This method helps overcome limitations in studying human hematopoietic development by providing a simple and tractable in vitro platform based on direct cell reprogramming. Visual demonstration of this method will provide ideal guidance in fundamental steps to generate human hemogenic cells namely during lentiviral production, fibroblast transduction and cell culture. By converting skin fibroblasts into hemogenic precursors, we hope to generate patient specific hematopoietic stem and progenitor cells in sufficient numbers for stem cell transplantation.
Begin by growing HEK293T cells in a 100 millimeter tissue culture treated dish with 10 milliliters of complete DMEM at 37 degrees Celsius and 5%carbon dioxide until confluency. On the day prior to transfection, after aspirating the medium, wash the dish carefully with five milliliters of PBS and detach the cells with 1.5 milliliters of dissociation solution. After incubating for five to 10 minutes at 37 degrees Celsius, inactivate the solution with three milliliters of complete DMEM and transfer the cell suspension to a 15 milliliter conical tube.
Wash the dish with 5 milliliters of complete DMEM to remove any remaining attached cells and pull the wash with the cell solution. Collect the cells by centrifugation, aspirate the supernatant and resuspend the pellet in six milliliters of complete DMEM. Then, split the suspension evenly between six 100 millimeter tissue culture treated dishes in a final volume of 10 milliliters of complete DMEM per dish.
The next day, add 10 micrograms total mass of the three transfer plasmids together to a new 15 milliliter conical tube. Next, add 10 micrograms of the second generation of psPAX2 packaging vector encoding the gag, pol, tat and rev genes followed by the addition of five micrograms of pMD2. G envelope vector encoding the VSV-G gene to the tube.
Finally, add water to bring the final volume to 500 microliters. In two new 15 milliliter conical tubes, add 10 micrograms of FUW-M2rtTA plasmid, 10 micrograms of the packaging vector, and five micrograms of the envelope vector to each tube. Then, add water up to a final volume of 500 microliters to each tube.
Next, add 62.5 microliters of two molar calcium chloride to each of the three tubes and use a pipette controller equipped with a Pasteur Pipette to release bubbles into each mixture. While the bubbles are forming, add 500 microliters of BES buffered saline dropwise against the Pasteur Pipette and onto the mixture. Incubate the tubes at room temperature for at least 15 minutes until the mixtures appear slightly cloudy.
During incubation, carefully replace the supernatant of the HEK293T cell cultures with 10 milliliters of complete DMEM without antibiotics. Distribute the DNA complexes dropwise onto individual HEK293T cell culture dishes and incubate for 24 hours. The next day, replace the supernatants with four milliliters of complete DMEM per culture and return the dishes to a 37 degrees Celsius 5%carbon dioxide cell culture incubator overnight.
The next morning, collect the supernatants into one 50 milliliter tube per dish and add four milliliters of fresh medium to each dish. After eight more hours of culture, pool the supernatant in each dish with the previously harvested supernatant and add four milliliters of fresh medium. Return the dishes to the cell culture incubator overnight and collect the supernatants one final time.
When all of the virus has been collected, filter each lentiviral supernatant through a 0.45 micrometer low protein binding filter into individual tubes and add a maximum of 15 milliliters of filtered supernatant to individual centrifugal filter units for centrifugation. Discard the flow through. A viscous lentivirus containing liquid will remain in the filter unit.
When all of the lentiviral supernatant has been filtered aliquot 50 to 200 microliters of the concentrated lentiviruses for cold storage. Before beginning the reprogramming procedure, code a six well tissue culture treated plate with 500 microliters of 0.1%gelatin per well and incubate the plate for 20 minutes at 37 degrees Celsius. At the end of the incubation, aspirate the remaining gelatin solution.
Plate human dermal fibroblasts at a density of 1.5 times ten to the fifth cells per plate in two milliliters of complete DMEM per well and incubate overnight. The next morning, replace the medium in each well with two milliliters of complete DMEM supplemented with eight micrograms per milliliter polybrene and add a one to one mixture of pool produced transcription factor lentiviruses and M2rtTA in a new microcentrifuge tube. Then, transduce the fibroblast with an optimal volume of the lentiviral mixture, between 10 to 100 microliters per well.
After 16 hours of incubation, replace the supernatants with complete DMEM and return the cells to the cell culture incubator for six to eight hours. After the recovery, replace the supernatants with two milliliters of complete DMEM supplemented with polybrene and perform a second transduction as just demonstrated. At the end of the second transduction incubation, replace the supernatants with complete DMEM supplemented with one microgram per milliliter of doxycycline and return the plate to the cell culture incubator for 48 hours.
At the end of the incubation, split each well at a one to two ratio and replate cells in two milliliters per well of hematopoietic medium supplemented with doxycycline in a new gelatin coated six well plate. To obtain a sufficient number of cells for chromatin immunoprecipitation sequencing analysis, plate three times 10 to the fifth fibroblasts in a gelatin coated six well plate and incubate overnight. At the end of the incubation, transduce cells twice in two consecutive days with 10 to 20 microliters of lentivirus containing the individual factors of interest or a pool of the three factors plus FUW-M2rtTA at a one to one ratio.
16 hours after the second transduction, remove the virus containing supernatant and incubate the cells in complete DMEM for 24 hours. At the end of the incubation, replate the contents of each well into individual gelatin coated 100 millimeter tissue culture treated dishes with complete DMEM to a final volume of 10 milliliters of medium per dish, and return the cells to the cell culture incubator. After six days, replace the supernatant with complete DMEM supplemented with doxycycline.
Return the cultures to the incubator for an additional two days. As these representative cytometry plots demonstrate, approximately 17%of reprogrammed cells express both CD49f and CD9 after 25 days of reprogramming. The majority of double positive cells express CD143 and a small population express CD34, suggesting a dynamic hemogenic fate induction.
These markers are not activated in M2rtTA transduced human dermal fibroblasts cultured for 25 days. Immunofluorescence imaging confirms the expression of CD9 and CD143 in adherent and round cells that are morphologically distinct from fibroblasts, which are negative for these markers. Brightfeild imaging is performed to visualize cell confluency, morphology, and colony formation throughout the reprogramming process.
Single-cell RNA sequencing analysis of reprogrammed cells reveals a stepwise increase in CD49f, CD9, and CD143 expression from days two to 25. After chromatin immunoprecipitation sequencing, Genome Browser profiles shows GATA2 binding to genomic regulatory regions of ITGA6 and ACE when fibroblasts are cotransduced with the three factors or GATA2 individually. The volume of antiviral particles added to the culture should be optimized for successful reprogramming without compromising cell viability.
This platform can be coupled with pharmacological inhibition and genome-scale screening technologies, such as CRISPR-Cas9, to define novel regulators of human definitive hematopoiesis. This direct reprogramming approach will allow researchers to explore new questions in the field of human developmental hematopoiesis and to decipher mechanisms underlying the specification of human hematopoietic stem cells. It is important to perform antiviral collections and transductions in a laminar flow hood dedicated for lentiviral work and to discard viral contaminated waste into an appropriate container.
