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 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.
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.
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.
2. Astrocyte isolation
3. Spinal cord tissue dissection
NOTE: Dissection of tissue can be performed outside of a biological safety cabinet.
4. Magnetic activated cell sorting (MACS)
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).
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.
7. Reprogramming of astrocytes (7 days analysis)
8. Reprogramming of astrocytes into mature neurons (long term cultures)
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 exclusively of astrocytes, while other cell types, such as neuroblasts, are virtually absent (Figure 1D)29.
Reprogramming factors can be delivered to astrocytes either via retroviral or lentiviral transduction or through transfection of DNA plasmids. Usually, viral transduction infects more cells as compared to transfection. As direct neuronal conversion causes a substantial amount of cell death34,35, retroviral or lentiviral transduction is preferred to maximize the number of cells for analysis. Different promoters can be used to control the expression of the reprogramming factors: constitutive (e.g., CMV, CAG)15,32, inducible (e.g., Tet-responsive elements, Tet-ON)21, or cell type specific (e.g., GFAP promoter)36,37. When using constitutive or cell type specific promoters, astrocytes start to express detectable levels of the transgenes, assessed by fluorescent reporter expression (e.g., GFP, DsRed), within 24 h after the gene delivery, with each cell independent to the others. Conversely, inducible promoters allow to synchronize the expression of the transgenes across the cells transduced, as they are activated following the addition of a small molecule (e.g., doxycycline) to the culture medium. Detectable levels of the transcription factors are usually reached 18-20 h after the activation of the promoter. In most cases, the peak of the reprogramming factor expression is reached at around 48 h, with lentivirus-mediated expression taking slightly longer.
While transcriptional changes following reprogramming factors expression can be detected as early as 4 h32, robust changes occur after 24 h and later29,32. Morphological changes follow transcriptional changes, and first signs of conversion can be observed around 3 days post-transduction/transfection (3 DPT). At 7 DPT, induced neuronal cells are clearly distinguishable from astrocytes: their soma is smaller than control or un-reprogrammed astrocytes, they have long processes, and they are positive for neuronal marker βIII-tub and are negative for astrocyte marker GFAP (Figure 1E). However, it is worth noting that some cells can be either positive for both GFAP and βIII-tub, suggesting that the neuronal program has been induced but the astrocyte identity has not been inhibited, or negative for both markers, indicating the repression of the astrocyte identity but the absence of induction of the neuronal cascade. In either case, the cells usually maintain an astrocyte morphology.
For more functional analyses, such as electrophysiology or the evaluation of the generated neuronal subtypes, cultures are generally maintained for a minimum of 21 DPT and treated with maturation medium. At 21 DPT, many induced neurons are capable of firing action potentials and are positive for the mature neuronal marker NeuN as well as for the pan-synaptic protein Synaptophysin29 (Figure 1F).
Figure 1: Overview of astrocyte culture and reprogramming. (A)Timeline of astrocyte-to-neuron direct conversion. Each black line represents an important step in the protocol. (B) Representative brightfield images of cultured spinal cord-derived astrocytes after 7 days in culture. Pictures were taken using a brightfield microscope and 10x objective. Scale bar represents 100 µm. (C) Representative brightfield images of spinal cord astrocytes 1 day after re-plating at a density of 5.5 x 104 cells per well in a 24-well plate. Images were taken using a brightfield microscope and a 10x objective. Scale bar represents 100 µm. (D) Immunofluorescence image of a βIII-tub, Sox9, GFAP triple staining on astrocytes fixed 1 day after plating to demonstrate culture purity. Cells were fixed in 4% paraformaldehyde for 10 min and washed twice with 1x PBS. Cells were blocked using a 3% BSA, 0.5% Triton-X 100 in 1x PBS solution. Primary antibodies were diluted at the proper concentration (e.g., anti-GFAP 1:250; anti-βIII-tub 1:250; anti-Syp1 1:500) in blocking solution and incubated for 2 h at room temperature. Cells were washed three times with 1x PBS and incubated with fluorophore-conjugated secondary antibodies for 1 h at room temperature. Coverslips were washed three times with 1x PBS before mounting with Aqua Poly/Mount. Images were acquired using an epifluorescence microscope and a 40x objective. Scale bar represents 20 µm. (E) Immunofluorescence image of a βIII-tub, DsRed double staining to demonstrate astrocyte to neuron conversion with Ascl1 after 7 DPT. Protocol of immunofluorescence and image acquisition was as described above. Scale bar represents 20 µm. (F) Immunofluorescence images of a βIII-tub, DsRed, Synaptophysin 1 (Syp1) triple staining to demonstrate neuronal maturity after 21 DPT of reprogramming with Ascl1. Protocol of immunofluorescence and image acquisition was as described above. Scale bar represents 20 µm. Please click here to view a larger version of this figure.
