The cerebral cortex of the brain, responsible for higher-level cognitive processes, movement control, and sensory input processing, is composed of a wide variety of neurons and glial cells. Radial glial progenitors (RGPs) produce all excitatory neurons in the developing cerebral cortex. Now, for the first time, a quantitative framework of RGP lineage progression has been revealed. The team of researchers from the Institute of Science and Technology Austria (ISTA) established Mosaic analysis with double markers (MADM)-based lineage tracing in vivo—in mouse embryonic stem cells in a self-organizing cortical organoid system. The findings present critical time windows in development when compared to the real mouse brain.
This work is published in Nature in the paper, “Temporal uncoupling of radial glia lineage progression in cortical organoids.”
“In our lab we study how the brain develops from stem cells,” Simon Hippenmeyer, PhD, professor at the ISTA explains. “How a brain reaches the right size, how stem cells know when and into which neurons they should develop, but also what happens when something goes wrong during development or disease—for example, in microcephaly or macrocephaly, where the brain is unusually small or large.”
Two mouse cortical organoids at 13 days in culture. Cortical rosettes, the actual portions of the organoids that resemble aspects of the developing brain, are seen as lumps along the outer edge of the organoid. [Stouffer et al. / Nature]
The researchers compared specific developmental stages of the mouse brain with those of the organoids. Using single-cell sequencing, they examined which cell types show up in both systems, their relative abundance, and at what point in time they emerge or disappear again.
“In the developmental stages we examined, we see very similar cell populations of the mouse brain with those of the organoids,” Hippenmeyer explains. “The molecular programs are similar.”
“Now that we had this rigorous organoid system, we were able to examine even more closely what happens to stem cells during cortical structure development—and compare these processes directly with our in vivo model, the mouse,” he continues.
Using MADM technology—a unique genetic method that makes it possible to track stem cell division during organogenesis—the group produced a clear roadmap of development in the mouse brain at the single progenitor cell level.
The team found that “RGPs exhibit a high level of plasticity in proliferative potential in organoids rather than strict temporally stereotyped lineage progression as observed in vivo.” In addition, RGPs in organoids showed “increased lineage restriction, diminishing cell-type diversity in clones of cortical projection neurons, despite uniform single-cell transcriptional signatures of RGPs and a unitary lineage trajectory.”
The researchers suspect that organoids lack certain external signals. The microstructures in Petri dishes form through self-organization, and due to being cultured in the lab, they lack many of the external influences that are present in the living organism.
Researchers from the Hippenmeyer group at ISTA who carried out the study. From left to right: Simon Hippenmeyer, Osvaldo Miranda Romero, Fabrizia Pipicelli, and Carmen Streicher. [ISTA]
“In our organoids, this does not seem to work perfectly,” says Hippenmeyer. “The physical force of self-organization alone is apparently not enough. Factors present in in vivo systems are missing—the so-called stem-cell niche.”
The stem-cell niche is the specific microenvironment in which stem cells live and are regulated. It includes, for example, neighboring cells, blood vessels, signaling molecules, and growth factors, as well as mechanical signals.
They write, “critical non-cell-autonomous cues that are absent in self-organizing systems and/or the genuine stem cell niche are essential for faithful temporal control of RGP lineage progression and the generation of clonal cortical cell-type diversity.
This work presents a robust protocol for producing cortical organoids from mouse cells. It also highlights the processes in development which are sensitive to changes to—or a lack of—the stem cell niche.
These findings are important for organoid research. They show how similarly certain developmental processes unfold in organoids and in the mouse brain—and up to what point, based on current knowledge, specific aspects of brain development can be reliably studied in an organoid.
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