Embryonic Stem Cells
What are Embryonic Stem Cells?
Pluripotent embryonic stem cells (ESCs) correspond to cells within a developing embryo that have the capacity to generate all the embryonic germ layers (i.e., endoderm, mesoderm and ectoderm), and are able to give rise to all cell types in the body. ESCs may be derived from developing embryos at the pre-implantation blastocyst stage, and specifically from cells within the inner cell mass (ICM). In mice, the pluripotent state of ICM cells (mESCs) is often referred to as a “naïve” state. Following blastocyst implantation, ICM derived cells or mouse epiblast stem cells (mEpiSCs) retain self-renewal capacity but are in a “primed” state of pluripotency. Similarly, conventional ICM derived human ESCs (hESCs) are characterized by “primed” pluripotent properties, a state that is thought to occur in association with the in vitro conditions used for their derivation and maintenance. However, cultivation of naïve hESCs has been achieved through 1) induced expression of pluripotency factors or 2) incubation with specific combinations of small modulating molecules and growth factors.
Trilineage differentiation of mouse embryonic stem cells. Immunocytochemistry/Immunofluorescence staining of v6.5 mouse embryonic stem cells grown in mouse pluripotent stem cell media. For differentiation, cells were cultured as embryoid bodies for 5 days, plated onto Cultrex BME-coated plates (Catalog # 3434-005-02) for an additional 5-10 days. (Left) Expression of the ectoderm marker, SOX1, was detected in day 10 cells using Goat Anti-Human/Mouse/Rat SOX1 (Catalog # AF3369) followed by NorthernLights NL557-conjugated Donkey Anti-Goat Secondary Antibody (Catalog # NL001). (Middle) Expression of the mesoderm marker, Smooth Muscle Actin, was detected in 15 days cells using Mouse Anti-Human/Mouse/Rat Smooth Muscle Actin (1A4/asm1) (Catalog # NBP2-33006). (Right) Expression of the endoderm marker, SOX17, was detected in 10 days cells using Goat Anti-SOX17 Affinity Purified Polyclonal Antibody (Catalog # AF1924) followed by NL557-conjugated Donkey Anti-Goat Secondary Antibody. Nuclei were counterstained with DAPI (Catalog # NBP2-31156).
How is Pluripotency Confirmed?
ESC pluripotency in vitro and in vivo may be confirmed through various approaches.
- Injection of ESCs into tissues of adult immune-deficient mice to confirm the ability of ESCs to differentiate and form teratomas.
- Spontaneous or directed differentiation of mouse ESCs in vitro to monitor the formation of embryoid bodies (EBs).
Histological analysis (e.g., Immunohistochemical or Immunocytochemical) of teratomas and EBs serves to confirm differentiation of ESCs into a variety of cell types from endoderm, mesoderm, and ectoderm origin. - Injection of ESCs into blastocysts for the generation of "chimeric mice" confirms germline capacity.
Functional identification (i.e., tri-lineage differentiation) is an efficient and standardized method for assessing pluripotency in vitro. This method uses growth factors to differentiate pluripotent stem cells into ectoderm, mesoderm, and endoderm.
BG01V human embryonic stem cells were differentiated to ectoderm, mesoderm, and endoderm using the media supplements included in the Human Pluripotent Stem Cell Functional Identification Kit (Catalog # SC027B). To verify differentiation to ectoderm, cells were stained with Goat Anti-Human Otx2. To verify differentiation to mesoderm, cells were stained with Goat Anti-Human Brachyury. To verify differentiation to endoderm, cells were stained with Goat Anti-Human SOX17. The cells were stained using the NorthernLights™557-conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog # NL001; red) and the nuclei were counterstained with DAPI (Catalog # NBP2-31156).
What is the Difference between Naïve and Primed ESCs?
Naïve and primed ESCs share the same capacity for self-renewal and ability to give rise to the three embryonic germ layers. However, only naïve ESCs generate germline chimeras. The expression of specific pluripotency factors differs between stem cells in a naïve vs primed state.
| Naïve mESCs Expressed Genes | Primed mESCs Expressed Genes | Naïve hESCs Expressed Genes | Primed hESCs Expressed Genes |
|---|---|---|---|
| Dppa3/Stella, Esrrb, Fgf5, Klf2, Klf4, Klf5, Nanog, Oct4, CD31 (PECAM-1), Prdm14, Sox2, Tbx3, Tfcp2l1, Zfp42 | Dnmt3a, Fgf5, Fgfr1, Foxa2, Pdgfra, Sox1, Twist2 | Dnmt3L, Dppa5, Klf4, Klf17, Nanog, Oct4, Sox2, Tbx3 | CD24, Nanog, Oct4, Sfrp2, Sox2, CD90 (Thy-1), Zic2 |
Embryonic Stem Cell Surface Markers
Pluripotent stem cells express a variety of cell surface proteins which may be used for their characterization and isolation from heterogeneous populations under culture conditions. The combined use of pluripotency markers to identify ESCs facilitates downstream applications aimed at the propagation of ESCs or their differentiation towards specific lineages.
