Formative pluripotency in livestock species

During early embryogenesis, pluripotency emerges as a naïve state in the blastocyst and transits towards a primed state when cells start to fix their fate through gastrulation. Formative pluripotency was recently proposed as a cell state that bridges naïve and primed state. We recently established the culture conditions required to capture mouse and human early embryonic cells with features of formative pluripotency.

Mouse formative stem (FS) cells have unique transcription factor dependency, chromatin landscape, germ cell differentiation potency and chimaera competency. FS cells satisfy all the criteria of a pluripotent stem cell, similar to embryonic stem (ES) cells. Mouse and human FS cells share the same culture conditions, however functional analysis of hFS cells is limited because of accessibility of early post-implantation human embryos.

Livestock species share features of early embryogenesis with humans that differ from mice. However, stem cell lines from livestock species have not been derived until recently. Our chemically defined culture is applicable to porcine, ovine and bovine stem cells.

Transcriptome analysis shows relatedness to the pre-gastrulation embryonic disc so we named them Embryonic Disc Stem Cells (EDSCs). EDSCs may be practically useful as donors for somatic cell nuclear transfer to create genetically modified animals. EDSCs, however, do not share all the pluripotent features of mouse FS cells, so FS cells from livestock remain to be established.

Most recently, we have succeeded in establishing self-renewing FS cell candidates from livestock embryos using combinations of growth factors and inhibitors different from those used for EDSCs. They have a different transcriptome from EDSCs. Based on our knowledge of mouse FS cells, we will determine the precise timing of the formative phase in livestock embryos, and establish and characterize livestock FS cells.

We will apply high throughput sequencing to elucidate the transcriptome and epigenetic modifications of livestock FS cells. These analyses will reveal the conserved features of in vitro formative pluripotency. Mouse FS cells have reliable chimaera contribution, which has not been achieved in livestock stem cells to date.

This is a key goal for this research. Taking advantage of reproductive technologies used in large animals, we will assess chimaera competency both in vitro and in vivo. We will assess chimaera competency both in vitro and in vivo.

The chimaera technology in these species will be a useful tool for creating transgenic and gene edited livestock.

The project will reveal the properties of formative pluripotency in livestock species. It generates new knowledge of pre-gastrulation stage embryogenesis in livestock. Livestock FS cells can directly benefit animal breeding, transgenic animal production, biomedical research and agriculture.

In addition, the findings will provide new insights into human biology. Conventional human ES and iPS cells are considered broadly equivalent to peri-gastrulation stage cells, but numerous cell lines and variable culture conditions makes them difficult to define. It is well known that these cells have biased differentiation potency so it is necessary to select the appropriate clone to work with.

Current human stem cell research is focused on the development of robust protocols for steering them to the desired tissue. However, the undifferentiated state and what causes variations have not been intensively studied. By addressing epigenetic difference between FS cells and EDSCs in livestock, this research aims to overcome this variation barrier and the laborious process of selecting cell lines, which will be directly translatable to human culture, benefiting production of target cells from patient-derived iPS cell lines, which will accelerate clinical translation.