Generating Neurons by Reprogramming Human Dermal Fibroblast Cells

Abstract

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.

Protocol

1. Preparation of Skin Fibroblasts for Reprogramming

1. Using an automated cell thawing system or a 37 °C water bath, thaw the adult human dermal fibroblasts (aHDFs) and plate 200,000 per T75 flask (count with an automated cell counter) in 10 mL of fibroblast medium (see Table 1) at 37 °C in 5% CO₂.
2. Perform a complete medium change with fibroblast medium on the next day.
3. Change the fibroblast medium every 3–4 days until the cells reach 95% confluency.
   NOTE: One confluent flask will contain approximately 1,000,000 cells.

Results

Figure 1: Evolution of the iN conversion over time. (A) Timeline of the experiment and map of the construct packaged in a lentivirus used to reprogram the adult human dermal fibroblasts. Each black arrow represents a medium change. (

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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