cells: Stroma-supported growth vs serum dependence and the roles of TGF-beta and M-CSF(1996) Experimental Hematology. 24, 6, p. 728-737 Abstract
Leukemia cell lines that do not proliferate in the absence of serum grow well when cultured with stromal cells. To study this growth dependence on stroma, we selected the M1 myeloblast clone, since its stroma dependence is reminiscent of that exhibited by hematopoietic stem cells. Conditioned medium from a stromal cell line, prepared under serum-free conditions, contained an activity that induced the proliferation of M1 cells and was therefore designated M1 myeloid activity (MMA). Among the various cytokines tested for MMA-like activity, only transforming growth factor-beta (TGF-beta) and macrophage colony-stimulating factor (M-CSF) were found to affect M1 cell survival, and the two cytokines acted synergistically to induce M1 cell growth. Antibodies to both TGF-beta and M-CSF abolished most, but not all, of the MMA in the medium conditioned by stromal cells, indicating that additional factors contribute to MMA. A subclone of M1 cells, M1/2, selected in medium conditioned by stroma, was found to respond to stromal stimulation but was unable to proliferate in fetal calf serum (FCS). Neutralization experiments indicated that M1/2 cell growth depended mainly on M-CSF and also partially on TGF-beta. By contrast, the same neutralizing antibodies did not affect the ability of serum to support M1 cell growth. The molecules that promoted leukemia cell growth in serum seemed therefore to differ from those provided by stroma. This model system may offer novel information on the interactions of normal and leukemic hematopoietic cells with their stromal microenvironment.
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Control of stroma-dependent hematopoiesis by basic fibroblast growth factor: Stromal phenotypic plasticity and modified myelopoietic functions(1996) Cytokines and Molecular Therapy. 2, 1, p. 29-38 Abstract
It has been suggested that basic fibroblast growth factor (bFGF) affects hematopoietic cells directly and that it may also act indirectly by modulating stromal cell functions. We tested the response of phenotypically and functionally distinct stromal cell clones to this cytokine. We studied cell phenotype, the composition and organization of cytoskeleton and extracellular matrix, the ability to repopulate 'wounded areas', the expression of cytokine genes, and the capacity of the stroma to support long-term hematopoiesis in vitro. Although the impact of bFGF on cell growth was small, it induced a prominent morphological change in three stromal cell types that we tested. We analyzed the molecular basis for this change: bFGF modified the protein expression of a-smooth muscle actin (alpha-SMA), tropomyosin, a-tubulin, fibronectin and paxillin in a distinct manner characteristic of each of the stromal cell types. Immunofluorescence analysis of these proteins revealed profound changes in the cytoskeleton and extracellular matrix (ECM) networks accompanied by increased ability of the 14F1.1 stromal cells to scatter in in vitro 'wounded' areas. Furthermore, although only limited changes were monitored in the expression of cytokine genes, the ability of the stromal cells to support hematopoiesis was markedly modified. Thus bFGF profoundly changes the cellular organization of stromal cells, their adhesion and their motility properties. These changes are associated with modified capacity to support hematopoiesis in culture.
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Selective adhesion of immature thymocytes to bone marrow stromal cells: Relevance to T cell lymphopoiesis(1996) Experimental Hematology. 24, 2, p. 386-391 Abstract
We investigated the interactions between the bone marrow microenvironment and T cell populations at different stages of maturation. Thymocytes were seeded onto confluent layers of bone marrow stromal cell lines (MBA-13 or 14F1.1). Within a few hours two main thymocyte populations were observed; one remained in the liquid phase and the other adhered to the stromal cells. After 24 hours of culture, most of the adhering cells expressed the phenotype of the precursors, double negative (DN) CD4(-)CD8(-), or of immature thymocytes, double positive (DP) CD4(+)CD8(+). The number of adhering DN cells did not change during the time of the culture, whereas that of the DP declined. The CD4(+)CD8(-) or CD4(-)CD8(+) cells did not adhere to any significant extent. The expression of CD3 antigen on adherent thymocytes was lower than that on nonadherent ones. Sorted thymocytes at a high level of purification (>96%) were cultured over stromal layer and, after 24 hours, 60% of the DN or 22% of the DP cells were found to adhere to the stroma. The culture medium was replaced every 24 hours or after 48 hours; no significant change was noted in the number of adhering DN and DP cells. The reappearance of immature T cells in the liquid phase suggested proliferation of this cell type. Thus, early thymocytes, phenotypically characterized as DN and DP, preferentially adhere to bone marrow stromal cells. This in vitro phenomenon may represent the function of the BM stroma as an extrathymic site of T cell lymphopoiesis.
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(1996) Principles of bone biology. p. 1267-1276 Abstract
Book: This comprehensive, authoritative, and timely work deals with all aspects of bone biology, including developmental and functional issues, pharmacology, and pathobiology. In addition, there is a workshop section which deals with actual methods and techniques relevant to investigators in this field. Principles of Bone Biology provides the reader with the most recent information, views, and references, focused on individual topics, contributed by the foremost authorities in the field.... [It] spans the spectrum from molecular biology to in vivo pharmacology. This chapter is in the Methods in Bone Research section.
