High-resolution transcriptional landscape of xeno-free human induced pluripotent stem cell-derived cerebellar organoids
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Abstract
Current protocols for producing cerebellar neurons from human pluripotent stem cells (hPSCs) often rely on animal co-culture and mostly exist as monolayers, limiting their capability to recapitulate the complex processes in the developing cerebellum. Here, we employed a robust method, without the need for mouse co-culture to generate three-dimensional cerebellar organoids from hPSCs that display hallmarks of in vivo cerebellar development. Single-cell profiling followed by comparison to human and mouse cerebellar atlases revealed the presence and maturity of transcriptionally distinct populations encompassing major cerebellar cell types. Encapsulation with Matrigel aimed to provide more physiologically-relevant conditions through recapitulation of basement-membrane signalling, influenced both growth dynamics and cellular composition of the organoids, altering developmentally relevant gene expression programmes. We identified enrichment of cerebellar disease genes in distinct cell populations in the hPSC-derived cerebellar organoids. These findings ascertain xeno-free human cerebellar organoids as a unique model to gain insight into cerebellar development and its associated disorders.
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###Reviewer #3:
Nayler et al. report methods to generate cerebellar organoids from human induced pluripotent stem cells and their characterization by single-cell sequencing and bioinformatic analysis. They further test the effect of adding Matrigel to the system, which has previously been useful in other organoid systems. The topic is important for the study of human cerebellar developmental and modeling of human disease. The paper suffers from a number of issues, especially the fact that the claims in the text are not supported by the data.
Specific comments:
The method is largely the same as developed by Muguruma et al, a methodology that has not proved to be very effective or reproducible. That said, it is not clear that cerebellar organoids generated in this report have differentiated as well as the original paper based on …
###Reviewer #3:
Nayler et al. report methods to generate cerebellar organoids from human induced pluripotent stem cells and their characterization by single-cell sequencing and bioinformatic analysis. They further test the effect of adding Matrigel to the system, which has previously been useful in other organoid systems. The topic is important for the study of human cerebellar developmental and modeling of human disease. The paper suffers from a number of issues, especially the fact that the claims in the text are not supported by the data.
Specific comments:
The method is largely the same as developed by Muguruma et al, a methodology that has not proved to be very effective or reproducible. That said, it is not clear that cerebellar organoids generated in this report have differentiated as well as the original paper based on immunolabeling, though this may be due to low power images. The authors repeatedly point out that their method does not need co-culture with mouse granule cells, however they show no maturation of Purkinje cells, which is what prior reports had used them for.
While this method is not entirely novel, single-cell sequencing has not previously been performed using this method. Unfortunately, their analysis of the scRNAsea data is qualitative and unconvincing.
Canonical markers are not associated with the expected populations. For example, PCP4 and IGF1 are found in the P0-choroid plexus group and not with P6 Purkinje cells (PCs), suggesting the markers or separation of populations used for classification are not sufficient. CXCL14 is used as an identifier for PCs, however the gene appears to be downregulated in the P6-PC expression table, while it is instead upregulated in the P0 expression table. These discrepancies between the text and the data do not give confidence in the overall analysis.
Fig S4A there is no legend describing what the dot plot shows (color scale, size scale)
To substantiate cell classification, the authors compare their data with previously published mouse datasets. Cell type clusters are generously suggested to have a "high degree of overlap" with mouse data, with a "high degree of confidence". These claims are not statistically supported nor upon close inspection do they appear to be accurate. While some cells types cluster with mouse cell types, others clearly do not. For example, of the two major cerebellar neurons, human granule cells are found in three clusters (granule cell precursors, granule cells (S-phase), and granule cells (G2M-phase), of which only one clusters with mouse granule cells. Human and mouse Purkinje cells do not cluster. The authors state that pseudotime trajectory reconstruction shows "a pattern reminiscent of the developmental cellular phylogeny of the cerebellum; progression from primitive CP/RP cell types to RL/VZ precursors and subsequently to committed neuronal progeny..." however the choroid plexus and roof plate do not give rise to rhombic lip or ventricular zone precursors (note, ventricular zone precursors are not depicted in the data).
Embedding of cerebellar organoids in Matrigel is novel, however a major finding of this report is that Matrigel increases organoid variability, which itself is already a significant issue in the organoid field. The role of Matrigel in promoting specification of rhombic lip over ventricular zone could be useful.
Have they looked at gene expression any earlier than DIV21? When is the timepoint at which each of the key cerebellar markers appear? This information is lacking for all markers assessed and it is not clear why the timepoints that they are showing were chosen. More characterization and perhaps even scRNA at multiple time-points would have given a clearer view of what they have induced.
