Changes in neural progenitor lineage constituent during astrocytic differentiation of human iPSCs
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eLife Assessment
This useful study differentiated pluripotent stem cells to astrocytes, using a genetic modification that allows the long-lasting tracing of cells that initially turn on the gene LMX1A, crucial for midbrain identity. After selecting the positive cells, there were differences in physiological responses and some cellular processes with negative cells. The presented results, however, are incomplete to fully support the conclusions.
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Abstract
Abstract
The regional specificity of stem cell-derived astrocytes is believed to be an important prerequisite for their application in disease modelling and cell-based therapies. Due to the lack of subtype-defining markers for astrocytes in different regions of the brain, the regional identity of in vitro-derived astrocytes is often declared by the dominant positional characteristics of their antecedent neural progenitors, patterned to a fate of interest, with the assumption that the positional trait is preserved by the derived astrocytes via linear descent. Using a human induced pluripotent stem cell line designed for tracing derivatives of LMX1A+ cells combined with a ventral midbrain induction paradigm, we show that astrocytes originating from LMX1A+ progenitors can only be generated if these progenitors are purified prior to the astrocyte differentiation process, or their progenies are gradually lost to progenies of LMX1A- progenitors. This finding indicates that the lineage composition of iPSC-derived astrocytes may not accurately recapitulate the founder progenitor population. Using deep single-cell RNA sequencing, we identified distinct transcriptomic signatures in astrocytes derived from the LMX1A+ progenitor cells. Our study highlights the need for rigorous characterization of pluripotent stem cell-derived regional astrocytes, and provides a resource for assessing LMX1A+ ventral midbrain progenitor-derived human astrocytes.
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eLife Assessment
This useful study differentiated pluripotent stem cells to astrocytes, using a genetic modification that allows the long-lasting tracing of cells that initially turn on the gene LMX1A, crucial for midbrain identity. After selecting the positive cells, there were differences in physiological responses and some cellular processes with negative cells. The presented results, however, are incomplete to fully support the conclusions.
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Joint Public Review:
Summary:
In this manuscript, Li and coworkers present experiments generated with human induced pluripotent stem cells (iPSCs) differentiated to astrocytes through a three-step protocol consisting of neural induction/midbrain patterning, switch to expansion of astrocytic progenitors, and terminal differentiation to astroglial cells. They used lineage tracing with a LMX1A-Cre/AAVS1-BFP iPSCs line, where the initial expression of LMX1A and Cre allows the long-lasting expression of BFP, yielding BFP+ and BFP- populations, that were sorted when in the astrocytic progenitor expansion. BFP+ showed significantly higher number of cells positive to NFIA and SOX9 than BFP- cells, at 45 and 98 DIV. However, no significant differences in other markers such as AQP4, EAAT2, GFAP (which show a proportion of less than 10% in all …
Joint Public Review:
Summary:
In this manuscript, Li and coworkers present experiments generated with human induced pluripotent stem cells (iPSCs) differentiated to astrocytes through a three-step protocol consisting of neural induction/midbrain patterning, switch to expansion of astrocytic progenitors, and terminal differentiation to astroglial cells. They used lineage tracing with a LMX1A-Cre/AAVS1-BFP iPSCs line, where the initial expression of LMX1A and Cre allows the long-lasting expression of BFP, yielding BFP+ and BFP- populations, that were sorted when in the astrocytic progenitor expansion. BFP+ showed significantly higher number of cells positive to NFIA and SOX9 than BFP- cells, at 45 and 98 DIV. However, no significant differences in other markers such as AQP4, EAAT2, GFAP (which show a proportion of less than 10% in all cases) and S100B were found between BFP-positive or -negative, at these differentiation times. Intriguingly, non-patterned astrocytes produced higher proportions of GFAP positive cells than the midbrain-induced and then sorted populations. BFP+ cells have enhanced calcium responses after ATP addition, compared to BFP- cells. Single-cell RNA-seq of early and late cells from BFP- and BFP+ populations were compared to non-patterned astrocytes and neurons differentiated from iPSCs. Bioinformatic analyses of the transcriptomes resulted in 9 astrocyte clusters, 2 precursor clusters and one neuronal cluster. DEG analysis between BFP+ and BFP- populations showed some genes enriched in each population, which were subject to GO analysis, resulting in biological processes that are different for BFP+ or BFP- cells.
