Ultra-Deep Bisulfite Sequencing to Detect Specific DNA Methylation Patterns of Minor Cell Types in Heterogeneous Cell Populations: An Example of the Pituitary Tissue

<div><p>DNA methylation is an epigenetic modification important for cell fate determination and cell type-specific gene expression. Transcriptional regulatory regions of the mammalian genome contain a large number of tissue/cell type-dependent differentially methylated regions (T-DMRs) with DNA methylation patterns crucial for transcription of the corresponding genes. In general, tissues consist of multiple cell types in various proportions, making it difficult to detect T-DMRs of minor cell types in tissues. The present study attempts to detect T-DMRs of minor cell types in tissues by ultra-deep bisulfite sequencing of cell type-restricted genes and to assume proportions of minor cell types based on DNA methylation patterns of sequenced reads. For this purpose, we focused on transcriptionally active hypomethylated alleles (Hypo-alleles), which can be recognized by the high ratio of unmethylated CpGs in each sequenced read (allele). The pituitary gland contains multiple cell types including five hormone-expressing cell types and stem/progenitor cells, each of which is a minor cell type in the pituitary tissue. By ultra-deep sequencing of more than 100 reads for detection of Hypo-alleles in pituitary cell type-specific genes, we identified T-DMRs specific to hormone-expressing cells and stem/progenitor cells and used them to estimate the proportions of each cell type based on the Hypo-allele ratio in pituitary tissue. Therefore, introduction of the novel Hypo-allele concept enabled us to detect T-DMRs of minor cell types with estimation of their proportions in the tissue by ultra-deep bisulfite sequencing.</p></div

Bisulfite analyses of relatively small numbers of clones (reads) for detection of Hypo-alleles by Sanger and MiSeq sequencing.

<p>Cell type-restricted genes (<i>Gh1</i>, <i>Prl</i>, and <i>Tbx19</i>) were analyzed by Sanger sequencing of 15–20 clones. Black and white circles indicate methylated and unmethylated CpGs, respectively. (B) Successive 20 reads were grouped as a trial and analyzed, and five trials were performed from the first read of MiSeq raw data of the <i>Gh1</i>, <i>Prl</i>, and <i>Tbx19</i> T-DMRs to mimic the conventional bisulfite sequencing analyses of 20 clones (reads) five times each. The Hypo-allele ratio of the <i>Gh1</i> T-DMR from the 3,304 total reads was 12.8%, whereas those of five groups of 20 reads were between 0–30% (average: 13.0%, SD: 11.0%). Similarly, the Hypo-allele ratios of the <i>Prl</i> and <i>Tbx19</i> T-DMRs from the 1,107 and 820 total reads were 22.7% and 23.2%, respectively, whereas those of five trials of 20 reads were between 15–30% (average: 22.0%, SD: 7.0%) for the <i>Prl</i> T-DMR and 10–35% (average: 27.0%, SD: 10.0%) for the <i>Tbx19</i> T-DMR. Methylated and unmethylated CpGs are shown as black and white bars, respectively. Asterisks indicate Hypo-alleles.</p

Validation of hypomethylated alleles (Hypo-alleles) by MiSeq ultra-deep sequencing.