1995
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The plasmacytoma growth inhibitor restrictin-P is an antagonist of interleukin 6 and interleukin 11: Identification as a stroma-derived activin A(1995) Journal of Biological Chemistry. 270, 49, p. 29594-29600 Abstract
A stromal protein, designated restrictin-P, that specifically kills plasma-like cells was purified to homogeneity and shown to be identical with activin A. The specificity to plasma-like cells stemmed from the ability of restrictin-P/activin A to competitively antagonize the proliferation-inducing effects of interleukin (IL) 6 and IL-11. Restrictin-P further interfered with the IL-6-induced secretion of acute phase proteins by HepG2 human hepatoma cells and with the IL-6-mediated differentiation of M1 myeloblasts. A competition binding assay indicated that restrictin-P did not interfere with the binding of IL-6 to its receptor on plasma-like cells, suggesting that it may act by intervening in the signal transduction pathway of the growth factor. Indeed, concomitant addition of restrictin-P and IL-6 to cytokine-deprived B9 hybridoma cells was followed by sustained overexpression of junB gene until cell death occurred, while IL-6 alone caused a transient increase only. This altered response to IL-6 stimulation was accompanied by a moderate increase in STAT protein activation. Thus, in this study, we identified the plasmacytoma growth inhibitor, restrictin-P, as being activin A of stromal origin. It is shown that activin A is an antagonist of IL-6-induced functions and that it modifies the IL-6 signaling pattern.
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(1995) Growth Factors. 12, 4, p. 277-287 Abstract
We have recently found that the inhibitor of plasmacytoma cell growth, restrictin-P, is a stroma derived activin A and that it is an antagonist of interleukin-6 and interleukin-11. The present study was aimed at determining the mode by which this cytokine kills its target cells. On addition of the cytokine there was little or no net increase in cell number, depending on the specific target cells. All plasmacytoma cell lines tested exhibited a similar time dependent inhibition of DNA synthesis and a G0/G1 shift in the cell cycle. Electron microscope examination revealed classical apoptotic features i.e. chromatin condensation and membrane blebbing. DNA fragmentation, measured qualitatively and quantitatively, occurred in all cytokine treated plasmacytoma cell lines. Bovine activin A had an identical capacity to reduce cell viability, to induce G0/G1 shift and to cause DNA fragmentation. X-ray microanalysis of intracellular ions revealed an increase in calcium ions, following exposure of plasmacytoma cells to restrictin-P, accompanied by a decrease in phosphor ions. The cytotoxicity of the inhibitor was augmented in an additive manner by cycloheximide (CHX) indicating that the process did not require de novo protein synthesis. This study thus shows that restrictin-P/stromal activin A kills its target cells by inducing apoptosis. This effect was mediated by subnanogram concentrations and therefore may represent one physiological function of this pleiotropic cytokine.
1994
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(1994) Journal of Cellular Biochemistry. 56, 3, p. 374-384 Abstract
Osteoclasts are derived from hemopoietic precursors in the marrow. Their differentiation pathway is still undefined, but an important role was observed for the marrow microenvironment in the regulation of osteoclasto-genesis. Various marrow stromal cell subtypes were used to study their possible role in the formation of osteoclasts from myeloblast (M1) cells. Interactions between M1 cells and the 14F1.1 endothelial-adipocyte stromal cell line were demonstrated in a coculture model. M1 cells attached to the adherent layer of 14F1.1 cells and formed distinct foci reminiscente of ''cobblestone areas.'' Following these interactions, M1 cells developed specific enzymatic activities and became multinucleated. Both mononuclear and multinuclear M1 cells became positive to tartrate-resistant acid phosphatase (TRaP) and ATPase, a feature characteristic of osteoclasts, and were also responsive to calcitonin. Furthermore, they attached to mineralized bone particles and their membrane changed into a ruffled border at the zone of interaction with the bone matrix. We thus demonstrated that marrow endothelial-adipocytes may play a role in regulating the differentiation of myeloblasts into osteoclasts. (C) 1994 Wiley-Liss, Inc.
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(1994) Calcified Tissue International. 55, 2, p. 120-127 Abstract
The present study describes a new three-dimensional(3-D) culture system that enables the maintenance and phenotypic expression of bone marrow stromal osteoblasts. This culture substratum is advantageous in that it provides suitable conditions for attachment, growth, and differentiation of cells forming 3-D layers. The MBA-15 cell line was grown in unlimited quantities on 3-D Fibro-Cel carriers. These cells mineralized when exposed to ascorbic acid and P-glycerophosphate (beta GP). Under these mineralization conditions, mRNA expressions of procollagen alpha(2)(I) and [H-3]-proline-labeled protein were increased. The expression of mRNA for osteonectin and to a lesser extent, for osteopontin was increased, whereas alkaline phosphatase and biglycan remained unaffected under similar conditions. Exposure of mineralizing cultures to dexamethasone reduced mRNA of procollagen alpha(2) (I) and osteonectin to control level. Scanning electron microscopy revealed that cells were grown along the fabric's fibers and produced collagen fiberils. Under appropriate conditions, extensive mineralization had taken place. The mineralization process involves the formation of calcospherites, and correlates with an increase in calcium content. The Fibro-Cel carriers enable formation of 3-D architecture and mineralized tissue in vitro.