Strengths:
The manuscript tries to tackle an important aspect in Neuroscience, namely the importance of patterning in astrocytes. Regionalization is crucial for neuronal differentiation and the presented experiments constitute a trackable system to analyze both transcriptional identities and functionality on astrocytes.
Weaknesses:
The presented results have several fundamental issues, to be resolved, as listed in the following major points:
(1) It is very intriguing that GFAP is not expressed in late BFP- nor in BFP+ cultures, when authors designated them as mature astrocytes.
(2) In Fig. 2D, authors need to change the designation "% of positive nuclei".
(3) In Fig. 2E, the text describes a decrease caused by 2APB on the rise elicited by ATP, but the graph shows an increase with ATP+2APB. However, in Fig. 2F, the peak amplitude for BFP+ cells is higher in ATP than in ATP+2APD, which is mentioned in the text, but this is inconsistent with the graph in 2E.
(4) The description of Results in the single-cell section is confusing, particularly in the sorted CD49 and unsorted cultures. Where do these cells come from? Are they BFP-, BFP+, unsorted for BFP, or non-patterned? Which are the "all three astrocyte populations"? A more complete description of the "iPSC-derived neurons" is required in this section to allow the reader to understand the type and maturation stage of neurons, and if they are patterned or not.
(5) A puzzling fact is that both BFP- and BFP- cells have similar levels of LMX1A, as shown in Fig. S6F. How do authors explain this observation?
(6) In Fig. 3B, the non-patterned cells cluster away from the BFP+ and BFP-; on the other hand, early and late BFP- are close and the same is true for early and late BFP+. A possible interpretation of these results is that patterned astrocytes have different paths for differentiation, compared to non-patterned cells. If that can be implied from these data, authors should discuss the alternative ways for astrocytes to differentiate.
(7) Fig. 3D shows that cluster 9 is the only one with detectable and coincident expression of both S100B and GFAP expression. Please discuss why these widely-accepted astrocyte transcripts are not found in the other astrocytes clusters. Also, Sox9 is expressed in neurons, astrocyte precursors and astrocytes. Why is that?
(8) Line 337, Why authors selected a log2 change of 0.25? Typically, 1 or a higher number is used to ensure at least a 2-fold increase, or a 50% decrease. A volcano plot generated by the comparison of BFP+ with BFP- cells would be appropriate. The validation of differences by immunocytochemistry, between BFP+ and BFP-, is inconclusive. The staining is blur in the images presented in Fig. S8C. Quantification of the positive cells, without significant background signal, in both populations is required.
(9) Lines 349-351: BFP+ cells did not show higher levels of transcripts for LMX1A nor FOXA2. This fact jeopardizes the claim that these cells are still patterned. In the same line, there are not significant differences with cortical astrocytes, indicating a wider repertoire of the initially patterned cells, that seems to lose the midbrain phenotype. Furthermore, common DGE shared by BFP- and BFP+ cells when compared to non-patterned cells indicate that after culture, the pre-pattern in BFP+ cells is somehow lost, and coincides with the progression of BFP- cells.
(10) For the GO analyses, How did authors select 1153 genes? The previous section mentioned 287 genes unique for BFP+ cells. The Results section should include a rationale for performing a wider search for the enriched processes.
(11) For Fig. 4C and 4D, both p values and the number of genes should be indicated in the graph. I would advise to select the 10 or 15 most significant categories, these panels are very difficult to read. Whereas the listed processes for BFP+ have a relation to Parkinson disease, the ones detected for BFP- cells are related to extracellular matrix and tissue development. Does it mean that BFP+ cells have impaired formation of this matrix, or defective tissue development? This is in contradiction of enhanced calcium responses of BFP+ cells compared to BFP- cells.
(12) Both the comparison between midbrain and cortical astrocytes in Fig. S8A, and the volcano plot in S8B do not show consistent changes. For example, RCAN2 in Fig. S8A has the same intensity for cortical and midbrain cells, but is marked as an enriched gene in midbrain in the p vs log2FC graph in Fig. S8B. -
Author response:
The following is the authors’ response to the original reviews
Reviewer 1:
In vitro nature of this work being the fundamental weakness of this paper
We disagree with this statement. As explained in the provisional response, the aim of this study was to test the validity of a general concept applied in pluripotent stem cell research that pluripotent stem cell-derived astrocytes faithfully represent the lineage heterogeneity of their ancestral neural progenitors and hence preserve the regionality of such progenitors. Our genetic lineage study is justified for addressing this in vitro-driven question. However, we have highlighted the rationale where appropriate in the revised paper.
