Human spinal cord in vitro differentiation pace is initially maintained in heterologous embryonic environments

  1. Alwyn Dady
  2. Lindsay Davidson
  3. Pamela A Halley
  4. Kate G Storey

  • Curated by eLife

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    Evaluation Summary:

    This manuscript is of potential interest to a large audience in the fields of stem cells, developmental biology and neural regeneration. The authors assess the roles of extrinsic versus intrinsic signalling on differentiation of human neural cells by comparing their differentiation rates across different environments (in vitro, in the human embryo and grafted into a chicken embryo). While the experimental design tests the role of environment on differentiation, some aspects of data analysis need to be clarified and extended to support the conclusions.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

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Abstract

Species-specific differentiation pace in vitro indicates that some aspects of neural differentiation are governed by cell intrinsic properties. Here we describe a novel in vitro human neural-rosette assay that recapitulates dorsal spinal cord differentiation but proceeds more rapidly than in the human embryo, suggesting that it lacks endogenous signalling dynamics. To test whether in vitro conditions represent an intrinsic differentiation pace, human iPSC-derived neural rosettes were challenged by grafting into the faster differentiating chicken embryonic neural tube iso-chronically, or hetero-chronically into older embryos. In both contexts in vitro differentiation pace was initially unchanged, while long-term analysis revealed iso-chronic slowed and hetero-chronic conditions promoted human neural differentiation. Moreover, hetero-chronic conditions did not alter the human neural differentiation programme, which progressed to neurogenesis, while the host embryo advanced into gliogenesis. This study demonstrates that intrinsic properties limit human differentiation pace, and that timely extrinsic signals are required for progression through an intrinsic human neural differentiation programme.

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  1. Version published to 10.7554/elife.67283 on eLife
  2. eLife

    Author Response:

    Reviewer #1 (Public Review):

    In this interesting paper by Dady and colleagues the nature of human neural progenitor differentiation is evaluated via transplantation studies. The first part of the paper establishes the timing of neural differentiation in human IPSC model systems and in human embryonic spinal cord, showing that the relative timing of neurogenesis and gliogenesis is maintained. In the second part of the paper these human IPSC neural rosettes are transplanted into the chick spinal cord during neurogenic stages (i.e. Isochronic transplantation) and they find that neurons are generated by these transplanted populations. Analysis of transplants at later stages reveals that neurogenesis has "stalled" and is relatively reduced within the transplanted population.

    Overall, this is an interesting paper that uses classic approaches to answer potentially interesting questions, however there are some issues that limit it's potential impact. The first two figures are recursive and show that the authors can implement an existing protocol.

    Our previous work established this differentiation protocol (Verrier et al 2018 Development), but this is its first use to analyse by immunofluorescence the timing of neural differentiation and the appearance of specific neuronal and glial cell types in an in vitro neural rosette assay. None of the data presented is recursive and it provides a quantitative timeline of human dorsal spinal cord differentiation. Moreover, our comparison with the human embryonic spinal cord indicates that neural differentiation progresses more rapidly in vitro.

    The transplantation studies are intriguing but do not offer sufficient new insights. The key finding seems to be that at later stages post-transplantation neuronal differential is "stalled". There are many other reasons (besides "stalling") that could explain their results. Suppose that stalling was indeed occurring, the authors offer no cellular or molecular insights into what regulates these intrinsic differences across species. At the end, it is not sufficiently clear what we have learned about the mechanisms that control the timing (pace seems to be another term for timing) of differentiation in human neural stem cells.

    Our study shows that differentiation of isochronic transplantation of human iPSC neural rosettes into the chick embryonic spinal cord initially follows that of neural rosettes cultured in vitro, rather than that of the faster differentiating host chicken embryo. This suggests that the human cells follow an intrinsic differentiation programme. However, after a longer period of culture the transplanted rosettes now lag behind their in vitro counterparts, suggesting that intrinsic cues are insufficient and that appropriate extrinsic cues are needed to promote differentiation progression. We discuss potential reasons for this stall of the differentiation programme in the Discussion and agree that some further investigation is of interest. Moreover, revision experiments now further extend our findings.

    Reviewer #2 (Public Review):

    In general, this manuscript provides new significant knowledge by comparing between neural differentiation rate within the same species (human) in vivo and in vitro and between species (human and chick). The quality of the data is excellent, and the combining of the in vivo chick model to compare between grafted and host cells is a fantastic idea, that can only be done in this experimental model. Yet, some controls and more in-depth analysis are missing and are required in my opinion before publication.

