generation of neuronal cells from blood-derived pluripotent stem cells

Generation of Neuronal Cells from Blood-Derived Pluripotent Stem Cells

Place glass coverslips in 4-well plates and coat them with 1-to-5 diluted poly-L-ornithine in double-distilled water. After incubating the coverslips at 37 degrees Celsius for one hour, wash them with double-distilled water.
Slowly thaw 0.5 to 2 milligrams per milliliter of laminin, and add it to the top of coverslips. Incubate them at 37 degrees Celsius for two hours, then remove excess laminin by pipetting, and add neuronal medium N2 to the culture dishes.
Culture BD-derived CD45 negative cells in N2 medium on laminin-ornithine-coated glass coverslips for two days in an incubator, with 5% carbon dioxide at 37 degrees Celsius to initiate a neuronal differentiation of newly BD generated cells. Next, culture cells in neuronal differentiation medium, and place the plates in an incubator at 37 degrees Celsius and 5% carbon dioxide for 16 days.

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JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

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Ethic approvals were obtained when performing the experiments.
1. Isolation of human peripheral blood mononuclear cells (PBMNCs)
<ol>
	<li>Ensure that all donors signed informed consent before blood withdrawing in compliance with institutional guidelines.</li>
	<li>Take 30 mL of blood from healthy donors by trained medical personnel according to the standard protocol.</li>
	<li>Isolate PBMNCs by density gradient media. Use 10 mL of media with 25 mL of 1:1 blood diluted with phosphate-buffered saline (PBS), and centrifuge at 300 x&#160;g&#160;for 30 min.</li>
	<li>Isolate the interphase layer between the plasma and the density gradient media by pipetting. Wash the isolated cells with 5 mL of sterile PBS and centrifuge at 300 x&#160;g&#160;for 10 min. Repeat twice.</li>
	<li>Count the number of cells by standard methods using a counting chamber.</li>
</ol>
2. Activation of human glycosylphosphatidylinositol (GPI)-anchored glycoprotein by antibody crosslinking on the surface of PBMNCs
<ol>
	<li>Place the 6 x 106&#160;mononuclear cells (MNCs) in 15 mL tubes and perform antibody crosslinking by incubating the cells with human GPI-linked membrane protein-specific antibody (30 &#181;g/mL) for 30 min in PBS with 1% bovine serum albumin (BSA) at 37 &#176;C.</li>
	<li>Replace incubation medium with Iscove&#39;s modified Dulbecco&#39;s medium supplemented with 10% fetal bovine serum (FBS).</li>
	<li>Grow cells in 15 mL polystyrene tubes, put the tubes in an incubator at 37 &#176;C and 5% CO2&#160;for 8-10&#160;days (without shaking). On day 5 (D5), add an additional 1-2 mL of Iscove&#39;s medium supplemented with 10% FBS to each 15 mL tube.</li>
</ol>
3. Sorting of newly generated dedifferentiated cells
<ol>
	<li>Count cells with an automated cell counter (18 &#181;L cell suspension + 2 &#181;L fluorescence dye) or in a counting chamber.</li>
	<li>Centrifuge cultured cell suspension (5-7 x 106) at 300 x&#160;g&#160;for 10 min and aspirate the resulting supernatant with a sterile Pasteur pipette.</li>
	<li>Re-suspend the cell pellet in 90 &#181;L of pre-cooled PBS pH 7.2, 0.5% BSA and 2 mM ethylenediaminetetraacetic acid (EDTA).</li>
	<li>Add CD45 positive nano-sized magnetic beads (80 &#181;L) to the cell suspension and incubate on ice for 15 min.</li>
	<li>Wash the cells by adding 2 mL of PBS buffer and centrifuge at 300 x&#160;g&#160;for 10 min.</li>
	<li>Re-suspend the cells in 500 &#181;L of PBS buffer.</li>
	<li>Wash the column with 500 &#181;L of pre-cooled PBS buffer and place it in the magnetic field.</li>
	<li>Place the cell suspension on the column and wash it with 500 &#181;L of PBS buffer (two times) and the centrifuge flow containing CD45 negative cells. Collect them in Iscove&#39;s medium supplemented with 1% BSA.</li>
	<li>Count the cells in the counting chamber.</li>
</ol>
4. Preparing cell culture dishes for neuronal differentiation of newly generated stem cells
<ol>
	<li>Coat the culture vessels with poly-L-ornithine and laminin for growing neuronal cells.</li>
	<li>Place the glass coverslips in 4-well plates and coat it with 1:5 diluted poly-L-ornithine (0.1 mg/mL in double distilled water [ddH2O]) in ddH2O. Place the coverslips into a 37 &#176;C incubator for 1 h. Then wash with ddH2O.</li>
	<li>Slowly thaw laminin (0.5-2.0 mg/mL) and add to the top of coverslips. Incubate it at 37 &#176;C for 2 h.</li>
	<li>Prepare neural induction medium N2 consisting of 49 mL of Dulbecco&#39;s modified Eagle medium/nutrient mixture F-12 (D-MEM/F12), 500 &#181;L of N2 supplement, 400 &#181;L of non-essential amino acids (NEAA), basic fibroblast growth factor (FGF) solution at 20 ng/mL final concentration (prepared from 100 &#181;g/mL stock solution), and heparin at 2 ng/mL final concentration.</li>
