Paola Spitalieri

In Vivo and In Vitro Studies Support That a New Splicing Isoform of OLR1 Gene Is Protective Against Acute Myocardial Infarction

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), encoded by the OLR1 gene, is a scavenger receptor that plays a fundamental role in the pathogenesis of atherosclerosis. LOX-1 activation is associated with apoptosis of endothelial cells, smooth muscle cells (SMCs), and macrophages. This process is an important underlying mechanism that contributes to plaque instability and subsequent development of acute coronary syndromes. Independent association genetic studies have implicated OLR1 gene variants in myocardial infarction (MI) susceptibility. Because single nucleotide polymorphisms (SNPs) linked to MI are located in intronic sequences of the gene, it remains unclear as to how they determine their biological effects. Using quantitative real-time PCR and minigene approach, we show that intronic SNPs, linked to MI, regulate the expression of a new functional splicing isoform of the OLR1 gene, LOXIN, which lacks exon 5. Macrophages from subjects carrying the “non-risk” disease haplotype at OLR1 gene have an increased expression of LOXIN at mRNA and protein level, which results in a significant reduction of apoptosis in response to oxLDL. Expression of LOXIN in different cell types results in loss of surface staining, indicating that truncation of the C-terminal portion of the protein has a profound effect on its cellular trafficking. Furthermore, the proapoptotic effect of LOX-1 receptor in cell culture is specifically rescued by the coexpression of LOXIN in a dose-dependent manner. The demonstration that increasing levels of LOXIN protect cells from LOX-1 induced apoptosis sets a groundwork for developing therapeutic approaches for prevention of plaque instability.

Rescue of murine silica-induced lung injury and fibrosis by human embryonic stem cells

Alveolar type II pneumocytes (ATII cells) are considered putative alveolar stem cells. Since no treatment is available to repair damaged epithelium and prevent lung fibrosis, novel approaches to induce regeneration of injured alveolar epithelium are desired. The objective of this study was to assess both the capacity of human embryonic stem cells (HUES-3) to differentiate in vitro into ATII cells and the ability of committed HUES-3 cells (HUES-3-ATII cells) to recover in vivo a pulmonary fibrosis model obtained by silica-induced damage. In vitro differentiated HUES-3-ATII cells displayed an alveolar phenotype characterised by multi-lamellar body and tight junction formation, by the expression of specific markers such as surfactant protein (SP)-B, SP-C and zonula occludens (ZO)-1 and the activity of cystic fibrosis transmembrane conductance regulator-mediated chloride ion transport. After transplantation of HUES-3-ATII cells into silica-damaged mice, histological and biomolecular analyses revealed a significant reduction of inflammation and fibrosis markers along with lung function improvement, weight recovery and increased survival. The persistence of human SP-C, human nuclear antigen and human DNA in the engrafted lungs indicates that differentiated cells remained engrafted up to 10 weeks. In conclusion, cell therapy using HUES-3 cells may be considered a promising approach to lung injury repair.

Identification of multipotent cytotrophoblast cells from human first trimester chorionic villi.

In this article we used immunohistochemistry and FACS analyses to show that cells expressing markers typical of human stem cells such as SSEA4, OCT-4, ALP, and CD117 are present within the cytotrophoblastic tissue of human fetal chorionic villus samples (CVSs). After immunoselection of CV cells for SSEA4, FACS analyses showed an increased number of cells positive for OCT-4 and ALP and a small percentage (around 4%) of side population (SP) cells. In the same cell population, RT-PCR indicated the presence of OCT-4, NANOG, and SOX2 transcripts, also typical of stem cells. Depending on the in vitro conditions, a subset of SSEA4+ cells formed colonies resembling hESCs, with limited self renewal ability. At the same time, these cells were able to differentiate in vitro into derivatives of all three germ layers. When inoculated into immunocompromised mice, SSEA4+ cells did not form teratomas but were able to populate depleted hematopoietic tissues. Moreover, after injection into mouse blastocysts, they were incorporated into the inner cell mass and could be traced into several tissues of the adult chimeric mice. Finally, we show that SSEA4+ cells isolated from fetuses affected by Spinal Muscular Atrophy (SMA) can be genetically corrected with high efficiency in culture by Small Fragment Homologous Recombination (SFHR), a gene targeting approach. Taken together, our results indicate that SSEA4+ cells obtained from human CVSs contain a subpopulation of multipotent cells that we propose to name Human Cytotrophoblastic-derived Multipotent Cells (hCTMCs). These cells may be a safe and convenient source of cells for cell-based therapy, as well as an ideal target for in utero fetal gene therapy.

