Genetic predisposition, environmental factors, and infectious agents interact in the development of gastric diseases. Helicobacter pylori (Hp) and Epstein–Barr virus (EBV) infection has recently been shown to be correlated with these diseases. A cross-sectional study was performed on 100 hospitalized Italian patients with and without gastric diseases. The patients were stratified into four groups. Significant methylation status differences among CDH1, DAPK, COX2, hMLH1 and CDKN2A were observed for coinfected (Hp-EBV group) patients; particularly, a significant presence of COX2 (p = 0.0179) was observed. For microsatellite instability, minor stability was described in the Hp-HBV group (69.23%, p = 0.0456). Finally, for p53 mutation in the EBV group, exon 6 was, significantly, most frequent in comparison to others (p = 0.0124), and in the Hp-EBV group exon 8 was, significantly, most frequent in comparison to others (p < 0.0001). A significant positive relationship was found between patients with infection (Hp, EBV or both) and p53 mutation (rho = 0.383, p = 0.0001), methylation status (rho = 0.432, p < 0.0001) and microsatellite instability (rho = 0.285, p = 0.004). Finally, we observed among infection and methylation status, microsatellite instability, and p53 mutation a significant positive relationship only between infection and methylation status (OR = 3.78, p = 0.0075) and infection and p53 mutation (OR = 6.21, p = 0.0082). According to our analysis, gastric disease in the Sicilian population has different pathways depending on the presence of various factors, including infectious agents such as Hp and EBV and genetic factors of the subject.

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 umbilical cord mesenchymal stem cells (hUC-MSCs) serve as a potential cell-based therapy for degenerative disease. They provide immunomodulatory and anti-inflammatory properties, multipotent differentiation potential and are harvested with no ethical concern. It is unknown whether MSCs collected from different areas of the human umbilical cord elicit more favorable effects than others. Three MSC populations were harvested from various regions of the human umbilical cord: cord lining (CL-MSCs), perivascular region (PV-MSCs), and Wharton's jelly (WJ-MSCs). Mesenchymal markers (CD90 and CD73) were expressed by all three cell populations. Stemness marker (OCT4), endothelial cell adhesion molecular marker (CD146), and monocyte-macrophage marker (CD14) were expressed by WJ-MSCs, PV-MSCs, and CL-MSCs, respectively. Stroke presents with oxygen and glucose deprivation and leads to dysfunctional mitochondria and consequently cell death. Targeting the restoration of mitochondrial function in the stroke brain through mitochondrial transfer may be effective in treating stroke. In vitro exposure to ambient and OGD conditions resulted in CL-MSCs number decreasing the least post-OGD/R exposure, and PV-MSCs exhibiting the greatest mitochondrial activity. All three hUC-MSC populations presented similar metabolic activity and survival in normal and pathologic environments. These characteristics indicate hUC-MSCs potential as a potent therapeutic in regenerative medicine.

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.

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.

The first complete description of the umbilical cord (UC) tissue was given by Thomas Wharton, whose book “Adenografia sive glandularum totius corporis descriptio,” was published in London in 1656. This allowed to first define the features of the peculiar tissue constituting the bulk of the cord, now known as Wharton’s jelly (WJ), that is nowadays classified as a mature mucoid connective tissue. The embryological origin of the UC is quite complex because different extraembryonic tissues concur to its formation: extraembryonic mesoderm, extraembryonic endoderm, amnioblasts. There is a vast literature on the development of the UC tissues since the first phases of differentiation and specification of its constituents. With respect to the difficulties to work with human embryos, studies on animal models shed new light on the developmental origin of the cells found in mature UC. The mesoblast is the key tissue that evolutionarily differentiates the triblastic embryos from the diblastic ones. And in human development, differently from well-known animal models as the mouse, the first 2 weeks are devoted principally to the development of extraembryonic tissues. During implantation, a key phenomenon occurs in the embryoblast cells: these cells organize themselves in a planar arrangement constituted by two epithelial layers, the epiblast, characterized by mainly columnar cells, and the hypoblast. The second half of the second week of development is characterized by the formation of other key extraembryonic tissues. At day 11, a deepithelization and cavitation process starts in the epiblast layer, giving rise to the formation of the first amnioblast cells. These will proliferate and cover the overlying cytotrophoblast to create the primordium of the amniotic cavity. Around days 12e13, another extraembryonic tissue appears, namely, extraembryonic mesoderm or mesoblast. The origin of this tissue in human embryo is still a debated

Among the potential sources of cells with attractive features for regenerative medicine, perinatal tissues have gained much attention in recent years. The umbilical cord (UC) is a fundamental part of placenta, the organ providing feto–maternal interface during pregnancy. The mature UC is constituted by three vessels, one vein, and two arteries, which are comprised into a meshwork formed by a mature mucous connective tissue, named Wharton's jelly (WJ). Recent data from research groups worldwide highlighted that WJ stromal cells possess unique features both in terms of differentiative ability and immunomodulation. These features are desirable for cellular therapy applications, thereby expanding the number of possible clinical uses of these cells. In this chapter, we provide insights on the developmental history of WJ cells and the recent developments for their use in both "replacement type" or "supportive" regenerative medicine applications in key diseases.

