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1. HiPSC Differentiation into Mixed Neurons and Glia
NOTE: The procedure takes approximately 28 days to complete, with the main steps outlined in Figure 1.
<ol>
	<li>Generation of embryoid bodies (EBs) (Days 0 &#8594; 1) 
	NOTE: This procedure requires good manual skills and precision. HiPSC (human induced pluripotent stem cells) colony fragments should be of equal size to obtain homogenous embryoid bodies (EBs) in the next steps. Morphologically differentiated colonies (with large cytoplasmic fractions and small nucleoli) should be discarded.
	<ol>
		<li>Refresh the hiPSC medium (3 mL/60-mm Petri dish) prior to cutting the undifferentiated hiPSC colonies (about 1 mm in diameter, see Figure 2A) under sterile conditions.</li>
		<li>Cut the undifferentiated colonies (as shown in Figure 2A and Figure 2B) into fragments of approximately 200 &#181;m x 200 &#181;m using a 1-mL syringe with a 30G needle. Use a stereoscopic microscope at 4X magnification in a laminar flow cabinet at room temperature.</li>
		<li>Detach the colony fragments from the dish surface using a 200-&#181;L pipette by gently pipetting medium underneath to lift the pieces.</li>
		<li>Transfer all detached fragments and the medium to a 15-mL tube using a 1, 2, or 5 mL pipette.</li>
		<li>Rinse the dish with 2 mL of complete hiPSC medium to recover all fragments.</li>
		<li>Centrifuge at 112 x g for 1 min.</li>
		<li>Aspirate the supernatant and gently resuspend the fragments in 5 mL of complete hiPSC EB medium (see the Table of Materials).</li>
		<li>Plate the colony fragments in a 60 mm ultra-low attachment Petri dish (5 mL/60-mm Petri dish).</li>
		<li>Incubate the Petri dish overnight at 37 &#176;C and 5% CO2.</li>
		<li>On the next day (Day 1), collect the EBs and their medium in a 15 mL tube using a 1, 2, or 5 mL pipette.</li>
		<li>Centrifuge the EBs at 112 x g for 1 min.</li>
		<li>Carefully aspirate the supernatant and gently resuspend the EBs in 5 mL of complete hiPSC EB medium using a 1, 2, or 5 mL pipette.</li>
		<li>Replate the EBs onto a new 60-mm ultra-low attachment Petri dish (5 mL/60 mm Petri dish).</li>
		<li>Incubate the Petri dish overnight at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>On Day 1, coat the dishes with basement membrane matrix (e.g., matrigel, referred to hereafter as &#34;standard matrix&#34;) or any other suitable protein substrate (e.g., laminin).
	<ol>
		<li>Store the standard matrix (see the Table of Materials) at -80 &#176;C in 200-&#181;L aliquots using cold 1.5 mL tubes and cold 5 or 10 mL pipettes.</li>
		<li>Thaw 200 &#181;L of standard matrix on ice.</li>
		<li>Dilute 200 &#181;L of standard matrix in 20 mL of basal (DMEM/F12) medium (1:100 dilution).</li>
		<li>Coat 60-mm Petri dishes with this solution (5 mL/dish).</li>
		<li>Incubate the coated dishes at 37 &#176;C overnight. 
		NOTE: These dishes will be used to plate the EBs (about 50 EBs/dish) and generate neuroepithelial aggregates (rosettes); see step 1.3.</li>
	</ol>
	</li>
	<li>Generation of neuroepithelial aggregates (rosettes) (Days 2 &#8594; 7)
	<ol>
		<li>On Day 2, remove the standard matrix coating solution from the 60-mm Petri dishes (no need to rinse the plates) and fill them with 5 mL/dish of complete neuroepithelial induction medium (NRI); see the Table of Materials.</li>
		<li>Transfer the floating EBs (from step 1.1.14) to coated dishes (~50 EBs/dish) using a 200 &#181;L pipette under a stereoscopic microscope at 4x magnification and placed in a laminar flow cabinet. 
		NOTE: It is critical to select homogenous, medium-sized EBs (~200-300 &#181;m in diameter). Too-small EBs may not survive well during neuroectodermal differentiation, while too-large EBs tend to undergo core necrosis.</li>
		<li>Incubate the dishes at 37 &#176;C and 5% CO2.</li>
		<li>The day after (Day 3), check the dishes under the microscope at 10x magnification to ensure that the EBs are all attached.</li>
		<li>Gently perform a total medium change with a complete NRI medium.</li>
		<li>Change the NRI medium every other day up to day 7, when neuroepithelial aggregates (rosettes) should be visible.</li>
		<li>On Day 7, coat standard matrix (or laminin), as described in step 1.2, onto any required plate or dish format: 96-well plates (100 &#181;L/well), 24-well plates (250 &#181;L/well), 12-well plates (500 &#181;L/well), MEA chips (for electrical activity, 1 mL/single-well chip), or 60 mm Petri dishes (4 mL/dish).</li>
		<li>Incubate the coated plates/dishes for at least 2 h at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>Rosette dissociation and neuronal differentiation (Days 8 &#8594; 28) 
	NOTE: This procedure requires good manual skills and precision. To avoid collecting mesodermal and endodermal cells, only ectoderm rosette-like structures should be dissociated and collected.
	<ol>
		<li>On Day 8, cut the rosette-like structures into fragments under a stereoscopic microscope at 10X magnification in sterile conditions. Use a 1 mL syringe with a 30G needle. Note that the rosettes tend to detach from the dish when touched with the needle easily.</li>
		<li>Complete the detachment of the rosette fragments using a 200-&#181;L pipette.</li>
		<li>Transfer the dish under the laminar flow hood and collect the rosette fragments and their medium into a 15 mL conical tube using a 1, 2, or 5 mL pipette. Rinse the dish with 2 mL of NRI medium to recover all fragments.</li>
		<li>Spin down the rosette fragments at 112 x g for 2 min.</li>
		<li>Aspirate the supernatant.</li>
