Behavior of Astrocytes Derived from Human Neural Stem Cells Flown onto Space and Their Progenies

Round 1

Reviewer 1 Report

The topic is very interesting and important. We need to know how the neuronal systems functions in microgravity with respect to human spaceflight and exploration. Furthermore, studies under altered mechanical stimulation provides us new knowledge on fundamental physiological processes under 1 g conditions and malfunctioning which might result in diseases. Correlations between functional deficits in space and health-related issues on Earth have been stated demonstrating the importance of space studies.

Thus, I was very interested and curious when reading the title of the manuscript. However, I have to state that I was disappointed after reading the paper, as I did not learn something really new. Spaceflight experiments are rare and unique and thus it is our responsibility to take out as much knowledge as possible. Sample size is low and experimental conditions restricted, thus ground controls are essential to support and discuss the space data. Furthermore, experiences taking from this unique experiment opportunity should be shared for lessons learned and designing future experiments. I am sure that you have all this information, but they have to be incorporated into the manuscript.

My criticism in detail:

NSCs were exposed for 39 days to space conditions. Was a medium change performed? Were the cultures continuously under 37 °C in an incubator? Your post experiments were performed after 21 days in 1g. One can expect that the cells have readapted. Postflight, after 3 weeks, astrocytes were induced. Why did you not start immediately after sample recovery?

What can we learn from this experiment scenario - that NGCs survive space travel? Data from 1g controls in the hardware and under normal laboratory conditions are missing and necessary to evaluate your data. I am sure that you performed these controls, but they should be demonstrated.

Some point your statements are unprecise, examples are:

L 18: proliferated slowly but significantly – compared to??

L 32: simulated microgravity – please add details on applied method, operational and experimental conditions. Did you perform a corresponding experiment – space versus simulation - to verify your simulation approach: NGCs for 39 days in simulated microgravity and post-experiments corresponding to your space experiment?

L L 35 under specific conditions – which ones?

L 66 the experiment has been very instrumental – what do you mean?

L 124: in time cells grew well, rather unprecise

L 214: in a shorter time than previously reported for astrocytes at 1 G. Who performed these studies under which conditions? In any case you need a 1 g control as mentioned above.

Figure 2: a control is missing. Scale bars are missing.

Figure 3: Magnification in red above the scale bar is not readable. Control is missing. Staining with DAPI of nuclei would be appreciate to discriminate the cells.

Figure 4: Control/comparison is missing.

Author Response

NSCs were exposed for 39 days to space conditions. Was a medium change performed?

We understand that this was not clear enough and have added an explanation to the text in section 2.2 that reads:

The hardware used for this study was called passive unit(s), because they travelled round trip with the same culture medium. We worked several years to choose the adequate hardware, cell densiity, culture medium and cell origin that would allow for a successful space flight. Thus, from our “Experimental Verification Tests” prior to the space flight, we learned and optimized the conditions for human NSCs for our study and determined how should the study be conducted.

Were the cultures continuously under 37 °C in an incubator?

Yes, figure 1 now shows the temperature at which this study was conducted. We have added the detailed information to the caption for figure 1.

Your post experiments were performed after 21 days in 1g. One can expect that the cells have readapted. Postflight, after 3 weeks, astrocytes were induced. Why did you not start immediately after sample recovery?

The main goal of the study was to ascertain the properties of NSCs upon return, yet, we had a limited number of cells, so we started time-lapse studies immediately and also allowed cells to propagate still as NSCs prior to phenotype induction.  We waited in order to increase the number of progenies.

 We demonstrated that the cells survived and proliferated upon return to Earth. Yes, we expected NSCs to re-adapt and so they did; however, readapted NSCs and their progenies also have ‘new’ properties as NSCs. This study is in progress. Thus, at this point we can only say that these astrocytes appear to be normal and still proliferate. We did not say they show enhanced proliferation, because we cannot show the comparison data.

What can we learn from this experiment scenario - that NGCs survive space travel? Data from 1g controls in the hardware and under normal laboratory conditions are missing and necessary to evaluate your data.

