Clinical application prospects and transformation value of dental follicle stem cells in oral and neurological diseases

Since dental pulp stem cells (DPSCs) were first reported, six types of dental SCs (DSCs) have been isolated and identified. DSCs originating from the craniofacial neural crest exhibit dental-like tissue differentiation potential and neuro-ectodermal features. As a member of DSCs, dental follicle SCs (DFSCs) are the only cell type obtained at the early developing stage of the tooth prior to eruption. Dental follicle tissue has the distinct advantage of large tissue volume compared with other dental tissues, which is a prerequisite for obtaining a sufficient number of cells to meet the needs of clinical applications. Furthermore, DFSCs exhibit a significantly higher cell proliferation rate, higher colony-formation capacity, and more primitive and better anti-inflammatory effects than other DSCs. In this respect, DFSCs have the potential to be of great clinical significance and translational value in oral and neurological diseases, with natural advantages based on their origin. Lastly, cryopreservation preserves the biological properties of DFSCs and enables them to be used as off-shelf products for clinical applications. This review summarizes and comments on the properties, application potential, and clinical transformation value of DFSCs, thereby inspiring novel perspectives in the future treatment of oral and neurological diseases.     

Comparison of umbilical cord tissue-derived mesenchymal stromal cells isolated from cryopreserved material and extracted by explantation and digestion methods utilizing a split manufacturing model

Background aimsUmbilical cord (UC) tissue is recognized as an advantageous source of mesenchymal stromal cells (MSCs), whose therapeutic properties are being actively evaluated in pre-clinical and clinical trials. In recognition of its potential value, storage of UC tissue or cells from UC tissue in newborn stem cell banks is now commonplace; however, strategies for isolating UC-derived MSCs (UCMSCs) from UC tissue have not been standardized. The majority of newborn stem cell banks take one of two approaches to cord tissue processing and cryopreservation: enzymatic digestion of the fresh tissue with cryopreservation of the subsequent cell suspension or cryopreservation of the tissue as a composite whole with later, post-thaw isolation of cells by explantation. Evaluation of UCMSCs derived by these two principal preparation and cryopreservation strategies is important to understanding whether the methods currently employed by newborn stem cell banks retain the desirable clinical attributes of UC cells.MethodsUCMSCs were isolated from 10 UC tissue samples by both explantation and enzymatic digestion methods to allow for comparison of cells from the same donor. Cell isolates from both methods were compared pre- and post-cryopreservation as well as after serial passaging. Cell viability, morphology, growth kinetics, immunophenotype, cytokine secretion and differentiation capacity were evaluated.ResultsUCMSCs could be derived from fresh UC tissue by both explantation and digestion methods and from thawed UC tissue by explantation. Initial cell populations isolated by digestion were heterogeneous and took longer to enrich for UCMSCs in culture than populations obtained by explantation. However, once isolated and enriched, UCMSCs obtained by either method showed no significant difference in viability, morphology, rate of proliferation, surface marker expression, levels of cytokine secretion or differentiation capacity.ConclusionsDerivation of UCMSCs by explantation after thawing UC cryopreserved as a composite tissue may be favorable in terms of initial purity and number of cells achievable by a specific passage. However, we observed no evidence of functional difference between UCMSCs derived by explanation or digestion, suggesting that cells isolated from cryopreserved material obtained by either method maintain their therapeutic properties.     

