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 using only autoclaved or sterile equipment. Dissection and dissociation reagents can be prepared outside of a biological safety cabinet.
<ol>
	<li>Prepare culture flasks by coating a T25 culture flask with poly-D-lysine (stock 1 mg/mL; working solution 20 &#181;g/mL) in H2O for a minimum of 2 h. Afterwards, rinse 3x with H2O and air dry.</li>
	<li>Prepare dissection buffer by adding 5 mL of 1 M HEPES buffer solution to 500 mL of Hank&#39;s balanced salt solution (HBSS).</li>
	<li>Prepare one C-tube (see Table of Materials) per six spinal cords by adding 1950 &#181;L of buffer X from the neural tissue dissociation kit (see Table of Materials). Store on ice during dissection. Prepare enzymatic digestion master mix by adding 20 &#181;L of buffer Y and 10 &#181;L of buffer A per C-tube (part of the neural tissue dissociation kit; see Table of Materials). Mix well and store at 4 &#176;C until needed.</li>
	<li>Prepare 1x phosphate buffered saline (1x PBS) with calcium and magnesium and place on ice. Prepare basic culture medium by adding 5 mL of penicillin-streptomycin (final concentration, 100 U/mL), 5 mL of 45% D-glucose, and 5 mL of glutamine supplement (final concentration 2 mM; see Table of Materials) to 485 mL of DMEM/F12. Basic medium is stable at 4 &#176;C for 4 weeks.</li>
	<li>Prepare astrocyte culture medium by adding 1 mL of B27-supplement and 5 mL of fetal bovine serum (FBS) to 44 mL of basic culture medium. Supplement medium with epidermal growth factor (EGF) and basic-fibroblast growth factor (bFGF) (10 ng/mL each). Add EGF and bFGF just before use to the appropriate amount of medium.</li>
	<li>For the dissection of spinal cord tissue, use a pair of large forceps with bent tip, a pair of small forceps with bent tip, one pair of small forceps, a pair of small scissors, and a small spatula.</li>
</ol>
2. Astrocyte isolation
<ol>
	<li>Isolate astrocytes from the spinal cord of postnatal mice for performing their direct reprogramming into functional neurons (Figure 1A). For this protocol, collect spinal cord tissue from mice at day 2-3 after birth. Collect six to eight spinal cords for astrocyte isolation. Use this same protocol to isolate astrocytes from other regions of the nervous system, such as the cortex, midbrain, and cerebellum. For these regions, obtain cells at five to seven days after birth. 
	&#8203;NOTE: When aiming to isolate astrocytes from regions that are not mentioned in this protocol, the age and input material should be experimentally determined.</li>
</ol>
3. Spinal cord tissue dissection
NOTE: Dissection of tissue can be performed outside of a biological safety cabinet.
<ol>
	<li>Sacrifice mice at P2-P3 by decapitation without anesthetizing the animal. Place the torso in a 35 mm Petri dish and keep on ice.</li>
	<li>Open the skin with scissors, remove vertebra with small scissors, extract the spinal cord, and place it in dissection buffer on ice. Under a stereotactic dissection microscope with 2x magnification, remove the meninges from the isolated spinal cords using forceps and transfer dissected and cleaned spinal cord tissue to a C-tube.</li>
</ol>
4. Magnetic activated cell sorting (MACS)
<ol>
	<li>Add 50 &#181;L of enzyme P from the neural tissue dissociation kit (see Table of Materials) and 30 &#181;L of previously prepared enzyme mix to each C-tube. Invert the C-tubes and place them on a heated dissociator (see Table of Materials), making sure that all tissue is collected in the lid of the tube.</li>
	<li>Run the 37_NTDK_1 dissociation program with a run time of approximately 22 min. Shortly before the end of the program, place a 70 &#181;m strainer (see Table of Materials) on a number of 15 mL tubes equal to the number of C-tubes used and pre-wet the strainer with 2 mL of ice-cold PBS. Store 1x PBS on ice during the entire MACS procedure.</li>
	<li>After completion of the program, remove C-tubes and briefly centrifuge them to collect the tissue at the bottom of the tubes. Transfer the dissociated tissue from the C-tubes to the 15 mL tubes prepared by passing it through the strainer. Leave the strainer on 15 mL tubes.</li>
	<li>Rinse C-tube with 10 mL of 1x PBS to collect leftover tissue and collect it in the same 15 mL tube by passing through the strainer once more. Centrifuge the 15 mL tubes containing dissociated tissue at 300 x g for 10 min at room temperature. 
	NOTE: Cool the centrifuge down to 4 &#176;C after this step.</li>
	<li>Remove the supernatant without disturbing the cell pellet before resuspending the cells in 80 &#181;L of 1x PBS; add&#160;10 &#181;L of blocking reagent from the mouse anti-ACSA-2 MicroBead kit (see Table of Materials). Gently mix by pipetting.</li>
	<li>Incubate at 4 &#176;C for 10 min in the dark (fridge). Add 10 &#181;L of anti-astrocyte cell surface antigen-2-coupled microbeads&#160;(see Table of Materials) and gently mix by pipetting. Incubate for 15 min at 4 &#176;C in the dark.</li>
