Dental Pulp Of Deciduous Teeth Research Articles

Cartilage-Specific Gene Expression and Extracellular Matrix Deposition in the Course of Mesenchymal Stromal Cell Chondrogenic Differentiation in 3D Spheroid Culture.

Articular cartilage damage still remains a major problem in orthopedical surgery. The development of tissue engineering techniques such as autologous chondrocyte implantation is a promising way to improve clinical outcomes. On the other hand, the clinical application of autologous chondrocytes has considerable limitations. Mesenchymal stromal cells (MSCs) from various tissues have been shown to possess chondrogenic differentiation potential, although to different degrees. In the present study, we assessed the alterations in chondrogenesis-related gene transcription rates and extracellular matrix deposition levels before and after the chondrogenic differentiation of MSCs in a 3D spheroid culture. MSCs were obtained from three different tissues: umbilical cord Wharton's jelly (WJMSC-Wharton's jelly mesenchymal stromal cells), adipose tissue (ATMSC-adipose tissue mesenchymal stromal cells), and the dental pulp of deciduous teeth (SHEDs-stem cells from human exfoliated deciduous teeth). Monolayer MSC cultures served as baseline controls. Newly formed 3D spheroids composed of MSCs previously grown in 2D cultures were precultured for 2 days in growth medium, and then, chondrogenic differentiation was induced by maintaining them in the TGF-β1-containing medium for 21 days. Among the MSC types studied, WJMSCs showed the most similarities with primary chondrocytes in terms of the upregulation of cartilage-specific gene expression. Interestingly, such upregulation occurred to some extent in all 3D spheroids, even prior to the addition of TGF-β1. These results confirm that the potential of Wharton's jelly is on par with adipose tissue as a valuable cell source for cartilage engineering applications as well as for the treatment of osteoarthritis. The 3D spheroid environment on its own acts as a trigger for the chondrogenic differentiation of MSCs.

Unique Transcriptional Signatures Observed in Stem Cells from the Dental Pulp of Deciduous Teeth Produced on a Large Scale

Unique Transcriptional Signatures Observed in Stem Cells from the Dental Pulp of Deciduous Teeth Produced on a Large Scale

SSEA-4 positive dental pulp stem cells from deciduous teeth and their induction to neural precursor cells

BackgroundStage-specific embryonic antigen-4 (SSEA-4) is a marker for the identification of multipotent embryonic cells. It is also positive in neuroepithelial cells, precursor neural cells (NPC), and human dental pulp cells. The aim of this study was to evaluate the potential morphodifferentiation and histodifferentiation to NPC of SSEA-4 positive stem cells from human exfoliated deciduous teeth (SHED).MethodsA SHED population in culture, positive to SSEA-4, was obtained by magnetic cell separation. The cells were characterized by immunohistochemistry and flow cytometry. Subsequently, a neurosphere assay was performed in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF); afterward, cells were neurodifferenciated with a neurobasal medium. Finally, indirect immunohistochemistry was performed to identify neuronal markers.ResultsThe morphological and histological changes in the SSEA-4 positive SHEDs were observed after induction with epidermal and fibroblast growth factors in neurobasal culture medium. At the end of induction, the markers Nestin, TuJ-1, and GFAP were identified.ConclusionsThe findings show that SSEA-4 positive SHEDs have a behavior similar to neuronal precursor cells. Our findings indicate that the dental pulp of deciduous teeth is a promising source for regeneration therapies associated with neurodegenerative diseases or peripheral nerve alterations.

Metformin enhances osteogenic differentiation of stem cells from human exfoliated deciduous teeth through AMPK pathway.

