In the present study, five hESC lines with normal karyotypes and two lines with abnormal karyotypes have been derived from poor quality blastocysts. Successful derivation of hESC lines from poor quality blastocysts has been previously reported by other authors (Hovatta et al., 2003
; Mitalipova et al., 2003
; Genbacew et al., 2005
; Chen et al., 2005
; Mateizel et al., 2006
; Lerou et al., 2008
): the derivation rate from poor quality blastocysts was <10%. In the present study, out of 42 blastocysts, seven ESC lines have been established with a rate of 16.7%. This higher rate may be attributed to the further culture of Day 5 blastocysts in the blastocyst optimum culture medium, which significantly increased the number of cells in the ICM, thus isolation of ICM became much easier. However, if the rate is calculated from Day 3 embryos, it is very low (2.6%) in the present study. This rate is the same as that reported previously (Chen et al., 2005
; Genbacew et al., 2005
). The low rate is due to poor quality of Day 3 embryos as many of them usually arrest during the subsequent culture due to chromosome abnormalities, such as aneuploidy, mosaicism, haploidy or polyploidy, as these are often found in poor human embryos (Magli et al., 2007
; Munne et al., 2007
Currently, there are standard culture protocols for human ESC culture and there are many poor quality human embryos being discarded from IVF clinics. Thus, if these embryos can be used correctly, it is possible to establish more and more hESC lines.
There are two possibilities for the origins of cell lines with normal karyotype. One is that it is derived from normal fertilized oocytes, thus all chromosomes are normal in the subsequent culture, and the other is that it is derived from embryos with chromosomal abnormalities, but the cells undergo a self-correction during subsequent culture, thus the chromosomes are normal in the cell lines. In the present study, because we did not examine the embryo's chromosomal constitutions at Day 3 or 5, we do not know if the cell lines with normal karyotype are derived from embryos with normal chromosomes or embryos with chromosomal abnormalities.
In the present study, self-correction was not observed in a hESC line (FY-3PN) that was derived from a triploid embryo. Triploid embryos used for hESC line derivation may be from a polyspermic oocyte or a diploid oocyte plus a fertilized sperm. In the present study, when we further examined STRs in this cell line, we found that it was a homogeneous triploid cell line (the peak area ratio of each STR locus except D7S820 is almost 1:2.) (Fig. G). Therefore, this cell line may result from duplication of the chromosomes in the oocyte. No mosaic in the chromosomal constitutions may indicate that the chromosomes are more stable in the cell lines derived from such triploid embryos than trisomy embryos.
Also, in the unbalanced Robertsonian translocation cell line, the two long arms of 13 chromosomes fused at the centromere and the two short arms were lost. This situation is also different from chromosome separation error in the aneuploid embryo. Thus, it may be difficult to self-correct such an error. From these results, it would appear that self-correction occurs in the ESCs derived from partial chromosomally abnormal embryos, but not in the ESC lines derived from complete triploid embryos or translocation embryos. It is also possible that some cell lines may undergo self-correction, but others may not.
Previous studies indicated that hESC from chromosomally abnormal embryos had all cell markers that hESC should have, and had the ability to differentiate (Heins et al., 2004
; Baharvand et al., 2006
). In the present study, we examined not only cell markers and ability to differentiate, but also other characteristics, such as imprinted genes, DNA methylation and X chromosome inactivation. Similar to previous studies, we did not find any difference in all characteristics in all seven lines, such as AP activity and cell surface markers, SSEA-4, TRA-1-60 and TRA-1-81. Oct-4, which is an important factor for early embryos and undifferentiated cells (Hay et al., 2004
: Lee et al., 2006
), was present in all cell lines.
Furthermore, all of these hESC lines are pluripotent. When the cells were injected into immunosuppressed mice to examine the formation of teratomas, we found that they formed cells of all three germ layers including mesoderm (cartilage), ectoderm (epithelium) and endoderm (muscle cells). Also when these cells were spontaneously differentiated in vitro, EBs formed muscular cells, nerve cells and other types of cells.
In order to practically use the established hESCs, the characteristics of each cell line should be clarified. Therefore, we developed a comprehensive database of DNA profiles for each cell line based on STR loci and HLA typing. The exploitation of STR elements in the genome is important in the field of genetic mapping, linkage analysis and human identity testing. STR loci have become the standard for identifying hESC lines (Plaia et al., 2006
). STR analysis is also useful in confirming and clarifying some of the anomalies. For example, STR map of FY-3PN showed different peak mapping when compared with others. In order to provide more major histocompatibility complex matched cell lines, HLA typing would be critical for stem cell-based therapies. HLA is a family of cell proteins found on the surface of white blood cells and other nucleated cells in the body. These proteins vary from person to person and are critical for the activation of immune responses. HLA-matched transplantation will minimize the possibility of rejection. Our results from seven cell lines revealed that all of these cells were heterozygous HLA genotype.
