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| Status |
Public on Jan 11, 2023 |
| Title |
Human iPSC Derived Enamel Organoid Guided by Single Cell Atlas of Human Tooth Development |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Tooth enamel secreted by ameloblasts is the hardest material in the human body, acting as a shield protecting the teeth. However, enamel is gradually damaged or partially lost in over 90% of adults and cannot be regenerated due to a lack of ameloblasts in erupted teeth. Here we use sci-RNA-seq to establish a spatiotemporal single cell atlas for the developing human tooth and identify regulatory mechanisms controlling the differentiation process of human ameloblasts. We reveal key signaling pathways involved between the support cells and ameloblasts during fetal development and recapitulate those findings in a novel human ameloblast in vitro differentiation from iPSCs. We furthermore develop a mineralizing enamel organ-like 3D organoid system. These studies pave the way for future regenerative dentistry and therapies toward genetic diseases affecting enamel formation.
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| Overall design |
To understand how the early differentiation processes of tooth and salivary gland differ and how the epithelial and mesenchymal cell lineages acquire the odontogenic competence, we analyzed the developmental gene expression profiles of human fetal stages by single cell sequencing approach. Tooth germ and salivary gland samples were collected from five fetal age groups. These age groups represented the following developmental stages for tooth differentiation: the bud stage (9-11gw), the cap stage (12-13gw), the early bell stage (14-16gw), and the late bell stage (17-19gw and 20-22gw). We also collected submandibular salivary glands from three matched timepoints (12-13gw, 14-16gw, 17-19gw) that cover the pseudo-glandular and canalicular stages for salivary gland development. A total of 12 samples were prepared for sci-RNA sequencing that consisted of specific tissue from multiple fetuses. For young fetuses, 9-11gw, dissecting individual tooth germs or salivary glands that were in the bud stage was not feasible due to the large number of cells required to perform sci-RNA-seq protocol, instead whole jaw segments were prepared for sequencing. Ten anterior segments (which span from canine tooth to canine tooth region) were pooled as the anterior jaw sample, and 12 posterior jaw segment pairs were pooled as the posterior jaw sample. For older fetuses from 12-22gw, individual tooth germs and salivary glands were dissected. The numbers of pooled samples varied per stage and tooth type, ranging from 40-66 for anterior teeth (including incisors and canines), and 17-51 for molar teeth, as molar teeth are generally larger in size. The salivary gland pooled samples ranged from 3-7 in the three age groups collected.
We utilized the inferred signaling pathways from the sci-RNA-seq data to develop a novel in vitro differentiation protocol that recapitulated the early stages of human ameloblast development from hiPSCs (iAM differentiation); To analyze the efficiency of the differentiation we performed sci-RNA-seq on the two time points of iPSC derived ameloblast differentiation (day10-OE and day16-Early-ameloblasts) and compared the gene expression data to the fetal tissue gene expression data. Bulk RNA-seq was also performed for day0 and day16 of the differentiation .
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| Contributor(s) |
Alghadeer A, Hanson-Drury S, Ehnes D, Zhao YT, Patni A, O'Day D, Spurrell C, Gogate A, Phal A, Zhang H, Devi A, Wang Y, Starita L, Doherty DA, Glass I, Shendure J, Baker D, Regier MC, Mathieu J, Ruohola-Baker H |
| Citation(s) |
36540608, 38259324 |
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| Submission date |
Sep 24, 2021 |
| Last update date |
Feb 09, 2024 |
| Contact name |
Hannele Ruohola-Baker |
| E-mail(s) |
hannele@uw.edu
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| Phone |
(206) 543-8468
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| Organization name |
University of Washington
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| Department |
Department of Biochemistry
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| Lab |
Ruohola-Baker
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| Street address |
UW Medicine at South Lake Union, 850 Republican Street
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
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| Platforms (2) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
| GPL30173 |
NextSeq 2000 (Homo sapiens) |
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| Samples (19)
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| Relations |
| BioProject |
PRJNA766055 |
| SRA |
SRP338604 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE184749_Day0_vs_Day16_Diff_bulk_RNAseq_featurecount_result.txt.gz |
5.0 Mb |
(ftp)(http) |
TXT |
| GSE184749_How_to_load_data_into_R.txt.gz |
1001 b |
(ftp)(http) |
TXT |
| GSE184749_RAW.tar |
397.0 Mb |
(http)(custom) |
TAR (of MATRIX, TXT) |
| GSE184749_all_data_cell_annotations.csv.gz |
458.6 Kb |
(ftp)(http) |
CSV |
| GSE184749_all_data_counts.mtx.gz |
108.2 Mb |
(ftp)(http) |
MTX |
| GSE184749_all_data_gene_annotations.csv.gz |
767.2 Kb |
(ftp)(http) |
CSV |
| GSE184749_day10_oe_diff_cell_annotations.csv.gz |
109.7 Kb |
(ftp)(http) |
CSV |
| GSE184749_day10_oe_diff_counts.mtx.gz |
36.6 Mb |
(ftp)(http) |
MTX |
| GSE184749_day10_oe_diff_gene_annotations.csv.gz |
767.2 Kb |
(ftp)(http) |
CSV |
| GSE184749_day16_am_diff_cell_annotations.csv.gz |
144.0 Kb |
(ftp)(http) |
CSV |
| GSE184749_day16_am_diff_counts.mtx.gz |
39.8 Mb |
(ftp)(http) |
MTX |
| GSE184749_day16_am_diff_gene_annotations.csv.gz |
767.2 Kb |
(ftp)(http) |
CSV |
| GSE184749_dental_epithelium_cell_annotations.csv.gz |
97.0 Kb |
(ftp)(http) |
CSV |
| GSE184749_dental_epithelium_counts.mtx.gz |
8.9 Mb |
(ftp)(http) |
MTX |
| GSE184749_dental_epithelium_gene_annotations.csv.gz |
652.8 Kb |
(ftp)(http) |
CSV |
| GSE184749_dental_mesenchyme_cell_annotations.csv.gz |
90.0 Kb |
(ftp)(http) |
CSV |
| GSE184749_dental_mesenchyme_counts.mtx.gz |
17.4 Mb |
(ftp)(http) |
MTX |
| GSE184749_dental_mesenchyme_gene_annotations.csv.gz |
781.7 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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