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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 transcription factors in definitive endoderm development
H9 hESCs (WiCell) were differentiated into definitive endoderm according to D’Amour, et al2 and monitored on a BD LSRFortessa™ flow cytometer using the BD Stemflow™ definitive and pancreatic endoderm analysis kit (Cat. No. 562496). As embryonic stem cells (A) differentiated toward definitive endoderm (B), more cells expressed both Sox17 and FoxA2, while Nanog expression decreased.
Changes in intracellular and surface markers in neural cell differentiation
Changes in intracellular and surface markers in neural cell differentiation
NSCs derived from H9 hESCs using the serum-free embryoid body (SFEB) method were differentiated into neurons and glia and monitored on a BD LSRFortessa flow cytometer using the BD Stemflow™ human neural lineage analysis kit (Cat. No. 561526). A. As differentiation progressed, cells expressed less Nestin and more DCX. B. Nestin+ cells were further delineated into a glial cell population that expressed both CD44 and Nestin over time.

References

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.