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Alternative methods to obtain mature oocytes are still needed for women with premature ovarian failure (POF). Oogonial stem cells (OSCs), found in adult ovaries, have provided insight into potential paths to treating infertility. Previously, the DDX4 antibody marker alone was utilized to isolate OSCs; however, extensive debate over its location in OSCs versus resulting oocytes (transmembrane or intracytoplasmic) has raised doubt about the identity of these cells. Separate groups, however, have efficiently isolated OSCs using another antibody marker Ifitm3 which is consistently recognized to be transmembrane in location. We hypothesized that by using anti-DDX4 and anti-IFITM3 antibodies, in combination, with MACS, we would improve the yield of isolated OSCs versus using anti-DDX4 antibodies alone. Our study supports earlier findings of OSCs in ovaries during the entire female lifespan: from reproductive age through post-menopausal age. MACS sorting ovarian cells using a the two-marker combination yielded a ~ twofold higher percentage of OSCs from a given mass of ovarian tissue compared to existing single marker methods while minimizing the debate surrounding germline marker selection. During in vitro culture, isolated cells retained the germline phenotype expression of DDX4 and IFITM3 as confirmed by gene expression analysis, demonstrated characteristic germline stem cell self-assembly into embryoid bodies, and formed > 40 µm “oocyte-like” structures that expressed the early oocyte markers GDF9, DAZL, and ZP1. This enhanced and novel method is clinically significant as it could be utilized in the future to more efficiently produce mature, secondary oocytes, for use with IVF/ICSI to treat POF patients.

This paper examines the challenges to judicial independence posed by measures that depart from normal standards of justice in countering terrorism with particular reference to the UK. Two particular contexts are examined: the Irish ‘troubles’-based terrorism that have stretched from the early 1970s until the present day and ‘jihadi’-based terrorism that has been taking place since 9/11. The chapter argues that while judicial independence has managed to remain unscathed throughout both these phases, there is a particular need today to preserve the principle of open justice as much as possible in order to counter any suggestion that judges are deferring too readily to the executive.

Ovaries are the primary physiological source of female sex hormones, which play a crucial role in maintaining ovarian cycle, determining secondary sexual characteristics and preparing the endometrium for implantation. In vitro follicle engineering has been used to investigate follicle development, including ovarian hormone production and gamete maturation. To engineer functional follicles, culture and expansion of the primary ovarian cells are essential. However, the phenotypic and functional characteristics of primary ovarian cells are often lost during culture. The objective of this study is to develop an optimized culture system for maintaining ovarian cell growth and functionality. Granulosa cells (GCs) and theca cells (TCs) were isolated from female rats. The addition of follicle-stimulating hormone (FSH) or luteinizing hormone (LH) to the basal culture media significantly enhanced the secretion of estradiol from GCs and androstenedione from TCs. Serum concentrations of 5% and 10% had a similar role in promoting ovarian cell expansion and secretion of estradiol and androstenedione hormones from both types of cells. Growth differentiation factor 9 (GDF9), bone morphogenic protein 15 (BMP15), BMP7 and basic fibroblast growth factor (bFGF) enhanced GC proliferation and estradiol production, respectively. Among them, the effect of bFGF was most significant. bFGF also enhanced TC proliferation. When GCs and TCs were cultured in 5% serum, gonadotropin and bFGF-containing medium, they proliferated exponentially throughout the culture period of up to 40 days while maintaining their functional characteristics. Taken together, these results indicate that our medium formula is optimal for maximizing proliferation of functionally differentiated ovarian cells.

Although menopausal hormone therapy (MHT) is the most effective approach to managing the loss of ovarian activity, serious side effects have been reported. Cell-based therapy is a promising alternative for MHT. This study constructed engineered ovarian cell spheroids and investigated their endocrine function. Theca and granulosa cells were isolated from ovaries of 10-week-old rats. Two types of engineered ovarian cell spheroids were fabricated through forced aggregation in microwells, multilayered spheroids with centralized granulosa aggregates surrounded by an outer layer of theca cells and mixed ovarian spheroids lacking spatial rearrangement. The ovarian cell spheroids were encapsulated into a collagen gel. Non-aggregated ovarian cells served as controls. The endocrine function of the engineered ovarian spheroids was assessed over 30 days. The structure of the spheroids was well maintained during culture. The secretion of 17β-estradiol from both types of engineered ovarian cell spheroids was higher than in the control group and increased continuously in a time-dependent manner. Secretion of 17β-estradiol in the multi-layered ovarian cell spheroids was higher than in the non-layered constructs. Increased secretion of progesterone was detected in the multi-layered ovarian cell spheroids at day 5 of culture and was sustained during the culture period. The initial secretion level of progesterone in the non-layered ovarian cell spheroids was similar to those from the controls and increased significantly from days 21 to 30. An in vitro rat model of engineered ovarian cell spheroids was developed that was capable of secreting sex steroid hormones, indicating that the hormone secreting function of ovaries can be recapitulated ex vivo and potentially adapted for MHT.

