Immunological isolation and characterization of neuronal progenitors from human dental pulp: A laboratory-based investigation

IF 5.4 1区医学 Q1 DENTISTRY, ORAL SURGERY & MEDICINE International endodontic journal Pub Date : 2024-05-07 DOI:10.1111/iej.14077

Hayley P. McMillan, Fionnuala T. Lundy, Orla M. Dunne, Kiran John McLoughlin, Imad About, T. M. Curtis, Ikhlas El Karim

{"title":"Immunological isolation and characterization of neuronal progenitors from human dental pulp: A laboratory-based investigation","authors":"Hayley P. McMillan, Fionnuala T. Lundy, Orla M. Dunne, Kiran John McLoughlin, Imad About, T. M. Curtis, Ikhlas El Karim","doi":"10.1111/iej.14077","DOIUrl":null,"url":null,"abstract":"<div>\n \n \n <section>\n \n <h3> Aims</h3>\n \n <p>Dental pulp stem cells (DPSCs) contain a population of stem cells with a broad range of differentiation potentials, as well as more lineage-committed progenitors. Such heterogeneity is a significant obstacle to experimental and clinical applications. The aim of this study is to isolate and characterize a homogenous neuronal progenitor cell population from human DPSCs.</p>\n </section>\n \n <section>\n \n <h3> Methodology</h3>\n \n <p>Polysialylated-neural cell adhesion molecule (PSA-NCAM+) neural progenitors were isolated from the dental pulp of three independent donors using magnetic-activated cell sorting (MACS) technology. Immunofluorescent staining with a panel of neural and non-neural markers was used to characterize the magnetically isolated PSA-NCAM+ fraction. PSA-NCAM+ cells were then cultured in Neurobasal A supplemented with neurotrophic factors: dibutyryl cyclic-AMP, neurotrophin-3, B27 and N2 supplements to induce neuronal differentiation. Both PSA-NCAM+ and differentiated PSA-NCAM+ cells were used in Ca<sup>2+</sup> imaging studies to assess the functionality of P2X3 receptors as well as membrane depolarization.</p>\n </section>\n \n <section>\n \n <h3> Results</h3>\n \n <p>PSA-NCAM+ neural progenitors were isolated from a heterogeneous population of hDPSCs using magnetic-activated cell sorting and anti-PSA-NCAM MicroBeads. Flow cytometry analysis demonstrated that immunomagnetic sorting significantly increased the purity of PSA-NCAM+ cells. Immunofluorescent staining revealed expression of pan-neuronal and mature neuronal markers, PGP9.5 and MAP2, respectively, as well as weak expression of the mature sensory markers, peripherin and islet1. ATP-induced response was mediated predominately by P2X3 receptors in both undifferentiated and differentiated cells, with a greater magnitude observed in the latter. In addition, membrane depolarizations were also detected in cells before and after differentiation when loaded with fast-voltage-responding fluorescent molecule, FluoVolt™ in response to potassium chloride. Interestingly, only differentiated PSA-NCAM+ cells were capable of spontaneous membrane oscillations.</p>\n </section>\n \n <section>\n \n <h3> Conclusions</h3>\n \n <p>In summary, DPSCs contain a population of neuronal progenitors with enhanced neural differentiation and functional neural-like properties that can be effectively isolated with magnetic-activated cell sorting (MACS).</p>\n </section>\n </div>","PeriodicalId":13724,"journal":{"name":"International endodontic journal","volume":null,"pages":null},"PeriodicalIF":5.4000,"publicationDate":"2024-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/iej.14077","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"International endodontic journal","FirstCategoryId":"3","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1111/iej.14077","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"DENTISTRY, ORAL SURGERY & MEDICINE","Score":null,"Total":0}

引用次数: 0

Abstract

Aims

Dental pulp stem cells (DPSCs) contain a population of stem cells with a broad range of differentiation potentials, as well as more lineage-committed progenitors. Such heterogeneity is a significant obstacle to experimental and clinical applications. The aim of this study is to isolate and characterize a homogenous neuronal progenitor cell population from human DPSCs.

