Decision letter: Paraxial mesoderm organoids model development of human somites

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics 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. Editor's evaluation 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, thus recapitulating the time course of expression markers typically observed along PSM and somite early differentiation. https://doi.org/10.7554/eLife.68925.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Humans are part of a group of animals called vertebrates, which are all the animals with backbones. Broadly, all vertebrates have a similar body shape with a head at one end and a left and right side that are similar to each other. Although this is not very obvious in humans, vertebrate bodies are derived from pairs of segments arranged from the head to the tail. Each of these segments or somites originates early in embryonic development. Cells from each somite then divide, grow and specialize to form bones such as the vertebrae of the vertebral column, muscles, skin, and other tissues that make up each segment. Studying different animals during embryonic development has provided insights into how somites form and grow, but it is technically difficult to do and only provides an approximate model of how somites develop in humans. Being able to make and study somites using human cells in the lab would help scientists learn more about how somite formation in humans is regulated. Budjan et al. grew human stem cells in the lab as three-dimensional structures called organoids, and used chemical signals similar to the ones produced in the embryo during development to make the cells form somites. Various combinations of signals were tested to find the best way to trigger somite formation. Once the somites formed, Budjan et al. measured them and studied their structure and the genes they used. They found that these lab-grown somites have the same size and structure as natural somites and use many of the same genes. This new organoid model provides a way to study human somite formation and development in the lab for the first time. This can provide insights into the development and evolution of humans and other animals that could then help scientists understand diseases such as the development of abnormal spinal curvature that affects around 1 in 10,000 newborns. Introduction Paraxial mesoderm (PM) development involves the formation of embryonic segments called somites, which are produced sequentially from the presomitic mesoderm (PSM) and arranged periodically along the anterior-posterior (AP) axis of the vertebrate embryo. Somites give rise and contribute to a variety of tissues including skeletal muscle, dermis, cartilage, and bone (Chal and Pourquié, 2017). Somite formation is controlled by a conserved molecular oscillator, the segmentation clock (Dequéant et al., 2006; Hubaud and Pourquié, 2014; Oates et al., 2012). Previous efforts have focused on how this oscillator controls somite formation using a variety of model systems such as mouse, zebrafish, and chick because of ethical and technical limitations of culturing human embryos. Recently, researchers were able to recapitulate PM development using human and mouse pluripotent stem cells cultured as 2D monolayers (Chu et al., 2019; Diaz-Cuadros et al., 2020; Matsuda et al., 2020). These cells undergo species-characteristic oscillations similar to their in vivo counterparts. However, final stages of somite development and vertebra formation were not observed in currently published human cell culture systems (Palla and Blau, 2020), suggesting that certain aspects of in vivo development are not recapitulated in these 2D systems. We reasoned that a 3D cell culture system may exhibit all the stages of PM development including morphogenetic processes associated with somite formation. Here, we describe an organoid system derived from human induced pluripotent stem cells (hiPSCs) called Somitoids, which faithfully recapitulates functional, morphological, and molecular features of PM development, including formation of somite-like structures in vitro. To identify the culture conditions that reproducibly yielded formation of somite-like structures, we developed a quantitative image-based screening platform for individual organoids. Our screening approach identified the optimal parameter values for the culture conditions such as the initial cell number and the concentration of the chemical modulators. We show using single-cell RNA-sequencing (scRNA-seq), immunofluorescence, and qRT-PCR that Somitoids resemble their in vivo counterparts both transcriptionally and morphologically and can be differentiated into somite derivatives such as sclerotome and dermomyotome in vitro. Our Somitoid system is reproducible and scalable, allowing for systematic and quantitative analysis of PM development and somite formation to study human spine development and disease in a dish. Finally, our approach can be used to systematically screen organoid cultures for desired phenotypes and reproducibility. Results Recently, protocols have been developed to differentiate mouse or human pluripotent stem cells (hPSCs) towards PM using a combination