	<li>Wash cells by adding 3 mL of 1x PBS and centrifuge at 300 x g for 10 min at 4 &#176;C. While centrifuging, assemble a magnetic separator (see Table of Materials) by placing the appropriate number of magnetic sorting columns (see Table of Materials) onto the separator with a 15 mL collection tube underneath. Rinse the columns with 500 &#181;L of 1x PBS.</li>
	<li>Remove supernatant and resuspend the cells in 500 &#181;L of 1x PBS before transferring the cell suspensions to the magnetic columns. Let drain by gravity. Wash the 15 mL tubes with 500 &#181;L of 1x PBS and apply to column. Wash columns with 500 &#181;L of 1x PBS for an additional two washes.</li>
	<li>Elute cells by removing the column from the separator, adding 800 &#181;L of astrocyte culture medium and pushing cells out of the column with the included plunger.</li>
	<li>Plate cells in the previously prepared culture flasks by adding 4.2 mL of astrocyte culture medium supplemented with EGF and bFGF and culture at 37 &#176;C and 5% CO2. 
	NOTE: Coating culture flasks is not necessary for astrocytes isolated from other brain regions but provides a better substrate for astrocytes isolated from the spinal cord.</li>
	<li>Culture cells for approximately 7 days until confluency (80%-90%). If the cells are not confluent after 7 days, wait up until 10 days before plating them. After 10 days, cells do not proliferate anymore, and the reprogramming efficiency declines.</li>
</ol>
5. Seeding of astrocytes for reprogramming
NOTE: The following steps have to be performed under a biological safety cabinet with a safety level 1 (SL1).
<ol>
	<li>Prepare 24-well plates with poly-D-lysine coated glass coverslips in the same way culture flasks were prepared previously. To determine the number of plates needed, consider that astrocytes are plated at a density of 5-5.5 x 104 cells per well in a 24-well plate. Usually, isolating six spinal cords from P2 mice yields around 1 x 106 cells.</li>
	<li>Aspirate media from the T25 culture flasks containing the cultured astrocytes and wash once with 1x PBS. Detach the astrocytes from the culture flask by adding 0.5 mL of 0.05% Trypsin/EDTA and incubate at 37 &#176;C for 5 min. Gently tap the side of the flask to release cells from the culture flask surface and check detachment under a brightfield microscope using a 10X magnification.</li>
	<li>Stop trypsinization with 2.5 mL of astrocyte culture medium and collect cell suspension in 15 mL tubes. Centrifuge at 300 x g for 5 min, aspirate supernatant, and resuspend cells in 1 mL of astrocyte culture medium. Calculate cell concentration using a haemocytometer or an automated cell counting system.</li>
	<li>Based on the number of cells, dilute the cell suspension with fresh astrocyte medium to obtain a solution of 1-1.1 x 105 cells per mL. Supplement the medium with EGF and bFGF at 10 ng/mL per factor. Add 500 &#181;L of cell suspension, equivalent to 5-5.5 x 104 cells, to each well of the previously prepared 24-well plates and culture cells at 37 &#176;C and 5% CO2.</li>
</ol>
6. Forced expression of transcription factors
NOTE: Before proceeding with the protocol, it is essential to properly design the experiment. In particular, it is important to always include a negative control for the reprogramming, namely a condition where no reprogramming factor is expressed. For instance, when using vectors carrying the cDNA for the reprogramming factor and a reporter (e.g., green fluorescent protein (GFP), DsRed), the negative control is represented by the same vector carrying only the reporter. When expressing multiple factors carrying different reporters, the negative control should be accordingly adjusted.
<ol>
	<li>The day after plating, inspect the 24-well plates to make sure cells have adhered to the coverslips.</li>
	<li>Based on the experimental aim and available resources, the forced expression of reprogramming factors can be achieved by viral transduction (see step 6.3) or DNA transfection (see step 6.4). 
	NOTE: The use of retrovirus or lentivirus requires the approval of government authorities and must be performed under a biological safety cabinet within a laboratory with safety level 2 (SL2).</li>
	<li>Reprogram the astrocytes by transducing the cells with the retrovirus or lentivirus carrying the genetic information to express the reprogramming factor(s) of interest as described below.
	<ol>
		<li>Transduce cells with a high virus titer of 1 x 1010-1 x 1012 particles/mL by adding 1 &#181;L of the cell suspension directly to the astrocyte medium. This ensures a high infection rate. Culture the cells with the astrocyte medium containing viral particles at 37 &#176;C for 24-36 h before proceeding with section 7 or section 8, depending on the purpose of the experiment. 
		NOTE: Different promoters can be used to drive the expression of the transgenes (see also Representative Results). Constitutive promoters (e.g., CMV, CAG) induce the expression of the transgene earlier than inducible promoters; however, both types of promoter types have been successfully used to reprogram cells into neurons.</li>
