New advances in organoids! Human-mouse hybrid brain organoids respond to visual stimuli for the first time!

With the continuous advancement of stem cell technology, brain organoids derived from human induced pluripotent stem cells (hiPSCs) have become a hot topic
in disease models.
Brain organoids are expected to bring new development opportunities
in the fields of drug screening, precision medicine, and neurorepair.

The advantages of brain organoids are reflected in the following two aspects:

- Compared to two-dimensional cell culture

Brain organoids can reflect the complex characteristics of neuronal tissue, such as cortical structure, cell type diversity, etc
.

- Compared to animal models

Brain organs preserve differences in the genetic background of individual patients
.
In addition, human brain organoids implanted in the mouse cortex can be vascularized to supply nutrients and oxygen, prevent cell necrosis in the organoid nucleus, and extend nerve axons into surrounding host tissues
.

On October 16, 2022, Biovalley interpreted a blockbuster paper
published by Stanford Pascal's team in Nature.
This paper, titled "Maturation and circuit integration of transplanted human cortical organoids", reports a human-mouse hybrid brain organoid, which provides a new strategy
for brain neurodegenerative disease research and new drug development.
(Article review: Nature blockbuster: human-mouse hybrid brain comes out, 'brain organoids' research adds new progress)

Image Credit: Nature 610, 319–326 (2022)

However, current in vivo studies of brain organoids have focused on local and acute responses
in a small number of cells.
How transplanted organoids receive external stimuli and produce a chronic functional response in the recipient has not been proven
.
However, existing detection technologies cannot support longitudinal studies
in both tissue structure and electrophysiology.

To answer these questions, the Duygu Kuzum team at UC San Diego and the team at Boston University Anna Devor collaborated
.
The research team combined transparent microelectrode arrays and two-photon imaging to perform longitudinal multimodal monitoring
of human brain organoids transplanted into the retrosplenial cortex of adult mice 。 On December 26, 2022, the results were published in the journal Nature communications as "Multimodal monitoring of human cortical organoids implanted in mice reveal functional connection with visual cortex
".

Image Credit: Nat Commun 13, 7945 (2022)

Organoid and microelectrode array co-implantation

Longitudinal monitoring of organoid grafts is possible

Duygu Kuzum's team and Anna Devor's team collaborated to develop a transparent graphene microelectrode that enables two-photon microscopy, optogenetic stimulation, and cortical recording-free crosstalk-free integration, which was published in the journal Nature communications in 2018
.
In this paper, the collaborative team used the graphene microelectrode technology described above to record the electrical and optical signals
of transplanted brain organoids and surrounding host tissues.

Schematic diagram of the structure of the microelectrode array

Image source: Nat Commun 9, 2035 (2018).

The team first reprogrammed human skin fibrous cells and obtained hiPSCs
.
After inducing hiPSCs to produce brain organoids and cultured for 7-9 weeks, individual brain organoids
were selected according to size and shape.
The team selected 8-12 week-old NOD/SCID mice as recipients, implanted human brain organoids into the left retrosplenial cortex (RSC), and then placed graphene microelectrodes on
them.
The team also used 3D printing to make a hat that protects the lines
on the electrodes.
After one week of postoperative recovery, mice were electrophysiologically tested every two weeks, and the entire experiment lasted 8-11 weeks
.

Schematic diagram of organoid and microelectrode array co-implantation

Image Credit: Nat Commun 13, 7945 (2022)

Organoid grafts produce a neural response to sensory stimuli

The research team first studied whether brain organoids could produce a functional response to sensory stimuli by applying visual stimuli and recorded the electrical response
that followed.
The team hypothesized that the organoids would begin to respond
to visual stimuli only after they integrated with the host's visual cortex tissue.

The researchers placed a fiber-coupled white LED light in front of the right eye of the experimental mice, and the light pulses emitted by the device can trigger a stimulus-induced electrical response
.
Visual stimuli are then clearly detected in organoids and cortical channels close to the visual cortex
.
From 3 weeks after organoid implantation until the end of the experiment, the researchers did observe local field potentials (LFPs) in the electrode channels, indicating that the organoid implant established a prominent connection to the surrounding cortical tissue and received functional input
from the mouse brain.

