The brain in the brain: when human neurons are transplanted into animal brains Nature long article

Originally written by Richard Van Noorden

Transplanting human cells into animal brains can inspire insights into life development and disease, but also raise new ethical questions
.

In a darkroom in a lab in London, a team of students and researchers are watching as clusters of human brain cells settle into their "new home," the brain of a living mouse
.
On a computer monitor next to the microscope, human nerve cells occasionally flash the light they release when synchronized activity occurs
.
Over time, cells gradually grow new connections several centimeters long, which connect to each other into a network
.
Vincenzo De Paola, who runs the Imperial College London lab, said his students were fascinated
.
"That's all they want to do, I can't let them go
.
" He said
.

The researchers were watching an extraordinary show
in the front row.
De Paola's team is one of the few labs that can study the work of human nerve cells in the living, developing brain.
The system is mostly banned
for ethical and technical reasons.
"We can't study these processes when they occur in the human fetal brain," he says, "Instead, we want to observe how neurons in the human cerebral cortex mature and form active neural networks in living animals
.
" ”

Human neuronal cells (green) prepared from stem cells two weeks after transplantation into mouse
brains.
Source: Pierre Vanderhaeghen

De Paola's research system is a specific type of neural chimera — a field that has boomed in the last five years and has sparked widespread debate
over the ethical issues surrounding "mixing human and animal brain tissue.
" Proponents argue that such research systems are necessary to manipulate living human neurons in experiments, and that research using them has shed some important light on
our understanding of health and disease.
For example, by using neural chimeras in research, scientists have found differences
in how neurons develop and behave in Down syndrome and Alzheimer's disease.

But others warn that such a chimera represents a moral gray area, as it could blur the lines between humans and other animals, or reproduce human-like perception or cognition
in animals.
Some researchers say such chimeras should only be used
if no other cell or animal model is suitable.
"Can the model really be used to answer a scientific question, or are we just crossing the line to use it?" Naomi Moris, a developmental biologist at the Francis Crick Institute
, asked.
Ethicists are asking when clusters of human neurons present in an animal's brain should be considered to have some unique moral status
.

While research using chimeras (entities made up of cells from different living bodies or species) has been around for decades, the emergence of neural chimeras raises new ethical questions
.
A special report on neural chimera research (see go.
nature.
com/3pii9q5), published by the US National Academies of Science, Engineering and Medicine in 2021, identified some problems, such as giving animals new cognitive abilities.
or human diseases that may cause suffering to animals, etc
.
The committee recommended that while stem cell and animal research is currently well regulated, the field of neural chimeras should be closely regulated
.
The Commission also encourages pre-experiments and close monitoring of animals to identify any emerging or unusual animal behaviour
.

For regulators, there will be a lot to focus on
in the future.
Rather than limiting themselves to transplanting a small number of isolated cells, the researchers are beginning to consider further creation of chimera animals
with human brain regions.
Several studies of transplanting human brain stem cells into monkey brains helped launch in 2018 of a clinical trial to test the feasibility of
human brain stem cell transplantation in the treatment of Parkinson's disease.
In 2019, Japan lifted a ban
on government funding for research using "human-animal" chimeric embryos.
Many countries, including the United States and the United Kingdom, legally allow research involving mixing human brain cells or brain tissue with the brain of another animal, and can receive government funding
after additional scrutiny.
The United States prohibits its government from funding
research involving "human-animal" chimeric embryos.

Observers expect progress in this area to be rapid
.
"We know it's going to grow
at a high rate.
" Insoo Hyun, director of research ethics at Harvard Medical School, said
.

Research mainstream

The history of biological development revolves around chimeras
.
Ali Brivanlou, a developmental biologist at Rockefeller University in the United States, said that as early as the early 20th century, embryologists cut and paste the components of xenomorph embryos together, such as fusing chickens and quails together, to explore the source of
embryonic development signals.