	</ol>
	</li>
	<li>DNA plasmids can also be introduced into astrocytes via DNA transfection as described below. 
	NOTE: This can be done under a biological safety cabinet approved for SL1.
	<ol>
		<li>Before transfection, obtain the transfection reagent, a plasmid DNA of the desired constructs, fresh astrocyte medium, and serum-reduced medium (see Table of Materials). Calculate the required amount of serum-reduced medium (see Table of Materials) by considering that each well of a 24-well plate requires 300 &#181;L of serum-reduced medium. Add the appropriate amount of serum-reduced medium to a 50 mL tube and warm it to 37 &#176;C.</li>
		<li>When the serum-reduced medium is warm, aspirate astrocyte medium from all wells and collect it in a 50 mL tube. Filter the collected astrocyte medium with a 0.45 &#181;M syringe filter to remove detached cells.</li>
		<li>Add an equal volume of fresh astrocyte medium to the filtered medium to obtain a solution sufficient to add 1 mL of astrocyte medium per well. Maintain the astrocyte conditioned medium in the incubator at 37 &#176;C until use (step 6.4.9). 
		NOTE: The culture medium is re-used as it contains several secreted factors that support the viability of the culture.</li>
		<li>Add 300 &#181;L of pre-warmed serum-reduced medium to each well and place the 24-well plate back to the incubator.</li>
		<li>Prepare solution A, consisting of DNA and serum-reduced medium. For each well, use a total of 0.6 &#181;g of DNA and add it to 50 &#181;L of serum-reduced medium. Store solution A at room temperature until use. 
		NOTE: Typically, a technical triplicate per condition is considered. Therefore, a mix sufficient for 3.5 reaction is prepared (e.g., 2.1 &#181;g of total DNA diluted in 175 &#181;L of serum-reduced medium) to ensure enough material to transfect three wells of a 24-well plate.</li>
		<li>Prepare solution B, composed of the transfection reagent and serum-reduced medium. For each well, add 0.75 &#181;L of transfection reagent to 50 &#181;L of serum-reduced medium. As this solution is common to all transfection conditions, prepare it in bulk to reduce the variability across transfections.</li>
		<li>Incubate solution B at room temperature for 5 min. Add solution B to solution A drop by drop at a 1:1 ratio and gently mix. Do not vortex. Incubate solution A+B for 20-30 min at room temperature under the hood.</li>
		<li>Repeat step 6.4.5-6.4.7 for all transfection conditions. After 20-30 min, add solution A+B to each well drop by drop for a final volume of 100 &#181;L. Gently shake the plate and place the cells back in the incubator at 37 &#176;C for 4 h.</li>
		<li>After 4 h, remove the transfection medium and add 1 mL of the pre-warmed astrocyte conditioned medium prepared in step 6.4.3. Maintain cells for 36-48 h before proceeding with section 7 or section 8, depending on the purpose of the experiment.</li>
	</ol>
	</li>
</ol>
7. Reprogramming of astrocytes (7 days analysis)
<ol>
	<li>Prepare neuronal differentiation medium by adding 1 mL of B27-supplement to 49 mL of basic culture medium (see step 1.4). After 24-48 h, depending on whether cells have been transduced or transfected (see steps 6.3 and 6.4, respectively), replace astrocyte medium with 1 mL of neuronal differentiation medium per well and culture cells at 37 &#176;C and 9% CO2. 
	NOTE: Cells can also be kept at 5% CO2 if no 9% CO2 incubator is available. However, neuronal reprogramming is more efficient under these conditions.</li>
	<li>Optional: To increase the reprogramming efficiency, supplement neuronal differentiation medium with Forskolin (final concentration of 30 &#181;M) and Dorsomorphin (final concentration of 1 &#181;M) when replacing the astrocyte medium to differentiation medium. When opting to treat cells with Forskolin and Dorsomorphin, provide a second dose of Dorsomorphin 2 days after the initial treatment. Add this second treatment directly to the culture medium.</li>
	<li>Direct reprogramming of murine astrocytes into neuronal cells normally occurs within 7 days after changing the medium. Hence, 7 days after the initiation of neuronal reprogramming, fix or collect cells for downstream analysis.</li>
</ol>
8. Reprogramming of astrocytes into mature neurons (long term cultures)
<ol>
	<li>Prepare neuronal differentiation medium by adding 1 mL of B27-supplement to 49 mL of basic culture medium (see step 1.4). After 24-48 h, depending on whether cells were transduced or transfected (see steps 6.3 and 6.4, respectively), replace astrocyte medium with 1 mL of neuronal differentiation medium supplemented with Forskolin (final concentration of 30 &#181;M) and Dorsomorphin (final concentration of 1 &#181;M) per well and culture cells at 37 &#176;C and 9% CO2. 
	NOTE: Cells can also be kept at 5% CO2 if no 9% CO2 incubator is available. However, neuronal reprogramming is more efficient under these conditions.</li>
