Single iPSC and hESC cardiomyocytes.

<p>Histograms of contraction force, beat width and beat rate of single iPSC-CM (a, top) and hESC-CM (a, bottom). Each curve in the plot is the smoothed histogram of the beats of a single cell measured at a single site on each cell. (b) Statistical analysis showing means of individual cells (dots), plus 25^th, 50^th, and 75^th percentile quantiles (box) and range of all points (whiskers). Statistical comparison by t test is shown.</p

AFM measurement of iPSC-CM cluster.

<p>(a) Bright field micrograph of cluster of iPSC-CMs. (b) Contraction force trajectory. The contraction of the CM cluster shows very regular beat force (c), frequency (d) and width (e).</p

Measurement of force of CMs.

<p>(a) The AFM cantilever is brought into gentle contact with the cardiomyocyte, placing 100 pN of pre-loaded force on it. The z-piezo is locked and the cantilever tip dwells on the top of the cardiomyocyte. (b) Shows a typical force trajectory where the green box shows indentation of the cell. The contraction of the cardiomyocyte appears as peaks in the trajectory. The height, full width at half maximum (FWHM) and reciprocal of beat-to-beat separation of peaks characterize the force, duration and frequency of cardiomyocyte beat, respectively. The fit of indentation curve by using Hertz model (red curve in (c)) produces the Young's modulus of the cell membrane at the contact point.</p

Measurement of drug effect on CMs.

<p>(a) Contraction force trajectories measured before and after treatment of iPSC- and hESC-CMs with norepinephrine. (b) Histograms of contraction force and frequency before and after treatment. (c) Beating force measured from a cluster of iPSC-CM in response to increasing doses of the adrenergic agonist epinephrine. (d) Beating force measured from a cluster of iPSC-CM in response to increasing doses of epinephrine, treated prior with the beta-blocker metoprolol.</p

AFM dwell map of dilated cardiomyopathy iPSC-CM.

<p>(<b>a</b>) Brightfield image of an iPSC-CM showing the AFM cantilever (black shadow). The yellow grid superimposed on the cell shows the range of the dwell map. (<b>b</b>) Dwell map showing contraction forces of a single iPSC-CM at various points on the grid. (<b>c</b>) Dwell map showing Young's modulus of a single iPSC-CM at various points on the grid. The periphery of the cell had higher contraction forces and elasticity compared to the central areas. (<b>d</b>) Beating force and Young's modulus (local elasticity) measurements were obtained from dwell mapping iPSC-CM derived from either a healthy subject (red) or from a subject with dilated cardiomyopathy (DCM, blue). Single points on the plot correspond to beat force and elasticity measured at each grid points of the dwell map. Points where no contraction force was measured (e.g., on the glass slide surrounding the cell) are not shown. The contour plot (middle) represents the probability distribution of dwell mapped points in the Young's modulus vs. beating force coordinate system. The contours show that beats measured from most portions of the healthy iPSC-CM fall in a region of moderate elasticity (50 – 5 kPa) and strong force (∼1 nN), whereas some points of the dwell map of DCM iPSC-CM showed comparatively lower beat forces and lower elasticity. Corresponding histograms that flank the contour plot are the distributions of beat force (above the contour plot) and elasticity (left of the contour plot), respectively.</p

Development of Poly(β-amino ester)-Based Biodegradable Nanoparticles for Nonviral Delivery of Minicircle DNA

Gene therapy provides a powerful tool for regulating cellular processes and tissue repair. Minicircle (MC) DNA are supercoiled DNA molecules free of bacterial plasmid backbone elements and have been reported to enhance prolonged gene expression compared to conventional plasmids. Despite the great promise of MC DNA for gene therapy, methods for safe and efficient MC DNA delivery remain lacking. To overcome this bottleneck, here we report the development of a poly(β-amino ester) (PBAE)-based, biodegradable nanoparticulate platform for efficient delivery of MC DNA driven by a Ubc promoter <i>in vitro</i> and <i>in vivo.</i> By synthesizing and screening a small library of 18 PBAE polymers with different backbone and end-group chemistry, we identified lead cationic PBAE structures that can complex with minicircle DNA to form nanoparticles, and delivery efficiency can be further modulated by tuning PBAE chemistry. Using human embryonic kidney 293 cells and mouse embryonic fibroblasts as model cell types, we identified a few PBAE polymers that allow efficient MC delivery at levels that are comparable or even surpassing Lipofectamine 2000. The biodegradable nature of PBAE-based nanoparticles facilitates <i>in vivo</i> applications and clinical translation. When injected <i>via</i> intraperitoneal route <i>in vivo</i>, MC alone resulted in high transgene expression, and a lead PBAE/MC nanoparticle formulation achieved a further 2-fold increase in protein expression compared to MC alone. Together, our results highlight the promise of PBAE-based nanoparticles as promising nonviral gene carriers for MC delivery, which may provide a valuable tool for broad applications of MC DNA-based gene therapy

