human pluripotent stem cell differentiation in neurons using engineered lentiviruses

Human Pluripotent Stem Cell Differentiation in Neurons Using Engineered Lentiviruses

Two days before induction of differentiation, detach ES cells by adding 1 milliliter of cell detachment solution, and incubating at room temperature for 10 minutes. Transfer the cells to a tube. Then, wash the well with 2 milliliters of media and add it to the same tube. Centrifuge the cells at 300 times g for 5 minutes. Then, resuspend the pellet in media and plate the cells onto matrix-coated six-well plates at the seating density of 1 times 10 to the fifth cells per well.
On the next day, add lentiviruses expressing Ngn2 plus PuroR and tetracycline transactivator together with polybrene in fresh ES cell maintenance medium. On day zero, add 2 micrograms per milliliter doxycycline in DMEM/F12 medium supplemented with N2 and without morphogens. On day one, add puromycin in fresh DMEM/F12 plus N2 and doxycycline to the final concentration of 1 microgram per milliliter.
Select the transduced cells in puromycin for at least 24 hours. On day two, detach differentiating neurons with cell detachment solution and replate them on 24-well plates coated with matrix solution. Maintain them in NBA/B27 medium without doxycycline. Culture pure induced neurons on the plates coated with extracellular matrix-based solutions.

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1. Human neuron induction from human pluripotent stem cells
<ol>
	<li>Lentivirus preparation (~5 days, detailed protocol as described previously)
	<ol>
		<li>Plate ~1 million HEK293T cells in each T75 flask to have them ~40% confluent when performing transfection. Transfect them with plasmids expressing tetracycline-inducible Ngn2 and puromycin-resistant gene (PuroR; under the same TetO promoter control), rtTA, and the three helper plasmids pRSV-REV, pMDLg/pRRE, and VSV-G (12 &#181;g of lentiviral vector DNA and 6 &#181;g of each of the helper plasmid DNA). Prepare at least three flasks per lentivirus preparation. Use PEI for transfection following the manufacturer&#8217;s instructions. Change the media after 16 h and discard.</li>
		<li>Harvest released viral particles by collecting culture media every day and replace with fresh media for 3 days. Pool the collected media containing viral particles for purification. Filter the virus through a 0.22 &#181;m filter and centrifuge at 49,000 x g for 90 min. Resuspend the pellet in the appropriate volume of PBS-glucose (~150 &#181;L).</li>
	</ol>
	</li>
	<li>Neuron Induction (~5 days) 
	NOTE: This induction protocol (Figure 1A; flow diagram) is highly effective for both iPS and ES cells of validated pluripotency (which can be assayed by immunohistochemistry staining of well-characterized pluripotency markers; Figure 1B).
	<ol>
		<li>Use commercially available H1 human ES cells at the passage of 52 (see Table of Materials). Culture the cells on extracellular matrix solution-coated 6-well plates (~0.5 mg of matrix solution per 6-well plate; see Table of Materials) using ES cell maintenance medium (see Table of Materials) and incubate the plates at 37 &#176;C with 5% CO2.</li>
		<li>On Day -2, detach ES cells (80% confluent) with 1 mL of cell detachment solution (see Table of Materials) and incubate at room temperature for 10 min. Transfer the cells to a tube; wash the well with 2 mL of media and combine in the same tube. Centrifuge at 300 x g for 5 min, resuspend the pellet in media, and plate the cells onto matrix-coated 6-well plates at the seeding density of 1 x 105 cells per well.</li>
		<li>On Day -1, add lentiviruses expressing Ngn2 plus PuroR and rtTA together with polybrene (8 &#181;g/ml) to the ES cells in fresh ES cell maintenance medium (see Table of Materials). The exact amount of viruses should be determined by actual titers or the titration. We typically add 5 &#181;L each virus per well in a 6-well plate.</li>
		<li>On Day 0, add Doxycycline (2 &#181;g/mL to activate Ngn2 expression) in DMEM-F12 medium with N2 supplement without morphogens.</li>
		<li>On Day 1, add Puromycin in fresh medium of DMEM-F12 plus N2 and doxycycline, to the final concentration of 1 &#181;g/mL medium. Select the transduced cells in Puromycin for at least 24 h. Higher Puromycin concentration (up to 5 &#181;g/mL) and longer selection period (up to 48 h) may be required to adequately remove the under-transduced cells if the virus titer is low.</li>
		<li>On Day 2, detach differentiating neurons with cell detachment solution (see Table of Materials), re-plate them on 24-well plates (between 80,000&#8211;200,000 cells/well) coated with matrix solution (see Table of Materials), and maintain them in NBA/B27 medium without doxycycline. The seeding density is critical.</li>
		<li>At this stage, detached neurons can be frozen in a specialized commercial freezing medium (see Table of Materials) and stored in liquid nitrogen for up to 3 months. Pure neurons can be plated accounting for the typical ~15%&#8211;20% cell death post-thaw, cultured alone or co-cultured with other brain cell types.</li>
		<li>Culture pure iNs on the plates coated with extracellular matrix-based solutions as instructed by the manufacturer (see Table of Materials). The characteristic pyramidal morphology should be apparent by Day 4 (and Day 6; Figure 1C). The synapse formation can be detected as early as Day 14 to 16 and is prominent at Day 24 by immunohistochemical staining with standard pre- and post-synaptic markers. (Figure 1D; labeled with the pre-synaptic marker Synapsin 1 and the dendritic marker Map2).</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accutase</td>
			<td>STEMCELL Technologies</td>
			<td>7920</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>ThermoFisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 medium</td>
			<td>STEMCELL Technologies</td>
			<td>36254</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>ThermoFisher</td>
			<td>D12345</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>MilliporeSigma</td>
			<td>D3072</td>
			<td></td>
		</tr>
		<tr>
			<td>H1 human ES cells</td>
			<td>WiCell</td>
			<td>WA01</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR plus</td>
			<td>STEMCELL Technologies</td>
			<td>5825</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>ThermoFisher</td>
			<td>17502001</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal A medium</td>
			<td>ThermoFisher</td>
			<td>10888-022</td>
			<td></td>
		</tr>
		<tr>
			<td>Non Essential Amino Acids</td>
			<td>ThermoFisher</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>PEI</td>
			<td>VWR</td>
			<td>71002-812</td>
			<td></td>
		</tr>
		<tr>
			<td>pMDLg/pRRE</td>
			<td>Addgene</td>
			<td>12251</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>MilliporeSigma</td>
			<td>TR-1003-G</td>
			<td></td>
		</tr>
		<tr>
			<td>pRSV-REV</td>
			<td>Addgene</td>
			<td>12253</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>ThermoFisher</td>
			<td>A1113803</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff Neural Progenitor Freezing Media</td>
			<td>STEMCELL Technologies</td>
			<td>5838</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff SMADi Neural Induction Kit</td>
			<td>STEMCELL Technologies</td>
			<td>8581</td>
			<td></td>
		</tr>
		<tr>
			<td>Tempo-iOlogo: Human iPSC-derived OPCs</td>
			<td>Tempo BioScience</td>
			<td>SKU102</td>
			<td></td>
		</tr>
		<tr>
			<td>TetO-Ng2-Puro</td>
			<td>Addgene</td>
			<td>52047</td>
			<td></td>
		</tr>
		<tr>
			<td>VSV-G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Assetta, B., et al., <a href="https://app.jove.com/v/61778">Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions.</a> J. Vis. Exp. (2020).
This video demonstrates the process of inducing human pluripotent stem cells to differentiate into neurons by transfecting them with engineered lentiviruses carrying a neurogenic transcription factor gene under the control of&#160; tetracycline-responsive element, followed by treatment with a tetracycline derivative and maturation medium.

