Single cell RNA sequencing analysis of human dental pulp stem cells and human periodontal ligament stem cells

Abstract Background Magnesium (Mg2+)-enriched microenvironment promotes odontogenic differentiation in human dental pulp stem cells (DPSCs), but the regulatory mechanisms remain undefined. The aim of this work was to assess magnesium’s function in the above process and to explore the associated signaling pathway. Methods DPSCs underwent culture in odontogenic medium with the addition of 0, 1, 5, or 10 mM MgCl2. Intracellular Mg2+ levels in DPSCs were evaluated flow cytometrically using Mag-Fluo-4-AM. Mg2+-entry was inhibited by TRPM7 inhibitor 2-aminoethoxydiphenyl borate (2-APB). RNA-Sequencing was carried out for assessing transcriptome alterations in DPSCs during odontogenic differentiation associated with high extracellular Mg2+. KEGG pathway analysis was performed to determine pathways related to the retrieved differentially expressed genes (DEGs). Immunoblot was performed for assessing magnesium’s role and exploring ERK/BMP2/Smads signaling. Results Mg2+-enriched microenvironment promoted odontogenic differentiation in DPSCs via intracellular Mg2+ increase. Consistently, the positive effect of high extracellular Mg2+ on odontogenic differentiation in DPSCs was blocked by 2-APB, which reduced Mg2+ entry. RNA-sequencing identified 734 DEGs related to odontogenic differentiation in DPSCs in the presence of high extracellular Mg2+. These DEGs participated in many cascades such as MAPK and TGF-β pathways. Consistently, ERK and BMP2/Smads pathways were activated in DPSCs treated with high extracellular Mg2+. In agreement, ERK signaling inhibition by U0126 blunted the effect of high extracellular Mg2+ on mineralization and odontogenic differentiation in DPSCs. Interestingly, BMP2, BMPR1, and phosphorylated Smad1/5/9 were significantly decreased by U0126, indicating that BMP2/Smads acted as downstream of ERK. Conclusions Mg2+-enriched microenvironment promotes odontogenic differentiation in DPSCs by activating ERK/BMP2/Smads signaling via intracellular Mg2+ increase. This study revealed that Mg2+-enriched microenvironment could be used as a new strategy for dental pulp regeneration.

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Análise bibliométrica do uso de células-tronco em pesquisas odontológicas

ARCHIVES OF HEALTH INVESTIGATION ◽

10.21270/archi.v8i12.4790 ◽

2020 ◽

Vol 8 (12) ◽

Author(s):

Jéssica Gomes Alcoforado de Melo ◽

Diego Moura Soares

Keyword(s):

