Single‐cell RNA‐seq of in vitro expanded cells from cranial neural crest reveals a rare odontogenic sub‐population

Abstract Ecto‐mesenchymal cells of mammalian tooth germ develops from cranial neural crest cells. These cells are recognised as a promising source for tooth development and regeneration. Despite the high heterogeneity of the neural crest, the cellular landscape of in vitro cultured cranial neural crest cells (CNCCs) for odontogenesis remains unclear. In this study, we used large‐scale single‐cell RNA sequencing to analyse the cellular landscape of in vitro cultured mouse CNCCs for odontogenesis. We revealed distinct cell trajectories from primary cells to passage 5 and identified a rare Alx3+/Barx1+ sub‐population in primary CNCCs that differentiated into two odontogenic clusters characterised by the up‐regulation of Pax9/Bmp3 and Lhx6/Dmp1. We successfully induced whole tooth‐like structures containing enamel, dentin, and pulp under the mouse renal capsule using in vitro cultured cells from both cranial and trunk neural crests with induction rates of 26.7% and 22.1%, respectively. Importantly, we confirmed only cells sorted from odontogenic path can induce tooth‐like structures. Cell cycle and DNA replication genes were concomitantly upregulated in the cultured NCCs of the tooth induction groups. Our data provide valuable insights into the cell heterogeneity of in vitro cultured CNCCs and their potential as a source for tooth regeneration.