Optimized cryopreservation method for human dental pulp-derived stem cells and their tissues of origin for banking and clinical use

Dental pulp is a promising source of mesenchymal stem cells with the potential for cell-mediated therapies and tissue engineering applications. We recently reported that isolation of dental pulp-derived stem cells (DPSC) is feasible for at least 120 h after tooth extraction, and that cryopreservation of early passage cultured DPSC leads to high-efficiency recovery post-thaw. This study investigated additional processing and cryobiological characteristics of DPSC, ending with development of procedures for banking. First, we aimed to optimize cryopreservation of established DPSC cultures, with regards to optimizing the cryoprotective agent (CPA), the CPA concentration, the concentration of cells frozen, and storage temperatures. Secondly, we focused on determining cryopreservation characteristics of enzymatically digested tissue as a cell suspension. Lastly, we evaluated the growth, surface markers and differentiation properties of DPSC obtained from intact teeth and undigested, whole dental tissue frozen and thawed using the optimized procedures. In these experiments it was determined that Me₂SO at a concentration between 1 and 1.5 M was the ideal cryopreservative of the three studied. It was also determined that DPSC viability after cryopreservation is not limited by the concentration of cells frozen, at least up to 2 × 10⁶ cells/mL. It was further established that DPSC can be stored at −85 °C or −196 °C for at least six months without loss of functionality. The optimal results with the least manipulation were achieved by isolating and cryopreserving the tooth pulp tissues, with digestion and culture performed post-thaw. A recovery of cells from >85% of the tissues frozen was achieved and cells isolated post-thaw from tissue processed and frozen with a serum free, defined cryopreservation medium maintained morphological and developmental competence and demonstrated MSC-hallmark trilineage differentiation under the appropriate culture conditions.     

Characterisation of human dental stem cells and buccal mucosa fibroblasts

Human craniofacial stem cells are recently discovered sources of putative mesenchymal stem cells that hold great promise for autogenic or allogenic cell therapy and tissue engineering. Prior to employing these cells in clinical applications, they must be thoroughly investigated and characterized. In this study, the surface marker expression was investigated on dental pulp stem cells (DPSCs), dental follicle cells (DFCs), periodontal ligament stem cells (PDLSCs), and buccal mucosa fibroblasts (BMFs) utilising surface markers for flow cytometry. The osteogenic potential was also examined by bone-associated markers alkaline phosphatase, Runx2, collagen type I, osteocalcin, and osteopontin. The results from our study demonstrate that the dental cell sources exhibit comparable surface marker and bone-associated marker profiles parallel to those of other mesenchymal stem cell sources, yet distinct from the buccal mucosa fibroblasts. Our data support evidence towards clinical applicability of dental stem cells in hard tissue regeneration.     

Dental stem cell banking: Techniques and protocols

Dental tissue-derived stem cells (DSCs) provide an easy, accessible, relatively noninvasive promising source of adult stem cells (ASCs), which brought encouraging prospective for their clinical applications. DSCs provide a perfect opportunity to apply for a patient's own ASC, which poses a low risk of immune rejection. However, problems associated with the long-term culture of stem cells, including loss of proliferation and differentiation capacities, senescence, genetic instability, and the possibility of microbial contamination, make cell banking necessary. With the rapid development of advanced cryopreservation technology, various international DSC banks have been established for both research and clinical applications around the world. However, few studies have been published that provide step-by-step guidance on DSCs isolation and banking methods. The purpose of this review is to present protocols and technical details for all steps of cryopreserved DSCs, from donor selection, isolation, cryopreservation, to characterization and quality control. Here, the emphasis is on presenting practical principles in accordance with the available valid guidelines.     

Viability of pulp stromal cells in cryopreserved deciduous teeth

The cryopreservation of exfoliated deciduous teeth and harvesting of stem cells from them as required would reduce the costs and efforts associated with banking stem cells from primary teeth. The aim of this study was determine whether the viability of pulp stromal cells from deciduous teeth was influenced by the cryopreservation process itself or the period of cryopreservation. In total, 126 deciduous teeth were divided into three groups: (1) fresh, (2) cryopreserved for <3 months (cryo<3), and (3) cryopreserved for 3–9 months (cryo3–9). The viability of the pulp tissues was compared among the three groups by evaluating the outgrowth from pulp tissues and cell activity within those pulp tissues. In addition, the terminal deoxynucleotidyl transferase-mediated dUTP–biotin nick end labeling (TUNEL) assay was performed to compare cell apoptosis within fresh pulp tissue and pulp tissue that had been cryopreserved for 4 months. The outgrowth from and cell activity within the pulp tissues did not differ significantly between the fresh and cryo<3 pulp tissues. However, these parameters were significantly reduced in the cryo3–9 pulp tissue. In TUNEL assay, 4-month cryopreserved pulp tissues has more apoptotic cells than fresh group. In conclusion, it is possible to acquire pulp stromal cells from cryopreserved deciduous teeth. However, as the period of cryopreservation becomes longer, it is difficult to get pulp cells due to reduced cell viability.     

