Laboratories and faculty

Organ Developmental Engineering

Assoc Prof. Isotani
Associate Professor
ISOTANI Ayako
Assistant Professor
YURI Shunsuke
Labs HP
https://bsw3.naist.jp/isotani/index-en.html

Outline of Research and Education

In mammals, until the eight-cell embryo stage, fertilized eggs have totipotency, meaning that each cell can differentiate into all kinds of cell. In blastocyst-stage embryos just before implantation, the cells’ fates are divided into the trophectoderm (TE), which will develop into placental tissue, and the inner cell mass (ICM), which has pluripotency in that its cells will develop into three germ layers, including germline cells. Embryonic stem cells (ESCs) were established from ICM, promoting the study of regenerative medicine, and led to the discovery of induced pluripotent stem cells (iPSCs). We combine these early embryos, ESCs/iPSCs, and developmental technology with the aim of performing basic studies that will lead to regenerative medicine using animal models.

Major Research Topics

Model of organ formation using xenogeneic chimeras

Xenogeneic chimeras containing both mouse and rat cells were generated using blastocysts and ESCs (Fig. 1). When we injected rat ES cells into blastocysts of nu/nu mice lacking a thymus, we could produce a rat thymus in chimeric animals. This indicates the formation of an organ from ES cells in xenogeneic conditions. Although this rat thymus could educate T-cells (Fig 2, REF5), it was smaller than that of a mouse, and the functions of the educated T-cells were unclear. On the other hand, we could detect rat spermatozoa in mouse←rat ES chimeric testes. Rat pups were generated from rat spermatozoa in the xenogeneic chimeric testis by intracytoplasmic injections, and the normal germline potential of rat spermatozoa in the xenogeneic chimeric testes was demonstrated. Findings of the functions of organs, tissues, and cells developed in xenogeneic chimeras are valuable for future translational research.

Trials of novel animal models

Gene knockout animals can easily be generated using genome editing systems such as the CRISPR/Cas9 system. Using the combination of this system and ESCs,complicated gene modification can be performed. However, ESC has no potency to differentiate into the placental lineage. The tetraploid embryo, which is produced by fusing two blastomeres at the 2-cell stage, can contribute placental lineage only, not fetus tissues. In the tetraploid complementation method, whose way is gene-modified ESCs injected into the tetraploid embryo, the fetus or pups are derived from ESCs only, and then it is possible to be F0 analysis (Fig. 3, REF 1 and 2).
We aim to produce novel animal models using these technologies.

Fig. 1
Fig. 1 Two kinds of mouse and rat xenogeneic chimeras
A rat-sized xenogeneic chimera which produced mouse ES cells injected into rat blastocysts (upper). A mouse-sized xenogeneic chimera which produced rat ES cells injected into mouse blastocysts (bottom).
Fig. 2
Fig. 2 The function of rat thymus in xenogeneic chimera
When rat thymus from a xenogeneic chimera was transplanted into renal subcutaneous tissues of nu/nu rat, rat T-cells were educated.
Fig. 3
Fig. 3 F0 analysis using the tetraploid complementation method.
When the gene-modified ESCs are injected into the tetraploid embryo, fetus or pups consist of ESC-derived cells only. We can directly analyze F0 fetuses or pups, not through the next generation.

References

  1. Hirata et al., Exp Anim 71, 82-89, 2022
  2. Kishimoto et al., Sci Rep 11, 8297, 2021
  3. Isotani et al., Biol Reprod 97, 61-68, 2017
  4. Isotani et al. Sci Rep 6, 24215, 2016
  5. Isotani et al. Genes Cells 16, 397-405, 2011
  6. Isotani et al. Proc Natl Acad Sci U S A 102, 4039-4044, 2005