Carob extract induces spermatogenesis in an infertile mouse model via upregulation of Prm1, Plzf, Bcl-6b, Dazl, Ngn3, Stra8, and Smc1b.

ETHNOPHARMACOLOGICAL RELEVANCE: Ethnopharmacological studies for drug discovery from natural compounds play an important role for developing current therapeutical platforms. Plants are a group of natural sources which have been served as the basis in the treatment of many diseases for centuries. In this regard, Ceratonia siliqua (carob) is one of the herbal medicine which is traditionally used for male infertility treatments. But so far the main mechanisms for effects of carob are unknown. Here, we intend to investigate the ability of carob extract to induce spermatogenesis in an azoospermia mouse model and determine the mechanisms that underlie its function. AIM OF THE STUDY: This is a pre-clinical animal model study to evaluate the effect of carob extract in spermatogenesis recovery. METHODS: We established an infertile mouse model with the intent to examine the ability of carob extract as a potential herbal medicine for restoration of male fertility. Sperm parameters, as well as gene expression dynamics and levels of spermatogenesis hormones, were evaluated 35 days after carob administration. RESULTS: Significant enhanced sperm parameters (P < 0.05) showed that the carob extract could induce spermatogenesis in the infertile mouse model. Our data suggested an anti-apototic and inducer role in the expressions of cell cycle regulating genes. Carob extract improved the spermatogenesis niche by considerable affecting Sertoli and Leydig cells (P < 0.05). The carob-treated mice were fertile and contributed to healthy offspring that matured. Our data confirmed that this extract triggered the hormonal system, the spermatogenesis-related gene expression network, and signaling pathways to induce and promote sperm production with notable level (P < 0.05). We found that the aqueous extract consisted of a polar and mainly well water-soluble substance. Carob extract might upregulate spermatogenesis hormones via its amino acid components, which were detected in the extract by liquid chromatography-mass spectrometry (LC-MS). CONCLUSION: Our results strongly suggest that carob extract might be a promising future treatment option for male infertility. This finding could pave the way for clinical trials in infertile men. This is the first study that has provided reliable, strong pre-clinical evidence for carob extract as an effective candidate for fertility recovery in cancer-related azoospermia.

Organoid technology in female reproductive biomedicine.

Recent developments in organoid technology are revolutionizing our knowledge about the biology, physiology, and function of various organs. Female reproductive biology and medicine also benefit from this technology. Organoids recapitulate features of different reproductive organs including the uterus, fallopian tubes, and ovaries, as well as trophoblasts. The genetic stability of organoids and long-lasting commitment to their tissue of origin during long-term culture makes them attractive substitutes for animal and in vitro models. Despite current limitations, organoids offer a promising platform to address fundamental questions regarding the reproductive system's physiology and pathology. They provide a human source to harness stem cells for regenerative medicine, heal damaged epithelia in specific diseases, and study biological processes in healthy and pathological conditions. The combination of male and female reproductive organoids with other technologies, such as microfluidics technology, would enable scientists to create a multi-organoid-on-a-chip platform for the next step to human-on-a-chip platforms for clinical applications, drug discovery, and toxicology studies. The present review discusses recent advances in producing organoid models of reproductive organs and highlights their applications, as well as technical challenges and future directions.

Assessment of the Efficacy of Tributylammonium Alginate Surface-Modified Polyurethane as an Antibacterial Elastomeric Wound Dressing for both Noninfected and Infected Full-Thickness Wounds.

Risk factors of nonhealing wounds include persistent bacterial infections and rapid onset of dehydration; therefore, wound dressings should be used to accelerate the healing process by helping to disinfect the wound bed and provide moisture. Herein, we introduce a transparent tributylammonium alginate surface-modified cationic polyurethane (CPU) wound dressing, which is appropriate for full-thickness wounds. We studied the physicochemical properties of the dressing using Fourier transform infrared, 1H NMR, and 13C NMR spectroscopies and scanning electron microscopy, energy-dispersive X-ray, and thermomechanical analyses. The surface-modified polyurethane demonstrated improved hydrophilicity and tensile Young's modulus that approximated natural skin, which was in the range of 1.5-3 MPa. Cell viability and in vitro wound closure, assessed by MTS and the scratch assay, confirmed that the dressing was cytocompatible and possessed fibroblast migratory-promoting activity. The surface-modified CPU had up to 100% antibacterial activity against Staphylococcus aureus and Escherichia coli as Gram-positive and Gram-negative bacteria, respectively. In vivo assessments of both noninfected and infected wounds revealed that the surface-modified CPU dressing resulted in a faster healing rate because it reduced the persistent inflammatory phase, enhanced collagen deposition, and improved the formation of mature blood vessels when compared with CPU and commercial Tegaderm wound dressing.

