It's no small feat to create a spinal column. In humans, 33 interlocking bones must come together to protect the spinal cord. These vertebrae are interconnected and have delicate attachments to muscles, nerves and ligaments. The arrangement of vertebrae must be carefully orchestrated on a molecular level through a series of sequential steps -- a process governed by the segmentation clock. While the segmentation clock has been documented in many animals, in humans, the spinal column and its vertebrae form during the third week of an embryo's development, often before a woman is aware she is pregnant, making it much more difficult to study. To reproduce the molecular steps that lead to the proper formation of the human spine, investigators from Brigham and Women's Hospital have developed a model in a dish, allowing them to study just what makes our segmentation clock tick. The work has led to important insights into our embryonic development. Results are published this week in Nature.
"We have developed an extremely powerful system with which to dissect the segmentation clock in humans," said corresponding author Olivier Pourquié, PhD, a principal investigator in the Department of Pathology at the Brigham. "Before this, it would have been impossible to capture the intricacies of vertebrae development, but now our system opens up a completely new spectrum of possibilities."
Twenty years ago, the Pourquié lab first provided evidence for a segmentation clock controlling the rhythmic production of vertebral precursors in chicken embryos. Over the years, Pourquie and others have shown its existence in mice, snakes, frogs, zebrafish and even insects. The segmentation clock controls the periodic activation of molecular signaling pathways, a bit like a metronome keeping the beat of vertebrae development. The temporal pulses delivered by the segmentation clock are then converted into the periodic series of vertebrae.
In this week's Nature publication, they report the existence and characterization of the human segmentation clock. (The same issue of the journal includes a paper that establishes models of congenital scoliosis, a severe spine segmentation disorder in which the segmentation clock has been implicated.) Following an approach similar to what has been developed to create mini-brains and other miniature organs in a dish, Pourquié and colleagues coaxed human-induced pluripotent stem cells (iPSCs) to develop into paraxial mesoderm cells, the cells which form the muscle and vertebrae. The team carefully characterized the molecular switches turned on and off as the cells developed.
The investigators report that the differentiation of human pluripotent cells to the musculo-skeletal lineages are remarkably efficient and can be achieved in 2 to 3 days with greater than 90 percent efficiency by adding just two compounds to the culture medium. They were also able to measure the clock period of the human segmentation clock. In mice, each tick of the clock -- representing the coming together of all the precursors of a single vertebrae -- is about two hours. In humans, each tick is about five hours, which corresponds to the longer time it takes for a human embryo to fully develop.
Having a laboratory model for vertebrae development may help investigators answer key questions about diseases and conditions related to both the spine and muscle tissue.
"This represents an ideal system to understand and study the defects in segmentation that lead to conditions like congenital scoliosis," said Pourquié. "Our lab is also very interested in Duchenne's muscular dystrophy and the potential to leverage stem cell therapies and a deeper understanding of muscle tissue development. Our research already shows us how critically important it will be to reconstruct every step of development for these cells."
This research was supported by the National Institutes of Health (5R01HD085121 and 1K99GM121852).
Paper cited: Diaz-Cuadros, M et al. "In vitro characterization of the human segmentation clock" Nature DOI: 10.1038/s41586-019-1885-9