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Ethic approvals were obtained when performing the experiments.
1. Cell Culture and Reagent Preparation
<ol>
	<li>Prepare coated plates for cell culture.
	<ol>
		<li>Dilute extracellular matrix (ECM) coating solution with Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) media to prepare a 1 mg/mL stock solution. Aliquot the diluted ECM stock solution into 30 conical tubes of 3 mL each and immediately store in -20 &#176;C. For a working solution, resuspend 3 mL of ECM stock into 33 mL of pre-chilled DMEM/F12 media, bringing the total volume to 36 mL for a concentration of 80 &#181;g/mL.</li>
		<li>Coat 1 mL per well of a 6-well cell culture plate with ECM working solution. Add an additional 1 mL DMEM/F12 per well (if necessary) to ensure that the surface of the well is fully covered. Allow the coated 6-well cell culture plates to sit at room temperature for at least 1 h before use. 
		NOTE: Coated plates may be stored in the incubator or at 4 &#176;C for up to 2 weeks.</li>
	</ol>
	</li>
	<li>Prepare media formulations, growth factors, and small molecules.
	<ol>
		<li>To prepare 500 mL of human pluripotent stem cell (hPSC) medium, add 20x and 500x supplements according to manufacturers&#39; instructions.</li>
		<li>To prepare 500 mL of neural medium (NM), add heparin to a final concentration of 2 mg/mL, 1x of antibiotic/antimycotic solution, 1x B27 supplement or 1x N2 supplement to a 500 mL bottle of DMEM/F12 with L-glutamine supplement as previously detailed.</li>
		<li>To prepare a 10 mM (1,000x) stock solution of Rho-Kinase Inhibitor Y27632 (Y), add 10 mg of powder into 3 mL of phosphate buffered saline (PBS). Filter sterilize, aliquot, and store at -20 &#176;C. Use at a 10 &#181;M working concentration in media.</li>
		<li>To prepare a 20 mM (10,000x) stock solution of SB431542, add 10 mg of powder into 1.3 mL of dimethyl sulfoxide (DMSO). To prepare a 2 mM (1,000x) stock solution of DMH1 (4-[6-(4-Isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline, 4-[6-[4-(1-Methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), add 10 mg of powder into 13.1 mL of DMSO. Filter sterilize, aliquot, and store each solution at -20 &#176;C. Use each at a 2 &#181;M working concentration in media (NM + SB431542 + DMH1). 
		NOTE: This protocol recommends 2 &#181;M working concentrations of both SB431542 and DMH1 based upon our observations and reports from others&#160;stating that this concentration effectively promotes neural induction from hPSCs. Higher concentrations have also been reported. Additionally, with alternative media, it has also been shown that small molecules are not necessary for high neural conversion. Thus, working concentrations will vary depending on alternative cell culture environments, and optimal concentrations should be tested by individual researchers.</li>
		<li>To prepare a stock solution of doxycycline hydrochloride (Dox), first dissolve powder in DMSO to 100 mg/mL and store at -20 &#176;C. Further dilute it to make a 2 mg/mL (1,000x) stock solution in PBS and store at -20 &#176;C. Use at 2 &#181;g/mL working concentration in media;&#160;e.g., add 50 &#181;L Dox to 50 mL of NM (NM + Dox). 
		Note: Do not re-freeze solutions after thawing.</li>
	</ol>
	</li>
</ol>
2. Generation of Neural Subtypes from Human Induced Pluripotent Cells (hPSCs)
NOTE: All cell cultures should be maintained in an incubator with 5% CO2&#160;at 37 &#176;C. These cultures are maintained at room oxygen levels, though lower levels may be utilized.
<ol>
	<li>Generate and maintain astrocyte progenitors (hAstros) from hPSCs. 
	NOTE: This section provides a brief and simplified differentiation protocol. (Figure 1A, dotted green box).
	<ol>
		<li>Seed clusters of hPSCs (Figure 1B) onto ECM-coated 6-well plates with 2 mL of hPSC medium + Y per well. Feed the stem cells every day until they are about 50% confluent and split as needed.
		<ol>
			<li>To split hPSCs, add 1 mL of passaging reagent to wells and aspirate after 1 min. Incubate cells at room temperature for 5 min, add 1 mL of hPSC medium, and split aggregates 1:10 onto new ECM-coated wells in 2 mL of hPSC medium + Y per well.</li>
		</ol>
		</li>
