Clinical trials using dental stem cells: 2022 update

For nearly 20 years, dental stem cells(DSCs) have been successfully isolated from mature/immature teeth and surrounding tissue, including dental pulp of permanent teeth and exfoliated deciduous teeth, periodontal ligaments, dental follicles, and gingival and apical papilla. They have several properties(such as self-renewal, multidirectional differentiation, and immunomodulation) and exhibit enormous potential for clinical applications. To date, many clinical articles and clinical trials using DS...     

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.     

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.     

Applications of single cell RNA sequencing to research of stem cells

Stem cells(SCs) with their self-renewal and pluripotent differentiation potential,show great promise for therapeutic applications to some refractory diseases such as stroke, Parkinsonism, myocardial infarction, and diabetes. Furthermore, as seed cells in tissue engineering, SCs have been applied widely to tissue and organ regeneration. However, previous studies have shown that SCs are heterogeneous and consist of many cell subpopulations. Owing to this heterogeneity of cell states, gene expression is highly diverse between cells even within a single tissue,making precise identification and analysis of biological properties difficult, which hinders their further research and applications. Therefore, a defined understanding of the heterogeneity is a key to research of SCs. Traditional ensemble-based sequencing approaches, such as microarrays, reflect an average of expression levels across a large population, which overlook unique biological behaviors of individual cells, conceal cell-to-cell variations, and cannot understand the heterogeneity of SCs radically. The development of high throughput single cell RNA sequencing(scRNA-seq) has provided a new research tool in biology, ranging from identification of novel cell types and exploration of cell markers to the analysis of gene expression and predicating developmental trajectories. scRNA-seq has profoundly changed our understanding of a series of biological phenomena. Currently, it has been used in research of SCs in many fields, particularly for the research of heterogeneity and cell subpopulations in early embryonic development. In this review, we focus on the scRNA-seq technique and its applications to research of SCs.     

犬牙囊干细胞膜片的构建及生物学特性的研究

目的:利用犬牙囊干细胞(Dental Follicle Stem Cells,DFSCs)构建细胞膜片并研究其生物学特性。方法:取4至6月龄犬尖牙牙胚,分离培养DFSCs,鉴定。用含抗坏血酸的培养基诱导2周构建细胞膜片,并通过倒置显微镜、HE染色、茜素红染色、油红染色、扫描电镜(SEM)对膜片进行形态学检测,检测成骨、成脂能力。结果:DFSCs于体外被成功分离、纯化、培养,细胞克隆形成率约为5.1%。流式鉴定为CD29+CD44+CD34-,增殖能力及克隆形成能力较强,并能成功构建成细胞膜片。光镜和电镜显示膜片细胞排列紧密,细胞基质分泌多,油红O染色后可见细胞内有大量脂滴形成。(B)茜素红染色后可见大量清晰的钙结节形成。结论:成功构建犬DFSCs膜片,并证明其具有较强的成骨能力,为进一步利用犬DFSCs膜片修复牙槽骨缺损的研究提供条件。     

Proteome of Human Stem Cells from Periodontal Ligament and Dental Pulp

Background

Many adult tissues contain a population of stem cells with the ability to regenerate structures similar to the microenvironments from which they are derived in vivo and represent a promising therapy for the regeneration of complex tissues in the clinical disorder. Human adult stem cells (SCs) including bone marrow stem cells (BMSCs), dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) have been characterized for their high proliferative potential, expression of characteristic SC-associated markers and for the plasticity to differentiate in different lineage in vitro.

Methodology/Principal Findings

The aim of this study is to define the molecular features of stem cells from oral tissue by comparing the proteomic profiles obtained with 2-DE followed by MALDI-TOF/TOF of ex-vivo cultured human PDLSCs, DPSCs and BMSCs. Our results showed qualitative similarities in the proteome profiles among the SCs examined including some significant quantitative differences. To enrich the knowledge of oral SCs proteome we performed an analysis in narrow range pH 4–7 and 6–9, and we found that DPSCs vs PDLSCs express differentially regulated proteins that are potentially related to growth, regulation and genesis of neuronal cells, suggesting that SCs derived from oral tissue source populations may possess the potential ability of neuronal differentiation which is very consistent with their neural crest origin.

Conclusion/Significance

This study identifies some differentially expressed proteins by using comparative analysis between DPSCs and PDLSCs and BMSCs and suggests that stem cells from oral tissue could have a different cell lineage potency compared to BMSCs.     