	<li>Repeat Dorsomorphin treatment 2 days after the initial treatment by adding it directly to the neuronal differentiation medium.</li>
	<li>After 7 days from the start of neuronal reprogramming, prepare maturation medium by adding 6 &#181;L of N2, 1.2 &#181;L of NT3 (stock 20 &#181;g/mL), 1.2 &#181;L of BDNF (stock 20 &#181;g/mL), 1.2 &#181;L of GDNF (stock 20 &#181;g/mL), and 1.2 &#181;L of cAMP (stock 100 mM) to 189.2 &#181;L of neuronal differentiation medium. Supplement neuronal differentiation medium present in each well with 200 &#181;L of maturation medium. 
	NOTE: Maturation medium is supplemented only for the first treatment. All subsequent treatments are done by partially replacing the neuronal differentiation medium as described below.</li>
	<li>Repeat the treatment with small molecules by removing 200 &#181;L of neuronal differentiation medium and adding 200 &#181;L of fresh maturation medium twice a week for up to 6 weeks. During the maturation, use cells for electrophysiological experiments (typically, at day 21 or later) or fix for downstream analysis (e.g., immunofluorescence).</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+aging+astrocyte+transcriptome+from+multiple+regions+of+the+mouse+brain.">The aging astrocyte transcriptome from multiple regions of the mouse brain.</a> Cell Reports. 22 (1), 269-285 (2018).</li><li>Ohlig, 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=Molecular+diversity+of+diencephalic+astrocytes+reveals+adult+astrogenesis+regulated+by+Smad4.">Molecular diversity of diencephalic astrocytes reveals adult astrogenesis regulated by Smad4.</a> The EMBO Journal. 40 (21), 107532 (2021).</li><li>Herrero-Navarro, A., 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=Astrocytes+and+neurons+share+region-specific+transcriptional+signatures+that+confer+regional+identity+to+neuronal+reprogramming.">Astrocytes and neurons share region-specific transcriptional signatures that confer regional identity to neuronal reprogramming.</a> Science Advances. 7 (15), (2021).</li><li>Kempf, J., 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=Heterogeneity+of+neurons+reprogrammed+from+spinal+cord+astrocytes+by+the+proneural+factors+Ascl1+and+Neurogenin2.">Heterogeneity of neurons reprogrammed from spinal cord astrocytes by the proneural factors Ascl1 and Neurogenin2.</a> Cell Reports. 36 (3), 109409 (2021).</li><li>Mattugini, N., 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=Inducing+Different+Neuronal+Subtypes+from+Astrocytes+in+the+Injured+Mouse+Cerebral+Cortex.">Inducing Different Neuronal Subtypes from Astrocytes in the Injured Mouse Cerebral Cortex.</a> Neuron. 103 (6), 1086-1095 (2019).</li><li>Heins, N., 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=Glial+cells+generate+neurons:+the+role+of+the+transcription+factor+Pax6.">Glial cells generate neurons: the role of the transcription factor Pax6.</a> Nature Neuroscience. 5 (4), 308-315 (2002).</li><li>Masserdotti, G., 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=Transcriptional+mechanisms+of+proneural+factors+and+REST+in+regulating+neuronal+reprogramming+of+astrocytes.">Transcriptional mechanisms of proneural factors and REST in regulating neuronal reprogramming of astrocytes.</a> Cell Stem Cell. 17 (1), 74-88 (2015).</li><li>Rao, Z., 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=Molecular+mechanisms+underlying+Ascl1-mediated+astrocyte-to-neuron+conversion.">Molecular mechanisms underlying Ascl1-mediated astrocyte-to-neuron conversion.</a> Stem Cell Reports. 16 (3), 534-547 (2021).</li><li>Gascon, 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=Identification+and+successful+negotiation+of+a+metabolic+checkpoint+in+direct+neuronal+reprogramming.">Identification and successful negotiation of a metabolic checkpoint in direct neuronal reprogramming.</a> Cell Stem Cell. 18 (3), 396-409 (2016).</li><li>Russo, G. 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. D., 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=The+Ink4a/Arf+locus+is+a+barrier+to+direct+neuronal+transdifferentiation.">The Ink4a/Arf locus is a barrier to direct neuronal transdifferentiation.</a> The Journal of Neuroscience. 34 (37), 12560-12567 (2014).</li><li>Heinrich, C., 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=Generation+of+subtype-specific+neurons+from+postnatal+astroglia+of+the+mouse+cerebral+cortex.">Generation of subtype-specific neurons from postnatal astroglia of the mouse cerebral cortex.</a> Nature Protocols. 6 (2), 214-228 (2011).</li><li>Batiuk, M. Y., 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=An+immunoaffinity-based+method+for+isolating+ultrapure+adult+astrocytes+based+on+ATP1B2+targeting+by+the+ACSA-2+antibody.">An immunoaffinity-based method for isolating ultrapure adult astrocytes based on ATP1B2 targeting by the ACSA-2 antibody.</a> The Journal of Biological Chemistry. 292 (21), 8874-8891 (2017).</li></ol>