		<li>Gently resuspend the pellet in 1 mL of 1x DPBS (without calcium and magnesium) and gently pipette the rosette fragments up and down using a 1,000-&#181;L pipette to partially dissociate them.</li>
		<li>Add 4 mL of complete NRI medium and count the cells using trypan blue and an automated cell counter (see the Table of Materials) 
		NOTE: Dilute 20 &#181;L of cell suspension in 20 &#181;L of Trypan blue. This step may be omitted if the cells cannot be brought into a single-cell suspension. If the rosette fragments do not look completely dissociated, rosette fragments derived from about 50 EBs/60-mm dish can be resuspended in 50 mL of complete NRI medium and plated, as indicated in Table 1.</li>
		<li>Aspirate the standard matrix (or laminin) coating solution from the Petri dishes, plates, and/or MEA chips (from step 1.3.7). Do not let them dry.</li>
		<li>Plate the cells in a complete NRI medium according to the study plan (about 15,000 cells/cm2; see Table 1 for plating volume indications).</li>
		<li>Incubate the plates overnight at 37 &#176;C and 5% CO2.</li>
		<li>On Day 10, perform a total medium change using a complete neuronal differentiation medium (ND); see the Table of Materials.</li>
		<li>Refresh the complete ND medium twice a week until Day 28.</li>
	</ol>
	</li>
</ol>
Table 1.
Note on dissociated rosette plating density: if rosette fragments do not look completely dissociated, in order to reach a cell plating density of about 15.000 cells/cm2, dissociated rosette fragments deriving from about 50 EBs/1 x 60mm-dish can be resuspended in 50 mL of complete NRI medium and plated as follows (depending on the plate format):
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Multiwell plates / MEA</td>
			<td>Growth area (cm2/well)</td>
			<td>Volume of the cell suspension to plate per well (or MEA chip)</td>
			<td>Maximum number of plates that can be plated (with 50 ml of cell suspension)</td>
		</tr>
		<tr>
			<td>96 wells</td>
			<td>0.3</td>
			<td>100 ul</td>
			<td>5</td>
		</tr>
		<tr>
			<td>48 wells</td>
			<td>0.7</td>
			<td>220 ul</td>
			<td>4</td>
		</tr>
		<tr>
			<td>24 wells</td>
			<td>2</td>
			<td>625 ul</td>
			<td>3</td>
		</tr>
		<tr>
			<td>12 wells</td>
			<td>4</td>
			<td>1.25 ml</td>
			<td>3</td>
		</tr>
		<tr>
			<td>6 wells</td>
			<td>10</td>
			<td>3.125 ml</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Single well MEA chip</td>
			<td>3.5</td>
			<td>1.1 ml</td>
			<td>45</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>hiPSC EB medium:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>Thermo-Fisher</td>
			<td>10829-018</td>
			<td>(Step 2.1.7)</td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement (KOSR)</td>
			<td>Thermo-Fisher</td>
			<td>10828-028</td>
			<td>20% final concentration (Step 2.1.7)</td>
		</tr>
		<tr>
			<td>Non-Essential Amino Acids</td>
			<td>Thermo-Fisher</td>
			<td>11140-035</td>
			<td>(Step 2.1.7)</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo-Fisher</td>
			<td>15140-122</td>
			<td>50 U/mL final concentration (Step 2.1.7)</td>
		</tr>
		<tr>
			<td>L-Glutamine 200 mM Solution</td>
			<td>Thermo-Fisher</td>
			<td>25030-081</td>
			<td>2 mM final concentration (Step 2.1.7)</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Thermo-Fisher</td>
			<td>31350-010</td>
			<td>50 &#181;M final concentration (Step 2.1.7)</td>
		</tr>
		<tr>
			<td>Complete neuroepithelial induction medium (NRI):</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo-Fisher</td>
			<td>3133-038</td>
			<td>(Step 2.3.1)</td>
		</tr>
		<tr>
			<td>Non-Essential Amino Acids</td>
			<td>Thermo-Fisher</td>
			<td>11140-035</td>
			<td>(Step 2.3.1)</td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>Thermo-Fisher</td>
			<td>17502-048</td>
			<td>(Step 2.3.1)</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo-Fisher</td>
			<td>15140-122</td>
			<td>50 U/mL final concentration (Step 2.3.1)</td>
		</tr>
		<tr>
			<td>Heparin Grade I-A, &#8805;180 USP units/mg</td>
			<td>Sigma-Aldrich</td>
			<td>H3149-100KU</td>
			<td>2 &#181;g/ml final concentration (Step 2.3.1)</td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>Thermo-Fisher</td>
			<td>13256-029</td>
			<td>20 ng/ml final concentration added before use (Step 2.3.1)</td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane Matrix</td>
			<td>Corning</td>
			<td>354234</td>
			<td>1:100 (Step 2.2). Thaw Matrigel on ice, prepare 200 ul aliquots and store them in -80&#176;C. For coating, dilute 200 ul aliquot in 20 ml of cold DMEM/F12 medium.</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td>1:100 (Step 2.2). Dilute in PBS 1X.</td>
		</tr>
		<tr>
			<td>Complete Neuronal Differentiation medium (ND):</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Thermo-Fisher</td>
			<td>21103049</td>
			<td>(Step 2.4.11)</td>
		</tr>
		<tr>
			<td>B-27 Supplements (50x)</td>
			<td>Thermo-Fisher</td>
			<td>17504044</td>
			<td>(Step 2.4.11)</td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>Thermo-Fisher</td>
			<td>17502-048</td>
			<td>(Step 2.4.11)</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo-Fisher</td>
			<td>15140-122</td>
			<td>50 U/mL final concentration (Step 2.4.11)</td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>Thermo-Fisher</td>
			<td>PHC7045</td>
			<td>1 ng/ml final concentration. Added before use. (Step 2.4.11)</td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>Thermo-Fisher</td>
			<td>PHC7074</td>
			<td>2.5 ng/ml final concentration. Added before use. (Step 2.4.11)</td>
		</tr>
	</tbody>
</table>

