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1. Preparation of human embryonic stem cells (hESC)-qualified Basement Membrane Matrix-coated Plates
<ol>
	<li>Slowly thaw the hESC-qualified basement membrane matrix (see&#160;Table of Materials) solution at 4 &#176;C for at least 2&#8211;3 h or overnight to avoid formation of a gel.</li>
	<li>To prepare basement membrane matrix-coated plates, dilute matrix in cold Dulbecco&#39;s Modified Eagle Medium with F12 (DMEM+F12) to a 2% final concentration. Mix well and coat each well of a 6 well plate with 1 mL of the diluted matrix solution.</li>
	<li>Incubate the basement membrane matrix-coated plates at room temperature (RT) for at least 3 h or at 4 &#176;C overnight.&#160; 
	NOTE: Plates with basement membrane matrix can be stored at 4 &#176;C for 1 week before the matrix solution is removed and plates are used.</li>
</ol>
2. Adaptation of Colony type hESCs to Single cell hESC Culture
<ol>
	<li>To passage the feeder-free cultures of colony type H9 (WA09) hESCs grown on the basement membrane matrix, aspirate the medium from the wells (Figure 1A).</li>
	<li>Wash 1x with 1 mL of DPBS. Add 1 mL of dispase solution (1 U/mL) per well and incubate at 37 &#176;C for 20 min.</li>
	<li>Remove the dispase, wash the cells 1x gently with 2 mL of DMEM/F12, remove the medium, and add 2 mL of DMEM/F12 to each plate.</li>
	<li>Gently detach colonies by gently pipetting up and down and transfer to a 15 mL tube.</li>
	<li>Centrifuge the pellets for 2 min at 370 x&#160;g&#160;and aspirate the medium.</li>
	<li>To dissociate the cell pellets into single cells, add 2 mL of cell detachment solution (1x concentration, see&#160;Table of Materials) and incubate at 37 &#176;C for 10 min.</li>
	<li>Centrifuge cells for 2 min at 370 x&#160;g&#160;and remove the detachment solution.</li>
	<li>Add fresh mTeSR1 human ESC medium and dissociate the cells into single cells by gentle pipetting up and down.</li>
	<li>To adapt colony type hESCs to a single-cell type culture, plate approximately 1.5&#8211;2.0 x 106&#160;hESCs into each well of the basement membrane matrix-coated 6 well plate in 2 mL of mTeSR1 containing 10 &#956;M ROCK inhibitor for 24 h (Figure 1A).</li>
	<li>After 24 h, replace the hESC medium with fresh mTeSR1 without ROCK inhibitor and allow the hESCs to grow as a single-cell type for 3 days. Change the medium daily.</li>
	<li>On day 4, when cultures reach nearly 100% confluency, dissociate cells in detachment solution, then replate as described in step 2.6. 
	NOTE: The ROCK inhibitor improves cell survival during the initial 24 h of single-cell type hESC culture.</li>
</ol>
3. Differentiation of Single-cell Type hESCs Into NPCs (Figure 2)
<ol>
	<li>To induce NPC differentiation, dissociate single-cell type hESCs with 1 mL of detachment solution (1x) and incubate for 10 min at 37 &#176;C.</li>
	<li>Centrifuge the cells for 2 min at 370 x&#160;g&#160;and remove the detachment solution supernatant. Add 1 mL of DMEM/F12 and resuspend cells by gentle pipetting.</li>
	<li>Plate cells on a basement membrane matrix-coated 6 well plate at a density of 2 x 105&#160;cells/well in 2 mL of mTeSR1 containing 10 &#956;M ROCK inhibitor.</li>
	<li>After 24 h, replace the culture medium with a neural induction medium (DMEM with 1% B27 minus vitamin A) supplemented with 1 &#956;M dorsomorphin and 5 &#956;M SB431542.</li>
	<li>Change the medium every other day during the first 4 days of neural induction, then every day until reaching confluence at day 7 (Figure 2B).&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: (1) Dorsomorphin inhibits the BMP pathway by targeting ALK2,3,6 receptors and also inhibits AMPK; SB431542 is an inhibitor of the TGF&#946;/Activin pathway by targeting ALK5,7. (2) The same protocol was tested with another ESC line (WA01), which yielded similar results as WA09. (3) We have generally used Matrigel for the basement membrane matrix; however, cells can be cultured on Geltrex and then differentiated into NPCs with very similar results as indicated by the expression of several NPC markers (Figure 3D, E). Handle Geltrex in the same manner as Matrigel (see section 1).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6-well plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104-500</td>
			<td>Cell detachment solution</td>
		</tr>
		<tr>
			<td>Activin A</td>
			<td>R&#38;D system</td>
			<td>338-AC-050</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement (-Vit A)</td>
			<td>Thermo Fisher</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>DAMON/ICE</td>
			<td>428-6759</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo Fisher</td>
			<td>4110</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning hESC-qulified Matrix (Magrigel)</td>
			<td>Corning</td>
			<td>354277</td>
			<td>Basement membrane matrix (used for most of the protocol here)</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Stemcell Technologies</td>
			<td>7923</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher</td>
			<td>10569-010</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo Fisher</td>
			<td>10565-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>Tocris</td>
			<td>3093</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Fisher Scientific</td>
			<td>SH3007003HI</td>
			<td></td>
		</tr>
		<tr>
			<td>Geltrex matrix</td>
			<td>Thermo Fisher</td>
			<td>A1569601</td>
			<td>Basement membrane matrix</td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Thermo Fisher</td>
			<td>35050061</td>
			<td>Glutamine supplement, 100X</td>
		</tr>
		<tr>
			<td>H9 (WA09) human embryonic stem cell line</td>
			<td>WiCell</td>
			<td>WA09</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma Aldrich</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>Stemcell Technologies</td>
			<td>85850</td>
			<td>hESC culture medium</td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Thermo Fisher</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK inhibitor</td>
			<td>Tocris</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Tocris</td>
			<td>1614</td>
			<td></td>
		</tr>
	</tbody>
</table>

