Sequencing-based study of neural induction of human dental pulp stem cell

Sequencing-based study of neural induction of human dental pulp stem cell | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sequencing-based study of neural induction of human dental pulp stem cell Shohei Takaoka, Fumihiko Uchida, Hiroshi Ishikawa, Junko Toyomura, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4574156/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Aug, 2024 Read the published version in Human Cell → Version 1 posted 4 You are reading this latest preprint version Abstract Techniques for triggering neural differentiation of embryonic and induced pluripotent stem cells into neural stem cells and neurons have been established. However, neural induction in mesenchymal stem cells, including dental pulp stem cells (DPSCs), has been assessed primarily based on neural-related gene regulation, and detailed research into characteristics and differentiation status of cells is lacking. Therefore, this study aimed to evaluate the cellular components and differentiation pathways of neural lineage cells obtained via neural induction of human DPSCs. Human DPSCs were induced to neural cells in monolayer culture and examined for gene expression and mechanisms using microarray-based ingenuity pathway analysis. Additionally, the neural lineage cells were subjected to single-cell RNA sequencing (scRNA-seq) to classify cell populations based on gene expression profiles and elucidate their differentiation pathways. Ingenuity pathway analysis revealed that genes exhibiting marked post-neuronal induction overexpression, such as FABP7 and ZIC1 , were associated with neurogenesis. Furthermore, in canonical pathway analysis, axon guidance signals demonstrated maximum activation. The scRNA-seq and cell type annotations evidenced the presence of neural progenitor cells, astrocytes, neurons, and a small number of non-neural lineage cells. Moreover, trajectory and pseudotime analyses demonstrated that the neural progenitor cells initially engendered neurons, which subsequently differentiated into astrocytes. This result indicates that the aforementioned neural induction strategy generated neural stem/progenitor cells from DPSCs, which might differentiate and proliferate to constitute neural lineage cells. Therefore, neural induction of DPSCs may present an alternative approach to pluripotent stem cell-based therapeutic interventions for nervous system disorder. mesenchymal stem cell neural stem cells neurons astrocytes neural induction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Neural stem cells (NSCs) are essential in the central nervous system during neurogenesis, which occurs throughout embryonic development and adulthood [ 1 ]. NSCs possess the ability to undergo self-renewal and progenitor cell-mediated differentiation into neurons, astrocytes, and oligodendrocytes. This property facilitates NSC cultivation in vitro, making them extremely attractive for investigating neurogenesis and the progenitor-mediated development of various cell lineages. Moreover, NSC transplantation is presumed to exert therapeutic effects in neurodegenerative disorders, cerebrovascular diseases, and traumatic brain or spinal cord injuries via regeneration, repair, or enhancement of central nervous system functions [ 2 – 5 ]. Furthermore, NSCs substantially contribute to the establishment of disease models and drug and toxicity screening research [ 6 ]. Pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), can generate NSCs in vitro. Recently, a myriad of protocols, comprising monolayer culture, neural rosettes, and three-dimensional aggregate organoids, have been established for the neural induction of pluripotent stem cells [ 6 – 9 ]. NSC-derived neural lineage cells have been meticulously characterized by their gene expression profiles, and differentiated cells have been compared with cells in vivo [ 10 – 12 ]. However, the risk of tumorigenesis in iPSCs and ethical concerns associated with ESCs warrant alternative sources of pluripotent stem cells for their clinical application. Mesenchymal stem cells (MSCs), a class of multipotent stem cells, harbor the potential to differentiate into mesodermal cell lineages, namely chondrocytes, osteocytes, and adipocytes, as well as ectoderm- or endoderm-derived neural lineage cells and hepatocytes [ 13 – 15 ]. Predominantly, MSCs manifest an intrinsic expression profile encompassing a diverse array of NSC markers, including nestin, alongside neuron-specific markers, such as doublecortin and β3-tubulin, thereby establishing their neurogenic propensity [ 16 – 18 ]. Notably, dental pulp stem cells (DPSCs) exhibit heightened neurogenic capabilities, as evidenced by their augmented expression of NSC and neuron-specific markers, compared with other MSCs because of originating from neural crest cells [ 18 , 19 ]. Consequently, DPSCs are considered an optimal reservoir for neural induction in MSCs. Nonetheless, it is challenging to evaluate the efficacy of neural induction with the inherent expression of these neural markers in MSCs, including DPSCs, rendering the comprehensive characterization of cells post-neuronal induction elusive. The neural induction of DPSCs has also been primarily evaluated by increased expression of neural markers [ 19 – 21 ]; however, the differentiation status of the cells was not identified. This study aimed to investigate neural induction of human DPSCs and the associated gene expression profiling using single-cell RNA sequencing (scRNA-seq) and microarray-based ingenuity pathway analysis (IPA). This study also aimed to delineate neural lineage cells originating from human DPSCs by conducting a thorough assessment of gene expression profiles at the single-cell level and categorizing them within the established cell type dataset. The corresponding findings demonstrate that our neural induction protocol yields neural lineage cells, including neural progenitors, astrocytes, and neurons, along with a limited presence of non-neural lineage cells from human DPSCs. Materials and methods Isolation and culture of DPSCs This study was approved by the University of Tsukuba Clinical Research Ethics Review Committee (Approval Number: H29-173), and informed consent was received from the study participants. DPSCs were isolated following a previously described methodology [ 2 , 22 ]. Dental pulp tissue was acquired from teeth extracted from healthy patients at the Department of Oral and Maxillofacial Surgery, University of Tsukuba Hospital; sectioned into diminutive fragments; and subjected to static cultivation to procure migrating cells from each fragment. Subsequently, 1 × 10 3 single cells were inoculated into 10-cm cell culture dishes and cultured for approximately 10 days. The colonies exhibiting maximum proliferation were identified and used as DPSCs in subsequent experiments. The cultivation was sustained in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F12 (DMEM/F12; Thermo Fisher Scientific, Waltham, MA, USA), fortified with 10% fetal bovine serum (FBS; Sigma-Aldrich Corporation, St. Louis, MO, USA), 100 µM glutamate (GlutaMAX I; Thermo Fisher Scientific), 0.1% MEM non-essential amino acids (MEM-NEAA; Thermo Fisher Scientific), 50 U/mL penicillin and 50 µg/mL streptomycin (both from Fujifilm Wako Pure Chemicals Corporation, Tokyo, Japan), and 0.25 µg/mL fungizone (Cytiva, Marlborough, MA, USA), and incubated at 37°C in the presence of 4.7% CO 2 . The DPSCs were passaged and perpetuated in a 1:3 ratio. Neural induction of DPSCs The DPSCs were induced to neural cells following the methodology outlined by Takahashi et al. [ 23 ]. DPSCs were aliquoted (1 × 10 4 ) into 60-mm culture dishes, and the neural induction protocol was extended for approximately 2 weeks. The neural induction medium comprised DMEM/F12 supplemented with 5% FBS, 10 mM MEM-NEAAs, 10 nM all-trans retinoic acid (Sigma-Aldrich), 2 mM glutamate (Sigma-Aldrich), 50 mM ascorbic acid (Sigma-Aldrich), 5 mM insulin (Sigma-Aldrich), 10 nM dexamethasone (Sigma-Aldrich), 20 nM progesterone (Sigma-Aldrich), 20 nM estradiol (Sigma-Aldrich), 10 nM neural growth factor-1 (Sigma-Aldrich), 10 ng/mL thyroxine (Sigma-Aldrich), 50 U/mL penicillin, and 50 µg/mL streptomycin. Distinctive colonies exhibiting morphological divergence from the surrounding DPSCs were identified under a phase-contrast microscope and retrieved using filter paper soaked in a solution containing 0.1% trypsin and 0.02% EDTA/phosphate-buffered saline (−). The harvested colonies were subsequently cultured in a neurobasal medium supplemented with B27 (Thermo Fisher Scientific), 20 µg/mL basic fibroblast growth factor (PeproTech, Cranberry, PA, USA), and 20 µg/mL epidermal growth factor (PeproTech) and