The blood-brain barrier (BBB) is a dynamic component of the brain-vascular interface that maintains brain homeostasis and regulates solute permeability into brain tissue. The expression of tight junction proteins between adjacent endothelial cells and the presence of efflux proteins prevents entry of foreign substances into the brain parenchyma. BBB dysfunction, however, is evident in many neurological disorders including ischemic stroke, trauma, and chronic neurodegenerative diseases. Currently, major contributors to BBB dysfunction are not well understood. Here, we employed a multicellular 3D neurovascular unit organoid containing human brain microvascular endothelial cells, pericytes, astrocytes, microglia, oligodendrocytes and neurons to model the effects of hypoxia and neuroinflammation on BBB function. Organoids were cultured in hypoxic chamber with 0.1% O2 for 24 hours. Organoids cultured under this hypoxic condition showed increased permeability, pro-inflammatory cytokine production, and increased oxidative stress. The anti-inflammatory agents, secoisolariciresinol diglucoside and 2-arachidonoyl glycerol, demonstrated protection by reducing inflammatory cytokine levels in the organoids under hypoxic conditions. Through the assessment of a free radical scavenger and an anti-inflammatory endocannabinoid, we hereby report the utility of the model in drug development for drug candidates that may reduce the effects of ROS and inflammation under disease conditions. This 3D organoid model recapitulates characteristics of BBB dysfunction under hypoxic physiological conditions and when exposed to exogenous neuroinflammatory mediators and hence may have potential in disease modeling and therapeutic development.

This study investigated the safety of a novel cell-labeling technology with mKATE and Renilla reniformis luciferase (mKATE-renLUC) and assessed the efficacy on tracking implanted human placental stromal cells (PSC) in an erectile dysfunction (ED) animal model. Human PSC were labeled with mKATE-renLUC using a lentivirus. Cell viability, apoptosis, proliferation, migration, surface marker expression and differentiation potential of the labeled PSC were evaluated and compared with non-labeled PSC. The paracrine profile of labeled cells was examined using an angiogenesis protein array. The brightness and duration of labeled cells with different densities were evaluated. An ED rat model was established and labeled PSC were injected into cavernosal tissue of the penis. The migration and distribution of transplanted PSC were monitored using an IVIS imaging system in real time. Implanted PSC were identified in isolated tissues via detection of mKATE fluorescence. The cell viability, morphology, proliferation, migration, surface marker expression and differentiation potential of mKATE-renLUC-labeled PSC were similar to those of non-labeled cells in vitro (no statistical difference p>0.05). Similar expressions of trophic factors were found between labeled and non-labeled PSC. The migration and distribution of PSC expressing renLUC were tracked in vivo using IVIS imaging system. mKATE-positive PSC were detected in penile, kidney, prostate and hepatic tissues using histological methods. This labeling technology provides a safe and effective cell-tracking approach with a brighter fluorophore and codon-optimized luciferase.

Kidney disease is a major medical problem globally. Chronic kidney disease (CKD) is a progressive loss of kidney function. It causes accumulation of waste and fluid in the body, eventually resulting in kidney failure as well as damaging other organs. Although dialysis and kidney transplantation have been used as primary treatments for renal disease, dialysis does not restore full renal function, and there is a shortage of donor kidneys for transplantation. Recent advances in cell-based therapies have offered a means to augment and restore renal function. Various types of cells have been tested to evaluate their therapeutic effects on injured kidneys. Among various types of cells, amniotic fluid stem cells (AFSCs) share advantages of both embryonic and adult stem cells, such as pluripotent activity, remarkable plasticity, and immunomodulatory effects, which may allow their future therapeutic use as an “off-the-shelf” cell source. AFSC presents advantages of both conventional pluripotent and adult stem cells, such as pluripotent activity, remarkable plasticity, and immunomodulatory effects. This study demonstrates that administration of human-derived AFSC facilitates functional and structural improvement in a rat model of CKD, and suggests that cell therapy with AFSC has potential as a therapeutic strategy to recover renal function in patients with CKD. IMPACT STATEMENT: Patients with chronic kidney disease (CKD) have limited treatment options, and renal transplantation is the only definitive treatment method that restores kidney function. However, challenges associated with transplantation, including donor organ shortage, rejection, and life-long immunosuppression, remain a problem. Recently, stem cell-based therapies have been proposed as an alternative approach to augment and restore renal function. In this study, we used human-derived amniotic fluid stem cells (AFSCs) to treat CKD in a rat model and demonstrated that AFSC treatment facilitated positive effects in terms of improvements of renal function.

