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.     

Chromosome instability and tumor lethality suppression in carcinogenesis

The maintenance and survival of each organism depends on its genome integrity. Alterations of essential genes, or aberrant chromosome number and structure lead to cell death. Paradoxically, cancer cells, especially in solid tumors, contain somatic gene mutations and are chromosome instability (CIN), suggesting a mechanism that cancer cells have acquired to suppress the lethal mutations and/or CIN. Herein we will discuss a tumor lethality suppression concept based on the studies of yeast genetic interactions and transgenic mice. During the early stages of the multistep process of tumorigenesis, incipient cancer cells probably have adopted genetic and epigenetic alterations to tolerate the lethal mutations of other genes that ensue, and to a larger extent CIN. In turn, CIN mediated massive gain and loss of genes provides a wider buffer for further genetic reshuffling, resulting in cancer cell heterogeneity, drug resistance and evasion of oncogene addiction, thus CIN may be both the effector and inducer of tumorigenesis. Accordingly, interfering with tumor lethality suppression could lead to cancer cell death or growth defects. Further validation of the tumor lethality suppression concept would help to elucidate the role of CIN in tumorigenesis, the relationship between CIN and somatic gene mutations, and would impact the design of anticancer drug development.     

Effect of Mechanical Forces on the Behavior of Dental Stem Cells: A Scoping Review of <i>In-Vitro</i> Studies

This article is a scoping review of the studies that assessed the effect of mechanical forces on the behavior of dental stem cells (DSCs). PubMed and Scopus searches were done for in-vitro studies evaluating the effect of tension, hydrostatic pressure (i.e., the pressure applied through an incompressible fluid), compression, simulated microgravity, and vibration on DSCs. The following factors were analyzed: osteogenic/odontogenic differentiation, proliferation, adhesion and migration. Articles were reviewed according to the Preferred Reporting Items for Systematic Reviews extension for scoping reviews (PRISMA-ScR) guideline. Included studies were evaluated based on the modified Consolidated Standards of Reporting Trials (CONSORT). A total of 18 studies published from 2008–2019 were included. Nine studies were focusing on Periodontal ligament Stem Cells (PDLSCs), eight studies on Dental Pulp Stem Cells (DPSCs) and one study on Stem Cells from Apical Papilla (SCAP). Results showed that tension, three-dimensional stress and simulated microgravity promoted the proliferation and osteogenic differentiation of PDLSCs. DPSCs proliferation increased after microgravity and tension exertion. In addition, dynamic hydrostatic pressure and compression promoted odontogenic differentiation of DPSCs. Besides, mechanical stimuli increased the osteogenic differentiation of DPSCs. One study analyzed the effect of carrier features on the response of DSCs to 3D-stress and showed that cells cultivated on scaffolds with 30% bioactive glass (BAG) had the highest osteogenic differentiation compared to other ratios of BAG. It has been shown that increasing the duration of tension (i.e., from 3 h to 24 h force application) enhanced the positive effect of force application on the osteogenic differentiation of DSCs. In conclusion, all types of mechanical forces except uniaxial tension increased the osteogenic/odontogenic differentiation of DSCs. In addition, the effect of mechanical stimulation on the proliferation of DSCs differs based on the type of stem cells and mechanical force.     

Linear Model of Colon Cancer Initiation

Cancer results if regulatory mechanisms of cell birth and death are disrupted. Colorectal tumorigenesis is initiated by somatic or inherited mutations in the APC tumor suppressor gene pathway. Several additional genetic hits in other tumor suppressor genes and oncogenes drive the progression from polyps to malignant, invasive cancer. The majority of colorectal cancers present chromosomal instability, CIN, which is caused by mutations in genes that are required to maintain chromosomal stability. A major question in cancer genetics is whether CIN is an early event and thus a driving force of tumor progression. We present a new mathematical model of colon cancer initiation assuming a linear flow from stem cells to differentiated cells to apoptosis. We study the consequences of mutations in different cell types and calculate the conditions for CIN to precede APC inactivation. We find that early emergence of CIN is very likely in colorectal tumorigenesis.     

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.     

Molecular hydrogen inhibits lipopolysaccharide/interferon γ-induced nitric oxide production through modulation of signal transduction in macrophages

Adipose-derived stem cells (ASCs) have been successfully applied in treating bone defects both in animals and humans and promoted osteogenesis in vivo significantly. However, the mechanism of in vivo osteogenesis of ASCs was still little known, we hypothesized that this was mediated in part by interaction between implanted ASCs and local vein endothelial cells. In this study, human adipose-derived stem cells (hASCs) and human umbilical vein endothelial cells (HUVEC) were isolated and characterized. Cells were then either cultured alone or cocultured. Alkaline phosphatase (ALP) staining, quantitative measurement of ALP activity and Alizarin staining of hASCs cultured alone, HUVEC cultured alone and cells cocultured demonstrated that osteogenic differentiation of cocultured cells increased obviously. Osteocalcin (OC) expression of hASCs cocultured with HUVEC showed an obvious raise than hASCs cultured alone. HUVEC cultured alone showed BMP-2 secretion and increased with culturing time. Real-time PCR of the cocultured cells showed four osteogenic differentiation related genes raised with culturing time, while two adipogenic differentiation related genes showed a slightly decrease with culturing time. Results of our study with different culture models showed that in vitro osteogenesis of hASCs was enhanced by coculture with HUVEC which secreted BMP-2. This study not only provided us with an in vitro model of studying interaction between cells, but also helped us to understand the in vivo therapeutic mechanisms of ASCs.     