Stem cells from human exfoliated deciduous teeth (SHEDs) are ideal seed cells in bone tissue engineering. As a first-line antidiabetic drug, metformin has recently been found to promote bone formation. The purpose of this study was to investigate the effect of metformin on the osteogenic differentiation of SHEDs and its underlying mechanism. SHEDs were isolated from the dental pulp of deciduous teeth from healthy children aged 6 to 12, and their surface antigen markers of stem cells were detected by flow cytometry. The effect of metformin (10-200 μM) treatment on SHEDs cell viability, proliferation, and osteogenic differentiation was analyzed. The activation of adenosine 5'-monophosphate-activated protein kinase (AMPK) phosphorylation Thr172 (p-AMPK) was determined by western blot assay. SHEDs were confirmed as mesenchymal stem cells (MSCs) on the basis of the expression of characteristic surface antigens. Metformin (10-200 μM) did not affect the viability and proliferation of SHEDs but significantly increased the expression of osteogenic genes, alkaline phosphatase activity, matrix mineralization, and p-AMPK level expression in SHEDs. Compound C, a specific inhibitor of the AMPK pathway, abolished metformin-induced osteogenic differentiation of SHEDs. Moreover, metformin treatment enhanced the expression of proangiogenic/osteogenic growth factors BMP2 and VEGF but reduced the osteoclastogenic factor RANKL/OPG expression in SHEDs. In conclusion, metformin could induce the osteogenic differentiation of SHEDs by activating the AMPK pathway and regulates the expression of proangiogenic/osteogenic growth factors and osteoclastogenic factors in SHEDs. Therefore, metformin-pretreated SHEDs could be a potential source of seed cells during stem cell-based bone tissue engineering.

Serine Metabolism Controls Dental Pulp Stem Cell Aging by Regulating the DNA Methylation of p16

To investigate the characteristics and molecular events of dental pulp stem cells (DPSCs) for tissue regeneration with aging, we isolated and analyzed the stem cells from human exfoliated deciduous teeth (SHED) and permanent teeth of young (Y-DPSCs) and old (A-DPSCs) adults. Results showed that the stemness and osteogenic differentiation capacity of DPSCs decreased with aging. The RNA sequencing results showed that glycine, serine, and threonine metabolism was one of the most enriched gene clusters among SHED, Y-DPSCs, and A-DPSCs, according to analysis based on the Kyoto Encyclopedia of Genes and Genomes. The expression of serine metabolism–related enzymes phosphoserine aminotransferase 1 (PSAT1) and phosphoglycerate (PHGDH) decreased in A-DPSCs and provided less methyl donor S-adenosylmethionine (SAM) for DNA methylation, leading to the hypomethylation of the senescence marker p16 (CDNK2A). Furthermore, the proliferation and differentiation capacity of Y-DPSCs and SHED decreased after PHGDH siRNA treatment, which reduced the level of SAM. Convincingly, the ratios of PSAT1-, PHGDH-, or proliferating cell nuclear antigen–positive cells in the dental pulp of old permanent teeth were less than those in the dental pulp of deciduous teeth and young permanent teeth. In summary, the stemness and differentiation capacity of DPSCs decreased with aging. The decreased serine metabolism in A-DPSCs upregulated the expression of p16 via attenuating its DNA methylation, resulting in DPSC aging. Our finding indicated that serine metabolism and 1 carbon unit participated in stem cell aging, which provided new direction for stem cell aging study and intervention.

Cartilage regeneration with human dental pulp stem cells - a systematic review

Background & Aim Background there is a high prevalence of patients with symptomatic cartilage lesions and it rises as a socioeconomic issue with a considerable economic problem. Despite the numerous techniques available today, complete healing of damaged or defective cartilage and the consistent reproduction of normal hyaline cartilage is not possible. Choice of harvesting site is motivated by MSCs abilities to modulate immunologic and inflammatory response. Recently, dental pulp has been shown to contain a stem cell niche and these dental pulp stem cells (DPSCs) maintain their self-renewal capacity due to the active environment in the dental pulp of deciduous teeth. Aim the aim of this study was to review the current literature on the potential and limitations of cell-based therapies in cartilage regeneration using dental pulp derived mesenchymal stem cells. Methods, Results & Conclusion Methods an customized electronic search of scientific articles was conducted using PubMed/ MEDLINE and EMBASE databases from their inception to December 2018. Inclusion criteria were applied and the manuscripts that described usage chondral treatment with dental pulp stem cells were elected for complete evaluation. The articles were classified according to scaffold used, experimental model, features of chondrogenic differentiation, local of the defect, cartilage evaluation and results. After application of eligibility criteria, a total of 9 studies were selected and fully analyzed. Results a variety of animal models were used in the studies analyzed in this systematic review, including mice, rat, rabbit and miniature pig. It was evidenced tissue engineering for articular cartilage defects and safety of using human DPSCs to repair cartilage defects. There are six studies that created local defects to receive scaffold and test for DPSCs efficacy. Two studies focused on preclinical models for cartilage tissue engineering. Conclusion once presented and ongoing studies confirm positive results of DPSCs for articular cartilage treatment, new boundaries of stem cell therapy are open for cartilage tissue bioengineering and subsequent clinical trials should be performed.

