A Guide to Pluripotency Markers

Pluripotency refers to the remarkable ability of a single cell to differentiate into all somatic lineages of the adult organism. This property can be harnessed in vitro using stem cells derived from embryos, which serve as valuable models for understanding developmental biology. Human pluripotent stem cell lines, with the capacity of indefinite self-renewal and differentiation potential, can serve as reservoirs of cells with promising applications, particularly in cell therapy and organ regenerative therapies. Aside from embryonic stem cells (ESCs), the reprogramming of somatic cells using key transcription factors can be used to generate induced PSCs (iPSCs).

The use of markers for pluripotent stem cells (PSCs) is essential for enabling and optimizing research on pluripotent stem cells, as well as in regenerative and cell-based clinical applications. Here, we review recent research to provide a general overview of markers used in identifying pluripotent stem cells.

Key Pluripotency Factors

The transcription factors OCT4, SOX2, KLF4, and c-MYC have been widely referenced as key pluripotency factors in stem cells. After all, it had been the ectopic expression of these proteins that gave rise to the term "induced pluripotency.” Other factors such as NANOG and STAT3 have also been found to play major roles. Acting as master regulators, these factors are essential in establishing and maintaining pluripotency. In somatic cells, they enable the induction of pluripotency by reprogramming differentiated cells back into a pluripotent state. Their collective functions include regulating genes necessary for self-renewal, suppressing differentiation pathways, enhancing cellular proliferation, and promoting the transcriptional reactivation of pluripotency genes. Expression levels of transcription factors are often considered in stem cell reprogramming strategies and in experiments characterizing pluripotent cells. 

Figure 1. Pluripotent stem cells (PSCs) generally arise from embryonic cells or reprogrammed adult cells. This diagram highlights how pluripotency markers are used to characterize different populations of PSCs. 

Markers of Naïve and Primed Pluripotent Stem Cells

Pluripotent stem cells can be described in two distinct states, naïve and primed, which reflect stages of embryonic development. Naïve PSCs correspond to the pre-implantation epiblast, capable of unbiased differentiation into all three germ layers. On the other hand, primed PSCs resemble the more developmentally advanced post-implantation epiblast and exhibit some lineage tendencies toward certain differentiation pathways. Differences between the two states also include variations in gene expression, epigenetic regulation, and metabolic activity. 

These states, first identified in mice, have also been observed in human PSCs, with standardized molecular criteria based on transcriptional and epigenetic profiles. Naïve PSCs can be derived directly from embryos, reprogrammed from somatic cells, or converted from primed PSCs, though such conversions often yield heterogeneous populations. Both naïve and primed PSCs maintain pluripotency and self-renewal capabilities, but their distinct molecular and functional properties influence their behavior in research and therapeutic applications. These differences have led researchers to seek out and establish markers using techniques such as flow cytometry and immunofluorescence for validation. 

Key naïve-specific surface markers include CD75, CD7, CD77, and CD130, which have been validated in multiplex antibody panels for distinguishing naïve from primed states. These markers are particularly valuable for isolating emerging naïve PSCs from heterogeneous populations during reprogramming and for monitoring the dynamics of state transitions. Additional naïve-associated proteins include LAMP2, CD229, CD320, alkaline phosphatase, as well as the transcription factors DNMT3L, DPPA2, DPPA3, DPPA5, KLF17, KLF5, and TFCP2L1. 

Common markers for primed PSCs include CD24, CD57, CD90, and HLA-A, B,C. Other markers associated with the primed state include CD172a, CDH3, NLGN4X, and the transcription factors DUSP6, OTX2, and ZIC2. Global DNA methylation is another defining characteristic, in which naïve human PSCs display hypomethylation, measured using H3K27me3, compared to hypermethylation in primed PSCs. 

