Cells isolated from cryopreserved dental follicle display similar characteristics to cryopreserved dental follicle cells

Dental follicle tissue is a promising resource of mesenchymal stem cells for cytotherapeutic approaches and tissue engineering applications. There are two procedures for banking of human dental follicle stem cells have been reported. Conventional method requires cell isolation, expansion and immediate cryopreservation. Whereas dental follicle stem cells can be isolated from cryopreserved dental follicle fragments. The aim of this study was to compare the characteristics of dental follicle cells isolated from cryopreserved fragments (DFCs-CF) with dental follicle cells recovered from cryopreserved cells (DFCs-CC). Dental follicle fragments obtained after mechanical disaggregation were divided into two parts, with one part maintained in culture, while another part underwent cryopreservation. Dental follicle fragments and dental follicle cells from fresh tissue were stored in liquid nitrogen for 3 months. After thawing, the isolation, morphology, proliferation, cell cycle, colony-forming-unit ability, stemness-related marker expression, apoptosis, and multi-lineage differentiation potential of DFCs-CF were tested compared with DFCs-CC. DFCs-CF expressed mesenchymal stem cells marker, proliferated well, showed similar levels of mRNA for stemness- and apoptosis-related genes and exhibited the capacity of multi-lineage differentiation similar to those of DFCs-CC. These results imply that cryopreservation of dental follicle fragments is an effective banking method for isolation of dental follicle cells.     

A two-step strategy for neuronal differentiation in vitro of human dental follicle cells

Human dental follicle cells (DFCs) derived from wisdom teeth are precursor cells for cementoblasts. In this study, we recognized that naïve DFCs express constitutively the early neural cell marker β-III-tubulin. Interestingly, DFCs formed β-III-tubulin-positive neurosphere-like cell clusters (NLCCs) on low-attachment cell culture dishes in serum-replacement medium (SRM). For a detailed examination of the neural differentiation potential, DFCs were cultivated in different compositions of SRM containing supplements such as N2, B27, G5 and the neural stem cell supplement. Moreover, these cell culture media were combined with different cell culture substrates such as gelatin, laminin, poly-l-ornithine or poly-l-lysine. After cultivation in SRM, DFCs differentiated into cells with small cell bodies and long cellular extrusions. The expression of nestin, β-III-tubulin, neuron-specific enolase (NSE) and neurofilament was up-regulated in SRM supplemented with G5, a cell culture supplement for glial cells, and the neural stem cell supplement. DFCs formed NLCCs and demonstrated an increased gene expression of neural cell markers β-III-tubulin, NSE, nestin and for small neuron markers such as neuropeptides galanin (GAL) and tachykinin (TAC1) after cultivation on poly-l-lysine. For a further neural differentiation NLCC-derived cells were sub-cultivated on laminin and poly-l-ornithine cell culture substrate. After 2 weeks of differentiation, DFCs exposed neural-like cell morphology with small neurite-like cell extrusions. These cells differentially express neurofilament and NSE, but only low levels of β-III-tubulin and nestin. In conclusion, we demonstrated the differentiation of human DFCs into neuron-like cells after a two-step strategy for neuronal differentiation.     

Neurogenic differentiation of murine and human adipose-derived stromal cells

The identification of cells capable of neuronal differentiation has great potential for cellular therapies. We examined whether murine and human adipose-derived adult stem (ADAS) cells can be induced to undergo neuronal differentiation. We isolated ADAS cells from the adipose tissue of adult BalbC mice or from human liposuction tissue and induced neuronal differentiation with valproic acid, butylated hydroxyanisole, insulin, and hydrocortisone. As early as 1-3 h after neuronal induction, the phenotype of ADAS cells changed towards neuronal morphology. Following neuronal induction, muADAS cells displayed immunocytochemical staining for GFAP, nestin and NeuN and huADAS cells displayed staining for intermediate filament M, nestin, and NeuN. Following neuronal induction of murine and human ADAS cells, Western blot analysis confirmed GFAP, nestin, and NeuN protein expression. Pretreatment with EGF and basic FGF augmented the neuronal differentiation of huADAS cells. The neuronal differentiation of stromal cells from adipose tissue has broad biological and clinical implications.     

