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Method Article
* These authors contributed equally
Here, we present a method for reproducible generation of ventral midbrain patterned astrocytes from hiPSCs and protocols for their characterization to assess phenotype and function.
In Parkinson's disease, progressive dysfunction and degeneration of dopamine neurons in the ventral midbrain cause life-changing symptoms. Neuronal degeneration has diverse causes in Parkinson's, including non-cell autonomous mechanisms mediated by astrocytes. Throughout the CNS, astrocytes are essential for neuronal survival and function, as they maintain metabolic homeostasis in the neural environment. Astrocytes interact with the immune cells of the CNS, microglia, to modulate neuroinflammation, which is observed from the earliest stages of Parkinson's, and has a direct impact on the progression of its pathology. In diseases with a chronic neuroinflammatory element, including Parkinson's, astrocytes acquire a neurotoxic phenotype, and thus enhance neurodegeneration. Consequently, astrocytes are a potential therapeutic target to slow or halt disease, but this will require a deeper understanding of their properties and roles in Parkinson's. Accurate models of human ventral midbrain astrocytes for in vitro study are therefore urgently required.
We have developed a protocol to generate high purity cultures of ventral midbrain-specific astrocytes (vmAstros) from hiPSCs that can be used for Parkinson's research. vmAstros can be routinely produced from multiple hiPSC lines, and express specific astrocytic and ventral midbrain markers. This protocol is scalable, and thus suitable for high-throughput applications, including for drug screening. Crucially, the hiPSC derived-vmAstros demonstrate immunomodulatory characteristics typical of their in vivo counterparts, enabling mechanistic studies of neuroinflammatory signaling in Parkinson's.
Parkinson's disease affects 2%-3% of people over 65 years of age, making it the most prevalent neurodegenerative movement disorder1. It is caused by degeneration of ventral midbrain dopamine neurons within the substantia nigra, resulting in debilitating motor symptoms, as well as frequent cognitive and psychiatric issues2. Parkinson's pathology is typified by aggregates of the protein, α-synuclein, which are toxic to neurons and result in their dysfunction and death1,2,3. As the dopaminergic neurons are the degenerating population in Parkinson's, they were historically the focus of research. However, it is apparent that another cell type in the brain, the astrocytes, also demonstrate abnormalities in Parkinson's, and are believed to contribute to degeneration in models of Parkinson's4,5,6,7.
Astrocytes are a heterogenous cell population that can transform both physically and functionally as required. They support neuronal function and health via a plethora of mechanisms, including the modulation of neuronal signaling, shaping of synaptic architecture, and trophic support of neuronal populations via secretion of specific factors6,8,9,10. However, astrocytes also have a substantial immunomodulatory role, integral to the development and propagation of neuroinflammation10,11. Neuroinflammation is observed in the brains of Parkinson’s patients, and significantly has recently been shown to pre-empt the onset of Parkinson's symptoms12,13,14,15, thereby taking the center stage in Parkinson's research.
At a cellular level, astrocytes are said to become reactive in response to injury, infection, or disease, as an attempt to facilitate neuroprotection9,6,10,16. Reactivity describes a shift in astrocyte phenotype characterized by changes in gene expression, secretome, morphology, and mechanisms of clearance of cell debris and toxic byproducts9,10,11,17. This reactive shift occurs in response to inductive signals from microglia, which are the immune cells of the CNS and the first responders to injury and disease9. Both astrocytes and microglia respond to inflammatory signals by moderating their own function and can transduce inflammatory signals and thus directly influence neuroinflammation9,10. However, the chronic nature of Parkinson's results in a transition where reactive astrocytes become toxic to neurons, and themselves promote degeneration and disease pathology6,9,10,18,19. Significantly it was recently demonstrated that blocking the transformation of astrocytes into the reactive neurotoxic phenotype prevents the progression of Parkinson's in animal models11. Astrocyte reactivity in the paradigm of neuroinflammation has therefore become a major focus of Parkinson's research, and similarly relates to a wide spectrum of diseases of the CNS. Together these findings build a picture of significant astrocytic involvement in the etiology of Parkinson's, emphasizing the need for accurate research models that recapitulate the phenotype of the human astrocyte populations that are involved in Parkinson's.
