Resistance of Human Liver Mesenchymal Stem Cells to FAS-Induced Cell Death

Round 1

Reviewer 1 Report

Kholodenko et al reported increasing resistance of human liver MSC to Fas induced cell death during inflammatory processes. Results from this study were sound, and the study design is appreciated. There are only minor issues that need to be addressed.

1. The authors isolated the L-MSC from biopsies and verified the expression of MSC markers by flowcytometry, did the authors also monitor the markers until the 5th passage as used (line 106)? 

2. TNFalpha is used as inflammatory pretreatment of the L-MSCs, TNFalpha is known to be effective in hepatocytes, did the authors check on other pro-inflammatory cytokins? Or do authors have evidence that L-MSC already aquired features of hepatocytes? Please could authors discuss on this?

3. The authors may consider a way to improve the statistical analysis, for example comparing detected events.

4. Please could authors add a positive control for the metabolic activity recorded by MTT assay -supplemental?

Fig 5 Isotype controls are missing

The discussion brings the findings in a broad context in comparison with resent literature and reporting honestly about study limitations.

 

Author Response

Dear Reviewer,

Thank you very much for studying our manuscript and valuable comments.

We tried to give the most complete answers and make appropriate corrections to the text and figures.

 

Kholodenko et al reported increasing resistance of human liver MSC to Fas induced cell death during inflammatory processes. Results from this study were sound, and the study design is appreciated. There are only minor issues that need to be addressed.

 

1. The authors isolated the L-MSC from biopsies and verified the expression of MSC markers by flowcytometry, did the authors also monitor the markers until the 5th passage as used (line 106)?

 

At the isolation stage, we did not perform a flow cytometry analysis of the surface marker expression. We usually carry out the first such analysis at passage 2-3. We usually do not systematically monitor the expression of surface markers at each subsequent passage. However, for individual cultures of L-MSC obtained from different donors, we periodically analyze the expression of MSC markers in order to monitor possible phenotypic changes. In these control experiments, we see that the MSC-like phenotype of L-MSCs remains stable over long passages (approximately 2 to 10 passages). In our work, we used cells no later than the 5th passage. Since there is a lot of data in the literature that MSCs can change their properties during long-term cultivation, in particular, they differentiate worse in vitro, their immunomodulatory properties and sensitivity to apoptosis-inducing signals can also change, the proliferation rate slows down, etc.

 

2. TNFalpha is used as inflammatory pretreatment of the L-MSCs, TNFalpha is known to be effective in hepatocytes, did the authors check on other pro-inflammatory cytokins? Or do authors have evidence that L-MSC already aquired features of hepatocytes? Please could authors discuss on this?

 

The main objective of these experiments was to create a pro-inflammatory environment in order to increase the sensitivity of cells to Fas-induced apoptosis. When choosing a proinflammatory cytokine for these studies, we primarily proceeded from the fact that TNFalpha plays a key role in the development of liver fibrosis (doi: 10.1007/s40139-015-0093-z). Since we isolated MSCs from the fibrotic liver, where the corresponding inflammatory process is already present, this cytokine seemed to us the most suitable. Moreover, several studies have shown that in various pathologies associated with the inflammatory process, TNFa plays a decisive role in the induction of apoptosis/autophagy of transplanted MSCs (DOI: 10.1038/nm.2542; DOI: 10.4161/auto.28771). In our preliminary studies in the MTT test, we analyzed the effect of cytokines such as TNFα, TGFβ on the viability of L-MSCs (both cytokines stimulate the development of liver fibrosis) and IFNγ (https://doi.org/10.1016/S1542-3565(05)00404-0), IL6 (DOI: 10.1016/j.cytogfr.2018.01.002) (both cytokines have anti-fibrotic properties); and did not find any negative effect on L-MSC viability. We have included these results in the Supplementary Files (Figure S7). We have also added to Section 3.5. relevant explanations and references (lines 292-300).