Systematic Review of Human Dental Pulp Stem Cells for Cartilage Regeneration.

Background: Symptomatic cartilage lesions and early osteoarthritis produce significant clinical and economic burdens. Cartilage repair can improve the symptoms and delay arthroplasty. The complete healing of damaged cartilage with the consistent reproduction of normal hyaline cartilage has not yet been achieved. The choice of harvesting site might influence the cells' abilities to modulate immunologic and inflammatory responses. Recently, dental pulp has been shown to contain a stem cell niche consisting of dental pulp stem cells (DPSCs) that maintain their self-renewal capacity due to the active environment in the dental pulp of deciduous teeth. Objective: The aim of this study was to critically review the current literature on the potential and limitations of the use of dental pulp-derived mesenchymal stem cells in cell-based therapies for cartilage regeneration. Methods: An electronic, customized search of scientific articles was conducted using the PubMed/MEDLINE and EMBASE databases from their inception to December 2018. The inclusion criteria were applied, and the articles that described the use of DPSC in cartilage treatment were selected for complete evaluation. The articles were classified according to the scaffold used, experimental model, chondrogenic differentiation features, defect location, cartilage evaluation, and results. After the application of the eligibility criteria, a total of nine studies were selected and fully analyzed. Results: A variety of animal models were used, including mice, rats, rabbits, and miniature pigs, to evaluate the quality and safety of human DPSCs in the repair of cartilage defects. Among the articles, two studies focused on preclinical models of cartilage tissue engineering. Five studies implanted DPSCs in other animal sites. Conclusion: The use of DPSCs is a potential new stem cell therapy for articular cartilage repair. The preclinical evidence discussed in this article provides a solid foundation for future clinical trials. Impact statement Osteoarthritis presents an ever-increasing clinical and socioeconomic burden. While cartilage repair has the potential to improve symptoms and delay joint replacement, complete regeneration of hyaline cartilage has been an elusive goal. Dental pulp has been shown to contain a niche that protects dental pulp stem cells (DPSCs) from the cumulative effects of genetic and environmental factors and maintains their self-renewal capacity due to the active environment. Transplantation and preclinical trials have demonstrated the strong potential of regenerative tissue-engineering protocols using DPSCs.

PiggyBac Transposon-Based Immortalization of Human Deciduous Tooth Dental Pulp Cells with Multipotency and Non-Tumorigenic Potential.

We aimed to immortalize primarily isolated human deciduous tooth-derived dental pulp cells (HDDPCs) by transfection with piggyBac (PB)-based transposon vectors carrying E7 from human papilloma virus 16 or complementary DNA (cDNA) encoding human telomerase reverse transcriptase (hTERT). HDDPCs were co-transfected with pTrans (conferring PB transposase expression) + pT-pac (conferring puromycin acetyltransferase expression) + pT-tdTomato (conferring tdTomato cDNA expression) and pT-E7 (conferring E7 expression) or pTrans + pT-pac + pT-EGFP (conferring enhanced green fluorescent protein cDNA expression) + pT-hTERT (conferring hTERT expression). After six days, these cells were selected in medium containing 5 μg/mL puromycin for one day, and then cultured in normal medium allowing cell survival. All resultant colonies were harvested and propagated as a pool. Stemness and tumorigenic properties of the established cell lines (“MT_E7” for E7 and “MT_hTERT” for hTERT) with untransfected parental cells (MT) were examined. Both lines exhibited proliferation similar to that of MT, with alkaline phosphatase activity and stemness-specific factor expression. They displayed differentiation potential into multi-lineage cells with no tumorigenic property. Overall, we successfully obtained HDDPC-derived immortalized cell lines using a PB-based transfection system. The resultant and parental cells were indistinguishable. Thus, E7 and hTERT could immortalize HDDPCs without causing cancer-associated changes or altering phenotypic properties.

Increased Expression of Cell Surface SSEA-1 is Closely Associated with Naïve-Like Conversion from Human Deciduous Teeth Dental Pulp Cells-Derived iPS Cells.

