Uncommitted Mesenchymal Stem Cells Research Articles

Glucocorticoids inhibit the maturation of committed osteoblasts via SOX2.

During bone formation, mesenchymal progenitor cells mature into bone-forming osteoblasts after undergoing several stages of differentiation. Impaired bone formation is a predominant finding in glucocorticoid (GC)-induced osteoporosis (GIO). Osteoblasts at different stages of maturation can be affected by excessive endogenous or therapeutic GCs. Sex-determining region Y-box 2 (SOX2) is normally expressed in immature osteoblasts, but its overexpression can suppress osteoblast differentiation. This study aimed to evaluate whether GC affects SOX2 expression in osteoblasts, and whether SOX2 contributes to GC-induced inhibition of osteoblast differentiation. Treatment with GCs such as dexamethasone (Dex) or hydrocortisone enhanced SOX2 expression. Silencing SOX2 improved inhibition of GC-induced osteoblast differentiation, whereas SOX2 overexpression decreased mineralized nodule formation and RUNX2 and Osterix expression in MC3T3-E1 cells. On the contrary, when C3H10T1/2 uncommitted mesenchymal stem cells were subjected to SOX2 overexpression, RUNX2 expression increased. As a mechanism of Dex-induced SOX2 upregulation in preosteoblasts, we found that the STAT3 pathway or GC receptor (GR) is involved, using a GR antagonist, STAT3 regulators, and chromatin immunoprecipitation assays. Moreover, mice treated with Dex for 4 weeks showed a notable increase in SOX2 expression in the bones and an increased ratio of procollagen type 1 N-terminal propeptide to osteocalcin in the plasma than in control mice. This study demonstrated that GC enhances SOX2 expression in vitro in osteoblast and in vivo in the mice bone, which affects bone-forming activity differently depending on the differentiation stage of osteoblast-lineage cells. Our results provide new insights into prevention and treatment against impaired bone formation in GIO.

Glucocorticoid receptor-mediated cis-repression of osteogenic genes requires BRM-SWI/SNF

Glucocorticoids are an effective therapy for a variety of severe inflammatory and autoimmune disorders; however, the therapeutic use of glucocorticoids is severely limited by their negative side effects, particularly on osteogenesis. Glucocorticoids regulate transcription by binding to the glucocorticoid receptor (GR), which then binds the promoters of target genes to induce either activation or repression. The gene activation effects of nuclear hormone receptors broadly require the cooperation of the chromatin remodeling complex known as SWI/SNF, which is powered by an ATPase core. The well-studied SWI/SNF ATPase, BRG1, is required for gene activation by a spectrum of nuclear hormone receptors including GR. However, glucocorticoid-induced side effects specifically related to impaired osteogenesis are mostly linked with GR-mediated repression. We have considered whether cis-repression of osteogenic genes by GR may be mediated by a distinct subclass of SWI/SNF powered by the alternative ATPase, BRM. BRM does not have an essential role in mammalian development, but plays a repressor role in osteoblast differentiation and favors adipogenic lineage selection over osteoblast commitment, effects that mirror the repressor effects of GR. The studies reported here examine three key GR cis-repression gene targets, and show that GR association with these promoters is sharply reduced in BRM deficient cells. Each of these GR-targeted genes act in a different way. Bglap encodes osteocalcin, which contributes to normal maturation of osteoblasts from committed pre-osteoblasts. The Per3 gene product acts in uncommitted mesenchymal stem cells to influence the osteoblast/adipocyte lineage selection point. Fas ligand, encoded by FasL, is a means by which osteoblasts can modulate bone degradation by osteoclasts. Repression of each of these genes by glucocorticoid favors bone loss. The essential role of BRM in cooperation with GR at each of these control points offers a novel mechanistic understanding of the role of GR in bone loss.

