eoe sub articles

EoE Sub Articles

1. Stem Cell Maintenance
NOTE: The cell maintenance protocol described below applies to pluripotent stem cells (PSCs) maintained in an adherent monolayer. Media, other reagents, and cell culture plates used prior to dimethyl sulfoxide (DMSO) treatment can be adjusted as needed. For all the following protocols in this manuscript, cells should be handled under a biological safety cabinet.
<ol>
	<li>Coat sterile, 6 well, tissue culture-treated plates with a pluripotent stem cell-qualified matrix or substrate prepared per the manufacturer&#39;s instructions and incubate for at least 1 h in a CO2&#160;incubator (5% CO2, humid atmosphere). Coated plates can be film wrapped and stored at 4 &#176;C for up to one week.</li>
	<li>Thaw the cryopreserved PSCs in a 37 &#176;C water bath. Sterilize vial with ethanol prior to introduction to the biological safety cabinet, then immediately transfer the cells by pipetting to a sterile conical tube containing 5-10 volumes of prewarmed stem cell media.</li>
	<li>Centrifuge the cells at 300 x&#160;g&#160;for 5 min at room temperature (RT).</li>
	<li>Aspirate the media and gently resuspend the cell pellet in 1 mL of stem cell media supplemented with a 10 &#181;M rho-associated protein kinase&#160;(ROCK) inhibitor, such as Y-27632.</li>
	<li>Aspirate the culture matrix from the plate and seed the cells at the desired density, typically 0.5-1 x 106&#160;cells per well in at least 2 mL of stem cell media per well. 
	NOTE:&#160;The plating density can vary across different cell lines and clones and should be optimized accordingly.</li>
	<li>Maintain the cells by replacing with prewarmed stem cell media daily. Split the cells at roughly 70%-80% confluency or when the cell colonies begin to make contact.</li>
	<li>For splitting cells, aspirate the media and wash the cells once with sterile phosphate-buffered saline (PBS). Incubate the cells with 1 mL of a dissociation enzyme solution per well for 5-10 minutes at 37 &#176;C.</li>
	<li>Wash and resuspend the cells with prewarmed stem cell media and transfer to a sterile conical tube with 5-10 volumes of stem cell media. Follow steps 1.3-1.7 to plate the cells.</li>
</ol>
2. DMSO Pretreatment
NOTE:&#160;When plating the cells for DMSO pretreatment prior to differentiation, the starting plating cell density should be optimized with consideration of the typical growth rate of the stem cell line as well as the differentiation protocol being used. Validate pluripotency using conventional markers, as necessary. Cells should be passaged at least 1x-2x after initial thawing prior to differentiation.
<ol>
	<li>2D culture differentiation
	<ol>
		<li>When the cells reach an appropriate confluency, prepare coated plates, dissociate the cells, and prepare a single-cell suspension as described above.</li>
		<li>Count the live cells using a hemocytometer or automatic cell counter including trypan blue or another viability marker.</li>
		<li>Plate the cells onto a coated 6 well plate at 0.5-1 x 106&#160;cells per well in stem cell media with the 10 &#181;M ROCK inhibitor.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
		NOTE:&#160;For the cell lines tested in our laboratory, these densities typically resulted in 80%-90% confluent cells within the 24 h DMSO pretreatment.</li>
		<li>Allow cells to incubate for 24 h at 37 &#176;C in a CO2&#160;incubator (5% CO2, humid atmosphere).</li>
		<li>Prepare 1%-2% DMSO in prewarmed stem cell media (e.g., 100 &#181;L of DMSO in 10 mL of media = 1% DMSO solution, or 200 &#181;L DMSO in 10 mL of media = 2% DMSO solution).</li>
		<li>After 24 h incubation, aspirate the media from cells and replace it with DMSO solution.</li>
		<li>Allow the cells to incubate for 24 h to 48 h at 37 &#176;C in a CO2&#160;incubator (5% CO2, humid atmosphere) prior to differentiation.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
		NOTE:&#160;Typically, a 24 h DMSO treatment is sufficient across a majority of human embryonic and induced pluripotent stem cell (ESC and iPSC) lines. Cell lines with very slow growth rates (long doubling times) can benefit from the 48 h incubation with DMSO. For a 48 h incubation with DMSO, media can be replaced with fresh stem cell media with 1%-2% DMSO after the first 24 h of treatment.</li>
	</ol>
	</li>
</ol>
3. Differentiation to Primary Germ Layers
NOTE:&#160;The following describes methods previously shown to be effective in our laboratory for PSCs grown in a monolayer on 6 well plates. Any differentiation protocol of choice should be used after the DMSO treatment to promote differentiation into desired lineages. Remove DMSO solution after a 24-48 h treatment and proceed with differentiation following standard protocols.
<ol>
	<li>Ectoderm differentiation
	<ol>
		<li>Pretreat the cells with DMSO as described above for 2D cultures.</li>
		<li>Prepare Noggin and SB431542 stock solutions.</li>
		<li>Prepare ectodermal differentiation base media by dissolving knockout serum replacement (KOSR) to a final concentration of 10% in knockout Dulbecco&#39;s Modified Eagle Medium (DMEM).&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
		NOTE:&#160;Prepare enough base media for 3-4 days of media change.</li>
		<li>Prepare ectodermal differentiation media by adding Noggin to a final concentration of 500 ng/mL and SB431542 to a final concentration of 10 &#181;M to the appropriate volume of prewarmed KOSR/knockout DMEM.</li>
		<li>After DMSO pretreatment, aspirate media from cells and replace with differentiation media (e.g., 2 mL per well of a 6 well plate).</li>
		<li>Allow cells to incubate for 3-4 days at 37 &#176;C in a CO2&#160;incubator (5% CO2, humid atmosphere), replacing media daily with freshly added differentiation factors.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II FL Automated Cell Counter</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>AMQAF1000</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>Gibco</td>
			<td>10829018</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Matrix</td>
			<td>Corning</td>
			<td>354277</td>
			<td></td>
		</tr>
		<tr>
			<td>Noggin Fc Chimera Protein</td>
			<td>R&#38;D Systems</td>
			<td>3344-NG-050</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Stemgent</td>
			<td>04-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Low Attachment Microplates</td>
			<td>Corning</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>StemCell Technologies</td>
			<td>72302</td>
			<td></td>
		</tr>
	</tbody>
</table>