There is a need for effective wound treatments that retain the bioactivity of a cellular treatment, but without the high costs and complexities associated with manufacturing, storing, and applying living biological products. Previously, we developed an amnion membrane-derived hydrogel and evaluated its wound healing properties using a mouse wound model. In this study, we used a full thickness porcine skin wound model to evaluate the wound-healing efficacy of the amnion hydrogel and a less-processed amnion product comprising a lyophilized amnion membrane powder. These products were compared with commercially available amnion and nonamnion wound healing products. We found that the amnion hydrogel and amnion powder treatments demonstrated significant and rapid wound healing, driven primarily by new epithelialization versus closure by contraction. Histological analysis demonstrated that these treatments promote the formation of a mature epidermis and dermis with similar composition to healthy skin. The positive skin regenerative outcomes using amnion hydrogel and amnion powder treatments in a large animal model further demonstrate their potential translational value for human wound treatments. Significance statement This study demonstrates the efficacy of amnion hydrogel and amnion powder wound healing products in a large animal model. This further demonstrates their potential translational value for human wound treatments.

Authenticity is a perennial issue in art markets. This paper investigates the character of some art identifying it as meta-credence goods and utilizing a formal Bayesian model of how experts (or buyers) play a role in evaluating art works, as suggested in a recent paper by Ginsburgh et al. (J Econ Behav Organ 159:36–50, 2019). Experts or a consensus of experts determines credence status where present and future falsification (in the sense of Karl Popper) is impossible. Credence is always a matter of probabilistic degree, and we define a class of extreme credence goods called meta-credence. Consensus of expert opinion serves as verification in the art world where consensus substitutes for falsification. These opinions are relied on by buyers and the art-loving public. This paper outlines the process that the art expert undergoes to render a verdict on the authenticity of art.

Introduction The prevalence of erectile dysfunction (ED) is substantial and continues to rise. Current therapeutics for ED consist of oral medications, intracavernosal injections, vacuum erection devices, and penile implants. While such options may manage the disease state, none of these modalities, however, restore function. Stem cell therapy has been evaluated for erectile restoration in animal models. These cells have been derived from multiple tissues, have varied potential, and may function via local engraftment or paracrine signaling. Bone marrow-derived stem cells (BMSC) and adipose-derived stem cells (ASC) have both been used in these models with noteworthy effects. Aim Herein, we will review the pathophysiology of ED, animal models, current and novel stem-cell based therapeutics, clinical trials and areas for future research. Methods The relevant literature and contemporary data using keywords, “stem cells and erectile dysfunction” was reviewed. Main outcome measure Examination of evidence supporting the association between erectile dysfunction and adipose derived stem cells, bone marrow derived stem cells, placental stem cells, urine stem cells and stem cell therapy respectively. Results Placental-derived stem cells and urine-derived stem cells possess many similar properties as BMSC and ASC, but the methods of acquisition are favorable. Human clinical trials have already demonstrated successful use of stem cells for improvement of erectile function. Conclusion The future of stem cell research is constantly being evaluated, although, the evidence suggests a place for stem cells in erectile dysfunction therapeutics. Matz EL, Terlecki R, Zhang Y, et al. Stem Cell Therapy for Erectile Dysfunction. Sex Med Rev 2019;7:321–328.

Diseases, disorders, and dysfunctions of the female reproductive tract tissues can result in either infertility and/or hormonal imbalance. Current treatment options are limited and often do not result in tissue function restoration, requiring alternative therapeutic approaches. Regenerative medicine offers potential new therapies through the bioengineering of female reproductive tissues. This review focuses on some of the current technologies that could address the restoration of functional female reproductive tissues, including the use of stem cells, biomaterial scaffolds, bio-printing, and bio-fabrication of tissues or organoids. The use of these approaches could also be used to address issues in infertility. Strategies such as cell-based hormone replacement therapy could provide a more natural means of restoring normal ovarian physiology. Engineering of reproductive tissues and organs could serve as a powerful tool for correcting developmental anomalies. Organ-on-a-chip technologies could be used to perform drug screening for personalized medicine approaches and scientific investigations of the complex physiological interactions between the female reproductive tissues and other organ systems. While some of these technologies have already been developed, others have not been translated for clinical application. The continuous evolution of biomaterials and techniques, advances in bioprinting, along with emerging ideas for new approaches, shows a promising future for treating female reproductive tract-related disorders and dysfunctions.

