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Use of a 3D Floating Sphere Culture System to Maintain the Neural Crest-Related Properties of Human Dental Pulp Stem Cells.

ABSTRACT: Human dental pulp is considered an interesting source of adult stem cells, due to the low-invasive isolation procedures, high content of stem cells and its peculiar embryological origin from neural crest. Based on our previous findings, a dental pulp stem cells sub-population, enriched for the expression of STRO-1, c-Kit, and CD34, showed a higher neural commitment. However, their biological properties were compromised when cells were cultured in adherent standard conditions. The aim of this study was to evaluate the ability of three dimensional floating spheres to preserve embryological and biological properties of this sub-population. In addition, the expression of the inwardly rectifying potassium channel Kir4.1, Fas and FasL was investigated in 3D-sphere derived hDPSCs. Our data showed that 3D sphere-derived hDPSCs maintained their fibroblast-like morphology, preserved stemness markers expression and proliferative capability. The expression of neural crest markers and Kir4.1 was observed in undifferentiated hDPSCs, furthermore this culture system also preserved hDPSCs differentiation potential. The expression of Fas and FasL was observed in undifferentiated hDPSCs derived from sphere culture and, noteworthy, FasL was maintained even after the neurogenic commitment was reached, with a significantly higher expression compared to osteogenic and myogenic commitments. These data demonstrate that 3D sphere culture provides a favorable micro-environment for neural crest-derived hDPSCs to preserve their biological properties.

SUBMITTER: Pisciotta A

PROVIDER: S-EPMC5985438 | biostudies-other | 2018

REPOSITORIES: biostudies-other

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Use of a 3D Floating Sphere Culture System to Maintain the Neural Crest-Related Properties of Human Dental Pulp Stem Cells.

Pisciotta Alessandra A Bertoni Laura L Riccio Massimo M Mapelli Jonathan J Bigiani Albertino A La Noce Marcella M Orciani Monia M de Pol Anto A Carnevale Gianluca G

Frontiers in physiology 20180528

Human dental pulp is considered an interesting source of adult stem cells, due to the low-invasive isolation procedures, high content of stem cells and its peculiar embryological origin from neural crest. Based on our previous findings, a dental pulp stem cells sub-population, enriched for the expression of STRO-1, c-Kit, and CD34, showed a higher neural commitment. However, their biological properties were compromised when cells were cultured in adherent standard conditions. The aim of this stu ...[more]

PMID: 29892229

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Project description:Introduction: Dental pulp cells, a unique source of ectomesenchymal pluripotent stem cells, are originated from the skull neural crest. They are considered as one ideal source of cells for the regenerative medicine applications. Cerebrospinal fluid (CSF), a transparent fluid found in the brain and spinal cord, is enriched with electrolytes, proteins, and growth factors such as EGF, bFGF, BDNF, GDNF, and neuropeptides and can be utilized as a trigger in order to induce the neural differentiation. On the other hand, photobiomodulation (PBM), with the ability to prevent cell apoptosis, can induce cell proliferation by means of increasing the ATP synthesis in mitochondria and facilitating the secretion of the growth factors. In this research, we first aimed to isolate and culture the dental pulp stem cells (DPSCs) and subsequently to investigate their potential for neural differentiation. Methods: Human dental pulp stem cells (hDPSCs) were isolated from the pulp tissues using an outgrowth method and subsequently cultured. In order to access the cells' differentiation potential, cells were firstly classified into four groups which were treated with CSF, gallium aluminum arsenide diode laser irradiation (808 nm; 30 mW power output) and a combination of both, while the fourth group was considered as the control. MTT assay was then used to examine the viability of cells following the treatments. After 4, 7, and 14 days the cell morphology in the treated groups was evaluated while RT-PCR was used in order to evaluate the Nestin and β-tubulinIII neural gene marker expressions. Results: It was shown that PBM has the ability to elevate the proliferation of DPSCs. Also, the differentiated morphology was obvious in the CSF treated group, especially on day 14 with the formation of three-dimensional (3D) structures. The results of gene expression analysis showed that on the fourth day of post-treatment, Nestin and β-tubulinIII gene expressions were reduced in all groups while a rising trend in their expression was observed subsequently on days 7 and 14. Conclusion: In accordance with previous studies, including functional and protein base researches, it has been demonstrated that CSF has a direct role in neural induction. Although past works have been significant, none of them shows a 3D structure. In this article, we investigated the dual effect of PBM and CSF. Initial results confirmed the upregulation of neural-related transcription factors. The 3D organization of the formed tissue could imply the initiation of organogenesis which has not been reported before. In sum, the dual effect of CSF and PBM has been shown to have the potential for contributing to the initiation of neurogenesis and organogenesis.

