Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The overall goal of this method is to identify the phenotypic properties and engraftment potential of clonal Hematopoietic Stem Cell or HSC precursors during murine embryonic development. This method can help answer key questions in the field of developmental hematopoiesis about the unique lineage contributions of clonal HSC precursors during embryonic development. The main advantage of this technique is that hemogenic precursors are analyzed at the single cell level.

This allows identification of the unique phenotypic properties that distinguish rare HSC precursors. Begin by adding Aorta-Gonad-Mesonephros, AGM, tissues dissected from embryos between 9.5 and 11.5 days post coitum to a 15 milliliter conical tube containing 10 milliliters of PBS supplemented with 10%FBS on ice. When the tissues have settled to the bottom of the tube, aspirate the supernatant and immediately add one milliliter of 0.25%collagenase to the samples.

After 25 minutes in a 37 degree Celsius water bath, stop the reaction with one milliliter of PBS plus FBS and use a one milliliter pipette tip to triturate the tissues 20 to 30 times until a single cell suspension is obtained. Then dilute the cells with eight more milliliters of PBS plus FBS and collect the cells by centrifugation. Resuspend the pellet in 500 microliters of blocking buffer for five minutes on ice.

Then add 500 microliters of the antibody cocktail of interest. After a 15 to 20-minute incubation on ice, wash the cells in eight milliliters of PBS plus FBS and resuspend the pellet in one milliliter of fresh PBS plus FBS. Filter the cells through a 35 micrometer cell strainer into a five milliliter tube, washing the strainer with an additional three milliliters of PBS plus FBS to collect all of the cells and centrifuge the cells again.

Then resuspend the pellet in 500 microliters of PBS plus FBS and place the cells on ice. To set the gates for sorting in single cell and index sorting mode, load a small aliquot of cells onto the flow cytometer and set the acquisition to the lowest flow rate. Open a forward scatter area versus side scatter area plot and set a gate to include cells of varying size.

Open a forward scatter area versus forward scatter width plot and a side scatter area versus side scatter width plot and set a gate in each plot to exclude the doublets. Set a forward scatter area versus DAPI plot to gate the live cells. Then create an anti-vascular endothelial Cadherin versus anti-endothelial protein C receptor plot and gate the vascular endothelial Cadherin positive and high endothelial protein C receptor expressing cells.

To index sort the aorta-gonad-mesonephros cells, place a 96-well plate precultured with AGM endothelial cells in AGM serum-free medium plus cytokines into the plate block of the flow cytometer and load the experimental sample. Begin the sample acquisition and confirm that the index sorting single cell mode is selected to sort a single cell into each well of the 96-well plate. When one cell has been sorted into each well of the plate, place the plate into a tissue culture incubator set to 37 degrees Celsius and 5%CO2 for up to seven days.

At the end of the co-culture period, hematopoietic colonies of various sizes and morphologies can be visualized using an inverted microscope at a 100X magnification. To analyze the clones by flow cytometry, use a multichannel pipette to dissociate the hematopoietic cells from the endothelial layers without detaching the endothelial cells. Then transfer 100 microliters of cells into individual wells of a new 96-well v-bottom plate and return the original 96-well plate containing the remaining 100 microliters of cells to the tissue culture incubator.

Pellet the cells in the v-bottom plate by centrifugation and remove the supernatant by inverting and flicking the plate in a single rapid motion. Resuspend the pellets in each well with 50 microliters of blocking buffer at four degrees Celsius. After five minutes, add the antibody cocktail of interest to each well for a 20-minute incubation at four degrees Celsius.

At the end of the incubation, wash each well with 200 microliters of PBS plus FBS, flicking the plate at the end of the centrifugation to discard the supernatant. Resuspend the pellets in 50 microliters of PBS plus FBS and analyze the cells according to standard flow analysis protocols to screen for colonies that contain hematopoietic stem cell potential. Selecting the wells that contain cells with hematopoietic stem cell phenotype by flow analysis, use a multichannel pipette to resuspend the remaining 100 microliters of cells from the original co-culture and add five times 10 to the fourth rescue bone marrow cells in 100 microliters of PBS plus 2%FBS to each well.

After adoptive transfer of each clone into individual lethally irradiated congenic recipient animals, collect peripheral blood samples from the engrafted animals at regular intervals post transplant. To identify the clones with long-term engraftment, analyze the samples by flow cytometry according to standard flow analysis protocols. The phenotypic properties of each clone can then be correlated with its engraftment properties.

Pre-hematopoietic stem cells can be enriched by vascular endothelial Cadherin positivity and a high level of endothelial protein C receptor expression. Staining with other fluorochrome conjugated antibodies allows the retrospective analysis of additional phenotypic parameters that can be recorded for each cell during index sorting. Following index sorting into aorta-gonad-mesonephros endothelial cell co-cultures, the individual clones integrate into the aorta-gonad-mesonephros endothelial cell layer before the clones begin to round up and form hematopoietic clusters.

After five to seven days of co-culture, colonies of various sizes and morphologies can be detected. Here, four different colonies obtained following co-culture are shown with three colonies demonstrating different proportions of phenotypic hematopoietic stem cells along with other more differentiated cell types and the fourth colony lacking cells with hematopoietic stem cell marker expression. Adoptive transfer of the clone cell progeny allows assessment of the long-term multi-lineage engraftment capability of each clone following donor contribution to the myeloid B and T cell compartments of the peripheral blood of the recipient over time.

Although most colonies containing cells with a hematopoietic stem cell phenotype achieved long-term engraftment, transplantation is necessary to confirm a functional multi-lineage engraftment as some clones lose engraftment over time. The functional hematopoietic stem cell potential of each clone can then be correlated back to that clone's original index sorting parameters to identify its precise phenotypic characteristics. Following this procedure, other methods like secondary culture of hematopoietic progeny on stroma supporting B and T cell development in vitro can be performed to answer additional questions about the lymphocyte potentials of individual hemogenic precursors.