Induced Pluripotent Stem Cell-Derived Organoids: Their Implication in COVID-19 Modeling.

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a significant global health issue. This novel virus's high morbidity and mortality rates have prompted the scientific community to quickly find the best COVID-19 model to investigate all pathological processes underlining its activity and, more importantly, search for optimal drug therapy with minimal toxicity risk. The gold standard in disease modeling involves animal and monolayer culture models; however, these models do not fully reflect the response to human tissues affected by the virus. However, more physiological 3D in vitro culture models, such as spheroids and organoids derived from induced pluripotent stem cells (iPSCs), could serve as promising alternatives. Different iPSC-derived organoids, such as lung, cardiac, brain, intestinal, kidney, liver, nasal, retinal, skin, and pancreatic organoids, have already shown immense potential in COVID-19 modeling. In the present comprehensive review article, we summarize the current knowledge on COVID-19 modeling and drug screening using selected iPSC-derived 3D culture models, including lung, brain, intestinal, cardiac, blood vessels, liver, kidney, and inner ear organoids. Undoubtedly, according to reviewed studies, organoids are the state-of-the-art approach to COVID-19 modeling.

The Neglected Uterine NK Cells/Hamperl Cells/Endometrial Stromal Granular Cell, or K Cells: A Narrative Review from History through Histology and to Medical Education.

Reproductive immunology is at the forefront of research interests, aiming to better understand the mechanisms of immune regulation during gestation. The relationship between the immune system and the implanting embryo is profound because the embryo is semi-allogenic but not targeted by the maternal immune system, as expected in graft-versus-host reactions. The most prominent cell population at the maternal-fetal interface is the population of uterine natural killer (uNK) cells. Uterine NK cells are two-faced immunologically active cells, bearing comparison with Janus, the ancient Roman god of beginnings and endings. Their first face can be seen as natural killer cells, namely lymphocytes, which are critical for host defense against viruses and tumors. Even though uNK cells contain cytolytic molecules, their cytotoxic effect is not applied to classical target cells in vivo, playing a permissive rather than a defensive role. Their second face is crucial in maintaining physiological gestation-uNK cells show critical immunomodulatory functions with the potential to control embryo implantation and trophoblast invasion, regulate placental vascular remodeling, and promote embryonic/fetal growth. Therefore, we believe that their current designation "natural killer cells" (the first "cytotoxic" Janus's face) is misleading and inappropriate, considering their principal function is supporting and maintaining pregnancy. In this narrative review, we will focus on three lesser-known areas of knowledge about uNK cells. First, from the point of view of histology, we will comprehensively map the history of the discovery of these cells, as well as the current histological possibilities of their identification within the endometrium. To be brief, the discovery of uNK cells is generally attributed to Herwig Hamperl, one of the most influential and prominent representatives of German pathology in the 20th century, and his co-worker, Gisela Hellweg. Secondly, we will discuss the interesting aspect of terminology, since uNK cells are probably one of the human cells with the highest number of synonymous names, leading to significant discrepancies in their descriptions in scientific literature. From the first description of this cell type, they were referred to as endometrial granulocytes, granular endometrial stromal cells, or large granular lymphocytes until the end of the 1980s and the beginning of the 1990s of the last century, when the first publications appeared where the name "uterine NK cells" was used. The third area of present review is medical teaching of histology and clinical embryology. We can confirm that uNK cells are, in most textbooks, overlooked and almost forgotten cells despite their enormous importance. In the present narrative review, we summarize the lesser-known historical and terminological facts about uNK cells. We can state that within the textbooks of histology and embryology, this important cell population is still "overlooked and neglected" and is not given the same importance as in fields of clinical research and clinical practice.

Traditional and contemporary views on the functional morphology of the fallopian tubes and their importance for gynecological practice.

The uterine tube, belonging to the female internal reproductive organs, is the only tubular organ in the human body that has, under physiological conditions, a transport function occurring in two opposite directions. It transports the picked-up oocyte released during ovulation and early embryo towards the uterine cavity. At the same time, it can transport spermatozoa towards the abdominal opening of the fallopian tube. Moreover, the uterine tube has many other vital functions as sperm selection (one of the crucial factors preventing polyspermy) and the production of tubal fluid. This unique secretion is essential not only for the process of fertilization but also for sperm activation and the nourishment of the early embryo during its transport into the uterine cavity. The first part of our review is focused on the historical introduction to the topic in which the reader will become familiar with the views and understanding of these peculiar organs by famous anatomists of the 16th and 17th centuries, namely Gabriele Falloppio and Renier de Graaf. The following section will cover the overview of the latest anatomical, embryological, and histological knowledge, which are also crucial for a better understanding of pathological processes affecting the fallopian tube, such as tubal infertility or tubal pregnancy. Interestingly, recent years have been very fruitful regarding uterine tube morphology, e. g. the discovery of an unique mechanism of lymphatic flow within the uterine tube mucosa, the first description of immunologically-active intraepithelial suppressor T-lymphocytes, or the observation of pacemaker cell population - telocytes - in the muscle layer.

