This work was supported by the Spastic Paraplegia Foundation, the Blazer Foundation and the NIH (R21NS109837).

1. Generation of telencephalic glutamatergic neurons from iPSCs
NOTE: The detailed protocol for maintaining iPSCs and their differentiation into telencephalic glutamatergic neurons are similar to those described previously18. Here, the critical process during the differentiation of human pluripotent stem cells is introduced and highlighted.
<ol>
	<li>Culture iPSCs on mouse embryonic fibroblast (MEF) feeders in human embryonic stem cell (hESC) medium supplemented with fibroblast growth factor (bFGF, 4 ng/mL).</li>
	<li>After incubation with dispase (1 mg/mL) for 3 min, dissociate the iPSCs into small clumps. Then, culture the iPSC aggregates in suspension in the hESC medium for 4 days. Refer to this timepoint, when the iPSCs are starting as the suspension culture, as day 1 (D1). Change the medium daily for 4 days.</li>
	<li>At D5, collect the iPSC aggregates after centrifugation at 200 x g for 2 min and culture in neural induction media (NIM) for 3 days. Culture the cell aggregates in suspension for 3 days by changing half of the media every 2 days. Add DMH-1 (2 &#956;M) and SB431542 (2 &#956;M) to NIM medium to increase neural induction efficiency.</li>
	<li>At D8, collect the iPSC aggregates after centrifugation at 200 x g for 2 min and let them adhere to 6 well plates (around 20&#8211;30 aggregates per well of the plate) by culturing in NIM with 10% FBS (or with laminin-coated plates) overnight. On the following day, remove the old medium and change to NIM every 2 days until D17. 
	NOTE: The generation of neuroepithelial cells, which is indicated by the formation of columnar cells with rosette structures, is observed in this period.</li>
	<li>At D17, mechanically isolate the neuroepithelial cells in the center of colonies (lifting off directly by applying a small pressure to the center of the colonies) or enzymatically (treated with dispase). Transfer the isolated neuroepithelial cells to non-treated tissue culture T25 flask containing 8 mL of NIM with addition of B27 (1x), cAMP (1 &#181;M), and IGF (10 ng/mL). 
	NOTE: The suspension culture allows cells to form neurospheres that are enriched with cortical projection neural progenitors.</li>
	<li>After D35, plate the neurospheres on the poly-ornithine and LDEV-free reduced growth factor basement membrane matrix (Table of Materials)-coated 35 mm glass bottom dishes to generate telencephalic glutamatergic neurons (cortical projection neurons). Before plating, dissociate the neurospheres to small clusters by incubating with 1 mg/mL cell detachment solution for 2 min at 37 &#176;C.</li>
	<li>Remove the cell detachment solution by centrifugation and resuspend in 1 mL of neural differentiation medium (NDM). Plate cells on the glass bottom dish (around five clusters in 100 &#181;L of medium per dish) to let them attach. Then, add NDM (1 mL per dish) and culture the cells in NDM containing B27 (1x), cAMP (1 &#181;M), IGF (10 ng/mL), hBDNF (10 ng/mL), and GDNF (10 ng/mL). 
	NOTE: Small clusters are plated because they survive well and give rise to long projection neurons. Alternatively, cells can be dissociated and plated at a density around 20,000 cells per dish for better separation.</li>
	<li>Characterize the telencephalic glutamatergic neurons by immunostaining the Tbr1 and &#946;III-tubulin markers.</li>
</ol>
2. Examination of mitochondrial transport along axons of telencephalic glutamatergic neurons
<ol>
	<li>Warm up the NDM and turn on the incubator for the fluorescence microscope set at 37 &#176;C and 5% CO2.</li>
	<li>To visualize mitochondria along the axons of forebrain neurons, incubate the neurons with 50 nM red fluorescent dye to stain mitochondria in live cells (e.g., MitoTracker CMXRos) in NDM for 3 min at 37 &#176;C. Next, wash cells 2x with warmed NDM. The neurons are incubated in NDM.</li>
	<li>In order to record the mitochondrial transport along the axons, take time-lapse images of mitochondrial movement using the 40x objective with a fluorescence microscope. 
	NOTE: To stabilize the culture, perform the live cell imaging when the cells are incubated in the incubator for 20 min following mitochondrial staining.</li>
	<li>Under phase field, identify the axons based on morphological characteristics (directly emerge from neuronal cell body, constant thin long neurites with no branching). Mitochondria move in anterograde (from cell body to distal axon) and retrograde directions (from axonal terminal to cell body). To distinguish the direction of mitochondrial movement along axons, clearly identify the cell bodies of neurons. In this step, focus axons under phase field to reduce photobleaching.</li>
	<li>After distinguishing the cell body and axon, adjust the exposure time and focus of mitochondria in axons. Then, capture the transport of mitochondria within axons every 5 s for a total duration of 5 min, yielding 60 frames. Randomly capture at least five locations for each dish and repeat 3x for each group.</li>
</ol>
3. Data analysis of mitochondrial transport and morphology in cortical neurons
NOTE: Analyze the collected data on mitochondrial transport using an image analysis software (e.g., ImageJ or MetaMorph19). Since ImageJ is readily available, perform the analysis of mitochondrial transport and morphology using ImageJ with the MultiKymograph, Macros, and Analyze particles plugins.
<ol>
	<li>Analyzing mitochondrial transport using ImageJ
	<ol>
		<li>After capturing the time-lapse images of mitochondrial motility, analyze the velocity and movement of mitochondria using the MultiKymograph plugin. The images are saved as .tiff format files, which are analyzed by Fiji software following a previously reported method20.</li>
