Few Single Nucleotide Variations in Exomes of Human Cord Blood Induced Pluripotent Stem Cells

<div><p>The effect of the cellular reprogramming process <i>per se</i> on mutation load remains unclear. To address this issue, we performed whole exome sequencing analysis of induced pluripotent stem cells (iPSCs) reprogrammed from human cord blood (CB) CD34⁺ cells. Cells from a single donor and improved lentiviral vectors for high-efficiency (2–14%) reprogramming were used to examine the effects of three different combinations of reprogramming factors: OCT4 and SOX2 (OS), OS and ZSCAN4 (OSZ), OS and MYC and KLF4 (OSMK). Five clones from each group were subject to whole exome sequencing analysis. We identified 14, 11, and 9 single nucleotide variations (SNVs), in exomes, including untranslated regions (UTR), in the five clones of OSMK, OS, and OSZ iPSC lines. Only 8, 7, and 4 of these, respectively, were protein-coding mutations. An average of 1.3 coding mutations per CB iPSC line is remarkably lower than previous studies using fibroblasts and low-efficiency reprogramming approaches. These data demonstrate that point nucleotide mutations during cord blood reprogramming are negligible and that the inclusion of genome stabilizers like ZSCAN4 during reprogramming may further decrease reprogramming-associated mutations. Our findings provide evidence that CB is a superior source of cells for iPSC banking.</p> </div

Palmitic acid is a toll-like receptor 4 ligand that induces human dendritic cell secretion of IL-1β

<div><p>Palmitic acid (PA) and other saturated fatty acids are known to stimulate pro-inflammatory responses in human immune cells via Toll-like receptor 4 (TLR4). However, the molecular mechanism responsible for fatty acid stimulation of TLR4 remains unknown. Here, we demonstrate that PA functions as a ligand for TLR4 on human monocyte derived dendritic cells (MoDCs). Hydrophobicity protein modeling indicated PA can associate with the hydrophobic binding pocket of TLR4 adaptor protein MD-2. Isothermal titration calorimetry quantified heat absorption that occurred during PA titration into TLR4/MD2, indicating that PA binds to TLR4/MD2. Treatment of human MoDCs with PA resulted in endocytosis of TLR4, further supporting the function of PA as a TLR4 agonist. In addition, PA stimulated DC maturation and activation based on the upregulation of DC costimulatory factors CD86 and CD83. Further experiments showed that PA induced TLR4 dependent secretion of the pro-inflammatory cytokine IL-1β. Lastly, our experimental data show that PA stimulation of NF-κB canonical pathway activation is regulated by TLR4 signaling and that reactive oxygen species may be important in upregulating this pro-inflammatory response. Our experiments demonstrate for the first time that PA activation of TLR4 occurs in response to direct molecular interactions between PA and MD-2. In summary, our findings suggest a likely molecular mechanism for PA induction of pro-inflammatory immune responses in human dendritic cells expressing TLR4.</p></div

Efficient generation of iPSCs from cord blood CD34⁺ cells.

<p>(<b>A</b>) Efficient reprogramming of cord blood with lentiviral vectors. Three different combinations of reprogramming factors were used: OCT4 and SOX2 (OS), OS+ZSCAN4 (OSZ), and OS+MYC and KLF4 (OSMK). After transduction, 2500–5000 cells were seeded in 6-well plates. iPSC colonies were counted 2 weeks later and reprogramming efficiencies were calculated accordingly. (<b>B</b>) iPSCs express pluripotency makers OCT4, NANOG and SSEA4. Representative pictures for each group (OS iPSC lines, OSZ iPSC lines, and MK iPSC lines (OSMK)) are presented (200×). Confocal imaging did not identify any differences in expression of pluripotency makers among 15 iPSC lines.</p

Genes found to be mutated in exomes of 15 CB iPSC lines.

<p>The full details of each SNV including reads of SNV and wildtype alleles are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059908#pone.0059908.s005" target="_blank">Table S1</a>.</p><p>MK: iPSC lines generated with OCT4, SOX2, MYC and KLF4; OS: iPSC lines generated with OCT4 and SOX2; Z: iPSC lines generated with OCT4, SOX2 and ZSCAN4.</p><p>CDS: coding sequence; UTR: untranslated region; Downstream: SNV is at downstream of 5′UTR; S: synonymous coding mutation; NS: nonsynonymous coding mutation.</p

Dendritic cell viability following high (300μM), medium (150μM) and, low (75μM) dose PA treatment.

