In order to apply human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) to regenerative medicine, the cells should be produced under restricted conditions conforming to GMP guidelines. Since the conventional culture system has some issues that need to be addressed to achieve this goal, we developed a novel culture system. We found that recombinant laminin-511 E8 fragments are useful matrices for maintaining hESCs and hiPSCs when used in combination with a completely xeno-free (Xf) medium, StemFit™. Using this system, hESCs and hiPSCs can be easily and stably passaged by dissociating the cells into single cells for long periods, without any karyotype abnormalities. Human iPSCs could be generated under feeder-free (Ff) and Xf culture systems from human primary fibroblasts and blood cells, and they possessed differentiation abilities. These results indicate that hiPSCs can be generated and maintained under this novel Ff and Xf culture system.

Mammalian cloning has been accomplished in several mammalian species by nuclear transfer. However, the production rate of cloned animals is quite low, and many cloned offspring die or show abnormal symptoms. A possible cause of the low success rate of cloning and abnormal symptoms in many cloned animals is the incomplete reestablishment of DNA methylation after nuclear transfer. We first analyzed tissue-specific methylation patterns in the placenta, skin, and kidney of normal B6D2F1 mice. There were seven spots/CpG islands (0.5% of the total CpG islands detected) methylated differently in the three different tissues examined. In the placenta and skin of two cloned fetuses, a total of four CpG islands were aberrantly methylated or unmethylated. Interestingly, three of these four loci corresponded to the tissue-specific loci in the normal control fetuses. The extent of aberrant methylation of genomic DNA varied between the cloned animals. In cloned animals, aberrant methylation occurred mainly at tissue-specific methylated loci. Individual cloned animals have different methylation aberrations. In other words, cloned animals are by no means perfect copies of the original animals as far as the methylation status of genomic DNA is concerned.

ABSTRACT

DNA methylation controls various developmental processes by silencing, switching and stabilizing genes as well as remodeling chromatin. Among various symptoms in cloned animals, placental hypertrophy is commonly observed. We identified the Spalt-like gene3 ( Sall3 ) locus as a hypermethylated region in the placental genome of cloned mice. The Sall3 locus has a CpG island containing a tissue-dependent differentially methylated region (T-DMR) specific to the trophoblast cell lineage. The T-DMR sequence is also conserved in the human genome at the SALL3 locus of chromosome 18q23, which has been suggested to be involved in the 18q deletion syndrome. Intriguingly, larger placentas were more heavily methylated at the Sall3 locus in cloned mice. This epigenetic error was found in all cloned mice examined regardless of sex, mouse strain and the type of donor cells. In contrast, the placentas of in vitro fertilized (IVF) and intracytoplasmic sperm injected (ICSI) mice did not show such hypermethylation, suggesting that aberrant hypermethylation at the Sall3 locus is associated with abnormal placental development caused by nuclear transfer of somatic cells. We concluded that the Sall3 locus is the area with frequent epigenetic errors in cloned mice. These data suggest that there exists at least genetic locus that is highly susceptible to epigenetic error caused by nuclear transfer.

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Cloned animals have various health problems. Aberrant DNA methylation is a possible cause of the problems. Restriction landmark genomic scanning (RLGS) that enabled us to analyze more than 1,000 CpG islands simultaneously demonstrated that all cloned newborns had aberrant DNA methylation. To study whether this aberration persists throughout the life of cloned individuals, we examined genome-wide DNA methylation status of newborn (19.5 dpc, n=2), adult (8-11 months old, n=3), and aged (23-27 months old, n=4) cloned mice using kidney cells as representatives. In the adult and aged groups, cloning was repeated using cumulus cells of the adult founder clone of each group as nucleus donor. Two newborn clones had three with aberrantly methylated loci, which is consistent with previous reports that all cloned newborns had DNA methylation aberrations. Interestingly, we could detect only one aberrantly methylated locus in two of the three adult clones in mid-age and none of four senescent clones, indicating that errors in DNA methylation disappear with advancement of animals' aging.