We would like to thank Ines M&#252;hlhahn for cloning the constructs for reprogramming, Paulina Chlebik for viral production, and Magdalena G&#246;tz and Judith Fischer-Sternjak for comments on the manuscript.

The following procedure follows the animal care guidelines of the Helmholtz Zentrum Munich in accordance with the directive 2010/63/EU on the protection of animals used for scientific purposes. Please make sure to comply with the animal care guidelines of the institution where the dissection is performed.
1. Preparation of dissection, dissociation, and culture materials
NOTE: Prepare all culture reagents within a biological safety cabinet and work usi...

<ol>
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H., Hess, H. H., Pope, A., Thalheimer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+cytoarchitectonic+distribution+of+neurons,+glia,+and+DNa+in+rat+cerebral+cortex.">Quantitative cytoarchitectonic distribution of neurons, glia, and DNa in rat cerebral cortex.</a> The Journal of Comparative Neurology. 143 (4), 481-490 (1971).</li><li>Yoon, H., Walters, G., Paulsen, A. R., Scarisbrick, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+heterogeneity+across+the+brain+and+spinal+cord+occurs+developmentally,+in+adulthood+and+in+response+to+demyelination.">Astrocyte heterogeneity across the brain and spinal cord occurs developmentally, in adulthood and in response to demyelination.</a> PLoS One. 12 (7), 0180697 (2017).</li><li>Taverna, E., Gotz, M., Huttner, W. 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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-mediated+induction+of+neuron-enriched+mitochondrial+proteins+boosts+direct+glia-to-neuron+conversion.">CRISPR-mediated induction of neuron-enriched mitochondrial proteins boosts direct glia-to-neuron conversion.</a> Cell Stem Cell. 28 (3), 524-534 (2021).</li><li>Guo, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonstochastic+reprogramming+from+a+privileged+somatic+cell+state.">Nonstochastic reprogramming from a privileged somatic cell state.</a> Cell. 156 (4), 649-662 (2014).</li><li>Hu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Region-restrict+astrocytes+exhibit+heterogeneous+susceptibility+to+neuronal+reprogramming.">Region-restrict astrocytes exhibit heterogeneous susceptibility to neuronal reprogramming.</a> Stem Cell Reports. 12 (2), 290-304 (2019).</li><li>Ge, W. P., Miyawaki, A., Gage, F. H., Jan, Y. N., Jan, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+generation+of+glia+is+a+major+astrocyte+source+in+postnatal+cortex.">Local generation of glia is a major astrocyte source in postnatal cortex.</a> Nature. 484 (7394), 376-380 (2012).</li><li>Price, J. 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Primary cultures of murine astrocytes are a remarkable in vitro model system to study direct neuronal reprogramming. In fact, despite being isolated at a postnatal stage, cells express typical astrocyte markers29, retain the expression of patterning genes28,29, and maintain the capacity to proliferate, similar to in vivo astrocytes at a comparable age38. After MACS-mediated isolation, cells first a...

