hsa_circ_0000417 downregulation suppresses androgen receptor expression and apoptotic signals in human foreskin fibroblasts via sponging miR-6756-5p.

BACKGROUND: Dysregulated apoptosis of penile mesenchymal cells during male urethragenesis has been previously demonstrated to underly hypospadiac urethral closure failure, and androgen receptor (AR) has been shown to play a central role in regulating penile mesenchyme cell proliferation and survival. However, the regulatory mechanisms upstream and downstream of AR remain poorly understood. Our clinical data and bioinformatics analysis previously indicated that hsa_circ_0000417, a circRNA significantly downregulated in hypospadias preputial specimens, may act as a ceRNA for AR via sequestering hsa_miR-6756-5p, and that the biological functions of hsa_circ_0000417 may significantly involve the PI3K/AKT pathway. In this study, we employed human foreskin fibroblasts (HFF-1) to experimentally validate this putative hsa_circ_0000417/miR-6756-5p/AR axis and its impact on penile mesenchymal cell proliferation and apoptosis. METHOD AND RESULTS: We showed that hsa_circ_0000417 knockdown significantly promoted proliferation and suppressed apoptosis of HFF-1 cells. Mechanistically, hsa_circ_0000417 functioned as a molecular sponge for miR-6756-5p in HFF-1 cells and relieved the latter's translational repression on AR mRNA, leading to decreased AKT activation and increased expression of pro-apoptotic proteins BAX and cleaved-caspase 9. Conversely, elevated levels of miR-6756-5p resulted in diminished AR expression concomitant with enhanced AKT activation and HFF-1 cell proliferation. CONCLUSIONS: Collectively, our data describe for the first time a circRNA-mediated post-transcriptional regulatory mechanism of AR and its functional consequences in penile mesenchymal cells in the context of hypospadias. These findings may contribute to advancing our current understanding of the roles of AR and mesenchymal cell fate decisions during penile morphogenesis.

Synthesis and Antiparasitic Activity of New Trithiolato-Bridged Dinuclear Ruthenium(II)-arene-carbohydrate Conjugates.

Eight novel carbohydrate-tethered trithiolato dinuclear ruthenium(II)-arene complexes were synthesized using CuAAC 'click' (Cu(I)-catalyzed azide-alkyne cycloaddition) reactions, and there in vitro activity against transgenic T. gondii tachyzoites constitutively expressing ß-galactosidase (T. gondii ß-gal) and in non-infected human foreskin fibroblasts, HFF, was determined at 0.1 and 1 µM. When evaluated at 1 µM, seven diruthenium-carbohydrate conjugates strongly impaired parasite proliferation by >90%, while HFF viability was retained at 50% or more, and they were further subjected to the half-maximal inhibitory concentration (IC50) measurement on T. gondii ß-gal. Results revealed that the biological activity of the hybrids was influenced both by the nature of the carbohydrate (glucose vs. galactose) appended on ruthenium complex and the type/length of the linker between the two units. 23 and 26, two galactose-based diruthenium conjugates, exhibited low IC50 values and reduced effect on HFF viability when applied at 2.5 µM (23: IC50 = 0.032 µM/HFF viability 92% and 26: IC50 = 0.153 µM/HFF viability 97%). Remarkably, compounds 23 and 26 performed significantly better than the corresponding carbohydrate non-modified diruthenium complexes, showing that this type of conjugates are a promising approach for obtaining new antiparasitic compounds with reduced toxicity.

Repression of Mafb promotes foreskin fibroblast proliferation through upregulation of CDK2, cyclin E and PCNA.

