Selected Ionotropic Receptors and Voltage-Gated Ion Channels: More Functional Competence for Human Induced Pluripotent Stem Cell (iPSC)-Derived Nociceptors

Round 1

Reviewer 1 Report

The study by Schoepf et al. is a detailed study on the generation of peripheral sensory neurons and nociceptors from iPS-cells. The authors use a modified small molecule protocol based on Chambers 2012, which is the most widely and most successfully used protocol. To date there is no in-depth characterization of the resulting sensory neurons which includes immunostaining and functional tests of a range of different ion channels. This is what the paper aims to address by assessing transcription factors, TrkA, HCN, TrpV1, formation of synapses, Cav channels, KCC3 and Gaba channels using immunostaining. The functional characterization includes assessment of spontaneous activity and reaction to capsaicin and GABA by Ca-imaging. Side by side, the authors compare their findings with adult mouse DRGs.

Overall, the results suggest that iPs-cell derived sensory neurons are expressing all necessary markers, which is an important and encouraging finding. Additionally, the authors describe synaptic interactions between neurites (varicosities), which cannot be observed in mice, suggesting a species difference which may be of important pathophysiological relevance. This aspect of the otherwise well written manuscript could be discussed in more detail.

Overall, this is a focused, well-presented study that extends recent work by showing expression patterns and the direct comparison between mouse nociceptors and human iDNs.

Questions:

1. Quantification of immunostaining is suggested to result in 100% BRNA3a and ISL1 positive neurons, although Fig. 1 B and C clearly shows DAPI staining without associated brn3a or isl1 staining. Please clarify. Are these also just the Tuj1 positive neurons?
2. The authors don’t provide data on antibody characterization. Although the supplier may claim their specificity, it needs to be shown at least rudimentary by the authors themselves (e.g. isotype controls, …). The discrepancy between staining and function of Trpv1 could also result from the use of a bad antibody.
3. How do you define spontaneous activity as depicted in Fig. 5E? Which cells were counted for the population analysis?
4. A table with the quantification of their stainings in iDNs and DRGs would make it easier to compare the two species and would provide a useful overview of the authors findings.

Author Response

Reviewer 1

We thank this reviewer very much for reviewing our manuscript and for the positive and constructive comments.

Question R1: Quantification of immunostaining is suggested to result in 100% BRNA3a and ISL1 positive neurons, although Fig. 1B and C clearly shows DAPI staining without associated brn3a or isl1 staining. Please clarify. Are these also just the Tuj1 positive neurons?

Answer 1: Neurons were selected based on the threshold of >10µm DAPI counterstaining for a positive cell and additionally selected by the presence of the surrounding neurite marker TUJ1. DAPI counterstainings without associated TUJ1 were excluded from the counting. Using these selection criteria we found expression in 100% of cells, however, the expression levels of cells were not homogenously distributed in iDNs. An individual quantification of single cells would be possible, however complicated and would not provide important additional information for the study. 

Question 2: The authors don’t provide data on antibody characterization. Although the supplier may claim their specificity, it needs to be shown at least rudimentary by the authors themselves (e.g. isotype controls). The discrepancy between staining and function of Trpv1 could also result from the use of a bad antibody.

Answer 2: All antibodies used in the study were used in previous publications and were routinely tested before the application in iDNs and DRGs. We now include the relevant publications in the material and methods part. The discrepancy of TRPV1 staining and function is likely to be due to TRPV1 sequestration in vesicles by the interaction with t-SNARES (Sondermann et al. 2019) or VAMP1 containing vesicle trafficking (Meng et al. 2016), thus chemical nociceptor stimulation might not fully target these vesicle fractions of TRPV1. We included this aspect in the discussion of the manuscript.

Question 3: How do you define spontaneous activity as depicted in Fig. 5E? Which cells were counted for the population analysis?

Answer 3: We added a paragraph to Figure 5E/F that describes how we defined spontaneous activity computationally to avoid an unbiased selection of calcium transient peaks and that we took iDNs into consideration that exhibit a general neuronal morphology.

