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Introduction Circulating fetal extravillous trophoblasts may offer a superior alternative to cell-free fetal DNA for noninvasive prenatal testing. Cells of fetal origin are a pure source of fetal genome; hence, unlike the cell-free noninvasive prenatal test, the fetal cell-based noninvasive prenatal test is not expected to be affected by maternal DNA. However, circulating fetal cells from previous pregnancies may lead to confounding results.Material and methods To study whether fetal trophoblast cells persist in maternal circulation postpartum, blood samples were collected from 11 women who had given birth to a boy, with blood sampling at 1-3 days (W0), 4-5 weeks (W4-5), around 8 weeks (W8) and around 12 weeks (W12) postpartum. The existence of fetal extravillous trophoblasts was verified either by X and Y chromosome fluorescence in situ hybridization analysis or by short tandem repeat analysis. To exclude technological bias in isolating fetal cells, blood samples were also collected from 10 pregnant women between a gestational age of 10 and 14 weeks, the optimal time frame for cell-based noninvasive prenatal test sampling. All the samples were processed according to protocols established by ARCEDI Biotech for fetal extravillous trophoblast enrichment and isolation.Results Fetal extravillous trophoblasts were found in all the 10 samples from pregnant women between a gestational age of 10 and 14 weeks. However, only 4 of 11 blood samples taken from women at 1-3 days postpartum rendered fetal extravillous trophoblasts, and only 2 of 11 samples rendered fetal extravillous trophoblasts at 4 weeks postpartum.Conclusions In this preliminary dataset on few pregnancies, none of the samples rendered any fetal cells at or after 8 weeks postpartum, showing that cell-based noninvasive prenatal testing based on fetal extravillous trophoblasts is unlikely to be influenced by circulating cells from previous pregnancies.

Hemophilia is an X-linked recessive bleeding disorder. In pregnant women carrier of hemophilia, the fetal sex can be determined by non-invasive analysis of fetal DNA circulating in the maternal blood. However, in case of a male fetus, conventional invasive procedures are required for the diagnosis of hemophilia. Fetal cells, circulating in the maternal bloodstream, are an ideal target for a safe non-invasive prenatal diagnosis. Nevertheless, the small number of cells and the lack of specific fetal markers have been the most limiting factors for their isolation. We aimed to develop monoclonal antibodies (mAbs) against the ribosomal protein RPS4Y1 expressed in male cells. By Western blotting, immunoprecipitation and immunofluorescence analyses performed on cell lysates from male human hepatoma (HepG2) and female human embryonic kidney (HEK293) we developed and characterized a specific monoclonal antibody against the native form of the male RPS4Y1 protein that can distinguish male from female cells. The availability of the RPS4Y1-targeting monoclonal antibody should facilitate the development of novel methods for the reliable isolation of male fetal cells from the maternal blood and their future use for non-invasive prenatal diagnosis of X-linked inherited disease such as hemophilia.

The reliability of non-invasive prenatal testing is highly dependent on accurate estimation of fetal fraction. Several methods have been proposed up to date, utilizing different attributes of analyzed genomic material, for example length and genomic location of sequenced DNA fragments. These two sources of information are relatively unrelated, but so far, there have been no published attempts to combine them to get an improved predictor. We collected 2454 single euploid male fetus samples from women undergoing NIPT testing. Fetal fractions were calculated using several proposed predictors and the state-of-the-art SeqFF method. Predictions were compared with the reference Y-based method. We demonstrate that prediction based on length of sequenced DNA fragments may achieve nearly the same precision as the state-of-the-art methods based on their genomic locations. We also show that combination of several sample attributes leads to a predictor that has superior prediction accuracy over any single approach. Finally, appropriate weighting of samples in the training process may achieve higher accuracy for samples with low fetal fraction and so allow more reliability for subsequent testing for genomic aberrations. We propose several improvements in fetal fraction estimation with a special focus on the samples most prone to wrong conclusion.

