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

82. Cftr Gene Targeting in Murine ES Cells Mediated by the SFHR Technique

Small Fragment Homologous Recombination (SFHR)-mediated targeting is a gene therapy strategy where a specific genomic locus is modified through a target exchange between a small DNA fragment (SDF) and genomic DNA. Here we demonstrate that SFHR can stably introduce a 3-bp deletion (corresponding to |[Delta]|F508) within Cftr (Cystic Fibrosis Transmembrane Conductance Regulator) locus in the genome of mouse embryonic stem (ES) cells. SDFs (about 6.4|[times]|105 molecules per cell) carrying the |[Delta]|F508 mutation were transfected by nucleofection protocol. About 12% of transcript corresponding to deleted allele was detected and about 60% of the electroporated cells no longer had measurable CFTR-dependent chloride efflux. The CFTR activity was also analyzed by measuring the chloride efflux by the fluorescence microscopy-coupled digital video imaging system in each ES cell colony, previously loaded with MQAE, a chloride sensitive dye. An average of 4-6 regions for each cell colony was analysed to verify the genotypic homogeneity of each colony. In fact all regions examined in each colony showed a similar significant chloride efflux after PKA activation. Moreover on twelve electroporated ES colonies analysed, eight were successfully mutated (Cl- efflux not significantly different from zero) while four colonies showed Cl-efflux CFTR-dependent not significantly different from the untreated ones

Two Different Therapeutic Approaches for SARS-CoV-2 in hiPSCs-Derived Lung Organoids

The global health emergency for SARS-CoV-2 (COVID-19) created an urgent need to develop new treatments and therapeutic drugs. In this study, we tested, for the first time on human cells, a new tetravalent neutralizing antibody (15033-7) targeting Spike protein and a synthetic peptide homologous to dipeptidyl peptidase-4 (DPP4) receptor on host cells. Both could represent powerful immunotherapeutic candidates for COVID-19 treatment. The infection begins in the proximal airways, namely the alveolar type 2 (AT2) cells of the distal lung, which express both ACE2 and DPP4 receptors. Thus, to evaluate the efficacy of both approaches, we developed three-dimensional (3D) complex lung organoid structures (hLORGs) derived from human-induced pluripotent stem cells (iPSCs) and resembling the in vivo organ. Afterward, hLORGs were infected by different SARS-CoV-2 S pseudovirus variants and treated by the Ab15033-7 or DPP4 peptide. Using both approaches, we observed a significant reduction of viral entry and a modulation of the expression of genes implicated in innate immunity and inflammatory response. These data demonstrate the efficacy of such approaches in strongly reducing the infection efficiency in vitro and, importantly, provide proof-of-principle evidence that hiPSC-derived hLORGs represent an ideal in vitro system for testing both therapeutic and preventive modalities against COVID-19

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

Organoid factory: The recent role of the human induced pluripotent stem cells (hiPSCs) in precision medicine

During the last decades, hiPSC-derived organoids have been extensively studied and used as in vitro models for several applications among which research studies. They can be considered as organ and tissue prototypes, especially for those difficult to obtain. Moreover, several diseases can be accurately modeled and studied. Hence, patient-derived organoids (PDOs) can be used to predict individual drug responses, thus paving the way toward personalized medicine. Lastly, by applying tissue engineering and 3D printing techniques, organoids could be used in the future to replace or regenerate damaged tissue. In this review, we will focus on hiPSC-derived 3D cultures and their ability to model human diseases with an in-depth analysis of gene editing applications, as well as tumor models. Furthermore, we will highlight the state-of-the-art of organoid facilities that around the world offer know-how and services. This is an increasing trend that shed the light on the need of bridging the publicand the private sector. Hence, in the context of drug discovery, Organoid Factories can offer biobanks of validated 3D organoid models that can be used in collaboration with pharmaceutical companies to speed up the drug screening process. Finally, we will discuss the limitations and the future development that will lead hiPSC-derived technology from bench to bedside, toward personalized medicine, such as maturity, organoid interconnections, costs, reproducibility and standardization, and ethics. hiPSC-derived organoid technology is now passing from a proof-of-principle to real applications in the clinic, also thanks to the applicability of techniques, such as CRISPR/Cas9 genome editing system, material engineering for the scaffolds, or microfluidic systems. The benefits will have a crucial role in the advance of both basic biological and translational research, particularly in the pharmacological field and drug development. In fact, in the near future, 3D organoids will guide the clinical decision-making process, having validated patient-specific drug screening platforms. This is particularly important in the context of rare genetic diseases or when testing cancer treatments that could in principle have severe side effects. Therefore, this technology has enabled the advancement of personalized medicine in a way never seen before

