Stanford Medicine researchers transplanted human brain organoids into genetically engineered mice missing most of their cerebral cortex. Published Sept. 16 in Nature, the human tissue integrated deeply into the rodent nervous system, and revealed previously unseen human brain cells.
For decades, researchers studying neuropsychiatric conditions faced an intractable biological wall. Growing human brain tissue in laboratory glassware offered a glimpse into early development, but those cell clusters lacked blood vessels and a body to send and receive signals. Transplanting human organoids into rats marked a significant leap, yet those earlier grafts competed for space with rapidly developing rodent brain cells, limiting how far the human tissue could grow and wire.
In a study published online Sept. 16 in Nature, the researchers succeeded in transplanting self-organizing bits of laboratory-grown human brain tissue called cortical organoids into mice specially bioengineered and bred so that almost all of their cerebral cortex was missing. The cerebral cortex is the outermost “rind” of the brain, to which much of our higher-level functioning such as cognition, language, attention and decision-making is attributed. The resulting vastly enlarged cavity in the mice’s brains proved to be a hospitable environment. The human tissue survived, thrived, grew — and developed working connections to the mice’s brain and beyond to the spinal cord. These animal models offer a unique opportunity to study how disease-associated alterations in human brain circuitry manifest in an intact nervous system,
said Sergiu Pasca, MD, the Kenneth T. Norris, Jr. Professor II of Psychiatry and Behavioral Sciences and a member of Bio-X as well as the Wu Tsai Neurosciences Institute. Sergiu Pasca, who is the Bonnie Uytengsu and Family Director of the Stanford Brain Organogenesis Program, a CZ BioHub Investigator and a science fellow of the Hoover Institution at Stanford, is the study’s senior author. Lead co-authorship is shared by postdoctoral scholar Konstantin Kaganovsky, PhD; assistant professor of psychiatry and behavioral sciences Kevin Kelley, MD, PhD; neurosurgery instructor Tilo Gschwind, PhD; and medical student Paul Harary.
Removing Spatial Competition in Genetically Engineered Mice
To overcome the barrier of overcrowding, a team led by Stanford scientists altered the developmental path of laboratory mice. Emx1-cre (JAX, B6.129S2-Emx1tm1(cre)Krj/J), Esco2fl/fl (JAX, B6N.129S-Esco2tm1.1Ge/J) and C57BL SCID (JAX, B6. Cg-Prkdcscid/SzJ) mice were used to generate the breeding scheme: male Esco2fl/flPrkdcscid/scid × female Emx1-cre+/−Esco2fl/+Prkdcscid/scid. Breeder and offspring cages were supplemented with breeder chow (Envigo Teklad 2919) and DietGel 76A (with plant protein) to help to increase pup survival. Some breeders were more prone to parental infanticide, and in these cases, an aunting strategy was implemented with Swiss Webster active dams from Charles River, while a subset of pups was euthanized during the second postnatal week to reduce competition for maternal care and milk.
We generated and cultured hCOs from hiPS cells as previously described. In brief, hiPS cells were treated with Accutase (Innovate Cell Technologies, AT-104) at 37 °C for 7 min to dissociate them into single cells. Approximately 3 × 106 cells in Essential 8 medium (Life Technologies, A1517001) supplemented with the ROCK inhibitor Y-27632 (10 μM; Selleckchem, S1049) were added to each well of the AggreWell 800 plate (StemCell Technologies, 34815), which were then centrifuged at 100g for 3 min and incubated at 37 °C with 5% CO2 (day −1). Spheroids were collected 24 h after cell aggregation (day 0) and transferred into ultra-low attachment plastic dishes (Corning, 3262) in Essential 6 medium (Life Technologies, A1516401) supplemented with dorsomorphin (2.5 μM; Sigma-Aldrich, P5499) and SB-431542 (10 μM; Tocris, 1614). From days 2 to 5, the Essential 6 medium was changed daily and supplemented with dorsomorphin and SB-431542, and on the sixth day in suspension, neural spheroids were transferred to neural medium composed of Neurobasal A (Life Technologies, 10888), B-27 supplement without vitamin A (Life Technologies, 12587), GlutaMax (1:100, Life Technologies, 35050) and 10 U ml−1 penicillin–streptomycin (Gibco, 15140122). From days 6 to 24, the neural medium was supplemented with 20 ng ml−1 epidermal growth factor (EGF; R&D Systems, 236-EG) and 20 ng ml−1 basic fibroblast growth factor (FGF; R&D Systems, 233-FB), with medium changes occurring daily from days 6 to 15 and every other day until day 24.
Transplanting self-organizing human cortical organoids provided a remarkably hospitable environment. The human tissue survived, thrived, and formed working connections to the mice’s brains and spinal cords. Developmental neurobiologists noted that the timing of the procedure — carried out days after the mouse pups’ birth, past the point at which the brain’s core wiring is already in place — also ensures that human cells cannot take over complex thinking, says Madeline Lancaster, a developmental neurobiologist at the University of Cambridge, UK, who was not involved in the study. In fact, behavioural tests revealed that the human tissue did not enhance the rodents’ intellect. The goal here is clearly not to make a mouse that’s super intelligent — nor would it be,
she says. The goal here is to have human brain tissue inside a realistic body setting so you can start using it to understand human neurobiology and human neurological diseases
.
Uncovering Uniquely Human Neurons and Vulnerabilities
Inside these hybrid animal models, the human tissue matured into specialized neural circuitry. The researchers were astonished to find, in these mice, an important nerve-cell type that hasn’t been previously glimpsed in laboratory culture and has been seen only in autopsied human brains. Although creating human–mouse hybrids, or chimeras, can cause discomfort and raise ethical questions for some people, researchers working in this field say that precautions are taken with investigations such as this one. For instance, the work underwent extensive oversight, including review by independent bioethics panels, according to Pașca.
In 2022, Pașca and his colleagues showed that transplanting human brain organoids into newborn rats allowed the structures’ neurons to mature and wire into sensory pathways, and two years later, the group used those rats to evaluate how effective drugs called antisense oligonucleotides were against Timothy syndrome, a severe genetic condition linked to autism and epilepsy. We now have a very powerful new system
for understanding what makes the human brain unique and uniquely susceptible to disease,
says study co-author Sergiu Pașca, a neuroscientist at Stanford University in California.
Implications for Severe Neurodevelopmental Research
The new methodology should speed research into the underlying biological causes of schizophrenia, epilepsy, profound autism and cerebral palsy, Pasca said. Neuroscientists will be able to learn much more about the causes and mechanisms of neurodevelopmental and pregnancy-incurred disorders and to test possible interventions to correct or prevent them.
Such conditions are exceedingly difficult to study at the molecular and cellular level because the brain is more complex than any other organ and because living human brain tissue is nearly always inaccessible. One in 20 American adults is troubled by a severe psychiatric illness; the associated medical, social and economic burdens are huge. More than 1 in 100 adults suffers from schizophrenia, believed to be largely the result of brain-circuit abnormalities predating birth.