In this article, we outline and discuss available information on the cellular site and mechanism of proteasome interaction with cytosolic polyubiquitinated proteins and heat-shock molecules. The particulate cytoplasmic structure (PaCS) formed by barrel-like particles, closely reproducing in vivo the high-resolution structure of 26S proteasome as isolated in vitro, has been detected in a variety of fetal and neoplastic cells, from living tissue or cultured cell lines. Specific trophic factors and interleukins were found to induce PaCS during in vitro differentiation of dendritic, natural killer (NK), or megakaryoblastic cells, apparently through activation of the MAPK-ERK pathway. Direct interaction of CagA bacterial oncoprotein with proteasome was shown inside the PaCSs of a Helicobacter pylori-infected gastric epithelium, a finding suggesting a role for PaCS in CagA-mediated gastric carcinogenesis. PaCS dissolution and autophagy were seen after withdrawal of inducing factors. PaCS-filled cell blebs and ectosomes were found in some cells and may represent a potential intercellular discharge and transport system of polyubiquitinated antigenic proteins. PaCS differs substantially from the inclusion bodies, sequestosomes, and aggresomes reported in proteinopathies like Huntington or Parkinson diseases, which usually lack PaCS. The latter seems more linked to conditions of increased cell proliferation/differentiation, implying an increased functional demand to the ubiquitin–proteasome system.

Objective: A non-invasive prenatal determination of the fetal RhD status might be useful for the management of pregnancies in RhD-negative women whose partners are RhD positive. Methods: Maternal peripheral blood of 32 RhD-negative women (17–24 weeks of gestation) was collected, and circulating fetal cells were enriched by CD71 mini-magnetic activated cell sorting. The RhD status of the fetuses was assessed using multiparametric flow cytometry, and results were compared to those of reverse transcriptase (RT)-polymerase chain reaction (PCR), or PCR, which acted as control. Flow-cytometric study of fetal cells employed monoclonal antibodies directed against CD71, glycophorin A (GPA) and RhD antigens. Results: The median percentage of CD71- and RhD-positive cells was 0.83% (range 0.14–6.44%), and that of CD71 and GPA-positive cells was 10.07% (range 0.52–45.84%). Flow-cytometric analysis correlated with RT-PCR results of RNA obtained from whole maternal blood. In 1 case, an incorrect result was due to the failure of the amplification of the specific RhD band on RNA extracted from the CD71-positive fraction. In two instances, we observed false-positive results for RhD in PCR of DNA obtained from maternal plasma. Conclusion: Based on our results, flow-cytometric analysis might be proposed as a clinical tool for the non-invasive prenatal determination of the fetal RhD status independently of fetal gender.

INTRODUCTION: Traumatic spinal cord injury is one of the most disabling conditions occurring in man and thus stimulates a strong interest in its histopathological, biochemical, and functional changes, primarily as we search for preventive and therapeutic methods. PURPOSE: To develop an experimental model for transplantation of cells from the fetal rat central nervous system to the site of an injured spinal cord of an adult rat in which the transplanted cells survive and become integrated. This experimental model will facilitate investigations of factors that promote regeneration and functional recovery after spinal cord trauma. MATERIAL AND METHODS: Fifteen adult Wistar rats underwent laminectomy, and an spinal cord lesion was made with microdissection. Fetal spinal cord tissue was then transplanted to the site of the injury. The rats were monitored over a 48-hour period, and then their vertebral column was completely removed for histological analysis. RESULTS: In 60% of transplanted rats, the fetal tissue at the injured site remained viable in the site of the lesion. INTRODUÇÃO: A lesão traumática da medula espinal consiste numa das mais incapacitantes lesões que o ser humano pode sofrer e tem despertado grande interesse no conhecimento das alterações histopatológicas, bioquímicas, funcionais e principalmente na busca de métodos de prevenção e tratamento. OBJETIVO: Propor um modelo experimental de transplante de células do sistema nervoso fetal de ratos para o sítio de lesão medular de ratos adultos que permitisse sua sobrevivência e integração para possibilitar protocolos de pesquisa para identificar outros fatores de regeneração e recuperação funcional pós trauma raquimedular. MATERIAL E MÉTODOS: Utilizaram-se 15 ratos adultos que foram submetidos a laminectomia e lesão de 5mm de hemimelula realizada com auxílio de microscópio óptico. Os ratos tiveram seu sítio de lesão medular transplantado com células do sistema nervoso central de fetos de rato. Os ratos foram monitorados por 48 horas e tiveram sua coluna vertebral extraída para análise histológica. RESULTADOS: Demostrou-se que em 60% dos casos as células transplantadas permaneciam viáveis no sítio da lesão.

