Lipotype acquisition during neural development in vivo is not recapitulated in stem cell-derived neurons

  1. Anusha B. Gopalan
  2. Lisa van Uden
  3. Richard R. Sprenger
  4. Nadine Fernandez-Novel Marx
  5. Helle Bogetofte
  6. Pierre Neveu
  7. Morten Meyer
  8. Kyung-Min Noh
  9. Alba Diz-Muñoz
  10. Christer S. Ejsing

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Abstract

During development, different tissues acquire distinct lipotypes that are coupled to tissue function and homeostasis. In the brain, where complex membrane trafficking systems are required for neural function, specific glycerophospholipids, sphingolipids, and cholesterol are highly abundant, and defective lipid metabolism is associated with abnormal neural development and neurodegenerative disease. Notably, the production of tissue-specific lipotypes requires appropriate programming of the underlying lipid metabolic machinery, but when and how this occurs is unclear. To address this, we used high-resolution mass spectrometry-based (MS ALL ) lipidomics to perform a quantitative and comprehensive analysis of mouse brain development covering early embryonic and postnatal stages. We discovered a distinct bifurcation in the establishment of the neural lipotype, whereby the canonical brain lipid biomarkers 22:6-glycerophospholipids and 18:0-sphingolipids begin to be produced in utero , whereas cholesterol attains its characteristic high levels after birth. In contrast, when profiling rodent and human stem cell-derived neurons, we observed that these do not acquire a brain lipotype per se . However, upon probing the lipid metabolic wiring by supplementing brain lipid precursors, we found that the stem cell-derived neurons were partially able to establish a brain-like lipotype, demonstrating that the cells are partially metabolically committed. Altogether, our report provides an extensive lipidomic resource for brain development and highlights a potential challenge in using stem cell-derived neurons for mechanistic studies of lipid biochemistry, membrane biology and biophysics that can be mitigated by further optimizing in vitro differentiation protocols.

Significance Statement

We report an extensive time-resolved resource of lipid molecule abundances across mouse brain development, starting as early as 10 days post-fertilization. The resource reveals a bifurcation in the establishment of the neural lipotype where the canonical 22:6-glycerophospholipid and 18:0-sphingolipid biomarkers are attained in utero , whereas cholesterol is attained after birth. Furthermore, we uncover that the neural lipotype is not established in rodent and human stem cell-derived neurons in vitro .

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    Reply to the reviewers

    We thank the Reviewers for their useful feedback on our manuscript. We have addressed the Reviewers’ comments and revised our manuscript accordingly. A point-by-point response is provided below.

    Reviewer comments:

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Gopalan et al use quantitative, comprehensive lipid mass spectrometry of mouse brain tissue isolated at various time points in embryonic and postnatal development. They then go on to use the same quantitative analysis of mouse and human stem cells differentiated in vitro into neurons to define the lipid composition of these cultures.

    Major Comments:

    1. As mentioned above, it is difficult to assess whether the discrepancy in the lipotype acquisition between in vivo mouse brain development and stem cell differentiation is due to metabolic differences in the in vitro differentiation as the authors state or is due to a lack of the stem cells to actually acquire a neuronal phenotype. Perhaps showing more clearly that the protocols for neuronal differentiation work efficiently and/or how they compare to brains dissected would be helpful in stating that the lipotype is different. The protocol referenced here (Bogetofte et al) only gives ~30% TH+ positive DA neurons in their manuscript. What cell type the other 70% of the cells are is something that could be discussed as means of "diluting" out the lipotype seen in these cultures. Perhaps the 30% TH+ DA neurons do attain the "correct" lipotype, but the lipidomic analysis can not detect this due to the contaminating effects of the non-differentiated cells. In this work, it would be nice to see what percentage of the cells differentiate into the expected cell type rather than referencing previous manuscripts. As differentiation protocols and originating cell sources are highly variable and error-prone, it's difficult to know what the lipotype results are actually reporting on. Furthermore, discussion about these differentiation techniques and how well they represent functional neurons is warranted. The papers referenced here don't show 100% differentiation into the phenotypes that are described in this work such that the lipotype finding is not the only suggestion of a "general failure of in vitro neuronal differentiation models". Maybe a discussion of how the lack of ability to attain the neuronal lipotype due to the metabolic deficiencies discussed here could be causative to the inability to full recapitulate the neuronal phenotype is useful for the reader.

    We thank the reviewer for this question and suggested experiments. Following the advice, we have now show immunofluorescence data of pan-neuronal markers (i.e., b-tub III or MAP2) in mESC and iPSCs. In agreement with previously published datasets from the Noh and Meyer labs (Gehre et al., 2020; Bogetofte et al.,2019), we show that the protocols we use generate a very high percentage of neurons. We have now included these images and quantifications in our manuscript as Figs. 2B and S6A,B.

