This study was supported by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research 17H05661 and 18K19903, Core-2-core program, and Beyond AI institute.

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In part of this protocol, a patent has been licensed to Jiksak Bioengineering, Inc. which was founded by Jiro Kawada.

This protocol describes the formation of a motor nerve organoid (MNO) which has an axon bundle extended from a motor neuron spheroid generated from human iPS cells. The formed axon bundle is thick, flexible, and well-organized in unidirectional structures. By dissecting the axon bundle, high-purity axonal protein and RNA can be obtained sufficiently for biochemical analyses. Neuronal activity can be measured in axon bundles and spheroids with calcium imaging. Contamination of nuclear and dendritic proteins in the axonal lysate was not detected by western blotting, demonstrating that our method efficiently separated axons from cell bodies and dendrites.
One of the advantages of this protocol is the rapid differentiation and generation of MNO equipped with an axon bundle, in which all processes can be done in 4 weeks with the 3D protocol and 5-6 weeks using the 2D protocol. This is short compared to other protocols which typically take 3-4 weeks to simply differentiate into MN from embryonic stem cells and iPS cells17 and it takes an additional 2-4 weeks to obtain axonal elongation. The 3D protocol is generally preferred over the 2D protocol because of the shorter differentiation time, fewer steps, and reduced technical risks without the dissociation step compared to the 2D protocol. The microfluidic-based tissue culture chip was designed in a manner so that the axons of MNS can elongate toward the other compartment through the microchannel, which facilitates formation of a bundle of axons by inducing axo-axonal interactions and affinity between axons. Because of the simple experimental set-up, all the protocols described here can not only be performed by bioengineers, who are familiar with the manipulation of a tissue culture chip, but also biologists and neuroscientists who are not familiar with microfluidics and microfabrication techniques. It should be noted that Steps 1 and 2 can be also performed by using an external fabrication service.
One of the critical steps to accomplish the protocol is a sequential change of culture medium. It is advised to completely change the culture medium at each step during the differentiation so that factors in the spent medium do not disturb the MN differentiation. Another critical point of this protocol is to maintain undifferentiated iPS cells in good quality. Quality of the initial iPS cell culture significantly affects the efficiency to obtain motor neurons and MNO. Another point is that the diameter of MNS should be smaller than the size of the hole of the chip (1.5 mm). Larger spheroids cannot enter the chamber, and potentially experience severe hypoxic necrosis in the center part. The size of MNSs can be controlled by changing an initial seeding number of iPS cells (in 3D protocol) or motor neurons (in 2D protocol). The seeding density of cells should be optimized for each iPS cell line.
Compartmentalized microfluidic devices with microgrooves and small pore filters have been widely used to separate axons from cell bodies and dendrites. This technique can also separate axons from cell bodies and dendrite, with superior abundance of axons in bundled tissues. Compared to the other methods, one major limitation of this method is that it cannot separate two different culture media in the current design of the tissue culture chip, which hinders capability of co-culture of two different cells that require two distinct media. Another limitation is that the PDMS chip put predetermined restriction on size of the tissue. A spheroid larger than the hole cannot enter the chamber, and the axon bundle cannot grow thicker than the width of microfluidic channel.
This method can be applied to other types of neurons. Our group has shown capability to model a cerebral tract by using a modified method combined with cerebral organoid techniques18. Cortical spheroids were introduced into both compartments and the axons spontaneously elongated reciprocally toward each spheroid, and subsequently an axon bundle formed spontaneously. As a result, two cortical spheroids can be connected through an axon bundle, and the tissue could be obtained as one piece. This demonstrates that the approach is highly versatile to form axon bundle tissues regardless of neuronal cell types. In this protocol, human iPS cells were used, however, other stem cells including human ES cells and human neural stem cells may be used with modifications to the presented protocol. 3D spheroids of neurons can be generated by diverse protocols19,20. This method of making tissues with an axon bundle can potentially be combined in the future with the other differentiation protocols for making 3D MN spheroid. In addition, the thickness and the length of the axon bundle can be controlled by simply changing the width and height of microchannels of the tissue culture chip for future developments.
We believe that this protocol can be used for drug testing and screening and can contribute to the understanding of mechanisms underlying the development and diseases of axon fascicles.

