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 δ), 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 polymerase active site cause the autosomal dominant Progeroid Syndrome called MDPL, Mandibular hypoplasia, Deafness and Progeroid features with concomitant Lipodystrophy Since DNA damage represents the main cause of ageing and age-related pathologies, an overview of critical Pol δ 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.

Indole-3-carbinol in vitro antiviral activity against SARS-Cov-2 virus and in vivo toxicity.

The effects of indole-3-carbinol (I3C) compound have been described deeply as antitumor drug in multiple cancers. Herein, I3C compound was tested for toxicity and antiviral activity against SARS-CoV-2 infection. Antiviral activity was assessed in vitro in both in VeroE6 cell line and human Lung Organoids (hLORGs) where I3C exhibited a direct anti-SARS-CoV-2 replication activity with an antiviral effect and a modulation of the expression of genes implicated in innate immunity and inflammatory response was observed at 16.67 µM. Importantly, we further show the I3C is also effective against the SARS-CoV-2 Omicron variant. In mouse model, instead, we assessed possible toxicity effects of I3C through two different routes of administration: intragastrically (i.g.) and intraperitoneally (i.p.). The LD50 (lethal dose 50%) values in mice were estimated to be: 1410 and 1759 mg/kg i.g.; while estimated values for i.p. administration were: 444.5 mg/kg and 375 mg/kg in male and female mice, respectively. Below these values, I3C (in particular at 550 mg/kg for i.g. and 250 mg/kg for i.p.) induces neither death, nor abnormal toxic symptoms as well as no histopathological lesions of the tissues analysed. These tolerated doses are much higher than those already proven effective in pre-clinical cancer models and in vitro experiments. In conclusion, I3C exhibits a significant antiviral activity, and no toxicity effects were recorded for this compound at the indicated doses, characterizing it as a safe and potential antiviral compound. The results presented in this study could provide experimental pre-clinical data necessary for the start of human clinical trials with I3C for the treatment of SARS-CoV-2 and beyond.

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.

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.

Characterization of MDPL Fibroblasts Carrying the Recurrent p.Ser605del Mutation in POLD1 Gene.

Mandibular hypoplasia, deafness, and progeroid features, with concomitant lipodystrophy, define a multisystem disorder named MDPL syndrome. MDPL has been associated with heterozygous mutations in POLD1 gene resulting in loss of DNA polymerase δ activity. In this study, we report clinical, genetic, and cellular studies of a 13-year-old Pakistani girl, presenting growth retardation, sensorineural deafness, altered distribution of subcutaneous adipose tissue, and insulin resistance. We performed Sanger sequencing of POLD1 gene in the proband and the healthy parents. Fibroblasts obtained from dermal biopsy were evaluated for the specific hallmarks of cellular senescence and for their response to the DNA-induced damage. Patient carried the recurrent heterozygous de novo in frame deletion (c.1812_1814delCTC, p.Ser605del ) within POLD1 gene, previously detected in 16 MDPL patients. In patient's fibroblasts we observed severe nuclear envelope anomalies, presence of micronuclei, accumulation of prelamin A, altered cell growth, and cellular senescence. In addition, we observed a persistence of DNA damage after cisplatin exposure, compared to control cells. In conclusion, the MDPL nuclear and cellular findings resemble features observed in other progeroid syndromes and familial lipodystrophies. Although further investigations will be necessary, these information could be used to establish targeted therapeutic approaches.

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.

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.

GC/MS-based Analysis of Volatile Metabolic Profile Along in vitro Differentiation of Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (hiPSCs) are a promising tool in cell-based therapies for degenerative diseases. A safe application of hiPSCs in vivo, requires the detection of the presence of residual undifferentiated pluripotent cells that can potentially cause the insurgence of teratomas. Several studies point out that metabolic products may provide an alternative method to identify the different steps of cells differentiation. In particular, the analysis of volatile organic compounds (VOCs) is gaining a growing interest in this context, thanks to its inherent noninvasiveness. Here, a protocol for VOCs analysis from human induced pluripotent stem cells (hiPSCs) is illustrated. It is based on Solid-Phase Microextraction (SPME) technique coupled with gas chromatography-mass spectrometry (GC/MS). The method is applied to measure the volatile metabolite modifications in cells headspace during cell reprogramming from chorionic villus samples (CVS) to hiPSCs, and along hiPSCs in vitro differentiation into early neural progenitors (NPs), passing through embryoid bodies (EBs) formation.

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), ß-thalassemia (ß-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.

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.

Cftr gene targeting in mouse embryonic stem cells mediated by Small Fragment Homologous Replacement (SFHR).

Different gene targeting approaches have been developed to modify endogenous genomic DNA in both human and mouse cells. Briefly, the process involves the targeting of a specific mutation in situ leading to the gene correction and the restoration of a normal gene function. Most of these protocols with therapeutic potential are oligonucleotide based, and rely on endogenous enzymatic pathways. One gene targeting approach, "Small Fragment Homologous Replacement (SFHR)", has been found to be effective in modifying genomic DNA. This approach uses small DNA fragments (SDF) to target specific genomic loci and induce sequence and subsequent phenotypic alterations. This study shows that SFHR can stably introduce a 3-bp deletion (deltaF508, the most frequent cystic fibrosis (CF) mutation) into the Cftr (CF Transmembrane Conductance Regulator) locus in the mouse embryonic stem (ES) cell genome. After transfection of deltaF508-SDF into murine ES cells, SFHR-mediated modification was evaluated at the molecular levels on DNA and mRNA obtained from transfected ES cells. About 12% of transcript corresponding to deleted allele was detected, while 60% of the electroporated cells completely lost any measurable CFTR-dependent chloride efflux. The data indicate that the SFHR technique can be used to effectively target and modify genomic sequences in ES cells. Once the SFHR-modified ES cells differentiate into different cell lineages they can be useful for elucidating tissue-specific gene function and for the development of transplantation-based cellular and therapeutic protocols.