The blood-brain barrier (BBB) is a dynamic component of the brain-vascular interface that maintains brain homeostasis and regulates solute permeability into brain tissue. The expression of tight junction proteins between adjacent endothelial cells and the presence of efflux proteins prevents entry of foreign substances into the brain parenchyma. BBB dysfunction, however, is evident in many neurological disorders including ischemic stroke, trauma, and chronic neurodegenerative diseases. Currently, major contributors to BBB dysfunction are not well understood. Here, we employed a multicellular 3D neurovascular unit organoid containing human brain microvascular endothelial cells, pericytes, astrocytes, microglia, oligodendrocytes and neurons to model the effects of hypoxia and neuroinflammation on BBB function. Organoids were cultured in hypoxic chamber with 0.1% O

Although menopausal hormone therapy (MHT) is the most effective approach to managing the loss of ovarian activity, serious side effects have been reported. Cell-based therapy is a promising alternative for MHT. This study constructed engineered ovarian cell spheroids and investigated their endocrine function. Theca and granulosa cells were isolated from ovaries of 10-week-old rats. Two types of engineered ovarian cell spheroids were fabricated through forced aggregation in microwells, multilayered spheroids with centralized granulosa aggregates surrounded by an outer layer of theca cells and mixed ovarian spheroids lacking spatial rearrangement. The ovarian cell spheroids were encapsulated into a collagen gel. Non-aggregated ovarian cells served as controls. The endocrine function of the engineered ovarian spheroids was assessed over 30 days. The structure of the spheroids was well maintained during culture. The secretion of 17β-estradiol from both types of engineered ovarian cell spheroids was higher than in the control group and increased continuously in a time-dependent manner. Secretion of 17β-estradiol in the multi-layered ovarian cell spheroids was higher than in the non-layered constructs. Increased secretion of progesterone was detected in the multi-layered ovarian cell spheroids at day 5 of culture and was sustained during the culture period. The initial secretion level of progesterone in the non-layered ovarian cell spheroids was similar to those from the controls and increased significantly from days 21 to 30. An in vitro rat model of engineered ovarian cell spheroids was developed that was capable of secreting sex steroid hormones, indicating that the hormone secreting function of ovaries can be recapitulated ex vivo and potentially adapted for MHT.

Human placental stem cells (PSCs) enhance histological and functional recovery in a rodent erectile dysfunction (ED) model. We tested the hypothesis that bioactive factors secreted by PSC (i.e., the secretome) mediate functional recovery and that acellular-conditioned media (CM) from PSC culture (PSC-CM) could be used independently to facilitate functional and histological recovery. To identify factors relative to efficacy of PSC, a comparison of CM from PSC and three additional human stem cell populations was performed. CM from human PSC, amniotic fluid stem cells (AFSCs), adipose-derived stem cells (ADSC), and human umbilical vein endothelial cells (HUVECs) was assayed using a semi-quantitative human cytokine antibody array. Male rats, after surgically created ED by neurovascular injury, were randomly divided into four groups: vehicle control (phosphate-buffered saline [PBS]), PSC, PSC-CM, and serum-free media control (SFM) as control. Functional data on intracorporal and mean arterial pressure were obtained, and histological architecture was examined 6 weeks after single injection. PSCs were found to secrete at least 27 cytokines and growth factors at a significantly higher level than the other three cell types. Either single injection of PSC-CM or PSC significantly improved erectile functional recovery and histological architecture compared with SFM or PBS. Injection of the secretome isolated from human PSC improves erectile functional recovery and histological structure in a rat model of neurovascular injury-induced ED. Further characterization of the unique protein expression within the PSC-CM may help to identify the potential for a novel injectable cell-free therapeutic for applicable patients.

Background Organoids play an important role in basic research, drug screening, and regenerative medicine. Here, we aimed to develop a novel kind of three-dimensional (3D) organoids generated from urine-derived stem cells (USCs) and to explore whether kidney-specific extracellular matrix (kECM) could enable such organoids for renal function in vitro. Methods USCs were isolated from human urine samples and cultured with kECM extraction to generate 3D organoids in vitro. Eight densities from 1000 to 8000 cells per organoids were prepared, and both ATP assay and Live/Dead staining were used to determine the optimal USC density in forming organoids and kECM additive concentration. The morphology and histology of as-made organoids were evaluated by hematoxylin and eosin (H.E.) staining, immunofluorescence staining and whole mount staining. Additionally, RT-qPCR was implemented to detect renal-related gene expression. Drug toxicity test was conducted to evaluate the potential application for drug screening. The renal organoids generated from whole adult kidney cells were used as a positive control in multiple assessments. Results The optimized cell density to generate ideal USC-derived organoids (USC-organoids) was 5000 cells/well, which was set as applying density in the following experiments. Besides, the optimal concentration of kECM was revealed to be 10%. On this condition, Live/Dead staining showed that USC-organoids were well self-organized without significant cell death. Moreover, H.E. staining showed that compact and viable organoids were generated without obvious necrosis inside organoids, which were very close to renal organoids morphologically. Furthermore, specific proximal tubule marker Aquaporin-1 (AQP1), kidney endocrine product erythropoietin (EPO), kidney glomerular markers Podocin and Synaptopodin were detected positively in USC-organoids with kECM. Nephrotoxicity testing showed that aspirin, penicillin G, and cisplatin could exert drug-induced toxicity on USC-organoids with kECM. Conclusions USC-organoids could be developed from USCs via an optimal procedure. Combining culture with kECM, USC-organoid properties including morphology, histology, and specific gene expression were identified to be similar with real renal organoids. Additionally, USC-organoids posed kECM in vitro showed the potential to be a drug screening tool which might take the place of renal organoids to some extent in the future.

