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1. Cell Culture and Reagent Preparation
<ol>
	<li>Prepare coated plates for cell culture.
	<ol>
		<li>Dilute extracellular matrix (ECM) coating solution with DMEM/F12 media to prepare a 1 mg/mL stock solution. Aliquot the diluted ECM stock solution into 30 conical tubes of 3 mL each and immediately store in -20 &#176;C. For a working solution, resuspend 3 mL of ECM stock into 33 mL of pre-chilled DMEM/F12 media, bringing the total volume to 36 mL for a concentration of 80 &#181;g/mL.</li>
		<li>Coat 1 mL per well of a 6-well cell culture plate with ECM working solution. Add an additional 1 mL DMEM/F12 per well (if necessary) to ensure that the surface of the well is fully covered. Allow the coated 6-well cell culture plates to sit at room temperature for at least 1 h before use. 
		&#8203;NOTE: Coated plates may be stored in the incubator or at 4 &#176;C for up to 2 weeks.</li>
	</ol>
	</li>
	<li>Prepare media formulations, growth factors, and small molecules.
	<ol>
		<li>To prepare 500 mL of human pluripotent stem cell (hPSC) medium, add 20x and 500x supplements according to manufacturers&#39; instructions.</li>
		<li>To prepare 500 mL of neural medium (NM), add heparin to a final concentration of 2 mg/mL, 1x of antibiotic/antimycotic solution, 1x B27 supplement or 1x N2 supplement to a 500 mL bottle of DMEM/F12 with L-glutamine supplement as previously detailed.</li>
		<li>To prepare a 10 mM (1,000x) stock solution of Rho-kinase inhibitor Y27632 (Y), add 10 mg of powder into 3 mL of phosphate-buffered saline (PBS). Filter, sterilize, aliquot, and store at -20 &#176;C. Use at a 10 &#181;M working concentration in media.</li>
		<li>To prepare a 20 mM (10,000x) stock solution of SB431542, add 10 mg of powder into 1.3 mL of dimethyl sulfoxide (DMSO). To prepare a 2 mM (1,000x) stock solution of DMH1, add 10 mg of powder into 13.1 mL of DMSO. Filter, sterilize, aliquot, and store each solution at -20 &#176;C. Use each at a 2 &#181;M working concentration in media (NM + SB431542 + DMH1). 
		NOTE: This protocol recommends 2 &#181;M working concentrations of both SB431542 and DMH1 based on our observations and reports from others stating that this concentration effectively promotes neural induction from hPSCs. Higher concentrations have also been reported30. Additionally, with alternative media, it has also been shown that small molecules are not necessary for high neural conversion. Thus, working concentrations will vary depending on alternative cell culture environments, and optimal concentrations should be tested by individual researchers.</li>
		<li>To prepare individual 100 &#181;g/mL (10,000x) stock solutions of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF2), add 1 mg of each into 10 mL of PBS + 0.1% BSA. Aliquot EGF and FGF2 stock solutions into 50 &#181;L aliquots and store at -80 &#176;C. Use each at a 10 ng/mL working concentration in media; e.g., add 5 &#181;L EGF and 5 &#181;L FGF2 to 50 mL of NM (NM + EGF + FGF2).</li>
		<li>To prepare a stock solution of doxycycline hydrochloride (Dox), first dissolve powder in DMSO to 100 mg/mL and store at -20 &#176;C. Further, dilute it to make a 2 mg/mL (1,000x) stock solution in PBS and store at -20 &#176;C. Use at 2 &#181;g/mL working concentration in media; e.g., add 50 &#181;L Dox to 50 mL of NM (NM + Dox). 
		NOTE: Do not re-freeze solutions after thawing.</li>
	</ol>
	</li>
</ol>
2. Generation of Neural Subtypes from Human Induced Pluripotent Cells (hPSCs)
NOTE: All cell cultures should be maintained in an incubator with 5% CO2 at 37 &#176;C. These cultures are maintained at room oxygen levels, though lower levels may be utilized.
<ol>
	<li>Generate and maintain astrocyte progenitors (hAstros) from hPSCs. 
	NOTE: This section provides a brief and simplified differentiation protocol. For a detailed description (with embryoid body formation, rosette selection, and regional patterning steps), refer to the previously described protocol (Figure 1A, dotted green box).
	<ol>
		<li>Seed clusters of hPSCs (Figure 1B) onto ECM-coated 6-well plates (see step 1.1) with 2 mL of hPSC medium + Y per well. Feed the stem cells every day until they are about 50% confluent and split as needed.
		<ol>
			<li>To split hPSCs, add 1 mL of passaging reagent to wells and aspirate after 1 min. Incubate cells at room temperature for 5 min, add 1 mL of hPSC medium, and split aggregates 1:10 onto new ECM-coated wells in 2 mL of hPSC medium + Y per well.</li>
		</ol>
		</li>
		<li>Maintain the stem cells until they are about 50% confluent, then change media to NM + SB431542 + DMH1 to induce neural differentiation (day 0). When cells are about 95% confluent, split 1:6 into new ECM-coated wells in the same medium.</li>