	<li>Remove excess laminin by pipetting and add neuronal medium N2 to culture dishes.</li>
</ol>
5. Culturing of neuronal dedifferentiated blood cells
<ol>
	<li>Culture BD-derived CD45 negative cells on laminin/ornithine-coated glass coverslips for 2 days in an incubator at 37 &#176;C and 5% CO2&#160;in N2 medium to initiate a neuronal differentiation of newly BD-generated cells.</li>
	<li>Culture cells further in neuronal differentiation medium consisting of 48 mL of Neurobasal medium, 500 &#181;L of L-glutamine, 1 mL of B27 Supplement, 500 &#181;L of NEAA, 50 &#181;L of recombinant human glial-derived neurotrophic factor (GDNF) at 5 &#181;g/250 &#181;L in PBS/0.1% BSA, and 50 &#181;L of recombinant human brain derived neurotrophic factor (BDNF) at 5 &#181;g/200 &#181;L in PBS/0.1% BSA and 50 &#181;L of ascorbic acid solution 2.9 g/50 mL in PBS. Place plates in an incubator at 37 &#176;C and 5% CO2.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Albumin Fraction V</td>
			<td>Roth</td>
			<td>T8444.4</td>
			<td></td>
		</tr>
		<tr>
			<td>AO/PI Cell Viability kit</td>
			<td>Biozym</td>
			<td>872045</td>
			<td>Biozym discontinued. The product produced by Logos Biosystems.</td>
		</tr>
		<tr>
			<td>Ascorbic acid 2-phosphate sequimagnes</td>
			<td>Sigma Aldrich</td>
			<td>A8960-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Serum free 50x</td>
			<td>Fisher Scientific (Gibco)</td>
			<td>11530536</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic FGF solution</td>
			<td>Fisher Scientific (Gibco)</td>
			<td>10647225</td>
			<td></td>
		</tr>
		<tr>
			<td>Biocoll</td>
			<td>Merck Millipore</td>
			<td>L6115-BC</td>
			<td>density gradient media</td>
		</tr>
		<tr>
			<td>BSA Frac V 7.5%</td>
			<td>Gibco</td>
			<td>15260037</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 MicroBeads</td>
			<td>Miltenyi</td>
			<td>130-045-801</td>
			<td>nano-sized magnetic beads</td>
		</tr>
		<tr>
			<td>Cell counting slides Luna</td>
			<td>Biozym</td>
			<td>872010</td>
			<td>Biozym discontinued. The product produced by Logos Biosystems.</td>
		</tr>
		<tr>
			<td>D-MEM/F12</td>
			<td>Merck Millipore</td>
			<td>FG4815-BC</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Merck Millipore</td>
			<td>S0115/1030B</td>
			<td>Discontinued. Available under: TMS-013-B</td>
		</tr>
		<tr>
			<td>GDNF recombinant human</td>
			<td>Fisher Scientific (Gibco)</td>
			<td>10679963</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMax 100x</td>
			<td>Gibco</td>
			<td>35050038</td>
			<td>L-glutamine</td>
		</tr>
		<tr>
			<td>Heparin sodium cell</td>
			<td>Sigma-Aldrich</td>
			<td>H3149-50KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Human BDNF</td>
			<td>Fisher Scientific (Gibco)</td>
			<td>11588836</td>
			<td></td>
		</tr>
		<tr>
			<td>Iscove (IMDM)</td>
			<td>Biochrom</td>
			<td>FG0465</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin mouse</td>
			<td>Fisher Scientific (Gibco)</td>
			<td>10267092</td>
			<td></td>
		</tr>
		<tr>
			<td>Luna FL Automated Cell Counter</td>
			<td>Biozym</td>
			<td>872040</td>
			<td>Biozym discontinued. The product produced by Logos Biosystems.</td>
		</tr>
		<tr>
			<td>MACS Buffer</td>
			<td>Miltenyi</td>
			<td>130-091-221</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM NEAA 100x</td>
			<td>Gibco</td>
			<td>11140035</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS Trenns&#228;ulen</td>
			<td>Miltenyi</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiplatte Nunclon 4 wells</td>
			<td>Fisher Scientific</td>
			<td>10507591</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 Supplement 100x</td>
			<td>Fisher Scientific (Gibco)</td>
			<td>11520536</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Gibco</td>
			<td>10888022</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS sterile</td>
			<td>Roth</td>
			<td>9143.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-ornithine</td>
			<td>Sigma-Aldrich</td>
			<td>P4957-50ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Becker-Koji&#263;, Z. A., et al. <a href="https://app.jove.com/v/61634">GM-Free Generation of Blood-Derived Neuronal Cells.</a> J. Vis. Exp. (2021).
This video demonstrates a technique to generate neuronal cells from blood-derived pluripotent stem cells (BD-PSCs). The BD-PSCs are obtained via reprogramming blood progenitor cells. The undifferentiated BD-PSCs are cultured on cell culture substrate-coated coverslips using a neural induction medium and a neural differentiation medium, triggering differentiation into cells with a neuronal phenotype.

