Larry W. Fisher

SHED: Stem cells from human exfoliated deciduous teeth

To isolate high-quality human postnatal stem cells from accessible resources is an important goal for stem-cell research. In this study we found that exfoliated human deciduous tooth contains multipotent stem cells [stem cells from human exfoliated deciduous teeth (SHED)]. SHED were identified to be a population of highly proliferative, clonogenic cells capable of differentiating into a variety of cell types including neural cells, adipocytes, and odontoblasts. After in vivo transplantation, SHED were found to be able to induce bone formation, generate dentin, and survive in mouse brain along with expression of neural markers. Here we show that a naturally exfoliated human organ contains a population of stem cells that are completely different from previously identified stem cells. SHED are not only derived from a very accessible tissue resource but are also capable of providing enough cells for potential clinical application. Thus, exfoliated teeth may be an unexpected unique resource for stem-cell therapies including autologous stem-cell transplantation and tissue engineering.

Expression and localization of the two small proteoglycans biglycan and decorin in developing human skeletal and non-skeletal tissues.

The messenger RNAs and core proteins of the two small chondroitin/dermatan sulfate proteoglycans, biglycan and decorin, were localized in developing human bone and other tissues by both 35S-labeled RNA probes and antibodies directed against synthetic peptides corresponding to nonhomologous regions of the two core proteins. Biglycan and decorin expression and localization were substantially divergent and sometimes mutually exclusive. In developing bones, spatially restricted patterns of gene expression and/or matrix localization of the two proteoglycans were identified in articular regions, epiphyseal cartilage, vascular canals, subperichondral regions, and periosteum, and indicated the association of each molecule with specific developmental events at specific sites. Study of non-skeletal tissues revealed that decorin was associated with all major type I (and type II) collagen-rich connective tissues. Conversely, biglycan was expressed and localized in a range of specialized cell types, including connective tissue (skeletal myofibers, endothelial cells) and epithelial cells (differentiating keratinocytes, renal tubular epithelia). Biglycan core protein was localized at the cell surface of certain cell types (e.g., keratinocytes). Whereas the distribution of decorin was consistent with matrix-centered functions, possibly related to regulation of growth of collagen fibers, the distribution of biglycan pointed to other function(s), perhaps related to cell regulation.

Targeted disruption of the biglycan gene leads to an osteoporosis-like phenotype in mice

The resilience and strength of bone is due to the orderly mineralization of a specialized extracellular matrix (ECM) composed of type I collagen (90%) and a host of non-collagenous proteins that are, in general, also found in other tissues. Biglycan (encoded by the gene Bgn) is an ECM proteoglycan that is enriched in bone and other non-skeletal connective tissues. In vitro studies indicate that Bgn may function in connective tissue metabolism by binding to collagen fibrils and TGF-ß (Refs 5,6), and may promote neuronal survival. To study the role of Bgn in vivo, we generated Bgn-deficient mice. Although apparently normal at birth, these mice display a phenotype characterized by a reduced growth rate and decreased bone mass due to the absence of Bgn. To our knowledge, this is the first report in which deficiency of a non-collagenous ECM protein leads to a skeletal phenotype that is marked by low bone mass that becomes more obvious with age. These mice may serve as an animal model to study the role of ECM proteins in osteoporosis.

Six Genes Expressed in Bones and Teeth Encode the Current Members of the SIBLING Family of Proteins

Bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), enamelin (ENAM), matrix extracellular phosphoglycoprotein (MEPE), and osteopontin (OPN) are glycophosphoproteins expressed in bones and/or teeth. Direct comparison of their amino acid sequences do not suggest that they belong to a single genetic family, but a detailed analysis of their chromosomal location and gene structure does. Analysis of human brain mRNA by RT-PCR has led to the discovery of two additional exons thereby making it more convincing that MEPE is a member of the SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family. We propose that the members of this SIBLING family are extended, flexible proteins in solution that can facilitate the formation of a number of different complexes. For example, OPN can bridge complement Factor H to either an RGD-dependent integrin or to CD44 forming a membrane-bound complex that actively suppresses the alternate complement pathway. Two possible mechanisms for inhibiting the lytic pathway of alternate complement are presented.

Expression of bone sialoprotein (BSP) in developing human tissues

SummaryBone sialoprotein (BSP) and its messenger RNA were localized in developing human skeletal and nonskeletal tissues by means of immunohistochemistry andin situ hybridization. Both protein and mRNA were found in mature, bone-forming cells but not in their immature precursors. In addition, osteoclasts displayed positive immunostaining and high densities of autoradiographic grains byin situ hybridization experiments. BSP was expressed in fetal epiphyseal cartilage cells, particularly in hypertrophic chondrocytes of growth plates. Though neither the protein nor the mRNA were identified in a variety of other connective and nonconnective tissues, an unexpected finding was the expression of BSP in the trophoblast cells of placenta. These findings show that BSP is primarily an osteoblast-derived component of the bone matrix expressed at late stages of differentiation. We have also found that osteoclasts produce BSP, possibly as a mediator of cell attachment to bone.