Microchimerism is the occurrence of small populations of cells with a different genetic background within an individual. Tissue microchimerism is considered to be primarily pregnancy-derived and is often studied relative to female-dominant autoimmune diseases, pregnancy complications, malignancies, response to injury, and transplantation outcomes. A particular distribution pattern of chimeric cells across various organs was recently described in a model of murine pregnancies. Our aim was to determine the frequency and distribution of tissue microchimerism across organs during and after pregnancy in humans. We performed in situ hybridization of the Y chromosome on paraffin-embedded autopsy samples of kidneys, livers, spleens, lungs, hearts and brains that were collected from 26 women who died while pregnant or within 1 month after delivery of a son. Frequencies of chimeric cells in various tissues were compared with those of a control group of non-pregnant women who had delivered sons. Tissue microchimerism occurred significantly more frequently in the lungs, spleens, livers, kidneys and hearts of pregnant women compared with non-pregnant women (all P < 0.01). We showed that some of the chimeric cells were CD3+ or CD34+. After correction for cell density, the lung was most chimeric (470 Y chromosome-positive nuclei per million nuclei scored), followed by the spleen (208 Y+/10(6) nuclei), liver (192 Y+/10(6) nuclei), kidney (135 Y+/10(6) nuclei), brain (85 Y+/10(6) nuclei) and heart (40 Y+/10(6) nuclei). Data from this unique study group of women who died while pregnant or shortly after delivery provide information about the number and physiologic distribution of chimeric cells in organs of pregnant women. We demonstrate that during pregnancy, a boost of chimeric cells is observed in women, with a distribution across organs, that parallels findings in mouse models.

More than 40% of the body mass is represented by muscle tissue, which possesses the innate ability to regenerate after damage through the activation of muscle-specific stem cells, namely satellite cells. Muscle diseases, in particular chronic degenerative states of skeletal muscle such as dystrophies, lead to a perturbation of the regenerative process, which causes the premature exhaustion of satellite cell reservoir due to continuous cycles of degeneration/regeneration. Nowadays, the research is focused on different therapeutic approaches, ranging from gene and cell to pharmacological therapy, but still there is no definitive cure in particular for genetic muscle disease. Keeping this in mind, in this article, we will give special consideration to muscle diseases and the use of fetal derived stem cells as a new approach for therapy. Cells of fetal origin, from cord blood to placenta and amniotic fluid, can be easily obtained without ethical concern, expanded and differentiated in culture, and possess immune-modulatory properties. The in vivo approach in animal models can be helpful to study the mechanism underneath the operating principle of the stem cell reservoir, namely the niche, which holds great potential to understand the onset of muscle pathologies.

The ability to capture and analyze fetal cells from maternal circulation or other sources during pregnancy has been a goal of prenatal diagnostics for over thirty years. The vision of replacing invasive prenatal diagnostic procedures with the prospect of having the entire fetal genome in hand non-invasively for chromosomal and molecular studies for both clinical and research use has brought many investigators and innovations into the effort. While the object of this desire, however, has remained elusive, the aspiration for this approach to non-invasive prenatal diagnosis remains and the inquiry has continued. With the advent of screening by cell-free DNA analysis, the standards for fetal cell based prenatal diagnostics have been sharpened. Relevant aspects of the history and the current status of investigations to meet the goal of having an accessible and reliable strategy for capturing and analyzing fetal cells during pregnancy are reviewed.