In recent decades, researchers have also been introducing human components, such as organs, cells or genes, into other animals
.
Brivanlou says this is often done to better understand how organisms' organ systems work, or to find treatments for
diseases.
Cancer researchers often transplant human tumors into mice, and since the late 80s, scientists have bred mice with human immune systems [1].

Scientists transplanted human organoids (bright green) into mouse brains to study the cellular behavior
of neuronal cells in the context of health and disease.
Source: Abed Mansour

Another purpose is more practical: to try to grow human-compatible organs in animals to alleviate the shortage
of donor organs in organ transplants.
Over the past few months, researchers have transplanted genetically modified pig kidneys and hearts into humans
.

But human neuronal cell transplantation, which enables long-term survival of cells, did not appear
until nearly a decade.
In 2013, Pierre Vanderhaeghen, a neuroscientist at the Vrije Universiteit Brussels, and colleagues perfected the delicate process of growing human neuronal cells from stem cells [2] so that they can thrive in mouse brains without growing out of control
.

Scientists use two types of human stem cells to make chimera neuronal cells: one is embryonic stem cells (ES cells) originally derived from embryos; The other is induced pluripotent stem cells (iPS cells), which are derived from adult cells
that have been reprogrammed to an "embryo-like" state.
Both types of cells have the potential to develop into any type of tissue in the body and can guide development into neurons
.
"These derived from human pluripotent stem cells are more plastic than other types of cells transplanted in the past
.
" Hyun says this may allow these human cells to better integrate into the animals
.

The National Institutes of Health proposed in 2016 that it lift a ban
on funding research using animal embryos containing human cells.
Public comment poured in against the majority (the funding ban remains in place
).
But a 2020 survey of 430 Americans found that 59% supported "human-pig" chimera embryo research, which aims to achieve the production of human tissue in pigs [3].

It is one thing to have human kidney or liver tissue in pigs, and it may not be so easy to accept neural tissue
.
"People associate
the brain with moral status.
" Hyun said
.
And the researchers say that while none of these studies are moving toward producing human-like cognition in animals, the connections are causing them to rethink — to what extent do animal brains approach boundaries that are too human-like, causing social concern?

Mix brain cells

Over the past five years, researchers have developed multiple ways
to make neural chimeras.
They vary in complexity, from transplanting a single human nerve cell or a piece of brain tissue cultured in vitro, to combining embryos from two species to try to generate chimeric brain tissue
from scratch.

The easiest way to observe human neuronal cells in working condition is to transplant several cells
at a time.
Vanderhaeghen's research team is currently working at the Center for Brain and Disease Research at the Flemish Institute of Biotechnology (VIB)-Catholic University of Leuven (KU Leuven) in Belgium, using pyramidal neurons (the most numerous type of neuronal cells in the human cerebral cortex) obtained from in vitro culture of ES cells
.
They wanted to know how these cells form connections
in living organisms over longer time scales.
"We wanted to know how neurons that were 'trained' in a petri dish would behave
when transferred to the 'battlefield' of the brain.
" Vanderhaeghen said
.

His team, working with the team of Vincent Bonin at the Flanders Institute of Neuroelectronics, transplanted a "cell soup" of some neuronal cells in the form of scattered single cells rather than cell clumps into the cerebral cortex of newborn mice [4].

Human brain cells and their nuclei
two months after transplantation into mouse brains.
Source: Raquel Real, Manuel Peter, Rick Livesey and Vincenzo De Paola

Human neuronal cells mature at a customary time, taking 6 to 12 months, compared to only 5 weeks
for mouse neuronal cells that are their neighbors.
Vanderhaeghen said that even in the mice's brain environment, these human cells persist in developing
according to their long timeline.
"This suggests that the longer developmental time is encoded internally, in the neurons themselves
.
"

The team found that human neuronal cells not only develop normally, but also integrate into and function in the visual circuits of mice, and respond in the
same way that mouse neuronal cells respond to the same visual stimulus, such as moving black and white bars.
The surprising finding that human neurons can colonize and function properly in other brains also means that damaged brain circuits
may be repaired with cell transplants in the future.