<p>MoDCs were inoculated with PA in a 2:1 molar ratio with BSA for (A) 12 hrs, (B) 24 hrs, or (C) 48 hrs. The cells were analyzed for loss of viability by flow cytometry using Annexin V and propidium iodide (PI) staining. LPS was used as a positive control to assess MoDC activation. The data is represented as the percent of total MoDCs negative for both Annexin V and PI normalized to the BSA negative control. (D) PA induces caspase 3/7 activity. MoDCs were treated with PA complexed 2:1 with BSA for 36 hrs. Caspase 3/7 activity was measured by the emitted fluorescence of cleaved DEVD-AMC. N = 3. Statistical analysis was calculated based on One way ANOVA (** = p<0.01, *** = p<0.001, **** = p<0.0001).</p

PA activation of MoDCs is regulated by ROS.

<p>(A) PA induces ROS in MoDCs. MoDCs were treated for 24hrs with PA. ROS was measured by DCF fluorescence and expressed as fold increase relative to MoDCs not incubated with DCF. The ROS scavenger MCI-186 was used as a negative control. N = 3. (B) Comparison of activation z-scores for cellular functions related to ROS production. After 24hr treatment, the DC proteome was assessed by mass spectrometry and protein changes analyzed using IPA. Transformed z-scores are displayed on the bar graph. The z-scores indicate the association of proteome changes with the indicated cellular functions in the MoDC after PA treatment. Non-transformed z-scores and p-values for association with cellular functions are in Supplement 4. (C) MoDCs were pre-incubated with the H₂O₂ probe PF6-AM. Upon addition of the indicated stimuli, PF6 fluorescence was detected by rapid-fire snap shots. The mean fluorescence intensity (MFI) of PF6 was normalized to baseline and plotted against time. (D) PA-induced MoDC activation is attenuated by MCI-186. MoDCs were treated with 300μM PA for 36 hrs. Cells were stained and analyzed by flow cytometry. MoDCs were gated by the phenotype (CD11c^hi) and histograms for CD86 were plotted. Data is representative of three separate experiments. (E) PA-induced activation of NF-κB is ROS dependent. MoDCs were treated with 300μM PA+/- MCI-186 or LPS+/- MCI-186 for 3hrs and analyzed by EMSA.</p

Coding mutations in each of the 15 CB iPSC lines.

<p>Number of coding SNVs (<b>A</b>) and nonsynonymous coding SNVs (<b>B</b>) in each line compared to the parent cord blood cells. MK: iPSC lines generated with OCT4, SOX2, MYC and KLF; OS: iPSC lines generated with OCT4 and SOX2; Z: iPSC lines generated with OCT4, SOX2; and ZSCAN4. The use of OSZ appears to decrease the coding SNV load in iPSC lines compared to the OSMK control (<i>P</i>>0.05) (<b>A</b>), while OSZ iPSC lines harbor significant fewer number of nonsynonymous coding SNVs relative to the OSMK control (<i>P</i><0.05) (<b>B</b>).</p

Summary of the exome sequencing data and the identified single nucleotide variants.

<p>The numbers of heterozygous variants are those that have a minimum of 5× coverage. The dbSNP percentage represents the portion of identified variants present in the Single Nucleotide Polymorphism Database.</p

Palmitic acid is a ligand for TLR4/MD-2.

<p>(A) XY plot of fatty acid carbon chain length vs Gibbs free energy derived from docking FAs to MD-2 via the SwissDock server. (B) Molecular model of 5 PA docked in the hydrophobic binding pocking of MD-2. Orange color indicates hydrophobicity of MD-2 and blue indicates hydropholicity. Each PA molecule is in a solid color (yellow, black, blue, green, and purple) with oxygen atoms in red. (C) Isothermal titration calorimetry. Isotherms and fitted area curve of PA into TLR4/MD-2 titration. Insert contains thermodynamic parameters for PA and TLR4/MD-2 binding interaction. (D) Control isotherm of PA titrated into buffer. (E-F) PA induces the internalization of TLR4. MoDCs were treated with 300μM PA (E) or 10ng/mL LPS (F) for indicated time intervals. Cells were stained for flow cytometry. CD14^-CD11c^hiHLA-DR^hi cells were assessed for TLR4. Data is presented as the percentage of baseline Geometric MFI. N = 3. Graphs display the mean ± SEM. Statistical analysis was calculated based on One way ANOVA (** = p<0.01, *** = p<0.001, **** = p<0.0001).</p

PA induced DC maturation and secretion of IL-1β.

<p>(A) PA upregulated DC activation and maturation markers. MoDCs were treated with 150μM PA for 12hrs. Representative histograms from 1 of 8 individual patient samples is presented, and the average geometric MFI for the indicated marker on CD11c^hi cells (MoDCs) from all 8 samples is graphed. (B) mRNA fold change in cytokines of MoDCs treated with 300μM PA (N = 8). (C) Quantification of cytokines secreted from MoDCs treated with 300μM PA (N = 4). ND = Not detectable.</p