If regional identity is not maintained, so what? Don't we already know that this can happen? The authors acknowledge that this is known in …
Author response:
The following is the authors’ response to the original reviews
Reviewer 1:
In vitro nature of this work being the fundamental weakness of this paper
We disagree with this statement. As explained in the provisional response, the aim of this study was to test the validity of a general concept applied in pluripotent stem cell research that pluripotent stem cell-derived astrocytes faithfully represent the lineage heterogeneity of their ancestral neural progenitors and hence preserve the regionality of such progenitors. Our genetic lineage study is justified for addressing this in vitro-driven question. However, we have highlighted the rationale where appropriate in the revised paper.
If regional identity is not maintained, so what? Don't we already know that this can happen? The authors acknowledge that this is known in the discussion.
Importance of regional identity: Growing evidence demonstrates the functional heterogeneity of brain astrocytes in health and disease. Therefore, for in vitro disease modeling, it is believed that one should use astrocytes represent the anatomy of disease pathology; for example, midbrain astrocytes for studying dopamine neurodegeneration and Parkinson’s disease. Understanding the dynamics of stem cell-derived astrocytes and identifying astrocyte subtypes is important for their biomedical applications.
Regional identity change/Discussion: It seems that the reviewer misunderstood the context in which the ‘identity change’ was discussed. The literature referred to (in the Discussion) concerns shifts in regional gene expression in bulk-cultured cells. In the days of pre-single-cell analysis/lineage tracking, one cannot distinguish whether this was due to a change in the transcriptomic landscape in progenies of the same lineage or alterations in lineage heterogeneity, but to interpret at face value as regional identity was not maintained. In the revised paper, we have made an effort to indicate that ‘regional identity’ is used broadly to refer to lineage relationships and/or traits rather than static gene expressioin.
validation of the markers/additional work
The scNAseq analysis performed in this study compared the profiles of astrocytes derived from LMX1A+ and LMX1A- ventral midbrain-patterned neural progenitors. Since it is not possible to perform genetic lineage tracking in humans and an analogous mouse lineage tracer line is not available, in vivo validation of these markers with respect to their lineage relationship is not currently feasible. However, we took advantage of abundant single-cell human astrocyte transcriptomic datasets and validated our genes in silico. We also validated the differential expression of selected markers in late BFP+ and BFP- astrocytes using immunocytochemistry, where reliable antibodies are available. The results of the additional analyses are presented in Figure S8 and Supplemental Data 5.
Knowledge gaps concerning astrocyte development
Reviewer 1 pointed out a number of knowledge gaps concerning astrocyte development, such as the transcriptomic landscape trajectories of midbrain floor plate cells as they progress towards astrocytes. Indeed, the limited knowledge on regional astrocyte molecule heterogeneity restricts the objective validation of in vitro-derived astrocyte subtypes and the development of novel approaches for their generation in vitro. We agree with the need for in-depth in vivo studies using model organisms, although these are beyond the scope of the current work.
Reviewer 2:
(1) The authors argue that the depletion of BFP seen in the unsorted population immediately after the onset of astrogenic induction is due to the growth advantage of the derivatives of the residual LMX1A- population. However, no objective data supporting this idea is provided, and one could also hypothesize that the residual LMX1A- cells could affect the overall LMX1A expression in the culture through negative paracrine regulation.
We acknowledge the lack of evidence-based explanation for the depletion of BFP+ cells in mixed cultures. We were unable to perform additional experiments because of resource limitations. The design of the LMX1A-Cre/AAVS1-BFP lineage tracer line determines that BFP is expressed irreversibly in LMX1A-expressing cells or their derivatives regardless of their LMX1A expression status. Therefore, the potential negative paracrine regulation of LMX1A by residual LMX1A- cells should not affect cells that have already turned on BFP. We have highlighted the working principles of the LMX1A tracer line in the revised manuscript.