Fate map of the dental mesenchyme: dynamic development of the dental papilla and follicle

At the bud stage of tooth development the neural crest derived mesenchyme condenses around the dental epithelium. As the tooth germ develops and proceeds to the cap stage, the epithelial cervical loops grow and appear to wrap around the condensed mesenchyme, enclosing the cells of the forming dental papilla. We have fate mapped the dental mesenchyme, using in vitro tissue culture combined with vital cell labelling and tissue grafting, and show that the dental mesenchyme is a much more dynamic population then previously suggested. At the bud stage the mesenchymal cells adjacent to the tip of the bud form both the dental papilla and dental follicle. At the early cap stage a small population of highly proliferative mesenchymal cells in close proximity to the inner dental epithelium and primary enamel knot provide the major contribution to the dental papilla. These cells are located between the cervical loops, within a region we have called the body of the enamel organ, and proliferate in concert with the epithelium to create the dental papilla. The condensed dental mesenchymal cells that are not located between the body of the enamel organ, and therefore are at a distance from the primary enamel knot, contribute to the dental follicle, and also the apical part of the papilla, where the roots will ultimately develop. Some cells in the presumptive dental papilla at the cap stage contribute to the follicle at the bell stage, indicating that the dental papilla and dental follicle are still not defined populations at this stage. These lineage-tracing experiments highlight the difficulty of targeting the papilla and presumptive odontoblasts at early stages of tooth development. We show that at the cap stage, cells destined to form the follicle are still competent to form dental papilla specific cell types, such as odontoblasts, and produce dentin, if placed in contact with the inner dental epithelium. Cell fate of the dental mesenchyme at this stage is therefore determined by the epithelium.     

Human dental pulp stem cells: Applications in future regenerative medicine

Stem cells are pluripotent cells, having a property of differentiating into various types of cells of human body. Several studies have developed mesenchymal stem cells (MSCs) from various human tissues, peripheral blood and body fluids. These cells are then characterized by cellular and molecular markers to understand their specific phenotypes. Dental pulp stem cells (DPSCs) are having a MSCs phenotype and they are differentiated into neuron, cardiomyocytes, chondrocytes, osteoblasts, liver cells and β cells of islet of pancreas. Thus, DPSCs have shown great potentiality to use in regenerative medicine for treatment of various human diseases including dental related problems. These cells can also be developed into induced pluripotent stem cells by incorporation of pluripotency markers and use for regenerative therapies of various diseases. The DPSCs are derived from various dental tissues such as human exfoliated deciduous teeth, apical papilla, periodontal ligament and dental follicle tissue. This review will overview the information about isolation, cellular and molecular characterization and differentiation of DPSCs into various types of human cells and thus these cells have important applications in regenerative therapies for various diseases. This review will be most useful for postgraduate dental students as well as scientists working in the field of oral pathology and oral medicine.     

