Paraxial mesoderm organoids model development of human somites

  1. Christoph Budjan
  2. Shichen Liu
  3. Adrian Ranga
  4. Senjuti Gayen
  5. Olivier Pourquié
  6. Sahand Hormoz

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    Evaluation Summary:

    Budjan et al. describe an organoid protocol to obtain somite-like structures from human iPSCs. Using defined culture media, the authors describe the formation after 5 days in vitro of organoids that express a variety of PSM differentiation markers, such as the segmentation clock gene Hes7 and Pax3. Optimization of their culture conditions and transcription analyses of what they name their "somitoid" system revealed that their culture system recapitulates the time course of expression markers typically observed along PSM and somite early differentiation. Furthermore, somitoid reacted to Shh activation by activating sclerotomal markers Pax1 and 9.

    (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. The reviewers remained anonymous to the authors.)

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Abstract

During the development of the vertebrate embryo, segmented structures called somites are periodically formed from the presomitic mesoderm (PSM) and give rise to the vertebral column. While somite formation has been studied in several animal models, it is less clear how well this process is conserved in humans. Recent progress has made it possible to study aspects of human paraxial mesoderm (PM) development such as the human segmentation clock in vitro using human pluripotent stem cells (hPSCs); however, somite formation has not been observed in these monolayer cultures. Here, we describe the generation of human PM organoids from hPSCs (termed Somitoids), which recapitulate the molecular, morphological, and functional features of PM development, including formation of somite-like structures in vitro . Using a quantitative image-based screen, we identify critical parameters such as initial cell number and signaling modulations that reproducibly yielded formation of somite-like structures in our organoid system. In addition, using single-cell RNA-sequencing and 3D imaging, we show that PM organoids both transcriptionally and morphologically resemble their in vivo counterparts and can be differentiated into somite derivatives. Our organoid system is reproducible and scalable, allowing for the systematic and quantitative analysis of human spine development and disease in vitro .

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  1. Version published to 10.7554/elife.68925 on eLife
  2. Version published to 10.1101/2021.03.22.436471v2 on bioRxiv
  3. eLife

    Author Response:

    We thank the reviewers and editor for the feedback on our manuscript. We now included high-magnification images of somite-like structures, which clearly show the shape of the somitic cells and the polarity of these epithelial cells by staining for multiple polarity markers (new Figure 3-Supplemental Fig 1; new Figure 3-Video 1). Consistent with the morphology and polarity of in vivo somites, we observe PAX3+/TCF15+ bottle-shaped cells radially arranged around a central cavity, which form rosette-like structures that are approximately the same size as the Carnegie stage 11 in vivo somites (Fig 5B). This can be observed in multiple new images added to the manuscript (Fig 3-Supplemental Fig 1A-A’’, Fig 3-Supplemental Fig 1B-B’’, Fig 2-Supplemental Fig 3A,B). Additionally, apical surface markers F-ACTIN (Fig 5A) and N-CADHERIN (Fig 3-Supplemental Fig 1) are both expressed around the central cavity, suggesting that the apical side of the somitic cells is facing the inside of the somite structure, again consistent with their in vivo counterparts. This is particularly evident in the newly added high-magnification images (Figure 3-Supplemental Fig 1) and accompanying movie showing a full confocal z-stack through the in vitro somites (Figure 3-Video 1). Together with our protein (Fig 3B,C and Fig 5A) and gene expression data (both in bulk (Fig 1C, Fig 5C, Fig 1-Supplemental Fig 1B) and at the single-cell level (Fig 4, Fig 4-Supplemental Fig 1-5)), and directed differentiation experiments of Somitoid-derived cells towards sclerotome (Fig 5C) and dermomyotome (newly added Figure 5-Supplemental Fig 1), we conclude that our in vitro somites are molecularly, morphologically, and functionally equivalent to in vivo somites.

    Reviewer #1:

    The foundational differentiation protocol up until day3 (formation of PSM) has been published previously in Diaz-Cuadros et al., 2020; Matsuda et al., 2020. The main difference between this manuscript and published protocol being the 2D (published) vs 3D differentiation. In this manuscript the authors were able to generate Pax3+ Somites (day4-5) from PSM. Both Diaz-Cuadros et al., 2020; Matsuda et al., 2020 were unable to generate Pax3+ somite in their 2D culture system but instead could only obtain a TCF15+ somatic mesoderm intermediate state. Moreover, the somites obtained in this manuscript could be further differentiated to sclerotome.

