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 |
Tags
Citar este artículo
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).