Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage

Abstract

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells.

Figure 1

Population structure of the human ES cell lines analyzed. Principal component (PC) analyses were conducted on the entire final merged data set. PC1 and PC2 are plotted on the y and x axes, respectively. (a) The overall distribution of the human ES cell lines studied compared to the major ethnic groups identified in the HapMap study, the human genome diversity panel (HGDP) and the Pan-Asian SNP Initiative. (b–d) The cell lines were further subdivided to show their relationships to European (b), East Asian and Indian (c) and Middle East-European–Central South Asian populations (c).

Figure 1

Population structure of the human ES cell lines analyzed. Principal component (PC) analyses were conducted on the entire final merged data set. PC1 and PC2 are plotted on the y and x axes, respectively. (a) The overall distribution of the human ES cell lines studied compared to the major ethnic groups identified in the HapMap study, the human genome diversity panel (HGDP) and the Pan-Asian SNP Initiative. (b–d) The cell lines were further subdivided to show their relationships to European (b), East Asian and Indian (c) and Middle East-European–Central South Asian populations (c).

Figure 1

Population structure of the human ES cell lines analyzed. Principal component (PC) analyses were conducted on the entire final merged data set. PC1 and PC2 are plotted on the y and x axes, respectively. (a) The overall distribution of the human ES cell lines studied compared to the major ethnic groups identified in the HapMap study, the human genome diversity panel (HGDP) and the Pan-Asian SNP Initiative. (b–d) The cell lines were further subdivided to show their relationships to European (b), East Asian and Indian (c) and Middle East-European–Central South Asian populations (c).

Figure 1

Population structure of the human ES cell lines analyzed. Principal component (PC) analyses were conducted on the entire final merged data set. PC1 and PC2 are plotted on the y and x axes, respectively. (a) The overall distribution of the human ES cell lines studied compared to the major ethnic groups identified in the HapMap study, the human genome diversity panel (HGDP) and the Pan-Asian SNP Initiative. (b–d) The cell lines were further subdivided to show their relationships to European (b), East Asian and Indian (c) and Middle East-European–Central South Asian populations (c).

Figure 2

Cytogenetic changes occurring during prolonged passage of human ES cells. (a) Percentage of human ES cell line pairs that exhibited a karyotypic abnormality in either early or late passages, or both. Cell lines were excluded if they were known to be derived from karyotypically abnormal embryos. The ES cell pairs are grouped according to whether the chromosome change was observed at late passage only (normal early, abnormal late), both at early and late passages (abnormal early, abnormal late) or early passage only (abnormal early, normal late) and no chromosomal change (normal early, abnormal late). The percentage of cell lines that have individual gains of chromosomes 1, 12, 17 and 20, gain of chromosomes 1 and 17, or gain of chromosomes 1, 12, 17 and 20 are highlighted. Chromosome changes not involving 1, 12, 17 and 20 are indicated as ‘Other’. The numbers above each bar indicate the total number of lines that fall into the four categories out of the total number of pairs of lines analyzed. *Two cell lines (C02 and CC05) in the ‘abnormal early, abnormal late’ category were known to be derived from karyotypically abnormal embryos (a trisomy 13 and ring chromosome 18). One abnormal cell line (AA06) has been excluded from this figure as only one passage was available for analysis. (b) Proportion of pairs of lines that acquired karyotypic abnormalities over different periods in culture. The pairs of lines are grouped according to ‘Delta’, the difference in estimated population doublings between the early and late passages. Only those lines that had a normal karyotype at the early-passage level were included in the analysis, and of those only 117 pairs could reliably be assigned an estimated population doubling time estimate.

Figure 2

Cytogenetic changes occurring during prolonged passage of human ES cells. (a) Percentage of human ES cell line pairs that exhibited a karyotypic abnormality in either early or late passages, or both. Cell lines were excluded if they were known to be derived from karyotypically abnormal embryos. The ES cell pairs are grouped according to whether the chromosome change was observed at late passage only (normal early, abnormal late), both at early and late passages (abnormal early, abnormal late) or early passage only (abnormal early, normal late) and no chromosomal change (normal early, abnormal late). The percentage of cell lines that have individual gains of chromosomes 1, 12, 17 and 20, gain of chromosomes 1 and 17, or gain of chromosomes 1, 12, 17 and 20 are highlighted. Chromosome changes not involving 1, 12, 17 and 20 are indicated as ‘Other’. The numbers above each bar indicate the total number of lines that fall into the four categories out of the total number of pairs of lines analyzed. *Two cell lines (C02 and CC05) in the ‘abnormal early, abnormal late’ category were known to be derived from karyotypically abnormal embryos (a trisomy 13 and ring chromosome 18). One abnormal cell line (AA06) has been excluded from this figure as only one passage was available for analysis. (b) Proportion of pairs of lines that acquired karyotypic abnormalities over different periods in culture. The pairs of lines are grouped according to ‘Delta’, the difference in estimated population doublings between the early and late passages. Only those lines that had a normal karyotype at the early-passage level were included in the analysis, and of those only 117 pairs could reliably be assigned an estimated population doubling time estimate.

