A team led by a BBRF grantee has reported another major advance in efforts to study how the human brain develops and how various dysfunctions and pathologies, especially those occurring before birth, give rise to major brain and behavior disorders ranging from autism and schizophrenia to epilepsy and cerebral palsy.

Sergiu P. Pasca, M.D., who received a BBRF Independent Investigator grant in 2017 and a Young Investigator grant in 2012, led a team at Stanford University in experiments that culminated in the successful development of what the team calls a “xenocortical mouse.” Also on the team were Karl Deisseroth, M.D., Ph.D., BBRF Scientific Council member, 2013 BBRF Goldman-Rakic Prize winner and 2-time BBRF Young Investigator grantee; Kevin W. Kelley, M.D., Ph.D., 2024 BBRF Young Investigator (a co-first author of the paper); and Felicity Gore, Ph.D., 2019 BBRF Young Investigator. Dr. Deisseroth is a co-winner of this year’s Nobel Prize in Physiology or Medicine.

Xenocortical mice are genetically engineered to lack most of the cerebral cortex—the focus, in mice and other mammals including humans, of higher-level operations including cognition, movement, language, and decision-making—as well as as the hippocampus, a region responsible for learning and memory. The prefix “xeno,” which means “foreign,” refers to the great innovation of the new research: implantation into the cavity in the mouse brain normally occupied by the cortex with human-derived cells comprising a cortical “organoid,” grown in the lab before being engrafted into the mouse.

As the new experiments, reported in Nature, demonstrate, the graft of human cortical cells finds a remarkably accepting host in the cortex and hippocampus-less brain of newborn mice, not only surviving, but thriving and forming complex functional connections and circuits.

“These animal models offer a unique opportunity to study how disease-associated alterations in human brain circuitry manifest in an intact, living nervous system,” Dr. Pasca commented. He is a pioneer in technologies that have enabled researchers to study human brain development and disease-related pathologies with progressively increasing sophistication and explanatory power. This series of innovations addresses the greatest problem facing those seeking to study the human brain, and particularly the critical period prior to birth when the brain emerges: the inaccessibility of human brain tissue for research, particularly in the context of living individuals.

Dr. Pasca has been an innovator in the technologies that led to the first regionalized brain organoids, grown in a lab dish from reprogrammed seed-cells (usually blood or skin cells) donated by humans. Such organoids bear all of the genetic anomalies in the donor, notably those with genetic-based neurodevelopmental illnesses. Dr. Pasca and colleagues later demonstrated the feasibility of combining separate human cell-derived-organoids each modeling a particular part of the brain and its specialized cells, into conglomerates he dubbed “assembloids.” Single organoids have been engrafted into living mice in yet another series of pathbreaking experiments in the Pasca lab. One of these, in 2024, using cells donated by a human patient, was able to replicate pathologies related to Timonthy’s syndrome, a severe neurodevelopmental disorder and sometimes considered part of the autism spectrum. Having the human-derived organoid functioning in the mouse brain enabled the Pasca lab to test a potential therapeutic, which is now moving toward a human clinical trial.

But, as the team notes in the paper reporting their newest advance, past organoid experiments have been limited by two principal factors. One is a lack of physical space in the cranial cavity in the mouse into which the human-derived organoids are implanted—“a competition for turf,” Dr. Pasca explains. The other factor is a competition for establishing neural connections between cells in the transplanted organoid and the host brain. The mouse brain develops more rapidly than its human equivalent, and so the implanted human-derived cells have tended after a time to lag and lose out to native mouse cells in forging connections. This has limited the fidelity of the models with the human brain and hence their potential utility.

In their novel xenocortical mouse, the team documented the graft of human-derived cortical cells into their new mouse environment. The graft developed extensive connectivity throughout the host brain and extended connections even into the mouse spial cord. Even before transplantation of the human cells, a variety of experiments indicated that despite the virtual absence of a cortex and hippocampus, many behaviors in the pre-transplant mice were somewhat preserved. The animals showed a near-normal gait and were a bit less able to remember being in recently encountered spaces; but they were fully viable.

The transplant of human organoids, each comprising about 100,000 cells, took place in 2-day-old animals. Three months later, over 90% by volume of the cortical tissue in these mice was human. During the next 3 months, in various behavioral tests, the mice performed about as well as their same-age normal peers.

Two experiments with the mice suggest how the new models may help to advance research and treatment of brain illnesses. Dr. Pasca’s team was surprised to find a very rare human-brain cell type proliferating in detectable numbers in the developing brains of the xenocortical mice. Called VEN cells (for von Enconomo neurons), these large cells had never been observed in prior versions of the transplantation technology or in lab dishes where cortical neurons were grown. These cells, now thought to occur only in large-brained highly social animals including apes, elephants, dolphins, and whales in addition to humans, are situated in brain regions involved in social awareness and decision-making. They are an important find in the xenocortical animal brains because “this cell type appears to be particularly vulnerable in frontotemporal dementia (FTD), a neurodegenerative disorder that can begin in midlife,” Dr. Pasca says.  “In some forms of the disease, these cells are markedly reduced and early symptoms can include changes in social behavior and personality or language difficulties, often before memory problems emerge.”

The new mouse model gives researchers a way to observe and experiment with the VEN cells in a living-brain environment. “Now we can generate these rare cells from a healthy person and study them in a living, behaving animal to learn more about what they’re doing. Or we can derive cells from patients with FTD or other neurological conditions, transfer them to these mice, test the animals for cellular or circuit-level correlates of behavioral disability, and then screen therapeutic candidates.”

Another aspect of the team’s research with the new mice involved demonstrating how they can be used to study specific illnesses—in this case, those caused by oxygen deprivation, which is devastating to the human brain. The xenocortical mice were exposed to 5 hours of low oxygen, which was observed to cause substantial damage to cortex (composed, in these animals, mostly of human cells). The mice had problems moving and with balance, not unlike those seen in children with cerebral palsy. Mice without a cortex but not implanted with human cells were relatively unaffected, in comparison.  “Finding out what accounts for this difference could yield clues about human neural susceptibility to oxygen deprivation, shed light on cerebral palsy, and provide a platform for testing potential therapies,” Dr. Pasca says.