Influence of Angiopoietin Treatment with Hypoxia and Normoxia on Human Intervertebral Disc Progenitor Cell’s Proliferation, Metabolic Activity, and Phenotype
, Andrea Oberli 1, Andreas S. Croft 1, Katharina A. C. Oswald 2, Christoph E. Albers 2, Benjamin Gantenbein 1,2,* and Julien Guerrero 1Round 1
Reviewer 1 Report
Bischof et al assessed and investigated the effect of angiopoietin growth factor on the IVD progenitor cells( particular Tie2+/- cell population) under both normoxic and hypoxic conditions.
The manuscript is well written and has an impeccable fluency. Some minor aspects to be addressed:
-P3, Line 119-111& Table 1. If possible, would be useful to include the Pfirrmann score for each patient in Table 1. Also, the abbreviations from Table 1 have to be explained.
- Were the cells isolated from b NP, AF and CEP? Or did the authors differentiate also between the components? As starting from the abstract the focus is placed on NP cells.
- I do strongly appreciate the transparency in the statistical analysis. If the data is not normally distributed data, why are the mean values displayed ( i.e. Figure1, 2, 3) and not the median( commonly used in this situations). Maybe in order compare the results with those of previous published works?!?
-Figure 1. -Any particular reason for the different “N”- Tie2+ (N=2) and Tie2- (N=3)? I believe this aspect has to be stated as it impacts the statistical analysis.
- I do salute the transparency with respect to the study limitations. Another limitation of the study is that there are actually no truly healthy samples?
Author Response
Reviewer 1:
The manuscript is well written and has an impeccable fluency.
Author response: We really appreciate the reviewer’s feedback concerning our manuscript. We thank the reviewer for his thoughtful and thorough review and believe his input has been invaluable to make our manuscript more accurate.
However, some minor aspects have to be addressed:
- P3, Line 119-111& Table 1. If possible, would be useful to include the Pfirrmann score for each patient in Table 1. Also, the abbreviations from Table 1 have to be explained.
Answer: Thank you for pointing this out. According to the Reviewer’s comment, we detailed the abbreviations present in the caption of Table 1 However, the Pfirrmann score was not available for the samples used in our study. According to the reviewer’s comment and to clarify our manuscript, the related part of the Materials and Methods was deleted to avoid confusion.
- Were the cells isolated from NP, AF and CEP ? Or did the authors differentiate also between the components? As from the abstract, the focus is placed on NP cells.
Answer: Indeed, we did not precise the fate of the AF and CEP cells in the Materials and Methods section. However, the isolated cells used in our study were exclusively isolated from the NP tissue. The cells extracted from AF and CEP were frozen and used for other studies. To improve the clarity of our manuscript the following sentence was added to the Materials and Methods section;
Page 4, Line 139; “The NP cell suspension was then transferred into T150 culture flasks (#90151; TPP, Trasadingen, Switzerland). Furthermore, cells from AF and CEP tissue were frozen.”
- I do strongly appreciate the transparency in the statistical analysis. If the data is not normally distributed data, why are the mean values displayed (e. Figure 1, 2, 3) and not the median (commonly used in this situations). Maybe in order compare the results with those of previous published works?!?.
Answer: Thank you for your feedback. It is true that in Figures 1, 2, and 3, data are represented as mean +/- SD. This representation was used as no outliers were present in our set of data and as a result, our representation was not suffering from skews. Moreover, the mean values were displayed mainly to compare the results of previously published work (Guerrero et al., 2020; Zhang et al., 2020; Frauchiger et al., 2019).
- Figure -Any particular reason for the different “N”- Tie2+ (N=2) and Tie2- (N=3)? I believe this aspect has to be stated as it impacts the statistical analysis.