sustained in cell culture dishes coated with BD Matrigel® Basement Membrane (BD Biosciences, San Jose, CA, USA). The cell cultures were maintained under controlled environmental conditions of 37°C and 4.7% CO 2 . The nervous system cells were passaged at a 1:3 ratio and defined as neural-induced DPSCs (Ni-DPSCs). Microarray-dependent IPA DPSCs procured from the teeth of three participants and individual DPSC-derived Ni-DPSCs were subjected to comprehensive genetic analyses using the Human Clariom S Assay (Thermo Fisher Scientific). Data on these cell populations were processed using the Expression Console 1.3.1 software (Thermo Fisher Scientific). Quality control assessment was conducted using Transcriptome Analysis Console software version 3.1.0.5 (Thermo Fisher Scientific). The data were used to enlist biological functions in the Ingenuity KnowledgeBase and are publicly available in GEO (Accession Number: GSE168399). Fluctuations in gene expression were recorded, and the values were entered into the IPA software version 01-20-04 (QIAGEN, Hilden, Germany) for analyses. A causal network analysis was performed to identify the master regulatory factors presumably implicated in the observed alterations in gene expression. The z-score was used to predict the activation status of the canonical pathway. Library preparation and scRNA-seq Ni-DPSCs were cultured and sub-cultured for 2 and 3 days in vitro (DIV), respectively, before being subjected to scRNA-seq. The adherent cells were dissociated using trypsin, and following singlet processing, they were filtered using a 40-µm cell strainer. Cells were stained with trypan blue and observed under a microscope to assess cell counts and viability; single-cell suspensions with viability > 80% were employed for library preparation. Single-cell libraries were engineered in accordance with the 10× Genomics protocol (Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1; Dual Index; CG000315 Rev E) and sequenced using NovaSeq 6000 (Illumina Inc., San Diego, CA, USA). Analysis of scRNA-seq data Cell Ranger (Version 7.1.0, 10× Genomics, Pleasanton, CA, USA) was used to perform unique molecular identifier (UMI) quantification in adherence to the default and recommended parameters to fashion a filtered gene-barcode matrix for each sample. Low-quality cells (number of UMIs 20%) were screened using Seurat version 4.2.1 (R software). Doublet cells were detected and eliminated from the analysis based on the parameters recommended by DoubletFinder (R software). UMI counts were normalized and scaled using the “logNormalize” method of the NormalizeData function. The nonlinear dimension was reduced using RunUMAP with principal components. The unique characteristics of each individual cell community and cluster were discerned using the FindNeighbors and FindClusters functions in Seurat (R software). Differentially expressed gene markers in each cluster were identified using the FindAllMarkers function in Seurat (R software). To annotate all cell clusters, the marker genes of each cluster and cell types were compared using CellMarker 2.0 and SCSA (Version 1.0), respectively. Trajectory and pseudotime analyses were performed using STREAM (Version 1.0), wherein the state of the cell at the branching and endpoints of cell differentiation is indicated by S [number]. Immunofluorescence staining Please refer to the Supplementary Information for a detailed account of this procedure. Results Morphological evaluation of Ni-DPSCs The DPSCs were spindle shaped and morphologically similar to fibroblasts (Fig. 1a). Ni-DPSCs, composed of cells of various morphologies, both large and small, were obtained after neural induction (Fig. 1b). The Ni-DPSCs comprised numerous cell layers, with minute cells in the top layer (Fig. 1c). Additionally, small bipolar cells were discovered in the top layer (Fig. 1d). Figure 1 Morphology of neural-induced dental pulp stem cells (Ni-DPSCs). (a) Phase-contrast micrograph of the DPSCS. (b) Phase-contrast micrograph of Ni-DPSCs. (c) Phase-contrast micrograph of Ni-DPSCs at high magnification. Many small cells are observed in the top layer. (d) A bipolar neuron-like cell was located in the top layer. A bipolar cell is indicated by a white arrow Comparison between genes expressed in DPSCs and Ni-DPSCs using IPA Compared with DPSCs, the most upregulated gene in Ni-DPSCs was the NSC and astrocyte marker FABP7 . Additionally, the expression of astrocytic markers A2B , S100B , and ZIC1 , prominent regulators of neurogenesis, was considerably enhanced (Fig. 2a). Regarding molecular and cellular biological functions, proliferation, organization, and development during neural induction were shown. Neurodevelopment was demonstrated in physiological system development and function. Embryonic development was also observed (Fig. 2b). During the differentiation of DPSCs into Ni-DPSCs, beta-estradiol, which is important for embryonic and neural development, and SOX2, which is important for maintaining stemness in NSCs, were predicted to be upstream master regulators (Fig. 2c). Among the top five master regulators, SOX2 and HEXIM1 were predicted inhibitors. Canonical pathway analysis predicted that axon guidance signaling was the most prominent signal (Fig. 2d). Figure 2 IPA analysis of DPSCs vs. Ni-DPSCs. (a) Top 10 upregulated and downregulated genes between Ni-DPSCs and DPSCs. (b) Top five biological, molecular, and cellular functions, and physiological system development and functions identified through ingenuity pathway analysis (IPA). (c) Top five predicted master regulators that control the expression of genes in our datasets identified through the causal network analysis of IPA. Factors predicted to behave as inhibitors are shown in red. (d) Top 10 canonical pathways identified using IPA. “Ratio” is the number of differentially expressed genes that fit into each pathway relative to the overall number of genes in that pathway Identification of Ni-DPSC subpopulations and gene expression signatures To assess the diversity of Ni-DPSCs, scRNA-seq was performed according to the 10× Genomics transcriptomic protocol. The 13 clusters identified based on the expression of genes across the cell population of 7,193 cells were visualized by uniform manifold approximation and projection (UMAP), and five cell types—neural progenitor cells, astrocytes, neurons, epithelial cells, smooth muscle cells, fibroblasts, and mesenchymal cells—were identified according to the expression matrix of the marker genes (Fig. 3a). The population of each cluster comprised approximately 42% neural progenitor cells, 31% astrocytes, 11% epithelial and/or smooth muscle cells, 9% fibroblasts and/or mesenchymal cells, and 7% neurons (Fig. 3b). Based on the differential gene expression analysis, a heat map was generated using the top 10 marker genes for each identified cluster (Fig. 3c, Supplementary Table 1). The top two expressed genes in each cluster are shown in UMAP (Supplementary Fig. 1). Many genes were expressed across these clusters. GFAP is a gene characteristic of two astrocyte clusters (astrocytes 1 and 3). Figure 3 Single-cell RNA sequencing analysis of Ni-DPSCs. (a) Two-dimensional UMAP depicting single cells, colored to represent 13 different transcriptionally distinct clusters. (b) Population of each cluster; total is shown as 100%. (c) Gene expression heatmap of the top 10 characteristic genes for each cluster Trajectory and pseudotime analyses of Ni-DPSCs Before performing trajectory and pseudotime analyses, Ni-DPSC clusters were broadly classified into astrocytes, neural progenitor cells, neurons, and non-neuronal cells (Fig. 4a). To assess the differentiation into increasingly specialized cell subtypes, a trajectory analysis was performed (Fig. 4b). S1–S7 and S13 were branching points for cell differentiation. The differentiation pathways of neural progenitor cells, astrocytes, and non-neural lineage cells were S1–S and S8–11, while S7 and S12–S15 were inferred for the differentiation pathways of neurons from neural progenitor cells. Flat tree and subway map plots revealed that in a series of differentiations beginning with neural progenitors (S12), the neural progenitors differentiated into neurons (S14) and then differentiated into astrocytes. In the final stage of differentiation, astrocytes transformed into non-neural lineage cells (Fig. 4c, 4d). Astrocytes and neural progenitor cells changed into clusters as differentiation progressed, indicating that these multiple clusters could be classified according to the degree of differentiation (Fig. 4d). Figure 4 Trajectory inference and pseudotime analysis. (a) The 13 clusters are divided into four groups: non-neural lineage cell, astrocyte, neural progenitor cell, and