| INTRODUCTION
Tooth regeneration is a promising strategy to restore the function and aesthetics of missing or damaged teeth.7][8][9] However, one of the major challenges is to obtain sufficient and suitable mesenchymal cells that can interact with epithelial cells to form tooth-like structures.Neural crest cells (NCCs) are multipotent stem cells that give rise to various craniofacial tissues, including the dental mesenchyme.Therefore, NCCs are expected as promising mesenchymal cell sources for tooth repair and regeneration.
1][12][13][14] In mice, the developing notochord, along with adjacent mesenchyme induces the ectoderm on its surface to form neural plate at around embryonic days 6 and 7 (E6 and E7), which gradually closes to form neural tube. 15[18] According to the in vivo localisations, neural crest can be divided into four types: cranial, cardiac, trunk and vagal NCCs.They can migrate and differentiate under the regulation of various signalling pathways, such as TGF-beta and Wnt signalling pathways, and eventually form different tissues and organs.It is worth mentioning that in the process of craniofacial formation, a small group of CNCCs can differentiate into dental mesenchymal cells under the influence of surrounding environment and extracellular signals. 19,20Therefore, cranial neural crest is recognised as the promising cell resource for tooth regeneration in vitro.
2][23] Besides, pluripotent cells derived neural crest-like cells were considered as a promising cell source for tooth regeneration. 24,25The dental markers could be activated in the induced neural crest-like cells derived from pluripotent stem cells by recombination with dental epithelium or in bioengineering materials.However, no successful regeneration of whole tooth with structures of dental pulp, dentin, and more importantly the enamel derived from neural crest-interacting non-odontogenic epithelium (other than E10.5-E12.5 dental epithelium) from neural crest-like cells has been reported yet.Meanwhile, scientists found that the dermal denticles on the back of cartilaginous fish have similar structures of enamel, dentin and pulp in teeth. 26Dermal denticles on the back are proved to be derived from the migration and differentiation of trunk neural crest (TNC), indicating the trunk neural crest cells (TNCCs) may have the odontogenesis potential as those from the cranial neural crest.
The NCCs become highly heterogeneous during the migration and differentiation. 27The dorsal neural tube, representing the origin area of NCCs, consists of several sub-populations with heterogeneous expression patterns containing neural and neural crest markers, pluripotency factors, differentiation markers and genes associated with cell proliferation or cell death.Recently, scientists have applied single-cell RNA sequencing to demonstrate the notable heterogeneity within the CNCC and TNCC in vivo, and their cell fate transitions during the embryonic development. 28,29However, the cellular heterogeneity of in vitro cultured CNCCs as the cell resource for tooth induction remains unclear.
In this study, we performed large-scale single-cell RNA sequencing ($59,000 cells) to demonstrate the cell trajectories of in vitro cultured and expanded mouse CNC cells from primary cells to passage 5.A rare sub-population in primary CNCCs is identified to differentiate from odontogenic clusters.Further, we applied SENIC analysis to identify the key factors to drive the odontogenic cell differentiation.Moreover, combined with post-bud dental epithelium, for the first time, we successfully induced the whole tooth-like structures in mouse kidney using in vitro cultured cells from both CNC and TNC.Cell cycle, DNA replication, and Wnt signalling pathway genes are upregulated for successful tooth induction.These findings might contribute to further studies of NCCs as a promising cell resource for tooth repair/regeneration.
dissected from the distal head between two red lines (Figure 1A, left), followed by removal of the surrounding tissues to keep the area of the light red square only (Figure 1A, middle).At E9.5, the TNC develops to a more mature state than the CNC, showing fusion of the neural folds on both sides with the appearance of closed neural tube and roof plate in the trunk part.However, the fusion of the neural folds just starts with the existence of neural groove in the cranial part (Figure 1A, middle and right).We isolated the TNCCs as well from the distal back which are denoted as ganglion 6-11 and showed between two blue lines in Figure 1A.The area of light blue square in TNC was prepared for further cell culturing (Figure 1A, middle, Figure S1A).Epithelial surface marker E-cad, neural progenitor cell marker PAX3 and NCC markers P75 were selected for immunofluorescence to suggest the in vivo localisations of the isolated neural tube (Figure 1A, right).
As expected, E-cad-positive cells could be generally detected in almost all cells of the dissected region of CNC and TNC, displaying in both neural tubes/folds/grooves and its surrounding region.Positive PAX3 expression is present in most cells and absent in a small number of cells around the neural folds (arrowheads) with surrounding thin layers in the CNC and roof plate (arrowheads) in the TNC.In particular, most P75 positive cells are found to slightly display surrounding the neural tube in CNC and widely spread surrounding the neural tube in TNC.These data indicate that the NCCs are migrating and undergoing the epithelial-mesenchymal transition (EMT), which confirms that migrating NCCs are isolated and cultured in vitro with neural tubes/folds in our study.With explant-attached method, we cultured and passaged the cells on Matrigel-coated plates with a slightly modified culture medium from the previous report, 30 containing N2, B27, bFGF, EGF in DMEM/F12 based medium (Figure 1B) (detailed in Section 4.2).In detail, cells climbed out from the cultured neural tubes at day 1 of passage 0 (P0D1).The morphologies of those cells implied their heterogeneities; some cells were spindle-shaped, while some cells were irregular quadrilateral.Neural tube and miscellaneous cells were removed at P0D3, and then the fully proliferated primary cells at P0D6 were passaged and cultured until P5.At P3, the cells showed a variety of shapes, including irregular quadrilateral, long spindle, triangle and circular.Besides, the cultured cells further displayed two types clearly, spindle-shaped or ellipse-shaped at P5.In addition, a small amount of nerve filaments (arrowheads) could be observed in some spindle-shaped cells (Figure 1B, Figure S1B), indicating part of cells at P5 differentiate towards neural lineages.
To further identify the heterogeneity of primary CNCCs, we performed single-cell RNA sequencing (scRNA-seq) of P0 cells using 10Â genomics platform.A total number of 5569 cells were detected.
Strikingly, a rare sub-population of Alx3 + /Barx1 + cells was identified in P0 primary CNCCs (Figure 1C left for cell type clustering, Figure 1C right for the gene expression levels of Alx3 and Barx1, respectively).Crabp1, Tfap2b, Prrx1 and Fli1 were also specifically expressed in this subset of cells (Figure 1D).According to our previous study, Alx3 is the critical transcriptional factor in incisor mesenchyme, while Barx1 and Tfap2b are crucial in molar mesenchyme. 31Moreover, Crabp1 and Fli1 were previously reported to be expressed in dental papilla. 32,33Furthermore, in situ expression of Alx3 was observed in cranial neural crest, while six genes (Ap2b, Pax9, Prrx1, Barx1, Crabp1 and Fli1) were observed in both CNC and TNC (Figure 1E).
We also examined the expression patterns of key genes for neural ①: Otx2 + NCCs could lead to Otx2 + /Igfbpl1 + axons; ② and ③: Sox8 + NCCs could lead to Sox2 + /Sox10 À NPC and osteogenic cells, respectively; ④: Alx3 + /Barx1 + NCCs could lead to Barx1 + odontogenic cells.Based on the dynamics of cell differentiation paths, the genes associated with lineage were clearly classified (Figure 2B bottom, Figure S2A).Furthermore, we performed pseudo-time analysis to draw the gene expression levels in four cell differentiation paths (Figure 2C): odontogenic genes, Barx1, Pax9 and Alx3, were expressed but low in early stage, and subsequently high upregulated in Barx1 + odontogenic path.Those factors are critical in E12.5-E13.5 dental mesenchymal cells, according to our previous report. 31Bmp3 was silenced at the early stage and activated in Barx1 + odontogenic cells.
Moreover, Lhx6 and Dmp1 were silenced at the early stage and expressed in Barx1 À odontogenic cells.Collagen genes (Col1a1, Col3a1 and Col4a1) and osteogenic genes (Xist, Dcn, H19 and Ogn) were also activated in odontogenic paths (illustrated as heat map in Figure 2D, and UMAP visualisations in Figure S2A).On the other hand, neural markers were dynamic during the multilineage landscape in neural paths.Astrocyte genes were activated in the late stage of the cell differentiations (Figure 2D, Figure S2A).Besides, novel genes specifically expressed in each cell type were shown in our scRNA-seq analysis (Figure 2E).
We further applied SCENIC analysis to gain insight into the gene regulatory networks (GRNs) and inferred critical transcription factors (TFs) for odontogenic differentiation of in vitro cultured CNCCs.
SCENIC can predict TFs, jointly named as regulons, through coexpression patterns and cis-regulatory motif analysis, along with their candidate target genes. 35Two GRNs, Prrx1 and Trap2b, were identified to drive the differentiation lineage specifically from Alx3 + /Barx1 + NCCs to Barx1 + odontogenic cells (Figure S2B,C).There were 32 common genes between Prrx1-GRN and Tfap2b-GRN, which related to Wnt signalling pathway and osteoblast differentiation according to gene ontology analysis.We analysed the expression dynamics of the potential targets of Prrx1-GRN and Trap2b-GRN during the NCCs to odontogenic cell transition.We used a heat map to visualise the scaled expression levels of these genes in the Barx1+ lineage along the pseudo-time (Figure S2D).The downstream genes of Prrx1-GRN were mostly active at the late stage and were involved in biological processes related to bone formation and tooth development.These In summary, there are two distinct developmental trajectories during CNC in vitro passages.Eventually, the cells divided into neurolineage and osteo/odonto-lineage in P3 and P5.Two odontogenic cell subgroups were identified during the in vitro culture and expansion.