The mammalian central nervous system (CNS) is highly complex, consisting of hundreds of different cell types, including a vast number of different neuronal subtypes1,2,3,4,5,6. Unlike other organs or tissues7,8,9, the mammalian CNS has a very limited regenerative capacity; neuronal loss following traumatic brain injury or neurodegeneration is irreversible and often results in motor and cognitive deficits10. Aiming to rescue brain functions, different strategies to replace lost neurons are under intense investigation11. Among them, direct reprogramming of somatic cells into functional neurons is emerging as a promising therapeutic approach12. Direct reprogramming, or transdifferentiation, is the process of converting one differentiated cell type into a new identity without passing through an intermediate proliferative or pluripotent state13,14,15,16. Pioneered by the identification of MyoD1 as a factor sufficient to convert fibroblasts into muscle cells17,18, this method has been successfully applied to reprogram several cell types into functional neurons19,20,21.
Astrocytes, the most abundant macroglia in the CNS22,23, are a particularly promising cell type for direct neuronal reprogramming for several reasons. First, they are widely and evenly distributed across the CNS, providing an abundant in loco source for new neurons. Second, astrocytes and neurons are developmentally closely related, as they share a common ancestor during embryonic development, the radial glial cells24. The common embryonic origin of the two cell types seems to facilitate neuronal conversion as compared to reprogramming of cells from different germ layers19,21. Furthermore, patterning information inherited by astrocytes through their radial glia origin is also maintained in adult astrocytes25,26,27, and seems to contribute to the generation of regionally appropriate neuronal subtypes28,29,30. Hence, investigating and understanding the conversion of astrocytes into neurons is an important part of achieving the full potential of this technique for cell-based replacement strategies.
The conversion of in vitro cultured astrocytes into neurons has led to several breakthroughs in the field of direct neuronal reprogramming, including: i) the identification of transcription factors sufficient to generate neurons from astrocytes15,19,31, ii) the unravelling of molecular mechanisms triggered by different reprogramming factors in the same cellular context32, and iii) highlighting the impact of developmental origin of the astrocytes on inducing different neuronal subtypes28,29,33. Furthermore, in vitro direct conversion of astrocytes unravelled several major hurdles that limit direct neuronal reprogramming34,35, such as increased reactive oxygen species (ROS) production34 and differences between the mitochondrial proteome of astrocytes and neurons35. Hence, these observations strongly support the use of primary cultures of astrocytes as a model for direct neuronal reprogramming to investigate several fundamental questions in biology12, related to cell identity maintenance, roadblocks preventing cell fate changes, as well as the role of metabolism in reprogramming.
Here we present a detailed protocol to isolate astrocytes from mice at postnatal (P) age with very high purity, as demonstrated by isolating astrocytes from the murine spinal cord29. We also provide protocols to reprogram astrocytes into neurons via viral transduction or DNA plasmid transfection. Reprogrammed cells can be analyzed at 7 days post-transduction (7 DPT) to assess various aspects, such as reprogramming efficiency and neuronal morphology, or can be maintained in culture for several weeks, to assess their maturation over time. Importantly, this protocol is not specific to spinal cord astrocytes and can be readily applied to isolate astrocytes from various other brain regions, including the cortical gray matter, midbrain, and cerebellum.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin/EDTA</td>
			<td>Life Technologies</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;, 6-Diamidino-2-phenyindole, dilactate (DAPI)</td>
			<td>Sigma-Aldrich</td>
			<td>D9564</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse IgG1 Alexa 647</td>
			<td>Thermo Fisher</td>
			<td>A21240</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Mouse IgG1 Biotin</td>
			<td>Southernbiotech</td>
			<td>Cat# 1070-08; RRID: AB_2794413</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse IgG2b Alexa 488</td>
			<td>Thermo Fisher</td>
			<td>A21121</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rabbit Alexa 546</td>
			<td>Thermo Fisher</td>
			<td>A11010</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqua Poly/Mount</td>
			<td>Polysciences</td>
			<td>Cat# 18606-20</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>Peprotech</td>
			<td>450-02</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>Life Technologies</td>
			<td>13256029</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumine (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>cAMP</td>
			<td>Sigma Aldrich</td>
			<td>D0260</td>
			<td></td>
		</tr>
		<tr>
			<td>C-Tubes</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Life Technologies</td>
			<td>21331020</td>
			<td></td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>Sigma Aldrich</td>
			<td>P5499</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Life Technologies</td>
			<td>PHG0311</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>PAN Biotech</td>
			<td>P30-3302</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma Aldrich</td>
			<td>F6886</td>
			<td></td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>Peprotech</td>
			<td>450-10</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS Octo Dissociator</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-427</td>
			<td></td>
		</tr>
		<tr>
			<td>GFAP</td>
			<td>Dako</td>
			<td>Cat# Z0334; RRID: AB_100013482</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G8769</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Life Technologies</td>
			<td>35050038</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Life Technologies</td>
			<td>14025050</td>
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			<td>Hepes</td>
			<td>Life Technologies</td>
			<td>15630056</td>
			<td></td>
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			<td>Lipofectamine 2000 (Transfection reagent)</td>
			<td>Thermo Fisher</td>
			<td>Cat# 11668019</td>
			<td></td>
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			<td>MACS SmartStrainer 70&#181;m</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-462</td>
			<td></td>
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			<td>MiniMACS Seperator</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
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			<td>Mouse anti-ACSA-2 MicroBeat Kit&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-678</td>
			<td></td>
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			<td>Mouse IgG1 anti-Synaptophysin 1</td>
			<td>Synaptic Systems</td>
			<td>Cat# 101 011 RRID:AB_887824)</td>
			<td></td>
		</tr>
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			<td>Mouse IgG2b anti-Tuj-1 (&#946;III-tub)</td>
			<td>Sigma Aldrich</td>
			<td>T8660</td>
			<td></td>
		</tr>
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			<td>MS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
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			<td>N2 Supplement</td>
			<td>Life Technologies</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Neural Tissue Dissociation Kit&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-628</td>
			<td></td>
		</tr>
		<tr>
			<td>NT3</td>
			<td>Peprotech</td>
			<td>450-03</td>
			<td></td>
		</tr>
		<tr>
			<td>octoMACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-109</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiMEM &#8211; GlutaMAX (serum-reduced medium)</td>
			<td>Thermo Fisher</td>
			<td>Cat# 51985-026</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma Aldrich</td>
			<td>P1149</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-RFP</td>
			<td>Rockland</td>
			<td>Cat# 600-401-379; RRID:AB_2209751</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-Sox9</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# AB5535; RRID:AB_2239761</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin Alexa 405</td>
			<td>Thermo Fisher</td>
			<td>Cat# S32351</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# T9284</td>
			<td></td>
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isolation and direct neuronal reprogramming of mouse astrocytes

