RSeT™
Defined Medium for Naïve-Like Human Pluripotent Stem Cells
Spectrum of Human Pluripotent Stem Cell States
During embryogenesis, cells produced within the first few rounds of cell division are typically considered totipotent, as they have the capacity to divide and generate all the tissues of an organism. As embryonic development progresses towards the pre-implantation blastocyst stage, the cells within the inner cell mass are considered pluripotent as these cells are more restricted in their potency and typically give rise to tissues of the embryo proper. Pluripotency is a dynamic state that exists briefly during embryonic development (Figure 1).
Figure 1. Spectrum of Human Pluripotent Stem Cell States
A schematic illustrating the pluripotency potential of cells at each developmental stage.
Human and mouse pluripotent stem cells are derived from the inner cell mass of the blastocyst embryo. However, gene expression patterns differ between human and mouse pre-implantation epiblasts, and this difference is also reflected in vitro as conventional human pluripotent stem cells (hPSCs) are considered to more closely resemble post-implantation mouse epiblast stem cells (EpiSCs) or primed state than mouse embryonic stem (ES) cells, which are considered to be a more naïve state of pluripotency.1 Recently, multiple groups have modified culture conditions to revert and maintain hPSCs closer to a naïve-like pluripotency state. However, although global transcriptome analysis shows similarities between naïve-like hPSCs and mouse ES cells, there are still distinct differences in gene expression patterns and different culture requirements.2-4 While our understanding of human naïve stem cells and their characteristics has improved (Table 1), there are still many unknowns in this rapidly expanding area of hPSC research.
Table 1. Characteristics of Naïve and Primed hPSCs
A comparison of naïve and primed hPSCs and the characteristics that are shared and distinct between these two stem cell states.
Culture Conditions to Maintain Naïve-Like hPSCs
In vitro, different cell culture media with specific combinations of cytokines or small molecules have been shown to maintain cells in a naïve or primed pluripotent state. Specifically, mouse ES cells are dependent on culture media that contain leukemia inhibitory factor (LIF), while conventional hPSCs and mouse EpiSCs are cultured in FGF and Activin.2 Multiple research groups have identified different culture conditions capable of shifting and maintaining hPSCs towards the "ground" or "naïve" state and away from the traditional primed state of hPSCs.3,5-7
RSeT™
RSeT™ is a feeder-dependent, defined medium that reverts primed pluripotent stem cells and maintains cells in a naïve-like state (Figure 2). Developed under license from the Weizmann Institute of Science5, this improved medium does not contain bFGF or TGFβ. With pre-screened quality components that ensure batch-to-batch consistency, RSeT™ produces robust cultures with phenotypes characteristic of naïve-like stem cells and markers associated with undifferentiated cells (Figures 3-5).
Why Use RSeT™ to Culture Human PSCs?
- Passage as single cells while maintaining normal cell karyotypes.
- Maintains pluripotency without inclusion of bFGF or TGFβ.
- Defined medium contains pre-screened quality components.
- No exogenous genes required for reversion to a naïve-like state.
Data
Figure 2. Schematic of Reversion of Primed to Naïve-Like hPSCs
Primed hPSCs are plated as aggregates in mTeSR™1 onto inactivated mouse embryonic fibroblasts (iMEFs). On day 1, mTeSR™1 is replaced with RSeT™, and the medium is exchanged daily. By day 5 or 6, the colonies are generally large enough to be passaged. During the initial culture in RSeT™ medium, colonies expand and begin to adopt domed shape characteristic of naïve-like stem cells and can continue to be propagated in RSeT™.
Figure 3. ES and iPS Cells Can Be Reverted to a Naïve-Like State
Representative images of human (A) ES cells (H1) and (B) iPS cells (STiPS-F016) that reverted to a naïve-like state after cultured in RSeT™ for 6 and 10 passages, respectively. (C) During reversion, colonies change from a flat morphology to a domed morphology characteristic of naïve-state hPSCs. Once naïve-like cultures are established, typically >80% of colonies have a dome-shaped morphology.
Figure 4. ES and iPS Cells Can Be Converted Back into a Primed State
Representative images of human ES cells (H1) cultured in (A) RSeT™ for 3 passages and then (B) reconverted into a primed state in mTeSR™1 for 5 days.
Figure 5. hPSCs Maintained in RSeT™ Express High Levels of Factors Associated with Naïve-Like PSCs
Expression of markers associated with naïve-like PSCs (DNMT3L, KLF17, KLF2, KLF4, NANOG, TBX3, and TCFP2L1) in (A) WLS-1C and (B) STiPS-F019 iPS cells that were reverted to a naïve-like state by culturing in RSeT™. Expression levels were measured by quantitative PCR (qPCR) and normalized to levels in primed STiPS-F019 iPS cells.
Figure 6. hPSCs Maintained in RSeT™ Display Normal Karyotype after Long-Term Passaging
Representative karyograms of (A) ES cell (H1) and (B) iPS cell (STiPS-F019) lines that were cultured in RSeT™ medium for 31 and 48 passages, respectively.
Figure 7. hPSCs Reverted to Naïve-Like State with RSeT™ Medium are Capable of Differentiation to All Three Germ Layers
Human ES cells (H1) maintained in RSeT™ were differentiated into endoderm, mesoderm and ectoderm lineages. Endoderm specification was achieved using STEMdiff™ Definitive Endoderm Kit (Catalog #05110), and mesoderm specification using STEMdiff™Mesoderm Induction Medium (Catalog #05220). Ectoderm specification was achieved by culturing the cells in mTeSR™1 (Catalog #05850) and then in STEMdiff™ Neural Induction Medium (Catalog #05835).
References
- Rossant J. (2008) Stem Cells and Early Lineage Development. Cell 132(4): 527–531.
- Nichols J et al. (1998) Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95(3): 379–391.
- Takashima Y et al. (2014) Resetting Transcription Factor Control Circuitry toward Ground-State Pluripotency in Human. Cell 158(6): 1254–1269.
- Marks H et al. (2012) The transcriptional and epigenomic foundations of ground state pluripotency. Cell 149(3): 590–604.
- Gafni O et al. (2013) Derivation of novel human ground state naive pluripotent stem cells. Nature 504(7479): 282–6.
- Theunissen TW et al. (2014) Systematic Identification of Defined Conditions for Induction and Maintenance of Naive Human Pluripotency. Cell Stem Cell 15(4): 471–487.
- Chan YS et al. (2013) Induction of a human pluripotent state with distinct regulatory circuitry that resembles preimplantation epiblast. Cell Stem Cell 13(6): 663–675.