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 adhere to the flask and then start to proliferate, giving rise to highly enriched astrocyte cultures29. Importantly, cultured astrocytes do not dedifferentiate into a multipotent cell state nor get immortalized. Furthermore, they do not spontaneously generate neurons following the expression of a reporter protein (e.g., DsRed or GFP), but maintain an astrocyte identity. Also, they do not proliferate indefinitely, but rather slow down their proliferation and transition into a more mature stage, which reduces their direct neuronal reprogramming potential32,39.
There are several critical steps in this protocol: first, it is essential to carefully isolate the region of interest and remove any contaminating tissues. For example, to prepare spinal cord astrocytes, the spinal cord is extracted from the vertebrae and the dorsal root ganglia (DRG) are carefully removed. Second, converting cells undergo significant cell death34, which has a negative impact on the transduced cells and the overall culture, due to the stimulation of phagocytosis by surrounding astrocytes as well as altered media osmolarity. Therefore, it is important to replace the astrocyte medium with an adequate volume of differentiation medium (usually 1 mL/well of a 24-well plate). Additionally, the transfection of plasmid DNA is an easy and more accessible approach compared to viral transduction, which requires an approved safety level 2 cell culture room. However, transfection rate and reprograming efficiency are lower compared to viral-mediated delivery of the reprogramming factors. Therefore, transfection can be used as a fast method to test the reprogramming potential of new candidate reprogramming factors or to screen pools of factors. Regarding neuronal maturation, reprogrammed cells usually become electrophysiologically active at around 3 weeks. Though not required, treating the cells with small molecules increases both survival as well as maturation of the reprogrammed cells, leading to a higher density of induced neurons and a more mature morphology.
Although the described method to isolate and culture astrocytes is robust and reliable, a few aspects need to be considered. First, while conventional methods based on mechanical dissociation of tissue yield an overall higher number of cells in culture per tissue dissected40, a MAC-sorted approach requires the dissection of tissue from six to eight pups to isolate an adequate number of cells for subsequent experiments. Furthermore, the isolation of astrocytes is based on the expression of the ATPase Na+/K+ transporting subunit Beta2 protein (Atp1b2), recognized by the antibody ACSA-241. In principle, astrocytes not expressing Atp1b2 would be lost in the preparation, therefore causing a bias in the preparation. Although we cannot exclude that this is the case, our analysis of MACS flow-through revealed that few cells in the negative fraction were immunoreactive for the astroglia markers Sox9, suggesting the high efficiency of MAC-sorting protocol. A second caveat regarding Atp1b2 is related to its expression. Atp1b2 is specifically expressed by astrocytes at postnatal stage, while in the mouse adult brain other cell types express it, in particular myelinating oligodendrocytes and ependymal cells27. Therefore, a careful dissection of the area of interest and a myelin removal step is required to isolate astrocytes from adult brains.
Compared to other methods for isolating astrocytes, the MACS-based approach ensures high purity of the cultures (>90% of Sox9+ cells) and provides a standardized procedure to isolate astrocytes from different regions of the CNS. This is particularly important when comparing cultures from different CNS regions, as the culture purity obtained by classical mechanical dissociation can vary remarkably (>80% GFAP+/DAPI from cortical gray matter, ~50% GFAP+/DAPI from spinal cord)15,29. A standardized protocol reduces such variability and provides a common starting point for in vitro reprogramming experiments. This allows, for instance, to systematically compare the molecular identity of astrocytes from different regions28,29 and to investigate the impact of the developmental origin on the reprogramming efficiency and the subtype identity of the induced neurons.
