When making brain organoids, researchers choose from many protocols and tweak them. For her latest project, Lancaster modified the lab’s existing protocol for making cerebral organoids. “In fact, we found that various approaches for generating a forebrain identity work fine as a starting point,” she says. That might involve a cerebral organoid kit such as the commercial one from Stemcell Technologies, she says, or homemade media and a scaffold of fibers made of polylactide-co-glycolide as her lab has done previously8. To promote a choroid plexus subregional identity, says Lancaster, it was key to use Wnt and Bmp pathway activators to help direct the tissue to a more dorsal identity, reflecting the area where the choroid plexus develops. Although a simple tweak, the “difficult part was figuring out the correct concentration and timing, but once we figured that out, we found this little nudge is highly efficient.”

As USC’s Quadrato says, vexing conditions such as autism spectrum disorder, bipolar disorder or schizophrenia have an array of symptoms that can vary considerably between individuals. The conditions are mainly polygenic, with heterogeneous combinations of many alleles acting together, and various anatomic and circuit changes may also play a role. Every patient might have a different genetic background. To model mechanisms underlying these disorders, one can start with a patient’s cells, induce them to become pluripotent stem cells, generate organoids from them and perform comprehensive characterization, including single-cell RNA sequencing. Cortical organoids have become more robust and reproducible, says Quadrato, thanks to work by a number of teams, including Paola Arlotta’s lab at Harvard University9,10. (Quadrato completed her postdoctoral fellowship in the Arlotta lab but did not lead the work in question.) The Arlotta team and colleagues at other institutions applied single-cell RNA sequencing to characterize cells from 21 organoids collected at three and six months. Cortical organoids from different human stem cell lines, both male and female, delivered almost identical compendia of cortical cell types. “You always get the same ratio of different cortical cell types in each individual organoid,” says Quadrato. Identity matters right at the start of brain organoid experiments. Stem cells grow into balls of cells called embryoid bodies, which can potentially differentiate into the three germ layers — ectoderm, mesoderm and endoderm — that give rise to all the human body’s tissues. Brain organoid researchers nudge this development. They can use signaling molecules such as morphogens to generate ectoderm, the layer from which the brain develops. “The default choice of the organoid is to become cortex,” says Quadrato. Lineage trajectories between human cortex and cortical organoids are similar. Some cellular subtypes can be missing in an organoid, which will not have all brain regions or sensory input from a ‘body’. Although cortical organoids have become quite reproducible, “if you want to make other brain regions, then it becomes more difficult,“ says Quadrato. The cerebellum holds her fascination as the brain structure with the most neurons in the brain. It has expanded the most over the course of evolution and appears to have played a big role in the acquisition of human-specific cognitive traits. Evidence is growing about its role in conditions such as autism. One of her lab’s projects is assuring that cerebellar organoids can be made reproducibly. A brain organoid will always have a tendency to make some forebrain, she says. That means organoids of other brain regions can vary from one batch to another and across different cell lines. This heterogeneity and variability can make it difficult for scientists to draw conclusions from their organoids about the mechanisms underlying disorders. High-throughput, high-resolution techniques are needed to reveal the identity of cells in a given organoid; “otherwise, it’s very difficult to understand what’s going on and to characterize them,” she says. She is concerned that some labs start working with organoids but underestimate how much time, effort and, ultimately, money, it takes to thoroughly characterize organoids. Costs will drop as technologies mature, but, using the wrong techniques or applying them to an in vitro model in the wrong way, “you can draw conclusions that are completely wrong,” she says. Among the techniques she is establishing in her lab is Patch-seq, which involves recording electrical activity from a neuron or groups of neurons through patch-clamping followed by RNA sequencing. When working with mice, location hints at the cell types one is recording from. An organoid lacks such anatomical reference points, and ‘blind’ recording from just anywhere in an organoid is a bad idea, she says. With Patch-seq, one can aspirate the RNA from the just patch-clamped cells and “then you look at that profile; it’s very helpful.”

The Pașca lab has developed assembloids, which combine different organoid types. Organoids can be characterized with many techniques.
Credit: Adapted with permission from ref. 3, Springer Nature

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