Effect of cryopreservation on biological and immunological properties of stem cells from apical papilla

Stem cells from apical papilla (SCAP) are a novel population of multipotent stem cells that, although similar to dental pulp stem cells, are a discrete source of dental stem cells. SCAP have potential roles in root development, apexogenesis, pulp/dentin regeneration, and bioroot engineering. However, procedures to store and preserve SCAP for future clinical applications have not been explored. In this study, we compared human freshly isolated SCAP (fSCAP) with cryopreserved SCAP (cSCAP) in terms of cell viability, colony‐forming efficiency, cell proliferation rate, multilineage differentiation potential, profiles of mesenchymal stem cell (MSC) markers, karyotype analysis, and immunological assays. cSCAP showed a similar viable cell ratio, colony‐forming efficiency, cell proliferation rate, multilineage differentiation potential, MSC surface markers, apoptotic rate, and G‐banded karyotype when compared to fSCAP. There was no significant difference between fSCAP and cSCAP with regard to immune properties. In addition, cSCAP of miniature pig possessed the similar proliferation rate, differentiation potential, and immunomodulatory function as seen in fSCAP. This study demonstrates that cryopreservation does not affect the biological and immunological properties of SCAP, supporting the feasibility of SCAP cryopreservation in nitrogen. J. Cell. Physiol. 223: 415–422, 2010. © 2010 Wiley‐Liss, Inc.     

Boron increases the cell viability of mesenchymal stem cells after long-term cryopreservation

The field of stem-cell biology has emerged as a key technology for the treatment of various disorders and tissue regeneration applications. However, a major problem remains in clinical practice, which is the question of whether stem cells preserve their self-renewal and differentiation potential in the culture conditions or not. In the current study, effects of boron on the cryopreservation of human tooth germ stem cells (hTGSCs) were evaluated for the first time. The impacts of various boron concentrations (sodium pentaborate pentahydrate (NaB)) were tested on characterized hTGSCs viability for different time intervals (24, 48, and 72 h). 20 μg/ml NaB with lower Me₂SO concentration was found to display positive effects on hTGSCs during repeated freezing and defrosting cycles, and long-term cryopreservation. After thawing, cells were analyzed for their surface antigens and differentiation capacity. hTGSCs were successfully cryopreserved without any change in their mesenchymal stem cell characteristics as they were treated with boron containing freezing medium. In addition, fatty acid composition was examined to demonstrate membrane fatty acid profiles after freeze-thawing. Besides, NaB treatment extended osteogenic and chondrogenic differentiation of hTGSCs remarkably after long-term cryopreservation with respect to control groups. The study clearly suggests that NaB has a protective role on the survival of hTGSCs in short- and long-term cryopreservation. Due to the possible storage of hTGSCs at early ages, development of a functional and reliable cryopreservation media can be designed as a future solution to the dental stem cell banking.     

Clonal multipotency and effect of long-term in vitro expansion on differentiation potential of human hair follicle derived mesenchymal stem cells

Hair follicle harbors a rich stem cell pool with mesenchymal lineage differentiation potential. Although previous studies with rodent cells demonstrated that hair follicle sheath and papilla cells possess multi-lineage differentiation potential, human hair follicle derived mesenchymal stem cells (hHF-MSCs) have not been characterized in detail in terms of their multipotency. In addition, it is not clear whether these cells are true stem cells that can differentiate along multiple lineages or whether they represent a collection of progenitor cells with restricted differentiation potential. Here we report that hHF-MSCs are highly proliferative cells that can be maintained in culture for ~ 45 population doublings before they start to show signs of cellular senescence. Under appropriate culture conditions, hHF-MSCs differentiated along the myogenic, osteogenic, adipogenic and chondrogenic lineages, as demonstrated by kinetic gene expression profiling and functional assays. Interestingly, the differentiation potential decreased with time in culture in a lineage-specific manner. Specifically, myogenesis and chondrogenesis showed a moderate decrease over time; osteogenesis was maximum at intermediate passages and adipogenesis was highly sensitive to long-term culture and was diminished at late passages. Finally, hHF-MSCs were clonally multipotent as the majority of hHF-MSCs clones (73%) demonstrated bi- or tri-lineage differentiation potential. These results suggest that hHF-MSCs may present as an alternative source of easily accessible, autologous stem cells for tissue engineering and regenerative medicine.     

