Towards Tissue-Specific Stem Cell Therapy for the Intervertebral Disc: PPARδ Agonist Increases the Yield of Human Nucleus Pulposus Progenitor Cells in Expansion

Round 1

Reviewer 1 Report

The authors describe the effect of the PPARδ agonist on the increase of the self-renewal ability of Tie2+ NPCs. The authors suggest that the PPARδ agonist GW501516 might increase the self-renewal ability of Tie2+ NPCs by the stimulation of the mitophagy pathway.

The manuscript is well written, clear, easy to understand and offering potentially important information. The following concerns should be addressed before the manuscript can be considered for publication.

Abstract:

• The authors should introduce the term “NPC” in the abstract.

Introduction:

• The authors should explain better why NPPCs could be a future cell source for therapy, as they refer this in the abstract.
• Moreover, it is important to highlight in the Introduction section why it is important to increase the Tie2+ NPCs yield during NPC expansion.
• The differences between NPPCs and NPCs should be better described.
• The authors state that the maintenance of NPPC’ stemness over in vitro culture time is needed. Thus, it would be important to explain how this can be applied in a clinical context.
• Since this work describes the effect of the PPARδ agonist on the increase of the self-renewal ability of Tie2+ NPCs, it would be important to explain better the mechanism of action of PPARδ agonist and Tie2+ cells.
• The authors need to explain better how stimulation of mitophagy can increase self-renewal of Tie2+ cells.

Materials and Methods:

• Why were cells cultured under normoxic conditions since the authors claim in the Introduction section (page 2, line 53) that these cells like hypoxic environments.
• Why did the authors choose the concentration of 0.1 uM GW501516? Was this concentration optimized?

Results:

• Quantification of cell circularity revealed that PPARδ agonist treatment resulted in a more rounded cell shape compared to cells cultured within the control. What is the importance of these results? Does the circularity of cells influence its behavior?
• The Tie2+ yield between PPARδ agonist treated group and vehicle group did not present any statistical significant difference. Thus, the authors should hypothesize why this result is relevant and why this is occurring.
• The authors should include more results related to a higher cell passage to compare the results between PPARδ agonist treated group and vehicle group.

Discussion:

• The authors should be more careful in drawing some conclusions (page 8, line 260). The authors state that if the culture time had been increased, a significant difference in gene expression between PPARδ agonist treatment and control group would have been observed, however this may not be true, since no experiments were performed. This paragraph should be rephrased.

Author Response

• Reviewer 1:

  1. The authors should introduce the term “NPC” in the abstract.

   Answer: Thank you for pointing this out. We introduced the term “NPC” on L21.

  2. The authors should explain better why NPPCs could be a future cell source for therapy, as they refer this in the abstract.

   Answer: We appreciate this comment. The NPPCs is one kind of adult progenitor cells. The adult stem cells give a regenerated possible to degeneration disease. We added this detail on L50-54.

  3. Moreover, it is important to highlight in the Introduction section why it is important to increase the Tie2+ NPCs yield during NPC expansion.

  Answer: Thank you for pointing this out. We added more detail on L73-77. In briefly, the primary NPPCs is limited. If only purified NPPCs were expended, they will lose their stemness soon. This can be avoided by expanding the whole NPCs population. To harvest more NPPC, an increased Tie2+ NPCs yield is needed.

  Another reason is, that this method of increasing Tie2+ NPCs yield could be translated in vivo and can be analyzed for its effect against IVDD.

  4. The differences between NPPCs and NPCs should be better described.

  Answer: Thank you for pointing this out. We better described the differences between NPPCs and NPCs on L47-49 “The NPPC (Tie2+ NPCs) are multipotential, but not the adult NPCs (Tie2- NPCs) [8-10]. Besides from the absence of Tie2, adult NPCs can be distinguished from multipotent NPPCs by their lower clonogenic ability [8-10]”.

  5. The authors state that the maintenance of NPPC’ stemness over in vitro culture time is needed. Thus, it would be important to explain how this can be applied in a clinical context.

  Answer: Thank you for pointing this out. On L59 -69, we added hypothesis therapy methods based on NPPC.

  6. Since this work describes the effect of the PPARδ agonist on the increase of the self-renewal ability of Tie2+ NPCs, it would be important to explain better the mechanism of action of PPARδ agonist and Tie2+ cells.

  Answer: Thank you to point out this. We added how PPARδ agonist increase the Tie2+ hematopoietic stem cells self-renewal on L94-102. “Moreover, with Tie2+ hematopoietic stem cells, PPARδ agonist can up-regulated gene expression of PINK1 and PARK2. The upregulation of PINK1 gene was through the upregulation of FOXO3 gene. Upregulation of these genes lead to an increase mitophagy. This mechanism enables self-renewal in Tie2 + -hematopoietic stem cells to be increased by PPARδ agonists. Additionally, hematopoietic Tie2 + stem cells lose their ability to renew themselves when prevent the mitophagy through the inhibition of the PINK1 and PARK2 proteins. On the contrary, this “self-renewal path of PPARδ agonist mitophagy” could not be demonstrated for hematopoietic Tie2- stem cells [20].”

  7. The authors need to explain better how stimulation of mitophagy can increase self-renewal of Tie2+ cells.

   Answer: We appreciate this comment. The mitochondria behavior regulates the stemness fate (L101-103), the mitophagy was a way to clean damage mitochondria (L83-84). In Tie2+ hematopoietic stem, if inhibit the mitophagy relative protein PINK1 and PARK2, the self-renewal could not increase (L98-101).

