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Method Article

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

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DOI:

10.3791/64175

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July 7th, 2022

 ,  , 
1Institute of Stem Cell Research, Helmholtz Zentrum München, German Research Center for Environmental Health, 2Department of Physiological Genomics, Biomedical Center Munich, Ludwig-Maximilians University, 3Graduate School of Systemic Neurosciences, BioCenter, Ludwig-Maximilians University

In This Article

Summary

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.

Abstract

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 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.

Introduction

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.

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Protocol

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.

  1. Prepare culture flasks by coating a T25 culture flask with poly-D-lysine (stock 1 mg/mL; working solution 20 µg/mL) in H2O for a minimum of 2 h. Afterwards, rinse 3x with H2O and air dry.
  2. Prepare dissection buffer by adding 5 mL of 1 M HEPES buffer solution to 500 mL of Hank's balanced salt solution (HBSS).
  3. Prepare one C-tube (see Table of Materials) per six spinal cords by adding 1950 µL 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 µL of buffer Y and 10 µL of buffer A per C-tube (part of the neural tissue dissociation kit; see Table of Materials). Mix well and store at 4 °C until needed.
  4. 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 °C for 4 weeks.
  5. 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.
  6. 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.

2. Astrocyte isolation

  1. 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.
    ​NOTE: When aiming to isolate astrocytes from regions that are not mentioned in this protocol, the age and input material should be experimentally determined.

3. Spinal cord tissue dissection

NOTE: Dissection of tissue can be performed outside of a biological safety cabinet.

  1. Sacrifice mice at P2-P3 by decapitation without anesthetizing the animal. Place the torso in a 35 mm Petri dish and keep on ice.
  2. 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.

4. Magnetic activated cell sorting (MACS)

  1. Add 50 µL of enzyme P from the neural tissue dissociation kit (see Table of Materials) and 30 µL 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.
  2. 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 µm 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.
  3. 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.
  4. 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 °C after this step.
  5. Remove the supernatant without disturbing the cell pellet before resuspending the cells in 80 µL of 1x PBS; add 10 µL of blocking reagent from the mouse anti-ACSA-2 MicroBead kit (see Table of Materials). Gently mix by pipetting.
  6. Incubate at 4 °C for 10 min in the dark (fridge). Add 10 µL of anti-astrocyte cell surface antigen-2-coupled microbeads (see Table of Materials) and gently mix by pipetting. Incubate for 15 min at 4 °C in the dark.
  7. Wash cells by adding 3 mL of 1x PBS and centrifuge at 300 x g for 10 min at 4 °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 µL of 1x PBS.
  8. Remove supernatant and resuspend the cells in 500 µL of 1x PBS before transferring the cell suspensions to the magnetic columns. Let drain by gravity. Wash the 15 mL tubes with 500 µL of 1x PBS and apply to column. Wash columns with 500 µL of 1x PBS for an additional two washes.
  9. Elute cells by removing the column from the separator, adding 800 µL of astrocyte culture medium and pushing cells out of the column with the included plunger.
  10. 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 °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.
  11. 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.

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).

  1. 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.
  2. 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 °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.
  3. 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.
  4. 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 µL 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 °C and 5% CO2.

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.

  1. The day after plating, inspect the 24-well plates to make sure cells have adhered to the coverslips.
  2. 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).
  3. 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.
    1. Transduce cells with a high virus titer of 1 x 1010-1 x 1012 particles/mL by adding 1 µL 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 °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.
  4. 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.
    1. 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 µL of serum-reduced medium. Add the appropriate amount of serum-reduced medium to a 50 mL tube and warm it to 37 °C.
    2. 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 µM syringe filter to remove detached cells.
    3. 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 °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.
    4. Add 300 µL of pre-warmed serum-reduced medium to each well and place the 24-well plate back to the incubator.
    5. Prepare solution A, consisting of DNA and serum-reduced medium. For each well, use a total of 0.6 µg of DNA and add it to 50 µL 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 µg of total DNA diluted in 175 µL of serum-reduced medium) to ensure enough material to transfect three wells of a 24-well plate.
    6. Prepare solution B, composed of the transfection reagent and serum-reduced medium. For each well, add 0.75 µL of transfection reagent to 50 µL of serum-reduced medium. As this solution is common to all transfection conditions, prepare it in bulk to reduce the variability across transfections.
    7. 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.
    8. 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 µL. Gently shake the plate and place the cells back in the incubator at 37 °C for 4 h.
    9. 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.