The authors declare no conflicts of interest.

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><tbody><tr><td>0.05% Trypsin/EDTA</td><td>Life Technologies</td><td>25300054</td><td></td></tr><tr><td>4&#039;, 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><td></td></tr><tr><td>Hepes</td><td>Life Technologies</td><td>15630056</td><td></td></tr><tr><td>Lipofectamine 2000 (Transfection reagent)</td><td>Thermo Fisher</td><td>Cat# 11668019</td><td></td></tr><tr><td>MACS SmartStrainer 70&amp;micro;m</td><td>Miltenyi Biotec</td><td>130-098-462</td><td></td></tr><tr><td>MiniMACS Seperator</td><td>Miltenyi Biotec</td><td>130-042-102</td><td></td></tr><tr><td>Mouse anti-ACSA-2 MicroBeat Kit&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-097-678</td><td></td></tr><tr><td>Mouse IgG1 anti-Synaptophysin 1</td><td>Synaptic Systems</td><td>Cat# 101 011 RRID:AB_887824)</td><td></td></tr><tr><td>Mouse IgG2b anti-Tuj-1 (&amp;beta;III-tub)</td><td>Sigma Aldrich</td><td>T8660</td><td></td></tr><tr><td>MS columns</td><td>Miltenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>N2 Supplement</td><td>Life Technologies</td><td>17502048</td><td></td></tr><tr><td>Neural Tissue Dissociation Kit&amp;nbsp;</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 &amp;ndash; 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></tr></tbody></table>

