The overall goal of this method is to direct the Differentiation of Human Pluripotent Stem Cells toward the Primitive or Definitive Hematopoietic Program using a stage specific signal manipulation approach. This method can address many key questions in the field, such as understanding the signal and transcriptional regulators of human definitive hematopoiesis. The main advantage of this technique is that it uses stage specific manipulation instantly.

Demonstrating this procedure is Carissa Dege, a post doc in my laboratory. To begin the protocol, obtain previously prepared Human Pluripotent Stem Cell Lines or HPSCs grown on mouse embryonic fiberblasts or MEFs to 70%to 80%confluency in a 37-degree Celsius incubator with five percent CO2. Aspirate the HPSC media and add one milliliter of 37-degree Celsius 0.25%Trypsin EDTA to each well for one minute.

After one minute, aspirate the Trypsin and add one milliliter of stock media to each well. Using a cell scraper, scrape the cells off the plate. Then, add one milliliter of wash buffer to each well.

With a two-milliliter serological pipette, triturate the cells three to five times to break up large clumps. And transfer the cells to a 50-milliliter conical tube. Centrifuge the HPSCs at 330 x G for five minutes.

After the spin, add HPSC media and re-suspend the cells. Next, add two milliliters of re-suspended HPSCs to each well of the MAT-coded plasticware and incubate the cells overnight. After 24 hours, aspirate the media and replace it with 37-degree Celsius 0.05%Trypsin EDTA for one minute.

Aspirate the Trypsin EDTA. Add one milliliter stock media and scrape the cells vigorously with a cell scraper. Then add one milliliter of wash media.

With a two-milliliter serological pipette, triturate the cells three times and transfer them to a 50-milliliter conical tube. Centrifuge the cells at 220 x G for five minutes. Then aspirate the media, add 20 milliliters of wash media and use a five milliliter serological pipette to gently re-suspend the cells.

After centrifugation and aspiration, carefully re-suspend the Embryoid Bodies or EBs in Day Zero media. Dispense two milliliters of differentiation media containing EBs into each well of a six-well low-adherence cell culture dish using a ten-milliliter serological pipette. Place the EBs into a 37-degree Celsius five percent CO2, five percent O2 multigas incubator.

Twenty four hours later, add two milliliters Day One media. Incubate the cells overnight in a multigas incubator. After 18 hours, gently harvest the EBs with a five-milliliter serological pipette and spin them at 100 x G for five minutes.

Wash and combine the entire culture with ten milliliters of Iscove's Modified Dulbecco Medium or IMDM. After spinning the culture again at 100 X G for five minutes to remove debris, aspirate the media. Gently re-suspend the EBs with Day Two Media using a P1000 pipette.

Then dispense two milliliters of the EBs per well into six-well low-adherence cell culture plates. Add three micromolar CHIR99021 or IWP2 to specify definitive or primitive hematopoetic progenitors respectively. Incubate the plate in a 37-degree Celsius multigas incubator for 30 hours.

After 30 hours, isolate the EBs with a two-milliliter serological pipette by transferring them to a 50-milliliter conical tube and centifuging them at 330 x G for five minutes. Gently re-suspend the EBs and wash them with 10 milliliters of IMDM. Re-suspend the EBs in Day Three Media and dispense two milliliters into each well of low-adherence six-well plates.

Incubate the cells in a 37-degree Celsius multigas incubator for 72 hours. After 72 hours of incubation, add two milliliters of Day Six Media to each well. Then incubate the EBs in a 37-degree Celsius multigas incubator for an additional 48 hours.

Isolate the CHIR99021-treated EBs on day eight of differentiation and proceed to the next section. Centrifuge the EBs at 330 x G for five minutes. Aspirate the supernatant.

Next add two milliliters of 0.25%Trypsin EDTA to the EBs. Vortex the EBs for five seconds and incubate the Ebs in a 37-degree Celsius water bath for eight minutes. Then add five milliliters of stop solution and centrifuge the EBs.

