Organoids are becoming more popular in research, consistency in their use is needed for more accurate results

By Charlene N. Rivera-Bonet | Waisman Science Writer

Brightfield image of organoids
Brightfield image of organoids. Photo provided by Soraya Sandoval.

To the naked eye, they look like tiny blobs free floating in a pink liquid. Under a light microscope, they look similar. But organoids, a 3-D mini version of an organ grown in a lab, may contain invaluable information about how the human brain develops.

The use of brain organoids in research is rapidly becoming more popular because of their unique characteristics in closely mimicking human brain development. However, they pose multiple challenges as the field grows and each lab uses their own method to form, grow, and analyze brain organoids. A more uniform and standardized way of using organoids is needed to be able to produce rigorous and replicable results. Soraya Sandoval, a graduate student in the lab of Xinyu Zhao, PhD, Jenni & Kyle Professor in Novel Neurodevelopmental Diseases and professor of neuroscience, and colleagues address these challenges and recommendations in a review article recently published in Stem Cell Reports, the journal for the International Society  for Stem Cell Research (ISSCR).

Brain organoids are derived from human induced pluripotent stem cells (iPSCs). These stem cells are acquired from an individual’s blood or skin, reprogrammed into a stem cell state, and turned into neurons, allowing researchers to study the process of brain development using human models. “What’s really nice about induced pluripotent stem cells is that we’re able to derive these from patients themselves,” Sandoval says. iPSCs taken from individuals with a neurodevelopmental condition such as autism spectrum disorder, fragile X syndrome or Down syndrome, can be turned into neurons for research, which can provide insight into how these disorders impact brain development.

iPSCs grow in monolayers or 2-D form. “To generate 3-D organoids, you allow iPSCs to follow almost an intrinsic program of development. They naturally generate this ball of cells,” Zhao says. These small, round cell aggregates can capture and preserve human genetics and aspects of human brain development. They can self-organize or be patterned to form aggregates of brain cells that resemble the diverse regions of the nervous system including the cerebral cortex, hippocampus, cerebellum, spinal cord, and more. These characteristics of organoids allows researchers to study the human brain in a way that would be impossible to do in live humans.

Soraya Sandoval
Soraya Sandoval holds flasks of organoids in culture. Photo provided by Xinyu Zhao.

Much of the knowledge we have about brain development has been obtained from model organisms such as mice. “But humans are really quite unique,” Zhao says. “In order to study human early brain development, organoids are among the best models out there.” However, although they have become quite popular in research, a few roadblocks stand in the way of their use. “There hasn’t really been a standardized way of analyzing the organoids because the field is so new,” Sandoval says.

Zhao together with a subgroup of the Cross-IDDRC Human Stem Cell Consortium, a multi-site group of stem cell researchers, have set two important goals to help address these roadblocks and standardize the field. The first one was ensuring organoids actually recapitulate human brain development. “If we develop a method to directly compare the organoid gene expression and the human brain gene expression during development, then we can actually say how close they are, how different they are,” Zhao says. Daifeng Wang, PhD, associate professor of biostatistics and medical informatic and computer sciences, developed a computational method to directly align organoids single-cell data with human single-cell data and determine how closely they match.

The second effort was to review all the research done using organoids to identify challenges in quantitative analyses and develop recommendations with the hope of moving toward more standardized methods.

The biggest challenge they encountered was the number of different protocols used to both generate and analyze organoids.

A lack of sufficient detail in reporting also makes it impossible for experiments to be replicated and results to be used for accurate comparisons. Organoids exhibit different characteristics at different timepoints of their development. Each week corresponds to different stages of prenatal brain development. “We want to make sure that people are reporting exactly what stage the organoids they analyze are in, and what compounds are they adding into their culture,” Sandoval says.

Xinyu Zhao, PhD
Xinyu Zhao, PhD (Photo © Andy Manis)

In addition to rigor and reproducibility, detailed reporting allows for more accurate studying of neurological disorders. This makes quantitative rather than qualitative study of organoids essential in order to have established baselines for what typical development looks like in organoids. “When you start to study diseases, you want to say ‘okay, fragile X organoids may have more or fewer glia or maybe more subtypes of neurons, you have to quantify those cell types.’ If you don’t quantify, you can’t really assess what happens in the disease model,” Zhao says.

Zhao explains that no protocol is overall better than another, but each lab must know the pros and cons of available protocols in order to choose the one that better fits their goals. “But it’s very important that you report what and how exactly you did it, and what method you used,” she urges.

In Zhao’s lab, Sandoval uses brain organoids to study fragile X syndrome, one of the most common causes of inherited intellectual disability. In this project funded by a competitive NIH R36 dissertation award, Sandoval is trying to characterize organoids derived from iPSCs obtained from individuals with fragile X to better understand brain development in individuals with the syndrome.

“The Waisman Center has been in a leadership role in organizing these efforts,” Zhao says. Her lab, along with Waisman Center investigators Wang, Anita Bhattacharyya, PhD, associate professor of cell and regenerative biology and a co-Lead of Cross-IDDRC Human Stem Cell Consortium, André Sousa, PhD, assistant professor of neuroscience, and Qiang Chang, PhD, professor of medical genetics and neurology, have been at the forefront of pushing for standardization of the use of human iPSCs in research to create a rigorous and reproducible processes that may lead to more accurate understanding of human brain development.