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EXPRESSION OF THE C-KIT LIGAND AND INTERLEUKIN-6 GENES IN MOUSE BONE-MARROW STROMAL CELL-LINES(1994) Stem Cells. 12, 4, p. 409-415 Abstract
The expression of c-kit ligand and interleukin 6 (IL-6) genes in mouse bone marrow-derived stromal cell lines was examined using quantitative polymerase chain reaction (PCR) analysis based on the design of an internal DNA control. The stromal cells studied included the 14F1.1 endothelial-adipocytes that support long-term hemopoiesis and two additional cell lines (MBA-1, MBA-13) which do not have this function. All the cell lines expressed c-kit ligand gene constitutively, and this expression was not increased by lectins. On the other hand, the expression of the IL-6 gene was markedly induced in all the lines by lipopolysaccharide (LPS) and by phorbol 12-myristate 13 acetate (PMA). The constitutive expression of c-kit ligand in 14F1.1 cells was the lowest among the three cell lines studied and could be increased by stimulation with IL-4. Thus, we observed some quantitative differences among the cell lines in their expression of cytokine genes. However, the unique capacity of 14F1.1 cells to support in vitro hemopoiesis cannot thus far be explained solely on the basis of the ability of these cells to secrete cytokines which are not produced by other stromal cell lines. c-kit ligand may be necessary, but its presence alone is not sufficient for 14F1.1 cells to support prolonged hemopoiesis.
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BONE-FORMATION BY MARROW OSTEOGENIC CELLS (MBA-15) IS NOT ACCOMPANIED BY OSTEOCLASTOGENESIS AND GENERATION OF HEMATOPOIETIC SUPPORTIVE MICROENVIRONMENT(1994) Journal of Bone and Mineral Research. 9, 7, p. 1107-1114 Abstract
This study was aimed at elucidating the relationship between osteogenic activity of marrow stromal cells and their ability to support hematopoiesis followed by the bone-remodeling process. We used the MBA-15 cell line, which expresses osteoblastic phenotype in vitro and forms bone in diffusion chamber. We have compared bone formation and hematopoietic responses elicited in vivo by these cells with the implantation of freshly isolated bone marrow cells (BMC) or demineralized tooth matrix (DTM). Both MBA-W cells and BMC, implanted under the kidney capsule, yielded intramembraneous bone, but DTM, implanted subcutaneously, elicited endochondral bone. MBA-15 formed primary bone, mimicking only the initial sequential stages of the ossification process. Neither histologic signs of bone resorption and remodeling nor tartrate-resistant acid phosphatase (TRAP)-positive cells and marrow formation were observed. Bone formation was monitored biochemically. Functions for hematopoietic stem and committed cell content were measured by GM-CFU and BFU-E assays that confirmed the morphologic observations. In both BMC and DTM implantation, bone formation was followed by hematopoietic activity, osteoclastogenesis, and remodeling. We conclude that MBA-15 osteoprogenitor cells, despite their extensive bone formation ability, are unable to form a microenvironment supportive for hematopoiesis and osteoclastogenesis or to initiate bone remodeling.
1993
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TRANSFECTION OF INTERFERON-GAMMA GENE IN A MOUSE BONE-MARROW STROMAL PREADIPOCYTE CELL-LINE CAUSES APOPTOTIC CELL-DEATH(1993) Experimental Hematology. 21, 11, p. 1498-1503 Abstract
It has been reported that bone marrow and serum of patients with aplastic anemia or chronic myeloproliferative disorders contain an abnormal concentration of cytokines. In the present study, we tried to isolate mouse bone marrow stromal cell lines that were stably transformed with a variety of cytokine genes and that expressed them constitutively. From mouse bone marrow stromal cell lines MBA-1, MBA-13, and 14F1.1, we isolated clones secreting interleukin-3 (IL-3), IL-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte (G)-CSF. Interferon-gamma (IFN-gamma)-producing stable transformants could not be established from 14F1.1 cells in spite of repeated transfection trials. At early stages of transfection, 14F1.1 cells did secrete IFN-gamma; however, exogenously added mouse IFN-I could not inhibit 14F1.1 cell growth. We discovered that chromosomal DNA isolated from 14F1.1 after transfection with the mouse IFN-gamma gene was fragmented. This is characteristic of cells undergoing apoptotic cell death. DNA fragmentation was also observed in 14F1.1 cells transfected with the human IFN-gamma gene. These results indicate that intracellular IFN-gamma induces apoptotic cell death of 14F1.1 stromal cells.
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HEMATOPOIESIS ON CELLULOSE ESTER MEMBRANES .13. A COMBINATION OF CLONED STROMAL CELLS IS NEEDED TO ESTABLISH A HEMATOPOIETIC MICROENVIRONMENT SUPPORTIVE OF TRILINEAL HEMATOPOIESIS(1993) Experimental Hematology. 21, 2, p. 257-262 Abstract
A mixture of stromal cells from murine bone marrow placed upon cellulose ester membranes (CEM) and then implanted intraperitoneally (i.p.) in mice results in a regenerated hematopoietic microenvironment which supports trilineal hematopoiesis. We used this model to study the capacity of 5 cloned murine stromal cell lines of marrow origin to support hematopoiesis in vivo: MBA-1 (fibroblast); MBA-2 (endothelial); MBA-13 (fibroendothelial; 14F1.1 (endothelial-adipose); and 14M1.4 (macrophage); 10(7) stromal cells of a single cell line were applied to 1.5 cm CEM, which were folded into tubes and implanted i.p. into mice. Similarly, combinations of 4, 3 and 2 stromal cell lines were applied to CEM and implanted i.p. Single lines were implanted into syngeneic hosts of the same murine strain from which the clone was derived and into nude mice. Combinations of stromal cells were implanted only in nude mice to avoid allogeneic incompatibility. CEM implants were removed after intervals of 5 to 36 weeks and examined histologically. 1) Stromal cells of a single phenotype did not develop hematopoiesis. 2) A combination of 4 stromal phenotypes (MBA-1, MBA-2, MBA-13 and 14F1.1) formed a hematopoietic microenvironment supportive of trilineal hematopoiesis and bone. 3) The combination of 14F1.1 (endothelial adipose) + a second stromal phenotype-MBA-1 (fibroblast) or MBA-2 (endothelial) or MBA-13 (fibroendothelial) also supported trilineal hematopoiesis and bone. 4) CEM coated with MBA-13 or MBA-1 developed bone but no hematopoiesis. The endothelial-adipose phenotype appears to be essential to support hematopoiesis but requires other types of stromal cells-fibroblast, fibroendothelial or endothelial phenotype.