In the embryonic brain, neurons appear first, with the astroglial lineage, namely, the astrocytes and oligodendrocytes, appearing later in development6. In vivo and in vitro studies have highlighted a number of signaling pathways that appear to control the potency of neural progenitor cells from neuronal to astroglial derivatives. In particular, JAK/STAT, EGF, and BMP signaling play roles in the proliferation, differentiation, and maturation of astroglia20,21. These pathways have been the focus of in vitro protocols for the generation of astrocytes from pluripotent cells, including hiPSC6,22,23. There have been many successful examples of generating astrocytes from hiPSCs6,24,25. However, it is apparent that in vivo astrocytes in the CNS possess specific regional identities, which relate directly to their function, in accordance with the specific requirements of those astrocytes in relation to their specialized neuronal neighbors17,24,25,26. For example, relating specifically to the ventral midbrain, it has been demonstrated that astrocytes in this region express specific sets of proteins, including receptors for dopamine enabling communication with the local population of midbrain dopamine neurons26. Furthermore, ventral midbrain astrocytes demonstrate unique signaling properties26. Therefore, to study the role of ventral midbrain astrocytes in Parkinson's, we require an in vitro model that reflects their unique set of characteristics.
To address this, we have developed a protocol to generate ventral midbrain astrocytes (vmAstros) from hiPSCs. The resulting vmAstros exhibit characteristics of their in vivo ventral midbrain counterparts such as expression of specific proteins, as well as immunomodulatory functions. The results presented are from the differentiation of the NAS2 and AST23 hiPSC lines, which were derived and gifted to us by Dr. Tilo Kunath27. NAS2 was generated from a healthy control subject whereas AST23 is derived from a Parkinson's patient carrying a triplication in the locus encoding α-Synuclein (SNCA). These hiPSC lines have been previously characterized and used in a number of published research papers, including for the generation of various neural cell types27,28,29,30,31.
1. Human hiPSC line thawing, maintenance, and cryopreservation
2. vmAstro Differentiation protocol
NOTE: A schematic summary of the vmAstros differentiation protocol is shown in Figure 1A. A detailed list of reagents required for the protocol and their preparation is given in Table 1.
3. Cryopreservation of vmNPCs, vmAPCs, and vmAstros
NOTE: Cryopreserve vmNPCs/vmAPCs/vmAstros at full confluency.
4. Characterization of vmAstro phenotype
Differentiation methodology and progression
Here we present the details of both the methods employed for the generation of vmAstros and the protocols used for their subsequent phenotypic characterization. The method for generation of vmAstros is made up of several distinct differentiation stages, which can be monitored by microscopy and identifying distinct morphological characteristics (Figure 1A-F). A feeder-free hiPSC culture (
This method for the generation of vmAstros from hiPSCs is highly efficient, generating pure cultures of vmAstros, and being reproducible for the generation of vmAstros from different hiPSC lines. This protocol was developed around the recapitulation of the developmental events required in the embryo to correctly pattern the developing midbrain and generate astrocytes and comprises three defined stages: 1) neural ventral midbrain induction to generate vmNPCs, 2) generation and expansion of vmAPCs, and finally 3) maturatio...
The authors have nothing to disclose.
This work was funded by a Parkinson's UK project grant (G-1402) and studentship. The authors gratefully acknowledge the Wolfson Bioimaging Facility for their support and assistance in this work.