		<li>Maintain the stem cells until they are about 50% confluent, then change media to NM + SB431542 + DMH1 to induce neural differentiation (day 0). When cells are about 95% confluent, split 1:6 into new ECM-coated wells in the same medium.</li>
		<li>On day 14, dissociate the cells with detachment solution and transfer to a non-coated flask with Y to promote formation of aggregates. 
		NOTE: The addition of Y is utilized to promote cell survival and sphere formation but is not included during every feed.</li>
		<li>For the generation of neuronal progenitors and neurons (hNeurons), use these day-14 cells to differentiate as described for transgenic cells.</li>
	</ol>
	</li>
	<li>Generate and maintain inducible neurons (iNeurons) from hPSCs.&#8203;
	<ol>
		<li>Seed transgenic hPSCs (with a stable doxycycline-inducible neurogenin 2 transgene) on ECM-coated 6-well plates with 2 mL of hPSC medium + Y per well (as described above for non-transgenic lines). Feed everyday with 2 mL of media per well until they are ready to lift at 70% confluency. Split cells as described above.</li>
		<li>When cells are about 35% confluent, add NM + Dox to induce differentiation into iNeurons. 
		NOTE: Cells will transition into neuronal progenitors but will not yet extend neurites.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6 well plate</td>
			<td>Fisher Scientific</td>
			<td>08-772-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Olympus Plastics</td>
			<td>28-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Sigma</td>
			<td>A6964-100ML</td>
			<td>Detachment solution</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Thermofisher</td>
			<td>17504044</td>
			<td>Media Supplement</td>
		</tr>
		<tr>
			<td>BrainPhys neuronal medium</td>
			<td>Stemcell Technologies</td>
			<td>5790</td>
			<td>Neurophysiological basal medium alternative</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermofisher</td>
			<td>10565-042</td>
			<td>With GlutaMAX supplement</td>
		</tr>
		<tr>
			<td>DMH-1</td>
			<td>Stemcell Technologies</td>
			<td>73634</td>
			<td>HAZARD: Toxic if swallowed. Working concentration: 2 &#956;M</td>
		</tr>
		<tr>
			<td>Doxycycline Hydrochloride (Dox)</td>
			<td>Sigma</td>
			<td>D3072-1ml</td>
			<td>HAZARD: Toxic for pregnant women. Working concentration: 2 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Hemacytometer or automatic cell counter</td>
			<td>Life Technologies</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149-50KU</td>
			<td>Working concentration: 2 mg/mL</td>
		</tr>
		<tr>
			<td>Matrigel membrane matrix</td>
			<td>Corning</td>
			<td>354230</td>
			<td>ECM coating solution. Working concentration: 80 &#956;g/ml. Prepare on ice and ensure that pipettes, tubes, and media are pre-chilled.</td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Thermofisher</td>
			<td>17502048</td>
			<td>Media Supplement</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Stemcell Technologies</td>
			<td>CA008-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-l-ornithine (PLO)</td>
			<td>Sigma</td>
			<td>P3655-100MG</td>
			<td>Working concentration: 0.5 mg/mL</td>
		</tr>
		<tr>
			<td>ReLeSR</td>
			<td>Stemcell Technologies</td>
			<td>5872</td>
			<td>Detachment and passaging reagent</td>
		</tr>
		<tr>
			<td>Rho-Kinase Inhibitor Y27632- (Y)</td>
			<td>Tocris</td>
			<td>1254</td>
			<td>Working concentration: 10 uM</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Stemcell Technologies</td>
			<td>72234</td>
			<td>Working concentration: 2 &#956;M</td>
		</tr>
		<tr>
			<td>T25 Culture Flask</td>
			<td>Olympus Plastics</td>
			<td>25-207</td>
			<td>Vented caps</td>
		</tr>
		<tr>
			<td>T75 Culture Flask</td>
			<td>Olympus Plastics</td>
			<td>25-209</td>
			<td>Vented caps</td>
		</tr>
		<tr>
			<td>TeSR-E8 basal medium</td>
			<td>Stemcell Technologies</td>
			<td>5940</td>
			<td>Human pluripotent stem cell (hPSC) medium</td>
		</tr>
		<tr>
			<td>TeSR-E8 supplements</td>
			<td>Stemcell Technologies</td>
			<td>5940</td>
			<td>Supplements for human pluripotent stem cell medium</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Invitrogen</td>
			<td>T10282</td>
			<td></td>
		</tr>
	</tbody>
</table>