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THE RESTRICTIVE ROLE OF STROMAL FACTORS IN HEMATOPOIESIS(1993) Negative Regulation Of Hematopoiesis: From Fundamental Aspects To Clinical Applications. 229, p. 243-250 Abstract
Keywords: Hematology
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Potentiation of myeloid colony-formation in bone marrow of intact and neonatally thymectomized mice by the thymic hormone THF-γ2(1993) Experimental Hematology. 21, 2, p. 277-282 Abstract
The effect of the thymic hormone THF-γ2 on committed stem cells of bone marrow (BM) origin was determined using the myeloid progenitor cell clonal assay. Preincubation of normal BM cells with THF-γ2 for 1 hour or 18 hours caused a 2- to 6-fold increase in the number of myeloid colonies in the presence of suboptimal concentrations of colony-stimulating factor (CSF). The optimal dose of THF-γ2 causing this enhancement was in the range of 25 to 100 ng/mL. THF-γ2 was not able to replace CSF as an inducer in these experiments. THF-γ2 neither induced IL-6 activity upon 24-hour incubation with bone marrow cells nor enhanced LPS-induced IL-6 secretion by bone marrow cells in vitro. Neonatal thymectomy (NTx) of Balb/c mice caused a decrease in myeloid progenitors, which was repaired by serial injections of THF-γ2. The repair of the stem cell compartment in the bone marrow correlated with an increased percentage of Thy1+ cells in the spleen of THF-γ2-treated NTx mice. These findings indicate that THF-γ2 is able to regulate committed stem cell functions in the bone marrow of immune-deprived NTx and of normal mice.
1992
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Absence of negative growth regulation in three new murine radiation-induced myeloid leukemia cell lines with deletion of chromosome 2(1992) Leukemia. 6, 12, p. 1288-1295 Abstract
Murine radiation-induced acute myeloid leukemia (RI-AML) may be considered as the experimental counterpart of human secondary leukemia. Three new myelomonocytic cell lines derived from RI-AML and carrying a partially deleted chromosome 2 are described. The RI-AML cells responded with increased proliferation after being incubated with the hemopoietic growth factors rG-CSF, rGM-CSF and IL-3. Increased proliferation of the same extent without any effect in differentiation, was also demonstrated in the RI-AML cells after incubation with IL-6 and with mouse lung conditioned medium (CM) and Krebs ascites tumor cells CM which induce differentiation in normal and most leukemic myeloid cells. Down-regulation of the c-myc gene and induction of (2'-5') oligoadenylate synthetase (reflecting autocrine interferon secretion), two essential mechanisms operating during arrest of growth and concomitant differentiation, were demonstrated to be absent in RI-AML cells. In contrast, the M1 cells responded to the above differentiating factors with growth arrest and differentiation and with appropriate c-myc down-regulation and synthetase induction. The genetic basis for the distinct RI-AML cells' behavior may be connected with the loss or structural and/or functional abnormalities of DNA sequences located in the deleted part of chromosome 2 or in the respective allele. The presently described new RI-AML cell lines may be used for studies concerning myeloid leukemogenesis in general and secondary leukemia in particular.
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(1992) Calcified Tissue International. 51, 3, p. 195-201 Abstract
Osteoblasts, members of the marrow stromal cellular network, may play an active role in the hemopoietic microenvironment as well as in bone remodeling. In this study, we examined the extent to which marrow-derived osteogenic cells (MBA-15) possess various stromal functions. This marrow stromal-derived cell line was shown by us to exhibit osteoblastic characteristics in culture and to form bone in vivo. These cells are shown here to constitutively produce and secrete cytokines identified as M-CSF, GM-CSF, and IL-6. MBA-15 cells modulate growth of normal and malignant myeloid and lymphoid cells as well as leukemia cell lines in vitro. Cell-cell interactions were studied in co-cultures with adherent MBA-15 cells and the target hemopoietic cells. Growth inhibition effects, observed under various experimental conditions, can be attributed to the presence of different soluble and membrane-bound inhibitory activities produced by MBA-15 cells. Thus, MBA-15 cells spontaneously produce both stimulators and inhibitors that can affect myeloid and lymphoid cell growth. Marrow osteogenic cells may therefore participate in the stromal regulation of hemopoiesis.
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THE RENEWAL AND DIFFERENTIATION OF HEMATOPOIETIC STEM-CELLS(1992) FASEB Journal. 6, 9, p. 2691-2697 Abstract
Blood-forming tissues are organized in well-defined microenvironments composed of hemopoietic cells and a supportive stroma of connective tissue and endothelium. Hemopoietic cells segregate to various lineages, all derived from a small population of pluripotent stem cells residing in the bone marrow. Regulation of growth and differentiation, particularly under conditions of perturbations, damage, and disease, is mediated by inducer colony-stimulating factors and interleukins counteracted by inhibitory cytokines. Whereas much is known about the mode of induction of differentiation, insufficient information is available to explain the process of stem cell renewal that is crucial for the longevity of the hemopoietic system. It is also only partially known how inhibition of hemopoietic processes occurs, and what molecules in blood-forming tissues signal organization into discrete patterns. This paper reviews recent progress that has opened new avenues to a better understanding of this highly complex issue.