Mafb plays a significant role in the development and differentiation of various organs, tissues and cells. Nevertheless, its role in the control of external genital cell proliferation and function in the mechanism of hypospadias remains unknown. In this study, the expression of Mafb in foreskin fibroblasts was inhibited by siRNA. The Cell Count Kit-8 (CCK-8) assay showed cell proliferation increased after transfection, and the number of cells entered the S phase significantly increased via flow cytometry. Both mRNA and protein levels of cyclin E, cyclin-dependent kinase 2 (CDK2) and proliferating cell nuclear antigen (PCNA) were significantly upregulated in the siRNA group. Meanwhile, twenty-five prepuce tissue samples were collected from hypospadias repair surgery. These samples were divided into two groups: the severe and mild groups. Normal prepuce tissue specimens were obtained during circumcision as the normal control. The upregulated expression of cyclin E, CDK2 and PCNA and downregulated Mafb expression were observed in the hypospadias group. This study reveals for the first time that the reduction in Mafb promotes the foreskin fibroblast proliferation. Thus, downregulated Mafb expression may cause hypospadias by upregulating CDK2, cyclin E and PCNA. These findings can shed new light on the embryonic development of the urethra.

New Nucleic Base-Tethered Trithiolato-Bridged Dinuclear Ruthenium(II)-Arene Compounds: Synthesis and Antiparasitic Activity.

Aiming toward compounds with improved anti-Toxoplasma activity by exploiting the parasite auxotrophies, a library of nucleobase-tethered trithiolato-bridged dinuclear ruthenium(II)-arene conjugates was synthesized and evaluated. Structural features such as the type of nucleobase and linking unit were progressively modified. For comparison, diruthenium hybrids with other type of molecules were also synthesized and assessed. A total of 37 compounds (diruthenium conjugates and intermediates) were evaluated in a primary screening for in vitro activity against transgenic Toxoplasma gondii tachyzoites constitutively expressing ß-galactosidase (T. gondii ß-gal) at 0.1 and 1 µM. In parallel, the cytotoxicity in non-infected host cells (human foreskin fibroblasts, HFF) was determined by alamarBlue assay. Twenty compounds strongly impairing parasite proliferation with little effect on HFF viability were subjected to T. gondii ß-gal half maximal inhibitory concentration determination (IC50) and their toxicity for HFF was assessed at 2.5 µM. Two promising compounds were identified: 14, ester conjugate with 9-(2-oxyethyl)adenine, and 36, a click conjugate bearing a 2-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl substituent, with IC50 values of 0.059 and 0.111 µM respectively, significantly lower compared to pyrimethamine standard (IC50 = 0.326 µM). Both 14 and 36 exhibited low toxicity against HFF when applied at 2.5 µM and are candidates for potential treatment options in a suitable in vivo model.

Comparison of two methods for isolation and culture of human foreskin fibroblasts. / ä¸¤ç§äººåŒ çš®æˆçº¤ç»´ç»†èƒžåˆ†ç¦»åŸ¹å »æ–¹æ³•çš„æ¯”è¾ƒ.