		<li>Download the Fiji software. Download plugins that will be needed for analyzing mitochondrial moving velocity from kymographs. Download the Bio-formats Package and Kymograph Plugin along with these four .class plugins, including: MultipleKymograph.class, MultipleOverlay.class, StackDifference.class, and WalkingAverage.class (Table of Materials). Move these files to the Fiji plugins folder. Download &#34;tsp050706.txt&#34; plugins to the plugins folder. Restart the Fiji software when the files are moved to the plugins folder.</li>
		<li>Open Fiji software. Choose plugins, then import images of the .tiff series through Bio-Formats Importer under Bio-Formats. Make sure to choose Standard ImageJ in Stack viewing, choose Open all series, check Autoscale, then click OK. Take note of the frame number and size of images in pixels, which are displayed on the top of the image. 
		NOTE: Take 60 frames per image.</li>
		<li>Consider making the images clearer by adjusting brightness/contrast under the Image menu for all 60 frames.</li>
		<li>Right-click the line tool to choose segmented line and draw a segmented line starting from the cell body and ending at the terminal axon. Generate the kymograph by choosing MultipleKymograph under Plugins. The selection of line width is prompted after choosing the MultipleKymograph. Ensure that this is an odd number. Choose 1 for the line width. A kymograph is generated after this step. 
		NOTE: Several important pieces of information can be read from the kymograph. The y-axis of kymograph is the time of duration (5 min) for the 60 frames. The x-axis represents the position of the selected axons.</li>
		<li>Use a diagonal line in the kymograph to determine the movement direction of mitochondria (anterograde, retrograde, or stable). For example, a line going down to the right along the y-axis indicates anterograde movement, and a line going down to the left along the y-axis indicates retrograde movement. A vertical line indicates that there was no movement in the mitochondrion.</li>
		<li>Measure the distance, time values, and velocity for the moving mitochondria using the Macros plugin in Fiji software. Go to Plugins | Macros | Install | tsp050607.txt. Draw a segmented line over the trace of mitochondrial movement on the kymograph, and always draw the line from the superior to inferior region (y-axis).</li>
		<li>After drawing the line, go to Plugins | Macros | read velocities from tsp. Since a segmented line along the trace is drawn, the plugin reads segmented velocities corresponding to the line. 
		NOTE: The unit for all data is pixels. dy sum is the time consumed from the starting point, and dx sum indicates the distance of the mitochondrion moving in the x-axis. dy now and dx now shows the period time and movement distance for every segment, respectively. actual speed shows the speed for every segment (dx now/dy now). average speed indicates the average speed for the mitochondrial moving (dx sum/dy sum).</li>
		<li>Change the unit of dx now from pixel to &#181;m as the ratio of pixel in &#181;m by measuring the scale bar. Convert the unit for distance from pixel to &#181;m by measuring scale bars in images. Use the line tool to draw a line along the scale bar and measure the length of the line by choose Measure&#39; under &#34;Analyze. Change the time from pixel to seconds, as 1 pixel = 5 seconds in this experiment.</li>
		<li>In the spreadsheet file, calculate the average velocity and correspondingly label retrograde or anterograde movement. Average the anterograde or retrograde movement velocity.</li>
		<li>Determine the percentage of stationary and motile mitochondria from the kymograph. The anterograde or retrograde moving mitochondria are defined as moving 5 &#181;m forward or backward from the origin during the entire period21. Mitochondria are considered stationary if they did not move more than 5 &#181;m during the 5 min.</li>
	</ol>
	</li>
	<li>Analysis of mitochondrial morphology using ImageJ 
	NOTE: Determine mitochondrial morphology by measuring mitochondrial length, area, and aspect ratio using ImageJ. To do so, follow the steps below.
	<ol>
		<li>In order to analyze the mitochondrial length and area within axons, download the Straighten_.jar plugin from ImageJ website (see Table of Materials) and move it to the folder of plugins. Restart ImageJ software.</li>
		<li>Open the picture through the open function under File and convert the 32 bit image to 8 bit using Type under Image.</li>
		<li>Draw a segmented line along the axons. Choose Straighten under Plugins and set 50 pixels for Width of Filament/Wide Line. This will generate a straightened axon.</li>
		<li>Adjust the threshold under Image and set the measurement under Analyze by choosing &#34;Area | Perimeter | Fit ellipse | Shape descriptors.</li>
		<li>Measure the scale bar using the line function and set the scale under Analyze by filling the distance in pixel, know distance, and the unit of length. Then, choose Global to set this scale setting.</li>
		<li>Use Analyze Particles under Analyze to determine the area. The prompt parameters are size (pixel^2) = 0.20&#8211;infinity, circularity = 0.00&#8211;1.00, and show = ellipses. Choose Display results. 
		NOTE: The measurement will yield the results of multiple mitochondrial morphology parameters including area, perimeters, length (major), width (minor), and aspect ratio (AR). The corresponding mitochondrial number is also listed.</li>
		<li>Calculate the mitochondrial number per axon (&#181;m) using the mitochondrial number divided by axonal length.</li>
	</ol>
	</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+mitochondrial+transport+in+neurons.">Characterization of mitochondrial transport in neurons.</a> Methods in Enzymology. 547, 75-96 (2014).</li></ol>