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LONG-TERM SURVIVAL OF HUMAN MYELOID PROGENITOR CELLS INDUCED BY A MOUSE BONE-MARROW STROMAL CELL-LINE(1992) Stem Cells. 10, 3, p. 153-160 Abstract
Mouse endothelial-adipocyte cell line (14F1.1), which induces proliferation of mouse stem cells in culture, is also capable of supporting long-term survival in culture of human myeloid progenitor cells; colony forming unit-granulocyte/macrophage (CFU-GM) was recovered from cultures incubated with the 14F1.1 cell line after over a month of incubation. The CFU-GM population increased beyond the input number, whereas, in control cultures initiated without stromal cells, the number of progenitors gradually declined. Addition of a relatively low concentration of human colony-stimulating factors (CSFs) into the cultures promoted the formation of "cobblestone areas," where mouse stroma and human hemopoietic cells closely interacted. 14F1.1 supernatant alone did not support the survival of human CFU-GM but synergized with the function of human granulocyte-macrophage colony-stimulating factor (GM-CSF) to stimulate adherent macrophage proliferation.
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(1992) Stem Cells. 10, 5, p. 299-308 Abstract
Stromal cells of bone marrow origin produce a variety of known cytokines and some factors exhibiting apparently new biological activities. Several of these were identified by the study of cell to cell interactions and were not found in detectable amounts in media conditioned by the cells. We describe here a culture system that enables the release of stromal cytokines into medium free of any added proteins and supplemented with peptides from casein hydrolysate (0.1%). The absence of serum proteins allows extensive concentration and monitoring of activities that are otherwise undetectable. Stromal cells of the MBA2.1 clonal cell line were seeded in a stationary bed reactor packed with a carrier of nonwoven fabric matrix. After a proliferation phase with serum containing medium, the cells were maintained for over 10 months in proteinfree medium. Throughout this extended incubation in the absence of serum or serum replacing proteins, stromal cells retained their viability and continuously released transforming growth factorβ (TGFβ), macrophagecolony stimulating factor (MCSF) and restrictinP, a cytotoxic factor that specifically arrested the growth of plasmacytoma cells. In addition, interleukin6 (IL6) was first undetectable, and later in culture its titer reached a maximum of 180,000 international units (IU)/ml. Concomitantly, the production of restrictinP diminished and reached its lowest levels at the end of 10 months. The results may imply a possible causal relationship between the expression of IL6 and restrictinP, since no similarly significant changes were observed in the titers of MCSF and TGFβ. This novel bioreactor system may be adaptable for efficient production of different cytokines under absolute serumfree conditions.
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(1992) Molecular Biology of Haematopoiesis: Proceedings of the International Conference on Molecular Biology of Haematopoiesis. p. 247-256 Abstract
1991
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(1991) Calcified Tissue International. 49, 3, p. 202-207 Abstract
A series of stromal cell lines derived from mouse bone marrow, representing subpopulations of putative stromal cell types, were examined for the expression of osteoblastic properties. The effects of dexamethasone and specific inhibitors on alkaline phosphatase activity, cAMP response to bone-seeking hormones, and the ability to mineralize extracellular matrix in vitro as well as collagen typing were used as osteoblastic markers. We found that all stromal cell types examined possess some osteoblastic features but differ in the degree of expression. The data provide support to the hypothesis of a common stem cell for marrow stromal cells.
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EXPRESSION OF ALPHA-SMOOTH MUSCLE ACTIN IN MURINE BONE-MARROW STROMAL CELLS(1991) Blood. 78, 2, p. 304-309 Abstract
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(1991) Annals of the New York Academy of Sciences. 628, p. 287-297 Abstract
MBA-2.1 cells produce an activity, designated restrictin-P, which is specifically inhibitory to the growth of plasmacytomas and mature B cell lymphomas. We examined whether the activity of this stromally derived glycoprotein could be attributed to a well-characterized growth factor. Restrictin-P-producing cells were therefore screened for the expression of transcripts of a variety of growth suppressors. With the exception of TGF-β1, none was produced in detectable amounts by these cells. Furthermore, recombinant forms of the inhibitory molecules tested did not exert a biological effect similar to that of restrictin-P. Restrictin-P was shown to elicit a G0/G1 arrest in the cell cycle of its target cells, as soon as 24 h after their exposure to the inhibitor. This effect could not be mimicked by TGF-β1. We suggest that restrictin-P is part of a novel family of inhibitors which are required for the maintenance of cell-type specificities in the hematopoietic microenvironment.
