differentiation of induced pluripotent stem cells into neural progenitor cells

Differentiation of Induced Pluripotent Stem Cells into Neural Progenitor Cells

For this protocol, begin with establishing feeder cells. To establish this culture, plate 600,000 feeder cells into each well of a six-well plate, with 300 milliliters of medium per well. After culturing the feeder cells for 48 hours, replace the medium, and incubate the plates for an hour, while preparing the stem cells.
Thaw the stem cells in a 37 degrees Celsius water bath. Once thawed, transfer the cells to a 15-milliliter conical tube and fill the tube to 5 milliliters with culture medium, added dropwise. Next, gently centrifuge the cells for four minutes. Aspirate the supernatant, and gently resuspend the pellet in 1 milliliter of stem cell medium with ROCK inhibitor.
After quantifying the cell density, plate 300,000 stem cells onto the feeder cells. Then, grow and expand the cultures, periodically selecting for healthy cells. After expansion and freezing cells for backup, grow the stem cells&#39; colonies on standard plates until they reach 50% to 70% confluence.
Then, use a mild enzymatic treatment and gentle titration with a 1-milliliter pipette tip to harvest the HIPSCs for neural precursor cell differentiation. Gently centrifuge the suspension, aspirate the supernatant, and resuspend the pellet in 5 milliliters human iPSC medium. Then, transfer the cell suspension to a 0.1% gelatin-coated 5-centimeter cell culture dish, and incubate the plate for one hour.
After an hour, transfer the non-adherent cells to a 15-milliliter centrifuge tube. Then, gently rinse the plate with 3 milliliters of medium, and transfer it to the same tube. Next, repeat the resuspension step with gentle centrifugation.
Then, determine the cell density and plate the cells into a 96-well, low-adhesion V-bottom plate at 9,000 cells per well. Now, spin the plate for three minutes and start culturing the cells. Over the next 14 days, every other day, replace half the medium with fresh differentiating medium one.

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1. Generation of Neural Progenitor Cells 
<ol>
	<li>Maintain hiPSCs derived from fibroblasts and peripheral blood mononuclear cells (PBMCs) in 6-well plates on a &#947;-irradiated mouse embryonic feeder (MEF) cell layer in human iPSC medium supplemented with small-molecules. 
	NOTE: Daily maintenance is a modified version of previously reported procedures.
	<ol>
		<li>Plate 6 x 105 MEFs onto each well of a 6-well tissue culture grade plate in 300 &#956;L/well recommended medium (following manufacturer&#39;s protocol).</li>
		<li>After 48 h, replace MEFs medium with 300 &#956;L/well hiPSC medium for 1 h before adding hiPSCs.</li>
		<li>Quickly thaw hiPSCs in a 37 &#176;C water bath and slowly add 1 mL hiPSC medium dropwise. Transfer the hiPSCs and medium to a 15 mL conical tube. Add medium to bring the total volume to 5 mL. Centrifuge for 4 min at 129 x g, aspirate the supernatant, gently resuspend the pellet in 1 mL hiPSC medium with Rho-associated protein kinase (ROCK) inhibitor at 1:1,000 concentration.</li>
		<li>Plate the cell suspension (typically seeded at 300,000 cells/well) onto the MEF cell layer and incubate at 37 &#176;C, 5% CO2, 95% humidity.</li>
		<li>Monitor iPSC culture closely using a light microscope. After 48 h, remove cultures that have uneven edges, have grown to become cystic-like or appear yellowish-brown under the light microscope to minimize spontaneous differentiation. 
		NOTE: After 7 - 10 days, hiPSC colonies should form. Colonies should be round, with clearly defined borders and uniform cell densities, and devoid of loosely packed non-uniform cells.</li>
		<li>Manually select round hiPSC colonies to clear the culture of spontaneously differentiated cells. Expand the colonies by placing them on fresh MEF layers. Expand 1:3 every 7 days when cultures near confluency and freeze.</li>
	</ol>
	</li>
	<li>After expansion and freezing cells (for backup), grow hiPSC colonies on plates until they reach 50 - 75% confluence.</li>
	<li>Seven-days post plating, use mild enzymatic treatment (5-10 min with 300 &#956;L of enzyme solution) and gentle trituration (2-4 times with a 1,000 &#181;L pipet) to harvest and prepare hiPSCs for neural precursor cell differentiation.</li>
	<li>Centrifuge the cell suspension at 129 x g for 4 min, aspirate the supernatant, and resuspend the pellet in 5 mL human iPSC medium. Transfer the cell suspension to a 0.1% gelatin coated 5 cm cell culture dish for 1 h at 37 &#176;C to eliminate MEFs and to maximize the hiPSC yield. Transfer the medium (with non-adherent cells) to another 5 cm gelatin coated dish.</li>
	<li>Transfer the non-adherent cells to a 15 mL centrifuge tube using a transfer pipet. Gently rinse the plates with 3 mL medium and add it to the 15 mL tube.</li>
	<li>Centrifuge the cell suspension at 129 x g for 4 min, aspirate the supernatant, and gently resuspend the pellet in medium. Determine the cell count using an automated cell counter. Plate 9,000 cells/well in a 96-well low-adhesion V-bottom plate.</li>
	<li>Centrifuge the plate at 163 x g for 3 min. Incubate the plate at 37 &#176;C, 95% humidity and 5% CO2. Using a pipette, change 50% medium every other day by removing half of the medium and replacing it with fresh chemically-defined differentiating medium 1 (DM1; Figure 1; Table of Materials) for 14 days.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>SFEB Neuronal Differentiation Cell culture Media. Reagents. Components&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff Neural Induction Medium (hiPSC Media)</td>
			<td>STEMCELL Technologies</td>
			<td>0-5835</td>
			<td>250ml</td>
		</tr>
		<tr>
			<td>PluriQ ES-DMEM Medium (MEF Media)</td>
			<td>GlobalStem</td>
			<td>GSM-2001</td>
			<td></td>
		</tr>
		<tr>
			<td>DM1 Media Components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D-MEM/F-12 (1X), Glutamax liquid, 1:1</td>
			<td>Invitrogen</td>
			<td>10565018</td>
			<td>385ml</td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement</td>
			<td>Invitrogen</td>
			<td>10828028</td>
			<td>20% 100ml</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Invitrogen</td>
			<td>15140122</td>
			<td>5ml</td>
		</tr>
		<tr>
			<td>Glutamax 200mM</td>
			<td>Invitrogen</td>
			<td>35050061</td>
			<td>5ml</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution 10 mM (100X), liquid</td>
			<td>Invitrogen</td>
			<td>11140050</td>
			<td>5ml</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol (1,000X), liquid</td>
			<td>Invitrogen</td>
			<td>21985023</td>
			<td>900ul</td>
		</tr>
		<tr>
			<td>Small Molecules</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Y27632 (ROCKi)</td>
			<td>Stemgent</td>
			<td>04-0012-10</td>
			<td>10uM</td>
		</tr>
		<tr>
			<td>Components/Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Embryonic Fibroblasts</td>
			<td>GlobalStem</td>
			<td>GSC-6301G</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well V bottom w/Lids</td>
			<td>Evergreen</td>
			<td>222-8031-01V</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell Dissociation Reagent</td>
			<td>ThermoFisher</td>
			<td>A1110501</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well flat bottom</td>
			<td>Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Phillips, A. W., et al. <a href="https://app.jove.com/v/55799">Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays.</a> J. Vis. Exp. (2017).
The video demonstrates a technique for differentiating human induced pluripotent stem cells (hiPSCs) into neural progenitor cells (NPCs). Initially, the hiPSCs are cultured on a feeder layer of mouse embryonic fibroblasts (MEFs). Subsequently, the cells are detached and transferred to a biopolymer-coated plate to allow the MEFs to adhere to and separate from the hiPSCs. The suspended hiPSCs are then transferred to a V-bottom plate and centrifuged to form aggregates. Finally, the addition of a differentiation medium induces the differentiation of hiPSCs within the aggregate into NPCs.

