Feb 29, 2024

Public workspaceLENTIVIRAL TRANSDUCTION OF HUMAN PLURIPOTENT STEM CELLS

This protocol is a draft, published without a DOI.
  • 1Garvan Institute of Medical Research, Sydney, NSW 2010, Australia;
  • 2Garvan Weizmann Center for Cellular Genomics, Garvan Institute of Medical Research, Sydney, NSW 2010, Australia;
  • 3Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA;
  • 4School of Medical Science, University of New South Wales, Sydney, NSW, 2052, Australia;
  • 5Graduate School of Biomedical Engineering, University of New South Wales, Sydney, NSW, 2052, Australia;
  • 6The Centre for Stem Cell Biology, Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY, USA;
  • 7Ann Romney Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA;
  • 8The New York Stem Cell Foundation Research Institute, New York, NY, USA
Renuka Ravi Gupta
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Protocol Citation: Renuka Ravi Gupta, Nona Farbehi, hendersa, Vikram Khurana, Gist Croft, Lorenz Studer, Joseph Powell 2024. LENTIVIRAL TRANSDUCTION OF HUMAN PLURIPOTENT STEM CELLS. protocols.io https://protocols.io/view/lentiviral-transduction-of-human-pluripotent-stem-c9wtz7en
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: February 28, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 95923
Keywords: ASAPCRN
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000472
Abstract
We have developed a protocol for lentiviral transduction of human pluripotent stem cells (hPSCs), including induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs). In this protocol, concentrated lentiviral supernatant with a Multiplicity Of Infection (MOI) ranging from 0.1 to 0.3 (equivalent to 10% to 30% Blue Fluorescent Protein (BFP) positive cells) is combined with E8 Flex media and added to adherent H9 CRISPRi dCAS9 cells in 48-well plates. Subsequent centrifugation (spinfection) is employed to ensure efficient transduction. Transduction efficiency is assessed by determining the percentage of cells expressing Blue Fluorescent Protein (BFP) using Fluorescence Activated Cell Sorting (FACS).
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LENTIVIRAL TRANSDUCT...
10KB
Materials


ABC
MATERIALCOMPANYCATALOG
48 well TC treated plateFalcon353078
15ml polypropylene centrifuge tubesFalcon352096
5ml serological pipettesCorning4487
10ml serological pipettesCorning4488
DNA Low-bind tubes 1.5mlEppendorf022431021
P1000 tipNeptuneBT1250
FBSBovogen2008A
DPBSThermo Fisher Scientific14040133
E8 Flex media KitThermo Fisher ScientificA2858501
RevitaCell Supplement(100x)Thermo Fisher ScientificA2644501
AccutaseStemCell Technologies7922
Vitronectin-N (VTN-N)Thermo Fisher ScientificA14700
REAGENT COMPOSITION
AB
FACS Buffer (PBS +2% FBS)
REAGENTVOLUME IN mL
PBS49
FBS1


Day 0: Coating wells with VTN-N and seeding hPSCs
Day 0: Coating wells with VTN-N and seeding hPSCs
Add 60 ul of VTN-N to 6 ml of DPBS.
Coat 100 ul per well in a 48 - well plate.
Incubate the plate at room temperature for an hour and the plate is ready to be used.
Seed 3x104 cells/cm2 in a 48 well plate with E8 flex media and RevitaCell after dissociating the cells with accutase.
Incubate the cells overnight at 37ºC with 5% CO2 and 20.9% O2.
Day 1: Transduction of hPSCs with Lentiviral CRISPRi library supernatant
Day 1: Transduction of hPSCs with Lentiviral CRISPRi library supernatant
The H9 dCAS9 CRISPRi cells were transduced with the pooled CRISPRi library to harvest genomic DNA (gDNA), after calculating the exact volume of viral supernatant needed to achieve the desired MOI (multiplicity of infection).
Prepare 15 ml tubes with E8 flex media and concentrated lentiviral supernatants for a MOI of 0.1-0.3.
Add 200 ul/well of the virus media cocktail with the cells from step i.
To increase the transduction efficiency, centrifuge the plate at 300g for 20 minutes at 25ºC.
Incubate the cells at 37ºC with 5% CO2 and 20.9% O2 for 16-18 hours.
Day 2: Replace media
Day 2: Replace media
Aspirate the viral supernatant media gently and immediately add maturation media.
Return the plate back to the incubator.
Day 3: FACs Sort
Day 3: FACs Sort
Aspirate the spent media.
Wash the cells 10 times with DPBS to remove the viral particles from the lentivirus transduced hPSCs.
Note
Be very gentle while doing the washes as the cells tend to lift off during the wash step

Add 100 ul accutase and incubate the cells for 10 mins in the incubator.
Note: Ideally the hPSCs should dissociate as single cells.
Neutralize the accutase with E8 flex media and collect the cells into 1.5ml eppendorf tubes.
Note
Use a P1000 tip to pipette the cells up and down to break them into single cell suspension.

Centrifuge the cells at 300 g for 4 minutes.
Aspirate the spent media gently without disturbing the pellet.
Resuspend the cells in 300 ul of FACs buffer.
Transfer the cells with the FACs buffer into FACs tubes.
Sort the H9 CRISPRi cells to obtain 10- 30% BFP positive cells and collect the cells in E8 flex media.
Centrifuge the cells at 300 g for 4 mins.
Aspirate the spent media to obtain a cell pellet to be frozen or freshly used for DNA extraction.