Single-cell transcriptomics-informed induced pluripotent stem cells differentiation to tenogenic lineage
- Orthopaedic Stem Cell Research Laboratory, Cedars-Sinai Medical Center, United States
- Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, United States
- Department of Biomedical Sciences, Cedars-Sinai Medical Center, United States
- Department of Orthopedics, Cedars-Sinai Medical Center, United States
- Department of Surgery, Cedars-Sinai Medical Center, United States
Figures
Figure 1 with 1 supplement
Stepwise induction of iPSCs to syndetome-like cells using chemically defined media and small molecules in vitro.
(A) Schematic of iPSC to SYN stepwise induction using chemically defined media and small molecules. (B) Brightfield micrographs of cells going through the differentiation stages at. Scale bars represent 200 μm. (C) Pluripotent markers were expressed in iPSCs and were downregulated in further stages. Gene expression analyses for stage-specific markers with the 007i iPSC line: upregulation of early mesoderm markers at the presomitic mesoderm (PSM, n = 8 replicates/group) (D), somitogenesis at SM (n = 8 replicates/group) (E), and sclerotome-related markers at SCL (n = 12) (F). (G) Tenogenic markers are significantly upregulated at the SYN stage (n = 4) compared to iPSC (n = 9) and SCL (n = 12) stages. Differentiation experiments were repeated independently n = 2 with the 007i line and n = 2 with a second iPSC line that was later tested (83i); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 1—figure supplement 1
Flow cytometry of DLL-1 at the presomitic mesoderm (PSM) stage for low and high seeding densities.
Left panel: low iPSC seeding density resulted in a high percentage of DLL-1+ cells. Right panel: high iPSC seeding density resulted in a comparably reduced DLL-1+ cell population.
Figure 2 with 1 supplement
Immunofluorescence co-staining for stage-specific markers for confirmation of protein expression at each induction stage.
(A, B) OCT4 and NANOG were observed in iPSCs but not at PSM. (C, D) Early mesoderm markers TBX1, TBX6, and DLL1 at PSM vs. SM. (E, F) Somitogenesis markers PARAXIS, MEOX1, and PAX3 at SM vs. SCL. (G, H) sclerotome-related markers PAX1 and PAX9 at SCL vs. SYN. (I) Tenogenic markers SCX, COL1, TNMD, MKX co-expression at the end of induction to SYN. Nuclei were stained with DAPI (blue). Scale bars represent 50 μm. Differentiation experiments were repeated independently n = 2 with the 007i line and n = 2 with a second iPSC line that was later tested (83i). IF staining was performed in n = 3 technical triplicates.
Figure 2—figure supplement 1
Immunofluorescence staining of selected markers and DAPI for nuclear staining from Figure 2 expanded to show all separate channels.
(B’) NANOG at PSM stage; (E’) MEOX1 and PAX3 at SCL stage, and (G’) PAX9 at SM stage.
Figure 3 with 4 supplements
Single-cell RNA sequencing reveals cellular heterogeneity at the end of induction of iPSC to syndetome-like cells and off-target differentiation to neural-like progenitors.
(A) UMAPs of each differentiation step sorted into 11 cell clusters from iPSC to SYN were annotated to 6 distinct cell populations: iPSC (OCT4+SOX2+NANOG+), Syndetome (SYN, MKX+TNMD+COL1A1+), Neuromesodermal Progenitors/Neural Crest cells (NMP/NC, PAX3+NRP2+COLEC12+), Mesoderm (Mes, DLL1+DLL3+PARAXIS+), Neuromesodermal Progenitors – Cranial (NMP-C, DLL1+DLL3+NOTCH1+CRAB1+), and Neural Lineage cells (NL, NRN1+DCX+NNAT+). During induction, pluripotent clusters gradually disappeared, and three main clusters emerged: SYN, NMP-C, and NL. Feature plots (B) and dot plots (C) of stage-specific genes displayed for all differentiation stages. (D) Expression of primary tenogenic markers COL1A1 (blue) and either SCX (red) or TNMD (red) on UMAP plot. (E, F) Original samples and clusters were ordered on pseudo-time developmental trajectory. (E) Trajectory analysis based on original samples showed transition from iPSC to SYN correlated with the samples; however, SYN cells were located within three endpoints. (F) Trajectory analysis based on clusters showed SYN cluster as the main differentiation endpoint with NMP-C and NL as off-target differentiation endpoints. Branching point heat maps (G, H) and gene expression were predominated by neural-related markers SYP, PCLO, DCX, and NRN1 (I). (J, K) IPA analysis revealed that the off-target clusters NMP-C and NL clusters were linked with increased Wnt pathway activity (K, L), while the SYN cluster was associated with tenocyte differentiation and linked to decreased Wnt pathway activity (J).