OBJECTIVES: The efficient acquisition and purification of fibroblasts as ideal seed cells are very important. For optimization of the isolation and culture of human foreskin fibroblasts (HFF), we compared the improved tissue culture method (ITCM) and the enzyme digestion method (EDM). METHODS: In ITCM, the skin tissue was digested with 0.1% Type II collagenase overnight at 4 ℃, the epidermis was separated from the dermis and digested again with 0.25% trypsin at room temperature for 15 min, and then the tissue block was attached to the culture dish. In EDM, the skin tissue was digested with 0.25% trypsin overnight at 4 ℃, the epidermis was separated from the dermis and digested with 0.1% Type II collagenase overnight at 4 ℃, the tissue block was filtered and squeezed together with the enzyme mixture, the filter was rinsed with medium containing fetal bovine serum, and the cell suspension was cultured. Both ITCM and EDM used 2 digestion enzymes, but the order, digestion time, and temperature of the 2 enzymes were different. The final inoculations of ITCM and EDM in the dishes for subsequent culture were tissue blocks and cell suspensions, respectively. In this study, HFF cells were isolated and cultured with ITCM and EDM, and the cell morphology was observed from Passage 0 to Passage 3 in the ITCM and EDM groups. The cell purity was identified by staining for vimentin, CD68, and Pan-keratin. The growth curves of Passage 3 were plotted to compare the proliferation ability of the 2 groups. Passage 3 HFF cells in the ITCM and EDM groups were irradiated with medium-wave ultraviolet (UVB) at an energy value of 120 mJ/cm2 to establish a light damage model. The experiments were grouped into an UVB group and a control group (Control) according to the presence or absence of UVB irradiation. Platelet-poor plasma (PPP) was extracted by secondary centrifugation, and the HFF cells of ITCM and EDM groups were cultured in groups using complete medium containing different concentrations (0, 2.5%, 5.0%, and 10.0%) of PPP, and the proliferation of damaged cells was detected by cell counting kit-8 after 24 h of PPP incubation. RESULTS: A large number of HFF could be observed in the ITCM group up to day 3, which was less affected by impurities; the observation of HFF morphology in the EDM group was affected by more impurities. By day 9, cells in both ITCM and EDM groups could be passaged; HFF isolated and cultured in vitro by the 2 methods showed long spindle-shaped, swirling growth. The positive rates of vimentin in the ITCM and EDM groups when HFF cells were cultured up to Passage 2 were significantly different [(97.36±0.76)% vs (99.4±0.56)%, P<0.01)]. The positive rates of CD68 were also significantly different [(70.8±0.46)% vs (78.37±0.75)%, P<0.01]. The expressions of pan-keratin in the ITCM group and the EDM group were positive and negative, respectively. There was no difference in vimentin and pan-keratin staining results between the ITCM group and the EDM group when HFF were cultured to Passage 3. The positive rates of CD68 between the ITCM group and the EDM group were significantly different [(74.73±1.37)% vs (85.27±2.63)%, P<0.001]. The proliferative capacity of HFF cells in Passage 3 was significantly higher in the EDM group than that in the ITCM group (P<0.05). After UVB (120 mJ/cm2) irradiation, HFFs procured by the 2 isolation methods showed damage. The damage repair test demonstrated that the 2.5% PPP+UVB irradiation group showed significantly higher repair competence than the other groups (all P<0.05). CONCLUSIONS: In contrast with HFFs isolated via ITCM, HFF cells isolated by EDM have a faster purification rate and a stronger proliferative capacity. Therapy with PPP can moderately repair UVB-induced damage to HFFs. The results provide a theoretical basis for clinical treatment studies in the future.

Comparative Proteomic Analysis Identifies EphA2 as a Specific Cell Surface Marker for Wharton's Jelly-Derived Mesenchymal Stem Cells.

Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) are a valuable tool in stem cell research due to their high proliferation rate, multi-lineage differentiation potential, and immunotolerance properties. However, fibroblast impurity during WJ-MSCs isolation is unavoidable because of morphological similarities and shared surface markers. Here, a proteomic approach was employed to identify specific proteins differentially expressed by WJ-MSCs in comparison to those by neonatal foreskin and adult skin fibroblasts (NFFs and ASFs, respectively). Mass spectrometry analysis identified 454 proteins with a transmembrane domain. These proteins were then compared across the different cell-lines and categorized based on their cellular localizations, biological processes, and molecular functions. The expression patterns of a selected set of proteins were further confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blotting, and immunofluorescence assays. As anticipated, most of the studied proteins had common expression patterns. However, EphA2, SLC25A4, and SOD2 were predominantly expressed by WJ-MSCs, while CDH2 and Talin2 were specific to NFFs and ASFs, respectively. Here, EphA2 was established as a potential surface-specific marker to distinguish WJ-MSCs from fibroblasts and for prospective use to prepare pure primary cultures of WJ-MSCs. Additionally, CDH2 could be used for a negative-selection isolation/depletion method to remove neonatal fibroblasts contaminating preparations of WJ-MSCs.