Stage-specific embryonic antigen 1 (SSEA-1) is an antigenic epitope (also called CD15 antigen) defined as a Lewis X carbohydrate structure and known to be expressed in murine embryonal carcinoma cells, mouse embryonic stem cells (ESCs), and murine and human germ cells, but not human ESCs/induced pluripotent stem cells (iPSCs). It is produced by α1,3-fucosyltransferase IX gene (FUT9), and F9 ECCs having a disrupted FUT9 locus by gene targeting are reported to exhibit loss of SSEA-1 expression on their cell surface. Mouse ESCs are pluripotent cells and therefore known as “naïve stem cells (NSCs).” In contrast, human ESCs/iPSCs are thought to be epiblast stem cells (EpiSCs) that are slightly more differentiated than NSCs. Recently, it has been demonstrated that treatment of EpiSCs with several reprograming-related drugs can convert EpiSCs to cells similar to NSCs, which led us to speculate that SSEA-1 may have been expressed in these NSC-like EpiSCs. Immunocytochemical staining of these cells with anti-SSEA-1 revealed increased expression of this epitope. RT-PCR analysis also confirmed increased expression of FUT9 transcripts as well as other stemness-related transcripts such as REX-1 (ZFP42). These results suggest that SSEA-1 can be an excellent marker for human NSCs.

Human exfoliated deciduous teeth and oral mucosa: promising applications in tissue regeneration

In the last three decades, the constantly increasing need for therapies, efficiently preventing and/or treating human diseases, has raised the interest in Regenerative Medicine (RM). RM is based on employing mesenchymal stem cells (MSCs), that showed to have great proliferation, self-renewal and multi-lineage differentiation potential, in vitro as well as in vivo. The opportunity of an accessible, painless and low-cost reservoir of MSCs constitutes the first important step of a successful regenerative therapy to include in the current clinical practice. Oral cavity has recently demonstrated to contain different MSCs niches: dental pulp from permanent and deciduous teeth, periodontal ligament, dental follicle, apical papilla and mucosa. MSCs from dental pulp of deciduous teeth, naturally lost in pediatric age, and the oral mucosa have shown to be easily harvested and to have a promising regenerative potential. Thus, the aim of the paper is to review the potentialities of human exfoliated deciduous teeth stem cells (SHEDs) and oral mucosa stem cells (OMSCs) in RM, with the purpose of their use as accessible source of MSCs for the future of pediatric patient.

Basic Fibroblast Growth Factor Regulates REX1 Expression Via IL-6 In Stem Cells Isolated From Human Exfoliated Deciduous Teeth.

Basic fibroblast growth factor (bFGF) regulates pluripotent marker expression and cellular differentiation in various cell types. However, the mechanism by which bFGF regulates REX1 expression in stem cells, isolated from human exfoliated deciduous teeth (SHEDs) remains unclear. The aim of the present study was to investigate the regulation of REX1 expression by bFGF in SHEDs. SHEDs were isolated and characterized. Their mRNA and protein expression levels were determined using real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. In some experiments, chemical inhibitors were added to the culture medium to impede specific signaling pathways. Cells isolated from human exfoliated deciduous tooth dental pulp tissue expressed mesenchymal stem cell surface markers (CD44, CD73, CD90, and CD105). These cells differentiated into osteogenic and adipogenic lineages, when appropriately induced. Treating SHEDs with bFGF induced REX1 mRNA expression and this effect was attenuated by pretreatment with FGFR or Akt inhibitors. Cycloheximide pretreatment also inhibited the bFGF-induced REX1 expression, implying the involvement of intermediate molecule(s). Further, the addition of an IL-6 neutralizing antibody attenuated the bFGF-induced REX1 expression by SHEDs. In conclusion, bFGF enhanced REX1 expression by SHEDs via the FGFR and Akt signaling pathways. Moreover, IL-6 participated in the bFGF-induced REX1 expression in SHEDs. J. Cell. Biochem. 118: 1480-1488, 2017. © 2016 Wiley Periodicals, Inc.

Structural and recovery mechanisms of 3D dental pulp cell microtissues challenged with Streptococcusmutans in extracellular matrix environment.