Figure 3—figure supplement 1
Distribution of cell subpopulations per sample.
Cell clusters from iPSC to SYN were annotated to 6 distinct cell populations: iPSC (OCT4+SOX2+NANOG+), Syndetome (SYN, MKX+TNMD+COL1A1+), Neuromesodermal Progenitors/Neural Crest (NMP/NC, PAX3+NRP2+COLEC12+), Mesoderm (Mes, DLL1+DLL3+PARAXIS+), Neuromesodermal Progenitors – Cranial (NMP-C, DLL1+DLL3+NOTCH1+CRAB1+), and Neural Lineage cells (NL, NRN1+DCX+NNAT+).
Figure 3—figure supplement 2
Addition of WNTi to the differentiation resulted in less off-targets.
(A) ScRNA-seq comparison of two different iPSC lines for SYNWNTi. Cell population annotation is shown in Figure 5 and in Supplementary file 1. (B) Distribution of cell subpopulations per sample, where the second cell line sample at the syndetome stage has also been included (designated as WNTi-83i). Cluster numbers and annotations are shown in Supplementary file 1.
Figure 3—figure supplement 3
A non-biased gene ontology (GO) analysis was performed.
Figure 3—figure supplement 4
Feature plots of NKX3.2 and MEOX1 displayed for all differentiation stages.
Figure 4
Inhibition of WNT signaling resulted in decreased expression of off-target markers and a more homogeneous population.
(A) Gene expression analysis of neural markers NRN1, DYNLL1, and FMN1, as well as tenogenic marker TNMD on day 10 of SYN induction (day 21 of the differentiation). (A1) Cells treated with WNTi had significantly downregulated neural marker expression compared to just SYN. n = 4 replicates/group. (A2) Tendon gene expression was significantly upregulated in the WNTi-treated group; n = 3/group; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (B, C) Immunofluorescence staining for neural markers (DCX, SYP, NRN1), SYN (B1–B3), and SYNWNTi (C1–C3) showed that they were present in SYN and almost disappeared in SYNWNTi. Scale bars represent 50 μm. (D–F) Immunofluorescence staining for tenogenic markers (SCX, MKX, TNMD, COL1) of SYN vs. SYNWNTi groups. Scale bars are 50 μm. Differentiation experiments were repeated independently n = 2 with the 007i line and n = 2 with a second iPSC line that was later tested (83i). IF staining was performed in n = 3 technical triplicates.
Figure 5 with 3 supplements
Addition of WNTi to the SCL and SYN induction stages of the differentiation improved differentiation to SYN and eliminated off-target clusters.