Earlier in our work (doi: 10.3390/cells8101127) we, as well as other teams studying L-MSCs, discussed a certain hepatic commitment of these cells. However, we do not claim that L-MSCs acquire the properties of hepatocytes. L-MSCs, like MSCs from other sources, can be differentiated into hepatocyte-like cells in vitro. According to the literature, they differentiate more efficiently than, for example, umbilical cord-derived MSCs (reference [29]).

 

3. The authors may consider a way to improve the statistical analysis, for example comparing detected events.

 

We improved the statistical analysis by plotting the statistical differences between the control values and the obtained effects.

4. Please could authors add a positive control for the metabolic activity recorded by MTT assay -supplemental?

 

We have added MTT test data to the supplementary data for positive control of metabolic activity with CoCl2 and STS (Figure S5 C, D).

 

Fig 5 Isotype controls are missing

This experiment does not suggest an isotype control as there is no antibody staining. Cells are stained with PI; the control is represented by the upper left histogram.

 

The discussion brings the findings in a broad context in comparison with resent literature and reporting honestly about study limitations.

Author Response File: Author Response.pdf

Reviewer 2 Report

Journal: Curr. Issues Mol. Biol

Title: Resistance of human liver mesenchymal stem cells to Fas-induced cell death

Ms No: cimb-1797767

 

This article demonstrate that human L-MSCs have an increased resistance to Fas receptor mediated cell death both in normal and under inflammatory conditions. However, there are many major concerns after carefully go through for this manuscript, in particular for “Results” section. Therefore, it makes hard for readers to understand as well as not scientific sound for publish before major revision.

 

Major concerns:

1.     Why choice biopsies of liver fibrosis patients instead of health people? Please explain?

2.     Please provide more detail description of MSC isolation in the “2.1. Isolation and cultivation of human liver MSCs” section.

3.     CD95 (APO-1/Fas) is a death receptor used by immune cells to kill cancer cells through induction of apoptosis.  Why highly expression of CD95 on MSCs ? Please explain and confirm this data?

4.     “the cells were preincubated with TNF-α (20 ng/mL) (Merck Life 128 Science LLC, Moscow, Russia) for 48 h” (Line 128-129), why treatment 48 hr instead of pre-treatment for 24 hr because it’s possible induce cell death for a longer incubation. Please explain and included citation reference.

5.     Please provide the calcein AM staining data to double confirm the MTT result and make it to be more convinence.

6.     Please move Figure S1 then combind to Figure 1. 

7.     Please provide the negative markers of MSCs (CD14, CD45 and CD34) in the Figure 1.

8.     Please provide the quantification data of fluorescence intensity in the Figure 1.

9.     Please provide the new data of Figure S2, the images of ORO and ARS staining seems not good.

10.  “Analysis of CD95 expression in three cultures of L-MSCs showed that all cell populations express it at a high level (Figure 1). This high and homogeneous Fas/CD95 expression may indicate the potential sensitivity of the cells to Fas-induced cell death due to the interaction of CD95 on the cell surface with FasL or anti-Fas mAb.” (Line 182-185). Please provide direct evidence for this description “his high and homogeneous Fas/CD95 expression may indicate the potential sensitivity of the cells to Fas-induced cell death due to the interaction of CD95 on the cell surface with FasL or anti-Fas mAb.”

11.  Please provide the new data of 24 hr and 48 hr in the “Figure 2”.

12.  Please provide the histogram data in the Figure 2 as well as include 24 hr and 48 hr. 

13.  Please provide the IC 50 data by MTT test and included into Figure 2A&2B.

14.  As we know, the normal working concentration of Fas mAb around (mg/ml), why author use ng/ml in your study. Please confirm and explain that to make it to be more convinence in your study.

15.  Please provide the statistical difference in the Figure 2C and Figure 2D.

16.  lease remove Figure S4, it’s seems not necessary include in this article.

17.  Why author use “short-term incubation (4 h) of the cells with these inducers at high doses (2 μg/mL anti-Fas mAb or 500 ng/mL FasL)” (line 227-line229) instead of 24 hr or 48 hr? Please explain?