CRISPR/dCas9-mediated activation of multiple endogenous target genes directly converts human foreskin fibroblasts into Leydig-like cells.

Recently, Leydig cell (LC) transplantation has been revealed as a promising strategy for treating male hypogonadism; however, the key problem restricting the application of LC transplantation is a severe lack of seed cells. It seems that targeted activation of endogenous genes may provide a potential alternative. Therefore, the aim of this study was to determine whether targeted activation of Nr5a1, Gata4 and Dmrt1 (NGD) via the CRISPR/dCas9 synergistic activation mediator system could convert human foreskin fibroblasts (HFFs) into functional Leydig-like cells. We first constructed the stable Hsd3b-dCas9-MPH-HFF cell line using the Hsd3b-EGFP, dCas9-VP64 and MS2-P65-HSF1 lentiviral vectors and then infected it with single guide RNAs. Next, we evaluated the reprogrammed cells for their reprogramming efficiency, testosterone production characteristics and expression levels of Leydig steroidogenic markers by quantitative real-time polymerase chain reaction or Western blotting. Our results showed that the reprogramming efficiency was close to 10% and that the reprogrammed Leydig-like cells secreted testosterone rapidly and, more importantly, responded effectively to stimulation with human chorionic gonadotropin and expressed Leydig steroidogenic markers. Our findings demonstrate that simultaneous targeted activation of the endogenous NGD genes directly reprograms HFFs into functional Leydig-like cells, providing an innovative technology that may have promising potential for the treatment of male androgen deficiency diseases.

Differentiation of human foreskin fibroblast-derived induced pluripotent stem cells into hepatocyte-like cells.

The aim of this study was to investigate the differentiation potential of induced pluripotent stem cells (iPSCs) derived from human foreskin fibroblasts (HFFs) into hepatocyte-like cells (HLCs). The iPSCs were firstly induced by transduction of OCT4, SOX2, KLF4, and c-MYC into HFFs using retrovirus. Afterwards, expressions of pluripotency factors were identified by semiquantitative reverse transcription-polymerase chain reaction and immunofluorescence staining, and karyotype, embryoid, and teratoma were observed by microscope. Then, iPSCs were gradually differentiated into endoderm cells, hepatic progenitor cells, and mature HLCs by special culture medium. During this process, differentiation efficiency into each kind of cells was evaluated by detecting SOX17, HNF4a, and ALB using flow cytometry, respectively. Besides, enzyme-linked immunosorbent assay was conducted to detect the secretion of ALB in iPSC-induced HLCs and quantitative reverse transcription-polymerase chain reaction was performed to detect the expression levels of hepatocyte-specific genes. The iPSCs were successfully induced by HFFs, which exhibited typical embryonic stem cells morphology, positive alkaline phosphatase staining, normal diploid karyotype, and positive expression of various pluripotency factors. Meanwhile, spherical embryoid and teratoma with 3 germ layers were formed by iPSCs. The iPSCs were consecutively induced into endoderm cells, hepatic progenitor cells and mature HLCs, and the differentiation efficiency was 55.7 ± 2.9%, 45.7 ± 4.8%, and 35.0 ± 3.9%, respectively. Besides, the secretion of ALB and expression of various hepatocyte-specific genes was highly detected in iPSC-induced HLCs. The iPSCs were successfully derived from HFFs and then differentiated into HLCs, which proved a new source for hepatocyte transplantation. HIGHLIGHTS: HFFs were successfully induced into iPSCs by transduction of OCT4, SOX2, KLF4, and c-MYC. Positive expressions of various pluripotency factors were exhibited in HFFs-induced iPSCs. The iPSCs were consecutively induced into endoderm cells, hepatic progenitor cells, and mature HLCs. Various hepatocyte-specific genes were highly expressed in iPSC-induced HLCs.

