PLURIPOTENCY AND THE UNDIFFERENTIATED STATE: DEBUNKING COMMON MISCONCEPTIONS

As the stem cell field continues to expand and evolve, clear and accurate communication of data has become more critical than ever. Unfortunately, the incorrect use of terminology has contributed to confusion and misconceptions when reporting results involving human pluripotent stem cells (hPSCs). Terms like "undifferentiated" and "pluripotent" are frequently used interchangeably, despite referring to distinct—though related—concepts. A cell that is undifferentiated is not necessarily pluripotent.

The introduction of induced pluripotent stem cells (iPSCs) has likely added to this confusion. Many of the same cell surface markers used to identify undifferentiated stem cells also function as reprogramming factors, or “pluripotency factors,” in iPSC generation. This overlap in terminology can blur the distinction between cells that are undifferentiated and those that are functionally pluripotent, which can lead to incorrect assumptions about the cells being studied. In a field where precision matters, recognizing the difference between these terms is not just semantic; it's necessary for ensuring the validity of experimental outcomes.

Defining Pluripotency and the Undifferentiated State

Human induced pluripotent stem cell colony stained to highlight OCT4 in red. Credit: National Eye Institute/NIH
Human induced pluripotent stem cell colony stained to highlight OCT4 in red. Credit: National Eye Institute/NIH
Pluripotent stem cells, including embryonic stem cells and iPSCs, are defined by their ability to self-renew and differentiate into cells from all three primary germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is a functional attribute that must be demonstrated experimentally1,2. In mouse embryonic stem cells, pluripotency is validated through their ability to form chimeras or contribute to the germline when injected into a blastocyst. For human pluripotent stem cells (PSCs), surrogate assays are used, such as the formation of teratomas containing tissues from all three germ layers in immune-deficient mice or through in vitro differentiation assays. The International Stem Cell Initiative confirms that in vitro embryoid body (EB) assays, are sufficient to evaluate the differentiation potential of PSCs3.

On the other hand, "undifferentiated" refers to stem cells that have not yet committed to a specific cell type or lineage. Researchers often measure the expression of specific cell surface markers and intracellular transcription factors to monitor differentiation and to identify pluripotent stem cells (PSCs). However, because these markers are not exclusively expressed by pluripotent stem cells, they alone are not definitive indicators of pluripotency4. For example, "nullipotent" embryonal carcinoma cells, which have lost their ability to differentiate altogether, express many of the same markers we associate with undifferentiated pluripotent cells, including OCT4 (POU5F1) and NANOG1. These cells are undifferentiated but not pluripotent, highlighting the need for careful use of terminology. As such, these so-called “pluripotency markers” are more accurately referred to as “markers of the undifferentiated state”. 

Building on earlier work characterizing cell surface antigens in human embryonal carcinoma cells2, the International Stem Cell Initiative identified specific markers (Table 1) commonly expressed by undifferentiated human embryonic stem cells (hESCs), which can be used for identification and monitoring5.
   
MARKER    
   
ANTIBODY   

Stage-specific embryonic antigen-3 (SSEA-3)
   
MC631   
   
Stage-specific embryonic antigen-4   (SSEA-4)    
   
MC813-70   
   
Trafalgar-1-60 (TRA-1-60)   
   
TRA-1-60   
   
Trafalgar -1-81 (TRA-1-81)   
   
TRA-1-81   
   
Germ Cell Tumor Marker 2 (GCTM2)     
   
GCTM2   
   
Liver-type Alkaline phosphatase (L-ALP)   
   
TRA-2-54   
   
Liver-type Alkaline phosphatase (L-ALP)   
   
TRA-2-49   
   
Cluster of Differentiation 90 (CD90 or Thy-1)   
   
F15-14-1   
   
Cluster of Differentiation 9 (CD9)   
   
TG30   
Table 1 (Source: The International Stem Cell Initiative, 2007)


When to Validate Cell Line Pluripotency?

Ultimately, the decision to validate pluripotency should be guided by the specific scope and goals of your research project, and resources such as the ISSCR ‘‘Standards for Human Stem Cell Use in Research’’ provide clear guidelines that can aid in decision-making. For example, if a cell line's pluripotency has been previously established and reported in peer-reviewed publications, there is generally no need to repeat comprehensive validation studies. However, routine monitoring of established cell lines should include quantitative marker analysis to monitor the undifferentiated status of the cells.   

For new pluripotent cell lines or those derived using novel reprogramming techniques or when untested culture methods are used, more comprehensive validation is necessary, including evaluating undifferentiated state markers and differentiation potential into the three germ layers. Due to ethical concerns and increasingly stringent regulations, xenograft (teratoma) assays are not required for confirming pluripotency. Instead, in vitro assays, such as organoid or 3D models, alongside the loss of markers associated with the undifferentiated state, can provide adequate evidence of pluripotency, though xenograft assays may still be used to assess potential malignancy risks1

The Importance of Terminology in Stem Cell Research

Precision in terminology is essential for minimizing misunderstandings and ensuring experimental integrity within the stem cell field and beyond. Distinguishing between terms like "pluripotent" and "undifferentiated" and applying them correctly upholds scientific integrity and minimizes misinterpretation of experimental data. In an ever-evolving field, clear communication of data facilitates knowledge transfer across the scientific community.



Acknowledgments:

Special thanks to Dr. Tenneille Ludwig and Dr. Peter Andrews for their valuable insights and contributions to this article.


References

  1. Ludwig TE, Andrews PW, Barbaric I, et al. ISSCR standards for the use of human stem cells in basic research. Stem Cell Reports. 2023;18(9):1744-1752. doi:10.1016/j.stemcr.2023.08.003
  2. 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
  3. International Stem Cell Initiative. Assessment of established techniques to determine developmental and malignant potential of human pluripotent stem cells. Nat Commun. 2018;9(1):1925. Published 2018 May 15. doi:10.1038/s41467-018-04011-3
  4. Andrews PW, Casper J, Damjanov I, et al. Comparative analysis of cell surface antigens expressed by cell lines derived from human germ cell tumours. Int J Cancer. 1996;66(6):806-816. doi:10.1002/(SICI)1097-0215(19960611)66:6<806::AID-IJC17>3.0.CO;2-0
  5. International Stem Cell Initiative, Adewumi O, Aflatoonian B, et al. Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nat Biotechnol. 2007;25(7):803-816. doi:10.1038/nbt1318