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

Published: April 30, 2023

Abstract

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 Preparation of Solution and Culture Medium 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. 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. To prepare 200 mL …

Divulgaciones

The authors have nothing to disclose.

Materials

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

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Generation of Induced Neural Stem Cells (iNSCs) from Peripheral Blood Mononuclear Cells (PBMNCs): A Sendai Virus-based Procedure to Reprogram PBMNCs into iNSCs. J. Vis. Exp. (Pending Publication), e20669, doi: (2023).

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