differentiating non-transgenic and transgenic human pluripotent stem cells into neural progenitor cells

Source: Cvetkovic, C., et al. <a href="https://app.jove.com/v/58034">Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells.</a> J. Vis. Exp. (2018).
This video demonstrates the generation of neural progenitor cells from non-transgenic and transgenic human pluripotent stem cells (hPSCs). hPSCs are differentiated into neural progenitor cells using a neuronal differentiation medium. While differentiation is induced in non-transgenic cells using small molecules, the transgenic hPSCs are differentiated by adding an antibiotic that induces the promoter for a neuronal differentiation-specific transcription factor.

<img alt="Figure 1" src="/files/ftp_upload/22391/22391_Figure1.jpg" /> 
Figure 1: Stepwise depiction of the differentiation and formation of 3D neural spheres derived from hPSCs.&#160;(A) Timeline of key steps in the protocol. (B) Pure populations of neural cells can be generated from human induced pluripotent stem cells (hPSCs). (C) Inducible neurons (iNeurons; see section 2.2) generated from transgenic hPSCs via in...

Differentiating Non-Transgenic and Transgenic Human Pluripotent Stem Cells into Neural Progenitor Cells

Ethic approvals were obtained when performing the experiments.
1. Cell Culture and Reagent Preparation

	Prepare coated plates for cell culture.
	
		...

Take a multi-well plate coated with an extracellular matrix, or ECM.
Into separate wells, introduce non-transgenic and transgenic human pluripotent stem cells, or hPSCs, in a culture medium.
Transgenic hPSCs carry a transgene under an antibiotic-inducible promoter. The transgene encodes a transcription factor that induces neural differentiation.
Incubate to facilitate cell adhesion to the ECM. Small molecules in the medium prevent apoptosis, promoting cell proliferation.
Add a detachment solution to dissociate the cells; add the medium to stop dissociation, and then transfer them to another ECM-coated microplate to allow cell proliferation.
Introduce a neural differentiation medium containing small molecules onto the non-transgenic cells and the inducer antibiotic onto the transgenic cells.
The small molecules induce non-transgenic cells to differentiate into neural progenitor cells or NPCs. In the transgenic cells, the antibiotic-induced expression of the transcription factor activates the genes required for differentiation into NPCs.
Maintain the NPCs in culture for downstream applications.

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Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

30 Views. Source: Cvetkovic, C., et al. Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells. J. Vis. Exp. (2018). This video demonstrates the generation of neural progenitor cells from non-transgenic and transgenic human pluripotent stem cells (hPSCs). hPSCs are differentiated into neural progenitor cells using a neuronal differentiation medium. While differentiation is induced in non-transgenic cells using small molecules, the transgenic hPSCs ar...

Video: Differentiating Non-Transgenic and Transgenic Human Pluripotent Stem Cells into Neural Progenitor Cells - Concept

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical movement. The architecture of the NMJ structure influences the functions of the neuron, the muscle and the mutual interaction. Previous studies have reported many strategies by co-culturing the motor neurons and myotubes to generate NMJ in vitro with complex induction process and long culture period but have struggled to recapitulate mature NMJ morphology and function. Our in vitro NMJ induction system is constructed by differentiating human iPSC in a single culture dish. By switching the myogenic and neurogenic induction medium for induction, the resulting NMJ contained pre- and post- synaptic components, including motor neurons, skeletal muscle and Schwann cells in the one month culture. The functional assay of NMJ also showed that the myotubes contraction can be triggered by Ca++ then inhibited by curare, an acetylcholine receptor (AChR) inhibitor, in which the stimulating signal is transmitted through NMJ. This simple and robust approach successfully derived the complex structure of NMJ with functional connectivity. This in vitro human NMJ, with its integrated structures and function, has promising potential for studying pathological mechanisms and compound screening.

Here we provide a protocol to generate in vitro NMJs from human induced pluripotent stem cells (iPSCs). This method can induce NMJs with mature morphology and function in 1 month in a single well. The resulting NMJs could potentially be used to model related diseases, to study pathological mechanisms or to screen drug compounds for therapy.

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical ...

JoVE Journal

Developmental Biology

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

Differentiating Non-Transgenic and Transgenic Human Pluripotent Stem Cells into Neural Progenitor Cells

Transcript

Differentiating Non-Transgenic and Transgenic Human Pluripotent Stem Cells into Neural Progenitor Cells

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions