Minoru Miyagoshi

Establishment of a tetraploid cell line from mouse H‐1 (ES) cells highly polyploidized with demecolcine

Abstract.  Objective: Establishment of tetraploid ES cells. Materials and methods: Mouse H‐1 (ES) cells were polyploidized by demecolcine and released from the drug. Results: A tetraploid cell line (4nH1 cells) was established from mouse H‐1 (ES) cells (2nH1 cells) highly polyploidized by treatment with demecolcine. Cell cycle parameters of 4nH1 cells were almost the same as those of 2nH1 cells, suggesting that the rate of DNA synthesis was about twice that of the diploid cells. Mode of chromosome number of 4nH1 cells was 76, about twice that of 2nH1 cells. Cell volume of 4nH1 cells was about twice of that of diploid cells, indicating that 4nH1 cells contained about twice as much total intracellular material as 2nH1 cells. Morphology of the 4nH1 cells was flagstone‐like, thus differing from that of the spindle‐shaped 2nH1 cells, suggesting that the transformation had occurred during the diploid–tetraploid transition. 4nH1 cells exhibited alkaline phosphatase activity and formed teratocarcinomas, implying that they would be pluripotent. Conclusion: A pluripotent tetraploid cell line (4nH1 cells) was established.

Establishment of a triploid V79 cell line from tetraploid cells obtained through polyploidization using K‐252a

Abstract. Triploid V79 cells were established from tetraploid cells. Diploid V79 cells were polyploidized by K‐252a, an inhibitor of protein kinases, and then released from the drug for 10 days. At that time, the cell population was a mixture of diploid and tetraploid cells. Triploid cells were obtained through the cloning of tetraploid cells. They had 33 chromosomes (1.5 times the diploid number) and showed a karyotype of three homologueous chromosomes. The duration of the G1, S and G2/M phases was almost the same as for diploid cells. The cell volume of triploid V79 cells was about two times that of the diploid cells. An explanation for the diploid‐tetraploid‐triploid transition is proposed.

Alteration and preservation of cellular characteristics in long‐term culture of tetraploid H‐1 (ES) cells

To examine the alteration in cellular characteristics of polyploid ES cells during long-term culturing, tetraploid H-1 (ES) cells were continuously cultured for 180 days. Cellular DNA content of the tetraploid cells decreased and reached a plateau of 3.3 C, where C represents the complement of haploid chromosomes. The chromosome number also decreased, indicating that the DNA loss was induced by chromosome loss. Cell volume was maintained, suggesting that the DNA loss did not involve cytoplasmic loss. The cell cycle parameters were almost the same during the DNA decay process, indicating that cell cycle progression was independent of the quantity of homologous chromosomes. Hypotetraploid cells showed alkaline phosphatase activity and formed teratocarcinomas in mouse abdomens, suggesting that the pluripotent potential was maintained. Cellular morphology was also retained, suggesting that the gene expression specifying morphological characteristics was conserved. We conclude that these initial cellular characteristics of tetraploid H1 (ES) cells were preserved in long-term culture, irrespective of chromosome loss.

Octaploid Meth-A cells are established from a highly polyploidized cell population

Abstract.  Tetraploid Meth‐A cells were polyploidized by demecolcin, an inhibitor of spindle fibre formation in M phase, and then released from the drug 1, 2, 3 and 4 days after the addition. Octaploid cells were successfully established from cell populations including hexadecaploid cells produced by 2, 3 and 4 days of exposure to demecolcin. One‐day‐treated cells were polyploidized octaploid cells, but they returned to tetraploid cells. All of the octaploid Meth‐A cells showed essentially the same features. The octaploid Meth‐A cells had eight homologous chromosomes and double the DNA content of the parent tetraploid cells. The doubling time of octaploid Meth‐A cells was 30.2 h, somewhat longer than the 28.3 and 24.0 h of tetraploid and diploid cells, respectively. The fractions of cells in the G1, S and G2/M phases were essentially the same in diploid, tetraploid and octaploid Meth‐A cells. The cell volume of octaploid Meth‐A cells was about two times that of the tetraploid cells. It was concluded that octaploid Meth‐A cells were established from transient hexadecaploid cells produced by the polyploidization of tetraploid cells that had been established from diploid cells.

DNA stable pentaploid H1 (ES) cells obtained from an octaploid cell induced from tetraploid cells polyploidized using demecolcine

