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, naïve hESCs isolation has been achieved through 1) induced expression of pluripotency factors and growth factors or 2) following incubation with specific combinations of small modulating molecules and growth factors.

Embryonic pluripotent stem cells are derived from the blastocyst inner cell mass and have the capacity to give rise to all embryonic germ layers.

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ES like Cells: Induced Pluripotent Stem Cells (iPSCs)

iPSCs originate from somatic cells which are reprogrammed to a pluripotent state by the re-expression of key pluripotency factors. Reprogramming of somatic cells was first demonstrated by Takahashi and Yamanaka in 2006, and was achieved through the induction of Oct4, Sox2, Klf4 and c-Myc expression in mouse fibroblasts. Since then, there has been a significant expansion in the range of adult somatic cells reprogrammed to a pluripotent state (e.g., human peripheral blood mononuclear cells, hematopoietic progenitors, hepatocytes, keratinocytes, muscle cells, and adipocyte cells) and in the efficiency of the reprogramming methodologies.


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Ectoderm Marker

Mesoderm Marker

Endoderm Marker

Embryonic pluripotent stem cells differentiated in culture into Sox1/ectoderm marker, Smooth muscle actin/mesoderm marker or Sox17/endoderm marker expressing cells. Embryonic pluripotent stem cells differentiated in culture into Sox1/ectoderm marker, Smooth muscle actin/mesoderm marker or Sox17/endoderm marker expressing cells. Embryonic pluripotent stem cells differentiated in culture into Sox1/ectoderm marker, Smooth muscle actin/mesoderm marker or Sox17/endoderm marker expressing cells.

v6.5 Mouse embryonic stem cells [NBP1-41162] - Embryonic Stem Cells Grown in Mouse Pluripotent Stem Cell Media Undergo Tri-Lineage Differentiation. v6.5 mouse embryonic stem cells were cultured in complete StemXVivo Mouse Pluripotent Stem Cell Media. For differentiation, cells were cultured as embryoid bodies for 5 days, plated onto Cultrex BME-coated plates (R&D Systems, 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-SOX1 Affinity Purified Polyclonal Antibody (AF3369) followed by NorthernLights NL557-conjugated Donkey Anti-Goat Secondary Antibody. (Middle) Expression of the mesoderm marker, Smooth Muscle Actin, was detected in 15 days cells using Mouse Anti-Smooth Muscle Actin monoclonal antibody (R&D Systems, MAB1420).  (Right) Expression of the endoderm marker, SOX17, was detected in 10 days cells using Goat Anti-SOX17 Affinity Purified Polyclonal Antibody (R&D Systems, AF1924) followed by NL557-conjugated Donkey Anti-Goat Secondary Antibody. Nuclei were counterstained with DAPI.


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 "chimera 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.

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JOY6 hiPSCs were differentiated towards ectoderm, mesoderm, and endoderm fate using Human Pluripotent Stem Cell Functional Identification Kit

JOY6 hiPSCs were differentiated towards ectoderm, mesoderm, and endoderm fate using Human Pluripotent Stem Cell Functional Identification Kit (R&D Systems, Catalog # SC027B). Flow cytometry, based on the protocol provided in the kit, was used for differentiation analysis. (orange) Differentiated cells have increased expression of lineage-specific markers for ectoderm - Otx2, mesoderm - Brachyury, or endoderm - SOX17 when compared to (blue) undifferentiated JOY6 hiPSCs.


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, Klf5Nanog, 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

This table contains only a subset of upregulated naïve and primed cell markers. See Ghimire et al. 2018 (mESCs) and Messmer et al. 2019 (hESCs) for a more complete list.


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.

mESCs Markers hESCs 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

SSEA-3, SSEA-4, TRA-1-60, TRA-1-81


Mouse D3 embryonic stem cells were analyzed for expression of pluripotent markers including SSEA-1, SSEA-4, Oct-3/4, and SOX2 by flow cytometry.

H/M Pluripotent Stem Cell Flow Cytometry Kit [FMC001-NOV] - Mouse D3 embryonic stem cells were stained using reagents included in the Human/Mouse Embryonic Stem Cell Multi-Color Flow Cytometry Kit. Cells were analyzed for expression of pluripotent 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 pluripotent marker SOX2.


BG01V human embryonic stem cells were analyzed for expression of pluripotent markers including SSEA-1, SSEA-4, Oct-3/4, and SOX2 by flow cytometry.

H/M Pluripotent Stem Cell Flow Cytometry Kit [FMC001-NOV] - BG01V human embryonic stem cells were stained using reagents included in the Human/Mouse Pluripotent Stem Cell Multi-Color Flow Cytometry Kit. Cells were simultaneously analyzed for expression of pluripotent 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 pluripotent marker SOX2.


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Select 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., Roost, M. S., Anckaert, J., Popovic, M., … Heindryckx, B. (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., Santos, F., Mohammed, H., Lun, A. T. L., … Reik, W. (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