Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue.

Human mesenchymal stem cells (MSC), that have been reported to be present in bone marrow, adipose tissues, dermis, muscles and peripheral blood, have the potential to differentiate along different lineages including those forming bone, cartilage, fat, muscle and neuron. This differentiation potential makes MSC excellent candidates for cell-based tissue engineering. In this study, we have examined phenotypes and gene expression profile of the human adipose tissue-derived stromal cells (ATSC) in the undifferentiated states, and compared with that of bone marrow stromal cells (BMSC). ATSC were enzymatically released from adipose tissues from adult human donors and were expanded in monolayer with serial passages at confluence. BMSC were harvested from the metaphysis of adult human femur. Flowcytometric analysis showed that ATSC have a marker expression that is similar to that of BMSC. ATSC expressed CD29, CD44, CD90, CD105 and were absent for HLA-DR and c-kit expression. Under appropriate culture conditions, MSC were induced to differentiate to the osteoblast, adipocyte, and chondrogenic lineages. ATSC were superior to BMSC in respect to maintenance of proliferating ability, and microarray analysis of gene expression revealed differentially expressed genes between ATSC and BMSC. The proliferating ability and differentiation potential of ATSC were variable according to the culture condition. The similarities of the phenotypes and the gene expression profiles between ATSC and BMSC could have broad implications for human tissue engineering.     

Trithorax- and Polycomb-group response elements within an Ultrabithorax transcription maintenance unit consist of closely situated but separable sequences.

In Drosophila, two classes of genes, the trithorax group and the Polycomb group, are required in concert to maintain gene expression by regulating chromatin structure. We have identified Trithorax protein (TRX) binding elements within the bithorax complex and have found that within the bxd/pbx regulatory region these elements are functionally relevant for normal expression patterns in embryos and confer TRX binding in vivo. TRX was localized to three closely situated sites within a 3-kb chromatin maintenance unit with a modular structure. Results of an in vivo analysis showed that these DNA fragments (each approximately 400 bp) contain both TRX- and Polycomb-group response elements (TREs and PREs) and that in the context of the endogenous Ultrabithorax gene, all of these elements are essential for proper maintenance of expression in embryos. Dissection of one of these maintenance modules showed that TRX- and Polycomb-group responsiveness is conferred by neighboring but separable DNA sequences, suggesting that independent protein complexes are formed at their respective response elements. Furthermore, we have found that the activity of this TRE requires a sequence (approximately 90 bp) which maps to within several tens of base pairs from the closest neighboring PRE and that the PRE activity in one of the elements may require a binding site for PHO, the protein product of the Polycomb-group gene pleiohomeotic. Our results show that long-range maintenance of Ultrabithorax expression requires a complex element composed of cooperating modules, each capable of interacting with both positive and negative chromatin regulators.     

Craniosynostosis and multiple skeletal anomalies in humans and zebrafish result from a defect in the localized degradation of retinoic acid

Excess exogenous retinoic acid (RA) has been well documented to have teratogenic effects in the limb and craniofacial skeleton. Malformations that have been observed in this context include craniosynostosis, a common developmental defect of the skull that occurs in 1 in 2500 individuals and results from premature fusion of the cranial sutures. Despite these observations, a physiological role for RA during suture formation has not been demonstrated. Here, we present evidence that genetically based alterations in RA signaling interfere with human development. We have identified human null and hypomorphic mutations in the gene encoding the RA-degrading enzyme CYP26B1 that lead to skeletal and craniofacial anomalies, including fusions of long bones, calvarial bone hypoplasia, and craniosynostosis. Analyses of murine embryos exposed to a chemical inhibitor of Cyp26 enzymes and zebrafish lines with mutations in cyp26b1 suggest that the endochondral bone fusions are due to unrestricted chondrogenesis at the presumptive sites of joint formation within cartilaginous templates, whereas craniosynostosis is induced by a defect in osteoblastic differentiation. Ultrastructural analysis, in situ expression studies, and in vitro quantitative RT-PCR experiments of cellular markers of osseous differentiation indicate that the most likely cause for these phenomena is aberrant osteoblast-osteocyte transitioning. This work reveals a physiological role for RA in partitioning skeletal elements and in the maintenance of cranial suture patency.     

Lack of evidence that hematopoietic stem cells depend on N-cadherin-mediated adhesion to osteoblasts for their maintenance

Recent studies have proposed that bone marrow hematopoietic stem cells (HSCs) are maintained via N-cadherin-mediated homophilic adhesion with osteoblasts. However, there is not yet any evidence that N-cadherin-expressing cells have HSC activity or that osteoblasts are required for HSC maintenance. We were unable to detect N-cadherin expression in highly purified HSCs by polymerase chain reaction, by using commercial anti-N-cadherin antibodies, or by beta-galactosidase staining of N-cadherin gene trap mice. Only N-cadherin-negative bone marrow cells exhibited HSC activity in irradiated mice. Finally, biglycan-deficient mice had significant reductions in trabecular bone and osteoblasts but showed no defects in hematopoiesis, HSC frequency, or function. Thus, reductions in osteoblasts do not necessarily lead to reductions in HSCs. Most bone marrow HSCs in wild-type and biglycan-deficient mice localized to sinusoids, and few localized within five cell diameters of the endosteum. These results question whether significant numbers of HSCs depend on N-cadherin-mediated adhesion to osteoblasts.     