differentiating human embryonic stem cells into neural progenitors

Source: Jeon, K. et al., <a href="https://app.jove.com/v/60571">Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells.</a> J. Vis. Exp.&#160;(2020)
This video demonstrates the differentiation of human embryonic stem cells, or hESCs, directly into neural progenitor cells without using feeder cells or any intermediate steps. The selective inhibition of bone morphogenetic protein or BMP and transforming growth factor-beta, or TGF-&#946;, signaling pathways leads to hESCs differentiation into neural progenitor cells, or NPCs.

<img alt="Figure 1" src="/files/ftp_upload/22374/22374_Figure1.jpg" /> 
Figure 1: Adaptation of colony type hESCs to single-cell type culture.&#160;(A) Representative phase contrast images of single cell cultures of H9 hESCs at different times after plating on 2% basement membrane matrix-coated dishes. Low (left) and high (right) magnification. Top panel: representative image of colony type hESCs. Other panels show representative images of cultures a...

Differentiating Human Embryonic Stem Cells into Neural Progenitors

1. Preparation of human embryonic stem cells (hESC)-qualified Basement Membrane Matrix-coated Plates

	Slowly thaw the hESC-qualified basement membrane ...

Begin with a multi-well plate coated with a basement membrane matrix that includes extracellular polymers and adhesion proteins.
Seed the wells with a single-cell suspension of human embryonic stem cells in a growth medium containing a Rho-kinase inhibitor.
Incubate to allow stem cell attachment to the matrix.
Cells absorb the inhibitor, which blocks the Rho-kinase enzymes and prevents apoptotic cell death.
This promotes the cells&#39; survival and maintains them in an undifferentiated state.
Replace the growth medium with a neural induction medium rich in growth factors and containing inhibitors that target specific ALK receptors.
These inhibitors interact with their receptors and block the BMP and TGF-&#946; signaling pathways, preventing the cell differentiation into endodermal or mesodermal precursors.
This leads to selective differentiation of stem cells into ectodermal precursors.
These ectodermal precursors utilize nutrients and growth factors from the medium to multiply and later differentiate into neural progenitor cells.

differentiating-human-embryonic-stem-cells-into-neural-progenitors

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

57 조회수. 출처: Jeon, K. et al., Efficient Neural Differentiation Using Single-Cell Culture of Human Embryonic Stem Cells. J. Vis. 특급.(2020년) 이 비디오는 인간 배아 줄기 세포(hESCs)가 영양 세포나 중간 단계를 사용하지 않고 신경 전구 세포로 직접 분화되는 과정을 보여줍니다. 뼈 형태 유전 단백질(BMP)의 선택적 억제와 성장 인자-베타 또는 TGF-β 신호 전달 경로의 변형은 hESC를 신경 전구 세포(NPC)로 분화시킵니?...

비디오: 인간 배아 줄기 세포를 신경 전구 세포로 분화 - 개념

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

혈청이없는 단층 문화 마우스 배아 줄기 세포의 신경 분화

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

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JoVE Journal

발생학

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

말초 단핵 세포에서 유도 된 신경 줄기 세포의 생성 및 이식 연구에 대 한 도파민성 신경 전구체로 분화

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative&#160;reverse transcription&#160;polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.

We describe the procedure for the in vitro differentiation of mouse embryonic stem cells into neuronal cells using the hanging drop method. Furthermore, we perform a comprehensive phenotypic analysis through RT-qPCR, immunofluorescence, RNA-seq, and flow cytometry.

마우스 배아 줄기 세포에서 신경 전조및 뉴런의 분화 및 특성화

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

Differentiating Human Embryonic Stem Cells into Neural Progenitors

내레이션 대본

Differentiating Human Embryonic Stem Cells into Neural Progenitors