Effects of Nanostructures and Mouse Embryonic Stem Cells on <i>In Vitro</i> Morphogenesis of Rat Testicular Cords

<div><p>Morphogenesis of tubular structures is a common event during embryonic development. The signals providing cells with topographical cues to define a cord axis and to form new compartments surrounded by a basement membrane are poorly understood. Male gonadal differentiation is a late event during organogenesis and continues into postnatal life. The cellular changes resemble the mechanisms during embryonic life leading to tubular structures in other organs. Testicular cord formation is dependent on and first recognized by SRY-dependent aggregation of Sertoli cells leading to the appearance of testis-specific cord-like structures. Here we explored whether testicular cells use topographical cues in the form of nanostructures to direct or stimulate cord formation and whether embryonic stem cells (ES) or soluble factors released from those cells have an impact on this process. Using primary cell cultures of immature rats we first revealed that variable nanogratings exerted effects on peritubular cells and on Sertoli cells (at less than <1000 cells/mm²) by aligning the cell bodies towards the direction of the nanogratings. After two weeks of culture testicular cells assembled into a network of cord-like structures. We revealed that Sertoli cells actively migrate towards existing clusters. Contractions of peritubular cells lead to the transformation of isolated clusters into cord-like structures. The addition of mouse ES cells or conditioned medium from ES cells accelerated this process. Our studies show that epithelial (Sertoli cell) and mesenchymal (peritubular cells) cells crosstalk and orchestrate the formation of cords in response to physical features of the underlying matrix as well as secretory factors from ES cells. We consider these data on testicular morphogenesis relevant for the better understanding of mechanisms in cord formation also in other organs which may help to create optimized in vitro tools for artificial organogenesis.</p> </div

Effects of mouse embryonic stem cells (OG2 cells expressing the GFP-transgene) on cord formation of rat testicular cells.

<p>3A) Micrographs showing immunofluorescence of GFP-signal (upper panels) and merged images (phase contrast plus immunofluorescence) of primary rat (7-day-old) Sertoli cells and peritubular cells seeded at an initial density of 10⁶ cells/cm² on flat PDMS substrates after culture for one day (upper two panels), three days (middle two panels) and six days (lower two panels) in DMEM. A variable number of OG2 cells (10, 10², 10³, 10⁴, 10⁵ cells/cm²) was added to the primary cells at the initiation of cell cultures. No OG2 cells were added to controls. No obvious change of cellular arrangements was observed at day 1. On days 3 and 6 of culture cord-like structure formation is observed which intensified with increasing numbers of OG2 cells. OG2 cells formed expanding colonies in contact with the cord-like structures. 3B) Analysis of the size of cord-like structures at day 6 of culture. Cord-like structures were significantly larger compared to all other experimental groups depending on the initial density of OG2 cells on flat PDMS substrates at day 6. In the control group and after addition of only 10 OG2 cells no cord like structures were encountered.</p

Combined effects of nanostructures and OG2 cell conditioned medium on cord formation of testicular cells.

<p>5A) Phase contrast micrographs of primary rat (7-day-old) testicular cells seeded at an initial density of 10⁶ cells/cm² on 350 nm (right panel) and flat (left panel) PDMS substrates in conditioned medium (Three day culture of OG2 cells at a density of 10⁵ cells/cm²) at day 1 (upper panel) and day 3 (lower panel) of culture. At day 1 orientation of cells in the direction of nanogrids was visible when compared to flat PDMS substrates. At day 3 of culture cellular aggregation and cord-like structure formation occurred on both flat and 350 nm PDMS substrates. The direction of cord-like structure was aligned with nanogratings on 350 nm PDMS substrates but not on flat PDMS substrates. Red arrows indicate the direction of nanogratings. Scale bar = 200 um. 5B) Analysis of the direction of cord-like structures in conditioned medium and after exposure to 350 nm or flat PDMS substrates at day 3 of culture. The quantitative results confirm the microscopic observation that nanogratings have an impact on the orientation of cord-like structures. Cord-like structures on flat PDMS show a random orientation. 5C) Analysis of the size of cord-like structures at day 3 of culture. Cord-like structures were of similar size irrespective of the exposure to nanogratings.</p

Cord like-structure formation in testicular cell cultures (see also Video S1).

<p>2A) Two series of three micrographs presenting the stages of cord-like structure formation in testicular cells cultured on 350 nm and flat PDMS substrates in DMEM at days 14, 14.5 and 15 of culture. Primary rat (7-day-old) Sertoli and peritubular cells were seeded at an initial density of 10⁶ cells/cm². At two weeks, cultures consisted of confluent mixed monolayers with several Sertoli cell clusters. Imaging was started at day 14 when first indications of cellular migration were observed. During the observation period active movements triggered a change of the confluent monolayers into cord-like structures on both 350 nm (upper panel) and flat PDMS substrates (lower panel). The orientation of cord-like structures did not follow the direction of nanogratings (red arrows). 2B) Testicular cell cultures performed under identical conditions as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060054#pone-0060054-g002" target="_blank">Fig. 2A</a> except that cells were seeded at a lower initial density of 10⁵ cells/cm² either on nanograting (left panel) or flat (right panel) PDMS substrates. Micrographs were taken at day 15 of culture. The orientation of cord-like structures was following the direction of nanogratings (red arrow). Scale bar = 200 um. 2C) Analysis of changes in the direction of cord-like structures seeded at high (10⁶ cells/cm²) or low (10⁵ cells/cm²) initial density on 350 nm PDMS substrates at day 15. 2D) Analysis of the size of individual cord-like structures seeded at high (10⁶ cells/cm²) or low (10⁵ cells/cm²) initial density on 350 nm and flat PDMS substrates at day 15.</p

Cell specific and density dependent response of testicular cells to nanogratings.

