Evidence of a Human Segmentation Clock Reveals How an Embryo’s Vertebrae Tick

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 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 the 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

Nanoparticle therapeutic restores tumor suppressor, sensitizes cancer cells to treatment

In preclinical experiments, investigators restored p53 using synthetic mRNA nanoparticles, making lung and liver cancer cells susceptible to available cancer drugs

The tumor suppressor gene p53, also known as the guardian of the genome, plays a critical function in preventing cancer. Because of its powerful role, it is one of the most commonly compromised genes in cancer.

Investigators have long sought a way to restore the activity of tumor suppressor genes such as p53. Most recently, attention has turned to an approach developed at Brigham and Women’s Hospital that uses nanotechnology to deliver synthetic messenger RNA (mRNA). Leveraging advancements in nanotechnology, investigators from the Brigham have found that restoring p53 not only delays the growth of p53-deficient liver and lung cancer cells but may also make tumors more vulnerable to cancer drugs known as mTOR inhibitors. The team’s findings are published in Science Translational Medicine.

“mTOR inhibitors have been approved for the treatment of certain types of cancers but have not worked as well in clinical trials for many common cancers,” said co-corresponding author Jinjun Shi, PhD, a faculty member in the Brigham’s Center for Nanomedicine and Department of Anesthesiology. “We present evidence here that using the lipid-polymer hybrid mRNA nanoparticle platform we’ve developed to restore p53 may sensitize cancer cells to mTOR inhibitors. This represents a potentially powerful combination for cancer treatment.”

Shi and colleagues, including co-corresponding authors Omid Farokhzad, MD, MBA, (director of Brigham’s Center for Nanomedicine) and Wei Tao, PhD, (faculty in the Center), and first author Na Kong, MD, used a redox-responsive nanoparticle platform to deliver p53-encoding synthetic mRNA. The synthetic p53 caused cell cycle arrest, cell death and delayed the growth of liver cancer cells and lung cancer cells in which p53 had been depleted. In addition, synthetic p53 made the cells more sensitive to everolimus, a drug which is an mTOR inhibitor. The team reports successful results in multiple in vitro and in vivo models.

Past clinical trials of everolimus failed to show a clinical benefit in advanced cases of liver and lung cancer but found that response to the drug varied greatly between patients. Studies have also found that p53 is altered in approximately 36 percent of hepatocellular carcinomas (the most common form of liver cancer) and 68 percent of non-small cell lung cancers.

The authors note that further preclinical development and evaluation will be needed to explore that translational potential and scalability of the approach as well as its applicability to other p53 mutations and other cancers.

“We expect that this mRNA nanoparticle approach could be applied to many other tumor suppressors and rationally combined with other therapeutic modalities for effective combinatorial cancer treatment,” the authors write.

Funding for this work was provided by the National Institutes of Health (grant R01 CA200900), Key Project of Zhejiang province Ministry of Science and Technology (no. 2015C03055), and Key Project of Hangzhou Ministry of Science and Technology (no. 20162013A07). Kong, Tao, Farokhzad and Shi are inventors on a U.S. patent application (62778215) filed by Brigham and Women’s Hospital related to the technology disclosed in the paper. Farokhzad declares financial interests in Selecta Biosciences, Tarveda Therapeutics, and Seer. A co-author has received honoraria from Bristol-Myers Squibb; consulting fees from Bayer, Tilos, and twoXAR; and research funding from Merrimack, Leap Therapeutics, Bayer, Bristol-Myers Squibb, and Exelixis.

Paper cited: Kong, N et al. “Synthetic mRNA nanoparticle-mediated restoration of p53 tumor suppressor sensitizes p53-deficient cancers to mTOR inhibition” Science Translational Medicine DOI: 10.1126/scitranslmed.eaaw1565

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