PPIA and YWHAZ Constitute a Stable Pair of Reference Genes during Electrical Stimulation in Mesenchymal Stem Cells
Round 1
Reviewer 1 Report
The authors of this article have tackled an important issue, that is often not taken into account in scientific research, which is utilization of standard housekeeping genes without proper optimization studies. In this article, the authors reveal that the most stable housekeeping genes for the analysis of electrical stimulation of MSCs often might differ from the most popularized housekeeping genes used in this type of research.
I have no major corrections, only a minor detail should be considered:
Q1) The authors stated that MSCs were exposed to an electric stimulation of 100 mV/mm. Considering that a cell viability test was performed (based on exposure time), please justify this choice of electric field strength.
Author Response
Dear reviewer,
Thank you for your comments.
Q1) In response to your comment, I have included the following sentence in the discussion:
"The electric field strength was selected primarily because the same ES device had also shown pro-osteogenic effects at 100 mV/mm in the literature, and it falls within the range of physiological EFs that are established in vivo for both development and regeneration [13],[37]."
Reviewer 2 Report
The authors presents a very well written and clearly conducted study. My only comment considers the Figure legend, namely use the same abbreviations in all of them (legends for Figure 4 and 5 needs to be changed).
Author Response
Thank you for your comments.
The Figure legends 4 and 5 have been modified for further clarity, as shown below. The abbreviation +ES only applies to Figure 5, so has not been included in the Figure 4 legend.
Figure 4: The NormFinder stability value results for the most stable gene across both unstimulated control cells and cells stimulated for 60 minutes per day. A separate NormFinder analysis was performed for cells seeded at (a) 5,000 cells/cm2, and (b) 10,000 cells/cm2.
Figure 5. Gene expression analysis performed using the ∆∆CT method in unstimulated controls (Control) and cells stimulated for 60 minutes per day (+ES). Expression is normalized to the control and to the gene determined to be the most stable for the respective seeding density by NormFinder. Each graph corresponds to the two seeding densities used: (a) 5,000 cells/cm2, and (b) 10,000 cells/cm2. *p<0.05 versus control of the same gene. Each gene was analyzed separately using an unpaired t-test; n=2 for Control, n=3 for +ES group.
Reviewer 3 Report
Interesting experimental design and well conducted.
Authors must present references throughout the text in accordance with the journal's rules.
The article must be reformatted, the spaces between the lines are not always the same.
NTRODUCTION
Line 36 – the reference is missing
Line 55 - the reference is missing
Line 56 - change “Articles have” to “some studies have”
MATERIALS AND METHODS
“…was used as 67 described previously…”; “…The ES device was as described previously…” - It is in the materials and methods that authors must do this description.
RESULTS
Line 367 – Results it’s results
DISCUSSION
Lines 381; 385; 391; 397; 399; 420; 425;
CONCLUSIONS
The real conclusions are missing. Authors must present them objectively.
Author Response
Thank you for your comments.
References were used where appropriate and relevant. The difference in spacing between sections was as per the journal template.
We have included a reference for lines 36 and 55, and Line 56 has been changed as follows:
Introduction:
Of the techniques used to gather these data, one of the most common is real-time quantitative reverse transcription polymerase chain reaction, qRT-PCR [1].
An accurate, rapid and convenient method for assessing the effect of ES on MSCs is to investigate changes in gene expression, including differentiation marker genes, through qRT-PCR [1].
Some studies have been published on the ideal reference genes for equine and porcine MSCs in growth conditions and multilineage differentiation [2], [18], bone marrow and umbilical cord-derived human MSCs in long-term culture [19], adipose-derived MSCs [20], and in MSCs undergoing osteogenic differentiation [21], and tenogenic differentiation [22].
We also thank the reviewer for raising our attention to the fact that ‘as previously described’ was not the correct wording for either the section on the Y201 cells or the ES device. The wording has now been changed in both instances.
An immortalized human MSC line, Y201, gifted by Dr. Stephen Richardson, was used [23].
An electric field of 100 mV/mm was applied to the cells in a 6-well plate through 2 x 0.5 mm 99.95% L-shaped platinum wire (Alfa Aesar) electrodes using a ‘9130’ programmable direct current (DC) power supply (BK Precision) [16].
In line 367, we have kept the capital R as this was used in the context of a proper noun.
The comment concerning line 381 has been addressed by adding a reference to the Supplementary Table S.1.
In general, most gene expression studies use only one reference gene, rather than a pair of reference genes (Table S.1).
Three references ([2], [19], and [22]) have been added to line 399, as follows:
Many other published reference or ‘housekeeping’ gene articles use BestKeeper, NormFinder, and also GeNorm [2], [19], [22].
The lack of a reference in lines 385, 391, 397, 420, and 425 was not resolved as these comments were either generally accepted facts in the field about standard experimental procedure, or discussion points surrounding the findings arising from this study.
Facts on experimental procedure:
Not only is RNA quantification often prone to error, but also the RNA-to-cDNA reverse transcription efficiency can be variable between samples.
The input concentration of cDNA is calculated by assuming a ‘perfect’ reverse transcription efficiency of 100% for all genes [29]; however, the actual efficiency is unknown and will differ, as already mentioned, between genes and between samples.
A further advantage of using a pair of reference genes is the ability to quickly identify a scenario in which either gene does change.
Facts on our own findings:
Evidently, the seeding density plays a role in the cells’ response to ES.
Based on our findings, the use of GAPDH as the only reference gene in ES studies should be verified before continued use, especially at lower seeding densities.
Please note that due to earlier changes in the manuscript, some line numbers have now changed.
We do not think it necessary to include a Conclusion section. As per the “Instructions for Authors” issued by the Applied Sciences journal, a conclusion is only necessary where the Discussion is overly complex, and this is not the case for this work.
Reviewer 4 Report
It is a novel in-vitro study aimed to constitute a stable pair of reference genes during electrical stimulation in mesenchymal stem cells other than GAPDH and ACTB. The authors used an immortalized human MSC line and primary bone-marrow human MSCs with a seeding density of 5,000 cells/cm2 or 10,000 cells/cm2, stimulated for 30 min or 60 min per day (m/d) for a period of 5 days. They support the use of PPIA and YWHAZ as an optimal reference gene pair and discourage the use of ACTB and GAPDH at lower seeding densities. The introduction has a rationale, design and methodology are sound and the results are clear. Only, the authors asked to specify exactly what is the actual period for electrical stimulation; 4 days or 5 days?
Author Response
Thank you for your comments.
Electrical stimulation was applied for 5 days. This has been explained in more detail in the RNA Extraction section:
The stimulated cells underwent 30 or 60 m/d of ES over 5 days; RNA was extracted from the cells immediately after the last period of ES on day 5.
And the Metabolic activity section of the Materials and Methods:
The stimulated cells underwent 30 or 60 m/d of ES over 5 days. The cellular metabolic activity was measured on day 5, prior to the final ES period, using the Deep Blue Cell Viability Kit (BioLegend).
A similar sentence was also included in Results section 3.2:
The cellular metabolic activity was measured on day 5, prior to the final ES period.
This was also further explained in Results section 3.3:
Part of this study was to determine if increasing the duration of the ES over 5 daily applications had an effect on the expression of the six potential reference genes in our study.
Round 2
Reviewer 1 Report
Mesenchymal stem cells (MSCs) are widely studied in regenerative medince, since they have a great potential in diverse application. The authors analyzed reported genes to find th e most proper reference genes. In conclusion, they could have presented the most stable reference genes, PPIA and YWHAZ. This will be helpful for basic stem cell research in this field.