Then aspirate the supernatant. Re-suspend the EBs in five milliliters of Collagenase II.Vortex the tube for five seconds and then incubate the EBs in a 37-degree Celsius water bath. After 60 minutes, vortex the EBs for five seconds and add five milliliters of stop solution.

After centrifugation, aspirate the supernatant and re-suspend the cells in one milliliter of FACS buffer. Pass the cells thru a 40-micron cell strainer to remove undissociated cell clumps. Proceed with antibody labelling and FACS isolation of CD34-positive hemogenic endothelial progenitors.

Following FACS isolation, resuspend the cells in HE media. Distribute the cells in 50-microliter aliquots in a 96-well low-adherence culture plate and then incubate the cells overnight. The next morning, the hematopoietic progenitors should aggregate into clumps of six to ten cells.

Gently transfer the 50-miroliter volumes of re-aggregated cells into the center of a 24-well MAT-coded plate. Gently place the plate in the multigas incubator for four to six hours until the cells are attached. After four to six hours, to allow for cells to attach, slowly add one milliliter of freshly prepared HE media to each well.

Incubate the cells in the multigas incubator until the hematopoetic cells are visible. Next harvest the definitive hematopoetic progenitors by gently removing the HE media from the cells and passing through a 40-micron cell strainer to collect flow-through. For the remaining adherent cells, add 0.5 milliliters 0.25%Trypsin EDTA to the cells and incubate them in a 37-degree Celsius incubator for five minutes.

After Trypsinization, add 0.5 milliliters of stop solution to each well. Pass the cells through the same 40-micron cell strainer to pool all adherent and non-adherent cells together and spin the cells. Add 2, 000 to 10, 000 FACS isolated cells to one well of the OP9 DL4 cells in OP9 media.

Incubate in a 37-degree Celsius, 5%CO2 incubator. To passage the cells every four to five days, triturate the cells with a one-milliliter pipette. Pass through a 40-micron filter to remove clumps.

After centrifugation, aspirate the media. Re-suspend the cells in two milliliters of fresh T-cell media and place them on fresh OP9 DL4 cells. After at least 21 days of cold culture, triturate the cells vigorously and pass through a 40-micron filter to remove cell debris.

Centrifuge at 330 x G for five minutes and aspirate the supernatant and then continue with standard flow cytometry. Flow cytometry shows that IWP2-treated differentiation cultures, yield a distinct CD34 positive, CD43 negative and CD34 low CD43 positive population. While CHIR-treated differentiation cultures have less than 1%CD43 positive cells on day eight of differentiation.

Primitive hematopoietic progenitors derived under IWP2 conditions isolated on day nine will give rise to predominantly primitive erythroid and myeloid progenitors in the methylcellulose assays. The CD34 positive CD43 negative population derived from the CHIR99021 treatment is a heterogeneous population containing endothelial progenitors as well as CD34 positive, CD43 negative, CD73 negative, CD184 negative hemogenic endothelium, which when isolated by FACS will initially form a monolayer of endothelial-like cells that then undergoes an endothelial to hematopoietic transition resulting in round, refractile, non-adherent hematopoietic cells. These hematopoietic cells can be assessed by flow cytometry for the expression of CD34 and CD45.

Hematopoietic progenitors isolated from the EHT assays can be used in methylcellulose assays and give rise to large, burst-like, erythroid colony forming units, small erythroid colony forming units and myeloid progenitors. These representative flow cytometric analyses of the T-lymphocyte assays potential from CHIR99021-derived CD34 positive, CD43 negative, CD73 negative, CD184 negative hematopoietic progenitors and IWP2-derived CD34 positive, CD43 negative hematopoietic progenitors show the presence of a CD45 positive, CD56 negative, CD4 positive, CD8 positive population in CHIR-derived progenitors indicative of the hematopoietic potential within the input CD34 positive population. After watching this video, you should have good understanding of how to differentiate human pluripotent stem cells towards primitive or definitive amount of hematopoietic progenitors.