Stem Cell Research Analysis and Isolation of Stem Cells Using Flow Cytometry

Stem Cell Research Analysis and Isolation of Stem Cells Using Flow Cytometry
Stem Cell Research

Analysis and Isolation of
Stem Cells Using Flow Cytometry
2   Cover Illustration: Fairman Studios, LLC.
Flow cytometry is a powerful methodology for
characterizing, analyzing, and isolating stem cells
and their derivatives.
        Recent discoveries in stem cell biology have amplified the importance
        of pluripotent stem cells in therapeutics and have improved researchers’
        understanding of normal and disease processes. Still, the inherent
        heterogeneous nature of differentiating cultures remains a primary
        challenge in stem cell research.
        Flow cytometry is unique in its ability        Researchers can use fluorochrome-
        to investigate large cell populations          conjugated antibodies to either cell surface
        at the single-cell level. In contrast to       or intracellular biomarkers to verify that
        methods such as Western blot and cellular      stem cells have maintained pluripotency.
        imaging, multicolor flow cytometry             Since stem cells differentiate into the three
        enables researchers to interrogate             primary germ layers and into differentiated
        heterogeneous cell populations and analyze     tissue, antibodies can monitor their
        their subpopulations. Using multiple           changing expression patterns. Analysis based
        fluorescent-labeled antibodies, researchers    on cell surface markers can preserve cell
        can obtain robust, multiparametric             viability for use in additional experiments.
        data and population-based statistics on        BD Lyoplate™ cell surface marker screening
        differentiating stem cell cultures—and         panels provide a powerful method for
        can isolate stem cells and their derivatives   discovering surface marker signatures that
        from primary tissue and diverse in vitro       can be used to explore these cells in depth.
        populations.
                                                       BD Biosciences offers a diverse set of
        Flow cytometry has been used for decades       tools including high-quality antibodies,
        by biologists studying hematopoietic           buffers, protocols, and instrumentation to
        stem cells to address the challenge of         support stem cell research. This evolving
        heterogeneity. New methods and tools           toolset combines the power of advanced
        are enabling researchers to employ this        technologies and world-class service to
        powerful technique to make key discoveries     support investigators in characterizing,
        about other stem cell types and their          analyzing, and sorting heterogeneous stem
        respective lineages.                           cell populations.




                                                                                                       3
Isolation of live cell populations

    Cell surface staining for analysis
    and sorting of live cells
      Each type of stem cell or derivative expresses characteristic                                digestion process, preventing antibody labeling, they must
      surface and intracellular proteins that can be used for                                      be evaluated for each surface marker being measured.
      identification (Tables 1 and 2). Because intracellular analysis                              BD Accutase tends to be more broadly applicable than
      requires permeabilization, surface markers are essential                                     trypsin and yields a more consistent single-cell suspension
      when researchers want to isolate live cell populations for                                   than does scraping.
      further analysis. Fluorescence-activated cell sorting, or FACS,                              Monoclonal antibodies
      can be used to sort cells of interest in bulk or in single-cell                              Once cells are harvested and the dissociation buffer is
      depositions for downstream applications. To analyze cells                                    removed, the cells are ready to be stained with antibodies.
      for surface marker expression, a single-cell suspension must                                 BD Biosciences offers an extensive reagent selection
      be stained with fluorescent-labeled antibodies and analyzed                                  of antibodies against hundreds of stem cell markers
      or sorted on a flow cytometer.                                                               conjugated to a variety of fluorochromes for flexibility in
      Sample preparation                                                                           experimental design. For analysis of rare events and low-
      Stem cells tend to be adherent and can grow as three-                                        density antigens, the BD Horizon™ Brilliant Violet™ family
      dimensional structures. To prepare a single-cell suspension                                  of reagents can increase brightness and resolution. For ease
      for flow cytometric analysis, enzymatic digestion (with                                      of use, BD Stemflow™ kits and cocktails contain standard
      BD™ Accutase cell detachment solution or trypsin) or                                         antibody panels for analysis or sorting of different stem cell
      mechanical scraping can be used. Since enzymatic methods                                     types, as shown in Table 1.
      might cleave or modify some protein epitopes during the