Spinal motor neurons (MN) extend axons to skeletal muscles to control body motion. Their axonal trajectories are highly organized and regulated in the developmental process. Despite many studies on axon extension and guidance1, mechanisms for organized axon bundle formation are still under investigation. Axons of motor neurons are often damaged by neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS)2, but pathophysiological mechanisms of the damage on axon fascicles are poorly understood. Thus, a physiological and pathological model to recapitulate axon bundle formation and regression is required in the field.
A human stem cell-derived motor neuron is a promising platform for understanding the development and diseases such as ALS3. Human induced pluripotent stem cells (iPS cells) can be used to model diseases using patient-derived cells. To date, various differentiation methods from pluripotent stem cells into MN have been reported4,5,6. However, axons of neurons in two-dimensional culture are randomly oriented and do not recapitulate in vivo microenvironment within developing nerves in which axons are unidirectionally assembled through dense axo-axonal interactions7. To overcome this issue, we have developed a technique to generate a three-dimensional tissue resembling motor nerve from human iPS cells8, and named the tissue as motor nerve organoid (MNO). The MNO consists of cell bodies located in a motor neuron spheroid (MNS) and an axonal fascicle extended out from the spheroid. The axons in the fascicle are unidirectionally oriented, which resembles axons in developing motor nerves. Hence, MNOs uniquely provide a physiological axonal microenvironment, which was not done by any other previously developed neuronal culture methods.
In this protocol, we describe methods for tissue culture chips fabrication, rapid motor neuron differentiation, and motor nerve organoid formation in developed chips. Our tissue culture chip is very simple, and it only contains a compartment for accepting a spheroid, a microchannel for forming an axon bundle, and a compartment for housing axon terminals. The device does not contain complex structures including microgrooves or micropore filters that are often used to separate axons and cell bodies by size9,10. Hence our devices can be easily fabricated by following the steps described in this protocol if a photolithography setup is available.
Rapid differentiation of human iPS cells is achieved with an optimized combination of inducing and patterning factors (SB431542, LDN-193189, retinoic acid (RA), and smoothened agonist (SAG)) and acceleration factors (SU5402 and DAPT). It has been reported that the combination of SU5402 and DAPT accelerates the differentiation of peripheral neurons and neural crest cells11. In this protocol, we offer three different methods to generate MNOs, so that readers can decide on a method most suited to their needs. We recommend performing differentiation of human iPS cells after forming a spheroid (the 3D method), since the differentiated MNS can be transferred directly into a tissue culture chip. Alternatively, human iPS cells can be differentiated into motor neurons in monolayer (2D) culture, and then created into three-dimensional motor neuron spheroids as we previously reported8. We have updated the protocol, and with the three-dimensional differentiation method described in this protocol, the transition from 2D to 3D can be avoided and MNOs can be obtained with shorter differentiation time, fewer steps, and reduced technical risks without the dissociation step. Commercially available neurons can be also used to generate MNS to reduce the time for differentiation.
To generate an MNO, we cultured an MNS in the tissue culture chip. The axons elongate from the spheroid and extend into the microchannel in which axons gather and align unidirectionally. This facilitates axo-axonal interaction and spontaneous formation of a tightly assembled unidirectional bundle tissue of axons in the microchannel, which is uniquely achieved by this protocol, whereas either spontaneous bundle formation or guided axonal orientation alone can be achieved by other protocols12,13,14. In a typical experiment, few cells migrate out from spheroids to the microchannel and most cells stay nearby spheroids. This method allows axons to be spontaneously separated out from the spheroids without using size-dependent physical barriers (e.g., microgrooves or micropore filters) to separate axons from cell bodies.
The resultant MNO can be subjected to various examinations, including morphological, biochemical, and physical analyses. The cell body and extended axon bundle can be physically isolated by cutting and can be separately analyzed for downstream experiments, e.g., biochemical assays. Biological materials including RNA and protein can be isolated from just a few axon bundles for regular biochemical assays including RT-PCR and western blotting. Here, we describe a protocol for generating motor nerve organoid from iPS cells, which offers an attractive physiological and pathological model to study the mechanism underlying development and disease of axon fascicles.