(2) Furthermore, on line 124 it is stated that: "Interestingly, the sorted BFP+ cells exhibited similar population growth rate to that of unsorted cultures...". In the face of the suggested growth disadvantage of those cells, this statement needs clarification.
To avoid confusion, we have removed the statement.
(3) Regarding the fidelity of the model system, it is not clear to me how the TagBFP expression was detected in the BFP+ population supposedly in d87 and d136 pooled astrocytes (Fig S6C) while no LMX1A expression was observed in the same cells (Fig S6F).
The TagBFP tracer is expressed in the progenies of LMX1A+ cells, regardless of their LMX1A expression status. We have gone through the MS text to ensure that this information has been provided.
(4) The generated single-cell RNASeq dataset is extremely valuable. However, given the number of conditions included in this study (i.e. early vs late astrocytes, BFP+ vs BFP-, sorted vs unsorted, plus non-patterned and neuronal samples) the resulting analysis lacks detail. For instance, from a developmental perspective and to better grasp the functional significance of astrocytic heterogeneity, it would be interesting to map the identified clusters to early vs late populations and to the BFP status.
We performed additional bioinformatics analysis, which provided independent support for the relative developmental maturity suggested by functional assays. The additional data are now provided in the revised Figure 3B, C, E.
Moreover, although comprehensive, Figure S7 is complex to understand given that citations rather than the reference populations are depicted.
The information provided in the revised Figure S7.
(5) Do the authors have any consideration regarding the morphology of the astrocytes obtained in this study? None of the late astrocyte images depict a prototypical stellate morphology, which is reported in many other studies involving the generation of iPSC-derived astrocytes and which is associated with the maturity status of the cell.
The morphology of our astrocytes was not unique to the present study. Many factors may influence the morphology of astrocytes, such as the culture media and supplements used, and maturity status. Based on the functional assays and limited GFAP expression, our astrocytes were relatively immature.
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eLife assessment
The manuscript by Li and coworkers analyzed astrocytic differentiation of midbrain floor plate-patterned neural cells originating from human iPS cells, with a LMX1A reporter. This valuable work identifies transcriptomic differences at the single-cell level, between astrocytes generated from LMX1A reporter positive or negative cells, as well as non-patterned astrocytes and neurons. The evidence is solid, but the paper can be strengthened by further analyses of the transcriptomic data, and astrocytic morphology; also, searching for some of the differentially expressed genes by immunohistochemistry in different regions of the mammalian brain, or in human specimens, would be very informative.
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Reviewer #1 (Public Review):
Summary:
In a previous study, the authors developed a human iPS cell line which expresses Cre under the control of the Lmx1a promoter in order to track, select for, and differentiate human dopamine neurons. In the manuscript under review, the authors are using methods which they have modified to generate astrocytes from the same cell line. The authors are interested in examining astrocytes which are derived from regionalized, floor plate progenitors.
The fundamental weakness of this paper is that the authors are making arguments about regional identity but their work is limited to experiments in vitro. Some of the claims that the authors make should be tested in vivo - ie, in sections, at least. Are floor plate markers or other ventral markers ever expressed in astrocytes or glial progenitors in the …
Reviewer #1 (Public Review):
Summary:
In a previous study, the authors developed a human iPS cell line which expresses Cre under the control of the Lmx1a promoter in order to track, select for, and differentiate human dopamine neurons. In the manuscript under review, the authors are using methods which they have modified to generate astrocytes from the same cell line. The authors are interested in examining astrocytes which are derived from regionalized, floor plate progenitors.
The fundamental weakness of this paper is that the authors are making arguments about regional identity but their work is limited to experiments in vitro. Some of the claims that the authors make should be tested in vivo - ie, in sections, at least. Are floor plate markers or other ventral markers ever expressed in astrocytes or glial progenitors in the mammalian fetus? When do astrocytes emerge in the floor plate? All of the data here are based on an overly simplified in vitro platform.
Lmx1a expression is not limited to the ventral midbrain; it is also expressed in other parts of the developing, ventral CNS and in the roof plate and dorsal CNS (Millonig et al, Nature 2000). Indeed, many of the phenotypes of the Lmx1a mutant mouse (dreher) have little to do with the ventral midbrain. The authors are making an assumption that regional identity is fixed when they begin their astrocyte differentiation protocol - not necessarily true. After astrocytic differentiation is initiated, the authors have done little to demonstrate that floor plate identity is maintained even in selected cells; in fact, the transcriptomic data suggests that the cells are released from a floor plate fate. The authors seem to realize this but do not make any attempt to prove their thesis. If regional identity is not maintained, the authors need a better experiment.