Dental stem cells: The role of biomaterials and scaffolds in developing novel therapeutic strategies

Dental stem cells(DSCs) are self-renewable cells that can be obtained easily from dental tissues, and are a desirable source of autologous stem cells. The use of DSCs for stem cell transplantation therapeutic approaches is attractive due to their simple isolation, high plasticity, immunomodulatory properties, and multipotential abilities. Using appropriate scaffolds loaded with favorable biomolecules, such as growth factors, and cytokines, can improve the proliferation, differentiation, migration, and functional capacity of DSCs and can optimize the cellular morphology to build tissue constructs for specific purposes. An enormous variety of scaffolds have been used for tissue engineering with DSCs. Of these, the scaffolds that particularly mimic tissue-specific micromilieu and loaded with biomolecules favorably regulate angiogenesis, cell-matrix interactions, degradation of extracellular matrix, organized matrix formation, and the mineralization abilities of DSCs in both in vitro and in vivo conditions. DSCs represent a promising cell source for tissue engineering, especially for tooth, bone, and neural tissue restoration. The purpose of the present review is to summarize the current developments in the major scaffolding approaches as crucial guidelines for tissue engineering using DSCs and compare their effects in tissue and organ regeneration.     

Notch1 signalling inhibits apoptosis of human dental follicle stem cells via both the cytoplasmic mitochondrial pathway and nuclear transcription regulation

Dental follicle stem cells (DFSCs) have been considered as promising candidate cells for periodontal tissue regeneration. Understanding the signalling pathways underlying the apoptosis of DFSCs will facilitate its biomedical application. Here we showed that Notch1 signalling could inhibit DFSCs apoptosis because the constitutive overexpression of the intracellular domain of Notch1 (ICN1) promoted proliferation and suppressed apoptosis by inhibiting cytoplasmic mitochondrial membrane depolarization, cytochrome c release and activation of caspase-9 and caspase-3. The survival-promoting effect of Notch1 was also accomplished by up-regulation of the anti-apoptotic proteins Bcl-2 and Mcl-1, down-regulation of the pro-apoptotic proteins Bax and Bad, and blockade of Bax multimerization. Moreover, p-Akt (S473) was significantly increased after ectopic Notch 1 activation. The expression of p53 was also inhibited in Notch1-overexpressing DFSCs, while the ectopic expression of p53 promoted apoptosis even when Notch1 was overexpressed. Meanwhile, all of the opposite phenomena were observed in Notch1 shRNA-silenced DFSCs. Our data strongly suggested that Notch1 signalling inhibited the apoptosis of DFSCs via the cytoplasmic mitochondrial pathway and ICN-Akt signalling pathway, together with nuclear gene expression regulation. These findings would provide molecular cues for the further medical application of DFSCs.     

Tooth-derived stem cells: Update and perspectives

Tissue engineering is an emerging field of science that focuses on creating suitable conditions for the regeneration of tissues. The basic components for tissue engineering involve an interactive triad of scaffolds, signaling molecules, and cells. In this context,stem cells(SCs) present the characteristics of selfrenewal and differentiation capacity, which make them promising candidates for tissue engineering. Although they present some common markers, such as cluster of differentiation(CD)105, CD146 and STRO-1, SCs derived from various tissues have different patterns in relation to proliferation, clonogenicity, and differentiation abilities in vitro and in vivo. Tooth-derived tissues have been proposed as an accessible source to obtain SCs with limited morbidity, and various tooth-derived SCs(TDSCs) have been isolated and characterized, such as dental pulp SCs, SCs from human exfoliated deciduous teeth, periodontal ligament SCs, dental follicle progenitor cells, SCs from apical papilla, and periodontal ligament of deciduous teeth SCs. However, heterogeneity among these populations has been observed, and the best method to select the most appropriate TDSCs for regeneration approaches has not yet been established. The objective of this review is to outline the current knowledge concerning the various types of TDSCs, and discuss the perspectives for their use in regenerative approaches.     

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.     