The authors declare no competing financial interests.

This article describes a method to analyze mitochondrial transport and morphology in neuronal axons using red fluorescent dye and ImageJ software, both of which provide a unique platform to study axonal degeneration and mitochondrial morphology in neurodegenerative disease. There are several critical steps in the protocol, including staining of mitochondria, live cell imaging, and analyzing the images. In this method, a fluorescent dye was used to stain mitochondria. Since human iPSC-derived neurons are easily detached f...

Mitochondrial motility and distribution play a vital role in fulfilling variable and specialized energetic demands in polarized neurons. Neurons can extend extremely long axons to connect with targets through the formation of synapses, which demand high levels of energy for Ca2+ buffering and ion currents. Transport of mitochondria from soma to axon is critical for supporting axonal and synaptic function of neurons. Spatially and temporally dynamic mitochondrial movement is conducted by fast axonal transport at rates of several micrometers per second1.
Specifically, motor or adaptor proteins, such as kinesin and dynein, participate in the fast organelle transport along microtubules to control the movement of mitochondria2,3. Normal neuronal activity requires proper transport of newly assembled mitochondria from neuronal soma to distal axon (anterograde axonal transport) and reverse transport of mitochondria from the distal axon back to the cell body (retrograde transport). Recent studies have indicated that improper mitochondrial allocation is strongly associated with neuronal defects and motor neuron degenerative diseases4,5. Therefore, to dissect the role of mitochondria in neurodegeneration, it is important to establish methods for examining mitochondrial movement along axons in live cultures.
There are two main challenges in examining and analyzing the tracking of mitochondria: (1) identifying mitochondria from the background in every frame, and (2) analyzing and generating the connections between every frame. In resolving the first challenge, a fluorescence labeling approach is used widely to distinguish mitochondria from the background, such as MitoTracker dye or transfection of fluorescence-fused mitochondrial targeting protein (e.g., mito-GFP)6,7,8. To analyze the association between frames, several algorithms and software tools have been described in previous studies9. In a recent paper, researchers compared four different automated tools (e.g., Volocity, Imaris, wrMTrck, and Difference Tracker) to quantify mitochondrial transport. The results showed that despite discrepancies in track length, mitochondrial displacement, movement duration, and velocity, these automated tools are suitable for evaluating transport difference after treatment10. In addition to these tools, an integrated plugin &#34;Macros&#34; for ImageJ (written by Rietdorf and Seitz) has been widely used for analyzing mitochondrial transport11. This method generates kymographs that can be used to analyze mitochondrial movement, including velocity in both anterograde and retrograde directions.
Mitochondria are highly dynamic organelles that constantly change in number and morphology in response to both physiological and pathological conditions. Mitochondrial fission and fusion tightly regulate mitochondrial morphology and homeostasis. The imbalance between mitochondrial fission and fusion can induce extremely short or long mitochondrial networks, which can impair mitochondrial function and result in abnormal neuronal activities and neurodegeneration. Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases, such as Alzheimer&#39;s disease, Parkinson&#39;s disease, Huntington&#39;s disease, and hereditary spastic paraplegia (HSP)12,13,14,15. HSP is a heterogeneous group of inherited neurological disorders characterized by the degeneration of the corticospinal tract and subsequent failure to control lower limb muscles16,17. In this study, iPSC-derived forebrain neurons are used to assess mitochondrial transport and morphology in HSP. This method provides a unique paradigm for examining mitochondrial dynamics of neuronal axons in live cultures.

