Name: analyzing mitochondrial transport and morphology in human induced pluripotent stem cell-derived neurons in hereditary spastic paraplegia
Uploaded: 2020-02-09T13:00:00
Description: 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

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 ...

<ol>
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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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>Accutase Cell Detachment Solution</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety hood</td>
			<td>Thermo Scientific</td>
			<td>1300 SERIES A2</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A-7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain derived neurotrophic factor (BDNF)</td>
			<td>Peprotech</td>
			<td>450-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>Sorvall Legend X1R/ 75004261</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Chemiglass Life Sciences</td>
			<td>1760-012</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclic AMP (cAMP)</td>
			<td>Sigma-Aldrich</td>
			<td>D0627</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td></td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>Selleckchem</td>
			<td>S7146</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle medium with F12 nutrient mixture (DMEM/F12)</td>
			<td>Corning</td>
			<td>10-092-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>16141-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast growth factor 2 (FGF2, bFGF)</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix</td>
			<td>Gibco</td>
			<td>A1413201</td>
			<td></td>
		</tr>
		<tr>
			<td>Gem21 NeuroPlex Serum-Free Supplement</td>
			<td>Gemini</td>
			<td>400-160</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Dishes</td>
			<td>MatTek</td>
			<td>P35G-0.170-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>9&#39;&#39; glass pipetes</td>
			<td>VWR</td>
			<td>14673-043</td>
			<td></td>
		</tr>
		<tr>
			<td>Glial derived neurotrophic factor (BDNF)</td>
			<td>Sigma-Aldrich</td>
			<td>D0627</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX-I</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin growth factor 1 (IGF1)</td>
			<td>Invitrogen</td>
			<td>M7512</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout Serum Replacer</td>
			<td>Gibco</td>
			<td>A31815</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma</td>
			<td>L-6274</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker CMXRos</td>
			<td>Invitrogen</td>
			<td>M7512</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Non Essential Amino Acids</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 NeuroPle Serum-Free Supplement</td>
			<td>Gemini</td>
			<td>400-163</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus microscope IX83</td>
			<td>Olympus</td>
			<td>IX83-ZDC2</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Phase contrast microscope</td>
			<td>Olympus</td>
			<td>CKX41/ IX2-SLP</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well plates</td>
			<td>Corning</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-ornithine hydrobromide (polyornithine))</td>
			<td>Sigma-Aldrich</td>
			<td>P3655</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Stemgent</td>
			<td>04-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 50ml Disposable Vacuum Filtration System 0.22 &#956;m Millipore Express&#174; Plus Membrane</td>
			<td>Millipore</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup 500/1000 ml Durapore 0.22 &#956;M PVDF</td>
			<td>Millipore</td>
			<td>SCGVU10RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Tbr1 antibody (1:2000)</td>
			<td>Chemicon</td>
			<td>AB9616</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin inhibitor</td>
			<td>Gibco</td>
			<td>17075029</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>Phenix</td>
			<td>SS-PH50R</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>Phenix</td>
			<td>SS-PH15R</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 flasks (untreated)</td>
			<td>VWR</td>
			<td>10861-572</td>
			<td></td>
		</tr>
		<tr>
			<td>Plugins for softwares</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-formats Package</td>
			<td><a href="http://downloads.openmicroscopy.org/bio-formats/5.1.0/" target="_blank">http://downloads.openmicroscopy.org/bio-formats/5.1.0/</a></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji software</td>
			<td><a href="https://fiji.sc/" target="_blank">https://fiji.sc/</a></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kymograph Plugin</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>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MultipleKymograph.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>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MultipleOverlay.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>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>WalkingAverage.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>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<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>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Straighten_.jar</td>
			<td><a href="https://imagej.nih.gov/ij/plugins/straighten.html" target="_blank">https://imagej.nih.gov/ij/plugins/straighten.html</a></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tsp050706.txt</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>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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 ...