Project description:BackgroundDental pulp stem cells (DPSCs) have been developed as a potential source of mesenchymal stem cells (MSCs) for regeneration of dental pulp and other tissues. However, further strategies to isolate highly functional DPSCs beyond the colony-forming methods are required. We have demonstrated the safety and efficacy of DPSCs isolated by G-CSF-induced mobilization and cultured under normoxia (mobilized DPSCs, MDPSCs) for pulp regeneration. The device for isolation of MDPSCs, however, is not cost-effective and requires a prolonged cell culture period. It is well known that MSCs cultured under hypoxic-preconditions improved MSC proliferation activity and stemness. Therefore, in this investigation, we attempted to improve the clinical utility of DPSCs by hypoxia-preconditioned DPSCs (hpDPSCs) compared with MDPSCs to improve the potential clinical utility for pulp regeneration in endodontic dentistry.MethodsColony-forming DPSCs were isolated and preconditioned with hypoxia in a stable closed cultured system and compared with MDPSCs isolated from the individual dog teeth. We examined the proliferation rate, migration potential, anti-apoptotic activity, and gene expression of the stem cell markers and angiogenic/neurotrophic factors. Trophic effects of the conditioned medium (CM) were also evaluated. In addition, the expression of immunomodulatory molecules upon stimulation with IFN-γ was investigated. The pulp regenerative potential and transplantation safety of hpDPSCs were further assessed in pulpectomized teeth in dogs by histological and immunohistochemical analyses and by chemistry of the blood and urine tests.ResultshpDPSCs demonstrated higher proliferation rate and expression of a major regulator of oxygen homeostasis, HIF-1α, and a stem cell marker, CXCR-4. The direct migratory activity of hpDPSCs in response to G-CSF was significantly higher than MDPSCs. The CM of hpDPSCs stimulated neurite extension. However, there were no changes in angiogenic, migration, and anti-apoptotic activities compared with the CM of MDPSCs. The expression of immunomodulatory gene, PTGE was significantly upregulated by IFN gamma in hpDPSCs compared with MDPSCs. However, no difference in nitric oxide was observed. The regenerated pulp tissue was quantitatively and qualitatively similar in hpDPSC transplants compared with MDPSC transplants in dog teeth. There was no evidence of toxicity or adverse events of the hpDPSC transplantation.ConclusionsThese results demonstrated that the efficacy of hpDPSCs for pulp regeneration was identical, although hpDPSCs improved stem cell properties compared to MDPSCs, suggesting their potential clinical utility for pulp regeneration.

Project description:Dental pulp stem cells (DPSCs) are increasingly being advocated for regenerative medicine-based therapies. However, significant heterogeneity in the genotypic/phenotypic properties of DPSC subpopulations exist, influencing their therapeutic potentials. As most studies have established DPSC heterogeneity using 2D culture approaches, we investigated whether heterogeneous DPSC proliferative and contraction/remodelling capabilities were further evident within 3D type I collagen gels in vitro. DPSC subpopulations were isolated from human third molars and identified as high/low proliferative and multipotent/unipotent, following in vitro culture expansion and population doubling (PD) analysis. High proliferative/multipotent DPSCs, such as A3 (30 PDs and 80 PDs), and low proliferative/unipotent DPSCs, such as A1 (17 PDs), were cultured in collagen gels for 12 days, either attached or detached from the surrounding culture plastic. Collagen architecture and high proliferative/multipotent DPSC morphologies were visualised by Scanning Electron Microscopy and FITC-phalloidin/Fluorescence Microscopy. DPSC proliferation (cell counts), contraction (% diameter reductions), and remodelling (MMP-2/MMP-9 gelatin zymography) of collagen gels were also evaluated. Unexpectedly, no proliferation differences existed between DPSCs, A3 (30 PDs) and A1 (17 PDs), although A3 (80 PDs) responses were significantly reduced. Despite rapid detached collagen gel contraction with A3 (30 PDs), similar contraction rates were determined with A1 (17 PDs), although A3 (80 PDs) contraction was significantly impaired. Gel contraction correlated to distinct gelatinase profiles. A3 (30 PDs) possessed superior MMP-9 and comparable MMP-2 activities to A1 (17 PDs), whereas A3 (80 PDs) had significantly reduced MMP-2/MMP-9. High proliferative/multipotent DPSCs, A3 (30 PDs), further exhibited fibroblast-like morphologies becoming polygonal within attached gels, whilst losing cytoskeletal organization and fibroblastic morphologies in detached gels. This study demonstrates that heterogeneity exists in the gel contraction and MMP expression/activity capabilities of DPSCs, potentially reflecting differences in their abilities to degrade biomaterial scaffolds and regulate cellular functions in 3D environments and their regenerative properties overall. Thus, such findings enhance our understanding of the molecular and phenotypic characteristics associated with high proliferative/multipotent DPSCs.

Dataset Information

Use of a 3D Floating Sphere Culture System to Maintain the Neural Crest-Related Properties of Human Dental Pulp Stem Cells.

Publications

Use of a 3D Floating Sphere Culture System to Maintain the Neural Crest-Related Properties of Human Dental Pulp Stem Cells.

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