    1. In the grating experiment, non-manipulated embryos serve as controls. Yet, the grafted rosettes are inserted near an injured area where a piece of the neural tube was moved. A better control would be to graft homologous cells from a donor chick embryo (GFP+ chick line is available in the UK) or quail embryo (which has a similar growing rate as chick at E2) and examining whether the injured area doesn't affect the grated cells to differentiate in a different pace as compared to the human grafts. This control is necessary to rule out the possibility that the human graft did not accelerate their differentiation rate and later stopped differentiating due to extrinsic signals/lack of signals form the manipulated environment.

    For clarity, we point out that the control is not an unmanipulated embryo, but an embryo subject to the same tissue removal experiment but lacking the graft. We found that these operated only embryos quickly regenerated lost tissue to such an extent that the neural tube appeared similar to the unoperated contralateral side of the neural tube after 2 days. We further note many previous studies using quail tissue in place of chick to fate map the embryo (for example the many excellent studies of Nicole Le Douarin) which suggest that such manipulations result in normal differentiation of the grafted tissue and so are unaffected by placement into an injury site in a developing embryo. However, we appreciate that it would be informative to demonstrate this for our precise experiment and in our hands.

    1. When examining the entire results of the manuscript some important points need to be addressed: On the one hand, the rosettes correspond to their in vitro growth conditions/extrinsic cues and display an accelerated differentiation pace, when compared to their in vivo counterpart human cells. On the other hand, the rosettes do not correspond initially to the chick environment and maintain their own intrinsic tempo.

    The human rosettes were matched for differentiation state with the tissue removed from the chick embryo, despite this and the local cues that support the chicken spinal cord development beyond this point, the human cells retained the differentiation timing of their in vitro counterparts rather than that of the host chick embryo. This is consistent with the slower cell cycle and cell metabolism characteristic of human cells.

    Later, they do change their developmental program and attenuate their differentiation. Therefore, the conclusion that the cells mostly obey to intrinsic regulation is confusing. It would be great if the authors could provide better experimental data to confirm their conclusion. Some ideas that the authors may consider are to determine whether there is a time window that sets the tempo of the rosettes that cannot be influenced later by extrinsic cues. Will the grafted cells correspond differently whether they would be grafted at a more/less advanced stages and domains? Is there an initial mechanistic elucidation to the different behavior of spinal cord progenitors in the three contexts? Is there a possibility somehow to obtain human spinal cord progenitors and grow them in the same in vitro conditions as the rosettes to compare their differentiation rate? I am aware that some of these experiments are very hard to perform and not expecting the authors to perform all the suggested ones, yet, some more in-depth analysis would enable this article to explain better the presented observations.

    These are interesting suggestions, heterochronic grafting of the human neural rosettes, for example into the same site in an older chicken embryo would further test whether they continue to operate an intrinsic differentiation programme in this temporary distinct embryonic environment.

    Reviewer #3 (Public Review):

    The authors have developed dorsal spinal cord rosette assays from human pluripotent stem cells (hPSCs) and also from human induced pluripotent stem cells (hiPSCs) in a minimal culture medium containing retinoic acid. They define the dorsal spinal cord identity of these cells based on the presence of SOX2, PAX6, SNAI2 and PAX7, and absence of OLIG2 (characteristic of more ventral neural tube). Assessment of markers for migrating neural crest-like cells (HNK1, SOX10 and TFAP2alpha), immature neurons (DCX) and glial progenitors (NFIA) at different time points was used to show that the in vitro model recapitulates sequential differentiation observed in the spinal cord of avian and mouse embryos. Next, by comparing these results with neural differentiation in the human embryo, the authors show that neural differentiation occurs faster in vitro than in vivo. The authors then asked how these hiPSC-derived neural rosettes would respond to the more rapidly developing chicken embryonic environment, by grafting the rosettes into the developing chick neural tube. By assessing expression of various neural markers in the graft-derived cells, authors conclude that after two days of culture, human cells continued differentiation at the rate of the in vitro hiPSCs rather than at the rate of the host chicken cells. After longer culture (5 days), authors say that neurogenesis rate among graft-derived human cells attenuates and that the cells stall in the neural progenitor phase. Authors conclude that while initially an intrinsic differentiation programme is followed by the human cells, appropriate extrinsic inputs are required to maintain the neural differentiation trajectory of human cells.