Question 4: A table with the quantification of their stainings in iDNs and DRGs would make it easier to compare the two species and would provide a useful overview of the authors findings.

Answer4: Thank you very much for this suggestion. Accordingly, a comparative table has been prepared and is included in the manuscript.

New references:

Geisler, S., C. L. Schopf, R. Stanika, M. Kalb, M. Campiglio, D. Repetto, L. Traxler, M. Missler and G. J. Obermair (2019). "Presynaptic alpha2delta-2 Calcium Channel Subunits Regulate Postsynaptic GABAA Receptor Abundance and Axonal Wiring." J Neurosci 39(14): 2581-2605.

Hortnagl, H., R. O. Tasan, A. Wieselthaler, E. Kirchmair, W. Sieghart and G. Sperk (2013). "Patterns of mRNA and protein expression for 12 GABAA receptor subunits in the mouse brain." Neuroscience 236: 345-372. 

Meng, J., J. Wang, M. Steinhoff and J. O. Dolly (2016). "TNFalpha induces co-trafficking of TRPV1/TRPA1 in VAMP1-containing vesicles to the plasmalemma via Munc18-1/syntaxin1/SNAP-25 mediated fusion." Sci Rep 6: 21226.

Sondermann, J. R., A. M. Barry, O. Jahn, N. Michel, R. Abdelaziz, S. Kugler, D. Gomez-Varela and M. Schmidt (2019). "Vti1b promotes TRPV1 sensitization during inflammatory pain." Pain 160(2): 508-527.

Reviewer 2 Report

Schoepf and collaborators characterized nociceptors (iDNs) derived from human-induced pluripotent stem cells (iPSCs) by immunofluorescence analysis and microfluorimetric Ca²⁺ measurements. The immunofluorescence markers used were early somatosensory transcription factors (BRN3A/ISL1/RUNX1), TrkA, HCN, high-voltage activated Cav2, TRPV1 channels and GABA_A receptors; results thus obtained in iDNs were found to be similar to those of mouse dorsal root ganglion (DRG) neurons. Moreover, it was found that Ca²⁺ transients in iDNs are produced by superfusing capsaicin-, GABA-containing and high K⁺ solutions. As a result, they concluded that iDNs are useful in studying pain mechanisms. Although this result appears to be of interest, there are many concerns that should be addressed and are many points that may serve to amend this manuscript, as follows:

Major points:

1. Title should be modified, because only a part of ionotropic receptors and voltage-gated ion channels have been examined in the present study.
2. In Materials and Methods, the authors should give more information for the other investigators to be able to mimic the results obtained by the authors, for example, information about companies (STEMCELL Technologies, Thermo Fischer, PeproTech, Roche, Invitrogen, Biochrom, Synaptic Systems and so on) from which the drugs used were obtained and about the compositions of medium (mTeSR1 medium, N2/B27 Neurobasal medium, KSR medium, TNB^TM medium and so on) used. Please explain shortly LDN-1931189, SB431542, CHIR99021, DAPT, SU5402, Liberase Blendzyme 1, DAPI and AML1/TLX3.
3. Page 5: each of Figs. 1A, B and C should be explained.
4. Page 7: each of Figs. 2A, B, C and D should be explained.
5. Page 8: each of Figs. 3A, B, C and D and of Figs. 4A and B should be explained.
6. Page 9: each of Figs. 5A, B, C, D and E should be explained.
7. Page 11: each of Figs. 5A, B, C, D, E and F should be explained.
8. First paragraph on page 13: each of Figs. 7A and B should be explained; the authors should mention why the expression of b-2/3 but not other subunits of GABA_A receptors was examined.
9. Second paragraph on page 13: each of Figs. 8A, B, C, D, E and F should be explained; please specify Figure 2. Moreover, the authors should mention which concentration of capsaicin, GABA or KCl was used. How many iDNs were examined in Figs. 8B and E? How did the authors judge an activation by GABA of GABA_A but not GABA_B receptors? Please address these questions.