neuron. (b) Trajectory inference of all single cells throughout differentiation reveals seven branches (S1, 2, 3, 6, 17, and 13). (c) Pseudotime flat tree map of each subpopulation generated by STREAM. From the blue plot to the red plot, the cellular hierarchy by pseudotime trajectory is shown. (d) Pathways of cell differentiation by pseudotime trajectories are shown for 13 clusters Assessment of neural-related marker proteins in cultured Ni-DPSCs Ni-DPSCs were characterized via immunocytochemical staining (Fig. 5). In many Ni-DPSCs, the NSC markers, Nestin, FABP7, and SOX2, as well as the glial cell marker GFAP, were expressed. Ki-67 is expressed in the nuclei of many cells, indicating active cell proliferation. However, the cells with small nuclei did not express Ki-67. The GFAP-positive cells were the neuronal progenitor cell marker DCX-positive cells at 2 DIV. Considerably developed MAP2-positive cells, a marker of mature neurons, were observed at 15 DIV. Additionally, αSMA-positive cells, a mesenchymal marker for smooth muscle cells, and fibroblasts were present. Figure 5 Assessment of the cultured Ni-DPSCs. Immunocytochemical staining of Ni-DPSCs at 2 days in vitro (DIV) with antibodies against Nestin, GAFP, SOX2, FABP7, Ki67, DCX, and αSMA. Ni-DPSCs at 15 DIV with antibodies against MAP2 Discussion Our neural induction protocol generated neural progenitor cells from human DPSCs and further demonstrated that neural progenitor cells differentiated into neurons and astrocytes. MSCs are stromal cells capable of self-renewal and differentiation into various cell types among adult stem cells, and their use is free of ethical concerns, teratoma development, and histocompatibility issues [ 24 ]. Furthermore, MSCs are attractive research targets because of their ease of extraction, isolation, and maintenance. Therefore, neural induction of MSCs has been widely attempted. Many neural-related marker genes and proteins are expressed or their expression increases when cultured under certain conditions [ 16 , 17 , 25 – 28 ]. Commonly used neural-related markers are the NSC markers (Nestin and SOX2), neural markers (β3-tubulin and NF200), and glial marker (GFAP). Recently, Gao et al. [ 24 ] reported on the induction of adipose stem cells toward neurons. This induction results in increased expression of neuron-associated proteins and electrophysiological activity. However, the morphology and localization of synaptic vesicles are not characteristic of neurons, suggesting that they may be differentiating into neurons. Karakaş et al. [ 29 ] reported the induction of bone marrow stem cell toward neurons. The induced cells were assessed for morphological changes, increases or decreases in neuro-related markers, and electrophysiological active. Furthermore, the induction of NSCs and oligodendrocytes from the MSCs of the human umbilical cord and placenta has been reported; however, only the expression of neuron-related markers and their increase or decrease were evaluated [ 30 ]. Thus, neural induction of MSCs is primarily assessed by the expression of neuron-related genes or proteins and their increase or decrease. However, MSCs are a heterozygous cell population, and there are many cells that originally express nestin as NSC markers, β3 tubulin as neuronal markers, and glial cell markers such as GFAP and A2B5 [ 16 – 18 ]. Additionally, MSCs have electrical activity originally [ 31 ]. Therefore, it is difficult to accurately determine the results of MSC neural induction. Gancheva et al. [ 32 ] collected RNA from whole cultured cells after neural induction of DPSCs and performed bioinformatics analysis along with transcriptomic analysis. After neural induction, cell type was restricted to the neuronal lineage but failed to show the stages of differentiation of cells. To accurately assess the characteristics and differentiation status of a cell, it is necessary to comprehensively evaluate gene expression in single cells. scRNA-seq is an approach used to elucidate RNA transcripts in individual cells and reveal the composition of different cell types and functions in highly complex tissues and cultured cells [ 33 ]. In the present study, single-cell analysis was used to accurately evaluate the results of our neural induction of DPSCs. Progenitor cells are intermediates between stem cells and differentiated cells; however, it is difficult to accurately distinguish multipotent NSCs from neural progenitors. In fact, differentiated cells do not differentiate directly from NSCs but differentiate through the progenitor cell stage [ 34 ]. Briefly, during neurogenesis, neuroepithelial cells release nascent neurons and differentiate into radial glia. Radial glia differentiate asymmetrically and generate neurons through progenitor cells. Furthermore, they convert into astrocytes [ 34 , 35 ]. In the trajectory inference and pseudotime analyses of Ni-DPSC differentiation in this study, the neural progenitor cells first gave rise to neurons and then differentiated into astrocytes. This is similar to the course of in vivo neurodevelopment, and our neural induction method may be a suitable protocol for generating neural progenitor cells similar to NSCs. scRNA-seq revealed that non-neuronal cells were present in Ni-DPSCs, and in the trajectory and pseudotime analyses, astrocytes differentiated into non-neural lineage cells. However, it is unlikely that the beyond-embryonic differentiation of astrocytes into mesenchymal cells occurs. Pseudotime analysis of STREAM was performed on the assumption that the clusters are in the same differentiation pathway. It is likely that differentiation from astrocytes to non-neuronal cells was indicated. There are two possible explanations for the presence of non-neural lineage cells. Early neural rosettes derived from human ESCs contain a mixture of non-neuronal cells [ 36 ]. A neural rosette is a structure found during the neuronal induction of a universal cell: radially organized columnar epithelial cells with a lumen in the center that resembles the cross-section of a developing neural tube [ 8 ]. In addition, neural rosettes are capable of generating neurons and glia and serve as a site for the proliferation and maintenance of NSCs and their maintenance. Considering retrospective differentiation, our neural induction may have produced cells with stem cell properties comparable with those of the early neural rosettes. Furthermore, the possibility of differentiation of a single cell must be considered. Kuroda et al. [ 37 ] identified cells with pluripotency in stromal cells and named them multilineage-differentiating stress-enduring (Muse) cells [ 37 , 38 ]. Muse cells present in DPSCs may gave rise to ectodermal neural lineages and mesodermal lineage cells. This study had certain limitations. First, DPSCs were isolated by initially culturing dental pulp component cells at a sparse concentration and subsequently selecting the largest colonies as the DPSC colonies [ 2 , 3 , 22 ]. Consequently, the possibility that DPSC colonies do not form from a single cell cannot be excluded, indicating that progenitor cells of alternative cell types may have contaminated the cell culture. Additionally, DPSCs represent a heterozygous cell population; nonetheless, the characteristics of the DPSCs used in the experiment were not stipulated. Technically, although it is difficult for current technology to specifically stimulate individual cells with predetermined traits, differentiation and proliferation simultaneously occur. Furthermore, future challenges lie in characterizing the DPSCs used in our experiments and ascertaining the DPSC characteristics most conducive to neural induction. In summary, the results revealed 13 clusters based on the expression of genes across the cell population, and five cell types were identified: neural progenitor cells, astrocytes, neurons, epithelial cells and/or smooth muscle cells, and fibroblasts and/or mesenchymal cells. Furthermore, pseudotime analysis showed that neural progenitor cells generated neurons, after which they differentiated into astrocytes. This study demonstrates the utility of scRNA-seq for the neural induction of MSCs. Ni-DPSCs have the potential to be an alternative option to pluripotent cells in cell-based therapies for neural diseases. Declarations Funding This work was supported in part by JSPS KAKENHI (Grant Number: JP22K21029, JP22K16035). Conflicts of interest The authors declare no potential conflicts of interest pertaining to the authorship and/or publication of this article. Ethics approval This study was approved by the University of Tsukuba Clinical Research Ethics Review Committee (Approval Number: H29-173). Informed consent Informed consent was received from the study participants. Author contributions S. Takaoka: Contributed to study conception and design, data acquisition and analysis, and manuscript drafting. F. Uchida: Contributed to the study conception and design, data analysis, and drafting of the manuscript. H. Ishikawa, E Ishikawa, and H. Bukawa: Contributed to the study conception and design, data analysis and interpretation, and drafting and critical revision of the manuscript. J. Toyomura, A. Ohyama, M. Hideaki, and K. Hirata: Contributed to data acquisition, analysis, interpretation, and drafting of the manuscript. A. Marushima, K. Yamagata T. Yanagawa, and Y. Matsumaru: Contributed to data interpretation and critical revision of the manuscript. K. I-Naomi and S. 