Induced Pluripotency of Human Prostatic Epithelial Cells

<div><p>Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells, the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here, we attempted to reprogram E-PZ cells by forced expression of Oct4, Sox2, c-Myc, and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81, SSEA-3, Nanog, Sox2, and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5, CK14, and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal, mesodermal, and endodermal cells in vitro, although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly, E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44, p63, MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR), and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen (PSA), a hallmark of secretory cells, suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally, when injected into mice, E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme, E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified epigenetic changes in key pathways and genes involved in prostatic differentiation as E-PZ-iPS-like cells converted to differentiated AR- and PSA-expressing cells. Our results suggest that iPS-like cells derived from prostatic epithelial cells are pluripotent and capable of prostatic differentiation; therefore, provide a novel model for investigating epigenetic changes involved in prostate cell lineage specification.</p></div

Generation of iPSCs as a Pooled Culture Using Magnetic Activated Cell Sorting of Newly Reprogrammed Cells

<div><p>Although significant advancement has been made in the induced pluripotent stem cell (iPSC) field, current methods for iPSC derivation are labor intensive and costly. These methods involve manual selection, expansion, and characterization of multiple clones for each reprogrammed cell sample and therefore significantly hampers the feasibility of studies where a large number of iPSCs need to be derived. To develop higher throughput iPSC reprogramming methods, we generated iPSCs as a pooled culture using rigorous cell surface pluripotent marker selection with TRA-1-60 or SSEA4 antibodies followed by Magnetic Activated Cell Sorting (MACS). We observed that pool-selected cells are similar or identical to clonally derived iPSC lines from the same donor by all criteria examined, including stable expression of endogenous pluripotency genes, normal karyotype, loss of exogenous reprogramming factors, and <i>in vitro</i> spontaneous and lineage directed differentiation potential. This strategy can be generalized for iPSC generation using both integrating and non-integrating reprogramming methods. Our studies provide an attractive alternative to clonal derivation of iPSCs using rigorously selected cell pools and is amenable to automation.</p></div

Characterization of tri-fusion transgenic mouse.

<p>(A) <i>In vivo</i> bioluminescence, micro-PET and fluorescence imaging of adult tri-fusion transgenic mice and non-transgenic littermate. Mice (N=15) with different expression levels of the tri-fusion reporter were scanned, and multimodality imaging results show that the expression level of all three genes are correlated with each other. No bioluminescence signal was found in non-transgenic littermate. Background micro-PET signal was found in heart and gastrointestinal system of non-transgenic littermate, and autofluorescence was also found in non-transgenic littermate. Transgenic positive mice show different signal levels in multimodality imaging due to the different gene expression levels. (B–C) <i>In vivo</i> bioluminescence and fluorescence imaging of strongly positive newborn and adult tri-fusion transgenic mice and non-transgenic littermates. Strong bioluminescence and fluorescence signals were found in tri-fusion transgenic mice, but not in non-transgenic littermates. p/s/cm² /sr=photons per second per cm² per steradian; % ID/g = percentage of injection dose per gram.</p

Characterization of MACS purified iPSC pool and clones.

<p>(<b>A</b>) FACS analysis showing pool selected iPSCs (SVPool) stably express pluripotency markers TRA-1-60 and SSEA4 through long-term passaging up to p74, similar to three clonally derived iPSC lines (SV7, SV10, SV20) from the same donor. (<b>B</b>) Immunocytochemistry and (<b>C</b>) qRT-PCR analyses of mRNA levels from iPSCs showing SVPool and iPSC clones (p15–20) express similar levels of a panel of pluripotency markers tested. (<b>D</b>) SVPool and iPSC clones both show Sendai viral reprogramming vector clearance at p15–20 by RT-PCR analysis. (<b>E</b>) G-banded karyotyping was performed on SVPool and clones at p28–33 and all cell lines were found to be karyotypically normal. The data presented are chromosomal banding patterns of SVPool and one of the clones (SV10) for comparison.</p