                                                                Human Markers                             Mouse Markers                  BD Stemflow™ or other Kit                     Cat. No.
                                                                Pluripotent Stem Cells (ESCs and iPSCs)
                                                                Positive: Alkaline Phosphatase, SSEA-4,   Positive: SSEA-1               Human iPSC Sorting and Analysis Kit           562626
                                                                SSEA-3, TRA-1-81, TRA-1-60
                                                                                                                                         Human Pluripotent Stem Cell Sorting and       560461
                                                                Negative: SSEA-1
                                                                                                                                         Analysis Kit
                                                                                                                                         Human and Mouse Pluripotent Stem Cell         560477
                                                                                                                                         Analysis Kit
                                                                Hematopoietic Stem Cells (HSCs)
                                                                Positive: CD34, CD49f, CD90               Positive: CD150, c-Kit, Sca1   BD Pharmingen™ Human Lineage Cocktail 4       562722
      Table 1.
                                                                Negative: CD38, CD45RA, Lineage*          Negative: CD34, CD41, CD48,
      Representative surface markers of selected                                                          Lineage                        Mouse Hematopoietic Stem Cell Isolation Kit   560492
      stem cells and derivatives.
                                                                Mesenchymal Stem Cells (MSCs)
                                                                Positive: CD44, CD73, CD90, CD105,        Positive: CD29, CD44, CD90,    Human MSC Analysis Kit                        562245
                                                                CD146, CD271                              CD105, CD106, Sca-1
                                                                                                                                         Human Mesenchymal Stem Cell Lineage           562530
                                                                Negative: CD11b, CD19, CD31, CD34,        Negative: CD11b, CD31,         Antibody Cocktail
                                                                CD45, CD144, HLA-DR                       CD45, Ter-119
                                                                Neural Stem Cells (NSCs)
                                                                Positive: CD15mid, CD24, CD184            –                              Human Neural Cell Sorting Kit                 562271
                                                                Negative: CD44, CD271
                                                                Neurons
                                                                Positive: CD15low, CD24                   –                              Human Neural Cell Sorting Kit                 562271
                                                                Negative: CD44, CD184
                                                                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


4
HETEROGENEITY


                                                                                                A                                                                                             B




                                                                               (x 1,000)
                                                                                  250




                                                                                                                                                                                   5
                                                                                                                                                                                         10
                                                                                        200




                                                                                                                                                                                   4
                                                                                                                                                                                         10
                                                                                                                                                                            7-AAD
                                                                                        150
                                                                            SSC-A
Hematopoietic stem cell phenotyping




                                                                                                                                                                                   3
                                                                                                                                                                                         10
Cells of the hematopoietic system are well characterized                                                                                                                                                Lineage-Viable




                                                                                        100
with respect to surface marker expression, which is often
used to isolate and characterize subsets of cells during




                                                                                                                                                                                   2
                                                                                                                                                                                         10
                                                                                        50
hematopoiesis. Hematopoietic stem cells (HSCs), the source




                                                                                                                                                                                         0
                                                                                                                                                      Scatter
of these hematopoietic cells, are currently a focus area in




                                                                                                                                                                                  -124
                                                                                                              50       100            150            200           250                            101          102         103      104             105
stem cell biology because they can be used to replenish                                                                      FSC-A                              (x 1,000)
                                                                                                                                                                                            0
                                                                                                                                                                                                                 FITC Lineage Cocktail 4
                                                                                                C                                                                                             D
normal bone marrow function.




                                                                                                                                                                                      5
                                                                                     5




                                                                                                                                                                                    10
Historically, among a pool of cells, HSCs were identified
                                                                                   10




as lineage-negative cells that expressed CD90 and CD34.1
                                                                                                                                                                                                           CD90+CD45RA-
Recently, researchers have used additional markers to




                                                                                                                                                                                    4
                                                                                                                                                                                         10
                                                                           BV421 CD38




                                                                                                                                                                            BV605 CD90
                                                                                   4
                                                                                        10




enrich pools of long-term HSCs (LT-HSCs) capable of self-
renewal. These markers include CD38,2 CD45RA,3 and most




                                                                                                                                                                                    3
                                                                                                                                                                                         10
recently CD49f.4 Reportedly, about 10% of cells with a
                                                                                   3
                                                                                        10




Lin–CD34+CD38–CD90+CD45RA–CD49f+ phenotype are able to
provide long-term repopulating capacity in mouse models.4



                                                                                                                                                                                             2
                                                                                                                                                                                    -10 0 10
                                                                                   2




                                                                                                                                                                                       2
                                                                                        10




