HSPC and MPP2 isolation, barcoding, EPO treatment, and transplantation

Protocol for lentiviral barcoding of EPO treated hematopoietic stem and progenitor cells: Cell purification, barcoding, and transplantation

Study overview:

Figure 1: Overview of the experimental design (a) HSPCs were sorted from the bone marrow of donor mice, lentivirally barcoded, cultured ex vivo with or without 1000 ng/ml EPO for 16 hr, and transplanted into sublethally irradiated mice. At week 4 post-transplantation, the erythroid (E), myeloid (M), and B-cells 

Material:

A full list of reagents and material used in this protocol is provided in the original article in the ‘Key resources’ table

Step 1- progenitor isolation

A. BM isolation: Bone collection and ckit enrichment

• Sacrifice mice
  • 6 BL6N D45.1 Males aged 8 weeks
• Recover BM (tibia, femur, iliac)
• Take some cells for unstained and single stain controls

B. Flushing bones

• Put bones and medium into a 10 cm petri dish. 
• Prepare the scissors and tweezer by rinsing with EtOH. 
  • Rinse before taking bone in petri dish medium to dilute EtOH. 
• Prepare a 15 ml tube or 50 ml with 4-5 ml/ mouse cold PBS+ to collect the cells.
• Prepare a 2ml syringe with a long green needle (21G 50mm) 
  • fill it with 2ml cold PBS+ from the collection tube 
• Put needle into tibia or non-star like end of femur. 
  • For iliac bone at V shaped end at the tip of the V. 
  • Take care to use pointiest part of needle. 
• Put entire bone into cold PBS+ and eject 1ml of cold PBS+. 
• For tibia and femur, turn the bone ¼ without detaching it, for iliac bone just put deeper into bone (should feel that is breaking a membrane), and eject other 1ml into the tube. 
• If bone is not white yet, restart, cold PBS+ taken is from the collection tube!
• Empty bones can be collected in lid of petri dish. 
• Between each bone, dissociate the red pulp by pipetting up and down with the syringe and ejecting against the tube wall. 
• Never put tweezer into collection tube. If lose bone try to get back with needle.
• Pass the cell through a 100um cell strainer with the pipette
• Spin down for 5 mins at 4°C at 1700 rpm
• Rinse the petri dish and the Falcon with medium.

C. cKit enrichment

Cell preparation

• Pool cells from 3 mice into a single samples (this will give 2 samples of 3 mice)
• Centrifuge cells at 1700rpm 5 min 4°C
• Take off the supernatant and leave the last drop
• Add 25 ul of a-CD117 beads in each tube, mix, add 125ul of cold 10%RPMI, mix.
• Incubate for 20 min on ice covered by aluminium
• Add 5ml of cold 10%RPMI to the cells
• Pass through a 100 um cell strainer into 50 ml tube.
• Wash tube and cell strainer with 5 ml of cold medium. 
• Take off the last drop of cells under the cell strainer with an 1000ul pipette. 
• Centrifuge cells at 1700 rpm 6min 4°C
• Resuspend in 3ml cold medium.

MACS for c-kit+ cells

• Wash the LS column with 3ml of cold 10%RPMI 
• Mix cells with an insulin syringe with short 25G needle
• Eject cells against the LS column wall. 
• Wash the tube and column with 3 ml of medium. [end of the ckit- collection]
• Detach the column, put into a new 15mL falcon (ckit+ collection tube) and fill up with medium (5mL)
• Insert the plunger and eject the cells [end of the ckit+ collection]
• Count cells

D. Sorting                                                                                     

Figure 1: Gating strategies and hematopoietic stem and progenitor cell (HSPC) marker expression after lentiviral transduction and ex vivo culture with or without erythropoietin (EPO). (a) HSPCs were gated as propidium iodide-negative single C-Kit⁺ Sca-1⁺ Flt3^- CD150⁺ cells of C-Kit⁺-enriched bone marrow cells. (b) Erythroblast cells were gated as Ter119⁺ CD44⁺ FSC^hi cells on Ter⁺ enriched cells. (c) Gating strategy for B-cells (CD19⁺ CD11b^-), dendritic cells (CD19 CD11b^- CD11c⁺), and myeloid cells (CD119^- CD11c^- CD11b⁺) on Ter119^- live single-donor cells. (d) Gating for MkP (C-Kit⁺ Sca-1^- CD150⁺ CD41⁺) from C-Kit⁺-enriched bone marrow cells. (e) Sort gating for GFP⁺ erythroid, myeloid, B-, and dendritic cells, respectively, used for barcoding analysis. (f) Representative flow cytometry plots of sorted HSPC pool after 6 hr lentiviral transduction and 16 hr ex vivo incubation with or without EPO.

Step 2-Introducing barcodes-transduction 

• Centrifuge the cells and remove the supernatant
• Resuspend in 100uL of Stem Span + 50ng/mL mSCF + 1,3uL of virus corresponding to the LG2.2 lentivirus library. The precise amount of virus was titrated to achieve a transduction efficiency of 10%. This can be reduced to 1% to further reduce multiple barcode usage.
• Mix with the pipette
• Centrifugate at RT 90 min at 300g (brake 2-2) 
• Remove the plate and put in the incubator at 37°C 5% CO2 during 4.5h
• Wash twice the cells with complete RPMI
• Resuspend in 500uL of Stem Span + 50ng/mL mSCF

Step 3-EPO treatment 

After transduction, cells were incubated with or without human recombinant EPO (Eprex, erythropoietin alpha, Janssen)  in Stem Span culture medium supplemented with 50ng/mL mSCF  (SS+) at a final concentration of 1000 or 160 ng/ml or PBS for 16 hr at 37°C. 

• Coat a 1,5 ml Eppendorf tube with 1 ml 10% RPMI.
• Transfer transduced cells into coated Eppendorf tubes
•  Add 1 ml of 10% RPMI, centrifuge 6 min 1700 rpm, 22°C. 
• Take off supernatant with 1 ml pipette. 
• Resuspend in 400µl SS+ or 420 if transduction control and mix well. 
• Take transduction control 20 ul in extra well on plate and add 100 ul or more of SS+.
• Take 200µl for the non-EPO condition and place it into a well. 
• Add EPO on the rest and place it into well. 
• Culture the cells for 16h at 37°C.

Step 4-injection of barcoded cells into irradiated recipients 

After EPO incubation, barcoded MPP2 and unbarcoded CD48^- HSPCs were mixed at a ratio of 32/45 (to be as close as possible to the original ratio of both populations in the HSPCs). On average, 2600 cells (mean 2684 cells ± 175 cells) were injected in the tail vein of each mouse in a volume of 100 microliters. Three hours prior to tail vein injection recipient mice (8 week old BL6N D45.1/1 male mice) were 6 Gy sublethally irradiated on a CIXD irradiator. 

• Coat a 1,5 ml Eppendorf tube with 1 ml 10% RPMI.
• Mix incubated cells well and transfer into coated Eppendorf tube. 
• Wash wells again with 200 ul of 10%RPMI. 
• Centrifuge 6 min 1700 rpm 22°C. 
• Remove supernatant and wash again in DPBS. 
• Resuspend cells in number of mice*100 ul+20 ul and take fraction (1%) for flow cytometry check -> transfer into FACS tubes with 100 ul (or 50ul) antibody mix (as previous day, without CD34) and keep for 35 min (flt3) or 15 (without flt3) in fridge.
• Keep all samples at 4°C till injection or flow cytometry check.