Pluripotency Verification by Immunostaining

Embryonic and pluripotent stem cells retain pluripotency and self-renewing ability due to both their inherent characteristics and the culture conditions. The ability to differentiate into all cell lineages and maintain an undifferentiated state during in vitro culture makes them holding significant promise for therapeutic applications. The key problem in clinical application is to distinguish them from other cell types, especially tumor cells, to avoid potential risks. There are many special molecules that can affect the pluripotency and self-renewal. Identification, characterization, and classification of these molecules will provide useful tools for the isolation and verification of stem cells and subsequent research.

Cell Surface Markers

Covered on the surface of cells are specialized proteins that can selectively bind or adhere to other signaling molecules. Some of the proteins are uniquely present or secreted in particular cell types. Therefore, specific cell surface proteins can serve as cell markers. Membrane proteins are the most important marker types for identifying pluripotent stem cells without destroying the cell membrane (Table 1), with SSEA4 and Tra-1-60 being the most commonly used cell surface markers for pluripotency validation assessment.

Table 1. Cell surface markers.

SSEAs markersCharacteristicsClassification
SSEA-1Murine embryos, mouse ES cells, mouse and human germ cells, embryonal carcinoma (EC) cellsCarbohydrate-associated molecules
SSEA-3Primate ES cells, human embryonic germ cells, human ES cells, embryonal carcinoma (EC) cellsCarbohydrate-associated molecules
SSEA-4Primate ES cells, human embryonic germ cells, human ES cells, embryonal carcinoma (EC) cellsCarbohydrate-associated molecules
TRA-1-60Human ES cells, teratocarcinoma, embryonic germ cells, embryonal carcinoma (EC) cellsSurface antigen
TRA-1-81Human ES cells, teratocarcinoma, embryonic germ cells, embryonal carcinoma (EC) cellsSurface antigen

Transcription Factors

Unique genes appear and do functions in the nucleus mean that the cell has responded to a certain condition. Therefore, tracking the expression of these genes can be used as a marker for a specific cellular situation. The transcription factors expressed in pluripotent stem cells are shown in Table 2.

Oct4, Sox2, and Nanog are the core factors responsible for pluripotent stem cell self-renewal, and maintenance of pluripotency or initiation of differentiation depends on a highly regulated equilibrium between Oct4, Nanog and Sox2. These factors will be rapidly down-regulated during cell differentiation.

Table 2. Transcription factors.

Core transcription factorsCharacteristicsClassification
Oct-3/4Mouse ES cells, human ES cells, embryonal carcinoma (EC) cellsPOU family transcription factors
Sox2Mouse ES cells, human ES cells, embryonal carcinoma (EC) cells, neural stem (NS) cellsPOU family binder transcription factors
KLF4Mouse ES cells, human ES cells, embryonal carcinoma (EC) cellsZinc-finger transcription factors
NanogMouse ES cells, human ES cells, embryonal carcinoma (EC) cellsTranscription factors


A panel of biochemical and molecular markers have been identified that are specific to pluripotent stem cell physiology and fundamental to maintain the undifferentiated state. The antibody combinations include nuclear and surface markers. We have combined antibodies to reduce the number of cells while avoiding cross-reaction between the secondary antibodies. The first primary antibody combination is Oct4 (mouse IgG) and SSEA3 (rat IgM), using secondary antibodies that have been pre-absorbed with the serum from the other species (mouse-specific IgG antibody pre-absorbed with rat serum and vice versa) to avoid cross-reaction. The second combination consists of Sox2 (rabbit IgG), SSEA4 (mouse IgG) and Tra-1-60 (mouse IgM). In this case, secondary antibodies specific to the µ- or γ-chain of the mouse immunoglobulin must be used. Finally, the third combination is Nanog (goat IgG) and Tra-1-81 (mouse IgM), in which non-goat-derived secondary antibodies (donkey or rabbit antibodies) must be used to avoid cross-reaction.

Briefly, pluripotent stem cells are fixed in 4% paraformaldehyde (PFA) for 20 minutes at room temperature (RT). Then the cells are washed three times for 5 minutes in PBS at RT and permeabilized in Triton-X for 8 minutes. After washing in PBS, they were kept in blocking solution (PBS + BSA + tween-20) for one hour at RT. Pluripotent stem cells are then incubated overnight in primary antibodies at 4°C. The following day, cells are washed and incubated with secondary antibodies for one hour and then counterstained with DAPI at RT for 15-30 minutes. All secondary antibodies are tested for cross reactivity and non-specific immunoreactivity.


  1. Wenxiu Zhao, et al. Embryonic stem cell markers. Molecules, 2012, 17: 6196-6236.
  2. Martí M. et al. Characterization of pluripotent stem cells, Nat Protoc, 2013, 8(2): 223-253.

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