<table><tbody><tr><td>(Tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane</td><td>Sigma</td><td>440302</td><td></td></tr><tr><td>16% Paraformaldehyde (formaldehyde) aqueous solution</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>200&amp;micro;l Wide Bore Pipet Tips</td><td>BMBio</td><td>BMT-200WRS</td><td></td></tr><tr><td>6-well plates</td><td>Violamo</td><td>2-8588-01</td><td></td></tr><tr><td>Accutase</td><td>ICT</td><td>AT104</td><td></td></tr><tr><td>B-27 Supplement (50X)</td><td>Gibco</td><td>17504044</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A6003</td><td></td></tr><tr><td>Brain-derived neurotrophic factor (BDNF)</td><td>Wako</td><td>020-12913</td><td></td></tr><tr><td>CO2 incubator</td><td>Panasonic</td><td>MCO-18AIC</td><td></td></tr><tr><td>Cryostor CS10</td><td>Stem Cell Technologies</td><td>07959</td><td></td></tr><tr><td>DAPT</td><td>Sigma</td><td>D5942</td><td></td></tr><tr><td>DMEM/F12</td><td>Sigma</td><td>D8437</td><td></td></tr><tr><td>Fluo-4 AM</td><td>Dojindo Laboratories</td><td>CS22</td><td></td></tr><tr><td>GlutaMAX Supplement</td><td>Gibco</td><td>35050-061</td><td></td></tr><tr><td>Growth factor reduced Matrigel (basement membrane matrix)</td><td>Corning</td><td>354230</td><td></td></tr><tr><td>HB9 Antibody</td><td>Santa Cruz</td><td>sc-22542</td><td></td></tr><tr><td>HBSS</td><td>Wako</td><td>085-09355</td><td></td></tr><tr><td>Hoechst 33342</td><td>Sigma</td><td>14533</td><td></td></tr><tr><td>iCell motor neuron (commercially available human iPS cell-derived motor neurons)</td><td>Cellular Dynamics</td><td>R1051</td><td></td></tr><tr><td>Isopropyl alcohol (IPA)</td><td>Wako</td><td>166-04836</td><td></td></tr><tr><td>Knock Out Serum Replacement</td><td>Gibco</td><td>10828028</td><td></td></tr><tr><td>LDN193189</td><td>Sigma</td><td>SML0559</td><td></td></tr><tr><td>MEA probe</td><td>Alpha MED Scientific inc</td><td>MED-P5004A</td><td></td></tr><tr><td>MEM Non-essential Amino Acid Solution (100x) (NEAA)</td><td>Sigma</td><td>M7145</td><td></td></tr><tr><td>Microscope Glass</td><td>Matsunami</td><td>S9111</td><td></td></tr><tr><td>mTeSR Plus</td><td>Stem Cell Technologies</td><td>05825</td><td></td></tr><tr><td>N2 supplement</td><td>Wako</td><td>141-08941</td><td></td></tr><tr><td>Neurobasal medium</td><td>Gibco</td><td>21103049</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Photoresist SU-8 2100</td><td>Microchem</td><td>#SU-8 2100</td><td></td></tr><tr><td>Prime surface 96U</td><td>Sumitomo Bakelite</td><td>MS-9096U</td><td></td></tr><tr><td>ReLeSR (passaging reagent)</td><td>Stem Cell Technologies</td><td>05872</td><td></td></tr><tr><td>Retinoic acid</td><td>Wako</td><td>186-01114</td><td></td></tr><tr><td>SAG</td><td>Sigma</td><td>SML1314</td><td></td></tr><tr><td>SB431542</td><td>Wako</td><td>192-16541</td><td></td></tr><tr><td>Silicon wafer</td><td>SUMCO</td><td>PW-100-100</td><td></td></tr><tr><td>Silpot 184 w/c kit</td><td>Dow Toray</td><td>Silpot 184 w/c kit</td><td></td></tr><tr><td>Smi32 Antibody</td><td>Biolegend</td><td>801701</td><td></td></tr><tr><td>SU5402</td><td>Sigma</td><td>SML0443</td><td></td></tr><tr><td>SU-8 Developer</td><td>Microchem</td><td>Y020100</td><td></td></tr><tr><td>Synapsin I Antibody</td><td>Millipore</td><td>Ab1543</td><td></td></tr><tr><td>TrypLE Express liquid without phenol red (dissociation solution)</td><td>Gibco</td><td>12604-021</td><td></td></tr><tr><td>Tuj1 Antibody</td><td>Biolegend</td><td>801202</td><td></td></tr><tr><td>Y-27632</td><td>Wako</td><td>030-24021</td><td></td></tr></tbody></table>

