The Truth Is Out There: Biological Features and Clinical Indications of Extracellular Vesicles from Human Perinatal Stem Cells.

The potential of perinatal tissues to provide cellular populations to be used in different applications of regenerative medicine is well established. Recently, the efforts of researchers are being addressed regarding the evaluation of cell products (secreted molecules or extracellular vesicles, EVs) to be used as an alternative to cellular infusion. The data regarding the effective recapitulation of most perinatal cells' properties by their secreted complement point in this direction. EVs secreted from perinatal cells exhibit key therapeutic effects such as tissue repair and regeneration, the suppression of inflammatory responses, immune system modulation, and a variety of other functions. Although the properties of EVs from perinatal derivatives and their significant potential for therapeutic success are amply recognized, several challenges still remain that need to be addressed. In the present review, we provide an up-to-date analysis of the most recent results in the field, which can be addressed in future research in order to overcome the challenges that are still present in the characterization and utilization of the secreted complement of perinatal cells and, in particular, mesenchymal stromal cells.

Facing the Challenges in the COVID-19 Pandemic Era: From Standard Treatments to the Umbilical Cord-Derived Mesenchymal Stromal Cells as a New Therapeutic Strategy.

Coronavirus disease 2019 (COVID-19), the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which counts more than 650 million cases and more than 6.6 million of deaths worldwide, affects the respiratory system with typical symptoms such as fever, cough, sore throat, acute respiratory distress syndrome (ARDS), and fatigue. Other nonpulmonary manifestations are related with abnormal inflammatory response, the "cytokine storm", that could lead to a multiorgan disease and to death. Evolution of effective vaccines against SARS-CoV-2 provided multiple options to prevent the infection, but the treatment of the severe forms remains difficult to manage. The cytokine storm is usually counteracted with standard medical care and anti-inflammatory drugs, but researchers moved forward their studies on new strategies based on cell therapy approaches. The perinatal tissues, such as placental membranes, amniotic fluid, and umbilical cord derivatives, are enriched in mesenchymal stromal cells (MSCs) that exert a well-known anti-inflammatory role, immune response modulation, and tissue repair. In this review, we focused on umbilical-cord-derived MSCs (UC-MSCs) used in in vitro and in vivo studies in order to evaluate the weakening of the severe symptoms, and on recent clinical trials from different databases, supporting the favorable potential of UC-MSCs as therapeutic strategy.

Current Perspectives on Adult Mesenchymal Stromal Cell-Derived Extracellular Vesicles: Biological Features and Clinical Indications.

Extracellular vesicles (EVs) constitute one of the main mechanisms by which cells communicate with the surrounding tissue or at distance. Vesicle secretion is featured by most cell types, and adult mesenchymal stromal cells (MSCs) of different tissue origins have shown the ability to produce them. In recent years, several reports disclosed the molecular composition and suggested clinical indications for EVs derived from adult MSCs. The parental cells were already known for their roles in different disease settings in regulating inflammation, immune modulation, or transdifferentiation to promote cell repopulation. Interestingly, most reports also suggested that part of the properties of parental cells were maintained by isolated EV populations. This review analyzes the recent development in the field of cell-free therapies, focusing on several adult tissues as a source of MSC-derived EVs and the available clinical data from in vivo models.

Human Amnion-Derived Mesenchymal Stromal Cells in Cirrhotic Patients with Refractory Ascites: A Possible Anti-Inflammatory Therapy for Preventing Spontaneous Bacterial Peritonitis.

Cirrhosis is associated with dysregulated immune cell activation and immune dysfunction. These conditions modify gut flora, facilitate bacterial translocation, and increase susceptibility to bacterial peritonitis and consequent systemic infections by dramatically affecting long-term patient survival. Human amnion-derived mesenchymal stromal cells (hA-MSCs) exert immunomodulatory potential benefit, and have the ability to modulate their actions, especially in situations requiring immune activation through mechanisms not fully understood. In this study, we aimed to investigate, in vitro, the immunostimulant or immunosuppressive effects of hA-MSCs on cellular components of ascitic fluid obtained from cirrhotic patients with refractory ascites. We found that hA-MSCs viability is not affected by ascitic fluid and, interestingly, hA-MSCs diminished the pro-inflammatory cytokine production, and promoted anti-inflammatory M2 macrophage polarization. Moreover, we found that there was no simultaneous significant decrease in the M1-like component, allowing a continual phagocytosis activity of macrophages and NK cells to restore a physiological condition. These data highlight the plasticity of hA-MSCs' immunomodulatory capacity, and pave the way to further understanding their role in conditions such as spontaneous bacterial peritonitis.