                                                                                                                    CD34+CD38-

                                                                                                                                                                                  -531
                                                                                                        102             103                  104            105                                          -102 0102        103       104             105
                                                                                                                                                                                         -650
                                                                                                                        APC CD34                                                                                     APC-H7 CD45RA
                                                                                                E                                                                                             F
                                                                                   5
                                                                                        10




                                                                                                                                                                                                  Tube: enriched
                                                                                   4
                                                                                        10




                                                                                                                                                                                                  Population                     #Events %Parent %Total
                                                                          BV605 CD90




                                                                                                                                                                                                    All Events                   201,698    ####      100.0
                                                                                                                                                                                                        Scatter                  145,671    72.2       72.2
                                                                                                                                                                                                           Lineage-Viable         48,194    33.1       23.9
                                                                                                                                                                                                               CD34+CD38-          4,650      9.6       2.3
                                                                                   3
                                                                                        10




                                                                                                                                                                                                                 CD90+CD45RA-        879    18.9        0.4
                                                                                                                                                                                                                    CD49f+           184     20.9       0.1
                                                                                                                                    CD49f+
                                                                                            2
                                                                                   -10 0 10
                                                                                  -5312




                                                                                         -105       0         102             103              104          105
                                                                                                                        PE CD49f
Gating strategy for LT-HSCs.
Frozen human cord blood mononuclear cells (Stem Cell 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.



                                                                                                                                                                                                                                                      5
Study of differentiation pathways

    Intracellular staining for
    transcription factor analysis
      Intracellular staining allows researchers to extend the                            Sample preparation
      speed and statistical relevance of flow cytometry to the                           For intracellular staining, as with surface staining, a
      investigation of functional proteins inside the cell. It can be                    single-cell suspension must be prepared using enzymatic
      used in combination with surface staining to identify critical                     or mechanical methods. BD Accutase is recommended
      time points, markers, and proportions of cells moving along                        since it helps to prevent cell clumping and can preserve
      particular differentiation pathways. Intracellular staining                        surface proteins for simultaneous analysis. After optional
      protocols require the cells to be fixed and permeabilized so                       surface staining, cells must be fixed and permeabilized to
      that antibodies can access the cytoplasm and nucleus. Since                        enable antibodies to enter. The cells are then stained with
      fixation effectively kills the cells, intracellular staining is not                fluorescent-labeled antibodies to intracellular antigens and
      compatible with live-cell sorting.                                                 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.




      Table 2.                                       Cell Type                                Intracellular Markers                BD Stemflow Kit                     Cat. No.
      Representative intracellular markers of        Embryonic stem cells (ESCs)              Nanog, Oct3/4, Sox2                  Human Pluripotent Stem Cell         560589
      selected stem cells and derivatives.           Induced pluripotent stem cells (iPSCs)                                        Transcription Factor Analysis Kit
                                                                                                                                   Mouse Pluripotent Stem Cell         560585
                                                                                                                                   Transcription Factor Analysis Kit
                                                     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     562496
                                                                                                                                   Endoderm Analysis Kit
                                                     Late pancreatic endoderm                 NeuroD1, Nkx6.1                      –                                   –
                                                     Cardiac                                  cTNI, GATA4, Islet-1, Myosin Heavy   –                                   –
                                                                                              Chain
                                                     Hepatic                                  AFP, GATA4                           –                                   –




6
INTRACELLULAR
                                                                                                                                                                                                               Day 0: H9 hESCs
                                                                                                                         A




                                                                                                                      5




                                                                                                                                                                                                                                                    5
                                                                                                                         10




                                                                                                                                                                                                                                                      10
                                                                                                                      4




                                                                                                                                                                                                                                                    4
                                                                                                                         10




                                                                                                                                                                                                                                                      10
                                                                                                                                                           Sox17+FoxA2+




                                                                                                                Sox17




                                                                                                                                                                                                                                            Sox17
                                                                                                                                                                                                               1%




                                                                                                                      3




                                                                                                                                                                                                                                                    3
                                                                                                                         10




                                                                                                                                                                                                                                                      10
                                                                                                                                                                                                                                                                                                                 Nanog+

                                                                                                                                                                                                                                                                                                                                   92%




                                                                                                                      2




                                                                                                                                                                                                                                                    2
                                                                                                                         10




                                                                                                                                                                                                                                                      10
                                                                                                                      1




                                                                                                                                                                                                                                                    1
                                                                                                                         10




