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New Pluripotent Stem Cell Type with Enhanced Therapeutic Value Described

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Review of “An alternative pluripotent state confers interspecies chimaeric competency” from Nature by Stuart P. Atkinson

A stellar cast of researchers from the laboratories of Alan SaghatelianBing RenJoseph R. Ecker & Juan Carlos Izpisua Belmonte have recently published a new study in Nature in which they describe the discovery of a new type of pluripotent stem cell (PSC). PSCs, such as embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs), are currently derived at specific time points during development, although we understand the spatial co-ordination of stem cells also plays an important role in development [1-3]. Therefore, researchers have used a cocktail of chemical factors to direct pluripotent stem cells (PSCs) to become spatially oriented and form region-selective pluripotent stem cells (rsPSCs) which are easier to grow in vitro, can engraft within mouse embryos, and are easily manipulated at the genetic level. They posit that these “primed” stem cells could not only provide a new model system for studying human development, but also lead to the growth of human organs for therapeutic purposes [4].

The group’s initial investigations concentrated on the optimization of epiblast culture conditions, and they discovered that epiblast explants grown on mitotically inactivated mouse embryonic fibroblasts (MEFs) using serum-free N2/B27 supplemented medium [5] in the presence of FGF2 and the Wnt inhibitor IWR1 (N2B27F/R1) mediated good EpiSC outgrowth and homogenous morphology, with little-to-no differentiation. This suggested that this medium captured all epiblast cells in a proliferative pluripotent state as cells also homogenously expressed OCT4 and SSEA-1. Further repetition of this strategy using epiblasts from different genetic backgrounds and different developmental stages of post implantation as well as pre-implantation epiblasts gave a derivation success rate of nearly 100%. This is in stark comparison to EpiSC derivation using FGF2/Activin-A (F/A) which generally has a much lower success rate.

Further analysis demonstrated that EpiSCs derived under N2/B27F/R1 conditions (EpiSCsF/R1) bore properties indicative of a “primed” pluripotent state akin to EpiSCs, but did display some notable differences. EpiSCsF/R1 had a high cloning efficiency at a comparable level to ESCs, they proliferated at a much faster pace and displayed a much shorter doubling time than EpiSCs, and they relied more on glycolysis than mitochondrial respiration than already highly glycolytic EpiSCs. These findings brought the researchers to hypothesize that the EpiSCsF/R1 actually existed in a primed pluripotent state distinct to that of EpiSCs. As expected, EpiSCsF/R1 did not contribute to chimeras when injected into blastocysts but did when grafted into post-implantation epiblasts, although EpiSCsF/R1 only integrated efficiently in the posterior region of the epiblast. Indeed, cells grafted in this area dispersed from the graft site, proliferated and differentiated into the three germ layers. For this reason, the group named these primed pluripotent cells, with a new spatial identity, region-selective EpiSCs (rsEpiSCs).

The group confirmed the distinct identity of rsEpiSCs from EpiSCs at the transcriptomic, epigenomic and metabolic level, even though both cell types exist in the primed pluripotent state. The differences included a transcriptional signal of epithelial to mesenchymal transition initiation, higher levels of glycolytic enzymes/complexes, and lower levels of mitochondrial enzymes/complexes in rsEpiSCs. Further, RNA-Seq analysis found that rsEpiSCs resembled the in vivo posterior-proximal region of the epiblast, while temporal rsEpiSCs resembled cells from the late-primitive streak/no-bud-stage epiblast undergoing epithelial to mesenchymal transition before lineage commitment.

They next attempted to grow human ESCs, which resemble mouse EpiSCs, in F/R1 conditions (human region-selective ESCs, or human rsESCs). They found that these conditions allowed for long-term self-renewal with karyotypic stability, an improved cloning efficiency, easier genome editing, and cell growth as larger homogenous pluripotent colonies. Additionally, F/R1 conditions also supported the long term region specific-like growth of non-human primate (NHP) PSCs including rhesus macaque PSC and chimpanzee iPSC. Interestingly both human rsESCs and rhesus macaque rsESCs efficiently integrated, proliferated and differentiated into all three germ layers following transplantation into the posterior region of the mouse post-implantation epiblast, unlike normal ESCs.

This is the first time that spatial identity has been used to distinguish pluripotent states in PSC populations and, in this study, the clever manipulation of culture parameters has led to the discovery and description of a potentially important new pluripotent state. Pluripotent cells in the primed state have multiple advantages which make them advantageous for large scale use in therapeutics or modeling, although heterogeneity and poor cloning efficiency associated with the conventional F/A culture usually makes their application more difficult. This new culture strategy removes these barriers and their ability to integrate and form cells of the three germ layers in chimaeric embryos may soon provide us with a way to study early developmental processes and advanced applications such as cell and tissue replacement therapies.

References
1. Chenoweth JG, McKay RD, and Tesar PJ Epiblast stem cells contribute new insight into pluripotency and gastrulation. Dev Growth Differ 2010;52:293-301.
2. Pera MF In search of naivety. Cell Stem Cell 2014;15:543-545.
3. Beddington RS and Robertson EJ Axis development and early asymmetry in mammals. Cell 1999;96:195-209.
4. Wu J, Okamura D, Li M, et al. An alternative pluripotent state confers interspecies chimaeric competency. Nature 2015;521:316-321.
5. Ying QL, Wray J, Nichols J, et al. The ground state of embryonic stem cell self-renewal. Nature 2008;453:519-523.