The overall goal of this procedure is to monitor chimerism in the stem cell transplant setting. This method can help address key questions in the transplant field such as, engraftment of the hematopoietic stem cell transplant for patients with hemoglobinopathies. The main advantage of this method is that it combines flow cytometry based cell sorting, column information assay and subsequent analysis of short term repeats to assess the proliferation and differentiation of progenitors in the erythroid compartment.
The implication of this technique extend out therapy of graft sub rejection because it may help identify patients susceptible to disease relapse and initiate subsequent immunomodulatory strategies. Further more, it also can be applied to future clinical studies, stem cell research and design of skin therapy trials. Visual demonstration of this method is critical as the process is difficult to learn and needs to be performed by trained personnel.
Demonstrating this procedure will be, Elisabeth Hoflehner, a technician in my lab. To begin, generate a single cell suspension of the bone marrow sample for fat staining. To do this, place a cell strainer containing the bone marrow and screw it on top of a centrifuge tube.
Use the syringe plunger, to force pieces of tissue through the mesh. To collect all material, rinse with a few milliliters of suspension buffer. Add three milliliters of gradient medium to a centrifuge tube and carefully layer the single cell suspension onto it.
Centrifuge the tube at 550 G at 20 degree celsius for 30 minutes, with acceleration set at nine and deceleration at zero. Remove the upper layer containing plasma and platelets with the pipette, making sure to leave the mononuclear cell layer undisturbed at the interface. Transfer this mononuclear cell layer to a centrifuge tube.
Next, wash the cells with 10 milliliters of suspension buffer. Centrifuge the tube at 250 G at four degree celsius, for five minutes. Discard the supernatant and re-suspend the cells in two milliliters of complete isokov's modified dulbecco's medium, by pipetting.
To determine the cell concentration with a hemocytometer, place 95 microliters of 2%trypan blue stain in a 96 well plate. Add five microliters of cell suspension, mix well and incubate at room temperature for a short time. Fill the hemocytometer and observe it under a microscope.
Count only on stained viable cells. After that, centrifuge the cell suspension at 250 G at 25 degree celsius for 10 minutes. To block the FC receptor, use 2.5 micrograms of block per 100 microliters.
Incubate on ice for 10 to 15 minutes. Add the combination of pre-titrated monoclonal antibodies appropriate for the volume used to stain the cells. Incubate at four degrees celsius in the dark for 20 minutes.
Wash the cells with three milliliters of staining buffer and then, centrifuge. After adjusting cell concentration, transfer the cell suspension to a standard flow cytometry tube, by pipetting it through a 70 microliter cell strainer to remove aggregates. Then, set up and optimize the cell sorter and software according to the manufacturer's instructions.
Set up a template that includes a bivariate plot to display forward scatter or FSC and side scatter or SSC. To place the cell population of interest on scale, run the cells on the sorter and adjust FSC and SSC. Begin with recording the data for the negative control tube, after that, record the data for each single stain compensation control tube and calculate compensation.
After that, gabe the experimental samples on a bivariate FSC, SSC dot plot to include both lymphocytic and myeloid cells. By comparing the different signals of FSC, exclude doublets and aggregates. Then visualize the singlet cells that display CD45 and CD36 on a bivariate dot plot.
CD45 positive events represent leukocytes and their progenitors. CD45 negative and CD36 positive events, represent erythroid progenitors. To define CD36 positive and CD45 positive cells, use gating tools and then visualize the CD45 positive events and another dot plot displaying CD34 and SSC parameters.
Next, use gating tools to define the CD34 positive cells or leukocyte progenitors and SSC high cells or myeloid cells at various stages of differentiation. After that, select gates for sorting into external collection tubes. Finally, continue with cell sorting at four degree celsius until acquiring desired number of cells.
Dilute the cells by adding complete IMDM medium supplemented with human recombinant erythropoietant or EPO. Transfer 300 microliters of the diluted cells to three milliliters of H4434 medium and incubate while shaking at room temperature for five minutes. Next, plate three 35 millimeter dishes with one milliliter of cell suspension each.
Place two of the culture dishes together with a third filled with water in a 100 millimeter plate. Culture these cells in a humidified incubator at 37 degree celsius, with 5%carbon dioxide for 14 days. Use an inverted microscope at 40x magnification and the culture dish marked with a scoring grid, to score the colonies according to their morphology.
Classify the colony forming units or CFU into multi-potential progenitor cells, granulocyte monocyte progenitor cells, burst forming unit erythroid and colony forming unit erythroid cells. To begin, recover the cells from fax or the CFU assay plate according to the text protocol and proceed with DNA extraction. After centrifuging the cells, use an automated blood DNA isolation instrument to isolate nucleic acid from the pellet.
Measure the DNA concentration using a fluorometer. Prepare the samples according to the manufacturer's instructions and then place the sample into the sample chamber of the fluorometer. Add diluted DNA to a mix of DNA polymerase, the primer set and the reaction mix to set up a PCR reaction according to the manual.
Include pre-transplant DNA from donor and recipient, as well as nuclease free water, as a negative control and 9947A is a positive control. To analyze PCR results, add 10 microliters of denatured DNA samples to the wells of a 96 well reaction plate. Seal the plate, briefly centrifuge and place the plate on ice.
Megakaryocyte erythroid progenitors assorted as population positive for CD36 and negative for CD45 antigen. Additionally, CT45 positive and CD34 positive cells are identified as lymphoid myeloid precursors and CD45 positive cells, with high SSC signal as mature myeloid cells. The purity of erythroid progenitor cells after sorting was more than 85%And the purity of myeloid cells was more than 95%Bone marrow cells separated by CD34 specific magnetic beads were cultured for 14 days.
Some of these cells appear to proliferate, differentiate and form colonies. Stimulation with various concentrations of EPO increased the formation of prominently red erythroid colonies. PCR analysis of short tandem repeats or STRs specific for recipient and donor DNA can detect chimerism in cells.
This method successfully determined the amount of recipient versus donor DNA in cells obtained from the cell sorting of bone marrow cells, as well as in cells obtained from the clonogenic assay. Once mastered, this technique can be done in four hours if it is performed properly. By attempting this procedure, it is important to remember that this technique needs to be standardized and performed by appropriately trained personnel.
Following this procedure, residual donor derived erythroid progenitor cells can be further analyzed in order to answer additional questions like, the selective advantage for genetically corrected erythroid stem cells. After it's development, this technique paved the way for researchers in the field of stem cell transplantation, to explore proliferation and differentiation of progenitors in the erythroid compartment. After watching this video, you should have a good understanding of how to monitor engraftment after stem cell transplantation in patients with hemonoglobinopathies.