If regional identity is not maintained, so what? Don't we already know that this can happen? The authors acknowledge that this is known in the discussion.
The authors have done transcriptomics studies to follow the changes in these cells but they have not told us very much that is meaningful. It would be useful to validate some of the new astrocytic markers that they have identified - Pax and Irx genes (Welle et al., Glia 2021) come quickly to mind. What about genes related to Shh and Wnt signaling that are prevalent in the floor plate? In particular, a lot of work has been done examining the role of Shh on the properties and lineage of astrocytes (Farmer et al., Science 2016; Hill et al., eLife 2019; Gingrich et al., Neural Dev 2022; Xie et al., Cell Rep 2022). There are a lot of stones which remain unturned, here, and the authors could actually tell us much more without doing an immense amount of work. These suggestions and criticisms are described in far greater detail in the confidential comments to the authors.
Work Cited:
Chizhikov et al., Mamm Genome 2006. https://pubmed.ncbi.nlm.nih.gov/17019651/
Chizhikov et al., Development 2004. https://journals.biologists.com/dev/article/131/11/2693/42269/Control-of-roof-plate-formation-by-Lmx1a-in-the
Chizhikov et al., PNAS 2010. https://pubmed.ncbi.nlm.nih.gov/20498066/
Emsley and Macklis. Neuron Glia Biol 2007. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1820889/
Farmer et al., Science 2016. https://pubmed.ncbi.nlm.nih.gov/26912893/
Gross et al., Development 2016. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4958331/
Hill et al., eLife 2019. https://pubmed.ncbi.nlm.nih.gov/31194676/
Gingrich et al., Neural Dev 2022. https://pubmed.ncbi.nlm.nih.gov/35027088/
Iskusnykh et al., eLife 2023. https://elifesciences.org/articles/84095
Millonig et al, Nature 2000. https://pubmed.ncbi.nlm.nih.gov/10693804/
Welle et al. Glia 2021. https://pubmed.ncbi.nlm.nih.gov/36342840/
Xie et al., Cell Rep 2022. https://pubmed.ncbi.nlm.nih.gov/35196485/
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Reviewer #2 (Public Review):
In the current manuscript Li et al., study the preservation of the regional identity during the process of astrocyte generation from pluripotent stem cells. More precisely, this work investigates if neural progenitor cells patterned for the ventral midbrain give rise to astrocytes with conserved regional specification, which could reflect the astrocytic heterogeneity in the brain. To this end, the authors utilized a previously generated reporter iPSC line in which the expression of introduced blue fluorescence protein (BFP) is subjacent to the activation of LMXA1, a ventral midbrain floor plate marker. The study reports that following a defined patterning protocol based on SHH and FGF8, over 90% of d19 cells, corresponding to a neural progenitor stage, acquired the midbrain floor plate identity. However, …
Reviewer #2 (Public Review):
In the current manuscript Li et al., study the preservation of the regional identity during the process of astrocyte generation from pluripotent stem cells. More precisely, this work investigates if neural progenitor cells patterned for the ventral midbrain give rise to astrocytes with conserved regional specification, which could reflect the astrocytic heterogeneity in the brain. To this end, the authors utilized a previously generated reporter iPSC line in which the expression of introduced blue fluorescence protein (BFP) is subjacent to the activation of LMXA1, a ventral midbrain floor plate marker. The study reports that following a defined patterning protocol based on SHH and FGF8, over 90% of d19 cells, corresponding to a neural progenitor stage, acquired the midbrain floor plate identity. However, during the subsequent astrogenic induction and glial progenitor expansion, this identity is gradually lost, supposedly due to the growth advantage of cells deriving from the residual LMX1A- neural progenitors. Contrariwise, if the LMX1A+ progenitors were purified, regional identity would be maintained throughout the astrocytic generation and incur an early astrogenic switch and maturation of derived astrocytes. By using single-cell RNA sequencing, the authors further identified distinct transcriptomic signatures on the astrocytic progeny of LMX1A- and LMX1A- progenitors.
