Fetal Stem Research Articles

#73 Single Cell RNA Sequencing of Zika Infection in Neural Tissues Reveals an Astrocyte-Driven Innate Immune Response Governed by Interferon Beta.

Abstract Background Congenital infection with Zika virus (ZIKV) has been linked to severe of fetal brain disruption and other developmental anomalies referred to collectively as congenital Zika syndrome (CZS). Long-term follow up of ZIKV-exposed infants has shown that even those without overt findings of CZS can have significant cognitive and developmental difficulties. However, the pathogenesis of ZIKV-related microcephaly and cognitive dysfunction remain poorly understood. To begin answering these questions, we sought to define the consequences of ZIKV infection on specific cell types in the developing brain. Method We have used primary human fetal brain cultures and human induced pluripotent stem cell-derived neural progenitor cell lines to probe innate immune signaling to ZIVK infection in vitro. We used single-cell RNA sequencing (10x Genomics) to identify the cell type-specific transcriptional changes induced by ZIKV infection. Results We found that neural progenitor cells (NPCs) support higher ZIKV RNA copy number than do differentiated neural cells. Moreover, while IFN-β is the predominant cytokine released by differentiated cells in response to ZIKV infection, NPCs do not produce IFN-β. By additionally characterizing the transcriptional response to IFN-β treatment, we have been able to distinguish virus-stimulated genes (VSGs) from interferon-stimulated genes (ISGs) across cell types. We find extensive heterogeneity in cell type-specific VSG and ISG profiles. Astrocytes undergo the largest transcriptional changes in response to infection and are the main producers of IFN-β. Our findings also identify genes whose expression is altered during viral infection specifically in NPCs and neurons, providing candidate pathways that may underlie neural dysfunction. Conclusion Across cell types, the response to Zika infection closely matches the response to IFN-β, arguing that astrocyte-derived IFN-β is the main driver of the innate immune response to ZIKV in the developing brain. Our findings provide insight into the pathogenesis of CZS and offer novel targets for improving developmental outcomes after fetal ZIKV infection.

MG-63 and FetMSC Cell Response on Atomic Layer Deposited TiO2 Nanolayers Prepared Using Titanium Tetrachloride and Tetraisopropoxide

Titanium oxide nanocoatings were synthesized on the surface of monocrystalline silicon and ultra-fine-grained titanium by atomic layer deposition (ALD) using titanium tetrachloride (TiCl4) and titanium tetraisopropoxide (TTIP). The morphology of the samples was studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The structure and composition were studied by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), contact angle measurements, and energy-dispersive spectroscopy (EDS). The cytological response of osteosarcoma MG-63 and human fetal mesenchymal stem cells (FetMSCs) were studied by analyzing their morphology, viability, and alkaline phosphatase activity with and without the use of medium-induced differentiation in the osteogenic direction. A significant influence of the precursor type and ALD temperature on the crystal structure, morphology, composition, and surface free energy of TiO2 nanocoatings was found. The biocompatibility of amorphous non-stoichiometric and partially crystalline stoichiometric TiO2 coatings was compared. Both types of cells showed faster adhesion and improved spreading on the surface for the samples from TTIP compared to those from TiCl4 at the early stages of cultivation (2 h) due to the difference in composition and higher surface free energy. No cytotoxic effect was found on both types of coatings, nor was there a noticeable difference in cell differentiation. All ALD coatings provided excellent biocompatibility and osteoconductive properties.

Cloning Efficiency of Porcine Somatic Cell Nuclear Transfer Compared with Fetal Fibroblasts and Mesenchymal Stem Cells

Cloning Efficiency of Porcine Somatic Cell Nuclear Transfer Compared with Fetal Fibroblasts and Mesenchymal Stem Cells

The Utilization of Human Placental, Fetal Mesenchymal Stem Cell Derived- Exosomes in Treating Keloid Scars

The Utilization of Human Placental, Fetal Mesenchymal Stem Cell Derived- Exosomes in Treating Keloid Scars

Multi-modal profiling of human fetal liver hematopoietic stem cells reveals the molecular signature of engraftment