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 ...

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

Research

JoVE Journal

Neuroscience

2.7K Views.  German Research Center for Environmental Health. Astrocyten zijn een interessante autogene populatie om te targeten voor directe neuronale programmering. Dit protocol biedt een betrouwbare techniek om cultuurastrocyten met hoge zuiverheid uit verschillende regio's of het centrale zenuwstelsel te isoleren. Dit protocol is ontworpen en geoptimaliseerd om het vermogen van astrocyten te onderzoeken om te worden geherprogrammeerd in functionele neuronen zonder verstorende factoren zoals verschillen in de zuiverheid van astrocyten van verschillen...

Video: Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

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.

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

Developmental Biology

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.

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

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies indicate that the severity of brain injury appear to be sex dependent, and that the male neonates are more susceptible to the effects of HI-related brain injury, resulting in more severe neurological outcomes as compared to females with comparable brain injuries. The development of reliable methods to isolate and maintain highly enriched populations of sexed hippocampal astrocytes is essential to understand the cellular basis of sex differences in the pathological consequences of neonatal HI. In this study, we describe a method for creating sex specific hippocampal astrocyte cultures that are subjected to a model of in-vitro ischemia, oxygen-glucose deprivation, followed by reoxygenation. Subsequent reactive astrogliosis was examined by immunostaining for the Glial Fibrillary Acidic Protein (GFAP) and S100B. This method provides a useful tool to study the role of male and female hippocampal astrocytes following neonatal HI, separately.

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrocytes are one of the most important key players in the central nervous system (CNS). Here, we are reporting a practical method of sexed hippocampal astrocyte culture protocol in order to study the mechanisms underlying the astrocyte function in male and female neonate pups after in-vitro ischemia.

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines.

In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes

Astrocytes tile the cerebral cortex uniformly, making the analysis of their complex morphology challenging at the cellular level. The protocol provided here uses multicolor labeling based on in utero electroporation to single out cortical astrocytes and analyze their volume and morphology with a user-friendly image analysis pipeline.

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at ...

Efficient Extraction of Astrocytes and Microglia from the Adult Mouse Spinal Cord

Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For In Vitro Assays and Transcriptomic Studies

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic cells exhibit rapid responses to environmental changes and display significant heterogeneity in terms of morphology, transcriptional profiles, and functions. While our understanding of the functions of glial cells in health and disease has advanced substantially, there remains a need for in vitro, cell-specific analyses conducted in the context of insults or injuries to comprehensively characterize distinct cell populations. Isolating cells from the adult mouse offers several advantages over cell lines or neonatal animals, as it allows for the analysis of cells under pathological conditions and at specific time points. Furthermore, focusing on spinal cord-specific isolation, excluding brain involvement, enables research into spinal cord pathologies, including experimental autoimmune encephalomyelitis, spinal cord injury, and amyotrophic lateral sclerosis. This protocol presents an efficient method for isolating astrocytes and microglia from the adult mouse spinal cord, facilitating immediate or future analysis with potential applications in functional, molecular, or proteomic downstream studies.

Author Spotlight: Isolation of Glial Cells from the Adult Mouse Spinal Cord 

This protocol outlines the isolation of purified astrocytes and microglia from the adult mouse spinal cord, facilitating subsequent applications such as RNA analysis and cell culture. It includes detailed cell dissociation methods and procedures designed to enhance both the quality and yield of isolated cells.

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic ...