(A) Schematic of optimized iPSC to SYN stepwise induction with WNTi addition implemented at SM to SCL and SCL to SYN stages. Informed by single cell transcriptomics, the addition of Wnt pathway inhibitor at the later stages of the differentiation resulted in a more specific differentiation of iPSCs to tenocytes. (B) UMAPs of each differentiation step sorted into 12 distinct clusters from iPSC to SYN were annotated into 6 distinct cell populations: Syndetome (SYN, MKX+TNMD+DCN+BGN+), iPSC (OCT4+NANOG+LIN28A+SOX2+), Neuromesodermal Progenitors/Neural Crest (NMP/NC, TBXT+TWIST1+SP5+SNAI2+), Neural Crest (NC, PTN+NTKR2+SOX4+SOX11+), Fibrocartilage (FC, COL2A1+SOX9+FN1+BGN+COL1a1+), and Neural Lineage (NL, SOX2+DCX+MAP2+UNCX+SOX4+). UMAP comparison of the SYN and SYNWNTi populations demonstrated increased size of the SYN cluster and elimination of NL cluster. (C) Dot plots of gene expression of stage-specific genes for SYN and SYNWNTi. (D) Expression of primary tenogenic markers Col1a1 (blue), Scx (red), and Tnmd (red) on UMAP plot for SYN and SYNWNTi. (E) Original samples and clusters were ordered on pseudo-time developmental trajectory. Trajectory analysis revealed one main endpoint, the SYN cell populations. (F) After addition of WNTi, the proportion of cells in the SYN clusters increased by 59% while the proportion of cells in the NL cluster was eliminated. (G) IPA network analysis showed that WNT pathway was enriched in the NMP/NC and NC clusters but not in NL.
Figure 5—figure supplement 1
Dot plot of stage-specific markers of SYNWNTi for each cluster.
Cell population annotation is presented in Figure 5.
Figure 5—figure supplement 2
Change in SCX expression throughout SYN induction with WNTi.
All timepoints are normalized to day 0 (iPSC). *p < 0.0001.
Figure 5—figure supplement 3
Separate and combined feature plots of SCX and TNMD expression at SCL through SYN stage with and without WNT inhibitor (WNTi).
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (human) | 007i-cntr-n5 | Cedars-Sinai iPSC Core Facility | N/A | https://biomanufacturing.cedars-sinai.org/?filter_cell-type=ipsc&filter_primary-tissue&filter_disease&filter_sex&filter_age-at-sampling&filter_ethnicity&filter_race&filter_gene&filter_mutation&filter_project&cs_product_search |
| Cell line (human) | 83i-cntr-22n1 | Cedars-Sinai iPSC Core Facility | N/A | See above |
| Antibody | Rabbit Polyclonal Anti-TBX1 | Abcam | Ab19530 | IF (1:25-1:250) |
| Antibody | Goat Polyclonal Anti-TBX6 | R&D Systems | AF4744 | IF (1:25-1:250) |
| Antibody | Mouse Monoclonal Anti-DLL1 | Miltenyi Biotec | 130-106-148 | IF (1:25-1:250) |
| Antibody | Mouse Monoclonal Anti-DLL1 | Miltenyi Biotec | 130-106-148 | FACS (10 µl per test) |
| Antibody | Rabbit Polyclonal Anti-PARAXIS | Sigma | HPA060221 | IF (1:25-1:250) |
| Antibody | Rabbit Polyclonal Anti-MEOX1 | Sigma | HPA045214-25UL | IF (1:25-1:250) |
| Antibody | Mouse Monoclonal Anti-PAX1 | Developmental Studies Hybridoma Bank | Clone: C2 | IF (1:25-1:250) |
| Antibody | Mouse Monoclonal Anti-PAX3 | Thermo Fisher | 60217-1-IG | IF (1:25-1:250) |
| Antibody | Mouse Monoclonal Anti-PAX9 | Thermo Fisher | H00005083-M03 | IF (1:25-1:250) |
| Antibody | Rabbit Polyclonal Anti-NKX3.2 | Sigma | HPA027564 | IF (1:25-1:250) |