The human hematopoietic stem cell harbors remarkable regenerative potential that can be harnessed therapeutically. During early development, hematopoietic stem cells in the fetal liver undergo active expansion while simultaneously retaining robust engraftment capacity, yet the underlying molecular program responsible for their efficient engraftment remains unclear. Here, we profile 26,407 fetal liver cells at both the transcriptional and protein level including ~7,000 highly enriched and functional fetal liver hematopoietic stem cells to establish a detailed molecular signature of engraftment potential. Integration of transcript and linked cell surface marker expression reveals a generalizable signature defining functional fetal liver hematopoietic stem cells and allows for the stratification of enrichment strategies with high translational potential. More precisely, our integrated analysis identifies CD201 (endothelial protein C receptor (EPCR), encoded by PROCR) as a marker that can specifically enrich for engraftment potential. This comprehensive, multi-modal profiling of engraftment capacity connects a critical biological function at a key developmental timepoint with its underlying molecular drivers. As such, it serves as a useful resource for the field and forms the basis for further biological exploration of strategies to retain the engraftment potential of hematopoietic stem cells ex vivo or induce this potential during in vitro hematopoietic stem cell generation.

Protective Effect of Conditioned Media of Human Fetal Dermal Mesenchymal Stem Cells Can Inhibit Burn-induced Microvascular Hyperpermeability.

Burns often cause loss of skin barrier protection, fluid exudation, and local tissue edema, which hinder functional recovery. Effectively improving the quality of deep burn wound healing, shortening the wound healing time, and reducing tissue fluid leakage are urgent problems in the medical field. Human mesenchymal stem cells (MSCs) can effectively stabilize vascular endothelial injury. Fetal dermal MSCs (FDMSCs) are a newly discovered source of MSCs derived from the skin of accidentally aborted fetuses. However, the effect of FDMSCs on vascular permeability remains poorly understood. In this study, conditioned media from FDMSCs (F-CM) extracted from fetal skin tissue was prepared. The effect of F-CM on vascular permeability was evaluated using the internal circulation method FITC-dextran in vivo, and several in vitro assays, including cell viability assay, transwell permeability test, immunofluorescence, and western blotting. Altogether, our results demonstrate that F-CM could inhibit burn-induced microvascular hyperpermeability by increasing the protein expression levels of occludin and VE-cadherin, while restoring the expression of endothelial F-actin, and providing the foundation of a novel therapy for the treatment of burns with F-CM.

Taspase1 orchestrates fetal liver hematopoietic stem cell and vertebrae fates by cleaving TFIIA.

Taspase1, a highly conserved threonine protease encoded by TASP1, cleaves nuclear histone-modifying factors and basal transcription regulators to orchestrate diverse transcription programs. Hereditary loss-of-function mutation of TASP1 has recently been reported in humans as resulting in an anomaly complex syndrome, which manifests with hematological, facial, and skeletal abnormalities. Here, we demonstrate that Taspase1-mediated cleavage of TFIIAα-β, rather than of MLL1 or MLL2, in mouse embryos was required for proper fetal liver hematopoiesis and correct segmental identities of the axial skeleton. Homozygous genetic deletion of Taspase1 disrupted embryonic hematopoietic stem cell self-renewal and quiescence states and axial skeleton fates. Strikingly, mice carrying knockin noncleavable mutations of TFIIAα-β, a well-characterized basal transcription factor, displayed more pronounced fetal liver and axial skeleton defects than those with noncleavable MLL1 and MLL2, 2 trithorax group histone H3 trimethyl transferases. Our study offers molecular insights into a syndrome in humans that results from loss of TASP1 and describes an unexpected role of TFIIAα-β cleavage in embryonic cell fate decisions.

Sirt7-p21 Signaling Pathway Mediates Glucocorticoid-Induced Inhibition of Mouse Neural Stem Cell Proliferation.

Prenatal glucocorticoid (GC) overexposure impacts fetal hippocampal neural stem cells (NSCs) and increases the risk for relative cognitive and mood disorders in offspring. However, the precise underlying mechanisms remain elusive. Here, we treated mouse hippocampal NSCs with dexamethasone (DEX) in vitro and found that DEX inhibited cell proliferation and Sirt7 expression. In addition, prenatal mouse overexposure to DEX induced the suppression of Sirt7 in the hippocampus of offspring. Sirt7 knockdown significantly decreased the percentage of proliferating cells but did not further reduce the NSC proliferation rate in the presence of DEX, whereas Sirt7 overexpression rescued DEX-induced inhibition of hippocampal NSC proliferation. Moreover, DEX inhibited Sirt7 expression through the glucocorticoid receptor (GR), and p21 was found to mediate the functional effect of DEX-induced Sirt7 suppression. In conclusion, our data demonstrate for the first time the effect of DEX on the Sirt7-p21 pathway in hippocampal NSCs, identifying a new potential therapeutic target for prenatal GC overexposure-related neurodevelopmental disorders in offspring.