Xeno-free generation of new Yazd human embryonic stem cell lines (Yazd4-7) as a prior stage toward good manufacturing practice of clinical-grade raw materials from discarded embryos: A lab resources report.

Background: Xeno-free generation of human embryonic stem cells (hESCs) is important to prevent potential animal contaminations in culture for advanced cell-based therapeutic applications. Xeno-free production of hESCs is the first step for manufacturing clinical-grade hESC lines. Objective: To produce new hESC lines in xeno-free condition. Materials and Methods: This lab resources report was conducted at Stem Cell Biology Research Center, Yazd, Iran from 2019-2022. 4 new hESC lines from 11 (10 fresh and 1 frozen) donated surplus discarded human embryos were established. In this study, we report the xeno-free derivation of new Yazd hESC lines (Yazd4-7), without using immunosurgery, by culturing intact zona-free blastocysts obtained from discarded embryos onto the YhFF#8 cells as a feeder layer in a microdrop culture system. The pluripotency gene expression profile of the cell lines was assessed by reverse transcription polymerase chain reaction and the expression of specific surface markers was detected using immunofluorescent staining. In vitro differentiation was induced using embryoid body formation and gene expression profile of 3 germ layers and germ cells. Reverse transcriptase polymerase chain reaction was investigated to prove their pluripotent capacity. Results: In sum, we have been able to generate 4 new hESC lines (Yazd4-7) from 11 discarded embryos in xeno-free culture conditions using a micro drop culture system and YhFF#8 as a human source feeder layer. Conclusion: The outcome of this work can be the foundation for the future allogeneic cell-based therapeutic application using clinical grade good manufacturing practice-derived hESC derivatives.

Comparative analysis of chlorambucil-induced DNA lesion formation and repair in a spectrum of different human cell systems.

Chlorambucil (CLB) belongs to the class of nitrogen mustards (NMs), which are highly reactive bifunctional alkylating agents and were the first chemotherapeutic agents developed. They form DNA interstrand crosslinks (ICLs), which cause a blockage of DNA strand separation, inhibiting essential processes in DNA metabolism like replication and transcription. In fast replicating cells, e.g., tumor cells, this can induce cell death. The upregulation of ICL repair is thought to be a key factor for the resistance of tumor cells to ICL-inducing cytostatic agents including NMs. To monitor induction and repair of CLB-induced ICLs, we adjusted the automated reversed fluorometric analysis of alkaline DNA unwinding assay (rFADU) for the detection of ICLs in adherent cells. For the detection of monoalkylated DNA bases we established an LC-MS/MS method. We performed a comparative analysis of adduct formation and removal in five human cell lines and in peripheral blood mononuclear cells (PBMCs) after treatment with CLB. Dose-dependent increases in adduct formation were observed, and suitable treatment concentrations were identified for each cell line, which were then used for monitoring the kinetics of adduct formation. We observed significant differences in the repair kinetics of the cell lines tested. For example, in A2780 cells, hTERT immortalized VH10 cells, and in PBMCs a time-dependent repair of the two main monoalkylated DNA-adducts was confirmed. Regarding ICLs, repair was observed in all cell systems except for PBMCs. In conclusion, LC-MS/MS analyses combined with the rFADU technique are powerful tools to study the molecular mechanisms of NM-induced DNA damage and repair. By applying these methods to a spectrum of human cell systems of different origin and transformation status, we obtained insight into the cell-type specific repair of different CLB-induced DNA lesions, which may help identify novel resistance mechanisms of tumors and define molecular targets for therapeutic interventions.

Damage prevention effect of milk-derived peptides on UVB irradiated human foreskin fibroblasts and regulation of photoaging related indicators.

