The overall goal of this procedure is to differentiate human hematopoietic stem and progenitor cells along the erythroid lineage and to knock down a particular transcription factor during ex vivo erythropoiesis. This is accomplished by first isolating CD 34 positive human stem and progenitor cells from cord blood, bone marrow, or peripheral blood mobilized with GCSF. The second step of the procedure is to differentiate these hematopoietic stem cells along the erythroid lineage using a four step cell culture procedure that combines specific cytokines and co-culture on a stromal cell layer to mimic the bone marrow microenvironment.
Next, prepare lentiviral vector particles expressing an HRNA molecule against the transcription factor of interest, a scrambled HRA molecule as a negative control and the GFP protein for controlling the efficiency of virus infection. The final step of the procedure is to infect the primary human erythroid cells at a given stage of differentiation with lentiviral particles. Ultimately, results can be obtained that show cell proliferation and erythroid differentiation through the analysis of cell morphology, colony forming capacity of progenitors, production of hemoglobin and expression of erythroid specific markers by facts.
Efficiency of virus infection is analyzed by measuring GFP expression by facts and knockdown of the transcription factor of interest is analyzed by R-T-Q-P-C-R. This method can help answer key questions in the field of hematopoesis, such as molecular mechanism by each transcription factor, regulate gene expression in a non transformed environment provided by primary human cells. This is important for understanding fundamental processes of hematopoiesis such as sulfate determination, self renewal proliferation, or apoptosis.
Although this method can provide insight into normal ROIs and its regulation at the molecular level, it can also be used as a model system to study targeted therapies for diseases arising from imbalance, ingrowth, and differentiation, including myeloproliferative disorders such as polycythemia Vera Andro leukemia. The protocol for ex vivo erythropoiesis of human hematopoietic stem and progenitor cells to mature hemoglobin containing red blood cells is comprised of two steps, the isolation of CD 34 positive human hematopoietic stem and progenitor cells, and serum free erythroid differentiation in cell culture. In step one, CD 34 positive cells are isolated from cord blood, bone marrow, or human peripheral blood mobilized with GCSF using positive immuno selection.
The second step serum free erythroid differentiation involves growing CD 34 positive cells in liquid culture. According to a four step protocol adapted from the method initially developed by the DU laboratory from day zero to day 11, hematopoietic cells are grown in supplemented ice cuffs, modified ECCOs, medium containing hydrocortisone stem cell factor interleukin three and erythropoietin. Then from day 11 to day 15, erythroid cells are co-culture on MS five mesenchymal cells In supplemented IMDM medium containing EPO in step three.
After harvesting summer erythroid cells for future use, the remaining cells are co cultured on MS five cells in supplemented IMD and medium with no cytokines. Lastly, from day 18 to day 26, erythroid cells are concentrated three to four fold and maintained on an MS five layer to allow maturation and 10%FBS is added in supplemented IMDM medium for better preservation of cultured red blood cells. For the purposes of this video article, we will only demonstrate step two of erythroid differentiation, the co-culture of erythroid cells on mesenchymal cells with erythropoietin.
The most difficult aspect of this procedure is to perform these steps on a large scale washing and replacing the medium of the flask containing AUS five cells for co culture and subsequently transferring art trade cells into this flask. Therefore, we process for flask at a time and have 2%organ simultaneously. To begin the co-culture of erythroid cells on mesenchymal cells with EPO count the erythroid cells using trian blue to exclude dead cells.
On day 11, the cell density should be about 1.5 times 10 of the six cells per milliliter reserve about 80 milliliters of the erythroid cell culture for co-culture on MS five mesenchymal cells. Prewarm PBS supplemented IMDM medium, and supplemented IMDM medium containing EPO at 37 degrees Celsius. Next, retrieve from the incubator four of the previously prepared flasks with confluent MS five cells.
Wash each flask with 20 milliliters of one XPBS and immediately add 80 milliliters of fresh supplemented IM DM medium containing EPO into each flask. Keep the cells in the incubator while the erythroid cells are being prepared. Now transfer the reserved erythroid cells to two 50 milliliter conical tubes and centrifuge at 1500 RRP M for 10 minutes.
After discarding the supernatant wash each of the two cell pellets with 20 milliliters of supplemented IMDM medium resus, suspend each cell pellet in 40 milliliters of supplemented IMDM medium containing EP.Then distribute 20 milliliters of this cell suspension to each of the four flasks containing MS five cells and 80 milliliters of medium. At this point, you should have four flasks with confluent MS five cells containing 100 milliliters of erythroid cells at 0.3 times 10 to the six cells per milliliter on the following day, counter erythroid cells with trian blue to exclude dead cells. Cell density should be about 0.6 times 10 to the six cells per milliliter.
Harvest the necessary number of cells for tests to verify proper cell proliferation and erythroid differentiation. Studying erythropoiesis at the transcriptional level also requires the ability to over express or knock down specific factors in primary erythroid cells. In this example, lentivirus mediated gene delivery system is used to knock down the transcription factor to one in primary human erythroid cells.
