Development of an All-in-One Inducible Lentiviral Vector for Gene Specific Analysis of Reprogramming

<div><p>Fair comparison of reprogramming efficiencies and <em>in vitro</em> differentiation capabilities among induced pluripotent stem cell (iPSC) lines has been hampered by the cellular and genetic heterogeneity of de novo infected somatic cells. In order to address this problem, we constructed a single cassette all-in-one inducible lentiviral vector (Ai-LV) for the expression of three reprogramming factors (<em>Oct3/4</em>, <em>Klf4</em> and <em>Sox2</em>). To obtain multiple types of somatic cells having the same genetic background, we generated reprogrammable chimeric mice using iPSCs derived from Ai-LV infected somatic cells. Then, hepatic cells, hematopoietic cells and fibroblasts were isolated at different developmental stages from the chimeric mice, and reprogrammed again to generate 2nd iPSCs. The results revealed that somatic cells, especially fetal hepatoblasts were reprogrammed 1200 times more efficiently than adult hepatocytes with maximum reprogramming efficiency reaching 12.5%. However, we found that forced expression of <em>c-Myc</em> compensated for the reduced reprogramming efficiency in aged somatic cells without affecting cell proliferation. All these findings suggest that the Ai-LV system enables us to generate a panel of iPSC clones derived from various tissues with the same genetic background, and thus provides an invaluable tool for iPSC research.</p> </div

Reprogramming efficiency of somatic cells in the presence of c-Myc.

<p>(A) Reprogramming efficiency of fibroblasts (MEF, NB fb, 1wk fb and Adult fb), hematopoietic cells (FL CD45, HSC, HPC, MP and Mac) by four reprogramming factors including <i>c-Myc</i>. (B) Cell proliferation rate of fibroblasts (MEF, NB fb, 1wk fb and Adult fb) at three, four and five days after seeded in the absence of Dox (left) and in the presence of Dox (right). (C) Reprogramming efficiency of MEF were compared between reprogrammed by three factors (Oct4, Klf4 and Sox2), four factors (<i>Oct4</i>, <i>Klf4</i>, <i>Sox2</i> and <i>c-Myc</i>) and three factors plus VPA (0.5 mM, 1 mM and 2 mM).</p

Characterization of mtDNA mutations in homozygous <i>Polg</i> mice with aging.

<p>(A). Comparison of mean number of germline mutations in wt and <i>Polg</i> mice at young and old age (n = 31 for young wt, n = 8 for young <i>Polg</i>, n = 44 for old wt and n = 14 for old <i>Polg</i>). Error bars, mean ± SEM. Asterisk indicates a significant increase in the number of mutations per tissue in old <i>Polg</i> compared to the old wt (P < 0.05, Student’s t-test). (B). Mean heteroplasmy levels of non-synonymous germline mutations with ≥2% heteroplasmy in <i>Polg</i> mice (mean ± SEM; asterisk, P < 0.05, Student’s t-test). (C) Pie charts showing gene distributions of non-synonymous germline mutations in young <i>Polg</i> mice (2 months, left) and old <i>Polg</i> mice (9 months, right). (D) Bar graphs representing the mean heteroplasmy levels of non-synonymous germline mutations in protein-coding and RNA-coding genes in <i>Polg</i> mice (asterisks, P < 0.05, Student’s t-test). (E) Pie charts showing the distribution of shared and unique mtDNA mutations detected in single skin fibroblast (SF) clones in young and old <i>Polg</i> mice. (F) Mean heteroplasmy changes for non-synonymous somatic mutations with ≥15% heteroplasmy in <i>Polg</i> mice. Error bars, mean ± SEM. Asterisk, P < 0.05, Student’s t-test. (G) Changes in number of non-synonymous somatic mutations with heteroplasmy levels ≥ 15% among different gene types with <i>Polg</i> mice aging. Error bars, mean ± SEM. Asterisks, P < 0.05, Student’s t-test.</p

Early embryonic and NCR mtDNA mutations in <i>Polg</i> mice.

<p>(A) Bar graphs representing changes in mean heteroplasmy of early embryonic mutations with <i>Polg</i> mice aging. Error Bars, mean ± SEM. (B) Distribution of non-synonymous early embryonic mtDNA mutations among different genes. Error bars, mean ± SEM. (C) Quantification of mtDNA mutations in the non-coding region (NCR) in <i>Polg</i> mice (n = 12 for 2 months; n = 24 for 9 months). Error bars, mean ± SEM. (D) Bar graphs representing mean heteroplasmy levels of NCR mtDNA mutations in <i>Polg</i> mice with aging. Error bars, mean ± SEM. (E) Summary of mtDNA mutations found in the NCR region (mtDNA15423-16299) in <i>Polg</i> mice. Dots represent mtDNA mutations and numbers under dots represent the heteroplasmy levels. ETAS indicates the extended termination associated sequence and CSB indicates the conserved sequence block.</p

Summary of mtDNA mutations detected in wild type and <i>Polg</i> mice.

<p>Summary of mtDNA mutations detected in wild type and <i>Polg</i> mice.</p

Reprogramming efficiency of rat somatic cells.

<p>(A) Reprogramming efficiency of rat fibroblasts (REF, r1wk fb and rAdult fb) by three reprogramming factors (<i>Oct4</i>, <i>Klf4</i> and <i>Sox2</i>) (left) and by four reprogramming factors (<i>Oct4</i>, <i>Klf4</i>, <i>Sox2</i> and <i>c-Myc</i>) (right). (B) Cell proliferation rate of rat fibroblasts in the presence of Dox.</p

Reprogramming efficiency from somatic cells at different developmental stages.

<p>(A) <i>Nanog</i> immunostaining of 2^nd iPS colonies derived from MEF (2000 cells), NB fb (2000 cells), 1wk fb (5000 cells) and Adult fb (10000 cells). Each cell were seeded on a feeder layer and cultured in the presence of Dox for two weeks. (B) Reprogramming efficiency of fibroblasts (MEF, NB fb, 1wk fb and Adult fb) *p<0.05 (left panel), hematopoietic cells (FL CD45, HSC, HPC, MP and Mac) *p<0.05 (middle panel) and liver cells (Fetal hep and Adult hep) *p<0.01 (right panel) were analyzed by dividing seeded cell number by the number of <i>Nanog</i> positive colonies. (C) Cell proliferation rate of fibroblasts (MEF, NB fb, 1wk fb and Adult fb) at three, four and five days after seeded in the absence of Dox (left) and in the presence of Dox (right).</p