Energy Metabolism Analysis of Three Different Mesenchymal Stem Cell Populations of Umbilical Cord Under Normal and Pathologic Conditions.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) are a pivotal source of therapeutically active cells for regenerative medicine due to their multipotent differentiation potential, immunomodulatory and anti-inflammatory proprieties, as well as logistical collection advantages without ethical concerns. However, it remains poorly understood whether MSCs from different compartments of the human umbilical cord are therapeutically superior than others. In this study, MSCs were isolated from Wharton's jelly (WJ-MSCs), perivascular region (PV-MSCs) and cord lining (CL-MSCs) of hUC. These cells expressed the mesenchymal markers (CD90, CD73), stemness marker (OCT4), endothelial cell adhesion molecular marker (CD146), and the monocyte/macrophage marker (CD14) found within the MSC population implicated as a key regulator of inflammatory responses to hypoxia, was displayed by WJ-, PV-, and CL-MSCs respectively. A direct consequence of oxygen and glucose deprivation during stroke and reperfusion is impaired mitochondrial function that contributes to cellular death. Emerging findings of mitochondria transfer provide the basis for the replenishment of healthy mitochondria as a strategy for the treatment of stroke. Cell Energy Phenotype and Mito Stress tests were performed the energy metabolic profile of the three MSC populations and their mitochondrial function in both ambient and OGD cell culture conditions. PV-MSCs showed the highest mitochondrial activity. CL-MSCs were the least affected by OGD/R condition, suggesting their robust survival in ischemic environment. In this study, MSC populations in UC possess comparable metabolic capacities and good survival under normal and hypoxic conditions suggesting their potential as transplantable cells for mitochondrial-based stem cell therapy in stroke and other ischemic diseases.

Wharton's Jelly Mesenchymal Stromal Cells from Human Umbilical Cord: a Close-up on Immunomodulatory Molecules Featured In Situ and In Vitro.

Therapeutic options for end-stage organ failure are often limited to whole organ transplantation. The tolerance or rejection of the transplanted organ is driven by both early non-specific innate and specific adaptive responses. The use of mesenchymal stromal cells (MSCs) is considered a promising tool in regenerative medicine. Human umbilical cord (HUC) is an easily available source of MSCs, without relevant ethical issues. Moreover, Wharton's jelly-derived MSCs (WJ-MSCs), showed consistent immunomodulatory features that may be useful to promote immune tolerance in the host after transplantation. Few data are available on the phenotype of WJ-MSCs in situ. We investigated the expression of immune-related molecules, such as HLAs, IDO, CD276/B7-H3, and others, both in situ (HUC) and in in vitro-cultured WJ-MSCs. Morphological and biochemical techniques were used to define the expression of such molecules. In addition, we focused on the possible role of CD276/B7-H3 on T cells proliferation inhibition. We assessed CD276/B7-H3 expression by WJ-MSCs both in situ and alongside cell culture. WJ-MSCs were able to suppress T cell proliferation in mixed lymphocyte reaction (MLR). Moreover, we describe for the first time a specific role for CD276/B7-H3, since the immunomodulatory ability of WJ-MSCs was abolished upon anti-CD276/B7-H3 antibody addition to the MLR. These results further detail the immune regulation properties and tolerance induction exerted by human WJ-MSCs, in particular pointing to CD276/B7-H3 as one of the main involved factors. These data further suggest WJ-MSCs as potent tools to modulate local immune response in "support-type" regenerative medicine approaches.

Hsp10 nuclear localization and changes in lung cells response to cigarette smoke suggest novel roles for this chaperonin.

Heat-shock protein (Hsp)10 is the co-chaperone for Hsp60 inside mitochondria, but it also resides outside the organelle. Variations in its levels and intracellular distribution have been documented in pathological conditions, e.g. cancer and chronic obstructive pulmonary disease (COPD). Here, we show that Hsp10 in COPD undergoes changes at the molecular and subcellular levels in bronchial cells from human specimens and derived cell lines, intact or subjected to stress induced by cigarette smoke extract (CSE). Noteworthy findings are: (i) Hsp10 occurred in nuclei of epithelial and lamina propria cells of bronchial mucosa from non-smokers and smokers; (ii) human bronchial epithelial (16HBE) and lung fibroblast (HFL-1) cells, in vitro, showed Hsp10 in the nucleus, before and after CSE exposure; (iii) CSE stimulation did not increase the levels of Hsp10 but did elicit qualitative changes as indicated by molecular weight and isoelectric point shifts; and (iv) Hsp10 nuclear levels increased after CSE stimulation in HFL-1, indicating cytosol to nucleus migration, and although Hsp10 did not bind DNA, it bound a DNA-associated protein.