  8. Why were cells cultured under normoxic conditions since the authors claim in the Introduction section (page 2, line 53) that these cells like hypoxic environments.

  Answer: We appreciate this comment. “The aim of our research was to increase the Tie2+ NPCs percentage of the whole NPC population during expansion. The classic and common culture conditions are based on 2D culture and normoxic environment [13-15]. In this research, we primarily wanted to compare the impact of chemical treatments on the Tie2+ NPPCs population [16,17]. For this reason, the cells were cultured within a normoxic environment. However, the comparison between hypoxia and normoxia could be taken into account for future analyses.” We added this on L335-340.

  9. Why did the authors choose the concentration of 0.1 uM GW501516? Was this concentration optimized?

  Answer: The concentration of GW501516 (0.1 uM) was used in accordance to already published data by Ito et al. in 2016, untitled “Self-renewal of a purified Tie2+ hematopoietic stem cell population relies on mitochondrial clearance”. We did not optimize it for the NPCs. We added this information on L150.

  10. Quantification of cell circularity revealed that PPARδ agonist treatment resulted in a more rounded cell shape compared to cells cultured within the control. What is the importance of these results? Does the circularity of cells influence its behavior?

  Answer:  Thank you for pointing this out. A higher circularity reflects a more naïve phenotype for NPCs (Barcellona, M.N.; Speer, J.E.; Fearing, B.V.; Jing, L.; Pathak, A.; Gupta, M.C.; Buchowski, J.M.; Kelly, M.; Setton, L.A. Control of adhesive ligand density for modulation of nucleus pulposus cell phenotype). With the increase of the degeneration process, the NPCs will show a more fibrotic morphology, and will be more deviate from a round-shaped phenotype. We add this information on L297-299.

  11. The Tie2+ yield between PPARδ agonist treated group and vehicle group did not present any statistically significant difference. Thus, the authors should hypothesize why this result is relevant and why this is occurring. The authors should include more results related to a higher cell passage to compare the results between PPARδ agonist treated group and the vehicle group.

  Answer:  We have added a supplementary figure that shows the gathered data from the group treated with PPARδ agonist and the control group. Thereby, the results showed a significant difference on the Tie2 yield. We added this as supplementary data, as the culture situations were showed differences compared to the main experiments, such as the passage number.

  12. The authors should be more careful in drawing some conclusions (page 8, line 260). The authors state that if the culture time had been increased, a significant difference in gene expression between PPARδ agonist treatment and control group would have been observed, however, this may not be true since no experiments were performed. This paragraph should be rephrased.

  Answer:  Thank you to point out. We decided to delete this overstatement.

Author Response File: Author Response.pdf

Reviewer 2 Report

Reviewer Comments

Thank you to the authors for performing these studies. Before the manuscript can be published, several items must be addressed:

1.3. Mitophagy in NPC67

Suggested edit: In this context, mitophagy relative gene and protein of PARKIN and PINK1 [16] also showed a difference between health and disordered NPCs

2.2. PPARδ agonist Treatment

Please clarify if the cells that were used are the P0 NPs. If cells beyond P0 were used, please state.

2.4 Flow Cytometry

Some immune system cells and endothelial cells also express Tie2. If the NP cells are sourced from injured patients, it may be possible that the Tie2 stain is detecting these extra cell types. Can the authors address this? Is there a significant heterogeneity in the NP cells after extraction? These answers should be discussed

2.7 Statistics

Why was a nonparametric distribution assumed? The normality of data should be checked before analysis. A Q-Q plot can help. After analyzing the distribution, the statistical analysis may need to be revisited.

Author Response

Reviewer 2:

1. Suggested edit: In this context, mitophagy relative gene and protein of PARKIN and PINK1 [16] also showed a difference between health and disordered NPCs.

Answer: We would like to thank you for your suggestion. We edited the manuscript in accordance to the reviewer’s comment on L106.

2. Please clarify if the cells that were used are the P0 NPs. If cells beyond P0 were used, please state.

Answer: We added the passage information on L150.

3. Some immune system cells and endothelial cells also express Tie2. If the NP cells are sourced from injured patients, it may be possible that the Tie2 stain is detecting these extra cell types. Can the authors address this? Is there a significant heterogeneity in the NP cells after extraction? These answers should be discussed.

Answer: We washed the NP tissues twice with PBS to remove potential cell contaminations that can occur during the trauma/injury and surgery. We added this information on L126-127. However, the NP tissue is an avascular tissue, therefore, no endothelial cells should be present in NP tissue. Also, during surgery, there do not have blood vessel contamination. We confirm the NP cells based on the NP tissue but not heterogeneity.

Why was a nonparametric distribution assumed? The normality of data should be checked before analysis. A Q-Q plot can help. After analyzing the distribution, the statistical analysis may need to be revisited.

Answer: Thanks for pointing this out. We performed the Shapiro-Wilk test prior to analysis to check for normally distributed data. According to the plied test, data were non-parametrically distributed. We added this information on L184.

Round 2

Reviewer 1 Report

The authors have addressed all comments suggested by the reviewer.

Reviewer 2 Report

The authors have address the concerns from the original peer review.