Nestin Modulates Glucocorticoid Receptor Function by Cytoplasmic Anchoring

Nestin is the characteristic intermediate filament (IF) protein of rapidly proliferating progenitor cells and regenerating tissue. Nestin copolymerizes with class III IF-proteins, mostly vimentin, into heteromeric filaments. Its expression is downregulated with differentiation. Here we show that a strong nestin expression in mouse embryo tissue coincides with a strong accumulation of the glucocorticoid receptor (GR), a key regulator of growth and differentiation in embryonic development. Microscopic studies on cultured cells show an association of GR with IFs composed of vimentin and nestin. Cells lacking nestin, but expressing vimentin, or cells expressing vimentin, but lacking nestin accumulate GR in the nucleus. Completing these networks with an exogenous nestin, respectively an exogenous vimentin restores cytoplasmic anchoring of GR to the IF system. Thus, heteromeric filaments provide the basis for anchoring of GR. The reaction pattern with phospho-GR specific antibodies and the presence of the chaperone HSC70 suggest that specifically the unliganded receptor is anchored to the IF system. Ligand addition releases GR from IFs and shifts the receptor into the nucleus. Suppression of nestin by specific shRNA abolishes anchoring of GR, induces its accumulation in the nucleus and provokes an irreversible G1/S cell cycle arrest. Suppression of GR prior to that of nestin prevents entry into the arrest. The data give evidence that nestin/vimentin specific anchoring modulates growth suppression by GR. We hypothesize that expression of nestin is a major determinant in suppression of anti-proliferative activity of GR in undifferentiated tissue and facilitates activation of this growth control in a precise tissue and differentiation dependent manner.     

Overexpression of HDAC1 induces cellular senescence by Sp1/PP2A/pRb pathway

The differentiation of stem cells can be directed by the grade of stiffness of the developed tissue cells. For example a rigid extracellular matrix supports the osteogenic differentiation in bone marrow derived mesenchymal stem cells (MSCs). However, less is known about the relation of extracellular matrix stiffness and cell differentiation of ectomesenchymal dental precursor cells. Our study examined for the first time the influence of the surface stiffness on the proliferation and osteogenic differentiation of human dental follicle cells (DFCs). Cell proliferation of DFCs was only slightly decreased on cell culture surfaces with a bone-like stiffness. The osteogenic differentiation in DFCs could only be initiated with a dexamethasone based differentiation medium after using varying stiffness. Here, the softest surface improved the induction of osteogenic differentiation in comparison to that with the highest stiffness. In conclusion, different to bone marrow derived MSCs, soft ECMs have a superior capacity to support the osteogenic differentiation of DFCs.     

Tartrate-resistant acid phosphatase in mononuclear and multinuclear cells during the bone resorption of tooth eruption

Tartrate-resistant acid phosphatase (TRAP) has been used as a cytochemical marker for the cell mediators of bone resorption, osteoclasts and their mononuclear precursors. We have applied a cytochemical method for TRAP to study the dependence of the osteoclast-mediated bone resorption of tooth eruption on the dental follicle, a connective tissue investment of the developing tooth, by analyzing the TRAP activity of mononuclear cells in the dental follicle before and during pre-molar eruption in dogs. The percentage of TRAP-positive monocyte cells increases until mid-eruption, slightly preceding a previously demonstrated rise in numbers of osteoclasts on adjacent bone surfaces. These data suggest an ontogenetic relationship between follicular mononuclear cells and osteoclasts on adjacent alveolar bone surfaces during tooth eruption. However, because TRAP occurs in other tissues and is not an exclusive indicator of pre-osteoclasts, proof of their relationship will have to await application of more definitive techniques.     

Rigid matrix supports osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED)

Stem cell fate can be induced by the grade of stiffness of the extracellular matrix, depending on the developed tissue or complex tissues. For example, a rigid extracellular matrix induces the osteogenic differentiation in bone marrow derived mesenchymal stem cells (MSCs), while a softer surface induces the osteogenic differentiation in dental follicle cells (DFCs). To determine whether differentiation of ectomesenchymal dental precursor cells is supported by similar grades of extracellular matrices (ECMs) stiffness, we examined the influence of the surface stiffness on the proliferation and osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED). Cell proliferation of SHED was significantly decreased on cell culture surfaces with a muscle-like stiffness. A dexamethasone-based differentiation medium induced the osteogenic differentiation of SHED on substrates of varying mechanical stiffness. Here, the hardest surface improved the induction of osteogenic differentiation in comparison to that with the softest stiffness. In conclusion, our study showed that the osteogenic differentiation of ectomesenchymal dental precursor cells SHED and DFCs are not supported by similar grades of ECM stiffness.     

Nestin structure and predicted function in cellular cytoskeletal organisation

Nestin is an intermediate filament protein expressed in dividing cells during the early stages of development in the CNS, PNS and in myogenic and other tissues. Upon differentiation, nestin becomes downregulated and is replaced by tissue-specific intermediate filament proteins. Interestingly, nestin expression is reinduced in the adult during pathological situations, such as the formation of the glial scar after CNS injury and during regeneration of injured muscle tissue. Although it is utilised as a marker of proliferating and migrating cells very little is known about its functions or regulation. In depth studies on the distribution and expression of nestin in mitotically active cells indicate a complex role in regulation of the assembly and disassembly of intermediate filaments which together with other structural proteins, participate in remodeling of the cell. The role of nestin in dynamic cells, particularly structural organisation of the cell, appears strictly regulated by phosphorylation, especially its integration into heterogeneous intermediate filaments together with vimentin or alpha-internexin.     