"We expected some connectivity, but we were very shocked by the specificity of these cellular responses," Bonin said, "and could have failed a million approaches
.
" ”

The team also transplanted healthy human neurons into the brains of mice with a genetic predisposition to Alzheimer's disease
.
The study [5] showed that human neuronal cells degenerate in the brains of diseased mice, but the original mouse neuronal cells remain alive
.
This work not only confirms that human neuronal cells are particularly vulnerable to Alzheimer's disease, but also provides researchers with a way to observe what happens
to human neuronal cells in diseased brains.

De Paola also runs a research team at the Duke-NUS School of Medicine in Singapore, working to study how human neuronal cells are connected to each other and how these connections are disrupted
in developmental disorders.
His team transplanted pyramidal neuronal cells made from human iPS cells into the somatosensory cortex of the brain of adult mice [6].

Unlike Vanderhaeghen's transplant, De Paola's transplant eventually grew into dense human tissue micrografts in mouse brains and survived until the end
of the experiment five months later.
"We were amazed at the amount of growth, it formed a huge network," De Paola said, "of course, 'massive' is relative, actually about the same as a large hamahama bean
.
" ”

De Paola said that most of the connections produced by the transplanted cells occur between the transplanted cells (more than 90% of the connections between the transplanted human cells are to each other), but these human cells also do send neural projections to other parts of the mouse cerebral cortex and receive some neural projections, blood vessels and immune cells
from the mouse brain 。 These supports allow the tissue to continue to develop for up to 5 months and perform the behaviors expected in the developing human fetal brain, i.
e.
pruning neuronal cell branches and cell-to-cell connections and starting to fire in a synergistic manner
.

De Paola's team also performed the same transplantation experiment using neurons made from cells from patients with Down syndrome [6].

They found that these neurons formed fewer dynamic networks and had lower neural activity, but it was unclear how, if any,
there was a connection between the two features.
The team is currently conducting follow-up research
.
"We can experiment
with this model.
Obviously, we can't do this experiment
in adult or fetal brains.
De Paola said
.

Could the transplants from these research teams somehow transform the visual or sensory perception of mice into something more human-like? Neither team tested the cognition or behavior of transplanted mice, but both reported that the mice behaved in much the same way
as their non-transplanted companions.
Both De Paola and Vanderhaeghen were skeptical that a small number of human neurons and neural connections might alter cognition in mice
.
"I don't think even stimulating thousands of human cells at the same time can stimulate human behavior or perception
.
" Vanderhaeghen said
.
But he and De Paola argue that those working in the field should try to identify turning points
that could lead to such changes.

Long-lived organoids

A major advance in the field of human brain tissue research in the lab is the rise of brain organoids, self-organizing structures
formed by the growth of brain stem cells in 3D culture.

Since Madeline Lancaster and Jürgen Knoblich[7] first created brain organoids in 2013, they have become increasingly complex
.
Some researchers even splice multiple organoids into "sembloids
.
"

Sergiu Pasca, a neuroscientist at Stanford University in the United States, believes that the complexity of organoids makes them sufficient to ask many scientific questions related to the human brain, but even the "assembly" is far from the complexity
of a real human brain.
He thinks this is because they lack sensory input signals, blood vessels, immune cells, and support cells, and they can't receive feedback
.
In addition, once the structural size of the organoid exceeds 3-4 mm, the cells in the middle will die
due to the lack of nutrients provided by the cell culture medium.
It is difficult to support their growth for more than a few months
.

To overcome these limitations, neuroscientists have begun experimenting with transplanting organoids into the brains of animals to more closely mimic the complexity of human brain circuitry, and the mistakes
they make in disease.