Role of Nanog in the maintenance of marrow stromal stem cells during post natal bone regeneration

Post natal bone repair elicits a regenerative mechanism that restores the injured tissue to its pre-injury cellular composition and structure and is believed to recapitulate the embryological processes of bone formation. Prior studies showed that Nanog, a central epigenetic regulator associated with the maintenance of embryonic stem cells (ESC) was transiently expressed during fracture healing, Bais et al. In this study, we show that murine bone marrow stromal cells (MSCs) before they are induced to undergo osteogenic differentiation express ～50× the background levels of Nanog seen in murine embryonic fibroblasts (MEFs) and the W20-17 murine marrow stromal cell line stably expresses Nanog at ～80× the MEF levels. Nanog expression in this cell line was inhibited by BMP7 treatment and Nanog lentivrial shRNA knockdown induced the expression of the terminal osteogenic gene osteocalcin. Lentivrial shRNA knockdown or lentiviral overexpression of Nanog in bone MSCs had inverse effects on proliferation, with knockdown decreasing and overexpression increasing MSC cell proliferation. Surgical marrow ablation of mouse tibia by medullary reaming led to a ～3-fold increase in Nanog that preceded osteogenic differentiation during intramembranous bone formation. Lentiviral shRNA knockdown of Nanog after surgical ablation led to an initial overexpression of osteogenic gene expression with no initial effect on bone formation but during subsequent remodeling of the newly formed bone a ～50% decrease was seen in the expression of terminal osteogenic gene expression and a ～50% loss in trabecular bone mass. This loss of bone mass was accompanied by an increased ～2- to 5-fold adipogenic gene expression and observed increase of fat cells in the marrow space. In summary these data show that Nanog is expressed during surgically induced marrow bone formation and is functionally involved in post natal marrow stromal cell maintenance and differentiation.     

Cellular and molecular aspects of muscle growth,adaptation and ageing

Although major advances have been made over the past few decades in prosthetic dentistry, deterioration in oral function and altered facial appearance are still common accompaniments of ageing. Molecular biology methods now allow us to understand these age-related changes at the level of gene expression. Muscle loss as well as bone loss still present major problems, the magnitude of which increases as the age profile of our society changes. Both muscle and bone tissue respond to mechanical signals for which bone depends on muscle and for muscle, stretch has been shown to be important as it induces protein synthesis and an increase in girth as well as length of the muscle fibres. The latter involves the production of more sarcomeres in series so that the jaw muscles adapt to a new functional length following changes in vertical dimension of occlusion. It also determines the postural position of the lower jaw. In our investigations into the control of muscle mass we have recently cloned a growth factor which is expressed in exercised and/or overloaded muscles. This comes in two forms: an autocrine or local form and a paracrine or systemic form. Both growth factors influence muscle growth markedly and it is probable that the systemic type is also involved in maintenance of bone. The discovery of these growth factors provides the mechanisms by which mechanical signals are transduced into chemical signals that in turn regulate gene expression and protein synthesis.     

Bone morphogenetic proteins in development and homeostasis of kidney

Bone morphogenetic proteins play a key role in kidney development and postnatal function. The kidney has been identified as a major site of bone morphogenetic protein (BMP)-7 synthesis during embryonic and postnatal development, which mediates differentiation and maintenance of metanephric mesenchyme. Targeted disruption of BMP-7 gene expression in mice resulted in dysgenic kidneys with hydroureters, causing uremia within 24h after birth. Several experimental animal models of acute and chronic renal injury have all unequivocally shown beneficial effect of BMP-7 in ameliorating the severity of damage by preventing inflammation and fibrosis. Apart from the beneficial effect on kidney disease itself, BMP-7 improves important complications of chronic renal impairment such as renal osteodystrophy and vascular calcification.     

RNA干扰与抗病毒感染

RNA干扰(RNAi)是由小干扰RNA(siRNA)引发的生物细胞内同源基因的转录后基因沉默(PTGS)现象,是一种古老的生物抵抗外在感染的防御机制。RNAi因其在维持基因组稳定、调控基因表达和保护基因组免受外源核酸侵入等方面发挥的重要作用,已被广泛用于探索基因功能、基因治疗和新药的研发。外源导入siRNA引发的RNAi可以特异性抑制病毒的复制与感染,为抗病毒感染治疗开辟了一条新的途径。     

2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits differentiation of normal diploid rat osteoblasts in vitro

The influence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent halogenated aromatic hydrocarbon, on the development of bone tissue-like organization in primary cultures of normal diploid calvarial-derived rat osteoblasts was examined. Initially, when placed in culture, these cells actively proliferate while expressing genes associated with biosynthesis of the bone extracellular matrix. Then, post-proliferatively, genes are expressed that render the osteoblast competent for extracellular matrix mineralization and maintenance of structural as well as functional properties of the mature bone-cell phenotype. Our results indicate that, in the presence of TCDD, proliferation of osteoblasts was not inhibited but post-confluent formation of multicellular nodules that develop bone tissue-like organization was dramatically suppressed. Consistent with TCDD-mediated abrogation of bone nodule formation, expression of alkaline phosphatase and osteocalcin was not upregulated post-proliferatively. These findings are discussed within the context of TCDD effects on estrogens and vitamin D-responsive developmental gene expression during osteoblast differentiation and, from a broader biological perspective, on steroid hormone control of differentiation.