Strengths and weaknesses:
(1) The main model utilized was engineered from the KOLF2 human iPSC line into an elegant LMX1A-reporter line based on the expression of BFP. This results in an attractive model for studies tracing the fate of LMX1A cells. However, consideration should be given to the fact that the parental line, exhibits a splice disruption in the COL3A1 gene encoding type III collagen (Pantazis 2022, doi:10.1016/j.stem.2022.11.004 ), which has been identified as being enriched in certain ventral astrocytic populations (Bradley 2019, doi:10.1242/dev.170910).
(2) The authors argue that the depletion of BFP seen in the unsorted population immediately after the onset of astrogenic induction is due to the growth advantage of the derivatives of the residual LMX1A- population. However, no objective data supporting this idea is provided, and one could also hypothesize that the residual LMX1A- cells could affect the overall LMX1A expression in the culture through negative paracrine regulation. Therefore, cell cycle or proliferation studies of these cells are needed to prove the authors' assumption. Furthermore, on line 124 it is stated that: "Interestingly, the sorted BFP+ cells exhibited similar population growth rate to that of unsorted cultures...". In the face of the suggested growth disadvantage of those cells, this statement needs clarification.
(3) Regarding the fidelity of the model system, it is not clear to me how the TagBFP expression was detected in the BFP+ population supposedly in d87 and d136 pooled astrocytes (Fig S6C) while no LMX1A expression was observed in the same cells (Fig S6F).
(4) The generated single-cell RNASeq dataset is extremely valuable. However, given the number of conditions included in this study (i.e. early vs late astrocytes, BFP+ vs BFP-, sorted vs unsorted, plus non-patterned and neuronal samples) the resulting analysis lacks detail. For instance, from a developmental perspective and to better grasp the functional significance of astrocytic heterogeneity, it would be interesting to map the identified clusters to early vs late populations and to the BFP status. Moreover, although comprehensive, Figure S7 is complex to understand given that citations rather than the reference populations are depicted.
(5) Do the authors have any consideration regarding the morphology of the astrocytes obtained in this study? None of the late astrocyte images depict a prototypical stellate morphology, which is reported in many other studies involving the generation of iPSC-derived astrocytes and which is associated with the maturity status of the cell.
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Author response:
(1) Rationale of the study and key finding
We respectively disagree with Reviewer #1’s comments on ‘the fundamental weakness of this paper … about regional identity ...’. We believe that they misunderstood the rationale and key message of the paper.
The rationale of the study stems from the increasing recognition of the importance of generating ‘regional-specific’ astrocytes from iPSCs for disease modelling, due to astrocyte heterogeneity and their region-specific involvement in disease pathology. Regional astrocytes are typically differentiated from neural progenitors (NPCs) that are ‘patterned’ to the desired fate during iPSC neural induction. While the efficiency is not 100%, it is nevertheless assumed that the initial lineage composition (%) of patterned NPCs is preserved during the course of astrocyte …
Author response:
(1) Rationale of the study and key finding
We respectively disagree with Reviewer #1’s comments on ‘the fundamental weakness of this paper … about regional identity ...’. We believe that they misunderstood the rationale and key message of the paper.
The rationale of the study stems from the increasing recognition of the importance of generating ‘regional-specific’ astrocytes from iPSCs for disease modelling, due to astrocyte heterogeneity and their region-specific involvement in disease pathology. Regional astrocytes are typically differentiated from neural progenitors (NPCs) that are ‘patterned’ to the desired fate during iPSC neural induction. While the efficiency is not 100%, it is nevertheless assumed that the initial lineage composition (%) of patterned NPCs is preserved during the course of astrocyte differentiation and hence that the derived astrocytes represent the intended regional fate.
We questioned this approach using genetic lineage tracing with ventral midbrain-patterned neural progenitors as an example. By monitoring astrocytic induction of purified BFP+ NPCs and unsorted ventral midbrain-patterned NPC (referred to as BFP- in the paper, line 154 submitted PDF), we demonstrate that despite BFP+ NPCs being the vast majority (>90% LMX1A+ and FOXA2+) at the onset of astrocytic induction, their derivatives were lost in the final astrocyte product unless BFP+ NPCs were purified prior to astrocytic induction and differentiation.
