Stepwise induction of iPSCs to syndetome 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 100 or 200 μm. (C) Pluripotent markers were expressed in iPSCs and were downregulated in further stages. (D-F) Gene expression analyses for stage-specific markers: upregulation of early mesoderm markers at the PSM (n=4 replicates/group) (D), somitogenesis at SM (n=6 replicates/group) (E), sclerotome-related markers at SCL (n=7 replicates/group) (F). (G) Tenogenic markers are significantly upregulated at the SYN stage (n=4) compared to iPSC (n=9) and SCL (n=11) stages. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Immunofluorescence co-staining for stage-specific markers for confirmation of protein expression at each induction stage.

(A-B) POU5F1 and NANOG were expressed in iPSCs but were downregulated 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 50μm.

Single-cell RNA sequencing reveals cellular heterogeneity at the end of induction of iPSC to syndetome 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. (B-C) 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. (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 SYN cluster was associated with tenocyte differentiation and linked to decreased Wnt pathway activity (J).

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; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (B-C) Immunofluorescence staining for neural markers (DCX, SYP, Neuritin), SYN (B1-B3) and SYNWNTi (C1-C3) showed that they were present in SYN and disappeared in SYNWNTi. Scale bars represent 50μm. (D-F) Immunofluorescence staining for tenogenic markers (SCX, MKX, TNMD, COL1) of SYN and SYNWNTi showed that more COL1+TNMD+ and SCX+MKX+ cells were observed in SYNWNTi compared to SYN. Scale bars are 50μm.

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+), 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.