		<li>On day 14, dissociate the cells with the detachment solution and transfer them to a non-coated flask with Y to promote the formation of aggregates. 
		NOTE: The addition of Y is utilized to promote cell survival and sphere formation but is not included during every feed.</li>
		<li>For the generation of neuronal progenitors and neurons (hNeurons), use these day-14 cells as described below. Alternatively, utilize these cells at later time points from monolayer or sphere cultures before neuronal maturation occurs or gliogenesis commences. 
		NOTE: By default, this protocol produces dorsal-cortical astrocytes. However, astrocyte subtypes can be regionally specified by the addition of patterning morphogens if desired. Retinoic acid (RA) may be added to caudalize cells into spinal cord phenotypes, while sonic hedgehog (SHH), smoothened agonist (SAG), or purmorphamine will produce ventral phenotypes.</li>
		<li>For the generation of astrocyte progenitors and astrocytes in spontaneously-formed 3D aggregates (hAstrospheres), switch to NM + EGF + FGF2 and feed weekly (or as needed to ensure stable pH) as previously detailed.
		<ol>
			<li>Gently dissociate hAstro aggregates with detachment solution when dark centers appear and remove spheres that spontaneously attach. Break spheres gently once a week to maintain sphere health and to avoid necrotic cores. 
			NOTE: Do not exceed 5 min of treatment with detachment solution.</li>
			<li>After 4-6 months of expansion, confirm cell identity and either freeze in cryopreservation medium (according to manufacturer&#39;s instructions) or alternative storage conditions for long-term preservation in liquid nitrogen or use immediately for experimentation.</li>
		</ol>
		</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 plate</td>
			<td>Fisher Scientific</td>
			<td>08-772-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>Olympus Plastics</td>
			<td>28-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Sigma</td>
			<td>A6964-100ML</td>
			<td>Detachment solution</td>
		</tr>
		<tr>
			<td>AggreWell plate</td>
			<td>Stemcell Technologies</td>
			<td>34850</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Adherence Rinsing Solution</td>
			<td>Stemcell Technologies</td>
			<td>7010</td>
			<td>Prevent cell adhesion to microwell plates</td>
		</tr>
		<tr>
			<td>Anti/anti</td>
			<td>Thermofisher</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Thermofisher</td>
			<td>17504044</td>
			<td>Media Supplement</td>
		</tr>
		<tr>
			<td>BrainPhys neuronal medium</td>
			<td>Stemcell Technologies</td>
			<td>5790</td>
			<td>Neurophysiological basal medium alternative</td>
		</tr>
		<tr>
			<td>Cryostor CS10</td>
			<td>Stemcell Technologies</td>
			<td>7930</td>
			<td>Cryopreservation medium with 10% DMSO</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermofisher</td>
			<td>10565-042</td>
			<td>With GlutaMAX supplement</td>
		</tr>
		<tr>
			<td>DMH-1</td>
			<td>Stemcell Technologies</td>
			<td>73634</td>
			<td>HAZARD: Toxic if swallowed. Working concentration: 2 uM</td>
		</tr>
		<tr>
			<td>Doxycycline Hydrochloride (Dox)</td>
			<td>Sigma</td>
			<td>D3072-1ml</td>
			<td>HAZARD: Toxic for pregnant women. Working concentration: 2 ug/mL</td>
		</tr>
		<tr>
			<td>Epidermal growth factor (EGF)</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td>Working concentration: 10 ng/mL</td>
		</tr>
		<tr>
			<td>Fibroblast growth factor-2 (FGF)</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td>Working concentration: 10 ng/mL</td>
		</tr>
		<tr>
			<td>Hemacytometer or automatic cell counter</td>
			<td>Life Technologies</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel membrane matrix</td>
			<td>Corning</td>
			<td>354230</td>
			<td>ECM coating solution. Working concentration: 80 ug/ml. Prepare on ice and ensure that pipettes, tubes, and media are pre-chilled.</td>
		</tr>
		<tr>
			<td>Mounting solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Thermofisher</td>
			<td>17502048</td>
			<td>Media Supplement</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Stemcell Technologies</td>
			<td>CA008-300</td>
			<td></td>
		</tr>
		<tr>
			<td>ReLeSR</td>
			<td>Stemcell Technologies</td>
			<td>5872</td>
			<td>Detachment and passaging reagent</td>
		</tr>
		<tr>
			<td>Rho-Kinase Inhibitor Y27632- (Y)</td>
			<td>Tocris</td>
			<td>1254</td>
			<td>Working concentration: 10 uM</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Stemcell Technologies</td>
			<td>72234</td>
			<td>Working concentration: 2 uM</td>
		</tr>
		<tr>
			<td>T25 Culture Flask</td>
			<td>Olympus Plastics</td>
			<td>25-207</td>
			<td>Vented caps</td>
		</tr>
		<tr>
			<td>T75 Culture Flask</td>
			<td>Olympus Plastics</td>
			<td>25-209</td>
			<td>Vented caps</td>
		</tr>
	</tbody>
</table>