Stem Cells ◽

Dental Pulp ◽

Periodontal Ligament ◽

Dental Pulp Stem Cells ◽

Deciduous Teeth ◽

Level Laser ◽

Human Dental Pulp ◽

Low Level Laser Irradiation ◽

Human Exfoliated Deciduous Teeth

As pesquisas com células-tronco, seja de origem dental ou não, vêm crescendo na área da odontologia nos últimos anos em decorrência das possibilidades terapêuticas que a utilização desse tipo celular oferece. Este estudo visa demonstrar um panorama brasileiro das pesquisas com células-tronco realizadas no país por pesquisadores da área da odontologia nos anos de 2014 até 2018, com base nos anais de trabalhos apresentados nas Reuniões Anuais da Sociedade Brasileira de Pesquisa Odontológica (SBPqO). Foi analisado aspectos como tipo de instituição, se as pesquisas foram financiadas e qual a agencia de fomento, tipo de estudo, estado e região que desenvolveu a pesquisa, tipo de célula e fonte da célula-tronco utilizada. Foram analisados um total de 15,214 resumos, deste total 96 estudos foram incluídos por se enquadrarem com os critérios de inclusão. A região Sudeste foi responsável por 65,7% dessa produção. As pesquisas realizadas nas instituições estaduais representaram 42,7% da produção nacional e 59,4% dos trabalhos foram financiados. As células-tronco humanas foram o tipo mais utiilizado, especialmente as originadas da polpa dentária (25%). Conclui-se que há uma escassez da produção científica voltada para as células-tronco na odontologia, bem como a necessidade de descentralização dessa produção nas demais regiões brasileiras.Descritores: Células-Tronco; Odontologia; Pesquisa em Odontologia.ReferênciasGronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci USA.2000;97(25):13625-630.Freshney IR, Stacey GN, Aurebach JM. Culture of human stem cells: culture of specialized cells. New York: Wisley-Liss; 2007.Serakinci N, Keith WN. Therapeutic potential of adult stem cells. Eur J Cancer.2006;42(9):1243-46.Bianco P, Riminucci M, Gronthos S, Robey PG. Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells.. 2001;19(3):180-92.Mvula B, Mathope T, Moore T, Abrahamse H. The effect of low-level laser irradiation on adult human adipose-derived stem cells. Lasers Med Sci. 2008;23(3):277-82.Kern S, Eichler H, Stoeve J, Klüter H, Bieback K. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells. 2006; 24(5):1294-301.Slack JM. Stem cell in epithelial tissue. Science. 2000; 287:1431-33.Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG et al. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci USA.2003; 100(10):5807-12.Chen SC, Marino V, Gronthos S, Bartold PM. Location of putative stem cells in human periodontal ligament. J Periodontal Res. 2006; 41(6):547-53.Nuti N, Corallo C, Chan BMF, Ferrari M, Gerami-Naini B. Multipotent differentiation of human dental pulp stem cells: a literature review. Stem Cell Rev. 2016;12(5):511-523.Barboza CAG, Ginani F, Soares DM, Henrique ACG, Freitas RA. Low-level laser irradiation induces in vitro proliferation of mesenchymal stem cells. Einstein (São Paulo) 2014;12(1):75-81.Soares DM, Ginani F, Henriques AG, Barboza CAG. Effects of laser therapy on the proliferation of human periodontal ligament stem cells. Lasers Med Sci. 2015;30(3):1171-74.Ginani F, Soares DM, Rabêlo LM, Rocha HAO, Souza LB, Barboza CAG. Effect of a cryopreservation protocol on the proliferation of stem cells from human exfoliated deciduous teeth. Acta Odontol Scand. 2016;74(8):598-604.Maciel MMSA, Silva KBN, Melo JGA, Soares DM. Metodologia ativa aplicada ao ensino odontológico: um panorama nacional a partir de um estudo bibliométrico. Arch Health Invest. 2019;8(2):74-78.Melo NB, Fernandes Neto JA, Catão MHCV, Bento PM. Metodologia da Problematização e Aprendizagem Baseada em Problemas na Odontologia: análise bibliométrica dos trabalhos apresentados nas Reuniões da SBPqO. Revista da ABENO 2017;17(2):60-7.Xavier AFC, Silva ALO, Cavalcanti AL. Análise da produção científica em Odontologia no nordeste brasileiro com base em um congresso odontológico. Arq Odontol.2011;47(3):127-34.Aquino SN, Martelli DR, Bonan PRF, Laranjeira AL, Martelli Júnior H. Produção científica odontológica e relação com agências de financiamento de pesquisa. Arq Odontol. 2009; 45(3):142-46.Pontes KT, Silva EL, Macedo Filho RA, Silva DR, Lima FJ. Estudo bilbiométrico da produção científica em endodontia. Arch Health Invest. 2017;6(9):435-38.Soares DM, Maciel MMSA, Figueiredo-Filho A, Melo JGA. Brazilian scientific production in periodontics: a national panorama from a bibliometric study. Rev Clin Periodoncia Implantol Rehabil Oral. 2019;12(2):66-9.Taumaturgo VM, Vasques EFL, Figueiredo VMG. A Importância Da Odontologia Nas Pesquisas Em Células-Tronco. Rev Bahiana Odontol. 2016;7(2):166-71.Primo BT, Grazziotin-Soares R, Bertuzzi D, Claudy MP, Hernandez PAG, Fontanella VRC. Produção científica da ULBRA: análise do número e do delineamento das pesquisas publicadas nos suplementos da Brazilian Oral Research (SBPqO). Stomatos. 2010;16(31):69-76.Zorzanelli RT, Speroni AV, Menezes RA, Leibing A. Pesquisa com células-tronco no Brasil: a produção de um novo campo científico. Hist ciênc saúde-Manguinhos 2017;24(1):129-44.Lan X, Sun Z, Chu C, Boltze J, Li S. Dental Pulp Stem Cells: An Attractive Alternative for Cell Therapy in Ischemic Stroke. Front Neurol. 2019;10:824.Aydin S, Sahin F. Stem cells derived from dental tissues. Adv Exp Med Biol. 2019;1144:123-32.