| NCCs cultured from both cranial and trunk neural tubes exhibited the tooth regeneration potential
It was demonstrated that TNCCs do give rise to odontoblasts of trunk dermal denticles in cartilaginous fish models by cell lineage tracing.
This cell population with the likely primitive skeletogenic potential

| Cells from odontogenic path can induce tooth-like structures
Next, we identified a cell marker based on scRNA-seq data, EMCN, which is specifically expressed in the odontogenic path in P3 and P5 (Figure 3C).We also drew the dot plots from scRNA data to show Emcn was expressed in odontogenic and osteogenic cells but not in neural cells, in both P3 and P5 (Figure 3D).We sorted the P3 cells into EMCN+ and EMCN-groups and performed tooth inductions.The EMCN+ and EMCNÀ cells accounted for 67.9% and 26.8% of the total P3 cells, respectively (Figure 3E).The EMCN+ cells showed a $50% success rate of tooth induction (6/13, Figure 3F), while the EMCNÀ cells failed to induce any tooth-like structures (0/6, Figure 3G).The success rate was increased.Moreover, the tooth-like structures derived from EMCN + cells contained dental pulp (dp), dentin (d) and enamel space (es), as confirmed by H&E staining (Figure 3F, left and middle), whereas the EMCNÀ cells only produced the cyst (Figure 3G).Importantly, the tooth-like structures show much better structures in each part, such as the dentin (d) and the odontoblast layer (od) derived from EMCN+ sorted cells, which displays DSPP positive reaction by immunohistochemistry (Figure 3F right, arrows).This result indicates a relatively pure cell population would greatly contribute to healthy tooth formation.

