Human ESCs predisposition to karyotypic instability: Is a matter of culture adaptation or differential vulnerability among hESC lines due to inherent properties?

Abstract

Background: The use of human embryonic stem cells (hESCs) in research is increasing and hESCs hold the promise for many biological, clinical and toxicological studies. Human ESCs are expected to be chromosomally stable since karyotypic changes represent a pitfall for potential future applications. Recently, several studies have analysed the genomic stability of several hESC lines maintained after prolonged in vitro culture but controversial data has been reported. Here, we prompted to compare the chromosomal stability of three hESC lines maintained in the same laboratory using identical culture conditions and passaging methods.

Results: Molecular cytogenetic analyses performed in three different hESC lines maintained in parallel in identical culture conditions revealed significant differences among them in regard to their chromosomal integrity. In feeders, the HS181, SHEF-1 and SHEF-3 hESC lines were chromosomally stable up to 185 passages using either mechanical or enzymatic dissection methods. Despite the three hESC lines were maintained under identical conditions, each hESC line behaved differently upon being transferred to a feeder-free culture system. The two younger hESC lines, HS181 (71 passages) and SHEF-3 (51 passages) became chromosomally unstable shortly after being cultured in feeder-free conditions. The HS181 line gained a chromosome 12 by passage 17 and a marker by passage 21, characterized as a gain of chromosome 20 by SKY. Importantly, the mosaicism for trisomy 12 gradually increased up to 89% by passage 30, suggesting that this karyotypic abnormality provides a selective advantage. Similarly, the SHEF-3 line also acquired a trisomy of chromosome 14 as early as passage 10. However, this karyotypic aberration did not confer selective advantage to the genetically abnormal cells within the bulk culture and the level of mosaicism for the trisomy 14 remained overtime between 15%-36%. Strikingly, however, a much older hESC line, SHEF-1, which was maintained for 185 passages in feeders did not undergo any numerical or structural chromosomal change after 30 passages in feeder-free culture and over 215 passages in total.

Conclusion: These results support the concept that feeder-free conditions may partially contribute to hESC chromosomal changes but also confirm the hypothesis that regardless of the culture conditions, culture duration or splitting methods, some hESC lines are inherently more prone than others to karyotypic instability.

Figure 1

Molecular cytogenetic analysis of HS181 hESC line. HS181 hESC line was genetically stable for 71 passages in feeders. Upon being transferred to feeder-free conditions and split using enzymatic methods, the HS181 hESC line gained a chromosome 12 as early as passage 17 and an additional marker, shown by SKY to be an extra chromosome 20 by passage 21. The level of mosaicism gradually increased from 26% (p17) to 89% (p30). In conventional karyotyping analysis (A) and CGH (C), the blue arrows indicate chromosomal gains. In the SKY images (B), the white arrows confirm the gain of chromosome 12 and characterized the marker seen by G-banding as a partial gain of chromosome 20.

Figure 2

Molecular cytogenetic analysis of SHEF-3 hESC line. SHEF-3 hESC line was genetically stable for 51 passages in feeders. Upon being transferred to feeder-free conditions and passaged using enzymatic methods, it quickly gained a trisomy of chromosome 14 as early as in p10. Opposite to the HS181 line, the degree of mosaicism for the extra chromosome 14 did not increase overtime in feeder-free conditions and remained between 23%–36% over the culture (see Table 1). In conventional karyotyping analysis (A), the blue arrows indicate chromosomal gains. B,C: SKY and CGH analyses showing the absence of further structural or numerical abnormalities.

Figure 3

Molecular cytogenetic analysis of SHEF-1 hESC line. In contrast to HS181 and SHEF-3 hESC lines, the SHEF-1 hESC line was genetically stable after as many as 185 passages in feeders. Upon being transferred to feeder-free conditions, the SHEF-1 hESC line displayed no karyotypic changes assessed by G-banding (A), SKY (B) and CGH (C) after further 30 passages in feeder-free conditions despite being split by means of enzymatic methods throughout over 215 passages (almost 4 years in in vitro culture). This data suggests that although culture conditions may partially contribute to chromosome stability, some hESC lines are inherently more predisposed than others to karyotypic changes.