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through multipotent intermediates. This technique not only holds great promises in the field of disease modeling, as it allows to convert, for example, fibroblasts for patients suffering neurodegenerative diseases into neurons, but also represents a promising alternative for cell-based replacement therapies. In this context, a major scientific breakthrough was the demonstration that differentiated non-neural cells within the central nervous system, such as astrocytes, could be converted into functional neurons in vitro. Since then, in vitro direct reprogramming of astrocytes into neurons has provided substantial insights into the molecular mechanisms underlying forced identity conversion and the hurdles that prevent efficient reprogramming. However, results from in vitro&#160;experiments performed in different labs are difficult to compare due to differences in the methods used to isolate, culture, and reprogram astrocytes. Here, we describe a detailed protocol to reliably isolate and culture astrocytes with high purity from different regions of the central nervous system of mice at postnatal ages via magnetic cell sorting. Furthermore, we provide protocols to reprogram cultured astrocytes into neurons via viral transduction or DNA transfection. This streamlined and standardized protocol can be used to investigate the molecular mechanisms underlying cell identity maintenance, the establishment of a new neuronal identity, as well as the generation of specific neuronal subtypes and their functional properties.

Primary cultures of astrocytes typically reach 80%-90% confluency between 7 to 10 days after MAC-sorting and plating (Figure 1B). Generally, a single T25 culture flask yields around 1-1.5 x 106 cells, which is sufficient for 20-30 coverslips when seeding cells at a density of 5-5.5 x 104 cells per well. The day after plating, cells typically cover 50%-60% of the coverslip surface (Figure 1C). At this stage, cultures consist almost exclusive...

Watch this Scientific Journal Video about Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes at JoVE.com

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

Here we describe a detailed protocol to generate highly enriched cultures of astrocytes derived from different regions of the central nervous system of postnatal mice and their direct conversion into functional neurons by the forced expression of transcription factors.

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through ...

