Single Cell Transcriptomics-Informed Induced Pluripotent Stem Cells Differentiation to Tenogenic Lineage

Reviewer #1 (Public Review):

Papalamprou et al. established a methodology to differentiate iPSCs to the syndetome stage and validated it by marker gene expression and scRNA-seq analysis. They further found that inhibition of WNT signaling enhanced the homogeneity of the cell population after identifying a group of braching-off cells that overexpressed WNT. Their results will be helpful in developing cell therapy systems for tendon injuries. However, there are several issues to improve the manuscript:

IPA analysis was performed after scRNA-seq. Although it is knowledge-based software with convenient graphic utilities, it is questionable whether an unbiased genome-level analysis was performed. Therefore, it is not convincing if WNT is the only and best signal for the branching-off marker. Perhaps independent approaches, such as GO, pathway, or module analyses, should be performed to validate the finding.

According to the method section, two iPSC lines were used for the study. However, throughout the manuscript, it is not clearly described which line was used for which experiment. Did they show similar efficiency in differentiation and in responses to WNTi? It is also worrisome if using only two lines is the norm in the stem cell field. Please provide a rationale for using only two lines, which will restrict the observation of individual-specific differential responses throughout the study.

How similar are syndetome cells with or without WNTi? It would be interesting to check if there are major DEGs that differentiate these two groups of cells.

Please discuss the improvement of the current study compared to previous ones (e.g., PMID 36203346, 35083031, 35372337).

Reviewer #2 (Public Review):

Summary:
Dr. Sheyn and colleagues report the step-wise induction of syndetome-like cells from human induced pluripotent stem cells (iPSCs), following a previously published protocol which they adjusted. The progression of the cells through each stage, i.e. presomitic mesoderm (PSM), somitic mesoderm (SM), sclerotome (SCL), and syndetome (SYN)) is characterized using FACS, RT-qPCR and immunofluorescence staining (IF). The authors also performed single-cell RNA sequencing (scRNAseq) analysis of their step-wise induced cells and identified signaling pathways which are potentially involved in and possibly necessary for syndetome induction. They then optimized their protocol by simultaneous inhibition of BMP and Wnt signaling pathways, which led to an increase in syndetome induction while inhibiting off-target differentiation into neural lineages.

Strengths:
The authors conducted scRNAseq analysis of each step of their protocol from iPSCs to syndetome-like cells and employed pathway analysis to uncover further insights into somitic mesoderm (SM) and syndetome (SYN) differentiation. They found that BMP inhibition, in conjunction with the inhibition of WNT signaling, plays a role in driving syndetome differentiation. Analyzing their scRNAseq results, they could improve the syndetome induction efficiency of their protocol from 47.6% to 67%-78% while off-target differentiation into neural lineages could be reduced.

Weaknesses:
The authors demonstrated the efficiency of syndetome induction solely by scRNA-seq data analysis before and after pathway inhibition, without using e.g. FACS analysis or immunofluorescence (IF)-staining based assessment. A functional assessment and validation of the induced cells is also completely missing.

The following points are not clear and need to be addressed by the authors:
1. Notably, in Figure 1D, certain PSM markers (TBXT, MSGN1, WNT3A) show higher expression on day 3. If the authors initiate SM induction on day 3 instead of day 4, could this potentially enhance the efficiency of syndetome-like cell induction?

2. In the third paragraph of the result section the authors note, "Interestingly, SCX, a prominent tenogenic transcription factor, was significantly downregulated at the SCL stage compared to iPSC, but upregulated during the differentiation from SCL to SYN." Despite this increase, the expression level of SCX in SYN remains lower than that in iPSCs in Fig.1G and Fig.3C. Can the authors provide an explanation for this? Can the authors provide IF data using iPSCs and compare it with in vitro-induced SYN cells? Can the authors provide e.g. additional scRNAseq data which could support this statement?

3. In the fourth paragraph of the result section the authors state, "SM markers (MEOX1, PAX3) and SCL markers (PAX1, PAX9, NKX3.2, SOX9) were upregulated in a stepwise manner." However, the data for MEOX1 and NKX3.2 seems to be missing from Figure 3B-C. The authors should provide this data and/or additional support for their claim.

4. In Figures 2B and 2E, the background of the red channel seems extremely high. Are there better images available, particularly for MEOX1? Given the expected high expression of MEOX1 in SM cells, the authors should observe a strong signal in the nucleus of the stained somitic mesoderm-like cells, but that is not the case in the shown figure. The authors should provide separate channel images instead of merged ones for clarity. The antibody which the authors used might not be specific. Can the authors provide images using an antibody which has been shown to work previously e.g. antibody by ATLAS (Cat#: HPA045214)?

