Stem Cell Research

Cell surface staining for analysis and sorting of live cells.

Each type of stem cell or derivative expresses characteristic surface and intracellular proteins that can be used for identification. Because intracellular analysis requires permeabilization, surface markers are essential when researchers want to isolate live cell populations for further analysis. Fluorescence-activated cell sorting, or FACS, can be used to sort cells of interest in bulk or in single-cell depositions for downstream applications. To analyze cells for surface marker expression, a single-cell suspension must be stained with fluorescent-labeled antibodies and analyzed or sorted on a flow cytometer.


Sample preparation

Stem cells tend to be adherent and can grow as three-dimensional structures. To prepare a single-cell suspension for flow cytometric analysis, enzymatic digestion (with BD™ Accutase cell detachment solution or trypsin) or mechanical scraping can be used. Since enzymatic methods might cleave or modify some protein epitopes during the digestion process, preventing antibody labeling, they must be evaluated for each surface marker being measured. BD Accutase tends to be more broadly applicable than trypsin and yields a more consistent single-cell suspension than does scraping.

Monoclonal antibodies

Once cells are harvested and the dissociation buffer is removed, the cells are ready to be stained with antibodies. BD Biosciences offers an extensive reagent selection of antibodies against hundreds of stem cell markers conjugated to a variety of fluorochromes for flexibility in experimental design. For analysis of rare events and low density antigens, the BD Horizon™ Brilliant Violet™ family of reagents can increase brightness and resolution. For ease of use, BD Stemflow™ kits and cocktails contain standard antibody panels for analysis or sorting of different stem cell types.

Representative surface markers of selected stem cells and derivatives

Human Markers Mouse Markers BD Stemflow™ or Other Kit Cat. No.
Pluripotent Stem Cells (ESCs and iPSCs)

Positive: Alkaline Phosphatase, SSEA-4, SSEA-3, TRA-1-81, TRA-1-60

Negative: SSEA-1

Positive: SSEA-1 Human iPSC Sorting and Analysis Kit 562626
Human Pluripotent Stem Cell Sorting and Analysis Kit 560461
Human and Mouse Pluripotent Stem Cell Analysis Kit 560477
Hematopoietic Stem Cells (HSCs)

Positive: CD34, CD49f, CD90

Negative: CD38, CD45RA, Lineage*

Positive: CD150, c-Kit, Sca1

Negative: CD34, CD41, CD48, Lineage

BD Pharmingen™ Human Lineage Cocktail 4 562722
Mouse Hematopoietic Stem Cell Isolation Kit 560492
Mesenchymal Stem Cells (MSCs)

Positive: CD44, CD73, CD90, CD105, CD146, CD271

Negative: CD11b, CD19, CD31, CD34, CD45, CD144, HLA-DR

Positive: CD29, CD44, CD90, CD105, CD106, Sca-1

Negative: CD11b, CD31, CD45, Ter-119

Human MSC Analysis Kit 562245
Human Mesenchymal Stem Cell Lineage Antibody Cocktail 562530
Neural Stem Cells (NSCs)

Positive: CD15mid, CD24, CD184

Negative: CD44, CD271

Human Neural Cell Sorting Kit 562271
Neurons

Positive: CD15low, CD24

Negative: CD44, CD184

Human Neural Cell Sorting Kit 562271
Cancer
CD15, CD24, CD34, CD44, CD45, CD49f, CD166, CD326, CD338, Her-2/Neu, Lgr5

* Human lineage (lin) markers: CD2, CD3, CD4, CD7, CD8, CD10, CD11b, CD14, CD19, CD20, CD56, CD235a

Hematopoietic stem cell phenotyping

Cells of the hematopoietic system are well characterized with respect to surface marker expression, which is often used to isolate and characterize subsets of cells during hematopoiesis. Hematopoietic stem cells (HSCs), the source of these hematopoietic cells, are currently a focus area in stem cell biology because they can be used to replenish normal bone marrow function.

Historically, among a pool of cells, HSCs were identified as lineage-negative cells that expressed CD90 and CD34.1 Recently, researchers have used additional markers to enrich pools of long-term HSCs (LT-HSCs) capable of self renewal. These markers include CD38,2 CD45RA,3 and most recently CD49f.4 Reportedly, about 10% of cells with a LinCD34+CD38CD90+CD45RACD49f+ phenotype are able to provide long-term repopulating capacity in mouse models.4

Gating strategy for LT-HSCs
Gating strategy for LT-HSCs
Frozen human cord blood mononuclear cells (StemCell Technologies) were enriched using the BD IMag™ human lineage cell depletion set – DM (Cat. No. 560030), stained with antibodies, and acquired and analyzed on a BD LSRFortessa™ flow cytometer. A. First, cells were gated based on light-scatter properties to screen out debris. B. Next, viable, lineage-negative (CD2, CD3, CD4, CD7, CD8, CD10, CD11b, CD14, CD19, CD20, CD56, CD235a) cells were gated based on the viability dye 7-AAD and the BD Pharmingen™ human lineage cocktail 4 kit (Cat. No. 562722). To identify a highly enriched LT-HSC population, (C) cells were gated on CD34+CD38; (D) then (in a child gate) on CD90+CD45RA; and (E) finally on CD49f+. This combination of cell surface markers, summarized in (F) the complete gating hierarchy, results in a population rich in LT-HSCs.

References

1. Baum CM, Weissman IL, Tsukamoto AS, Buckle AM, Peault B. Isolation of a candidate human hematopoietic stemcell population. Proc Natl Acad Sci U S A. 1992;89:2804-2808.

2. Bhatia M, Wang JC, Kapp U, Bonnet D, Dick JE. Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proc Natl Acad Sci U S A. 1997;94:5320- 5325.

3. Majeti R, Park CY, Weissman IL. Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood. Cell Stem Cell. 2007;1:635-645.

4. Notta F, Doulatov S, Laurenti E, Poeppl A, Jurisica I, Dick JE. Isolation of single human hematopoietic stem cells capable of long-term multilineage engraftment. Science. 2011;333:218-221.