Figure 3

Ideogram demonstrating the chromosome changes found in this study. Each colored bar represents one chromosome change occurrence in one cell line. Chromosome losses and gains are shown to the left and right of the ideogram, respectively, except that those instances where a single chromosome rearrangement results in a gain and a loss the colored bars are shown together for clarity. The cytogenetic changes are color coded: Maroon, loss of a whole chromosome (monopsony); red, loss via a structural chromosome rearrangement (unbalanced translocation or interstitial deletion); dark green, gain of a whole chromosome (trisomy); light green is gain via a structural chromosome rearrangement (unbalanced translocation or interstitial duplication); blue represents the occurrence of an apparently balanced rearrangement the nature of which is labeled. Instances in which a change affected only a single chromosome are denoted by ●, whereas changes associated with complex karyotypes (>5 unrelated chromosome aberrations) are denoted by ★. Two cell lines (C02 and CC05) were known to be derived from karyotypically abnormal embryos and contain a trisomy 13 and ring chromosome 18 respectively. iPS cell lines are excluded from this figure. Based upon these studies the minimal critical chromosomal regions subject to gain in culture adapted human ES cell lines were 1q21-qter, 12p11-pter, 17q21.3-qter and 20q11.2. The minimal regions subject to loss were 10p13-pter, 18q21-qter and 22q13-qter.

Figure 4

Copy number variation occurrence in human ES cell lines during prolonged passage. (a) 20q11.21 gain. The region on chromosome 20 frequently found to experience gain over extended human ES cell culture is indicated by the red boxed region in the chromosome ideogram. Also shown are the B allele frequency and logR ratio plots representing instances of one of the longest and one of the shortest 20q11.21 structural variants. (b) Length representation of all individual occurrences of gains in the 20q11.21 region. Samples from which the structural variant was derived are indicated on the left-hand column. The invariant 5′ region and the variable 3′ positions are indicated. Position of genes outside of the minimal amplicon that show greater than 20 RPKM level of expression in human ES cells are shown (RPKM = number of reads that map per kilobase of exon model per million mapped reads for each gene). (c) Expression, RefSeq gene, and regulation tracks in the minimal amplicon. Positive and negative strand mRNA-Seq data from H1 human ES cells indicating polyA RNA transcripts expressed within the minimal amplicon region (chr20:29,267,954-29,853,264) are shown together with H1 human ES cell ChIP-Seq data of histone modifications considered universal predictors of enhancer and promoter activity. (d) Comparison of expression levels of three genes (HM13, ID1, BCL2L1) contained within the identified minimal 20q11.2 amplicon between early- (normal) and late-passage (20q11.2 CNV carrying) samples. MM01 and FF02 are genetically identical sub-lines from two separate laboratories, MM01 has no amplification at 20q11.2, whereas FF02 possesses a copy number change at 20q11.2 that includes the identified minimal amplicon (b).

Figure 5

Cumulative distribution function of methylation changes in human ES cells in this study. The change in DNA methylation is represented by empirical CDF curves of the absolute difference in DNA methylation between early- and late-passage cell-line pairs for all 1,536 analyzed probes. The black curves denote genetically stable lines; the red curves denote genetically unstable lines. All analyzed lines were divided into quartiles based on the passage-number difference between the early and late member of each pair. The first quartile contains the lines with the lowest difference in passage number between the early and late sample (range 4 to 47), whereas the fourth quartile contains the lines with the highest difference in estimated population doublings (range 210 to 1,482).

Figure 6

Recent pericentric inversion associated with 20q11.21 susceptibility to gain. (a) The ancestral condition of chromosome 20 before a pericentric inversion in the last common ancestor of the gorilla, chimp and human. (b) Structure of human chromosome 20 with the location of the gap indicated in which the proximal end of all 20q11.21 amplicons lie.