Cariopathogen Streptococcus mutans exists in infected dental pulp of deciduous teeth and is frequently linked with heart diseases. Organotypic (3D) dental pulp stem cell (DPSC) cultures/microtissues, developed to mimic the physiological conditions in vivo, were utilized to assess the bacterial impact on their (i) 3D structural configuration and (ii) recovery mechanisms. The cultures, developed in extracellular matrix (ECM) bio-scaffold (Matrigel™), interacted with WT and GFP-tagged bacterial biofilms by permitting their infiltration through the ECM. Challenged cell constructs were visualized by F-actin/nuclei staining. Their pluripotency (Sox2) and differentiation (osteocalcin) markers were assessed by immunocytochemistry. Secreted mineral was detected by alizarin red, and 3D structural arrangements were analysed by epi-fluorescence and confocal scanning microscopy. Bacterial biofilm/ECM-embedded DPSC interactions appeared in distinct areas of the microtissues. Bacterial attachment to the cell surface occurred without evidence of invasion. Surface architecture of the challenged versus unchallenged microtissues was apparently unaltered. However, significant increases in thickness (138.42 vs 106.51 µm) and bacterial penetration were detected in challenged structures causing canal-like microstructures with various diameters (12.94 -42.88 µm) and average diameter of 20.66 to 33.42 µm per microtissue. Challenged constructs expressed pluripotency and differentiation markers and secreted the mineral. Presented model shows strong potential for assessing pulp-pathogen interactions in vivo. S. mutans infiltrated and penetrated the microtissues but did not invade the cells or compromise major cell repair mechanisms. These findings would suggest reexamining the role of S. mutans as an endodontic pathogen and investigating DPSC resistance to its pathogenicity.

Effect of low-level laser irradiation on proliferation of human dental mesenchymal stem cells; a systemic review

ContextIdentification of factors that enhance the proliferation of human dental mesenchymal stem cells (DMSCs) is vital to facilitate tissue regeneration. The role of low-level laser irradiation (LLLI) on proliferation of human DMSCs has not been well established. ObjectiveTo assess the effect of LLLI on proliferation of human DMSCs when applied in-vitro. Data SourcesElectronic search of literature was conducted (2000–2016) on PubMed, Web of Science, and Scopus databases. Search terms included low-level light therapy, low-level laser irradiation, low-level light irradiation, LLLT, humans, adolescent, adult, cells, cultured, periodontal ligament, dental pulp, stem cells, dental pulp stem cells, mesenchymal stem cells, periodontal ligament stem cell, deciduous teeth, cell proliferation, adult stem cells, radiation, and proliferation. ResultsThe literature search identified 165 studies with 6 being eligible for inclusion; all used diode lasers; 5 studies used InGaAIP diode lasers; 4 used 660nm, and the other two applied 810nm or 980nm wavelength LLLI. The distance between the DMSCs and the laser spot ranged between 0.5mm to 2mm. The time intervals of cell proliferation analysis ranged from 0h to 7days after LLLI. After 660nm LLLI, an increase in the DMSC's proliferation was reported [DMSCs extracted from dental pulp of deciduous teeth (two irradiations, 3J/cm2, 20mW was more effective than 40mW), adult teeth (two irradiations, 0.5 and 1.0J/cm2, 30mW), and from adult periodontal ligament (two irradiations, 1.0J/cm2 was more effective than 0.5J/cm2, 30mW)]. Similarly, an increase in the proliferation of DMSCs extracted from dental pulp of adult teeth was reported after 810nm LLLI (7 irradiations in 7days, 0.1 and 0.2J/cm2, 60mW) or 980nm LLLI (single irradiation, 3J/cm2, 100mW). However, 660nm LLLI in one study did not increase the proliferation of DMSCs (single irradiation, energy densities of 0.05, 0.30, 7, and 42J/cm2, 28mW). ConclusionThere is limited evidence that in-vitro LLLI (660/810/980nm, with energy densities of 0.1-3J/cm2) increases the proliferation of DMSCs. Considering the limited evidence and their method heterogeneity it is difficult to reach a firm conclusion. Further research is necessary to identify the optimal characteristics of the LLLI setting (wave length, energy density, power output, frequency/duration of irradiations, distance between the cells and the laser spot/probe) to increase proliferation of DMSCs, and assess its impact on replicative senescence, as well as determine feasibility of the use in the clinical setting.