Application of CRISPR/Cas9 to human-induced pluripotent stem cells: from gene editing to drug discovery

Human-induced pluripotent stem cells (hiPSCs) and CRISPR/Cas9 gene editing system represent two instruments of basic and translational research, which both allow to acquire deep insight about the molecular bases of many diseases but also to develop pharmacological research. This review is focused to draw up the latest technique of gene editing applied on hiPSCs, exploiting some of the genetic manipulation directed to the discovery of innovative therapeutic strategies. There are many expediencies provided by the use of hiPSCs, which can represent a disease model clinically relevant and predictive, with a great potential if associated to CRISPR/Cas9 technology, a gene editing tool powered by ease and precision never seen before. Here, we describe the possible applications of CRISPR/Cas9 to hiPSCs: from drug development to drug screening and from gene therapy to the induction of the immunological response to specific virus infection, such as HIV and SARS-Cov-2

From cue to meaning: The involvement of POLD1 gene in DNA replication, repair and aging

Aging is an extremely complex biological process. Aging, cancer and inflammation represent a trinity, object of many interesting researches. The accumulation of DNA damage and its consequences progressively interfere with cellular function and increase susceptibility to developing aging condition.DNA Polymerase delta (Pol 8), encoded by POLD1 gene (MIM#174761) on 19q13.3, is well implicated in many steps of the replication program and repair. Thanks to its exonuclease and polymerase activities, the enzyme is involved in the regulation of the cell cycle, DNA synthesis, and DNA damage repair processes. Damaging variants within the exonuclease domain predispose to cancers, while those occurring in the poly-merase active site cause the autosomal dominant Progeroid Syndrome called MDPL, Mandibular hypoplasia, Deafness and Progeroid features with concomitant LipodystrophySince DNA damage represents the main cause of ageing and age-related pathologies, an overview of critical Pol 8 activities will allow to better understand the associations between DNA damage and nearly every aspect of the ageing process, helping the researchers to counteract all the ageing-pathologies at the same time

Epidermal growth factor-like domain 7 promotes migration and invasion of human trophoblast cells through activation of MAPK, PI3K and NOTCH signaling pathways

Epidermal growth factor-like domain 7 (Egfl7) is a gene that encodes a partially secreted protein and whose expression is largely restricted to the endothelia. We recently reported that EGFL7 is also expressed by trophoblast cells in mouse and human placentas. Here, we investigated the molecular pathways that are regulated by EGFL7 in trophoblast cells. Stable EGFL7 overexpression in a Jeg3 human choriocarcinoma cell line resulted in significantly increased cell migration and invasiveness, while cell proliferation was unaffected. Analysis of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways showed that EGFL7 promotes Jeg3 cell motility by activating both pathways. We show that EGFL7 activates the epidermal growth factor receptor (EGFR) in Jeg3 cells, resulting in downstream activation of extracellular regulated kinases (ERKs). In addition, we provide evidence that EGFL7-triggered migration of Jeg3 cells involves activation of NOTCH signaling. EGFL7 and NOTCH1 are co-expressed in Jeg3 cells, and blocking of NOTCH activation abrogates enhanced migration of Jeg3 cells overexpressing EGFL7. We also demonstrate that signaling through EGFR and NOTCH converged to mediate EGFL7 effects. Reduction of endogenous EGFL7 expression in Jeg3 cells significantly decreased cell migration. We further confirmed that EGFL7 stimulates cell migration by using primary human first trimester trophoblast (PTB) cells overexpressing EGFL7. In conclusion, our data suggest that in trophoblast cells, EGFL7 regulates cell migration and invasion by activating multiple signaling pathways. Our results provide a possible explanation for the correlation between reduced expression of EGFL7 and inadequate trophoblast invasion observed in placentopathies

