Source: Shrigley, S., et al. Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts. J. Vis. Exp. (2018)
The video demonstrates the generation of neurons from adult human dermal fibroblasts (aHDFs). The aHDFs are reprogrammed using a lentiviral vector carrying genes for neuronal transcription factors and short hairpin RNAs (shRNAs). The shRNAs inhibit the expression of a neuronal transcriptional repressor, and the neuronal transcription factors activate genes to differentiate aHDFs into neurons. These reprogrammed aHDFs are cultured in an early and a late neuronal conversion medium to generate mature neurons.
The authors have nothing to disclose.
Cell Lines | |||
Adult human dermal fibroblasts | [C2 passage #7] Donor was a 67 year old female. Cells obtained from the Parkinson’s Disease Research and Huntington’s disease clinics at the John van Geest Centre for Brain Repair (Cambridge, UK). | ||
Reagents for Fibroblast Culture, Transduction and Conversion | |||
Dulbecco's phosphate-buffered saline (DPBS) [-CaCl2, -MgCl2] | Gibco | 14190094 | |
Trypsin-EDTA [0.5%] | Gibco | 15400-054 | Dilute to 0.05% in DPBS. |
Virkon (agent used to neutralize virus) | Viroderm | 7511 | Dilute to 1% solution with warm water. |
Milli-Q Water | Millipore | ||
Basal medium – Dulbecco’s Modified Eagle Medium (DMEM) + GlutaMax | Gibco | 61965059 | |
Penicillin/Streptomycin [10,000 U/ mL] | Gibco | 15140-122 | |
Fetal Bovine Serum (FBS) | Gibco | 10270-106 | |
CryoMACS® dimethyl sulfoxide (DMSO) 10 | Miltenyi | 170-076-303 | |
Neural differentiation medium – NDiff 227 | Takara-Clontech | Y40002 | |
LM-22A4 | Tocris | 4607 | Dilute 10 mg in 1450 µL DMSO. Stock concentration: 20 mM. |
Glial cell line-derived neurotrophic factor (GDNF) [recombinant human] | R&D systems | 212-GD-010 | Dilute 10 ug in 500 µL 0,1% BSA in DPBS. Stock concentration: 20 µg/mL. |
NT3 [recombinant human] | R&D systems | 267-N3-025 | Dilute 25 µg in 2,5 mL 0,1% BSA in DPBS. Stock concentration: 10 µg/mL. |
db-cAMP | Sigma Aldrich | D0627 | Dilute 1 g in 40,7 mL Milli-Q water. Filter and make 500 µL aliquots or stock tubes of 10 mL. Stock concentration: 50 mM. |
CHIR99021 | Axon | 1386 | Dilute 2 µg in 429,8 µL DMSO. Stock concentration: 10 mM. |
LDN-193189 | Axon | 1509 | Dilute 2 mg in 360 µL DMSO. Stock concentration: 10 mM. |
Valproic acid sodium salt (VPA) | Merck Millipore | 676380 | Dilute 5 g in Milli-Q water to acheive a stock concentration of 1 M. CAUTION: Avoid ingestion, contact with skin, and breathing dust formation. |
Reagents for Coatings | |||
Gelatin | Sigma Aldrich | G2500 | Dilute to 0.1% in Milli-Q water. |
Poly-L-ornithine | Sigma Aldrich | P3655 | Dissolve in Milli-Q water. Use at 15µg/mL. |
Fibronectin | ThermoFisher Scientific | 33010-018 | 2 mL of Milli-Q water + 70 µL 0,25 M NaOH. Use at 5 µg/mL. |
Laminin | ThermoFisher Scientific | 23017-015 | Store at -80°C. Thaw on ice, keep cool and aliquot 30 µL. Use at 5 µg/mL. |
Equipment | |||
T75 flask [Nunclon Delta Surface] | ThermoFisher Scientific | 156499 | |
24-well plate [Nunc] | ThermoFisher Scientific | 142485 | |
1.5 mL polypropylene tube | Sigma Aldrich | Z336769 | |
15 mL falcon tube | Sarstedt | 6,25,54,502 | |
50 mL falcon tube | Sarstedt | 6,25,47,254 | |
Pippette controller | For pipetting volumes 1-25 mL. | ||
Sterile serological pipettes: 5, 10 and 25 mL | Sarstedt | 86.1253.001, 86.1254.001, 86.1685.001 | |
Adjustable volume pipettors: 5, 20, 200, and 1,000 µL | |||
Sterile pipette tips | For pipetting volumes of 0.5 – 1,000 µL. | ||
ThawSTAR Automated Cell Thawing System | BioCision | BCS-601 | |
Countess II Automated Cell Counter | ThermoFisher Scientific | AMQAX1000 | |
Cell counting chambers [50 slides] and trypan blue [0.4%] | ThermoFisher Scientific | C10228 | For use with Countess II Automated Cell Counter. |
Laminar flow hood | |||
Humidified 5% CO2 37 °C incubator | |||
Centrifuge | Suitable for 1,5, 15 and 50 mL tubes. | ||
Orbital shaker | |||
Inverted fluorescence microscope | Leica | DMI6000 B |