<p>≥75% unmethylated CpG sites (3 or 4 out of the 4 CpGs shown) are defined as Hypo-alleles. Among the five reads, reads 1 and 2 are Hypo-alleles, and the Hypo-allele ratio is 40% (2 reads/5 reads). Left panel: conventional CpG methylation analysis data; white and black circles indicate unmethylated and methylated CpGs, respectively. Right panel: MiSeq ultra-deep bisulfite analysis; white and black bars indicate unmethylated and methylated CpGs, respectively. (B) Validation of Hypo-allele ratio analysis by MiSeq ultra-deep bisulfite sequencing at the <i>Sall4</i> T-DMR using mixtures of genomic DNAs from PFF and iPSC. Sequenced reads above the dotted lines are Hypo-alleles. White and black bars indicate unmethylated and methylated CpGs, respectively. Mixtures of genomic DNAs of PFF and iPSC (0:100, 25:75, 50:50, 75:25, or 100:0) would exhibit the expected respective Hypo-allele ratios (100%, 75%, 50%, 25%, or 0%) for the <i>Sall4</i> T-DMR. (C) Hypo-allele ratios of the <i>Sall4</i> T-DMR analyzed by ultra-deep sequencing. Hypo-allele ratios were calculated from the PFF/iPSC mixtures (expected Hypo-allele ratios of 100%, 75%, 50%, 25%, and 0%) shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146498#pone.0146498.g001" target="_blank">Fig 1B</a>. The Hypo-allele ratios from three independent experiments are shown as mean ± SE (n = 3). (D) Accuracy of detecting Hypo-allele ratio (10%) in relation to sequenced read numbers based on the MiSeq data at the <i>Sall4</i> T-DMR. For this analysis, genomic DNAs of PFF and iPSC were mixed at 90:10 for a Hypo-allele ratio of 10%. From two independent MiSeq analyses, 1,109 reads (Exp. 1) and 1,447 reads (Exp. 2) were obtained. Hypo-allele ratios of each trial are plotted (filled circles, n = 5), and mean ± SD for five trials each for 10, 20, 50, 100, or 200 reads are plotted as triangles with lines. (E) Examination of detection accuracy of Hypo-allele (10% or 50%) by conventional analysis of small numbers of sequencing reads based on the MiSeq data at the <i>Sall4</i> T-DMR. Genomic DNAs of PFF and iPSC were mixed at 90:10 or 50:50 for samples exhibiting Hypo-allele ratios of 10% or 50%, respectively. From the raw data, 20 successive reads were grouped from the first through 100th read. The Hypo-allele ratios of 20-read groups were calculated. Asterisks indicate Hypo-alleles. White and black bars indicate unmethylated CpGs and methylated CpGs, respectively.</p

Comparison of DNA methylation profiles of pituitary-related genes in porcine tissues by ultra-deep bisulfite sequencing between the conventional DNA methylation analysis and a novel Hypo-allele ratio analysis.

<p>Hypo-allele ratios of 37 pituitary-related genes were analyzed in pituitary (#1 and #2), liver, brain, and PFF samples using a MiSeq sequencer. The Hypo-allele data for each tissue are shown as a heatmap after hierarchical clustering based on Euclidean distance (left panel). Using the same bisulfite sequencing data, the conventional DNA methylation degrees calculated by methyl-CpGs/total CpGs are also shown as a heatmap with hierarchical clustering (right panel). ND, No data.</p

Schematic diagram of predicted proportions of pituitary cell types in adult porcine pituitary tissue.

<p>Proportions of several pituitary cell types were estimated based on the Hypo-allele ratios of the pituitary cell type-restricted genes (white).</p

Detection of Hypo-alleles of endogenous imprinted genes in porcine pituitary and liver.

<p>Bisulfite ultra-deep sequencing was performed for differentially methylated regions of imprinted <i>Meg3</i> and <i>Peg10</i> genes, and their Hypo-allele ratios were calculated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146498#pone.0146498.g001" target="_blank">Fig 1</a>. Sequenced reads above the dotted lines are Hypo-alleles. White and black bars indicate unmethylated and methylated CpGs, respectively.</p

MHC-Matched Induced Pluripotent Stem Cells Can Attenuate Cellular and Humoral Immune Responses but Are Still Susceptible to Innate Immunity in Pigs

<div><p>Recent studies have revealed negligible immunogenicity of induced pluripotent stem (iPS) cells in syngeneic mice and in autologous monkeys. Therefore, human iPS cells would not elicit immune responses in the autologous setting. However, given that human leukocyte antigen (HLA)-matched allogeneic iPS cells would likely be used for medical applications, a more faithful model system is needed to reflect HLA-matched allogeneic settings. Here we examined whether iPS cells induce immune responses in the swine leukocyte antigen (SLA)-matched setting. iPS cells were generated from the SLA-defined C1 strain of Clawn miniature swine, which were confirmed to develop teratomas in mice, and transplanted into the testes (<i>n</i> = 4) and ovary (<i>n</i> = 1) of C1 pigs. No teratomas were found in pigs on 47 to 125 days after transplantation. A Mixed lymphocyte reaction revealed that T-cell responses to the transplanted MHC-matched (C1) iPS cells were significantly lower compared to allogeneic cells. The humoral immune responses were also attenuated in the C1-to-C1 setting. More importantly, even MHC-matched iPS cells were susceptible to innate immunity, NK cells and serum complement. iPS cells lacked the expression of SLA class I and sialic acids. The in vitro cytotoxic assay showed that C1 iPS cells were targeted by NK cells and serum complement of C1. In vivo, the C1 iPS cells developed larger teratomas in NK-deficient NOG (T-B-NK-) mice (<i>n</i> = 10) than in NK-competent NOD/SCID (T-B-NK+) mice (<i>n</i> = 8) (<i>p</i><0.01). In addition, C1 iPS cell failed to form teratomas after incubation with the porcine complement-active serum. Taken together, MHC-matched iPS cells can attenuate cellular and humoral immune responses, but still susceptible to innate immunity in pigs.</p></div

Complement-mediated cytotoxicity to C1 iPS cells.