Astrocytes are an interesting autogenous population to target for direct neuronal programming. This protocol provides a reliable technique to isolating culture astrocytes with high purity from different region or the central nervous system. This protocol is designed and optimized to investigate the ability of astrocytes to be reprogrammed into functional neurons without confounding factors such as differences in astrocytes purity of different startup population.Demonstrating the procedure will be done by Bob Hersbach, Postdoc in the lab, and Tatiana Simon a technical assistance in our laboratory. To begin, place the torso of a euthanized mouse in a 35 millimeter Petri dish and keep it on ice. Open the skin with scissors and remove the vertebra with small scissors.Then extract the spinal cord and place it in a dissection buffer on ice. Under a stereotactic dissection microscope with two times magnification, remove the meninges from the isolated spinal cords using forceps and transferred dissected and cleaned spinal cord tissue to a C tube. Add 50 microliters of enzyme P from the neural tissue dissociation kit and 30 microliters of previously prepared enzyme mix to each C tube.Invert the C tubes and place them on a heated dissociated ensuring that all tissue is collected in the tube's lid. Elude cells by removing the column from the separator adding 800 microliters of astrocyte culture medium and pushing cells out of the column with the included plunger. Plate cells in the previously prepared culture flasks by adding 4.2 milliliters of astrocyte culture medium supplemented with 10 nanograms per milliliters of EGF and bFGF.It is not necessary to coat the culture flasks for astrocytes isolated from other brain regions but it provides a better substrate for astrocytes isolated from the spinal cord. Culture the cells at 37 degrees Celsius and 5%carbon dioxide. Perform astrocyte seating under a biological safety cabinet with a safety level one and prepare 24 well plates with Poly-D-Lysine coated glass cover slips as described in the text manuscript.For plating, isolate six spinal cords from P2 mice yields around 1 million cells. Aspirate media from the T-12.5 culture flasks containing the cultured astrocytes and wash once with 1x PBS. Detach the astrocytes from the culture flask by adding 0.5 milliliters of 0.05%trypsin EDTA and incubate at 37 degrees Celsius for five minutes.Gently tap the side of the flask to release cells from the culture flask surface and check detachment under a brightfield microscope using a 10 times magnification. Stop trypsinazation with 2.5 milliliters of astrocyte culture medium and collect cell suspension in 15 milliliter tubes. Centrifuge at 300 RCF for five minutes at four degrees Celsius.Aspirate supernatant and resend cells in one milliliter of astrocyte culture medium. Calculate cell concentration using a hemocytometer or an automated cell counting system. Based on the number of cells, dilute the cell suspension with fresh astrocyte medium supplemented with EGF and bFGF at 10 nanograms per milliliter per factor.Add 500 microliters of the cell suspension to each well of the previously prepared 24 well plates and culture cells at 37 degrees Celsius and 5%carbon dioxide. Prepare neuronal differentiation medium by adding one milliliter B27 supplement to 49 milliliters of basic culture medium. After 24 to 48 hours, depending on whether cells have been transduced or transfected, replace astrocyte medium with one milliliter neuronal differentiation medium per well.Culture the cells at 37 degrees Celsius with 9%carbon dioxide. To increase the reprogramming efficiency, supplement the neuronal differentiation medium with forskolin and dorsomorphin to a final concentration of 30 micromolar and one micromolar respectively when replacing the astrocyte medium with the differentiation medium. When opting to treat cells with forskolin and dorsomorphin provide a second dose of dorsomorphin two days after the initial treatment.Add this second treatment directly to the culture medium. Primary cultures of astrocytes reached 80 to 90%confluency between seven to 10 days after magnetic assisted cell sorting and plating. The day after plating, cells typically covered 50 to 60%of the cover slip surface.Cultures majorly consisted of astrocytes at this stage, while other cell types such as neuro blasts were virtually absent. At seven days post transduction or transfection, induced neuronal cells were clearly distinguishable from astrocytes. The neuronal cell soma was smaller than unreprogrammed Astrocytes had long processes and was positive for neuronal marker, beta three tubulin, and negative for astrocyte marker GFAP.At 21 days post transduction or transfection, many induced neurons were capable of firing action potentials and were positive for the mature neuronal marker NeuN and the pan synaptic protein Synaptophysin. It is essential to properly dissect the region of interest. Avoiding contamination from surrounding tissue.Care must be taken to remove the meninges and the dorso root ganglia from the isolated spinal cord. This method can be used to isolate astrocytes from various regions of the central nervous system including the cerebral cortex, dorsal midbrain, and cerebellum. In the future, this technique will allow us to expand our knowledge on regional astrocytes and their potential to be reprogrammed into functional neurons.

isolation-and-direct-neuronal-reprogramming-of-mouse-astrocytes

Research

JoVE Journal

Neuroscience

2.5K visualizações.  German Research Center for Environmental Health. Os astrócitos são uma população autógena interessante para atingir a programação neuronal direta. Este protocolo fornece uma técnica confiável para isolar a cultura astrócitos com alta pureza de diferentes regiões ou do sistema nervoso central. Este protocolo é projetado e otimizado para investigar a capacidade dos astrócitos de serem reprogramados em neurônios funcionais sem confundir fatores como diferenças na pureza dos astrócitos de diferentes populações de startups....