Age-Dependent Impaired Neurogenic Differentiation Capacity of Dental Stem Cell is Associated with Wnt/β-Catenin Signaling

Two kinds of dental stem cells (DSCs), dental pulp stem cells (DPSCs) and stem cells from human-exfoliated deciduous teeth (SHED), have been identified as novel populations of mesenchymal stem cells that can be induced to differentiate into osteoblasts, chondrocytes, adipocytes, and neuron-like cells in vitro. As we know, both of them originate from the neural crest, but have distinct characteristics and functions in vitro and in vivo. The regeneration potential of DSCs declines with advanced age; however, the mechanism of the impaired potential in DSCs has not been fully explored. In this study, we investigated whether declined neurogenic differentiation capacity is associated with an altered expression of Wnt signaling-related proteins in vitro. We compared stem cells isolated from human dental pulp in two age groups: the exfoliated deciduous teeth (5–12 years), and the third permanent teeth (45–50 years). We found that the expression levels of neuron markers, such as βIII-tubulin, microtubule-associated protein 2(MAP2), tyrosine hydroxylase (TH), and Nestin were lower in the DPSCs group compared with that in the SHED group; however, in supplementation with human recombinant Wnt1 in the medium, the DPSCs were prone to neural differentiation and expressed higher levels of neurogenic markers. In summary, our study demonstrated that Wnt/β-catenin signaling may play a vital role in the age-dependent neural differentiation of DSCs. Therefore, DSCs may provide an ideal source of stem cells that can further extend their therapeutic application in nerve injury and neurodegenerative diseases.     

Adult human neural stem cell therapeutics: Current developmental status and prospect

Over the past two decades, regenerative therapies using stem cell technologies have been developed for various neurological diseases. Although stem cell therapy is an attractive option to reverse neural tissue damage and to recover neurological deficits, it is still under development so as not to show significant treatment effects in clinical settings. In this review, we discuss the scientific and clinical basics of adult neural stem cells (aNSCs), and their current developmental status as cell therapeutics for neurological disease. Compared with other types of stem cells, aNSCs have clinical advantages, such as limited proliferation, inborn differentiation potential into functional neural cells, and no ethical issues. In spite of the merits of aNSCs, difficulties in the isolation from the normal brain, and in the in vitro expansion, have blocked preclinical and clinical study using aNSCs. However, several groups have recently developed novel techniques to isolate and expand aNSCs from normal adult brains, and showed successful applications of aNSCs to neurological diseases. With new technologies for aNSCs and their clinical strengths, previous hurdles in stem cell therapies for neurological diseases could be overcome, to realize clinically efficacious regenerative stem cell therapeutics.     

人体牙髓干细胞的分离与鉴定

目的探讨牙髓干细胞（DPSC）对牙周病,外伤及肿瘤等造成下颌骨缺损、口腔软组织与神经损伤的修复治疗作用。方法本研究利用组织块培养法分离出人体DPSC,用流式细胞仪进行了鉴定,并进行DPSC成骨、成脂、成神经的分化研究。结果分离出3株DPSC,流式细胞分析表明DPSC表达CD73和CD90标志物,但不表达生血干细胞标志物CD34。用茜素红染色表明DPSC能分化成骨细胞,油红O染色表明DPSC能分化成脂肪细胞,免疫免疫荧光染色表明DPSC分化的细胞表达神经细胞特异标志物TUJ1。结论组织块培养能够高效快速分离表达CD73和CD90的DPSC,在体外诱导条件下DPSC能分化为成骨细胞、脂肪细胞和神经细胞,此研究为DPSC在治疗和修复骨组织缺损和神经损伤中的临床应用提供了实验依据。     

Hair follicle stem cells: walking the maze

The discovery of epithelial stem cells (eSCs) in the bulge region of the outer root sheath of hair follicles in mice and man has encouraged research into utilizing the hair follicle as a therapeutic source of stem cells (SCs) for regenerative medicine, and has called attention to the hair follicle as a highly instructive model system for SC biology. Under physiological circumstances, bulge eSCs serve as cell pool for the cyclic regeneration of the anagen hair bulb, while they can also regenerate the sebaceous gland and the epidermis after injury. More recently, melanocyte SCs, nestin+, mesenchymal and additional, as yet ill-defined "stem cell" populations, have also been identified in or immediately adjacent to the hair follicle epithelium, including in the specialized hair follicle mesenchyme (connective tissue sheath), which is crucial to wound healing. Thus the hair follicle and its adjacent tissue environment contain unipotent, multipotent, and possibly even pluripotent SC populations of different developmental origin. It provides an ideal model system for the study of central issues in SC biology such as plasticity and SC niches, and for the identification of reliable, specific SC markers, which distinguish them from their immediate progeny (e.g. transient amplifying cells). The current review attempts to provide some guidance in this growing maze of hair follicle-associated SCs and their progeny, critically reviews potential or claimed hair follicle SC markers, highlights related differences between murine and human hair follicles, and defines major unanswered questions in this rapidly advancing field.     