Our findings demonstrate that iPSC-derived astrocytes may not faithfully represent the antecedent neural progenitor pool in terms of lineage, and that the regionality of PSC-derived astrocytes should not be assumed based on the (dominant) NPC identity. We believe that this finding is important for iPSC disease modelling research, especially where disease pathophysiology concerns astrocytes of specific brain regions.
Reviewer #1 raised several interesting questions concerning floor plate marker expression during astrocytic induction and astrocyte differentiation in normal development. These are important outstanding questions in developmental neurobiology, but they are outside the scope of this in vitro study. Indeed, the approach taken by published PSC-astrocyte studies - such as assigning regional identity of PSC-derived astrocytes based on the starting NPC fate or validating PSC-astrocyte using regional markers defined in the developing embryo - is partly due to our limited knowledge about the developing and mature astrocytes in different brain regions. This knowledge gap consequently restricts a thorough characterisation of the regional identity of PSC-astrocytes in such cases.
(2) LMX1A expression in the brain and LMX1A-BFP lineage tracer line
We thank Reviewer #1 for highlighting the wider expression of LMX1A. We are fully aware of this consideration and hence the thorough examination of PSC-derived ventral midbrain-patterned NPCs by immunostaining and single cell RNA-sequencing in this and a previous study (PMID: 38132179). All LMX1A+ cells produced in our protocol exhibit ventral midbrain progenitor gene expression profiles when compared to dataset obtained from human fetal ventral midbrain.
Some of the comments give us the impression that there might be some confusion regarding the lineage tracing system used in this study. The LMX1A-Cre/AAVS1-BFP line is not a classic reporter line that mark LMX1A-expressing cells in real time. Instead, it was designed as a tracer line that expresses BFP in the derivatives of LMX1A+ cells as well as cells expressing LMX1A at the time of analysis.
(3) Is regional identity fixed?
We feel that Reviewer #1 misunderstood the paper in their comments ‘The authors are making an assumption that regional identity is fixed when they begin their astrocyte differentiation protocol - not necessarily true…’. We in fact pointed out in the paper that expression of LMX1A and FOXA2, a signature of midbrain floor plate progenitors, is lost in our BFP+ astrocytes. In this paper, ‘regional identity’ was loosely used to also refer to lineage identity and genetic traits, not just gene expression. We will consider alternative wording during revision to avoid potential confusion.
(4) Splice disruption in the COL3A1 gene and potential effect on astrocyte differentiation of Kolf2 iPSCs
We thank Reviewer #2 for highlighting the variations in KOLF2C1 hiPSCs and the study by Bradley et al. (2019) on differential COL3A1 expression in some ventral astrocytes. We noted that the progenitors produced by Bradley et al. were NKX2.1+ ventral forebrain cells, rather than the LMX1A+ ventral midbrain progenitors investigated in our study. Our scRNAseq data show that all three populations of astrocytes exhibit low levels of COL3A1 expression. While we will continue to examine astrocyte COL3A1 expression in publicly available gene expression datasets, we feel that a selective impairment in astrocyte differentiation of BFP+ cells is unlikely.
(5) Additional data analysis and validation of potential new markers
We will carefully consider the reviewers’ suggestions on further analysis of our single-cell RNA sequencing dada during revision. Regarding eLife’s assessment of validating differential gene expression in different brain regions, it is worth noting that both BFP+ and BFP- cells mapped to the published midbrain scRNAseq data set (La Manno et al, Cell 2016, PMID: 27716510), supporting their midbrain fate. We agree in principle that all single-cell RNA sequencing findings should be validated by immunostaining. It would be beneficial to experimentally verify that our candidate BFP+ differentially expressed genes indeed mark astrocytes derived from LMX1A+ NPCs in vivo, as opposed to other midbrain NPCs. However, this verification cannot be realistically performed in a human setting, but only in an analogous mouse tracer line.
The current eLife assessment nicely summarised part of our findings, in a sense secondary output of this work. We would appreciate a revised eLife assessment that include the message that iPSC-derived astrocytes, in terms of genetic lineage, can deviate greatly from the starting progenitor pool. We would be very happy to provide further information or clarification if it would be helpful. We are committed to doing our best as authors to enhance reader experience and support the continued success of eLife.
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