Kardiomyogene Differenzierung von germline derived pluripotent stem cells auf dem Biomaterial Resomer® LR704

Stem cells with broad differentiation potential, such as the recently described germline-derived pluripotent stem cells (gPS cells), are an appealing source for tissue engineering strategies. Biomaterials can inhibit, support or induce proliferation and differentiation of stem cells. The present study identified (i) polymers which maintain self-renewal and differentiation potential of gPS cells for feeder-free expansion and (ii) polymers supporting the cardiomyogenic fate of gPS cells by analyzing a panel of polymers of an established biomaterial bank previously used to assess growth of diverse stem cell types. Identification of cytocompatible gPS cell/biomaterial combinations required analysis of several parameters including morphology, viability, cytotoxicity, apoptosis, proliferation and differentiation potential. Pluripotency of gPS cells was visualized by the endogenous Oct4-promoter-driven GFP and by Sox2 and Nanog immunofluorescence. Viability assay, proliferation assay and flow cytometry showed that gPS cells efficiently adhere and are viable on synthetic polymers, such as Resomer LR704 (poly(L-lactic-D,L-lactic acid), PTFE (poly(tetrafluor ethylene), PVDF (poly(vinylidene fluoride), and on gelatine-coated TCPS (tissue culture polystyrene). Expansion experiments showed that Resomer LR704 is an alternative substrate for feeder-free gPS cell maintenance. Resomer LR704, PTFE and PVDF were found to be suitable for gPS cell differentiation. Spontaneous beating in embryoid bodies cultured on Resomer LR704 occurred already on day 8 of differentiation, much earlier compared to the other surfaces. This indicates that Resomer LR704 supports spontaneous cardiomyogenic differentiation of gPS cells, which was also confirmed on molecular, protein and functional level

Chloroquine augments radiation-induced apoptosis in NPC cells.

<p>A) Cell cycle analysis. Radiation (6 Gy) induces a G2M arrest in all 5 NPC cell lines and the immortalized nasoepithelial cell line NP69. Radiation also induces apoptosis measured by an increase in subG1 in all NPC cell lines except of C666-1. Treatment with chloroquine (20 μM) significantly augments apoptosis in radiation-sensitive NPC cell lines compared to cells radiated only (CNE-2 and HNE-1: P<0.001; CNE-1: P<0.01; HONE-1: P<0.05; Student’s t-test). The data represent the means of three independent experiments and the corresponding standard error. (B) Combined treatment with chloroquine and radiation increases the number of cells with activated caspase-3 in radiation-sensitive NPC-cell lines. Quantitative data are reported as means ± S.E.M. (triplicate samples) (Student’s t- test; <b>*</b> = P<0.05; <b>**</b> = P<0.01; <b>***</b> = P<0.001). (C) Hoechst 33258 staining. Pretreatment with chloroquine before radiation increases the percentage of cells with morphological signs of apoptosis (condensed and fragmented nuclei) in radiation-sensitive NPC cell lines CNE-2 and HNE-1, but not in the radioresistant cell line C666-1 and the immortalized nasoepithelial cell line NP69. Morphologic changes were examined under a fluorescence microscope at 200x magnification, phase contrast images are shown for cell density comparison. Data of all experiments were shown at 72h after radiation.</p

Knock-down of ATG3, ATG5, ATG6 or ATG7 by siRNA substitutes for the enhancing effect of chloroquine on radiation-induced apoptosis in NPC cells.

<p>Silencing of ATG3, ATG5, ATG6 or ATG7 by specific siRNA enhances radiation-induced apoptosis to a similar extent as chloroquine in radiation-sensitive NPC cell lines treated with scrambled siRNA as shown by an increase in subG1-DNA-content (A) or increase of active caspase-3 (B) both measured by flow cytometry. After transfection the cells were treated as described before. Quantitative data are reported as means ± S.E.M. (triplicate samples) (Student ‘s t- test; <b>*</b> = P<0.05).</p

Chloroquine blocks radiation-induced autophagy in NPC cells.

<p>(A) Immunoblot for LC-I and LC-II. Preincubation with chloroquine before radiation increases expression of LC-II in radiation-sensitive NPC cells 8h post treatment (B) Immunofluorescence for autophagic vacuoles. Combined treatment of chloroquine and radiation increases the formation of autophagic vacuoles 8h post treatment in cell lines CNE-2 and HNE-1. Autophagic vacuoles were examined under a fluorescence microscope at 200x and 400x magnification. (C) Flow cytometric analysis of autophagic vacuoles. Combined treatment of chloroquine and radiation increases the number of cells with autophagic vacuoles 8h post treatment in radiation-sensitive NPC cell lines. (Student’s t- test; <b>*</b> = P<0.05; <b>**</b> = P<0.01; <b>***</b> = P<0.001) (D) Transmission electron microscopy. Photomicrographs show normal nuclear and mitochondrial morphologies in untreated cells and cells treated with chloroquine. Especially, in irradiated CNE-2 and HNE-1 cells the number of autophagosomes is significantly increased 8h following radiotherapy and further augmented by pretreatment with chloroquine.</p

Fibroblasts facilitate the engraftment of embryonic stem cell-derived cardiomyocytes on three-dimensional collagen matrices and aggregation in hanging drops

There is growing interest in the use of cardiomyocytes purified from embryonic stem (ES) cells for tissue engineering and cardiomyoplasty. However, most transplanted cells are lost shortly after transplantation due to the lack of integration into the host tissue and subsequent apoptosis. Here we examine whether murine embryonic fibroblasts (MEFs) can support the integration of purified murine ES cell-derived cardiomyocytes in a 3-dimensional tissue culture model based on a freezed-dryed collagen matrix with tubular structure. Collagen matrix was seeded either with cardiomyocytes alone or in combination with MEFs. The collagen sponges that were transplanted with cardiomyocytes alone showed neither morphological nor functional integration of viable cells. Cardiomyocytes also did not appear to be capable of attaching quantitatively to any of 16 different 2-dimensional biomaterials. However, cardiomyocytes co-cultured with MEFs formed fiber-like structures of rod-shaped cells with organized sarcomeric structure that contracted spontaneously. Electrical coupling between cardiomyocytes was suggested by strong expression of connexin 43. In addition, MEFs as well as cardiac fibroblasts supported re-aggregation of dissociated cardiomyocytes in hanging drops in the absence of collagen matrix. We conclude that fibroblasts promote cardiomyocyte engraftment and formation of functional 3-dimensional tissue in vitro. Elucidation of the mechanism of this phenomenon may help improve the integration of cardiomyocytes in vivo