Umbilical cord revisited: from Wharton's jelly myofibroblasts to mesenchymal stem cells.

The umbilical cord (UC) is an essential part of the placenta, contributing to foetal development by ensuring the blood flow between mother and foetus. The UC is formed within the first weeks of gestation by the enclosure of the vessels (one vein and two arteries) into a bulk of mucous connective tissue, named Wharton's jelly (WJ) and lined by the umbilical epithelium. Since their first identification, cells populating WJ were described as unusual fibroblasts (or myofibroblasts). Recent literature data further highlighted the functional interconnection between UC and the resident cells. The UC represents a reservoir of progenitor populations which are collectively grouped into MSCs (mesenchymal stem cells). Such cells have been sourced from each component of the cord, namely the sub-amnion layer, the WJ, the perivascular region, and the vessels. These cells mainly show adherence to the phenotype of adult MSCs (as bone marrow-derived ones) and can differentiate towards mature cell types belonging to all the three germ layers. In addition, cells from human UC are derived from an immunoprivileged organ, namely the placenta: in fact, its development and function depend on the elusion of the maternal immune response towards the semi-allogeneic embryo. This is reflected in the expression of immunomodulatory molecules by UC-derived MSCs. The present paper describes UC structural features and the cell types which can be derived, with a focus on their phenotype and the novel results which boosted the use of UC-derived cells for regenerative medicine applications.

Hsp10: anatomic distribution, functions, and involvement in human disease.

There is growing evidence that molecular chaperones/heat shock proteins are involved in the pathogenesis of a number of human diseases, known as chaperonopathies. A better molecular understanding of the pathogenetic mechanisms is essential for addressing new strategies in diagnostics, therapeutics and clinical management of chaperonopathies, including those in which Hsp10 is involved. This chaperonin has been studied for a long time as a member of the mitochondrial protein-folding machine. However, although in normal cells Hsp10 is mainly localized in the mitochondrial matrix, it has also been found during and after stress in other subcellular compartments, such as cytosol, vesicles and secretory granules, alone or in combination with other proteins. In these extramitochondrial locales, Hsp10 plays an active role in cell signalling. For example, cancer cells often show altered levels of Hsp10, compared to normal cells. Hsp10 may also be found in the extracellular space and in the bloodstream, with a possible immunomodulatory activity. This minireview focuses on some studies to date on the involvement of Hsp10 in human disease pathogenesis.

Human Wharton's jelly mesenchymal stem cells maintain the expression of key immunomodulatory molecules when subjected to osteogenic, adipogenic and chondrogenic differentiation in vitro: new perspectives for cellular therapy.

Rheumatoid arthritis and osteoarthritis are the main diseases that imply an inflammatory process at the joints involving the articular cartilage. Recently, mesenchymal stem cells (MSCs) derived from perinatal tissues were considered good candidates for cellular therapy of musculoskeletal and orthopaedic diseases, since they can differentiate into multiple cell types and are an easily accessible cellular source. Therefore, several protocols exist on the differentiation of mesenchymal stem cells of different origins into osteoblasts and chondrocytes. Another key feature of MSCs is their capacity to modulate the immune system responses in vitro and in vivo. This may have critical outcomes in diseases of the musculoskeletal system where an inflammatory or autoimmune process is at the basis of the main disease. In the present paper, after isolation of MSCs from Wharton's Jelly (WJ-MSCs), we performed the three standard differentiation protocols. The acquisition of the differentiated phenotype was demonstrated by the specific histological stains. As the main objective of this work, we determined the expression of immunomodulatory molecules (by immunohistochemistry and qualitative RT-PCR), both in undifferentiated cells and after differentiation. We demonstrated for the first time that immune-related molecules (as B7-H3/CD276 and HLA-E) which have been characterized in undifferentiated MSCs, are also expressed by the differentiated progeny. This strongly suggests that also after the acquisition of a mature phenotype, WJ-MSCs-derived cells may maintain their immune privilege. This evidence, which deserves much work to be confirmed in vivo and in other MSCs populations, may provide a formal proof of the good results globally achieved with WJMSCs as cellular therapy vehicle.