We have previously reported (Cepeda et al., 2019) in the same special issue, that NSCs, survived space flight, that they proliferate and self-renew as expected. Moreover, we also showed that they also gave rise to young neurons. Another point we reported in that manuscript that NSCs proliferated in space, in contrast to Silvano et al., 2015, that used simulated microgravity and reported a transient arrest in proliferation of mouse NSCs. Thus, we have learned and reported a lot. Unfortunately, we cannot show our 1G data as it is still at our university and, for the time being, inaccessible due to COVID-19 closure.

 

 

 

Originally, we had the astrocyte data as part of another manuscript, nonetheless it had become very large, thus, we separated the data. In the parallel manuscript entitled: “Human Neural Stem Cells Proliferate More While in Space, Have a Shorter Cell Cycle, and are Larger than Ground Control Cells After Space Flight”, to be published in International Journal of Molecular Sciences Special Issue "Microgravity and Space Medicine", we show that human space-flown neural stem cells proliferate, more in space and upon return to Earth confirming our previous report  on the effects simulated microgravity on these cells. Moreover, we also show that some features are different when compared to cells cultured in simulate microgravity.

I am sure that you performed these controls, but they should be demonstrated.

This reviewer is right, we performed the pertinent controls. Nonetheless, as stated above, we will be able to show the control data when we can process and analyze the information. Long-term time-lapse microscopy requires more than one microscope and we gave priority to Space-returning cells consequently, we could analyze the data sooner. We performed the 1G time-lapse imaging after the space-flown cells, yet we cannot access the data because UCLA is still closed.

Some point your statements are unprecise, examples are:

L 18: proliferated slowly but significantly – compared to??,

We compared across time points and the differences were significant. Data could have been different, for example, it could have been that after a while, cells would proliferate slower or stop proliferating.

We have added: “showed a tendency to continue proliferating at the same pace, in a steady manner”.

L 32: simulated microgravity – please add details on applied method, operational and experimental conditions. Did you perform a corresponding experiment – space versus simulation - to verify your simulation approach: NGCs for 39 days in simulated microgravity and post-experiments corresponding to your space experiment?

I must say that the goal of our study was to examine the effects of space microgravity on NSCs. Thus, our grant does not include funding for parallel studies using simulated microgravity. Moreover, simulated microgravity and space microgravity are not control for each other, the effects may vary depending on the features subject of study.

L L 35 under specific conditions – which ones?

Our sentence in the introduction refers to our previous work and it reads:

“We more recently reported that NSCs flown onto space (SPC) survived the space flight and proliferated while in the International Space Station (ISS). They also kept their stemness and under specific conditions as previously described gave rise to neurons [3]”.

We prefer not to confuse the reader indicating how we made neurons in the current paper, since the subject matter of our research is NSCs and their specification to the astrocyte phenotype. Neuronal specification was the subject in our previous paper (Cepeda et al., 2019 # 13). Should the reader be interested in further information on the specification of NSCs into neurons with our defined culture medium, we have added the reference to our original paper we added #14 after reference #3.

L 66 the experiment has been very instrumental – what do you mean?

It has allowed us to bring back to Earth live NSCs to study their behavior as they re-adapt to 1G.

L 124: in time cells grew well, rather unprecise

We changed the word well to: “healthy”

L 214: in a shorter time than previously reported for astrocytes at 1G. Who performed these studies under which conditions? In any case you need a 1 g control as mentioned above.

As replied above the 1G data will be shown as soon as COVID-related clsure of our university is finished.

Figure 2: a control is missing.

Also added: “Ground control cells were grown in the same conditions and hardware, as space flown NSCs, in the investigator’s laboratory”.

Scale bars are missing.

Corrected

Figure 3: Magnification in red above the scale bar is not readable. Control is missing. Staining with DAPI of nuclei would be appreciate to discriminate the cells.

Data not accessible at the present time

Figure 4: Control/comparison is missing.