| Antibody | Rabbit Polyclonal Anti-SCX | Thermo Fisher | PA5-115874 | IF (1:25-1:250) |
| Antibody | Goat Polyclonal Anti-COL1 | Bio-Rad | 131001 | IF (1:25-1:250) |
| Antibody | Rabbit Polyclonal Anti-TNMD | Sigma | HPA055634 | IF (1:25-1:250) |
| Antibody | Mouse Monoclonal Anti-MKX | Thermo Fisher | MA5-26976 | IF (1:25-1:250) |
| Antibody | Monoclonal Mouse Anti-Neuritin | R&D Systems | AF283 | IF (1:25-1:250) |
| Antibody | Mouse Monoclonal Anti-SYP | Biolegend | 837104 | IF (1:25-1:250) |
| Antibody | Polyclonal Sheep Anti-DCX | R&D Systems | AF10025-100 | IF (1:25-1:250) |
| Antibody | Monoclonal Mouse Anti-NANOG | Sigma-Aldrich | AMAB91393 | IF (1:25-1:250) |
| Antibody | Monoclonal Mouse Anti-OCT4 | Cell Signalling | D705Z | IF (1:25-1:250) |
| Sequence-based reagent | Dynein light chain LC8-type 1 | Thermo Fisher | Hs04378026_m1 | qPCR primers |
| Sequence-based reagent | Neuritin 1 | Thermo Fisher | Hs00213192_m1 | qPCR primers |
| Sequence-based reagent | One cut homeobox 2 | Thermo Fisher | Hs00191477_m1 | qPCR primers |
| Sequence-based reagent | Formin 1 | Thermo Fisher | Hs05010770_m1 | qPCR primers |
| Sequence-based reagent | Nanog homeobox | Thermo Fisher | Hs02387400_g1 | qPCR primers |
| Sequence-based reagent | POU class 5 homeobox1 | Thermo Fisher | HS04260367_gH | qPCR primers |
| Sequence-based reagent | T-box transcription factor T | Thermo Fisher | HS00610080_m1 | qPCR primers |
| Sequence-based reagent | SRY-box transcription factor 2 | Thermo Fisher | HS04260357_g1 | qPCR primers |
| Sequence-based reagent | Delta like canonical notch ligand 1 | Thermo Fisher | HS01011330_m1 | qPCR primers |
| Sequence-based reagent | T-box transcription factor 6 | Thermo Fisher | Hs00365539_m1 | qPCR primers |
| Sequence-based reagent | Mesogenin 1 | Thermo Fisher | Hs03405514_s1 | qPCR primers |
| Sequence-based reagent | Wnt family member 3A | Thermo Fisher | Hs00263977_m1 | qPCR primers |
| Sequence-based reagent | Mesenchyme homeobox 1 | Thermo Fisher | Hs00244943_m1 | qPCR primers |
| Sequence-based reagent | Transcription factor 15 | Thermo Fisher | Hs00231821_m1 | qPCR primers |
| Sequence-based reagent | Paired box 3 | Thermo Fisher | Hs00240950_m1 | qPCR primers |
| Sequence-based reagent | Paired box 9 | Thermo Fisher | Hs00196354_m1 | qPCR primers |
| Sequence-based reagent | Paired box 1 | Thermo Fisher | Hs01071292_m1 | qPCR primers |
| Sequence-based reagent | NK3 homeobox 2 | Thermo Fisher | Hs00154168_m1 | qPCR primers |
| Sequence-based reagent | Scleraxis | Thermo Fisher | Hs03054634_g1 | qPCR primers |
| Sequence-based reagent | Tenomodulin | Thermo Fisher | Hs00223332_m1 | qPCR primers |
| Sequence-based reagent | Tubulin polymerization promoting protein family member 3 | Thermo Fisher | Hs03043892_m1 | qPCR primers |
| Sequence-based reagent | Platelet derived growth factor receptor alpha | Thermo Fisher | Hs00998018_m1 | qPCR primers |
| Sequence-based reagent | Early growth response 1 | Thermo Fisher | Hs00152928_m1 | qPCR primers |
| Sequence-based reagent | Collagen type III alpha 1 chain | Thermo Fisher | HS00943809_m1 | qPCR primers |
| Sequence-based reagent | Collagen type I alpha 1 chain | Thermo Fisher | HS00164004_m1 | qPCR primers |