N-Acetyl-l-cysteine enhances ex-vivo amplification of deciduous teeth dental pulp stem cells

ObjectiveObtaining high number of stem cells is of interest for cell based therapies. N-Acetyl-l-cysteine (NAC) acts as a source of sulfhydryl groups and an anti-oxidative agent. The aim of this study was to test different NAC concentration on proliferation and differentiation of deciduous teeth dental pulp stem cells (DTSCs) in vitro as well as to define the possible underlining mechanism of its effect. DesignNumber of viable, apoptotic and senescent DTSCs was determined after addition of NAC (0.1mM, 1.0mM, 2.0mM). Also, cell cycle analysis, HIF1-α expression, LDH isoenzymes, superoxide-dismutase (SOD) and catalase (CAT) activity, sulfhydryl groups content, the level of lipids’ and proteins’ oxidative damage and differentiation capacity of NAC treated DTSCs was determined. ResultsDTSCs expressed HIF-1α in all conditions. The lowest NAC dose (0.1mM) increased the number of DTSCs by one fifth comparing to the control, most likely stimulating entry of cells into S phase of cell cycle and enhancing the activity of LDH5 isoenzyme. The highest NAC dose (2mM) inhibited DTSCs proliferation. Also, DTSCs had the lowest level of oxidative damage with 0.1mM NAC. All tested NAC concentrations enhanced DTSCs osteo-chondrogenesis. ConclusionThe lowest NAC dose exerted significant positive effect on DTSCs proliferation as well as antioxidative protection creating beneficial environment for stem cells in vitro cultivation especially when their clinical use is important for stimulation of osteo-chondrogenesis.

Creating a Future of Transplantation.

On behalf of the Japan Society for Organ Preservation and Medical Biology (JSOPMB), I express my sincere appreciation to Dr. David J. Eve and Professor Cesar V. Borlongan, Coeditors of Cell Medicine, for providing us with such an excellent opportunity to publish the data that were presented at the annual meeting of the JSOPMB. I also thank Dr. David J. Eve for the editing of our articles in detail. I am very sure that the relationship between Cell Medicine and JSOPMB has enhanced the motivation of JSOPMB members as well as board members and will continue to do so in the future, while also encouraging young Japanese researchers to join this organization. One of the extremely important missions of the annual meeting of the JSOPMB is to exchange new research outcomes and create new therapeutic concepts. JSOPMB always encourages and motivates young investigators. JSOPMB was started in 1974 for the study of organ preservation and developed widely in the 1990s with the participation of researchers in various fields of medicine, pharmacology, engineering, veterinary medicine, and basic science. Currently, JSOPMB has more than 700 members and is run under the direction of Professor Takashi Kondo, the president of the JSOPMB. Excellent presentations conducted at the 41th annual meeting of the JSOPMB held on November 28–29, 2014, in Osaka, Japan, under the supervision of Dr. Yoshiki Sawa (Professor, Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, Japan), were selected and given an opportunity to be published in this special issue of Cell Medicine. Seven of these presentations are herein published in this special JSOPMB issue. Stem cell research was a major topic of interest. There were two articles regarding stem cells. Miyagi-Shiohira et al. reviewed cryopreservation of mesenchymal stem cells, especially adipose-derived stem cells. Dimethyl sulfoxide (DMSO) is commonly used as a cryopreservation medium as it diffuses into the cell through the plasma membrane and protects the cells from the damage caused by freezing. As substitutes for DMSO or animal-derived serum, cell banker series, polyvinylpyrrolidone (PVP), sericin and maltose, and methyl cellulose (MC) have been investigated for their clinical applications. Saitoh et al. evaluated feeder cells for establishing induced pluripotent stem cells (iPSCs) from human deciduous tooth dental pulp cells (HDDPCs). Their data showed that mouse embryonic fibroblasts (MEFs) were better feeder cells than an immortalized line established from Santos inbred mouse (SIM) embryonic fibroblasts resistant to 6-thioguanine and ouabain known as STO cells for establishing iPSCs. There were three articles regarding pancreatic islets. Noguchi et al. reviewed islet culture/preservation before islet transplantation. In the current clinical culture conditions, it seems that islets cultured at 37°C cannot receive enough oxygen because central necrosis of islets during culture was frequently observed. Low-temperature storage may be better than culture, although a future study should carefully evaluate the effects of islet equivalent and tissue volume in relation to the antigenicity of 4°C-preserved islets. Hanayama et al. showed efficient gene transduction of dispersed islet cells in culture using fiber-modified adenoviral vectors harboring a polylysine (K7) peptide in the C-terminus of the fiber knob. The fiber-modified adenoviral vector yielded higher transduction efficiencies than conventional adenoviral vector at a multiplicity of infection (MOI) of 5 and 10. Yamashita et al. evaluated human laminin (HL) isotype coating for creating islet cell sheet. HL-332 was an optimal human-derived extracellular matrix (ECM) for coating temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm) surfaces. Miyamoto et al. showed a Tapered Stencil for Cluster Culture (TASCL) device developed to create liver spheroids in vitro. The TASCL device will be useful for application as a toxicity evaluation system for drug testing. Pillai et al. have achieved the fluorescence off state of quantum dots (QDs) by the conjugation of black hole quencher (BHQ) molecules intermediated with a peptide using streptavidin-QDs585 and biotin-pep-BHQ-1. The technology is expected to be useful as molecular imaging with on–off control of QD fluorescence. The theme of this JSOPMB issue is “Creating a Future of Transplantation.” The board members and I are looking forward to seeing further progress in JSOPMB in conjunction with Cell Medicine.

