Pamela Gehron Robey

Investigation of multipotent postnatal stem cells from human periodontal ligament

BACKGROUND Periodontal diseases that lead to the destruction of periodontal tissues--including periodontal ligament (PDL), cementum, and bone--are a major cause of tooth loss in adults and are a substantial public-health burden worldwide. PDL is a specialised connective tissue that connects cementum and alveolar bone to maintain and support teeth in situ and preserve tissue homoeostasis. We investigated the notion that human PDL contains stem cells that could be used to regenerate periodontal tissue. METHODS PDL tissue was obtained from 25 surgically extracted human third molars and used to isolate PDL stem cells (PDLSCs) by single-colony selection and magnetic activated cell sorting. Immunohistochemical staining, RT-PCR, and northern and western blot analyses were used to identify putative stem-cell markers. Human PDLSCs were transplanted into immunocompromised mice (n=12) and rats (n=6) to assess capacity for tissue regeneration and periodontal repair. Findings PDLSCs expressed the mesenchymal stem-cell markers STRO-1 and CD146/MUC18. Under defined culture conditions, PDLSCs differentiated into cementoblast-like cells, adipocytes, and collagen-forming cells. When transplanted into immunocompromised rodents, PDLSCs showed the capacity to generate a cementum/PDL-like structure and contribute to periodontal tissue repair. INTERPRETATION Our findings suggest that PDL contains stem cells that have the potential to generate cementum/PDL-like tissue in vivo. Transplantation of these cells, which can be obtained from an easily accessible tissue resource and expanded ex vivo, might hold promise as a therapeutic approach for reconstruction of tissues destroyed by periodontal diseases.

Bone marrow stromal stem cells: Nature, biology, and potential applications

Bone marrow stromal cells are progenitors of skeletal tissue components such as bone, cartilage, the hematopoiesis‐supporting stroma, and adipocytes. In addition, they may be experimentally induced to undergo unorthodox differentiation, possibly forming neural and myogenic cells. As such, they represent an important paradigm of post‐natal nonhematopoietic stem cells, and an easy source for potential therapeutic use. Along with an overview of the basics of their biology, we discuss here their potential nature as components of the vascular wall, and the prospects for their use in local and systemic transplantation and gene therapy.

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.

Bone marrow stromal cells attenuate sepsis via prostaglandin E 2 –dependent reprogramming of host macrophages to increase their interleukin-10 production

Sepsis causes over 200,000 deaths yearly in the US; better treatments are urgently needed. Administering bone marrow stromal cells (BMSCs—also known as mesenchymal stem cells) to mice before or shortly after inducing sepsis by cecal ligation and puncture reduced mortality and improved organ function. The beneficial effect of BMSCs was eliminated by macrophage depletion or pretreatment with antibodies specific for interleukin-10 (IL-10) or IL-10 receptor. Monocytes and/or macrophages from septic lungs made more IL-10 when prepared from mice treated with BMSCs versus untreated mice. Lipopolysaccharide (LPS)-stimulated macrophages produced more IL-10 when cultured with BMSCs, but this effect was eliminated if the BMSCs lacked the genes encoding Toll-like receptor 4, myeloid differentiation primary response gene-88, tumor necrosis factor (TNF) receptor-1a or cyclooxygenase-2. Our results suggest that BMSCs (activated by LPS or TNF-α) reprogram macrophages by releasing prostaglandin E2 that acts on the macrophages through the prostaglandin EP2 and EP4 receptors. Because BMSCs have been successfully given to humans and can easily be cultured and might be used without human leukocyte antigen matching, we suggest that cultured, banked human BMSCs may be effective in treating sepsis in high-risk patient groups.

MT1-MMP-Deficient Mice Develop Dwarfism, Osteopenia, Arthritis, and Connective Tissue Disease due to Inadequate Collagen Turnover

MT1-MMP is a membrane-bound matrix metalloproteinase (MT-MMP) capable of mediating pericellular proteolysis of extracellular matrix components. MT1-MMP is therefore thought to be an important molecular tool for cellular remodeling of the surrounding matrix. To establish the biological role of this membrane proteinase we generated MT1-MMP-deficient mice by gene targeting. MT1-MMP deficiency causes craniofacial dysmorphism, arthritis, osteopenia, dwarfism, and fibrosis of soft tissues due to ablation of a collagenolytic activity that is essential for modeling of skeletal and extraskeletal connective tissues. Our findings demonstrate the pivotal function of MT1-MMP in connective tissue metabolism, and illustrate that modeling of the soft connective tissue matrix by resident cells is essential for the development and maintenance of the hard tissues of the skeleton.

