Novel Ultrastructural Insights into the Clear-Cell Carcinoma of the Pancreas: A Case Report.

Pancreatic cancer, most frequently as ductal adenocarcinoma (PDAC), is the third leading cause of cancer death. Clear-cell primary adenocarcinoma of the pancreas (CCCP) is a rare, aggressive, still poorly characterized subtype of PDAC. We report here a case of a 65-year-old male presenting with pancreatic neoplasia. A histochemical examination of the tumor showed large cells with clear and abundant intracytoplasmic vacuoles. The clear-cell foamy appearance was not related to the hyperproduction of mucins. Ultrastructural characterization with transmission electron microscopy revealed the massive presence of mitochondria in the clear-cell cytoplasm. The mitochondria showed disordered cristae and various degrees of loss of structural integrity. Immunohistochemistry staining for NADH dehydrogenase [ubiquinone] 1 alpha subcomplex, 4-like 2 (NDUFA4L2) proved specifically negative for the clear-cell tumor. Our ultrastructural and molecular data indicate that the clear-cell nature in CCCP is linked to the accumulation of disrupted mitochondria. We propose that this may impact on the origin and progression of this PDAC subtype.

Changes Induced by P2X7 Receptor Stimulation of Human Glioblastoma Stem Cells in the Proteome of Extracellular Vesicles Isolated from Their Secretome.

Extracellular vesicles (EVs) are secreted from many tumors, including glioblastoma multiforme (GBM), the most common and lethal brain tumor in adults, which shows high resistance to current therapies and poor patient prognosis. Given the high relevance of the information provided by cancer cell secretome, we performed a proteomic analysis of microvesicles (MVs) and exosomes (EXOs) released from GBM-derived stem cells (GSCs). The latter, obtained from the brain of GBM patients, expressed P2X7 receptors (P2X7Rs), which positively correlate with GBM growth and invasiveness. P2X7R stimulation of GSCs caused significant changes in the EV content, mostly ex novo inducing or upregulating the expression of proteins related to cytoskeleton reorganization, cell motility/spreading, energy supply, protection against oxidative stress, chromatin remodeling, and transcriptional regulation. Most of the induced/upregulated proteins have already been identified as GBM diagnostic/prognostic factors, while others have only been reported in peripheral tumors. Our findings indicate that P2X7R stimulation enhances the transport and, therefore, possible intercellular exchange of GBM aggressiveness-increasing proteins by GSC-derived EVs. Thus, P2X7Rs could be considered a new druggable target of human GBM, although these data need to be confirmed in larger experimental sets.

Recommendations from the COST action CA17116 (SPRINT) for the standardization of perinatal derivative preparation and in vitro testing.

Many preclinical studies have shown that birth-associated tissues, cells and their secreted factors, otherwise known as perinatal derivatives (PnD), possess various biological properties that make them suitable therapeutic candidates for the treatment of numerous pathological conditions. Nevertheless, in the field of PnD research, there is a lack of critical evaluation of the PnD standardization process: from preparation to in vitro testing, an issue that may ultimately delay clinical translation. In this paper, we present the PnD e-questionnaire developed to assess the current state of the art of methods used in the published literature for the procurement, isolation, culturing preservation and characterization of PnD in vitro. Furthermore, we also propose a consensus for the scientific community on the minimal criteria that should be reported to facilitate standardization, reproducibility and transparency of data in PnD research. Lastly, based on the data from the PnD e-questionnaire, we recommend to provide adequate information on the characterization of the PnD. The PnD e-questionnaire is now freely available to the scientific community in order to guide researchers on the minimal criteria that should be clearly reported in their manuscripts. This review is a collaborative effort from the COST SPRINT action (CA17116), which aims to guide future research to facilitate the translation of basic research findings on PnD into clinical practice.

The Dual Role of Necroptosis in Pancreatic Ductal Adenocarcinoma.