Ethic approvals were obtained when performing the experiments.
1. Isolation of human peripheral blood mononuclear cells (PBMNCs)

	Ensure that all donors ...

Take a microplate with glass coverslips placed at the bottom of the wells.
Introduce a positively charged polymer to coat the coverslips and wash to remove any unbound polymer. Treat with an extracellular matrix protein to coat the coverslips and aspirate the unbound proteins.
The coating acts as a culture substrate that facilitates cell adhesion.
Add an induction medium to the wells and introduce a suspension of blood-derived pluripotent stem cells, or BD-PSCs. These cells, capable of differentiating into various cell types, are generated via reprogramming blood progenitor cells.
Incubate for the cells to adhere to the substrate. Growth factors and nutrients in the induction medium transform BD-PSCs into neural progenitor cells.
Introduce a differentiation medium containing neuronal growth factors and incubate, triggering differentiation of the neural progenitors into a neuronal phenotype, exhibiting extended cellular processes and cell-cell contacts.

20 次观看. 从血源性多能干细胞生成神经元细胞

视频: 从血源性多能干细胞生成神经元细胞 - 实验

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

利用人类胚胎干细胞的单细胞培养进行高效神经分化

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

科研

JoVE Journal

发育生物学

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.

小鼠胚胎干细胞中神经祖细胞和神经元的分化与表征

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

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.

小鼠胚胎干细胞体外神经元分化

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

Generation of Neuronal Cells from Blood-Derived Pluripotent Stem Cells

成績單

Generation of Neuronal Cells from Blood-Derived Pluripotent Stem Cells