HSCs give rise to all blood lineages and are capable of self-renewal. Transplant studies have shown that only a few of these cells are necessary to repopulate the entire hematopoietic system in mice. Multipotent progenitor cells (MPPs), on the other hand, can give rise to multiple lineages, but are limited in their capacity for self-renewal.

Scientists at BD Biosciences set out to isolate HSCs from human umbilical cord blood using the cell surface signature Lin-CD34+CD38-CD90+CD45RA- defined by Park, Majeti, and Weissman. They collected data on both the pre-sort and post-sort samples, and uploaded the data to Cytobank. This public experiment can be found at https://www.cytobank.org/cytobank/experiments/8414.

In this HSC experiment, the channels were labeled with their corresponding reagents when the samples were collected on the flow cytometer. We can see these labels by clicking “Setup” on the Channels figure dimension from within the Working Illustration. If the channels were not labeled during sample acquisition, the labels can be edited at this stage by clicking in the appropriate Reagent box and editing the text.

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File-Internal Compensation is used in this experiment, meaning the channels were compensated during collection on the flow cytometer. You can learn more about compensation from our video tutorials. Cell populations were then defined from within the gating applet, which can be entered by clicking “Gate” on the Populations dimension. Once the populations have been defined, you can view the gating hierarchy by clicking “View” in the “Populations” box.

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The final step is to create an Illustration containing plots. There are two useful ways to view these data. First, from your Working Illustration, you can view the Gating Hierarchy (click the arrowhead to expand the section) for the HSC (Lin-CD34+CD38-CD90+CD45RA-) and MPP (Lin-CD34+CD38-CD90-CD45RA-) populations. The Gating Hierarchy shows the gating strategy used to define these populations. In this example, the Gating Hierarchy also corresponds to the gating scheme used during the sorting process on the flow cytometer. Make sure your populations of interest are selected in the Populations dimension before clicking to show the file hierarchy.

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The second way to view the data is to build an Illustration with plots comparing the pre-sort and post-sort samples, with the goal being to compare HSC purity levels. To generate the plots shown below, enable the Sample Types dimension. Choose “pre-sort” and “post-sort” as the Sample Types to display, and choose the “Lin-CD34+CD38-” Population to display. Under Plot Controls, display CD90 – PE on the Y-Axis and CD45RA-Horizon V450 on the X-Axis. Set Show Gates to Yes: this will display the CD90 and CD45RA gates defining the HSC and MPP populations on the Lin-CD34+CD38- parent population. Set Show Gate Statistic to Percentages to display the percentages of the populations in each gate. Set Show Plot Statistics to Yes to show a data table below the plots. Set Plot Statistic to Percent in Gate and Selected Gate to Lin-CD34+CD38-CD90+CD45RA- (HSCs): this will set the data table to show the percent of HSCs in the pre-sort and post-sort conditions. Finally rearrange the figure dimensions in this order: Sample Types, Populations, Channels. Remember to click to update your Illustrations after making all the changes. We can see here that HSCs were enriched from 43.05% pre-sort to 99.55% post-sort.

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