Pancreatic cancer (PC) is the seventh leading cause of cancer-related death. PC incidence has continued to increase by about 1% each year in both men and women. Although the 5-year relative survival rate of PC has increased from 3% to 12%, it is still the lowest among cancers. Hence, novel therapeutic strategies are urgently needed. Challenges in PC-targeted therapeutic strategies stem from the high PC heterogeneity and from the poorly understood interplay between cancer cells and the surrounding microenvironment. Signaling pathways that drive PC cell growth have been the subject of intense scrutiny and interest has been attracted by necroptosis, a distinct type of programmed cell death. In this review, we provide a historical background on necroptosis and a detailed analysis of the ongoing debate on the role of necroptosis in PC malignant progression.

A non-mutated TROP2 fingerprint in cancer genetics.

The advent of high throughput DNA sequencing is providing massive amounts of tumor-associated mutation data. Implicit in these analyses is the assumption that, by acquiring a series of hallmark changes, normal cells evolve along a neoplastic path. However, the lack of correlation between cancer risk and global exposure to mutagenic factors provides arguments against this model. This suggested that additional, non-mutagenic factors are at work in cancer development. A candidate determinant is TROP2, that stands out for its expression in the majority of solid tumors in human, for its impact on the prognosis of most solid cancers and for its role as driver of cancer growth and metastatic diffusion, through overexpression as a wild-type form. The Trop-2 signaling network encompasses CREB1, Jun, NF-κB, Rb, STAT1 and STAT3, through induction of cyclin D1 and MAPK/ERK. Notably, Trop-2-driven pathways vastly overlap with those activated by most functionally relevant/most frequently mutated RAS and TP53, and are co-expressed in a large fraction of individual tumor cases, suggesting functional overlap. Mutated Ras was shown to synergize with the TROP2-CYCLIND1 mRNA chimera in transforming primary cells into tumorigenic ones. Genomic loss of TROP2 was found to promote carcinogenesis in squamous cell carcinomas through modulation of Src and mutated Ras pathways. DNA methylation and TP53 status were shown to cause genome instability and TROP gene amplification, together with Trop-2 protein overexpression. These findings suggest that mutagenic and the TROP2 non-mutagenic pathways deeply intertwine in driving transformed cell growth and malignant progression of solid cancers.

The 2EF Antibody Targets a Unique N-Terminal Epitope of Trop-2 and Enhances the In Vivo Activity of the Cancer-Selective 2G10 Antibody.

Trop-2 proteolytic processing in cancer cells exposes epitopes that were specifically targeted by the 2G10 antibody. We sought additional recognition of Trop-2 within difficult-to-reach, densely packed tumor sites. Trop-2 deletion mutants were employed in immunization and screening procedures, and these led to the recognition of a novel epitope in the N-terminal region of Trop-2, by the 2EF antibody. The 2EF mAb was shown to bind Trop-2 at cell-cell junctions in MCF-7 breast cancer cells, and in deeply seated sites in prostate cancer, that were inaccessible to benchmark anti-Trop-2 antibodies. The 2EF antibody was shown to inhibit the growth of HT29 colon tumor cells in vitro, with the highest activity at high cell density. In vivo, 2EF showed anticancer activity against SKOv3 ovarian, Colo205, HT29, HCT116 colon and DU-145 prostate tumors, with the highest impact on densely packed tumor sites, whereby 2EF outcompeted benchmark anti-Trop-2 antibodies. Given the different recognition modes of Trop-2 by 2EF and 2G10, we hypothesized the effective interaction of the two mAb in vivo. The 2EF mAb was indeed demonstrated to enhance the activity of 2G10 against tumor xenotransplants, opening novel avenues for Trop-2-targeted therapy. We humanized 2EF by state-of-the-art CDR grafting/re-modeling, yielding the Hu2EF for therapy of Trop-2-expressing tumors in patients.

Mapping of the Human Amniotic Membrane: In Situ Detection of Microvesicles Secreted by Amniotic Epithelial Cells.

The potential clinical applications of human amniotic membrane (hAM) and human amniotic epithelial cells (hAECs) in the field of regenerative medicine have been known in literature since long. However, it has yet to be elucidated whether hAM contains different anatomical regions with different plasticity and differentiation potential. Recently, for the first time, we highlighted many differences in terms of morphology, marker expression, and differentiation capabilities among four distinct anatomical regions of hAM, demonstrating peculiar functional features in hAEC populations. The aim of this study was to investigate in situ the ultrastructure of the four different regions of hAM by means of transmission electron microscopy (TEM) to deeply understand their peculiar characteristics and to investigate the presence and localization of secretory products because to our knowledge, there are no similar studies in the literature. The results of this study confirm our previous observations of hAM heterogeneity and highlight for the first time that hAM can produce extracellular vesicles (EVs) in a heterogeneous manner. These findings should be considered to increase efficiency of hAM applications within a therapeutic context.

Concern about the Effectiveness of mRNA Vaccination Technology and Its Long-Term Safety: Potential Interference on miRNA Machinery.

After the outbreak of the pandemic due to COVID-19 infection, several vaccines were developed on short timelines to counteract the public health crisis. To allow the administration of mRNA vaccines through a faster-paced approval process, the Emergency Use Authorization (EUA) was applied. The Ba.5 (omicron) variant of SARS-CoV-2 is the predominant one at this moment. Its highly mutable single-stranded RNA genome, along with its high transmissivity, generated concern about the effectiveness of vaccination. The interaction between the vaccine and the host cell is finely regulated by miRNA machinery, a complex network that oversees a wide range of biological processes. The dysregulation of miRNA machinery has been associated with the development of clinical complications during COVID-19 infection and, moreover, to several human pathologies, among which is cancer disease. Now that in some areas, four doses of mRNA vaccine have been administered, it is natural to wonder about its effectiveness and long-term safety.

Phylogenetic conservation of Trop-2 across species-rodent and primate genomics model anti-Trop-2 therapy for pre-clinical benchmarks.

A phylogenetic conservation analysis of Trop-2 across vertebrate species showed a high degree of sequence conservation, permitting to explore multiple models as pre-clinical benchmarks. Sequence divergence and incomplete conservation of expression patterns were observed in mouse and rat. Primate Trop-2 sequences were found to be 95%-100% identical to the human sequence. Comparative three-dimension primate Trop-2 structures were obtained with AlphaFold and homology modeling. This revealed high structure conservation of Trop-2 (0.66 ProMod3 GMQE, 0.80-0.86 ± 0.05 QMEANDisCo scores), with conservative amino acid changes at variant sites. Primate TACSTD2/TROP2 cDNAs were cloned and transfectants for individual ORF were shown to be efficiently recognized by humanized anti-Trop-2 monoclonal antibodies (Hu2G10, Hu2EF). Immunohistochemistry analysis of Macaca mulatta (rhesus monkey) tissues showed Trop-2 expression patterns that closely followed those in human tissues. This led us to test Trop-2 targeting in vivo in Macaca fascicularis (cynomolgus monkey). Intravenously injected Hu2G10 and Hu2EF were well tolerated from 5 to 10 mg/kg. Neither neurological, respiratory, digestive, urinary symptoms, nor biochemical or hematological toxicities were detected during 28-day observation. Blood serum pharmacokinetic (PK) studies were conducted utilizing anti-idiotypic antibodies in capture-ELISA assays. Hu2G10 (t1/2 = 6.5 days) and Hu2EF (t1/2 = 5.5 days) were stable in plasma, and were detectable in the circulation up to 3 weeks after the infusion. These findings validate primates as reliable models for Hu2G10 and Hu2EF toxicity and PK, and support the use of these antibodies as next-generation anti-Trop-2 immunotherapy tools.