The protocol for preparing lentiviral vector particles is available in the manuscript. This video will demonstrate the procedure for lentiviral infection of primary erythroid cells for gene delivery. Nucleated erythroid cells can be efficiently infected at any stage of differentiation using a 50 to 100 multiplicity of infection.
Here lentivirus expressing a T one or scramble S-H-R-N-A will be used to infect erythroid cells at days eight and nine of differentiation using an MOI of 52 use per cell. To begin this procedure, prepare three 15 milliliter conical tubes containing one times 10th of the sixth erythroid cells, each one tube for TAL one knockdown, one tube for scrambled negative control and one tube for GFP expression. Control wash cells with five milliliters of supplemented IMDM medium containing EPO spin at 1000 RPM for five minutes and remove as much supernatant as possible.
Infection should be carried out in minimal amounts of media to allow maximum absorption of lentivirus onto the cells. Add poly brain at a final concentration of 40 micrograms per milliliter to each tube of concentrated lentiviruses. Add 100 microliters of prepared concentrated virus containing poly brain to each tube of cells.
Incubate for one hour in the incubator at 37 degrees Celsius mixed by finger tapping every 10 minutes. This enables the even distribution of virus across the cells at the completion of the one hour incubation at 1.9 milliliters of supplemented IMDM medium containing EPO to each tube. Transfer each sample to a six well plate and place in the 37 degree Celsius incubator for 24 hours.
After 24 hours, harvest the cells in separate 15 milliliter tubes. Spin them at 1000 RRP M for five minutes and remove the supernatant. Repeat the infection, add virus and poly brain and incubate for one hour 37 degrees Celsius with finger tapping.
Then add supplemented IMDM medium containing EPO to the cells. Transfer each sample to a six well plate and incubate for 24 hours at 37 degrees Celsius. On the following day, transfer the cells to three 15 milliliter conical tubes and wash by centrifugation, removal of supernatant and resus suspension of cells and fresh medium centrifuge.
Again, aspirate the supernatant and resuspend the cells at a concentration of 0.2 times 10 of the six cells per milliliter into supplemented IMDM containing EPO. After that, proceed with the procedure for ex vivo erythroid differentiation cells are counted every second day during the ex vivo erythroid differentiation protocol. A representative result in logarithmic scale shows that cells are highly proliferative as illustrated by the 10, 000 fold amplification.
In addition, may grunwald gem, so morphology staining is done every second day using 0.05 times 10 of the six cells for day zero to eight and 0.2 times 10 of the six cells for the other days. As illustrated in this figure cells at particular stages of differentiation, display recognizable sizes and morphologies. Notice the process of enucleation that occurs at the end of differentiation starting at day 20.
At day 26, all cells have lost their nuclei. Colony forming assays are used to detect early and late hematopoietic progenitors during the first days of differentiation, and the results are shown here at day zero. In addition to erythroid progenitors, granulocyte, macrophage progenitors are significantly represented within the early CD 34 positive cells.
As differentiation proceeds see a few GM decreased significantly, and at day six they have been completely replaced by erythroid progenitors, BAUE and CAUE. It is also important to note that from day four to day six, the early erythroid progenitors, BAUE are progressively replaced by their more differentiated counterparts, CAUE. By day 10, the erythroid cells have essentially lost their colony forming capacity.
Shown here are typical pictures of BFUE and CFUE. The red brown color is due to hemoglobin of BFUE and CFUE. Erythropoiesis is monitored during exvivo differentiation by harvesting cells at the indicated time points and analyzing them by facts or by microscopy for expression of cell surface markers.
Here, the bars represent percentages of positive cells. Significant observations include the progressive loss of the hematopoietic stem progenitor marker CD 34, the progressive acquisition and loss of the progenitor marker CD 36, the progressive acquisition and loss of the erythroid, progenitor, and reticulocyte marker CD 71, and the increase of the erythroid precursor and erythrocyte marker GPA. Other markers of erythroid differentiation include hemoglobin synthesis starting at day eight as measured by benadine staining and cell enucleation starting at day 20.
As measured by the loss of the DNA stain LDS, the results of lentiviral mediated gene transfer in human primary pro erythroblasts are shown next knockdown of the transcription factor. T one was mediated by lentivirus delivered anti T one SHRA or a scrambled control SHRA measurement of the kinetics of T one mRNA transcript level by RT qPCR R and expression of T one transcript relative to 18. Srna showed that 75%of T one transcripts were successfully knocked down in primary erythroid cells.
The increase of T one transcript level from day nine to day 12 and the scrambled control underscores the importance of this transcription factor during erythroid differentiation. Finally, in another experiment, erythroid cells were infected with lentivirus expressing the GFP protein. This figure shows that three days after infection, most erythroid cells are expressing GFP as measured by fluorescence under the microscope and by facts.
This indicates an efficiency of infection in primary erythroid cells close to 100%After its development. This technique paved by for researchers in the field of hematopoiesis to explore transcription regulation in human erythropoiesis. After watching this video, you should have a good understanding of how to differentiate hematopoietic stem and progenitor cells into cytes in culture, as well as how to use lentiviral mediated gene delivery in order to induce efficient gene knockout at any stage of this differentiation process.
Thank you for watching and good luck with your experiments.