Choice of Feeders Is Important When First Establishing iPSCs Derived From Primarily Cultured Human Deciduous Tooth Dental Pulp Cells.

Feeder cells are generally required to maintain embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs). Mouse embryonic fibroblasts (MEFs) isolated from fetuses and STO mouse stromal cell line are the most widely used feeder cells. The aim of this study was to determine which cells are suitable for establishing iPSCs from human deciduous tooth dental pulp cells (HDDPCs). Primary cultures of HDDPCs were cotransfected with three plasmids containing human OCT3/4, SOX2/KLF4, or LMYC/LIN28 and pmaxGFP by using a novel electroporation method, and then cultured in an ESC qualified medium for 15 days. Emerging colonies were reseeded onto mitomycin C-treated MEFs or STO cells. The colonies were serially passaged for up to 26 passages. During this period, colony morphology was assessed to determine whether cells exhibited ESC-like morphology and alkaline phosphatase activity to evaluate the state of cellular reprogramming. HDDPCs maintained on MEFs were successfully reprogrammed into iPSCs, whereas those maintained on STO cells were not. Once established, the iPSCs were maintained on STO cells without loss of pluripotency. Our results indicate that MEFs are better feeder cells than STO cells for establishing iPSCs. Feeder choice is a key factor enabling efficient generation of iPSCs.

PiggyBac transposon-mediated gene delivery efficiently generates stable transfectants derived from cultured primary human deciduous tooth dental pulp cells (HDDPCs) and HDDPC-derived iPS cells.

The ability of human deciduous tooth dental pulp cells (HDDPCs) to differentiate into odontoblasts that generate mineralized tissue holds immense potential for therapeutic use in the field of tooth regenerative medicine. Realization of this potential depends on efficient and optimized protocols for the genetic manipulation of HDDPCs. In this study, we demonstrate the use of a PiggyBac (PB)-based gene transfer system as a method for introducing nonviral transposon DNA into HDDPCs and HDDPC-derived inducible pluripotent stem cells. The transfection efficiency of the PB-based system was significantly greater than previously reported for electroporation-based transfection of plasmid DNA. Using the neomycin resistance gene as a selection marker, HDDPCs were stably transfected at a rate nearly 40-fold higher than that achieved using conventional methods. Using this system, it was also possible to introduce two constructs simultaneously into a single cell. The resulting stable transfectants, expressing tdTomato and enhanced green fluorescent protein, exhibited both red and green fluorescence. The established cell line did not lose the acquired phenotype over three months of culture. Based on our results, we concluded that PB is superior to currently available methods for introducing plasmid DNA into HDDPCs. There may be significant challenges in the direct clinical application of this method for human dental tissue engineering due to safety risks and ethical concerns. However, the high level of transfection achieved with PB may have significant advantages in basic scientific research for dental tissue engineering applications, such as functional studies of genes and proteins. Furthermore, it is a useful tool for the isolation of genetically engineered HDDPC-derived stem cells for studies in tooth regenerative medicine.

Recruitment of mesenchymal stem cells by stromal cell-derived factor 1α in pulp cells from deciduous teeth.

