Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

The overall goal of this procedure is to generate erythroid and myeloid blood cells from human pluripotent stem cells by forced expression of transcription factors. This method is applicable for the generation of homogenic endothelium in blood cells for studies of endothelial to hepo transition and transcriptional regulation of hepo development and specification. The main advantage of this technique is that the method provides inefficient and rapid means of homogenic endothelium induction from human preport stem cells, and allows for observation of the endothelial hemmie transition in a cell culture dish.

Dr.Bruske, a research assistant in Dr.Lin's laboratory, will demonstrate the procedure Before beginning preco six wall plates with an extracellular matrix such as mat gel by diluting it according to the manufacturer's instructions and pouring it into the wells. Incubate the plates for 30 to 60 minutes at 37 degrees Celsius for the transduction medium. First, prepare lentiviral aliquots for each transduction set such that the estimated multiplicity of infection or MOI of virus to cells is one virus per cell and keep them at four degrees Celsius.

Next, combine complete serum free medium for hbsc viruses poly brain to a final concentration of six micrograms per milliliter and rock inhibitor Y 2 7 6 3 2 to a final concentration of 10 micromolar in a total volume of 1.3 milliliters for each transduction set and set the mixes on ice to prepare the cells. Start with a healthy batch of pluripotent stem cells at 70%confluence about four days after the last passage. If human pluripotent stem cells were maintained in maps, they must be transferred to feeder free conditions, two to three passages prior to transduction experiments and represent healthy pluripotent stem cell cultures.

Free of spontaneous differentiation. Aspirate the HPSC medium and add two milliliters of salt detachment solutions such as Accutane per well of a six Well plate incubating the plate at 37 degrees Celsius and 5%carbon dioxide for five to seven minutes. Then add two milliliters of complete medium with 10 micromolar rock inhibitor and collect the detached cells.

After preparing a homogenous cell suspension, take a small aliquot and count the viable cells by the Trian blue exclusion method. Next, centrifuge the cells at 200 times G for five minutes and resuspend the pellet in complete medium with 10 micromolar rock inhibitor at a final concentration of 3.4 times 10 to the six live cells per milliliter. Then add 200 microliters of cell suspension containing 0.68 times 10 to the six cells to each of the 1.3 milliliter transduction medium prepared earlier.

Remove the matrix coated six wall plate from 37 to degrees Celsius. Next, aspirate the matrix solution and transfer each of the transduction mixes into a well and swirl the plate to distribute the cells evenly in the well. 24 hours.

After transduction, visualize the wells under the microscope to make sure that at least 80%of the cells are adhered. Discard the virus solution and wash the attached cells with incomplete medium without any growth factors. Next, add three milliliters of the medium supplemented with cytokines referred to as three F.Medium to the cells.

Incubate the cells overnight on the following day. Change the medium with fresh three F medium containing one microgram per milliliter of pur mycin. To eliminate the residual nont transduced cells.

Replace this medium every 24 hours to remove the dead cells. After 48 hours of the treatment, discontinue pur mycin and use only fresh three F medium on day four. After transduction, observe the cells under the microscope for the appearance of clustered cells with endothelial morphology to determine the formation of hemo endothelial cells.

First associate the cells between days three or four from the culture dish with the cell detachment solution. Collect the detached cells in complete media and centrifuge them, resuspend the cells in max buffer at room temperature and count the cells in a hemo cytometer, then incubate one times 10 to the fifth cells per staining reaction with suitable antibodies for the detection of v catrin, CD 2 26, CD 73, and CD 43. Cell surface markers.

Perform flow cytometry is described in the text protocol from day four. After transduction, continue differentiation of cells by replacing half of the medium from each well, every 48 hours between five to seven days round blood cells will appear loosely aggregated and refile above the endothelial cell layer. Maintain the cultures for up to 14 days during which time the floating cells should expand significantly.

Flow cytometric analysis of ETV two, gata one and ETV two. Gata two transduced cells shows the formation of terin positive hemo endothelium, which can be identified by the expression of CD 2 26 and lack of CD 73 expression at day three to four of differentiation flow cytometric analysis of ETV two, gata one and ETV two. Gata two transduced cells shows induction of CD 43 expressing blood cells at day seven.

After transduction flow cytometric analysis of gata two TAL one LMO two induced cells on day 14 after transduction shows formation of blood cells that express CD 2 35 A and CD 41 A right stained cytosine preparations of gata two TAL one lmo two induced hematopoietic cells showed erythroid macrophage and mega cario acidic cell morphologies in colony forming cell assays. The transduction of human pluripotent stem cells can be done in less than an hour while performing the transduction procedure. It is important to consider stem cell viability always to include rock inhibitor in the reaction mixture and to use an optimal amount of virus per cell following the first three days.

Post transduction are the methods like immuno staining, colon forming asay, and flow cytometry can be performed in order to answer questions about the efficiency of differentiation and the type of hemato dease is induced. After watching this video, you should have a good understanding of how to ize and infect human pluripotent stem cells and what to expect in differentiating cultures within the first seven to 10 days. After transcription factors were successfully delivered into human purport stem cells, cell lose their per potency and gradually commit to a hematopoietic fate.

Thus, researchers will have a convenient in vitro model to study molecular mechanisms of the endothelial to hematopoietic transition. Don't forget that working with lentivirus containing regions can be extremely hazardous and precautions such as wearing personal protection equipment and proper disposal of regions containing viruses should always be taking while performing this procedure.