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Supplementary Files ESM.docx Cite Share Download PDF Status: Published Journal Publication published 29 Aug, 2024 Read the published version in Human Cell → Version 1 posted Reviewers agreed at journal 19 Jun, 2024 Reviewers invited by journal 19 Jun, 2024 Editor assigned by journal 15 Jun, 2024 First submitted to journal 12 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4574156","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":316256432,"identity":"b786e338-5b2c-4dc3-8c27-780250b0ebb5","order_by":0,"name":"Shohei Takaoka","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYDACCTDJzMAPohIKiNVyAKhFsgGkxYAULQYHQDxitMjPbj7A/DHHOnHz+dWJHx4YMMjzix3Ar8XgzrEEhoPb0hO33Xi7WQLoMMOZsxMIaJHIMQBqOQzUcnYDSEuCwW0CWuRnQLVsnnF28w+itDDcgGrZwN+7jThbDG6kJRw4uy3deMYN3m0WCQYShP0iPyP54IPKbday/f1nN9/8UWEjzy9NyGEMoGgBAQmwSgnCyhGA/wApqkfBKBgFo2AkAQCAQ0nOEpKatQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-7690-9120","institution":"University of Tsukuba Faculty of Medicine: Tsukuba Daigaku Igaku Iryokei","correspondingAuthor":true,"prefix":"","firstName":"Shohei","middleName":"","lastName":"Takaoka","suffix":""},{"id":316256433,"identity":"cf2cc7d0-61ee-428c-bd13-bf24abae0a58","order_by":1,"name":"Fumihiko Uchida","email":"","orcid":"","institution":"Tsukuba Daigaku Igaku Iryokei","correspondingAuthor":false,"prefix":"","firstName":"Fumihiko","middleName":"","lastName":"Uchida","suffix":""},{"id":316256434,"identity":"5c9be76b-28a8-4cc3-82eb-a80052e84a59","order_by":2,"name":"Hiroshi Ishikawa","email":"","orcid":"","institution":"Tsukuba Daigaku Igaku Iryokei","correspondingAuthor":false,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Ishikawa","suffix":""},{"id":316256435,"identity":"1dd27fc5-1cc7-4f9d-9f4b-14fb032e42d2","order_by":3,"name":"Junko Toyomura","email":"","orcid":"","institution":"Tsukuba Daigaku Igaku Iryokei","correspondingAuthor":false,"prefix":"","firstName":"Junko","middleName":"","lastName":"Toyomura","suffix":""},{"id":316256436,"identity":"b9c727cc-d920-40de-af40-2bb597f44920","order_by":4,"name":"Akihiro Ohyama","email":"","orcid":"","institution":"Tsukuba Daigaku Igaku Iryokei","correspondingAuthor":false,"prefix":"","firstName":"Akihiro","middleName":"","lastName":"Ohyama","suffix":""},{"id":316256437,"identity":"a8bd8a32-e918-4609-acb0-81c45f30c862","order_by":5,"name":"Hideaki Matsumura","email":"","orcid":"","institution":"Tsukuba Daigaku Igaku Iryokei","correspondingAuthor":false,"prefix":"","firstName":"Hideaki","middleName":"","lastName":"Matsumura","suffix":""},{"id":316256438,"identity":"c1b0344c-24ba-44fc-8a64-094593d37cec","order_by":6,"name":"Koji Hiorata","email":"","orcid":"","institution":"Tsukuba Daigaku Igaku Iryokei","correspondingAuthor":false,"prefix":"","firstName":"Koji","middleName":"","lastName":"Hiorata","suffix":""},{"id":316256439,"identity":"80b50dec-d6b6-4830-9103-29b6fdac786e","order_by":7,"name":"Satoshi Fukuzawa","email":"","orcid":"","institution":"Tsukuba Daigaku Igaku Iryokei","correspondingAuthor":false,"prefix":"","firstName":"Satoshi","middleName":"","lastName":"Fukuzawa","suffix":""},{"id":316256440,"identity":"b5b9ea8c-a4e9-4c80-a91c-62850f6d1d70","order_by":8,"name":"Naomi Ishibashi Kanno","email":"","orcid":"","institution":"Tsukuba Daigaku Igaku Iryokei","correspondingAuthor":false,"prefix":"","firstName":"Naomi","middleName":"Ishibashi","lastName":"Kanno","suffix":""},{"id":316256441,"identity":"6dfaf2f5-7b35-4d44-aa10-7fe874fb9663","order_by":9,"name":"Aiki Marushima","email":"","orcid":"","institution":"Tsukuba Daigaku Igaku Iryokei","correspondingAuthor":false,"prefix":"","firstName":"Aiki","middleName":"","lastName":"Marushima","suffix":""},{"id":316256442,"identity":"3171fce4-33bb-4da4-9e78-2e8cd5b1f3d9","order_by":10,"name":"Kenji Yamagata","email":"","orcid":"","institution":"Tsukuba Daigaku Igaku Iryokei","correspondingAuthor":false,"prefix":"","firstName":"Kenji","middleName":"","lastName":"Yamagata","suffix":""},{"id":316256443,"identity":"6f9d65d9-0fee-423a-8ea6-fd56aa6ae1e3","order_by":11,"name":"Toru Yanagawa","email":"","orcid":"","institution":"Tsukuba Daigaku Igaku Iryokei","correspondingAuthor":false,"prefix":"","firstName":"Toru","middleName":"","lastName":"Yanagawa","suffix":""},{"id":316256444,"identity":"1116ee01-a872-4f63-b72e-6ce8c6132260","order_by":12,"name":"Yuji Matsumaru","email":"","orcid":"","institution":"Tsukuba Daigaku Igaku Iryokei","correspondingAuthor":false,"prefix":"","firstName":"Yuji","middleName":"","lastName":"Matsumaru","suffix":""},{"id":316256445,"identity":"146d75bf-3ded-4ef8-8ffb-d2a0a2723e44","order_by":13,"name":"Eiichi Ishikawa","email":"","orcid":"","institution":"Tsukuba Daigaku Igaku Iryokei","correspondingAuthor":false,"prefix":"","firstName":"Eiichi","middleName":"","lastName":"Ishikawa","suffix":""},{"id":316256446,"identity":"a99a3545-eff6-433e-807d-e0410034dc0b","order_by":14,"name":"Hiroki Bukawa","email":"","orcid":"","institution":"Tsukuba Daigaku Igaku Iryokei","correspondingAuthor":false,"prefix":"","firstName":"Hiroki","middleName":"","lastName":"Bukawa","suffix":""}],"badges":[],"createdAt":"2024-06-13 07:06:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4574156/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4574156/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s13577-024-01121-7","type":"published","date":"2024-08-29T15:58:05+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59744432,"identity":"ca832c26-15e1-4174-a283-a4508b2a26e7","added_by":"auto","created_at":"2024-07-05 16:23:21","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1359006,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphology of neural-induced dental pulp stem cells (Ni-DPSCs). \u003c/strong\u003e(a) Phase-contrast micrograph of the DPSCS. (b) Phase-contrast micrograph of Ni-DPSCs. (c) Phase-contrast micrograph of Ni-DPSCs at high magnification. Many small cells are observed in the top layer. (d) A bipolar neuron-like cell was located in the top layer. A bipolar cell is indicated by a white arrow\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4574156/v1/e5964dad537da04e30861deb.jpg"},{"id":59744426,"identity":"c78370d9-9703-44db-b6a2-56e23dd14308","added_by":"auto","created_at":"2024-07-05 16:23:21","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":531708,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIPA analysis of DPSCs vs. Ni-DPSCs. \u003c/strong\u003e(a) Top 10 upregulated and downregulated genes between Ni-DPSCs and DPSCs. (b) Top five biological, molecular, and cellular functions, and physiological system development and functions identified through ingenuity pathway analysis (IPA). (c) Top five predicted master regulators that control the expression of genes in our datasets identified through the causal network analysis of IPA. Factors predicted to behave as inhibitors are shown in red. (d) Top 10 canonical pathways identified using IPA. “Ratio” is the number of differentially expressed genes that fit into each pathway relative to the overall number of genes in that pathway\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4574156/v1/799b36750540540730188918.jpg"},{"id":59744624,"identity":"b714d6ba-4e98-492a-bdc1-9864709a215e","added_by":"auto","created_at":"2024-07-05 16:31:21","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1028185,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle-cell RNA sequencing analysis of Ni-DPSCs. \u003c/strong\u003e(a) Two-dimensional UMAP depicting single cells, colored to represent 13 different transcriptionally distinct clusters. (b) Population of each cluster; total is shown as 100%. (c) Gene expression heatmap of the top 10 characteristic genes for each cluster\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4574156/v1/13773a0965fdfde1070b7f56.jpg"},{"id":59744427,"identity":"a398fa09-dc24-48d7-bd53-d9c97761aa94","added_by":"auto","created_at":"2024-07-05 16:23:21","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1585841,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTrajectory inference and pseudotime analysis. \u003c/strong\u003e(a) The 13 clusters are divided into four groups: non-neural lineage cell, astrocyte, neural progenitor cell, and neuron. (b) Trajectory inference of all single cells throughout differentiation reveals seven branches (S1, 2, 3, 6, 17, and 13). (c) Pseudotime flat tree map of each subpopulation generated by STREAM. From the blue plot to the red plot, the cellular hierarchy by pseudotime trajectory is shown. (d) Pathways of cell differentiation by pseudotime trajectories are shown for 13 clusters\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4574156/v1/ac4060fa561c33317c1797c1.jpg"},{"id":59744429,"identity":"a1cb2d11-43cf-452f-8ec3-ad2d7f19571b","added_by":"auto","created_at":"2024-07-05 16:23:21","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2197451,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of the cultured Ni-DPSCs. \u003c/strong\u003eImmunocytochemical staining of Ni-DPSCs at 2 days in vitro (DIV) with antibodies againstNestin, GAFP, SOX2, FABP7, Ki67, DCX, and αSMA. Ni-DPSCs at 15 DIV with antibodies against MAP2\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4574156/v1/3755715e9b7b5f1cf0c3aba5.jpg"},{"id":63821117,"identity":"0405b0c4-330e-4986-b866-1e1772e8fd6b","added_by":"auto","created_at":"2024-09-02 16:12:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7300010,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4574156/v1/beab17ca-ed41-4066-9efa-ae0d0876c905.pdf"},{"id":59744430,"identity":"5a49f475-600e-4c80-879e-4b4e00cfd41a","added_by":"auto","created_at":"2024-07-05 16:23:21","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":594931,"visible":true,"origin":"","legend":"","description":"","filename":"ESM.docx","url":"https://assets-eu.researchsquare.com/files/rs-4574156/v1/884978bded39af4afb51ac13.docx"}],"financialInterests":"","formattedTitle":"Sequencing-based study of neural induction of human dental pulp stem cell","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNeural stem cells (NSCs) are essential in the central nervous system during neurogenesis, which occurs throughout embryonic development and adulthood [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. NSCs possess the ability to undergo self-renewal and progenitor cell-mediated differentiation into neurons, astrocytes, and oligodendrocytes. This property facilitates NSC cultivation in vitro, making them extremely attractive for investigating neurogenesis and the progenitor-mediated development of various cell lineages. Moreover, NSC transplantation is presumed to exert therapeutic effects in neurodegenerative disorders, cerebrovascular diseases, and traumatic brain or spinal cord injuries via regeneration, repair, or enhancement of central nervous system functions [\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Furthermore, NSCs substantially contribute to the establishment of disease models and drug and toxicity screening research [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), can generate NSCs in vitro. Recently, a myriad of protocols, comprising monolayer culture, neural rosettes, and three-dimensional aggregate organoids, have been established for the neural induction of pluripotent stem cells [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. NSC-derived neural lineage cells have been meticulously characterized by their gene expression profiles, and differentiated cells have been compared with cells in vivo [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, the risk of tumorigenesis in iPSCs and ethical concerns associated with ESCs warrant alternative sources of pluripotent stem cells for their clinical application.\u003c/p\u003e \u003cp\u003eMesenchymal stem cells (MSCs), a class of multipotent stem cells, harbor the potential to differentiate into mesodermal cell lineages, namely chondrocytes, osteocytes, and adipocytes, as well as ectoderm- or endoderm-derived neural lineage cells and hepatocytes [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Predominantly, MSCs manifest an intrinsic expression profile encompassing a diverse array of NSC markers, including nestin, alongside neuron-specific markers, such as doublecortin and β3-tubulin, thereby establishing their neurogenic propensity [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Notably, dental pulp stem cells (DPSCs) exhibit heightened neurogenic capabilities, as evidenced by their augmented expression of NSC and neuron-specific markers, compared with other MSCs because of originating from neural crest cells [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Consequently, DPSCs are considered an optimal reservoir for neural induction in MSCs. Nonetheless, it is challenging to evaluate the efficacy of neural induction with the inherent expression of these neural markers in MSCs, including DPSCs, rendering the comprehensive characterization of cells post-neuronal induction elusive. The neural induction of DPSCs has also been primarily evaluated by increased expression of neural markers [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]; however, the differentiation status of the cells was not identified.\u003c/p\u003e \u003cp\u003eThis study aimed to investigate neural induction of human DPSCs and the associated gene expression profiling using single-cell RNA sequencing (scRNA-seq) and microarray-based ingenuity pathway analysis (IPA). This study also aimed to delineate neural lineage cells originating from human DPSCs by conducting a thorough assessment of gene expression profiles at the single-cell level and categorizing them within the established cell type dataset. The corresponding findings demonstrate that our neural induction protocol yields neural lineage cells, including neural progenitors, astrocytes, and neurons, along with a limited presence of non-neural lineage cells from human DPSCs.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and culture of DPSCs\u003c/h2\u003e \u003cp\u003eThis study was approved by the University of Tsukuba Clinical Research Ethics Review Committee (Approval Number: H29-173), and informed consent was received from the study participants. DPSCs were isolated following a previously described methodology [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Dental pulp tissue was acquired from teeth extracted from healthy patients at the Department of Oral and Maxillofacial Surgery, University of Tsukuba Hospital; sectioned into diminutive fragments; and subjected to static cultivation to procure migrating cells from each fragment. Subsequently, 1 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e single cells were inoculated into 10-cm cell culture dishes and cultured for approximately 10 days. The colonies exhibiting maximum proliferation were identified and used as DPSCs in subsequent experiments. The cultivation was sustained in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium/Ham\u0026rsquo;s nutrient mixture F12 (DMEM/F12; Thermo Fisher Scientific, Waltham, MA, USA), fortified with 10% fetal bovine serum (FBS; Sigma-Aldrich Corporation, St. Louis, MO, USA), 100 \u0026micro;M glutamate (GlutaMAX I; Thermo Fisher Scientific), 0.1% MEM non-essential amino acids (MEM-NEAA; Thermo Fisher Scientific), 50 U/mL penicillin and 50 \u0026micro;g/mL streptomycin (both from Fujifilm Wako Pure Chemicals Corporation, Tokyo, Japan), and 0.25 \u0026micro;g/mL fungizone (Cytiva, Marlborough, MA, USA), and incubated at 37\u0026deg;C in the presence of 4.7% CO\u003csub\u003e2\u003c/sub\u003e. The DPSCs were passaged and perpetuated in a 1:3 ratio.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eNeural induction of DPSCs\u003c/h2\u003e \u003cp\u003eThe DPSCs were induced to neural cells following the methodology outlined by Takahashi et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. DPSCs were aliquoted (1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e) into 60-mm culture dishes, and the neural induction protocol was extended for approximately 2 weeks. The neural induction medium comprised DMEM/F12 supplemented with 5% FBS, 10 mM MEM-NEAAs, 10 nM all-trans retinoic acid (Sigma-Aldrich), 2 mM glutamate (Sigma-Aldrich), 50 mM ascorbic acid (Sigma-Aldrich), 5 mM insulin (Sigma-Aldrich), 10 nM dexamethasone (Sigma-Aldrich), 20 nM progesterone (Sigma-Aldrich), 20 nM estradiol (Sigma-Aldrich), 10 nM neural growth factor-1 (Sigma-Aldrich), 10 ng/mL thyroxine (Sigma-Aldrich), 50 U/mL penicillin, and 50 \u0026micro;g/mL streptomycin. Distinctive colonies exhibiting morphological divergence from the surrounding DPSCs were identified under a phase-contrast microscope and retrieved using filter paper soaked in a solution containing 0.1% trypsin and 0.02% EDTA/phosphate-buffered saline (\u0026minus;). The harvested colonies were subsequently cultured in a neurobasal medium supplemented with B27 (Thermo Fisher Scientific), 20 \u0026micro;g/mL basic fibroblast growth factor (PeproTech, Cranberry, PA, USA), and 20 \u0026micro;g/mL epidermal growth factor (PeproTech) and sustained in cell culture dishes coated with BD Matrigel\u0026reg; Basement Membrane (BD Biosciences, San Jose, CA, USA). The cell cultures were maintained under controlled environmental conditions of 37\u0026deg;C and 4.7% CO\u003csub\u003e2\u003c/sub\u003e. The nervous system cells were passaged at a 1:3 ratio and defined as neural-induced DPSCs (Ni-DPSCs).