Name | Company | Catalog Number | Comments |
Reagents | |||
0.2M Tris-Cl (pH 8.5) | n/a | n/a | Made up from Tris base and plus HCl |
0.5M EDTA, PH 8 | ThermoFisher | 15575-020 | 1:1000 in D-PBS to 0.5 mM final |
1,4-diazabicylo[2.2.2]octane (DABCO) | Sigma | D27802- | 25 mg/mL in Mowiol mounting solution |
13 mm coverslips | VWR | 631-0149 | |
2-Mercaptoethanol (50 mM) | ThermoFisher | 31350010 | |
Accutase | ThermoFisher | 13151014 | |
Advanced DMEM/F12 | ThermoFisher | 12634010 | Has 1x NEAA but we add to final concentration of 2x (0.2 mM) |
Ascorbic acid | Sigma | A5960 | 200 mM stock, 1:1000 to 200 µM final |
B27 Supplement | ThermoFisher | 17504-044 | 50x stock |
BSA | Sigma | 5470 | |
Cell freezing media | Sigma | C2874 | Cryostor CS10 |
Cell freezing vessel | Nalgene | 5100-0001 | |
CHIR99021 | Axon Medchem | 1386 | 0.8 mM stock, 1:1000 dilution to 0.8 µM final |
Cryovials | Sigma | CLS430487 | |
DAPI | Sigma | D9542 | 1 mg/mL, 1:10,000 to 100ng/mL final (in PBS) |
DMEM/F12 + Glutamax | ThermoFisher | 10565018 | |
Dulbeccos-PBS (D-PBS without Mg or Ca) | ThermoFisher | 14190144 | pH 7.2 |
E8 Flex medium kit | ThermoFisher | A2858501 | |
Formaldehyde (36% solution) | Sigma | 47608 | |
Geltrex | ThermoFisher | A1413302 | 1:100 or 1:400 in ice-cold DMEM/F12 |
Glutamax | ThermoFisher | 35050038 | 2 mM stock (1:200 in N2B27, 1:100 in ASTRO media to 20 µM final) |
Glycerol | Sigma | G5516 | |
Human BDNF | Peprotech | 450-02 | 20 µg/mL stock, 1:1000 to 20 ng/mL final |
Human BMP4 | Peprotech | 120-05 | 20 µg/mL stock, 1:1000 to 20 ng/mL final |
Human EGF | Peprotech | AF-100-15 | 20 µg/mL stock, 1:1000 to 20 ng/mL final |
Human GDNF | Peprotech | 450-10 | 20 µg/mL stock, 1:1000 to 20 ng/mL final |
Human insulin solution | Sigma | I9278 | 10 mg/mL stock, 1:2000 to 5 µg/mL final |
Human LIF | Peprotech | 300-05 | 20 µg/mL stock, 1:1000 to 20 ng/mL final |
IL-6 ELISA kit | Biotechne | DY206 | |
Isopropanol | Sigma | I9516-4L | For filling Mr Frosty cryostorage vessel |
LDN193189 | Sigma | SML0559 | 100 µM stock, 1:10,000 dilution to 10 nM final |
Mowiol 40-88 | Sigma | 324590 | |
N2 Supplement | ThermoFisher | 17502048 | 100x stock |
NEAA | ThermoFisher | 11140035 | 10 mM stock, 1:100 to 0.1 mM final |
Neurobasal media | ThermoFisher | 21103049 | |
Normal Goat serum | Vector Labs | S-1000-20 | |
Revitacell | ThermoFisher | A2644501 | 100x stock, 1:100 to 1x final |
SB431542 | Tocris | 1614 | 10 mM stock, 1:1000 dilution to 10 µM final |
SHH-C24ii | Biotechne | 1845-SH-025 | 200 µg/mL stock, 1:1000 to 200 ng/mL final |
Tris-HCl | Sigma | PHG0002 | |
Triton-X | Sigma | X100 | |
Tween-20 | Sigma | P7949 | |
Vitronectin | ThermoFisher | A14700 | 1:50 in D-PBS |
Antibodies for immunocytochemistry | Company | Catalogue Number | Host species |
Antibody against S100b | Sigma | SAB4200671 | Mouse; 1:200 |
Antibody against FOXA2 | SCBT | NB600501 | Mouse; 1:50 |
Antibody against LMX1A | ProSci | 7087 | Rabbit; 1:300 |
Antibody against LMX1A | Millipore | AB10533 | Rabbit; 1:2000 |
Antibody against LMX1B | Proteintech | 18278-1-AP | Rabbit; 1:300 |
Antibody against GLAST | Proteintech | 20785-1-AP | Rabbit; 1:300 |
Antibody against GFAP | Dako | Z0334 | Rabbit; 1:400 |
Antibody against CD49f | Proteintech | 27189-1-AP | Rabbit; 1:100 |
Antibody against MSI1 | Abcam | ab52865 | Rabbit; 1:400 |
Alexa Fluor 488 Goat Anti-Rabbit | ThermoFisher | A32731 | Goat; 1:500 |
Alexa Fluor 488 Goat Anti-Mouse | ThermoFisher | A32723 | Goat; 1:500 |
Alexa Fluor 568 Goat Anti-Rabbit | ThermoFisher | A11036 | Goat; 1:500 |
Alexa Fluor 488 Goat Anti-Mouse | ThermoFisher | A11031 | Goat; 1:500 |
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