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1990
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DETECTION OF INTERLEUKIN-3 IN THE SERUM OF MICE INFECTED WITH MYCOBACTERIUM-LEPRAEMURIUM(1990) Journal of Infectious Diseases. 162, 5, p. 1202-1204 Abstract
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REGULATION OF HEMATOPOIESIS BY CYTOKINES THAT RESTRICT OPTIONS FOR GROWTH AND DIFFERENTIATION(1990) Cancer Cells-A Monthly Review. 2, 7, p. 205-211 Abstract
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SELECTIVE ACCUMULATION OF LYMPHOCYTE PRECURSOR CELLS MEDIATED BY STROMAL CELLS OF HEMATOPOIETIC ORIGIN(1990) Experimental Hematology. 18, 4, p. 332-340 Abstract
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ROLE OF STROMAL CELL FACTORS (RESTRICTINS) IN MICROORGANIZATION OF HEMATOPOIETIC TISSUES(1990) Biology Of Hematopoiesis. 352, p. 115-122 Abstract
Keywords: Oncology; Hematology; Medicine, Research & Experimental
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1989
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MULTILINEAGE HEMATOPOIESIS INDUCED BY CLONED STROMAL CELLS(1989) Stem Cells. 7, 6, p. 373-384 Abstract
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LONG-TERM PROLIFERATION OF HUMAN-LEUKEMIA CELLS INDUCED BY MOUSE STROMA(1989) Experimental Hematology. 17, 5, p. 398-404 Abstract
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(1989) Mechanisms of Ageing and Development. 48, 1, p. 91-99 Abstract
The effect of aging on the hemopoietic capacity of bone marrow (BM) cells was investigated. No difference was found in the incidence of colony forming units-spleen (CFU-S) and granulocyte-macrophage colony forming cells (GM-CFC) present in the BM of young (2-4 months) or old (24-30 months) mice. However, increased proliferations (× 3) of the old BM cells was observed when cultured in the presence of L-cell conditioned medium. The cells were also cultivated over an adherent layer of a BM stroma cell line (14F1.1, endothelial-adipocytes) and the non-adherent cells removed and tested weekly. Cells orginating from the old BM proliferated to a greater extent and produced more GM-CFC than those from the young. Differentiation into T-cells after colonizing fetal thymus explants was also measured and found to be reduced in both groups, though to a greater extent in the old. Thus, under the present experimental conditions, myeloid progenitors in the old BM manifest a more pronounced self renewal and differentiation capacity than the young while for the T-cell pregenitors the situation is reversed.
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STROMAL CELLS FROM THE BONE-MARROW - EVIDENCE FOR A RESTRICTIVE ROLE IN REGULATION OF HEMATOPOIESIS(1989) European Journal of Haematology. 42, 3, p. 225-232 Abstract
1988
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MODULATION OF HEMATOPOIESIS BY NOVEL STROMAL CELL FACTORS(1988) Leukemia. 2, 12, p. S9-S15 Abstract
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RESTRICTINS - STROMAL CELL ASSOCIATED FACTORS THAT CONTROL CELL ORGANIZATION IN HEMATOPOIETIC TISSUES(1988) Natural Immunity and Cell Growth Regulation. 7, 3, p. 185-192 Abstract
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INTRODUCTION OF INTERLEUKIN-3 GENE INTO STROMAL CELLS FROM THE BONE-MARROW ALTERS HEMATOPOIETIC DIFFERENTIATION BUT DOES NOT MODIFY STEM-CELL RENEWAL(1988) Blood. 71, 3, p. 586-596 Abstract
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Interleukin-4 (b Cell Stimulating Factor) Enhances the Antigen Presenting Ability of Thymic and Bone Marrow Macrophages(1988) Antigen presenting cells. Tew J. G. & Schook L. B.(eds.). New York: . p. 243-249 (trueProgress in leukocyte biology). Abstract
1987
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(1987) Journal of Immunology. 138, 12, p. 4275-4279 Abstract
We have studied the effects of recombinant mouse interleukin 4 (IL 4) (previously known as B cell stimulatory factor 1) on the antigen-presenting ability of murine splenic B cells and bone marrow macrophages. Our assay is based on the induction of antigen-presenting ability in these cells after incubation with IL 4 for 24 hr. The presenting cells were then used to stimulate IL 2 production by antigen-specific, I-A(d)-restricted T cell hybridomas, a response mainly dependent on the induction of Ia antigens. Consistent with our previously published data using partially purified natural IL 4, we show here that recombinant IL 4 (but not interferon-γ (IFN-γ) or IL 1) induces antigen-presenting ability in B cells. Recombinant IL 4 was also found to induce antigen-presenting ability in a cloned, bone marrow derived-macrophage cell line (14M1.4), and in normal bone marrow-derived macrophages. These macrophage populations also respond to IFN-γ showing enhanced antigen-presenting ability (mediated by increased Ia antigen expression). A small but significant increase in Ia antigen expression was also detected in 14M1.4 macrophages induced with IL 4. However, additional analysis suggested that the effect of IL 4 on 14M1.4 is different from that of IFN-γ, because IL 4 (but not IFN-γ) is able to maintain the viability and increase the size of and metabolic activity of bone marrow macrophages. However, IL 4 may not affect all macrophages because the macrophage cell line P388D1, which responds to IFN-γ, failed to show enhanced antigen-presenting function after stimulation with IL 4. These observations indicate that IL 4, a lymphokine previously considered to be B cell lineage specific, has effects on macrophages and may be involved in their activation.
1986
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(1986) Cell. 46, 1, p. 31-40 Abstract
Different hematopoietic cells produce minute amounts of β-related interferon (IFN) following induction of differentiation by chemical or natural inducers. The endogenous IFN binds to type I cell surface receptors and modulates gene expression in the producer ceils. We show that self-induction of two members of the IFN-induced gene family differs in the dose response sensitivity and the prolonged kinetics of mRNA accumulation from the response to exogenous IFN-β1. Production and response to endogenous IFN are also detected when bone marrow precursor cells differentiate to macrophages after exposure to colony stimulating factor 1. In M1 myeloid cells induced to differentiate by lung-conditioned medium, addition of antibodies against IFN-β partially abrogates the reduction of c-myc mRNA and the loss in cell proliferative activity, which both occur during differentiation. The endogenous IFN therefore functions as an autocrine growth inhibitor that participates in controlling c-myc suppression and the specific G0 G1 arrest during terminal differentiation of hematopoietic cells.