Aims: Transcervical samples following endocervical lavage from 28 women undergoing pregnancy termination were analysed in terms of fetal cells content and suitability for in situ hybridization analysis. A non fluorescent protocol of hybridization coupled to specific cytological procedures was used for simultaneous recognition and investigation on syncitiotrophoblast fragments. Methods: A series of endocervical washings was randomised to cytobrushing samples. Cervical washings were performed using variable volumes of physiologic saline solution (2ml, 5ml, 10ml). Single target in situ hybridization for chromosomes 1 and 18 was applied for determining a model of efficiency. Syncitial cells were recognized by means of cytoplasmic and nuclear stainings obtained with Eosin G (Diff-Quik) and haematoxylin respectively. Results: When 5 - 10ml of sterile solution were flushed multiple fragments of syncitio-trophoblast were almost constantly recovered. Conversely using 2ml of flushing solution no syncitia at all could be retrieved. Fetal cells were collected only in 50% of cervical cytobrushing. The per cell hybridization efficiency was on average 50%. Following in situ hybridization, a normal number of signal-products (2) was found in all nuclei analysed. Conclusion: During endocervical washing the amount of volume used is a critical factor to allow successful recovery of fetal cells. By using 5 - 10 ml of injecting solution, enough fetal material was generally obtained from each individual to guarantee the cytogenetic investigation for two chromosomes. The use of Diff-Quik and haematoxylin staining coupled to bright-field microscope facilitate the recognition of syncitia among a highly heterogeneous population of maternal cells. Further studies are ongoing to prospectively evaluate safety of the transcervical approach for sampling and analysis of fetal cells.

Margherita Maioli,1–3 Giovanni Contini,1 Sara Santaniello,1,2 Pasquale Bandiera,1 Gianfranco Pigliaru,1,2 Raimonda Sanna,5 Salvatore Rinaldi,3 Alessandro P Delitala,1 Andrea Montella,1,5 Luigi Bagella,1,6 Carlo Ventura2–41Department of Biomedical Sciences, University of Sassari, Sassari, 2Laboratory of Molecular Biology and Stem Cell Engineering, National Institute of Biostructures and Biosystems, Bologna, 3Department of Regenerative Medicine, Rinaldi Fontani Institute, Florence, 4Cardiovascular Department, S Orsola-Malpighi Hospital, University of Bologna, Bologna, 5Facility of Genetic and Developmental Biology, AOU Sassari, Sassari, Italy; 6Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA, USAAbstract: Mouse embryonic stem cells were previously observed along with mesenchymal stem cells from different sources, after being treated with a mixed ester of hyaluronan with butyric and retinoic acids, to show a significant increase in the yield of cardiogenic and vascular differentiated elements. The aim of the present study was to determine if stem cells derived from primitive fetal cells present in human amniotic fluid (hAFSCs) and cultured in the presence of a mixture of hyaluronic (HA), butyric (BU), and retinoic (RA) acids show a higher yield of differentiation toward the cardiovascular phenotype as compared with untreated cells. During the differentiation process elicited by exposure to HA + BU + RA, genes controlling pluripotency and plasticity of stem cells, such as Sox2, Nanog, and Oct4, were significantly downregulated at the transcriptional level. At this point, a significant increase in expression of genes controlling the appearance of cardiogenic and vascular lineages in HA + BU + RA-treated cells was observed. The protein expression levels typical of cardiac and vascular phenotypes, evaluated by Western blotting, immunofluorescence, and flow cytometry, were higher in hAFSCs cultured in the presence of HA + BU + RA, as compared with untreated control cells. Appearance of the cardiac phenotype was further inferred by ultrastructural analysis using transmission and scanning electron microscopy. These results demonstrate that a mixture of HA + BU + RA significantly increased the yield of elements committed toward cardiac and vascular phenotypes, confirming what we have previously observed in other cellular types.Keywords: fetal cells, human amniotic fluid, hyaluronic acid, butyric acid, retinoic acid, prodynorphin, Nkx-2.5, GATA-4, von Willebrand factor