Rheumatoid arthritis: From synovium biology to cell-based therapy.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that affects the synovial joints and, if not treated properly, can lead to multiple progressive articular and extra-articular damage. Its pathogenesis is primarily associated with an inadequate immune response and dysregulated cytokine production. However, RA is also linked to disruption in oxygen metabolism, impaired redox signaling, acidosis and aberrant intercellular communication. Even though treatment modalities have made RA a manageable disease, a significant number of patients still do not respond satisfactorily or suffer considerably from the adverse events of conventional therapy. In recent years, cell-based strategies, especially the administration of the mesenchymal/medicinal stem/signaling cells (MSCs), have been proposed as a novel and very promising therapeutic approach. RA patients may benefit from the potent anti-inflammatory and immunomodulatory properties and tissue-repair potential of MSCs. Furthermore, the satisfactory safety profile of MSC therapy has been already demonstrated in several clinical studies. This review summarizes current understanding of the pathomechanism behind RA at the molecular and cellular level and focuses on MSC-based clinical research and applications of MSCs for RA treatment.

Implication of Mesenchymal Stem Cells and Their Derivates for Osteochondral Regeneration.

Healing of articular cartilage defects presents a challenging issue, due to its regenerative shortcomings. Lacking vascularity and innervation of cartilage and low proliferative potential of chondrocytes are the main reasons for the limited healing potential of articular cartilage. Traditional reparative approaches are limited in their efficiency, hence there is a demand for novel reparative treatments. Mesenchymal stromal cells, preferred for clinical uses, can be readily derived from various sources and have been proven to have a therapeutic effect on cartilage and subchondral bone. Therefore, mesenchymal stromal cells, their derivates, and scaffolds have been utilized in research targeting osteochondral regeneration. The present review aims to comprehensively outline and discuss literature considering this topic published within last 5 years.

Cell-Based and Selected Cell-Free Therapies for Myocardial Infarction: How Do They Compare to the Current Treatment Options?

Because of cardiomyocyte death or dysfunction frequently caused by myocardial infarction (MI), heart failure is a leading cause of morbidity and mortality in modern society. Paradoxically, only limited and non-curative therapies for heart failure or MI are currently available. As a result, over the past two decades research has focused on developing cell-based approaches promoting the regeneration of infarcted tissue. Cell-based therapies for myocardial regeneration include powerful candidates, such as multipotent stem cells (mesenchymal stem cells (MSCs), bone-marrow-derived stem cells, endothelial progenitor cells, and hematopoietic stem cells) and induced pluripotent stem cells (iPSCs). These possess unique properties, such as potency to differentiate into desired cell types, proliferation capacity, and patient specificity. Preclinical and clinical studies have demonstrated modest improvement in the myocardial regeneration and reduced infarcted areas upon transplantation of pluripotent or multipotent stem cells. Another cell population that need to be considered as a potential source for cardiac regeneration are telocytes found in different organs, including the heart. Their therapeutic effect has been studied in various heart pathologies, such as MI, arrhythmias, or atrial amyloidosis. The most recent cell-free therapeutic tool relies on the cardioprotective effect of complex cargo carried by small membrane-bound vesicles-exosomes-released from stem cells via exocytosis. The MSC/iPSC-derived exosomes could be considered a novel exosome-based therapy for cardiovascular diseases thanks to their unique content. There are also other cell-free approaches, e.g., gene therapy, or acellular cardiac patches. Therefore, our review provides the most recent insights into the novel strategies for myocardial repair based on the regenerative potential of different cell types and cell-free approaches.

Cardiac Telocytes 16 Years on-What Have We Learned So Far, and How Close Are We to Routine Application of the Knowledge in Cardiovascular Regenerative Medicine?