New frontiers in regenerative medicine in cardiology: the potential of Wharton's jelly mesenchymal stem cells.

Cardiomyopathies are still the first cause of death in the world. The identification of resident stem cells, comprising those derived from sub-endocardial stroma, suggests the possible self regeneration of the heart under autocrine/paracrine modulation in the cardiac microenvironment. Nevertheless, because of the limited in vivo regeneration potential of damaged cardiac tissue, the use of drugs and ultimately cardiac transplantation remain the common treatments of heart diseases and defects. The differentiative potential of embryonic and mesenchymal stem cells (MSCs) derived from different tissues (such as bone marrow and adipose tissue) was extensively explored in cell therapy for regenerative medicine. Many groups have been focused, in recent years, on isolation, characterization, and differentiation potential of MSCs derived from perinatal (or extraembryonic) tissues, mainly the placenta and the human umbilical cord. In this review, we summarized recent works about the stemness of Wharton's jelly stromal cells and their potential in cardiac regeneration with favourable use in cell therapy and regenerative medicine. The peculiar features of these cells, as the expression of cardiac-specific transcription factors and immunomodulatory molecules suggest that human umbilical cord may be considered as a reliable alternative source of MSC useful for advanced therapy in cardiac regenerative medicine.

Isolation and characterization of CD276+/HLA-E+ human subendocardial mesenchymal stem cells from chronic heart failure patients: analysis of differentiative potential and immunomodulatory markers expression.

Mesenchymal stem cells (MSCs) are virtually present in all postnatal organs as well as in perinatal tissues. MSCs can be differentiated toward several mature cytotypes and interestingly hold potentially relevant immunomodulatory features. Myocardial infarction results in severe tissue damage, cardiomyocyte loss, and eventually heart failure. Cellular cardiomyoplasty represents a promising approach for myocardial repair. Clinical trials using MSCs are underway for a number of heart diseases, even if their outcomes are hampered by low long-term improvements and the possible presence of complications related to cellular therapy administration. Therefore, elucidating the presence and role of MSCs that reside in the post-infarct human heart should provide essential alternatives for therapy. In the current article we show a novel method to reproducibly isolate and culture MSCs from the subendocardial zone of human left ventricle from patients undergoing heart transplant for post-infarct chronic heart failure (HSE-MSCs, human subendocardial mesenchymal stem cells). By using both immunocytochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR), we demonstrated that these cells do express key MSCs markers and do express heart-specific transcription factors in their undifferentiated state, while lacking strictly cardiomyocyte-specific proteins. Moreover, these cells do express immunomodulatory molecules that should disclose their further potential in immune modulation processes in the post-infarct microenvironment. Another novel datum of potentially relevant interest is the expression of cardiac myosin heavy chain at nucclear level in HSE-MSCs. Standard MSCs trilineage differentiation experiments were also performed. The present paper adds new data on the basic biological features of heart-resident MSCs that populate the organ following myocardial infarction. The use of heart-derived MSCs to promote in-organ repair or as a cellular source for cardiomyoplasty is a fascinating and challenging task, which deserves further research efforts.

Hsp60 and heme oxygenase-1 (Hsp32) in acute myocardial infarction.

Heat shock proteins (Hsps) are produced in response to various stressors, including ischemia-reperfusion, and they can exit cells and reach the blood. In this pilot study, we determined serum levels of Hsp60 and heme-oxygenase-1 (HO-1; also named Hsp32) in subjects with acute myocardial infarction (AMI) to assess their clinical significance and potential prognostic value. We also performed a bioinformatics analysis of the 2 molecules in search of structural clues on the mechanism of their release from cells. We studied 40 patients consecutively admitted for AMI (male:female patient ratio=20:20, mean age: 64 ± 13 years) and 40 matched controls. A blood sample was drawn for biochemical analyses within 24 h of symptoms onset, and Hsp60 and HO-1 concentrations were determined by enzyme-linked immunosorbent assay (ELISA). All patients were followed up for 6 months to register adverse post-AMI cardiovascular events. A multivariate analysis demonstrated that elevated Hsp60 (P=0.0361), creatine phosphokinase-muscle brain (CK-MB) (P=0.0446), and troponin (P=0.0490) were predictive of post-AMI adverse events. In contrast, increased HO-1 showed a significant association with less severity of coronary artery diseases (P=0.0223). These findings suggest that Hsp60 and HO-1 play distinct roles in the pathogenesis of AMI and subsequent AMI-related pathology. The possibility that these proteins differ in their roles and mechanisms of action in AMI and post-AMI pathology was supported also by the bioinformatics estimates of probability of their localization in various subcellular compartments. The results clear the way for subsequent investigation on the pathogenetic role and clinical significance of Hsp60 and HO-1 in AMI.

