Challenges and Considerations of Preclinical Development for iPSC-Based Myogenic Cell Therapy

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is well prepared review focusing at the pluripotent stem cells and their possible application in research and possible in clinics. Since the review covers not only iPSCs but also ESCs I would recommend adjustment of the title to PSCs. However, this is not a major issue.

While reading the manuscript I had a strong impression that Authors are not trained developmental biologists. Thus, some statement might origin from simplification of certain processes and phenomena. This issues should be corrected:

1) Both ESCs and iPSCs are natural - they are living cells, derived either from ICMs or somatic cells. Thus, the sentence in introduction stating that ESCs are natural but iPSCs are not should be corrected;

2) I am not sure if one can state that iPSCs do not rise any ethical issues. They rise different issues, however, especially when they could be used to generate so called artificial embryos, they are not ethically neutral;

3) Neither ESCs nor iPSCs were discovered. One can discover something what existed but was not known. These type of cells were not present before their derivation. Thus, I would suggest word derived, derivation but not discovered, discovery;

4) Neither ESCs nor iPSCs (if not generated with the use of c-Myc) are more tumorigenic than normal somatic cells! Danger of their use comes from the fact that transplantation of pluripotent stem cells may lead to the development of teratomas. But teratomas do not contain tumorigenic cells. Their cells do not divide infinitively like cancer cells, instead they differentiate into terminal tissues, which of course could be dangerous developing in ectopic sites in the recipient body. Of course they can transform as any other cell - and form teratocarcinomas, but this is completely different story. In case iPSCs are generated with the use of c-Myc - yes, they can be tumorigenic - but this issue has to be clearly explained in the manuscript. Thus, the word "tumorigenic" should not be used in the current context within the manuscript.

Besides, I do not see any serious problems with the manuscript.

Author Response

We thank the reviewer 1 for your positive feedback and comments. We have revised our manuscript accordingly and please check the attached word documents for the point-to-point response. 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This review highlights some important aspects of the literature but in its present form it displays several weaknesses. The major issue is the lack of focus. The review is all over the place, but it does not really discuss anything specific about translational studies as alluded on the title. The authors start by introducing the different available protocols to generate iPSC-derived myogenic progenitors.  Then they go on and list genetic modification strategies to correct patient iPSCs. These two parts could make sense in the context of a review. However in the 3^rd part, the authors listed some of the muscular dystrophy models available but not in the context of cell therapy. Some of the models listed are not relevant for cell therapy studies. A list of models developed to study muscular dystrophy disease pathogenesis seems irrelevant for a review focused on cell therapy. There is no discussion whatsoever of preclinical studies showing engraftment of iPSC-derived myogenic progenitors in vivo, outcomes, hurdles, etc. The manufacturing aspect is the weakest part of this review. Based on the senior author’s background, this could have been the most valuable contribution by this review, but it is mostly speculative. They propose cell suspension cultures to scale up cell production, but they cited only one study involving myogenic progenitors, and in this study, it was reported that “the majority of cells exit the cell cycle and do not proliferate”. Is this feasible for translational applications? The last paragraph on future directions mentions gene therapy, which is clearly not in the scope of this review.

In summary, the title “Challenges and benefits for iPSC-based myogenic cell therapy development: from bench to clinic” is not appropriate. The authors addressed the bench aspect but not the clinic aspect, which is too succinct and does not provide any insightful information to the readers. The actual process to bring iPSC derived myogenic progenitors to the clinic is not described and the rare comments are just speculative. Therefore, I suggest the authors revise this review to focus on the generation of myogenic cells from iPSCs, and their applicability (gene editing, therapeutic application, etc), making the manuscript more focused and comprehensive on the bench aspect, and change the title to eliminate the suggestion that this manuscript is about clinical work.

 Minor comments:

1- References are missing for the line 69-70: “The second advancement is made by using nonintegrating virus like adenovirus or sendai virus as reprogramming vehicles, which are diluted out of cells by passaging”

2- Line 85- 87: “So far, although few, there has been clinical trials initiated for hiPSC cell therapies to treat spinal cord injury, Parkinson’s Disease, macular degeneration, retinitis pigmentosa, etc. However, there has been no record of trials using hiPSC in muscular disease therapies.” References and/or clinical trial numbers of the different ongoing clinical trials should be provided.

3- Line 92: “tumorgenicity” should be tumorigenicity.

4- Line 142-144: “When the cells were transplanted into immunodeficient and injured mice, cells were shown to participate in muscle regeneration with a subset of transplanted cells homing to the local satellite cell niche area”. This study also showed engraftment in the non-injured mdx (~10%). A review should be comprehensive.

5- Line 214-217: a lot of “So far”. Please avoid excessive repetitions of the same word.

6- Table 1: the reference number should appear in the table

7- Line 214-255: “So far, fluorescence activated cell sorting (FACS) has been mainly used as the purification method. As each line-age specific method induces the lineage specification slightly different, the surface protein presentations are different (Table 1). So far, several surface markers have been identified in enriching the myogenic progenitor population which have been reported to possess superior myogenic regeneration capability measured by in vitro myotube fusion and in vivo engraftment. When it comes to isolating desired cell populations, FACS and magnetic activated cell sorting (MACS) are usually considered [34]. FACS, which is used in the research setting, could select the cells more precisely with gating, however, it lacks the scalability due to the limited sorting speed (~5 million cells per hour) [35].”

The reference for FACS should be present on line 214

8- Line 295: “DNAsuch” space is missing

9- Line 660: Arpke et al, 2013 should be cited for the generation of the NSG-mdx4cv mouse model.

 

 

Author Response

We thank the reviewer 2 for your feedback and comments. We have revised our manuscript accordingly and please check the attached word documents for the point-to-point response. 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors The authors have addressed several of previous issues. However, there is still concern with the biased manner this review is presented since the translational part focuses only on 3D and transgene free cultures.  

Author Response

The response to the review in the attached file. 

Author Response File: Author Response.pdf