three-dimensional motor nerve organoid generation

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and neurodegenerative diseases. Although numerous studies of axons have been conducted, our understanding of formation and dysfunction of axon fascicles is still limited due to the lack of robust three-dimensional in vitro models. Here, we describe a step-by-step protocol for the rapid generation of a motor nerve organoid (MNO) from human induced pluripotent stem (iPS) cells in a microfluidic-based tissue culture chip. First, fabrication of chips used for the method is described. From human iPS cells, a motor neuron spheroid (MNS) is formed. Next, the differentiated MNS is transferred into the chip. Thereafter, axons spontaneously grow out of the spheroid and assemble into a fascicle within a microchannel equipped in the chip, which generates an MNO tissue carrying a bundle of axons extended from the spheroid. For the downstream analysis, MNOs can be taken out of the chip to be fixed for morphological analyses or dissected for biochemical analyses, as well as calcium imaging and multi-electrode array recordings. MNOs generated with this protocol can facilitate drug testing and screening and can contribute to understanding of mechanisms underlying development and diseases of axon fascicles.

Motor neurons were differentiated within 12-14 days in 3D differentiation procedures (Figure 4 and Figure 5). Importantly, more than 60% of cells expressed motor neuron marker HB9 during the differentiation. Immunohistochemistry revealed that approximately 80% of the cells in the MNS were SMI32-positive motor neurons. HB9 and SMI32 are the established early-stage motor neuron markers15,16. The expression of HB9 and SMI32 are the key parameters that need to be confirmed to ensure cellular identity of motor neurons. After the introduction of an MNS into the culture chip, axons extend into the channel and an axon bundle forms. Due to microchannels serving as physical guides, axons elongate from the MNS and form a bundle by axo-axonal interaction (Figure 6A). It is essential to confirm the formation of the axon bundle by microscopic observation to confirm the generation of an MNO. A successful MNO bears an axon bundle wider than 50 &#956;m and few isolated axons out of the bundle in the channel. Initial elongation of axons can be observed 24 hours after the introduction of the spheroid. Within the next 3-4 days, the axons reached to the center of the microchannel and then reaches to the other end within an additional 10 days (Figure 6A). Consequently, the axons assembled and formed a straight and unidirectional bundle in 2-3 weeks in a chip, and neuronal activities were observed thereafter.
Motor nerve organoids can be collected from the chip by detaching the PDMS from the microscope glass for biological analysis (Figure 6B). Axon bundles and cell bodies can be dissected and isolated by cutting using a surgical knife or tweezer under a microscope (Figure 6B). These biological materials including RNA and protein can be used for regular biochemical assays such as RT-PCR and western blotting. In axon bundles of MNOs, nuclear or dendritic maker proteins are not detected in western blotting (Figure 6C).
In combination with a calcium indicator (Fluo-4 AM), neuronal activity can be captured in the tissue culture chip. Spontaneous activity of motor neurons in the spheroid and the axon bundle were observed within the MNO. Also, the neural activities were observed by using a multi-electrode array system.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61544/61544fig01.jpg" /> 
Figure 1: The dimension of PDMS tissue culture chip. 
(A) Photomask of the tissue culture chip. (B) Dimensions of microchannel in the tissue culture chip. The diameter of the base chamber for holding motor neuron spheroid is 2 mm and the hole of PDMS above the chamber is 1.5 mm. The width and height of a microchannel bridging two chambers are both 150 &#956;m. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61544/61544fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61544/61544fig02.jpg" /> 
Figure 2: Schematic illustration of motor neuron differentiation. 
(A) The differentiation steps involved neural induction, patterning into motor neuron lineage, and maturation of motor neurons. (B) Two options to create motor neuron spheroid (MNS) from iPS cells: 3D protocol, and a 2D protocol with dissociation step of motor neurons. Motor nerve organoid (MNO) can be obtained by both protocols. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61544/61544fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61544/61544fig03.jpg" /> 
Figure 3: Step by step protocol for basement membrane matrix coating and motor neuron spheroid introduction. 
(A) Basement membrane matrix coating in the channel of the tissue culture chip. (B) MNS introduction into the hole of the chip. (C) Culture medium change by aspiration of exhausted medium. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61544/61544fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61544/61544fig04.jpg" /> 
Figure 4: 2D and 3D motor neuron differentiation. 
(A) Time course of representative 3D MNS differentiation (3D protocol). The size of the MNS gradually increased over time. Scale bar: 500 &#956;m. (B) Time course of 2D differentiation on -D2, D0, D1, D6, D12 (2D protocol). Scale bar: 500 &#956;m. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61544/61544fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61544/61544fig05.jpg" /> 
Figure 5: Characterization of motor neurons. 
(A) (Left) A cryosection of a MNS stained with SMI-32 antibody and DAPI. (Middle) A phase-contrast image of replated MNS on a basement membrane matrix-coated surface. Axonal elongation was observed. (Right) Axons of replated MNS stained with Synapsin I and Tuj1 antibodies. Scale bar: 500 &#956;m (Left and Middle) and 50&#956;m (Right). (B) A representative image of 2D motor neurons immunostained with Tuj and HB9 antibodies. Scale bar: 200 &#956;m. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61544/61544fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61544/61544fig06.jpg" /> 
Figure 6: Characterization of a motor nerve organoid (MNO) generated in a culture chip. 
(A) Representative images of axon elongation and thick axon bundle formation on D32. Scale bar: 500 &#956;m. (B) Immunostaining of motor nerve organoid (MNO) by SMI-32 and DAPI. Axons and cell bodies can be isolated by physically cutting. Scale bar: 1 mm. (C) Purity of the protein from axons and cell bodies quantified by western blotting. MAP2, a dendritic marker, was not detected in axonal protein, whereas axonal marker Tau1 is enriched in the axonal protein. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61544/61544fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Three-Dimensional Motor Nerve Organoid Generation at JoVE.com