Wharton’s jelly mesenchymal stromal cells (WJ-MSCs) have been recently exploited as a feeder layer in coculture systems to expand umbilical cord blood–hematopoietic stem/progenitor cells (UCB-HSPCs). Here, we investigated the role of WJ-MSCs in supporting ex vivo UCB-HSPC expansion either when cultured in direct contact (DC) with WJ-MSCs or separated by a transwell system or in the presence of WJ-MSC–conditioned medium. We found, in short-term culture, a greater degree of expansion of UCB-CD34 + cells in a DC system (15.7 ± 4.1-fold increase) with respect to the other conditions. Moreover, in DC, we evidenced two different CD34 + cell populations (one floating and one adherent to WJ-MSCs) with different phenotypic and functional characteristics. Both multipotent CD34 + /CD38 − and lineage-committed CD34 + /CD38 + hematopoietic progenitors were expanded in a DC system. The former were significantly more represented in the adherent cell fraction than in the floating one (18.7 ± 11.2% vs. 9.7 ± 7.9% over the total CD34 + cells). Short-term colony forming unit (CFU) assays showed that HSPCs adherent to the stromal layer were able to generate a higher frequency of immature colonies (CFU-granulocyte/macrophage and burst-forming unit erythroid/large colonies) with respect to the floating cells. In the attempt to identify molecules that may play a role in supporting the observed ex vivo HSPC growth, we performed secretome analyses. We found a number of proteins involved in the HSPC homing, self-renewal, and differentiation in all tested conditions. It is important to note that a set of sixteen proteins, which are only in part reported to be expressed in any hematopoietic niche, were exclusively found in the DC system secretome. In conclusion, WJ-MSCs allowed a significant ex vivo expansion of multipotent as well as committed HSPCs. This may be relevant for future clinical applications.

In recent years, umbilical cord blood (UCB) has been widely used as an alternative source to bone marrow (BM) for transplantation of hematopoietic stem and progenitor cells (HSPCs) in a variety of hematological and non-hematological disorders. Nevertheless, the insufficient number of UCB-HSPCs for graft represents a major challenge. HSPCs ex vivo expansion prior to transplantation is a valid strategy to overcome this limit. Several attempts to optimize the expansion conditions have been reported, including the use of mesenchymal stromal cells (MSCs) as feeder layer. Wharton's Jelly (WJ), the main component of umbilical cord (UC) matrix, is especially rich in MSCs, which are considered ideal candidates for feeder layer in co-culture systems. In fact, they can be easily harvested and grow robustly in culture, producing a confluent monolayer in a short time. Similarly to bone marrow-mesenchymal stromal cells (BM-MSCs), WJ-derived MSCs (WJ-MSCs) have been used to support hematopoiesis in vitro and in vivo. Here, we review the rationale for using MSCs, particularly WJ-MSCs, as a feeder layer for UCB-HSPCs ex vivo expansion. In addition, we report the main findings attesting the use of these MSCs as a support in hematopoiesis.

This chapter focuses on the immunomodulatory properties of placental mesenchymal stromal cells (MSCs) derived from the amniotic membrane, umbilical cord, and chorionic villi. In accordance with the in vitro data, some in vivo studies clearly support that the immunomodulatory effects of human Wharton's jelly MSCs/human umbilical cord MSCs during the course of cellular therapy can be attributed, at least in part, to the disturbance of Dendritic cells differentiation and maturation processes. The response of T cells to their cognate antigens is principally governed by two distinct molecular signals. Natural killer cells are important cytolytic effector cells that play a relevant role in innate immunity, participating in the first line of defense against viruses and pathogens, and also play a role in immune surveillance against tumors. Neutrophils are the professional phagocytosing cells present in both blood and tissues that have the capacity to recognize, engage, phagocytose, and kill their targets by multiple cytotoxic mechanisms.