Comparison of Stem-cell-mediated Osteogenesis and Dentinogenesis

The difference between stem-cell-mediated bone and dentin regeneration is not yet well-understood. Here we use an in vivo stem cell transplantation system to investigate differential regulation mechanisms of bone marrow stromal stem cells (BMSSCs) and dental pulp stem cells (DPSCs). Elevated expression of basic fibroblast growth factor (bFGF) and matrix metalloproteinase 9 (MMP-9, gelatinase B) was found to be associated with the formation of hematopoietic marrow in BMSSC transplants, but not in the connective tissue of DPSC transplants. The expression of dentin sialoprotein (DSP) specifically marked dentin synthesis in DPSC transplants. Moreover, DPSCs were found to be able to generate reparative dentin-like tissue on the surface of human dentin in vivo. This study provided direct evidence to suggest that osteogenesis and dentinogenesis mediated by BMSSCs and DPSCs, respectively, may be regulated by distinct mechanisms, leading to the different organization of the mineralized and non-mineralized tissues.

cDNA cloning, mRNA distribution and heterogeneity, chromosomal location, and RFLP analysis of human osteopontin (OPN) ☆

A human osteopontin (OP) cDNA was isolated from a library made from primary cultures of human bone cells. The distribution of osteopontin mRNA in human tissues was investigated by Northern analysis and showed that the human message was predominant in cultures of bone cells and in decidua cells isolated at 6-12 weeks of gestation. Immunohistochemical analysis confirmed that OP expression is high in decidua cells as well as in the endometrial glands of a non-pregnant secretory-phase human uterus. Two variants of the OP message were evident on the basis of DNA sequencing and polymerase chain reaction amplification of bone and decidua cell mRNA. The peptides potentially translated by the variant messages differ by the presence (OP1b) or absence (OP1a) of 14 amino acids at residue 58 of the molecule. The deduced human protein sequence shows a conservation between species in the position of the Arg-Gly-Asp (RGD) cell attachment site. Chromosomal mapping of the osteopontin gene (OPN) using human-rodent cell hybrids demonstrated a location on chromosome 4 in the human genome. In situ hybridization of metaphase chromosomes using radiolabeled OP1a as a probe indicated that the gene is located on a region of 4q that is near the centromere. A high-frequency restriction fragment length polymorphism was evident in the DNA from 29 unrelated individuals using the enzyme BglII. Analysis of total genomic DNA by digestion with several restriction enzymes, Southern blotting, and hybridization with the human osteopontin cDNA indicated that the gene is a single copy with an approximate length of 5.4-8.2 kb.

Small Integrin-Binding LIgand N-linked Glycoproteins (SIBLINGs): Multifunctional proteins in cancer

Numerous components and pathways are involved in the complex interplay between cancer cells and their environment. The family of glycophosphoproteins comprising osteopontin, bone sialoprotein, dentin matrix protein 1, dentin sialophosphoprotein and matrix extracellular phosphoglycoprotein — small integrin-binding ligand N-linked glycoproteins (SIBLINGs) — are emerging as important players in many stages of cancer progression. From their detection in various human cancers to the demonstration of their key functional roles during malignant transformation, invasion and metastasis, the SIBLINGs are proteins with potential as diagnostic and prognostic tools, as well as new therapeutic targets.

The small leucine-rich proteoglycan biglycan modulates BMP-4-induced osteoblast differentiation

Biglycan (bgn) is a small leucine‐rich proteoglycan enriched in extracellular matrices of skeletal tissues. Bgn‐deficient mice develop age‐related osteopenia with a phenotype that resembles osteoporosis and premature arthritis. In the present study, we have examined the differentiation of ftgw‐deficient osteoblasts from neonatal murine calvariae and found that the absence of bgn caused less BMP‐4 binding, which reduced the sensitivity of osteoblasts to BMP‐4 stimulation. The loss of sensitivity resulted in a reduction of Cbfa1 expression, which ultimately led to a defect in the differentiation of osteoblasts. However, the response of ftgw‐deficient osteoblasts to BMP‐4 was completely rescued by reintroduction of biglycan by viral transfection. We propose that biglycan modulates BMP‐4‐induced signaling to control osteoblast differentiation.—Chen, X.‐D., Fisher, L. W., Robey, P. G., Young, M. F. The small leucine‐rich proteoglycan biglycan modulates BMP‐4‐induced osteoblast differentiation. FASEB J. 18, 948–958 (2004)

Mutations of the GNAS1 Gene, Stromal Cell Dysfunction, and Osteomalacic Changes in Non–McCune–Albright Fibrous Dysplasia of Bone

Activating missense mutations of the GNAS1 gene, encoding the α subunit of the stimulatory G protein (Gs), have been identified in patients with the McCune–Albright syndrome (MAS; characterized by polyostotic fibrous dysplasia, café au lait skin pigmentation, and endocrine disorders). Because fibrous dysplasia (FD) of bone also commonly occurs outside of the context of typical MAS, we asked whether the same mutations could be identified routinely in non‐MAS FD lesions. We analyzed a series of 8 randomly obtained, consecutive cases of non‐MAS FD and identified R201 mutations in the GNAS1 gene in all of them by sequencing cDNA generated by amplification of genomic DNA using a standard primer set and by using a novel, highly sensitive method that uses a protein nucleic acid (PNA) primer to block amplification of the normal allele. Histologic findings were not distinguishable from those observed in MAS‐related FD and included subtle changes in cell shape and collagen texture putatively ascribed to excess endogenous cyclic adenosine monophosphate (cAMP). Osteomalacic changes (unmineralized osteoid) were prominent in lesional FD bone. In an in vivo transplantation assay, stromal cells isolated from FD failed to recapitulate a normal ossicle; instead, they generated a miniature replica of fibrous dysplasia. These data provide evidence that occurrence of GNAS1 mutations, previously noted in individual cases of FD, is a common and perhaps constant finding in non‐MAS FD. These findings support the view that FD, MAS, and nonskeletal isolated endocrine lesions associated with GNAS1 mutations represent a spectrum of phenotypic expressions (likely reflecting different patterns of somatic mosaicism) of the same basic disorder. We conclude that mechanisms underlying the development of the FD lesions, and hopefully mechanism‐targeted therapeutic approaches to be developed, must also be the same in MAS and non‐MAS FD.