Leaders in Pharmaceutical Business Intelligence (LPBI) Group

Naïve Human Pluripotency

Larry H. Bernstein, MD, FCAP, Curator

LPBI

The Current State of Naïve Human Pluripotency
Benjamin T. Dodsworth, Rowan Flynn,  Sally A. Cowley 

Stem Cells Nov 2015; 33(11): 3181–3186   DOI: http://dx.doi.org:/10.1002/stem.2085

The newly discovered state of ‘naïve’™ human pluripotency is not only an extremely interesting biological phenomenon, but also promises to overcome some of the problems posed by conventional ‘primed’™ human pluripotent stem cells. These problems include variable differentiation capability, difficult single-cell passaging, and low gene editing efficiency – all bottlenecks for applications such as regenerative medicine. A flurry of recent papers describe new ways of accessing naïve human pluripotency. However, there are important differences between these protocols, making this concise yet comprehensive review a timely necessity to navigate the complexities of this emerging field.

Naïve or ground state pluripotency is a cellular state in vitro which resembles cells of the preimplantation epiblast in vivo. This state was first observed in mouse embryonic stem cells and is characterized by high rates of proliferation, the ability to differentiate widely, and global hypomethylation. Human pluripotent stem cells (hPSCs) correspond to a later or “primed” stage of embryonic development. The conversion of hPSCs to a naïve state is desirable as their features should facilitate techniques such as gene editing and more efficient differentiation. Here we review protocols which now allow derivation of naïve human pluripotent stem cells by transgene expression or the use of media formulations containing inhibitors and growth factors and correlate this with pathways involved. Maintenance of these ground state cells is possible using a combination of basic fibroblast growth factor and human leukemia inhibitory factor together with dual inhibition of glycogen synthase kinase 3 beta, and mitogen-activated protein kinase kinase (MEK). Close similarity between the ground state hPSC and the in vivo preimplantation epiblast have been shown both by demonstrating similar upregulation of endogenous retroviruses and correlation of global RNA-seq data. This suggests that the human naïve state is not an in vitro artifact. Stem Cells 2015;33:3181–3186

In mice, two pluripotent states have been captured in vitro. Mouse embryonic stem cells (mESCs) are sourced from the inner cell mass (ICM) of the preimplantation blastocyst [1, 2]. When derived and maintained using a combination of leukemia inhibitory factor (LIF) and 2i (dual inhibition of extracellular signal-regulated protein kinases 1/2 [ERK1/2] pathway and glycogen synthase kinase 3 beta [GSK3β]) they are described as being in a naïve or ground state [3]. When injected back into an early embryo, these cells can contribute to all lineages without tumorigenesis [4]. A more recent discovery has been mouse epiblast stem cells (mEpiSCs—Fig. 1). These are sourced from postimplantation epiblast cells [5, 6] and are termed primed, due to their inability to integrate into a preimplantation blastocyst. They can, however, be differentiated into all three germ layers in vitro. The most striking difference is the very high expression of de novo methyltransferases, which leads to condensing of chromatin [7]. Additionally, these cells require basic fibroblast growth factor (bFGF also known as FGF2) and transforming growth factor beta (TGFβ) for self-renewal, instead of 2i and LIF [3, 5]. mEpiSCs can be converted back to the naïve state by transfection with Klf4 or other reprogramming factors or using small molecules [8, 9].

Naïve pluripotent stem cells have been successfully captured in vitro from primed rhesus monkey induced pluripotent stem cell (iPSC) lines using specialized media containing 2i and LIF [10]. Since naïve pluripotent stem cells can be generated from primates, this suggests that the state of naivety might be conserved across species. Using primate cells also allows dissection of genetic background and species to species differences. Primate naïve iPSCs require bFGF, whereas bFGF causes differentiation in mESCs. Additionally, TGFβ is not required for maintenance of primate naïve iPSCs, indicating that TGFβ might not be essential in the human system [10].

Embryogenesis is inherently different between species, which is reflected by the difficulties in generating truly naïve human pluripotent stem cells (hPSCs) in vitro. For ethical reasons, information on human embryogenesis is lacking and many assumptions are made based on the mouse model [11]. Despite being sourced from the same point in development as mESCs, hESCs resemble mEpiSCs. Both form large, flat, 2D colonies and require bFGF for self-renewal. The ability to convert mEpiSCs to mESCs has led to the prediction that naïve hPSCs might also be accessible by reverting primed hESCs. This has prompted several recent publications of strategies to capture the human naïve state, either relying on transgene overexpression [12-14] or different combinations of small molecule inhibitors [15-20]. Here we review and compare all these published protocols, including a protocol devised by Duggal et al. [16] published in this issue.

• Key Characteristics of the Naïve State
• Gene Editing Efficiency
• Strategies of Derivation and the Pluripotency Network

The concept of naïve hPSCs has been contentious. Pera [41] argues that since this state was actively searched for in humans, it is highly likely that it is purely an artifact generated in the lab. However, Wang et al. used RNA-seq data which was available from cells taken directly from the ICM of early embryos and showed a tight correlation to naïve cells generated in vitro [27].

This was confirmed when Huang, Maruyama, and Fan took a systems biology approach and compared datasets from many previous publications [42]. Their analysis revealed poor conservation of gene networks between mPSCs and hPSCs but a high resemblance to the ICM of their respective blastocysts. They also found variations in transcriptomes from different naïve conversion protocols, but all established naïve cells showed clear resemblance to human late preimplantation embryos. According to this study, naïve cells generated by Takashima et al. [14] and Theunissen et al. [19] most closely resembled the human preimplantation blastocyst. The protocols by Valamehr et al. [18] and Duggal et al. [16] were not included in the study. In conclusion, the authors propose comparing the combination of transcriptome analysis and epigenetic characterization to in vivo data from embryogenesis as a gold standard for naivety [42].

The description of just two states, naïve and primed, is an oversimplification [11, 27, 43, 44]. Two studies [27, 43] used single-cell RNA-seq and reported a polyclonal spectrum of cell states ranging between these extremes and that naïve PSCs are present as a subpopulation in cultures previously considered entirely primed. Wang et al. [27] used a reporter system based on the endogenous retrovirus HERVH’s LTR7 promoter which is only active in naïve cells. This approach showed a consistent 4% of cells with naïve reporter expression which can be selected for using 2i and LIF and do not need prior conversion. Recently, Wu et al. were able to capture another alternative state designated “region-selective primed” pluripotency in vitro in both mouse and human which are distinct from both naïve and primed states [44].

There remain many challenges in the field of naïve pluripotency. All protocols for generating human naïve PSCs yield slightly different cellular states. It is still unclear which of these is closest to its in vivo counterpart. The in vivo naïve state is inherently transient, so continuous in vitro culture may be detrimental. For example, female cells maintained in the naïve state that do not exhibit X-inactivation might suffer from double dosage effects. With protocols now readily available which allow the generation and maintenance of naïve cells, these questions can be addressed. Meanwhile, their faster rate of growth, single cell survival, and enhanced gene editing efficiency will be used. In the near future, naïve hPSCs may be useful for accessing paths of differentiation which have been previously unreachable.

Siân Gregory, Sushma Swamy, Zoe Hewitt, Andrew Wood, Richard Weightman, Harry Moore, Autophagic response to cell culture stress in pluripotent stem cells, Biochemical and Biophysical Research Communications, 2015