Methodology

Polysialylated-neural cell adhesion molecule (PSA-NCAM+) neural progenitors were isolated from the dental pulp of three independent donors using magnetic-activated cell sorting (MACS) technology. Immunofluorescent staining with a panel of neural and non-neural markers was used to characterize the magnetically isolated PSA-NCAM+ fraction. PSA-NCAM+ cells were then cultured in Neurobasal A supplemented with neurotrophic factors: dibutyryl cyclic-AMP, neurotrophin-3, B27 and N2 supplements to induce neuronal differentiation. Both PSA-NCAM+ and differentiated PSA-NCAM+ cells were used in Ca²⁺ imaging studies to assess the functionality of P2X3 receptors as well as membrane depolarization.

Results

PSA-NCAM+ neural progenitors were isolated from a heterogeneous population of hDPSCs using magnetic-activated cell sorting and anti-PSA-NCAM MicroBeads. Flow cytometry analysis demonstrated that immunomagnetic sorting significantly increased the purity of PSA-NCAM+ cells. Immunofluorescent staining revealed expression of pan-neuronal and mature neuronal markers, PGP9.5 and MAP2, respectively, as well as weak expression of the mature sensory markers, peripherin and islet1. ATP-induced response was mediated predominately by P2X3 receptors in both undifferentiated and differentiated cells, with a greater magnitude observed in the latter. In addition, membrane depolarizations were also detected in cells before and after differentiation when loaded with fast-voltage-responding fluorescent molecule, FluoVolt™ in response to potassium chloride. Interestingly, only differentiated PSA-NCAM+ cells were capable of spontaneous membrane oscillations.

Conclusions

In summary, DPSCs contain a population of neuronal progenitors with enhanced neural differentiation and functional neural-like properties that can be effectively isolated with magnetic-activated cell sorting (MACS).

Abstract Image

查看原文

微信好友朋友圈 QQ好友复制链接

本刊更多论文

从人类牙髓中免疫学分离和鉴定神经元祖细胞：基于实验室的研究。

目的：牙髓干细胞（DPSCs）包含一群具有广泛分化潜能的干细胞，以及更多的系定向祖细胞。这种异质性是实验和临床应用的一大障碍。本研究旨在从人类 DPSCs 中分离出同源神经元祖细胞群并确定其特征：方法：使用磁激活细胞分拣（MACS）技术从三名独立供体的牙髓中分离出聚ialylated-神经细胞粘附分子（PSA-NCAM+）神经祖细胞。用一组神经和非神经标记物进行免疫荧光染色，以确定磁性分离的 PSA-NCAM+ 部分的特征。然后将 PSA-NCAM+ 细胞置于添加了神经营养因子（二丁烯环-AMP、神经营养素-3、B27 和 N2 补充剂）的 Neurobasal A 中培养，以诱导神经元分化。PSA-NCAM+细胞和分化的PSA-NCAM+细胞都被用于Ca2+成像研究，以评估P2X3受体的功能和膜去极化：结果：利用磁激活细胞分选和抗PSA-NCAM MicroBeads技术，从异质的hDPSCs群体中分离出了PSA-NCAM+神经祖细胞。流式细胞仪分析表明，免疫磁性分选显著提高了 PSA-NCAM+ 细胞的纯度。免疫荧光染色显示了泛神经元和成熟神经元标记物 PGP9.5 和 MAP2 的表达，以及成熟感觉标记物 peripherin 和 islet1 的微弱表达。在未分化细胞和分化细胞中，ATP 诱导的反应主要由 P2X3 受体介导，后者的反应幅度更大。此外，分化前后的细胞在氯化钾作用下装载快速电压反应荧光分子 FluoVolt™ 时也检测到膜去极化。有趣的是，只有分化后的 PSA-NCAM+ 细胞才能发生自发膜振荡：总之，DPSCs含有神经元祖细胞群，它们具有增强的神经分化能力和类似神经元的功能特性，可通过磁激活细胞分拣（MACS）有效地分离出来。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文去求助

来源期刊

International endodontic journal 医学-牙科与口腔外科

CiteScore

10.20

自引率

28.00%

发文量

195

审稿时长

4-8 weeks

期刊介绍： The International Endodontic Journal is published monthly and strives to publish original articles of the highest quality to disseminate scientific and clinical knowledge; all manuscripts are subjected to peer review. Original scientific articles are published in the areas of biomedical science, applied materials science, bioengineering, epidemiology and social science relevant to endodontic disease and its management, and to the restoration of root-treated teeth. In addition, review articles, reports of clinical cases, book reviews, summaries and abstracts of scientific meetings and news items are accepted. The International Endodontic Journal is essential reading for general dental practitioners, specialist endodontists, research, scientists and dental teachers.