2017 – EXPLOITING THE FOETAL ORIGIN OF MLL-AF4-DRIVEN ACUTE LYMPHOBLASTIC LEUKAEMIA

Infant MLL-AF4-driven Acute Lymphoblastic Leukaemia (ALL) is a devastating disease with dismal prognosis which has not improved with the advances of medicine. The lack of progress towards successfully treating these patients has been attributed to a lack of understanding of the unique biology of this disease, particularly its prenatal origin. To better understand the prenatal origin of the disease, we performed multiple RNA sequencing experiments on foetal, neonatal and adult haematopoietic stem and progenitor cells (HSPCs) from human and mouse. With this approach we identified a conserved foetal transcriptional signature that was characterised by a prominent proliferative and oncogenic nature, suggesting that the foetal origin of the disease could be critical for the initiation of this aggressive leukaemia. Interestingly, multiple aspects of the foetal transcriptome persisted into the blasts derived from infant patients. By performing multiple comparisons, we obtained a list of 20 conserved genes that were highly expressed in the healthy foetal tissues and in the infant blasts whereas there was little expression in neonatal and adult HSPCs. Pharmacological and genetic targeting of these genes had a dramatic effect on the survival of SEM cells, a cell line derived from an infant/paediatric patient with MLL-AF4-driven ALL. Amongst the more attractive targets were genes PLK1, which is involved in cell division, and ELOVL1 which is required for the elongation of very long fatty acid chains. PLK1 inhibition of SEM cells with the FDA approved inhibitor Volasertib resulted in a dramatic decrease in the proliferation potential of these cells which lead to apoptosis. Similarly, genetic targeting of ELOVL1 with CrispR-Cas9 showed a tremendous decrease in the survival of SEM cells. To identify the mechanisms of action of ELOVL1 we performed a lipidomics experiment where it was shown that there was a dramatic decrease in lipids that are involved in the maintenance of cell membranes. As expression of ELOVL1 was very low in healthy bone marrow haematopoietic cells, it seems that this requirement is unique to the infant disease and therefore a novel therapeutic avenue of treating the infant patients. In summary, in this study we describe a novel approach that allowed us to identify the Achilles heel of infant MLL-AF4-driven ALL which its foetal origin.

Abstract ES10-2: Understanding breast cancer using a developmental perspective

Abstract Parallels among embryonic development, stem cells, and cancer have long been recognized. We identified, isolated, and characterized stem cells that first become committed to a mammary fate during embryogenesis; we refer to these cells as fetal mammary stem cells (fMaSCs). Lineage tracing, in vitro sphere formation, and in vivo transplantation studies by our group and many others all confirm that cells in the embryo are the bipotent progenitors of the mammary gland. There is debate, however, on whether such bipotent cells persist into the adult, or whether the luminal and basal lineages are maintained by unipotent progenitors. To gain insight into the relationships between fMaSCs and breast cancer, and to investigate their potential persistence in the adult, we have applied bulk and single cell RNA-sequencing (sc-RNA-seq) and single nucleus ATAC-sequencing (snATAC-seq) throughout mammary development. The results to be discussed demonstrate that fMaSC transcriptomes are heterogeneous, but all share co-expression of genes associated with luminal and basal cell fates. This fits a model in which the bipotent state is created by a balance of lineage specifiers. We also find that the fMaSC transcriptome is highly enriched in basal-like human breast cancers and identify potential embryonic pathways that correlate with poor prognosis. We used a variety of computational tools to infer the gene expression programs that ensue when fMaSCs commit to luminal and basal states. The data from scRNA-seq and snATAC-seq demonstrate that the transitions are gradual, not precipitous, and that luminal and basal cells exhibit significant transcriptomic and epigenetic heterogeneity. This challenges the notion that the mammary gland consists of discrete cell types defined by rigid transcriptomic parameters, and reveals a potential for intrinsic phenotypic plasticity of normal mammary cells. Using the combined databases, we identified Sox10 as a significantly differentially expressed cell state regulator. We show that tumors are heterogeneous with regard to Sox10 expression, and that locally invasive cells tend to express high Sox10 levels. Elevated Sox10 correlates with acquisition of a neural-crest like, EMT-related state. Implications for interception of metastasis by targeting neural crest-like cells will be discussed. Finally, we have generated a web resource that is available to the scientific community to enable the transcription and epigenetic characteristics of any gene of interest to be tracked through mammary development (https://wahl-labsalk.shinyapps.io/Mammary_snATAC/). Citation Format: GM Wahl, Z Ma, C Chung, C Dravis, BT Spike, RR Giraddi, O Balcioglu, C Fan, B Hagos, R Heinz, Herrera-Valdez J, X Hou, J Hwang, R Lasken, G Luna, NE Lytle, EM Mehrabad, M Novotny, CM Perou, O Poirion, S Preissl, B Ren, T Reya, CL Trejo, KT Varley. Understanding breast cancer using a developmental perspective [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr ES10-2.

