Name: three-dimensional motor nerve organoid generation
Uploaded: 2020-09-24T13:00:00
Duration: 9 min 57 s
Description: Watch this Scientific Journal Video about Three-Dimensional Motor Nerve Organoid Generation at JoVE.com

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&#181;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://www.jove.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://www.jove.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://www.jove.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://www.jove.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://www.jove.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://www.jove.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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