| Cell cycle and DNA replication genes are critical in tooth induction
To investigate the critical genes in tooth formation, we performed RNA-seq experiments of CNC in vitro cultured cells and analysed the differential expressed genes between success and fail groups in tooth formation (T vs. NT) (Figure 4A,B).Cell cycle genes Ccnd1/2 were both upregulated in CNCC and TNCC groups.In addition, GO analysis suggested genes involved in DNA replication, RNA metabolism, as well as cell cycling/nuclear divisions are critical for the tooth formation.Moreover, we observed 138 genes overexpressed in the successful tooth formation group of CNCC sample and 1270 genes in TNCC sample, with 110 common genes (Figure 4C).Ccnd1/2, Tgfbr2, Fzd4/10, Peg12 and Ruvbl1 are the respective genes of T groups (Figure 4D).These genes are receptors of Wnt signalling pathway or Wnt target genes.On the other hand, neural genes Sox9, Gfap and We further check the expression levels of odontogenic gene markers identified from scRNA data, and the expression of odontogenic markers between T and NT groups.Among those markers, only Lhx6, Sp7, Dmp1 and Ogn showed the differences between T and NT groups with no statistical significance (Figure 3E).Furthermore, we found that the genes downregulated in T group were mainly related to neural development, while the genes upregulated in T group were associated with cell cycle, nuclear division and DNA replication processes.These findings suggest that the optimal cell proliferation state with appropriate levels of odontogenic markers is the key factor for successful tooth induction in kidney experiments.

| DISCUSSION
Tooth organ develops from the interaction between the oral epithelia cells and the dental mesenchymal cells originating from the cranial neural crest.Therefore, NCCs are expected as promising mesenchymal cell sources for tooth repair and regeneration.The amount of post-migratory CNCCs is limited when isolated from neural tube.
Besides, CNC differentiation in vitro is highly dynamic.The in vivo development of CNCCs has been profiled and reported previously. 29Moreover, our previous study 31 and another recent study, 36    with mandibular arch epithelium at E9 and E10, 19 it is ambiguous that mandibular arch epithelium at early stage are odontogenic thus can induce non-odontogenic mesenchyme to form the teeth. 2 In the same article, the authors also reported that these freshly dissected neural crests from both origins could not produce teeth (CNC, n = 0/18; TNC, n = 0/5) when recombined with limb epithelium (E9-E11).In

| Cell isolation and cell culture
The CNCC and TNCC were separated from embyonic day 9.5 (E9.5)ICR mice.The pregnant ICR mice were sacrificed and the neural grooves or neural tubes of the embryos were separated.The neural grooves or neural tubes with surrounding thin layers of tissue were dissected from the cranial and trunk parts, respectively (Figure 1A).All the dissected specimens were collected and digested in Dispase Grant/Award Number: 202201020379; Guangzhou Key Medical Disciplines 2021-2023; National Key Research and Development Program of China Stem Cell and Translational Research, Grant/Award Number: 2017YFA0104800; Science and Technology Projects in Guangzhou, Grant/Award Number: 2023A03J0818; Guangzhou Medical University; Key Laboratory of Guangdong Higher Education Institutes, Grant/Award Number: 2021KSYS009 structures.Cell cycle and DNA replication genes were concomitantly upregulated in the cultured NCCs of the tooth induction groups.Our data provide valuable insights into the cell heterogeneity of in vitro cultured CNCCs and their potential as a source for tooth regeneration.