Three-Dimensional Motor Nerve Organoid Generation

This protocol provides a comprehensive procedure to fabricate human iPS cell-derived motor nerve organoid through spontaneous assembly of a robust bundle of axons extended from a spheroid in a tissue culture chip.

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and ...

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Universidad de Cartagena UDEC

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Neuroscience

9.0K Views.  The University of Tokyo. This protocol provides a comprehensive procedure to fabricate human iPS cell-derived motor nerve organoid through spontaneous assembly of a robust bundle of axons extended from a spheroid in a tissue culture chip.

Video: Three-Dimensional Motor Nerve Organoid Generation

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids represent useful models to study the mechanisms that control stem cell self-renewal and differentiation in mammals, including primary ciliogenesis and ciliary signaling. Primary ciliogenesis is the dynamic process of assembling the primary cilium, a key cell signaling center that controls stem cell self-renewal and/or differentiation in various tissues. Here we present a comprehensive protocol for the immunofluorescence staining of cell lineage and primary cilia markers, in whole-mount mouse mammary organoids, for light sheet microscopy. We describe the microscopy imaging method and an image processing technique for the quantitative analysis of primary cilium assembly and length in organoids. This protocol enables a precise analysis of primary cilia in complex three-dimensional structures at the single cell level. This method is applicable for immunofluorescence staining and imaging of primary cilia and ciliary signaling in mammary organoids derived from normal and genetically modified stem cells, from healthy and pathological tissues, to study the biology of the primary cilium in health and disease.

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Stem cell-derived organoids facilitate the analysis of molecular and cellular processes that regulate stem cell self-renewal and differentiation during organogenesis in mammalian tissues. Here we present a protocol for the analysis of the biology of the primary cilium in mouse mammary organoids.

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids ...

Genetics

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology has emerged recently. It is still in its infancy and has some limitations unsolved yet. The current protocols do not allow obtaining organoids to be consistent enough for drug discovery and preclinical studies. The maturation of organoids can take up to a year, pushing the researchers to launch multiple differentiation processes simultaneously. It imposes additional costs for the laboratory in terms of space and equipment. In addition, brain organoids often have a necrotic zone in the center, which suffers from nutrient and oxygen deficiency. Hence, most current protocols use a circulating system for culture medium to improve nutrition.
Meanwhile, there are no inexpensive dynamic systems or bioreactors for organoid cultivation. This paper describes a protocol for producing brain organoids in compact and inexpensive home-made mini bioreactors. This protocol allows obtaining high quality organoids in large quantities.

Here we describe a protocol for generating brain organoids from human induced pluripotent stem cells (iPSCs). To obtain brain organoids in large quantities and of high quality, we use home-made mini bioreactors.

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology ...