18.  Please provide Annexin/PI staining data by flow cytometery assay and include to Figure 2.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

Thank you very much for studying our manuscript and valuable comments.

We tried to give the most complete answers and make appropriate corrections to the text and figures.

 

This article demonstrate that human L-MSCs have an increased resistance to Fas receptor mediated cell death both in normal and under inflammatory conditions. However, there are many major concerns after carefully go through for this manuscript, in particular for “Results” section. Therefore, it makes hard for readers to understand as well as not scientific sound for publish before major revision.

 

Major concerns:

 

1. Why choice biopsies of liver fibrosis patients instead of health people? Please explain?

The sampling of liver biopsy from patients is carried out exclusively for medical reasons during the corresponding surgical operation. There are no grounds for such manipulations in healthy people.

 

2. Please provide more detail description of MSC isolation in the “2.1. Isolation and cultivation of human liver MSCs” section.

We added a detailed description of the method for isolating MSCs (lines 101-106) to section 2.1.

 

3. CD95 (APO-1/Fas) is a death receptor used by immune cells to kill cancer cells through induction of apoptosis. Why highly expression of CD95 on MSCs ? Please explain and confirm this data?

Many studies on the phenotyping of MSCs isolated from different sources show high expression of CD95 (DOI: 10.1159/000129013, https://doi.org/10.1016/j.cellbi.2007.08.002, doi: 10.1002/cyto.a.23574). We added these references in section “Discussion” ([56-58]). In an early work, Mazar et al. (DOI: 10.1074/jbc.M109.032235) showed that despite the high expression of Fas by bone marrow MSCs, the cells are resistant to Fas-induced apoptosis ([59]). And one of the immunosuppression mechanisms is Fas-mediated induction of apoptosis in activated lymphocytes by bone marrow MSCs ([59]).

 

4. “the cells were preincubated with TNF-α (20 ng/mL) (Merck Life 128 Science LLC, Moscow, Russia) for 48 h” (Line 128-129), why treatment 48 hr instead of pre-treatment for 24 hr because it’s possible induce cell death for a longer incubation. Please explain and included citation reference.

Since TNF-α by itself did not induce L-MSC cell death (added the results of the MTT test to the Figure S7), for perhaps better sensitization to the subsequent induction of apoptosis through Fas, we chose such a long time. Faletti et al. ([43]) showed that TNFα itself did neither induce caspase-3 activity nor apoptosis in mouse primary hepatocyte, mouse hepatoma Hepa1-6 cells, immortalized mouse AML12 hepatocytes and primary and SV40-immortalized mouse embryo fibroblasts. In the PI test, we also showed that TNFα does not induce cell death of L-MSC (Figure 5). According to Rex et al. ([44]), after treatment of hepatocytes with TNFα, pro-apoptotic proteins up-regulate, and anti-apoptotic proteins downregulate over time (1-30h).

 

5. Please provide the calcein AM staining data to double confirm the MTT result and make it to be more convinence.

In our work, we usually use CalceinAM to analyze the lysis of target cells by immune cells by effectors, for example, in co-culture of activated allogenic lymphocytes with L-MSCs. To assess the viability of individual cell cultures, the MTT method is used, since under our conditions it exhibits greater sensitivity and is characterized by better reproducibility than the method with CalceinAM. Also, the greater sensitivity of the MTT method compared to CalceinAM is shown in various works, for example, in [DOI: 10.1177/1087057104265386]. In addition, in the MTT test, we increased the starting anti-Fas mAbconcentration to 2 μg/mL, which did not lead to a decrease in cell viability (Figure S5). Therefore, if there are no anti-Fas mAb effects (at high concentrations) in MTT and PI tests, it is difficult to assume that they can be registered using the CalceinAM.

 

6. Please move Figure S1 then combind to Figure 1.

We combined Figure S1 with Figure 1.