Osteogenic differentiation of GFP-labeled human umbilical cord blood derived mesenchymal stem cells after cryopreservation

The osteogenic capacity of human umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) has been demonstrated both in vitro and in vivo. Therefore, cell labeling and storage are becoming necessary for researching the potential therapeutic use of UCB-MSCs for bone tissue engineering. The aim of this study was to determine the effect of cryopreservation on the osteogenic differentiation of green fluorescent protein (GFP)-marked UCB-MSCs in vitro. MSCs were isolated from full-term human UCB, expanded, transfected with the GFP gene, and then cryopreserved in liquid nitrogen for 4 weeks. After thawing, cell surface antigen markers and osteogenic potential were analyzed, and the luminescence of these cells was observed by fluorescence microscopy. The results demonstrate that cryopreservation has no effect on the cell phenotype, GFP expression or osteogenic differentiation of UCB-MSCs, showing that cryopreserved GFP-labeled UCB-MSCs might be applied for bone tissue engineering.     

In vitro differentiation of human dental follicle cells with dexamethasone and insulin

The dental follicle is an ectomesenchymally derived connective tissue harboring precursor cells for the tooth supporting apparatus. In this study, we examined gene expression of freshly isolated human dental follicle cells during osteogenic differentiation in vitro. These plastic adherent fibroblastic cells express Notch-1, nestin and vimentin. We differentiated dental follicle cells with dexamethasone or insulin-based protocols into membrane-like structures containing mineralizing foci. An analysis of mineralized tissue with atomic force microscopy illustrated a bone and cementum-like structure. A real-time RT-PCR analysis was developed to investigate expression of typical osteoblast or cementoblast related genes during differentiation. Gene expressions of osteocalcin (OCN), bone morphogenic protein (BMP)-2 and nestin were increased during the both differentiation approaches. Our work demonstrates differentiation of dental follicle cells with an insulin-based protocol for the first time.     

Comparison of murine dental follicle precursor and retinal progenitor cells after neural differentiation in vitro

Human dental stem or precursor cells can differentiate into multiple cell types like adipocytes, osteoblasts or chondrocytes. Recently, a number of different human dental stem cell lines were differentiated into neurons. This makes dental stem cells interesting as possible cell-based therapeutics for neural degenerative diseases. To test this hypothesis, we have investigated the neural differentiation potential of murine dental follicle precursor cells (mDFPCs). The mDFPC cell line was newly established without cell immortalization. After differentiation, neural cell marker expression in mDFPCs was checked and compared with that of murine retinal progenitor cells (mRPCs). Differentiated mDFPCs became neuron-like cells with small cell bodies and long/branching neurites, similar to differentiated mRPCs. However, mRPCs showed more complete neural differentiation. Furthermore, 5% of the differentiated mDFPCs and 37% of the differentiated mRPCs were positive for the glia cell marker GFAP (glial fibrillary acidic protein). The data presents new evidence of neural differentiation of mDFPCs, but only a small percentage of mDFPCs differentiated into glia cells, unlike mRPCs.     

Application of dental stem cells in three-dimensional tissue regeneration

Dental stem cells can differentiate into different types of cells. Dental pulp stem cells, stem cells from human exfoliated deciduous teeth, periodontal ligament stem cells, stem cells from apical papilla, and dental follicle progenitor cells are five different types of dental stem cells that have been identified during different stages of tooth development. The availability of dental stem cells from discarded or removed teeth makes them promising candidates for tissue engineering. In recent years, three-dimensional (3D) tissue scaffolds have been used to reconstruct and restore different anatomical defects. With rapid advances in 3D tissue engineering, dental stem cells have been used in the regeneration of 3D engineered tissue. This review presents an overview of different types of dental stem cells used in 3D tissue regeneration, which are currently the most common type of stem cells used to treat human tissue conditions.