New emerging potentials for human Wharton's jelly mesenchymal stem cells: immunological features and hepatocyte-like differentiative capacity.

In recent years, human mesenchymal stem cells (MSC) have been extensively studied. Their key characteristics of long-term self-renewal and a capacity to differentiate into diverse mature tissues favor their use in regenerative medicine applications. Stem cells can be found in embryonic and extraembryonic tissues as well as in adult organs. Several reports indicate that cells of Wharton's jelly (WJ), the main component of umbilical cord extracellular matrix, are multipotent stem cells, expressing markers of bone marrow mesenchymal stem cells (BM-MSC), and giving rise to different cellular types of both connective and nervous tissues. Wharton's jelly mesenchymal stem cells (WJ-MSC) express markers previously characterized in embryonic stem cells (ESC), such as Nanog and Oct3/4A. WJ-MSC further emerge as promising hypoimmunogenic cells, due to the expression of molecules able to modulate NK cells and expand regulatory T-cell populations. Moreover, it is now accepted that the differentiative capacities of such cells span all the mesoderm-derived tissues, extending to neuroectodermal as well as endodermal lineages. In this review, we compare very recent data on the potential of WJ-MSC to undergo hepatocyte-like differentiation with the results obtained from other adult MSC populations. Data in the literature strongly suggest that WJ-MSC can differentiate into diverse cell types, showing a unique ability to cross lineage borders. This, together with their in vitro proliferative potential and their immunoregulatory features, renders these cells extremely promising for regenerative medicine applications in different pathological settings.

Human Hsp10 and Early Pregnancy Factor (EPF) and their relationship and involvement in cancer and immunity: current knowledge and perspectives.

This article is about Hsp10 and its intracellular and extracellular forms focusing on the relationship of the latter with Early Pregnancy Factor and on their roles in cancer and immunity. Cellular physiology and survival are finely regulated and depend on the correct functioning of the entire set of proteins. Misfolded or unfolded proteins can cause deleterious effects and even cell death. The chaperonins Hsp10 and Hsp60 act together inside the mitochondria to assist protein folding. Recent studies demonstrated that these proteins have other roles inside and outside the cell, either together or independently of each other. For example, Hsp10 was found increased in the cytosol of different tumors (although in other tumors it was found decreased). Moreover, Hsp10 localizes extracellularly during pregnancy and is often indicated as Early Pregnancy Factor (EPF), which is released during the first stages of gestation and is involved in the establishment of pregnancy. Various reports show that extracellular Hsp10 and EPF modulate certain aspects of the immune response with anti-inflammatory effects in patients with autoimmune conditions improving clinically after treatment with recombinant Hsp10. Moreover, Hsp10 and EPF are involved in embryonic development, acting as a growth factor, and in cell proliferation/differentiation mechanisms. Therefore, it becomes evident that Hsp10 is not only a co-chaperonin, but an active player in its own right in various cellular functions. In this article, we present an overview of various aspects of Hsp10 and EPF as they participate in physiological and pathological processes such as the antitumor response and autoimmune diseases.

Role of endothelial cell stress in the pathogenesis of chronic heart failure.

Endothelial cells are key modulators of diverse physiological processes, and their impaired function is a cause of numerous cardiovascular diseases. Under physiologic condition, the reactive oxygen and nitrogen mediators in endothelia lead to the signal propagation of the initial stimulus, by forming molecules with a longer half-life like hydrogen peroxide. Hydrogen peroxide is the focus of growing attention in endothelial biology, and consequently the enzymes involved in its generation and clearance are viewed as novel mediators of great importance. In particular, among peroxidases, myeloperoxidase is recognized as a key enzyme, capable of impairing intracellular NO reservoirs as well as producing oxidized amino acids such as 3-chlorotyrosine or 3-nitrotyrosine. This process switches the functional pathways from normal signalling to a condition characterized by oxidative and/or nitrosative stress. Understanding the molecular mechanisms involved in these stress responses in endothelium will lead to better therapeutic strategies for oxidative stress-driven cardiovascular diseases.