The regeneration of a diseased heart is one of the principal challenges of modern cardiovascular medicine. There has been ongoing research on stem-cell-based therapeutic approaches. A cell population called telocytes (TCs) described only 16 years ago largely contributed to the research area of cardiovascular regeneration. TCs are cells with small bodies and extremely long cytoplasmic projections called telopodes, described in all layers of the heart wall. Their functions include cell-to-cell signaling, stem-cell nursing, mechanical support, and immunoregulation, to name but a few. The functional derangement or quantitative loss of TCs has been implicated in the pathogenesis of myocardial infarction, heart failure, arrhythmias, and many other conditions. The exact pathomechanisms are still unknown, but the loss of regulative, integrative, and nursing functions of TCs may provide important clues. Therefore, a viable avenue in the future modern management of these conditions is TC-based cell therapy. TCs have been previously transplanted into a mouse model of myocardial infarction with promising results. Tandem transplantation with stem cells may provide additional benefit; however, many underresearched areas need to be addressed in future research before routine application of TC-based cell therapy in human subjects. These include the standardization of protocols for isolation, cultivation, and transplantation, quantitative optimization of TC transplants, cost-effectivity analysis, and many others.

Stem Cells and Their Derivatives-Implications for Alveolar Bone Regeneration: A Comprehensive Review.

Oral and craniofacial bone defects caused by congenital disease or trauma are widespread. In the case of severe alveolar bone defect, autologous bone grafting has been considered a "gold standard"; however, the procedure has several disadvantages, including limited supply, resorption, donor site morbidity, deformity, infection, and bone graft rejection. In the last few decades, bone tissue engineering combined with stem cell-based therapy may represent a possible alternative to current bone augmentation techniques. The number of studies investigating different cell-based bone tissue engineering methods to reconstruct alveolar bone damage is rapidly rising. As an interdisciplinary field, bone tissue engineering combines the use of osteogenic cells (stem cells/progenitor cells), bioactive molecules, and biocompatible scaffolds, whereas stem cells play a pivotal role. Therefore, our work highlights the osteogenic potential of various dental tissue-derived stem cells and induced pluripotent stem cells (iPSCs), the progress in differentiation techniques of iPSCs into osteoprogenitor cells, and the efforts that have been made to fabricate the most suitable and biocompatible scaffold material with osteoinductive properties for successful bone graft generation. Moreover, we discuss the application of stem cell-derived exosomes as a compelling new form of "stem-cell free" therapy.

Recent Overview of the Use of iPSCs Huntington's Disease Modeling and Therapy.

Huntington's disease (HD) is an inherited, autosomal dominant, degenerative disease characterized by involuntary movements, cognitive decline, and behavioral impairment ending in death. HD is caused by an expansion in the number of CAG repeats in the huntingtin gene on chromosome 4. To date, no effective therapy for preventing the onset or progression of the disease has been found, and many symptoms do not respond to pharmacologic treatment. However, recent results of pre-clinical trials suggest a beneficial effect of stem-cell-based therapy. Induced pluripotent stem cells (iPSCs) represent an unlimited cell source and are the most suitable among the various types of autologous stem cells due to their patient specificity and ability to differentiate into a variety of cell types both in vitro and in vivo. Furthermore, the cultivation of iPSC-derived neural cells offers the possibility of studying the etiopathology of neurodegenerative diseases, such as HD. Moreover, differentiated neural cells can organize into three-dimensional (3D) organoids, mimicking the complex architecture of the brain. In this article, we present a comprehensive review of recent HD models, the methods for differentiating HD-iPSCs into the desired neural cell types, and the progress in gene editing techniques leading toward stem-cell-based therapy.

Recent Progress in the Regeneration of Spinal Cord Injuries by Induced Pluripotent Stem Cells.

Regeneration of injuries occurring in the central nervous system, particularly spinal cord injuries (SCIs), is extremely difficult. The complex pathological events following a SCI often restrict regeneration of nervous tissue at the injury site and frequently lead to irreversible loss of motor and sensory function. Neural stem/progenitor cells (NSCs/NPCs) possess neuroregenerative and neuroprotective features, and transplantation of such cells into the site of damaged tissue is a promising stem cell-based therapy for SCI. However, NSC/NPCs have mostly been induced from embryonic stem cells or fetal tissue, leading to ethical concerns. The pioneering work of Yamanaka and colleagues gave rise to the technology to induce pluripotent stem cells (iPSCs) from somatic cells, overcoming these ethical issues. The advent of iPSCs technology has meant significant progress in the therapy of neurodegenerative disease and nerve tissue damage. A number of published studies have described the successful differentiation of NSCs/NPCs from iPSCs and their subsequent engraftment into SCI animal models, followed by functional recovery of injury. The aim of this present review is to summarize various iPSC- NPCs differentiation methods, SCI modelling, and the current status of possible iPSC- NPCs- based therapy of SCI.