We explained the predicament we are in by not having access to our files. Nonetheless, we have now added comparative images of the cells have been submitted as Supplemental figure 1 .It allows to see the structures and nuclei. That is all the material available to us at the moment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The present report from Drs. Shaka and colleagues continues their important work conducted as part of the BioScience-4 mission, launched onboard the Space-X 16 Dragon capsule in November of 2018, which was the first study to examine the impact of spaceflight/microgravity on neural stem cells (NSCs).  In prior studies, the authors explored the ability of spaceflown NSCs (derived from human iPSC) to survive, proliferate, and differentiate into neurons upon return to Earth. In the present study, the authors extend their analysis of the spaceflown NSCs, testing their ability to undergo astrocytic differentiation/lineage commitment.   Given the critical role astroglia play in the normal function of the CNS as well as its response to injury/stress, the possibility that spaceflight might alter commitment to this lineage is an important issue.  The fact that deregulated astrocyte maturation and/or proliferation has also been implicated in several neurodegenerative diseases adds to the significance of the question being posed.  

The studies described are quite straightforward and the authors provide convincing evidence that the spaceflown NSCs maintain the ability to differentiate into astrocytes that express appropriate markers (GFAP S100b) upon return to Earth.  The paper is, for the most part well written, and the argument made for the importance of the data generated is compelling.

With that said, there are several issues the authors need to address to clarify the justification for some of the methods they employed and to better place their findings int eh context of what is actually happening during spaceflight within the astrocyte population of the CNS.

1. It seems odd that the only “induction factor” the authors provided to drive astrocyte differentiation of the spaceflown NSCs was FCS. There are countless publications describing specific factors that greatly enhance this process.  For example:

• [PMID: 32509845] BMP4/LIF promoted the astrocyte-like differentiation of monkey NSCs
• [PMID: 31810286] Flavonoids induce astocytogenesis from NSC
• [PMID: 31733648] endothelin-1 over-expression promotes NSC differentiation into astrocytes
• [PMID: 31562895] The MEK inhibitor PD98059 induces astrocyte differentiation of NSCs

It is not clear why the authors did not use any of these approaches and relied solely on FCS to slowly drive differentiation of the NSCs along the astrocytic lineage. Can the authors please comment on this choice?

2. Why did authors allow the NSC to re-equilibrate to 1G for 3 weeks prior to initiating astrocytic differentiation? If their goal is to ascertain what is happening to NSC differentiation potential during spaceflight, allowing 3 weeks for the cells to readjust to 1G prior to analysis is hard to understand their rationale for this experimental design.
3. The authors hypothesize that the enhanced proliferation of the NSC-derived astrocytes could contribute to observed intracranial pressure in astronauts during spaceflight, but they didn’t show that astrocytes could be generated or proliferate more rapidly under conditions of uG, only after 3 weeks of re-equilibration to 1G. As such, it is hard to assess the validity of this interesting hypothesis.

Author Response

1. It seems odd that the only “induction factor” the authors provided to drive astrocyte differentiation of the space flown NSCs was FCS. There are countless publications describing specific factors that greatly enhance this process. For example:

• [PMID: 32509845] BMP4/LIF promoted the astrocyte-like differentiation of monkey NSCs

• [PMID: 31810286] Flavonoids induce astocytogenesis from NSC

• [PMID: 31733648] endothelin-1 over-expression promotes NSC differentiation

  into astrocytes

• [PMID: 31562895] The MEK inhibitor PD98059 induces astrocyte differentiation of NSCs

  We agree with this reviewer, yet each method varies when performed in different laboratories and, in some cases, by different people at the same lab. We must say that the astrocyte induction method we used was adequate for demonstrating that NSCs flown to space kept their ability to choose the astrocyte phenotype when cultured in the appropriate conditions.

  It is not clear why the authors did not use any of these approaches and relied solely on FCS to slowly drive differentiation of the NSCs along the astrocytic lineage. Can the authors please comment on this choice?

  Our goal was achieved with the method chosen. Should we have the opportunity to work more in-depth with astrocytes and microgravity in the future, we will keep the reviewer’s knowledge and suggestions in mind when writing future grant proposals.

  2. WhydidauthorsallowtheNSCtore-equilibrateto1Gfor3weekspriorto initiating astrocytic differentiation? If their goal is to ascertain what is happening to NSC differentiation potential during spaceflight, allowing 3 weeks for the cells to readjust to 1G prior to analysis is hard to understand their rationale for this experimental design.