Generation of Human Induced Pluripotent Stem Cells from Extraembryonic Tissues of Fetuses Affected by Monogenic Diseases

The generation of human induced pluripotent stem cells (hiPSCs) derived from an autologous extraembryonic fetal source is an innovative personalized regenerative technology that can transform own-self cells into embryonic stem-like ones. These cells are regarded as a promising candidate for cell-based therapy, as well as an ideal target for disease modeling and drug discovery. Thus, hiPSCs enable researchers to undertake studies for treating diseases or for future applications of in utero therapy. We used a polycistronic lentiviral vector (hSTEMCCA-loxP) encoding OCT4, SOX2, KLF4, and cMYC genes and containing loxP sites, excisible by Cre recombinase, to reprogram patient-specific fetal cells derived from prenatal diagnosis for several genetic disorders, such as myotonic dystrophy type 1 (DM1), β-thalassemia (β-Thal), lymphedema-distichiasis syndrome (LDS), spinal muscular atrophy (SMA), cystic fibrosis (CF), as well as from wild-type (WT) fetal cells. Because cell types tested to create hiPSCs influence both the reprogramming process efficiency and the kinetics, we used chorionic villus (CV) and amniotic fluid (AF) cells, demonstrating how they represent an ideal cell resource for a more efficient generation of hiPSCs. The successful reprogramming of both CV and AF cells into hiPSCs was confirmed by specific morphological, molecular, and immunocytochemical markers and also by their teratogenic potential when inoculated in vivo. We further demonstrated the stability of reprogrammed cells over 10 and more passages and their capability to differentiate into the three embryonic germ layers, as well as into neural cells. These data suggest that hiPSCs-CV/AF can be considered a valid cellular model to accomplish pathogenesis studies and therapeutic applications.

Modelling the pathogenesis of Myotonic Dystrophy type 1 cardiac phenotype through human iPSC-derived cardiomyocytes

Myotonic Dystrophy type 1 (DM1) is a multisystemic disease, autosomal dominant, caused by a CTG repeat expansion in DMPK gene. We assessed the appropriateness of patient-specific induced pluripotent stem cell-derived cardiomyocytes (CMs) as a model to recapitulate some aspects of the pathogenetic mechanism involving cardiac manifestations in DM1 patients. Once obtained in vitro, CMs have been characterized for their morphology and their functionality. CMs DM1 show intranuclear foci and transcript markers abnormally spliced respect to WT ones, as well as several irregularities in nuclear morphology, probably caused by an unbalanced lamin A/C ratio. Electrophysiological characterization evidences an abnormal profile only in CMs DM1 such that the administration of antiarrythmic drugs to these cells highlights even more the functional defect linked to the disease. Finally, Atomic Force Measurements reveal differences in the biomechanical behaviour of CMs DM1, in terms of frequencies and synchronicity of the beats. Altogether the complex phenotype described in this work, strongly reproduces some aspects of the human DM1 cardiac phenotype. Therefore, the present study provides an in vitro model suggesting novel insights into the mechanisms leading to the development of arrhythmogenesis and dilatative cardiomyopathy to consider when approaching to DM1 patients, especially for the risk assessment of sudden cardiac death (SCD). These data could be also useful in identifying novel biomarkers effective in clinical settings and patient-tailored therapies.

A preliminary analysis of volatile metabolites of human induced pluripotent stem cells along the in vitro differentiation

Cellular metabolism of stem cell biology is still an unexplored field. However, considering the amount of information carried by metabolomes, this is a promising field for a fast identification of stem cells itself and during the differentiation process. One of the goals of such application is the identification of residual pluripotent cells before cell transplantation to avoid the occurrence of teratomas. In this paper, we investigated in vitro the volatile compounds (VOCs) released during human induced pluripotent stem cells (hiPSCs) reprogramming. In particular, we studied hiPSCs differentiation to floating and adherent embryoid bodies until early neural progenitor cells. A preliminary Gas Chromatographic/Mass Spectrometer (GC/MS) analysis, based on a single extraction method and chromatographic separation, indicated 17 volatile compounds whose relative abundance is altered in each step of the differentiation process. The pattern of VOCs shown by hiPSCs is well distinct and makes these cells sharply separated from the other steps of differentiations. Similar behaviour has also been observed with an array of metalloporphyrins based gas sensors. The use of electronic sensors to control the process of differentiation of pluripotent stem cells might suggest a novel perspective for a fast and on-line control of differentiation processes.