<p>(A) Cytochemical assay for sialic acids using lectins. The expression of sialic acids was examined with SNA lectin and MAL lectin, both of which specifically bind to sialic acids. C1 iPS cells exhibited very low levels of sialic acids compared to fibroblasts. C1 iPS cells, but not PEFs, expressed Oct3/4. (B) Cell injury by complement was assessed by measuring LDH released in the supernatants described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098319#s2" target="_blank">Materials and Methods</a>. Triton-X (2%) was used as a positive control, and 30% of heat-inactivated (complement-inactivated) serum was used as a negative control. The percent cytotoxicity was indicated as average values of triplicate (^*<i>p</i><0.01, ^**<i>p</i><0.05). Three independent experiments were conducted and similar results were obtained.</p

Cellular and humoral immune responses in an SLA-matched setting.

<p>(A) Mixed lymphocyte reactions (MLRs) against allogeneic (C2) porcine embryonic fibroblasts (PEFs), C1 iPS cells and STO feeder cells. Peripheral blood mononuclear cells (PBMCs) supplemented with concanavalin A (Con A) and C2 PEFs were used as positive controls, and autologous PBMCs (C1) were used as a negative control. The stimulation index of C1 iPS cells was significantly lower than that of allogeneic cells (^*<i>p</i><0.01), but significantly higher than that of autologous cells (^*<i>p</i><0.01). MLR was performed in triplicate and repeated three times and a typical result was shown. (B) Immunohistochemical staining with anti-CD3 and anti-CD79 antibodies. Pig spleen was stained with the anti-CD3 antibody as a positive control. Slight infiltration of CD3+ T-cells and CD79+ B cells was detected at the transplantation site in the SLA-matched pig CT19. (C) Porcine IgG antibodies against C1 iPS cells were determined by flow cytometry. As SLA-mismatched recipients, miniature pigs that were not C1 or C2 were used. The porcine IgG against C1 iPS cells was detected at much lower levels in the SLA-matched C1 pigs (CT19 and CU65) than in the SLA-mismatched allogeneic pigs. Cells labeled with secondary antibody without porcine serum as a negative control.</p

Susceptibility of C1 iPS cells to NK cells.

<p>(A) Immunocytochemical staining with an anti-porcine SLA class I antibody. Porcine PEFs showed positive staining for SLA class I, but C1 iPS cells were negative. (B) Reverse-transcription polymerase chain reaction (RT-PCR) analysis of the expression of <i>SLA class I</i> and ligands for NK cells. Lane 1, PEF; 2, C1 iPS cells; 3, STO feeder cells; 4, no reverse transcriptase; and 5, RT-PCR buffer alone. <i>MICA</i> and <i>ULBP1</i>, ligands for NK cells, were expressed on C1 iPS cells. <i>GAPDH</i> was used as a loading control. (C) Cell injury by NK cells was assessed by measuring LDH released in the supernatants as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098319#s2" target="_blank">Materials and Methods</a>. Triton-X (2%) was used as a positive control. The percent cytotoxicity was quantified and shown as mean of triplicate. Two independent experiments were performed and similar results were obtained. (D) Estimated volumes of the teratomas in immunodeficient mice and SLA-matched pigs. C1 iPS cells were transplanted into NK-competent NOD/SCID (<i>n</i> = 14) and NK-deficient NOG (<i>n</i> = 14) mice, and C1 pigs (<i>n</i> = 5). After transplantation of C1 iPS cells (1×10⁶ cells/site) into immunodeficient mice, the teratomas were dissected and their size (diameter) was measured (^*<i>p</i><0.01). After transplantation of C1 iPS cells (3×10⁷ cells/site) into C1 pigs, no teratomas were observed.</p