Organoids, such as those shown in the figure (an organoid made from human neurons), have been transplanted into animal brains to study how neuronal cells connect and communicate
with each other.
Source: Ilaria Chiaradia

The group of neuroscientist Rusty Gage at the Salk Institute of Biological Research in the United States successfully transplanted human organoids into mouse brains and kept them alive for up to 11 months, which is almost the length of the entire life cycle of mice [8].

Some of the unpublished findings they obtained using the system show that human neurons mature from an embryo-like state, reaching a more complex state similar to that of infant neurons, and eventually exhibiting some of the characteristics of
adult neurons.
Human brain tissue is integrated into the mouse brain, where blood vessels grow, nerve cells in the tissue mature and respond to stimuli, and even form sparse but effective connections
with mouse neurons.

Abed Mansour, a postdoctoral researcher who worked with Gage and was involved in the establishment of organoid transplants, said the research system has advantages
for studying the changes in neurons in neurodegenerative diseases such as Alzheimer's disease.
Human neurons in organoid transplants emit long projections
to the host brain.
"This has the potential to be a good research system for exploring how healthy human neurons differ from those affected by disease in how this process occurs
.
" Mansour says he now leads his research team
at the Hebrew University Institute of Medicine in Israel.

Gage's team is currently planning to transplant brain organoids made from Alzheimer's patient-derived cells into healthy mouse brains and, conversely, to transplant organoids made from healthy human-derived cells into Alzheimer's
disease-modeled mouse brains.
The goal of the study was to find out which cells (neuronal cells themselves, or other cells such as astrocytes) are responsible for the inflammation
in the disease.

"This is the first time we have been able to monitor living human brain tissue
in the context of disease.
" Gage said
.
In the future, he said, this research may lead to individualized organoid transplant treatments to replace diseased or damaged brain tissue
.

In Lancaster's view, she is now a developmental biologist at the MRC Molecular Biology Laboratory in Cambridge, UK, organoid transplants have a place, but she urges researchers to scrutinize the animal experiments they are conducting and make sure they are legitimate
.
"As researchers, we need to be
careful.
The field is currently very hot and a large number of papers
are being published.
She said
.

As for the ethical status of organoids, when they were still in a petri dish outside the body, they were essentially considered just a complex class of 3D cell cultures
.
Lancaster, Gage, and others agree that they don't have the human ability
to perceive, feel, or cognitive.
And Gage says that even organoids transplanted into the body are not enough to confer any meaningful "humanity"
because they do not integrate well with the organism.

Chimera embryos

Another way to study human brain development in living animals is to add human components to the embryo of another animal at the earliest stages of development
.
Some research teams have tried to create human-animal chimeric embryos to study organ development in the hope that one day they will make organs
for transplantation.

One way to make it is to add human stem cells to animal embryos just a few days after fertilization, when these embryos are just small spheroids
in cell division.
Scientists have experimented
with rodents, livestock, and monkeys that are more closely related to humans (a controversial 2021 study) [9].
However, either these chimera embryos fail to develop beyond the very early stages, or the human cells in them die
rapidly.
Scientists believe that the cells from these xenomorphs are too different from each other to allow for the close coexistence and communication
necessary for embryonic cell development.

There is another way to make transplantable organs, which may one day be used to make brain tissue
needed for scientific research and disease treatment.
In a method called blastocyst complementation, scientists use a slightly later stage embryo called a blastocyst in which some genetic mutation is introduced to prevent it from forming a specific organ, such as the pancreas
.
They then took stem cells from another animal that could produce the organ and injected them into blastocysts
.

Stem cell biologist Hiromitsu Nakauchi's research team, split between Stanford University and Tokyo Medical and Dental University, has used blastocyst complementation to grow mouse pancreas in rat embryos [10].

"In a sense, we are using embryos as bioreactors because these embryos should 'know' how to generate organs," Nakauchi said
.