7. Reprogramming of astrocytes (7 days analysis)

  1. 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 °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.
  2. Optional: To increase the reprogramming efficiency, supplement neuronal differentiation medium with Forskolin (final concentration of 30 µM) and Dorsomorphin (final concentration of 1 µM) 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.
  3. 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.

8. Reprogramming of astrocytes into mature neurons (long term cultures)

  1. 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 µM) and Dorsomorphin (final concentration of 1 µM) per well and culture cells at 37 °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.
  2. Repeat Dorsomorphin treatment 2 days after the initial treatment by adding it directly to the neuronal differentiation medium.
  3. After 7 days from the start of neuronal reprogramming, prepare maturation medium by adding 6 µL of N2, 1.2 µL of NT3 (stock 20 µg/mL), 1.2 µL of BDNF (stock 20 µg/mL), 1.2 µL of GDNF (stock 20 µg/mL), and 1.2 µL of cAMP (stock 100 mM) to 189.2 µL of neuronal differentiation medium. Supplement neuronal differentiation medium present in each well with 200 µL 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.
  4. Repeat the treatment with small molecules by removing 200 µL of neuronal differentiation medium and adding 200 µL 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).

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Results

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...

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Discussion

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...

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Disclosures

The authors declare no conflicts of interest.

Acknowledgements

We would like to thank Ines Mühlhahn for cloning the constructs for reprogramming, Paulina Chlebik for viral production, and Magdalena Götz and Judith Fischer-Sternjak for comments on the manuscript.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.05% Trypsin/EDTALife Technologies25300054
4', 6-Diamidino-2-phenyindole, dilactate (DAPI)Sigma-AldrichD9564
anti-mouse IgG1 Alexa 647Thermo FisherA21240
anti-Mouse IgG1 BiotinSouthernbiotechCat# 1070-08; RRID: AB_2794413
anti-mouse IgG2b Alexa 488Thermo FisherA21121
anti-rabbit Alexa 546Thermo FisherA11010
Aqua Poly/MountPolysciencesCat# 18606-20
B27 SupplementLife Technologies17504044
BDNFPeprotech450-02
bFGFLife Technologies13256029
Bovine Serum Albumine (BSA)Sigma-AldrichCat# A9418
cAMPSigma AldrichD0260
C-TubesMiltenyi Biotec130-093-237
DMEM/F12Life Technologies21331020
DorsomorphinSigma AldrichP5499
EGFLife TechnologiesPHG0311
Fetal Bovine SerumPAN BiotechP30-3302
ForskolinSigma AldrichF6886
GDNFPeprotech450-10
gentleMACS Octo DissociatorMiltenyi Biotec130-096-427
GFAPDakoCat# Z0334; RRID: AB_100013482
GlucoseSigma AldrichG8769
GlutaMaxLife Technologies35050038
HBSSLife Technologies14025050
HepesLife Technologies15630056
Lipofectamine 2000 (Transfection reagent)Thermo FisherCat# 11668019
MACS SmartStrainer 70µmMiltenyi Biotec130-098-462
MiniMACS SeperatorMiltenyi Biotec130-042-102
Mouse anti-ACSA-2 MicroBeat Kit Miltenyi Biotec130-097-678
Mouse IgG1 anti-Synaptophysin 1Synaptic SystemsCat# 101 011 RRID:AB_887824)
Mouse IgG2b anti-Tuj-1 (βIII-tub)Sigma AldrichT8660
MS columnsMiltenyi Biotec130-042-201
N2 SupplementLife Technologies17502048
Neural Tissue Dissociation Kit Miltenyi Biotec130-092-628
NT3Peprotech450-03
octoMACS SeparatorMiltenyi Biotec130-042-109
OptiMEM – GlutaMAX (serum-reduced medium)Thermo FisherCat# 51985-026
Penicillin/StreptomycinLife Technologies15140122
Poly-D-LysineSigma AldrichP1149
Rabbit anti-RFPRocklandCat# 600-401-379; RRID:AB_2209751
Rabbit anti-Sox9Sigma-AldrichCat# AB5535; RRID:AB_2239761
Streptavidin Alexa 405Thermo FisherCat# S32351
Triton X-100Sigma-AldrichCat# T9284

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