generating and maintaining astrospheres using human pluripotent stem cells

Source: Cvetkovic, C., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/58034">Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells.</a> J. Vis. Exp. (2018).
The video demonstrates a method to generate astrospheres from progenitors from human pluripotent stem cells. It involves the growth and proliferation of stem cells with nutrient media containing growth factors and kinase inhibitors. These cells then aggregate and differentiate into astrocytes, forming astrospheres.

<img alt="Figure 1" src="/files/ftp_upload/22390/22390_Figure1.jpg" /> 
Figure 1: Stepwise depiction of the differentiation and formation of 3D neural spheres derived from hPSCs. (A) Timeline of key steps in the protocol. (B) Pure populations of neural cells can be generated from human induced pluripotent stem cells (hPSCs). For the generation of astrocyte progenitors (dotted green box), see step 2.1. (C) Inducible neurons (iNeurons; see section 2.2) generated from t...

Generating and Maintaining Astrospheres Using Human Pluripotent Stem Cells

1. Cell Culture and Reagent Preparation

	Prepare coated plates for cell culture.
	
		Dilute extracellular matrix (ECM) coating solution with DMEM/F12 ...

Begin with an ECM-coated plate and seed clusters of human pluripotent stem cells in a growth medium containing Rho-kinase inhibitors.
ECM enhances cell attachment, while the inhibitors block Rho-kinases, preventing apoptotic cell death and promoting cell survival and proliferation.
Introduce a neural differentiation medium to induce stem cell differentiation into neural progenitor cells.
Treat with a detachment solution; the enzymes in this solution degrade the ECM and cell contact, releasing the cells.
Transfer them into a non-coated flask containing Rho-kinase inhibitors.
The inhibitors facilitate cell survival and spontaneous aggregation into three-dimensional structures or spheroids.
Supplement the spheroids with a growth factor-rich neural differentiation medium, allowing neural progenitor cells to differentiate into astrocyte progenitors and form astrospheres.
Regularly, add a detachment solution to dissociate the astrospheres into smaller clusters. This allows nutrients to reach the inner cells of the spheroid and maintain their viability.
Maintain the astrospheres in a fresh medium for progenitors to mature into astrocytes.

generating-maintaining-astrospheres-using-human-pluripotent-stem

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

43 visualizações. Fonte: Cvetkovic, C., et al. Modelagem de microcircuitos sinápticos com coculturas 3D de astrócitos e neurônios de células-tronco pluripotentes humanas. J. Vis. Exp. (2018). O vídeo demonstra um método para gerar astrosferas a partir de progenitores a partir de células-tronco pluripotentes humanas. Envolve o crescimento e proliferação de células-tronco com meios nutrientes contendo fatores de crescimento e inibidores de quinase. Essas células então se agregam e se diferenciam em ast...

Vídeo: Geração e manutenção de astrosferas usando células-tronco pluripotentes humanas - Conceito

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

Isolamento e Cultura de neuroesferas Zebrafish Adulto derivado do cérebro

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

JoVE Journal

Biologia do Desenvolvimento

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

Isolamento e Cultura de astrócitos corticais de rato

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Neurociência

A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long period of disease progression prior to the onset of symptoms. Because fibroblasts from patients and healthy controls can be efficiently reprogrammed into human induced pluripotent stem cells (hiPSCs), and subsequently differentiated into neural progenitor cells (NPCs) and neurons for the study of these diseases, it is now possible to recapitulate the developmental events that occurred prior to symptom onset in patients. We present a method by which to efficiently differentiate hiPSCs into NPCs, which in addition to being capable of further differentiation into functional neurons, can also be robustly passaged, freeze-thawed or transitioned to grow as neurospheres, enabling rapid genetic screening to identify the molecular factors that impact cellular phenotypes including replication, migration, oxidative stress and/or apoptosis. Patient derived hiPSC NPCs are a unique platform, ideally suited for the empirical testing of the cellular or molecular consequences of manipulating gene expression.

This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.

Um Guia para a geração e uso hiPSC NPCs derivados para o Estudo das Doenças Neurológicas

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long ...

Generating and Maintaining Astrospheres Using Human Pluripotent Stem Cells

Transcrição

Generating and Maintaining Astrospheres Using Human Pluripotent Stem Cells