of the WNT agonist CHIR and BMP inhibitor LDN (Chal et al., 2015; Diaz-Cuadros et al., 2020). To adapt the protocol for a 3D model of human somitogenesis, we first optimized the initial conditions of our cultures by generating pluripotent spheroids of defined cell numbers. hiPSCs were allowed to aggregate for 24 hr as suspension cultures in pluripotency media (mTeSR1) in the presence of ROCK inhibitor and polyvinyl alcohol (PVA) to promote aggregation (Figure 1A). These pluripotent spheroids resemble cavity-stage epiblast embryos as previously described for kidney organoid cultures (Freedman et al., 2015; Figure 1B, Figure 1—figure supplement 1A). Next, spheroids were cultured in media containing CHIR and LDN (CL), similarly as done for the monolayer cultures (Chal et al., 2015; Diaz-Cuadros et al., 2020), but with CHIR at a higher concentration (10 µM). After 24 hr in CL media, epiblast-stage cells transition to a neuromesodermal progenitor (NMp) or primitive streak cell fate, characterized by co-expression of T/BRA and SOX2 (Tzouanacou et al., 2009; Figure 1B). By 48 hr, cells rapidly downregulate SOX2 and express PSM markers, including TBX6 and MSGN1 (Figure 1B). This PSM state persists from day 2 to day 4 and is also characterized by the expression of segmentation clock genes such as HES7 (Figure 1C). On day 5, organoids showed expression of marker genes associated with somite fate as characterized by qPCR (Figure 1C, Figure 1—figure supplement 1B). Taken together, the order of activation of marker genes in the PM organoids followed the expected stages of differentiation observed during PM development. Figure 1 with 2 supplements see all Download asset Open asset Human pluripotent stem cell (hPSC)-derived paraxial mesoderm (PM) organoids turn on marker genes associated with PM differentiation. (A) Schematic overview of PM organoid differentiation protocol from hPSCs. hPSCs aggregated and formed spheroids for 24 hr prior to differentiation. For differentiation, spheroids were exposed to Wnt agonist (CHIR) and BMP inhibitor (LDN) for 72 hr. On day 3, FGF2 was added to the media in addition to CHIR and LDN. (B) Immunofluorescence analysis of cell fate-specific marker genes shows progressive differentiation towards presomitic mesoderm (PSM) fate (top and middle rows). Organoids derived from human induced pluripotent stem cells (hiPSCs) harboring an MSGN1-Venus reporter express TBX6 at the same time as the reporter is activated (bottom row). Scale bar represents 100 µm. Representative images shown from n = 3 independent experiments. Cell lines used: NCRM1 hiPSCs and MSGN1-Venus hiPS reporter cells. (C) qRT-PCR analysis of PSM and somite markers reveals PSM-to-somite transition from day 4 to day 5. Relative gene expression levels are shown as Z-scores, expressed as fold-change relative to undifferentiated iPSCs (see Materials and methods). Source data is available in Figure 1—source data 1. Figure 1—source data 1 qPCR raw data of paraxial mesoderm (PM) organoid differentiation. https://cdn.elifesciences.org/articles/68925/elife-68925-fig1-data1-v2.txt Download elife-68925-fig1-data1-v2.txt We observed that following the above protocol resulted in a heterogeneous activation of somite marker genes across cells within the same organoid and across different replicates, as well as a low number of somite-like structures (Figure 1—figure supplement 2). To improve reproducibility, we set out to screen for the optimal initial cell number, the concentration of the signaling factors, and the timing of their additions during culture. For our primary screen, we compared organoids with an initial cell number of 500 and 1000 as well as combinations of modulators of candidate signaling pathways that have previously been involved in PM development and somite formation, including FGF, WNT, BMP, and TGF-β (Aulehla et al., 2008; Chal et al., 2015; Hubaud and Pourquié, 2014; Tonegawa et al., 1997; Xi et al., 2017). We primarily focused on the FGF and WNT signaling pathways since their critical role during somitogenesis is well established both in vivo (Aulehla et al., 2008; Aulehla et al., 2003; Delfini et al., 2005; Dubrulle et al., 2001; Dunty et al., 2008; Greco et al., 1996; Yamaguchi et al., 1999) and in vitro (Chal et al., 2015; Sakurai et al., 2012; Tan et al., 2013). Furthermore, dual inhibition of FGF and WNT signaling has been used with some success to generate PM derivatives in vitro (Loh et al., 2016; Matsuda et al., 2020). Finally, the BMP and TGF-β signaling pathways have been shown in vitro and in vivo to have a role in human somitogenesis (Loh et al., 2016; Xi et al., 2017). PSM-stage organoids on day 3 were treated with signaling modulators for 24 and 48 hr, and somite fate and morphogenesis was assessed using PAX3, a somite fate marker, and F-ACTIN, a structural marker of somite formation (Figure 2A). We chose day 3 as a starting point for our systematic screen because PSM marker gene expression was more uniform compared to day 2 organoids (Figure 1B), and day 4 organoids are not significantly different from day 3 organoids based on a previous study and our own immunostaining and qPCR data (Figure 1B and C; Diaz-Cuadros et al., 