Cardioprotective effects of omega-3 fatty acids and ascorbic acid improve regenerative capacity of embryonic stem cell-derived cardiac lineage cells.

One of the major issues in cell therapy of myocardial infarction (MI) is early death of engrafted cells in a harsh oxidative stress environment, which limits the potential therapeutic utility of this strategy in the clinical setting. Increasing evidence implicates beneficial effects of omega-3 fatty acids including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and ascorbic acid (AA) in cardiovascular diseases, in particular their role in ameliorating fibrosis. In the current study, we aim to assess the cytoprotective role of EPA + DHA and AA in protecting embryonic stem cell (ESC)-derived cardiac lineage cells and amelioration of fibrosis. Herein, we have shown that preincubation of the cells with EPA + DHA + AA prior to H2 O2 treatment attenuated generation of reactive oxygen species (ROS) and enhanced cell viability. Gene expression analysis revealed that preincubation with EPA + DHA + AA followed by H2 O2 treatment, upregulated heme oxygenase-1 (HO-1) along with cardiac markers (GATA4, myosin heavy chain, α isoform [MYH6]), connexin 43 [CX43]) and attenuated oxidative stress-induced upregulation of fibroblast markers (vimentin and collagen type 1 [Col1]). Alterations in gene expression patterns were followed by marked elevation of cardiac troponin (TNNT2) positive cells and reduced numbers of vimentin positive cells. An injection of EPA + DHA + AA-pretreated ESC-derived cardiac lineage cells into the ischemic myocardium of a rat model of MI significantly reduced fibrosis compared to the vehicle group. This study provided evidence that EPA + DHA + AA may be an appropriate preincubation regimen for regenerative purposes. © 2019 BioFactors, 45(3):427-438, 2019.

Fabrication of microporous inorganic microneedles by centrifugal casting method for transdermal extraction and delivery.

Microneedle patches have been widely used as transdermal transport systems because of their painless and easy application. Marked rigidity, strength, biocompatibility, and physiological stability are unique features of microneedles fabricated from ceramic materials to be used as microneedle patches. However, the conventional ceramic microneedles are typically dense structures with limited free space for biomolecule loading. A facile method is required for fabrication of biocompatible ceramic microneedles with interconnected porosity. Herein, the simple method of centrifugal casting was developed for fabrication of microporous microneedles from alumina suspensions. The slurry or resin-based alumina suspensions were casted into micromolds under centrifugal force, followed by sintering at high temperatures. The effects of particle size, solvent type, binder amount, resin content and sintering temperature on the microstructure and mechanical properties of microneedles were investigated. By optimizing the process parameters, highly porous (up to 60%) microneedles with interconnected micropores (of diameter ∼1-1.5 µm) were produced. The microporous microneedles were biocompatible and mechanically strong for skin penetration. The potential use of the microneedles for transdermal transportation of biomolecules was shown by fast and accurate extraction of glucose from a skin model and efficient loading and fast release of insulin under physiological conditions. The results suggested that the microporous alumina microneedles may serve as molecular transport systems in transdermal biosensing and drug delivery.

Smart liposomal drug delivery for treatment of oxidative stress model in human embryonic stem cell-derived retinal pigment epithelial cells.