A Simple Technique to Isolate and Culture Murine Astrocytes

Source: Wolfes, A. C., et al., <a href="https://app.jove.com/v/56092">Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol.</a> J. Vis. Exp. (2018)
The video demonstrates a simple method to culture murine astrocytes in isolation from other brain cells. During culturing, by using astrocyte-specific media and inhibiting the adherence of non-astrocytic cells, the method selectively cultures astrocytes. These cultured astrocytes exhibit stellate morphology with fine processes.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Rat or Mouse Brain Dissection
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JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

Direct Neuronal Reprogramming of Mouse Astrocytes into Neurons

Source: Hersbach, B. A. et al., <a href="https://app.jove.com/v/64175">Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes.</a> J. Vis. Exp. (2022) 
This video showcases the direct reprogramming of astrocytes expressing transforming factors into neurons. Transformation factors trigger a cascade of molecular events facilitating astrocyte-to-neuron conversion. The process is further supported by supplying specific agents and supplements through a neuronal differentiation medium.

1. Seeding of astrocytes for reprogramming
NOTE: The following steps have to be performed under a biological safety cabinet with a safety level 1 (SL1).

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Advanced Neuronal Models

Astrocyte Isolation: A Method to Obtain Pure Preparation of Mouse Cortical Astrocytes

Transfer the dish containing the brains to a stereomicroscope. Next, to isolate the cortices, grab the posterior end of each brain with fine forceps and perform a midline incision between the hemispheres.
Then, insert a second set of forceps to the created groove and peel away the plate-like structure of the cortex from the rest of the brain.
Once all cortices have been isolated, carefully peel the meninges away from the cortex by pulling with the fine forceps. This step avoids contamination of the final astrocyte culture by meningeal cells and fibroblasts.
Transfer the prepared cortical hemispheres into a second dish filled with chilled HBSS and place on ice. Continue accordingly with all four cortices. Finally, cut each hemisphere into four to eight small pieces using sharp blades.
Under sterile conditions, transfer the pieces of cortex into one 50-milliliter Falcon tube and add HBSS to a final volume of 22.5 milliliters. Then, add 2.5 milliliters of 2.5% trypsin.
Replace the cap, mix, and incubate the tissue in the water bath at 37 degrees Celsius for 30 minutes. Mix by shaking every 10 minutes. Then, after centrifuging for 5 minutes at 300 x g to pellet the cortex tissue pieces, carefully decant the supernatant.
Add 10 milliliters of astrocyte plating medium to the pellet, and use a 10-milliliter pipette to vigorously pipette the medium containing the tissue up and down 20 to 30 times until a single cell suspension is obtained.
Next, add astrocyte plating medium to a final volume of 20 milliliters and count the cells using a hemocytometer or automated cell counter according to standard procedures.
One preparation of four mouse pup cortices should yield 10 to 15 times 10 to the sixth dissociated single cells. Finally, aspirate the poly-D-lysine from a previously prepared flask and transfer the dissociated cell suspension to the flask.
Incubate the culture at 37 degrees Celsius in the tissue culture incubator for 2 days and then change the media. The media should then be changed every 3 days thereafter.
The astrocyte should appear confluent with an overlying layer of microglia after 7 to 8 days in culture. At this point, set the T75 flask on an orbital shaker set at 180 rotations per minute for 30 minutes to remove microglia.
Discard the supernatant containing microglia or spin it down and plate for culture. Then, add 20 milliliters of fresh astrocyte culture medium and continue by shaking the flask at 240 rpm for 6 hours to remove oligodendrocyte precursor cells.
Since some OPCs will not completely detach from the astrocyte layer, continue to shake vigorously by hand for 1 minute in order to prevent OPC contamination. Again, discard the supernatant or spin it down on a plate to culture OPCs.
Rinse the remaining confluent astrocyte layer twice with PBS. Aspirate the PBS and add 5 milliliters of trypsin-EDTA. Incubate in the tissue culture incubator at 37 degrees Celsius.
Check for detachment of astrocytes every 5 minutes and enforce the detachment of astrocytes by hitting the flask against the palm of your hand two to three times.
Once the astrocytes have detached from the flask, add 5 milliliters of astrocyte culture medium. Spin the cells at 180 x g for 5 minutes, aspirate the supernatant, and add 40 milliliters of fresh astrocyte plating medium.
One T75 flask of mixed cortical cells should yield around 1 times 10 to the sixth cells enriched for astrocytes after the first passage.

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

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