enhancing stem cell differentiation with a transient dmso treatment

Source: Sambo, D. et al., <a href="https://app.jove.com/v/59833">Transient Treatment of Human Pluripotent Stem Cells with DMSO to Promote Differentiation.</a> J. Vis. Exp. (2019)
This video demonstrates the method for enhancing stem cell differentiation with transient DMSO treatment, which arrests cells in the G1 phase. Post-treatment, an ectodermal differentiation medium with BMP and TGF-&#946; inhibitors is added to promote neural precursor formation.

Enhancing Stem Cell Differentiation with a Transient DMSO Treatment

1. Stem Cell Maintenance
NOTE: The cell maintenance protocol described below applies to pluripotent stem cells (PSCs) maintained in an adherent monolayer. ...

Begin with a multi-well plate containing adhered human pluripotent stem cells with multilineage differentiation ability.&#160;
Introduce a stem cell medium containing dimethyl sulfoxide or DMSO.
DMSO enters the cells and prevents the progression of the cell cycle from the Gap 1, or G1 phase, an initial stage of the cell cycle, to the synthesis, or S phase.
This creates a uniform population of cells in the G1 phase, enhancing their differentiation ability.
Replace the medium with a nutrient-rich ectodermal differentiation medium containing inhibitors of the BMP and TGF-&#946; signaling pathways.
These inhibitors bind to specific ALK receptors on the stem cells, blocking the BMP and TGF-&#946; signaling pathways and preventing their differentiation into endoderm and mesoderm precursors.
This allows the selective differentiation of G1-arrested stem cells into ectodermal precursors possessing neural differentiation ability.
Regularly replace the medium with a fresh medium to maintain the nutrient and differentiation factor levels, ensuring cell survival.

enhancing-stem-cell-differentiation-with-a-transient-dmso-treatment

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

64 Views. Fonte: Sambo, D. et al., Trattamento transitorio di cellule staminali pluripotenti umane con DMSO per promuovere la differenziazione. J. Vis. Exp. (2019) Questo video dimostra il metodo per migliorare la differenziazione delle cellule staminali con il trattamento transitorio DMSO, che arresta le cellule nella fase G1. Dopo il trattamento, viene aggiunto un mezzo di differenziazione ectodermica con inibitori di BMP e TGF-β per promuovere la formazione di precursori neurali. 

Video: Miglioramento della differenziazione delle cellule staminali con un trattamento transitorio con DMSO - Concept

Production and Characterization of Human Macrophages from Pluripotent Stem Cells

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key players in health and disease, acting as phagocytes during immune defense and mediating trophic, maintenance, and repair functions. Although it has been possible to study some of the molecular processes involved in human macrophage function, it has proved difficult to apply genetic engineering techniques to primary human macrophages. This has significantly hampered our ability to interrogate the complex genetic pathways involved in macrophage biology and to generate models for specific disease states. An off-the-shelf source of human macrophages that is amenable to the vast arsenal of genetic manipulation techniques would, therefore, provide a valuable tool in this field. We present an optimized protocol that allows for the generation of macrophages from human induced pluripotent stem cells (iPSCs) in vitro. These iPSC-derived macrophages (iPSC-DMs) express human macrophage cell surface markers, including CD45, 25F9, CD163, and CD169, and our live-cell imaging functional assay demonstrates that they exhibit robust phagocytic activity. Cultured iPSC-DMs can be activated to different&#160;macrophage states that display altered gene expression and phagocytic activity by the addition of LPS and IFNg, IL4, or IL10. Thus, this system provides a platform to generate human macrophages carrying genetic alterations that model specific human disease and a source of cells for drug screening or cell therapy to treat these diseases.

This protocol describes the robust generation of macrophages from human induced pluripotent stem cells, and methods for their subsequent characterization. Cell surface marker expression, gene expression, and functional assays are used to assess the phenotype and function of these iPSC-derived macrophages.

Produzione e caratterizzazione di macrofagi umani da cellule staminali pluripotenti

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key ...

Ricerca

JoVE Journal

Immunologia e infezioni

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings.

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible piggyBac vectors.

Conversione di cellule staminali pluripotenti indotte dall'uomo (IPSC) in neuroni motore spinale e cranico funzionale utilizzando vettori PiggyBac

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

Enhancing Stem Cell Differentiation with a Transient DMSO Treatment

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Enhancing Stem Cell Differentiation with a Transient DMSO Treatment