Mohammed T Al Samri,1,* Ankush V Biradar,2,3,* Ahmed R Alsuwaidi,1 Ghazala Balhaj,1 Suleiman Al-Hammadi,1 Safa Shehab,4 Suhail Al-Salam,5 Saeed Tariq,4 Thachillath Pramathan,1 Sheela Benedict,1 Tewodros Asefa,2,3 Abdul-Kader Souid11Department of Pediatrics, Abu Dhabi, United Arab Emirates; 2Department of Chemistry and Chemical Biology, 3Department of Chemical Engineering and Biochemical Engineering, Rutgers, The State University of New Jersey, Piscataway, NJ, USA; 4Department of Anatomy, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al Ain, Abu Dhabi, United Arab Emirates; 5Department of Pathology Faculty of Medicine and Health Sciences, United Arab Emirates University, Al ain, Abu Dhabi, United Arab Emirates*These authors contributed equally to this workBackground: The biocompatibility of two forms of calcined mesoporous silica particles, labeled as MCM41-cal and SBA15-cal, with fetal blood mononuclear cells was assessed in vitro.Methods and results: Fetal mononuclear cells were isolated from umbilical cord blood and exposed to 0.5 mg/mL of MCM41-cal or SBA15-cal for several hours. Transmission electron micrographs confirmed the presence of particles in the cytosol of macrophages, neutrophils, and lymphocytes without noticeable damage to the cellular organelles. The particles (especially MCM41-cal) were in close proximity to plasma, and nuclear and mitochondrial membranes. Biocompatibility was assessed by a functional assay that measured cellular respiration, ie, mitochondrial O2 consumption. The rate of respiration (kc, in µM O2 per minute per 107 cells) for untreated cells was 0.42 ± 0.16 (n = 10), for cells treated with MCM41-cal was 0.39 ± 0.22 (n = 5, P > 0.966) and for cells treated with SBA15-cal was 0.44 ± 0.13 (n = 5, P >0.981).Conclusion: The results show reasonable biocompatibility of MCM41-cal and SBA15-cal in fetal blood mononuclear cells. Future studies are needed to determine the potential of collecting fetal cells from a fetus or neonate, loading the cells in vitro with therapeutic MCM41-cal or SBA15-cal, and reinfusing them into the fetus or neonate.Keywords: mesoporous silica, nanomaterials, biocompatibility, bioenergetics, in vitro, fetal cells

The chemical functionalization of cell-surface proteins of human primary fetal bone cells with hydrophilic bioorthogonal intermediates was investigated. Toward this goal, chemical pathways were developed for click reaction-mediated coupling of alkyne derivatives with cellular azido-expressing proteins. The incorporation via a tetraethylene glycol linker of a dipeptide and a reporter biotin allowed the proof of concept for the introduction of cell-specific peptide ligands and allowed us to follow the reaction in living cells. Tuning the conditions of the click reaction resulted in chemical functionalization of living human fetal osteoblasts with excellent cell survival.

Myoblast transfer therapy has been extensively studied for a wide range of clinical applications, such as tissue engineering for muscular loss, cardiac surgery or Duchenne Muscular Dystrophy treatment. However, this approach has been hindered by numerous limitations, including early myoblast death after injection and specific immune response after transplantation with allogenic cells. Different cell sources have been analyzed to overcome some of these limitations. The object of our study was to investigate the growth potential, characterization and integration in vivo of human primary fetal skeletal muscle cells. These data together show the potential for the creation of a cell bank to be used as a cell source for muscle cell therapy and tissue engineering. For this purpose, we developed primary muscular cell cultures from biopsies of human male thigh muscle from a 16-week-old fetus and from donors of 13 and 30 years old. We show that fetal myogenic cells can be successfully isolated and expanded in vitro from human fetal muscle biopsies, and that fetal cells have higher growth capacities when compared to young and adult cells. We confirm lineage specificity by comparing fetal muscle cells to fetal skin and bone cells in vitro by immunohistochemistry with desmin and 5.1 H11 antibodies. For the feasibility of the cell bank, we ensured that fetal muscle cells retained intrinsic characteristics after 5 years cryopreservation. Finally, human fetal muscle cells marked with PKH26 were injected in normal C57BL/6 mice and were found to be present up to 4 days. In conclusion we estimate that a human fetal skeletal muscle cell bank can be created for potential muscle cell therapy and tissue engineering.