Long-term exposure to UVB can trigger acute inflammation of the skin and lead to skin photoaging. To scrutinize the anti-photoaging functions of peptides obtained from milk, the physicochemical including molecular weight and amino acid compositions were first analyzed. Totally 267 peptides were screened out and identified by PEAKS X software, and then evaluated through Peptide Ranker and BIOPEP-UMW. Six peptides with the highest antioxidant ability and relative abundance were selected. This study was then conducted in UVB-damaged human foreskin fibroblasts with proadministration of peptides. The results indicated that at concentrations of 0.08-0.10 mg/mL, milk-derived peptides could realize a damage prevention effect through inhibiting the generation of reactive oxygen species (ROS) and lipid peroxidation malondialdehyde (MDA). Also, these peptides were found to promote the photoaging related enzyme activities of superoxide dismutase (SOD) and catalase (CAT), while to block the production of matrix metalloproteinases-1. Through this study, we found that milk-derived peptide mixture is effective in preventing photoaging damage. Milk-derived peptides found in this study could serve as raw materials for future development of antioxidant functional foods.

Silibinin treatment protects human skin cells from UVB injury through upregulation of estrogen receptors.

Ultraviolet B (UVB) from the sunlight is a major environmental cause for human skin damages, inducing cell death, inflammation, senescence and even carcinogenesis. The natural flavonoid silibinin, clinically used as liver protectant, has protective effects against UVB-caused skin injury in vivo and in vitro. Silibinin is often classified as a phytoestrogen, because it modulates the activation of estrogen receptors (ERs). However, whether silibinin's estrogenic effect contributes to the skin protection against UVB injury remains to be elucidated. The issue was explored in this study by using the human foreskin dermal fibroblasts (HFF) and human non-malignant immortalized keratinocytes (HaCaT). In HFF, pre-treatment with silibinin rescued UVB-irradiated cells from apoptosis. Interestingly, silibinin increased the whole cellular and nuclear levels of ERα and ERß in UVB-irradiated cells. Activation of ERs by treatment with estradiol elevated the cell survival and reduced apoptosis in UVB-treated cells. ERα agonist increased cell survival, while its antagonist decreased it. ERß agonist also increased cell survival, but the antagonist had no effect on cell survival. Transfection of the cells with the small interfering RNAs (si-RNAs) to ERα or ERß diminished the protective effect of silibinin on UVB-irradiated cells. In UVB-treated HaCaT cells, both ERα and ERß were increased by silibinin treatment. Inhibition of activation and expression of ERα or ERß by specific antagonists and si-RNAs, respectively, reduced cell survival in UVB-treated HaCaT cells regardless of silibinin treatment. Taken together, it is summarized that silibinin up-regulates both ERα and ERß pathways in UVB-treated dermal HFF cells and epidermal HaCaT cells, leading to protection of skin from UVB-damage.

Lineage reprogramming of fibroblasts into induced cardiac progenitor cells by CRISPR/Cas9-based transcriptional activators.

Overexpression of exogenous lineage-determining factors succeeds in directly reprogramming fibroblasts to various cell types. Several studies have reported reprogramming of fibroblasts into induced cardiac progenitor cells (iCPCs). CRISPR/Cas9-mediated gene activation is a potential approach for cellular reprogramming due to its high precision and multiplexing capacity. Here we show lineage reprogramming to iCPCs through a dead Cas9 (dCas9)-based transcription activation system. Targeted and robust activation of endogenous cardiac factors, including GATA4, HAND2, MEF2C and TBX5 (G, H, M and T; GHMT), can reprogram human fibroblasts toward iCPCs. The iCPCs show potentials to differentiate into cardiomyocytes, smooth muscle cells and endothelial cells in vitro. Addition of MEIS1 to GHMT induces cell cycle arrest in G2/M and facilitates cardiac reprogramming. Lineage reprogramming of human fibroblasts into iCPCs provides a promising cellular resource for disease modeling, drug discovery and individualized cardiac cell therapy.

Inorganic polyphosphate in platelet rich plasma accelerates re-epithelialization in vitro and in vivo.