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 dis- tribution have been documented in pathological conditions, e.g. cancer and chronic obstructive pulmonary disease (COPD). Cigarette smoke (CS) is a potent stressor for the respiratory system, but its effects on the expression, function, and cellular locali- zation of mitochondrial chaperonins are still largely unknown. We studied in vivo (airways biopsies) the localization of Hsp10 and Hsp60 in patients (smokers and non-smokers) affected by mild-moderate COPD, and charac- terized the effects of non-lethal doses of CS extract (CSE) on the expression of these molecules in two human cell lines: lung fibroblasts (HFL-1) and bronchial epithelial cells (16HBE). We applied various in vitro methods: IHC, subcellular fractionation analyses (SFA), western blotting (WB), ICC, transmission electron microscopy (TEM) immunogold, chromati protein extracts (CPE), as well as 2D-gel based proteomics analyses. Bioinformatics was used to gather structural in silico data. IHC showed that Hsp10 occurred in nuclei of epithelial and lamina propria cells of bronchial mucosa from non-smokers and smokers. ICC, SFA, and WB showed that 16HBE and HFL-1 cells featured nuclear Hsp10, before and after CSE exposure; TEM immunogold further confirmed this observation. Proteomics data showed that CSE stimulation did not increase the levels of Hsp10 but did elicit qualitative changes as indicated by molecular weight and isoelectric point shifts. Bioinformatics analyses indicated that Hsp10 can localize in extramitochondrial sites, such as the nucleus, even if Hsp10 lacks known DNA-binding motifs or nuclear import signals. 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 as suggested by CPE/gel retardation experiments. Data reported here indicate that in human cells of the respiratory mucosa there are at least three different intracellular locales for Hsp10: mitochondrial, nuclear, and cyto- solic. Further experiments are en route for the definition of the mechanisms underlying the transfer of Hsp10 to the nucleus and other cellular/extracellular compartments. This work was supported by grants from University of Palermo (FFR 2012) to GLR.

Wharton’s jelly mesenchymal stromal cells (WJ-MSCs) have a unique ability to cross lineage borders. Their immunomodulatory and anti-inflammatory features, further render these cells promising for regenerative medicine applications. Few data are present in literature on the expression of immunomodulatory molecules in umbilical cord (UC) tissue and their maintenance in paired cultured WJ-MSCs, an important aspect in cellular therapy applications. In addition, few data exist on the maintenance of expression of immunomodulatory molecules in mature cell types differentiated from MSCs. Therefore we investigated, in vivo (in UC at full term) and in vitro (in either undifferentiated or differentiated WJ-MSCs), the expression of different markers and their maintenance alongside cell culture, ex vivo expansion and differentiation. IHC, ICC, RT-PCR and flow cytometry were used to detect expression of markers in both paired UC sections and WJ-MSCs. Differentiation was performed towards the standard mesenchymal lineages as well as hepatocyte-like cells. Paired ICC and IHC analyses showed that for most of the analyzed molecules the expression at the protein level is maintained in both UC tissue and WJ-MSCs. Structural molecules were expressed in both WJ and umbilical epithelium (UE), as well as in WJ-MSCs. We showed for the first time that UE and WJ were positive for both HLA-ABC and HLA-E, while HLA-DR was not detectable. The same data were confirmed on WJ-MSCs. Both B7-1 and B7-2 were absent in UC and WJ-MSCs, while we showed for the first time that B7-H3 was highly expressed in both WJ and WJMSCs. Differentiation experiments showed that immunomodulatory molecules were expressed upon application of complex differentiation protocols, in parallel to the acquisition of mature markers or functions. Some important conclusions may be drawn from the current experiments: i) WJMSCs mostly maintain the expression of molecules just present in their “niche”, under standard culture conditions; ii) the parallel expression of immunomodulatory molecules sheds new light on the ability of WJ-MSCs to modulate host immune responses; iii) the in vivo expression of molecules such as HLA-E and B7-H3 opens new questions on the role of WJ during pregnancy; iv) the expression of these molecules in differentiated cells provides key features for in vivo applications, in particular for hepatocyte-like cells.

Mesenchymal stem cells derived from Wharton’s jelly (WJ-MSCs) recently emerged as promising tools for cellular therapy due to their ability to differentiate into diverse cell types and their immunomodulatory features. Little is known on the expression of immunomodulatory molecules in mature cells differentiated from WJ-MSCs, therefore we aimed to characterize the extent of maintenance of the naive traits of these cells also in a highly specialized differentiated counterpart. WJ-MSCs were differentiated into hepatocyte-like cells (HLCs) with a four weeks protocol. RT-PCR, flow cytometry, IHC and ICC were performed to assess expression of key markers in both undifferentiated and differentiated cells. Hepatocyte specific assays such as PAS staining, CYP3A4 induction and activity, G6Pase activity, were performed to demonstrate the acquisition of traits of the mature hepatocyte phenotype. WJ-MSCs were successfully differentiated into HLCs using a multi-step protocol. These cells were able to store glycogen, incorporate specific live cells stains, perform enzymatic reactions and metabolic activities which are usually featured by mature hepatocytes. In addition, WJ-MSCs did express several immune-related molecules, for which an immunomodulatory role has been demonstrated both in vitro and in vivo. We demonstrated for the first time that key molecules as HLA-E and B7-H3 are expressed also by HLCs derived from WJ-MSCs. Our data showed for the first time that HLCs mostly maintain the expression of immune-modulating molecules, together with new markers and functional features of mature cells. In multiple pathologic conditions the intrinsic immunomodulatory ability of differentiated cells may help survive the interaction with the host immune system, even in the absence of a specific immunosuppressive therapy. This, together with the acquisition of key mature hepatocyte functions, may render WJ-MSCs promising in cell therapy applications for liver diseases.

