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.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Updated main text to improve clarity and include new analyses; Fig 3 and Fig 5 revised/updated; Added high-resolution images of in vitro somites (Fig 2-Supplemental Fig 3, Fig 3-Supplemental Fig 1); added figures to show reproducibility of optimized Somitoid differentiation protocol across experiments and cell lines (Fig 3-Supplemental Fig 3); added additional analysis of single-cell RNA-seq data of FGF, WNT, and NOTCH signaling pathways (Fig 4-Supplemental Fig 5); added new experimental data showing differentiation of Somitoid-derived cells to skeletal muscle (Fig 5-Supplemental Fig 1)