| mESC Markers | hESC Markers |
|---|---|
| CD9, CD24, CD29(b1 Integrin), CD31 (PECAM-1), CD49f (Integrina6), CD59, CD90 (Thy-1), CD117 (c-Kit), CD133, CD324 (E-Cadherin), CD326/EpCAM, Cripto, Frizzled5, SSEA-1 | CD9, CD24, CD29(b1 Integrin), CD31 (PECAM-1), CD49f (Integrina6), CD59, CD90 (Thy-1), CD117 (c-Kit), CD133, CD324 (E-Cadherin), CD326/EpCAM, Cripto, Frizzled5, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 |
Mouse D3 embryonic stem cells were analyzed for expression of pluripotency markers including SSEA-1, SSEA-4, Oct-3/4, and SOX2 by flow cytometry. A. Flow cytometric analysis shows that 91.1% of mouse D3 embryonic stem cells are positive for both Oct-3/4 and SSEA1 expression. B. Flow cytometric analysis data shows that 82.6% of mouse D3 embryonic stem cells are positive for SSEA-1 and negative for SSEA-4 a phenotype consistent with mouse embryonic stem cells. C. Flow cytometric analysis shows that mouse D3 embryonic stem cells express the pluripotency marker SOX2.
BG01V human embryonic stem cells were simultaneously analyzed for expression of pluripotency markers including SSEA-1, SSEA-4, Oct-3/4, and SOX2 by flow cytometry. A. Flow cytometry data shows that 91.9% of BG01V human embryonic stem cells are positive for both Oct-3/4 and SSEA4 expression. B. Flow cytometry data shows that 88.5% of BG01V human embryonic stem cells are positive for SSEA-4 and negative for SSEA-1, a phenotype consistent with human embryonic stem cells. C. Flow cytometric analysis shows that BG01V human embryonic stem cells express the pluripotency marker SOX2.
Selected References
Bieberich, E., & Wang, G. (2013). Molecular Mechanisms Underlying Pluripotency. In Pluripotent Stem Cells. https://doi.org/10.5772/55596
Boroviak, T., & Nichols, J. (2017). Primate embryogenesis predicts the hallmarks of human naïve pluripotency. Development (Cambridge). https://doi.org/10.1242/dev.145177
Carstea, A. C. (2009). Germline competence of mouse ES and iPS cell lines: Chimera technologies and genetic background. World Journal of Stem Cells. https://doi.org/10.4252/wjsc.v1.i1.22
Ghimire, S., Van Der Jeught, M., Neupane, J., et al. (2018). Comparative analysis of naive, primed and ground state pluripotency in mouse embryonic stem cells originating from the same genetic background. Scientific Reports. https://doi.org/10.1038/s41598-018-24051-5
Kim, J. S., Choi, H. W., Choi, S., & Do, J. T. (2011). Reprogrammed pluripotent stem cells from somatic cells. International Journal of Stem Cells. https://doi.org/10.15283/ijsc.2011.4.1.1
Kumari, D. (2016). States of Pluripotency: Naïve and Primed Pluripotent Stem Cells. In Pluripotent Stem Cells - From the Bench to the Clinic. https://doi.org/10.5772/63202
Messmer, T., von Meyenn, F., Savino, A., et al. (2019). Transcriptional Heterogeneity in Naive and Primed Human Pluripotent Stem Cells at Single-Cell Resolution. Cell Reports. https://doi.org/10.1016/j.celrep.2018.12.099
Nichols, J., & Smith, A. (2009). Naive and Primed Pluripotent States. Cell Stem Cell. https://doi.org/10.1016/j.stem.2009.05.015
Trusler, O., Huang, Z., Goodwin, J., & Laslett, A. L. (2018). Cell surface markers for the identification and study of human naive pluripotent stem cells. Stem Cell Research. https://doi.org/10.1016/j.scr.2017.11.017
Weinberger, L., Ayyash, M., Novershtern, N., & Hanna, J. H. (2016). Dynamic stem cell states: Naive to primed pluripotency in rodents and humans. Nature Reviews Molecular Cell Biology. https://doi.org/10.1038/nrm.2015.28
Zhao, W., Ji, X., Zhang, F., Li, L., & Ma, L. (2012). Embryonic Stem Cell Markers. Molecules. https://doi.org/10.3390/molecules17066196