In this paper, we have attempted to provide new evidence regarding American art as an investment vehicle, using tools typically employed to evaluate the investment worthiness of financial assets. We provided a brief review of the CAPM, and then estimated a general version of it allowing betas to differ between early American and contemporary American art using auction data on paired sales during the period 1987–2013. Our results indicate that early American art and contemporary American art are two distinct asset classes; that contemporary American art is probably a good investment on strictly financial grounds, but early American art may not be; that incorporating transactions costs is crucial to evaluating whether art is a good investment in some cases; that it is in principle possible to design an asset portfolio containing only American art that mimics the investment characteristics of the market portfolio; and that what appeared to be an art bubble bursting in the fall of 2015 may simply have been a predictable response of art prices to a drop in the stock market.

Host stem/progenitor cells can be mobilized and recruited to a target location using biomaterials, and these cells may be used for in situ tissue regeneration. The objective of this study was to investigate whether host biologic resources could be used to regenerate renal tissue in situ. Collagen hydrogel was injected into the kidneys of normal mice, and rat kidneys that had sustained ischemia/reperfusion injury. After injection, the kidneys of both animal models were examined up to 4 weeks for host tissue response. The infiltrating host cells present within the injection regions expressed renal stem/progenitor cell markers, PAX-2, CD24, and CD133, as well as mesenchymal stem cell marker, CD44. The regenerated renal structures were identified by immunohistochemistry for renal cell specific markers, including synaptopodin and CD31 for glomeruli and cytokeratin and neprilysin for tubules. Quantitatively, the number of glomeruli found in the injected regions was significantly higher when compared to normal regions of renal cortex. This phenomenon occurred in normal and ischemic injured kidneys. Furthermore, the renal function after ischemia/reperfusion injury was recovered after collagen hydrogel injection. These results demonstrate that introduction of biomaterials into the kidney is able to facilitate the regeneration of glomerular and tubular structures in normal and injured kidneys. Such an approach has the potential to become a simple and effective treatment for patients with renal failure.

The Consensus Coding Sequence (CCDS) project provides a dataset of protein-coding regions that are identically annotated on the human and mouse reference genome assembly in genome annotations produced independently by NCBI and the Ensembl group at EMBL-EBI. This dataset is the product of an international collaboration that includes NCBI, Ensembl, HUGO Gene Nomenclature Committee, Mouse Genome Informatics and University of California, Santa Cruz. Identically annotated coding regions, which are generated using an automated pipeline and pass multiple quality assurance checks, are assigned a stable and tracked identifier (CCDS ID). Additionally, coordinated manual review by expert curators from the CCDS collaboration helps in maintaining the integrity and high quality of the dataset. The CCDS data are available through an interactive web page (https://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi) and an FTP site (ftp://ftp.ncbi.nlm.nih.gov/pub/CCDS/). In this paper, we outline the ongoing work, growth and stability of the CCDS dataset and provide updates on new collaboration members and new features added to the CCDS user interface. We also present expert curation scenarios, with specific examples highlighting the importance of an accurate reference genome assembly and the crucial role played by input from the research community.

The early and effective treatment of wounds is vital to ensure proper wound closure and healing with appropriate functional and cosmetic outcomes. The use of human amnion membranes for wound care has been shown to be safe and effective. However, the difficulty in handling and placing thin sheets of membrane, and the high costs associated with the use of living cellularized tissue has limited the clinical application of amniotic membrane wound healing products. Here, we describe a novel amnion membrane-derived product, processed to result in a cell-free solution, while maintaining high concentrations of cell-derived cytokines and growth factors. The solubilized amnion membrane (SAM) combined with the carrier hyaluronic acid (HA) hydrogel (HA-SAM) is easy to produce, store, and apply to wounds. We demonstrated the efficacy of HA-SAM as a wound treatment using a full-thickness murine wound model. HA-SAM significantly accelerated wound closure through re-epithelialization and prevented wound contraction. HA-SAM-treated wounds had thicker regenerated skin, increased total number of blood vessels, and greater numbers of proliferating keratinocytes within the epidermis. Overall, this study confirms the efficacy of the amnion membrane as a wound treatment/dressing, and overcomes many of the limitations associated with using fresh, cryopreserved, or dehydrated tissue by providing a hydrogel delivery system for SAM.