7)There is huge variability in gene expression even before the Matrigel addition step. It is therefore unclear how this is an advancement in making cerebellar organoids compared to the original Muruguma paper in 2015 (which was a very qualitative paper itself).
- Low power images of immunolabeling make it impossible to assess the localization of labelling and distinguish between real and background staining. eg: Fig S1 and Fig 1A. This is critical in the stem cells field where spatial organization cannot be relied upon.
Their interpretation and their data don't always match with regard to their defined cell types and scRNAseq data. For example, ATOH1 only appears in group 5 yet they mention that more groups are graule cell precursors. Also, they say that a major impact of MG encapsulation is the expansion of the GC lineage, yet earlier in the paper they say that ATOH1 expression levels, a marker of the GC lineage, were unchanged, making it very difficult to get a clear picture of what they have found.
A major issue (along the same vein as their incorrect data interpretation) upon which the paper is framed is the assumption that the human cell types are like their mouse counterparts. No experiments were carried out to show the validity of this assumption. Figure 3B overlays the human and mouse data. Why such low representation of the human cells? Is it because of low sequencing depth (technical issue) or vastly different molecular composition of these organoids when compared to the mouse cerebellum?
Overall the execution is poor, and the data are not analyzed in any depth. Critically, there is a complete mismatch between what is stated in the text and what is shown in the figures. The claim to have produced all major cerebellar cell types would have been the novel aspect of the paper, but the data are unconvincing.
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###Reviewer #2:
In this Tools and Resources manuscript, Nayler and colleagues demonstrate a robust and reproducible protocol for hIPSC derived cerebellar organoids which do not require feeder populations. In general development of reliable pluripotent cell derived cerebellar cell types and organoids have been lagging compared to other regions of the brain and this paper represents a new resource. Given that the manuscript is presented as a resource, more detailed explanation of the generation of the organoids should be provided and their reproducibility should be demonstrated in more detail. Further histological characterization of the organoids with additional markers is needed to really see the reproducibility and the robustness of the methodology.
Major comments:
Authors mention that the PCs have bipolar morphology (data not shown). …
###Reviewer #2:
In this Tools and Resources manuscript, Nayler and colleagues demonstrate a robust and reproducible protocol for hIPSC derived cerebellar organoids which do not require feeder populations. In general development of reliable pluripotent cell derived cerebellar cell types and organoids have been lagging compared to other regions of the brain and this paper represents a new resource. Given that the manuscript is presented as a resource, more detailed explanation of the generation of the organoids should be provided and their reproducibility should be demonstrated in more detail. Further histological characterization of the organoids with additional markers is needed to really see the reproducibility and the robustness of the methodology.
Major comments:
Authors mention that the PCs have bipolar morphology (data not shown). I think this is one of the critical pieces of data that demonstrates the quality of the organoids and should be shown. In general, more IF analysis of the organoids with additional markers would have been helpful to understand the variabilities and the composition of the cerebellar organoids that were generated with their method.
Did the authors observe a delay in the maturation of the Matrigel embedded organoids? It is curious that there is an increase in the earlier progenitor cells (based on the increase in the OLIG2 expression as opposed to PTF1A). Based on the data later in the paper, authors suggest that Matrigel increases the expansion of GCPs. How does the non-significant enrichment of the ATOH1 expression shown in Figure 1G relate to the data presented later in the manuscript? It looks like only one of the organoid had upregulation of ATOH1 where other two didn't show any change?
Authors should report the relative proportions of the VZ- derived vs. RL-derived cell types within each organoid.
Were there any astrocytes (other than Bg) and OPC/oligodentrocytes observed in the organoids? Or do they need to culture them longer to observe those cells.
Why is there very low expression of PCP4 in the PCs and the cluster with most PCP4 expression is classified as Choroid plexus? Based on the in situ in figure S4, there is no PCP4 in the CP. Is this a species difference? In general characterization of the PCs are confusing to me based on the markers used. Please elaborate.
Based on the clustering shown in figure 3, is there a particular age from the mouse data that showed higher enrichment for overlapping human cerebellar organoid cells. The way the data is presented is hard to interpret and understand. Also, the ranges of the ages in the mouse data that overlaps with the respective human data is a lot larger than I would have expected (page 9 first paragraph). I am not an expert on integrating such multi age/species data however, I wonder if some additional pseudotime analysis like monocle could be performed on the combined data set represented in Figure S7 and Figure 3 would reveal finer temporal resolution of the human organoid with respect to the mouse developmental data.
Were there differences in the pseudotime ordering of the cells from Matrigel embedded compared to the ones from the control organoids (related to point 2).