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.     

体外培养的人牙胚上皮细胞的成釉分化

人表皮干细胞可以作为牙齿再生中上皮源性的种子细胞,但是其成釉分化的效率低下. 本研究分离培养了人牙胚上皮细胞,利用E13.5的小鼠牙间充质与其重组,构建重组牙胚,对其成釉分化的潜能和机制进行研究. 研究结果发现,体外培养的P1代人牙胚上皮细胞成釉率高达50%. 随着传代次数的增加,成釉率明显下降. 通过对牙上皮发育分化相关基因的表达检测和分析表明,重组牙胚成牙分化能力和成釉潜能的下降与牙上皮发育相关基因的表达状态密切相关. 特别是FGF8表达水平的下调以及PITX2不同亚型在人牙胚细胞中表达量的不均衡,可能是导致人牙胚细胞成釉潜能下降并丧失的主要原因. 本研究结果为理解牙齿再生过程中上皮源性的种子细胞的成釉机制提供了新的实验数据,对进一步提高表皮干细胞在牙齿再生过程中的成釉率有指导意义.     

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...     

Epigenetic therapy of cancer stem and progenitor cells by targeting DNA methylation machineries

Recent advances in stem cell biology have shed light on how normal stem and progenitor cells can evolve to acquire malignant characteristics during tumorigenesis. The cancer counterparts of normal stem and progenitor cells might be occurred through alterations of stem cell fates including an increase in self-renewal capability and a decrease in differentiation and/or apoptosis. This oncogenic evolution of cancer stem and progenitor cells, which often associates with aggressive phenotypes of the tumorigenic cells, is controlled in part by dysregulated epigenetic mechanisms including aberrant DNA methylation leading to abnormal epigenetic memory. Epigenetic therapy by targeting DNA methyltransferases (DNMT) 1, DNMT3A and DNMT3B via 5-Azacytidine (Aza) and 5-Aza-2’-deoxycytidine (Aza-dC) has proved to be successful toward treatment of hematologic neoplasms especially for patients with myelodysplastic syndrome. In this review, I summarize the current knowledge of mechanisms underlying the inhibition of DNA methylation by Aza and Aza-dC, and of their apoptotic- and differentiation-inducing effects on cancer stem and progenitor cells in leukemia, medulloblastoma, glioblastoma, neuroblastoma, prostate cancer, pancreatic cancer and testicular germ cell tumors. Since cancer stem and progenitor cells are implicated in cancer aggressiveness such as tumor formation, progression, metastasis and recurrence, I propose that effective therapeutic strategies might be achieved through eradication of cancer stem and progenitor cells by targeting the DNA methylation machineries to interfere their “malignant memory”.     

Comparative characterization of mesenchymal bone marrow stromal cells at early and late stages of culturing

The mesenchymal stromal cell is a multipotent precursor of osteoblasts, adipocytes, and some other cell types. In this study, a comparative analysis of cultured mesenchymal stromal cells from the rat bone marrow at the early and late stages of subculturing has been performed using molecular genetic and cytological methods. The culture has undergone 11 passages during 140 days. Upon long-term culturing, the mesenchymal stromal cells have proved to lose their potential for adipogenic differentiation but preserve the potential for osteogenesis. Morphological characters typical of osteogenic differentiation can be observed at the earlier stages of culturing (passages 1–4) but disappear at later stages (passages 9–11), despite mineralization of the extracellular matrix and the expression of osteogenic differentiation markers. A comparative analysis of the proliferation potential of stromal cells has shown that differences in the period of cell population doubling at the early and later stages of culturing are insignificant. An almost complete arrest of cell growth has been observed in the middle of the culture period (passages 5 and 6).     