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Comparison of the Expression of Periodontal Markers in Dental and Bone Marrow-derived Mesenchymal Stem Cells.

The Open Dentistry Journal ◽

10.2174/1874210602014010196 ◽

2020 ◽

Vol 14 (1) ◽

pp. 196-202

Author(s):

Devy Garna ◽

Manmeet Kaur ◽

Francis J Hughes ◽

Mandeep Ghuman

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Dental Pulp ◽

Periodontal Ligament ◽

Bone Marrow Cells ◽

Dental Pulp Stem Cells ◽

Periodontal Ligament Stem Cells ◽

Osteogenic Induction ◽

Pulp Cells

Background: Periodontal ligament stem cells are a source of mesenchymal stem cells, but it is unclear whether their phenotype is distinct from mesenchymal stem cells derived from different tissues, such as those derived from bone marrow. Objective: To investigate the expression of the putative PDL markers asporin, periostin, nestin and cementum protein 1, by periodontal ligament stem cells both constitutively and during osteogenic differentiation when compared to bone marrow-derived mesenchymal stem cells, and dental pulp stem cells. Methods: The primary human periodontal ligament, bone marrow, and dental pulp stem cells, and osteoblasts from different donors were cultured in vitro. The expression of periodontal marker associated genes during osteogenic induction was tested by qRT-PCR and immunofluorescence staining. Results: Asporin expression was detected in periodontal ligament stem cells and increased markedly during the time in culture (upregulated x53 fold at 21 days post-induction). During osteogenic differentiation, asporin expression significantly decreased in periodontal ligament cells whereas periostin significantly decreased in dental pulp cells. Periostin expression was absent in osteoblasts, but expression gradually increased in all other cells with time in culture. Nestin expression was mainly seen in the periodontal ligament and dental pulp cells and was largely absent in osteoblasts and bone marrow cells. Cementum protein-1 was most highly expressed in bone marrow cells and osteoblasts following osteogenic induction. Conclusions: The results provide further evidence that periodontal ligament-derived and bone marrow derived mesenchymal stem cells are phenotypically distinct. Periodontal markers are also expressed in dental pulp stem cells.

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Effects of co-culture of dental pulp stem cells and periodontal ligament stem cells on assembled dual disc scaffolds

Tissue Engineering and Regenerative Medicine ◽

10.1007/s13770-013-1109-6 ◽

2014 ◽

Vol 11 (1) ◽

pp. 47-58 ◽

Cited By ~ 3

Author(s):

Je-Duck Suh ◽

Ki Taek Lim ◽

Hexiu Jin ◽

Jangho Kim ◽

Pill-Hoon Choung ◽

...

Keyword(s):

Stem Cells ◽

Dental Pulp ◽

Periodontal Ligament ◽

Dental Pulp Stem Cells ◽

Periodontal Ligament Stem Cells

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Comparison of the differentiation of dental pulp stem cells and periodontal ligament stem cells into neuron-like cells and their effects on focal cerebral ischemia

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/gmaa082 ◽

2020 ◽

Vol 52 (9) ◽

pp. 1016-1029

Author(s):

Tingting Wu ◽

Wanting Xu ◽

Hanlin Chen ◽

Shasha Li ◽

Rengang Dou ◽

...