Osteogenic lineage commitment of mesenchymal stem cells from patients with ossification of the posterior longitudinal ligament

Ectopic bone formation is thought to be responsible for ossification of the posterior longitudinal ligament of the spine (OPLL). Mesenchymal stem cells (MSCs) were isolated from spinal ligaments and shown to play a key role in the process of ectopic ossification. The purpose of this study was to explore the capacity of these MSCs to undergo lineage commitment and to assess the gene expression changes between these committed and uncommitted MSCs between OPLL and non-OPLL patients. Spinal ligament-derived cells were obtained from OPLL patients or patients with cervical spondylotic myelopathy (non-ossified) for comparison (n=8 in each group). MSCs from the two patient cohorts were evaluated for changes in colony forming ability; osteogenic, adipogenic and chondrogenic differentiation potential; and changes in gene expression following induction with lineage-specific conditions. We show that the osteogenic differentiation potential was significantly higher in MSCs from OPLL patients than in those from non-OPLL patients. In addition, alkaline phosphatase activity and several osteogenic-related genes expressions (bone morphogenetic protein 2, runt-related transcription factor 2 and alkaline phosphatase) were significantly higher in the OPLL group than in the non-OPLL group. However, single cell cloning efficiency, adipogenic and chondrogenic differentiation, and the expression of adipogenic and chondrogenic-related genes were equivalent between MSCs harvested from OPLL and non-OPLL patient samples. These findings suggest an increase in the osteogenic differentiation potential of MSCs from OPLL patients and that this propensity toward the osteogenic lineage may be a causal factor in the ossification in these ligaments.

Silent Corticotroph Adenoma with Adrenal Cortical Choristoma: a Rare but Distinct Morphological Entity

This report describes a case of pituitary adenoma with interspersed adrenal cortical cells. The pituitary cells were confirmed to be corticotrophs with Tpit and adrenocorticotropic hormone immunohistochemistry, whereas the adrenal cortical cells were verified to be such with steroidogenic factor-1 (SF-1), inhibin, calretinin, and Melan A staining. The presence of normal adrenal cortical cells in the heterotopic location of the sella fulfills the definition of choristoma. The origin of adrenal cortical cells within a pituitary adenoma remains unexplained. The important role of SF-1 in both pituitary and adrenal cortex may explain a relationship that supports the possibility of an abnormal proliferation and differentiation of uncommitted mesenchymal stem cells within the sella. However, it remains possible that misplaced adrenal cortical cells derived during embryogenesis give rise to this rare but distinct morphological entity that can pose a difficult diagnostic dilemma. The approach to differential diagnosis is discussed.

Nonstimulated Human Uncommitted Mesenchymal Stem Cells Express Cell Markers of Mesenchymal and Neural Lineages

Ex vivo cultures of human bone marrow-derived mesenchymal stem cells (MSCs) contain subsets of progenitors exhibiting dissimilar properties. One of these subsets comprises uncommitted progenitors displaying distinctive features, such as morphology, a quiescent condition, growth factor production, and restricted tissue biodistribution after transplantation. In this study, we assessed the competence of these cells to express, in the absence of differentiation stimuli, markers of mesoderm and ectodermic (neural) cell lineages. Fluorescence microscopy analysis showed a unique pattern of expression of osteogenic, chondrogenic, muscle, and neural markers. The depicted "molecular signature" of these early uncommitted progenitors, in the absence of differentiation stimuli, is consistent with their multipotentiality and plasticity as suggested by several in vitro and in vivo studies.

Chondroma of the cheek: A case report

An extremely rare case of soft tissue chondroma occurring in the right cheek of a 47-year-old woman is reported. The localized nodular tumor was encapsulated and composed of hyalinized cartilage with fine calcifications. Most tumor cells were positive for vimentin and S-100 protein, but negative for cytokeratin, factor VIII, and smooth muscle actin. It seems likely that the tumor cells arise from uncommitted mesenchymal stem cells by metaplastic process.

