G418-selected PSC-derived cardiomyocytes are functionally integrated with the host myocardium.

<p>(a) The presence of EGFP⁺ PSC-derived cardiomyocyte in the host heart at 2 weeks post transplant. (b) α-actinin immunofluorescence of the same donor cell as in (a). (c) Merge of (a) and (b). (d-f) Two-photon laser scanning microscopy revealed synchronous action potential-evoked calcium transients in the EGFP⁺ donor cell and its adjacent EGFP^-host cardiomyocytes. (d) 2-D images taken at 16-μm <i>z</i>-steps across an EGFP⁺ donor (D) myocyte and its surrounding host (H) myocytes. The heart was being paced at a frequency of 3 Hz during image acquisition. Periodic increases in rhod-2 fluoresence intensity reflect increases in cytosolic calcium in response to propagating action potentials and span the entire width of the image, including the EGFP⁺ donor cell. The dotted line denotes the position of the scan line for line-scan imaging. (e) Line average as a function of time for the portions of the dotted scan line indicated by brackets in (d). Line scans were obtained during spontaneous sinus rhythm and during electrical pacing at 3 Hz. Action potential-evoked calcium transients in the host myocyte are in phase with those in the neighboring host myocytes. (f) Superimposition of normalized calcium transients for the donor and host myocytes, demonstrating similar kinetics of the transients in both cell types.</p

Characterization of growth and transfection efficiency of parthenogenetic stem cells (PSCs).

<p>(a, b) Effects of STO-conditioned medium (CM) on maintaining undifferentiated status (a; n = 12) and cell viability of PSCs (b; n = 12) after 2 days’ culture. (c) Viability and transfection efficiency of PSCs under electroporation at different voltages (n = 8). Results were evaluated according to ds-Red expressing reporter.</p

Transfection of PSCs with the MHC-neo^r/pGK-hygro^r construct.

<p>(a) After hygromycin selection for 1 week, 7 MHC-neo^r/pGK-hygro^r PSC sublines were generated. As indicated by PCR, PSC subline #4 and subline #7 exhibited stable integration of MHC-neo^r and MHC-EGFP in the genome. (b) Even after consecutive passages, PCR analysis showed stable integration of the MHC-neo^r and MHC-EGFP in the genome of PSC subline #4. (c) RT-PCR showed that PSC subline #4 retained robust expression of <i>Nanog</i> and <i>Oct4</i> transcript after passages, at levels similar to the parental cells. (d) Schematic diagram of the cardiac induction protocol. (e, f) Cell viability (e; n = 12) and frequency of cardiomyogenic differentiation (f; n = 12) in embryoid bodies (EBs) with initially variable cell input of PSC subline #4. The PSCs were cultured for 10 days (as illustrated in d, 5 days’ hanging drop culture followed by 5 days’ adhesive culture, also designated as “5+5 process”). * <i>p</i> < 0.05 vs. any other group.</p

Cardiomyocyte enrichment.

<p>(a) Schematic diagram of enrichment process; (b-e) Progressive enrichment of EGFP⁺ cardiomyocytes over time (as indicated) with G418 selection. (f) Purity of EGFP⁺ cardiomyocytes at day 13, day 17 and day 24 during G418 selection (n = 12).</p

Schematic diagram of differentiation of parthenogenetic stem cells and cardiomyocyte enrichment.

<p>Schematic diagram of differentiation of parthenogenetic stem cells and cardiomyocyte enrichment.</p

Primer sequences and product sizes of targeted genes

<p>Primer sequences and product sizes of targeted genes</p

Cardiomyogenic differentiation and selection of PSCs.

<p>(a) RT-PCR analysis for stemness markers (Oct-4 and Nanog) and cardiac lineage-specific markers (Nkx2.5, GATA-4, MHC, MLC-2v) through the differentiation and selection processes. (b) Co-localization of EGFP and α-actinin immune reactivity confirmed the cardiomyogenic identity of derived EGFP⁺ cells (at day 17).</p

RhoA Ambivalently Controls Prominent Myofibroblast Characteritics by Involving Distinct Signaling Routes

<div><p>Introduction</p><p>RhoA has been shown to be beneficial in cardiac disease models when overexpressed in cardiomyocytes, whereas its role in cardiac fibroblasts (CF) is still poorly understood. During cardiac remodeling CF undergo a transition towards a myofibroblast phenotype thereby showing an increased proliferation and migration rate. Both processes involve the remodeling of the cytoskeleton. Since RhoA is known to be a major regulator of the cytoskeleton, we analyzed its role in CF and its effect on myofibroblast characteristics in 2 D and 3D models.</p><p>Results</p><p>Downregulation of RhoA was shown to strongly affect the actin cytoskeleton. It decreased the myofibroblast marker α-sm-actin, but increased certain fibrosis-associated factors like TGF-β and collagens. Also, the detailed analysis of CTGF expression demonstrated that the outcome of RhoA signaling strongly depends on the involved stimulus. Furthermore, we show that proliferation of myofibroblasts rely on RhoA and tubulin acetylation. In assays accessing three different types of migration, we demonstrate that RhoA/ROCK/Dia1 are important for 2D migration and the repression of RhoA and Dia1 signaling accelerates 3D migration. Finally, we show that a downregulation of RhoA in CF impacts the viscoelastic and contractile properties of engineered tissues.</p><p>Conclusion</p><p>RhoA positively and negatively influences myofibroblast characteristics by differential signaling cascades and depending on environmental conditions. These include gene expression, migration and proliferation. Reduction of RhoA leads to an increased viscoelasticity and a decrease in contractile force in engineered cardiac tissue.</p></div

Summary scheme of proposed mechanism.

<p>Summary scheme of proposed mechanism.</p

Knockdown of RhoA and actin depolymerization by Latrunculin A (LatA) results in changes in the Golgi apparatus structure.

<p>A) Representative immunofluorescence staining of the Golgi apparatus (640x, left) and structural analysis of Golgi apparatus size and density (right) of shControl and shRhoA NRCF (means ± SEM, n = 30, *p < 0.05). B) Immunofluorescence staining of actin (left) and Golgi apparatus (middle) in control and LatA treated NRCF (8.5 μM, 200x) In addition, higher magnifications are shown (640x). C) Bar graph summary of immunoblot analysis of β-actin normalized to total cell lysate protein, α-sm-actin, tubulin normalized to β-actin and acetylated tubulin normalized to total tubulin. Whole cell lysates were obtained from control and LatA treated NRCF. The relative change of expression in LatA treated NRCF to control NRCF is given (8.5 μM, n = 7).</p