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(1986) Proceedings of the National Academy of Sciences of the United States of America. 83, 12, p. 4547-4551 Abstract
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(1986) Annual Reports in Medicinal Chemistry. 21, p. 263-272 Abstract
Clonal growth of blood cells was achieved by seeding bone marrow cells embedded in agar medium supplemented with serum onto a \u201cfeeder layer\u201d of mouse embryo fibroblasts Following a week of incubation hemopoietic colonies developed. Feeder cell layers could be replaced by media conditioned by embryo fibroblasts or extracts of other cells and tissues. The biologically active substance(s) present in those conditioned media termed \u201ccolony stimulating factor\u201d (CSF), induced the formation of colonies containing granulocytes and macrophages. Hemopoietic system is composed of a hierarchy of cells. The pluripotent stem cell gives rise to independent progenitor cells, committed to the various differentiation lineages and capable of forming colonies. The colony stimulating factor was one of a family of glycoprotein molecules having different although overlapping target cell specificities. The clonal growth and differentiation of the various types of CFCs depended upon the presence of an appropriate CSF. Many of these stimulating factors have been purified and molecularly cloned. The purpose of this chapter is to summarize and compare the properties of these molecules, describe newly discovered hemopoietic factors, and evaluate the in vivo roles and potential medical uses of CSF's. The chapter discusses multipotential colony stimulating factors, colony stimulating factors for granulocytes and macrophages, MG-1 and MG-2. Certain myeloid leukemia cell lines that are independent with respect to their capacity to proliferate in culture have been shown to terminally differentiate and stop growing upon the addition of a factor designated MG-2. In normal cells, MGI-2 is endogenously produced following induction of growth by colony stimulating factors. These latter factors are referred to by the collective name MGI-1newly identified factors with activities overlapping those of CSFsthat includes role of CSFs in the regulation of hemopoiesis, relationship between CSFs or their receptors and cancer, and potential clinical utility of CSF.
1985
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PHENOTYPIC HETEROGENEITY AMONG STROMAL CELL-LINES FROM MOUSE BONE-MARROW DISCLOSED IN THEIR EXTRACELLULAR-MATRIX COMPOSITION AND INTERACTIONS WITH NORMAL AND LEUKEMIC-CELLS(1985) Blood. 66, 2, p. 447-456 Abstract
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In vitro functions of stromal cells from human and mouse bone marrow(1985) Experimental Hematology. 13, 7, p. 603-609 Abstract
Human fibroblastoid cell strains obtained from primary bone marrow cultures and continuous stromal cell lines recently derived from mouse bone marrow were studied. The incidence of fibroblastoid precursors (CFU-F) varied considerably in human bone marrow samples, and no differences could be detected between marrows from a group of myelodysplastic patients (age range 70-82 years) and groups of age-matched controls or younger individuals. A lack of direct correlation between initial clonogenicity and ultimate capacity of fibroblastoid cells to grow in continuous culture was observed in both the normal and the myelodysplastic groups. Despite the apparently normal clonogenicity of CFU-F in patients with myelodysplastic syndromes, some of these marrows failed to grow when subcultured. Normal fibroblastoid cells at 104 per culture exhibited myelopoietic activities when cocultured with fresh bone marrow cells. At higher concentrations, these cells inhibited myeloid colony formation. Fibroblastoid cells from only one out of four myelodysplastic patients examined exhibited comparable inhibitory activity. The specificity of the inhibitor(s) was demonstrated by the lack of effect of fibroblastoid cells from normal human bone marrow on the clonogenicity of mouse erythroleukemia cells. Moreover, human foreskin fibroblasts were devoid of such inhibitory activity. These functions of cultured stromal cells may correlate with some of their activities in the bone marrow microenvironment.
1984
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(1984) Annales de l'Institut Pasteur - Immunology. 135, 2, p. 299-302 Abstract
The complex structure of the haemopoietic system, i. e. distinct organs physically separated from each other and interconnected by circulatory networks, requires inter-organ feedback loops to control cell production. The need for such control mechanisms is obvious in the case of the thymus, which is both a site of extensive cell proliferation and an obligatory differentiation inducer of T-cell lineage. These processes are strictly dependent upon the migration, into the thymus, of cells produced in the microenvironment of the bone marrow. Is there evidence for an active role of the thymus in the control of stem cell pools within the marrow? An overview of the experimental data presented in this Forum by various investigators points to a number of mechanisms involving the activity of both thymusderived cells and secreted factors. Figure 1A schematically summarizes the implications of these experimental systems.
1982
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(1982) Experimental Hematology Today 1982. Ledney G. D., Baum S. J. & Thierfelder S.(eds.). Basel: . p. 19-29 (trueExperimental Hematology Today). Abstract
Although it is well established that for myelopoiesis in vitro CSF (colony-stimulating factor) is required, it has not been conclusively demonstrated that in vivo CSF is obligatory. The authors found that although adherent stromal cells from mouse bone marrow support the proliferation of myeloid progenitors in short-term bone marrow cultures, they inhibit the formation of G/M colonies (30).