Induction of pluripotency in long-term cryopreserved human neonatal fibroblasts in feeder-free condition.

A novel approach for stem cell generation is the attempt to induce conversion of the adult somatic cells into pluripotent stem cells so called induced pluripotent stem cells (iPSCs) by introducing specific transcription factors. iPSCs have two essential cell characteristics, they are pluripotent and posses long term cell-renewal capacity. Additionally, iPSCs can be derived from patient-specific somatic cells, thus bypassing ethical and immunological issues. The aim of our study was to reprogram long-term cryopreserved human neonatal fibroblasts by new method using lipid nano-particle technology (Lipofectamine 3000 reagent transfection system) in combination with Epi 5 reprogramming vectors. Obtained iPSCs were characterized by several sophisticated methods of molecular biology and microscopy. Distinct colonies of iPSCs started to appear by day 20 after reprogramming. The presence of iPSCs colonies was proved by alkaline phosphatase (AP) live staining. After manual picking the colonies and their subsequent passaging, they did not lose ability to form embryoid bodies, they were positive for AP, Tra-1-60, and SSEA-5. Moreover, obtained iPSCs expressed pluripotency markers Oct4, Sox2 and Nanog, and the expression levels of chondrogenic, osteogenic and adipogenic markers were significantly higher in comparison to control (p < 0.05). In summary, we have demonstrated that long-term cryopreserved human neonatal fibroblasts can be reprogrammed into iPSCs and after further analysis concerns on their biological safety they may be used as patient-specific cells in regenerative medicine.

Toxicity testing and drug screening using iPSC-derived hepatocytes, cardiomyocytes, and neural cells.

Unexpected toxicity in areas such as cardiotoxicity, hepatotoxicity, and neurotoxicity is a serious complication of clinical therapy and one of the key causes for failure of promising drug candidates in development. Animal studies have been widely used for toxicology research to provide preclinical security evaluation of various therapeutic agents under development. Species differences in drug penetration of the blood-brain barrier, drug metabolism, and related toxicity contribute to failure of drug trials from animal models to human. The existing system for drug discovery has relied on immortalized cell lines, animal models of human disease, and clinical trials in humans. Moreover, drug candidates that are passed as being safe in the preclinical stage often show toxic effects during the clinical stage. Only around 16% drugs are approved for human use. Research on induced pluripotent stem cells (iPSCs) promises to enhance drug discovery and development by providing simple, reproducible, and economically effective tools for drug toxicity screening under development and, on the other hand, for studying the disease mechanism and pathways. In this review, we provide an overview of basic information about iPSCs, and discuss efforts aimed at the use of iPSC-derived hepatocytes, cardiomyocytes, and neural cells in drug discovery and toxicity testing.

Recent advances in iPSC technologies involving cardiovascular and neurodegenerative disease modeling.

Cardiovascular and neurodegenerative diseases are the most common health threats in developed countries. Limited cell derivation and cell number in cardiac tissue makes it difficult to study the cardiovascular disease using the existing cardiac cell model. Regarding the neurodegenerative disorders, the most potential sources of cell therapeutics such as fetal-derived primary neurons and human embryonic stem cells (ESCs) are associated with ethical or technical limitations. The successful derivation of human-induced pluripotent stem cells (iPSCs) by de-differentiation of somatic cells offers significant potential to overcome hurdles in the field of the replacement therapy. Human iPSCs are functionally similar to human embryonic stem cells, and can be derived autologously without the ethical challenges associated with human ESCs. The iPSCs can, in turn, be differentiated into all cell types including neurons, cardiac cells, blood and liver cells, etc. Recently, target tissues derived from human iPSCs such as cardiomyocytes (CMs) or neurons have been used for new disease modeling and regenerative medicine therapies. Diseases models could be advantageous in the development of personalized medicine of various pathological conditions. This paper reviews efforts aimed at both the practical development of iPSCs, differentiation to neural/cardiac lineages, and the further use of these iPSCs-derived cells for disease modeling, as well as drug toxicity testing.

Comparative analysis of mesenchymal stromal cells from different tissue sources in respect to articular cartilage tissue engineering.