    However, it is difficult to assess whether all conclusions by the authors for the human-into-chicken graft experiments are supported by their data, as some details of analysis are unclear (1) or experimental design was not conducive to the questions being asked (2). Some aspects of data analysis therefore need to be clarified and extended.

    1. Position of graft derived cells within the chicken host is very important when analysing presence/absence of a marker, but it is not always clear whether this has been taken into account by the authors. It appears that authors are assessing expression of markers in graft derived cells that are present outside OR inside domains in the chick host that would normally express that marker, and are not separating out such analysis. This will confuse interpretation of results and affect conclusions.

    One example where this would affect major conclusions of the manuscript is in the case of Islet-1 expression in human graft derived cells in the chicken host. Authors say that no Islet-1 was found in single graft derived cells in the chick embryo after two days of culture and use this to support their conclusion that the "pace of neural differentiation in the grafted human rosettes is unaltered in a more rapidly differentiating environment". However, Islet-1 expression in the chick is restricted to specific domains, therefore it would be important to know whether the graft-derived cells that the authors were analysing were within these Islet-1 positive host domains. Lack of Islet-1 in graft derived cells within such Islet-1 positive domains in chick would suggest that the graft derived cells have not responded to the host's timing of differentiation, and would support the authors' conclusions. However, lack of Islet-1 in graft derived cells outside of such Islet-1 positive domains could not be used to conclude the same thing as cells would be receiving different signals from the host. It appears that the graft used by the authors to show absence of Islet-1 in Fig 4G is outside of chick Islet-1 positive domains. Therefore, lack of Islet-1 in graft derived cells cannot be used to suggest that pace of human neural differentiation is initially directed by cell intrinsic factors, unless the location of the human cells in the chick is clearly shown to be within Islet-1 expressing domains in the chick.

    Human rosette cells were grafted into the chicken neural tube following removal of the dorsal half of the host neural tube at E2 and grafted cells were assessed in at least 3 sections from grafts in 3 different embryos for each marker analysed (see meta-data tables S1-7 and Methods). The reviewer is correct in that in a subset of sections graft cells were not in precisely the same position as chick endogenous Islet-1 expressing cells. We can provide the data which just includes only those with cells in this precise domain (3 sections from 3 different graft embryos), but also note that none of the sections analysed included human cells expressing ISLET1. This is the only marker analysed where this is an issue, other neuronal markers, such as P27 are expressed throughout the dorsal extent of the neural tube.

    1. Size of the graft used when transplanting human iPSCs into the chick will also affect the interpretation of results, as human cells will be exposed to varying levels of host signal depending on how much of their surface is exposed to host cells. Since the authors are using this experiment to test the effects of the chicken environment on human cells, this is a crucial point. After grafting hiPSC derived neural rosettes into the chick and culturing the chick embryo, authors assess expression of various markers in the graft-derived cells and separate out their analysis of marker expression across three different categories; cells found in 'cell rosettes', 'cell groups' or as 'single cells'. However, it remains unknown for how long these groupings were true during the culture time. For example, while it is known that at the time of grafting the cells were in a rosette structure, it is unknown at what time cells detached to incorporate as single cells (it could have been directly after grafting, or just prior to analysis) and is therefore not consistent across cells being analysed.

    One way to go around this would be not to graft the entire rosettes, but rather to dissociate the rosette and graft single cells/small groups of cells into the chick. With single cells the community effect (Gurdon 1988) would be avoided and the experiment would be testing the influence of only the host environment on this cell (rather than a combined influence of host environment and environment created by neighbouring graft derived cells as is the case in the current manuscript). This is particularly important as the data presented in the manuscript appear to show a difference between marker expression in single cells versus groups of cells and rosettes (plots in Fig 4 and 5).

    Details of rosette graft preparation are provided in the paper and this includes a gentle cell dissociation step, so we grafted human rosette cells that then reformed a rosette structure (which may reflect that human cells have greater affinity for each other), and some single cells were also initially available for insertion into the chicken neuroepithelium. It is likely that this cell mixing takes place early on while the chick dorsal neural tube reforms following the operation. For this reason, we analysed cell type specific markers in the human cells in large cell groups (reformed rosettes), smaller cell groups incorporated into the chick dorsal neural tube and single cells within this chick neuroepithelium. We appreciate that without the ability to monitor single cells throughout the experiment it is not possible to account fully for the environment experienced by a grafted cell. We agree that smaller grafts or other approaches may increase the number of cases of single human cells surrounded by chick neuroepithelial cells. We note the reviewer has taken up our consideration of the Community effect in the paper, which is of course why we have analysed marker expression in the three cell configurations. We also make clear in the paper that the apparent increase in P27 expression in single cells is not statistically significant and that this reflects the small number of single/isolated human cells within the chick neuroepithelium available for analysis (see metadata provided).