2020). To quantitatively compare conditions, we developed an image analysis pipeline to determine organoid diameter and normalized average PAX3 expression intensity per organoid in an automated manner (Figure 2C and D, Figure 2—figure supplement 2B). We analyzed three organoids per condition. Strikingly, all organoids that were treated with any combination of FGF or WNT inhibitor reproducibly expressed PAX3 within 24 hr of treatment (Figure 2B and D). However, organoids that were initiated from 1000 cells displayed a higher fraction of PAX3-negative cells compared with organoids initiated from 500 cells, even though the average PAX3 expression levels across the entire organoid were comparable (Figure 2D, Figure 2—figure supplement 2A). Additional staining for SOX2, a neural marker, showed that PAX3-negative cells expressed SOX2, suggesting that our PM organoids derive from NMps (Figure 1B, Figure 2—figure supplement 2A). In addition, staining for F-ACTIN, together with nuclear expression of PAX3, more consistently revealed somite-like structures (radial arrangement of PAX3+ columnar cells with expression of apical F-ACTIN in the central cavity) in organoids made from 500 cells compared with organoids made from 1000 cells (Figure 2B, Figure 2—figure supplement 3A and B). Taken together, based on these observations, we used 500 cells as the initial cell number going forward. Figure 2 with 3 supplements see all Download asset Open asset Pilot screen to optimize differentiation conditions for somite phenotype in paraxial mesoderm (PM) organoids reveals optimal initial number of cells and duration of treatment. (A) Schematic overview of systematic screen in PM organoids (Somitoids). Presomitic mesoderm (PSM)-stage organoids were treated on day 3 for 24 hr or 48 hr with signaling agonists/antagonists. Treated organoids were cultured in basal media with inhibitors as indicated. Control organoids were maintained in CL media with FGF added. NCRM1-derived organoids were used for the screen. (B) Representative immunofluorescent images of day 4 and day 5 organoids after treatment for 24 hr or 48 hr, respectively, stained for somite marker PAX3 and F-ACTIN to visualize rosette-like somite structures. Organoids generated from 1000 cells generally show a more diffuse F-ACTIN pattern compared to organoids made from 500 cells, which exhibit bright foci, consistent with somite formation. Confocal images are shown as maximum intensity z-projections. Scale bar represents 100 µm. Small-molecule inhibitors used are indicated in brackets. FGFRi, FGF receptor inhibitor (PD173074); WNTi; Wnt inhibitor (C59 or XAV939); Bi, BMP inhibitor (LDN); TGF-βi, TGF-β inhibitor (A-83-01). Representative image shown for each condition from three organoid replicates. (C) Automated quantification of organoid diameter for each organoid/replicate treated as indicated (see Materials and methods for details). Three organoids per condition were characterized except where indicated with gray boxes. Organoids initiated from 500 cells show a decreased diameter when treated for 48 hr compared with 24 hr. Source data is available in Figure 2—source data 1. (D) Automated quantification of normalized average PAX3 intensity for each organoid/replicate treated as indicated. Three organoids are shown per condition except where indicated with gray boxes. Organoids initiated from both 500 and 1000 cells show higher average normalized PAX3 levels when treated for 24 hr compared with 48 hr. Source data is available in Figure 2—source data 2. Figure 2—source data 1 Quantification of organoid diameter from primary screen. https://cdn.elifesciences.org/articles/68925/elife-68925-fig2-data1-v2.csv Download elife-68925-fig2-data1-v2.csv Figure 2—source data 2 Quantification of average PAX3 levels per organoid from primary screen. https://cdn.elifesciences.org/articles/68925/elife-68925-fig2-data2-v2.csv Download elife-68925-fig2-data2-v2.csv Organoids that were treated for 48 hr with signaling pathway modulators showed an overall decrease of PAX3 expression compared with organoids treated for only 24 hr across replicates, indicating that prolonged signaling manipulation does not improve the somite phenotype (Figure 2D). Additionally, organoids initiated from 500 cells and then treated for 48 hr had a smaller diameter compared with organoids of the same initial cell number that were treated for only 24 hr (Figure 2C). This suggests that long-term inhibition of WNT and/or FGF, known mitogenic signaling pathways, has detrimental effects on proliferation or cell survival. These results indicate that treatment of PSM-stage organoids with pathway modulators for 24 hr is sufficient to induce somite fate. Next, we set out to optimize the culture conditions to increase the number of somite-like structures in addition to the expression levels of the somite marker genes. We looked for morphological hallmarks of somite formation, specifically the formation of rosette-like structures consisting of radially arranged bottle-shaped PAX3+ epithelial cells with their NCAD+ apical surface facing a central cavity (Figure 3—figure supplement 1A and B, Figure 3—video 1). We used 500-cell spheroids as an initial