generating neurons and glial cells from human induced pluripotent stem cell-derived embryoid bodies

Source: Pistollato, F., et al. <a href="https://app.jove.com/v/55702">Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing.</a> J. Vis. Exp.(2017)
This video demonstrates the process of differentiating embryoid bodies (EBs) into neuroepithelial aggregates and their subsequent division into neuronal and glial cells. This method allows the efficient generation of neuronal and glial cells in vitro, which serve as test systems for toxicity testing as well as assessment of different pathways involved in neurotoxicity.

<img alt="Figure 1" src="/files/ftp_upload/22426/22426_Figure1.jpg" /> 
Figure 1: Schematic Representation of the Neuronal Differentiation Protocol. (Upper part) IMR90-hiPSC colonies can be cut into fragments to form embryoid bodies (EBs). After 2 days in vitro (DIV), EBs can be plated onto laminin- or standard matrix-coated dishes and cultured in the presence of neuroepithelial induction (NRI) medium to generate neuroectodermal derivatives (rosettes, here s...

Generating Neurons and Glial Cells from Human Induced Pluripotent Stem Cell-Derived Embryoid Bodies

1. HiPSC Differentiation into Mixed Neurons and Glia
NOTE: The procedure takes approximately 28 days to complete, with the main steps outlined in Figure ...