General consensus on multimodal functions and validation analysis of perinatal derivatives for regenerative medicine applications.

Perinatal tissues, such as placenta and umbilical cord contain a variety of somatic stem cell types, spanning from the largely used hematopoietic stem and progenitor cells to the most recently described broadly multipotent epithelial and stromal cells. As perinatal derivatives (PnD), several of these cell types and related products provide an interesting regenerative potential for a variety of diseases. Within COST SPRINT Action, we continue our review series, revising and summarizing the modalities of action and proposed medical approaches using PnD products: cells, secretome, extracellular vesicles, and decellularized tissues. Focusing on the brain, bone, skeletal muscle, heart, intestinal, liver, and lung pathologies, we discuss the importance of potency testing in validating PnD therapeutics, and critically evaluate the concept of PnD application in the field of tissue regeneration. Hereby we aim to shed light on the actual therapeutic properties of PnD, with an open eye for future clinical application. This review is part of a quadrinomial series on functional/potency assays for validation of PnD, spanning biological functions, such as immunomodulation, anti-microbial/anti-cancer, anti-inflammation, wound healing, angiogenesis, and regeneration.

Tips and Tricks and Clinical Outcome of Cryopreserved Human Amniotic Membrane Application for the Management of Medication-Related Osteonecrosis of the Jaw (MRONJ): A Pilot Study.

Medication-related osteonecrosis of the jaw (MRONJ) is a complication of certain pharmacological treatments such as bisphosphonates, denosumab, and angiogenesis inhibitors. There are currently no guidelines on its management, particularly in advanced stages. The human amniotic membrane (hAM) has low immunogenicity and exerts anti-inflammatory, antifibrotic, antimicrobial, antiviral, and analgesic effects. It is a source of stem cells and growth factors promoting tissue regeneration. hAM acts as an anatomical barrier with suitable mechanical properties (permeability, stability, elasticity, flexibility, and resorbability) to prevent the proliferation of fibrous tissue and promote early neovascularization at the surgical site. In oral surgery, hAM stimulates healing and facilitates the proliferation and differentiation of epithelial cells in the oral mucosa and therefore its regeneration. We proposed using cryopreserved hAM to eight patients suffering from cancer (11 lesions) with stage 2-3 MRONJ on a compassionate use basis. A collagen sponge was added in some cases to facilitate hAM grafting. One or three hAMs were applied and one patient had a reapplication. Three patients had complete closure of the surgical site with proper epithelialization at 2 weeks, and two of them maintained it until the last follow-up. At 1 week after surgery, three patients had partial wound dehiscence with partial healing 3 months later and two patients had complete wound dehiscence. hAM reapplication led to complete healing. All patients remained asymptomatic with excellent immediate significant pain relief, no infections, and a truly positive impact on the patients' quality of life. No adverse events occurred. At 6 months of follow-up, 80% of lesions had complete or partial wound healing (30 and 50%, respectively), while 62.5% of patients were in stage 3. Radiological evaluations found that 85.7% of patients had stable bone lesions (n = 5) or new bone formation (n = 1). One patient had a worsening MRONJ but remained asymptomatic. One patient did not attend his follow-up radiological examination. For the first time, this prospective pilot study extensively illustrates both the handling and surgical application of hAM in MRONJ, its possible association with a collagen sponge scaffold, its outcome at the site, the application of multiple hAM patches at the same time, and its reapplication.

Development and Characterization of Curcumin-Silver Nanoparticles as a Promising Formulation to Test on Human Pterygium-Derived Keratinocytes.