For treatments requiring split-thickness skin grafts, it is preferable to mesh the grafts. This reduces the amount of excised skin and covers more wound area. The mesh technique, however, destroys surface continuity, which results in scarring. Strain-based bioreactors, on the other hand, have successfully expanded split-thickness skin grafts in vitro within a 7-day period, increasing graft coverage. After in vitro expansion, the expanded skin grafts were tested in a porcine full-thickness excisional wound model. Expanded graft take rate was 100%. Volumetric, histologic, and mechanical assessments indicated that expanded grafts were comparable to unexpanded grafts (positive control). While there was considerable variation in expansion (31% to -3.1%), this technique has the potential to enhance the coverage area of skin grafts while reducing or eliminating scarring.

The organs of the male reproductive system play a role in the production, maintenance, and transportation of sperm and semen to the female reproductive tract during sexual intercourse, as well as the production and secretion of male sex hormones responsible for maintaining the male reproductive system. Andrology is defined as the branches of science and medicine dealing with male reproductive functions under physiological and pathological conditions. Central topics of andrology are infertility, hypogonadism, and erectile dysfunction. Although the science of male reproductive function and dysfunction has experienced significant progress in recent years, regenerative medicine has opened new avenues for treating patients with severe andrology disorders, such as congenital abnormalities, cancer, trauma, infection, inflammation, and iatrogenic injuries. Here we summarize recent advances in the use of cell, tissue, and organ-based regenerative medicine strategies for clinical application in male reproductive system disorders.

Alternative methods to obtain mature oocytes are still needed for women with premature ovarian failure (POF). Oogonial stem cells (OSCs), found in adult ovaries, have provided insight into potential paths to treating infertility. Previously, the DDX4 antibody marker alone was utilized to isolate OSCs; however, extensive debate over its location in OSCs versus resulting oocytes (transmembrane or intracytoplasmic) has raised doubt about the identity of these cells. Separate groups, however, have efficiently isolated OSCs using another antibody marker Ifitm3 which is consistently recognized to be transmembrane in location. We hypothesized that by using anti-DDX4 and anti-IFITM3 antibodies, in combination, with MACS, we would improve the yield of isolated OSCs versus using anti-DDX4 antibodies alone. Our study supports earlier findings of OSCs in ovaries during the entire female lifespan: from reproductive age through post-menopausal age. MACS sorting ovarian cells using a the two-marker combination yielded a ~ twofold higher percentage of OSCs from a given mass of ovarian tissue compared to existing single marker methods while minimizing the debate surrounding germline marker selection. During in vitro culture, isolated cells retained the germline phenotype expression of DDX4 and IFITM3 as confirmed by gene expression analysis, demonstrated characteristic germline stem cell self-assembly into embryoid bodies, and formed 40 µm "oocyte-like" structures that expressed the early oocyte markers GDF9, DAZL, and ZP1. This enhanced and novel method is clinically significant as it could be utilized in the future to more efficiently produce mature, secondary oocytes, for use with IVF/ICSI to treat POF patients.

Ovaries are the primary physiological source of female sex hormones, which play a crucial role in maintaining ovarian cycle, determining secondary sexual characteristics and preparing the endometrium for implantation. In vitro follicle engineering has been used to investigate follicle development, including ovarian hormone production and gamete maturation. To engineer functional follicles, culture and expansion of the primary ovarian cells are essential. However, the phenotypic and functional characteristics of primary ovarian cells are often lost during culture. The objective of this study is to develop an optimized culture system for maintaining ovarian cell growth and functionality. Granulosa cells (GCs) and theca cells (TCs) were isolated from female rats. The addition of follicle-stimulating hormone (FSH) or luteinizing hormone (LH) to the basal culture media significantly enhanced the secretion of estradiol from GCs and androstenedione from TCs. Serum concentrations of 5% and 10% had a similar role in promoting ovarian cell expansion and secretion of estradiol and androstenedione hormones from both types of cells. Growth differentiation factor 9 (GDF9), bone morphogenic protein 15 (BMP15), BMP7 and basic fibroblast growth factor (bFGF) enhanced GC proliferation and estradiol production, respectively. Among them, the effect of bFGF was most significant. bFGF also enhanced TC proliferation. When GCs and TCs were cultured in 5% serum, gonadotropin and bFGF-containing medium, they proliferated exponentially throughout the culture period of up to 40 days while maintaining their functional characteristics. Taken together, these results indicate that our medium formula is optimal for maximizing proliferation of functionally differentiated ovarian cells.

This article charts how security‐cleared counsel have been constructed as a mechanism for managing the tension between security and fairness in secret trials and transferred across national boundaries as an example of ‘best practice', before going on to evaluate recent cross‐cultural and transnational research on this ‘best practice'. Particular attention is paid to the central role played by the European Court of Human Rights (ECtHR) in promoting the role of ‘special advocates’ and a contrast is made between the methodologies deployed by the Court and those used in recent research to identify and problematize ‘best practice’ within the closed world of security‐cleared counsel. The article then goes on to explore the relationship between ‘best practice’ and procedural tradition and argues that normative solutions advancing ‘best practice’ need to pay careful attention to the procedural contexts and cultures in which they are embedded.