Keyword(s):

Stem Cells ◽

Ischemic Stroke ◽

Cerebral Ischemia ◽

Neuronal Differentiation ◽

Dental Pulp ◽

Periodontal Ligament ◽

Treated Group ◽

Dental Pulp Stem Cells ◽

Therapeutic Effects ◽

Periodontal Ligament Stem Cells

Abstract Recent studies have reported an increasing incidence of ischemic stroke, particularly in younger age groups. Dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) are the most common stem cells acquired from the teeth of adults, even elderly people. However, there are no detailed reports on whether DPSCs or PDLSCs are suitable for the treatment of ischemic stroke. In this study, the in vitro differentiation of DPSCs and PDLSCs into neuron-like cells was evaluated. Then, we established a rat model of cerebral ischemia. DPSCs or PDLSCs were administered to animals, and the therapeutic effects of these two types of cells were investigated. The results showed that PDLSCs had a higher differentiation rate than DPSCs. Immunofluorescence studies showed that the expression of the neuronal differentiation marker Thy-1 was higher in PDLSCs than in DPSCs, and other gene markers of neuronal differentiation showed corresponding trends, which were confirmed by western blot analysis. In this process, the Notch and Wnt signaling pathways were inhibited and activated, respectively. Finally, rats with transient occlusion of the right middle cerebral artery were used as a model to assess the therapeutic effect of PDLSCs and DPSCs on ischemia. The results showed that rats in the PDLSC-treated group emitted significantly greater red fluorescence signal than the DPSC-treated group. PDLSC transplantation promoted the recovery of neurological function more effectively than DPSC transplantation. Hence, PDLSCs represent an autogenous source of adult mesenchymal stem cells with desirable biological properties and may be an ideal candidate for clinical applications.

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Characterization of Adipose Stem Cells through Single Cell RNA‐sequencing Analysis

The FASEB Journal ◽

10.1096/fasebj.2021.35.s1.05297 ◽

2021 ◽

Vol 35 (S1) ◽

Author(s):

Hahn Nahmgoong ◽

Yong Geun Jeon ◽

Eun Seo Park ◽

Jong Kyoung Kim ◽

Jae Bum Kim

Keyword(s):

Stem Cells ◽

Single Cell ◽

Rna Sequencing ◽

Adipose Stem Cells ◽

Sequencing Analysis ◽

Single Cell Rna Sequencing

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Single-cell characterization of monolayer cultured human dental pulp stem cells with enhanced differentiation capacity

International Journal of Oral Science ◽

10.1038/s41368-021-00140-6 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Yu Cui ◽

Wei Ji ◽

Yongyan Gao ◽

Yao Xiao ◽

Huan Liu ◽

...

Keyword(s):

Stem Cells ◽

Single Cell ◽

Dental Pulp ◽

Dental Pulp Stem Cells ◽

Immunofluorescent Staining ◽

Tissue Formation ◽

Cellular Composition ◽

Functional Changes ◽

Human Dental Pulp

AbstractHuman dental pulp stem cells (hDPSCs) are easily obtained multipotent cells, however, their potential value in regenerative medicine is hindered by the phenotypic and functional changes after conventional monolayer expansion. Here, we employed single-cell RNA sequencing (scRNA-seq) to comprehensively study the transcriptional difference between the freshly isolated and monolayer cultured DPSCs. The cell cluster analysis based on our scRNA-seq data showed that monolayer culture resulted in a significant cellular composition switch compared to the freshly isolated DPSCs. However, one subpopulation, characterized as MCAM(+)JAG(+)PDGFRA(−), maintained the most transcriptional characteristics compared to their freshly isolated counterparts. Notably, immunofluorescent staining revealed that the MCAM(+)JAG(+)PDGFRA(−) hDPSCs uniquely located in the perivascular region of human dental pulp tissue. Flow-cytometry analysis confirmed that their proportion remained relatively stable (~2%) regardless of physiological senescence or dental caries. Consistent with the annotation of scRNA-seq data, MCAM(+)JAG(+)PDGFRA(−) hDPSCs showed higher proliferation capacity and enhanced in vitro multilineage differentiation potentials (osteogenic, chondrogenic and adipogenic) compared with their counterparts PDGFRA(+) subpopulation. Furthermore, the MCAM(+)JAG(+)PDGFRA(−) hDPSCs showed enhanced bone tissue formation and adipose tissue formation after 4-week subcutaneous implantation in nude mice. Taken together, our study for the first time revealed the cellular composition switch of monolayer cultured hDPSCs compared to the freshly isolated hDPSCs. After in vitro expansion, the MCAM(+)JAG(+)PDGFRA(−) subpopulation resembled the most transcriptional characteristics of fresh hDPSCs which may be beneficial for further tissue regeneration applications.

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