Identifying PSCs Using Cell Surface Markers  

Current PSC cultures often exhibit heterogeneity, which limits their therapeutic potential and increases the risk of tumorigenicity in clinical settings. Pluripotency markers specific to PSCs can enable precise identification and selection of viable cells in defined states of pluripotency through techniques such as flow cytometry-based sorting and magnetic sorting. Such markers can also be used to create homogenous populations and remove residual cells from preparations. By enhancing homogeneity, researchers can improve the quality of reprogrammed cell lines and create promising new human cell lines for both developmental research and clinical interventions.

Cell surface markers, which are often used in characterizing and sorting many somatic cell types, are likewise invaluable in identifying PSC populations. Antibody panels designed for immunophenotyping rely on a detailed understanding of surface antigen expression, facilitating precise cell identification and functional analysis. Profiling surface markers allows for the standardized assessment of PSCs under various culture conditions, optimizing differentiation protocols, studying molecular mechanisms of pluripotency or reprogramming, and isolating specific target cell populations.

Through various proteomic and immunodetection methods, researchers have identified several cell surface markers notably expressed by pluripotent cells, including ESCs and PSCs. These markers include receptors, transmembrane and membrane-associated proteins, as well as oligosaccharide chains on the outer surface of the cell membrane. Among these are TRA-1-60, TRA-1-81, ADGRG2, Basigin, TDGF1, F11R, PODXL, PCDH1, SSEA-1 (in mice), SSEA-3, SSEA-4, SSEA-5, and L1CAM. 

While these markers can be useful in characterizing PSCs, it is important to acknowledge that they are not unique to PSCs; they are also expressed in other cell types. Pluripotency is a term defined by the functional ability to differentiate and PSC lines must be demonstrated functionally using appropriate differentiation assays.

Table of Pluripotency Markers

The table below lists markers of pluripotent stem cells as described by recent literature. Marker information is accompanied by links to relevant antibody and ELISA kit products, as these are commonly used in marker immunodetection. The associated products are offered by a variety of manufacturers and, when used in combination with other markers of pluripotency, can serve as a useful reference for identifying or isolating PSC populations.

References

Morgani S, Nichols J, Hadjantonakis AK. The many faces of Pluripotency: in vitro adaptations of a continuum of in vivo states. BMC Dev Biol. 2017;17(1):7. Published 2017 Jun 13. doi:10.1186/s12861-017-0150-4

Collier AJ, Panula SP, Schell JP, et al. Comprehensive Cell Surface Protein Profiling Identifies Specific Markers of Human Naive and Primed Pluripotent States. Cell Stem Cell. 2017;20(6):874-890.e7. doi:10.1016/j.stem.2017.02.014

Collier AJ, Rugg-Gunn PJ. Identifying Human Naïve Pluripotent Stem Cells - Evaluating State-Specific Reporter Lines and Cell-Surface Markers. Bioessays. 2018;40(5):e1700239. doi:10.1002/bies.201700239

Trusler O, Huang Z, Goodwin J, Laslett AL. Cell surface markers for the identification and study of human naive pluripotent stem cells. Stem Cell Res. 2018;26:36-43. doi:10.1016/j.scr.2017.11.017

Yilmaz A, Benvenisty N. Defining Human Pluripotency. Cell Stem Cell. 2019;25(1):9-22. doi:10.1016/j.stem.2019.06.010

Meyfour A, Pahlavan S, Mirzaei M, Krijgsveld J, Baharvand H, Salekdeh GH. The quest of cell surface markers for stem cell therapy. Cell Mol Life Sci. 2021 Jan;78(2):469-495. doi: 10.1007/s00018-020-03602-y. Epub 2020 Jul 24. PMID: 32710154; PMCID: PMC11073434.

Goodwin J, Laslett AL, Rugg-Gunn PJ. The application of cell surface markers to demarcate distinct human pluripotent states. Exp Cell Res. 2020 Feb 1;387(1):111749. doi: 10.1016/j.yexcr.2019.111749. Epub 2019 Nov 30. PMID: 31790696; PMCID: PMC6983944.

Andrews PW, Gokhale PJ. A short history of pluripotent stem cells markers. Stem Cell Reports. 2024;19(1):1-10. doi:10.1016/j.stemcr.2023.11.012