４倍体iPS細胞の樹立とその心筋細胞分化への応用についての成果を発表 ／ Establishment of Tetraploid iPS Cells and Their Application to Cardiomyocyte Differentiation, Published

　私達の身体を構成する細胞のほとんどは、各染色体を両親から１本ずつ受け継いだ２倍体ですが、心筋細胞や肝細胞などの一部の細胞は、成熟するにつれて染色体数を倍加させることが知られています。人間では思春期を過ぎた頃に、心筋細胞の80%以上が各染色体を４本ずつ持つ４倍体になっていますし、肝細胞では８倍体や１６倍体細胞も存在します。心筋細胞も肝細胞もiPS細胞から分化させて医療に応用されることが期待されていますが、これまでこの倍数性にはほとんど注意が払われてきませんでした。iPS細胞から分化させた細胞は、一般的にどの細胞種でも未熟で胎児期の状態に近いと考えられていますが、心筋細胞もまたほとんどが２倍体に留まり、４倍体化はほとんど起きていませんでした。

　そこで私達は、発想を完全に逆転させました。２倍体iPS細胞から分化させた心筋細胞を４倍体へと成熟させるのではなく、iPS細胞自体を４倍体にしてから心筋細胞に分化させれば４倍体心筋細胞が得られるはずだと考えたのです。同時に、これまで多倍体化は、心筋細胞や肝細胞が成熟した原因なのか結果なのかは不明でした。４倍体iPS細胞由来心筋細胞と２倍体iPS細胞由来心筋細胞を比較すれば、４倍体化によって成熟が促されるかも検証できると考えたのです。

　考え方はシンプルで、通常の２倍体iPS細胞同士を融合させれれば、４倍体になるだろうと考えました。実際に樹立に成功しました。さらに、心筋細胞に分化させて、通常の２倍体iPS細胞由来心筋細胞と比較したところ、ミトコンドリアの量や機能の向上、細胞周期関連遺伝子の発現低下、心収縮の強度と速度の向上など、４倍体化により成熟した特徴が認められました。これらの結果から、４倍体iPS細胞を用いることで、これまで得ることができなかった、増殖期を終えた状態に近い４倍体心筋細胞を得られるようになりました。また、４倍体化は成熟の原因である、ということも示唆されました。今年卒業した中島さんが主導してくれました。

　私は大学院生から助教になった頃に、マウスで肝再生の研究をしていました。その頃から、肝細胞には多様な倍数性を持った集団がいることを不思議に思っていました。また、肝臓を切除して、そこから再生する際に、肝細胞がその倍数性によって異なる反応性を示し、再生前後で肝臓全体の倍数性は増加するということも知られており、それも不思議だとずっと思っておりました。さらに遡って、学部の卒業研究時代には分裂酵母を使って研究をしており、その際に興味のある遺伝型を持つ酵母を、接合型などと関係なく細胞壁を酵素で消化して物理的に半ば無理やり融合し、目的の株を得られることが強く印象に残っていました。iPS細胞を使った疾患モデルなどの研究を経験して、また倍数性という観点に舞い戻り、さらには学部生の頃の経験から細胞融合を思いついたので、勝手に集大成的な研究内容だったと思っています。

　ただ本当のことを言うと、細胞融合をしたのは細胞の成熟を促すためだけではありませんでした。もっとやりたいことがあります。いずれはそれらも論文にしたいです。

Most of the cells that make up our bodies are diploid, having inherited one copy of each chromosome from each parent; however, it is known that certain cells, such as cardiomyocytes and hepatocytes, double their chromosome number as they mature. In humans, by the time puberty is over, more than 80% of cardiomyocytes have become tetraploid, possessing four copies of each chromosome, and hepatocytes can become even octaploid and hexaploid cells. Although both cardiomyocytes and hepatocytes are expected to be differentiated from iPS cells for medical applications, little attention has been paid to this ploidy. Cells differentiated from iPS cells are generally considered to be immature and close to a fetal state, regardless of cell type; consequently, most cardiomyocytes remained diploid, and tetraploidy rarely occurred.

　Therefore, we completely reversed our approach. Rather than maturing cardiomyocytes differentiated from diploid iPS cells into tetraploid cells, we reasoned that if we first made the iPS cells themselves tetraploid and then differentiated them into cardiomyocytes, we should be able to obtain tetraploid cardiomyocytes. At the same time, it had previously been unclear whether polyploidization was the cause or the result of the maturation of cardiomyocytes and hepatocytes. We reasoned that by comparing cardiomyocytes derived from tetraploid iPS cells with those derived from diploid iPS cells, we could verify whether tetraploidy promotes maturation.

　The concept was simple: we reasoned that fusing two diploid iPS cells would result in a tetraploid cell. We successfully established such a cell line. Furthermore, when we differentiated these cells into cardiomyocytes and compared them with cardiomyocytes derived from standard diploid iPS cells, we observed characteristics indicative of maturation due to tetraploidy, including increased mitochondrial quantity and function, reduced expression of cell cycle-related genes, and improved strength and speed of cardiac contraction. Based on these results, the use of tetraploid iPS cells has made it possible to obtain tetraploid cardiomyocytes that are close to the state of having completed their proliferative phase—a state that had previously been unattainable. Furthermore, the findings suggest that tetraploidy itself is the cause of this maturation. Ittetsu Nakajima, who graduated this year, led this work.

　When I transitioned from being a graduate student to a staff scientist, I was conducting research on liver regeneration in mice. Even back then, I found it intriguing that liver cells comprised populations with diverse ploidy levels. It is also known that when the liver is resected and regenerates, hepatocytes exhibit different responses depending on their ploidy, and the overall ploidy of the liver increases after regeneration—a fact I have long found puzzling as well. Going even further back, during my undergraduate thesis research, I worked with fission yeast. I was deeply impressed by the fact that I could obtain the desired strain by physically—almost forcibly—fusing yeast cells with the genotype I was interested in by enzymatically digesting their cell walls, regardless of their mating type. After gaining experience with research such as disease models using iPS cells, I returned to the concept of ploidy, and my undergraduate experience led me to the idea of cell fusion—so I personally consider this research to be a culmination of my work.

To be honest, however, I didn’t perform cell fusion solely to promote cell maturation. There are other things I want to explore. Eventually, I hope to publish papers on those as well.