    The experiments across the paper were validated using three repeats using appropriate quantitative microscopy. Imaging data are high quality and mostly presented in a clear manner. However, it is unclear exactly what the authors are scoring as a somite. Moreover, for each figure it is not clear whether technical or biological replicates are presented. Similarly, the heatmap block presented in Fig2C,D, 3C,D apparently represents just one organoid/replicate. Authors should comment on the efficiency of the protocol over the different cell lines used. Transcriptome data presented strongly support the reproducibility and accuracy of the 3D differentiation, although comparison with the in vivo situation is highly limited - in part due to lack of availability of human in vivo data at these developmental stages.

    We thank the reviewer for the insightful comments and questions. Following the suggestion of the reviewer, we generated new organoids with somite structures and imaged them at a higher magnification (shown in the newly added Figure 3-Supplement Figure 1). The higher magnification clearly shows the ‘bottle-shaped’ morphology of the cells that comprise the somites in that the apical surface of these columnar cells is typically smaller than their basal side. These higher-magnification images also more clearly show the rosette structure (radial arrangement of columnar cells) with a central cavity in which NCAD is highly expressed. Nuclear expression of PAX3 is also clearly visible in these cells. These empirical observations were the criteria used to manually identify somites in the images acquired from the organoid screen. Additionally, we generated movies of z-stacks acquired on a confocal microscope showing examples of organoids with and without somite-like structures based on our scoring criteria using PAX3 and NCAD staining (Newly added Figure 3-Video 1).

    Regarding the number of replicates used and the questions related to variability/efficiency of our optimized protocol, we have made the following changes and performed new experiments to address these questions. To clarify which cell lines were used for each figure, we have added the information to each figure caption in the manuscript. We have also specified the number of organoids used to quantify variation for each experiment in the figure captions. As described below, we have conducted new experiments to further quantify the technical variability across experiments, as well as quantify the efficacy of our optimized differentiation protocol when applied to genetically independent cell lines.

    For our initial screens (Figures 2 and 3), we only used one cell line (NCRM1 hiPSCs). As mentioned by the reviewer, we measured variability across individual organoids for each condition of the screen to help identify the conditions that minimized variability and produced the most reproducible organoids (Figure 2-Supplemental Fig 2B; Figure 3-Supplemental Fig 1B). To analyze technical variability of our optimized protocol, we have repeated our optimized protocol two more times and quantified the number of somites across 10 organoids for each experiment (see new Figure 3-Supplemental Fig 3). Inter-organoid variability was comparable to our initial results from the secondary screen (CV for Experiment 1 = 17% and CV for Experiment 2 = 9.8%; see also Fig 3-Supplemental Fig 2B). Furthermore, mean and median are very comparable between the two additional experiments (Experiment 1: 39+/-8 (mean+/-std), median=40; Experiment 2: 43+/-4 (mean+/-std), median=41, p-val = 0.16). Please note that the absolute number of somites in our new experiments has increased compared to our initial screen (see Figure 3). This is a result of improvements in both our immunostaining protocol as well as the image acquisition workflow. The two new experiments both used the same improved staining and image acquisition workflow and are therefore comparable with each other.

    To extend our variability analysis to other cell lines, we tested the following cell lines: our original cell line (NCRM1), the WTC cell line released by the Conklin Laboratory at the Gladstone Institute, and a reporter cell line, ACTB-GFP, from the Allen Cell Collection. We tested our optimized protocol alongside another high scoring condition in all three cell lines:

    • CL+FGF2 for 24h, basal media for 24h (our optimized protocol)
    • WNTihi(C59, 2 µM) for 48h

    Applying our optimized protocol to two other cell lines confirmed that our protocol is reproducible across different genetic backgrounds/cell lines. NCRM1, average somite number = 43+/-4 (mean+/-std); ACTB-GFP, average somite number = 40+/-6; WTC, average somite number = 33+/-4. We also tested one additional top scoring condition (C59, 2µM for 48 hours) in all three cell lines. Notably, for this condition, the ACTB-GFP derived Somitoids (32+/-4 (mean+/-std)) showed a higher average number of somites compared with the Somitoids derived from the other cell lines (NCRM1, 20+/-3; WTC, 17+/-4). However, our optimized protocol resulted in the highest number of somites across all cell lines.

