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

  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 solution, 2 mL of nonessential amino acid (NEAA), 2 mL of N2 supplement and 2 mL of B27 supplement. Add 10 ng/mL recombinant human leukemia inhibitory factor, 3 μM CHIR99021 and 2 μM SB431542 prior to use. Filter the medium and store it at 4 °C.
      NOTE: Use the medium within 2 weeks. Add recombinant human leukemia inhibitory factor, CHIR99021 and SB431542 immediately before use.
  2. Reprogramming of PBMNCs to iNSCs by SeV Infection
    1. Ultraviolet-sterilize a clean bench prior to use. Sterilize all surfaces and equipment with 75% alcohol. Sterilize all tips using an autoclave.
    2. On day 0, collect the cells in MNC medium and transfer to a 15 mL conical tube. Centrifuge the cells at 200 x g for 5 min. Aspirate the supernatant and resuspend the cells with 1 mL of pre-warmed MNC medium.
    3. Count the viable cells with trypan blue. Resuspend the cells with pre-warmed (37 °C) MNC medium to a concentration of 2 x 105 cells per well in 24-well plates.
    4. After removing the SeV tubes from -80 °C storage, thaw the tubes containing SeV in 37 °C water bath for 5–10 s, and then allow them to thaw at RT. Once thawed, place them on ice immediately.
    5. Add the SeV encoding human Klf4, Oct3/4, SOX2, and c-MyC to the wells, at a multiplicity of infection (MOI) of 10. Centrifuge cells with plates at 1,000 x g for 30 min to facilitate the attachment of cells. Leave the cells and supernatant in the plates. Place the plates in the incubator at 37 °C, 5% CO2.
      CAUTION: All procedures involving SeV must be performed in a safety cabinet, and all tips and tubes should be treated with ethanol or bleach before disposal.
    6. On day 1, transfer the medium and cells to a 15 mL centrifuge tube. Rinse the well with 1 mL of MNC medium. Centrifuge the cell suspension at 200 x g for 5 min. Aspirate the supernatant and resuspend the cells with 500 μL of fresh pre-warmed MNC medium in 24-well plates.
      NOTE: Use a low attachment 24-well plate to prevent attachment of any cells before plating on PDL/laminin.
    7. On day 2, dilute 1 mL of 1 mg/mL PDL with 19 mL of D-PBS to a concentration of 50 μg/mL. Coat 6-well plates with 50 μg/mL PDL for at least 2 h at RT.
    8. Dilute 200 μL of 0.5 mg/mL laminin with 20 mL of D-PBS to a concentration of 5 µg/mL. Aspirate PDL in the 6-well plates, and dry on the vertical clean bench.
    9. Coat 6-well plates with 5 μg/mL laminin and incubate for 4–6 h at 37°C. Wash with D-PBS before use.
    10. On day 3, plate the transduced cells obtained in step 1.2.6 in iNSC medium on PDL/laminin-coated 6-well plates.   
      NOTE: Move the plates gently if needed after the cells are placed on PDL/laminin-coated plates, trying not to disturb the attachment of the cells.
    11. On day 5, add 1 mL of pre-warmed (37 °C) iNSC medium in each well in 6-well plates gently. 
      NOTE: It is expected that the cells will undergo drastic death (>60%).
    12. On day 7, add 1 mL of pre-warmed (37 °C) iNSC medium in each well in 6-well plates gently.
    13. From day 9 to day 28, replace spent medium with fresh pre-warmed (37 °C) iNSC medium every day. Monitor the emergence of iNSC colonies. Pick and transfer iNSC clones for expansion in about 2–3 weeks. Pick up colonies with appropriate morphology using burned glass pipettes, excluding any possibly contaminating cells, and aspirate the colonies with 200 μL tips.

Disclosures

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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Cite This Article
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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