Transduction of the reporter vector containing miRNA targets does not grossly affect expression of hESC-specific markers.

<p>(A and B) Single-cell suspensions of hESC (H1), hiPSCs transduced with a lentiviral vector encoding EGFP miR-T/mCherry miR-T (E/m#1, E/m#5, and E/m#101) or untransduced (hiPSC#19) were analyzed for the expression of EGFP and mCherry (A) and that of hESC-specific markers (SSEA1, SSEA3, TRA1-60, and TRA-1-81) (B) by flow cytometry. The number (%) in each quadrant is listed on each plot. (C) hESCs (H1), hiPSCs transduced with a lentiviral vector encoding EGFP miR-T/mCherry miR-T (E/m#1, E/m#5, and E/m#101) or untransduced (hiPSC#19) were plated on poly-L-lysine and Matrigel coated glass coverslips and expanded for a week. Cells were then fixed with 1% formaldehyde, permeabilized with 0.2% Triton X-100 for 5 min on ice, and stained with anti-Nanog antibody and DyLight488 conjugated anti-rabbit IgGs. 7-amino-actinomycin D (7-AAD) was used for nuclear staining.</p

Ectopic expression of the miRNAs specifically suppresses expression of the reporter vector containing miRNA targets in 293T cells.

<p>(A) Map of transcriptional units of the reporter vector used in this study. CMVmini: CMV minimal promoter. UbiC: ubiquitin C promoter. CCR5 target: siRNA against CCR5 target sequence (5′-GAGCAAGCTCAGTTTACACC-3′) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011834#pone.0011834-Shimizu1" target="_blank">[59]</a>. (B) 293T cells were infected with lentiviral vectors encoding various reporters shown in (A). Expression levels of EGFP and mCherry were analyzed by flow cytometry 2 days post-infection. 293T cells infected with a lentiviral vector encoding EGFP miR-T/mCherry miR-T were super-infected by a lentiviral vector encoding either <i>miR-302a</i>, <i>miR-302b</i>, <i>miR-302c</i>, and <i>miR-302d</i> (miR-302 a-d) or <i>miR-155</i> 2 days post-infection. Cells were then further cultured for 4 days and analyzed for EGFP and mCherry expression by flow cytometry. The number (%) in each quadrant is listed on each plot.</p

Primers for RT-PCR.

<p>Primers for RT-PCR.</p

Reprogramming state specific expression of the reporter vector containing miRNA targets during hiPSC formation from human fetal fibroblasts (HFFs).

<p>(A) HFFs were infected with lentiviral vectors encoding various reporters shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011834#pone-0011834-g001" target="_blank">Fig. 1A</a>. Expression levels of EGFP and mCherry were analyzed by flow cytometry 2 days post-infection. (B and C) HFFs were infected with lentiviral vectors encoding 4 different hiPSC factors (<i>OCT4, SOX2, KLF4,</i> and <i>cMYC</i>) and with or without a lentiviral vector encoding EGFP miR-T/mCherry miR-T. Cells were then cultured for 3 days and replated on irradiated mouse embryonic fibroblast (iMEF) feeder cells at 5×10⁴ cells/60 mm plate. Expression levels of EGFP and mCherry were analyzed by flow cytometry on days 0, 7, 14, 21, and 28 (B), and by fluorescence microscopy on days 0, 7, 14, and 21 (C). iMEF feeder cells were labeled with PE-Cy7 conjugated mouse CD29 antibody and excluded from the flow cytometry analysis in (B). (D) hiPSC colonies expressing mCherry were picked on Matrigel coated plate on days 21–25 and propagated in mTeSR medium. Expression levels of EGFP and mCherry were analyzed by fluorescence microscopy and by flow cytometry. The number (%) in each quadrant is listed on each plot. hiPSC #1: hiPSC clone without transduction of EGFP miR-T/mCherry miR-T reporter vector. E/m#8: hiPSC clone with transduction of EGFP miR-T/mCherry miR-T reporter vector.</p

Molecular characterization of hiPSCs transduced with the reporter vector containing miRNA targets.

<p>Total RNA was isolated using QIAGEN's RNeasy Mini kit from HFFs transduced with (4Fs/HFF) or without 4 reprogramming factors (HFF), hESCs (H1), and 4 different hiPSC clones transduced with (E/m#1, E/m#5, and E/m#101) or without (hiPSC#19) the reporter vector encoding EGFP miR-T/mCherry miR-T. Total RNA (250 ng) was reverse-transcribed using QIAGEN's Omniscript reverse transcription kit and used as a template in subsequent PCR with 5-PRIME's HotMaster Taq DNA polymerase. PCR products were analyzed on a 2% agarose gel. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.</p

hiPSCs transduced with the reporter vector containing miRNA targets indicate differentiation-specific reporter expression in neural lineages.