    We agree with the reviewer that our transcriptome analysis strongly supports the reproducibility and accuracy of our optimized 3D differentiation protocol. We unfortunately were not able to obtain human in vivo data at the relevant developmental stages to make a direct comparison. However, we did extend our analysis to compare our single-cell RNA-seq dataset with the previously published transcriptome data from 2D paraxial mesoderm differentiation protocols (Diaz-Cuadros et al., 2020, Matsuda et al., 2020), which we have included in our updated Discussion section.

    We compared the single-cell RNA-seq data from Diaz-Cuadros et al. with our own single-cell RNA-seq data. The 2D differentiated cells in Diaz-Cuadros et al. at the final time point of their experiment do not show a clear somitic cell state signature. PAX3, MEOX1, MEOX2, FOXC2, UNCX, and TBX18 are not expressed (compared for example to FOXC1). FOXC1 does not appear to be a somite-specific marker as it is expressed in early and late PSM-like cells as seen in our own data, starting on day 2 (Figure 4-Supplemental Fig 3). In the Diaz-Cuadros et al. dataset, TCF15 is not expressed uniformly in all cells nor specifically in the late-stage cells. Conversely, TCF15 is specifically expressed in day 5 somitic cells in our dataset both in a uniform manner and at high levels (as shown in the figure below and Figure 4C).

    We also analyzed the bulk RNA-seq data in Matsuda et al. 2020 as shown in Figure 1B of their paper. They show the expression of 4 somite markers (TCF15, MEOX1, PAX3, and RIPPLY1). As can be seen in the figure below (and Figure 4-Supplemental Fig 3), all markers highlighted in their paper are specifically expressed in our day 5 somitic cell population, including RIPPLY1 (see also updated Figure 4-Supplemental Fig 3). Beyond comparison of marker gene expression, it is difficult to assess the similarities and differences with the Matsuda dataset since their data lacks single-cell resolution. Thus, heterogeneity and efficiency of somitic fate induction within their cell population is unclear. Finally, neither of these two papers report the formation of somite-like structures.

    Reviewer #2:

    This study is interesting in the sense that it brings us one step closer to the formation of complex structures (such as somites) from human iPSCs. Markers that are typical of somites are indeed present at the end of the (rather complex) culture protocol. There is also quite a lot of work involved and the illustrations are of good quality.

    However, the spatial organization that is typical of somites is lacking in a number of important ways. Early somites in amniotes are epithelial (in fact it is a pseudo-stratified epithelium made of bottle shaped cells), apical facing the somitocoele (a cavity filled with a loose mesenchyme) and basal to the outside. Quite similar to the organization of the neural tube. This organization is initiated in the anterior last third of the PSM and amplified concomitant to segmentation. It would be important to show that somitoids display such structure. It does not seem that there is a central cavity. It is also unclear from the picture whether somitoid cells are bottle-shaped. Importantly, one sees (Figure 5a) that F-actin (which labels the apical side of epithelial cells) is facing the outside of the somitoid, and not the inside as it should. In this condition, the term "somitoid" seems quite inaccurate in comparison to other organoid systems that faithfully reproduce their in vivo counterparts, not only the "classical" intestinal crypts but also the more recently published neural tube organoids. Aggregates of somite-like cells may be more accurate.

