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

Generation of human induced pluripotent stem cells from extraembryonic tissues of fetuses affected by monogenic diseases

Introduction: The generation of human induced pluripotent stem cells (hiPSCs) derived from autologous extra-embryonic fetal source is an innovative personalized-regenerative technology, which 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 enables researchers to undertake studies for treating diseases or for future applications of in utero therapy. Methods: we use 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 deriving from prenatal diagnosis for several genetic disorder, such as Myotonic Dystrophy type 1 (DM1), β-Thalassemia (β-Thal), Lymphema-Distichiasis Syndrome (LDS), Spinal Muscular Atrophy (SMA), Cystic Fibrosis (CF), as well as from wild type (WT) fetal cells. Since cell type used for create iPSCs influence both the reprogramming process efficiency and the kinetics, we use chorionic villus (CVS) and amniotic fluid (AF) cells, demonstrating how they represent an ideal cell resource for efficient generation of hiPSCs. Results: The successful reprogramming of both CV and AF cells into hiPSCs is confirmed by specific morphological, molecular and immunocytochemical markers, and by their teratogenic potential when inoculated in vivo. We further demonstrate the stability of reprogrammed cells over 10 and more passages and their ability to differentiate into the three embryonic germ layers . Conclusions: These data suggest that hiPSCs-CVS/AF can be considered a valid cellular model to accomplish pathogenesis studies and therapeutic applications

AFM nano-mechanical study of the beating profile of hiPSC-derived cardiomyocytes beating bodies WT and DM1

Myotonic Dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults, characterized by a variety of multisystemic features and associated with cardiac anomalies. Among cardiac phenomena, conduction defects, ventricular arrhythmias, and dilated cardiomyopathy represent the main cause of sudden death in DM1 patients. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful in vitro model for molecular, biochemical, and physiological studies of disease in the target cells. Here, we used an Atomic Force Microscope (AFM) to measure the beating profiles of a large number of cells, organized in CM clusters (Beating Bodies, BBs), obtained from wild type (WT) and DM1 patients. We monitored the evolution over time of the frequency and intensity of the beating. We determined the variations between different BBs and over various areas of a single BB, caused by morphological and biomechanical variations. We exploited the AFM tip to apply a controlled force over the BBs, to carefully assess the biomechanical reaction of the different cell clusters over time, both in terms of beating frequency and intensity. Our measurements demonstrated differences between the WT and DM1 clusters highlighting, for the DM1 samples, an instability which was not observed in WT cells. We measured differences in the cellular response to the applied mechanical stimulus in terms of beating synchronicity over time and cell tenacity, which are in good agreement with the cellular behavior in vivo. Overall, the combination of hiPSC-CMs with AFM characterization can become a new tool to study the collective movements of cell clusters in different conditions and can be extended to the characterization of the BB response to chemical and pharmacological stimuli

Functional analysis of POLD1 p.ser605del variant: the aging phenotype of MDPL syndrome is associated with an impaired DNA repair capacity

Mandibular hypoplasia, Deafness and Progeroid features with concomitant Lipodystrophy define a rare systemic disorder, named MDPL Syndrome, due to almost always a de novo variant in POLD1 gene, encoding the DNA polymerase δ. We report a MDPL female heterozygote for the recurrent p.Ser605del variant. In order to deepen the functional role of the in frame deletion affecting the polymerase catalytic site of the protein, cellular phenotype has been characterised. MDPL fibroblasts exhibit in vitro nuclear envelope anomalies, accumulation of prelamin A and presence of micronuclei. A decline of cell growth, cellular senescence and a blockage of proliferation in G0/G1 phase complete the aged cellular picture. The evaluation of the genomic instability reveals a delayed recovery from DNA induced-damage. Moreover, the rate of telomere shortening was greater in pathological cells, suggesting the telomere dysfunction as an emerging key feature in MDPL. Our results suggest an alteration in DNA replication/repair function of POLD1 as a primary pathogenetic cause of MDPL. The understanding of the mechanisms linking these cellular characteristics to the accelerated aging and to the wide spectrum of affected tissues and clinical symptoms in the MDPL patients may provide opportunities to develop therapeutic treatments for progeroid syndromes

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. © 2018, The Author(s)

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