Minor points:

1. Line 101: please give references here.
2. Line 104: what is the concentration of Trypsin-EDTA used?
3. Line 105: not “L-Glutamin” but “L-Glutamine”.
4. Lines 105 and 106: please give the concentrations of drugs used.
5. Lines 121 and 193: p75 should be shortly explained with respect to TrkA.
6. Lines 193 and 342: not “TRKA” but “TrkA”.
7. Line 121: what is NKCC1? Did the authors examine the expression of NKCC1?
8. Line 132: not “NaCL” but “NaCl”; not “KCL” but “KCl”.
9. Line 307: not “Capsaicin” but “capsaicin”; not “KCL” but “KCl”.
10. Line 366: it is unnecessary to repeatedly define TRP (see line 206).
11. Line 388: it is unnecessary to repeatedly define KCC (see line 291).
12. There appear to be more mistakes than pointed out above. Please check your manuscripts very carefully.

Author Response

Reviewer2

We thank this reviewer very much for reviewing our manuscript and for the positive and constructive comments.

Major point 1: Title should be modified, because only a part of ionotropic receptors and voltage-gated ion channels have been examined in the present study.

Answer: Thank you for this suggestion we adapted the title.

Major points 2-7: In Materials and Methods, the authors should give more information for the other investigators to be able to mimic the results obtained by the authors, for example, information about companies (STEMCELL Technologies, Thermo Fischer, PeproTech, Roche, Invitrogen, Biochrom, Synaptic Systems and so on) from which the drugs used were obtained and about the compositions of medium (mTeSR1 medium, N2/B27 Neurobasal medium, KSR medium, TNBTM medium and so on) used. Please explain shortly LDN-1931189, SB431542, CHIR99021, DAPT, SU5402, Liberase Blendzyme 1, DAPI and AML1/TLX3

.Page 5: each of Figs. 1A, B and C should be explained.

Page 7: each of Figs. 2A, B, C and D should be explained.

Page 8: each of Figs. 3A, B, C and D and of Figs. 4A and B should be explained.

Page 9: each of Figs. 5A, B, C, D and E should be explained.

Page 11: each of Figs. 5A, B, C, D, E and F should be explained. Page 11: each of Figs. 5A, B, C, D, E and F should be explained.

Answer2-7: Thank you very much for your comments and suggestions. The according changes are included in the material and methods part, and highlighted in yellow.

Question 8: Why was the expression of β-2/3 but not other subunits of GABAA receptors was examined.

Answer 8: Because these are to our knowledge the most abundant subunits of GABA_AR in neurons. Additionally the β-2/3 is a well established antibody and well characterized in one of our previous studies (Geisler et al. 2019). We would also have access to other GABA_AR antibodies previously described in (Hortnagl et al. 2013, Geisler et al. 2019)targeting other subunits like α1N, α2C, α3N, α4N, β2L, β3L, which could be included in further studies, however this would go beyond the scope of the current manuscript.

Question 9a: Second paragraph on page 13: each of Figs. 8A, B, C, D, E and F should be explained; please specify Figure 2.

Answer 9a: Figure descriptions are adapted and marked in yellow

Question 9b Moreover, the authors should mention which concentration of capsaicin, GABA or KCl was used. How many iDNs were examined in Figs. 8B and E? How did the authors judge an activation by GABA of GABA_A but not GABA_B receptors?

Answer 9b

Day 40: 189 iDNs in 6 experiments, selected and defined based on morphology. Day 60: 98 iDNs in 4 Experiments and concentrations of each substance applied were added to the text and figure legend. Application of GABA does not allow us tounequivocally distinguish between the activation of different GABA receptor types but gives a general overview of functional GABA receptors expressed in iDNs that exhibit depolarizations comparable to those found in rodent DRGs. The time course of calcium transients, however, is more consistent with calcium influx through ionotropic GABA receptors. The GABA_B receptor is a G-protein coupled receptor that associates with G-proteins of the pertussis toxin sensitive Gi/o family that regulate voltage-gated calcium channels, G-protein activated inwardly rectifying K(+) (GIRK) channels, and adenylyl cyclase (PMID:20655481). As a result of GABA_B receptor activation, calcium influx or release from intracellular stores appears rather unlikely. 