Pterygium is a progressive disease of the human eye arising from sub-conjunctival tissue and extending onto the cornea. Due to its invasive growth, pterygium can reach the pupil compromising visual function. Currently available medical treatments have limited success in suppressing efficiently the disease. Previous studies have demonstrated that curcumin, polyphenol isolated from the rhizome of Curcuma longa, induces apoptosis of human pterygium fibroblasts in a dose- and time-dependent manner showing promising activity in the treatment of this ophthalmic disease. However, this molecule is not very soluble in water in either neutral or acidic pH and is only slightly more soluble in alkaline conditions, while its dissolving in organic solvents drastically reduces its potential use for biomedical applications. A nanoformulation of curcumin stabilized silver nanoparticles (Cur-AgNPs) seems an effective strategy to increase the bioavailability of curcumin without inducing toxic effects. In fact, silver nitrates have been used safely for the treatment of many ophthalmic conditions and diseases for a long time and the concentration of AgNPs in this formulation is quite low. The synthesis of this new compound was achieved through a modified Bettini's method adapted to improve the quality of the product intended for human use. Indeed, the pH of the reaction was changed to 9, the temperature of the reaction was increased from 90 °C to 100 °C and after the synthesis the Cur-AgNPs were dispersed in Borax buffer using a dialysis step to improve the biocompatibility of the formulation. This new compound will be able to deliver both components (curcumin and silver) at the same time to the affected tissue, representing an alternative and a more sophisticated strategy for the treatment of human pterygium. Further in vitro and in vivo assays will be required to validate this formulation.

The Role of the CREB Protein Family Members and the Related Transcription Factors in Radioresistance Mechanisms.

In the framework of space flight, the risk of radiation carcinogenesis is considered a "red" risk due to the high likelihood of occurrence as well as the high potential impact on the quality of life in terms of disease-free survival after space missions. The cyclic AMP response element-binding protein (CREB) is overexpressed both in haematological malignancies and solid tumours and its expression and function are modulated following irradiation. The CREB protein is a transcription factor and member of the CREB/activating transcription factor (ATF) family. As such, it has an essential role in a wide range of cell processes, including cell survival, proliferation, and differentiation. Among the CREB-related nuclear transcription factors, NF-κB and p53 have a relevant role in cell response to ionising radiation. Their expression and function can decide the fate of the cell by choosing between death or survival. The aim of this review was to define the role of the CREB/ATF family members and the related transcription factors in the response to ionising radiation of human haematological malignancies and solid tumours.

Curcuma longa Hepatotoxicity: A Baseless Accusation. Cases Assessed for Causality Using RUCAM Method.

Curcuma longa is a perennial herb that belongs to the Zingiberaceae family. To date, literature includes more than 11.000 scientific articles describing all its beneficial properties. In the last 3 decades various surveys by the U.S. Food and Drug Administration (FDA) concluded that curcumin, the most active ingredient of the drug, is a "generally safe" compound with strong anti-oxidant effects. Curcuma longa was introduced in the daily diet by ayurvedic teachers due to its beneficial effects on health. Nonetheless, recently several reports, from the various global surveillance systems on the safety of plant products, pointed out cases of hepatotoxicity linked to consumption of food supplements containing powdered extract and preparations of Curcuma longa. The latest trend is the use of Curcuma longa as a weight-loss product in combination with piperine, which is used to increase its very low systemic bioavailability. Indeed, only 20 mg piperine, one of the alkaloids found in black pepper (Piper nigrum), assumed at the same time with 2 g curcumin increased 20-fold serum curcumin bioavailability. This combination of natural products is now present in several weight loss supplements containing Curcuma longa. The enhanced drug bioavailability caused by piperine is due to its potent inhibition of drug metabolism, being able to inhibit human P-glycoprotein and CYP3A4, while it interferes with UDP-glucose dehydrogenase and glucuronidation activities in liver. While only few cases of hepatotoxicity, assessed using Roussel Uclaf Causality Assessment Method (RUCAM) method, from prolonged intake of piperine and curcumin have been reported, it would be reasonable to speculate that the suspected toxicity of Curcuma longa could be due to the concomitant presence of piperine itself. Hence, not only there is the need of more basic research to understand the etiopathology of curcumin-related hepatotoxicity and of the combination curcumin-piperine, but human trials will be necessary to settle this dispute.