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			<td>Accutase Cell Detachment Solution</td>
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			<td>Glial derived neurotrophic factor (BDNF)</td>
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			<td>Knockout Serum Replacer</td>
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			<td>Laminin</td>
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			<td>2-Mercaptoethanol</td>
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			<td>Neurobasal medium</td>
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			<td>Non Essential Amino Acids</td>
			<td>Gibco</td>
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			<td>N2 NeuroPle Serum-Free Supplement</td>
			<td>Gemini</td>
			<td>400-163</td>
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			<td>Olympus microscope IX83</td>
			<td>Olympus</td>
			<td>IX83-ZDC2</td>
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			<td>PBS</td>
			<td>Corning</td>
			<td>21-031-CV</td>
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			<td>Phase contrast microscope</td>
			<td>Olympus</td>
			<td>CKX41/ IX2-SLP</td>
			<td></td>
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			<td>6 well plates</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
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			<td>24 well plates</td>
			<td>Corning</td>
			<td>353047</td>
			<td></td>
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			<td>Poly-L-ornithine hydrobromide (polyornithine))</td>
			<td>Sigma-Aldrich</td>
			<td>P3655</td>
			<td></td>
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		<tr>
			<td>SB431542</td>
			<td>Stemgent</td>
			<td>04-0010</td>
			<td></td>
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			<td>Sterile 50ml Disposable Vacuum Filtration System 0.22 &#956;m Millipore Express&#174; Plus Membrane</td>
			<td>Millipore</td>
			<td>SCGP00525</td>
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			<td>Stericup 500/1000 ml Durapore 0.22 &#956;M PVDF</td>
			<td>Millipore</td>
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			<td>Tbr1 antibody (1:2000)</td>
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			<td>Gibco</td>
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			<td>Phenix</td>
			<td>SS-PH50R</td>
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			<td>MultipleOverlay.class</td>
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			<td>WalkingAverage.class</td>
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			<td>StackDifference.class</td>
			<td><a href="https://www.embl.de/eamnet/html/body_kymograph.html" target="_blank">https://www.embl.de/eamnet/html/body_kymograph.html</a></td>
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			<td><a href="https://imagej.nih.gov/ij/plugins/straighten.html" target="_blank">https://imagej.nih.gov/ij/plugins/straighten.html</a></td>
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			<td>tsp050706.txt</td>
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analyzing mitochondrial transport and morphology in human induced pluripotent stem cell-derived neurons in hereditary spastic paraplegia

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human neurons, which may contribute to neurodegeneration in various disease states. Although it is challenging to examine mitochondrial dynamics in live human nerves, such paradigms are critical for studying the role of mitochondria in neurodegeneration. Described here is a protocol for analyzing mitochondrial transport and mitochondrial morphology in forebrain neuron axons derived from human induced pluripotent stem cells (iPSCs). The iPSCs are differentiated into telencephalic glutamatergic neurons using well-established methods. Mitochondria of the neurons are stained with MitoTracker CMXRos, and mitochondrial movement within the axons are captured using a live-cell imaging microscope equipped with an incubator for cell culture. Time-lapse images are analyzed using software with &#34;MultiKymograph&#34;, &#34;Bioformat importer&#34;, and &#34;Macros&#34; plugins. Kymographs of mitochondrial transport are generated, and average mitochondrial velocity in the anterograde and retrograde directions is read from the kymograph. Regarding mitochondrial morphology analysis, mitochondrial length, area, and aspect ratio are obtained using the ImageJ. In summary, this protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology to facilitate studies of neurodegenerative diseases.