The main goal of this study was a comparison of biological properties of mesenchymal stromal cells (MSCs) obtained from bone marrow, adipose tissue and umbilical cord with respect to articular cartilage regeneration. MSCs were isolated and expanded in vitro up to the third passage. The kinetics of proliferation was analyzed by cell analyzer CEDEX XS and expression of selected markers was assessed by flow cytometry. The morphology was analyzed by inverted microscope and TEM. Pellet culture system and chondrogenic medium containing TGF-ß1 was used to induce chondrogenic differentiation. Chondrogenesis was analyzed by real-time PCR; the expression of collagen type I and type II was compared. MSCs from all sources showed similar kinetics of proliferation and shared expression of CD73, CD90 and CD105; and were negative for CD14, CD20, CD34 and CD45. Observation under inverted microscope and TEM showed similar morphology of all analyzed MSCs. Cells from all sources underwent chondrogenic differentiation - they expressed collagen type II and acid mucopolysaccharides typical for hyaline cartilage. On the basis of obtained results it should be emphasized that MSCs from bone marrow, adipose tissue and umbilical cord share biological properties. They possess the chondrogenic potential and may be utilized in cartilage tissue engineering.

Stem cell regenerative potential for plastic and reconstructive surgery.

Stem cells represent heterogeneous population of undifferentiated cells with unique characteristics of long term self renewal and plasticity. Moreover, they are capable of active migration to diseased tissues, secretion of different bioactive molecules, and they have immunosuppressive potential as well. They occur in all tissues through life and are involved in process of embryogenesis and regeneration. During last decades stem cells attracted significant attention in each field of medicine, including plastic and reconstructive surgery. The main goal of the present review article is to present and discuss the potential of stem cells and to provide information about their safe utilization in chronic wounds and fistulae healing, scar management, breast reconstruction, as well as in bone, tendon and peripheral nerve regeneration.

Induced pluripotent stem cells and their implication for regenerative medicine.

In 2006 Yamanaka's group showed that stem cells with properties similar to embryonic stem cells could be generated from mouse fibroblasts by introducing four genes. These cells were termed induced pluripotent stem cells (iPSCs). Because iPSCs avoid many of ethical concerns associated with the use of embryonic material, they have great potential in cell-based regenerative medicine. They are suitable also for other various purposes, including disease modelling, personalized cell therapy, drug or toxicity screening and basic research. Moreover, in the future, there might become possible to generate organs for human transplantation. Despite these progresses, several studies have raised the concern for genetic and epigenetic abnormalities of iPSCs that could contribute to immunogenicity of some cells differentiated from iPSCs. Recent methodological improvements are increasing the ease and efficacy of reprogramming, and reducing the genomic modification. However, to minimize or eliminate genetic alternations in the derived iPSC line creation, factor-free human iPSCs are necessary. In this review we discuss recent possibilities of using iPSCs for clinical applications and new advances in field of their reprogramming methods. The main goal of present article was to review the current knowledge about iPSCs and to discuss their potential for regenerative medicine.

Therapeutic applications of mesenchymal/medicinal stem/signaling cells preconditioned with external factors: Are there more efficient approaches to utilize their regenerative potential?

Mesenchymal/medicinal stem/signaling cells (MSCs) have emerged as a promising treatment option for various disorders. However, the donor's age, advanced stage of disease, and prolonged in vitro expansion often diminish the innate regenerative potential of MSCs. Besides that, the absence of MSCs' comprehensive "pre-admission testing" can result in the injection of cells with reduced viability and function, which may negatively affect the overall outcome of MSC-based therapies. It is, therefore, essential to develop effective strategies to improve the impaired biological performance of MSCs. This review focuses on the comprehensive characterization of various methods of external MSCs stimulation (hypoxia, heat shock, caloric restriction, acidosis, 3D culture, and application of extracellular matrix) that augment their medicinal potential. To emphasize the significance of MSCs priming, we summarize the effects of individual and combined preconditioning approaches, highlighting their impact on MSCs' response to either physiological or pathological conditions. We further investigate the synergic action of exogenous factors to maximize MSCs' therapeutic potential. Not to omit the field of tissue engineering, the application of pretreated MSCs seeded on scaffolds is discussed as well.

Stem Cells and Their Derivatives: An Implication for the Regeneration of Nonunion Fractures.