Wound healing requires well-coordinated events including hemostasis, inflammation, proliferation, and remodeling. Delays in any of these stages leads to chronic wounds, infections, and hypertrophic scarring. Burn wounds are particularly problematic, and may require intervention to ensure timely progression to reduce morbidity and mortality. To accelerate burn wound healing, Platelet-Rich Plasma (PRP)1 can be of value, since platelets release growth factor proteins and inorganic polyphosphates (polyP) that may be integral to wound healing. We used polyP-depleted keratinocyte (HaCaT) and fibroblast cell culture models to determine cell proliferation and scratch-wound repair to determine if polyP, platelet lysate, or combined treatment could accelerate wound healing. While polyP and PRP significantly reduced the open scratch-wound area in fibroblasts and keratinocytes, polyP had no effect on keratinocyte or fibroblast proliferation. PRP was also evaluated as a treatment in a murine model of full thickness wound healing in vivo, including a treatment in which PRP was supplemented with purified polyP. PRP induced significantly more rapid re-epithelialization by Day 3. Pure polyP enhanced the effects of PRP on epithelial tongues, which were significantly elongated in the PRP + high-dose polyP treatment groups compared to PRP alone. Thus, PRP and polyP may serve as an effective therapeutic combination for treating wounds.

Attempts for Generation of Embryonic Stem Cells from Human Embryos Following In Vitro Embryo Twinning.

In vitro embryo twinning can be used to increase the number of the human embryos available for production of human embryonic stem cell (hESC) lines. The aim of this study was to generate hESCs following the production of the twin embryos by in vitro embryo splitting procedures. In total 21 chromosomally abnormal (three pronuclei) embryos underwent in vitro embryo twinning and were allowed to develop to the blastocyst stage. As a result, 42 twin embryos were obtained, of which 24 developed to blastocyst stage. Using micromanipulation technique, the zona-free blastocysts were recovered and plated onto mitotically inactivated Yazd human foreskin fibroblast (Batch18; YhFF#18) feeder layers in microdrops. After 3 to 5 days of blastocyst culture onto human foreskin fibroblast feeder layers, the hESC-like outgrowths were passaged onto new feeders in microdrops. The initial outgrowths of hESC-like cells were generated, and cells were proliferated, passaged, and some of them expressed hESC and trophoblastic markers; however, no cell lines were established. This might be due to the low cell number and poor quality of inner cell mass within these twin blastocysts. In vitro embryo twinning by increasing the number of the human embryos could be useful in the future for the generation of new pluripotent stem cell lines. However, the challenge remains to optimize the methods.

Organelle dynamics and viral infections: at cross roads.

Viruses are obligate intracellular parasites of the host cells. A commonly accepted view is the requirement of internal membranous structures for various aspects of viral life cycle. Organelles enable favourable intracellular environment for several viruses. However, studies reporting organelle dynamics upon viral infections are scant. In this review, we aim to summarize and highlight modulations caused to various organelles upon viral infection or expression of its proteins.

Direct conversion of human fibroblasts into functional Leydig-like cells by SF-1, GATA4 and NGFI-B.

The reprogramming of fibroblasts to induced pluripotent stem cells raises the possibility that a somatic cell can be reprogrammed to an alternative, differentiated fate without first becoming a stem/progenitor cell. Recent work has shown that fibroblasts can be reprogrammed to other, terminally differentiated cells with a combination of several transcription factors. Here, we report that a combination of four developmental transcription factors; GATA4, SF-1, NGFI-B, and COUP TF2; efficiently reprogrammed human foreskin fibroblasts into functional induced Leydig-like cells (iLCs). The iLCs expressed Leydig-specific markers and secreted testosterone in vitro. We found that GATA4 and SF-1 were particularly critical for Leydig-specific markers expression and that GATA4, SF-1, and NGFI-B were necessary to generate functional iLCs that secreted testosterone. These findings demonstrate that fibroblasts can be directly converted into iLCs with a few, defined factors and may provide insight into potential therapies to treat testosterone deficiency.