     The answer is very simple: we wanted to study if NSCs and their progenies were normal prior to embarking on phenotype specification.

  3. TheauthorshypothesizethattheenhancedproliferationoftheNSC-derived astrocytes could contribute to observed intracranial pressure in astronauts during spaceflight, but they didn’t show that astrocytes could be generated or proliferate more rapidly under conditions of uG, only after 3 weeks of re-equilibration to 1G. As such, it is hard to assess the validity of this interesting hypothesis.

     There are three reasons for waiting to induce the astrocyte phenotype: 1) we had to wait in order to obtain enough cells for all the proposed studies in our grant; 2) we have previously reported evidence that the NSCs and their progenies grown in both, simulated and space microgravity, “remember” having been in space and proliferate more. While we could not directly demonstrate that they proliferated more when compared with 1G astrocytes, we showed that our astrocytes proliferated faster versus data we could find in the literature. Reports in the literature are shown using either different astrocyte species or embryonic stem cells; however, we feel that this would not be an appropriate comparison.

We would like to respectfully ask this reviewer if he/she has knowledge of reports that may show that human normal astrocytes in culture proliferate within 17h or less without genetic manipulation, it would be extremely helpful to our paper and for the reader. We welcome any information on this matter.

The third reason is that we had performed the work to show a comparative cell growth, 1G vs. SPC derived astrocytes. Unfortunately, our institution has been closed since March 2020, and it is not clear when we will be able to access our laboratories to retrieve the time-lapse data to complete the analysis of cell numbers for 1G cells that is currently missing in fig. 4. Nonetheless, we have now added supplemental figure S1 comparing the morphological features and general aspect of astrocytes derived from naïve-1G NSCs, versus astrocytes derived from SPC-flown NSCs.

“NSC-derived astrocytes could contribute to observed intracranial pressure in astronauts during spaceflight, but they didn’t show that astrocytes could be generated or proliferate more rapidly under conditions of uG, only after 3 weeks of re-equilibration to 1G”.

In order to address the concern of this reviewer, we have changed this part of the discussion to read as follows:

While it is true that with the data analyzed so far, we cannot assert that there is an increased division of these cells in comparison to1G NSCs derived astrocytes, it is also true that we found preliminary evidence that SPC-flown NSCs generate younger-like astrocytes and radial-glial cells evocative of neural development, where the latter are an abundant progenitor population, in particular for cortical expansion (Hansen et al., 2010; Matsumoto et al., 2020). Our analysis will continue when the pandemic allows us to go back to the laboratory to complete this ongoing study. Nonetheless, one could hypothesize that these glial cells may play a significant role in the intracranial hypertension, or pressure inside the skull, that has been observed during human spaceflight. On one hand, our data confirmed the moderate contribution that one could expect from astrocytes generating new astrocytes in non-injury conditions. Yet, on the other hand, four questions come to mind: 1) Are astrocytes in the brain of astronauts increasing in number, even if slowly and therefore, contributing to intracranial hypertension? 2) Are space flown NSCs giving rise to radial-glia in addition to astrocytes? 3) Are space-flown-NSCs transmitting their proliferative potential to cells that are specified to the astrocyte phenotype in 1G? or, 4) Are these newly specified astrocytes in a state of ‘inherited stress” resulting from the space flight or the return of

their mother NSCs re-entry and splash down? Currently, we do not have answers to these important questions. A subsequent space flight would be necessary to generate enough material to pursue studies aimed at answering them. Moreover, studies in samples containing other cell types, such as organotypic cultures or animal work where the entire CNS experiences space microgravity are of the essence to understand if the regulation of proliferation in space differs when cells are in homogeneous cultures as opposed to the entire brain.

Despite not having the 1G data analysis, we feel that our study should be made known to the Scientific Community, as space research is a dynamic field, even during the pandemic.

Our studies will continue after the pandemic gets under control, but for the moment, we must comply with health protocols and work off Campus until further notice.

Finally, we were careful not to use the term “enhanced proliferation”, because we could not ascertain 1G astrocytes proliferation rates. Nonetheless, our work is ongoing, and we will elucidate this and other behaviors of NSCs and astrocytes in future papers.