| Peptide, recombinant protein | Insulin | Sigma-Aldrich | 674889 | |
| Peptide, recombinant protein | Apo-Transferrin human | Sigma-Aldrich | T1147 | |
| Peptide, recombinant protein | 1-Thioglycerol | Sigma-Aldrich | M6145 | |
| Peptide, recombinant protein | Recombinant human FGF-basic (146 a.a.) | PeproTech | 100-18C | |
| Peptide, recombinant protein | FGF-8 | PeproTech | AF-100-25 | |
| Peptide, recombinant protein | Human/mouse recombinant TGF-beta 3 | STEMCELL Technologies | 78131 | |
| Peptide, recombinant protein | Recombinant Human BMP-7 | PeproTech | 120-03P | |
| Commercial assay or kit | Chromium Single-cell 3′ Reagent Kits | 10x Genomics | N/A | |
| Chemical compound, drug | DAPI (4′,6-Diamidino-2-Phenylindole, Dihydrochloride) | Invitrogen | D1306 | |
| Chemical compound, drug | IMDM, no phenol red | Thermo Fisher | 21056023 | |
| Chemical compound, drug | Ham’s F-12 nutrient mix | Thermo Fisher | 11765047 | |
| Chemical compound, drug | Chemically defined lipid concentrate | Thermo Fisher | 11905031 | |
| Chemical compound, drug | Antibiotic-antimycotic solution | Thermo Fisher | 15240096 | |
| Chemical compound, drug | SB 431542 hydrate | Sigma-Aldrich | S4317 | |
| Chemical compound, drug | DMH-1 (CAS 1206711-16-1) | Santa Cruz Biotechnology | Sc-361171 | |
| Chemical compound, drug | CHIR99021 | Biogems | 2520691 | |
| Chemical compound, drug | Y-27632 Dihydrochloride | PeproTech/Biogems | 683093 | |
| Chemical compound, drug | SAG | PeproTech/Biogems | 9128694 | |
| Chemical compound, drug | LDN193189 | PeproTech/Biogems | 1062443 | |
| Chemical compound, drug | Matrigel | Corning | 354230 | |
| Chemical compound, drug | Geltrex | Thermo Fisher | A1413301 | |
| Chemical compound, drug | DMSO | Sigma-Aldrich | D2650 | |
| Chemical compound, drug | Wnt-C59 | Cayman Chemical | 16644 | |
| Chemical compound, drug | Poly-D-lysine | Thermo Fisher | A3890401 | |
| Software, algorithm | R (v4.1.2) | R Development Core Team | https://www.R-project.org | |
| Software, algorithm | RStudio (v1.4.1103) | RStudio Team (2020). RStudio: Integrated Development for R. RStudio, PBC | https://www.rstudio.com/products/rstudio/ | |
| Software, algorithm | Cell Ranger (v3.0.0) | 10x Genomics | ||
| Software, algorithm | Loupe Cell Browser (v3.0.0) | 10x Genomics | ||
| Software, algorithm | Seurat (v4.1.0) | Satija et al., 2015; Butler et al., 2018; Hao et al., 2021; Satija et al., 2015; Stuart and Satija, 2019 | https://satijalab.org/seurat | |
| Software, algorithm | Monocle (v2.18.0) | Qiu et al., 2017a; Qiu et al., 2017b; Trapnell et al., 2014 | http://cole-trapnell-lab.github.io/monocle-release/ |
Additional files
-
Supplementary file 1
Normalized cell counts (expressed as %) per cluster following WNTi treatment, shown for the two cell lines, 007i and 83i.
- https://cdn.elifesciences.org/articles/89652/elife-89652-supp1-v1.docx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/89652/elife-89652-mdarchecklist1-v1.pdf
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Single-cell transcriptomics-informed induced pluripotent stem cells differentiation to tenogenic lineage
eLife 12:RP89652.
https://doi.org/10.7554/eLife.89652.3
