Direct lineage reprogramming from fibroblasts to erythroid progenitor cells. It's a new method we have developed in our lab in order to better study the processes of transcriptional regulation determining erythroid cell fate. The main advantage with using direct lineage reprogramming to study development is that it's a very straightforward technique.
So compared to, for example, loss of function studies within knockout mice, here we can very easily manipulate the factor, over-express it at the certain time point, and then easily get enough material to study downstream effects such as chromatin modifications or gene expression changes. While this method focuses on the reprogramming of mouse tail-tip fibroblasts to erythroid progenitors, it can also be applied to other fibroblast cell types, and to other organisms including humans. The implications of this technique extend towards transfusion medicine in that it paves the way towards the production of red blood cells in vitro.
Demonstrating the procedure will be Alban Johansson from my research group. Begin by covering the surface of the dishes with 0.1%gelatin. Incubate them at 37 degrees Celsius for approximately 20 minutes.
Aspirate the gelatin solution and allow the dishes to dry. Use scissors to cut at the base of the tail of a euthanized mouse to remove it. Place the tail in DPBS with 2%FBS until ready to use.
In a tissue culture hood under sterile conditions, prepare a diluted trypsin solution of 0.02%trypsin EDTA in DPBS and add 5 milliliters into an uncoated 10-centimeter dish. In a tissue culture hood, wash the tail in enough 70%ethanol in a 15-milliliter tube to cover it and then in DPBS. Place the tail flat in a dish and use forceps to hold it in place.
Use a scalpel to make an incision on the tail along its longitudinal axis from the base to the tip. Use a pair of forceps to grasp the tail and hold it vertically and use a second pair to grip the skin beside the incision at the base of the tail and peel it back. Perform this at both sides of the incision until the skin can be peeled off by pulling downwards toward the tip of the tail.
Hold the peeled tail with forceps over the dish containing the trypsin solution. Use dissection scissors to cut the tail into small pieces. Use scissors to fragment these pieces in the trypsin solution into even smaller pieces and then incubate at 37 degrees Celsius for 10 minutes.
To quench the trypsin, add two volumes of FEX medium, DMEM with supplements and antibiotics. Collect entire contents of the dish into a 50-milliliter tube. Centrifuge the tube at 350 times G for five minutes at four degrees Celsius.
Aspirate the supernatant and resuspend the pellet containing the tail fragments in 10 milliliters of fresh FEX medium. Transfer this suspension to a gelatin-coated dish. Incubate at 37 degrees Celsius in 5%carbon dioxide and 4%oxygen, adding fresh FEX medium every two days.
Observe the culture after five to seven days, in which the tail fragments have attached at the bottom of the dish, and fibroblasts are moving away from them. Once clusters of fibroblasts are visible, gently shake the dish to dislodge tail pieces. Aspirate the medium with all the bone fragments, leaving the fibroblasts attached to the plate.
Add 10 milliliters of fresh FEX medium and culture the fibroblasts until confluent. To ensure that there is no contamination by hematopoietic progenitors in the fibroblast culture, dissociate the cells from the plate using trypsin EDTA as described in the manuscript. Add medium and collect the cells, and add magnetic beads with antibodies.
Use a magnetic separation system to remove cells expressing hematopoietic markers. Including this step is absolutely vital because it eliminates any HSPCs that might be present in the culture and therefore the likelihood of any false positives on the day of the FACS analysis. Seed retroviral packaging cells at approximately 25, 000 cells per square centimeter on a tissue culture treated dish.
Incubate the cells in high-glucose DMEM at 37 degrees Celsius and 5%carbon dioxide overnight. On the following morning, remove the medium and add half the volume used to culture overnight of DMEM with no additives. In the afternoon, check the cells for 70 to 80%confluency required for transfection.
Begin the transfection by preparing a two to one mixture of six micrograms of the expression vector PMX and three micrograms of the helper vector containing Gag, Pol, and envelope genes for each reprogramming factor. Next, for each reprogramming factor, add 300 microliters of room-temperature DMEM in a sterile polystyrene tube. Then carefully add 27 microliters of room-temperature commercial transfection reagent directly into the medium to avoid contact with the tube wall.
Add the plasmid mix into the transfection reagent containing tube. Vortex briefly and incubate the mixture for 15 minutes at room temperature. Vortex the transfection reagent DNA mixture again.
Add it dropwise to the retroviral packaging cells so that it is evenly spread over the culture and incubate at 37 degrees Celsius. 24 hours after transfection, remove the medium and add DMEM with 20%FBS and 100 units per milliliter penicillin streptomycin. 48 hours after transfection, collect the supernatant and filter it through a 0.22 micrometer pore size syringe filter.
Begin the transduction by seeding the tail tip fibroblasts at 10, 000 cells per square centimeter on 0.1%gelatin pre-coated dishes in FEX medium. Incubate at 37 degrees Celsius for 24 hours. When carrying out this step, it is important to add the four retroviral supernatants at an equal ratio to ensure that all four transgenes contribute equally to the reprogramming process.
On the following day, prepare a transduction mixture by first adding one volume of viral supernatant for each reprogramming factor supplemented with four micrograms per milliliter of retroviral infection reagent to six volumes of FEX medium. After that, aspirate the FEX medium from the fibroblast culture and add the transduction mixture. Incubate the transduction reaction for four hours at 37 degrees Celsius in hypoxic conditions at 5%carbon dioxide and 4%oxygen.
After four hours, aspirate the transduction mixture and add fresh reprogramming medium. Incubate at 37 degrees Celsius in hypoxic conditions for eight days, adding fresh reprogramming medium every two days. Observe successful reprogramming with yielded clusters of cells detached from the plate.
To harvest reprogrammed cells from the dish for further analysis, gently pipette them up and down and then collect the cells. After successful reprogramming of iEPs, YFP-positive cells are observable as early as five days after transduction. They become round, lift from the surface of the plate, begin forming clusters, and display an erythroid precursor-like morphology.
Hemoglobinization of some cells is evident by positive benzidine staining. A small fraction expresses the erythroid-specific surface marker TER-119. By day eight, large YFP-positive clusters can be seen.
Day eight iEPs have a more differentiated erythroid phenotype than day five iEPs. They are significantly smaller, have accumulated more hemoglobin, and show increased expression of TER-119. Gene expression analysis by qPCR of iEPs collected at day eight shows that they have almost shut down expression of fibroblast genes and have upregulated many erythroid genes.
After performing BFU-e colony-forming assays on the reprogrammed cells, day eight iEPs form two types of colonies, distinctly red and not visibly red. While cells from red colonies displayed erythroblast morphology, cells from non-red colonies did not. Approximately one in 1, 000 day five iEPs formed red colonies while only approximately one in 10, 000 colonies formed from day eight iEPs.
Reprogramming efficiency of tail-tip fibroblasts or TTFs is influenced by the passage number. TTFs that had been passaged nine times showed a dramatic reduction in the ability to produce clusters of iEPs compared to the cells that had been passaged three times. Furthermore, different culture conditions can affect efficiency of reprogramming.
Transduced TTFs cultured in normoxia are much slower to reprogram and IEP clusters are observed after 10 days instead of five to eight days. Following this procedure, methods such as colony-forming assays, qPCR and FACS analysis can be applied in order to determine the cell differentiation status. Since its development, this technique is paving the way for researchers in the field of erythropoiesis in understanding the switch between primitive and definitive erythroid cells in mouse and human.