Menin Is Required for Bone Morphogenetic Protein 2- and Transforming Growth Factor β-regulated Osteoblastic Differentiation through Interaction with Smads and Runx2

Menin, the product of the multiple endocrine neoplasia type 1 (MEN1) gene, is required for commitment of multipotential mesenchymal stem cells to the osteoblast lineage, however, it inhibits their later differentiation (Sowa, H., Kaji, H., Canaff, L., Hendy, G.N., Tsukamoto, T., Yamaguchi, T., Miyazono, K., Sugimoto, T., and Chihara, K. (2003) J. Biol. Chem. 278, 21058-21069). Here, we have examined the mechanism of action of menin in regulating osteoblast differentiation using the mouse bone marrow stromal ST2 and osteoblast MC3T3-E1 cell lines. In ST2 cells, reduced menin expression achieved by transfection of menin antisense DNA (AS) antagonized bone morphogenetic protein (BMP)-2-induced alkaline phosphatase activity and osteocalcin and Runx2 mRNA expression. Menin was co-immunoprecipitated with Smad1/5 in ST2 and MC3T3-E1 cells, and inactivation of menin antagonized BMP-2-induced transcriptional activity of Smad1/5 in ST2 cells, but not MC3T3-E1 cells. Menin was co-immunoprecipitated with the key osteoblast regulator, Runx2, and AS antagonized Runx2 transcriptional activity and the ability of Runx2 to stimulate alkaline phosphatase activity only in ST2 cells but not in MC3T3-E1 cells. In the osteoblast MC3T3-E1 cells, transforming growth factor-beta and its signaling molecule, Smad3, negatively regulated Runx2 transcriptional activity. Menin and Smad3 were co-immunoprecipitated, and combined menin and Smad3 overexpression antagonized, whereas menin and the dominant-negative Smad3DeltaC together enhanced BMP-2-induced transcriptional activity of Smad1/5 and Runx2. Smad3 alone had no effect. Therefore, menin interacts physically and functionally with Runx2 in uncommitted mesenchymal stem cells, but not in well differentiated osteoblasts. In osteoblasts the interaction of menin and the transforming growth factor-beta/Smad3 pathway negatively regulates the BMP-2/Smad1/5- and Runx2-induced transcriptional activities leading to inhibition of late-stage differentiation.

Subcellular Distribution and Mitogenic Effect of Basic Fibroblast Growth Factor in Mesenchymal Uncommitted Stem Cells

Uncommitted mesenchymal stem cells (MSC), upon commitment and differentiation give rise to several mature mesenchymal lineages. Although the involvement of specific growth factors, including FGF2, in the development of committed MSC is known, the effect of FGF2 on uncommitted progenitors remains unclear. We have analyzed on a comparative basis, the subcellular distribution and mitogenic effect of FGF2 in committed and uncommitted MSC prepared from human bone marrow. Indirect immunofluorescence studies showed strong nuclear FGF2 staining in both progenitors; however, cytoplasmic staining was only detected in committed cells. Western blot analysis revealed the presence of 22.5 and 21-22 kDa forms of FGF2 in the nucleus of both progenitors; however, their relative content was higher in uncommitted than in committed cells. Exogenous FGF2 stimulated proliferation and sustained quiescence in committed and uncommitted cells, respectively. These results show that both type of progenitors, apart from morphological and proliferative differences, display specific patterns of response to FGF2.

Critical windows in development of the rodent immune system.

The immune system of rodents, like that in humans, develops from a population of pluripotential hematopoietic stem cells (HSC) that are generated early in gestation from uncommitted mesenchymal stem cells in the intraembryonic splanchnoplure surrounding the heart. This early population of HSC gives rise to all circulating blood cell lineages, including cells of the innate and acquired immune system. To access the impact of chemical exposure on the developing immune system and establish developmental windows of potential vulnerability to these exposures, it is essential to first consider the anatomical development of hematopoietic and lymphopoietic tissues and the sequence of appearance of cells that give rise to the immune system. This is particularly true in embryonic development because, after they initially appear in intraembryonic mesenchyme early in gestation, HSC migrate through an orderly series of tissues before establishing residence in the bone marrow and thymus. The effect of exposure to chemical insults in utero, then, may differ depending on the specific timing of exposure and anatomical location of hematopoiesis. Mechanisms and consequences of developmental immunotoxicity in experimental animals will need to be considered in that context. This review presents an overview of developmental hematopoiesis and a working hypothesis of critical developmental windows of vulnerability of this developmental system to toxic insult by chemical exposure.