BRAIN-MAKING 101 As blobs of two types of brainlike tissue fuse, interneurons (green) migrate from the left clump to the right, linking with neurons (not stained) in the right blob. On both sides, neural support cells called glia appear in purple.
In a white lab coat and blue latex gloves, Neda Vishlaghi peers through a light microscope at six milky-white blobs. Each is about the size of a couscous grain, bathed in the pale orange broth of a petri dish. With tweezers in one hand and surgical scissors in the other, she deftly snips one tiny clump in half.
When growing human brains, sometimes you need to do some pruning.
The blobs are 8-week-old bits of brainlike tissue. While they wouldn’t be mistaken for Lilliputian-sized brains, some of their fine-grained features bear a remarkable resemblance to the human cerebral cortex, home to our memories, decision making and other high-level cognitive powers.
Vishlaghi created these “minibrains” at the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, where she’s a research assistant. First she immersed batches of human pluripotent stem cells — which can morph into any cell type in the body — in a special mix of chemicals.
The free-floating cells multiplied and coalesced into itty-bitty balls of neural tissue. Nurtured with meticulously timed doses of growth-supporting ingredients, the cell clumps were eventually transferred to petri dishes of broth laced with Matrigel, a gelatin-like matrix of proteins.
On day 56, the blobs display shadowy clusters of neural “rosettes.” Under a laser scanning microscope, razor-thin slices of those rosettes reveal loose-knit layers of a variety of dividing neural stem cells and the nerve cells, or neurons, they give rise to. The layered structures look similar to the architecture of a human fetal brain at 14 weeks of gestation.
Day 0: Around 9,000 stem cells are transferred into V-shaped wells and suspended in a cocktail of vitamins, amino acids and (for the first six days) Y-27632, a chemical to prevent the stem cells from committing suicide. Within a few days, the multiplying cells self-aggregate into tiny balls of neural tissue. The broth is refreshed every two to three days.
Day 18: The enlarging neural balls are moved into petri dishes of broth with CDLC, a supplement that provides fats. High oxygen levels (40 percent oxygen, 5 percent carbon dioxide) help the tissue absorb enough oxygen. Well-defined clusters, or rosettes, show early layers of various types of developing neural cells by weeks four to five.
Day 35: At 2 to 3 millimeters wide, the brainlike organoids are cut in half to give cells inside more access to nutrients and oxygen. The tissue clumps are nurtured in new broth infused with growth enhancers: a gelatin-like matrix of support proteins called Matrigel, B27 vitamin supplement, heparin and the growth promoter LIF.
Day 56: Organoids are again snipped in half and transferred to new petri dishes made of oxygen-permeable plastic to optimize access to oxygen for healthy growth. From here on, the organoids are halved every two weeks or so, routinely surviving as long as 150 days.
Source: M. Watanabe et al/Cell Reports 2017