Here, human iPSCs were differentiated into telencephalic glutamatergic neurons, which were characterized by immunostaining with Tbr1 and &#946;III tubulin markers (Figure 1A). To examine the axonal transport of mitochondria, these cells were stained with red fluorescent dye, and time-lapse imaging was performed. Since ImageJ is readily available and easier to obtain, mitochondrial transport was further analyzed with the &#34;MultiKymograph&#34; and &#34;Macros&#34; ImageJ pl...

Watch this Scientific Journal Video about Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia at JoVE.com

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...

analyzing-mitochondrial-transport-morphology-human-induced

Research

JoVE Journal

Neuroscience

7.7K Views.  University of Illinois College of Medicine Rockford. Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.

Video: Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven therapies for TBI have been developed. This paper presents a novel method for pre-clinical neurotrauma research intended to complement existing pre-clinical models. It introduces human pathophysiology through the use of human induced pluripotent stem cell-derived neurons (hiPSCNs). It achieves loading pulse duration similar to the loading durations of clinical closed head impact injury. It employs a 96-well format that facilitates high throughput experiments and makes efficient use of expensive cells and culture reagents. Silicone membranes are first treated to remove neurotoxic uncured polymer and then bonded to commercial 96-well plate bodies to create stretchable 96-well plates. A custom-built device is used to indent some or all of the well bottoms from beneath, inducing equibiaxial mechanical strain that mechanically injures cells in culture in the wells. The relationship between indentation depth and mechanical strain is determined empirically using high speed videography of well bottoms during indentation. Cells, including hiPSCNs, can be cultured on these silicone membranes using modified versions of conventional cell culture protocols. Fluorescent microscopic images of cell cultures are acquired and analyzed after injury in a semi-automated fashion to quantify the level of injury in each well. The model presented is optimized for hiPSCNs but could in theory be applied to other cell types.

Here we present a method for a human in vitro model of stretch injury in a 96-well format on a timescale relevant to impact trauma. This includes methods for fabricating stretchable plates, quantifying the mechanical insult, culturing and injuring cells, imaging, and high content analysis to quantify injury.

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven ...

Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a pre-assembled multi-compartment chip made in a cyclic olefin copolymer (COC) to compartmentalize neurons differentiated from human stem cells. The footprint of these COC chips are the same as a standard microscope slide and are equally compatible with high resolution microscopy. Neurons are differentiated from human neural stem cells (NSCs) into glutamatergic neurons within the chip and maintained for 5 weeks, allowing sufficient time for these neurons to develop synapses and dendritic spines. Further, we demonstrate multiple common experimental procedures using these multi-compartment chips, including viral labeling, establishing microenvironments, axotomy, and immunocytochemistry.

This protocol demonstrates the use of compartmentalized microfluidic chips, injection molded in a cyclic olefin copolymer to cultured neurons differentiated from human stem cells. These chips are preassembled and easier-to-use than traditional compartmentalized poly(dimethylsiloxane) devices. Multiple common experimental paradigms are described here, including viral labeling, fluidic isolation, axotomy, and immunostaining.

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a ...

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings.

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible piggyBac vectors.

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) pathophysiology in vitro. Multi-electrode array (MEA) recordings are a means to record electrical field potentials from large populations of neurons and analyze network activity over time. It was previously demonstrated that the presence of hiPSC-A that are differentiated using techniques to promote a spinal cord astrocyte phenotype improved maturation and electrophysiological activity of regionally specific spinal cord hiPSC-motor neurons (MN) when compared to those cultured without hiPSC-A or in the presence of rodent astrocytes. Described here is a method to co-culture spinal cord hiPSC-A with hiPSC-MN and record electrophysiological activity using MEA recordings. While the differentiation protocols described here are particular to astrocytes and neurons that are regionally specific to the spinal cord, the co-culturing platform can be applied to astrocytes and neurons differentiated with techniques specific to other fates, including cortical hiPSC-A and hiPSC-N. These protocols aim to provide an electrophysiological assay to inform about glia-neuron interactions and provide a platform for testing drugs with therapeutic potential in ALS.

We describe a method for differentiating spinal cord human induced pluripotent-derived astrocytes and neurons and their co-culture for electrophysiological recording.

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) ...