Comparative characterization of human fetal neural stem cells and induced neural stem cells from peripheral blood mononuclear cells

Comparative characterization of human fetal neural stem cells and induced neural stem cells from peripheral blood mononuclear cells

Human fetal skin-derived stem cell secretome enhances radiation-induced skin injury therapeutic effects by promoting angiogenesis

BackgroundRadiation dermatitis is a refractory skin injury caused by radiotherapy. Human fetal skin-derived stem cell (hFSSC) is a preferable source for cell therapy and skin tissue regeneration. In the present study, we investigated the repair effect of using hFSSC secretome on a radiation skin injury model in rats.MethodsWe prepared the hFSSC secretome and studied its effects on the proliferation and tube formation of human umbilical vein endothelial cell (HUVEC) in vitro. Furthermore, we used a Sr-90 radiation-induced skin injury model of rats and evaluated the effects of hFSSC secretome on radiation skin injury in vivo.ResultsThe results showed that hFSSC secretome significantly promoted the proliferation and tube formation of HUVEC in vitro; in addition, hFSSC secretome-treated rats exhibited higher healing quality and faster healing rate than the other two control groups; the expression level of collagen type III α 1 (Col3A1), transforming growth factor β3 (TGF-β3), angiotensin 1 (Ang-1), angiotensin 2 (Ang-2), vascular endothelial growth factor (VEGF), and placental growth factor (PLGF) was significantly increased, while collagen type I α 2 (Col1A2) and transforming growth factor β1 (TGF-β1) were decreased in hFSSC secretome group.ConclusionsIn conclusion, our results provided the first evidence on the effects of hFSSC secretome towards radiation-induced skin injury. We found that hFSSC secretome significantly enhanced radiation dermatitis angiogenesis, and the therapeutic effects could match with the characteristics of fetal skin. It may act as a kind of novel cell-free therapeutic approach for radiation-induced cutaneous wound healing.

Characterization of a Novel JAK1 Pseudokinase Mutation in the First Case of Trisomy 21-Independent GATA1-Mutated Transient Abnormal Myelopoiesis