Use of Amniotic Membrane and Its Derived Products for Bone Regeneration: A Systematic Review.

Thanks to their biological properties, amniotic membrane (AM), and its derivatives are considered as an attractive reservoir of stem cells and biological scaffolds for bone regenerative medicine. The objective of this systematic review was to assess the benefit of using AM and amniotic membrane-derived products for bone regeneration. An electronic search of the MEDLINE-Pubmed database and the Scopus database was carried out and the selection of articles was performed following PRISMA guidelines. This systematic review included 42 articles taking into consideration the studies in which AM, amniotic-derived epithelial cells (AECs), and amniotic mesenchymal stromal cells (AMSCs) show promising results for bone regeneration in animal models. Moreover, this review also presents some commercialized products derived from AM and discusses their application modalities. Finally, AM therapeutic benefit is highlighted in the reported clinical studies. This study is the first one to systematically review the therapeutic benefits of AM and amniotic membrane-derived products for bone defect healing. The AM is a promising alternative to the commercially available membranes used for guided bone regeneration. Additionally, AECs and AMSCs associated with an appropriate scaffold may also be ideal candidates for tissue engineering strategies applied to bone healing. Here, we summarized these findings and highlighted the relevance of these different products for bone regeneration.

A Clinical and Histological Study about the Socket Preservation in a Patient under Oral Bisphosphonates Treatment: A Case Report.

The aim of the present study is to assess the clinical and histological healing of a post-extractive alveolus following the procedure for socket preservation, in a patient receiving oral bisphosphonates for more than 6 years. After the extraction, enzymatically-deantigenated horse bone granules and an equine pericardium membrane were used to preserve the tooth socket. The patient was placed on a monthly follow-up in order to monitor the healing process. A 3 mm trephine bur was used to drill the bone for implant site preparation and to collect the bone sample. No signs and symptoms related to osteonecrosis of the jaws were reported. Histological data showed that, after 5 months, the mean percentages of trabecular bone, bone marrow and residual bone graft were respectively 45.74 ± 0.09%, 48.09 ± 0.08%, and 6.16 ± 0.01%. The residual graft material appeared to be osteointegrated and none of the particles appeared to be encapsulated. The present case report supports the guidelines that assume that patients undergoing oral bisphosphonate therapy can be eligible for surgical therapy. More clinical studies with larger sample sizes are needed to support this clinical evidence.

A Retrospective Longitudinal Study in a Cohort of Children With Dyskinetic Cerebral Palsy Treated With Tetrabenazine.

Tetrabenazine has been studied with a variety of hyperkinetic movement disorders, but there is limited and empirical literature on the potential efficacy of tetrabenazine in children with dyskinetic cerebral palsy (DCP). The purpose of this study was to evaluate the efficacy of tetrabenazine in a sample of children with DCP using the Movement Disorders-Childhood Rating Scale 4-18 Revised (MD-CRS 4-18 R). The study is a multicenter retrospective longitudinal study in which the participants were selected from the databases of each Center involved, according to detailed inclusion criteria. The study was performed on 23 children and adolescents (19 male and 4 females; mean age 8.28 years, SD 3.59) with DCP having been evaluated before starting the treatment (baseline), after 6 and 12 months of treatment and in a sub-cohort after >2 years follow-up. A linear mixed model was used to evaluate the effects of the different timings on each MD-CRS 4-18 R Index (Index I, Index II, and Global Index) adding age and type of movement disorder as random effect. A significant clinical improvement related to a reduction of MD-CRS 4-18 R Indexes was detected between the baseline and after 6 and 12 months of treatment. Findings support the efficacy of tetrabenazine in children with DCP through a standardized outcome measure (MD-CRS 4-18 R) and confirm the use of this scale as a suitable tool to detect changes in further randomized clinical trials.