Pentaploid H1 (ES) cells (5H1 cells) were accidentally obtained through one‐cell cloning of octaploid H1 (ES) cells (8H1 cells) that were established from tetraploid H1 (ES) cells (4H1 cells) polyploidized using demecolcine. The number of chromosomes of 5H1 cells was 100, unlike the 40 of diploid H1 (ES) cells (2H1 cells), 80 of 4H1, and 160 of 8H1 cells. The durations of G1, S, and G2/M phases of 5H1 cells were 3, 7, and 6 h, respectively, almost the same as those of 2H1, 4H1, and 8H1 cells. The cell volume of 5H1 cells was half of that of 8H1 cells, suggesting that 5H1 cells were created through abnormal cell divisions of 8H1 cells. The morphology of growing 5H1 cells was a spherical cluster similar to that of 2H1 cells and differing from the flagstone‐like shape of 4H1 and 8H1 cells. Pentaploid solid tumors were formed from 5H1 cells after interperitoneal injection into the mouse abdomen, and they contained endodermal, mesodermal, and ectodermal cells as well as undifferentiated cells, suggesting both that the DNA content of 5H1 cells was retained during tumor formation and that the 5H1 cells were pluripotent. The DNA content of 5H1 cells was stable in long‐term culturing as 2H1 cells, meaning that 5H1 and 2H1 cells shared similarities in DNA structure. The excellent stability of the DNA content of 5H1 cells was explained using a hypothesis for the DNA structure of polyploid cells because the pairing of homologous chromosomes in 5H1 cells is spatially forbidden. J. Cell. Physiol. 223: 369–375, 2010.

Cell cycle, morphology and pluripotency of octaploid embryonic stem cells in comparison with those of tetraploid and diploid cells

To examine the alteration of cellular characteristics on ploidy transition of embryonic stem (ES) cells, octaploid cells (8H1 cells) were established from tetraploid H-1 (ES) cells, and compared with tetraploid and diploid H-1 (ES) cells (4H1 and 2H1 cells, respectively). The duration of G1, S, and G2/M phases were essentially the same among 2H1, 4H1, and 8H1 cells, suggesting that cell cycle progression is conserved. The ratio of cell volume of 2H1, 4H1, and 8H1 cells was about 1 : 2 : 4, indicating that these polyploid cells were generated through cell cycle progression without cell division. The morphology of 8H1 cells was flagstone-like and flatter than that of 4H1 cells, and differed from the spindle-like shape of 2H1 cells, suggesting that transformation occurred during the ploidy transitions. Alkaline phosphatase activity was expressed equivalently in 2H1, 4H1, and 8H1 cells, and solid tumors that contained endodermal, mesodermal, and ectodermal cells were formed by 2H1, 4H1 or 8H1 cells after interperitoneal injection into the mouse abdomen, suggesting that pluripotency was preserved in the ploidy transition.

The reversion to diploid cells from established triploid V79 cells.

Abstract.  The triploid V79 cells are stable under usual culture conditions, and do not revert to being diploid. Here, the triploid–diploid transition of triploid V79 cells has been successfully induced in suspension culture in culture dishes with untreated surfaces. The diploid cells began to appear in a population of triploid V79 cells cultured under these conditions for 4 weeks. All of the triploid cells were transformed to diploid through subsequent monolayer culture for 5 weeks. It was confirmed that the revertant diploid cells had the same characteristics as original diploid V79 cells, with respect to DNA histograms, cell volume and chromosome number. Thus, it seems that suspension culturing is an important factor that induces the triploid–diploid transition.

DNA-unstable decaploid mouse H1 (ES) cells established from DNA-stable pentaploid H1 (ES) cells polyploidized using demecolcine

Objectives:  DNA content of diploid H1 (ES) cells (2H1 cells) has been shown to be stable in long‐term culture; however, tetraploid and octaploid H1 (ES) cells (4H1 and 8H1 cells, respectively) were DNA‐unstable. Pentaploid H1 (ES) cells (5H1 cells) established recently have been found to be DNA‐stable; how, then is cell DNA stability determined? To discuss ploidy stability, decaploid H1 (ES) cells (10H1 cells) were established from 5H1 cells and examined for DNA stability.

Pluripotency of a polyploid H1 (ES) cell system without leukaemia inhibitory factor.

Objectives:  Tetraploid cells are strictly biologically inhibited from composition of embryos; by the same token, only diploid cells compose embryos. However, the distinction between diploid and tetraploid cells in development has not been well explained. To examine pluripotency of polyploid ES cells, a polyploid embryonic stem (ES)‐cell system was prepared.

Different responses between diploid and tetraploid H1 embryonic stem cells appeared during long‐term culturing in L15F10 medium without leukemia inhibitory factor

To examine the alteration in cellular characteristics of polyploid embryonic stem (ES) cells during long-term culturing without leukemia inhibitory factor (LIF), mouse diploid and tetraploid H-1 (ES) cells (2H1 and 4H1 cells, respectively) were cultured without LIF for approximately 5 months. 2H1 and 4H1 cells were adapted to the medium without LIF by decreasing the concentration for several passages, and they were denoted as 2H1(−) and 4H1(−) cells, respectively. DNA content of 4H1(−) cells decreased gradually in the early stage, increased abruptly in the second stage, and then was maintained for a long time. 4H1(−) cells exhibited longer doubling time and equivalent phase fraction compared with those of 2H1(−) cells. The G1 phase fractions of 2H1(−) and 4H1(−) cells were increased compared with that of 2H1 cells. Cellular morphology and pluripotency were maintained in 4H1(−) cells but not in 2H1(−) cells. 2H1(−) cells showed a cell population consisting of several kinds of cells, and they lost alkaline phosphatase activity, suggesting that the cells had differentiated. 4H1(−) cells, however, exhibited alkaline phosphatase activity and formed teratocarcinoma in mouse abdomen, suggesting that the cells maintained their pluripotency in the medium without LIF.