Clonal proliferation of megakaryoblasts, called transient abnormal myelopoiesis (TAM), is a rare disease of newborns triggered by trisomy 21 (constitutional or somatic) together with acquired mutations of GATA1 resulting in the exclusive production of its short variant - GATA1s. No other TAM drivers have been described so far. We have diagnosed a unique TAM case with a typical clinical and laboratory manifestation but without the gain (or any other aberration) of chromosome 21. Thorough genomic profiling revealed 4 somatic mutations: GATA1 D65_C228del, JAK1 F636del, FN1 R2420C and SPIRE2 R471W. With respect to the generally accepted 2-hit theory, we hypothesized that this TAM arose from a collaboration of the atypical GATA1 mutation (not inducing GATA1s) with (at least) one of the other identified mutations. Unlike SPIRE2 and FN1 aberrations, various mutations of the JAK1 kinase have been previously described as leukemia drivers, suggesting JAK1 F636del as a top candidate for the second hit. Moreover, JAK1 mutations have been associated with the transformation of TAM into acute megakaryoblastic leukemia (Nikolaev et al., Blood, 2013). The aim of our project was to functionally characterize this novel JAK1 mutation. Phenylalanine 636 belongs to a phylogenetically conserved triad of amino acids suggested to control catalytic activity of JAK1 via mediating a switch between the supposedly active and inactive conformations (Toms et al., Nat Struct Mol Biol, 2013). Hence, F636 seems to be essential for JAK1 function. Surprisingly, homology modeling showed that loss of F636 is compatible with both functionally opposite conformations. Indeed, Western blot analysis of JAK/STAT signaling in transiently transformed HEK293T cells showed that catalytic activity is preserved in JAK1 F636del. However, we observed lower levels of auto- and STATs- phosphorylation compared to wild-type (wt) JAK1 suggesting decreased kinase activity of JAK1 F636del. Subsequently, we tested the oncogenic potential of JAK1 F636del in the Ba/F3 cell assay; unlike the known oncogenic JAK1 variant (JAK1 V658I), JAK1 F636del did not induce IL3-independent growth. To further assess phenotypic impact of F636del, we introduced JAK1 F636del into murine bone marrow and fetal liver hematopoietic stem and progenitor cells (HSPCs) using lentiviruses and performed colony forming assays. The number and morphology of colonies did not differ in JAK1 F636del compared to wt JAK1. Furthermore, we assessed the impact of JAK1 F636del in the context of mutated GATA1. We utilized the in-vitro model recently described by Labuhn et al. (Cancer Cell, 2019), in which the CRISPR/Cas9-mediated induction of Gata1s expression leads to the expansion and sustained proliferation of fetal liver HSPCs from embryonic day 13.5 ROSA26:Cas9-EGFPki/wt mice. Similar to wt JAK1, lentivirally introduced JAK1 F636del had no impact on the proliferation and maturation status of such Gata1-edited HPSCs irrespective of the timing of its introduction (simultaneously with Gata1 editing versus into fully established Gata1-edited culture) or of culturing conditions (fully cytokine-supplemented growth-supportive versus cytokine-depleted growth-restrictive medium). Altogether, we show that unlike known oncogenic variants, F636del identified in the first case of trisomy 21-independent TAM attenuates JAK1 kinase activity. The results of our phenotypic studies question the potential contribution of this mutation to TAM development. Interestingly, Labuhn et al. (2019) recently showed that non-activating JAK mutations occur at higher than random frequency in trisomy 21-dependent TAM. This tempts us to speculate that JAK1 mutations may still play a role in TAM. Yet, this role may significantly differ from that of known oncogenic mutations; it may result from attenuation/modulation instead of activation of downstream signaling and it may remain unrevealed utilizing the currently available sophisticated, yet still imperfect experimental models. Support: GAUK 86218, EHA Research Mobility Grant Disclosures No relevant conflicts of interest to declare.

Myeloid-Lineage Enhancers Drive Oncogene Synergy and Represent a Novel Therapeutic Target in CSF3R-CEBPA Mutant AML

Myeloid-Lineage Enhancers Drive Oncogene Synergy and Represent a Novel Therapeutic Target in CSF3R-CEBPA Mutant AML

Effects of eicosapentaenoic acid and docosahexaenoic acid on C2C12 cell adipogenesis and inhibition of myotube formation

ABSTRACTEicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) modulate cellular metabolic functions and gene expression. This study investigated the impacts of EPA and DHA on gene expression and morphological changes during adipogenic inducement in C2C12 myoblasts. Cells were cultured and treated with differentiation medium with and without 50 μM EPA and DHA. Cells treated with fatty acids had noticeable lipid droplets, but no formation of myotubes compared to control group cells. The expression levels of key genes relevant to adipogenesis and inflammation were significantly higher (P < 0.05) in cells treated with fatty acids. Genes associated with myogenesis and mitochondrial biosynthesis and function had lower (P < 0.05) expression with fatty acids supplementation. Moreover, fatty acid treatment reduced (P < 0.05) oxygen consumption rate in the differentiated cells. This suggested blocking myotube formation through supplementation with EPA and DHA drove myoblasts to enter the quiescent state and enabled adipogenic trans-differentiation of the myoblasts. Data also suggested that overdosage of EPA and DHA during gestation may drive fetal mesenchymal stem cell differentiation to the fate of adipogenesis and have a long-term effect on childhood obesity.