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.

Cigarette smoke is a potent stressor for the respiratory system, contributing to pathogenesis, for instance in chronic obstructive pulmonary disease (COPD), but its effects on the expression, function, and cellular localization of mitochondrial chaperonins are still largely unknown. We studied in vivo (airways biopsies) the localization of Hsp10 and Hsp60 in patients (smokers and non-smokers) affected by mild-moderate COPD, and characterized the effects of non-lethal doses of cigarette smoke extract (CSE) on the expression of these molecules in two human cell lines: lung fibroblasts (HFL-1) and bronchial epithelial (16HBE). We applied various in vitro methods: immunohistochemistry (IHC), subcellular fractionation analyses (SFA), Western blotting (WB), immunocytochemistry (ICC), and transmission electron microscopy (TEM) immunogold, and used bioinformatics and databases searches to gather structural in silico data for interpreting and complementing the in vitro results. IHC showed that in smokers and non-smokers COPD patients Hsp10 was localized in both, the cytoplasm and the nucleus of epithelial and lamina propria cells, while Hsp60 was present only in the cytosol. ICC, SFA, and WB on both CSE-exposed cell lines confirmed the presence of nuclear Hsp10, with an increasing trend in parallel to CSE concentration. TEM immunogold further confirmed Hsp10 in the nucleus, in addition to its presence in the cytoplasm and mitochondria, on both cell lines. Bioinformatics and in silico structural analyses indicated that Hsp10 can localize in extramitochondrial sites, such as the nucleus, even if Hsp10 lacks known DNA-binding motifs or nuclear import signals in its primary sequence. Our data suggest a link between exposure to exogenous oxidative stress and cell response, involving Hsp10, which would play roles different from its canonical functions. It is known that Hsp10 can display an array of functions depending on its location: cytoplasm, mitochondria, or extracellular. Here, we show for the first time the presence of Hsp10 in the nucleus of epithelial and stromal human-lung cell lines, paralleling the observations in vivo in COPD patients, and indicating that intranuclear Hsp10 levels are affected by oxidative stress due to an exogenous stressor like cigarette-smoke. The questions now are by what mechanism Hsp10 becomes a resident of the nucleus and what are its functions there.

Mesenchymal stem cells derived from Wharton’s jelly (WJ-MSCs) recently emerged as promising tools for cellular therapy due to their ability to differentiate into diverse cell types and their immunomodulatory features. Little is known on the expression of immunomodulatory molecules in mature cells differentiated from WJ-MSCs, therefore we aimed to characterize the extent of maintenance of the naive traits of these cells also in a highly specialized differentiated counterpart. WJ-MSCs were differentiated into hepatocyte-like cells (HLCs) with a four weeks protocol. RT-PCR, flow cytometry, IHC and ICC were performed to assess expression of key markers in both undifferentiated and differentiated cells. Hepatocyte specific assays such as PAS staining, CYP3A4 induction and activity, G6Pase activity, were performed to demonstrate the acquisition of traits of the mature hepatocyte phenotype. WJ-MSCs were successfully differentiated into HLCs using a multi-step protocol. These cells were able to store glycogen, incorporate specific live cells stains, perform enzymatic reactions and metabolic activities which are usually featured by mature hepatocytes. In addition, WJ-MSCs did express several immune-related molecules, for which an immunomodulatory role has been demonstrated both in vitro and in vivo. We demonstrated for the first time that key molecules as HLA-E and B7-H3 are expressed also by HLCs derived from WJ-MSCs. Our data showed for the first time that HLCs mostly maintain the expression of immune-modulating molecules, together with new markers and functional features of mature cells. In multiple pathologic conditions the intrinsic immunomodulatory ability of differentiated cells may help survive the interaction with the host immune system, even in the absence of a specific immunosuppressive therapy. This, together with the acquisition of key mature hepatocyte functions, may render WJ-MSCs promising in cell therapy applications for liver diseases.