Generation of Induced Neural Stem Cells (iNSCs) from Peripheral Blood Mononuclear Cells (PBMNCs): A Sendai Virus-based Procedure to Reprogram PBMNCs into iNSCs

Samenvatting

Source: Zheng, W. et al. Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies. J. Vis. Exp. (2019)

This video demonstrates the reprogramming of peripheral blood mononuclear cells (PBMNCs) into induced neural stem cells (iNSCs) using Sendai virus. Sendai virus is a non-integrating virus that offers an efficient and safe vehicle for reprogramming cells.

Protocol

1. Reprogramming of PBMNCs to iNSCs by SeV Infection

1. Preparation of Solution and Culture Medium
   1. Prepare a poly-D-lysine (PDL) stock solution by dissolving 100 mg of PDL with 100 mL of H₂O to a concentration of 1 mg/mL. Store at -20 °C in 1 mL aliquots.
   2. Prepare an insulin stock solution by dissolving 100 mg of insulin in 20 mL of 0.01 N HCl to a concentration of 5 mg/mL. Store at -20 °C in 1 mL aliquots.
   3. To prepare 200 mL of iNSC basal medium, combine 96 mL of DMEM-F12 and 96 mL of basic medium (Table of Materials) with 2 mL of 100x glutamine stock solu....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
B27 supplement	Invitrogen	17504044
CHIR99021	Gene Operation	04-0004
DMEM-F12	Gibco	11330
DMEM-F12	Gibco	11320
Laminin	Roche	11243217001
N2 supplement	Invitrogen	17502048
NEAA	Invitrogen	11140050	Non-essential amino acid
Neurobasal	Gibco	10888	Basic medium
PDL	Sigma-Aldrich	P7280	Poly-D-lysine
SB431542	Gene Operation	04-0010-10mg	Store from light at -20?
Sendai virus	Life Technologies	MAN0009378
Insulin	Roche	12585014
24-well plate	Corning	3337
15-ml conical tube	Corning	430052
6-well plate	Corning	3516
EPO	Peprotech	100-64-50UG	Human Erythropoietin
Human leukemia inhibitory factor	Millpore	LIF1010
Human recombinant SCF	Peprotech	300-07-100UG
IGF-1	Peprotech	100-11-100UG	Human insulin-like growth factor
IL-3	Peprotech	200-03-10UG	Human interleukin 3
Dexamethasone	Sigma-Aldrich	D2915-100MG
IMDM	Gibco	215056-023	Iscove's modified Dulbecco's medium
Insulin	Roche	12585014
ITS-X	Invitrogen	51500-056	Insulin-transferrin-selenium-X supplement
GlutaMAX	Invitrogen	21051024	100 × Glutamine stock solution
Ham's-F12	Gibco	11765-054
1-Thioglycerol	Sigma-Aldrich	M6145	Toxic for inhalation and skin contact
Ascorbic acid	Sigma-Aldrich	A92902	Toxic with skin contact
Knockout serum replacement	Gibco	10828028	Serum free basal medium
Chemically defined lipid concentrate	Invitrogen	11905031
Transferrin	R&D Systems	2914-HT-100G
Trypan blue	Gibco	T10282