2 | RESULTS 2 . 1 |
Single-cell RNA sequencing revealed the heterogeneities of primary CNCCs and identified a rare Alx3+/Barx1+ sub-population To investigate the heterogeneity of Primary CNCCs at the single-cell transcriptome level, the neural tube in mouse E9.5 embryos was -cell RNA sequencing NCCs culture and passage Neural tube isolation and seed (DMEM/F12 + N2, B27 supplement + bFGF, EGF, plate coated with matrigel)

2. 2 |
In vitro cultured CNCCs divided into distinct differentiation trajectories during passages and led to two odontogenic sub-populations Detected by immunofluorescence (Figure 2A, left), NCC marker P75 were downregulated gradually from P3 to P5 during the passaging of primary CNCCs in vitro.It was speculated that the cells began to differentiate.Meanwhile, neural progenitor cell (NPC) markers Sox2/ Pax3 were down regulated, while CNS cell marker Hnk1 in mice was upregulated (Figure 2A, right).Those results suggested the cell fates of primary CNCCs transited from the neural crest to neural progenitor/CNS during in vitro passages.Still, $50% of P5 cells were Sox2 negative, which indicated other cell lineages during the differentiations.To demonstrate the lineage specifications of primary CNCCs during in vitro culture and passaging, P3 and P5 cells were collected and scRNA-seq was performed, respectively.Together with P0 cells, F I G U R E 1 Isolation and identification of primary neural crest cells.(A) Left, cranial and trunk neural tubes in the embryo; Middle, schematic diagram of the reserved part of the neural tube, and the part outside the red frame or blue frame is removed; Right, immunofluorescence staining of neural tube and surrounding tissues with E-cadherin, Pax3 and p75.DAPI (4 0 ,6-diamidino-2-phenylindole) was used for nuclear staining.Scale bar: 100 μm.(B) Flow chart of NCCs isolation and culture, including schematic diagram of sampling points for immunofluorescence and single-cell RNA sequencing.Scale bar: embryo and neural tube 500 μm, cell culture 200 μm.(C) UMAP visualisations of single-cell RNA-seq analysis of primary CNCCs.(D) Violin plots of top marker genes of each cell type of CNCCs primary cells.(E) Expression patterns of Alx3, Ap2b, Pax9, Prrx1, Barx1, Crabp1 and Fli1 in E9.5 mouse cranial and trunk neural tube.Scale bars correspond to 200 μm.CNCC, cranial neural crest cell; NCC, neural crest cell.

F
I G U R E 2 Legend on next page.58,493 cells in total were detected to draw the single-cell resolution cell landscape of CNC lineage specifications in vitro.Clustering and marker analysis of the transcriptomes of P0, P3 and P5 cells revealed the expansion of a variety of cell types over the differentiation time.

(
Sox2, Sox10, Pax3, Pax6, Gfap and Igfbpl1) and osteogenic/ odontogenic (Ogn, Dcn, Bmp2, Pax9, Barx1 and Dmp1) differentiation at P3 and P5 stages (FigureS1E,F).These results revealed two distinct cell differentiation trajectories: neural lineage and osteogenic/ odontogenic lineage.To better investigate the multilineage differentiation landscape of primary CNCCs in vitro, we applied RNA velocity analysis to recover the directed dynamic information by leveraging splicing kinetics of in vitro cultured CNCCs combined P0, P3 and P5 (Figure2B, top left).The predicted future state of cells was visualised based on their gene expression profiles using arrows in the FA embedding (Force Atlas 2 algorithm).The arrows indicated the direction and magnitude of the transcriptional changes in each cell.The arrows were computed from the ratio of unspliced and spliced mRNA abundances, which represented the rate of change of each gene.34The figure revealed the transcriptional dynamics of NCCs along the differentiation trajectories, as well as the potential fate of intermediate cell states.From five groups of NCC cells at P0 (Sox10 + , Alx3 + /Barx1 + , Cnn2 + , Sox8 + and Otx2 + , respectively), four possible cell differentiation paths from NCC to neural or osteogenic/odontogenic lineages (Figure2B, top right): genes were enriched for specific gene ontology (GO) terms in the Receptor Tyrosine Kinases (RSK) signalling pathway, ossification and odontogenesis (Figure S2C, right).The genes that were downstream of Trap2b-GRN were mostly active at the intermediate stage and were involved in biological processes related to tissue specification and growth.These genes were enriched for specific GO terms in cell fate commitment, epithelial cell proliferation and myofibril assembly (Figure S2C, right).