Purpose: Drug-induced nephrotoxicity can occur in patients with pre-existing renal dysfunction or renal ischemia, potentially leading to chronic kidney disease (CKD) and end-stage renal disease (ESRD). Prompt treatment of CKD and the related side effects is critical in preventing progression to ESRD. The goal of this study was to demonstrate the therapeutic potential of urine-derived stem cells (USC) to treat chronic kidney disease-induced by nephrotoxic drugs and renal ischemia. Materials and methods: Human USC were collected, expanded and characterized by flow cytometry. A CKD model was induced by creating an ischemia-reperfusion injury and gentamicin administration. Twenty-eight adult immunodeficient rats were divided into three groups: PBS-treated group (n=9), USC-treated group (n=9), and sham group with age-matched control animals (n=10). Cell suspension of USC (5 x 106 / 100µl / kidney) or PBS was injected bilaterally into the renal parenchyma 9 weeks after CKD model creation. Renal function was evaluated by collection blood and urine samples to measure serum creatinine and glomerulus filtration rate. The kidneys were harvested 12 weeks after cell injection. Histologically, the extent of glomerulosclerosis and tubular atrophy, the amount of collagen deposition, interstitial fibrosis, inflammatory monocyte infiltration, and expression of transforming growth factor beta 1 (TGF-s1), and superoxide dismutase 1 (SOD-1) were examined. Results: USC expressed renal parietal epithelial cells (CD24, CD29 and CD44). Renal function, measured by GFR and serum Cr in USC-treated group were significantly improved compared to PBS-treated animals (p

Introduction The human placenta provides a bountiful and noncontroversial source of stem cells which have the potential for regeneration of injured tissue. These cells may restore erectile function after neurovascular tissue injury such as that seen in radical pelvic surgeries and pelvic trauma. Aim To determine the effect of human placenta–derived stem cells on erectile function recovery and histological changes at various time points in a cavernous nerve injury rat model and to study the fate of injected stem cells throughout the regenerative process. Methods Human placental stem cells (PSCs) were dual labeled with monomeric Katushka far red fluorescent protein (mKATE)-renLUC using a lentivirus vector. A pelvic neurovascular injury–induced erectile dysfunction model was established in male, athymic rats by crushing the cavernous nerves and ligating the internal pudendal neurovascular bundles, bilaterally. At the time of defect creation, nonlabeled PSCs were injected into the corpus cavernosum at a concentration of 2.5 × 106 cells/0.2 mL. The phosphate-buffered saline–treated group served as the negative control group, and age-matched rats (age-matched controls) were used as the control group. Erectile function, histomorphological analyses, and Western blot were assessed at 1, 6, and 12 weeks after model creation. The distribution of implanted, dual-labeled PSCs was monitored using an in vivo imaging system (IVIS). Implanted cells were further tracked by detection of mKATE fluorescence in histological sections. Main Outcome Measure The main outcome measure includes intracavernous pressure/mean arterial pressure ratio, neural, endothelial, smooth muscle cell regeneration, mKATE fluorescence, and IVIS imaging. Results The ratio of intracavernous pressure to mean arterial pressure significantly increased in PSC-injected rats compared with phosphate-buffered saline controls (P < 0.05) at the 6- and 12-week time points, reaching 72% and 68% of the age-matched control group, respectively. Immunofluorescence staining and Western blot analysis showed significant increases in markers of neurons (84.3%), endothelial cells (70.2%), and smooth muscle cells (70.3%) by 6 weeks in treatment groups compared with negative controls. These results were maintained through 12 weeks. IVIS analysis showed luminescence of implanted PSCs in the injected corpora immediately after injection and migration of cells to the sites of injury, including the incision site and periprostatic vasculature by day 1. mKATE fluorescence data revealed the presence of PSCs in the penile corpora and major pelvic ganglion at 1 and 3 days postoperatively. At 7 days, immunofluorescence of penile PSCs had disappeared and was diminished in the major pelvic ganglion. Clinical Implications Placenta-derived stem cells may represent a future “off-the-shelf” treatment to mitigate against development of erectile dysfunction after radical prostatectomy or other forms of pelvic injury. Strength & Limitations Single dose injection of PSCs after injury resulted in maximal functional recovery and tissue regeneration at 6 weeks, and the results were maintained through 12 weeks. Strategies to optimize adult stem cell therapy might achieve more effective outcomes for human clinical trials. Conclusion Human PSC therapy effectively restores the erectile tissue and function in this animal model. Thus, PSC therapy may provide an attractive modality to lessen the incidence of erectile dysfunction after pelvic neurovascular injury. Further improvement in tissue regeneration and functional recovery may be possible using multiple injections or systemic introduction of stem cells.