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMicroarray-dependent IPA\u003c/h2\u003e \u003cp\u003eDPSCs procured from the teeth of three participants and individual DPSC-derived Ni-DPSCs were subjected to comprehensive genetic analyses using the Human Clariom S Assay (Thermo Fisher Scientific). Data on these cell populations were processed using the Expression Console 1.3.1 software (Thermo Fisher Scientific). Quality control assessment was conducted using Transcriptome Analysis Console software version 3.1.0.5 (Thermo Fisher Scientific). The data were used to enlist biological functions in the Ingenuity KnowledgeBase and are publicly available in GEO (Accession Number: GSE168399). Fluctuations in gene expression were recorded, and the values were entered into the IPA software version 01-20-04 (QIAGEN, Hilden, Germany) for analyses. A causal network analysis was performed to identify the master regulatory factors presumably implicated in the observed alterations in gene expression. The z-score was used to predict the activation status of the canonical pathway.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eLibrary preparation and scRNA-seq\u003c/h2\u003e \u003cp\u003eNi-DPSCs were cultured and sub-cultured for 2 and 3 days in vitro (DIV), respectively, before being subjected to scRNA-seq.\u0026nbsp;The adherent cells were dissociated using trypsin, and following singlet processing, they were filtered using a 40-\u0026micro;m cell strainer. Cells were stained with trypan blue and observed under a microscope to assess cell counts and viability; single-cell suspensions with viability\u0026thinsp;\u0026gt;\u0026thinsp;80% were employed for library preparation. Single-cell libraries were engineered in accordance with the 10\u0026times; Genomics protocol (Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1; Dual Index; CG000315 Rev E) and sequenced using NovaSeq 6000 (Illumina Inc., San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of scRNA-seq data\u003c/h2\u003e \u003cp\u003eCell Ranger (Version 7.1.0, 10\u0026times; Genomics, Pleasanton, CA, USA) was used to perform unique molecular identifier (UMI) quantification in adherence to the default and recommended parameters to fashion a filtered gene-barcode matrix for each sample. Low-quality cells (number of UMIs\u0026thinsp;\u0026lt;\u0026thinsp;10,000 and percentage of mitochondrial reads\u0026thinsp;\u0026gt;\u0026thinsp;20%) were screened using Seurat version 4.2.1 (R software). Doublet cells were detected and eliminated from the analysis based on the parameters recommended by DoubletFinder (R software). UMI counts were normalized and scaled using the \u0026ldquo;logNormalize\u0026rdquo; method of the NormalizeData function. The nonlinear dimension was reduced using RunUMAP with principal components. The unique characteristics of each individual cell community and cluster were discerned using the FindNeighbors and FindClusters functions in Seurat (R software). Differentially expressed gene markers in each cluster were identified using the FindAllMarkers function in Seurat (R software). To annotate all cell clusters, the marker genes of each cluster and cell types were compared using CellMarker 2.0 and SCSA (Version 1.0), respectively. Trajectory and pseudotime analyses were performed using STREAM (Version 1.0), wherein the state of the cell at the branching and endpoints of cell differentiation is indicated by S [number].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e \u003cp\u003ePlease refer to the Supplementary Information for a detailed account of this procedure.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMorphological evaluation of Ni-DPSCs\u003c/h2\u003e \u003cp\u003eThe DPSCs were spindle shaped and morphologically similar to fibroblasts (Fig.\u0026nbsp;1a). Ni-DPSCs, composed of cells of various morphologies, both large and small, were obtained after neural induction (Fig.\u0026nbsp;1b). The Ni-DPSCs comprised numerous cell layers, with minute cells in the top layer (Fig.\u0026nbsp;1c). Additionally, small bipolar cells were discovered in the top layer (Fig.\u0026nbsp;1d).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;1 Morphology of neural-induced dental pulp stem cells (Ni-DPSCs).\u003c/b\u003e (a) Phase-contrast micrograph of the DPSCS. (b) Phase-contrast micrograph of Ni-DPSCs. (c) Phase-contrast micrograph of Ni-DPSCs at high magnification. Many small cells are observed in the top layer. (d) A bipolar neuron-like cell was located in the top layer. A bipolar cell is indicated by a white arrow\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eComparison between genes expressed in DPSCs and Ni-DPSCs using IPA\u003c/h2\u003e \u003cp\u003eCompared with DPSCs, the most upregulated gene in Ni-DPSCs was the NSC and astrocyte marker \u003cem\u003eFABP7\u003c/em\u003e. Additionally, the expression of astrocytic markers \u003cem\u003eA2B\u003c/em\u003e, \u003cem\u003eS100B\u003c/em\u003e, and \u003cem\u003eZIC1\u003c/em\u003e, prominent regulators of neurogenesis, was considerably enhanced (Fig.\u0026nbsp;2a). Regarding molecular and cellular biological functions, proliferation, organization, and development during neural induction were shown. Neurodevelopment was demonstrated in physiological system development and function. Embryonic development was also observed (Fig.\u0026nbsp;2b). During the differentiation of DPSCs into Ni-DPSCs, beta-estradiol, which is important for embryonic and neural development, and SOX2, which is important for maintaining stemness in NSCs, were predicted to be upstream master regulators (Fig.\u0026nbsp;2c). Among the top five master regulators, SOX2 and HEXIM1 were predicted inhibitors. Canonical pathway analysis predicted that axon guidance signaling was the most prominent signal (Fig.\u0026nbsp;2d).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;2 IPA analysis of DPSCs vs. Ni-DPSCs.\u003c/b\u003e (a) Top 10 upregulated and downregulated genes between Ni-DPSCs and DPSCs. (b) Top five biological, molecular, and cellular functions, and physiological system development and functions identified through ingenuity pathway analysis (IPA). (c) Top five predicted master regulators that control the expression of genes in our datasets identified through the causal network analysis of IPA. Factors predicted to behave as inhibitors are shown in red. (d) Top 10 canonical pathways identified using IPA. \u0026ldquo;Ratio\u0026rdquo; is the number of differentially expressed genes that fit into each pathway relative to the overall number of genes in that pathway\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of Ni-DPSC subpopulations and gene expression signatures\u003c/h2\u003e \u003cp\u003eTo assess the diversity of Ni-DPSCs, scRNA-seq was performed according to the 10\u0026times; Genomics transcriptomic protocol. The 13 clusters identified based on the expression of genes across the cell population of 7,193 cells were visualized by uniform manifold approximation and projection (UMAP), and five cell types\u0026mdash;neural progenitor cells, astrocytes, neurons, epithelial cells, smooth muscle cells, fibroblasts, and mesenchymal cells\u0026mdash;were identified according to the expression matrix of the marker genes (Fig.\u0026nbsp;3a). The population of each cluster comprised approximately 42% neural progenitor cells, 31% astrocytes, 11% epithelial and/or smooth muscle cells, 9% fibroblasts and/or mesenchymal cells, and 7% neurons (Fig.\u0026nbsp;3b). Based on the differential gene expression analysis, a heat map was generated using the top 10 marker genes for each identified cluster (Fig.\u0026nbsp;3c, Supplementary Table\u0026nbsp;1). The top two expressed genes in each cluster are shown in UMAP (Supplementary Fig.\u0026nbsp;1). Many genes were expressed across these clusters. \u003cem\u003eGFAP\u003c/em\u003e is a gene characteristic of two astrocyte clusters (astrocytes 1 and 3).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;3 Single-cell RNA sequencing analysis of Ni-DPSCs.