Proteomic Characterization of Two Extracellular Vesicle Subtypes Isolated from Human Glioblastoma Stem Cell Secretome by Sequential Centrifugal Ultrafiltration.

Extracellular vesicles (EVs) released from tumor cells are actively investigated, since molecules therein contained and likely transferred to neighboring cells, supplying them with oncogenic information/functions, may represent cancer biomarkers and/or druggable targets. Here, we characterized by a proteomic point of view two EV subtypes isolated by sequential centrifugal ultrafiltration technique from culture medium of glioblastoma (GBM)-derived stem-like cells (GSCs) obtained from surgical specimens of human GBM, the most aggressive and lethal primary brain tumor. Electron microscopy and western blot analysis distinguished them into microvesicles (MVs) and exosomes (Exos). Two-dimensional electrophoresis followed by MALDI TOF analysis allowed us to identify, besides a common pool, sets of proteins specific for each EV subtypes with peculiar differences in their molecular/biological functions. Such a diversity was confirmed by identification of some top proteins selected in MVs and Exos. They were mainly chaperone or metabolic enzymes in MVs, whereas, in Exos, molecules are involved in cell-matrix adhesion, cell migration/aggressiveness, and chemotherapy resistance. These proteins, identified by EVs from primary GSCs and not GBM cell lines, could be regarded as new possible prognostic markers/druggable targets of the human tumor, although data need to be confirmed in EVs isolated from a greater GSC number.

Cancer-Homing CAR-T Cells and Endogenous Immune Population Dynamics.

Chimeric antigen receptor (CAR) therapy is based on patient blood-derived T cells and natural killer cells, which are engineered in vitro to recognize a target antigen in cancer cells. Most CAR-T recognize target antigens through immunoglobulin antigen-binding regions. Hence, CAR-T cells do not require the major histocompatibility complex presentation of a target peptide. CAR-T therapy has been tremendously successful in the treatment of leukemias. On the other hand, the clinical efficacy of CAR-T cells is rarely detected against solid tumors. CAR-T-cell therapy of cancer faces many hurdles, starting from the administration of engineered cells, wherein CAR-T cells must encounter the correct chemotactic signals to traffic to the tumor in sufficient numbers. Additional obstacles arise from the hostile environment that cancers provide to CAR-T cells. Intense efforts have gone into tackling these pitfalls. However, we argue that some CAR-engineering strategies may risk missing the bigger picture, i.e., that a successful CAR-T-cell therapy must efficiently intertwine with the complex and heterogeneous responses that the body has already mounted against the tumor. Recent findings lend support to this model.

Human amniotic fluid stem cells are able to form embryoid body-like aggregates which performs specific functions: morphological evidences.

Human second trimester Amniotic Fluid Stem Cells (hAFSCs) harbour the potential to differentiate into cells of each of the three germ layers and to form Embryoid Body (EB)-like aggregates, without inducing teratoma formation and with no ethical concerns. However, in spite of the number of reports on hAFSCs-EBs and their characterization, a thorough evaluation in light and electron microscopy of morphological and morphometric features of hAFSCs-EBs development in vitro has not been reported yet. Apart from a superficial layer of epithelial-like flat cells, displaying rare microvilli on the free surface, hAFSCs-EBs enclose inner material, abundant in vesicles and secretory granules, showing early characteristics of connective extracellular matrix dispersed among different types of inner cells. The observation of a number of microvesicles mainly represented by microparticles and, to a lower extent, by exosomes indicates the presence of a complex cellular communication system within this structure. According to morphological analysis, after 7 days of in vitro culture hAFSCs-EB appears as a well-organized corpuscle, sufficiently young to be a carrier of stemness and at the same time, when appropriately stimulated, able to differentiate. In fact, 7-day hAFSCs-EB represents itself an initial cellular transformation towards a specialized structure both in recording and in providing different stimuli from the surrounding environment, organizing structures and cells towards a differentiation fate.