Fig S1:

Figure S1. Comparison of the morphological features and general aspect of astrocytes derived from naïve-1G NSCs vs. astrocytes derived from SPC-flown NSCs 50 days after being in DF-10% FCS. A) Astrocytes derived from hiPS-NSCs grown solely in 1G, display their typical flat morphology. B) Astrocytes derived from hiPS-derived NSCs flow onto space, displayed a less flat morphology, presented elongated cell processes and some preserved the bipolar elongated radial glia-like morphology frequently observed during CNS development (inset). Bar = 50μ.

Quantitative data and longitudinal study data will be submitted upon re-opening of UCLA.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Reviewer´s question on the original manuscript and Author's Notes with reviewer´s answer

NSCs were exposed for 39 days to space conditions. Was a medium change performed?

We understand that this was not clear enough and have added an explanation to the text in section 2.2 that reads:

The hardware used for this study was called passive unit(s), because they travelled round trip with the same culture medium. We worked several years to choose the adequate hardware, cell density, culture medium and cell origin that would allow for a successful space flight. Thus, from our “Experimental Verification Tests” prior to the space flight, we learned and optimized the conditions for human NSCs for our study and determined how should the study be conducted.

Reviewer´s answer: Due to your preparation work for several years, the behavior of the samples in the flight hardware as well as under normal laboratory conditions are known and serve as control to judge and discuss the impact of microgravity.

 

Were the cultures continuously under 37 °C in an incubator?

Yes, figure 1 now shows the temperature at which this study was conducted. We have added the detailed information to the caption for figure 1.

Reviewer´s answer: thanks for the information

 

Your post experiments were performed after 21 days in 1g. One can expect that the cells have readapted. Postflight, after 3 weeks, astrocytes were induced. Why did you not start immediately after sample recovery?

The main goal of the study was to ascertain the properties of NSCs upon return, yet, we had a limited number of cells, so we started time-lapse studies immediately and also allowed cells to propagate still as NSCs prior to phenotype induction.  We waited in order to increase the number of progenies.

 We demonstrated that the cells survived and proliferated upon return to Earth. Yes, we expected NSCs to re-adapt and so they did; however, readapted NSCs and their progenies also have ‘new’ properties as NSCs. This study is in progress. Thus, at this point we can only say that these astrocytes appear to be normal and still proliferate. We did not say they show enhanced proliferation, because we cannot show the comparison data.

Reviewer´s answer: Due to my opinion, your 1g controls are necessary. Corona is for all of us a catastrophic situation, which we have to overcome. Science and the publications will persist and will be readable for the future. You had the unique opportunity to do a space experiment which binds a lot of work of years and money. Thus, best science output is your responsibility. Sorry, but I think you should wait with this publication and add the necessary controls. Otherwise, the output is incomplete and less meaningful.

 

What can we learn from this experiment scenario - that NGCs survive space travel? Data from 1g controls in the hardware and under normal laboratory conditions are missing and necessary to evaluate your data.

We have previously reported (Cepeda et al., 2019) in the same special issue, that NSCs, survived space flight, that they proliferate and self-renew as expected. Moreover, we also showed that they also gave rise to young neurons. Another point we reported in that manuscript that NSCs proliferated in space, in contrast to Silvano et al., 2015, that used simulated microgravity and reported a transient arrest in proliferation of mouse NSCs. Thus, we have learned and reported a lot. Unfortunately, we cannot show our 1G data as it is still at our university and, for the time being, inaccessible due to COVID-19 closure.

Reviewer´s answer: Concerning Corona see my comment above. You state that Silvano et al. performed experiments in simulated microgravity and the results were differently to your data from Cepeda et al. 2019. This statement is too much generalized and it is essential to get information how the simulation was performed (clinostat, Rotating Wall Vessel, Random Positioning Machine, etc., which operational mode was used, which hardware, experiment duration etc., see corresponding literature).

