Establishment of Homozygote Mutant Human Embryonic Stem Cells by Parthenogenesis

<div><p>We report on the derivation of a diploid 46(XX) human embryonic stem cell (HESC) line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA) from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.</p></div

Whole genome view of Cytoscan SNP Array data.

<p>Genome wide SNP array results obtained from (A) reference DNA from a male with a normal karyotype (46; XY); and (B) SZ-SMA5 HESC line DNA. The X axis represents chromosomes 1–22, X and Y. (I) The Y axis represents the copy number, determined by the log2 ratio (grey dots) on the left side of the graph, and it's smoothed ratio (red line) on the right. The expected copy number is 2 for autosomal chromosomes (log2 of 0 and smooth signal of 2). The log2 ratio and the smooth signal are determined from both the nonpolymorphic copy number probes and the polymorphic SNP probes. (II) The Y-axis corresponds to homozygote calls (AA or BB) and heterozygote calls (AB). Allele peaks of 1, 0, and -1 indicate a copy number of two, while allele peaks of 0.5 and -0.5 indicate a copy number of one. Allele peaks are calculated from SNP probes. The distinction between XY (reference DNA) and XX (SZ-SMA5) cells is clearly illustrated by the difference in X chromosome copy number. In addition, the overall 0.49% inherent heterozygote call error rate in SZ-SMA5 is below even the expected array genotyping error of ~1% (as determined by dividing the number of heterozygous calls by the total number of SNP probes on the array). Therefore, these data indicate that SZ-SMA5 features a completely homozygous diploid genome. (C) Fraction of SNP heterozygote calls in WT male reference and SZ-SMA5 DNA. Chromosomes are indicated in the X axis and the Y axis indicates the fraction of heterozygous SNP calls per total SNP calls on each chromosome.</p

Characterization of SZ-SMA5.

<p>(A) Expression of undifferentiated cell specific markers by immunostaining for <i>OCT4</i> (red nuclear staining, merged onto Hoechst (blue)), for the cell surface marker <i>Tra 1–60</i> (red, merged onto Hoechst (blue)); and (B) for alkaline phosphatase activity (AP). (C) Expression of undifferentiated cell specific markers: <i>OCT4</i>, <i>REX1</i>, <i>NANOG</i> and <i>SOX2</i> in SZ-SMA5 and a wild-type (WT) HESC control by RT-PCR. <i>GAPDH</i> expression served as a loading control. (D) Teratoma sections stained by H&E from SZ-SMA5 demonstrating multi-cellular structures derived from the three different embryonic germ layers. (E) A representative normal 46(XX) karyotype in SZ-SMA5 as identified by Giemsa staining of 20 different spreads of metaphase chromosomes.</p

Methylation levels at <i>H19</i>, <i>SNRPN</i> and <i>MEST</i> imprinted loci.

<p>Bisulfite single colony sequencing was performed on 3 different imprinted regions (<i>H19</i>, SNRPN and <i>MEST</i>) to determine methylation levels in normal (WT) and SZ-SMA5 HESC lines. Each line represents a single DNA molecule. Full circles correspond to methylated CpGs while empty circles represent unmethylated CpGs.</p