In summary, in vitro direct neuronal reprogramming of optimized cultures of astrocytes is a very powerful approach to unravel universal as well as region-specific molecular mechanisms of astrocyte-to-neuron conversion, providing essential information to design better and more effective strategies for in vivo direct conversion of resident CNS astrocytes.
The authors have nothing to disclose.
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.
0.05% Trypsin/EDTA | Life Technologies | 25300054 | |
4', 6-Diamidino-2-phenyindole, dilactate (DAPI) | Sigma-Aldrich | D9564 | |
anti-mouse IgG1 Alexa 647 | Thermo Fisher | A21240 | |
anti-Mouse IgG1 Biotin | Southernbiotech | Cat# 1070-08; RRID: AB_2794413 | |
anti-mouse IgG2b Alexa 488 | Thermo Fisher | A21121 | |
anti-rabbit Alexa 546 | Thermo Fisher | A11010 | |
Aqua Poly/Mount | Polysciences | Cat# 18606-20 | |
B27 Supplement | Life Technologies | 17504044 | |
BDNF | Peprotech | 450-02 | |
bFGF | Life Technologies | 13256029 | |
Bovine Serum Albumine (BSA) | Sigma-Aldrich | Cat# A9418 | |
cAMP | Sigma Aldrich | D0260 | |
C-Tubes | Miltenyi Biotec | 130-093-237 | |
DMEM/F12 | Life Technologies | 21331020 | |
Dorsomorphin | Sigma Aldrich | P5499 | |
EGF | Life Technologies | PHG0311 | |
Fetal Bovine Serum | PAN Biotech | P30-3302 | |
Forskolin | Sigma Aldrich | F6886 | |
GDNF | Peprotech | 450-10 | |
gentleMACS Octo Dissociator | Miltenyi Biotec | 130-096-427 | |
GFAP | Dako | Cat# Z0334; RRID: AB_100013482 | |
Glucose | Sigma Aldrich | G8769 | |
GlutaMax | Life Technologies | 35050038 | |
HBSS | Life Technologies | 14025050 | |
Hepes | Life Technologies | 15630056 | |
Lipofectamine 2000 (Transfection reagent) | Thermo Fisher | Cat# 11668019 | |
MACS SmartStrainer 70µm | Miltenyi Biotec | 130-098-462 | |
MiniMACS Seperator | Miltenyi Biotec | 130-042-102 | |
Mouse anti-ACSA-2 MicroBeat Kit | Miltenyi Biotec | 130-097-678 | |
Mouse IgG1 anti-Synaptophysin 1 | Synaptic Systems | Cat# 101 011 RRID:AB_887824) | |
Mouse IgG2b anti-Tuj-1 (βIII-tub) | Sigma Aldrich | T8660 | |
MS columns | Miltenyi Biotec | 130-042-201 | |
N2 Supplement | Life Technologies | 17502048 | |
Neural Tissue Dissociation Kit | Miltenyi Biotec | 130-092-628 | |
NT3 | Peprotech | 450-03 | |
octoMACS Separator | Miltenyi Biotec | 130-042-109 | |
OptiMEM – GlutaMAX (serum-reduced medium) | Thermo Fisher | Cat# 51985-026 | |
Penicillin/Streptomycin | Life Technologies | 15140122 | |
Poly-D-Lysine | Sigma Aldrich | P1149 | |
Rabbit anti-RFP | Rockland | Cat# 600-401-379; RRID:AB_2209751 | |
Rabbit anti-Sox9 | Sigma-Aldrich | Cat# AB5535; RRID:AB_2239761 | |
Streptavidin Alexa 405 | Thermo Fisher | Cat# S32351 | |
Triton X-100 | Sigma-Aldrich | Cat# T9284 |