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###Reviewer #1:
In this manuscript, Nayler et al present a new protocol to generate cerebellar organoids that they differentiate from human iPSCs. Using this system and single-cell sequencing, the authors show that most major cerebellar cell types develop in these organoids. They also find that the micro-environment of the developing organoids changes growth dynamics and cellular differentiation, which motivated the authors to suggest that this organoid approach may be a good model for studying human cerebellar development and disease. The strength, and indeed the motivation, of this manuscript is the description of a novel model system with which to study multiple human cerebellar subtypes in an ex vivo system. In general, this work is a timely addition to several other recent studies on the transcriptomics of mouse cerebellar …
###Reviewer #1:
In this manuscript, Nayler et al present a new protocol to generate cerebellar organoids that they differentiate from human iPSCs. Using this system and single-cell sequencing, the authors show that most major cerebellar cell types develop in these organoids. They also find that the micro-environment of the developing organoids changes growth dynamics and cellular differentiation, which motivated the authors to suggest that this organoid approach may be a good model for studying human cerebellar development and disease. The strength, and indeed the motivation, of this manuscript is the description of a novel model system with which to study multiple human cerebellar subtypes in an ex vivo system. In general, this work is a timely addition to several other recent studies on the transcriptomics of mouse cerebellar development, transcriptomics of human cerebellar development, and the use of hPSC derived Purkinje cells grown in co-cultures with mouse granule cells. The data in this manuscript are strong and likely of broad interest to the neuroscience community. However, below I outline several concerns that, if addressed, would help improve the clarity, readability, and impact of the manuscript:
Comments:
In the title, the authors state "...cerebellar organoids shows recapitulation of cerebellar development". Development in what? human? model systems? Some specificity will be needed in this title, especially since recent work from the Millen group has unveiled some specific differences between mouse and human cerebellar development.
In the Abstract, the authors state "However, this was at the expense of reproducibility." What do you mean? There are issues with reproducibility? If yes, the authors need to provide a thorough discussion about this, as this issue would be essential for researchers to know about if they were to adopt this approach.
Also in the Abstract, the authors state "...conditions, representing a more biologically relevant..." More biologically relevant than what? What about the counter argument that studying the cerebellum would be "more biologically relevant" in vivo in an animal model?
In the Introduction, the authors state "Specifically, abnormal cerebellar development is an emerging theme contributing to many brain disorders (Sathyanesan et al., 2019)." Do you mean to many non-motor brain disorders?
A couple of times in the Introduction the authors use the Manto et al. 2012 reference. This is in fact a very large online book consisting of several dozen chapters. Rather than using such a broad sweep approach, I would highly recommend using the primary original references for such key statements. It's also slightly misleading since Manto himself did not have any involvement in these developmental studies.
The authors state that "Current models have mainly focussed on the differentiation of hPSC-derived Purkinje cells through co-culture with mouse cerebellar progenitors." Okay, but what is your argument against such methodology? Some context and motivation for this statement should be provided.
In the Introduction, the authors frame their case by stating "As a proof of principle,..." But, what is this method proof of principle for?
In the Introduction, the authors state "...we show perturbation analysis of the organoids..." Please state what the perturbation was, and what problem was this perturbation used to test?
Based on the Introduction of the paper, it is very hard to see what motivates this work. Also, related, why focus on the basement membrane? What led to this? The authors need to provide a much stronger rationale for the study upfront, and in particular for the specific concepts that they tackle using their new approach.
The authors state that induction of GBX2 was observed at the expense of the anterior marker OTX2. Apologies if I have missed it, but what was the experiment that shows directly in your organoids that OTX2 was initially high and then lowered due to GBX2?
The authors state "...EBs to MG treatment, we encapsulated these at three different timepoints during differentiation..." What was the justification for picking these timepoints?
The authors state "Overall, the relative effect of MG encapsulation resulted in distinct responses in the various cerebellar populations..." So, what does it mean that each cell type has a different response? Please expand on this.
The authors state "using the murine cerebellum as a close developmental blueprint, most signatures indicate a mixture of mid-late embryonic temporal maturity, suggesting that the cerebellar organoids recapitulate developmental stages of the normally developing cerebellum. An exception to this was overlap of human GCs with murine GCs of postnatal maturity, suggesting that this cell type was more mature than its counterparts." AND "human PCs clustered more closely to murine progenitors and astroglia, suggesting that by day 90 organoid-derived PCs were still developmentally immature, compared with murine PCs. In further support of this, we did not detect appreciable levels of SHH."
The sentences in this statement raise several questions. First, PCs normally develop before the GCs. Thus, the finding that PCs in the organoids are less mature than GCs is surprising and may even be concerning as it suggests that the organoids do not fully (or reliably) replicate the temporal order of normal cell development that is so characteristic for cerebellar development. Second, the relationship between PC SHH secretion and the responding GC is now well established and has been shown to be an important, if not essential, mechanism for GCs proliferation in vivo. It is therefore surprising that GCs form and proliferate in the organoid without proper SHH signaling. What may be the mechanism for this? The authors need to account for this issue and provide a discussion to address all of these points as well. Moreover, the authors should discuss how the maturation state of PCs in the organoids is different between this paper and the recently published Buchholz et al paper (2020 - DOI: 10.1073/pnas.2000102117).