Natriuretic peptides are neuroprotective on in vitro models of PD and promote dopaminergic differentiation of hiPSCs-derived neurons via the Wnt/β-catenin signaling

Abstract Over the last 20 years, the efforts to develop new therapies for Parkinson’s disease (PD) have focused not only on the improvement of symptomatic therapy for motor and non-motor symptoms but also on the discovering of the potential causes of PD, in order to develop disease-modifying treatments. The emerging role of dysregulation of the Wnt/β-catenin signaling in the onset and progression of PD, as well as of other neurodegenerative diseases (NDs), renders the targeting of this signaling an attractive therapeutic opportunity for curing this brain disorder. The natriuretic peptides (NPs) atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), are cardiac and vascular-derived hormones also widely expressed in mammalian CNS, where they seem to participate in numerous brain functions including neural development/differentiation and neuroprotection. We recently demonstrated that ANP affects the Wnt/β-catenin pathway possibly through a Frizzled receptor-mediated mechanism and that it acts as a neuroprotective agent in in vitro models of PD by upregulating this signaling. Here we provide further evidence supporting the therapeutic potential of this class of natriuretic hormones. Specifically, we demonstrate that all the three natriuretic peptides are neuroprotective for SHSY5Y cells and primary cultures of DA neurons from mouse brain, subjected to neurotoxin insult with 6-hydroxydopamine (6-OHDA) for mimicking the neurodegeneration of PD, and these effects are associated with the activation of the Wnt/β-catenin pathway. Moreover, ANP, BNP, CNP are able to improve and accelerate the dopaminergic differentiation and maturation of hiPSCs-derived neural population obtained from two differed healthy donors, concomitantly affecting the canonical Wnt signaling. Our results support the relevance of exogenous ANP, BNP, and CNP as attractive molecules for both neuroprotection and neurorepair in PD, and more in general, in NDs for which aberrant Wnt signaling seems to be the leading pathogenetic mechanism

Generation and Neuronal Differentiation of hiPSCs From Patients With Myotonic Dystrophy Type 2

Human induced pluripotent stem cells (hiPSCs)-patient specific are an innovative tool to reproduce a model of disease in vitro and summarize the pathological phenotype and the disease etiopathology. Myotonic dystrophy type 2 (DM2) is caused by an unstable (CCTG)n expansion in intron 1 of the CNBP gene, leading to a progressive multisystemic disease with muscle, heart and central nervous dysfunctions. The pathogenesis of CNS involvement in DM2 is poorly understood since no cellular or animal models fully recapitulate the molecular and clinical neurodegenerative phenotype of patients. In this study, we generated for the first time, two DM2 and two wild type hiPSC lines from dermal fibroblasts by polycistronic lentiviral vector (hSTEMCCA-loxP) expressing OCT4, SOX2, KLF4, and cMYC genes and containing loxP-sites, excisable by Cre recombinase. Specific morphological, molecular and immunocytochemical markers have confirmed the stemness of DM2 and wild type-derived hiPSCs. These cells are able to differentiate into neuronal population (NP) expressing tissue specific markers. hiPSCs-derived NP cells maintain (CCTG)n repeat expansion and intranuclear RNA foci exhibiting sequestration of MBNL1 protein, which are pathognomonic of the disease. DM2 hiPSCs represent an important tool for the study of CNS pathogenesis in patients, opening new perspectives for the development of cell-based therapies in the field of personalized medicine and drug screening