September 7, 2011  |  Cytobank, Flow Cytometry  |  By  |  0 Comments

Analyzing Hematopoietic Stem Cell Enrichment Data in Cytobank

Dataset #8414: Human Cord Blood – HSC isolation

Background

Hematopoietic Stem Cells (HSCs) give rise to all blood lineages and are capable of self-renewal. Clinically, HSC transplantation is under investigation for the treatment of diseases of the blood and bone marrow, including cancer, where a patient’s blood cells are wiped out and replaced with healthy cells that arise from transplanted donor HSCs. Transplant studies in mice have shown that only a few of these cells are necessary to repopulate the entire hematopoietic system.

Human umbilical cord blood is a rich source of stem cells, including HSCs. However, a variety of other cell types populate cord blood and must be removed from HSC preparations used for transplantation. Multipotent progenitor cells (MPPs) are one such population. Derived from HSCs, MPPs give rise to multiple lineages and are present in significant quantities in cord blood, though they are limited in their capacity for self-renewal. Purification of HSCs can be achieved by staining and running cord blood through a FACS sorter and isolating cells with a Lin-CD34+CD38-CD90+CD45RA- surface signature (as defined by Park, Majeti, and Weissman). MPPs can be quantified or isolated by their Lin-CD34+CD38-CD90-CD45RA- signature.

Sample Data

If you would like to try your hand at analyzing HSC enrichment data on Cytobank, we have made available an HSC dataset provided to us by scientists at BD Biosciences. You can find a tutorial to guide your analysis on our documentation site.

Here are a few Cytobank features that will facilitate your analysis (click on the images to enlarge them):

View Populations

In the gating applet, you can view the gating hierarchy you have drawn by clicking View in the Populations box. Click the arrows next to the populations to reveal their descendents.

Gating Hierarchy

With the HSC and MPP populations selected in the Populations Figure Dimension, maximizing the Gating Hierarchy box in the Working Illustration will lay out the gating hierarchy, which directly corresponds to the FACS sort scheme used to isolate the cells.

Illustrations and Percent in Gate

Build an Illustration to compare pre-sort and post-sort samples, with the goal being to compare HSC purity levels. The Percent in Gate features enable you to display the percentage of cells falling within gates. In the example HSC dataset, we can see that HSCs were enriched from 43.05% pre-sort to 99.55% post-sort.

You can learn more about these and other features by viewing the HSC Isolation from Human Cord Blood analysis tutorial on our documentation site.

Forward Thinking

Cytobank can help with data management and sharing, in addition to data analysis. When you upload to Cytobank, the data are securely backed up in case of a personal computer failure and are available to be shared with colleagues or collaborators. Also, the annotation features in Cytobank enables descriptive information for the experiment to be saved. For example, flow facility operators running sorts for users can upload the data to Cytobank, share the pre-sort and post-sort purities, and draw gates and annotate files for their users.

Do you have questions about this dataset or other tutorials? Email us at helpdesk@cytobank.org.

– Angela

Reference:

Park CY, Majeti R, Weissman IL. In vivo evaluation of human hematopoiesis through xenotransplantation of purified hematopoietic stem cells from umbilical cord blood. Nat Protoc. 2008;3(12):1932-40. PMID: 19180077