The early treatment and rapid closure of acute or chronic wounds is essential for normal healing and prevention of hypertrophic scarring. The use of split thickness autografts is often limited by the availability of a suitable area of healthy donor skin to harvest. Cellular and non-cellular biological skin-equivalents are commonly used as an alternative treatment option for these patients, however these treatments usually involve multiple surgical procedures and associated with high costs of production and repeated wound treatment. Here we describe a novel design and a proof-of-concept validation of a mobile skin bioprinting system that provides rapid on-site management of extensive wounds. Integrated imaging technology facilitated the precise delivery of either autologous or allogeneic dermal fibroblasts and epidermal keratinocytes directly into an injured area, replicating the layered skin structure. Excisional wounds bioprinted with layered autologous dermal fibroblasts and epidermal keratinocytes in a hydrogel carrier showed rapid wound closure, reduced contraction and accelerated re-epithelialization. These regenerated tissues had a dermal structure and composition similar to healthy skin, with extensive collagen deposition arranged in large, organized fibers, extensive mature vascular formation and proliferating keratinocytes.

This chapter offers a critique of the “common law model” of the Law of Evidence and calls for a new organizing principle that “reimagines” evidence law as forensic science, particularly in the context of criminal adjudication. It first provides an overview of the orthodox common law model of Evidence Law before deconstructing it, arguing that it adopts a very narrow doctrinal focus, thus undermining the dynamic processes through which evidence is collected, organized, presented, tested, and evaluated in legal proceedings. It also suggests that the model is difficult to defend in terms of robust disciplinary boundaries differentiating that which is specifically evidentiary from broader aspects of substantive and procedural law. Finally, it considers the so-called “New Evidence Scholarship” on evidence law, the impact of the new cosmopolitanism on common law evidence, and the rationale for reconceptualizing evidence law as part of an interdisciplinary “forensic science” that goes “beyond common law.”

This article examines the responses of national courts to the ECtHR's decision in Salduz v Turkey that suspects be provided with access to a lawyer before they are first interrogated by the police. It argues that harmonious application of human rights standards in criminal proceedings should build upon common values underpinning the procedural traditions of member states. ECtHR success in gaining acceptance for the principle of access to a lawyer during police interrogation, anchoring it in the privilege against incrimination, is contrasted with resistance towards giving the defence any active role during criminal investigations. It is argued that this resistance can be overcome by an appeal to safeguards that have long dominated the trial process. As the investigation phase increasingly determines the outcome of criminal proceedings, standards of fairness traditionally reserved for the trial process should be applied also to this phase in order to provide suspects with an effective defence.

The rise in secret courts or ‘closed material proceedings’ (CMPs) in recent years has called into question our commitment to long-cherished principles of open justice and due process. This debate has somewhat overshadowed the role of special advocates, who are appointed to represent the interests of parties excluded from such hearings. These advocates pose a challenge to the traditions of advocacy in the adversarial system but an international consensus across the common law world appears to be emerging that they may be justified on human rights grounds of fairness in that they bring a measure of procedural fairness to closed material proceedings. This paper examines this claim and considers the extent to which the rise of special advocates poses a threat to the adversarial tradition.

Magnetic Resonance Imaging (MRI) is a commonly used, non-invasive imaging technique that provides visualization of soft tissues with high spatial resolution. In both a research and clinical setting, the major challenge has been identifying a non-invasive and safe method for longitudinal tracking of delivered cells in vivo. The labeling and tracking of contrast agent labeled cells using MRI has the potential to fulfill this need. Contrast agents are often used to enhance the image contrast between the tissue of interest and surrounding tissues with MRI. The most commonly used MRI contrast agents contain Gd(III) ions. However, Gd(III) ions are highly toxic in their ionic form, as they tend to accumulate in the liver, spleen, kidney and bones and block calcium channels. Endohedral metallofullerenes such as trimetallic nitride endohedral metallofullerenes (Trimetasphere®) are one unique class of fullerene molecules where a Gd3N cluster is encapsulated inside a C80 carbon cage referred to as Gd3N@C80. These endohedral metallofullerenes have several advantages over small chelated Gd(III) complexes such as increased stability of the Gd(III) ion, minimal toxic effects, high solubility in water and high proton relativity. In this study, we describe the evaluation of gadolinium-based Trimetasphere® positive contrast agent for the ​in vitro labeling and in vivo tracking of human amniotic fluid-derived stem cells within lung tissue. In addition, we conducted a 'proof-of-concept' experiment demonstrating that this methodology can be used to track the homing of stem cells to injured lung tissue and provide longitudinal analysis of cell localization over an extended time course.