Derivation of Transgene-Free Human Induced Pluripotent Stem Cells from Human Peripheral T Cells in Defined Culture Conditions

<div><p>Recently, induced pluripotent stem cells (iPSCs) were established as promising cell sources for revolutionary regenerative therapies. The initial culture system used for iPSC generation needed fetal calf serum in the culture medium and mouse embryonic fibroblast as a feeder layer, both of which could possibly transfer unknown exogenous antigens and pathogens into the iPSC population. Therefore, the development of culture systems designed to minimize such potential risks has become increasingly vital for future applications of iPSCs for clinical use. On another front, although donor cell types for generating iPSCs are wide-ranging, T cells have attracted attention as unique cell sources for iPSCs generation because T cell-derived iPSCs (TiPSCs) have a unique monoclonal T cell receptor genomic rearrangement that enables their differentiation into antigen-specific T cells, which can be applied to novel immunotherapies. In the present study, we generated transgene-free human TiPSCs using a combination of activated human T cells and Sendai virus under defined culture conditions. These TiPSCs expressed pluripotent markers by quantitative PCR and immunostaining, had a normal karyotype, and were capable of differentiating into cells from all three germ layers. This method of TiPSCs generation is more suitable for the therapeutic application of iPSC technology because it lowers the risks associated with the presence of undefined, animal-derived feeder cells and serum. Therefore this work will lead to establishment of safer iPSCs and extended clinical application.</p></div

Analysis of TiPSCs genome modification and karyotype.

<p>(A): Bisulfite sequencing analysis of the <i>NANOG</i> and <i>OCT3/4</i> promoter regions in peripheral T cells, ESCs, and M-TiPSCs. Each row of circles for a given amplicon represents the methylation status of the CpG dinucleotides in one bacterial clone for that region. Open circles represent unmethylated CpGs and closed circles represent methylated CpGs. (B): G-band analysis for karyotypes of M-TiPSCs generated under a defined culture condition. M-TiPSCs1 and M-TiPSCs2 at passages 6 and 15, respectively, were used for G-band analysis. (C): Analysis of TCR rearrangements. V, D, and J segment usages in the <i>TCRB</i> gene locus were sequenced and identified by comparison to the international ImMunoGeneTics information system database. M-TiPSCs showed rearrangements of Vβ/Dβ1,2 and Dβ1,2/Jβ2.</p

Structure-function correlations.

<p><b>A</b>. Representative 2,3,5-triphenyltetrazoliumchloride (TTC) sections covering the LV chamber from the apex to the base from all three groups tested. The blue arrowhead indicates the section level that corresponds to the MR images in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144523#pone.0144523.g003" target="_blank">3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144523#pone.0144523.g004" target="_blank">4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144523#pone.0144523.g005" target="_blank">5</a>. <b>B</b>. Correlation between the scar mass (g) evaluated separately by LGE images (JD) and by TTC staining (ET). <b>C</b>. Representative tagged CMR images with infarct zone (IZ) defined by the presence of enhancing myocardium, neighboring myocardium as border zone (BZ), and normal myocardium as remote zone (RZ) for the analysis of regional strain, highlighting the contractility differences among the groups. Blue arrowheads indicate the RV insertion and the location of the first segment of each map. <b>D., E., F</b>. Representative diagrams of the averaged strain included in the analysis (the more negative, the greater the contractility). <b>G</b>. Better contractility was observed in the CDC-treated group compared to the placebo. <b>H</b>. Trend towards better effect of CDC therapy on mid-ventricular wall synchrony assessed by CURE ratio. Error bars indicate SEM. * p<0.05, ** p<0.01</p

In vitro and in-TiPSCs.

<p>(A): Immunofluorescence staining for Sox17 (endodermal marker), αSMA (mesodermal marker), and Nestin (ectodermal marker) in each TiPSCs1-derived differentiated cell in vitro. (B): Gross morphology of representative teratomas derived from TiPSCs1 in vivo (hematoxylin and eosin staining).</p

Vessel density and cardiomyocyte hypertrophy.

<p><b>A</b>. Representative immunostaining sections with sma, isolectin B4 and DAPI, 1 month post treatment. <b>B</b>. Vessel density within the IZ (p = 0.05), <b>C</b>., the BZ and <b>D</b>., the RZ in the CDC-treated compared to placebo (Scale bar = 75μm). <b>E</b>. Representative sections immunostained with α-sa and WGA. <b>F., G</b>. Similar cardiomyocyte diameters in all three groups, both in the BZ and in the RZ. Scale bar = 50μm. Abbreviations: IZ, infarct zone; BZ, border zone; RZ, remote zone; sma, smooth muscle actin (red); isolectin B4 (green); DAPI, blue, 4',6-diamidino-2-phenylindole; asa, α-sarcomeric actinin; WGA, wheat germ agglutinin to define the cell borders. Error bars indicate SEM.</p

MRI results in triple-vessel protocol.