<p>Micrographs showing immunostaining of primary rat (7-day-old) Sertoli cells and peritubular cells. The cells were seeded at an initial density of 10⁶ cells/cm² in DMEM and cultured for one week. Cells were fixed at day 7. Peritubular cells are marked for α-smooth muscle actin (brown precipitate). Nuclei were stained blue with hematoxylin. 1A–D) Cells cultured on flat PDMS substrate (1A), and substrates carrying nanogratings of 200 nm (1B), 350 nm (1C) and 5 um (1D) dimensions. Red arrows indicate the direction of nanogratings. 1E) Quantitatitive analysis of directional changes in peritubular cells on different substrates. All nanogratings evoked changes in the orientation of peritubular cells. No visible influence on Sertoli cells was noted. 1F–H) Cells cultured on 350 nm PDMS substrate after inhomogeneous seeding creating diversity in plating density. Areas of high cell density (1F) revealed a random distribution of Sertoli cells in contrast to an aligned orientation towards nanogratings in low cell density zones (1G) on the same substrate in the same well. Peritubular cells were always oriented in accordance with the direction of the nanogratings. Quantification of cellular orientation in Sertoli cells (1H). Sertoli cell density was determined in randomly selected microscopic frames (as seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060054#pone-0060054-g001" target="_blank">Figs. 1F–G</a>) and the predominant cellular characteristic (either randomly shaped or spindle shaped (aligned)) was recorded for each frame. Seven recordings were performed per experiment and seven independent experiments were analyzed. We established that the threshold to respond to the nanogratings occurred at a density of approximately 1000 cells/mm². Scale bar = 50 um.</p

Irradiation induced molecular and phenotypic changes of EMT.

<p>(A) Radiation cell survival curves and the clonogenic figures of the control KYSE-150 cells without irradiation and radioresistance subclone KYSE-150/RR cells. (B) Morphology of KYSE-150 and KYSE-150/RR cells was examined with phase-contrast microscopy. (C) Expression of EMT markers (E-cadherin and vimentin) and transcription repressors of E-cadherin (Snail and Slug) were detected by qRT-PCR, data shown as mean ±SD, *<i>P</i> <0.05. Data represent means with standard deviation from three independent experiments. (D) Representative western blots of E-cadherin, vimentin, Snail and Slug were showed.</p

Irradiation-induced EMT enhanced cellular mobility.

<p>(A) KYSE-150 cells were subjected to a wound-healing assay with or without radiation at 100× magnification. Representative images were photographed right and 24 h after the scratch. (B) Representative images of migration assay and invasion assay of KYSE-150 cells with or without radiation were photographed after 24 h with crystal violet stain. (C) Summary graphs for migration and invasion (data shown as mean ±SD, * <i>P</i> <0.05).</p

Firefly-like Water Splitting Cells Based on FRET Phenomena with Ultrahigh Performance over 12%

A firefly-like chemiluminescence reaction was utilized in a ZrO₂ nanoparticle matrix of water splitting cells, where the chlorophyll of <i>Lantana camara</i> was used as the major photosensitizer to excite electrons to the conduction band of ZrO₂. The fluorescence resonance energy transfer (FRET) was induced by rubrene, a firefly-like chemiluminescence molecule, and <i>Lantana camara</i> chlorophyll combined with 9,10-diphenylanthracene. The ZrO₂ nanoparticle film coated by the chlorophyll of <i>Lantana camara</i> and 9,10-diphenylanthracene under chemiluminescence irradiation in 1 M KHCO₃ water solution demonstrated the highest photocurrent density (88.1 A/m²) and the highest water splitting efficiency (12.77%)

PTEN deficiency is required for irradiation-induced EMT.

<p>(A) Radiation significantly decreases the expression of PTEN in the KESE-150/RR cells no matter in mRNA level (data presented as mean ±SD, **<i>P</i> <0.01) and protein level (representative western blots). (B) The transfected efficiency of siPTEN in KYSE-150 (left) and pcDNA-PTEN in KYSE-150/RR cells (right) (data presented as mean ±SD, **<i>P</i> <0.01). (C) Representative western blot analysis showed expression of PTEN, E-cadherin and vimentin in KYSE-150 cells transfected with siPTEN or vehicle, or KYSE-150/RR cells transfected with pcDNA-PTEN or pcDNA3.0. Data shown represent three different experiments. (D) Representative images of migration and invasion assay of KYSE-150 cells transfected with siPTEN or vehicle after 48 h (left); Summary graphs is for migration and invasion (right, data shown as mean ±SD, *<i>P</i> <0.05). (E) Representative images of migration assay and invasion assay of KYSE-150/RR cells transfected with pcDNA-PTEN or pcDNA3.0 were photographed after 24 h with crystal violet stain (left); Summary graphs for migration and invasion (right, data shown as mean ±SD, *<i>P</i> <0.05). siPTEN is short for siRNA-PTEN.</p

Proposed pathway for irradiation induced EMT.

<p>PTEN acts as an upstream regulator of the PI3K/Akt/GSK-3β signaling network to evoke the cascades of EMT. Slug regulates E-cadherin expression in a PTEN independent way.</p