\u003c/b\u003e (a) Two-dimensional UMAP depicting single cells, colored to represent 13 different transcriptionally distinct clusters. (b) Population of each cluster; total is shown as 100%. (c) Gene expression heatmap of the top 10 characteristic genes for each cluster\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTrajectory and pseudotime analyses of Ni-DPSCs\u003c/h2\u003e \u003cp\u003eBefore performing trajectory and pseudotime analyses, Ni-DPSC clusters were broadly classified into astrocytes, neural progenitor cells, neurons, and non-neuronal cells (Fig.\u0026nbsp;4a). To assess the differentiation into increasingly specialized cell subtypes, a trajectory analysis was performed (Fig.\u0026nbsp;4b). S1\u0026ndash;S7 and S13 were branching points for cell differentiation. The differentiation pathways of neural progenitor cells, astrocytes, and non-neural lineage cells were S1\u0026ndash;S and S8\u0026ndash;11, while S7 and S12\u0026ndash;S15 were inferred for the differentiation pathways of neurons from neural progenitor cells. Flat tree and subway map plots revealed that in a series of differentiations beginning with neural progenitors (S12), the neural progenitors differentiated into neurons (S14) and then differentiated into astrocytes. In the final stage of differentiation, astrocytes transformed into non-neural lineage cells (Fig.\u0026nbsp;4c, 4d). Astrocytes and neural progenitor cells changed into clusters as differentiation progressed, indicating that these multiple clusters could be classified according to the degree of differentiation (Fig.\u0026nbsp;4d).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;4 Trajectory inference and pseudotime analysis.\u003c/b\u003e (a) The 13 clusters are divided into four groups: non-neural lineage cell, astrocyte, neural progenitor cell, and neuron. (b) Trajectory inference of all single cells throughout differentiation reveals seven branches (S1, 2, 3, 6, 17, and 13). (c) Pseudotime flat tree map of each subpopulation generated by STREAM. From the blue plot to the red plot, the cellular hierarchy by pseudotime trajectory is shown. (d) Pathways of cell differentiation by pseudotime trajectories are shown for 13 clusters\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of neural-related marker proteins in cultured Ni-DPSCs\u003c/h2\u003e \u003cp\u003eNi-DPSCs were characterized via immunocytochemical staining (Fig.\u0026nbsp;5). In many Ni-DPSCs, the NSC markers, Nestin, FABP7, and SOX2, as well as the glial cell marker GFAP, were expressed. Ki-67 is expressed in the nuclei of many cells, indicating active cell proliferation. However, the cells with small nuclei did not express Ki-67. The GFAP-positive cells were the neuronal progenitor cell marker DCX-positive cells at 2 DIV. Considerably developed MAP2-positive cells, a marker of mature neurons, were observed at 15 DIV. Additionally, αSMA-positive cells, a mesenchymal marker for smooth muscle cells, and fibroblasts were present.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;5 Assessment of the cultured Ni-DPSCs.\u003c/b\u003e Immunocytochemical staining of Ni-DPSCs at 2 days in vitro (DIV) with antibodies against Nestin, GAFP, SOX2, FABP7, Ki67, DCX, and αSMA. Ni-DPSCs at 15 DIV with antibodies against MAP2\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur neural induction protocol generated neural progenitor cells from human DPSCs and further demonstrated that neural progenitor cells differentiated into neurons and astrocytes. MSCs are stromal cells capable of self-renewal and differentiation into various cell types among adult stem cells, and their use is free of ethical concerns, teratoma development, and histocompatibility issues [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Furthermore, MSCs are attractive research targets because of their ease of extraction, isolation, and maintenance. Therefore, neural induction of MSCs has been widely attempted. Many neural-related marker genes and proteins are expressed or their expression increases when cultured under certain conditions [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Commonly used neural-related markers are the NSC markers (Nestin and SOX2), neural markers (β3-tubulin and NF200), and glial marker (GFAP). Recently, Gao et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] reported on the induction of adipose stem cells toward neurons. This induction results in increased expression of neuron-associated proteins and electrophysiological activity. However, the morphology and localization of synaptic vesicles are not characteristic of neurons, suggesting that they may be differentiating into neurons.\u003c/p\u003e \u003cp\u003eKarakaş et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] reported the induction of bone marrow stem cell toward neurons. The induced cells were assessed for morphological changes, increases or decreases in neuro-related markers, and electrophysiological active. Furthermore, the induction of NSCs and oligodendrocytes from the MSCs of the human umbilical cord and placenta has been reported; however, only the expression of neuron-related markers and their increase or decrease were evaluated [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Thus, neural induction of MSCs is primarily assessed by the expression of neuron-related genes or proteins and their increase or decrease. However, MSCs are a heterozygous cell population, and there are many cells that originally express nestin as NSC markers, β3 tubulin as neuronal markers, and glial cell markers such as GFAP and A2B5 [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Additionally, MSCs have electrical activity originally [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Therefore, it is difficult to accurately determine the results of MSC neural induction.\u003c/p\u003e \u003cp\u003eGancheva et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] collected RNA from whole cultured cells after neural induction of DPSCs and performed bioinformatics analysis along with transcriptomic analysis. After neural induction, cell type was restricted to the neuronal lineage but failed to show the stages of differentiation of cells. To accurately assess the characteristics and differentiation status of a cell, it is necessary to comprehensively evaluate gene expression in single cells. scRNA-seq is an approach used to elucidate RNA transcripts in individual cells and reveal the composition of different cell types and functions in highly complex tissues and cultured cells [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In the present study, single-cell analysis was used to accurately evaluate the results of our neural induction of DPSCs.\u003c/p\u003e \u003cp\u003eProgenitor cells are intermediates between stem cells and differentiated cells; however, it is difficult to accurately distinguish multipotent NSCs from neural progenitors. In fact, differentiated cells do not differentiate directly from NSCs but differentiate through the progenitor cell stage [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Briefly, during neurogenesis, neuroepithelial cells release nascent neurons and differentiate into radial glia. Radial glia differentiate asymmetrically and generate neurons through progenitor cells. Furthermore, they convert into astrocytes [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In the trajectory inference and pseudotime analyses of Ni-DPSC differentiation in this study, the neural progenitor cells first gave rise to neurons and then differentiated into astrocytes. This is similar to the course of in vivo neurodevelopment, and our neural induction method may be a suitable protocol for generating neural progenitor cells similar to NSCs.\u003c/p\u003e \u003cp\u003escRNA-seq revealed that non-neuronal cells were present in Ni-DPSCs, and in the trajectory and pseudotime analyses, astrocytes differentiated into non-neural lineage cells. However, it is unlikely that the beyond-embryonic differentiation of astrocytes into mesenchymal cells occurs. Pseudotime analysis of STREAM was performed on the assumption that the clusters are in the same differentiation pathway. It is likely that differentiation from astrocytes to non-neuronal cells was indicated. There are two possible explanations for the presence of non-neural lineage cells. Early neural rosettes derived from human ESCs contain a mixture of non-neuronal cells [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. A neural rosette is a structure found during the neuronal induction of a universal cell: radially organized columnar epithelial cells with a lumen in the center that resembles the cross-section of a developing neural tube [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In addition, neural rosettes are capable of generating neurons and glia and serve as a site for the proliferation and maintenance of NSCs and their maintenance. Considering retrospective differentiation, our neural induction may have produced cells with stem cell properties comparable with those of the early neural rosettes. Furthermore, the possibility of differentiation of a single cell must be considered. Kuroda et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] identified cells with pluripotency in stromal cells and named them multilineage-differentiating stress-enduring (Muse) cells [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Muse cells present in DPSCs may gave rise to ectodermal neural lineages and mesodermal lineage cells.