<p>EBs generated from pooled hiPSCs were differentiated into neural lineages using Noggin and SB431542. EBs were then transferred onto fibronectin coated 6-well plates and further differentiated in N2 medium. (A) Total RNA was isolated using QIAGEN's RNeasy Mini kit from the hiPSCs on day 0 (undifferentiated population: Undiff.) and day 30 (differentiated population: Diff.) after induction of differentiation. Total RNA (250 ng) was reverse-transcribed using QIAGEN's Omniscript reverse transcription kit and used as a template in subsequent PCR with 5-PRIME's HotMaster Taq DNA polymerase. PCR products were analyzed on a 2% agarose gel. GAPDH was used as an internal control. (B) Neural tube-like rosettes observed after the differentiation. (C) Dark pigmented melanocyte-like cells surrounding neural tube-like structures.</p

hiPSCs transduced with the reporter vector containing miRNA targets show differentiation-specific reporter expression in EBs.

<p>hESC (H1) and 4 different hiPSC clones (E/m#1, E/m#5, E/m#101, and hiPSC#19) were differentiated into EBs and maintained 25 days in IMDM containing 10% FBS. EBs were then dissociated with 0.25% trypsin/EDTA and the reporter expression was analyzed by flow cytometry. Histograms filled with black are undifferentiated controls. Histograms filled with blue (EGFP) and pink (mCherry) are differentiated cells, respectively. The numbers indicated in histogram show percentage of positive cells (EGFP) and negative cells (mCherry). MFI: mean fluorescence intensity. U: MFI of undifferentiated cells. D: MFI of differentiated cells.</p

The reporter vectors containing miRNA targets show the lineage-specific expression.

<p>(A) U937 cells were infected with lentiviral vectors encoding various reporters showed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011834#pone-0011834-g001" target="_blank">Fig. 1</a> A. Expression levels of EGFP and mCherry were analyzed by flow cytometry 2 days post-infection. (B and C) CEM and Ramos cells (B) and CD34+ HPSCs derived from 3 independent donors (C) were infected with a lentiviral vector encoding EGFP miR-T/mCherry miR-T. Expression levels of EGFP and mCherry were analyzed by flow cytometry 2 days post-infection. (D) hESCs (H1) were infected with a lentiviral vector encoding EGFP miR-T/mCherry miR-T. Single cell clone was isolated by culturing transduced cells in the presence of 10 µM Y27632 for 14 days. Expression levels of EGFP and mCherry were analyzed by fluorescence microscopy and by flow cytometry. The number (%) in each quadrant is listed on each plot.</p

Modulation of Gene Expression by Polymer Nanocapsule Delivery of DNA Cassettes Encoding Small RNAs

<div><p>Small RNAs, including siRNAs, gRNAs and miRNAs, modulate gene expression and serve as potential therapies for human diseases. Delivery to target cells remains the fundamental limitation for use of these RNAs in humans. To address this challenge, we have developed a nanocapsule delivery technology that encapsulates small DNA molecules encoding RNAs into a small (30nm) polymer nanocapsule. For proof of concept, we transduced DNA expression cassettes for three small RNAs. In one application, the DNA cassette encodes an shRNA transcriptional unit that downregulates CCR5 and protects from HIV-1 infection. The DNA cassette nanocapsules were further engineered for timed release of the DNA cargo for prolonged knockdown of CCR5. Secondly, the nanocapsules provide an efficient means for delivery of gRNAs in the CRISPR/Cas9 system to mutate integrated HIV-1. Finally, delivery of microRNA-125b to mobilized human CD34+ cells enhances survival and expansion of the CD34+ cells in culture.</p></div

Nanocapsule delivery of miRNA to increase survival and expansion of hematopoietic stem/progenitor cells (HSPC).

<p>a) Flow cytometry of mobilized CD34+ cells incubated with fluorescence-labeled miR-125b DNA nanocapsules for 4 hours; b) <i>Ex-vivo</i> expansion of cytokine-mobilized CD34+ cells cultured with 50ng/mL SCF, 50ng/mL Flt-3L, and 50ng/mL TPO for 6 days after delivery of miR-125b DNA nanocapsules and control DNA nanocapsules. Annexin V staining of mobilized CD34+ cells c) without nanocapsules, d) with miR-125b DNA nanocapsules and e) with control DNA nanocapsules 24 hours later after treatment of staurosporine for 4 hours. Microscopic images of mobilized CD34+ cells f) without nanocapsules, g) with miR-125b DNA nanocapsules and h) transduced with miR-125b expressing lentiviral vector, cultured and expanded for 7 days.</p