<img alt="Figure 1" src="/files/ftp_upload/22599/22599_Figure 1.jpg" /> 
Figure 1: Direct Generation of human-induced neurons (iNs) from hPSCs. (A) Flow diagram of iN generation. (B) Representative bright field and immunofluorescence images of the starting culture of human pluripotent stem cells (H1) to confirm the pluripotency. Oct4 is shown in red and Sox2 in green. (C) Representative bright field images of iNs at ...

1. Human neuron induction from human pluripotent stem cells

	Lentivirus preparation (~5 days, detailed protocol as described previously)
	
		Plate ~1 ...

Treat the adhered human pluripotent stem cells with engineered lentiviruses in a transfection reagent.
The virus carries genes for antibiotic resistance and the neurogenic transcription factor, controlled by the tetracycline-responsive and regulatory elements.
The positively charged transfection reagent enhances viral fusion and its content release.
Viral RNA reverse-transcribes into DNA and integrates into the stem cell DNA, enabling the expression of regulatory elements that produce activator proteins.
Add a basal medium with tetracycline derivative, which forms a complex with the activator proteins.
This complex binds to the tetracycline-responsive element, promoting gene expression and producing the neurogenic transcription factors that induce stem cell differentiation into neurons.
Add a basal medium with an antibiotic drug to eliminate the untransfected cells.
Treat the cells with a detachment solution containing proteolytic enzymes that degrade the extracellular matrix or ECM and release the cells.
Remove the enzymes and re-plate the cells onto ECM-coated wells with a maturation medium, promoting neuronal maturation.

16 ビュー. 人工レンチウイルスを用いたニューロンにおけるヒト多能性幹細胞分化

ビデオ: 人工レンチウイルスを用いたニューロンにおけるヒト多能性幹細胞分化 - 実験

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

マイクロ電極アレイ上のネットワーク・アクティビティを測定するための人工多能性幹細胞の急速な神経分化

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

JoVE Journal

発生生物学

Transient Treatment of Human Pluripotent Stem Cells with DMSO to Promote Differentiation

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and iPSCs) across various lineages remain. Numerous differentiation protocols have been developed, yet variability across cell lines and low rates of differentiation impart challenges in successfully implementing these protocols. Described here is an easy and inexpensive means to enhance the differentiation capacity of PSCs. It has been previously shown that treatment of stem cells with a low concentration of dimethyl sulfoxide (DMSO) significantly increases the propensity of a variety of PSCs to differentiate to different cell types following directed differentiation. This technique has now been shown to be effective across different species (e.g., mouse, primate, and human) into multiple lineages, ranging from neurons and cortical spheroids to smooth muscle cells and hepatocytes. The DMSO pretreatment improves PSC differentiation by regulating the cell cycle and priming stem cells to be more responsive to differentiation signals. Provided here is the detailed methodology for using this simple tool as a reproducible and widely applicable means to more efficiently differentiate PSCs to any lineage of choice.

Generating differentiated cell types from human pluripotent stem cells (hPSCs) holds great therapeutic promise but remains challenging. PSCs often exhibit an inherent inability to differentiate even when stimulated with a proper set of signals. Described here is a simple tool to enhance multilineage differentiation across a variety of PSC lines.

DMSOを用したヒト多能性幹細胞の一過性治療による分化を促進

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Human Pluripotent Stem Cell Differentiation in Neurons Using Engineered Lentiviruses

記録

Human Pluripotent Stem Cell Differentiation in Neurons Using Engineered Lentiviruses