Oxidative stress induces myeloperoxidase expression in endocardial endothelial cells from patients with chronic heart failure.

Increased oxidative stress has been implicated in the pathogenesis of a number of cardiovascular diseases. Recent findings suggest that myeloperoxidase (MPO) may play a key role in the initiation and maintenance of chronic heart failure (CHF) by contributing to the depletion of the intracellular reservoir of nitric oxide (NO). NO consumption through MPO activity may lead to protein chlorination or nitration, leading to tissue damage. Primary cultures of human endocardial endothelial cells (EEC) obtained at heart transplantation of patients with CHF and human umbilical vein endothelial cells (HUVEC) were subjected to oxidative stress by incubation with hydrogen peroxide at non lethal (60 microM) dose for different exposure times (3 and 6 h). Treated and control cells were tested by immunohistochemistry and RT-PCR for MPO and 3-chlorotyrosine expression. Both endothelial cell types expressed myeloperoxidase following oxidative stress, with higher levels in EEC. Moreover, 3-chlorotyrosine accumulation in treated cells alone indicated the presence of MPO-derived hypochlorous acid. Immunohistochemistry on sections from post-infarcted heart confirmed in vivo the endothelial positivity to MPO, 3-chlorotyrosine and, to a minor extent, nitrotyrosine. Immunohistochemical observations were confirmed by detection of MPO mRNA in both stimulated EEC and HUVEC cells. This study demonstrates for the first time that EEC can express MPO after oxidative stress, both in vitro and in vivo, followed by accumulation of 3-chlorotyrosine, an end product of oxidative stress. Deregulation of endothelial functions may contribute to the development of a number of cardiovascular diseases, including CHF. The results also highlight the notion that endothelium is not only a target but also a key player in oxidative-driven cardiovascular stress.

Isolation and characterization of Oct-4+/HLA-G+ mesenchymal stem cells from human umbilical cord matrix: differentiation potential and detection of new markers.

The presence of multipotent cells in several adult and embryo-related tissues opened new paths for their use in regenerative medicine. Extraembryonic tissues such as umbilical cord are considered a promising source of stem cells, potentially useful in therapy. The characterization of cells from the umbilical cord matrix (Wharton's Jelly) and amniotic membrane revealed the presence of a population of mesenchymal-like cells, sharing a set of core-markers expressed by "mesenchymal stem cells". Several reports enlightened the differentiation capabilities of these cells, even if at times the lack of an extensive characterization of surface markers and immune co-stimulators expression revealed hidden pitfalls when in vivo transplantation was performed. The present work describes a novel isolation protocol for obtaining mesenchymal stem cells from the umbilical cord matrix. These cells are clonogenic, retain long telomeres, can undergo several population doublings in vitro, and can be differentiated in mature mesenchymal tissues as bone and adipose. We describe for the first time that these cells, besides expressing all of the core-markers for mesenchymal stem cells, feature also the expression, at both protein and mRNA level, of tolerogenic molecules and markers of all the three main lineages, potentially important for both their differentiative potential as well as immunological features.

Heterozygous nonsense SCN5A mutation W822X explains a simultaneous sudden infant death syndrome.

The sudden, unexpected, and unexplained death of both members of a set of healthy twins (simultaneous sudden infant death syndrome (SSIDS)) is defined as a case in which both infants meet the definition of sudden infant death syndrome individually. A search of the world medical literature resulted in only 42 reported cases of SSIDS. We report the case of a pair of identical, male, monozygotic twins, 138 days old, who suddenly died, meeting the full criteria of SSIDS and where a genetic screen was performed, resulting in a heterozygous nonsense SCN5A mutation (W822X) in both twins. Immunohistochemistry was performed on cardiac tissue samples utilizing polyclonal antibodies anti-Na+ CP type Valpha (C-20) and a terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling assay. The cellular localization of the Na+ CP type Valpha (C-20) demonstrated by confocal microscopy on staining pattern of myocytes was concentrated in the intercalated disks of ventricular myocytes. These findings suggest that defective ion channels represent viable candidates for the pathogenesis of SIDS and, obviously, of SSIDS, supporting a link between sudden infant death syndrome and cardiac channelopathies.