3
Single-cell landscape of cultured CNCCs lineage specifications in vitro.(A) Left: Characterisation of P3 and P5 CNCCs with immunofluorescence.Scale bar corresponds to 100 μm.Right: Bar plots of positive cells P3 and P5 CNCCs with immunofluorescence as statistical results.(B) Single-cell RNA-seq analysis of in vitro cultured CNCCs in P0, P3, and P5: (top) Heterogeneous cell types and landscape of in vitro cultured CNCCs; (bottom) markers gene expressions and velocity in each lineage.(C) Trends of marker genes expressed in specific lineages as pseudo time.(D) Heat map showing the marker genes expressed in odontogenic path and astrocyte path.(E) Dot plots of top marker genes of each cell type in CNCCs differentiation landscapes.CNCC, cranial neural crest cell.waspinpointed to be a neural crest origin of dentine throughout the ancestral vertebrate dermal skeleton.Thus, we also isolated NCCs from the trunk part at somite 6th to 11th in E9.5 mouse embryos (Figure1A, tissues between blue lines in left).The cells isolated from the trunk part exhibit similar cell shape to those of the cranial part during the passages (FigureS1A).To confirm whether these cultured cells from both CNCCs and TNCCs could induce tooth regeneration, we pelleted cells randomly harvested from P3, P4 or P5 by centrifugation, respectively, and then each cell pellet was recombined with a piece of epithelium from E14.5 to E16.5 mouse tooth germ, followed by transplantation under the mouse renal capsule.After 3 weeks, we observed tooth-like structures in the kidney from both cranial and trunk groups (Figure3A).The induction rate of tooth formation is 26.7% (N = 36/135) and 22.1% (N = 17/77) in the cranial and trunk groups, respectively.The sizes of most tooth-like structures in both groups are smaller than those derived from tooth germs transplanted after 3 weeks.After the decalcification, confirmed by haematoxylin and eosin (H&E) staining in sections, the tooth-like structures contain dental pulp (dp), dentin (d) and enamel space (es) in sections.Nevertheless, the cells in the regenerated structures look unhealthy with loose arrangement, including the dental pulp cells, surrounding tissues and kidney tissues of host (kd).In particular, some irregular structures were detected in the local areas of the regenerated dentin (Figure 3A, Characterisation of NCCs as seed for tooth regeneration from cranial (upper row) and trunk neural tube (bottom row).(A) Left, regenerated tooth-like structures from the recombinants of NCCs and E14.5 DE after transplantation under renal capsule for 3 weeks.Scale bar corresponds to 100 μm.Right, H&E staining of relative tooth sections showing the tooth-like structures containing dental pulp (dp), dentin (d), enamel space (es) and kidney (kd).Scale bar corresponds to 200 μm.(B) Upper row, EGFP labelled neural crest cells and labelling efficiency detected by flow cytometry.Lower row, sections of the regenerated tooth germ constructed by EGFP NCCs and results of tooth formation.Scale bar corresponds to 200 μm.(C) scRNA-seq analysis of EMCN expression in cells derived from NCCs.(D) Dot plots to show the expression levels of Emcn in each cell groups in P3 and P5.(E) Proportions of EMCN+ and EMCNÀ cells sorted at P3. (F and G) Tooth inductions with EMCN+ and EMCNÀ cells sorted from P3; dental pulp (dp), dentin (d), enamel space (es) and bone (B); Scale bar: 100 μm.DE, dental epithelium; H&E, haematoxylin and eosin; NCC, neural crest cell.To confirm the origin of the mesenchymal component, we used lentivirus system to encode enhanced green fluorescent protein (EGFP) with the cultured cells from CNCs at P3 or P5 (Figure 3B).The EGFP infection rate is 72.5%.All mesenchymal cells in the 2-day recombinant germs are EGFP positive, indicating the odontogenesis induction is from the cultured NCCs.As expected, the rate of tooth formation in GFP signal-containing group is 23.1% (n = 9/39), which represents the mesenchymal-derived dentin, dental pulp, and the surrounding bone tissues.