starting point and compared two different inhibitor doses for FGF and WNT in addition to the other pathway modulators applied to PSM-stage organoids on day 3. We characterized the organoids after treating them for 24 hr, 48 hr, and 24 hr followed by culture in basal media without any added factor for an additional 24 hr (five organoids per condition, Figure 3A). In addition to quantifying PAX3 levels (Figure 3D, Figure 3—figure supplement 2B), we also counted the number of somite-like structures per organoid (Figure 3B and E, Figure 3—figure supplement 2A and B; see Materials and methods for a description of the scoring criteria). Comparing PAX3 expression levels in our treated organoids, we observed that somite fate can be broadly induced across a range of treatment regimes, concentrations, and types of WNT and/or FGF inhibitors (Figure 3C and D, Figure 3—figure supplement 2C). However, the number of somite-like structures is not necessarily correlated with average PAX3 expression levels. For example, the numbers of somite-like structures in several conditions (FGFRihi/PD173, WNTihi/C59, WNThi/C59+ FGFRilo, WNTihi/XAV) were lower in organoids treated for 24 hr followed by culture in basal media for 24 hr compared with organoids that were treated with the same inhibitors for 48 hr, even though they exhibited higher PAX3 expression levels on average (Figure 3D and E, Figure 3—figure supplement 2A). This suggests that marker gene expression alone may not be the best predictor when screening for morphologically complex phenotypes such as somite formation. Figure 3 with 4 supplements see all Download asset Open asset Secondary screen of paraxial mesoderm (PM) organoids identifies optimal differentiation protocol for somite formation. (A) Schematic overview of secondary screen in PM organoids. Presomitic mesoderm (PSM)-stage organoids were treated on day 3 for 24 hr followed by measurement, 24 hr of treatment followed by 24 hr culture in basal media (no added factors) and then measured (24 hr + 24 hr), and 48 hr of treatment followed by measurement. Treated organoids were cultured in basal media with inhibitors as indicated. WNT and FGF inhibitors were tested at two different concentrations. Control organoids were maintained in CL media with FGF added. NCRM1-derived organoids were used for the screen. (B) Representative immunofluorescent images of day 5 organoids stained for somite markers PAX3 and NCAD showing the rosette-like structures that were scored as somite-like structures based on expression of somite fate markers and structural features (scoring criteria detailed in Materials and methods). Images are shown as individual z-sections. (C) Representative immunofluorescent images of day 4 and day 5 organoids stained for somite markers PAX3 and NCAD to visualize rosette-like somite structures. Confocal images are shown as maximum intensity z-projections. Scale bar represents 100 µm. Small-molecule inhibitors used are indicated in brackets. FGFRi, FGF receptor inhibitor (PD173074); WNTi, Wnt inhibitor (C59 or XAV939); Bi, BMP inhibitor (LDN); TGF-βi, TGF-β inhibitor (A-83-01). Representative image shown for each condition from five organoid replicates. (D) Automated quantification of normalized average PAX3 intensity for each organoid/replicate treated as indicated (see Materials and methods for details). Five organoids are shown per condition except as indicated with gray boxes. Several inhibitor combinations with a treatment regime of 24 hr treatment followed by 24 hr cultured in basal media show highest average PAX3 levels. Source data is available in Figure 3—source data 1. (E) Quantification of the number of somite-like structures for each condition. Each row represents one organoid replicate. Five organoids are shown per condition except where indicated with gray boxes. Organoids that were maintained in CL media with added FGF for 24 hr followed by culture in basal media for 24 hr reproducibly exhibit the highest number of somite-like structures per organoid. Source data is available in Figure 3—source data 2. Figure 3—source data 1 Quantification of average PAX3 levels per organoid from secondary screen. https://cdn.elifesciences.org/articles/68925/elife-68925-fig3-data1-v2.csv Download elife-68925-fig3-data1-v2.csv Figure 3—source data 2 Quantification of somite-like structures of secondary screen. https://cdn.elifesciences.org/articles/68925/elife-68925-fig3-data2-v2.csv Download elife-68925-fig3-data2-v2.csv Surprisingly, organoids that were cultured for an additional 24 hr (day 3 to day 4) in FGF, WNT pathway agonist, and BMP inhibitor, considered a treatment control, followed by culture in basal media only for another 24 hr, consistently exhibited the highest number of somite-like structures across all organoid replicates (Figure 3E, Figure 3—figure supplement 1A and B, Figure 3—figure supplement 2A and B, Figure 3—video 1) as well as technical replicates (Figure 3—figure supplement 3A). Additionally, the average PAX3 expression was among the highest of all conditions tested (Figure 3D). This suggests that simply removing FGF/WNT pathway agonists and BMP inhibitor, which maintain cells in a PSM state, is sufficient