Epigenetic stability has profound implications for the use of hESCs in regenerative medicine. Genomic imprinting is erased in the primordial germ cells during development and is reestablished during gametogenesis. Aberrant expression of imprinted genes can cause inherited diseases and induce tumors. For example, the imprinting domain on human chromosome 15q11–13 contains a large cluster of imprinted genes, including paternally expressed SNRPN. Improper regulation of imprinted genes in this cluster results in PWS and AS (Glenn et al., 1997
). Loss of imprinting of H19 gene or IGF2 gene, which is normally located at 11p15.5, is related to embryonic cancers, such as BWS [Beckwith–Wiedemann syndrome and Wilm's tumor, Neu-roblastomas and Yolk sac carcinomas (Rainier et al., 1995
)]. In our study, no different expression in H19, IGF2, SNRPN and GNAS was found in these cell lines.
DNA methylation is essential for normal mammalian development (Herman et al., 1996
). It is not clear whether the potential epigenetic changes occur during long-term ESC culture. Thus, in order to reveal epigenetic stability of imprinted genetic regions of SNRPN in undifferentiated hES cells, we examined DNA methylation status via MSP (Zeschnigk et al., 1997
). The SNRPN critical region, such as the maternal allele, is methylated and the paternal allele is not methylated and is transcriptionally active. MSP analysis of these regions demonstrated that all of the hESCs have a normal SNRPN methylation status, indicating that there are no deletions, uniparental disomy or imprinting mutations of SNRPN methylation in these normal and abnormal karyotype hESC lines.
In mice, establishing a stable XX ESC line is not easy due to loss of one X chromosome. The unstable X chrosomosome and DNA methylation have been found in diploid parthenogenetic ESC lines, which results in only one X chromosome (XO genotype) in the cells (Robertson et al., 1983
; Zvetkova et al., 2005
). Failures of X chromosome inactivation in different hESC lines has also been reported (Dhara and Benvenisty, 2004
; Hoffman et al., 2005
). Epigenetic variation between hESC lines may also perturb X chromosome inactivation. In order that female embryos express similar levels of X-linked genes to males, one of the two X chromosomes is inactivated at an early embryonic stage. It has been revealed by genetic studies that X chromosome selection is influenced by the X controlling element (Simmler et al., 1993
). The X chromosome is randomly inactivated in a 50:50 ratio in most females whereas ~10% females have a non-random X chromosome inactivation (Kubota et al., 1999
). Asymptomatic female carriers of X-linked diseases have the preferential selection of the normal non-mutated X chromosome, which causes extremely non-random inactivation, such as X-linked hyper-IgM syndrome (97:3), Pai syndrome (89:11), multiple congential anomalies (4:96) and unbalanced X autosome translocation (91:9) (Kubota et al., 1999
). Therefore, determination of the X-inactivation pattern is important for the detection of carriers of X-linked diseases. Our data showed that all of our XX hESC lines have both active and inactive X chromosomes. Almost extremely non-random X chromosome inactivation patterns (>95:5) were also found in FY-hES-5, FY-hES-8 and FY-3PN. Our data indicate that the patterns of XX hESC with extremely non-random X chromosome inactivation are similar to the patterns of X-linked diseases with skewed X chromosome inactivation. Whether this phenotype means a relationship between XX hESC lines and X-linked disease is unknown, thus further study on the mechanism of non-random X inactivation may be necessary to explain epigenetic states and developmental competence in hESC lines. X chromosome inactivation should be affected in the hESC lines derived from triploidy embryos since there were three X chromosomes. However, we did not find significant differences in X chromosome inactivation in the cell line with XX chromosomes and three X chromosomes. Further studies are necessary to address these issues and hESC lines with abnormal karyotypes are useful materials for these studies.
In conclusion, our results indicate that new hESC lines can be successfully established from poor quality human embryos. All hESC lines established in our laboratory showed all hESC characteristics and could be differentiated into three germ layers, regardless of their karyotype. Through the detailed examination of ESC biological characterizations, gene expression, DNA methylation and X-inactivation, we found that hESC lines with abnormal karyotype are also useful experimental materials for developmental biology and genetic research. Our results also indicate that these ESC lines have potential application in human cell therapy.