Dental pulp cells (DPCs), including dental pulp (DP) stem cells, play a role in dentine repair under certain conditions caused by bacterial infections associated with caries, tooth fracture and injury. Mesenchymal stem cells (MSCs) have also been shown to be involved in this process of repair. However, the mechanisms through which MSCs are recruited to the DP have not yet been elucidated. Therefore, the aim of the present in vitro study was to investigate whether stromal cell-derived factor 1α (SDF1)-C-X-C chemokine receptor type 4 (CXCR4) signaling is involved in tissue repair in the DP of deciduous teeth. A single-cell clone from DPCs (SDP11) and UE7T-13 cells were used as pulp cells and MSCs, respectively. The MG-63 and HuO9 cells, two osteosarcoma cell lines, were used as positive control cells. Reverse transcription polymerase chain reaction (RT-PCR) revealed that all cell lines (SDP11, UE7T-13 MG-63 and HuO9) were positive for both SDF1 and CXCR4 mRNA expression. Moreover, immunocytochemical analysis indicated that SDF1 and CXCR4 proteins were expressed in the SDP11 and UE7T-13 cells. SDF1 was also detected in the cell lysates (CLs) and conditioned medium (CM) collected from the SDP11 and UE7T-13 cells, and AMD3100, a specific antagonist of CXCR4, inhibited the migration of the UE7T-13 cells; this migration was induced by treatment with CM, which was collected from the SDP11 cells. In addition, real-time PCR showed that the expression of SDF1 in the SDP11 cells was inhibited by treatment with 20 ng/ml fibroblast growth factor (FGF)-2, and exposure to AZD4547, an inhibitor of the FGF receptor, blocked this inhibition. Collectively, these data suggest that SDF1 produced by DP plays an important role in homeostasis, repair and regeneration via the recruitment of MSCs.

Isolation and Culture of Postnatal Stem Cells from Deciduous Teeth

Background: Currently, degenerative diseases represent a public health problem; therefore, the development and implementation of strategies to fully or partially recover of damaged tissues has a special interest in the biomedical field. Therapeutic strategies based on mesenchymal stem cells transplantation from dental pulp have been proposed as an alternative. Purpose: To develop a mesenchymal stem cells culture isolated from dental pulp of deciduous teeth. Methods: The mesenchymal stem cells isolation was performed from dental pulp of two deciduous canines, freshly extracted with therapeutic indication. Specimens were cultured in D-MEM medium with 20% FBS at 37 °C and 5% CO2. The medium was changed every three days. 80-90% confluence was achieved after three weeks. Cells were stained with DAPI and STRO-1 and analyzed through flow cytometry and fluorescence microscopy. Results: The analysis showed that adherent cells had a fusiform fibroblast-like morphology and colony forming units (CFUs) were observed. 17% of the whole population was STRO-1+ and 26% of the larger and more complex population was positive to this antigen. Additionally, STRO-1+ cells were localized preferential in UFCs. Conclusions: The protocol described here could be used to enriching mesenchymal stem cells from dental pulp extracted from deciduous teeth and as a starting point for future therapeutic trials and their application in tissue regeneration. KEYWORDS cultured cells; stem cells; deciduous teeth

Nanofiber Scaffolds Support Bone Regeneration Associated with Pulp Stem Cells

Currently, there are a number of alternatives for bone grafting, though when used correctly they present physical, chemical or biological limitations, which justifies the pursuit for new alternatives for bone regeneration. This study gives a report on the potential for bone regeneration in the use of biodegradable nanofibers from poly (lactic-co-glycolic acid) (PLGA) in association with human mesenchymal stem cells from dental pulp of deciduous teeth (SCDT). Five samples of SCDT were seeded with scaffolds (test) or without scaffolds (control) for cell adhesion and viability assay. To evaluate the ability of the association in promoting bone formation, critical defects were made in the calvarium of rats (n=20), which were then divided into the following groups: I--sham group; II--implant of scaffolds; III--scaffolds/ SCDT; and IV--scaffolds/SCDT. They were kept for 13 days in osteogenic media. After 60 days, the histomorphometric analysis was performed. It was observed that the adherence and viability of SCDT in the control and test group were similar throughout the experiment (p>0.05). The association of scaffolds/SCDT maintained in osteogenic media, showed greater bone formation than the other groups (p<0.05). The study demonstrated that the association of SCDT seeded in biodegradable PLGA scaffolds has the ability to promote bone regeneration in rats, which is a promising alternative for application in regenerative medicine.