Despite advances in biomedical research, fracture nonunion rates have remained stable throughout the years. Long-bone fractures have a high likelihood of nonunion, but the specific biological pathways involved in this severe consequence are unknown. Fractures often heal in an organized sequence, including the production of a hematoma and an early stage of inflammation, the development of a soft callus and hard callus, and eventually the stage of bone remodeling. Deficient healing can result in a persistent bone defect with instability, discomfort, and loss of function. In the treatment of nonunions, mesenchymal stem cells (MSCs) prove to be a promising and safe alternative to the standard therapeutic strategies. Moreover, novel scaffolds are being created in order to use a synergistic biomimetic technique to rapidly generate bone tissue. MSCs respond to acellular biomimetic matrices by regenerating bone. Extracellular vesicles (EVs) derived from MSCs have recently gained interest in the field of musculoskeletal regeneration. Although many of these techniques and technologies are still in the preclinical stage and have not yet been approved for use in humans, novel approaches to accelerate bone healing via MSCs and/or MSC derivatives have the potential to reduce the physical, economic, and social burdens associated with nonhealing fractures and bone defects. In this review, we focus on providing an up-to-date summary of recent scientific studies dealing with the treatment of nonunion fractures in clinical and preclinical settings employing MSC-based therapeutic techniques.

The Immunomodulatory Role of Cell-Free Approaches in SARS-CoV-2-Induced Cytokine Storm-A Powerful Therapeutic Tool for COVID-19 Patients.

Currently, there is still no effective and definitive cure for the coronavirus disease 2019 (COVID-19) caused by the infection of the novel highly contagious severe acute respiratory syndrome virus (SARS-CoV-2), whose sudden outbreak was recorded for the first time in China in late December 2019. Soon after, COVID-19 affected not only the vast majority of China's population but the whole world and caused a global health public crisis as a new pandemic. It is well known that viral infection can cause acute respiratory distress syndrome (ARDS) and, in severe cases, can even be lethal. Behind the inflammatory process lies the so-called cytokine storm (CS), which activates various inflammatory cytokines that damage numerous organ tissues. Since the first outbreak of SARS-CoV-2, various research groups have been intensively trying to investigate the best treatment options; however, only limited outcomes have been achieved. One of the most promising strategies represents using either stem cells, such as mesenchymal stem cells (MSCs)/induced pluripotent stem cells (iPSCs), or, more recently, using cell-free approaches involving conditioned media (CMs) and their content, such as extracellular vesicles (EVs) (e.g., exosomes or miRNAs) derived from stem cells. As key mediators of intracellular communication, exosomes carry a cocktail of different molecules with anti-inflammatory effects and immunomodulatory capacity. Our comprehensive review outlines the complex inflammatory process responsible for the CS, summarizes the present results of cell-free-based pre-clinical and clinical studies for COVID-19 treatment, and discusses their future perspectives for therapeutic applications.

Decellularization of the human urethra for tissue engineering applications.

Recently, several scaffolds have been introduced for urethral tissue engineering. However, acellular human urethral scaffold harvested from deceased donors may provide significant advantages compared to synthetic, composite, or other biological scaffolds. This study aims to develop the protocol for decellularization of the human urethra that preserves substantial extracellular matrix (ECM) components, which are essential for subsequent recellularization mimicking the natural environment of the native ECM. A total of 12 human urethras were harvested from deceased donors. An equal part of every harvested urethra was used as a control sample for analyses. The protocol design was based on the enzyme-detergent-enzyme method. Trypsin and Triton X-100 were used to remove cells, followed by DNase treatment to remove DNA residues. Subsequently, the specimens were continually rinsed in deionized water for seven days. The efficiency of decellularization was determined by histochemistry, immunohistochemical staining, scanning electron microscopy (SEM), and DNA quantification. Histological analysis confirmed cell removal and preservation of urethral structure after decellularization. The preservation of collagen IV and fibronectin was confirmed by histologic examination and immunohistochemical staining. SEM confirmed the maintenance of the ultrastructural architecture of ECM and fibers. DNA content in decellularized urethra was significantly lower compared to the native sample (P < 0.001), and so the criteria for decellularized tissue were met. Cytotoxicity analysis data showed that the matrix-conditioned medium did not contain soluble toxins and had no significant inhibitory effect on cell proliferation, providing evidence that the decellularized samples are not toxic. This study demonstrates the feasibility of the enzyme-detergent-enzyme-based decellularization protocol for removing cellular components and maintaining urethral ECM and its ultrastructure. Moreover, obtained results provide solid ground for recellularization and urethral tissue engineering, which will follow.