 

7. Please provide the negative markers of MSCs (CD14, CD45 and CD34) in the Figure 1.

We provided negative markers of L-MSCs in the Figure 1.

 

8. Please provide the quantification data of fluorescence intensity in the Figure 1.

In Figure 1, we added RFI for quantification data of fluorescence intensity.

 

9. Please provide the new data of Figure S2, the images of ORO and ARS staining seems not good.

We replaced the data with new ones.

 

10. “Analysis of CD95 expression in three cultures of L-MSCs showed that all cell populations express it at a high level (Figure 1). This high and homogeneous Fas/CD95 expression may indicate the potential sensitivity of the cells to Fas-induced cell death due to the interaction of CD95 on the cell surface with FasL or anti-Fas mAb.” (Line 182-185). Please provide direct evidence for this description “his high and homogeneous Fas/CD95 expression may indicate the potential sensitivity of the cells to Fas-induced cell death due to the interaction of CD95 on the cell surface with FasL or anti-Fas mAb.”

In this case, we do not state that high expression of CD95 is necessarily associated with cell sensitivity to Fas-induced apoptosis. We suggest that the high expression of CD95 may be indicative of this. We have corrected the text to make it more obvious (line 197).

 

11. Please provide the new data of 24 hr and 48 hr in the “Figure 2”.

Figure 2 shows the results of the PI test averaged over three cultures of MSCs obtained from three different donors. Representative data at 24h and 48h presented as histograms in the Supplementary Files (Figures S2-S4).

12. Please provide the histogram data in the Figure 2 as well as include 24 hr and 48 hr.

The histograms presented in the Supplementary Files (Figures S2-S4).

13. Please provide the IC 50 data by MTT test and included into Figure 2A&2B.

In the MTT test, we did not reach the IC50 for the anti-Fas mAb and FasL. In Figure S5 indicated IC 50>2µg/mL and IC50>500ng/mL for anti-Fas mAb and FasL, respectively, and IC50 for CoCl2 and STS.

 

14. As we know, the normal working concentration of Fas mAb around (mg/ml), why author use ng/ml in your study. Please confirm and explain that to make it to be more convinence in your study.

The selection of the concentration range of anti-Fas mAb was carried out on the basis of literature data. Anti-Fas mAb clone DX2 was used in this work, for which the working apoptosis-inducing concentration is 1 µg/mL (DOI: 10.1084/jem.180.4.1547). The maximum concentration that we used to induce cell death was 2 µg/ml. In the MTT test, the maximum concentration of anti-Fas mAb was increased to 2 µg/ml. The new data is presented in Figure S5.

 

15. Please provide the statistical difference in the Figure 2C and Figure 2D.

Statistical differences between the control values and the obtained effects were noted on the graphs.

 

16. lease remove Figure S4, it’s seems not necessary include in this article.

Figure S4 (S6) demonstrates no effect of low doses of FasL and anti-Fas mAb on mitochondrial membrane potential. In the manuscript it is written about this and given a link to this figure (lines 235-237).

 

17. Why author use “short-term incubation (4 h) of the cells with these inducers at high doses (2 μg/mL anti-Fas mAb or 500 ng/mL FasL)” (line 227-line229) instead of 24 hr or 48 hr? Please explain?

In this series of experiments, we used short-term incubation (4 h) with inducers, since changes in the mitochondrial membrane potential usually occur within a short time after Fas binding on the cell surface (10.1016/j.bpj.2009.07.056, 10.1074/jbc.275.16.11814).

 

18. Please provide Annexin/PI staining data by flow cytometery assay and include to Figure 2.

In our studies of MSC apoptosis, we found that, in the case of L-MSC, Annexin/PI staining is not applicable to assess apoptosis, since these cells normally exhibit phosphatidyl serine on the outer monolayer of the plasma membrane (our unpublished data), i.e. L-MSCs cultured under standard conditions without the addition of cell death inducers stain positively with AnnexinV. This phenomenon is the subject of our further study.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Authors were adequately responded all comments.