Newswise — Research led by scientists at UC San Francisco and Case Western Reserve University School of Medicine has used brain “organoids” — tiny 3D models of human organs that scientists grow in a dish to study disease — to identify root causes of Miller-Dieker Syndrome (MDS), a rare genetic disorder that causes fatal brain malformations.

MDS is caused by a deletion of a section of human chromosome 17 containing genes important for neural development. The result is a brain whose outer layer, the neocortex, which is normally folded and furrowed to fit more brain into a limited skull, instead has a smooth appearance (lissencephaly) and is often smaller than normal (microcephaly). The disease is accompanied by severe seizures and intellectual disabilities, and few infants born with MDS survive beyond childhood.

In the new study — published online January 19, 2017 in Cell Stem Cell — the research team transformed skin cells from MDS patients and normal adults into neural stem cells, which they placed in a 3 dimensional culture system to grow organoid models of the human neocortex with and without the genetic defect that causes MDS.

Closely observing the development of these MDS organoids over time revealed that many neural stem cells die off at early stages of development, and others exhibit defects in cell movement and cell division. These findings could help explain how the genetics of MDS leads to lissencephaly, the authors say, while also offering valuable insights into normal brain development.

“The development of cortical organoid models is a breakthrough in researchers’ ability to study how human brain development can go awry, especially diseases such as MDS,” said Tony Wynshaw-Boris, MD, PhD, chair of the Department of Genetics and Genome Studies at Case Western Reserve University School of Medicine, and co-senior author of the new study. “This has allowed us to identify novel cellular factors that contribute to Miller-Dieker syndrome, which has not been modeled before.”

‘Smooth brain’ organoids reveal defects in stem cells key to human brain development Previous research on the causes of lissencephaly has relied on mouse models of the disease, which suggested that the main driver of the disorder was a defect in the ability of young neurons to migrate to the correct location in the brain. But Arnold Kriegstein, MD, PhD, professor of neurology, director of the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at UCSF, and co-senior author, says there are significant drawbacks to this approach.

“Unlike the human brain, the mouse brain is naturally smooth,” Kriegstein said. “If you are studying a disease that leads to a smooth brain in humans, it’s a challenge to study it in an animal that normally has a smooth brain.”

The mouse brain also lacks a type of neural stem cell called outer radial glia, which were discovered by Kriegstein’s group in 2010. These cells are thought to have played a crucial role in the massive expansion in size and complexity of the primate brain relative to other mammals over the course of evolution.

“There are just fundamental differences in how mouse and human brains grow and develop,” said Marina Bershteyn, PhD, who led the new study. Formerly a postdoctoral researcher in the Wynshaw-Boris and Kriegstein labs, Bershteyn is now a researcher at Neurona Therapeutics, a company founded by Kriegstein and colleagues to develop stem cell therapies for neurological diseases. “We hypothesized in this study that part of explanation is different types of neural stem cells that are abundant in human but rare in mouse.”

In order to more accurately model the progression of MDS in the embryonic human brain, Bershteyn spearheaded the development of MDS cortical organoids and techniques to observe how stem cells within these organoids developed in the laboratory into the different cell types seen in first-trimester embryonic human brains.

Bershteyn and her team found that outer radial glia cells readily grew in patient-derived organoids, but time-lapse images revealed a defect in these cells’ ability to divide and multiply — potentially contributing to the small, smooth brains seen in MDS patients.

In addition, the team found that early neural stem cells called neuroepithelial cells – which are present in both mice and humans – die at surprisingly high rates in MDS organoids, and when they do survive, divide in an abnormal way — as if they are prematurely transforming into neurons, cutting short important early stages of brain development.

Consistent with prior mouse studies, these time-lapse images also revealed that newborn neurons are unable to migrate properly through the 3-D cell culture system used by the researchers, pausing and “tumbling” on the tracks that ought to take them into the brain’s growing cortex, and potentially contributing to the failure of MDS brains to properly form outer brain structures. Organoid research opens doors to studying human brain diseases in lab

Newswise — Together, these observations helped the team pinpoint developmental stages and specific neural stem cell types that are impaired in MDS. The next step to understanding lissencephaly more broadly, the authors say, will be to test cells from patients with different genetic forms of the disease, so researchers can begin to link specific mutations with the cellular defects that drive brain malformation.

The study is also a demonstration of the utility of patient-derived brain organoids as a way to bridge the gap between animal models and human disease, the authors say. In particular, the finding that human outer radial glia cells readily grow in organoid models opens the door for scientists worldwide to study the role of these cells in both normal human brain development and disease.

“Patient-derived cortical organoids are creating huge amount of excitement,” Kriegstein said. “We are now able to study human brain development experimentally in lab in ways that were not possible before.”

### Additional authors on the paper were Tomasz J. Nowakowski, PhD, Alex A. Pollen, PhD, and Elizabeth Di Lullo, PhD, of UCSF, and Aishwarya Nene, PhD, of Caltech.

Funding for the study was provided by National Institutes of Health grants NS075998 and MH105989, and a California Institute for Regenerative Medicine (CIRM) award (GCIR-06673) to A.R.K. M.B. was supported by a postdoctoral fellowship from CIRM (grant TG2-01153), and a K99 career development award from the National Institute for Neurological Disorders and Stroke (grant 5K99NS088572).

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Journal Link: Cell Stem Cell, Jan-2017