5. In Fig. 2C and Supplementary Fig. 1, the authors present data from immunofluorescence (IF) staining and FACS analysis using a DLL1 antibody. While FACS analysis indicates an efficiency of 96.2% for DLL1+ cells, this was not clearly observed in their IF data. How can the authors explain this discrepancy? Could the authors quantify their IF data and compare it with the corresponding FACS data?

6. In Fig. 2G, PAX9 is expected to be expressed in the nucleus, but the shown IF staining does not appear to be localized to the nucleus. Could the authors provide improved or alternative images to clarify this? The authors should use antibodies shown to work with high specificity as already reported by other groups.

7. Why did the authors choose to display day 10 data for SYN induction in Fig. 4A? Could they provide information about the endpoint of their culture at day 21?

8. In Supplementary Fig. 5, the authors depicted the expression level of SCX, a SYN marker, which peaked at day 14 and then decreased. By day 21, it reached a level comparable to that of iPSCs. Given this observation, could the authors provide a characterization of the cells at day 21 during SYN induction using IF? What was the rationale behind selecting 21 days for SYN induction? The authors also need to show 'n numbers'; how many times were the experiments repeated independently (independent experiments)?
9. Overall the shown immunofluorescence (IF) data does not appear convincing. Could the authors please provide clearer images, including separate channel images, a bright field image, and magnified views of each staining?

10. As stated by the authors in the manuscript, another research group performed FACS analysis to assess the efficiency of syndetome induction using SCX antibody, and/or quantification of immunofluorescence (IF) with SCX, MKX, COL1A1, or COL2A1 antibodies. Could the authors conduct a comparative analysis of syndetome induction efficiency both before and after protocol optimization, utilizing FACS analysis in conjunction with an SCX reporter line or antibody staining, e.g. quantifying induction efficiency via immunofluorescence (IF) staining with syndetome-specific marker genes?

11. To enhance the paper's significance, the authors should conduct functional validation experiments and proper assessment of their induced syndetome-like cells. They could perform e.g. xeno-transplantation experiments with syndetome cells into SCID-mice or injury models. They could also assess whether the in vitro induced cells could be applied for in vitro tendon/ligament formation.

12. The authors should also compare their scRNA-seq data with actual human embryo data sets, something which could be done given the recent increase in available human embryo scRNA-seq data sets.

Reviewer #3 (Public Review):

Papalamprou et al sought to fine-tune existing tenogenic differentiation protocols to develop a robust multi-step differentiation protocol to induce tendon cells from human GMP-ready iPSCs. In so doing, they found that while existing protocols are capable of driving cells towards a syndetome-like fate, the resultant cultures contain highly heterogeneous cell populations with sub-optimal cell survival. Through single-cell transcriptomic analysis, they identify WNT signaling as a potential driver of an off-target neural population and show that inhibition of WNT signaling at the later 2 stages of differentiation can be used to promote higher efficiency of generation of syndetome-like cells.

This paper includes a useful paradigm for identifying transcriptional modulators of cell fate during differentiation and a clear example where transcriptional data can be used to guide the chemical modulation of a differentiation protocol to improve cell output. The paper's conclusions are mostly well supported by the data, but the image analysis and figure presentation need to be improved to strengthen the impact.

The data outlining the differences between the differentiation outcome of the two tested iPSCs is intriguing, but the authors fail to comment on potential differences between the two iPSC lines that could result in drastically different cell outputs from the same differentiation protocol. This is a critically important point, as the majority of the SCX+ cells generated from the 007i cells using their WNTi protocol were found in the FC subpopulation that failed to form from the 83i line under the same protocol. From the analysis of only these 2 cell lines in vitro, it is difficult to assess whether this WNTi protocol can be broadly used to generate tenogenic cells.

The authors make claims to changes in protein expression but fail to quantify either fluorescence intensity or percent cell expression from their immunofluorescence analyses to substantiate these claims. These claims are not fully supported by the data as presented as it is unclear whether there is increased expression of tendon markers at the protein level or more cells surviving the protocol. Additionally, in images where 3 channels are merged, it would be helpful to show individual channels where genes are shown in similar spectra (ie. Fig 2I SCX/MKX). Furthermore, the current layout and labelling scheme of Figure 4 makes it very difficult to compare conditions between SYN and SYNWNTi protocols.

Individual data points should also be presented for all qPCR experiments (ie. Fig 4A). Biological replicate information is missing from several experiments, particularly the immunofluorescence data, and it is unclear whether the qPCR data was generated from technical or biological replicates.