 

 Originally, we had the astrocyte data as part of another manuscript, nonetheless it had become very large, thus, we separated the data. In the parallel manuscript entitled: “Human Neural Stem Cells Proliferate More While in Space, Have a Shorter Cell Cycle, and are Larger than Ground Control Cells After Space Flight”, to be published in International Journal of Molecular Sciences Special Issue "Microgravity and Space Medicine", we show that human space-flown neural stem cells proliferate, more in space and upon return to Earth confirming our previous report on the effects simulated microgravity on these cells. Moreover, we also show that some features are different when compared to cells cultured in simulate microgravity.

 

I am sure that you performed these controls, but they should be demonstrated.

This reviewer is right, we performed the pertinent controls. Nonetheless, as stated above, we will be able to show the control data when we can process and analyze the information. Long-term time-lapse microscopy requires more than one microscope and we gave priority to Space-returning cells consequently, we could analyze the data sooner. We performed the 1G time-lapse imaging after the space-flown cells, yet we cannot access the data because UCLA is still closed.

See my comment above

 

Some point your statements are unprecise, examples are:

L 18: proliferated slowly but significantly – compared to??,

We compared across time points and the differences were significant. Data could have been different, for example, it could have been that after a while, cells would proliferate slower or stop proliferating.

We have added: “showed a tendency to continue proliferating at the same pace, in a steady manner”. thanks

L 32: simulated microgravity – please add details on applied method, operational and experimental conditions. Did you perform a corresponding experiment – space versus simulation - to verify your simulation approach: NGCs for 39 days in simulated microgravity and post-experiments corresponding to your space experiment?

I must say that the goal of our study was to examine the effects of space microgravity on NSCs. Thus, our grant does not include funding for parallel studies using simulated microgravity. Moreover, simulated microgravity and space microgravity are not control for each other, the effects may vary depending on the features subject of study.

Reviewer´s answer: There is a misunderstanding; I only asked about information not for further experiments in simulated microgravity. Information on the kind of simulation approach and experimental scenario are necessary for discussing and comparison to space data.

 

L 35 under specific conditions – which ones?

Our sentence in the introduction refers to our previous work and it reads:

“We more recently reported that NSCs flown onto space (SPC) survived the space flight and proliferated while in the International Space Station (ISS). They also kept their stemness and under specific conditions as previously described gave rise to neurons [3]”.

We prefer not to confuse the reader indicating how we made neurons in the current paper, since the subject matter of our research is NSCs and their specification to the astrocyte phenotype. Neuronal specification was the subject in our previous paper (Cepeda et al., 2019 # 13). Should the reader be interested in further information on the specification of NSCs into neurons with our defined culture medium, we have added the reference to our original paper we added #14 after reference #3.

L 66 the experiment has been very instrumental – what do you mean?

It has allowed us to bring back to Earth live NSCs to study their behavior as they re-adapt to 1G.

L 124: in time cells grew well, rather unprecise

We changed the word well to: “healthy”

L 214: in a shorter time than previously reported for astrocytes at 1G. Who performed these studies under which conditions? In any case you need a 1 g control as mentioned above.

As replied above the 1G data will be shown as soon as COVID-related clsure of our university is finished.

 

Figure 2: a control is missing.

Also added: “Ground control cells were grown in the same conditions and hardware, as space flown NSCs, in the investigator’s laboratory”.

Scale bars are missing.

Corrected

Figure 3: Magnification in red above the scale bar is not readable. Control is missing. Staining with DAPI of nuclei would be appreciate to discriminate the cells.

Data not accessible at the present time

Figure 4: Control/comparison is missing.

We explained the predicament we are in by not having access to our files. Nonetheless, we have now added comparative images of the cells have been submitted as Supplemental figure 1 .It allows to see the structures and nuclei. That is all the material available to us at the moment.

 

In summary, I do not see the progress on my basic criticism – lack of appropriate 1g controls – and suggest to reject the manuscript and resubmit it, when the necessary data are available.

Author Response

See attachment please

Author Response File: Author Response.pdf

Reviewer 2 Report

I would like to thank the authors for their thorough and well thought-out responses to the questions posed during the first review. All issues have now been satisfactorily addressed.

Author Response

We thanks Reviewer 2 for  kind time and comments to help improve our manuscript.