                                                                                                                                                                                                                                                      10
                                                                                                                         0




                                                                                                                                                                                                                                                      0
                                                                                                                        -63




                                                                                                                                                                                                                                                    -63
                                                                                                                                                2                                 3                 4                     5                                                          2               3                    4           5
                                                                                                                              -68     0       10                                10                10             10                                         -149       0 10                        10                   10          10

                                                                                                                                                                        FoxA2                                                                                                                      Nanog

Stem cell differentiation
                                                                                                                              B                                                      Day 3: Definitive Endoderm
The ability of human pluripotent stem cells to differentiate
into various cell lineages is a central topic in developmental




                                                                                                                                                                                                                                                    5
                                                                                                                      5




                                                                                                                                                                                                                                                     10
                                                                                                                         10
biology and has applications for regenerative medicine
and cellular therapy. As pluripotent cells differentiate into
                                                                                                                      4
                                                                                                                         10




                                                                                                                                                                                                                                                    4
                                                                                                                                                              Sox17+FoxA2+




                                                                                                                                                                                                                                                     10
different lineages, the expression of transcription factors                                                     Sox17
                                                                                                                                                                                                                57%




                                                                                                                                                                                                                                            Sox17
and other proteins can change. Multiparametric flow
                                                                                                                      3




                                                                                                                                                                                                                                                    3
                                                                                                                         10




                                                                                                                                                                                                                                                     10
cytometry is an excellent method for determining                                                                                                                                                                                                                                                                     Nanog+


the relative numbers of cells expressing markers of interest,                                                                                                                                                                                                                                                                      24%
                                                                                                                      2




                                                                                                                                                                                                                                                    2
                                                                                                                         10




                                                                                                                                                                                                                                                     10
and can be used to optimize, quantitate, and compare
                                                                                                                      1




                                                                                                                                                                                                                                                    1
                                                                                                                         10




                                                                                                                                                                                                                                                     10
                                                                                                                         0




                                                                                                                                                                                                                                                     0
differentiation protocols and differentiation potential.




                                                                                                                                                                                                                                                    -52
                                                                                                                      -52




                                                                                                                                          1        2                                 3                     4                  5                                                     2                   3                      4          5
                                                                                                                              -42 10
                                                                                                                                  0           10                                10                 10                     10                                -113       0        10                 10                    10          10
For example, in mammalian embryonic development, the
                                                                                                                                                           FoxA2                                                                                                                          Nanog
definitive endoderm generates the liver, pancreas, and
intestine.5 During lineage specification into definitive                                                              Changes in transcription factors in definitive endoderm
endoderm, the levels of transcription factors Sox17 and                                                               development.
FoxA2 increase, while pluripotency markers such as Nanog                                                              H9 hESCs (WiCell) were differentiated into definitive endoderm
                                                                                                                      according to D’Amour, et al6 and monitored on a BD LSRFortessa
decrease.6
                                                                                                                      flow cytometer using the BD Stemflow™ definitive and pancreatic
The differentiation of neural stem cells to neural lineages                                                           endoderm analysis kit (Cat. No. 562496). As embryonic stem cells (A)
                                                                                                                      differentiated toward definitive endoderm (B), more cells expressed
can also be monitored using multicolor flow analysis. As
                                                                                                                      both Sox17 and FoxA2, while Nanog expression decreased.
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.


                                                  A                                                Day 0                                                                                          Day 14                                                                                 Day 27

                                                                                                                                                                        17.3%                                                                                     40.0%
                                                                       5




                                                                                                                                                       5




                                                                                                                                                                                                                                                      5
                                                                     10




                                                                                                                                                           10




                                                                                                                                                                                                                                                          10




                                                                                   6.5%
                                                                       4




                                                                                                                                                       4




                                                                                                                                                                                                                                                      4
                                                                     10




                                                                                                                                                           10




                                                                                                                                                                                                                                                          10
                                                                                                                                                       3




                                                                                                                                                                                                                                                      3
                                                                     3




                                                                                                                                                           10




                                                                                                                                                                                                                                                          10
                                                                        10




Changes in intracellular and surface
                                                                                                                                                       2




                                                                                                                90.5%
                                                                              2




                                                                                                                                                           10




                                                                                                                                                                                                                                                      2
                                                                     -10 0 10




                                                                                                                                                                                                                                                          10
                                                 DCX




markers in neural cell differentiation.                                                                                                                                                                        73.7%                                                                               49.8%
                                                                        2