Oxidative stress has been implicated in the progression of age-related macular degeneration (AMD). Treatment with antioxidants seems to delay progression of AMD. In this study, we suggested an antioxidant delivery system based on redox-sensitive liposome composed of phospholipids and a diselenide centered alkyl chain. Dynamic light scattering assessment indicated that the liposomes had an average size of 140 nm with a polydispersity index below 0.2. The percentage of encapsulation efficiency of the liposomes was calculated by high-performance liquid chromatography. The carriers were loaded with N-acetyl cysteine as a model antioxidant drug. We demonstrated responsiveness of the nanocarrier and its efficiency in drug delivery in an oxidative stress model of human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells. The modeled cells treated with diselenide containing liposomes loaded with 10 mM NAC, showed a better therapeutic effect with a cell metabolic activity of 90%, which was significantly higher compared to insensitive liposomes or NAC treated groups (P < 0.05). In addition, the expression of oxidative-sensitive gene markers in diselenide containing liposomes groups were improved. Our results demonstrated fabricated smart liposomes opens new opportunity for targeted treatment of retinal degeneration.

Effects of hawthorn ( Crataegus pentagyna) leaf extract on electrophysiologic properties of cardiomyocytes derived from human cardiac arrhythmia-specific induced pluripotent stem cells.

Cardiac arrhythmias are major life-threatening conditions. The landmark discovery of induced pluripotent stem cells has provided a promising in vitro system for modeling hereditary cardiac arrhythmias as well as drug development and toxicity testing. Nowadays, nutraceuticals are frequently used as supplements for cardiovascular therapy. Here we studied the cardiac effects of hawthorn ( Crataegus pentagyna) leaf extract using cardiomyocytes (CMs) differentiated from healthy human embryonic stem cells, long QT syndrome type 2 (LQTS2), and catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) patient-specific induced pluripotent stem cells. The hydroalcoholic extract resulted in a dose-dependent negative chronotropic effect in all CM preparations leading to a significant reduction at 1000 µg/ml. This was accompanied by prolongation of field potential durations, although with different magnitudes in CMs from different human embryonic stem cell and iPSC lines. Hawthorn further prolonged field potential durations in LQTS2 CMs but reduced the beating frequencies and occurrence of immature field potentials triggered by ß1-adrenergic stimulation in CPVT1 CMs at 300 and 1000 µg/ml. Furthermore, isoquercetin and vitexin flavonoids significantly slowed down isoproterenol (5 µM)-induced beating frequencies at 3 and 10 µg/ml. Therefore, C. pentagyna leaf extract and its isoquercetin and vitexin flavonoids may be introduced as a novel nutraceutical with antiarrhythmic potential for CPVT1 patients.-Pahlavan, S., Tousi, M. S., Ayyari, M., Alirezalu, A., Ansari, H., Saric, T., Baharvand, H. Effects of hawthorn ( Crataegus pentagyna) leaf extract on electrophysiologic properties of cardiomyocytes derived from human cardiac arrhythmia-specific induced pluripotent stem cells.

Exogenous treatment with eicosapentaenoic acid supports maturation of cardiomyocytes derived from embryonic stem cells.

Embryonic stem cells offer multiple advantages over adult stem cells in terms of achieving acceptable number of functional cardiomyocytes to be exploited in cell therapy. However, differentiation efficacy is still a major issue to be solved before moving to regenerative medicine. Although a vast number of chemical compounds have been tested on efficiency of cardiac differentiation, the effect of fish oil components, such as eicosapentaenoic acid (EPA) on developmental bioenergetics, and hence cardiac differentiation, remained unstudied. EPA has been reported to have several cardioprotective effects, but there is no study addressing its role in cardiac differentiation. After mesoderm induction of embryoid bodies (EBs) derived from mouse embryonic stem cells (mESCs) in hanging drops initiated by ascorbic acid, they were treated with various concentrations of EPA. Gene and protein expression and functional properties of cardiomyocytes derived from ESCs were evaluated following treatment with various concentrations of EPA. Exposure to low concentrations of EPA (10 µM) increased percentage of beating colonies and beating area. This treatment also resulted in up to 3 fold increase in expression of NKX2-5, MEF2C, MYH6, TNNT2 and CX43. FACS analysis confirmed gene expression analysis with increased percentage of MYH6 positive cells in EPA-treated group compared to the control group. In contrast, the expression of genes coding for cardiac differentiation, remained constant or even declined with higher concentrations of EPA. In conclusion, we have demonstrated that treatment of mESCs undergoing cardiac differentiation with low concentration, but not high concentration of EPA up-regulate transcription of genes associated with cardiac development.

Long-term self-renewable feeder-free human induced pluripotent stem cell-derived neural progenitors.