<img alt="Figure 1" src="/files/ftp_upload/22635/22635_Figure1.jpg" /> 
Figure 1: Schematic Outlining the Early Steps in Preparing SFEBs. After iPSCs have been harvested using an enzymatic dissociation, they are plated in 96-well plates and centrifuged at 163 x g for 3 min to induce quick aggregation. After 14 days in vitro (DIV) cell aggregates can be transferred from 96-well plates via wide-bore pipets onto cell culture insert-containing 6-well plates.

1. Generation of Neural Progenitor Cells 

	Maintain hiPSCs derived from fibroblasts and peripheral blood mononuclear cells (PBMCs) in 6-well plates on a ...

Take a microplate containing an adherent culture of mouse embryonic fibroblasts, or MEFs, in a medium.
The adhered MEFs function as a feeder layer, providing a supportive cell culture microenvironment.
Remove the medium and introduce human induced pluripotent stem cells, or hiPSCs, in an hiPSC medium supplemented with small molecules that enhance cell survival.
Incubate to allow hiPSCs to adhere to the feeder layer, which secretes growth factors that facilitate hiPSC proliferation.
Remove the medium and add an enzyme solution to detach the cells. Transfer the cells and centrifuge them, then remove the supernatant and resuspend them in the hiPSC medium.
Transfer the cells to a biopolymer-coated plate. Incubate to allow the MEFs to adhere.&#160;
Transfer the suspended hiPSCs to a V-bottom microplate. Centrifuge the cells and incubate them, inducing cell aggregate formation.
Replenish with a differentiation medium. The medium&#39;s nutrients and cell-cell interactions within the aggregate facilitate hiPSC differentiation into neural progenitor cells.

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视频: 诱导多能干细胞分化为神经祖细胞 - 实验

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

脐带血诱导的多能干细胞的软骨形成颗粒形成

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

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A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides reliable analysis of neuronal morphology together with quantitative measurements of modifications on neurite length and synaptic protein localization and abundance upon treatment with small molecule compounds. In addition to the application of the presented method in neurite outgrowth studies, neurotoxicity assessment can be performed to assess, distinguish and rank commercial chemical compounds based on their potential developmental neurotoxicity effect.
Even though cell lines are nowadays widely used in compound screening assays in neuroscience, they often differ genetically and phenotypically from their tissue origin. Primary cells, on the other hand, maintain important markers and functions observed in vivo. Therefore, due to the translation potential and physiological relevance that these cells could offer neurite outgrowth assay and neurotoxicity assessment can considerably benefit from using human neural progenitor cells (hNPCs) as the primary human cell model.
The presented method herein can be utilized to screen for the ability of compounds to induce neurite outgrowth and neurotoxicity by taking advantage of the human neural progenitor cell-derived neurons, a cell model closely representing human biology.&#34;

The presented protocol describes a method for a neurite outgrowth assay and neurotoxicity assessment of small molecule compounds.

神经石生长分析与神经毒性评估与人类神经原生细胞衍生神经元

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides ...

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.

从多能干细胞生成人类神经元和少突胶质细胞，用于模拟神经元-少突胶质细胞相互作用

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

Differentiation of Induced Pluripotent Stem Cells into Neural Progenitor Cells

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Differentiation of Induced Pluripotent Stem Cells into Neural Progenitor Cells