Take embryoid bodies, or EBs, derived from human induced pluripotent stem cells. These EBs are composed of three germ layers.
Transfer the EBs to a matrix-coated culture dish containing a suitable medium and incubate.
The matrix enables the EBs to attach to the culture dish.
The growth factors and nutrients in the medium facilitate the ectodermal cells of the EBs to form neuroepithelial aggregates arranged in a circular pattern termed a rosette.
Cut the rosette fragments and detach them from the dish.
Collect the fragments and spin them down.
Discard the supernatant and resuspend the fragments.
Using a pipette, move the fragments up and down to dissociate them.
Add the desired number of cells to a matrix-coated well plate containing a differentiation medium.
The components of the differentiation medium facilitate the neuroepithelial cells to transform into fully functional neuronal and glial cells, the primary cells of the nervous system.

generating-neurons-glial-cells-from-human-induced-pluripotent-stem

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JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

36 ビュー. 出典:Pistollato, F., et al.神経毒性試験のためのニューロンとグリアの混合培養物へのヒト誘導多能性幹細胞の分化のためのプロトコル。J. Vis. Exp.(2017年) このビデオは、胚様体(EB)を神経上皮凝集体に分化させ、その後のニューロン細胞とグリア細胞への分裂のプロセスを示しています。この方法により、in vitroで神経細胞およびグリア細胞を効率的に生成することができ、毒?...

ビデオ: ヒト人工多能性幹細胞由来胚様体からのニューロンおよびグリア細胞の生成 - 概念

Nerve Stem Cell Differentiation by a One-step Cold Atmospheric Plasma Treatment In Vitro

As the development of physical plasma technology, cold atmospheric plasmas (CAPs) have been widely investigated in decontamination, cancer treatment, wound healing, root canal treatment, etc., forming a new research field named plasma medicine. Being a mixture of electrical, chemical, and biological reactive species, CAPs have shown their abilities to enhance nerve stem cells differentiation both in vitro and in vivo and are becoming a promising way for neurological disease treatment in the future. The much more exciting news is that using CAPs may realize one-step, and safely directed, differentiation of neural stem cells (NSCs) for tissue transportation. We demonstrate here the detailed experimental protocol of using a self-made CAP jet device to enhance NSC differentiation in C17.2-NSCs and primary rat neural stem cells, as well as observing the cell fate by inverted and fluorescence microscopy. With the help of immunofluorescence staining technology, we found both the NSCs showed an accelerated differential rate than the untreated group, and ~75% of the NSCs selectively differentiated into neurons, which are mainly mature, cholinergic, and motor neurons.

Nerve Stem Cell Differentiation by a One-step Cold Atmospheric Plasma Treatment In Vitro

This protocol aims to provide detailed experimental steps of a cold atmospheric plasma treatment on neural stem cells and immunofluorescence detection for differentiation enhancement.

ワンステップ低温大気プラズマ治療体外で神経幹細胞の分化

As the development of physical plasma technology, cold atmospheric plasmas (CAPs) have been widely investigated in decontamination, cancer treatment, ...

JoVE Journal

工学

Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment. However, challenges remain in obtaining stem cell-derived beta cells that adequately mimic native human beta cells. Building upon previous studies, hPSC-derived islet cells have been generated to create a protocol with improved differentiation outcomes and consistency. The protocol described here utilizes a pancreatic progenitor kit during Stages 1-4, followed by a protocol modified from a paper previously published in 2014 (termed "R-protocol" hereafter) during Stages 5-7. Detailed procedures for using the pancreatic progenitor kit and 400 &#181;m diameter microwell plates to generate pancreatic progenitor clusters, R-protocol for endocrine differentiation in a 96-well static suspension format, and in vitro characterization and functional evaluation of hPSC-derived islets, are included. The complete protocol takes 1 week for initial hPSC expansion followed by ~5 weeks to obtain insulin-producing hPSC islets. Personnel with basic stem cell culture techniques and training in biological assays can reproduce this protocol.

The differentiation of stem cells into islet cells provides an alternative solution to conventional diabetes treatment and disease modeling. We describe a detailed stem cell culture protocol that combines a commercial differentiation kit with a previously validated method to aid in producing insulin-secreting, stem cell-derived islets in a dish.

ヒト多能性幹細胞のインスリン産生膵島クラスターへの分化

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and ...

発生生物学

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.

ニューウトジェネシスとシナプス成熟の高含有量スクリーニングに対するヒト多能性幹細胞由来ニューロンの分化後再形成

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

Generating Neurons and Glial Cells from Human Induced Pluripotent Stem Cell-Derived Embryoid Bodies

記録

Generating Neurons and Glial Cells from Human Induced Pluripotent Stem Cell-Derived Embryoid Bodies