Developmental Biology

Innervation of Human Intestinal Organoids

The complexity of intestinal cytoarchitecture and function poses significant challenges for the creation of the bioengineered small intestine. Techniques for generating human intestinal organoids (HIOs) resembling human small intestine have been previously reported. HIOs contain epithelium and mesenchyme but lack other critical components of functional intestine such as the enteric nervous system (ENS), immune cells, vasculature, and microbiome. Two independent research groups have published distinct methods to innervate HIOs with an ENS. Here we discuss a unique method of incorporating the ENS into an HIO-derived bioengineered small intestine, which utilizes components of these prior reports to optimize progenitor cell identity as well as developmental timing.
Human pluripotent stem cells (hPSCs) are differentiated to independently generate HIOs and enteric neural crest cells (ENCCs) by temporal regulation of differentiation markers over a period of several days per published protocols. Once HIOs reach the mid-hindgut spheroid stage (approximately day 8), day 15-21 ENCC spheroids are dissociated, co-cultured with HIOs, and suspended within clear three-dimensional (3D) basement membrane matrix droplets. HIO + ENCC co-cultures are maintained in vitro for 28-40 days before transplantation into &#62;9-week-old immunodeficient mice for further development and maturation. Transplanted HIOs (tHIOs) with ENS can be harvested 4-20 weeks later. This method integrates elements from two previously published techniques by utilizing ENCCs generated from hPSCs and co-culturing them with HIOs at an early stage of development to maximize exposure to early developmental cues that likely contribute to the formation of a more mature intestinal morphology.

Human intestinal organoids must be innervated to better recapitulate the structure and function of the native human intestine. Here, we present one method for incorporating an enteric nervous system into these constructs.

The complexity of intestinal cytoarchitecture and function poses significant challenges for the creation of the bioengineered small intestine. Techniques ...

Generation of 3D Midbrain Organoids from Human-Induced Pluripotent Stem Cells

The development of midbrain organoids (MOs) from human pluripotent stem cells (hPSCs) represents a significant advancement in understanding brain development, facilitating precise disease modeling, and advancing therapeutic research. This protocol outlines a method for generating midbrain-specific organoids using induced pluripotent stem cells (iPSCs), employing a strategic differentiation approach. Key techniques include dual-SMAD inhibition to suppress SMAD signaling, administration of fibroblast growth factor 8b (FGF-8b), and activation of the Sonic Hedgehog pathway using the agonist purmorphamine, guiding iPSCs towards a midbrain fate.
The organoids produced by this method achieve diameters up to 2 mm and incorporate a diverse array of neuroepithelial cell types, reflecting the midbrain&#39;s inherent cellular diversity. Validation of these organoids as authentic midbrain structures involves the expression of midbrain-specific markers, confirming their identity. A notable outcome of this methodology is the effective differentiation of iPSCs into dopaminergic neurons, which are characteristic of the midbrain.
The significance of this protocol lies in its ability to produce functionally mature, midbrain-specific organoids that closely replicate essential aspects of the midbrain, offering a valuable model for in-depth exploration of midbrain developmental processes and the pathophysiology of related disorders such as Parkinson&#39;s disease. Thus, this protocol serves as a crucial resource for researchers seeking to enhance our understanding of the human brain and develop new treatments for neurodegenerative diseases, making it an indispensable tool in the field of neurological research.

This protocol describes a novel method for creating 3D midbrain organoids from human induced pluripotent stem cells, guiding their formation to mimic native midbrain tissue, thereby aiding in the study of development and disorders.

The development of midbrain organoids (MOs) from human pluripotent stem cells (hPSCs) represents a significant advancement in understanding brain ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Bioengineering

Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle contraction. In motor neuron disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), NMJs degenerate, resulting in muscle atrophy and progressive paralysis. The underlying mechanism of NMJ degeneration is unknown, largely due to the lack of translatable research models. This study aimed to create a versatile and reproducible in vitro model of a human motor unit with functional NMJs. Therefore, human induced pluripotent stem cell (hiPSC)-derived motor neurons and human primary mesoangioblast (MAB)-derived myotubes were co-cultured in commercially available microfluidic devices. The use of fluidically isolated micro-compartments allows for the maintenance of cell-specific microenvironments while permitting cell-to-cell contact through microgrooves. By applying a chemotactic and volumetric gradient, the growth of motor neuron-neurites through the microgrooves promoting myotube interaction and the formation of NMJs were stimulated. These NMJs were identified immunocytochemically through co-localization of motor neuron presynaptic marker synaptophysin (SYP) and postsynaptic acetylcholine receptor (AChR) marker &#945;-bungarotoxin (Btx) on myotubes and characterized morphologically using scanning electron microscopy (SEM). The functionality of the NMJs was confirmed by measuring calcium responses in myotubes upon depolarization of the motor neurons. The motor unit generated using standard microfluidic devices and stem cell technology can aid future research focusing on NMJs in health and disease.