    From the discussion and work here is unclear why the stearate feeding of the stem cells did not result in an increase in the 18:0-containing sphingolipids. The authors state that the appropriate metabolic pathways are not fully established and go on to look at the CerS expression levels across the differentiation timeline. It appears that the results presented in Fig. S7 counter the authors' interpretation of the lipotype and more discussion here would be nice to clarify this discrepancy.

    We thank the reviewer for highlighting this seemingly counterintuitive observation. We have now included a quantification of CerS mRNA from commercially available mouse tissues analysed in Sladitschek and Neveu, 2019 and compared this to the data from Gehre et al, 2020. In the mouse brain tissue, CerS1 expression is upregulated dramatically, while CerS5 and 6 are downregulated (see new panel A in Fig. S8). In contrast, during in vitro differentiation of mESCs, CerS5 is not downregulated and CerS6 is upregulated (Fig. S8B). Accordingly, we have expanded our discussion in the revised manuscript as follows:

    “On the other hand, supplementing the cells with stearic acid (18:0) does not result in high levels of 18:0-sphingolipids. It is known that the Cer synthase CerS1 is specific for stearoyl-CoA (Venkataraman et al., 2002), which results in the production of 18:0-sphingolipids, while the synthases CerS5 and CerS6 are responsible for 16:0-sphingolipid production. During brain development, one observes a 35-fold increase in the expression of CerS1 and a downregulation of CerS5 and CerS6 compared to embryonic tissue (Sladitschek & Neveu, 2019) (Fig. S8A). In contrast, during* in vitro* neuronal differentiation, between day 8 and 12, CerS1 expression increases only by 5-fold and, contrary to expectation, CerS6 expression is upregulated and CerS5 expression is unchanged (Gehre et al., 2020) (Fig. S8B). This could underpin the observation that 16:0-sphingolipids remain elevated whereas brain-specific 18:0-sphingolipids only increase marginally, despite supplementation with stearic acid. Overall, this suggests that appropriate programming of the sphingolipid metabolic machinery is not fully established in stem cell-derived neurons.”

    Minor comments:

    1. I find the data presentation of the LENA analysis to be difficult to follow (Fig. 1E). In my opinion, the p-value is not the most important bit of information in this graph, though having it on the y-axis with other pertinent information encoded by colors or arrows being disguised. I would rather see the data on the x-axis that is above a certain p-value (denoted in the figure legend) plotted with the direction and magnitude of change shown.

    We thank the reviewer for this suggestion. In the revised manuscript, we now plot log2(odds ratio) on the y-axis instead of the p-value. Moreover, we have dimensioned the size and color intensity of each point as function of the p-value (Fig. 1E and 2E, shown below).

    In the PCA in Fig 1, what are the loadings that define the variable PC1 and PC2? What is predominantly changing the P21 samples that lead to such a large shift if most of the data shown in the subsequent panels are not changing much between P2 and P21.

    In the revised manuscript, we now include a plot of the PCA loadings of the lipids majorly influencing principal components 1 and 2 as supplemental Fig. S3.

    Reviewer #1 (Significance (Required)):

    This work provides a nice reference for the complex lipidomes in embryonic and postnatal murine brain development. The details of the lipotype changes during development are well laid out and will of no doubt be of great use across a variety of scientific fields. While I found the in vivo data to be compelling, interesting, and useful, the lack of controls for the in vitro stem cell differentiation work makes this particular data set and comparison less useful. Further work to identify the limitations of the stem cell differentiation protocols as a valid comparison to in vivo brain development need to be done and/or the discussion of the direct comparisons between the two toned down.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    The study used a quantitative lipidomics approach which I am very familiar with. The results should be highly reproducible.

    Reviewer #2 (Significance (Required)):

    The manuscript submitted by Gopalan et al. reported a quantitative and comparative lipidomics study between mouse brain samples from early embryonic to postnatal stages, and rodent and human stem cell-derived neurons. The authors found a couple of very unique characters only existing in brain samples, but not in stem cell-derived neurons, including 22:6-containing glycerophospholipids and 18:0-containing sphingolipids. The authors further found the brain-like lipotypes can only be partially established in stem cell-derived neurons after supplementing brain lipid precursors. These findings clearly suggest that stem cell-derived neurons might not be appropriately used to mechanistically study lipid biochemistry, membrane biology, and biophysics in brains. The study was well designed. and the manuscript was very informative and resourceful. I would suggest to accept the manuscript for publication.

    We thank the Reviewer for the positive assessment of our work.