Immunohistochemical analysis of two stem cell markers of α-smooth muscle actin and STRO-1 during wound healing of human dental pulp

Recent studies have employed two markers, alpha-smooth muscle actin (α-SMA) and STRO-1, to detect cells with mesenchymal stem cell properties in dental pulp. The present study aimed to explore the expression profile of α-SMA and STRO-1 in intact dental pulp as well as during wound healing in adult dental pulp tissue. Healthy pulps were mechanically exposed and capped with the clinically used materials MTA (ProRoot White MTA) or Ca(OH)(2) to induce a mineralized barrier at the exposed surface. After 7-42?days, the teeth were extracted and processed for immunohistochemical analysis using antibodies against α-SMA, STRO-1 and nestin (a neurogenic cytoskeletal protein expressed in odontoblasts). In normal pulp, α-SMA was detected in vascular smooth muscle cells and pericytes. Double immunofluorescent staining with STRO-1 and α-SMA showed that STRO-1 was localized in vascular smooth muscle cells, pericytes and endothelial cells, in addition to nerve fibers. During the process of dental pulp healing, numerous α-SMA-positive cells emerged at the wound margin at 14?days, and the initially formed mineralized barrier was lined with α-SMA-positive cells similar in appearance to reparative odontoblasts, some of which co-expressed nestin. STRO-1 was abundant in nerve fibers. In the advanced stage of mineralized barrier formation at 42?days, cells lining the barrier were stained with nestin, and no staining of α-SMA was detected in those cells. These observations indicate that α-SMA-positive cells temporarily appear along the wound margin during the earlier phase of mineralized barrier formation and STRO-1 is confined in vascular and neuronal elements.     

The cytology of the dental follicle and adjacent alveolar bone during tooth eruption in the dog

Previous studies from our laboratories have shown that premolar eruption in dogs depends upon the presence of the dental follicle and is independent of root or crown growth or attachment to the oral epithelium. The present study is an analysis of the cellular composition of the dental follicle and the cellular investment of the adjacent walls of the bony crypt before and during eruption of the third and fourth mandibular permanent premolars in young beagle dogs. Four premolar follicles and their adjacent bony crypts were examined at 2-week intervals over 12 weeks before and during eruption of these teeth. Tissues were removed, fixed, processed, and oriented so that each follicle and the adjacent crypt wall could be reproducibly examined in vertical and horizontal planes. Mononuclear cells with abundant cytoplasm, euchromatic nuclei, and prominent nucleoli were present in juxtavascular location in the coronal part of the dental follicle; and these cells increased in number immediately preceding and during tooth eruption in parallel with an increase in osteoclasts on the adjacent crypt wall. These data are interpreted to mean that the coronal part of the dental follicle may coordinate the alveolar bone resorption required for tooth eruption by attracting and directing to the crypt wall a population of mononuclear cells, which either become osteoclasts and/or direct osteoclastic activity during tooth eruption.     

Directing the differentiation of human dental follicle cells into cementoblasts and/or osteoblasts by a combination of HERS and pulp cells

It is known that the dental follicle (DF) consists of progenitor cells that give rise to the cementum, periodontal ligament, and alveolar bone; but little information is available about the regulation of DF cell differentiation into either cementogenic or osteogenic cell lineages for the regeneration of diseased periodontal tissue. Here, we investigated the roles of DF, Hertwig’s epithelial root sheath (HERS), and pulp cells in the cementum and during alveolar bone formation. We cultured these cells; transplanted them alone or in combination into immunocompromised mice; and observed their effects at 6 and 12 weeks. Histological and immunohistochemical results revealed that DF cells formed cementum-like tissues with immunoreactivity to cementum-derived attached protein, bone sialoprotein, type I collagen, and alkaline phosphatase. In addition, HERS cells played a role in the induction and maturation of cementum-like tissues formed by DF cells. In contrast, implants of DF cells in the presence of pulp cells led to the formation of bone-like tissues. Interestingly, in the presence of both HERS and pulp cells, DF cells formed both cementum-like and bone-like tissues. We demonstrated that while HERS cells are able to induce DF cell differentiation into cementoblasts and promote cementum formation, pulp cells could direct DF cell differentiation into osteoblasts and enhance alveolar bone formation. These results suggest that the combined use of DF, HERS, and pulp cells could direct DF cell differentiation into cementoblasts and/or osteoblasts in vivo, thus providing a novel strategy for the successful repair and regeneration of diseased periodontal tissue.