                                                                                                                                                                                                                                                          0
                                                                                                                                                       1
                                                                                                                                                           010




NSCs derived from H9 hESCs using the
                                                                     -384




                                                                                                                                                       -124




                                                                                                                                                                                                                                                     -384




                                                                            M                  2            3             4               5                                 1                2             3          4                 5                                       2              3                 4             5
                                                                                   0         10           10            10            10                                10             10           10           10                10                              0       10             10                10               10
                                                                             -42                                                                                   -3                                                                                       -42

serum-free embryoid body (SFEB) method
were differentiated into neurons and glia         B                  Nestin
and monitored on a BD LSRFortessa flow
                                                          5




                                                                                                                                                      5




                                                                                                                                                                                                                                                        5




cytometer using the BD Stemflow human
                                                        10




                                                                                                                                                    10




                                                                                                                                                                                                                                                      10




neural lineage analysis kit (Cat. No. 561526).
                                                                                                                                                                                                 5.3%                                                                  35.4%
                                                        4




                                                                                                                                                    4




                                                                                                                                                                                                                                                      4
                                                           10




                                                                                                                                                       10




                                                                                                                                                                                                                                                       10




A. As differentiation progressed, cells
expressed less Nestin and more DCX.
                                                                                                                                                                                                                                                      3
                                                                                                                                                    3
                                                          3




                                                                                                                                                                                                                                                       10
                                                                                                                                                       10
                                                        10




B. Nestin+ cells were further delineated into
a glial cell population that expressed both
                                                 CD44
                                                                 2




                                                                                                                                                                                                                                                      2
                                                                                                                                                    2
                                                        -10 0 10




                                                                                                                                                                                                                                                       10
                                                                                                                                                       10




CD44 and Nestin over time.
                                                           2




                                                                                                                                                                                                                                                       0
                                                                                                                                                       0
                                                        -384




                                                                                                                                                    -124




                                                                                                                                                                                                                                                     -384




                                                               M                         2            3           4               5                                     1                2             3          4                 5                                        2              3                 4               5
                                                                         0             10           10          10             10                                   10                10           10           10                10                               0       10             10                10            10
                                                               -42                                                                                            -3                                                                                            -42




                                                                     Nestin

                                                                                                                                                                                                                                                                                                                                              7
Unique multimarker signatures

    Rapid and efficient
    surface marker screening
     A major challenge facing stem cell biology is the                     Both the human (Cat. No. 560747) and mouse (Cat. No.
     heterogeneous nature of cultures and differentiations. To             562208) panels contain three plates. Each well contains
     sort viable cells to purify for use in later experiments, one         lyophilized, purified antibody to one cell surface marker
     must know the cell surface signature for the particular               or isotype control. Following reconstitution, the cellular
     cell of interest. Since different types of related cells may          samples are stained with purified antibodies, and detection
     share markers, researchers must find a unique multimarker             reagents included with the panel are added. Finally,
     signature for each, while other markers may be useful in              samples are analyzed by flow cytometry or imaging.
     distinguishing subpopulations of a particular type of cell.
                                                                           To provide flexibility while simplifying workflow, open
     BD Lyoplate cell surface marker screening panels provide              wells allow the panel to be expanded to include additional
     a comprehensive and efficient solution for profiling stem             markers. Powerful BD Biosciences analysis tools facilitate
     cells and their derivatives for hundreds of human or mouse            data mining and heatmap generation.
     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                Table 3.
     targeted cells from heterogeneous populations for                     Considerations for flow cytometry vs image screening with
                                                                           BD Lyoplate panels.
     functional studies, drug screening, in vivo animal studies,
     and cell therapy research.                                            Property of sample                               Flow cytometry                             Imaging
                                                                           Suspension cells                                              X
     The hundreds of monoclonal antibodies in each panel
                                                                           Rare cell populations                                         X
     constitute one of the most cost-effective screening tools
                                                                           Subpopulation analysis
     available for cellular analysis. To simplify the transition                                                                         X
                                                                           Co-staining with multiple markers
     to more targeted, larger-scale experiments, all antibodies            Reporter lines                                                X                                  X
     included in the screening panels are available in the                 Specific morphology changes                                                                      X
     BD Biosciences catalog.                                               Limited number of cells                                                                          X




                                                    A                                              Hits                                          FACS
     Using surface marker screening to                  Heterogeneous                             Flow screen
                                                                                                            (-) t
                                                                                                          te an
                                                                                                        et in