Pax6,
which could lead to glial cell development and axongenesis, were upregulated in NT groups.Therefore, our result suggested cell cycle and DNA replication genes Ccnd1/2 and Wnt signalling pathway are crucial for the tooth formation led by in vitro expanded NCC cells.
Scientists have been facing the challenge of inducing the odontogenic lineage efficiently from neural crest cell culture and expansion for years.Therefore, it is essential to understand the heterogeneity of in vitro cultured NCCs in high resolution and identify the odontogenic subpopulation and differentiation landscape, which can interact with epithelium to form the whole tooth-like structures.scRNA-seq is a powerful technology for resolving heterogeneous cell types and provides insights into cell functional heterogeneity.In this study, we demonstrated the heterogeneity and differentiation potential of in vitro cultured CNCCs for odontogenesis by scRNA-seq analysis.The key finding from our result is the identification of odontogenic differentiation landscape of in vitro cultured and expanded CNCCs in single-cell resolution.A rare Alx3 + /Barx1 + cell sub-population of primary CNCCs can differentiate into two odontogenic cell clusters (Barx1+ vs. Barx1-cells) at P3 and P5.Our results suggest that EMCN is a marker of cells from the odontogenic path.
which examined the scRNA profiles of molar dental pulp, provided the cell landscape and regulatory atlas during the dental pulp development.Although neural and osteogenic/odontogenic cell lineages have been observed in both in vivo and in vitro cell differentiation, the in vitro cell lineages are less heterogeneous than the in vivo development.This may be due to our efforts to optimise the culture medium to enrich the odontogenic cells.For the rare cell sub-populations in other lineages during in vivo CNCC development, they may not survive in our medium.The researchers need to develop

4
Legend on next page.their own medium to expand the relevant cell sub-populations for other applications.Another important finding is we succeeded to form the whole tooth-like structure using both CNCCs and TNCCs in vitro.Although a previous publication suggested that teeth could be formed in combinations of fresh dissected CNC (n = 36/117) or TNC (n = 5/40) at E8

4 |
Figure 2D.This indicates that Msx1 may regulate other odontogenic genes as a trigger in temporal manner.Another finding is the identification of two odontogenic Barx1+ and Barx1À cell populations, which

( 1 .
5 mg/mL, Gibco, 17105-041) at 37ºC for 10 min.After digestion, the specimens were washed three times gently with DMEM/F12 (Hyclone, SH30023.01) and seeded on Matrigel (Corning, 354277) coated six-well plate.Samples were incubated in the culture medium containing DMEM/F12 supplemented with 200Â N2 supplement (Gibco, 17502-048), 100Â B27 supplement (Gibco, 17504-044), 20 ng/μL bFGF and 20 ng/μL EGF.After incubation at 37ºC and 5% CO 2 for 48 h, the rest tissue blocks were picked up and the media in the wells were changed every two days.The cultured cells were F I G U R E 4 RNA-seq analysis and differentiated expressed genes (DEGs) of CNCs and TNCCs which succeeded or failed to induce the whole tooth-like structures (T vs. NT).(A and B) Volcano plots (left) and gene ontology analysis (right) to show the DEGs of CNCCs (A) or TNCCs (B) which succeeded or failed to induce the whole tooth-like structures (T vs. NT).(C) Venn diagram of genes upregulated in CNCCs and TNCCs which succeed in inducing the whole tooth-like structures.(D) Heat map of the expression patterns of the most significant DEGs.(E) Bar plots of the expression levels of Lhx6, Sp7, Dmp1 and Dgn between T and NT groups from bulk RNA-seq.CNC, cranial neural crest; CNCC, cranial neural crest cell; TNCC, trunk neural crest cell.