1981
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MYELOPOIESIS IN THE PRESENCE OF STROMAL CELLS FROM MOUSE BONE-MARROW .2. MECHANISM OF GLUCOSE DEPENDENT COLONY FORMATION(1981) Experimental Hematology. 9, 6, p. 663-673 Abstract
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MYELOPOIESIS IN THE PRESENCE OF STROMAL CELLS FROM MOUSE BONE-MARROW .1. MONOSACCHARIDES REGULATE COLONY FORMATION(1981) Experimental Hematology. 9, 6, p. 656-662 Abstract
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1980
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(1980) Cell Proliferation. 13, 3, p. 287-298 Abstract
The in vitro growth pattern of a number of mouse lymphoblastoid tumour cell lines was modified in the presence of adherent cell layers from various sources. The AVRij1 and ST4b cell lines exhibited a concentrationdependent growth pattern, i.e., they would only grow well when seeded at high starting cell concentrations. Better growth of these cells from low cell concentrations was observed in the presence of adherent cell layers from syngeneic or allogeneic bone marrow. Adherent cell layers derived from mouse spleen and pleural or peritoneal cavity could also promote the growth of the above tumour cells, but in a narrower range of cell concentrations and to a lower extent. Moreover, confluent adherent layers from the pleural and peritoneal cavities completely inhibited the growth of AVRij1 and ST4b cells, while adherent cell layers from the bone marrow did not inhibit growth at any cell concentration tested. The in vitro growth of concentrationindependent cell lines was also affected by the presence of adherent cells from the bone marrow. Under syngeneic conditions, a slight increase in the growth of the null or preB lymphoma cell line ABLS8.1 was observed. On the other hand, the growth of tumour cells expressing more differentiated properties, such as the thymus T lymphoma tumour cell line ST1.3 and the plasma cell tumour MPC11.45.6.2.4, was inhibited in the presence of syngeneic bone marrow derived adherent cell layers. This inhibition was more pronounced under allogeneic conditions. Growth inhibition was also observed when concentrationindependent cell lines were cocultured with adherent cells from the pleural and peritoneal cavities. Thus, adherent cell layers from nonhaemopoietic sources inhibited the growth of all cell lines tested. On the other hand, adherent cells from the bone marrow had a differential effect on growth of lymphoblastoid tumour cell lines. This depended on the in vitro growth properties of each tumour cell line and on some additional specific tumour cell properties. The latter could relate to the differentiation stage characterizing each tumour cell line. The culture method described here may serve as a model system for studies on interaction of leukaemic cell and the haemopoietic microenvironment.
1979
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The role of fibroblastoid cells and macrophages from mouse bone marrow in the in vitro growth promotion of haemopoietic tumour cells(1979) Experimental Hematology. 7, 4, p. 206-218 Abstract
Adherent cells from mouse bone marrow have been shown to promote the in vitro growth of the AVRij-1 tumour cell line. The experiments presented here suggest that the adherent cells involved in this phenomenon are the progeny of bone marrow derived fibroblastoid colony forming units. The latter were characterized by means of cell density distribution analysis. They had a broad distribution pattern and an average peak cell density of 1.069 g.cm-3. The growth promotion activity exerted by adherent cell layers from the various density fractions on the AVRij-1 tumour cell line coincided with the distribution of fibroblastoid colony forming units. On the other hand, the presence of macrophages in the adherent layers seemed to be non-essential for the in vitro promotion of growth of the AVRij-1 cell line.
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Changes in the fibroblastoid colony forming unit population from mouse bone marrow in early stages of Soule virus induced murine leukemia(1979) Experimental Hematology. 7, 3, p. 137-144 Abstract
Fibroblastoid cells from mouse bone marrow belong to the hemopoietic inductive microenvironment involved in the regulation of hemopoiesis. We observed a decline in the incidence of precursors of these cells (fibroblastoid colony forming units) in the early stages of viral leukemogenesis. This was not accompanied by similar changes in bone marrow derived hemopoietic colony forming cell populations (CFU-s and CFU-c). Cultured fibroblastoid colonies from the bone marrow of young AKR mice or Soule murine leukemia virus inoculated BALB/c mice were found to produce type-C ecotropic virus. No such production was observed when similarly cultured granulocyte macrophage colonies (CFU-c) from the bone marrow of these mice were examined. The possibility that the fibroblastoid cell population is a major source of ecotropic leukemia viruses in the early stages of viral leukemogenesis is discussed.
1977
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CHARACTERIZATION OF HUMAN LYMPHATIC LEUKEMIAS - T AND B CELL PROLIFERATIONS(1977) Israel Medical Association Journal. 13, 8, p. 753-757 Abstract
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BINDING OF F(AB')2 OF NORMAL HUMAN IGG TO HUMAN LYMPHOCYTES(1977) Journal of Immunology. 119, 6, p. 2209-2211 Abstract
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(1977) Advances in Comparative Leukemia Research. Yohn D. S., Hilgers JO. & Bentvelzen P.(eds.). Amsterdam: . p. 314-315 Abstract
1975
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ROLE OF A THYMUS HUMORAL FACTOR IN PROLIFERATION OF BONE-MARROW CFU-S FROM THYMECTOMIZED MICE(1975) Experimental Hematology. 3, 6, p. 389-398 Abstract
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RECENT STUDIES ON ROLE OF THYMUS IN EARLY STAGES OF LYMPHOPOIESIS AND IMMUNE DIFFERENTIATION(1975) Bollettino Dell Istituto Sieroterapico Milanese. 54, 3, p. 211-218 Abstract
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IMPAIRED RADIOPROTECTIVE CAPACITY AND REDUCED PROLIFERATIVE RATE OF BONE-MARROW FROM NEONATALLY THYMECTOMIZED MICE(1975) Experimental Hematology. 3, 1, p. 1-11 Abstract
1973
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DEFECTIVE CAPACITY OF BONE-MARROW FROM NUDE MICE TO RESTORE LETHALLY IRRADIATED RECIPIENTS(1973) Blood. 42, 5, p. 671-678 Abstract