The authors argue about the cell structure and expression of cell markers in the organoids. However, based on what is shown, it is not clear how robust these features are in the organoids. The authors need to provide additional images of the organoids at much higher magnification in order to properly demonstrate cell structure and identity. In this regard, based on their argument, it would be important for authors to show the bipolar morphology of Calbindin-positive cells and excessive neural outgrowth at the periphery of the organoids (currently referred to in text as "data not shown"). Finally, it would be interesting to see whether different cell types are intermingling or spatially segregated in the organoids. That is, what does the cellular organization in the organoid actually look like?
Along the same lines as above, it seems to me that the authors should present more details about the anatomical architecture of the organoids. One of the major arguments raised by the authors is that the organoids recapitulate many features of normal cerebellar development. Of course, the organoids likely don't show all the intricacies of in vivo cerebellar development, but given that the 3-dimensional assembly of the cerebellum is essential for all aspects of cellular and circuit formation, one needs to fully appreciate exactly what aspects of the cerebellum the organoid is able to reflect. Only then can one predict its full utility towards studying different aspects of development or disease.
There are several cases that the authors state "data not shown". In every one of these cases the data seems essential to me and it should be presented in full.
The authors use the fact that the cell types from the human organoids cluster with mouse cerebellar cell types as an argument that the human organoids have a good representation of the cerebellar cell types. But, the authors also go on to state that the human organoids are advantageous over model organisms because they may better model human genetic background. These two statements are contradictory, especially given the previous issue raised about the organoids not reproducing the temporal sequence of cellular development. Do the authors have additional data to support their statements about the biological relevance of their xeno-free conditions? For example, did they find any human specific genes or developmental pathways? The statements presented by the authors creates a circular argument that needs to be revised and/or supported by additional data. What would help is a much deeper comparison between the organoids, human cerebellar development, and mouse cerebellar development.
What is the fold-change of RNA expression in figure 1 based on? What is the statistical test actually testing? What is the control that this fold-change is compared to?
On the issue of statistics, the section describing the statistics in the methods is rather brief. It would help tremendously if the authors expanded this section by describing which test goes with which experiment and some level of justification for the use of the different statistical tests would be very useful as well.
The authors use a lot of abbreviations. Some of these abbreviations hinder the readability of the text, which would be especially problematic for an audience not as closely acquainted with these terms. It may help to limit the use of abbreviations to cell-types and gene names. For example, Matrigel and embryonic bodies do not have to be abbreviated.
The size of the text in all figures is too small, including gene names, axis labels, and legends.
In the Discussion, the authors state "...this includes proximally located territories in which adjacent signalling is required for cerebellar maturation and development." I am not sure whether enough direct evidence is presented to make this conclusion. As commented on before, additional anatomy should be presented, and based on those data, inter-cellular signaling could then be examined with more confidence. Otherwise, the authors would have to tone down and/or revise this conclusion.
The authors conclude that "hiPSC-derived organoid models offer unprecedented opportunities to model brain development and disorders and for therapeutic development..." I agree, but as a general comment, I found it very hard to know what exactly the authors are comparing in this paper. It appears that the comparisons are mainly to mouse development, although it seems that a more thorough and direct side by side comparison should be made. I suppose some kind of detailed developmental timeline-based model is warranted.
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##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary:
Based on several recent molecular studies, the strength of the current manuscript is the establishment of an organoid approach that could potentially add to our knowledge of normal and abnormal cerebellar development by providing a flexible technique with which to resolve cellular mechanisms. However, there was overall agreement that while the approach has promise, the data presented are lacking in terms of a concrete comparison to known milestones in cerebellar development (in animal models or human). …
##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary:
Based on several recent molecular studies, the strength of the current manuscript is the establishment of an organoid approach that could potentially add to our knowledge of normal and abnormal cerebellar development by providing a flexible technique with which to resolve cellular mechanisms. However, there was overall agreement that while the approach has promise, the data presented are lacking in terms of a concrete comparison to known milestones in cerebellar development (in animal models or human). Moreover, given the technical nature of the manuscript, it was deemed necessary that a more complete characterization of the organoid "anatomy" would be required in order to convince the reader of the claims. There was also a concern that the quantitative aspects and interpretation of the scRNA-seq experiments, particularly the characterization of the clusters obtained and the analysis performed to compare the human organoid data to the mouse developmental data, could have been carried out with greater depth.
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