to reproducibly induce somite fate and morphological formation of somite-like structures (Figure 3—figure supplement 2A, Figure 3—figure supplement 3A). Computing the variation of the number of somite-like structures across the five organoids confirmed that this phenotype was highly reproducible (coefficient of variation = 11.1%; Figure 3—figure supplement 2B). Finally, our optimized protocol reproducibly yielded efficient induction of somite-like structures in multiple genetically independent hiPSC lines (Figure 3—figure supplement 3B). Taken together, we determined that initiating the protocol with 500 cells and treating day 3 organoids with CL + FGF for 24 hr followed by culture in basal media for an additional 24 hr yield the most robust induction of somite-like structures while minimizing variation between experiments (technical variation) and different cell lines (biological variation). This optimized differentiation protocol was therefore used for all subsequent experiments. To further characterize the developmental trajectory and transcriptional states of our Somitoid system, we collected 15,558 cells (after postprocessing) over the course of the optimized 5-day differentiation protocol at timepoints that capture the key transition steps (day 1, day 2, day 3, day 5) and performed scRNA-seq (Figure 4A). Multiple organoids were used to obtain the required number of cells at each timepoint (see Materials and methods). We first combined all the cells across the timepoints and clustered them using the Leiden clustering algorithm (Traag et al., 2019). Predominantly, the four major clusters to cells from the four different cells at each timepoint have transcriptional states that are different compared with cells from the other In addition, within each cells exhibit similar transcriptional as indicated by the of the expression levels of marker genes across individual cells (Figure Figure supplements Figure 4 with 5 supplements see all Download asset Open asset RNA-sequencing analysis of paraxial mesoderm PM organoids reveals differentiation trajectory from cells to (A) and of single-cell of human PM organoids, by timepoint NCRM1-derived organoids were used to cells. (B) of human PM organoids, by Leiden based on marker gene expression (see Materials and methods). (C) of marker genes of PM differentiation. timepoint and Leiden are indicated. genes are based on primary associated cell fate as indicated. (D) of single-cell gene expression levels. Cells are by Leiden genes are by with of indicated on the (E) with normalized of cell fate marker genes. Cells collected on day 1 exhibited gene expression similar to primitive streak or genes such as SOX2, and (Figure and E, Figure supplement 1). on day 2, cells expressed PSM marker genes such as and clock genes of the signaling pathway including and (Figure and E, Figure supplement 2). 5 cells expressed somite marker genes such as PAX3, and (Figure and E, Figure supplement a of the day 5 cells also expressed somite markers, and which suggests of somite in Somitoids (Figure Figure supplement Furthermore, two of the which are characterized by co-expression of PSM and somite marker both day 3 and day 5 cells, indicating that the PSM-to-somite transition is in our in vitro system (Figure and B). one somite was for genes and sclerotome genes suggesting that these cells more of cells (Figure Figure supplement 4A). Finally, we observed the expected activation pattern of the genes in our Somitoid system starting with on day 1, followed by other and genes on days to a in the organoids (day Figure Figure supplement Taken together, our scRNA-seq show that our Somitoid system faithfully recapitulates the gene of human PM development and somite-like cells, which express the of known marker genes. Furthermore, we not cells of different developmental suggesting that we are generating organoids containing only PM derivatives. We assessed our in somite-like structures show similar and size to their in vivo counterparts. To some of the somite marker genes that we identified in our scRNA-seq we measured expression levels of PAX3, and F-ACTIN in day 5 Somitoids using immunostaining (Figure 5 cells both somite marker genes and PAX3 the and somite-like structures displayed apical of To determine in somite-like structures were similar in size to human embryonic we compared them with early human somites (Figure see Materials and methods for a description of how somite were somite-like structures were similar in size = range = to somites = = but human somites somites, = = somites, = = see Figure these results that our somite-like structures and molecular features as well as overall size with their in vivo counterparts. Figure 5 with 1 supplement see all Download asset Open asset Somitoids express known marker genes and can differentiate to sclerotome fate. (A) Immunofluorescence analysis of day 5 Somitoids reveals co-expression of somite markers and PAX3 and rosette-like structures as indicated by F-ACTIN suggesting that somite-like structures resemble in vivo counterparts in both a molecular and morphological Representative images are shown as maximum intensity from three organoid replicates. NCRM1 human in