A biocompatible sodium alginate/povidone iodine film enhances wound healing.

In the last few years, there has been an increasing tendency to use natural polymers for the fabrication of dressings for wound and burn management. Among them, alginate, a polysaccharide extracted primarily from marine algae, exhibits attractive properties being non-toxic, hydrophilic and biodegradable. The aim of this study was to characterize the in vitro biocompatibility and the efficacy of a composite polymeric material based on sodium alginate (NaAlg) and povidone iodine (PVPI) complex in a mouse model of wound healing. The developed material combines the excellent wound healing properties of alginates with the bactericidal and fungicidal properties of PVPI, providing a controlled antiseptic release. We demonstrated that the NaAlg/PVPI films are able to reduce the inflammatory response both in human foreskin fibroblasts after lipopolysaccharide (LPS) stimulus and in rodents after wound induction. Furthermore, the NaAlg/PVPI film-treated animals showed a significantly higher wound closure compared to untreated animals at each time point considered. Interestingly, the complete wound closure was achieved within 12 days only in the film-treated group, indicating that the full-thickness wounds healed more rapidly in these animals. The results demonstrate that the NaAlg/PVPI films are biocompatible and possess healing properties that accelerate the wound closure.

Surface Treatment of PEOT/PBT (55/45) with a Dielectric Barrier Discharge in Air, Helium, Argon and Nitrogen at Medium Pressure.

This work describes the surface modification of 300PEO-PEOT/PBT 55/45 thin films using a medium pressure dielectric barrier discharge system operated in argon, helium, nitrogen or dry air to improve cell-surface interactions of this established biomaterial. The first part of the paper describes the optimization of the plasma processing parameters using water contact angle goniometry. The optimized samples are then characterized for changes in surface topography and surface chemical composition using atomic force microscopy (AFM) and X-ray fluorescence spectroscopy (XPS) respectively. For all plasma treatments, a pronounced increase in surface wettability was observed, of which the extent is dependent on the used plasma discharge gas. Except for dry air, only minor changes in surface topography were noted, while XPS confirmed that the changes in wettability were mainly chemical in nature with the incorporation of 5-10% of extra oxygen as a variety of polar groups. Similarly, for the nitrogen plasma, 3.8% of nitrogen polar groups were additionally incorporated. Human foreskin fibroblast (HFF) in vitro analysis showed that within the first 24 h after cell seeding, the effects on cell-surface interactivity were highly dependent on the used discharge gas, nitrogen plasma treatment being the most efficient. Differences between untreated and plasma-treated samples were less pronounced compared to other biodegradable materials, but a positive influence on cell adhesion and proliferation was still observed.

Development and Characterization of a Porcine Mitral Valve Scaffold for Tissue Engineering.

Decellularized scaffolds represent a promising alternative for mitral valve (MV) replacement. This work developed and characterized a protocol for the decellularization of whole MVs. Porcine MVs were decellularized with 0.5% (w/v) SDS and 0.5% (w/v) SD and sterilized with 0.1% (v/v) PAA. Decellularized samples were seeded with human foreskin fibroblasts and human adipose-derived stem cells to investigate cellular repopulation and infiltration, and with human colony-forming endothelial cells to investigate collagen IV formation. Histology revealed an acellular scaffold with a generally conserved histoarchitecture, but collagen IV loss. Following decellularization, no significant changes were observed in the hydroxyproline content, but there was a significant reduction in the glycosaminoglycan content. SEM/TEM analysis confirmed cellular removal and loss of some extracellular matrix components. Collagen and elastin were generally preserved. The endothelial cells produced newly formed collagen IV on the non-cytotoxic scaffold. The protocol produced acellular scaffolds with generally preserved histoarchitecture, biochemistry, and biomechanics.