Mitochondrial dysfunction underlies many neurodegenerative diseases. Our protocol provides an important tool for examining mitochondrial dynamics in the axons, facilitating the study of neurological diseases involving axonal degeneration. By combining mitochondrial labeling, live cell imaging and induced pluripotent stem cell technology our protocol can be used to characterize mitochondrial trafficking along human axons and to analyze their morphologies.Impaired mitochondrial transport and morphology can be observed in stem cell cultures and animal models of neurodegenerative diseases providing potential therapeutic targets for treating these diseases. After day thirty five of culture, dissociate the neurospheres differentiated from human induced pluripotent stem cells into small clusters with 1 mg/ml of cell detachment solution for two minutes at 37 degrees Celsius. At the end of the incubation collect the cell clusters by centrifugation and resuspend the pellet in one milliliter of NDM.Plate about five clusters of cells in one hundred microliters of medium per polyornithine and lactose dehydrogenase elevating virus-free reduced growth factor basement membrane matrix coated 35 millimeter glass bottom dishes. Then add one milliliter of NDM supplemented with B27, cyclic adenosine monophosphate, insulin-like growth factor, human brain-derived neurotrophic factor and glial cell-derived neurotrophic factor to each culture. To visualize mitochondria along the axons of forebrain neurons first stain the neurons with 50 nanomolar red fluorescent dye in NDM for three minutes at 37 degree Celsius followed by two washes in warm NDM.Next, place the culture on the stage of a fluorescence microscope and select the 40X objective. Under the phase-field identify the axons based on their morphological characteristics. To distinguish the direction of the mitochondrial movement along the axons clearly identify the cell bodies of neurons.After distinguishing the cell body and axon adjust the exposure time and focus of the mitochondria in the axons and capture the mitochondrial transport within the axons every five seconds for a total of five minutes. To analyze mitochondrial transport in ImageJ open Fiji and select the Bio-Formats plugin. Use the Bio-Formats importer to import the time-lapse images of the tiff series and select standard ImageJ and Open all series.Check autoscale split channels and click OK taking care to note the frame number and size of the images in pixels. After adjusting the brightness and contrast for all 60 frames use the segmented line to draw a line from the cell body to the terminal axon. To generate the Kymograph select the multiple Kymograph plugin and select the line width.To measure the distance, time values and velocity for the moving mitochondria open tsp050607. txt in the macros plugin. And draw a segmented line over the trace of mitochondrial movement on the kymograph from the superior to the inferior region.After drawing the line open read velocities from tsp in the macros plugin to read the segmented velocities corresponding to the line. Next open the original image and use the line tool to draw a line along the scale bar. Select analyze to measure the length of the line and convert the dx now and the distance units from pixels to micrometers.Then change the time from pixel to seconds. To analyze the mitochondrial length and area within axons in ImageJ install the straighten underscore dot JAR plugin and open the axon image of interest. Convert the 32 bit image to eight bit and use the segmented line tool to trace the axon.Select the Straighten underscore JAR plugin and set the width of filament wide line to 50 pixels. Trace the axon again to generate a straightened axon and adjust the image threshold. Select analyze set measurements perimeter fit ellipse and shape descriptors to set the measurement and use the line function to measure the scale bar in the original image as demonstrated.Under analyze and to set scale enter the distance in pixels known distance and the unit of length to set the scale. Then select global to set the scale setting to all of the images. Finally select analyze and analyze particles to determine the area and select display results to view the resulting measurement.After differentiation into telencephalic glutamatergic neurons the cells can be characterized by their T-B-R one and beta three tubulin marker expression. Staining with red fluorescent dye and time-lapse imaging allows evaluation of the axonal transport of mitochondria. A single mitochondrion can remain static or move in an anterograde or retrograde direction within an axon.The velocity of the mitochondrial movement along the axon can be quantified as illustrated. For example using commercially available analysis software the percentage of motile mitochondria is significantly reduced in S-P-G-3-A neurons compared to wild type neurons. To analyze the mitochondrial area length and aspect ratio axons can straightened in the image analysis software and selected by adjusting the threshold.The mitochondrial area perimeter length width and aspect ratio can then be obtained from the straightened axon. For example both the mitochondrial length and the aspect ratio are significantly reduced in S-P-G 15 neuron axons compared to control wild type axons. It is important to warm the medium and the microscope incubator to wash the cells gently and to allow the cells to stabilize for twenty minutes before imaging.After live cell imaging the cultures can be fixed and subjected to immunostaining to examine the expression of other proteins of interest. It is challenging to examine mitochondrial dynamics in live human nerves. This technique provides the unique tool for the study of mitochondrial dynamics and nerve degeneration in neurological disease.

analyzing-mitochondrial-transport-morphology-human-induced

Research

JoVE Journal

Neuroscience

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) ...