    __ References__

    Gehre, M., Bunina, D., Sidoli, S., Lübke, M. J., Diaz, N., Trovato, M., Garcia, B. A., Zaugg, J. B., & Noh, K. M. (2020). Lysine 4 of histone H3.3 is required for embryonic stem cell differentiation, histone enrichment at regulatory regions and transcription accuracy. Nature Genetics, 52(3), 273–282. https://doi.org/10.1038/s41588-020-0586-5

    Levy, M., & Futerman, A. H. (2010). Mammalian ceramide synthases. IUBMB Life, 62(5), 347–356. https://doi.org/10.1002/iub.319

    Sladitschek, H. L., & Neveu, P. A. (2019). A gene regulatory network controls the balance between mesendoderm and ectoderm at pluripotency exit. Molecular Systems Biology, 15(12), 1–13. https://doi.org/10.15252/msb.20199043

    Venkataraman, K., Riebeling, C., Bodennec, J., Riezman, H., Allegood, J. C., Cameron Sullards, M., Merrill, A. H., & Futerman, A. H. (2002). Upstream of growth and differentiation factor 1 (uog1), a mammalian homolog of the yeast longevity assurance gene 1 (LAG1), regulates N-stearoyl-sphinganine (C18-(dihydro)ceramide) synthesis in a fumonisin B1-independent manner in mammalian cells. Journal of Biological Chemistry, 277(38), 35642–35649. https://doi.org/10.1074/jbc.M205211200

  5. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    The study used a quantitative lipidomics approach which I am very familiar with. The results should be highly reproducible.

    Significance

    The manuscript submitted by Gopalan et al. reported a quantitative and comparative lipidomics study between mouse brain samples from early embryonic to postnatal stages, and rodent and human stem cell-derived neurons. The authors found a couple of very unique characters only existing in brain samples, but not in stem cell-derived neurons, including 22:6-containing glycerophospholipids and 18:0-containing sphingolipids. The authors further found the brain-like lipotypes can only be partially established in stem cell-derived neurons after supplementing brain lipid precursors. These findings clearly suggest that stem cell-derived neurons might not be appropriately used to mechanistically study lipid biochemistry, membrane biology, and biophysics in brains. The study was well designed. and the manuscript was very informative and resourceful. I would suggest to accept the manuscript for publication.

  6. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Gopalan et al use quantitative, comprehensive lipid mass spectrometry of mouse brain tissue isolated at various time points in embryonic and postnatal development. They then go on to use the same quantitative analysis of mouse and human stem cells differentiated in vitro into neurons to define the lipid composition of these cultures.

    Major Comments:

    1. As mentioned above, it is difficult to assess whether the discrepancy in the lipotype acquisition between in vivo mouse brain development and stem cell differentiation is due to metabolic differences in the in vitro differentiation as the authors state or is due to a lack of the stem cells to actually acquire a neuronal phenotype. Perhaps showing more clearly that the protocols for neuronal differentiation work efficiently and/or how they compare to brains dissected would be helpful in stating that the lipotype is different. The protocol referenced here (Bogetofte et al) only gives ~30% TH+ positive DA neurons in their manuscript. What cell type the other 70% of the cells are is something that could be discussed as means of "diluting" out the lipotype seen in these cultures. Perhaps the 30% TH+ DA neurons do attain the "correct" lipotype, but the lipidomic analysis can not detect this due to the contaminating effects of the non-differentiated cells. In this work, it would be nice to see what percentage of the cells differentiate into the expected cell type rather than referencing previous manuscripts. As differentiation protocols and originating cell sources are highly variable and error-prone, it's difficult to know what the lipotype results are actually reporting on.

    Furthermore, discussion about these differentiation techniques and how well they represent functional neurons is warranted. The papers referenced here don't show 100% differentiation into the phenotypes that are described in this work such that the lipotype finding is not the only suggestion of a "general failure of in vitro neuronal differentiation models". Maybe a discussion of how the lack of ability to attain the neuronal lipotype due to the metabolic deficiencies discussed here could be causative to the inability to full recapitulate the neuronal phenotype is useful for the reader.

    1. From the discussion and work here is unclear why the stearate feeding of the stem cells did not result in an increase in the 18:0-containing sphingolipids. The authors state that the appropriate metabolic pathways are not fully established and go on to look at the CerS expression levels across the differentiation timeline. It appears that the results presented in Fig S7 counter the authors' interpretation of the lipotype and more discussion here would be nice to clarify this discrepancy.

    Minor comments:

    1. I find the data presentation of the LENA analysis to be difficult to follow (Fig 1E). In my opinion, the p-value is not the most important bit of information in this graph, though having it on the y-axis with other pertinent information encoded by colors or arrows being disguised. I would rather see the data on the x-axis that is above a certain p-value (denoted in the figure legend) plotted with the direction and magnitude of change shown.
    2. In the PCA in Fig 1, what are the loadings that define the variable PC1 and PC2? What is predominantly changing the P21 samples that lead to such a large shift if most of the data shown in the subsequent panels are not changing much between P2 and P21.

    Significance

    This work provides a nice reference for the complex lipidomes in embryonic and postnatal murine brain development. The details of the lipotype changes during development are well laid out and will of no doubt be of great use across a variety of scientific fields. While I found the in vivo data to be compelling, interesting, and useful, the lack of controls for the in vitro stem cell differentiation work makes this particular data set and comparison less useful. Further work to identify the limitations of the stem cell differentiation protocols as a valid comparison to in vivo brain development need to be done and/or the discussion of the direct comparisons between the two toned down.

  7. Version published to 10.1101/2022.08.31.505694 on bioRxiv