Cell therapy for nucleus pulposus regeneration is an attractive treatment for early disc degeneration as shown by studies using autologous nucleus pulposus cells or stem cells. Another potential source of cells is fetal cells. We investigated the feasibility of isolating fetal cells from human fetal spine tissues and assessed their chondrogenic potential in alginate bead cultures. Histology and immunohistochemistry of fetal tissues showed that the structure and the matrix composition (aggrecan, type I and II collagen) of fetal intervertebral disc were similar to adult intervertebral disc. Isolated fetal cells were cultured in monolayer in basic media supplemented with 10% FBS and from each fetal tissue donation, a cell bank of fetal spine cells at passage 2 was established and was composed of around 2,000 vials of 5 million cells. Gene expression and immunohistochemistry of fetal spine cells cultured in alginate beads during 28 days showed that cells were able to produce aggrecan and type II collagen and very low level of type I and type X collagen, indicating chondrogenic differentiation. However variability in matrix synthesis was observed between donors. In conclusion, fetal cells could be isolated from human fetal spine tissues and since these cells showed chondrogenic potential, they could be a potential cell source for intervertebral disc regeneration.

Intervertebral disc (IVD) degeneration is one of the prevailing medical concern and actual health cost. Industries and academic institutions research for therapeutic solutions going from drugs to complete and partial arthroplasty devices as well as engineered tissues. Most research focus on the nucleus pulposus (NP) of the IVD because it is believed that, in the degeneration process, the NP is the scene of the first (irreversible) biochemical and mechanical changes. Mechanical, biochemical, nutritional and genetic factors may induce the degenerative process. Authors agree that (dynamic) mechanical stimuli are essential to preserve the integrity of the NP tissue. Indeed, sustained static loading and joint immobilization are proven to accelerate tissue degeneration. One possible explanation is that dynamic stimuli are required to transport (by convection) macromolecules, whereas smaller molecules are solely driven by passive diffusion. Another explanation is the requirement of direct cell-solid and/or cell-fluid interactions, namely mechanotransduction. In reality, both mechanisms are believed to influence tissue maintenance and should be considered evenly. It is, however, difficult to find a quantitative variable that unifies both theories. Viscoelasticity is the result of imperfect processes in which energy is dissipated. These imperfections arise from microstructural interactions and internal transport phenomena. Integrated, they result in time dependent and observable macroscopic behaviour. Although, interactions between phases are probably dominant, it makes sense to assume that cell-phase interactions are proportionally related. Therefore, investigating correlations between viscoelastic properties of the NP with cell synthesis may provide an experimental and theoretical mean to investigate the effect of dynamic mechanical stimuli on living tissues. The objective of this project is to verify this hypothesis on a hydrogel-cell construct model. Because the following step is to localize the viscoelastic description, extracellular matrix production is to be assessed with a 3D spatial resolution.

Current restrictions for human cell-based therapies have been related to technological limitations with regards to cellular proliferation capacity and maintenance of differentiated phenotype for primary human cell culture. In our experience, we have seen that cultured primary fetal cells meet the exigent and stringent technical aspects for development of therapeutic products. As fetal skin cells show rapid growth, Master and Working Cell Banks have been prepared from fetal tissue in short periods of time compared to other primary cell types; one month compared to three or four. Working Cell Banks are then used for subsequent batches of cells for use in research and Tissue Engineering projects. This study focuses on the requirements for fetal tissue selection and testing, cell banking process and testing and growth of cells in production for fetal skin fibroblasts. Depending on the initial concentration of fetal cells seeded, life-span can range from 11-16 weeks of passages in a semi-industrial production protocol. Repetitive cultures from the same tissue show consistent cell culture development when tested for cell growth, protein concentrations, RNA content and gene mRNA expression for genes involved in tissue repair (PTN, MDK, OPG, TGF-β1). As it is the intention that banked primary fetal cells can profit from the prospected treatment of hundreds of thousands of patients with only one organ donation, it is imperative to show consistency, tracability and safety of the process including donor tissue selection, cell banking, cell testing and growth of cells in up-scaling for the preparation of cell transplantation.