Effects of tricalcium phosphate in polycaprolactone scaffold for mesenchymal stem cell-based bone tissue engineering

ABSTRACTPolycaprolactone (PCL) – tricalcium phosphate (TCP) is an established composite material for bone tissue engineering. However, the effects of TCP inclusion in PCL for cell attachment, viability, proliferation and differentiation have not been defined. In this study, PCL and PCL-TCP scaffolds are seeded with human fetal mesenchymal stem cells (hfMSC), cultured in osteogenic differentiation medium, and cell attachment, viability, cellular proliferation, differentiation and extracellular mineral depositions were compared. Both materials allowed for good cell attachment and viability, while the final cellularity in PCL-TCP scaffolds was 1.25-fold higher than PCL scaffolds. Mineralisation on both scaffolds was similar, after correcting for the initial calcium in the PCL-TCP scaffolds. In conclusion, the addition of 20% TCP into the macroporous PCL scaffold resulted in 1.25-fold higher cellularity, which may be advantageous for transplantation purposes.

Novel Chiral 1,3,4‐Oxadiazole Derivatives Inducing Astrocyte Differentiation of Rat Fetal Neural Stem Cells

Novel Chiral 1,3,4‐Oxadiazole Derivatives Inducing Astrocyte Differentiation of Rat Fetal Neural Stem Cells

Isolation and characterization of neural stem cells from ovine foetal cerebral cortex

Neural stem cells are a potential source in cell based therapy for neurodegenerative diseases and traumatic brain injuries. The aim of this study is to isolate the ovine (Ovis aries) fetal neural stem cells and to analyze their neurogenic characteristics in vitro. Cerebral cortex pieces harvested from ovine fetal brain tissue were triturated for dispersal into single cells, cultured in neuronal stem cells culture medium and expanded in vitro in adherent culture. The morphological changes and the expression of neuron-specific gene markers and proteins were evaluated. These isolated NSCs were plastic adherent, self-renewing cells maintained the capacity to differentiate into brain specific cell types and they were found positive for neuronal stem cell markers, Nestin and â III tubulin by Reverse Transcription-PCR. Immunocytochemistry of the isolated NSCs also showed positive for Nestin, an established marker for neural and glial precursors as well as MSC marker CD90 and negative for hematopoietic CD34 markers.

Cardiac Restoration Stemming From the Placenta Tree: Insights From Fetal and Perinatal Cell Biology.

Efficient cardiac repair and ultimate regeneration still represents one of the main challenges of modern medicine. Indeed, cardiovascular disease can derive from independent conditions upsetting heart structure and performance: myocardial ischemia and infarction (MI), pharmacological cardiotoxicity, and congenital heart defects, just to name a few. All these disorders have profound consequences on cardiac tissue, inducing the onset of heart failure over time. Since the cure is currently represented by heart transplantation, which is extremely difficult due to the shortage of donors, much effort is being dedicated to developing innovative therapeutic strategies based on stem cell exploitation. Among the broad scenario of stem/progenitor cell subpopulations, fetal and perinatal sources, namely amniotic fluid and term placenta, have gained interest due to their peculiar regenerative capacity, high self-renewal capability, and ease of collection from clinical waste material. In this review, we will provide the state-of-the-art on fetal perinatal stem cells for cardiac repair and regeneration. We will discuss different pathological conditions and the main therapeutic strategies proposed, including cell transplantation, putative paracrine therapy, reprogramming, and tissue engineering approaches.

Amniotic Fluid Cells Biobank for Research on Fetal Mesenchymal Stem Cells

The research biobank “Anna Maria Ferraro Cutino” AOR-villa Sofia-Cervello, started in 2010 by a grant of the Sicilian Department of Health. Within the biobank are collected, characterized, stored and distributed amniotic fluids (AF) from amniocentesis performed for maternal age. Patients choose to donate to the biobank the first 3 ml of AF that cannot be used for prenatal diagnosis. AF and donor data are anonymously archived in a database, according to the standard ethical principles. Collateral biobanks are present to collect, storage and manage donor sera and DNA samples, to perform microbiological and genetic tests necessary for the unit release. Funding Statement: From 2009 to date this work is supported by: 1. “Franco e Piera Cutino” onlus Foundation, Palermo (Italy); 2. Health Department Sicilian Region; 3. Productive Activities Department Sicilian Region; 4. AOR “Villa Sofia –Cervello” (Palermo).