Stimulation-induced local field potential recordings of organoids and cerebral cortex

Image Credit: Nat Commun 13, 7945 (2022)

Feel stimulated

Multi-unit activity is regulated

Next, the team investigated whether the organoids' spiking activity was regulated
by peripheral cortical nerve activity.
Because multi-unit activity (MUA) reflects local neuron firing, the researchers evaluated
multi-unit activity in 0.
5–3 kHz bandpass filtered data.
The researchers selected 2 representative channels (organoids: O1 and O4, mice: C2 and C7)
in brain organoids and mouse cerebral cortex, respectively.
In all channels, spontaneous multi-unit activity
was observed.
And when visual stimulation began, increased multiunit activity was observed in both the organoids and the mouse cerebral cortex
.

Multiunit activity of organoids and cerebral cortex

Image Credit: Nat Commun 13, 7945 (2022)

Compared to the host cortex

Transplanted organoids respond differently to anesthesia

The researchers further hypothesized that organoid grafts primarily receive local signal input, while long-distance projections like those from the thalamic nucleus and neuromodulation centers are absent
.
Since anesthesia has been shown to affect long-distance projection activity in the cerebral cortex, the team observed changes in spontaneous neural activity between organoids and surrounding host cortex after anesthetizing mice with 1.
5% isoflurane
.

Isoflurane anesthesia results in a significant decrease
in organoid neuronal activity compared to the peripheral cortex.
Moreover, in the awake state, organoid activity is usually lower than that of the peripheral cortex, and there is no selectivity
for specific frequency bands.
The results showed that the organoids were innervated by local neurons but lacked long-range projections
.

After anesthetizing mice with 1.
5% isoflurane,

Analysis of local field potentials of organoids and mouse brains

Image Credit: Nat Commun 13, 7945 (2022)

Endovascularization of organoid transplants

and integrates with the surrounding cortex

Using in vivo two-photon imaging and immunostaining, the researchers observed
the integration of organoid grafts with surrounding host tissue.

Vascularization has always been a difficult problem in organoid construction, and mice are injected with the intravascular tracer Alexa Fluor 680 glucan
9 to 10 weeks after organoid implantation.
Through two-photon microscopy, it was found that the organoid implantation areas all contained the mouse vasculature
.
Also, the density of blood vessels in organoid areas is lower
compared to the peripheral cortex.

In vivo imaging of organoid vascularization

Image Credit: Nat Commun 13, 7945 (2022)

Animals are sacrificed 8 to 11 weeks after organoid implantation and the brain is immunohistochemically stained to detect and analyze human cells (NM-95), endothelial cells (CD31), and neuronal nuclei (NeuN).

The organoid implantation area showed positive for NM-95, indicating that the implanted cells survived throughout the experimental cycle
.
Moreover, some NM-95-positive cells underwent cell migration
.
Consistent with two-photon microscopy observations, the researchers also observed vascular endothelial cells
passing through organoid implants.
By costaining NeuN and hematoxylin , it was found that about 48% of the cells of organoid grafts had a neuronal phenotype
.

Immunostaining for organoid vascularization

Image Credit: Nat Commun 13, 7945 (2022)

The next research direction

The experimental setup developed in this study provides an unprecedented opportunity
to study the dysfunction of human neural networks at the level behind developmental brain diseases.
Next, the research team needed to build a neurological disease model, integrate calcium imaging analyses, and visualize
the firing activity of organoid neurons.

Duygu Kuzum said in an interview that in the future, stem cell technology will be combined with neural recording technology for multiple brain disease research scenarios
.
For example, building disease models under physiological conditions, validating specific treatments based on patient-derived organoids, and assessing the potential
of organoids to repair specific lost, degenerated or damaged brain regions.

Although the current function of human brain organoids is too primitive, with the increase of research related to human brain organoid transplantation, it is worth paying attention to
whether organoid transplantation will affect animal brain function or animal behavior.
The screenwriter of "Rise of the Planet of the Apes 4", can you consider it and integrate into the current hot topic?

Resources:

1.
Revah, O.
, Gore, F.
, Kelley, K.
W.
et al.
Maturation and circuit integration of transplanted human cortical organoids.
Nature 610, 319–326 (2022).

2.
Wilson, M.
N.
, Thunemann, M.
, Liu, X.
et al.
Multimodal monitoring of human cortical organoids implanted in mice reveal functional connection with visual cortex.
Nat Commun 13, 7945 (2022).

3.
 Thunemann, M.
, Lu, Y.
, Liu, X.
et al.
Deep 2-photon imaging and artifact-free optogenetics through transparent graphene microelectrode arrays.
Nat Commun 9, 2035 (2018).

4.
https://medicalxpress.
com/news/2022-12-human-brain-organoids-implanted-mouse.
html