Mesenchymal Stem Cells: Revisiting History, Concepts, and Assays

The concept of mesenchymal stem cells has gained wide popularity. Despite the rapid growth of the field, uncertainties remain with respect to the defining characteristics of these cells, including their potency and self-renewal. These uncertainties are reflected in a growing tendency to question the very use of the term. This commentary revisits the experimental origin of the concept of the population(s) referred to as mesenchymal stem cells and the experimental framework required to assess their stemness and function.

Surface protein characterization of human adipose tissue-derived stromal cells.

Human bone marrow stromal cells are a multipotent population of cells capable of differentiating into a number of mesodermal lineages as well as supporting hematopoeisis. Their distinct protein and gene expression phenotype is well characterized in the literature. Human adipose tissue presents an alternative source of multipotent stromal cells. In this study, we have defined the phenotype of the human adipose tissue‐derived stromal cells in both the differentiated and undifferentiated states. Flow cytometry and immunohistochemistry show that human adipose tissue‐derived stromal cells have a protein expression phenotype that is similar to that of human bone marrow stromal cells. Expressed proteins include CD9, CD10, CD13, CD29, CD34, CD44, CD 49d, CD 49e, CD54, CD55, CD59, CD105, CD106, CD146, and CD166. Expression of some of these proteins was further confirmed by PCR and immunoblot detection. Unlike human bone marrow‐derived stromal cells, we did not detect the STRO‐1 antigen on human adipose tissue‐derived stromal cells. Cells cultured under adipogenic conditions uniquely expressed C/EBPα and PPARδ, two transcriptional regulators of adipogenesis. Cells cultured under osteogenic conditions were more likely to be in the proliferative phases of the cell cycle based on flow cytometric analysis of PCNA and Ki67. The similarities between the phenotypes of human adipose tissue‐derived and human bone marrow‐derived stromal cells could have broad implications for human tissue engineering.

Human Bone Cells In Vitro

SummaryHuman bone cell cultures were established by maintaining collagenase-treated, bone fragments in low Ca++ medium. The resulting cell cultures exhibited a high level of alkaline phosphatase activity and produced a significant increase in intracellular cAMP when exposed to the 1–34 fragment of human parathyroid hormone. With continued culture, the cells formed a thick, extracellular matrix that mineralized when cultures were provided daily with normal levels of calcium, fresh ascorbic acid (50 μg/ml) and 10 mM β-glycerol phosphate. Biosynthetically, these cells produced type I collagen (without any type III collagen), and the bone-specific protein, osteonectin. In addition, the cells produced sulfated macromolecules electrophoretically identical to those positively identified as the bone proteoglycan in parallel cultures of fetal bovine bone cells. This technique provides a useful system for the study of osteoblast metabolismin vitro

Single‐Colony Derived Strains of Human Marrow Stromal Fibroblasts Form Bone After Transplantation In Vivo

Populations of marrow stromal fibroblasts (MSFs) can differentiate into functional osteoblasts and form bone in vivo. It is not known, however, what proportion of MSF precursor cells, colony forming units‐fibroblast (CFU‐Fs), have osteogenic potential. In the present study, analysis of bone formation in vivo by single‐colony derived strains of human marrow stromal fibroblasts (HMSFs) has been performed for the first time. Each strain originated from an individual CFU‐F and underwent four passages in vitro prior to subcutaneous implantation into immunodeficient mice within vehicles containing hydroxyapatite‐tricalcium phosphate ceramic. Multicolony derived HMSF strains were also transplanted to serve as positive controls. After 8 weeks, abundant bone formation was found in the transplants of all multicolony derived HMSF strains, whereas 20 out of 34 (58.8%) single‐colony derived strains from four donors formed bone. Immunostaining with antibody directed against human osteonectin and in situ hybridization for human‐specific alu sequences demonstrated that cells forming new bone were of human origin and were vital for at least 45 weeks post‐transplantation. Both the incidence of bone‐forming colonies and the extent of bone formation by single‐colony derived HMSF strains were increased by cultivation with dexamethasone and ascorbic acid phosphate. Other factors, including type of transplantation vehicle, morphology, size, and structure of the original HMSF colonies showed no obvious correlation with the incidence or extent of bone formation. Hematopoietic tissue within the newly formed bone was developed in the transplants exhibiting exuberant bone formation. These results provide evidence that individual human CFU‐Fs have osteogenic potential and yet differ from each other with respect to their osteogenic capacity.

The meaning, the sense and the significance: translating the science of mesenchymal stem cells into medicine

Mesenchymal stem cells (MSCs) are the focus of intensive efforts worldwide directed not only at elucidating their nature and unique properties but also developing cell-based therapies for a diverse range of diseases. More than three decades have passed since the original formulation of the concept, revolutionary at the time, that multiple connective tissues could emanate from a common progenitor or stem cell retained in the postnatal bone marrow. Despite the many important advances made since that time, substantial ambiguities still plague the field regarding the nature, identity, function, mode of isolation and experimental handling of MSCs. These uncertainties have a major impact on their envisioned therapeutic use.