\u003c/p\u003e \u003cp\u003eThis study had certain limitations. First, DPSCs were isolated by initially culturing dental pulp component cells at a sparse concentration and subsequently selecting the largest colonies as the DPSC colonies [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Consequently, the possibility that DPSC colonies do not form from a single cell cannot be excluded, indicating that progenitor cells of alternative cell types may have contaminated the cell culture. Additionally, DPSCs represent a heterozygous cell population; nonetheless, the characteristics of the DPSCs used in the experiment were not stipulated. Technically, although it is difficult for current technology to specifically stimulate individual cells with predetermined traits, differentiation and proliferation simultaneously occur. Furthermore, future challenges lie in characterizing the DPSCs used in our experiments and ascertaining the DPSC characteristics most conducive to neural induction.\u003c/p\u003e \u003cp\u003eIn summary, the results revealed 13 clusters based on the expression of genes across the cell population, and five cell types were identified: neural progenitor cells, astrocytes, neurons, epithelial cells and/or smooth muscle cells, and fibroblasts and/or mesenchymal cells. Furthermore, pseudotime analysis showed that neural progenitor cells generated neurons, after which they differentiated into astrocytes. This study demonstrates the utility of scRNA-seq for the neural induction of MSCs. Ni-DPSCs have the potential to be an alternative option to pluripotent cells in cell-based therapies for neural diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported in part by JSPS KAKENHI (Grant Number: JP22K21029, JP22K16035).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest pertaining to the authorship and/or publication of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the University of Tsukuba Clinical Research Ethics Review Committee (Approval Number: H29-173).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was received from the study participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS. Takaoka: Contributed to study conception and design, data acquisition and analysis, and manuscript\u0026nbsp;drafting.\u003c/p\u003e\n\u003cp\u003eF. Uchida: Contributed to the study conception and design, data analysis, and drafting of the manuscript.\u003c/p\u003e\n\u003cp\u003eH. Ishikawa, E Ishikawa, and H. Bukawa: Contributed to the study conception and design, data analysis and interpretation, and drafting and critical revision of the manuscript.\u003c/p\u003e\n\u003cp\u003eJ. Toyomura, A. Ohyama, M. Hideaki, and K. Hirata: Contributed to data acquisition, analysis, interpretation, and drafting of the manuscript.\u003c/p\u003e\n\u003cp\u003eA. Marushima, K. Yamagata T. Yanagawa, and Y. Matsumaru: Contributed to data interpretation and critical revision of the manuscript.\u003c/p\u003e\n\u003cp\u003eK. I-Naomi and S. Fukuzawa: Contributed to data analysis and interpretation and critical revision of the manuscript.\u003c/p\u003e\n\u003cp\u003eAll authors have approved the final version of the manuscript and agreed to be accountable for all aspects of the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJohansson CB, Momma S, Clarke DL, Risling M, Lendahl U, Fris\u0026eacute;n J. Identification of a neural stem cell in the adult mammalian central nervous system. Cell. 1999;96:25\u0026ndash;34.\u003c/li\u003e\n\u003cli\u003eHirata K, Marushima A, Nagasaki Y, et al. Efficacy of redox nanoparticles for improving survival of transplanted cells in a mouse model of ischemic stroke. Hum Cell. 2023;36:1703\u0026ndash;15.\u003c/li\u003e\n\u003cli\u003eMatsumura H, Marushima A, Ishikawa H, et al. Induced neural cells from human dental pulp ameliorate functional recovery in a murine model of cerebral infarction. 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Cell Transplant. 2016;25:849\u0026ndash;61.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"human-cell","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huce","sideBox":"Learn more about [Human Cell](http://link.springer.com/journal/13577)","snPcode":"13577","submissionUrl":"https://www.editorialmanager.com/huce/default2.aspx","title":"Human Cell","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"mesenchymal stem cell, neural stem cells, neurons, astrocytes, neural induction","lastPublishedDoi":"10.21203/rs.3.rs-4574156/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4574156/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTechniques for triggering neural differentiation of embryonic and induced pluripotent stem cells into neural stem cells and neurons have been established. However, neural induction in mesenchymal stem cells, including dental pulp stem cells (DPSCs), has been assessed primarily based on neural-related gene regulation, and detailed research into characteristics and differentiation status of cells is lacking. Therefore, this study aimed to evaluate the cellular components and differentiation pathways of neural lineage cells obtained via neural induction of human DPSCs. Human DPSCs were induced to neural cells in monolayer culture and examined for gene expression and mechanisms using microarray-based ingenuity pathway analysis. Additionally, the neural lineage cells were subjected to single-cell RNA sequencing (scRNA-seq) to classify cell populations based on gene expression profiles and elucidate their differentiation pathways. Ingenuity pathway analysis revealed that genes exhibiting marked post-neuronal induction overexpression, such as \u003cem\u003eFABP7 \u003c/em\u003eand \u003cem\u003eZIC1\u003c/em\u003e, were associated with neurogenesis. Furthermore, in canonical pathway analysis, axon guidance signals demonstrated maximum activation. The scRNA-seq and cell type annotations evidenced the presence of neural progenitor cells, astrocytes, neurons, and a small number of non-neural lineage cells. Moreover, trajectory and pseudotime analyses demonstrated that the neural progenitor cells initially engendered neurons, which subsequently differentiated into astrocytes. This result indicates that the aforementioned neural induction strategy generated neural stem/progenitor cells from DPSCs, which might differentiate and proliferate to constitute neural lineage cells. Therefore, neural induction of DPSCs may present an alternative approach to pluripotent stem cell-based therapeutic interventions for nervous system disorder.\u003c/p\u003e","manuscriptTitle":"Sequencing-based study of neural induction of human dental pulp stem cell","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-05 16:23:16","doi":"10.21203/rs.3.rs-4574156/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-06-19T12:01:41+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-19T07:20:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-15T08:29:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Human Cell","date":"2024-06-13T03:06:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"human-cell","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huce","sideBox":"Learn more about [Human Cell](http://link.springer.com/journal/13577)","snPcode":"13577","submissionUrl":"https://www.editorialmanager.com/huce/default2.aspx","title":"Human Cell","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ba311312-5f22-4fb4-a782-9fb944e10020","owner":[],"postedDate":"July 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-02T16:04:26+00:00","versionOfRecord":{"articleIdentity":"rs-4574156","link":"https://doi.org/10.1007/s13577-024-01121-7","journal":{"identity":"human-cell","isVorOnly":false,"title":"Human Cell"},"publishedOn":"2024-08-29 15:58:05","publishedOnDateReadable":"August 29th, 2024"},"versionCreatedAt":"2024-07-05 16:23:16","video":"","vorDoi":"10.1007/s13577-024-01121-7","vorDoiUrl":"https://doi.org/10.1007/s13577-024-01121-7","workflowStages":[]},"version":"v1","identity":"rs-4574156","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4574156","identity":"rs-4574156","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}