Whether animal blastocysts can develop normally while carrying human pancreas, kidneys or brain regions has been an open question
.
Bjoern Schwer, a molecular biologist at the University of California, San Francisco, believes that when human cells are mixed into embryos from the beginning, living organisms often fail to develop normally; But when a specific microenvironment is created for an organ or tissue, it can make it easier for human cells to contribute to the development
of living organisms.
Blastocyst complementation has not yet been tried on human neural stem cells, but Schwer's team and others are considering preliminary experiments
.
His first research question was whether a piece of brain tissue derived from a nonhuman primate could grow
in a rodent embryo.

Schwer's research group has used blastocyst complementarity techniques to create mouse-mouse chimeric brains
.
In a 2018 collaborative study with Frederick Alt's research group at Harvard Medical School, researchers used the technique to grow the entire forebrain region of one strain of mouse in another strain of mouse embryos [11].

"It's a fairly modular research system, and we're able to use it in different ways to remove and replace a variety of different brain regions
.
" Schwer said
.
For example, replacing a portion of a mouse brain with a brain region from a non-human primate instead of a human brain cell could provide researchers with a simpler and more ethical way to study in vivo monkey brain region development that closely resembles its human brain region
.
Schwer has received approval from his university to try using embryonic stem cells from macaques and marmosets to create a small piece of primate forebrain
in mouse brains.

He is also thinking about how to use blastocyst complementarity to grow a piece of human brain
tissue in the brains of developing mice, provided that it is ethical and technically feasible.
Such experiments could test how certain mutations promote the growth of human brain tumors, and perhaps find ways
to turn off these mutations.

"We're not there yet, but there's hope
.
" Schwer said
.
Like other researchers of neural chimera, he doesn't think a small piece of human brain tissue in a mouse brain can lead to human-like cognition
.

Crossing cognitive boundaries

However, it is precisely this possibility that worries
ethicists and the general public.
Alta Charo, a bioethicist and professor emeritus at the University of Wisconsin-Madison, said: "The combination of nerves touches on what makes us 'human' essence – our mind, our memories and our sense of self"
.
She argues that the public will be uncomfortable
with the concept of human minds imprisoned in animals or with half-human brains.

She and other ethicists argue that there is a wide gap
in public perception of why such research is conducted.
Researchers in the field of neurochimeras should often share their work with the public, for example by referencing Pasca's TED-style talks
, she said.
Charo believes that as research progresses, researchers will have to consider how high the proportion of human brain tissue may begin to move towards cognitive function in vitro culture, or trigger human-like traits in mice, and when it may add emotional stress or pain
to animals.

Human brain cells made from stem cells (purple) grow
in parts of the mouse brain (blue).
Source: Raquel Real, Manuel Peter, Rick Livesey and Vincenzo De Paola

Another concern is the unpredictable cellular behavior of human embryonic cells after they are placed in animal embryos, and whether their growth will spiral out of control
.
"The difficulty is the uncertainty
of the proportion of embryos being taken over.
" "We believe embryos will 'do their own thing,' but that may not be what we think
," Moris said.
”

Of course, neural chimera research requires biomaterial from human donors, which therefore involves questions about informed consent and how to properly inform people that their cells will be reprogrammed into neuronal cells and reborn
in petri dishes, mice, or chimeric embryos.

Schwer felt that anyone who provided their own cells for the scientific study of iPS cells should consult with them before they were used to make neural tissue
.
"I want to know, won't you?"

Brivanlou, for his part, is optimistic that future gains from this work could transform the complexities
of the moment.
"When you cure a disease with this — cure Alzheimer's disease for a child with Huntington's disease or a grandmother — everyone will agree
.
" But the road to that goal has been bumpy, and that's where we are now
.
”

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The original article was published in the August 3, 2022 issue of Nature's news feature section as Hybrid brains: the ethics of transplanting human neurons into animals

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doi: 10.
1038/d41586-022-02073-4

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