                                                                                                                                                                                       iii
                                                        neural induction
                                                                                                              +)




                                                                                                                                    i                         i
     characterize and enrich neural stem
                                                                                                      os m
                                                                                                           e(


                                                                                                    -r ta
                                                                                                    -r 9)




                                                                                                                            CD184
                                                                                                   SC tt




                                                                                                                                                  CD44




                                                                                                                                                                                CD24
                                                                                                EB con
                                                                                                EB (H
                                                                                                N ose
                                                                                                    SC



                                                                                                   SC




                                                                                                                                                                                                10% of
                                                                                                  its




     cells and neurons.
                                                                                                hE




                                                                                                N




                                                                                                H




                                                                                                                                                         ii                                      total
                                                                                        % +ve                   CD184 pos
                                                                                          0
                                                                                                                CD15 pos
     A. H9 hESCs were induced using the SFEB                                  Flow        5
                                                                                         10
                                                                                         15                     CD54 neg
                                                                                                                                        FSC-A                      CD271                     CD15
     method,7 and the BD Lyoplate human cell                                 screen      20
                                                                                         25
                                                                                         30
                                                                                                                CD29
                                                                                                                CD71
                                                                                         35
                                                                                         40                     CD49c
     surface marker screening panel (Cat. No.
                                                                                         45
                                                                                         50                     CD49e
                                                                                         55
                                                                                         60                     CD63

     560747) was used to screen the resulting                                            65
                                                                                         70
                                                                                         75
                                                                                                                CD57
                                                                                                                CD47
                                                                                         80

     heterogeneous neural cultures on a                                                  85
                                                                                         90
                                                                                         95
                                                                                                                CD81
                                                                                                                CD59
                                                                                        100

     BD™ LSR II flow cytometer to identify a                                                                    CD58
                                                                                                                CD56
                                                                                                                CD46
     surface marker signature for NSCs. After                                                                   CD24 pos
                                                                                                                CD55
     identifying potential hits, NSC cell surface                                                               CD271neg
                                                                                                                CD50
     phenotypes were verified and a CD184+CD44–
     CD271–CD24+CD15mid sorting profile was used
     on a BD FACSAria™ II cell sorter to obtain a
                                                                                                                                                Enriched NSCs
     near-pure subpopulation of NSCs.

8                                                                                                                                                             FACS
                                                                                                         Hits
                                                                                                                                                              P3

                                                                                                                                                                           P5
DISCOVERY




Screening of neural populations                                           In panel B, the purified NSCs were differentiated into
Neural cell populations derived from pluripotent stem                     neuronal and glial cell populations, which were screened by
cells are important for studying human disease and                        imaging using the same panel to identify surface markers
development. Pluripotent stem cells can be differentiated                 for isolating neurons. An imaging screen was chosen due
into self-renewing NSCs, which can be further differentiated              to the unique morphology of neurons and the ability
into heterogeneous populations of neurons and glia.7 A key                to co-stain with a neuronal-specific marker. A potential
to further research is to identify surface marker signatures              neuronal surface phenotype of CD44–CD184–CD24+CD15low
for each of these cell types.                                             was verified by flow cytometry and used to purify neurons.

In the example, the BD Lyoplate human cell surface marker                 In addition to neural cells, the BD Lyoplate human cell
screening panel (Cat. No. 560747) was used to identify                    surface marker screening panel has also been used to
cell surface phenotypes for NSCs and neurons. In panel A,                 identify cell surface markers of cardiomyocytes derived
heterogeneous neural induction cultures were screened by                  from pluripotent stem cells.8 Most recently, this powerful
flow cytometry, and potential NSC markers were identified                 methodology was used to develop a human stem cell model
on a heatmap. A resulting NSC cell surface phenotype                      of Alzheimer’s disease.9
of CD184+CD44–CD271–CD24+CD15mid 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
                                                                                                   Hits                                              FACS
confirmed.                                     Heterogeneous                                       Flow screen
                                                                                                           (-) t
                                                                                                         te an
                                                                                                       et in




                                                                                                                                                                                            iii
                                                       neural induction
                                                                                                             +)




                                                                                                                                      i                           i
                                                                                                     os m
                                                                                                          e(


                                                                                                   -r ta
                                                                                                   -r 9)




                                                                                                                             CD184
                                                                                                  SC tt