牙髓干细胞的研究进展

牙髓干细胞是来源于牙髓组织中的一种成体干细胞,该种细胞具有高度增殖、自我更新的能力和多向分化潜能。牙髓干细胞的研究对牙髓再生、牙体修复等牙组织工程将产生重要的意义。该文就牙髓干细胞的研究现状作一综述,并对其应用前景进行探讨。     

Identification of Rabbit Annulus Fibrosus-Derived Stem Cells

Annulus fibrosus (AF) injuries can lead to substantial deterioration of intervertebral disc (IVD) which characterizes degenerative disc disease (DDD). However, treatments for AF repair/regeneration remain challenging due to the intrinsic heterogeneity of AF tissue at cellular, biochemical, and biomechanical levels. In this study, we isolated and characterized a sub-population of cells from rabbit AF tissue which formed colonies in vitro and could self-renew. These cells showed gene expression of typical surface antigen molecules characterizing mesenchymal stem cells (MSCs), including CD29, CD44, and CD166. Meanwhile, they did not express negative markers of MSCs such as CD4, CD8, and CD14. They also expressed Oct-4, nucleostemin, and SSEA-4 proteins. Upon induced differentiation they showed typical osteogenesis, chondrogenesis, and adipogenesis potential. Together, these AF-derived colony-forming cells possessed clonogenicity, self-renewal, and multi-potential differentiation capability, the three criteria characterizing MSCs. Such AF-derived stem cells may potentially be an ideal candidate for DDD treatments using cell therapies or tissue engineering approaches.     

Inducing human induced pluripotent stem cell differentiation through embryoid bodies: A practical and stable approach

Human induced pluripotent stem cells (hiPSCs) are invaluable resources for producing high-quality differentiated cells in unlimited quantities for both basic research and clinical use. They are particularly useful for studying human disease mechanisms in vitro by making it possible to circumvent the ethical issues of human embryonic stem cell research. However, significant limitations exist when using conventional flat culturing methods especially concerning cell expansion, differentiation efficiency, stability maintenance and multicellular 3D structure establishment, differentiation prediction. Embryoid bodies (EBs), the multicellular aggregates spontaneously generated from iPSCs in the suspension system, might help to address these issues. Due to the unique microenvironment and cell communication in EB structure that a 2D culture system cannot achieve, EBs have been widely applied in hiPSC-derived differentiation and show significant advantages especially in scaling up culturing, differentiation efficiency enhancement, ex vivo simulation, and organoid establishment. EBs can potentially also be used in early prediction of iPSC differentiation capability. To improve the stability and feasibility of EB-mediated differentiation and generate high quality EBs, critical factors including iPSC pluripotency maintenance, generation of uniform morphology using micro-pattern 3D culture systems, proper cellular density inoculation, and EB size control are discussed on the basis of both published data and our own laboratory experiences. Collectively, the production of a large quantity of homogeneous EBs with high quality is important for the stability and feasibility of many PSCs related studies.     

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.     

Proliferation of dental follicle-derived cell populations in heat-stress conditions

Objectives: Isolation and purification of adult stem cells (ASC) are a great challenge. Our objectives were to determine whether ASC are more heat‐tolerant than non‐stem cells, and to explore if ASC could be enriched by heat‐stress treatments. Materials and methods: Rat dental follicle cells were cultured in a variety of media to obtain either a heterogeneous cell population (H‐DFC) consisting of stem cells and non‐stem cells, or a homogenous cell population (DFC) containing non‐stem cells only. Real‐time RT‐PCR was conducted to compare expression of heat‐shock proteins (HSPs) between the two populations. To study heat tolerance, H‐DFC and DFC were incubated under heat‐stress conditions and cell proliferation was evaluated by alamar blue reduction assay. Furthermore, cells resulting from heat‐stress treatments were evaluated for differentiation capability and expression of stem cell markers. Results: H‐DFC expressed higher levels of HSP110, HSP70s and HSP27s than did DFC. H‐DFC increased levels of proliferation at 40 °C compared to controls grown at 37 °C; no significant reduction in proliferation occurred at temperatures below 40.5 °C. In contrast, DFC showed significant reduction in proliferation under all heat‐stress treatments. Heat‐stressed H‐DFC had increased differentiation capability and increased expression of stem cell markers. Conclusion: Stem cells appear to be more tolerant to heat stress than non‐stem cells. Incubation of a heterogeneous cell population in heat‐stress conditions resulted in increased stem cell numbers.     

Intra-tumor heterogeneity of MLH1 promoter methylation revealed by deep single molecule bisulfite sequencing

A single tumor may contain cells with different somatic mutations. By characterizing this genetic heterogeneity within tumors, advances have been made in the prognosis, treatment and understanding of tumorigenesis. In contrast, the extent of epigenetic intra-tumor heterogeneity and how it influences tumor biology is under-explored. We have characterized epigenetic heterogeneity within individual tumors using next-generation sequencing. We used deep single molecule bisulfite sequencing and sample-specific DNA barcodes to determine the spectrum of MLH1 promoter methylation across an average of 1000 molecules in each of 33 individual samples in parallel, including endometrial cancer, matched blood and normal endometrium. This first glimpse, deep into each tumor, revealed unexpectedly heterogeneous patterns of methylation at the MLH1 promoter within a subset of endometrial tumors. This high-resolution analysis allowed us to measure the clonality of methylation in individual tumors and gain insight into the accumulation of aberrant promoter methylation on both alleles during tumorigenesis.