The telomeric protein AKTIP interacts with A- and B-type lamins and is involved in regulation of cellular senescence

AKTIP is a shelterin-interacting protein required for replication of telomeric DNA. Here, we show that AKTIP biochemically interacts with A- and B-type lamins and affects lamin A, but not lamin C or B, expression. In interphase cells, AKTIP localizes at the nuclear rim and in discrete regions of the nucleoplasm just like lamins. Double immunostaining revealed that AKTIP partially co-localizes with lamin B1 and lamin A/C in interphase cells, and that proper AKTIP localization requires functional lamin A. In mitotic cells, AKTIP is enriched at the spindle poles and at the midbody of late telophase cells similar to lamin B1. AKTIP-depleted cells show senescence-associated markers and recapitulate several aspects of the progeroid phenotype. Collectively, our results indicate that AKTIP is a new player in lamin-related processes, including those that govern nuclear architecture, telomere homeostasis and cellular senescence.

SMA Human iPSC-Derived Motor Neurons Show Perturbed Differentiation and Reduced miR-335-5p Expression

Spinal Muscular Atrophy (SMA) is a neuromuscular disease caused by mutations in the Survival Motor Neuron 1 gene, resulting in very low levels of functional Survival of Motor Neuron (SMN) protein. SMA human induced Pluripotent Stem Cells (hiPSCs) represent a useful and valid model for the study of the disorder, as they provide in vitro the target cells. MicroRNAs (miRNAs) are often reported as playing a key role in regulating neuronal differentiation and fate specification. In this study SMA hiPSCs have been differentiated towards early motor neurons and their molecular and immunocytochemical profile were compared to those of wild type cells. Cell cycle proliferation was also evaluated by fluorescence-activated cell sorting (FACS). SMA hiPSCs showed an increased proliferation rate and also higher levels of stem cell markers. Moreover; when differentiated towards early motor neurons they expressed lower levels of NCAM and MN specific markers. The expression of miR-335-5p; already identified to control self-renewal or differentiation of mouse embryonic stem cells (mESCs); resulted to be reduced during the early steps of differentiation of SMA hiPSCs compared to wild type cells. These results suggest that we should speculate a role of this miRNA both in stemness characteristic and in differentiation efficiency of these cells.

Transabdominal coelocentesis as early source of fetal DNA for chromosomal and molecular diagnosis

Abstract This study reports a comparative analysis between results of transabdominal coelocentesis and traditional invasive procedure in order to assess the usefulness of coelocentesis as a source of fetal DNA for molecular and chromosomal analysis. A number of 28 women were included in the study. A successful sampling of coelomic fluid was obtained in 25 women by transabdominal procedure. A positive amplification of DNA with QF-PCR techniques was obtained in 90% of cases, while 10% of cases failed to reveal interpretable results. Although all samples were cultured, the growth rate was not sufficient to determine karyotypes within 2 weeks. Five samples were selected to be analyzed by array-based comparative genomic hybridization (a-CGH) but the interpretation of these results was difficult and ambiguous. Our results suggest that transabdominal coelocentesis is suitable for the detection of single DNA variation and for QF-PCR analysis, while further experiments are needed to develop optimized protocols for traditional karyotyping and array-analysis.

Volatile compounds emission from teratogenic human pluripotent stem cells observed during their differentiation in vivo

Several investigations point out that the volatile fraction of metabolites, often called volatilome, might signal the difference processes occurring in living beings, both in vitro and in vivo. These studies have been recently applied to stem cells biology, and preliminary results show that the composition of the volatilome of stem cells in vitro changes along the differentiation processes leading from pluripotency to full differentiation. The identification of pluripotent stem cells is of great importance to improve safety in regenerative medicine avoiding the formation of teratomas. In this paper, we applied gas chromatography and gas sensor array to the study of the volatilome released by mice transplanted with human induced pluripotent stem cells (hiPSCs) or embryoid bodies (EBs) derived from hiPSCs at 5 days and spontaneously differentiated cells at 27 day. Gas chromatography analysis finds that, in mice transplanted with hiPSCs, the abundance of 13 volatile compounds increases four weeks after the implant and immediately before the formation of malignant teratomas (grade 3) become observable. The same behaviour is also followed by the signals of the gas sensors. Besides this event, the gas-chromatograms and the sensors signals do not show any appreciable variation related neither among the groups of transplanted mice nor respect to a placebo population. This is the first in vivo observation of the change of volatile metabolites released by human induced pluripotent stem cells and hiPSCs-derived cells during the differentiation process. These results shed further light on the differentiation mechanisms of stem cells and suggest possible applications for diagnostic purposes for an early detection of tumor relapse after surgery.