                                                                                                                                                      CD44




                                                                                                                                                                                     CD24
                                                                                               EB con
                                                                                               EB (H
                                                                                               N ose
                                                                                                   SC



                                                                                                  SC




                                                                                                                                                                                                     10% of
                                                                                                 its
                                                                                              hE




                                                                                               N




                                                                                               H




                                                                                                                                                             ii                                       total
                                                                                      % +ve                      CD184 pos
                                                                                        0
                                                                                                                 CD15 pos
                                                                              Flow      5
                                                                                       10
                                                                                       15                        CD54 neg
                                                                                                                                          FSC-A                        CD271                      CD15
                                                                             screen    20
                                                                                       25
                                                                                       30
                                                                                                                 CD29
                                                                                                                 CD71
                                                                                       35
                                                                                       40                        CD49c
                                                                                       45
                                                                                       50                        CD49e
                                                                                       55
                                                                                       60                        CD63
                                                                                       65                        CD57
                                                                                       70
                                                                                       75                        CD47
                                                                                       80
                                                                                       85                        CD81
                                                                                       90
                                                                                       95                        CD59
                                                                                      100
                                                                                                                 CD58
                                                                                                                 CD56
                                                                                                                 CD46
                                                                                                                 CD24 pos
                                                                                                                 CD55
                                                                                                                 CD271neg
                                                                                                                 CD50




                                                                                                                                                    Enriched NSCs


                                                B
                                                                                                                                                                  FACS
B. The purified NSCs were differentiated                                                                  Hits
                                                                                                                                                                  P3



into a mixed culture of neuronal and glial
                                                                                                                                                                                P5


                                                    Heterogeneous neuronal
                                                                                                                                     CD44




cell populations, which was screened using               differentiation
                                                                                                                                                                         CD24




                                                                                                                                              P4



the same panel to identify a surface marker
signature for neurons. An imaging screen                                     Image
was chosen due to the unique morphology                                      screen
                                                                                                                                          M


                                                                                                                                                   CD184                                    CD15
of neurons and the ability to co-stain with a
neuron-specific marker. Potential hits were
verified by flow cytometry, and a CD44–
CD184–CD24+CD15low sorting profile was used
to purify neurons.7




                                                                                                                                                     Near-pure neurons

                                                                                                                                                                                                              9
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      References
      1. Baum CM, Weissman IL, Tsukamoto              4. Notta F, Doulatov S, Laurenti E, Poeppl       7. Yuan SH, Martin J, Elia J, et al. Cell-
          AS, Buckle AM, Peault B. Isolation of a          A, Jurisica I, Dick JE. Isolation of single       surface marker signatures for the
          candidate human hematopoietic stem-              human hematopoietic stem cells capable            isolation of neural stem cells, glia and
          cell population. Proc Natl Acad Sci U S A.       of long-term multilineage engraftment.            neurons derived from human pluripotent
          1992;89:2804-2808.                               Science. 2011;333:218-221.                        stem cells. PLoS One. 2011;6:e17540.
      2. B
          hatia M, Wang JC, Kapp U, Bonnet            5. Murry CE, Keller G. Differentiation of        8. Uosaki H, Fukushima H, Takeuchi A, et
         D, Dick JE. Purification of primitive             embryonic stem cells to clinically relevant       al. Efficient and scalable purification of
         human hematopoietic cells capable of              populations: lessons from embryonic               cardiomyocytes from human embryonic
         repopulating immune-deficient mice.               development. Cell. 2008;132:661-680.              and induced pluripotent stem cells by
         Proc Natl Acad Sci U S A. 1997;94:5320-                                                             VCAM1 surface expression. PLoS One.
                                                       6. D’Amour KA, Agulnick AD, Eliazer S,
         5325.                                                                                               2011;6:e23657.
                                                           Kelly OG, Kroon E, Baetge EE. Efficient
      3. M
          ajeti R, Park CY, Weissman IL.                  differentiation of human embryonic            9. Israel MA, Yuan SH, Bardy C, et al.
         Identification of a hierarchy of                  stem cells to definitive endoderm. Nat            Probing sporadic and familial Alzheimer’s
         multipotent hematopoietic progenitors             Biotechnol. 2005;23:1534-1541.                    disease using induced pluripotent stem
         in human cord blood. Cell Stem Cell.                                                                cells. Nature. 2012;482:216-220.
         2007;1:635-645.

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