Technical Notes: Assessing the Pluripotent Status of Stem Cells
Embryonic stem cells have the potential to differentiate into multiple cell types and are widely recognized as holding significant promise for therapeutic applications. The quality of a stem cell culture determines its ability to give rise to differentiated cell types efficiently, and can be measured by analysis of the expression of molecular markers of the pluripotent phenotype. Markers of early lineage committed cells are also useful to determine whether differentiated cells are present in a stem cell culture, and can indicate which lineage pathway the differentiated cells represent.
Figure 1. PCR products amplified by primer sets specific for 14 different markers of stem cell pluripotency and lineage commitment visualized by gel electrophoresis. cDNAs used for amplification were derived from a variety of embryonic tissues. Markers in lanes 3-6 are expressed in undifferentiated ES cells, lanes 6-9 ectodermal lineage, lanes 10-14 endodermal lineage, lane 15 mesodermal lineage, and lane 16 contains a marker for germ cells. Lanes 1, 2, and 17-19 show controls.
The R&D Systems Human Pluripotent Stem Cell Assessment Primer Pair Panel (Catalog # SC012; Figure 1) profiles the mRNA transcripts of fourteen genes that are frequently used as markers for molecular characterization of undifferentiated and early lineage-committed human ES cells. A primer pair for human GAPDH is included and can be used as a control for successful cDNA synthesis. A positive control is also included. In addition, R&D Systems offers a mouse/rat version of this kit (Catalog # SC015).
Figure 2. Assessment of Oct3/4 expression in NTera2 cells by intracellular flow cytometry using rat anti-human/mouse Oct3/4 monoclonal antibody (Catalog # MAB1759; orange) or isotype control (Catalog # MAB0061; gray) followed by APC-conjugated secondary antibody.
Additional reagents to assess stem cell phenotype are available from R&D Systems. For example, Oct 3/4 expression is detected in undifferentiated NTera2 human teratocarcinoma cells by flow cytometry (Figure 2), and DPPA4 expression is shown by immunocytochemistry in the nuclei of mouse D3 embryonic stem cells (Figure 3).
Figure 3. Detection of DPPA4 in the nuclei of D3 cells using R&D Systems anti-mouse DPPA4 polyclonal antibody (Catalog # AF3730) and R&D Systems NorthernLights™ 557-conjugated secondary antibody (Catalog # NL001).