Paraxial mesoderm organoids model development of human somites

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 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 paraxial mesoderm (PM) organoids from hPSCs (termed Somitoids), which recapitulate the molecular, morphological and functional features of paraxial mesoderm 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 somite formation 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 spinal cord development and disease in vitro.

HOX genes in stem cells: Maintaining cellular identity and regulation of differentiation

“…Concerning the signaling it is known that the paraxial regions of the mesoderm become specified following the action of BMPs, members of the FGF family and WNT molecules. Cross-regulation of these pathways, e.g., by the WNT effector molecule NOGGIN that inhibits BMP signaling, is not only important for the paraxial mesoderm specification but also for maintaining this identity (Aulehla and Pourquie, 2010;Budjan et al, 2022). As a model of human somitogenesis, paraxial mesoderm organoids were developed by differentiating human pluripotent stem cells towards paraxial mesoderm using a combination of WNT signaling activation together with BMP inhibition and following FGF2 treatment (Budjan et al, 2022).…”

“…Cross-regulation of these pathways, e.g., by the WNT effector molecule NOGGIN that inhibits BMP signaling, is not only important for the paraxial mesoderm specification but also for maintaining this identity (Aulehla and Pourquie, 2010;Budjan et al, 2022). As a model of human somitogenesis, paraxial mesoderm organoids were developed by differentiating human pluripotent stem cells towards paraxial mesoderm using a combination of WNT signaling activation together with BMP inhibition and following FGF2 treatment (Budjan et al, 2022). Thereby, the sequential activation pattern of the HOX genes was elegantly recapitulated.…”

“…Thereby, the sequential activation pattern of the HOX genes was elegantly recapitulated. HOXA1 expression was early detected following WNT activation and BMP inhibition (with 24 h), followed by other cervical (HOX1-HOX5 paralogues) and thoracic (HOX6-HOX9 paralogues) HOX genes within the next 48-72 h, to HOXD9, a lumbosacral HOX gene in the somite-stage organoids of day 4-5, which was 24-48 h after the successive FGF2 treatment (Budjan et al, 2022). Concerning the mechanism it was revealed that WNT molecules activate HOX gene expression in a temporally collinear way as WNTdependent enhancers, namely interaction regions with HOXA1 called "HOXA developmental early side (ADES)" that are located within the posterior region.…”

Advanced models of human skeletal muscle differentiation, development and disease: Three-dimensional cultures, organoids and beyond

“…Excitingly, recent studies are also focussing on closely studying and modelling developmental myogenesis and early (i.e. foetal) muscle disease pathogenesis taking advantage of emerging technologies [ 37 , 71 , 72 ]. Looking forward, we see the need for better integration of the two main methodologies used to differentiate human iPSCs into functional skeletal myofibres (i.e.…”