Stem Cell Research

To support stem cell research, BD Biosciences offers a diverse set of tools including highquality antibodies, buffers, protocols, and instrumentation. This evolving toolset offers a powerful base to support investigators in characterizing, analyzing, and sorting heterogeneous stem cell populations.

From identifying stem cell types and their lineages to monitoring changing cell expression patterns, flow cytometry techniques are advancing researchers’ understanding of normal and disease processes.

Overview Surface Staining Intracellular Staining Screening

Flow cytometry has been used for decades by biologists studying hematopoietic stem cells to address the challenge of heterogeneity.

New methods and tools are enabling researchers to employ this powerful technique to make key discoveries about other stem cell types and their respective lineages.

Researchers can use fluorochrome-conjugated antibodies to either cell surface or intracellular biomarkers to verify that stem cells have maintained pluripotency. Since stem cells differentiate into the three primary germ layers and into differentiated tissue, antibodies can monitor their changing expression patterns. Analysis based on cell surface markers can preserve cell viability for use in additional experiments. BD Lyoplate™ cell surface marker screening panels provide a powerful method for discovering surface marker signatures that can be used to explore these cells in depth.

BD Biosciences offers a diverse set of tools including high-quality antibodies, buffers, protocols, and instrumentation to support stem cell research. This evolving toolset combines the power of advanced technologies and world-class service to support investigators in characterizing, analyzing, and sorting heterogeneous stem cell populations.

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: CD15 mid, CD24, CD184

Negative: CD44, CD271

Human Neural Cell Sorting Kit 562271

Positive: CD15 low, CD24

Negative: CD44, CD184

Human Neural Cell Sorting Kit 562271
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 Lin CD34 +CD38 CD90 +CD45RA CD49f + phenotype are able to provide long-term repopulating capacity in mouse models. 4

Gating strategy for LT-HSCs


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.

Intracellular staining for transcription factor analysis

Intracellular staining allows researchers to extend the speed and statistical relevance of flow cytometry to the investigation of functional proteins inside the cell. It can be used in combination with surface staining to identify critical time points, markers, and proportions of cells moving along particular differentiation pathways. Intracellular staining protocols require the cells to be fixed and permeabilized so that antibodies can access the cytoplasm and nucleus. Since fixation effectively kills the cells, intracellular staining is not compatible with live-cell sorting.

Sample preparation

For intracellular staining, as with surface staining, a single-cell suspension must be prepared using enzymatic or mechanical methods. BD Accutase is recommended since it helps to prevent cell clumping and can preserve surface proteins for simultaneous analysis. After optional surface staining, cells must be fixed and permeabilized to enable antibodies to enter. The cells are then stained with fluorescent-labeled antibodies to intracellular antigens and analyzed on a flow cytometer.

To optimize permeabilization and staining conditions, BD has developed several kits for the detection of key stem cell transcription factors. The kits contain optimized antibodies and buffer systems to characterize stem cells as well as their differentiation into various lineages.

Representative intracellular markers of selected stem cells and derivatives

Cell Type Intracellular Markers BD Stemflow™ Kit Cat. No.
Embryonic stem cells (ESCs) Induced pluripotent stem cells (iPSCs) Nanog, Oct3/4, Sox2 Human Pluripotent Stem Cell Transcription Factor Analysis Kit 560589
Mouse Pluripotent Stem Cell Transcription Factor Analysis Kit 560585
Neural stem cells (NSCs) Nestin, Pax6, Sox1, Sox2 Human Neural Lineage Analysis Kit 561526
Astrocytes GFAP Human Neural Lineage Analysis Kit 561526
Neurons Doublecortin Human Neural Lineage Analysis Kit 561526
Early pancreatic endoderm FoxA2, Pax6, Pdx1, Sox17 Human Definitive and Pancreatic Endoderm Analysis Kit 562496
Late pancreatic endoderm NeuroD1, Nkx6.1
Cardiac cTNI, GATA4, Islet-1, Myosin Heavy Chain
Hepatic AFP, GATA4

Stem cell differentiation

The ability of human pluripotent stem cells to differentiate into various cell lineages is a central topic in developmental biology and has applications for regenerative medicine and cellular therapy. As pluripotent cells differentiate into different lineages, the expression of transcription factors and other proteins can change. Multiparametric flow cytometry is an excellent method for determining the relative numbers of cells expressing markers of interest, and can be used to optimize, quantitate, and compare differentiation protocols and differentiation potential.

For example, in mammalian embryonic development, the definitive endoderm generates the liver, pancreas, and intestine. 1 During lineage specification into definitive endoderm, the levels of transcription factors Sox17 and FoxA2 increase, while pluripotency markers such as Nanog decrease. 2

The differentiation of neural stem cells to neural lineages can also be monitored using multicolor flow analysis. As neural stem cells (NSCs) differentiate into neurons, they gradually express less Nestin and more of the early neuronal marker doublecortin (DCX). A subpopulation of cells that continues to express Nestin further delineates into a glial cell population that expresses CD44.