<p><b>A</b>. Representative baseline (upper row) and 1 month post-infusion (lower row) short-axis images in all three groups highlighting the decrease in the scar size and the scar thickness. Yellow arrowheads indicate the infarct zone borders. <b>B</b>. Ejection fraction (EF) decreased significantly in the placebo group compared to both CDC-treated groups (p = 0.1 placebo vs CDCs stop-flow and p = 0.05 placebo vs CDCs continuous-flow). <b>C., D., E</b>. Changes in end-systolic and end-diastolic volumes and in scar mass in the treated groups compared to placebo. <b>F</b>. Infarct wall thickening (p = 0.01 placebo vs CDCs stop-flow and p = 0.05 placebo vs CDCs continuous-flow) and <b>G</b>., infarct wall thickness (p = 0.1 placebo vs CDCs stop-flow and p<0.01 placebo vs CDCs continuous-flow) in the placebo compared to both treated groups. Error bars indicate SEM. * p<0.05, ** p<0.01</p

Side-by-side evaluation of intramyocardial injections and intracoronary infusions of CDCs data.

<p><b>A</b>. The cell treatment effect of both intramyocardial and intracoronary delivery was superior to placebo with no difference between the 2 treatment groups. <b>B</b>. Similar changes were observed in scar size reduction. <b>C</b>. More cells were detected post intramyocardial injections compared to intracoronary delivery at 24hr of evaluation. Intracoronary data from the present study; intramyocardial data replotted from ref. 32. Error bars indicate SEM.</p

Generation of human TiPSCs under defined conditions.

<p>(A): Strategy used in the present study for reprogramming T cells. (B): Typical ESC-like TiPSC colony on day 25 after blood sampling under the defined culture condition. (C): Comparison of reprogramming efficiencies between the culture system using a feeder cell layer and that using defined culture conditions. Data show the mean ± s.d. (D): Comparison of representative 10-cm dishes stained for ALP (red spots) between feeder layer condition and defined culture condition (Matrigel) on day 25. (E): Comparison of reprogramming efficiencies between a culture system using a feeder cell layer and one using Matrigel and mTeSR1 medium for samples from five donors. Data show the mean ± s.d.</p

Multi-vessel study design and safety.

<p><b>A</b>. Timeline of the study. <b>B</b>., Allogeneic CDC isolation and manufacturing from 2 male donor hearts resulted in a master cell bank and bags of frozen CDCs. <b>C</b>. Effects of cryopreservation on CD105 and CD45 expression of CDCs. <b>D., E., F</b>. TIMI flow post-infusion in each vessel. <b>G., H</b>. TnI bump (7.4ng/ml) was observed at 24hrs in one animal after the stop-flow protocol. Error bars indicate SEM. Abbreviations: MI, myocardial infarction; CDCs, cardiosphere-derived cells; MRI, magnetic resonance imaging; LAD: left anterior descending artery.</p

Single-vessel Study.

<p><b>A</b>. Illustration of stop-flow protocol and <b>B</b>., the continuous-flow protocol. <b>C, D</b>. Box Plots showing serum [TnI] before infusion and 24hrs post infusion (p = ns; normal <0.05ng/ml). <b>E</b>. Short-axis contrast-enhanced images at baseline and 4 weeks after CDC or vehicle infusion showing <b>F</b>. significant changes of ejection fraction in placebo versus either treated group (p = 0.03 placebo vs CDCs stop-flow and p<0.01 placebo vs CDCs continuous-flow). <b>G</b>. Changes in end-systolic volume between baseline and 4 weeks post-infusion (p = 0.02 placebo vs CDCs stop-flow and p = 0.05 placebo vs CDCs continuous-flow), and <b>H</b>., in end-diastolic volume (p = 0.06 placebo vs CDCs stop-flow and p = 0.2 placebo vs CDCs continuous-flow). Changes in scar mass (<b>I</b>, p = 0.08 placebo vs CDCs stop-flow and p<0.01 placebo vs CDCs continuous-flow), <b>J</b>., infarct wall motion (p = 0.04 placebo vs CDCs stop-flow and p = 0.02 placebo vs CDCs continuous-flow) and <b>K</b>., infarct wall thickness between placebo and treated groups (p = ns). Error bars indicate SEM. * p<0.05, ** p<0.01</p