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

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

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

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

Optimization of the PBMNC reprogramming protocol.

<p>(<b>A</b>) Depletion of CD3⁺ and 19⁺ lymphoid cells increases reprogramming efficiency. Whole PBMNCs or CD3⁻/CD19⁻ cells (T/B cell-depleted PBMNCs) were cultured for 4 days before nucleofection with episomal vectors expressing OS (OCT4 and SOX2), MK (MYC and KLF4) and B (BCL-XL). 1×10⁶ cells were used for nucleofection. The numbers of iPSC colonies were counted at 3–4 weeks after nucleofection and numbers of iPSC colonies per 1 ml of PB were calculated by normalization to the amount of starting peripheral blood. Data shown are presented as mean ± SEM (n = 4). * indicates <i>P</i><0.05. (<b>B</b>) Culturing PB CD3/19⁻ cells for 4 days allows maximum reprogramming. CD3/19⁻ cells (T/B cells-depleted) PBMNCs were cultured for 0 to 8 days before nucleofection with episomal vectors expressing OS, MK and B. 1×10⁶ cells were used for nucleofection. The numbers of iPSC colonies were counted at 3–4 weeks after nucleofection. Graphed data are presented as mean ± SEM (n = 6). * indicates <i>P</i><0.05.</p

In vitro multilineage differentiation of integration-free PB iPSCs.

<p>(<b>A</b>) Differentiation of PB iPSCs into MSCs. iPSC-MSCs show a typical MSC morphology and are capable of differentiation into adipocytes, osteoblasts and chondrocytes. Oil Red O stains the oil droplets of adipocytes. Alizarin Red stains the bone nodules formed by osteoblasts. Alcian Blue stains acid mucopolysaccharides synthesized and secreted by chondrocytes. (<b>B</b>) PB iPSC-derived hepatocytes show a typical morphology of hepatocytes at 25 days after hepatocytic differentiation. These cells also express markers AFP, albumin (ALB), and alpha 1-antitrypsin (α1-AT). The differentiated cells were stained with monoclonal antibody against AFP, goat anti-albumin, and goat anti-alpha 1-antitrypsin at 18 days after differentiation culture. (<b>C</b>) PB iPSC can be induced to differentiation into cardiomyocytes that express Troponin I marker. Cell nuclei were counterstained with DAPI.</p

BCL-XL significantly enhances OS-mediated reprogramming of cord blood and peripheral blood cells with lentiviral vectors.

<p>(<b>A</b>) Differential effects of BCL2 family members on enhancing OS-mediated reprogramming of CB cells. CB CD34⁺ cells were cultured for 2 days before lentiviral transduction. CB iPSC colonies were enumerated at 2 weeks after transduction of reprogramming factors. Data shown are presented as mean ± SEM (n = 4). OS: OCT4 and SOX2. * indicates <i>P</i><0.05. (<b>B</b>) Differential effects of BCL2 family members on enhancing OS-mediated reprogramming of PBMNCs. PBMNCs were cultured for 4–6 days before lentiviral transduction. PB iPSC colonies were enumerated at 3 weeks after transduction of reprogramming factors. Data shown are presented as mean ± SEM (n = 4). OS: OCT4 and SOX2. PBMNCs, peripheral blood mononuclear cells. * indicates <i>P</i><0.05. BCL2 and BCL-XL significantly increased reprogramming of both CB CD34⁺ cells and PBMNCs.</p

Generation of integration-free iPSCs from adult PBMNCs with episomal vectors.

<p>(<b>A</b>) ALP staining of iPSCs at 4 weeks after nucleofection of PBMNCs with reprogramming factor-expressing episomal vectors. OS, OCT4 and SOX2; MK, MYC and KLF4; B, BCL-XL. PBMNCs were cultured for 4–8 days before nucleofection. 1×10⁶ PBMNCs were nucleofected and then seeded into each well. (<b>B</b>) Inclusion of BCL-XL increases PB reprogramming efficiency by up to 10-fold. PBMNCs were cultured for 4–8 days before nucleofection. ALP-positive iPSC colonies were enumerated at 3–4 weeks after nucleofection. Data are presented as mean ± SEM (n = 6). In all 3 conditions, BCL-XL significantly increased reprogramming efficiency. * indicates <i>P</i><0.05. (<b>C</b>) iPSC are generated from PBMNCs expressing the myeloid lineage marker, CD33, but not lymphoid cells (CD3⁺ and CD19⁺ cells) in PBMNCs. ALP staining of iPSCs at 4 weeks after nucleofection of fractionated PBMNCs with episomal vectors OS+MK+B. CD33, myeloid marker; CD3, T cell marker; CD19, B cell marker. 1×10⁶ indicated cells were nucleofected and then seeded into each well. ALP staining was conducted at 4 weeks after nucleofection.</p