Minor points 1-12:

Response: Thank you very much for pointing out these points. We have added the respective reference and amended the spellings of the diverse proteins.

New references:

Geisler, S., C. L. Schopf, R. Stanika, M. Kalb, M. Campiglio, D. Repetto, L. Traxler, M. Missler and G. J. Obermair (2019). "Presynaptic alpha2delta-2 Calcium Channel Subunits Regulate Postsynaptic GABAA Receptor Abundance and Axonal Wiring." J Neurosci 39(14): 2581-2605.

Hortnagl, H., R. O. Tasan, A. Wieselthaler, E. Kirchmair, W. Sieghart and G. Sperk (2013). "Patterns of mRNA and protein expression for 12 GABAA receptor subunits in the mouse brain." Neuroscience 236: 345-372. 

Meng, J., J. Wang, M. Steinhoff and J. O. Dolly (2016). "TNFalpha induces co-trafficking of TRPV1/TRPA1 in VAMP1-containing vesicles to the plasmalemma via Munc18-1/syntaxin1/SNAP-25 mediated fusion." Sci Rep 6: 21226. 

Sondermann, J. R., A. M. Barry, O. Jahn, N. Michel, R. Abdelaziz, S. Kugler, D. Gomez-Varela and M. Schmidt (2019). "Vti1b promotes TRPV1 sensitization during inflammatory pain." Pain 160(2): 508-527.

Round 2

Reviewer 1 Report

The authors have adressed all questions. The new table is a very helpful addition to the ms. Citing relevant literature for antibody staining is ok. It would have been nicer to see the in house testing of the antibodies, as, espcially with ion channels, quite often the published results can be challenging to reproduce. I am a little bit confused with the p75 antibody – is it directed against p75 or Trka, or the combination?

Author Response

Comments, Reviewer 1, Round 2

The authors have addressed all questions. The new table is a very helpful addition to the ms. Citing relevant literature for antibody staining is ok. It would have been nicer to see the in house testing of the antibodies, as, especially with ion channels, quite often the published results can be challenging to reproduce. I am a little bit confused with the p75 antibody – is it directed against p75 or Trka, or the combination?

We thank this reviewer very much for reviewing our manuscript and for the positive and constructive comments, especially concerning the antibody issue for the low-affinity nerve growth factor receptor p75.

Answer 1: Our lab has a long time experience with ion channel antibodies, especially with calcium channels. We only use antibodies, which do not show off targeting, and background contamination and most of the Cav antibodies were previously published, also by our laboratory.

We double-checked the p75 antibody with the #AB52987 from Abcam. Indeed, it only targets the p75 low affinity nerve growth factor receptor, but not the high affinity nerve growth factor receptor TrkA! We changed the labeling accordingly in Figure2. Additionally we also changed the text for Figure2. p75 was shown to be required for the sensory neuron diversity by potentiating RET signaling and RET signaling was shown to be activated subsequently after RUNX1 in the Chambers protocol. Thus this marker even fits better to prove the robustness of the differentiation protocol. Additionally p75 is a well-accepted nociceptive marker and we included the relevant literature.

Reviewer 2 Report

This revised manuscript has been amended according to my comments. There are no further concerns except for the following minor comments:

1. Line 57: please put “(HCN)”, because HCN is used without its definition in line 66.
2. Line 190: please move “(mDRG)” to line 189.
3. Line 411: please expand TRPA1 with its short explanation.

Author Response

Comments, Reviewer 2, Round 2

We thank this reviewer very much for reviewing our manuscript and for the positive and constructive comments.

This revised manuscript has been amended according to my comments. There are no further concerns except for the following minor comments:

1. Line 57: please put “(HCN)”, because HCN is used without its definition in line 66.
2. Line 190: please move “(mDRG)” to line 189.
3. Line 411: please expand TRPA1 with its short explanation.

Answer 1: We have corrected these points, and included an additional labelling for Figure1 (iDN/mDRG)