    We are glad the reviewer found our study interesting and a step towards formation of complex structures in vitro. We believe that the reviewer has misunderstood the structure of the somites that we observe in vitro. We now include high magnification images that more clearly show the shape of the cells and the locations at which the epithelial polarity markers are expressed (new Figure 3-Supplemental Fig 1; new Figure 3-Video 1). Consistent with in vivo somites, we do observe bottle-shaped cells radially arranged around a central cavity forming rosette-like structures that are the same size as their in vivo counterparts (Fig 5B). This can be observed in multiple images shown in our manuscript (Fig 3-Supplemental Fig 1A-A’’, Fig 3-Supplemental Fig 1B-B’’, Fig 2-Supplemental Fig 3A,B). Importantly, both F-ACTIN (Fig 5A) and N-CADHERIN (Fig 3-Supplemental Fig 1) are indeed expressed around the central cavity, suggesting that the apical side of the PAX3+/TCF15+ somitic cells is facing the inside of the somite structures. This is especially evident in the newly added high-magnification images (Figure 3-Supplemental Fig 1) and accompanying movie showing a full confocal z-stack through the in vitro somites (Figure 3-Video 1). Taken together with our protein (Fig 3B,C and Fig 5A) and gene expression data (both in bulk (Fig 1C, Fig 5C, Fig 1-Supplemental Fig 1B) and at the single-cell level (Fig 4, Fig 4-Supplemental Fig 1-5) and our directed differentiation experiments of our Somitoid cells to dermomyotome (Fig 5-Supplemental Fig 1) and sclerotome fates (Fig 5C), we believe that the somite structures generated by our optimized in vitro protocol are indeed equivalent to their in vivo counterparts.

  4. eLife

    Evaluation Summary:

    Budjan et al. describe an organoid protocol to obtain somite-like structures from human iPSCs. Using defined culture media, the authors describe the formation after 5 days in vitro of organoids that express a variety of PSM differentiation markers, such as the segmentation clock gene Hes7 and Pax3. Optimization of their culture conditions and transcription analyses of what they name their "somitoid" system revealed that their culture system recapitulates the time course of expression markers typically observed along PSM and somite early differentiation. Furthermore, somitoid reacted to Shh activation by activating sclerotomal markers Pax1 and 9.

    (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. The reviewers remained anonymous to the authors.)

  5. eLife

    Reviewer #1 (Public Review):

    The foundational differentiation protocol up until day3 (formation of PSM) has been published previously in Diaz-Cuadros et al., 2020; Matsuda et al., 2020. The main difference between this manuscript and published protocol being the 2D (published) vs 3D differentiation. In this manuscript the authors were able to generate Pax3+ Somites (day4-5) from PSM. Both Diaz-Cuadros et al., 2020; Matsuda et al., 2020 were unable to generate Pax3+ somite in their 2D culture system but instead could only obtain a TCF15+ somatic mesoderm intermediate state. Moreover, the somites obtained in this manuscript could be further differentiated to sclerotome.

    The experiments across the paper were validated using three repeats using appropriate quantitative microscopy. Imaging data are high quality and mostly presented in a clear manner. However, it is unclear exactly what the authors are scoring as a somite. Moreover, for each figure it is not clear whether technical or biological replicates are presented. Similarly, the heatmap block presented in Fig2C,D, 3C,D apparently represents just one organoid/replicate. Authors should comment on the efficiency of the protocol over the different cell lines used. Transcriptome data presented strongly support the reproducibility and accuracy of the 3D differentiation, although comparison with the in vivo situation is highly limited - in part due to lack of availability of human in vivo data at these developmental stages.

  6. eLife

    Reviewer #2 (Public Review):

    This study is interesting in the sense that it brings us one step closer to the formation of complex structures (such as somites) from human iPSCs. Markers that are typical of somites are indeed present at the end of the (rather complex) culture protocol. There is also quite a lot of work involved and the illustrations are of good quality.

    However, the spatial organization that is typical of somites is lacking in a number of important ways. Early somites in amniotes are epithelial (in fact it is a pseudo-stratified epithelium made of bottle shaped cells), apical facing the somitocoele (a cavity filled with a loose mesenchyme) and basal to the outside. Quite similar to the organization of the neural tube. This organization is initiated in the anterior last third of the PSM and amplified concomitant to segmentation. It would be important to show that somitoids display such structure. It does not seem that there is a central cavity. It is also unclear from the picture whether somitoid cells are bottle-shaped. Importantly, one sees (Figure 5a) that F-actin (which labels the apical side of epithelial cells) is facing the outside of the somitoid, and not the inside as it should. In this condition, the term "somitoid" seems quite inaccurate in comparison to other organoid systems that faithfully reproduce their in vivo counterparts, not only the "classical" intestinal crypts but also the more recently published neural tube organoids. Aggregates of somite-like cells may be more accurate.

  7. Version published to 10.1101/2021.03.22.436471v1 on bioRxiv