Endometrial stromal cells and decidualized stromal cells: Origins,transformation and functions

Decidualization of endometrium, which is characterized by endometrial stromal cell (ESC) decidualization, vascular reconstruction, immune cell recruitment, and plentiful molecule production, is a crucial step for uterus to become receptive for embryo. When implantation takes place, ESCs surround and directly interact with embryo. Decidualized stromal cells (DSCs) are of great importance in endometrial decidualization, having a broad function in regulating immune activity and vascular remodeling of uterus. DSCs are shown to have a higher metabolic level and looser cytoskeleton than ESCs. What's the origin of ESCs and how ESCs successfully transform into DSCs had puzzled scientists in the last decades. Breakthrough had been achieved recently, and many studies had elucidated some of the characters and functions of DSCs. However, several questions still remain unclear. This paper reviews current understanding of where ESCs come from and how ESCs differentiate into DSCs, summarizes some characters and functions of DSCs, analyzes current studies and their limitations and points out research areas that need further investigation.     

The potential of mesenchymal stem cells derived from amniotic membrane and amniotic fluid for neuronal regenerative therapy

The mesenchymal stem cells (MSCs), which are derived from the mesoderm, are considered as a readily available source for tissue engineering. They have multipotent differentiation capacity and can be differentiated into various cell types. Many studies have demonstrated that the MSCs identified from amniotic membrane (AM-MSCs) and amniotic fluid (AF-MSCs) are shows advantages for many reasons, including the possibility of noninvasive isolation, multipotency, self-renewal, low immunogenicity, anti-inflammatory and nontumorigenicity properties, and minimal ethical problem. The AF-MSCs and AM-MSCs may be appropriate sources of mesenchymal stem cells for regenerative medicine, as an alternative to embryonic stem cells (ESCs). Recently, regenerative treatments such as tissue engineering and cell transplantation have shown potential in clinical applications for degenerative diseases. Therefore, amnion and MSCs derived from amnion can be applied to cell therapy in neuro-degeneration diseases. In this review, we will describe the potential of AM-MSCs and AF-MSCs, with particular focus on cures for neuronal degenerative diseases. [BMB Reports 2014; 47(3): 135-140]     

Hair follicle stem cells are specified and function in early skin morphogenesis

In adult skin, epithelial hair follicle stem cells (SCs) reside in a quiescent niche and are essential for cyclic bouts of hair growth. Niche architecture becomes pronounced postnatally at the start of the first hair cycle. Whether SCs exist or function earlier is unknown. Here we show that slow-cycling cells appear early in skin development, express SC markers, and later give rise to the adult SC population. To test whether these early slow-cycling cells function as SCs, we use Sox9-Cre for genetic marking and K14-Cre to embryonically ablate Sox9, an essential adult SC gene. We find that the progeny of Sox9-expressing cells contribute to all skin epithelial lineages and Sox9 is required for SC specification. In the absence of early SCs, hair follicle and sebaceous gland morphogenesis is blocked, and epidermal wound repair is compromised. These findings establish the existence of early hair follicle SCs and reveal their physiological importance in tissue morphogenesis.     

Advances of tooth‐derived stem cells in neural diseases treatments and nerve tissue regeneration

Nerous system diseases, both central and peripheral, bring an incredible burden onto patients and enormously reduce their quality of life. Currently, there are still no effective treatments to repair nerve lesions that do not have side effects. Stem cell–based therapies, especially those using dental stem cells, bring new hope to neural diseases. Dental stem cells, derived from the neural crest, have many characteristics that are similar to neural cells, indicating that they can be an ideal source of cells for neural regeneration and repair. This review summarizes the neural traits of all the dental cell types, including DPSCs, PDLCs, DFCs, APSCs and their potential applications in nervous system diseases. We have summed up the advantages of dental stem cells in neural repair, such as their neurotrophic and neuroprotective traits, easy harvest and low rejective reaction rate, among others. Taken together, dental stem cells are an ideal cell source for neural tissue regeneration and repair.