Changes in transcription factors in definitive endoderm development

Changes in intracellular and surface markers in neural cell differentiation


1. Murry CE, Keller G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell. 2008;132:661-680.

2. D’Amour KA, Agulnick AD, Eliazer S, Kelly OG, Kroon E, Baetge EE. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol. 2005;23:1534-1541.

Rapid and efficient surface marker screening

A major challenge facing stem cell biology is the heterogeneous nature of cultures and differentiations. To sort viable cells to purify for use in later experiments, one must know the cell surface signature for the particular cell of interest. Since different types of related cells may share markers, researchers must find a unique multimarker signature for each, while other markers may be useful in distinguishing subpopulations of a particular type of cell.

BD Lyoplate™ cell surface marker screening panels provide a comprehensive and efficient solution for profiling stem cells and their derivatives for hundreds of human or mouse cell surface markers by flow cytometry or cellular imaging. Deciphering the cell surface proteome enables researchers to define strategies for the analysis and isolation of targeted cells from heterogeneous populations for functional studies, drug screening, in vivo animal studies, and cell therapy research.

The hundreds of monoclonal antibodies in each panel constitute one of the most cost-effective screening tools available for cellular analysis. To simplify the transition to more targeted, larger-scale experiments, all antibodies included in the screening panels are available in the BD Biosciences catalog.

Both the human (Cat. No. 560747) and mouse (Cat. No. 562208) panels contain three plates. Each well contains lyophilized, purified antibody to one cell surface marker or isotype control. Following reconstitution, the cellular samples are stained with purified antibodies, and detection reagents included with the panel are added. Finally, samples are analyzed by flow cytometry or imaging.

To provide flexibility while simplifying workflow, open wells allow the panel to be expanded to include additional markers. Powerful BD Biosciences analysis tools facilitate data mining and heatmap generation.

Considerations for flow cytometry vs image screening with BD Lyoplate panels

Property of sample Flow cytometry Imaging
Suspension cells X  
Rare cell populations X  
Subpopulation analysis
Co-staining with multiple markers
Reporter lines X X
Specific morphology changes   X
Limited number of cells   X

Using surface marker screening to characterize and enrich neural stem cells and neurons

Using surface marker screening to characterize and enrich neural stem cells and neurons

Screening of neural populations

Neural cell populations derived from pluripotent stem cells are important for studying human disease and development. Pluripotent stem cells can be differentiated into self-renewing NSCs, which can be further differentiated into heterogeneous populations of neurons and glia. 1 A key to further research is to identify surface marker signatures for each of these cell types.

In the example, the BD Lyoplate human cell surface marker screening panel (Cat. No. 560747) was used to identify cell surface phenotypes for NSCs and neurons. In panel A, heterogeneous neural induction cultures were screened by flow cytometry, and potential NSC markers were identified on a heatmap. A resulting NSC cell surface phenotype of CD184 +CD44 CD271 CD24 +CD15 mid was verified using intracellular NSC markers. The surface phenotype was used to sort a near-pure subpopulation of NSCs, the ability of which to differentiate both in vivo and in vitro was later confirmed.

In panel B, the purified NSCs were differentiated into neuronal and glial cell populations, which were screened by imaging using the same panel to identify surface markers for isolating neurons. An imaging screen was chosen due to the unique morphology of neurons and the ability to co-stain with a neuronal-specific marker. A potential neuronal surface phenotype of CD44 CD184 CD24 +CD15 low was verified by flow cytometry and used to purify neurons. In addition to neural cells, the BD Lyoplate human cell surface marker screening panel has also been used to identify cell surface markers of cardiomyocytes derived from pluripotent stem cells. 2 Most recently, this powerful methodology was used to develop a human stem cell model of Alzheimer’s disease. 3


1. Yuan SH, Martin J, Elia J, et al. Cell surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells. PLoS One. 2011;6:e17540.

2. Uosaki H, Fukushima H, Takeuchi A, et al. Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression. PLoS One. 2011;6:e23657.

3. Israel MA, Yuan SH, Bardy C, et al. Probing sporadic and familial Alzheimer’s disease using induced pluripotent stem cells. Nature. 2012;482:216-220.

Gating strategy for LT-HSCs

Changes in transcription factors in definitive endoderm development

Changes in intracellular and surface markers in neural cell differentiation

Using surface marker screening to characterize and enrich neural stem cells and neurons

Using surface marker screening to characterize and enrich neural stem cells and neurons

Viability of H9 hESCs following sorting

Viability of H9 hESCs following sorting

Viability of H9 hESCs following sorting

Pluripotency of H9 hESCs following sorting

Pluripotency of H9 hESCs following sorting

Bioimaging of hESCs cultured on BD Matrigel matrix


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For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Alexa Fluor® is a registered trademark of Life Technologies Corporation.
mTeSR™1 is a trademark of StemCell Technologies.
WiCell™ is a trademark of WiCell Research Institute.

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