Enhancing human naive pluripotency induction


Conventional human embryonic stem cells (hESCs) retain a variety of epigenetic properties that are consistent with possessing a primed pluripotent state. These include an inability to tolerate MEK/ERK inhibition (MEKi/ERKi), predominant utilization of the proximal enhancer element to maintain OCT4 expression, X-chromosome inactivation, and high levels of DNA methylation. Several groups have previously established conditions for deriving naive MEK/ERK signaling-independent genetically unmodified human pluripotent stem cells (hPSCs). For example, naive human stem cell medium (NHSM) conditions do not require the use of exogenous transgenes or feeder cells, maintain teratoma formation competence, and contain the following components: 2iLIF, P38i/JNKi, protein kinase C inhibitor (PKCi), ROCKi, ACTIVIN, and FGF2. Under NHSM conditions, hPSCs exhibit some naive features, most notably pluripotency preservation while MEK/ERK signaling is inhibited, which is the hallmark of mammalian naive pluripotency, predominant TFE3 nuclear localization, in vitro reconstitution of human primordial germ cells (PGCs), and a mild reduction in DNA methylation. The latter effect is profoundly weaker than the one observed in mouse pluripotent cells, suggesting sub-optimal human naive pluripotency growth conditions.

Realizing the vision of culturing organs for use in life-saving transplantation procedures is still a long way off. However, the work of Prof. Jacob Hanna on stem cells is paving the way for this to become a reality. Hanna and his team from the Weizmann Institute of Science’s Molecular Genetics Department have found a way to culture human stem cells in a much earlier state than was previously possible. Not only that, the stem cells they created are far more competent, meaning that they are able to integrate more efficiently with their host environment. This substantially improves the chances of obtaining what is called a cross-species chimera—allowing cells from one creature to play a substantial role in the development of another.

The recently published findings demonstrate that very early human cells can be created and then successfully integrated into mice, owing to their undifferentiated (or “naïve”) state, wherein they can develop into any type of cell in the body, including other stem cells. Additionally, the researchers lay out a protocol for significantly increasing the efficiency (or competence) with which these cells can integrate. Improving our ability to create and study these cell types could be used in the future to transfer cells—if not organs—from one animal to another, humans included.

Hanna’s lab broke ground in 2013 when they were the first to inject human stem cells into mice and show that they can successfully integrate into the latter’s developing embryos. Eight years after this study was first published, Hanna and his team felt that they could go one step further by attempting to produce an even earlier, “fully” naïve form of stem cells for use in similar procedures. As they were mulling over the idea, Hanna knew that this might be nearly—if not altogether—impossible to achieve.

These cells normally suffer from genetic as well as epigenetic instability, and in the end they don’t differentiate too well, which is key to proper embryonic development and a prerequisite for their integration into another animal’s embryo. In fact, only about 1-3 percent of cells that have been transferred between species actually manage to integrate and contribute to development.

To boost these numbers, the researchers in the new study inhibited two additional signaling pathways to produce naïve human stem cells having a stable genome, relatively few gene regulation glitches, and most importantly, the ability to differentiate perfectly. The researchers also mutated an important gene that contributes to genome stability, which resulted in not only competent but also competitive stem cells that can integrate well without causing damage to the host.  The authors found a way to make human stem cells more competent, and competitive, increasing the chances for a successful transfer by about fivefold compared to what we were able to do in the past.

While the previous study showed that human naïve stem cells can differentiate into primordial germ cells—the progenitors of egg or sperm cells—the fully naïve stem cells produced in the present study can also differentiate into extraembryonic tissues, the placenta and yolk sac cells that sustain the developing embryo. Such cells could be used, for example, as the source for developing synthetic embryos without the need for donor eggs. Reaching this state with mouse stem cells is particularly difficult to accomplish. This is perhaps the most surprising finding that the researchers made—highlighting the differences between the behavior of human and mouse stem cells, and between the different states of naïve cells. These differences expose the work that still needs to be done in making the dream of developing “made-to-order” organs a real-world actuality.

According to the authors, understanding these differences will be pivotal for overcoming myriad issues still facing the field of stem cell research and application If in the future we should wish to grow a pancreas in pigs for human transplantation, for example, we will have to take into account these massive evolutionary differences between species, beginning with mice and humans. For now, it would seem that Hanna and his team have taken a constructive leap in that direction.

The authors findings in mice and human indicate that the ability to tolerate permanent ablation of such repressors may be one of the key features of naive pluripotency across different species and might prove to be an efficient method for inducing naive pluripotency from other species in vitro.

Enhancing human naive pluripotency induction - Medicine Innovates

About the author

Prof. Yaqub (Jacob) Hanna

Being able to generate all cell types, mouse embryonic stem cells are a most valuable tool for research. They can be found in the developing mouse embryo in two distinct states: naïve – in the blastocyst, and primed – in the post-implantation epiblast. These two states are distinct in various aspects, most notable, only naïve cells can contribute efficiently to chimera. Naïve and primed cells can be sustained in-vitro, and are dependent on distinct signaling. In human, naïve stem cells were out of reach for a long time. The lab investigates the regulation of naïve and primed pluripotent stem cell in mouse and human. Specifically, we were able to maintain human stem cells in a “naive” state, with distinct molecular and functional properties, including enhanced ability to contribute to cross-species mouse chimeric embryos (Gafni et al, 2013). In addition, we found that mRNA methylation has a critical role in facilitating degradation of pluripotent genes, an essential step during the switch from naïve to primed states, both in-vitro and in-vivo (Geula et al, 2014). Our current studies involve elucidating molecular regulation of these states across different species, and define how their molecular architecture dictates their functional competence.


Jonathan Bayerl, Muneef Ayyash , Tom Shani, Noa Novershtern, Sergey Viukov, Jacob H. Hanna. Principles of signaling pathway modulation for enhancing human naive pluripotency induction. Cell Stem Cell, VOLUME 28, ISSUE 9, P1549-1565.E12, 2021