  3. eLife

    Evaluation Summary:

    This manuscript is of potential interest to a large audience in the fields of stem cells, developmental biology and neural regeneration. The authors assess the roles of extrinsic versus intrinsic signalling on differentiation of human neural cells by comparing their differentiation rates across different environments (in vitro, in the human embryo and grafted into a chicken embryo). While the experimental design tests the role of environment on differentiation, some aspects of data analysis need to be clarified and extended to support the conclusions.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    In this interesting paper by Dady and colleagues the nature of human neural progenitor differentiation is evaluated via transplantation studies. The first part of the paper establishes the timing of neural differentiation in human IPSC model systems and in human embryonic spinal cord, showing that the relative timing of neurogenesis and gliogenesis is maintained. In the second part of the paper these human IPSC neural rosettes are transplanted into the chick spinal cord during neurogenic stages (i.e. Isochronic transplantation) and they find that neurons are generated by these transplanted populations. Analysis of transplants at later stages reveals that neurogenesis has "stalled" and is relatively reduced within the transplanted population.

    Overall, this is an interesting paper that uses classic approaches to answer potentially interesting questions, however there are some issues that limit it's potential impact. The first two figures are recursive and show that the authors can implement an existing protocol. The transplantation studies are intriguing but do not offer sufficient new insights. The key finding seems to be that at later stages post-transplantation neuronal differential is "stalled". There are many other reasons (besides "stalling") that could explain their results. Suppose that stalling was indeed occurring, the authors offer no cellular or molecular insights into what regulates these intrinsic differences across species. At the end, it is not sufficiently clear what we have learned about the mechanisms that control the timing (pace seems to be another term for timing) of differentiation in human neural stem cells.

  5. eLife

    Reviewer #2 (Public Review):

    In general, this manuscript provides new significant knowledge by comparing between neural differentiation rate within the same species (human) in vivo and in vitro and between species (human and chick). The quality of the data is excellent, and the combining of the in vivo chick model to compare between grafted and host cells is a fantastic idea, that can only be done in this experimental model. Yet, some controls and more in-depth analysis are missing and are required in my opinion before publication.

    1. In the grating experiment, non-manipulated embryos serve as controls. Yet, the grafted rosettes are inserted near an injured area where a piece of the neural tube was moved. A better control would be to graft homologous cells from a donor chick embryo (GFP+ chick line is available in the UK) or quail embryo (which has a similar growing rate as chick at E2) and examining whether the injured area doesn't affect the grated cells to differentiate in a different pace as compared to the human grafts. This control is necessary to rule out the possibility that the human graft did not accelerate their differentiation rate and later stopped differentiating due to extrinsic signals/lack of signals form the manipulated environment.

    2. When examining the entire results of the manuscript some important points need to be addressed: On the one hand, the rosettes correspond to their in vitro growth conditions/extrinsic cues and display an accelerated differentiation pace, when compared to their in vivo counterpart human cells. On the other hand, the rosettes do not correspond initially to the chick environment and maintain their own intrinsic tempo. Later, they do change their developmental program and attenuate their differentiation. Therefore, the conclusion that the cells mostly obey to intrinsic regulation is confusing. It would be great if the authors could provide better experimental data to confirm their conclusion. Some ideas that the authors may consider are to determine whether there is a time window that sets the tempo of the rosettes that cannot be influenced later by extrinsic cues. Will the grafted cells correspond differently whether they would be grafted at a more/less advanced stages and domains? Is there an initial mechanistic elucidation to the different behavior of spinal cord progenitors in the three contexts? Is there a possibility somehow to obtain human spinal cord progenitors and grow them in the same in vitro conditions as the rosettes to compare their differentiation rate? I am aware that some of these experiments are very hard to perform and not expecting the authors to perform all the suggested ones, yet, some more in-depth analysis would enable this article to explain better the presented observations.