We describe a method to generate human motor units in commercially available microfluidic devices by co-culturing human induced pluripotent stem cell-derived motor neurons with human primary mesoangioblast-derived myotubes resulting in the formation of functionally active neuromuscular junctions.

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle ...

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

In this study, we provide a detailed technique for a simple yet robust cortical organoid culture system using standard feeder-free hPSC cultures. This is a rapid, efficient, and reproducible protocol for generating organoids that model aspects of brain senescence in vitro.

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and ...

A High-Throughput Platform for Culture and 3D Imaging of Organoids

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by standard imaging approaches. This protocol describes a way to prepare microfabricated organoid culture chips, which enable multiscale, 3D live imaging on a user-friendly instrument requiring minimal manipulations and capable of up to 300 organoids/h imaging throughput. These culture chips are compatible with both air and immersion objectives (air, water, oil, and silicone) and a wide range of common microscopes (e.g., spinning disk, point scanner confocal, wide field, and brightfield). Moreover, they can be used with light-sheet modalities such as the single-objective, single-plane illumination microscopy (SPIM) technology (soSPIM). 
The protocol described here gives detailed steps for the preparation of the microfabricated culture chips and the culture and staining of organoids. Only a short length of time is required to become familiar with, and consumables and equipment can be easily found in normal biolabs. Here, the 3D imaging capabilities will be demonstrated only with commercial standard microscopes (e.g., spinning disk for 3D reconstruction and wide field microscopy for routine monitoring).

This paper presents a fabrication protocol for a new type of culture substrate with hundreds of microcontainers per mm2, in which organoids can be cultured and observed using high-resolution microscopy. The cell seeding and immunostaining protocols are also detailed.

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by ...

Biology

Generating Neuromuscular Junctions Using a Microfluidic Device

Source: Stoklund Dittlau, K. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/62959">Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices.</a>&#160;J. Vis. Exp. (2021)
The video demonstrates the formation of neuromuscular junctions using a microfluidic device. Neural precursor cells and muscle progenitor cells are seeded into different wells within the device and left to mature into motor neurons and myotubes, respectively. The creation of a volume and chemical gradient leads to the development of active neuromuscular junctions.

Generowanie połączeń nerwowo-mięśniowych za pomocą urządzenia mikroprzepływowego

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
All reagents and equipment used in ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Advanced Neuronal Models

Generation of Dopaminergic Neuronal Organoids from Human Embryonic Stem Cells

Source: Cosset, E. et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/59682">Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases.</a> J. Vis. Exp. (2019)
This video demonstrates the differentiation of human embryonic stem cells (hESCs) into dopaminergic neurons, to provide a model for studying Parkinson&#39;s disease. The hESCs are cultured in a neural induction medium in a microwell plate to form 3D neurospheres. These are then cultured in a neural induction medium to promote the differentiation into dopaminergic progenitor neurons. The final maturation of the neurons occurs in a culture insert under air-liquid interface conditions, resulting in 3D organoids consisting of mature dopaminergic neurons.

Wytwarzanie dopaminergicznych organoidów neuronalnych z ludzkich embrionalnych komórek macierzystych

1. hESC-derived dopaminergic organoids for Parkinson&#39;s Disease (PD) studies

	Day 0: Amplify hESCs in 2D culture up to 60% confluency (day 0), then ...

Three-Dimensional Motor Nerve Organoid Generation

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Rozdziały w tym wideo

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

Innervation of Human Intestinal Organoids

Generation of 3D Midbrain Organoids from Human-Induced Pluripotent Stem Cells

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

A High-Throughput Platform for Culture and 3D Imaging of Organoids

Generowanie połączeń nerwowo-mięśniowych za pomocą urządzenia mikroprzepływowego

Wytwarzanie dopaminergicznych organoidów neuronalnych z ludzkich embrionalnych komórek macierzystych