Human induced pluripotent stem cells (hiPSCs) have led to an important revolution in stem cell research and regenerative medicine. To create patient-specific neural progenitors (NPs), we have established a homogenous, expandable, and self-renewable population of multipotent NPs from hiPSCs, using an adherent system and defined medium supplemented with a combination of factors. The established hiPSC-NPs highly expressed Nestin and Sox1. These NPs were continuously propagated for ~1 year without losing their potential to generate astrocytes, oligodendrocytes, and functional neurons and maintained a stable chromosome number. Voltage clamp analysis revealed outward potassium currents in hiPSC-NPs. The self-renewal characteristic of the NPs was demonstrated by a symmetrical mode of Nestin-positive cell division. Additionally, these hiPSC-NPs can be easily frozen and thawed in the presence of Rho-associated kinase (ROCK) inhibitor without losing their proliferation, karyotype stability, and developmental potential. The characteristics of our generated hiPSC-NPs provide the opportunity to use patient-specific or ready-to-use hiPSC-NPs in future biomedical applications.

Feeder- and serum-free establishment and expansion of human induced pluripotent stem cells.

Although human induced pluripotent stem cells (hiPSCs) hold great promise as a source of differentiated cells for vast therapeutic implications, many obstacles still need to be surmounted before this can become a reality. One obstacle, a robust feeder- and serum-free system to generate and expand hiPSCs in culture is still unavailable. Here, for the first time, we describe a novel establishment and maintenance culture technique that uses human dermal fibroblasts to generate hiPSCs by introducing four factors, Klf4, Oct4, Sox2, and c-Myc under serum- and feeder-independent conditions. We have used a serum replacement product, conditioned medium (CM), or feeder-free medium (FFM) supplemented with high elevated basic-fibroblast growth factor in the absence or presence of Matrigel. Our FFM system in the presence of Matrigel enhanced the efficiency of alkaline phosphatase-positive colonies at a frequency at least 10-fold greater than the conventional method on feeder cells. The established hiPSCs are similar to human embryonic stem cells in many aspects including morphology, passaging, surface and pluripotency markers, normal karyotype, gene expression, ultrastructure, and in vitro differentiation. Such hiPSCs could be useful particularly in the context of in vitro disease modeling, pharmaceutical screening and in cellular replacement therapies once the safety issues have been overcome.

Rat marrow-derived mesenchymal stem cells developed in a medium supplemented with the autologous versus bovine serum.

The controversial effect of autologous serum (AS) on human mesenchymal stem cells (MSC) was studied in rat MSC culture. Rat bone marrow cells were plated in a medium containing either FBS (fetal bovine serum) or AS were cultured to passage 3, during which the population doubling number (PDN) of both cultures were measured and compared statistically. The number of viable cells, the cell colonogic activity, and cell growth rate were also compared. In addition, mineralization in the osteogenic cultures from each system was measured. Our data indicated that AS enriched medium provided a microenvironment in which growth rate as well as bone differentiation of the isolated MSCs were significantly higher than in FBS enriched medium.

Differentiation of human embryonic stem cells into functional hepatocyte-like cells in a serum-free adherent culture condition.

Embryonic stem cells (ESCs) are considered one promising new approach to generate a transplantable cell source for the treatment of liver diseases. Because traditional methods, such as the initial formation of embryoid body in the presence of serum result in all three germ layer derivatives, strategies have been utilized that favor cell-specific differentiation to generate more uniformity. Here, we have presented the use of a multistep protocol with growth factors in a serum-free adherent culture configuration to mediate the hepatocyte differentiation of human ESCs (hESCs). The differentiated cells exhibited characteristic hepatocyte morphology, ultrastructure, and expressed hepatic-related genes as shown by reverse transcription-polymerase chain reaction and displayed antibody detectable expression of markers specific for hepatic maturation. These hepatocyte-like cells also demonstrated evidence of albumin and alpha-fetoprotein secretion, glycogen storage, urea production, uptake of low-density lipoprotein, and indocyanine green. Therefore, we propose that the hepatocyte-like cells derived from hESCs by the present method may provide a useful model for the studies of key events during early liver development and a potential source of drug screening and transplantable cells for cell-replacement therapies.