  6. eLife

    Reviewer #3 (Public Review):

    The authors have developed dorsal spinal cord rosette assays from human pluripotent stem cells (hPSCs) and also from human induced pluripotent stem cells (hiPSCs) in a minimal culture medium containing retinoic acid. They define the dorsal spinal cord identity of these cells based on the presence of SOX2, PAX6, SNAI2 and PAX7, and absence of OLIG2 (characteristic of more ventral neural tube). Assessment of markers for migrating neural crest-like cells (HNK1, SOX10 and TFAP2alpha), immature neurons (DCX) and glial progenitors (NFIA) at different time points was used to show that the in vitro model recapitulates sequential differentiation observed in the spinal cord of avian and mouse embryos. Next, by comparing these results with neural differentiation in the human embryo, the authors show that neural differentiation occurs faster in vitro than in vivo. The authors then asked how these hiPSC-derived neural rosettes would respond to the more rapidly developing chicken embryonic environment, by grafting the rosettes into the developing chick neural tube. By assessing expression of various neural markers in the graft-derived cells, authors conclude that after two days of culture, human cells continued differentiation at the rate of the in vitro hiPSCs rather than at the rate of the host chicken cells. After longer culture (5 days), authors say that neurogenesis rate among graft-derived human cells attenuates and that the cells stall in the neural progenitor phase. Authors conclude that while initially an intrinsic differentiation programme is followed by the human cells, appropriate extrinsic inputs are required to maintain the neural differentiation trajectory of human cells.

    However, it is difficult to assess whether all conclusions by the authors for the human-into-chicken graft experiments are supported by their data, as some details of analysis are unclear (1) or experimental design was not conducive to the questions being asked (2). Some aspects of data analysis therefore need to be clarified and extended.

    1. Position of graft derived cells within the chicken host is very important when analysing presence/absence of a marker, but it is not always clear whether this has been taken into account by the authors. It appears that authors are assessing expression of markers in graft derived cells that are present outside OR inside domains in the chick host that would normally express that marker, and are not separating out such analysis. This will confuse interpretation of results and affect conclusions.

    One example where this would affect major conclusions of the manuscript is in the case of Islet-1 expression in human graft derived cells in the chicken host. Authors say that no Islet-1 was found in single graft derived cells in the chick embryo after two days of culture and use this to support their conclusion that the "pace of neural differentiation in the grafted human rosettes is unaltered in a more rapidly differentiating environment". However, Islet-1 expression in the chick is restricted to specific domains, therefore it would be important to know whether the graft-derived cells that the authors were analysing were within these Islet-1 positive host domains. Lack of Islet-1 in graft derived cells within such Islet-1 positive domains in chick would suggest that the graft derived cells have not responded to the host's timing of differentiation, and would support the authors' conclusions. However, lack of Islet-1 in graft derived cells outside of such Islet-1 positive domains could not be used to conclude the same thing as cells would be receiving different signals from the host. It appears that the graft used by the authors to show absence of Islet-1 in Fig 4G is outside of chick Islet-1 positive domains. Therefore, lack of Islet-1 in graft derived cells cannot be used to suggest that pace of human neural differentiation is initially directed by cell intrinsic factors, unless the location of the human cells in the chick is clearly shown to be within Islet-1 expressing domains in the chick.

    2. Size of the graft used when transplanting human iPSCs into the chick will also affect the interpretation of results, as human cells will be exposed to varying levels of host signal depending on how much of their surface is exposed to host cells. Since the authors are using this experiment to test the effects of the chicken environment on human cells, this is a crucial point. After grafting hiPSC derived neural rosettes into the chick and culturing the chick embryo, authors assess expression of various markers in the graft-derived cells and separate out their analysis of marker expression across three different categories; cells found in 'cell rosettes', 'cell groups' or as 'single cells'. However, it remains unknown for how long these groupings were true during the culture time. For example, while it is known that at the time of grafting the cells were in a rosette structure, it is unknown at what time cells detached to incorporate as single cells (it could have been directly after grafting, or just prior to analysis) and is therefore not consistent across cells being analysed.

    One way to go around this would be not to graft the entire rosettes, but rather to dissociate the rosette and graft single cells/small groups of cells into the chick. With single cells the community effect (Gurdon 1988) would be avoided and the experiment would be testing the influence of only the host environment on this cell (rather than a combined influence of host environment and environment created by neighbouring graft derived cells as is the case in the current manuscript). This is particularly important as the data presented in the manuscript appear to show a difference between marker expression in single cells versus groups of cells and rosettes (plots in Fig 4 and 5).

  7. Version published to 10.1101/2021.02.06.429972v2 on bioRxiv
  8. Version published to 10.1101/2021.02.06.429972v1 on bioRxiv