Answer: Thank you for this note. We agree with the reviewer's comment that the difference in “N” between the Tie2+population and the Tie2- population has to be stated in the manuscript. The low amount of “N” in the Tie2+population is essentially due to the difficulty in expanding these NP progenitor cells as stated in previous work (aka. Guerrero et al., 2020). To clarify and to improve the manuscript, the following sentence was added to the document;
Page 15, Line 600; “Moreover, previous studies were demonstrating the difficulties to expand in vitro this population [16,44].”
- I do salute the transparency with respect to the study limitations. Another limitation of the study is that there are actually no truly healthy samples?
Answer: Thank you for bringing this to our attention. We agree with the reviewer’s comment. However, IVD isolated from trauma patients is the closest tissue from a healthy state that is available in our site of research. However, this limitation from our study is already stated in the manuscript;
Page 15, Line 601; “Additionally, the “controls” used in the study were not truly healthy IVDs, as they were traumatic discs.”
Reviewer 2 Report
[Abstract] I suggest adding more specific results in the abstract, especially the word “variation” (24 and 26). Please indicate the information of NP cells such as source (human, pathologic?)
[Materials and Methods] Do you have any reason to isolate AF and CEP cells? There is no result for these cells.
How do you prepare the mixed population? Just mixed 50% Tie2- and 50% Tie+?
What is Tie+ population before cell sorting?
[Conclusions] Please summary your results and add clinical implication if possible.
Author Response
Reviewer 2:
We thank the reviewer for their careful reading of the manuscript and their constructive remarks. We have taken the comments on board to improve and clarify the manuscript. Please find below a detailed point-by-point response to all comments.
- [Abstract] I suggest adding more specific results in the abstract, especially the word “variation” (24 and 26). Please indicate the information of NP cells such as source (human, pathologic?)
Answer: Thank you for reporting it. We agree with the Reviewer’s comment that the addition of more specific results is needed in the abstract. For this reason, the term “variation” was replaced in the abstract to add more specificity in the result’s description. Additionally, the NP cell source was added.
- [Materials and Methods] Do you have any reason to isolate AF and CEP cells? There is no result for these cells.
Answer: Thank you for pointing this out. The AF and CEP cells were isolated for others studies ongoing in the lab. We precised that they were just simply frozen to clarify the manuscript.
- How do you prepare the mixed population? Just mixed 50% Tie2- and 50% Tie2+?
Answer: Thank you for this question. The mixed population was indeed the population just before sorting them into Tie2+ and Tie2- cell populations (aka. Native NP population). To clarify and to improve our manuscript we added the following sentence;
Page 4, Line 159; “The NP cell population prior to the sorting process was considered as mixed NP cell population, and the NP cell population after the sorting process was considered as Tie2+ or Tie2- NP cell populations.”
- What is Tie2+ population before cell sorting?
Answer:, The percentage and the phenotype of the Tie2+ cells present in the native NP tissue (aka. progenitor cells) are already well described in the literature (Sakai et al., 2012).
- [Conclusions] Please summary your results and add clinical implication if possible.
Answer: We agree with the reviewer’s comment that the results needed to be summarized and that a clinical implication needs to be stated. For this reason, the following sentences were added to the manuscript.
Page 16, Line 627; “In this study, we used human NP cells isolated from trauma patients’ IVDs. We demonstrated the significant impact of culturing NP cells under hypoxic conditions to improve proliferation, metabolism and gene expression of NP progenitor cells. However, no impact or effects were observed concerning the implementation of Ang-1 or Ang-2 in our culture medium.
The ensuing findings could directly impact clinical and therapeutic strategies in contrast to preclinical studies in other species that often need to be verified in humans first. Additionally, we provided novel evidence that culture within a hypoxic environment can drastically influence the proliferation, metabolism, and gene expression of the different cell populations present in the human NP tissue. Moreover, we demonstrate that the hypoxic conditions affect more the progenitor cell populations from the human NP tissue (aka. Tie2+) than the two other populations (mixed and Tie2-). All those findings could lead to a new way to culture NP progenitors cells prior to clinical applications.”
Round 2
Reviewer 2 Report
Thank you for editing. Now, it is much clear.
